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} | s2 | Production of Functional High-protein Beverage Fermented with Lactic Acid Bacteria Isolated from Korean Traditional Fermented Food
The aim of this study was to manufacture functional high protein fermented beverage, using whey protein concentrate (WPC) and Lactobacillus plantarum DK211 isolated from kimchi, and to evaluate the physicochemical, functional, and sensory properties of the resulting product. The fermented whey beverage (FWB) was formulated with whey protein concentrate 80 (WPC 80), skim milk powder, and sucrose; and fermented with Lactobacillus plantarum DK211 as single, or mixed with Lactococcus lactis R704, a commercial starter culture. The pH, titratable acidity, and viable cell counts during fermentation and storage were evaluated. It was found that the mixed culture showed faster acid development than the single culture. The resulting FWB had high protein (9%) and low fat content (0.2%). Increased viscosity, and antioxidant and antimicrobial activity were observed after fermentation. A viable cell count of 109 CFU/mL in FWB was achieved within 10 h fermentation, and it remained throughout storage at 15℃ for 28 d. Sensory analysis was also conducted, and compared to that of a commercial protein drink. The sensory scores of FWB were similar to those of the commercial protein drink in most attributes, except sourness. The sourness was highly related with the high lactic acid content produced during fermentation. The results showed that WPC and vegetable origin lactic acid bacteria isolated from kimchi might be used for the development of a high protein fermented beverage, with improved functionality and organoleptic properties.
Introduction
Probiotic bacteria has been shown to have effects in enhancement of the immune system, and in prevention of gut, vaginal, and urogenital infections, diarrhea, and gastritis, by inhibiting enteric and foodborne microbial pathogens (Walsh et al., 2010). Consumers' interest in functional foods containing probiotic bacteria and prebiotics has created a huge market, and their market share is still expanding (Rathore et al., 2012;Walsh et al., 2010). The consumer request for food with nutritional benefits is also quickly improved, which has generated the manufacture of various value-added dairy products (Shiby et al., 2013). Recently, the sales of ready-to-drink (RTD) protein drinks have been increased through mainstream supermarket distribution.
The value of proteins as an essential source of amino acids is well documented, but recently it has been recog-nized that dietary proteins exert many other functionalities in vivo, by means of biologically active peptides. Such peptides are inactive within the sequence of the parent protein, and can be released by digestive enzymes during gastrointestinal transit, or by fermentation or ripening during food processing (Korhonen, 2009). In particular, milk proteins are regarded as a source of energy and of essential amino acids, which are needed for growth and maintenance of physiological functions (Unal and Akalm, 2012).
Whey protein, a by-product recognized as a valuable food ingredient with important nutritional and functional properties, is gaining acceptance as a functional food ingredient. Commercial whey proteins are considered a GRAS (Generally Recognized As Safe) substance for food product applications (Sinha et al., 2007). A wide variety of whey ingredients are available for use in the manufacture of yogurt and fermented beverages, including sweet whey powder (SWP), whey protein concentrate (WPC), whey protein isolate (WPI) and specialized WPCs (Hugunin, 2008). Whey proteins possess high biological value, and are superior to other proteins, such as those of egg, soy and caseins of milk, mainly due to their high content of branched essential amino acids (Pescuma et Whey-based lactic beverages represent an emerging segment of non-conventional dairy products that require sensory, physical, and chemical characterization for quality control and product development (Almeida et al., 2009). However, consumer acceptance of these health drinks depends on the development of nutritional beverages that maintain their desirable appearance, texture, and flavor characteristics during storage and consumption (Shiby et al., 2013). Numerous formulations of liquid products based on whey proteins have been developed to improve their characteristics (Almeida et al., 2009;Athanasiadis et al., 2004;Djurić et al., 2004;Pescuma et al., 2010: Shiby et al., 2013. However, the protein contents of products in their studies are less than 5%. Whey proteins that are not modified to have more heat stability will not be stable as the sole ingredient at levels above 3% protein, that is, they will gel or precipitate under high heat treatment (Rittmanic, 2008). A typical yogurt and Greek yogurt provides on average 3 and 6.7 g protein/100 g serving, respectively. The protein contents in commercial protein drinks or shakes are mostly between 6 and 10%.
The aim of this study was to formulate a novel functional fermented whey beverage containing high protein content, by using whey protein, and lactic acid bacteria isolated from Korean traditional fermented foods. However, a potential issue with culture is the use of vegetableorigin probiotic in a milk-based product containing high protein content; consequently, this probiotic would be potentially unsuited to growth in milk-based product. Moreover, the incubation time is highly related with the production capacity in plant. The reduction in fermentation time could increase the production capacity of plant, and significantly reduce production costs (Hugunin, 2008). A commercial strain, Lactococcus lactis R704 was selected for mixture use with vegetable-origin lactic acid bacteria isolated from kimchi in previous study, as it is extensively used for the industrial production of fermented milk, cheese, and yogurt (Khalid et al., 2011).
Hence, this study was conducted (1) to optimize the conditions to develop a fermented whey beverage with acceptable organoleptic properties, (2) to determine functional properties, such as antioxidant activity and antimicrobial activity, and (3) to assess the shelf life of the product.
Strains and materials
The strain, Lactobacillus plantarum DKL 121 was iso- Whey protein concentrate (WPC 80) and skim milk were purchased from Sung Poon Co. (Korea) and Seoul milk (Korea), respectively. Table 1 shows the compositions of these dairy ingredients. Sucrose was obtained from CJ Co. (Korea). All other chemicals and reagents used were of analytical grade, and were purchased from Sigma-Aldrich (USA).
Fermentation condition
Samples were withdrawn every 2 h during 24 h incubation, for the measurements of pH, titratable acidity, and viable cell counts. Bacterial growth was observed with enumerated viable colony at the MRS agar plates incubated at 37°C for 48 h.
Manufacture of fermented whey beverage
Fermented whey beverage was prepared, using 11% (w/ v) WPC 80, 2% (w/v) skim milk powder, and 10.3% sugar. Each strain was subcultured triplicate in MRS broth at 37°C. All dry ingredients were dissolved in sterile water, and homogenized with a homomixer (IKA, Japan) at 10,000 rpm. This mixture was then pasteurized at 70°C for 30 min, cooled to about 40°C, inoculated culture at a rate of 20 mL/L (10 8 CFU/mL), and fermented at 37°C for 10 h. The resulting FWB was distributed in sterile glass bottles, and stored at 4, 10, and 15°C for 28 d. Viable cell count, pH, and titratable acidity were measured after 0, 7, 14, 21, and 28 d of storage. The FWBs containing commercial strains (LH 166 or ST-Body 1) were also prepared as above, except for the fermentation time. The fermentation time of FWB with Lactobacillus helveticus LH 166 and that with ST-body were 13 h and 14.5 h, respectively. Assay for proteolytic activity Proteolytic activity was assessed by an agar diffusion method. Milk agar plates containing 1.6%(w/v) skim milk and 1.5% (w/v) agar were prepared. Three hundred μL of each strain at a final concentration of 10 8 CFU/mL was inoculated onto plates. After incubation at 37°C for 48 h, absence of an inhibitory zone was observed. Each plate was examined for clear zones.
Measurement of physical properties
The measurement of pH was conducted at room temperature, using a digital pH meter (Orion 3 star, Thermo Scientific, Korea). Titratable acidity expressed as a percentage of lactic acid was measured by titrating 9 mL of the sample with 0.1 N NaOH, until the substance reached to the end point of the phenolphthalein. The viscosity was measured by a dynamic viscometer (RVDI, Brookfield, USA).
Measurement of chemical analysis
The organic acid contents were determined using highperformance liquid chromatography (HPLC), consisting of UV/visible detector (Waters 410, Waters, USA). Five mL of samples were mixed with 5 mL of 0.0085 N H 2 SO 4 , and stood at room temperature for 2 h. This mixture was centrifuged at 4,000 g for 10 min, and then filtered with a syringe filter (pore size 0.22 μm). The chromatographic analysis was conducted using hydrosphere C 18 column (4.6 mm × 150 mm, particle size 5 μm, YMC, Japan). The mobile phase used was 0.0085 N H 2 SO 4 . The chromatographic separation of samples (20 μL) was performed at a constant flow rate of 0.6 mL/min. The concentration was calculated by using the peak area obtained with standard organic acids (acetic acid and lactic acid).
Measurement of DPPH radical scavenging activity
This method is based on reduction of the free radical DPPH (1,1-diphenyl-2-picrylhydrzyl). A 0.2 mM/L DPPH radical solution in 99% ethanol was prepared. A 100 μL aliquot of each sample was added to 1 mL of 0.2 mM DPPH solution containing 50 μL of ethanol. After gentle mixing, the solution was incubated at 37°C for 30 min. The absorbance was measured at 515 nm, using an UV spectrophotometer (X-ma 1200, Human Co., Korea). Trolox was used as a reference antioxidant, at a concentration of 0.1 mg/mL. Free radical scavenging activities were calculated as
Measurement of ferric reducing antioxidant power (FRAP)
This method was used to measure the reductive power of samples. The FRAP reagent was prepared by mixing 300 mM sodium acetate buffer (pH 3.6), 10 mM 2,4,6tripyridyl-s-triazine (TPTZ), and 20 mM FeCl 3 at a ratio of 10:1:1 (v/v/v). One mL of each sample was mixed with 100 μL of trichloroacetic acid with vortexing, then centrifuged for 10 min at 9,300 g. The supernatant (40 μL) was mixed with 1.2 mL of FRAP reagent and 20 μL of distilled water; and then this mixture was incubated at 37°C for 5 min. The absorbance was measured at 593 nm using a spectrophotometer, using deionized water as the blank. Trolox was used as the positive control. The results were expressed as micromoles of Fe(II), using a linear calibration curve obtained with different concentrations of FeSO 4 .
Antimicrobial activity assay
Staphylococcus aureus and Salmonella enterica were used as test strains, to evaluate the antimicrobial effects of FWB samples. These strains were incubated in LB broth for 18 h at 37°C, then 0.15 mL of cell culture was spread onto LB agar. The paper disc (diameter, 10 mm) was soaked with 100 μL of each FWB sample. The soaked paper discs were placed on the surface of each plate. After incubation at 37°C for 18 h under aerobic condition, each plate was examined for the clear inhibition zones around the paper disc.
Sensory evaluation
The samples were subjected to sensory evaluation. The control was a commercial protein drink, a banana-flavored processed milk fortified with milk protein concentrate (MPC). Thirty-five panelists were employed, and the members of this panel were students selected for their familiarity with yogurt products, but who received no training at all, in order to represent the average consumer. The FWB and commercial product were presented to panels in identical plastic cups labeled with random numbers, and in random order. Each panelist was asked to rate the samples for the four attributes (overall taste, flavor, offflavor, freshness, and aftertaste) using a 7-point hedonic scale (1="I dislike extremely", 7="I like extremely"). The intensities of sweetness, sourness, and off-flavor were evaluated using a 7-point "just-about-right" scale, ranging from 1="much too mild", to 7="much too strong". Statistical analysis was conducted using ANOVA, where the significant effect was found. Significance of differences between samples was determined on the basis of Turkey's test, at a significance level of p<0.05. The statistical analysis was performed using Statistica 8.0 software (StatSoft Inc., USA).
Results and Discussion
Selection of culture In previous study, a total of 106 colonies were isolated from Kimchi samples. Of them, 60 colonies were identified as Lactobacillus species, through observation of phenotypic characters, and identification using API kit and 16S rRNA gene sequencing. Further studies on acid tolerance, bile tolerance, and proteolytic activity were conducted, to select strains for commercial use as probiotics and starter culture in dairy products. It was found that L. plantarum DK 211 had tolerance under acid and bile conditions, as its viable cell count remained at pH 2.0 and under 1.0 % bile salt condition, after 2 h incubation time (data not shown). Other studies (Lee et al., 2011; Lim and Im, 2009) conducted on L. plantarum strain isolated from kimchi, also indicated that L. plantarum strain had acid and bile tolerance, compared with other species.
The proteolytic system of lactic acid bacteria is essential for their growth in milk and ripening of fermented milk products, because it leads to the liberation of peptides and amino acids, which consequently has considerable effects on the physical structure, taste, and flavor development (Beganović et al., 2013;Cho et al., 2013;Khalid and Marth, 1990). Skimmed milk agar plate assays allow principally for qualitative determinations of protease activity, as the hydrolysis zone produced on the skim milk agar could be related to the amount of protease produced by the bacteria (Vijayaraghavan and Vincent, 2013). For the determination of proteolytic activity, a zone of proteolysis was detected on the skimmed milk agar plates. As shown in Fig. 1, this strain possesses strong proteolytic activity, as it produced larger clear zones on milk agar than commercial Lactobacillus plantarum strain, LP-5. It showed similar proteolytic activity to 911 LC. The high proteolytic activity of 911LC was proven by the study of Ko et al. (2005). The proteolytic activities of all cultures studied in this study followed a timedependent pattern.
Growth patterns of culture Whey proteins will gel or precipitate under high heat treatment, when they are used at levels above 3% protein as the sole ingredient. Therefore, whey protein beverages are generally formulated to a pH of less than 4.5, because they are subjected to a mild thermal pasteurization process (Rittmanic, 2008). The current minimum standard for yogurt manufacture is about 0.9% acidity, at a final pH between 4.2 and 4.5 (Campagne et al., 2010).
The growth kinetic of L. plantarum DK 211 was estimated by measuring the pH, titratable acidity, and viable cell count during fermentation. The growth curve obtained by measuring the pH and titratable acidity was used to estimate at what point in time the bacteria reached to pH 4.5. As shown in Fig. 2, the pH of culture media sharply decreased, until it reached to pH 4.5 at 18 h of incubation. When mixture culture of L. plantarum DK 211 and Lactococcus lactis was used, there was almost 30% reduction High viable counts are necessary to get the desired acid production and reduction in pH, which affect organoleptic properties of the final product and its shelf-life, and prevent product contamination (Rathore et al., 2012). During fermentation, the population of L. plantarum DK 211 rapidly increased at the beginning, and the growth reached a stationary phase after 10 h (Fig. 3). The viable cell counts increased from an initial value of 6.5 Log CFU/mL, to above 9.0 Log CFU/mL at 10 h of incubation time. On the other hand, the population of mixed culture of L. plantarum DK 211 and L. lactis R704 reached above 9 Log CFU/mL at 6 h of incubation time. An ideal starter culture should be quick and steady in acid production (Cho et al., 2013). Campagne et al. (2010) also stated that mixing strains could affect the growth and acidification rates, which are important benefits for sanitary and economic standpoints. According to these results, mixed culture of L. plantarum DK 211 and L. lactis R704 with 10 h of fermentation time was selected for the preparation of FWB in the further study. Table 2, the protein, carbohydrate, and fat of FWB were 9.0, 11.5, and 0.2%, respectively. The protein content of FWB showed almost twice higher than that of commercial yogurt products, and was similar with that in commercial protein drinks or shakes. Regarding viscosity, fermentation by lactic acid bacteria caused increasing viscosity of the FWB produced. According to Hassan et al. (2012), the viscosity behavior is related to the pH, so the increase in viscosity takes place at acidic value.
Characteristics of FWB As shown in
One of the characteristic features of fermentation by lactic acid bacteria is the production of different organic acids, due to the degradation of some components in the raw material, and the resultant decrease in pH (Wu et al., 2011). Therefore, the organic acid profile in fermented dairy foods is an indicator of the metabolic activity of added bacterial cultures. These acids act as natural preservatives, and contribute to the characteristic sensory properties (Adhikari et al., 2002). Lactic acid contents greatly increased as much as 3.5 times during fermentation, while there was less increase in the content of citric acid. These results were similar with those of Adhikari et al. (2002), in which there was a 3-fold increase in the content of lactic acid during fermentation. However, in their study, the unfermented mix had a higher amount of citric acid than the fermented product. They explicated that it was because of the utilization of citric acid by the yogurt starter during fermentation. FWB sample stored at 4°C had slight acid development during the storage period, while the samples stored at 10 and 15°C showed post-fermentation acidification, as the pH decreased from 4.5, to around 3.7 at 28 d of storage (Fig. 4). A similar behavior was suggested by Almeida et al. (2009), who observed that the post-acidification behavior of most co-cultures was faster in a milk-whey base.
To exert beneficial health effects, the amount of probiotic bacteria in the food product should be adequately high, i.e., 10 6 -10 8 CFU/mL throughout the entire shelf life (Najgebauer-Lejk, 2014). Table 3 shows that the level of probiotics in this study maintained 10 9 CFU/mL during 28 d of the storage period, regardless of the storage temperature, which was beyond this criterion.
Functional properties of FWB Antioxidant activity has been reported for milk proteins, sodium caseinate and whey protein concentrate, yoghurt, and lactic acid bacteria (Unal and Akalm, 2012). Two antioxidant assays were employed to measure the antioxidant activity of FWB, i.e., DPPH and FRAP assay. DPPH assay is used to evaluate the ability of antioxidant to scavenge free radicals, which is known to give reliable information concerning the antioxidant ability of the tested compounds (Zha et al., 2009). Transition metal ions are also involved in many oxidation reactions in vivo, as the reduced form of iron causes oxygen toxicity, by converting the less reactive hydrogen peroxide to the more reactive oxygen species, such as hydroxyl radical, via the Fenton reaction (Unal and AKalm, 2012;Zha et al., 2009). The FRAP assay measures the reduction of ferric to ferrous ion at low pH in the presence of antioxidants, which causes a colored ferrous-tripyridyltriazine complex to form (Gliszczynska-Swiglo, 2006).
To ensure that the measured antioxidant activity was produced during fermentation, the antioxidant activity of non-fermented whey beverage was measured. The DPPH scavenging activity of samples ranged from 10.2% (nonfermented sample) to 42.07%, when Trolox at a concentration of 0.1 mg/mL showed a DPPH scavenging activity of 68.93% (Fig. 5A). Regardless of the starter culture, all fermented whey beverage samples had a higher DPPH scavenging activity than the non-fermented one, indicating that fermentation by lactic acid bacteria increased antioxidant levels. FRAP values in all fermented products were also 3 times higher than in the non-fermented one (Fig. 5B). Increased scavenging effect of FWB might be due to antioxidant peptides released during the fermentation of milk by lactic acid bacteria.
Unal and Akalm (2012) who studied the antioxidant activity of yoghurt fortified with different types of milk protein, revealed that whey protein concentrate showed higher scavenging activity than sodium calcium caseinate. They stated that high scavenging activity of the whey proteins could be attributed to lactoferrin, α-lactalbumin, and β-lactoglobulin. Species of Lactobacillus being widely All values are mean±SD of triplicates. used as starter cultures in the dairy industry have been reported to play a significant role in the production of bioactive peptides in fermented dairy products (Ramesh et al., 2012). Farvin et al. (2010) also indicated that the milk peptides act as electron donors, and could react with free radicals, to convert them to more stable products. The strong proteolytic activity of L. plantarum DK 211 was already observed in Fig. 1. Therefore, the antioxidant activity of FWB fermented with the mixture culture of L. plantarum DK 211 and Lactococcus lactis could be due to the peptides produced by the action of L. plantarum DK 211. The antibacterial activity of FWB prepared with selected strains was estimated by the agar diffusion method. A strong inhibitory effect of FWB on the growth of Stapylococcus aureus and Salmonella enteritica was detected (Table 4). It has recently been reported that Lactobacillus sp. strains inhibit the growth of Gram-negative pathogenic bacteria, which has generally been attributed to the production of lactic acid during the Lactobacillus growth (Nikolova et al., 2009).
Sensory evaluation
By the addition of a high amount of whey protein, FWB had higher protein content as much as twice, and significantly lower fat content, than the commercial product. However, FWB had a low acceptability score, because of the unappealing taste and excessive acidity of whey protein, which consequently affects the overall taste of the product in preliminary study (data not shown). Djurić et al. (2004) also stated that numerous procedures have been developed for characteristics of whey protein. This defect can be overcome by adding flavor components, such as vanilla or fruit flavor. Vanilla flavor was selected to add to the final product, because this flavor was found through the preliminary sensory test to be better matched with FWB than fruit flavors, such as banana and strawberry (data not shown). Table 5 shows the mean panelists rating of the sensory attributes of the FWB and control. There were no significant differences in the ratings of all attributes, except sourness. The FWB was found to contain sour characteristics, which seemed highly correlated with the lactic acid content. The stinky smell of whey protein in FWB seems to have been improved by the addition of vanilla flavor, because there were no significant differences in the ratings of aftertaste and off-flavor with the commercial product. Sensory evaluation of FWB showed that it was a favorable product, as the panelist ratings for overall taste and aftertaste were above the mid-point.
The results obtained in this study provide evidence that a novel high-protein beverage having functional properties and acceptable organoleptic character could be produced by fermentation of whey protein using L. plantarum DK 211, a vegetable-origin lactic acid bacterium. | v3-fos |
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} | s2 | Objects in Contact with Classical Scrapie Sheep Act as a Reservoir for Scrapie Transmission
Classical scrapie is an environmentally transmissible prion disease of sheep and goats. Prions can persist and remain potentially infectious in the environment for many years and thus pose a risk of infecting animals after re-stocking. In vitro studies using serial protein misfolding cyclic amplification (sPMCA) have suggested that objects on a scrapie-affected sheep farm could contribute to disease transmission. This in vivo study aimed to determine the role of field furniture (water troughs, feeding troughs, fencing, and other objects that sheep may rub against) used by a scrapie-infected sheep flock as a vector for disease transmission to scrapie-free lambs with the prion protein genotype VRQ/VRQ, which is associated with high susceptibility to classical scrapie. When the field furniture was placed in clean accommodation, sheep became infected when exposed to either a water trough (four out of five) or to objects used for rubbing (four out of seven). This field furniture had been used by the scrapie-infected flock 8 weeks earlier and had previously been shown to harbor scrapie prions by sPMCA. Sheep also became infected (20 out of 23) through exposure to contaminated field furniture placed within pasture not used by scrapie-infected sheep for 40 months, even though swabs from this furniture tested negative by PMCA. This infection rate decreased (1 out of 12) on the same paddock after replacement with clean field furniture. Twelve grazing sheep exposed to field furniture not in contact with scrapie-infected sheep for 18 months remained scrapie free. The findings of this study highlight the role of field furniture used by scrapie-infected sheep to act as a reservoir for disease re-introduction although infectivity declines considerably if the field furniture has not been in contact with scrapie-infected sheep for several months. PMCA may not be as sensitive as VRQ/VRQ sheep to test for environmental contamination.
Introduction
Classical scrapie is a transmissible spongiform encephalopathy (TSE) of sheep and goats, which is caused by infection with a proteinacious infectious particle, the prion (1). Prions are particularly resistant to commonly used disinfectants and inactivating methods and can remain biologically active, which increases the risk of re-infection after re-stocking.
Soil has been implicated as plausible reservoir of scrapie prions because laboratory studies have shown that scrapie-associated prion protein (PrP Sc ) can bind to soil particles, which may remain close to the surface where it was released so that it is accessible to grazing animals (2). Indeed, pastures not used by a scrapieinfected flock for more than a month remained contaminated and led to infection of genetically fully susceptible "clean" sheep, even if exposed for only 6 weeks (3). However, the scrapie agent was reportedly also able to re-infect sheep in a building that had not been used for 16 years (4); thus, reservoirs other than soil contribute to scrapie transmission.
While contamination of the environment had historically been mainly attributed to prions in placentae from scrapie-infected ewes (5), it has now been established by PrP Sc detection methods that various sources may contribute to environmental contamination, such as urine (6), feces (7), saliva (8), and possibly rubbed off skin (9), which explains the lateral transmission of scrapie to sheep where no lambing has occurred (10,11). However, less is known about the vectors that facilitate disease transmission, particularly in buildings that have previously housed scrapie-infected sheep.
Prions were detected by serial protein misfolding cyclic amplification (sPMCA) in swabs taken from water troughs and fences that had been present on a scrapie-infected sheep farm, and it was hypothesized that they could act as environmental reservoirs to contribute to scrapie transmission (12). The current study was designed to investigate the persistence of environmental scrapie infectivity using a sheep bioassay. As there was surprisingly little evidence of environmental contamination on pastures used previously by a scrapie-infected flock after re-introduction of new sheep, this study aimed to confirm the relevance of field furniture as a vector for scrapie transmission by exposing sheep to objects that had been in contact with a scrapie-infected flock.
Materials and Methods
All procedures were in accordance with the United Kingdom (UK) Animal (Scientific Procedures) Act 1986, under license from the UK Government Home Office (project licenses 70/6774 and 70/7782) following approval by the institutional ethical review process.
Scrapie-Infected Flock
A scrapie research flock was established in 1998 by purchasing clinically healthy sheep from scrapie-affected flocks with scrapiesusceptible genotypes and maintained through breeding sheep of susceptible genotypes (10). At the time of the project, more than 50% of the sheep in the flock were homozygous VRQ (valine at codon 136, arginine at 154, and glutamine at 171 of the ovine prion protein gene), which is associated with high susceptibility to classical scrapie (13), and developed clinical disease from 18 months of age. Sheep were kept on pastures for many years, being kept in sheds just before lambing and occasionally in severe weather. The pasture was managed on a rotation basis and was plowed and reseeded as required. Manure and composted bedding from the lambing sheds was incinerated and not spread on the pasture. For logistical reasons, sheep with clinical signs suggestive of scrapie were predominantly kept on one particular pasture to enable closer monitoring until cull at clinical end-stage.
Classical Scrapie-Free Flock
Testing for infectivity was carried out using Cheviot sheep from a flock that was originally generated from imported sheep from New Zealand and kept free of classical scrapie through strict biosecurity measures (14), which was confirmed routinely by testing of culled sheep's brains for TSEs (Bio-Rad TeSeE; Bio-Rad Laboratories, UK). All sheep were homozygous VRQ.
Level of Contamination of Field Furniture in Clean Housing
Field furniture that had been in contact with the scrapie flock 8 weeks earlier was used. Selection of items was to some degree influenced by the results from an earlier study where environmental sources of scrapie prions were investigated by sPMCA and which had demonstrated the presence of PrP Sc on a fire extinguisher box (referred to as plastic scratching post in Reference 12), wooden fence post, and fencing (12) 2 years earlier. Sample extractions from objects and sPMCA methodology were carried out as described previously (12). Briefly, eight swabs were taken for each surface, a swab sample consisted of a wetted foam swab that had been rubbed 10 times over an area of approximately 10 cm × 2 cm, and these were stored frozen at −80°C until extraction. Samples were extracted using a silicon dioxide enrichment methodology (15), the extracts from two swabs being finally eluted in 200 µl of 0.1% w/v SDS solution, of which 10 µl was then used to seed each sPMCA reaction. Each extracted sample was amplified in triplicate or duplicate reactions for nine sPMCA rounds using a substrate consisting of a 10% healthy VRQ/VRQ sheep brain homogenate [a different brain substrate to that used in Ref. (12)]. The 0.2-ml PCR tubes containing the extracts and substrate were placed in an ultrasonicating water bath (model S4000; Misonix, USA) at 37°C. Sonications were performed for 40 s at 200 W and these were repeated once every 30 min for 24 h (one PMCA round). After each PMCA round, the samples were diluted 1 in 3 with fresh substrate in a final volume of 100 µl and the process repeated up to a total of nine rounds. Reaction products were then digested with 50 µg/ml proteinase K (PK) with the addition of 0.045% (wt/vol) SDS for 1 h at 37°C before western blot analysis using 12% (wt/vol) NuPAGE precast Bis-Tris gels. Reactions were scored positive if a PK-resistant triplet was visible on the western blot after probing with the antibody SHa31-and HRP-based chemiluminescent detection.
For in vivo testing of the infectivity of objects from pastures grazed by the scrapie flock, 24 VRQ/VRQ lambs 1-5 days of age (83% aged 2 days) were housed in a building that had never been used for any classical scrapie-infected sheep. The lambs, which were kept with their dams until 3-4 months of age, were housed in four pens with group sizes determined by availability of sheep and housing capacity as follows: Pen 1. Five lambs in a pen with clean furniture (building controls). Pen 2. Six lambs in a pen with a water trough from a pasture used mainly by subclinically infected sheep of the scrapie flock. Pen 3. Six lambs in a pen with a water trough from a pasture, which was used by clinically affected sheep prior to cull. Pen 4. Seven lambs in a pen with a wooden fence post, fencing with traces of fleece from sheep attached to it and a fire extinguisher box. This box had been fixed outside an area where the sheep from the scrapie-infected flock would gather just before entering the handling area and had been used by sheep to rub their backs and head.
None of the items were cleaned but the water troughs were emptied and filled with new water in the animal accommodation. All items placed into pens 2-4 were swabbed and analyzed by sPMCA in triplicate before arrival of the sheep. Swabs were taken from analogous surfaces to the scrapie-contaminated farm, e.g., water troughs, metal hurdles, etc., at the farm housing the classical scrapie-free flock to serve as control samples, which were tested in parallel with the samples from the scrapie-contaminated objects.
The pens shared common air space but were separated by 3 m high concrete walls, and each pen had its own entrance and separate equipment and protective clothing to avoid crosscontamination.
Scrapie infection was monitored by rectal biopsy at approximately 6, 9, 13, and 19 months post exposure (mpe). Biopsies were taken under local anesthesia with a prilocaine and lidocaine mixture (EMLA cream; AstraZeneca, UK) and the recto-anal mucosaassociated lymphoid tissue (RAMALT) examined for presence of disease-associated prion protein (PrP Sc ) with rat monoclonal antibody R145 (APHA, UK) according to previously published methods (16). For the rectal biopsy at 6 mpe, lambs were sedated with 0.8 ml acepromazine (ACP injection 2 mg/ml; Novartis Animal Health, UK) given intramuscularly approximately 30 min prior to the biopsy due to their restlessness. Sheep were examined for signs of scrapie according to a short clinical protocol (17) prior to cull from 23 mpe, whereby sheep were assessed by a veterinarian for signs of abnormal behavior (e.g. separation from others), gait (e.g. ataxia), and sensation (e.g. reaction to scratching with head or lip movements, impaired menace response) and -depending on the display of signs -classified as clinical suspect, inconclusive, or clinically healthy with regards to scrapie.
Scrapie infection was determined postmortem by immunohistochemical examination of lymphoreticular tissue (RAMALT, distal ileum, mesenteric lymph node) in all animals and -in all sheep over 12 months of age -additional examination of brain tissue (right and left half of the obex) for PrP Sc by immunohistochemistry (18) and for the proteinase-resistant form of PrP Sc (PrP res ) by ELISA (Bio-Rad TeSeE; Bio-Rad Laboratories, UK) according to the manufacturer's instructions.
Exposure of Sheep to Contaminated Furniture on Pasture in Paddock 1
For the field study, a 5.4 ha-sized pasture that had been occupied by scrapie sheep was divided into four equal paddocks, with two paddocks (termed 1 and 2) used for the reported study. Each paddock, measured approximately 13,000 m 2 , was double fenced with new fencing, with a minimum of 2 m between the fences to prevent nose-to-nose contact between sheep in different paddocks. Each paddock had its own set of protective clothing for farm workers and equipment.
Contamination of the soil in both paddocks used for the reported study was previously investigated by moving groups of six 2-day-old lambs with their dams to these paddocks, each equipped with a new water trough. None of the six lambs in each group had detectable PrP Sc in a rectal biopsy taken at 6 and 9 mpe and although they were subsequently moved to a different paddock at 10 mpe, none of the sheep presented with detectable PrP Sc in brain and lymphoid tissue when culled at 34 months of age.
Contaminated field furniture added to paddock 1, which by that time had not been grazed by scrapie-affected sheep for 40 months, were metal hurdles, a metal lamb creep and a water trough, replacing the previous one, which all had been in contact with the scrapie flock 8 weeks earlier.
The paddock was occupied by 24 lambs aged 1-4 days (67% aged 2 days) with their dams, with the exception of one ewe that arrived with a 14-day-old lamb delivered by cesarean section. Dams were removed 3-4 months later and not tested for scrapie. Rectal biopsies were taken at approximately 9 mpe and examined as described above. Based on the RAMALT results (see Results), all sheep were culled from 11 mpe and examined for scrapie by postmortem tests as before.
Swabs were taken as described above from water trough, fence, wooden post, hurdles, and lamb creep 5 days after the first sheep were introduced in the paddock and examined for PrP Sc by sPMCA in duplicate reactions.
Effect of Replacement of Contaminated Furniture on Pasture in Paddock 1
Twelve 18-to 28-day-old lambs (median 21 days) were moved with their dams to paddock 1, which had been occupied up to 7 days previously by sheep to test for infectivity of contaminated furniture (see above), but clean furniture was now provided (water trough from a paddock not used by scrapie sheep before, fencing replaced). The increase in age was due to poor weather conditions at the time of lambing. Dams were removed as before after 3-4 months. The sheep were culled from 11 mpe and tissues examined by TSE postmortem tests, as described above.
Exposure of Sheep to Weathered Contaminated Furniture on Pasture in Paddock 2
Concurrently with the furniture replacement study, 13 12-to 25-day-old lambs (median 21 days) moved with their dams to paddock 2, which had been grazed by the natural scrapie flock 53 months previously, and contained a water trough and additional objects (lamb creep and hurdles used during lambing of the scrapie flock) from the scrapie-infected flock, which had not been used by these sheep for 18 months. Removal of dams and TSE testing protocol was identical to the furniture replacement study.
Statistical Tests
Where appropriate infection rates between groups were compared by Fisher's exact test (Prism 6, GraphPad Software, USA), with P < 0.05 classified as statistically significant (adjusted in the case of multiple comparisons by multiplying the P value following each test by the number of comparisons).
Results
The results of the sheep studies are summarized in Table 1.
Level of Contamination of Field Furniture in Clean Housing
Infection of sheep was first demonstrated in RAMALT at 9 mpe in a water trough-exposed group (water trough previously exposed to clinically infected sheep) and at 13 mpe in the rubbing objectsexposed group. Scrapie was confirmed in four sheep on brain examination by immunohistochemistry (three exposed to the water trough, one to rubbing objects) but only one was also confirmed by ELISA. This sheep was the only sheep that presented with clinical signs prior to cull at 23 mpe. Evidence of infection in the other sheep was based on presence of PrP Sc in lymphoid tissue only.
Despite evidence of infection in the sheep exposed to one of the water troughs and the scratch objects, the difference compared to the control group was statistically not significant (P = 0.14 and P = 0.24, respectively, after adjusting for multiple comparisons).
PrP Sc was detected by sPMCA in swabs taken from water trough and fencing (see Table 2) in an assay that could consistently detect PrP Sc within 10 pg of ovine brain from a clinically affected animal. However, PrP Sc was also detected in 2 out of 139 reactions from environmental swab samples taken from the classical scrapie-free farm, and the difference in the sPMCA results between scrapie-affected and scrapie-free farm was statistically not significant (P = 0.124).
Exposure of Sheep to Contaminated Furniture on Pasture in Paddock 1
PrP Sc was first detected in RAMALT of 18 (78%) sheep. When the sheep were culled 3 months later at 11-12 mpe, scrapie was confirmed by postmortem tests on lymphoid tissue in 20 (87%), six of which also had PrP Sc in the obex (no detectable PrP res by ELISA). Figure 1 gives an example of a sheep with confirmed scrapie infection compared to a sheep in the same paddock with no evidence of infection. None of the swabs taken from the furniture yielded detectable PrP Sc by sPMCA ( Table 2), even though other analogous environmental samples (from inside farm buildings) run concurrently for a different study yielded positive results (data not presented).
Effect of Replacement of Contaminated Furniture on Pasture in Paddock 1
Scrapie infection was confirmed in a single sheep (8%), which presented with PrP Sc in lymphoid tissue only. Compared with exposure of sheep to contaminated furniture in the same paddock, this was a significant reduction in the infection rate (P < 0.0001).
Exposure of Sheep to Weathered Contaminated Furniture on Pasture in Paddock 2
Exposure to objects not used by scrapie-infected sheep for 18 months did not result in any infection.
Discussion
Classical scrapie is an environmentally transmissible disease because it has been reported in naïve, supposedly previously unexposed sheep placed in pastures formerly occupied by scrapie-infected sheep (4,19,20). Although the vector for disease transmission is not known, soil is likely to be an important reservoir for prions (2) where -based on studies in rodents -prions can adhere to minerals as a biologically active form (21) and remain infectious for more than 2 years (22). Similarly, chronic wasting disease (CWD) has re-occurred in mule deer housed in paddocks used by infected deer 2 years earlier, which was assumed to be through foraging and soil consumption (23).
Our study suggested that the risk of acquiring scrapie infection was greater through exposure to contaminated wooden, plastic, and metal surfaces via water or food troughs, fencing, and hurdles than through grazing. Drinking from a water trough used by the scrapie flock was sufficient to cause infection in sheep in a clean building. Exposure to fences and other objects used for rubbing also led to infection, which supported the hypothesis that skin may be a vector for disease transmission (9). The risk of these objects to cause infection was further demonstrated when 87% of 23 sheep presented with PrP Sc in lymphoid tissue after grazing on one of the paddocks, which contained metal hurdles, a metal lamb creep and a water trough in contact with the scrapie flock up to 8 weeks earlier, whereas no infection had been demonstrated previously in sheep grazing on this paddock, when equipped with new fencing and field furniture. When the contaminated furniture and fencing were removed, the infection rate dropped significantly to 8% of 12 sheep, with soil of the paddock as the most likely source of infection caused by shedding of prions from the scrapie-infected sheep in this paddock up to a week earlier.
This study also indicated that the level of contamination of field furniture sufficient to cause infection was dependent on two factors: stage of incubation period and time of last use by scrapieinfected sheep. Drinking from a water trough that had been used by scrapie sheep in the predominantly pre-clinical phase did not appear to cause infection, whereas infection was shown in sheep drinking from the water trough used by scrapie sheep in the later stage of the disease. It is possible that contamination occurred through shedding of prions in saliva, which may have contaminated the surface of the water trough and subsequently the water when it was refilled. Contamination appeared to be sufficient to cause infection only if the trough was in contact with sheep that included clinical cases. Indeed, there is an increased risk of bodily fluid infectivity with disease progression in scrapie (24) and CWD (25) based on PrP Sc detection by sPMCA. Although ultraviolet light and heat under natural conditions do not inactivate prions (26), furniture in contact with the scrapie flock, which was assumed to be sufficiently contaminated to cause infection, did not act as vector for disease if not used for 18 months, which suggest that the weathering process alone was sufficient to inactivate prions.
PrP Sc detection by sPMCA is increasingly used as a surrogate for infectivity measurements by bioassay in sheep or mice. In this reported study, however, the levels of PrP Sc present in the environment were below the limit of detection of the sPMCA method, yet were still sufficient to cause infection of in-contact animals. In the present study, the outdoor objects were removed from the infected flock 8 weeks prior to sampling and were positive by sPMCA at very low levels (2 out of 37 reactions). As this sPMCA assay also yielded 2 positive reactions out of 139 in samples from the scrapie-free farm, the sPMCA assay could not detect PrP Sc on any of the objects above the background of the assay. False positive reactions with sPMCA at a low frequency associated with de novo formation of infectious prions have been reported (27,28). This is in contrast to our previous study where we demonstrated that outdoor objects that had been in contact with the scrapie-infected flock up to 20 days prior to sampling harbored PrP Sc that was detectable by sPMCA analysis [4 out of 15 reactions (12)] and was significantly more positive by the assay compared to analogous samples from the scrapie-free farm. This discrepancy could be due to the use of a different sPMCA substrate between the studies that may alter the efficiency of amplification of the environmental PrP Sc . In addition, the present study had a longer timeframe between the objects being in contact with the infected flock and sampling, which may affect the levels of extractable PrP Sc . Alternatively, there may be potentially patchy contamination of this furniture with PrP Sc , which may have been missed by swabbing. The failure of sPMCA to detect CWD-associated PrP in saliva from clinically affected deer despite confirmation of infectivity in saliva-inoculated transgenic mice was associated with as yet unidentified inhibitors in saliva (29), and it is possible that the sensitivity of sPMCA is affected by other substances in the tested material. In addition, sampling of amplifiable PrP Sc and subsequent detection by sPMCA may be more difficult from furniture exposed to weather, which is supported by the observation that PrP Sc was detected by sPMCA more frequently in indoor than outdoor furniture (12). A recent experimental study has demonstrated that repeated cycles of drying and wetting of prion-contaminated soil, equivalent to what is expected under natural weathering conditions, could reduce PMCA amplification efficiency and extend the incubation period in hamsters inoculated with soil samples (30). This seems to apply also to this study even though the reduction in infectivity was more dramatic in the sPMCA assays than in the sheep model. Sheep were not kept until clinical end-point, which would have enabled us to compare incubation periods, but the lack of infection in sheep exposed to furniture that had not been in contact with scrapie sheep for a longer time period supports the hypothesis that prion degradation and subsequent loss of infectivity occurs even under natural conditions.
In conclusion, the results in the current study indicate that removal of furniture that had been in contact with scrapie-infected animals should be recommended, particularly since cleaning and decontamination may not effectively remove scrapie infectivity (31), even though infectivity declines considerably if the pasture and the field furniture have not been in contact with scrapieinfected sheep for several months. As sPMCA failed to detect PrP Sc in furniture that was subjected to weathering, even though exposure led to infection in sheep, this method may not always be reliable in predicting the risk of scrapie infection through environmental contamination. These results suggest that the VRQ/VRQ sheep model may be more sensitive than sPMCA for the detection of environmentally associated scrapie, and suggest that extremely low levels of scrapie contamination are able to cause infection in susceptible sheep genotypes.
Author Contributions
TK analyzed the data and drafted the manuscript. HS designed and managed the study, supported by SH, LT, and TD who participated in the coordination of different parts of the study. BM and KG were responsible for the sPMCA investigations. All authors contributed to the final draft of this manuscript and read and approved the final manuscript.
Funding
The study was funded by the UK Department for Environment, Food and Rural Affairs (project SE1861). | v3-fos |
2015-09-18T23:22:04.000Z | {
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} | s2 | Unmasking of Olive Oil Adulteration Via a Multi-Sensor Platform
Methods for the chemical and sensorial evaluation of olive oil are frequently changed and tuned to oppose the increasingly sophisticated frauds. Although a plethora of promising alternatives has been developed, chromatographic techniques remain the more reliable yet, even at the expense of their related execution time and costs. In perspective of a continuous increment in the number of the analyses as a result of the global market, more rapid and effective methods to guarantee the safety of the olive oil trade are required. In this study, a novel artificial sensorial system, based on gas and liquid analysis, has been employed to deal with olive oil genuineness and authenticity issues. Despite these sensors having been widely used in the field of food science, the innovative electronic interface of the device is able to provide a higher reproducibility and sensitivity of the analysis. The multi-parametric platform demonstrated the capability to evaluate the organoleptic properties of extra-virgin olive oils as well as to highlight the presence of adulterants at blending concentrations usually not detectable through other methods.
Introduction
Olive oil is the most popular vegetable oil produced and consumed in Mediterranean countries. According to international standards [1], olive oils have to be obtained exclusively from the fruit of the olive tree (Olea europaea) using cold pressing techniques and in conditions that do not alter the organoleptic properties of the oil at all. Current European Union regulation [2] and the International Olive Committee (IOC) require olive oils to be graded in function of sensory assessment and three fundamental chemical parameters: free acidity, peroxide value, and UV absorbance [2]. By comparing oils scores with threshold values, these are classified as extra virgin olive oil (EVOO), virgin olive oil, and other low-quality olive oil typologies. Olive oil is a very complex matrix [3,4]. The main compounds are triacylglycerols and fatty acids contributing to 94%-96% of their total weight. However, triacylglycerols and fatty acid contents show a broad variability in olive oils chemical composition and this is largely dependent on both cultivar and geographical origin [5]. Recently, the authentication of products labeled as olive oil has become a fundamental issue for either commercial or health aspects [6,7]. In fact, the high price of olive oil and its increased popularity as a potential health food have made it an ideal target for frauds [8]. Common olive oil adulterations include accidental contaminations during production stages, deliberate mislabeling of less expensive oil categories and, more often, the admixtures of expensive olive oils with low quality oils. Although advances in knowledge and technology have undoubtedly led to greater success over frauds, even more complex forms of adulteration have been developed to invalidate the usefulness of official methods, thus leaving the authenticity verification still an unsolved matter [9]. Actually, no rapid and universal method exists that is officially recognized for all the authenticity issues [10]. Liquid and gas chromatographic techniques represent the elective methods for the authentication and characterization of individual olive oil compounds [11][12][13][14][15]. Nevertheless, these analytic verifications require valuable instrumentation and highly-qualified staff. All of these features together make authentication a time consuming and expensive process which is not applicable as routine analysis. In this context the BIONOTE (BIOsensor-based multisensorial system for mimicking Nose, Tongue and Eyes), a recently developed sensor platform [16], has been employed. The system, which embeds gas and liquid sensors having a common biologically-derived sensing interface, allows the simultaneous analysis of the vapor and liquid phase of the samples. As a consequence, the integrated multi-sensorial platform led different sensors to catch more comprehensive information which, in turn, requires a further elaboration through multivariate data analysis techniques. At the end of the analytical procedure, similarities and differences between the samples are highlighted. In this multi-parametric study, the correct discrimination of twelve EVOOs made up of dissimilar olive cultivars and having different geographical origin has been achieved. Furthermore, the high sensitivity and reproducibility of the analysis, which were guaranteed by the innovative electronic interface of the system, permitted the detection of fraudulent admixing of extraneous vegetable oils (pomace, soybean, sunflower seeds, and peanut oils) up to concentrations lower than 5%. These promising results altogether present BIONOTE as a rapid and economic tool for high-throughput screening analysis.
Oil Samples
Twelve EVOO samples, indicated in the paper as EVOO #1, #2, #3, and so on, were obtained from twelve different Italian orchards. Several characteristics of the oils are reported ( Table 1). The commercial EVOO as well as the pomace, soybean, sunflower seeds, and peanut oils were bought at a local market.
Gas Analysis
Quartz Micro Balances (QMBs) with six functionalized piezoelectric sensors were used as transducers for the gas sensor array as already described [16]. In order to perform homogeneous gas measurements the following experimental set-up was used. A volume of 2 mL for each olive oil sample was placed in a 50 mL glass flask and kept for 10 min at room temperature to obtain an adequate headspace. Dehumidified reference air was pumped into the sensors chamber at a flow rate of 3 L/min for 10 min to desorb any volatile trace from sensors surface before every measure. Oil samples were analyzed five times, setting a sampling interval of 90 s.
Liquid Analysis
Electronic interface and sensors employed in the liquid analyses were the same described in Santonico et al. Cyclic voltammetry in the range from −1 to 1 V was performed using a triangular function at 10 mHz and a sampling interval of 1 second. Olive oil samples for liquid sensor analysis were prepared following the procedure reported below. Briefly, a volume of 1 mL of oil was poured into a tube with 3 mL of methanol 70% (v/v) and mixed vigorously for 1 min. The vial containing the oil-alcohol emulsion was centrifuged for 5 min at 1000 RCF and 4 °C to separate the two phases efficiently. Finally, the methanol phase was collected and stocked in ice up until the analysis.
Chemical Quality Control Analyses
Polyphenol content, free acidity, peroxide value, ∆K, and refractive index of olive oil samples have been assessed following the standard chemical testing methods [15]. Briefly, polyphenol content was evaluated by means of Folin-Ciocalteu method, according to the procedure reported by Singleton and Rossi [17]. Free acidity content [18] was evaluated, dissolving the samples in a mixture of equal parts by volume of ethyl ether (95%) and ethyl alcohol, thus titrating with an ethanolic solution of potassium hydroxide, using phenolphthalein as indicator. Results were reported as grams of oleic acid per 100 g of oil.
To determine the peroxide value [19], oil samples were dissolved in chloroform and glacial acetic acid, then a solution of potassium iodide was added, leaving the mixture incubating for five minutes in the dark, and finally a titration of the generated iodine with a standard sodium thiosulphate solution, using starch solution as indicator, was performed. The peroxide value was expressed in terms of milliequivalents of active oxygen per kilogram able to oxidize potassium iodide under the operating conditions. The quality of the olive oils employed in this study was also assessed measuring the absorption bands between 200 and 300 nm [20]. Samples were dissolved in iso-octane to obtain 1% (w/v) solutions and the specific absorbance at 232 and 270 nm with reference to pure solvent was determined. These absorptions were expressed as specific extinctions, conventionally indicated by K. Finally, a ∆K value was calculated relating the maximum recorded absorbance at 270 nm against the absorption of surrounding spectral region (±4 nm). The refractometric index of olive oils was determined using the Abbé refractometer, paying attention to correct the recorded value on a temperature basis. Three independent parameter's determinations were carried out for each test sample. All the reagents used in this study were of certified analytical quality.
Data Analysis
Multivariate data analysis: Principal Component Analysis (PCA) and Partial Least Square Discriminant Analysis (PLS-DA), was performed using PLS-Toolbox (Eigenvector Research Inc., Manson, WA, USA) in the Matlab Environment (The MathWorks, Natick, MA, USA). PLS-DA models have been calculated in order to detect EVO adulteration and investigate BIONOTE relevance to the chemical parameters.
Olive Oil BIONOTE Characterization
Twelve Italian EVOOs having different geographical origin and olive variety compositions have been characterized through the BIONOTE system, performing five measuring cycles each. Gas analysis was performed on EVOOs without any modification of the samples. Volatile compounds released in the system headspace at room temperature were characterized through their interaction with the functionalized sensors, resulting in a reproducible pattern response (Figure 1). Olive oil as such is not applicable for electrochemical analysis due to the absence of conductivity and the high viscosity of the media. Therefore, oil samples underwent liquid extraction with methanol and the deriving alcoholic fractions were analyzed by the liquid sensor ( Figure 1). Cyclic voltammetry in the range from −1 to 1 V was performed using a triangular function at 10 mHz and a sampling interval of 1 second. By means of this setup, an array of 100 virtual sensor responses has been obtained from one physical sensor for each voltammetric measuring cycle. Finally, a data fusion of the information deriving from the last three measuring cycles of gas and liquid sensors was accomplished. The obtained data set has been evaluated by Principal Component Analysis (PCA) and the ability of the system to sharply discriminate the twelve EVOOs was demonstrated. The score plot of the first two Principal Components (PCs), accounting for 76.94% of the explained variance, is reported ( Figure 2). Ten of the twelve oil samples clustered in three separate regions along the Principal Component 2 (PC2). EVOOs #1, #6, and #12 formed a group in the bottom part of the plane. EVOOs #5, #8, #10, and #11 distributed in a second area at the interception of the two PCs. EVOOs #2, #4, and #9 clustered in the upper portion of the plane (Figure 2). Nevertheless, within the groups almost every oil sample can be discriminated from the others along the Principal Component 1 (PC1). EVOOs #3 and #7 were distinguished from the rest of the analyzed samples by positioning at the upper end and at the left edge of the plane, respectively ( Figure 2). Additionally, a Partial Least Square Discriminant Analysis (PLS-DA) model using the leave one out criterion has been calculated showing a correct classification rate of 100% for the twelve different EVOOs (five independent repetitions each).
Olive Oil Chemical Characterization
To assess the quality of the EVOOs, common chemical analyses were also performed. All the EVOOs got parameters satisfying the imposed normative limits, even though some slight differences between the samples were found (Table 2), thus supporting BIONOTE discrimination evidence. Free acidity and ∆K values were significantly lower than normative standard ones being, however, slightly different among each other. The refractive index of the twelve oil samples was almost the same, while the peroxide parameter showed the greatest variability. The obtained results confirmed the excellent quality of the oil samples, highlighting the absence (in terms of usual parameters) of significant differences between the EVOOs themselves.
Olive Oil Adulteration
A commercial EVOO was bought at local market and mixed with four vegetable oils (pomace, soybean, sunflower seeds, and peanut oils) at different blending concentrations (1.25%, 5%, 10%, and 25% (v/v)). The prepared EVOO's admixtures were characterized through the BIONOTE system, performing five measuring cycles each. Sophisticated EVOO samples were treated as already described (see Materials & Methods section) before being analyzed through either the liquid or the gas sensors. A comprehensive array containing the overall sensors' responses was built for each EVOO sophistication independently and the collected data were further analyzed using multivariate data analysis techniques. The calculated PLS-DA models highlighted the ability of the system to distinguish an authentic EVOO from an adulterated one in all the tested cases, showing also a rather high degree of efficiency in the concentration discrimination ( Figure 3). BIONOTE was able to predict the presence of contaminating lower-grade oils up to concentration values lower than 10% (v/v). The Root Mean Square Error in Cross Validation (RMSECV), using the Leave One Out criterion, was slightly different among the four kinds of sophistication. System performance was almost the same for the soybean, sunflower seeds, and peanut oils with RMSECV ranging from 2.1% to 4.4%, while the discrimination of the pomace oil sophistications resulted less precise accounting for an error of 8.3% (v/v) (Figure 3).
BIONOTE Relevance to the Chemical Parameters
BIONOTE relevance to the measured chemical parameters have been investigated by calculating four different models to predict polyphenols content, free acidity, peroxide value, and TEAC on the gas and liquid sensor array data. The results obtained are very promising (see Figure 4
Discussion
Adulteration is a common problem usually related to high-value products. As a consequence of the fundamental role in the Mediterranean diet and the documented nutraceutical effect [6], EVOO represents a clear target for sophistication aimed to trade. According to recent studies, adulteration is becoming an escalating issue for olive oil in the market with consequences undermining the quality attributes of the product and sometimes even its safety consumption [21]. Although reliable and accurate analyses intended to guarantee olive oil quality in the broadest sense already exist, these are not routinely used. While chemical parameters as free acidity, peroxide value, ∆K, and refractive index are necessary to define if an olive oil fulfills the requirements to be labeled and marketed as EVOO, these constraints are not sufficient for authenticity verification in the most of cases [1,22]. Fraudulent olive oil admixtures are usually chemically corrected to meet international standards, thus requiring more complex analyses to be recognized as adulterations. Nevertheless, even when official analytical methods are applied to screen olive oil samples, olives' biological differences, due to geographical origin and genetic aspects, sometimes generate problems to distinguish between sophistications and authentic EVOOs [23]. So far, numerous modern techniques have been proposed to support or replace official standard methods in the task of olive oil authentication [10,[24][25][26][27][28]. However, those do not offer clear advantages yet, because their adulteration detection limits, being usually greater than 10% of contamination, are worse in comparison with chromatographic techniques' ones. In this study, a novel system able to characterize EVOOs in terms of genuineness and authenticity has been presented. The BIONOTE platform takes advantage of either liquid and gas analysis to accomplish a multi-parametric characterization, giving comprehensive information about the sample [17]. The overall sensors' responses are elaborated through multivariate data analysis techniques to highlight similarities and differences, resulting in a correct classification rate of 100%, even when similar EVOOs have been analyzed. Hence, BIONOTE showed the ability to discriminate between twelve Italian EVOOs originating from different Apulian neighboring olive tree orchards. The result highlighted the capability of BIONOTE not only to identify EVOOs against lower grade olive oils, but also to discriminate between EVOOs obtained from different olive cultivars. This is a notable outcome because this issue is usually addressed via more complex genetic approaches. The innovative electronic interface, providing to the system a higher reproducibility and sensitivity comparable to similar devices [29][30][31][32], allowed BIONOTE to be also successfully employed in the authenticity verification process, with admixtures percentage thresholds below the best levels reported by literature. BIONOTE was challenged with different kind of EVOO sophistications, covering concentrations lower than 10% (v/v), and in all cases it was able to distinguish authentic oil from an adulterated one. The system detected the presence of fraudulent admixing of extraneous vegetable oils (soybean, sunflower seeds and peanut oils) up to concentrations lower than 5%. However, when the pomace oil was used, system performance decreased. This discrepancy, leading to an increment of the detection limit to about 8%, could be probably explained by the shared origin between EVOO and pomace oil. Considering the demand of EVOO traceability and safety claimed by both producers and consumers, BIONOTE represents a potential solution. In fact, the BIONOTE system is able to address the EVOO authenticity issue focusing not only on the labeling control but also the genuineness of the oil, accounting for geographical origin and olive varieties composition at the same time.
Conclusions
Nowadays, global markets and international regulations have increased significantly the number of samples that require validation, raising the necessity of rapid analytical methods. In this context, BIONOTE could represent a real opportunity thanks to its reduced time of analysis. However, due to the profiling approach on which the system is based on, BIONOTE has not been intended to replace the high specificity of the official chromatographic methods. Hence, it is proposed as a rapid tool for preliminary high-throughput screening, aimed to detect samples that require further analytical verifications. This workflow has been designed to reduce the employment of high-value instrumentation and qualified personnel only to specific cases, thus decreasing the costs, while maintaining the elevated number of samples analyzed.
Author Contributions
Marco Santonico and Giorgio Pennazza conceived and designed the experiments and analyzed the data; Simone Grasso and Francesco Genova performed the experiments and wrote the paper; Alessandro Zompanti and Francesca Romana Parente contributed analysis tools. | v3-fos |
2018-04-03T00:50:38.551Z | {
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} | s2 | Slowing the Spread of Grapevine Leafroll-Associated Viruses in Commercial Vineyards With Insecticide Control of the Vector, Pseudococcus maritimus (Hemiptera: Pseudococcidae)
Vineyards were surveyed for grapevine leafroll-associated viruses and their insect vectors in New York State’s Finger Lakes region in 2006–2008. Grape mealybug, Pseudococcus maritimus (Erhorn) (Hemiptera: Pseudococcidae), European Fruit Lecanium, Parthenolecanium corni (Bouche), and Cottony Maple Scale, Pulvinaria acericola (Walsh and Riley) (Hemiptera: Coccidae) were identified as vector species in this region. An increase in the incidence of Grapevine leafroll-associated virus 1 (GLRaV-1) and GLRaV-3 was observed in 8 of the 20 vineyards surveyed, which implies transmission by these insect vectors. Two of the vineyards for which a temporal increase in disease incidence was documented were then used to evaluate the efficacy of foliar applications of horticultural oil and two classes of insecticides for control of P. maritimus and for slowing virus spread over 2 years of vine protection. Delayed dormant applications of horticultural oil contributed to control of early season crawlers; however, this was not the case for control of summer populations. Applications of acetamiprid and spirotetramat achieved control in summer populations; however, spirotetramat outperformed acetamiprid in percent reduction of treated compared with control vines and in a side-by-side trial. Vines treated with spirotetramat had a lower percentage of new vines testing positive for GLRaV-1 than control vines after 2 years, while no other spray program altered the increase in incidence of GLRaV-1 or -3.
Grapevine leafroll disease negatively affects vineyard productivity and quality of wine and table grapes in all major grape-growing regions worldwide (Charles et al. 2006, Rayapati et al. 2014. Disease symptoms such as downward rolling of leaf margins and interveinal discoloration are most likely to be observed late in the season and are more conspicuous on red varieties than on white varieties. Infection delays fruit maturity, decreases the productive life of the vineyard, and reduces fruit yield and quality by way of altered sugar content, acidity, pigmentation, and phenolic profiles (Goheen and Cook 1959, Over de Linden and Chamberlain 1970, Credi and Babini 1997, Martelli 2014, Rayapati et al. 2014. Several phloem-limited filamentous viruses in the family Closteroviridae have been identified in leafroll-diseased vines as grapevine leafroll-associated viruses (GLRaVs) including GLRaV-1, -3, and -4 from the genus Ampelovirus, GLRaV-2 from the genus Closterovirus, and GLRaV-7 from the genus Velarivirus (Martelli 2014, Rayapati et al. 2014). Considered to be the main etiological agent contributing to the disease, GLRaV-3 is the most ubiquitous species worldwide (Maree et al. 2013) as well as the primary GLRaV species in New York vineyards (Fuchs et al. 2009b). Viruses are disseminated through propagation material, so infected planting material is often the initial source of infection in vineyards. However, ampeloviruses are also spread by phloem-feeding insects, such as soft scales and mealybugs (Coccidae and Pseudococcidae, respectively; Golino et al. 2002. Infection is spread when insects acquire the pathogen from feeding on infected vines then walk to neighboring vines or are spread by vineyard equipment or wind dispersion (Habili et al. 1995, Habili and Nutter 1997, Cabaleiro et al. 2008, Grasswitz and James 2008). The primary vector species in North America is the grape mealybug, Pseudococcus maritimus (Erhorn) (Hemiptera: Pseudococcidae) . P. maritimus overwinters as first-instar nymphs or eggs on canes and trunks and emerges around bud swell to feed and develop to adulthood and their offspring hatch in mid-summer (Grimes andCone 1985, Geiger andDaane 2001). Although all life stages are capable of transmitting GLRaVs, the first-instar "crawlers" are considered the most efficient vectors because of their high relative mobility compared with older stages, which are typically sessile upon reaching adulthood (Mahfoudhi et al. 2009, Tsai et al. 2010). There are two generations of P. maritimus in most regions, therefore two periods of high crawler activity which are the times of greatest concern for virus management .
Historically, foliar applications of organophosphate and carbamate insecticides were recommended during peaks of crawler activity, but many of these materials have lost their efficacy due to resistance or are no longer commercially available in the United States (Flaherty 1982, Grimes and Cone 1985, Bentley et al. 2006. Moreover, mealybugs can be particularly difficult to control with contact insecticides since they are often in protected areas under loose bark. Horticultural oils or broad-spectrum foliar insecticides are somewhat limited in their efficacy due to this behavior (Geiger andDaane 2001, Grasswitz andJames 2008). Broad-spectrum insecticides also negatively affect nontarget insects, natural enemies, and pollinators (Walton and Pringle 1999). More recent work has investigated alternatives for control of mealybugs including biological control, mating disruption, insect growth regulators, and systemic insecticides , Franco et al. 2009).
Current management options of leafroll disease incur high costs in labor and materials, i.e., removing infected vines (roguing) and replacing with vines derived from certified virus-tested stock to reduce sources of inoculum (Atallah et al. 2012, Pereira-Crespo et al. 2012. Effective insecticidal control of mealybug vectors could slow the progression of the disease through affected vineyard blocks, thereby reducing the need for expensive vine removal and replacement initiatives, although this approach has not been rigorously tested. New York's Finger Lakes region is an area of significant wine and grape juice production with a known history of grapevine leafroll disease (Fuchs et al. 2009b). Here we report on field surveys of more than 20 commercial vineyards that confirm P. maritimus as the primary mealybug species in New York's Finger Lakes region. We also report on field trials evaluating the efficacy of horticultural oil and two classes of systemic insecticides for control of P. maritimus and the effect of P. maritimus control on the rate of GLRaV spread in two commercial vineyards.
Materials and Methods
Vector Surveys. Twenty-three commercial vineyards in New York's Finger Lakes Region were scouted for soft scale insects and mealybugs from mid-June to mid-July 2006, and 17 vineyards were scouted in June 2007. A range of cultivars was sampled within V. vinifera, Vitis labrusca, and interspecific hybrids. All vineyards were in production and variable in age. Canopy management varied by cultivar and location. Sixteen to 20 sampling points were recorded following a systematic sampling scheme for each vineyard site in 2006. Based on the relatively low counts the previous year, in 2007 we increased the number of assessment points to 25-100 for a more comprehensive sampling effort. Sampling points were evaluated for 2 min by multiple observers who counted insects by continuously scanning under loose bark on trunks and canes. Observers used 10X optical glass binocular magnifiers (Opti Visor, Donegan Optical Company, Lenexa, KS) to aid in recognizing small instars. The presence and total number of soft scale insects and mealybugs were recorded, independent of lifestage. When found, samples of soft scale or mealybug specimens were collected and returned to the laboratory for identification (Kosztarab 1996). Specimens were also sent for confirmation to the USDA Systematic Identification Lab (Beltsville, MD).
Virus Surveys. Commercial vineyards in New York's Finger Lakes Region were surveyed for GLRaV-1 and GLRaV-3 from late August to October in 2006-2008 using a 4 by 5 quadrat sampling approach where leaf samples are collected from vines at four equally spaced locations along a row (near start of row, 1/3 down the row, 2/3 down the row, and near end of row), and this was repeated every five rows for a maximum of 20 quadrats per vineyard block (Fuchs et al. 2009a). There was partial overlap between vineyards surveyed for vectors and for GLRaV-1 and GLRaV-3, but each survey included vineyard blocks not included in the other. Composite samples of three leaves per vine and five vines per quadrat were collected, making a total of 15 leaves per sample. Leaf samples were processed and tested by double-antibody sandwich (DAS) enzyme-linked immunosorbent assay (ELISA) using specific antibodies (Bioreba, Reinach, Switzerland) as previously described (Fuchs et al. 2009a). Substrate hydrolysis was recorded at 405 nm with an absorbance BioTek ELx808TM microplate reader (BioTek, Winooski, VT). Samples were considered positive if their optical density values at 405 nm were at least twice those of healthy controls. A total of 95 vineyard blocks were surveyed in 2006. Based on a low to moderate incidence (<20%) of GLRaV-1 and/or GLRaV-3 documented in 2006 (Fuchs et al. 2009b), 20 out of the 95 vineyards were further surveyed in 2007 and 2008. The number of quadrats with samples testing positive for GLRaV-1 or GLRaV-3 in DAS-ELISA was determined for every vineyard surveyed and 2007 data were compared with 2006 data; similarly, 2008 data were compared with 2007 data. An increase over time in the number of quadrats with infected samples was evidence of an increase in virus incidence in a given vineyard, likely as a result of vector-mediated virus spread.
Vector Management. Insecticide spray programs were evaluated for efficacy on P. maritimus at two commercial vineyards with a history of P. maritimus infestation and an increasing GLRaV incidence over time. The vineyards were managed by the cooperating growers for diseases following standard practices (Weigle and Muza 2013). Growers did not apply insecticides to blocks of grapes used in vector management experiments described below during the study period. An approximately 3 hectare plot of Vitis vinifera, "Chardonnay" at vineyard A (41 41' 5.21" N, 76 44' 36.64" W) was used to evaluate horticultural oil (PureSpray 10E, Petro-Canada, Mississauga, ON), acetamiprid (Assail 30SG, United Phosphorus Inc., King of Prussia, PA), and a combination of the two materials versus a water control during the 2009 and 2010 growing seasons. Vineyard A was cane-pruned using an umbrella kniffin training system (Reynolds and Vanden Heuvel 2009) and comprised older vines (>10 years in age) interspersed with less than 10% replacement vines of younger age. Treatments were assigned to plots using a randomized block experimental design (36 vines per plot ¼ 4 rows by 9 vines/row), replicated four times per treatment, with horticultural oil applied to vines at delayed dormancy (late April) at a rate of 28 L/ha in 1,870 L/ha of water, and acetamiprid applied at a rate of 0.175 kg/ha in 935 liters/ha of water when the first new summer generation of crawlers was observed on vines (early July). Treatments included oil only, acetamiprid only, oil plus acetamiprid, and water control and were applied with a small plot sprayer and hand gun. Another approximately 3 hectare plot of V. vinifera "Chardonnay" at vineyard B (42 o 28' 22.80" N, 77 o 10' 56.1" W) was used to evaluate a newly labeled systemic insecticide, spirotetramat (Movento, Bayer CropScience, Research Triangle Park, NC) during the 2011 and 2012 growing seasons. Vineyard B was cane pruned using an umbrella kniffin training system and comprised older vines (>10 years in age) interspersed with less than 10% replacement vines of younger age. Treatments (spirotetramat or water control) were assigned to six plots using a randomized block experimental design (64 vines per plot ¼ 4 rows by 16 vines/row), and spirotetramat was applied at a rate of 0.457 liter/ha in 935 liters/ha of water after bud break (early June) and again when the first new generation of crawlers was observed (early July) using a ATV-mounted sprayer with flat fan nozzles on a hand boom. An additional experiment was conducted in a section of vines at vineyard A in 2010 to assess efficacy of acetamiprid and spirotetramat using the same per ha rate of insecticide and amount of water per ha as used in larger plot experiments in a side-by-side comparison using a back pack sprayer with flat fan nozzle with six vine plots and five replicates per treatment. A nonionic surfactant (LI 700, Loveland Products, Greeley, CO) was included in all treatments at a rate of 0.25% by volume.
P. maritimus abundance was recorded after treatments. Spring P. maritimus populations were measured a week after horticultural oil applications at vineyard A by four different observers who counted the number of crawlers present under or nearby the cracked bark of one vine stem at 20 locations per plot. We only sampled vines in the middle sections of the center two rows of each plot thereby avoiding edge vines. At this time of year, crawlers are in exposed sites out on canes and therefore more readily enumerated compared with less-exposed mid-summer crawlers. Summer P. maritimus abundance was measured at all sites in early August by four different observers who conducted 10 timed counts per plot at vineyards A and B where the number of adults and nymphs were counted anywhere on vines for 5 min (Geiger and Daane 2001). We only sampled vines in the middle sections of the center two rows of each plot thereby avoiding edge vines. All observers conducted timed counts in every replicate plot. Observers used optivisors when necessary to confirm presence of small instars.
Effect of Vector Management on Virus Spread. All vines were tested for GLRaV-1 and GLRaV-3 at vineyard A and for GLRaV-1 in vineyard B by DAS-ELISA in September of each year using composite samples of four to six lower canopy leaves per vine. Samples were processed and tested as previously described (Fuchs et al. 2009a). Spirotetramat has been reported to control scale insects for up to 18 months (McKenna et al. 2013). Although no insecticide treatments were applied in the third year at vineyard B, these vines were tested for GLRaV, using the same methods as previously described, to evaluate this spray program for residual effects on disease spread.
Statistical Analysis. All statistical analyses were conducted using JMP (SAS Institute, Cary, NC). Count data from observations of P. maritimus were highly skewed and did not respond to transformation, so effect of year and spray program were evaluated in a generalized log-linear model, assuming a Poisson distribution (O'Hara 2009). There were no statistical differences among observers in the number of P. maritimus recorded at any date, so counts were summed across observer in spring counts (n ¼ 80), summer counts (n ¼ 40), and counts from the side-by-side trial (n ¼ 15). Effects found to be significant were followed by pairwise contrast analysis, using a Bonferroni corrected P value to determine significant difference between means. An analysis of variance followed by Tukey's HSD means separation was used to determine significant difference in the proportion of newly infected vines between treatments in year 2 at vineyard A (four replicates per treatment). A Student's t-test was used to determine significant difference in the proportion of newly infected vines between treatments in year 2 at vineyard B (6 replicates per treatment). Newly infected vines were those that tested positive for GLRaV in year 2 (or three) but were not positive for the virus in year 1.
Results
Only one species of mealybug, confirmed as P. Virus surveys indicated a temporal increase of GLRaV-1 and GLRaV-3 in seven and 2 of the 20 vineyards surveyed, respectively (Table 1). No increase in virus incidence was documented in 12 of the 20 vineyards surveyed from 2006 to 2008. Vineyards A and B were selected for further mealybug management studies because virus spread and the presence of P. maritimus were documented, and virus incidence was moderate to high (25-50%) in 2008. Vines in vineyard A were coinfected with GLRaV-1 and GLRaV-3, while vines in vineyard B were infected with GLRaV-1 (Table 1).
The interaction between year and spray program was found to have a significant effect on the number of P. maritimus observed in the spring at vineyard A (v 2 ¼ 37.70, df ¼ 3, P < 0.0001). Subsequent pairwise contrast tests found no differences between P. maritimus abundance on controls between the two experimental years, but acetamiprid-treated vines had fewer P. maritimus in the second year of the experiment, while there was an increase in the number of P. maritimus observed on vines treated with horticultural oil only. Fewer P. maritimus were observed on vines treated with any spray program than controls in each year, and fewer early season P. maritimus were observed on vines treated with oil and acetamiprid than vines of any other treatment in year 2 ( Table 2).
At vineyard A, the mean percentage of untreated vines that tested positive for GLRaV-1 increased from 29% in 2009 to 42% in 2010 and untreated vines testing positive for GLRaV-3 increased from 69% in 2009 to 90% in 2010. At vineyard B, the mean percent of untreated vines testing positive for GLRaV-1 increased from 26% in 2011 to 35% in 2012. There was no difference among treatments in the increase of newly GLRaV-infected vines in the second year of screening at vineyard A. Spirotetramat treatments slowed the spread of the virus at vineyard B as treated vines had a lower percentage of new vines testing positive for GLRaV-1 than control vines (t ¼ 3.40, df ¼ 5, P ¼ 0.0030; Fig. 1). There was no difference among treated blocks in the percent increase of GLRaV-infected vines in the third year of screening (no insecticide application) at vineyard B.
Discussion
All materials (horticultural oil and two classes of insecticides) tested showed some efficacy against P. maritimus but varied in the level of control. Summertime P. maritimus populations were reduced by 45-66% in vines treated with acetamiprid, and the treatments may have even contributed to reductions in springtime populations in year 2 at vineyard A, before that year's application (Tables 2 and 3). Neonicotinoids are reported to provide multi-year control of sucking insects in woody hosts when used as a soil drench (Szczepaniec and Raupp 2000); however, the long-term residual life of acetamiprid when applied as a foliar is not known. Although horticultural oil did not contribute to the control of summertime P. maritimus populations, this treatment provided some control in the spring of the first year at vineyard A, and combination of horticultural oil followed by acetamiprid was the most successful in reducing early season P. maritimus populations after 2 years of application (Table 2). Vines treated with spirotetramat had a 68-100% reduction in summer P. maritimus populations, and spirotetramat treatments were the only spray program found to slow virus spread (Fig. 1). Spirotetramat outperformed acetamiprid in efficacy against P. maritimus in the side-by-side trial, and both spirotetramat-and acetamipridtreated vines had numerically higher percent P. maritimus reduction than those treated with horticultural oil alone (Tables 2 and 3). Similar control has been reported for the vine mealybug, Plannococcus ficus (Signoret), where both acetamiprid and spirotetramat in combination provided significant reductions in pest population, but spirotetramat generally outperformed the neonicotinoid (Haviland et al. , 2011Sial et al. 2012). Both classes of insecticides have translaminar systemic activity, which increases the probability of phytophagous insect exposure to a foliar insecticide application (Horowitz et al. 1998 , Table 3. Summertime P. maritimus abundance (mean 6 SE mealybugs per observation) in vineyard A for year 1 (2009) and year 2 (2010), in the side-by-side trial (2010) and in vineyard B for year 1 (2011) and year 2 (2012), and percent change in P. maritimus abundance compared with the mean number of mealybugs observed on control vines Bucholz and Nauen 2002, Brück et al. 2009, Gaskin et al. 2010. However, the translaminar transport of acetamiprid does not likely match that of spirotetramat, as spirotetramat is the only product labeled on grapes that is fully systemic when applied to foliage while acetamiprid is generally considered most effective as a systemic insecticide in soil drench applications. Spirotetramat is also reported to reduce fecundity in sublethal doses and was found to provide control of scale for 18 months (Brück et al. 2009, McKenna et al. 2013. However, in our experiment, any extended control from this material did not extend beyond the season of application as indicated by no differences in virus spread at Vineyard B in the third year of testing, a year after spirotetramat was applied. We found that low vector abundance can result in spread of GLRaVs. The mean percent infected vines increased 13% in controls at vineyard A, where 5-10 P. maritimus were observed per 5 min observation. Vineyard B saw a 21% increase in untreated vines, where 3-4 P. maritimus were observed per 5-min observation. Similar rates of spread were seen by Golino et al. (2008) in a Napa Valley, CA. "Cabernet Sauvignon" vineyard where P. maritimus were found at low levels and the number of infected vines increased by an average of 10% per year. Even with very good control of vectors within a vineyard block, virus spread may occur due to migration from outside areas if the population size of vectors and level of disease in surrounding vineyards are high. These results are consistent with insecticidal control of the glassy-winged sharpshooter to limit spread of Xylella fastidiosa in California vineyards (Daugherty et al. 2015). In the case of our experiments, vector populations are relatively low, and there is little evidence of vector movement of virus from vineyard block to vineyard block based on high nucleotide sequence similarity of virus in the vector and the vine from which it is collected (Fuchs et al. 2009a).
P. maritimus was the focus vector species in this study because mealybugs are considered the primary vector in most GLRaV-affected regions; however, each region will have different challenges depending on the vector complex. Although potential vectors were found in the majority of vineyards surveyed in our study, P. maritimus populations were generally low. Moreover, a majority of vineyards in this region (12 out of 20) did not show increases in the incidence of GLRaV infection over three consecutive years (Table 1), suggesting at many sites, vector abundance was below some critical level that promotes measurable disease spread. Improved knowledge of this critical vector population size will be important in assessing the potential of using chemical control of vector populations to prevent spread of leafroll disease. Although P. maritimus rarely reaches economically damaging levels as a direct pest in any region, mealybug species in other regions, which complete more generations in a year, will often reach levels exceeding economic damage to grape clusters (Charles 1982. Higher rates of spread of grapevine leafroll disease are reported in some areas where the vector complexes are made up of these species (i.e., Planaococcus ficus, P. longispinus, and Pl. citri), which further differentiates the Finger Lakes region from other grapevine leafroll disease-affected areas (Engelbrecht and Kasdorf 1990, Jordan 1993, Cabaleiro and Segura 1997, Habili and Nutter 1997. Additionally, our survey found scale more frequently than mealybug in Finger Lake vineyards; more than half of the surveyed sites in both years compared with roughly a quarter where mealybugs were observed. The residual efficacy of the materials used in these experiments should provide control of related vector species as the life histories of these groups would indicate some temporal overlap of crawler periods (Kosztarab 1996, Geiger andDaane 2001), but the control of all species should be considered in each region.
An economic analysis of grapevine leafroll disease management strategies reported by Atallah et al. (2012) suggested greater than the 25% infection as the economic cut-off point for complete vineyard replacement when factoring in the costs of roguing against the cost of removing the entire vineyard and replacing vines with certified virusfree stock. Although this economic analysis did not include costs or benefits of insecticidal control of vectors, the benefit of using insecticidal control in combination with roguing for control of grapevine leafroll disease has been indicated in other studies. Control of vine mealybug (Pl. ficus) using insecticides and roguing with herbicide to kill remnant roots resulted in very good control of grapevine leafroll disease in South African vineyards; however, roguing alone may not remove 100% of infected plant material from blocks as virus may remain in root tissue for several years .
Insecticide use may play a critical role in protecting new grapevines in or near grapevine leafroll disease-affected blocks, and acetamiprid or spirotetramat applications would also provide control of other key pests of grapevine in the Finger Lakes, such as Japanese beetle and other hemipteran pests like grape phylloxera and leafhoppers (Weigle and Muza 2013). These materials are also relatively safer than broadspectrum insecticides for nontarget insects like predators and parasitoids, particularly spirotetramat which could be an integrated pest management compatible insecticide (Broughton et al. 2013, Garcera et al. 2013, Beers and Schmidt 2014.
In conclusion, this study found that insecticidal control of vector insects can slow the spread of GLRaVs when vector abundance was relatively low. However, clearly more research is required to determine under what situations chemical control of vectors, on its own or in combination with other measures such as roguing, should be recommended for managing grape leafroll disease. Early intervention in the life of the vineyard is likely a critical factor, further elevating the importance of using planting material derived from certified virus-tested stock and monitoring to maintain healthy and productive vineyards. | v3-fos |
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} | s2 | Development of the First Cisgenic Apple with Increased Resistance to Fire Blight
The generation and selection of novel fire blight resistant apple genotypes would greatly improve the management of this devastating disease, caused by Erwinia amylovora. Such resistant genotypes are currently developed by conventional breeding, but novel breeding technologies including cisgenesis could be an alternative approach. A cisgenic apple line C44.4.146 was regenerated using the cisgene FB_MR5 from wild apple Malus ×robusta 5 (Mr5), and the previously established method involving A. tumefaciens-mediated transformation of the fire blight susceptible cultivar ‘Gala Galaxy’ using the binary vector p9-Dao-FLPi. The line C44.4.146 was shown to carry only the cisgene FB_MR5, controlled by its native regulatory sequences and no transgenes were detected by PCR or Southern blot following heat induced recombinase-mediated elimination of the selectable markers. Although this line contains up to 452 bp of vector sequences, it still matches the original definition of cisgenesis. A single insertion of T-DNA into the genome of 'Gala Galaxy' in chromosome 16 was identified. Transcription of FB_MR5 in line C44.4.146 was similar to the transcription in classically bred descendants of Mr5. Three independent shoot inoculation experiments with a Mr5 avirulent strain of Erwinia amylovora were performed using scissors or syringe. Significantly lower disease symptoms were detected on shoots of the cisgenic line compared to those of untransformed 'Gala Galaxy'. Despite the fact that the pathogen can overcome this resistance by a single nucleotide mutation, this is, to our knowledge, the first prototype of a cisgenic apple with increased resistance to fire blight.
Introduction
Apple is one of the most important fruit crops worldwide considering its production level of 80.8 million tons per year [1]. Apple production relies on a small number of commercial cultivars. Most of today's successful cultivars such as 'Braeburn', 'Fuji', 'Gala', 'Golden Delicious', 'Jonagold' and 'Cripps Pink' are susceptible to fire blight [2,3] and to other major plant diseases like apple scab. For a long time resistance to diseases was often neglected in many breeding programs as the main focus was on fruit quality combined with optimal agronomical properties [4] and only in the last decades of the previous century, did disease resistance begin to gain relevance [5][6][7][8][9]. This occurred with increasing awareness of the ecological and economical costs of disease management strategies relying on plant protective chemicals only.
High levels of resistance to diseases are often found in wild Malus accessions, e.g. the Rvi6 (alias Vf) resistance gene from M. floribunda clone 821 conferring resistance to apple scab [10] caused by Venturia inaequalis, or the FB_MR5 of M. ×robusta 5 which confers resistance to fire blight [11] caused by Erwinia amylovora. When such resistances are introduced into a breeding program, at least 5 pseudo backcrosses are then necessary to remove the unwanted properties inherited from the wild ancestor (e.g. small fruits) together with the resistance. Considering that the juvenile phase of the domesticated apple lasts between three and twelve years (and linkage drag), the introduction of a resistance from a wild source takes between 20 to 50 years until a cultivar with fruit of marketable quality can be released [12].
The rapid advancement in gene cloning in apple and in genetic transformation methods allows the deployment of resistances by means of genetic modification [13]. In particular cisgenesis may be considered for the goal of adding a particular resistance gene to cultivars of commercial value [14]. Schouten et al. [14] defined a cisgenic plant as a plant that does not contain any transgenes and that has been genetically modified with one or more genes (containing introns and flanking regions such as native promoter and terminator regions in sense orientation), isolated from a crossable donor plant. The recent identification and cloning of several apple resistance genes like Rvi6 [15], Rvi15 [16,17], Pl2 [18], FB_MR5 [19], as well as the development of methods to generate marker-free genetically modified apple plants [20][21][22] are the basic requirements for developing cisgenic apple lines. The first cisgenic apple lines with scab resistance were developed by Vanblaere et al. [23]. Würdig et al. [24] and Krens et al. [25] later developed further cisgenic apple lines with Rvi6.
The most problematic bacterial disease in apple orchards is fire blight, caused by the Gramnegative bacterium Erwinia amylovora (Burrill) Winslow et al. [26]. It causes severe losses in years of heavy epidemics, with economical costs reaching up to several million Euros per year in single European countries or US states [27][28][29].
The use of fire blight resistant cultivars is a tool that is currently not popular in the management of the disease. This is because the very few commercial cultivars showing high levels of resistance to the disease including 'Rewena' [30] and 'Enterprise' [31], do not fully meet retailers and consumers demand, because their level of fruit and tree quality is not comparable with the top world varieties. Good levels of fire blight resistance were found in individual accessions of different wild apple species like M. fusca [32], M. baccata [33], M. prunifolia [34], M. ×atrosanguinea [35], M. ×robusta var. persicifolia [36] and M. sieversii [37] as well as in 'Evereste' [38,39], M. floribunda 821 [38] and Malus ×robusta 5 [11].
In the last decade the resistance of the crab apple genotype Malus ×robusta 5 has been studied in depth. Peil et al. [11] identified a major QTL on linkage group 3 of M. ×robusta 5. Later on, Fahrentrapp et al. [40] identified a candidate resistance gene in this region, which was designated as FB_MR5, and predicted to code for a CC-NBS-LRR resistance protein. The functionality of the FB_MR5 gene under its native promotor and terminator sequences and under the constitutive CaMV 35S promoter and ocs terminator, respectively, was demonstrated [19]. Vogt et al. [41] suggested that this resistance undergoes a gene-for-gene interaction, as a single amino acid substitution in the AvrRpt2 EA gene of E. amylovora, is sufficient to allow the pathogen to become virulent and to overcome this resistance.
In the present study the generation of a cisgenic 'Gala Galaxy' line carrying the FB_MR5 gene showing increased resistance to fire blight is presented. In vitro shoot cultures of 'Gala Galaxy' were transformed with the novel binary transformation vector p9-Dao-FLPi [24], allowing the post-transformation heat induced flippase (Flp) based removal of the excisable cassette containing three transgenes (NptII, Flp and dao1). Although it was not possible to use the selectable marker dao1 to increase the number of regenerants of lines without transgenes, a cisgenic line C44.4.146 was identified. Being FB_MR5 controlled by its native promotor and terminator, this line matches the definition of cisgenic plants of Schouten et al. [14]. The level of fire blight resistance of this line was assessed by means of two different shoot inoculation methods and compared with untransformed 'Gala Galaxy' plants. Further characterization of this line involved the assessment of the number of T-DNA integrations, site of integration in the genome, and transcription level of the FB_MR5 gene in this line compared to conventionally bred genotypes carrying the FB_MR5 gene. To our knowledge, this is the first report of a cisgenic apple with increased resistance to fire blight.
Results
Generation of the cisgenic line C44. 4
.146
From two Agrobacterium-mediated transformation experiments T44 and T45, with vector p9-Dao-FLPi-FB_MR5 (Fig 1), eleven and two transgenic lines were obtained, corresponding to transformation efficiencies (transformants per explants) of 13.8% and 0.7%, respectively. Integration of FB_MR5 (amplicon D, Fig 2) and NptII (amplicon B, Fig 2) was confirmed in all 13 lines and in two cases of T44, integration of backbone sequences was detected (S1 Fig). Those two lines were removed, as lines with backbone usually do not lead to cisgenic plants. Nine out of the eleven remaining transgenic lines (seven from T44 and two from T45) produced sufficient leaf material to be subjected to heat shock. About 3,200 explants were subjected to heat shock activation of the Flp recombinase. The negative D-amino acid / dao1 selection system proposed by Hättasch et al. [42] could not be applied, as it was recently found that the selective medium containing D-Ile hinders the regeneration [24]. On this account PCR was used to screen the obtained putative cisgenic regenerants. Four out of 447 regenerants contained FB_MR5 and were free of selection marker (S2 Fig). Because all four regenerants originated from calli from the same transgenic mother line T44.4, and assuming a consistent excision process, we consider them to be the same genotype. Nevertheless only one shoot of the four regenerants was designated as line C44.4.146 and used for micrografting on 'Golden Delicious' seedlings for further characterization. PCR confirmed the presence of FB_MR5 (amplicon D, Fig 3), absence of both transgenes NptII (amplicon B, Fig 3) and Flp (amplicon C, Fig 3) plus absence of backbone sequences beyond the left border in this line after micrografting (amplicon A, Fig 1).
Copy number of C44.4.146
Using Southern hybridization a single specific band was detected with an NptII probe in the transgenic motherline T44.4, indicating that originally one copy of the T-DNA had been inserted ( Fig 4A). In C44.4.146 this band was not detected confirming excision of the excisable cassette ( Fig 4A). As expected 'Gala Galaxy' showed no hybridization with this probe. Southern hybridization with an FB_MR5 probe allowed the identification of an unspecific band in 'Gala Galaxy', while C44.4.146 and T44.4 showed a single additional FB_MR5-specific band (Fig 4B).
Integration site of C44.4.146
The genomic region flanking the insertion site in line C44.4.146 was identified using iPCR. The sequence from the RB site showed highest level of sequence identity to a sequence of the 'Golden Delicious' contig MDC012271.181 located on chromosome 16. No gene was predicted at the insertion site. Furthermore, mapping the reads generated by Gusberti et al. [43] did not reveal any novel transcript at the insertion site. The closest gene was MDP0000141330 (coding for a transmembrane transporter) at about 600 bp. Primer pairs IS146-1163F/IS146-321R (amplicon F, Fig 5), and IS146-1391R/iPCR-RBout (amplicon G, Fig 5) were designed to characterize the insertion site in line C44.4.146 at left border (LB) and right border (RB), respectively (Fig 2). These primers can be used to discriminate between C44.4.146 or T44.4 lines and the not genetically modified 'Gala Galaxy'. Using the primers IS146-1163F/IS146-1391R (amplicon E, Fig 2), which flank the insertion site, no large fragments (eight kb in C44.4.146 or
Transcription of FB_MR5
Transcription level of FB_MR5 was estimated and compared with two classically bred FB_MR5 accessions (ACW 22161 and ACW 22176). It was observed that the CT values of both target amplicons, EF1α and FB_MR5, differed largely, with CT values for EF1α in the range between 20 and 28, while for FB_MR5 they were between 30 and 50, whereas only few samples showed CT FB_MR5 values above 40 (S1 Table). However we observed no significant difference between transcription ratio in line C44.4.146 and in accession ACW 22176 and only one replicate of 6). This replicate of ACW 22161 (I) also differed significantly from its own biological replicate (III, Fig 6). As a negative control the fire blight susceptible 'Gala Galaxy' was used. No amplification of the FB_MR5 probe was observed (S1 Table).
Fire blight resistance of the cisgenic line C44. 4
Discussion
In the present study a cisgenic apple of the cultivar 'Gala Galaxy' with increased resistance to fire blight was developed. The susceptible cultivar 'Gala Galaxy' acquired fire blight resistance by insertion of the resistance gene FB_MR5 flanked by its 1995 bp native 5'-UTR and 1547 bp It is presumed that E amplifies in 'Gala Galaxy' two alleles while only one is amplified in T44.4 and C44.4.146 (Fig 2 and results) as the second amplicon, containing the inserted T-DNA sequence, is too large to be amplified.
doi:10.1371/journal.pone.0143980.g005 native 3'-UTR. The same regulatory sequences were shown to effectively regulate the function of FB_MR5 in transgenic lines developed by Broggini et al. [19]. Cisgenic transformation was achieved using a novel vector p9-Dao-FLPi [24] for Agrobacterium-mediated transformation allowing the post-transformation heat-induced Flp-based excision of the transgene cassette. As a negative selection marker to exclude cells in which recombination did not occur, the gene dao1, encoding D-aminooxidase 1 [42], is present in the excisable cassette of p9-Dao-FLPi. However, this negative selection marker could not be applied, as it has recently been shown that the use of D-Ile containing medium (to remove cells in which the recombination did not occur) completely hinders the formation of cisgenic shoots and moreover transgenic shoots survived for several months on it [24]. Therefore we decided to regenerate lines without application of the D-Ile / dao1 system, using a regeneration medium without D-Ile and selecting the regenerants by PCR. Thereby cisgenic regenerants can be identified as they result in no PCR product after amplification with primers for the selectable marker cassette, in contrast to transgenic regenerants. Chimeric cisgenic-transgenic plants should lead to amplification of NptII and Flp according to the detection limit of PCR. Despite the hindering circumstances, a cisgenic genotype was identified (Fig 3). Nevertheless, a transformation vector with an effective negative selection would have been of great help to increase the number of regenerating cisgenic genotypes. Substitution of dao1 in p9-Dao-FLPi with codA could result in a pMF1-alternative vector with functional negative selection. This strategy was also proposed by Würdig et al. [24]. An approach with an alternative selectable marker could be the use of the MdMYB10 gene [44] originating from the apple gene pool, leading to visually detectable, red colored calli, shoots and fruits [25].
Line C44.4.146 was regenerated and molecularly investigated. We consider C44.4.146 as a cisgenic line, as PCR revealed that C44.4.146 amplified FB_MR5 but no transgenic selection markers (Fig 3). Southern blot hybridization indicated a single T-DNA insertion in this genotype, as well as the absence of NptII in the final cisgenic line C44.4.146 (Fig 4). On this account we consider the cisgenic line C44.4.146 to carry a single T-DNA insertion. FB_MR5 transcription analysis revealed that the transcription in this line was not different from the transcription in the FB_MR5 accessions ACW 22161 and ACW 22176 (Fig 6). Cisgene insertion in C44.4.146 occurred as a single copy on chromosome 16 (Fig 4). Gene prediction on contig MDC012271.181 indicates that no apple endogenous gene has been disrupted by the T-DNA insertion in line C44.4.146. 85 bps of T-DNA ends were trimmed away (63 bps on LB side, 22 bps on RB side) and T-DNA insertion resulted in the deletion of 36 bps genomic sequences (Fig 2). Trimming of T-DNA ends was also observed in cisgenic apple developed by Vanblaere et al. [45]. The extent of foreign sequences resulting in the final product generated using p9-Dao-FLPi without this trimming is larger than using pMF1 (538 bps vs 140 bps, [20]). While designing novel vectors to generate cisgenic genotypes only foreign sequences shorter than 20 bps can be present in the final product [46] to match the EFSA definition of cisgenic crops used to formulate their safety assessment on cisgenesis [47]. However, a product matching this definition can hardly be achieved when using recombinase recognition sequences (of which one copy remains in the final cisgenic product) that are at least 34 bps long for the Flp / FRT system [48] in p9-Dao-FLPi and 58 bps long for the R / Rs system in pMF1 [20]).
Three different inoculation experiments, using scissors and/or syringe shoot inoculation, showed consistently that line C44.4.146 is significantly more fire blight resistant than the wild type (Figs 7 and 8). The comparison of the results of experiments 1 and 2 seems to indicate that while inoculating using a syringe, more severe fire blight symptoms are induced in the cisgenic line. However, when C44.4.146 and 'Gala Galaxy' plants were inoculated with the two inoculation methods in the same experiment (experiment 3), this effect was no longer observed (Fig 7). Flower susceptibility is very important for controlling the fire blight disease, as most of the infections occur during flowering. Resistance of this cisgenic line upon inoculation of the flowers with the pathogen has still to be investigated.
We discourage the single use of the introduced fire blight resistance gene FB_MR5 by conventional breeding or by cisgenesis for cultivation, as it is known that a single point mutation leading to amino acid substitution in the pathogen can result in a virulent strain [41]. FB_MR5 virulent strains of E. amylovora have already been identified in North America, while such strains have not yet been identified in Europe. The mutation rate of the codon 43 switch of rpsL gene from K to R altering the ribosomal protein S12 leading to streptomycin resistance in most of the strains of E. amylovora was estimated to 4 x 10 −9 per bacterial generation [49]. If we assume the same mutation rate for the switch from C-Allele to the S-Allele of AvrRpt2 EA (leading to FB_MR5 virulent strains) and we consider that E. amylovora populations ranging from 10 3 to 10 7 cfu have been detected on stigmas of flowers of different rosaceous hosts after natural infection [50,51], it can be assumed that Mr5-virulent E. amylovora strains are already present in Europe and can rise very quickly once we start selecting for them (i. e. by planting FB_MR5 genotypes at commercial scale). Therefore it can be anticipated that if used alone, this resistance would not be durable. Whatever use (e.g. classical breeding, transgenesis or cisgenesis) is made of FB_MR5, this resistance must be further combined with other (vertical or horizontal) resistances as suggested by McDonald and Linde [52] and Emeriewen et al. [53].
Experimental Procedures
Generation of the cisgenic line C44. 4
.146
Binary vector p9-Dao-FLPi-FB_MR5 (Fig 1) was constructed by a DNA cloning company (DNA Cloning Service, Hamburg, Germany) and transformed into Agrobacterium tumefaciens strain GV3101pMP90RK. This vector was derived from P9 and was designed to carry on the T-DNA an excisable cassette flanked by FRT recombination sites. This excisable cassette contains the NptII kanamycin resistance selection gene for plant selection, the D-amino oxidase 1 gene (dao1), originally planned to be used as negative selection marker, and the Flp recombinase under control of a heat shock promoter [42]. The same vector carrying HcrVf2 was recently published by Würdig et al. [24]. Instead of HcrVf2 and its native regulatory elements, the FB_MR5 gene (4167 bps) flanked by its 1995 bps native 5'-UTR and 1547 bps native 3'-UTR was cloned, using the same primers and restrictions sites as described by Broggini et al. [19] for generating vector 390p95N-Mr5FB1.
Transgenic plants were regenerated by transforming in vitro explants of the cultivar 'Gala Galaxy' following the protocol as described by Szankowski et al. [54] and Vanblaere et al. [23]. Young leaves of the in vitro plants were cut in explants (a total of 80 in T44 and 300 in T45), co-cultured with the transformed agrobacteria carrying the vector p9-Dao-FLPi-FB_MR5 and regenerated on a medium containing ticarcilin and kanamycin to select for pure (agrobacteriafree), transformed, transgenic calli. Once the plants started to regenerate they were transferred to elongation medium [23] and propagated every four to six weeks. To test if the shoots were successfully transformed and thus to confirm integration of FB_MR5 and NptII, all 13 lines growing on selective medium containing kanamycin were investigated by PCR with the primer pairs FB_MR5q1 F/FB_MR5q1 R (amplicon D, Fig 2) for FB_MR5 and 167nptII-for/367nptIIrev (amplicon B, Fig 2) for NptII. In order to identify lines that contain backbone sequences beyond the left border, PCR using primer pair Bb_LB1/Bb_LB2 (Amplicon A, Fig 1) was performed.
As soon as sufficient differentiated shoots were available, about 3,200 explants of nine transgenic lines were subjected to heat shock in an incubator (four hours at 42°C on regeneration medium without kanamycin) to activate excision of the trangenic cassette containing the selectable marker genes NptII, dao1 and the Flp recombinase gene as well. Following heat treatment explants were cultured on regeneration medium without kanamycin for two weeks in the dark. After this period, explants were put on non-selective elongation medium, like during the transformation procedure, and started to form calli. Elongating shoots were transferred to elongation medium as soon as shoots were detectable or cells started to differentiate. To detect the cisgenic regenerants, they were screened for absence of NptII and Flp and for presence of FB_MR5 by PCR using the method of Frey et al. [55]. For this purpose primers 167nptII-for/367nptII-rev [54], FlpF/FlpR [22] and FB_MR5q1 F/FB_MR5q1 R [56] were used (Table 1 and Fig 2).
Fire blight resistance test of the cisgenic line C44. 4
.146
Between six and twenty plants of each genotype grafted on M9T337, were subjected to fire blight resistance tests with E. amylovora strain EA222_JKI in each of the three independent A shoot of C44.4.146 and non-transformed 'Gala Galaxy' control plants, which simultaneously experienced in vitro culturing were then micrografted on 'Golden Delicious' seedlings following the procedure described by Joshi [57] and acclimatized to greenhouse conditions. This latter genotype is described in this manuscript as "'Gala Galaxy' in vitro". DNA of the micrografted C44.4.146 plant, its motherline T44.4, Malus ×robusta 5 and 'Gala Galaxy' in vitro, was retested by PCR for absence of sequences beyond the left border, presence of FB_MR5 and absence of the selectable marker genes NptII and Flp using the corresponding primers (Figs 2 and 3). experiments performed. Only actively growing plants that reached at least a shoot length of 13.0 cm were considered. Two inoculation methods were used: scissor inoculations (experiments 1 and 3) were performed as described by Peil et al. [11] and syringe inoculation (experiments 2 and 3) as described by Khan et al. [58]. In all three experiments (experiments 1-3) an E. amylovora suspension in phosphate buffered saline with an optical density (OD 600 ) adjusted to about 1.0 (1.06, 0.99, 1.09, respectively), corresponding to about 10 9 cfu / ml, was used. The percentage of lesion length (PLL) was recorded 21 days post inoculation.
Copy number of C44.4.146 Southern hybridization was performed as described by Broggini et al. [19] with minor changes. DNA (10 μg) from each line and plasmid p9-Dao-FLPi-FB_MR5 was digested with 100 units BsaI (Thermo Fisher Scientific Inc. ©, Waltham, USA). Cleaved DNA was separated on a 0.8% agarose gel and transferred onto a nylon membrane (Roche Diagnostics, Mannheim, Germany). A DIG-labelled NptII probe was amplified by PCR using primers (nptII_F/nptII_R) and an FB_MR5 specific probe using primers (FbMr5_SondeF / FbMr5_SondeR, Table 1). Hybridization with each of the probes was performed using the ECF-Random-Prime-Labeling and Detection Kit (Amersham Biosciences, Freiburg, Germany) according to the manufacturer's manual.
Integration site of C44.4.146 Molecular characterization and iPCR for determination of insertion site was performed as described by Vanblaere et al. [45] with the following modifications: i) Backbone integration was assessed by PCR amplification (Fig 3) using primers Bb_LB1 and Bb_LB2 (Fig 1 and Table 1); ii) For iPCR, XbaI-digested DNA of cisgenic genotypes was subjected to ligase reaction and the primers iPCR-RB-XbaI-closer and iPCR-RBout (Table 1) were used to amplify by PCR the junction between T-DNA and genomic sequence at the right border (Fig 2). The resulting PCR product was then sequenced and BLAST analysis against the apple genome [59] was performed. Primers IS146-1163F and IS146-1391R (amplicon E, Fig 5) that flank the insertion site were designed using the sequence of contig MDC012271.181 and used in combination with the primers IS146-LB-321R (amplicon F, Fig 5) or iPCR-RBout (amplicon G, Fig 5) to characterize the T-DNA junctions in the cisgenic line C44.4.146 (Fig 2). Primer sequences are summarized in Table 1.
Transcription of FB_MR5
Transcript levels of FB_MR5 in different genotypes were determined by RT-qPCR. A Taqman Probe with the sequence YYE-TGGCTTCCATTTCAAACGGATCACAGA-BHQ1 was designed to specifically detect FB_MR5 in combination with primers FB_MR5q1 F and FB_MR5q1 R [56]. As reference primers pairs EF1α and relative Taqman probe developed by Gusberti et al. [60] were used. RNA was extracted from three young unfolded leaves of different plants of genotypes C44.4.146, 'Gala Galaxy' in vitro, and the classically bred FB_MR5-carrying accessions ACW 22161 and ACW 22176, using Zymo QuickRNA extraction kit (Zymo Research Corporation, Irvine, USA). Extracted RNA was then subjected to a second DNAse treatment (DNAse Ambion 1 Life Technologies, Carlsbad, USA), after which first strand synthesis was performed using the Fermenta's first strand minus H cDNA synthesis kit. cDNA was then diluted 1 / 10 and 5 μl were used for qPCR on ViiA Ruo qPCR device (Thermo Fisher scientific Inc. ©, Waltham, USA) in a total reaction volume of 20 μl using the Taqman Fast Universal Master Mix (Thermo Fisher Scientific Inc. ©, Waltham, USA). Each reaction had following primer / probe final concentration: EF1α primers at 600 nM, EF1α probe at 400 nM, FB_MR5q1 primers at 900 nM and FB_MR5q1 probe at 250 nM. The same reaction protocol was used to generate standard curves using both dilution series of DNA as well as cDNA from the cisgenic line C44.4.146. Relative expression ratio of FB_MR5 / EF1α was calculated according to Pfaffl [61]. For transcription comparison we excluded three data points with CT FB_MR5 > 40 (two from ACW 22161 II) and one from ACW 22161 III)) as relative expression ratio in standard curves was no longer linear and standard deviation increased above a CT FB_MR5 value of 40.
Statistical analysis
Statistical analysis was performed using software JMP 1 10.0 and 11.0 (SAS Institute INC., Cary, NC) and R [62]. As datasets did not follow normal distribution nonparametric tests were used to compare groups. Transcription level data were compared using the Steel Dwass test. In fire blight inoculation experiments 1 and 2, the Wilcoxon test was performed and in experiment 3 all groups were compared using the Steel Dwass test. Additionally all groups in all three experiments were compared simultaneously using the Steel Dwass test with corrected p-values (Fig 7). One outlier in experiment 2 (C44.4.146 inoculated by syringe) and one in experiment 3 ('Gala Galaxy' inoculated by scissors) were removed from the dataset for the statistical analysis as they differed more than 1.5 interquartile ranges from the corresponding group median ( Fig 7). All statistical analysis were performed with a significance level of α = 0.05. Fig 1). T45.5 did not amplify FB_MR5 (data not shown) and was not further considered. (TIF) S2 Fig. PCR tests to identify cisgenic lines. Amplicons after PCR with primer pair 167nptIIfor / 367nptII-rev (Table 1) indicated that most of the investigated lines still carry NptII (amplicon B, Fig 1) and are therefore transgenic. Three cisgenic lines (239, 240 and 243) were identified and all three originated from the same transgenic motherline (T44.4) as C.44.4.146 (Fig 3). (TIF) S1 Table. Overview of RT-qPCR results. Date when experiment was performed (date). Sample: 'Gala Galaxy' (Gala) Cisgenic line C44.4.146 (Cis), conventionally bred genotypes ACW 22161 (22161) and ACW 22176 (22176). Biological replicates are indicated (A, B or C). Target: Specific probe for Elongation Factor 1 α (EF1) or FB_MR5 (Mr5). CT value: If no target was identified "Undetermined" is indicated. Transcription ratios were calculated in comparison to the average CT value of CisA on 4 th of May (21.209 for EF1 and 31.594 for Mr5) according to Pfaffl [61]. Gala plants showed no amplification with Mr5 (indicated in bold). Ã : Data was not included for | v3-fos |
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} | s2 | Molecular Characterisation and Diagnosis of Root-Knot Nematodes (Meloidogyne spp.) from Turfgrasses in North Carolina, USA
Root-knot nematodes (Meloidogyne spp.) are the most common and destructive plant-parasitic nematode group worldwide and adversely influence both crop quality and yield. In this study, a total of 51 root-knot nematode populations from turfgrasses were tested, of which 44 were from North Carolina, 6 from South Carolina and 1 from Virginia. Molecular characterisation was performed on these samples by DNA sequencing on the ribosomal DNA 18S, ITS and 28S D2/D3. Species-specific primers were developed to identify turfgrass root-knot nematode through simplex or duplex PCR. Four species were identified, including M. marylandi Jepson & Golden in Jepson, 1987, M. graminis (Sledge & Golden, 1964) Whitehead, 1968, M. incognita (Kofoid & White, 1919) Chitwood, 1949 and M. naasi Franklin, 1965 through a combined analysis of DNA sequencing and PCR by species-specific primers. M. marylandi has been reported from North Carolina and South Carolina for the first time. Molecular diagnosis using PCR by species-specific primers provides a rapid and cheap species identification approach for turfgrass root-knot nematodes.
Introduction
Turfgrasses are used worldwide for lawns of home and office buildings, athletic fields, other recreational facilities, and roadsides. In the United States, there are more than 50,000,000 lawns and 16,000 golf courses and the turfgrass area was estimated to be 30 million acres in 2007 [1,2]. In North Carolina (NC), there are 664 golf courses [http://www.golflink.com/golfcourses/state.aspx?state=NC] and the turfgrass industry is a 2.3 billion dollar a year industry (http://www.golf2020.com/media/32940/nc_golf_full_rpt_sri_final_29apr2013.pdf). However, maintenance of turfgrass is very challenging due to damage by various pests, including nematodes. During a survey from 2010 to 2013, 29 species of plant-parasitic nematodes belonging to 22 genera in 15 families were found associated with bermudagrass (Cynodon dactylon), creeping bentgrass (Agrostis stolonifera), and zoysiagrass (Zoysia japonica) in NC and South 18S, ITS and 28S D2/D3, then to develop and validate turfgrass RKN species-specific primers for a reliable and rapid PCR assay to support our diagnostic services and to allow species identification of RKNs through a combined analysis of DNA sequencing and PCR by species-specific primers. The specificity and application of the assay were demonstrated.
Nematode samples
A total of 51 RKN populations from turfgrasses were tested in this study, which comprised of 44 from NC, 6 from SC and 1 from Virginia (Va) ( Table 1). These samples were submitted to the Nematode Assay Laboratory of the Agronomic Division, NCDA&CS voluntarily from golf courses, sod farms and homeowners' lawns. Some of the samples were collected during a plantparasitic nematode survey of 111 golf courses in 39 counties in NC and SC in the summer 2011 [3]. No specific permissions were required in sampling for plant-parasitic nematodes and no endangered or protected species were involved. In addition, nine non-turfgrass nematode populations belonging to M. arenaria Table 1). The identification of these reference species had already confirmed by DNA sequencing and PCR by speciesspecific primers in other projects (data not shown herein). Nematodes were extracted from soil samples by a combination of elutriation [21] and centrifugation [22] methods. The nematode sample was poured into a counting dish (7.5 cm L × 3 cm W × 1.5 cm H) and the nematodes were identified and counted under a Nikon Diaphot 200 inverted microscope (Tokyo, Japan). Further species confirmation was performed with a Leica DM2500 compound microscope (Leica Microsystems Inc., Buffalo Grove, IL, USA) with interference contrast at up to 1,000× magnification.
DNA extraction. For molecular analysis, a single or up to 10 nematodes of the J2 from the same sample were hand-picked into 10-μl AE buffer (10 mM Tris-Cl, 0.5 mM EDTA; pH 9.0) on a glass microscope slide (7.5 cm x 2.5 cm). The nematodes were then macerated with a pipette tip into pieces and collected in 50-μl AE buffer and stored at -20°C.
DNA amplification, cleaning and sequencing. The primers used for PCR and DNA sequencing are given in Table 2. The primers SSUF07/SSUR26 [23], 18S965/18S1573R [24], and 18SnF/18SnR [25] were used to amplify the ribosomal DNA near-full-length 18S gene. The primers rDNA2/ rDNA1.58S [26,27] were used to amplify the ITS1 rDNA region. The primers D2a/D3b [28] were used to amplify the partial rDNA 28S gene D2/D3 domain. PCR for these genes was also conducted using various combinations of universal forward and reverse primers designed for Meloidogyne to ensure high success in PCR (Table 2). These primers were based on the conserved sites from a multiple alignment of many representative Meloidogyne species from the GenBank and their approximate positions are shown in Fig 1. The primer selection criteria were as follows: Tm (melting temperature) 55 to 60°C, primer length 18 to 22 bp, and absence of secondary structure when possible. These primers were synthesized by Integrated DNA Technologies, Inc. (Coralville, Iowa, USA). The 25-μl PCR was performed using 12.5-μl 2X Apex Taq red master mix DNA polymerase (Genesee Scientific Corporation, San Diego, CA, USA), 9.5-μl water, 1-μl each of 10-μM forward and reverse primers, and 1μl of DNA template according to the manufacturer's protocol in a Veriti 1 thermocycler (Life Technologies, Carlsbad, CA, USA). The thermal cycler program for PCR was as follows: denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 45 s, and extension at 72°C for 1 min. A final extension was performed at 72°C for 10 min. PCR products were cleaned using ExoSap-IT (Affymetrix, Inc., Santa Clara, CA, USA) . The sequences used in phylogenetic analysis were chosen from the highest match based on BlastN result in GenBank against the four RKN species recovered from this study. The model of base substitution in the DNA sequence data was evaluated using MOD-ELTEST version 3.06 [29]. The Akaike-supported model [30], the proportion of invariable sites, and the gamma distribution shape parameters and substitution rates were used in phylogenetic analyses using DNA sequence data. Bayesian analysis was performed to confirm the tree topology for each gene separately using MrBayes 3.1.0 [31], running the chain for 1,000,000 generations and setting the 'burnin' at 1,000. Markov Chain Monte Carlo (MCMC) methods were used within a Bayesian framework to estimate the posterior probabilities (pp) of the phylogenetic trees [32] using the 50% majority-rule. The λ2 test for homogeneity of base frequencies and phylogenetic trees was performed using PAUP Ã version 4.0 (Sinauer Associates, Inc. Publishers, Sunderland, MA, USA).
Simplex PCR by species-specific primers
The species identification of M. incognita was confirmed using PCR by species-specific SCAR primers Inc-K14-F/Inc-K14-R which produce a 399-bp DNA fragment [33]. Mn28SFs/ RK28SUR in 28S D2/D3 were designed specific for M. naasi producing a 272-bp DNA fragment based on JN019291. Primers Mg28SFs/RK28SUR and Mm28SFs/RK28SUR in 28S D2/ Table 1) to test the scenario if a mixed species was present. The PCR condition is the same as described above. Duplex PCR by ITS species-specific primers and 28S universal primers The 25-μl duplex PCR was performed using 12.5-μl 2X Apex Taq red master mix DNA polymerase, 7.5-μl water, 1-μl each of 10-μM forward and reverse primers specific for M. graminis and M. marylandi, plus 1-μl each of 10-μM primers RK28SF/MR as internal positive control, and 1-μl of DNA template. The PCR condition is the same as described above.
Root-knot nematode identification
The J2s of RKNs were recovered from the turfgrass soil samples. Species identification in this study was based on the combined analysis of DNA sequencing on the rDNA 18S, ITS and 28S D2/D3 (Table 1) and PCR by species-specific primers ( Table 3). Four species were recovered including M. marylandi, M. graminis, M. incognita and M. naasi; the results are given in Table 1.
DNA sequencing
The rDNA 18S, ITS and 28S D2/D3 were successfully sequenced; their accession numbers from the GenBank are presented in Table 1 [7,[35][36][37][38], ITS [7,39], 28S [7,39] and IGS [40]. Therefore, the conserved ribosomal DNA can't separate these tropical RKNs. The mitochondrial DNA has a faster rate of evolution than the corresponding nuclear genes, creating sufficient nucleotide variation for species-level analyses [15]. The region of the mitochondrial genome flanked by the COII gene and the large (16S) ribosomal gene were successfully applied in large-scale regional RKN survey through PCR and RFLP [14]. Unfortunately, numerous attempts using the same primers [14] or designing new primers for turfgrass RKNs in this project were not successful, with a low rate of success in PCR and insufficient DNA sequence data to generate any meaningful results. Thus, the use of mitochondrial genome on molecular identification for turfgrass RKNs needs further study.
Simplex PCR by species-specific primers
Results of simplex PCR by species-specific primers are given in Table 3 for M. marylandi. Results of 28S primers and ITS primers agree with each other. Primer set Mn28SFs/RK28SUR are positive only for M. naasi. However, M. naasi is rather rare in this study and only one population was available for further PCR testing by specie-specific primers. Two other populations (11-30383 and 11-30385) The simplex PCR results for testing four common Meloidogyne species from turfgrass using species-specific primers are presented in Fig 4. Fig 4A amplified a 198-bp DNA fragment in 28S D2/D3 using Mg28SFs/RK28SUR for M. graminis, but the other three species failed to get any PCR products. Fig 4B amplified a 198-bp DNA fragment in 28S D2/D3 using Mm28SFs/ RK28SUR for M. marylandi, but the other three species failed to get any PCR products. Fig 4C amplified a 272-bp DNA fragment in 28S D2/D3 using Mn28SFs/RK28SUR for M. naasi, but the other three species failed to get any PCR products. Fig 4D amplified a 399-bp DNA fragment in SCAR using Inc-K14-F/Inc-K14-R for M. incognita, but the other three species failed to get any PCR products. All these four samples produced a 612-bp DNA fragment using RK28SF/MR. All results are positive if the DNA is from a mixture of four species. Water used as a negative control in all these assays was negative.
Duplex PCR by ITS species-specific primers and 28S universal primers
Results of duplex PCR by ITS species-specific primers and 28S universal primers are given in Table 3 and agree with simplex PCR results. The duplex PCR results for testing two most common Meloidogyne species (M. marylandi and M. graminis) from turfgrass using ITS speciesspecific primers and 28S universal primers are presented in Fig 5. Fig 5A amplified a 267-bp DNA fragment using MgmITSF/MgITSRs and a 612-bp DNA fragment using RK28SF/MR for M. graminis, but the other three species only amplified a 612-bp DNA fragment by RK28SF/ MR. Fig 5B amplified a 323-bp DNA fragment in ITS using MgmITSF/MmITSRs and a 612-bp DNA fragment using RK28SF/MR for M. marylandi, but the other three species only amplified a 612-bp DNA fragment by RK28SF/MR. Water used as a negative control in all these assays was negative. The duplex PCR provides any assay to detect the target species and any RKNs in a single reaction to prevent false negatives caused by failure of the PCR for any reason.
In conclusion, this study characterized DNA sequences on rDNA 18S, ITS and 28S D2/D3 on a wide range of RKN populations from turfgrasses mainly from NC. Universal primers were also developed for PCR on the genus Meloidogyne for these three gene fragments. Analysis of the sequences through BlastN search and phylogenetic analysis revealed four distinct species, namely M. marylandi, M. graminis, M. incognita and M. naasi, the first two being the predominant species in NC. This result is different from the western United States where M. naasi was determined to be the most common species [7,12]. In this same study [7,12],.M. minor was only detected from Washington and M. chitwoodi and M. fallax only from California, but none of these three species were detected in the current study.
Species-specific primers on rDNA 28S D2/D3 were developed to identify turfgrass RKN through simplex PCR by species-specific primers on M. marylandi, M. graminis and M. naasi. Species-specific primers on ITS were also developed to identify two most common species M. marylandi and M. graminis to allow species confirmation using an additional marker through simplex or duplex PCR. SCAR primers Inc-K14-F/Inc-K14-R [33] were employed to identify M. incognita which produces a 399-bp DNA fragment. In addition, the RKN-universal primers RK28SF/MR were designed and included to amplify a 612-bp DNA fragment as a RKN endogenous control to detect the presence of RKN rDNA 28S gene, so that a RKN-negative sample can still be evaluated to exclude false negatives due to instrument, pipetting, reagent, and/or reaction failure. Compared with other molecular diagnosis [7,14,16,17,19,20,49], this assay only requires routine PCR and electrophoresis and is simple, cheap and rapid (<4 h), without further restriction digestion, DNA sequencing or expensive real-time PCR equipment and reagents. This molecular diagnosis using PCR by species-specific primers provides a rapid species identification approach for turfgrass RKN independent of morphology. | v3-fos |
2019-04-01T13:11:37.993Z | {
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} | s2 | Substrate Water Content and Fertilizer Rate Affect Growth and Flowering of Potted Petunia
. Rising concerns over environmental impacts of excessive water and fertilizer use in the horticultural industry necessitate more efficient use of water and nutrients. Both substrate volumetric water content ( u ) and fertilizer affect plant growth, but their interactive effect is poorly understood. The objective of this study was to determine the optimal fertilizer rates for petunia ( Petunia 3 hybrida ) ‘Dreams White’ grown at different u levels. Petunia seedlings were grown at four levels of u (0.10, 0.20, 0.30, and 0.40 m 3 · m L 3 ) with eight different rates of controlled-release fertilizer (CRF) (Osmocote 14-14-14; 14N–6.1P–11.6K; rates of 0 to 2.5 g/plant, equivalent to 0 to 6.25 kg · m L 3 substrate). Shoot dry weight increased as the CRF rate increased from 0 to 1.67 g/plant but decreased again at even higher CRF rates. The effect of CRF rate on growth was more pronounced at higher u . Leaf size doubled as the u thresholds increased from 0.10 to 0.40 m 3 · m L 3 . Flowering was reduced by a combination of high CRF rates (greater than 0.63 g/plant) and high u (0.30 and 0.40 m 3 · m L 3 ), indicating that optimal conditions for vegetative growth are different from those for maximal flowering. These results suggest that without leaching, high-quality petunias can be grown with lower CRF rates than commercially recommended rates.
Fertilizer and irrigation are inseparable components of ornamental plant production. Nutrient availability to the roots is dependent on q, because nutrient movement is driven by mass flow and diffusion (Nye and Tinker, 1977). Greenhouse crops are typically grown in containers with a limited amount of substrate from which water and nutrients are rapidly depleted (Rouphael et al., 2008). Thus, frequent irrigation and fertilization may be required to sustain high growth rates (Cabrera, 2005). However, this does not always improve crop quality (Zheng et al., 2004), because plant growth is not enhanced by fertilizer application above an optimum rate (van Iersel et al., 1998) and growth rates may actually decline (Dole et al., 1994;James and van Iersel, 2001) as a result of increased salinity (Kang and van Iersel, 2009;Rouphael et al., 2008). Excessive fertilization also inhibits flowering in ornamentals such as Rosa ·hybrida 'Baccara' (Plaut et al., 1973) and Dianthus caryophyllus 'Santorini' (Taylor et al., 2004).
Efficient water and nutrient management is an increasingly important aspect of greenhouse production, because excessive irrigation leads to nutrient leaching and runoff, which have serious environmental consequences. Government regulations increasingly require growers to minimize their environmental impact (Lea- Majsztrik et al., 2011). Efficient fertilizer management is also economical, because of the increasing price of fertilizer (Majsztrik et al., 2011). Furthermore, leaching compromises production efficiency by removing fertilizer from the root zone (Mikkelsen et al., 1994;Ristvey et al., 2001) and lowering nutrient use efficiency (Bilderback, 2002;Shaviv and Mikkelsen, 1993).
Controlled-release fertilizer can be used to provide plants with adequate nutrition and minimize runoff because CRFs release nutrients gradually (Broschat and Moore, 2007;Cox, 1993) and are less prone to leaching than water-soluble fertilizers (Chen et al., 2001;Hershey and Paul, 1982). However, leaching can still occur with CRFs (Broschat, 1995), because initial nutrient release rates are often high and increase as substrate temperature rises (Adams et al., 2013;Birrenkott et al., 2005;Broschat, 1996;Merhaut et al., 2006).
Timers are commonly used to control greenhouse irrigation and provide water according to a predetermined schedule. This may result in unnecessary water applications and leaching because a set irrigation volume is provided regardless of q or plant water status. Applying irrigation water in the needed amount is especially difficult when environmental conditions, and especially light levels, fluctuate from day to day (e.g., Baille et al., 1994;Garland et al., 2012;Kim et al., 2011;O'Meara et al., 2013). Precision irrigation systems, based on realtime measurements of q, can be used instead of timer-controlled systems to maintain a consistent q with little or no leaching (Nemali and van Iersel, 2006). The use of q measurements to trigger irrigation can reduce water use and CRF requirements (Bayer et al., 2014) in ornamental production and could be used to control plant morphology by altering q and fertilization rates (Groves et al., 1998).
Little is known about the interactive effects of q and fertilization in containergrown plants. Relatively low q and high fertilization rates increase substrate electrical conductivity (EC) and inhibit growth by imposing osmotic stress in New Guinea impatiens (Impatiens hawkeri) (Haver and Schuch, 1996). Nutrient availability may also be affected by irrigation practices, because more leaching occurs as irrigation volume increases . Drawing on previous studies (Alem et al., 2014;Bayer et al., 2014;Chappell et al., 2013;Nemali and van Iersel, 2006), we hypothesize that by minimizing or eliminating leaching through precision irrigation control, growers can reduce CRF application rates. The objectives of this experiment were to: 1) quantify the interactive effects of CRF rate and q on the growth of petunia; 2) determine the optimum q and CRF rate for the production of highquality plants without leaching, and 3) to quantify the effect of environmental conditions on plant water use.
Materials and Methods
Plant material and treatments. Petunia 'Dreams White' seedlings were obtained from a commercial greenhouse (Tagawa Greenhouses, Brighton, CO; 512 seedlings/ flat). Controlled-release fertilizer (Osmocote 14-14-14; 14N-6.1P-11.6K; The Scotts Co., Marysville, OH) was incorporated at eight different rates (0, 0.21, 0.42, 0.63, 0.83, 1.25, 1.67 or 2.50 g/plant; 1 g/plant is equivalent to a nitrogen rate of 0.14 g/plant and a fertilizer rate of 2.5 kg · m -3 ) into a peat:perlite (80:20) substrate (Fafard 1P; Fafard, Agawam, MA; pH range: 5.5 to 6.5 after wetting). The substrate also contained starter nutrients, a wetting agent, and dolomitic limestone, which was the main source of magnesium and calcium for the plants. Thirty-two rectangular trays (36 · 24.4 · 10 cm) were filled with substrate after which CRF was incorporated at the eight different rates. Twenty-four uniform seedlings (5 cm tall) were transplanted into each of the 32 trays and hand-watered for 1 week to allow for root establishment.
Subsequent irrigation was controlled using an automated soil moisture sensor-controlled system based on the design by Nemali and van Iersel (2006). Threshold q levels of 0.10, 0.20, 0.30, or 0.40 m 3 · m -3 and the eight CRF treatments were assigned to the 32 trays in a factorial design. Two capacitance soil moisture sensors (EC-5; Decagon, Pullman, WA) were inserted diagonally into the substrate of each tray and connected to a data logger (CR10; Campbell Scientific, Logan, UT) through two multiplexers (AM16/32; Campbell Scientific). Measurements were taken every 10 min using a 2.5-VDC excitation voltage supplied by the data logger. The sensors' raw voltage output was converted to q using a substrate-specific calibration (q = voltage · 1.8862 -0.5624, R 2 = 0.95) developed using the methodology described by Nemali et al. (2007). Whenever the average q for an individual tray fell below the threshold q, the data logger sent a signal to a relay driver (SDM-CD16AC/DC controller; Campbell Scientific), which opened a solenoid valve (X-13551-72; Dayton Electric Co., Niles, IL) for 20 s to irrigate the tray through two pressure-compensating emitters (WPCJ20; Netafim USA, Fresno, CA) and a custom-made irrigation grid, supplying 3.7 mL/plant during each irrigation event.
Data collection. The data logger recorded the number of irrigation events daily and the average q for each tray every 2 h throughout the study. Daily and total irrigation volumes were calculated using the known volume per irrigation event. Total evapotranspiration (ET) from each tray was calculated as the sum of the total amount of irrigation water applied and the change in the amount of water in the substrate over the course of the study [total ET = total irrigation + (initial q -final q) · substrate volume]. Pore water EC was determined on Days 5,6,7,8,12,13,14,20, and 23 after transplanting using a handheld probe (SigmaProbe; Delta T Devices, Cambridge, U.K.). This probe measures bulk EC, the dielectric permittivity of the substrate (a proxy for water content), and substrate temperature to calculate pore water EC (Hilhorst, 2000). Leaf chlorophyll index was measured twice on four fully expanded leaves from the upper part of the canopy of two plants per treatment using a chlorophyll meter (SPAD 502; Konica Minolta Sensing Americas, Ramsey, NJ).
At the end of the experiment (Day 23), plants were visually evaluated for flower abundance and 10 fully expanded leaves were randomly sampled from the plants in each tray and measured using a leaf area meter (LI-3100; LI-COR, Lincoln, NE). The shoots were then cut off at the substrate surface and dried in an oven for 1 week at 80°C, and dry weight was determined. Nutrient concentrations of the dried shoots were subsequently analyzed at the USDA-ARS Application Technology Research Unit (Toledo, OH). The trays with substrate were weighed after harvesting the shoots, dried, and reweighed to gravimetrically quantify their water content. These data were compared with the final q measurements to confirm differences in q among the treatments. Note that both the soil moisture sensors and the gravimetric measurements measure water in the roots in addition to water held in the substrate.
Environmental conditions. Plants were grown on a bench in a glass-covered greenhouse at the University of Georgia, Athens, GA. Temperature and relative humidity (RH) in the greenhouse were measured every 20 s using a temperature and humidity probe Pore water electrical conductivity (EC) as a function of the amount of controlled-release fertilizer incorporated into the substrate. Each data point represents the pore water EC averaged overall four substrate water content thresholds and all 9 measurement days. Substrate water content did not affect pore water EC, whereas EC decreased over time (pore water EC = 1.56 + 0.0776 · fertilizer rate -0.00221 · day 2 -0.000619 · fertilizer rate 2 ; R 2 = 0.77, P < 0.0001).
| SOIL MANAGEMENT, FERTILIZATION, AND IRRIGATION (HMP60; Vaisala Inc., Woburn, MA). Light [photosynthetic photon flux (PPF)] was measured using a quantum sensor (QSO-sun; Apogee Instruments, Logan, UT). All sensors were connected to the data logger, which stored daily temperature extremes and average PPF and RH values and calculated saturation vapor pressure and vapor pressure deficit (VPD) using the temperature and RH data and daily light integral (DLI) from the PPF data. Daily maximum and minimum temperature averaged 28.3 ± 2.6 and 19.0 ± 2.1°C, maximum and minimum VPD averaged 1.65 ± 0.59 and 0.30 ± 0.08 kPa, whereas the DLI averaged 24.3 ± 8.0 mol · m -2 · d -1 (means ± SD). Experimental design and data analysis. The study consisted of a completely randomized factorial design with one replication of each treatment (four q treatments · eight CRF rates). The experimental unit was a container with 24 plants. The data were subjected to multiple regression analysis using SAS (SAS Institute, Cary, NC). To quantify the effects of q and CRF rate on the measured parameters, linear and quadratic effects of q and CRF rate as well as the interactive effect were included in the model. For variables that were measured multiple times throughout the study, time (days after transplanting) and its interactions with q and CRF rate were included in the model as well. Significant effects were then determined using stepwise forward selection at P < 0.05 (proc REG; SAS Institute). We used partial R 2 values to determine how much of the variability in the data was explained by the significant components of the resulting regression model.
The effects of environmental conditions on plant water use were determined using the data from plants grown with a q threshold of 0.40 m 3 · m -3 and a CRF rate of 1.67 g/plant. This treatment was chosen because it produced the largest plants with the highest total irrigation volume. Stepwise forward selection regression analysis (P < 0.05, proc REG; SAS Institute) was used to evaluate the effects of DLI, days after transplanting, average daily VPD, temperature, and their two-way interactions on daily water use.
Results and Discussion
Substrate q. Automatic irrigation commenced once the substrate dried out to below the threshold q, which took 1 (0.40 m 3 · m -3 q threshold) to 2 weeks (0.10 m 3 · m -3 q threshold). Thereafter, q was consistently maintained close to threshold levels ( Fig. 1), despite changes in environmental conditions and increased transpiration rates resulting from plant growth over time, similar to previous findings (Nemali and van Iersel, 2006). Moisture sensor readings were strongly correlated to gravimetrically determined q at the end of the study (r = 0.94, results not shown).
Pore water EC. Stepwise regression analysis indicated that pore water EC was unaffected by the q threshold, whereas there was a quadratic effect of both CRF rate and time (days after transplanting). There were no significant interactions among time, CRF rate, and q threshold (data not shown). Averaged over the course of the study, pore water EC increased from 1.2 to 3.6 dS · m -1 as CRF rate increased from 0 to 2.5 g/plant (Fig. 2). Fig. 3. Response of leaf size (area of the upper-most, fully expanded leaf) and shoot dry weight of petunias to different rates of controlled-release fertilizer at four different substrate water contents (q). Curves indicate multiple regression results (see Table 1 for coefficients). There was no quadratic effect of q on any of the measured variables. Responses were analyzed using y = x 0 + x 1 · q + x 2 · fertilizer + x 3 · q · fertilizer + x 4 · q 2 + x 5 · fertilizer 2 , where y is the measured variable of interest and x 0 , ., x 5 are regression coefficients. y Nonsignificant.
Regardless of q and CRF rate, pore water EC decreased by 1.2 dS · m -1 from transplanting to the end of the experiment (23 d; results not shown). Because there was no leaching, this decrease in EC was at least partly the result of plant nutrient uptake. Approximately 63% of the variation in pore water EC could be explained based on the effects of CRF rate, whereas the decrease in EC over time accounted for an additional 13% of the variation. Pore water EC may decline with increasing q because of dilution (Scoggins and van Iersel, 2006) or as the result of increased plant nutrient uptake attributable to faster growth. Substrate EC, determined from saturated substrate paste (Hershey, 1989), has been found to increase under low q conditions when high rates of CRF are applied (Haver and Schuch, 1996). We observed no effect of q on pore water EC, possibly as a result of enhanced nutrient release from the CRF at higher q thresholds (Du et al., 2006), although Adams et al. (2013) concluded that this effect was minimal at best.
Shoot dry weight. There was a quadratic effect of CRF rate, a positive, linear effect of q, and an interactive effect of q and CRF rate on shoot dry weight ( Fig. 3; Table 1). Shoot dry weight increased as CRF rates increased from 0 to 1.67 g/plant at all q thresholds and decreased again as the CRF rate increased further to 2.5 g/plant. Maximum shoot dry weights were achieved with CRF rates of 1.3 to 1.67 g/plant, corresponding to an average pore water EC of 3.1 to 3.4 dS · m -1 (Fig. 2). The decrease in shoot dry weight at high CRF concentration was possibly caused by an increase in salt concentration in the substrate pore water (average pore water EC of 3.6 dS · m -1 at the 2.5-g/plant CRF rate; Fig. 2), causing osmotic stress (Morgan and Reed, 1998). The interactive effect of q and CRF rate on shoot dry weight indicates that increasing CRF rates stimulated plant growth more as the q threshold increased. Fertilizer concentration (Frett et al., 1985;James and van Iersel, 2001) and q threshold (Kim et al., 2011;van Iersel et al., 2010) are known to affect petunia shoot dry weight, but interactive effects have not been previously reported.
Leaf size. The area of the uppermost fully expanded leaves increased with increasing q threshold and this effect was more pronounced at higher CRF rates (Fig. 3). Most of the variation in leaf size was explained by q threshold (partial R 2 = 0.71; Table 1). There was a weak interactive effect of CRF rate and q (partial R 2 = 0.092; Table 1). Leaf size approximately doubled as the q threshold increased from 0.10 to 0.40 m 3 · m -3 (Fig. 3). A decrease in leaf expansion is among the first indicators of drought stress and is very sensitive to substrate water availability (Fernandez et al., 2002;Kalapos et al., 1996).
Shoot nutrient concentrations. There was no significant main effect of q on shoot concentrations of any nutrient (data not shown). Shoot nitrogen (N) concentrations increased with CRF rate up to 1.75 g/plant with little response to further increases in CRF rate (Fig. 4). More than 91% of the variation in shoot N concentration was explained by CRF effects (Table 2). There was also an interactive effect of CRF rate and q on shoot N concentration ( Fig. 4; Table 2). Decreases in shoot N concentration in response to increasing q were larger at higher CRF rates (Fig. 4). This effect was highly significant but explained only 3% of the variation in shoot N (Table 2) and may be the result of N dilution resulting from the positive effect of q on shoot dry weight (Fig. 3).
Fertilizer rate had a quadratic effect on shoot phosphorus (P), potassium (K), and magnesium (Mg) concentrations ( Fig. 4; Table 2), which increased with increasing Fig. 4. Shoot nutrient concentrations of nitrogen (N), phosphorus (P), potassium (K), and magnesium (Mg) as affected by substrate volumetric water content (q) and fertilizer rate. Symbols indicate measured tissue nutrient concentrations, whereas the curves show regression results (see Table 2 for regression equations). There was no effect of q or interactive effect of q and fertilizer rate on tissue P, K, and Mg and the regression curves for those nutrients show the quadratic effect of fertilizer rate. Table 2. Regression coefficients, P values, and R 2 values from multiple regression analysis of the effects of substrate volumetric water content (q), fertilizer rate, and their interaction on shoot nutrient concentrations (y = x 0 + x 1 · q + x 2 · fertilizer + x 3 · q · fertilizer + x 4 · q 2 + x 5 · Fertilizer 2 , where x 0 , ., x 5 are regression coefficients 0.457 0.457 z There was no significant effect of q on any of the nutrients, whereas there were no significant treatment effects at all on Ca, S, Fe, and Mo (P > 0.05). N = nitrogen; P = phosphorus; K = potassium; Mg = magnesium; Cu = copper; Mn = manganese; B = boron; Zn = zinc; Ca = calcium; S = sulfur; Fe = iron; Mo = molybdenum. fertilization rates. Higher CRF rates above 1.25 g/plant had little additional effect on shoot P and K concentrations. Shoot Mg concentrations decreased as CRF rates increased from 1.25 to 2.5 g/plant. The CRF did not contain Mg and increasing the CRF rate therefore did not increase the Mg supply to the plant. There was no effect of q, CRF rate, or their interaction on calcium (Ca) and sulfur (S) concentrations (results not shown). Shoot Ca and S concentrations averaged 1.35 and 0.41 mg · g -1 , respectively.
Micronutrient shoot concentrations had no (iron and molybdenum) or a weak [zinc (Zn), boron (B), manganese (Mn), and copper (Cu)] relationship (total R 2 < 0.50; Table 2) with CRF rate. There was a weak interaction of CRF rate and q on the concentration of Cu in the shoot (partial R 2 = 0.13). Zinc, B, Mn, and Cu concentrations ranged from 61.7 to 90.1, 18.0 to 18.7, 99 to 147, and 4.1 to 3.4 mg · g -1 , respectively, at CRF rates of 0 to 2.5 g/plant. The lack of strong relationships between micronutrients and CRF rate may be because the CRF contained no micronutrients. Most of the micronutrients in the plant tissue likely came from the starter fertilizer incorporated into the substrate during manufacturing. Tissue nutrient concentrations for most of the macro-and micronutrients were above the minimum of the range reported for petunia (Mills and Jones, 1996), except for the plants that did not receive CRF. The low micronutrient concentrations in the plants that did not receive CRF were surprising, because these plants received the same amount of micronutrients as those in the other treatments. This suggests that a lack of macronutrients inhibited micronutrient uptake.
Water use. Total irrigation volume was affected by q (partial R 2 = 0.60) and CRF rate (partial R 2 = 0.11; Table 1). Total irrigation volume increased by 215 mL/plant as the irrigation q threshold increased from 0.10 to 0.40 m 3 · m -3 (Table 1; Fig. 5). Previous work with similar irrigation systems also showed an increase in total irrigation volume with an increase in q threshold (Burnett and van Iersel, 2008;van Iersel et al., 2010). Increasing the CRF rate from 0 to 1.67 g/plant increased the total irrigation volume by 91 mL/plant, regardless of the q threshold. Total irrigation volume decreased slightly as the CRF rate increased from 1.67 to 2.5 g/plant (Table 1; Fig. 5).
For the plants grown with a 0.40 m 3 · m -3 q threshold and 1.67 g/plant CRF rate, most of the day-to-day variation in irrigation volume was explained by the interaction between days after transplanting (a proxy for plant age or size) and DLI (R 2 = 0.96). Daily irrigation volume generally increased over time because plant size increased (Fig. 6). The effect of DLI on daily irrigation volume increased as the plants grew, because canopy light interception, which depends on DLI and canopy size, has a strong impact on plant water use (Kim et al., 2011). van Iersel et al. (2010 also reported an interactive effect of DLI and plant age on the water use of petunia. Other environmental variables such as temperature and VPD also can affect water use of ornamental greenhouse crops (Baille et al., 1994;Kim and van Iersel, 2009) but were not statistically significant in this study. However, we cannot rule out the possibility that these variables affected daily irrigation volume, because there were strong correlations between VPD and DLI (r = 0.92) and temperature and days after transplanting (r = -0.90). Thus, it was difficult to distinguish between the effects of temperature and days after transplanting or of VPD and DLI.
Consistent with previous findings (Kim et al., 2011;van Iersel et al., 2010), shoot dry Fig. 5. The effect of controlled-release fertilizer rate and substrate water content (q) on the total irrigation volume of petunias over a 23-d growing period. There was a quadratic effect of fertilizer rate and a linear effect of q on irrigation volume, but no interactive effect (see Table 2 for regression results). Fig. 6. The daily light integral (DLI) and daily irrigation volume of petunia over a 23-d growing period. The plants were grown with a substrate water content (q) threshold of 0.40 m 3 ·m -3 and a controlled-release fertilizer rate of 1.25 g/plant. Daily irrigation volume = 1.44 + 0.06569 · day · DLI (R 2 = 0.96, P < 0.0001).
weight was strongly correlated with both total irrigation volume and total ET with slopes of 2.51 and 3.51 g · L -1 , respectively (Fig. 7). The slope of the dry weight vs. irrigation volume was similar to previous findings (2.54 and 2.45 g · L -1 ; Kim et al., 2011;van Iersel et al., 2010). Although we have previously referred to the slope of this regression line as water use efficiency (Kim et al., 2011;van Iersel et al., 2010), this slope does not reflect water use efficiency in the traditional sense (dry weight produced per unit water applied). It does, for example, not take into account possible differences in water use efficiency among the treatments.
Flowering. Flowering is an important ornamental quality trait of petunia. Plants grown at lower CRF rates and q thresholds had more flowers than those with the highest shoot dry weights and leaf areas (Fig. 8). Maximum shoot dry weight was achieved with 1.3 to 1.67 g/plant CRF and a q threshold of 0.40 m 3 · m -3 (Fig. 3), but maximum flowering occurred with the 0.21 to 0.63 g/plant CRF rates and q thresholds of 0.20 m 3. m -3 . A similar relationship between flowering and fertilizer rate was described by James and van Iersel (2001); flowering of subirrigated petunia and wax begonia (Begonia ·semperflorens) increased with an increase in fertilizer solution EC (from 0.15 to 1.8 dS · m -1 ), whereas flower number decreased (begonia) or was similar (petunia) at even higher fertilizer solution EC. The CRF rates that produced maximum flowering (0.21 to 0.63 g/plant) were much lower than the commercially recommended rates (1.01 to 3.26 g/plant). The use of precision irrigation may have prevented nutrient leaching from the substrate, thus reducing the fertilizer requirements of the crop.
Leaf chlorophyll index. The average leaf chlorophyll index increased with an increase in CRF rate (partial R 2 = 0.15). However, the leaf chlorophyll index also decreased over time, but the decrease was dependent on CRF rate and q thresholds (partial R 2 = 0.38). The chlorophyll index increased with increasing CRF rate at the 0.10 m 3 · m -3 q threshold, but this response was less pronounced at higher q thresholds and absent at 0.40 m 3 · m -3 (Fig. 9). The leaf chlorophyll index is a common indicator of plant health and leaf nitrogen content (e.g., Yoder and Pettigrew-Crosby, 1995). The leaf chlorophyll index was correlated with shoot N (r = 0.54, P = 0.0015), P (r = 0.49, P = 0.0048), K (r = 0.46, P = 0.0086), S (r = 0.53, P = 0.0013), and Zn (r = 0.40, P = 0.023). Given the correlation of the leaf chlorophyll index with multiple nutrients, it is not clear which nutrient(s) were limiting the leaf chlorophyll index. However, given the typical nutrient deficiency symptoms of petunia (Pitchay et al., 2002), low chlorophyll levels were most likely caused by N and/or S deficiency.
Conclusions
The sensor-controlled irrigation system effectively maintained q close to treatment threshold levels with little or no leaching. High-quality petunias were grown with CRF rates much lower than commercially recommended. Shoot dry weight increased with an increase in CRF rate, but the increase depended on q. Shoot dry weight of plants grown at higher q thresholds responded more strongly to an increase in CRF rate. Leaf size also increased with increasing CRF rate and this effect was more pronounced at higher q thresholds. However, lower CRF rates (0.21 to 0.63 g/plant) resulted in more flowers. Because vegetative growth responded differently to CRF and q than flowering, growers can adjust fertilizer rates and q to manipulate growth and development of petunia. Fig. 7. The shoot dry weight as a function of total irrigation volume (left) and total evapotranspiration (right) from the start of the irrigation treatments to the end of the experiment (23 d). Fig. 8. Appearance of petunias at harvest (23 d after transplanting) as affected by the rate of controlledrelease fertilizer and substrate water content (q). Flowering was reduced by the combination of high fertilizer rates (> 0.63 g/plant) and high q (0.20 to 0.40 m 3 ·m -3 ). | v3-fos |
2018-04-03T06:07:11.078Z | {
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} | s2 | Near-Infrared Spectroscopy as an Analytical Process Technology for the On-Line Quantification of Water Precipitation Processes during Danhong Injection
This paper used near-infrared (NIR) spectroscopy for the on-line quantitative monitoring of water precipitation during Danhong injection. For these NIR measurements, two fiber optic probes designed to transmit NIR radiation through a 2 mm flow cell were used to collect spectra in real-time. Partial least squares regression (PLSR) was developed as the preferred chemometrics quantitative analysis of the critical intermediate qualities: the danshensu (DSS, (R)-3, 4-dihydroxyphenyllactic acid), protocatechuic aldehyde (PA), rosmarinic acid (RA), and salvianolic acid B (SAB) concentrations. Optimized PLSR models were successfully built and used for on-line detecting of the concentrations of DSS, PA, RA, and SAB of water precipitation during Danhong injection. Besides, the information of DSS, PA, RA, and SAB concentrations would be instantly fed back to site technical personnel for control and adjustment timely. The verification experiments determined that the predicted values agreed with the actual homologic value.
Introduction
Danshen and Honghua are two traditional herbal medicines commonly used in China for the treatment of cardiovascular diseases. Danhong injections are prepared from aqueous Danshen (Salvia miltiorrhiza Bunge) and Honghua (Carthamus tinctorius L.) extracts. Danhong injections are applied to activate circulation of the blood and resolve stasis to promote regeneration [1], and the formula is applied extensively to tens of millions of patients for the prevention and treatment of coronary artery disease [2] in clinics in China. The crude extracts of Danshen and Honghua contain both fat-soluble and water-soluble compounds. The fat-soluble compounds, such as tanshinone, are primarily antibacterial and antiphlogosis [3]. Meanwhile, the watersoluble ingredients, such as phenolic acids, are the medicinal components in the Danhong injection and promote blood circulation and resolve stasis to promote regeneration [4]. The Danhong injection quality depends on separating and purifying the crude Danshen and Honghua extracts. The industrial production of the Danhong injection uses alcohol precipitation initially to purify the crude extract. This process removes some macromolecular substances such as proteins, polysaccharides, tannins, and pigments [5]. However, certain fat-soluble compounds, primarily tanshinones, are removed via subsequent water precipitations [6].
As a separation technology for producing botanical medicines, water precipitation has been researched less than ethanol precipitation. This work uses water precipitation to purify the ethanol precipitation solution for a Danhong injection to describe its separation characteristics. During the water precipitation process, several active ingredients more or less precipitated with impurities via encapsulation. To data, few studies have examined the loss of active ingredients, such as danshensu (DSS), protocatechuic aldehyde (PA), rosmarinic acid (RA), and salvianolic acid B (SAB) [7,8]. Danshensu [3-(3,4-dihydroxyphenyl)-2-hydroxy-propanoic acid; DSS] is the major biologically active and water soluble component in Danshen. In vitro and in vivo experiments have demonstrated that DSS has antioxidant, antiatherosclerotic, and endothelial cell protective effects [9,10]. PA inhibits migration and proliferation of vascular smooth muscle cells and intravascular thrombosis [11]. RA prevents against memory deficits in ischemic mice [12] and has potent 2 International Journal of Analytical Chemistry antiviral activity against enterovirus 71 infections [13]. SAB has antipulmonary fibrotic activity [14] and attenuates lung inflammation induced by cigarette smoke in mice [15]. Guaranteeing the final Danhong injection quality requires water precipitation research. Detection using on-line, real-time measurements would greatly improve the process efficiency for water precipitation.
Radiation in the near-infrared (NIR) energetic range excites overtone and combination vibrations in the sample material, revealing a high content of chemical as well as physical information at the same time dispersed in the whole spectral area [16]. NIR region covers wavelengths from 780 nm up to 2.5 m and mainly reflects the overtones and combinations of fundamental vibrations of C-H, N-H, O-H, and S-H bond in organic molecules [17,18]. Multivariate data evaluation methods were employed to extract the NIR spectral features and to investigate the correlation between the spectral data and the concentration (content) variables measured by the reference assays [19,20]. Together with multivariate data evaluation methods, NIR spectroscopy has become a popular process control tool with a broad range of applications for precise material characterization [21]. In 2004, the Food and Drug Administration (FDA) issued a guidance document to the pharmaceutical industry regarding the implementation of process analytical technology (PAT) [22]. NIR spectroscopy was applied to develop a fast and reliable quality control system for a pharmaceutical substance to support information obtained through PAT surveillance of its manufacturing process. NIR spectroscopy with multivariate classification as a PAT production control supplement was applied to reliably determine the quality of the end product at minimum measuring effort and to at least partially replace laborious, conventional analysis methods in a long term [23].
As a commonly used PAT tool, NIR spectroscopy is a fast and nondestructive technique; it requires no sample preparation and will not produce any waste products. NIR spectroscopy can be considered as a powerful tool for raw material testing, product quality control, and process monitoring [24][25][26] in the pharmaceutical industry and, moreover, it has gained wide acceptance by pharmaceutical manufacturers and regulatory agencies. NIR spectroscopy has been investigated and successfully applied to on-line detection during the alcohol precipitation processes for Danhong injections [27]. The variations between the NIR spectra and intermediate quality attributes were investigated. The results indicate the NIR models were suitable for the realtime on-line of alcohol precipitation. A method [28] based on high-performance liquid chromatography combined with chromatographic fingerprint analysis was developed to quantitatively analyze ten Danhong injection components. This outstanding and precise method simultaneously detects multi-indexes, including DSS, PA, RA, and SAB. However, investigation of the variations in the important intermediate quality attributes during water precipitation processes for Danhong injections was not performed. This paper used NIR spectroscopy for the on-line monitoring of water precipitation processes for Danhong injections. As an analytical process technology, NIR spectroscopy was tested to monitor the quality during water precipitation.
On-Line NIR Monitoring
Device. The on-line NIR monitoring device for the water precipitation process is shown in Figure 1. This device consisted of a Bruker Matrix-F Fourier transform NIR spectrometer (Bruker Optic Inc., Germany), peristaltic pump, duplex filters (100-mesh sieve for removing deposit and reducing velocity), two hand valves, and one sampling valve. The on-line measurements used two fiber optic probes designed to transmit NIR radiation through a 2 mm flow cell (Solvias, Germany) connected to the Matrix-F Fourier transform NIR spectrometer. When the water precipitation solution passed through the flow cell, the flow rate, solid impurities, and bubbles significantly influenced the collected NIR spectra [30]. However, avoiding such influences is difficult for in-line methods, for example, inserting the NIR probe into the reactor, as described in [31]. To eliminate these influences during this study, the solution was passed through the duplex filters before entering the circulation loop to ensure an even distribution of the chemical components in the reactor. The peristaltic pump maintained the solution flow rate through the flow cell and reduced the effect of the flow rate on the NIR spectra.
Hand valves and sampling valves were designed for sampling during the NIR spectra collection.
On-Line Collection of NIR Spectra and Samples.
Spectra were collected from 4500 to 12000 cm −1 with a resolution of 8 cm −1 . Each spectrum was collected in the absorbance mode using the average of 32 scans. To capture the variation during water precipitation, NIR spectra were collected every 30 seconds in real-time. However, samples were added every International Journal of Analytical Chemistry 3 2 minutes via the sampling valve. Meanwhile, the frequency parameter for the peristaltic pump was set to 20.00 Hz to continuously pump water precipitation solution through the flow cell. At the moment, the flow rate in the flow cell was 360 L/h. The water precipitation experiment was performed five times, and 146 samples were collected. The first batch was used for the preexperiment, the fifth batch was used as a prediction set for validation, and remaining batches were identified as calibration sets for the model. Number of samples in calibration set and in validation set was 89 and 32, respectively. In total, all spectral pretreatments and chemometrics analyses were performed using OPUS software (version 7.0, Bruker, Germany).
High Performance Liquid Chromatography (HPLC) Analysis
Method. This paper used HPLC as the reference method to simultaneously quantify the DSS, PA, RA, and SAB.
HPLC Conditions.
The chromatographic analysis was performed on an Agilent 1200 HPLC system (Agilent Technologies, USA) equipped with a diode array detector (wavelength range from 190 nm to 949 nm) and an Agilent Chem-Station software used for recording chromatograms. All samples were separated on an Agilent Eclipse-C18 analytical column (4.6 mm × 250 mm, 5 m particle size) at 35 ∘ C. The mobile phase was a mixture of (A) methanol and (B) aqueous solutions containing 0.5% (v/v) formic acid. The gradient elution procedure was as follows: initial 9% (A); 0-20 min, linear change from 9% to 39% (A); 20-36 min, linear change from 39% to 47% (A); 36-39 min, linear change from 47% to 90% (A); hold at 90% (A) for 39-45 min. The reequilibration duration between individual runs was 10 min. The mobile phase flow rate was fixed to 1.0 mL/min. The detection wavelength was 280 nm at 0-13 min, was converted to 403 nm at 13-21 min, and reconverted into 280 nm at 21-45 min. The samples during this research were diluted before centrifuging at a rotating speed of 1500 rpm for 10 min, the supernatant was filtered with a 0.45 m Nylon microfiltration membrane (Beijing Envta Technology CO., Ltd., the location of the producer is room 210 C, Jiaxin Business Building, No. 59, Annei street, Dongcheng district, Beijing, China), and 5 L of the filtrate was injected into the HPLC system for analysis. One chromatogram including peak assignment is shown in Figure 2.
HPLC Method Validation.
The developed HPLC method was validated based on its linearity, precision, stability, and accuracy. Parameters of HPLC method validation for DSS, PA, RA, and SAB were presented in Table 1. To determine the method repeatability, a sample (number 12 from the second batch) was randomly selected and analyzed by consecutively injecting six needles under the above HPLC conditions. The relative standard deviation (RSD) in the peak areas for DSS, PA, RA, and SAB was 0.18%, 1.08%, 0.46%, and 0.10%, respectively. These results suggest that the instrument precision was acceptable. Additionally, the stability was tested by analyzing the same sample every 2 h for 12 h at room temperature. The RSD in the peak areas for DSS, PA, RA, and SAB were 0.88%, 0.38%, 0.60%, and 0.17%, respectively, which indicates that samples were stable for 12 h. The accuracy was evaluated using a recovery test via the standard addition method at three concentrations. Accordingly, previously analyzed samples (number 12 from the second batch) were spiked with DSS, PA, RA, and SAB to
Chemometrics and Data
Analysis. The spectral data were manipulated by identifying the usable spectral regions selecting appropriate preprocessing methods and correlating to the quantitative HPLC data with PLSR to develop the calibration models. During this work, the raw spectra were pretreated using derivatives, straight line subtraction (SLS), vector normalization (VN), standard normal variate (SNV), and multiplicative scatter correction (MSC). Derivatives, including the first derivative (1st Der) and second derivative (2nd Der), were introduced to remove any spectral baseline drift [32]. The SLS corrected the baseline. The VN and MSC are commonly used to eliminate irrelevant information in the spectra from unknown sources such as surface irregularities, distance variation of sample, and detector [33]. Specifically, a spectrum undergoes VN by subtracting the average intensity, and the MSC corrects any multiplicative effects due to scattering via the linear transformation of each spectrum. The SNV was also considered as a scatter correction method [34]. The partial least squares regression (PLSR) helped correlate the pretreated spectral data to the indicator contents to construct the calibration models [35]. To avoid under-or overfitting, the optimum number of latent variables (LVs) in the PLSR models was determined via the Leave-One-Out cross-validation method [36]. The PLSR computations were performed using the OPUS software (version 7.0, Bruker, Germany). The predictive capabilities of the developed PLSR models were estimated via the coefficient of determination ( 2 ), root mean square errors of calibration and prediction (RMSEC and RMSEP, resp.), relative standard errors of calibration and prediction (RSEC and RSEP, resp.), root mean squares error of cross-validation (RMSECV), and ratio of prediction to deviation (RPD) for the calibration set. An excellent model generally has low RMSEC, RMSEP, and RMSECV; high 2 ; and a small difference between the RMSEC and RMSECV. Moreover, the RMSEP value should be close to the RMSEC value.
Results of HPLC Determination.
The dynamic course of the DSS, PA, RA, and SAB concentrations during the water precipitation process for Danhong injection is shown in Figure 3. The time evolution curves from the water precipitation process were divided into two phases based on the zero minute. One phase referenced the water-adding stage before 0 min, while the other was the thermostatic stage from 0 to 100 min. For the five batches, the first batch began its water precipitation process two days later than the others. Thus, the first batch was different from the other four batches for DSS and SAB and indicated the water precipitation process should be performed immediately after the alcohol precipitation. The DSS, PA, RA, and SAB concentrations at the end of the water precipitation are summarized in Table 2. The average DSS, PA, RA, and SAB concentrations for the four parallel batches were 1.28, 0.260, 0.564, and 2.07 mg/mL, respectively. The RSD values were 2.19%, 4.54%, 3.13%, and 7.35% for the DSS, PA, RA, and SAB concentrations, respectively.
Selection of NIR Spectral Regions.
Raw NIR spectra for the water precipitation solutions ranging from 4000 to 12000 cm −1 were acquired during the monitoring period as shown in Figure 4. The 4000 to 4600 cm −1 region had some noise, which was caused by the optical fiber absorption [37]. Simultaneously, the noise was significantly enhanced from 4600 to 5450 cm −1 and 6100 to 7500 cm −1 (strong signals from water) [38] due to the flow rate of the water precipitation solution through the flow cell. Additionally, the region from 9400 to 12000 cm −1 exhibited low intensities and a low signal-to-noise ratio [39]. Furthermore, spectral regions with absorbances equal to or high than 1.5, such as 4000 to 5450 cm −1 , were considered inappropriate for the spectral analysis due to their zero transmissivity, and they were considered saturated [36]. Hence, removing the abovementioned regions may improve the calibration model accuracy. The remaining regions, 5450-6100 cm −1 and 7400-9500 cm −1 , were utilized for the DSS, PA, RA, and SAB models. To confirm the choice of spectral region was valid, the correlation coefficients for the spectra were investigated as shown in Figure 5. The coefficients for the selected regions were generally above 0.6.
Spectral Data Pretreatments.
Different spectral pretreatments were investigated to optimize the calibration performance. Several preprocessing methods were tested with the NIR spectra, including VN, MSC, constant offset elimination, first derivative, SLS, and their combinations. The VN and MSC are commonly used to eliminate irrelevant information in the spectra from unknown sources such as surface irregularities, distance variation of sample, and detector. The SNV transformation was applied to correct for International Journal of Analytical Chemistry light scattering and to reduce the changes in the path length. The 1st Der can reduce peak overlap and eliminate constant and linear baseline drifts. The SLS corrected the baseline. The detailed description of the techniques can be found in [40,41]. During the water precipitation process, the flow rate influences the spectral baseline, and derivatives not only reduce peak overlap, but also eliminate linear baseline drifts [42]. However, the second derivative operation decreased the signal-to-noise ratio. Therefore, the first derivative was used to eliminate the spectral differences from baseline shifts. To avoid enhancing the noise, all of the derivative spectra were smoothed with a 17-point Savitzky-Golay filter [43]. The first derivative spectra with a 17-point Savitzky-Golay smoothing pretreatment, obviously reflected the quality attributes investigated in the 5450-6100 cm −1 and 7700-8700 cm −1 regions. The pretreated spectra were depicted in Figure 6. Figure 6(a) showed the first derivative spectra with a 17-point Savitzky-Golay smoothing pretreatment from 4000 to 12000 cm −1 . The spectral regions of wavenumbers (1) and wavenumbers (2) in Figure 6(a) were from 5450 to 6100 cm −1 and 7700 to 8700 cm −1 , respectively, as shown in the Figures 6(b) and 6(c).
Comparing several different preprocessing techniques for modeling (results summarized in Table 3 derivative preprocessing method yielded the best DSS, PA, RA, and SAB models.
Establishing the Calibration Models.
Four calibration models were established using the first derivative pretreatment in the regions from 5450 to 6100 cm −1 and 7700 to 8700 cm −1 . The regression plots between the measured and predicted values using HPLC and NIR, respectively, for the DSS, PA, RA, and SAB concentrations in calibration and validation sets are depicted in Figure 7. Based on the data in Table 3, the 2 , RMSECV, and RMSEC values for the four first derivative pretreatment models were superior to the spectra using other preprocessing methods. Parameters of selected NIR models for DSS, PA, RA, and SAB were shown in [44,45]. Based on the above results, the four models were reliable and could accurately predict the quality attributes, and the water precipitation process can be monitored via on-line NIR using the developed quantitative models.
On-Line Quantitative
Monitoring. The established NIR models were used to monitor the water precipitation process and predict the on-line DSS, PA, RA, and SAB concentrations in real-time. The RSEP and RPD values were used to assess the accuracy of the predicted results. If the RSEP value was below 20% and the RPD value was above 3.0, the established model accuracy was acceptable [46]. To validate the applicability, the fifth sample batch (the prediction set) was scanned on-line and predicted using the established models. Figure 8 shows the concentration trends predicted by the NIR in real-time and those obtained from the reference assay agreed well. In addition, the RSEP and RPD values (presented in Table 4) were calculated as 3.86% and 8.83 for DSS, 3.26% and 8.39 for PA, 3.02% and 9.25 for RA, and 4.02% and 8.22 for SAB, respectively. Validated results with RSEP values below 10% and RPD values above 3.0 met the practical requirements. All of these data demonstrated the developed models had high predictive accuracy during the on-line monitoring DSS, PA, RA, and SAB for the water precipitation process in Danhong injection. Hence, NIR spectroscopy is a suitable on-line quality control analysis technology for practical applications.
Conclusions
The present study has demonstrated that NIR spectroscopy provided us with important advantages to determine the concentrations of DSS, PA, RA, and SAB in a quantitative and nondestructive manner and in an extremely short time. Based on the HPLC reference method, the combination of NIR spectroscopy with PLSR was investigated for on-line detection of the compositions of Danhong injection during the water precipitation process. When the established models were used for prediction, these excellent results proved that NIR spectroscopy was valuable for on-line detecting the concentrations of DSS, PA, RA, and SAB. Finally, application of on-line NIR spectroscopy to Danhong injection production enabled us to detect, in real-time, changes in the compositions of Danhong injection during the water precipitation process.
Generally, industrial on-line applications have only rarely been described in scientific publications, although there is a need of further large-scale on-line studies in the pharmaceutical industry to verify reliability and accuracy of NIR spectroscopy under process conditions. However, many of the published studies appear to prove the suitability of NIR spectroscopy as a valuable tool for on-line process control and quality management or at least underline its potential for industrial applications. | v3-fos |
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} | s2 | Genome-wide identification and characterization of auxin response factor (ARF) family genes related to flower and fruit development in papaya (Carica papaya L.)
Background Auxin and auxin signaling are involved in a series of developmental processes in plants. Auxin Response Factors (ARFs) is reported to modulate the expression of target genes by binding to auxin response elements (AuxREs) and influence the transcriptional activation of down-stream target genes. However, how ARF genes function in flower development and fruit ripening of papaya (Carica papaya L.) is largely unknown. In this study, a comprehensive characterization and expression profiling analysis of 11 C. papaya ARF (CpARF) genes was performed using the newly updated papaya reference genome data. Results We analyzed CpARF expression patterns at different developmental stages. CpARF1, CpARF2, CpARF4, CpARF5, and CpARF10 showed the highest expression at the initial stage of flower development, but decreased during the following developmental stages. CpARF6 expression increased during the developmental process and reached its peak level at the final stage of flower development. The expression of CpARF1 increased significantly during the fruit ripening stages. Many AuxREs were included in the promoters of two ethylene signaling genes (CpETR1 and CpETR2) and three ethylene-synthesis-related genes (CpACS1, CpACS2, and CpACO1), suggesting that CpARFs might be involved in fruit ripening via the regulation of ethylene signaling. Conclusions Our study provided comprehensive information on ARF family in papaya, including gene structures, chromosome locations, phylogenetic relationships, and expression patterns. The involvement of CpARF gene expression changes in flower and fruit development allowed us to understand the role of ARF-mediated auxin signaling in the maturation of reproductive organs in papaya. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-2182-0) contains supplementary material, which is available to authorized users.
Background
Auxin is a plant hormone that plays pivotal roles in the regulation of plant growth in response to diverse developmental and environmental events such as embryogenesis, organogenesis, tropic growth, root architecture, flower and fruit development, tissue and organ patterning, and vascular development [1][2][3]. It has been shown that auxin coordinates plant development essentially through the transcriptional regulation of some gene families, such as auxin/indole-3-acetic acid (Aux/IAA), Gretchen Hagen3 (GH3), small auxin up RNA (SAUR), and auxin response factor (ARF) [4,5]. It was subsequently found that these so-called early auxin-responsive genes are characterized by conserved promoter elements, including the TGA element (AACGAC), core element of the auxin response region (AuxRE-core; GGTCCAT), and auxin response element (AuxRE; TGTCTC) [6,7]. Being an important component of auxin signaling pathway, ARFs activate or repress the expression of auxin response genes by binding to AuxRE in their promoter [8].
A typical ARF contains a highly conserved N-terminal B3-like DNA binding domain (DBD) that recognizes AuxRE in the promoter of auxin-responsive genes [8]. The C-terminal dimerization domain (CTD) contains two motifs, called III and IV, that are also found in Aux/IAA and enable the formation of homo-and hetero-dimers among ARFs and Aux/IAAs [9,10]. The middle region (MR), located between DBD and CTD, confers transcriptional activation or repression depending on its amino acid composition [8,11].
The functions of ARFs are well studied. In Arabidopsis thaliana, arf1 and arf2 loss-of-function mutations affect leaf senescence and floral organ abscission [12]. Lossof-function arf3 mutants display defects in gynoecium and floral meristem patterning [13,14], while mutant arf5 is characterized by abnormal vascular strands and embryo axis [15]. AtARF7 is involved in the conditional regulation of differential growth in aerial tissues, and a mutation in AtARF7 impairs hypocotyl response to blue light and auxin stimuli [16]. AtARF8 regulates hypocotyl elongation, auxin homeostasis, and fruit development [12,17]. Furthermore, the flowers of arf6/arf8 double mutant are infertile closed buds with short petals, short stamen filaments, and undehisced anthers [18]. The double mutation, arf7/arf19 affects auxin mediated lateral root development [19]. In rice (Oryza sativa L.), transgenic plants that express an antisense OsARF1 show extremely low growth, poor vigor, curled leaves, and sterility, suggesting that this gene is essential for vegetative and reproductive development [20]. Previous studies have shown that OsARF16, a transcription factor regulating auxin redistribution, is required for iron and phosphate deficiency responses in rice [21][22][23]. Another auxin response factor, OsARF19, controls rice leaf angles through the positive regulation of OsGH3-5 and OsBRI1 [24]. In tomato (Solanum lycopersicon), recent studies have shown the involvement of SlARF genes in flower development and fruit set, development, and ripening [25][26][27].
Papaya (Carica papaya L.) is an economically important fruit crop in tropical and subtropical countries [28]. Sex type in this trioecious species is determined by a pair of sex chromosomes, and plants have either female (XX), male (XY), or hermaphrodite [XY(h)] flowers [29]. Papaya often exhibits male and imperfect hermaphrodite flowers, which are influenced by environmental and hormonal factors [30][31][32]. Under high summer temperatures, the flowers have been observed to change from hermaphrodite to male because of ovary abortion and stamen carpelloid. Some endohormones, such as auxin, may play important roles in this change process [28,33]. Despite the various causes of malformation in papaya fruit, the pear-shaped fruits from hermaphrodite flowers are commercially preferred, and hermaphrodite papayas are favored worldwide for economic production [28,34]. Papaya fruits are very susceptible to deterioration and postharvest losses mainly by fungal decay and physiological disorders such as chilling injury, pests, mechanical injury, and over-ripeness. Therefore, there are several critical problems in breeding and cultivation of hermaphrodite plants that need to be solved [35]. Auxin has a positive role in the quality maintenance and shelf life of harvested papaya fruits [36]. Application of exogenous auxin can delay fruit ripening in many crop species [34]; however, the underlying mechanism linking auxin signaling and reproduction of papaya is largely unknown.
As an important segment of the auxin-signaling pathway, ARFs are encoded by a multi-gene family in many different plant species. There are 23 members in Arabidopsis, 22 in tomato, 31 in maize (Zea mays L.), 15 in cucumber (Cucumis sativus), 39 in poplar (Populus trichocarpa), 25 in rice (Oryza sativa L.), 24 in Medicago (Medicago truncatula), 19 in sweet orange (Citrus sinensis), and 51 in soybean (Glycine max L.) [5,7,21,[37][38][39][40][41][42]. In this study, we used the existing data in public databases to perform domain analysis and identify genes encoding ARFs in papaya. We also aimed to reveal comprehensive information on the gene structure, protein motif architecture, and sequence homology of 11 CpARFs.
Results
Genome-wide identification of CpARF genes A total of 11 ARFs were identified in C. papaya. These genes were named according to the phylogenetic relationships between C. papaya and Arabidopsis. Comprehensive information on these 11 CpARF genes, including gene name, locus ID, open reading frame (ORF) length, number of introns, location on supercontigs and deduced polypeptide sequences, is presented in Table 1. The size of deduced CpARFs ranged from 311 (CpARF6) to 938 amino acids (CpARF5), the corresponding molecular masses from 34.83 to 103.7 kDa, and the predicted isoelectric points from 5.16 (CpARF5) to 9.03 (CpARF6). All the nucleic acid sequences were listed in the Additional file 1: Table S1.
Analysis of phylogenetic relationships and gene structure
The phylogenetic distribution suggested that ARFs could be grouped into four major subclasses, including Ia, Ib, II, and III (Fig. 1a). Based on the phylogenetic tree, seven sister gene pairs were identified between Arabidopsis and C. papaya: CpARF2/AtARF2, CpARF3/AtARF3, CpARF4/ AtARF4, CpARF5/AtARF5, CpARF10/AtARF10, CpARF16/ AtARF16, and CpARF17/AtARF17. No sister gene pairs were found between C. papaya and rice. Most CpARFs contained three typical domains: DBD, domain II, and AUX/IAA family domain. CpARF2, CpARF3, CpARF6, and CpARF17 contained DBD and domain II, but no AUX/IAA family domain (Fig. 1b). The exon-intron structure of each CpARF was revealed by comparing the full-length cDNA sequences with the corresponding genomic DNA sequences. The number of introns in CpARF genes ranged from 1 to 13 (Fig. 1c). CpARF genes, even with close phylogenetic relationship, displayed complex distribution patterns of introns-exons.
Analysis of amino-acid composition and classification of CpARFs
The 11 CpARFs were classified into three groups based on their MR amino-acid composition and the presence or absence of CTDs: (1) ARFs with a DBD, activator MR and a CTD; (2) ARF with a DBD, repressor MR and a CTD; and (3) ARFs with a DBD, repressor MR, but no CTD ( Fig. 2a and Additional file 2: Figure S1). The domain position in these 11 CpARFs is presented in Additional file 3: Table S2, and the amino acid composition of MRs is shown in Fig. 2b and Additional file 4: Table S3. CpARFs contained four putative transcriptional activators, CpARF5, seven, ten, and 16 (QSL-rich MR), and three putative transcriptional repressors, CpARF1, four, and 11 (SLPG-rich MR). Three CpARFs (CpARF2, three, and 17) were putative transcriptional repressors that did not contain a CTD. Only one CpARF, CpARF6, contained only a DBD.
Expression patterns for CpARF genes in different plant tissues
To study the physiological function of CpARF genes, the spatial-specific expression pattern of the 11 CpARF genes was detected in different tissues and organs, including shoots, leaves, flowers, fruits and roots. The expression of most CpARF genes was ubiquitous in all studied tissues and organs, suggesting that they might have a putative function in many aspects of plant growth and development. Some CpARF genes (CpARF2, CpARF6, CpARF10, CpARF16, and CpARF17) showed fruit-specific expression, which indicated that they might play a role in fruit ripening. CpARF1 was highly expressed in flowers, while CpARF3, CpARF5, and CpARF11 were highly expressed in roots. Many CpARF genes, including CpARF1, CpARF2, CpARF3, CpARF6, CpARF7, CpARF16, and CpARF17, were hardly detectable in leaves and shoots (Fig. 3). In our study, we focused on the expression pattern of CpARF genes in flowers during eight different developmental stages. Except for CpARF17 that showed the lowest expression level in all flowering stages, the remaining CpARF genes exhibited dynamic expression patterns. CpARF1, CpARF2, CpARF4, CpARF5, and CpARF10 showed the peak expression in flower developmental stage one and decreased during the following developmental stages, while CpARF6 increased during the developmental process and reached the peak at stage seven.
In addition, the expression pattern of CpARF genes that belonged to the same phylogenetic branch also varied significantly. The expression of CpARF3 did not change significantly during the developmental process, while the expression of its sister pair gene, CpARF4, showed a clear decrease ( Fig. 4 and Additional file 5: Table S4).
Tissue-specific expression analysis showed that some CpARF genes were highly expressed in the reproductive organs (Fig. 3). These results prompted us to investigate the expression of CpARF genes during various fruit ripening stages. The data indicated that the expression of most CpARF genes underwent a significant change associated with fruit ripening. The expression of CpARF1 showed a significant increase during the fruit ripening stages; while the expression of CpARF7 and CpARF11 decreased from stage one to stage six ( Fig. 5 and Additional file 6: Table S5).
Auxin regulation of CpARF genes in the flower and fruit
The qRT-PCR data showed that most of CpARF genes were responsive to IAA and TIBA treatment.
In flowers, the expression of CpARF1 was significantly down regulated by IAA treatment and up regulated by TIBA treatment. CpARF2 and CpARF3 expression levels were significantly increased by IAA treatment and remained stable after TIBA treatment. CpARF6 showed no response to IAA treatment and was largely induced by TIBA treatment. CpARF10 also showed no response to IAA treatment and was significantly induced by TIBA treatment. CpARF5 showed opposite expression patterns between IAA treatment and TIBA treatment. The expression of CpARF5 was up regulated by IAA treatment and down regulated by TIBA treatment (Additional file 7: Figure S2).
Expression of CpARF genes involved in malehermaphrodite differentiation
To understand the regulatory mechanisms of auxin signaling involved in sex determination, we analyzed the expression abundance of CpARF genes in the three different sex types. Most CpARF genes showed higher expression abundance in male and hermaphrodite flowers than in female flowers. For example, CpARF3, CpARF6, CpARF11, CpARF16, and CpARF17 showed the highest expression abundance (>50 %) in male flowers. However, CpARF10 showed the highest expression abundance in hermaphrodite flowers, while it was almost undetectable in male flowers ( Fig. 6 and Additional file 9: Table S6).
Endogenous IAA measurement
To reveal the involvement of auxin in the development of flowers and fruits in papaya, endogenous IAA contents were measured. The data showed that the endogenous IAA contents were much lower in the flowers under later stages than in the flowers under early stages. In the fruits, the endogenous IAA contents keep on a high level from stage one to stage four, and then significantly declined in the stages five and six. Furthermore, three different sex type flowers were collected for endogenous IAA measurements. The highest IAA contents were detected in the male flowers. The IAA contents in the female and hermaphrodite flowers were lower than that in the male flowers (Additional file 12: Figure S4).
Discussion
Auxin is a key signaling molecule for most organogenesis and patterning processes occurring during plant development [50]. The auxin transduction pathway is mainly comprised of two transcriptional regulator families: ARFs and Aux/IAAs [37,51]. ARFs directly bind to down-stream target genes and regulate their expression during development [52]. ARFs are also involved in the reproduction of various plant species [3,53]. Characterization and analysis of CpARFs allowed us to reveal the mechanisms behind auxin involvement in fruit and flower development of papaya [54].
In this study, the reference genome sequence of papaya, which is relatively small in size (372 Mbp) [55], was used to identify the complete CpARF family. The number of CpARF genes was less than that in Arabidopsis (23 ARFs) [37]. Protein domain analysis provided us useful information on the biological function of ARFs. A typical ARF contains a DBD, an MR, and a CTD [37]. Aux/IAAs bind to CTDs of ARFs and form heterodimers. The presence of a large number of CpARFs without CTD suggested that some auxin-responsive genes in papaya can be regulated in an auxin independent manner [51]. The percentage of CTD-truncated CpARFs (36.4 %) was higher than that in other plant species, such as soybean (15.68 %), Arabidopsis (17.39 %), Brassica rapa (22.58 %), rice (24 %), and tomato (28.57 %) [10,56]. Based on the amino acid composition of MR domains, CpARFs were classified into two groups: transcriptional activators and repressors [8]. The average activator/repressor ratio of CpARFs was 0.57 (Fig. 2), similar to Arabidopsis (0.59) and rice (0.56), and almost double compared to that in tomato (0.27) [5]. Only one ARF in papaya, CpARF6, contained only a DBD. These data provided insight into the potential functions of CpARF genes in plant developmental regulation. We also built a phylogenetic tree to analyze the relationship of ARF families between papaya, Arabidopsis, and rice. The results showed that seven sister gene pairs with high bootstrap values (≥99 %) were identified between papaya and Arabidopsis, suggesting that ARFs in papaya were highly homologous to those in Arabidopsis (Fig. 1a). Many AtARFs have been already reported in previous reports [53,[57][58][59]; therefore, comparative studies may reveal useful information on the respective biological functions in papaya.
In Arabidopsis, transcription factors ARF6 and ARF8 regulate a complex process by promoting expansion, stamen filament elongation, anther dehiscence, and gynoecium maturation [18,50]. The expression of CpARF6, a homologous gene of AtARF6 and AtARF8, was increased more than six folds from flower developmental stage 1 to stage 8 (Fig. 4), indicating a putative function of this gene in flower development and maturation. The double mutant arf6/arf8 in Arabidopsis delays the elongation of floral organs and subsequently delays the opening of flower buds and petal growth [18,60]. Most defects in arf6/arf8 are attributed to the abnormal expression of class one KNOXs [61]. The promoters of some KNOX genes in papaya, such as CpKNOX2, 3, 5, 7, and 8, contain several AuxRE elements, suggesting that these genes may be negatively regulated by CpARF6 in the developing floral organs (Fig. 7).
AtARF4 was also reported to be involved in flower patterning [62]. CpARF4 (homologous gene of AtARF4) showed high expression levels in the flowers (Fig. 3). However, the expression of CpARF4 gradually declined from flower developmental stage one to stage eight, suggesting that it might play a different role compared to CpARF6 during flower development, especially at the initial stage. The Arabidopsis mutant arf2 has a delayed flowering and ripening, while a double mutant arf1/arf2 has an enhanced arf2 phenotype, indicating that AtARF1 acts in a partially redundant manner with AtARF2 [53]. In papaya, CpARF1 (homologous gene of AtARF1) was highly expressed in flowers, while CpARF2 (homologous gene of AtARF2) showed a fruit-specific expression. Furthermore, the expression level of CpARF1 was much higher in female flowers than in male flowers, and CpARF2 showed an opposite expression pattern to CpARF1. The expression level of CpARF2 was eight-fold higher in male flowers than in female flowers (Fig. 6). Additionally, the expression of CpARF1 and CpARF2 also declined during flower development (Fig. 4). The preferred expression in early stages suggested that CpARF1 and CpARF2 participated in flower bud formation, which is a key step for flower development. TIR1/AFB-mediated auxin-responsive gene expression is controlled by the interaction between Aux/IAA repressors and ARF transcription factors [63]. CpARF1, CpARF2, and CpARF4-related auxin expression regulation was decreased, while CpARF6-mediated auxin expression regulation was activated in the mature flowers.
Fruit development is a complex interplay of cell division, differentiation, and expansion that occurs in a temporally and spatially coordinated manner in the reproductive organs [64]. Auxin triggers and/or promotes the unpollinated, quiescent ovary to undergo cell division and elongation, and hence it is considered to play a major role in fruit set and development [65,66]. In tomato, SlARFs are involved in the regulation of various aspects of fruit development [67]. SlARF7 acts as a negative regulator of fruit set after pollination and fertilization, and moderates auxin response during fruit growth [68]. Another tomato gene, SlARF4, an auxin response factor involved in the control of sugar metabolism during fruit development, expresses in pericarp tissues of immature fruit [26]. In papaya, several CpARF genes, including CpARF2, CpARF6, CpARF7, CpARF10, CpARF16, and CpARF17, displayed fruit-specific expression patterns, suggesting their importance in improving fruit-related agronomic traits in papaya [29]. Goetz et al. suggested that AtARF8 restricts auxin signal transduction in ovules and pistil until the initiation of fruit development [12]. However, no homologous gene of AtARF8 was identified in papaya.
It is well studied that reproductive organs of plants reacted differently to different plant hormones. Many previous researches have presumed that auxin might play important roles in flower differentiation in papaya, and delay fruit ripening in other plant species [34,36]. However, there is still no decisive evidence revealing that endogenous IAA plays roles in the flower and fruit development in papaya. The endogenous IAA contents showed a decline during both the flower and fruit development, suggesting that a high level of endogenous IAA might contribute to the initiation of reproductive organs in papaya.
Ethylene-auxin crosstalk regulates a variety of developmental and growth processes in plants, including fruit development and ripening [69][70][71][72][73]. Auxin plays a key role in progressing of fruit development towards the transition phase that leads to the initiation of autocatalytic ethylene production in an auxin-and ethylene-dependent manner [73][74][75]. In Arabidopsis, AtARF7 and AtARF19 are involved in ethylene response, indicating an interaction between auxin and ethylene [58]. SlARF7, a homolog of AtARF7 in tomato, was also found to be involved in auxin signaling transduction during tomato fruit set and development [76]. In our study, the expression of CpARF7 (homologous gene of AtARF7) was significantly inhibited during fruit ripening (Fig. 5). High expression levels of CpARF6 and CpARF7 in mature flowers and early fruit developmental stages indicated that these two genes might be involved in fruit set and early cell division stage of the fruit. To get the putative targets for CpARFs during fruit ripening, we analyzed the promoter regions of several ethylene-signaling-and ethylene-synthesis-related genes in papaya [43][44][45][46][47][48][49]. The results showed that many AuxREs were contained in the promoters of two selected ethylene-signaling-related genes (CpETR1 and CpETR2) and three ethylene-synthesis-related genes (CpACS1, CpACS2 and CpACO1) (Fig. 7). In papaya, ARFs may be also involved in fruit ripening by regulating ethylene-signaling-related and ethylene-synthesisrelated genes.
Conclusions
In conclusion, our study provided comprehensive information on ARF family in papaya, including gene structures, chromosome locations, phylogenetic relationships, and expression patterns. The involvement of CpARF gene expressions in flower and fruit development allowed us to understand the role of ARFmediated auxin signaling in the maturation of reproductive organs in papaya.
Plant materials and growth conditions
Two-year-old C. papaya cv. 'Sunrise' trees were planted in a 3 m × 3-m plot with drip irrigation at the Lingnan Normal University field experimental station in Zhanjiang City, Guangdong Province, China. Agronomic practices and fertilizer applications were applied as needed. Our experimental station has a gentle tropical oceanic monsoon climate with an average daily temperature of 22.8°C, minimum temperature of 15.7°C, and maximum temperature of 28.8°C. The total yearly rainfall ranges between 1100 and 1800 mm [77]. The environmental conditions were strictly recorded during the sampling period. No extreme events and bad weather occurred in our experiment period.
Genome-wide identification of CpARF genes
Arabidopsis ARFs (AtARFs) were used to blast against the C. papaya genome database on Phytozome 10.1 using TBLASTN (http://phytozome.jgi.doe.gov). Information on AtARFs used in this study is presented in Additional file 13: Table S9. Furthermore, the hidden Markov model (HMM) profiles of the ARF family [Pfam 02309: AUX/IAA family; Pfam 06507: ARF (AUX_RESP); Pfam 02362: DBD] were employed to identify ARFs from the C. papaya genome. All the obtained sequences were sorted as unique sequences for further protein domain search using InterProScan (http://www.ebi.ac.uk/Tools/pfa/iprscan/).
Sequence analysis and phylogenetic tree
Multiple sequence alignment of CpARFs was performed using ClustalW (http://www.ebi.ac.uk/Tools/msa/ clustalw2/) with the default parameters and adjusted manually. Four classical domains were identified in most CpARFs based on alignment results. DNA and cDNA sequences corresponding to each predicted gene were obtained from the C. papaya genome. Arabidopsis and rice ARFs (OsARFs) were used for the construction of a phylogenetic tree. Information on AtARFs and OsARFs is presented in Additional file 13: Table S9. Gene structure was analyzed using Gene Structure Display Server (http://gsds.cbi.pku.edu.cn/ index.php), and the phylogenetic tree was constructed with 11 aligned CpARF sequences, 23 AtARF sequences, and 25 OsARF sequences using MEGA5.1 (http://www.megasoftware.net/) employing the neighborjoining (NJ) method. Bootstrap values were calculated using 1000 iterations. The constructed phylogenetic tree was visualized using TreeView1.6 (http://www.brc.dcs. gla.ac.uk/services/).
Prediction of amino-acid content and protein classification
Amino-acid content of the MR domain in CpARFs was calculated using MEGA 5.1, and the histogram was constructed using Excel 2010. The classification of CpARFs was based on the respective amino acid content [Domains with CTD: Glutamine/serine/leucine (QSL)rich MR; Repressor with a carboxyl terminal domain (CTD); Serine/proline/glycine/leucine (SPGL)-rich MR; Repressor without CTD: Glycine-rich MR].
RNA isolation and quantitative real time polymerase chain reaction (qRT-PCR)
Total RNA from different tissues, such as shoots, leaves, flowers, fruits, and roots, was extracted using Plant RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The criterion of flowers and fruits under different developmental stages was described as follows. In total, flowers of eight different developmental stages were collected in this experiment, including five stages of flower buds based on their diameters (1 mm, stage 1; 3 mm, stage 2; 5 mm, stage 3; 7 mm, stage 4 and 9-10 mm, stage 5), young flower with closed petals (stage 6), mature flower with partially opened petals (stage 7) and mature flower with opened petals (stage 8). In addition, papaya fruit samples of different developmental stages were harvested at 20, 40, 60, 80, 100 and 120 days after anthesis, respectively. For all the fruit samples, fruit core was excluded, and the flesh with peel were chopped up, frozen in liquid nitrogen and stored at−80°C for further test. Flowers used in tissue-specific expression experiment were a mixture of male, female, and hermaphrodite types. To avoid the affects of environmental factors, the fruit and flower samples were collected from fifteen of uniform, well growth and disease free trees that distributed in different places in our field. Then, the samples were mixed and divided into several independent groups for further analysis.
DNase I was used to remove any genomic DNA contamination from total RNA. CpActin (evm.model.-supercontig_18.238) was used as an internal standard to calculate the relative fold differences based on the comparative cycle threshold (2 -ΔΔCt ) values. Briefly, 1 μl of 1/20 dilution of cDNA was mixed with 5 μl of 2 × SYBGreen and 100 nM of each primer (forward and reverse), and then water was added to a final volume of 10 μl. PCR conditions were as follows: 95°C for 10 min, 40 cycles at 95°C for 15 s, and 60°C for 60 s. All the primer sequences are listed in Additional file 14: Table S10. To visualize qRT-PCR data, heat map was constructed by ClustalW and Treeview using the average Ct value. In the heat map, red color represented up regulation, black color represented unchanged expression, and green color represented down regulation. In this experiment, a specific fold change value (2×) was used to identify any significant differences between different treatments. Expression analysis was carried out using five biological repeats, and the values shown in figures represent the average values of the five repeats.
IAA treatment and cis-elements analysis
Flower and fruit samples were soaked in liquid Murashige and Skoog (MS) medium with or without (mock treatment) 10 μM IAA or 10 ìM 2, 3, 5triodobenzoic acid (TIBA) for 1 h. Samples from each treatment were collected, and total RNA was isolated as previously described. Experiments were repeated five times with similar results. The promoters (1500 bp) of reproduction-related genes were obtained from Phytozome 10.1. AUX1 (TGTCTC core sequence) and a less stringent variant called AUX2 (TGTVYS) were used to manually scan promoter regions.
IAA content measurement
The fruit and flower samples were collected and washed five times in deionized water to clean the surface of the tissues. The plant tissues were blotted dry with a paper towel and weighed using an electronic balance. After the addition of 500 pg of the 13 C6-IAA internal standard, five independent biological replicates of each 50 mg sample were purified using ProElu C18 (http://www.dikma.com.cn). IAA contents were determined by a FOCUS GC-DSQII (Thermo Fisher Scientific Inc., Austin, TX, USA). | v3-fos |
2019-04-14T13:02:29.408Z | {
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} | 0 | [] | 2015-07-01T00:00:00.000Z | 111506412 | {
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} | s2 | Biocementation Potential of Tropical Residue Soil Infused with Facultative Anaerobe Bacteria
Biomediated soil improvement, a promising new branch of microbial geotechnology; which involved multi disciplines has successfully attracted abundant attentions among researchers, geotechnical engineers, and other industries practitioners. Few of the researches were conducted to examine the potential implementation of this technique on tropical residue soil. However, the uncertainties outcomes and inconsistency of bio mediated soil improvement, especially on the clayed soil have made this technique remained at the laboratory stage. Therefore, this paper intended to provide better understanding of this technique by investigating the relation between the bacteria, cementation reagents, and tropical residue soil. The residual soil was mixed with facultative anaerobe bacteria, Bacillus Subtilis before it was compacted into a prefabricated PVC mould. The soil samples were treated with different treatment condition such as (1) control or untreated, (2) treated with cementation solution, (3) treated with bacteria only, and (4) treated with bacteria and Cementation reagent. A worth noting finding showed that the sample treated with bacteria and nutrient only has produced the highest increment of shear strength. This phenomenon might have been caused by the effect of the chemical reagent to the mineralogy of residue soil. The presence of the chemical reagents is believed to have weakened the shear strength of the tropical residual soil.
Background of study/Introduction
Biomediated soil stabilization or similarly known as Microbial-induced calcite precipitation (MICP), an emerging discipline of soil improvement in geotechnical engineering. This MICP technique integrated with microorganism and chemical reagents to create a more environmental friendly soil improvement technique by forming the microbial induced calcite precipitation.
However, exposure to the byproduct of this MICP technique such as concentrated ammonium, and oxidized by-product nitrate are dangerous and may impair the environmental and mankind [1,2]; Therefore, in order to address its limitations, many effort and researches have been conducted and developed rapidly over the years [3][4][5][6]. Initially, most of the MICP researches were merely focused on the fine sand until recently [3,7]; some of the researches have conducted to investigate the potential and feasibility of MICP on the tropical residue soil [8,9].
The objective of this paper is to provide better understanding of this technique by investigating the relation between the bacteria, cementation reagents, and tropical residue soil as well as to investigate the effectives and feasibility of Bacillus subtilis in this biomediated soil improvement.
Biological Processes in Biomediated Soil Improvement
The Biomediated soil improvement started when both of the bacteria and chemical reagents were mixed together into the soil. The role of the bacteria in this process is to perform the urea hydrolysis where the supplied urea was decomposed into ammonium ion by its urease enzyme.
The pH of the soil was increased with the presence of the ammonium ion, and created a favorable environment for the calcite precipitation. Meanwhile, the carbonate ion combined with the supplied calcium ion to form the calcite precipitation.
The formation of calcite (CaCO 3 ) precipitation improved the shear strength and permeability of the treated soil; the former is achieved by biocementation which binds the soil particles together and the latter is achieved by bioclogging which fills the void between the soil particles. Both of the biocementation and bioclogging happen concurrently during the MICP treatment.
Material and Methodology
Type of Bacteria. Bacillus subtilis (ATCC 55422) is a type of facultative anaerobe bacteria which is capable to survive with and without oxygen. The diameter of the bacterium is about 2um of a round shaped, and with gram positive bacterium.
Cementation reagent. The cementation reagents were essential material in forming the Microbial induced Calcite Precipitation (MICP). Analytical Reagents grade was adopted throughout the process to ensure the quality and consistency of the research. Cementation reagents such as urea, and calcium carbonate were prepared at 0.25 M concentration and 3g/L nutrient broth was added as the supplement in this study.
Laboratory Setup /Placement of microorganism. Chemical reagents were filled on the top of the special prefabricated PVC mould (as illustrated in the Fig.1). The cementation reagents were absorbed and infiltrated into the compacted soil with help of the atmosphere pressure. The soil samples were always kept overtopped with the chemical reagent to ensure it was always in the saturated condition and avoid any potential loss due to evaporation or leakages.
Soil Specimen. The tropical residual soil specimen used in this research was retrieved at Faculty of Electrical Engineering, University Technology Malaysia. The physical and engineering properties of the tropical residual soil specimens were determined in accordance to the British Standard (BS1377) as tabulated in table 1. hours (approximately at 6 x 10 5 cfu/ml) prior to the microorganism placement process. The tropical residual soil was mixed thoroughly with the cultivated bacteria and compacted into a prefabricated 52mm diameter of PVC mould. It was compacted in three layers by hand tamping method at the desired density of 1188kg/m 3 (90% of maximum dry density). Treatment was started by filling the chemical reagents at the top of the mould (Fig. 1) for 2 days to ensure the sample was entirely saturated. After 2 days of treatment, the treated soil samples were extruded for shear strength determination by using unconfined compression test (UCT).
Experimental Variables. 4 different treatment conditions were considered in this study. 1) Control specimen in which the specimen was supplied and saturated with distilled water only, 2) Specimen treated with chemical reagents only, 3) specimen was treated with bacteria only, 4) both bacteria and chemical reagents ware added to the soil. All of the samples were treated at 0.25M of urea and calcium chloride.
Result and Discussions
Fig .2 showed the shear strength of treated soil under various treatment conditions. The unconfined compressive strength of the untreated residual tropical soil (control) was 10.02kPa.As Seen in Fig.2, the injection of the chemical reagent to the tropical residue soil has literally improved the shear strength of the treated soil about 31.3% from 10.02kPa to 13.16 kPa, which correspondingly implying that the existing bacteria inhabited in the tropical residue soil has favored the treatment process. Meanwhile, the presence of both bacteria and chemical reagent literally increase the shear strength of the biotreated soil with 78.5% of increment as compared to the control specimen. This outcome also suggested that the introduction of Bacillus subtilis is evidently favorable and feasible to the MICP process. The most striking result to emerge from the data is that the soil sample treated with bacteria only has exhibited the highest strength among the other, with 115.8% of increment of the shear strength after the treatment. This result is different with other published research in which the highest shear strength was achieved with the presence of both bacteria and cementation reagent [6,8].it was believed that the injected chemical reagents might have caused some undesirable effect which hindered the growth of this particular bacterium and subsequently affected on the development of MICP. Another possible explanation for this phenomenon is that the component of the chemical reagents might have weakened the bonding and mineralogy of tropical residue soil particles and resulting in a slightly reduction of its shear strength after the treatment.
Besides that, the retained dead cell of the bacteria in the sample could have increased the shear strength of the treated soil [10]. Further investigation on the chemical effect to the soil mineralogy, dead bacteria cell and bonding should be conducted to clarify the aforementioned matter.
Conclusion
In this present preliminary test, the introduction of the Bacillus Subtilis in this biomediated soil improvement has proven its feasibility and effectiveness with significant improvement of shear strength. The results also indicated that the presence of the chemical reagents is effective in this technique. The presence of both bacteria and chemical reagents are favorable in this process; although, the highest improvement of shear strength was achieved with sample which was treated with bacteria only .For that reason, further profound study is required to examine the effect of the chemical reagents to the bacteria and the mineralogy of tropical residual soil.
Acknowledgement.
This | v3-fos |
2018-04-03T00:05:42.754Z | {
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} | 0 | [] | 2014-12-18T00:00:00.000Z | 8143092 | {
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} | s2 | PECTIN METHYLESTERASE48 is involved in Arabidopsis pollen grain germination.
65 Germination of pollen grains is a crucial step in plant reproduction. However, the molecular 66 mechanisms involved remain unclear. We investigated the role of PECTIN 67 METHYLESTERASE48 (PME48), an enzyme implicated in the remodeling of pectins in 68 Arabidopsis thaliana pollen. A combination of functional genomics, gene expression, in vivo 69 and in vitro pollen germination, immunolabeling and biochemical analyses was used on wild- 70 type and Atpme48 mutant plants. We showed that AtPME48 is specifically expressed in the 71 male gametophyte and is the second most expressed PME in dry and imbibed pollen grains. 72 Pollen grains from homozygous mutant lines displayed a significant delay in imbibition and 73 germination in vitro and in vivo . Moreover, numerous pollen grains showed two tips emerging 74 instead of one in the wild-type. Immunolabeling and FT-IR analyses showed that the degree 75 of methylesterification of the homogalacturonan (HG) was higher in pme48-/- pollen grains. 76 In contrast, the PME activity was lower in pme48-/- partly due to a reduction of PME48 77 activity revealed by zymogram. Interestingly, the wild-type phenotype was restored in pme48- 78 /- with the optimum germination medium supplemented with 2.5 mM calcium chloride 79 suggesting that in the wild-type pollen, the weakly methylesterified HG is a source of Ca 2+ 80 necessary for pollen germination. Although pollen specific PMEs are traditionally associated 81 with pollen tube elongation, this study provides strong evidence that PME48 impacts the 82 mechanical properties of the intine wall during maturation of the pollen grain which, in turn, 83 influence pollen grain germination. Here, we report the study of a homozygous knock-down mutant for AtPME48 during imbibition and germination of pollen grains and pollen tube growth. We have investigated in vitro and in vivo pollen germination, pollen tube morphology and growth as well as the level 161 of PME activity and the degree of methylesterification of the HG by immunolabeling and FT- 162 IR in mutant and wild-type plants. Our results show that the group 1 PME48, the second most 163 expressed PME gene in pollen, plays a major role in remodeling the HG of the intine cell wall 164 during Arabidopsis pollen grain maturation resulting, after rehydration, in a normal pollen 165 grain germination.
shank. Back from the tip, the inner layer is mainly composed of callose, a (1,3)-β-glucan, 1 0 2 which is not detectable in the tip region in normal growth conditions (Ferguson et al., 1998;1 0 3 Derksen et al., 2002;Parre and Geitmann, 2005b;Dardelle et al., 2010;Chebli et al., 2012). Moreover, callose is also deposited periodically within the pollen tube to form plugs that In eudicot species, pectins constitute a major portion of the primary cell wall. Pectins In order to assess further the biochemical differences between the wild-type and pme48-/-, we 2 7 7 investigated the total PME activity in pollen grains by using enzymatic assays. The data 2 7 8 showed a 50 % reduction of the total PME activity in pme48-/-pollen grains compared to the 2 7 9 wild-type (Fig. 5A). Moreover, zymogram after isoelectrofocusing (IEF) of total proteins 2 8 0 extracted from pollen grains revealed a disappearance of a diffuse band in the pI range 2 8 1 between 8.2 and 9 in pme48-/- (Fig. 5B) that may correspond at least in part to PME48 which 2 8 2 has a predicted pI of 8.3. Two spots, at pI ranging from 9.8 to 10.2 and from 9.5 to 9.7, in the was higher in pme48-/-pollen grains. The DM of the HG was consistently higher in the 2 9 0 pme48-/-(32.5 ± 1.7 %) compared to the wild type (13.4 ± 1.1 %) pollen grains (Fig. 5D). As 2 9 1 de-esterified HG binds more Ca 2+ than esterified HG, our data may suggest that HGs in the 2 9 2 intine wall of pme48-/-are less associated with Ca 2+ compared to wild-type pollen grains. To assess if the germination defect of pme48-/-pollen grains was related to the possible lower 2 9 5 Ca 2+ sink in the intine wall due to the higher DM of the HG, the optimum culture medium 2 9 6 containing 5 mM CaCl 2 was supplemented with 2.5 mM CaCl 2 to reach a final concentration 2 9 7 of 7.5 mM or with the Ca 2+ chelator, EDTA. In the presence of 1 mM EDTA, none of the 2 9 8 wild-type and pme48-/-pollen grains germinated, even after 24h of culture (n>1,000) (Fig. 6A-B). In the supplemented medium with Ca 2+ , pme48-/-pollen grain germination rates were 1 4 restored reaching the levels of the ones observed with wild-type pollen grains (n>1,000). The 6A-B). Moreover, the speed of imbibition of pme48-/-pollen grains was as fast as the wild-3 0 3 type pollen grains (Fig. 6C). In addition, a significant reduction of the abnormal phenotypes 3 0 4 such as burst tubes (from 33% to 13%) (Fig. 3F, 6D) and the double-tipped tubes (from 18% 3 0 5 to 2%) (Fig. 3G, 6E) was observed in pme48-/-pollen grains cultured in the supplemented 3 0 6 GM. Finally, in the CaCl 2 supplemented medium, the rates of burst tubes increased in wild-3 0 7 type pollen grains (32%) (Fig. 6D) compared to that observed when pollen grains were grown 3 0 8 in the optimum GM (~10%) (Fig. 3F). The regulation of the DM of HG has been implicated in many aspects of plant development predicted PME genes in the genome of Arabidopsis, 14 are specifically expressed in the male 2007). Two of them have already been studied using functional genomics approaches. The 3 2 3 first one is VANGUARD1 (At2g47040), the alteration of which led to unstable pollen tubes 3 1 5 and retarded growth of the pollen tube in the style and transmitting tract, resulting in a 3 2 5 significant reduction of male fertility (Jiang et al., 2005). The second pollen specific PME 3 2 6 characterized to date is PPME1 (At1g69940) (Tian et al., 2006). The homozygous mutant 3 2 7 atppme1 displayed reduced growth and irregular shape of pollen tube grown in vitro. In the 3 2 8 present study, we show that the alteration of the expression of PME48, the second most 3 2 9 expressed PMEs in pollen grains, results in a strong delay in imbibition and in germination 3 3 0 both in vivo and in vitro as well as altered phenotypes with abnormal rates of burst tubes and 3 3 1 two pollen tube tips emerging from the same pollen grain. The phenotype is rescued by These data suggest that PME48 is mainly involved in the remodeling of the intine cell wall simulations (Chebli and Geitmann, 2007;Zerzour et al., 2009;Fayant et al., 2010). The more 3 4 0 abundant highly methylesterified HG in the intine wall of pme48-/-pollen grains and at the tip intine. In many species such as Nicotiana tabacum (Li et al., 1995), Arabidopsis thaliana HGs, the latest being more abundant. The almost absent labelling of weakly methylesterified 3 5 2 HG with JIM5 at the pollen mother cell and tetrad stages (Rhee and Somerville, 1998) but its 3 5 3 strong detection in the intine at the late microspore stage and mature dry Arabidopsis pollen Golgi apparatus and may be secreted under a highly methylesterified form and then processed The early de-methylesterification of HG by PME48 during the pollen formation and pollen grain hydration (Fig. 7 ). The DM of HG can affect the water holding capacity of the methylesterified HG increased compared to the wild-type suggesting that i) β -galactosidase 3 7 5 may be required for PME activity (Western et al., 2001;Walker et al., 2011) and ii) PME DM of the HG in the intine of pme48-/-is almost 2.5-fold higher than in the wild-type. Second, upon the block-wise action of PMEs, de-esterified blocks of HG chains can be cross- the unesterified HG in the maturing pollen grain may act as a reservoir of Ca 2+ that will be 3 8 5 used later during rehydration and germination (Fig. 7 ). The reduction of PME activity in prepare the future protrusion of the pollen tube tip by weakening the intine wall ( Fig. 7 b ).
9 8
Recently, Rafińska et al., (2014) suggested that the cell wall of the female tissues can also act 3 9 9 as a reservoir of Ca 2+ in order to ensure correct germination and pollen tube growth in Larix However, we cannot rule out that PMEs originating from the stigma may also participate in Although pollen specific PMEs are traditionally associated with pollen tube 4 3 0 elongation, the present study provides substantial evidence that PMEs, especially PME48, are phase with 60% humidity with daily watering. Valencia, CA) following the instructions of the supplier. the accumulated fluorescence signal to cross a threshold above the background) were acquired 4 6 0 with the LightCycler 480 software (Roche) using the second derivative maximum method.
7 0
PCR was performed on the cDNA generated from total RNA extracted from 6 h-old pollen 4 7 1 tubes grown in vitro. Primers used for the RT-PCR are in bold and underlined in Figure S1D. were then cleared with several washes in 70% ethanol. The promoter of the PME48 gene (At5g07410) was PCR-amplified from a plasmid construct In vitro pollen tube growth 5 0 0 Pollen grains were grown in vitro in a liquid medium according to the method described by with ImageJ (n=126 and n=172 for the wild type and pme48-/-pollen tubes, respectively from 5 2 2 4 independent experiments).
2 3
Pollen grains were also cultured in the GM supplemented with 1 mM EDTA or 2.5 In vivo pollen tube growth 5 2 7 Emasculated mature flowers from wild-type plants were hand-pollinated with wild-type or Viability test and DAPI staining 5 3 7 The viability of pollen grains was assessed using fluorescein diacetate (FDA) dissolved in 5 3 8 acetone at 10 mg mL -1 and stored at -20°C. Prior to each experiment, FDA was diluted in a 5 3 9 10% sucrose solution to a final concentration of 0.2 mg mL -1 . Hydrated pollen grains were
4
The nuclei of the pollen grains were stained with 10 µg mL -1 DAPI (4',6-diamidino-2- Immunolocalization was performed by cell surface labelling as previously described by serum albumin as a standard. PME activity was measured using the alcool oxidase method 6 1 0 according to Klavons and Bennett (1986). One IU of PME activity induces the production of Isoelectric focalisation (IEF) and zymogram were performed as described by Paynel et al. according to Bertheau et al. (1984). The gel was incubated for 1 h at 25 °C and the de- Data were statistically treated using the graphpad software (www.graphpad.com). Table S1. List of primer pairs used for the qRT-PCR analyses. 2 8 Figure S4. Estimation of the growth speed in liquid medium of wild-type (black bars) and pme48-/-(grey bars) pollen tubes.
3 7
Movie S1. Time-lapse imaging of germination and tube growth of wild-type pollen grains. Wageningen University (The Netherlands) for the gift of the pPLV06 plasmid. Arabinogalactan-proteins in root and pollen tube cells: distribution and functional thaliana is associated with persistence of pectic polysaccharides of the pollen mother wall PME and PMEI at the pollen tube tip involves PMEI endocytosis, and reflects the improves the ultrastructural preservation of in vivo grown lily pollen tubes. Table S1. List of primer pairs used for the qRT-PCR analyses. | v3-fos |
2019-04-01T13:16:02.203Z | {
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} | s2 | Effect of Explant, Genotype and Plant Growth Regulators on Regeneration and Agrobacterium-Mediated Transformation of Potato
The objective of the present study is to develop an efficient protocol for plant regeneration and Agrobacterium-mediated transformation of potato cultivars Desiree, Agria and Marfona grown under Iranian agricultural conditions. The regeneration efficiency from internode, leaf and petiole explants was examined. The results from all Shoot Induction Medium (SIM) combinations and all cultivars together indicate highly efficient shoot regeneration from internode (4.56 shoots per explant). The response of petiole and leaf explants was lower, 3.83 and 2.55 shoots per explant, respectively. The highest efficiency of shoot regeneration was achieved with internode explants of cultivar Desiree (6.64 shoots per explant) on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ) 1 mg LG + 6-benzyladenine (BAP) 1 mg LG. For plant transformation, internode explants were inoculated and co-cultivated with Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pBI-121 containing β-glucuronidase (GUS) and nptII genes. Reverse transcriptase-polymerase chain reaction analysis and histochemical assay for β-glucuronidase indicated that the gene coding for this enzyme was integrated in the potato genome and could be expressed in potato tissues. The presence of nptII gene in the kanamycin resistant plants was verified by polymerase chain reaction analysis. The transformation frequency ranged from 22-42%.
INTRODUCTION
Potato (Solanum tuberosum) is one of the most economically important annual vegetable crops of Solanaceae family. It is the fourth most cultivated food crop in the world after wheat, rice, maize and is the most important dicotyledonous tuber crop. The edible part of plant is tuber used as cheap food, industrial raw material, animal feed and seed tuber. Potato is sensitive to a number of biotic and abiotic factors. Several approaches including traditional breeding techniques have been made in the past to overcome these constraints. But traditional methods are considered to be complicated in nature and consumed comparatively a longer period to produce a variety with desired characters. To address some of these limitations, plant genetic engineering using desirable genes from various organisms has provided promising results. Most of the transformation studies were conducted with the intention to transfer genes/traits expected to increase the resistance of potato against predators and pathogens (Chye et al., 2005) or to modify common metabolic pathways, such as starch and sucrose synthesis (Edwards et al., 1999).
Genetic engineering of potato cultivars requires the development of transformation protocols and the establishment of efficient regeneration systems. Various important factors such as genotype, source of explants and plant growth regulators significantly influence regeneration of potato. Among the factors that influence growth, the type of cytokinin, J. Agron., 14 (4): 227-233, 2015 such as thidiazuron (TDZ) and 6-benzyladenine (BAP), is critical for initiation of shoot primordia from cultured explants. TDZ, a phenylurea-type cytokinin, has been used as a plant growth regulator to induce organogenesis, including adventitious regeneration of transformed plants in many plant species that were thought to be recalcitrant to regeneration (Cuenca et al., 2000;Corredoira et al., 2008;Sriskandarajah and Lundquist, 2009).
Transformation efficacy in potato is actually highly genotype-dependent, which is the main reason for the existence of many different protocols. The objective of this study is to establish an effective protocol for plant regeneration and transformation of local Iranian varieties, using different types of explants that would allow genetic engineering for important traits. Three cultivars, Agria, Marfona and Desiree were used for transformation with an Agrobacterium binary vector (pBIl21) which included genes for GUS and nptII.
Plant materials and culture conditions:
In vitro grown plants of potato cultivars Agria, Marfona and Desiree prepared from Genetic Engineering Laboratory, University of Tabriz, Tabriz, Iran. These cultivars were propagated using single-node segments on MS medium (Murashige and Skoog, 1962) containing 2% w/v sucrose and 0.8% w/v agar. The pH of the medium was adjusted to 5.8 before autoclaving and the cultures were grown under a 16 h dayG 1 photoperiod with a light intensity of 56 μmol mG 2 secG 1 provided by cool white fluorescent tubes and incubated at 22±2°C ambient temperature.
Regeneration experiments: Internode, petiole and leaf segments of in vitro grown potato cultivars were cut into sections (1 cm each) or small pieces (1 cm 2 ) as explants for callus induction. The explants were cultured on callus induction medium [MS agar medium containing 2% sucrose supplemented with 2 mg LG 1 6-benzyladenine (BAP) and 1 mg LG 1 α-naphthalene acetic (NAA)] and incubated at 22±2°C under a light intensity of 56 μmol mG 2 secG 1 . The calli were transferred to the fresh callus induction medium about 21 days interval for further proliferation and maintenance. After 2-3 weeks of incubation, the well-developed compact calli were cultured in Shoot Induction Media (SIM) containing MS basal medium supplemented with various combinations of BAP (1 and 2 mg LG 1 ), kinetin (1 and 2 mg LG 1 ) and thidiazuron (1 and 2 mg LG 1 ). Approximately 40-45 explants were cultured on each of the 8 types of SIM combination. Each experiment was repeated three times. The cultures were incubated in a growth chamber under same conditions of light and temperature, as that of callus induction. After 4-6 weeks, shoot formation was observed. The shoots were excised and transferred to root induction medium (MS medium containing 0.1 mg LG 1 NAA) for root induction.
Bacterial strains: Agrobacterium tumefaciens strain LBA4404 possessing binary vector pBI121 was inoculated on YEB medium and used for gene transfer into potato.
Agrobacterium tumefaciens-mediated transformation:
Internodal (5-6 mm) explants were prepared from 4 weeks old plants for transformation. The internodes were wounded several times leaving about a one mm gap between the cuts. The internode pieces were placed in MS liquid medium containing 1 mg LG 1 NAA, 2 mg LG 1 BAP and 2% sucrose at pH 5.7, in Petri dishes for 48 h at room temperature for co-cultivation. After the dark incubation period, internode pieces were washed three times in sterile water and in a cefotaxime solution (250 mg LG 1 ) to remove bacterial excess. The explants were blotted dry with a sterile filter paper and then placed in callus induction medium (MS medium containing 1 mg LG 1 NAA, 2 mg LG 1 BAP, 50 mg LG 1 kanamycin, 250 mg LG 1 cefotaxime, 3% sucrose and 0.8% agar at pH 5.7). After 7 days of incubation, the explants were transferred to the shoot induction medium (MS medium, supplemented with 1 mg LG 1 TDZ+2 mg LG 1 BAP, 75 mg LG 1 kanamycin, 250 mg LG 1 cefotaxime, 3% sucrose at pH 5.7 solidified with 0.8% agar). The explants were transferred every week to fresh shoot induction medium. After four weeks, shoots formed at the cut end of the internodes. Regenerated shoots of 2.0-2.5 cm in length were then excised and transferred into rooting medium (MS solidified medium supplemented with 0.1 mg LG 1 NAA, 2% sucrose, 100 mg LG 1 cefotaxime and 75 mg LG 1 kanamycin).
β-glucuronidase (GUS) histochemical assay:
The β-glucuronidase assay was carried out essentially as described by Jefferson et al. (1987). Leaves from kanamycin resistant shoots were tested for histochemical GUS expression in X-gluc solution consisting of 2 mM X-gluc, 0.1 M phosphate buffer (pH 7.0) and 5 mM each of potassium ferricyanide and ferrocyanide with 0.1% (v/v) Triton X-100. After incubating overnight in the substrate solution at 37°C, the stained tissues were rinsed several times with 70% ethanol to remove chlorophyll.
Polymerase Chain Reaction (PCR) analysis: Genomic DNA was isolated from 100 mg of young leaves of transformed and untransformed potatoes by the method of Dellaporta et al. (1983). The PCR amplification was carried out using the gene specific primers (5'-GAACAAGATGGATTGCACGC-3') and (5'-GAAGAACTCGTCAAGAAGGC-3') for nptII generating 786 bp product. PCR was performed in a 25 µL reaction mixture containing 50 ng of genomic DNA as template, 1xTaq DNA polymerase buffer, 400 µM each dNTPs, 10 pmol of each oligonucleotide primer and 0.3 U of Taq DNA polymerase (ASTEC PC-818; Fukuka, Japan). DNA amplifications were done in a thermal cycler (Takara, Japan) using the following program: Initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 45 sec, annealing at 55°C for 45 sec and extension at 72°C for 2 min followed by final extension for 5 min at 72°C. The PCR products were separated by electrophoresis in a 1% agarose gel containing ethidium bromide and visualized under UV using Gel Documentation System OptiGo 600/650 (ISOGEN).
Reverse Transcriptase-PCR (RT-PCR): Total RNA was isolated from the transgenic as well as from untransformed control plants using TRIZOL reagent (Invitrogen, USA). Five milligram of total RNA was taken in a nuclease free tube. One microliter of oligo(dT) 20 primer (50 µM), 1 µL of annealing buffer and DEPC treated water were added to it so that the final volume was made up to 8 µL. This mixture was incubated at 65°C for 5 min and then slowly cooled to room temperature so that the oligo(dT)s bind to polyA tail. About 10 µL of 2X First-Strand Reaction Mix (Invitrogen, USA) and 2 µL of SuperScript R III/RNaseOUT™ Enzyme Mix were added. The enzyme mixture was gently mixed and incubated at 50°C for 1 h. The enzyme was deactivated by heating the reaction mixture at 85°C for 5 min. The cDNA (1 µL) synthesized from the transgenic and untransformed control lines were taken as template for PCR using the GUS gene specific primers (5'-CCCTTACGCTGAAGAGATGC-3' and 5'-GAGCGTCGCAGAACATTACA-3') with same PCR condition. Actin was used as a loading control with PCR primers described by Nicot et al. (2005). Reaction products were separated on 1% agarose gel stained with ethidium bromide and visualized under UV light.
Statistical analysis:
The data pertaining to shoot regeneration was analyzed using two-way analysis of variance to examine the main and interaction effects of cultivar and SIM combination. The differences among means were compared by Least Significant Difference (LSD) at p<0.05 significance level. Analysis was performed using programs in MSTAT-C (Michigan State University, Ann Arbor, MI).
Plant regeneration:
It is important to optimize the regeneration frequency of potato to increase the likelihood of recovery of transformants; therefore, different combinations of BAP with TDZ and Kin were evaluated for their regeneration ability of the leaf, internode and petiole explants of Marfona, Agria and Desiree cultivars. Figure 1 shows different developmental stages of regeneration of potato. Callus formation was observed from all types of explants after they were cultured on callus induction medium (Fig. 1a). After 2 weeks, calli were transferred to Shoot Induction Media (SIM). Shoots were regenerated from nodular callus of all type of explants (Fig. 1b). The evaluation of shoot induction was based on the mean number of shoots per explant. Adventitious roots could be seen forming within 1 week after the regenerated shoots were placed on root induction medium (Fig. 1c). Finally, rooted plants were transferred to pots and grown under greenhouse conditions (Fig. 1d). Table 1 gives the results of two-way ANOVA for the differences between SIM combinations and between cultivars with regard to number of shoots per explant from particular explants. All null hypotheses regarding the effect of SIM combination, cultivar and explants type were rejected at the 0.01 probability level. The SIM combination×cultivar interaction was significant for all explant sources, indicating that the cultivars differed in the various SIM combinations.
Averaging the results from all SIM combinations and all cultivars together indicates highly efficient shoot regeneration from internode (4.59 shoots per explant). Shoot regeneration from petiole and leaf explants were 3.83 and 2.55 shoots per explant, respectively ( Table 2). The highest efficiency of shoot regeneration was achieved on MS medium supplemented with1 mg LG 1 BAP+1 mg LG 1 TDZ and 2 mg LG 1 BAP+1 mg LG 1 TDZ for all cultivars, respectively (Table 2).
Plant transformation:
Among the different explants tried, internode explants were found to be best for shoot induction. For transformation, MS medium supplemented with 2 mg LG 1 BAP and 1 mg LG 1 NAA (callus induction medium) followed by MS medium supplemented with 2 mg LG 1 BAP and 1 mg LG 1 TDZ for subsequent shoot proliferation was used routinely. A concentration of 75 mg LG 1 kanamycin was chosen for selection of transformants. The same parameters were used routinely for transformation studies in all the three varieties. A total of 150 internode explants were cocultivated in the three varieties (Table 3). The highest number of J. Agron., 14 (4): 227-233, 2015 kanamycin resistant shoots was recorded with Desiree (122) followed by Marfona (108) and Agria (86). Although, the explants remained green, some of the shoots did not grow more than 10-15 mm. Only those shoots measuring 2.0-2.5 cm were transferred to rooting medium (MS medium supplemented with 0.1 mg LG 1 NAA and 75 mg LG 1 kanamycin). Similarly, highest transformation efficiency were recorded in Desiree (42%), followed by Marfona (30%) and lowest in Agria (22%) ( Table 3). All rooted plants were planted in pots and molecular analysis was carried out to confirm the transgenic nature.
Confirmation of transgenic plants:
In comparison with untransformed wild-type plants which did not contain any expression of GUS, the transgenic plant lines showed the typical dark-blue after the GUS assay. Upon histochemical staining of leaves from kanamycin resistant rooted plants, all lines showed expression of GUS gene (Fig. 2). Besides the GUS assay, the expression of GUS gene was also carried out by RT-PCR analysis. The presence of a band of 362 bp was amplified from the cDNA products of transformed plants while the cDNA products of untransformed plants (control) cDNA showed no amplification (Fig. 3). The presence of the
DISCUSSION
The objective of the present study is to develop an efficient Agrobacterium tumefaciens-mediated transformation protocol for important Iranian potato cultivars Agria and Marfona. For this purpose, two separate experiments, plant regeneration and transformation were carried out.
In plant regeneration experiment, we studied the effects of various concentrations and combinations of plant growth regulators on plant regeneration from different explant sources (leaf, petiole and internode) of potato cultivars. First, all explants were placed on callus induction medium (1 mg LG 1 NAA and 2 mg LG 1 BAP). Calli were obtained from all three types of explant of the three investigated cultivars. The BAP and NAA combination have been shown to be an efficient combination for induction of callus in potato (Beaujean et al., 1998). Medium combination and explant source had an important influence on plant regeneration. In the present study, BAP in combination with Kin induced a lower plant regeneration but it promoted the highest plant regeneration in combination with TDZ (4.86 shoot per explant). The highest efficiency of shoot regeneration was obtained in internode explants of cultivar Marfona on MS medium supplemented with 1 mg LG 1 TDZ+1 mg LG 1 BAP (Table 2). Abd Elaleem et al. (2009) and Khalafalla et al. (2010) reported that MS medium containing 5 mg LG 1 TDZ was the best for days to shoot initiation, the highest percentage of callus with shoot and highest number of shoot per callus in potato. Deore and Johnson (2008) suggested that TDZ and BAP together had a synergistic effect in adventitious shoot-bud induction in Jatrophacurcas. Also they reported that TDZ promoted higher regeneration from leaf explants than Kinetin. Huetteman and Preece (1993) reported that TDZ, a synthetic phenylurea, is considered to be one of the most active cytokinins for shoot induction in plant tissue culture. Husain et al. (2007) suggested that TDZ induces shoot regeneration better than other cytokinins. Apart from cytokinin-like activity, TDZ has been suggested to be a modulator of the endogenous auxin levels. There is experimental evidence that TDZ stimulates de novo synthesis of auxins by increasing the levels of IAA and its precursor, tryptophan (Murthy et al., 1995). Increases in endogenous auxin, cytokinin and ethylene have been seen in response to TDZ treatment (Murthy et al., 1995). Also, TDZ has been shown to be useful for rapid plant regeneration in several recalcitrant species through organogenesis (Malik and Saxena, 1992). Similarly, in our studies, it was observed that TDZ is essential for the high-frequency induction of adventitious shoots from all kind of explants.
Transformation efficiency of 22-42% was observed and true transformed shoots were rooted within 1 week in selection medium. This efficiency is high, as can be seen when comparing this internode transformation protocol with direct selection in kanamycin to other potato transformation protocols that employ leaf segments as explants (Barrell et al., 2002;Soto et al., 2007). Soto et al. (2007) reported higher transformation efficiency (68%) in potato cultivar Desiree using internodal stems as explant and phosphinothricin as a selection agent. While De Block (1988) obtained a transformation efficiency of only 30% with the cultivars Bintje and Desiree using phosphinothricin as selection marker. Moreover, the leaf explants were easily injured during the manipulation, which resulted in a low percentage of transformation (De Block, 1988) while the internodal explants are much more resistant during manipulation and more amenable to in vitro conditions. It was also observed that the transformed cells were located essentially at the section and in the cambium/pericycle cell (Higgins et al., 1992 Longitudinal sectioning of internodes considerably increased the section, with the vascular bundle zone being fully accessible to Agrobacterium and this allowed a large number of transgenic buds to be obtained (Beaujean et al., 1998). Analysis of all putative transgenic lines using PCR established the presence of the nptII gene in all regenerated lines (Fig. 4), thereby confirming their transgenic status and a high rate of success for the Agrobacterium-mediated gene transfer system of potato using kanamycin resistance as a selectable marker (Barrell et al., 2002). Based on the data of the present study, it was concluded that the reported regeneration system is repeatable and can be easily used to regenerate transgenic potato plants expressing the genes present in the Agrobacterium binary vector T-DNA. Using this regeneration and transformation protocol, transgenic potato with higher contents of pharmaceutical compounds or modified secondary metabolic profiles will be produced in the future by approaches of specific metabolic engineering. | v3-fos |
2019-03-20T13:03:33.223Z | {
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} | s2 | Prevalence of Wax Moth in Modern Hive with Colonies in Kafta Humera
A cross-sectional study was carried out from April 28 to May 30, 2009 in four village of Kafta Humera to determine the prevalence of wax moth in modern hive with colonies. A total of 307 modern bee hives with colonies were inspected during the study period. Physical inspection and observation were the methods of the study used. According to the study the overall prevalence of wax moth in modern bee hive was found to be 27.4%. During the study period larval stage of wax-moth was only detected. The nature of severity was differentiated and categorized in to three groups as 35(11.4%), 47(15.3%) and 2(0.65%) as light, moderate and severely affected respectively. Infestation rate of wax moth based on supering was found to have statistically significant difference as determined as 8(16%), 65(27.7%) and 11(47.7%) in base only, base plus one box and base plus two boxes. To combat the problem, awareness creation should be done on proper management of bee keeping in the area.
Introduction
The contrasting geomorphic land scopes of Ethiopia create favorable environment for the existence of a large and unique biodiversity both in plants and animals including honey bee sub species. The ideal climate conditions and diversity of floral resources allow the country to sustain around 10 million honey bee colonies of which 7.5 million are kept in local bee hives around 30 thousand in top bar hives by farmers and the remaining exist in the forest as wild colonies. This makes the country to have the highest bee density in Africa [1,2,3].Beekeeping preserves nature, agriculture, sustains livelihoods, and provides food security [4]. Beekeeping in Tigray is most traditional and has long years of experience which is parallel to the history of agriculture, but still a recent time, Beekeeping and honey production for that matter has been in state of stagnation.
The honey bee could be affected by many infectious diseases which are parasites, bacteria, virus, protozoa, fungus, pests, and poisons and attacked by predators. Transmission of bee diseases from one colony to another can be by adult bee, reuse of contaminated comb, bee keepers, beekeeping equipment, feeding of infected honey and pollen [5,6] Wax moths are potentially very troublesome to bee keepers all over the world. These are of two species of wax moth namely Galleria mellonnella and Achroing grisella. All occur naturally or have been introduced by man in almost all regions of the world. Wax moth is a serious destructive pest cause considerable damage to both normal and abandoned combs of bee and brings considerable loss to beekeeping industry [7].The weak colonies or abandoned combs are easily attacked more often by wax moth [8,9]. Throughout the world today they are an ever present cause of economic loss to bee keepers who have capital invested in framed combs built on comb foundation.
Wax moth emerge as adults in the hive and the female may live a few days or a few weeks after dark she flies out to a tree and mates, she then re-enters to hive and lays about 500 eggs. The newly hatched larvae can run very fast and thus distribute themselves around the hive, they then burrow in to comb, damaging it in a characteristics way by constructing a feeding tunnel of silk through it [10]. Damage may be done to combs containing honey to freshly extracted combs, and to stored combs. The market demand for beeswax both in the domestic and international trade is very high. Beeswax from Ethiopia has higher demand and also earns higher price in EU [11].The wax moth is also the major problem in the whole country and regions which causes damages and loses. Other than its presence the magnitude, type and amount of lose are not well studied in depth. Therefore the objective of this paper was to assess wax moth infection rate in modern bee hives in Kaft Humera district of Tigray.
Study Area
The study was conducted in Kafta Humera district ,Tigray regional state of Ethiopia. The study was conducted on four selected 'kebeles'/lockal administrative region / of the Kafta Humera district namely, Baecker, Adhrdi, Mayweyni and Adi-goshu. The geographical location of the research site is 130 42' to 140 28' North latitude and 360 23' to 370 31' East longitude and its elevation is 550-2800 m.a.s.l. The mean monthly maximum temperature remains high throughout the year ranging from 30 0 c in August to 40 0 c in March and May in Humera.
Study Design
Across sectional epidemiological study was conducted from April 28 to June 30, 2009. During the study period data was collected through gross hive inspection of each hive. The data collected from the study area were summarized and analyzed by the possible categories and variables. The nature of severity was differentiated and categorized in to three groups as light, moderate and sever infestation based on the criteria of [12].
A total of 307 modern hives with colonies have been randomly sampled for wax moth infestation. Sample size taken for each study sites includes, Adhrdi from total 114 modern hives 50 were sampled; Baeker from 80 modern hives 36 were sampled; Adigoshu from 373 modern hives 163 were sampled and in Mayweyni from 130 modern hives 58 were randomly inspected (44-45% of the total modern hives with colony in the selected village were included in the study).
Degree of Wax Moth Infection
The total numbers of 307 modern bee hives with colonies were inspected during the study period. The routine internal inspection revealed that 27.4%(84) of the modern lives with colonies had visible larval stage of wax moth. Degree of severity based on Burges criteria was differentiated and categorized in to three groups as 35(11.4%), 47(15.3%) and 2(0.65%) light, moderate and severely affected respectively (Table 1). During the study period egg and adult stage of wax moth were not detected.
Comparison Based on Hive Positioning
Out of 307 modern hive inspected 50(16.3%), 234(76.2%) and 23(7.5%) were base only, base plus one box and base plus two boxes respectively. The infection rate of wax moth based on their supering was determined as 8(16%), 65(27.7%) and 11(47.8%) in base only, base plus one box and base plus two boxes respectively (Table-2
Discussion
An overall prevalence of wax moth in modern bee hive in the study area was found to be 27.4%. The nature of severity was differentiated in to three categorized as light, moderate and severely affected. The larvae of was moth silken tunnels throughout the comb and when hives are heavily infested the combs are completely consumed and only the larval silk, droppings and cocoons remain in the hive [10]. Based on Burges criteria only 2(0.65%) from the total affected modern hives were severely affected. Based on positioning of hives the infection rate was determined in base only, base plus one box and base plus two boxes. The infection rate was statistically significant increasing as supering of hive increases and this current result is agreed with the idea of [11] as bee colonies become weak the chance of getting infection by wax moth is high and the combs are vulnerable to wax moth when in store, as well as inner parts of hives not occupied by bees. Supers of empty combs are sometimes left above the brood box for storage, but they are safe from wax moths only where the colony can be kept strong enough throughout the year [12].
The current result was indicating that infestation of wax moth in the study area was high. This may be due to the fact that lack of proper management in the study site. This can be due to lack of extension services in the district and lack of training to the farmers to create awareness about proper management. There is no method for killing of wax moth can take the place of management that prevents infestation in the hive [13,14]. The apiaries are to ensure that all colonies are strong and healthy. During the study period it was observed that larval stage of wax moth was responsible for destroying the combs. There for wax moth is mainly responsible for damaging the combs and feeds completely, and cause for the colony to be absconding. This is congruent to the idea of Crane [13] that combs are vulnerable to wax moth when in store as well as in parts of hives not occupied by bees. Estimated the annual loss in the USA as $500,000 and the damage is likely to be relatively more in other parts of the world. The other important contribution for this higher wax moth infestation recorded in the area may be due to lack of food resources in the season and poor site selection. Weak colonies are easily susceptible to wax moth infestation. Burges [11]indicates that as adult wax moth emerge in the hive and the female may live a few days, in the hive a few weeks later she flies out to a tree and mates she then re-enters to hive and lays about 500 eggs in dark time in a weak colony.
Conclusion and Recommendations
The infestations of honey comb by wax moth are potentially very troublesome to bee keepers all over the world and the studied area. During the study period observation and inspection the combs were vulnerable to wax moth as in parts of hives not occupied by bees. The fast moving larval stage of wax moth was responsible in destroying the comb and its contents. It is must to keep colony of hive to be strong and healthy to avoid infestation of wax moth.
In general we can conclude that poor management of bee keeping is the main reason for wax moth to invade modern bee hive. In light of the above facts the following recommendations can be made with respect to the status of the infestation of wax moth and then possible measures to be taken at the study area.
awareness should be created to wards management of bees.
Apiary inspectors, beekeepers and researchers must be able to recognize possible intervention, option that could improve on the control of wax moth. Site selection should be assisted by experts Flowering plants should be planted to increases bee forage | v3-fos |
2016-05-04T20:20:58.661Z | {
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"year": 2015
} | s2 | Hypoglycin A Content in Blood and Urine Discriminates Horses with Atypical Myopathy from Clinically Normal Horses Grazing on the Same Pasture
Hypoglycin A (HGA) in seeds of Acer spp. is suspected to cause seasonal pasture myopathy in North America and equine atypical myopathy (AM) in Europe, fatal diseases in horses on pasture. In previous studies, this suspicion was substantiated by the correlation of seed HGA content with the concentrations of toxic metabolites in urine and serum (MCPA-conjugates) of affected horses. However, seed sampling was conducted after rather than during an outbreak of the disease. The aim of this study was to further confirm the causality between HGA occurrence and disease outbreak by seed sampling during an outbreak and the determination of i) HGA in seeds and of ii) HGA and MCPA-conjugates in urine and serum of diseased horses. Furthermore, cograzing healthy horses, which were present on AM affected pastures, were also investigated. AM-pastures in Germany were visited to identify seeds of Acer pseudoplatanus and serum (n = 8) as well as urine (n = 6) from a total of 16 diseased horses were analyzed for amino acid composition by LC-ESI-MS/MS, with a special focus on the content of HGA. Additionally, the content of its toxic metabolite was measured in its conjugated form in body fluids (UPLC-MS/MS). The seeds contained 1.7–319.8 μg HGA/g seed. The content of HGA in serum of affected horses ranged from 387.8–8493.8 μg/L (controls < 10 μg/L), and in urine from 143.8–926.4 μg/L (controls < 10 μg/L), respectively. Healthy cograzing horses on AM-pastures showed higher serum (108.8 ± 83.76 μg/L) and urine concentrations (26.9 ± 7.39 μg/L) compared to control horses, but lower concentrations compared to diseased horses. The range of MCPA-carnitine and creatinine concentrations found in diseased horses in serum and urine were 0.17–0.65 mmol/L (controls < 0.01), and 0.34–2.05 μmol/mmoL (controls < 0.001), respectively. MCPA-glycine levels in urine of cograzing horses were higher compared to controls. Thus, the causal link between HGA intoxication and disease outbreak could be further substantiated, and the early detection of HGA in cograzing horses, which are clinically normal, might be a promising step in prophylaxis.
Introduction
In horses on pasture, atypical myopathy (AM) and the similar disorder called seasonal pasture myopathy (SPM), are highly fatal forms of non exertional rhabdomyolysis occurring mainly in autumn but also less frequently in the subsequent spring [1][2][3][4]. Particular, season-dependent pasture characteristics seem to promote the incidence of AM or SPM [4], [5]. The first cases of AM were reported in 1939 in the North of Wales [6] and since the 1980s, also in various continental European countries [7][8][9][10]. The similar disease, SPM, is mainly described in North America [11], [12]. The first continental European outbreak occurred in Northern Germany in 1995 [12], whereby nearly all of the AM-affected horses died (111/115). However, the quantity of affected horses on the respective pasture is not uniform worldwide. In Europe large outbreaks with many affected horses on the same pasture are described, whereas in North America only a few horses with the same conditions develop the disease [2], [11], [13], [14]. Thus, cograzing horses exhibiting no disease symptoms have not been studied alongside affected horses.
In addition to the presence of Acer spp. (Acer negundo, Acer pseudoplatanus), other pasturerelated risk factors exist [14], [15]. Gusts of wind have been reported to coincide with an outbreak or were discussed to be a forerunner, respectively [4], [11], [16]. Predisposed horses for AM or SPM have been found to be predominantly young males (< 3 years; [14]) that were kept on poorly maintained pastures (often more than 12 hours a day) without supplementary feeding. Previous studies revealed increased prevalence of AM in females, but this is probably due to the more frequent exposure to pastures compared to males [4]. The prevalence in young horses might be due to the fact, that horses until the age of three years spend more time on pastures until horse owners start to work with them [4]. Several hypotheses about the biochemistry underlying disease development have been communicated in the past, but the discovery of the disruption of fatty acid ß-oxidation and amino acid metabolism by an acquired enzymatic deficiency of multiple acyl-CoA dehydrogenases (MADD) was a major step in diagnostic investigation [17]. As a consequence, there is excessive myofiber lipid storage and an abnormal production of blood acylcarnitines and urine organic acids [2], [17]. MADD is also described in humans. Clinical signs of inherited forms in humans range from a fatal neonatal condition to an adult onset mild lipid storage myopathy [18]. After ingestion of unripe Jamaican ackee fruit (Blighia sapida), whose seeds contain a nonproteinogenic amino acid called hypoglycin A (HGA, L-α-amino-methylenecyclopropylpropionicacid), an acquired form of MADD in humans can develop, with hypoglycaemia, continuing vomiting, and possible fatal outcome. HGA is metabolized to MCPA (methylencyclopropylacetic acid) and binds to CoA and is consequently a potent inhibitor of Acyl-CoA-dehydrogenases [19]- [20]. In horses with MADD, clinical manifestations include muscular weakness, stiffness, trembling, sweating, and myoglobinuria [1], [17]. Very indicative signs are severe acute myonecrosis of respiratory and postural muscles and less frequently the myocardium [4], [21], [22]. The serum activities of the creatine kinase (CK) and lactate dehydrogenase (LDH) are extremely elevated. The majority of horses developed recumbency and respiratory difficulties. In 75% of all cases the outcome was lethal [4], [7], [12], [15], [23].
Chase et al. [24] determined HGA concentrations in unripe ackee fruit components as high as 939, 711, and 41.6 mg/100 g of seed, aril, and husk components, respectively. Analysis of the ripe fruit components showed a remarkable decrease by about 70% in seeds. A current study conducted by Bowen-Forbes et al. [25] across two different harvest seasons of ackee fruits shows a strong inverse relationship between HGA and hypoglycin B (HGB), a dipeptide of glutamic acid and hypoglycin, which is also toxic. HGA decreased from 80 mg/100 g in green seeds to 14 mg/100 g in ripened seeds whereas HGB increased from 16 mg/100 g to 118 mg/ 100 g. The authors reasoned that the rise in HGB content represents a possible detoxification mechanism of the fruit.
HGA was also found in seeds of box elder trees (Acer negundo, Sapindaceae), which belongs to the same taxonomic plant family as Blighia sapida. Interestingly, box elder trees were present on pastures in North America with an increased incidence of SPM in horses [16]. Additionally toxic HGA metabolites were found in urine (MCPA-glycine) and in serum (MCPA-carnitine) of affected horses suggesting the ingestion of seeds of Acer spp. [16]. In Europe another Acer spp. called Acer pseudoplatanus (Sycamore maple) was identified on all pastures with horses affected with AM [26]. Subsequent (> 1 year later) analysis of HGA-content in Sycamore maple seeds of AM-affected farms could detect a HGA content (3.6-252.9 μg/seed [27]; 0.74 and 7.2 mg/g seed [28]) similar to previous studies (3.0-160.0 μg/seed; [16]). In addition to the ackee fruit (Blighia sapida) hypoglycin A or hypoglycin-like compounds were also detected in different chestnuts by Bressler et al. [29], for example Aesculus parviflora, Aesculus glabra as well as in many other species. All studies noticed a high variation of HGA content in different plants according to the level of maturity. Unger et al. [27] also showed a high variation of HGA content in the seeds of Acer pseudoplatanus, but a direct link between maturity and measured HGA concentrations is missing. In order to provide toxicological evidence of ackee, Fincham [30] measured HGA in blood and plasma of human patients. However, the detection limit was too high (1.4 mg/L) because HGA is already toxic at lower concentrations. A more sensitive HGA detection method to improve the prevention of intoxication of ackee or maple poisoning was presented approximately 40 years later [28] with a detection limit for HGA of 0.35 μg/L blood. The first quantitative determination of HGA in serum samples of two AM affected horses showed a HGA content of 446.9 and 87.8 μg/L [28]. However, there was no proof between the consumption of HGA containing maple seeds and the disease.
We hypothesized that the outbreak of AM in horses on pastures in Germany was caused by the ingestion of seeds of sycamore maple tree that contained high amounts of HGA. Toxic HGA and its metabolites (MCPA-conjugates) could be identified in the serum and urine of the affected horses. Cograzing healthy horses coexisting on affected pastures were also tested to determine the concentration of HGA in order to establish a link between HGA levels in serum and urine and their clinical disease.
Case selection
In autumn of 2013 and 2014, 16 horses (7 Warmbloods, 4 Haflinger, 5 ponies; comprising 7 geldings, 2 stallions, 7 mares) were studied, which were turned out on pastures in Germany and exhibited acute clinical signs of muscle pain and weakness. The farm veterinarians conducted the initial treatment and diagnosed AM based on the clinical signs and clinicopathological results. Five horses were admitted to the equine clinic of the Department of Large Animals Medicine, Faculty of Veterinary Medicine Leipzig, Germany, for intensive care where they died or were euthanized. In addition to the preliminary report, clinical signs included the sudden onset of indicator signs of acute rhabdomyolysis (e.g. myoglobinuria, stiffness, trembling, sweating), weakness, recumbency and depression, with rapid progression of signs or even unexpected death. Of the investigated cases 15 horses died and one survived. Eight serum samples and 6 ante-mortem urine samples were collected from these affected horses during the disease. Additionally 12 horses that were clinically normal and cograzing pastures with AM cases were included in the study.
Ethics statement
Animal Care and Use Committee approval was not obtained for this study because no animals were handled specifically for this experiment. Blood and urine samples were collected by qualified veterinarians through their routine practice, in the framework of official programs aimed at the diagnosis and a possible treatment. Total duration of the blood sampling procedure did not exceed 1 min, and non-painful sampling was confirmed by the absence of any retreat behavior of the horses during the procedure. Urine samples were collected as free-catch urine. The detection of the contents of HGA and MCPA-conjugates was conducted with the owner's consent. An experienced botanist and a veterinarian executed the botanical rating and collection of seed samples on affected pastures. Therefore, the legal restrictions do not apply, as they are waived in the case of non-experimental procedures and routine veterinary practices with livestock species (no laboratory animals).
Botanical inspection and owner interview
The AM and cograzing horses originated from 11 different pastures (pasture A-L, Fig 1). Nine affected pastures were visited directly after the occurrence of AM or at latest 14 days after the outbreak to collect seed samples. An experienced botanist and a veterinarian, with the owner´s consent, conducted the botanical rating. Digital images were taken and available trees and plants were obtained and cataloged. Samples of grass, leaves and seeds were collected. The focus was directed towards Acer spp., especially sycamore maple trees (Acer pseudoplatanus) to determine the presence of the seeds and the availability of those to the horses. Individual seed sampling in 2013 from soil was conducted according to the protocol of Valberg et al. [16] (pasture B, C, E, F). In 2014 the sampling method was adapted according to the protocol of Unger et al. [27] with pooled samples from the trees (pasture G, H, J, K, L). Pasture A and D were not visited, because it was too late for seed sampling (information about these cases more than 4 weeks later). Additionally the owners were asked about the development of AM (first admission to the pasture, date of first clinical signs, composition of the herd, hours on pasture, additional food stuffs).
MCPA-conjugates analysis
Samples of affected horses obtained immediately before demise and within 3 days of the onset of AM disease were available for analysis. Acylconjugates concentrations were analyzed by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) to identify MCPA-carnitine and-glycine in equine serum and urine in a special screening laboratory in Hannover, Germany [31]. Samples of cograzing horses were tested as well. Owners gave permission for measuring MCPA-conjugates in available body fluids. Serum samples of 14 horses without any symptoms of AM and without contact to seeds of Acer spp. on pastures were used as controls. In these cases blood was drawn as part of routine screening or in the diagnostic work up of cases unrelated to AM. Five urine samples of horses were collected as free-catch urine from healthy horses as negative controls. All samples were frozen at -20°C until they were sent to the laboratory for analysis.
Hypoglycin A analysis
Authentic S-Hypoglycin A was from Toronto Research Chemicals (Toronto, ON). Plant tissue (between 100 and 300 mg) were ground to a fine powder with a mortar and pestle under liquid nitrogen and subsequently further homogenized with a mixer mill MM301 (Retsch, Haan, Germany) at a frequency of 25/s for 50 s with a single 5 mm diameter steel bead in a 2 ml Eppendorf cup. The resulting powder was extracted with 750 μl 70% (v/v) methanol containing 10 nmol of norvaline with vigorous shaking and the slurry was centrifuged twice at 10,000 g for 5 min.
HGA was analyzed by LC-ESI-MS/MS and quantified as its Fmoc-derivative according to the protocol for amino acid determination published by Ziegler et al. [32] using 25 μl of plant extract or 5 μl of serum and urine samples, respectively. HGA contents in affected and cograzing horses were determined. Owners gave permission for measuring HGA in available body fluids. Serum (n = 5) and urine (n = 4) samples of horses without any symptoms and without contact to Acer pseudoplatanus were used as controls. Blood in these cases was drawn as part of routine screening or in the diagnostic work up of cases unrelated to AM. Four urine samples of horses were collected as free-catch urine from healthy horses as negative controls. All samples were frozen at -20°C until analysis.
MS parameters describing the MRMs for the HGA are shown in the Supporting information (S1 Table).
Hypoglycin A calculation in mg HGA/horse
For calculation of HGA concentration in mg HGA/horse to reveal the maximal tolerated dosage (MTD) the following parameters were used: 1. measured HGA concentration in serum (μg/L) of affected, cograzing and control horses 2. estimated total blood volumes ( [33], 101 ml/kg bwt), which was calculated with the bwt.
Statistical analysis
For the subsequent multiple comparison post hoc test a SAS based macro according to the protocol of Elliott and Hynan [34] was used to reveal differences between the concentrations of HGA in serum and urine of affected and cograzing horses in comparison to the controls. The macro takes into account different group sizes according to Dunn's multiple comparison procedure [34]. Since the data from HGA in urine and serum are not normally distributed, ANOVA could not be used. However, since the data are independent and continuous, and the null hypothesis of Levene's Test to test the homogeneity of variance could not be rejected, the nonparametric Kruskal-Wallis Test to test for difference among groups was applied. The null hypothesis of the Kruskal-Wallis Test was rejected for both traits, i.e. that at least 2 groups are different.
Case history, outcome of owner interview and clinical findings
The affected horses comprised 3 different breeds (Warmblood, Haflinger, German Riding Pony) and included 7 geldings, 2 stallions and 7 mares aged from 1.5-16 years (Fig 1). All affected horses had a good body condition (ranged from 5.0-5.5/9) and the turnout time onto the pasture was variable between the cases (6-24 hrs/d, Fig 1). Trees were present on or around all pastures and horses had access to dead wood and leaves. Pastures were poorly maintained and the grassland was in most cases very sparse, the turf was often damaged with soil showing through and a lot of dead leaves (Fig 2). All horses (except horse No. B1) got additional feed such as hay and cereals (oat grains) or a mixed feed. Hay was offered ad libitum at stable with free access to the pastures (personal communication with the owner) or was additionally provided at pasture with a hayrack. Cereals or mixed feeds were portioned once or twice a day. All had access to a salt or mineral block and all were routinely dewormed. A total of three horses were used for riding (B1, J1; 2-3 time per week) or driving (D1; 1-2 times per week). The other horses had not been exercised, mainly because of their age (< 3 years).
All horses exhibited typical clinical symptoms of AM, as it was described in several previous studies [1][2][3][4][5], [13][14][15][16]. Interestingly the horses C1, C2, C3 and C4 from the same pasture developed AM consecutively within 48 hours, although access to pasture was constrained after the first horse showed the onset of disease. Hence, although the seeds containing toxic amino acid HGA were no longer available, the horses succumbed to disease within two days (Fig 1). Horse C4 was the last horse from this pasture becoming ill and the blood sample showed a moderate increase in CK. However, this horse was euthanized at a very early stage of the disease while it was still standing. One horse died within 6 hours (F1), owners found the horse dead on pasture. The second and oldest (16 years) horse on this pasture (F2) survived, and the concentrations of CK and LDH were only moderately increased. After giving the horse intensive care (treatment included intravenous administration of fluids 70-100 mL/kg bwt/day, flunixin-meglumin over 7 days) a paralysis of the tongue and a slightly lameness was observed, but this was reversible after 4-6 weeks.
MCPA-carnitine and acylcarnitines in serum and urine
Serum and urine acylcarnitines profiles resembled that of MADD in all analysed samples (Tables 1 and 2). Concentrations of medium chain acyl conjugates in serum were at least ten times higher compared to controls, whereas urine samples of affected horses exhibited a 1,00-1,000 times higher level compared to control samples. High MCPA-carnitine was found in all sera of severely diseased horses, but MCPA-glycine was present at very low concentrations. However, in urine the MCPA-glycine concentrations were high in all samples obtained from diseased animals. In all cases, high concentrations of acylcarnitines (e.g. Butyryl-, Isovaleryl-, Valeryl-carnitine) and MCPA-conjugates (MCPA-carnitine and-glycine) in serum corresponded to elevated levels in urine. Especially horse C4 showed a moderate increase beyond all parameters. However, this horse was euthanized at a very early stage of the disease and therefore detected concentrations are much lower in comparison to the other affected horses.
In healthy, cograzing horses increased concentrations of the glycine-fraction (Valerylglycine, Hexanoylglycine, MCPA-glycine) in urine were observed. The levels of Valeryl-carnitine in body fluids were higher than the concentrations in controls, but much lower compared to the concentrations of affected horses. MCPA-carnitine in serum was detectable only in very low concentrations.
Hypoglycin A assay in seeds and bodyfluids
There was a strong relationship between the presence of Sycamore maple trees (Acer pseudoplatanus) and the incidence of AM. Trees were either found right on the pasture or as in the case of pasture B the tree was about 50 meters away. However, HGA containing seeds were consistently found on all pastures (Fig 3).
Seed sampling was performed on 9 pastures with AM occurrence and the HGA concentrations of the seeds were analysed. HGA concentrations in sycamore maple seeds varied highly from seed to seed between 0.7-111.6 μg/seed. The average weight of the collected seeds was 314 mg (range from 191-428 mg). Both chosen sampling methods showed a very high variation of HGA contents (1.7-319.8 μg/g seed; Fig 4). Body fluids of all affected horses contained the toxic amino acid HGA (Table 3). Measured concentrations in serum ranged from 387.8-8,493.8 μg/L and in urine from 143.8-926.4 μg/L. Cograzing horses showed HGA concentrations between the controls and diseased horses, ranging from 37.8 and 328.5 μg/L (n = 12; p < 0.01,) in serum, and between 18.9 and 35.1 μg/L (n = 4; p < 0.01,) in urine. HGA content in serum and urine of control horses (no access to AM-pastures) was below the limit of detection (< 10 μg/L). The results with statistical analysis are summarized in Table 4, wherein significance is indicated by p-values < 0.05.
Hypoglycin A in mg HGA/horse
Referring measured serum HGA concentration to the bwt of the horses and their estimated total blood volumes revealed a minimal MTD in horse A1 with 17.47 mg HGA/horse and a maximal MTD in horse G1 with 128.68 mg HGA/horse (Table 5). Within the set of cograzing horses the mean of the determined HGA concentrations was used for comparison. Cograzing horses showed HGA concentrations between the controls and diseased horses with a mean of 5.99 mg HGA/horse, thereby in close proximity to the minimal MTD of horse A1. The HGA content in serum of control horses was calculated with the detection line (< 10 μg HGA/ L) and revealed amounts of < 0.5 mg HGA/horse.
Discussion
In the present study acquired MADD was diagnosed in AM horses by increases in short and medium chain serum acylcarnitines [2], [17]. In addition to the presence of MCPA-conjugates in blood or urine of affected horses, HGA, as the possible cause for developing a MADD, was measured in the seeds of Acer pseudoplatanus from affected pastures and in body fluids of AM horses. To our knowledge HGA levels in body fluids of affected horses during an outbreak of AM have not been reported yet. In previous studies the presence of serum MCPA-carnitine was a consistent feature [16] and was absent in horses without contact to Acer spp. on pastures. In this study MCPA-carnitine concentrations in serum of affected horses were highly variable and ranged from 166-652 nmol/L. The highest concentration (4.8-102.4 nmol/L) reported by Valberg et al. [16] is within the range of our results. Votion et al. [13] measured lower concentrations of 20.4 μ 17.2 nmol/L. Since the use of the same internal standard for analysis (octanoylcarnitine) allows the comparison of the results between the studies these differences are most likely due to the amount of ingested toxic material and metabolism. The lowest concentration of MCPA-carnitine in this study was found in a horse (C4) exhibiting the lowest concentration of short and medium chain acylcarnitines (Valeryl-, Hexanoyl-, Isovaleryl-, Valeryl-, Octanoyl-and Decenoylcarnitine), but a high concentration of HGA in serum, respectively. Possibly the early euthanasia of this horse led to an immediate stop of metabolism of the toxic amino acid right after the beginning of intoxication. At this pasture C with 4 Haflinger horses, one after another developed an AM, although the horses did not have access to AM affected pasture after the first horse became ill. Within 3 days all horses died (C1-C3) or were euthanized (C4), respectively. Possibly different diet preferences of the horses on pastures increased the toxicity of HGA. Previous studies revealed different amounts of HGA as an acute toxic dose (130-150 mg HGA/kg bwt [36], 100 mg HGA/kg bwt [37]) or as the MTD (1.5 mg HGA/kg bwt [38]) leading to the conclusion that the susceptibility of animals to HGA is influenced by carbohydrate/protein ratio in their diet or the composition of the administered hypoglycin preparation. Concentrations of HGA in seeds of Acer pseudoplatanus from AM pastures ranging from 0.7 to 111.6 μg/seed are similar to previously reported values with 4-56 μg/seed [16] and 3.6-252.9 μg/seed [27]. The latter showed high variation of HGA content in individual seeds collected from a single tree. Even though pooled samples (five seeds, triplicated samples) were used, the interpretation of the results remains difficult. A direct link between measured HGA amounts in seeds and the number of affected horses cannot be drawn. Farms without reported AM cases showed the highest concentrations of HGA in the collected seeds from these pastures [27]. The level of HGA in affected horses on AM-pastures calculated with their bwt and the estimated total blood volume is similar to the MTD in rats as determined by Blake et al. [38] using ackee as the source. Valberg et al. [16] converted the MTD for rats to a MTD for horses by using body surface area and the equation 1.5 mg HGA/kg bwt à bwt 0.75 /6 (rat km value) and determined a MTD of 26.5 mg HGA/500 kg horse. This calculation can be confirmed by our results because the level of HGA in the present study ranged from 17.47 mg HGA/horse and 128.68 mg HGA/horse and especially the minimal dosage is in the field of the calculation of Valberg et al. [16]. Extrapolating 26.5 mg HGA/horse on the highest measured HGA concentration (820.8 μg/seed, [27]) 32 seeds would be sufficient to poison a 500 kg horse. Especially in autumn, when the first seeds drop to the ground, for example after a windstorm, the percentage of seeds with high HGA content available on the surface of the pasture is very high, and horses would probably not hesitate or even would not notice the ingestion of a limited number of seeds. However, to evaluate the HGA concentrations in the horse it is important to also look at the measured concentrations of the MCPA-conjugates. Since HGA is rapidly metabolized to MCPA, both values comprise essential information about HGA ingestion. Thus, looking at only body fluid HGA levels possibly underestimates the amount of HGA ingested by the horse.
Valberg et al. [16] included horses without clinical symptoms cograzing on the same pasture as an "ideal" control group. However, these horses incorporate HGA, although at lower concentrations (~5.99 mg HGA/horse, Table 5). The ingestion of seeds of Acer pseudoplatanus from cograzing horses on pastures was demonstrated [39]. Since the seeds have no characteristics which make them unpalatable to horses, there is a chance that they will be consumed during grazing. If these consumed seeds contain a high concentration of HGA, it could result in poisoning [40]. An ideal control group consists of horses without access to seeds of Acer spp. This distinction is necessary because both groups are clinically unaffected, but the likelihood of developing an AM-disease is higher within the cograzing horses because of detectable concentrations of HGA and MCPA-conjugates.
Currently we are unable to standardise the MTD of HGA for horses, but suggestions about possible quantities can be deduced based on our results. Concentrations of HGA in cograzing horses were higher than in controls but always lower compared to affected horses, although some of these horses (Table 3) showed HGA-contents in close proximity to affected horses. This assumption leads to the conclusion that the detected HGA concentrations in horses and therefore a possible intoxication mainly depends on i) the high variation of HGA levels in seeds and thus ii) the availability of highly toxic seeds and iii) the different feed preferences of the individual horse as well as iv) the time of blood sampling (before or after a peak). Obviously, cograzing horses as well as affected horses ingested HGA containing material (most likely Acer pseudoplatanus seeds, [39]), but the cograzing group did not acquire the necessary toxic amount of HGA at the time of sampling. The detection of low but elevated HGA concentration in blood samples of cograzing horses should result in a complete closing off the pasture to all horses. Still, clinical symptoms may develop shortly afterwards, but an early initiated therapy may decelerate the metabolism of HGA to the toxic MCPA-conjugates and prognosis could be improved.
Conclusions
This study correlated an outbreak of AM with high contents of HGA in seeds of Acer pseudoplatanus on affected pastures and in body fluids of diseased horses. Although the horses in our study obtained additional foodstuffs (hay, mixed feeds) and had in most cases only limited access to the pasture, they ingested enough HGA to be poisoned. The recommendation of preventing horses from grazing on affected pastures with Acer pseudoplatanus is indispensable, however, the analysis of HGA in horses grazing on pastures with Acer pseudoplatanus without reported cases allows a quick survey, whether or not HGA was ingested. We suggest that the availability of a test for early detection of HGA in clinically normal horses might represent a valuable diagnostic tool for prophylaxis, providing information on the HGA-status of horses at risk, similar to a diagnostic marker. The causal chain of HGA availability by Acer pseudoplatanus seeds, HGA ingestion and HGA as well as toxic metabolites in body fluids of affected horses, was further substantiated by our study. Although sycamore maple seeds were the likely source of toxin in all horses in the present study other related trees or plants as a cause for the disease cannot be excluded. | v3-fos |
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} | s2 | Two new bioactive polyketides from Curvularia trifolii, an endolichenic fungus isolated from Usnea sp., in Sri Lanka
Two new polyketides (compounds 1 and 2) were isolated from the EtOAc extract of an endolichenic fungus, Curvularia trifolii obtained from Usnea sp. in Sri Lanka. The structures of these compounds were elucidated on the basis of spectroscopic methods (UV, IR, MS, 1D NMR and 2D NMR). The bioactivity of the compounds 1 and 2 were studied using DPPH antioxidant assays and they showed radical scavenging activity with IC 50 values of 4.0 ± 2.6 and 1.3 ± 0.2 mg/mL, respectively. The radical scavenging activity of compound 2 was higher than that of compound 1 and it was comparable to BHT. Moreover, the new compounds 1 and 2 were evaluated for their anti-inflammatory activity, and only compound 2 exhibited a significant activity comparable to the standard antiinflammatory drug, aspirin. Compound 1 was evaluated for the inhibition of cell proliferation in a panel of five cancer cell lines NCI-H460, MCF-7, SF-268, PC-3M and MIA Pa Ca-2, and exhibited > 90 % inhibitory activity at 5 μg/mL with all of the above cell lines.
INTRODUCTION
Endolichenic fungi are considered as a new source of bioactive fungal metabolites (Paranagama et al., 2007). Only a few investigations have been reported on the isolation and characterisation of secondary metabolites of endolichenic fungi, but they have shown great potential to be a new source for structurally diverse and biologically active natural products (Paranagama et al., 2007;Ding et al., 2009;Zhang et al., 2009;Wang et al., 2010). The endolichenic fungi available in Sri Lanka are still an untapped source of bioactive natural products since their identity and the chemistry of their secondary metabolites have not been explored thoroughly. The first report on isolation and identification of endolichenic fungi from the lichens in Sri Lanka was published by a research team from the University of Kelaniya (Kannangara et al., 2009). New bioactive compounds from endolichenic fungi from Sri Lanka have been isolated and identified (Kannangara et al., 2009;Kulasekera et al., 2013;Pary et al., 2013;Samanthi et al., 2013a;2013b;2014;2015). In the ongoing study on isolation of bioactive secondary metabolites from endolichenic fungi, the fungal strain US/US/06 was found inhabiting the lichen, Usnea sp. collected from the Hakgala Botanical Garden, Central province, Sri Lanka in December 2010. This study was aimed at determining the bioactive compounds present in the ethyl acetate (EtOAc) extract of US/US/06 cultured on potato dextrose agar (PDA) and their potential for the production of bioactive secondary metabolites. This led to the isolation of two new polyketides (compounds 1 and 2). The details of isolation, structure elucidation, and bioactivity screening of compounds 1 and 2 are described here.
Isolation of the fungal strain
Usnea sp. was collected from the Hakgala Botanical Garden in Sri Lanka. Sterilised polythene bags were used to store the lichen samples for transport to the laboratory of the Department of Chemistry, University of Kelaniya. Surface sterilisation method was used to isolate the fungi from the lichen (Kannagara et al., September 2015 Journal of the National Science Foundation of Sri Lanka 43 (3) 2009). Samples of Usnea sp. were cleaned in tap water and surface sterilised by consecutive immersion for 10 s in 95 % ethanol, 3 min in 0.5 % sodium hypochloride and 30 s in 75 % ethanol. Sterile filter papers were used to dry the cleaned lichens, which were cut aseptically into small segments (1 mm × 1 mm). Twenty pieces of the lichen were placed on 2 % malt extract agar (MEA) supplemented with 0.01 % streptomycin. The plates were sealed with parafilm and incubated up to 14 d at room temperature under ambient light. Pure cultures were prepared using fungi growing from each lichen particle. The identification of fungi was initially carried out using the sticky tape method (Felgel, 1980) and identification keys (Barron, 1988). The pure culture of the fungus was photographed and deposited as a living voucher at the Department of Chemistry, University of Kelaniya, Sri Lanka under the accession number US/US/06.
Molecular identification of isolated endolichenic fungus
Molecular identification of the isolated pure strain of the endolichenic fungus was performed using a molecular biological protocol by genomic DNA extraction, amplification and sequencing as described by Samanthi et al. (2015). The extraction of fungal DNA was carried out using Promega Wizard ® Genomic DNA Purification Kit, and the fungal strain was identified by analysis of the nuclear internal transcribed spacer (ITS) region of the extracted DNA and selective amplification by polymerase chain reaction (PCR) using ITS 1 and ITS 4 primers (Arnold & Lutzoni, 2007). Before being subjected to sequencing, excess nucleotides and the remaining primers and enzymes in the PCR product were removed according to a gel purification protocol. Agarose gel electrophoresis was carried out to separate the desired amplified DNA bands from non specific bands.
Extraction and isolation
A culture of C. trifolii was grown in PDA for ten days. The secondary metabolites were extracted with EtOAc (6 × 500 mL) and the solvent was evaporated under reduced pressure. This afforded a dark brown solid (1.3 g), which was found to be active in antioxidant assay using the 2,2-diphenyl-1-picrylhydrdrazyl (DPPH) method. taken any anti-inflammatory drugs for 2 months prior to the experiment. The blood was transferred to heparinised centrifuge tubes and centrifuged at 3,000 rpm followed by washing three times with an equal volume of normal saline and reconstituted as 10 % v/v suspension with normal saline. The reaction mixture (5.5 mL) consisted of 5 mL of the test solution and 0.5 mL of 10 % RBCs suspension. Saline was also added to the control test tube instead of the sample. Aspirin was taken as the standard drug. All the centrifuge tubes containing the reaction mixtures were incubated in a water bath at 56 o C for 30 min and cooled under running tap water. The reaction mixtures were centrifuged at 3000 rpm for 10 min and the absorbances of the supernatants were taken at 560 nm. The test was performed in triplicate. Percentage membrane stabilisation activity was calculated (Sakat et al., 2010) and plotted against test concentrations to determine the IC 50 .
Cytotoxicity assay
The in vitro assay of cytotoxicity to human nonsmall cell lung cancer (NCI-H460), CNS glioma (SF-268), breast cancer (MCF-7), human metastatic breast adenocarcinoma (MDAMB-231), prostate adenocarcinoma (PC-3), metastatic prostate adenocarcinoma (PC-3M) and pancreatic cancer (MIAPaCa-2), and normal human primary fibroblast cells (WI-38) was carried out using the resazurin-based colourimetric (alamarBlue) assay (Wang et al., 2011). The cancer cells were all cultured under standard culture conditions and the test compound or vehicle control (DMSO) was added to appropriate wells, and the cells were incubated for 72 h. Then 20 μL/well of alamarBlue solution was added into the assay plates for a final assay volume of 200 μL/well, yielding a final concentration of 10 % alamarBlue. After they were shaken for 10 s, the plates were returned to the incubator and kept for 4 h. The plates were then exposed to an excitation wavelength of 560 nm, and the fluorescence emitted at 590 nm was read. The percentage viability was expressed as fluorescence counts in the presence of the test compound as a percentage of that in the vehicle control. Doxorubicin and DMSO were used as positive and negative controls, respectively. 1 H-and 13 C-NMR spectra were recorded on Bruker Avance III 400 instrument using CDCl 3 as a solvent. Shimadzu LCMS-QP8000α was used for MS analysis. Analytical and preparative thin layer chromatography (TLC) were performed on precoated 0.25 mm thick plates of silica gel 60 F254 sprayed with a solution of anisaldehyde in EtOH, followed by heating to visualise the compounds on analytical TLC. The melting point of compound 1 was determined using a melting point apparatus (MEL-TEMP ® , USA).
RESULTS AND DISCUSSION
The fungus, US/US/06 was identified based on the morphological and molecular characteristics. The ITS sequence matched with C. trifolii sequences from Gene-Bank with a sequence identity of 99 %. This endolichenic fungus was identified as Curvularia trifolii. The 1 H NMR spectrum of compound 1 indicated the occurrence of one 3H singlet due to CH 3 groups (δ H 1.2) and three 1H double doublets at δ H 7.33, 5.80 and 5.22 due to three olefinic protons attached to C2, C3, C11, and the olefinic C attached to C12 showed a multiplet as it was coupled with the two protons attached to C13 at δ C 130.2. DEPT spectrum of compound 1 indicated the occurrence of five CH 2 (δ C 34, 31.8, 26.7, 41.0 and 42.9) groups accounting for 17 protons in compound 1 ( Table 1) (Table 3). However compound 1 did not show any significant activity against the antioxidant and anti-inflammatory assays (Figure 3). Compound 2 obtained as a fluorescent active semi -solid when analysed for C 23 H 30 O 6 by a combination of FABMS and NMR data indicated 9 degrees of unsaturation with an isocoumarin ring. The UV λ max at 238 nm indicated the presence of an α,β-unsaturated lactone carbonyl chromophore (Schubert & Sweeney, 1955). Its IR spectrum had absorption bands at 3438, 1706 and 1641 cm −1 , suggesting the presence of a OH group, carboxylic acid carbonyl, and α,β-unsaturated lactone functionalities.
The 1 H NMR spectrum of compound 2 ( Table 2) indicated the occurrence of four 3H singlets, of which three were due to CH 3 groups attached to carbons 4, 8 and 21 (δ H 1.1, 1.4 and 2.1) and the fourth due to a OCH 3 group (δ H 3.8), and one 1H singlet at δ H 6.5 due to an aromatic proton. DEPT spectrum of compound 2 indicated the occurrence of seven CH 2 groups accounting for 27 out of 30 protons. The remaining three protons were suspected to be that of the two CH and COOH groups. (2) its 1 H− 1 H COSY spectrum ( Figure 4). The connectivity of the above two spin systems, tertiary methyls, and nonprotonated carbons was established by the analysis of the HMBC correlations ( Figure 2) to constitute a structure with a twelve membered ring connected to a dihydroisocoumarin ring skeleton. | v3-fos |
2016-05-18T04:15:48.073Z | {
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} | s2 | Efficacy of pine leaves as an alternative bedding material for broiler chicks during summer season
Aim: The aim was to assess the efficacy of pine leaves as an alternative bedding material on the performance of broiler chicks. Materials and Methods: The present study was conducted in summer. Total 120, day old Vencobb straight run chicks were procured, and after 5 days of brooding, chicks were randomly distributed into four treatment groups viz. paddy husk (Group I), paddy straw (Group II), pine leaves (Group III), and combination of paddy straw and pine leaves (Group IV), each having 30 chicks with 3 replicates of 10 chicks each. Chicks were reared under intensive conditions in houses that have a semi-controlled environment, with optimum temperature and adequate ventilation. Food and water were provided as per NRC (1994) requirement. Results: The average body weight after 6 weeks of the experiment was 2018.83±31.11, 1983.80±33.27, 2007.36±35.73, and 1938.43±36.35 g. The bedding type had no significant effect on the carcass characteristics viz. evisceration rate and proportion of cut-up parts of the carcass except giblet yield. The experiment suggested that performance of broiler chicks reared on paddy straw and pine leaves as litter material, had improved body weight and feed conversion ratio as compared to rearing on paddy husk as bedding material. Bacterial count, parasitic load and the N, P, K value of manure of different bedding material shows no significant difference. Conclusion: Pine leaves have a potential to be used as an alternative source of litter material to economize poultry production in a sustainable way, so as to make poultry farming as a profitable entrepreneur.
Introduction
The poultry industry is one of the largest and fastest growing sectors of livestock production in the world with a 35% increase in meat and egg production in the period from 2000 to 2008 [1]. The 2010 world annual census data estimated the world flock to be over 18 billion birds with an estimated yearly output of 22 million tons of manure [1]. The deep litter system is the most popular system of housing in broiler production throughout the world. A variety of paper products [2], gypsum [3], hardwood bark [4], kenaf [5], peanut hulls [6], sand [7,8], rice hulls [9], rice hull ash [10], rice and wheat straw [11], and soft wood chipping fines [12] have occasionally been used as substitute bedding materials with various degree of success. Total replacement of the litter after every flock results in considerable environmental impact due to the high amounts of substrate required (e.g., wood shavings, straw, or sawdust), and the destination of this residue in the environment [13][14][15]. Therefore, the selection of litter material type, quantity and quality and availability of that have an important role in controlling environment within poultry house and bird performance [16]. Chicken litter is a mixture of feces, wasted feeds, bedding materials, and feathers [17,18]. Litter type affects litter consumption and litter bacteria [2], thus may affect body weight and carcass characteristics of broiler chicks. The three wastes of primary concern in poultry production are the bedding materials used for poultry housing, the manure resulting from poultry production and dead birds, common to all operations [19].
Litter material play a crucial role in the rearing of poultry. Common litter material used in the poultry industry includes wood shavings, sawdust, sand, pine shaving, shredded papers or paper chips, dry straw, rice hulls, maize cobs, corn silage; peat has alternative litter materials [20,21]. Pine shavings have been the preferred material for broiler production in the southeastern US for many years [22]. About 60% of farmers use saw dust as the bedding material, 16.7% use wood chippings, 20% use both wood chipping and saw dust while 3.3% of farmers do not use bedding materials. The type of bedding material used by farmers depends on locality and availability [23]. Paddy husk is most preferred and commonly used bedding source for poultry in northern India. In recent year, paddy husk has extensively used material by other industries as raw material for biofuel. However, due to diversified use of these materials, their availability is decreasing, and cost is increasing day by day. With the banning of cage system by EU and launching of National Program for Organic Production [24] in India, requirement of bedding material is expected to increase many folds in near future and make the poultry industry less profitable entrepreneur. Therefore, it is necessary to seek alternative sources of bedding material to make the poultry industry as a sustainable entrepreneur. State like J & K is blessed with pine forests (1825 km 2 ) as per Directorate of Economics and Statistics [25] which could be exploit in poultry production by utilizing its wood shaving [26] or pine leaves as litter material in a sustainable way.
The pine leaves are utilizing as a bedding material in pet animals in many countries because of its anti-microbial property and could be a good litter material for raising of poultry. Keeping this in view an experiment was conducted to compare the efficacy of paddy husk, paddy straw, and pine leaves as a litter material for raising of broilers.
Ethical approval
Permission of the Institutional Animal Ethics Committee was taken prior to the start of the experimental study.
Location and preparation of poultry shed
The experiment was carried out at the Poultry shed having semi-controlled environment, with optimum temperature and adequate ventilation, SKUAST-J, R. S. Pura, Jammu is located at 32°38′N 74°44′ E/32.63°N 74.73°E at an average elevation of 270 m (886 ft) during the summer season (10 th May-21 st June, 2013). The pens were cleaned and disinfected with phenyl solution before spreading litter and placing birds at random.
Collection and processing of bedding material
The bedding materials used were collected from a local area of Jammu region in the month of December-March 2013. After proper drying under the sun, they were processed for the preparation of bedding material. The bedding materials were provided with a depth of 5 cm during the rearing period.
Experimental design
The experiment was conducted in completely randomized design, a total of 120, day-old commercial (Vencobb) broiler chicks were procured and after 5 days of brooding were randomly divided into four groups having total 30 chicks in each group with their 3 replicates, each having 10 chicks and kept in a deep litter system. The litter material used was paddy husk (Group I), paddy straw (Group II), pine leaves (Group III), and (Group IV) with combination of paddy straw and pine leaves (50:50). The ration fed to the broiler birds were procured from commercial out let having nutritive value mentioned in Table-1 and other managerial conditions were similar throughout the study period.
Data recording
The data pertaining to body weights, feed consumption, carcass characteristics, bacterial and parasitic load, moisture content and temperature, relative humidity, and THI were collected up to 6 weeks of age. At the end of the experiment, litter samples were collected and examined for manorial value by estimating the nitrogen (N), phosphorus (P) content [27] and potassium (K) content by flame photometer. Ammonia estimation was also conducted at end of the experiment [28].
Statistical analysis
The results obtained were subjected to analysis of variance and treatment means were ranked using Duncan's multiple range test. Significance was declared at p<0.05 unless otherwise stated.
Results
The performance of broilers on different bedding types are summarized in Table-2. At the age of 42 days, body weight of broilers was not significantly (p<0.05) affected by the litter type. Broilers grown in Group I had the highest body weight and Group IV had the lowest body weight (Table-3). Accordingly, body weight gain per day was highest in Group I and lowest in Group IV. Total feed intake in different (Table-4). Feed conversion ratio (FCR) of Group I and Group III did not differ significantly and were lower when compared to other two groups. Percentage of eviscerated weight, breast, and thigh were not affected by litter type (p<0.05), but significant differences were observed for giblet yield (Table-5). Percentage of eviscerated weight and breast were highest in Group I and lowest in Group IV and percentage of the thigh was highest in Group II and lowest in Group III. Percentage of giblet yield was significantly different and tended to be highest in Group I and lowest in Group II. The bacterial load (Staphylococcus and Escherichia coli counts) and parasitic load were slightly higher in Group IV and lower in Group III showing a non-significant difference (Table-6). The data recorded for N, P, and K content of the manure in the experiment (Table-7) indicated that there was no significant difference in N, P, and K content of Group I=Chicks reared on paddy husk, Group II=Chicks reared on paddy straw, Group III=Chicks reared on pine leaves, Group IV=Chicks reared on combination of paddy straw and pine leaves, SE=Standard error Group I=Chicks reared on paddy husk, Group II=Chicks reared on paddy straw, Group III=Chicks reared on pine leaves, Group IV=Chicks reared on combination of paddy straw and pine leaves, SE=Standard error Mean values bearing different superscripts in a row differ significantly (p<0.05). Group I=Chicks reared on paddy husk, Group II=Chicks reared on paddy straw, Group III=Chicks reared on pine leaves, Group IV=Chicks reared on combination of paddy straw and pine leaves. Note: Expressed as ( 1 ) per cent of body weight, ( 2 ) per cent of eviscerated weight the litter among various groups. The nitrogen content was higher in Group III and lowest in Group I, the phosphorus content was higher in Group III and lowest in Group II and the potassium content was higher in Group III and lowest in Group II. Ammonia concentration was calculated by following the procedure as employed by Moum et al. [28]. For the purpose of ammonia estimation, each pen was covered with a transparent plastic sheet. A pH paper strip having a range of 6-7.7 and 9.5, moistened with sterile neutral water was held for 15 s at the level of birds in each pen. It was found that the ammonia production was similar in all the treatment groups and all the treatment groups show a change in orange color of the pH paper strip which indicates that the ammonia concentration in the different treatment groups was between 25 and 50 ppm (parts per million).
Discussion
In the present study, broilers reared in Group IV had the lowest body weight in comparison to Group I, II, and III (Table-2). The observed differences in body weight may be attributed to depression of feed intake in birds reared in Group IV and increasing feed intake on others litter especially on paddy husk (Table-3 & 4). Our result were in agreement with the finding of Davis et al. [21] that the type of litter material had no significant effect on body weight of broiler and Navneet et al. [29] observed that paddy straw can also be used as an alternate bedding material for broilers. The findings of Mahmoud et al. [30], and Farhadi, [31] that litter type have significant effect on body weight at 4, 5, and 6 weeks of age and rice hulls due to its favorable properties could be successfully used as alternative poultry litter material were contradictory with the result. Many studies, in which alternative materials were tested, have reported that the type of litter material used does not affect body weight [31]. FCR of broilers was not affected by litter type (Table-2). Other researchers have reported similar findings regarding the influence of various litter material on FCR [3,[32][33][34]. The bedding type had no influence on the carcass characteristics viz. evisceration rate and proportion of cut-up parts of the carcass except the giblet yield. Our results were in accordance with Atapattu and Wickramasinghe [34], and Grimes [35] who also did not find any significant effect of the type of litter on the carcass characteristics.
The bacterial load were slightly higher in Group IV and lower in Group III showing a non-significant difference (Table-6) which is similar to Estevez [36] who also observed higher incidence of E. coli infection in a combination of ammonia and wet litter. The coccidial load in Group IV was higher and lower in Group III. Coccidial infection was mainly influenced by the moisture content of the litter. Moisture content was highest in Group IV (25.69±1.79) and lowest in Group III (22.53±1.40). Waldenstedt et al., [37] also reported that the frequency of outbreaks of coccidiosis in chicks reared under damp and moist conditions was higher.
The nitrogen content reported in our finding has no significant difference among various litter material which was consistent with the Biswas et al., [38] who also reported no significant effect of bedding material viz. sugarcane bagase, sawdust and wheat straw on the N content of the litter. Whereas K and P contents in wheat straw were reported to be significantly higher as compared with other litter materials which may be due to the difference in the initial content of these nutrients in the type of bedding materials used by these workers. However, our result was contradicted in relation to P and K content in litter material.
Conclusion
In conclusion, it may be stated that paddy straw and pine leaves could be used as an alternate litter to commonly used paddy husk to promote the economical intensive poultry production system. Group I=Chicks reared on paddy husk, Group II=Chicks reared on paddy straw, Group III=Chicks reared on pine leaves, Group IV=Chicks reared on combination of paddy straw and pine leaves, E. coli=Escherichia coli, SE=Standard error Group I=Chicks reared on paddy husk, Group II=Chicks reared on paddy straw, Group III=Chicks reared on pine leaves, Group IV=Chicks reared on combination of paddy straw and pine leaves, SE=Standard error | v3-fos |
2016-05-04T20:20:58.661Z | {
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} | s2 | Livestock-Associated Staphylococcus aureus: The United States Experience
Staphylococcus aureus is a gram-positive bacterium that colonizes a variety of animal species [1]. S. aureus infections in animals are most commonly reported as a cause of mastitis in dairyproducing animals (including cattle and goats) and “bumblefoot” in chickens [2], as well as being identified as a pathogen of farmed rabbits [3]. Most reports characterizing animalassociated S. aureus have demonstrated that strains affecting animals are distinct from those infecting humans, suggesting that there are host-specific lineages which only rarely cross species boundaries [4]. Livestock-associated strains may evolve on farms because of the use of antibiotics in animal husbandry. These may be used as feed additives for growth promotion in industrial livestock and poultry [5], for prevention of disease within a herd, or for treatment of an existing disease outbreak. Agricultural-use antibiotics include many classes that are relevant for human health, including tetracyclines, macrolides, penicillins, and sulfonamides, among others. Antimicrobial resistance generated during animal husbandry may then be spread to the general human population in a number of different manners: contact with contaminated meat products (via handling or ingestion); occupational contact (farmers, meat packers, butchers, etc.) and potential secondary spread into the larger community from those who are occupationally exposed; entry into and transmission via hospitals or other health care facilities; or spread via environmental routes including air, water, or manure in areas in proximity to live animal farms or crop farms where manure has been used as a fertilizer (Fig. 1). While methicillin resistance has been the most commonly investigated phenomenon and will be the main topic of this review, resistance to any of these antibiotics can occur and can potentially be a threat to successful treatment of S. aureus infections and therefore to human health outcomes. As such, my research group and others have begun to look more broadly at any S. aureus present on farms, including those that may be susceptible to methicillin but resistant to other antibiotics.
discovery of this strain led to the addition of livestock-associated MRSA (LA-MRSA) to the lexicon, to complement hospital-associated (HA) and community-associated (CA) strains.
In most European countries, CC398 remains the most commonly identified type of LA-MRSA [6][7][8][9], leading to a presumption that the terms LA-MRSA and CC398 are practically interchangeable. However, while CC398 strains have been found in livestock across the globe, the epidemiology of livestock-associated S. aureus has been found to differ in other geographic areas. Several Asian studies have demonstrated that a different strain of MRSA, ST9, appears to be the prominent type of LA-MRSA in several Asian countries [10][11][12][13][14]. Poultry may harbor CC398 strains [15][16][17] but also other types unrelated to CC398, including CC5 [15,18] or other types [17]. In the United States, the diversity of livestock-associated S. aureus appears to be higher than that identified in Europe or Asia, with reports of both CC398 as well as a variety of "human" types of S. aureus in live animals, as described below. Antibiotic-resistant Staphylococcus aureus is a growing public health concern, but tracing the origins of the bacterium is complicated. Evidence suggests that antibiotic-resistant strains of S. aureus can spread in livestock operations and hospitals where antibiotics are regularly used. These antibiotic-resistant organisms can then spread into communities and the environment. More research is needed to determine exactly how these transfers occur. Image by Kalliopi Monoyios. The epidemiology of CC398 and other strains found in both animals and humans [12] has led to a reexamination of the idea of host specificity in S. aureus. CC398 appears to be frequently shared between animals and humans and is capable of causing active symptomatic infections in both species [19,20]. Furthermore, both CC398 and a poultry-adapted S. aureus strains of CCT5 have been phylogenetically analyzed and appear to have originated in humans, who transmitted strains to animals, in which the strains subsequently spread and evolved a variety of host adaptations [21,22]. As such, there exist both human-associated CC398 strains as well as true livestock strains, complicating studies of origin or host association based only on knowledge of sequence type.
Epidemiology of CC398 and Other Livestock-Associated S. aureus in the US
The epidemiology of LA-SA in the US appears to be notably different than in European countries, where the bulk of LA-SA research has been carried out. While early studies on farms and of meat-identified CC398 strains in animals, farm workers, and meat products, [23,24], contemporaneous studies also documented CC398 in populations with no obvious livestock contact [25][26][27]. In one Texas publication carried out in a jail setting rather than on a farm, CC398 isolates made up a significant portion (13.2%) of all methicillin-susceptible S. aureus (MSSA) identified within this population. Clearly, the association of CC398 exclusively with an agricultural reservoir did not appear to hold in the US.
While CC398 can have LA as well as human versions, other human strains of S. aureus have also been found in US livestock. Studies carried out on swine farms in the US have identified human strains within the noses of live animals [28][29][30] or as components of environmental samples of farm dust [31]. Several papers have found CC5 strains rather than CC398-associated types to be the dominant strain isolated from pig farms in both Iowa and Ohio [31,32], while others have found CC398 to be the most common molecular type [23,33]. Three studies in North Carolina examining workers on pig farms and in processing plants similarly found substantial diversity among S. aureus isolated from workers, including CC398, CC5, and CC8 strains, among others [34][35][36].
Transmission between Animals and Humans in the Farming Setting
Studies of individuals living in proximity to concentrated animal feeding operations (CAFOs) support the idea that nonlivestock strains may be spreading within areas proximal to farms. Two independent studies carried out in Iowa and Pennsylvania that examined the relationship between animal farms and MRSA found an increased risk of MRSA colonization or infection in those living close to farms or in areas where manure was spread on fields [37,38]. In both studies, however, no classic LA strains were found when molecular typing was carried out on isolates collected. This suggests that either strains other than LA isolates are evolving on farms (consistent with on-farm sampling described above) or that it may be the presence of antibiotic resistance genes and antibiotic residues on farms that are moving to the subjects' own bacterial flora and causing a shift toward antibiotic-resistant strains in these populations, or perhaps a combination of both mechanisms. Firm conclusions are difficult to make in the absence of a concerted, national-level on-farm sampling effort, which is difficult to carry out in the US because of private/ corporate ownership of many farms and laws in several states that are unfriendly to farm visitors.
Human Infections with Livestock-Associated S. aureus Strains
A number of human infections with CC398 strains have been reported. Most of these have been documented in Europe [39][40][41]; however, CC398 infections from the US [26,42,43] and Canada [44] have been reported as well. Because many infection reports were published prior to the recognition of distinct lineages of CC398, it is not always clear, particularly for individuals lacking exposure to livestock, whether the CC398 strains identified are ancestral human strains, or derived livestock types. This has significance for prevention and treatment, as human-origin strains appear to be more virulent than true livestock strains but may also be less likely to be multidrug resistant (and as such, more easily treatable) [22]. Nonetheless, the majority of reported infections with CC398 strains appear to be similar in scope to community-associated S. aureus strains, causing skin and soft tissue infections and, more rarely, serious invasive infections and death.
Potential for Meat Products as a Source of LA-SA in the Community
Just as a variety of human and livestock strains have been found in live animals on farms, so have they been found in meat products sampled in the US [18,24,[45][46][47][48][49]. CC398 strains have been found in pork and chicken products in the US and appear to be the dominant contaminating strains in raw turkey meat. S. aureus may be transmitted to humans from meat products by handling of contaminated products or by the cross contamination of household surfaces (such as countertops and sinks), which are then touched by family members. While antibiotic use on farms may drive selection of antibiotic-resistant strains of S. aureus that eventually end up in meat products, eliminating consumer exposure to such bacteria is not as straightforward as simply purchasing products raised in an antibiotic-free environment. In a study examining conventional versus antibiotic-free pork products, no difference was found in prevalence of MRSA between these types of samples [46]. This was a different result obtained from sampling results on conventional versus antibiotic-free farms [33], suggesting the potential for either contamination of pigs with MRSA in the lairage area prior to slaughter or contamination of meat products during processing or packaging, either via humans in the plants who may spread MRSA to meat products or from bacterial residues present from conventional products. It is currently not known what the risk is to consumers from S. aureus-contaminated meat products.
Conclusions, Significance, and Future Studies
Livestock-associated S. aureus is an emerging category of S. aureus throughout the world. Currently, the research carried out has focused more closely on carriage than on transmission and infection, but these strains appear to be less likely to cause human infections and to spread person-to-person than typical human strains [50]. However, these conclusions should be noted with caution, as few well-designed prospective studies have been conducted to answer these questions to date.
Recent research suggests that bidirectional transmission of strains of S. aureus between humans and livestock is not a rare occurrence. In addition to the movement of CC398 between animals and humans, studies have suggested that a human pandemic clone, CC97, had its origin in cattle [51]. Additionally, antibiotic resistance genes, including mecA [52,53] and mecC [54,55], have been suggested to have an animal origin.
Currently, we are limited in the ways we can distinguish whether any particular strain of S. aureus is a human or livestock-adapted isolate. We can use the presence of marker genes, including the loss scn and presence of tet(M), both of which are genotypes associated with livestock adaptation of CC398 lineages [22,56] or examine the presence of a single-nucleotide polymorphism (SNP) that has also been identified in this clade [56]. However, large-scale studies validating these markers in other lineages (CC5, CC8, and more) are lacking. Additional large-scale studies in both human and animal populations are necessary in order to gather isolates that are epidemiologically well characterized. These isolates can then be analyzed in order to validate current genomic markers, as well as to identify novel ones in lineages besides CC398.
S. aureus surveillance is most commonly carried out within a human clinical or hospital setting, with far fewer research dollars devoted to analysis of carriage within communities, particularly in a rural setting, and very little research examining animal strains. As such, it is likely we are missing other spillover events of S. aureus from livestock to humans or vice versa. To track such events and facilitate both surveillance and source tracking of novel isolates, the buyin of industry is needed. All too often, the relationship between public health and the agricultural and food industry is one of antagonism rather than assistance. Working together will mean both safer food products and well-protected workers. More attention to this type of research is needed, as we are rapidly approaching a "post-antibiotic era" [57]. The effectiveness of antimicrobial stewardship in the clinical setting may be reduced if pathogens and resistance genes from the agricultural environment are repeatedly, but silently, being introduced into the human population [58]. | v3-fos |
2019-05-28T13:15:33.102Z | {
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} | s2 | ASSESSMENT OF SELECTED FOOD PRODUCTS FOR PESTICIDE RESIDUE IN MAJOR MARKETS OF OYO STATE, NIGERIA
. A study was carried out on the assessment of selected food produce for pesticide residue in Oyo State. With the aid of multi stage sampling technique, 8 selected markets were surveyed in the major constituencies of Oyo State which included Oje and Bodija Market in Ibadan, Arada and Sabo Markets in Ogbomoso, Ajegunle and Araromi Market in Oyo, Owode and Sango Markets in Igboho and Saki respectively in Oke-Ogun. Information on type of pesticide use, reason for choosing the pesticide, method of applying the pesticide and duration of post application storage, were collected. Food produce sampled were yam chips, cowpea, maize and smoked fish for residual analysis. The residual analysis includes extraction of samples, cleaning of extraction and screening of the pesticide using GC-MS to detect any chemical metabolites. The survey reveals that majority (87.5%) of respondents were involved in selling of maize, yam chips and cowpea and the most used pesticide in the market was Aluminium phosphide marketed as phostoxin. Many of respondents applied pesticide by fumigation and the choice of these pesticides was base on customary practices. These food produce are stored for less than a month before being disposed of to consumers. Result from residual analysis shows sample from all the markets contains high percentage of alkane compounds especially the heptadecanes, eicosane, hexadecane and octadecane. Organochlorines were also found in the samples at high percentage. Banned pesticides are still in use in the visited markets and the use of banned pesticide should
INTRODUCTION
In the recent time, there has been news of food poisoning in many household in Nigeria. The bulk of the blame is laid on the farmers' table as the farming communities were been accused of using pesticides which are tagged to be dangerous to human health. Food storage plays an important role in the ability of any nation to feed its citizens, there is usually an abundance of food immediately after harvest, if appropriate storage has been perfected [1]. It is estimated that in the tropics between 25 and 40% of stored agricultural products is lost because of inadequate farm-and village-level storage every year [2]. In the field and during storage the products are threatened by insects, rodents, birds and other pests. These products may be spoiled by infection from fungi, yeasts or bacteria.
The United State Census Beaureu (USCB) estimates that the world population exceeds 7 billion on March 12, 2012. The world population is expected to become between 8.3 -10.9 billion by 2050. The high population growth rate, particularly in the developing countries, and the changing diets will lead to a much higher food demand by 2050 [3]. To meet this increasing rise in population there is the need to store food product with pesticides to reduce losses due to pest.
The frequent incidence of pests in agricultural commodities such as insects, fungi, rodent etc, has a potential negative impact. This is because harvest and post-harvest techniques adequate for the prevention of pest infestation are seldom practiced coupled with inadequate storage facilities [4,5].
Pesticides are an important management tool in agricultural enterprise, they protect produce against pests at post-harvest and storage, it has continued to be the bedrock of agriculture in modern times because of its unquantifiable benefits one of which include enhancement of shelf life of stored agricultural products [6]. [7] 2007 maintained that for every dollar spent on pesticide for crop yield and storage four dollars is saved. In Nigeria, insect related to post-harvest food losses are estimated at around 30%, insects control have been by the use of synthetic insecticides which have adverse effects on human health and the undesirable consequences of pesticides use on human health have become more evident [8].
A global scale of acute poisoning by plant protection chemicals is not exactly known and all data are based on estimates. In 2002, the number of deaths due to acute poisoning by pesticides was estimated at about 220,000 annually, which represent only a small percentage of the total number of such poisonings of roughly 26 million a year [9]. The threat of the use of chemicals in agriculture is greatest in developing countries, since the awareness of the local population about the negative impact of pesticides on health is still relatively low. This is confirmed by recent research carried out in rural regions of Asia where the number of deaths due to pesticide poisoning is estimated at 300,000 cases a year [10].
Recently concern about the health effects of pesticides have increased over the years considering the rates at which scholars have put into the study of the phenomenon. Most often, scholars have written so much on the use of dangerous chemicals, banned pesticides, pesticide overdose, safe practices and the like but little has been written on the waiting period between application and consumption. This dearth of scholarly information is inexcusable, scarce resources and time have often been channelled to investigate other cause of pesticide contamination in stored produce, and the silence on withholding period which leaves residues that are poisonous to the consumer of the treated products.
For example it was reported that four out of a family of 9 died after eating yam flour treated with pesticides in Isua-Akoko Area of Ondo State, Nigeria. It was diagnosed at the Federal Medical Centre Owo, Nigeria, that they have eaten poison [11]. These adverse effects of pesticides are caused by pesticide abuse and misuse, a recent report indicated that two children died and 112 people were hospitalised after consuming cowpea treated with pesticide in Cross River state, test shows that the consequence is as a result of pesticide poisoning [12].
The presence of the pesticide residue is a concern for consumer because pesticides are known to have potential harmful effects to other non-target organisms than pests and disease. Exposing consumers to food items treated with pesticide before the chemical fully break down could pose a serious threat to health and life. Therefore, this work will looks into the types of pesticide used, reasons for choosing the pesticide, duration of storage before and after application as well as mode of application.
Therefore, the objectives of this study therefore are to evaluate the different types of pesticides used by market men and women to preserve selected agriculture produce and analyze these produce for chemical residues.
SURVEY AREA
A survey was carried out in major markets in Oyo State which included Bodija and Oje Markets in Ibadan, Araada and Sabo Markets in Ogbomoso, Akeesan and Ajegunle Markets in Oyo, Sango Market in Saki, and Owode market in Igboho. The selected markets were known for storage and merchandise of agricultural produce in the state.
FIGURE 1: MAP OF OYO STATE
COLLECTION OF PRIMARY DATA Questionnaires were used to conduct oral interviews for market sellers on the type of pesticide use, the mode of application, choice for choosing pesticide and length of storage after pesticide application.
International Letters of Chemistry, Physics and Astronomy Vol. 54 49
COLLECTION OF SAMPLES FOR CHEMICAL ANALYSIS Three food samples (yam chips, cowpea and smoked fish) were collected at the above mentioned markets from fifteen randomly selected merchants. From each market, a kilogram of each food item was collected from each merchants and mixed together, out of which 2 kg sample was taken for laboratory analysis.
PREPARATION OF SAMPLE FOR EXTRACTION
These samples were carefully crushed with mortar and pestle to expose the inner portions of the food samples. A portion of the crushed sample (50 g) was weighed using a sensitive weighing balance and tied in filter paper for the extraction. Reflux extraction was done with the use of Soxhket extractor. The samples were assembled in the extractor. After extraction, the solvent was removed by means of rotary evaporator yielding the extracted compound. The non-soluble portion of the extracted solid was discarded.
GAS LIQUID CHROMATOGRAPHIC ANALYSIS
The extract was then analysed by Gas Liquid Chromatography Mass Spectrometer (GC-MS) using Ai Cambridge Ltd. Model GC94FID operated at these conditions: FID column 12 ft x 2 mm I.D wit OV17 on 100/120 H.P. chromosorb G: N2 at 30 cc/ min, injector 190°C, column, 180°C Detector 200°C. Standards of all pesticides of all pesticide detected were prepared at 100µI / ml using Hamilton Microsyringe 10 µl i.e 10 ppm of appropriate standards were first injected into the GC to establish their retention times. Identification of the unknown sample was based on a comparison of the retention time of the unknown component with that obtained from a known standard compound analyzed under identical conditions according to [13]. DATA ANALYSIS Data collected from the questionnaire were summarised using frequencies percentage and description statistics, with the aid of the Statistical Package for Social Sciences (SPSS version 15.0).
RESULTS
Most of the Respondents sell; maize, yam chips, cowpea and smoked fish (30.8%, 27.5%, 29.2% and 12.5% respectively) ( Table 1). Table 2 shows the pesticide use and its duration in the selected markets of Oyo State. Most of the respondents 66 (55.0%) used phostoxin, 26 (21.6%) of the respondents does not know the type of pesticide used or those not use pesticide at all to preserve the food produce, 24 (20.0%) of the respondents use DDforce while 4 (3.3%) of the respondents use dichlorvos. Some of the respondents 35 (29.2%) does not have any reason for choosing the selected pesticide. Thirty four (28.7%) of the respondents chose pesticide to use based on custom, about 30 (25.0%) of the respondents chose the selected pesticide based on availability and 21 (17.5%) of the respondents used the selected pesticide due to its ease of application.
About 59 (49.1%) of the respondents stored their food produce for less than a month after pesticide application, 30 (25.0%) of the respondents stored food produce for more than 6 months after application, 16 (13.3%) respondents stored food produce for 3 months while least proportion of respondents stored for 2 months (7.5%), 1 month (4.2%) and 4-6 months (0.8%) after application. Fifty-nine (49.2%) respondents applied pesticide by fumigation, 31 (25.8%) of the respondents did not know the method used, 27 (22.5%) of the respondents applied by spraying while least proportion of the respondents 3(2.5%) applied pesticide by retting.
DISCUSSION
Maize, yam chips and cowpea were the major staple food in the markets, this is in accordance with the report of [14] (2010) which states that the most staple foods are derived from either cereals such as maize, rice or root tubers such as yam and yam products or legumes such as cowpea and groundnut. Majority of the respondents use phostoxin which can be used as rodenticide or fumigants in preserving their produce. This was in line with what [15,16] reported that, the commonly used aluminium phosphide are phostoxin, Quickphos and Fumitox. The most commonly used Organophosphates include; DDforce, chlorpyrifos, parathion and malathion. Majority of the respondents store produce for less than a month before disposing it to the consumer, which could cause food poisoning or contamination. This might be because many do not understand the concept of withholding period which is the maximum length of time that must elapse or a person must wait after application of pesticide on food crop before it is safe for consumption. It was reported that 20 fast food outlets were closed in Nigeria because of fatalities traced to pesticide residue in their food [17].
In all the markets visited from the survey, different chemicals (alkane, organochlorine, pyrethrum, ester, akyl, alkene, sesquiterpene, alkanol, alkanal, phenols, acids) were detected in all the food samples ready for sale to the consumers. Cowpea and fish samples have the highest number of alkane in all the market sampled, although studies have investigated alkane in the environment [18;19], dust [20], human serum [21], human milk [22], and animal tissues [23,24], few have examined alkane contamination of food. Toxicity studies suggest that the alkane is an endocrine disruptor and developmental neurotoxicant; specifically, some alkane compound have been associated with changes in rat thyroid systems [25,26], altered function of human natural killer cells [27;28], and neurotoxic effects such as decreased fine manipulative abilities and lower attention in children [29].
ILCPA Volume 54
Organochlorine has highest proportion in fish samples and yam chips. Many studies conducted so far have revealed the presence of detectable level of pesticide especially organochlorines in fruits, vegetables, fish and fish products. The studies pointed out that majority of the samples contaminated by chlorinated pesticides exceeded the maximum limits which could cause pesticide hazard to the consumer [30,31]. It was observed that organochlorines persisted in the environment long after being banned and may be involved in the causation of breast cancer as a result of estrogenic activity [32]. Alkene, alkyne, esters, acids, phenols and pyrethrum were found in small quantity in all the food samples taken in all sampled markets.
CONCLUSION AND RECOMMENDATION
Most of the respondents deal in selling maize, cowpea and yam chips and the most common insecticide used were phostoxin and DDforce. Many of the respondents sell not long after application of insecticide to their products without considering the withholding period. It can be concluded that in all the food samples assessed in the visited markets, cowpea and fish samples have the highest number of alkane and organochlorine was high in fish samples and yam chips. There is therefore, the need to educate produce merchants on safe use of pesticides in preservation of consumable meant for immediate sale. | v3-fos |
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} | s2 | Effects of protein sources for milk replacers on growth performance and serum biochemical indexes of suckling calves
This study investigated the effects of protein sources for milk replacers on growth performance and serum biochemical indexes of suckling calves. Fifty Chinese Holstein bull calves with similar BW and age were randomly allocated to 5 groups (1 control and 4 treatments) of 10 calves in each group. Five types of milk replacers were designed to have the same level of energy and protein. The protein source for milk replacers of the control group was full milk protein (MP). The protein source of milk replacers of the 4 treatment groups was composed of MP and one vegetable protein (VP) (30 and 70% of total protein). The 4 types of VP were soybean protein concentrate (SP), hydrolyzed wheat protein (WP), peanut protein concentrate (PP), and rice protein isolate (RP). Results of the experiment showed: 1) there was no significant difference on average daily gain (ADG) and feed:gain ratio (F:G) among the MP, SP and RP groups (P > 0.05), whereas the ADG and F:G of the WP and PP groups were significantly lower compared with the MP group (P < 0.05); 2) there was not a significant difference in withers height, body length and heart girth among treatment groups compared with the MP group (P > 0.05). Thereby the 4 VP milk replacers had no adverse effects on body size of calves; 3) all groups showed no significant difference in the serum contents of urea nitrogen, total protein, albumin, globulin, β-hydroxybutyrate, growth hormone, insulin-like growth factor-1, and the ratio of albumin to globulin (A:G) (P > 0.05). In conclusion, SP or RP (accounts for 70% of the total protein) as calf milk replacers could substitute MP, whereas wheat gluten and PP had a significant adverse effect on growth performance in this experiment.
Introduction
Using milk replacers to feed early-weaned calves is an effective way to raise replacement cattle in modern dairy industry (Heinrichs, 1993). Protein is one of the most important nutrients in milk replacers, and choosing appropriate protein sources becomes an important factor affecting feedstuff quality and animal production costs (Erickson et al., 1989). Protein sources usually used as milk replacers fall into two types: milk protein (MP) and non-milk protein. Milk protein is an excellent protein source as a milk replacer because it has balanced amino acid constituents, a low level of anti-nutritional factors, and a high digestibility value. However, China has a relative shortage of MP source as a result of a high demand for dairy products, which keeps MP price high. Research has showed soybean protein as a protein source for milk replacers to be comparable to MP on growth performance of suckling calves (Lalles et al., 1995;Tomkins et al., 1994a,b. Furthermore, the nutritional value of vegetable proteins (VP) from wheat, peanut and rice is similar to that of soybean protein, and China produces considerable amounts of these crops. Thus, exploring the potential of these VP will open up novel sources of milk replacers. Studies on protein sources for milk replacers in the past focused mainly on adding low levels of soybean protein (replacement level lower than 50% crude protein). Using high levels of VP as milk replacers (replacement level higher than 50%) has rarely been reported.
This study tested 4 types of VP as main protein sources of milk replacers with the main essential amino acids in relative balance. Growth performance and serum biochemical indexes of suckling calves were studied.
Experimental animals
The experiment was conducted at Zhuochen Livestock Co. Ltd., Beijing. Fifty Holstein bull calves (21 ± 6 d, 46 ± 6 kg) with similar age and BW were selected and randomly allocated to 5 groups of 10 calves per group.
Experiment design and diets
In this experiment, a single-factor completely randomized design was used to study the effects of different VP sources on calves. These VP sources were soybean protein concentrate (SP), hydrolyzed wheat protein (WP), peanut protein concentrate (PP), and rice protein isolate (RP). Five milk replacers were formulated using 30% MP and 70% VP. The milk replacers used in this experiment had the same CP (22%), GE (19.66 MJ/kg), Lys (1.84%), and Lys:Met:Thr:Trp ratio (100:29.5:65:20.5). The amino acid levels of the milk replacers were adjusted by adding crystalline amino acids to the basal ration. Calves in the controlled group (MP) were fed a full MP milk replacer. Calves in treatment groups were fed 4 types of VP milk replacers, which were derived from SP (CP ¼ 65.2%), WP (CP ¼ 77.8%), PP (CP ¼ 54.7%) and RP (CP ¼ 82.0%). All calves in this experiment were fed the same starter up to 42 days of age. The nutrient levels and composition of milk replacers and starter in this experiment are shown in Table 1 and Table 2.
Feeding management
All selected calves were fed 3 L colostrum within 1.5 h after birth. Twelve hours after birth, they were fed colostrum and ordinary milk, which amounted to 10% of their BW, by an esophageal feeder. The transitional period of milk replacer was from 15 to 20 days of age. The ratio of milk to milk replacer (3:1) gradually decreased to 1:3 during the transitional period, and eventually to 0:1 on 21 days of age.
One portion of milk replacer was completely dissolved into 7 portions of warm water (wt/wt) which had been boiled and then cooled to 40 to 50 C. The calves were fed the milk replacers twice daily (0800 and 1830) using individual bowls. All calves had ad libitum access to water 0.5 h after feeding. Milk replacer allowance per day per calf was 100 g/kg BW, and the amount was adjusted as their BW increased.
With restricted feeding, the daily dry matter intake (DMI) of a starter calf was set at 400 g during weeks 4 to 5, and was set at 800 g in week 6. Each calf was housed in a 2.25 m 2 single pen. In order to ensure calf health, lime was used to disinfect the pen once a week.
Measurement of DMI, BW, and body size
The amounts of milk replacer and starter that were supplied and remained were accurately recorded daily. The milk replacer and starter feed were sampled before morning meals daily. Feed samples of each group were collected and thoroughly mixed, and average daily intake was calculated every week.
Calves were weighed weekly and the body sizes (withers height, heart girth and body length) were determined every 2 weeks prior to morning meals during the experiment period.
Blood serum sample collection
From week 3, four calves per each group were selected for blood serum sampling per 2 weeks prior to morning meals. An 8-mL blood sample was taken from the jugular vein and then stood in vacuum centrifuge tube at room temperature for 30 min. The blood serum was acquired by centrifugation at 1,485 Â g for 20 min and immediately stored at À20 C.
Sample determination
Samples of milk replacers and starter were analyzed for dry matter (DM), total CP, ash, total P, total Ca (AOAC, 1980), and total ether extract (EE) by supercritical fluid extraction (TFE 2000 Leco Fat Extractor, St. Joseph, MI), and gross energy (GE) by bomb calorimeter (Parr 6300 Automatic Bomb Calorimeter, Parr Instrument Company, Moline, IL). The content of total protein (TP), albumin, globulin, blood urea nitrogen (BUN), b-hydroxybutyric acid (b-HB) in blood sera was determined using the standard kit (Biosino biotechnology and science incorporation) by biochemical analyzer (Model 7600; Hitachi, Tokyo, Japan). The contents of growth hormone (GH) and insulin-like growth factor-1 (IGF-1) in blood sera were determined according to the radioimmunoassay kit.
Statistical analysis
We carried out the statistical analysis using the model of MIXED and ANOVA in SAS 9.2 to test P-value and SEM. The difference was considered to be significant when the P-value was less than 0.05.
Results and analyses
3.1. Growth performance 3.1.1. Dry matter intake and feed conversion ratio Table 3 shows that no significant difference for DMI of the starter exists among groups from 22 to 56 days (P > 0.05), and it was significantly higher for the SP and RP groups than for other groups from 57 to 63 days (P < 0.05). Table 3 also indicates that the ratio of feed to gain (F:G) was significantly higher for the PP group than for the MP group (control group) from 22 to 63 days (P < 0.05), and F:G was significantly higher for the PP group than for the SP group during the whole experiment period except from 29 to 35 days (P < 0.05), and F:G was significantly higher for the RP group than for the PP group during the whole experiment period except from 29 to 42 days (P < 0.05). Table 4 shows no significant differences existed for initial BW among all groups on day 21 with average BW 45.2 kg (P > 0.05). From 22 to 28 days, no significant difference existed for average daily gain (ADG) among all groups (P > 0.05); form 29 to 35 days, groups showed the following sequence: MP > SP > RP > WP > PP in terms of ADG and the MP group had a significantly superior ADG than the WP, PP, RP groups (P < 0.05); from 36 to 42 days, the PP group had a significantly inferior ADG than the MP, SP, RP groups, and the WP group had a significantly inferior ADG than the MP group (P < 0.05), and differences for ADG were not significantly different among the SP, RP, and MP groups (P > 0.05); ADG of all groups showed similar patterns for the following 4 periods: 36 to 42 days, 43 to 49 days, 50 to 56 days, and 57 to 63 days. Table 5 shows no significant differences existed for withers height, body length and heart girth among groups from 21 to 63 days (P > 0.05). All body size indexes significantly increased with age (P < 0.01). There was no significant interaction between treatments and age for body height, body length and heart girth among all groups (P > 0.05). Table 6 shows the change of serum BUN with age was not significant for all groups (P > 0.05), it showed a tendency to ascend and then to descend for all groups and it was significantly lower for the WP and PP groups than for the other 3 groups during the whole experiment period, and it was significantly lower for the PP group than for the MP and SP groups on day 35 (P > 0.05). The age had a significant influence on the level of TP in blood sera (P > 0.05) and no significant differences were shown among groups (P > 0.05), and the average TP concentrations of the WP and PP groups, 54.48 and 52.78 g/L respectively, were less than 57.18 g/L of the MP group. The effects of age on the level of ALB in the blood sera were not significant (P > 0.05), and differences among groups were not significant (P > 0.05) during the whole experiment period. The ALB concentrations of all groups showed a tendency to decline at first and then to rise gradually with age which is similar to TP. Results of statistical analysis showed the level of b-HB in sera significantly increased with age (P < 0.05), whereas the effects of protein sources on the b-HB concentration in sera of calves were not significant for all groups (P > 0.05).
Concentrations of hormones
As Table 7 shows, age had a significant impact on the level of GH, IGF-1 in the sera of calves in all groups (P > 0.05). The concentrations of GH, IGF-1 showed a same tendency to decline (21 to 35 days) and then to rise (35 to 49 days) and then to decline (49 to 63 days). During the whole experiment period, the levels of GH and IGF-1 showed no significant differences among all groups (P > 0.05), but the levels of the WP and PP groups were slightly less than those of the MP group.
Effects of protein sources for milk replacers on the growth performance of suckling calves
Different VP sources and proportions for milk replacers had different effects on the growth performance of suckling calves (Drackley and Davis, 1998). Using unprocessed full-fat soybean powder in milk replacer significantly reduced the growth performance of calves (Smith and Sissons, 1975). However, SP, soybean protein isolated and rumen fermentative soybean flour as the main protein sources for milk replacers were equivalent to MP in gaining calf growth rate (Lalles et al., 1995). Hill et al. (2008) used wheat gluten and rice protein concentrate (CP content 80%) as protein sources for calf diets to substitute MP, and their result showed, as the proportions of VP continuously increased, ADG continuously declined. Li et al. (2008) used soybean protein as a protein source for milk replacers to provide 20, 50, 80% of total dietary protein, and reported that BW gain showed a downward trend as the soybean protein proportion increased during the whole experimental period. Tomkins et al. (1994a,b) also indicated that, although effects of the utilization of VP was not very ideal for 2-week-old calves, the ADG of calves in VP group was only 6.4% lower than that in the full MP group during a 6-week test period. The present study indicated that the 4 VP had biggish differences in affecting the growth performance of suckling calves compared with MP, and this had a direct relationship with different protein sources.
The SP and RP groups showed growth performance similar to that of the MP group. However, the WP and PP groups showed lower growth performance than the MP group. The starter intakes were significant higher in the SP and RP groups than in the MP group from 50 to 63 days, the reason could be that the rumens of calves in the SP and RP groups were fully developed in this period. It was in accordance with the report of Lesmeister and Heinrichs (2004) that feeding milk replacers from MP source was beneficial for rumen and reticulum development of calves. During the whole experiment period, the 4 groups of VP had better performance for F:G but worse performance for ADG, and the SP, WP, RP groups showed no significant differences in F:G, but the PP group had significant higher F:G compared with the MP group. The 5 types of milk replacer in this experiment were formulated to have a same protein level using a same amino acid model, thus it virtually eliminated effects of nutrient levels on the growth performance of calves. The favorable growth performance of the SP and RP groups indicated that soybean and rice were excellent protein sources in daily ration of calves. In this study, the starter DMI of the WP and PP groups were similar to that of the MP group, but the ADG of the WP and PP groups, 626.7 and 554.2 g/d, respectively, were significant lower than 775.6 g/d of the MP group (P < 0.05), which was opposite to the conclusion of Ortigues-Marty et al. (2003) that feeding wheat protein milk replacers to calves gained a growth performance similar to that of feeding calves full MP milk replacers.
However in Ortigues-Marty's study, wheat protein accounted for 49% TP, which was lower than 70% of this experiment. This indicates that at the nutrient level of the present study, wheat protein or peanut protein, which accounted for 70% TP, had detrimental effects on growth performance of suckling calves. The SP and RP selected in this experiment were likely to better satisfy the digestibility and nutritional needs of suckling calves using the same level of main essential amino acids and nutrient in the milk replacers, whereas WP and PP reduced the protein digestibility and production performance of calves due to anti-nutritional factors etc.
Effects of protein sources for milk replacers on body sizes of suckling calves
Animal growth is a coordinated and developmental process in terms of various aspects. The condition and appearance of an animal body directly reflect the condition of body growth (Zhang et al., 2010). A close relationship exists between BW and body size (Heinrichs et al., 1992). The most significant relationship was between chest circumference and BW (Davis et al., 1961). The body size of calves that fed full MP was regarded as normal growth and development in this study. The body size of calves significantly increased with age in each group, and no significant difference was shown between the VP groups and the MP group which indicates that MP and VP that were selected in this experiment had similar functions in ensuring physical growth.
The withers height, body length and chest circumference of an animal could accurately reflect the growth and development levels of bones (Wickersham and Schultz, 1963). The present experiment showed that these indexes were higher of the MP, SP, RP groups than of the WP and PP groups, and DMI showed no significant differences among groups from 21 to 56 days, because the calves at this age had very limited abilities to digest nutrition of the starter due to underdeveloped rumen, thus they mainly depend on liquid feed before weaning. Compared with the other 3 groups, the WP and PP groups likely had lower protein digestion levels, thus the digestible nutrients of the 2 groups may be insufficient, which might cause a difference in the growth of bones, but the difference was not significant in statistics.
Effects of protein sources for milk replacers on serum biochemical indexes of suckling calves
The concentration of BUN in blood sera can accurately reflect the conditions of protein metabolism and dietary amino acid balance in animal bodies (Stanley et al., 2002). In this experiment, 4 protein sources for milk replacers had no significant effects on the BUN concentration but the BUN concentration of all VP groups was lower than that of the MP group, and the lowest BUN concentration was in the WP and PP groups. This result indicated that the digestibility is lower of VP than of MP, and different VP have different digestibilities. The low digestibility of protein means a low level of digestible protein in the daily ration, which was supported by the report of Li et al. (2008) that the BUN concentration in blood sera of calves was lower for low protein level daily rations than for high protein level daily rations. Therefore, milk replacers should contain a higher level of crude protein in VP milk replacers than in full MP (Sanz et al., 1997).
Inadequate nutrient intake or stress would cause the TP concentration in the blood sera of calves to decline (Sampelayo et al., 1997). Effects of protein sources on TP, ALB, ALOB concentrations, and albumin:globulin ratio (A:G) in blood sera of calves were not significant in this experiment. Calves in the MP group had higher ALB concentrations and A:G than those in the other 4 groups. The WP and PP groups had lowest values of ALB, TP concentrations in blood sera and A:G among the 4 VP groups. This is an indication that calves in the WP and PP groups were likely to have higher immune activation reactions than the other 3 groups, and accordingly the proportions of nutrient intakes in order to ensure growth were lower in the WP and PP groups than in the other 3 groups (Jianhong et al., 2012). This conclusion was in line with the poor growth performance of the WP and PP groups in this experiment.
When the metabolism of glucose and lipid was abnormal in an animal body, it would cause the concentration of b-HB to rise in the serum (Gilbert et al., 2000). The concentrations of b-HB had no significant differences among groups, and basically maintained between 50 and 61 mmol/L, thus the effects of protein sources on calf glucose and lipid metabolism could be ignored.
Effects of protein sources for milk replacers on hormone indexes of suckling calves
Growth hormone can promote amino acids in muscle tissues to move into cells and enhance the synthesis of protein (Breier, 1999). The effects of protein sources on the concentration of GH in sera were not significant in this experiment. The GH concentrations of the MP, SP, and RP groups continuously increased with age, however, they firstly declined and then rose to normal levels with age in the WP, PP groups during the whole experiment period. The result indicated that protein sources had a little effect on relevant protein anabolism but the effect didn't reach study significance. Adding soybean or rice protein to milk replacers up to 70% TP could achieve a similar level of protein anabolism as of using full MP. Adding wheat or peanut protein to milk replacer up to 70% TP likely had detrimental effects on protein anabolism of calves. However, calves enhanced the utilization ability of wheat and peanut protein with age and the functions of protein anabolism gradually restored to the level of the MP group.
Insulin-like growth factor-1 was a peptide mainly secreted by the liver cells and bone marrow cells and played an important role in protein retention, and it could reduce protein oxidation and enhance protein synthesis in body tissues, and the secretion of Table 7 Concentrations of growth hormone (GH) and insulin-like growth factor-1 (IGF-1) of control and vegetable protein groups. IGF-1 was controlled by GH (Brameld et al., 1996). Kita et al. (2000) verified that the concentration of IGF-1 in the serum partly affected the velocity of muscle protein syntheses. Studies also have shown that the nutrient level, protein quality and source of daily ration would affect the level of IGF-1 gene expression, thereby control body protein metabolism (Pell et al., 1993;Pell and Bates, 1987;Bartke, 1999). Therefore the IGF-1 concentration in the serum likely reflected the change of body protein metabolism and nitrogen balances. The effects of protein sources on the IGF-1 concentrations of calves were not significant in this experiment. During the whole experiment period, the IGF-1 concentration did not show big fluctuations among all groups although it was higher in the MP group than in the other 4 groups with the lowest in the WP, PP groups. The present study controlled essential amino acids in milk replacers to a same nutrient level by adding crystalline amino acids, this probably improved the quality and digestibility of VP. However, it is not satisfactory for weight gains in the WP and PP groups. This indicated that different protein sources gave rise to the different growth and development of calves. This warrants further studies. As the IGF-1 concentration in sera of this experiment showed, it was entirely feasible to use VP as main proteins for milk replacers under the current condition of nutrition regulation.
Conclusions
Basing on the nutrition level of this experiment, we draw the following conclusions: 1) Under basically the same condition of nutrient levels (including the 4 types of essential amino acids), SP and RP as main protein sources for milk replacers have similar effects on weight gain performance and feed conversion ratio of suckling calves as full MP, but WP and PP have worse effects. Different VP as main protein sources for milk replacers have different effects on the growth performance of sucking calves. 2) The effects of different protein sources for milk replacers on metabolite in calf sera are not significant (no stress beyond the affordable range of calf body appeared).
In summary, high-quality VP can be used as main protein sources for milk replacers by using the nutritional manipulation technique of this study. | v3-fos |
2019-04-15T13:04:05.460Z | {
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} | s2 | Perspectives of Using Ultra-Fine Metals as Universal Safe BioStimulators to Get Cattle Breeding Quality Products
We have conducted investigations of ultra-fine metals biological activity with lab non-pedigree white rats, rabbits breed “Soviet chinchilla” and cattle young stock of the black and white breed as the most widely spread in the central part of Russia. One can see the possibility of using microelements of ultra-fine iron, cobalt and copper as cheap, non-toxic and highly effective biological catalyst of biochemical processes in the organism that improve physiological state, morphological and biochemical blood parameters increasing activity of the experimental animals’ ferment systems and their productivity and meat biological value. We have proved the ultra-fine powders safety when adding them to the animals’ diet.
Introduction
Modern vet and livestock technologies are closely connected with nano-technologies providing potential vector of solving many actual problems of mankind, mainly food industry. The aim of agriculture and livestock breeding in particular is providing people with high quality full value food products. Healthy animals' products consumption is the basis for human diseases prevention, improving lifetime and its quality.
As far as transferring the livestock sector to the industrial ground and increasing production it is necessary to pay more attention to full value, balanced animal nutrition and increasing the index of fodder efficiency. Being the necessary component of many biologically active compounds like proteins, ferments, hormones, vitamins and pigments or influencing their functions microelements participate in different processes of animals' life activity and metabolism. Bio-drugs of new generation, i.e. microelements in the form of metals ultra-fine powders (MUFP) arouse great interest. Ultra-fine powders of iron, cobalt, copper, manganese, selenium and others have higher biological activity. Investigations conducted in last years have shown their efficiency in crop breeding, fodder production and livestock raising [1,2,3,11].
Methods
We have considered the multi-factorial variant of using nanomaterials, i.e. ultra-fine metals as highly active bio-stimulators of animals' physiological processes. This optimizes the hormone, immune and bio-chemical status of animals and increases the young stock livability.
We have studied these bio-drugs in different regions of Russia (Moscow, Ryazan, Tula and other oblasts, Krasnodar and Stavropol territories). We have been studying ultra-fine powders at Ryazan State The degustation commission has discovered that organoleptic parameters (taste, aroma, delicacy, juiciness and others) of broth, fried and boiled meat of the experimental and control groups have not differed greatly and can be eaten without any restrictions.
3.2.
Influence of nano-crystal metals on physiological state of lab and agricultural animals and safety of livestock products when adding them to the diet Nano-powders are in meta-stable state and therefore have higher chemical activity subjected both dimensioned and structural factors and changed mechanical, electrical, optical and other properties of substances. Nano-particles possess more energy than traditional powders. So they have got a number of unique physical and chemical properties. Nano-particles are digested and produce physiological action with the organism several times stronger than familiar natural mineral substances or their chemical analogues. Biological activity of nano-powders is due to small sizes of particles and their possibility to penetrate into the organism. As opposed to metal ions nano-particles before their penetration into the plant and animals cells do not have any charge therefore they do not form large complexes with transmitting protein and can easily pass through pores of plasmodesma in a bi-lipid layer of cell membranes sized 50 nm that is comparable with the size of nano-particles (25-35 nm) and get into cytoplasm. One can see the nano-powders effect on the organism in nano-particles on the cell and micro-molecular levels [4,5].
For full development and safe implementation of biologically active drugs based on metals nanopowders sized 25-45 nm in livestock raising we have developed their veterinary and toxicological characteristic. As a result of the conducted experiments we have estimated that nano-powders of iron, cobalt, copper and copper oxide do not possess any cumulative properties and differ by lower toxicity than inorganic salt of these metals. This allows using them in premixes as a source of micro-elements [6,7]. We have tested their efficiency for prophylaxis of cows' postnatal complications and newborn calves' enteric infections. The primary evaluation of safety and efficiency has shown high preventive properties of the drug and its safety.
As a result of some preventive measures the cows' lochial period has passed without any complications. The cows have been hogging timely that has proved the normal revival of the reproductive system after calving. 80 % of animals getting the drug have not got any postnatal complications (endometritis). 20 % of the experiment cows have had postnatal endometritis in a benignant catarrhal form. More than 60 % of cows have been inseminated in one hog without being stimulated by hormones.
The newborn calves' prophylaxis has left out young stock mortality from enteric infections. The use of nano-materials having high biological activity can make a considerable contribution to solving this problem and offer an efficient system of diseases prophylaxis by activating own protective strength of the organism.
We have studied the influence of metals ultra-fine powders on immune, bio-chemical and hormone parameters of the organism so that physiologically justify the use of metals nano-particles as nonmedicinal drugs of animals' prophylaxis and correction.
We have chosen animals to the groups for the experiment according to the principle of the balance group-analogues considering gender, age, breed, live weight and the same feeding and housing conditions. We have taken blood from the side ear vein four-timely at interval of 10 days before morning feeding. We have determined all blood parameters (hemoglobin, erythrocytes, leucocytes, leucocytic formula and blood sedimentation rate) according to unitized methods of clinical diagnostics [9]. We have analyzed the biochemical parameters of blood with spectrophotometer "Spectrum" firm "Abbot" according to unitized methods of clinical lab investigations.
Determining optimal doses of metals nano-powders when adding them to the diet of lab animals
We have had experiments with rabbits breed "Soviet chinchilla" aged 30 days. We have got 10 experiment groups 16 rabbits weighing 800±0.5 g each. We have used the mixed fodder treated with the drugs suspension to add metals nano-powders to the diet. We kept the rabbits in recommended housing and 2 times feeding conditions. The influence of different doses of nano-crystal metals on live weight in the control and experiment groups of animals is shown in figure 1. One can consider 0.08 mg/kg of live weight as the optimal dose of iron nano-powder. The increase of the animals' weight has been 25% after 30 days of giving the drug. The optimal cobalt nano-powder concentration has been 0.02 mg/kg that has allowed increasing the live weight by 27 %. The corresponding data for copper have been 0.04 mg/kg and 26 %. The cancellation of MUFP has not led to decline of the experiment animals' weight. Moreover the tendency of mass increase has remained even after 10 days after the cancellation of metals ultra-fine powders ( Figure 1). We have studied the influence of these concentrations nano-powders on rabbits' physiological parameters and morphobiochemical parameters of blood. The experiment lasted for 60 days and the age of the animals at the beginning of the experiment was 30 days. The animals of all 4 groups have regularly gained the weight. They have been active and got a good appetite. Adding iron nano-powder to the diet has increased the rabbits' live weight by 11.7 % by the end of the experiment as compared with that of the control, cobalt nano-powder by 17.8 % and copper by 16.3 %. We have also investigated morphological and biochemical blood parameters of the experiment animals.
All parameters of blood serum when adding nano-crystal metals have been within the limits of the physiological norm. But there have been authentic increase of erythrocytes and hemoglobin, crude protein at the expense of the globulin fraction and α 1 -and β-globulins in particular. Increasing these fractions leads to strengthening the active transfer of carbohydrates and lipids to tissues that influences the globulins number and activity that activates metabolism. One can see the same character of changes with γ-globulins. At the expense of γ-globulins increase as compared with the control there has been an increase of immune-biological reactivity as they are defensive antibodies (immunoglobulins) and are responsible for specific immune reply. The immune system that is structurally and functionally organized complex of lymphoid cells interacting with each other and accessorial cell elements on some stages of immunogenesis can serve an indicator reflecting the nanomaterials affect on the organism. If consider that microelements in ultra-fine state activate the immune, ferment and humoral systems of the organism promoting the increase of metabolism and better digestion and nutrition then one can use them as bio-additives [10,11].
The influence of nano-crystal iron, cobalt and copper on the physiological state of the young stock (heifers)
We have had experiments with black and white cattle as most widely spread in Ryazan oblast at farm JSC "Starozhilovo stud farm". We have treated the mixed fodder with water powders suspension before feeding the animals. Suspension consumption has been 1 l per 1 t of mixed fodder. They have added the treated mixed fodder to the diet of the control and experiment animals on the basis of 2 kg of mixed fodder per 1 animal a day during the whole experiment. The age of experiment and control animals at the beginning of the experiment has been approximately 4 months. The experiment lasted for 8 months. We weighed all animals once a month. The first group (control) did not get ultra-fine powders, the second one (experiment) got nano-crystal iron (0.08 mg/kg of live weight a day), the third experiment group has got nano-crystal cobalt (0.02 mg/kg), the forth experiment group has got nano-crystal copper (0.04 mg/kg).
The experiment animals during the whole experiment have regularly exceeded the control animals in live weight beginning from the 1 st month of the experiment. At that, every next month the weight gain of the live weight of the experiment heifers has increased. Adding nano-crystal iron to the heifers' diet has promoted the live weight increase in 8 months by 22.4 as compared with the control, cobalt by 13.7 % and copper by 10.7 % as compared with the control. Daily average gain has also increased ( Figure 2). The increase of live weight, daily average and gross gain in a case of adding nano-crystal metals proves catalytical action of these bio-drugs that manifests in metabolism strengthening on the whole and protein metabolism in particular. Copper nano-powder influence on blood morphological content ( Table 2) has manifested in little rise of erythrocytes (by 10.7 %) and hemaglobin (by 12.4 %) as compared with the control. All other blood parameters have remained within the norm and differed inconsiderably from the control.
Influence of nano-crystal iron, cobalt and copper on morphological parameters of heifers' blood
One could see leucocytes increase in the 8 th month of the experiment by 7.6 % (iron) and 5.1 % (cobalt) and the change of the leucogram, i.e. lymphocytes increase for iron cobalt and copper has been 11.4 %, 8.2 % and 5% higher than the control. These changes prove the increase of the immune system of animals as the main function of В-lymphotcytes is producing antibodies or protective immunoglobulins. The lymphocytes increase has caused the significant decline of granulocytes and neutrophils lower than the control in particular.
The lymphocytes increase in the leucogram has proved strengthening protective functions, whereas the increase of hemoglobin and erythrocytes has proved the improvement of the hemopoietic system of the organism. With nano-powders there is growth and renewal of blood cells and strengthening of oxidation processes that is one of the criteria in diagnosing and evaluating the nano-materials safety.
Influence of nano-crystal iron, cobalt and copper on bio-chemical blood parameters of the experiment animals
Bio-chemical blood parameters reflect metabolism of protein, fat, carbohydrates vitamins, hormones and water-mineral characteristics of the organism. They let interpret the growth and development of the organism, help to discover latent diseases.
Using ultra-fine iron has considerably activated much ferment. So by the end of the experiment ALT content in blood serum has increased by 12.5 % and AST by 7.6 % as compared with the control. At that de Rittis coefficient (correlation of AST to ALT) has changed from 1.65 in the control to 1.40 in the first experiment group of animals that got nano-iron and up to 1.55 in the 2 nd and 3 rd experiment groups that got nano-cobalt and nano-copper correspondingly. This parameter in the experiment groups has reached the physiological norm (1.2 -1.4) that proves stabilization of animals' metabolisms. These ferment activation in the blood of the experimental animals is connected with strengthening the protein metabolism that, in its turn, leads to intensive gain of the live weight (Table 5). This is also connected with the increase of γ-glutamyltransferase in blood serum by 14.2 -21.4 % when using nano-metals as compared with the control. This ferment participates in amino acids metabolism and its activity also reflects protein metabolism intensity. The uric acid has increased by 13.3-23.3 %, urea by 13.6 -18.2 %, creatinine by 5.5 -9.5 % higher than the control one. These substances are products of protein metabolism and the fall under residual nitrogen and the increase of their amount are connected with strengthening protein metabolism intensity in animals. Cholesterol in blood of the experimental animals as compared with the control has declined by 30% that positively influences the animals' physiological state. This is proved by the increase of αamylase by 17.1 -28.6 % as compared with the control. The intensive growth of the organism also proves the activity of alkaline phosphatase. Its content is 24.2 -29.3 % higher than the control. This ferment participates in regulating the processes of bone tissue formation and catalyzes phosphorus metabolism in animals and the increase of alkaline phosphatase activity is particularly important for the growing organism.
Such a considerable change of ferment system activity is connected with using some inconsiderable amount of nanocrystal metals that has presupposed some additional increase of micro-elements at the expense of these elements better digestion in animals' digestive duct.
Nano-metals influence on mineral substances in animals' blood, muscle tissue and liver
The results of our investigations have proved that nano-metals stimulate accumulation of other macroand micro-elements (Table 6). That has been proved by the analysis of blood, muscles and liver mineral composition. After 8 months of the experiment there has been authentic increase of kalium by 6.6-11.0 %, natrium by 3.5 -5.1%, calcium by 14-19 % and phosphorus by 4.4-13.0 % in blood of animals that have got ferum nano-powder. The content of chlorine ions has increased by 5.1-8.8 %. There has also been 6.0 % increase of iron and 9.0 % of copper as compared with the control.
In this case the iron increase has promoted copper accumulation. This proves that we have selected correct doses as iron surplus in the organism could have led to copper and calcium deficit. Ultra-fine cobalt and copper have also contributed to kalium, calcium and phosphor increase in blood. Other substances content has not changed ( Table 5).
Iron nano-powder in the diet has increased the content of calcium, copper, kalium, magnesium and manganese in muscles 20%-30 % higher than those of the control. Iron in muscle tissue has not exceeded the control one that shows the absence of cumulative properties of the drug and its ecological safety. Strengthening digestion and metabolism processes has not led to heavy metals accumulation in muscles that has influenced positively the general physiological state of the animals. Cobalt and copper nano-powders have also changed the content of mineral substances in animals' muscles but the level of cobalt and copper themselves has not exceeded the control one. At that heavy metals have not accumulated. One can find similar results when analyzing the mineral composition of the control and experiment animals' liver. We have not seen any metals including iron, copper and cobalt accumulation in blood, liver and muscles of the animals.
Biochemical investigations of internal tissues and organs
We have determined the protein value of meat as for the correlation between essential and nonessential amino acids. The number of essential amino acids in meat protein of animals that have got iron nano-powder has increased by 3.9 %, in case with cobalt by 4.2 % and copper by 4.8 % that means the increase of the meat food value of the experiment heifers. There has also been some increase of glycine, histidine, methionine and serine, in particular stimulating anti-oxidants accumulation.
The content of fatty acids in subcutis of the experiment animals has not differed from that of the control one that proves the absence of pathological biochemical processes in the organism and the normal development of lipid metabolism. There has been an increase of vitamin A up to 11-35%, vitamin C up to 10-20 % and E (up to 33 %) in the liver and in muscles (А 17-42 %; С 8-16 %; Е to15 %) of the experiment animals that has also increased meat value as a food product.
If at the beginning of the experiment the amino acid composition has been approximately the same in the control and experiment groups, by the end of the experiment there has been a considerable common increase of amino acids content in blood of the animals getting cobalt nanopowder by 26.8 % and copper by 21.9 % as compared with the control (Figure3,4). We have got similar conclusions when studying the influence of cobalt and copper nano-particles on physiological and productive parameters of black and white bull calves (2008)(2009)(2010). We have used the suspension on the basis of 1 liter per 1 ton of the mixed fodder. The metals nano-powders activity has remained during 1 month. The animals getting metals nano-powders have had higher digestive activity than the control ones. The dry matter digestion index has been 3.9 % and 2.5 % higher than the control for cobalt and copper nano-powders. The organic matter content has been 6.8 % and 3.5 % higher, protein 5.6 % and 3.8 % higher, fat 4.4 % and 3.8 % higher, fiber 3.6 % and 4.0 % higher and anazotic extractive matters 3.8 % and 4.0 % higher than those of the control for cobalt and copper nano-powders correspondingly. Adding cobalt and copper nano-powders has caused the increase of nitrogen and calcium balance in the organism and the decline of their fecal and urinary excretion.
There have been some changes of the experiment animals' meat productivity parameters. As a result of the slaughter the cows having nano-cobalt in the diet have had the hot carcass mass increased by 18.7 %, the dead weight by 16.9 % and the slaughter yield by 4.0 % as compared with the control. For copper the hot carcass mass has increased by 14.0 % and the slaughter yield by 3.5 %. At that the meat energetic value has increased by 10.1 -12.5 %.
Immunity increase under the influence of iron, copper and cobat nano-particles
We have conducted investigations during the postpartum period characterized by the organism immune depression. In many instances one can consider it to be the reason for most cows having complications as a result of necessary protective strengths absence. In comparison with ill animals the cows getting metals nano-particles safely escaped complications of the postpartum period. At that they have had Т indexes, total lymphocytes, considerably lower, only 38.3 % as opposed to 53.1 % of ill animals, whereas healthy cows have had this index equal to 44 %. This shows the absence of total lymphocytosis and low specific immunity in the experiment group. Т-killers of the tested cows have been considerably higher (24.7 %), especially under the influence of nano-particles as compared with sick animals having 3.7 %. This fraction provides activation of the specific cell immunity. As the animals having such high exponents under nano-particles have not fallen ill we can consider it a favorable factor. В-lymphocytes have been vice versa lower in the experiment group that also shows the absence of complications development threat. In this situation the organism of the animal with the help of nano-particles copes with the activation of only the cell link not including the humoral one that is, as you know, increases when acute bacterial infections. One can also connect with this Т-helpers lowering of cows having got nano-particles and stable high level of Тsuppressors. We suppose stimulating the immunity is connected with the immune protective action of metals nano-particles. Copper and iron are important bio-metals participating in immune reactions. Their deficit weakens the function of the immune system: the decline of antimicrobial activity of macrophages, tissue saturation with granulocytes, cell eating suppression and anti-bodies formation. Production of lactoferrin that is an immune response modifier and an important component of supporting homeostase of the organism systems, participating in transporting iron and possessing antimicrobial, antitoxic and antiphlogistic activity is also connected with iron. All these facts prove the immune stimulating action of iron, copper and cobalt nano-particles in cows' bodies.
Milk productivity
We have studied milk productivity and milk quality of the first calf black and white heifers having iron ultra-fine powder in winter housing. We have formed 2 groups of animals numbering 20 animals in each of them. While doing that we have considered the animals' age, live weight and the cows' pregnancy period. All the experiment animals have weighed 470... 475 kg being 5…6 months pregnant and been kept in the same housing and feeding conditions. As for feeding difference the experiment first calf heifers got not only the balanced diet (according to norms) but the mixed fodder with 0.009 mg of iron per 1 kg of the mixed fodder a day.
Note: Р ≤0,05; Adding iron ultra-fine powder do the diet of the experiment animals has increased the daily average milk yield of the first calf heifers by 13.3 % as compared with the control (Table 7). For the first 100 days of the experiment group first calf heifers' lactation we have got 147 kg more milk as compared with the control group. Fat in the milk of the experiment animals has been 4.25 % that is 9.0 % higher than that of the control group. Milk fat in the milk for 100 days of lactation in the experiment group has been considerably higher (5.6 kg) than that in the control group as they have got more milk from the experiment cows and fat mass percentage in it has been a little higher.
Organoleptic estimation of the experiment first calf heifers' milk has not shown any differences. It has met the requirements of GOST R 52054-2003 in taste, color and smell.
All parameters under study of the experiment group have to some extent exceeded the analogues of the control group. Calcium and phosphorus in milk of the experiment group first calf heifers has been 9.0% higher than those of the control group (Table 7).
Physical-chemical properties of milk determined by concentration and the dispersiveness degree of the compounds are of great importance for the processing industry. To physical-chemical properties of milk one can attribute density and acidity. Milk density of the experiment first calf heifers has been 0.1-0.9º А higher that that of the control. The base-titratable acidity of all experiment animals' milk has not practically differed and corresponded to the norm. Thus, adding ultra-fine iron to the diet has increased milk yield, milk fat and improved milk chemical composition.
Conclusions
Ultra-fine microelements affect the organism mainly indirectly changing activity of ferments, hormones, protein, vitamins and other biologically active substances including metals or sensitive to their concentration change in the environment. Adding nano-crystal metals especially iron and cobalt promotes intensive accumulation of the animals' live weight due to the positive influence of protein, carbohydrates and mineral substances digestion. This supposition has been proved by morphological and biochemical investigations of blood and the content of mineral substances in blood serum. Ultrafine microelements activate immune, enzymatic and humoral systems of the organism promoting metabolism increase and better digestion and fixation of the diet. The increase of protein, carbohydrates and mineral metabolism leads to considerable increase of the experiment bull calves' and black and white heifers' meat productivity and the quality parameters of meat (chemical and biochemical composition, vitamins, essential amino acids, macro-and microelements) and milk. | v3-fos |
2019-04-01T13:15:50.272Z | {
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} | s2 | The Pathogenicity of Sclerotium rolfsii on Cyperus difformis and its Potential Host Specificity among the Genus Cyperus
Sclerotium rolfsii is a versatile soil borne pathogen commonly occurs in the tropics, subtropics, and other warm regions, especially at high humidity and warm temperatures. It may cause a variety of diseases, for example, damping off of seedlings, collar or stem rot, foot rot, crown rot, Sclerotium wilt and blight [1]. Previous studies have reported that S. roflsii infects more than 500 species of monocotyledonous and dicotylednous plants, especially severe on vegetables, flowers, legumes, cereals, forage plants and weeds [2,3]. The signs and symptoms of Sclerotinia minor were observed on yellow nutsedge (Cyperus esculentus L.) in Bertie County [4], but infection of S. rolfsii in Cyperus spp. has not been reported yet.
Introduction
Sclerotium rolfsii is a versatile soil borne pathogen commonly occurs in the tropics, subtropics, and other warm regions, especially at high humidity and warm temperatures. It may cause a variety of diseases, for example, damping off of seedlings, collar or stem rot, foot rot, crown rot, Sclerotium wilt and blight [1]. Previous studies have reported that S. roflsii infects more than 500 species of monocotyledonous and dicotylednous plants, especially severe on vegetables, flowers, legumes, cereals, forage plants and weeds [2,3]. The signs and symptoms of Sclerotinia minor were observed on yellow nutsedge (Cyperus esculentus L.) in Bertie County [4], but infection of S. rolfsii in Cyperus spp. has not been reported yet.
The histopathology of infection by Sclerotium spp. has been studied in considerable detail. Previous histopathological studies have reported that S. rolfsii penetrates host tissue by formation of appressoria [5,6], followed by apparent tissue necrosis in advance of the mycelium [6]. Phytotoxins such as oxalic acid and cell wall degrading enzymes play a key role in the infection of a host [1,7] and a multi-enzyme system for the degradation of different polysaccharides was discovered in the host tissue [8]. Although "hyphal aggregates" have been reported to form during infection by S. rolfsii, the role of these aggregates in pathogenesis has not been determined, and tissue death in advance of mycelial growth has also not been conclusively demonstrated.
S. rolfsii isolate SC64, a fungus indigenous in Jiangsu province, was isolated from an alien invasive weed Solidago canadensis L. (Canadian goldenrod, Asteraceae) [9]. The fungus caused basal stem rot lesions on S. canadensis and was found capable of controlling some dicotyledon weeds and Cyperus difformis, which was unrecognized as the host before, in a host range test and field trials [10]. Understanding the infection differences between C. difformis and other species of the Cyperus family to S. rolfsii isolate SC64 is imperative to estimating the host range of this isolate as a biocontrol agent and enriching the infection mechanism of S. rolfsii. Therefore, the objectives of this research were to 1) determine the host specificity of S. rolfsii isolate SC64 among 7 species in genus Cyperus 2) study in detail the performance of S. rolfsii isolate SC64 on C. difformis stem surface by using light and scanning electron microscopy and 3) compare the transverse section of C. difformis with six other Cyperus species by using paraffin section observation and try to elucidate the selective mechanism of S. rolfsii among Cyperus spp. based on anatomical structure differences.
Materials and Methods
Pathogen and host plant: Isolate SC64 of S. rolfsii from S. canadensis in Nanjing city, Jiangsu province of China, was used throughout this study. For inoculum preparation, the fungus was grown in Petri dishes on potato dextrose agar (PDA) at 28°C in the dark for 2-3 days.
Greenhouse-grown host plants included in this study were C. difformis L.; C. rotundus L.; C. iria Linn.; C. glomeratus L.; C. amuricus Maxim. and C. cuspidatus H.B.K. Plants were transplanted from wild field (near a lake in Xiamafang Park, Nanjing city) into plastic pots (14 cm in diameter) and grown in the greenhouse under natural photoperiod and temperature conditions. Host specificity tests: A starter culture of isolate SC64 was produced by placing five agar plugs (5 mm diameter), cutting from the actively growing margin of the PDA culture, into 100 ml (250 ml flask) potato dextrose broth (PDB, potato extract, 20 g D-glucose and water to Abstract Sclerotium rolfsii Sacc. infects more than 500 species of monocotyledonous and dicotyledonous plants except Cyperaceae family. The pathogenicity of a S. rolfsii isolate was evaluated by seven Cyperus species in order to explicate host specificity to Cyperaceae family. The results showed that only C. dofformis L. was infected with typical water-soaked lesions of the basal stem, which progressed to rotting, wilting, blighting, and eventually death. The performance of hyphae on the surface of Cyperus plants was compared and found that only stomata of C. difformis were adhered by hyphae of S. rolfsii. The infection process of S. rolfsii on leaf sheath of stem base in C. difformis showed that dense mycelial networks and ramifying hyphae were usually formed on the inoculated tissues, then growing hyphal tips were observed to spread wavelike on the stem surface, reaching the stomata between the leaf veins accurately and directly enter the host through stomata. Differences of the main micro-morphology characters of leaf sheath abaxial epidermis among the seven species were compared. The stomata of C. difformis were always presented between the leaf veins (3 or 4 rows of cells from the leaf veins), while the stomata of tolerant Cyperus species were close to the leaf veins. Underneath the stromata of C. difformis were air chambers, however vascular bundles were always present underneath the stomata of the tolerant Cyperus. Our study indicates that different anatomical structures in genus Cyperus may be associated with resistance to S. rolfsii infection. make 1 L, pH 5.0). The starter culture was cultured (28°C) in an orbital shaking incubator (110 rpm) for 7d. and aseptically blended. The starter culture was then used to inoculate plants growing in pots. Each pot contained three plants and each species was replicated four times. The experiments were performed twice. Each plant was inoculated with 0.5 ml of starter culture to the basal stem by 1 ml pipettor. After inoculation, plants were moved to greenhouse with natural light and temperature of 25~35°C. The soil was maintained wet throughout the experiment by adding water to saucer under the pots. Control plants were inoculated with autoclaved water only. Plants were monitored every day for two weeks to detect the characteristic symptoms of basal stem water soaked lesions and wilt caused by S. rolfsii. Monitoring was conducted to record the presence or absence of these symptoms.
A dose response test of C. difformis to S. rolfsii SC64 was also carried out. Seedlings with 3.5~5.5 leaf stages were inoculated with fresh fungus-infested cotton seed hulls at 60~120 g•m -2 . Plant mortality and fresh weight reduction were evaluated 14 days after inoculation. Surviving plants were excised at soil surface level, weighed and the percentage of biomass reduction was determined as compared to the control plants. The experiment included four replications for each treatment and was repeated once. The control treatment was treated with autoclaved cotton seed hulls.
Inoculation of stems of Cyperus: Mature Cyperus plants stem were excised at the soil surface level, rinsed in tap water and blotted dry. Leaves were peeled off carefully from the stems and placed on moistened filter paper in Petri dishes (9 cm in diameter). Tissues were inoculated with agar disks (5 mm diameter) cut from the advancing margin of 3-day old cultures of the pathogen grown on PDA. Dishes were sealed in polyethylene bags and incubated at 25~28°C with a 12hr photoperiod under cool-white fluorescent lights.
Light microscopy: At approximately 4-hr time intervals from 12 to 32 hr after inoculation, pieces of infected tissues from appropriate regions were fixed for 24 hr in FAA (70% ethanol︰38% formaldehyde︰glacial acetic acid, 90:5:5, v/v/v). The growth of S. rolfsii SC64 was estimated by lactophenol aniline blue staining as describe previously (Govrin and Levine, 2000). The other part of uninoculated stem of the 7 Cyperus species was fixed in FAA for observing the surface morphological characteristics and transverse sections with light microscopy. The transverse sections of stems were made through usual paraffin method in thickness of 8 μm, and stained with safranine and fast green. The pictures were taken using the image analysis software Motic Images Plus version 2.0.
Scanning electron microscopy: Leaf and stem Samples from C. dofformis were taken 8, 12, 16, 20 and 24 hr after inoculation (hai). Samples were first fixed with 4% (v/v) glutaraldehyde in 50 mM phosphate buffer (pH 6.8) for 8~10 hr at 4°C, then rinsed with the same buffer for 3 hr. After dehydration in a graded acetone series, the samples were critical-point dried, mounted on stubs, sputter coated with gold-palladium, and viewed using a HITACHI S-3000 scanning electron microscope operating at 15 kV.
Results
Host specificity and pathogenicity to Cyperus difformis: Two days after inoculation of S. rolfsii, characteristic lesions were observed on the basal stems C. difformis. Leaves collapsed 2~5 days after inoculation, and then 2~4 cm basal stem rot lesions appeared and the whole plant began to wilt and die. Meanwhile white sclerotia appeared around the basal stem and soil surface and quickly turned brown in 1~2 days ( Figure 1). Infested tissues and mature scleotia were collected for re-isolation of the fungus. Microscopic examination and culture of isolate from C. difformis confirmed that it had been infected by S. rolfsii SC64. None of other plants inoculated with S. rolfsii showed any signs of pathogenicity and therefore are considered to be immune to this pathogen ( (Figure 2a). Hyphae frequently ramified towards the stomata (Figure 2b-2d), where more intense staining was found. Ramified hyphae or adhering upon stomata were not observed on the non-susceptible Cyperus species (Figure 2e and 2f).
The infection process on C. difformis stem surface by S. rolfsii SC64: The scanning electron microscopy (SEM) observations showed that the running hyphae grew from the inoculum disks over the stem surface and formed a dense hyphal network, especially between the leaf veins ( Figure 3a) within 12 hai. The host surface was covered by the ramifying hyphae, which were relatively smaller (Figure 3b). Growing hyphal tips on the root surface were also observed to spread wavelike on the stem surface, reaching the stomata between the leaf veins accurately ( Figure 3c and 3d). Slime material (mucilage) covering hyphae, hyphal tips and extending between the hyphae, was also deposited on the plant surface. Small changes in cuticle integrity were observed (Figure3f) and the infection hyphae entered host tissue through the open stomatal ( Figure 3e). Appressorium structure was also observed on the stomata. Water soaked lesions were visible in the leaf and stem parts which underneath the inoculum disks and the host tissue turned soft at this developmental stage. Hyphae also penetrated the host surface directly through cracks. The host surface was depressed and penetrating hyphae grew into the host tissue (Figure 4g and 4h). characters, e.g. the shape of long-cells long-tubular or short-tubular, rarely sub-tetragonal, the margin of cell walls sinuous or deeply sinuous; short cells absent; stomatal subsidiary cells triangular, domeshaped to triangular, tall dome-shaped and dome-shaped; papillaes present over the veins (Figure 4). However, the presence, shape, and distribution of stromata in the epidermal surface were different from other six Cyperus species. The stomata of C. difformis were always present between the leaf veins (3 or 4 rows of cells from the leaf veins). The density of stromata was relatively higher and the length/width of stomata was relatively lower than other six Cyperus species. We propose that the micro-morphological characteristics of stromata may play an important role in the fungal infectivity.
The comparison of transverse section of Cyperus species: The differences of stromata characteristics between C. difformis and the non-susceptible Cyperus species were visually apparent in the examination of basal leaf cross-sections ( Figure 5). Vascular bundle was always present underneath the leaf veins. The stromata of the non-susceptible Cyperus species were always close to the leaf veins, so under the stromata were mesophyll cell, bundle sheath cell and vascular bundle. However, the case was different in C. difformis, where the stromata presented in the middle of vascular bundles and below the stromata was air space. From this point of view, the structure of C. difformis was more beneficial for fungus infection.
Discussion
Sclerotium rolfsii is a polyphagous pathogen in the world and new record of host species are reported continually, which includes some monocotyledonous plants e.g. Poa annua L. [11], garlic (Allium sativum) [12]; Asiatic dayflower (Commelina communis L.) [13]. Our experiment on the stem rot of C. difformis caused by S. rolfsii contributes one more new host specie for this fungus. Further studies on the fungus have shown that it is an effective agent of biological control of the weed in dry direct-seeded rice [10]. In the host specificity test of our study the primary nutrient sources of S. rolfsii was mycelium grown from PDB medium. Hyphae growing from the PDB suspension extended and ramified on the stem surfaces. However, there is substantial hyphae growth and extension difference under the light microscopy. Only the atomata of C. difformis were adhered by ramified hyphae of S. rolfsii. The pathogen directly penetrates the host surface via stomata. Therefore, stomata play an important role for S. rolfsii infection of C. difformis. However, stomatapenetrating pathogens need appropriate cues to locate stomata pores [14]. The development of wavelike hyphae may be assumed to increase the adhesion of the pathogen to the host surface in order to effectively reach the stomata. Leaf veins located close to stomata of the resistant Cyperus species might a natural fence, making a false angle for the eruptive hyphae.
Infection cushions are produced by many plant pathogenic fungi (e.g. Sclerotinia sclerotiorum; Rhizoctonia solani) and these structures were reported to facilitate infection of the host [15][16][17]. Previous studies have reported the occurrence or presumed functions of hyphal aggregates which were formed during infection of host tissue by S. rolfsii [5,18]. Infection was also reported to occur from appressoria produced by germinating basidiospores of the teleomorph of S. rolfsii [19]. In this study, neither multicellular compound appressoria (infection cushions) nor flattened hyphae was observed on the host surface. After penetration into mesophyll via stomata, the host tissue turned yellowish-brown soft and the cuticle became disintegrated. Then hyphae entered into the host tissue through the rifts. S. rolfsii produce extracellular enzymes including pectin methyleasterase [7], cutinase [20], phosphatidase [21], arabanase [22], galatanase, mannanase, xylanse [23], oxalic acid and polygalacturonase [24]. It is assumed that tissue death in advance of mycelial growth during infection of C. difformis by S. rolfsii.
Plant defense against pathogen attack is complex, with many local and systemic aspects [25]. The internal anatomy and surface features of the leaves often determine plant resistance to biotrophic pathogen infection [26]. Among such characters, aspects of stromata, cuticle and trichome morphology can influence disease resistance [27]. We compared the transverse section of C. difformis and some other resistant Cyperus species and found that the variation of stomata distribution was in relation to the stem anatomy. The air chamber underneath the stomata of C. difformis provides the weakest mechanical obstruction to fungal penetration, while the vascular bundle underneath the stomata of resistant Cyperus may be a natural barrier.
In rust fungi, the emerging germ tubes adhere first to the leaf surface; subsequently, they grow and encounter stomata through directional growth [28], which in turn triggers appressorium formation [29]. Directional growth of the germ tube and formation of appressorium are controlled by the stimuli originating from the host [30]. It seems that, for the first time, an alternative 'avoidance' or prepenetration mechanism is apparent in Cyperus -S. rolfsii interaction, which operates after the contact of parasite on the host epidermal cell [31,32]. However, we failed to observe the extension of hyphae inter-and intracellularly. More details of the infection process, the effect of mechanical obstacles of epidermis (eg. waxy deposition) and the mechanism of directional growth of hyphae all require further investigation. | v3-fos |
2019-03-21T13:08:22.841Z | {
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} | s2 | Effects of Different Levels of Copper Sulfate on Growth and Reproductive Performances in Guppy (P. reticulate)
Copper (Cu) like other trace elements [zinc (Zn), iron (Fe), manganese (Mn), etc. serves important functions in living cells and is essential for fish [1]. Copper sulfate (CuSO4) is the sulfated form of copper which routinely used as an algicide in commercial and recreational fish ponds. It is generally recognized that copper can be highly toxic to teleosts [2,3]. In aquaculture, copper is being used as eternal parasites, bacterial and fungal disease preventer and also weeds cleaning in sulfated form [4]. The ideal concentration of copper sulfate for weed termination has been suggested to be as much as 1mg.l-1 in which has lower poisonous effect on fish but highly affects the invertebrates [5]. According to the records of United States Environmental Protection Agency (EPA), copper sulfate is an ordinary compound which is being used broadly in aquaculture. Moreover, the Food and Drug Administration (FDA) has barred the medicinal use of copper in aquaculture.
Introduction
Copper (Cu) like other trace elements [zinc (Zn), iron (Fe), manganese (Mn), etc. serves important functions in living cells and is essential for fish [1]. Copper sulfate (CuSO 4 ) is the sulfated form of copper which routinely used as an algicide in commercial and recreational fish ponds. It is generally recognized that copper can be highly toxic to teleosts [2,3]. In aquaculture, copper is being used as eternal parasites, bacterial and fungal disease preventer and also weeds cleaning in sulfated form [4]. The ideal concentration of copper sulfate for weed termination has been suggested to be as much as 1mg.l -1 in which has lower poisonous effect on fish but highly affects the invertebrates [5]. According to the records of United States Environmental Protection Agency (EPA), copper sulfate is an ordinary compound which is being used broadly in aquaculture. Moreover, the Food and Drug Administration (FDA) has barred the medicinal use of copper in aquaculture.
It should be considered that copper could be accumulated in water body and gradually increases the concentration will raise to lethal concentration for fishes [4]. Toxicity of copper to aquatic species depends on factors such as organism sensitivity, concentration of copper and its bioavailability [6], total hardness, pH [7], organic particles or various other inorganic cations and anions [8]. So it is notable to avoid using copper in waters with lower alkalinity than 50 mg CaCo 3 .l -1 . The range of copper sulfate is in aquaculture as much as 0.025-2 mg.l -1 according to alkalinity and total hardness [9] but the usual effective use in aquaculture is reported as much as 0.01 of total alkalinity [10]. Guppy (P. reticulata) (or rainbow fish) is one of the most widely distributed tropical fish in the world. It is a member of the Poeciliidae family and, like all other members of the family, is live-bearing. Northeast South America is the native habitat for guppies but now, they could be found all around the world. High adaption ability makes them live in many different environmental and ecological conditions. Guppies exhibit sexual dimorphism and omnivorous feed habit (algal remains, diatoms, invertebrates, plant fragments, mineral particles, aquatic insect larva, etc.). Females produce offspring from first 10 weeks to 34 months of age, but first reproduction occur in 10-20 weeks of age. They are used as a model organism in the field of ecology, evolution, and behavioral studies [11]. Due to copper sulfate therapeutic trait which made it an ordinary drug for use in ornamental fish's hatcheries and personal aquariums despite its toxic effects and shortage of focused study on reproductive performance in guppy (P. reticulate), this study carried out.
Methods and Materials
This study performed at the aquatic laboratory of Shahid Fazli Bar Abadi located in Gorgan university of Agricultural Science and natural Resources, Golestan, Iran. Experimental fishes were bought from a local hatchery (Shast Kolah road, Gorgan province, Iran). Upon arrival, 480 individuals of 2.5-3 months aged guppies were acclimated to laboratory conditions for 2 weeks in a 1000 L round fiberglass tank measuring 1 m in diameter and fed a commercial diet (0.5-0.8 mm in size, Pars Kilka Corp, Babolsar, Iran) as much as 3% of body weight twice a day with two equal meals at 0800 and 1700 h. The experiment was conducted in a completely randomized design with six replications per treatment for 56 days. At the beginning of the experiment, 16 fish (N: 12 female and 4 male; mean length: 3.59 ± 0.11 cm; mean weight: 0.36 ± 0.01 g) were stocked in each aquarium (50×35×30 cm) designed to contain 35 L tap water (pH: 7.4 ± 0.12; salinity; 0.35 ± 0.12 ppt; DO: 7.36 ± 0.98 mg.l -1 ; total hardness: 270-300 mg.l -1 CaCO 3 ) and equipped with airstones to maintain dissolved oxygen levels as much as possible. The water temperature was kept 28 ± 1°C by electrical heaters. Water quality variables were checked daily. Handmade plastic Happas were the intestine can damage the tissue and by means of that, reduces the nutrition intake from intestine. These researchers found out that, the existence of copper sulfate around catfishes reduced their growth performance due to interruption in intestine tissue. Furthermore, copper sulfate decreases the Zn [24] and Se [25] intake from intestine.
2) High energy consumption for copper detoxification in liver [26] reported that, in freshwater prawn (Macrobrachium rosenbergii) copper usually transported by the haemolymph to other organs primarily hepatopancreas for storage and detoxification. Ignoring the physiological differences, at the same polluted condition (Here by copper sulfate) guppies transfer copper to liver for detoxification and for fulfilling the detoxification process, body needs extra energy which is provided by more food consumption. Lack of energy for other metabolic processes result in low growth performance.
3) Chronic stress [27][28][29]: Chronic stress reduced the growth too [30]. The increase in blood glucose and cortisol concentrations is known as a general secondary response to stress of fish to toxic effects [31]. Griffin et al. [32] reported that, copper sulfate raised blood cortisol level (as stressor factor) in channel catfish (I. punctatus). These researches showed that the initial signs of stress (cortisol level) decreased consequently as copper sulfate diminished in water. At present study, a combination of three mentioned hypothesis is reliable reason for low growth performance. These results were in accordance with Berntssen et al. [33] on Atlantic salmon (Salmo salar), [34] on channel catfish (I. Punctatus). These researchers noted that increase in copper sulfate lead to low growth in fish.
Copper sulfate also had negative effects on reproduction performance (Table 1). In sensitive species of teleosts, copper adversely affects reproduction and survival from 0.01-0.02 mg Cu.l -1 . At present study, surviving rate, gonadosomatic index, relative fecundity and finally offspring production decreased by increase in copper sulfate levels (P<0.05) and best performance of mentioned factors resulted in control group (with no copper sulfate). Although relative fecundity between first two treatments (0 and 0.004 CuSO 4 . l -1 ) were not analytically significant; but it would be due to rare concentration of experimental treatment (0.004 CuSO 4 . l -1 ). Copper may affect reproductive success of fish through disruption of hatch coordination with food availability or through adverse effects on larval fishes. Chronic exposure of representative species of teleosts to low concentrations (0.005 to 0.04 mg.l -1 ) of copper in water containing low concentrations of organic material adversely affects survival and spawning. Dethloff et al. reported that cortisol is released to the blood via stimulation of the Hypathalamo-Pituitary-Adrenal (HPI) axis by heavy metal exposure. Cortisol has depressive effects on a number of immune responses in fish, including phagocytosis and lymphocyte mitogenesis [35]. Despite the valuable role of HPI hormones in reproductive processes of fishes, cortisol hormone which made in response to stress resistance in fish would interrupt HPI hormones. It will be probably the reason for lowering slope gonadosomatic index in response of rise in copper sulfate concentrations.
Pulsford et al. [36] showed that, cortisol restricts the metabolic activities of macrophage and lymphocyte cells which especially spread in kidney and spleen; on one side, negative effects of copper on intestine inhibited absorption of nutrients like electrolytes and fatty acid intake [37]; on the other side, inducing hypertrophy in gill cells [38], blocking calcium transport in gills through interference with chloride cells and ionic and gas exchanges [39] make fishes weak to stand with situation. That's why low survival rate appears in high doses of copper sulfate. put in aquariums to act as shelters for new born offsprings from predation by parents.
In this study, five levels -one as blank and four as experimental groups -of copper sulfate penta-hydrate (CuSO 4 5H 2 O, Merck, Germany) concentrations including 0 (as control), 0.004, 0.013, 0.019 and 0.026 mg CuSO 4 .l -1 . Prior to start the trial, the mean 96 h LC50 in guppies determined as much as 0.046 mg Cu.l -1 then, four concentrations were selected randomly. According to each level of copper sulfate, selected concentration prepared [12] into four reserved tanks to renew the replaced water volume. All experimental groups have been monitored daily by atomic absorption [13] to ensure the determined concentrations achieved [14].
Acute median lethal concentration (LC50) and their 95% confidence limits for all tests obtained by Finney's method were calculated with the formula of Mohapatra and Rengarajan. LC50 values of 24, 48, 72 and 96h were determined using Finney's method of probit analysis'' and with SPSS computer statistical software. The mean lethal concentration LC50 for an exposure period of 24, 48, 72 and 96 hr was designated by trial and error. Amounts of LC1, LC10, LC30, LC50, LC70, LC90, LC99 were calculated by probit tables, mortality and probit regression.
All data were reported as mean ± standard deviation. Statistics were performed by using one-way analysis of variance (ANOVA) followed by Duncan multiple comparisons test if significant differences were found. A Kolmogrov-Smirnov test was used to assess normality of distribution and abnormal data were log transformed. Significance was set at P<0.05 level.
Results and Discussion
Liver, brain, heart, kidney, and muscle are the main storing places for copper; in tissues and blood cells, it bounds to proteins, including many enzymes. It exerts a wide range of physiological effects on vital), and other hematopoietic tissues in which threats the present fishes in area [19]. Copper sulfate toxicity differs among fish species [20]; For instance, the amounts of 8, 8.97 and 16 mg Cu.l -1 reported to have negative effects on tilapia (Oreochromis niloticus × O-aureus), grupper (Epinephelus malabaricus) and channel catfish (Ictalurus punctatus) respectively [21,22].
In 8 weeks, toxic effects of copper sulfate exposure were recorded ( Table 1). As seen, the best growth performance resulted in control group (0 mg CuSO 4 .l -1 ) and growth diminished as copper sulfate increased.
Despite the same initial weight, age and feed consumption, rising amounts of copper had negative effects on weight gain, specific growth rate and feed conversion ratio (Figure 1). There are hypothesis for reduction in growth performance including: 1) Intestine tissue interruption [23]: Heavy metals have their special recipients in which act as a stimulator to them. Intestine can be considered as a target tissue for metals. Copper which enter into | v3-fos |
2019-03-31T13:43:41.744Z | {
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} | s2 | Comparison of the Biochemical Activities of Commercial Yogurts and Lactobacillus acidophilus-containing Yogurt
Lactobacillus spp.같은 유산균은 탄수화물을 발효하여 ATP를 생성하며, 시판되는 요구르트 생산에 이용되고 있다. Lactobacillus spp.는 장관계 건강에 유익한 균으로, 특히 Lactobacillus acidophilus를 함유하는 요구르트는 대장암 초기단계를 예방하는 효과가 있는 것으로 나타나 관심이 모아지고 있다. 본 연구에서는 ...
Introduction
Lactic acid-producing bacteria such as Lactobacillus spp., which ferment carbohydrates and produce ATP (Kandler, 1983) are known for their considerable benefits to the health of the human intestine. Indeed, probiotic substances pro- In the food industry, Lactobacillus spp. are used extensively for producing cheese, bean paste, fermented milk, kimchi, soy sauce, yogurt, and yogurt-based products (Sugiyama, 1984;Lücke, 1996 Although many commercial yogurts have been used to promote health, the content and activities of lactic acid bacteria in solid and liquid yogurts have not been fully elucidated. Therefore, in this study, we aimed to compare the lactic acid-producing capacities and antibacterial activities of commercial solid and liquid yogurts and a homemade yogurt containing L. acidophilus. We expect that our results will provide important insights into the production of more effective probiotic yogurts.
Materials
Solid and liquid-type yogurts from three different companies purchased from local supermarkets and a single strain of L. acidophilus were included in this study. All materials were analyzed within 24 h after storage at 10 o C in a refrigerator.
Culture and preprocessing
One milliliter of each sample type (solid yogurt, liquid yogurt and L. acidophilus-containing yogurt) was cultured in with Tween 80 by centrifugation at 3000 rpm for 10 min.
Statistical analysis
All experiments were repeated at least three times. The results are presented as the mean±standard deviation (SD).
All experimental data were analyzed using the SPSS program (SPSS 18.0, SPSS Institute, Chicago, IL, USA). One-way analysis of variance (ANOVA) with Duncan's multiple range tests was used to examine the differences between groups. Differences with p values of less than ＜0.05 were considered statistically significant.
Turbidity test
Increased turbidity suggests the growth of bacteria. Liquid yogurt was found to have a significantly higher absorbance than solid yogurt, while L. acidophilus-containing yogurt had a significantly higher absorbance than liquid yogurt (Fig. 1).
These data implied that L. acidophilus-containing yogurt had the highest growth ability among the three groups.
pH meter analysis
pH is indicative of acid-producing activity. The lowest pH was in the L. acidophilus-containing yogurt followed by liquid yogurt and solid yogurt (Fig. 2). Thus, the L. acidophilus-containing yogurt had the highest lactic acid production, while solid yogurt had the lowest. However, the pH values of the liquid yogurt and L. acidophilus-containing yogurt were not significantly different.
HPLC analysis
HPLC analysis revealed that lactic acid production was in the order of L. acidophilus-containing yogurt ＞ liquid yogurt www.kjcls.org ＞ solid yogurt (Fig. 3).
Antibacterial activity
Culture supernatants from the commercial yogurts and L.
acidophilus-containing yogurt did not show any antibacterial activity against S. aureus (data not shown). However, L.
acidophilus-derived supernatants showed an inhibitory zone of 1.8 mm in response to E. coli in a paper disk diffusion test (Fig. 4).
Discussion
In this study, culture turbidity and pH were measured to compare the growth ability and lactic acid production among solid-type, liquid-type, and L. acidophilus-containing yogurt.
L. acidophilus-containing yogurt had the highest turbidity and lowest pH. Additionally, L. acidophilus-containing yogurt exhibited the highest lactic acid production, followed by liquid yogurt and solid yogurt. L. acidophilus-containing yogurt also showed the highest antibacterial activity. Thus, our data provide important insights into the production of more effective probiotic yogurts.
Lactic acid is a weak-organic acid and plays a role in diverse biochemical processes (Ibrahim et al., 2008 | v3-fos |
2018-05-20T02:48:00.454Z | {
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} | s2 | Regeneration of Plantlets via Direct Somatic Embryogenesis from Different Explants of Murraya koenigii. (L.) Spreng
An in vitro plant regeneration system was developed via direct somatic embryogenesis from different seedling explants of an important medicinal plant Murraya koenigii (L.) Spreng Cotyledons (COT), Hypocotyl (HYP) (10 to 15 mm) and Root (RT) segments (10 to 20 mm) were excised from 60 days old seedling as explants. The somatic embryos induction was achieved on Murashige and Skoog (MS) basal medium augmented with different concentrations of 6-benzyleaminopurine (BAP) 1.33 to 8.40 μM and thidiazuron (TDZ) 1.08 to 9.82 μM. The globular embryos originated from cut ends and entire surface of the root, hypocotyl explants and margins of cotyledons within 30-40 days. The percentage of somatic embryos induction per explant was significantly higher in HYP explants (94.21±5.77%) in the MS basal medium supplemented with 6.20 μM BAP and 8.64 μM TDZ. The highest rate of conversion of torpedo, heart and cotyledonary stages from globular stage was obtained in MS medium supplemented with 8.64 μM TDZ. The matured somatic embryos were transferred to the MS basal medium without Plant Growth Regulators (PGRs). Highest 88% of the matured embryos were germinated on transfer to 1⁄2 MS basal medium without PGR, where they grew for a further 3-4 weeks. Out of seventy six hardened plants seventy (92%) plantlets were found healthy under field conditions.
Introduction
Murraya koenigii (L.) Spreng. locally known as "Curry patta" or " Meetha neem" belonging to the family Rutaceae that grows wildly in Southern Asia. Its aromatic leaves are commonly used as the raw material for traditional medicinal formulations in India. The leaves contain essential oil and also used as a condiment. Several monomeric and binary carbazol alkaloids present in the various plant parts [1]. These alkaloids have been reported to possess antimicrobial [2], [3], mosquitocidal, topo-isomerase inhibition [4]- [7] and antioxidant properties [8], [9]. Some of the alkaloids reported in this plant have showed anti carcinogenic [10], [11] and antidiabetic properties [12]. The conventional method of propagation of M. koenigii is by seeds only, which retain their viability for a short period. The biotechnological approach for genetic improvement and development of regeneration protocol is prerequisite for germplasm conservation through micropropagation and the development of transgenic plants. An efficient method of somatic embryogenesis is required for genetic improvement of this valuable medicinal plant. However, in vitro propagation of curry leaf plant through adventitious shoot proliferation from inter-nodal cuttings was reported by Joshi et al. [13]. Also in Citrus, close relative of M. koenigii, methods for genetic improvement by somatic hybridization have been reported by Grosser et al. [14]. Although few reports are available for in vitro studies of this plant which are restricted to the nodal and stem segments [15], [16] and from intact seedling [17]. Paul et al. [18] reported a regeneration system of direct and indirect somatic embryogenesis in this plant but they have used Zygotic embryonic axis and Cotyledon as explants only. The present communication reports an efficient translational protocol for regeneration of plants through direct somatic embryogenesis from Hypocotyl (HYP), Cotyledon (COT) and Root (RT) explants.
Plant material, explant preparation and cultures establishment
Ripe blackish purple colour fruits collected from matured tree of Murraya koenigii grown in campus and surrounding area of Ajmer were washed with running tap water for 15 minutes. Seeds were removed from fruits and washed free of pulp. Seeds were first washed with liquid detergent (Teepol; Qualigen, India) and then soaked with 0.1% solution of Bavistin fungicide (BASF, India) and rinsed with distilled water. The seeds were surface sterilized with an aqueous solution of 0.1% (w/v) HgCl 2 (Hi Media, India) for 5-6 minutes followed by four to five autoclaved distilled water rinses. Disinfected seeds were germinated in 200 ml screwcapped glass jars containing 40 ml seed germinating half strength micro and macronutrient Murashige and Skoog (MS) basal medium [19] devoid of plant growth regulators. Cotyledons (COT), Hypocotyl (10 to 15 mm) and root segments (10 to 20 mm) were excised from 60 days old seedling as explants.
Culture media and experimental conditions
For induction of direct somatic embryogenesis the explants were inoculated on MS medium supplemented with 1.33 to 6.20 μM 6-benzylaminopurine (BAP) and 1.08 to 9.82 μM thidiazuron (TDZ). Somatic embryos were further transferred to another set of MS medium supplemented with 1.08 to 9.82 μM TDZ for their maturation. Thus, for germination the matured somatic embryos were sub-cultured to ½ basal MS medium free of plant growth regulators (PGR). All the cultures were maintained regular sub-culture after three to four weeks on the same medium. Media were solidified by adding 0.8% agar powder (Qualigen, India) and 3% sucrose were added as carbon source (Qualigen, India). The pH of media was adjusted to 5.8 (± 0.1) before autoclaving. The media were autoclaved at 121 Psi for
Acclimatization and field transfer
In vitro developed plantlets with 40-50 mm shoot length and strong root were washed with running tap water and were transferred into 200 ml jars 1/3 filled with a pasteurized mixture of vermiculite, perlite and peat moss in equal ratio. The plantlets in the screw capped jars were kept under a hardening unit for one week and then the screw caps were removed from bottle. They were later gradually transferred to the low humidity and high light intensity zone of hardening unit in the interval of one week. The plantlets were finally transferred to poly bags and exposed to field conditions.
Statistical analysis
All experimental data were subjected to analysis of variance (ANOVA) and significant (P>0.05) means were determine with Duncan"s Multiple-range test (DMRT) to distinguish differences between treatments means at the α = 0.05 level using SPSS or Windows version 16.0.
Induction of direct somatic embryos from COT, HYP and RT explants
The somatic embryos induction was achieved on MS basal medium supplemented with different concentrations of BAP 1.33 to 8.40 μM and TDZ 1.08 to 9.82 μM. The embryos originated from cut ends as well as from surface of the root, hypocotyl and margins of cotyledons within 30-40 days. However, the frequency of responding explants and number of somatic embryos per explants varied in different concentrations of PGRs and type of the explants. The percentage of somatic embryos induction per explant was significantly higher in HYP explants (94.21±5.77%) whereas the lowest percentage of embryos (79.25±1.66%) were formed from cotyledon explants in the MS basal medium supplemented with 6.20 μM BAP and 8.64 μM TDZ ( Table 1). The somatic embryos formed in these media were of globular stages (Fig. 1a) only and they could not attain the advanced stages of somatic embryogenesis i.e. heart and torpedo stages even after culturing them for 60 days in the same medium.
Maturation and germination of somatic embryos
The globular stages of somatic embryos did not convert into next stages. The conversion percentage varied widely from 37 to 98% depending on the concentrations of growth regulators used. The highest rate of conversion of globular stage of embryos to torpedo, heart and cotyledonary stages was obtained from MS medium supplemented with 8.64 μM TDZ (Fig. 2) which converted the highest 98% of globular embryo into further heart, torpedo (Fig 1. b) and cotyledonary stages (Fig. 1c) of somatic embryogenesis within three to four weeks The same basal medium when supplemented with higher concentrations of TDZ did not show any significant differences on the percentage of conversion. The matured somatic embryos, either in clusters or individually, were transferred to the MS basal medium without PGRs, for their germination. Some of the (10%) embryos germinated even when they were still attached to the mother tissues (Fig. 1d) and the highest 88% of the matured embryos were germinated on transfer to the PGR free medium where they grew for a further 4-6 weeks (Fig. 1e). Six weeks old plantlets with 5-6 cm shoots and well developed tap roots (Fig. 1f) were transferred to poly bags containing a mixture of perlite, vermiculite and peat moss in equal ratio. Out of seventy six hardened plants ninety two percent plantlets were found healthy under field conditions (Fig. 1g).
Discussion
In present study an improved regeneration system through direct somatic embryogenesis in M. koenigii was developed. The induction of direct somatic embryogenesis and their subsequent development are influenced by culture media, choice of explants and concentration and combinations of PGRs. Culture medium is one of the most important factors to be considered for in vitro plant cell culture. The MS medium is most common medium which has been reported to be used in plant regeneration via direct and indirect somatic embryogenesis [18] and by axillary and adventitious bud culture [13], [18], [15]. Alternatively B5 medium has been used for in vitro regeneration of Arabidopsis thaliana [20]. However, present study suggested that MS medium responded better than other culture media.
In general, relatively high auxin concentrations favor callus formation and induction of cell polarity. Afterwards, when somatic embryo induction stage has been achieved, it is necessary to reduce or eliminate the auxins because the embryos begin to synthesize their own auxin, possibly via an alternative pathway [21], [22]. In the species such as Zoysia japonica [23], Begonia gracilis [24] and Oncidium spp. [25], the use of cytokinins favors the induction of somatic embryos. In our study the BAP 6.20 μM and TDZ 8.64 μM were found more effective for induction of globular embryos directly from HYP, COT and RT explants.
The TDZ a phenyl urea derivative (N-Phenyle-N1 -1, 2, 3thidiazol-5-ylurea) has a unique mode of action with intrinsic cytokinin like activity [26] and is known to be more effective than all adenine type cytokinins in inducing high frequency organogenesis in a number of plant species [27], [28] . Application of TDZ may increase the levels of endogenous cytokinins by inhibiting the action of cytokinin oxidase [29]. Present investigation revealed that the TDZ alone in the concentration of 8.64μM is prerequisite for conversion of 98% of globular embryos into torpedo and heart shape embryos. The germination or conversion of somatic embryo into plantlet is difficult during somatic embryogenesis [30]. For woody species in particular, the efficiency of germination is relatively low, which restricts application of somatic embryogenesis system for commercial purpose [31].
In our study it was observed that the matured somatic embryos could not germinate and even prolonged exposure to TDZ supplemented medium resulted in their distortion. Similar results have also been reported by Murch et al. [32] and Khurana et al. [33]. Inhibition of germination of somatic embryos by TDZ may be due to its high cytokinin activity Licensed Under Creative Commons Attribution CC BY [34]. In this study we have found that within 20 to 30 days the matured somatic embryos, recovered from HYP, COT and RT explants, were germinated on ½ MS basal medium without PGRs.
The in vitro plantlets developed under low light, aseptic conditions and on the media containing sample of sugar and nutrients cannot survive in the external environmental conditions when directly placed in green house or field [35], [36]. The physiological and anatomical characteristics of micropropagated plantlets necessitate that they should be gradually acclimatized to the green house or field conditions [37]. The in vitro plantlets developed during the study program were successfully hardened and transfer to the field where 92% plants were found healthy.
In conclusion, we have developed an improved and viable regeneration system based on direct somatic embryogenesis from HYP, COT and RT explants of M. koenigii which can suffice the need of translational studies for lab to land technology. Furthermore, the investigation will potentially address the issues of large scale micropropagation and genetic transformation of M. koenigii and its close relatives. | v3-fos |
2019-04-05T03:38:57.935Z | {
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} | s2 | Application of RSM and Multivariate Statistics in Predicting Antioxidant Property of Ethanolic Extracts of Tea-Ginger Blend
The optimum conditions for ethanolic extraction of antioxidants from tea-ginger blend were determined using response surface modelling. The relationship between the colour, hue index and antioxidant properties of the extracts were also expressed as multivariate models using ordinary least square, principal component and partial least square regressions (OLSR, PCR, and PLSR). Results from the multi-response optimisation revealed the optimum conditions for the extraction as temperature of 50.16°C, concentration of 2.1 g (100 ml) and time of 5 minutes with a desirability of 0.68. The PLSR gave the most preferable model among the three multivariate regression techniques investigated. Hue index, A510 and a* were able to predict total flavonoid content (R 2 = 0.933, Q = 0.905) and diphenyl-picrylhydrazyl (DPPH) radical activity (R = 0.945, Q = 0.919). The a*, A510, hue Index and hue were able to predict iron chelating activity (R 2 = 0.854, Q 2 = 0.794). The study revealed that colour and hue index property could give an indication of some antioxidant properties of ethanolic extracts of tea-ginger blend. Original Research Article Makanjuola et al.; EJMP, 6(4): 200-211, 2015; Article no.EJMP.2015.056 201
INTRODUCTION
Ginger has gained popularity worldwide for its culinary and nutraceutical usage. Ginger root is one of the most heavily consumed dietary substances in the world [1,2]. The health benefits of ginger are derived mainly from its antioxidant property. Rats fed with ginger extract and methotrexate have been reported to have enhanced antioxidant levels compared with rats fed with methothrexate only which experienced a decline in antioxidant levels [3]. This indicates that ginger could play a role in reducing the effect of oxidative stress. Ginger contains many bioactive phenolic compounds, including nonvolatile pungent compounds such as gingerols, paradols, shogaols and gingerones [4]. Tea (Camellia sinensis) is the most widely consumed beverage in the world after water [5]. Flavonoids are one of the major antioxidant components of tea. Tea flavonoid consumption has been linked to lower incidences of chronic diseases such as cardiovascular disease and cancer [6].
Antioxidants came to public attention in the 1990s, when scientists began to understand that free radical damage was involved in the early stages of artery clogging atherosclerosis and may contribute to cancer, vision loss, and a host of other chronic conditions [7]. Antioxidants have been known to prevent degenerative oxidative reactions. The antioxidant property confers on ginger and tea their ability to prevent oxidation of cells thus hindering malignant reactions. With the increase in oxidative stress in humans as a result of globalization and industrialization, the need to increase the consumption of antioxidants is quite germane. A combination of different antioxidants can help increase protection against free radical reaction. According to Halvorsen et al. [8], a combination of different redox-active compounds (ie, antioxidants) may be needed for proper protection against oxidative stresses.
Various novel techniques have been employed to recover phenolics from plant matrices but from an industrial production point of view, solvent extraction is commonly chosen due to simplicity, efficiency of the procedure, and low investment costs required in terms of equipment [9]. Parameters having a great impact on the amount and composition of antioxidants in extracts, and thus on the measured antioxidant capacity, notably include the extraction solvent composition, temperature, extraction time (duration), solvent-to-solid ratio, and storage conditions [10].
Quality control is an essential part of the food manufacturing chain. An important quality check for ginger and tea is their antioxidant property. The measurement of quality parameters (i.e. antioxidants) is generally, carried out using traditional analytical techniques whose application in the food industry poses several problems: they require very long duration, are expensive and destructive [11]. Colour can be an important indication of the antioxidant properties of foods. An understanding of this relationship can help present a rapid analytical technique for the evaluation of the antioxidant content of teaginger extracts. This is possible because many food components -such as xanthophylls, lycopenes, tannins, anthocyanins and βcarotenes -are responsible for the colour of the food. The colour of foods will usually change when these food pigments undergo degradation. Degradation of these pigments can occur due to storage method used and processing method applied. It was reported that canned whole tomatoes packed in CaCl 2 juice were lighter than tomatoes packed in ordinary juice [12]. The advantage of relating colour property of food to their antioxidant property centres on the opportunity of doing rapid online in-process check in the factory to have an indication of the antioxidant property of the extract being produced. This means that the time for reagent preparation, sample preparation and incubation time are eliminated. The other advantage that would be presented by this new approach will be the reduced frequency in the use of analytical reagents. This means a reduced cost of evaluation. Another positive this approach offers is environmental friendliness -as the volume of reagent that will be used for antioxidant analysis will be reduced. It has been demonstrated that the antioxidant activity and total phenol content of carrots can be predicted from their colour [11]. Also colour measurements of intact tomatoes have been used as a non-destructive method to assess total antioxidant capacity of tomatoes [13].
In this study, we seek to: i) determine the optimum condition for ethanolic extraction of antioxidants from tea-ginger blend using response surface methodology (RSM), ii) investigate the relationship between colour, hue index and antioxidant properties of the ethanolic tea-ginger extracts using multivariate statistics (ordinary least square regression -OLSR, principal component regression, PCR and partial least square regression -PLSR).
To our knowledge, this is the first study looking at extraction of antioxidants from tea-ginger blend. Futhermore we are not aware of studies that have tried to predict antioxidant properties of tea, ginger and tea-ginger extracts from their colour property.
Plant Material and Processing
Tea leaves were obtained from Obudu Mountain in Cross River state in Nigeria. The tea leaves were sun-dried, ground and passed through a 1.4 mm sieve. Ginger rhizomes were obtained from Kaduna state. Kaduna state is the leading ginger producing state in Nigeria. The ginger rhizomes were peeled, sun-dried and ground. The powder samples were passed through a 1.4 mm sieve. The obtained powders were packed in aluminium foil and stored under refrigerated condition until analysis.
Extraction
The extraction was done in a conical flask placed on temperature controlled magnetic stirrer (UC 152, Bibby Scientific, UK). The stirrer speed was set at scale 3. Ethanol was then introduced into the conical flask. The flask was covered with aluminium foil to minimize light penetration. To ensure the accuracy of the extraction temperature, a temperature controller (SCT 1, Bibby Scientific, UK) was placed inside the conical flask and connected to the temperature controlled magnetic stirrer. Once the required extraction temperature was reached, the required weight of blended powder sample of tea-ginger (2:1) was introduced into the conical flask. Teaginger (2:1) powder was selected after some preliminary investigation which revealed that the tea-ginger (2:1) powder had a higher total flavonoid content compared to the tea-ginger (1:1) and tea-ginger (1:2) extracts. The extraction was continued until the required extraction time was achieved. The extract was then filtered to remove the residues.
Response Surface Methodology
A face centered central composite design with three independent variables was used. The design consisted of 20 experiments: 8 factorial points, 6 axial points and 6 central points. The range of the independent variables investigated were: extraction temperature (TEM: 30-70 o C), powder to solvent ratio (CON: 0.12-2.10 g/100 ml), extraction time (TIM: 5-90 min). The response variables consisted of selected antioxidant properties of the extracts. The antioxidant properties were: total flavonoid content (TFC), total phenol content (TPC), 2,2′azinobis (3-ethylbenzothiazoline sulfonate (ABTS) radical activity, diphenyl-picrylhydrazyl (DPPH) radical activity, peroxide scavenging activity (PSA) and iron chelating activity (ICA). Data were fitted to different models. Models considered were linear, 2 factor Interaction and quadratic. Analysis of variance (ANOVA) was carried out to select the best model. The best model selected was further subjected to backward regression to remove redundant variables. Both single response and multiresponse optimisation were done using the desirability concept. The optimisation was set to maximise all the antioxidant properties and the process conditions were set to be within the experimental range. The antioxidant properties were all given an equal weighting of 1 for the optimisation. The quality of the model was determined by evaluating the lack-of-fit, the coefficient of determination (R 2 ), Adjusted R 2 , Predicted R 2 , and adequate precision.
Prediction of Antioxidant Properties from Colour and Hue Index Properties of the Extract
Colour (CIE L*, a*, b*), sample absorbance at 510 nm (A510) and 610 nm (A610) of the extracts were determined. L* is a measure of lightness with value ranging from 0 to 100. The a* and b* are chromaticity coordinates. From the a* and b* values, the hue and chroma of the extract were estimated. The hue index value was also estimated from A510 and A610. The hue index has been used in the caramel industry as an indicator of its colour [14]. The suitability of hue index in evaluating colour of tea has also been reported [15]. A multivariate regression was conducted on the obtained data. The dependent variables were the antioxidant properties. The independent variables were: L*, a*, b*, hue, chroma, A510, A610, A510/A610 and hue index. The multivariate statistics used were: ordinary least square regression (OLSR), principal component regression (PCR) and partial least square regression (PLSR). The data were scaled and centered before running the regression analysis. In the PCR analysis, the regression was run for components that explain between 90 to 99% of the variation in the independent variables. The dependent variables were also subjected to some transformation (log 10 , square root and inverse square root) to check if it improves the quality of the model.
Antioxidant Analysis
ABTS was assayed using the improved technique of Miliauskas et al. [16], as described by Spradling [17]. A phosphate buffer solution (PBS) was prepared by mixing 95 ml of sodium phosphate monobasic (2.98 g 100 ml -1 ) and 405 ml of sodium phosphate dibasic (15.6 g 500 ml -1 ), followed by 8.04 g of sodium chloride and filled to volume (1 l), lastly the pH was adjusted to 7.4 with 2M NaOH. The ABTS mother solution was prepared by mixing 44.8 mg of ABTS, 8.12 mg potassium persulfate, and 20 ml of distilled water. The solution was allowed to react in the dark for 12 h. The ABTS working solution was prepared by mixing 145 ml of PBS with 5 ml of the ABTS mother solution. Trolox was used as standard. To 2900 μl of the ABTS working solution, 100 µL of each extract or standard was added and allowed to react for 15 min before reading spectrophotometrically (Spectrumlab 23A, England) at 734 nm against a blank solution. DPPH was measured as described by Sompong et al. [18]. The reaction mixture contained 1.5 ml DPPH working solution (4.73 mg of DPPH in 100 ml ethanol HPLC-grade) and 300 µl extract. The mixture was shaken and left to stand for 40 min in the dark at room temperature. The absorbance was read at 515 nm relative to the control (as 100%) using a spectrophotometer. The percentage of radical-scavenging ability was calculated by using the formula: where A control = Absorbance at 515 nm of control, A sample = Absorbance at 515 nm of sample.
Iron chelating activity was measured by the method of Dinis et al. [19] as described by Ozena et al. [20]. The samples were added to a solution of 2 mM FeCl 2 (0.05 ml). The reaction was initiated by the addition of 5 mM ferrozine (0.2 ml) and the mixture was shaken vigorously and incubated at room temperature for 10 min. The absorbance of the resulting solution was then measured at 562 nm. The iron chelating activity was calculated by the given formula: where A control = Absorbance at 562 nm of control, A sample = Absorbance at 562 nm of sample.
Peroxide scavenging activity was measured by the method of Smirnoff and Cumbes [21] as described by Ozena et al. [20]. Peroxide radicals were generated by mixing of FeSO 4 and H 2 O 2 .
The reaction mixture contained 1 ml FeSO 4 (1.5 mM), 0.7 ml H 2 O 2 (6 mM), 0.3 ml sodium salicylate (20 mM) and appropriate volume of extracts. This was followed by incubation for 1 h at room temperature. The absorbance of the hydroxylated salicylate complex was measured at 562 nm. The percentage scavenging activity was calculated as: The peroxide scavenging activity (%) = Where A 0 is the absorbance of the control (without extract or standards), A 1 is the absorbance including the extract or standard and A 2 is the absorbance without sodium salicylate.
Total flavonoid content was measured as described by Prommuaka et al. [22]. A 0.5 ml of the extracted samples or catechin solutions was mixed with 1.5 ml of 95% ethanol (v/v), 0.1 ml of 10% aluminum chloride -AlCl 3 .6H 2 O (m/v), 0.1 ml of 1 M of potassium acetate, and 2.8 ml of distilled water, and the mixture was incubated at room temperature for 30 min. The absorbance of the mixture was then measured against a blank using a spectrophotometer at 415 nm. The blank contained all the reagents except the extract. Catechin was used as standard.
Total phenol was measured as described by Waterhouse [23], using the method of Slinkard and Singleton [24]. From the calibration solution, extract, or blank, 50 µl volume was taken and added to 1.58 mL water, and 100 µl of Folin-Ciocalteu reagent, and mixed well. After 8 min, 300 µl of the sodium carbonate solution was added. The solutions were left at room temperature for 1 h and absorbance of each solution was determined at 765 nm against the blank. The sodium carbonate solution was prepared by dissolving 200 g of anhydrous sodium carbonate in 800 ml of water and brought to boil. After cooling, a few crystals of sodium carbonate powder were added. After 24 h, the solution was filtered and made up to 1 l. Gallic acid was used as standard.
Colour and Hue Index Analysis
Colour was measured with a spectrophotometer CM-700d (Konica Minolta Sensing). The spectrophotometer was calibrated against a white plate. The extracts were placed in a cuvette for the measurement. The CIE L*, a* and b* values were read from the spectrophotometer. Readings were taken in triplicate. Hue was calculated as θ using eq. 4.
The hue index was calculated from eq. 10.
Hue index= (10*log (A510/A610)) The A610 and A510 values were determined by measuring the absorbance of the extracts against a distilled water blank in a spectrophotometer.
Software
The response surface analysis was carried out using Design Expert v 7.0.0 (Stat-Ease). The multivariate statistics was done with XLSTAT Pro, 2013 (Addinsoft) Table 1 shows the regression parameters for the extraction of antioxidants. The very low P-values (P < 0.05) demonstrate that all the antioxidant extraction models were significant. An insignificant lack-of-fit (P > 0.05) indicates that the entire extraction model fits the data well. The R 2 is a measure of the ratio of the mean sum of squares to total sum of squares (MSS/TSS). This gives a measure of the amount of variation in the data explained by the model. A value closer to one indicates that the model has a very good fit. However, the R 2 tends to increase with an increase in the number of variables in the model. With this situation a case may arise such that a model may have a very high R 2 but a low predictive quality. To compensate for the weakness in R 2 , another parameter used to assess a model is the adjusted R 2 . The adjusted R 2 does not necessarily increase with an increase in the number of variables in the models. The adjusted R 2 only increases if the new variable added to the model has a significant contribution to the model. The R 2 for the extraction models in this investigation range from 0.5410 to 0.9212 and the adjusted R 2 range from 0.4550 to 0.8848. This means that the extraction model with the least adjusted R 2 was able to explain 45.5% of the extraction process. The adjusted R 2 from this study indicates that the parameters included in the different extraction models could explain the various extraction process but this does not in any way tell us about the predictive ability or quality of the model. The predicted R 2 is a measure of the predictive quality of model. It measures the ability of a model to predict from new data. From Table 1, it is observed that the predictive quality of TPC and ABTS are very low with predicted R 2 of 0.1271 and 0.1779, respectively. The extraction models for TFC, PSA. ICA and DPPH have good predictive quality, with predicted R 2 of 0.8325, 0.5379, 0.6333 and 0.7396, respectively. The adequate precision is a measure of signal to noise ratio of the model. It is a measure that indicates if the model generated can be used to navigate the design space. The Design Expert software suggested that a value greater than 4.0 can be used to navigate the design space.
Single Response Optimisation
A square root transformation was used for the extraction model for TFC, as it gave a better model (R 2 = 0.8982, adjusted R 2 = 0.8791, predicted R 2 = 0.8325) compared to the untransformed model (R 2 = 0.8326, adjusted R 2 = 0.8129, predicted R 2 = 0.7545). Similar pattern was also observed in the TPC models and a square root transformation was found to give the best model. Temperature, concentration and quadratic effect of concentration had significant influence (P < 0.05) on the extraction of flavonoids. Temperature, concentration and interaction of temperature and concentration had significant influence on extraction of phenolics.
The ABTS of the extract was influenced by temperature, concentration, time and quadratic effect of temperature. Temperature, concentration, time, temperature-concentration interaction, temperature-time interaction and quadratic effect of temperature had significant influence on peroxide scavenging activity of the extracts. The iron chelating activity of the extracts was significantly influenced by temperature, concentration, temperatureconcentration interaction and quadratic effect of temperature. Temperature, concentration, time, temperature-concentration interaction, concentration-time interaction and quadratic effect of concentration had significant influence on the DPPH activity of the extracts. A look at the regression coefficients for all the extraction models indicated that concentration had the most significant impact on the antioxidant property of the extracts.
The response surface graphs for the models are shown in Fig. 1. From Figs. 1a and c, the optimum temperature to maximise TFC and ABTS are around 60°C and 50°C, respectively. A numerical optimisation approach using the desirability factor approach was used to obtain the optimum conditions for the antioxidant extractions (Table 2). Using a central composite design model [26], the optimal conditions for extraction of total flavonoid from green tea using the desirability function was achieved at the extraction time of 32.5 min, ethanol concentration of 100% (v/v) and solid-to-liquid ratio of 1:32.5 (m/v). This extraction was performed at the boiling point of ethanol. In our study, the optimum condition for extraction of total flavonoids from tea-ginger was 58.14°C, 2.10 and a time of 9.99 min. The desirability for the single response optimisation extraction was defined to maximise each of the antioxidant properties in this study.
Multi-Response Optimisation
The different antioxidant properties have varied optimum regions (Table 2). This has an implication such that a particular antioxidant property might have reached its maximum and begins to degrade, whereby another antioxidant property may just start approaching its maximum.
To resolve this kind of issue, the multi-response optimisation using the desirability approach was used. The desirability for the multi-response optimisation was defined to maximise all the antioxidant properties. The process variables were set to be within the range used in the investigation. The response surface graph for the multi-response optimisation is shown in Fig. 2.
The required process condition for the multiresponse optimisation is a temperature of 50.16°C, concentration of 2.1 g 100 ml -1 and time of 5 min. This process condition resulted in a desirability of 0.68.
The confirmation run was done at 50.00°C, concentration of 2.1 g 100 ml -1 and time of 5 min. The temperature was approximated to the nearest whole number by taking into account the operating convenience of the temperature controller. The values obtained from the confirmation runs were similar to the predicted values (Table 3).
Prediction of Antioxidant Properties from Colour and Hue Index Properties of the Extract
The OLSR, PCR and PLSR were run for all the antioxidant properties and a comparative analysis was done between the regression models. Multivariate model obtained by multiple linear regressions has been used for the prediction of antioxidant properties [11]. The PCR model having the highest adjusted R 2 was selected as the most preferred model among the PCR models. A sample of a PCR analysis is shown in , because the independent variables are selected in such a way that these selected variables are also able to explain the dependent variables [28]. The OLSR could be regarded as an all inclusive supervised technique because all the measured independent and dependent variables are involved in building the regression model. The OLSR extraction models of TPC and PSA had a good R 2 of 0.755 and 0.789, respectively, but a low Q 2 of 0.068 and -0.530, respectively. This infers that we have an OLSR model that is able to predict the current data well but has poor predictive quality with new data. This is a demerit of the OLSR technique. Hence, there is need to use other quality parameters (other than R 2 ) to assess the quality of regression models. The PLSR models had the lowest RMSE for the TFC and DPPH prediction and the OLSR model had the lowest RMSE for the prediction of ICA (Table 5). In terms of model simplicity, the PCR and the PLSR gave the most parsimonious models. One of the considerations in model building is simplicity. In most cases, the rule of Occam's Razor, which states that the simpler explanation is the preferable one, is very useful, and is now applied to data analysis or data mining techniques in building models [27]. The PCR was able to predict TFC with a combination of: a*, hue index, hue, though with a very high RMSE of 1404.897 mg CE l -1 . The PLSR was able to predict TFC with a combination of: a*, A510, hue index with a low RMSE of 552.706 mg CE l -1 .
Pace et al. [11], were able to predict the antioxidant activity and total phenol content of pigmented carrots using a regression equation built from L*, a* and b* properties of the carrots. Also Wold [13] reported that colour measurements of intact tomatoes can be used as a non-destructive method to assess total antioxidant capacity of tomatoes. They reported that a high negative correlation existed between high values of L*, b* and ferric reducing ability of plasma (FRAP), and a high positive correlation between a*, hue, a*/b and FRAP values. A comparative analysis of the 3 regression techniques revealed the PLSR models as the most preferred model due to its higher R Q 2 , and low RMSE and less number of 208 ferric reducing ability of , and a high positive correlation between a*, hue, a*/b and FRAP values. A comparative analysis of the 3 regression techniques revealed the PLSR models as the igher R 2 , higher , and low RMSE and less number of independent variables in the model. We have chosen the word preferred and not the best models because all the models have their merits and demerits. For example a look at the ICA extract models showed that the PLSR gave the highest R 2 and Q 2 , the OLSR gave the lowest RMSE.
CONCLUSION
This work has identified optimum conditions for extraction of antioxidants from tea-ginger (2:1) blend using ethanol. Rapid procedures that could be useful for predicting the TFC, DPPH and ICA of ethanolic tea-ginger (2:1) extract have also been identified. A comparative analysis of the regression techniques indicated that the PLSR gave the most preferred models.
CONSENT
Not applicable.
ETHICAL APPROVAL
Not applicable. | v3-fos |
2019-04-24T13:07:17.753Z | {
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} | s2 | EVALUATION OF QUALITY PROTEIN MAIZE AND DROUGHT TOLERANT MAIZE IN FAR WESTERN HILLS OF NEPAL
The coordinated varietal trial of quality protein maize was carried out in research station of regional agricultural research station, Doti during 2011/12 and 2012/13. A total of fourteen entries including standard and local checks were included in the experiment of both years. Out of tested entries of the year 2011/12, S99TLYQ-A produced the highest grain yield (2814 kg/ha) followed by SO3TLYQ-AB-01 (2765 kg/ha) and SO3TLYQ-AB-02 (2293 kg/ha). MSTATC software was used in data analysis. Flowering days, plant height and grain yield due to genotypes was found significant at 1-5% level. Amongst the tested entries include in the experiment of the year 2012/13, S99TLYQ-AB recorded the highest grain yield (6006 kg/ha) followed by SO3TLYQ-AB-01 (5409 kg/ha) and S99TLYQ-A (5330 kg/ha). There was significant difference in flowering days, plant height, ear height and grain yield due to genotypes at 1-5 % level. Combined analysis over year was also carried out. Genotypes SO3TLYQ-AB-01 (4087 kg/ha), S99TLYQ-A (4072 kg/ha) and S99TLYQ-AB (3750 kg/ha) identified as promising genotypes for lower hills and river basin agro-environment of far western hills. Similarly, experiment on selection of drought tolerance maize genotypes was also carried out at this research station during 2011/12 and 2012/13. Total 105 genotypes of maize were planted for selection of drought tolerance maize genotypes during 2011/12 and it was replicated twice. Quantitative and qualitative traits were recorded for screening against drought. Out of the genotypes included in the experiment, Manakamana-4 was found super variety in terms of grain yield (4561 kg/ha), leaf senescence (1) and leaf roll (1). Statistically, plant height, ear height, flowering days and leaf senescence due to genotypes were found significantly different. Total eight genotypes were selected for second year's drought tolerance genotypes selection experiment and were planted in three replications. Out of the eight genotypes included in the experiment, Upahar (6897 kg/ha), TLBRSO7F16 (6216 kg/ha) and BLSBSO7F12 (6215 kg/ha) were found superior in terms of grain yield.
Introduction
Maize comes in third position in cultivated area coverage after wheat and rice in far western hills of Nepal. Maize cultivation is a way of life for most farmers in the hills of Nepal. It is a traditional crop cultivated as food, feed and fodder on slopping Bari land (rainfed upland) in the hills. It is grown under rainfed conditions during the summer (April-August) as a single crop or relayed with millet later in the season.
The centre of origin of maize is the Mesoamerican region, probably in the Mexican highlands, from where it spread rapidly. Archaeological records and phylogenetic analysis suggest that domestication began at least 6,000 years ago (Piperno and Flannery 2001;Matsuoka et al., 2002). Maize spread around the world after European discovery of the Americas in the 15 th century, particularly in temperate zones (Paliwal 2000;Farnham et al., 2003).
Maize can be grown in a number of environments (Farnham et al., 2003) from 58° North to 40° South. Generally, tropical maize is grown between 30°North and 30° South, subtropical maize between 30 and 34° both North or South, and temperate maize beyond 34° latitudes. It can be grown in a range of altitudes from sea level up to 3,800 meters and with growing seasons between 42 and 400 days. This ability to grow in a wide range of environments is reflected in the high diversity of morphological and physiological traits (Edmeads, 2008).
Maize grain yields in the temperate developed world average 8.2 ton/ha, vs. 3.5 t/ha in tropical less developed countries. In both production environments drought is the most important abiotic stresses constraining and destabilizing maize grain production, and is one of several reasons for the differences between mean production levels of temperate vs. tropical regions. In both regions water deficits occur unpredictably throughout the season. Withinfield variability in soil texture and depth means that plant-Research Article available soil water also varies, and this can result in yield variation of up to 10-fold in a relatively dry year. Since farmers usually plant a single variety in any given field, this implies a need for a good level of drought tolerance in the large majority of hybrids and varieties grown under rainfed conditions. An investigation on chemical composition of QPM and normal maize was carried out by Martinez et al. (1996). It was reported that QPM contained higher amounts of protein, fat, dietary fibre and ash (10.56, 3.56 and 1.72%, respectively) when compared to normal maize (10.44, 2.66 and 1.28%, respectively). Starch content was lower in QPM than normal maize (82.6 Vs 84.2%). However, QPM showed better lysine content (42 g/kg of protein) than normal maize (35 g/kg of protein). Similar higher lysine content of QPM varieties was reported by Ahenkora et al. (1999). A range 3.7 to 4.2 g/100 g of protein was reported which was significantly higher than that of normal maize (2.6 to 3.1 g/100 g protein). A comparative study on QPM cultivars and commercial maize varieties was conducted by Zarkadas et al. (2000). It was observed that QPM genotypes contained high levels of lysine (3.43 to 4.21 g/100 g of protein) compared to commercial maize varieties which ranged from 2.9 to 3.1 g/100 g of protein.
Study site description
Coordinated varietal trial of quality protein maize was carried out in research station of Regional Agricultural Research Station, Doti during 2011/12 and 2012/13. The experiments was located at altitude of 610 m above mean sea level on 29 o 15' north latitude and 80 o 55' east longitudes. The soil was light texture, low organic matter (1-2 %) and acidic in nature containing pH 6.
Similarly, the experiment on selection of drought tolerance maize genotypes was also carried out at RARS, Doti during 2011/12 and 2012/13. Total 105 genotypes of maize were planted for selection of drought tolerance maize genotypes during 2011/12 and it was replicated twice. This experiment was also planted during 2012/13, but only eight genotypes which were selected from first year's (2011/12) experiment were planted with three replication. These selected genotypes includes TLBRSO7F16, Upahar, Manakamana-4, RPOP-3, ACROSS 9331, BLSBSO7F10, BLSBSO7F12 and TLBRSO7F14. The experiment was conducted in RCBD and spacing of 75 cm x 25 cm was maintained. Fertilizer was applied @120:60:40 NPK kg/ha with basal application@60:60:40 NPK kg/ha. Quantitative data such as tasseling days, silking days, plant height, ear height and grain yield (kg/ha) were recorded.. Besides this, for indirect selection to drought tolerance, qualitative data such as tassel size, leaf senescence and leaf rolling were also recorded in both the year.
Data Analysis
Analysis of variance for grain yield and other ancillary characters of maize were analyzed with MSTATC software. Treatments (variety) were compared using the "F-test" and any significant differences between treatments were compared by Least Significant Difference (LSD) at 5% level of probability.
Results and Discussion
In coordinated varietal trial of quality protein maize; out of tested fourteen entries of the year 2011/12, S99TLYQ-A produced the highest grain yield (2814 kg/ha) followed by SO3TLYQ-AB-01 (2765 kg/ha) and SO3TLYQ-AB-02 (2293 kg/ha). Statistically, the difference in flowering days, plant height and grain yield due to genotypes was found significant at 1-5% level (Table 1). Amongst the tested entries include in the experiment of the year 2012/13, S99TLYQ-AB recorded the highest grain yield (6006 kg/ha) followed by SO3TLYQ-AB-01 (5409 kg/ha) and S99TLYQ-A (5330 kg/ha). Statistically significant difference was observed in flowering days, plant height, ear height and grain yield (Table 2). Combined analysis over year was also carried out and genotypes SO3TLYQ-AB-01 (4087 kg/ha), S99TLYQ-A (4072 kg/ha) and S99TLYQ-AB (3750 kg/ha) were identified promising for lower hills and river basin agroenvironment of far western hills. Statistically, effect of treatment and year for grain yield was found significant at 1% level whereas interaction effect of treatment and year was found non-significant from combined analysis over year (Table 3 and Figure 1).
Similarly, out of the genotypes included in the first year of drought tolerance maize experiment (2011/12), Manakamana-4 was found super variety in terms of grain yield (4561 kg/ha), leaf senescence (1) and leaf roll (1). Out of 105 genotypes, only eight genotypes were selected for next year's drought tolerance genotypes selection experiment and data of only these selected eight genotypes have been presented in the following tables of the FY 2011/12. Statistically, difference in tested traits like plant height, ear height, flowering days and leaf senescence due to genotypes was found significant whereas difference in leaf rolling, tassel size and grain yield due to genotypes was found non-significant (Table 4 and Table 5).
Out of the eight genotypes included in experiment of the year 2012/13, Upahar (6897 kg/ha), TLBRSO7F16 (6216 kg/ha) and BLSBSO7F12 (6215 kg/ha) genotypes were found superior in terms of grain yield. Statistically, the difference in tested traits like plant height, ear height, leaf rolling, leaf senescence, tassel size and grain yield due to genotypes was found non-significant (Table 6 and Table 7).
Conclusion
Selection of high yielding genotypes for a particular location is the most important task in varietal development program. The QPM genotypes namely SO3TLYQ-AB-01, S99TLYQ-A and S99TLYQ-AB were identified as promising genotypes for lower hills and river basin agroenvironment of far western hills. Similarly, maize genotypes UPAHAR, TLBRSO7F16 and BLSBSO7F12 were identified as better drought tolerant genotypes. These experiments need to be continued for one more year for final conclusion. | v3-fos |
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} | s2 | Designer synthetic media for studying microbial-catalyzed biofuel production
Background The fermentation inhibition of yeast or bacteria by lignocellulose-derived degradation products, during hexose/pentose co-fermentation, is a major bottleneck for cost-effective lignocellulosic biorefineries. To engineer microbial strains for improved performance, it is critical to understand the mechanisms of inhibition that affect fermentative organisms in the presence of major components of a lignocellulosic hydrolysate. The development of a synthetic lignocellulosic hydrolysate (SH) media with a composition similar to the actual biomass hydrolysate will be an important advancement to facilitate these studies. In this work, we characterized the nutrients and plant-derived decomposition products present in AFEX™ pretreated corn stover hydrolysate (ACH). The SH was formulated based on the ACH composition and was further used to evaluate the inhibitory effects of various families of decomposition products during Saccharomyces cerevisiae 424A (LNH-ST) fermentation. Results The ACH contained high levels of nitrogenous compounds, notably amides, pyrazines, and imidazoles. In contrast, a relatively low content of furans and aromatic and aliphatic acids were found in the ACH. Though most of the families of decomposition products were inhibitory to xylose fermentation, due to their abundance, the nitrogenous compounds showed the most inhibition. From these compounds, amides (products of the ammonolysis reaction) contributed the most to the reduction of the fermentation performance. However, this result is associated to a concentration effect, as the corresponding carboxylic acids (products of hydrolysis) promoted greater inhibition when present at the same molar concentration as the amides. Due to its complexity, the formulated SH did not perfectly match the fermentation profile of the actual hydrolysate, especially the growth curve. However, the SH formulation was effective for studying the inhibitory effect of various compounds on yeast fermentation. Conclusions The formulation of SHs is an important advancement for future multi-omics studies and for better understanding the mechanisms of fermentation inhibition in lignocellulosic hydrolysates. The SH formulated in this work was instrumental for defining the most important inhibitors in the ACH. Major AFEX decomposition products are less inhibitory to yeast fermentation than the products of dilute acid or steam explosion pretreatments; thus, ACH is readily fermentable by yeast without any detoxification. Electronic supplementary material The online version of this article (doi:10.1186/s13068-014-0179-6) contains supplementary material, which is available to authorized users.
Background
Increasing fossil fuel utilization by industrialized economies has been a subject of intense debate within the scientific and political communities worldwide. Increasing energy demand, depleting petroleum reserves, the negative environmental repercussions due to increased greenhouse gas (GHG) emissions, and the control of fossil fuel production by a limited number of nations are among the main reasons why there is an ongoing effort to restructure our energy sector towards greater sustainability via utilization of renewable sources of energy [1].
Lignocellulosic biofuels are projected to play a substantial role in the replacement of current-generation fossil-derived liquid fuels such as gasoline and diesel [2,3]. In second generation biorefineries, ethanol production from lignocellulosic substrates involves enzymatic digestion of cellulose and hemicellulose sugar polymers into fermentable sugars, which can be converted to ethanol during microbial fermentation. However, the plant cell wall structure has naturally evolved to be highly recalcitrant to enzymatic deconstruction by fungi and bacteria. In order to improve enzyme accessibility to the polysaccharides embedded in plant cell walls, some form of pretreatment is necessary to reduce biomass recalcitrance to enzymatic hydrolysis.
Among the pretreatment technologies available today, thermochemical pretreatments are considered to be the most promising [4]. Most of these pretreatment processes use either acids (such as sulfuric acid, phosphoric acid, and maleic acid) or bases (such as ammonium hydroxide, sodium hydroxide, and potassium hydroxide) to pretreat plant cell walls, often resulting in the formation of cell wall-derived decomposition products that can inhibit both enzymes and microbes [5][6][7].
Ammonia Fiber Expansion (AFEX™) a is a well-established pretreatment technology that utilizes concentrated ammonia at relatively low temperatures (60 to 140°C) and short residence times (5 to 45 min) to pretreat biomass [4]. AFEX has proven to be particularly effective on monocot-based grasses (for example, corn stover), improving cellulose hydrolysis rates by up to fivefold and generating highly fermentable hydrolysates [8]. Moreover, AFEX produces much lower concentrations of sugar-derived decomposition products compared to acidic pretreatments, while preserving the native nutrient content for more efficient fermentation [9][10][11]. Therefore, AFEX-based biomass hydrolysates do not require detoxification, exogenous nutrient supplementation, and extensive water washing of the pretreated substrate for efficient glucose fermentation by yeast or bacteria [10,12]. However, the efficiency of xylose consumption during co-fermentation of AFEX pretreated biomass hydrolysates (enriched in both pentoses and hexoses) still requires improvement. Some of the issues faced during mixed hexose/pentose fermentation are the low xylose consumption rate and the lower ability of yeast and bacteria to co-ferment hexose/pentose mixtures [10,12]. Our recent work in Escherichia coli KO11 and Saccharomyces cerevisiae 424A (LNH-ST) demonstrated that the xylose consumption rate is related to the presence of pretreatment-derived biomass decomposition products, ethanol, and other fermentation metabolites [13]. In the case of E. coli KO11, the ability to consume xylose from AFEX hydrolysate was severely affected by the presence of pretreatment-derived biomass degradation products in combination with high concentrations of ethanol. On the other hand, a 22% reduction of cell growth and 13% reduction of specific xylose consumption rate was observed for S. cerevisiae 424A (LNH-ST) due to the presence of AFEX decomposition products in the hydrolysate. However, very little is known about the nature of pretreatment-based biomass decomposition products that inhibit xylose consumption, their mechanism of action, and their overall effect on the metabolism of sugars by yeast and bacteria. Answering these questions is an important step toward developing new microbial strains with improved performance on lignocellulosic hydrolysates, and hence increasing the economic competitiveness of liquid biofuels as a viable substitute to conventional gasoline and diesel.
One approach for gaining a deeper understanding of the interactions between inhibitory components present in biomass hydrolysates and microorganisms, including inhibition synergies, levels of inhibition, and metabolic effects, involves using a synthetic medium that mimics the composition of authentic lignocellulosic hydrolysates, that is, a synthetic hydrolysate (SH). The importance of such SHs for these studies is supported by the work published by [10], who observed that the inhibition of xylose fermentation is closely dependent on the nutrient availability in the culture medium. The formulation of an SH will enable the inclusion of precisely defined positive and negative controls in experimental designs, which represent a current limitation of directly using complex lignocellulosic hydrolysates. Also, using an SH will allow the manipulation of relative concentrations and ratios between the different components of the hydrolysate, according to the objective of each study. Furthermore, the SH will facilitate the integration of isotope-labeled components in the medium (for example, 13 C-labeled xylose or glucose) to conduct metabolomics-based experiments, aiming to trace potential deviations in the metabolic flux during xylose consumption in the presence and absence of compounds of interest.
In this work, we have attempted to establish a platform for conducting the above-mentioned studies, by characterizing a highly complex lignocellulosic hydrolysate derived from AFEX pretreated corn stover (AFEX-CS) and formulating a well-defined SH using both commercially available and custom-synthesized reagents/chemicals. This SH platform was also implemented here to screen the effect of different classes of AFEX pretreatment-based biomass decomposition products on xylose fermentation using a recombinant S. cerevisiae 424A (LNH-ST) strain.
Biomass
Corn stover (CS) was harvested at Field 570-C Arlington Research Station, University of Wisconsin, in the year 2008. Pioneer 36H56 (triple stack -corn borer/rootworm/ Roundup Ready) seeds were used for planting. The CS sample containing leaves, stem, and cobs was dried to < 8% moisture (dry weight basis) using a 60°C oven and milled to 4-mesh size and stored in sealed polythene bags at room temperature until further use. The composition of the untreated corn stover (UT-CS) was 35.7% glucan, 21.2% xylan, 2.6% arabinan, 17.4% lignin, 5.9% ash, and 2.4% acetyl content. AFEX pretreatment was carried out using the procedure reported by Balan et al. [14]. The pretreatment condition in this study was 1:1 ammonia to biomass ratio (dry weight), 60% moisture loading, and 140°C for 15 min total residence time. After pretreatment, the residual ammonia was allowed to evaporate in the hood overnight, before being bagged and stored at 4°C prior to further usage. The composition of the pretreated biomass did not change appreciably as a result of the AFEX pretreatment [15].
Feruloyl amide
Feruloyl amide was synthesized via a one-step ammonolysis reaction, using ethyl 4-hydroxy-3-methoxycinnamate (AK Scientific, Inc., Mountain View, CA, USA) as the starting reagent. For the reaction, 3 g of ethyl 4-hydroxy-3methoxycinnamate was dissolved in 150 mL of 28 to 30% ammonium hydroxide (EMD, Gibbstown, NJ, USA) solution in a high pressure reactor (HEL, Inc., Lawrenceville, NJ, USA). The reaction was carried out at 100°C for 5 h at 300 rpm mixing speed. Under these conditions feruloyl amide was the major product, followed by ferulic acid. The purification and recovery of feruloyl amide were conducted by preparative-scale HPLC using a Waters XBridgeTM Prep C18 column (5.0 μm, 10 mm × 100 mm; Waters Co., Milford, MA, USA). The HPLC system was equipped with a Waters 600 Controller, Waters Delta 600 pump, and a Shimadzu SPD-M10A VP Diode Array Detector and connected to a Waters Fraction Collector (Waters Co., Milford, MA, USA). The solution was diluted to about 5 g/L of feruloyl amide in methanol before injection. The injection volume was 5 mL, and the HPLC flow rate was 0.25 mL/min using the gradient shown in Table 1. The feruloyl amide enriched fractions were freeze dried to a powder and stored in a desiccator. Fractions with purity >95% (as determined by LC-MS and described in Chundawat et al. [15]) were pooled together before utilization in fermentation experiments.
Coumaroyl amide
Coumaroyl amide was also synthesized using the same methodology as for feruloyl amide. However, in this case the ammonolysis reaction was carried out on methyl 4hydroxycinnamate (Frinton Laboratories, Inc., Vineland, NJ, USA). The purification and recovery of coumaroyl amide were performed using the same methodology as for feruloyl amide. Fractions with purity >95% (as determined by LC-MS and described in Chundawat et al. [15]) were pooled together before utilization in fermentation experiments.
Xylo-oligomers
The xylo-oligomer mixture was produced by enzymatic hydrolysis of Birchwood xylan (Sigma-Aldrich, St. Louis, MO, USA) using an NS50014 series endoxylanase enzyme (5 mg/mL protein concentration estimated by the Kjeldahl method) provided by Novozymes (Davis, CA, USA). The enzyme composition and substrate specificity for this cocktail have been provided elsewhere [16]. The reaction was carried out at 6% (w/v) solids loading in a 250-mL flask at pH 4.8 (0.05 M phosphate buffer), 150 rpm, and 50°C for 48 h, with an enzyme loading of 2 mL/g xylan. The supernatant was separated from the undissolved solids by centrifugation to further isolate the soluble xylo-oligomers. A Thermo Scientific Hypersep Hypercarb PGC 453 column (Thermo Scientific, Bellefonte, PA, USA) was employed to separate the xylo-oligomers from other soluble products. The column was first conditioned with 30 mL of methanol followed by 30 mL of distilled water. A sample volume of 3.5 mL of hydrolysate was then added to the column. After washing the column with 45 mL of water, 60 mL of methanol was added to elute xylo-oligomers from the sorbent. Methanol was removed using a rotary evaporator (BUCHI, Switzerland) and the xylo-oligomer solution was adjusted to appropriate concentrations in distilled water.
Other chemicals
With the exception of the compounds described above, all other chemicals used in the SH were purchased from various commercial vendors: formic acid, 4hydroxybenzaldehyde, trans-aconitic acid, vanillic acid, and vanillin were obtained from Fluka (St. Louis, MO, USA), and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cellobiose was used as the only gluco-oligomeric component.
Preparation of AFEX-CS hydrolysate (ACH)
AFEX-CS was enzymatically hydrolyzed with a commercial enzyme mixture as previously described [10]. Compositional analysis of the ACH Glucose, xylose, arabinose, acetate, formate, and lactate in this hydrolysate mixture were analyzed using an HPLC system equipped with a Bio-Rad Aminex HPX-87H column (Bio-Rad Co., Hercules, CA, USA) as previously described [12]. The mobile phase was 5 mM H 2 SO4 at a flow rate of 0.6 mL/min, and the column temperature was maintained at 50°C. Oligosaccharides were determined by acid hydrolysis following the NREL protocol (LAP-014; www.nrel.gov/ biomass/analytical_procedures.html), and the monomeric sugars produced after acid hydrolysis were quantified using HPLC (LAP-002). Protein-derived amino acids quantification was conducted on an LC-MS system in the Department of Biochemistry and Molecular Biology at Michigan State University. The analytical methodology details have been reported elsewhere [17]. For total amino acids analysis, 100 μL of CS hydrolysate was hydrolyzed with 1 mL 6 M HCl at 110°C overnight and then dried under vacuum (SpeedVac, Eppendorf, Germany). The hydrolyzed dry sample was solubilized in 10 mL of water. Valine-d8 (1 μM) was added into the solution as an internal standard. For free amino acid analysis, the same procedure was followed with the exception of the 6 M HCl hydrolysis step.
Protein and ammonium nitrogen contents in the biomass were determined using Kjeldahl assays and a Timberline TL-1800 ammonia analyzer, respectively, at Dairy One Cooperative Inc. (Ithaca, NY). Nitrogen incorporated in the biomass during ammonolysis reactions was estimated by subtracting the total nitrogen (w/w) present in AFEX-CS from the nitrogen in UT-CS, as described previously [15].
Trace element analysis was carried out with inductively coupled plasma mass spectrometry (ICP-MS) in the ICPMS & XRF Laboratory at Michigan State University [18]. Approximately 1 mL of liquid sample was digested on a hot plate, sub-boiling in acid-cleaned Teflon Savillex beakers using 1.9 mL Optima nitric acid and 0.1 mL trace metal clean hydrofluoric acid for 24 h. After digestion, 250 μL of trace metal clean 30% hydrogen peroxide was added, and the sample was evaporated to near dryness on a hotplate. Samples were then brought up to final volume with 5 mL of 2% Optima nitric acid: visual inspection showed a complete digestion of all samples. This solution was run in the ICP-MS for full mass scan analyses. For the major element analysis, potassium (K), magnesium (Mg), calcium (Ca), phosphorus (P), and sodium (Na) samples were diluted 1:300 prior to analysis. For the trace element analysis, cobalt (Co), copper (Cu), manganese (Mn), zinc (Zn), and iron (Fe) samples were run without dilution.
Organic acids and aromatic aldehyde/ketone analyses were conducted by LC-MS/MS at Baylor University. Instrumentation and details of the applied methodology have been published elsewhere [19]. The nitrogenous compounds were identified and quantified by LC-MS/ MS and GC-MS for AFEX-CS hot water extracts as reported previously by Chundawat et al. [15]. The composition of the ACH is presented in Table 2.
Microorganism and seed culture S. cerevisiae 424A (LNH-ST) [20], a xylose-fermenting yeast strain obtained from Purdue University, was used in this study. The seed culture was prepared by inoculating a frozen glycerol stock into 50 mL of synthetic medium (described below) using 50 g/L glucose as the sole carbon source in a 125 mL flask. The culture typically reached a cell density of 6.0 to 6.2 OD 600 (optical density at 600 nm wavelength) after 18 h incubation at 30°C and 150 rpm. The cells were then harvested by centrifugation at 4,000 rpm for 5 min at room temperature and used as the inoculum for all reported experiments.
Synthetic medium and fermentation
A synthetic medium (SM) with a well-defined composition was used as a seed culture and fermentation medium. The composition of the SM, as shown in Table 3, was designed to closely match the nutrient composition of AFEX-CS hydrolysate (ACH) at 6% (w/w) glucan loading ( Table 2). Concentrated stock solutions of sugars, peptone (BD Bacto™ Tryptone, Franklin Lakes, NJ, USA), vitamins (Sigma-Aldrich, St. Louis, MO, USA), ammonium sulfate, and mineral salts were prepared separately and sterilized by vacuum filtration (Millipore Stericup®, 0.2 μm). The medium was adjusted to an initial pH of 5.5 with KOH and sterile filtered after the addition of all relevant components.
Fermentations were performed in a 25 mL Erlenmeyer flask with a working volume of 10 mL. The flasks were capped with rubber stoppers, which were pierced with a needle to vent CO 2 . Fermentations were not performed under strict anaerobic conditions; however, the air initially present in the head space of the fermentation flasks was displaced by the CO 2 generated during fermentation. The seed was inoculated into the medium at an initial OD 600 of 0.5, corresponding to 0.24 g/L cell mass concentration (dry weight). All fermentations were conducted in triplicate at 30°C and 150 rpm in a shaking incubator and the pH was maintained around 5.5 by periodic manual additions of 6 M KOH. As the fermentation media did not contain buffer, the pH had a tendency to decrease in the first 24 h. Therefore, we adjusted the pH to 5.5 before every sampling time. Approximately 300 μL of samples were withdrawn at designated times (0, 4, 8, 18, 24, 48, and 72 h) and frozen immediately for subsequent analysis.
Cell mass was estimated using a UV/Vis spectrophotometer (Beckman Coulter, Brea, CA) at 600-nm wavelength. One unit of absorbance is approximately equal to 0.48 g/L yeast cell biomass (dry weight). Sugars, ethanol, organic acids, glycerol, and xylitol were determined by HPLC, using the method described for compositional analysis of the hydrolysate.
The ethanol productivities for the various fermentation experiments were calculated for 24 h and/or 48 h time points, dividing the produced ethanol concentration by the respective residence time. Fermentation product yields were calculated for the first 48 h period, dividing the mass of product formed by the mass of total sugar consumed during that period. Statistical analyses for the fermentation results included standard deviations (shown in the respective tables) and a two-tailed t-test analysis (see Additional file 1) performed in Microsoft Excel (Microsoft, Seattle, WA).
Effect of ACH components on fermentation
To simplify this study, all characterized AFEX pretreatment-derived biomass decomposition products were divided into five groups ( Table 4): 1) nitrogenous compounds, 2) furans, 3) aliphatic acids, 4) aromatic compounds, and 5) carbohydrates. The effect of these five groups of compounds on xylose fermentation was tested individually and in combination (five groups in combination) in order to investigate their synergistic inhibitory effect. The fermentations were conducted in SM supplemented with 60 g/L glucose and 26 g/L xylose. The decomposition products in each group and their concentrations are given in Table 2, and matched their absolute abundance as found in 6% glucan loading-based ACHs. To make stock solutions of decomposition products, all compounds were dissolved in water according to the categories of aliphatic acids, aromatic acids, aromatic aldehyde/ketones, furans, imidazoles, and pyrazines at 50-fold higher concentrations and the stock solutions were sterile filtered prior to their addition into the SM. Ferulic acid, p-coumaric acid, amides, and carbohydrates were directly added to the fermentation media at the desired concentrations (Table 2) due to their lower solubility in water. Fermentations of SM without any decomposition products (blank) and ACHs were used as negative and positive controls, respectively. The ACH was adjusted to pH 5.5 before inoculum addition.
Effect of nitrogenous compounds on fermentation
Fermentations were carried out in SM supplemented with 60 g/L glucose and 26 g/L xylose, respectively. The nitrogenous compounds investigated are classified into three subgroups: 1) amides, 2) pyrazines, and 3) imidazoles. All compounds in each subgroup and their concentrations tested are listed in Table 2. Fermentation of SM without any decomposition products was the control experiment (blank).
Effect of amides and corresponding acids on fermentation
In order to compare the effect of amides and their corresponding acids on hexose/pentose sugars co-fermentation performance, feruloyl amide (6.2 mM), ferulic acid (6.2 mM), p-coumaroyl amide (7.5 mM), p-coumaric acid (7.5 mM), acetamide (28.8 mM), and acetic acid (28.8 mM) were selected for this study. The molar concentration of each compound was the total sum of the amide and its corresponding acid found in ACH at 18% solids loading ( Table 2). The compound was directly dissolved in SM and the initial pH of the medium was adjusted to 5.5 before filter sterilization. The fermentation media and control experiments were as described above.
Results and discussion
The major objective of this work is the formulation of a synthetic lignocellulosic hydrolysate (SH), as a tool to understand the effect of various components from pretreated biomass on microbial fermentation. This work provides guidelines and a methodology to formulate a detailed SH, based on the composition of industrially relevant lignocellulosic hydrolysates. The SH described in this work was designed based on the composition of ACH and was used to determine the impact of various major biomass-derived products on the performance of S. cerevisiae 424A (LNH-ST) fermentation. The details concerning 1) characterization of the AFEX-CS hydrolysate, 2) formulation of an SH, and 3) the impact of major hydrolysate components on yeast fermentation will be discussed here.
Characterization of the ACH
Characterization of the ACH involves identification and quantification of 1) natively available microbial nutrients, 2) plant-derived chemicals, and 3) pretreatment-specific decomposition products. The nutrients available in the ACH are listed in Table 2 and comprise various forms of carbohydrates, nitrogenous compounds, vitamins, and minerals. The carbohydrates which could be consumed by S. cerevisiae 424A (LNH-ST) as a carbon source were glucose (60 g/L) and xylose (26 g/L). Other carbohydrates were found in ACH at lower abundances, including arabinose (5 g/L), glucan-derived oligomers (12 g/L), and xylan-derived oligomers (18 g/L). However, these were not categorized as nutrients, as S. cerevisiae 424A (LNH-ST) is not capable of using these sugars as a primary carbon source [21]. A total of 1.44 g/L of protein was estimated by LC-MS during the amino acid analysis of the ACH. Individual amino acid concentrations are shown in Additional file 1: Table S1. While the total protein concentration is fairly similar to the results of a previous study [18], the relative proportions of individual amino acids were significantly different. In this work, aspartate, valine, and proline were the most abundant amino acids, as opposed to glutamate, glycine, and alanine reported by Lau et al. [18]. These differences are likely related to the fact that, in this study, enzymatic hydrolysis was performed on a different source of corn stover, using different commercial enzymes. However, these changes in amino acid proportions, due to differences in feedstock and enzyme sources, did not affect the overall fermentation profiles of S. cerevisiae 424A (LNH-ST) grown on the hydrolysate, as the results obtained in this study are comparable to those of our previous work [10].
The free ammonium concentration found in the hydrolysate was the same as the total protein (1.44 g/L) and significantly different from the value reported previously (0.75 g/L) [18]. The concentration of free ammonium in the hydrolysate is dependent on the levels of residual ammonia left adsorbed on the biomass after AFEX pretreatment, which may vary due to differences in the relative organic acid content of the feedstock and the efficiency of ammonia removal by evaporation in the fume hood following pretreatment. However, these variations in ammonium concentration between pretreatment batches did not have significant effects on the fermentation profiles of S. cerevisiae 424A (LNH-ST) compared to previous studies [10,13]. This observation suggests that nitrogen is not a limiting factor for efficient fermentation of ACH. Vitamin concentrations reported in this manuscript ( Table 2) were based on results previously reported by our laboratory [18]. A sensitivity analysis was carried out to evaluate the impact of vitamin concentrations on the fermentability of SH and verify if using previously reported values is a reasonable assumption for this work. For this purpose, experiments using ACH with and without 50% vitamin supplementation (based on values from Table 2) were performed. As no significant differences were observed on the fermentation profiles (data not shown) during this sensitivity analysis, it was reasonable to assume that the values obtained in the previous study could be used to estimate the vitamin composition of the SH (Table 2). It is also important to note that the goal of this study is to formulate a SH based on a typical composition of an industrially relevant biomass hydrolysate, which can vary significantly depending on the origin of the feedstock and enzymes used. Therefore, using values from our previous study was a reasonable assumption for achieving the aforementioned goals of the current study.
The mineral content of ACH was quantified by ICP-MS. Macro-elements such as P, K, and Mg were present in concentrations above the optimum range required for yeast growth defined by Jones and Greenfield (1984) [22]. These minerals are essential to all yeast and must be present in millimolar concentrations for optimal cell growth [22]. From Table 2, it is possible to observe an extremely high level of K (3886.50 mg/L), which resulted from the utilization of KOH for pH adjustment to 4.8 during enzymatic hydrolysis. Unfortunately, pH maintenance is essential to maximize enzymatic hydrolysis conversions; therefore, little could be done to avoid the accumulation of this macro-element.
Elements such as Na, Ca, and Mn are also available in concentrations above the optimum for yeast growth [22]. However, the effect of high levels of trace elements in combination with other components found in ACHs is not yet understood. Chelation effects and ionic interactions with hydrolysate components may affect the optimum range of these minerals for yeast growth [23].
Characterization of pretreatment-derived decomposition products and potential plant-derived inhibitory compounds present in the hydrolysate was performed using targeted LC-and GC-MS analysis. In our previous work, the most abundant compounds produced during AFEX pretreatment of CS were identified and quantified [15]. That work served as the platform to characterize the hydrolysate composition described here. In Table 4, these products were categorized into nitrogenous compounds, furans, aliphatic acids, aromatic compounds, and carbohydrates.
The concentration of nitrogenous compounds and furans was calculated based on the amounts present in AFEX-CS, as previously reported by Chundawat et al. [15]. High levels of sugars in the hydrolysate interfered with the direct quantification of these products by GC-MS, including acetamide, pyrazines, imidazoles, and furans. We considered removing those monomeric sugars from the hydrolysate prior to GC-MS analysis; however, this would have required extensive sample preparation and would thus affect the accuracy of absolute quantification of each target compound (without extensive method development, see previous study for issues encountered during typical GC-MS analysis in presence of high soluble sugar background [24]). For achieving the major goals of our current study, we have assumed that all nitrogenous compounds and furans found in AFEX-CS were totally solubilized (with 100% recovery) in the supernatant during enzymatic hydrolysis, as they are highly soluble at those concentration levels (see Table 4 for details). All other compounds presented in Table 4 were directly quantified in the hydrolysate using HPLC and LC-MS analysis.
Carbohydrates are by far the most abundant compounds in the hydrolysate, where 60 g/L glucose, 26 g/L xylose, and 5 g/L arabinose were quantified as the major carbohydrate monomers ( Table 4). The concentration of carbohydrates (and other compounds) depends on the solids loading used during enzymatic hydrolysis of the pretreated biomass. In this work, 18% solids loading enzymatic hydrolysis was performed to create the ACH, giving a sufficient concentration of sugars to produce approximately 4 wt% ethanol after fermentation. This enzymatic hydrolysis condition is considered to be industrially relevant; therefore, results from this study have practical industrial relevance. However, typically under such high solids loadings, the enzymes are inhibited by high concentrations of soluble sugars [25,26], leading to the progressive accumulation of sugar oligomers derived from xylan and glucan. This likely explains the presence of 12 g/L and 18 g/L of gluco-and xylo-oligomers, respectively, in the ACH.
From Table 4, it is clear that, besides carbohydrates, the major water soluble plant-derived compounds present in ACH are feruloyl amide, p-coumaroyl amide, acetamide, acetic acid, trans-aconitic acid, formic acid, and p-coumaric acid. All these components are present in the hydrolysate in concentrations above 300 mg/L and, therefore, their presence at such levels can potentially impact the performance of yeast fermentation during biofuel production. The nitrogenous compounds presented in Table 4 are products of reactions between plant cell wall components and ammonia, which are produced during AFEX pretreatment [15]. For example, acetamide, feruloyl amide, and pcoumaroyl amide are products of ammonolysis reactions that cleave ester-bound acetates, coumarates, and ferulates, which are abundantly present in the plant cell wall of CS [27,28]. These reactions are thought to be important for the efficacy of the pretreatment, by disrupting the ester cross-links between carbohydrates and lignin, or by deacetylating the xylan backbone of hemicellulose [15,28,29]. The acid counterparts of these amides, that is, acetic acid, ferulic acid, and p-coumaric acid, are products of hydrolysis of the same esters, which also occur during AFEX due to the presence of hydroxyl ions in the pretreatment media [15]. Similarly to dilute acid pretreatment, formic acid is also widely produced during AFEX; however, it is formed by a different mechanism, likely via alkaline peeling reactions of polysaccharides [15,30,31]. On the other hand, trans-aconitic acid is not regarded as a typical AFEX pretreatment-derived decomposition product, but it is a well-known plant metabolite that is particularly abundant in grasses, including maize [15,32,33]. Therefore, its presence in a CS-derived hydrolysate at these levels is expected.
Other less abundant products present in the hydrolysate, also listed in Table 4, are included in various categories such as nitrogenous compounds, furans, aliphatic acids, and aromatic compounds. Though they are present in lower amounts in the hydrolysate, their inclusion in the SH is important because their cumulative and synergistic inhibitory effects may be significant during microbial fermentation [34].
Formulation of a control synthetic medium
As mentioned above, ACH contains nutrients, as well as plant-derived compounds that are potentially inhibitory to microorganisms. The control synthetic medium was formulated to contain a similar level of nutrients as the biomass-derived hydrolysate, without the plant-derived inhibitory components. Table 3 summarizes the nutrient formulation of the control synthetic medium used in this work. Specifically, (NH 4 ) 2 SO 4, peptone, and vitamins were used to match the concentrations of ammonia, protein, and vitamins, respectively, present in the hydrolysate. The concentrations of mineral elements added to the control synthetic medium were largely matched by adding a selection of salts as described in Table 3. The salts were carefully selected to avoid solubility problems during media preparation. In general, chlorine-based salts showed higher solubility in the synthetic hydrolysate than the sulfate, phosphate, or carbonate counterparts. However, a recent study by Casey et al. [35] revealed that chloride salts can be more detrimental to the specific xylose consumption rate of S. cerevisiae 424 A (LNH-ST) compared to their sulfate counterparts, for example. Therefore, high concentrations of chlorine anions in solution could negatively affect xylose fermentation. To avoid the presence of high levels of chlorine-based salts in the synthetic medium, we selected potassium salts with three different anion pairs and sodium salts with two different anion pairs (Table 3). For the same reason, we also chose to use Ca(NO 3 ) 2 instead of CaCl 2 .
In this synthetic control medium, S. cerevisiae 424A completely consumed glucose and xylose in 18 and 72 h, respectively, generating ethanol at a concentration of around 35 g/L (about 80% metabolic yield) and a cell density (OD 600 nm) of approximately 12 (Figure 1). These results suggest that ACH is not limited by nitrogen, protein, or micronutrients for consuming glucose and xylose during ethanol production (though the rate of xylose uptake is significantly slower than that of glucose). However, determining the nutrient composition of AFEX pretreated biomass hydrolysates and formulating a control synthetic medium is critical to further improving microbial co-fermentations for more efficient and rapid conversion of lignocellulosic hydrolysates in the presence of inhibitory compounds. This is highlighted by the fact that xylose fermentation is affected by the nutrients level in the medium and that individual decomposition products in ACH are not very inhibitory for robust yeast species such as S. cerevisiae [10]. Thus, it is likely that the inhibitory effect of many of the plant-derived compounds present in the hydrolysate would not be observed in nutrient-rich media (such as Yeast Extract Peptone, YEP medium). Though the control synthetic medium formulated in this work is not exactly comparable with the actual biomass hydrolysate due to experimental limitations, the nutrient value is close enough to be considered acceptable for studying the effect of different inhibitors on strain performance. A sensitivity analysis was performed to determine how the concentration of amino acids affects cell growth and the fermentation performance. It was found that a variation of the amino acid concentration by up to two times the amount detected in the hydrolysate did not affect cell growth; however, it did improve the xylose consumption rate (data not shown).
Inhibitory effect of different classes of compounds from ACH on S. cerevisiae 424A fermentation
As previously mentioned, the plant-derived compounds and pretreatment decomposition products present in ACH were divided into five groups: nitrogenous compounds, organic acids, aromatic compounds, carbohydrates, and furans ( Table 4). The effect of the different groups of lignocellulose decomposition products on S. cerevisiae 424A fermentation was investigated and compared with the control synthetic medium (blank) formulated in this work ( Table 5). The various classes of compounds identified in ACH were added to the blank medium at an abundance comparable to that in the actual hydrolysate to determine their individual and combinatorial inhibitory contribution to yeast fermentation.
From the results presented in Table 5, the nitrogenous compounds caused a significant decrease in cell biomass yield, xylose consumption rate, and 24 h ethanol productivity compared to the blank control SM (Additional file 1: Table S2-1, S2-2, and S2-4). Though this class of compounds is not usually found in most lignocellulosic hydrolysates, certain amides are produced by a variety of plants and are known to have anti-fungal effects [36]. As nitrogenous compounds are quite abundant in AFEX biomass-derived hydrolysates and limited information about their inhibitory effect on microbes is currently available in the literature, we will discuss this in more detail in the subsequent section.
Similarly to the effect of nitrogenous compounds, the xylose consumption rate and cell biomass yields were also negatively affected by the addition of aliphatic acids and aromatic compounds (Table 5). On the other hand, the ethanol metabolic yield was enhanced by the presence of these two classes of compounds, which is consistent with earlier findings in the literature [37]. As these weak acids will be present in the hydrolysate solution predominantly in their non-dissociated form, they will be permeable through the yeast cell membrane [38]. Once they enter the cytosol, the acids will dissociate and the cell will be forced to pump excess protons through the membrane to maintain homeostasis. Though low concentrations of organic acid have been observed to increase ethanol yields and fermentation rates, this benefit is lost at higher acid concentrations [39][40][41][42]. High levels of anionic acid species are also toxic to the cell and can result in cessation of growth or cell death [41,43], which does not seem to be the case for ACH. Lignin-derived aromatic compounds, such as phenols, are also known to inhibit S. cerevisiae growth, especially lower molecular weight phenolics. The toxicity of these compounds is dependent on the relative position (ortho, meta, or para) of the functional group in the benzene ring [44] and also on the type of functional group (for example, aldehydes, ketones, or acids). The phenolic compounds may interact with biological membranes, interfering with their function. However, the inhibition mechanism of this family of compounds is not well understood [45].
The oligomeric carbohydrates (particularly xylooligomers) also negatively affected xylose consumption rate in the first 24 h (18% of the control xylose consumption was reduced in the first 24 h). However, at the 48 h time point this difference was reduced to 2.7% of the control xylose consumption. As a result, the 48 h ethanol metabolic yield was only reduced by 2.5% of the control in the first 48 h. To our knowledge, xylose consumption inhibition by oligomeric carbohydrates has never been reported in the literature, and it would be interesting to determine the possible reason for this observation in a future study.
Addition of furans did not affect fermentation kinetics in great extent compared to the control (blank). The results from Table 5 show that there were no significant differences in biomass yield, 24 h ethanol productivity, and 48 h acetate yields compared to the blank SM (see Additional file 1: S2). Although the other parameters shown in Table 5 related to furan addition were statistically different from those of the blank SM, the observed difference was not very pronounced. The inhibitory effects of furans (such as furfural and hydroxymethyl furfural) on cellular metabolism have been thoroughly studied by several researchers [37,46]. These effects include oxidative damage of yeast cells by lower abundance of reducing agent concentrations (such as NADPH and NADH) and reduced activities of enzymes involved in the glycolysis pathway. From the most common furans found in lignocellulosic hydrolysates, furfural seems to be more inhibitory when compared to 5-HMF, at equivalent concentrations [47]. As AFEX pretreatment produces a low level of 5-HMF (Table 4) and practically no furfural, the concentration of this class of compounds in the hydrolysate appears to be low enough to avoid oxidative damage during yeast fermentation.
The synergistic inhibitory effect of the various classes of decomposition products (DP) was observed on xylose fermentation. The combination of all compounds (blank + DP in combination) showed a higher inhibitory effect than the aggregate value of individually added products (48 h data). This result agrees with previous reports that also observed synergies on the inhibitory effect of different compounds during yeast fermentation [47].
Among all the classes of decomposition products tested herein, nitrogenous compounds were the most inhibitory to xylose fermentation (Table 5), which could potentially be explained by their relatively higher concentration in the hydrolysate.
In the presence of aliphatic acids, about 70% decrease in acetate production was observed compared to the blank SM (Table 5). This result may be related to end-product (acetate) inhibition of the acetate synthesis pathway in yeast. Moreover, when all the decomposition products were added together, acetate was consumed by the yeast after 48 h fermentation, instead of being produced. It is possible that the yeast cells consume acetate to equilibrate the redox imbalance caused by the xylose metabolic pathway and due to the presence of high concentrations of other inhibitory compounds [48]. However, to better understand this finding, more detailed metabolomic experiments will need to be carried out in the future using SHs.
The carbon mass balance closures for the various synthetic media evaluated in Table 5 Table 5 Fermentation parameters for synthetic media (SM) in presence of various groups of lignocellulose decomposition products (DP) e Biomass yield b (g/g) Xylose consumption (%) Ethanol productivity (g/L/h) Ethanol yield c (g/g) Glycerol yield (g/g) Xylitol yield (g/g) Acetate yield (g/g) The blank was the synthetic medium without the addition of decomposition products (DP).
b Biomass yield was based on both glucose and xylose consumed at 18 h fermentation, when the cell density reached the maximum. One unit of absorbance at 600 nm is approximately equal to 0.48 g dry cell wt/L. c Theoretical metabolic yield of ethanol for both sugars was 0.51 g EtOH/g consumed sugar.
d The initial concentration of acetate in the hydrolysate and synthetic medium with the addition of aliphatic acids and DP in combination was 1.9 g/L. e The t-test results for determining statistically significant differences between the different results are presented in Additional file 1, S2, Tables S2-1 -S2-9.
equal to 1. However, for the actual hydrolysate the carbon mass balance closes at 1.06, which means that there is 6% more carbon being formed than the carbon consumed. This observation may suggest that there are other carbon sources present in small quantities in the actual hydrolysate, which were not detected or analyzed in this study. More in-depth characterization is required to determine the minor carbon sources that contribute to this carbon mass balance closure.
Inhibitory effect of individual families of nitrogenous compounds
Since the effects of nitrogenous products on fermentation are particularly less well understood than the remaining categories of compounds, and because these products are specifically linked to ammonia-based pretreatment, we decided to further investigate their individual effect on the fermentation profile of S. cerevisiae 424A. Here, we evaluated in more detail the effect of various 1) pyrazines, 2) imidazoles, and 3) amides on xylose consumption and ethanol production rates (Table 6 and Figure 2). The results show that the addition of pyrazines or imidazoles to a well-defined SM did not significantly affect the kinetics of xylose consumption and ethanol production (P-value ≥ 0.05, see Additional file 1, S3, Tables S3-2 -S3-4). These two families of compounds are not present in the hydrolysate at high concentrations and therefore are likely not to have any major inhibitory effect on yeast fermentation. However, amides are present at much higher concentration in the hydrolysate, and their addition to the blank media resulted in decrease of biomass yield, xylose consumption rate, and ethanol productivity. Specifically, with the addition of amides the biomass yield, xylose consumption, and ethanol productivity were reduced from 0.068 g/g, 95%, and 0.71 g/L/h to 0.061 g/g, 80%, and 0.65 g/L/h, respectively. Though we see some level of inhibition on xylose consumption and ethanol production, the mechanisms of amide inhibition are not well understood. It is possible that phenolic amides have a similar mechanism of inhibition to the lignin-derived phenolic compounds, which tend to impact the integrity of the cell membranes when present at high concentrations [44,45].
Comparison between the inhibitory effects of amides and the corresponding carboxylic acids
Three amides (feruloyl amide, coumaroyl amide, and acetamide) present in the hydrolysate were further studied individually and their inhibition profiles were compared to their corresponding acid forms (ferulic acid, coumaric acid, and acetic acid) in the blank synthetic medium (Figure 3). Unlike the previous experiments reported herein, the concentration of amides and acids chosen for this study was not based on their actual amount in the ACH. In this case, it was assumed that all the reacting esters present in the biomass were cleaved by ammonolysis or hydrolysis reactions, respectively. As a result, the same exact molar concentrations of the acid and amide counterparts were used for each comparative inhibition experiment. In contrast to our previous results for when the amides were added together, the individual amides did not show a substantial inhibitory effect on fermentation compared to the control. Xylose was completely consumed to undetectable levels within 72 h with maximum OD 600 of around 12 for all the amides tested in this work ( Figure 3). Therefore, the inhibitory behavior of amides is likely a synergistic effect, coupled with the fact that the total concentration of amides was higher than when present individually. The corresponding acid forms of those amide compounds, however, all showed substantial inhibition on cell growth and xylose fermentation. Among all acids, ferulic acid showed the highest inhibitory effect followed by acetic acid and coumaric acid, which was consistent with their relative abundance in the ACH (Table 4). Furthermore, ferulic acid is known to be a more potent inhibitor of yeast growth than coumaric acid, when present at similar concentrations. From the results presented in Figure 3B, the presence of ferulic acid in the fermentation media reduced the cell density by 45%. The average xylose consumption rate decreased to a very low 0.09 g/L/h (0 to 24 h), a much larger decrease than that caused by feruloyl amide (reduced to 0.55 g/L/h). Ferulic acid even affected the glucose consumption rate, which was not observed for any other decomposition product tested herein. Complete glucose consumption was only achieved after 48 h fermentation instead of 18 h, as it was in the case of the control blank medium.
From these results, it is evident that amides are less inhibitory than their corresponding acid forms, based on the same molar concentration, on yeast fermentations. Carboxylic acids permeate into the cytosol in their undissociated form when performing fermentations at pH 5.5. While in the cytosol, the acids dissociate due to the near-neutral conditions of the cytosol, decreasing the intracellular pH [38]. This effect will not be observed for amides, which typically have pKa values greater than 10. This could partially explain why AFEX pretreated biomass has greater fermentability compared to dilute acid pretreated biomass [9]. Ester hydrolysis reactions that occur during dilute acid and steam explosion pretreatments result in the formation of the organic acids studied herein, probably at similar concentrations to the ones used in this study. However, in the case of AFEX pretreatment (under the presently employed conditions) only about one third of the total available esters are hydrolyzed to yield acids, while the remaining are ammonolyzed to the less inhibitory amides. One possible way to enhance the fermentability of AFEX pretreated biomass is to further reduce the hydrolysis reaction products during pretreatment and promote conditions that improve the selectivity toward the less inhibitory ammonolysis reaction-derived products.
Comparison between SH and ACH
The fermentation profile of S. cerevisiae 424A in SH was compared side by side to the actual ACH as shown in Figure 4. The cell growth during fermentation in the control synthetic medium (blank) was comparable to that of the actual hydrolysate, achieving a cell density of OD 600 11.5 after 18 h ( Figure 4D). However, cell growth in the SH, in the presence of all the decomposition products from Table 4, was greatly reduced, showing a cell density of around OD 600 8 after 18 h fermentation. This value represents just 68% of the cell density obtained using the blank medium.
As expected, xylose was almost completely consumed after 48 h fermentation in the control synthetic medium. The average xylose consumption rate was 0.70 g/L/h (0 to 24 h). However, the xylose consumption rates in the SH and ACH were 0.23 g/L/h and 0.28 g/L/h, respectively, which were much lower than the control rate. The lower cell density in the SH was one of the possible causes of the decreased xylose consumption rates. DP inhibition of specific xylose consumption rate and decreasing viable cell density were probably the other two reasons for the slow xylose fermentation [13]. Regarding the ethanol yield, SH and ACH results were statistically different (0.439 g/g and 0.486 g/g, respectively) and this difference represents about 10% of the ACH ethanol yield. In both these cases, the ethanol yields were significantly higher than the control (0.405 g/g). This increased ethanol metabolic yield in the presence of AFEX pretreatment-derived decomposition products is consistent with our previous observations and other reports [10,13,49]. The final ethanol concentrations achieved in the ACH, control synthetic medium, and SH were 38 g/L, 35 g/L, and 32 g/L, respectively. During the first 18 h period, glucose and xylose consumption were equivalent for both media, and only after 18 h fermentation was it possible to observe significant differences in xylose consumption and, consequently, in ethanol production. Therefore, the higher ethanol yields observed for the actual biomass hydrolysate seem to be related to better xylose fermentation. Except for biomass yield, the differences between all other blank (SM) results from Tables 5 and 6 are not statistically significant (P > 0.05). For an ideal SH, one would expect identical cell growth behavior, sugar consumption rates and ethanol yields to those observed for the actual hydrolysate. The differences in cell growth profile between the SH and the ACH may be due to incomplete evaluation of the composition of the actual hydrolysate, which is very complex and presents various analytical challenges. Possible improvements for future versions of the SH may include the analysis of redox co-factors present in plant biomass (for example, NAD(P)H), which could potentially help the yeast cells to improve their fermentation performance. Also, the higher concentration of chlorine-containing salts in the SH might be another possible factor that could have caused such a negative impact (Tables 2 and 3). Therefore, optimizing the choice of salts to closely match the mineral content of the ACH would help improve the performance of the SH. Nevertheless, the SH presented in this study was successfully used to evaluate the relative levels of inhibition associated with the various classes of compounds that are present in the actual ACH. Moreover, as we performed a detailed characterization of the amino acids present in the ACH (Additional file 1: Table S1), it is possible to formulate a well-defined synthetic medium by the addition of individual amino acids, at the respective concentrations, in contrast to peptone. The utilization of defined synthetic media will be important for future multi-omics studies that will help us understand the mechanisms of inhibition under well-controlled experimental conditions.
Conclusion
In this work, nutrients and decomposition products present in ACH were characterized with the goal of formulating a synthetic hydrolysate, which will be used in multi-omics analysis for understanding the inhibition mechanisms of the lignocellulosic hydrolysate. This work also provides an example showing how synthetic lignocellulosic hydrolysates derived from other pretreatment technologies (such as dilute acid and steam explosion) can be formulated.
The ACH contained high levels of nitrogenous compounds, notably phenolic amides and acetamide. Due to their presence at high concentrations, their observed inhibitory effect on xylose consumption and ethanol production was the most significant among all the families of compounds tested herein, which included aliphatic acids, furans, lignin-derived phenolic compounds, and oligomeric carbohydrates. However, when comparing the inhibition due to amides at the same molar concentrations as their acid counterparts, we observed that amides are significantly less inhibitory to both glucose and xylose fermentation than the acids. The reduced inhibitory effect of amides is a major advantage of AFEX-and ammonia-based pretreatments over other pretreatment technologies that mainly produce carboxylic acids as decomposition products. Because of the reduced production of carboxylic acids and furans, notably furfural and 5-HMF, the ACH is easily fermentable without any detoxification.
Although we were able to identify the major groups of inhibitory compounds present in the ACH, the SH did not exactly match the performance of the actual hydrolysate. The cell density in SH was considerably lower than in the actual hydrolysate and, as a consequence, the xylose consumption rate was also slightly reduced. However, the proposed SH was instrumental in identifying the inhibitory effect of various classes of compounds present in the hydrolysate and their relative contribution to the overall inhibition. Due to the complexity of the lignocellulosic hydrolysate composition, we will likely develop more representative versions of the SH as we learn more about the composition of actual hydrolysates. The SH formulation will be instrumental in future multi-omics studies to understand the nature of AFEX pretreatment-specific decomposition products and how they inhibit yeast and bacteria, so that we can engineer better strains to maximize biofuel yields and productivity.
Additional file
and pretreatment decomposition product analyses. XT also contributed to the formulation of the SH, the experimental design, and the preparation of the manuscript. LDCS contributed in the synthesis of the amides used in the SH, the experimental design, formulation of the SH, data interpretation, and preparation of the manuscript. MJ contributed to the design of the fermentation experiments, data interpretation, and preparation of the manuscript. SPSC contributed to the methodology development, data interpretation, and preparation of the manuscript. CKC contributed by analyzing/quantifying the pretreatment decomposition products in ACH and provided important insight during the preparation of the manuscript. MWL helped with the methodology development and contributed by providing guidelines to formulate the SH. ZX and BED participated on the identification of the problem and helped with the preparation of the manuscript. VB also participated in the identification of the problem, helped with the experimental design, and coordinated this work. All authors read and approved the final manuscript. | v3-fos |
2016-05-04T20:20:58.661Z | {
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} | s2 | Phytochemicals from Mangifera pajang Kosterm and their biological activities
Background Mangifera pajang Kosterm is a plant species from the mango family (Anacardiaceae). The fruits are edible and have been reported to have high antioxidant content. However, the detailed phytochemical studies of the plant have not been reported previously. This study investigates the phytochemicals and biological activities of different parts of Mangifera pajang. Methods The plant samples were extracted with solvents of different polarity to obtain the crude extracts. The isolated compounds were characterized using spectroscopic methods. The extracts and isolated compounds were subjected to cytotoxicity tests using human breast cancer (MCF-7), human cervical cancer (HeLa) and human colon cancer (HT-29) cells. The free radical scavenging activity test was conducted using the DPPH assay. Antimicrobial activity tests were carried out by using the disc diffusion method. Results Phytochemical investigation on the kernel, stem bark and leaves of Mangifera pajang led to the isolation of methyl gallate (1), mixture of benzaldehyde (2) and benzyl alcohol (3), mangiferonic acid (4), 3β-hydroxy-cycloart-24-ene-26-oic acid (5), 3β,23-dihydroxy-cycloart-24-ene-26-oic acid (6), lupeol(7) lupenone(8), β-sitosterol(9), stigmasterol(10), trans-sobrerol(11) and quercitrin (12). Crude ethyl acetate and methanol extracts from the kernel indicated strong cytotoxic activity towards MCF-7 and HeLa cells with IC50 values of less than 10 μg/mL, while petroleum ether, chloroform and ethyl acetate extracts of the stem bark showed strong to moderate activity against MCF-7, HeLa and HT-29 cancer cell lines with IC50 values ranging from 5 to 30 μg/mL. As for the antimicrobial assays, only the ethyl acetate and methanol extracts from the kernel displayed some inhibition against the microbes in the antibacterial assays. The kernel extracts showed highest free radical scavenging activity with IC50 values of less than 10 μg/mL, while the ethyl acetate and methanol extracts of leaves displayed only weak activity in the DPPH assays. Conclusions Phytochemical investigations on various parts of Mangifera pajang have identified terpenoids and a flavonol derivative as major constituents. Bioassay studies have indicated that the crude extracts and isolated compounds have potential as naturally-derived anticancer and antimicrobial agents, besides possess high free radical scavenging activity.
Background
Mangifera pajang Kosterm is also known as 'bambangan'; a plant species from the mango group which can be found in Borneo Island (Malaysia -Sabah and Sarawak, Brunei, and Indonesia -Kalimantan) [1]. Unlike commercial mangoes (Mangifera indica), the fruit of Mangifera pajang are rarely eaten as it is unpopular among the public in Peninsula Malaysia and it is relatively unknown as antioxidant source. The fruits are ovoid and light-brown coloured, while the peels are thick which constitute approximately 27% of the whole bambangan fruit. The bambangan fruit is also about three times larger than its more common counterpart, mango [2,3]. The pulp is fibrous, juicy, has a specific aromatic flavour and strong smell and can be eaten fresh while the peel is used for cooking curries [3]. In Sabah, the kernel and flesh are used to make 'jerok bambangan' among Kadazans, the young leaves are sold as vegetables in Sarawak, while the thick rind can be sun-dried and preserved to be used for preparation of 'sambel' [4]. Previous studies of the fruit parts (peel, pulp and kernel) have reported high antioxidant and cytotoxic activity towards cancer cell lines [5,6]. However, there are no reports on the isolation of chemical constituents from the kernel, leaves and stem bark of the plant. In continuation of our research on local underutilised medicinal plants, we wish to report herein on the phytochemical properties and biological activities of Mangifera pajang.
Plant collection
The plant was collected from Sabah, Malaysia and was identified by Dr Mohd Fadzelly Abu Bakar from Universiti Tun Hussein Onn Malaysia (UTHM). The voucher specimen was deposited at the herbarium, BORNEENSIS, Universiti Malaysia Sabah. The plant samples (kernel, leaves and stem bark) were air-dried and ground into a fine powder prior to being used.
Cytotoxic assays
Various crude extracts of Mangifera pajang were screened for cytotoxic activity against MCF-7 (human breast cancer), HeLa (human cervical cancer) and HT-29 (human colon cancer) cells according to the method described previously [17]. The stock solutions of 100 mg/mL were prepared by using dimethyl sulphoxide (DMSO) of concentrations ranging from 0.1 μg/mL -30.0 μg/mL. Working solutions were made up by two fold dilution of the stock and 20 μL of each concentration was added to each well in triplicates. The control wells of the untreated population were treated with highest concentration DMSO as the negative control. After 3 days, the cell viability was determined by introducing 20 μL of MTT solution (5 mg/mL in PBS) to each well, followed by 4 hours of incubation. The blue formazan crystals that formed were dissolved in DMSO and the absorbance was read using the Elisa reader with wavelength of 570 nm and a reference wavelength of 630 nm. The cytotoxic activity was determined by the IC 50 values, which were defined as the concentration of the test samples that resulted in a 50% reduction of absorbance or a measure of 50% concentration of tested samples that was required to inhibit the growth of cancer cells. Extracts and isolated compounds that indicated IC 50 values < 10 μg/mL, are considered to have significant cytotoxic activity against that particular cell line.
DPPH assay
The scavenging activity of the crude extracts and constituents were determined by using 1,1-diphenyl-2pycrylhydrazyl (DPPH) according to a procedure described previously [18]. DPPH was used as the stable free radical agent while DMSO was used as the blank. The sample was dissolved in DMSO in the ratio of 1:1 (w/v) and diluted to achieve concentrations of 500, 250, 125, 62.50, 31.25, 15.63 and 7.81 μg/mL. The IC 50 values were determined by plotting the percentage of inhibition against sample concentration, ranging from 500 to 7.81 μg/mL. The IC 50 values are defined as the amount of antioxidants needed to decrease the initial concentration of DPPH by 50%.
Antibacterial and antifungal assays
All crude extracts from Mangifera pajang (kernel, leaves and stem bark) were subjected to antibacterial and antifungal assays towards several targeted microbes including Methicillin resistant staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Salmonella choleraesuis and Bacillus subtilis for antibacterial screening while Candida albican, Aspergillus ochraceaus and Sacchoromyces cerevisiae were used for antifungal screening. The assays were carried out using the disc diffusion method [17]. This involved placing paper discs of 6 mm in diameter that contained the samples onto a plate where the microbes were growing. Ampicillin (Gram-negative Bacteria) and Streptomycin (Gram-positive Bacteria) standards were used for the bacteria while nystatin was used as the positive control for the fungi. The plates were inverted and incubated at 30-37°C for 18-24 hours for bacteria and 24-48 hours for fungi or until sufficient growth had occurred. After incubation, each plate was examined. The diameters of the zones of complete inhibition were measured.
Isolated constituents
Phytochemical investigations on the kernel, stem bark and leaves of Mangifera pajang have afforded several classes of constituents including aromatic esters, cycloartane and lupane triterpenes, a monoterpene, steroids and a flavonol glycoside.
was obtained from the ethyl acetate and methanol extracts of the leaves and appeared as a yellow solid with a melting point of 188 -190°C. The compound was identified by comparison of its spectral data with that available literature of the compound isolated previously from the leaves of Myrsine seguinii [16].
Cytotoxicity of crude extracts and isolated compounds
All crude extracts from the kernel, stem bark and leaves were subjected to cytotoxic screening against MCF-7, HeLa and HT-29 cancer cells and the results are summarized in Table 1.
Ethyl acetate and methanol extracts of the kernel showed strong cytotoxic activity towards MCF-7 and HeLa cell lines with IC 50 values less than 10 μg/ mL, and displayed strong to moderate activities towards the HT-29 cell line. In addition, petroleum ether and chloroform extracts of the stem bark also showed high cytotoxic activity towards MCF-7, HeLa and HT-29 cells with IC 50 values of less than 15 μg/mL. In contrast, the ethyl acetate extract of the stem bark and the petroleum ether extract of the leaves displayed rather weak activity with IC 50 values of more than 20 μg/mL. Similarly, most of the extracts from the leaves were not active with IC 50 values being more than 30 μg/mL. Previous studies on the cytotoxic activity of Mangifera pajang have been reported on the ethanolic extract of its kernel towards MCF-7 (hormone-dependent breast cancer cells) and MDA-MB-231 (non-hormone dependent breast cancer cells) with IC 50 values of 23 and 30.5 μg/mL, respectively [20]. For cytotoxic screening of the isolated compounds in this work, only six compounds with sufficient amounts were selected for the anticancer tests. Cytotoxic assay of the compounds against MCF-7 cell line (Table 1) indicated strong activity as shown by methyl gallate (1) with an IC 50 value of 10.5 ± 0.29 μg/mL. Meanwhile, 3β-hydroxy-cycloart-24-ene-26-oic acid (5) displayed moderate cytotoxic activity towards MCF-7 with an IC 50 value of 13.03 ± 0.81 μg/mL. In contrast, weak cytotoxic activities were shown by lupeol (7), lupenone (8) and quercitrin (12) (4) have also demonstrated moderate cytotoxic activity with IC 50 values of 18.07 ± 0.37 and 18.03 ± 0.75 μg/mL, respectively. However, no cytotoxic activity were shown by 3β-hydroxy-cycloart-24ene-26-oic (5), lupeol(7) and lupenone (8) where the IC 50 values were more than 30 μg/mL. Some of the isolated compounds have been reported to show cytotoxic activities against several cancer cell lines. Methyl gallate (1) was shown to possess weak anticancer effects (IC 50 value of more than 70 μg/mL) against human cervix adenocarcinoma cells (HeLa) and human fibroblast cells (L-132) [21]. However, a study by Lee et al. [22] demonstrated that methyl gallate (1) successfully enhanced antitumor effects through modulation of CD4+ CD25+ Treg cell functions, delaying tumour growth even though this compound was known to have low anticancer activity. Mangiferonic acid (4) reported by Li et al. [23] showed insignificant anticancer activity with IC 50 values of around 100 μg/mL towards three murine cancer cell lines (colon 26-L5 carcinoma, B16-BL6 melanoma and Lewis lung carcinoma) and also towards three human cancer cell lines (lung A549 adecarcenoma, cervix HeLa adecarcinoma and HT-1080 fibrosarcoma). Meanwhile, anticancer activities of lupeol (7) as reviewed by Gallo and Sarachine, [24] found that lupeol (7) exhibited weak cytotoxicity in human melanoma SK-MEL-2, human lung carcinoma A549 and murine melanoma B16-F10 cells. Lupeol (7) also inhibited the proliferation of MDA-MB-231 human breast cancer cells and has been tested previously against many different numbers of cancer cells and showed range of activities towards the tested cells according to Gallo and Sarachine, [24]. These findings further corroborated with that reported in this work [25], where lupeol (7) and lupenone (8) exhibited weak cytotoxicity with IC 50 values more than 30 μg/mL against human colorectal cancer (HT-29) and mammary breast cancer (MDA-MB) cell lines. However, lupeol (7) was slightly toxic against the normal cell line (3 T3) with an IC 50 value of 38.92 μg/mL while lupenone (8) did not demonstrate any cytotoxic effect against the 3 T3 cell line with IC 50 values of more than 100 μg/mL [26]. Meanwhile, quercitrin (12) was found to have a weak inhibitory effect on the human prostate cancer cell line PC-3 [26].
Structure-activity relationships
The moderate to strong anticancer activities of the constituents of the plant have contributed to the overall cytotoxic properties of the plant extracts.
Among the tested crude extracts, the methanol extract from kernel was the most potent with IC 50 values less than 6 μg/mL for the three cancer cell lines. This may due to the presence of the cytotoxic methyl gallate (1) constituent. Methyl gallate (1) which is an ester (Figure 1), has a carbonyl and three hydroxyl groups attached to a benzene ring which probably synergistically enhanced the anticancer properties of the compound [21]. Meanwhile, mangiferonic acid (4) and 3β-hydroxycycloart-24-ene-26-oic acid (5) which were isolated from the hexane and ethyl acetate extracts of the stem bark of the plant, displayed various ranges of cytotoxicity against the three cancer cell lines. In the current study, 3β-hydroxy-cycloart-24-ene-26oic acid (5) was more cytotoxic compared to mangiferonic acid (4). In contrast to the results described by Li et al. [23], the anticancer potency of the cycloartane skeleton was suggested to be depending on the substituent at C-3; in order of α-OH > C = O > β-OH [23]. Quercitrin (12) obtained from extracts of leaves in polar solvents showed good anticancer activity with IC 50 values around 10 μg/mL for HeLa and HT-29 cancer cell lines but simultaneously reduced the cytotoxicity of the crude extracts, suggesting the antagonistic effect of quercitrin (12). Quercitrin (12) is a glycoside of quercetin with a rhamnose group attached to the hydroxyl group at C-3. However, the glycoside substituent on the ring did not enhance the cytotoxicity of quercitrin (12) as its parent flavonol structure; quercetin significantly inhibited human prostate cancer cell line PC-3 cell proliferation, whereas quercitrin (12) itself did not have any antiproliferation effect on the cancer cells [26]. In addition, lupeol (7) and lupenone (8) together with most of leaf extracts exhibited weak cytotoxicity towards all cancer cell lines. Lupeol (7) is slightly more cytotoxic compared to lupenone (8) due to the presence of a hydroxyl group at C-3.
Antimicrobial activity
The results of the agar diffusion assay indicated that most of the crude extracts did not show significant inhibition activity towards targeted microbes. Some crude extracts of different parts of the plant displayed either weak or moderate activities with inhibition zones between 6 and 13 mm.
In this study, only the isolated compound methyl gallate (1) demonstrated strong antibacterial activity towards MRSA with an inhibition zone of 21.5 mm, as compared with an inhibition zone of 23.0 mm given by the standard compound (streptomycin). In addition, methyl gallate (1) also exhibited moderate antimicrobial activities towards P. aeruginosa, S. choleraesuis and B. subtilis with inhibition zones 12.0, 15.5, and 12.5 mm, respectively. This was in agreement with previous reports on the potent antimicrobial properties of methyl gallate (1) (12), mangiferonic acid (4), 3βhydroxy-cycloart-24-ene-26-oic (5) and lupeol (7) towards the targeted microbes. As for the antifungal tests, none of extracts and isolated compounds showed activity against Candida albican, Aspergillus ochraceaus and Sacchoromyces cerevisiae. To our knowledge, this is the first antimicrobial activity study reported on Mangifera pajang.
Conclusions
Extracts of the kernel, stem bark and leaves of Mangifera pajang have demonstrated potential cytotoxic activity towards MCF-7, HeLa and HT-29 cancer cells, and the extract of kernel also displayed strong free radical scavenging activity. These are assumed to be due to the presence of bioactive constituents that were isolated including cycloartane and lupane triterpenes, together with the major constituent, methyl gallate. The plant species, Mangifera pajang could become a potential source for natural anticancer (especially for breast, liver and colon cancers) and antioxidant agents. In particular, the major isolated constituent, methyl gallate could become a potential anticancer, antioxidant and antimicrobial agent. | v3-fos |
2016-05-04T20:20:58.661Z | {
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} | s2 | How to get the best deal
Floral scents and nectar attract both pollinators and other animals that may reduce the plant's fitness, and therefore put flowering plants in a challenging situation.
eLife 4:e07641. doi: 10.7554/eLife.07641 Image Coyote tobacco flowers being visited by a hawkmoth M ost flowering plants rely on animals to spread their pollen. However, plants that rely on easily perceived signals, such as brightly coloured petals and floral scents, to attract pollinators are also advertising themselves to other animals that cause damage. These so-called 'floral antagonists' include animals that eat plant tissues (herbivores and florivores) and animals that steal nectar and pollen without helping with pollination.
These different interactions mean that flowering plants are subjected to a range of selection pressures. However, while most published research has focused on seemingly mutually beneficial relationships, little is known about how a plant can attract beneficial visitors and at the same time hide from floral antagonists that might cause harm. Plants attempt to address these challenges in multiple ways to maximize their fitness (Galen and Cuba, 2001;Chen et al., 2009;Kessler et al., 2008Kessler et al., , 2013Schiestl et al., 2014). The picture is complicated further when a single animal can act as both a pollinator and a floral antagonist (e.g., by wasting pollen, robbing nectar, or switching roles at different life stages; Adler and Bronstein, 2004). This puts the plant in a difficult situation, since the animal is responding to the same signals despite playing different roles. Any attempt by the plant to change its strategy to avoid the antagonist will also reduce pollination. Now, in eLife, Danny Kessler, Ian Baldwin and colleagues at the Max Planck Institute for Chemical Ecology have assessed the roles played by a range of pollinator and antagonist species to develop a more complete picture of plantpollinator interactions (Kessler et al., 2015). The MPI team used coyote tobacco, Nicotiana attenuata, to investigate how floral scent and nectar affect this plant's interactions with three of its pollinators: a hummingbird (Archilochus alexandri) and two hawkmoths (Hyles lineata and Manduca sexta). The first two species appear to act as mutualists, trading pollination for a nectar reward. However, M. sexta plays contrasting roles; the adult moths pollinate the flowers, but the females also lay eggs on plants and the caterpillars eat the leaves ( Figure 1).
Coyote tobacco attracts its pollinators with floral scent and rewards them with nectar. Kessler et al. studied these interactions using an approach that is innovative in a number of ways. First, they used RNA interference to silence the genes underlying the production of floral scent or nectar, either alone or in combination. This allowed them to evaluate specific floral traits in living plants, without too many confounding changes in other traits. Second, the approach is also unusual because few previous studies have combined plant-pollinator or plant-herbivore interactions and genetic manipulation in the study of floral scent (but see Kessler et al., 2008;Klahre et al., 2011;Kessler et al., 2013;Byers et al., 2015). Finally, it is also uncommon to combine field Copyright Byers and Schiestl. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. studies with more controlled greenhouse studies. This is important because while greenhouse studies can be more sensitive, their results do not always translate to the field (Obrycki and Tauber, 1984).
Pollinators are often classified into "guilds" of species that are presumed to interact with plants in similar fashions. However, little experimental work has studied the responses of different pollinator species within a guild. Kessler, Baldwin and colleagues address this issue, perhaps in an unforeseen way, by testing three different pollinators of coyote tobacco. Although M. sexta and H. lineata are both hawkmoths, they behave differently. When acting as a pollinator, M. sexta prefers wild-type plants to those lacking in scent or nectar or both, with all three alternatives being equally unattractive. H. lineata, on the other hand, treats wild-type plants and plants that lack scent or nectar the same, and prefers all three to plants that lack both scent and nectar. Hummingbirds, meanwhile, do not visit plants that lack nectar, and also appear to display a weak preference for plants that produce scent. This is perhaps unexpected because the flowers of coyote tobacco give off little scent during the day when the hummingbirds are foraging; hummingbirds also have a poor sense of smell and a limited ability to learn floral scent (Byers et al., 2015). These results -in particular, the fact that M. sexta and H. lineata behave differently, despite being members of the same guild -are also unexpected and argue for a more complex and nuanced picture of plant-pollinator interactions.
Kessler et al. found that M. sexta moths show different preferences when acting as pollinators compared to when they act as a floral antagonists. As a pollinator, M. sexta responds equally strongly to the loss of both scent and nectar. However, as an antagonist, this moth responds more strongly to the loss of nectar than it does to the loss of floral scent.
It is difficult to include multiple floral phenotypes and floral interactors in the study of plantpollinator interactions, and as such this area remains largely unexplored. By addressing some of the related questions, Kessler et al. remind us of the value of an integrative approach. Their findings also suggest that future research in this area should consider whether model pollinators are representative of the real visitor community, and whether aspects such as learning play a role in these interactions. Flowers rarely occur alone, and thus considering the role of the surrounding floral community and background scents will also be important (Riffell et al., 2014). Research that combines floral scent and other phenotypes, their underlying genes, and their role in interactions with specific pollinators in a community context will, in the future, broaden our understanding of the field of plant-visitor interactions. | v3-fos |
2019-04-11T13:16:36.790Z | {
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} | s2 | Bioethanol Production from Indica IR.64 Rice Straw Biomass by Direct Saccharification and Fermentation
Lignocellulosic substances such as agricultural wastes are attractive feed stocks for bioethanol production. Indica IR.64 rice straw is one of abundant agricultural wastes in Indonesia and could be used to bioethanol production. It has several characteristics such as high content of cellulose and hemicelluloses that can be readily hydrolyzed into fermentable sugars. A simple process (the direct saccharification and fermentation process) to produce ethanol from rice straw was developed in order to establish an efficient bioethanol production. In this work, no harsh pre-treatment steps were applied and also use a simple one-vat reactor without the risk of losing liberated carbohydrate. The first step in using rice straw for bioethanol production is size reduction through milling and sieving process prior to enzymatic hydrolysis. Direct saccharification and fermentation (DSF) of Indica IR.64 rice straw was examined and compared with two type of control (systems devoid of yeast and enzyme). The experiment were carried out under anaerobic condition, where the cellulase crude enzyme and cellulosic substrates (rice straw) produced glucose from the cellulose and Saccharomyces cerevisiae directly assimilated the glucose to bioethanol. The faster rate of bioethanol production during DSF by Saccharomyces cerevisiae was obtained within the first 12h. The maximum ethanol concentration, ethanol yield, and theoretical ethanol yield of untreated rice straw were 0.25 g/L, 10 and 14.88%, respectively. Nevertheless, the direct saccharification and fermentation shows the potential for lower cost and higher efficiency for bioethanol production. Keywords— Bioethanol; Rice straw; Direct saccharification and fermentation; Lignocellulosic waste.
I. INTRODUCTION
At present were are living in a situation where the world's demand for energy continues to increase at a predicted rate of 1.8%, especially as countries develop, while at the same time the supply of energy appears limited. To prevent a looming energy crisis, the development of renewable energy sources is becoming a priority. Renewable energy sources can provide a constant supply of energy, and examples are hydroelectricity, wind, wave power, and biological-based fuels.
In term of biological based fuel, bioethanol is an increasingly important alternative for the replacement of fossil fuel, with a world production of 19,535 millions of gallons in 2009 [1]. Bioethanol made from lignocellulosic biomass including agricultural, forestry residues and woody crops, is finally being recognized widely as a unique alternative fuel with powerful economic, environmental and strategic attributes [2] and currently became a focus in the bioenergy research field.
Lignocellulosic ethanol has recently been produced from agricultural wastes (straw and baggase), which are low cost feedstock and available in plenty and also does not have the ethical concerns associated with the use of potential food resources [2].
Rice straw is one of the most favourable feed stocks for bioethanol production due to their availability throughout the year. Indonesia is one of the major producers of rice straw in the world. The utilization fraction of rice straw is too low and varies with geographic region. Each year a large portion of agricultural residues (rice straw) is disposed as a waste. For instance, approximately 600-900 million tons per year rice straw is produced globally [3]. The options for the disposal of rice straw are limited by great bulk of material, slow degradation in the soil, harbouring of rice stem diseases, and high mineral content. Only a small portion of globally produced rice straw is used as animal feed, the rest is removed from the field by burning, a common practice all over the world. This could increase air pollution and affecting human health. As climate change is extensively recognized as a threat to development there is growing interest in alternative uses of agro-industrial residues for energy applications. In this context, rice straw would be a potential candidate for our future energy needs.
The technologies for the conversion of lignocellulosic feedstocks to ethanol have been grouped into two broad platforms, which can be referred to as the sugar platform (biochemical conversion) and the syngas platform (thermochemical conversion). In this study, we focus to applied biochemical conversion to produce bioethanol from rice straw. The biochemical conversion consists of three main process elements: production of saccharolytic enzymes, hydrolysis to monomeric sugars and fermentation.
The objective of this study is to utilize abundant lignocellulosic biomass (rice straw) for production of bioethanol. Another objective of this study is to obtain costeffective processing and to improve the product yield. This condition can be achieved by applying direct saccharification and fermentation (DSF) process. The saccharification of the lignocellulosic biomass by the enzymes and the subsequent fermentation of the sugars to ethanol by yeast take place in a single reactor in DSF process. The DSF gives higher reported yield and requires lower amounts of enzyme because end-product inhibition from cellobiose and glucose formed during enzymatic hydrolysis is relieved by the yeast fermentation [9], [10]. It should be noted that in this process no harsh pretreatment steps and use a simple one reactor without the risk of losing liberated carbohydrates.
A. Rice Straw
The Indica IR.64 rice straw was obtained from commercial farm in Bogor, Jawa Barat, Indonesia and was harvested at the mature stage. A wet sample of approximately 5 kg was collected in June 2014 and immediately dried at 65 o C for 3 days (to the final moisture 3-5%). The rice straw was milled with a high-speed milling machine (Black & Decker, FX 350, England). Then, the milled samples gradually screened with a sieve shaker 80, 100 and 200-µm-mesh, respectively. The screened materials were stored in desiccators until their use. The composition of rice straw was determined before the experiment.
1) Culture Collection
Cellulolytic isolate Saccharopolyspora flava used in this experiment from Biotechnology Culture Collection (BTCC). The medium for reviving is ISP2 and the composition for 1 Liter medium are 10 g malt extract, 4 g yeast extract, 5 g cellulose substrate (Carboxymetyl cellulose, Sigma).
2) Crude Enzyme Production.
The potential isolate Saccharopolyspora flava were produce spesific crude enzyme such as cellulase inoculated into ISP2 medium with spesific carbon sources (5 g/L Carboxymetyl cellulose, Sigma). The preculture process was incubated in shaker incubator with condition 30°C, 150 rpm for 24 hours and the production culture was incubated for 72 hours in the same medium and condition. Crude enzymes resulted after centrifuge the medium at 10.000 rpm, 4°C for 10 min and collect the supernatant.
3) Enzyme Assays
Enzyme activity were assayed as follows: a reaction mixture containing 0.5 mL of 0.5% Xylan substrate in 50 mM phosphate buffer pH 6.0 as well as 0.5 mL of enzyme solution in the same buffer was incubated at room temperature for 30 min, the addition of 1.5 mL DNS reagent for deactivated the enzyme catalytic activity then measured the solution by spectrophotometer (Shimadzu, Kyoto Japan) at 540 nm in wavelength. The resulting reducing power was determined by dinitrosalicylic acid (DNS) modification method [11] and using xylose as a standard. One unit of the cellulase activity was defined as the amount of enzyme liberating 1 µmol of reducing sugar per minute under the above condition.
C. Yeast Pre-culture Medium
Pre-culture medium (yeast medium) contains 0.3 g/L malt extract, 0.3 g/L peptone, 0.3 g/L yeast extract, 10 g/L glucose. Culture medium then incubated at 30 o C, 200 rpm, for 48 hours.
D. Fermentation
The hydrolysis broth medium was modified by using rice straw as the substrate with final concentration 5 g/L, 4 YP medium, phosphate buffer 200 mM pH 6.0 with final concentration 50 mM and yeast cell OD 600 = 20. Ethanol production was performed under oxygen limited conditions at 30 o C with mild agitation (150 rpm) in 100 mL bottles equipped with an outlet for CO 2 . The ethanol productions were checked for several sampling times (0, 2, 4, 6, 8, 12, 24, 48, 72, 96). The fermentation process was performed in triplicate, and the experimental data are average values. Respectively, two control systems devoid of yeast and enzyme were also prepared. Fermentation yield (g ethanol/g dry substrate) was calculated by the following equation: (1) where E is the ethanol concentration (g/L), V is volume of the reactor (L), and M is the substrate mass in the culture (g). The maximum theoretical ethanol yield was calculated using the following equation: where, E max = maximum produced ethanol (g/L), d = dry weight of biomass (g/L), F = cellulose fraction in biomass (for pretreated rice straw = 0.593) [12], [13].
E. Analysis
The ethanol produce during the sampling times of fermentation were measured by gas chromatography -mass spectrometry (GC-MS) (QP2010UltraPlus, Shimadzu, Japan). This instrument equipped with the DB-FFAP column (60 m x 0.25 mm i.d., 0.5 µm film thickness; Agilent J&W GC Columns). The injection temperature was 250 o C and the flow rate of helium gas through the column was 1 mL/min. The column temperature was held at 40 o C for 5 min isothermally before being increased by 30 o C/min to 230 o C, and held at 230 o C for 10 min. The interface and the ion source temperatures were 250 o C and 230 o C, respectively.
The first step in using rice straw for bioethanol production is size reduction through milling and sieving process (Fig. 1). Milling and size reduction were applied prior to enzymatic hydrolysis. This step can improve susceptibility to enzymatic hydrolysis by reducing the size of the materials and degree of crystallinity of lignocelluloses, which improves enzymatic degradation of rice straw toward ethanol [15], [16], [17]. Unlike woody biomass, the lignocellulosic materials from agricultural residues treatment does not requires as much energy as recalcitrant woody materials to reach size reduction for further hydrolysis process [18]. Size reduction enhanced the susceptibility of substrate to enzymatic hydrolysis. By applied the smallest straw particles in bioethanol production will increase the amount of glucose release for the hydrolysis process compared to untreated substrate [19].
B. Crude Enzyme Production
Cellulolytic microbes can produce cellulase enzymes which have ability to hydrolyze complex polysaccharides into simple molecules such as xylose and glucose as a monomer. Cellulase of microbial origin have been reported to be both induced as well as constitutive enzymes and are usually being secreted extracellular into the medium in which the microorganism is cultured (Fig. 2a).
Further quantitative analyses for these bacteria were conducted such as activity of cellulase, and the cellulase activity is 0.88 U/mL in the third day of fermentation. The optimal pH and temperature range for enzyme production from Saccharopolyspora flava and cellulase activity assay were 6 at room temperature (Fig. 2).
C. Yeast Cells
The yeast strain used in this study is Saccharomyces cereviceae NBRC 1440 from NITE Biological Resource Center (NBRC) collection (Fig. 3). The cells were produced by using fermentation process and collected with centrifugation method at 5000 rpm in 5 minute duration time and wash the cells using milli-Q water for 3 times for eliminate the remain medium. The final condition of yeast cells were adjusted at 600 nm (OD 600nm ) for culture optical density equal to 20 as initial concentration. The low concentration of yeast cells has intention to determine the ethanol production based on short time duration of fermentation in optimum condition.
D. Bioethanol Production by Direct Saccharification and Fermentation
Direct saccharification and fermentation (DSF) of Indica IR.64 rice straw was examined and compared with two type of control (systems devoid of yeast and enzyme). The experiment were carried out under anaerobic condition, where the cellulase crude enzyme and cellulosic substrates (rice straw) produced glucose from the cellulose and S. cerevisiae directly assimilated the glucose to bioethanol.
Bioethanol fermentation media were incubated at 30ºC with agitation at 150 rpm for several sampling times (0, 6, 12, 24, 48, 72, 96 h). The batch culture summarizes the important parameter of ethanol fermentation under the initial cell concentration and size of rice straw biomass (Fig. 5). As shown in Fig. 5, the faster rate of bioethanol production during DSF by S. cerevisiae was obtained within the first 12h. The experiments lasted for 4 days. However, the bioethanol concentrations decrease sharply after 2 days. It gives general conclusion that we need around 2 days to reach the maximum bioethanol concentration under anaerobic conditions (Fig. 5). In addition, it was found that the physical observation of the rice straw biomass has merge in second day (48h) of fermentation and indicating the enzyme works suitable in saccharification process. The concentrations of bioethanol for several sampling times were measured with GC-MS by using isopropylalcohol as an internal standard. The maximum bioethanol concentration during DSF is 0.25 g/L at 24h as an optimum producing time. The chromatogram for GCMS analysis is shown in Fig. 6. The internal standard isopropyl-alcohol with concentration 100 ppm was showed the positive peak of ethanol at retention time 8.712 and identified the amount of ethanol concentration at 0.25g/L.
The fermentation yield and maximum theoretical ethanol yield were 10 and 14.88%, respectively (calculated according to eq. 1 and 2). These values were 9.0 times higher than the control system. The low level of fermentation and theoretical ethanol yield might be due to untreated substrate (there are no appropriate pretreatment steps were applied to remove lignin from rice straw). Consequently, cellulose and hemicelluloses might still be bundled by a great amount of lignin, which would harm the enzymatic hydrolysis and ethanol fermentation [20]. In addition, the use of cellulase crude enzyme and rice straw as cellulosic substrate makes the saccharification and fermentation process could not optimum. However, some other factors may also affect the ethanol yield. Physical properties and cellulose microstructure are among the potential factors influencing enzymatic hydrolysis [21]. The direct saccharification and fermentation process is an extremely simple and environmentally-friendly system without harsh pretreatment. Further integrated research on this process including the optimization/stabilization of feedstock quality and development of harvesting/storage technologies will be indispensable for commercialization of this technology.
IV. CONCLUSIONS
Rice straw could provide us with a great opportunity for bioethanol production. In DSF process, the saccharification of rice straw by the enzyme and the subsequent fermentation of the sugars to ethanol by yeast take place in a single reactor. Despite of this system in this work gave the lower level yields, nevertheless, the value of yield which are greater than in the control system, shows the DSF process are potential to applied. In order to obtain high ethanol yield, the suitable pretreatment method, and the use of commercial enzyme are necessary to consider. | v3-fos |
2016-05-12T22:15:10.714Z | {
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} | s2 | Macrominerals and Trace Element Requirements for Beef Cattle
Eighty-seven Nellore animals were utilized in this study to estimate net requirements for the maintenance and growth of beef cattle as well as the retention coefficients of 13 minerals: macrominerals (Ca, P, Mg, K, Na, and S) and trace elements (Cu, Fe, Mn, Se, Zn, Co, and Cr). The net requirements for maintenance and the true retention coefficient were estimated by using the regression between apparent retention and intake for each mineral. The net requirement for maintenance (μg/kg BW) and retention coefficients (%) were 163 and 85 for Cu, 2,097 and 53 for Fe, 32.3 and 24 for Mn, 3.72 and 48 for Se, 669 and 0.80 for Zn, 18.4 and 86 for Co, and 22.9 and 78 for Cr. The dietary requirements of macrominerals (g/kg DMI) were 5.12 for Ca, 2.38 for P, 0.96 for Mg, 2.40 for K, 0.79 for Na, and 1.47 for S. This is the first study using Nellore cattle to estimate mineral requirements; considering that Nellore cattle are the most common breed in Brazil and that Brazil is a major beef producer globally, this knowledge can help producers to improve animal performance by supplying the correct amount of minerals.
Introduction
Minerals play a role in four types of functions in animals: structural, physiological, catalytic, and regulatory [1]. Thus, information regarding the mineral requirements for body maintenance and growth are essential for beef cattle to reach their maximum productive potential [2]. The beef cattle National Research Council (NRC) [3] suggested that at least 17 minerals are required by beef cattle; however, the requirements of the majority of minerals are suggested in grams or milligrams per kilogram of dry matter intake (DMI) without discrimination between body maintenance or growth status, and the absorption coefficient is not taken into account for trace elements and sulfur. Additionally, requirements for certain minerals are not listed because the available research results are inadequate for accurate determination [3]. Moreover, the available systems for the determination of nutrient requirements were developed using breeds that are uncommon in Brazil, such as Angus and Hereford cattle. Thus, it is necessary to undertake studies using Nellore cattle to determine nutrient requirements for this breed, which is the most utilized cattle breed in Brazil. mm screen (Wiley mill; A. H. Thomas, Philadelphia, PA). At the end of each 7-d period, a composite sample of orts was made for each 42-d period proportionally to weight (DM basis) from each week. For the corn silage, the composite samples were made every 21 d.
Experiment II-heifers and steers. Eighteen Nellore steers (150 ± 44.2 kg) and 32 Nellore heifers (180 ± 41.0 kg) were used in this experiment. Four animals per gender were used as REF animals and were slaughtered at the beginning of the experiment. Four animals from each gender were fed at MAI, and 10 steers and 24 heifers were assigned to the ADL group. The ADL and MAI heifers were further divided into four groups and assigned to the following dietary treatments: 1) Ca and P fed at requirements (CaPR) with a 50:50 roughage:concentrate (R: C) diet, 2) CaPR with a 70:30 R:C diet, 3) 43% Ca and 80% P requirements (CaPL) with a 50:50 R:C diet, and 4) CaPL with a 70:30 R:C diet. The nutrient requirements were considered to be those recognized by the BR CORTE [6]. The ADL and MAI steers were fed CaPR with a 50:50 R:C diet. Half of the ADL animals were slaughtered at d 50 and the other animals were slaughtered at d 100 of the feeding period. All of the MAI-fed cattle were slaughtered at d 100. The values of 43% Ca and 80% P were the lowest values that were achieved when this diet was provided to the animals. The animals were housed in individual pens with concrete floors with a total area of 30 m 2 . This experiment was a completely randomized design with a 2 × 2 + 1 factorial arrangement of treatments which considered two roughage/concentrate ratios, two Ca and P levels, plus steers. The diet was formulated according to the BR CORTE system [6] for a daily BW gain of 0.3 kg. The diet consisted of fresh sugarcane and concentrate, which was formulated with ground corn, soybean meal, limestone, common salt, and a mineral mix ( Table 2). The DM of sugarcane was assessed three times weekly to adjust the amount of urea and ammonium sulfate that was supplied to the animals. The urea/ammonium sulfate (U/AS) mixture was used to adjust the crude protein (CP) content of the diets to 124.0 g/kg DM (19.8 g of N/kg DM).
Feed was supplied twice daily to the animals and adjusted to keep orts at approximately 5 to 10% of the total supplied feed. Water was continuously available. The MAI animals were fed at 11 g/kg BW. The amount of supplied feed was recorded daily. The ingredients in the concentrate were sampled directly from the silos of the feed manufacturing facility each time they were mixed. Feed samples were obtained and composited as described for experiment I.
Digestibility trial
For both experiments, a digestibility trial was conducted immediately before each slaughter period; total feces and urine were collected for three consecutive days from animals that were maintained in tie stalls [7]. At the end of each collection day, feces were weighed and homogenized, and a sample was collected. The samples were weighed, dried in a forced-ventilation oven (55°C) for 72 h, and ground through a 1 mm screen (Wiley mill; A. H. Thomas, Philadelphia, PA). One composite sample per animal, based on the DM weight for every collection day, was prepared. Urine collection was performed via collecting funnels attached to the bulls and steers, while hoses carried the urine to tanks that were kept in polyethylene boxes with ice and 20% H 2 SO 4 to reduce N loss. For heifers, a 2-way Foley catheter (No. 22, Rush Amber, Kamuting, Malaysia) with a 30-mL balloon was utilized to collect urine. A polyethylene tube was attached to the free end of the catheter, through which the urine flowed into a lidded plastic container that held 200 mL of 20% H 2 SO 4 . After each 24 h collection period, the total excreted weight of the urine was determined. The contents of all tanks were then homogenized. A 50-mL sample was obtained and was stored at −20°C for further laboratory analyses.
Slaughter and Samplings
Before slaughter, all animals were fasted for 16 h to obtain the shrunk body weight (SBW). Animals were then slaughtered via captive bolt stunning followed by bleeding. After bleeding, the digesta was removed and discarded. The heart, lungs, liver, spleen, kidneys, the fat around the kidney, pelvis, and heart (KPH fat), diaphragm, mesentery, tails, trimmings, and washed gastrointestinal tracts were weighed. These values were added to the other parts of the body (i.e., carcasses, head, hide, limbs, and blood) to determine the empty body weight (EBW).
The rumen, reticulum, omasum, abomasum, small and large intestines, KPH fat, mesentery, liver, heart, kidneys, lung, tongue, spleen, diaphragm, esophagus, trachea, and reproductive tract were homogenized in an industrial cutter for 20 min. After removing the hide, the head and limbs were also ground in a bone crusher for 20 min. The hide was sampled in two parts to represent the shoulder, three parts to represent the dorsal line, two parts to represent the ventral line, two parts to represent the rear, one part to represent each foot, and one part to represent the head, which altogether represented the entire hide. A composite sample of non-carcass components was constructed in which blood, head, limbs, hide, organs, and viscera were sampled based on the relative proportions of each component after summing all weights of the components.
After slaughter, the carcasses of each animal were split into two half-carcasses which were chilled at 4°C for 18 h. After the 18 h-period, the half-carcasses were weighed again. The left half-carcass was completely separated into muscle, fat, and bone. Muscle and fat were ground together, and the bones were ground separately. A composite sample of the carcass was constructed by using relative individual proportions in the carcass. The non-carcass and carcass samples were lyophilized and ground in a ball mill for mineral analyses.
Mineral Analyses
Corn silage (experiment I), sugarcane (experiment II), the concentrate, feces, urine, non-carcass, and carcass samples were analyzed for macrominerals (Ca, P, Mg, K, Na, and S) and trace minerals (Cu, Fe, Mn, Se, Zn, Co, and Cr). Calcium and Mg were determined by adding up lanthanum [8], and the readings were performed through atomic absorption spectrometry. For Na and K, concentrations were determined using flame emission spectrometry. Inductively coupled plasma-atomic emission spectroscopy with ultrasonic nebulization [9] was used for the determination of the following mineral concentrations: Co, Cr, Cu, Fe, Mn, Se, and Zn.
Procedures Used to Calculate Mineral Requirements
Net requirements for maintenance and the true retention coefficient. The retained minerals in the body were determined for each mineral by subtracting the fecal and urinary mineral content from the mineral intake. The true retention coefficient and the net requirement of each mineral for maintenance were calculated based on regressions between the amounts of each macromineral (mg/kg EBW) and trace element mineral (μg/kg EBW) that was retained in the body and their intakes (Eq 1). Thus, a linear regression between the retained mineral content and its intake was performed by using the following regression model: where MR is the retained mineral in the body, MI is the mineral intake, β 0 is the intercept and is considered to be the net requirement for the maintenance of each mineral, β 1 is the slope and is considered to be the true retention coefficient, and ε i is residual term.
Net requirements for growth. The amounts of each mineral in the body from each animal were estimated as a function of EBW according to the following model proposed by Brody [10]: where Mi = the macromineral (grams) or trace element (milligrams) content in the animal's body, β 0 and β 1 = regression parameters from Eq 2 and ε i is random error. Based on the model parameters presented above, the net growth requirements of each mineral per kilogram of empty body gain (EBG) were calculated by using the derivative of Eq 2 with respect to EBW as follows: where Y = net growth requirement of each mineral (macronutrient = g/kg EBG and trace element = mg/kg EBG). Dietary requirements. After calculating the net requirements for maintenance and growth and the true retention coefficient for each mineral, the dietary requirements were calculated. The sum of the net requirements for maintenance and growth were divided by the true retention coefficient to estimate the dietary requirements.
Running Analysis
To obtain the net requirements for maintenance and the true retention coefficient, the data for retained minerals were analyzed as in Eq 1 through the MIXED procedure of SAS (SAS version 9.3, Institute Inc., 2011, Cary, NC, USA). In instances where the parameters were different from zero, a Variance Component structure of the variance-covariance matrix was used to fit the equations. The NLIN procedure of SAS was used to fit the net requirements for growth as nonlinear models, and the Gauss method was selected for convergence. The parameter was considered to be different from zero if the P-value was less than 0.05.
Results and Discussion
The descriptive statistics of the animals that were used in this study are shown in Table 3, while intake, excretion, and retention of each mineral are shown in Table 4. The study group was composed of animals with SBW between 121.0 and 591.5 kg; average daily gain (ADG) between 0 and 1.95 kg/d, and DMI between 1.18 and 11.1 kg/d.
The first step to meet dietary requirements is to estimate the empty body weight based on the shrunk body weight. In this context, the BR CORTE system [6] and the beef cattle NRC system [3] reported that the relationship between EBW and SBW is constant. However, our study indicated that this relationship might be different since the gastrointestinal content in the whole body decreased as the animal grew. Thus, we regressed the gastrointestinal tract content (GTC, g/kg SBW) on SBW (kg), from which GTC was calculated based on the difference between SBW and EBW (Fig 1). Furthermore, we assumed that the relationship between EBW and SBW is not constant and can be estimated by the following equations: where GTC is the gastrointestinal tract content (g/kg SBW), SBW is shrunk body weight (kg), and EBW is empty body weight (kg). The BR CORTE system [6] and the beef cattle NRC system [3] suggest values of 0.891 and 0.895 for the relationship between EBW and BW, respectively. Young animals have a greater proportion of gastrointestinal tract contributing to BW. For example, a 100 kg animal would have 89.1 and 89.5 kg of EBW according to the BR CORTE system [6] and the beef cattle NRC [6], the Beef cattle NRC system [3], and our calculations, respectively.
Calcium
Calcium is the most abundant mineral in the body [3]. Due to the importance of this mineral in the body, there is a need to estimate dietary Ca requirements. The net Ca requirement that was estimated in the present study for maintenance was 20.0 mg/kg BW (Fig 2A), which was similar to values reported in the beef cattle NRC system [3], the dairy cattle NRC system [11], and the ARC system [4] recommendations ( Table 5). The true absorption coefficient of Ca observed the present study was similar to those suggested by the dairy cattle NRC system [11] and the AFRC system [5] (Table 5), although it was 72% greater (Fig 2A) than those reported in the beef cattle NRC system [3] and the BR CORTE system [6] (Table 5) However, the beef cattle NRC [3] and the BR CORTE [6] publications used the Ca absorption coefficients while discarding urinary losses. In our study, there was considerable mineral excretion in the urine for all of the assayed minerals (Table 4). Calcium excretion in the urine (3.8%) was lower than excretion of other minerals. Thus, we considered the retention coefficient to correctly estimate the mineral bioavailability. We therefore recommend 72% as the true retention coefficient for beef cattle. The net Ca requirement for growth can be estimated by the following equation: Ca = 0.21 × EBW -0.94 , in which the amount of Ca is calculated as grams of Ca per kilogram of EBG. The negative exponent indicates that deposition of Ca is reduced when the animals grow and that younger animals require more Ca than older animals. The dietary requirement for Ca was obtained from the sum of the net Ca requirements for maintenance and growth, and the subsequent division by the retention coefficient. Therefore, for a 300 kg beef cattle with 1.00 kg of ADG, the dietary Ca requirement would be 23.6 g/d ( Table 6).
Phosphorus
The net P requirement for maintenance estimated in the present study was 16.1 mg/kg BW (Fig 2B), which similar values recommended in the beef cattle NRC system [3] and ARC system [4] ( Table 5). The true retention coefficient that was reported in this study (82%; Table 5) was greater than those presented elsewhere [3,5,9]. The absorption of P can vary based on the P content of forage and concentrate, as well as the mineral sources that are used to feed animals [5]. According to the beef cattle NRC system [3], in terms of availability, supplemental sources of phosphorus were ranked (greatest to least) as follows: dicalcium phosphate, defluorinated phosphate, and bone meal [12]. As we used dicalcium phosphate in this study as the main source of phosphorus, the higher true retention coefficient was expected.
The net P requirement for growth can be calculated by the following equation: P = 6.10 × EBW 0.04 , in which the amount of P in the body is calculated as grams of P per kilogram of EBG. The exponent was close to zero, which indicates that the net P requirement for growth is almost constant over time. A different response was reported in the BR CORTE system [6] where the net P requirement for growth decreases as the animal grows. The discrepancy between our results and values reported in the BR CORTE [6] publication may be due to Absorption or retention coefficient (%) the BR CORTE [6] using data from finishing animals in which bone and muscle deposition are low, and considering that phosphorus performs a structural function [3], the requirement for growth in this category of animal is also low. The dietary P requirement for 300 kg beef cattle with 1.00 kg/d of ADG was calculated as 15.4 g/d (Table 6), which was close to those values recommended by the beef cattle NRC system [6] (15.91 g/d). Erickson et al. [13] reported that the dietary P requirement is lower than 1.60 g/kg DM. However, the dietary P requirement that was observed in this experiment was 2.38 g/kg DM ( Table 5). The ARC system [4] also reported that the ratio between Ca and P for ruminant diets is important because both minerals function together in bone formation, and they recommend that the Ca:P ratio is between 1:1 and 2:1. However, the beef cattle NRC system [3] highlighted that the effect of the Ca:P ratio on ruminant performance has been overemphasized in the past [14,15] and that dietary Ca:P ratios between 1:1 and 7:1 result in similar animal performance. The Ca:P ratio that was observed in the present study was 2.15:1.
Magnesium
The estimated net Mg requirement for maintenance was 17.2 mg/kg (Fig 2C). This value is greater than those suggested by the ARC system [4], the dairy cattle NRC system [3], and the BR CORTE system [6] (Table 5). According to the ARC system [4], the urinary endogenous loss of Mg has been ignored. However, our results (Table 4) show that the urinary excretion of Mg represented 21.4% of the total consumed Mg. Thus, we believe that the urinary excretion is important and it should be considered. Based on this data, the true retention coefficient was calculated as 98.3% (Fig 2C). The ARC system [4] reported an overall mean value of 29.4%, but for the calculation of allowances which provide a margin of safety, the lower value of 17.0% was recommended and used by the beef cattle NRC system [3] and the dairy cattle NRC system [11] as the absorption coefficient of Mg. The major difference observed between our results and requirements suggested by the ARC system [4] could be due to the amount of Mg that is released in urine and the breed of the animals that were used in the experiments.
The net Mg requirement for growth can be estimated by the following equation: Mg = 0.35 × EBW -0.02 , in which the exponent is close to zero. This indicates that the net
Potassium
The ARC system [4] separated endogenous losses such as feces (2.6 g/kg DMI), urine (37.5 mg/kg BW), saliva (0.7 g/100 kg BW), and skin (1.1 g/d) by reporting different estimates for each loss for K. Therefore, the net K requirements for maintenance can be achieved by considering the sum of each loss. However, the data used by the ARC system [4] were from one study [17] that utilized nine heifers in a 3 × 3 Latin square to evaluate mineral balance. Thus, when aiming to standardize this estimate, we estimated the net K requirements for maintenance to be 33.0 mg/kg BW/d while accounting for all losses (Fig 2D). When considering a 300-kg animal with a DMI of 7.5 kg/d, we estimated the net K requirement for maintenance using the ARC system [4] to be 33.95 g/d. This value was greater than those observed in this study (9.9 g/d) and those proposed by the dairy cattle NRC system [11] at 11.4 g/d (38 mg/kg BW). These differences can be explained by the low number of observations that generated the estimates for the ARC system [4], while in this study, we utilized 87 observations, which provided a greater precision of the estimate. Ward [18] reported that K is absorbed from the rumen and omasum as well as from the intestine. The ARC system [4] used the absorption coefficient to convert net to dietary requirements and assumed 100% as the K absorption coefficient. However, in this study, the retention coefficient was calculated as 69.8% (Fig 2D; Table 5). Ward [18] also indicated that urine is the major route of K excretion, and body reserves of K are minimal. Our results suggest that this cannot be accurate, as we have shown that the urine excretion represents only 16% of the animal's intake, while the fecal excretion and the amount that is retained by the cattle are 49 and 35%, respectively. The dairy cattle NRC system [11] reports a true absorption coefficient of 90%. Therefore, we recommend the use of 69.8% as the true retention coefficient for K.
The net K requirement for growth can be estimated by the following equation: K = 1.43 × EBW 0.03 , in which the exponent is close to zero indicating that the net requirement for growth does not vary considerably when the animal grows. However, the BR CORTE system [6] found that the net K requirement for growth increases when the animal grows. For a 300-kg beef cattle with 1.00 kg/d of ADG, the dietary K requirement would be 46.5 g/d ( Table 6). This value is greater than the value of 34.4 g/d recommended for the same animal category by the BR CORTE system [6].
Sodium
The ARC system [4] suggests that dietary Na can be freely and completely absorbed by cattle, and that the concept of strict endogenous fecal loss does not apply to Na. However, our data shows that there was a considerable amount of Na lost in the feces and urine (Table 4; 63.1 and 34.6% of the total consumed amount, respectively). Based on this data, the net Na requirement for maintenance was 8.51 mg/kg BW (Fig 2E), which was close to those values recommended by the ARC system [4] and the BR CORTE system [6] (Table 5); however, it was lower than those suggested by the dairy cattle NRC system [11](15 mg/kg BW). The beef cattle NRC system [3] and the dairy cattle NRC system [11] recommend 91 and 90% as the absorption coefficient for Na, respectively. However, Bankir et al. [19] reported that vasopressin increased Na reabsorption, and between 65 to 80% of the filtrate is reabsorbed. In the present study, we estimated the true retention coefficient for Na to be 57.6%, which was greater than the value proposed by the BR CORTE system [6], which was 19%.
The net Na requirement for growth can be estimated by the following equation: Na = 0.89 × EBW 0.05 , in which the exponent approaches close to zero indicating that the net requirement for growth does not vary considerably when the animal grows. When the dietary requirement is represented as grams per kilogram of DMI, the value that was presented in this study was 0.96, which was close to those suggested by the beef cattle NRC system [3] and the CSIRO system [16], which were both 0.80 mg/kg DMI.
Sulfur
Despite the fact that the beef cattle NRC system [3] and the dairy cattle NRC system [11] reported that S requirements of beef cattle were not well defined, they recommended that dietary S requirements should be between 1.5 and 2.0 g/kg DMI. In fact, the net S requirement for beef cattle maintenance and the true retention coefficient have not been evaluated. In this study, maintenance requirements and the retention coefficients were 9.4 mg/kg BW and 67%, respectively. The net S requirement for growth was estimated by the following equation: S = 0.03 × EBW 0.89 . The net S requirement for growth had different characteristics than those of other macronutrient minerals, because most of the macronutrient mineral requirements decline or do not change when the animal grows. For a 300 kg beef cattle with 1.00 kg of ADG, the dietary S requirement would be 10.5 g/d (Table 6). When expressed as gram per kilogram of DMI, the dietary S requirement is 1.47 g/kg DMI, which was close to the recommendations of the beef cattle NRC system [3] but lower than that of the dairy cattle NRC system [11]. This difference might be because data from the dairy cattle NRC system [11] was provided by only one study [20] that used mid-lactation dairy cows producing 30 to 37 kg milk/d.
Copper
The ARC system [4] reported that the net Cu requirement for maintenance was 7.1 μg/kg BW; however, this value was generated from an equation with variables such as Cu intake, hepatic Cu loss, and change in BW. Each of these components in the equation has certain assumptions. Also, two studies involving cattle yielded estimates of 1.8 and 0.8 μg/kg BW, respectively [21,22]. The CSIRO system [16] adopted 4.0 μg/kg BW as the net Cu requirement for maintenance based on a study developed by Suttle [23]. On the contrary, the net Cu requirement for maintenance in this study was 163 μg/kg BW, which is greater than those reported by the ARC system [4] and the CSIRO system [16]. Also, the ARC system [4] estimated the Cu absorption coefficient as 6% when using the hepatic Cu retention technique. In this study, the true retention coefficient when using the mineral balance technique was 84.7% (Fig 3A), thus showing a value that is greater than those proposed by the ARC system [4]. However, the ARC system [4] reported that the efficiency of absorption and the hepatic retention of copper have not been detected in cattle, and that the recommendation of this council was provided based on data using sheep. Thus, we recommend the use of 84.7% as the true retention coefficient for beef cattle. Also, the net Cu requirement for growth can be estimated by the following equation: Cu = 1.25 × EBW 0.33 in which Cu had a similar response to that observed for S, since the Cu requirement for growth increases as an animal grows.
Mullis et al. [24] estimated Cu requirements of Angus and Simmental heifers to be 7 mg/kg DMI. However, these authors did not consider the amount of Cu in the diet. Therefore, the Cu requirement might be lower than the value that was suggested in this study. A dietary Cu requirement of 9.53 mg/kg DMI should be adequate (Table 5). Iron Thomas [25] reported that insufficient dietary Fe will reduce body stores as well as plasma Fe and blood hemoglobin concentrations. However, no studies have attempted to estimate the net Fe requirement for maintenance and the true retention coefficient. The net Fe requirement for maintenance that was estimated in this study was 2,097 μg/kg BW, while the true retention coefficient was 52.7% (Fig 3B). The net Fe requirement for growth can be estimated by the following equation: Fe = 15.5 × EBW 0.43 .
Bremner and Dalgarno [26] evaluated Fe requirements in calves and recommended that a dietary intake of 40 mg of soluble Fe/kg DMI is enough to prevent the development of anemia. Bernier et al. [27] recommended an additional Fe supplementation between 30 and 50 mg/kg DMI to avoid anemia. The large difference between the beef cattle NRC system [3] recommendation and our data (50 vs. 218 mg/kg DMI; Table 5) may be due to differences in basal diet Fe concentrations, physiological status of the animals, and breed differences across studies.
Manganese
The body of a normal 70 kg animal is estimated to contain a total of 10 to 20 mg Mn [28]. The amount of Mn in the body is distributed widely throughout the tissues and fluids and may vary with age, species, organs, and in relation to other dietary trace elements. Schroeder et al. [29] assumed that 20 to 25 mg Mn/kg DMI is necessary for an animal to have optimum skeletal development. However, no study has evaluated the net Mn requirement for maintenance. The net Mn requirement for maintenance in this study was 32.3 μg/kg BW and the true retention coefficient was estimated to be 23.6% (Fig 3C). The dairy cattle NRC system [11] suggested that the net Mn requirement for maintenance and the true absorption coefficient is 2.00 μg/kg BW and 75%, respectively. Although, some authors [30][31][32] have reported that approximately 1 to 4% of dietary Mn is absorbed, irrespective of dietary concentration. Hurley and Keen [33] reported that several factors, including a high concentration of Ca, P, and Fe in the diet may decrease Mn absorption.
The net Mn requirement for growth can be estimated by the following equation: Mn = 0.07 × EBW 0.80 . Bentley and Phillips [34] concluded that 10 mg/kg DMI can meet the Mn requirements for growth in young heifers. This value was reported by the BR CORTE system [6] as the Mn requirement for growth. Hartmans [35] fed cows for 2.5 to 3.5 years with diets containing 16 to 21 mg of Mn/kg diet DM, and did not report any signs of Mn deficiency or improvements in animal performance when supplementing Mn. In this study, the dietary Mn requirement was estimated to be 9.59 mg/kg DMI, which was lower than the recommendation of 20 mg/kg DMI by the beef cattle in the NRC system [3].
Selenium
The Se concentration in the body is dependent upon the amount and chemical form of Se in the diet, as well as the type of tissue that is evaluated. High concentrations can occur in the liver and kidneys, but the largest amount of Se is sequestered by muscles [36]. There are no published experiments that have evaluated the net Se requirement for maintenance or the retention coefficient for beef cattle. The net Se requirement for maintenance was estimated to be 3.72 μg/kg BW in the current study. Wright and Bell [37] evaluated the absorption coefficient in sheep and swine and found that 35% of ingested isotopic Se was absorbed in sheep. The value in this study (48.7%; Fig 3D) is greater than values reported by Wright and Bell [37] and the CSIRO system [16]; however, it was close to the dairy cattle NRC system [11] recommendation ( Table 5). The net Se requirement for growth can be estimated by the following equation: Se = 1.07 × EBW -0.07 .
Oh et al. [38] fed lambs with milk and increasing Se concentrations from 0.01 to 0.05 mg/kg DMI, and observed an increase in glutathione peroxidase activity, but the maximal enzyme activity was not obtained until the diet provided at least 0.1 mg Se/kg DMI. However, these authors did not consider the amount of Se provided by milk. The dietary Se requirement was estimated to be 0.57 mg/kg DMI, which is greater than those values observed by Oh et al. [38] and reported in the CSIRO system [16], though they were close to those suggested by the dairy cattle NRC system [11] (Table 5).
Zinc
Some researchers [39][40][41] have reported that the Zn concentrations in plants and animals are often comparable to those of Fe and that are usually greater than those of most other trace elements [42]. The beef cattle NRC system [3] used an average of several studies [43][44][45] to estimate the endogenous urinary Zn loss of 12 μg/kg BW (ranging from 4 to 19 μg/kg BW). Weigand and Kirchgessner [46] evaluated the Zn requirements of lactating dairy cows and estimated that the net Zn requirement for maintenance to be 53 μg/kg BW. The dairy cattle NRC system [11] and ARC system [4] estimated the net Zn requirement for maintenance to be 55 μg/kg BW, while the CSIRO system [16] estimate the net Zn requirement to be 45 μg/kg BW. These values are lower than those that were estimated in the present study (669 μg/kg BW; Fig 3E). The ARC system [4] also used an absorption coefficient for Zn of 30% for young growing ruminants, and a value of 20% for mature animals based on data from several studies [44][45][46][47][48]. The CSIRO system [16] adopted a true absorption coefficient of 60% for pre-ruminant calves and 40% for older animals with a functional rumen [49]. Our estimate, based on the retention coefficient, was calculated to be 80%.
The net Zn requirement for growth can be estimated by the following equation: Zn = 1.16 × EBW 0.86 . The ARC system [4] suggests that 16 to 31 mg Zn/kg BW may be incorporated into body tissue for each kilogram of body weight gained. The values for dietary Zn requirements in this study were greater than the values reported in the beef cattle NRC system [3] recommendations (Table 5). However, the data that provided the beef cattle NRC system [3] with estimates were from studies [50,51] that evaluated the growth response to additional Zn supplementation where the Zn content in the basal diet was unknown in certain studies.
Cobalt
Smith [52] reported that the efficiency in which animals obtain vitamin B 12 from dietary Co is very low. Some studies [53,54] verified that 84 to 98% of the dietary Co appeared in the feces within 5 to 14 days. However, the net Co requirement for maintenance was 18.4 μg/kg BW, while the true retention coefficient was 85.6% (Fig 3F), thus showing a different response from the previous studies. The net Co requirement for growth can be estimated by the following equation: Co = 0.04 × EBW 1.00 . The net Co requirement for growth increases proportionally to increases in animal body weight.
Smith [52] also suggested that the dietary Co requirement was 0.11 mg/kg DMI. This value was adopted by the beef cattle NRC system [3] and the dairy cattle NRC system [11], but they did not consider the absorption coefficient and feed composition. In the present study, the average dietary Co requirement was 2.78 mg/kg DMI (Table 5).
Chromium
No study was specifically conducted to evaluate the net Cr requirements for maintenance and growth or the true retention coefficient. However, other studies [55,56] evaluated Cr supplementation in calves and suggested that the addition of Cr at 0.4 mg/kg DMI increased the glucose clearance rate. Bernhard et al. [57] evaluated the effects of Cr supplementation on the performance of steers and observed a difference in ADG between steers without Cr supplementation and those that were supplemented with 0.3 mg of Cr per kilogram of DMI. In our study, the net Cr requirement for maintenance was 22.9 μg/kg BW, while the true retention coefficient was calculated as 78.4% (Fig 3G). The net Cr requirement for growth can be estimated by the following equation: Cr = 0.23 × EBW 0.61 , in which the amount of Cr is calculated as milligram of Cr per kilogram of EBG. For a 300 kg beef cattle with 1.00 kg of ADG, the dietary Cr requirement is 18.6 mg/d ( Table 6). This value represents 2.53 mg/kg DMI, which is greater than published data (0.2-0.4 mg/kg DMI; Table 5).
Conclusions
The use of the true retention coefficient improves the estimates of dietary requirements for Na, K, Mg, and S due to the considerable excretion of these minerals in urine. Also, the use of the net requirements for maintenance and growth and the true retention coefficient for the majority of the trace minerals resulted in different estimates than those obtained from the literature. This study provides information about mineral nutrition of Nellore cattle and would be useful in dietary formulations in countries that use this breed, such as Brazil.
Supporting Information S1 Spreadsheet. The spreadsheet included as supporting information can be used to meet mineral requirements for maintenance and growth such as dietary requirements using different body weights and average daily gain. | v3-fos |
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} | s2 | Mutagenic activity of cadmium chloride on the genetic variability of soft wheat
: The action of the chemical compound – CdC1 2 on soft wheat varieties resulted in plant modifications on a number of qualitative and quantitative traits. Genetic analysis conducted on the basis of reciprocal crosses showed that the inheritance of altered traits in mutants is independent of the direction of crossing. Modification of habitus and phenotypes of mutant plants is accompanied by a violation of cell division in meiosis.
Introduction
Chemical mutagens are an effective means of formative processes in wheat breeding and receiving selection and significant deviations [1,2]. Obtaining of mutants and using them for hybridization are required to study genetic nature of appearing changes which have great importance for the selection of effective and specific action of mutagens, and for extension and deepening of understanding the nature of the evolution of wheat. In this work we present some results of research on genetic analysis of the mutant wheat. New forms, such as dwarf mutants in wheat and barley, ultra-fast mutants in barley, resistant to fungal diseases, high-leasing and highly productive mutants might be obtained qualitatively by chemical mutagenesis [3]. These facts show that the mutants obtained by chemical mutagenesis can successfully serve as progenitors of new high-yielding varieties. However, to obtain mutants and study them -this is only the first stage of the selection work. More important is the using mutants in the hybridization to obtain positive transgressions. The hybridization makes possibility to better use mutations in wheat breeding [4][5][6]. Cytological studies were carried out using a microscope LOMO Mikmed-1. Genetic analysis of qualitative and quantitative traits of wheat F 1 and F 2 hybrids were conducted. Statistical analysis was limited to estimation of the arithmetic mean and in order to determine the reliability of the difference between the arithmetic means of quantitative traits using the Student's t test, genetic -finding a significant χ2 value [7,8]. Accounting of chromosomal abnormalities in MI, AI and AII of meiosis was performed on temporary acetocarmine preparations under the microscope MBI-3. The representativeness of research result provided an adequate sample size -60-100 plants.
Results and their discussion
Genetic analysis of mutant wheat. Chemical mutagenesis in plant breeding is used as an effective method in order to enhance the variability of the starting material. In the world literature there is sufficient information about the creation of commercial varieties derived from experimental mutagenesis. To apply selected mutants in selection process is necessary to examine their genetic nature. For this, in genetic research are using two methods: analyzes and reciprocal crosses.
Analyzing cross. In order to establish the nature of any mutational change by variables usually used carrying reciprocal crosses between the original form and receiving on the basis of its mutant subsequent analysis of the hybrids F 1 . In our studies M 2 generation plants with modified number of quantitative and qualitative characteristics was preserved the properties displayed in M 1 . To establish the homo and het-https://doi.org/10.26577/2218-7979-2015-8-2-56-59 erozygous genotype of mutant plants was carried out analyzing cross with an initial variety.
Mutant forms with signs of anthocyanin coloration of the stem, pubescent leaf surface, lengthening with spike crossed with an initial variety of Kazakhstanskaya 3. In BC 1 splitting signs to change and corresponds to the normal ratio of 1:1, and in F 2 is 3: 1 ((χ2 = 1.89). Similar results were obtained with the mutant varieties of Shagala on the grounds of anthocyanin coloration of stem and leaf axils. BC 1 and F 2 hybrids were observed splitting on the grounds of lengthening the stem and normal nodes in the ratio of 1:1 and 3:1, respectively, which indicates that the heterozygous nature of the mutant and monogenic inheritance of this trait.
In contrast, cleavage by productive tillering, length and density of the spike in BC 1 corresponded to 3:1, and an F 2 population of 15:1, 13:9 and 3:7, respectively. This shows that the treats of mutant lines are inherited by a polymer, and complementary mechanisms of epistatic interactions of non-allelic genes. This shows that plants reaction by chemical compounds depends on wheat genotype. Further studies displayed arising changes in M 1 by the elements of productivity of Kazakhstanskaya 3 and Shagala varieties appeared in subsequent M 2 -M 6 generations. It was proved to conduct reciprocal crossing, where the modified attributes are inherited independently from direction of the crossing. Phenotypic variation of plants was accompanied by a violation of the process of meiosis.
Cytological analysis of M 2 mutant plants.
Chemical mutagens because of its ability to induce a higher frequency of mutations are used in many countries around the world to create a breeding material. Chromosomal aberrations and violation of cell division during meiosis is one of the major tests for mutagenicity. Most notable in this regard is a meiotic cell division, especially in subjects like wheat, having a large number of hardly identifiable chromosomes. Moreover, violations of meiotic division are more likely to be transmitted to the next generation.
In mutant plants of M 2 generation percentage of damaged cells in MI meiosis equals 35, and at anaphase AI and AII -20, which indicates a significant reduction in percentage of cells with disorders compared to M 1 mutant plants (64% AI and 68% -AII). Violation of this phenomenon is cytomixis -transition of contents to neighboring cells, M 1 amounts 20-30% of all the studied cells, while the percentage of such cells in M 2 decreases to 7-9%. So, the percentage of abnormalities in mutant forms of Kazakhstanskaya 3 M 2 equals 55%, in contrast, violation in M 1 -90-95%.
Violation of meiosis in mutant plants of Kazakhstanskaya 3 variety is shown on Fig. 1-4.
Same decrease in percentage of violations is observed in mutants of Jenis, Lutescens 32 and Shagala varieties. In AI and AII some minor violations as a lagging chromosome fragments on the pole, bridge, asynchronous division. Occasionally cells with no content are observed. | v3-fos |
2017-11-08T18:54:46.649Z | {
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} | s2 | Ethylene resistance in flowering ornamental plants – improvements and future perspectives
Various strategies of plant breeding have been attempted in order to improve the ethylene resistance of flowering ornamental plants. These approaches span from conventional techniques such as simple cross-pollination to new breeding techniques which modify the plants genetically such as precise genome-editing. The main strategies target the ethylene pathway directly; others focus on changing the ethylene pathway indirectly via pathways that are known to be antagonistic to the ethylene pathway, e.g. increasing cytokinin levels. Many of the known elements of the ethylene pathway have been addressed experimentally with the aim of modulating the overall response of the plant to ethylene. Elements of the ethylene pathway that appear particularly promising in this respect include ethylene receptors as ETR1, and transcription factors such as EIN3. Both direct and indirect approaches seem to be successful, nevertheless, although genetic transformation using recombinant DNA has the ability to save much time in the breeding process, they are not readily used by breeders yet. This is primarily due to legislative issues, economic issues, difficulties of implementing this technology in some ornamental plants, as well as how these techniques are publically perceived, particularly in Europe. Recently, newer and more precise genome-editing techniques have become available and they are already being implemented in some crops. New breeding techniques may help change the current situation and pave the way toward a legal and public acceptance if products of these technologies are indistinguishable from plants obtained by conventional techniques.
INTRODUCTION
The production of potted ornamental plants and cut flowers is a growing industry with annual turnover in the tens of billions of dollars, 1 with the European Union being the single largest producer and consumer. 2 Due to the competitive nature of the ornamental plant industry, research and development of new products and improvement of quality in existing products have become essential. The quality of flowering ornamentals, often judged by the longevity of their flowers, is an extremely important parameter in assessing quality. 3 During the postharvest period, plants will likely experience stress as a result of poor lighting, temperature, and suboptimal humidity or watering. 4,5 This stress often leads to visual symptoms such as wilting, color change, and abscission of various plant parts including flowers, petals, and buds. In climacteric plants, stress triggers the production of the phytohormone ethylene, which quickly deteriorates plants visually. Ethylene of exogenous origin can also affect the plants in closed spaces 6 and plants that have been exposed to ethylene will often no longer be sellable.
In recent years, the biological significance of ethylene in ornamental production, its signaling pathway within the plant, and methods to alleviate its consequence to the aesthetic value of ornamental plants have been extensively reviewed. [7][8][9][10][11][12][13] However, many reviews only consider a limited number of plant species, or focus on a particular perspective for solving the problem in only one or two of the levels of the ethylene pathway. In order to acquire an overview of the problems associated with ethylene-induced quality losses in ornamental plant production, recent research and advances in the field of ethylene biology and breeding techniques must be considered. The present review aims to compare results from past approaches in order to discuss future breeding strategies.
It will also point out avenues not yet explored in the area of ornamental plant breeding which may be essential for reducing ethylene responses that remains a decisive factor for high-quality production of many ornamental plants.
REGULATION OF THE ETHYLENE PATHWAY
The ethylene biosynthesis and signaling pathway can be presented in a linear model ( Figure 1). Ethylene is essential for many processes in the plant and thus, there is a constant, low ethylene production ( Figure 1a). 14 Under certain conditions, however, ethylene biosynthesis and sensitivity increase in specific tissues and this triggers the ethylene signaling pathway (Figure 1b). 15 This initially starts as an increase in expression of some of the enzymes responsible for ethylene biosynthesis [16][17][18] which leads to higher ethylene production 19 that may amplify itself in an autocatalytic fashion in some cases. 20 Recently, it was shown that different ACS homologs of Dianthus caryophyllus 18 and Dianthus superbus 21 were expressed in association with either the basal or the climacteric phase. In Petunia 22 and Paeonia suffruticosa, 23 the same was observed for certain ACS and ACO homologs. It therefore seems that these enzymes, central for ethylene biosynthesis (Figure 1), may hold the key for the ability of the plant to transition from basal to climacteric ethylene production ( Figure 1). 24 Furthermore in Arabidopsis, ACS activity was demonstrated to be affected by the formation of hetero-and homo-dimers, 24,25 and phosphorylation by the mitogen-activated protein kinase (MAPK) MPK6, which itself is expressed in response to stress. 26 Recently, a similar role for MPK6 was demonstrated in Rosa hybrida. 27 The function of ethylene receptors too, is subject to modification from various proteins such as RESPONSIVE TO ANTAGONIST1 (RAN1), 28,29 REVERSION TO ETHYLENE SENSITIVITY1 (RTE1), [30][31][32] and their own histidine autokinase activity. 33 Receptors often act simultaneously, and in physical association with each other, 34,35 but as with the ethylene biosynthesis enzymes, different receptor homologs are expressed depending on the developmental stage of the flower, as has been documented in D. caryophyllus 36 , R. hybrida, 37 Oncidium, 38 Delphinium, 39 and Pelargonium hortonum. 40 The function of EIN2 can be inhibited not only by CTR1 but also ETHYLENE RESISTANT/ETHYLENE RECEPTOR1 (ETR1) 41 and EIN2 TARGETING PROTEIN1 (ETP1) and ETP2. ETP1 and ETP2 were down-regulated in the presence of ethylene, 42,43 but activated CTR1 in the absence of ethylene. 44 Unlike most of the other genes of the ethylene pathway, EIN2 has no homologs and no functional overlap with other genes. [45][46][47] There is some indication that EIN2 expression is influenced by auxin and abscisic acid (ABA), 48 which means that EIN2 could be a point of crosstalk for several different pathways. Interestingly, increase in EIN2 protein levels has been shown to be concomitant with expression of the transcription factor ORESARA1 (ORE1) in Arabidopsis, associated with age-induced programmed cell death. ORE1 is suppressed by miRNA164, which declines with cell age. 49 In R. hybrida flowers exposed to ethylene, different miRNAs including miRNA164, exhibited a change in expression level in petals, 50 indicating a possible new level for expression control of ethylene genes.
EIN3/EIL homologs also change in the transition of the plant tissue to the climacteric phase. In D. caryophyllus, DcEIL3 increased 51 and DcEIL1 decreased 52 in expression as flower development occurred. Tanase et al. 53 on the other hand documented that DcEIL3 expression did not change with age in petals, but DcEIL4 was expressed less in older flowers. In P. suffruticosa, PsEIL1 accumulated from the flower opening stage to senescence, while PsEIL2 and PsEIL3 decreased after flower opening. 54 PsEIL2 and PsEIL3 mRNA levels increased in response to exogenous ethylene, while PsEIL1 was unaffected by this treatment and plants treated with 1methylcyclopropene (1-MCP) and exposed to ethylene, exhibited a decrease in PsEIL3 expression. 54 In Oncidium gardneri, OgEIL1 and OgEIL2 were constitutively expressed in flower buds, but when the buds were exposed to ethylene, OgEIL1 clearly peaked in expression relative to OgEIL2. 55 Collectively, these genes present numerous targets for modifying ethylene regulation molecularly.
REDUCING ETHYLENE-PROMOTED SENESCENCE
Approaches addressing the problem of ethylene-induced senescence can be broadly divided into two main groups: interventions directly addressing the ethylene pathway and those indirectly targeting it as described below. Because the ethylene pathway is fundamentally integrated into plant metabolism, changing a seemingly unrelated pathway of the plant can result in some effect on the ethylene pathway. Techniques to combat senescence such as changes in the gas composition of the atmosphere or temperature of storage places for plants have also been examined and gave mixed results. [56][57][58] These techniques, although successful to a point, lead to unsustainable production of ornamental plants because they extend the time needed for production and cost in terms of consumption of electricity, water, personnel, and specialized equipment. Silver thiosulfate (STS) and 1-MCP have been proven to be powerful inhibitors of ethylene responses and STS is commonly used in ornamental plant production. However, silver is harmful to humans and the environment and thus its use should be avoided. Plant breeding on the other hand strives to improve the intrinsic quality of ornamental plants and thereby produce sustainable products. 59
TARGETING THE ETHYLENE PATHWAY DIRECTLY
Cross-pollination coupled with ethylene screening The simple strategy of choosing individuals in a population that display superior flower ethylene tolerance and crossing these individuals with each other, will result in progeny with a lower ethylene sensitivity than the original population, if the trait is heritable. 60 Success in such breeding has been reported in Begonia 61 and D. caryophyllus. 62 It has long been known that there are vast differences among cultivars of D. caryophyllus in their ethylene biosynthetic ability and affinities of receptors to ethylene. 63 By using only simple pollination, Onozaki and coworkers 62 achieved a significant improvement in the longevity of D. caryophyllus flowers from 1992 to 2004. Their original material had flower longevity of 5.7 days, 64,65 but through repeated crossing, the sixth generation had increased its mean of flower life to 15.9 days. 62 An investigation into the cause of this increase, revealed that the ethylene biosynthesis enzyme genes DcACS1, DcACS2, and DcACO1 were all expressed in extremely low levels in cultivars with a good longevity and ethylene production was as low. 66,67 This approach involves screening for ethylene insensitivity and will only be applicable if a population exhibits significant differences in response between individuals. The number of ornamental flower cultures showing such diversity is, however, very large. Differences in longevity of flowers have been noted for Paeonia, 68 Delospermum, Campanula, Sedum, Cephalaria, Lobelia, Armeria, Primula, Penstemon, 69 Lisianthus, Trachelium, Zinnia, 70 Potentilla, Lysimachia, Veronica, Chelone, 71 and Rosa 11,72,73 which also exhibited varying ethylene production levels. [73][74][75][76] Similar observations has been made for Pelargonium, 77 where heritability has also been documented, 78 as it has been for Impatiens walleriana, 79 Antirrhinum majus, [80][81][82] Dianthus barbatus, 83 and Petunia. 84 In many ornamental plant genera, cultivar-specific variation in ethylene sensitivity has been demonstrated including Phalaenopsis 85,86 and Kalanchoë. 87 Hybridization Many of the abovementioned genera are typically represented by hybrids in ornamental plant production. Hybridization is achieved by interspecific cross-pollination and it thus produces extremely heterogeneous progeny, which may be good for producing cultivars with higher ethylene tolerance. A well-documented example of this is found in Australian waxflower breeding where Chamelaucium species were hybridized with Verticordia plumosa, forming more ethylene-insensitive plants. 88 Hybrids of Leptospermum species 89 and Grevillea 90 similarly have been reported to have longer longevity than the parental species, which may be correlated with higher ethylene tolerance as both genera are climacteric. This aspect, however, has not yet been investigated.
The major disadvantage of both intra-and interspecific crosspollination is that it cannot be used directly for improving existing cultivars. Considerable back-crossing to the original plant may be necessary. 91 This problem can be solved to some extent by employing marker-assisted selection, if reliable markers are found 92 and have been used successfully in ornamentals such as D. caryophyllus with regards to bacterial wilt resistance. 93 Hybridization can be more demanding as manipulation of the style as well as embryo rescue or ovule and ovary culture may be necessary in order to yield any progeny, as exemplified in Kalanchoë species hybridization. 94 Conventional mutagenesis Techniques that increase genetic variability where no natural variation exists are found in conventional breeding with the use of mutagenic chemicals or radiation. 95 Such breeding strives to change the cultivar in a certain qualitative aspect and leaves its agronomical traits unchanged, making it easier for producers to handle, and saves time that would otherwise be spent on backcrossing. 71,96 However, as it is impossible to control where the mutation occurs in the genome, many plants will have to be screened before a plant exhibiting any change in the relevant trait is found. 97 Targeting Induced Local Lesions In Genomes (TILLING) can be used to save time that would otherwise be needed for phenotyping the plants and has been used successfully in the field of ethylene-response improvement. Dahmani-Mardas et al. 98 produced Cucumis melo with a knockout mutation in the CmACO1 gene which displayed a longer shelf life and better firmness than nonemutated plants. TILLING is readily applicable to ornamental plants as is exemplified in Petunia. 99 Genetic transformation Genetic transformation is the transfer of foreign or native genes and promoters to a target genome by Agrobacterium-mediated transformation, particle bombardment, or infiltration. Agrobacterium tumefaciens-mediated transformation with recombinant DNA has been used in various ways to modify ethylene biosynthesis and signaling. These approaches have improved understanding of ethylene biosynthesis and signaling; however, their use in commercial breeding is limited and this technique's products are considered genetically modified. 59,100 Antisense or sense transformations of ACS and ACO genes have been successfully attempted in various ornamental species including D. caryophyllus, [101][102][103][104][105][106][107][108][109][110][111][112][113] Torenia, 104 Petunia, 22,105 and Begonia. 60,106 All transformed plants exhibited longer shelf life, presumably due to lower production of ethylene. Klee et al. 107 conducted an alternative modification of the biosynthesis pathway with the removal of ACC by the addition of the enzyme ACC deaminase, found in bacteria, to Solanum lycopersicum which significantly delayed ripening of the fruit. However, even though plants with lower ethylene production have a longer flower life, their quality can still be negatively affected by exogenous ethylene.
Transformations of Petunia with the Atert1-1 mutated gene from Arabidopsis using CaMV 35S constitutive promoter, resulted in plants with considerably higher ethylene tolerance but also with severely hampered growth. 108 Succeeding studies used a flowerspecific FBP1 promoter in Kalanchoë, 109 Campanula, 110,111 and D. caryophyllus, 112,113 which provided a better flower longevity without other developmental effects, since the promoter ensured expression solely in flowers. Transformations of mutated genes other than Atetr1-1 have also been studied in Nemesia strumosa 114 and Torenia fournieri. In both cases, ethylene insensitivity was increased, but not as much as with Atert1-1 ( Table 1). 8 Other promoters such as FBP3, 115 P SAG-12 , 116 FS19, and FS26 8 that are solely associated with flower tissue, also have potential to be used in this context and other promoters are still being investigated. 117 Chemically inducible promotor systems are also within the reach of today's technology, and are exemplified by the DEX-inducible system, demonstrated in Petunia hybrida 118 (Table 1), and more famously, the ethanol-inducible system 119 which has been successfully used in different crops. 120 Inducible systems seem not to affect the plant adversely, 121 but the inducing chemical still needs to be applied to the plant in due time, which makes such a system commercially less appealing.
Great care needs to be exercised when choosing a promoter, as a study by Cobb et al. 122 demonstrated that Petunia transformed with Atetr1-1 did not have delay in senescence under the flower-specific APETALA3 (AP3) promoter, as this promoter only drives expression in buds and young flowers, but not in mature flowers (Table 1).
Overexpressing PhEIN2 in Petunia using the CaMV 35S promoter has also been attempted. This produced plants with significant delays in petal senescence in response to exogenous ethylene or pollination, as well as inhibited root development and a shorter life span. 126 When this silencing transformation was combined with the Atetr1-1 mutation, ethylene was inhibited even more, 127 demonstrating the possibility of increasing insensitivity by stacking transformations of several components simultaneously.
Petunia plants transformed with a sense PhEIN3 exhibited delayed petal senescence, but no increase of flower longevity, 8,127 which may possibly be due to the redundancy found among EIN3/EIL members. S. lycopersicum was transformed with a construct silencing EIN3 BINDING F-BOX1 (EBF1) and EBF2, which rapidly target EIN3/EILs for degradation that significantly increased the rate of senescence in S. lycopersicum. 45 This presents an opportunity for future research, potentially by overexpression of EBFs.
ERFs are a sizable and diverse group of genes, many of which are up-regulated in response to ethylene, but have largely been overlooked by researchers so far. Chang et al. 128 observed that by silencing the ERF Petunia transcription factor homeodomain-leucine zipper protein (PhHDZip), PhACS and PhACO were decreased in expression in transformed flowers and led to an increase of flower longevity by 20%.
Genome-editing technologies During the last decades, several innovative biotechnologies have been developed which appear to have potential in breeding toward ethylene tolerance. 100,129,130 These technologies are based on engineered nucleases that cleave DNA in a sequence-specific manner, thus enabling targeted genome-editing. Modifying or inactivating specific gene function is possible due to sequence-specific DNA binding domains or RNA sequences. The oldest of these techniques is zinc finger nucleases (ZNFs), which relies on an engineered endonucleases that are able to attach to a specific target sequence and induce a double-strand break. This is perceived by the cell, which repairs the break by mechanisms which may cause sequence alterations or introduce small templates. 131,132 A newer alternative to ZNFs is transcription activator-like effector nucleases (TALENs), which functions in much the same manner as ZNFs. Recently, however, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein9 (CRISPR/Cas9) system has been developed. The CRISPR/Cas9 system functions via a mechanism similar to RNA interference, which can recognize and cleave foreign DNA. 100 Both TALEN and CRISP/Cas9 have been demonstrated to be functional for targeted mutagenesis in S. lycopersicum, 133,134 however, no modifications of the ethylene pathway have yet been conducted. It is important to mention that all these methods rely on a technology in order to deliver the construct to the genome of the plant, and are therefore still limited to plants where a genetic transformation, regeneration or virus-based delivery system is reliable. In order to bypass negative effects on growth of plants due to defective ethylene signaling, the genes that should be targeted are those which have homologs that are clearly associated with the climacteric phase. Using this approach, the pathway should function normally under growth and development. Therefore, certain homologs of ACS, ACO, ethylene receptors, and EIN3/EIL may present good targets for knockouts.
Hormonal interaction
Application of cytokinins such as kinetin and zeatin to petals of D. caryophyllus delayed the conversion of ACC to ethylene. 135 Cytokinin oxidase/dehydrogenase, responsible for cytokinin degradation, was up-regulated during senescence in D. caryophyllus 136 and Petunia, 137 and when it was inhibited in D. caryophyllus petals, the senescence phase was prevented. 138 This knowledge has been applied by transformations with isopentenyl transferase, important for the synthesis of many cytokinins, under the control of senescence-associated promoter P SAG12 . Transformed Petunia plants exhibited elevated levels of cytokinins in flowers and significant delay in senescence and ethylene production as well as higher tolerance for exogenous ethylene. 139 Similarly in Rosa, ethylene sensitivity decreased in leaves, but flowers were not studied. 140 Exogenous application of hormones and other substances have also been shown to decrease the expression of genes of the ethylene pathway including ABA, which inhibited ACS and ACO in Hibiscus 141 and D. caryophyllus. 142 Moreover, nitric oxide downregulated the activity of RhACO and lowered the production of ethylene resulting in longer shelf life of R. hybrida flowers 143 and glucose down-regulated PsACS1 in P. suffruticosa flowers. 144 Other sugars have also been shown to increase flower longevity. 136,[145][146][147][148] These studies demonstrate that there is a vast potential in exploring new ways to achieve products of higher quality.
Senescence-related and non-ethylene pathway genes Exploration of different genes which is seemingly not connected to the ethylene pathway has also been pursued. There are three main strategies in this area. The first is identification of genes that are highly expressed in young tissue but not during senescence, and constitutively overexpressing those. The second approach goes the other way and starts with the identification of genes highly expressed in senescing tissue, and silencing their expression. The third tactic is targeting protein synthesis.
The first strategy can be exemplified by the gene FOREVER YOUNG FLOWER (FYF) from Arabidopsis, which was highly expressed in young flowers but not in old flowers. Transformation using this gene under constitutive expression in S. lycopersicum resulted in Ethylene resistance in ornamentals A Olsen et al down-regulation of various ACS and ACO homologs. The same transformation in Eustoma grandiflorum caused a delay in senescence and down-regulation of ERFs. 149 Furthermore, when combined with etr1, ein2, or ctr1 mutations, it further enhanced flower longevity in Arabidopsis. 149 Investigating a MADS-domain transcriptional regulator, AGAMOUS-LIKE-15 (AGL15), Fernandez et al. 150 noted its accumulation in young tissue and developing organs. Constitutive overexpression of AGL15 increased the longevity of petals significantly in Arabidopsis 151 and caused a repression of GmACO1 in Glycine max. 152 It is now established that AGL15 has similar activity as AGL18, both serving as repressors of early flowering and in this way leading to better longevity if still expressed when flowering does occur. 153 Another MADS-domain transcriptional regulator that has been recognized to be associated with young flowers of Phalaenopsis equestris is PeMADS6. Arabidopsis plants transformed with a construct overexpressing PeMADS6 exhibited flower longevity up to four times longer than wild type plants. 154 Examples from the second strategy of silencing senescencerelated genes, include ACTIN-RELATED PROTEIN 4 (ARP4), a chromatin modification gene, that has been silenced using RNAi in Arabidopsis, 155 which resulted in longer flower life. The gene MjXB3, coding for a RING zinc finger ankyrin repeat protein, has been demonstrated to be highly expressed in senescing flowers of Mirabilis jalapa, P. hybrida, and D. caryophyllus. Using virusinduced gene silencing for MjXB3 in P. hybrida, Xu et al. 156 demonstrated that flower longevity was extended by two days, corresponding to a 20% increase compared to wild type flowers.
The third strategy of targeting protein synthesis owes its inspiration to ethylene-insensitive flowers, 13 where complete inhibition of protein synthesis in flowers increases flower longevity. 157 The relevance of this to climacteric flowers has been attested by silencing PBB2 (coding for the beta subunit of the 26S proteasome) using an inducible system in Petunia. 158 Mature cut flowers that were induced lasted considerably longer than uninduced flowers. Reid and Jiang 13 demonstrated the same concept by silencing one of the ribosomal subunit genes (RPL2), again using an inducible system. Once more, the longevity of cut Petunia flowers placed in water with the inducing agent was much better than those uninduced. This approach, however, still relies on the application of an inducer, and is not realistic for use in commercial potted-plant production. For the cut-flower industry, however, there may be great potential.
Natural transformation Transformation with the naturally occurring soil bacterium Agrobacterium rhizogenes has been termed natural transformation 159 due to the fact that no recombinant DNA is used and the infection is a natural process. Authorities in Denmark have confirmed that naturally transformed plants are not covered by the GMO legislation in Europe. 59 Using a wild strain of A. rhizogenes, Lü tken et al. 59,160 transformed Kalanchoë plants and observed that plants which contained rol-genes from A. rhizogenes exhibited significantly increased postharvest quality, 159 an ability which was maintained through two generations along with the rol-genes. 161 The mechanism behind this response is yet unknown and many open reading frames of the plasmids in question still remain uninvestigated. Changes in longevity of flowers transformed with rolgenes may be due to altered hormone homeostasis and/or sugar metabolism and transport. 159
DISCUSSION
Strategies targeting the ethylene pathway indirectly seem to be as effective as those which target it directly. Of the indirect strategies, ectopic overexpression of genes associated with juvenile tissue, such as FYF or AGL15, seems to be particularly promising, as is the prospect of overproduction of hormones that are antagonistic to ethylene, such as cytokinins and ABA. Strategies that focus on the ethylene pathway directly have been more numerous and have targeted nearly all known elements of the ethylene pathway. Research comprising flower-specific promotor sequences in genetic transformations seems to be especially propitious in providing the ability to express the transferred gene only in flowers or even more specifically, only in senescing flowers. Silencing several genes simultaneously in the flowers of plants as in Petunia, 119 may be the ultimate answer, and is a feature that is well within the grasp of current technology and methodology. Various combinations of overexpression of certain genes associated with development and silencing genes associated with senescence may very well be a worthwhile strategy as well.
It is remarkable that both conventional techniques such as simple cross-pollination and genetic transformation using recombinant DNA succeeded in breeding ornamental plants which have higher tolerance for ethylene and thus higher intrinsic quality. Agrobacterium-mediated transformation, where reliable, has the advantage that breeding can be much faster when transferring specific target genes. Breeding based on genetic transformation is more precise and can easily cross incompatibility boundaries in comparison with conventional crossing. 91 This is a particularly important advantage in ornamental plants that have long development cycles such as orchids, 162 however, in fast growing plants, this advantage is less substantial. 60 Although conventional and genetic transformation are not mutually exclusive, the fact remains that ornamental plant products that are genetically modified are conspicuously missing on the market, especially in Europe, with only a handful of products sold worldwide. 163 The basic reason for this is the status of such product as genetically modified organisms (GMOs). Europe, the biggest consumer, producer and breeder of ornamentals has some of the most comprehensive and strict legislation concerning GMOs. 1 At the moment, the approval of GMO products is conducted on a caseby-case basis and is both expensive and time-consuming. 164 Moreover, the technology and training required for producing GMOs are themselves expensive, which brings a substantial economic burden. 60 Producers not operating in Europe also face difficulties as GMO legislation may be quite different from country to country. 165 However, the emergence of new genome-editing techniques presents new challenges particularly for European legislation in the area of GMOs, which could lead the way to broader acceptance of new breeding techniques and their products.
Techniques such as ZFN, TALENs, and CRISPR/Cas9 that can be used to achieve precise mutagenesis and silencing or overexpressing genes are now readily available, and can be used in such a way that does not introduce foreign elements to the genome of the final product. 127,166 The question of whether the products of these techniques are considered GMOs or not has not yet been settled, and jurisdictions that consider the methods alone rather than the actual properties of the final plant product may end up with ineffective legislation that cannot be enforced, as pointed out in Seeds of Change. 167 Certainly such techniques have the ability to greatly reduce the time, and thus the bulk of the cost which is associated with plant breeding. The first commercially available plant derived from a genome-editing technique, a herbicide-resistant oilseed rape, has already been announced. 168 There are still technical difficulties associated with the actual transformation step for many ornamental plants, which can only be overcome by further research. For this reason, it is must be of the utmost importance for any plant breeding company to devote some of its resources to research in such new breeding techniques.
CONCLUSIONS
Strategies targeting the ethylene pathway directly seem to have as much potential as those targeting indirect components, and very Ethylene resistance in ornamentals A Olsen et al diverse species of ornamental plants suggest great potential in ethylene tolerance breeding. Although conventional breeding techniques are slower than newer breeding techniques, they remain the most used in ornamental plant breeding for higher ethylene tolerance. This is most probably due to the legal status of genetic modification approaches. However, as newer precise genome-editing techniques become available, it is very likely that the products of such techniques will be accepted worldwide. The reason for this is that such plants would be indistinguishable from plants derived by the use of conventional breeding techniques. It is therefore recommendable for ornamental plant breeders to begin the implementation of such new breeding technologies, as they have great to potential to lead to superior plant products needed by the ornamental plant industry. | v3-fos |
2018-04-03T05:18:09.232Z | {
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} | s2 | Data on antioxidant activity in grapevine (Vitis vinifera L.) following cryopreservation by vitrification
Cryopreservation is used for the long-term conservation of plant genetic resources. This technique very often induces lethal injury or tissue damage. In this study, we measured indicators of viability and cell damage following cryopreservation and vitrification-cryopreservation in Vitis vinifera L. axillary buds cv. “Flame seedless” stored in liquid nitrogen (LN) for: three seconds, one hour, one day, one week and one month; after LN thawed at 38 °C for three minutes. The enzymatic activity of catalase (CAT) and superoxide dismutase (SOD), as well as the amount of malondialdehyde (MDA), total protein and viability were assayed.
Value of the data
This data provides information on the effect of cryopreservation in axillary grapevine buds, and in tissue antioxidant activity. The data obtained shows the behavior of the antioxidant system cryopreservation-vitrification in different times of storage in liquid nitrogen.
Information of the antioxidant effects in cryopreserved buds produced here provides a tool to understand how the tissues adapt to this extreme environment.
This data can be used to evaluate different preservation techniques.
Data
The data shared in this article is the viability and tissue antioxidant activity of 'Flame seedless' grapevine axillary buds in several cryopreservation conditions, which were stored at different times.
Viability
Cryopreserved and vitrified-cryopreserved buds showed differences in viability with treatment. No differences (P 40.05) were found between cryopreserved (Fig. 1a) and vitrified-cryopreserved ( Fig. 1b) treatments, but both treatments were significantly different compared to the control.
Antioxidant activity
No significant effects (P 40.05) were detected in the CAT activity due to the thawing step (Fig. 2b). However, the CAT activity showed a large decrease with respect to the control (Tukey, P o0.05). A reduction in the enzymatic activity of the vitrified-cryopreserved buds compared with the cryopreserved buds (Fig. 2a) was observed. No significant differences were observed in the SOD activity of the cryopreserved buds with or without a thawing step (Fig. 2c), and the enzymatic activity showed a tendency to decline compared to the control as the storage time increased. In contrast, the SOD activity in the vitrified-cryopreserved tissues (Fig. 2d) showed a large decrease compared with the control (Tukey, P o0.05). The SOD activity in the cryopreserved buds ( Fig. 2c) was higher than in the vitrified-cryopreserved buds (Fig. 2d). No differences were found in the treatments without thawing ( Fig. 3a) with respect to the controls (C) and (P). Comparison between the cryopreserved and vitrified-cryopreserved buds after 1 h in LN revealed that the highest level of MDA was observed in the vitrified-cryopreserved buds (0.11 mmol g À 1 FW) compared to the cryopreserved buds (0.025 mmol g À 1 FW). No significant differences were found among the treatments for the other storage times (Tukey, P 40.05). Significant differences were detected between the vitrified-cryopreserved and cryopreserved buds at the initial time (I) and after one month of storage in LN (Fig. 3b).
Protein content significant differences were found between the vitrification-cryopreservation treatments at the initial time with thawing and after one month in LN and for the treatments with or without the thawing step ( Table 1). The highest concentration of protein (48.5 mg g À 1 FW) was found in the treatment with no thawing step after one month of storage in liquid nitrogen. No significant differences (Tukey, P4 0.05) were observed among the other treatments; for each storage time in LN, a higher amount of total protein was obtained in the buds that were in contact with the PVS2 solution compared with cryopreservation either with or without thawing. In addition, a higher amount of total protein was recorded in the tissues treated with vitrification-cryopreservation compared to the control (Tukey, Po 0.05).
Cryopreservation procedure
The rootstocks were randomly selected and washed three times with water. Axillary buds were dissected with a sterile razor blade and disinfected in commercial chlorine solution at 25% (1.3% NaOCl) with 0.1% Tween-20 for 5 min and then rinsed three times with sterile distilled water. The samples were then treated with the systemic fungicide benomyl (100 ppm) for 3 min, washed again with sterile distilled water and with 70% ethanol (v/v), and then washed with sterile distilled water three times. For cryopreservation, the disinfected buds (five replicates per treatment with five buds each, n ¼25) were transferred to sterile 2-mL polypropylene cryovials and immersed directly into LN. The sampling was carried out at the initial time of freezing (3 s) and after storage for an hour, a day, a week, and a month in LN. After each freezing period, cryovials containing frozen buds were obtained without thawing and then thawed rapidly in a water-bath at 38°C for 3 min.
Vitrification-cryopreservation procedure
For vitrification-cryopreservation, the disinfected buds were treated according to the procedure described by Matsumoto and Sakai [1] with some modifications. The plant vitrification solution N°2 (PVS2) contained 30% (w/v) glycerol, 15% (w/v) ethylene-glycol, and 15% (w/v) dimethyl-sulfoxide in MS medium with 0.4 M sucrose at pH 5.8 [2]. The buds were disinfected as described above and transferred into 2-mL cryovials (five replicas per treatment with five buds each, n¼ 25) containing 1 mL of PVS2 solution previously sterilized by filtration. The control treatment did not include the PVS2 solution. The samples were incubated at 2572°C with agitation for 180 min. This was the best incubation time according to a previous viability assay using grapevine buds (data not shown). The control treatment samples were not frozen. The cryovials were directly immersed in LN, and an initial sample was taken immediately after 3 s of freezing; the remaining buds were stored for an hour, a day, a week, and a month. After each treatment, one sample was obtained without thawing and another was thawed in a water-bath at 38°C for 3 min. The PVS2 solution was removed, and the buds were washed with sterile distilled water.
Viability assay
Viability was estimated using a triphenyltetrazolium chloride (TTC) reduction assay [3]. Five buds were incubated in 5 mL of 0.1% TTC solution in 0.05 M potassium phosphate buffer (pH 7.5). The Table 1 Effect of thawing on total protein content (mg g À 1 ) of fresh weight in 'Flame seedless' grapevine buds treated for different times with vitrification or vitrification-cryopreservation. reaction was performed at 30°C for 24 h, after which the buds were washed with sterile distilled water. Formazan was extracted from viable cells with 5 mL of 95% ethanol at 80°C for 10 min in the dark. The supernatant was separated by centrifugation (10,000 rpm for 5 min) [4]. The absorbance of the extracted formazan was measured at 530 nm in a Cary 50 UV-visible spectrophotometer. The tissue viability was expressed as the percentage reduction in TTC activity compared to that of control cells (non-cryopreserved fresh tissue) and was calculated as described by Alotto et al. [5] [6,7] by quantifying the photochemical reduction of nitro blue tetrazolium by the change in absorbance at 550 nm using a Cary 50 UV-visible spectrophotometer. CAT (EC 1.11.1.6) activity was determined at 25°C according to the method described by Aebi [8] by monitoring the decrease in absorbance of hydrogen peroxide (H 2 O 2 ) at 240 nm during 1 min at 25°C using a Cary 50 UV-Visible spectrophotometer from Varian.
Malondialdehyde concentration
Lipid peroxidation was determined by quantifying the concentration of malondialdehyde. For the extraction of MDA from the treated buds and controls, the buds were ground with liquid nitrogen to a fine powder, which was then homogenized with 50 mM phosphate buffer (pH 8.3) containing 2.8% NaH 2 PO 4 Á H 2 O and 1.8% of Na 2 HPO 4 and centrifuged at 12,000 rpm for 50 min. After centrifugation, the supernatant was used for the quantification of MDA [9,10] using an "Oxitek TBARS assay" kit, which contains the reactive substance thiobarbituric acid. The entire procedure was carried out by carefully following the manufacturer's instructions (ZeptoMetrix Corporation). The MDA level was expressed as mmol per gram of fresh weight (FW).
Total protein content
The amount of total protein was determined according to the Bradford method [11]. Bovine serum albumin from Sigma (USA) was used as the standard, and the enzymatic activities were expressed as specific activity by determining the protein concentration in each sample.
Data analysis
The data obtained were analyzed using an analysis of variance (ANOVA). Significant differences among the treatments were determined by the Tukey-Kramer multiple range test at a significance level of 95%. All data were analyzed using the statistical package NCSS (Statistical Number System) version 2007. | v3-fos |
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} | s2 | Concentrations of Morphine and Codeine in Paired Oral Fluid and Urine Specimens Following Ingestion of a Poppy Seed Roll and Raw Poppy Seeds
Interpretation of opiate drug test results can be challenging due to casual dietary consumption of poppy seeds, which may contain variable opiate content. Opiate concentrations in paired oral fluid (OF), collected with the Oral-Eze® Oral Fluid Collection System, and urine were analyzed after ingestion of poppy seeds from the same source, consumed raw or contained in a roll. In Part 1, 12 individuals consumed equal portions of a poppy seed roll. For Part 2, the same individuals consumed an equivalent quantity of raw poppy seeds, containing ∼3.2 mg of morphine and 0.6 mg of codeine. Specimens were analyzed both by enzyme immunoassay (opiates) and by GC–MS (morphine/codeine). Urinary morphine was between 155–1,408 (roll) and 294–4,213 ng/mL (raw), measured at 2, 4, 6 and 20 h post-ingestion. Urinary codeine concentrations between 140–194 (roll) and 121–664 ng/mL (raw) were observed up to 6 h post-ingestion. Following consumption of raw poppy seeds, OF specimens were positive, above LOQ, from 0.25 to 3.0 h with morphine ranging from 7 to 600 ng/mL and codeine from 8 to 112 ng/mL. After poppy seed roll consumption, morphine concentrations of 7–143 ng/mL were observed up to 1.5 h with codeine detected in only 5.5% of OF specimens and ranging from 8 to 28 ng/mL. Combined with the existing poppy seed literature, these results support previous findings and provide guidance for interpretation of OF opiate testing.
Introduction
Morphine and codeine are naturally occurring substituents of the poppy plant, Papaver somniferum (1). Since the 1980s, concerns regarding positive opiate drug tests following the ingestion of poppy seeds in bagels, pastries and food prepared according to various cultural traditions have been expressed (2)(3)(4)(5)(6)(7)(8)(9)(10). This has led to the so-called 'poppy seed defense' as an explanation for positive morphine and codeine findings in urine drug tests. Therefore, distinguishing between dietary poppy seed ingestion and legitimate versus non-prescribed opiate or heroin abuse is important for individuals performing safety-sensitive duties as well as other workers subject to routine drug testing for opiates. Previous studies have shown that ingestion of poppy seeds can result in urinary concentrations of morphine in excess of the established Department of Health and Human Services' (DHHS) Substance and Abuse Mental Health Services Administration (SAMHSA) drugtesting cutoff of 2,000 ng/mL [see Lachenmeier et al. (11)]. The opiate content of poppy seeds varies greatly and is dependent on the seed origin and method of processing, with documented morphine concentrations ranging from 0.1 to 294 mg/g (4-6, 9, 12, 13). To minimize the number of positive opiate tests resulting from poppy seed consumption (i.e., 'incidental exposure' from food products), SAMHSA raised the federally mandated cutoff concentration for morphine and codeine from 300 to 2,000 ng/ mL in November 1998 (14).
Urine has historically been used in both federally regulated and non-regulated (company policy) workplace drug testing. However, oral fluid (OF) is also increasingly being used in clinical and nonregulated forensic drug testing settings. OF is a suitable alternative specimen due to its ease of collection, difficulty adulterating or substituting and detection window which better reflects potential impairment or more recent drug use (15 -17). In July 2012, SAMHSA's Drug Testing Advisory Board (DTAB), a scientific council which advises SAMHSA's Federal workplace drug-testing program, issued recommendations to evaluate OF as an approved alternative specimen for federally regulated workplace drug testing programs.
To date, there are limited studies in OF using commercially available collection devices to evaluate the impact of consumption of poppy seed-containing products (bagel, cakes, rolls, etc.) on opiate test results. Detection of morphine and codeine subsequent to poppy seed exposure has been explored primarily in urine (2,3,6,9,11,13,18,19) and serum (3,13,20); yet fewer studies have been published in alternative matrices such as hair (21) and OF (22 -24). The purpose of the present study is to investigate opiate analytical results in paired urine and OF specimens, following ingestion of poppy seeds of known opiate content, using the Oral-Eze w Oral Fluid Collection System and the CEDIA w Opiate OFT Assay, an FDA-cleared collection and testing system, and GC-MS testing. Morphine and codeine concentrations were monitored in urine and OF collected from participants after consumption of a Ukrainian-style poppy seed roll and raw poppy seeds in a two-part study.
Study design
Twelve (seven male/five female) healthy volunteers, 26 -64 years old and weighing 49.0-88.6 kg participated in a two-part study. In the first part of the study each participant consumed one traditionally baked Ukrainian-style poppy seed roll, which was prepared with 15 g of poppy seeds per serving. During preparation, the poppy seeds were rinsed using room temperature water, then soaked in hot water (.808C) to allow for seed expansion. The swollen seeds were ground with recipe ingredients (nuts, sugar, etc.), spread on flattened dough and formed. The rolls were allowed to rise for 3 -4 h (25 -358C) prior to baking for 35 -50 min at 3508F (1758C). For the second part of the study, 2 days later, participants ingested 15 g of raw poppy seeds from the same source used to prepare the rolls. Three participants completed Part 2 within 7 days from Part 1.
All study volunteers provided written informed consent to Quest Diagnostics Incorporated to participate in the research study. Each participant was assigned a unique coded identifier, which was used to document the date and time on the custody and control form used during specimen collection for Parts 1 and 2 of the study. Baseline ( pre-ingestion) OF and urine specimens were collected up to an hour before the start of each part of the study. Participants were permitted to drink up to 180 mL of fluid (evenly divided, where possible) during the first 2 h, then ad libitum for the remainder of each part of the study. Participants were instructed to consume the poppy seeds within 15 min and were permitted to drink during this time; OF collection began 15 min after the completion of poppy seed ingestion. OF specimens were collected at 0.25, 0.5, 1, 1.5, 2 and 3 h after poppy seed ingestion (baked roll and raw) and also at 4, 5 and 6 h post-ingestion. Urine was collected at 2, 4 and 6 h after ingestion. Approximately 20 h (upon arising the following morning) after poppy seed ingestion, the last OF and urine void was obtained.
Specimen testing
Coded specimens were transported, accessioned and processed for routine drug testing at Quest Diagnostics Laboratory (Lenexa, KS, USA). All OF and urine specimens collected were screened by enzyme immunoassay and confirmed for morphine and codeine via gas chromatography -mass spectrometry (GC -MS), using previously validated methods established by the NLCP certified and CAP-FDT accredited toxicology laboratory.
Poppy seed opiate concentration and serving size Poppy seed concentrations of morphine and codeine were determined by sonication of methanolic extracts containing 0.1% acetic acid for 2 h (25). Quantitative values obtained by GC-MS analysis of poppy seed aliquots were run in duplicate using three representative samples from the same source of seeds used during the study (n ¼ 6), which were purchased from City Market (Kansas City, MO, USA). The poppy seeds contained a mean concentration of 219.6 mg/g of morphine and 39.3 mg/g of codeine, with coefficients of variation calculated at 8.2 and 6.1%, respectively. In Part 1, individual participants consumed a roll containing 14.58 g (dry weight before preparation) of poppy seeds per serving, for a total of 3.2 mg of morphine and 0.6 mg of codeine. In Part 2, the total serving of morphine was 3.3 mg and codeine was 0.6 mg after consumption of 15 g of raw poppy seeds.
Immunoassay
Initial testing for OF and urine was performed on a 5400 Beckman Coulter automated chemistry analyzer (Beckman Coulter, Brea, CA, USA) with methods validated for workplace drug testing analyses, using controls targeted at +25 and +50% of cutoff concentration for urine and OF, respectively. For OF screening, morphine and codeine concentrations were determined by b-galactosidase recombinant DNA technology using Thermo Scientific CEDIA w Opiate OFT Assay (Thermo Fisher Scientific, Fremont, CA, USA), implementing a 30 ng/mL cutoff in neat OF. Drug concentrations in urine were determined using the DRI w Opiate Assay (Thermo Fisher Scientific), which utilizes a decision point of 2,000 ng/mL for opiates (morphine).
Measurement of creatinine
Creatinine concentration was determined using a kinetically modified Jaffe procedure (Beckman Coulter Urine TOX reagent), with the rate of change proportional to creatinine concentration, measured bichromatically at 520 and 800 nm wavelengths. Urinary drug concentrations are expressed as raw quantitative values or normalized to 100 mg/dL of creatinine and reported as ng of drug per 100 mg of creatinine, designated as Creat 100 . Urine samples were normalized to creatinine to minimize the impact of urine dilution effects on drug concentrations.
Urine
All collected samples, whether the initial screening was presumptive positive or negative, were quantified for morphine and codeine by GC -MS using a cutoff concentration of 2,000 ng/mL. Each calibrator, quality control (QC) and participant sample contained a final concentration of 2,000 ng/mL of morphine and codeine deuterated internal standards (ISTDs). To 1 mL of urine, 2 mL of 2.0 M sodium acetate buffer ( pH 5.0) was added prior to the addition of 100 mL of b-glucuronidase (Campbell Science, Rockford, IL, USA). Samples were hydrolyzed for 2.5 h at 608C. Three milliliters of 0.1 M phosphate buffer ( pH 6.0) was added to the samples, which were centrifuged (2,500 Â g), then extracted using UCT Clean Screen TM DAU Solid Phase Extraction (SPE) Columns (Waltham, MA, USA) and eluted with methylene chloride : isopropyl alcohol (80 : 20, v/v) containing 2% ammonium hydroxide. Eluates were concentrated under nitrogen and derivatized with N-methyl-bis(trifluoroacetamide).
Quantitative testing was performed on an Agilent 5975C gas chromatograph-mass spectrometer utilizing previously validated analytical methods. For each confirmation run, calibration was achieved using a single-point calibrator, target concentration of 2,000 ng/mL, for both morphine and codeine. Negative, 40 and 125% cutoff QC samples were analyzed with each analytical run. Chromatographic separation was achieved with a 3.0 mg/mL injection of extracts (split 40 : 1) on a 5% phenylmethyl silicone DB5 cross-linked capillary column (15 m, 0.25 mm; Agilent Technologies, Santa Clara, CA, USA) with an inlet temperature of 2808C and gradient temperature programing. Extracts were monitored in SIM mode at (quantification ions in bold) m/z 364, 477 and 311 for morphine (m/z 367, 480 morphine-d 3 ) and m/z 282, 395 and 283 for codeine (m/z 288, 401 codeine-d 6 ). GC/MSD Chemstation software (Agilent Technologies) was used for data processing. Extracts were considered acceptable if the peaks were symmetrical with valid (+20%) ion ratios, both 40 and 125% control were within +20% of the established range, negative control(s) had a quantitative value less than the limit of detection (LOD) and peaks were within 2% of the retention time standard.
Oral fluid OF was collected using the Oral-Eze Oral Fluid Collection system, which implements a 3-fold dilution of original (neat) OF with buffer preservative solution contained in the collection/transport tube. All participant samples, whether the initial screening was presumptive positive or negative, were quantified for morphine and codeine by a previously validated GC -MS method using a cutoff concentration of 30 ng/mL (neat OF equivalent). Morphine and codeine deuterated ISTDs were added to each calibrator, QC and participant sample to achieve a final concentration of 39.9 ng/mL (equivalent concentration in neat OF). One milliliter of 0.1 M sodium acetate buffer ( pH 4.5) and 500 mL of 10% methoxamine were added to each 300 mL aliquot of calibrator, QC and participant sample. After 1 h incubation, samples were extracted with CEREX Trace B solid phase extraction columns (SPEware, CA, USA) and eluted with ethyl acetate containing 2% ammonium hydroxide. Eluates were evaporated to dryness and derivatized using N,O-Bis (trimethylsilyl)trifluoroacetamide containing 1% trimethylchlorosilane. Extracts were injected onto a 6890N Agilent GC System equipped with a Deans Switch and coupled to a 5975 Agilent Mass Selective Detector using negative chemical ionization and utilizing deuterated ISTDs for retention time and quantification reference. Calibration was performed for each confirmation run using a single-point calibrator, target concentration of 30 ng/mL, for both morphine and codeine. Negative, 50 and 125% cutoff QC samples were included with each analytical run.
Chromatographic separation was achieved with a 5.0 mL injection volume ( pulsed splitless) on column 1 consisting of a DB-1 column (15 m, 0.25 mm, 0.25 mm; Agilent Technologies) with an inlet temperature of 2508C and a pulse pressure of 40.0 psi and column 2 consisting of a DB-17 column (15 m, 0.25 mm, 0.25 mm; Agilent Technologies) using ultra-pure helium carrier gas. SIM acquisition mode was used to monitor the following ions (quantitation ions in bold): m/z 429, 430, 401 for morphine (m/z 435, 420 morphine-d 6 ) and m/z 371, 343, 372 for codeine (m/z 377, 349 codeine-d 6 ). GC/MSD Chemstation software was used for data acquisition, and extracts were considered acceptable if peaks were symmetrical, the negative control was less than the LOD, both 50 and 125% cutoff QC controls were within +25% of the established mean and peaks were symmetrical with valid (+20%) ion ratios within 2% of the retention time standard. Table I were calculated using sample (n 2 1) standard deviation.
Results
Poppy seeds were obtained from a local spice market, analyzed and determined to contain 3.2 mg of morphine and 0.6 mg of codeine per 15 g (dry weight) portion of poppy seeds. For Part 1, participants consumed equal servings of a traditional Ukrainianstyle poppy seed roll prepared with 15 mg of poppy seeds per serving. Part 1 of the study (roll consumption) was started on experimental Day 1, with Part 2 (raw poppy seed ingestion) performed on the second day (i.e., Day 3) after the Part 1, with the exception of the three participants referenced in the Study Design section above. Urine and OF specimens confirmed by GC-MS at or above the cutoff of 2,000 ng/mL (urine) and 30 ng/mL (OF) were qualitatively positive by enzyme immunoassay. All participants' baseline ( pre-ingestion) urine and OF specimens (Studies 1 and 2) were negative.
Urine specimens
Urine specimens were analyzed by enzyme immunoassay for amphetamines (1,000 ng/mL), cocaine metabolites (300 ng/mL), THC (50 ng/mL), opiates (2,000 ng/mL) and phencyclidine (25 ng/mL) with confirmation of morphine, codeine, hydromorphone, hydrocodone and 6-acetylmorphine performed by GC -MS with a limit of quantitation (LOQ) of 75 ng/mL for morphine and codeine. A total of 120 urine specimens were collected for baseline testing and post-ingestion analysis at 2, 4, 6 and 20 h. Overall, 95.8% (46/48) of specimens were positive for morphine, with 6.2% (3/48) of specimens positive for codeine at or above the LOQ in Part 1 (roll) of the study. In comparison, 100% of samples were morphine-positive and 47.9% codeine-positive after Part 2 (seeds). Table I provides the mean and range of raw urinary opiate concentrations, along with standard deviation and sample size for each time point after roll and raw poppy seed ingestion. Morphine C min was 155 ng/mL (6 h) with C max at 1,408 ng/mL (2 h) after consumption of the poppy seed roll. Codeine C min was 140 ng/mL (4 h) with a C max of 194 ng/mL (4 h). After raw poppy seed ingestion, the C min was 188 ng/mL for morphine and 121 ng/mL for codeine, at 2 and 6 h, respectively. C max after raw poppy seeds was 4,213 ng/mL for morphine (4 h) and 664 ng/mL for codeine (4 h). The percent of morphine and codeine-positive urine specimens at each collection time and cutoff level are shown in Table II. Based on the GC -MS testing and using a 300 ng/mL cutoff, 75% (36/48) of the total specimens collected at 2, 4, 6 and 20 h after Part 1 (roll) contained morphine concentrations above cutoff, whereas no specimens were positive for morphine when a 2,000 ng/mL cutoff was utilized. In Part 2 (raw), depending on the time after collection, up to 100% of specimens were positive for morphine (300 ng/mL), with 33.3% of specimens positive at 2 h (2,000 ng/mL). Conversely, 66.7% of specimens were codeine-positive 4 h after raw poppy seed ingestion. Figure 1 depicts normalized urinary concentrations of morphine and codeine after poppy seed roll or raw poppy seed ingestion with 300 and 2,000 ng/mL cutoffs represented (dashed lines). Using a 300 ng/mL cutoff, morphine concentrations after roll consumption were above cutoff at 2, 4, and 6 h, while codeine concentrations remained below cutoff at every time point. After raw poppy seeds, morphine concentration exceeded the 300 ng/mL threshold from 2 -20 h, with codeine concentrations above the cutoff at 2 and 4 h. Following raw poppy seed ingestion, morphine concentrations were greater than the 2,000 ng/mL cutoff at 2 and 4 h, with codeine concentrations below the threshold across experimental time points.
OF specimens OF specimens were analyzed by enzyme immunoassay for methamphetamines (120 ng/mL), amphetamine (150 ng/mL), cocaine metabolites (15 ng/mL), marijuana ( parent-THC) (3 ng/ mL), opiates (30 ng/mL) and phencyclidine (3 ng/mL) with confirmation of morphine, codeine, hydrocodone and hydromorphone performed by GC-MS operating with a LOQ of 7.5 ng/ mL for morphine and codeine. OF was collected prior to the start of each study and at 0.25, 0.5, 1, 1.5, 2, 3, 4, 5, 6 and 20 h after poppy seed ingestion. Since no OF specimens in Part 1 or 2 were positive for morphine or codeine after 3 h, data collected at 4, 5, 6 and 20 h were excluded from the final analyses. A total of 246 OF specimens were collected using the Oral-Eze device. Of those, analytical results after poppy seed ingestion were not obtained from Participant 102 at 1.5 h and Participant 106 at 1 h. Based on the GC -MS testing, 20.8% (15/72) of specimens were positive for morphine and 1.3% (1/72) were positive for codeine at or above method LOQ for study Part 1 (roll). In Part 2 (seeds), 61.4% (43/70) of samples were morphine-positive with 45.7% (32/70) codeine-positive at or above the LOQ. OF mean and range concentrations (ng/mL), along with standard deviation and sample size for each time point, are summarized in Table I. After the roll, morphine C min was 7 ng/mL with a C max of 143 ng/mL, both observed at 0.25 h. Codeine C min was 8 ng/mL (0.25 and 1.5 h) with a C max of 28 ng/mL observed at 0.25 h. Following raw poppy seed ingestion, specimens exhibited a morphine C min of 7 ng/mL (3 h) and a C max of .600 ng/mL (0.25 h; data not shown), with the second highest concentration of 333 ng/mL observed at 0.5 h. Furthermore, codeine C min was 8 ng/mL (1.5 and 2 h), with a C max of 112 ng/mL at 0.25 h. Table II summarizes morphine and codeine concentrations after poppy seed roll or raw poppy seed ingestion relative to a 30 ng/mL (neat OF) cutoff, illustrated by a dashed line in Figure 2. After poppy seed roll consumption, positivity, near
Discussion
The goal of this study was to investigate differences in morphine and codeine concentrations after ingestion of poppy seeds from the same source, consumed raw or contained in a traditionally prepared Ukrainian-style poppy seed roll. The morphine content in poppy seeds from around the world is variable (0.1-294 mg/g), and exposure concentrations depend on poppy seed origin, harvesting procedure and the method of poppy seed foodstuffs preparation (3 -6, 9, 12, 13, 20, 21). Significant reductions in opiate content ( 80 -90%) have been documented after food preparative processes, with decreased drug concentration shown after washing, soaking, grinding and baking (4,5,11,25). In fact, complete removal of detectable concentrations of morphine was reported after commercial preparation (11). A limitation of this study is that morphine and codeine loss in the baked poppy seed roll was not quantified as part of this study; however, the results provided in Tables I and II demonstrate how processing of poppy seeds in the roll greatly reduced the percentage of urine (cutoff 2,000 ng/mL) and OF (cutoff 30 ng/mL) positivity. As previously discussed (2,11), poppy seed studies have often failed to document the opiate content of poppy seeds used or have modeled a 'worst-case' scenario using large doses of raw poppy seeds. This study attempts to address such concerns by using the same poppy seeds and participants for both raw and prepared seeds, which permits comparison of opiate concentrations between studies. While anecdotal in nature, bolus ingestion of raw poppy seeds has been described as unpleasant, having little palatability (6,21). Moreover, during our study, participants felt that 15 g of poppy seeds was close to the maximum tolerable limit of ingestion, which corresponds roughly to ad libitum ingestion by three volunteers in a study published by Rohrig and Moore (23). While people may use the so-called 'poppy seed defense', the quantity of poppy seeds used in previous studies are much larger than in diets that consist of casual exposure to poppy seed-containing foods (bagels, cakes, curries, etc.). Our results demonstrate that consumption of poppy seeds in a Ukrainian-style roll versus raw poppy seed resulted in urinary morphine concentrations below 2,000 ng/mL as given in Tables I and II. In one investigation, a physician demonstrated that a typical poppy seed bagel produces urine concentrations of 336 and 446 ng/mL after 2 and 5 h of consumption, respectively (26). Conversely, Mule and Casella (10) described morphine concentrations in hydrolyzed urine in excess of 2,000 ng/mL after two poppy seed bagels. After eating a curry dish prepared with 25 g of poppy seeds (1,002 mg morphine/ 479.5 mg codeine), maximal urine concentration was 1,270 ng/ mL (4). Furthermore, other publications report morphine urinary concentrations below the 2,000 ng/mL cutoff, following consumption of poppy seed-containing cakes (5,9) and rolls (19). Our study revealed concentrations of morphine above the 2,000 ng/mL cutoff in urine specimens at 2, 4 and/or 6 h following ingestion of raw poppy seeds (Tables I and II). Taking into account potential dilution effects, specimens remained positive (2 and 4 h) after urine specimens were normalized to creatinine (Figure 1). Similar to findings of Smith et al., after raw poppy seeds, no specimens were positive for codeine at the 2,000 ng/ mL cutoff, despite an approximate 5-fold difference in total codeine dose between studies (0.6 versus 3.1 mg). Previous studies have documented urinary morphine concentrations above 2,000 ng/mL after ingestion of 9 -21 g (23) and 40 g (63 + 15 mg/g of morphine) of raw poppy seeds (3). After ingestion of a poppy seed cake containing 2.4 -7.7 mg morphine (151.6 mg/g morphine), 37.5% (30/80) of urine specimens were .2,000 ng/mL (2 -21 h post-ingestion), with a morphine concentration of 10,040 ng/mL (6 h post-ingestion) in one volunteer (12).
After raw poppy seed ingestion, OF specimens had morphine and codeine concentrations of 7 -333 and 8 -112 ng/mL, respectively (Table I and Figure 2). These data are consistent with Niedbala et al. (22) who demonstrated positive opiate immunoassay results 15 min following consumption of up to a 40-g dose of commercially available, uncooked poppy seeds. Furthermore, Rohrig and Moore (23) detected morphine in OF up to 1 h after combined ingestion of a poppy seed bagel and canned poppy seed filling, with the total amount ingested ranging from 9.82 to 20.82 g. The maximum concentration of morphine detected in this study was 205 ng/mL with the OF max occurring 15 min post-ingestion (23), which is consistent with our data of C max of 284 ng/mL at 0.25 h (Table I). Most recently, Conchiero et al. published a study in OF using poppy seeds whereby the opiate content was quantified prior to ingestion. Subjects received two equal doses of raw poppy seeds 8 h apart, for a total ingested dose of 31.4 mg of morphine and 6.2 mg of codeine (24). After the first dose, peak morphine and codeine concentrations were 177 and 32.6 ng/mL, reported from 0.5 to 1 h and 0.5 to 2 h, respectively (24). Differences in maximum morphine (333 ng/mL) and codeine (112 ng/mL) concentrations from our study, compared with Conchiero et al., may be explained by differences in ingestion, route of administration (raw versus suspension) and time after first collection (0.5 versus 0.25 h). Furthermore, positivity rates were more pronounced in both frequency and duration (Table II) after raw poppy seed ingestion (Part 2) when compared with consumption of the poppy seed roll (Part 1). While participants were permitted to drink during ingestion of poppy seeds, additional measures (rinsing oral cavity or brushing) were not performed prior to the start of OF collection(s). As a result, the initial morphine and codeine concentrations observed after raw poppy seed ingestion are likely affected by poppy seed residue in the oral cavity. In one study, opiate concentrations of 22 and 24 ng/mL were documented after ingestion of a poppy seed muffin by two volunteers; after the oral cavity was rinsed with mouthwash, opiate value was 12 ng/mL (27). Consequently, it cannot be determined in this study whether drug concentrations are wholly reflective of the systemic circulation.
After consumption of a poppy seed-containing roll, morphine concentrations of 7 -143 ng/mL were observed up to 1.5 h postingestion, with codeine detected in only 5.5% of OF specimens. Importantly, if an opiate cutoff of 30 ng/mL is considered, consumption of a poppy seed roll does not produce a positive OF result 1 -3 h after ingestion, with only one specimen observed above cutoff at 0.5 h (Table II). Thirty-three percent (4/12) of OF specimens were positive 0.25 h post-ingestion at this cutoff; however, individuals are routinely instructed to refrain from eating for at least 10 min prior to OF collection. Lastly, OF specimens were below the proposed cutoff (30 ng/mL) at 1 h, indicating there is a decreased likelihood of a positive morphine result in OF versus urine after bolus ingestion of raw poppy seeds.
Conclusions
OF drug testing is advantageous because collection is noninvasive, can be performed outside traditional testing facilities without gender-specific personnel requirements, is easily observed and is not easily adulterated or substituted. Morphine and codeine are detected in OF for 2 h after raw poppy seed ingestion, and only 0.5 h after consumption of a poppy seed-containing roll.
These data suggest that OF, due to its narrower window of detection, may be a specimen less susceptible to the 'poppy seed defense' after casual dietary poppy seed consumption. | v3-fos |
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} | s2 | Characteristics of Korean ginseng varieties of Gumpoong, Sunun, Sunpoong, Sunone, Cheongsun, and Sunhyang
Background Ginseng (Panax ginseng Meyer) is an important medicinal herbs in Asia. However, ginseng varieties are less developed. Method To developed ginseng varieties, a pure line selection method was applied in this study. Results Gumpoong was testing of 4-yr-old specimens in 2002, the proportions of the below-ground roots that were rusty colored for Gumpoong was 1.29 in Daejeon and 1.45 in Eumseong, whereas the proportions for its yellow berry variant were 2.60 and 2.45 in the two regions, respectively. Thus the Gumpoong was resistant to root rust. Sunpoong has a high yielding property. Its average root weight is 70.6 g for 6-yr-old roots. Its yield is 2.9 kg/1.62m2 and the rate of heaven- and earth-grade product is 20.9%, which is very high compared to 9.4% for Yunpoong. Sunone is resistance to root rot and the survival rate of 4-yr-old roots was 44.4% in 1997, whereas that of the violet-stem variant landrace was 21.7%. Sunhyang has content of arginyl-fructosyl-glucose (AFG), which produces the unique scent of red ginseng, is 95.1 μmol/g and greater than the 30.8 μmol/g of Chunpoong in 6-yr-old plants. Sunun and Cheongsun are being nurtured to protect genetic resources. Conclusion Developed ginsneg varieties will be used as the basis for the protection of genetic resources and breeding.
Introduction
The study of ginseng (Panax ginseng Meyer) breeding began at the Gwacheon Experimental Station of the Central Research Institute, Monopoly Bureau (Gwacheon, Korea) in 1968 [1]. Early breeding studies concentrated on the growth characteristics of the aerial and below-ground sections of violet-stem variants according to the number of stems [2], the prevalence of multi-stems according to the region and the location of ginseng cultivation, the direct and indirect effects of the number of stems on the characteristics of the below-ground section (root weight, diameter, and length), and the fact that multi-stem variants are more significantly affected by genetic factors than by environmental factors [3].
In a study of the yellow berry variant of ginseng, the characteristics of the aerial and below-ground sections of the violet-stem variant and yellow berry variant were compared, whereas the lengths of the peduncles were compared to the relative growth rates and the changes in the mineral contents of the leaves and stems (N, P, K) (N, nitrogen; P, phosphate; K, potassium). Additionally, the saponin contents were compared by age. In another study, the mineral contents, photosynthesis, respiration, and diseases of the leaves and roots of the yellow berry variant and violet-stem variant were examined based on the amounts of fertilizers used during cultivation. The seed setting rates of the breeding yellow berry variant and violet-stem variant were examined and the characteristics of the aerial and below-ground sections of F1 were studied [4]. In the F2 of the yellow berry variant and violet-stem variant, the ratio of stem colors was purple (3):green (1) and the ratio of seed colors was red (3):yellow (1). It was identified that purple and red were dominant to green and yellow for both stems and seeds. There was no reciprocal effect of the yellow berry variant  the violet-stem variant or the violet-stem variant  the yellow berry variant F1. The aerial section characteristics of the red berry and the below-ground section characteristics of the green stem variant were first studied in 1983, and the separation of the stem colors of the two entities of the second generation offspring and 14 entities of the third generation offspring were studied in 1988. The stem color of eight third generation offspring was green, but the six entities were divided into purple and green stems, indicating that it was undergoing homogenization [5]. Superior entities were selected through a pure line selection breeding method and the five superior varieties were named KG101, 102, 103, 104, and 105 after Korean Ginseng (KG) in 1985 to initiate productivity tests and adaptation tests. It was reported that these varieties increased the yield of ginseng varieties by 15%. Among them, KG101 and KG102 were named and registered as Panax ginseng var. Chunpoong and var. Yunpoong, respectively, in 1998.
The major characteristics of the ginseng varieties that are currently grown by the Korea Ginseng Corporation (KGC)d Gumpoong, Sunun, Sunpoong, Sunone, Cheongsun, and Sunhyangdhave not yet been fully reported. This study reports on the characteristics of the aerial and below-ground sections, and the red ginseng quality characteristics of all six varieties.
Study of the aerial section, below-ground section, and red ginseng quality
The study of the characteristics of the aerial and below-ground sections of the six varieties followed the guidelines set out by the Ginseng International Union for the protection of new varieties of plants [6]. Based on these guidelines, the anthocyanin color of the stem on the aerial section, the type of the inflorescence, the shape of the berry, and the presence of stolon roots on the below-ground section were studied among the qualitative characteristics. Among the pseudoqualitative characteristics, the anthocyanin distribution of the stem on the aerial section, the shape of the leaflet, the color of the fruit, and the color of the autumn leaves were studied, and the color of the roots was examined. Among the below-ground section characteristics, root length, root diameter, and root weight were indicated as an average based on 2e3 yr of productivity testing. The characteristics of the aerial sections of ginseng Fig. 1. Red ginseng grades are divided into four grades. (A) Heaven-grade product should not have cracks and scratches. For rootlets, one or more should be well developed and the rootlet length should be three quarters or less of the main root. On the rootlet, there must be a diameter of 0.5 mm of whitening and pitting and an affected length of 10 mm. The color of the textures must be maroon, brown, or dark brown. (B) Earth-grade product has cracks and scratches on the body of the total surface area of less than a quarter. Earthgrade rootlet development is the same as that of heaven-grade. There must be a diameter of 2.0 mm of whitening and pitting and the affected portion length of a quarter. The earth-grade textures/colors are similar to heaven-grade. (C) The yang-grade standard is not limited to the body. Rootlets are unbalanced. Whitening of the body length is one third or less, and the pitting of the body length is one half or less. Like the other two grades, the Yang-grade color is shiny, but the color is not uniform. (D) The jab-grade has no standard and grade. The heaven-, earth-, yang-, and jab-grades are given based on the parameters provided in the Korea Ginseng Industry Act.
were investigated in 4-yr-olds. Below-ground section characteristics were investigated in 6-yr-olds. For the quality of red ginseng, the roots that could be used as ingredients for the 2-or 3-yr productivity tests were gathered to make red ginseng according to the red ginseng production process of the KGC, and an expert appraiser judged the red ginseng quality (Fig. 1). The survey items examined were 6-yr-old ginseng roots.
Rusty root examination
For rusty root examination, 1-to 4-yr-old ginseng fields were drenched with 10 L distilled water per 1.62 m 2 once a month every AprileAugust from 1996 to artificially cause rusty roots. These entities were studied by classifying the rusty root index into four grades (degree of rusty root, 0 ¼ noninfected, 1 ¼ 1e10%, 2 ¼ 11e25%, and 3 ¼ above 26%). The reappearance testing of rusty roots was carried out in Daejeon and Punggi in 2000 using the same method.
Root rot resistance examination
To select the varieties that are resistant to root rot, Sunone and the violet-stem variants were transplanted to root rot-infected soil at the Daejeon Experimental Center three times using a randomized block design method on April 5, 1996, and the survival rate of the aerial sections was examined on June 4, 1996. For the reappearance test of the selected varieties, they were transplanted to root rot-infected soil at the Daejeon Experimental Centers on April 4, 1997, and the survival rates of the aerial sections were examined on July 25, 1997. They were again transplanted to root rot-infected soil at the Suwon Experimental Center on March 28, 2004 and harvested on June 15, 2004 to study the root rot infection rate. This study was performed on 2-yr-old ginseng.
Analysis of arginyl-fructosyl-glucose
The analysis was performed using a Dionex ion chromatography system (ICS3000; Dionex, USA). The chromatographic separation was accomplished on a CarboPac PA-1 column (4 Â 250 mm; Dionex, USA) and the column temperature was 30 C. The elution was performed with water and 250mM sodium hydroxide (50:50, v/v) using isocratic elution. The flow rate was 1.0 mL/min and the injection volume was 20 mL. The arginyl-fructosyl-glucose (AFG) was detected using an electron capture detector equipped with a gold working electrode and an Ag/AgCl reference electrode operating a quadruple potential waveform.
Standard solutions were obtained from AMBO (Ambo Institute, Seoul, Korea). Stock solutions were prepared in water at a concentration level of 1 mg/mL and stored at 4 C. Two standard stock solutions were mixed to make standard working solutions and serially diluted with water to obtain concentrations for calibration curve standards.
The red ginseng of Sunhyang and Chunpoong, 6-yr-old roots cultivated at the KGC Central Research Institute's Suwon Experimental Center were harvested in 2005, freeze-dried to make samples, and ground into powder for the 40-sieve (2.0 m/m; CISA, Barcelona, Spain). Approximately 0.1 mg of powder was weighed and dissolved in 10 mL of distilled water. The sample solutions were diluted with water to be within the standard calibration range. After sonication for 30 min without any sample clean-up, the supernatant was filtered with a 0.2-mm polyvinylidene fluoride membrane filter and injected (20 mL) into an HPAEC-PAD (High Performance Anion Exchange Chromatography-Pulsed Amperometric Detector) system.
Breeding of Panax ginseng var. Gumpoong
A variety with green stems and yellow matured fruits was discovered in Gaepung-gun, Gyeonggi-do in 1926 and in Jangdangun, Gyeonggi-do in 1928. This variety has been called the yellow berry variant [4]. In order to further understand the characteristics of the yellow berry variant, continuous studies have been conducted on the aerial section characteristics of each year and at the flowering period, the relative elongation and saponin content, the chlorophyll content related to heavy and low fertilizing, and the mineral contents and photosynthetic characteristics of the leaves and roots. Substantial testing at the production site began in 1985. However, the damage of rusty roots to 5-yr-old plants was 100% in Pocheon, 60% in Icheon, and 20% in Jeollabuk-do as a result of substance testing in 1994. Therefore, the importance of the yellow berry variant was revealed as it is resistant to rusty roots. Gumpoong was selected to tolerance of rust in the Punggi region in 1979 and it was named the Punggi Yellow Berry Variant. The characteristics were examined through the growth of variants from 1979 to 1992. Productivity and substance tests were performed from 1996 through 2012 and the variant was named "KG111" and registered as Gumpoong during farm substance testing in 1996.
Distinction of aerial sections of Gumpoong and Chunpoong for comparison
Among the qualitative characteristics of the aerial section, Gumpoong showed no purple in the anthocyanin color of stems, whereas Chunpoong distinctively showed purple in the stems. The type of the inflorescence and berries were simple and round for both Gumpoong and Chunpoong. The existence of stolon roots on the below-ground section was found in both Gumpoong and Chunpoong. As for the distribution of the anthocyanin color of stems, which is the similar qualitative characteristic of the aerial section, the color was green for Gumpoong with no anthocyanin, whereas Chunpoong showed purple only on the basal section. The color of berries was yellow for Gumpoong and orange for Chunpoong. The color of autumn leaves was yellow for Gumpoong and orange for Chunpoong. However, the shape of the aerial section leaflets and the color of the roots on the below-ground section were broad elliptical and cream, respectively, for both Gumpoong and Chunpoong (Table 1; Fig. 2A, 2G).
Major characteristics of the aerial sections of Gumpoong and Chunpoong
The major characteristics of the aerial sections of Gumpoong and Chunpoong based on 3 yr of substance testing at the production site are shown in Table 2. The average stem diameter was 7.6 mm for Gumpoong and 7.4 mm for Chunpoong. The average stem length was 35.5 cm for Gumpoong and 38.5 cm for Chunpoong: Gumpoong was 5 cm shorter than Chunpoong. The average leaflet length was 16.3 cm for Gumpoong and 15.9 cm for Chunpoong; Gumpoong was 0.6 cm longer than Chunpoong. The average leaflet width was 7.1 cm for Gumpoong and 6.3 cm for Chunpoong; Gumpoong was 0.8 cm longer than Chunpoong. The average number of palmately compound leaves was 5.5 for Gumpoong and 5.6 for Chunpoong with no distinction. The average number of leaflets was 23.6 for Gumpoong and 22.7 for Chunpoong. Table 3 shows the characteristics of the below-ground sections and the average quality of the red ginseng of Gumpoong and Chunpoong. The average yield was 2.1 kg/1.62 m 2 for Gumpoong and 2.0 kg/1.62 m 2 for Chunpoong. The average length of the main root was 7.6 cm for Gumpoong and 8.3 cm for Chunpoong. The average diameter of the main root was 26.8 cm for Gumpoong and 27.0 cm for Chunpoong with no significant difference between the two variants. By contrast, the root weight was 70.2 g for Gumpoong and 61.0 g for Chunpoong: Gumpoong was 9.2 g heavier than Chunpoong. In terms of the quality of red ginseng, the average rate of heaven-grade ginseng was 21.2% for Gumpoong and 21.8% for Chunpoong. The average rate of earth-grade ginseng was 12.8% for Gumpoong and 16.2% for Chunpoong. The average rate of yanggrade ginseng was 17.6% for Gumpoong and 20.9% for Chunpoong. Overall, for these grades, the rates of Gumpoong were lower than those of Chunpoong. The average rate of jab-grade ginseng was 48.3% for Gumpoong and 41.0% for Chunpoong; the rate of Gumpoong was higher than the rate of Chunpoong.
Gumpoong's prevalence of rusty roots
The average primary rate of rusty roots in 1996 was 0.8 for Gumpoong and 1.6 for the yellow-berry variant in comparison. As a result of the reappearance test in 2000, the reproductivity rates were 1.29 for Gumpoong and 2.66 for the yellow-berry variant in Daejeon. These rates were 1.45, and 2.46, respectively, in Eumseong. Fully-grown Gumpoong showed lower rates than those of the yellow-berry variant (Table 4).
Breeding of Panax ginseng var. Sunun
Ginseng is a self-fertilizing crop and its genetic resources are very limited. Therefore, the breeding of ginseng variants focused on the distinction of appearance, and the characteristics of the aerial and below-ground sections. The color of fully-ripened berries of Chunpoong is orange [7], whereas Gumpoong is green with no purple on the stems and yellow for the color of fully-ripened berries [8]. Chengsun has as green a stem as Gumpoong, but the color of fullyripened berries is red [9]. Also, a variant whose color of fullyripened berries are pink was recently bred, and is currently being tested for cultivation [10]. Other variants grown include Sunone whose shape of the palmately compound leaf is a bushy type [11], Yunpoong shows two or more stems from the 4th year with many stipules [12], and Gopoong shows a rich purple color across the stems, peduncle, and the length of the petiole [13]. The direction of ginseng breeding is turning to functionality, resistance to diseases/ insects, and tolerance to environmental conditions such as drought and excessive moisture injury. For example, Sunhyang is a variant with a high content of unique ginseng flavor [14]. Sunil has a tolerance to damage from high temperature [15]. In order to breed these variants, however, the quality characteristics should vary by one grade and the quantitative characteristics should vary by two grades compared to the check variety for comparison when registering a new variant. Sunun was registered as a new variant because it is Values are expressed as mean AE standard deviation of three plants ADT, adaptation testing; PDT, productivity testing 1) The characteristics of the aerial sections were investigated in a 4-yr-old plant Values are expressed as mean AE standard deviation of three repetitions per 1.62 m 2 ADT, adaptation testing; PDT, productivity testing 1),2) Investigated in 6-yr-old ginseng 3) Data was not investigated clearly distinguished from other variants with the strong development of blistering of the surface of the leaflets compared to the genetic resources of ginseng possessed by the KGC [16].
A variant with very strongly developed blistering of the surface of the leaflets was selected at the production site, used in a purple-stem population breeding in 1985, and named "85822". In 1989, it underwent the breeding test to name the breed "KG114". As a result of productivity and adaptation testing from 2000, the genetic stability of the distinctively strong blistering of the surface of the leaflets was verified and the institute registered the new variant as "Sunun".
Distinction of aerial sections of Sunun and Chunpoong for comparison
Among the qualitative characteristics of the aerial sections, Sunun showed purple in the anthocyanin color of stems, much the same as Chunpoong. The shapes of inflorescence and berries were simple type and round for both Sunun and Chunpoong. The existence of stolon roots in the below-ground section was found in both Sunun and Chunpoong. As for the distribution of the anthocyanin color of stems, which is the similar qualitative characteristic in the aerial section, Sunun showed purple along the whole stems, whereas Chunpoong showed purple only on the lower section. The color of berries was red for Sunun, whereas it was orange for Chunpoong. The color of autumn leaves was red for Sunun and it was distinctively orange for Chunpoong. The shape of the aerial section leaflets was broad elliptical for both Sunun and Chunpoong. The root color of the below-ground section was cream for both varieties. Among the characteristics of the aerial sections, Sunun showed strong development of the blistering of the surface of the leaflets (Table 1; Fig. 2B, 2G).
Major characteristics of the aerial sections of Sunun and Chunpoong
Among the characteristics of the aerial sections of Sunun and Chunpoong based on 2 yr of substance testing at the production site, the average stem diameter was 6.9 mm for Sunun and 7.4 mm for Chunpoong, revealing that Sunun was slightly smaller than Chunpoong. The average stem length was 29.0 cm for Sunun and 31.8 cm for Chunpoong; Sunun was slightly shorter than Chunpoong. The average leaflet length was 16.2 cm for Sunun and 15.6 cm for Chunpoong. The average leaflet width was 7.0 cm for Sunun and 6.2 cm for Chunpoong; Sunun was slightly longer than Chunpoong. The average number of palmately compound leaves was 4.5 for Sunun and 4.6 for Chunpoong, and the average number of leaflets was 24.3 for Sunun and 21.7 for Chunpoong. In this regard, there was no significant difference between the two varieties ( Table 2). Table 3 shows the characteristics of below-ground sections and the average quality of red ginseng. The average length of the main root was 8.2 cm for Sunun and 8.1 cm for Chunpoong and the average diameter of the main root was 26.5 mm for Sunun and 26.3 mm for Chunpoong with no significant difference between the two varieties. The average root weight was 67.8 g for Sunun and 60.1 g for Chunpoong and Sunun was slightly heavier than Chunpoong. In terms of the quality of red ginseng, the average rate of heaven-grade ginseng was 16.4% for Sunun and 32.3% for Chunpoong, the average rate of earth-grade ginseng was 18.0% for Sunun and 25.6% for Chunpoong, and the average rate of yang-grade ginseng was 24.2% for Sunun and 27.9% for Chunpoong. The average rate of jab-grade ginseng was 41.5% for Sunun and 13.7% for Chunpoong. Overall, in terms of the quality of red ginseng, the rates of heaven-and earth-grade ginseng were lower for Sunun than those of Chunpoong.
Breeding of Panax ginseng var. Sunpoong
The characteristics of Chunpoong include that the average rates of heaven-and earth-grade ginseng being 6e13% higher than those of the violet-stem variant when produced using grade-two fresh ginseng [17]. However, the average root weight was only 70 g when the root weight of grade-one fresh ginseng should be 75 g or heavier according to the raw ginseng quality standards [18]. It is a concern that the grade of raw ginseng may become lower when the root weight of Chunpoong is low. Yunpoong was bred as a highyield variant because its average yield is 2.8 kg/1.62 m 2 , and 0.6 kg heavier than 2.2 kg/1.62 m 2 of the violet-stem landrace. However, the rates of heaven-and earth-grade ginseng was 9.4% and lower than the 11.7% of the violet-stem landrace. This is because the root weight is heavy and the diameter of the main root is large, thereby increasing the rate of interior vanity and the rate of undergrade ginseng when producing red ginseng [19]. For these reasons, it was necessary to develop a variant with a heavy root weight and high rates of heaven-and earth-grade ginseng. Among the high-yield populations possessed by the KGC, "7224-1-1" with a very long length of peduncle was tested for breeding and named "KG104". As a result of performing the productivity testing from 1981, the adaptation test in 1984, and the substance testing at the farm in 1991, its average root weight was 15.4% heavier than that of Chunpoong and the new variety was registered as Sunpoong.
Distinction of aerial sections of Sunpoong and Chunpoong for comparison
Among the qualitative characteristics of the aerial sections, both Sunpoong and Chunpoong showed purple in the anthocyanin color of stems. The shapes of the inflorescence and berries were simple type and round for both Sunpoong and Chunpoong. The existence of stolon roots in the below-ground section was found in both Sunpoong and Chunpoong. Therefore, there was no significant distinction in the qualitative characteristics of the aerial sections. By contrast, as for the distribution of the anthocyanin color of stems, which is the similar qualitative characteristic of the aerial section, Sunpoong showed purple along the whole stem, whereas Chunpoong showed purple only on the lower section. The color of berries was red for Sunpoong, whereas it was orange for Chunpoong. The color of the autumn leaves was red for Sunpoong and it was distinctively orange for Chunpoong. The shape of aerial section leaflets was broad elliptical for both Sunpoong and Chunpoong. The root color of the below-ground section was cream for both varieties (Table 1; Fig. 1C, 1G).
Major characteristics of the aerial sections of Sunpoong and Chunpoong
Among the characteristics of the aerial sections of Sunpoong and Chunpoong based on 3 yr of substance testing at the production Table 4 Prevalence of rusty root in the field for "Gumpoong" (Table 2). In particular, the average length of the peduncle was 28.5 cm for Sunpoong and 22.9 cm for Chunpoong; Sunpoong was 5.7 cm longer (date not shown). Table 3 shows the characteristics of the below-ground sections and the average quality of red ginseng. The average quantity of Sunpoong and Chunpoong was 2.9 kg/1.62 m 2 and 3.0 kg/1.62 m 2 , respectively. The average length of the main root was 6.5 cm for Sunpoong and 6.7 cm for Chunpoong, and the average diameter of the main root was 30.3 mm for Sunpoong and 28.3 mm for Chunpoong with no significant difference between the two varieties. The average root weight was 70.6 g for Sunpoong and 59.7 g for Chunpoong; Sunpoong was 10.9 g heavier than Chunpoong. In the study of the quality of red ginseng, the average rate of heaven-grade ginseng was 8.7% for Sunpoong and 17.5% for Chunpoong, the average rate of earth-grade ginseng was 12.2% for Sunpoong and 20.5% for Chunpoong, and the average rate of yang-grade ginseng was 43.2% for Sunpoong and 36.9% for Chunpoong. The average rate of jab-grade ginseng was 35.9% for Sunpoong and 33.1% for Chunpoong.
Breeding of Panax ginseng var. Sunone
Root rot (Cylindrocarpon destructans) is the disease that rots the roots of ginseng during cultivation. This disease is a major cause of yield reduction in ginseng cultivation areas because it is the cause of damage to continuous cropping. For the selection of a variant resistant to root rot through breeding, packaging and indoor testing of root rot began in 1982 with 50 excellent variants including 680-29-1. The selection of variants resistant to root rot continued, but most of the variants were found to be not resistant in the reappearance testing. Sunone was cultivated by population breeding to purple-stem landrace in 1978 and one entity with a broad leaf, purple stem, and six rounds, palmately compound leaves was selected among the entities packaged at the production site. It was named "78135" and the productivity testing was performed in 1992 after the breeding testing in 1982. It was the first variant selected for resistance to root rot in 1996 and its resistance was verified during the reappearance testing in 1997 and 2002. The variant was named KG112 in 2000, and an adaptation test was performed to register the new variety as Sunone.
Distinction of aerial sections of Sunone and Chunpoong for comparison
Among the qualitative characteristics of the aerial sections, both Sunone and Chunpoong showed purple in the anthocyanin color of stems and the shapes of inflorescence and berries were simple type and round for both Sunone and Chunpoong. The existence of stolon roots in the below-ground section was found in both Sunone and Chunpoong. Therefore, there was no significant distinction in the qualitative characteristics of the aerial sections. However, as for the distribution of anthocyanin color in the stems, which is the similar qualitative characteristic of the aerial section, Sunone showed purple along the whole stem, whereas Chunpoong showed purple only on the lower section. The color of berries was red for Sunone, whereas it was orange for Chunpoong. It was previously reported that for Sunone the color of berries was orange-red; however, this was done in error [20]. The color of the senescence of leaves was red for Sunone and it was distinctively orange for Chunpoong. The shape of the aerial section leaflets was broad elliptical for both Sunone and Chunpoong. The root color of the below-ground section was cream for both variants (Table 1; Fig. 2D, 2G).
Major characteristics of aerial sections of Sunone and Chunpoong
Among the characteristics of the aerial sections of Sunone and Chunpoong based on 2 yr of substance testing at the production site, the average stem diameter was 7.5 mm for Sunone and 7.5 mm for Chunpoong. The average stem length was 35.4 cm for Sunone and 33.6 cm for Chunpoong; Sunone was 1.8 cm longer. The average leaf length was 15.7 cm for Sunone and 15.1 cm for Chunpoong and the average leaf width was 6.8 cm for Sunone and 6.3 cm for Chunpoong. The average number of palmately compound leaves was 4.5 for Sunone and 4.5 for Chunpoong, and the average number of leaflets was 24.4 for Sunone and 23.4 for Chunpoong, with no significant difference between the two varieties ( Table 2). Table 3 shows the characteristics of the below-ground sections and the average quality of red ginseng. The average yield of Sunone and Chunpoong was 3.3 kg/1.62 m 2 for Sunone and 1.9 kg/1.62 m 2 for Chunpoong. Sunone produced 42.4% more than Chunpoong did. The average length of the main root was 8.5 cm for Sunone and 7.3 cm for Chunpoong; Sunone was slightly longer than Chunpoong. The average diameter of the main root was 29.3 mm for Sunone and 25.3 mm for Chunpoong, and Sunone was a little thicker than Chunpoong. The average root weight was 84.1 g for Sunone and 52.5 g for Chunpoong; Sunone was 31.6 g heavier than Chunpoong. In the study of the quality of red ginseng, the average rate of heaven-grade ginseng was 1.9% for Sunone and 14.0% for Chunpoong, the average rate of earth-grade ginseng was 6.8% for Sunone and 14.9% for Chunpoong, and the average rate of yanggrade ginseng was 32.0% for Sunone and 56.7% for Chunpoong. The average rate of jab-grade ginseng was 59.5% for Sunone and 15.3% for Chunpoong.
Sunone's resistance to root rot
The average survival rate of the 2-yr-old aerial sections against root rot was 61.1% for Sunone and 20.5% for the violet-stem variant in testing in 1996. The average survival rate of the 4-yr-old areal section was 44.4% for Sunone and 21.7% for the violet-stem variant in 1997. Later, the average prevalence of root rot for the 2-yr-old Sunone and violet-stem variant in 2004 was 32.2% and 15.5%, respectively, to prove the reappearance of Sunone's resistance to root rot (Table 5). In 2002, the average yield of 6-yr-old ginseng per section of farmland in the package affected by root rot was 1.6 kg for Sunone and 1.3 kg for the violet-stem variant (data not shown).
Breeding of Panax ginseng var. Cheongsun
The green-stem variant was first discovered in Jangdan-gun, Gyeonggi-do in 1927 as a variant with green stems and red berries. The same entity was discovered at the Korea Ginseng & Tobacco Research Institute's Jeungpyeong Experimental Station in Chungcheongbuk-do in 1978. The yearly study of the characteristics of the aerial section and below-ground section of this ginseng began in 1983. In 1988, the distribution of the color of stems was studied for 14 entities of third-generation offspring and two entities of second-generation offspring. Among the third-generation offspring, eight entities showed green stems and red berries, but the remaining six entities showed a 3:1 distribution of green and purple stems. Also, the characteristics of photosynthesis and respiration according to temperature were studied for the violetstem variant, yellow berry variant, and green stem variant. There was no significant difference among them [21]. The rusty roots, cracking, and root rot rates of the below-ground sections were studied and it was reported that there were no significant differences between the violet-stem variant and the yellow berry variant. For the green stem variant, an entity with green stems, petiole, and peduncle similar to Gumpoong, and red fully-ripened berries was selected at the Korea Ginseng & Tobacco Research Institute's Jeungpyeong Experimental Station in 1978. After performing the breeding tests on the aerial section and below-ground section of the green stem variant, the new variety was registered as Cheongsun through productivity testing in 1991 and adaptation testing in 1999.
Distinction of aerial sections of Cheongsun and Chunpoong for comparison
Among the qualitative characteristics of the aerial section, Cheongsun distinctively showed no purple in the anthocyanin color of stems, unlike Chunpoong. The shapes of the inflorescence and berries were of simple type and round for both Cheongsun and Chunpoong. The existence of stolon roots in the below-ground section was found in both Cheongsun and Chunpoong. As for the distribution of the anthocyanin color of stems, which is the similar qualitative characteristic of the aerial section, Cheongsun showed green across the stems with no purple, whereas Chunpoong showed purple only on the lower sections. The color of the berries was red for Cheongsun, whereas it was orange for Chunpoong. The color of autumn leaves was red for Chengsun and it was distinctively orange for Chunpoong. The shape of aerial section leaflets was broad elliptical for both Cheongsun and Chunpoong. The root color of the below-ground sections was cream for both variants (Table 1; Fig. 2E, 2G).
Major characteristics of aerial sections of Cheongsun and Chunpoong
Among the characteristics of the aerial sections of Cheongsun and Chunpoong based on 3 yr of substance testing at the production site, the average stem diameter was 7.0 mm for Chengsun and 6.8 mm for Chunpoong, the average stem length was 32.2 cm for Cheongsun and 32.9 cm for Chunpoong, the average leaf length was 14.7 cm for Chengsun and 14.8 cm for Chunpoong, and the average leaf width was 6.7 cm for Cheongsun and 6.2 cm for Chunpoong. The average number of palmately compound leaves was 4.8 for Cheongsun and 4.9 for Chunpoong, and the average number of leaflets was 23.8 for Cheongsun and 24.2 for Chunpoong. There were no significant differences between the two variants in terms of the characteristics of the aerial section (Table 2). Table 3 shows the characteristics of the below-ground sections and the average quality of red ginseng. The length of the main root was 8.6 cm and 9.5 cm for Cheongsun and Chunpoong, respectively. The diameter of the main root was 25.4 mm and 26.9 mm, respectively; Cheongsun was slightly smaller than Chunpoong. The root weight was 73.7 g and 67.4 g, respectively; Cheongsun was 6.3 g heavier than Chunpoong. In the study of the quality of the red ginseng, the average rate of heaven-grade ginseng was 14.6% for Cheongsun and 24.9% for Chunpoong, the average rate of earthgrade ginseng was 7.2% for Cheongsun and 15.0% for Chunpoong, and the average rate of yang-grade ginseng was 34.1% for Cheongsun and 28.8% for Chunpoong. The average rate of jab-grade ginseng was 44.1% for Cheongsun and 31.4% for Chunpoong. For the characteristics of the below-ground section, the root weight varied between the two varieties and the quality of red ginseng was lower for Cheongsun compared to Chunpoong.
Breeding of Panax ginseng var. Sunhyang
The breeding of ginseng variants resulted in the creation of Chunpoong with excellent physical characteristics [17]; in this case, Yunpoong with its high-yielding properties [19], and Gopoong with its high saponin content [22]. However, more functional variants are being developed for ginseng as more people are interested in a healthy lifestyle. For the specifics of the active substances of ginseng, the content and distribution of saponin in each section and the root of ginseng [23] and the saponin content of each variant of ginseng have been reported. For the study of the red ginseng flavor, the primary identification of raw ginseng's volatile aroma [24] and the aromatic substances of gas chromatograph/mass spectrophotometry (GC/MS) have been studied [25]. In 1978, an entity was selected and named 78093. The characteristics were studied through breeding testing from 1979 to 1981. The productivity testing was performed from 1983 through 1991 and the substance testing at the production site was performed from 1999. This variant was named KG110. During the substance testing, it was found that the quantity of the below-ground section was outstanding and the high content of Aroma-1 in red ginseng was acknowledged in 2006, which led to the registration of the new variety as Sunhyang.
Distinction of aerial sections of Sunhyang and Chunpoong for comparison
Among the qualitative characteristics of the aerial sections, both Sunhyang and Chunpoong showed purple in the anthocyanin color of the stems. The shapes of the inflorescence and berries were of simple type and round for both Sunhyang and Chunpoong. The existence of stolon roots in the below-ground section was found in both Sunhyang and Chunpoong. As for the distribution of anthocyanin color in the stems, which is the similar qualitative characteristic of the aerial section, both Sunhyang and Chunpoong showed purple only on the lower section. The color of berries was red for Sunhyang, whereas it was red-orange for Chunpoong. The color of the senescence of the leaves was red for Sunhyang and Values are expressed as percentages 1) Ginseng growth stage orange for Chunpoong. The shape of the aerial section leaflets was broad elliptical for Sunhyang and distinctively broad elliptical for Chunpoong. The root color of the below-ground sections was cream for both variants (Table 1; Fig. 2F, G).
Major characteristics of the aerial sections of Sunhyang and Chunpoong
Among the characteristics of the aerial sections of Sunhyang and Chunpoong based on 2 yr of substance testing at the production site, the average stem diameter was 6.6 mm for Sunhyang and 6.4 mm for Chunpoong, the average stem length was 39.6 cm for Sunhyang and 38.6 cm for Chunpoong, the average leaf length was 14.9 cm for Sunhyang and 14.1 cm for Chunpoong, and the average leaf width was 6.5 cm for Sunhyang and 6.4 cm for Chunpoong. The average number of palmately compound leaves was 4.3 for Sunhyang and 4.5 for Chunpoong, and the average number of leaflets was 22.8 for Sunhyang and 23.5 for Chunpoong. There were no significant differences between the two varieties in the characteristics of the aerial sections (Table 2). Table 3 shows the characteristics of the below-ground sections and the average quality of red ginseng based on 3 yr of substance testing at the production site. The average yield was 1.8 kg/1.62 m 2 for both Sunhyang and Chunpoong and the average length of the main root was 7.4 cm and 8.4 cm, respectively. The average diameter of the main root was 29.0 mm and 27.1 mm, respectively. There was no significant difference between the two varieties, but the root weight was 78.8 g and 70.8 g, respectively; Sunhyang was 1.0 g heavier than Chunpoong. In the study of the quality of red ginseng, the average rate of heaven-grade ginseng was 13.3% for Sunhyang and 32.3% for Chunpoong, the average rate of earth-grade ginseng was 14.1% for Sunhyang and 26.0% for Chunpoong, and the average rate of yang-grade ginseng was 15.5% for Sunhyang and 28.1% for Chunpoong. The average rate of jab-grade ginseng was 57.2% for Sunhyang and 13.7% for Chunpoong.
Sunhyang's unique flavor of red ginseng
As a result of harvesting the ginseng in mid-October 2005 and steaming it to compare the content of AFG, the precursor of Aroma-1, the measurements for AFG were 95.1 mmol/g for Sunhyang and 30.8 mmol/g for Chunpoong. In fresh ginseng, AFG, the precursor of Aroma-1, is produced by the combination of maltose and arginine. Comparing the arginine contents of the fresh ginseng and red ginseng of the two variants, Sunhyang showed 121.3 mmol/g in the fresh ginseng and 226.9 mmol/g in the red ginseng, whereas Chunpoong showed 101.4 mmol/g in raw ginseng and 112.4 mmol/g in red ginseng ( Table 6).
As shown above, the KGC has developed the new ginseng variants of Gumpoong, Sunun, Sunpoong, Sunone, Chengsun, and Sunhyang. Among the violet-stem variants, the yellow berry variant and the green stem variant were very weak against rusty roots. Gumpoong, which is strong against rusty roots, showed no purple stem in the characteristics of the aerial section and distinctively had yellow berries and senescence of leaves. In 2002, the rate of rusty roots in the below-ground section was 1.29 in Daejeon and 1.45 in Eumseong, whereas that of the yellow berry variant was 2.66 and 2.45, respectively, and it was proven that Gumpoong was tolerant to rusty roots (Table 4; Fig. 2A). However, there is little information on the physiological mechanism of rusty roots in ginseng, therefore studies are needed in the future. Chunpoong shows higher rates of heaven-and earth-grade, but a lower root weight than the other varieties. Therefore, it was necessary to breed a variant with heavier root weight and high rates of heaven-and earth-grade ginseng. Sunpoong distinctively has a long peduncle among the characteristics of the aerial section. It is a high-yielding variant with the average root weight of 70.6 g and yield of 2.9 kg/1.6 m 2 . Its rate of heaven-and earth-grade was 20.9% and very high compared to the 9.4% of Yunpoong. It is a high-yielding variant with high rates of heaven-and earth-grade ginseng (Table 3). In ginseng cultivation, root rot makes it impossible to do continuous cropping. Therefore, it was necessary to breed a variant with resistance to root rot. Sunone distinctively has bushy-type, palmately compound leaves among the characteristics of the aerial section. The survival rate of 4-yr old roots in 1997 was 44.4%, whereas only 21.7% of the violetstem variant survived. In 2004, the prevalence of root rot in 2-yrold roots was 61.1%, whereas it was 20.5% for the violet-stem variant ( Table 5). The focus of breeding shifted from high-yield and outstanding physical properties to resistance and functionality. The purpose of breeding functional variants of ginseng was to breed a variant with high saponin content. The long storage time of red ginseng increases the more specific flavor. Also, the specific flavor of red ginseng on the market has a significant impact on sales. Sunhyang showed no clear distinction in the characteristics of the aerial section, but Sunhyang's content of AFG, the unique scent of red ginseng, was 95.1 mmol/g and greater than the 30.8 mmol/g of Chunpoong (Table 6). Ginseng does not have much genetic diversity and distinctiveness is very low when breeding new variants. In order to breed a new variant, it is necessary to develop a variant with a clearly distinctive aerial section. A clearly distinctive variant can be used as an intermediate parent. Sunun has a distinctively strongly developed blistering of the surface on the leaflets among the characteristics of the aerial section (Fig. 2B). Chengsun has no purple on the stems and distinctively red berries and senescence of leaves among the characteristics of the aerial section (Fig. 2E). Chunpoong and Chengsun have reportedly been derived from the Chunkyung landrace; also Gumpoong was derived from the Hwangsook landrace [20]. However, Cheon and Kim [21] reported that Chengsun was derived from the Jakyung landrace. Takahasi & Ousmi first reported samples of green-stems and red berry, and the green-stems and yellow berry variant was first discovered as one line of the Jakyung landrace in 1927 [26].
The saponin content of 6-yr-old red ginseng in the water extraction was Sunhyang > Sunun > Gumpoong > Sunpoong > Sunone > Cheongsun. For the different forms of ginsenoside, Sunhyang contained 2.4 mg/g of Rg1, Cheongsun contained 0.9 mg/ g of Re, Sunun contained 0.9 mg/g of Rf, and Sunhyang contained 0.7, 0.6, 2.4, 0.8, 0.8, and 0.6 mg/g of Rh1, Rg2, Rb1, Rc, Rd and Rb2, respectively. However, there were no significant differences in Rd among the variants (data not shown). The saponin content of 6-yrold red ginseng in the ethanol extraction was Sunun > Sunhyang > Sunone > Sunpoong > Gumpoong > Cheongsun and Sunun; Sunun contained 3.6 mg/g of Rg1, Cheongsun contained 1.6 mg/g of Re, Sunun contained 1.1 mg/g of Rf, Sunpoong contained 5.5 mg/g of Rb1, and Sunhyang contained 1.7 mg/g of Rc and Rb2. However, Table 6 Arginyl-fructosyl-glucose (AFG) and arginine contents of "Sunhyang" there were no differences in Rh1, Rg2, Rd, and Rg2 levels among the variants (data not shown). The major purpose of breeding ginseng focused on the quality of red ginseng, high-yielding property, out-standing physical properties, resistance to disease, and high saponin content from 1970 through to the late 1990s. Since 2000, however, due to the increasing interest in red ginseng for its health properties, the focus has shifted to developing variants with a high content of nonsaponin substances and resistance to the damage of high temperature. Ginseng is a half-shade plant and its growth is lowered by the damage of high temperature and intense light. In order to overcome these environmental conditions, a variant was selected with tolerance to the damage of high temperature and the characteristics of this variant were reported [27e29]. The genetic diversity of ginseng variants is very limited as it is a self-fertilized plant. Therefore, it is necessary to introduce cross-breeding and mutantbreeding methods to overcome this. By doing so, variants with a high content of active substances and tolerance to environmental conditions, such as drought and humidity, and resistance to diseases/insects should be developed. These are among the goals that the KGC works towards in striving to maintain the quality of plant output and improve certain characteristics to meet the realities and demands of the modern world.
Conflicts of interest
None of the authors have any conflicts of interest to declare. | v3-fos |
2017-08-03T02:30:12.982Z | {
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} | s2 | Survey of physicochemical characteristics and microbial contamination in selected food locally vended in Morogoro Municipality, Tanzania
Background Raw milk, raw fruit juice and raw fish are enriched with essential nutrients for human diet but are prone to microbial contamination along the value chain. This cross sectional study was conducted to assess physicochemical characteristics and microbial quality of raw milk, fruit juice and fish from food vendors in Morogoro Municipality, Tanzania. The physicochemical assessment of food samples was done by smell, colour, presence of debris, turbidity, consistence, pH and clot on alcohol test. Hygiene of food containers, personnel and the vending environment was also assessed. Qualitative and quantitative microbial assessment of food was done using standard laboratory protocols as described by Tanzania Bureau of Standards and International Systems of Standards. Results Raw milk sold in Morogoro was of poor quality since was adulterated with water, contained sediments and clotted on alcohol test. Up to 63 % of the milk samples were contaminated with Escherichia coli and 60 % had higher total viable count (TVC) than the recommended values. Raw fruit juice was stored in dirty containers and sold under unhygienic environment. Seventy-three percent of juice samples had TVC beyond the recommendations while E. coli contamination rate was 63.3 %. The raw fish samples had started spoiling as depicted through sensory evaluation. E. coli contamination rate was 55 % and that of Campylobacter jejuni was 0.5 %. The mean TVC of raw fish was 8.1 (Log cfu/g) and 96.2 % of the fish samples had TVC beyond the recommended limits of 5.0 Log cfu/g. Conclusions The physicochemical characteristics of food vended in Morogoro Municipality were of poor quality. The food had high bacterial contaminations. This situation poses health risks to the public and losses to food vendors due to spoilage. Stakeholders in food value chain should be educated on safe production and good hygienic practices. Routine quality and safety assessment of locally vended food, inspection of selling premises and regular health check-up of the personnel involved in food vending industry should be instituted.
Introduction
Background Food-and water-borne pathogens are leading causes of illnesses in developing countries, causing deaths to about 1.9 million people annually at the global level [1]. In developed countries, an estimated one-third of the population is affected by microbiological food-borne diseases each year [2]. In Africa, food-and water-borne diseases are responsible for 33-90 % cases of mortality in children [3]. Most of the cases are due to microbial contamination in food which is associated with poor hygiene in handling along food chain especially the street food. However, street food industry plays an important role in developing countries in meeting the food demands of the urban dwellers. Street foods feed millions of people daily with a wide variety of foods that are relatively cheap and easily accessible [4]. According to FAO, street foods have significant nutritional implications for consumers, particularly from middle and low-income sectors of the population [5]. Street food vendors in developing countries are often unlicensed, untrained in food hygiene and sanitation, and work under crude unsanitary conditions [5][6][7][8]. In Tanzania, urbanization accompanied with low wages offered to employees and labourers has led to proliferation of street food vendors who offer cheap meals that is always contaminated by microbes [9][10][11]. In a study to evaluate microbiological quality of ready-to-eat foods in Pemba Island, coliform bacteria, Salmonella spp. and Vibrio alginolyticus were the common contaminants [9].
Milk as a nutritious food is prone to microbial contamination and many milk-borne epidemics of human are spread through contaminated milk. The primary sources of microbial contamination in milk are the infected or sick lactating animals [12,13]. The secondary causes of microbial contamination occur along the milk value chain from milk handlers, unsanitary utensils and/ or milking equipments and water supplies used in sanitary activities [12,13]. Studies show that up to 90 % of all dairy-related diseases are due to pathogenic bacteria like Brucella abortus, Escherichia coli 0157:H7, Mycobacterium bovis, Campylobacter jejuni and Staphylococcus aureus [14][15][16]. Moreover, fish is one of the most important protein sources for the rapid growing population in Tanzania. The per capita fish consumption has been estimated to be 25-30 kg [17]. A large population in Tanzania lives nearby water bodies and the consumption of fish constitutes more than 50 % of the total animal protein source [18]. However, fish are prone to microbial contamination from the sources when the water bodies are contaminated and along the value chain [19,20]. When the fish are contaminated, the microbes accelerate spoilage and are the cause of food-borne diseases to consumers.
In addition, unpasteurized fruit juices pose a high public health risk especially in developing countries where strict food hygiene is limited [21][22][23]. Studies have reported pathogens like Salmonella spp., Shigella spp., E. coli O157:H7 and Listeria monocytogenes as common contaminants in juices with low pH [24,25]. Most of the locally vended raw fruit juices in Tanzania are characterized with low pH [8,11], a condition which favour survival of bacteria. The number of documented outbreaks of human infections associated with the consumption of raw fruits, vegetables, and unpasteurized fruit juices has increased in recent years [26,27]. Unhygienic preparation, processing and handling of the raw fruit juices are the key sources of bacterial contaminations [8,22]. A study by Simforian et al. [11] reported high microbial contamination in raw fruit juices locally vendered in Dar es Salaam city, something that poses a threat of foodborne diseases to consumers.
There has been a big number of food-borne diseases in Tanzania including diarrhoea, dysentery, typhoid and cholera [28], but it is not known how much of these diseases have been contributed by the consumption of contaminated street vended food like milk, fish and raw fruit juices. Milk, fish and fruits are nutritious food which nutritionists recommend them in the daily diets of humans but when are not well handled; they are the potential sources of food-borne diseases. Over 90 % of the people in Tanzania consume raw milk that predisposes them to infections [29]. In cities like Morogoro, there has been an increase in consumption of freshly extracted fruit juices because of availability of variety of fruits throughout the year [30] but their safety is not known. This study aimed to assess the physical characteristics and establish the bacterial status of the selected food that is locally sold in Morogoro Municipality, Tanzania. The baseline data of this work will provide added information on status of microbial contaminations in food marketed in Tanzania. This will also be useful to public health officials in instituting control measures of food-borne diseases in Morogoro Municipality and Tanzania at large.
Study area
The study was conducted in Morogoro Municipality, Tanzania. Selection of the municipality, as the study area was based on convenience of accessibility from the laboratory and it generally represents food vending systems in urban areas of Tanzania.
Ethical approval
The permission to carry out this study was granted by the Morogoro Municipal Director. The Vice Chancellor of Sokoine University of Agriculture (SUA) issued a research permit letter on behalf of The Tanzania Commission for Science and Technology (COSTECH) that permitted the researchers from SUA to conduct their research in Morogoro Municipality. Before commencement with the research, the study protocol was prepared, submitted to, and approved by the SUA ethical committee. Verbal consent was obtained from each of the food vendor after explaining the purpose and importance of the study prior to start of data collection. Participation in the study was on voluntary basis. In addition, before the manuscript was submitted to journal for publication, the data were submitted to the local ethics committee at SUA and to the Morogoro Municipal Director and permission to publish the results was granted.
Sampling and sample handling
Milk samples were collected from farmers, vendors and restaurants/milk kiosks in different wards and streets. At farm level, 20 raw milk samples were collected directly from the storage containers with the pooled milk. From vendors, nine raw milk samples were collected directly from the storage containers. Eleven boiled milk samples were collected from 11 randomly selected restaurants/ milk kiosks. Before sampling, milk assessment for smell, colour, any dirty, turbidity, cleanliness of containers and milk handlers was done. The smell of milk was assessed after the farmer or vendor had opened the lid of the container. The colour was examined visually by putting well stirred milk in the clean glass container as described by Kurwijila et al. [29]. About 250 ml of milk sample was collected from each source, put in a sterile glass bottles and placed in a cool box with ice packs during the field work. A total of 40 milk samples were collected and analysed.
The raw juice samples were collected from vendors in different places including 11 samples from the bus terminals, 10 samples from selected cafeterias and 9 samples along roadsides. Before sampling, juice assessment for smell, colour, dirtiness, cleanliness of containers and assessment of the environment was done. The juice samples were collected directly from the storage containers into sterile falcon tubes and placed in a cool box with ice packs before shipment to the laboratory for analysis. A total of 30 juice samples were collected and analysed.
1. Cleanliness of the fruit juice containers: • Clean: A container is obviously free from dirty substances such as abnormal stains, dried juice spillover on surfaces and has not changed its original colour due to wear and tear. • Satisfactory: no dried juice spillover, presence abnormal stains that cannot be removed by cleaning, container appears to have irreversibly changed its original colour but appears clean. • Dirty: refers to presence of dried juice spillover, change of original container colour and presence of abnormal stains that may be removable by cleaning.
2. Cleanliness of the environment at fruit juice selling points: • Dirty: the area around where juice is sold has discarded litter materials such as plastic bags, papers, poured water on the ground, sold nearby municipal drainage systems, near dump sites or any other materials that should not be discarded irregularly such as maize cobs, mango and orange peelings. • Clean: the area is free from above mentioned factors dirty environment.
3. Presence of dirty in fruit juice: • Yes: refers to presence of abnormal suspended particles such as sand, fruit peelings or any other materials that should not be present • No: refers to lack of obvious abnormal suspended materials. • Note that the following criteria were developed and used in assessment of hygienic practices in the juice handling environment as per FAO [31].
Freshwater and marine fish samples were collected from 14 fish shops and Mindu dam shoreline selling point. Before collection of samples, information on the type of fish (freshwater or marine), fish sources and means of package were collected. From the fish shop freezers, four representative fish samples were taken from different corners, each was put into separate sterile zipped plastic bags and placed in a cool box with ice packs. A total of 185 fish samples were collected from different fish selling points in Morogoro of which, 79 (42.7 %) were marine and 106 (57.3 %) freshwater fish. All the collected samples were being transported in a cool box with ice packs to the Faculty of Veterinary Medicine laboratory at Sokoine University of Agriculture for analysis within 2-3 h of collection.
Laboratory analysis of food samples Milk samples
Physicochemical quality assessment of milk samples Part of the milk samples was filtered using a clean white cloth and assessed for physical dirt/contaminants [32]. In case of any debris remained on the white cloth; the milk was regarded as dirty. Determination of pH of milk was done by using Jenway 3540 pH and Conductivity Meter (Bibby Scientific Ltd, Staffordshire, UK) which was first calibrated using standard buffer solutions of pH 7.0 and pH 4.0 (ISO 7218:2007) [33]. The used reference pH was 6.6-6.8 for raw cow milk as given by East African Community standards [34]. Alcohol test was done to ascertain the milk acidity and it involved mixing a 5 ml aliquot of the milk sample with an equal volume of 70 % ethanol in a clean test tube and examined for the presence clots. Specific gravity was measured by use of a lactometer at standardized temperature of 20 °C [29]. The used reference relative density for whole milk was 1.027-1.030 as given by East African Community standards [34].
Assessment of Escherichia coli and total viable count (TVC) in milk samples
Total viable count (TVC) for enumeration of microorganism in raw milk at 37 °C applied a protocol described by Tanzania Bureau of standards and ISO/FDIS 8261 (E) [35,36]. Briefly, tenfold serial dilution of milk sample from 10 −1 to 10 −10 in sterile normal saline solution was done, using disposable pipettes. From each dilution, 1 ml of diluted sample was placed in a sterile Petri dish followed by the addition of 20 ml of molten nutrient agar (Oxoid Ltd, Basingstoke, UK), gently shaken and left to solidify. Incubation was done under aerobic condition at 37 ± 1 °C for 24 ± 3 h. Microbial colon count on the plates was done with the aid of portable magnifying lens and colonies in the culture plate were countered by using colony counter. Two consecutive plates with 30-300 colony forming units (cfu/ml) were considered for record [37]. The countable microbial colonies from two consecutive plates of each sample were converted into colony forming units per millilitre (cfu/ml) using a for- where N-number of microbial colonies counted, C-sum of colonies counted in two successful dilutions, v-volume of sample and d-dilution in the first plate counted [33].
Escherichia coli was determined in milk samples to assess for the possibilities of faecal contaminations. Briefly, 1 ml of milk sample was added into universal bottle containing 9 ml of buffered brilliant green bile glucose broth (EE broth) (Oxoid Ltd, Basingstoke, UK) then incubated at 37 °C for 24 ± 2 h under aerobic condition for enrichment. Thereafter, the sample was subcultured into tryptone water and incubated under the same conditions. Presence of E. coli was confirmed by addition of indole reagent in tryptone water and observed for presence of a red ring in the alcoholic phase which indicated indole production signifying a positive test. Isolation and identification of pure colonies of E. coli was done as described in ISO 7251:2005 protocol [38]. Briefly, the enriched sample in EE broth was striped onto MacConkey agar and incubated at 37 °C for 24 ± 2 h under aerobic condition. Typical colonies of E. coli grown on MacConkey agar were dry, medium in size, pink in colour and appeared singular or in groups. Gram staining and biochemical reactions like oxidase, nitrate and indole tests were performed to confirm the presence of E. coli.
Juice samples
pH and microbiological determination in juice samples The pH of juice samples was measured by using Jenway 3540 pH and Conductivity Meter (Bibby Scientific Ltd, Staffordshire, UK) after calibration using standard buffer solutions of pH 7.0 and pH 4.0 [33]. Microbiological evaluation was done by establishing TVC and most probable number (MPN). Total viable count for enumeration of microorganism in raw juice at 37 and 45 °C applied a protocol described by Tanzania Bureau of standards and ISO/FDIS 8261 (E) [35,36] as was described for the raw milk. For the determination of total coliform count (TCC) (coliform and faecal coliform bacteria), MPN, the presumptive test for coliforms was carried out using MacConkey broth (Oxoid Ltd, Basingstoke, UK) in the three test tube method as described by WHO [39]. The three tubes with 10 ml of broth were inoculated with 1 ml from 10 −1 , 10 −2 , 10 −3 , 10 −4 and 10 −5 prepared dilutions and incubated at 37 °C for 24 h for coliforms and at 45 °C for faecal coliforms as described by WHO [39]. All tubes showing gas production were observed and recorded. The MPN tables for three tubes dilution were used to report the result of the presumptive MPN of coliform bacteria per ml. E. coli was confirmed in samples as described above for the milk samples.
Fish samples
Physical assessment of fish samples Upon arrival in the laboratory, fish samples were identified to species level using standard identification keys. Biodata details of fish like sex, weight, age and length was not determined. Fish samples from the freezers were allowed to thaw followed by physical assessment. Physical evaluations undertaken included smell, sight (appearance) and touch (consistence) as described by Codex guidelines for the sensory evaluation of fish and shellfish in laboratories [40]. A special attention was directed to the eyes, gills, skin, texture and attachment of scales for the scaled fish like tilapia.
Microbiological assessment of fish samples For the purpose of qualitative analysis, three bacteria groups were targeted namely E. coli, thermophilic Campylobacter and Salmonella spp. Isolation and identification of E. coli from fish swab samples started with enrichment in buffered brilliant green bile glucose broth (EE broth) (Oxoid Ltd, Basingstoke, UK) and all the other procedures were done as for the milk samples.
Fish swabs with samples for Campylobacter isolation were immediately placed in a sterile universal bottle containing 10 ml of Bolton broth (Oxoid Ltd, Basingstoke, UK) with antibiotic (cefoperazone 32 mg/l) supplements (Oxoid Ltd, Basingstoke, UK). The samples in the Preston broth were loaded in the microaerophilic candle jars (Coldstream Engineering Ltd, Arista, Sweden) with a lighting candle and incubated at 37 °C for 24 h for enrichment as described by Atabay and Corry [41]. Isolation and identification of thermophilic Campylobacter was carried out according to the method described by Karmali et al. [42]. Briefly, after incubation, the universal bottles with enriched samples were properly shaken and sub-cultured onto modified charcoal cefoperazone deoxycholate agar (Oxoid Ltd, Basingstoke, UK) for primary isolation of Campylobacter. The inoculated Petri dishes were incubated at 42 °C for 48 h under microaerophilic conditions and colonies resembling Campylobacter were sub-cultured onto blood agar (Oxoid Ltd, Basingstoke, UK) at 37 °C under microaerophilic conditions for 24 h. Suspected Campylobacter colonies on blood agar that were Gram negative, curved, or spiral rods and showed corkscrew-like motion; positive to catalase, oxidase and nitrate reduction tests were further tested for hippurate hydrolysis, H 2 S production, and susceptibility to nalidixic acid and cephalothin. These parameters formed the basis for the identification of C. jejuni, C. coli, or C. lari as described by On [43].
The swab samples for Salmonella isolation were put in sterile universal bottles containing 10 ml selenite cysteine broth (Oxoid Ltd, Basingstoke, UK). Isolation and identification of Salmonella species was carried out by using ISO 6579:2002 protocol [44].
Total viable count (TVC) of microorganisms in fish at 37 °C was performed using a protocol described previously [35]. Briefly, 1 g of fish sample was grinded using sterile mortar and pestles and 10 ml of maximum recovery diluents (MRD) (Oxoid Ltd, Basingstoke, UK) was added and thoroughly mixed as described in EAS 217-1-3:2008 protocol [45]. The TVC in fish sample at 37 °C applied a protocol described by Tanzania Bureau of standards and ISO/FDIS 8261 (E) [35,36] as was described for the raw milk. After incubation all colonies were counted and the results reported as cfu/g.
Statistical analysis
The TVC values were log transformed before statistical analysis in order to make the frequency distribution more symmetrical. The Epi info version 2000 (approved by CDC, Atlanta, GA, US) was used for statistical analysis. Differences on means of TVC between groups of samples were compared by Mann-Whitney/Wilcoxon two-sample test. Means were considered significantly different at p < 0.05.
Physicochemical and microbiological quality of milk samples
Total of 40 milk samples were collected, whereby 11 were boiled milk and 29 were raw milk from Morogoro Municipality. It was found that the milk sold in Morogoro Municipality was of poor quality as depicted through physical and microbiological assessment. Three milk samples (7.5 %) had bad smell, 20 % were contaminated with cow dung, hairs and unidentified sediments, 25 % clotted on alcohol test and were the milk which had pH between 5.7 and 6.1. Majority (70 %) of the milk samples had low specific gravity (Table 1).
Microbiological results showed that the mean TVC of raw milk was 4.7 (Log cfu/ml) with the highest count recorded in raw unboiled milk samples (5.5-5.6 Log cfu/ml) particularly those which had bad smell and sediments ( Table 2). Lowest microbial count (3.3-4.1 Log cfu/ml) was depicted in five boiled milk samples and three samples which had a pH of 5.7. Assessment of different sources of milk samples against differences in the means TVC showed the significant differences (p = 0.0000) with the highest mean TVC (4.9 Log cfu/ml) recorded in milk from vendors. The mean TVC (4.9 Log cfu/ml) in unboiled milk was higher than that of boiled milk (4.3 Log cfu/ml) and the difference was statistically significant (p = 0.0000). The figures described herewith are averages over all target categories of milk sources in this study as were depicted in Table 2. The overall results indicated that 60 % of all milk handled had higher TVC than the maximum recommended of 5.3 Log cfu/ml for raw milk as given by East Africa Community standards [34]. A total of 25 (62.5 %) of the milk samples were contaminated with E. coli. This included all the eight milk samples from the farms which had sediments of dung and hairs.
Physicochemical and microbiological quality of juice samples
A total of 30 samples of locally vendered raw fruit juices from: cafeteria 10, along roadside 9 and from bus
Physical and microbiological quality of fish samples
A total of 185 fish samples were collected from different fish selling points in Morogoro of which, 79 (42.7 %) were marine and 106 (57.3 %) freshwater fish. Fourteen fish shops and Mindu dam shore fish selling points were involved in the study. The marine fish species were Siganus, Sardine, Caranx, Tuna and Lethrinus while freshwater fish species were tilapia (Oreochromis niloticus and O. urolepsis) and Clarias gariepinus. The physical appearance of the slime layer on fish skin to most fish (62 %) had started becoming turbid, opaque and milky. All the assessed fish had soft muscles with pitting characteristics on touch. The tilapia had their scales easily peeling off. Majority of the fish (80 %) generally appeared dull with loss of bloom, their eyeballs were sunken and cloudy, their gills appeared brown or gray covered with some mucus and others were greenish in colour. Fortythree percent of the fish had sour smell.
The prevalence of water-borne bacteria in fish was 54.6, 0.5 and 0.0 % for E. coli, C. jejuni and Salmonella spp. respectively. The only C. jejuni isolated was from freshwater fish (Tilapia) sourced from Mtera dam. Freshwater fish and unfrozen fish were more contaminated with E. coli than the marine fish and frozen fish respectively (p < 0.05) ( Table 5). The mean TVC of raw fish was 8.1 (Log cfu/g) with the highest count recorded in freshwater unfrozen fish samples (8.9 Log cfu/g) from Mindu dam ( Table 5). The lowest microbial count (4.2 Log cfu/g) was observed in frozen marine fish from Tanga. Comparisons of fish types, storage conditions and sources against differences in the means TVC showed significant differences (p = 0.0000). According to Tanzania criteria/ guidelines established by Fisheries Act No. 22 of 2003 and Fisheries Regulations of 2003 [47] it recommends that the TVC should not exceed 5.0 Log cfu/g for raw fish. With these results, 96.2 % the fish samples had TVC beyond the recommended limits [47].
Discussion
The current study aimed to assess the physical quality and establish the microbial status in raw milk, raw fish and raw unpasteurized fruit juices locally sold in Morogoro Municipality, Tanzania. It was generally found that the milk sold in Morogoro Municipality was of poor quality as depicted through physical and bacteriological assessment. Unpasteurized raw fruit juice indicated high bacterial load which may be sources of spoilage and endangers the health of consumers. Similarly, fish bacterial analysis indicated high prevalence of E. coli as an indicator of faecal pollution in water and also isolated C. jejuni from freshwater fish. High proportion of food samples with poor physical quality and high bacterial loads as was observed during this study suggests that there is high risk of public exposure to food-borne diseases in Morogoro Municipality. Food producers and vendors should be educated on good and safe food preparations accompanied with good hygienic practices so as to safeguard the public from eating contaminated food. It is further stressed that there is a need for enforcement of regulations regarding the safety of locally vended food and routine inspection of food, selling premises and regular health check-up of the personnel involved in food vending industry.
Assessment of milk quality
Presence of physical dirt in raw milk serves as potential sources of microbial contamination and cause poor eye appeal to consumers that may lead to lowered marketability. Abnormally bad smell of milk observed during this study partly may be contributed by spoiled milk, dirty containers, contamination with dirty like cow dung, hairs and general poor milk hygiene. Similar observations have been reported by Bukuku et al. [48]. Contaminations accelerate spoilage which may lead to milk-borne diseases to consumers. Large amount (70 %) of the milk sold had low specific gravity (SG) below standard of 1.028 g/ ml as given by East African community [34] which suggests adulterations.
The results of the present study showed that (60 %) of all milk handled had higher TVC than the maximum recommended of 5.3 Log cfu/ml as given by East Africa Community standards [34]. The mean TVC of raw milk was 3.4 Log cfu/ml with the highest count recorded in raw unboiled milk samples (5.5-5.6 Log cfu/ml). This suggests a considerable proportion of milk consumers are at risk of milk-borne diseases since majority of the people in Tanzania consume raw unboiled milk [29]. Bacterial contamination of raw milk can occur from three main sources; within the udder, outside the udder, and from the surface coming into contact with the milk [12,13,48]. It was observed that among the milk samples with highest TVC were those with sediments (cow dung and hairs) which are potential sources of microbes acquired from the animal itself. In addition, farmers and other milk dealers may incur losses due to rejection of spoiled milk as was observed during this study, 25 % of the milk samples tested clotted on alcohol test. The results of this study are inline to those previously published in Tanzania by Bukuku et al. [48] and Kivaria et al. [49] in which most of the samples tested had higher bacterial count above the standards.
Surprisingly, a high mean TVC of 4.3 Log cfu/ml in boiled milk was recorded implying that boiling is not the only critical step for improving the microbiological quality of milk products. The unsatisfactory quality of boiled milk is the consequence of the poor quality of raw milk used and/or a high level of recontamination after boiling. Poor handling and storage of boiled milk in restaurants/ kiosks observed during this study give high possibilities for post-boiling contaminations. These findings highlight the fact that boiled milk of such poor microbiological quality poses a threat to consumers. E. coli was isolated from 62.5 % of the milk samples analyzed. According to WHO guidelines, E. coli should not be present in any raw or processed milk since the bacteria are used as an indicator of faecal contamination. In addition, strains like E. coli O157:H7 are also pathogenic to humans and their occurrence has been reported in Tanzania [50].
Assessment of fruit juice quality
The general finding on fruit juice showed that it was prepared and served in unhygienic environments. Most of the containers (43.3 %) used to store juice were dirty while 20 % of the selling points were also dirty suggesting of contamination. Disregard of sanitary practices during juice preparation, storage in clean containers and vending in clean environment might be the possible cause of high bacterial load in fruit juices sold along roadsides and bus terminals as previously reported by Nonga et al. and Simforian et al. [8,11]. This was supported by the high bacterial load in fruit juice samples beyond the recommendations. According to the Gulf standards, the allowable TVC levels for raw unpasteurized fruit juices should be within 3.7-4.0 Log cfu/ml [46]. Majority of the fruit juices (73.3 %) in the current study had TVC above the recommended levels which imply poor quality. The TVC recorded in the current study varied from 4.2 to 6.3 Log cfu/ml at 37 °C and 1-5.8 Log cfu/ml at 44.5 °C which indicate a serious case of poor hygiene and the juice was unfit for consumption. This range is comparable to the findings reported by Simforian et al. [11]; it is however, lower than those reported by Kumar et al. [51]. With these results, it calls for the routine screening and control of locally extracted fresh fruit juice which is being sold in streets by the local vendors in different town centers in Tanzania.
It was further found that majority of the samples were contaminated with coliforms in particular E. coli which indicates evidence of faecal contamination or poor hygiene and the juice was unfit for consumption. The main source of coliform contamination might be through contaminated water supplies which are used in processing of juice or the personnel [52]. Assessment of food preparation areas indicated that majority of vendors prepared and sold juices in unhygienic conditions that attracted houseflies and dust. According to Tanzania Specifications (TZS 585:2003), coliforms like E. coli must not be present in ready to drink beverages [53]. The E. coli prevalence recorded in the current study indicates that most of the raw unpasteurized fruit juice vended in Morogoro is not fit for human consumption and predisposes consumers to food-borne diseases.
Assessment of raw fish quality
Results on raw fish indicated that most of the fish were of poor quality since most of them were at different stages of spoilage and therefore unfitness for human consumption. High prevalence of E. coli (54.6 %) was recorded which indicates evidence of faecal contamination either from the water body, rapture of intestines during eviscerations or contamination along the fish value chain mainly due to poor hygiene and storage conditions. Some strains of E. coli are known to cause diarrhea, urinary infections, pyogenic infections and septicemia in humans [50,54,55]. Meanwhile, isolation of C. jejuni from tilapia sourced from Mtera dam poses more danger to fish consumers. Campylobacter jejuni has been reported in Tanzania as among the major causes enteritis in humans [55,56].
Findings on bacterial enumeration in fish indicated that the mean TVC in freshwater fish was 8.3 Log cfu/g while in marine fish was 6.2 Log cfu/g. According to Tanzania criteria/guidelines established by Fisheries Act No. 22 of 2003 and Fisheries Regulations of 2005 [47] it recommends that the TVC should not exceed 5.0 Log cfu/g meaning that 96.2 % of the fish samples had TVC beyond the recommendations. Other studies in Tanzania have also reported high bacteria count in freshwater and marine fish [20,57]. High bacterial load in fish predisposes them to fast spoilage and possibly become potential sources of food-borne poisoning to consumers. This was evidenced by the results of physical assessment which showed majority of the fish were spoiled. In addition, presence of pathogenic bacteria like C. jejuni in raw fish poses a health risk to the consumers.
Conclusions
It is concluded that most of food vended in the Morogoro Municipality is of poor quality physically and microbiologically, which poses health risks to public and losses to food owners due to spoilage. Stakeholders in food value chain should be educated on safe production and good hygienic practices. Routine safety assessment of locally vended food, inspection of selling premises and regular health check-up of the personnel involved in food vending industry should be done. Authors' contributions HEN developed the research proposal, collected data, compiled all data for raw milk, raw fruit juice and raw fish, analysed the data and drafted the manuscript. HAN planned the study, supervised data collection, participated in data analysis, interpretation and proof reading the final version of manuscript before submission. RHM was the overall supervisor in all activities involving study planning, data collection and he participated in providing scientific and technical advice in laboratory work and proof read the final version of manuscript before submission. EM participated in fruit juice sample collection, laboratory analysis and writing of report. GBN and JAM participated in raw fish sample collection physical and laboratory analysis, and writing of report. RW participated in raw milk sample collection, physical and laboratory analysis, and writing of report. All authors read and approved the final manuscript | v3-fos |
2022-11-30T14:32:52.792Z | {
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} | s2 | Hydrolysis with Cucurbita ficifolia serine protease reduces antigenic response to bovine whey protein concentrate and αs-casein
In the present study the effect of hydrolysis with non-commercial Cucurbita ficifolia serine protease on a reduction of the IgE and IgG binding capacity of whey protein concentrate and αs-casein was investigated. The intensity of the protein degradation was analyzed by the degree of hydrolysis, the free amino groups content and RP-HPLC. The ability to bind the antibodies by native proteins and their hydrolysates was determined using a competitive ELISA test. Deep hydrolysis contributed to a significant reduction of immunoreactive epitopes present in WPC. In the case of IgE and IgG present in the serum pool of children with CMA, the lowest binding capacity was detected in the 24 h WPC hydrolysate, where the inhibition of the reaction with native WPC was ≤23 and ≤60 %, respectively. The analysis of the IgG reactivity in the antiserum of the immunized goat showed that the lowest antibody binding capacity was exhibited also by 24 h WPC hydrolysate at a concentration of 1000 μg/ml where the inhibition of the reaction with nWPC was ≤47 %. One-hour hydrolysis of α-casein was sufficient to significant reduction of the protein antigenicity, while the longer time (5 h) of hydrolysis probably lead to the appearance of new epitopes reactive with polyclonal.
Introduction
Food allergies, i.e. adverse reactions to food are an abnormal immune response to a specific food component. The reaction are known to cause many health problems (Koletzko et al. 2012) starting from skin reactions and hives, even to an anaphylactic shock. One of the most common is allergy that to milk and dairy products (cow's milk allergy-CMA), which affects mainly children (Wal 2004;Kneepkens and Meijer 2009). It is reported that 0.6-2.5 % of preschoolers, 0.3 % of older children and teens, and up to 0.5 % of adults suffer from CMA (Fiocchi et al. 2010). When breast-feeding is not available or possible, cow's milk is usually used as a natural substitute for human milk. Because milk proteins are the first exogenous proteins, such a substitution can lead to nutritional and immunological problems (Wal 2004;Kneepkens and Meijer 2009). CMA is mediated both through IgE and non-IgE mechanisms, where the non-IgE reactions are less readily recognized due to a less distinct temporal relationship between exposure and symptoms (Turnbull et al. 2015).
Cow's milk contains two major protein fractions: casein and whey proteins. Casein is composed of α-S1-, α-S2-, βand κ-fractions. While among whey proteins β-lactoglobulin (BLG, 55-60 %) and α-lactalbumin (15-20 %) are in the majority. Other minor proteins are bovine serum albumin, immunoglobulins and lactoferrin. Due Abstract In the present study the effect of hydrolysis with non-commercial Cucurbita ficifolia serine protease on a reduction of the IgE and IgG binding capacity of whey protein concentrate and αs-casein was investigated. The intensity of the protein degradation was analyzed by the degree of hydrolysis, the free amino groups content and RP-HPLC. The ability to bind the antibodies by native proteins and their hydrolysates was determined using a competitive ELISA test. Deep hydrolysis contributed to a significant reduction of immunoreactive epitopes present in WPC. In the case of IgE and IgG present in the serum pool of children with CMA, the lowest binding capacity was detected in the 24 h WPC hydrolysate, where the inhibition of the reaction with native WPC was ≤23 and ≤60 %, respectively. The analysis of the IgG reactivity in the antiserum of the immunized goat showed that the lowest antibody binding capacity was exhibited also by 24 h WPC hydrolysate at a concentration of 1000 μg/ml where the inhibition of the reaction with nWPC was ≤47 %. Onehour hydrolysis of α-casein was sufficient to significant reduction of the protein antigenicity, while the longer time to their outstanding nutritional traits and properties whey proteins are used as food ingredients in functional food products, such as infant formula, yogurt, meat and bakery products.
Studies conducted on large population of infants with CMA have shown, that the major milk allergens are BLG and α-S1-casein (Wal 2004;Mine and Yang 2008;Schulmeister et al. 2009). BLG-which is absent in human milk-may induce allergies in infants because of their underdeveloped gastrointestinal tract and immune system (Kattan et al. 2011). The BLG monomer is a globular protein composed of 162 amino acids with a molecular weight of 18.3 kDa. The tertiary structure of this globular protein, is stabilized by two disulfide bonds, makes it stable at low pH and resistant to gastric digestion (Lovegrove et al. 1993). In patients with persistent allergic reaction, seven IgE and six IgG binding epitopes were detected on BLG (Järvinen et al. 2001). Casein is, in general, easily digested, although some experiments (Schulmeister et al. 2009) have shown that some intact IgE-reactive fragments α-S1-casein that forms the core of the micelle are responsible for the induction of allergic reactions. It was also shown that the α-S1-casein found in human milk differs from the bovine casein (Otani et al. 1987).
Reduction or elimination of the major milk allergens by application of effective methods and technologies is essential to patients allergic to milk. However, it is very difficult to entirely remove the protein components from diet, and it is even harder to perform in case of infants who cannot be breast-fed (Kim et al. 2007).
Different attempts have been made to reduce the allergenicity of cow's milk proteins and various technological processes have been applied for this purpose. Extensively hydrolyzed cow's milk based formulas have been recommended; however, they have major drawbacks, such as an unacceptable bitter taste and high production costs (Bu et al. 2013). The remaining allergenicity depends on the degree of hydrolysis, the enzyme used and the technological processes such as filtration and heating (Bu et al. 2013). Thus, a study of the IgE responses to native, denatured and hydrolyzed cow's milk proteins is critical for the development of new milk derivatives or replacements for sensitive patients.
Whey proteins are significantly resistant to being hydrolyzed. Proteases cleave milk proteins into peptides and may therefore have crucial effects on further gastrointestinal milk digestibility, release of the bioactive peptides, and exposure of antigenic epitopes (Raikos and Dassios 2014). The application of enzymes can increase the cost of the process, therefore cheap sources are preferred. Introducing enzymes derived from easily accessible sources may result in obtaining hydrolysates exhibiting potentially attractive properties, and simultaneously reducing production costs.
For example, plant serine protease isolated from C. ficifolia fruit exhibits attractive proteolytic properties which have been analyzed towards e.g. casein or protein from corn gluten meal (CGM) (Illanes et al. 1985;Curotto et al. 1989).
The aim of this study was to investigate the effect of hydrolysis with non-commercial C. ficifolia serine protease on a reduction of the IgE and IgG binding capacity of whey protein concentrate and αs-casein.
Materials and methods
The enzyme Serine protease was isolated from C. ficifolia fruit by the method of Dryjański et al. (1990). After separating peel and seeds, the pulp was homogenized and centrifuged 5000×g, 20 min. To the clear supernatant solid ammonium sulfate was added to the 30 % saturation. The final precipitate was collected by centrifugation at 5000 × g, 20 min. Desalting was conducted by dialysis in water. The specific activity of the enzyme preparation was 4411 U/g.
Substrates Whey protein concentrate (WPC-80) manufactured from sweet whey and spray dried was provided by Davisco Foods International, Inc. Alpha-s-casein was obtained according to the method of Thompson and Kiddy (1964) by a urea-calcium chloride procedure with a final precipitation in ethanol-ammonium acetate solution.
Sera All sera from patients were kindly provided by med. dr Ewa Willak-Janc from the 1st Department of Pediatrics, Allergy and Cardiology, Medical University in Wrocław.
Hydrolysis of WPC-80 and αs-casein Enzymatic hydrolysis of 1 % protein solution was conducted using serine protease isolated from C. ficifolia at the dose of 150 U/mg of hydrolyzed protein. The reaction was carried at 37 °C for 1, 3, 5, 24 h in 0.1 M Tris-HCl buffer at pH 8.0. The hydrolysis was terminated by thermal inactivation (for biological activity determinations) or by the addition of 10 % trichloroacetic acid (TCA) (1:1 V/V).
The degree of hydrolysis The course of the hydrolysis was monitored by the determination of soluble peptide concentration in 5 % TCA in relation to total protein. The concentration of the trichloroacetic acid-soluble product in the supernatant was measured spectrophotometrically at λ 280 nm (Spellman et al. 2003).
The free amino groups' concentration The concentration of free amino groups (μmol Gly/g) was determined using trinitrobenzene sulfonic acid (TNBS, Sigma) according to the method described by Kuchro et al. (1983).
Reversed-phase high-performance liquid chromatography (RP-HPLC) Peptide profiles were determined by RP-HPLC with an Agilent 1100 Series system. The peptide preparations were solubilized in the even volume of phase A (0.1 % TFA in H 2 O) before loading on the chromatographic HPLC column (Zorbax Eclipse XDB-C18 Agilent column (50 × 4.6 mm). Separation was performed at a flow rate of 1 ml/min at 30 °C. Peptide fractions, varying in hydrophobicity, equal from the column in linear gradient of phase B (0.1 % TFA in acetonitrile). Absorbance measurement was made at λ = 230 nm (DAD, G1315B).
Determination of protein content Protein content was determined by colorimetric method of Lowry et al. (1951), using BSA (Sigma, P0834) as a standard.
Determination of the binding capacity of IgG and IgE antibodies by WPC-80 hydrolysates
Analysis of antigenicity of WPC-80 hydrolysates. The ability to bind IgE and IgG antibodies by native WPC-80 (nWPC) and WPC-80 hydrolysates was determined using a competitive ELISA test according to the modified method described by Pescuma et al. (2011).
A pool of sera from 20 children (3-5 years old) with persistent cow's milk allergy, as defined by a positive result in specific serum IgE was used in the study. Sera were selected from 37 analyzed samples and were characterized by the highest absorbance levels (OD 450nm ≥0.5) in the reaction with WPC. A pool of 5 sera, with high IgG reactivity (OD 450nm ≥0.5) with nWPC, taken from children not exhibiting any clinical symptoms of CMA was used as a control.
A pool of sera with various protein solutions was mixed at a ratio of 1:1 and incubated for 2 h at room temperature on rotary platform in 48-well plates. After incubation the mixture was loaded in triplicates on plates previously coated with nWPC solution and blocked and incubated for approximately 18 h at 4 °C on a rotary platform. As the secondary antibodies goat anti-human IgE conjugated to HRPO (Sigma-Aldrich A9667, dilution 1: 3000), or rabbit antihuman IgG conjugated to HRPO (Sigma-Aldrich A8792, dilution 1:60 000) were used in amount of 100 μl of conjugate per well. Afterwards the mixture was incubated for 1.5 h at room temperature with gentle rotation. The reaction was developed for 30 min using the TMB Super Sensitive substrate (Sigma-Aldrich, T4444). Readings were performed at 450 nm using a Quantum reader (Biotek).
The percentage of antigen-binding inhibition was calculated using the following equation: Inhibition rate (%) = [(Abs o −Abs x )/Abs o ] × 100, where Abs o was the mean absorbance value of no-antigen triplicates (serum was incubated with PBST at a ratio of 1:1); Absx was the mean absorbance value of triplicates obtained with different antigen concentrations.
Determination of the binding capacity of goat anti-whey IgG/immune sera by WPC hydrolysates
The binding capacity of goat anti-whey IgG by WPC-80 hydrolysates was determined using the competitive ELISA test. The analyzed immune serum obtained as a result of 5-time, intradermal immunization of goats with whey proteins. The obtained and purified antigenic preparations, emulsified with Freund's complete adjuvant (Calbiochem) were administered every 14 days. Twelve days after the last injection, blood was collected from the cervical vein and the obtained serum was stored at −20 °C until use (Stefaniak et al. 2000).
Determination of the binding capacity of IgG and IgE antibodies by αs-casein hydrolysates
The binding capacity of IgE and IgG by native αs-casein (nα-c) and αs-casein hydrolysates was determined using a competitive ELISA.
A pool of sera from 20 children (3-5 years old) with persistent cow's milk allergy, as defined by a positive result in a specific serum IgE was used in the study. Sera were selected from 37 analyzed samples and were characterized by the highest absorbance levels (OD 450nm ≥ 0.5) in the reaction with nα-c. A pool of 5 sera, with high IgG reactivity (OD 450nm ≥ 0.5) with nα-c was taken from children not exhibiting any clinical symptoms of CMA was used as a control.
The experiment was conducted as described above. In the pre-incubation of sera with nα-c protein solutions/α-c hydrolysates after 1, 5, 24 h of hydrolysis, the following starting dilutions of serum pool were used: 1:10 in the case of determining the IgE reactivity and 1:3000 for IgG in the group of children with CMA, and 1:2000 in the group of healthy children.
Statistical analysis
All assays were conducted in triplicate. The results were analyzed using an Statistica 7.0 program analysis of variance (ANOVA), followed by a Duncan multiple range test to determine the significant difference between sample at p ≤ 0.05.
Results and discussion
The elimination of all cow's milk products, without appropriate substitutions, can lead to malnutrition and/or specific nutrient deficiencies at a time when infants and children are growing. Hence there is a need for reduction or elimination these major milk allergens by effective methods and technologies. Proteolysis may be considered an efficient way of removing allergenic epitopes in proteins and increasing their digestibility (Jędrychowski and Wróblewska 1999; Wróblewska et al. 2004). Based on the results of a randomized controlled study, only an extensively hydrolyzed formula was able to significantly decrease the prevalence of CMA (Businco et al. 1999). Extensively hydrolyzed protein formula, rather than an amino acid formula, is recommended for infants with IgE mediated CMA at low risk of anaphylactic reactions (no prior history of anaphylaxis or currently receiving an extensively hydrolyzed protein formula) (Koletzko et al. 2012).
Extent of hydrolysis
The course of the enzymatic hydrolysis of WPC-80 and αs-casein was monitored by determination of the degree of hydrolysis (DH) (%) ( Table 1). The determined DH for αs-casein and WPC-80 after 24 h hydrolysis reached the values of 61.3 and 43.2 %, respectively. During the protein degradation in all hydrolysates, a proportional increase in free amino group concentration (FAG) was also observed ( Table 1). The final concentrations of determined FAG reached 4725 µmol Gly/g for αs-casein hydrolysate and 4142 µmol Gly/g for WPC-80 hydrolysate. The progress of hydrolysis was also confirmed by RP-HPLC peptide profiles analysis (Figs. 1, 2). The presence of peptide fractions, eluted from the column at low concentration of acetonitrile and differing in terms of hydrophobicity, was noted on all hydrolysates chromatograms. BLG, the most abundant protein present in bovine whey, is known for its low susceptibility to enzymatic degradation. The RP-HPLC separation showed that even 1 h of hydrolysis with C. ficifolia serine protease resulted in almost complete disappearance of the peaks originating from the substrate. The use of this enzyme offers the possibility for protein degradation without other additional technological treatments, such as thermal and/or high pressure processing. Similar effects were observed by Ena et al. (1995), who studied conformation changes of proteins in WPC preparations after their hydrolytic degradation. During 3 h hydrolysis with Corolase 7092 (peptidases from Aspergillus strains) particular fractions were almost undetectable with the SDS-PAGE method. 24 h hydrolysis resulted in DH exceeding 21 %. Also Vázquez-Lara et al. (2003) demonstrated that 2 h hydrolysis of native β-lactoglobulin with the plant protease Actinidin lead to a DH of 43 %.
Antigenicity of WPC hydrolysates
Analysis of the IgE reactivity in the sera of children with cow's milk allergy (Fig. 3a.1) showed that a 2-hour preincubation of antibodies with nWPC (at a concentration ≥10 μg/ml) induced the binding of all WPC-specific IgE antibodies. Hydrolysis resulted in a reduction (approx. 24-77 %) in IgE-binding capacity by peptides as compared to the control (nWPC). It was also observed that prolonged hydrolysis did not result in a decrease in binding capacity of IgE by ≤50 μg/ml solution of WPC hydrolysates. For all hydrolysates with those concentrations we observed less than 20 % inhibition of IgE antibody reactivity with nWPC.
This observation may suggest that the obtained hydrolysates have the ability to bind similar amounts of antibodies, and thus the degree of degradation did not reduce the amount of reactive epitopes. It should be noted however, that this observation may be due to the competition for binding sites (epitopes) between antibodies of different classes, i.e.: IgE, IgG and IgM present in the analyzed samples. The competition for epitopes may interfere with the reactivity of IgE antibodies, particularly as the concentration of these antibodies in the serum is much lower than the concentration of IgG. At higher hydrolysates concentrations (>50 μg/ml), where the number of potential sites for antibody binding was higher, the lowest IgE-binding capacity was detected in the 24 h WPC hydrolysate, where the inhibition of the reaction with nWPC was ≤23 %. Deep hydrolysis contributed to a reduction of higher amounts of reactive epitopes present in WPC.
The research by Pescuma et al. (2011) indicates that the hydrolysis of BLG by Lactobacillus strain, at the concentration of 1 mg/ml, considerably reduced (32 %) its recognition by the IgE of allergic children as compared to heatdenatured BLG. Although the degradation of this protein was incomplete, the IgE-binding reaction was still observed.
In the case of IgG present in the serum pool of children with CMA ( Fig. 3b.1) a high binding capacity of this antibody by both the control and the obtained hydrolysates was determined. In the case of the highest concentrations of analyzed WPC hydrolysates (1000 µg/ml), we found no significant differences in the IgG-binding capacity between the control and the hydrolysates obtained after 1, 3 and 5 h of hydrolysis. Inhibition of reactivity with nWPC did not exceed 78 %. The lowest IgG-binding capacity was determined in 24 h nWPC hydrolysate characterized by the highest degree of degradation, where the inhibition of the reaction with nWPC was ≤60 %.
Analysis of the reactivity of IgG present in the sera of healthy children (Fig. 3c.1) showed, as in the case of children with allergy, a high binding capacity for IgG by nWPC. There was a significant reduction (in the range of 21.5-84 %) in binding capacity for IgG by 1 h and 24 h hydrolysates compared to the control. Hydrolysate obtained after 24H of proteolysis, at a concentration of ≤10 μg/ml, showed no significant reactivity for IgG in the ELISA. It can be assumed that the application of this type of hydrolysate to an infant would not raise their immune system response.
It has been shown that exposure to cow milk protein during the first 3 months of life results in high levels of IgG subclass antibodies, detected even at 8 years of age (Jenmalm and Björkstén 1998). Similar findings were reported by Nentwich et al. 2004 who demonstrated the presence of specific IgG antibodies against cow's milk protein not only in the serum of children and an adult with CMA, but also in the serum of healthy people consuming cow's milk. It was demonstrated that CMA patients are characterized by elevated levels of specific IgE levels as well as the specific IgG levels (Shek et al. 2005;Meulenbroek et al. 2013). The IgG antibody could play an important role in allergen-Ab complex formation. The presence of IgG in allergen-IgE complexes may result in binding to B cells which may affect Ag processing and presentation, and thereby influencing the allergic response (Meulenbroek et al. 2013). Oldaeus et al. 1999 analyzed the presence of IgG and IgE antibodies specific to BLG in serum samples of 94 infants with a family allergy medical history, who had been fed with cow's milk formulas of different DH. They showed that the IgG responses to BLG were very low in the group exposed to extensively hydrolyzed formula, intermediate in the partially hydrolyzed formula, and high in case of a regular cow's milk formula. High immune responses connected to high concentration of specific IgE and IgG antibodies were associated with the development of atopic disease. The low antigenicity and allergenicity of the extensively hydrolyzed formula supports its use in allergy prophylaxis.
The analysis of the IgG antibodies reactivity in the antiserum of the immunized goat ( Fig. 3d.1) showed that a 2-h pre-incubation with nWPC antibodies (at a protein concentration = 1000 μg/ml) contributed to nearly 100 % of inhibition of their reactivity, while a 24-h pre-incubation with the hydrolysate at the same concentration caused inhibition of the reaction at the two times lower level.
In the case of using low concentrations of hydrolysates (<5 μg/ml) longer hydrolysis did not decrease the binding capacity for anti-WPC antibodies by the resulting peptides. For all hydrolysates it was found that the inhibition ▸ of antibody reactivity for nWPC at a similar level (≤30 %). A difference was found for higher concentrations of hydrolysates (≥5 μg/ml), where the progress of hydrolysis reduced the antigenicity of the whey proteins. The lowest antibody binding capacity was exhibited by hydrolysates 5 h and 24 h, at a concentration of 1000 μg/ml the inhibition of the reaction with nWPC was ≤63.4 and ≤47 %, respectively. This experiment confirmed that the hydrolysis procedure caused a partial reduction in reactive epitopes, which lead to a reduction of antigenicity of the WPC proteins.
Antigenicity of αs-casein hydrolysates
Analysis of the reactivity of IgE antibodies present in the serum pool of children with an allergy to cow's milk ( Fig. 3a.2) showed that 2-hour pre-incubation of antibodies with nα-c did not lead to a binding of all IgE specific for that protein. The inhibition of the reaction was ≤60 %. The observed phenomenon could have been caused by the strong competition for free epitopes between different classes of antibodies; some epitopes were bound by both IgG and IgM.
The time of hydrolysis caused a decrease in IgE-binding capacity of the resulting peptides compared to control, but the most preferred effect was observed after the 1 and 24 h hydrolyses, but not after the 5-hour hydrolysis (inhibition of reaction with nα-c was respectively ≤12, ≤21 ≤ 56.4 %). One-hour hydrolysis of α-casein was sufficient to significant reduction of the protein antigenicity, while the longer time (5 h) of hydrolysis probably lead to the appearance of new epitopes reactive with polyclonal anti-αs-casein IgE which were then degraded by further hydrolysis (24 h).
To date, the presence of nα-c specific IgG and IgE has been demonstrated in the serum of patients with CMA, as well as IgG antibodies in healthy individuals (Nentwich et al. 2004;Meulenbroek et al. 2013). It has been proven, however, that among the three fractions of casein, alphacasein has the lowest reactivity with IgG antibodies present in the serum of patients with CMA (Nentwich et al. 2004).
As shown by other researchers, hydrolysis can either decrease or increase cow's milk protein immunoreactivity as it is directly dependent on the specific activities of the different enzymes (Wróblewska and Kaliszewska 2012). During hydrolysis the protein is cleaved to different in size peptide fragments. Their properties are dependent from the amino acid sequence of the protein and also from their secondary structure. Hydrolysis of BLG by trypsin/chymotrypsin reduces its allergenicity, but also exposes hidden allergenic peptides, which are recognized by the specific IgE of allergic patients (Selo et al. 1999).
Analysis of the reactivity of IgG present in the serum pool of children with an allergy to cow's milk (Fig. 3b.2) showed a high binding capacity for this class of antibodies by nα-c. The resulting hydrolysates showed lower in comparison to control inhibition of reactivity with nα-c. However, the prolonged hydrolysis did not result in the decrease of binding capacity for IgG antibodies by the peptides (maximum difference in the inhibition of reactivity with nα-c between the 1H and 24H hydrolysates did not exceed 20 %). At a hydrolysates concentration of 1000 μg/ml the lowest binding capacity of IgG antibodies was found in hydrolysates after 1 and 5 h of the process, where reactivity inhibition in those samples was about 25 % lower than the controls. This can be partly explained by the altered aggregation and immunologic behavior of peptides in the entire hydrolysate mixture, where especially in high concentrations, they tend to re-associate and form macromolecular complexes.
In our study, analysis of the reactivity of IgG present in the serum pool of healthy children (Fig. 3c.2) showed a high binding capacity for this class of antibodies by nα-c. The lowest binding capacity of IgG antibodies specific for nα-c was shown by hydrolysate 24H, and the observed inhibition of the reaction with nα-c compared to control was at least 35 % lower. In the research by Nentwich et al. (2004), casein hydrolysate demonstrated only about 18.5 % casein antigenicity. No significant difference in IgG binding was detected between the patient and control sera in casein hydrolysate.
Conclusion
The results obtained in the present study demonstrate that hydrolysis using a non-commercial serine protease isolated from C. ficifolia led to a significant decrease in the antigenic IgE and IgG response to major allergens from bovine milk (WPC and αs-casein). These findings suggest that hydrolysates with such traits may have the potential to be administrated in the prophylactic treatment of infants with a high risk of allergy. appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. | v3-fos |
2019-05-30T13:20:29.009Z | {
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} | s2 | Willingness to initiate spice-tourism in the Kolonna District Secretariat of Ratnapura district in Sri Lanka: Farmers’ perspective
A study was conducted to assess the factors affecting the establishment of spice tourism in spice gardens in Sri Lanka, based on farmers’ perception including their willingness to start agro-tourism destinations. A sample of 60spice-growing farmers was randomly selected for the study from among those who have registered under the Department of Export Agriculture (DEA) in the Kolonna District Secretariat (DS) division in the Ratnapura district of Sri Lanka. A field survey was conducted using a pre-tested questionnaire. Data collection was conducted during from January to April 2014. Descriptive statistics and Chi Square test were used to analyze the data. Results revealed that the majority of the spicegrowing farmers were males and above 50 years old. They had education up to G.C.E. Ordinary Level (O/L), more than five members in a family, and long term experience in farming. The main reasons for farmers to start spice tourism destinations were better usage of the farm’s resources, employment opportunities for the family members, support from other existing tourist location and the interest or being a hobby. Furthermore, middle-age farmers having a significant level of education, large size of farm, higher annual income of the family, high profit from farm, large family size and long term experiences had associations with willingness to start spice tourism destinations. The study concluded that there is a significant number of positive factors for farmers to establish spice tourism in the Kolonna DS division. The government of Sri Lanka could assist this endeavor by giving education and training, improving infrastructure facilities and also motivating and assisting farmers to establish spice tourism destinations in the Kolonna DS division as an extra income generating activity.
Introduction
"Agro tourism is a commercial enterprise on a working farm, ranch, or agricultural plant conducted for the enjoyment of visitors that generates supplement income for the owner" (Mahaliyanaarachchi, 2014).In addition, agro tourism is an important option for farmers to diversify their farming operations that will bring more economic activities to rural areas. Agro tourism can be viewed as small scale, low impact, education-focused and recreational activity (Mnguni, 2010) Agriculture has been the oldest vocation in all countries since the Neolithic revolution (Senanayakeand Wimalaratana, 2008). Agro-tourism is all about unravelling various aspects of village life including opening up farms to tourists from cities and abroad, and letting them spend some time in the lap of nature (Barbieri, 2013). Apart from telling them about the various crops and how they are sown and harvested, agro-tourism exposes tourists to authentic food, handicraft, dress, culture, music and language. Tourists get to involve in rural activities such as bullockcart rides, milking cows and goats and picking farm-fresh fruits and vegetables. These activities may vary from village to village. Introduction of agro-tourism under different themes would be of great significance and would improve the overall economic, social, cultural, and aesthetic level of the areas (Das and Rainey, 2010;Colton and Bissix (2005). Wet Zone (WZ) of Sri Lanka has been the home for the major plantation crops and spices for centuries (Punyawardena, 2007). Though agrotourism in Sri Lanka is still at its infant stage, the country has a huge potential to expand the agro-tourism sector (Malkanthi and Routry, 2012).
Sri Lanka is famous for its spices, exporting a range of products including cinnamon, pepper, cloves, nutmeg and cardamom. The Ratnapura district of the Sabaragamuwa province contributes to 10 % of the total cultivation of cinnamon and 13 % of pepper (DEA, 2014). Many spices such as cinnamon, pepper, cardamom, citrus, cloves, fennel, garlic and nutmeg are grown in the Kolonna District Secretariat (DS) division in the Ratnapura district, providing a significant income to the people. The cultivation and processing of spice crops are assisted by the Department of Export Agriculture. This DS division is also a famous tourism destination with sites such as the Maduwanwela Walauwwa, Panamura Ethgala, Waulpane cave, etc.
Though most of the people in the Kolonna area cultivate one or more spice crops, harvesting of spice crops is mainly done once a year.These spice farmers face difficulties in having a significant source of income during the rest of the period during a year. Furthermore, they face financial difficulties when price reductions take place in the world market. Thus, there is a need for alternative income-generating opportunities. In tourism industry, agro (spice)-tourism is a sub sector that is gradually gaining popularity at present. Tea tourism, wine tourism, spices tourism, etc. are among the many sub divisions in agro-tourism. This study focuses on initiation of spice tourism in the Kolonna area by evaluating the potential of the area and the farmers' perception on it.
Conceptual Framework of the Study
Conceptual framework (Figure 1
Study area
The study area that was selected for the research study was the Kolonna DS division in Ratnapura district ( Figure 2). The area was selected purposively due to being a suitable area to promote spice tourism within the Ratnapura district considering the extent of spice cultivation. The agro-climatic features and the soil conditions are also favourable for spice cultivation.
Research design
There were 240 farmers in the Kolonna DS division who have registered under the DEA. They were considered as the sampling frame and out of them, 60 farmers (25%) were randomly selected for the sample of the study. Primary data including quantitative and qualitative variables were collected from the farmers during January-April 2014. Few group discussions with farmers were conducted to obtain detailed information on important aspects such as farmer attitude, opporutnities, issues and chellenges of agrotourism. Descriptive analysis was conducted to assess the demographic factors of the farmers. To investigate the reasons and limitations for initiating spice tourism, respondents were asked to mark their level of agreement by ranking the given statements using number in a Likert scale. The statement received the smallest value in ranking (from 1 to 5), was considered as the most important statement (Reason or limitation). Chi square analysis was applied to find the association between the demographic factors of farmers with their willingness to initiate spice tourism.
Results and Discussion
Results of the study are presented under four sections namely, socio-economic factors and other useful information of farmers, reasons to start and not to start spice tourism destination in the area, factor association with willingness of farmers to start spice tourism, and strengths, weaknesses, opportunities and threats (SWOT) for starting spice tourism in the Kolonna DS division.
Socio-economic factors of farmers
Socio-economic characteristics of the farmers in the area are shown in Table 1. The majority of farmers in the study sample were above 50 years old (80 %; Table 1), followed by the age groups 41-50 (12 %) and 31-40 (8 %). Most of the farmers had a considerable level of experience, interest and knowledge in farming due to long years of farming. Past experience in farming is highly useful for successful cultivations in future, too. Participation of males in farming was higher than females. Sri Lanka is still a male dominant society. However, females help males in farming and allied activities to a greater extent. About 51 % of farmers had education up to G.C.E O/L and overall, 97 % of farmers had primary school education. Therefore, the study sample had a significant level of education to conduct good farming practices as well as tourism operations. Furthermore, it is significant that a higher number (67 %) of farm families have more than 5 members in their families. This is a significant opportunity for farming as well as for tourism especially in securing labour for those activities. The land ownership was high however, the majority of farmers (67 %) have farm lands between 1.1 (0.455 ha) to 4 acres (1.62 ha). A small percentage of farmers in the sample had land sizes above 10 acres (4.04 ha) indicating the small and medium scale farming within the study sample. Moreover, the majority of farmers were (87 %) earned above Rs. 50,000 per annum indicating that spice cultivating farmers earn a significant level of income annually whereas 65 % of the study sample felt that it is a profitable business.
Other useful information of the area
The distance to spice gardens and type of spice crops cultivated by the farmers are considered as other important information of the stud area. Information related to these two aspects is presented in the Table 2. About 37 % of farmers had their farm lands close to Kolonna town (less than 5km distance) and 58 % had their lands 5-9 km away from the city indicating that most of the spice gardens are close to the town. This provides a good opportunity for agrotourism. Results presented in Table 2 also shows the crop combinations used by farmers in cultivating spice crops in the area thus indicating another opportunity for initiation of spice tourism in the area.
Reasons to start spice tourism in the Kolonna DS division
The first reason for stating spice tourism in the area (Table 3) was "Kolonna is a major spices producing area in Sri Lanka" followed by (2) "Generating employment for family members", (3) "As interests or hobbies" and (4) "Agro-tourism is a high profit-generating business" respectively. Moreover, fifth and sixth reasons were "There is a high potential to establish spices gardens" and "Other tourists places are already exist to support agro-tourism". Other important reasons are "Kolonna is already established as one of the top tourist destination", "Better use of the farm's resources" and "There are good communication and transport facilities". Therefore, it is clear that there are many opportunities to initiate spice tourism in the study area.
Limitations to start spice tourism by farmers in the Kolonna DS division
The first limitation to start spice tourism in the Kolonna DS division is "Ignorance of the farmers" (Table 4), suggesting that farmers have enough knowledge regarding such type of activities. "Lack of capital to develop basic infrastructure for the spice tourism", "Fear of destruction of the rural culture of society", "Lack of support from the government", "Difficult to ensure hygiene and basic requirements considering urban visitors" and "It destroys the beauty of nature" are the other main limitations faced by the farmers. Attention should be paid to overcome these constraints.
Factors association with willingness of farmers to start spice tourism in the Kolonna DS division
Nine important variables on willingness to start agro tourism operations were identified by referring to literature (Table 5). All the variables listed in Table 5 had a significant association (p<0.05) with willingness to start spice tourism. Thus, there is a clear trend to initiate spice tourism in the area.
Strength, weaknesses, opportunities and threats (SWOT) in starting spice tourism in the Kolonna DS division
Strength, weaknesses, opportunities and threats (SWOT) in starting spice tourism in the area were studied in detail. The findings are presented in the Table 6clealry indicating that there are a significant number of strengths and opportunities in the study area to initiate spice tourism. Although few weaknesses and threats were identified, they can easily be managed through the intervention of local and national government institutions.
Kolonna is a major spice-producing area in Sri Lanka, generating employment for family members and agro-tourism would be a high profit-generating business. There are several reasons behind farmers' decision on initiating agro (spice)-tourism that have add negative pressure on initiating and developing spice tourism in the area successfully. Furthermore, lack of knowledge was the main limitation to initiate such an activity. Hence, the study shows that the spice-growing farmers need specific assistance and resources form respective local and government institutions to develop spice tourism successfully. Despite these limitations, there is a willingness to start spice tourism, which needs to be exploited.
Conclusion
The study revealed that the socio-economic conditions of the farmers in the Kolonna DS division in Ratnapura district of Sri Lanka favours initiation of spice tourism in the area. Majority of farmers are educated, with more than 10 years of farming experiences, owned spice lands and farm lands located closer to the Kolonna town and some other historical tourist attractions. The majority of farmers in Kolonna were pepper and cinnamon growers while few were growing tea and banana in combination with pepper or cinnamon. These mono-cropping as well as multi cropping situations would provide attractive models in spice-tourism. Hence, though there is no spice-tourism conducted by even a single farmer yet in the Kolonna DS division, the majority of spice growers are willing to start spice-tourism.
Recommendations
Following recommendations can be made to farmers and government to initiate and develop spice-tourism in the Kolonna DS division.
Financial facilities for the farmers
As the availability of finances is a serious problem, it is important to introduce a suitable loan or credit facility for the farmers to overcome this constraint. Government could intervene in this situation by introducing a subsidiary loan scheme for these farmers to initiate spice-tourism in the study region.
Education and training for the farmers
Although some farmers had the basic knowledge, skills and experiences on farming, and willingness to start spice tourism, they are poor in business management skills, entrepreneurship skills, management skills, interpersonal and communication skills, (especially English language). Therefore, education and training programmes for the farmers in these areas would be crucial for a successful implementation of the spicetourism program in the Kolonna DS division.
Development of infrastructure facilities in the area
Good infrastructure facilities are essential for the development of any tourism industry. Therefore, government should pay high attention on establishment of basic infrastructure facilities such as a good road network, transport facilities, electricity, telecommunication, safety and protection in the study area to attract visitors to the spice-tourism areas.
Formulation of proper policies
As development of tourism industry creates several social, cultural, environmental issues, there should be suitable policies to handle these issues effectively. Relevant government institutions should provide the policy and regulatory frameworks on these matters to support efficient and effective implementation of spice-tourism in the study area.
Help in tourism marketing and promotional activities
Although it is costly, development of tourism industry heavily depends on proper marketing and promotional activities. However, the spice-growing farmers in the study region are poor, without the ability to carry out an effective marketing and promotional campaign for spice-tourism on their own. Hence, the local government or other institutions should assist the farming community in this regard, to support the spice-tourism industry. | v3-fos |
2018-12-12T19:54:38.531Z | {
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} | s2 | The Potential of Lumbricus Rubellus as a Bioaccumulator of Excess Pb and Cd in Organic Media
Lead (Pb) and cadmium (Cd) are sources of serious problems in the environment due to their reactivity and toxicity. Lumbricus rubellus is an earthworm reared by people is expected to reduce Pb and Cd concentrations in the environments. The aim of this study was to explore the ability of Lumbricus rubellus in reducing excess of Pb and Cd in organic media generated from urban waste. Sixteen treatments (four levels of Pb concentration and four levels of Cd concentration) were arranged in a completely randomized design with three replications. Each treatment was placed in a wooden pot of 20 cm x 20 cm x 25 cm, and supplied with 40 Lumbricus rubellus for 30 days. Results of this study showed that 20 and 40% of the earthworm could survive until day 30 in organic media contaminated with Pb and Cd, respectively. Pb accumulated in the earthworm bodies ranged from 0.03 to 211.42 mg/kg, while the Cd accumulated in the earthworm body ranged from 0.57 to 22.11 mg/kg. The bioaccumulation factor for Pb was 46.98%, while that of Cd was 53.83%t. The content of Pb in vermicompost ranged from 0.04 to 19.41 mg/kg, while that of Cd ranged from 0.01 to 1.58 mg/kg.
Introduction
Lead (Pb) and cadmium (Cd) are sources of serious problems in the environment because these metals are not easily degraded (Sati et al., 2014). Lead is a toxic heavy metal that is often found in sewage of many industries such as electricity, battery manufacturing, mineral processing and paint formulations (Han et al., 2006). Lead accumulates mainly in the human bones, brain, kidneys, and muscles and can cause many serious disorders, including anemia, kidney disease, nerve disorders, and even death (Chua et al., 2012). Cadmium is a potentially toxic metal (Kuriakose and Prasad, 2008), which is classified as a source of carcinogens that cause cancer disease in humans (IARC, 1993).
Therefore, it is important to remove these heavy metals from urban waste (Kumar and Rao, 2011). There is also an urgent need to find alternative measures for the sustainable processing of municipal solid waste in the landfill (Sim and Wu, 2010). Methods used to remove Pb and Cd should be relatively efficient, and inexpensive. One promising alternative is to use biosorption organism as an adsorbent for removing heavy metals (Ozturk, 2007). The main advantage of biosorption technology is its effectiveness in reducing the concentration of heavy metal ions to a very low level (Wong et al., 2000). Rapid metal uptake and maximum loading capacity are some important factors to consider when choosing biosorption technology (Akhtar et al., 2007).
Earthworms can be employed as bioindicators of environmental damage to the toxicity of a chemical compound, because earthworms are able to accumulate the chemical compounds in the environment into their body (Frund et al., 2011). Earthworms are solution to overcome the urban organic waste disposal and meet the needs of organic fertilizer for sustainable agriculture (Wani, 2002). Earthworms can also reduce significant concentrations of heavy metals (Suthar and Singh, 2009). The use of earthworms in waste management has long been documented. The organic material will undergo a process conducted by earthworm to produce vermicompost (Ansari and Ismail, 2008). Some epigeic earthworm species are often used as biological agents in the process of vermicompost (Ansari and Ismail, 2008).
Vermicompost contains active microorganisms and enzymes that can increase fertility of biochemically degraded soils (Fernandez-Gomez et al., 2010). Therefore, the use of vermicompost as the organic amendment material is a promising alternative, suitable and inexpensive technology to remediate the heavy metal contaminated sites (Fernández-Gómez et al., 2012).
The aims of this study were (1) to explore the potential of Lumbricus rubellus earthworm as a bioaccumulator in reducing excess of Pb and Cd in organic media, and (2) to measure the Pb and Cd contents in the vermicompost produced by the earthworm.
Materials and Methods
The study was conducted in a greenhouse of UPN Veteran East Java in Surabaya from March to May 2015. The treatments tested were application of earthworm (Lumbricus rubellus) on urban organic waste supplied with 0, 150, 300, and 450 mg Pb / kg and 0, 15, 30, and 45 mg Cd / kg. Pb was supplied in the form of Pb (C 2 H 3 O 2 ) 2 , and Cd was in the form of Cd (NO 3 ) 2 . Sixteen treatments were arranged in a completely randomized design with three replications. The experiment was conducted in glass pots measuring 20 cm x 20 cm x 25 cm containing 1 kg of urban organic waste that previously had been incubated for 30 days. At each pot were applied 40 earthworms of known weight.
The experiment was conducted for 30 days. At the end of the experiment (30 days after application earthworms), the number and gross weight (weight + food in the body) of the earthworms that were still alive in the media were measured. The earthworms were placed in bottles for 24 hours under cold temperature (OECD, 2009). The earthworms were weighed and then dried in an oven at 60°C-70°C until a constant weight. The dried earthworms were then crushed and sieved to pass through 5 mesh sieve. A sample of 0.5 g was then mixed with 5 ml HNO 3 and 1 ml HClO 4 and left overnight. On the next day, the earthworm sample was destructed by slowly heating from 100 o C to 200 o C until yellow vapour discharged enhanced heat 200 o C until white smoke was released. After cooling, 20 mL of distilled water was added into the bottle. The solution was then filtered through 42 Whatman filter paper and diluted to 50 ml. The contents of Pb and Cd in the 'earthworm' solution were measured using AAS (Atomic Absorption Spectrometer).
The remaining organic media (yield of vermicompost) was air-dried and sieved for measuring water, organic C, N, available P, available K, Pb and Cd contents in the remaining media. Water content was measured by gravimetric method. Pb and Cd contents were measured using a wet digestion method with 5 ml and 1 ml HNO 3 HClO 4 . Organic-C content was measured using Walkey-Black method. Total-N content was measured by the Kjeldahl method. Available P content was measured by the method of Bray I. Available K content was measured using NH 4 OAc spectrophotometry method. Statistical analysis was performed using the F test at a level of 95% ( = 5%), followed by the least significant difference test (LSD 5%) to see the difference in metal accumulation between treatments. .
Results and Discussion
The percentage of dead earthworms.
The increase of Pb and Cd doses applied to the media significantly affected the dead percentage of earthworms (Table 1). The highest percentage of dead earthworms (48.54%) due to application of Pb in the media was observed for P3 treatment. Application of Cd also significantly affected the percentage of dead earthworms. The highest percentage of dead earthworm (51.04%) due to application of Cd was observed for C3 treatment. Heavy metals in the environment will affect the lives of earthworms by accumulating heavy metals into the tissues of the body, which in turn will cause the death of the earthworms (Kizilkaya, 2004).
Lethal dosage-50.
Lethal dosage-50 (LD-50) is the concentration or dose causing the death of more that 50% of the organisms tested. Results of LD 50 of Pb and Cd doses are presented in Table 2. In general, the results of tests for dose of metal applied that caused the death of more than 50% (LD 50) decreased when it was combined with other metals. Data presented in Table 2 indicate that the toxicities of Pb which caused the death of more than 50% for Pb in C0, C1, C2 and C3 were 1069.26 mg/kg, 710.24 mg/kg, 461.08 mg/kg, and 381.68 mg/kg, respectively. The toxicities of Cd metal that caused the death of more than 50% for P0, P1, P2, and P3 were 68.79 mg/kg, 46.65 mg/kg, 38.61 mg/kg, and 35.37 mg/kg, respectively. The increase of dose of heavy metals would lead to the increase of toxicity that can kill the earthworms. Addition of other metals in the organic media would increase the toxicity that lead to the death of earthworms. The existence of some heavy metals in a biological medium can interact with organisms in the environment; so that the organism will suffer poisoning caused by some of these metals (Sati, et al. 2014).
Earthworm biomass
The treatments that affected earthworm biomass were only doses of each applied Pb and Cd (Table 3). There was no significant interaction between Pb and Cd affecting the earthworm biomass (fresh weight and dry weight). Fresh weight of earthworms from P1, P2, and P3 treatments were 0.258 g/earthworm, 0.286 g/earthworm, and 0.291 g/earthworm, respectively. These figures were higher than that in the control treatment (P0) amounting to 0.334 g/earthworm. A similar trend was also noted for the dry weight of earthworms indicating that the increase of heavy metal dose would reduce fresh weight of earthworms. The increase of Pb doses significantly affected the dry weight of earthworms, i.e. 0.055 g/earthworm, 0.058 g/earthworm, and 0.066 g/earthworm for P1, P2 and P3, respectively, compared with the dry weight of earthworms in the control treatment (P0) that amounting to 0.079 g/earthworm. The increase of dose of Cd applied also significantly affected the fresh weight and dry weight of earthworms compared to those in the control treatment (P0) of 0.364 g/earthworm. The dose of P3 yielded the lowest fresh weight (0.236 g/earthworm) that was not significantly different with the P2 treatment (0.273 g/earthworm), but was significantly different with the P1 treatment (0.296 g/earthworm). Similar trends were also observed for the earthworm dry weight. The increase of Cd dose also significantly reduced the earthworm dry weight. Application of Cd doses (C1, C2 and C3) yielded lower dry weight of earthworm than in the control treatment (C0), i.e. 0.079 g/earthworm. The lowest dry weight of earthworms (0.048 g/earthworm) was observed at a treatment supplied with 45 mg Cd/kg (C3 treatment). The P1 and P2 treatments yielded earthworm dry weight of 0.065 g/earthworm, and 0.065 g/earthworm, respectively. Quality of earthworm media and environmental conditions will lead to increased eating activity of earthworms, so there will be an increase in weight of earthworms during vermicomposting process that in turn affects the increase in biomass of earthworms (Suthar and Singh, 2009).
Pb and Cd accumulation by earthworms
The interaction of Pb and Cd doses significantly affected Pb and Cd contents in the body of earthworms ( According to Butt and Lowe (2011), earthworms have properties of resistant and sensitive to pollutants, so that earthworms are able to accumulate chemical compounds at high concentrations in the environment. According to Langdon et al. (2003), Lumbricus rubellus can be used to detect the presence of heavy metals in the mining area by looking at the metal content in body tissue of the earthworms. Earthworms can serve as indicators of soil contaminated by heavy metals lead, cadmium, zinc and copper that are accumulated in the body of the earthworms (Spurgeon and Hopkins, 1999).
The treatment combinations showed that the increased doses of Pb decreased Pb accumulation in the body of the worms as Cd metal treatment dose increased. Instead, accumulation of Cd in the body of earthworms increased with increasing dose combination of Cd and Pb. According to Prasad (2004), the presence of Cd will reduce the disruption of transport of other heavy metals in the environment, so an increase in Cd dose will decrease the solubility of Pb, which in turn decreased the amount Pb consumed and accumulated by earthworms. There was a positive correlation between the increase in the dosage of Pb and Cd applied against the Pb and Cd contents in the body of the earthworm, which is expressed by the percentage of the relationship R> 0.90.
Bioaccumulation factor (BAF) of earthworms
Bioaccumulation factor (BAF) is the concentration of the chemical compounds in the earthworm's body compared to the amount of the chemical compounds in the soil (Frund et al., 2011).
Experiments on earthworms in environments contaminated by heavy metals can be used to determine the ability of bioaccumulation of metals in the process of vermicomposting (Suthar and Singh, 2009). The value of bioaccumulation factors demonstrates the ability of earthworms in accumulating compounds in the environment. Data presented in Table 5 show that the value of BAF of earthworms to Pb increased with increasing doses of Pb applied into the organic media ranging from 0.36 (P1) to 0.47 (P3). Application of Cd to the organic media reduced the value of BAF of earthworms of Cd that ranged from 0.62 (P1C1) to 0.17 (P3C3). The increased application of Cd also reduced BAF of Cd by earthworms from 0.54 (C1) to 0.49 (C2).
The addition of Pb sharply reduced the BAF value from 0.73 (P3 C3) to 0.27. Bioaccumulation factor of a metal will decrease with increasing concentration of the metals in the environment of earthworms, as well as any other metals that are also present in the environment (Zhao, et al., 2003). Table 5 show that the value of BAF of Lumbricus rubellus against Pb and Cd increased in the provision of these metals as a single metal. The toxicity of a metal on the organism can increase with increasing concentrations and the presence of other heavy metals in the environment (Kabata-Pendias and Pendias, 2001).
Vermicompost yield
The treatments that significantly affected the content of Pb in vermicompost were Pb doses, whereas treatments that significantly affected the content of Cd in vermicompost were Cd (Table 6). Application of 450 mg Pb/kg and 45 mg Cd /kg in the organic media produced vermicompost having the largest content of Pb (19.41 mg/kg) and Cd (11.13 mg/kg). Treatments that affected Cd content in vermicompost produced were C2 and C3 treatments. The highest content of Cd in the vermicompost was 1.58 mg/kg of the C3 treatment that was significantly different from other treatments (C0, C1 and C2).
Earthworms can affect the concentration of heavy metals in organic media by consuming and accumulating the metals into their body tissues (Kizilkaya, 2004;Karaca et al., 2010). In the process of consumption of contaminated metal organic media, earthworms reduce the heavy metals into their body the tissues and in vermicompost produced (Nahmani et al., 2007). Pb and Cd that given to organic media were consumed by earthworms to produce bio-products such as vermicompost. Heavy metals can be present in the body tissues of earthworms in high concentrations, while their residues can contain a lower amount of metal (Kizilkaya, 2004).
Conclusion
Earthworms have ability to accumulate 211.73 mg Pb/kg in the treatment of the provision of Pb 450 mg/kg (P3) and 22.11 mg Cd/kg in the treatment of 45 mg Cd/kg (C3). The produced vermicompost contained lower Pb but larger Cd than standard of organic fertilizer No. 70 / Permentan / SR.140 / 10/2011. | v3-fos |
2018-04-03T02:52:39.294Z | {
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} | s2 | Characterization of a Sorghum mosaic virus (SrMV) isolate in China
Sorghum mosaic virus (SrMV), a causal agent of the destructive sugarcane mosaic disease, has a global presence. An isolate of SrMV infecting a commercially-grown sugarcane plant was recovered from the Hainan province of China. The virions were visualized by an electron microscope, and the coat proteins (CPs) were sequenced by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and tandem mass spectrometry. Discrepancies between the CP predicted and actual amino acid sequences were noted, which confounded the phylogenetic assignment of the isolate. The apparent variations may have physiological effects on the pathogenicity and virulence of SrMV.
Introduction
The main source of sugar production in China is from sugarcane (Saccharum spp.) grown in the southern part of the country, especially in the Yunnan and Guangxi provinces (Li et al., 2014;Lin et al., 2012;Luo et al., 2014). ROC22 is the most popular hybrid cultivar, covering more than 85% of the sugarcane planting area (Xu et al., 2008), and it is susceptible to one of the most common and destructive diseases of sugarcane, sugarcane mosaic disease (SMD). This disease occurs in sugarcane-growing countries worldwide and has significant economic impacts. Major yield losses caused by SMD have been reported as high as 21% in the United States (Grisham, 2000) and up to 42% in South Africa (Balarabe et al., 2014). In China, the incidence of mosaic disease ranges from 30% to 100% leading to a 3-50% decrease in cane yield and a 6-14% decrease in sucrose content; this corresponds to an annual loss of over US$30 million (Li et al., 2014). A characteristic symptom of SMD is the development of mosaic-like patterns of green and yellow shades along the leaves due to chlorosis. Intensity of the mosaic pattern depends on the sugarcane cultivar, growth conditions, age of the leaf and viral strain (Grisham, 2000). Disease control is primarily through the use of resistant cultivars (Wu et al., 2012).
Known natural causal agents of SMD are members of the Potyviridae family: Sugarcane mosaic virus (SCMV) and Sorghum mosaic virus (SrMV) in the genus Potyvirus, and Sugarcane streak mosaic virus in Poacevirus (Grisham, 2000;Hall et al., 1998). These are transmitted via several aphid species in a non-persistent manner (Grisham, 2000). SrMV is the most common pathogen found in Chinese sugarcanes suffering from SMD (Li et al., 2014), though co-infections of SCMV and SrMV seem to increase virulence and yield loss compared to hosts infected with a single virus (Grisham, 2000;Xu et al., 2008).
The SCMV Potyvirus subgroup includes Johnsongrass mosaic virus, Maize dwarf mosaic virus, Zea mosaic virus, Cocksfoot streak virus and Pennisetum mosaic virus which infect other economically important Poaceous plants and crops (Seifers et al., 2000;Wang et al., 2010). A Potyvirus species has a single-stranded positive sense RNA genome which is polyadenylated and covalently linked to a viral protein. The genome is contained within a filamentous capsid consisting of about 2000 identical coat proteins (CPs) (Adams et al., 2005a;Urcuqui-Inchima et al., 2001). Most Potyvirus genomes are monopartite, around 10 kb in size and code for a single polyprotein which self-cleaves into ten mature proteins (Adams et al., 2005a).
We characterized a Chinese isolate of SrMV by its virulence, morphology and partial CP sequence. In doing so, we observed multiple discrepancies between the CP predicted and actual amino acid sequences, suggesting variations and modifications of the viral capsid.
SrMV isolation, purification, biological identification and observation
SrMV isolate HN-lg-1 was detected in Saccharum hybrid cultivar ROC22 during a screen of sugarcane plants suffering from severe mosaic symptoms during the 2011 growing season in Hainan province of China (Zhang et al., unpublished). SrMV serological tests were performed as described elsewhere (Mohammadi et al., 2006). Fresh sugarcane leaves (600 g) from plants infected with SrMV HN-lg-1 were ground with extraction buffer (0.2 M sodium acetate, pH 5.0) containing 0.2% of b-mercaptoethanol to reduce oxidation. The purification procedure involved precipitation with polyethylene glycol, centrifugation with a 30% sucrose density gradient and isopycnic centrifugation with CsCl 2 as previously described (Hull, 2001). The purified SrMV particles were treated with 2% phosphotungstic acid solution for negative staining prior to observation under a JEM 2100 electron microscope (Johnson and Gregory, 1993).
Differential maize and sugarcane hosts were inoculated with isolate HN-lg-1 for viral species identification and maintenance of hosts infected with only SrMV for later SrMV CP purifications. Isolated HN-lg-1 in a buffer containing 1% K 2 HPO 4 , 0.1% Na 2 SO 3 and small amounts of silicon carbide was added to sterile soil (sterilized at 120°C, 0.11 MPa, 20 min) in which host seedlings were potted. Symptoms were observed and recorded every day. The control group was inoculated with sterile water.
RT-PCR
RNA was extracted from ground sugarcane leaf samples frozen in liquid nitrogen using the Plant RNA Kit (Omega Bio-Tek, USA) following the provided instructions. After treatment with DNase I (Takara, Japan), RT-PCR was performed to synthesize cDNA. Reverse transcriptase (M-MLV; TransGen, China), and random and oligo-dT primers were used following the manufacturer's instructions.
SrMV CP gene specific primers (Table 1) were used to detect the virus by PCR. These were constructed based on the SrMV complete genome (strain H, NCBI GenBank Accession No. U57358). Reaction mixtures consisted of 1 ll of cDNA (10-20 ng/ll), 0.5 ll or each primer (10 lM), 2 ll of 10· PCR buffer, 1 ll of 2.5 mM dNTPs, 0.25 units of Ex-Taq DNA polymerase (Takara, Japan) and RNase-free water to a final volume of 20 ll. The amplification reaction parameters were 94°C for 3 min, 35 cycles at 94°C for 30 s, 50°C for 30 s and 72°C for 1 min, and 72°C for a final 10 min. PCR products were subjected to gel electrophoresis on a 1% agarose gel at 80 V for 20 min and visualized with Goldview dye (Beijing SBS Genetech, China).
The RT-PCR products were purified using a Gel Extraction Kit (TianGen, China) and cloned into pMD19-T (Takara, Japan) by TA ligation following the manufacturer's instructions. The cloned vector was transformed into Escherichia coli DH5a as described elsewhere (Froger and Hall, 2007). Colonies containing the cloned vector were detected using the universal primers M13-47 and RV-M (Table 1) by colony PCR (protocol as described for amplification PCR). Vectors from three colonies were purified using the QIAGEN Plasmid Midi Kit (QIAGEN, Germany). These were separately sequenced using the automated ABI 3130xl Genetic Analyzer (Applied (Kollerova´et al., 2008). Following electrophoresis, viral peptides were removed from the gel and subjected to matrix-assisted laser desorption/ ionization tandem time-of-flight (MALDI-TOF/TOF) and tandem mass spectrometry (MS/MS) performed at the Beijing Genomics Institute, China. Three independent samples were sent for analysis. The results were analyzed using the Mascot database search tool (Matrix Science).
SrMV isolation, purification, biological identification and observation
Sugarcane hybrid cultivar ROC22, infected with SrMV exhibited serious mosaic symptoms in the field, including dwarfing (Fig. 1A). ID-ELISA tests confirmed SrMV as a causal agent in 13 out of 20 field samples (Table 2). SrMV isolated from Sample No. HN-lg-1 was selected for further characterization. After CsCl 2 density-gradient centrifugation, a single light-scattering zone was observed in the upper middle part of the tube; its position coincided with a peak UV absorbance at 260 nm. The standard sedimentation constant was 160-175 S, and the buoyant density was 1.285-1.342 g/ml (Fig. 1B).
Purified particles were inoculated in differential host maize and sugarcane cultivars. The maize leaves showed the systemic streak and mottled symptoms seven days post inoculation (Fig. 1D) while sugarcane showed atypical symptoms (data not shown) even after 30 days. Electron microscopic examinations of purified virions revealed a high concentration of long rod-shaped virus particles with sizes 800 ± 50 nm · 13 ± 2 nm (Fig. 1E). This was consistent with previous observations of SrMV and other members of Potyvirus (Edwardson et al., 1968;Hema et al., 1999;Lesemann et al., 1992;Pirone and Anazalone, 1966). SDS-PAGE and western immunoblotting indicated that the SrMV CP consisted of multiple copies of a peptide with a molecular weight of 35.68 kDa, which is close to the predicted size of about 36 kDa (Fig. 2).
SrMV coat protein nucleotide sequence
The 913 bp cDNA product from RT-PCR was found to have a nucleotide sequence nearly identical to other SrMV CP sequences, further confirming the species identification. Potyvirus species can be further differentiated into strains by their CP sequences (Shukla and Ward, 1988; Viswanathan (Adams et al., 2005b;Shukla and Ward, 1988). A BLAST search of the CP predicted amino acid sequence of the HN-lg-1 isolate (Accession No. AGU12592) revealed that the isolate shared 100% identity with one other Chinese SrMV isolate from the Yunnan province (isolate YN11, Accession No. CAX36847). Up to 99% identity was achieved with several other SrMV isolates found in southern China (Wang et al., 2010), including those found by our group along with HN-lg-1. However, as noted in Section 3.3, the predicted and actual amino acid sequences of the HN-lg-1 isolate were considerably different, with only an 87% identity to each other. Thus, we performed a second BLAST search with the actual amino acid sequence. This resulted in a different list of SrMV CP sequences with 100% query coverage and identities ranging from 78% to 98% with one sequence scoring 100% identity (strain FJ10, Accession No. ABP68394). At this time, we are not able to definitively identify the strain in which HN-lg-1 belongs.
Discrepancies between SrMV coat protein predicted and actual amino acid sequences
The majority of the SrMV HN-lg-1 CP amino acid sequence was revealed by MALDI-TOF/TOF. A third of it was verified by MS/MS analysis (Fig. 3). A comparison of the CP predicted and actual amino acid sequences revealed multiple discrepancies between the two (Fig. 4). For residue identities determined by both MALDI-TOF/TOF and MS/MS, eight residues were not as expected. Furthermore, these mismatches were not consistent. For example, predicted lysine residues were found to be glutamine, arginine or were as predicted. We do not know the reasons for these variations. However, we suspect that the apparent variations in the SrMV CP were mostly the result of post-translational modifications formed either in vivo or during sample processing. We also considered codon bias to be a possible cause. Lower concentrations of certain tRNA molecules in the host may result in amino acid substitutions during translation. However, the apparent variations were consistent and only affected select amino acids, suggesting a more controlled mechanism such as post-translational modification. Additionally, there is a lack of evidence demonstrating that plant viral codon usage is adapted to that of the host; it instead seems to stem from mutational biases, possibly caused by viral genomic structure (Adams and Antoniw, 2004;Cardinale et al., 2013).
The physiological effects of these modifications and/or variations are not known. The CP plays many roles in cellto-cell and systemic movement, encapsulation of the genome, association with aphid vectors and regulation of replication (Urcuqui-Inchima et al., 2001). Previous studies have shown that several key residues and regions of Potyvirus CPs are vital for proper functioning. When the N-terminal DAG motif or proximal residues are varied, aphid transmission of Tobacco vein mottling virus and Tobacco etch virus is no longer possible or significantly impaired (Atreya et al., 1991(Atreya et al., , 1995Lo´pez-Moya et al., 1999). Select mutations in the core of the Plum pox virus CP rendered the virus unable to move systemically throughout a host or assemble new virions (Varrelmann and Maiss, 2000). While we did not conduct any mutational studies, it is possible that the modified or varied residues play vital roles in SrMV pathogenicity and virulence. Moreover, we did not inspect the full CP. There may be other modifications or variations which have important implications. Future studies will have to further investigate the causes and effects of the discrepancies between the predicted and actual amino acid sequences.
Author contributions
G. Yin designed the experiments. Y.L. Zhang and H. Wang performed the experiments. All authors co-prepared the manuscript. | v3-fos |
2016-03-22T00:56:01.885Z | {
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} | s2 | Irrigation Water Quality for Leafy Crops: A Perspective of Risks and Potential Solutions
There is increasing evidence of the contribution of irrigation water in the contamination of produce leading to subsequent outbreaks of foodborne illness. This is a particular risk in the production of leafy vegetables that will be eaten raw without cooking. Retailers selling leafy vegetables are increasingly targeting zero-risk production systems and the associated requirements for irrigation water quality have become more stringent in regulations and quality assurance schemes (QAS) followed by growers. Growers can identify water sources that are contaminated with potential pathogens through a monitoring regime and only use water free of pathogens, but the low prevalence of pathogens makes the use of faecal indicators, particularly E. coli, a more practical approach. Where growers have to utilise water sources of moderate quality, they can reduce the risk of contamination of the edible portion of the crop (i.e., the leaves) by treating irrigation water before use through physical or chemical disinfection systems, or avoid contact between the leaves and irrigation water through the use of drip or furrow irrigation, or the use of hydroponic growing systems. This study gives an overview of the main problems in the production of leafy vegetables associated with irrigation water, including microbial risk and difficulties in water monitoring, compliance with evolving regulations and quality standards, and summarises the current alternatives available for growers to reduce microbial risks.
Introduction
Water of inadequate quality has the potential to be a direct source of contamination and a vehicle for spreading localized contamination in the field, facility or transportation environments in the production of fresh produce crops [1]. A number of comprehensive review articles have been published which highlight irrigation water as a source of pathogenic microorganisms in produce [2][3][4][5][6][7][8]. Pathogenic microorganisms associated with irrigation water include bacteria, viruses and parasites (protozoa and helminths). Although viruses and parasites are of paramount importance and can be transmitted to fresh produce via irrigation water, this paper focuses on pathogenic bacteria.
The increasing evidence of contamination of produce from irrigation water has been reviewed recently by Uyttendaele et al. [9] and contamination events were identified where water is a risk factor in the production and harvesting of fresh produce. However, the authors made clear that identification of the implicated food vehicle and/or the location of the point of food contamination in fresh produce-associated outbreaks are a recurrent challenge.
Despite this evidence, research focused on microbial quality of agricultural water is relatively limited. Most research regarding microbial quality of water (e.g., pathogen prevalence or indicator organisms) has been conducted for objectives related to reclaimed water, drinking and recreational water supplies and the effects of agriculture on the environment [10]. In 2011, Pachepsky et al. [5] highlighted that no databases on microbial quality of irrigation water had been compiled. However, there is an increasing number of recent research papers that focus on the evaluation of the microbial quality of irrigation water used for the production of fresh produce and its significance as a source of contamination e.g., [11][12][13][14][15][16][17][18][19][20]. Several of these published papers show the results obtained within the frame of a research project consortium of the FP7 project Veg-i-Trade (www.vegitrade.org) focused on the impact of climate change and globalisation on safety of fresh produce.
Growers use a variety of water sources for field operations and irrigation and much knowledge is needed to relate risk factors associated with the transfer coefficients for pathogens by source, concentration and use [10]. Quantitative microbial risk assessment (QMRA) is being applied to establish the links between concentrations of pathogenic microorganisms in agricultural water and the probability of illness [21][22][23]. Lately, as a result of several longitudinal microbiological surveys, different scenarios have been evaluated to estimate the potential impact of agricultural practices, including water treatments and the application of potable water, on the E. coli levels and prevalence of pathogenic microorganisms in fresh produce [24,25].
Based on this evidence, standards and guidelines for Quality Assurance Schemes (QAS) have been designed to cultivate prevention and control of food safety hazards [26] but QAS have become not only a prevention strategy but also a marketing strategy used by the industry, which is pushing towards zero-risk for many factors including irrigation water sources, in response to consumers concerns over food safety [27]. In many cases, current QAS require growers to develop and implement management systems and to risk assess the irrigation water sources to reduce microbial risks. Increasingly QAS are requiring stringent testing regimes of water coming into contact with the edible portion of leafy vegetables [28]. However, some studies highlight that the required monitoring programs can be self-defeating and create additional uncompensated monitoring costs [26].
Growers should also consider other alternatives to guarantee the safety of the produce they produced under any circumstance. Surface water represents one of the most risky water sources, thus where growers are utilising surface water for agricultural applications, particularly for leafy greens or produce that is intended to be consumed raw, and mitigation strategies may be required [29]. Several strategies have been proposed to reduce the risk of produce contamination with pathogens during irrigation [5]. The main aims of these strategies is to reduce the use of uncontrolled sources of water and to establish distance limits for water resources used for irrigation from livestock housing, stored effluents, and/or land spread with manure [30].
Water treatment is also a feasible strategy to guarantee the microbial quality of irrigation water. Treating water during storage and while in the delivery systems may represent a good alternative to high frequency microbial testing [8] and probably the only possibility for growers using irrigation water with microbial loads above regulatory thresholds. Another option for growers using water sources of poor microbiological quality is the use of different production systems, such as hydroponic systems and drip irrigation, which avoid contact between the edible part of the crop and the irrigation water.
This study gives an overview of the main problems associated with irrigation water, including microbial risk and difficulties in water monitoring, compliance with evolving regulations and quality standards, and summarises the current alternatives available for growers to reduce microbial risks.
Irrigation Water as a Risk Factor
Agricultural water has been defined as a major risk factor in the contamination of leafy crops eaten raw as salads [30]. When available, fresh water is consumed for agricultural production but water scarcity is becoming a major threat to the sustainability of agriculture, which needs to rely much more on marginal water sources, including treated wastewater [31]. However, little is known regarding the microbial quality of irrigation water used for leafy crop production. Systematic sampling focused on determining the risks of microbial contamination associated with different water sources are rare and most of the existing information comes from U.S. The available literature indicates that generic E. coli levels and prevalence of pathogenic foodborne bacteria in irrigation water significantly varies depending on several factors including seasonality, geographical location and weather conditions among others (reviewed by [8,9]). The Center for Produce Safety [10] have published a report to provide a summary of the scientific and technical information related to factors that affect the microbiological safety of agricultural water. This report summarizes the most recent research on irrigation water and gives a good overview of the needs for future research including sampling strategies that provide an estimate of the true underlying distribution of bacteria in a water system, correlation of field and water system management practices with pathogen prevalence in agricultural water samples, as well as a better understanding of risk factors leading to survival and/or growth of pathogens on fresh produce following application of contaminated water.
Current Situation in Europe
Recently, a comprehensive review has summarized the main irrigation water sources used in Europe highlighting municipal water, rainwater, groundwater and surface water as the most relevant [9]. In a temperate production area, such as the UK, the primary sources of water for irrigation are reported to be surface water (54%) and groundwater (41%), with the remainder coming from public mains water, rainwater and other sources [32,33]. However, in arid and semi-arid parts of Europe, such as the South of Spain, Italy and Greece, which are confronting increasing water shortages, treated municipal wastewater is a valuable water source for recycling and reuse in agriculture. As an example, 347 hm 3 of treated wastewater were reused in 2010 in Spain, particularly in the South East of Spain where almost 60% of the reclaimed water produced was reused [34] and 233 hm 3 in Italy [35]. Although the use of untreated wastewater for crop production is not a recommended practice, research results indicate that tertiary water treatment, including final disinfection using UV light, chlorination and/or ultrasound have been shown to be effective in removal of indicator microorganisms and pathogens to below limits of detection [5,36].
Research related to microbial quality of agricultural water in Europe is limited, although recent research studies have highlighted the microbial risks associated with different water sources (Table 1). In most cases, longitudinal microbiological surveys have been carried out to evaluate the microbiological quality of produce samples, excluding the monitoring of risk factors such as irrigation water. It should be taken into account that the performance of irrigation water-quality monitoring and management practices are highly variable within countries as well as among different countries, with large differences across Europe. In 2006, Tyrrel et al. [37] reported that in UK, the majority of growers do not irrigate with water that would conform the European Union Drinking Water Standard [38] and used water that was faecally contaminated, but that typical faecal coliform concentrations were ≤1000/100 mL. More recently, Holvoet et al. [16] monitored eight Belgian lettuce farms to establish the relationships between levels of indicator bacteria and detection of enteric zoonotic pathogens. A high prevalence (75%, n = 120) of E. coli was found in the irrigation water sources with 65% of the positive samples having E. coli levels ≥1 log cfu/100 mL while 26% of the samples showed E. coli counts ≥2 log cfu/100 mL, which is above most of the irrigation water-quality standards. Additionally, 35% of the collected samples were positive for at least one pathogen (Salmonella, Campylobacter or Shiga toxin-producing E. coli (STEC)). In this study, most of the monitored farms used open wells to hold collected rainfall water. Previously reported E. coli values for surface water collected from rivers within an agricultural landscape were within the range 1.5-3.3 log cfu/mL [39], confirming the evidence that surface water, including rivers, streams, and creeks have unpredictable water quality and activities upstream can rapidly change the levels of contaminants [9]. A study focused on Belgian strawberry production reported significant differences in the microbiological quality of irrigation water obtained from different water sources including groundwater and collected rainfall water stored in ponds [19]. They reported that water obtained from the ponds was positive for E. coli (40/56) with an average level of 1.6 log cfu/100 mL and almost 20% of these samples were positive for STEC. However, groundwater samples showed much lower levels of E. coli and these samples did not contain STEC. Water stored in reservoirs or lagoons provides both an opportunity for pathogen die-off through natural UV but also for new contamination associated with the use of reservoirs as wildlife habitats [37].
In many European countries, a significant proportion of irrigation water is abstracted and stored in farm reservoirs. Castro-Ibañez et al. [17] monitored several water reservoirs used to irrigate growing fields of leafy greens. Samples were analysed for presence of foodborne pathogens and only one sample was positive for Salmonella spp. Reported E. coli prevalence and concentration in irrigation water samples from water reservoirs was lower than that reported from open wells in Belgium. Observed differences between Belgium and Spain might be due to differences in weather conditions as solar radiation has been highlighted as an important mechanism for bacterial decline in environmental samples including irrigation water [18]. Urban wastewater has been used in agriculture as a way to overcome water scarcity in the South of Europe. The European Water Framework Directive [40], specifies that treated wastewater should be used in agriculture where and whenever possible. Currently, urban wastewater is mainly used for irrigation in combination with production systems that avoid direct contact between the water and the edible part of the fresh produce. In countries such as Spain, Greece and Italy, commercial production using urban wastewater as irrigation water usually involves production of tomatoes, peppers and cucumbers grown hydroponically in greenhouses. Sometimes, wastewater treatment plants, consisting of a train of individual unit processes, are located close to the greenhouse ( Figure 1).
Figure 1.
A secondary treatment wastewater plant located in a greenhouse production unit to provide irrigation water for tomato production. Reprinted with permission of Quality and Safety Lab CEBAS-CSIC.
Studies focusing on the microbial quality of reclaimed water used for irrigation reported presence of faecal contamination within the range 2-4 log E. coli cfu/100 mL and also presence of pathogenic microorganisms such as Salmonella spp. [41,42] (Table 1). Production systems that minimize irrigation water contact with the edible portion of the crop seem to reduce the risk of contamination. Codex Alimentarius [43] reported that plants grown in hydroponic systems absorb nutrients and water at varying rates, constantly changing the composition of the re-circulated nutrient solution and because of this water used in hydroponic culture should be changed frequently, or if recycled, should be treated to minimize microbial and chemical contamination.
Regulations, Guidelines and Microbial Quality Standards for Water Used in Primary Production
The microbial quality of irrigation water has been related with food safety for more than 25 years. In Europe, specific microbial criteria are only currently established in guidelines and quality assurance standards (QAS), but specific microbial criteria have been introduced in U.S. legislation and these are starting to have an impact on European standards.
Legislation-The European Commission
Regulation (EC) No 852/2004 on the hygiene of foodstuffs defines potable water as "meeting the minimum requirements laid down in Council Directive 98/83/EC [38] on the quality of water intended for human consumption", clean water is defined in Regulation (EC) No. 852/2004 [44], as "clean seawater and fresh water of a similar quality" and clean seawater is defined as "natural, artificial or purified seawater or brackish water that does not contain micro-organisms, harmful substances or toxic marine plankton in quantities capable of directly or indirectly affecting the health quality of food" [44]. Specific microbiological criteria are not defined but growers must be able to demonstrate that their operations are managed in a way that controls food safety risks, including those associated with the use of water.
World Health Organization (WHO)
Whilst it was clear that care was needed in the safety of water used to irrigate crops that are eaten raw, such as leafy salads; prior to 1973 there was no generally accepted standard of microbial water quality. In 1973 the World Health Organization (WHO) addressed the issue that drinking water microbial quality standards (i.e., ≤2.2 cfu/100 mL coliforms) were not a realistic target and produced a guideline value of ≤100 cfu/100 mL coliforms for unrestricted irrigation water (i.e., water used to irrigate crops that will be eaten uncooked) derived from wastewater [45]. Following consideration of what was achievable by wastewater treatment processes and associated epidemiological studies of wastewater use, these guidelines were revised in 1989 to a geometric mean of ≤1000 cfu/100 mL faecal coliforms and an arithmetic mean of ≤1/L intestinal nematode during the irrigation period [46]. These standards have been criticised, particularly from those developing guidelines with the aim for "zero-risk" irrigation water [47]. The WHO has more recently changed the approach and there are now no definitive values for microbiological guidelines for irrigation water. Instead, irrigation water safety should be based upon risk assessment as recommended in WHO documents and water guidelines in advanced economies should rely on in-country standards [48,49].
Codex Alimentarius Commission
Baseline guidance on safety requirements of irrigation water is provided through Codex Alimentarius: both the General Principles of Food Hygiene-CAC/RCP 1-1969 [50] and the Code of hygienic practice for fresh fruit and vegetables-CAC/RCP 53-2003 [42] address the issue. However this only provides a "general framework of recommendations […] rather than providing detailed recommendations for specific agricultural practices…" [43] i.e., the water used in primary production must be safe. However the microbial requirements are not defined. For example, in the Leafy Green annex of the Code of hygienic practice for fresh fruit and vegetables-CAC/RCP growers are required to "seek appropriate guidance on water quality and delivery methods to minimize the potential for contamination with microbial pathogens" [43]. And where water comes in to "substantial contact with the edible portion of the leafy vegetable should meet the standards for potable or clean water" [43] where these are defined as: Potable water-water which meets the quality standards of drinking water such as described in the WHO Guidelines for Drinking Water Quality; Clean water-water that does not compromise food safety in the circumstances of its use [43].
QAS-1st Generation
In the early 1990s the UK fresh produce supply chain started to develop Quality Assurance Schemes (QAS) also known as Retailer Codes of Practice partly in response to the 1990 UK Food Safety Act that required growers to be treated as running food businesses within a consolidated supply and as a consequence growers were exposed to due diligence imposed from the retail end of the supply chain [51,52]. Amongst these QAS were Assured Produce-1991; Tesco Natures Choice-1992 and, more widely relevant, EurepGAP-1997 [53]. In essence these QAS followed Codex guidelines and required systems to be in place to ensure that the water used in production was safe. The approach followed Hazard Analysis and Critical Control Point (HACCP) principles such as to risk assess water sources and use but did not define water quality criteria. In 2004 Marks and Spencer (M&S) released a new QAS "Field to Fork" that required growers to test their irrigation water for E. coli, but again no criteria were stipulated as to what an acceptable level of indicator organism would be [53].
QAS-2nd Generation
The QAS increased their focus on microbial risks associated with irrigation water and a new generation of schemes (e.g., Red Tractor Fresh Produce Scheme, GlobalGAP, Tesco Nurture, M&S Field to Fork version 2) was developed in the 2000s [51]. As with the first generation of schemes, growers were still required to develop and implement management systems along HACCP principles and to risk assess all water sources. There was also more guidance on risk assessment methodology but critical levels of indicator organisms (with some confusion between E. coli and faecal coliforms) in water used in primary production were now defined. Generally, the criteria were based on the WHO guidance for water for irrigation of produce that can be consumed uncooked of ≤1000 faecal coliforms cfu/100 mL with an additional reporting level of ≥400 cfu/100 mL in Tesco Nurture and M&S Field to Fork version 2, which has become more stringent in later revisions for leafy crops.
QAS-3rd Generation
In 2011 the Food Safety Modernization Act (FSMA) became US law [54]. The FSMA proposed microbial quality standards for irrigation water and wash water that came into contact with the edible portion of fresh produce crops. Although the original microbial quality standards are being revised to include rules on produce safety that are more flexible and less burdensome in key areas [55], these standards have been applied in Good Agricultural Practices (GAP) used in the USA and also more widely. One example is the McDonalds GAP [28] where the grower is required to implement systems to ensure food safety, based on HACCP principles and risk assessment as with previous generations of QAS, but clear metrics and microbial criteria are now defined. Irrigation water that may contact the edible portion of the crop must have no more than 235 cfu (or most probable number (MPN), as appropriate) generic E. coli per 100 mL for any single sample, or a rolling geometric mean (n = 5) of more than 126 cfu (or MPN, as appropriate) generic E. coli per 100 mL of water [28]. A pre-planting water sampling programme is needed for any irrigation water used (and stored rain water). Each water source must have a minimum of one sample analyzed from the point of application closest to the crop to establish that water quality is acceptable before being used for irrigating the crop (i.e., ≤235 cfu generic E. coli per 100 mL). Subsequently, during crop growth, a set of at least five irrigation water samples must be collected prior to harvest to establish the rolling geometric mean. The sampling frequency is not stipulated but five samples must be taken within the crop growth period or 30 days, whichever is the shorter [28].
More recently the FDA has revised the upper limit of the irrigation water quality criteria to a statistical threshold value (i.e., a value that should not be exceeded by more than 10 percent of the samples taken) ≤410 CFU of generic E. coli in 100 mL of water [55] and it would be anticipated that this standard would start to be used in some QAS.
It has become clear that the industry is being pushed towards zero-risk irrigation water sources in response to supply chain concerns over food safety and that there is a greater requirement for frequent water testing regimes with complex calculations of critical values. These high standards will be difficult to achieve with open water sources such as rivers or stored water that are regularly tested, and for some producers their water sources will not comply with the requirements. This may lead to alternative strategies being required.
Monitoring Pathogen and Indicator Species in Water Sources
Testing water can be used to establish a history of microbiological quality and inform a risk ranking for a water source [9]. The prohibitive cost and time requirement of pathogen detection make microbial indicators a good strategy to characterize microbial contamination in agricultural water [56]. Several studies report that indicator bacteria, and particularly generic E. coli concentrations, are not correlated with the presence of pathogens such as E. coli O157 or Salmonella spp. in water samples, suggesting that E. coli data might not be suitable to predict the risk of exposure to pathogenic strains [10,57,58]. However, a logistic regression analysis of samples taken monthly for 12 months from 18 locations throughout Central Florida showed that E. coli concentration can predict the probability of enumerating selected Salmonella levels, indicating that E. coli provides a reasonable way to predict Salmonella levels in surface water. According to this, in recent longitudinal microbial surveys carried out in Europe, presence of elevated levels of E. coli increased the probability of presence of pathogens (STEC and Salmonella spp.) [16,18]. Additionally, Wilkes et al. [59] reported that faecal indicators such as E. coli spp. were conservative surrogates for a variety of pathogenic microorganisms in surface waters within an agricultural landscape. Based on these reports, E. coli spp. could be identified as suitable for a hygiene criterion at primary production of leafy greens and can be applied for validation and verification studies of GAP.
However, factors such as the dynamic nature of agricultural water microbial quality, the time lag between obtaining agricultural water testing results and water use, and the fact that current water sampling strategies are based on an assumption that bacteria are floating as single cells in water, make water monitoring limited in its ability to monitor microbial risks [10]. In addition, Won et al. [60] reported that n > 5 canal and n > 14 reservoir samples were needed to calculate E. coli concentrations at a precision level of 85% with 95% confidence interval under the same environmental conditions during the testing period; a frequency much greater than required by the most stringent QAS. The frequency of testing will vary depending on the water source and the risk of environmental contamination [3]. For example, Holvoet et al. [61] observed in a study of leafy vegetable farms in Belgium that contamination of irrigation water was more prevalent in open-field production compared to greenhouse production. An additional limitation of the testing approach is the costs of an effective monitoring program which are usually too expensive for most small to medium growers [9]. Hence, alternatives to monitoring sampling plans such as water treatments and the selection of less risky production systems, which avoid contact between the edible part of the plant and irrigation water, may be an alternative option in reducing microbial risks to leafy vegetables.
Water Treatments
Physical and chemical disinfection systems have been explored as methods to remove human pathogens from agricultural water sources (Table 2), although disinfection treatment of irrigation water is still a very limited practice [4]. Nowadays, chemical sanitizers are the most commonly used water treatments, although environmentally friendly alternatives are being demanded, particularly for organic production. In fact, concerns have risen recently regarding both the absence of water treatment and the excessive use of potentially toxic chemicals to treat irrigation water. Norton-Brandão et al. [35] presented a critical review of a wide range of urban reclamation technologies that could be applied to water; of these, the treatments relevant to agricultural irrigation water included coagulation, flocculation, filtration and chemical disinfection [8].
Among commercially available water treatments, chlorine-based sanitizer remains the most common water treatment used for the removal of biohazards from irrigation water [4,8]. The advantages of using water treatment are generally associated with microbial reductions of both foodborne pathogens as well as phytopathogens and the reduction of contamination from biofilm formation in the irrigation pipes (Table 2) [61]. There are many inexpensive commercial applications of chlorine-based sanitizers available for growers. When using this type of water disinfectants, a key step is the estimation of the peak chlorine demand over a range of typical operating conditions, where the peak chlorine demand is defined as the maximum amount of free chlorine in a batch of water that is "used up" by soil and organic materials added with product during washing [62]. Two of the more widespread treatments in Europe and also U.S., are calcium hypochlorite or chlorine dioxide but limitations of chlorine-based disinfectant in terms of formation of disinfection by-products and the potential negative effect on the environment have limited its use in agricultural water. Nevertheless, the goal of the dosing system is, in most cases, to reduce E. coli levels to be within a compliant range, for which a minimal dose of disinfectant is usually enough. This reduces the potential detrimental effects on the farm soil or environment from disinfection by-products [63] in the short-term but there are still concerns for chronic effects of large-scale use over long periods of time [4]. Additionally, the volumes of water that are commonly used in a medium-large production field are very high (50-100 m 3 ), and the cost associated to the treatment of irrigation water can be substantial.
Greener technologies based on physical treatments such as ultrasound (US), ultraviolet light (UV-C) and filtration have been successfully tested to reduce microbial loads of irrigation water [4,29,36]. US technology has the advantage of reducing microbial loads, including algae, without the detrimental effects of the formation of disinfection by-products [36]. Other reported advantages of US include potential simultaneous oxidation, thermolysis, shear degradation, and enhanced mass transfer processes [64]. UV-C light has also been used frequently to disinfect irrigation water and requires the installation of a UV-C treatment system where water passes through a vessel while it is illuminated by UV-C lamps located in the vessel (Figure 2). These systems are usually relatively cheap and represent a good treatment option if water turbidity is low [65]. However, a pre-treatment usually based on sand filtration and also regular maintenance of the lamp are needed to ensure proper efficacy of the system. Membrane and sand filtration have been reported as effective technologies to remove pathogens from water [35]. Different pore sizes are applied for different applications. Thus, membrane filters classified as micro-(0.1-10 µm), ultra (0.002-0.1 µm), nano (0.0005-0.002 µm) filtration or reverse osmosis (<0.0005 µm) are used for removal of microorganisms while larger pore sizes and rapid sand filters are used to reduce soil and plant material that may clog the membrane filters [66]. Membrane filtration is more effective at controlling pathogens when combined in a series of multi-stage filters and in combination with other treatments that have other modes of action such as chlorine [67]. Innovative filtration systems such filters containing sand and/or materials with reactive components have been explored as potential water treatments [10]. Slow sand filtration controls microorganisms present in the water by biological, physical and chemical reactions. In these systems, microbial removal is carried out by a complex microbial community located in the upper layer on the sand bed but also by physically entrapping the pathogens and debris [67]. Biosand filter zero-valent iron incorporated (ZVI) treatments, which have been used in permeable reactive barriers to remove a broad range of chemical contaminants in groundwater, have also been reported as potential water treatments [68]. Ingram et al. [69] have recently proposed ZVI treatment as a cost-effective mitigation option for irrigation water to help small farmers reduce risk of foodborne E. coli infections associated with contamination of leafy greens.
Whilst alternative systems are being proposed in scientific studies, the evaluation of the site-specific applicability of any of these technologies, such as maintenance costs, safety, and biological effects on crops and humans, is missing [4,5]. In general, water treatment technologies for agricultural water have not been deeply evaluated and in most of the cases, operational and maintenance costs are knowledge gaps crucial to the decision making process of farmers [4]. On the other hand, process control for water treatment technologies is mandatory to guarantee the efficacy of the treatment. A good understanding and consistent implementation of systems to monitor, control, and document water treatment performance are needed because without the process control, all water treatment systems may give a "false confidence".
Alternative Production Systems
An alternative strategy to managing the risk of contaminating leafy vegetables through faecally contaminated irrigation water is to avoid direct contact with the edible parts of the crop. This can be achieved through selecting the irrigation method and/or the production system used to the crops. Application of microbial-contaminated irrigation water using subsurface drip irrigation has been shown to reduce contamination of crops including lettuce at harvest compared to furrow irrigation [70]. However, even if direct contact between irrigation water and the edible part of the leafy crop is avoided, irrigation water may contaminate the soil or substrate, where the bacteria can survive for some time [30] and irrigation or rainfall splash may contaminate the crop [11].
Soilless systems, such as hydroponic floating systems [71] or nutrient film techniques (NFT) [72], are being used for leafy vegetables with short production cycles allowing a better control and standardization of the cultivation process ( Figure 3). Many advantages have been attributed to the use of soilless systems in greenhouses to produce leafy greens, but reductions in product quality and shelf life have been observed [73] which may limit the use of these systems. However, recent studies carried out in commercial agricultural production sites showed that the use of poor quality irrigation water combined with the use of soilless production systems considerably reduced microbial contamination risks to fresh produce [42,74]. This reduction can be attributed to preventing contact between the irrigation water and the edible part of the plant. In a non-commercial hydroponic system lettuce grown using diluted effluent from secondary UV treated grey-water was shown to have an acceptable level of E. coli spp. on the harvested leaves, even though levels on the roots were high [75]. The use of alternative production systems such as greenhouses and hydroponics are assumed to be safer than open field production from the microbiological point of view due to the minimization of some risk factors associated with sources of pre-harvest contamination, but also a greater control of water disinfection with water being recirculated and cleaned periodically [76]. The nutrient solution, and hence water, used in soilless systems is one of the most important aspects for the success of leafy greens production [77] and the quality of water can be better controlled in soilless systems [63] both from a plant pathogen and human pathogen perspective. Nevertheless, before hydroponics could be used to grow crops using lower quality water more knowledge is required on the risks posed from root contamination and internalization of bacteria (e.g., [78]) from contaminated hydroponic water sources.
Conclusions
There is an increasing evidence of contamination of produce from irrigation water, but scarce information on the microbial quality of agricultural water is available. Despite this, retailers selling leafy vegetables are risk averse and are targeting zero-risk production systems. As a consequence the requirements for irrigation water quality and safety in QAS have evolved and become more stringent. One approach is for growers to identify water sources that are contaminated with potential pathogens through a monitoring regime and only use water free of pathogens, but this approach is very costly and ultimately does not ensure safe water. The use of faecal indicators, particularly E. coli, allows growers to identify water sources where there is a route of faecal contamination, allowing risk categorization of water sources [9,30]. Where growers only have access to water sources of moderate quality, with low levels of faecal contamination, they have two main options available to produce leafy vegetables that will not pose unacceptable risks to the consumer: treat water before use, using physical or chemical disinfection systems, with chlorine the most commonly used system currently; or reduce/eliminate contact with the leaves from irrigation water through irrigation water placement i.e., drip or furrow, or the use of soilless growing systems. It is likely that a combination of approaches will be needed to meet the microbial requirements of leafy vegetables and ensure safe food for consumers. | v3-fos |
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} | s2 | Optimization of Trypsin-like Protease Production by Lactobacillus plantarum FNCC 0270 using Response Surface Methodology
The purpose of this study was to get optimum medium co position and agitation to trypsin-like protease production by Lactobacillus plantarum FNCC 0270. The medium composition and agitation fo r enzyme production was optimized using Central Composite Design and Response Surface Metho d with Design Expert software version 7.1.5. Fermen tation was carried out in erlenmeyer flask at initial pH 8, 37 °C, with shaker incubator at 87.5 rpm. The results o f the best of enzyme activity 1.0 mU/mL, protein levels of 0.557 mg/mL a nd desirability value of 0.740. Numerical optimizat on was performed to approach the ideal state of the fermentation or desirability value of 1. The medium composition of fermentation used was: 3.64% baker's yeast, 1.21% glucose, and 0.13% skim milk. The enzyme activity reached was 1.51 mU/ L and protein levels of 0.205 mg/mL. After numerical opti mization, the fermentation process was verified usi ng 125 mL Erlenmeyer in shaking incubator at 77 rpm, initial pH 8, 37 °C, 15 h of fermentation. The verification results s howed that the enzyme activity and protein levels was 1.273 ± 0.227 mU/mL and 0.248 ± 0.012 mg/mL, respectively.
Introduction
The value of pharmaceutical enzyme imported in 2007 was $ 2.988 billion and the estimated value in 2008 and 2011 were $3.91 billion and $ 4.55 billion, respectively [1], approximately 90% of the demand, while the world enzyme demand also continued to increase by 6.5% per year, up to $ 5.1 billion in the year 2009 [2]. Trypsin is a protease enzyme produced by the pancreas and secreted to the duodenum, where it hydrolyses proteins into peptides during the digestion of food. Trypsin is memberof the serine protease family, which digest protein from the carboxyl terminal (C-terminal) of the amino acid lysine (Lys) and arginine (Arg) [3].
Trypsin deficiency can also cause a variety of problems in the physiology of the body as impaired amino acid absorption in case of inflammation of the pancreas. One effort to address them is by giving intake digestive enzyme (trypsin) from outside the body [4]. Until now a lot of research has been done on the isolation of trypsin from various species of fish including pomfret fish, bigeye snapper, red snapper, chinook salmon, sardine monterey, mandarin fish and skipack [5].
Trypsin can also be isolated from pork and beef. It can be a bit problematic because there is a fear the spread of bovine spongiform enchephalopathy (mad cow disease). This led to increased interest in the proteases of microbial because microorganisms can be produced on a large scale, relatively short production time, and production can be run continuously.
Lactic acid bacteria (LAB) is one among microbes received GRAS status (Generally Recognized As Safe) from the FDA and safe for food products. One source of microbes potential as trypsin-like protease producer is lactic acid bacteria (LAB). LAB is commonly used in food productions, thus has GRAS status (Generally Recognized as Safe). This is, of course, important if the enzyme is intended for human consumption.
Media composition and growth condition play important roles in providing the culture's growth rate and productivity, as well as the activity of the product. To obtain optimum media composition and growth conditions, such as temperature, pH, or agitation, requires good experimental design and reproducible empirical data. To design more focused and well targeted experiments, statistical methods are often used for example, the Response Surface Methodology (RSM). RSM is a statistical method used to explore the relationships between several explanatory variables and one or more response variables. Response surface methodology is a collection of mathematical and statistical techniques that are useful for the modeling and analysis of problems, in which a response of interest is influenced by several variables. Using RSM, we can determine which explanatory variables (e.g. medium components or culture conditions) have impacts to the response variables of interests (e.g. growth rate, protein level or productivity) [6]. CCD technique contains an imbedded factorial or fractional factorial design with 'center points' that is augmented with a group of `star points' allowing estimation of curvature [7]. Response Surface Methodology (RSM) and Central Composite Design (CCD) with four numerical factors (temperature, intensity, pH, aeration) and one response (µmax) was performed to optimize the growth of Nannochloropsis oculata [8]. Response surface methodology (RSM) was applied to optimize the medium constituents. A 2 4 fullfactorial central composite design (CCD) was chosen to explain the combined effects of the four medium constituents, viz. moisture content, particle size of the substrate, di-potassium hydrogen phosphate and trace ion solution concentration, for the production of lovastatin by using strain of Aspergillus terreus UV 1718 [9]. In this study, we performed medium optimization for the production of trypsin-like protease using the RSM and 2 4 full-factorial CCD with 4 numerical factors (concentration of baker's yeast, glucose, skim milk and the agitation speed) with two responses, trypsin activity and protein contents.
The best medium composition and agitation will be used to scale up and purification as well as characterization of trypsin-like protease.
Growth medium. The growth medium used in this study was 5.2% de Man Rogosa Sharpe (MRS) medium, sterilized by autoclave at 121 °C for 15 min. All fermentations were performed at 37 °C, pH 8.
Production medium and fermentation. Baker's yeast was used instead of yeast extract because it was a cheap and edible alternative of nitrogen source. Each medium composition was prepared separately. Glucose of carbon source was dissolved in water and was adjusted to pH 8 using HCl 0.1M. Baker's yeast was dissolved in water and then centrifuged for 15 min at 25 °C 3000 rpm to obtain the supernatant. Glucose solution and baker's yeast supernatant were mixed and homogenized, then autoclaved at 121 °C and 2 atm for 15 min. Skim milk of inducer was suspended in water and then pasteurized at 110 °C and 2 atm for 15 min. Prior to use, glucosebaker's yeast solution and skim milk suspension were mixed aseptically. Fermentation was carried out in erlenmeyer flask 125 mL at initial pH 8, 37 °C, with shaker incubator. The results of a preliminary study to determine the number of cells is recommended in this study ≥10 8 cells/mL.
To prepare starter culture, glycerol stock of lactic acid bacteria isolates FNCC 0270 was inoculated into 40µl growth medium and incubated at 37 °C 150 rpm for 5 h. 100µL starter culture was used to be inoculate in 5mL growth medium and then incubated at 37 °C 150 rpm. After 5 h, cells were harvested by centrifugation at 4 °C 6000 rpm for 5 min, and then used to inoculate the production medium. Production culture was then incubated for 24 h at 37 °C with agitation. After 24 h, The preliminary experiments to define the glucose and skim milk concentration range for the production of trypsin-like protease from L. plantarum FNCC 0270 was performed using medium with the following compositions; 2% w/v baker's yeast, 0.5% and 1% w/v glucose, and 0.25, 0.5, 0.75, and 1.0% w/v skim milk. Agitation speed used was 87.5 rpm. Observation of trypsin-like activities and protein levels expressed at various concentrations was performed every 6 hours for 24 hours. From eight combination experiments obtained by a five combination consisting of medium composition baker's yeast 2% w/v, glucose 1% w/v, skim milk 0, 25% w/v and 87.5 rpm agitation the best enzyme activity was obtained so based on a five combination determined value range of media composition and agitation baker's yeast is 1.5-2.5% (w / v), glucose from 0.75 to 1.25% (w/v), skim milk from 0.15 to 0.35% (w/v) and agitation varies between 50-125 rpm, to put the software Design Expert 7.1.5. (Stat Ease Inc., USA). The determination of the value of the middle point can be seen in Table 1, used in this experimental plan.
Analitycal methods. The number of cells used as starter of fermentation was estimated using spectroscopy at 560 nm (OD 560 ). Trypsin -like protease activity and protein content from crude enzyme fraction were analyzed using Khantaphant and Benjakul method [5] and Bradford method [11], respectively. Preliminary studies for analysis of proteins using Bovine Serum Albumin, the protein content 0.293 mg/mL using centrifuge and 0.326 mg/mL without centrifugation, whereas the trypsin-like proteases production of L plantarum FNCC 0270, the protein content 0.167 mg/mL at fermented 7 hours.
Experimental design and optimization. The present study used program Design Expert version 7.1.5., with study type: Response Surface, using Central Composite Design (CCD) as the initial design and Quadratic design model, by four numerical factors or independent variable, which were A: baker's yeast (%w/v), B: glucose (%w/v), C: skim milk (%w/v), D: agitation (rpm) Star po-int Cen-ter po-int and two responses, which were enzyme activity (Y 1, U/mL) and protein contents (Y 2, mg/mL).
The CCD matrix employed for four numerical factors is presented in Table 2, consisting of: 2 4 (16) factorial points with eight (8) star points and six (6) replications at the center points leading to a total number of 30 experiments. Activity and protein content were determined and assigned as responses 1 and 2, respectively.
Experiments on each condition expressed in factorial and star points were performed in duplicates. A total of 54 runs were performed.
The experimental results were plotted on a second-order polynomial equation and processed with the program Design Expert version 7.1.5. The mathematical model is as expressed in equation (1) . Y = a 0 + a 1 A + a 2 B + a 3 C + a 4 D + a 12 AB + a 13 AC + a 14 AD + a 23 BC + a 24 BD+ a 34 CD+ a 11 A 2 + a 22 B 2 + a 33 C 2 + a 44 D 2 (1) where, Y : the response that comes from each treatment, in this case is the enzyme activity (Y 1 ) and protein content (Y 2 ).
A, B, C, D, : numeric factors a 0 : intercept a 1 , a 2 , a 3 , a 4 : coefficients estimate of linear effect of factor a 12 , a 13 , a 14 , a 23 , a 24 , a 34 : coefficients estimates of interaction terms a 11 , a 22 , a 33 , a 44 : coefficients for quadratic terms
Results and Discussion
The results of preliminary studies of trypsin-like proteases production of L. plantarum FNCC 0270 baker's yeast as a source of N and replacement of yeast extract gives almost the same specific activity 0.68 mU/mg and 0.75 mU/mg respectively. Design Expert 7.1.5 provide the three models (Sequential Model Sum of Squares, Lack of Fit Test and Model Summary Statistics) were presented influence of different experimental variables, i.e. the percentage of baker's yeast, glucose, skim milk and agitation towards response of enzyme activities. The Model Summary Statistics towards response of enzyme activities at Table 3.
The Model Summary Statistics indicated that amongst the available models, i.e. linier, 2FI, quadratic and cubical, the Qudratic Model was the one most suitable representing the relationship between the experimental variables and enzyme activities.
The Analysis of variance (ANOVA
Model accuracy was predicted by comparing the actual experimental results and model prediction using paired Student's t test using online GraphPad software (http:// www.graphpad.com/quickcalcs/ttest2.cfm). The twotailed p value of the t-test was 1, indicating that at 95% confidence any difference between the predicted and actual enzyme acivities occured by chance (not repoted).
From Design Expert version 7.1.5, there are six pictures of three-dimensional interaction between media composition baker's yeast, glucose, skim milk and agitation on the response of the enzyme activity, only four pictures are presented in Figure 1. Figures 1 (a, b, c, d) show that the enzyme activity increases with increasing concentrations of baker's yeast as a source of N, the concentration of glucose as a source of C and a reduction in the concentration of skim milk as an inducer while agitation speed do not play a significant role in the increase enzyme activity. Figure 2 demonstrates the distribution of actual and predicted protein contents. The predicted values were expressed as straight line and the actual values were expressed in squares. Model accuracy was predicted by comparing the actual experimental results and model prediction using paired Student's t test using online GraphPad software (http://www.graphpad.com/quick calcs/ttest2.cfm). The two -tailed p value of the t-test was 1, indicating that at 95% confidence, any difference between the predicted and actual protein contents was not significant and occured only by chance. Figure 2 shows that the actual and predicted values are spread away from the straight line, indicating the low standard deviation (0.091) as calculated based on ANO--VA. The low standard deviation indicated that the model has good accuracy and the model is not suitable. From Design Expert version 7.1.5, there are six pictures of three-dimensional interaction between media composition baker's yeast, glucose, skim milk and agitation on the response of the protein content, only four pictures are presented in Figure 3. To verify the selected condition, fermentation was performed in 125 mL erlenmeyer flask using incubator shaker and medium composition and growth conditions as follows; baker's yeast, glucose and skim milk concentrations 3.64%, 1.21%, and 0.13%, respectively, agitation speed 77 rpm, initial pH 8. The results of the verification of enzyme activity and protein content 0.001273 ± 0.000227 U / mL and 0.248167 ± 0.011805 mg /mL, respectively. The ratio of C/N 0.043 in the fermentation substrate, baker yeast and glucose as a source of N and C respectively, while skim milk as an inducer. This value of C/N ratio then be used for largerscale fermentation substrates in future studies.
Figure 2. Distribution of Actual and Predicted Protein Contents
In particular the response surface method (RSM) is a sequential procedure with an initial objective of leading the experimenter rapidly and efficiently to the general vicinity of the optimum. RSM has already been successfully employed for the optimization of microalgae cultures [6]. Central composite designs which maximize both the precision and the accuracy of estimates of the external point of a second-order response surface for fixed values of the model parameters are constructed [12].
The results of this study indicated that numerical factors or variables; baker yeast, glucose and skim milk and agitation may not be the only factors affecting the enzyme activity and protein levels. This was indicated by the R squared value of only 65.92% and 42.11% respectively. Another study conducted by Qi-he et al. [13] using RSM with 6 variables (T, t, the volume of media, inoculation volume, seed age, shaking speed) on the enhanced production of elastase B.licheniformis ZJUEL31410 also obtained R squared values below 50%, and so was study by Fucinos et al. (2011) [14] using RSM with 2 variables (pH and T) on the model of stability obtained KLEST-3S, which showed R squared values 0.797.
To explore whether the optimized model from RSM could be applied to a large scale synthesis, the reaction volume should be increased by a factor of 10. This had been done by Liu et al. (2007) [15], who demonstrated that there was no significant difference between large and small scale syntheses.
Conclusions
The experiments indicated that medium containing 3.64% baker's yeast, 1.21% glucose, 0.13% skim milk and 76.79 rpm agitation speed was the best for this purpose, the enzyme activity was 1.273 ± 0.227 mU/mL and protein levels 0.248 ± 0.012 mg/mL.
The best media composition with C/N ratio 0.043, skim milk as an inducer and 77 rpm agitation might be applied to scaled-up fermentation to produce trypsin -like protease and purification as well as characterization of trypsin-like protease from L plantarum FNCC0270 in future studies. | v3-fos |
2016-05-14T18:07:42.550Z | {
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} | s2 | Fungal Endophyte Diversity and Bioactivity in the Indian Medicinal Plant Ocimum sanctum Linn
Endophytic mycopopulation isolated from India’s Queen of herbs Tulsi (Ocimum sanctum) were explored and investigated for their diversity and antiphytopathogenic activity against widespread plant pathogens Botrytis cinerea, Sclerotinia sclerotiorum, Rhizoctonia solani and Fusarium oxysporum. 90 fungal isolates, representing 17 genera were recovered from 313 disease-free and surface sterilised plant segments (leaf and stem tissues) from three different geographic locations (Delhi, Hyderabad and Mukteshwar) during distinct sampling times in consequent years 2010 and 2011 in India. Fungal endophytes were subjected to molecular identification based on rDNA ITS sequence analysis. Plant pathogens such as F. verticillioides, B. maydis, C. coarctatum, R. bataticola, Hypoxylon sp., Diaporthe phaseolorum, Alternaria tenuissima and A. alternata have occurred as endophyte only during second sampling (second sampling in 2011) in the present study. Bi-plot generated by principal component analysis suggested tissue specificity of certain fungal endophytes. Dendrogram revealed species abundance as a function of mean temperature of the location at the time of sampling. Shannon diversity in the first collection is highest in Hyderabad leaf tissues (H' = 1.907) whereas in second collection it was highest from leaf tissues of Delhi (H' = 1.846). Mukteshwar (altitude: 7500 feet) reported least isolation rate in second collection. Nearly 23% of the total fungal isolates were considered as potent biocontrol agent. Hexane extract of M. phaseolina recovered from Hyderabad in first collection demonstrated highest activity against S. sclerotiorum with IC50 value of 0.38 mg/ml. Additionally, its components 2H-pyran-2-one, 5,6-dihydro-6-pentyl and palmitic acid, methyl ester as reported by GC-MS Chromatogram upon evaluation for their antiphytopathogenic activity exhibited IC50 value of 1.002 and 0.662 against respectively S. sclerotiorum indicating their significant role in antiphytopathogenic activity of hexane extract. The production of 2H-pyran-2-one, 5,6-dihydro-6-pentyl from M. phaseolina, an endophytic fungus is being reported for the first time.
Introduction
Endophytic fungi inhabit a unique biological niche and are categorized as highly diverse, polyphyletic group of primarily ascomycetous fungi, capable of colonizing tissues of plants asymptomatically without initiating any disease or overt negative symptoms [1], [2], [3]. Endophytes have been recovered from all plants explored for their presence till date [4], [5]. 420,000 plant species exist in nature and only a few have been completely studied relative to their endophytic biology [6]. Diversity of fungal endophytes is 7% out of total of 1.5 million fungi on earth [7], [8]. Fungal endophytes are believed to be a treasure of structurally and biologically unique natural products as documented previously in several reviews [9], [10], [11], [12], [13], [14]. Numerous antifungal metabolites have been reported from endophytic fungi belonging to different structural classes such as alkaloids, peptides, steroids, terpenoids, phenols, quinines and flavonoids [15].
Ocimum sanctum is ubiquitous in Indian culture and tradition, called Tulsi in Hindi [16]. Because of its well documented therapeutic potential, Ayurveda (Indigenous system of Indian medicine) describes O. sanctum as Sashemani Shwasaharani (antiasthmatic), Kaphaghna (suppressant drug) and believed to promote longevity [17], [18], [19]. It is mentioned as adaptogenic in nature, balancing different processes in the body and helpful for adopting to stress in Charaka Samhita [20].
Besides, it's numerous pharmacological benefits, Ocimum sanctum extracts and essential oils particularly eugenol have been known to be highly effective against a plethora of plant pathogens. Essential oils extracted from Uttarakand grown Ocimum sanctum displayed antifungal activity against Rhizoctonia solani with MIC of 62.5 μg/ml [21]. O. sanctum plant extracts have also shown 38.15% inhibition against Sclerotinia sclerotiorumde Bary causing stem rot of Indian mustard [22]. Similarily leaves of Ocimum sanctum has been reportedly found effective in inhibition of alfatoxin producing fungi Aspergillus parasiticus by 7-20% [23].
Therefore, the objective of the work reported in this manuscript was to do systematic study of diversity and distribution of endophytic fungi isolated from different tissues of Ocimum sanctum collected from three different geographic locations and in two variable sampling times (seasons), and further screen them as potential biocontrol agents against four broad spectrum plant fungal pathogens and finally to scrutinize their metabolite profile with GC-MS Chromatography.
Collection of host plants
In order to maximize the chances of getting bioactive endophytic fungi, collection of host plants was done in different agroclimatic zones varying widely in climatic conditions, namely Southern plateau and hills region: Hyderabad (17.3660°N, 78.4760°E); Western Himalayan region: Mukteshwar (29.4722°N, 79.6479°E) and Trans Gangetic plains: Delhi and Gual pahari (28.4700°N,77.0300°E) in two different sampling time (autumn: (August-September) and summer (April-June) between two consequent years 2010 and 2011 with recorded variations in mean temperature°C and rainfall as mentioned in Table 1. Host plant samples were collected from healthy, disease free cultivated plant hosts from the above mentioned places for isolation of endophytic fungi. All the individuals of medicinal host plant Ocimum sanctum were collected from cultivated herbal gardens in all the locations. No particular variety of O. sanctum was selected. Same individuals were investigated in different seasons/sampling times. Endophytic fungi were isolated only from leaf and stem parts. Plants were freshly transported in moist ziplocked polybags from collection site to laboratory in Delhi. Plant parts (leaf and stem) were thoroughly washed in running tap water and processed for isolation of endophytic fungi immediately (within 1-2 days) after collection of whole plant.
Ethics statement. No specific permissions were required for collection of host plants from above stated locations. At Mukteshwar and Delhi, O.sanctum plants were procured by first author Ms. Kanika Chowdhary from Host Institution's (The Energy and Resources Institute) Herbal Garden, being in the capacity of its Research student. Whereas in Hyderabad O.sanctum plants were procured from Herbal Garden of a regional Agricultural University and specific permission was not required as they were available for sale for public. (Cash bill from Herbal Garden, Hyderabad is appended as S1 Fig). It is hereby confirmed that field studies didn't involve endangered or protected species.
Data availability. All relevant data are within the paper and its supporting information files. Also, minimal dataset needed for replication is available within the manuscript, supporting information and stored in a stable repository as well.
Isolation of endophytic fungi and maintenance
Surface Sterilization for isolation of endophytic fungi was done following previously established procedures [24]. Washed plant parts were treated by the following immersion sequence: 70% ethanol for 2 mins followed by 1% sodium hypochlorite (NaOCl) solution for 3 mins. Thereupon, samples were rinsed in double distilled, sterilized water for a couple of minutes. Then samples were dried on a blotting sheet. Imprints of dried and sterilized samples were taken on media plates; finally samples were chopped into 8 mm diameter segments and placed (3-4 segments on each plate) onto petri dishes (Tarsons, Kolkata) containing malt agar as medium. Malt extract-Agar medium {Malt extract (Himedia, Mumbai)15g, Agar powder (Himedia, Mumbai)15 g dissolved in 1000ml distilled water} amended with antibiotics chloramphenicol (Himedia, Mumbai) @ 0.2g/l and streptomycin sulphate (Himedia, Mumbai) @ 0.1 g/l of media at 7.4-7.8 pH was used as an medium for isolation, purification and maintenance of endophytic fungi. The plates were incubated at 24 ± 2°C for upto three weeks. The plant segments were observed once a day for the growth of the endophytic fungi. Hyphal tips of the endophytic fungi growing from the plant segments were isolated from isolation plate and maintained on fresh malt agar plates and coded with a unique number until identified. Total genomic DNA extraction, PCR amplification and sequencing Total genomic DNA of the endophytic fungi was isolated directly from freshly subcultured endophytic fungi grown on Malt extract-agar medium, using the DNeasy plant minikit (Qiagen), according to manufacturers' protocol. PCR amplification of isolated DNA was carried out by performing slight modification in previously described method [24]. The universal primers ITS1 5 0 TCCGTAGGTGAACCTGCGG 3 0 and ITS4 5 0 TCCTCCGCTTATTGATATGC 3 0 (Sigma Aldrich) were used to amplify the ITS region in rDNA. PCR mixture (
Identification of endophytic fungi and phylogenetic evaluation
To identify the isolates, sequences were subjected to the BLAST search with the NCBI database. Multiple sequence alignment of approximately 500 bp sequences was performed using CLUS-TAL W version 1.8. The phylogenetic tree was reconstructed and the evolutionary history inferred using the Neighbor-Joining method [25]. Tree topologies were evaluated by performing bootstrap analysis of 1,000 dataset [26] with the updated MEGA 6 [27], [28]. The sequences of this study were deposited at GenBank. The accession numbers are detailed in Table 2.
Dual culture bioassay
After purification of each endophytic fungal species, dual culture bioassay was conducted against economically significant plant pathogenic fungi e.g. Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium oxysporum, Botrytis cinerea; to test the antiphytopathogenic activity of the given endophyte fungi. Potato dextrose agar medium (Fresh Potato 15gms, distilled water 1000ml, D-glucose 15gms and Agar powder 15gms) was selected for dual culture as it favours growth of plant pathogenic fungi. The pathogen cultures were obtained from Indian Type Culture Collection, Indian Agricultural Research Institute, New Delhi. Both endophytic fungi (under investigation) and plant pathogenic fungi were placed with the aid of cork borer (8mm diameter) on PDA plate respectively opposite to each other and left for incubation at 24±2°C. After 6-7 days of incubation plates were observed and antagonism expressed and the various modes of interactions possible between endophytic fungi and pathogenic fungi were duly recorded. The experiments were conducted with 3 set of replication plates.
Multiplication of selected endophytic fungal isolates
Thirteen endophytic fungal isolates showing antagonistic activity at least with 3 pathogenic fungi were further explored for antiphytopathogenic activity of their crude extracts by multiplication on rice media. Rice medium was prepared in 1L of Erlenmeyer flasks containing 300gms of Rice grain (autoclaved) [29]. The flasks were inoculated with 1-2ml distilled water suspension of hyphae scrapped from freshly cultivated culture of potential fungal endophyte under static conditions in daylight at 25±2°C. filtered under vacuum using a buchner funnel and extraction was repeated twice with ethylacetate for complete extraction of metabolites. The pooled extract was then dried up with vacuum rotary evaporator (Heidolph, Germany) under reduced pressure at 40°C which served as crude extract for further evaluation [29].
Preliminary phytochemical analysis of crude fungal extract for presence of terpenoids
Since plant parts of O. sanctum are reservoir of essential oils, fungal endophytes isolated from them were investigated for presence of terpenoids which are primary constituents of essential oils. To the crude extract chloroform (Qualigens, India) and conc. H 2 SO 4 (Rankem, India) were added to form a lower layer [30], [31]. Formation of reddish brown color confirms the presence of terpenoids. Fungal crude extracts showing presence of terpenoids were further subjected to partitioning between n-hexane (Rankem, India) and 90% methanol (Qualigens, India). Confirmation of presence of terpenoids in hexane extracts was done by GC-MS Chromatography.
Gas chromatography mass spectroscopy (GC-MS)analysis of hexane extract
The GC-MS analysis of hexane extracts was carried on GC-MS (Agilent Technologies 7890A). For this 1mg of extract was dissolved 1 ml LR-grade dichloromethane (Qualigens, India). DB-WAX column (30 m × 250μm × 0.25 μm) was used with helium as a carrier gas at a flow rate of 1 ml/min at pressure of 11.654 psi. The GC oven temperature was kept at 120°C for 2 min and programmed to 5°C/min to 130°C for 1 min; 5°C/min to 150°C for 2 min; 2°C/min to 180°C for 3 min; 2°C/min to 200°C for 3 min; 5°C/min to 240°C for 20 min. Splitless injections were done with liquid injection method in this study. A library search was carried out using NIST library.
Poisoned food bioassay of endophyte fungal crude extract and pure compounds
Poisoned food bioassay of endophytic crude extracts was conducted by means of biometric agar dilution method [32]. Dried crude extract/pure compound was dissolved in methanol (Stock solution: 40mg/ml). From the stock solution 125, 62.5, 12.5,6.25 and 1.25 μl were dissolved in 5ml of PDA medium to obtain five different concentrations i.e. 1mg/ml, 0.5mg/ml, 0.1mg/ml, 0.05 mg/ml and 0.01 mg/ml respectively. Intoxicated media plates were inoculated with 8 cork borer plugs of plant pathogenic fungi measuring 3mm 2 placed in such a way that seven are put in periphery and one in the centre of the plate. The control experiment consisted of only methanol solvent in PDA medium [33]. % GI was calculated for each set of bioassay using following formula: Where A = radial diameter of plant pathogenic fungi in check plate with methanol solvent only. B = radial diameter of plant pathogenic fungi in extract /fraction intoxicated plates IC 50 was calculated by regression equation analysis. For calculating IC 50 value, %GI at five different concentrations (mg/ml) were put in excel 2010 and scatter plot was generated. Linear trendline was produced on it displaying regression equation. Finally, mathematically (where, Y = 50) solving the equation provides the IC 50 value.
Data analysis
Isolation rate. Isolation rate was calculated as the number of isolates obtained from segments/pieces divided by the total number of segments/pieces [34].
Relative frequency. Relative frequency was calculated as the number of isolates of one species divided by the total number of isolates, and expressed as percentage [34].
Menhinick's index. Species richness among the isolated endophytic fungi was determined by calculating the Menhinick's index (Dmn) using the following equation [35]: where, s is the number of different endophytic species in a sample (plant tissue) and N is the total number of isolated endophytic fungi in a given sample.
Camargo's index. The fungal dominance can be determined by Camargo's index (1/D mn ), where D mn represents species richness.
Shannon diversity index. Furthermore, to quantify the endophytic fungal diversity the Shannon diversity index (H 0 ) was calculated using the following equation: Where, Pi is relative abundance of a species is in a given sample H 0 values could start from 0 (only one species present with no uncertainty as to what species each individual will be) and go higher revealing high uncertainty as species are relatively evenly distributed.
where, H 0 is the Shannon diversity of the endophytic fungi in a given sample and S is the total number of endophytic fungal individuals present in the given sample [36], [37], [38], [39]. The similarity and of endophytic fungal assemblages among both tissues was compared using the following similarity indices: where 'a' is the number of common species in both communities, while 'b' and 'c' are the number of species specified to each community under investigation, respectively [40].
Jaccard's index of similarity. JS was calculated using the formula: JS = a/(a + b + c) where 'a' is the number of common species in both communities, while 'b' and 'c' are the number of species specified to each community, respectively [41].
Principal component and cluster analysis. Principal component analysis studied interrelationships between endophytic fungi recovered from O. sanctum different plants parts in distinct sampling time, whilst cluster analysis deduced concurrence between species richness and temperature of geographical locations at the time of sampling. For this, software Unsrcambler X: version 10, CAMO, USA was used [42].
Identification of fungal endophytes
90 endophytic fungi were isolated based on culture dependent technique. Fungal isolates were grouped into 43 different morphospecies according to their morphological characteristics: colony colour and texture, border type, and radial growth rate on MA media. All isolates of each morphospecies group were submitted to molecular identification based on rDNA ITS sequence analysis. Based on the molecular identification these morphospecies were grouped into 23 species. The detailed description of 23 different endophytic fungal isolates with respective identification, accession number from GenBank, % similarity, % query coverage and number of base pairs sequence analysed are summarised in Table 2. The evolutionary history was inferred using the Neighbor-Joining method (Fig 1). The optimal tree with the sum of branch length = 72.08408877 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum composite likelihood method and are in the units of the number of base substitutions per site. All the fungal endophytes recovered in the present study belonged to phylum Ascomycota besides Rhizopus oryzae (4.4%: Mucormycotina) that belongs to phylum Zygomycota. Within Ascomycota, endophytic fungal isolates obtained are in six families of class Sordariomycetes (40%: Hypocreaceae, Glomerellaceae, Xylariaceae, Nectriaceae, Diaporthaceae and Chaetomiaceae), two families of class Dothideomycetes (33.3%: Pleosporaceae, Botryosphaeriaceae), two families of class Saccharomycetes (4.4%: Debaryomycetaceae, Trichomonascaceae) and single family of class Eurotiomycetes (16.6%:Trichocomaceae) (Fig 1). Aspergillus niger was observed as the most dominant species with 10% relative abundance (RA), followed by D. phaseolorum, Hypocrea sp. and M. phaseolina (RA = 7.86%) ( Table 2). Only 3 endophytic fungal isolates namely, Bipolaris maydis with relative abundance (RA) of 2.22%, Meyerozyma guilliermondii (RA = 2.2%) and Fusarium verticillioides (RA = 1.11%) were exclusively isolated from stem tissues and rest were present in both the tissues (S1 Table and Table 2)
Spatial and temporal variations in endophytic fungal isolates
Since medicinal value of O.sanctum resides largely inside leaf tissues, we incubated more leaf tissues for endophytic fungal isolation. A total of 313 plant tissue segments (222 leaf and 91 stem tissues) of O.sanctum collected from all geographical locations in both sampling times for isolation of endophytic fungi were incubated (Table 3). We characteristically observed both spatial and temporal variations in endophytic mycobiota.). In leaf, highest isolation rate of fungal endophytes in O. sanctum (40%) was observed in samples collected from Hyderabad during first sampling time and from Delhi in second collection in 2011. While, in stem tissues Mukteshwar samples collected during first sampling had highest isolation rate (71.4%) followed by isolation rate (50%) from Hyderabad during first collection. O. sanctum collected in second sampling from Mukteshwar recorded lowest isolation rate of fungal endophytes both for leaf (10.7%) and stem (10%) tissues (Table 3). Bi-plot and correlation loading plot generated by principal component analysis showed that fungal endophytes such as Alternaria tenuissima, A. alternaria, Macrophomina phaseolina and Penicillium sp. were more abundant in leaf tissues. While Fusarium verticillioides, Bipolaris maydis, Chaetomium coarctatum, Rhizoctonia bataticola, Hypoxylon sp., Diaporthe phaseolorum, Alternaria tenuissima were exclusively recovered from second sampling in 2011 suggesting strong seasonal implications on endophytic communities (Fig 2). In addition, when species richness recovered from both plant parts from each site in different sampling time were considered alongwith temperature at the time of sampling for cluster analysis, the resulting Dendrogram revealed concurrence between species richness and mean temperature of the geographical location at the time of plant sampling (Fig 3).
Diversity, richness and similarity index
Shannon diversity in the first collection is highest in Hyderabad leaf tissues (H' = 1.907) whereas in second collection it was highest from leaf tissues of Delhi (H' = 1.846). Shannon diversity has been recorded to be higher in leaf tissues as compared to stem tissues at all places besides Hyderabad in second sampling time ( Table 4). The species richness determined by calculating the Menhinick's index (Dmn) revealed that the leaves collected from Hyderabad in first sampling were richest in endophytic fungal species (Dmn 3.46), followed by the leaves from Delhi in second sampling in 2011 (Dmn 1.87), and finally amongst stem tissues Hyderabad in second sampling in 2011 (Dmn 1.656) had a rich endophytic assemblage. Camargo's index depicting the tissue-specific fungal dominance was 0.746 for the Delhi stem tissues in first sampling (highest), followed by that of leaves of Hyderabad in second sampling 0.709 (Table 4). Combined data obtained by these mathematical tests indicate that diversity of fungal endophytes harbored inside leaf tissues was higher than in stem tissues of O. sanctum. Pielou's eveness index, measuring the variation in endophytic mycopopulation differs among geographic locations ranged between 0.456 to 1. Pielou's eveness index differs correspondingly amongst geographical locations but is nearly similar when compared between plant tissues solitary (Table 4). Species shared between Delhi and Hyderabad was highest in second sampling which resulted in high Sorenson's similarity index (0.33). Collectively analysed, lower similarity index amongst three sites indicates uniqueness of endophytic mycobiota in different sampling times from different locations, observations which are further substantiated by PCA (Table 5).
Antiphytopathogenic activity
Vital observations were made to gain insight into the biocontrol potential of endophytic fungi outside its plant source on an artificial media in vitro against four widespread phytopathogens in India namely Botrytis cinerea, Sclerotinia sclerotiorum, Fusarium oxysporum and Rhizoctonia solani. After doing necessary macroscopic evaluation of the interaction, we followed literature sources available on endophyte-pathogen interaction types and categorised dual culture bioassay results accordingly [43], [39]. Mostly results obtained were similar within the replication plates between all endophytic fungi against all four pathogenic fungi, but in certain cases of conflict, we accepted the observations as appeared in at least 2 out of 3 replication plates. Based on our results we categorized mode of interactions into nine (I-IX) types. The key purpose of the dual culture bioassay was based on bioprospecting strategy to select potential endophytes with having antiphytopathogenic activity. The means of achieving the objective was to carry out qualitative screening of plethora of fungal endophytes recovered from different medicinal plant hosts in distinct sampling times on the basis of their ability to exhibit antagonism against broad spectrum phytopathogens. The selected fungal endophytes were marked and maintained for further evaluation ( Table 6, Fig 4). Nearly 23% of the total fungal isolates were considered as potent biocontrol agent, as they showed antifungal activity against at least three of the broad spectrum plant pathogens (S2 Table). Fungal Endophyte Diversity and Bioactivity in Ocimum sanctum
Bioactivity of crude extracts of fungal endophytes
Based on the results of dual culture bioassay (S2 Table) Table 7. Extract of Chaetomium coarctatum recorded the highest activity against all phytopathogens with IC 50 value of < 1mg/ml ranging from 0.262mg/ml-0.553 mg/ml. Second best activity was reported with Fusarium proliferatum obtained from Hyderabad with IC 50 value ranging from 0.299 to 1.04 mg/ml. Strain specificity for bioactivity was strongly observed. For example, Fusarium proliferatum obtained from Hyderabad exhibited IC 50 value of 0.51mg/ml against F.oxysporum which was stronger than another isolate of F. proliferatum recovered from Delhi in second sampling (Table 7). Likewise, Macrophomina phaseolina recovered from stem tissue displayed IC 50 Table 6. Different modes of interaction observed between isolated endophytic fungi and the four plant pathogens in dual culture/Confrontation bioassay.
Class I Both pathogen and endophyte grow towards each other, growth stopped at equidistant at physical contact but mycelial boundary is maintained by both even after two weeks of observation
Class II
Both pathogen and endophyte grow towards each other, but endophyte grows over the pathogenic fungi mycelia; pushing it backwards
Class III Both pathogen and endophyte grow towards each other, zone of inhibition is formed which is maintained for another week
Class IV Pathogenic fungi's growth is completely stunted by endophytic fungi at its periphery Class V Both pathogen and endophyte grow towards each other and Pathogenic fungi overgrows endophytic and stops its growth at physical contact Class VI Pathogenic fungi completely stunts the growth of endophytic fungi at its periphery Class VII Both pathogen and endophyte grow towards each other, but pathogenic fungi mycelia overgrows endophytic fungi's mycelia
Bioassay of pure compounds
Among all the compounds recorded in GC-MS Chromatography of hexane extracts of fungal endophytes 2H-pyran-2-one, 5,6-dihydro-6-pentyl, palmitic acid, methyl ester, oleic acid are reported to have antifungal activity [44], [45]. Therefore, in order to confirm the contributory effect of these compounds on the antifungal activity of hexane extracts of endophytic fungi, they were procured from commercial sources and their antifungal activity was evaluated against phytopathogens. 2H-pyran-2-one, 5, 6-dihydro-6-pentyl and palmitic acid, methyl ester exhibited antiphytopathogenic activity exhibited IC 50 value of 1.002 and 0.662 respectively against S. sclerotiorum confirming their antifungal activity (Table 8, Fig 6).
Discussion
All the fungal endophytes reported from O. sanctum in present study belong to Ascomycota besides Rhizopus oryzae (Zygomycota). Rhizopus oryzae has been reportedly primary causal agent of tuber rot disease in sweetpotato in India and in this study has been isolated from Delhi and Hyderabad in first sampling season [46]. Ascomycota is the largest phylum of fungi, one of the most diverse and ubiquitous phyla of eukaryotes covering approximately 8% of the Earth's landmasses [47]. Interestingly, all the fungal endophytes recovered from O. sanctum have been known as plant pathogens. The study for the first time reports following three plant pathogens as endophytes, Bipolaris maydis, Rhizoctonia bataticola and Chaetomium coarctatum harbored inside O. sanctum. Earlier literature suggests that endophytes have evolved directly from plant pathogenic fungi [48], [49]. It has been established for a plethora of fungi that pathogenicendophytic lifestyles are interchangeable and are due to a number of environmental, chemical and/or molecular triggers [50], [51], [52]. Thus, even a fungus that is pathogenic in one ecological niche can be endophytic to plant hosts in another ecosystem. Photita et al in 2004 confirmed that fungal endophytes isolated from wild banana (Musa acuminata) were in vitro latent pathogens [53]. Fungal endophytes reported from present study have been previously described in their endophytic nature producing bioactive secondary metabolites. For instance, Fusarium proliferatum causing root rot on Soybean (Glycine max) [54], has been reported to produce a plethora of secondary metabolites with different biological activities. It has been known to produce sanguinarine possessing antibacterial, anthelmintic, and anti-inflammatory properties when isolated from Macleaya cordata [55]. 240ng/l of paclitaxel was produced byFusarium proliferatum when isolated from paclitaxel producing plant Taxus x media [56]. Two new tricyclic sesterterpenes, fusaprolifins A and B were reportedly produced from the same fungi when isolated from marine mangrove plant Bruguiera sexangula [57]. Alternaria alternata is a well known necrotrophic fungus [58]. Altenuene derivatives, isocoumarin alongwith other metabolites were produced by Alternaria alternata isolated from Camellia sinensis [59]. Diketopiperazines extracted from grapevine endophyte Alternaria alternata effectively inhibited Plasmopara viticola [60]. Also, one of the endophytic strain of Alternaria alternata harbored inside fruit and seeds of Miquelia dentate Bedd. produced camptothecin, 9-methoxy camptothecin and 10-hydroxy camptothecin [61]. Similarily, Penicillium crustosum recently described as causal agent of blue mold in stored apples [62]. In its endophytic state, when recovered from seeds of coffee beans it reportedly produced mycophenolic acid and a new phthalide, 5-hydroxy-7-methoxy-4-methylphthalide [63].
In the present study Mukteshwar (altitude: 7500 feet) recorded least isolation rates in second sampling time which is in line with the report of Carroll and Carroll [64] who reported low isolation rates at high elevations that could result from delayed onset of endophytic infections. Highest isolation frequency (75%) was recovered from stem tissues from Hyderabad collected in second sampling season. One possible explanation could be that recently penetrated epiphytic hyphae could have survived surface sterilization procedures [65]. Bertoni and Cabral [66] found that epiphytic fungi may form limited colonizations through substomatal chambers and also avoid the effects of surface sterilization. In this study, only the culturable endophytic fungi could be isolated and the assemblages do not represent non-culturable endophytic fungal isolates of the O.sanctum plants investigated. It should also be mentioned here that rDNA ITS analysis can sometimes underestimate the endophytic fungal 'species diversity', and additional parameters should be coupled to ITS rDNA sequence [67]. Increased abundance of A. tenuissima, A.alternaria, M. phaseolina and Penicillium sp. in leaf tissues could be described as tissue preference. Tissue preference has been previously reported for endophytic fungi, highlighting their surviving ability within a specific substrate [34]. Occurrence of eight phytopathogens viz. F. verticillioides, B. maydis, C. coarctatum, R. bataticola, Hypoxylon sp., D. phaseolorum and A. tenuissima together in second sampling as explained in principle component analysis, could be hypothesised that may be during high temperature and low humidity plant pathogens are less virulent and are better established as endophyte within plant tissues coping with medicinal plant's defence mechanism [68].
Endophytic fungal diversity is higher in tropical and subtropical plants than other climatic zones likewise reported from Delhi and Hyderabad regions in the present study [69], [70], [71], and [72].
Additionally, cluster analysis inferred distribution and abundance of endophytic fungal species from different geographical locations as a function of temperature. It grouped together species richness of Delhi (second sampling) and Hyderabad (first sampling) when temperature ranged from 23-24°C into cluster 1, cluster 2 comprises of Delhi (first sampling) and Hyderabad (second sampling) when temperature ranged from 30-31°C and cluster 3 belongs to Fungal Endophyte Diversity and Bioactivity in Ocimum sanctum endophytic mycobiota of Mukteshwar where temperature range was 14-17°C during both sampling times. This revelation is in accordance with previous studies elucidating higher influence of temperature, precipitation and other climatic factors than geography on endophytic communities of plants [73], [74]. Fungal Endophyte Diversity and Bioactivity in Ocimum sanctum Following on the lines of "bioprospecting" and "biological diversity can lead to novel chemistry" from endophytic fungi of medicinal plants [1], [9], [24] all the endophytic fungal isolates recovered were tested for antiphytopathogenic activity by means of dual culture/confrontation bioassay against four economically significant plant pathogens. S. Sclerotiorum causes heavy yield losses up to the tune of 40% in Brassicas in India [75]. Fusarium wilt is one of the major diseases of chickpea and at national level the yield losses encountered was reported to the tune of 60 per cent in India [76]. Botrytis grey mould (BGM), caused by the fungus Botrytis cinerea Pers. ex Fr., is an important disease of chickpea causing economic losses across the world in chickpea-growing regions. There are no available resistance sources in cultivated chickpea against this disease [77]. R. solani causes economically important root and hypocotyl diseases in common bean throughout the world [78]. Different degrees and types of interactions were observed between phytopathogens and endophytic fungal isolates. Each one of the 90 endophytic fungal isolate of O. sanctum exhibited antagonism against atleast one plant pathogen. For instance, all the endophytic fungal isolates obtained from Delhi in first sampling in 2010 showed antiphytopathogenic activity against Fusarium oxysporum. Best antiphytopathogenic activity was depicted from endophytic fungal isolates of Hyderabad and Mukteshwar in first sampling in 2010. 50% of the endophytic fungal isolates were found to be active against Rhizoctonia solani majority of them identified as Diaporthe phaseolorum collected from Hyderabad in second sampling in 2011. In addition, Penicillium sp.isolated from Mukteshwar in first sampling in 2010 demonstrated antiphytopathogenic activity against all of plant pathogens tested whereas other isolate identified Penicillium sp. recovered from Hyderabad in first sampling in 2010 could only be antagonistic against S. sclerotiorum. Thus, our report corroborates that biology of endophytes may be influenced by many factors such as localization, season and environment [79].
Hexadecanoic acid and 9, 12 octadecadienoic acid identified from extracts of T. crispa were found to be strongly active against C. albicans [84]. The major bioactive compound oleic acid found in L. cristata was reported be highly effective to phytopathogens Colletotrichum fulcatum NCBT 146, Fusarium oxysporum NCBT 156 and Rhizoctonia solani NCBT 196 [85]. Another study described inhibitory activity of linolenic and linoleic acids against phytopathogenic fungi including R. solani [45].
Conclusion
The study reports noted plant pathogens such as Bipolaris maydis, Rhizoctonia bataticola and Chaetomium coarctatum in endophytic state harbored inside O. sanctum for the first time. In present study metabolites produced by M. phaseolina recovered from Hyderabad in first collection has exhibited promising antifungal activity against S. sclerotiorum and F. oxysporum broad spectrum phytopathogens. Metabolites originated from fungal endophytes hold promise to be further developed as greener and safer biocontrol agent in crop disease management. It can be concluded that fungal endophytes harbored inside leaf and stem tissues of Ocimum sanctum collected from different geographical locations in different sampling times hold great promise not only as biocontrol agents against broad spectrum and economically significant phytopathogens, but also as sustainable resource of novel antifungal secondary metabolites. | v3-fos |
2019-03-12T13:04:09.170Z | {
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} | s2 | The Dark Chocolate against Angiogenesis?
Aimed to study the connection of dark chocolate and different phases of angiogenesis resulting in cancer pathogenesis. Dark chocolate is derived from cocoa (cacao), may have speckled polyphenol and flavonoids contents retains different levels of antioxidant abilities. The existence of methylxanthines, peptides and polyphenols like flavonoids may synergistically augment or diminish antioxidant properties of dark chocolate (cocoa). The Dark chocolate has certain capacity to famish tumors development and fighting against angiogenesis.
grown in rainy forests in tropics, ordinarily on large farmsteads, where it essentially sheltered from passionate sunlight and wind. Different parts of cacao tree have been consumed; explicitly cocoa beans primed as chocolate, cocoa butter, cocoa bark, cocoa pulp, cocoa flower and cocoa leaf. In 1505, Spanish fetched Cocoa to Europe. In 1653, in Europe cocoa used in terms of medicine comparatively than that of pleasant foodstuff. Practice of chocolate was renowned as an inspiring vigorous utility of spleen and other gastrointestinal functions. Revisions upon wellbeing profits of the cocoa food stuffs have been steered over past span with a topmost emphasis on deteriorating diseases. These doles could be in result of their momentous amounts of catechin and epicatechin (flavonoid monomers) which have copious favorable biological actions in anticipation of angiogenesis and cancer [2]. Most of revisions have scrutinized the charities of flavonoids in cocoa as well as cocoa foodstuffs towards health doles, but it should be renowned that cocoa and its foodstuffs are also ironic in methylxanthines (theophylline, caffeine, and Theo bromine) [3]. Revisions have confirmed which methylxanthines can retain equally positive and negative vigor effects (Table 1) [4].
Introduction
Timeline of cocoa arose in 2,000 B.C, the date recognized by historians to hoariest swallowing cups and plates that has endlessly revealed in Latin America, Ulúa valley (Honduras), where cocoa has a vital role. Cacao is plagiaristic from Olmec and consequent Mayan languages kakaw, whereas chocolateassociated tenure cacahuatl is Nahuatl, Aztec language, resulting from Olmec-Mayan etymology [1]. An elusive tree, cacao is merely methylxanthines are liable for cravings of chocolate [16]. Theobromine, the foremost methylxanthine found in cocoa, which is 4%, and caffeine is up to 0.2% [17]. The percentage of theobromine is higher than that of theophylline in cocoa beans [17]. Major Methylxanthine in cocoa and cocoa foodstuffs is caffeine [3]. The studies specified that bioactivity as well as importance of cocoa and cocoa foodstuffs were due to polyphenols [13]. It has been identified that theobromine has therapeutic results on cancer and angiogenesis, like; theobromine potentially obstruct angiogenesis prompted by ovarian cancerous cells through mechanism to reticence the production of VEGF (vascular endothelial growth factor) [18].
Peptides
Cocoa contains large number of proteins. Peptides in cocoa are mainly accountable for flavor [19,20]. It consists of four different kinds of proteins, which are, albumins, prolamin, globulins, and glutelin. Among them albumin is in foremost protein ratio [21]. Protein Albumin is 52% whereas globulin is about 43% of protein present in cocoa bean [22].
Angiogenesis
The process of developing new blood vessels resulting from pre-surviving blood vessels called as angiogenesis [23]. It is vigorous and common process in wound healing, development and growth. It is important for tumor conversion from resting condition into malignant, resulting in usage of angiogenesis inhibitors [24].
Sprouting angiogenesis
This type of angiogenesis carried out in different phases that are; biological signaling phase in which angiogenic factors motivate receptors located on surface of endothelial cells in blood vessels that are pre-existing, these motivated endothelial cells instigate to proclamate enzymes known proteases which worsen cellar membrane that allowing endothelial cells and seepage them from paternal vessel walls. This mechanism results in proliferation of endothelial cells into adjacent matrix to form sprouts, after which endothelial cells proliferate into adjacent matrix to form solid sprouts linking nearby vessels [25,26].
Intussusceptions angiogenesis
Intussusceptions angiogenesis is also called splitting angiogenesis. During this type of angiogenesis, capillary wall spreads into lumen results in splitting of single vessel into two. Its mechanism takes place in four different phases. In first phase of Intussuscepted angiogenesis, two conflicting capillary walls institute a region of communication followed by second phase in which the endothelial cell confluences are rationalized that allow vessel bilayer to Puncture to permit growth factor and thus cells are breach in lumen. In third phase, there is formation of core between two newly developed vessels at region of connection occupied by pericytes and myofibroblasts. Cells initiate to lay collagen gibers within core to afford extra cellular for progression of vessel lumen [26].
Normal angiogenesis
In process of vasculogenesis, there is proliferation of angioblasts that merge in embryonic grid of vessels called as capillary plexus (primary). Endothelial cell matrix functions as a gibbet for angiogenesis generated by vasculogenesis [27]. There is development and splitting of new vessels resulting from preexisting vessels in angiogenesis once capillary plexus (primary) formed. In embryo where is the most of normal angiogenesis happens and inaugurates primary vascular pyramid and tolerable vasculature to support growth and development of organs [28]. Angiogenesis process in adults carried out through ovarian cycle as well as in physiological repair mechanisms like; healing of wound [29]. There is minute endothelial cells revenue that happens in adult vasculature [30]. There are many sundry processes in micro vessels that results in fruition and renovation of newly bent micro vessels [29], pericytes should be detached from forking vessel to produce newly blood sprouts. Proteases like matrix metalloproteinases sulliedas well as refashioned extracellular matrix and endothelial cell cellar membrane, thus new matrix produced via stromal cells [31]. This whole process results the flow of blood in newly synthesized vessels.
Factors regulating normal angiogenesis [32]
The factors that regulate normal angiogenesis are consists of three main groups including soluble factors, membrane -bound proteins and biomechanical forces. There mechanisms are shown in
Tumor-persuaded angiogenesis
Tumors are inhabitants of host-imitative cells, which have vanished ability to legalize growth resulting in aberrant proliferate. Yet numerous landscapes differentiate them from non-renovated foils, many features of tumor cells that are analogous to normal ones [32]. One foremost resemblance is obligation for a sufficient hoard of oxygen as well as nutrients and an operative means to eradicate trashes in mandate for metabolic procedures to befall and being to be conserved. Propinquity to vascular stream fulfills these needs for mammalian cells. Ordinary cells and tissues trust on functional vasculogenesis and angiogenesis to deliver them with vasculature, which fulfills the metabolic needs [32].
Tumor Vasculature
Tumors can begin their personal blood supply by numerous revenues. In tumor-persuaded angiogenesis, a tumor may provoke development of blood vessels resulting from pre-surviving capillaries. Furth more, tumors cells are capable to develop around a surviving vessel and later, at least primarily, do not want to persuade angiogenesis for passable vascularization [33]. In addition, (CEPs), angioblast-identical cells that result from bone marrow tissue but conveyed to be exist in adult circulation, have freshly been recommended to donate to a tumor-imitated blood vessels [34]. Among them numerous dearth functional pericytes [35], which are distended and elaborated, as well as they are remarkably permanent due to existence of fenestrae and transcellular slums and dearth of a thorough basement membrane [36]. Additionally, walls of tumor may be composed up of endothelial cells as well as tumor cells mutually [37]. These structural aberrations in tumor's vessels reproduce pathological flora of their generation, however their aptitude to upkeep cell growth may also inspires use of physiological appliances of angiogenesis, which tumors requisition for their propagation [32] (Table 3). [32].
Antioxidant effects
Reactive oxygen species (ROS) are potential carcinogens that facilitate mutagenesis, tumor promotion and progression [38]. Food derived products exist universally and are expected to be safe, they are highly interesting for development as chemo preventive agents to treat reactive oxygen free radicals that results in cancer pathogenesis [39,40]. Natural compounds has the ability to induce cytotoxicity thereby protects against cancer and many researcher's developing chemotherapeutic by based on its ability to induce apoptosis [41][42][43][44]. Well-known character of polyphenols in cocoa is their ability to show there action as antioxidants. Polyphenols (flavones and catechins) are powerful flavonoids for defensive the body alongside reactive oxygen radicals. Cells and tissues of body constantly endangered by the injury affected by free radicals as well as reactive oxygen radicals that are bent in usual oxygen metabolism or are persuaded by exogenous injury [45]. Sequences and mechanisms of trials thru which free radicals restrict with cellular roles are not entirely known but most significant trial appears to be lipid peroxidation resulting in damage of cellular membrane. This cellular damage causes a shift in the net charge of cell that changes the osmotic pressure, resulting in swelling and ultimately cell death. Free radicals can entice numerous inflammatory mediators, subsidizing to common inflammatory retort and tissue injury [32].
Living bodies have established numerous effective procedures order to prevent there selves from free oxygen radicals [46]. Antioxidant-defensive mechanisms of body comprise certain enzymes like; catalase, glutathione peroxidase, and superoxide dismutase, but have no enzymatic foils like; ascorbic acid, tocopherol, and glutathione. Neovascularization that includes angiogenesis is mandatory for advancement of metastasis [32].
Anti-Angiogenesis Effects
Secretion of neovascularization/angiogenesis mediators via mast cells and motivation of mast cell migration by tumorplagiaristic peptides specify that mast cells can be tangle in metastasis migration of tumor [47][48][49]. Persuasive reticence of the mast cell stimulation and propagation by numerous flavonoids can also donate antitumor effects [49]. The mast cells discharge TNF that persuades molecule expression of endothelial adhesion [49,50]. There is reduction in stages of plasminogen activators, bFGF induction and their functional inhibitors [51]. bFGF motivates manufacturing of urokinase-type, plasminogen activator and PAI-1 in vascular-endothelial cells. Plasminogen synthesized plasmin that showing stepwise proteolytic deprivation of matrix protein. This is the key phase in mechanism of neovascularization.
Conclusion
Components of dark chocolate analytically impulse numerous cellular, immunological and a series of biological measures related with angiogenesis, cancer development and growth, like; the cell proliferation, cell differentiation, apoptosis, and neovascularization. The studies in vitro have recognized a connotation between flavonoid-persuaded impulsion of MMP and protein kinase actions with tumor cell invasion, apoptosis and cellular proliferation conduct. Certain dark chocolate components, such as flavonoids pageant antitumor activity and reduce angiogenesis in vivo. | v3-fos |
2019-03-28T13:42:23.876Z | {
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} | s2 | Effect of cutting type and IBA on rooting and growth of Citron ( Citrus medica
This study was conducted during 2012 and 2013 season to investigate the effect of IBA on rooting of citron stem cuttings ( Citrus medica Linnaeus ) Corsian cultivar. The cutting referred to the location from which it was taken (tip , medial and bottom ). Cuttings were taken and exposed to different IBA doses ( 0, 500, 1000, 2000 ppm).They were planted in greenhouse in mixed media (1 part peat moss + 2 parts sand ) to increase rooting success percentage in the stem cuttings of ( Citrus medica Linnaeus ) Corsian cultivar , improving their vegetative growth , and investigate the response of stem cuttings of citron to Auxin IBA concentrations. The results indicated that the type of cuttings were different in rooting capacity , shoot length content. Also increase the concentrations of IBA increased rooting percentage , shoot diameter, number of leaves ,length of roots and leaves relative chlorophyll content, significantly form compared with control treatment under level 5%. The result indicated that the medial cutting parts with 500 and 1000 ppm perform better in terms of root percentage(100%),and medial type cutting with500 IBA ppm in length of shoots(23.08 cm), shoot diameter(16.33 mm) , number of leaves (16.44 leaves .plant ),length of roots(17.92cm).
Introduction
Citron (Citrus medica L) is a species of citrus fruit. It usually has a thick rind and small sections. Originally, the tree came from South east Asia that the real citron grows in the Garo Hills of Assam (Woodford , 2005) .Today it is mainly grown in Sicily, Morocco, Crete, and Corsica, as well as Puerto Rico. The tree can grow to a height of about 3 meters. The fruit can grow to a size of about 25 cm in length, and about 4 kg in weight. The pulp of the fruit is hardly ever used. It is made into an additive for cooking. Jam can also be made from the rind. The rind is also used to make vegetable oil, which is used for perfumes.
The wild forms of citron along with those of the mandarin and pomelo Cutting propagation is often the preferred method for plant propagation. The stem cutting is suitable method for regeneration for the species. It is inexpensive, rapid and simple and does not require the special techniques as required in other vegetative methods. Other methods that are more difficult or more expensive may exist but are often not chosen. Major factors to be considered are the parent plant or stock plant and the propagating conditions along with the techniques and tools of cutting propagation (Bose ,1985 ) . This cutting may be referred to by the location from which it is taken. Rooting of the cutting is one of the possible techniques for vegetative propagation and it was observed that cuttings play important roles in the rooting of important species of some fruit species and colonal rootstocks .The stimulation of adventitious root formation in stem cuttings treated with auxins is well known ( Blazich, 1988).Exogenous plant growth regulators are one of the most commonly used methods in propagation (Polat and Kamilolu, 2007).But, there are some endogenous and exogenous factors affecting rooting of cuttings such as growth substances (Hartmann et al.,2002,Awang et al.,2009). Sabbah and et al (1991 reported that the treatment with 1000 and 3000 ppm NAA ,1000 and 3000 ppm IBA , Rooton powder (acommorchial formulation containing 2000 ppm NAA and 1000ppm IBA ),and control that stem cuttings of the different clones responded significantly in root production to NAA and IBA treatments. The concentration of 1000 NAA ppm and 3000 IBA ppm yielded the maximum rooting percentage (75% ) and produced higher numbers of roots that were longer and thicker than those of control across all selection . Daoud et al.(1995) took cuttings of eight citrus rootstock and used indole butiric acid at concentrations zero -1250-2500-5000 and 10000 ppm. Cuttings were planted in greenhouse in sandy soil . The results obtained indicated that some rootstock reopened very well to IBA treatment and the rooting percentage reached 61.1-91.7% but some other rooting percentages were only 13.3 -38.9 % and IBA at concentrations 500-2500 ppm increased significantly the number of rootstocks and the number of new shoots per rooted cutting of new rootstocks. Al-Safi (1996) showed in the study the effect of indole butiric acid at concentrations 0 ,1000 and 2000 ppm used of IBA treatment had significant effect on the rooting percentage ,and length roots/cutting .The present study deals with the use of cutting types and IBA for rooting nature and success percentage in stem cuttings of Citron. Bhusa and et al.(2001) showed in the study the effect of indole butiric acid at concentrations 4000 ppm , that the stem cuttings of Citrus treated with IBA increased booth root number and length relative to non-treated cuttings, but did not differ significantly among IBA treatments and control in the final rooting percentages . The aims of this study are to determine the rooting percentage of various types of citrus stem cuttings by using some concentrations of auxin IBA , to increase rooting success percentage in the stem cuttings of (Citrus medica Linnaeus ),Corsian cultivar, improving their vegetative growth and investigate the response of stem cuttings of citron to concentrations of auxin IBA.
Materials and Methods
The experiment was carried out during the year of 2012-2013 in the Nursery of Department of Horticulture, Faculty of Agriculture and Forestry, Duhok University, to investigate the effect of IBA at -0-500 -1000 and 2000 ppm on different types stem cutting of Citron ( Citrus medica ) Corsian cultivar. Stem cuttings (15-17 cm length ± 1) of Citron Corsian cultivar, were taken in 23 January with three types T1 (Tip cutting ),T2 (Medial cutting), T3 ( Bottom cutting).The bottom parts of cuttings were treatments different IBA doses ( 0 , 500 ,1000 and 2000 ppm ).Then the cuttings (5-6 nodes with 0.7 -0.9 cm diameter ) were planted in mixed media (1part peat moss to 2 parts sand ). One part of Peat moss was mixed thoroughly with the 2 parts of well dried sand . This prepared media was filed in perforated wood boxes under air condition in greenhouse in order to root (buried about 2/3 their height) by 7 x 10 cm rows. Cuttings were irrigated properly immediately after planting .Approximately 9 months after the beginning of the experiment, in the mid October, the Percentage success of rooting, plant diameters ,plants length and length of roots were measured. The experiment was laid out in Randomized Complete Block Design (RCBD) with three replications, using 30 cuttings for experimental units for the experiment. Statistical analysis systems were performed using SAS program (SAS , 2000).
Results and Discussion
Table (1) shows that the type of cutting have no significant effect on percentage of rooting and the highest percentage (90.0%) where found in T2(Medial type) and the least Percentage (85.0%) was in T1(Tip cutting),also show that IBA significantly affected the percentage of rooting cutting. The highest percentage of rooting cutting found in 1000 ppm IBA reach (93.33%) and the least percentage of rooting cutting was in (control) (71.11%) This is in agreement with what has been found by Kako (2011) in fig (Ficus carica ) cultivars and Kako(2013) in Mulberry .While The interaction between diameter and IBA was significantly effective on percentage of rooting cutting, the highest percentage of rooting cutting was in T2 and 1000 ppm IBA (100 %), and T1 and Zero ppm IBA gave the least percentage of rooting cutting(70.00%), might be due to the co-effect of both the type and the auxin . Table (2) indicate that the type of cutting had no significant effects on length of shoots, the highest length was in T2 (Medial cutting ) 16.29 cm, and the least length was in treatment T3(Bottom cutting ) 14.0 cm. The results in the Table (2) also indicate that IBA ppm had significantly affected on the length of shoots, the highest length of shoots were in 1000 ppm IBA (16.55 cm) and the least length was in control treatment (12.11 cm ) .The interaction between diameter and IBA was significantly affected the length of shoots, the highest length of transplants was in T2 +500 ppm IBA (23.08cm), and T2 + zero ppm IBA gave the least length (10.42cm), might be due to the co-effect of both the type of cutting and the auxin .
Results in the
The result of in the Table (3) show that the type of cutting had significant effects on diameter of shoots, the highest diameter of transplants in treatment T2 (Medial cutting )10.88 mm, and treatment T1(Tip cutting ) had the least diameter 9.20 mm, the treated cuttings with IBA also had significantly effected on the diameter of cutting ,the maximum diameter in 2000 ppm IBA (10.54 mm) while the treatment 500 ppm IBA had least value (8.99 mm) .This is in agreement with what has been found by Kako (2011) Table(3) showed increasing diameter of cuttings in interaction T2+500 ppm IBA reach 16.33 mm ,the treatment T3 + 500 ppm IBA had least value (4.91 mm) this mish be due to the co-effect of both the diameter and the auxin. Mean of IBA 9.52ab 8.99b 10.32ab 10.54a Any two numbers followed by the same letter are not significantly different at P≤0.05 level Results in the Table (4) show no significant difference between numbers of leaves of shoots with the type of cutting .On the other hand IBA had significant effects on numbers of leaves of shoots, since 1000 ppm IBA gave (12.72 leaves. shoots) while the control treatment had least value (8.36 leaves. shoots). Regarding the effect of interaction between type of cutting and IBA, the treatment of (500 ppm IBA + Medial cutting type) gave ( 16,33 leaves. shoots )which was significantly higher than other numbers of leaves actions in Table(4) ,the lowest value of number of leaves was recorded in T1(Bottom type cutting ) and 500 ppm IBA treatment (7.33 leaves . shoots) .The reason might be due to the co effect of both the type of cutting and of auxin IBA . (6) declare a significant effect of type cutting on relative chlorophyll content, the use of T3 (Bottom type cutting) gave the highest chlorophyll content in leaves estimated at (62.08 %) whereas the least content (46.41%) was recorded for (Tip type cutting ).While the use of IBA refers to there were no significant differences among the tested in chlorophyll leaves content. The interaction treatment between (Bottom type cutting) and auxin IBA (zero ppm IBA gave the higher chlorophyll content in leaves (64.70%) while the least content (42.60%) was recorded for the interaction treatment between the( Tip type cutting )and auxin IBA (Zero ppm in leaves) . This might be due to the co effect of both the type of cutting and of auxin .
CONCLUSION
The results of this investigation indicated that the medial stem cuttings of Citron ( Citrus medica ) Corsian cultivar effect significantly on shoots diameter , length of roots and leaves relative chlorophyll content , also the concentrations of auxin effect significantly form (IBA 500and 1000 ppm on rooting percentage , IBA 1000 ppm on the length shoots and number of leaves, IBA 2000 on the shoot diameter, length of roots and leaves relative chlorophyll content ) compared with control treatment .The best treatment was ( Medial type cutting in the concentrations 500 and 1000 ppm of auxin IBA in root percentage and the concentrations 500 ppm in shoots length , diameter shoots, number of leaves and length of roots and leaves relative chlorophyll content. | v3-fos |
2019-08-17T00:00:06.846Z | {
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} | s2 | Cell Viability, Acrosomal Status and DNA Integrity in Porcine Sperm Permeabilized with Streptolysin O
The aim of this study is to determine the use of streptolysin O (SLO), to open pores on the plasmatic membrane of the swine sperm, to see the effect on the viability, functional status and integrity of nuclear chromatin. 30 ejaculates were used; immediately after collection, semen was diluted in a solution of BTS (2:1, v/v preheated to 35ºC). Two aliquots of semen samples were assigned to each group, of which: Group 1. 0.3 IU/ml SLO, Group 2. 0.6 IU/ml, Group 3 1.2 IU/ml, and Group 4 Control (PBS). All groups were incubated for 5 minutes at 37ºC, in SLO; after this period, the samples were incubated in 5% fetal bovine serum (FBS) for 15 minutes at 37ºC. Each treatment were evaluated before and after incubation with FBS (to close the pores), in duplicate. Sperm viability, acrosome status and integrity of the nuclear chromatin were evaluated with eosin nigrosine (NE), chlortetracycline (CTC) and acridine orange (AO) stains, respectively. The data obtained was analyzed by comparing proportions in each treatment using non-parametric module of STATISTICA V10.0 program. The Kruskal Wallis H test was used for the analysis. In assessing sperm viability each of the treatments were compared in between, and the resulting differences were also compared with the control group (iv). There was no difference in the proportion of viable sperm, using the concentration of 0.6 IU/ml, before and after sealing of pores (83.15±9.10 vs 91.52±4.83) (p>0.05). And mathematical trend indicates that, after sealing the pores, a higher sperm proportion were viable in both treatments. However, no statistical differences were found (P>0.05). Also no changes were found in patterns of acrosomal status and DNA integrity, suggesting that the SLO executes its permeabilizing effect at the level of sperm membrane.
INTRODUCTION
The Streptolysin O (SLO) is a toxin produced by Streptococcus pyogenes, it's a protein with a molecular weight of 60 kDa; belonging to group A of cytolytic toxins β-haemolytica, known as oxygen-labile and produced by a gram positive bacteria. Besides its pathogenic interest, SLO has been reported as an ideal tool for cell pores forming [1]. The SLO is toxic for eukaryotic cells, because of its affinity for cholesterol, but in controlled doses, has proven to be useful for live cell permeabilization [2,3]. In the case of sperm, the SLO has been used to study the mechanisms that regulate some physiological processes of the sperm as capacitation, the acrosome reaction and motility [4,5]. The SLO binds to cholesterol in the cytoplasmic membranes of animal cells [6], and subsequently participates in reversible pore formation, in the plasma membrane [6,7,8]. The poration with SLO has been used extensively to introduce external molecules, such as proteins, DNA, and RNA, into the intracellular compartment [9,10,11]. Fawcett et al. [3] conducted a research with SLO (0.2 IU / ml) to form pores in the cells membrane of mouse myocyte and introduce fluorescent macromolecules [3]. Other work with mouse sperm, shown that these cells remain viable in SLO medium with concentrations up to 0.6 IU / ml for short periods of time, showing greater viability than cells treated with lower concentrations and a longer exposure [4]. Other studies mention the use of SLO as a former of pores in the membrane, to introduce proteins in different cell types of the immune system; Moreover, after the sealing of pores, such cells showed similar viability rates to those of control cells that were not treated with SLO [9]. In a more recent work, Bo-Woong et al. [12] used SLO a dose of 5 IU/ ml as a medium for the introduction of exogenous DNA into mouse sperm and showed that there was an increase in the rate of transgenic embryo production and later litters than with intracytoplasmic sperm injection. The sperm cell is a complex and a good biological model to several studies; the opening of the pores can modify their structure and, therefore, serve as a vehicle for cryopreservation studies and biotechnology. The aim of this study is to determine the effect of streptolysin on cell viability, functional status and integrity of nuclear chromatin of swine sperm.
Reagents
Unless otherwise stated, all of the chemicals used in the experiments were analytical grade and were purchased from Sigma-Aldrich Co. (
Biological Material
The work took place from February to July 2014. Thirty ejaculates, which were analyzed before use, to check that met inclusion criteria. Procedures involving animals were approved by the Institutional Animal Care and Use Committee at the University of Veracruz, Mexico. Two-four years old clinically healthy boars without pathologies associated to the male reproductive tract and with a proven history of fertility after conventional AI with fresh extended semen were selected. In addition, boars followed a routine schedule of vaccination, de-worming and vitamins (mainly ADE) appropriate to the species. Boars were housed in individual pens, fed twice daily with 1.5 kg of concentrate that contained 15% of crude protein and 1.2 Mcal of metabolizable energy.
Collection
Semen was collected by the gloved hand technique. During the collection, semen was filtered through sterile gauze and only the spermrich fraction was used. Semen samples with 70% progressive motility and 80% normal sperm morphology and intact acrosome status (assessed by phase contrast microscopy, the sperm samples were fixed in 2% buffered glutaraldehyde).
Motility evaluation
To evaluate mass motility, a semen drop was placed in a glass slide at 37ºC, and was observed under microscope (Olympus CX41) 100 x magnification, for one minute. The estimate is based on intensity of waves, sorting have values 0-5; samples with less than 3 degrees, were not considered for this study. For individual motility analysis, a drop of diluted semen in saline, was placed on a glass slide at 37ºC, covered with a microscope was observed under microscope at 400 x magnification; was evaluated for progressively motile spermatozoa (scale 0-100%) [13].
Morphology
The morphology was evaluated with the technique of chinese ink, which is prepared by placing a volume 1: 8 v/v semen-ink and it was observed with a microscope at 400 x counting 200 cells per replicate [14].
Dilution
Immediately after the collection, the semen was diluted in a BTS solution (2: 1, v / v) preheated to 35ºC. Subsequently, the semen was transported in a container between 20-22ºC, to the Laboratory of Biology of Animal Reproduction FMVZ UV.
Sample preparation and groups description
Two aliquots with semen samples were assigned to each group with: Group 1. 0.3 IU/ml SLO, Group 2. 0.6 IU/ml, Group 3 1.2 IU/ml, and Group 4 Control (FBS). All samples were incubated for 5 minutes at 37ºC, in SLO; after this period, the samples were incubated in fetal bovine serum (FBS) at 5% for 15 minutes at 37ºC.
Cell permeabilization
Cells were permeabilized, using the protocol described by Fawcett et al. [3]. For the membrane permeabilization, three treatments were used with Streptolysin O (SLO) lyophilized Sigma Aldrich brand. The lyophilized streptolysin was dissolved in cold distilled water to 60 IU / ml and activated with 4 mM DTT at for 2 h at 37ºC and aliquots were stored at -20ºC until use. 100 ul of semen was added to each treatment and incubated for five minutes at 37ºC, washed twice by centrifugation at 1200 rpm for 3 minutes, the supernatant removed and resuspended in 1 ml PBS. Concentrations for the treatment with SLO were 0.3, 0.6 and 1.2 IU / ml.
Sealed method
For closing the pores, Fetal Bovine Serum (FBS) at 5% in PBS was used, to restore the integrity of cell membrane. The incubation was performed at 37ºC for 15 minutes [3].
Cell evaluation
Each experimental group was evaluated before and after incubation with FBS, this was done in duplicate. The sperm viability was evaluated by eosin nigrosine stain (NE), the acrosomal status of sperm by chlortetracycline technique (CTC), and the integrity of nuclear chromatin was evaluated by fluorescence technique with acridine orange (NA).
Sperm viability
The assessment of sperm viability was performed with eosin nigrosine staining (EN) (HYSEL ® ). Using a 1:8 dilution of the semen sample and dye, the sample mixture was incubated for 5 minutes at 37ºC and a smear was made on a slide. The slides were observed under phase contrast microscope at 400x, several fields were reviewed to count 200 cells, which showed the following pattern of coloration. [14,15].
Images of Eosin Nigrosine staining (EN)
A
Acrosomal status
To determine the status of capacitation and acrosome reaction, It was used the technique described in 1993 by Das Gupta et al, which must presented the following fluorescence patterns: [16]. i) Uniform fluorescence over the whole head: Un-Capacitated and -acrosome-reacted. ii) Fluorescence-free band in the postacrosomal region: Capacitated. iii) Dull fluorescence over the entire head: capacitated and acrosome-reacted.
DNA integrity assessement
DNA integrity was assessed by fluorescence technique with acridine orange (OA). It was used as described by Tejada (1984). Covered the slide and immediately observed under the fluorescence microscope (Leica DM 020-518500 / LS) with filter blue excitation 405-455 nm, 400x, reviewed several fields to count 200 cells which must present the following fluorescence patterns: Green sperm = intact DNA. Orange sperm or red Sperm = denatured DNA [17].
Statistical analysis
The data obtained were analyzed using the nonparametric module V10.0 STATISTICA program; the parameters evaluated include: the percentage of the acrosomal status CTC technique, the percentage of plasma membrane integrity by eosin nigrosin technique and the percentage of the integrity of the nuclear chromatin and acridine orange technique. Fig. 1 shows the percentage of live sperm in each of the treatments and there was no statistically significant differences (P>0.05), among the groups. The percentage of live spermatozoa increased in all treatments after incubation with 5% FBS; however this increase was not significant (P>0.05). Moreover, as shown in Table 1, the value of the lowest sperm viability was 83.15±9.10 with the SLO 0.6 IU / ml treatment, before incubation with FBS, while the highest value (91.52±4.83) was found in the same concentration of SLO but after being incubated with SFB to seal the pores; the differences were not significant (P>0.05).
Acrosomal Status
A not significant (P>0.05) decrease in the percentage of un-capacitated and -acrosome reacted spermatozoa only in 1.2 IU / ml of SLO treatment group in comparison to control before and after incubation with FBS was observed. The differences among the groups are shown in Fig. 2. The highest value (83.68%) was for control treatment before incubation with FBS and the lowest (79.42%) was in the 1.2 IU / ml of SLO treatment group, after sealing of pores; However, these results are not significant (P<0.05) for any of the treatments due to the standard deviations are > 4.7% (Table 2). Fig. 3 shows the effect of treatment on the integrity of the nuclear chromatin of spermatozoa. There was no statistically significant difference (P<0.05) between treatments before or after incubation with FBS. The percentages of cells with chromatin integrity remained > 98% in all treatments (Table 3) before and after sealing of pores. Although standard deviations were lower than in other techniques, but still no significant (P<0.05) difference was observed between control and treatment groups.
DISCUSION
In this study the effect of incubation with streptolysin O on sperm viability, indicated that there is no difference between treatments before and after sealing of the pores. The observed decrease in the percentage of viable sperm incubated with SLO for the permeabilization (Fig. 1), followed by an increase of the percentage of viable sperm after incubation with FBS for sealing pores, indicates SLO acts on the sperm plasma membrane, this allows the entry of eosin dye into the cell, similar to that described by Tartakoff [18], which states that certain dyes waterproof such as trypan blue and eosin, can be able to cross the plasma membrane of cells treated with toxins pores forming as the α-toxin, and the digitionina SLO and that prolonged exposure to such dyes causes staining in intact cells; however, in this study no statistical differences between treatments were found before and after the sealing of pores, contrary to what was reported by Johnson et al. [4], that using the same technique at a dose of 0.6 SLO IU / ml achieved 80% permeabilization in mouse sperm. Moreover, Michaut et al. [21] and Yunes et al. [19,20] conducted a study in human sperm and achieved to permeabilize 100% cells. These differences can be associated to the cholesterol found in the membrane, which differs significantly between species, being the lowest in pigs than in mouse or human and as mentioned above, the SLO is a toxin that acts by binding to cholesterol in plasma membranes, which have great concentration in these species. Fawcett et al. [3] found that the temperature, the dose and the period of exposure to the toxin, are factors that are associated with the effect of this on the membranes. Worked with mouse myocytes at a dose of 0.2 IU / ml at 37ºC for 5 min, and a higher percentage of viable cells when higher exposure periods and higher doses of the toxin used was found; In our study the incubation temperature was 37ºC and the time of 5 minutes, and it was found that the viability, status of acrosome integrity and integrity of nuclear chromatin, were maintained in high percentages in all treatments, similar to reported by these authors. To determine the effect of SLO on sperm physiology technique CTC, which indicates the state of capacitation and acrosome reaction was used to perform the study. No statistically significant differences between treatments before and after incubation with FBS for sealing; however, a decreasing trend is observed in the percentage of un-capacitated and acrosome reacted. This decrease can be attributed to the contact of the sperm and the seminal plasma and begins to develop such processes naturally [22].
In this study, porcine sperm permeabilization for 5 minutes with SLO 0.3, 0.6 and 1.2 IU / ml was found that the percentages of cells without capacitation and without acrosome reaction were of 80.34±7.8, 83.67±5.6 and 81.71±8.18 respectively, similar to those reported by Johnson et al. [4]; to permeabilize SLO mouse spermatozoa with 0.6 IU / ml for 5 minutes found that most of the cells remained with intact acrosome (75%) and that increasing the exposure time of 15 minutes the percentage decreased (44%). These results indicate that permeabilization with SLO performed in the plasma membrane and not in acrosomal membrane.
Until now there are no reports about the effect of the use upon the integrity of the nuclear chromatin of sperm cells SLO as permeabilizer; However, many authors report that individuals with spermatozoa exihibiting > 50% full chromatin are considered normally fertile. In our study, > 98% of spermatozoa were with intact chromatin in all treatments before and after incubation with FBS, which indicates that the SLO has no negative effect on the structural integrity of nuclear chromatin and permeabilized and sealed cells are able to fertilize.
CONCLUSION
In this work it was found that the SLO acts on the plasma membrane while the use of FBS as pore sealant does not affect sperm physiology. Finally no negative effect was observed in the use of the SLO in the structural integrity of DNA. | v3-fos |
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} | s2 | APA-style human milk fat analogue from silkworm pupae oil: Enzymatic production and improving storage stability using alkyl caffeates
Silkworm pupae oil derived from reeling waste is a rich source of α-linolenic acid (ALA), which has multipal applications. ALAs were added in sn-1, 3 positions in a triacylglycerol (TAG) to produce an APA-human milk fat analogues (APA-HMFAs, A: α-linolenic acid, P: palmitic acid). The optimum condition is that tripalmitin to free fatty acids of 1:12 (mole ratio) at 65 °C for 48 h using lipase Lipozyme RM IM. Results show that, the major TAG species that comprised APA-HMFAs were rich in ALA and palmitic acid, which contained 64.52% total unsaturated fatty acids (UFAs) and 97.05% PA at the sn-2 position. The melting point of APA was −27.5 °C which is much lower than tripalmitin (40.5 °C) indicating more plastic character. In addition, the practical application of alkyl caffeates as liposoluble antioxidants in APA was developed. Alkyl caffeate showed a superior IC50 (1.25–1.66 μg/mL) compared to butyl hydroxy anisd (1.67 μg/mL) and L-ascorbic acid-6-palmitate (L-AP) (1.87 μg/mL) in DPPH analysis. The addition of ethyl caffeate to oil achieved a higher UFAs content (73.58%) at high temperatures. Overall, APA was obtained from silkworm pupae oil successfully, and the addition of caffeates extended storage ranges for APA-HMFAs.
Results and Discussion
Fatty acid profiles from desilked silkworm pupae oil. Based on GC analysis, all major components in the samples were separated from each other and identified. Desilked silkworm pupae oil was characterized as the initial material. Table 1 shows the FA compositions of the saponified silkworm pupae oil that was used to estimate the average molecular weight of the desilked silkworm pupae oil FFA (MW = 275.95 g/mol). The major FAs contents in silkworm pupae oil were ALA (34.73 ± 1.74%), oleic acid (31.16 ± 1.56%), palmitic acid (19.50 ± 0.97%), linoleic acid (7.55 ± 0.38%) and stearic acid (7.05 ± 0.35%). The saturated fatty acid content was 26.55%. Compared with other materials like olive oil and fish oil, silkworm pupae oil has more ALA which can serve as the DHA and EPA precursor in human body. In addition, the cost of materials containing DHA and EPA are much higher than silkworm oil containing ALA. The aim of this research was to produce structured TAGs with immobilized lipases by modifying the palmitin TAG, increase the UFAs, especially ALA esterified at the sn-1, 3 positions, and to physically and chemically characterize the resulting structured TAGs.
However, saturated fatty acids were not beneficial to the reaction. A rapid, simple and inexpensive procedure recently developed for urea fractionation was applied to isolate polyunsaturated FFA from the FFA extract derived from desilked silkworm pupae oil. This process removed saturated and monounsaturated FFA 22,23 . Table 1 shows the comparison of FFA content before and after the urea inclusion method from crude silkworm pupae oil, after the urea-based fractionation. These data were used to estimate the average molecular weight of the FFA (MW = 279.04 g/mol) after the process. The content of palmitic acid, stearic acid and oleic acid were decreased to 0.77%, 0% and 13.96%, respectively. The linoleic acid and ALA were increased to 12.10% and 73.16%. The total content UFAs reached to 99.22% after the urea-based fractionation, which was a favorable acyl donor for our further reactions.
Desilked silkworm pupae oil is a good source of ALA, but selectively enriching ALA is a technical challenge due to the presence of multiple analogues such as linoleic acid and oleic acid. A two-stage combinative inclusion process by β-cyclodextrin (β-CD) and urea was compared in Table 1, reported by Wang et al. 11 . Compared with a two-stage combinative inclusion process, a higher ALA content (73.16%) from crude oil of silkworm pupae was obtained by the urea inclusion method. Urea molecules readily form solid-phase complexes with saturated FFA, however, the characteristics in the hydrocarbon chains, such as double bonds, branching, or bulky constituents, greatly reduce the propensity for urea complex (UC) formation. PUFAs (e.g. C18:3) have been isolated through removal of saturated (e.g. C16:0) and monounsaturated FFA (e.g. C18:1) by UC formation.
Compared with other oils and fats, such as soybean oil (8.77% of ALA), the silkworm pupae is a superior source for ALA production. The FA content of these animal oils and fats is: lauric acid (0.10%), myristic acid (1.00%), PA (23.90%), stearic acid (15.40%), OA (41.60%), linoleic acid (13.20%) and ALA (0.40%). Despite this, ALA is the first precursor in the metabolic pathway leading to EPA by desaturases and elongases 24 , and is an essential fatty acid for humans that just could obtain from food. Silkworm-based sources of n-3 PUFAs are easy to access, highly efficient, green and safe, which make them more advantageous and promising as a source of n-3 PUFAs. Thus, silkworm-containing n-3 PUFAs could be an ideal source for the supply of these fatty acids.
Interesterification reaction. Figure 2 shows the effect of different conditions on the incorporation of UFAs during the acidolysis of PPP with UFFA from silkworm pupae oil. Enzymatic interesterification is often used to produce structured TAGs with the improved functionality by incorporating fresh FAs into the TAG. The sn-1, 3 specific lipases, Lipozyme RM IM and Lipozyme TL IM, were used to screen for optimum reaction conditions, and the results were showed in Fig. 2A. The lipase has more specific catalytic activity than other enzymes like Novozym 435 which usually used for interesterification. With the lipase, the UFAs were combined at sn-1, 3 more precisely. This figure showed that the initial rate of incorporation of UFAs in PPP was relatively higher with both lipases tested, but the maximum incorporation rates were obtained with Lipozyme RM IM (41.52%). Figure 2B shows the PA content at the sn-2 position of APA. This work aimed to incorporate UFAs at sn-1, 3 positions and maintain high PA incorporation at the sn-2 position. It was observed that the selectivity of Lipozyme RM IM was remarkably higher than Lipozyme TL IM, This is in consistence with previous reports 25 . Therefore, Lipozyme RM IM was selected for the catalyst in our investigation. Figure 2C shows the effect of reaction temperature on the incorporation of UFAs during acidolysis of the PPP with UFFA. When the reaction temperature was below 60 °C, the total UFAs content increased as the temperature increased at the same substrate concentration. However, when the temperature increased to 80 °C, the total UFAs content significantly decreased. In general, the temperature of an enzymatic reaction is chosen according to the activity and stability of the enzyme, but the fact that PPP has a melting point of 66 °C must be also considered in this reaction. The substrates rich in PPP must remain in liquid form at the chosen temperature chosen, but the addition of organic solvent can reduce this temperature. Theoretically, an increase of reaction temperature results in an acceleration of the reaction based on the Arrhenius law. However, lipase will be deactivated at high temperature resulting to lower reaction rate 26 . Also, as shown in Fig. 2D, higher temperatures increases the rate of acyl migration, which may reduce the sn-1, 3 specificity of Lipozyme RM IM. Therefore, in the n-hexane system, 40, 50, 60, 70 and 80 °C were chosen as the experimental temperature, and the result shows 60 °C as the best reaction temperature. Figure 2E shows the effect of the substrate molar ratio on the incorporation of UFAs during the acidolysis of PPP with UFFA. When the substrate molar ratio of UFFA to PPP was set at 12:1, the content of ALA was 61.85%, which was significantly different from the other reaction conditions. However, when the substrate molar ratio of UFFA to PPP rose to 15:1, the total UFAs content was essentially unchanged. Provided that the lipase shows the same selectivity toward all FAs and also the same regiospecificity toward sn-1 and sn-3 positions, the theoretical equilibrium or maximum incorporation of all acyl donors can be calculated using the following equation, if no side reactions occur: Where Inc max is the equilibrium acyl incorporation and S r is the substrate mole ratio of FFA to PPP. In this study, the effect of acyl donor substrate concentration on the incorporation of UFAs was investigated. Theoretically, when the S r is 3, 5 and 7, Inc max is 39.92, 47.62 and 51.88, respectively. However, when S r increased from 3 to 7, ALA incorporation also increased (Fig. 2E). The results are also consistent with Eq. (1) with respect to maximum incorporation. Under the conditions of high-substrate ratios, substrate inhibition, enzyme saturation or partial loss of lipase activity may occur. High levels of FFA could produce high levels of free or ionized carboxylic acid groups, which would acidify the micro aqueous phase surrounding the lipase or cause adsorptions of water from the interface. Also, higher FFA may increase the rate of acyl migration (Fig. 2F). However, in this interesterification reaction, more UFAs were added into the sn-2 position rather than being introduced into the sn-1, 3 positiondue to the acyl migration effect. Acyl migration is complex and could not be completely avoided, but it can be minimized 27 . Compared to solvent-free systems, solvent systems are much more stable, because of the increased solubility and mobility of dissolved substrate 28 . A high rate of incorporation and low rate of acyl migration for Lipozyme RM IM were observed under these reaction conditions. Therefore, the possible reason for the decrease in incorporation of ALA may be ascribed to inhibition of Lipozyme RM IM at higher substrate (FFA) concentrations, and therefore the substrate molar ratio of UFFA to PPP at 12:1 was used for future investigation. Figure 2G shows the effect of reaction time on the incorporation of UFAs during the acidolysis of PPP with UFFA. The UFAs content increased with the reaction time. In the first 8 h, reaction rate is relatively high. However, the reaction rate began to decrease during the 8 th h to 36 th h. From 36 h to 48 h, the rate was practically constant. The incorporation of UFAs increased slowly until 48 h with highest content of UFAs of 58.78%. In addition, high PA content of 97.07% in the sn-2 position was decreased all the time in the transesterification reaction (Fig. 2H).
Overall, two widely used immobilized sn-1, 3 regiospecific lipases Lipozyme RM IM and Lipozyme TL IM perform distinctly in different solvent systems. The best results were achieved with 10% (total weight) Lipozyme RM IM in n-hexane with a substrate molar ratio of 12:1 (UFAs: PPP) at 60 °C. After a 48 h reaction, 97.07% PA at the sn-2 position and 64.52% UFAs in the APA-product were obtained. In addition, the total content of ALA in the product reached 48.59%, which contains with 70.93% sn-1, 3 APA-style HMFA. Most of the ALA (96.22%) was esterified in the sn-1, 3 positions, which could be used as nutrition intensifying agent.
Compared with the previous work in our lab (Table 2), the APA-style HMFA provided essential fatty acids in the sn-1, 3 positions of the TAG and contained high content of PA in the sn-2 position 29 . The results reveals that APA-style HMFA has the most PA at sn-2 and ALA at sn-1, 3 positions, which shows higher reaction efficiency compared to others like algae oil SLs and olive oil SLs. Several studies have pointed to fat digestion and absorption of dietary TAG to explain the efficient absorption of PA when located at the sn-2 position 30, 31 . It has been suggested that pancreatic lipase attack the TAGs in the intestinal lumen with a high degree of positional specificity. Lipolysis occurs predominantly at the sn-1 and sn-3 positions, and has showed to transform a 2-monoglyceride (2-MG) and two FFAs. The 2-MGs formed efficiently, diffused into the enterocytes from micelles and well absorbed 32 . This is the major reason why HMF is well absorbed, and provides 50% of the dietary energy requirements for the infant. Other sources of similar FA composition (e.g. plant oil, lard) contained the saturated FA esterified at the sn-1, 3 positions. Consequently, the FA composition and the distribution of dietary TAG have been the targets in infant formula studies.
Model fitting.
Response surface applications is an effective method to analyze the effect of many factors at the same time. Figure 3 shows the contour plots comparing two parameters for UFAs incorporation. The effects of: time (44, 48 and 52 h) (Fig. 3A), reaction temperature (50, 60 and 70 °C) (Fig. 3B) and substrate mole ratio of UFFA fraction to PPP (9:1, 12:1 and 15:1) (Fig. 3C) on UFAs content (Y) were considered in the present study. Table 3 shows the observed responses (Y) from each of the 17 experimental conditions generated by response surface methodology (RSM). Multiple linear regression (MLR) was used to fit a linear equation for a response using reaction factors. To provide the best fit to the model, a backward-elimination procedure was applied to remove variables with higher P-values (P > 0.05) that not make a significant contribution to a prediction. Table 4 displays the analysis of variance (ANOVA) of response surface for the model of UFAs content, showed that the model is significant (P value < 0.05); there is only a 3.06% chance that a "Model F-Value" this large could occur due to noise, and the lack-of-fit was not significant (P = 0.117). The R 2 values (the fraction of variation of the response explained by the model) and R 2 Adj (the fraction of variation of the response that can be predicted by the model) for measuring the goodness of the fit were 0.852 and 0.661, respectively 33 . The values were closed to 1 for both R 2 and R 2 Adj indicated a good model with excellent predictive power. Thus, the statistical values suggest that the response surface models for ALA content were adequate and well fitted. Regression coefficients and significance Scientific RepoRts | 5:17909 | DOI: 10.1038/srep17909 (P) values of the generated models for ALA acid incorporation are given in Table 5, and the predictive response surface equations are: According to the predictive response surface equations, the optimum reaction conditions are as follows: 48 h reaction time, 60 °C reaction temperature and a substrate mole ratio of UFFA to PPP of 12:1. The predicted UFAs incorporation was 64.52%, which was consistent with experimental verification. Under optimal reaction conditions, all the fatty acids were identified by GC analysis. Table 2 shows the FA composition of APA-style structured lipids from silkworm pupae oil under the optimum conditions, with a comparison of this product with other structured lipids (SLs). The major FAs in APA-SLs were ALA (48.59%), palmitic acid (35.48%), linoleic acid (8.94%) and oleic acid (6.99%). Compared with our previous study 29 , silkworm pupae oil SLs (SPOSLs), with low PA and high ALA, oleic acid (OA) and linoleic acid (LA) contents were synthesized from SPO using a solvent-free system catalyzed by Lipozyme RM IM. The APA-SLs maintained higher ALA content than SPOSLs. For the sn-2 FAs, 97.05% of PA content in the sn-2 position had been obtained. Figure 4 also compared the fatty acid content of FFA from silkworm pupae oil (Silkworm pupae oil FFA), purified UFFA after the urea inclusion method from crude silkworm pupae oil (Purified UFFA), total FA content of APA-style HMFA from silkworm pupae oil (Total FA content of APA) and sn-2 fatty acid content of APA-style HMFA from silkworm pupae oil (sn-2 FA content of APA). These structured TAGs are rich in sn-2 PA and containing high ALA at sn-1, 3 positions, which is the principal TAG of HMF, and these APA-style TAGs can be used to achieve nutritional fortification for infants.
Reuse of lipase.
The reusability of the Lipozyme RM IM was studied with the optimal condition. Figure 5 shows the enzyme can be used for 16 times (32 d) with the activity of that is beyond 80%. The results show that the catalytic stability of the Lipozyme RM IM is so high that can efficiently decrease the cost of reaction.
Melting and crystallization profiles. Figures 6A,B show the melting and crystallization profiles of product APA-style TAGs and PPP. The melting and crystallization profiles of the fats were closely correlated with their chemical compositions. The melting curves are important for investigating physical state of the fat within the human body. Only fat with a melting point below the physiological temperature (36.6-37.3 °C) 34 , can be quickly emulsified and absorbed. The melting and crystallization curves of APA-style TAGs were lower than the physiological temperature. Compared with PPP (40.09 °C), the majority of the components in the structured TAGs melted in the middle of the melting point (− 26.45 °C), which may indicate that LC-PUFAs was dispersed in the structured TAGs products. In addition, the crystallization properties of the fat are of great importance for application. High initial milk fat crystallization temperatures were correlated with high final melting temperatures and vice versa. Reaction products showed the highest starting crystallization temperatures of −12.12 °C, with a corresponding highest final melting temperature of −20.12 °C of these APA-style TAGs. The same as the previous discussion, fat with a lower melting point than the physiological temperature (36.6-37.3 °C) can be emulsified and absorbed quickly. After the reaction, the thermograms peaks of structured TAGs shifted to the right and broadened indicating the improved plasticity and storage range of structured TAGs. Figure 7A shows the half-inhibitory concentration (IC 50 ) for the radical-scavenging activity of CAPE and alkyl caffeates with different chain lengths. The traditional antioxidants for oil and fat, BHA and L-AP, were used for comparison. The inhibition ratio of alkyl caffeates for DPPH radical-scavenging was as follows: CAPE > MC > IpC > IaC > EC > BuC > HexC > AC > PC > BHA > L-AP. This result indicated that the alkyl caffeates were potent antioxidants because a higher DPPH scavenging ability was obtained, compared to commercial BHA and L-AP fat-soluble antioxidants. The IC 50 values for CAPE and MC were 1.25 and 1.29 μg/ml, respectively, while the IC 50 values of BHA and L-AP were 1.66 and 1.87 μg/ml, respectively. Thus, CAPE and MC were more efficient antioxidants than BHA and L-AP. In addition, IpC, IaC, EC, BuC, HexC, AC and PC showed more significant antioxidant activity than BHA and L-AP (P < 0.05). Among these antioxidants, CAPE was the most efficient in eliminating free radicals in this product, which agreed with earlier studies. Therefore, the alkyl caffeates esterified from CA and the alkyl alcohols, especially CAPE, can prevent structured TAGs oxidation during the processing and storage period. In a practical application, alkyl caffeates esterificated from CA and the alkyl alcohols could completely replace BHA and L-AP, especially in infant formula. APA-style TAGs contain a large quantity of UFAs (64.52%), which can effectively prevent a variety of diseases. Unfortunately, these UFAs are liable to be oxidized. Increasing attention has been attracted to stabilize edible oil, especially for its storage, to improve the quality and safety of the oil. The resistance to oxidants is assessed by several factors: antioxidant type, antioxidant amount, storage conditions and oil fatty acid profile, especially the UFAs content 35 . The oxidative products degraded the quality of oil, shorten its storage time and threaten human health. Higher antioxidant activity is necessary within structured TAG products to inhibit oxidation. This is essential in maintaining product safety, especially for infants. Therefore, more attention should be focused on the development of natural sources of liposoluble antioxidants. In the present study, alkyl caffeates were investigated as antioxidants to demonstrate their practical value with our APA-style HMFA. Figure 7B shows the UFAs (C18:1, C18:2 and C18:3) content of APA-STAGs with the addition of alkyl caffeates, BHA and L-AP or no antioxidants addition (BC: blank control) at 180 °C. Overall, the content of UFAs increased with the addition of alkyl caffeates, BHA and L-AP. For the total content of unsaturated fatty acids in APA-style TAGs (Fig. 7C), the highest effective antioxidant was AC at 180 °C for 4.5 h. However, the antioxidant effect of some antioxidants, such as BuC, IpC, IaC and CAPE was not significantly different from AC (P < 0.05), which was shown in Table 6, suggesting that the natural sources of antioxidants could improve the storage ability of STAGs.
Antioxidant activity of alkyl caffeates.
In conclusion, APA-style HMFA was successfully produced by silkworm pupae oil. The structured TAGs with 94.31% of UFAs at the sn-1, 3 positions and the palmitic acid of 97.05% were obtained via the interestrification. The result shows the higher reaction efficiency that other similar work like the olive oil SLs recently cannot reach 12 . The ALA content in the modified oil reached 48.59%. Characterization showed that APA-style HMFA with improved plasticity and storage ranges may be used for nutritional enhancement. In addition, alkyl caffeates demonstrated obvious antioxidant activity on APA-style HMFA. Despite the advancements of our technique, the cost of enzymes and the time-consuming process limited the industrial utilization of this process. Future work will focus on the increase of the enzymatic efficiency and reduce the cost of this process to assure an economic industrial application.
Methods
Preparation of free fatty acids (FFAs) from the silkworm pupae. The desilked silkworm pupae were dried at 60 °C in a DHG-9240A drying oven (Shanghai Yiheng Scientific instruments, Shanghai, China), powered by an herb disintegrator (Qinzhou Sanyang Package Equipment, Qinzhou, China) and then sieved (60 meshes). The oil was extracted from the silkworm pupae powder using ultrasonic-assisted (60 watts) solvent extraction (n-hexane). The offscourings were removed by filtration, and the oil-n-hexane mixture was subjected to vacuum rotatory evaporation until no n-hexane remained. The crude desilked silkworm pupae oil (500 g) was then saponified by refluxing it for 6 h at 65 °C under a nitrogen atmosphere using a mixture of NaOH (8.0 g) and 95% v/v aqueous ethanol (2 L). This was performed until the whole mixture became a clear, homogeneous solution. The remaining ethanol was recovered under reduced pressure. To dissolve the saponified mixture, distilled water (500 mL) was added and the aqueous layer, containing saponified material, was acidified (pH 3-4) with 10% HCl. The solution was shaken and held for 1 h, and the water layer was removed. Petroleum ether (boiling point 60-90 °C, 500 mL) was added to extract the oil layer. Distilled water (500 mL, 3 times) was added to wash the oil layer until it was neutral. The petroleum ether layer, containing free fatty acids, was then dried over anhydrous magnesium sulfate and the solvent was removed at 50 °C to recover crude FFAs (300 g). Thus, from FFAs preparation to APA-style HMFA production via enzymatic transesterfication using desilked silkworm pupae as feedstock, which whole process was shown in Fig. 8.
Enrichment of ALA from crude FFAs. The separation of ALA from FFAs was carried out by urea complexation 11,36 . Crude FFAs extracted from the desilked silkworm pupae oil were dissolved in ethanol, and then slowly added to a separatory funnel over 10-15 min. The rest of the oil was washed by 95% of ethanol. The ratio of saturated urea/EtOH solution to FFAs was 2:1 (v/v). The mixture was stirred at 4 °C for 2 h until the solution was clear under a slow flow of nitrogen. The mixture was transferred into conical flasks when the reaction was completed.
Interesterification Reaction. The mixtures of reactants (PPP and FFAs) that were prepared at different substrate mass ratios were placed in a 50 mL conical flask and treated with enzymes in a 220 rpm orbital shaking To remove FFAs in the final products, 50 mL of hexane and 10 drops of phenolphthalein solution were added and the final products were titrated with 0.5 M KOH in 95% ethanol until a pink color appeared. The reaction mixture was washed several times with water until the pink color disappeared. The saponated FFAs were recovered and acidified again, to enrich for the ALA which had not participated in prior reaction. The organic layer was passed through an anhydrous sodium sulfate column to remove moisture, and the solvent was completely evaporated under nitrogen at 40 °C.
Experimental design for response surface method. A three-factor, rotatable three level BBD was employed to generate factor combinations by using Design Expert 8.05 b (Stat-Ease) software. The three factors chosen were substrate molar ratio (Sr, UFFA/PPP, 9-15 mol/mol), temperature (T, °C, 50-70 °C), and reaction time (t, hours, 44-52 h). A total number of 17 runs were generated by the software. The independent variables and experimental design are shown in Table 3. Experiments were run randomly, and duplicate reactions were carried out at all design points. Technologies gas chromatograph equipped with a Hewlett Packard 3396 Series II integrator and 7673 controller, a flame ionization detector, QA/QC nds cerity system and split injector (Agilent Technologies Inc., Santa Clara, CA). The gas chromatographic conditions were as described by Wang et al. but were revised 39 . The initial oven temperature was 200 °C, and held for 1 min, and then the temperature was increased to 220 °C at a rate of 1.5 °C/ min. The column head pressure was 280 kPa. The injector was set to 250 °C and the detector to 280 °C. The split ratio was 50:1; the carrier gas was nitrogen with a flow rate of 1mL/min. FAs were identified by comparing their retention times with FAME standards. GC-MS analysis was performed using an Agilent 6890 gas chromatograph with a 5973 MS detector equipped with 60 m × 0.25-mm i.d. 0.25-μ m/MS capillary column (DB-5). The following temperature program was used: the injector was set at 250 °C, the oven was initially set at 200 °C, held for 1 min and then heated to 230 °C (1.5 °C/ min, then held for 10 min). The characterization and identification of FAMEs was completed in scan mode 40 . The selected ion-monitoring mode was from 35 to 320 m/z.
Melting and crystallization profile. Melting and crystallization profiles were determined for structured
TAGs and PPP using a differential scanning calorimeter (DSC model DSC7, Perkin-Elmer Co., Norwalk, CT). The method of Lee and others was used 41 , with minor modifications: indium and n-decane were used as standards. Samples were weighed in aluminum pans and ranged from 8 to 12 mg. To destroy any previous crystalline structures, samples were heated from 25 to 80 °C at 50 °C /min and held for 10 min. For crystallization profiles samples were then cooled from 80 to 55 °C at 10 °C/min and held for 30 min. To get melting profiles the samples were then heated from 55 to 80 °C at 5 °C/min. Dry ice and acetone were used as the coolants. The thermograms were analyzed using DSC (Pyris software, Perkin-Elmer, Shelton, CT). DPPH Assay. The DPPH-scavenging assay was performed to elucidate the radical-scavenging properties of CA and its alkyl derivatives 42 . An ethanol solution of DPPH is purple and has an absorption maximum at 517 nm 43 . In the presence of antioxidants, DPPH captures an electron causing the molecule to lose its absorbance at 517 nm 44 . The alkyl caffeates were dissolved using ethanol, and different concentrations were mixed with a methanolic solution of DPPH. The mixtures were then incubated for 30 min in the dark at room temperature. The 96 well micro-plates were read in a SpectraMax i3 Multi-mode detection platform (Molecular Devices Co., San Francisco, US).
Effect of different antioxidants. Eight alkyl caffeates, CAPE, BHA and L-AP were added to the product structured TAGs. The antioxidant concentrations were set at 0.02% (molar ratio). The samples were heated in an oil bath at 180 °C for 5 h. After the reactions, 0.2 g sample was dissolved with 0.5 mL of n-hexane in a 10 mL tube. 2 mol/L KOH-CH 3 OH solution (0.2 mL) was added to the tube 18 . The structured TAGs were transformed into FAMEs, and the FAME content was determined using the GC methods described above.
Statistical analysis.
All experiments were performed in triplicate and the standard deviation was calculated to check the reliability of the results. Statistical analysis was performed using ANOVA. Statistical comparison between data was based on the Pearson correlation coefficient, with P < 0.05 considered significant. Statistical analysis was performed using SPSS 19.0, and response surface applications were performed using Design-Expert 8.05 b (Stat-Ease, Inc., US). Duncan's multiple range tests were performed to determine the significance of difference at P < 0.05. | v3-fos |
2019-03-18T14:07:28.351Z | {
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} | s2 | Relationship of resistance to sudden death syndrome with yield and other important agronomic traits in a recombinant inbred soybean population
The objective of this study was to evaluate a recombinant inbred line population derived from a cross between a recombinant inbred line (RIL) resistant to sudden death syndrome (SDS). ‘LS90-1920’ with a susceptible line, ‘Spencer’ in order to identify any significant association between yield and important agronomic traits with SDS, estimate heritability of these traits and determine whether there are traits that can be used as predictors for SDS resistance. Correlation coefficients for yield and agronomic traits (maturity, lodging, and plant height) were moderately to highly significant but there was no significant association between these traits and SDS resistance. Genotype by environment interaction was significant for all traits studied except of plant height. Maturity, lodging, plant height and SDS resistance were moderately to highly heritable whereas yield showed very low heritability. Our findings showed that environment plays a very crucial role in selection. It is showed that genotypic selection can speed up but cannot replace phenotypic selection across environments and time. Environment is important for the development and production of crop plants because it optimizes the association between the genotype and the phenotype. Highlights: Created Recombinant Inbred Line; Tested for agronomic traits including yield; Tested for disease resistance; Analyzed results to determine if Recombinant Inbred Line differed from the parental lines; Determined if traits were inherited from parents.
Introduction
Soybeans [Glycine max (L.) Merr.] are an important agronomic crop with worldwide production topping two hundred and fifty million metric tons [1]. Plant yield is the most important characteristic for soybeans but at the same time, there is a clear need to prevent losses due to disease. In 2009 there was an estimated loss of over nine hundred thousand metric tons of soybeans due to SDS caused by Fusarium verguliforme [2]. Because of this high loss of yield, a conceited effort must be undertaken in order to minimize the loss.
Yield loss due to SDS tends to be either pre-emergence or post-emergence [3]. Pre-emergent SDS can be treated via a seed treatment in order to minimize yield loss [4]. However, due to the environmental factors that play into the post-emergence appearance of SDS, it is easier to have resistant parents than to deal with spray fields with fungicide. The categorization of soybean lines for SDS resistance is also vital due to the high number of new lines of soybeans released each year [5,6]. Symptoms of SDS post-emergence affect the leaf, forming a necrosis on the leaf. Therefore, it is important to understand how SDS interacts with agronomic traits. By understanding how the disease interacts with agronomic traits we can better understand how SDS affects crop yield and agronomic traits.
Njiti et al [7] described a method for determining the reaction of plants to SDS disease symptoms by looking at a combination of two factors, disease incidence and disease severity and using them for calculating disease index (DX) [7]. This method ensures that lines have resistance to SDS, as heavy disease presence can affect yield [8]. It was shown that agronomic traits have either a positive or negative correlation to yield [9]. A better understanding of the effect of SDS on agronomic traits will allow for a better management practices for fields. Similarly, the interaction of agronomic traits and how they affect each other is also a vital understand how they affect the recombinant inbred line (RIL) [10].
Study of quantitative traits is challenging because they are affected by environmental conditions and their heritability is reduced, facts that makes genetic improvement difficult [11]. Thus, correlation studies are important because they bring up hidden genetic patterns and interrelationship of quantitative traits. Results of these studies can be useful for designing successful breeding programs and helpful for trait evaluation and selection.
The objectives of this study were to use a RIL population (n=94) derived from a cross between a high-yield line resistant to SDS, 'LS90-1920' with a susceptible line, 'Spencer' to (i) identify any significant association between yield and important agronomic traits with SDS (ii) estimate heritability of these traits and (iii) determine whether there are traits that can be used as predictors for SDS resistance.
Plant Material and Field Evaluation
Ninety-four RIL lines were developed from a cross of LS90-1920 and Spencer that was made in 2002 at Agriculture Research Center of Southern Illinois University in Carbondale, IL. The LS90-1920 soybean line was released in 1996 because of its high yield and its resistance to SDS [12]. The soybean line Spencer [13] is a line that used as a check in SDS studies because it is highly vulnerable to F. virguliforme. The lines were advanced to the F 6 generation without any selection using single-pod descent method [14]. The F 6 and F 7 generation were evaluated for their yield performance and data for agronomic traits (maturity, lodging and plant height) were collected in 2011 across two locations in southern Illinois (Dowell and Harrisburg).
Reaction to SDS was scored in comparison to LS90-1920, Spencer, and 'Ripley' (PI 536636; [15]; resistant check) for two years (2010 and 2011) in Carbondale and Valmeyer, IL. The SDS evaluation method used was the same as was described by Njiti et al. [7], which uses the formula DI*DS/9 to calculate DX. DI is the SDS disease incidence recorded per plot as the percentage of plants showing visible leaf symptoms and DS is the disease severity that was rated only from plants that showed symptoms, following a scale from 1 to 9 (1=0-10% death of the plant with 1-5% of leaf area necrotic/chlorotic, 2=10-20% death of the plant with 6-10% of leaf area necrotic/chlorotic, 3=20-40% death of the plant with 10-20% of leaf area necrotic/chlorotic, 4=40-60% death of the plant with 20-40% of leaf area necrotic/chlorotic, 5=Over 60% death of the plant with over 40% of leaf area necrotic/chlorotic, 6=Up to 33% of leaf loss due to premature defoliation, 7=Between 33% and 66% leaf loss due to premature defoliation, 8=Over 66% leaf loss due to premature defoliation, and 9=premature death). Relative resistance (RR) is calculated as the percentage of the susceptible check's (Spencer) DX (RR=DX of line/DX of Spencer x 100). RR is useful in statistical analysis because it allows the comparison of lines across different environments.
In all experiments, RIL population along parent lines were planted in randomized complete block designs with two blocks. Plots were 2 rows wide and 6 m long, with 0.76-m space between rows. Seed yield was expressed as kg ha -1 . Data for maturity were collected when approximately 95% of the pods in a plot had reached mature color (R8; [16]) after September 1. Lodging was rated at maturity using a scale from 1 to 5, where 1 means that all plants standing erect and 5 that all plants prostrate. Plant height is expressed in cm.
Statistical Analysis
The mean, standard deviation, and Shapiro-Wilks test for normality was analyzed using JMP 11 (SAS Institute, Cary, NC). Means, standard deviations, Pearson's coefficient and regression with multiple predictors were calculated from RIL lines and their parents from collected data. The broad sense heritability of the RR was determined from the analysis of variance (ANOVA) where years and locations were treated as random effects. Broad sense heritability was calculated using the website pbstat. The mean, standard deviation, and Shapiro-Wilks test for normality of the RR, yield, maturity, plant height, and lodging were measured for RIL and compared against LS90-1920 and Spencer (Table 1). Significant differences were found between Spencer and RIL for RR, yield, plant height, lodging and maturity. There were significant differences between LS90-1920 and RIL for plant height ( Table 1). The distribution was significantly different from normal for RR, maturity, plant height, and lodging (Table 1). Also, there was no significant difference of normality for yield (Table 1).
Analysis of Variance and Heritability
A summary of ANOVA and broad sense heritability estimates for RR, yield, maturity, plant height, and lodging are presented in Table 2. There were significant differences between 94 RIL for all agronomic traits (P<0.01). All the traits, except of plant height had significant differences (P<0.01) in the interaction of RIL and testing locations. As for RR, differences between RIL and in the interaction of the year and location were also significant at P<0.01**.
Broad sense heritability for RR was determined across locations and time of testing using ANOVA results ( Table 2). Broad sense heritability of RIL for RR (62%), plant height (85%), maturity (95%) and lodging (84%) showed that these traits are controlled by genetics more than environmental components. Broad sense heritability for yield was very low (2%) which means that is highly influenced by the environment.
Correlations Coefficients
Pearson's coefficient was determined to the relationship between RR, yield, maturity, plant height, and lodging for RIL population ( Table 3). The highest relationship was found between plant height and lodging (r=0.6079***) and the lowest relationship that was still significantly different was between lodging and yield (r=0.2276***). Moderate but significant was correlation between yield with maturity (r=0.5028***), yield with plant height (r=0.3508***), maturity with plant height (r=0.5403***) and maturity with lodging (r=0.4911***). No significant was identified between RR and yield or other agronomic traits.
Discussion
In this study we analyzed data for SDS resistance using the method as described by Njiti et al. [7] in combination with agronomic characteristics included yield, maturity, lodging and plant height in a RIL population from the cross LS90-1920 and Spencer. The parental lines were chosen for their significantly different reaction to SDS. LS90-1920 was released and registered for its resistance to SDS [12] while Spencer is used as susceptible check to most SDS experiments [17].
The RR for RIL was significantly lower than the susceptible parent, but had no significantly differences than the resistant parent (Table 1). This shows that the RIL retained the resistant characteristic. More that 50% of RIL inherited the resistance trait from LS90-1920. This appears to be the case, as the broad sense heritability shows that 61% of RIL would have inherited the resistance (Table 2). However, there was no significant relationship between RR and any of the agronomic traits (Table 3). Since RR has no direct connection to the agronomic traits, than selections can be made for more disease resistant individuals without having to worry about the affect that it will have on the agronomic traits in the soybeans.
Yield for RIL differed significantly from the parental line Spencer ( Table 1). Lodging of RIL did not differ significantly from LS90-1920 but was significantly different than Spencer (Table 1). Plant height was both significantly different and higher from both parents (Table 1). Maturity for RIL was not significantly different from the LS90-1920, but was significantly different than the Spencer ( Table 1). The mean values for the RIL for RR, yield, and maturity were either between the mean values of the parents or not significantly different from the upper value (Table 1). The mean values for RIL agronomic traits for plant height and lodging were higher than the parents. (Table 1) There was a very high genotype by environment interaction for yield (Table 2). Similar results were reported for traits as yield, maturity and plant height in soybean from other researchers that identified the effect of environment to genotype and their strong interaction [18,11]. This indicates that a line can only produce significant yield when both genotype and environment are favorable.
Even though there were significant differences for yield between RIL and parents, heritability was very low (Table 2). When yield is analyzed for variance components, environmental effects are considered random. The genotype by location effect was highly significant, with a large mean square, and this was a major contributing factor for the low heritability. However, when environments were treated as fixed effects, yield differences became significant. Therefore, it can be concluded that there were significant differences for genetic effects, but only under the particular conditions of evaluation. All other agronomic traits that were tested showed a moderate to high heritability, as shown in Table 2.
Significant correlations were identified between yield and agronomic traits (plant height, maturity, and lodging) as presented in Table 3. Significant correlations of agronomic traits, both positive and negative were reported previously in soybean [19,11]. In our study, strong positive correlation (0.5028***) between maturity and yield is reasonable, as the longer a plant has to mature, the more it will yield. Similarly, a moderate positive correlation (0.3508***) between plant height and yield is also reasonable while a taller plant overall will be more productive. Yield and lodging had a weak positive correlation (0.2276 ns). Increased weight on soybean may increase the weight on the plant and subsequently increase the lodging score. The reason this may be not significant is due to the loss of yield due to the increased lodging. [20] However, because there is a strong positive correlation (0.6079***) between lodging and plant height, as well as the moderate correlation between height and yield described before, some of the lower values of correlation between lodging and yield may be explained. Increased height causes more lodging as well as higher yield, however, the higher plants cause increased lodging, which can lower yield recovered from plants [20]. The strong positive correlation (0.4911***) between lodging and maturity would indicate that the more lodged a plant would become the longer it would take to mature. Strong positive correlation between plant height and maturity (0.5043***) would indicate that a taller plant would take longer to mature. This makes sense, since a taller plant would have more leaves and therefore take longer to mature.
However, when an analysis of the interaction between plant height, maturity, and lodging was done using regression with multiple predictors, the interaction between height and lodging and maturity and lodging is significant at P<0.001, but the interaction of all three is not significant at P<0.05. This shows that, while the agronomic traits are correlated together, there is no connection linking all the traits at the same time.
In crop breeding, yield is considered as the absolute priority when selecting for new lines. Because of this, yield should be valued over other agronomic traits. Although other traits are not as important, they should not be disregarded as traits that are more helpful to the grower can be helpful as well.
Absence of significant correlation between yield and SDS resistance shows that selecting both traits is strenuous. Our research in this RIL population showed that environment plays a very crucial role in selection. Once more, it is showed that genotypic selection can accelerate but cannot replace phenotypic selection across environments and time. Environment is important for the development and production of crop plants because it optimizes the association between the genotype and the phenotype (Prof. Fasoulas, 2006, personal communication). | v3-fos |
2018-04-03T01:40:35.560Z | {
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} | 0 | [] | 2015-07-23T00:00:00.000Z | 15486572 | {
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} | s2 | Genome Sequence of Torulaspora delbrueckii NRRL Y-50541, Isolated from Mezcal Fermentation
Torulaspora delbrueckii presents metabolic features interesting for biotechnological applications (in the dairy and wine industries). Recently, the T. delbrueckii CBS 1146 genome, which has been maintained under laboratory conditions since 1970, was published. Thus, a genome of a new mezcal yeast was sequenced and characterized and showed genetic differences and a higher genome assembly quality, offering a better reference genome.
tolerance and high freeze tolerance (1)(2)(3)(4). These properties make this yeast an interesting organism, with potential biotechnological applications in bakery, wine, and dairy industrial processes. In the last few years, studies about enzyme production in winemaking have been carried out, but only a few genes have been characterized (5). Recently, it has been reported that T. delbrueckii yeast strains isolated from the mezcal-fermenting process produced -fructofuranosidase enzymes with fructosyltransferase activity (6). Therefore, the genome sequencing of T. delbrueckii can lead to the discovery of new genes with biotechnological application. Lately, the genome of T. delbrueckii CBS 1146 was obtained (using 454 sequencing), with the main purpose of studying the sex chromosome evolution in the Saccharomycetaceae family (7). However, the characterized strain has been maintained under laboratory conditions since 1970. Thus, in order to find the differences between the published reference and our mezcal isolate, we sequenced, assembled, and characterized it in order to find genes and variations associated with the fermentation process.
Genomic DNA from T. delbrueckii NRRL Y-50540 was isolated and prepared as Illumina sequencing libraries to generate a total of 20,514,013 paired-end reads (estimated coverage,~328ϫ) with a length of 72 bases, using the Illumina GAIIx platform. The assembly was performed with Velvet version 1.2.10 using a k-mer size of 35 (8). An assembly of 11,236,894 bp in 374 contigs with length Ն1,000 bp was obtained, with N 50 and N 90 values of 82,617 and 23,849 bp, respectively. The average contig length was 30,012 bp, giving a considerable space to search for genes. Finally, we ordered and scaffolded the assembly using ABACAS (9) against the available T. delbrueckii reference genome (7) of a different strain to leave the whole assembly in 8 scaffolds corresponding to 8 chromosomes. The average GϩC content was 42%, which is consistent with the reported genome. Gene prediction was performed using AUGUSTUS version 2.7, and using several different yeast species profiles, we predicted 4,714 protein-coding genes by intersecting all predictions (10). Using CEGMA version 2.5, we obtained a 97% genome completeness (11).
In contrast to the genome published by Gordon et al. (7), we found a slightly better value for completeness and fewer open reading frames (ORFs) (4,714 versus 4,972, respectively) in our assembly. Both assemblies presented gaps, but we were able to remove some of them, which led to a more complete genome. Although these differences are not of concern, they are expected, since each genome was assembled using different sequencing technologies and assembly strategies. Currently, we are working on a hybrid assembly strategy using the information from both strains in order to obtain a better assembly, gene prediction, and annotation.
We believe that the T. delbrueckii genome sequence presented here can be used as a better reference to perform further analyses, such as differential gene expression of enzymes related to the synthesis and degradation of biotechnological molecules of interest, for example, under different fermentation conditions analyzed using RNA sequencing (RNA-seq) data.
Nucleotide sequence accession numbers. This whole-genome shotgun project has been deposited at the NCBI GenBank database under the accession numbers CP011778 to CP011785.
ACKNOWLEDGMENTS
We thank the "Unidad de Secuenciación Masiva y Bioinformática, Instituto de Biotecnología (USMB), UNAM," for DNA sequencing advice and bioinformatics analysis. The USMB is part of the "Laboratorio Nacional de Respuesta a Enfermedades Emergentes," which has been created and funded by the "CONACYT-Programa de Laboratorios Nacionales." We also thank Veronica Jimenez-Jacinto for preparing and submitting the sequencing data to the NCBI SRA and GenBank repositories. | v3-fos |
2018-12-05T11:58:07.174Z | {
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} | 0 | [] | 2015-01-01T00:00:00.000Z | 55594012 | {
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} | s2 | Determination of reproductive parameters of sows at rural areas of Bangladesh
The aim of this study was to determine the reproductive parameters of sows in selected area of Bangladesh. A total of 51 sows from 21 farms were selected for this study. The data on reproductive parameters were collected from the owners by interviewing with pretested questionnaire. We found that the age at puberty, estrus cycle length, estrus duration, interval between farrowing and onset of estrus and gestation length were 254.5±34.4 and 21.2±1.2 days, 34.2±13.2 h, 51.8±10.9 and 114.3±0.9 days, respectively. The number of required services for each pregnancy in native sows was 1.4 ±0.6, and the first service pregnancy rate using natural mating was 63.8%. Moreover, the number of piglets born per sow was 7.6±3.4. The highest piglet production (10.6±3.1) was in parity 5 and the lowest (4.1±0.6) was in parity 1. Estrus was detected in 34.1% sows on the basis of observing standing reflex when boar mounted. In conclusion, reproductive parameters in native sows of Bangladesh need to be improved for ensuring satisfactory reproductive performances.
INTRODUCTION
The pig is considered as a highly prolific species having high ovulation rate, and if mated at the right moment, there is high probability of pregnancy in sows. (Peltoniemi et al., 2007). It is a highly productive animal compared with other farm animals (Anderson, 1974). However the most of the people in Bangladesh are Muslim religion. Therefore the pork is not popular in this country. But the environmental condition is suitable for pig reproduction. It is very prospective to export the pork to others country.
Reproductive performance of the sow herd is the key factor, if not the major factor, in controlling the efficiency of swine production (Hodson, 1980;Rekwot et al., 2001) and considered economically important to the swine industry (Day, 1974). It is measured primarily by the number of living pigs at birth or by the total farrowing or weaning weight of pigs produced by the dam within one year. Low levels of reproductive performance may not only result in low profit per sow, but will also limit attempts to improve the herd genetically (Rekwot et al., 2001).
There are several measures of reproductive performance including farrowing rate and litter size at birth and fertility index. Other parameters used include, pounds of pork/sow/yr and non -productive sow days ( Van der leek and Becker, 1993). The goal of reproductive performance is to have 2.0 L per female annually with an average of 8.0 pigs per litter at weaning, that is, a total of 16 pigs weaned per year for each female maintained in the herd (Day, 1974). Government has formulated "Poverty reduction strategy papers" (PRSP) to alleviate poverty. Accordingly, all national level policy planning has taken a sharp turn towards pro-poor approach reducing 50% poverty level by 2015 as indicated by the Millennium Development Goals (MDG) (United Nations, 2000).
In Bangladesh, there live about 3.5-5.5 million Dalits who exist far below the poverty line with extremely limited access to health services, education and employment (International Dalit Soliderity Network). About 31% of the people of Bangladesh are poor (Bangladesh Economic Census, 2011) and more than 60% Dalits have trapped them in poverty (Higgett, 2009). Though, Bangladesh is an agricultural country, these people are not engaged in agriculture. They cultivate only swine traditionally. In different cultural and social occasions, they slaughter boar for consumption. Besides, swine is the only protein source of them. Swine farming has an important role on their livelihood. Some of them have initiated farming in semi-ranging system. But, they rare swine traditionally without applying any technology. If they begin swine farming using modern technology, it will contribute to their economy as well as to the GDP of Bangladesh.
It is imperative to know the reproductive parameters of the existing swine breed in Bangladesh. Therefore, it seems rationale to determine the reproductive parameters of sows resulting in increased productivity and decreased poverty in Bangladesh. Although there is large population of pig at rural areas, few reports exist on the reproductive performance of smallholder piggeries at rural areas. Therefore, the objective of the present study was to determine the common reproductive parameters of local sows in selected area of Bangladesh.
MATERIALS AND METHODS
Selection of the study area: Two villages and two wards of Sadar upazila of Thakurgaon district namely-Gobidonagar, Khochabari, Fakirpara and Nischintopur were selected for the study. The data of indigenous sows were collected from the mentioned area. A large number of Dalits live there. Most of them work as sweepers. Rising of indigenous sows were the main source of their livelihood.
Management of sows:
The sows were managed extensively or traditionally. In extensively managed small hold farms, most sows were under grazing freely daily. All sows had free access to drinking water. There was no restriction of sucking by piglets. Weaning is occurred naturally in these sows. Boars and sows were kept together and natural mating occurred among them. They were kept in open houses made by bamboo. Floor of some houses was made of concrete and some were made of sand. Most of the houses of swine were not clean and the site of housing was not clean also. The houses of sows were located near the drain or garbage. Farmers are not careful of the general health condition of swine. No deworming and vaccination were in practice there. By day, sows were kept free and they took food from here and there. At night, they were kept in houses. Sows were fed with rice husk, rice polish, rice gruel and waste product of hotels.
Collection of data: Data were collected during September 2011 to April 2012 by direct interview. Information given by owners of sows was recorded on pre-tested questionnaire for analysis.
Preparation of the schedule:
The questionnaire was prepared according to the objective of the investigation and was designed in a simple way so that the farmers can understand easily. The questionnaire included questions to collect following information. age at puberty of sows, estrus cycle length of sows, estrus period of sows, prominent estrus signs of sows, gestation period of sows, number of piglets born per sow, parity of sows, interval between farrowing and onset of estrus, number of services required for each pregnancy.
Statistical analysis:
The collected data were entered into the Excel datasheet and descriptive statistics were performed. The values for age at puberty, estrus cycle length, estrus period, prominent estrus signs, gestation length, and number of piglets born per sow, interval between farrowing and onset of estrus, number of services for each pregnancy were expressed as mean ± standard deviation.
RESULTS AND DISCUSSION
The mean age at puberty, estrus duration and interval between farrowing and onset of estrus of indigenous sows is presented in Table 1. The mean age at puberty of indigenous sows was 254.5 ± 34.4 days. The minimum age at puberty was 209 days and the maximum age at puberty of indigenous sows was 360 days.
The mean estrus duration of indigenous sows was 34.2±13.2 h. The lowest duration was found 15 h and the highest was 70 h. The mean interval between farrowing and onset of estrus in indigenous sows was 51.8±10.9 days. The lowest interval between farrowing and onset of estrus was 37 days and the highest interval between farrowing and onset of estrus was 77 days.
The estrus cycle length in indigenous sows is presented in Table 2. The range of the estrus cycle length was from 19 to 24 days. The mean estrus cycle length was 21.2±1.2 days. Estrus cycle length of 24 days was found in the lowest proportion of sows (2.9%) and estrus cycle length of 19 days was fond in the highest proportion of sows (42.9%). The gestation length of indigenous sows is presented in Table 3. The gestation length in sows ranged from 113 to 116 days. The mean gestation length was 114.3±0.9 days. The lowest proportion of sows (12.9%) had the gestation period of 116 days and the highest proportion of sows (41.9%) had the gestation length of 114 days.
The number of piglets delivered by individual sows is presented in Table 4. The number of piglets furrowed by indigenous sows ranged from 3 to 14. The mean number of piglets delivered was 7.6±3.4. The lowest number of piglet production was 4.1±0.6 in parity 1 and the highest number of piglet production was 10.6±3.1 in parity 5. The minimum number of piglet production was 3 in parity 1 and the maximum number of piglet production was 14 in parity 5. 10.0 ± 2.0 8 12 9 (n=3) 9.6 ± 2.1 8 12 Total=35 Mean=7.6±3.4 (100) The number of natural services required for each pregnancy of indigenous sows is presented in Table 5. It was observed that the mean number of services required for each pregnancy of indigenous sows was 1.4±0.6. The lowest proportion of sows (5.5%) required 3 services for pregnancy and the highest proportion of sows (63.8%) required one service for pregnancy.
The prominent estrus signs observed in indigenous sows are presented in Table 6. The physical and behaviuoral sings in sows during estrus was almost common to all sows. The lowest proportion of sows (18.2%) was detected in estrus by observing pacing and grunting of sows and the highest proportion of sows The study was conducted to assess the reproductive parameters of indigenous sows in selected area of Bangladesh. For this purpose a survey work was conducted because it is thought to be more advantageous than other data collection methods (Hughes, 1982). Since the farmers do not keep proper records of their operation, in this study like other survey works, information from individual farmers was collected.
The average age at puberty of indigenous sows was 254.5±34.4 days. Similarly, the age at puberty in European Large White gilts was 8 months (Bon et al., 1979). The age at puberty of present study is a little bit higher than that of a previous study where the age at puberty was 204.1±28.0 days (Tummaruk et al., 2003). Similarly, occurrence of puberty between 5 and 8 months of age has been documented in European breeds (Christenson and Ford, 1979;Hughes, 1982). Moreover, Sterning et al. (1998) reported that the mean age at puberty of gilts was 206 days. Further, Camous et al. (1985) found that ages at puberty at different photoperiods were 193.4, 175.6 and 177.1 days, respectively. Contrasting to the present and previous findings, occurrence of puberty at the age of 3 months has been reported in Chinese Meishan breed elsewhere (Cheng, 1983). The variations at the age of puberty among different studies may be due to variations in breeds of swine, feeding practices and agro-climatic conditions among studies.
In the present study, the mean duration of estrus in indigenous sows was 34.2±13.2 h and it ranged from 15 to 70 h. Similarly, Singleton and Diekman (1984) reported that duration of estrus or heat is variable which may last only 12 h in gilts or up to 60 h or more in sows. The variation in duration of estrus among studies may be due to variation in breed of sows and agro-climatic conditions among studies. Since the actual time of the onset of estrus is rarely known, it is recommended that a female receives at least two mating during estrus. This helps ensuring that sperm are present at an optimum time relative to ovulation for fertilization to occur.
The mean estrus cycle length in indigenous sows of the present study was 21.2±1.2 days and the range of estrus cycle length varied from 19 to 24 days. The finding of the present study is consistent with previous study where the mean estrus cycle length was 21 days and it ranged from 18 to 24 days.
In the present study, the mean interval between farrowing and onset of oestrus in indigenous sows was 51.8±10.9 days and it ranged from 37 to 77 days. Adshell (1946) stated that sows do not usually come into heat during lactation. Sows generally lack of cyclic ovarian activity during the lactation period (Melampy et al., 1966;Crighton and Lamming, 1969). An experiment was conducted in confinement during 1997 to 1999 using 174 large white × landrace sows by Knox and Zas (2001). In that study, after weaning, the average percentage of sows returning to estrus within 8 days of weaning was 86.2. There are reports that a small percentage of sow exhibits anovulatory oestrus about 1 to 5 days postpartum (Warnick et al., 1950;Heitman and Cole, 1956). If oestrus occurs after the first week postpartum, ovulation usually also occurs. Normally, plasma progesterone remains at very low concentrations (less than 1 ng/mL) until late lactation or after weaning when estrus and ovulation occur (Ash and Heap, 1975;Aherne et al., 1976;Parvizi et al., 1976). Nevertheless, in the present study no weaning practice is done resulting in having long interval between farrowing and onset of estrus in indigenous sows. This emphasizes the importance of weaning of piglets for shortening of postpartum period. In the present study, the highest proportion of estrus detection was performed by standing of sows when boar mounted. This result supports the result of Sterning et al. (1998) who found that the 77.7% (351 out of 452) sows showed standing reflex at estrus period.
In the present study, the mean gestation length was 114.3 ± 0.9 days. Similarly, average gestation length of 114±2.0 has been reported elsewhere (Pitcher and Springer, 1997). Cole and Foxcroft (1982) also reported a gestation length of 114 days in domestic sows and 119 days in wild sows. In a study, the number of corpora lutea (CL) was adjusted to 4, 5, 6, 7 or 8 by unilateral ovariectomy at day 30 to 40 of gestation and it was observed that the number of CL or the level of progesterone and estrogen had no effect on the length of gestation. Moreover, there was an inverse relationship between litter size and length of gestation in sows.
In the present study, the mean number of piglet production was 7.6±3.4. Contrasting to the present finding, Zaleski et al. (1993) obtained 12.2 piglets per litter after evaluating 98 farrowings. Cole and Foxcroft (1982) also reported to have 12 piglets per litter in domestic sows and 5 piglets in wild sows. The variation between studies may be due to lower body weight and smaller size of the sows in present study compared to previous studies. In the present study, litter size increases gradually with the increasing number of parity and the highest litter size was in parity 5. This result is consistent with the earlier study where litter size usually increased from first to second litter and again from second to third litter, but then plateaus until approximately the seventh or eighth litter (Hughes and Varley, 1980).
In the present study, it was observed that the mean number of services required for each pregnancy in indigenous sows was 1.4±0.6. The first service pregnancy rate was 63.8% in indigenous sows. Compared to the present study, higher pregnancy rate (71.9%) has been documented in swine received natural service in Illinoise, USA. A number of factors contribute to the success or failure of breeding in sows. Age, mode of insemination and the number of insemination attempts have been shown to affect rates of successful pregnancy in swine (Scofield and Penny, 1969;Wrathall, 1975;Flowers and Alhusen, 1992). Lee et al. (1997) examined the effect of birth litter sex ratio of sows and gilts on reproductive performance using a 13 years database of breeding and litter data. Gilts that failed to become pregnant on the first breeding attempt came from litters with significantly higher proportions of males than gilts that successfully conceived on the first breeding attempt. Overall, female swine were significantly more likely to exhibit lower rates of successful breeding during their first four breeding attempts if they had been born in a male-biased litter. A total of 2,696 mating attempts were included in an analysis and examination of the data revealed that young females of approximately 1.5 years or less in age were more likely to have an unsuccessful mating, whereas the oldest groups of sows were more likely to conceive (Lee et al., 1997). However, in the present study, I used very few numbers of sows for investigation and the age of sows and birth litter sex ratio was not exactly known due to lack of records.
It is likely that disease or disorder free sows should be conceived at first mating by fertile boars as multiple ovulations occur in them. However, a minimum of 4 embryos are needed for maintaining pregnancy in sows (Pitcher and Springer, 1997). Because, if fewer than 4 embryos are present in the uterus, luteolysis occurs probably due to insufficient estrogen production by trophoblasts to prevent release of prostaglandin (Pitcher and Springer, 1997). Even if pregnancy occurs in one horn of sows (as in segmental aplasia of oviduct), luteolysis occurs and the pregnancy is terminated (Pitcher and Springer, 1997). Moreover, a minimum level of progesterone required for maintaining pregnancy in sows is 6 ng/mL (Cole and Foxcroft, 1982).
CONCLUSION
For native sows of Bangladesh, it may be concluded that the age at puberty, estrus cycle length, estrus duration are 254.5±34.4 and 21.2±1.2 days, and 34.2±13.2 h, respectively. The interval between farrowing and onset of estrus is 51.8±10.9 days. The gestation length is 114.3±0.9 days. The number of required services for each pregnancy is 1.4 ± 0.6. The first service pregnancy rate using natural mating is 63.8%. The number of piglet born per sow is 7.6±3.4. Standing reflex at estrus was observed in 34.1% females. | v3-fos |
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} | s2 | Identifying and confirming quantitative trait loci associated with heat tolerance at flowering stage in different rice populations
Background Climate change is affecting rice production in many countries. Developing new rice varieties with heat tolerance is an essential way to sustain rice production in future global warming. We have previously reported four quantitative trait loci (QTLs) responsible for rice spikelet fertility under high temperature at flowering stage from an IR64/N22 population. To further explore additional QTL from other varieties, two bi-parental F2 populations and one three-way F2 population derived from heat tolerant variety Giza178 were used for indentifying and confirming QTLs for heat tolerance at flowering stage. Results Four QTLs (qHTSF1.2, qHTSF2.1, qHTSF3.1 and qHTSF4.1) were identified in the IR64/Giza178 population, and two other QTLs (qHTSF6.1 and qHTSF11.2) were identified in the Milyang23/Giza178 population. To confirm the identified QTLs, another three-way-cross population derived from IR64//Milyang23/Giza178 was genotyped using 6K SNP chips. Five QTLs were identified in the three-way-cross population, and three of those QTLs (qHTSF1.2, qHTSF4.1 and qHTSF6.1) were overlapped with the QTLs identified in the bi-parental populations. The tolerance alleles of these QTLs were from the tolerant parent Giza178 except for qHTSF3.1. The QTL on chromosome 4 (qHTSF4.1) is the same QTL previously identified in the IR64/N22 population. Conclusion The results from different populations suggest that heat tolerance in rice at flowering stage is controlled by several QTLs with small effects and stronger heat tolerance could be attained through pyramiding validated heat tolerance QTLs. QTL qHTSF4.1 was consistently detected across different genetic backgrounds and could be an important source for enhancing heat tolerance in rice at flowering stage. Polymorphic SNP markers in these QTL regions can be used for future fine mapping and developing SNP chips for marker-assisted breeding. Electronic supplementary material The online version of this article (doi:10.1186/s12863-015-0199-7) contains supplementary material, which is available to authorized users.
Background
Global warming caused by greenhouse gases has a huge effect on sustaining agricultural development. Since the early 20th century, the earth's mean surface temperature has increased by 0.8°C, with about 0.6°C of this hike occurring since 1980 [1]. Global mean surface temperatures for 2081-2100, relative to 1986-2005, is likely to increase by 0.3°C to 1.7°C for the lowest and by 2.6°C to 4.8°C for the highest greenhouse gas emission scenarios [2]. This temperature change will not be regionally uniform [3]. It is virtually certain that, in most places, there will be more hot and few cold temperature extremes as global mean temperature increases. Of the 13 warmest years since 1880, 11 [4,5]. These drastic changes in temperature in recent years have caused more frequent occurrence of extremeweather events such as heat waves and drought, with serious consequences on rice yield. Rice yield losses due to high temperature have been reported in many tropical and subtropical countries, such as in Pakistan, India, Bangladesh, China, Thailand, Japan, Australia and the U.S. [6][7][8][9]. Significant yield losses have also been predicted by using different crop models. Short term predictions indicated that, by 2030, rice production in South Asia could decrease by up to 10% [10]. Medium to long term predictions, i.e. by 2080, estimated rice yields in developing countries to decrease by 10% to 25%, on average, while yields in India could drop by 30% to 40% [11]. By 2100, rice and maize yields in the tropics are expected to decrease by 20-40% because of higher temperatures without accounting for the decrease in yields as a result of drought enhanced by temperature increases [12]. Spatial model simulation indicated that yield of boro rice in Bangladesh could decrease by 20% and 50% by 2050 and 2070, respectively [13], and on average, rice yields could be reduced by up to 33% by 2081-2100 [14]. As the global temperature continuously rises, incorporation of heat tolerance in new rice varieties is therefore, becoming more and more important in rice breeding programs.
Flowering is the most sensitive stage to high temperature in the rice life cycle [15,16]. High temperature of over 35°C at flowering stage increases pollen and spikelet sterility, which leads to significant yield losses, low grain quality, and low harvest index [6,7,[17][18][19][20]. Large cultivar variation exists in the spikelet sensitivity to high temperature damage, and the primary cause of this cultivar variation in high temperature (heat) tolerance at flowering is the number of viable pollen grains shed on the stigma, resulting from the changes in the extent of anther dehiscence [21], which directly affect the spikelet fertility and grain yield [17]. Thus, spikelet fertility under high temperature has been widely used as a screening index for heat tolerance at reproductive stage [19].
In recent years, rice varieties tolerant to high temperature have been identified [17,22,23]. QTL mapping studies for heat tolerance at flowering stage have been conducted on various rice populations [24][25][26][27][28][29][30][31]. However, while QTLs for heat tolerance at flowering stage have been mapped on almost all rice chromosomes, improving heat tolerance in rice varieties using the identified genetic resources and QTLs has not yet been achieved. Further studies on QTL mapping using diverse genetic resources and populations are needed for addressing the means to enhance heat tolerance in rice. We have developed a precise method for phenotyping rice heat tolerance and employed the Illumina GoldenGate SNP assay technology for mapping QTLs for heat tolerance using IR64/N22 population [30]. The phenotyping protocol was shown to be repeatable and reliable in our ongoing fine mapping of the previously identified QTLs (qHTSF1.1 and qHTSF4.1) using different backcross populations. Here, we report the identification of QTLs for rice heat tolerance at the flowering stage using three other populations derived from Giza178, identified to be one of the most heat-tolerant rice varieties at flowering. The main objectives of this study were to identify QTLs controlling heat tolerance in Giza178 at flowering stage using different susceptible recipients, and to confirm the identified QTLs in an advanced population.
Developing mapping populations
To select novel heat tolerant donors for QTL mapping, we evaluated the heat tolerance of 455 rice varieties from different countries [23,32] and a set of over 40 promising varieties for heat tolerance at booting and flowering stages [23]. Preliminary analysis showed that the spikelet fertility of IR64, a heat sensitive variety, decreased significantly even with 1°C increase between 36-39°C. At 39°C, the spikelet fertility of IR64 dropped to less than 5%, but the tolerant varieties, such as N22 and Giza178, maintained fertility levels of over 60% (unpublished data). IR64 (susceptible) and Milyang23 (moderately susceptible) were crossed with Giza178 (tolerant) to develop two different mapping populations. IR64 is a mega-variety cultivated widely in Asia with low to moderate tolerance to high temperature at flowering stages [33,34]. Milyang23 is a Korean variety developed from the cross IR1317-316-5-1/ IR24, while Giza178 is an Egyptian variety developed from the cross Giza175/Milyang49. Both Milyang23 and Giza178 have Indica and Japonica introgressions in their earlier pedigrees. The F 1 plants were self-pollinated to produce F 2 populations for QTL identification. No hybrid sterility was observed in the F 1 plants. Due to the limited space in the growth chambers, relatively small populations were used to identify QTL with larger effects, rather than using large population to detect small-effect QTL. One hundred F 2 progenies from each cross were phenotyped for heat tolerance at flowering stage in 2012; and 86 and 96 progenies from IR64/Giza178 and Milyang23/Giza178 populations, respectively, were genotyped using a custom 384-plex Illumina GoldenGate SNP assay (Indica-Indica).
Based on the phenotyping results of the Milyang23/ Giza178 F 2 population, the top ten heat tolerant plants were crossed with IR64 to develop a three-way breeding population (Additional file 1). The three-way F 1 lines (IR64//Milyang23/Giza178) were preliminarily screened in a temperature-controlled phytotron (35/24°C day/night temperature, 80% relative humidity) at flowering stage. From each line, 2-3 plants with good agronomic traits and high spikelet fertility were harvested, and 10 seeds from each plant were grown in a net house for phenotyping in 2013 as described below.
Phenotyping of F 2 progeny
The seeds of the parents IR64, Milyang23, Giza178 and the F 2 progenies were germinated and sown in plastic pots (L × W × H = 9.5 × 9.5 × 8.5 cm with draining holes) filled with natural clay loam soil, one plant per pot. The pots were randomly arranged in trays (L × W × H = 75 × 50 × 20 cm) to ensure that plants were grown under the same water and nutrient conditions. The plants were grown in a net house under natural temperature and sunlight as described by [30]. Twenty-one days after sowing, a young leaf (about 10 cm long) from each plant was collected and frozen for DNA extraction, and 10 g of PerfectGro® composite fertilizer 14-14-14 (AFC Fert. & Chem., Inc) was applied to each tray. The position of the pot in the tray was changed within the tray every week to reduce any potential micro-climate effects of light and temperature, and to avoid interweaving of the roots that protruded from the bottom of the pots. Only the main stem and two tillers were maintained, all the other tillers were removed to prevent overcrowding. When each plant started heading, the plant was transferred into a new tray and moved into an indoor growth chamber (IGC, Thermoline, Australia). The temperature regime in the IGC was the same as described by [30] with 6 hours of high temperature (38°C) each day during the flowering time (08: 30-14:30). After 14 days of temperature exposure the plants were moved back to the net house and grown to maturity. The date of high temperature treatment commencement (heading date) was recorded. At physiological maturity, the number of fully filled spikelets (including partially filled) and empty spikelets were counted. The mean spikelet fertility of the three panicles maintained in each pot was used to evaluate the heat tolerance of the plant.
Genotyping using SNP markers
Genomic DNA of IR64, Milyang23, Giza178 and all the F 2 plants was extracted using SDS extraction buffer (100 mM Tris, 50 mM EDTA, 500 mM NaCl, 1.25% SDS and 1% v/v 2-mercaptoethanol) and chloroform/ isoamylalcohol (24:1) solution followed by ethanol precipitation. The RNA was digested by RNase at 37°C for 30 minutes. The final concentration of the DNA samples was normalized to 50 ng/μl for genotyping. The SNP genotyping was done by using Illumina BeadXpress 384plex SNP plates GS0011861 (customized for Indica-Indica) for the bi-parental populations and Illumina Infinium 6 K SNP beadchip for the -three-way F 2 population. The custom oligo pool assay (OPA) containing 384 well-distributed SNPs per assay was designed by Cornell University [35,36] from a high quality subset of the SNPs discovered in 20 diverse O. sativa landraces [37]. The 6 K beadchip was designed for SNP fingerprinting, high-resolution mapping and genome-wide SNP scans by Dr Susan MacCouch's group at Cornell University. It contains about 6,000 bead types, and 5,274 SNPs are high quality in the dataset, 247 of which are overlapped with the BeadXpress 384-plex SNP set (Indica-Indica). The PCR amplification and hybridization were carried out following the GoldenGate genotyping assay for VeraCode manual protocol (Illumina, San Diego, CA). If the SNP genotype is the same as in IR64, it was coded as AA, otherwise it was coded as BB, and the heterozygote was coded as AB.
Statistical analysis
The average spikelet fertility of different genotypes was compared by one-way ANOVA using MINITAB V14.0 (Minitab Inc.). The VeraScan raw data from BeadXpress Reader was initially analyzed using the GenomeStudio software V1.1.0 (Illumina Co.) and Alchemy [38]. The generated report and map file were used for QTL analysis by using composite interval mapping (CIM) in QGene V4.3.8 [39]. The genetic distance between SNP markers was estimated from the physical map based on the genomic sequence available at GRAMENE (www.gramene.org), with genetic distance (cM) = Physical distance (kb)/260. CIM was performed using the standard model with a walk speed of 2 cM. Cofactor selection was set at auto. Permutation tests [40] were performed for each trait with composite interval mapping and 1,000 permutations. For the 6 K SNP genotyping, we firstly checked the markertrait association by using the Trait Analysis by Association Evolution and Linkage (TASSEL) program [41]. The sites were filtered at a maximum count of 150 of the 167 F 2 plants, which accounts for sites where 90% of the lines have a call and a minimum frequency of 0.05 for the minor allele. The above criteria resulted in 4497 filtered sites. Among them, 1373 SNPs are polymorphic among the parents. These polymorphic SNPs were used for association analysis using general linear model (GLM) [42]. In this study, we set a significant threshold of p < 0.0001 (−log 10 p-value > 4.0). The genotypic and phenotypic data were then used for QTL mapping using Qgene software as described above. The identified QTLs were named using the CGSNL nomenclature [43]. The correlation between QTL alleles and spikelet fertility, and the interaction among QTLs were further analyzed by using MINITAB V14.0 (Minitab Inc.).
QTLs identified from IR64/Giza178 and Milyang23/Giza178 populations Among the screened 384 SNP markers, 133 (34.6%) markers showed polymorphism between IR64 and Giza178. The results from CIM showed four potential QTLs (qHTSF1.2, qHTSF2.1, qHTSF3.1 and qHTSF4.1) on chromosome 1, 2, 3 and 4 ( Figure 1a). These QTLs explained around 15 -22% of the variation in spikelet fertility caused by high temperature treatment, with both additive and dominance effects. The tolerant alleles for the QTLs are from the tolerant parent Giza178, except for qHTSF3.1 (Table 2). At marker id3001137 on chromosome 3, the mean spikelet fertility of AA genotype (41.8 ± 24.4%) was significantly higher than that of BB genotype (24.8 ± 23.9%) and heterozygote AB (21.7 ± 20.0%), and the spikelet fertility of BB and AB genotypes are not significantly different. The allele increasing spikelet fertility comes from the susceptible parent IR64 controlled by a recessive gene (Figure 2).
Among the same 384 SNP markers, 178 (46.4%) markers showed polymorphism between Milyang23 and Giza178. The results from CIM showed two potential QTLs (qHTSF6.1 and qHTSF11.2) on chromosome 6 and 11 ( Figure 1b). The QTL on chromosome 6 (LOD = 4.81) was located between SNP markers id6004481 and id6008973 at about 12.09 Mb. This QTL explained 20.6% of the variation in spikelet fertility caused by high temperature treatment. The QTL on chromosome 11 (LOD = 3.42) was located between SNP markers id11002639 and id11003924 at about 8.66 Mb, which explained 15.2% of the variation in spikelet fertility caused by high temperature treatment. The tolerant alleles for qHTSF6.1 and qHTSF11.2 are from the tolerant parent Giza178 (Table 2).
QTL identified from IR64//Milyang23/Giza178 population
For the genome-wide SNP scans using the 6 K beadchip, a dataset with 5,274 SNPs was obtained. Among them, 4,492 high quality SNPs have a call on 90% of the progeny and 1373 SNPs are polymorphic among the parental varieties. Most of the SNPs are evenly distributed on the chromosomes, with only a few small gaps on chromosome 6, 7 and 8. The largest gap is about 8.95 Mb on chromosome 6.
Through Cladogram analysis using TASSEL, one plant showed unusual segregation with others. This plant was removed, and a total of 166 plants were included in subsequent analysis. Results from the GLM analysis showed that some SNPs on chromosome 1, 2, 4, 6 and 11 are significantly associated with spikelet fertility under high temperature (−ln (p-value) > 4.0) (Additional file 2).
To conduct CIM analysis using Qgene, only the genotypes of the 1373 polymorphic SNPs were used. The results showed that five QTLs are potentially controlling the spikelet fertility under high temperature conditions (Figure 1c), which is consistent with the results from association analysis by using TASSEL. The LOD score of these QTLs ranged from 4.45 to 5.38 (permutation threshold α 0.05 = 3.86, α 0.01 = 4.60). These QTLs explained 11.6 -13.9% of the variation in spikelet fertility caused by high temperature stress treatment ( Table 2).
The spikelet fertility of different genotypic classes (AA, AB and BB) were significantly different for the SNP markers close to the peaks of the QTLs. qHTSF2.1 is controlled by a dominant gene (d = 5.9), the other QTLs are all controlled by recessive genes (Figure 2). The tolerant alleles of all QTLs are from the tolerant parent Giza178.
Among the five QTLs identified, qHTSF1.2 and qHTSF6.1 are overlapped with the QTLs identified in IR64/Giza178 and Milyang23/Giza178 populations, and qHTSF4.1 is located within the QTL region identified in IR64/Giza178 population. The other two QTLs (qHTSF2.2 and qHTSF11.3) are located nearby but not overlapped with the QTLs identified on the same chromosomes from the bi-parental populations (Table 2). There are interactions among the confirmed three QTLs, qHTSF1.2 can maintain relatively high spikelet fertility without qHTSF4.1, and qHTSF4.1 can maintain relatively high spikelet fertility without qHTSF6.1. The spikelet fertility even slightly decreased with both QTLs. The interaction of qHTSF1.2 and qHTSF6.1 could enhance heat tolerance when both QTLs are working together ( Figure 4).
Discussions
Giza178 is an Indica-Japonica rice variety from Egypt, which showed consistent heat tolerance in our experiments in the past few years (Tenorio et al. 2013). By using the F 2 population derived from the IR64/Giza178 and Milyang23/Giza178 crosses, six QTL were identified that are associated with spikelet fertility under high temperature conditions. However, different QTLs were identified in the two populations, though most of the tolerant alleles are from Giza178 except that for QTL on chromosome 3. QTLs for heat tolerance were located on chromosome 1, 2, 3 and 4 in IR64/Giza178 population, while those in Milyang23/Giza178 population were located on chromosome 6 and 11. The LOD scores of qHTSF1.2, qHTSF3.1, and qHTSF11.2 are slightly lower than the threshold from permutation, but these QTLs explained 14.6-17.3% of the variation of spikelet fertility under heat stress, and qHTSF1.2 was confirmed to be overlapped with a QTL identified in the three-way population. Thus, they were considered as potential QTLs. Among these six QTLs, three were confirmed in the three-way-cross population. Different QTLs were identified in different populations suggests that a strong background (or population) dependence exists among the mapping populations, and some of the important tolerant alleles may not be distinguished in a bi-parental population. To identify most of the tolerant alleles in a donor variety, it is necessary to cross the donor with different susceptible recipients. Nested association mapping (NAM) populations, in which a set of diverse lines is crossed with a common reference line, can be used as next generation mapping populations [44]. Alternatively, a multiparental population, such as multi-parent advanced generation inter-cross (MAGIC) population [42,45], can be used for identifying tolerant alleles associated with the trait by association mapping, which provides a platform for the discovery and characterization of genes responsible for complex traits like heat tolerance.
The tolerant alleles for the identified QTLs are from Giza178 except qHTSF3.1 on chromosome 3. For QTL qHTSF3.1, the spikelet fertility of the genotypes of the SNP marker close to the QTL showed that the heat tolerant allele is from the susceptible parent IR64. A QTL (qhr3-1) for heat tolerance was previously identified on the short arm of chromosome 3 [24]. QTL qhr3-1 may be located in the same interval as qHTSF3.1. However, qHTSF3.1 was not identified in the three-way cross population, this QTL may not be contributing to increasing heat tolerance when some of the heat tolerance alleles from Giza178 were introduced into IR64 background. The QTLs on chromosome 1 (qHTSF1.2) and 11 (qHTSF11.2 and qHTSF11.3) are different from those previously identified (qHTSF1.1 and qHTSF11.1) in the IR64/ N22 population [30]. But qHTSF1.2 and qHTSF11.2 are located in similar regions with qhr1 and qhr11-1 reported by [24]. The QTLs qHTSF2.1, qHTSF6.1 and qHTSF11.3 are newly identified loci related to spikelet fertility under high temperature stress conditions.
In the QTL region of qHTSF4.1 on chromosome 4, there is no polymorphism between Milyang23 and Giza178. Both Milyang23 and Giza178 have the tolerant allele for this QTL. This also explained why qHTSF4.1 was not identified in the Milyang23/Giza178 population. The same 384-plex SNP markers were used for genotyping IR64/N22 [30] and IR64/Giza178 populations. The QTLs identified in both populations are located in the same chromosomal regions and both of them are closely linked to SNP marker id4005120. We named this QTL as qHTSF4.1, the same name with the QTL identified from IR64/N22 population [30]. QTL qHTSF4.1 explained a similar percentage of the variation in spikelet fertility under high temperature conditions, and showed very similar genetic effects in both IR64/ Giza178 and IR64/N22 populations. This QTL is also confirmed in the three-way-cross population IR64// Milyang23/Giza178. At this locus, the mean spikelet fertility of Giza178 genotype (AA = 66.5 ± 19.5%) was significantly higher than those of IR64 genotype (aa = 55.1 ± 21.4%) and heterozygote (Aa = 50.8 ± 19.1%), and there was no significant difference between IR64 genotype and heterozygote ( Figure 2). The allele increasing spikelet fertility is from the tolerant parent Giza178 and qHTSF4.1 is controlled by a recessive gene. The same QTL controlling spikelet fertility under high temperature was also identified in a similar chromosomal region [29]. This confirms that qHTSF4.1 exists in different heat tolerant rice varieties; and therefore, could be considered important for increasing rice spikelet fertility under high temperature. Our fine mapping results also showed that qHTSF4.1 increased spikelet fertility under high temperature conditions in different backcross populations. In a BC 5 F 2 population with clean background of IR64, QTL qHTSF4.1 increased spikelet fertility by about 15% (Additional file 3).
Three QTLs identified on chromosome 1, 4 and 6 in the bi-parental populations were confirmed in the three-way population. These QTLs explained 11.6-13.0% of the variation in spikelet fertility caused by high temperature treatment. The additive effect of each QTL ranged from 4.1 to 11.7, indicating that heat tolerance at flowering stage is controlled by several QTLs with small genetic effects. There is a positive correlation between spikelet fertility and the number of alleles from Giza178 in the 5 QTL regions (r = 0.441, p < 0.0005, n = 166). The effects of these QTLs are accumulative (Figure 3). Increasing the number of QTLs in the plant will therefore, increase its heat tolerance. Thus, it is possible to pyramid these QTLs in breeding programs to improve the heat tolerance of new breeding lines.
By using two small populations (IR64/Giza178 and Milyang23/Giza178) with low marker density, we were able to identify most of the QTLs with relatively higher genetic effects. However, the QTL intervals are quite large (3.9-8.3 Mb), and the genetic effects of these QTLs were overestimated. When high-density SNP markers were used for genotyping a larger three-way population IR64// Miyang23/Giza178, big gaps were overcome. However, the recombination in the QTL regions of qHTSF1.2 and qHTSF2.1 was still low, hindering the possibility of getting higher resolution for these QTLs. On the other hand, the confidence intervals of the QTLs on chromosome 4 (qHTSF4.1), 6 (qHTSF6.1) and 11 (qHTSF11.3) were reduced to around 0.8-1.8 Mb. Though there are still many genes in the QTL regions, interestingly some heat shock protein (HSP) related genes are located in the QTL regions of qHTSF1.2 (Os01g42190) and qHTSF6.1 (Os06g13060 and Os06g14490). HSPs play fundamental roles in protecting plants against abiotic stresses, such as heat stress [46]. This may be important information for us to identify candidate genes for heat tolerance in the future.
QTLs for rice heat tolerance at flowering stage have been mapped on all chromosomes by using various rice populations and high temperature treatment methods [24][25][26][27][28][29][30][31] (Additional file 4). However, as the confidence intervals for some of the QTLs are quite large, it is still difficult to use linked markers of those QTLs for marker-assisted selection in breeding programs. At the same time, the additive effect of each QTL is low. Introducing one or a few QTLs into a plant may not be sufficient to significantly increase its heat tolerance. Introgression of more tolerant alleles will be needed to achieve higher heat tolerance. Furthermore, after high temperature treatment, the overall spikelet fertility of the Milyang23/Giza178 population (38.6 ± 25.7%) was higher than that of IR64/Giza178 population (29.6 ± 24.0%). This is because Milyang23 is more tolerant to high temperature than IR64. This suggests that using heat tolerant varieties as parents in a breeding program will increase the frequency of heat tolerant progeny in the populations. The average spikelet fertility of the three-way population was higher than those of the two bi-parental populations. This is because the three-way F 1 population was preliminarily selected after exposure to temperature closer to known critical thresholds of 35°C in the phytotron. It suggests that selection for heat tolerance at early generations is effective. Since it is difficult to accommodate large number of breeding lines in temperature-controlled conditions, it is possible to do the pre-selection using F 2 or backcrossed F 1 populations as an effective and practical strategy to save space. In this study, we identified large number of SNP markers in the QTL regions. It is possible to design specific SNP chips using those SNP markers, such as Fluidigm Dynamic Array, and using SNP chips for marker assisted selection. This will help to improve the efficiency of heat tolerance breeding.
Conclusions
In this study, we identified 8 QTLs controlling spikelet fertility under high temperature, and three of them were indentified in at least two populations. The results from different populations suggest that heat tolerance in rice at flowering stage is controlled by several QTLs with small effects and stronger heat tolerance could be attained through pyramiding validated heat tolerance QTLs. QTL qHTSF4.1 was consistently detected across different genetic backgrounds and could be an important source for enhancing heat tolerance in rice at flowering stage. Polymorphic SNP markers in these QTL regions can be used for future fine mapping and developing SNP chips for marker-assisted breeding. We are developing backcross populations to introduce the QTLs into IR64 background for further validation and fine mapping. | v3-fos |
2018-12-07T13:00:06.025Z | {
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} | s2 | Effectiveness of Endophytic Bacterial Consortium of Coffee Plant on Mortality of Pratylenchus Coffeae in Vitro
Bacteria live in wild in form of a consortium. Use of microbial consortium tends to give better results than single isolate, because the action of enzyme of each type of microbe can complement each other in order to survive. This study aimed to study the effectiveness of bacterial endophytic consortium from coffee plant on plant growth and mortality of parasitic nematodes in coffee. Isolation of bacteria is conducted by growing the crushed roots, stems and leaves of coffee on 20% TSA media, then testing their hemolysis and hypersensitivity reaction. Selected isolates were tested on their effect on the growth of seedling and Pratylenchus coffeae mortality, as well as their chitinolytic, proteolytic, lipolytic, HCN production, dissolution of phosphate (P) and fixation of nitrogen (N2) abilities. The results showed that from 27 isolates of the consortium, 23 isolates showed negative reaction to hypersensitive test and 9 isolates to hemolysis test. The highest mortality rate was shown by K6 isolate (65.8%). The highest growth was shown by K15 and K 21 isolates while the highest root length by K21 isolate. Further analisys showed that 100% of the isolates could hydrolyze proteases, lipid, and produce HCN, while chitinolytic activity was shown by 78% isolates which could fix N2 and 11% of isolates could dissolve phosphate.
INTRODUCTION
Pratylenchus coffeae nematodes is the most harmful nematodes on coffee plantations, both on Arabica coffee and Robusta coffee. P. coffeae is classified as endoparasitic sedentary or sedentary endoparasites (migratory). Female nematodes lay eggs in the root cortex tissue, especially young root tissue and eventualy damage the roots. Root damage will be more widespread with the growing population of nematodes, and finally the nematodes will move out of the roots and look for other healthy roots (CABI, 2015). P. coffeae attack on Robusta coffee and may cause a decrease in production up to 57%.
R. similis attack together with P. coffeae on Arabica coffee and can result in more severe damage reached to 80% and the plant will die at the age of less than three years (Wiyadiputra, 1998). This is due to that P. coffeae lives in the area of soil depth of 30 cm while R. similis at a depth of 50 cm (Hulupi & Mulyadi, 2007). According to Wiryadiputra & Atmawinata (1998), the decline in production by P. coffea on Robusta coffee ranged from 28.7 to 78.4%. Until the age of five years, the plant mortality rate can reach 30% of the population of coffee plantations in certain farms (Nursol et al., 2006). P. coffeae distribution in Indonesia is quite extensive, including all main coffee producing areas (Wiryadiputra & Atmawinata, 1998).
Endophyte is a micro organism that live inside plant tissues which provide a neutral, beneficial or detrimental effecs (Sikora et al., 2007). Use of endophytic bacteria as biological control has been studied in many plants, including coffee plants. Harni & Khaerati (2013) found that from 442 isolates, 12.3% were antagonistic and 14.2% can stimulate plant growth. In addition, 3 obtained isolates were potentially suppress population of P. coffeae with mortality about 94.8%. Vega et al. (2005) have isolated Bacillus, Burkholderia, Clavibacter, Curtobacterium, Escherichia, Micrococcus, Pantoea, Pseudomonas, Serratia, and Stenotrophomonas from fruits, leaves and stems of coffee.
The previous research on the ability of endophytic bacteria as biocontrol agents as described above, was the result of exploration in form of single isolate. According to Baker & Cook (1974), microbes in nature, live in a niche or the same niche, as a group of endophytic bacteria in form of a consortium, to create biological buffer area which supports biological balance, resulting in stable relationship and mutual benefit and ecosystem balance. Munif et al. (2015) had isolated and tested the potential of consortium of endophytic bacteria, from a forest, plant as biocontrol agents and plant growth promoters of tomatoes. The exploration of endophytic bacterial consortium from coffee plant and their potential as growth promoton and biological agents has not been carried out. Therefore, the aim of this research was to explore and use of endophytic bacterial consortium to suppress nematode on coffee.
MATERIALS AND METHODS
Samples of plant roots infected by P. coffeae parasitic nematode, were obtained from a multiplication carried out in Indonesian Coffee and Cocoa Research Institute, Jember. Two months old coffee seedlings were inoculated with P. coffeae as many as 50 nematodes/plant which was later used as source of nematodes for inoculation. P. coffeae nematodes were extracted from nematode infected roots with mist chamber method. Infected roots were cut about 1-2 cm and placed in a funnel covered with a filter cloth. Under the funnel a nematode reservoir container was placed connected with sprayer. The funnel filled with roots was put in a mist chamber equipped with a nozzle which produced water mist continuously. Harvesting performance of nematodes took 2-3 days after nebulization in a mist chamber. Harvesting was done by taking the solution in the container and filtered with a 500 mesh sieve. The nematodes were then counted as required.
Isolation of Endophytic Bacterial
Isolation of a consortium endophytic bacteria was conducted following the modified method of Hallmann et al. (1997). Samples of the coffee plants in form of roots, stems and leaves were washed with water and weighed as mach as one g fresh. Sterilization of tissue surface by soaking in 70% alcohol for 2 minutes, followed by immersion in NaOCl 3% + 3 drops of Tween 20 for 4 minutes, and rinsed with sterile distilled water three times. The samples were then crushed using a sterile mortar until fine size and diluted up to10 -3 . Suspension of 10 -2 dilution was grown on TSA 20% and incubated for 24 hours at room temperature. As a comparison, or control, is conducted an etching at the roots, stems and leaves were PELITA PERKEBUNAN, Volume 31, Number 3, December 2015 Edition that has been surface sterilized (before the ground) on the TSA media 20%. If in the control there was no growth of bacteria then the bacterial colony was assumed as a result of endophytic isolation.
Harvesting bacterial endophytic consortium from each source (roots, stems and leaves) was done by pouring 2 mL of TSB medium 100% + glycerol 20% on a petri dish. The colonies were isolated from solid medium using a sterile ose needle. The suspension of consortium endophytic bacteria was then stored in 1.5 mL Eppendorf tube at 4 O C.
Hypersensitivity and Hemolysis Tests
Hypersensitivity test of the endophytic bacterial consortium was conducted to determine its pathogenicity using a healthy tobacco plant. The endophytic bacterial consortium was propagated in TSB medium 100%, incubated in a shaker with the speed of 100 rpm for 24 hours. Suspension of bacterial endophytic consortium was injected using a syringe on tobacco leaves each 2 mL with three replicates for each endophytic bacteria, then incubated for 24 hours to 48 hours and observed the occurrence of chlorosis/ necrosis on the leaves. The bacterial consortium which showed a negative reaction (no necrosis) was used for further testing.
Consortium of endophytic bacteria that showed a negative reaction to the hypersensitive, was tested for its ability to hydrolyze red blood cell. The 24 hours old culture of consortium bacteria was grown at Blood Agar media, then incubated for 24 hours and observed for its clear zone formation.
Plant Growth Promoting Test
Selection of endophytic bacterial consortium as a plant growth promotor was carried out by modified method of Harni & Khaerati (2013). Hot water was applied on tomato seeds (as control). Tomato seeds were soaked in sterile distilled water at a temperature of about 55 O C for 20 minutes, followed by immersion in a suspension of endophytic bacteria consortium for approximately one hour, then drained on sterile tissue paper. Further, the seeds were planted on moistened sterile filter paper and stored in a closed sterile tray. As a control, the tomato seeds were soaked with sterile water and planted on the same medium. Observations carried out 4 days after planting.
Nematodes Mortality Test
The ability of endophytic bacterial consortium in inhibiting P. coffeae was carried out by following the modified method of Harni & Khaerati (2013). Nematode test inoculum was prepared by following the procedure of nematode preparation for in vitro endophytic test. Endophytic bacterial consortium was propagated by growing a 100 mL of culture in 100 mL TSB medium and shaked with speed 100 rpm for 24 hours. Secondary metabolites produced by the endophytic consortium bacteria was obtained by centrifugating at a speed of 6500 rpm, the supernatant was filtered with milipore 0.25 mm diameter and pore size of 0.2 µm. The filtrate was used to test the ability of the endophytic bacterial consortium on nematodes mortality. Fifty parasitic nematodes were put in 5 mL of secondary metabolites placed in a petri dish, stored at room temperature and observed after 24 hours. Each treatment was repeated three times.
Chitinolytic activity
Chitinolytic test was conducted following the modified method of Marin et al. (2013). Consortium of bacterial isolates were multiplied PELITA PERKEBUNAN, Volume 31, Number 3, December 2015 Edition in trypsic soybean broth (TSB) media and shaken for 24 hours at a speed of 100 rpm. Sterile filter paper diameter of 0.5 cm was placed on a nutrient agar medium (NA) enriched by 0.4% colloidal chitin in a petridish. A total 4 µL of culture was inoculated on the filter paper. Cultures were incubated at room temperature for 72-96 hours. Chitinolytic activity was measured based on diameter of clear zone that is formed.
Enzymes activities
Protease activity test was carried out by the modified procedure of Denizci et al. (2004). Liquid culture isolates were inoculated onto sterile paper blank disc that had been placed on the skim milk agar media (SMA), with a composition of 900 mL of 100% trypsic soy agar media (TSA) sterile plus 100 mL of sterile skim milk concentration of 10%. Incubation was done at room temperature for 24-72 hours. Proteolytic activity was shown by the formation of a clear zone surrounding the bacterial colonies (Baehaki & Budiman, 2011).
Lipolytic test used rhodamine-B mediaagar with 20 mL of liquid medium Hass Bushnell, 0.6 mL (3%) of cooking oil and 0.04 mL rhodamine-B 0.1% in water. Further, the isolates were streaked onto medium and incubated at room temperature for 48 hours. Lipolytic activity was characterized by the appearance of reddish orange luminescence zone under UV light.
Phosphatase test was carried out following the modified method of Gupta et al. (2012). Consortium isolates were inoculated on sterile paper disc in selective media Pikovskaya and observed for 48 hours at 30 O C. The ability of bacteria live in the phosphate solvent in media was characterized by the formation of a clear zone around the bacteria colony.
N 2 fixation and HCN production
N fixation ability was tested by growing isolates as much as 1 mL in semi-solid NFB (Nitrogen Fixing Bacteria) in 10 mL medium (Yim et al., 2009). Incubation was performed 4-7 days until the color changes from greenish blue media, as well as emerging pellicle/ring bacteria in media.
Production of cyanide was tested following the modified procedures of Wei et al. (1991). Bacterial culture medium was 100% nutrient agar (NA) containing 4.4 g glysin. Cyanide production was detected using cyanide detection solution (CDS), which consisted of 200 mL of sterile distilled water containing 2 g of picric acid and 8 g of sodium carbonate. Filter paper was cut to a size of 1 cm x 1 cm and sterilized. Bacterial consortium isolates was streaked in quadrant on NA media enriched with glysin. On the petri dish lid, a sterile filter paper was placed and soaked in a solvent of CDS and dried. Observation was made on day 4 after inoculation. HCN production was characterized by color changing of the filter paper from bright yellow to red orange.
RESULTS AND DISCUSSION
Isolation of endophytic bacteria consortium from coffee plants obtained 27 isolates, where 9 isolates from leaves, 9 isolates from roots, and 9 isolates from stem. Isolates were selected to obtain potential ones as biological agents. One absolute requirement of a bacterium can be used as a biological agent is that the microbe is not a potential pathogen or pathogens to plants. Baker & Cook (1974) stated that antibiotics (secondary metabolites) produced by an antagonist agent may not cause damage to host. Hypersensitivity test is conducted to collect isolates that are non pathogenic. As a result, from 27 isolates, PELITA PERKEBUNAN, Volume 31, Number 3, December 2015 Edition 23 non pathogenic isolates were obtained (Table 1, Figure 1).
Further, a microbe can be grouped as an antagonist agent if it is not pathogenic to human, therefore hemolysis test was conducted. Hemolysis is a destruction process of human erythrocytes (Segel, 2011). Hemolysin enzyme may cause lysis of erythrocytes, that are produced by several pathogenic bacteria, which is gram-negative bacteria such a Escherichia coli, Pseudomonas aeroginosa, Serratia spp., Proteus spp. and gram-positive such as Streptococcus spp., Staphylococcus aureus, Listeria spp. (Bauman, 2007). The results showed that there were 9 isolates that are gamma hemolysis, namely K6, K7, K8, K10, K12, K15, K19, K20, and 21 (Table 1, Figure 2) Nine selected isolates were tested for their ability on the mortality of P. coffeae in vitro. The high mortality percentages were shown by K6, K15 and K8 isolates in the range of 43-66% (Table 3). These results are in line with Harni & Khaerati (2013) that 12.3% single endophytic bacteria isolates tested was able to kill 5-80% P. coffeae.
The best shoot growth of tomato was shown by K21, K15 and K8 isolates followed, while for root length was shown by K21 followed with K8 and K10 isolates. The isolates were also significantly different with controls (Table 3). According to Sutariati et al. (2006), seed treatment with biological agents can improve seed germination and seedling growth of chili.
The ability of endophytic bacteria to increase the mortality of nematodes was higher than the ability of increasing the tomato shoot growth. Mechanism of endophytic bacteria in suppressing nematodes and plant growth can increase through antibiosis and induced resistance to promote plant growth. This is shown by the ability of the consortium to produce proteolytic, chitinolytic, and lipolytic enzymes, HCN production, dissolving phosphate and fixing N (Table 4 & Figure 3). (2011) mentioned that almost all of endophytic microorganism are able to perform enzymatic activity. The enzymatic activity of all tested isolates were proteolytic amylolytic, cellulolytic, esterolitik, lipolytic and asparaginase, further, that endophytic bacteria had enzymatic activity larger than the endophytic fungi.
Jalgaonwala & Mahajan
The results showed that 100% of isolates could hydrolyze proteins (proteolysis), fat (lipolytic), and produce HCN. Chitinolytic activity was show by 78% isolates meanwhile 67% isolates could fix N 2 and 11.1% of isolates could dissolve phosphates. Three (Table 4). This study showed that an endophytic bacteria can control plant pathogens via several mechanisms, including antibiosis and promoting plant growth. Jain et al. (2012) mentioned that a consortium of microbes in the rhizosphere can mediate induced systemic resistance (ISR). Chitinolytic and proteolytic bacteria has been widely reported to inhibit the development of pathogenic Bacillus spp. (Bressan & Figueiredo, 2010), Streptomyces tendae (Abdulkhair, 2012), Bacillus cereus QQ308 (Chang et al., 2006). Chitinolytic and proteolytic activity of endophytic bacteria also play a role in antibiosis against nematodes P. brachyurus (Harni et al., 2007). Carrim et al. (2006) reported that the endophytic bacteria isolated from Jacaranda decurrens Cham showed proteolytic (60%) and lipolytic (60%) activities. Proteolytic activity by endophytic bacteria can stimulate immune system of plants. Bertham & Nusantara (2011) stated that interactions with P inorganic cations (Fe, Al, and Ca) and immobilization into an organic compound by microorganisms cause low availability of P in the soil. Applications of Pseudomonas synxantha, Burkholderia gladioli, Enterobacter hormaechei and Serratia marcescens bacteria that are phosphate solubilizers can increase the availability of phosphate in soil, the absorption of phosphate in plants and plant growth and Aloe production up to 673% (Gupta et al., 2012). Bacteria of the genus Azotobacter, Bacillus, Micrococcus, Pseudomonas, Staphylococcus, Acetobacter, Escherichia, Flavobacterium, and Paracoccus are main phosphate solubilizers (Marista et al., 2013;Trivedi et al., 2007). Nitrogen is a macronutrient that plants need in large amount, so that N 2 fixation by endophytic bacteria provide more benefits because of endophytic bacteria located in the plant tissue, so the results of N 2 fixation are not easily separated. Adriano-Anaya et al. (2011) reported that the application of free-living N 2 -fixing bacteria can suppress nematode populations and Radopholus similis attack symptoms.
All consortium isolates test could produce HCN in glycine media. Heydaril et al. (2011) found that 37% of the tested fluorescent Pseudomonas bacteria were capable of producing hydrogen cyanide gas. Pseudomonas bacteria normally can produce secondary metabolites such as cyanide wich attack P. coffeae through respiratory disorder. Gallangher & Manoil (2001) found that secondary metabolites such as hydrogen cyanide was a major toxin produced by P. aeruginosa and kill Caenorhabditis elegans, a cosmopolitan soil nematode.
The consortium endophytic bacteria showed high ability in phosphate dissolution, promoting growth and N 2 fixation (Gupta et al., 2012;Suriaman, 2010). It is thought to be caused by a type of bacteria that are more variable in the consortium than single isolate, allowing colonization was faster in rhizosphere root and dominated the root. Sarma et al. (2015) in his review mentioned that biocontrol mechanisms are diverse from each bacteria in a consortium also can increase the suppression of plant diseases.
CONCLUSION
Consortium of endophytic bacteria can control P. coffeae nematodes through antibiosis mechanism of proteolytic, lipolytic, HCN production and chitinolytic, and the mechanism of induction of resistance by promoting growth through N fixation and phosphate dissolution. Consortium of endophytic bacteria is more profitable than a single isolate because of its kind that is more varied, so the ability to colonization and dominance rhizosphere and root is more quickly, as well as the mechanism of action of biocontrol agents are more variedcan improve the suppression of disease. | v3-fos |
2017-06-04T09:03:41.562Z | {
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} | 0 | [] | 2015-04-01T00:00:00.000Z | 8731072 | {
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} | s2 | Organochlorine and organophosphorus pesticide residues in fodder and milk samples along Musi river belt, India
Aim: The present study was conducted to find the organochlorine pesticide (OCP) and organophosphorus pesticide (OPP) residues in fodder and milk samples along Musi river belt, India. Materials and Methods: Fodder and milk samples collected from the six zones of Musi river belt, Hyderabad India were analyzed by gas chromatography with electron capture detector for OCP residues and pulsated flame photometric detector for the presence of OPP residues. Results: The gas chromatographic analysis of fodder samples of Zone 5 of Musi river showed the residues of dicofol at concentration of 0.07±0.0007 (0.071-0.077). Among organophosphorus compounds, dimetheoate was present in milk samples collected from Zone 6 at a level of 0.13±0.006 (0.111-0.167). The residues of OCPs, OPPs and cyclodies were below the detection limit in the remaining fodder and milk samples collected from Musi river belt in the present study. Conclusion: The results indicate that the pesticide residues in fodder and milk samples were well below the maximum residue level (MRL) values, whereas dicofol in fodder and dimethoate in milk were slightly above the MRL values specified by EU and CODEX.
Introduction
Nowadays more than 800 different kinds of pesticides are used for control of insects, rodents, fungi and unwanted plants in the process of agricultural production. Although most of them leave the products or degrade in soil, water and atmosphere, these chemicals can be transferred to human via food chain [1]. Furthermore, breeding animals and their accommodation can themselves be sprayed with pesticide solution to prevent pest infestations. Consequently, both these contamination routes can lead to bioaccumulation of persistent pesticides in food products of animal origin such as meat, fat, fish eggs and milk [2,3]. Moreover, health departments also used these chemicals for controlling various insects having vector role in spreading the disease like malaria, dengue fever and plague [4,5]. Many pesticides and their residues have been reported as contributory factors in several diseases such as heart disease, cancers, Alzheimer's disease and parkinsonism [6,7]. Pesticide residues in feed and fodders may transfer into herbivores through food chain of the animals [8]. Due to the lipophilic nature of these pesticides, milk and other fat-rich substances are the key items for their accumulation. These toxicants get into the human body through the food chain and cause serious health problems [7].
Musi river is located on the Deccan platue in the state of Andhra Pradesh, India. However, now the water is highly polluted as 600 million liters per day of untreated sewage water is discharged into Musi river, additionally 14 industrial estates drain their untreated effluents into this river. The agricultural drained water is another source of pollution and this river water is rich in heavy metals, pesticide residues, phenols, oils, grease, alkalis and acids [9]. The self-purifying property of river water is unable to clear the pollution, and the polluted water poses a serious risk to public health especially in areas where river water is used for irrigation.
Keeping this in view of the Musi river pollution and its direct or indirect effect on environment, animal and human system, a study was conducted to analyze the fodder and milk samples on the banks of river Musi for the presence of pesticide residues. The study has been conducted on river Musi, Located in Andhra Pradesh, India.
Ethical approval
No animals were harmed or given stress during the collection of milk samples.
Collection of samples
This study was based on 48 fodder and 48 milk samples collected from six divided zones (8 from each zone) (Table-1) on the downstream of Musi river belt, Andhra Pradesh, India in 2013. Zones were divided based on earlier reports on Musi river pollution by Pullaiah [9]. Approximately, 250 g of fodder samples were collected in sterilized polyethylene packs, packed and transported to lab. Sterilized glass bottles were used to collect 250 ml milk samples, labeled and transported to lab in ice pack, they were kept at 4°C until analysis. Samples were subjected to analysis within 24 h from their arrival.
Chemicals and reagents
Acetonitrile, acetone, dichloromethane, graphitized carbon black, hexane, magnesium sulfate, silica gel, sodium chloride, sodium sulfate, prostate specific antigen (PSA) of high-performance liquid chromatography residue grade obtained from Qualigens and Merck specialties Pvt. Ltd. Analytical standards with >99% purity were obtained from Dr. Ehrenstorfer, Germany during 2012 and stored in deep freeze maintained at −40°C.
Method validation
The required quantity of (organochlorine and organophosphorus) international standards prepared from certified reference materials were added to each 15 g sample to get fortification levels of 0.05 ppm and 0.1 ppm in three replications each. The AOAC official method 2007.01 with slight modifications was validated for the estimation of the limit of quantification (LOQ) of organochlorine and organophosphorus in fodder and milk. Fodder samples were chopped, and 7.5 g of sample was taken into 50 ml centrifuge tubes and 30 ml of acetonitrile was added and shaken well. The sample was homogenized at 14000-15000 RPM for 2-3 min using heidolp silent crusher then 3 g of sodium chloride was added, mixed well by shaking gently then it was centrifuged at 2500-3000 RPM for 3 min to separate the organic layer, approx. 16 ml of organic layer was taken into a test tube and 9 g of anhydrous sodium sulfate was added to remove moisture [1]. Taken about 0.4 g PSA sorbent and 1.2 g anhydrous magnesium sulfate into 15 ml centrifuge tubes. The 8 ml of organic layer extract was transferred into this 15 ml centrifuge tube, capped and vortex for 30 s, then tubes were centrifuged at 2500-3000 RPM for 5 min then 2 ml of extract was transferred into test tubes and the solvent (acetonitrile) was evaporated turbovap concentrator for GC analysis. Whereas for milk samples, 5 g of milk was taken into 250 ml beaker and 20 g of silica gel and 20 g of anhydrous sodium sulfate was added. Glass column was prepared with 40 ml of dichloromethane over cotton plug, sample was made into slurry with dichloromethane then this was transfer to column and allowed to stand for 90 min then dichloromethane was eluted dropwise, again the sample column was eluted with a mixture of 150 ml acetone: Dichloromethane (2:1 v/w) and anhydrous sodium sulfate was added to the elute, then concentrated to 2-3 ml, 10-15 ml of hexane was added to the concentrate to remove dichloromethane completely, volume was made with n-hexane. Finally, an aliquot of each extract was transferred to 2 ml injection vials to be ready for the analysis.
A Schimadzu 2010 gas chromatography (GC) equipment with a VF-1MS capillary column and with electron capture detector (ECD) and flame photometric detector. All the chemicals were purchased from M/s. Merck specialties Pvt. Ltd and were pesticide residue grade and all pesticide residue standards were purchased from Dr. Erhenstorfer, Germany during 2012. The gas chromatographic analysis was performed under the following conditions (Table-2). A volume of 1 ml sample was injected into the GC; peaks were identified by comparing their retention times with those of standards under the same injection conditions (Table-3). The peak areas of the various peaks whose retention times coincide with the standards were extracted on their corresponding calibration curves to obtain the concentrations.
Results and Discussion
A total of 48 fodder samples and 48 milk samples collected from all the six zones of Musi river belt and were analyzed for OCPs and OPPs residues. Concentration of various residues in each sample was calculated (in mg/kg sample). In the present study, the average recoveries of OCPs in fodder were from 88.05% at 0.05 ppm and 86.71% at 0.1 ppm and in milk were from 88.45% at 0.05 ppm and 91.25% at 0.1 ppm. Average recoveries of OPPs in fodder were 91.27% at 0.05 ppm and 94.67% at 0.1 ppm and in milk were from 91.25% at 0.05 ppm and 86.77% at 0.1 ppm. The efficiency of extraction methodologies were evaluated based on the recoveries of residues, and a recovery of 75-102% is considered as acceptable [10]. Hence, the extraction procedures employed in these experiments were efficient in recovering the maximum amount of residues present in the samples. The elute pattern of various OCPs (0.01 ppm) (Figure-1) and OPPs (0.05 ppm) along with specific retention time are depicted in Figure-2 for ECD and Figure-3 for pulsated flame photometric detector (PFPD). The limit of detection and LOQ for OCPs was 0.01 ppm and 0.05 ppm respectively and for OPPS was 0.05 ppm and 0.05 ppm respectively for both ECD and PFPD.
The prevalence of residues of p,p' DDE, p,p' DDT and total DDT in fodder was 5%, 60% and 3% respectively, was reported by Nagra [11]. Residue levels of o,p' DDT of 0.006 ppm in fodder was reported by Panseri [12]. A residue levels of total DDT of 0.17 ppm in fodder was reported by Nagra [11].
Residue levels of aldrin of 0.004 ppm and 0.03 ppm in fodder were reported by Panseri [12] and Nagra [11] respectively. A residue level of 0.007 ppm and 0.045 ppm was reported by Panseri [12] and Deka [13] in fodder samples, which is far below the specified MRL value by EU (CE: 698: 2005) is 0.1 ppm, whereas higher levels (0.42 ppm) was reported by Aulakh [14] for endosulphan sulfate. Residual concentration of 0.001 ppm and 0.02 ppm were reported by Panseri [12] and Aulakh [14] respectively in the fodder samples, which are far below the MRL value (0.01 ppm) specified by EU (CE:398: 2005) in fodder for heptachlor. Fagnani et al. [15] reported the residual concentration of dimethoate in fodder sample as 0.01 μgl −1 .
For milk samples also the same OCP compounds, cyclodiene compounds and organophosphorus compounds conducted for fodder samples were analyzed. Milk samples from zone V contain the residual concentration of dimithioate of 0.13 ppm (Figure-5), which is higher than concentrations of 0.01 μgl −1 for dimithioate reported by Fagnani et al. [15]. In the Table-4 present study except dimithioate all other OCPs compounds, cyclodiene compounds and organophosphorus compounds were below the detection limit in milk samples.
The levels of PP DDE were 0.028 ppm in cow milk [16] and 0.039 ppm in mixed milk [17]. Donia [16] reported 0.014 ppm and 0.009 ppm levels of o,p' DDD in buffalo and cow milk respectively. These findings are far below than the MRL value of 1.25 ppm for milk specified by PFA [18]. The residues of o,p' DDD of 0.002 ppm was reported by Panseri [12], which is far below than the MRL level (0.05 ppm) specified by EU (CE: 698: 2005). Residue levels of P'P'-DDT of 0.038 and 0.033 ppm were reported by Mohd Aslam [19] and Kampire et al. [20] respectively.
Donia [16] reported 0.022 ppm and 0.032 ppm in buffalo and cow milk respectively, whereas Waliszewski et al. [21] reported residue levels of 0.0372 and 0.078 in mixed milk for p,p' DDT. Residue levels of 0.007 ppm and 0.056 ppm in milk were reported by Cerkvenik [22] and Radzyminska [23] respectively, which were below than the MRL value (0.04 ppm) of EU (CE:698: 2005) for o,p' DDT.
Residue levels of aldrin at a level of 0.004 ppm were reported by Ahmed and Zaki [24] in mixed milk, whereas 0.066 ppm and 0.036 ppm were reported by Donia [16] in buffalo milk and cow milk respectively. All the values reported by the above scientists were below than the MRL value (0.2 ppm) of EU (CE: 698: 2005). For endosulphan sulfate, the residue levels of 0.002 ppm and 0.26 ppm were reported by Ahmed and Zaki [24] and Muhammad [25] respectively and prevalence of 9.09% was reported by Ahmed and Zaki [24] in mixed milk sample. The residue levels of heptachlor of 0.003 ppm and 0.022 ppm were reported by Cerkvenik [22] and Donia et al. [16] respectively, A prevalence of 16.16% of heptachlor was reported by Ahmad and Zaki [24] in mixed milk.
Conclusion
From this study, it can be concluded that all the pesticide residues in fodder and milk samples were below the MRL except dicofol in fodder and dimethoate in milk were slightly above the MRL values specified by EU and CODEX it might be due to use of these pesticides on vegetable crops grown on the banks of Musi river belt. However, the results of OPPs in different samples were detected by ECD and confirmed by PFPD, whereas O.C.'s were detected by only ECD but not confirmed by Mass Spectrometry due to non-availability of equipment. Owing to effects on human, animal and environmental health of pesticide residues need for education and awareness among farmers about extensive use of pesticide was envisaged. | v3-fos |
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} | s2 | Genomic data as the "hitchhiker's guide" to cattle adaptation: tracking the milestones of past selection in the bovine genome.
The bovine species have witnessed and played a major role in the drastic socio-economical changes that shaped our culture over the last 10,000 years. During this journey, cattle "hitchhiked" on human development and colonized the world, facing strong selective pressures such as dramatic environmental changes and disease challenge. Consequently, hundreds of specialized cattle breeds emerged and spread around the globe, making up a rich spectrum of genomic resources. Their DNA still carry the scars left from adapting to this wide range of conditions, and we are now empowered with data and analytical tools to track the milestones of past selection in their genomes. In this review paper, we provide a summary of the reconstructed demographic events that shaped cattle diversity, offer a critical synthesis of popular methodologies applied to the search for signatures of selection (SS) in genomic data, and give examples of recent SS studies in cattle. Then, we outline the potential and challenges of the application of SS analysis in cattle, and discuss the future directions in this field.
INTRODUCTION
The ability of domestic cattle to convert low-quality forage into meat, milk, and draft power is of direct importance to the livelihood of the human species. This ability is tightly linked to the adaptation of indigenous and exotic cattle to the diverse environments found around the world, which may result from complex-mostly untold-stories of migration, expansion, exposure to diseases, admixture, climate changes, and selective pressures (Ajmone-Marsan, 2010). These past events have shaped the genetic diversity of domestic cattle throughout history, and their present genomes may shelter tractable signatures of these phenomena.
Footprints of selection, such as specific patterns of change in allele frequencies, diversity loss, and haplotype structure, are currently detectable from single nucleotide polymorphism (SNP) data by well-established methodologies (Sabeti et al., 2006;Oleksyk et al., 2010), and can unravel past responses of the cattle genome to natural and human-driven selection, as well as evidences of loci and variants underlying adaptive and economically important traits. Detecting these selection signatures (SS) may not only help to shed some light on the key adaptive events that have generated the enormous phenotypic variation observed between cattle breeds today, but can also be of biotechnological relevance.
In recent years, SS studies are becoming increasingly popular because they offer a complementary strategy to genome-wide association (GWA) studies on mapping variants impacting traits of interest, helping to link phenotypes to gene function. In a typical GWA analysis, one starts from a phenotype and scan genotypes to identify underlying large and moderate effect variants (Bush and Moore, 2012). Generally, SS studies take the opposite direction: one starts with an evidence of selection in samples sharing geographical proximity, environmental factors or a common phenotype, and attempts to find selected mutations (Sabeti et al., 2006). The major motivation for SS studies is that this type of approach can pinpoint chromosomal segments sheltering large effect mutations even if they no longer segregate in a population. In such cases, these variants cannot be detected by classical quantitative genetics methods unless linkage experiments are designed using crosses (Ramey et al., 2013). Another appealing feature of SS studies is that they typically require over 10 fold smaller sample sizes in comparison to GWA studies. Moreover, SS can reveal signals on genes controlling traits that are difficult, expensive or even impossible to measure on a large population (i.e., disease resistance).
Although identifying SS is of paramount importance to uncover variants responsible for adaptive traits, its application to cattle data must be carefully interpreted as important demographic events such as severe population bottlenecks, genetic drift, and admixture, as well as confounding effects derived from the development of SNP panels, may give rise to false signals. Furthermore, SS have serious limitations in targeting specific traits, so assigning signals to phenotypes is a non-trivial problem.
Here, we provide a brief review of the demographic history of cattle as it is known, present a critical review of some of the main methodologies available for the detection of putative loci under selection, and provide examples of recently published results in cattle. Then, we outline the potential and the challenges of the application of these methods to cattle data, and discuss the future directions in this field.
CATTLE DEMOGRAPHIC HISTORY DIFFERENTIATION AND DOMESTICATION
The humpless taurine (Bos taurus) and the humped indicine or zebu cattle (Bos indicus) descend from the wild ox or auroch (Bos primigenius), which has been extinct since 1627 (Mona et al., 2010). The two populations of wild aurochsen that formed the ancestral pool for these interfertile cattle species may have diverged some 280,000 years ago (Murray et al., 2010), and were subjected to many demographic events before being domesticated by our species, including severe bottlenecks, admixture and natural selection.
Although the domestication of taurine and zebu cattle is still an open question and an active field of investigation, evidences collected over 20 years of research on molecular genetic diversity of cattle (see Groeneveld et al., 2010, for further review), combined with historic and archaeological data, support that these species were independently domesticated in at least two episodes (Loftus et al., 1994;Bruford et al., 2003): some 10,000 years ago, taurine animals were captured from the wild in the Fertile Crescent (modern-day countries of Israel, Jordan, Lebanon, Cyprus and Syria, and parts from Egypt, Turkey, Iraq, Iran, and Kuwait); 1500 years later, zebu cattle were domesticated in the Indus valley (present Northeastern Afghanistan, Pakistan, and Northwestern India).
EXPANSION
Taurine cattle have an almost cosmopolitan distribution today. From the domestication centre in Southwestern Asia, they followed human migrations and slowly expanded over Asia and Europe. An independent domestication episode of taurine cattle in Northeastern Africa is disputed, but as molecular data are not conclusive, the divergence of African and Eurasian taurine populations from a common ancestor domesticated in the Fertile Crescent is well supported (Hanotte et al., 2002;Ajmone-Marsan, 2010;Decker et al., 2014). Other isolated micro-events of domestication are also not discarded, not even interbreeding of domestic animals with wild aurochsen (Bonfiglio et al., 2010). Taurine cattle reached the New World by European importations after the discovery of America in the late fifteenth century, and their descendants living today are broadly referred as Creole cattle.
Zebu cattle also spread around the world accompanying human migrations, but became more endemic in tropical and subtropical regions due to their adaptability to these environments. Zebu cattle were probably introduced in the African continent in the seventh century by imports of B. indicus sires during Arabian invasions (Bradley et al., 1996). Indian zebu cattle were only introduced in Central and South America in the early twentieth century, and started to be massively imported to the American continent after 1950.
FORMATION OF SPECIALIZED BREEDS AND FURTHER CATTLE ADAPTATION
After domestication, farmers started to control cattle mating according to their interest in traits such as size, behavior, and milk production. This "breed the best to the best and hope for the best" strategy exerted a high artificial selective pressure, triggering a severe decline in cattle effective population size. Estimates from a genome-wide linkage disequilibrium analysis using a medium density SNP panel suggested that domestication was responsible for a 50 to 70-fold decline in the effective population size in comparison to the wild ancestors of taurine and zebu cattle (The Bovine HapMap Consortium, 2009).
About 200 years ago, farmers invented the concept of breed and started to limit germplasm exchange in order to standardize cattle populations based on morphology and performance, giving rise to over 1200 cataloged cattle breeds today (Taberlet et al., 2008; see also: http://www.ansi.okstate.edu/breeds/cattle/). In spite of this apparent rich source of genetic resources, over 200 additional breeds are already extinct, and many others are at risk (FAO, 2007).
Due to their high productive performance for milk and meat traits, some cattle breeds were adopted worldwide, such as the Holstein-Friesian, which is present in 128 countries (FAO, 2007). However, most of these specialized groups of genetically distinct animals are local, and even though they are not under the spotlights, they exhibit high adaption to their environments. Therefore, small local breeds should be considered as primary targets of SS studies, as several of them are endangered and their genomes shelter footprints of adaptation.
IMPLICATIONS TO POSITIVE SELECTION MAPPING
In theory, neutrally evolving alleles form the majority of the genome variation within and between species, and events such as population contraction, expansion, migration, isolation and admixture are responsible for random drift of such alleles. While the demographic history of a population determines its neutral allele frequency spectrum, alleles that impact fitness, survival, reproduction, or traits of human interest are subjected to natural or artificial selection, such that their frequencies deviate from the distribution of neutral alleles. Therefore, mapping loci under selection implicates searching for outlier alleles that substantially differ from the genome background.
The idea of outlier loci is dependent upon knowledge about the frequency distribution of neutrally evolving alleles. As different populations have distinct demographic histories, their allele frequency spectrum also differ. Consequently, statistical methods designed to map loci under selection must be calibrated to either a demographic model for the population under study or to the empirical distribution of scores across the genome, assuming most of the genome is evolving under neutrality. As most of the events that shaped cattle diversity at the species and breed level are still obscure, methods that are robust across different demographic scenarios that take advantage of genome-wide scores to detect outlier loci are more appropriate to analyze cattle data. Nevertheless, one can use the demographic model for cattle differentiation, domestication and expansion provided in this section and combine with specific models for specialized cattle breeds to simulate the distribution under neutrality and compare with empirical data.
METHODS FOR DETECTING POSITIVE SELECTION IN THE CATTLE GENOME
As proposed by Darwin and Wallace (1858), positive selection is the phenomenon where phenotypes that increase the likelihood of survival and reproduction (i.e., that increase an individual's fitness) become more prevalent in populations over time. In the context of the genome sequence, if a specific allele confers advantage, its carrier is more likely to thrive and leave more offspring than non-carriers, causing the haplotype containing that beneficial allele to spread quickly and increase in frequency in the population (Sabeti et al., 2002(Sabeti et al., , 2006. The majority of the genetic variation found within and between populations is deemed to have little or no effect on fitness, so that haplotype frequencies follow Hardy-Weinberg expectations (Kimura, 1968;Hellmann et al., 2003). Therefore, positive selection leaves distinctive tractable patterns of genetic variation that differ from the neutrally evolving background DNA sequence. These patterns are broadly referred as "signatures" or "footprints" of selection (Oleksyk et al., 2010).
In this section, we describe classes of signatures in terms of different signals. In each class, whenever convenient, we describe the basis of some popularly adopted methods to highlight their strengths and weaknesses for cattle studies. For a broader overview, we suggest the reader to consult other previously published reviews on the topic (Sabeti et al., 2006;Oleksyk et al., 2010;Vitti et al., 2013;Qanbari and Simianer, 2014). Additionally, we provide examples of studies reporting putative loci under selection in different cattle breeds.
LOCAL GENETIC DIVERSITY DEPRESSION
At each generation, recombination shuffles and breaks haplotypes down, producing linkage equilibrium. When selection increases the frequency of an advantageous allele, neighbor variants "hitchhike" and rise in frequency together so quickly that recombination does not prevent linkage disequilibrium, causing an entire chromosome segment to lose diversity. Therefore, positive selection can be probed by searching for chromosome regions where heterozygosity is much lower than expected under neutrality (Oleksyk et al., 2008). Ramey et al. (2013) scanned Illumina® BovineSNP50 (50 k) genotypes of over 6000 animals from 13 taurine and one zebu breed using an ad-hoc method to identify selective sweeps reaching fixation. Briefly, they considered a candidate locus under selection if at least five contiguous SNPs presenting a minor allele frequency (MAF) > 0.01 spanned a chromosome segment of 200 kb or more. Although the strategy was successful to find validated selective sweeps, such as the POLL locus that controls horn development (Georges et al., 1993), they showed that the SNP ascertainment bias of the 50 k assay incurred in false positives in breeds that are genetically distant from the SNP discovery breeds. This is not unexpected, as the influence of the 50 k bias on population genetics parameters, such as heterozygosity, genetic structure and differentiation, is well documented (Decker et al., 2009). Therefore, methods relying on heterozygosity are largely prone to type I errors generated from the SNP discovery process. Ideally, genotype data should be generated from less biased commercial arrays, such as the Illumina® BovineHD (HD), or from re-sequencing efforts.
Another approach to search for deficit of heterozygosity is the identification of islands of runs of homozygosity (ROH). As long stretches of consecutive homozygous genotypes indicate identical-by-descent haplotypes (Gibson et al., 2006;Lencz et al., 2007), ROH have been recently used to characterize genomewide inbreeding in cattle (Purfield et al., 2012;Ferenčaković et al., 2013;Kim et al., 2013). However, as recombination is random, the distribution of ROH across samples is expected to be highly heterogeneous under neutrality, so genomic hotspots of ROH can be a signal of selective sweep (Curik et al., 2014). Interestingly, the length of the run is negatively correlated with the number of generations back to the selective pressure or inbreeding event (Howrigan et al., 2011), so ROH size can be used to date the age of the selective sweep.
Focusing on the identification of ROH islands produced by recent artificial selection in U.S. Holstein cattle, Kim et al. (2013) proposed a new statistic, namely F RL , that compares local homozygosity between a population under artificial selection and a control population. Briefly, for each SNP, animals are scored as 1 if the locus is encompassed by a ROH or 0 otherwise. Then, F L is computed as the proportion of animals with scores equal to 1. Finally, F RL is obtained as: All scores are standardized (i.e., subtracting each value by the average score and dividing by the standard deviation) to produce a distribution with mean zero and variance one. Extreme positive values of F RL represent changes in allele frequency in the artificially selected population in comparison to the control group, and therefore reflect homozygosity attributable to recent inbreeding, drift or selection at the analyzed locus. As for the approach adopted by Ramey et al. (2013), SNP density and ascertainment bias are important confounders that can produce false positive ROH (Ferenčaković et al., 2013), so commercial SNP assays must be used with caution here. Methods that search for local absence of heterozygosity are most powerful to detect haplotypes that hitchhiked to fixation. However, allowing for some heterozygosity can be of help to detect partial sweeps, i.e., haplotypes under ongoing selection that have not reached fixation yet. For a given bi-allelic site, let n REF be the number of observed reference alleles, n ALT the number of observed alternative alleles, and p and q their respective frequencies. The expected frequency of heterozygote genotypes under Hardy-Weinberg Equilibrium is given by: This expectation can be easily extended for a chromosomal segment containing multiple bi-allelic sites as: Rubin et al. (2010) proposed running sliding windows across the genome, calculating H for these windows and then standardizing the obtained values. The method named as ZH p produces standard deviation scores, and extremely negative values represent chromosome windows where the regional diversity is substantially lower than the average genome diversity. Although the method has been successfully used to map candidate variants under selection in chickens (Rubin et al., 2010), dogs (Axelsson et al., 2013) and pigs (Rubin et al., 2012), no studies applying ZH p to cattle data have been published to date.
CHANGE IN THE ALLELE FREQUENCY SPECTRUM
After a complete selective sweep (i.e., the haplotype containing the selected variant reaches fixation), new mutations slowly restore local diversity in the course of many generations. As mutations are generally rare and may take a large number of generations to drift to high frequency under neutral evolution, the local heterozygosity depression signal is deemed to remain robust for several generations after the selective pressure has occurred.
Newly acquired mutations or derived alleles (i.e., variants that differ from the original or ancestral allele) occur in lower frequency in comparison to ancestral alleles under neutrality, but when they arise within a selective sweep they will hitchhike to high frequency quickly in the selected population. Therefore, another class of signals that emerge after the depression in local diversity is the enrichment for moderate to high frequency derived alleles. Several methods have been proposed in this category, including Tajima's (1983), Fay and Wu's (2000), and DAF (Grossman et al., 2010).
The limitation of the use of these methods in cattle data is that inference of ancestral alleles should be preferably performed by comparison of domesticated cattle genomes with wild type genomes. As these are not available, ancestral allele information for cattle SNP assays were derived from genotypes of outgroup species, such as Gaur (Bos gaurus), Buffalo (Bubalus bubalis) and Yak (Bos grunniens), which are assumed to descend from a common founder Bovinae species (Matukumalli et al., 2009;Utsunomiya et al., 2013). However, as not all SNP probes hybridize against the genomes of these outgroup species, ancestral allele information for cattle is incomplete. Future availability of genome assemblies for these outgroup species may help to better infer ancestral status for common SNPs.
One way to bypass the limitation of ancestral allele information is by using samples from several genetically distinct populations to estimate average allele frequencies that could represent the spectrum in the common ancestral population. In this approach, instead of searching for enrichment of high frequency derived or rare alleles, one may look for a shift in the allele frequency spectrum in one population in comparison to the average across populations. Stella et al. (2010) successfully applied the negative composite log-likelihood (CLL) approach, an extension of the composite likelihood ratio (CLR) test (Kim and Stephan, 2002;Nielsen et al., 2005) to 13 taurine, 4 zebu and 2 synthetic breeds, and reported several candidate loci under selection. These included KIT (mast/stem cell growth factor receptor gene), responsible for the piebald and color sidedness phenotype (Durkin et al., 2012), and MC1R (melanocortin 1 receptor gene), incriminated in the black/red coat color in Holstein and Angus (Klungland et al., 1995). These loci were further confirmed using whole genome sequence data of German Fleckvieh .
Briefly, CLL is computed for each SNP window as: Where, relative to SNP i, d is any random value from the theoretical distribution of allele frequencies with mean μ i = p i , p i is the reference allele frequency averaged across populations, and p i is the allele frequency in the population being investigated. The theoretical distribution of allele frequencies can be modeled as a binomial or a normal approximation to the binomial distribution. As calculations are very straightforward and only require a dataset with multiple breeds instead of ancestral allele information, it is general enough for cattle SNP data. Another extension of the CLR statistic was proposed by Chen et al. (2010), namely XPCLR (cross-population composite likelihood ratio test), which attempts to detect a shift in allele frequency in a target population in respect to a reference population. Lee et al. (2014) applied XPCLR to Holstein and Hanwoo whole genome sequence data and reported that the chromosome segment encompassing the kappa-casein gene (CSN3) exhibited high XPCLR scores in Holstein cattle.
LONG-RANGE HAPLOTYPES
The concept of Extended Haplotype Homozygosity (EHH), first introduced by Sabeti et al. (2002), attempts to identify haplotypes that increased so rapidly in frequency that recombination could not substantially break them down, so linkage disequilibrium presents a long-range persistency. Briefly, consider N chromosomes in a sample, and G unique haplotypes extending from a core SNP site to an upstream or downstream position x, with each group g having n g observations. The EHH score for the entire sample is calculated as: This score serves as a proxy for the probability of identity-bydescent of haplotypes within the chromosome segment being investigated. Generally, EHH is calculated at varying distances from the core SNP position, so that the decay of EHH as a
Frontiers in Genetics | Livestock Genomics
February 2015 | Volume 6 | Article 36 | 4 function of physical distance can be assessed to determine the extension of the haplotype homozygosity. From this seminal concept, a family of statistical methods was developed in order to scan entire genomes in the search for evidence of selection. Voight et al. (2006) proposed to measure how rapidly EHH decays from a core SNP site by calculating the area under the EHH curve, where iHH represents the definite integral of EHH evaluated over the domain of the chromosome segment delimited by upstream position a and downstream position b where EHH decays to some arbitrary small value (originally 0.05). As the area under the curve is not tractable analytically, a trapezoid quadrature with nonuniform grid can be adopted as a deterministic approximation: A within population score, namely Integrated Haplotype Score (iHS), for a given site i, was introduced by Voight et al. (2006) as the log-ratio between the integrated EHH for the haplotypes containing the ancestral allele (iHH A ) and the derived allele (iHH D ): These scores are then standardized to have mean zero and variance one. Extremely negative standardized iHS values have been of particular interest in human genetics, as they represent a recently acquired mutation that increased very rapidly in frequency (i.e., there is a partial sweep due to ongoing selection) or a haplotype that hitchhiked to fixation and then became enriched for derived alleles (Voight et al., 2006). However, a sweep can also produce large positive iHS values at nearby SNPs if ancestral alleles hitchhike with the selected site, so the chromosome region surrounding the selected variant typically exhibits a cluster of extreme positive and negative iHS values. Furthermore, in the context of cattle data, artificial selection and domestication probably favored "beneficial" alleles in the sense of human interest, regardless if it is ancestral or derived. Therefore, both positive and negative values should be investigated in cattle data. This implicates that the absolute value of standardized iHS scores should be preferred over the signed values, or, equivalently, that a two tailed hypothesis test should be assumed. As only partial ancestral allele information is available for cattle SNP assays, and the search for footprints of selection by iHS in cattle should disregard the direction of the sweep, a more appropriate generalized version of iHS can be postulated as the log-ratio between the integrated EHH for an arbitrary reference allele (iHH REF ) and for the alternative allele (iHH ALT ).
One of the limitations of this method is that if a given marker presents a nearly or completely fixed allele in the population being analyzed, this allele will have no integral to be calculated or an integral close to zero, so the log-ratio will result in a positive or negative infinite value. In this scenario, the calculation of iHS must be conditioned by iHH REF > 0 and iHH ALT > 0, which indirectly leads to a minor allele frequency (MAF) constraint. This limitation renders iHS underpowered to detect very recent nearly fixed selective sweeps, which are of primary interest in the cattle community. However, as discussed earlier, a crucial point to be considered is that contiguous chromosomal segments containing SNPs with MAF = 0 can also result from SNP chip ascertainment bias, which may produce false positive signals. Tang et al. (2007) and Sabeti et al. (2007) have independently developed equivalent methods, Rsb and XPEHH, respectively, which attempt to compare long-range haplotypes between populations in order to increase the power of selective sweep detection. The most crucial improvement is that, for each population being analyzed iHH is calculated for the entire sample, instead of being partitioned between derived and ancestral alleles. This eliminates the MAF constraint and recovers the power to detect sweeps reaching fixation. The comparison with a population where the selective sweep may not have occurred adds extra power to the method. Calculations are performed as follows: Where, relative to SNP i, iHH pop1,i is the integrated EHH in the first population and iHH pop2,i is the integrated EHH in the second population. Scores are also standardized to produce a distribution of standard deviates. Positive values indicate selective sweeps in the population used in the numerator, while negative values indicate selection in the population used in the denominator. Here, it is easy to keep track of the signals by using one-tailed hypothesis tests. Studies applying EHH-based methods to cattle data are numerous (for instance, Hayes et al., 2008;Gautier and Naves, 2011;Qanbari et al., 2011Flori et al., 2012;Utsunomiya et al., 2013;Huson et al., 2014). The reported loci are deemed to be genome responses to a variety of different selective pressures, such as milk and meat production, coat color, heat stress, and reproductive performance. Among these, one particularly interesting selective sweep, most likely related to adaptation to heat stress, has been reported in Creole cattle, including Senepol, Carora, Romosinuano, and cross-bred lineages (Flori et al., 2012;Huson et al., 2014). These cattle breeds present the slick hair coat phenotype, a dominant trait associated to heat tolerance in tropically adapted cattle that descend from Spanish cattle introduced to the New World. The chromosome segment containing the selective sweep ranges from 37.5 to 39.6 Mb on chromosome 20, with a variable peaking position (39.5 or 37.7 Mb) depending on the SNP panel (BovineSNP50 or BovineHD) and dataset analyzed (Flori et al., 2012;Huson et al., 2014). The disputed positional candidate genes are the retinoic acid induced 14 (RAI14 or NORPEG), prolactin receptor (PRLR), and S-phase kinaseassociated protein 2 (SKP2). A strong candidate mutation has been recently proposed for PRLR, a single base deletion in exon 10 (ss1067289408) predicted to cause a frameshift that introduces a premature stop codon (p.Leu462 * ) and consequent loss of 120 C-terminal amino acids from the long isoform of the prolactin receptor (Littlejohn et al., 2014).
POPULATION DIFFERENTIATION
Following the same principle as in Rsb/XPEHH, XPCLR and F RL , although positive selection may act across populations sharing geographical proximity, environmental factors or a common phenotype, outgroup populations may not share the same selective pressure. Therefore, changes in allele frequency promoted by selection in one group will not be detectable in the other, and large differences in allele frequencies between populations will be observed.
The fixation index F ST (Wright, 1950;Weir and Cockerham, 1984) and its abundant estimators is the gold standard method for detecting highly differentiated loci between populations. In essence, given the average allele frequency p across subpopulations, F ST is simply the ratio between the variance in the allele frequency in different subpopulations σ 2 S = k j = 1 p j − p 2 and the variance in the total population σ 2 T = p(1 − p). In pair-wise comparisons, calculations simplify to: Scores can be averaged across SNP windows or smoothed against genomic positions using a local variable bandwidth kernel estimator (Porto-Neto et al., 2013). Flori et al. (2009) applied F ST to three French dairy cattle breeds (Holstein, Normande, and Montbéliarde), and found that some of the putative loci under selection in that study overlapped genes that strongly affect milk production traits, such as the growth hormone receptor (GHR), and coat color, for instance MC1R. Porto-Neto et al. (2013) generated a comprehensive map of divergent loci between taurine and zebu cattle using over 777,000 SNPs and 13 cattle breeds, and reported that the highest scoring locus in the F ST analysis maps to chromosome 7:47.2-53.7 Mb, which shelters a cluster of immune-related genes and SPOCK1, a gene previously implicated in puberty (Fortes et al., 2010).
Extensions of F ST led to the development of the FLK test (Lewontin and Krakauer, 1973;Bonhomme et al., 2010;Fariello et al., 2013), which not only account for effective population size and hierarchical structure among populations, but also have known distributional properties under neutrality. Briefly, let q i be the vector of reference allele frequencies at marker i for the populations being compared, and q 0 the ancestral allele frequency for the same allele. The FLK method relies on the linear model: where the residual term e is assumed N (0, V i ), and V i is the expected variance-covariance matrix for vector q i . This matrix is modeled as: where F is a kinship matrix. The diagonal elements of F represent the expected inbreeding coefficients in each respective population, and off-diagonal elements represent the amount of drift accumulated on the different branches of the population tree. A weighted least squares estimate of q 0 is then obtained as: The FLK score is a measure of goodness-of-fit of this model, and is calculated as the deviance (i.e., residual sum of squares): Under the assumption of a star-like tree-pure drift model (i.e., the populations being compared evolved in parallel from a single ancestral population, with no mutations or admixture), matrix F can be simplified to F = I n F ST , where I n is an identity matrix and F ST is the average F ST over all SNP loci. In this case, the average allele frequency across populations is an unbiased estimator of q 0 , and the deviance is simplified to F ST (n − 1)/F ST , which gives a test statistic that is linearly correlated with F ST . As discussed later, while F ST has no known theoretical distribution under the neutral model, FLK can be modeled as a chi-squared distribution. Moreover, FLK outperformed F ST in simulations, especially when scores were computed based on haplotypes (hapFLK) instead of single markers (Fariello et al., 2013). This method is a suitable alternative to F ST for cattle data.
DISTRIBUTIONS UNDER THE NULL HYPOSTHESIS AND p-VALUES
Although different in formulation and assumptions, all methods presented so far attempt to address the same null and alternative hypothesis: H 0 = locus is neutral; H 1 = locus is not neutral. Under the hypothesis of neutrality, EHH-based methods are standard normal deviates (Voight et al., 2006;Sabeti et al., 2007;Tang et al., 2007). Hence, the probability that SNP i with Rsb or XPEHH score x i is neutral in the population used as numerator can be approximated by an upper tail p-value derived from the normal cumulative density function (CDF) : Likewise, the probability that SNP i with iHS score x i is neutral, given that both reference and alternative alleles are of interest, can be approximated by a two-tailed p-value: Recently, concerns have emerged on the interpretation of p-values in signatures of selection analyses . It is argued that, at least for the cases of EHH-based methods, scores standardized using genome-wide data are not test statistics in the classical sense but only deviates from an average (Voight et al., 2006), so p-values would only represent quantiles from an empirical distribution, rather than formal significance values. This implies that the probability of obtaining an arbitrary score given the locus is neutral cannot be exactly computed once the underlying true probability distribution may vary according to different demographic scenarios. The major caveat here is, for any given test discussed, the proposed asymptotic distribution under the hypothesis of neutrality is still largely based on coalescent simulations with demographic models calibrated for human data. However, if the majority of the genome is under neutrality and the null distribution is robust to a wide range of demographic models, one may expect that genome-wide distribution of scores in cattle data should mimic the simulations performed for the human model, serving as a control (Gianola et al., 2010). This has been observed in SS studies in cattle (Gautier and Naves, 2011;Flori et al., 2012;Utsunomiya et al., 2013), and therefore these theoretical distributions under neutrality are suitable for cattle data.
In the case of F ST , although approximate distributions are available (e.g., exponential or beta), such approximations are sub-optimal. One advantage of FLK over F ST is that scores are asymptotically distributed as χ 2 ν under the null hypothesis, where degrees of freedom ν can be equivalently calculated as number of subpopulations − 1 or as the average FLK across all loci. Upper tail p-values can be computed as: where F ν is the χ 2 CDF with ν degrees of freedom.
Although F ST and CLL scores have unknown distributions under neutrality, both rely on population comparisons. The use of datasets consisting of multiple populations allows for permutation tests via random sampling of individuals or random sorting of population labels in order to compute an empirical null distribution. Nevertheless, permutation tests are computationally intensive and may be impracticable in re-sequencing data.
The ZHp method suffers from the same problem, with the additional limitation of not allowing data permutations when a single population is surveyed. Although a truncated normal distribution could be suggested to approximate its null distribution, this has not been properly assessed in practice. Another challenge is deriving significance values when scores are averaged across SNP windows. Following the assumption that the majority of the loci (single markers or SNP windows for that matter) are under neutrality, an empirical CDF derived from genome-wide scores should converge to the underlying true CDF, so probability values could be empirically obtained from a step function: The probability that SNP i with score x i in a given test is selected is not as trivial to approximate. While the distributions under the hypothesis of neutrality seems to be robust across a wide range of demographic models, the distribution under the hypothesis of selection may vary widely depending upon the demographic history that shaped the allele frequency spectrum. Therefore, there is no unique theoretical distribution to represent selected variants, and coalescent simulations using well calibrated demographic models are required in order to generate empirical distributions. This has been successfully done for human data (Grossman et al., 2010), but is highly challenging in cattle as most of the demographic history of the bovine species is yet to be uncovered. Nevertheless, promising methods to infer demographic scenarios from the data, including estimates of population sizes and population separations over time, are now emerging (Schiffels and Durbin, 2014), which could be useful to elucidate cattle history and customize coalescent simulations based on empirical data. Table 1 summarizes all the software mentioned below. The first step to be considered before starting a SS analysis is filtering out poor quality data and markers and samples that are not informative or that may confound the analysis. Discussing the particularities of measures of quality control is beyond the scope of the present article, but some of the best practices in SS studies can emerge from common sense. First, metrics such as Hardy-Weinberg Equilibrium (HWE) and MAF, highly used in GWA studies, should be applied with caution. Elimination of markers with extreme deviations from HWE expectations may counteract the whole SS enterprise, as in this type of study outlier loci are being sought. Likewise, MAF controls may cause signals that are reaching fixation to be completely lost. A situation where HWE and MAF thresholds can be benign is when only markers with extreme excess of heterozygotes are eliminated, and MAF constraints are applied to the pooled allele frequencies across all populations in the study. We suggest PLINK v1.07 (Purcell et al., 2007) or PLINK v1.90 for this first data screening. Other usual filters such as GenCall and GenTrain scores and call rates should follow the same guidelines as in GWA studies.
AVAILABLE SOFTWARE AND ANALYSIS BEST PRACTICES
Another important issue is cryptic relatedness. Eliminating samples of closely related animals is of paramount importance to reduce false positive signals. We have previously reported an algorithm to find the maximum independent set based on identity-by-descent, i.e., maximize the number of samples while eliminating first and second degree relationships using SNP data (Utsunomiya et al., 2013). Other software such as PLINK v1.90 and GCTA v1.24.2 (Yang et al., 2011) also provide means to find the optimal set of independent individuals. The next key step is producing high quality phased data. There are several methods and implementations for phasing, such as fastPHASE v1.2 (Scheet and Stephens, 2006), Beagle v3.3 or later (Browning and Browning, 2008), and SHAPEIT2 (O'Connell et al., 2014). Although fastPHASE exhibits high phasing accuracy, it is orders of magnitude more computationally intensive than Beagle or SHAPEIT2. It is important to notice that phasing is not a straightforward procedure, and is highly prone to errors. Consequently, results from haplotype-based methods should be assessed with caution. The effect of haplotype errors on SS results remains underexplored and at some extent neglected.
Regarding the SS analysis per se, EHH-based methods can be computed using Sweep, selscan (Szpiech and Hernandez, 2014) or the R package rehh (Gautier and Vitalis, 2012). F RL has a dedicated software, as well as CLR (available at: and XPCLR. For ROH-based tests, runs can be computed using either PLINK or SNP & Variation Suite v7.6.8 or later, and F RL can be easily computed with home grown scripts. In the cases of F ST , CLL, ZH p and Ewing and Hermisson, 2010 the method reported by Ramey et al. (2013), allele frequencies and genotype counts can be obtained from either PLINK or SNP & Variation Suite, and calculation of scores can be easily implemented in R (available at: http://www.r-project.org/) or other languages. Simulations of population genetics datasets under specific demographic models, including neutral and selected loci, can be performed using coalescent simulators such cosi (Schaffner et al., 2005), cosi2 (Shlyakhter et al., 2014), or MSMS (Ewing and Hermisson, 2010).
COMBINING SELECTION SIGNALS
The available methodologies to detect positive selection differ substantially from each other in terms of the pattern of genetic variation encrypting a "signal." However, all of them have a shared objective: to identify loci that have undergone positive selection. Indeed, at least for recent selective pressures (up to a few thousand generations back), a selected variant is expected to be in a chromosome segment where there has been loss of diversity, enrichment for derived or rare alleles, population differentiation, and highly frequent long-range haplotypes. Therefore, collecting evidence across different methodologies targeting distinct classes of signals may help in identifying loci under positive selection. This section explores the statistical properties and limitations of some of the available methods designed to combine different methods for signatures of selection.
COMPOSITE OF MULTIPLE SIGNALS (CMS)
In the original implementation of the method (Grossman et al., 2010), CMS is a local test designed to narrow down signals detected from s distinct genome-wide scans, and is defined as the approximate joint posterior probability that a given variant is selected: The genome-wide extension focuses on the product of the Bayes Factors for each one of the tests to be combined. For each test, BF is computed as the ratio between the posterior and prior odds: Pr(x ij |selected)Pr(selected) Pr(x ij |neutral)Pr(neutral) In the absence of prior information on the number of loci under selection across the genome, CMS scores simplify to: Pr(x ij |selected) Pr(x ij |neutral) A challenging aspect of the implementation of these methods is computing Pr(x ij |selected), which requires simulations under clear demographic assumptions. In contrast, as discussed earlier, many methods designed to detect markers under positive selection allow for approximating Pr(x ij |neutral) from asymptotic theoretical or empirical distributions. Therefore, composite tests considering only the distributions under neutrality are more appropriate for cattle data. The original CMS score can be modified to take advantage of the assumed distributions under neutrality in order to relax demographic assumptions and avoid expensive simulations. The most essential modification involves reformulating the problem of detecting markers departing from neutrality. Instead of considering the assessment of whether a marker has been selected or not, one can look for support from the data against the null model, i.e., that the marker does not fit well to the neutral model. First, let the new CMS score be the approximate joint posterior probability of a given variant not being neutral: Here, Pr (x ij | neutral) is computed directly from its theoretical distribution, and Pr x ij not neutral is computed as 1 − Pr(x ij |neutral). Also, it can be assumed that the prior Pr(not neutral) = 1 − Pr(neutral). Therefore, the new CMS score can be re-written as: Likewise, the genome-wide modified CMS scores can be reformulated as: It is important to note that this modification does not allow for the same interpretation as the original CMS method: the composite likelihood does not indicate selection, but rather, that a marker does not fit well the neutral model.
META ANALYSIS OF SELECTION SIGNALS (META-SS)
Following the ideas expanded from the landmark publication of Grossman et al. (2010), given the probabilities under the null hypothesis for each test, our interest is to identify loci presenting consistent rejection of the neutral model across the different tests. For any given statistic, p-values are uniformly distributed in the interval between 0 and 1 under the null hypothesis. This property makes possible to use an inverse CDF, such as the Gaussian density, to produce scores for each test derived from a single theoretical distribution. Therefore, for each SNP i and test j, a new score can be computed as Z ij = −1 (1 − P ij ), where P ij is the p-value. These Z-transformed p-values can be then averaged and standardized to produce a composite score.
We have previously described meta − SS (Utsunomiya et al., 2013), an abstraction of the Stouffer Z-transformation for combining different selection signals using the aforementioned framework. As the Stouffer method assumes the tests are uncorrelated under the shared null hypothesis and the use of pair-wise comparisons produce correlated scores, a weighted average was originally proposed to penalize dependent tests: where ω j is the weight for test j. In this setting, a uniform penalization can be applied to control for the inflation of correlated tests. As this penalization does not incorporate the strength of correlations among tests, the meta − SS test can be modified to explicitly account for the magnitude of correlations between scores. Considering all scores are equally weighted, the corrected composite score can be computed as: where R is the sum of all pairwise Pearson's product-moment correlations. Under the hypothesis of neutrality, these composite scores are distributed as N(0, 1), so the higher is the Z-transformed value, the worse the marker fits the neutral model. Upper tail p-values can then be obtained from the standard normal CDF. Obvious limitations from meta − SS and CMS is the inability to incorporate statistics for which p-values cannot be derived. Randhawa et al. (2014) proposed Composite Selection Signals (CSS), a nonparametric interpretation of meta − SS, where fractional ranking is used instead of p-values to combine different tests. Briefly, the vector of test statistics for method j is first sorted and then ranked, taking values 1, . . . , k. Next, the vector of ranks is re-scaled by dividing all elements by k + 1, thus producing a variable ranging from 0 to 1. These re-scaled ranks are treated as they were p-values for the test statistics, and then combined as in the meta − SS approach. This strategy is equivalent to computing probabilities from an empirical CDF using a step function, as discussed earlier, which has an appealing feature: as fractional ranking can be generated for any particular test, signature blending is made feasible even if the theoretical distributions are unknown or if scores have been averaged in chromosome windows. However, a caveat is that the magnitude of the actual test statistics may be lost, so one may expect loss of power compared to the use of theoretical or simulated distributions. Simianer et al. (2014) proposed combining different tests by applying an eigendecomposition of the correlation matrix of the scores. The attractive feature of this method is the possibility of using standardized scores instead of approximate probabilities. However, as each principal component has heterogeneous loadings from each test, deriving a single synthetic score that summarizes all different tests remains a challenge in this framework.
CHALLENGES AND FUTURE DIRECTIONS
In theory, genome-wide genotypes are a vast source of information that can be explored in the search for large effect mutations that underwent selection. However, the existing data and methods still suffer from power issues and confounding effects that can give rise to false positive and false negative signals.
Although simulations suggest that only marginal gains in power are obtained when the sample size is increased from tens to hundreds of unrelated samples, marker density and allele frequency spectrum seems to impact power dramatically (Lappalainen et al., 2010;Simianer et al., 2014). Genotypes derived from commercial SNP arrays have two important limitations in this context: (1) incomplete genome coverage by markers; and (2) ascertainment bias. The search for SS must be preferentially performed using high density SNP panels, although optimal average intermarker distances to detect a sweep may vary depending on the effective population size, extent of linkage disequilibrium and the nature of the signal. Regarding ascertainment bias, commercial SNP arrays are suitable for cattle populations that are closely related to the breeds used in the SNP discovery process, but there is no guarantee they will be informative in genetically distant populations. Indeed, with a few exceptions, little congruence has been reported between candidate selected loci identified using whole genome sequence and different commercial genotyping platforms in African humans not included in the HapMap data (Lachance and Tishkoff, 2013). Altogether, these arguments suggest that re-sequencing data is the optimal choice in SS studies in cattle. At some extent, the HD assay is appropriate, as it has a high-density coverage of the genome with SNPs that are less biased than competing panels.
Another important source of confounding comes from the methods available to detect SS. First, all methods assume that individuals have no recent relationships in their pedigrees, a condition that is hardly true and generally ignored. It is essential to filter the data for cryptic relationships and assure to include only samples that are unrelated for at least two generations. Second, most of the methods rely on haplotypes and SNP coordinates, so further improvement of phasing strategies and of the bovine reference genome assembly is crucial to assure high quality results. Third, variants can depart from neutrality not only due to positive selection, but also as consequence of demographic events such as bottlenecks, genetic drift and admixture. Distinguishing loci under selection from neutrally evolving loci remains a major challenge in the field, and will require refinement of existing methods and development of new tests. Nevertheless, combining signals across different methods seems to be a promising approach to mitigate the individual methodological limitations. Also, when available, the concomitant analysis of environmental data (e.g., temperature, humidity, precipitation, disease prevalence, etc.) may be of great help in distinguishing true positives and accelerating the link between signal and phenotype (Lv et al., 2014).
Well-planned study designs will be crucial to exploit the full potential of SS in the detection of large effect mutations favored by selection. The identification of common adaptive phenotypes, together with geographical information data, should be an important player in sampling and decisions of population comparisons. Cattle breeds that are not highly productive but that exhibit genetic local adaptation should be considered as priority targets, as their environmental fitness was probably forged by hundreds of years of natural and artificial selection. In the context of artificial selection for complex traits, as large cattle pedigree cohorts for genomic selection become available, it will be soon possible to actually assess rapid changes in allele frequency using historical data, rather than present date data only. First demonstrations of such ideas were presented by Decker et al. (2012), and are likely to be incorporated as routine monitoring tools of genomic resources in breeding programs in the future.
Recently, comparing candidate loci across GWA studies has been facilitated in cattle with the advent of the Animal QTLdb (Hu and Reecy, 2007). Similarly, results from a SS scan on the human 1000 Genomes data reported by Grossman et al. (2013) were made available through the CMS viewer tool (available at: http://www.broadinstitute.org/mpg/cmsviewer/). Pybus et al. (2014) have also launched a comprehensive database of SS in the 1000 Genomes data (available at: http://hsb.upf.edu/). The research community would highly benefit from the development of a SS database for livestock species, which would not only facilitate cross-referencing, but would also help researchers willing to dig deep into the functional meaning of the signals to select promising candidates emerging from multiple preexisting studies.
Finally, similarly to the recent developments in human SS (Kamberov et al., 2013), unraveling the functional relevance of the putative selected variants will demand interdisciplinary reasoning, compilation of a wide range of data types (e.g., transcriptomic, proteomic), and assemblage of an arsenal of post-hoc assays, such as genomic editing, culture, phenotyping and challenge of specific cell lines, production of knock-out models, and generation of cross-bred lines for confirmatory segregation analyses. | v3-fos |
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} | s2 | Sensory Characterization and Consumer Preference Mapping of Fresh Sausages Manufactured with Goat and Sheep Meat
The main objective of this study was the sensory characterization, by a taste and a consumers’ panel, of fresh sausages from 140 culled goats and 140 culled ewes. Species and type of preparation effects were studied. All data were previously analyzed by analysis of variance. Taste panel data were analyzed by a Generalized Procrustes Analysis (GPA). Consumers’ panel data were analyzed by Preference Mapping. The 1st 2 factors explained 88.22% of total variation in GPA. Different sausages samples were perfectly differentiated by assessors. Goat sausages have been considered harder, more fibrous, and less juicy than sheep sausages. The panelists observed that sheep sausages without paprika had greater intensity of flavor, tasted spicy, and had an off-odor, while goat sausages with paprika were considered sweeter. Consumers’ panel did not show any preference for the different types of sausages. This means that all types of sausages can have market
Introduction
Quality of goats and sheep meat is directly related to its sensory characteristics, such as tenderness, juiciness, taste, and odor (Rodrigues and others 2009;. Meat from young sheep and goats has special sensory characteristics with good market value in contrast to adult animals, particularly the culled ones that have a very low acceptability and market price. This kind of meat is tougher and normally has an unpleasant taste and odor, and usually is transformed by processes as cured with salts or smoked and dried or also as fresh sausages after grinding, mixing with salt, spices, and other ingredients and casing. In the last years, there have been several studies concerning the incorporation of meat from culled sheep and goats in processed products (Nassu and others 2002;Pellegrini and others 2008). These studies have demonstrated the feasibility of using the meat of adult's sheep and goats in the manufacture of sausages and its good acceptance by consumers.
The consumption of sheep and goat meat as well as the consumption of sausages is of great importance and tradition in all MS 20141924 Submitted 11/21/2014, Accepted 5/5/2015. Authors Paulos, Oliveira, Leite, and Mediterranean countries. Particularly in North Portugal, there is a long tradition to consume several kinds of sausages. Taking advantage of these 2 factors, and to grant a higher commercial value to a low price product, a project to create a new product, a raw fresh meat sausage from Serrana goats and Churra Galega Bragançana ewes, was developed among a research center (Laboratório de Tecnologia e Qualidade da Carcaça e da Carne da Escola Superior Agrária de Bragança-Carcass and Meat Technology and Quality Laboratory of Agriculture School of Bragança), 2 breeder associations (ANCRAS-Associação Nacional de Criadores da Raça Serrana-Serrana Breed Natl. Assn., and ACOB-Associação de Criadores de Ovinos Bragançanos-Bragançana Breeders Assn.), and a meat manufacturing industry (Bísaro Salsicharia Tradicional). There is an important commercial issue related to this new product since the manufacturing industry is willing to expand their sale to other European Countries where they already have established market. During the process of developing, a new product is imperative to optimize parameters such as shape, color, appearance, odor, taste, texture, consistency, and interaction of all these components in order to achieve a complete balance, leading to an excellent quality and its good acceptability (Penna 1999) once consumers become more exigent with the characteristics of the products they choose and the industry should find the right way to satisfy their requirements. Sensory analysis allows evaluating the consumers' acceptability and quality of products, acting as an inherent part of the plan to create new products. Except for the hearing, all 4 senses as appearance, taste, smell, and texture, are used by meat consumers, which makes this methodology highly representative of human perceptions of food.
New product sensory analysis . . .
The objective of this study was the sensory characterization of a new product (fresh sausages made from goat and sheep meat) by a taste panel using the Quantitative Descriptive method referred by Meilgaard and others (2007), and the consumers' preference evaluation using the preference mapping procedure.
Sausages fabrication and sampling
A total of 280 culled females (140 Serrana goats and 140 Churra Galega Bragançana ewes breed) between 5 and 7 years old, with an average of 20 kg carcass weight were used from November 2010 until May 2011. Animals were slaughtered in the official slaughter house of Bragança (Trás-os-Montes region -northeast Portugal). Carcasses were deboned and cleaned from nerves, tendons, and connective tissues to be processed at the manufacturing meat industry. Raw meat (75% to 80% of goat or ewe) was mixed and minced with 15% to 20% of fatty cuts of pork belly of Bísaro (a local pig breed), and then the following ingredients: salt, peppers, bay leaves, water, garlic, rendimix R , and flavorex 4000 were added. The mixture was stuffed into 34 to 36 mm pork casings, hung for 24 h, and stabilized in a climate chamber at 13°C and 80% of relative humidity. And then packaged in a polyamidepolyethylene bag and stored in a freezer until sensory analysis.
Sensory analysis
For the sensory evaluation of goats and sheep fresh sausages, with or without paprika, a trained taste panel of 9 elements and consumers' panel with 82 elements were constituted, in accordance with the Portuguese Standard (NP- ISO-8586-1, 2001).
A taste panel, with about 10 y of experience that had already participated in previous research of our work team, was used. To form the panel, a recruitment of teachers, students, and staff of the Polytechnic Inst. of Bragança was made. About 40 people were recruited, to select 20, in order to obtain a panel of 9 people, according to the Portuguese Standard NP ISO 8586-1 2001. At recruitment, and before training, a preliminary selection of participants was carried out with the completion of a questionnaire, in order to eliminate the volunteers who were totally unsuitable for sensory analysis. Through selection tests, the following skills of the candidates were investigated: normal acuity to detect the different senses, the ability to discriminate different stimuli in quality and intensity, and recognition memory, verbal expression or descriptive capacity of sensory perception, and the ability in dealing with more complex food (Issanchou and others 1995;Nicod and others 1998;Eguía 2001;NP ISO 8586-1 2001). In the selection step, tests for the recognition of the taste, smell, texture, and vision were made, in which candidates discriminated and described the differences between diverse stimuli. The next step was training, which aimed to familiarize the taster with the procedures to carry out the tests in order to improve their ability to recognize, identify, and quantify the sensory attributes of a particular product. Also to improve its sensitivity and memory to different attributes so that they could provide an accurate answer, consistent and reproducible over time (Cross and others 1978;Costell and Durán 1981;Nicod and others 1998;Eguía 2001). In this step, the assays were based primarily on sensory memory. However, for the experienced taster further aims the ability to identify and recognize the characteristics of a product, the development of a set of descriptors, the identification and establishment of product standards, recognition factors of evolution within the products, and the evaluation of marketing factors (NP ISO 8586-1 2001). To train the assessors in the use of scales, the concepts of quotation category and scales intervals and proportionality were presented initially classifying series of simple stimuli, although related to the products that were evaluated, odor, taste or texture, as regards the intensity of a specific property (NP ISO 8586-1 2001).
In this study, training consisted of 4 specific sessions to promote panelists adaptation to the product characteristics. In the 1st training session, panelists were asked to describe the product characteristics taking into account the 5 senses. There was an analysis of the attributes referred by the panelists to eliminate synonyms and homogenize terms. In the next training sessions, established attributes were specifically trained. Then, each sample was evaluated for the following sensory attributes: odor intensity (odor associated with raw meat, animal species), off-odor presence (odor that is not natural or up to standard for this type of product), flavor intensity (flavor of raw meat, associated with the animal species), off-flavor (flavor different from the natural for this type of product), hardness (the force needed to chew), juiciness (water perceived during mastication), fibers presence (or stringy-fibers perceived during mastication), spiciness (spicy flavor), and sweetness (flavor of sugar) using Quantitative Descriptive method, in which the tasters identify and quantify the intensity of each attribute present in sausages (NP ISO 8586-1 2001). The products were evaluated by assessors in 8 sessions. In each session, 8 samples were evaluated. A structured but unnumbered scale of 10 cm, with the extremes representing either the minimum (no sensation) or maximum (extremely intense sensation) was used.
The consumers' panel was constituted by teachers, staff, and students from Polytechnic Inst. of Bragança, with no training. Consumers were between 18 and 58 years old, since the consumer analyses were carried out on a higher education institution, as has been said before. The consumers' panel corresponded mainly to young people. Giving the opportunity to study the preferences of this population group, supposedly more reactionary to consume goat and sheep meat or based products. Most consumers who participated in the study are female (62%) and 38% are male. For each sample, the following sensory attributes were evaluated: taste liking, texture liking, spiciness liking, and overall acceptability. In this panel, a scale of 10 cm, unstructured, with intervals (from 0 cm-"do not like" to 10 cm-"like very much") was used for each of the attributes.
The cooking and serving conditions were identical for both panels. Samples were individually wrapped in aluminum foil and cooked in the oven until the internal temperature reached about 75°C (NP-ISO-8586-1 2001). Once grilled, sausages were divided into pieces of 0.5 cm thick, wrapped in aluminum foil, labeled with random codes of 2 digits and 1 letter, and stored at 60 to 70°C for maintaining the temperature of the samples for evaluation. Before and between each sample, evaluation panelists must rinse their mouths with mineral water and no salt toasts and/or Golden apple.
Statistical analysis
In 1st place, an analysis of variance (ANOVA) was made to the taste panel and the consumers' panel data, using 2 species (sheep, goat) and 2 types of formulation (with or without paprika) as fixed factors, with SPSS. The analysis aims basically to check if there is a significant difference between the mean and the factors influence on some dependent variable.
After, the data from the taste panel sensory evaluation were analyzed by Generalized Procrustes Analysis (GPA). GPA is a powerful multivariate technique extensively used in sensory evaluation.
New product sensory analysis . . . .36 ± 1.60 abc * * P ࣘ 0.05; * * * P ࣘ 0.001. a to c Means with different superscript letters in the same row are significantly different. NS, not significant; SP, sheep with paprika; S/P, sheep without paprika; GP, goat with paprika; G/P, goat.
The analysis uses translation, rotation, and isotropic scaling to minimize differences among panelists (Gower 1975;Carlucci and others 1998), identifies agreement between them, and summarizes the sets of 3-dimensional data (samples, characteristics, and assessors). The data matrices of 4 (sausages samples) by 9 (sensory attributes) for the 9 assessors (configurations) were matched to find a consensus using the XLSTAT (Addinsoft 2012) software, an add on of Microsoft Office Excel. Results correspond to the average evaluation established by each panelist for each group of sausages and the respective parameter (sheep with paprika-SP, sheep without paprika-S/P, goat with paprika-GP, and goat without paprika-G/P). To relate consumers' preferences with meat sensory characteristics, external preferences maps (Schlich and McEwan 1992) were established for each of the following variables: taste, texture, spiciness, and overall acceptability. This method requires the use of the previous results from the GPA to describe products as a series of criteria. This 1st step consists in mapping products based on its characteristics, to obtain a sensory map. With a high number of consumers, it was decided to sort them into homogeneous groups in order to have a perceivable result when interpreting the preferences maps. The agglomerative hierarchical clustering was chosen, and the Ward method was used. To determine the number of Clusters relatively to each sensory attribute, we started to truncate the clustering into 4 classes and then evaluate the significance of the same classes and respective R 2 value. After, it was decided to maintain the least number of classes with the higher significance and R 2 , as suggested by McEwan (1998). Aiming consumers' representation in the sensory map, PREFMAP procedure from XLSTAT (Addinsoft 2012) software was finally applied, and products evaluations for each consumer (or group of consumers) were modeled, using products characteristics as explanatory variables.
Taste panel
In the 1st place, an ANOVA (Table 1) for taste panel data was made. In this analysis, significant differences were found between the 4 products for hardness, fibers presence, spiciness, and sweetness. The higher differences were for spiciness. S/P presented the higher value, and GP the lowest. This may be due to the typical bitter taste of sheep meat, and the camouflage produced by paprika. Although this type of statistical analysis can elucidate about the differences between samples, it was chosen to use the GPA, which is a widespread statistical analysis in sensory evaluation and can provide additional information. GPA was used to minimize the differences between panel assessors, identify the consensus between them, and summarize the results in a 3-dimensional representation, thereby making it easier to interpret and identify main conclusions. The assessors profiled 9 terms to describe the differences between products. Their training period promoted similar assessment methods among each other. Indeed, the residual analysis of each tester indicated low levels of variance, confirming the reliability of the panel (Table 2). However, no training could completely eliminate variation among assessors as predicted by Stone and Sidel (2004). The 1st 2 dimensions of GPA explained the greatest percentage of variability for each panelist. The variation may have occurred because some of our panelists used a large part of the scale, whereas others only used a small part of the same scale, as shown by the scaling factors in Table 2. Assessors 1, 2, 4, 6, 7, and 8, tended to use a larger range because scaling factors were higher than 1. Despite of the occurrence of the variation, it is not an issue as the GPA corrected it. Residuals, by treatment, indicated that the fresh sausages of sheep meat without paprika (S/P) had the lowest values (Table 3) and, therefore, were the most consensual. The 1st 2 main axes of the consensus configuration (Figure 1) explained 88.22% of the variability, a quite higher result when comparing with the 68.2% reported by Rodrigues and others (2009), in Terrincho fresh meat of lambs, but lower than the 93% found by , in Serrano fresh meat in young goats.
The correlation between sensory parameters and GPA factors (1 to 3) indicated that hardness, juiciness, and fibers' presence were highly correlated with factor 1 (Table 4). Factor 2 was highly correlated with the odor intensity, flavor intensity, off-flavor, spiciness, and sweetness. As for factor 3, it generally presents very low correlations, except for the off-odor presence, which highly correlates with this factor. However, their correlation with F1 (r = 0.660) is proximate to the value found for the correlation with F3 (r = -0.672). So, only the 1st 2 factors will be considered in Figure 1, which also shows the coordinates of objects (sausages samples), obtained in the principal components analysis, and the correlation between sensory parameters and the 1st 2 dimensions. In Figure 1, it is possible to notice that there is a perfect distribution of the different types of meat used in the preparation of sausages (sheep and goats) and condiment used in their manufacture (with or without paprika). The fresh sausages of sheep meat are positioned in the positive part of factor 1, while the fresh sausages of goats' meat are in the negative part of the same factor, indicating that the sausages are separated by sheep tenderness and juiciness compared to goat sausages. On the other hand, the fresh sausages with paprika are on the negative part of factor 2, while fresh sausages without paprika are in the positive, indicating that the condiment influences taste, odor, and flavor attributes.
Species had great influence on the sausage's texture with the hardness and fibrousness associated to goat sausages, while juiciness was more associated with sheep's, according to the panelists. This fact was expected as goat meat is harder and more fibrous than sheep's meat, having less percentage of intramuscular and subcutaneous fat, which increases with age and weight of the animal, improving meat tenderness (Sañudo and others 2000). But , in a study to characterize the carcass and meat quality of Serrano goat kids, observed that heavier animals had higher hardness and fibrousness. Condiment had influence on the presence and intensity of flavor, spiciness, and off-odor. Sausages without paprika presented higher spicy intensity, flavor intensity, and off-flavor than sausages with paprika, which had higher odor intensity and sweetness. Paprika masks the less pleasant sensory characteristics of this type of meat. The masking effect was also found by Nassu and others (2002) in a study on using goat meat in processing of fermented sausage, salami type, in which the incorporation of rosemary minimized goat odor and flavor.
Consumers' panel
Also for the consumers' panel, an ANOVA (Table 5) was performed. Averages show a median (5 to 6) preference for this type of product, except for spiciness. In this analysis, it was found that the means are very close to each other and only spiciness presented significant differences among sausages. Therefore, we chose to perform a multivariate analysis as Preference Mapping that can extract more information from data. The 1st step to obtain preferences maps correspond to the representation of the sensory map and can be observed in Figure 2. The figure shows the coordinates of the different types of sausages, and the correlation between sensory attributes and the 1st 2 factors obtained in the GPA, which can allow the determination of the sausages characteristics identified Vol. 80, Nr. 7, 2015 r Journal of Food Science S1571 New product sensory analysis . . . by the taste panel. Results from the ANOVA for each sensory variable are shown in Table 6 and indicate that none of the established classes was significant, but using more or less classes would not change their significance. Therefore, any conclusion about the classes will be uncertain. Anyway, that fact can be an indication that no preference differences were really detected by consumers, concerning species, or condiment considered in this work.
Even though not significant, an interpretation of the results can be suggested. So, for taste parameter (Figure 2A), consumers' class 1 (39 individuals) prefer tender and juicier sausages, namely the sheep sausages without paprika, as characterized by the taste panel. On the other hand, consumers' class 2 (33 individuals) prefer goat sausages with paprika characterized by being sweeter and with intense odor, but with less flavor intensity and less spicy. And consumer's class 3 (10 individuals) prefers sausages without paprika, which have higher flavor intensity and spicier.
Relatively to spiciness ( Figure 2B), goat sausages without paprika, characterized as harder and fibrous, and less juicy, are preferred by class 3 (28 individuals), and less appreciated by class 1 (25 individuals) and 2 (13 individuals). Sheep sausages without paprika, considered less tough and fibrous, spiciest and juiciest, and with a less intense odor, are more appreciated by classes 1 and 2 and less appreciated by class 3.
For the texture parameter ( Figure 2C), we can perceive that goat sausages without paprika, with a harder texture, and fibrous and less succulent were preferred by classes 2 (35 individuals) and 3 (14 individuals). On the other hand, class 1 (32 individuals) consumers preferred sausages with paprika, particularly sheep, characterized by low hardness and fibrousness.
Regarding overall acceptability ( Figure 2D), classes 1 (23 individuals) and 2 (31 individuals) preferred juicier and spicier sausages. Note that the vectors which represent them are directed to the coordinates of sheep sausages. Class 3 (27 individuals) shows a marked preference for goat sausages with paprika, considered as having higher odor intensity and sweetness.
In summary, as we have found in many preferences maps obtained by consumers assessments for taste, spiciness, texture, and overall acceptability, there were no significant differences in consumers preferences for sheep and goats fresh sausages, with or without paprika. In other words, there can be market for all types of fresh sausages, as the degree of preference is great for the 4 types (SP, S/P, GP, G/P).
Our results agree with the good acceptance by consumers for similar products studied by other authors (Souza and others 2005; Duarte and others 2007; Francois and others 2009) who used sheep and goat meat for the elaboration of sausages.
Conclusions
Tasters were able to distinguish the difference between fresh sausages of the 2 species and different formulations. Sheep fresh sausages were juicier, while the goat sausages were defined as harder and more fibrous. Sausages with paprika were evaluated as having a higher odor intensity and sweetness than sausages without paprika. Regarding consumers' panel, it was possible to realize that there is no significant preference for any of the fresh sausages, which means there is market for all types of sausages. This seems to be a product with excellent sensory characteristics, great acceptance by the various types of consumers. This product is an excellent alternative to add value to the sheep and goat meat from culled animals that have very low commercial price. Our study may be useful for producers, since, there is a description of the products, as well as the study of consumer mapping preferences, which will give a feedback acceptance of this new product by consumers, and thus meet the market needs. | v3-fos |
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} | s2 | Evaluation of pine kraft cellulosic pulps and fines from papermaking as potential feedstocks for biofuel production
Results of enzymatic hydrolysis of pine kraft cellulosic pulps (Kappa numbers ranging from 17.2 to 86.2) and waste fines from paper production line suggest that they are potential feedstocks for biofuel production. Glucose (61–68 % d.w.) and reducing sugars (around 90 % d.w.) yields from the pine cellulosic pulps with Kappa numbers ranging between 17.2 and 47.7 were higher than from the two pulps with Kappa numbers of 60.9 and 86.2. Glucose and reducing sugars yields from the latter pulps were around 54 and 45 % d.w. and 78 and 67 % d.w., respectively. The highest yields of glucose and total reducing sugars on a pine wood dry weight basis (29.3 and 43.1 %, respectively) were derived from the pulp with the Kappa number of 47.7. Thus the complete lignin removal from pine wood was not necessary to obtain maximum yields of glucose and other reducing sugars. Enzymatic hydrolysis of the fines from pine wood processing in a paper mill yielded around 75 % glucose and 78 % total reducing sugars on a dry weight basis that corresponded to around 34 and 36 % on a pine wood dry weight basis.
Introduction
Eventual depletion of fossil fuels and their contributions to greenhouse gasses have focused attention on renewable vegetal biomass as a principal alternative energy resource (Alvira et al. 2010). Since conversion of starch and plant oils into first generation biofuels may compete with food production, these raw materials have been increasingly replaced with lignocellulosic biomass, including wastes from forestry, agriculture, wood processing and papermaking, like woodchips and fines from paper mills. Lignocellulosic materials are an excellent source of polysaccharides since they contain around 70 % holocellulose on a dry weight basis. Production of biofuels via environmentally friendly technologies requires employing enzymes for polysaccharide saccharification. However, enzymatic degradation of cellulose and hemicelluloses contained in lignocellulosic biomass to simple sugars is not as easy as starch hydrolysis because of the complex, tightly bound and crosslinked, three dimensional structure of plant tissues and protective impact of lignin, resinous substances and waxes. The highly cross-linked lignin coating, surrounding fibers of cellulose and hemicelluloses is considered one of main causes of plant biomass recalcitrance since it restricts access of enzymes catalyzing depolymerization of polysaccharides (Alvira et al. 2010). Furthermore, the unproductive adsorption of cellulases and hemicellulases on lignin reduces the rate and degree of saccharification. Therefore, lignocellulosic biomass must be pretreated before enzymatic digestion to remove lignin and increase the accessible surface area of cellulose. Also crystallinity of the latter needs to be reduced since crystalline cellulose regions are much less susceptible to enzymatic degradation than the amorphous ones.
Wood is an attractive lignocellulosic feedstocks, despite the high mechanical durability and recalcitrance to enzymatic attack. It is estimated that global wood production reaches 3,410,357 thousand m 3 (of debarked logs) [www.bdl.lasy.gov.pl]. Around a half of this biomass is used to produce heat and energy while the second half is used in papermaking, production of furniture and other goods, in construction etc. Pine wood is an abundant biomass, widely applied in various branches of industry, including papermaking. Like other sorts of softwood, it contains high amounts of lignin (around 28 % d.w.) and extractives (up to 5 % d.w.) that reduce the access of depolymerizing enzymes to cellulose (around 42 % d.w.) and hemicelluloses (around 27 % d.w.). The hemicelluloses from pine wood contain residues of xylose, arabinose, mannose, galactose, and uronic acids as well as acyl groups (Girio et al. 2010). Because of the high contents of cellulose and hemicelluloses, common availability and relatively low costs of cultivation and processing, pine wood is a promising feedstock for biotechnology despite strong resistance towards enzymatic depolymerization (Agarwal et al. 2013).
In papermaking, wood processing begins with comminution to chips, which undergo chemical delignification by the sulfate method, yielding cellulosic pulps of various residual lignin contents. Sulfate method of wood processing is a fully mature technology. Nowadays, more than 150 million metric tons of cellulosic pulps are produced in this way. The sulfate method fulfills most of 12 principles of green chemistry and enables full recovery of chemicals used in the process. Cellulosic pulps obtained from low quality wood, e.g., partly degraded by microorganisms, may have insufficient characteristics to be used for production of paper because of shortened cellulose fibers.
However, these pulps are excellent sources of virtually pure cellulose for biotechnology since they are almost deprived of lignin and extractives, inhibiting not only enzymatic hydrolysis but also fermentation processes. Another attractive raw material for biotechnology is the waste from papermaking, consisting of small particles (mainly shortened cellulose fibers), named fines. This fraction contains almost completely delignified cellulose and is characterized by a high specific surface area, facilitating an access of cellulases to cellulose chains.
The objective of this study was to evaluate the potential of pine kraft pulps with Kappa numbers ranging between 17.2 and 86.2 as well as fines from pine wood processing in a paper mill as feedstocks for biofuels production. These materials were subjected to enzymatic hydrolysis, mediated by a multienzyme commercial preparation, containing cellulases and hemicellulases. To assess the effect of wood delignification by the sulfate method on the efficiency of enzymatic hydrolysis, pine woodchips (0.43-0.8 mm) that were obtained from the same wood, which was used to produce the pulps, were hydrolyzed under the same conditions as the latter.
Woodchips
Pine woodchips (particle size of 0.43-0.8 mm) were prepared by drilling of larger pieces of pine wood using an electric 6388AA Skil driller (Lowe's Co., USA) equipped with a Metabo drill (Poland) no 10 (10.0 mm in diameter). Then the chips were sieved through a series of sieves to separate the 0.43-0.8 mm fraction. This fraction was subjected to enzymatic saccharification.
Kraft wood pulps
Pine cellulosic pulps (Kappa numbers of 17.2-86.2) were prepared by the sulfate method as described in Modrzejewski et al. (1969) from woodchips (25 mm 9 15 mm 9 8 mm), containing 7-8 % humidity. The chips were obtained from wood after the inner and outer barks and knots had been mechanically removed. The disintegrated materials were kept in hermetically closed vials to avoid any changes in the humidity before the treatment with NaOH and Na 2 S solutions [at chips:solvent ratio of 1:4 (w/v)], which were prepared freshly before the usage. Delignification processes were conducted in 15 l stainless steel reactor with regulation of temperature. Suspensions of woodchips were heated for 120 min to achieve the temperature of 172°C and incubated at this temperature for the next 120 min. Then the temperature was decreased to the ambient one (22 ± 1°C) using cold tap water and the insoluble residue was separated by filtration on Buchner funnel, washed several times with demineralized water and incubated overnight in demineralized water to remove residues of the alkali-soluble fractions. The solids were disintegrated for 4 min (12 000 revolutions) in a laboratory propeller pulp disintegrator (type R1 from Labor-Meks, Poland), and the fibers were screened using membrane screener (gap 0.2 mm). Fibers were collected and left for 48 h so as to dry in ambient conditions (22 ± 1°C) and then weighed. Triplicate samples of these fibers were analyzed for the humidity and contents of cellulose, hemicelluloses and residual lignin (Kappa number) using standard methods as described in Modrzejewski et al. (1969). The yields of production of six pine cellulosic pulps with Kappa numbers ranging between 17.2 and 86.2 varied from 39.1 to 54 % on a wood dry weight basis, respectively (Table 1).
Industrial fines fraction
Residual fines fraction, which was subjected to enzymatic hydrolysis, was obtained from technological line in one of Polish paper mills. White water (30 l), containing fines, was sampled from a wire tank. Due to the low concentration of fines, this sample was placed in a vessel and left for 30 min so as to sediment the solids. Than the upper water layer was discarded and the decanted fraction was filtered. The concentration of filtered fines fraction was 8-10 %. The size of particles was measured using Kajaani FS-200 device and in all samples its maximal value was below 0.2 mm.
Enzyme preparation
A commercial, industrial grade enzyme preparation NS-22086, containing activities of cellulases and xylanases was kindly supplied by Novozymes A/S (Denmark). Activities of these enzymes were assayed by the 3,5-dinitrosalicylic acid (DNS) method (Miller 1959) at pH 5.0 and 50°C for 0.5 % carboxymethylcellulose and 0.5 % birch xylan, respectively (reaction time of 5 min). Activities of both the glycosidases were expressed as micromoles of reducing sugars released from the polysaccharide substrates in 1 min (U). The filter paper activity was determined at pH 5.0 and 50°C according to Adney and Baker (2008) and expressed as FPU/ml. Total reducing sugars and glucose concentrations in NS-22086 were assayed as described below.
Enzymatic hydrolysis
Substrates (around 0.3 g d.w. each) were suspended in 0.1 M sodium-acetate buffer solution (pH 5.0, 20 ml) and incubated for 15 min in a water bath at 50°C. Then 1 ml aliquot of the NS-22086 (diluted sixfold in the same buffer) was added (with vigorous mixing) to each of these suspensions to initiate enzymatic digestion, which was conducted at 50°C. The hydrolysates were sampled just after addition of the enzyme (to determine initial concentrations of glucose and total reducing sugars) and after 1, 3, 6, 24, 48 and 72 h of the process (to estimate the progress of enzymatic hydrolysis). Hydrolysis of pulps and fines was terminated after 48 h while the chips required 72 h digestion. All samples of enzymatic hydrolysates were filtered through a medium-fast filter paper and the filtrates were subjected to analyses. Enzymatic hydrolysis of the pine pulp with Kappa number of 17.2 was also conducted at its concentration around 7.4 % w/w, which was the highest initial concentration of this pulp at which mixing of the dense slurry was possible. Enzymatic hydrolysis was carried out at enzyme:substrate ratio of around 8.06 U:1 g dry weight for cellulases and 19.25 U:1 g dry weight for xylanases (around 1 ml of the NS-22086 preparation per 10 g d.w. pulp). The dense suspension of the pulp in demineralized water was thoroughly mixed with the enzyme preparation just after its addition and the process was conducted for 48 h at 50°C with intermittent mixing. Results of enzymatic attack on wood pulp fibers were monitored using a MPI3/SK12 PZO (Poland) optical microscope (at 2009 magnification). Changes in the appearance of the surface of pine woodchips during their enzymatic digestion were observed using an electron scanning microscope S-4700 Hitachi SEM/EDS (at 2009 magnification).
Analytical methods
Reducing sugars concentration was determined according to Miller (1959) using the alkaline DNS solution. Mono-and disaccharide profiles of the hydrolysates were determined by HPLC using an Ultimata 3000 Dionex liquid chromatograph equipped with a Rezex RPM-Monosaccharide Pb 2? column (8 lm, 7.8 9 300 mm) and a Shodex-RI-10 refractive index detector. The temperature of the column and RI detector was 80 and 40°C, respectively. Samples of hydrolysates were filtered through a nylon syringe filter (0.45 lm) before HPLC analysis. The volume of injected samples was 10 ll. HPLC grade water (Sigma) was used as the mobile phase at a flow rate of 0.6 ml/min. Results of sugar resolution were recorded over 35 min. To verify results of HPLC analysis, glucose concentration was also determined according to Barham and Trinder (1972), using a commercial diagnostic kit employing glucose oxidase and peroxidase (Biomaxima, Poland). The assay was conducted according to the instruction from the manufacturer of the kit.
Both hydrolysis processes and analyses of the hydrolysates were carried out in at least triplicate. Their results are presented as mean ± standard deviation (SD).
Yield calculations
Glucose and total reducing sugars yields from the pulps, woodchips and fines dry weight were calculated according to the equations: glucose yield ¼ glucose in hydrolysate g ð Þ Â 0:9=initial dry weight of the sample g ð Þ total reducing sugars yield ¼ hexosesin hydrolysate g ð Þ Â 0:9 þ cellobiose in hydrolysate g ð Þ Â 0:95 þ pentoses in hydrolysate g ð Þ Â 0:88=initial dry weight of the sample g ð Þ The correction factors of 0.9, 0.88 and 0.95, corresponding to hexoses, pentoses and cellobiose (Van Dyk and Pletschke 2012), were used in the calculations to compensate for the addition of a water molecule during hydrolysis of each glycosidic bond.
To determine the impact of lignin content on the degree of cellulose saccharification, glucose yields from cellulose contained in the pulps and woodchips were calculated according to Kumar and Wyman (2009): Â initial amount of glucan g ð Þ
Enzyme preparation
The preparation NS-22086, which was used in this study for hydrolysis of pine cellulosic pulps with Kappa numbers ranging from 17.2 to 86.2, pine woodchips (0.43-0.8 mm) and fines from paper mill, was selected as described by Buzała et al. (2015). It displayed the activities of cellulases and xylanases (Table 2). Our previous study showed that NS-22086 preparation rapidly degraded cellulosic fibers contained in pine pulp, which was confirmed by microscopic observations. Like many other enzyme preparations NS-22086 contains reducing sugars,
Enzymatic hydrolysis of pine woodchips
To assess the effect of lignin removal from pine wood by the sulfate method on saccharification efficiency, pine woodchips that were obtained from the same wood, which was used to produce the six cellulosic pulps of different residual lignin content, were digested under the same conditions as the pulps. The presence of lignin in the woodchips caused that the degree of cellulose and hemicelluloses hydrolysis was relatively low, which was reflected by small yields of glucose and total reducing sugars, of only 3.4 and 20.0 % on a wood dry weight basis, respectively (Table 4). The increase in glucose content in the hydrolysates of woodchips was observed for the first 30 h of enzymatic digestion (Fig. 1a, b). Glucose concentration was constant after 30 h. HPLC analysis of enzymatic hydrolysates obtained by 72 h digestion of the pine woodchips revealed that glucose accounted for around 27 % of the sum of 5 detected sugars while the level of its reducing dimercellobiose was more than twice higher (64 % w/w) ( Table 5). This might be a consequence of a relatively high activity of cellobiohydrolase in the preparation NS-22086 and inhibition of b-glucosidase despite very low glucose concentration (around 0.8 mg/ml). Apart from these two sugars, also mannose (8.8 % w/w) and xylose (0.4 % w/w) were contained in the hydrolysates.
Changes in the appearance of the surface of pine chips, caused by the attack of enzymes on accessible polysaccharide fibers, were visible under the electron scanning microscope (Fig. 2). The size of pores in their surface increased with time.
Enzymatic hydrolysis of pine pulps
Pine pulps rank among most popular softwood pulps in papermaking. To determine the effect of residual lignin on their digestibility and glucose yields, six pine pulps with Kappa numbers ranging from 17.2 to 86.2 were digested by the preparation NS-22086. Concentrations of glucose and total reducing sugars in the hydrolysates as well as their yields were monitored over 48 h (Table 4). These yields depended on the Kappa numbers of the six pulps. The largest amounts of glucose (61-68 % d.w.) and reducing sugars (around 90 % d.w.) were derived from the cellulosic pulps with Kappa numbers ranging between 17.2 and 47.7 (Table 4). The maximum yields of glucose and Sugars released by the preparation NS-22086 from polysaccharides into the liquid fraction were identified and quantified by HPLC (Table 5). HPLC analyses revealed that apart from glucose (71-77 % w/w) the hydrolysates of pulps contained some cellobiose (16-21 % w/w) and mannose (around 7 % w/w). The presence of the latter hexose provides evidence of the occurrence of some hemicellulose residues in the pulps. Interestingly, mannose was not detected in the hydrolysates of pine woodchips and fines, which contained xylose and arabinose.
The curves presenting the course of enzymatic hydrolysis of the pulps with Kappa numbers ranging from 17.2 to 86.2 and pine woodchips are shown in Fig. 1a. The hydrolysis of the six pine pulps was much more dynamic than the hydrolysis of woodchips. The yields of glucose released within the first 6 h ranged between 18 and 44 % on a dry weight basis. All these Fig. 1 Dynamics of hydrolysis of pine cellulosic pulps and woodchips (a) and residual fines from paper mill (b) by NS-22086 preparation (based on yields of glucose released from the substrates) pulps were completely converted into soluble sugars within 24 h. The further small increase in glucose and total reducing sugars levels (about 3 %) until 48 h is thought to be caused by hydrolysis of soluble oligosaccharides and cellobiose.
High yields of glucose and total reducing sugars released by the preparation NS-22086 from the pine pulp of Kappa number of 34.5 (Table 4) were correlated with visible changes in the appearance of fibers contained in this pulp. Microscopic images shown in Fig. 3, which present successive steps of gradual deconstruction of cellulosic fibers, provide evidence that the attack of cellulolytic and hemicellulolytic enzymes caused their complete degradation within 48 h.
As it was mentioned above, pine wood is characterized by the high recalcitrance to enzymatic attack. Mou et al. (2013) pretreated milled birch and pine wood by ionic liquid, hydrothermal and hydrotropic methods before enzymatic hydrolysis using a mixture of commercial cellulolytic enzymes (Celluclast 1.5 and Novozym 188). The most efficient pretreatment in terms of lignin removal was its extraction with a hydrotrope (30 % sodium xylenesulphonate, 120 min, 150°C), giving rise to glucose yields from pine and birch woods of 15.5 and 84 % d.w., respectively. Zhu et al. (2009) who pretreated pine wood by the sulfite method (SPORL) before mechanical size reduction using disc refining and enzymatic treatment for 48 h, obtained glucose yields of 37 % on a wood dry weight basis. These hydrolysates obtained this way contained low amounts of fermentation inhibitors such as furfural and 5-hydroxymethylfurfural. Agarwal et al. (2013) applied acid chlorite treatment (using a mixture of glacial acetic acid and sodium chlorite at 70°C) to remove lignin from pine wood and obtained cellulosic pulps with Klason lignin concentrations of 5.5-22.6 %. Sugar yields derived by 72 h enzymatic hydrolysis of these pulps (using Celluclast 1.5 and Novozyme 188) ranged between 9.5 and 93.3 % d.w., respectively. It was found that lignin removal by the applied pretreatment method caused also the loss of a part of hemicelluloses.
Enzymatic hydrolysis of the pine pulp at 7.4 % (w/ w) concentration Enzymatic hydrolysis of the pine pulp (Kappa number of 17.2) was also conducted at its higher concentration (around 7.4 % w/w) that enabled to increase glucose concentration in the hydrolysate from around 11 to 81 mg/ml (Table 7). Enzymatic hydrolysis was carried out for 48 h at 50°C with intermittent mixing at enzyme:substrate ratio of around 8.06 U:1 g dry weight for cellulases and 19.25 U:1 g dry weight for xylanases (around 1 ml of the NS-22086 preparation per 10 g d.w. pulp). The yield of glucose from the first step was almost 41 % d.w. of the pulp (around 16 % d.w. of pine wood). The insoluble residue was washed twice with demineralized water to remove soluble sugars adsorbed to the insoluble fraction. Noteworthy, the amount of glucose eluted from the insoluble residue reached around 16 % d.w. of the pulp. It was comparable with the yield of glucose from the second step of hydrolysis (conducted in the same conditions as the first one with an exception of around 6.6 % w/w substrate concentration) which was around 17.6 % d.w. of the pulp. The total glucose yield was around 75 % d.w. (from the two steps and washing) while the insoluble fraction accounted for around 14 % of the pine pulp dry weight. Thus around 5.7 kg glucose may be produced from 10 kg pulp d.w. by only one-step enzymatic hydrolysis, conducted at around 7.4 % w/w initial concentration of this pine pulp, using 1 l NS-22086 preparation, and followed by washing of the insoluble residue to release the absorbed soluble sugars. Theoretically, alcohol fermentation may yield around 2.9 kg ethanol from 5.7 kg glucose. Because around 3.9 kg of this pulp is derived from 10 kg d.w. of pine woodchips so as much as 1.1 kg ethanol may be obtained from 10 kg wood d.w. Another product will be energy derived from incineration of the lignin-rich fraction from the kraft pulping. Also the insoluble residue from enzymatic digestion may be used to produce energy. Another solution, enabling to reduce the overall process costs, consists of recycling the enzymes depolymerizing cellulose and hemicelluloses. These proteins may be separated from much smaller molecules of mono-and disaccharides, which are hydrolysis products, using membranes or liquid chromatography techniques and applied again for cellulosic pulp saccharification. The assays of residual activities of cellulases and xylanases from the preparation NS-22086 at the end of cellulosic pulps hydrolysis, which were performed in this study, showed that these enzymes retained around 80 % of the initial activities. The high residual activity of these hydrolases suggests that their recycling is feasible.
Enzymatic hydrolysis of the waste fines fraction
The fines derived from pine wood in one of Polish paper-mills represent an abundant and under-utilized waste material. Their hydrolysis by the NS-22086 preparation was very dynamic (Fig. 1b) that resulted in almost complete hydrolysis to soluble saccharides within the first 6 h. Glucose and total reducing sugars yields after 24 h hydrolysis were around 75 and 78 % d.w. (Table 4). Glucose accounted for around 84 % w/w of the sum of four detectable sugars while cellobiose content was relatively low (around 9 % w/w) ( Table 5). The presence of arabinose (around 4 % w/w) and xylose (around 3 % w/w) in the hydrolysate meant that the fines contained residual hemicelluloses apart from cellulose. Because of the sugar profile, these hydrolysates may be used as feedstocks for fermentation processes.
Conclusions
Pine cellulosic pulps, characterized by the Kappa numbers ranging between 17.2 and 86.2, and the waste fines from papermaking were found to be attractive substrates for enzyme-mediated production of highglucose hydrolysates (glucose content above 70 % w/w soluble sugars) using an appropriate multienzyme preparation, containing cellulases and hemicellulases, like the NS-22086 preparation from Novozymes. Enzymatic digestion of the pulps caused almost complete degradation of cellulosic fibers that was visible under optical microscope. The yields of production of these six pulps from pine wood increased with the Kappa number and ranged from around 39 to 54 % d.w. Determination of cellulose, hemicelluloses and lignin contents in the tested pulps showed that apart from lignin also a part of hemicelluloses was removed by kraft pulping. The contents of hemicelluloses in the pulps increased from around 2.54 to 9.11 % d.w. with the rise in Kappa number from 17.2 to 86.2. The outcomes of enzymatic hydrolysis depended on the residual lignin contents in the tested pulps that may be ascribed to either restricted access of cellulases and hemicellulases to the polysaccharides, protected by lignin, or binding the enzymes by the latter polymer. The higher amounts of glucose (61-68 % d.w.) and reducing sugars (around 90 % d.w.) were derived from the cellulosic pulps with Kappa numbers ranging between 17.2 and 47.7 compared to the two pulps containing the highest levels of residual lignin (Kappa numbers of 60.9 and 86.2, the glucose and reducing sugars yields below 55 and 79 % d.w. pulp, respectively). The maximum yields of glucose and other reducing sugars on the pine wood dry weight basis (29.3 and 43.1 %, respectively) were obtained from the pulp with the Kappa number of 47.7. Interestingly, also the glucose yield from cellulose (98.9 % d.w.) was the highest in case of this pulp. These results demonstrate that the complete lignin removal from wood is not necessary to obtain maximum glucose yields from cellulose. Reassuming, kraft pulping proved to be an attractive alternative to lignocellulosic biomass pretreatment since cellulosic pulps obtained by this method are susceptible to enzymatic saccharification and around twice more soluble sugars (on a wood dry weigh basis) may be obtained from the pulps than from pine woodchips under the same hydrolysis conditions. Enzymatic saccharification of low quality pulps, which cannot be used for paper production, and recycling of enzymes depolymerizing cellulose and hemicelluloses may reduce costs of conversion of wood biomass to simple sugars. Also enzymatic hydrolysis of the residual fines from pine wood processing, which were virtually deprived of lignin, was highly efficient and yielded around 75 % d.w. glucose and 78 % d.w. total reducing sugars (around 34 and 36 % on a pine wood dry weight basis, respectively). Biofuel production from hydrolysates of this waste may be an attractive route of its valorization. | v3-fos |
2017-06-17T19:57:26.771Z | {
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} | 0 | [] | 2015-09-01T00:00:00.000Z | 16479562 | {
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} | s2 | Effects of mannan oligosaccharide supplementation on growth, some immune responses and gut lactic acid bacteria of common carp (Cyprinus Carpio) fingerlings.
This study was conducted to determine the effects of mannan oligosaccharide (MOS) on growth, some immune responses and gut lactic acid bacteria of common carp. Four experimental diets containing 0%, 0.05%, 0.10% and 0.20% MOS were prepared. Each diet was randomly allocated to triplicate groups of fish with initial average weight of approximately 14 g. After eight weeks, survival rate was high in all treatments with no significant difference (p > 0.05). Growth performance including final weight, weight gain (WG) and specific growth rate (SGR) did not differ among the treatments. Feed conversion ratio (FCR) was better when the fish were fed 0.05 to 0.20% MOS diets. The alternative complement activity, lysozyme activity and serum total Ig were found to be significantly (p < 0.05) greater in fish fed 0.20% MOS diets. Although the total intestinal bacterial counts were not affected by dietary treatment (p > 0.05), the lactic acid bacteria levels were significantly elevated in fish fed MOS diets (p < 0.05). These results indicated that oral administration of MOS at 0.20% elevated the immune response, improved FCR and modulated intestinal microbiota of common carp.
Introduction
Antibiotics, for the past decades, have been included at the sub-therapeutic concentrations in the aqua-feeds due to their potential effects on survival, feed utilization and weight gain. 1 However, with the recent ban on the use of antibiotic growth promoters as additives in fish feed, alternative immunonutriens to enhance production and health status is a research topic of increasing interest. Prebiotics which are defined as non-digestible food ingredients beneficially affect the host by stimulating growth and/or activity of a limited number of beneficial bacteria in the gastrointestinal tract and have proved to be effective at enhancing health and growth performance of terrestrial and aquatic animals. 2 Among the most common prebiotics, mannan oligosaccharide (MOS) has been recently receiving heightening application in aquaculture. 1 The MOS are complex carbohydrates derived from yeast cell walls. These compounds contain mannose as the primary carbohydrate element. 3 This prebiotic has been demonstrated to have a variety of beneficial effects on livestock ranging from growth enhancing to immunostimulation in many species. 4 The growth promotion [5][6][7] and immunostimulatory benefits 8,9 of dietary MOS have also been studied in fish but often with contrasting findings.
The culture of warm-water species is an important aquaculture industry in Iran. Warm-water fish farming in Iran is based on Chinese carps including common carp (Cyprinus carpio). Annual production of farmed carps in Iran was 100,430 tons in 2009. 10 The present study was carried out to investigate the effects of dietary MOS supplementation on the growth performance and immunological parameters of common carp.
Materials and Methods
Fish and experimental conditions. Three hundred common carp fingerlings were obtained from a commercial carp farm in Shoushtar, Iran. The fish were transported to the laboratory, acclimated to laboratory conditions and fed the basal experimental diet without dietary MOS for one week. At the end of the acclimation period, fish with an average weight of approximately 14 g were randomly selected and stocked in twelve 300-L tanks (triplicate groups per dietary treatment) at a density of 20 fish per tank. The mean water quality parameters were recorded as follows: temperature 27 °C , dissolved oxygen 8.2 mg L -1 and pH 7.9.
Feed and feeding. Basal diet formulation and proximate composition analysis are shown in Table 1. The experimental diet was formulated to have 0% (basal), 0.05%, 0.10% and 0.20% of mannan oligosaccaride (MOS; Alltech Inc., Nicholasville, USA). Dietary ingredients were thoroughly mixed in a mixer, made into pellets and air-dried at room temperature. The pellets were broken into small pieces, packed and stored in a freezer until used.
Fish were fed by hand to apparent satiation two times per day with one of the four experimental diets over eight weeks.
Growth measurements. All fish in the different experimental groups were weighed at the end of 8-week feeding trial for estimation of growth. Based on recording the weight of each fish, specific growth rate (SGR), percentage of body weight gain (WG) and feed conversion ratio (FCR) were calculated for each group as follows: In addition, survival rate was calculated at the end of the experiment as: where, N0 is the initial number of fish and Nf is the final number of fish. Immunological assays. After eight weeks of feeding, blood samples for serum biochemical analysis were collected from the caudal vein of nine fish per each dietary treatment into Eppendorf tubes (Eppendorf, Hamburg, Germany) without anticoagulant. Blood samples in Eppendorf tubes were allowed to clot for 30 min at room temperature. The tubes were then centrifuged at 3500 g for 5 min and the supernatant serum was collected. The serum was kept frozen at -20 ˚C until analysis of selected biochemical parameters.
Alternative complement activity (ACH50). The 50% hemolysis was determined using the method of Sunyer and Tort 11 with the following modifications as described by Yeh et al. 12 The volume of serum complement producing ACH50 was determined, and the number of ACH50 unit mL -1 was calculated for the sample.
Lysozyme activity. Lysozyme activity in serum was determined according to the method of Demers and Bayne 13 based on the lysis of the lysozyme sensitive Gram positive bacterium, Micrococcus lysodeikticus (Sigma Chemical Co., St Louis, MO, USA). The dilutions of hen egg white lysozyme (Sigma Chemical Co.) ranging from 0 to 20 µL mL -1 in 0.1 M phosphate citrate buffer (Merck, Darmstadt, Germany), pH 5.8, were taken as the standard. This along with the undiluted serum sample (25 µL) was placed into wells of a 96-well plate in triplicate. A volume of 175 µL of M. lysodeikticus suspension (75 mg mL -1 ) prepared in the same buffer was then added to each well. After rapid mixing, the change in turbidity was measured every 30 sec for 5 min at 450 nm at approximately 20 °C using a microplate reader (Model Stat Fax-2100; Awareness Technology, Palm City, USA).
Total immunoglobulin content. Plasma total immunoglobulin (Ig) level was determined according to the method described by Puangkaew et al. 14 The assay was based on the measurement of total protein content in plasma prior to and after precipitating the Ig molecules employing a 12.0% solution of polyethylene glycol (Sigma Chemical Co.). The difference in protein content represents the Ig content.
Intestinal lactic acid bacteria analysis. The analysis of intestinal microbiota was performed at the end of the feeding trial as described by Hoseinifar et al. 15 with some modifications. Four fish per tank were randomly sampled from each treatment for microbiological sampling. The fish were killed by physical destruction of the brain, and the skin was then washed in a solution of 0.10% benzalkonium chloride before opening the ventral surface with sterile scissors. Intestinal tract samples of fish were removed, weighed, and suspended in sterile 0.85% NaCl solution (Merck, Darmstadt, Germany) and homogenized. The suspension, serially diluted to 10 -6 and 20 µL of the solution was spread in triplicate on to nutrient agar (NA; Acumedia Neogen Co., Lansing, USA). Man Rogosa and Sharpe media (MRS; Quelab, Montreal, Canada) was also used to detect lactic acid bacteria (LAB). All of the plates were incubated at 36 ˚C for three days and colony forming units (CFU) per g were calculated.
Statistical analysis. The data (Mean ± standard error) were analyzed by one way analysis of variance (ANOVA) followed by Tukey's test to compare the means between individual treatments with SPSS (Version 16; SPSS Inc., Chicago, USA) at p < 0.05 level.
Results
Survival and growth measurements. At the end of trial, survival rate was high in all treatments with no significant difference (p > 0.05). The growth performance of common carp fingerlings fed diets supplemented with different levels of dietary MOS is presented in Table 2. After eight weeks of feeding, growth performance including final weight, WG and SGR did not differ among the dietary treatments, however, FCR was better when the fish were fed 0.05 to 0.20% MOS supplemented diets.
Immunological assays. The effects of the MOS on the humoral innate immune responses of common carp are shown in Figs. 1, 2, and 3. All immune parameters measured (lysozyme activity, Ig and alternative pathway of complement activity) were significantly higher (p < 0.05) in 0.2% MOS fed fish compared to the control group. Fish fed 0.20% MOS showed significantly elevated lysozyme activity compared to the control, but the activity was not significantly higher than the 0.05% and 0.10% MOS groups, as was also the case for plasma Ig and ACH50 levels.
Intestinal microbiota. Intestinal microbiota data are shown in Fig. 4. At the end of feeding trial, total bacterial levels were not affected by MOS (p > 0.05). Levels of LAB were significantly elevated in MOS fed fish compared to the control group (p < 0.05).
Discussion
This study was conducted in order to investigate the effects of MOS on growth, survival and immune responses of common carp. From previous studies it appears that the effects of MOS on growth performance of aquatic species are contradictory. The results of the current study indicate In commercial aquaculture, the farmed species are vulnerable to ubiquitous opportunistic bacterial pathogens and stimulation of the innate immune system through dietary supplements is of great interest. The alternative pathway of complement activity acts as a powerful nonspecific defense mechanism, protecting fish from a wide range of potentially invasive organisms, such as bacteria, fungi, viruses, and parasites. 21 Lysozyme, being an enzyme with antimicrobial activity, can split peptidoglycan in Control 0.05 0.1 0.2 bacterial cell walls especially of the Gram positive species and can cause lysis of the cells. 22 As presented in Figs. 1 and 3, fish fed 0.20% MOS for eight weeks showed increased lysozyme activity and ACH50. Additionally, increased level of serum total Ig as the major components of the humoral immune system was detected in the serum of fish fed 0.20% MOS (Fig. 2). The results are consistent with findings of previous studies on shellfish 1,23 and fish species. Staykov et al. reported higher levels of bactericidal activity, lysozyme, antibody levels and alternative complement activity in rainbow trout fed MOS. 19 In addition, similar results have also been demonstrated in European sea bass fed 0.40% MOS supplemented diets. 20 Similarly, other prebiotics such as inulin and fructo-oligosaccharide (FOS) have been demonstrated to improve the innate immune response in other fish species. 15,24,25 The positive effects of prebiotics on humoral immune responses may be attributable to improved growth of health-promoting bacteria such as Lactobacillus and Bifidobacter spp. that consequently limit potentially pathogenic bacteria. 2 Our results based on analysis of intestinal microbiota revealed that LAB levels were significantly elevated in fish fed dietary MOS (p < 0.05). Prebiotics including MOS have been reported to enhance the growth of beneficial bacteria such as LAB. 2 Regarding the improvement of innate immune parameters measured in our study, it can be concluded that modulation of the intestinal microbiota towards a potentially more beneficial microbial community (i.e. elevated LAB levels) can be achieved through administration of dietary MOS.
In conclusion, the results of this study show that oral administration of MOS at 0.05-0.20% improves FCR, modulates intestinal microbiota and at 0.20% elevates the humoral immune response of common carp by increasing the alternative complement activity, lysozyme activity and serum total immunoglobin. | v3-fos |
2017-04-28T17:59:17.134Z | {
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} | 0 | [] | 2015-07-14T00:00:00.000Z | 16551502 | {
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} | s2 | Shifts in Abundance and Diversity of Soil Ammonia-Oxidizing Bacteria and Archaea Associated with Land Restoration in a Semi-Arid Ecosystem
The Grain to Green Project (GGP) is an unprecedented land restoration action in China. The project converted large areas (ca 10 million ha) of steep-sloped/degraded farmland and barren land into forest and grassland resulting in ecological benefits such as a reduction in severe soil erosion. It may also affect soil microorganisms involved in ammonia oxidization, which is a key step in the global nitrogen cycle. The methods for restoration that are typically adopted in semi-arid regions include abandoning farmland and growing drought tolerant grass (Lolium perenne L.) or shrubs (Caragana korshinskii Kom.). In the present study, the effects of these methods on the abundance and diversity of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) were evaluated via quantitative real-time PCR, terminal restriction fragment length polymorphism and clone library analysis of amoA genes. Comparisons were made between soil samples from three restored lands and the adjacent farmland in Inner Mongolia. Both the abundance and community composition of AOB were significantly different between the restored lands and the adjacent control. Significantly lower nitrification activity was observed for the restored land. Clone library analysis revealed that all AOB amoA gene sequences were affiliated with Nitrosospira. Abundance of the populations that were associated with Nitrosospira sp. Nv6 which had possibly adapted to high concentrations of inorganic nitrogen, decreased on the restored land. Only a slight difference in the AOB communities was observed between the restored land with and without the shrub (Caragana korshinskii Kom.). A minor effect of land restoration on AOA was observed. In summary, land restoration negatively affected the abundance of AOB and soil nitrification activities, suggesting the potential role of GGP in the leaching of nitrates, and in the emission of N2O in related terrestrial ecosystems.
Introduction
China implemented the Grain to Green Program (GGP) in 1897 counties of 25 provinces, including Inner Mongolia, to increase vegetation coverage [1]. By the end of 2012, approximately 9.7 million hectares of cropland had been converted to forest or grassland [2]. This greatly improved soil conditions across China through decreasing soil erosion [3], and increasing soil organic carbon content [4]. There are three typical restoration methods in Inner Mongolia: abandoning farmland, growing grass (Lolium perenne L.), and growing shrubs (Caragana korshinskii Kom.). Both Lolium perenne L. and Caragana korshinskii Kom. are drought-tolerant plants suitable for land restoration in arid or semi-arid regions [5]. Converting farmland into grassland or scrubland alters cover vegetation, which determines the quality and quantity of root exudates serving as nutrients for microorganisms [6]. Changes in fertilization, tillage, irrigation and other agricultural practices possibly altered soil physicochemical properties which could influence soil microbial communities as well [7]. The influences of land use on the diversity and community composition of soil microorganisms have been evaluated in several studies [8][9][10]. Despite numerous environmental benefits, the effects of land restoration on the soil nitrogen cycle, as well as on participating microorganisms, remain poorly understood.
Aerobic ammonia oxidation mediated by bacteria within βand γ-Proteobacteria [11] and archaea within Thaumarchaeota [12,13] is the rate-limiting step in nitrification, which influences plant nitrogen availability, the leaching of nitrates into ground water, greenhouse gas N 2 O emission [14]. Various studies attempted to discern the relative contribution of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) in nitrification across different ecological niches [15,16]. In terrestrial ecosystems, the abundance of AOA frequently exceed that of AOB, which points to the potential role of AOA in nitrification [17]. The abundance ratios of AOA to AOB in a number of studies negatively correlated with pH values [16,18,19], and ex-situ culture of AOB has failed in acidic conditions [20]. This indicates the importance of AOA to nitrification in acidic soils. In the study of Lu et al. [21], urea fertilization was found to stimulate nitrification activity and the growth of AOA from the marine Group 1.1a-associated lineage in the acidic soil. While in an alkaline soil the abundance and composition of AOB, not AOA, was significantly impacted by long-term fertilization [22]. In addition to pH, the ammonia concentration also determined the differential growth of AOA and AOB, and growth of AOB could be promoted at high ammonia levels [23]. In dryland ecosystems, the availability of water played an important role in the activities of ammonia oxidizers [24], and AOB may responded faster than AOA to the addition of water [25].
In this study, we hypothesized that land restoration would affect both AOA and AOB and the effects varied between different restoration methods. We then compared the nitrification activity and the abundance and diversity of the two ammonia oxidizers between degraded farmland and three adjacent restoration lands using the three typical methods used in semiarid Inner Mongolia.
Field site description and soil sampling design
The study area is in Wuchuan County (40°59'N, 110°33'E) in Inner Mongolia Province, China. This area is characterized by typical semi-arid characteristics with a temperate continental climate and with mean annual temperature at 2.7°C and mean annual precipitation at 250 mm-400 mm, over 80% of which is between June and September [26]. The soil is classified as Haplic Calcisol (FAO/UNESCO soil classification system). The farmland in Wuchuan was mainly converted from pasture during the 1950s [27], with wheat, sunflower and potato as main crops. The GGP has been implemented here since 2002 using three major restoration methods. Farmlands were (1) abandoned and allowed to be naturally recolonized with indigenous species, (2) sown with Lolium perenne L., and (3) planted with Caragana korshinskii Kom. This resulted in three land cover types including grassland with indigenous species (e.g. Agropyron cristatum L. Gaertn., Leymus chinensis Trin. Tzvel., Heteropappus altaicus Willd. Novopokr. and Artemisia frigida Willd.), grassland dominated by Lolium perenne L., and scrubland dominated by Caragana korshinskii Kom. According to our survey (data not published), 56.9 thousand ha of land had been converted to forest or grassland in Wuchuan County until 2013. Four land cover types were selected, including farmland (sunflower-potato-wheat three-year rotation, one crop per year, with urea at 300 kg N ha -1 per year and superphosphate at 150 kg P 2 O 5 ha -1 per year), abandoned farmland, Lolium perenne L. land, and Caragana korshinskii Kom. land, with the latter three converted from farmland in 2002. No specific permits were required for sampling. The location is not protected in any way, and there are no impacts on endangered species. Soil samples were taken on 9 July 2013 during the optimal growth period of native vegetation. Five replicates per cover were sampled and each replicate consists of six cores of soil from 0-20 cm depth. The different plots were 1.5-5 km apart from each other. Samples were then transported inside an ice box to the laboratory, sieved through 2 mm mesh, and stored under -80°C condition before DNA extraction.
Soil physico-chemical properties
All soil parameters were analyzed in the laboratory using standard methods. Soil pH was determined in 1:2.5 (wt/vol) diluted water suspension [28]. Soil moisture content was measured by drying fresh soil at 105°C for 24 hours. Phosphate was measured according to Olsen et al. [29]. Ammonia and nitrate were extracted with 1M KCl and measured using a continuous flow analyzer (Bran-Luebbe, Norderstedt, Germany). Soil organic carbon and total N were measured by CNS analyzer (Thermo Fischer scientific Inc., Sunnyvale, CA, USA). The soil chemical properties are listed in Table 1.
Potential nitrification rate
The potential nitrification rate (PNR) was determined via the chlorate inhibition method [30]. Fifty grams of soil sample were placed in the Erlenmeyer flask with 60 ml phosphate buffer solution (g l -1 : NaCl, 8.0; KCl, 0.2; Na 2 HPO 4 , 0.2; NaH 2 PO 4 , 0.2; pH 7.4) with 1 mM (NH 4 ) 2 SO 4 . Potassium chlorate with a final concentration of 10 mg l -1 was added to the flask to inhibit nitrite oxidation. The slurry was then shaken on an orbital shaker at 180 rpm for 24 h under 25°C to maintain an aerobic environment in the dark. The concentration of nitrite was measured at 6, 12, 18 and 24 h after incubation according to Shen et al. [22]. PNR was finally calculated from the rate of linear regression for nitrite concentrations with time.
Soil DNA extraction and quantitative real-time PCR Soil DNA was extracted from 0.25 g of soil using the PowerSoil DNA Isolation Kit (Mobio Laboratories Inc., Carlsbad, CA, USA) following the manufacturer's instructions. Primer pairs of amoA-1F/amoA-2R [31] and CrenamoA23f/CrenamoA616r [32] were used for quantitative real-time PCR (qPCR) analyses of AOB and AOA amoA genes, respectively. The qPCR assays were performed using an ABI 7500 Real-time PCR instrument (Applied Biosystems, Foster City, CA, USA). The reaction mixture (20 μl) contained 10 μl of SYBR Premix Ex Taq (Takara Biotechnology, Dalian, China), 1 μM of respective primer, 2 μg of BSA, and approximately 20 ng template or standard DNA. The specificity and efficiency of amplification was checked by melting curve analysis. Templates to generate standard curve were prepared by the serial dilution of purified plasmid DNA with amoA genes for AOB or AOA respectively.
Terminal restriction fragment length polymorphism analysis of AOA and AOB
For terminal restriction fragment length polymorphism (tRFLP) analysis of the bacterial amoA genes, PCR amplification was performed using amoA-1F and amoA-2R [31] with forward primer 6-carboxyfluorescein (FAM) labeled. The thermal profile consisted of 5 min at 94°C, 35 cycles of 30 s at 94°C, 30 s at 57°C and 60 s at 72°C, and 10 min at 72°C for extension. The PCR product was checked by agarose gel electrophoresis and purified using PCR Clean-Up System (Axygen Scientific, Union City, CA, USA). The purified products were digested with the restriction enzyme Msp I (NEB, Beijing, China). For tRFLP analysis of the archaea amoA genes, the PCR amplification was performed using CrenamoA23f and CrenamoA616r [32] with forward primer FAM labeled. The terminal profile for PCR amplification included 5 min at 94°C, 35 cycles of 45 s at 94°C, 30 s at 60°C and 60 s at 72°C, and 10 min at 72°C. The PCR products were checked and purified similarly as bacterial amoA genes. The purified products of AOA were digested with Mbo I (NEB, Beijing, China). After digestion, the sample of AOB and AOA was mixed with formamide and the internal standard GeneScanTM-500 LIZ (Applied Biosystems, Foster City, CA, USA). The mixed solutions were size-separated using an ABI-PRISM 3030XL Genetic Analyzer (Applied Biosystems, Warrington, UK). The relative abundance of each peak was quantified with GeneMarker version 2.2 (http://www.softgenetics. com).
Cloning, sequencing and phylogenetic analysis
AmoA genes of AOA and AOB were amplified using the same PCR conditions as the tRFLP analysis with non-FAM labeled primer. For each treatment, an equal volume of PCR products of five replicates were pooled to construct clone libraries. The purified PCR products were cloned into the pEASY-T1 vector (TransGen Biotech, Beijing, China). Thirty clones for each treatment were randomly selected for sequencing.
After truncating the primer regions, all sequences were subjected to BLAST-N and BLAST-X analyses to identify amoA gene sequences. Phylogenetic analysis of amoA genes was performed separately for AOA and AOB. The cloned gene sequences as well as known amoA genes were aligned and the phylogenetic tree was constructed using neighbor-joining method and tested by bootstrap analysis using MEGA version 4.0 [33]. The aligned sequences were assigned to operational taxonomic units (OTUs) by Mothur [34] at the 97% sequence similarity level. All the amoA gene sequences have been deposited in the GenBank under accession numbers KJ652991 to KJ653018 for AOB and KJ699396 to KJ699421 for AOA.
Statistical analysis
Two-way ANOVA in conjunction with Duncan's protected least significant difference tests (p < 0.05) were performed to examine the significance of treatment effects on the abundance of AOA and AOB using a log-transformed copy number of amoA genes. Spearman's correlation coefficients were calculated to assess the significant relations between the gene copy numbers, PNA, and soil chemical parameters. Principal component analysis (PCA) and Cluster analysis were applied to compare the tRFLP profiles of AOA and AOB. Analysis of similarity (ANOSIM) [35] was applied to examine the significance of treatment effects on the tRFLP profiles of AOA and AOB.
Soil physico-chemical properties
To compare soil physico-chemical characteristics between different treatments, total organic carbon and nitrogen, pH, ammonium, nitrate, phosphate, and water content were quantified.
The pH values of soils from both grasslands were slightly but significantly higher than that of the cropland ( Table 1). As expected, the concentrations of ammonium, nitrate and phosphate were lower in the restored land soils than that of in the cropland, in which additional fertilizer was applied annually (Table 1). However, two-way ANOVA analysis revealed non-significant differences for total nitrogen content amongst all treatments (Table 1). Soil organic carbon was higher in the restored land than in the cropland, with the exception of abandoned farmland ( Table 1). The carbon-to-nitrogen ratio of abandoned farmland was significantly higher than that of cropland ( Table 1). The lowest carbon-to-nitrogen ratio and water content were measured in land growing Caragana korshinskii Kom. (Table 1).
PNR measurement and abundances of AOB and AOA in soils
To study the effect of land restoration on the nitrogen cycle, the overall activities and abundances of ammonia-oxidizing bacteria and archaea were explored. Potential nitrification rates were measured for soils from the cropland and restored lands (Fig 1A). The highest activity was detected for cropland, while the lowest was found for soil in the Caragana korshinskii Kom. land (Fig 1A). However, the difference of PNR in soil between Caragana korshinskii Kom. land and Lolium perenne L. land was non-significant ( Fig 1A). Interestingly, PNR showed a positive correlation with soil water content (r = 0.629, n = 20, p <0.01) and NO 3 -N concentration (r = 0.719, n = 20, p < 0.001), while a negative correlation with total nitrogen (r = -0.480, n = 20, p < 0.05) and total organic carbon (r = -0.443, n = 20, p < 0.05).
The abundance of AOB and AOA were measured by quantitative PCR analysis of the amoA gene. The abundance of AOB was significantly higher in cropland than that of the restored land ( Fig 1B). Surprisingly, the AOB abundance for abandoned farmland and Lolium perenne L. land was significantly lower than that of Caragana korshinskii Kom. land (Fig 1B), where the lowest PNR was detected (Fig 1A). Nevertheless, the PNR still positively correlated with the abundance of AOB amoA gene copies (r = 0. 736, n = 20, p < 0.01). Based on the qPCR analysis of amoA, AOA was more abundant than AOB in all land types (Fig 1B, as the AOA to AOB ratio were 23.19-230.37), but no significant differences of AOA amoA gene copy numbers were observed between any treatments (Fig 1B).
Genetic profiling of bacterial and archaea amoA genes by tRFLP
To compare the community composition of soil ammonia oxidizing bacteria and archaea between the cropland and restored lands, the amoA gene amplicons of AOA and AOB were analyzed by tRFLP.
Totally, six terminal restriction fragments (tRFs) of AOB were detected from all the soil samples by the enzyme of Msp I (Fig 2). The relative abundance of different tRFs varied between restored and non-restored lands. Notably, the relative abundance of tRF 156 and 140 for the cropland was significantly lower than those for the restored land (Fig 2), while the relative abundance of tRF 256 for the cropland was significantly higher than those for the restored land (Fig 2). Both Cluster and PCA analyses revealed that the community composition of AOB was different between the restored and non-restored lands (S1 Fig). This was further confirmed by an ANOSIM of the restored and non-restored lands ( Table 2). On average, higher values of ANOSIM's R were observed for restored and non-restored land comparisons (ANOSIM's R values 0.56-0.87, all P values < 0.05). Although less pronounced, there were also differences in community composition between restoration methods evidenced by the ANOSIM's R values for abandoned farmland and Caragana korshinskii Kom. land at 0.45 and P values < 0.05. Redundancy analysis was performed to identify the correlation between soil geochemical characteristics and variations in the community compositions of AOB. Nitrate and phosphate were the two main factors which influenced the community composition of AOB (Fig 3). In total, 42% of the variation of the AOB community could be significantly explained by nitrate and phosphate concentrations. (Table 2).
Diversity of AOB and AOA in different soils as revealed by clone library analysis
Four clone libraries were constructed with the amoA amplifications for AOB and AOA respectively. Thirty clones per library were selected for sequencing. All acquired sequences were subjected to in silico digestion analysis. Four tRFs (tRF256, tRF235, tRF156 and tRF62) were detected from the clone library analysis for AOB (Fig 4). The relative abundance of dominant tRFs were comparable with their corresponding tRFs by tRFLP analysis (S1 Table). Interestingly, the sequences with tRF size of 256 bp were only detected for the cropland (Fig 4). Correspondingly, the highest relative abundance of tRF256 was found for cropland by tRFLP analysis (Fig 2). Among them, four sequences share high similarity (98% sequence identity) with the amoA gene sequence for Nitrosospira sp. Nv6 (Cluster 3b), and two sequences share 97% sequence identity with the amoA genes of Nitrosospira sp. Nsp58 (Cluster 2), and the other two sequences were similar to the amoA genes of Nitrosovibrio sp. RY3C (Cluster 3a.2 and Fig 4). More sequences with tRF size of 156 bp (13 for abandoned farmland, 16 for Lolium perenne L. land and 12 for Caragana korshinskii Kom. land) were detected for restored lands than the cropland (5 for farmland). All sequences with tRF size of 156 bp were closely (>95% sequence identity) related to the amoA genes of Nitrosospira sp. Nsp2 (Cluster 3a.1). Despite that the relative abundance of tRF62 was comparable between different soils, as suggested by both tRFLP and clone library analysis. Further sequence analysis revealed that 7 sequences (> 97% sequence identity) with tRF size 62 were only observed in the restored lands (S1 Table). Fewer OTUs were detected for restored soil with grass (8 for abandoned farmland and 7 for Lolium perenne L. land), while 20 OTUs were detected for the cropland (S1 Table). Table). All the four dominant OTUs shared a relative low similarity with those amoA genes from cultivable strains, such as Nitrososphaera viennensis (S4 Fig and S2 Table).
Discussion
Grain to Green Project has restored large areas of vegetation from cropland in semi-arid regions such as Inner Mongolia, resulting in several environmental benefits. In the present study, we explored the influence of three typical land restoration methods applied in semi-arid regions on the abundance and diversity of AOA and AOB in soils. Both the abundance and community composition of AOB were significantly different between the restored lands and the adjacent cropland, indicating that AOB, rather than AOA, was affected by land restoration in this type of ecosystem.
Soil pH is a prerequisite for determining the different responses of AOA and AOB to changes of land use [36]. The pH values in the present study ranged from 8.11 to 8.26, which are suitable for AOB. Laboratory cultures of AOB could only grow above neutral pH [20]. This finding is in agreement with results from previous researches [22,24]. In the study of Nicol et al. [19], the bacterial transcript abundance of amoA increased with increasing pH and the highest pH recorded was only 7.5, which is lower than those in our study. Shen et al. [22] found that long-term fertilization significantly influenced the abundance and composition of AOB in a sandy loam soil with pH ranging from 8.3 to 8.7. In acidic soil conditions, however, Hu et al. [37] found that AOA was increasingly involved in ammonia oxidation after adding water to dryland soils. In a microcosm experiment with soil (pH 7.0), Denaturing Gradient Gel Electrophoresis analyses of both 16S rRNA genes and amoA genes suggested a dramatic response of AOA to different temperature in nitrifications [32]. The pH could possibly affect the bioavailability of ammonia, as NH 3 would be ionized exponentially to NH 4 + when pH decreased [19]. In the study by Khademi and Stroud [38], the ammonia transporter (amtB) on the membrane of E.coli could only conduct the transfer NH 3 but not the protons. Recently, Offre et al. [39] discovered that the ammonia transporter for AOA was highly variable and clearly distinct from those for AOB. However, the specifics of how ammonia transporters of both ammonia oxidizers relate to pH remains to be explored. Both the abundance and community composition of AOB significantly differed between the restored land and the cropland, for which the highest abundance of AOB and PNR activities were detected (Fig 1). This result suggested that bacterial ammonia oxidation was reduced after land restoration. In contrast to the restored land, urea and superphosphate were applied annually in the croplands. Mineral nitrogen fertilizations often lead to increased soil PNR and an abundance of AOB in alkaline soils [22,24]. However, the effect of phosphate amendment on AOB has been rarely studied. Clone library analysis of amoA genes for AOB revealed that all sequences were Nitrosospira-like sequences (Fig 4). The dominance of Nitrosospira-like amoA sequences was also detected in several studies for terrestrial environments [22,24,40]. Seven of the AOB amoA gene sequences, detected exclusively in cropland soils, shared high similarity with the amoA genes of Nitrosospira sp. Nv6 (Fig 4). Nitrosospira sp. Nv6-like amoA genes were often detected in soils with elevated levels of mineral nitrogen fertilizer input [41,42]. Compared with cropland, the proportion of Nitrosospira sp. Nsp2-like amoA gene sequences increased after restoration (Fig 4, S1 Table), suggesting that these populations possibly adapted to low level of ammonia in the restored soils. The soil water content was significantly lower in the scrubland soil than in other treatments (Table 1), possibly due to the high ratio of bare land in the Caragana korshinskii Kom. land, where the lowest PNR was detected. In a recent study by Hu et al. [37], water addition was found to enhance PNR and the abundance of AOA and AOB in dry ecosystems. However, the abundance of AOB in the Caragana korshinskii Kom land was significantly higher than those in the other two restored lands. The symbiotic of Caragana korshinskii Kom. with nitrogen-fixing microorganisms could increase the soil nitrogen source and possibly promote the growth of AOB [43]. Restoration associated changes with plant diversity and composition may have both directly and indirectly effects on AOB community. Plant could compete for ammonia with ammonia oxidizing microorganisms [6], while root exudates produced by plant also could boost the proliferation of ammonia oxidizing microorganisms [44]. However, the effect of legume plants on the abundance of AOB was not consistent [45]. Further studies on the effect of Caragana korshinskii Kom. on the AOB community are still needed.
In the present study, both the abundance and community composition of AOA were comparable between different soils, suggesting that land restoration might not affect the quality and quantities of microniches for AOA. However, real time qPCR results, which revealed that the abundance of AOA was much higher than that of AOB in all the soils studied (Fig 1), coincided with several studies [17,22,24]. These findings indicated that AOA was still predominant in these soils despite that theirs high pH values were thought to be unfavorable for AOA. Soil networks are highly heterogeneous and complex, possibly providing microniches suitable for the survival of AOA [46]. The oligotrophic life style, which only needs low levels of maintenance energy [47,48], and the small cell size and small genome of most AOA [49] possibly contributed to its high abundance in soil as less energy is needed for its replication. The low water content in all the studied soils could reduce soil pore connectivity, which affects the diffusion of key factors. This might result in microniches with very limited ammonia. However, it is possibly that land restoration did not affect the quantities and qualities of microniches for AOA in this study.
Land restoration in Inner Mongolia, which was selected for its representation of semi-arid ecosystems, resulted in a significantly decreased abundance of AOB and ammonia oxidation activities, A significant difference in the community compositions of AOB was also observed between restored lands and the adjacent control, and AOB populations that possibly adapted to high concentration of inorganic N were negatively affected by the land restoration. Only a slight difference in AOB communities was observed between the restored land with the grass and with the shrub. These results indicate the potential role of GGP in reducing the emission of greenhouse gas N 2 O and in the leaching of nitrates into ground water. Table. OTU distribution of bacterial amoA gene sequences. | v3-fos |
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} | s2 | A Genome Scan for Selection Signatures in Pigs
Identifying signatures of selection can provide a straightforward insight into the mechanism of artificial selection and further uncover the causal genes related to the phenotypic variation. Based on Illumina Porcine60KSNP chip data, four complementary methods, Long-Range Haplotype (LRH), Tajima’s D, Cross Population Extend Haplotype Homozygosity Test (XPEHH) and FST, were implemented in this study to detect the selection signatures in the whole genome of one typical Chinese indigenous breed, Rongchang, one Chinese cultivated breed, Songliao, and two western breeds, Landrace and Yorkshire. False Discovery Rate (FDR) was implemented to control the false positive rates. In our study, a total of 159, 127, 179 and 159 candidate selection regions with average length of 0.80 Mb, 0.73 Mb, 0.78 Mb and 0.73 Mb were identified in Landrace, Rongchang, Songliao and Yorkshire, respectively, that span approximately 128.00 Mb, 92.38 Mb, 130.30 Mb and 115.40 Mb and account for approximately 3.74–5.33% of genome across all autosomes. The selection regions of 11.52 Mb shared by Landrace and Yorkshire were the longest when chosen pairs from the pool of the four breeds were examined. The overlaps between Yorkshire and Songliao, approximately 9.20 Mb, were greater than those of Yorkshire and Rongchang. Meanwhile, the overlaps between Landrace and Songliao were greater than those of Landrace and Rongchang but less than those of Songliao and Ronchang. Bioinformatics analysis showed that the genes/QTLs relevant to fertility, coat color, and ear morphology were found in candidate selection regions. Some genes, such as LEMD3, MC1R, KIT, TRHR etc. that were reported under selection, were confirmed in our study, and this analysis also demonstrated the diversity of breeds.
Introduction
Looking back on the evolutionary history of the pig, the domestication process began almost simultaneously in separate areas of the Asia and Europe [1,2]. Since then, the pig has experienced evolution over a very long time under natural and artificial selection. Domestic pig phenotypes, including both production-relevant traits and behavior, have been largely transformed compared to its wild counterpart, and the phenotypic variation in the breeds also becomes more distinctive [3].
To better understand the underlying genetic mechanism for phenotypic distinction caused by selection in pig, the hunt for genomic evidence of selection has been performed in various pig breeds using whole genome genotype data or pool sequencing data [4][5][6][7][8][9]. The studies associated with growth traits, reproduction traits and even coat color, to which breeders had attached great importance, have shown that selection facilitates the homozygosity of beneficially allelic genes [4,5]. Several genes with major effects on growth, reproduction trait and coat color have already been identified under selection, like insulin-like growth factor 2 (IGF2), relevant to muscle growth [10], parathyroid hormone-like hormone (PTHLH), associated with litter size and the number of teats in European pig [5,11] and v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) and melanocortin 1 receptor (MC1R), related with a series of pig breed color types [4].
Theoretically, a novel beneficial variant that has been under selection pressure usually shows long-range linkage disequilibrium (LD) and a high population frequency over a long period of time [12]. Thus, selection signatures could be detected through the decay of linkage disequilibrium and the variation of allele frequency. These methods for detecting selection signatures can be grouped into three categories according to the information used: population differentiation, site-frequency spectrum and linkage disequilibrium [13,14]. Corresponding to these groups, the F ST , the Tajima's D test, the Cross Population Extend Haplotype Homozygosity Test (XPEHH) and the long range haplotype (LRH) are the representative methods widely used in identifying selection signatures. Among them, F ST was initially used to assess population differentiation according to the DNA polymorphism in populations, which was attributed to geographically variable selection [15][16][17]. Tajima's D is the most famous method for detecting selection signatures based on segregating sites frequency, and it is sensitive to purifying selection and balancing selection [18]. LRH was developed to measure the degree of LD in one region with long range haplotypes with an adjustment to local variation in recombination rates, which is sensitive to the regions with a rapidly increased frequency of the derived allele at selected sites but may not detect selection at the fixed regions because of the elimination of variation at those sites [19]. Although both LRH and XPEHH are typical haplotype methods, XPEHH assumes that the occurrence of selection can be traced by measuring LD or observing overrepresented haplotypes in the observed population, making it possible to detect entirely or approximately fixed sites [19]. Recently, Rubin et al. [20] proposed a method ZH P utilizing the pooled heterozygosity in small window to detect selection signatures, mainly dealing with pooling chip/sequencing data.
Although some studies have been carried out to detect selection signatures in the pig, the findings have not been totally concordant due to the limitations of sample size and statistical methodology. In addition, the selection patterns in pig breeds are different as their different evolution histories. Therefore, it is necessary to explore selection signatures in more pig breeds, which will be helpful to better understand the genetic variation in different pig breeds and identify common variants in traits of interest. In this study, we detected the selection signatures at the whole genome level in one Chinese indigenous breed, Rongchang, one Chinese cultivated breed, Songliao, and two western breeds, Landrace and Yorkshire, using Illumina porci-neSNP60K BeadChip. Four methods, LRH, Tajima's D, XPEHH and F ST were implemented to identify selection signatures. Bioinformatics analysis was also performed to explain the biological function of the selection signatures.
Materials and Methods Populations
A total of 338 individuals from four pig breeds were collected for the experimental population in our study, including 72 Rongchang (Chinese indigenous breed), 86 Songliao (Chinese cultivated breed), 97 Yorkshire and 83 Landrace pigs. As a typical representative of indigenous breed, Rongchang was breed in the southwest of China and has a special coat color in comparison with other Chinese indigenous breeds. In the past decades, Songliao was cultivated through hybridization of the Duroc, Landrace and Chinese Minzhu, which originated in Northeast of China. Landrace and Yorkshire are superior to Chinese pig breeds with fast growth rate and high feed efficiency. Landrace originates from Denmark, and Yorkshire was bred through the cross between Asian breeds and British local breeds in past centuries [21]. Although Landrace and Yorkshire can be distinguished each other according to body size and ear morphology, they have similar selection direction in most economic traits. In this study, the principal component analysis (PCA) followed Paschou et al. [22] showed four breeds in this study are generally independent except that Songliao is genetically close to Landrace (S1 Fig.).
SNP genotyping and data filtering
Genomic DNA samples from all of the pigs were extracted from ear tissue using a standard phenol/chloroform method. All of the DNA samples were analyzed by spectrophotometry and agarose gel electrophoresis. The genotyping platform used was Infinium II Multisample assay (Illumina, San Diego, CA). SNP arrays were scanned using iScan (Illumina, San Diego, CA) and analyzed using BeadStudio (Version 3.2.2, Illumina, San Diego, CA). The entire tissue sampling procedure was carried out in strict accordance with the protocol approved by the Animal Welfare Committee of China Agricultural University (Permit Number: DK996).
We implemented a quality control procedure to ensure the high data quality by (1) removing SNP loci with call rate less than 0.95 and unknown position, (2) removing SNP loci with minor allele frequency (MAF) less than 0.05 and (3) discarding the individuals with call rate less than 0.90. Following quality control, we imputed the missing genotypes and inferred haplotypes for the haplotype-based methods (LRH and XPEHH) using BEAGLE [23]. In addition, HAPLOVIEW v4.1 [24] was used to estimate linkage disequilibrium (LD) in four breeds, respectively.
Methods for Detection of selection signature
Four methods, LRH, Tajima's D, XPEHH and F ST , were implemented to detect the selection signatures. Tajima's D and F ST directly handle the SNP genotype, while XPEHH and LRH mainly use phased data. Among them, XPEHH and F ST need to first define observed and reference population. In this study, Landrace was selected as the common reference population as it was involved in the cultivation of Songliao. Accordingly, three breed pairs of Yorkshire-Landrace (Y-L), Songliao-Landrace (S-L) and Rongchang-Landrace (R-L) were used for further analysis. For each breed pair, the common SNPs for one breed pair were unified before implementing XPEHH and F ST .
LRH and XPEHH Analyses. Both LRH and XPEHH are based on the 'Extended Haplotype Homozygosity' (EHH) algorithm to detect selection signatures. For LRH, the program Sweep v.1.1 was implemented to compute the 'Relative Extended Haplotype Homozygosity' (REHH) statistic and the core regions were defined as the chromosome fragments including at least 3 SNPs [25]. For XPEHH, the program at http://hgdp.uchicago.edu (coded by Joe Pickrell) was employed. The negative XPEHH scores suggest that selection occurred in the reference population, whereas the positive scores suggest the same about observed population. As the genetic distance between adjacent SNPs is needed for the calculation of XPEHH, a chromosome segment of 1Mb was straightly converted as 1 centiMorgan (cM).
Tajima's D and F ST Analyses. As a typical method for detecting selection signature [18], Tajima's D considers the difference between the mean pairwise difference and the number of segregating sites in nucleotide polymorphism data. The test statistic equals zero for neutral variation, is positive when an excess of rare polymorphism is caused by recent balancing selection for multiple alleles and is negative when the excess of high-frequency variants suggests selective sweep. To reveal the divergent selection in this study, a two-step process of F ST proposed by Gianola et al. [17,26] was employed to identify selection signatures based on population differentiation. The F ST value ranges from 0 (identical population) to 1 (complete differentiation).
Identifying potential selection signatures
As a widely used test of neutrality, Tajima's D has the appealing property that its empirical distribution approximately follows a standard normal distribution after normalization [27]. Similarly, in this study, the empirical distribution of F ST also approximately follows a standard normal distribution after the normalization of the square root of F ST values [26]. For the LRH test, the across genome REHH values were ordered into 20 bins according to their frequency. After normalizing each bin by log-transformation, the LRH scores approximately follows a standard normal distribution [25]. In likewise, XPEHH approximately follows a standard normal distribution after normalization as well [19]. Hence, the significance test based on normal distribution was performed in these four methods to hunt the potential selection signatures in this study. Considering the multiple testing, False Discovery Rate (FDR) was implemented to control the false positive rate [28][29][30]. The test statistic values with FDR less than 0.1 for each method were outlier signals in this study, and extending 250 kb towards the upstream and downstream directions of one outlier would be a potential selection region (PSR).
Gene annotation
To further control the false positive rates of the detection of selection signatures, we include the potential selection region as a candidate if FDR in one employed method is below 0.05 or if FDR in two or more employed methods is below 0.1. Bioinformatics analyses were then carried out to reveal the potential biological function of genes harbored in candidate selection regions through the NCBI database (http://www.ncbi.nlm.nih.gov/gene/). To further explore the biological function of those candidate selection regions, the QTLs enrolled in Pig QTLdb (www. animalgenome.org) were gathered and compared with those candidate selection regions based on the putative location of the QTLs.
Information of chip data
Following quality control and principal component analysis (S1 Fig.), 83, 72, 86 and 97 individuals and 47114, 28997, 45945 and 47569 SNPs corresponding to Landrace, Rongchang, Songliao and Yorkshire were retained for this analysis ( Table 1). The genome heterozygosity for Landrace, Rongchang, Songliao and Yorkshire is 0.345, 0.320, 0.347 and 0.335 on average, respectively. There is no significant difference in heterozygosity between Chinese local breeds and Western breeds. As a typical representative of Chinese indigenous pig breed, Rongchang has the minimum number of qualified SNPs, which is in agreement with the investigation by Ai et al. (2013) [6], in which most of Chinese indigenous pig breeds have lower number of SNPs. Further linkage disequilibrium analysis indicates that average r 2 in Rongchang (0.150) is lower than it in Landrace (0.251), Songliao (0.261) and Yorkshire (0.248). This might attribute that Illumina Porcine60KSNP chip was designed mainly according to the genomic information of European pig breeds. Correspondingly, the number of unified SNPs for the Rongchang-Landrace pair was lowest as well. As shown in Table 2, for the F ST and XPEHH analyses, a total of 43890, 45500 and 27100 common SNPs, were used for the Songliao-Landrace (S-L), Yorkshire-Landrace (Y-L) and Rongchang-Landrace (R-L) breed pairs, respectively. The average distance between adjacent SNPs for Y-L and S-L was approximately 50 kb, while 79 kb for R-L.
Empirical distribution of four test statistics
The empirical distributions of all test statistics for each breed/breed-pair are clearly illustrated. [19,25]. Similarly, the test statistics of Tajima's D and F ST after normalization roughly followed a standard normal distribution with a small skew. In addition, the distributions of the four test statistics indicated similar tendencies in other breeds/breed-pairs (S2 Fig.). We divided the assigned allele/core haplotypes into a series of 0.05 bins according to their frequencies and used box-and-whisker plots to depict the distribution of the outliers (Fig. 3 the locus at the genome level was very close to that in outlier for Tajima's D, the standard deviation of the absolute allele frequency difference was double in outlier, which resulted in a coefficient of variation in outlier windows two times higher than the one in the genome (Table 1). This reflected that the alleles under selection usually presented a high frequency variation towards beneficial mutation fixation compared with the other alleles in genome. Table 2 shows the selection signatures detected by XPEHH and F ST in the three breed pairs of Y-L, S-L, and R-L when Landrace was treated as a common reference population. For XPEHH analysis, 379, 412 and 248 outliers were detected in three breed pairs, Y-L, S-L and R-L, respectively. Taking Y-L as an example, 240 positive XPEHH values out of 379 outliers indicated that selection occurred in observed population (Yorkshire), and the other 139 outliers with negative XPEHH values suggested selection in the reference population (Landrace). For further analysis, the outliers detected in each breed pair were assigned to each breed, and the potential selection region was defined as a region of 500kb around an outlier identified by XPEHH. After merging regions of overlap, 28, 30 and 57 potential selection regions were identified in three observed breeds, Yorkshire, Songliao and Rongchang, respectively. For the Landrace selection signatures, a total of 139 outliers fell into 30 potential selection regions when Yorkshire was treated as the reference population. For F ST , a total of 77, 88 and 63 potential selection regions were detected from the three breed pairs, Y-L, S-L and R-L, respectively. Table 2 also shows that both the mean and standard deviation of the absolute frequency difference on one assigned allele (allele_1 in Table 2 and Fig. 3) in two of the populations in outlier regions/windows were higher than those in all regions/windows for XPEHH and F ST . Particularly, the breed pair of Rongchang-Landrace (R-L) indicated larger difference compared with other breed pairs, implying different selection direction in Rongchang and Landrace.
Candidate selection regions
To correctly reflect the footprints of selection, the potential selection region with FDR less than 0.05 for one method or FDR less than 0.1 for two or more methods were defined as a candidate selection region in this study. Finally, a total of 159, 127, 179 and 159 candidate selection regions with average lengths of 0.80 Mb, 0.73 Mb, 0.78 Mb and 0.73 Mb were identified in Landrace, Rongchang, Songliao and Yorkshire, respectively (Table 3, S9 Fig.). Overall, approximately 128.00 Mb, 92.38 Mb, 130.30 Mb and 115.40 Mb selection regions were detected in the four pig breeds, accounting for approximately 3.74-5.33 percent of the genome across all autosomes. Note that Rongchang, a representative Chinese indigenous pig breed, shared approximately 10.63 Mb candidate selection regions with the representative Chinese cultivated breed, Songliao. This is longer than the overlapping regions between Rongchang and Yorkshire. In addition, there are approximately 9.20 Mb candidate selection regions shared by Songliao and Yorkshire, which is longer than the length shared by Yorkshire and Rongchang but shorter than that shared by Rongchang and Songliao.
Genomic annotation
Based on the findings of selection regions, the candidate genes and QTLs harbored in the selection regions were revealed. The results of enrichment analysis did not show any intuitive information on selection. We noted that 33, 24, 26 and 27 candidate selection regions corresponding to Landrace, Rongchang, Songliao and Yorkshire were mapped in the gene deserts, which accounted for approximately 30 percent of all candidate selection regions in four breeds, respectively (S1 Table). In addition, some genes identified in the candidate selection region are yet not annotated. Nonetheless, many genes and QTLs identified under selection were still observed in our list (S1 Table). Meanwhile, Table 4 and Table 5 shows that a series of genes and QTLs associated with economic and appearance traits were not only identified in candidate selection regions but also in potential selection regions.
Genes overlap with candidate/potential selection regions. With the available annotation of the pig genome, Table 4 summarizes a part of the candidate genes falling into or overlapping [43] with the candidate/potential selection regions in this study. Among them, the 30.819-30.823 Mb selection region on SSC4 was detected by F ST in Yorkshire and Landrace. This region harbors the thyrotropin-releasing hormone receptor (TRHR) gene, which plays an important role in regulating the hypothalamic-pituitary-thyroid axis and, as a G-protein-coupled receptor, is relevant with average backfat thickness, daily gain, and carcass and meat quality [31]. The 21.52-21.55 Mb selection region on SSC16 and 49. 16-49.17 Mb selection region on SSC5 were only identified in Rongchang, and these two regions overlapped with the prolactin receptor (PRLR) gene and the parathyroid hormone-like hormone (PTHLH) gene, which were reported to be relevant with litter size and teat shape in pig [5,32]. Several genes related to pig coat color were also identified in our study, e.g. the solute carrier family 45 member 2 (SLC45A2) gene [5] located in the 20.71-20.75 Mb region of SSC16 was only found in Rongchang, while the KIT gene [4] located in the 43.55-43.59 Mb region of SSC8 was detected in Landrace, Songliao and Yorkshire, respectively. Another well-known coat color gene, MC1R [33], which could influence the synthesis of coat color in pigs, was found to overlap with the selection region in Songliao. In addition to genes influencing coat color suffering from selection, the genes related Complete QTL list see S1 with ear morphology were also overlapped with our selection regions, e.g. the LEM domain containing 3 (LEMD3) gene associated with flat-eared morphology [5] was separately overlapped with the 32.50-33.50 Mb selection region in Yorkshire and 32.10-33.01 Mb in Landrace on SSC5. Additionally, the region of 35.67-36.17 Mb on SSC7, identified in Yorkshire and Landrace by F ST , overlapped with the peroxisome proliferator-activated receptor delta (PPARD) gene, which not only affects ear size but also plays an important role in backfat thickness [34,35].
QTLs overlap with selection regions. The Pig QTLdb database (http://www.animalgenome. org/cgi-bin/QTLdb/index) has collected almost all of the QTLs reported in the past decades and is now being updated. Table 5 summarized some of the QTLs located in or overlapped with the selection regions in our study, more details see S1 Table. Taking the 0-4.41 Mb selection region of SSC2 detected in Songliao, Yorkshire and Rongchang for instance, two QTLs influencing backfat thickness and leaf fat in the carcass and meat and carcass quality were mapped in this region. Simultaneously, this region also overlapped with the carnitine palmitoyltransferase 1A (CPT1A) gene, which plays an important role in the regulation of fatty acid metabolism in newborn pig [36], as shown in Table 4. Again for ear morphology, a series of QTLs related to ear erectness in Yorkshire, ear size in Landrace and ear weight in Rongchang were separately found to overlap with the 32.
Discussion
Most studies of selection signatures have only implemented a single method, but different methods emphasize different information in the data and are sensitive to different categories of selection signatures [13,14]. Hence, only applying a single method to detect selection signatures might result in some unknown bias. In this study, we applied four methods, LRH, Tajima's D, F ST and XPEHH, to explore the selection signatures in Landrace, Rongchang, Songliao and Yorkshire pig breeds. In accordance with previous research [19], our results (Fig. 2) showed that the LRH test is effective for detecting ongoing selection signatures with low to moderate frequency, while the other three methods are efficient in revealing approximate or fixed selection signatures. The Tajima's D test focuses on selection signatures where the change in allele frequency under selection occurred quickly in comparison with the unselected loci in the same population. This feature was evident in the large difference in allele frequency at SNPs in the outlier windows and the genome (see Table 1). XPEHH is sensitive to detect approximately completed selection signatures in which the selected haplotype/allele has approached or achieved fixation in one population but remains polymorphic in the other one [19].
In addition, we also noted that the low density SNPs and the short-range LD pattern in Ronchang may result in low efficiency of haplotype-based methods to detect selection signatures. Our results shows LRH detected much less outliers in Rongchang than in other three breeds, while those detected by Tajima's D are very close (Table 1). Similarly, XPEHH detected less outlier in Rongchang-Landrace than in Yorkshire-Landrace, and close to those in Songliao-Landrace, which should have relatively less outliers considering the genetic connectedness of Songliao and Landrace. Comparing with Rongchang, Songliao is a recently cultivated pig breed, conceivably resulting in long-rang LD pattern. It makes Songliao was detected more outliers by Tajima's D and F ST in comparison with LRH and XPEHH (Table 1 and 2), likely bring higher false positive rates. It should be careful to use such methods in this situation.
According to the selection signature findings, our study exhibited the genetic diversity of Rongchang, Songliao, Landrace and Yorkshire, which was caused by geographic difference, introgression and demographic history [1,2,8]. Comparing with the overlapping selection regions between breeds, Landrace and Yorkshire shared the longest overlap of 11.52 Mb in selection regions as they both originated from Europe. As two well-known in commercial pig breeds, they have already experienced a relative long period of adaptive evolution to meet similarly commercial requirements. Rongchang is a typical Chinese indigenous breed that was domesticated in Sichuan basin in the southwest of China, and its white coat makes Rongchang different from most Chinese indigenous breeds that mainly have black coats. The isolated environment reduces the genetic connection with other breeds and maintains the unique Rongchang characteristics, which results in the fewer overlaps of selection signatures with the other western pig breeds. Songliao was cultivated through hybridization of the Duroc, Landrace and Chinese Minzhu (another famous Chinese indigenous breed with a black coat in northeast of China) in past decades. Therefore, the overlapping selection regions of Songliao with Landrace were longer than Landrace shared with Rongchang. Meanwhile, Rongchang shared longer selection regions with Songliao than with Landrace and Yorkshire due to the evolutionary trajectory of Chinese pig. This conclusion agrees with previous studies [37,38] and implies that wide diversity exist in various populations due to geographic difference and various selection preference.
Most of the important economic traits in pig have been suffering from strong selection pressure according to previous research [39], which is also supported by our findings. A series of genes relevant to growth, meat quality, fatty metabolism and fertility were found to be under selection in this study (S1 Table, Tables 4, 5). Among them, the CA3 gene that located in 56. 19-56.20 Mb on SSC4 was relevant to the intramuscular fat content and percentage of ham of pigs in accordance with previous research [40]. Correspondingly, the candidate selection region of 55.50-56. 25 Mb in Landrace was identified by LRH and Tajima's D, respectively. Similarly, the HMGA1 gene [41] related with the fat deposition was found in the candidate selection region in Rongchang, which was also detected by LRH and Tajima's D, respectively. Note that these two genes, the CA3 gene and the HMGA1 gene, separately correspond to the typical characteristics in Landrace and Rongchang, especially the fat deposition in Chinese local breed. Additionally, the genes harbored in potential selection regions were also relevant to the economic features of pig breeds. For example, three candidate genes (FSHB [42], PTHLH [11] and PRLR [43]) associated with reproduction traits were only identified in Rongchang, implying that the genetic mechanism for reproductive capacity in Rongchang may be different from others ( Table 4). As an establised sweep, IGF2 should have been detected under selection in this research, but unfortunately, our results could not provide further support as reported in other studies [9] because the fine location of this gene is still not clear in the current pig genome.
Ear morphology and body length are two important indicators that distinguish Landrace and Yorkshire. Accordingly, the 32.09-33.50 Mb candidate selection region on SSC5 in Yorkshire and Landrace harbored two candidate genes (WIF1 and LEMD3) that were reported to be relevant to bone development and ear morphology [44,45]. Fig. 4A demonstrates the selection regions that contains LEMD3 gene, the selection regions were identified by Tajima's D, LRH and F ST (P-value <0.05) in Landrace and Yorkshire, respectively. After the correction of multiple testing, the potential selection regions identified by F ST is still significant and this method is properly sensitive in divergent selection in according to previous research [15]. This phenomenon not only demonstrates the efficiency of the strategy of multiple methods, but also suggests that the ear morphology has been suffered different selection pressure between Landrace and Yorkshire. Ren et al. (2011) investigated PPARD related with ear morphology and underwent a selective sweep signal in Erhualian, a famous Chinese indigenous breed with large and floppy ears, simultaneously, they detected PPARD gene associated with backfat thickness due to the pleiotropism [34]. However, in this study, we only detected PPARD gene under selection in Landrace and Yorkshire by F ST without correction of multiple testing. In addition, the ADAM metallopeptidase with thrombospondin type 1 motif, 3 (ADAMTS3) gene [5], which is involved in body size, were found in the potential selection region in Yorkshire. Correspondingly, the ADAMTS12 gene [5] that was also relevant with body size was detected in Landrace with extreme statistical values when XPEHH and F ST were separately performed. This may provide support for the difference in their body size.
Coat color is one of important features for pig breeds as well. KIT and MC1R that associated with coat color have been investigated to suffer selection in pig and chicken [4,46]. In this study, KIT gene overlaps with or closes to the potential selection region in Yorkshire, Landrace and Songliao through the detection of Tajima's D (Fig. 4C). However, there is no potential selection region overlapping with KIT gene in Rongchang. As to MC1R, the mutation at amino acid 240 deduced Duroc's distinctive red coat, implying that only pigs with Duroc ancestry will contain this mutation allele in the MC1R gene [33,46]. Coincidently, MC1R gene was only detected under selection in Songliao in our study possibly attributed to the introgression of Duroc in the cultivation of Songliao (Fig. 4B). Wilkinson et al. (2013) collected 14 pig breeds with 24-34 individuals per breed to detect selection signatures using PorcineSNP60 chip, while they did not find KIT and MC1R gene under selection in European pigs. They attributed it to the poor coverage of the PorcineSNP60 chip [5]. This may be one explanation for the phenomenon that the KIT and MC1R genes were not detected under selection in Rongchang, only 28997 SNPs available. Another reason could be that the genetic mechanisms of white color in Rongchang are different from that in Yorkshire and Landrace (Fig. 4). Correspondingly, SLC45A2 gene was detected under selection by LRH in Rongchang, while it was not identified in Yorkshire and Landrace. However, when FDR was not carried out, SLC45A2 gene was identifed suffering from selection in these three white coat color breeds by three methods of LRH, F ST and XPEHH respectively (Fig. 4D), which is in accordance with Wilkinson et al. (2013) [5].
So far, several researches have been carried out to identify selection signatures in pig [4][5][6][7][8][9]. . Patterns of genetic variation associated with LEMD3, MC1R, KIT and SLC45A2 genes. Variation in breed allele frequencies of SNPs at the potential selection region for ear morphology/coat color on SSC5, SSC6, SSC8 and SSC16 (color coded by the pig breeds to which they belong), Genomic distribution of potential selection region as measured by four methods, each symbol represents a candidate selection region identified by corresponding methods.
doi:10.1371/journal.pone.0116850.g004 [7] only reveal a few potential selection signatures. On the contrary, Wilkinson et al. (2013) reveal selection signatures completely through widely between-population and within-population analysis [5]. Accordingly, a series of well-known candidate genes were found and most of them are reproductive in this study, such as WIF1 and LEMD3. In addition, we note that those researches only collected about 30 individuals in each breed, and the small sample size may make them inefficient to detect selection signatures, e.g. MC1R gene were not identified under selection by Wilkinson et al. (2013) [5], while identified in our study. As to the selection signatures relevant to economic traits, the low reproducibility across different researches is a common phenomenon in farm animal chip data analysis. This may be caused by the high marker distance of Porcine SNP60BeadChips. Rubin et al. (2010) [20] and Amaral et al. (2010) [8] employed the pool sequencing data to detect selection signatures in pig and they also found a series of interest candidate genes. However, the analysis of pool sequencing data only can make use of the information from allele frequency, which is one of elements in detecting selection signature. We know the long range haplotype is also an important element in detecting selection signatures. Despite this, Rubin et al. (2010) also highlighted a few established selection signatures, such as KIT gene, and displayed a series of reliable evidences to support the inferences [20]. With the development of sequencing technique, it becomes promising to detect selection signatures using sequencing data, especially the individual resequencing data, which may improve the accuracy of selection signature detection through improving the density of SNPs [4]. While the sample size and coverage of sequencing also need to be taken into consideration as point out by Cutler et al. (2010) [47]. | v3-fos |
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} | s2 | Attack Pattern and Reproductive Ecology of Tomicus brevipilosus (Coleoptera: Curculionidae) on Pinus yunnanensis in Southwestern China
Tomicus brevipilosus (Eggers) (Coleoptera: Curculionidae, Scolytinae) was recently discovered as a new pest of Yunnan pine (Pinus yunnanensis Franchet) in Yunnan Province in southwestern China. However, little was known on its reproductive biology and pattern of trunk attack on Yunnan pine. The objectives of this study were to better understand the reproductive biology of T. brevipilosus by investigating the seasonality of trunk attacks by parent adults for the purpose of reproduction (i.e., breeding attacks) and the within-tree pattern of these attacks. Our results showed that T. brevipilosus breeding attacks in P. yunnanensis generally started in early March and ended in early June in Anning County, Yunnan. T. brevipilosus exhibited two general patterns of infestation. From early March to mid-April, T. brevipilosus bred preferentially in the trunks of Yunnan pine trees that were already infested by Tomicus yunnanensis Kirkendall and Faccoli and Tomicus minor (Hartig), colonizing spaces along the trunk (mostly in the mid- and lower trunk) that were not already occupied by the other two Tomicus species. Later, from about mid-April to early June, when there were no Yunnan pine trees newly infested by T. yunnanensis and T. minor, T. brevipilosus attacked Yunnan pine by itself, infesting the lower parts of the trunk first and then infesting progressively upward along the trunk into the crown. Infestation by T. brevipilosus extends the total period that P. yunnanensis trees are under attack by Tomicus beetles in southwestern China, which helps explain why Yunnan pine has suffered high levels of tree mortality in recent decades.
Bark beetles in the genus Tomicus (Coleoptera: Curculionidae, Scolytinae), with the exception of Tomicus puellus (Reitter) that infests primarily Picea trees, are well-known pests of pine (Pinus) trees in most Eurasian countries where pine is native (Bakke 1968, Ye 1991, Faccoli 2007, Kirkendall et al. 2008, Lieutier et al. 2015. These univoltine bark beetles are commonly called shoot beetles because the newly emerged adults feed inside living shoots of their host trees during the summer months as they become sexually mature, which often results in death of the infested shoots (Långström 1983, Ye 1994a, Kohlmayr et al. 2002. The Palaearctic genus Tomicus contains eight recognized species worldwide, of which five occur only in Asia, one only in Europe, and two in Eurasia (Kirkendall et al. 2008, Lieutier et al. 2015. The Eurasian species, Tomicus piniperda (L.) is the only Tomicus species to be introduced beyond its native range, being first found in the Great Lakes region of North America in 1992 (Czokajlo et al. 1997, Haack and Poland 2001, Lieutier et al. 2015.
Tomicus brevipilosus (Eggers) is native to Asia where it has been reported to occur in China, India, Japan, Korea, and Philippines (Kirkendall et al. 2008, Lu et al. 2014). This bark beetle has been recorded to infest several species of Pinus, including Pinus yunnanensis (Franchet), Pinus koraiensis Siebold & Zucc., and Pinus kesiya Royle ex Gordon, but severe damage has seldom been reported (Murayama 1959, Kirkendall et al. 2008, Lu et al. 2014). In China, T. brevipilosus has been generally regarded as a secondary pine pest, mainly infesting pine trees weakened by drought, defoliation, and other environmental stressors (Kirkendall et al. 2008, Lu et al. 2014. However, since the late 1990s, T. brevipilosus along with Tomicus yunnanensis Kirkendall and Faccoli and Tomicus minor (Hartig) have reached outbreak levels, resulting in widespread tree mortality with more than 200,000 ha of infested P. yunnanensis forests in southwestern China (Ye 1991, Duan et al. 2004, Lu et al. 2014.
In Yunnan, T. brevipilosus completes its life cycle in Yunnan pine (P. yunnanensis) (Lu et al. 2014). After exiting the host material in which the brood developed, newly emerged adults fly to the crowns of nearby pine trees, usually beginning in June, where they feed in shoots for the next 9-10 mo and become sexually mature (Lu et al. 2014). Each adult usually feeds inside and kills 3-5 shoots (Lu 2011). Extensive shoot feeding by Tomicus adults can cause growth loss and lower a tree's natural resistance to the point where individual trees are predisposed to infestation (i.e., breeding attacks) during the beetles' reproductive phase (Lieutier et al. 2003, Lu et al. 2014.
Sexually mature adult beetles eventually depart the shoots to reproduce in the trunks of P. yunnanensis trees, primarily in April and May (Lu et al. 2014). It is not known what initiates the reproductive phase in T. brevipilosus. Adult females initiate attack by constructing individual longitudinal galleries in the phloem tissue and depositing eggs in niches along the gallery walls. After hatching, larvae feed transversely in individual galleries in the phloem and outer sapwood and then pupate at the ends of the galleries. Larval feeding disrupts nutrient flow within the phloem tissue (Långström 1983, Fernández et al. 1999, Ye and Ding 1999, Långström et al. 2002. Gallery construction and reproduction by T. brevipilosus and at times other Tomicus species are regarded as the direct causes for P. yunnanensis tree mortality, particularly when mass attack occurs on individual trees Ding 1999, Lu et al. 2014). As one aspect of developing a T. brevipilosis management program, the objective of our study was to clarify the timing and duration of the beetle's reproductive period and within-tree attack pattern, as well as other aspects of the beetle's reproductive biology. A further aim of this study was to improve our understanding of the reproductive ecology of T. brevipilosus with respect to other Tomicus species that infest P. yunnanensis in southwestern China.
Materials and Methods
Study Area. The field study was carried out (primarily by P.C., J.L., and H.Y.) in a P. yunnanensis stand in Anning County (24.97 N, 102.33 E, 1,800 m a.s.l), approximately 80 km west of Kunming, the capital of Yunnan, in southwestern China. The stand was along the lower slope of a hillside and covered about 300 ha. The P. yunnanensis trees in this stand were originally planted by aerial seeding in the mid-1970s. Most of the pine trees were 24-28 cm in diameter and 7.5-8.5 m tall at the time of this field study (2007)(2008)(2009). Low numbers of T. yunnanensis and T. minor were first recorded infesting trees in this stand in the early 1990s, followed by T. brevipilosus within a few years (Duan et al. 2004). All these three Tomicus species have been active in this stand since the late 1990s (Duan et al. 2004, Lu et al. 2014. It is important to note that in Yunnan, T. yunnanensis was considered as T. piniperda due to their morphological similarities before 2008 (e.g., Ye 1991Ye , 1994aYe ,b, 1995Ye and Lieutier 1997;Ye and Ding 1999;Långström et al. 2002;Lieutier et al. 2003;Duan et al. 2004), when T. yunnanensis was first described as a new Tomicus species (Kirkendall et al. 2008).
Experimental Procedures. During January to July in each year from 2007 to 2009, 3-4 P. yunnanensis trees with evidence of recent bark beetle breeding attacks were sampled every 10 d, for a total of 10 trees per month. Most of the sampled trees were cut from the interior portion of the stand and were among the larger trees present. We selected trees that appeared to have been recently infested based on the texture and color of the frass and resin that was present near the entry hole where the Tomicus beetles entered the bark along the trunk surface (Lu 2011). The sampled trees were felled by cutting the trunk near the ground with a chainsaw, and then we cut the trunk into 50-cm-long logs, starting at the base and stopping near mid-crown. The logs were marked in the field to identify from which tree and which part of the trunk they were cut and then transported to the laboratory. In the laboratory, usually within 1-2 d after cutting, we measured the surface area of each log and then carefully removed the outer bark to look for bark beetles and their galleries. We recorded several parameters related to beetle reproduction and development, including length and width of the maternal gallery using a Vernier calliper (HMCT 6202-01, Harbin, China), number of maternal galleries for each Tomicus species, and the number of eggs, larvae and at times pupae for each maternal gallery. All parent bark beetles were collected from the maternal galleries, placed individually into labeled plastic bags, and identified to species under a stereomicroscope (Nikon-smz 500) based on morphological characteristics of the various Tomicus species (Kirkendall et al. 2008. We recorded our observations for each Tomicus species on a per log basis, using data from all the bark beetles galleries that were present, no matter their age. In addition, detailed observations were made on 106 T. brevipilosus maternal galleries (in which the parent female was still present) at 10-d intervals from March to July 2009, from which the timing of first occurrence of eggs, larvae, and pupae were recorded. These parameters were used to estimate the duration of egg, larval, and pupal development in the field. At the same time, several other maternal gallery parameters were measured and recorded including gallery length, gallery width, and numbers of eggs or larvae present (for details see Lu 2011).
Data Analysis. Correlation analysis was conducted between the length of the maternal egg gallery and the number of eggs present in the galleries constructed by T. brevipilosus. Statistical analyses were performed using SPSS (version 13 for Windows) with an alpha level of 0.05. Temperature data were obtained from Yunnan Meteorological Bureau, which had a weather recording station about 8 km from our field site.
Results
Breeding Attacks. T. brevipilosus breeding attacks, including sister broods, on P. yunnanensis trees were initiated in early March and ended in early June, spanning a period of about 3 mo (Fig. 1). The timing and duration of breeding attacks were similar over the 3-yr study period from 2007 to 2009 (Fig. 1).
The attacking population of T. brevipilosus parent females varied from month to month, based on the total number of maternal galleries found each year for each Tomicus species while debarking the logs but formed an approximately normal distribution curve each year during the period from March to June (Fig. 1). Typically, breeding attacks in March accounted for about 10-25% of the annual attacks (10.25% in 2007, 24.44% in 2008, and 18.39% in 2009). The daily minimum and maximum air temperatures for the recording station about 8 km from our field site are shown in Fig. 2 for 1 January through 30 June during 2007-2009. Considering areas of the world where Tomicus species experience cold winters, spring flight is usually initiated when air temperatures exceed 10-12 C (Bakke 1968;Salonen 1973;Långström 1983;Haack et al. 2000Haack and Poland 2001;Ye et al. 2002;Lieutier et al. 2015). Considering a flight threshold of 12 C at our study site, it is clear from the data presented in Fig. 2 that this temperature was exceeded on almost every day from January through June during 2007-2009. Even air temperatures of 15 C were exceeded nearly every month (Fig. 2).
T. brevipilosus Breeding Attacks in Relation to Other Tomicus Species. The log dissections during this 3-yr study indicated that breeding attacks by T. brevipilosus occurred in P. yunnanensis trees both with and without coinfestation by the other two Tomicus species that were present in the area (Figs. 3 and 4). For example, in March, when T. brevipilosus commenced breeding attacks, there already existed several Yunnan pine trees that were infested by T. yunnanensis and T. minor, which had initiated breeding earlier in the year than T. brevipilosus (Lu et al. 2014). In this situation, T. brevipilosus only attacked trees that were already infested by T. yunnanensis and T. minor (Fig. 3). In April, as the number of trees newly infested by T. yunnanensis and T. minor decreased, T. brevipilosus continued to infest pine trees in which T. yunnanensis and T. minor already occurred as well as initiated attack by itself on other pine trees (Fig 3). During May and June, when there were no other pine trees with new T. yunnanensis or T. minor breeding attacks, T. brevipilosus attacked only by itself on previously uninfested pine trees (Fig. 3).
Within-Tree Distribution on Breeding Attacks. T. brevipilosus was able to colonize the entire trunk of P. yunnanensis trees, usually starting about 50 cm above the ground level and reaching the mid-crown level (Fig.3). However, the colonization pattern varied dramatically, depending if the other two Tomicus species were already present on the tree trunks or not. In those cases when both T. yunnanensis and T. minor were already present along the trunk at the time of T. brevipilosus infestation, which usually happened in March to April (Fig. 3), T. brevipilosus tended to colonize the open spaces of inner bark (phloem) where the other two Tomicus species and their brood had not already occupied. In most cases, T. yunnanensis appeared to be the first species to colonize the trees, occupying primarily the upper portions of the trunk, whereas T. minor tended to colonize trees already infested by T. yunnanensis, infesting primarily the lower portions of the trunk (Ye and Ding 1999) (Fig. 4A). There was some overlap of both T. yunnanensis and T. minor in the mid-trunk region of most trees (Fig. 4A). Given this situation, the early-season T. brevipilosus attacks were scattered along the entire trunk between the gallery systems of the two other Tomicus species but being concentrated mostly in the mid-and lowertrunk samples (Figs. 1 and 4A). Therefore, during the early portion of the T. brevipilosus flight season, T. brevipilosus attacks tended to overlap mostly with T. minor galleries in the lower trunk and to a much lesser degree with T. yunnanensis galleries in upper trunk and with both of these Tomicus species in the mid-trunk sections (Fig. 4A).
Daily air temperature (°C)
In those cases where T. brevipilosus was the only Tomicus species present to initiate attack, which usually happened from mid-April to early June (Fig. 3), T. brevipilosus tended to initiate attack along the lower trunk first and then move progressively upward along the trunk (Fig. 4B). When the tree trunks were fully colonized by T. brevipilosus, the attack density was broadly similar over much of the trunk surface area (Fig. 4B).
Reproduction. T. brevipilosus adult females initiated oviposition within a few days of starting gallery construction on the trunks of P. yunnanensis trees given that the first eggs were observed in early March. Overall, considering all T. brevipilosus breeding attacks observed in 2009, oviposition occurred from early March to early June (Table 1), about 3-3.5 mo, peaking in April to May. The period of active oviposition closely matched the period of tree colonization (i.e., breeding attacks; Fig. 1).
In 2009, the first T. brevipilosus eggs were found in early March and the first larvae in mid-March, indicating that the egg incubation period was less than 2 wk in the field during March. The first pupae were recorded in early May, with most pupating by mid-May, when average daily air temperature was about 18 C (Fig. 2). Therefore, T. brevipilosus larvae appeared to require about 40-50 d to complete larval development. The pupation period lasted about 10-20 d at the study site. Callow adults of T. brevipilosus were first observed under the bark of the sampled trees in late May, with emergence of the new brood adults starting in early June and peaking during mid-to late June (Table 1).
The typical maternal gallery of T. brevipilosus is a single longitudinal tunnel with an entrance hole at the bark surface that leads to a slightly enlarged mating chamber and then a single gallery in which eggs are deposited along the gallery walls. The average length of T. yunnanensis, Ty) based on sampling 10 trees per month in Yunnan that were cut from January to July, 2007July, -2009 apparently fully constructed T. brevipilosus maternal galleries was 7.2 6 2.3 (mean 6 SE, n ¼ 106) cm long (14.0 cm maximum) and varied in width from 4.7 to 5.4 mm. The average number of eggs laid per gallery was 50.9 6 18.7 (mean 6 SE, n ¼ 106) and ranged from 22 to 128 eggs. The number of the eggs per gallery was significantly and positively correlated with maternal gallery length (R 2 ¼ 0.486, n ¼ 106, P < 0.0001) (Fig. 5).
Discussion
The presence of two Tomicus species co-occurring in the same pine forest has been commonly reported in many Eurasian countries (Bakke 1968, Långström 1983, Ye and Ding 1999, but the co-occurrence of three Tomicus species infesting the same pine trees has seldom been reported in the world (Lu et al. 2014). In Yunnan, in southwestern China, where three Tomicus species can coexist in the same P. yunnanensis forest, it was not known how these three Tomicus species would interact. That is, would they compete with each other for the same resource or would they reduce competition by partitioning their breeding resource either spatially or seasonally (Paine et al. 1981, Flamm et al. 1987, Wilkinson and Haack 1987, Haack et al. 1989, Amezaga and Rodríguez 1998, Ayres et al. 2001? Obviously, answering this question will help in understanding the damage potential caused by the various Tomicus species in southwestern China. This study, along with the results presented by Lu et al. (2014), indicated that the three Tomicus species that coexist in P. yunnanensis forests are in large part separated seasonally in their reproductive cycles, and when they do overlap seasonally, they partition themselves spatially along the main trunk. For example, Lu et al. (2014) noted that T. yunnanensis reproduces from early November to March of the next year, whereas T. minor started to reproduce about 1-2 wk later, from late November to late March or early April, and usually only infested trees already under attack by T. yunnanensis Ding 1999, Chen et al. 2010). In contrast, breeding duration of T. brevipilosus was primarily from early March to early June, thus overlapping with that of the other two Tomicus species by only about 1 mo (Fig. 3). These differences in the peak breeding season for the above three Tomicus species in Yunnan likely reduce interspecific competition among these Tomicus species on the one hand but also lengthen the total period of time that pine trees can be under attack by Tomicus beetles each year and thereby can result in more serious damage to Yunnan pine stands (Lu et al. 2014).
The initiation of Tomicus breeding attacks in northern latitudes that experience cold winters is closely related to maximum daily air temperatures. For example, in northern Europe and North America, T. piniperda typically initiates flight and breeding attacks when daily air temperatures reach 10-12 C, and T. minor when temperatures reach 12-14 C (Bakke 1968;Salonen 1973;Långström 1983;Schlyter and Löfqvist 1990;Haack et al. 2000Haack and Poland 2001;Ye et al. 2002). In northeastern Italy, Tomicus destruens (Wollaston) initiates spring flight when daily mean temperatures exceed 12 C (Faccoli et al. 2005), but in central Italy, T. destruens flies to breeding sites in October and November when the maximum daily temperatures are 18-22 C and daily minimum temperatures are 7-14 C (Sabbatini Peverieri et al. 2008).
In contrast, in Yunnan, little is known about the circumstances that trigger spring flight in the four local Tomicus species. In the case of T. yunnanensis, adults initiate breeding attacks in November apparently by simply departing the shoots and walking along the branches to the main trunk of the same tree on which they shoot fed . Later, usually beginning in January, T. yunnanensis adults will fly to new hosts after first experiencing periods of cold and then having mean maximum air temperatures once again reaching or exceeding 12 C . The threshold temperature for spring flight of T. minor has not been studied in Yunnan, but it is likely slightly higher than that of T. yunnanensis given that T. minor initiates spring flight soon after T. yunnanensis (Lu et al. 2014).
The threshold temperature for spring flight of T. brevipilosus is not known, but it is likely considerably higher than that of T. yunnanensis and T. minor given that T. brevipilosus spring flight starts about 2 mo later, in March (Lu et al. 2014;Fig. 3). Perhaps initiation of T. brevipilosus spring flight is more closely linked to daily mean temperature rather than the daily maximum as reported for T. destruens in Italy (Faccoli et al. 2005). An examination of the temperature data in Fig. 2 shows that in March of each year, the daily mean air temperature ranged over 11. 8-19.6 C in 2007, 8.7-18.5 C in 2008, and 11.9-18.2 C in 2009. If we consider a mean air temperature of 15 C as an arbitrary threshold value for T. brevipilosus flight, then there were 15 d that could have supported flight in March and 16 d in April 2007, 10 and 25 d in 2008, and 18 and 26 d in 2009. Of course, other factors besides simply air temperature could influence initiation of spring flight in T. brevipilosus, such as day length, ovarian development, or some physiological change within the host tree that is detected by adults as they overwinter in the shoots.
As shown in Fig. 1, T. brevipilosus peak flight was apparently shifted 1 mo later in 2008 when compared with 2007 and 2009. It is not clear if this shift is simply an artifact of the trees that were sampled in the different years or if this apparent shift reflects some major temperature differences among the three sampling years. If the difference in the flight season is related to temperature, it is difficult to identify a major difference in the 2008 temperature data shown in Fig. 2 when compared with the data from 2007 and 2009. One difference, as mentioned above, is that there were fewer days in March 2008 when the mean daily temperature exceeded 15 C when compared with March in 2007 or 2009. However, mean temperatures in April 2008 were broadly similar to April 2009 but warmer than April 2007. Such year to year variation in the timing of the initial spring flight of overwintering Tomicus adults is common and has been shown to be related to annual differences in spring temperatures (Bakke 1968;Salonen 1973;Långström 1983Långström , 1986Haack and Lawrence 1995;Haack et al. 2000;Faccoli et al. 2005).
Among the eight species of Tomicus worldwide, T. piniperda usually breeds in wind-blown trees, fire-damaged trees, stumps, and other severely stressed pine trees (Bakke 1968;Ye 1991;Haack et al. 2000Kirkendall et al. 2008). Tomicus armandii Li and Zhang may infest weakened Pinus armandii Franchet trees in Yunnan . T. yunnanensis is regarded as one of the most aggressive species of Tomicus, being able to attack and kill live, apparently healthy P. yunnanensis trees Lieutier 1997, Lu et al. 2014). T. minor is often considered to be more of an opportunist, usually infesting host trees that are already infested by other Tomicus species such as T. piniperda or T. yunnanensis, which could facilitate tree death (Bakke 1968, Långström 1983, Eidmann 1992, Ye and Ding 1999, Lieutier et al. 2003, Chen et al. 2009). In this study, we discovered that T. brevipilosus preferred to breed in trunks of P. yunnanensis trees that were already infested by both T. yunnanensis and T. minor. However, later in the season, T. brevipilosus was able to attack apparently healthy P. yunnanensis trees on its own, indicating that T. brevipilosus is more similar to T. yunnanensis in its aggressiveness when compared with T. minor (Chen et al. 2009(Chen et al. , 2010. The spatial distribution of T. brevipilosus breeding attacks in the trunks of P. yunnanensis trees was greatly influenced by the presence or absence of T. yunnanensis and T. minor breeding in the same tree (Fig. 4). In trees that were already infested by the other two Tomicus species, T. brevipilosus colonized areas of the trunk that were not already occupied, resulting in a scattered distribution of T. brevipilosus attacks along the trunks. The ability of T. brevipilosus to adjust its infestation pattern in response to other Tomicus species likely decreases interspecific competition among Tomicus species, as well as better utilizes the limited inner bark resources. In addition, it is possible that the early-season attack pattern of T. brevipilosus could enhance the reproductive success of T. yunnanensis and T. minor by attacking unoccupied areas of the tree trunk and thereby further reducing any residual host tree resistance. However, later in the flight season, when T. yunnanensis and T. minor brood are already well advanced, the arrival of T. brevipilosus would likely not help much in further reducing host vigor.
In those cases when T. brevipilosus initiates attack by itself, the first attacks are generally located along the lower trunk with subsequent attacks occurring throughout the entire trunk. The within-tree attack pattern varies among other Chinese Tomicus species as well (Ye and Ding 1999). In Yunnan, the first attacks by T. yunnanensis tend to be along the upper trunk, whereas T. minor attacks are more concentrated along the lower trunk (Ye 1995, Ye and Ding 1999, Chen et al. 2010. In contrast, in northern Europe, Långström (1983) reported that T. piniperda tended to initiate attack along the lower trunk of local pines (mostly Pinus sylvestris L.), where the bark was thicker, whereas T. minor, which flies later, tended to colonize the upper portions of the trunk and branches where the bark is thinner. In the above two situations, it appears that T. minor is simply colonizing trees already infested by other Tomicus species but concentrating its attack on those portions of the trunk that are less occupied and thereby reducing interspecific competition. It is reasonable to assume that the attack pattern in any one world area is a reflection of the Tomicus and host-tree species present, as well as differences in beetle aggressiveness, threshold temperatures for flight, and adult body size (Kirkendall et al. 2008, Lieutier et al. 2015. For the three Tomicus species infesting P. yunnanensis in Yunnan, the adult beetles are broadly similar in body size, with T. yunnanensis being slightly larger, and T. brevipilosus and T. minor being very similar (Kirkendall et al. 2008, Lieutier et al. 2015. It is unclear why the attack pattern differs between T. yunnanensis (starting in the upper trunk) and T. brevipilosus (starting in the lower trunk) when each is the first bark beetle to infest the same species of pine, although such variation in attack pattern is well-recognized among bark beetles (Rudinsky 1962). Nevertheless, both attack patterns can result in tree death. This topic still requires further exploration.
The average length and number of eggs in T. brevipilosus maternal galleries reported in this study are broadly similar to findings reported for other Tomicus species that make longitudinal galleries (all species except for T. minor). For example, in comparison to the 7.2-cm-long average T. brevipilosus maternal gallery in this study, others have reported mean values of 4-8 cm for T. destruens (Faccoli 2007), 7.1-11.0 cm for T. piniperda (Långström and Hellqvist 1985), and 9.6-10.6 cm for T. yunnanensis (Ye and Ding 1999). However, an inverse relationship has been reported between attack density and average length of maternal galleries in some Tomicus species (Salonen 1973, Långström 1984, Faccoli 2009), but this relationship has not yet been investigated in T. brevipilosus. Clearly much more research is needed to fully elucidate the life history of T. brevipilosus and its interactions with other Tomicus species in Yunnan. | v3-fos |
2019-04-01T13:16:04.277Z | {
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} | s2 | Effect of Traditional Fish Processing Methods on the Proximate and Microbiological Characteristics of Laubuka dadiburjori During Storage at Room Temperature
Fish is a major source of protein and post-harvest loss of fish is a key factor of economic and protein wastages in developing countries like Bangladesh. This study was carried out to know the effects of sun drying, smoking, freezing and canning on proximate, biochemical and microbiological characteristics of chela (Laubuka dadiburjori) fish stored at room temperature for 60 days. The proximate compositions of the fish samples were determined. Moisture, protein, lipid, ash and carbohydrate contents of the fresh fish were 76.56, 13.74, 4.25, 2.37 and 1.41%, respectively. Total volatile base nitrogen content was 7.10 mg/100 g. Total plate count was 1.13×104 CFU gG1. The proximate compositions found in the different processing methods were statistically different to the fresh fish samples. Water activity and pH value increased significantly in sundried and smoked chela at the end of 60 days of storage. Salt content decreased significantly (p<0.05) in sundried fish than other three processing methods. Total protein content was high in smoke dried fish (61.31%) and low in frozen fish (11.85%) at the end of storage period. The TVB-N was found high in sundried and smoked fish samples after storage period. Smoking demonstrated efficient method of fish processing in terms of the retention of protein value and reduction in the moisture content but canning was the best method for benefit and retention of the product. The information obtained in this study could be useful to fish consumers, processors and nutritionists in the efficient management of fish resources.
INTRODUCTION
Fish is a good source of animal protein and minerals (Tidwell and Allan, 2001). Fish is widely consumed in many parts of the world because it has high protein content. The quality of fish protein is very high because of its low saturated fat, its riches with essential amino acids and also it's containing ω-3 and ω-6 fatty acids that known to support good health. According to FAO. (2008) and Gandotra et al. (2012), fish provides 20% of animal protein intake to about 2.6 billion people globally and at least 50% of animal protein intake for over 400 million in Asia and Africa. In developing countries, it provides only 13% of animal protein intake. Fish constitute the major source of animal protein intake in Bangladesh due to its availability and low cost. Although, fish J. Fish. Aquat. Sci., 10 (4): 232-243, 2015 Total Volatile Base Nitrogen (TVB-N): The total volatile base nitrogen (TVB-N) was determined by Conway's micro-diffusion analysis (Osman et al., 2001). In this procedure, the Trichloro Acetic Acid (TCA) extract prepared sample was treated with potassium carbonate, ammonia liberated and absorbed by boric acid. The quantity of ammonia absorbed was volumetrically determined by titrating the ammonium borate against standard sulphuric acid. The results were expressed as mg TVB-N kgG 1 muscle. Sodium chloride content: Salt content of the sample was determined by the method pre-scribed for salted fishery products (FAO, 2008). The 5 g of the muscle sample was treated with distilled water against standard silver nitrate solution using potassium chromate as indicator.
Water activity (a w ): In order to enumerate the influence of water on the flourishment of microbial fishery, the water activity of the sample was determined at room temperature using water activity value analyzer (model no. 5803, Glufft Gmbh and Co, Stuttgart-1). The instrument consists of a thermocole case, holding two sample containers each with a detachable sensor head, which indicates water activity directly on the dials at the required level of temperature. Calibration of the a w meter was carried out by keeping a filter paper soaked with the standard solution of barium chloride in the sample container (a w = 0.9 at 20°C). The holding period was 3 h to get the a w at room temperature. The instrument was manually calibrated to get the correct water activity. pH: The 1 g sample of the fish flesh was homogenized in 10 mL of distilled water and the mixture was filtered. The pH of the filtrate was measured using a pH meter (Mettler Toledo 320-s, Shanghi, China).
Microbiological analysis Total Plate Count (TPC):
The 10 g of muscle tissue samples from each fish was aseptically cut out and homogenized aseptically with 90 mL of Buffered Peptone Water (BPW) solution (Oxoid, Hampshire, UK). The tissues were stomached for 30 sec in a stomacher. Appropriate dilutions were made according to decimal dilution method and planted onto nutrient (plate count agar) agar plates by spread plate technique (BAM., 1995). The plates were incubated at 30±1°C for 24 h. Total Plate Count (TPC) was enumerated and expressed as number of colony forming U gG 1 . Strict aseptic procedures were followed in every step of analysis.
Halophillic bacterial count: Halophilic bacterial count was determined using 3.5% sodium chloride solution as diluent. Planting was done onto nutrient (plate count) agar with 10% salt plates by spread plate technique. The colonies developed in the planter were counted and expressed as number of colony forming units/g of the sample.
Total fungal count: Total Fungal Count (TFC) was enumerated using potato dextrose agar by BAM. (1995). The 10 g of fish sample was weighed aseptically and homogenized with 90 mL of physiological saline solution. Appropriate dilutions were made from the 9.0 mL physiological saline and plated onto Potato dextrose agar plate containing antibiotics or tartaric acid solution. The plates were incubated at room temperature for four days and all colonies were counted and the data was reported as Colony Forming Units CFU gG 1 . J. Fish. Aquat. Sci., 10 (4): 232-243, 2015 The pathogenic bacteria like Staphylococcus, Streptococcus, Pseudomonas, Vibrio etc and fungi were enumerated by following the method of BAM. (1995). All culture media were purchased from Oxoid (Hampshire, UK).
Sensory evaluation:
The sensory evaluation was carried out by a group of panelist according to the method described by Potter (1968). The boiled samples of sundried, smoked, frozen and canned fish were evaluated for quality attributes include texture, flavor, color, appearance, general taste and overall acceptability by using 9-point hedonic scales (9 = excellent, 8-9 very good, 6.5-7.9 good, 5-6.4 fair, <5 bad) and score 5.0 was considered the borderline of fish acceptability. A sensory panel formed consist of ten experienced judges (5 males and 5 females; 25-55 years old) who had been involved in sensory analysis of different kinds of fish and fish foods. Previously to the present experiment, a special training was carried out concerning different quality conditions and to check the panelists' understanding of the descriptors. The sensory evaluation was conducted between 10:30 and 11:30 am and Panelist received four samples per session. Fishes were cooked in a glass bowl covered with a cap in a microwave oven (Samsung) during 1.8 min at 600 W. Samples were coded with 3-digit random numbers and a randomized complete block design was used in which the samples were randomly assigned to each panelist. Panelists were asked to consume the dorsal part of the fillet. Sessions were performed in individual partitioned booths. These conditions were conducive to concentration and avoided communication between assessors and disturbance by external factors (ISO., 1988). Scores among panelists were averaged.
Processing techniques Sun drying: The fishes were aseptically gutted, eviscerated and washed in chilled water. Before drying, fishes were salted by dry commercial salt (NaCl) (fish weight: salt weight, 3:1) in clean plastic basins. The salting was done by placing crude salt in the gut cavity and outside the fish. The salting process was allowed to continue for several hours. Then fishes were dried by exposing to ambient sunlight at temperatures of 35-42°C on drying racks made of plastic coated metallic wire mesh racks. The racks with fishes were covered with fishnets during day time to prevent insects and other pests. At night, the racks were covered with plastic sheets to prevent water condensing on the drying fishes. After drying, they were allowed to cool naturally to ambient temperatures of 23-25°C. Sun-dried product was packaging with plastic bag maintaining aseptic condition as far as possible and was stored at room temperature.
Smoke drying:
The fish samples were gutted, washed thoroughly with clean water and prepared for smoke curing. Fishes were dip in freshly prepared salt solution (mix four parts of clean water and one part of salt) for 15 min followed by draining and then. The fishes were smoked in a mechanical kiln (AFOS-Torry Mini Kiln). Heat was generated by the burning of charcoal from tamarind wood chips. Pre-heating for 15 min and then loading the fish samples onto removable wire mesh trays in its central chamber for the smoking process. The desired temperature (75-80°C) was maintained manually by using a thermometer. Smoking was done approximately for 4 h. During smoking, fish samples were turned upside down in middle period, to make the sample smooth and steady in texture and appearance. Then the samples were cooled for 20-30 min at ambient temperature. The cooled smoked fish samples were then packed and sealed in vacuum condition in polythene bags. Smoke-dried fish product were then kept for storage at room temperature for further analysis of sensory and biochemical compositions.
Freezing:
The fishes were eviscerated and washed with chilled water to remove traces of blood. It was wrapped tightly in plastic wrap then kept in air tight plastic container and immediately stored in the freezer (model: Haier Thermocool BD-428A) for 60 days at below -16±2°C. Before analysis, frozen fish were thawed using running water (25-26°C).
Canning: The fishes were eviscerated and washed with chilled water, then precooked in steam pressure of 10 psi for 60 min to attain a final backbone temperature of 90-92°C. Precooking removes the fish oils and coagulates the protein in the fish to loosen the meat. The fish were then cooled at room temperature (15-18°C) for about 2 h. The 100 g fish were placed in small flat rectangular cans (105×60×25 mm; 150 mL). Can was then filled with brine (mixing four parts of clean water and one part of salt) as coating medium. The cans were vacuum-sealed and sterilized in a horizontal steam heated retort (115°C, 45 min, F o = 7 min). When the heating time was completed, steam was cut off and air was used to flush away the remaining steam. Cans cooling was carried out at reduced pressure.
Statistical analysis:
The obtained data were statistically analyzed using the Statistical Package SPSS 20.0 version. The differences in fish sample parameters due to time of storage (BS, 30 and 60 days) were analyzed using an analysis of variance (ANOVA) procedure. The significance level was set at the probability level of p<0.05.
RESULTS AND DISCUSSION
Biochemical characteristics of raw chela fish: Fresh chela used in this study contained 76.56% moisture, 13.74% protein, 4.25% lipid, 2.37% ash and 1.41% carbohydrate (Table 1). Sankar and Ramachandra (2001) mentioned that proximate composition of fish varying with species, body size, season, environmental factors and nutritional status. The TVB-N content of fresh chela was found 7.10 mg/100 g of sample, which below the level of 35 mg/100 g, has been suggested as border line for various fish and fish products (Ghaly et al., 2010). The carbohydrate content in fresh chela was less in amount as compared to protein and lipid. Table 1 show that the Total Plate Count (TPC) and total fungal count in fresh chela were 1.13×10 4 and 1.50×10 2 CFU gG 1 , respectively. Some dominant genera of bacteria were identified. Among those Pseudomonas spp., Micrococcus spp., Streptococcus spp. and Vibrio spp. were found in higher percentage. Surendran and Thampurah (2002), previously mentioned that Aeromonas spp. and Pseudo-monas spp. were the main microorganisms associated with fresh water fish. The dominant fungi isolated from the samples of chela were Aspergillus spp. and Mucor spp. Effect of traditional processing methods on the proximate composition of chela fish before storage: Proximate composition of the chela fish subjected to different processing methods (sun drying, smoke drying, freezing and canning) is presented in Table 2. Smoke dried fish recorded the lowest (1.41%) moisture content than sundried (5.88%), canned (67.15%) and frozen fish (76.25%). Protein contents were high in the smoke dried fish (67.32%) and then sundried fish (62.80%) compared to canned (16.68%) and frozen (13.21%) fish, this finding was in conformity with the findings of Chukwu (2009) and Kumolu-Johnson et al. (2010). According to Tidwell and Allan (2001) fish is a good source of protein. Indeed, the highest crude protein content in smoked and sundried chela fish compared with the raw, canned and frozen chela fish, suggests that protein nitrogen was not lost during drying. The increase in crude protein level can be explained by Kumolu-Johnson et al. (2010) who stated that smoking resulted in concentrating crude protein components of fish. This concentration was resulted from the loss of moisture by the smoking process as opined by Koral et al. (2009). The highest lipid content observed in smoked fish (19.27%) and then sundried fish (14.00%).The difference in their lipid contents was owing to oxidation of fat during the period of sun drying as mentioned by McGill et al. (1974).
Microbiological characteristics of raw chela fish: Results in
On the other hand, canned and frozen fish had the lowest lipid content (5.46 and 4.08%, for them, respectively). This reduction was related to their highest content of moisture. Aberoumad and Pourshafi (2010) stated that the lower the percentage of water, the greater the lipids and protein content and the higher the energy density of the fish.
High value of total ash content (8.91%) in sundried chela and then canned chela (8.15%) was attributed to high salt content. Similar levels of ash content in salted fish were recorded by Kiin-Kabari et al. (2011). Sundried fish also recorded the highest carbohydrate content (4.01%), meanwhile the lowest carbohydrate content (1.17%) was observed in frozen storage fish.
Effect of traditional processing methods on changes in proximate composition of chela fish after stored for 30 days and 60 days: A significant (p<0.05) increase in moisture content was observed of all post-storage fish products compared with the pre-storage products except frozen and canned storage fish (Table 2). Increase in moisture content was attributed to the difference in the moisture of the processed fish relative to the surroundings mentioned by Daramola et al. (2007) and moisture absorption during monsoon due to high relative humidity difference. This increase in moisture uptake during the storage period was higher in sundried and smoked fish than canned fish. The low moisture uptake by canned products indicates the advantage of processing chela fish. Reversely, a negative trend was observed in frozen fish.
Reduction in crude protein during the storage period may be due to gradual degradation of the initial crude protein to more volatile products, such as Total Volatile Bases (TVB). Low protein content recorded in 60 days stored sample may be due to denaturation of fish protein associated with frozen fish (Reay, 1933) and leaching out of some extractable soluble protein fraction (Daramola et al., 2007). Sundried, Smoked and frozen chela fish showed a reduction in crude fat during storage period. This may be due to breakdown of tissue cells during salting, followed by the heating effect of drying (Pace et al., 1989) and oxidation of Poly-Unsaturated Fatty Acids (PUFA) to products such as peroxides, aldehydes, ketones and the free fatty acids (Daramola et al., 2007). The highly susceptible of fish to oxidative rancidity resulted from the high degree of unsaturation in the form of multiple double bonds in fatty acids Obemeata et al. (2011). By comparing prestorage and post-storage crude fat content, a significant difference (p<0.05) was recorded in both of sundried and smoked chela fish.
Sun drying recorded the highest ash content (13.64%) after 60 days storage. The significant (p<0.05) increases in ash content recorded in sundried, smoke dried and canned fish due to the crude salt. According to Beauchamp and Engelman (1991), fish muscle absorbed more salt which was put into their gut during processing and same the finding was also observed in smoked fish samples during the storage period (Cardinal et al., 2001). Table 2 indicates that there was slightly increase in the post-storage carbohydrate content of the frozen and canned chela fish and a significant (p<0.05) reduction in sundried and smoked chela fish.
Changes in physiochemical characteristics: Initial salt content of sundried, smoke dried and canned chela was 17.54, 19.57 and 28.02%; these values decreased to 15.67, 18.20 and 27.24% respectively after 60 days of storage (Table 3). Dewi et al. (2011) showed that decrease in salt content attributed to the uptake of moisture during the storage period due to hydrostatic nature of salt.
There was an increase in water activity of chela fish except that in both frozen (0.70) and canned (0.59) fish, which almost remained constant. These value is lower than 0.70 and according to Brewer (1999) bacteria can't grow at a level of 0.70. So, frozen and canned fish remained safe. Significant (p<0.05) upper trends in a W observed in sun dried and smoke dried fish. Bacteria should not grow at a a W level of 0.70 (Brewer, 1999). pH is an indicator of the degree of freshness or spoilage. The pH in fresh fish flesh is almost neutral. In our study the pH value of fresh chela fish was 6.8. Drop off pH value after addition of salt was 6.6, 6.2 and 5.9 for sundried, smoked and canned fish respectively and after that pH value increased with time. In the post-mortem period, decomposition of nitrogenous compounds leads to an increase in pH in the fish flesh (Shenderyuk and Bykowski, 1989). The pH value of sundried and smoke dried chela fish were increased significantly (p<0.05) with storage period. According to Simeonidou et al. (1997) increase in pH values associated with the production of basic components induced by the growth of bacteria. Most microorganisms grow best at pH values between 6.6 and 7.5, whereas only a few grow at a pH below 4.
Microbiological changes Total Plate Counts (TPC):
The Total Plate Count (TPC) shows an overall increasing trend in sundried and smoke dried chela and increase was greater in sundried stored chela (Table 4).
No substantial TPC was detected in the frozen and canned fish samples of BS, 30 days and 60 days stored chela. This was due to their low water activity comparing to the other two species. The considerable counts were recorded during storage probably due to increase in water activity as well as storage duration. Hood et al. (1983) mentioned that microbial load increased with duration of storage and temperature. However, TPC is positively correlate with a w and TVB-N but negatively correlate with overall acceptability.
Halophilic count: Halophiles require salt for growth and grow mostly in salted dried fish products (Khan et al., 2005). They reported that halophiles are aerobic and usually not found in canned fish where limited oxygen. As shown in Table 4, the halophilic bacteria could not be detected initially but gradually increased from 60 days of storage due to higher moisture and salinity in the product. The counts of more than 1×10 2 cells gG 1 could be due to their higher water activity. AOAC. (2005) observed that a w for a saturated salt solution is 0.75 and if the equilibrium relative humidity is greater than 75% of the salted products it will take up moisture from the atmosphere increasing the O 2 and consequently increase the possibility of contamination by microbes. (Reilly, 1986). Results in Table 4 show an increasing trend in the salt content during the storage period. A considerable higher fungal growth was recorded in sundried and smoke dried chela after 30 and 60 days of storage compared to frozen and canned chela. This may be due to increase in a w and moisture content. According to Gandotra et al. (2012), products deteriorate by growth of moulds when water content is about 15%. This observation supports our present study. Generally, the rapid reduction in water activity (a w <0.75) is a controlling factor of fungi/mould contamination during storage (Kolakowska, 2002).
Changes in Total Volatile Bases (TVB-N):
The TVB-N in fresh chela recorded 7.10 mg N/100 g. TVB-N represented very small quantity in fresh fish and was produced by putrefaction process in spoiling fish. According to Wallace (2000) TVB-N is better index of spoilage. The TVB-N presence in the fish can be explained by means of two different pathways: (1) As a result of bacterial catalysis breakdown of trimethylamine oxide (TMAO) during the storage and (2) From thermal breakdown of proteins, amino acids and other nitrogenous compounds during the heat processing steps (Chia et al., 1983). The TVB-N content quantifies a wide range of basic volatile compounds (NH3, methylamine, dimethylamine, trimethylamine, etc). The TVB-N values were found to vary from 27.14 mg N/100 g in BS to 32.57 mg N/100 g in 30 days and 36.73 mg N/100 g in 60 days for sundried chela (Fig. 1). For smoke dried chela, it increased from 2 0.63 mg N/100 g in BS to 29.31 mg N/100 g in 30 days and 33.77 mg N/100 g in 60 days. The TVB-N values were high in sun and smoke dried chela after storage has exerted a higher bacterial and thermal action during storage period. The TVB-N values of frozen and canned chela were 12.63 mg N/100 g and 15.50 mg N/100 g, respectively in BS condition. It increased up to 21.14 mg N/100 g and 17.68 mg N/100 g after 30 days. After that it continued to increase to 25.14 mg N/100 g and 21.95 mg N/100 g after 60 days of storage period. The rate of spoilage increases with the time but few values exceeded the recommended value set for fish regarded as acceptable condition. The increase in TVB-N throughout the storage period may be due to microbial activity, storage temperature, absorption of moisture and relative decrease in salt content. However, the limiting level for rejection of TVB-N is 30-40 mg N/100 g for storage at ambient temperature (Connell, 1995).
Dominant bacterial and fungal flora:
Drying of fish is reported to impart a degree of microbiological stability to the product, which is a function of reduced water activity and heating. The dominant bacterial and fungal flora during storage period is shown in Table 5. Fortunately, E. coli and salmonella bacteria were not detected during the storage period. The previous observation of Sur Micrococcus spp and Pseudomonas spp are the main micro-organisms associated with fresh water fish.
The dominant fungi found during storage were Aspergillus sp. and Pencellium. Turkkan et al. (2008) reported that the production of aflatoxin when dried and smoked fish was inoculated with Aspergillus flavus. Although, there are no reported poisoned by mycotoxins in fishery products; there is a definite risk to human health considering how fish are traditionally processed.
Sensory evaluation results:
The sensory evaluation result expressed in mean value ( Table 6). The result shows that, there were changes in all the sensory parameters after subjecting the fish to different processing methods and storage period. In this study, high quality canned and frozen fish with excellent sensory and physical properties were obtained through storage period. The sensory properties of canned and frozen fish were in more acceptable condition throughout storage period than sundried and smoke dried fish.
CONCLUSION
In conclusion, this study shows that chela fish (Laubuka dadiburjori) can serve as a good source of dietary protein and lipid. Smoking and sun drying demonstrated efficient method of fish processing in terms of the retention of protein value and reduction in the moisture content. The results from microbiological study demonstrated that after sun drying, smoking, freezing and canning processing of chela, the fish was safe to be consumed after 60 days of storage period. Canning and freezing demonstrated efficient method of chela fish processing in terms of the retention of wholesomeness of stored product. | v3-fos |
2018-04-03T02:07:04.351Z | {
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} | s2 | Effect of Growth on Fatty Acid Composition of Total Intramuscular Lipid and Phospholipids in Ira Rabbits
The changes in fatty acid composition of total intramuscular lipid and phospholipids were investigated in the longissimus dorsi, left-hind leg muscle, and abdominal muscle of male Ira rabbits. Changes were monitored at 35, 45, 60, 75, and 90 d. Analysis using gas chromatography identified 21 types of fatty acids. Results showed that the intramuscular lipid increased and the intramuscular phospholipids (total intramuscular lipid %) decreased in all muscles with increasing age (p<0.05). An abundant amount of unsaturated fatty acids, especially polyunsaturated fatty acids, was distributed in male Ira rabbits at different ages and muscles. Palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), and arachidonic acid (C20:4) were the major fatty acids, which account to the dynamic changes of the n-6/n-3 value in Ira rabbit meat.
Introduction
In the last 50 years, the world production of rabbit meat has increased by 2.5-fold to 1.6 million tons in 2009. China is currently the world's leading producer of rabbit meat (700,000 t/year). Italy (230,000 t/year), Spain (74,161 t/year), and France (51,400 t/year) are the main rabbit-meat producers in Europe (FAOSTAT). Rabbit meat offers excellent nutritive and dietetic properties (Hernàndez and Gondret, 2006) and is characterized by very low monounsaturated fatty acids (MUFA) levels, high levels of polyunsaturated fatty acids (PUFA) and n-3 fatty acids. The meat is also a significant source of vitamin B family (vitamins B2, B5, B6, B3, and B12) and has very high phosphorus contents. Compared with other animal meats, rabbit meat has lower fat levels. The protein content of rabbit meat offers essential amino acids (lysine and tryptophan), especially at the longissimus dorsi muscle, which also has high digestibility (Hernández, 2008). Rabbit meat can be used to produce specific functional food (Dalle Zotte and Szendró´ , 2010; Hernández, 2008) since its good properties (e.g., fatty acid pro-file, mineral, and vitamin contents) and can be modified and further enriched via feeding (Bianchi et Nowadays, current research is directed toward developing feeding strategies to increase the nutritional value of rabbit meat as a "functional food" by including vitamins, antioxidants and essential fatty acids in rabbit diets and assessing their effects on quality of both raw and stored/ processed meat Petracci et al., 2009). However, limited information is available on the effect of ages and muscles on fatty acid profile of total intramuscular lipids and phospholipids from one breed of rabbit. Several factors, such as age (or weight), gender, genotype, and other external factors (transportation, slaughter and season), can influence both the quantity and quality of lipids in animal products (Kouba and Mourot, 2011). The lipid composition, especially the amount of intramuscular phospholipids in meat, is an important factor for flavor development and nutritive quality of freshcooked and dry-cured meat products (Cappelli and Vannucchi, 2005;Gray et al., 1996). Phospholipids are found in significant amount in muscles, which contain a high PUFA content to perform its function as a constituent of cellular membranes (Wood et al., 2008).
Ira rabbits are imported from France and are of high breeding efficiency. However, limited information is available on the fatty acid profile of total intramuscular lipids and phospholipids during the growth of Ira rabbits. Therefore, this study was conducted to examine the changes of fatty acid composition of total intramuscular lipid and phospholipids by gas chromatography to provide a database for the subsequent in-depth study of Ira rabbits.
Materials and Methods
Animals and diets A total of 150 35-d-old weaned male Ira rabbits (30 per age) were provided by College of Animal Science and Technology, Southwest University. The ingredients and proximate chemical composition of the diet were shown in the Table 1. They were maintained in a closed building under natural environmental conditions in individual wire mesh cages, equipped with metal troughs and automatic nipple drinkers. The rabbits had free access to feed and water.
The rabbits were bred under similar production system and slaughtered at the age of 35, 45, 60, 75, and 90 d in a local commercial slaughterhouse. During the experiment, the slaughter weights were recorded as 1.00±0.06, 1.31± 0.01, 2.15±0.12, 2.57±0.08, 3.18±0.07 kg. The facilities of the slaughterhouse met the requirements of the Institute of Animal Care and Use Committee. After 24 h postmortem, the longissimus dorsi muscle (LD), left-hind leg muscle (LL) (left biceps femoris muscle), and abdominal muscle (AM) (ventral musculus) of the carcass were removed and immediately vacuum-packed and frozen at 20°C until treatment.
Total intramuscular lipid content and fatty acid composition analysis Total intramuscular lipids were extracted according to Folch et al. (1957). Total lipid content was measured by weighing the lipid extracts after solvent evaporation. Fractions of intramuscular phospholipids were prepared with silica cartridges (Sep-Pack, Waters, USA) by using the method of Juaneda and Rocquelin (1985). Phospholipids were quantified by phosphorous determination (Bartlett, 1959). The total lipids and phospholipids were methylated with boron fluoride-methanol (Sigma Aldrich) according to Morrison and Smith (1964). The fatty acid methyl esters were analyzed by a QP-2010 gas chromatograph (Shimadzu, Japan) equipped with a flame ionization detector and a split injector. One microliter of fatty acid methyl esters was injected in split mode (5:1) onto a Rtx-Wax capillary column (Restek, Bellefonte, USA; 30 m × 0.25 mm id × 0.25 µm film thickness). The temperature of the column was programmed as follows: 1 min at 140°C, increments of 8°C/min to 180°C and held at 180°C for 2 min; increments of 3°C/min to 210°C; and increments of 5°C/min to 230°C and held at 230°C for 10 min. The temperature of the injector and the detector were both 250°C. The flow rate of the carrier gas (N 2 ) was 1.5 mL/min. Fatty acids were identified by comparing the retention time of the samples with those of the standards (Sigma). Results were expressed as percent of the total fatty acid methyl esters.
Statistical analysis
Statistical Analysis System (1996) was used to determine the means, standard errors, and analysis of variance. Duncan's multiple range test was used to compare differences among means. Values at p<0.05 were considered significant.
Results and Discussion
Effect of age and muscle on total intramuscular lipid and phospholipids content in male Ira rabbits Variation of intramuscular lipid content Fig. 1 shows the effect of age on total intramuscular lipid content among the muscles from male Ira rabbits. The total intramuscular lipid content (muscle weight %) of LD, LL, and AM significantly increased with increasing age of rabbits (p<0.05). The intramuscular lipid content among muscle sections (LD, LL, and AM) in the male rabbits increased from 0.77, 1.16, and 2.41 g/100 g muscle at 35 d to 1.21, 1.66, and 5.16 g/100 g muscle at 90 d, respectively. Among the three muscles, AM had the highest intramuscular lipid content, followed by LL and LD at each tested age. This result is consistent with the study of Hernàndez and Dalle Zotte (2010), who reported that the leanest cut of meat in the rabbit carcass was the loin, besides the hind leg was the most quantitatively important cut because of its low lipid content compared with other muscles. Therefore, lipid content depends greatly on the age and muscle of the rabbit. During the 35 d to 90 d Ira rabbit growth period, the total intramuscular lipid deposition of AM was significantly higher than LL and LD, of which the lipid content of the three sections increased faster during the 60 d to 75 d period than during the other growth stages. Thus, this period was characterized with the most expressed intramuscular fat deposition of male Ira rabbits. However, there was no significant difference in intramuscular lipid content of LD between 60 d (bc) and 75 d (ab). Fig. 2 shows that there is no significant reduction in phospholipid content of the muscles. Only the percentage of phospholipid in total muscle lipid decreases with age (p<0.05) (Fig. 3). This result may be attributed to the increasing deposition of triglycerides in the adipose tissues (Raes et al., 2004).
Variation of intramuscular phospholipids content
During the 35 d to 90 d growth period, LL showed the fastest reduction in phospholipid content, which was significantly higher than that in the AM and LD. In the 35 d to 45 d growth period, the reduction in phospholipids on all three muscles was more obvious than in the other growth stages. During the 35 d to 90 d growth period, the intramuscular phospholipids content decreased from 43.47, 53.81, and 42.21 g/100 g intramuscular lipids at 35 d to 26.84, 28.89, and 25.40 g/100 g intramuscular lipids at 90 d, respectively. Among the muscles at different ages, the LL total lipid accounted for the highest relative phospholipid content, followed by LD and AM. However, compared with the absolute percentage of intramuscular phospholipids (muscle weight %), the change of intramuscular phospholipids among the three muscles of Ira rabbit were not significant (Fig. 1). Therefore, the content of phospholipids in muscle was relatively stable during the 35 d to 60 d growth period. Meanwhile, among the three muscles, AM accounted for the maximum absolute percentage of intramuscular phospholipids (muscle weight %), followed by LL and LD.
Effect of age and muscle on fatty acid composition of total intramuscular lipid Table 2 showed the comparative total intramuscular lipid in LD, LL, and AM of male Ira rabbit meat of different ages. All the total lipids obtained high unsaturated fatty acid (UFA) proportion (the sum of PUFA and MUFA), especially PUFA. In rabbit meat, UFA represents around 60% of the total FA. By comparison, PUFA comprises 32.5% of the total FA, which is the highest among other meats, such as pork (13.8%), beef (8.79%), veal (13.3%), and chicken (27.4%) (Salma et al., 2007;Wood et al., 2008). The result of this previous study was consistent with our present investigation. Saturated fatty acids (SFA) were mainly composed of palmitic acid (C16:0) and stearic acid (C18:0). MUFA consisted of oleic acid (C18:1). PUFA, such as linoleic acid (C18:2n-6) and arachidonic acid (C20:4), were observed at high levels. In the total intramuscular lipid, C18:2n-6 represents 22±4.7% of the total FAs (average of 20 referenced studies, all meat portions combined together). Among the long-chain (C20-22) PUFAs, the eicosapentaenoic acid (EPA, 20:5n-3) percentage in rabbit loin meat constitutes 0.15±0.12%, and docosahexaenoic acid (DHA, 22:6n-3) is 0.31±0.31% of the total FAs (Hernàndez and Dalle Zotte, 2010). These reported data are lower than those of the present study. There is an increasing recognition of the health benets of polyunsaturated fatty acids (PUFA), and of n-3 fatty acids in particular, because these fatty acids are essential for humans (Conquer and Holub, 1998;Kouba et al., 2011;Simopoulos, 2001).
With increasing age of rabbits, the percentage of SFA in LD and LL did not increase significantly (p>0.05), whereas the SFA proportion from AM increased significantly (p<0.05). This result is attributed to the changes of C16:0 content in total intramuscular lipids, corresponding to the significant reduction of PUFA in the muscles (p< 0.05) and the significant increase of MUFA in LD and LL (p<0.05). In addition, during the growth of Ira rabbits, a significant reduction of PUFA/SFA ratio (1.10 to 0.90, 1.10 to 0.94, 0.98 to 0.80) and a significant increase in SFA + MUFA (57.94% to 65.06%, 55.94% to 61.61%, 60.74% to 66.20%) were observed in the LD, LL and AM, respectively.
Effect of age and muscle on fatty acid composition of intramuscular phospholipids
The comparative intramuscular phospholipids in LD, LL, and AM of male Ira rabbit meat at different ages were shown in Table 3. The percentage of SFA and MUFA in LD, LL, and AM increased significantly (p<0.05) with increasing age of rabbits, whereas the percentage of PUFA among the muscles was significantly decreased (p <0.05). In addition, as shown in Fig. 4, during the growth of Ira rabbits, a significant reduction of PUFA/SFA ratio (A) and a significant increase in SFA + MUFA (B) were observed (p<0.05). High levels of UFA (the sum of PUFA and MUFA) (C), especially the abundance of PUFA, including the long chain (C20-22) PUFA in muscle (D), were observed in all samples. The PUFA proportion of the phospholipids in LL was significantly higher than that in total intramuscular lipid, indicating that the phospholipids contributed more PUFA in the total lipid rather than in the total triglycerides.
In terms of Ira rabbit meat at different ages, the SFAs among the muscles are mainly composed of C16:0 and C18:0. MUFA is mainly represented by C18:1, whereas PUFA consists of C18:2 and C20:4. These results agree with the results of the total intramuscular lipid composition of Ira rabbits. As the major ingredient of feeds for quite a lot species, the incorporation of C18:2n-6 into the muscles, in relation to the amount in the diet, is greatest among other fatty acids. C18:2n-6 is deposited in muscle phospholipids at a high level. Along with its long chain products (e.g., arachidonic acid (C20:4n-6)), C18:2n-6 incorporates well for insertion into phospholipids molecules (Wood et al., 2008).
Long chain n-3 and n-6 PUFA are mainly found in phospholipid (Cooper et al., 2004;Enser et al., 2000). However, the fatty acids composition is seldom detected in different rabbit muscles during different feeding days. By comparing the variety of intramuscular phospholipids from LD, LL, and AM, we determined that C16:0, C14: 1n-6, C16:1n-7, C18:1n-9, C18:3n-3, C20:2n-6, and C20: 3n-6 were increased significantly (p<0.05). Both C20:4n-6 and C20:5n-3 decreased significantly (p<0.05). No significant differences were observed in C15:0 and C17:1. In terms of C18:0, LL was sequentially higher than LD and AM. For the C18:1n-9 proportion, LD was the highest, whereas AM was the lowest. These results were consistent with the characteristics of the obtained intramuscular lipid composition. The content of C18:2n-6 in LL was the highest. No significant variation was found among other fatty acids components. During the growth of Ira rabbits, the values of n-6/n-3 values in LD, LL, and AM was 3.93 to 7.38, 4.32 to 6.67, and 3.97 to 6.33, respectively. According to FAO/WHO, the recommended ratio of essential PUFA in a healthy daily diet was 5/1 to 10/1 (n-6/n-3). Nutritional value is determined primarily by the ratio between SFA and PUFA in meat and the balance between fatty acids of the n-6 and n-3 series. In general, a ratio of PUFA to SFA (termed P:S) above about 0.45 and a ratio of n-6:n-3 below about 4.0 are required in the diet tocombat various ''lifestyle diseases'' such as coronary heart disease and cancers (Simopoulos, 2004;Williams, 2000). Besides, a lower ratio is more desirable in reducing the risk of many chronic diseases. The optimal ratio varies depending on the disease under consideration (Simopoulos, 2002). Therefore, Ira rabbits may have excellent fatty acid profile and have potential nutritive value.
Conclusions
Compared with other animal meats, Ira rabbits are a meat source of excellent quality and contain low fat, low n-6/n-3 ratio, high phospholipids, and PUFA. As for the total intramuscular fatty acids, SFA consists of C16:0 and C18:0; MUFA consists of C18:1; and PUFA consists of C18:2 and C20:4. The content of total intramuscular lipid increases with age, whereas the phospholipids content (total intramuscular lipid weight%) decreases with the progressing age. However, the absolute percentage of intramuscular phospholipids in muscles does not significantly vary. During the 35 d to 90 d growth period, significant differences existed in the fatty acid composition of total intramuscular lipid and phospholipids in the LD, LL, and AM of male Ira rabbits. Considering these differences, the nutritional characteristic of the different muscles of Ira rabbits is a promising research topic. Further investigation is necessary to explore the interaction between meat quality and fatty acids composition. | v3-fos |
2019-04-25T13:04:31.748Z | {
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} | s2 | Salt Stress Genotypic Response: Wheat Cultivars Relative Tolerance of Certain to Salinity
Forty two wheat (Triticum aestivum L) cultivars screened for their relative salt resistance raising seedlings in halfHoagland solution (control) salinized with NaCl and maintained at 4, 8, 12 and 16 dsm-1 showed a wide range of salt resistance. The growth response to salinity, judged by the shoot and root lengths, ranged from a stimulation in the case of some cultivars at lower salinity levels (4 and 8 EC) to a severe suppression in most of the cultivars at higher levels (12 and 16 EC). It was further observed that the shoot growth was often suppressed more than the root growth with this a level of 12 EC also found to be critical for most of the cultivars except HD–2160 which showed good stand even at a salinity level of 16 EC. Based on these observations, cultivar IWP–72 of the 42 cultivars tested was found to have the maximum sensitivity to salt stress whereas cultivar HD–2160 showed highest salt tolerance. The remaining 40 cultivars fell between the two extremes and were categorized into salt–sensitive, moderately salt– tolerant and salt–tolerant groups exhibiting more than 60% , 40–60% and less than 40% reduction respectively in shoot length at 12 EC dsm-1 over control.
Introduction
The complexion of salt tolerance and the multitude of ways in which plants adapt to it have caused much confusion. Sodium (Na + ) and chloride (Cl -) are among the most common ions found in excess in saline soils, and some plant species are especially sensitive to one or both of these ions [1][2][3][4][5][6][7][8][9]. A general suppression of growth is probably the most common plant response to salinity [10]. Crop plants differ greatly in their tolerance to salinity. Differences between species and varieties in regard to salt tolerance have been reported by several workers [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. In saline soils [2,5,9,[21][22][23] the control of water, the proper techniques of planting and the choice of tolerant crops are essential for their successful use in crop production. The choice of crops is based on: (1) the tolerance to salt; (2) adaptability to climatic or soil characteristics and (3) value of the crop in the individual farm activity. The chances of a crop failure are less if an adequately salt tolerant crop or its variety is selected. The key to improving salt tolerance in plants and studying its inheritance lies in finding sufficient variation within breeding populations and devising a screening procedure capable of identifying resistant or tolerant genotypes.
Further, as the period of seed germination and early seedling stage is the most crucial and important stage in the life cycle of species growing in saline environment [24] the present investigation was, therefore, undertaken to analyze the relative salt tolerance in wheat (Triticum aestivum L) at the early seedling stage and to select varieties that could withstand varying concentrations of the salts in their environment.
Materials and Methods
Forty two wheat cultivars (Triticum aestivum L) were procured from Wheat Directorate, Cummings Laboratory, Division of Genetics and Plant Breeding, Indian Agricultural Research Institute, New Delhi and Chandra Sekhar Azad University of Agriculture and Technology, Kanpur (UP), India. Screening of wheat cultivars for salt resistance was made by Garrad's Technique [25] as modified by Sarin and Rao [26] and Sharma [2] and as per method of Sheoran and Garg [27] wherein shoot and root lengths of seedlings were recorded at definite intervals. Here test tubes of uniform size (30 ml capacity) were fitted with rolls of filter paper folded at the top into a cone to support the seeds. The tubes were filled to one-third part with the test solutions so that the solution might not come in direct contact with the growing roots, the salt solution being supplied to the roots through capillary action of the filter paper. Distilled water (represented the mean loss of water from the blanks) was added to each test tube after every 24 hr of interval in order to maintain salt concentration near the target levels throughout the germination period. The seeds were initially sterilized with 0.1% mercuric chloride (HgCl 2 ) solution and later washed thoroughly with distilled water. Three seeds per tube were then transferred to the edge of the filter paper cone and were allowed to grow between the filter paper roll and the wall of the test tube in dark growth chamber at 25 ± 20C. Fifteen replicates (five tubes each having three seeds) were maintained for each treatment including the controls (half-strength Hoagland solution grown). Observations on the influence of salinity levels at 4, 8, 12 and 16 EC dsm-1 of salt solution and the controls on the total length of coleoptile and root at early seedling stage were recorded at 24 hr intervals from 48 hr after sowing up to the end of 120 hr under green safe light. The relative tolerance of different cultivars was evaluated on the basis of the% age reduction in shoot growth at 12 EC.
All parameters were analyzed by ' Analysis of Variance' (ANOVA) method as given by Panse and Sukhatme [28] wherein Critical Differences (CD at 1 and 5% probability were calculated wherever the results were significant.
Seed lots of 42 wheat cultivars screened for salinity tolerance at the early seedling stage for shoot and root lengths under varying salinity levels (0, 4, 8, 12 and 16 dsm-1) induced by NaCl as indicated (ANOVA Table 1), all the main effects viz., variety, treatment and seedling age and their interactions (V x D, V x T, D x T and V x D x T) were highly significant at 0.01 probability with significant differences noticed in the shoot and root growth of all the cultivars studied ( Figure 1). (Table 2). Similarly, significant differences were also noticed in the root growth of the cultivars studied. As indicated in the Table 3 only 11 cultivars showed less than 60% reduction in shoot growth while majority of the 31 cultivars had more than 60% reduction at 16 EC. This is in contrast with root growth (Table 3) where almost a reverse trend was noticed, i.e, out of the 42 cultivars only 15 showed more than 60% reduction at 16 EC whereas 27 had less than 60% reduction. This clearly showed that the shoot is more to salinity than the root growth. This differential response of shoot and root growth is shown in Table 4 and Figure 2 where the mean shoot growth was found to be more adversely affected than the root growth. Thus, it was interesting to find that not all plant parts were equally affected. In spite of the fact that the roots were directly exposed to the saline environment it seemed significant that shoot growth was affected more adversely than the root growth. With this also 12 EC was found to be a critical level for most of the cultivars. Thus, shoot growth seemed to be better criterion for relative salt tolerance of the cultivars of the same species at early seedling stage. Based on these observations all the 42 wheat (Triticum aestivum L) cultivars were categorized into three groups viz., salt-tolerant, moderately salt-tolerant and salt-sensitive, showing <40% , 40-60% and >60% reduction in shoot growth at 12 EC over respective controls (Table 3). Further, the different rates of shoot growth of the three groups (Figures 3 and 4) as affected by increasing level of salinity showed a gradual decline in both the salt-tolerant and moderately salt-tolerant cultivars. On the other hand, the salt-sensitive cultivars had a sharp decline in growth with increasing salt concentrations. A significant reduction in shoot and root growth with increasing salinity levels was observed irrespective of cultivars and seedling age (Table 4 and Figure 2). The reduction was more pronounced after 8 EC salinity level. It was observed that the cultivars showed the first sign of germination at 48 hr after sowing irrespective of salinity level and thereafter shoot growth increased significantly with seedling age till 120 hr (Table 4 and Figure 2).
Interaction Duration
Interaction Treatment In the significant interaction of varieties with treatment the cultivars showed a decrease in shoot growth with salinity levels; however, the varietal variations were quite evident. All the cultivars except HD-2160, Sharbati sonora, Sonalika, WL-171, K-7634, Raj-1556, UP-154, HD-1977, K-7631, UP-115, and HD-1980 showed more than 60% reduction in shoot growth at 16 EC salinity level (Table 3). Like shoot growth, salinity in general, resulted in a reduction in root growth irrespective of cultivars and duration. This decline in root growth was significant at all EC levels. On the other hand, root growth increased significantly with the age of the seedling (Table 5 and Figure 3). Further, it was observed that the cultivars differed significantly in their response to increasing salinity levels and all other cultivars except HD-2160, UP-154, Sonalika, and WL-2200 showed less than 60% root growth at 16 EC level ( Table 6).
The relative comparisons of seedling growth between different wheat cultivars indicated better performance of HD-2160 at almost all levels of salinity when compared with controls. It showed highest tolerance to salinity (i.e., 82.60% shoot growth at 16 EC over control) and IWP-72 showing highest inhibition in shoot growth (i.e., only 5.14% growth at 16 EC over control). The next cultivars which were relatively lesser tolerant but close to HD-2160 were K-7634, WL-711, WL-1531, HD-2260, UP-115, HD-2252 and UP-154. Based on these growth responses other cultivars of wheat followed a sequence of decrease as shown in Table 3 as far as their resistance to salt stress was concerned. On the other hand, all the cultivars showed an increase in shoot growth with seedling age. It was evident that the different cultivars exhibited marked differences in their early seedling growth with increasing age of the seedling and that with advancement of seedling age the effect of salt declined and that, in general, tolerance to salinity increased. It was observed that root length increased with age of the seedlings in all the 42 cultivars studied irrespective of the salinity levels. This table also shows that the cultivars differed significantly in their relative root growth. Like shoot, it was observed in the present investigation that irrespective of the cultivars studied the seedlings exhibited increase in salt tolerance with the advancement of age (Tables 4 and 5 and Figures 2 and 3).
Conclusion
The observations recorded clearly indicated that the shoot is more sensitive to salt stress than the root and that shoot growth is a better index of relative salt tolerance of different cultivars of the same species at early seedling stage with this also 12 EC salinity level was found to be a critical level for majority of the cultivars. Thus, on the basis of the % reduction in shoot growth at 12 EC salinity level over respective control all the cultivars were categorized into three groups viz., salttolerant, moderately salt-tolerant and salt-sensitive, showing less than 40% , 40-60% and more than 60% reduction respectively. | v3-fos |
2019-03-31T13:43:28.728Z | {
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} | s2 | SOME FEATURES OF THE SPECIES OF THE GENUS JUGLANS L IN THE CONDITIONS OF BRYANSK OBLAST
The genus Juglans, family Juglandaceae, comprises several species. It is widely distributed throughout the world. Green walnuts, shells, seeds, barks, and leaves are used in the pharmaceutical and cosmetic industries. Leaves are easily available in abundant amounts. The analysis was conducted for several purposes. Firstly, the elemental compositions of various parts of the leaves and immature fruits of four species of the genus Juglans L, growing in the conditions of Bryansk region was determined. Ten elements were detected by using electron-ion raster scanning microscope «Quanta 200 3D» in nanocenter NIU "BSU" and percentages of macronutrients and micronutrients in the leaves and immature fruits were determined. The analysis was also conducted to determine quantification of various substances: flavonoids, organic acids, vitamin C, antioxidant activity, dry matter, chlorophyll, carotenoid, free and bound water. This data can be used in the pharmaceutical industry. Secondly, the morphology and anatomy of the leaf and water-holding capacity of four species of the genus Juglans L were also identified. Therefore, this result can prove the difference among the species of the genus Juglans L.
Introduction
It is well known that the species of the genus Juglans L are valuable fruit trees example [6, p.95-98; 15]. They have high economic value. They are widely used in medical and pharmaceutical industries. The fruits contain up to 76% fat, 22% protein and carbohydrates, as well as vitamins A, C, E and F [11]. Activated carbons were prepared from walnut shells. Foliage, barks, roots and green shells are used for dyeing fabrics (wool, silk, yarn), as well as for hair coloring [9, p.212-214]. Vitamin concentrate is prepared from immature fruits [3; 12 p.54]. Therefore, it is necessary to conduct a study of the chemical compositions of leaves and immature fruits to search for an additional source of medicinal plants. Additionally, species of the genus Juglans L are moisture-loving plants, but they differ in drought resistance [13]. Drought resistance is determined in the driest growing season [14]. We use physiological methods to identify drought resistance features of the species of the genus Juglans L in the conditions of the Bryansk region.
Materials and methods
The leaves and immature fruits of four species of the genus Juglans L were used as research materials: walnut (Juglans regia), Manchurian walnut (Juglans mandshurica), back walnut (Juglans nigra), butterwalnut (Juglans cinerea). Anatomical study and elemental compositions of the leaves and immature fruits were carried out using the ionscanning electron microscope. Anatomy and morphological parameters of the epidermal cells of Impact Factor ISRA (India) = 1.344 Impact Factor ISI (Dubai, UAE) = 0.829 based on International Citation Report (ICR) Impact Factor GIF (Australia) = 0.356 Impact Factor JIF = 1.500 Impact Factor SIS (USA) = 0.438 Impact Factor РИНЦ (Russia) = 0.179 ISPC The Best of European Innovations, Gothenburg, Sweden 50 the leaves of various species of the genus Juglans L were studied using the SEM Quanta 200 3D, the Carl Zeiss microscope and the program VideoTest 5.0. Chemical compositions of the leaves and immature fruits were analyzed following the instructions in GOST 5900-73 (for dry matter and water content ), differential spectrophotometry (for flavonoid content), in titrimetry (content of ascorbic acid; content of free organic acids ), in the Levantalya method (for determination of antioxidant activity of plant materials), in spectrophotometry (for chlorophyll content) in refractometer (for content of free and bound water). Drought resistance was evaluated by the degree of damage of the leaves.
Results and discussions 1. The chemical composition of leaves and immature fruits of some species of the genus Juglans L in the conditions of Bryansk region.
The data obtained allows us to conclude that the leaves of four species of the genus Juglans L contain a large amount of magnesium, which has a calming effect on the brain, and a large amount of this macro -element promotes anti-stress action [9, p.33-44]. The content of magnesium 1.18% (Table -1) is determined in the walnut Juglans nigra. The leaves of the four species of the genus Juglans L also contain a large amount of calcium. It is well known that the species of the genus Juglans L have the properties of drought resistance and winter hardiness [10], which are enhanced due to the presence of high calcium content. This is explained by the role of calcium because calcium and magnesium are the basis of pectin of medial plates of cells (intercellular substance) in the plant body. Calcium ensures the integrity of cell membranes has a water retention capacity of protoplasm, involves in the construction of the cell wall of plants; increases plant tissues' hardness and improves plants' endurance. Its contents are 0.15% (Table-1), which is found in the black walnut leaves and 4.03% in the Manchurian walnut immature fruits ( Table 2). It is known that calcium is an essential element in the human body. It plays an important role in the regulation of the blood clotting, in the heart rhythm regulation. It also plays a vital role in immune processes, in the synthesis and enzyme works [11]. Being present in every cell of the body, calcium regulates the reproduction of cells and protein synthesis. For these reasons, it is important to find affordable medicinal raw materials with a high content of calcium. A large amount of potassium is also found in the leaves and immature fruits. The content of potassium is 0.51-1.02% (Table 1) in the leaves and its content is revealed from 2.55% -3.87% (Table-2) in the immature fruit. Potassium is involved in the regulation of cellular osmotic potential. Potassium plays an important role in the drought resistance and winter hardiness. It also plays an essential role in the regulation of water balance and the process of normalization of cardiac rhythm [9, p.33-44]. It can be suggested that the leaves and immature fruits of the genus Juglans L are an available source of calcium, potassium and magnesium due to the high content of potassium according to our data (Table 1-2). Silicon is found in all four species of the genus Juglans L and its content is up to 2.34% in the leaves of Juglans regia that increases plant resistance to powdery mildew. Iodine is also found in the immature fruits. Tincture of pericarp is recommended in the treatment of hyperthyroidism Immature fruits of the walnut Juglans mandshurica contain a small amount of vitamin C. Fruits of other species contain sufficiently high amount of vitamin C and 0,811% vitamin C is found in immature fruits of the walnut Juglans nigra. To conclude that, unripe fruits of various species of the genus Juglans L can be used as a source of vitamin C to make jams and fortified wine [14]. The high Impact Factor ISRA (India) Stomata play an important role in adaptation processes in different conditions and also in the plant productivity.The stomatal apparatus of four species of the genus Juglans L has an anomotsitny type. Stomata are randomly arranged on the leaf surface. Guard cells dominated the principal cells of the epidermis is observed on the leaf surface of the walnut Juglans mandchurica. Stomata of the walnut Juglans regia, Juglans nigra, Juglans cinerea are located in the same level with the main cells of the epidermis ( Fig. 1 to 4). Additionally, stomata are found only on the lower side of the leaf. The number of stomata per unit area on the leaf surface varies between 81-115. It depends on the type species. The highest number of stomata is observed on the leaf surface of the walnut Juglans cinerea, they also have the largest size of stomata (Table 5).
According to data obtained by studying of the structural features of the epidermis of the leaf four species of the genus Juglans L that cells of the abaxial side are smaller than the cells of the adaxial side of the leaf ( Table 6). The shape of the upper epidermal cells is: quadrilateral and polygonal. These cells are densely arranged to each other, and the cells of the lower epidermis have a substantially quadrangular shape. The cells of the upper epidermis Juglans regia mostly have a quadrangular shape. The length of the epidermal cells is greater than their width. Two types of trichomes were found: simple and glandular trichomes. Glandular trichomes are found only on the veins. The study of leaf morphology of some species of the genus Juglans L in the conditions of Bryansk region showed that leaf of the walnut Juglans nigra has the smallest size, while the leaf of the walnut Juglans mandchurica has the largest size (Table-7 Qualitative and quantitative composition of pigments is considered as indicator of plant adaptation to environmental conditions. The amount of chlorophyll and carotenoid of four species of the genus Juglans L is decreased; this is due to adaptation of plants to high temperature and water shortages ( Table-8). However, the amount of bound water is increased. The amount of bound water of the walnut Juglans mandchurica is not practically changed; it can be explained by the fact that their leaves are damaged by drought. The results obtained from ( Table-9) shows that the walnut Juglans mandchurica and Juglans cinerea are the greatest water loss but they are the least drought -resistant plants. Leaves and immature fruits of four species of the genus Juglans L are highly valued due to their high content of calcium and potassium, magnesium, and vitamin C and high antioxidant activity. In conclusion, that they can be used as sources of additional medicinal herbs.
2. Four species of the genus Juglans L are drought resistant and winter resistant plants in the conditions of Bryansk region, due to high contain of calcium, potassium and magnesium. | v3-fos |
2019-03-19T13:05:00.668Z | {
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} | s2 | Distribution and Seroprevalence of PPR Virus in Chad During 2004-2014
Peste des petits ruminants (PPR) is an acute febrile viral disease of goats and sheep characterized by mucopurulent nasal and ocular discharge, erosive and necrotizing stomatitis, enteritis and pneumonia. It is widespread, virulent and devastating causing significant impacts to the economy, food security and livelihoods of poor marginalized farmers. This study focused on clinical observations of 233 suspected cases of PPR and provided detailed information on the prevalence of antibodies against the PPR virus in sera from 986 small ruminants and 31 from camels. Most of specimens for this study were obtained from the slaughterhouse in N'Djamena. Sera for the detection of antibodies against the PPR virus were not necessarily representative of the target population, even if they were from a larger number of regions. According to information provided by the epidemiological surveillance field agents, the foci of the disease were more severe in goats than in sheep and the outbreaks frequency was higher during the Cold dry season (December to June) than during the other months of the year. Analysis of 1017 sera collected in small ruminants and camels revealed that the prevalence of antibodies against the PPR virus was 34.48%. The prevalence of antibodies against the PPR virus varied by species (goats, 15.21%, sheep, 19.25% and camels, 0%), age groups and geographic areas. The rate of infection by the PPR virus was higher in the sheep population (34. 67%) than in goats (27.37%). The study showed seroprevalence higher in sheep than in goats. In contrast, no serum from camels revealed positive vis-à-vis the PPR virus. Another serological survey would need to be undertaken in the population of camels in order to clarify their role in the epidemiology of PPR.
Introduction
Peste des petits ruminants (PPR) is a disease of small ruminants widespread [26,27], virulent and devastating causing significant impacts to the economy, food security and livelihoods of marginal farmers in Africa and south Asia. It is an important concern for food security and poverty alleviation. The disease is caused by a morbillivirus [9] closely related to rinderpest virus eradicated worldwide in 2011. PPR is considered as one of the most damaging animal diseases in Africa, the Middle East and Asia. It is also one of the priority diseases identified in the World 5-year Action Plan (2013-2017) of the FAO-OIE Global Framework for the Progressive Control of Transboundary Animal Diseases (GF-TADs).
Formerly it was supposed to be confined in West Africa, but now rampant in Oman [27], Sudan and the United Arab Emirates [8]. The isolation of the virus in the gazelle (Gazella dorcas) poses the problem of the role of wildlife in the epidemiology of this disease [13]. It was described for the first time in 1942 by Gargadennec and Lalanne in Ivory Coast [6]. The disease is characterized by fever, the onset of clinical signs, state of exhaustion, hyperthermia, oculo-nasal discharge, oral lesions, breathing difficulty with cough, foul-smelling diarrhea, and ends in many cases by the death of the animal [28].
In Chad, the disease is endemic and ca economic losses each year related to high morbidity (90%) and mortality (80%) in infected goats and sheep [2,23].
This disease is particularly prevalent in areas where the conditions are ripe for hatching and remain in the animals whose maintenance is usually not satisfactory. This is the case in Chad where the disease has long time existed [22,23].
All African countries located between the Sahara and the equator in the Atlantic Ocean to the Red Sea, are in the endemic area of PPR [20]. North Africa, excepted Egypt and Morocco, is not affected by PPR virus. It is the same for Southern Africa.
In Chad, the population of small ruminants has been no mass vaccination campaign against the disease. Yet the suspicion of the existence of the disease confirmed by serological surveys [22] and then by the isolation of the pathogen [3] requires a prophylactic action throughout the national territory. If the research has been undertaken in the meantime on small ruminants, there is no information relate to the sensitivity of the Chadian camels to PPR virus. In the light of the information available in Ethiopia and Sudan, a serological survey is needed to determine the status of Chadian camels vis-à-vis the PPR virus.
Pneumonia episodes of camels found confirmed as PPR [17] reinforce the first viral identification work in Ethiopia where was observed an emerging disease of camels, characterized by a fever and respiratory syndrome with very high morbidity and variable mortality. The similarity of these syndromes with those of the PPR pushed research towards morbillivirus of ruminants, peste des petits ruminants and rinderpest virus [24]. This pathology of camel has since been confirmed in Sudan as the PPR [17].
This pathology similar to rinderpest clinically and antigenically [12,18,12] reveals of paramount importance and growing because the economic losses associated with high morbidity (about 100%) and mortality (about 90%) in infected goats and sheep are considerable. The pathogen is a vir belonging to family Paramyxoviridae and Morbillivirus [10]. The PPR virus was isolated for the first time in cell culture of sheep kidney by Gilbert et al. (1962) [11]. For a long time the virus was considered as a mutant of rinderpest virus adapted to small ruminants and which has lost its pathogenicity in this adaptation process for cattle. But soon after, with the development of molecular biology technologies, it has been shown that the PPR virus is a separate entity, but closely related antigenically to Rinderpest virus [7,10].
The purpose of this work is to know the distribution of PPR in the Republic of Chad and present the seroprevalence of PPR in the areas of concentration of small ruminants in the country in order to provide basic information for launching a mass vaccination campaign against this formidable virus.
Materials and Methods
Sera were collected from sheep (548), goats (438) and camels (31). To obtain the serum, blood was collected from the jugular vein in dry tubes " venoject » then left at room temperature for 2 Animal and Veterinary Sciences 2015; 3(3): 89-93 In Chad, the disease is endemic and causes significant economic losses each year related to high morbidity (90%) and mortality (80%) in infected goats and sheep [2,23].
This disease is particularly prevalent in areas where the conditions are ripe for hatching and remain in the animals intenance is usually not satisfactory. This is the case in Chad where the disease has long time existed [22,23].
All African countries located between the Sahara and the equator in the Atlantic Ocean to the Red Sea, are in the orth Africa, excepted Egypt and Morocco, is not affected by PPR virus. It is the same for In Chad, the population of small ruminants has been no mass vaccination campaign against the disease. Yet the ase confirmed by serological surveys [22] and then by the isolation of the pathogen [3] requires a prophylactic action throughout the national territory. If the research has been undertaken in the meantime on small ruminants, there is no information related to the sensitivity of the Chadian camels to PPR virus. In the light of the information available in Ethiopia and Sudan, a serological survey is needed to determine the status of Pneumonia episodes of camels found in Sudan and confirmed as PPR [17] reinforce the first viral identification work in Ethiopia where was observed an emerging disease of camels, characterized by a fever and respiratory syndrome with very high morbidity and variable mortality. The y of these syndromes with those of the PPR pushed of ruminants, peste des petits ruminants and rinderpest virus [24]. This pathology of camel has since been confirmed in Sudan as the PPR [17]. rpest clinically and ] reveals of paramount importance and economic losses associated with high morbidity (about 100%) and mortality (about 90%) in infected The pathogen is a virus Paramyxoviridae and the genus [10]. The PPR virus was isolated for the first time in cell culture of sheep kidney by Gilbert et al. (1962) [11]. For a long time the virus was considered as a mutant of dapted to small ruminants and which has lost its pathogenicity in this adaptation process for cattle. But soon after, with the development of molecular biology technologies, it has been shown that the PPR virus is a separate entity, but igenically to Rinderpest virus [7,10]. The purpose of this work is to know the distribution of PPR in the Republic of Chad and present the seroprevalence of PPR in the areas of concentration of small ruminants in the country ormation for launching a mass vaccination campaign against this formidable virus.
), goats (438) and camels (31). To obtain the serum, blood was collected from the jugular » then left at room temperature for 2 hours. After the clots are detached from the tube wall and removed. After removing the clots, the contents of the tubes are transported to the laboratory in cold where the serum is obtained by centrifugation at 3000 rev / min for 15 minutes. The sera are then frozen at minus 20 ° This study focused on suspected cases of PPR based on clinical signs. Information on suspicions were provided by field workers involved in the of epidemiological sur network of animal diseases in Chad abbreviated REPIMAT [14]. The study provided also detailed information on the prevalence of antibodies directed against the PPR virus. Most clinical indications for this study were obtained from 11 regions out of 23 that account Chad (Map 1). These are the following areas: Chari-Baguirmi, Hadjer East and West, Tandjilé, Logone Oriental Mandoul, Moyen chari, Guera, Wadi-Fira and Ouaddaï. Sera were collected mainly from the slaughterhouse in N'Dja sera was performed by competitive ELISA according to the protocol specified by the manufacturer. The ELISA kit used for this work is that marketed by by the International Atomic Energy Agency (IAEA). Information relating to the PPR was collected from 2004 to 2014 in the field through epidemiological surveillance agents trained on the recognition of clinical signs of the disease. Samples taken during these suspicions were sent to the 90 hours. After the clots are detached from the tube wall and removed. After removing the clots, the contents of the tubes are transported to the laboratory in cold where the serum is 000 rev / min for 15 minutes. The sera are then frozen at minus 20 ° C until further use.
This study focused on suspected cases of PPR based on clinical signs. Information on suspicions were provided by field workers involved in the of epidemiological surveillance network of animal diseases in Chad abbreviated REPIMAT [14]. The study provided also detailed information on the prevalence of antibodies directed against the PPR virus. Most clinical indications for this study were obtained from 11 f 23 that account Chad (Map 1). These are the Baguirmi, Hadjer-Lamis, Mayo-Kebbi East and West, Tandjilé, Logone Oriental Mandoul, Moyen Fira and Ouaddaï. Sera were collected mainly from the slaughterhouse in N'Djamena. Analysis of sera was performed by competitive ELISA according to the protocol specified by the manufacturer. The ELISA kit used for this work is that marketed by IDvet. It was kindly provided by the International Atomic Energy Agency (IAEA).
Administrative division of Chad: The colored areas indicate areas where there has been suspected the PPR virus.
Information relating to the PPR was collected from 2004 to 2014 in the field through epidemiological surveillance agents trained on the recognition of clinical signs of the disease. Samples taken during these suspicions were sent to the laboratory for diagnostic confirmation. Table 1 shows the number of suspected cases of the disease by region and year. From this table we can see that it is mostly in 2010 and 2011 when there were more suspicions of the disease. According to field workers, clinical signs are mostly seen on goats and lesser extent on sheep. These suspicions are based mainly on clinical signs of disease, but all are not confirmed by the laboratory for various reasons: Non-compliant and / or poorly preserved specimens. As it can be seen in Map 1, a total of 11 regions out of 23 (18, 18%) in the country are affected by the disease. These regions are located in the eastern, central, south and southwest of the country.
Results
Out of 233 suspicion cases recorded from 2004 to 2014, only 38 (16, 30%) were confirmed by the laboratory.
In total 1017 sera with 548 from sheep, 438 from goats and 31 from camels were collected at the slaughterhouse in N'Djamena. The analysis of these sera showed a prevalence rate of 34.48% in small ruminants which 19.26% for sheep and 15.21% for goats.
Considering the number of sera collected from sheep and goats, we observe the PPR prevalence rate of 34.67% and 27.37% respectively. No camel serum was positive vis-à-vis the PPR virus.
Discussion
The data collected from the field indicate that the clinical signs are mostly seen on goats. This is explained by the fact that the goats are more sensitive to the PPR virus than sheep. These observations are similar to those described by Bidjeh et al. (1995) [3] and Lefèvre et al. (1990) [16].
According to the annual reports of the Ministry of Livestock [8], frequency of occurrence of outbreaks of PPR is often seen between December and June. These observations support those reported by Wosu et al. (1990) [29] and Ban-bo et al. (2014) [4] indicating that the incidence of PPR is higher during the cold dry season (December and January) that during the rainy season.
From 2004 to 2014, 233 suspected PPR cases have been recorded by the REPIMAT of which 38 (16, 30%) were confirmed by the laboratory diagnosis. The small laboratory confirmation rate mentioned here would be due to non-availability of diagnostic reagents in the laboratory or inadequate or non-compliance samples collected in the field. Often the samples arrive at the laboratory in state of putrefaction, which does not allow the smooth running of the diagnosis. Hence the need to train field workers not only on the recognition of the disease, but also on how to perform the sampling, preservation and sending them to the laboratory. This study showed a global seroprevalence of 34. 48%, with 34.67% for sheep, 27.37% for goats and 0% for camels. These results are different from those obtained by Lancelot [15] in sahelian zone of Chad indicating a global seroprevalence varying between 50 and 80%. But it was similar to those mentioned by Chauhan [25]. These authors noted that the prevalence of PPR virus is higher in sheep than in goats. This is due to the fact that goats are more sensitive to the virus and making a severe form of the disease die quickly before developing antibodies against the virus.
Of 31 sera collected on camels, none was positive vis-à-vis the PPR virus. This result differs from that reported by [19], who detected antibodies directed against the PPR virus in camels. The explanation that can be given to this result of 0% prevalence of PPR in camels is that these animals were perhaps not in contact with the virus because out of an area where the disease does has not yet appeared; it can also be due to the low number of samples collected.
Conclusion
The information collected in the field from 2004 to 2014 showed that PPR has spread across much of territory of Chad. However, it remains endemic in central, eastern and southern part of countries.
The study showed a higher seroprevalence in sheep than in goats. By cons, no sera of camels were positive vis-à-vis the PPR virus.
Another serological survey would need to be undertaken in the population of camels in order to clarify their role in the epidemiology of PPR. | v3-fos |
2016-03-14T22:51:50.573Z | {
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} | s2 | Infant Milk Formulas: Effect of Storage Conditions on the Stability of Powdered Products towards Autoxidation
Thirty samples of powdered infant milk formulas containing polyunsaturated fatty acids (PUFAs) have been stored at four different temperatures (20, 28, 40 and 55 °C) and periodically monitored for their malondialdehyde (MDA) content up to one year. MDA levels ranged between 250 and 350 ng/kg in sealed samples with a maximum of 566 ng/kg in samples stored at 28 °C for three weeks after opening of their original packages, previously maintained for ten months at 20 °C. Sample stored at 40° and 55 °C were also submitted to CIE (Commission Internationale de l’Eclairage) colorimetric analysis, since color is the first sensorial property that consumers may evaluate. Overall, the results demonstrated a good stability of PUFA-enriched infant milk formulas in terms of MDA content. However, some care has to be paid when these products are not promptly consumed and stored for a long time after first opening.
Introduction
Infant milk formulas represent the only food for non-breast-fed infants and premature babies, playing a very important nutritional role, since they represent the only way for providing newborns with all of the nutrients they need for healthy growth. Due to the complex nature of infant milk formulas and the simulating the conditions of their domestic use, in order to assess the real exposure risk of newborns to oxidation and other degradation products.
All of the tested infant milk formulas were provided by the international company Kraft-Heinz. They were enriched with LC-PUFAs, Fe and vitamins E, C and A [490 µg retinol equivalents (RE)/100 g powder]. They were received just after manufacturing, sealed in an aluminum cardboard-coated box under a nitrogen modified atmosphere and kept, as received, in a dry place until the analysis. Some of the samples were monitored for three weeks after opening, maintaining them in their original packaging, as in their ordinary use.
Detection of MDA
MDA standard solutions, sample preparation, as well as recovery experiments were performed as previously reported [18].
Briefly, MDA was detected by the TBA test, where one molecule of MDA reacts with two molecules of TBA with the formation of a pink pigment, which can be quantified on the basis of the third derivative of its absorption spectrum.
Stock solutions of MDA were obtained stirring TMP (70 mg) dissolved in 0.1 N HCl (10 mL), for 5 min at 100 °C. Working solutions of MDA were prepared by opportune dilution of the stock solution with distilled water and used for calibration and recovery experiments.
The calibration curve was obtained by plotting the peak height at 526 nm vs. MDA concentration in the range 100-2000 ng· g −1 . It had a correlation coefficient R 2 = 0.9997 and was expressed by: y = 9 × 10 −6 x + 1.0 × 10 −5 , where y is the peak height in V and x is the MDA concentration in ng· g −1 .
The detection limit was 25 ng· g −1 , and the quantization limit was 80 ng· g −1 .
Aliquots of MDA stock solution were used also for the recovery experiments. Infant milk formulas were spiked with different amounts of MDA and the recovery yields were calculated as the ratio [(ppb found in the spiked sample)-(initial ppb found in the sample)]/(added ppb), according to Fenaille et al. [20]. A mean recovery yield of 70% was obtained.
For the quantification of MDA in infant milk formulas, samples (2.0 g) of milk powder were mixed in a test tube with 5% aqueous TCA (8 mL) and 0.8% BHT in n-hexane (5 mL). The mixture was stirred at room temperature for 10 min, and then, it was centrifuged. After separation, the top hexane phase was discarded, whereas the cloudy aqueous phase was heated for 20 min at 70 °C and further centrifuged. The aqueous layer was filtered and diluted to a final volume of 25 mL. Aliquots (1 mL) of the obtained solution were mixed with 0.8% TBA in n-hexane (1.5 mL) and 5% aqueous TCA (1.5 mL). After incubation carried out for 30 min at 70 °C in screw-capped bottles, the solutions were analyzed by third derivative spectrophotometry carried out with a UV spectrophotometer lambda 40 (Perkin Elmer, Milan, Italy), setting a scan speed of 120 nm· min −1 with 1-cm absorption cells. All of the experiments were performed in quadruplicate by two different operators.
Colorimetric Analysis
Infant milk formulas were monitored for color modification. Samples of the milk powder were withdrawn from their packages, placed on Petri dishes and the color measured with an X-Rite SP62 colorimeter, equipped with an integration sphere to determine the color reflectance. The L* (lightness), a* (red/green value) and b* (yellow/blue value) values are related to the illuminant D65 at 10 degrees. Each reported value is the median of ten measurements performed randomly on the surface of the Petri dish. All of the experiments were performed in duplicate.
Statistical Analysis
Each measurement was repeated at least four times, and the results were expressed as the mean ± the relative standard deviations (%RSD). Statistical significance was determined using the GraphPad Prism 4 statistical software package (GraphPad, San Diego, CA, USA). Comparison between experimental data was evaluated by Student's t-test, and statistical significance was defined as p < 0.05.
Experimental Planning
All of the samples were stored in a dry place sealed in their original boxes and maintained at three different temperatures of 20, 40 and 55 ± 2 °C, which were regularly monitored throughout the study. At the same time, some of the samples were analyzed for MDA content after opening, simulating the conditions of the ordinary use of milk. To this end, they were stored in their original packages for three weeks at two different temperatures (20 and 28 °C) and analyzed weekly.
The experimental planning is reported in Table 1. Samples 1-3 were analyzed for MDA content, according to the above-described procedures, immediately after opening.
Sample 2 was stored for three weeks at 20 °C after opening and analyzed weekly. Sample 3 was stored for three weeks at 28 °C after opening and analyzed weekly. Samples 4-7 were stored sealed at 55 °C and opened just before the analysis, which was performed twice a week up to 14 days. were stored sealed at 40 °C and opened just before the analysis, which was performed weekly up to three months.
Samples 20-30 were stored for one year at 20 °C and opened just before the analysis, which was performed monthly.
Sample 25 was stored sealed for six months at 20 °C, then it was opened and kept in its original packaging for three weeks at 20 °C and analyzed weekly.
Sample 29 was stored sealed for ten months at 20 °C, then it was opened and kept in its original packaging for three weeks at 28 °C and analyzed weekly.
Samples stored at 40 and 55 °C were also submitted to colorimetric analysis.
Results and Discussion
Thirty samples of just manufactured powdered infant milk formulas, all derived from the same batch, were stored at different temperatures and analyzed for their malondialdehyde content and colorimetric modifications.
According to the Italian and European laws, the analyzed formulas were prepared following the principles of good manufacturing practice (GMP) using powder demineralized whey milk, vegetable oils, powder skim milk, milk proteins, maltodextrins, lactose, minerals and vitamins. All of the samples were also fortified with microencapsulated LC-PUFAs (arachidonic and docosahexaenoic acids) derived from algal and fish oils.
All of the samples were kept in a dry place sealed in their original boxes and stored at three different temperatures of 20, 40 and 55 ± 2 °C, which were regularly monitored throughout the study. At the same time, some of the samples were analyzed for MDA content after opening and storage in their original packaging for three weeks at 20 and 28 °C, simulating the conditions of the domestic use of milk. Three weeks is a reasonable period for consumption and also the maximum recommended by manufacturers after opening, whereas 20 and 28 °C were chosen considering seasonal in-house temperature variations.
A set of preliminary experiments was run with the aim to determine the data reproducibility: three samples (labeled 1-3) were opened immediately after production and analyzed in order to evaluate the intra-and inter-sample variability. Recovery experiments were also carried out on the same specimens [19]. Seventy percent of MDA was recovered in these experiments; therefore, all of the following results were corrected taking into account this value. Each sample was analyzed in quadruplicate by two different operators.
Mean values and relative standard deviations (RSD) were calculated for each sample separately and for the three samples together: for the first one (226, 243, 289, 210), a mean value of 242 ng was obtained with an RSD of 14%; for the second one (286, 244, 266, 221), 254 ng with an RSD of 11%; for the third one (289,271,213,197), 242 ng with an RSD of 18%. Considering all twelve data together, a mean value of 246 ± 33 ng of MDA per g of sample, with an RSD of 13%, was obtained. This mean error was always confirmed in all of the following experiments.
These preliminary results show how important homogenization before sampling is; in fact, when infant milk formulas are properly homogenized, no differences among specimens from the same batch are obtained.
Infant milk formulas were stored at 55 °C (Samples 4-7) and monitored for MDA content during two weeks; on the other hand, Samples 8-19 were stored at 40 °C and monitored weekly up to three months. The data obtained from the experiments carried out at 55 °C are reported in Figure 1. As already described, each result is the mean of four values, previously corrected considering a recovery yield of 70%.
Results from samples stored at 55 °C for three days (240 ng/g MDA) perfectly overlap those obtained from the variability studies; then, a rapid and linear increase of the MDA content can be observed (Figure 1). The samples were always stored in their original packages, which were sealed during the manufacturing process under a modified atmosphere. Under these conditions, MDA significantly increased by 30% (from approximately 240-320 ng, p < 0.05) within 14 days of storage at 55 ± 2 °C , with a linear 10% increment for each data point after the first three days. It is likely that this value is the reduced induction time at 55 °C.
On the contrary, when the samples were stored at 40 ± 2 °C and monitored once a week up to three months, the MDA level varied around a mean value of 255 ng/g, with a mean RSD of 10%, in perfect agreement with the values found in the initial variability experiments.
These results evidenced no significant variation of the MDA content of milk formulas stored at 40 °C for three months, thus indicating a good stability of the analyzed products. At the same time, longer exposition times may be needed under these conditions, in order to have useful information about the shelf life of infant milk formulas, which can be more easily achieved storing the same samples at 55 °C.
Samples 20-30 were stored up to one year at the constantly-monitored temperature of 20 ± 2 °C , and every month, one of the samples was opened and analyzed. The results are reported in Figure 2. These data showed a weak, but constant increase of MDA, and following the same trend, an increment of about 40% in a year can be assumed. Therefore, a slight increase of MDA levels from a mean value of approximately 240 at time zero (t0) to 340 ng/g after one year of storage at 20 ± 2 °C can be supposed.
In the experiments performed at both 20 and 40 °C, MDA levels seem to vary around a mean value with a fluctuating trend that could be explained by the complexity of the matrix [21], and the slight increase becomes evident only after a very long period of storage. This means that 40 °C is too low of a temperature for performing accelerated stability tests and also that the analyzed milks are quite stable towards oxidation.
A set of experiments was also carried out with the aim of simulating the domestic use of infant milk formulas. To this end, Samples 2 and 3 were opened and stored in their original packaging at 20 and 28 ± 2 °C, respectively, and analyzed once a week up to three weeks. These values were chosen considering seasonal variations of in-home temperatures, since they can be considered the normal room temperature and the maximum temperature of a non-conditioned room in summer. At the same time, Sample 25 was stored in its original hermetically-sealed package for six months at 20 °C, and then, it was opened and stored at the same temperature for three weeks. On the other hand, Sample 29 was stored closed for ten months at 20 °C, then it was opened and stored at 28 °C for three weeks.
The results of these experiments are reported in Figure 3. Initial MDA levels for Sample 25 at t6 months and 29 at t10 months are reported as the mean values estimated from the trend line of the data reported in Figure 2.
As is well shown in Figure 3, the MDA levels of Sample 2 fall in the range of the variability study, even if a small increase of 10% can be evidenced with respect to the mean value. On the contrary, Sample 25, analyzed in the same conditions of Sample 2, but after six months of storage, is stable in the first week; then, it shows a more marked increase of 25% of the MDA content (p < 0.05). Sample 3 shows a rapid increase of MDA, which raised weekly by 20% till a final value of 432 ng (75% of the initial value, p < 0.05). Sample 29, analyzed under the same conditions of Sample 3, but after 10 months of storage sealed in its original package, is almost stable over the first week; then, its MDA content increases by 75%, behaving exactly like Sample 3 (p < 0.05). As a whole, these experiments show that great care should be taken during the storage of infant milk formulas, with particular attention to temperature, but also that significant changes can occur during their use, especially if they are maintained at unsuitable temperatures. In fact, the combination of prolonged storage times (10 months; infant milk formulas expire after two years from manufacturing) at 20 °C and the use over a period of three weeks at 28 °C leads to very high levels (over 550 ng/g) of malondialdehyde per gram of milk powder.
Comparing data obtained from samples stored under different conditions, some correlations can be obtained between samples stored closed in their original packaging at 20 °C and 55 °C and those stored closed at 20 °C and opened at 28 °C. In fact, an exposition time of one or two months at 20 °C is almost comparable to three or seven days at 55 °C, in the same way that a hermetically-sealed milk sample stored for seven months at 20 °C shows the same MDA content of a sample kept open for one week at 28 °C. The projection of these results evidences the dependence of autoxidation from temperature and air exposition, and assuming a linear increase of MDA concentration, one may expect to find the following values: after 30 days, 250 ng/g of MDA in a sealed sample stored at 20 °C, 450 ng/g in a sealed sample stored at 55 °C, 550 ng/g in a sample stored at 28 °C after opening and 700 ng/g in an open sample stored at 28 °C after keeping it for ten months at 20 °C sealed in its original packaging. The obtained results clearly show how 55 °C could be considered a valid temperature for accelerated stability tests, and they also give an idea of the significant changes that milk powder formulas undergo after opening, especially if they are stored after opening at unsuitable temperatures, and, therefore, how mandatory their consumption in a very short time is.
To complete and support the data obtained from the MDA analysis, the colorimetric changes suffered by milk powder formulas stored at 40 and 55 °C were evaluated. Interesting results were obtained from the preliminary colorimetric data reported in Figure 4.
A B
Both samples maintained at 40 °C and 55 °C in their original sealed packages show the same behavior: they undergo an initial bleaching, corresponding to a significant increase of both L* (Table 2) and of the reflectance values (p < 0.05). This change could be attributed to the bleaching of carotenoid pigments, which were added to the analyzed samples in the form of retinol equivalents (490 µg per 100 g of powder). Similar results have been already reported in the literature [22,23]. In particular, colorimetric analysis of milk powder was previously carried out by Nielsen et al. [23] showing a behavior similar to that observed in this work, even if the initial increasing of the L* value was not considered and discussed in depth.
The profile obtained after 17 days at 55 °C corresponds to that recorded after 12 weeks at 40 °C. In fact, the reflectance curve of the samples stored for 17 days at 55 °C completely overlaps that obtained after 12 weeks at 40 °C. After the initial bleaching, the samples maintained at 55 °C undergo a browning process, so that after 20 days of storage, the colorimetric parameters correspond to those obtained at Day 6. After 33 days, the weak decreases of both a* and b* are backed by a significant decrease of L* and of the resulting reflectance (p < 0.05). On the other hand, experiments performed at 40 °C for 12 weeks did not show any browning process after the initial bleaching, maybe because the exposition time at this temperature was not long enough for inducing this color change and longer periods of time are required in order to get more complete and useful information. Anyway, although interesting, the obtained results are not sufficient to state whether this temperature is able to start the browning process observed at 55 °C. This is the first time, to our knowledge, that two different steps in the colorimetric analysis of milk powder stored at different temperatures were taken into consideration: in fact, no bleaching of pigments was discussed before [23][24][25]. The slight modification observed is probably due to the bleaching of carotenoids (the first sign of the beginning of an oxidation process) and not to the Maillard reaction, like browning is. Therefore, this observation could be used to provide information about the shelf life of milk powder formulas in a very short time, even if more investigations are needed to support these results.
As in the case of malondialdehyde detection, also for the colorimetric results, it seems that 40 °C is not a suitable temperature for the prevision of the shelf-life of milk powder formulas, whereas more complete information can be obtained working at 55 °C. The exposition time of 12 weeks at 40 °C was too short to know if the bleaching process was stopped and the time requested to have information appear too long to be compatible with accelerated tests of stability.
To our knowledge, this modification due to the carotenoids' bleaching, was never evidenced before, as only the browning process due to the Maillard reaction is reported.
Conclusions
Stability studies performed on just-manufactured LC-PUFAs-enriched infant milk formulas evidenced the good stability of these products towards autoxidation when stored for three months at 40 °C. On the contrary, a significant increase in the MDA content was measured in samples stored for two weeks at 55 °C. Similar MDA levels were obtained after one year of storage at 20 °C. In the same way, preliminary studies on colorimetric changes of milk powder evidenced an initial bleaching of milk followed by a browning process in samples stored at 55 °C for one month, whereas only the bleaching was observed after storage at 40 °C for three months.
Overall, these results suggest that 55 °C may be a suitable temperature to have insight into the stability of infant milk powder formulas, and it might be proposed to predict the shelf life of these products, which have an expiration time of two years, as established by European laws.
In addition, stability studies performed on infant milk formulas after opening of their original packaging evidenced that not only the storage conditions of the sealed products are important, but also the time and temperature after opening have to be considered. In fact, even if low levels of MDA were found in sealed samples stored for a long time at 20 °C, dangerous levels were detected in samples stored open for three weeks at 28 °C. These findings should be used to stress the importance of the storage conditions of LC-PUFA-enriched infant milk formulas, especially in their domestic use. | v3-fos |
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} | s2 | Integration of production and financial models to analyse the financial impact of livestock diseases: a case study of Schmallenberg virus disease on British and French dairy farms
Aims and objectives The aim of the study was to investigate and compare the financial impact of Schmallenberg disease for different dairy production types in the United Kingdom and France. Materials and methods Integrated production and financial models for dairy cattle were developed and applied to Schmallenberg virus (SBV) disease in a British and French context. The five main production systems that prevail in these two countries were considered. Their respective gross margins measuring the holding's profitability were calculated based on public benchmarking, literature and expert opinion data. A partial budget analysis was performed within each production model to estimate the impact of SBV in the systems modelled. Two disease scenarios were simulated: low impact and high impact. Results The model gross margin obtained per cow space and year ranged from £1014 to £1484 for the UK and from £1037 to £1890 for France depending on the production system considered. In the UK, the net SBV disease costs in £/cow space/year for an average dairy farm with 100 milking spaces were estimated between £16.3 and £51.4 in the high-impact scenario and between £8.2 and £25.9 in the low-impact scenario. For France, the net SBV disease costs in £/cow space/year ranged from £19.6 to £48.6 in the high-impact scenario and £9.7 to £22.8 in the low-impact scenario, respectively. Conclusion The study illustrates how the combination of production and financial models allows assessing disease impact taking into account differing management and husbandry practices and associated price structures in the dairy sector. It supports decision-making of farmers and veterinarians who are considering disease control measures as it provides an approach to estimate baseline disease impact in common dairy production systems in the UK and France.
INTRODUCTION
Each year millions of pounds are spent globally on managing, preventing and researching animal diseases. The current allocation of money is largely based on biological processes of transmission and infection, and the application of biological knowledge to improve disease management. There is limited reference to animal disease impacts, which should be a fundamental aspect of prioritisation and efficient resource allocation, be it at the farm, national, international or global level. A principal reason for this is the lack of standardised, rigorous methods for the financial impact assessment of animal disease. At the farm level, estimates on the financial impact of disease provide important information for farm decision-making. However, disease impact studies at the farm level are highly variable due to the complex nature of the effects a disease exerts on production (Jericho 1974), augmented by numerous variables such as species, breed or management.
Several authors elaborated a theoretical methodological basis to estimate disease impact. In the conceptual framework of Mclnerney and others (1992), disease costs are split into output losses following disease occurrence and expenditures made to treat disease or prevent its occurrence. The economically optimal level of disease costs is obtained using a loss-expenditure frontier. This concept was applied (Yalcin and others 1999, Chi and others 2002, Alarcon and others 2013, at times criticised (e.g. Tisdell 1995) and further developed (e.g. Bennett andIJpelaar 2005, Howe andothers 2013) in a range of studies. Commonly, disease impact models only look at one or very few specified production types (e. g. Alarcon and others 2013) and do not consider finer details such as differences in production type (e.g. dairy v beef), calving patterns (e.g. block calving v all-year-round calving), reproductive services (e.g. use of bull, synchronised insemination), feeding practices (e.g. intensive v extensive) or breed and associated price structures (e.g. different market prices due to product differentiations, distinct geographic locations or selling times). Because farm-specific factors influence disease impact and consequently the most appropriate decision on cost-effective disease management, disease cost estimates should reflect the farm situation as closely as possible. One way of providing tailored information for farmers is to establish a farmlevel decision support system where the user can change herd input data (e.g. herd size, cull rate) and diseasespecific assumptions (e.g. mortality rate due to disease). Such disease control calculators for selected diseases were developed and made available online by the University of Reading (www.fhpmodels.reading.ac.uk) and the Royal Veterinary College (http://www.bpex.org.uk/articles/ 303060, Alarcon and others 2013) in the UK.
Schmallenberg virus (SBV) affects ruminants and appears exclusively transmitted by insect vectors of the Culicoides species group and vertically in utero others 2012, Garigliany andothers 2012). Virus detection in blood lasts up to 15 days after natural infection (Claine and others 2013), and animals infected with SBV could develop immunoprotection, which may prevent repeated infection for at least two months (Wernike and others 2013). In ruminants, clinical signs are mainly associated with reproductive disorders. Depending on the time of infection, abortion, stillborn animals, premature deliveries and various intrauterine congenital malformations may occur (Conraths and others 2012, Steukers and others 2012, Veldhuis and others 2014. SBV has been detected in malformed fetuses, stillborn or newborn calves born at term but with signs of neurological disorders, such as blindness, deafness, recumbency, an inability to suck and convulsions (Lievaart-Peterson and others 2012, Steukers and others 2012). Dystocia in cattle may lead to negative impacts on animal welfare, culling and cow deaths (European Food Safety Authority 2012). In adult cows, the acute infection can result in transient and non-specific symptoms, namely diarrhoea, inappetence, fever and a reduction in milk yield, usually followed by a full recovery (Hoffmann and others 2012, Muskens and others 2012). While several studies were published on the biological effects of the disease, to date no scientific literature is available that estimates the financial impact of SBV at the farm level. Moreover, there is no information on how different production systems in the dairy sector are affected. Servicing or parturition in certain periods of the year may coincide with increased susceptibility of herds and/or other factors such as vector presence that may impact on exposure to pathogens and disease transmission (e.g. cows in autumn-calving herds are expected to be in middle to late gestation during the period of high-vector activity-a time when the fetus is more susceptible to malformations and abortions). The quantity of milk produced fluctuates during the year depending on the production system, that is, it is more or less constant for all-year-round-calving herds, whereas highest production occurs in autumn for autumn-calving herds and in spring/early summer for spring-calving herds. Thus, effects on milk yield due to clinical disease in adult animals during the period of high-vector activity would be more prominent in spring-calving herds. Costs and revenues are further influenced by seasonal changes in prices for milk and concentrates. These effects are schematically illustrated in online supplement 1. Finally, management practices impact on the flexibility of farmers' decision-making. For example, a reproductive disease in closed herds may result in higher numbers of 'empty' breeding animals, thus the herd fails to achieve self-sustaining replacement rates, thereby causing suboptimal production. The same problem in an open herd can be balanced by buying in replacement stock.
To examine disease effects in different production systems and support decision-making on the farm, this study presents integrated production and financial models to estimate SBV disease costs in dairy cattle in the UK and France. The objectives were (1) to develop production models as a basis for financial analysis of SBV in dairy farms, (2) to apply the models to estimate the impact of SBV in the UK and France, and (3) to investigate potential differences in model variables and disease estimates between the two countries. This study provides a first step towards the development of a farm decision calculator for the control of SBV.
Development of production models
Available benchmarking data and expert opinion was used to identify the most common and representative dairy cattle systems in the UK and France. In total, five dairy production systems each were identified for the UK and France (Table 1) (Vignau-Loustau and Huyghe 2008). Literature data were complemented by the authors' knowledge and experience of these systems and information shared by expert colleagues.
For the UK, only Holstein farm models were developed as these account for over 95 per cent of dairy production in the UK (Agro Business Consultants 2012). Autumn and all-year-round dairy systems were stratified into open and closed herds, while spring-calving herds were assumed to be mainly closed herds. In France, the different dairy production systems are described primarily by location and feeding type (Table 1). All French systems apart from the Upland with grass Franche-Comté system (UGFC) match the UK all-year-round open dairy system. The UGFC system matches the UK dairy closed system that uses all-year-round calving.
For the development of the production models, which simulated one production cycle of one year, benchmarking data from different independent sources based on farm surveys and actual expenditures made by farmers were used for both the UK ( The majority of cows (80%) calve between August and November. These farms require an essential housing infrastructure and an increased amount of concentrate. Cows have a higher than national average milk yield, and milk price during the period of highest production is at the annual peak. Replacement 50% from own heifers and 50% buying heifers from outside. The first two services a cow receives are done using dairy bull semen; subsequent services using beef bull semen (about 40% of calves born from beef bulls) Autumn-calving closed farms (ACC) Similar to AOC, but with the difference that all replacements are bred and raised in the same farm. Therefore, all cows are inseminated by dairy bulls and nearly all heifers used for replacement. The 365-day calving rate in these farms is assumed to be higher than in open herds.
All-year-round open farms (AYOC)
Cows calve anytime during the year. Fertility is poorer than in the autumn-and spring-calving systems and the replacement rate is consequently higher. Replacement through own heifers (50% of replacements) and by purchasing heifers from other farms. The first two services a cow receives are done using dairy bull semen; subsequent services using beef bull semen (about 40% of calves born are from beef bulls) All-year-round closed farms (AYCC) Similar to AYOC, but with the difference that the replacement is done using only own heifers. All cows are mated with dairy bulls and almost all heifers are used for replacement. These farms are assumed to have better breeding performance and therefore a higher calving rate than open farms France Lowland with corn silage (LC) All-year-round calving; sometimes a calving peak may be seen in a proportion of the herd. High milk yield and high cost of production. Corn silage as main forage, normally all year long. Often zero-grazing system for cows in milk. A big part of concentrates needed is soy bean meal or derivates (bought). Poor fertility and high replacement rate. Replacement is mainly done using own heifers, but also purchasing of heifers from other farms. Only cows with reproduction issues are mated with beef artificial insemination (AI) (negligible) Lowland with corn silage and grass (LCG) Similar to LC, but grass is used in spring and fall, and sometimes all summer long (depending on area). It allows reducing the feed costs. Production remains high Upland with corn silage and grass (UCG) Relies predominantly on grass, even if corn silage is available at least for winter (quantity available is limited). Production level is lower compared with LCG. Beef AI can be used to increase the calf selling prices. Variable price of milk compared with national level Upland with grass Massif Central (UGMC) Located in mountains, centre of France. Mainly based on grass/hay. None or limited use of grass or corn silage. Both Holstein (with low production) and Montbéliarde breeds are used. Standard production (i.e. the same price applies for milk here and in the lowlands) and high-quality production (Protected Area Designation cheese, higher price of milk). Use of beef bull or bull AI is important (often up to 50% of cows) Upland with grass Franche-Comté (UGFC) Located in mountains, east of France. Mainly based on grass/hay. Silage forbidden for cheese production. The production is highly specialised in high-quality products (Protected Area Designation cheese Comté) with Montbéliarde only and no use of beef bull crossing or AI. Higher price of milk compared with national level disaggregated by the different classes of animals and thus needed to be broken down using the authors' professional judgement.
Estimation of annual gross margins
The production models were used to estimate the annual gross margins for the different production systems (Eq. 1): Gross margin ¼ Revenue À Replacement costs À Feed costs À Veterinary costs À other variable costs ð1Þ A summary of calculations of revenues and costs is given in Table 2. All input parameters associated with each system are shown in online supplement 2. Detailed calculations of revenues and costs are given in online supplement 3. The revenues, that is, the amount of money a farm receives in exchange for its goods or outputs, included the return from selling milk and animals. Milk price took into account accuracy bonus, forecast bonus and penalties when production drops below a certain threshold. Animal outputs included calves for fattening, calves for breeding, culled cows and, for the UK, non-calving heifers. The latter represents those heifers that failed to conceive, and that therefore have to be sold (blockcalving herds only). For France, non-calving heifers were not considered because of the absence of block-calving herds. Feed costs included the costs of concentrate, forage, bulk feed and milk replacer for calves sold. Forage costs were calculated differently for the UK and France (Table 2). For the UK, forage costs were calculated my multiplying the 'forage costs per hectare' by the number of cows per hectare. For France, estimations of forage costs for cows were based on the average daily dry matter intake depending on the lactation stage, the nature of the main forages used and the forage price. In France, disposing costs are paid through a tax when slaughtering animals and were therefore not included in the French models. Other variable costs included the costs of artificial insemination (AI), bedding, veterinary and miscellaneous costs, as reported in benchmarking data of both countries. Replacement costs accounted for ( Input values are specific to each production system. Number (N) and quantities of animals/products indicated in the equations are obtained from the production models C, cow; CV, calf; H, heifer the costs of purchasing new heifers-in calf (open farms) or raising own heifers (closed farms). For France, heifers purchased were young animals (not in calf ) in accordance with the main practices observed. The residual value of cows was accounted for as 'revenues from cows culled'. The costs of raising own heifers were calculated by estimating the number of heifers to be raised in the herd (accounting for heifer mortality) and multiplying this by the variable costs of heifer rearing (VCHR) (Eq. 2):
Revenues and costs Equations
VCHR ¼Heifer feeding costs (including forage) þ Heifer veterinary costs þ Heifer bedding costs þ Heifer miscellaneous cost þ Cost of artificial insemination ð2Þ The heifer feeding costs were the average daily dry matter intake of concentrates and forage multiplied by the respective costs, for each rearing period from weaning to the first calving.
Assessment of disease impact using partial budget models The scientific literature was screened to identify the biological effects of SBV in cattle, which included clinical manifestations (diarrhoea, milk drop and fever) and reproductive disorders (late abortion or malformations and related dystocia). Assumptions were made on general management practices and farmers' reactions to these biological effects (Box 1). In terms of disease effects, it was assumed that milk drop would occur during the duration of the clinical episode and that milk production would return to normal after recovery. SBV reproduction problems were assumed to occur in the last trimester only (stillborn or malformed calves or abortion) when cows are already dry. Due to the lack of scientific evidence of early abortion or empty cows, these effects were not included. The production and gross margin models were run with and without SBV disease parameters (Table 3), and the differences obtained between a herd with SBV and a herd without SBV were used to estimate the net value using standard partial budget analysis (PBA), an economic method used to calculate the extra cost or benefit of a change (Eq. 3): BOX 1: Assumptions made on general management practices and reactions to Schmallenberg virus (SBV)-related disorders in the UK and France (FR) to estimate the financial impact of SBV General management practices (without SBV) UK-FR: 100 milk cow spaces used to full capacity. Lactation period will be extended for some of the cows not conceiving to cover empty spaces. UK: Farmers need to purchase extra in-calf heifers or use their own heifers to reach 80 calvings per 100 milk cow spaces because of low calving rates. Assumption: fertility higher in closed than open herds. FR: The high replacement rate of dairy farms in France leads to consider 85 calvings per year for 100 inseminated cows. In open herds, a proportion of young heifers are purchased for replacement, whereas in closed herds replacement is done using own calves only. UK-FR: All cows in closed herds inseminated with dairy bulls. UK: 40 per cent of cows in open herds inseminated with beef bulls. Fifty per cent own stock replacement, 50 per cent purchase from outside. FR: 0 per cent (LC, UCG), 20 per cent (lowland with corn silage and grass) and 40 per cent (upland with grass Massif Central) of cows in open herds inseminated with beef bulls. Low percentage: no replacement heifers are bought as not needed; high percentage: young heifers can be purchased if needed. No purchasing of in calf heifers. Farmers' reaction to clinical disease UK-FR: A very small proportion of SBV affected cows will receive treatment (anti-inflammatory) to suppress fever. Reproductive SBV disorders and related management practices UK-FR: In case of abortion, the cow will be culled. Open farms will buy in-calf cows to replace those who have aborted. Closed farms will cull the cow and ▸ replace it the following year (the UK, block calving) with the exception of all-year-round farms ▸ replace it with older cows kept longer (FR and the UK, all-year-round calving) UK-FR: When the number of heifers produced is not enough to achieve the required replacement rate, ▸ in closed herds, farmers will keep older cows longer; the farmer has no longer the possibility of disposing cows with poor milk yield and therefore a reduction in milk production will occur; ▸ in open herds, in-calf heifers are bought and replacement heifers not sold (the UK). FR-UK: In case of late abortion, the veterinarian will be called out if the fetus presents signs of malformation. Antibiotic treatment will be applied to aborted cows. FR-UK: When malformations lead to dystocia, the veterinarian will be called out. In few cases of dystocia, farmers will agree to conduct a caesarean. Milk loss due to dystocia was considered negligible and was not included. UK-FR: When there is no dystocia, the veterinarian will not be called out and there will not be any medical treatment. FR-UK: The costs of culling a malformed calf are negligible, but not the disposal costs (the UK). FR-UK: A small proportion of aborted fetuses and calves stillborn and malformed will be submitted for SBV testing.
Net value (or net SBV disease costs) represents the financial impact of SBV for a year cycle and a defined disease scenario i. The individual partial budget items can be found in the results tables and the associated calculations in online supplement 3. For the PBAs, disease parameters were introduced in the production models and the differences between gross margin parameters of disease and no disease situations were obtained. For example, the number of 'dairy x dairy male calves sold' in a herd without SBV was higher than in an SBV-affected herd as SBV infection can cause reproductive problems. Consequently, the difference between the two was recorded as 'dairy x dairy male calves not sold' under revenues foregone in the PBA.
Data on the within-herd SBV incidence, the incidence of various disease effects (e.g. rate of diarrhoea, drop in milk yield, fever) and the magnitude of those effects (e. g. proportion of milk loss) are sparse. Consequently, only two disease scenarios were considered: 1. Scenario 1: A high impact in a herd that is highly susceptible to disease, which may, for example, be a management system where the susceptible gestation period falls into a season of high-vector activity. 2. Scenario 2: A low impact in a herd that is less susceptible to disease, which may, for example, be a management system in an area with low-vector density.
To complement the values derived from the scientific literature, the input values for the model were discussed and agreed on in the workshop described in the section 'Software, input values, sensitivity analysis and validation'. For the most variable and uncertain parameters, minimum, most likely and maximum values were agreed upon (Table 3).
Software, input values, sensitivity analysis and validation
All models were built in Microsoft Excel. Apart from the parameter values derived from published literature, a workshop with 10 experts representing members of the SBV surveillance team at the Animal Health and Veterinary Laboratories Agency, industry representatives, veterinary clinicians and academic researchers was held to present and discuss the structure of the production models, input variables and assumptions. Before the meeting, experts were requested to complete a table with their opinion on the values of specific disease parameters (Table 3), and their ranges, for high-and low-impact scenarios. The different expert estimates obtained and their averages were presented to the experts during the workshop for discussion. For the parameters with major differences and uncertainties, all workshop participants were encouraged to explain why they disagreed and a discussion was stimulated to get to an agreement on the most appropriate values. Further, the structure of the production models, gross margin and partial budget analyses was presented and discussed until an agreement was reached. The second workshop was held at the end of the study, where the models developed and their results were presented. Experts were asked for their opinion on the validity of the results obtained. Similarly, animal health professionals in France looked at the inputs and the assumptions made and provided recommendations for improvement if deemed necessary. Gross margin results were compared with literature estimates for validation purposes.
Sensitivity analysis
The sensitivity analyses were done by varying simultaneously the two variables' proportion of SBV abortions (varied between 0 and 3.5 per cent in steps of 0.5 per cent) and proportion of stillborn and malformed calves (varied at values from 0 to 5 per cent in steps of 1 per cent). These variables were selected taking into account the uncertainty attached to them and their hierarchical position in the model. Uncertainty was determined considering the range of estimates collated from the literature and experts, and the input from discussions during the first expert workshop. In addition, the models were run with all lowest and all highest values as defined in Table 3 to estimate the range of disease impact. (Fig 1). Main differences observed between the model gross margin and the industry gross margin were largely caused by differences in the estimation of replacement and feed costs (Fig 1). Overall model estimates were higher than the estimates reported in the literature.
Production models and gross margin analyses
The model gross margins obtained for the French lowland with corn silage (LC), lowland with corn silage and grass (LCG), upland with corn silage and grass (UCG), upland with grass Massif Central (UGMC) and UGFC systems were £1431, £1428, £1291, £1037 and £1890 per cow space and year, respectively. The model gross margin estimations for the LC, LCG and UCG systems showed differences of 3 per cent and 8 per cent, respectively, to published gross margins (Fig 2). The differences for the UGMC and UGFC systems were 13 per cent and 21 per cent, respectively, due to lower operational costs in the present work compared with references for these two upland systems (Fig 2).
Financial impact of SBV
Results of the SBV disease costs for dairy farms are shown in Tables 4 and 5.
In the high-impact scenario in the UK, the net SBV disease costs in £/cow space/year for an average dairy In the low-impact scenario in the UK, the net SBV disease costs in £/cow space/year for an average dairy cattle farm were estimated at 19.5 in SC, 11.9 in AOC, 25.9 in ACC, 11.6 in AYOC and 8.2 in AYCC systems (Table 4).
In the high-impact scenario in France, the net SBV disease costs in £/cow space/year were estimated at 19.9 in LC,19.6 in LCG,20.9 in UCG,21.0 in UGMC and 48.6 in UGFC systems. In the low-impact scenario in France, the net SBV disease costs in £/cow space/year were estimated at 9.7 in LC,9.6 in LCG,10.3 in UCG,10.5 in UGMC and 22.8 in UGFC systems (Table 5). If LC, LCG, UCG and UGMC were considered as closed, the net SBV disease costs in £/cow space/year were estimated at 42.5, 50.5, 39.9 and 55.4 for the high-impact scenario and 20.7, 25.6, 19.7 and 28.0 for the low-impact scenario, respectively. If UGFC was considered as open, the net total costs £/cow space/year were estimated at 20.8 and 10.2 for the high-and low-impact scenarios, respectively.
For all open systems (the UK and France), the main costs of disease were associated with purchasing or raising extra heifers for replacement (74-89 per cent of the sum of costs). For closed farms in the UK, milk loss due to culling cows with abortion represented the major costs of disease (50-61 per cent of the sum of costs). For the UGFC closed system in France, the replacement costs and the revenues foregone from cows not culled (32 and 27 per cent, respectively). Sensitivity analyses performed for two of the most sensitive and uncertain disease parameters expressed as net SBV disease costs in £/cow space/year showed that an increase in abortion rate influenced disease costs more than a rise in number of calves stillborn or malformed (see online supplement 4). For the UK, the net SBV disease costs per cow space and year ranged from £0.1 to £69.9 for SC, £0.1 to £44.1 for AOC, £0.1 to £91.6 for ACC, £0.1 to £42.8 for AYOC and £0.1 to £41.0 for AYCC farms. The ranges from the best case (using the minimum values for all disease inputs as defined in Table 3) to the worst case (using the maximum values for all disease inputs as defined in Table 3) for the highimpact scenario were £2.2 to £56 for SC, £2.4 to £42.5 for AOC, £2.2 to £68.6 for ACC, £2.2 to £40.6 for AYOC and £2 to £41.30 for AYCC farms. For the low-impact scenario, the net costs per cow and year ranged from £0 to £19.5 for SC, £0 to £11.9 for AOC, £0 to £25.9 for ACC, £0 to £11.6 for AYOC and £0 to £8.2 for AYCC farms.
For France, the net SBV disease costs per cow space and year ranged from £0 to £35.2 for LC, £0 to £34.5 for LCG, £0 to £36.8 for UCG, £0 to £38.4 for UGMC and £0 to £86.6 for UGFC farms. The net costs ranges from the best case to the worst case for the high-impact scenario per cow space and year were between £1.9 and £54.2 for LC, £2.1 and £53.2 for LCG, £2.0 and £55.9 for UCG, £2.9 and £75.8 for UGMC, and £2.6 and £86.6 for UGFC systems. For the low-impact scenario, the net costs per cow and year ranged from £0.7 to £9.7 for LC, £0.7 to £9.7 for LCG, £0.7 to £10.3 for UCG, £0.7 to £10.5 for UGMC and £0.7 to £22.8 for UGFC systems.
The impact of SBV on the farm profitability was assessed by comparing the gross margins without SBV and low-and high-impact SBV infection, respectively. In the UK, the reduction in gross margin per cow space and year was between 1.6 per cent (AYCC system) and 4.3 per cent (SC system) for the high-impact scenario, and between 0.9 per cent (AOC system) and 1.3 per cent (SC and AYOC systems) for the low-impact scenario. In France, the reduction in gross margin per cow space and year was between 1.4 per cent (open LC and UCG) and 2.6 per cent (closed UGFC) for the highimpact scenario and between 0.7 per cent (open LC and LCG) and 1.2 per cent (closed UGFC) for the lowimpact scenario. Detailed results are given in online supplement 5.
DISCUSSION
In the present study, farm-level models were developed that combine herd dynamics and gross margin analysis of different production systems as a basis for PBA to estimate SBV disease costs in the UK and France. The production models developed allow to account for financial consequences that are related to specific management practices that can be difficult to capture otherwise. The results clearly illustrate that the disease impact differs substantially depending on the underlying production system, based on closed or open farms, block calving and all-year-round calving, heifer replacement management and replacement rate, and various combinations thereof. The production models proved particularly useful in estimating the effects of disease parameters that have a cascade effect. For example, an increase in abortions means that fewer calves are born, which does not only accrue a loss in the form of less calves sold, but also reflects a cost due to more adult animals dying or being culled because of the abortion. In the case of a closed farm, this meant that either more calves needed to be kept for replacement in the model or, if the calving rate was not high enough, adult cattle earmarked for removal from the herd due to production reasons were kept longer. Consequently, block-calving closed herds were the systems with higher disease impact due to limited flexibility in maintaining the same level of milk production and the need to retain dairy heifer calves for the extra replacement needed due to SBV (revenue foregone from sales lost). On the other hand, major costs associated with open farms accrued from purchasing or raising heifers for replacement, which was almost double for open farms compared with closed farms due to the high market prices of replacement stock. Disease impact was found to be generally higher for autumn-calving herds due to higher replacement rates and milk price.
For France, the disease impact was also considerably higher when closed herds were considered. This was mainly due to the high replacement rate of French herds (between 28 and 35 per cent compared with the 20 and 25 per cent replacement rate of UK herds). In disease situations that affect the number of calves being raised, French closed farms cannot produce enough heifers to achieve target replacement rates. Consequently, they have to keep old cows that leads to inefficient production and a higher disease impact in such herds. While closed management does exist, it is likely that such farms will switch to open management to be able to use the existing milking spaces when facing replacement difficulties. Certain situations, particularly the scarcity of affordable replacement heifer or cows, mimic the challenges closed herds face. This could, for example, happen when demand for replacement animals is high because of high milk prices and/or there is limited supply of specific breeds (e.g. only few farmers sell Montbéliarde animals). The LCG and UGMC systems in France are open systems that are heavily reliant on purchasing young heifers because they commonly use beef bull or beef AI in their herds. The LC and UCG systems may be open or closed, depending on farmers' behaviour.
Building on the gross margin models, costs saved, new revenues, new costs and revenues foregone could be calculated by comparing the values between farms modelled to be healthy and those modelled to be diseased. Importantly, the net value obtained in the PBA is equivalent to the difference of the gross margins with and without disease. The partial budget approach helps to understand what the magnitude of the additional costs and benefit items are due to disease incursion and which items most influence the impact and therefore should be the focus of potential intervention measures. The impact of SBV on the gross margin, on the other hand, shows how profitability changes. This information is also important for farmers as it may indicate sustainability issues, reduction in capacity for investment and determine the importance farmers will attribute to disease. Consequently, both the PBA and gross margin analysis provide critical information in relation to SBV (or disease in general).
The partial budget could have been calculated without the underlying production model by, for example, using equations that take into account animal numbers, prices and the cascade effects of disease parameters. However, validation of such calculations would be difficult in the absence of empirical data. In this study, the creation of the production models allowed straightforward validation of the costs, revenues and gross margins by comparing them to published values on gross margins. For some production systems, particularly in the UK models, some model costs (e.g. replacement costs) were found to be higher than those calculated by the industry, and therefore the overall gross margins were higher. One main reason for this was that milk prices used were taken from the DairyCo benchmarking data, which were considerably higher than those used by other benchmarking reports. The DairyCo milk prices were used because they reflected the actual prices, whereas other references used projected estimates. The second main reason was that the calculations of replacement costs were similar to the latter references, but dissimilar to DairyCo data. Consequently, the UK models showed higher revenues for milk than most references and lower replacement costs than the DairyCo benchmarking report, which may have led to a slight overestimation of SBV disease impact. Nonetheless, the method and comparative approach used represent a useful way to understand the possible model deviations and their repercussion on the results.
The present models could also include situations that were not accounted for in the example used. For example, revenues foregone due to milk penalties associated with low milk production did not apply because milk drop and percentage of cows affected were too low for that. However, such effects could be substantial in the case of diseases that have a strong negative effect on milk yield.
The SBV disease costs were presented for a low-impact scenario and a high-impact scenario without providing any information about the likelihood of a farm being in the high-impact or low-impact category. The likelihood of being in the SBV high-or low-impact category may be different for the different production systems. For example, all-year-round-calving farms are expected to be less at risk of suffering from high impact due to their flexibility on the time of calving. The French systems, which are based on all-year-round calving, have therefore greater scope to influence the risk of exposure. However, only observational studies and/or epidemiological transmission models would allow estimating the probability of disease on a farm with some level of precision; this was not within the scope of this study and remains open to further research.
One of the main limitations of this study was the lack of data available in the literature on SBV disease effects, which may be partly due to a lack of reporting and the absence of incentives for reporting. Most of the published scientific literature described the situation on Schmallenberg-affected farms, but only in some exceptional cases compared them with non-affected farms or previous years before SBV emergence. In consequence, attribution of disease estimates was not possible from those studies. Experimental studies or epidemiological studies comparing affected and non-affected farms are needed in order to obtain more accurate disease estimates. The disease estimates used in this study were derived from scientific publications when possible and complemented by expert opinion consultation. Sensitivity analyses on disease estimates were used to account for this uncertainty and demonstrate the influence of the most uncertain input values used.
Because of the biological and economic consequences of SBV, there is a demand for effective animal health interventions (Trickett 2013). Given the epidemiology of the virus and infection in wildlife, elimination of the virus from populations may prove challenging and the focus therefore lies on interventions to avoid the negative impact of disease. Vaccines for SBV in ruminants have been developed in Europe and licences have been approved in some EU countries for marketing of the vaccine (Anonymous 2013a, b). The purpose of SBV vaccines is to induce an immune response that prevents the virus from reaching the fetus. Farmers will need to make a judgement whether the additional investment needed to protect their herds or flocks is justified by the resulting loss avoidance. Apart from vaccination, there are limited options to control the disease effectively. Some authors suggested measures to reduce exposure to the vector through disruption of vector breeding sites, pesticide use, housing and protection of ruminants by repellents (Baylis andothers 2010, British Cattle Veterinary Association 2012). Additional measures could be a breeding system that manages the timing of service or insemination of animals depending on the season and concentration of midges and thereby reduces exposure to the virus in the critical period. However, such a strategy may prove difficult where production and management systems are targeted towards the seasonality of grass growth and market demand.
The integrated production and partial budget models could be used to assess the economic efficiency of potential interventions at the farm level. There is scope to convert them into a farm-level decision tool where veterinarians or farmers could enter farm-level-specific data and thereby estimate disease impact and the net value of potential interventions for their farms. | v3-fos |
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} | s2 | Ethnobotany and Pharmacognostical studies of Jasminum sambac Linn.
In the present study of Jasminum sambac ethnobotanical and pharmacognostical activity of Jasminum sambac leaf .These species of highly medicinal purpose flowers, leaves act as lactifuge, arrest the secretion of milk in the puerperal state in case of threatened abscess pharmacognostical study of leaf parts of Jasminum sambac has been undertaken, hence it is establish. Pharmacognostical and quality control parameters of leaf, of Jasminum sambac was carried out . Various microscopic characters viz., transverse section of leaf, quantitative parameters (stomatal number, stomatal index, and vein-islet and termination number) were determined for the identification of plant material. Powder microscopic studies, were also performed. Physicochemical parameters such as total Ash(13.5%), water soluble ash (6.7%) and acid-insoluble ash (8.2 %)Alcohol soluble extractive(30%) Water content(6.19%)Crude fibre content(15.1%)Swelling index (1)and Foaming index (Lessthan 100). published. Till date, no ethno botany and pharmacognostical studies have been reported for Jasminum sambac . In the present investigations, ethnobotany and pharmacognostical studies viz., macroscopic, microscopic, physicochemical parameters was determined.
INTRODUCTION
Jasminum sambac Linn. (Family-Oleaceae) commonly known as Motia or lily Jasmine is a scandent or sub-erect shrub with young pubescent branches, broadly ovate or elliptic, opposite leaves, white, very fragrant flowers cultivated nearly throughout the tropical and sub-tropical parts of the world. The plant is much valued for its exquisitely fragrant flowers and it is estimated that nearly 400md of flowers are annually used for the extraction of perfumed oils and 250md for the preparation of attar .The plant is considered cool and sweet used as a remedy in case of insanity, in weakness of sight and affections of the mouth. The flowers are bitter, pungent, cooling, tonic to brain, purgative, cure tridosha, biliousness, itching sensation, allays fever, stop vomiting, useful in the diseases of eye, ear, mouth, good for skin diseases, leprosy and ulcers Kiritikar KR et al., (2003). Traditionally leaves are used in fever, cough, indolent ulcer, abdominal distention, diarrhoea, lowering the blood glucose level, regulating menstrual flow, to clean kidney waste, inflamed and blood shot eyes. Root, flowers, leaves are galactogogues therefore act as lactifuge, Kiritikar KR et al., (2003) and Nadkarni KM (2007
World Wide Distribution
Jasminum sambac (Linn.) Probably originated in India and was brought to Malaysia and Java around the 3rd century; since then widely cultivated throughout the Malaysian region for its heavily scented flowers and tropics of both Hemispheres. In Tamilnadu, Andhra Pradesh, Kannad, Chazipur, Sikanderpur and Karnataka, the Jasmine particularly Jasminum sambac (L.)Are commercially cultivated for flowers (Kiritikar KR et al., (2003) and ethnobotany Jasminum sambac 2010) .
ETHNOBOTANY
Ethno botanical survey of plant has been conducted in Solan, Una, Kangra, Sirmour and Mandi district of Himachal Pradesh, India. Ethno botanical Information about the Jasminum sambac has been collected through interview and discussion with the people having the age 30-85 years. Jasminum sambac are used in various diseases like as spinal cord injury, menstrual disorder, migraine and others, mentioned in Table 1.
Plant material
Fresh leaves and stem were collected from Villupuram District Alagramam (TamilNadu) in the month of March and were authenticated by Dr. N.Rajasulochana Madras University (Presidency College Autonomous) Chennai.
Instrument, Chemicals and reagents
Chloral hydrate, iodine and sodium hydroxide were procured from RFCL, Mumbai, India. Compound microscope, Camera Lucida, Stage and eyepiece micrometer, glass slides, cover slips,
ILNS Volume 37
watch glass and other common glassware were the basic apparatus and instruments used for the study. Microphotographs were taken using Labomed ATC-200 microscope attached with Sony digital camera.
Macroscopy of leaf
Morphological studies were done by using simple microscope to determine the shape, apex, base, margins, taste and odor of the leaves .
Microscopic studies
Microscopy was done by taking the thin hand sections of the midrib and lamina region of the leaves. The thin sections were cleared with chloral hydrate solution and stained with phloroglucinol and hydrochloric acid, then mounted in glycerin for the identification of various regions. Powder of the dried leaves was separately treated with phloroglucinol, hydrochloric acid and glycerin to study various characteristics. Similarly, the powder was also stained in iodine solution, ruthenium red solution for the identification of starch grains, calcium oxalate crystals etc. As a part of quantitative microscopy stomata number, stomata index, veinislets number and vein termination number were determined by using fresh leaves of the plant Evans WC (2005)
Physicochemical parameters
The moisture content, total ash, water soluble ash, acid insoluble ash, alcohol and water soluble extractive values were determined as a part of its physicochemical parameters Who Quality control methods for medicinal plant materials ABTS publisher and distributor (1998).
Powder analysis
The shade dried aerial parts of the plant were powdered and powder was passed through 100 # sieve. A small amount of powder was taken on to a microscopic slide, cleared from chlorophyll by heating with chloral hydrate solution and was mounted in 50% v/v glycerol in water. This was then observed under microscope to study the characteristic featuresWallice TE (2005) and Betty PJ et al., (2000).
Macroscopical characteristics
The leaf has prominent midrib, uniformly smooth and even lamina. Morphologically the leaf appeared simple in composition, opposite in arrangement, variable in shape usually ovate or elliptic, glabrous or nearly so, with acute apex, entire margin, petiolated (3-6mm), 4-12cm (length) by 2.4-6.5cm (breadth). The fresh leaf was green in colour with characteristic odour and slightly bitter taste ( Figure.1)
Microscopical characteristics The Transvers section ( T.S.) of leaf
In transverse section the leaf appeared dorsiventral in nature showing three layers (Fig .2). It showed the presence of single layered epidermis composed of flat rectangular cells covered by thin cuticle while lower epidermis covered by thick cuticle . The uniseriate, unicellular and multicellular covering trichomes were present in the upper and lower epidermis. The glandular trichomes were multicellular with single stalk . Stomata were present only on the lower epidermis . Below the epidermis layer in the lamina the next region was mesophyll which consisted of single layered long elongated palisade cells followed by spongy parenchymatous cells. The midrib region consisted of closely packed multilayered collenchymatous cells present below the upper epidermis and above the lower epidermis. Below and above the collenchymatous cells loosely packed with intracellular spaces parenchymatous cells were present. In the centre (midrib region) 'C' shaped or half moon shaped vascular bundles were present composed of xylem and phloem cells International Letters of Natural Sciences Vol. 37
Powder microscopy
The powder microscopy of plant (leaves) was light green in colour and showed the anomocytic stomata, spiral vessel, starch grain and calcium oxalate crystal.
Quantitative microscopy
Various quantitative microscopic parameters viz., stomatal number and index, vein islet and termination number and were determined as per the WHO guidelines for quality control methods for medicinal plant material. The results are given in Table 3.
Physicochemical parameter
Physicochemical parameter-Various parameters such as ash and extractive values, moisture content and foreign matter were established and the results are summarized in Table 5.
CONCLUSION
The methods carried out in the present research work namely .The pharmacognostical and physicochemical characteristics of Jasminum sambac Linn , which could be used in identification and to distinguish the plant material, were determined and established. The ethnobotanical study of plant material revels about its potential to cure various ailments. The present study may be useful information with regards to its, ethno-pharmacological potential, screening and isolation of phytoconstituent to carrying out further research. cooling, tonic to brain, purgative, cure tridosha, biliousness, itching sensation, fever, stop vomiting, skin diseases, leprosy and ulcers Foaming index Less than 100 | v3-fos |
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} | s2 | Compost mulch no-tillage systems in organic vegetable production
With growing agricultural demands from both conventional and organic systems comes the need for sustainable practices to ensure long-term productivity. In Hungary small scale vegetable growers face challenges in producing their crops due to the lack of effective weed control practices and viable methods of sustainable soil fertility management based on local or regional soil amendment resources. There is a demand for cultural practices that reduce hand labor requirements and black plastic mulches whereas long-term productivity is held or increased. To identify effective alternative weed control and soil fertility management options for the management of intensive organic vegetable systems, our research focuses on the evaluation of compost and paper mulches, in conjunction with reduced-tillage practices. In 2015 determinate tomato (cv. Roma) is grown in five different soil treatments using yard waste compost (YWC) mulch and combination of YWC and paper mulch (PM) plus bare ground control under intensive and reduced tillage variants to evaluate their effectiveness on organic tomato marketable yield and weed suppression. Since most organic tomatoes at present are grown on small acreage in Hungary, and are direct-marketed, the application of organic mulches can be assumed financially feasible. Our results will show if the combination of organic mulching materials together with reduced tillage may be a viable option for organic vegetable growers.
Introduction
The objective of this publication is to review the effects of composted organic mulches, and the combination of mulches and different tillage intensity systems as alternative weed control methods and viable soil fertility management solutions. Practices for vegetable and fruit production need to focus on decreasing synthetic inputs, sustainably managing disease and weed control, reducing soil erosion, and maintaining soil structure while producing highquality fruit and profitable yields (Grassbaugh et al. 2004). Although research on the benefit and use of mulches is extensive, little is known about how to optimize their application in organically managed system (Law et al. 2006).
Weed control
Production losses from weed competition are among the most important crop management concerns for organic growers, and the ability to control weeds is considered a major limiting factor for farmers wishing to transition to organic production systems (Bond and Gandy 2001). Organic vegetable production relies heavily on intensive tillage to reduce weeds and to create a fine seedbed for planting or seeding. This intensive tillage has been shown to be detrimental to long term soil quality, and often leads to contamination of the environment through on-site and offsite losses of organic matter, nutrients and sediments (Magdoff and van Es, 2000). It also reduces soil microbial activity, destructs soil structure, while increasing emission of greenhouse gases, and the potential for nitrate leaching to groundwater (Jackson et al. 2003). Reduced tillage systems are spreading practice especially on the American continent, largely as a result of growing concerns about soil quality, costs of tillage operations, fossil fuel and labor use, and environmental resources. On the other hand, decreased intensity of tillage may cause serious problems for growers because of increased weed pressure.
Organic mulches
Application of organic mulches is an alternative method to suppress weeds through blocking light and prevent weed establishment without mechanical or manual weeding (Brault et al. 2002) and can be as effective as herbicides in suppressing weeds (Ozores-Hampton, 1998). Organic mulches such as straw, wood chips or compost can conserve soil moisture, reduce soil erosion and may also have advantages of low-cost, with no removal requirement compared to black polyethylene mulch, commonly used among organic growers (Ozores-Hampton 1998;Feldman et al. 2000). These mulches have also been shown to improve soil quality and stimulate soil microbial communities due to the addition of organic matter. Possible disadvantages of organic mulches include nutrient tie-up and lowering, if soil temperatures decrease to sub-optimum level (Schonbeck and Evanylo, 1998). Also, organic mulch alone is not always sufficient to control perennial weeds, and may even pose a risk of weed infection, especially if its material of origin contained weed propagules, and was not fully hot composted (Merfield, 2002). Straw and hay mulches improve soil properties degradation and are used widely. However, they keep soil cooler which can delay early season growth. Surface-applied yard waste compost (YWC) substantially increases underlying soil nutrient levels (Feldman et al., 2010) and also increases yields (Gallaher and McSorley, 1994), whereas it does not have a cooling effect on the soil due to its dark colour. Composting is a biological decomposition process in which microorganisms convert organic materials into relatively stable humus like material. During decomposition, microorganisms assimilate complex organic substances and release inorganic nutrients. An adequate composting process should kill pathogens and stabilize organic carbon before the material is applied as mulch. The end-product of the composting process is optimal as soil amendment and mulch as well. YWC is easily accessible in many regions in Hungary where there are composting operations next to landfills.
Paper mulch
Paper mulches may offer another viable solution for weed control without the problem of disposal of plastic mulches, since they decompose fully after use (Radics and Bognár, 2004;Merfield 2002). Paper mulch as soil cover for special agricultural use is even produced and used in commercial scale in some countries and is a permissible product for weed control in organic farming certification systems (Harrington and Bedford, 2004). Its main disadvantage is rapid degradation; it tends to tear from the edges, may be lifted by wind and degrades too quickly, resulting in ahigh weed population on the field where applied. No scientific study analyzed an integrated approach where paper mulch and compost mulch is applied at the same time, on the same bed. According our hypothesis if paper mulch is covered by weed seed free compost and vegetable seedlings are transplanted into this mulch layer, the advantages of both mulch types may be utilized. Paper mulch effectively suppresses weed emergence during the first several weeks after transplanting, and the weed-free compost serves as optimal media for transplants. If drip irrigation applied, the sodden paper mulch will be penetrable for the growing roots of the vegetable plant, so it can reach the soil under the paper mulch ensuring its continuous development.
Material and Methods
Five tillage and organic mulch treatments (Table 1) in four replications are compared in a randomized complete block design in frame of a small scale organic vegetable production system. Treatments are compared regarding their weed control efficacy, effect on soil properties and influence on yields. YWC, PM and CM are applied by hand.
Each plot comprises of a 15 m long and 1,2 m wide bed that was prepared on a clay loam Luvisoil at the MagosVölgy Organic Farm, Terény, Hungary. In late April 2015 each plot was © Slovak University of Agriculture in Nitra Faculty of Agrobiology and Food Resources http://www.acta.fapz.uniag.sk 140 planted with tomato seedlings (cv. Roma), using three rows of plants 40 cm apart and with 40 cm spacing within the rows. Weed infestation will be measured using 1 x 1 m quadrates in four replications and four times per plot, assessed by counting number of weeds and coverage of plants. Height and development of tomato plants in different phonological phases after transplanting will be measured as well, as total plant biomass (dry weight) after the harvest season. Crop yields will be measured throughout the harvesting season several times according to the fruit ripening from 20 selected plants from the middle row of the beds. The YWC purchased from the Zöld-Híd Nonprofit Ltd. Company's Nógrádmarcal Facility will be analyzed for dry matter, organic matter, C, N, P, K and pH, and minerals before application using standard procedures.
Results
Preliminary results for weed infestation and yield will be available in November 2015 and will be published.
Conclusions
In literature no relevant scientific data has been found on mixed application of paper mulch and compost mulch on small scale, intensively managed organic vegetable production systems. According to the data available, and based on some practical (unstudied) examples of successful vegetable operations in California (US), it is assumed that a combined system of compost mulch and paper mulch methods may be competitive in terms if weed control and tomato yields, compared with conventionally tilled uncovered systems. First results of the study will be presented at ICOAS 2015. | v3-fos |
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} | s2 | Cashew apple extract inhibition of fat storage and insulin resistance in the diet-induced obesity mouse model
The cashew apple is an unvalued by-product from the cashew nut industry, of which millions of tonnes are simply discarded globally. Interestingly, however, cashew apple nutrients may have beneficial effects for health even if these are still poorly described. The present study was designed to evaluate the effect of a hydro-alcoholic extract of cashew apple (cashew apple extract; CAE; Cashewin™) on obesity and diabetes, in two experimental designs using the diet-induced obesity (DIO) mouse model. First, in the preventive design, mice were treated orally with the CAE at the dose of 200 mg/kg body weight from the first day under a high-fat diet (HFD) and during 8 weeks thereafter. Second, in the curative design, the animals were first maintained under the HFD for 4 weeks and then treated with the CAE for a further 4 weeks under the same regimen. For both experimental designs, body weight, peri-epididymal adipose tissue, liver weight, food consumption, glycaemia, insulinaemia and insulin resistance were assessed. In both designs, the CAE significantly reduced body-weight gain and fat storage in both the peri-epididymal adipose tissue and the liver for mice under the HFD. This was achieved without modifying their energy consumption. Furthermore, glycaemia, insulinaemia and insulin resistance (homeostasis model assessment-insulin resistance) of the DIO mice were significantly lowered compared with the control group. Thus, a well-designed hydro-alcoholic extract of cashew apple could provide an attractive nutritional food ingredient to help support the management of body weight and associated metabolic parameters such as blood glucose and insulin levels.
According to the WHO, obesity and overweight are considered to be the fifth largest risk factor for global deaths. The number of obese people has nearly doubled since 1980 and the WHO estimated in 2014 that more than 1·9 billion adults over 18 years of age were overweight, with more than 600 million being obese (1) . Scientific publications have showed that overweight is one of the major risks factors for metabolic syndrome disorders. Elevated BMI (>30 kg/m 2 ) increases the risk of developing type 2 diabetes, hypertension, cholesterol abnormalities, CVD, Alzheimer's disease and inflammationbased pathologies (2)(3)(4)(5) .
In the struggle to manage body weight, different therapeutic approaches have been investigated. Synthetic anti-obesity drugs have emerged, each having their own effects on fat absorption and metabolism, but all showing adverse effects on humans (6,7) . For example, Orlistat (tetrahydrolipstatin) is Abbreviations: CAE, cashew apple extract (Cashewin ™ ); DIO, diet-induced obesity; HFD, high-fat diet; HOMA-IR, homeostasis model assessment-insulin resistance; ND, normal diet; PEAT, peri-epididymal adipose tissue. the only gastrointestinal lipase inhibitor drug approved by the Food and Drug Administration and the European Agency for treating people suffering from obesity and hypercholesterolaemia, even if it causes some gastrointestinal side effects (8)(9)(10)(11) . Consequently, alternative nutritional approaches have been investigated and are gaining scientific and public interest (2,12,13) .
Cashew trees (Anacardium occidentale L.), while native to Brazil, are now widely spread across many tropical countries. They are mainly cultivated for their nuts, the 'true fruit', which is considered to be the main economic product. However, along with the nuts, a pseudo fruit is also collected, known as cashew apple and weighing around 90 % of the harvested mass (14) . Cashew apples are considered to be a by-product of the cashew nut industry. The ripe apples, although known to be edible, are not widely appreciated by consumers because of their limited shelf life as well as their sour and astringent taste (15,16) . Several industries have tried to develop suitable processes for giving a more widely accepted taste to this fruit either in juice (15) or wine (17) forms. Currently only 12 % of Brazilian cashew apples are being processed for food or beverage, with the remaining apples being either left on the ground to rot or used for animal feed (18,19) .
Few publications have reported the phytochemical constituents of the cashew apple (20)(21)(22)(23) . Michodjehoun-Mestres et al. have identified and quantified several glycosylated flavonols in the cashew apple (20) next to a new cinnamic acid derivative called 1-O-trans cinnamoyl-β-D-glucopyranose (22) . To date, some literature has discussed the hypoglycaemic properties of extracts obtained either from cashew tree stem-barks (24)(25)(26) , leaves (27)(28)(29) or nuts (30) in animal models. Prasertsri et al. suggested in a clinical study that cashew apple juice supplementation increased fat utilisation during high-intensity exercise in both trained and untrained subjects, yet with no observed effects on metabolic profiles (31) . To our knowledge, however, no data presenting the effect of cashew apple extracts (CAE) on metabolic syndrome parameters have been published to date.
The present study was carried out to evaluate the efficacy of a specific CAE (Cashewin ™ ) on C57BL/6 diet-induced obesity (DIO) mice in two different experimental designs to assess the preventive and curative effects on the reduction of bodyweight gain and fat storage, hyperglycaemia, hyperinsulinaemia and insulin resistance in an animal model of the metabolic syndrome.
Reagents
Chemical standards and other analytical grade reagents and solvents were from Sigma-Aldrich Chemical Co. and Extrasynthese.
Cashew apple extract
The CAE (Cashewin ™ ) tested in this study is manufactured by Dialpha. It is a hydro-alcoholic extract of cashew apple residues (Anacardium occidentale L.). Cashew apples were crushed and pressed to remove their juice. The press cake was used to prepare the extract. A quantity of 350 g of the press cake was extracted with 3·5 litres of a 50:50 water-ethanol solution (v/v) for 2 h at 50°C. After a first solid-liquid separation, the solid residue was extracted a second time in the same conditions with 2·45 litres of solvent. The grouped filtrates were concentrated at 50°C in a rotary evaporator and then converted into powder by freeze-drying. The average yield of extraction was about 5 % from the cashew apple residues.
Phenolic constituents of CAE were measured by the colorimetric method using Folin-Ciocalteu reagent (ISO 14502-1) for its total phenolic content and by HPLC for the aromatic compounds.
Analytical HPLC analysis of the CAE was performed on an Ultimate 3000 Dionex system fitted with a reversed phase C-18 column (250 × 4·6 mm internal diameter × 5 μm; ACE ® ) and with a guard column, operated at 30°C. The mobile phases used were 0·1 % formic acid in high-purity water (solvent A) and acetonitrile (solvent B), utilising the gradient as shown in Table 1 over a total run of 86 min with a flow rate of 0·7 ml/min. Samples were prepared at a concentration of 10 g/l in 80 % (v/v) methanol and injected at a level of 20 μl. Aromatic compounds in the extract were detected at 280 nm.
The identification of phenol constituents in the CAE by HPLC was based on the literature (20,22) . The main flavonoids, i.e. myricetin and quercetin derivatives, were quantified using their aglycone moieties as standards. 1-O-trans-cinnamoyl-β-D-glucopyranose was quantified using trans-cinnamic acid as standard.
Animals and experimental design
Male C57BL/6NCrl mice aged 5 weeks old weighing about 20 g were purchased from the Charles River Laboratories. Three mice were housed per cage. The animal room environment was controlled with a temperature of 22 ± 2°C, 60 % humidity, and day-night cycles of 12 h light-12 h dark The first study evaluated the preventive effect of chronic oral administration during 8 weeks of the CAE in the C57BL/6 DIO mice. After the acclimatisation period, mice were randomly assigned to the three different study groups according to their body-weight values in order to obtain homogeneous groups (nine animals per group). The ND-control group was kept under the ND and treated with the vehicle (Milli-Q water), the HFD-control group was switched to high-fat diet (HFD; EF R/M with 30 % fat, ref. E15126; Ssniff; see Table 2) and treated with the vehicle, and the HFD-CAE group was switched to the HFD and treated with the CAE at the dose of 200 mg/kg body weight. The extrapolation of this animal dose to human equivalent dose was based on the body surface area (Guidance for Industry -Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers; http:// www.fda.gov/downloads/Drugs/.../Guidances/UCM078932. pdf) normalisation method and resulted in a dose of 1200 mg CAE for a 70 kg person. This corresponded to a consumption of around 200 g of cashew apple, i.e. two to three fruits daily.
The CAE prepared in solution in Milli-Q water or the vehicle (Milli-Q water) were given by oral administration every morning between 08.00 and 10.00 hours. The dose volume was 10 ml/kg body weight and the actual volumes administered were calculated and adjusted based on the most recent body weight of each animal. The diets and filtered tap water were provided ad libitum.
The second study assessed the curative effect of chronic oral administration during 4 weeks of the CAE in the C57BL/6 DIO mice. After the acclimatisation period, mice were first submitted to a HFD for 4 weeks to induce obesity and pre-diabetes. The ND-control group was kept under the ND. At the end of this induction period, mice were randomly assigned to the two remaining study groups according to their bodyweight values in order to obtain homogeneous groups (nine animals per group). The ND-control group was kept under the ND and treated with the vehicle (Milli-Q water), the HFD-control group was kept under HFD and treated with the vehicle, and the HFD-CAE group was kept under the HFD and treated with the CAE at the dose of 200 mg/kg body weight.
In the two studies, mice were weighed three times per week and food consumption was monitored per cage once per week. Glycaemia of the mice was measured weekly in animals which had fasted for 5 h. One drop of blood was collected via the tail vein for glucose determination using a hand-held glucometer (OneTouch Ultra 2; LifeScan). At the end of the study, after a 5 h fast, animals were anaesthetised by an intraperitoneal injection of a mix of ketamin (Imalgen ® 1000) and xylazine (Rompun ® 2 %). A terminal blood sample was collected via cardiac puncture using heparin as anticoagulant for plasma preparation. This blood sampling resulted in the death of the animals. The peri-epididymal adipose tissue (PEAT) and the liver were harvested to measure their weights. Fasting plasma insulin levels were measured using an ELISA kit (Mercodia). Then the insulin resistance index (homeostasis model assessment-insulin resistance; HOMA-IR) was calculated using equation (1): The liver TAG content was measured in the preventive design. The livers were homogenised in a mixture of chloroform-methanol (2/1, v/v) and lipids were extracted using the Folch method (32) . The TAG content was determined using the respective ELITech kits ( TGML-0250 and CALI-0550; ELITech).
Statistical analysis
Results were expressed as means and standard deviations (n 9). The statistical analysis was conducted using Microsoft Excel software. One-way or repeated ANOVA tests as well as the Fisher's least square difference post hoc test were executed. Values of P < 0·05 were considered statistically significant when comparing two different groups.
Cashew apple extract
The results obtained showed that the CAE was composed of about 5 % total polyphenols, 13 % humidity, 7 % proteins, 7 % lipids, 3 % minerals and 65 % total carbohydrates. The HPLC profile for aromatic molecules is presented in Fig. 1, where peaks A to F represent the myricetin derivatives, peaks G, H and J the quercetin derivatives, and peak I the cinnamic acid derivative. The phytochemical content of the CAE expressed in g/kg dry extract was 2·7 g myricetin derivatives, 1·2 g quercetin derivatives and 1·4 g 1-O-trans-cinnamoyl-β-D-glucopyranose.
Preventive effects of the cashew apple extract on diet-induced obesity mice Preventive effects of the cashew apple extract on body weight, fat storage and energy consumption. Mice under the HFD (HFD-control) gained much more weight than those under the ND (ND-control; Fig. 2(a)). The difference in body weight between the two groups became statistically significant from the fourth day under the diets. At the end of the study, after 8 weeks of treatment, the HFD-control group reached an average body weight of 38 (SD 1) g, whereas the ND-control group reached an average weight of 24·6 (SD 0·3) g. Thus, the HFD-control group gained almost three times more weight than the ND-control group: 19 (SD 0·92) g for HFD v. 6·8 (SD 0·32) g for ND mice. This increase in body weight induced by the diet is primarily due to the storage of energy under the form of perivisceral fat. Indeed, PEAT weights of mice under the HFD were multiplied by six during the study compared with those of mice under the ND: 2·01 (SD 0·13) and 0·34 (SD 0·05) g for the HFD and ND, respectively ( Fig. 2(b)). Next to that, the HFD also induced the storage of fat in the liver (hepatic steatosis). The colour of the liver became white instead of red for mice under the ND and the liver weight was increased by 67 %: 1·72 (SD 0·14) g for HFD v. 1·03 (SD 0·04) g for ND mice (Fig. 2(b)). Furthermore, the liver TAG level was increased by 34·2 % in the HFD-control group: 2·16 (SD 0·24) mmol/l for HFD v. 1·61 (SD 0·22) mmol/l for ND mice (Fig. 2(c)). Combined, these results show that the HFD containing 30 % fat with long-chain SFA (beef tallow) induced a well-established model of obesity and related disorders.
As shown in Fig. 2(a), the CAE at the dose of 200 mg/kg body weight reduced by almost half the body-weight gain induced by the HFD (48·4 % at the end of the study). This effect was statistically significant from the fourth day of treatment (P < 0·01) and increased all along the study. Mice under the HFD treated with the CAE gained 13·1 (SD 1·09) g, while HFD-control mice treated with water gained 19 (SD 0·92) g and ND-control mice treated with water gained 6·8 (SD 0·32) g. This reduction in body-weight gain is at least partly due to a reduction of fat storage in the perivisceral fat. Indeed, the CAE reduced by 37·2 % fat storage into PEAT induced by the HFD (P < 0·01). PEAT weight of CAE-treated mice was 1·39 (SD 0·11) g, compared with 2·01 (SD 0·13) and 0·34 (SD 0·05) g for HFD-control mice and ND-control mice, respectively ( Fig. 2(b)). Also, the CAE reduced fat storage induced by the HFD in the liver by 63·8 % (P < 0·01). Liver weight of CAE-treated mice was 1·28 (SD 0·04) g, compared with 1·72 (SD 0·14) and 1·03 (SD 0·04) g for HFD-control mice and ND-control mice, respectively ( Fig. 2(b)). This reduction in liver weight was correlated with a significant reduction in liver TAG content of 138 % (P < 0·05). In fact, the liver TAG levels for the CAE-treated mice was 1·40 (SD 0·18) g, compared with 2·16 (SD 0·24) and 1·61 (SD 0·22) g for HFD-control mice and ND-control mice, respectively ( Fig. 2(c)).
After a stabilisation period of 2 weeks, the energy intake of the different groups remained stable throughout the experimental duration. Animals under the HFD consumed more energy than those under the ND. No statistically significant difference in energy consumption was observed between the HFD-control group and the HFD-CAE group (Fig. 2(d)).
Chronic oral administration of the CAE significantly reduced blood glucose levels compared with the HFD-control group treated with water. This effect was significant from the second week of treatment onwards, and lasted throughout the study (with a mean 44·8 % reduction of hyperglycaemia induced by the HFD; P < 0·001).
By the end of the study, fasting insulinaemia was also significantly increased by the HFD. Indeed, the blood insulin level of the HFD-control group reached 4·29 (SD 0·08) ng/ml, whereas that of the ND-control group reached 1·65 (SD 0·14) ng/ml ( Fig. 3(b)). In line with the fasting glycaemia and fasting insulinaemia increases, the insulin resistance index (HOMA-IR) was four times higher in the HFD-control mice compared with the ND-control mice (Fig. 3(c)).
As presented in Fig. 3(b), treatment of the mice with the CAE prevented the increase in fasting blood insulin level induced by the HFD. Insulinaemia reached 3·54 (SD 0·35) and 4·29 (SD 0·08) ng/ml for HFD-CAE mice and HFD-control mice, respectively. This effect was close to being statistically significant (P = 0·051). Therefore, according to the HOMA-IR insulin resistance index, the CAE significantly reduced insulin resistance induced by the HFD in DIO mice by 39·5 % (P < 0·01; Fig. 3(c)).
Curative effects of the cashew apple extract on diet-induced obesity mice
Curative effects of the cashew apple extract on body weight, fat storage and energy consumption. After 4 weeks under the diets, mice submitted to the HFD presented a significant difference in body weight compared with mice submitted to the ND (P < 0·001), showing that obesity was well established before the beginning of the treatment. The HFD-control group reached 30·4 (SD 0·5) g body weight, whereas the ND-control group reached 23·6 (SD 0·4) g ( Fig. 4(a)).
As for energy consumption, mice under the HFD consumed more energy than mice under the ND, but no significant differences were observed between the HFD-control and HFD-CAE groups (Fig. 4(c)).
As of the first week of treatment, and throughout the study, the CAE significantly reduced the hyperglycaemia induced by the HFD in mice by a mean of 22·4 % (P < 0·05 to P < 0·001, depending on the time point; Fig. 5(a)). At the end of the protocol, insulinaemia and insulin resistance were also reduced by the treatment with the CAE. The blood insulin level of CAE-treated mice reached 5·03 (SD 0·43) ng/ml, whereas the levels for the HFD-control group reached 6·30 (SD 0·29) ng/ml and the levels for the ND-control group reached 2·43 (SD 0·20) ng/ml (Fig. 5(b)). According to the HOMA-IR insulin resistance index, the CAE reduced insulin resistance induced by HFD in DIO mice by 34·4 % (P < 0·01; Fig. 5(c)).
Discussion
The metabolic syndrome is a pre-disease condition without any known remedy other than improving diet and increasing physical exercise. Overweight and obesity are common characteristics of the metabolic syndrome, increasing the risk of type 2 diabetes occurring. With our objective to identify active plant extracts for the prevention of overweight and obesity in mind, we screened various plant raw materials for that Values are means (n 9), with standard errors represented by vertical bars. Mean value was significantly different from that of the HFD-control group: * P < 0·05, ** P < 0·01, *** P < 0·001.
purpose. As part of our approach we gathered various agricultural by-products as they constitute sustainable materials, for some very rich in polyphenols and other potentially active molecules. The cashew apple is a poorly valued by-product of the cashew nut industry which is available in significant volumes. It was selected for our research amongst other raw material candidates. From here, we developed and optimised a specific CAE which was tested in a DIO animal model.
The C57BL/6 DIO mouse model provides a wellrecognised nutritional model of obesity and pre-diabetes. It is widely used to test drugs as well as complex plant extracts (33,34) . For our work, we used two different designs: a 'prevention' design so as to evaluate the ability of the extract to prevent the development of obesity, hyperglycaemia and insulin resistance, and a 'curative' design to test its capacity to reverse an established disease state.
The daily oral intake of 200 mg/kg body weight of the CAE, in both the preventive and the curative study, resulted in a significant body-weight-gain reduction of the mice submitted to a HFD. This reduction in body-weight gain was at least partly due to a decrease in the peri-epididymal (perivisceral) adipose tissue mass. Increase in perivisceral fat is recognised to be associated with, or to be a strong risk factor for, insulin resistance and the metabolic syndrome (35) . Hence the prevention of this tissue's increase, as it has been observed in mice treated with CAE, should contribute to the improvement in insulin resistance. Next to that, fat was also stored under the form of TAG in the liver (hepatic steatosis), resulting from the imbalance between energy intake and expenditure, combined with higher insulin resistance, oxidative stress and inflammation (36) . Liver weight and TAG content analysis performed in the preventive study showed that CAE treatment also prevented fat storage in the liver and, consequently, should improve liver functions. It is interesting to note that the CAE reduced body-weight gain without affecting food consumption as the CAE-treated and water-treated animals ate the same amount of HFD and then the same level of energy. Hence the CAE may reduce fat storage by reducing fat digestion and absorption, increasing fat metabolism, energy expenditure or thermogenesis. Further investigations are needed to clarify the CAE mechanism of action.
Next to its effect on body weight, CAE supplementation also reduced hyperglycaemia, hyperinsulinaemia and insulin resistance of DIO mice in both study designs. The reduction in body weight and the reduction in fat storage in the perivisceral adipose tissue as well as in the liver was probably linked to the improvement of insulin sensitivity and glycaemic control as it is recognised that even a small reduction in body weight can improve insulin action (37) . However, we cannot exclude a direct action of the CAE on insulin resistance at this stage of analysis.
The CAE is enriched in flavonoids and more precisely in myricetin and quercetin derivatives. Flavonoids are plant polyphenols that are recognised as effective antioxidants (38) and may protect against several chronic diseases (39) . Among flavonoids, diets enriched in myricetin and quercetin tend to lower the risk of developing type 2 diabetes (39) . Choi et al. showed that myricetin supplementation had a protective effect on obesity and insulin resistance in C57BL/6 mice fed with a high-fat and high-sucrose diet (40) . Similar results were obtained by Henagan et al. by using quercetin and a quercetin-rich onion extract in the same mice submitted to a HFD (41) . In vitro studies indicated that myricetin and quercetin flavonoids may inhibit intestinal glucose transporters (42) and/or glucose uptake in adipocytes (43) . Combined, these data suggest that myricetin and quercetin derivatives contained in plant extracts such as CAE may participate in the effect of the extract on DIO mice. Also, a poorly described cinnamic To convert glycaemia from mg/dl to mmol/l, multiply by 0·0555. Values are means (n 9), with standard errors represented by vertical bars. Mean value was significantly different from that of the HFD-control group: * P < 0·05, ** P < 0·01, *** P < 0·001.
acid derivative recently identified in cashew apple (22) , 1-O-trans-cinnamoyl-β-D-glucopyranose, is present in the CAE at almost the same amount as quercetin derivatives. The role of this phytochemical in the CAE activity observed in this study is not known. At last, the phenolic and nonphenolic compounds may also participate in the protective effect of the CAE against DIO and insulin resistance.
Conclusion
We reported here for the first time that the oral administration of a water-alcoholic CAE (Cashewin ™ ) can reduce bodyweight gain, fat storage, hyperglycaemia, hyperinsulinaemia and insulin resistance in DIO mice. This has been evidenced in both a preventive and a curative in vivo study design. Further investigations have to be carried out to identify more specifically which cashew apple molecules, and through which mechanisms of action, are responsible for achieving these protective effects. | v3-fos |
2019-04-28T13:02:13.937Z | {
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} | 0 | [] | 2015-03-02T00:00:00.000Z | 73687012 | {
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} | s2 | Application of Jute Fiber for the Improvement of Subgrade Characteristics
Improvement in subgrade has always been an area of concern to highway and geotechnical engineers. In case of a highway, a weak subgrade results in greater thickness of pavement layer which increases the cost of pavement construction. To strengthen the subgrade soil, the use of jute fiber is advantageous because they are cheap, locally available, biodegradable, and eco-friendly. Jute fiber mixed randomly with subgrade soil has significant impact on the improvement of subgrade characteristics over the last decade. Keeping this in view an experimental study was conducted on locally available soil reinforced with jute fiber. In this study the soil samples were prepared at its maximum dry density corresponding to optimum moisture content in the CBR mold with and without reinforcement. The percentage of jute fiber by dry weight of soil was taken as 0.3%, 0.6%, 0.9%, and 1.2%. In the present investigation the length of fiber was taken as 15 mm and 30 mm, and two different diameters (4 mm and 8 mm) were considered for each fiber length. From the laboratory tests the effect of jute fiber content on density of soil (optimum moisture content and maximum dry density) was observed for each length and diameter of jute fiber. It was observed that the optimum moisture content increases and the maximum dry density decreases with the increase in jute fiber content for each length and diameter of jute fiber. The laboratory CBR values of soil and soil reinforced with fiber were determined. The effects of aspect ratio (length/diameter ratio) of jute fiber on CBR value of soil were also investigated. Test results indicate that CBR value of soil increases with the increase in length of jute fiber. It was also observed that increasing the diameter of jute fiber further increases the CBR value of reinforced soil, and this increase is substantial at fiber content of 1.2% for aspect ratio of 3.75 (length = 30 mm, diameter = 8 mm).
Introduction
Transportation plays a vital rule in the development of economy, industry, society and culture of any country. To achieve development in those fields, a satisfactory mode of transport and communication is indispensable. This can be achieved through a transportation system, which is economically viable. Economy in road network can be achieved through economical pavement design. Practically it is not possible to have good subgrade always. Poor subgrade necessitates a greater pavement thickness resulting in increased construction cost. With the aim of reducing pavement thickness on poor subgrade new techniques of construction and soil stabilization have been continuously explored. Poor natural soils make them practically unsuitable for many civil engineering construction activities including road pavements. In such cases natural soils are being treated with different kinds of materials to improve their engineering properties. Aggarwal and Sharma (2010) studied the application of jute fiber in the improvement of subgrade characteristics. From this study it was concluded that jute fiber reinforcement reduces the maximum dry density and increases the optimum moisture content of the subgrade soil. The CBR value of the subgrade soil increases up to 250% with the inclusion of bitumen coated jute fiber [1]. Fiber reinforced soil has been used in many countries in the recent past and further research is in progress for many hidden aspects of it. Fiber reinforced soil is effective in all types of soil (i.e. sand, silt, clay). Use of natural material such as jute, coir, sisal and bamboo, as reinforcing materials in soil is prevalent for a long time and they are abundantly used in many countries like India, Philippines, and Bangladesh etc. The main advantage of these materials is they are locally available and are very cheap. They are biodegradable hence do not create disposal problem in environment. Processing of these materials into a usable form is an employment generation activity in rural areas of these countries. If these materials are used effectively, the rural economy can get uplift and also the cost of construction can be reduced, if the material use leads to beneficial effects in engineering construction. Singh and Bagra (2013) studied the application of jute fiber for the improvement of subgrade soil. They concluded that the CBR value of subgrade soil increases with the increase in jute fiber content. They also conclude that the CBR value also increases with increase in length and diameter of jute thread [2]. Tera et al. (2011) proposed that of all the natural fiber jute has highest tensile strength and withstand rotting and heat [3]. Gray and Ohashi (1983) conducted a series of direct shear tests on dry sand reinforced with different synthetic, natural and metallic fiber to evaluate the effects of parameters such as fiber orientation, fiber content, fiber area ratios, and fiber stiffness on contribution to shear strength [4]. We have several examples of reinforcing the soil like Great Wall of China (earliest example of reinforced earth using branches of trees as tensile materials), Ziggurats of Babylon (woven mats of read were used) etc. In USA & Europe, the erosion & stability of slopes for highway and railway embankment is controlled using reinforced soil [5]. Singh (2011) studied the strength characteristics of soil by using Geosynthetic [6]. Sivakumar and Vasudevan (2008) studied the strength and stiffness response of soil reinforced with coir-fiber [7]. Singh (2012) used the coir fiber to improve the laboratory CBR value of soil [8]. Sharma et al. (2011) studied the strength characteristics of soil reinforced with coir fiber [9]. Lawton et al. (1993) used multioriented geosynthetic to improve the laboratory CBR value of soil [10]. Consoli et al. (2002) used plastic waste to improve the engineering behavior of soil [11]. Ranjan et al. (1996) studied the probabilistic analysis of soil mixed randomly with fiber [12]. Rao et al. (2006) studied the behavior of load deformation of fiber reinforced gravel beds overlying soft clay [13]. Michalowski et al. (2002) studied the strength anisotropy of sand reinforced with fiber [14]. Gray et al. (1986) studied the comparative behavior of sand with fabric reinforcement and fiber reinforcement [15]. This paper presents the influence of jute fiber on the CBR value of the studied soil. A number of CBR value tests have been conducted on soil, and soil reinforced with varying amount of jute fiber. The effects of aspect ratio (length/diameter ratio) of jute fiber on CBR value of reinforced soil have also been investigated. The laboratory test results of reinforced and unreinforced soils were also compared.
Material Used
The soil used in this study is collected from Rajapur of Chapai Nawabgonj district, Bangladesh. The soil sample was collected from a depth of 60 cm after removing the top surface soil from natural ground surface. The jute fibers used in this study was purchased from the market in the diameter of 4 mm and 8 mm and in the length of 15 mm and 30 mm.
The fiber was coated with bituminous layer in order to protect it from decay. The bitumen used for the coating of jute fiber was of 80-100 grades.
The materials used for the present study are soil, jute fiber, and bitumen.
Soil
Index properties of the soil were determined ( Table 1) and classification of soil was done as per Textural Classification. The soil is classified as Clay Loam. The jute fiber used was procured from the local market. The diameter of the thread was 4 mm and 8 mm and length was 15 mm and 30 mm. These fibers were generally available in the threaded form. These were mechanically woven fibers with very fine threads. The physical appearance of jute thread is shown in Figure 2. Jute industry is one of the oldest industries in Bangladesh, has traditionally been used for packaging. However its versatility is coming to light now after the world had started looking for natural options to save the environment. Jute is a coarse natural hast fiber. The major components of jute fiber are shown in Table 2.
Bitumen
Bitumen was used for coating the jute fiber to protect them from microbial attack & degradation. Bitumen coating was done in the hot state at a temperature of 220 °C.
Grade and some of the other properties of bitumen are tabulated in Table 3.
A series of proctor compaction tests and California
Bearing Ratio tests have been carried out on soil mixed with jute fiber. The detailed procedure and results are as under.
Proctor Compaction Test
To assess maximum dry density (MDD) and optimum moisture content (OMC) Modified Proctor test is performed as per ASTMD-1557.
The jute fiber purchased from the market in diameter of 4 mm and 8 mm had been cut in the pieces of length nearly 1 m. These pieces of the jute were coated with bitumen in order to protect it from microbial attack & degradation. To coat the bitumen over jute fiber, bitumen was heated up to a temperature of nearly 220 °C. The thread of jute was dipped in it for about 2 to 3 second & extra bitumen was streamed off with the help of fork. The pieces in the way were left for cooling of 24 hour. After 24 hour the threads were further cut in the small pieces of length equal to 15 mm and 30 mm.
A series of modified proctor tests were carried out on the soil sample with 0.3% to 1.2% by weight of jute fiber coated with bitumen. While adding the jute fiber coated with bitumen in the soil mass, appropriate correction was applied to cater for the amount of bitumen.
From the result of modified proctor test it was observed that inclusion of jute fiber reduces the maximum dry density and increases the optimum moisture content for each length and diameter of jute fiber.
California Bearing Ratio Test
A series of California Bearing Ratio (CBR) tests were performed on the soil without jute fiber reinforcement and with different proportion of jute fiber based on the modified proctor test results. The test was performed for all the combination of the lengths and diameters of jute fiber and percentage.
Results and Discussions
Results of jute fiber reinforced soil are compared with that of unreinforced soil in the following sections.
Effect of Jute Content on the MDD and OMC
The Proctor tests results obtained from the tests conducted on the soil sample without jute fiber and with different percentage of jute fiber for various aspect ratios are discussed as below: It is observed that inclusion of jute fiber reduces the MDD and increases the OMC.
Similar affects are observed for aspect ratio 1.875 (length = 15 mm, diameter = 8 mm), 7.50 (length = 30 mm, diameter = 4 mm) and 3.75 (length = 30 mm, diameter = 8 mm) (see Figure 3). Table 4 presents the variation of CBR values with respect to aspect ratio (length/diameter ratio) of different percentage of jute fiber. The obtained results of Table 4 are plotted in Figure 4. It is observed from Figure 4 that when aspect ratio decreases the CBR value of soil increases for same percentage of jute content. For instance the CBR value of soil reinforced with jute fiber at 0.3% are 5.76% (aspect ratio = 3.75, length = 15 mm, diameter = 4 mm) and 6.30% (aspect ratio = 1.875, length = 15 mm, diameter = 8 mm). Similarly the CBR values of soil reinforced with fiber contents of 0.6%, 0.9 % and 1.2 %, increases from 7.92%, 8.46% and 9.36% to 8.82%, 9.96% and 10.56% respectively when aspect ratio decreases from 7.50 (length = 30 mm, diameter = 4 mm) to 3.75 (length = 30 mm, diameter = 8 mm).
Conclusions
A series of CBR tests and Modified Proctor tests were conducted in the present study. The laboratory test results are presented and discussed in the sections above. From the above discussion the following conclusions can be drawn: (i) Jute fiber reinforcement reduces the maximum dry density and increases the optimum moisture content of the subgrade soil for each aspect ratio.
(ii) The CBR value of soil increases when the aspect ratio decreases for same percentage of jute fiber. | v3-fos |
2017-06-28T17:11:17.767Z | {
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} | s2 | Peste des petits ruminants in Pakistan; past, present and future perspectives
Peste des petits ruminants (PPR) is considered to be one of the main constraints to enhancing the productivity of goats and sheep in regions where it is present and becoming endemic. PPR was recognized in Pakistan in early 1990s but got importance during the Participatory Disease Surveillance (PDS) of Rinderpest Eradication Campaign. Lot of research work has been initiated during last decade towards disease epidemiology, risk factor recognition, laboratory diagnosis, vaccination and demonstration of control strategies. Although there are ongoing projects working towards the progressive control of the disease in country yet there is need to have a national level control program for PPR. Also there is need to have comprehensive social economic surveys, disease hot spot recognition and identification of role of other species in disease transmission. With combined efforts of local and national authorities and political will, there is high likelihood that this devastating disease can be controlled and eventually eradicated in near future.
Introduction
Peste des petits ruminants (PPR) is an acute and highly contagious viral disease of small ruminants such as goats and sheep. PPR virus (PPRV) is a member of the genus Morbillivirus, and family Paramyxoviridae. Similar to other morbilliviruses, PPR virus is capable of destroying entire populations of immunologically immature/innate hosts by causing epidemics that may spoil the economy of a country and weaken both food security and the livelihoods of farmers. Abubakar et al. [3] have reported dramatic consequences with morbidity of 80-90 % and mortality between 50 and 80 % due to infection of PPR virus in small ruminants. In Pakistan, it causes economic losses of Rs 20.5 billion (US$ 0.24 billion) annually. The main transmission routes of PPR virus are oral and aerosol; the oral, nasal and ocular excretions being the key sources of infection [33].
For many years, PPR was considered as an African disease localized mainly in western and central Africa [62]. More recently, it has become endemic across Sub-Saharan Africa, the Middle East, the Arabian Peninsula, Turkey, Iran, Iraq, Pakistan, India, Bangladesh, Tajikistan and Kazakhstan in Central Asia [83]. The presence of PPR virus has been reported in China [87].
PPR, also known as goat plague, is an important disease in Africa [25,74] and Asia [78], where small ruminants form a considerable portion of livestock population. It mainly affects goats but involvement of sheep is not exceptional. The disease was once thought to be a fairly restricted problem in West Africa, but is now known to exist in most of the West, Central and East Africa, reaching eastwards through Western and South Asia [42]. However, variation in prevalence and severity of PPR outbreaks can be seen due to variations in the sheep and goat husbandry practices within different geographic regions, topography of different areas and other factors.
PPR should be reported to the World Animal Health Organization (OIE) [37]. Due to its rapid spread nature and consequent capacity, it is earlier regarded as List A disease by the Office of International Des Epizooties [70]. In this review, PPR's history, current status and future perspectives in Pakistan are discussed.
Review
History PPR disease was first noticed in Ivory Coast in West Africa during 2 nd World War [46] and was named as pseudo-rinderpest, stomatitis-pneumoenteritis syndrome and pneumoenteritis complex [30]. PPR has been recognized in Pakistan since 1991 when rinderpest like disease in goats was reported in the province of Punjab [24,71]. This report was based on clinical signs and post mortem findings without laboratory confirmation [18]. Three of these documentations were based on laboratory confirmation [49,50,70]; others were based on clinicoepidemiological observations. More recently, other workers have demonstrated the continuing presence of PPRV inflicting substantial economic losses [2,11,49,57,58,90,91].
Hosts range
Goats and sheep are natural host of PPR virus but goats affected more severely than sheep [61]. Sheep rarely suffer clinical disease [41,74] although high morbidity and mortality has been reported but exceptionally is assumed that sheep hold innate resistance to clinical disease [77]. Field outbreaks are reported from a zoological collection in Alain [45].
Transmission
Being an acute and highly contagious viral disease of small ruminants, the transmission of PPRV in healthy animals is matter of dire attention. Transmission PPR virus in healthy animals occurred by direct contact with infected animal and contaminated materials i.e. oculonasal and oral discharges, the loose faeces, hold large amount of the virus. Small infective droplets release into the air from these secretions and excretions, especially when affected animals cough or sneeze [7,10,31,81]. Likewise, movement of animals play a key role in transmission i.e., purchases, nomads, infected migratory animals etc. Apart from these nutritional deficiencies which lead to poor immunity of animal might be a cause of rapid transmission of PPR virus which results in heavy outbreaks.
Disease pattern
Movement of animals is determining factor of disease occurrence. In dry season, animals usually travel long distance in search of fodder and water [65]. In humid areas, PPR always occurred in an epizootic form with 80-90 % morbidity and 50-80 % mortality. PPR is often fatal and usually occur as a subclinical in arid and semi arid areas [61]. Young animals between age of three to four months are more susceptible to PPR virus infection [80] due to decrease in natural immunity (maternal anitibodies) [76]. There is constant circulation of virus between ages of 4 to 24 months [82]. High morbidity and mortality has been reported in all of the age groups [13]. Abubakar et al. [5] reported that prevalence of PPR in small ruminants in Pakistan is 40.98 % and disease is severe in goats mostly. Taylor and Abegunde [82] has recorded a prevalence of 57 % in sheep and 44 % in goats during a field survey in Nigeria. Taylor and Barrett [83] have reported that the disease rate of PPR in sheep appears to be more than that in goats. Singh et al. [79] reported an almost similar prevalence for sheep (36.3 %) and goats (32.4 %) in India while Zahur et al. [92] found a higher prevalence in goats (52.9) than in sheep (37.7 %). So in short a regions discrepancy about disease severity is present across the globe and more is linked to regional environment as well as animal breeds.
Clinical signs
The clinical signs associated with this disease are pyrexia, purulent mucous discharge from the eyes and nose, necrotizing and erosive stomatitis, gastroenteritis, diarrhoea and bronchopneumonia [27].
Clinical examination of affected goats revealed disturbed breathing and cough, muco-purulent discharge from eyes and nose severe diarrhea in young-ones, ulceration on mucous membrane of mouth, fever and depression. The postmortem examination revealed dark red areas (congestion) in different lobes of lungs, small and large intestines [2,4].
Sometimes PPR misdiagnosed as contagious caprine pleuropneumonia (CCPP), contagious ecthyma or pasteurellosis. This is partially due to lack of awareness, as it is a new disease, but also due to lack of diagnostic tools available to the ordinary district laboratories in Pakistan. PPR virus affects goats severely but mild form of disease in sheep while cause subclinical infection in cattle [20].
Abortions
Abubakar et al. [2] has reported that serum samples from the aborted dams found positive for PPR antibodies so the PPR disease has a possible association of mortality and prevalence with high rate of abortions in goat. Moreover, if the animal is infected with PPR virus abortions may occur at any stage of gestation.
Morbidity and mortality
The morbidity rate is 100 % and in severe outbreaks mortality reaches to 100 % [72]. Morbidity and mortality rates vary but may reach up to 100 % [61]. These rates are usually lower in endemic areas (mortality 20 % or less) and sero-surveillance is sometimes the only indicator of the infection [75]. Diallo et al. [38] has reported that in acute cases, mortality varies from 70 to 80 % with death between 10 and 12 days. Moreover the morbidity and mortality rates were higher in sucklers than in adult animals [3].
Seasonal occurrence
Climatic factors affects PPR occurrence. In rainy season outbreaks minimized due to decreased movement of animals as more fodder availability and increase nutritional and health status. In Dec-Feb the dry and dusty season combine with poor nutrition cause disease spread and cases get peak in April. In Pakistan, Khan et al. [57] reported high PPR seroprevalence in December to February and September and October while Abubakar et al. [5] reported the disease frequency greater in January to April and 33 % of cases reported in March. So we may say that the disease occurrence is throughout the year with the severity variation in different weathers.
Temporal and spatial distribution of ppr outbreaks
In Pakistan, during the last decade, PPR outbreaks have increased to an alarming level involving newer areas [17]. As, on the basis of clinical and serological methods, an outbreak of PPR was reported in goat flocks of Livestock Production and Research Institute (LPRI), Bahadurnagar, Okara, Pakistan [15] but amazingly no serological evidence of PPR was found in healthy sheep on same form. According to another reports based on observations from 50 laboratory confirmed outbreaks of PPR and provides details of the presence or otherwise of PPR virus (PPRV) in 427 tissue/organ samples from small ruminants in Pakistan. It was concluded that the disease outbreaks were more severe in goats than sheep and the frequency of disease outbreaks was greater between the months of January to April.
Based on the data of 50 outbreaks (427 samples), Abubakar et al. [3] reported the prevalence of PPR in small ruminants in Pakistan was 40.98 %. A greater number of positive cases were observed in the southern and northern parts of the country (30-60 %) as compared to west and south-west (10-30 %).
The OIE World Animal Health in 2000 also confirmed the outbreak of PPR with IcELISA at a wildlife breeding center of Faisalabad, Punjab, Pakistan. Similarly, in district Chitral, North West Frontier Province (NWFP), Pakistan in June 2006, an outbreak of Peste Des Petites Ruminants (PPR) was investigated in goat flocks. Based on competitive and immuno-capture ELISA, 09 (39.15) animal were positive for PPR antibodies [4]. Apart from these detailed reports, details of some significant outbreaks have been mentioned in Table 1.
Risk factors of ppr for sheep and goats population Age
Young animals are more affected by the disease and morbidity and mortality rates are much higher. The highest sero-prevalence is usually recorded in animals over 2 years of age. Moreover these older animals are more likely to be seropositive for PPR than young ones [92].
Specie
Although it is known as the disease affects both species but its prevalence and severity varies among sheep and goats. Prevalence of PPR seen and reported higher in goats as compared to sheep in many studies in various regions of Paksitan [5,34,40,54,59,92]. Wild ruminants can also be affected with PPR and a significant outbreak has been documented by Abubakar et al. [8] in which the Sindh Ibex was severely affected with PPR.
Sex
Jalees et al. [54] has reported that sheep showed higher seropositivity in ewe than in ram. Moreover, this phenomenon has been supported in village based production system in Pakistan by Khan et al. [55] who reported that male are usually slaughtered at early ages and female sheep and goats are retained.
Season
Although the disease is considered to be endemic in Pakistan yet there are few reports of its seasonal occurrence. In rainy season, PPR incidence decreased due to ample amount of fodder availability lead to increased resistance against disease [5]. Large flock size, animals that visit animal market and inadequate veterinary services are risk factors for PPR disease to occur [92].
Spatial distribution of ppr in various locations of pakistan
The disease pattern, although both goats and sheep are susceptible to infection and may show disease yet they are not always affected simultaneously, for example, in Africa PPR is seen most commonly in goats, while in western and South Asia sheep are usually the most noticeable victims [42]. But if we see the picture in Pakistan, as somewhere else, PPR affect both goats and sheep but in many villages it is seen that only goats are affected usually [84] and this concept is much supported by findings of Abubakar et al. [2].
In different districts of Sindh province, overall PPR seroprevalence in sheep is 49.5 % as compare to goats which is 56.3 %. According to Obi et al. [68], Durojaiye et al. [39] and Abubakar et al. [7], most cases of PPR emerge with the start of summer season and cases get peak during the months of April to July and then the prevalence drop again.
Khan et al. [57] reported the antibody prevalence of PPR virus in small ruminants in Punjab 51.3 %. The antibodies frequency against PPR virus recorded 67.7 %, 71.1 % and 60.2 % in the months of December, January and February and 50.7 % and 53.0 % in the months of September and October, respectively. Less local fodder availability and poor nutritional status of the animals may play a key role in the transmission of disease. Zahur et al. [92] has reported distribution of PPR virus in different districts of Pakistan that is over all 48.30 %. Jalees et al. [54] investigated that disease is more prevalent in young sheep and goats than adult and predilection site of the PPR virus remained the lymph nodes.
Sero-prevalence
The true sero-prevalence of PPR in Pakistan estimated to be 48.5 % (95 % CI, 46.6-50.3), and 52.9 % (95 % CI, 50.7-55.1) and 37.7 (95 % CI, 34.4-41.0) for goats and sheep, respectively. The sheep and goats exhibited a different seroprevalence pattern with a quite higher prevalence in goats. The highest prevalence was recorded in animals over 2 years of age: 49.29 % of sheep and 65.94 % of goats were seropositive for PPR [92].
As per report by Abubakar et al. [5] the specie wise PPR antibody seroprevalence recorded in sheep was 54.9 % as compared to goats 44.15 %. The area wise highest seroprevalence was 55.10 % in sheep and goats of Sindh province. The second highest prevalence ( According to Zahur et al. [92] PPRV is circulating in the small ruminant population throughout Pakistan. It was found that 49.3 % of sheep and 65.9 % of goats were infected by the third year of their life.
Comparison of diagnostic options
In general practices, PPR can be diagnosed from its clinical signs, pathological lesions, and specific detection of virus antigen/antibodies/genome in the clinical samples by different serological tests and molecular assays [27]. World widely, diagnostic tests which are used for the detection of PPRV, including isolation on cell culture, agar gel immunodiffusion (AGID), haemagglutination (HA) tests, immunocapture enzyme-linked immunosorbent assay (IC-ELISA), competitive ELISA, virus neutralization test (VNT) and reverse transcriptase polymerase chain reaction (RT-PCR) [19,44,67] while in Pakistan, diagnosis of said diseases is done by following methods;
Conventional methods and ELISA
At first stage of diagnosis process, following clinical sign and symptom including rapid and labored breathing and cough, muco-purulent discharge from eyes and nose severe diarrhea in young-ones, emaciation, leision in Attock, Texilla, Islamabad Capital Territory 2015 Goat ELISA and PCR [12] mouth, high fever, lassitude, dyspnoea, anorexia and depression has been varified in various studies/outbreak of PPR in sheep, goat and Sindh ibex in Pakistan. There is possible association of abortions to PPR [2]. The postmortem findings assocated with PPR in Pakistan includes dark red areas and congestin in different lobes of lungs with pnemonetic change, small and large intestines, enlargement of spleen and lymph nodes and erosion of abomasums [2,15,56]. A number of serological tests has been practiced in Pakistan for diagnosis of PPR using detecting antigen and antibodies. Among these tests Agar gel immunodiffusion test (AGID), haemagglutination (HA) tests [6,63], modified Counter immuno-electrophoresis, Immunocapture enzyme linked im-munosorbent assay (IcELISA) [3,56], Competitive Enzyme Linked Immunosorbent Assay (cELISA) [56,63,92], single radial haemolysis test (SRH) and countercurrent immunoelectroosmophoresis (CIEOP), Precipitinogen Inhibition Test (PIT) [63].
Abubakar et al. [5] reported that the competitive ELISA has high diagnostic specificity (99.8 %) and sensitivity (90.5 %) for the detection of PPR virus antibody in convalescent sera when compared with the gold standard VNT [20,60,79]. HA test is more sensitive than AGID for detection of PPRV antigen [6]. This result is in concordance with Nussieba et al. [67]. Moreover, the HA test is quick, simple, economical and reliable confirmatory test for the diagnosis of PPR virus.
Munir et al. [63] investigated the comparative efficiency of competitive ELISA (c-ELISA), standard agar gel immunodiffusion (AGID) and precipitinogen inhibition test (PIT) and Aslam et al. [23] investigated comparative efficiency between c-ELISA and AGID for the diagnosis of PPR in Pakistan and concluded that c-ELISA is used as standard since it has the best sensitivity and specificity and can be utilized for samples which are not kept under ideal conditions.
Serological and molecular diagnostic tools
Serological tests based monitoring; it is difficult to determine the level of vaccine failure and thus aggravates disease epidemiology and its control. As a result, determining the nature of circulating strains in different parts of Pakistan is essential to not only help in disease identification and but also to plan better control strategies in future. For this purpose isolation and molecular characterization of PPR virus is crucial step. Moreover, Genetic characterization of PPRV is very important to understand the epidemiology of PPR outbreaks in Pakistan [16]. These efforts also have been done in Pakistan, as a part of control and eradication of said disease.
As serological tests don't essentially point out the existing persistence of the disease, it is essential to execute molecular diagnosis along with characterization [21]. That's way, in recent years, various molecular tools i.e. conventional PCR, real-time PCR [10,16,21,40] has been evaluated for optimal detection of PPR in Pakistan. Genetic analysis of virus proved that lineage IV of PPRV is currently circulating in the country, with certain level of genetic diversity. These Pakistani samples clustered with Chinese, Tajikistani and Iranian isolates [16,21].
Based on phylogenetic analysis PPR virus has categorized into four lineages as I, II, III and IV. Pakistani PPR virus falls in lineage IV closely related to viruses from regions like Middle East, Arabia, south Asia and China [21,64,78,87,88].
Vaccine and vaccination
The PPR considered as endemic diseases of sheep and goat in Pakistan especially in the Punjab province where the population of sheep and goat is higher as compared to other provinces. Different vaccination programs were introduced with the live attenuated Virus belonging to Lineage I. Despite of the strict vaccination programs and other preventive and clinical measures the PPR outbreaks are frequent. Moreover, different type of PPR vaccines including conventional, thermostable, recombinant and edible vaccines has been developed and used from control/eradication of said disease world widely [9,10,12,69].
Currently, vaccination is recommended in certain areas of the country. This vaccination is based on Nig75/1, which belong to lineage II, while field isolates from Pakistan are grouped in lineage IV. Genetic characterization of field strains will provide foundations for construction of vaccines from domestic strains as has recently been practiced in India [21]. An overview of studies based on PPR vaccination has been mentioned below. Rashid et al. (2010) studies the response of locally prepared live attenuated PPR cell culture vaccine in sheep and goats of Pakistan. Vaccine produced high serological titre within 21 days post vaccine and was safe while vaccine titre persisted high for one year postvaccine. Moreover, [56] reported that PPR vaccination during the face of out break showed significant response to control the problem. The findings are useful towards planning appropriate control of the disease in subsistence farming of small ruminants in NWFP. A homologous vaccine has been developed and tested in field trials. The use of this PPR vaccine is strongly recommended to avoid confusion with Rinderpest during serological survey. It is now commercially available. Furthermore, Asim et al. [22] produced and evaluated a live attenuated cell culture vaccine for providing protection against PPR disease to small ruminants (sheep/goat) which are the species most susceptible to PPR virus. No annoying reactions were observed following vaccination. All vaccinated animals developed high titre of antibodies (PI > 50). So this live attenuated PPR cell culture vaccine can be safely used to immunize small ruminants against PPR disease to minimize the huge economical losses.
While, tissue culture based live freeze-dried PPR virus (PPR 75-1) vaccine has been produced by Abbas et al. [1] using Vero cell line and checked for validation, safety, sterility and efficacy. They concluded that this PPR vaccine would be an effective tool to limit PPR disease in goats as well as to reduce economic losses due to this disease in Pakistan.
The efficiency of PPRV vaccines available in Pakistan on the basis of the humoral immune response measured by haemagglutination inhibition (HI) and agar gel immunodiffusion (AGID) tests in sheep (n = 60) and goats (n = 60). Geometric mean titer (GMT) of antibodies against locally manufactured PPRV vaccine was higher (207.9) in comparison with Pestivec (73.3), a vaccine imported from Jordan at 63 rd day post vaccination in sheep; the corresponding values in goats were 147.0 and 48.5, respectively. All animals of control group were negative for antibodies by both of the diagnostic tests. Moreover, it was determined that efficacy of PPR virus vaccines depend on proper storage temperature, pH of buffer and immune response is better in sheep than in goats [51].
Control and prevention
Different control and preventive strategies can be used from PPR in animal. At very first stage separate the infected animals from healthy animal to minimise the chance of transmission of PPR virus from infected animals to healthy animals. Secondly slaughtering of apprent diseases animals and seropositive animals, moreover proper dispose off all infected material and decontamination of items of infected sheep/goat flock is crucial for control/ eradication of PPR. Moreover, vaccination of animals is good option to minimize the risk of occurrence in any healthy animal ppulation. In worldwide different immunization strategies against PPR has been used i.e. earlier immunization of small ruminants was done with lymph node and spleen materials containing virulent virus inactivated with 1.5-5 % chloroform, attenuated tissue culture rinderpest vaccine (TCRV) but now PPR homologous vaccine is available which is prepared by a new freeze-drying process and addition of stabilizing agents [30,35,89].
Economic impact
PPR is considered as one of the major constraints in augmenting the productivity of small ruminants in developing countries and mostly severely affects poor farmer's economy [27,52]. According to the Food and Agriculture Organization (FAO), 62.5 % of global domestic small ruminant's population is at risk of being infected by PPR virus. PPR is a target animal disease for poverty alleviation [43]. In one state of India, Maharashtra, annual losses due to PPR were estimated as Rs. 918 and Rs. 945 millions in sheep and goats, respectively [85].
Abubakar and Munir [11] reported the economic loss due to three outbreaks of PPR in Punjab. Disease caused mortality and morbidity of 10-15 % and 20-40 %, respectively, within a time period of 01-03 weeks. At these three farms, 116 of 365 animals exhibited the clinical disease, with an overall morbidity rate of 31.78 %. A total of 43 animals died with mortality rate of 11.78 % (43/365) causing a direct financial loss of $4300 (Pakistan Rupees 430,000/-), while the indirect cost due to treatment, loss of animal body condition, reduction in market value, increase veterinary services and labour was $7911 (Pak Rs. 791,100/-).
Future perspectives
Currently, although the PPR vaccine production capacity is present in the two places in the country yet there is no organized PPR vaccination campaign going on. With the current population of more than 90 millions of small ruminants and endemic situation of PPR, there is continuous threat from PPR in terms of food security. International authorities working on animal health (OIE and FAO) have recognized PPR as the next target disease for control and possible eradication from the world. So there is need of the time to have national PPR control program in the country. After the successful Rinderpest eradication campaign, OIE has officially declared PPR as next candidate disease, to be eradicated. Therefore serious efforts have been started towards the disease understanding and possible measures for its eradication.
Conclusion
Although there is a project launched by the FAO for the progressive control of PPR in Pakistan yet there is need to have comprehensive national program to combat this menace. This could only be achieved by the combined efforts of local and national authorities as well as political will; along with continuous support and strengthening by international agencies. | v3-fos |
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} | s2 | Evaluating the role of vaccine to combat peste des petits ruminants outbreaks in endemic disease situation
Among the main intimidation to the sheep and goat population, PPR outbreaks are causing huge losses especially in endemic areas. During recent times, six outbreaks of PPR were confirmed at semi-organized goat farms/herds in various regions of Punjab province and Islamabad capital territory (ICT), Pakistan. The disease started after introduction of new animals at these farms with no history of previous PPR vaccination. The clinical signs appeared affecting respiratory and enteric systems and spread quickly. Disease caused mortality of 10-20% and morbidity of 20-40% within a time period of four weeks. Morbidity and mortality rates were 30.38% (86/283) and 15.55% (44/283), respectively. Three treatment regimes were executed to demonstrate the role of vaccination during outbreak at these farms. First was to use only the broad spectrum antibiotics (Penicillin & Streptomycin and/ or Trimethoprim and Sulfadiazine) at two farms (Texilla and Attock). Second treatment regime was to use the same broad spectrum antibiotic along with extensive fluid therapy (Farms at ICT-1 and ICT-2). The third regime was to use of broad spectrum antibiotic plus fluid therapy along with vaccinating the herd against PPR during first week of outbreak (ICT-3 and ICT-4). The third scheme of treatment gave the better results as there was no mortality in third week post-outbreak. Therefore, it is suggested to give proper importance to PPR vaccination along with conventional symptomatic treatment when dealing the PPR outbreaks in endemic disease conditions.
Background
Pakistan at present is having more than 60 million head of goats, which consist of about 37 well-recognized breeds found in different regions of the country. Goats are important animals for humans for providing food in terms of milk and meat. They play main role in supporting millions of people who are poor and living in the rural areas. The main stock occurs in the form of nomadic and transhumant production system but the goat farming for commercial meat production is growing as a successful business.
PPR has caused significant economic loss in many parts of Africa and Asia that contain high densities of small ruminant population. The disease is characterized by sever pyrexia, anorexia, ulcerative necrotic stomatitis, diarrhea due to purulent oculo-nasal discharge and respiratory distress [1] which may be associated with coughing, foul offensive breath, pneumonia and death. Due to respiratory signs, the disease can be confused with contagious caprine pleuropneumonia (CCPP) and pasteurellosis. The disease was first time reported in Pakistan in 1991 [2].
Goats are the main victims of this disease but also involve sheep. The transmission of virus requires close contact between susceptible and infected animals in the febrile stage. The discharge from eyes, nose, mouth and the loose feces contain large amount of virus. Fine infected droplets are released into the air from these secretions and excretions particularly when infected animals cough and sneeze [3].
In Pakistan, during the last few years, PPR outbreaks have increased to an alarming level involving newer areas [4]. Keeping in view all the discussion, this report is designed to highlight the importance of various treatment strategies including vaccination while combating the PPR outbreaks in endemic disease situations to point out the possibilities to save goat farming from this risk.
Outbreaks description
Outbreaks were investigated at six semi-organized commercial farms of goats in Islamabad Capital Territory (ICT) and two nearby areas of Punjab province (Attock, Taxilla) ( Figure 1). The map of area is showing that the outbreaks occurred in close proximity. All the farms were having similar housing conditions with no history of previous PPR vaccination. The disease started after introduction of new animals to these farms. The animals (6-10 months of age) were purchased from different livestock markets with no history of PPRV vaccination.
Study plan for treatment regimes
The study was planned to demonstrate the affects of various treatment strategies especially role of vaccination in the natural outbreak situations. There was three treatment regimes were selected and executed with each treatment regime was carried out at two farms.
First scheme was to use only broad spectrum antibiotics (Penicillin-Streptomycin and/or trimethoprim and sulfadiazine) at two farms (Texilla and Attock). The antibiotics were given intramuscularly daily at recommended dose.
Second treatment regime was to use the broad spectrum antibiotic along with extensive fluid therapy (Oral and intravenous) at two farms (ICT-1 and ICT-2). Some salt and sugar preparation (ORS) were added in drinking water while Ringer-lactate was given intravenously.
The third regime was to use of broad spectrum antibiotic plus fluid therapy as in second group along with vaccinating the herd against PPR (Pestivec R ; Jordon) during first week of outbreak (ICT-3 and ICT-4). The vaccine was administered subcutaneously at dose rate of 1ml in adults and 0.5ml in young. Each treatment was started as soon as the disease was recognized at the farm.
Collection of samples and laboratory confirmation
Nasal and ocular swabs were taken from sick animals while tissues from lungs, liver, spleen, intestines and lymph-nodes were collected from dead animals. Blood serum was also collected from sick animals.
Thirty swabs and sixty serum samples, collected from clinically suspected animals, were tested for PPRV confirmation by using Immuno-capture enzyme linked immune-sorbant assay (IcELISA) and Competitive ELISA (cELISA), respectively. Representative samples of dead animals from each infected herds (4 from each herd) were also taken and tested through Ic ELISA and RT-PCR. In group-wise comparison, five swabs and ten serum samples were collected in each herd while all morbid animals were sampled for the confirmation of disease.
Tests used for the confirmation of outbreaks PPR antigen detection was performed using (Ic-ELISA) kit imported from World Reference Laboratory (Pirbright, UK). As recommended by the kit manufacturer, the final absorbance was measured at a wavelength of 492 nm [5].
The presence of viral nucleic acid from tissue samples was confirmed by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Total cellular RNA was extracted using the Qiagen-RNAeasy kit as per the manufacturer's instructions. RT-PCR was performed for the F-gene of PPRV using one step RT-PCR kit (Invitrogen) as per the manufacturer's instructions. PCR was carried out using PCR primers and conditions as described previously [6,7].
PPR antibodies were detected using cELISA kit (collectively produced by Biological Diagnostic Supplies Ltd, Flow Laboratories and The Pirbright Institute (formerly Institute for Animal Health). The OD values were converted to percentage inhibition and the samples with PI >50% were considered as positive [8].
Results
Disease started with sudden onset of fever, respiratory and enteric clinical signs and spread quickly. At three farms, 86 out of 283 animals exhibited the clinical disease, giving morbidity rate of 30.38%. A total of 44 animals died with mortality rate of 15.55% (44/283) ( Table 1).
Among the samples tested, swab and tissue samples were positive with both Ic-ELISA and RT-PCR while all the serum samples were found positive for PPR antibodies (Table 2).
There was a variation of mortality pattern in groups under different treatment regimes. In group I, the mortality continued up-to fourth week of clinical outbreak while the clinical disease sustained up to sixth week of its onset. In group II, the mortality stopped in third week and still the clinical disease continued up to fourth week post clinical disease.
In group-III, the mortality stopped in second week while the clinical disease continued up to fourth week but its symptoms were mild (Table 3). Mortality rate on first week of the outbreak was similarly high in all groups but it dropped appreciably in second week in group 2 and 3. It further dropped in group-2 in third week but there was no mortality in group 3 while the mortality continued in group-1 till fourth week.
Discussion
The study presents an important scenario for PPR disease and its treatment in endemic disease situation in Pakistan. Due to lack of awareness of disease and no organized vaccination program, the disease has become endemic in Pakistan [9][10][11]. In the past PPR outbreaks were diagnosed only on the basis of clinical signs because of laborious laboratory procedures, cost [12][13][14]. The implementation of cELISA aided in tracking the outbreaks of PPR disease in different geographical regions, measuring economic losses from the disease, epidemiology of the disease in different population of animals [1,15]. The mortality in PPR goats can be up to 100% in severe infections, but during milder outbreaks less than 50% mortality may be seen [16]. However, published information on the survivability of goats diagnosed with clinical PPR, under different antimicrobial therapies are sparse, if not absent. The results of the present study revealed that survivability in different treatment groups varies (Table 3). These treatment strategies are usually applied to combat PPR outbreaks in the field. Narayanan et al. [17] treated clinical cases of PPR were treated with broad-spectrum antibiotics like enrofloxcin, @5mg per kg body weight. Intestinal astringents like creta and kaolin were administered. Intravenous fluids like dextrose normal saline (10ml/ kg body weight), was administered for the treatment of diarrhea and restoration of body fluid ionic balance for seven days as described by Wosu [18] and Abubakar and Irfan [19]. In contrast, we used above strategies along with use of PPR vaccine which proved the best in combating the outbreaks.
Virus induced immune-suppression attributable to leucopenia predisposes secondary bacterial infections, where bronchopneumonia is the most frequently observed bacterial complication in peste des petits ruminants virus (PPRV)-infected animals [16,20,21]. Common secondary infections include Pasteurella species [21]. The disease is highly endemic in South Asia, caused by lineage 4 of PPRV unlike Sub-Saharan countries where circulation of lineages 1-3 are reported [2]. Because the outcome of PPRV infections can be linked to secondary bacterial infections, it is important to treat them appropriately, subsequently an increased survivability might be achieved.
A possible explanation for outbreaks could be the excretion of PPR viral antigen in some body secretion. Abubakar et al. [22] explained the possible excretion of PPRV antigen in fecal material in which they described a possible mechanism of virus transmission following natural infection. This idea may demonstrate a potential method by which PPRV outbreaks occur spontaneously in areas not previously known to have circulating virus. Shedding of PPRV antigen in the fecal material of the recovered goats following a disease incursion put forward the possibility that goats may be shedding the PPR virus in their fecal matter. These findings are also in agreement with Ezeibe et al. [23] and may reinforce the idea that virus can subclinically infect animals and excrete and/or transmit virus to naïve 'in contact' animals.
Conclusion
The main reason for these outbreaks could be the endemic nature of PPR disease in Pakistan and as there is no organized program for its vaccination. Therefore, it is recommended that proper importance should be given to treatment regimes as mentioned above as well as vaccination during the face of a PPR outbreak. | v3-fos |
2017-04-26T03:13:08.977Z | {
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} | 0 | [] | 2015-05-28T00:00:00.000Z | 12077292 | {
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} | s2 | Utility of Acute Phase Proteins as Biomarkers of Transport Stress in Ewes and Beef Cattle
The effect of transport on serum amyloid A (SAA), haptoglobin (Hp), Fibrinogen and white blood cells (WBC) was evaluated in 10 ewes and 10 beef cattle. All animals were transported by road for 6 h over a distance of about 490 km with an average speed of 80 km/h. Blood samples, collected via jugular venepuncture, were obtained before and after transport as well as after 12, 24 and 48 h rest time. One-way repeated measures analysis of variance showed a statistically significant effect of sampling time on SAA, Hp, and WBC in ewes and beef cattle. Based on these results, Hp and SAA levels, together with WBC, may be useful indicators of animal health and welfare and in predicting the risk assessment in meat inspection.
Introduction
The transport is an inevitable husbandry practice that animals unexpectedly encounter in the livestock industry and can have implications for their welfare. In fact, transported animals are exposed to a variety of physical and psychological stimuli that disrupt their homeostasis and metabolism. Road transport, considered as one of the main causes of stress, may be more or less severe and affect a large number of systems. The effects of transport stress on animal health and welfare have been evaluated through behavioural, physiological and haematological variables (Adenkola and Ayo, 2010;Broom, 2003Broom, , 2008, mobilisation of energy and protein metabolism (Todd et al., 2000), activity of enzymes and hormones (Adenkola and Ayo, 2010;Stull and Rodiek, 2000), and the changes of immune system (Early and O'Riordan, 2006). There is great scientific interest aimed at ensuring the welfare of transported animals and identifying easily obtainable biomarkers in relation to transport stress. In fact, stress experienced by farm animals during the transport may influ-ence the acute phase proteins (APPs) in beef cattle and ewes (Giannetto et al., 2011;Piccione et al., 2012) and also cause economic losses due to decreased carcass and meat quality (Teke et al., 2014). In the case of increased physiological stress or physical activity during the transport, muscle glycogen reserves may be used before slaughter. This can lead to higher ultimate meat pH, darker meat colour, tougher meat and greater water holding capacity (Gregory, 1998). Acute phase proteins are a group of blood proteins linked to stress because their concentrations decrease (negative APPs) or increase (positive APPs) in response to external or internal challenges (Gonzàlez et al., 2008;Petersen et al., 2004;Ceron et al., 2005;Eckersall and Bell, 2010). In particular, serum amyloid A (SAA), haptoglobin (Hp) and fibrinogen (Fbg) in health monitoring programmes in livestock are useful for the identification of diseases or subclinical diseases. After considering that the linkage among animal health, welfare and APPs becomes more and more important, the aim of this study was to evaluate the modifications of serum concentrations of Hp, SAA and Fbg, together with white blood cell (WBC), in order to identify the impact of transport on biomarkers. This will be increasingly useful to reduce transport stress that influences health, welfare and final quality of the meat of of ewes and beef cattle.
Materials and Methods
The study was carried out on 10 ewes and 10 beef cattle. Alla animals were clinically healthy. They were transported by road for 6 h over a distance of about 490 km with an average speed of 80 km/h, involving a combination of road surfaces ranging from small country lanes (10 km) through secondary roads (60 km) to motorways (420 km). All animals had no previous experience of road transport. The journey started at 08:00 a.m. and lasted 6 h. Particularly, transport took place during spring, with an outside temperature of 18-20°C and 50-60% relative humidity. After road transport the animals were confined to paddock where environmental temperature was between 18 and 23°C, and relative humidity was 50-65%. After the transport the animals were fed hay (2 kg), wheat straw (1 kg) and wheat concentrate (0.5 kg). Water was available ad libitum. All animals were transported in accordance with Directive 1/2005 CEE (European Commission, 2005).
Blood samples were collected from each animal by jugular venipuncture into evacuated glass tubes (Venoject; Terumo Europe, Leuven, Belgium) before and after the transport as well as after 12, 24 and 48 h rest time to determine the APPs. Each animal was sampled by experienced and skilled operators in less than 1 min to minimise handling stress affecting the results. The SAA concentration was performed with an enzyme-linked immunosorbent assay using ELISA kits (Tridelta Development, Maynooth, Ireland). The concentration of Hp was assessed using commercial colorimetric kits (Tridelta Development) in microplates, based on Hp-haemoglobin binding and preservation of the peroxidase activity of the bound haemoglobin at low pH. The reading of absorbancies and the consecutive calculation of final concentrations of both APPs were performed on automatic microplate reader Opsys MR (Dynex Technologies, Denkendorf, Germany).
The concentration of Fbg was assessed on blood samples containing citrated sodium, after centrifugation, using a coagulometer (Clot 2S; SEAC, Florence, Italy). The WBC count was assessed on blood samples containing ethylenediaminetetraacetic acid using a multiparametric automatic analyser (HecoVet; SEAC).
One-way repeated measure analysis of variance (ANOVA), followed Bonferroni's multiple post-hoc comparison, was performed to determine the significant effect of sampling time in ewes and beef cattle. The level of significance was set at <0.05. Data were analysed using the software STATISTICA 8 (Stat Soft Inc.). beef cattle. Tables 1 and 2 show average values of all studied parameters, expressed in conventional units of measurement with standard deviations and statistical significances, measured during the experimental period in ewes and beef cattle.
Discussion
All data obtained before transport were within the physiological range referred to in the literature for ewes and beef cattle (Eckersall and Bell, 2010;Ganheim et al., 2003;Jain et al., 2011). The results of this study confirmed that a linkage between stress and APP response exists. Particularly, SAA and Hp increased both in sheep and beef cattle during the rest time relative to changes from pre-transportation values. In sheep SAA increased significantly after 24 and 48 h of road transport, while Hp increased significantly after 48 h of road transport only. In beef cattle the results showed a statistically significant increase of Hp 24 h after transport and of SAA compared to the previous data points, and 48 h after transport. Both parameters reached statistically significant higher values. As previously demonstrated, the SAA concentration tends to increase rapidly, whereas Hp concentration increases at a slower rate during the acute phase response (Colditz et al., 2005;Eckersall et al., 2007), which is consistent with the findings of the present study. It has also been reported that the stimuli can induce different APPs response which may account for the different SAA concentration compared to the hp concentration observed (Lepherd et al., 2011). Lomborg et al. (2008) demonstrated marked SAA responses in healthy adult cattle after exposure to complex stressors as road transport. In fact, these results are in agreement with studies of other researchers who emphasised the role of APPs not only with inflammation but also with some conditions such as road transport which can be highly stressful and compromise welfare (Petersen et al., 2004). When ewes and beef cattle are stressed, there is a rapid relase of cathecolamines which results in glycogen depletion causing a lower rate of post-mortem lactic acid synthesis and hight ultimate pH, undesirable colour, making such dry beef (Marenčić et al., 2012). The APPs assay may have a potential role for monitoring adverse environmental and/or management stressors, thus enabling better animal welfare practice (Piñeiro et al., 2007;Murata, 2007). Consequently, the APPs represent important biomarkers of stress during the road transport of ewes and beef cattle. This is interesting not only to monitor the health status and welfare of transported animals but also to improve meat quality characteristics of ewes and beef cattle.
Conclusions
Modern veterinay medicine is increasingly focusing on prevention rather than cure and these biomarkers are important factors for the animal's environment and welfare. In particular, the results of this study suggest that SAA and Hp levels, together with WBC, may be useful indicators of animal health and welfare and good predictors of risk assessment in meat inspection. | v3-fos |
2018-04-03T03:38:16.651Z | {
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} | 0 | [] | 2015-12-15T00:00:00.000Z | 7560562 | {
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} | s2 | Active aggregation among sexes in bean flower thrips (Megalurothrips sjostedti) on cowpea (Vigna unguiculata)
Male sexual aggregations are a common territorial, mating-related or resource-based, behaviour observed in diverse organisms, including insects such as thrips. The influence of factors such as plant substrate, time of day, and geographic location on aggregation of thrips is uncertain, therefore we monitored the dispersion of male and female bean flower thrips (BFT), Megalurothrips sjostedti (Trybom) (Thysanoptera: Thripidae), on cowpea, Vigna unguiculata (L.) Walp. (Fabaceae), over three cowpea growth stages and across three cowpea-growing areas of Kenya. Our results indicated that for all the crop growth stages, the density of BFTs varied over the time of day, with higher densities at 10:00, 13:00, and 16:00 hours than at 07:00 hours. Thrips densities did not differ among blocks at the budding stage, but they did at peak flowering and podding stages. Dispersion indices suggested that both male and female BFTs were aggregated. Active male aggregation occurred only on green plant parts and it varied across blocks, crop stages, and locations. Similarly, active female aggregation was observed in peak flowering and podding stages. Such active aggregation indicates a semiochemical or behaviour-mediated aggregation. Identification of such a semiochemical may offer new opportunities for refining monitoring and management strategies for BFT on cowpea, the most important grain legume in sub-Saharan Africa.
Ecological studies on population dynamics over seasons have revealed that the BFT numbers greatly increase during specific crop growth stages, especially at flowering and podding (Ezueh, 1981;Salifu & Hodgson, 1987;Nyasani et al., 2013). However, prior to this study, in field surveys for thrips on grain legume crops such as dolichos, we noted that BFT males were highly aggregated. Many dolichos plants recorded very few or no males, whereas in very few dolichos plants high numbers of BFT males (sometimes up to 15 per plant) were observed (S. Subramanian, unpubl.). Based on this observation, we investigated the aggregation pattern among sexes of BFT on cowpea and assessed whether aggregation behaviour is active or passive and whether it is influenced by location, time of the day, crop growth stage, or plant part.
Materials and methods
Monitoring of male and female BFT densities on cowpea Variation in male and female BFT densities in relation to time of day and crop growth stage was studied on a cowpea farm at the International Centre of Insect Physiology and Ecology (icipe) headquarters in Nairobi, Kenya.
Cowpea (var. Kakamega) was planted, after the land was ploughed, harrowed, and ridged. Planting was completed in March 2013 corresponding to the long rains season in Kenya. The farm (40 9 45 m) was divided into four planting date blocks (10 9 10 m) separated by a corridor of 0.5 m. In each block, planting was undertaken successively at 2-week intervals between May and June 2013 with intra-and inter-row spacing of 0.25 and 0.45 m, respectively. Standard agronomic practices were adopted (Dugje et al., 2009). Observations in each block with different planting dates were undertaken at three crop growth stages: budding (BD) (5 weeks after planting when mostly unopened buds and few first flowers were seen), peak flowering (PF) (8 weeks after planting when more than 90% of plants were flowering), and podding stage (PS) (10 weeks after planting when most of the plants had young pods with a few flowers). Within each crop growth stage, observations were taken on the number of male and female BFT for four consecutive sampling days. On each sampling date, 25 randomly selected cowpea plants were sampled at 3-h intervals, from 07:00 to 16:00 hours using the whole-plant tapping method. This involved tapping the plants gently for 59 on white enamel tray (25 9 45 cm) kept right below. The number of thrips which fell on the tray were quickly estimated (Pearsall & Myers, 2000). Further samples of 10-25 flower buds based on the availability were randomly collected from different cowpea plants separated by a minimal distance of 2 m and placed in separate glass vials containing 70% ethanol for further observation of thrips in the laboratory. The sex of adult BFT emerging from flowers was identified and the total number of thrips per flower was recorded.
Statistical analysis
Analyses were performed using R v. 3.1.0 (R Development Core Team, 2014) using R Commander v. 1.6-3 (Fox et al., 2009) and Agricolae v. 1.1-4 packages (De-Mendiburu, 2013). To study the predictive factors for the number of BFTs, a negative binomial regression model was fitted because we had overdispersed count data as suggested by O'hara & Kotze (2010). The likelihood ratio (LR) test strongly suggested that the negative binomial model was more appropriate than the Poisson model (LR test: P<0.0001). To start with, a full model including sex, block, stage, their interaction terms, and time of the day was fitted. The dispersion parameter for this model was 1.154 indicating that the data were overdispersed, meaning that the variability encountered in the data is not equal to the mean, as with the Poisson distribution. The negative binomial model was further fitted for each stage separately.
To study the thrips dispersion on plants and flowers, two indices were calculated: (1) Lloyd's index of patchiness (LIP) (Lloyd, 1967) where s 2 represents sample variance and x is the sample mean; and (2) variance-to-mean ratio (VMR). If LIP>1, the population is considered aggregated and as LIP increases, the degree of aggregation also increases. If LIP = 1, then distribution is considered random. If LIP<1, the distribution is considered regular. To test whether the distribution was significantly different from that of a random population, a v 2 test was performed for VMR using the expression with (N À 1) degrees of freedom, where N is the number of observations (Hurlbert, 1990). In situations with aggregation, the cause of aggregation was determined (i.e., whether it was activebehaviour-mediatedaggregation or passive, influenced more by environmental factors). In this regard, the criteria outlined by Arbous & Kerrick (1951) were used to estimate the mean 'aggregation' size k (Southwood & Henderson, 2000): where k is the mean number of individuals in the aggregation for the probability level allocated to υ, x is the sample mean, k is the dispersion parameter obtained from the negative binomial model, and υ is a function with v 2 distribution with 2k degrees of freedom. In interpreting k, we note that k<2 indicates that aggregation is more due to an environmental effect and not to active behaviour of the insect, whereas k>2 is an indication that the cause of aggregation is due to either factor, but especially active processes by the thrips (Salifu & Hodgson, 1987;Verghese et al., 1988). Field data collected in Matuu and Mbita were subjected to similar analyses. We used v 2 tests to differentiate the proportion of male and female BFTs at each time of the day, block, and plant stage. All tests were performed with a = 0.05.
Density of male and female BFT on cowpea plants
The mean number of male and female BFT by crop stages and time of the day are summarized in Figure 1A. As the sex-by-block-by-stage interaction term was significant in the full/initial model (LR test: P<0.0001), a negative binomial model was fit for each stage separately with time, sex, block, and sex-by-block in the model (Table 1). The results indicated that for all the stages, the sex-by-block interaction term was significant (P<0.0001), implying the densities of male and female BFT varied across the blocks (Figure 2A). After controlling for sex and blocks, time of the day was significantly associated with the density of BFT at all the plant stages and significantly more insects were collected at 10:00, 13:00, and 16:00 hours than at 07:00 hours ( Figure 2B). Also, after controlling for time and block, it was four times more likely to get a male than a female BFT at the budding stage. At the flowering and podding stages, it was significantly less likely to get male thrips as compared to females ( Figure 2C).
Density of BFT on cowpea flowers
Negative binomial model results indicated that the difference in female densities per flower was not significant across block or time of day, whereas it differed across crop stages: female density per flower was higher at peak flowering (mean AE SE = 0.3 AE 0.1; Z = 2.3, P = 0.018) and podding (3.0 AE 0.4; Z = 2.7, P = 0.006) than at budding (0.1 AE 0.0). At all observation times, males were mostly absent in flowers over the crop growth stages ( Figure 1B).
Aggregated dispersion of BFT on cowpea plants and flowers
Overall LIPs for male and female BFT were 10.9 and 2.8, respectively, and the respective VMR were 8.26 and 3.32. VMRs for males and females were significantly greater than 1 (P<0.0001; Table 2). Among males, the LIP values were >1 except for blocks 2 and 4 in the budding stage and block 4 in the peak flowering (Table 2). Similarly the VMR were also >1 except for blocks 2 and 4 in the budding stage and block 4 in peak flowering stage. The VMR for females were also significantly >1, except for blocks 1 and 3 at the budding stage. In general, LIPs for males were higher than for females (Table 2). On cowpea flowers, the LIP for female BFTs was >1 at the peak flowering and podding stages (Table 3). The analysis of the cause of male aggregation indicated that k was >2 in block 1 at the budding stage and in all blocks at podding. At flowering, k was <2 in all blocks. In female BFT, k was >2 at peak flowering and podding. In flowers, k was >2 only at podding (Tables 2 and 3).
Dispersion indices indicated that in all the locations, both male and female LIPs were significantly higher than 1 (Table 4). Male LIPs were always higher than female LIPs. In the field, the values of k for both male and female BFT were >2 in Mbita and Nairobi only. As earlier noted in Nairobi, although males were occasionally spotted on green plant parts, they were mostly seen on the leaves ( Figure 3A); flowers of cowpea were mostly populated with female BFT ( Figure 3B).
Discussion
Previous studies of M. sjostedti have revealed that at higher densities the adults are highly aggregated (Salifu & Hodgson, 1987), which is suggestive of aggregation behaviour. However, beyond this indication and to the best of our knowledge, no detailed studies on aggregation in sexes of BFT or on factors associated with such aggregated dispersion are available in the literature. Female BFT occur in cowpea flowers in low numbers during the budding stage, but increased with the crop growth stage. Several authors have observed such increase in BFT population from the initial infestation at the budding stage (macroscopic flower buds to anthesis) to full infestation at later flowering and podding phases (Ezueh, 1981;Salifu & Hodgson, 1987;Salifu & Singh, 1987). Recently, Nyasani et al. (2013) also observed that BFT population on French bean, Phaseolus vulgaris L., was significantly lower before flowering than after. Our observation concurs with their findings and provides additional information with regard to sex-specific differences in infestation of cowpea growth stages.
We also found out that both male and female BFT infested the green plant parts of the cowpea crop, whereas flowers were only infested by females. The density of BFT on plants differed across blocks, crop growth stage, and time of day. Although both sexes presented aggregation patterns, males were more aggregated than females and aggregations were mainly observed on the green plant parts, especially the leaves. Similar aggregation of males on terminal leaves were also observed in Pezothrips kellyanus Bagnall (Mound & Jackman, 1998;Mound, 2004;Navarro-Campos, 2013). However, previous reports on male aggregation of other thrips species indicated that it takes place mainly on the corolla of flowers (Kirk, 1985;Terry & Dyreson, 1996). Frequent mating behaviour by P. kellyanus on ripe citrus fruits has been reported to occur during the late afternoon around 17:00 hours (Webster et al., 2006).
Generally, females were observed in flowers and their numbers significantly increased from budding to podding stages. This could be for feeding or oviposition, as reported in BFT and other thrips (Childers & Achor, 1995;Gahukar, 2004). In addition to higher LIP values observed in both sexes, the mean 'aggregation' size k values were >2, indicating active aggregation of both male and female, as suggested by Salifu & Hodgson (1987) and Verghese et al. (1988). Our results also reveal that factors such as block, crop growth stage, and location could influence BFT aggregation behaviour. In many thrips species, similar aggregations have been ascribed to the possibility of a semiochemical-mediated interaction between the sexes (Mound & Jackman, 1998;Milne et al., 2002). Aggregation pheromones responsible for such behaviour in thrips species such as Frankliniella occidentalis (Pergande) (Kirk & Hamilton, 2004;Hamilton et al., 2005), Frankliniella intonsa (Trybom) (Zhang et al., 2011), and Thrips palmi Karny (Akella et al., 2014) have been identified. The active aggregation observed in this study and recent report on the presence of male sternal glands associated with pheromone production in BFT (Krueger et al., 2015), underlines the need to identify possible pheromones associated with BFT aggregations. Sexual communication in a diverse array of phytophagous insects is strongly influenced by host plant chemistry (Landolt & Phillips, 1997). The increase in BFT populations as determined by cowpea growth stage, and the aggregation of males and females in leaves and flowers suggest interactions between host plant cues and pheromones which need to be investigated. The densities of adult BFT varied with time of day with more insects collected at 10:00, 13:00, and 16:00 hours than at 07:00 hours. This variation could be attributed to variation in temperature and time of the day, which also affect flower opening and closing in the host plant (Ekesi et al., 1999;Ige et al., 2011).
Among the three cowpea-growing areas, higher numbers of BFT, especially males with higher aggregation indices, were observed at Mbita than at Nairobi or Matuu. This difference could be ascribed to the lower altitude in Mbita, with higher temperature and higher humidity (Murage et al., 2012), which are among the preferred growth conditions for BFT and its host (Bottenberg et al., 1997;Ekesi et al., 1999).
Aggregation of sexes in BFT may be influenced by factors including crop growth stage, time of day, and biogeographical parameters. However, further research should focus on defining factors associated with this aggregation, such as mating/defence behaviour (Terry & Dyreson, 1996), role of male aggregation pheromones (Milne et al., 2002;Hamilton et al., 2005), and their interaction with plant growth stages (Sampson & Kirk, 2013). Defining the factors associated with the aggregation and identification of such aggregation pheromones may aid in refining monitoring strategies for BFT and in appropriate timing of management interventions. | v3-fos |
2016-05-14T13:18:25.360Z | {
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} | s2 | Thermochemical Pretreatments of Organic Fraction of Municipal Solid Waste from a Mechanical-Biological Treatment Plant
The organic fraction of municipal solid waste (OFMSW) usually contains high lignocellulosic and fatty fractions. These fractions are well-known to be a hard biodegradable substrate for biological treatments and its presence involves limitations on the performance of anaerobic processes. To avoid this, thermochemical pretreatments have been applied on the OFMSW coming from a full-scale mechanical-biological treatment (MBT) plant, in order to pre-hydrolyze the waste and improve the organic matter solubilisation. To study the solubilisation yield, the increments of soluble organic matter have been measured in terms of dissolved organic carbon (DOC), soluble chemical oxygen demand (sCOD), total volatile fatty acids (TVFA) and acidogenic substrate as carbon (ASC). The process variables analyzed were temperature, pressure and NaOH dosage. The levels of work for each variable were three: 160–180–200 °C, 3.5–5.0–6.5 bar and 2–3–4 g NaOH/L. In addition, the pretreatment time was also modified among 15 and 120 min. The best conditions for organic matter solubilisation were 160 °C, 3 g NaOH/L, 6.5 bar and 30 min, with yields in terms of DOC, sCOD, TVFA and ASC of 176%, 123%, 119% and 178% respectively. Thus, predictably the application of this pretreatment in these optimum conditions could improve the H2 production during the subsequent Dark Fermentation process.
Introduction
Anaerobic digestion is a widely applied technology for the treatment of organic wastes. This process is mainly developed through four main stages: hydrolysis, acidogenesis, acetogenesis and methanogenesis. Generally, hydrolysis is considered the rate-limiting step of anaerobic digestion of solid wastes [1]. Hence, the hydrolysis stage is decisive for the OFMSW biodegradation and it determines the overall rate of the process. Furthermore, if the hydrolysis stage is faster, the acidogenesis step (VFA and rich-H2 biogas) starts-up earlier and, hence, the global rate of the anaerobic digestion is higher [2]. It must be noted that nowadays, the H2 is considered the energetic vector of the future and, therefore, the increase of its generation in the acidogenic step by means of pretreatment may be an interesting target. To reach this goal, thermal, chemical (or combination of them) or biological pretreatments have been commonly developed in the literature as possible processes to accelerate the organic matter solubilisation process from organic wastes, as the sludge coming from wastewater treatments plants (WWTP), or complex wastes with small particle size [3][4][5][6]. The organic matter solubilisation is referred to the process in which the non-solubilized organic matter (organic matter contained inside particles for example) is solubilized to the liquid phase through the pretreatments discussed above.
About the thermal pretreatments, it must be highlighted that they have been successfully scaled to industrial scale [7]. Furthermore, the application of high temperatures allows the removal of pathogenic microorganisms and improves the properties of the digestate as its dehydration due to a decrease in its viscosity [8]. Most of the scientific references are focused on improving the performance of anaerobic digestion of solid waste, such as sludge from WWTP and lignocellulosic waste, by applying thermal pretreatments using temperatures between 50 and 250 °C. In this regard, it should be noted that very little research has been developed to improve the anaerobic digestion of OFMSW.
Analogously, most of the studies developed with chemical pretreatments were applied to sewage sludge and lignocellulosic waste [9]. Among the limited information regarding the application of chemical pretreatments to OFMSW are the results obtained by Lopez-Torres and Llorens. These authors reported improvements of 11.5% in the methane productivity by applying alkaline agents [10]. Furthermore, Neves et al. [11] reported improvements of 100% by applying NaOH as alkaline agent in concentrations of 0.3 g/gTS pretreating barley waste, and Patil et al. [12] reported the implementation of this type of pretreatment on waste such as water hyacinth with low lignocellulose content. The results of their study showed that, if the waste has low lignin content, this type of pretreatment (acid or alkaline) has little effect compared to mechanical pretreatments.
Finally, with respect to biological pretreatments, these may be developed in aerobic or anaerobic conditions and through the application of specific enzymes (such as peptidase, lipase and carbohydrolase) to the anaerobic reactors to pre-hydrolyze the waste. Similarly to the thermal and chemical pretreatments, biological pretreatments have been little studied to improve the anaerobic digestion of OFMSW. Most published works are focused on sewage sludge from WWTP and pulp and paper industries. It has been reported that the application of precomposting as aerobic biological pretreatment improves the hydrolysis of the hardly biodegradable solid waste due to the high concentration of hydrolytic enzymes that are provided by the biological agent, mature compost in this case [13].
However, the literature about the applications of pretreatments on organic wastes processed in full-scale plants and with high particle size, as the OFMSW coming from a full-scale mechanical-biological treatment (MBT) plant used in this study, is very limited. In fact, few references have been found about this topic [1,[14][15][16] in which the best organic matter solubilisation was achieved by means of thermochemical pretreatments at 180 °C, 5 bar and 3 g NaOH/L as alkaline agent.
About the NaOH dosage, Kim et al. [17] tested different alkaline agents and the best solubilisation yield (SY), 85% expressed in terms of COD, was obtained applying a dose of 7 g NaOH/L. On the other hand, Penaud et al. [18] reported 5 g NaOH/L as the optimum concentration to reach the maximum organic matter solubilisation.
In the above mentioned papers [1,[14][15][16], the range of the operational conditions tested must be considered as preliminary since there was not specific literature about the applications of pretreatments on OMFSW coming from a full-scale MBT plant. Therefore and based on the previous information, the main goals of this paper are three: (1) To determine with more accuracy, (while reducing the operational variables ranges), the best conditions for improving the organic matter solubilisation according to preliminary results obtained by Fdez-Güelfo et al. [1] in order to provide further detailed information that will complement the conclusions obtained by these authors. [25], the "acidogenic substrate as carbon (ASC)", in order to evaluate the possible effect of the pretreatments on the H2 production during the acidogenic phase of a sequential anaerobic process.
Results and Discussions
In order to determine the best conditions to develop the thermochemical pretreatments among those tested in this study, it is necessary to analyze the effect of each variable (temperature, pressure, NaOH dosage and operation time) individually taking into account the SY calculated according to Equation (1) (see Experimental section below).
Optimizing the Operational Variables
With regard to the temperature, according to the SY presented in Figure 1, it can be stated that the most successful thermochemical pretreatment has been generally performed at the lower temperature among those tested, 160 °C.
This result is in accordance with the results obtained by Delgenés et al. [26] who reported that temperatures ranging between 90 and 160 °C cause an increase in the organic matter solubilisation. For temperatures higher than 200 °C, the SY is considerably lower. It seems to be linked to the maintainence of high temperature during long operation periods, which may induce losses by thermal decomposition, polymerization or even caramelization processes of an important fraction of organic matter solubilized to the liquid phase and, therefore, the final increments of the pretreatment on the organic matter solubilisation could decrease.
Fdez-Güelfo et al. [1] reported that the most efficient temperature was 180 °C when the temperatures tested were 120, 150 and 180 °C but it is was not possible to fence the optimum. In this sense, the present work has increased the accuracy of the results reported by these authors, since the optimum temperature, in which SY measured as DOC, sCOD, TVFA and ASC is higher than 100%, has been found at 160 °C and therefore it could be restricted between 150 and 180 °C.
In regard to the influence of the NaOH additions, as it can be seen in Figure 2, at a temperature of 160 °C the optimum NaOH concentration is generally the intermediate between 2 and 4 g/L (3 g/L) for the different pressures tested, i.e., the higher NaOH concentration is not required to reach the maximum SY. In this sense, it is very important to highlight that the best SY has been obtained at lower temperature (160 °C). The above results may be associated to the fact that the hydrolytic capacity of the NaOH decreases when the operational temperature is very high. In fact, several authors have observed a decrease in this synergic effect between NaOH and temperatures over 180 °C [26]. This fact may be due to high temperature enhancing the formation of refractory compounds which are difficult to solubilize. In addition, at these conditions some intermolecular reactions occur between solubilized and non-solubilized compounds that lead to the formation of complex substances.
In addition, this result is in accordance with the conclusion obtained by Fdez-Güelfo et al. [1]. These authors reported that the most efficient NaOH concentration was 3 g/L when the tested NaOH concentrations were 1, 3 and 5 g/L. A narrower range (2-4 g/L) for the optimum had been established in this study.
With respect to the influence of the pressure, according to Figure 3, at 160 °C and 3 g NaOH/L, the best SY among those tested was obtained for 6.5 bar of pressure. If this result is compared with the conclusions reported by Fdez-Güelfo et al. [1], which found that the most efficient pressure was 5 bar when the tested pressures were 1, 5 and 10 bar, this study indicates that the optimum pressure must be placed between 5 and 10 bar. Again, this work has increased the accuracy of the results reported by these authors, but further studies must be faced to check if the optimum is higher or lower than 6.5 bar. Thus, as it can be seen in Figure 1, high pressures appear to have positive effects on the SY when the NaOH additions are low (2 to 3 g/L) while it is negative when the NaOH additions are higher (4 g/L). Finally, with regard to the influence of the operation time on the SY, after selecting the optimum conditions (160 °C, 6.5 bar and 3 g NaOH/L), three additional operation times (15,60 and 120 min) were tested in order to determine the effect of this variable on the SY. The results obtained are shown in Table 1. Table 1. Solubilisation yield (SY), expressed in terms of DOC, sCOD, TVFA and ASC, at 160 °C, 6.5 bar and 3 g NaOH/L, for the different operation times. As can be seen, the optimum operation time for this work is 30 min, the same as for sewage sludge and complex wastes with small particle size [1,[17][18][19]. In fact, according to these results it seems to be linked to long operation periods (60-120 min) which may induce the effects of Maillard reaction (caramelization processes previously described) generally reported by several authors whose studies were focused on thermal pretreatments applied to solid waste such as the OFMSW, municipal biomass waste, barley waste or food waste [7][8][9][10][11][12][13].
Expected Effect of Pretreatment on the Extent of Acidogenesis and H2 Production
ASC is the fraction of solubilized organic matter that has not been transformed into VFA and therefore, this indirect parameter may be used to study the behavior of the acidogenesis phase [25]. Thus, ASC may be used to describe how this pretreatment may affect the acidogenic phase of a Dark Fermentation process and, therefore, on the H2 production. If the solubilisation yield in terms of ASC is high, higher amounts of organic matter could be transformed to VFA during the acidogenesis step and, therefore, higher H2 production could be expected during a sequential anaerobic digestion process of pretreated waste.
As can be seen in Table 1, when the optimum conditions are applied (160 °C, 6.5 bar, 3 g NaOH/L and 30 min of operation time) the maximum increment of ASC (178%) is achieved. Therefore, it may be expected that implementation of this thermochemical pretreatment under these optimum conditions could promote the VFA and H2 generation during the acidogenic phase in a subsequent anaerobic process.
Statistical Analysis
A statistical study of the data was developed by means of the statistical software IBM ® SPSS ® Statistics version 19.0. Firstly, Tukey test (significant difference) was applied to determine if the experimental data have a normal distribution [27]. Values of the significance (S) higher than 0.05 imply that experimental data have a normal distribution. For all variables, the Tukey test backs ANOVA analysis.
Secondly, an analysis of variance (One-Way ANOVA) was carried out in order to estimate if each factor has a significant effect on the analyzed parameters. Similarly, following the analysis of variance, if the significance value is less than 0.05 indicates that there are significant differences among the data.
As it can be seen in Table 2, the temperature is the only factor that unequivocally shows a significant effect on the solubilisation yield when the organic matter is expressed in terms of DOC, TVFA and ASC. Statistical results for pressure and NaOH dosage are not unequivocally conclusive. Finally, a specific statistical analysis of temperature factor by Tukey tests allows corroboration that there are significant differences among the levels (160, 180 and 200 °C) tested in this study (Table 3). Thus, if 160 °C is the temperature in which the solubilisation yields measured experimentally are generally higher (Figure 1), it may be concluded that this level of temperature is the best from the experimental and statistical point of view. Bold values indicate that factor affects significantly (S < 0.05).
Methodology
Thermochemical pretreatments assays were carried out in inert atmosphere (N2). The employed waste was OFMSW coming from a 15-mm screen (trommel) of a full-scale 880 t/day MBT plant (named Las Calandrias) placed in Jerez de la Frontera (Cadiz, Spain). The following operative variables were studied: temperature, pressure and sodium hydroxide (NaOH) dosage as alkaline agent. In addition, different operation times were tested in this work in order to study its possible effect on the organic matter solubilisation.
The average particle size of the waste was lower than 15 mm and its characterization is shown in Table 4. It must be highlighted that the morphology of the waste used in the experiments was not altered in any manner, i.e., the waste was not processed through drying or milling. In addition, the composition of the OFMSW (44% organic matter) was not altered in the laboratory and, therefore, it had the complete, original features of real waste.
Design of Experiments
The experiment was conducted according to a 3 n -type factorial design (3 being the number of the variable and "n" being the number of levels of each variable tested). The variables were temperature, pressure and alkaline reagent dosage, as described in Table 5. The factorial design established 27 experiments. The assays were carried out in a 1-L non-stirred pressure vessel (Parr™, series 4600-4620) equipped with two proportional-integral-derivative (PID) controllers for temperature and pressure fitting. The pressure was regulated by means of a pneumatic over-pressure valve activated by a compressor. A muffla heating jacket was also employed to heat the system.
The reactor was filled till 75% of its capacity with OFMSW for each test. The waste was diluted by means of the addition of tap water until it reached a total solids (TS) concentration of 20%. The alkaline reagent was added from a 10 M-NaOH solution.
It is very important to highlight that in this study, (unlike the methodology followed by Fdez-Güelfo et al. [1], in which the operation time was set just on 30 min), four different operation times (15,30,60 and 120 min) were tested, from the optimization of the temperature, pressure and dosage variables, in order to study the possible effect of this variable on the solubilisation yield.
Determining the Solubilisation Yield (SY)
The effect of the pretreatments on the organic matter solubilisation has been measured in terms of dissolved organic carbon (DOC), total volatile fatty acids (TVFA), soluble chemical oxygen demand (sCOD) and acidogenic substrate as carbon (ASC). Solubilisation yields, in terms of increments of DOC, TVFA, sCOD in the liquid phase, were calculated as shown in Equation (1): Where SOMF and SOMI are the final and initial soluble organic matter respectively expressed as DOC, TVFA, sCOD or ASC, the last one calculated indirectly from classical parameters Equations (2) and (3) according to Fdez-Güelfo et al. [25]. where: DAC, the dissolved acid carbon and it represents an average of carbon considering the "carbon/molecular weight" ratios of each VFA independently measured by gas chromatography. DOC [M/L 3 ], the dissolved organic carbon measured by carbon analyzer. AiH [M/L 3 ], represents the concentration of every individual VFA measured by gas chromatography. ni, the number of carbons of AiH. MWi, the molecular weight of AiH.
In this study, the pretreatment has been considered successful when the organic matter solubilisation yield is higher than 100%, maximum value of efficiency obtained by other authors [17][18][19].
Analytical Techniques
TVFA, sCOD and DOC were measured in supernatant after a lixiviation step. Lixiviation was performed in a flask with continuous stirring by adding 10 g of sample to 100 mL deionized water for 20 min, according to Romero-Aguilar et al. [28]. Individual VFA (from C2 to C7, including iC4, iC5 and iC6) levels were determined by gas chromatography (SHIMADZU GC-17 A) with a flame ionization detector (FID) and a capillary column filled with Nukol (polyethylene glycol modified by nitro-terephthalic acid). The temperatures of the injection port and detector were 200 and 250 °C, respectively. Helium was the carrier gas at 50 mL·min −1 . In addition, nitrogen gas was used as make up at 30 mL·min −1 flow rate. On the other hand, DOC was measured according to 5310B standard method [29] in a combustion-infrared carbon analyzer (Shimadzu TOC5050A). Finally, sCOD was determined according to the colorimetric standard method 5220C [29].
Conclusions
Based on the above discussion, the following conclusions may be stated: (1) With respect to the influence of the factors temperature, pressure and NaOH dosage on the SY, it can be concluded that the best results have been obtained at 160 °C, 3 g NaOH/L, 6.5 bar and 30 min of operation time. In these conditions, the solubilisation yield obtained in terms of DOC, sCOD, TVFA and ASC were 176%, 123%, 119% and 178% respectively. Hence, the optimum conditions reported by Fdez-Güelfo et al. [1], 180 °C, 3 g NaOH/L and 5 bar, may be redefined with higher accuracy in order to improve the efficiency of this pretreatment for applying it to OFMSW coming from full-scale MBT plants. (2) According to the statistical analysis, the temperature is the only factor that unequivocally shows a significant effect on the solubilisation yield when the organic matter is expressed in terms of DOC, TVFA and ASC. | v3-fos |
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} | s2 | Assessment of the Safety and Efficacy of an Attenuated Live Vaccine Based on Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus
ABSTRACT The safety and efficacy of the JXA1-R vaccine, an attenuated strain of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV), were examined using an intramuscular challenge model in piglets. The JXA1-R vaccine was obtained by passing HP-PRRSV JXA1 through Marc-145 cells (82nd passage). Genomic sequence comparisons showed that strain JXA1-R and its parental strain, JXA1, differ by 47 amino acids, and most of these differences are scattered throughout the PRRSV genome. Four-week-old PRRSV-free piglets were inoculated intramuscularly with JXA1-R vaccine (103.0, 104.0, 105.0, 106.0, and 107.0 50% tissue culture infective doses [TCID50]/ml for groups 1 to 5, respectively) and then challenged intramuscularly with the 5th passage virus of JXA1 virus (JXA1-F5, 3 ml × 104.5 TCID50/ml) 28 days after inoculation. The humoral immune response, swine growth, clinical signs, and differential organ lesions were monitored. The results showed that all vaccinated piglets had a perceptible humoral immune response to vaccination after day 7, which then promptly increased, almost reaching the maximum sample/positive (S/P) ratio value at 28 days postimmunization. Viremia detection indicated that the viral replication levels of the challenge virus in the immunized groups (immunization doses ≥104.0/ml) were significantly lower than that of the virus-challenged unvaccinated control group. Piglets in groups 2 to 5 were effectively protected against lethal HP-PRRSV infection and did not show any obvious changes in body temperature or clinical signs of disease at any point during the experiment. However, two of five piglets in group 1 showed mild pathological lesions and transitory high fever. These results suggest that JXA1-R (TCID50/ml ≥104.0) is sufficiently attenuated and can provide effective protection against the lethal wild-type HP-PRRSV.
P orcine reproductive and respiratory syndrome (PRRS) was first discovered in the United States in 1987 (1,2). It is characterized by reproductive failure in pregnant sows and respiratory disorder in growing swine. PRRS has spread through most of the world's swine-producing regions and has caused substantial economic losses to the swine industry worldwide.
In June 2006, a highly pathogenic strain of PRRSV (HP-PRRSV) with a unique 30-amino-acid deletion in its Nsp2 coding region was isolated from diseased swine in China. Infections were characterized by high fever, high morbidity (50% to 100%), and high mortality (20% to 100%) (9,10). Since then, HP-PRRSV has been the major epidemic viral strain in pigs in China, causing enormous economic losses. Subsequently, HP-PRRSV was de-tected in countries neighboring China, including Vietnam and Laos (11,12).
Two types of commercial vaccines, modified live-attenuated vaccines (MLVs) and inactivated vaccines, are currently available against PRRSV (13,14). Inoculation with vaccines (especially live attenuated vaccines) can provide protection against PRRSV infections and prevent the spread of this disease. Currently, many modified live-attenuated PRRSV vaccines, such as Ingelvac ATP, RespPRRS/Repro1 ATP, RespPRRS MLV, and CH-1R, have been successfully employed (15). All of the vaccines against PRRSV are generally effective against homologous strains but 10 15 20 25 30 35 40 45 50 60 70 80 82 90 100 Nucleotide ORF1a NSP1 749 1904 T T T T T T T T T T T show varying and sometimes no effectiveness against heterologous strains (13)(14)(15). In addition, acute PRRS-like disease and atypical PRRS, clinical consequences of PRRS characterized by abortion and high mortality in pregnant gilts, were reported for MLV-vaccinated pigs (16,17). Notably, the HP-PRRSV strain emerged and rapidly became prevalent in China and other countries and severely affected their pork industries (11,12). Therefore, it is imperative to develop safe and effective vaccines against HP-PRRSV. In a previous study, we reported the outbreak of pig high fever syndrome in 2006. This syndrome is caused by the current epidemic strain of HP-PRRSV, called JXA1. We then established pathogenicity assays to characterize this strain (9,18). In this study, JXA1-R, a genetically stable, live attenuated vaccine strain against HP-PRRSV, was obtained by sequential passage of the HP-PRRSV JXA1 strain through Marc-145 cells (82nd passage). This vaccine provides safe and effective protection against a lethal HP-PRRSV JXA1-F5 challenge and can serve as an adequate vaccine against HP-PRRSV infection in herds. T T T T T T T T T T T T T T 1 1 8 I I I I I I I I I I I T T T T T T T T T T T T T A 174 S I I I I I I I I I I I I I I I ORF5 59
Serial
Boldface type indicates mutations of the nucleotides and amino acids of the JXA1-R strain.
propagation and titration. The virus was serially passaged through Marc-145 cells using Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and incubated at 37°C with 5% CO 2 .
When ϳ80% of the virus-infected Marc-145 cells showed a cytopathic effect (CPE), the virus was harvested using freeze-thaw techniques. To isolate the virus, plaque purification was performed after every five passages. The titers of different passages of HP-PRRSV JXA1 were measured by seeding Marc-145 cells into 96-well cell culture plates 1 day before infection. The 50% tissue culture infective dose (TCID 50 ) was calculated according to the Reed-Muench method (19). RNA isolation and genome sequencing. Passages of JXA1, F10, F15, F20, F25, F30, F35, F40, F45, F50, F60, F70, F80, F82, F90, and F100 strains were subjected to whole-genome sequence determination. Viral RNA was extracted using a Qiagen RNeasy minikit (Qiagen, Germany) according to the manufacturer's instructions. Eighteen pairs of PCR primers were used to amplify 18 overlapping cDNA fragments to cover the complete genome of the different JXA1 strains (18). Reverse transcription PCR (RT-PCR) was performed using SuperScript III reverse transcriptase (Invitrogen, USA) for reverse transcription and PfuUltra high-fidelity DNA polymerase (Stratagene, USA) for PCR amplification. Each fragment was amplified at least three times. The amplicons were purified with an E.Z.N.A. gel extraction kit (Omega, USA) and cloned into pGEM-T Easy vector systems (Promega, USA). Recombinant clones were sequenced with an automatic DNA sequencer (ABI, USA) and spliced artificially. Clustal X 1.83 and DNAStar software were used to analyze the sequences.
Animal studies and clinical examinations. In vivo studies were carried out at Guangdong Dahuanong Animal Health Produce Co., Ltd. Thirty-five healthy 4-week-old piglets were obtained from a PRRSV-negative farm and divided randomly into seven groups (five piglets per group). The piglets in groups 1 to 5 were immunized intramuscularly with the passage 82 (JXA1-R) harvest of the JXA1 strain at doses of 1 ml per piglet. The challenge control (group 6) and strict negative control (group 7) groups received only dilution medium. Twenty-eight days after vaccination, each piglet (except negative-control piglets from group 7) was challenged intramuscularly with 3 ml (1 ϫ 10 4.5 TCID 50 /ml) of strain JXA1-F5. The pathogenicity of strain JXA1-F5 was confirmed by our research group as described previously (9). The different treatment groups were kept in separate rooms, and the piglets were monitored for 49 consecutive days. Every day from the day of immunization to 49 days postimmunization (DPI), the clinical observations of each animal were recorded. These clinical parameters included fever, coughing, shivering, diarrhea, and changes in appetite. In addition, animals were weighed on the day of immunization (day 0), at challenge (day 28), and at necropsy (day 49) for assessments of average daily weight gain. All the animals were euthanized 21 days after the challenge, and tissue samples were collected. Protocols of all animal experiments performed in this study were approved by the CADC ethics committee (permit no. CADC-AEC-2009002).
Viremia detection by virus isolation and quantitative real-time RT-PCR analysis.
To determine if the piglets were viremic during vaccination and challenge, virus isolation was performed on all serum samples from all collection days. Briefly, sera were inoculated separately into Marc-145 cells, and the CPEs were observed microscopically for 5 days. The percentage of CPE-positive wells was calculated for each group. To determine the duration of viremia of the JXA1-R vaccine strain, serum samples collected on DPI 0, 3, 5, 7, 14, 21, 28, 35, 42, and 49 were used to detect the RNA copy number of PRRSV by TaqMan-based quantitative real-time RT- PCR. In brief, multiple-sequence alignment was carried out using 15 differential passages of JXA1 strains. Relative to the JXA1 strain, the JXA1-R strain contained two steady nucleotide mutations at sites 3572 and 3573 of the Nsp2 gene (Table 1). After JXA1-R inoculation, nucleotide mutations remained genetically stable, as determined by sequence analysis (data not shown). According to this specific nucleotide mutant, the real-time RT-PCR assay using minor groove binder (MGB) probes for rapid detection of the JXA1-R PRRSV strain in virus-infected cell cultures and clinical samples was established. The primers for the detection of JXA1-R were a forward primer at position 3513 to 3528 (5=-TCCACGCATCCTCGGG-3=) and reverse primer at position 3630 to 3649 (5=-TGCTCTCGTCAGA CTCCCGT-3=). The sequence of the TaqMan probe was 5=-CCTCGGCT CCCTCCA-MGB-3= at position 3566 to 3580. The RT-PCR assay using MGB probes for identifying the JXA1 HP-PRRSV strain was designed in the gene segment encoding NSP2 protein as described previously (20). For quantitation, the standard curve was generated as described previously (20). Each serum sample was checked in triplicate, and the mean RNA copy per milliliter of the differential samples was calculated using these values.
Histological observations. To evaluate histological lesions in the tissues of the different treatment groups, hematoxylin and eosin (H&E) staining was used to evaluate a series of collected specimens, including samples from the lungs, tonsils, lymph nodes, heart, liver, spleen, kidneys, brain, stomach, and intestine jejunum. These samples were fixed in 10% neutral buffered formalin, routinely processed, embedded in paraffin, and stained using Harris's H&E (21,22). Statistical analysis. Normally distributed data are shown as mean Ϯ standard deviation (SD) and were analyzed using one-way analysis of variance and the Student-Newman-Keuls post hoc test.
RESULTS
Amino acid mutations of different JXA1 passages. RNA was extracted from different JXA1 passages, and RT-PCR was performed as described previously (18). Compared to the parental virus JXA1, there were a total of 108 nucleotide changes at the 100th passage. Among these mutations were 2 nucleotide changes (at position 116[T-C] and 124[A-G]) in the 3=-untranslated region (UTR). No nucleotide mutations were observed in the 5=-UTR of any of the virus passages. Other nucleotide mutations were observed in nonstructural and structural coding regions, 50 of which were missense mutations, causing a change of 47 amino acids in ORFs 1a, 1b, 2, 3, 4, and 5 ( Table 1). All of these mutations were observed at the 80th passage. No nucleotide or amino acid changes were observed from passages 80 through 100 ( Table 1).
Immunogenicity of JXA1-R and parental viruses in piglets.
The serum samples were collected from all pigs at 0, 3, 5, 7, 14, 21, 28, 35, 42, and 49 DPI. These samples were assayed for anti-PRRSV antibody with ELISA, and the S/P ratio was averaged for each group (Fig. 1). All animals were seronegative to PRRSV within 7 DPI. The antibody response in the ELISA showed that the piglets in groups 2 to 5 had seroconverted on DPI 14, while three of the five piglets in group 1 had seroconverted by DPI 14. The remaining piglets had seroconverted by DPI 21. No PRRSV-specific antibodies were detected in group 6 (challenge control) or 7 (negative control) during the 28-day postimmunization period. On day 28, the piglets in groups 1 to 6 were challenged with the 5th passage of JXA1 virus. Mean S/P ratios of all virus-treated groups were higher than those of the challenge control group (Student's t test, P Ͻ 0.05) (1)(2)(3)(4)(5). In the challenge control group, only one piglet became seropositive on DPI 42 (14 days postchallenge). The other piglets had died by then. Swine in the negative control group remained seronegative throughout the study (Fig. 1). Viral isolation. After JXA1-R inoculation, viral isolation analysis showed that one of the animals in group 1, two of the animals in groups 2 and 3 (four total), and three of the animals in groups 4 and 5 (six total) were viremic by DPI 3. On DPI 28, one of the piglets in groups 1 and 2 (two total) and two of the piglets in group 4 were PRRSV positive ( Table 2). Following challenge, two of the piglets in group 2 and one piglet in group 4 were viremic on DPI 35. Four of the five piglets in group 1 were viremic on DPI 35. On DPI 42, viremia was not detected in any animal in group 2, 3, 4, or 5, but two of the piglets in group 1 were viremic. On DPI 49, viremia was detected in group 1 only, in which one of the piglets was viremic and two pigs showed positive staining in the lung samples by immunohistochemistry (see Table 4 below). However, virus was recovered from all living piglets in group 6 at 35, 42, and 49 DPI ( Table 2).
Detection of viral RNA. To further evaluate the duration of viral RNA of the JXA1-R vaccine strain, serum samples from 0, 3, (ADG) of 5 piglets per group was calculated for days 0 to 28 and days 28 to 49 postinfection. Error bars ϭ SD. *, Average ADG of a given set is higher than the challenge control (P Ͻ 0.05); **, average ADG of a given set is significantly higher than the challenge control (P Ͻ 0.01). 5,7,14,21,28,35,42, and 49 DPI were evaluated using real-time RT-PCR. The RNA copy numbers in groups 1 to 5 reached their highest levels on DPI 7 and then gradually declined in all groups. On DPI 42, the RNA copy numbers in groups 1 to 5 were not detectable by real-time RT-PCR ( Fig. 2A). This was taken as indicative of JXA1-R strain replication levels in the piglets. To further assay quantitative levels of challenge virus replication in the various immunization groups, serum samples from 7, 14, and 21 days postchallenge were analyzed for viral RNA by real-time RT-PCR ( Fig. 2B). At 7 days postchallenge, no viral RNA was detected in group 3 or 5, and the RNA copy numbers were significantly lower in groups 2 and 4 than in groups 1 and 6 (Student's t test, P Ͻ 0.01). At 14 days postchallenge, no viral RNA was detected in group 2 or 4, and the RNA copy numbers were significantly lower in group 1 than in group 6 (Student's t test, P Ͻ 0.01) (Fig. 2B). These results indicated that the viral replication levels of the immunized groups (doses of Ն10 4.0 /ml) were significantly different from those of the challenge control group. Thus, the level of challenge virus RNA in the immunization groups was significantly reduced by the vaccine. Clinical signs after immunization and challenge. General observations and abnormal signs postimmunization and postchallenge were recorded. No piglet immunized with JXA1-R vaccine developed the typical clinical signs of PRRS. After the challenge, the piglets in group 6 exhibited clinical signs typical of PRRS, such as persistently high fever (40.5°C to 42°C) (Fig. 3), depression, anorexia, cough, asthma, lameness, and shivering. Four of the five piglets in group 6 died between days 7 and 14 postchallenge (Table 3). In group 1, only soft coughing and transitory high fever were observed and in only two piglets; both piglets eventually recovered (Fig. 3). None of the piglets in groups 2, 3, 4, 5, and 7 showed high fever or clinical signs of PRRS at any point during the experiment (Table 3).
To assess the gross clinical effects of JXA1-R vaccination and HP-PRRSV challenge on piglets, all animals were weighed on DPI 0, 28, and 49. The average daily weight gain (ADG) in each group was evaluated between 0 to 28 and 28 to 49 DPI (Fig. 4). There was no significant difference in the weight of different treatment groups during days 0 to 28. However, after the challenge, piglets in group 1 gained weight faster than piglets in group 6 (Student's t test, P Ͻ 0.05), and piglets in the other groups gained weight even faster than piglets in group 1 (Student's t test, P Ͻ 0.01) (Fig. 4).
Pathological characterization. To further assess the efficacy of JXA1-R attenuated live vaccine, pathological changes in samples from each group were examined. Typical lesions were found in all piglets in group 6 at necropsy, as were hyperplasia in the lungs, lung hemorrhagic spots, lung edema, blood spots in the kidneys, enlarged lymph nodes with hemorrhagic spots, spleen infarction, and hyperemia of the meninges. In group 1, mild lesions were mainly localized to the lungs, tonsils, and lymph nodes in two of five piglets. No obvious pathological changes were observed in the other immunization groups (Table 4). In addition, histopathological examination was used to observe microscopic lesions. In the challenge control group, animals presented with disease manifestations, including nonsuppurative encephalitis, massive lymphomononuclear infiltration resulting in perivascular cuffing in the brain, and severe inflammation of the lungs, tonsils, lymph nodes, and spleen tissues characterized by infiltrating lymphocytes, macrophages, and necrocytosis. However, there was mild interstitial pneumonitis in the lungs of two of the piglets in group 1 and slight lymphomononuclear infiltration in lymph nodes and tonsils (Fig. 5). In groups 2 to 5, such histopathological changes were not evident.
DISCUSSION
In June 2006, a highly pathogenic PRRSV (HP-PRRSV) called JXA1, which has a unique 30-amino-acid deletion within its Nsp2 coding region, was isolated from diseased piglets in China, where it brought significant economic loss (9). Since then, this PRRSV has become the major epidemic viral strain in China. HP-PRRSV has 89.4% to 97.2% nucleotide identity with North American (NA) PRRSV strains VR-2332, BJ-4, CH-1a, HB-1(sh)/2002, and HB-2(sh)/2002. HP-PRRSVs share 98.2% to 100% nucleotide identity overall. These results indicate significant genetic diversity across the HP-PRRSV and the NA-type PRRSV strains. Studies have shown that vaccines protect pigs against homologous or very closely related strains of PRRSV only (23)(24)(25). This means that vaccines derived from the NA-type PRRSVs cannot provide completely effective protection against HP-PRRSV. After the outbreak of HP-PRRSV, the HUN4 and TJM vaccine strains provided effective protection in young pigs against the virulent parental HP-PRRS strain (15,26). Here, we described a newly developed HP-PRRSV vaccine candidate, JXA1-R, which is efficacious in the prevention of clinical infection caused by the parental JXA1 virus. Whether the JXA1-R can provide cross-protective immunization or be used in a multivalent vaccine remains an interesting question that requires further investigation.
In this report, JXA1-R, a genetically stable attenuated viral strain, was obtained by serially passaging the HP-PRRSV JXA1 viral strain through Marc-145 cells. All nucleotide changes and amino acid mutations relative to JXA1 were observed before the 80th passage. No nucleotide changes or amino acid mutations were observed between the 80th and 100th passages. This indi- ϩ ϩϩϩ ϩ ϩϩϩ ϩ 1 0 2 0 2 0 2 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 3 0 0 0 0 0 0 0 0 0 0 0 cated that the 80th JXA1 had adapted to the Marc-145 cells and was be stably passaged through them. Viruses from the 80th passage were used as seed viruses, and those of the 82nd passage were used for attenuated vaccine development and production.
Our findings indicate that the JXA1-R vaccine can successfully protect piglets against a challenge with HP-PRRSV. First, all piglets in groups 2 to 5 were protected, and all of these animals survived without any major clinical signs at any point in the experimental period. In group 1, two of five piglets exhibited mild coughing and transitory high fever; each piglet eventually recovered. These data suggested that a low dose (10 3.0 TCID 50 /ml per piglet) of JXA1-R vaccine provided incomplete protection from HP-PRRSV challenge. However, doses of Ն10 4.0 TCID 50 /ml per piglet provided full protection against HP-PRRSV. Even high doses of JXA1-R vaccine were found to be safe and effective in these piglets. Second, in this study, the time between vaccination and challenge was 4 weeks. The humoral immune response was assessed by ELISA. All vaccinated piglets had a perceptible humoral immune response to vaccination after day 7. This response promptly increased, almost reaching the maximum S/P ratio value at 28 days. Note also that the neutralizing antibodies generated in some piglets were present only at low levels, and no neutralizing antibodies were detected in certain piglets from groups 1 to 5 (data not shown). These piglets were still effectively protected, yet this suggested that the neutralizing antibodies were not essential for protective immunity. Some researchers reported that the levels of neutralizing antibodies were not always correlated with protection and that animals lacking neutralizing antibodies were nonetheless resistant to reinfection (27,28). Third, detection of viremia showed that the duration of viremia was about 42 days. HP-PRRSV JXA1 challenge took place 28 days postimmunization. Results indicated that challenge virus replication levels of the immunized groups (doses of Ն10 4.0 /ml) were significantly different from those of the challenge control group. The level of viremia in the immunized groups (doses of Ն10 4.0 /ml) was dramatically reduced during the study (Fig. 2B). Here, JXA1-R vaccination was clearly shown to provide effective protection against challenge with the same virus. The protective response was observed in immunized piglets during the experiment, although the mechanisms of immune protection are still not fully understood. In addition, other experimental studies showed that preventive vaccination with attenuated PRRSV or inoculation with virulent field virus provided a high level of protection against challenge with the same or nearly the same virus (29,30). Altogether, these results suggest that JXA1-R vaccine is effective against HP-PRRSV infection, but the immunization doses should be Ն10 4.0 TCID 50 /ml per piglet. It should be noted when drawing comparisons that the amount of time between vaccination and challenge was 4 weeks in this study versus 3 weeks in other studies (26,31).
In other studies, an intermediate JXA1 passage (JXA1-F49) was used to inoculate piglets at a dose of 10 6.5 TCID 50 /ml. Two of the five piglets developed clinical symptoms, including rubefaction, cough, and anorexia. Pathological changes included light hyper- in group 1 (1 ϫ 10 3.0 TCID 50 /ml) (a to e) and group 6 (challenge control) (A to E) by H&E staining postchallenge with HP-PRRSV. Arrows, disease manifestations. (a) Lung tissues of two of five piglets in group 1 show mild interstitial pneumonitis and alveolar septal thickening. (A) Dead piglet lung tissue from group 6 shows interstitial pneumonitis with alveolar septa thickened by lymphomononuclear infiltration and severe hemorrhage. In group 1, slightly lymphomononuclear infiltration in lymph (b) and tonsil (c) and nearly normal spleen and brain (d and e) are seen. In group 6, massive lymphomononuclear infiltration and hemorrhage in lymph (B), massive lymphomononuclear infiltration and lymphatic follicle scalloping in tonsil (C), massive hemorrhage in spleen (D), and massive lymphomononuclear infiltration surrounding perivascular cuffing (E) are seen. | v3-fos |
2019-08-18T09:31:37.868Z | {
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} | s2 | Strongylid Nematode Infections of Humans, Ruminants and Pigs in Kumasi, Ashanti Region of Ghana
Aim: Human Oesophagostomum infections are considered zoonotic. In Ghana, the human infections are known to be focally distributed in the north-eastern parts of the Northern and the Upper East regions. Factors involved in the distribution of the human infection are not clear. It is also not known whether the human and animal infections occur outside these regions. The present study, therefore, sought to determine the types of strongylid nematode parasites infecting ruminants and pigs and also, whether human Oesophagostomum infections occur in Kumasi. Study Design and Methodology: Stool samples were obtained from Hospital outpatients, abattoir workers and dealers in ruminants and pigs in Kumasi, processed by coproculture and examined microscopically for strongylid nematode larvae. Results: No human Oesophagostomum infections were detected in the sampled Hospital outpatients, abattoir workers and dealers in ruminants and pigs. However, there were infections with Necator sp. and Strongyloides stercoralis at prevalences of 8.4% and 7.4%, respectively . Prevalences of Oesophagostomum in the animals sampled, during the rainy and dry seasons were 39.9% and 49.5% respectively for pigs: 41.9% and 16.1% for cattle: 44.1% and 15.1% for sheep: 37.1% and 38.2% for goats. These prevalence levels were comparable to prevalences obtained in a study conducted in sampled population of human, ruminants and pigs at the Bolgatanga slaughterhouse in the Upper East Region of Ghana. Conclusion: The results may be indicative of different species of Oesophagostomum being infective to humans and the various animals. Further investigations into the conditions that determine the presence and transmission of the human and animal parasites are recommended.
INTRODUCTION
Helminth infections are endemic in areas where environmental sanitation is poor and health education virtually non-existent [1]. Studies in Latin America [2] indicate that intestinal parasites are prevalent in both rural and urban populations. High prevalence in urbanized developing countries has been attributed to poor sanitary conditions of low income populations, being worse in overcrowded cities than in most rural communities [3]. Intestinal parasitic infections are encountered as occupational hazards by workers whose job brings them into contact with soil, soil contaminated materials or contaminated water.
Three species of Oesophagostomum have been recognised as causing occasional infections in humans, Namely, O. oculeatum, O bifurcum and O. stephanostomum. Polderman et al. [8] demonstrated that man was a definitive host to O. bifurcum in northern Togo and Ghana. Further studies of the human infection in parts of the northern region of Ghana [10,11] yielded prevalence values ranging up to 87%. Studies on the taxonomy, treatment and transmission of O. bifurcum have also been undertaken [12,13]. In humans, the infection is noticed as an epigastric or peri-umbilical mass, described as "The Dapaong Tumor" after the town in northern Togo where it was first described [7].
Oesophagostomum columbianum is reported as a pathogen of sheep, whilst O. radiatum and O. dentatum occur in the colon of cattle and pigs respectively [14]. Transmission is believed to occur by ingestion of infective third stage larvae (L 3 ). Thus, poor hygienic conditions of communities especially where pigs and ruminants live close to human dwelling could facilitate the transmission. It was, however, not known which ecological and behavioural factors are key to the focal distribution and the high local prevalence of the infection. Nevertheless, the role of pigs as carriers of O. bifurcum and Necator americanus have been documented [15].
The relationship between the human and animal infections is not clear as different species are reported to infect humans, ruminants and pigs. Oesophagostomum infections have not been reported in Kumasi although Prof. Amon Kotei (personal communication) reported seeing nodular swellings on the intestines of three humans during separate autopsies at the Komfo Anokye Teaching Hospital in Kumasi. The present study was, therefore, undertaken to determine whether human Oesophagostomum infections occur in Kumasi and to determine the types and prevalence levels of related strongylid nematode infections in pigs and ruminants slaughtered at the Kumasi Abattoir. In a related study conducted in Bolgatanga, (community endemic to human Oesophagostomum) in the Upper East Region of Ghana, prevalences and intensities of Oesophagostomum and related strongylid nematode infections were determined in the abattoir workers as well as in sampled groups of ruminants and pigs [16].
Humans
The study population consisted of three groups of people. The first group was individuals who reported at the major health centers in Kumasi with stool samples for laboratory examination. The second group were workers at the Kumasi abattoir and the third group were cattle dealers and herdsmen. All persons investigated were registered by name and identification codes on questionnaire forms. Each questionnaire had two parts. The first part dealt with the background information on the individual. Information relating to residence, ethnicity, age, and occupation was obtained by interview. The second part dealt with the results of the parasitological examination.
Ruminants and pigs
Animals sampled in were those brought to the abattoir for slaughter where every second cattle, sheep and goats to be slaughtered in a day was sampled. All pigs brought for slaughter during a visit was sampled (due to low numbers).
Collection of Stool/Fecal Samples
Individuals recruited for the study at the slaughterhouses and cattle markets were volunteers who consented and willingly provided samples of their stool for examination after the purpose of the study was explained to them. At the hospitals, samples were collected from patients who presented at the laboratory, on request by the medical officer, stool for diagnostic examination. Each person sampled was given a plastic container with identification code number for their stool samples. A total of 1428 stool samples were collected; 1379 from the hospitals, 428 from Kumasi abattoir workers, cattle dealers and herdsmen.
At the slaughterhouse samples were taken directly from the colon of each animal at slaughter with clean short sticks into clean, individually labeled, plastic containers. Sample sizes were based on the availability of the animals during the sampling period and are presented the Table 1: Approximately 3 grams of stool were collected and taken to the laboratory for culture. Each stool sample was cultured in duplicate (1 gm each) on the day of collection. Each gram (1 gm) of feaces was mixed with same weight of vermiculite and processed for coproculture.
Coproculture of Fecal Samples
A plastic disc (40 mm x 4 mm) was placed in the centre of a 9 cm diameter petri-dish (Fig. 1). A Whatman filter paper of 8 cm diameter was placed on the plastic disc and a little distilled water was introduced into the petri-dish to the level just below the top of the plastic disc. One gram of stool was mixed with an equal quantity of vermiculite and transferred onto the moist filter paper. The stool-vermiculite mixture was left in culture at room temperature for seven days and stirred every other day to aerate it. Eggs of nematodes present in the stool hatched into larvae, wriggled out of the stool-vermiculate mixture into the clean water in the petri dish. On the eighth (8 th ) day of culture, the fluid containing L 3 larvae was harvested into separate conical tubes. The petri-dishes were rinsed once with distilled water and added to the content of the conical tubes. A sedimentation time of at least two (2) hours was allowed for the larvae to settle and 100 µl of sediment was observed using x10 and x40 objectives of the light microscope after staining with a drop of diluted Lugol's iodine. Two readings were made for each sample and the mean larval counts calculated. Photographs of larval specimens were taken at x100 or x400 magnification. Identification of filariform larvae was based on their respective characteristic features as indicated by Little [17].
STATISTICAL ANALYSIS
The data collected were entered into SPSS (windows) and the prevalence and intensity of infections computed. Prevalences of infection were given as the percentage of individuals infected with a parasite out of the number examined and the intensity of infection computed from mean larval counts in 100 µl of sediment of the culture fluid. Significant differences were determined at 5% level, using one-way ANOVA (LSD) after outliers were removed using the Pearson and Hartley, Biometric Tables for statisticians [18].
Human Strongylid Nematode Infections
No human Oesophagostomum infection was detected in this study. However, there were Necator americanus and Strongyloides stercoralis infections. Table 2 gives details of the prevalence of hookworm and Strongyloides infections recorded in hospital outpatients. Prevalences were generally low. Ranging from 2.0% to 8.6% for Necator americanus and 3.0% to 12.3% for Strongyloides stercoralis. The highest prevalence for hookworm infections were recorded at the Kumasi South urban health center (8.6%).
At the cattle market and the Kumasi abattoir also, no human Oesophagostomum infection was detected in the sampled population of dealers and abattoir workers. However, prevalences of Necator americanus recorded in the dealers and the abattoir workers were 56% and 36.9% respectively, and for S. stercoralis it was 24% and 23.9% respectively (Table 3). Higher prevalence of infection was observed for both N. americanus and S.stercoralis among workers at the abattoir and cattle market than among the hospital out-patients (Table 2). Intensity values (mean no. of larvae/100 µl of stool culture sediment) recorded for both N. americanus and S. stercoralis infections were generally low ( Table 3). prevalence values were higher during the rainy season. In pigs and goats, higher prevalences were recorded in the dry season.
Hookworm infection was detected in goats and pigs in the wet season and only in pigs in the dry season, in each case, at low prevalence. Strongyloides infections were, however, detected both in the dry and rainy seasons in the ruminants and pigs sampled ( Table 4). Prevalence of Trichostrongylus infection was lower in the dry season than in the rainy season in the ruminants and pigs.
Intensity of strongylid nematode infections in ruminants and pigs
Intensities of Oesophagostomum infections were generally higher in the rainy season (Fig. 3). Pigs* showed the highest intensity of Oesophagostomum infection in the rainy season and this differed significantly from intensity in cattle (P = .006). Significant differences were also observed between goats* and cattle ( .002) in the rainy season. During the dry season, differences were observed between pigs* and all the other animal hosts (P = .000) and between goats* and cattle (P = .004). Intensity of Strongyloides infection were higher during the rainy season than in the dry season with pigs again recording the hi larvae/100 µl; Table 5). However, significant differences were only observed in infection between sheep* and cattle ( and between goat* and cattle (P = .003). In the dry season, Significant differences were observed between goats* and cattle ( and also between goats* and sheep ( Intensities of Trichostrongylus infections were also generally higher during the rainy season. Significant differences were observed in intensity
Intensity of strongylid nematode infections in ruminants and pigs
infections were generally higher in the rainy season (Fig. 3). Pigs* showed the highest intensity of in the rainy season and this differed significantly from intensity in = .006). Significant differences were also observed between goats* and cattle (P = .002) in the rainy season. During the dry season, observed between pigs* and all = .000) and between Table 5 presents intensities of hookworm, Trichostrongylus infections recorded. A high intensity of hookworm infection was recorded in pigs (32.0 larvae/100 µl) during infection were higher during the rainy season than in the dry season with pigs again recording the highest (24.1 larvae/100 µl; Table 5). However, significant differences were only observed in Strongyloides infection between sheep* and cattle (P = .012) = .003). In the dry season, Significant differences were en goats* and cattle (P = .015) and also between goats* and sheep (P = .031).
infections were also generally higher during the rainy season. Significant differences were observed in intensity of Trichostrongylus infection between sheep* and cattle (P = .011), goat* and cattle ( pig* and cattle (P =.035). In the dry season however, differences were observed between pigs* and cattle (P =.027).
Human Strongylid Nematodes
Results of the study indicate that Strongyloides species are the main strongylid nematodes infecting the study populations in Kumasi and is comparable to prevalences observed at the Bolgatanga slaughter house [16]. The comparatively higher prevalence observed for hookworm among the workers at the Kumasi abattoir and cattle markets could be explained by the fact that most of the workers are of northern origin where hookworm infections are reported to be endemic [10,11]. Nevertheless, the possibility of these workers contracting the infection in Kumasi cannot be ruled out since hookworm infections have been reported in Kumasi as well [19]. From responses to questionnaires administered it became apparent that majority of these workers live in compound houses and squatter settlements where basic sanitary facilities are virtually lacking. These facilities are also nonexistent at the cattle market. Since hookworm infections are found whenever unsanitary disposal of human excr [20], this could explain the high prevalence observed (Table 3). etween sheep* and = .011), goat* and cattle (P =.000) and =.035). In the dry season however, differences were observed between
Human Strongylid Nematodes
icate that Necator and species are the main strongylid nematodes infecting the study populations in Kumasi and is comparable to prevalences observed at the Bolgatanga slaughter house [16]. The comparatively higher prevalence observed worm among the workers at the Kumasi abattoir and cattle markets could be explained by the fact that most of the workers are of northern origin where hookworm infections are reported to . Nevertheless, the possibility of these workers contracting the infection in Kumasi cannot be ruled out since hookworm infections have been reported in Kumasi as well From responses to questionnaires administered it became apparent that majority of these workers live in compound houses and squatter settlements where basic sanitary facilities are virtually lacking. These facilities are also nonexistent at the cattle market. Since hookworm infections are found whenever unsanitary disposal of human excreta is common , this could explain the high prevalence
infection in ruminants and pigs
Wet season Dry season None of the workers interviewed at the abattoirs (both in Kumasi and Bolgatanga) admitted suffering frequent ailments. Forty-eight percent of the workers at the Bolgatanga slaughterhouse also engage in farm work ranging from millet to paddy-rice farming and 44% of them also kept domestic animals [16].
Majority of the hospital outpatients are females who reported for routine antenatal check-ups. Only 41% of the patients including one male suffer frequent ailments ranging from fevers to stomach pains; 69% of the outpatients also work on farms and 93% keep domestic animals. Majority of the people interviewed do not take dewormers.
Generally, the prevalences determined for these strongylid infections in the human samples were low and confirm similar results from earlier studies [21]. Strongyloidiasis is quite rare [22]. According to Goldsmid [23] the prevalence of Strongyloides could be underestimated if it is based on a single specimen. On the other hand, hookworm larvae have longer life and greater resistance to adverse conditions than Strongyloides [24]. This could explain the higher frequencies of hookworm infection compared to Strongyloides. Also, Strongyloides species are known to have short latent periods [22] though they are capable of autoinfection. The ability to auto infect could affect the numbers of the parasite in each host. In worm endemic communities most individuals harbour few parasites and a few harbour heavy burdens [25].
Strongylid Nematodes of Ruminants and Pigs
Oesophagostomum infection in livestocks is contracted through contaminated food and water [14]. The differences in prevalences recorded may be related to environmental or behavioral factors playing a role in transmission. It is known that the requirements of the infective larva and its previous stages determine the geographical distribution of the adult nematode [13].
Ingested L 3 larvae of Oesophagostomum penetrate the intestinal wall where they develop to young adults. The duration of this tissuedwelling phase may be influenced by various factors notable among them being harsh environmental conditions [26]. It has been postulated that larval stages of some Oesophagostomum species do not normally continue their development to adulthood after the start of the dry season. The tissue-dwelling stage enters a dormant phase, to continue much later. Larvae of O. radiatum and O. columbianum have been shown to remain dormant for up to one year [26]. This postponement of development offers certain advantages to the parasite, so that the production of eggs would be highest during the season when conditions outside the host would be favorable for transmission. Also, it has been postulated that L 3 larvae undergo some form of aestivation during dry seasons. The rainy season, thus, provides suitable environmental conditions for the survival and transmission of the L 3 larvae, since the first and second stage larvae (L 1 and L 2 ) require abundant moisture to develop to the L 3 .
Desiccated L 3 larvae collected from defaecation grounds have been shown to regain their motility and infectivity after they have been rehydrated under laboratory conditions [13]. An infective larva under favorable conditions can complete its cycle to adulthood within 41 days after infection implying that Oesophagostomum species can develop through a number of generations during the rainy season. This could account for the higher prevalences observed during the rainy season.
Prevalence of Strongyloides was also generally higher during the rainy season. Strongyloides larvae develop best in water-saturated soils with abundant organic material [27]. These conditions become available during the rainy seasons, thus favouring the transmission which occurs both orally and percutaneously. Also, Strongyloides larvae have only slight resistance to desiccation or marked changes in temperature. This suggests that the rate of transmission may be reduced during the dry season.
The isolated cases of hookworm infections in the ruminants sampled suggested they are not natural hosts to the parasite. However, when they are exposed to a constant source of infection, they could become infected. Pigs, on the other hand, are known to feed on human excreta, so that a pig that feeds on hookworm infested stool will develop the infection. This was demonstrated by Steenhard et al. [15] when four parasite-free pigs were fed with fresh faeces from people heavily infected with Oesophagostomum and Necator whereupon third stage larvae of the two parasites were detected from the faeces of the exposed pigs after coproculture.
Ova and larvae of Trichostrongylus species require high humidity, abundant shade and the presence of grass or carpet vegetation for their survival. Infection with Trichostrongylus in animals thus occurs by ingestion of infective larvae on grass or vegetation. The rainy season, thus, provides the favorable condition for transmission (Table 4). However, if the favorable conditions are present in the local rearing environment of these animals during the dry season, high prevalences can be observed. This was observed in Bolgatanga, where higher prevalences were recorded during the dry season in cattle, sheep and pigs [16].
Generally, filariform larvae show tropisms which are characteristic of all strongylid larvae [28]. They are negatively geotropic, where they climb to the top of soil particles and positively phototropic to mild light. Thus, they will crawl up the grass blades in the early morning, towards the evening and at other times of the day during dull weather [14]. They are also markedly thermostatic and are rapidly stimulated into activity by the warmth of a nearby animal [28]. Moisture is necessary for these movements. All these tropisms would tend to increase the chance of infecting a passing host.
The intensity of Oesophagostomum infection (no. of larvae/100 µl of sample) derived from the geometric mean values shows pigs as having the highest intensity during the wet season (Fig. 3).
Similarly higest intensity was observed during the wet season in pigs in Bolgatanga [16]. This comparatively high intensity value, however, may not be attributed only to the coprophagic habits of pigs since the endemicity of a community to human oesophagostomiasis has not been shown to bear any correlation to the level of infection in pigs. It is also known that pigs harbour certain host-specific The intensity of Strongyloides infection was highest in pigs during the wet season. Significant differences were only observed between sheep* and cattle (P=.012) as well as goats* and cattle (P=.003) ( Table 5). Pigs also showed the highest intensity in Bolgatanga [16] in the rainy season and it differed significantly from intensity in cattle (P=.005), sheep (P=.003) and goats (P=.002), [16]. Sheep and goats are usually penned together, so that the differences observed could be due to differential host susceptibility of sheep and goats.
Again no specific reason could be given for the varying levels of Trichostrongylus infections in the animals except that this could be a result of the specific behavioural or animal husbandry practices in the communities. Significant differences were observed between infections in sheep* and cattle (P=011), goats* and cattle (P=.000) as well as pig* and cattle (P= 035) during the rainy season.
Conclusion
Human Oesophagostomum infections were not detected in Kumasi during the study period. Infections with the other strongylid nematodes such as Necator and Strongyloides were, however, detected. An observation, confirming that made in an earlier study in Bolgatanga in the Upper East Region of Ghana.
Oesophagostomum infections occurred in cattle, sheep and goats, and in pigs in Kumasi.
The possibility of zoonotic infection exists if the species infecting ruminants or pigs are also infective to humans.
Recommendation
A further study on the species of Oesophagostomum infecting the various animal hosts is recommended in order to ascertain their similarity and/or ability to infect humans. Such studies should include DNA analysis and characterization of the various species.
Control measures need be put in place through healthy animal husbandry practices, personal hygiene and health education. | v3-fos |
2019-02-15T14:21:21.084Z | {
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} | s2 | Optimization of Egg Tofu Formulations Containing Carrageenan, Gum Arabic and Corn Starch by Descriptive Sensory Analysis
Corresponding Author: Aminah Abdullah School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia Email: [email protected] Abstract: The objectives of this study was to determine the effects of carrageenan, gum Arabic and corn starch on the sensory characteristics of egg tofu and to optimize egg tofu formulations using Response Surface Methodology (RSM). A Central Composite Design (CCD) with three factors was used to study the effects of carrageenan (0.1-0.2%), gum Arabic (0.1-1.0%) and corn starch (1.5-2.5%) on the sensory characteristics of egg tofu. Eight trained panelists performed quantitative descriptive analysis. Analysis of Variance (ANOVA) showed that several of the response variables (egg aroma, starchiness, cohesiveness, astringency and aftertaste) were significantly different (p<0.05) and that the coefficients of determination (R 2 ) were in the range of 0.77-0.96. Based on the superimposed plot to determine the overlay region for sensory attributes, the formulation of egg tofu with desired sensory quality was obtained by incorporating 0.12% carrageenan, 0.61% gum Arabic and 2.00% corn starch. The best sensory performances were set as a goal for each response and the results showed that the optimum predicted response values obtained for egg aroma, starchiness, cohesiveness, astringency and aftertaste were 62, 88, 37, 34 and 60 mm respectively.
Introduction
There are various types of soy tofu available in the market such as dry tofu, firm tofu, soft tofu, silken tofu and filled (packed) tofu. Soy tofu has been consumed in Asia for thousands of years and it is known as traditional foods. Nowadays, egg tofu is getting popular among Asians. Egg tofu, which is also known as "Japanese tofu", is a savory tofu that is yellowish in color and possesses a strong egg aroma. Egg tofu is formed by the gelling process of whole eggs and soymilk (or seasoning) in the presence of a coagulant, such as Glucono-Delta-Lactone (GDL). The effect of hydrocolloids on the physico-chemical and sensory properties of soy tofu have been studied (Karim et al., 1999;Chang et al., 2003). However, there is less literature available on the incorporation of hydrocolloids in egg tofu. Therefore, it is desirable to determine the effects of the addition of carrageenan, gum Arabic and corn starch on the sensory characteristics of egg tofu.
A previous study has shown that the soy tofu gel becomes more stable with synergistic effect between carrageenan and soy protein (Baeza et al., 2002) and that 0.2% carrageenan enhances the water holding capacity of soy tofu (Karim et al., 1999). Gum Arabic is a complex branched hetero polyelectrolyte (Goycoolea et al., 1995). An addition of gum Arabic in an orange beverage formulation can trap the volatile components (Mirhosseinia et al., 2008). Starch is used in food applications because it is easy to cook at high concentrations and it can control moisture migration (Sajilata and Singhal, 2005). Moreover, starch can trap the flavor of food and reduce the astringent taste of phenolic acid (Naknean and Meenune, 2010;Troszynska et al., 2010). The astringent taste of egg tofu is due to the phenolic acids and isoflavones compounds in soymilk (Labbe et al., 2006). In the present study increased amount of cornstarch had significantly (p<0.05) reduce the astringent taste of egg tofu. However, the utilization of starch is limited due to its cohesive texture, heat sensitivity and opacity appearance in food (Sajilata and Singhal, 2005).
Sensory characteristics of food products are crucial for providing products with desired sensory properties. Sensory descriptive analysis can specify the intensity of sensory attributes in food products (Cartier et al., 2006). Several studies (Dutcosky et al., 2006;Gan et al., 2007;Brannan, 2009;Monaco et al., 2010;Yoon et al., 2011) have analyzed food properties using sensory descriptive analysis. The application of descriptive analysis requires extensive training to make sure that the panel agrees with the terminology and rating scales, so that the terms and scales can be used consistently to discriminate the samples.
Response Surface Methodology (RSM) is an effective statistical technique in optimizing product formulations because it can minimize the number of experiments and it is less time consuming. It consists of a statistical procedure that can be used to determine the relationships between the ingredients (independent variables or factors) and the response (dependent variables) (Castro et al., 2003). The optimization process is achieved by employing regression for the construction of empirical models that describe the product characteristics. Several studies have successfully used RSM to optimize ingredients in chestnut-based chips (Monaco et al., 2010), orange beverage emulsion (Mirhosseinia et al., 2008), pistachio nut roasting process (Kahyaoglu, 2008), traditional baked cassava cake (Gan et al., 2007), chocolate-flavored peanut-soy beverage (Deshpande et al., 2008), tropical mixed fruit jam (Abdullah and Cheng, 2001) and peanut milk-based whipped topping (Aminah et al., 1993). The objectives of this study was to determine the effects of carrageenan, gum Arabic and corn starch on the sensory characteristics of egg tofu using sensory descriptive analysis and to optimize the best formulation of egg tofu leading to the best sensory performance using RSM.
Experimental Design
Response Surface Methodology (RSM) was applied to determine the experimental design and the formulations of egg tofu were selected according to a Central Composite Design (CCD). Three independent variables, including carrageenan (X 1 ), gum Arabic (X 2 ) and corn starch (X 3 ), were tested against the following 14 response variables: Yellow color, egg aroma, soymilk aroma, egg taste, soy taste, starchiness, saltiness, cohesiveness, oiliness, firmness, surface roughness, astringency, aftertaste and overall acceptability. Coded variables and actual percentages of the ingredients used in the egg tofu formulations are shown in Table 1. Each independent variable had five coded levels (-1,682, -1, 0, 1 and 1.682) and 19 formulations including five replicated central points were required for the three variables.
Preparation of Egg Tofu
Soybean seeds were soaked in water, rinsed, ground with water at a bean: Water ratio of 1:3 and filtered with muslin cloth. The slurry was cooked at 100°C for 15 min. Egg tofu was prepared from a mixture of soymilk and fresh egg (2:1). Initially, carrageenan (0.1-0.2% w/w), gum Arabic (0.1-1.0% w/w) and corn starch (1.5-2.5% w/w) were added to the soymilk, which was then heated to a temperature of 80°C with continuous stirring until these components were completely dissolved. The mixture was then cooled to 40°C followed by the addition of a filtered fresh whole egg and Glucono-Delta-Lactone (GDL) (0.4% w/w) and the mixture was mixed well. The mixture was poured into a plastic container (5.5 cm diameter and 4.0 cm high) and it was steamed at 90°C for 20 min. Samples were cooled to room temperature and stored at 4°C prior to being used for sensory evaluation.
Descriptive Sensory Analysis
Eight panelists (1 male and 7 female) ages 24-39 year old who were students and staffs of Faculty of Science and Technology at Universiti Kebangsaan Malaysia, participated in the sensory evaluation test. The panelists were selected based on previous experience in descriptive sensory analysis, availability, motivated to participate and consumption of egg tofu. Panelists were trained over 12, 2 h per session, 2 times per week, for 3 weeks. In the training sessions, the Spectrum TM Descriptive Analysis Method (Meilgaard et al., 2006) was used to train panel and evaluate samples. Panelists were asked to generate terminology for both the commercial egg tofu and experimental samples in terms of sensory attributes (color, aroma, taste and texture) and fourteen sensory attributes were generated as shown in Table 2. The reference standard for each attributes and intensity selected were based on the Spectrum Intensity Scales for Descriptive Analysis (Meilgaard et al., 2006) ( Table 2). The intensity for each reference was vertically marked on a 150 mm unstructured line scale before rating the actual sample intensity according to the specific sensory attributes. The panelists then familiarized themselves with the references until they can consistently identify the intensity rate for each sensory attributes prior to actual sample evaluation. Adapted from (Meilgaard et al., 2006). Reference intensities were calculated as the mean rating of the group of eight panelists using a 150 mm unstructured line scale where 0 = none and 150 = very high intensity For the sensory evaluation, samples of egg tofu (5.5 cm diameter ×2.0 cm high) were taken from the refrigerator (4°C) and deep-fat fried using cooking palm oil obtained from Sime Darby Food and Beverages Marketing Sdn. Bhd. (Selangor, Malaysia) at 180°C for 3 min. Fried samples (5.2 cm diameter ×1.8 cm high) were then cooled to room temperature before they were cut into three portions. Samples were served on white translucent plates, coded with 3-digit random numbers. References for each sensory attributes were given to the panelists. Distilled water was provided for the panelist to rinse their palates between samples. Panelists then test the samples in individual booth in the sensory room.
Four or five samples were evaluated in a random order during each of the 4 sessions. Each sample was evaluated in duplicate on separate testing days.
Statistical Analysis
A central composite design was used and statistical analyses of the data for egg tofu containing carrageenan, gum Arabic and corn starch were performed using the Design Expert software package (version 6.0.10; State-Ease Inc., Minneapolis, MN, USA). The three-dimensional response surface showed the effects of independent variables (carrageenan, gum Arabic and corn starch) on the response variables (sensory attributes) using the regression polynomial equation. The following equation was obtained using RSM for predicting the response variables: Y X X X X X X X X X X X X = β + β + β + β + β +β + β + β + β + β Where: Yi = The predicted response; β 0 = The value of the fixed response at the central point of the experiment; β 1, β 2 and β 3 = The regression coefficients for linear effect terms; β 11, β 22 and β 33 = The regression coefficients for quadratic effect terms; and β 12, β 13 and β 23 = The regression coefficients for the interaction effect terms.
The statistical analysis of the models was evaluated with analysis of variance (ANOVA). The criteria of the model used to generate the response surface plot should be significant (p<0.05). The lack-of-fit must not be significant (p>0.05) and a coefficient of determination (R 2 ) greater than 0.70.
Optimization Procedure and Verification of Models
Numerical optimization was performed to determine the optimum level of independent variables (carrageenan, gum Arabic and corn starch) that produced desirable response variables (sensory attributes) of egg tofu. The contour plots for the response variables were created and they were superimposed to obtain the optimum formulation. The optimum formulation was selected and it was used to calculate the predicted values of response variables using the prediction equations. For verification, the predicted value of the response variables was statistically compared to the experimental value, which was the mean of three replicates.
Results
Based on visual observation, all produced egg tofu samples were light yellow. Different amounts of carrageenan, gum Arabic and corn starch added to the egg tofu significantly (p<0.05) affected five sensory attributes (egg aroma, starchiness, cohesiveness, astringency and aftertaste) based on the response quadratic model. Table 3 shows the predicted equation for each sensory attribute. Statistical analysis suggested that the model was adequate and had no significant lack-of-fit. Moreover, the model had a coefficient of determination (R 2 ) greater than 0.70, which was considered acceptable for sensory analysis (Abdullah et al., 1993) indicating that the variability of all responses was well explained by the models.
However, no significant differences (p>0.05) were observed in the other sensory attributes (yellow color, oiliness, soymilk aroma, firmness, surface roughness, soy taste, egg taste and saltiness). This can be due to the amount of carrageenan, gum Arabic and corn starch used in egg tofu formulations were not high enough to have a significant effects on these attributes.
Sensory Attributes of Egg Tofu
The sensory scores obtained for the egg aroma from 15 for mulations of egg tofu was in the range of 42 to 99 mm ( Table 1). The effect of carrageenan, gum Arabic and corn starch on the egg aroma can be visualized by the response surface plot shown in Fig. 1. An increased amount of gum Arabic caused an increased egg aroma (Fig. 1b), as its linear effect was positive at p<0.05. In addition, an increased amount of gum Arabic or carrageenan showed positive quadratic effect (p<0.05) on the egg aroma of egg tofu (Fig. 1a).
The sensory score range obtained for starchiness attribute of egg tofu containing carrageenan, gum Arabic and corn starch was 69 to 117 mm ( Table 1). The effect of carrageenan, gum Arabic and corn starch on the starchiness can be seen by the response surface plot shown in Fig. 2. An increased amount of corn starch showed a positive linear effect with the starchiness of egg tofu (Fig. 2b) and the positive quadratic effect of carrageenan on the starchiness of egg tofu is shown in Fig. 2a. Table 1 shows the mean sensory score for the cohesiveness, which was in the range of 18 to 68 mm. Table 3 clearly shows that cohesiveness was significantly (p<0.05) influenced by the following effects: Linear effect of carrageenan, gum Arabic and corn starch; quadratic effect of carrageenan; and interaction effects between carrageenan and gum Arabic and between carrageenan and corn starch. Figure 3 shows the effect of carrageenan, gum Arabic and corn starch on the cohesiveness of egg tofu through the response surface plot. An increased amount of carrageenan or corn starch resulted in higher cohesiveness of egg tofu (Fig. 3c).
The range of sensory scores for astringency attributes was 16 to 41 mm (Table 1). The response surface plot shows the effect of carrageenan, gum Arabic and corn starch on the astringent properties of egg tofu (Fig. 4). An increased amount of corn starch shows a negative quadratic effect on the intensity of the astringency of egg tofu ( Fig. 4b and 4c).
The range of sensory scores for aftertaste attribute was 53 to 94 mm (Table 1). The effect of carrageenan, gum Arabic and corn starch on the aftertaste properties of egg tofu is shown in the response surface plot (Fig. 5). An increased amount of gum Arabic or corn starch showed a positive quadratic effect on aftertaste of egg tofu (Fig. 5b).
Optimization of the Egg Tofu Formulation and Verification of the Model
The formulation of egg tofu was optimized to reduce the undesirable sensory properties such as aftertaste, astringency, starchiness and cohesiveness while inducing the egg aroma of egg tofu. Higher amount of corn starch had decreased (p<0.05) the astringent taste of egg tofu, however, it had significantly (p<0.05) increased the aftertaste, starchiness and cohesiveness properties of egg tofu. Meanwhile, increased amount of gum Arabic or carrageenan was found to increase (p<0.05) the egg aroma of egg tofu. Therefore, to obtain the optimum formulation of egg tofu, the region of the optimum condition was created by superimposing the contour plots of all the responses (aftertaste, astringency, starchiness, cohesiveness and egg aroma) in which the limits of each response have been determined (Fig. 6). The selection of any point within this region represented a combination of carrageenan, gum Arabic and corn starch that produced desirable sensory properties for each attribute of egg tofu.
Point A, which was selected in the superimposed region (Fig 6), indicated that the optimum formulation of egg tofu was 0.12% carrageenan, 0.61% gum Arabic and 2.00% corn starch. The predicted response and experimental values for egg aroma, starchiness, cohesiveness, astringency and aftertaste are shown in Table 4. For verification, the egg tofu with the optimum formulation was produced and analyzed. The experimental values for each response were statistically compared to those predicted by the equations of the model and the experimental and predicted values were not significantly (p>0.05) different.
Point B (0.12% carrageenan, 0.64% gum Arabic and 1.80% corn starch) was outside of the optimum region and there were no significant (p>0.05) differences observed between the predicted and experimental values (Table 4) for all response for this tofu formulation. Egg tofu formulations at point A (inside the region) and B (outside the region) had significantly (p<0.05) different results for the starchiness, cohesiveness and aftertaste response variables.
Discussion
Aroma substances are volatile compounds that are perceived by the odor receptor sites of the smell organ. The presence of polysaccharides in food could affect the release and retention of the volatile compounds (Naknean and Meenune, 2010). The linear and quadratic terms of gum Arabic and quadratic term of carrageenan showed a significant (p<0.05) positive effect which induced the egg aroma (Table 3). It is suggested that the presence of gum Arabic and carrageenan in egg tofu had trapped the volatile molecules by preventing the migration process of the molecules from the inside to the surface of food and thus retaining the egg aroma throughout the preparation of egg tofu. However, during the frying proses, the volatile molecules were released and contributed to the higher egg aroma. This is consistent with the results of (Terta et al., 2006), who reported that increased gum Arabic concentration had decreases the diffusion rate of aroma compounds through the interface of the solution. However, there was no change observed for egg aroma with increasing amounts of corn starch.
Starch is the most common form of carbohydrate in our diet. It is used in various food products as thickener and gelling agent (McWilliams, 1997). Generally, food containing starchform a thicken starch paste or a stiff gel and contributes to starchy taste. The linear, quadratic and interaction effect of carrageenan, gum Arabic and corn starch on the starchiness of egg tofu is presented in Table 3. The incorporation of corn starch and carrageenan in egg tofu formulation markedly affects the starchiness properties of egg tofu. Results showed that corn starch was the main contributor to the starchiness as evident by the significant (p<0.05) positive linear effect on the starchiness properties of egg tofu. Carrageenan also contributed to the starchiness of egg tofu where the predicted regression equation coefficient shows a significant (p<0.05) positive quadratic effect. Gum Arabic is non-starch polysaccharides (Annison et al., 1995) and therefore, incorporated of gum Arabic did not significantly (p>0.05) contribute to the starchiness of egg tofu.
Cohesiveness was measured by the degree of difficulty in breaking down the internal structure of egg tofu, which was varied with the amount of polysaccharides used. The linear term of carrageenan or corn starch had a significant (p<0.05) positive effect on cohesiveness. The aggregation and gelation behavior of these polysaccharides contributed to the changes of textural properties as similarly proposed by (Dickinson, 2003). Previous studies (Karim et al., 1999) have found that 0.2% carrageenan had result in the increase of cohesiveness of tofu. According to Venugopal (2008), the incorporation of protein in carrageenan gels increases the cohesiveness and reduces gel syneresis. This could be due to the excluded volume effects that increased the concentration of the hydrocolloid and electrostatic interaction between protein and carrageenan in solution (Baeza et al., 2002). In addition, starch also contributed to the cohesiveness as similarly reported by (Sajilata and Sighal, 2005). On the other hand, an increased amount of gum Arabic have a negative linear effect (p<0.05) on cohesiveness.
Astringency is a sensory attribute that is describe as a drying out, roughening and puckering mouth feel (Gawell et al., 2000). The intensity of astringency plays an important role towards acceptance of food products (Monteleone et al., 2004). The detection of astringency in egg tofu is due to the presence of phenolic acids and isoflavones compounds such as daidzein and genistein in soymilk (Lesschaeve and Nobel, 2005;Labbe et al., 2006). The quadratic term of corn starch showed a significant (p<0.05) negative effect on astringency of egg tofu (Tables 3). It indicates that, an increased amount of corn starch can effectively reduce astringency. Polysaccharides are well known for their ability to lower the intensity of taste and aroma when their concentration is above the coil overlap concentration (Troszynska et al., 2010). In this study, it seems that corn starch had reduced the intensity of astringency via binding the astringent compounds in the helical amylose through hydrophobic bonding as stated by (Boutboul et al., 2002). However, the presence of carrageenan and gum Arabic did not significantly (p>0.05) affect the astringent properties of egg tofu.
Aftertaste is described as the taste intensity that is perceived immediately after a food product is removed from the mouth (Meilgaard et al., 2006). In this study, the resulting aftertaste after the egg tofu had been swallows were a mixture of astringent, sour and stickiness feel inside the whole mouth which are not preferred by the panelist.The astringency felt can be cause by fenolic acids and isoflavon component found in soy milk (Labbe et al., 2006), while the sour taste was caused by glocoronic acid that was produced by GDL. Table 3 shows a positive significant (p<0.05) quadratic effect of gum Arabic or corn starch on the aftertaste of egg tofu. It shows that gum Arabic or corn starch had traped the astringent and sour taste in egg tofu and were released in mouth after the egg tofu had been swallows. The presence of carrageenan in egg tofu, however do not affect the aftertaste of egg tofu.
Conclusion
The application of a Central Composite Design (CCD) was useful in optimizing the formulations of egg tofu because it allowed the optimization of sensory properties when testing combinations of different amounts of carrageenan, gum Arabic and corn starch. Increased gum Arabic and carrageenan contents enhanced the egg aroma of egg tofu. The starchiness, cohesiveness and aftertaste properties of egg tofu were attributed mainly to corn starch. The overlap area with desired sensory attributes was generated and the optimum formulation of egg tofu contained 0.12% carrageenan, 0.61% gum Arabic and 2.00% corn starch. The experimental values were not significantly different (p>0.05) from the predicted value, thereby, indicating that the optimization equation obtained through CCD was valid and acceptable. | v3-fos |
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} | s2 | Determining the Prevalence and Seasonality of Fasciola hepatica in Pasture-based Dairy herds in Ireland using a Bulk Tank Milk ELISA
Background Fasciola hepatica is a helminth parasite of global importance in livestock, with major economic impact. However information on F. hepatica infections in Irish pasture-based dairy herds is limited. Therefore this study was conducted in order to determine the prevalence, seasonality and management factors associated with F. hepatica. A total of 319 Irish dairy herds were selected for this study. Bulk tank milk (BTM) samples were collected from 290 dairy farms on a quarter year basis, while from a further 29 dairy farms BTM samples were collected on a monthly basis to provide a more detailed pattern of F. hepatica exposure in Irish herds. BTM samples were analysed using a commercially available F. hepatica antibody detection ELISA. Furthermore, within-herd prevalence of F. hepatica was assessed in a subset of these 29 herds (n = 17); both individual serum samples and bulk tank milk samples were collected. Results A within-herd prevalence of ≤ 50 % was found for herds with negative bulk tank milk samples. The mean prevalence of the 290 study herds was 75.4 % (Range 52 %–75.1 %), with the highest prevalence being observed in November (75.1 %). The seasonal pattern of F. hepatica shows elevated antibodies as the grazing season progressed, reaching a peak in January. A significant association was found between F. hepatica and age at first calving. Conclusion This study demonstrates that F. hepatica is present in a large proportion of Irish dairy herds and provides a basis on which control practices, particularly in adult dairy cows, can be reviewed.
Background
The trematode Fasciola hepatica (liver fluke) is an important helminth parasite in livestock worldwide. The lifecycle of F. hepatica, similar to other trematodes, involves both a final (e.g. cattle, sheep) and an intermediate snail (Galba truncatula) host. F. hepatica outbreaks have a seasonal pattern with two waves of infection in summer and winter [1]. Infections with F. hepatica in livestock can result in significant economic losses, from decreased productivity, liver condemnations and mortality [2]. More specifically, in dairy cows and breeding heifers, these infections often remain subclinical, but result in reduced milk productivity and fertility [3].
Diagnosis of F. hepatica was previously based on coprological techniques alone, but with the advent of coproantigen, copro-PCR and enzyme linked immunosorbent assays (ELISA) for the diagnosis of F. hepatica in sera and milk, detection of F. hepatica has become more sensitive [4,5]. The use of ELISA on bulk tank milk (BTM) samples has allowed monitoring of an entire herd for F. hepatica status, and-when applied to individual milk samplesdetermination of the within-herd prevalence of F. hepatica [5,6]. Currently, control of F. hepatica is generally achieved using anthelmintic treatment, however more widespread application of herd ELISA status data will allow greater evidence-based control of F. hepatica at farm level [6]. This in turn will contribute to more appropriate and sustainable use of flukicide treatments in dairy herds [7], which could result in a reduction in anthelmintic use and reduce selection for anthelmintic resistance [8].
Previous international studies have been conducted to document the prevalence of F. hepatica infection in a number of Western European countries. The prevalence ranged from 37.3 % in Flanders, Belgium, to 76 % in the UK [9,10]. An Irish abattoir study reported that F. hepatica was present in 65 % of the livers of culled dairy and beef cows [11]. In a more recent study by Selemetas et al. [12] a prevalence of 67 % was found. The high prevalence of fluke in Irish dairy cows is not surprising given that Irish climatic conditions include abundant rainfall and lack of temperature extremes. This favours both the survival of F. hepatica and its intermediate host G. truncatula [13]. Additionally, the vast majority of Irish dairy farmers operate pasture-based seasonal-calving milk production systems [14] with the majority of cows calving during a compact period in spring from February to April [15], thereby maximizing milk production from low-cost grazed grass [16]. In general, Irish dairy cows are grazed outdoors on pasture from as early as February until as late as December, when weather conditions allow [17]. Irish cattle therefore have greater potential for exposure to and infection with F. hepatica compared to cattle reared in different livestock systems and climatic conditions. As only limited studies regarding the prevalence of F. hepatica infections exist in Ireland and heretofore no nationally representative study has been completed, the primary objective of this study was to determine the prevalence of F. hepatica in a geographically representative group of Irish dairy herds. Secondary objectives included determination of the within herd prevalence detectable by a specific bulk milk ELISA kit, and also investigation of the usefulness of this kit in highlighting seasonal patterns of liver fluke infestation in Irish herds.
Prevalence Study-selection of farms and sample collection
Dairy herds were selected from the HerdPlus® database containing 3,500 members, which represented 18 % of the Irish national dairy population in 2009. HerdPlus® contains records from dairy herds and is a breeding information decision support tool coordinated by the Irish Cattle Breeding Federation (ICBF). To yield sufficient study power a total of 500 dairy farms were randomly selected from HerdPlus®, with the prospect of yielding 300 dairy farmers. To join the 'HerdAhead' program a stratified sampling procedure based on herd size and geographical location was applied to select HerdPlus® dairy farms. The study population has previously been shown to geographically represent the Irish dairy farm population O'Doherty et al. [18]. A total of 312 farms volunteered to participate in the study, resulting in study sample size that yielded a 95 % confidence level and 5 % confidence interval based on a herd prevalence of 70 % (i.e. there is 95 % confidence that the results generated in the current study are representative of the national population of dairy herds).
In 2009, the bulk of these farms (n = 290 'Herdhead') were asked to submit a BTM sample on a quarterly year basis (23rd March, 8th June, 31st August, 2nd November), while 22 herds were selected to participate in a monthly BTM sampling programme. The 22 herds were selected on the basis that the farmers were members of the Dairy Management Information System ('DairyMIS') discussion group coordinated by Teagasc (Irish Agriculture and Food Development authority). These commercial farms were located in Munster in the south-west of Ireland. An additional 7 Teagasc herds were included in this sub-group called 'DairyMIS'.
BTM samples were collected using a standardized kit, which has previously been described in detail by O'Doherty et al. [18]. Briefly, this sampling kit contained a 500 ml jug, a 250 ml sampling container containing five milk preservative tablets (Broad spectrum Microtabs 2, D & F Control systems inc., USA), a submission form, sampling instructions and a cover letter informing the farmer of the required sampling date. To remind the dairy farmers of the sampling date a text message was forwarded the day prior to and on the day of sampling. The acquired bulk tank milk sample was then returned by express post with all samples received within 48 hours of sampling.
Between 2010 and 2012 BTM samples continued to be collected from the 'DairyMIS' herds (n = 29) using a slightly modified sampling kit. The sample kit contained a 50 ml sample bottle (Sarstedt, Germany), which contained a Broad Spectrum Microtab milk preservative tablet (D&F Control systems inc., USA) a submission form, sampling instructions and a cover letter informing the farmer of the required sampling date. The sampling dates were generally planned on the day of the 'DairyMIS' meeting on the first Wednesday of the month, allowing farmers to hand in the samples, otherwise express post was used for rapid delivery. On arrival to the laboratory, bulk tank milk samples were aliquoted into duplicate 2.5 ml micro tubes (Sarstedt, Germany), centrifuged at 20,000 g for 1 minute, de-fatted and the supernatant transferred to 1.5 ml microtubes (Sarstedt, Germany) and frozen at-80°C until further analysis.
Within herd prevalence: study population: selection of farms and sample collection A subset of these 'DairyMIS' herds referred to as 'Dairy17' herds (n = 7 in 2010 and n = 10 in 2012, with 5 herds collected in both years), were chosen to examine within-herd prevalence of F. hepatica. Herds were chosen on the basis of ELISA results from BTM samples which represent negative, low positive, moderate positive and high positive bulk tank milk readings. All milking cows were blood sampled by coccygeal venepuncture using a standard 18 gauge needle into plain vacutainers with no anticoagulant, within 15 days (mean = 9 days) of the bulk milk sample collection. Blood samples were centrifuged at 3000 rpm for 3 min within 12 hours of collection. Serum was aliquoted into 1.5 ml microtubes and frozen at-20°C degrees until further use.
Sample preparation and ELISA
F. hepatica analysis was completed using a commercially available ELISA kit, with a sensitivity [Se] and specificity [Sp] of 98 % (Ildana Biotech, Dublin, Ireland). All tests were carried out "in-house" according to kit manufacturer's instructions and previously described in detail by Selemetas et al. [12]. The assay is based on a recombinant mutant F. hepatica cathepsin L1 antigen (CL1) [19], which is produced as an inactive enzyme in Pichia pastoris. Plates are coated with 0.01 mg/ml antigen in carbonate coating buffer in alternate columns on a 96-well ELISA plate (EIA/RIA stripwell plates, Sigma-Aldrich, St, Louis, MO, USA) leaving each alternate strip uncoated. Samples were done in duplicate. Positive and negative bovine serum controls were supplied with the kit and used for S/P determination. The optical density (OD) reading of the uncoated well was subtracted from the reading of the coated well to yield a corrected OD. The ratio of the sample OD to the positive control OD was subsequently calculated to yield the S/P ratio. Likewise antibodies in serum were analysed using the same F. hepatica ELISA kit. A 1:20 ratio of bovine sera to sample buffer was used, 190 μl of sample buffer was mixed with 10 μl of control serum in each well.
A positive cut-off of 15 and 20 S/P was used for BTM and serum samples respectively.
Management and herd classification
A questionnaire was used to collect management data from 'HerdAhead' farms and has previously been described by Bloemhoff et al., [20]. Briefly farmers supplied information regarding general farm management, dosing and grazing management of cows, in-calf heifers, and calves (Table 1). Additionally herd size was downloaded from the ICBF database.
Study farms were allocated to three regions, described as Region-1 (West; counties, Donegal, Monaghan, Cavan, Longford, Leitrim, Sligo, Roscommon, Mayo, Galway, Clare, Kerry), Region-2 (East; counties, Louth, Meath, Westmeath, Kildare, Dublin, Offaly, Laois, Carlow, Wicklow, Wexford, Kilkenny) and Region-3 (South; counties, Tipperary, Limerick, Waterford, Cork) and outlined in Fig. 1. The map was created using ESRI Arcview 3.2 (Redlands, California, USA). The location of study herds was attributed to the centroid of the largest fragment of land for each herd according to the Land Parcel Identification System (LPIS) for 2008 (Fig. 1). The calving period of study herds was split into two categories i.e. spring-calving (i.e. majority of the herd calved between January and April) and mixed-calving (i.e. a proportion of the herd calved between January and April with remaining cows calved at other times of year). Livestock enterprise on the study herds was divided into two categories, namely dairy only (only dairy animals on the farm) or mixed enterprise (dairy plus beef and/or sheep). Flukicide treatment of adult cows, in-calf heifers and calves was previously described by Bloemhoff et al. [20], as was grazing season length.
Data analysis
Descriptive analysis was performed using PROC FREQ and PROC UNIVARATE (SAS version 9.3, USA). Graphical representations were generated and Student T-Test using Excel (Version MS Office 2003). Univariable and multivariable generalized estimating equation (GEE) analysis was completed using PROC GENMOD (SAS, version 9.3, USA).
The apparent prevalence (Ap) of 'HerdAhead' herds was calculated for each sampling date for F. hepatica ( Table 2). The overall annual prevalence for 'HerdAhead' herds is tabulated by recording at least one positive result of F. hepatica at any one sampling date. The true prevalence (Tp) for each herd was calculated using Rogan Gladen Epitools [21]. The seasonal trend in both Ap and Tp was tabulated. In addition, the distribution of low positive, moderate positive and high positive at each sampling point was completed. Student T-test using a one tailed distribution with two sample unequal variance, was used to compare the proportion of F. hepatica positive 'HerdAhead' herds in each Region (-1,-2,-3) at each sampling date (Table 3, Fig. 1).
As a first step a univariable analysis was performed using binomial dependent variable F. hepatica bulk tank milk result and independent variables, with herd included as a repeated measure. A binomial distribution was assumed and a logit link function used. An exchangeable correlation was applied for the analysis. Independent variables significant at
Results
The herds are a national representative sample spread across three regions (West, East and South) (Fig. 1). Due to missing data, 13 'HerdAhead' herds were excluded, leaving 277 'HerdAhead' herds for statistical analysis.
General management
All of the study herds had full access to pasture throughout most of the year (average 8 months, range 5-10 months). The mean herd size of study herds was 97.5 cows (range 28 to 400 cows). February (n = 170, 74.6 %) was the month when the majority of farmers turned out their adult cows, while housing was mostly performed in November (n = 137, 60.1 %). Close to 50 % of the study herds had a livestock enterprise of dairy only. An average of 24.5 first lactation heifers, were added to the adult herds (range 3-150) with an age of approximately 24.9 months (range 22-36 months).
Within-herd prevalence study
The 'Dairy17' herds were used to evaluate the relationship between the proportion of positive blood samples and the matching bulk tank milk S/P value (Fig. 2). In addition the individual blood samples of each herd were plotted in a boxplot, with attached bulk tank milk sample S/P result (Fig. 3). Each of the herds was categorized relative to the bulk tank milk result (Negative/None, low positive, moderate positive and high positive). The within-herd prevalence of F. hepatica of the 'Dairy17' herds was calculated as the number of individual cows positive at the >20 S/P value as a percentage of all cows serum sampled. Both Figs. 2 and 3 show a good agreement between the BTM S/P range and the percentage of cows positive to F. hepatica. The three herds that had a negative BTM result had less than half of the cows' serum samples positive to F. hepatica. In addition these negative BTM herds had considerably lower mean S/P values (S/P = 21), compared to positive BTM herds (S/P = 73). There was a good correlation between BTM S/P results and the individual blood S/P results in the subset of BTM positive herds (Fig. 3). BTM positive herds had a mean within-herd prevalence of approximately 88 % of cows seropositive to F. hepatica.
Prevalence
Results on prevalence from the 'HerdAhead herds (n = 277) are shown in Table 2 and Fig. 4 respectively. 12 farms were omitted from the study because of missing data. Positive herds were characterized as low (15- Monthly bulk milk sample S/P values were plotted for each herd, with the mean S/P value of all 29 farms also calculated and plotted (Fig. 5). The seasonality of F. hepatica infections in 'DairyMIS' herds was also calculated. A reduced number of herds were sampled in December, January and February, as cows were in the dry period. The majority of 'DairyMIS' herds were spring-calving herds
Multivariable analysis
The results of the multivariable analyses are shown in Table 4. Herds with heifers calving over 30 months of age were more likely (OR = 3.46) to be positive to F. hepatica compared to herds with heifers calving <24 months (p = 0.0269). No statistical regional differences were found in the multivariable analysis, however region was found to be significant in the univariable analysis. Table 3 shows that the proportion of herds positive to F. hepatica in Region-1 is higher than Regions-2 and-3. However the Student Ttest did not find a significant difference between Regions-1 vs.-2 (p = 0.075) and-2 vs.-3 (p = 0.393), although there was a significant difference between Region 1 vs. 3 (p = 0.0296).
Discussion
This study was used to determine the prevalence of F. hepatica in Irish dairy herds using a BTM F. hepatica ELISA. This study revealed that a large proportion (75 %) of the 'HerdAhead' study herds were positive to F. hepatica across the 2009 lactation. This is slightly higher than previously reported in an Irish abattoir study, which found that 65 % of the livers of culled dairy and beef cows were infected, but is consistent with a recent study carried out by Selemetas et al. [12], which used similar BTM ELISA methodology. In many countries in mainland Europe, F. hepatica prevalence is considerably lower [3,9,10,22]. In two abattoir studies a prevalence of 18 % and 28 % were found in Switzerland and in regions in Portugal and Spain, respectively [3,22]. Moreover in studies using bulk tank milk a prevalence of 37 % to F. hepatica was found in Belgium, while in Germany a prevalence of 23.6 % was found in adult cows [23,24]. However in the United Kingdom, the prevalence of F. hepatica (76 %) based on BTM samples was similar to that found in this study [2]. Ireland and the UK have a similar climate, which is predominantly influenced by the Atlantic gulf stream [25], which can result in abundant rain, moisture and limited temperature ranges that are favourable for the development and survival of both F. hepatica and its intermediate host Galba truncatula [26]. Moreover the management system used on many Irish dairy farms is mostly pasturebased. The majority of Irish dairy herds graze pastures fulltime for up to 10 months of the year, with grass comprising around 70 % of the diet [27]. These management factors have previously been found to be directly linked to the exposure to metacercariae [23]. The late housing (up to December) of dairy cows could also contribute to the exposure to F. hepatica as pasture infectivity is the highest during the autumn [23]. The UK study also established regional differences between F. hepatica prevalence [2]. In the current study, a significant difference was found between Region-1 (West) and-3 (South). This is not surprising given that the soil type is generally quite different between the two regions, with Region-1 containing a higher proportion of less well-drained soil compared to Region-3, where soil types are less heavy. It has also shown that farmers in Region-1 are more likely to use flukicides and anthelmintics for the treatment of F. hepatica [20] which suggests that these farmers in Region-1 are aware of F. hepatica as a problem in their area. The seasonal pattern found in this study shows a rise in September towards the end of the grazing season, which has previously been described in literature [23,28]. The late season rise in September could be explained due to increased infected snails in summer, which infect the pasture with metacercariae in summer/ autumn. Cows start picking up metacercariae around May or June, which could explain the increase of antibodies from August onwards. When these herds are housed and dried off, these antibodies generally drop. This might be explained due to the lack of metacercariae exposure during the dry period when spring-calving dairy cows are generally housed, and control of F. hepatica infection with an anthelmintic can be achieved [6]. However, from an epidemiological point of view, treatment might be more advantageously administered in August. However, due to the lack of availability of appropriate control measures for lactating dairy cows, treatment can often be difficult or impractical [11]. The limited number of flukicides available for dairy cattle all have withdrawal periods, which makes it more difficult to be used during lactation as valuable milk has to be discarded [23]. In addition the majority of flukicides available for dairy cows can only treat mature F. hepatica and therefore a subsequent treatment is required. To adhere to withdrawal periods the main time for flukicide treatments is during the dry period for spring-calving [3,10,29]. Moreover F. hepatica infections can reduce the reproductive performance and lifetime milk production of heifers. In-calf heifers calving around 2 years of age have a considerable advantage over heifers that calve later, as they are more likely to produce more milk and calves over their productive lifetime [30]. In this study, heifers that calved over 30 months of age were more likely to be in a herd positive for F. hepatica, compared to heifers that calved at 24 months and younger. Although reproductive performance in high-input highoutput systems is a very important factor, it is of more importance in seasonal calving systems. This is to maintain a compact calving pattern. Ideally breeding of the majority of the cows in such systems should be achieved within a 6 week breeding period in May and June [31]. While management and nutritional factors are the main determinants of reproductive performance in Irish dairy herds, optimal performance may also be hampered by F. hepatica infections during the breeding period. In this study, over half of the dairy herds were positive to F. hepatica during the crucial breeding month of June [31]. Therefore more research should be performed on the production losses due to F. hepatica infections in heifers and dairy cows. The ELISA clearly showed seasonal changes in bulkmilk F. hepatica antibodies. In addition there was a good correlation between the negative BTM results and the within-herd prevalence of F. hepatica positive animals in these herds. Therefore, the F. hepatica ELISA is a useful and easy to use tool for farmers and veterinarians.
Conclusion
This study shows a high prevalence of F. hepatica in Irish dairy herds at the end of the grazing season, but also a considerable amount during the grazing season. There is a need for appropriate control measures in adult dairy cows, especially during the lactation. Additionally the Ildana ELISA showed clearly the seasonal changes of F. hepatica and is therefore a useful and easy to use diagnostic tool. | v3-fos |
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} | s2 | Assessing sustainability and improvements in US Midwestern soybean production systems using a PCA–DEA approach
Abstract Documentation of on-farm sustainability in agricultural sectors is becoming an essential element to ensure market access. An assessment process was developed to help soybean farmers document practices and verifiable advances in community, environmental and economic sustainability. Technical difficulties in analyzing and summarizing such assessment data include a large number of practices, correlation in variables, and use of discrete measures. By combining non-negative principal components analysis and common-weight data envelopment analysis, we overcame these difficulties to calculate a composite sustainability index for each individual farm and for the farm group as a whole. Applying this method to assessment data from 410 US Midwestern soybean farmers gave average sustainability scores of 0.846 and 0.842 for the soybean-specific and whole-farm assessments, respectively. Scenario analysis examined the impact if the bottom 10% of growers adopted the top ten sustainability drivers identified by the analysis. The average sustainability score only increased by 2%, but the minimum score increased from 0.515 to 0.647 for the soybean-specific assessment, and from 0.624 to 0.685 for the whole-farm assessment, while the lowest 10th percentile increased from 0.635 to 0.819 for the soybean-specific assessment, and from 0.634 to 0.920 for the whole-farm assessment. These results suggest that significant advancements could be made through focused efforts to improve adoption of sustainable practices by soybean farmers at the lower end of the spectrum.
Introduction
While agricultural productivity has increased dramatically over the last half-century, meeting current and projected world food demand in a sustainable manner will be a major challenge for agriculture while facing global climate change (Beddington et al., 2012). In this context, sustainable agriculture continues to garner interest and increased investment in sustainability and agricultural intensification will be part of the response (Beddington et al., 2012;Garnett et al., 2013;Sustainable Agriculture Research and Education Program, 2015). Research and Education Program, 2015). For example, the 1977 Farm Bill officially defined sustainable agriculture as "[A]n integrated system of plant and animal production practices having a site-specific application that will over the longterm-(A) satisfy human food and fiber needs; (B) enhance environmental quality and the natural resource base upon which the agriculture economy depends; (C) make the most efficient use of nonrenewable resources and on-farm resources and integrate, where appropriate, natural biological cycles and controls; (D) sustain the economic viability of farm operations; and (E) enhance the quality of life for farmers and society as a whole." (National Agricultural Research, Extension, and Teaching Policy Act of 1977 (7 U.S.C. 3103 (19)). 1977. Available at http://www.gpo.gov/fdsys/pkg/ USCODE-2011-title7/html/USCODE-2011 This definition was reaffirmed in the 1990 Farm Bill (Food, Agriculture, Conservation, and Trade Act of 1990, Public Law 101-624. Title XVI, Subtitle A, Section 1603. Available at http://thomas.loc.gov/cgi-bin/ query/F?c101:4:./temp/~c101jiTXK1:e1108836) and the National Research Council adopted a similar definition in 2010 (National Research Council, 2010).
Consumers commonly express willingness to pay premiums for products with sustainability attributes (Blend and van Ravenswaay, 1999;Nimon and Beghin, 1999;Teisl, Roe and Levy, 1999;Onozaka and McFadden, 2011;Dong, Mitchell and Colquhoun, 2015), which reflects their concern about society and the environment. In order to capture potential price premiums or maintain market access, supply chain partners push for documentation of the current status of and improvements in agricultural sustainability and moreover for sustainability assessments so that they can make claims to differentiate their products from others (Lendle and Schaus, 2010;Murphy et al., 2014;Gibbs et al., 2015). Documenting input use and environmental outcomes at the farm level is often time consuming, seems overly intrusive to farmers and, consequently, is difficult and expensive to collect (Pennings et al., 2002). A more practical approach to assess on-farm sustainability should engage a large number of farmers in the process by using a practicebased, self-assessment that is easy to complete, not intrusive or cost prohibitive, and yet able to establish an accurate baseline of adopted practices. Such an approach can serve as an entry-point into a multi-tiered agricultural sustainability assessment as described in Figure 1.
A Tier 1, entry-level assessment of practices ( Fig. 1) is a foundational component of a multi-tiered approach to measuring sustainability that can then be linked with higher-tiered, outcome-based sustainability programs. It is a low-cost way to introduce sustainability to a large number of farmers and help them understand that sustainability is consistent with many of their own goals. Furthermore, it can ensure market access (Lendle and Schaus, 2010;Gibbs et al., 2015) or serve as a way to recruit farmers interested in more intensive approaches to pursuing sustainability.
Tier 2 agricultural sustainability programs pursue potential market premiums, but require more intensive data on input use and practices to provide accurate estimates of sustainability outcomes that reflect regional variability (Fig. 1). Such programs would collect relevant information from a smaller segment of farmers, but could use practice data from Tier 1 participants. Tier 3 programs represent a validation phase of assessing sustainability that builds a scientific foundation by experimentally measuring the linkages between practices and sustainability outcomes on a small number of farms. Because these research or show-case farms would be expensive to run, as they would collect and analyze a substantial amount of data, they would be few in number (Fig. 1).
In this context, our approach centers on the Tier 1 program. It develops a comprehensive set of good farming practices for Midwestern soybean farmers as a practical and concrete definition of agricultural sustainability for these farmers. Science-based practices are chosen that broadly address the three aspects of sustainability (environmental, economic and social) and are consistent with more official definitions of sustainability (e.g., those defined in the 1977 and1990 Farm Bill;and by National Research Council, 2010). After using this set of practices as a self-assessment survey to collect farmer adoption data, we apply a process to calculate an index number for each farmer; a composite indicator that measures practice adoption intensity as a sustainability metric. Such a composite indicator informs individual farmers how their sustainability practices and/or outcomes compare with their peers and identifies specific practices or outcomes to improve their sustainability performance. In our interactions with farmers who completed the assessment, they especially like the scorebased peer comparisons and specific feedback regarding practices they can adopt to improve. At the aggregate level, the distribution properties of the composite indicator describe how the farm population is performing as a whole and over time, which can be useful for developing and evaluating policies and programs to improve wholefarm sustainability.
The objectives of this study are first to develop and implement an entry-level sustainability self-assessment tool to document on-farm practices in Midwestern soybean production systems. We then use these data to: (1) evaluate a composite sustainability scoring system for Midwestern soybean farmers; (2) identify the practices that drive Midwestern soybean on-farm sustainability; and (3) illustrate how increased farmer adoption of these key practices affects individual farmer sustainability scores and the distribution of all sustainability scores.
Assessment tool development
Some key factors that guided development of the sustainability self-assessment included farmer engagement, a focus on science-based practices, cost-effective use of farmer time and anonymity. An important part of getting farmers to engage with the assessment process was to involve them directly in the development of the survey assessment, so that they became active participants in defining agricultural sustainability for themselves. Not only is this consistent with the social component of sustainability, but it also helps improve survey design and increase participation (Dillman et al., 1993). Farmers generally support a practice-based approach to agricultural sustainability that is consistent with most other agricultural programs, but the practices must be science-based with demonstrated sustainability benefits. Thus, the researchers, farmers and agricultural professionals involved in developing the assessment, focused on good management practices with positive outcomes that enhanced sustainability as documented in the scientific literature. Anonymity was important, as many farmers were concerned that the information would be used against them by government agencies for regulatory purposes, in the media by activists, or by companies to extract gain (Pennings et al., 2002;Bunge, 2014). Finally, the farmers involved emphasized that the assessment could not be time consuming or ask for information that required looking for information in farm records (Pennings et al., 2002), and so an entry-level assessment was developed as a low-cost approach to achieve wider participation (Fig. 1).
University research and Extension state specialists first developed a list of whole-farm and soybean-specific practices, guided by the expected outcomes for sustainable cropping practices described by the National Initiative for Sustainable Agriculture (NISA) (NISA, 2014a, b). All of the practices were drawn from accepted 'best management practices' in cash grain and soybean production guides and validated in peer-reviewed literature. These best management practices are promoted by Extension faculty and industry professionals annually in the Midwest, and are published annually in Extension recommendations (Schulte et al., 2005;Conley et al., 2011a, b;Laboski and Peters, 2012;Cullen et al., 2014) and regularly in refereed journal articles describing optimal crop and soybean management (Alexander et al., 2007;Conley et al., , 2011aJohnson et al., 2007;Hanna et al., 2008;Robinson et al., 2009;Davis and Conley, 2011;Furseth et al., 2011Furseth et al., , 2012Esker and Conley, 2012;Rowntree et al., 2013Rowntree et al., , 2014Rincker et al., 2014;Marburger et al., 2014).
These practices were converted into questions for a sustainability self-assessment survey, and then this survey was emailed to farmer leaders in state and national soybean associations around the US Midwest for feedback. Farmers were invited to provide specific feedback on the clarity and appropriateness of each question, whether the practice made sense as a 'good' practice for Midwestern soybean farmers, and to suggest additional practices that should be included. Next, a focus group was conducted in conjunction with a conference that most of these farmers attended. At this focus group, farmer leaders who were chosen with the help of state soybean associations, the North Central Soybean Research Program, and the United Soybean Board, provided specific comments and feedback, and as a result, several questions were changed, some dropped and new ones added. After that, a revised version of the sustainability self-assessment survey was sent around again for additional comments from farmers, researchers and industry leaders.
Following feedback from the focus group farmers and academic research (Pennings et al., 2002) on the types of sustainability questions that farmers would complete, the questions were constructed, so that a farmer would not have to consult records or give detailed information about input use. Also, most of the questions required 'yes' or 'no' answers, or choosing options from a list, so that the entire survey took less than an hour to complete.
Many practices do not pertain to a specific crop, but to many crops or to the farm operation in general, while others are specific to soybeans. As a result, the assessment included a soybean-specific section and a whole-farm section and was administered separately. This choice proved appropriate, since fewer farmers were willing to complete the substantially longer whole-farm assessment.
Based on farmer feedback, most farmers would not be willing to voluntarily share detailed information on their use of specific inputs, costs and profitability. As a result, some questions focused on maintenance of written plans or long-term records and attendance at farmer educational meetings and use of information. These practices are used as indicators of farmers who carefully planned input use, understood general agronomic principles and were aware of non-target and off-site impacts of their decisions, under the assumption that this planning and awareness led to appropriate use of inputs. Similarly, rather than asking for estimated carbon footprints, profitability, or other key outcomes, the self-assessment only asked if they had completed such estimates, under the assumption that these farmers would use this information appropriately. The final self-assessment contained questions about use of more than 200 practices and is available online (National Sustainable Soybean Initiative, 2014; National Initiative for Sustainable Agriculture, 2014a, b). Because the full set of questions and practices is quite long, only a short overview of each category is provided here. A complete list of the specific questions and practices is available (Dong et al., 2012).
Tables 1 and 2 list the main assessment categories along with example questions for the whole-farm section and the soybean-specific assessment, respectively. The wholefarm section contains questions on farming practices in eleven categories: farm soil and nutrient management, crop scouting, farm pest management, resistance management, chemical and worker safety, ecosystem restoration, farm production and management, farm learning and research, farm sustainability and community, farm economics and farm energy management. The soybean-specific assessment contains questions on farming practices in four categories: soybean production and management, weed management, insect management and disease management.
For the whole-farm assessment, farm soil and nutrient management focused on practices to manage wind and water erosion, such as conservation tillage, wind breaks, cover crops and filter strips, having nutrient management plans and following University nutrient use guidelines.
Controlling soil erosion to maintain long-term land productivity and appropriate use of crop nutrients to reduce off-site losses of nutrients are keystones of agricultural sustainability (Tillman et al., 2002;Montgomery, 2007). Crop scouting focused on scouting methods (i.e., formal versus informal) and the purposes of the scouting (i.e., to reduce pesticide use, or to monitor efficacy and known 'hot spots'), as well as the quality and long-term maintenance of records, and use of advanced scouting practices such as remote sensing. Scouting is a cornerstone of integrated pest management (IPM), a key part of agricultural sustainability (Tillman et al., 2002;Castle and Naranjo, 2009). Farm pest management was a long check list of best management practices for weed, insect and disease management promoted by Extension faculty and industry professionals, such as controlling weed escapes, preserving natural enemies and rotating crops to manage soil-borne diseases (Cullen et al., 2014). Resistance management to maintain the effectiveness of pest control focused not only on rotating modes of action, but also on planning, record keeping, and efficacy monitoring, as well as use of cultural control methods. Resistance management and IPM are important, not only for reducing excessive and unnecessary pesticide use, but also for maintaining the effectiveness of pest management practices besides pesticides (Tillman et al., 2002).
Chemical and worker safety focused on equipment maintenance, worker certification and safety training, use of protective clothing and equipment, the adequacy of on-farm storage facilities and practices to mitigate drift, non-target and other off-site impacts. Human safety is an important part of the social component of sustainability, often emphasized in corporate sustainability assessment systems (Labuschagne et al., 2005).
Ecosystem restoration not only included specific practices such as planting or protecting native ecosystems and pollinator habitat, but also activities such as meeting with ecologists, having a written restoration or pollinator protection plan, attending educational meetings, and knowing the natural ecosystems on the land they manage. Ecosystem restoration is important for enhancing biodiversity to increase ecosystem services (Tillman et al., 1996(Tillman et al., , 2002. Farm production management examined crop rotations, calibration and maintenance of field equipment and limiting soil compaction. These are commonly recommended practices to maintain soil productivity and prevent wasted or inefficient field operations (Schulte et al., 2005;Conley et al., 2011a, b). Farm learning and research focused on continuing education activities (educational meetings, field days and newsletters), conducting on-farm research and tracking practice efficiency using long-term field records and precision agriculture data. Continuing education activities and maintaining written plans and records indicate farmers, who carefully plan input use, understand general agronomic principles and are aware of non-target and off-site impacts of their decisions, which contribute to appropriate use of inputs. Farm sustainability and community included conducting sustainability assessments, use of on-farm recycling and involvement in a range of community activities, both agriculturally focused and more general. Recycling and conducting sustainability assessments demonstrate farms that have already been pursing sustainability, while community involvement shows a farmer's commitment to enhancing the local community as part of the social component of sustainability.
Farm economics focused on tracking costs and returns, use of marketing plans and strategies, as well as buying insurance and having disaster plans. These practices indicate farms that take the time to understand their farm and field level economics and so are more likely to achieve a higher level of economic sustainability, while insurance and disaster plans improve a farm's capacity to economically survive bad events (Nivens et al., 2002). Farm energy management focused on use of practices to improve energy efficiency, conducting energy audits, and use of renewable energy, and other practices to reduce net greenhouse gas emissions.
For the soybean-specific assessment, soybean production and management focused on crop rotation, soybean planting and establishment practices, practices to reduce soil erosion and improve soybean nutrient management, and key soybean harvest and storage practices to prevent storage losses. Soybean weed, insect and disease management used long check lists of best management practices promoted by Extension faculty and industry professionals, such as scouting to make control decisions, monitoring to determine control efficacy and to identify escapes, rotating modes of action and use of a variety of cultural control practices. These commonly recommended management practices provide a range of benefits, such as preventing wasted or inefficient use of inputs or field operations and maintaining the efficacy of pest control (Schulte et al., 2005;Conley et al., 2011a, b;Laboski and Peters, 2012;Cullen et al., 2014).
Data collection and summary
Farmer self-assessment response data were collected in two ways-using paper copies at extension meetings in Wisconsin and online. University of Wisconsin Extension regularly holds winter education meetings on soybean production around the state. At meetings in January-February, 2013, attendees were given paper copies of the self-assessments and time to complete them during the meeting. The meeting agenda included an explanation of what the self-assessment was, why they were being asked to complete it, assurances that it was voluntary and that their data would remain anonymous, and that the project was funded by the United Soybean Board (a grower funded organization) (Conley, 2013). Online versions of the self-assessments (National Sustainable Soybean Initiative, 2014a, b; National Initiative for Sustainable Agriculture, 2014) were publicized by the state soybean associations. The Illinois Soybean Association offered incentives for Illinois soybean growers to complete the assessments-US$25 each for the whole-farm and soybean-specific assessments. As a result of the data collection process, most of the responses were from Illinois and Wisconsin farmers, but Table 2. Categories and example questions for the soybean-specific sustainability assessment.
Category
Example practices and response options Soybean production and management (39 practices) . Which of the following practices did you use to maintain soil quality and quantity? (Check all that apply) • Managed pH levels to encourage optimal productivity of soybeans (target pH levels at least 6.2) • Used tillage and management practices which maintain residues on soil surface • Fields were worked perpendicular to dominant slopes of greater than 4% (e.g., contour strips) • Soybeans were planted no-till Soybean weed management (13 practices) . Which of the following practices did you use during this growing season? (Check all that apply) [select options shown] • Reviewed previous scouting records and planned a weed management strategy focused on key weed challenges • Reviewed previous herbicide records to ensure that herbicide carryover from a previous crop is not a concern • Rotated herbicide mode-of-action and/or utilized tank-mixtures to slow the development of herbicide resistance in accordance with manufacture and Extension recommendations • Monitored and managed field edges to limit weed seed migration into fields Soybean insect management (11 practices) . Which of the following practices did you use to manage insects? (Check all that apply) [select options shown] • Rotated classes of insecticides specifically to avoid the emergence of insect resistance • Used thresholds for soybean aphid (when 80% of the field averages 250 aphids per plant and population is increasing) • Biocontrol methods (beneficial insects, augmentative releases, or biological products) were used for insect control • Scouted soybean for insect pests at least weekly in a systematic pattern throughout the field and over the growing season • Culturally managed a soybean insect pest (e.g., wireworm, white grubs) with the crop planted prior to soybean in this field
Soybean disease management (seven practices)
. Which of the following practices did you use to control diseases? (Check all that apply) [select options shown] • Sampled for soybean cyst nematode populations (one sample per each 10 acres) • Planted a disease tolerant variety • Scouted soybean for disease weekly in a systematic pattern throughout the growing season • Used foliar fungicides only when risk to plant for disease infection was high because the assessment tool was online and mentioned at regional and national meetings, some of the farms are from other states. The final data used for this analysis included 410 completed soybean-specific assessments and 80 whole-farm assessments. Of the 410 completed soybean-specific assessments, 296 were from Illinois, 79 from Wisconsin, 16 from Minnesota, four from Iowa, three from Missouri, one each from Alabama, Indiana, Kansas, North Dakota and Pennsylvania, and seven unknown. Similarly, of the 80 completed whole-farm assessments, 33 were from Wisconsin, 31 from Illinois, nine from Minnesota, one each from California, Iowa, Kansas, Kentucky, North Dakota, Virginia and one unknown.
Given these data collection methods, these responses are a convenience sample, but generally representative of commercial cash-grain farmers. The average size of the farms for the analysis was 531 ha for the soybean-specific assessments and 603 ha for the whole-farm assessment. An average of 250 ha of soybeans was planted by farms in soybean-specific assessments. In 2012, 77% of the US soybean planted area was on farms planting at least 100 ha of soybeans, with an average of 272 ha of soybeans per farm (USDA, 2014), which is roughly consistent with the average for the soybean-specific assessments. In total, the soybean-specific assessments represent information from about 210,000 ha of farm land and more than 102,000 ha of soybeans, while the whole-farm assessments capture information from about 48,000 ha of land.
Summaries of responses for the all the practices is beyond the scope of this paper. A summary of assessment highlights prepared for a general audience is available (Knuteson et al., 2014), and so here we highlight responses to some practices from each category. Of the respondents, 92% reported following recommended nutrient management guidelines and 91% reported using reduced and no-till practices, but only 66% had a soil and water conservation plan. In addition, 88% used scouting practices and 85% used threshold for pest management decisions, but only 38% maintained scouting records for more than 2 years. However, 95% reported using cultural practices for insect, weed and disease management, yet only 48% reported managing pests in previous years to avoid insect pests in the current crop. In terms of resistance management, 80% reported rotating modes of action and 75% used diverse modes of action in rotational crops. Among respondents, 84% used technologies to minimize drift and 83% calibrated spray equipment to limit over application.
Ecosystem restoration practices were not highly adopted-34% were enrolled in conservation incentive programs, 23% attended ecological training sessions and 28% had developed an ecological management plan for their land, yet 73% used practices to conserve native wildlife. Of the respondents, 90% reported having a rotation at least 2 years, but only 42% reported cleaning and sanitizing their equipment at least twice per year. For learning and research, 78% attended informational sessions, 60% attended University-sponsored field days, and 68% conducted on-farm research with science-based partners. Of the respondents, 82% bought inputs locally, with 66% involved in community service organizations and 33% serving in local community leadership positions.
For farm economics, 82% completed a cost of production analysis by crop, but only 53% had a marketing plan and 47% had a farm succession plan, with 85% having property insurance and 79% having business liability insurance. Finally, 90% used practices to limit energy use to improve efficiency, while 53% had improved fuel efficiency of farm vehicles and equipment over time.
Methodology for data analysis
The soybean sustainability assessment includes a large number of discrete variables measuring practice adoption. In addition, these variables are highly correlated, since practices tend to be adopted together (e.g., scouting for insects, for weeds and for diseases or use of soil nutrient tests and of plant tissue tests to guide nutrient applications). Given the discrete and correlated nature of the data, we use the method developed by that combines principal component analysis (PCA) with data envelopment analysis (DEA) to construct a composite indicator.
PCA is a well-known multivariate analysis technique introduced and developed in the early 20th century (Pearson, 1901;Hotelling, 1933;Jolliffe, 2002). PCA is used to reduce a data set having a large number of interrelated variables into a smaller data set of uncorrelated variables, called principal components, that retains as much of the variation in the original data as possible (Jolliffe, 2002). Common-weight DEA has been used to construct composite indicators (Despotis, 2005;Hatefi and Torabi, 2010). However, the method of first uses non-negative polychoric PCA to process the data in order to address weaknesses of DEA. DEA is a widely used linear programming process. It allows benchmarking the performance of individual decision making units against a frontier based on the observed behavior and/or outcomes of other decision-making units (Cooper et al., 2007). The large dimension of the data set and correlations among variables reduce the discrimination power of DEA and introduces bias (Nunamaker, 1985;Dyson et al., 2001). The non-negative polychoric PCA reduces the number of variables, removes correlation, transforms discrete variables to continuous principal components, and ensures that all principal component elements are non-negative. The resulting principal components are suitable for the common-weight DEA, which further increases the discrimination power of DEA (Despotis, 2002).
The specific mathematics of the non-negative polychoric PCA and common-weight DEA process are briefly summarized here. More details about the method can be found in . First, the PCA with a nonnegativity constraint on individual elements of the principal vectors is conducted on the polychoric correlations of practice variables. In the second step, common-weight DEA is conducted using the principal components obtained from PCA. The common-weight DEA approach finds the set of common weights by solving a mathematical programming model that minimizes the weighted sum of the average deviation of the common-weight DEA scores over all farms and the maximum deviation of the common-weight DEA scores from the basic DEA score among all farms. In contrast to basic DEA which chooses a farm-specific weight for each principal component, common-weight DEA chooses a single weight for each principal component that is equal for all farms, which improves the discriminating power of DEA (Despotis, 2002(Despotis, , 2005.
The method utilizing PCA for common-weight DEA generates a weight for each practice and a composite sustainability score for each farm that can be used to evaluate two factors, the importance of each practice in sustainability assessment and the level of each farm's sustainability level.
Alternative scenario
The weight for each practice in sustainability assessment indicates the relative contribution of the practice to the final sustainability scores. Non-adoption of the practices with the highest weights is a major reason some farms have low scores, and as such, these practices indicate to producer organizations, policy makers and other stakeholders sustainability practices to target for higher adoption. To illustrate this advantage, the analysis here first identifies the ten practices with the highest weights for the soybean-specific and for the whole-farm assessments. Next, an alternative scenario is developed in which the farms in the lowest 10th percentile of scores adopt these ten practices, and then the PCA-DEA analysis is repeated for both data sets with this alternative practice adoption profile.
Comparing results for this alternative scenario to the original results indicates how increased practice adoption by the lowest performing farms affects not only the scores for all farms, but also the distribution of scores for the sample population. This alternative scenario illustrates how a producer group could use this assessment process to identify practices to target for adoption, and then how analysis results would change if the group were successful in encouraging their lowest performing members to adopt these targeted practices.
Results and Discussion
Non-negative polychoric PCA and common-weight DEA were separately conducted on the 70 practice variables in the soybean-specific sustainability assessment metrics and on the 145 practice variables in the whole-farm sustainability assessment. This analysis gave each farm a sustainability score on each assessment. The average score was 0.846, with a standard deviation of 0.099 and range from 0.515 to 1.000 for the soybean-specific data (Table 3). Similarly, for the whole-farm data, the average score was 0.842, the standard deviation was 0.129, and the range was from 0.624 to 1.000. Though the average scores are essentially equal for the two assessments, the standard deviations and ranges differ.
To show how the distributions differ, Figure 2 depicts histograms of these scores separately for each data set. The histogram for the 410 soybean-specific scores shows a fairly smooth distribution with a mode just above the mean and a long lower tail that diminishes, while the histogram for the 80 whole-farm scores shows a large peak near 1.0 with over 25% of the farms scoring over 0.95, then a relatively flat tail stretching to the observed minimum. Some of the relative smoothness of the histogram for the soybean-specific scores is likely due to having 410 observations, versus only 80 for the wholefarm scores.
In terms of interpretation, the highest scores (1.000) are for those farms adopting the most practices on the assessments and all other farms are scored relative to these high adopters. The results for both the whole-farm and the soybean-specific assessments show that a substantial portion of farms have relatively similar levels of practice adoption, as most scores exceed 0.85 (Table 3). These farms could adopt a few more sustainable practices to further increase their scores. More importantly, however, a lower tail exists for both assessments, indicating substantial potential exists for these relatively low-performing farms to increase their adoption of practices. Because these low-scoring farms have the greatest potential for improvement, we illustrate the ability of this analysis method to document sustainability improvements by assuming the lowest performing farms in the lower tails Table 3. Statistical description of soybean-specific and wholefarm sustainability scores from farmer self-assessment conducted in 2012/13 in the US Midwest. Scores potentially range from 0 for the lowest level of sustainable practice adoption to 1 for the greatest level of sustainable practice adoption. adopt more sustainable practices, and then repeating the sustainability analysis for both assessments. An advantage of the PCA-DEA process used here is that it gives each practice in the survey a weight for its contribution to individual farm sustainability scores. Tables 4 and 5 respectively report the ten practices with the highest weights for the soybean-specific and the whole-farm sustainability assessments. For farms with high sustainability scores, adoption of these practices substantially contributes to their high scores, while farms with low scores rarely adopt these practices and could increase their scores by adopting these highly weighted sustainability practices.
Statistic
For the soybean-specific assessment (Table 4), six of the practices identified as important involve management practices associated with integrated pest and disease management, including different scouting activities and practices for judicious and appropriate use of pesticides. These practices are important for sustainability, as poorly managed pests can greatly reduce crop yields, but excessive or wasteful pesticide use generates unneeded human and environmental health impacts, while cultural control options can reduce the need for pesticides (Tillman et al., 2002;Cullen et al., 2014). Crop rotation practices were also identified as key drivers of sustainability, with farmers using longer and/or more diversified rotations given higher scores. Crop rotation has a variety of benefits, from increasing soil productivity to improved pest control (Laboski and Peters, 2012;Cullen et al., 2014). The final two practices, which were given the greatest weights, focus on planting and post-harvest activities. No-till planting has several advantages, such as lower energy use, less erosion and improved soil structure, but requires enhanced management to reduce yield loss, such as adjusting the planter to soil conditions to ensure good stand establishment (Conley et al., 2011a, b;Pittelkow et al., 2015). Grain dryers that recirculate air or stir grain should be avoided as they increase the risk for grain damage and loss during storage (Iowa State University Extension, 2008).
In the whole-farm assessment, practices associated with IPM, especially scouting, were again important. In addition, practices to enhance economic diversity were identified as key sustainability drivers, including longer crop rotations for a more diverse crop mix, livestock production and even maintaining hunting/tourist uses of land. Maintaining diverse sources of livelihood is recognized as important for economic sustainability (Robinson et al., 2015). Practices to keep management current were also important, such as receiving industry newsletters and conducting on-farm research with university specialists. Finally, the analysis identified efforts to reduce wind erosion as important for sustainability, a type of erosion usually not addressed in the Midwest where sheet and rill erosion are more of a problem (USDA-NRCS, 2010).
Regarding the other practices on the assessments, many soybean farmers had already adopted crop rotation and practices that conserve soil, including reduced and/or no-till cultivation and using contour strips to limit soil loss. In addition, many already used cultural control practices for insect, disease and weed management, and practices that limit energy use. Completing a cost of production analysis was also very common among soybean farmers. However, the analysis gave these practices less weight since so many farmers had already adopted them. The final weight for each practice is determined by the PCA weight, the DEA weight and the standard deviation of practice adoption among farmers . Though many practices such as crop rotation, reduced tillage and use of cultural control for pest management are clearly also important for sustainability, the analysis gives less weight to them because most farmers have already adopted them. Practices that most farmers already use have little variation among farmers and do not help differentiate farmers from one another, and so have lower weights.
To illustrate how changing the practice adoption profile of the farmer population impacts the sustainability scores, a hypothetical scenario was developed in which farms at the lowest 10th percentile were assumed to adopt the ten practices listed in Table 4 for the soybean-specific assessment and ten in Table 5 for the whole-farm assessment, and then the analysis repeated. Comparing results to the original scores indicates the effects of a targeted policy focused on improving sustainability of farms Table 4. Top ten practices driving sustainability scores for the soybean-specific assessment (the ten practices in the soybean-specific assessment with the greatest weights).
Practice Final weight
Avoided grain dryers that re-circulate air 0.1436 Correctly adjusted no-till planter for planting conditions 0.0973 Adjusted insect treatment for field conditions and weather 0.0876 Alfalfa part of cropping history during the last 6 yr 0.0661 Number of years between soybean crops in typical rotation 0.0593 Consulted seed supplier to determine growing condition and/or disease concerns when choosing varieties to plant 0.0579 Assessed bean leaf beetle populations prior to movement into soybeans using surveys of overwintering sites and legume spring feeding sites 0.0477 Used insecticidal seed treatment only if early season pests were likely 0.0468 Scouted soybeans for disease weekly in a systematic pattern 0.0425 Minor insect pests controlled only when threshold levels reached 0.0415 Table 5. Top ten practices driving sustainability scores for the whole-farm assessment (the ten practices in the whole-farm assessment with the greatest weights).
Practice Final weights
Maintained hunting or tourist lands to be economically diverse 0.1185 Kept written or electronic records of scouting information 0.0835 Had livestock species to be economically diverse 0.0792 Used scouting data from farm dealer/co-op to make field management decisions 0.0724 Received industry-sponsored crop updates or internet newsletters 0.0637 Had an approved pesticide mixing and loading facility 0.0587 Number of years in the farm's average crop rotation 0.0552 Used existing windbreaks to prevent wind erosion 0.0529 Used formal crop scouting methods that followed specific patterns 0.0509 Conducted on-farm research in collaboration with university specialists 0.0489 ranked at the bottom by the original assessment. Different farms and different practices could be selected for a hypothetical scenario depending on proposed policies and goals. Results for the original and hypothetical scenarios are first compared by examining the shift in the distribution of scores for the farm population. In terms of population statistics, the sample average score for the hypothetical scenario relative to the original analysis increased from 0.846 to 0.862 for the soybean-specific assessment, and from 0.842 to 0.862 for the whole-farm assessment, about a 2% increase in both cases. Similarly, the minimum score increased from 0.515 to 0.647 for the soybean-specific assessment, and from 0.624 to 0.685 for the whole-farm assessment, which decreased the standard deviation, from 0.099 to 0.072 for the soybean-specific assessment and from 0.129 to 0.101 for the whole-farm assessment. The changes in the mean of the lowest 10th percentile farms are much larger, which increased from 0.635 to 0.819 for the soybean-specific assessment and from 0.634 to 0.920 for the whole-farm assessment.
To make shifts in score distributions easier to discern, empirical histograms are converted to parametric probability distributions. Specifically, a beta probability density function is assumed, since it is a flexible distribution ranging between 0 and 1. The observed sample mean and standard deviation of the scores are used to calculate the implied shape parameters of the beta density with a range of 0-1 using the equations reported in Evans et al. (2000). Figure 3 plots the probability density functions for the scores for the original and the hypothetical scenarios for both the soybean-specific and the wholefarm assessments. The changes in the hypothetical scenario affect most farm scores. As expected, the lower tail shrinks, with a subsequent increase in density in and around the mean and mode, as indicated by the arrows, and the upper end remains anchored at 1.0. However, there is a small reduction in the density just below 1.0, Figure 3. Estimated beta probability density functions for the distribution of sustainability assessment scores for the soybean-specific and whole-farm assessments for the current and alternative scenarios.
implying that farmers with relatively high scores in the current scenario would have slightly reduced scores for this alternative hypothetical scenario. This result occurs because of the relative nature of the scores and weights. Some farmers in the lowest 10th percentile surpass those who previously had relatively high scores after adopting the ten practices, causing those farmers' ranks and scores to decrease. In addition, with certain practices now more widely adopted by farmers, the weights for these practices decrease because these practices do not help differentiate farmers from one another as much as before, although the effect is relatively small.
To better illustrate the impact of the alternative scenario at the individual farm level, Figure 4 plots the sustainability score change for each farm relative to its original score for both the soybean-specific and the whole-farm assessments. As expected, farms with lowest scores for the current scenario had the largest increase in their sustainability score, with this score increase becoming smaller as the original score increased. As a result, both plots in Figure 4 show a strong downward slope. This score increase for those currently with the lowest scores drives the rightward shift of the lower tail in Figure 3. Figure 4 also shows that for many farms, their scores actually decrease, with the magnitude of this decrease generally being larger for those with greater scores for the original scenario. These changes account for the increased density of scores in and around the mean and the slight leftward shift of the upper end of the distribution evident in Figure 3. Finally, Figure 4 shows that there remain a few farms with a score of 1.0 for the current scenario that do not have a score change for the alternative scenario, so that the distribution remains anchored at 1.0 as evident in Figure 3.
Conclusions
This paper evaluated a data collection process and novel analysis method for calculating a composite sustainability Figure 4. Change in the sustainability assessment score for individual farms under the alternative scenario relative to the current scenario plotted against the original sustainability assessment score for the soybean-specific and the whole-farm sustainability.
scoring system, using it to identify the top practices for improving the sustainability of Midwest soybean production, and illustrated how sustainability scores would change if the lowest performing farmers adopted these top practices. The bottom-up approach used in setting up the sustainability assessment tool required participation of various stakeholders. A key to enhancing grower engagement in sustainability assessment and improvement is the simplicity of the process coupled with providing solid, data-driven results. Extensive voluntary data collection from Midwestern soybean farms in Illinois and Wisconsin is a strong indication of farmers' willingness to participate in such an assessment, as long as they are engaged at the beginning of the process. By identifying and highlighting specific practices growers could implement to increase their sustainability, this analysis gives growers credible feedback and helps advance sustainability systems. Furthermore, the analysis illustrated how targeted policies might impact farmers with lower scores.
It is important to note that this sustainability metric is a relative measure, quantifying the adoption intensity of good farming practices relative to the best farmers in the sample; it is not an absolute measure. To document progress over time, an 'ideal farm' that takes all practices at the most sustainable level could be set up as a frontier benchmark. Farms could then be compared with the ideal farm over time, which would allow the examination of improvement of their sustainability scores . Since sustainability-practice weights are determined to differentiate farmers from one another, widely used and unused practices receive less weight, even though they may be important for sustainability. As a result, this approach should be combined with other approaches to anchor results with some absolute measures, even as simple as adoption percentages for practices (Knuteson et al., 2014).
In terms of research, an important issue is how to best incorporate measures or estimates of key outcomes such as soil erosion, nutrient leaching or greenhouse gas emissions. Indeed, because farmers commonly mention their desire for a process that connects sustainability results to economic outcomes such as cost or net returns, this is an active area of research .
Nevertheless, these results suggest that Midwestern soybean farmers have successfully integrated many best management practices into their farm operations that advance them along the sustainability continuum, but key practices exist that farm leaders currently use and that many farmers could adopt to improve their sustainability. Many of these practices concern improving pest and disease management, with practices to improve crop rotation, to enhance farm economic diversity, and to reduce soil erosion also identified. Educational efforts and other policies should be targeted at increasing adoption of these top whole-farm and soybean-specific practices, as they are currently the key drivers for moving Midwestern soybean growers forward along the sustainability continuum. | v3-fos |
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} | s2 | Effect of Different Inclusion Level of Condensed Distillers Solubles Ratios and Oil Content on Amino Acid Digestibility of Corn Distillers Dried Grains with Solubles in Growing Pigs
The purpose of this experiment was to determine and compare the digestibility of crude protein (CP) and amino acids (AA) in full-oil (no oil extracted) and de-oiled (oil extracted) corn distillers dried grains with solubles (DDGS) with different condensed distillers solubles (CDS) ratios. Six barrows (29.6±2.3 kg) fitted with ileal T-cannula were allotted into a 6×6 Latin square design. Each period was comprised of a 5-d adaption period followed by a 2-d collection of ileal digesta. The five test diets contained 62% DDGS as the sole source of AA. A nitrogen-free diet was used to measure the basal endogenous losses of CP and AA. Chromic oxide (0.3%) was used as an index in each diet. The results showed that CP and AA were very similar in 5 DDGS, but the standardized ileal digestibility (SID) of lysine (from 56.16% to 71.15%) and tryptophan (from 54.90% to 68.38%) had the lowest values and largest variation within the essential AA, which suggests reduced availability of AA and different levels of Maillard reactions in the five DDGS. The apparent ileal digestibility and SID of CP and most of AA in full-oil DDGS (sources 1 and 2) were greater (p<0.05) than de-oiled DDGS (sources 3, 4, and 5). Comparing the AA SID in the 5 DDGS, full-oil with low CDS ratio DDGS (source 1) had non-significantly higher values (p >0.05) than full-oil with high CDS ratio DDGS (source 2); however, the SID of most AA of de-oiled with low CDS ratios DDGS (source 3) were non-significantly lower (p>0.05) than de-oiled with high CDS ratio DDGS (source 4); and the de-oiled DDGS with middle CDS ratio (source 5) but with different drying processing had the lowest SID AA values. In conclusion, de-oiled DDGS had lower SID of CP and AA than full-oil DDGS; a higher CDS ratio tended to decrease the SID of AA in full-oil DDGS but not in de-oiled DDGS; and compared with CDS ratio, processing, especially drying, may have more of an effect on AA digestibility of DDGS.
INTRODUCTION
Corn distillers dried grains with solubles (DDGS) is a rich source of protein and amino acids (AA) for pig feed (Stein and Shurson, 2009b). However, the high variation in DDGS AA composition and relatively low AA digestibility may play a role in its limited use in swine diets (Fastinger and Mahan, 2006;Liu, 2011).
The ratio of wet distillers grains (WDG) and condensed distillers solubles (CDS) was considered to be one of most 2011; Ren et al., 2011). Furthermore, there have been no reports of AA digestibility in de-oiled corn DDGS with different CDS ratios.
Therefore, the objectives of the present experiment were to determine the AID and SID of AA and CP in de-oiled and full-oil DDGS with different CDS ratios when fed to growing pigs and to compare the difference of these values by their processing and chemical composition.
General
The China Agricultural University Laboratory Animal Welfare and Animal Experimental Ethical Inspection Committee (Beijing, China) reviewed and approved the protocols used in the study. Two of full-oil corn DDGS and three of de-oiled corn DDGS samples were collected at the same time from a beverage plant and a fuel ethanol plant, respectively. The DDGS (source 5) was dried by tube bundle dryer and cage mill flash dryer (about 1/2, respectively), while the DDGS (sources 3 and 4) were only dried by tube bundle dryer. The information and chemical composition of the samples are shown in Table 1 and 2, respectively.
Animals and experimental design
The experiment was conducted in the Metabolism Laboratory of Ministry of Agriculture Feed Industry Centre (China Agricultural University, Beijing, China), and was designed to measure the AID and SID values for CP and AA in the five sources of corn DDGS. Eight crossbred (Duroc× Landrace×Yorkshire) barrows (initial body weight [BW] = 29.6±2.3 kg) were fitted with a simple T-cannula near the distal ileum according to the procedures described by Stein et al. (1998). After surgery, pigs were housed individually in stainless steel metabolism cages (1.4×0.7×0.6 m) in an environmentally controlled room (20±2°C). Pigs were allowed a 2 week recovery before the experiment was initiated. During the recovery period, a nutrient balanced grower diet (18% CP) was provided. After the recuperation period, six healthy pigs with similar physiological status were chosen and allotted to a 6×6 Latin square design with 6 periods and 6 diets.
Six diets were prepared before the initiation of the experiment (Tables 3 and 4). In five DDGS diets, DDGS was the sole source of CP and AA. A N-free diet was used to estimate basal endogenous losses of CP and AA. Chromic oxide (0.3%) was included in the six diets as an indigestible index.
Sucrose was included at 10% in the DDGS diets and at 15% in the N-free diets to increase palatability. Soybean oil was added to increase energy density and reduce dust. Acetate cellulose (Chemical Reagents Company, Beijing, China), a synthetic fiber, was included in the N-free diet (4%) to mimic the concentration of crude fiber in the DDGS diets. As potassium and magnesium were lacking in the Nfree diets, these minerals were provided in the form of potassium carbonate and magnesium oxide, respectively. Vitamins and minerals were supplemented in all diets to meet or exceed the estimated nutrient requirements for growing pigs recommended by NRC (1998).
The daily feed allowance was equivalent to 4% of BW at the beginning of each experimental period and was divided into two equal amounts at 0830 and 1630 h and fed in mash form. The pigs had unlimited access to water through low-pressure drinking nipples. Each experimental period lasted 7 days. The initial 5 days of each period were considered as an adaptation period to the diets, and the remaining 2 days were used for ileal digesta collection in 9 h periods as described by Stein et al. (2006). A 200 mL plastic bag was attached to the ileal cannula barrel using a cable tie, and digesta flowing into the bag were collected. During the collection period, bags were removed whenever they were filled with digesta, and immediately stored at -20°C to prevent bacterial degradation of AA in the digesta.
At the end of the experiment, the ileal samples were thawed, mixed within animal and diet, and a subsample was taken and lyophilised in a vacuum-freeze dryer (Tofflon Freezing Drying Systems, Minhang District, Shanghai, China), ground through a 1 mm screen, and thoroughly mixed before a subsample was collected for chemical analysis.
Chemical analysis
All chemical analyses were conducted in duplicate and repeated if the results differed by more than 5%. Dry matter (DM), ether extract (EE), CP (N×6.25), ash, calcium (Ca) and phosphorus (P) content of the diets and corn DDGS were analyzed according to the procedures of the Association of Official Analytical Chemists (AOAC, 2007). Crude fiber, neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined using fiber bags (Model F57, Ankom Technology, Macedon, NY, USA) and a Ankom A200 fiber analyzer (Ankom Technology, USA) after an adaptation procedure as described by Van Soest et al. (1991). The concentration of NDF was analyzed using heat stable α-amylase and sodium sulphite without correction for insoluble ash. The gross energy (GE) of corn DDGS samples were measured using an Automatic Isoperibol Oxygen Bomb Calorimeter (Parr 1281 Calorimeter, Moline, IL, USA). Total starch was obtained using the glucoamylase procedure (AOAC, 2007). Color scores (L*, a* and b*) of all DDGS samples were obtained using Minolta Chroma Meter CR-400 (Minolta, Osaka, Japan). The mean color score was the average of 10 measurements. Low values for L* indicate a dark color, whereas high scores indicate a light color (0 = black; 100 = white). Greater values of a* and b* indicate a greater degree of redness and yellowness, respectively.
Amino acids in corn DDGS, diets and digesta were hydrolyzed with 6 N HCl at 110°C for 24 h and analyzed for 15 AA using an Amino Acid Analyzer (Hitachi L-8900, Tokyo, Japan). Methionine and cysteine were determined as methionine sulfone and cysteic acid after cold performic acid oxidation overnight and hydrolyzing with 7.5 N HCl at 110°C for 24 h using an Amino Acid Analyzer (Hitachi L-8800, Tokyo, Japan). Tryptophan was determined after LiOH hydrolysis for 22 h at 110°C using HPLC (Agilent 1200 Series, Santa Clara, CA, USA). The chromium content in the diets and digesta was measured using an Atomic Absorption Spectrophotometer (Hitachi Z-5000 Automatic Absorption Spectrophotometer, Tokyo, Japan) according to the procedure of Williams et al. (1962).
Calculations
Apparent ileal digestibility values for AA were calculated as described by Stein et al. (2007): where AA digesta is the AA concentration in the ileal digesta DM (g/kg), AA diet is the AA concentration in the diet DM (g/kg), Cr diet is the chromium concentration in the diet DM (g/kg), and Cr digesta is the chromium concentration in the ileal digesta DM (g/kg).
The endogenous loss/kg of dry matter intake (DMI, IAA end ) of each AA was determined from pigs fed the Nfree diet based on the following equation: where IAA end is the basal endogenous loss of an AA (g/kg of DMI), AA digesta is the concentration of that AA in the digesta, and Cr diet and Cr digesta are the chromium concentration in diet and digesta, respectively (g/kg of DM). The average IAA end for the 6 pigs was used to calculate the SID of AA in all diets.
The SID was calculated by the following equation:
Statistical analysis
Data were analyzed using PROC GLM (SAS Inst. Inc., Cary, NC, USA). Each pig was the experimental unit. Pig and period were considered random effects. The CONTRAST statement of SAS was used to compare the values from the full-oil DDGS source with the de-oiled DDGS source. Results were expressed as standard error of the mean. In all analyses, the differences were considered significant if p<0.05.
Ileal amino acid digestibility
The AID and SID of CP and AA in the 5 corn DDGS are summarized in Table 5 and 6, respectively. The AID for CP was 71.96% in DDGS source 1 (Table 5), this value was higher (p<0.05) than the values obtained for sources 3 and 5 (63.50% and 62.12%, respectively) but no different from sources 2 and 4 (67.76% and 66.34%, respectively). There were no differences among the AID of 5 DDGS in Arg, Phe, Thr, Trp, Val, Asp, and Tyr. For AID of most AA, DDGS source 1 was the greatest and source 5 was the lowest; sources 2, 3, and 4 showed intermediate values relative to sources 1 and 5. There were no differences for most AA among the two full-oil DDGS sources and separately among the three de-oiled DDGS sources, but there were significant differences in AID CP and AA (p<0.05) between full-oil (sources 1 and 2) and de-oiled DDGS (sources 3, 4, and 5) except threonine, asparagine, and tyrosine. The trend of SID for CP and AA was similar to AID. Among all of the essential AA analyzed, lysine and tryptophan had the lowest digestibility value. The SID for CP was 78.79% in DDGS source 1 (Table 6), this value was greater (p<0.05) than the value obtained for source 5 (68.62%) but no different from sources 2, 3, and 4 (75.14%, 70.69%, and 72.97%, respectively). There were no differences among the SID of 5 DDGS in Arg, Phe, Thr, Trp, Cys, and Tyr. For the SID of most AA, DDGS sources 1 and 5 had the greatest and lowest values, respectively, and sources 2, 3, and 4 were intermediate in respect to them. For the SID values of most AA, there were no differences between DDGS sources 1 and 2, and no differences among sources 3, 4, and 5. However, there were significant differences (p<0.05) between the full-oil DDGS (sources 1 and 2) and de-oiled DDGS (sources 3, 4, and 5).
Composition of distillers dried grains with solubles samples
The concentrations of CP, EE, NDF, ADF, P, ash, Lys, and the other AA in the DDGS used in this experiment were very similar or within the range of the previously published values (Cromwell et al., 1993;Pedersen et al., 2007;Stein et al., 2009a;Almeida et al., 2011;Anderson et al., 2012;NRC, 2012).
As expected, the concentration of EE in full-oil DDGS (8.66% to 10.40%) was greater than the de-oiled DDGS (2.50% to 3.22%). The relative low oil content of de-oiled DDGS were similar to the 2.7% fat content (DM basis) reported by Saunders and Rosentrater (2009). As the oil was removed in de-oiled DDGS, GE was decreased and concentrations of CP and fiber were increased slightly or proportionately (Jacela et al., 2011).
In this study, as the CDS ratio increased, the color of DDGS sources 2, 4, and 5 were darker (L* decreased) than sources 1 and 3, respectively. It was not expected that DDGS source 5 was a little darker than source 4 although its CDS ratio was lower than source 4, and the reason may be that the processing, fermentation and drying equipment of source 5 were different with that of sources 3 and 4, and caused more heat damage (Maillard reactions). The fiber content in sources 2, 4, and 5 were lower than in sources 1 and 3. The above results agree with several published studies that DDGS would become lighter in color, and AA increased while fat decreased as the CDS content added to WDG reduced; the reason being that CDS contain more fat and total soluble sugars but less fiber than WDG (Kingsly and Ileleji, 2009;Kingsly et al., 2010;Probst et al., 2013). CDS contains more minerals than WDG (Jacela et al., 2011), therefore the concentrations of P and ash in sources 2, 4, and 5 were greater than sources 1 and 3, respectively. In this experiment, there were two "unusual" findings in de-oiled DDGS compared with the previous results in traditional full-oil DDGS. The processing and equipment between DDGS source 3 and 4 were similar to each other but different from source 5, so comparisons just focus on source 3 and 4. Firstly, DDGS source 4 was not greater and even had a less fat than source 3 as the CDS ratio increased, which does not agree with the published reports that more CDS resulted in a greater fat concentration in DDGS (Ganesan et al., 2008;Kingsly et al., 2010). The other finding was that the AA concentrations in DDGS sources 3 and 4 were nearly equal, which was not in agreement with previous results for full-oil DDGS where the AA and CP concentration would decrease as the CDS increased (Ganesan et al., 2008;Kingsly et al., 2010). To our knowledge, this is the first report of the comparison of composition in de-oiled DDGS with different CDS ratios. The reason of the unusual findings we suspect is the removal of oil makes it much less concentrated in CDS than that of no oil extracted process, which then makes the AA increase slightly in CDS of de-oiled DDGS.
Ileal amino acid digestibility
The average AID and SID of CP and AA for the 5 DDGS in this study are close to the values in NRC (2012). However, there was only one average value for the three kinds of corn DDGS (based on their oil level) in NRC (2012), which indicated that no trusted data for middle oil and low oil DDGS had been reported before the NRC (2012) published. In our study, the AID and SID of CP and most of AA in full-oil DDGS are greater than the NRC (2012), but for that of de-oiled DDGS, they were similar or less than the NRC (2012) values.
Similar to Stein et al. (2009a), of all the AA analyzed, the SID of lysine (from 56.16% to 71.15%) and tryptophan (from 54.90% to 68.38%) had the lowest values and largest variation, which suggests reduced availability of these two AA and the different levels of Maillard reactions in the five DDGS. The order of SID Lys from large to small in 5 DDGS sources trended according with the color (L*, a*, and b*) values within full-oil DDGS (source 1 vs 2) and deoiled DDGS (source 3 vs 4 or source 3 vs 5), respectively, but was not applicable for all the DDGS. This result indicated that DDGS color could be correlated with nutritional components, CDS ratio and AA digestibility of DDGS if the DDGS came from the same plant or plants had similar processing (Ganesan et al., 2008;Kingsly et al., 2010). However, color cannot accurately predict digestible lysine and other AA contents among DDGS sources which come from different processing (Urriola et al., 2013).
There were many reports on AA digestibility of full-oil (traditional) corn DDGS in pigs, and the values of sources 1 and 2 in our study had similar or in the range of previously reported values (Fastinger and Mahan, 2006;Almeida et al., 2011). However, for the de-oiled DDGS, very limited data can be referenced (Jacela et al., 2011;Ren et al., 2011).
Although there only 5 DDGS samples, considerable variation could be found in their AA digestibility. For the mean AID and SID of CP and most (more than 10) of AA, the sequence from greatest to lowest is DDGS source 1>source 2>source 4>source 3>source 5. There were no or almost no significant difference within the full-oil DDGS (source 1 and 2) and de-oiled DDGS (source 3, 4, and 5) in AA digestibility, respectively. But the AID and SID CP and most of AA in full-oil DDGS (source 1 and 2) were greater (p<0.05) than the de-oiled DDGS (source 3, 4, and 5). The AA concentrations in the 5 DDGS were very similar, so there are three possible reasons to explain the difference for SID of AA between full-oil DDGS and de-oiled DDGS. One is the difference of processing. To be compared with full-oil DDGS production, de-oil DDGS had undergone the oil extraction procedure and were exposed to more heat damage and Maillard reactions. The second reason may be due to the greater fiber content in de-oiled DDGS, as often reported, de-oiled DDGS have a higher fiber content, and high fiber such as NDF may reduce AA absorption and increase endogenous nitrogen and AA excretion, consequently decreasing the AA digestibility (Schulze et al., 1995;Lenis et al., 1996). The third possible reason is the inclusion of additional fat in the diet may delay gastric emptying, which could increase the AID of AA in growing pigs (Li and Sauer, 1994;Kil et al., 2011).
The AID and SID of CP and most AA for DDGS source 1 were greater than source 2 as the CDS ratio increased. These results are in agreement with previous research. The theory is that an increase CDS would increase total soluble sugars (reducing sugars) content (Kingsly et al., 2010) and the dryer temperature could lead to more Maillard reactions and lower AA digestibility (Martinez-Amezcua et al., 2007;Pahm et al., 2008;Stein et al., 2009a;Soares et al., 2012). But this theory maybe not be suitable for the de-oiled DDGS as indicated by the results of the present study. It is not clear why most SID AA increased as the CDS ratio increased in de-oiled DDGS (source 3 vs 4). We speculate that the reason also may lie in the oil extraction process. Because the oil extraction procedure removes the oil in the corn germ that then undergoes steaming and roasting (98±2°C), pre-pressing (95±2°C), desolventizing (110°C) etc., which leads to more heat damage in de-oiled corn protein and germ meal than that of full-oil DDGS, therefore, WDG may cause lower AA digestibility in de-oiled DDGS and could relatively reduce the negative effect of CDS on AA digestibility.
As for the DDGS source 5, it had a middle CDS ratio compared with sources 3 and 4, but the AID and SID CP and AA were the lowest in the three de-oiled DDGS. From the analyzed composition of sources 3, 4, and 5, we find that source 5 had the lowest EE concentration and L*, which indicated it had the highest oil extract efficiency and the most heat damage (Fastinger and Mahan, 2006). We suspect the reason for the lowest SID AA in source 5 was the processing and equipment used to produce source 5 which were different from sources 3 and 4, especially for the drying procedure. Source 5 was dried by both tube bundle dryer (about 1/2, using superheated steam, 285°C) and cage mill flash dryer (about 1/2, using saturated steam, 180°C to 200°C) to drying the WDG and CDS to DDGS, and then mixed together, while sources 3 and 4 were dried only by tube bundle dryer.
In conclusion, full-oil corn DDGS in this study had greater SID of CP and AA than de-oiled DDGS; higher CDS ratios tended to decrease the SID AA in full-oil DDGS but not in de-oiled DDGS; and compared to CDS ratio, processing, especially drying, may have a greater effect on AA digestibility of DDGS. | v3-fos |
2019-04-14T13:04:25.808Z | {
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} | s2 | Multivariate Statistical Analysis Applied in Wine Quality Evaluation
This study applies multivariate statistical approaches to wine quality evaluation. With 27 red wine samples, four factors were identified out of 12 parameters by principal component analysis, explaining 89.06% of the total variance of data. As iterative weights calculated by the BP neural network revealed little difference from weights determined by information entropy method, the latter was chosen to measure the importance of indicators. Weighted cluster analysis performs well in classifying the sample group further into two sub-clusters. The second cluster of red wine samples, compared with its first, was lighter in color, tasted thinner and had fainter bouquet. Weighted TOPSIS method was used to evaluate the quality of wine in each sub-cluster. With scores obtained, each sub-cluster was divided into three grades. On the whole, the quality of lighter red wine was slightly better than the darker category. This study shows the necessity and usefulness of multivariate statistical techniques in both wine quality evaluation and parameter selection.
INTRODUCTION
Wine is widely consumed in many countries around the world (Bentlin et al., 2012) and people are increasingly concerned with the quality of the wine. Some appraise wine quality by sensory tasting while others evaluate quality of wine by physicochemical analysis. Measurement of physicochemical index technology such as heterosexual natural isotopic fractionation and nuclear magnetic resonance technology, have been gradually developed (Jiang et al., 2008). With the improvement of measurement techniques, physicochemical analysis is being widely used.
The methods of physicochemical specifications analysis mainly include traditional statistical methods such as Principal Component Analysis (PCA), Cluster Analysis (CA), Discriminate Analysis (DA) and Decision Trees (DT), Artificial Neural Networks (ANN) and Support Vector Machine (SVM), which have been frequently used in the field of classification (Hernanz et al., 2007;Aly, 2005;Kavuri and Kundu, 2011;Jin, 2005;Osorio et al., 2008). Two principal components were grasped by using PCA and then wine samples were clearly clustered into two homogenous groups by using CA, which was sufficient to differentiate the wines produced with different clones (Burin et al., 2011). But previous researchers didn't take the clustering index weights into account. The quality of cluster is largely under the influence of index weights. Cluster weights reflect the importance of the index, which is the advantage of weighted cluster analysis. In addition, the new fuzzy clustering algorithm which defines indexes weights in the framework of Axiomatic Fuzzy Set (AFS) theory is based on Shannon Entropy (Zhang et al., 2009). With three-layer feed forward architecture, ANN of back propagation learning was applied to update weights (Shoemaker et al., 1991). The method of DA was used to distinguish wines from different countries based on a minimal number of the most important parameters (Römisch et al., 2006). ANN methods were used for the classification of Slovak white varietal wines with the aim to classify wines by different variety, producer, location and the year of production (Kruzlicova et al., 2009).
Technique for Order Performance by Similarity to Ideal Solution (TOPSIS), a Distance Comprehensive Evaluation Method, is one of the most common methods for problems involving multi-criteria decisions (Cruz-Ramiaírez et al., 2010). To achieve competitive edge in the market, TOPSIS method was performed to select fruits from superior locations in terms of total natural antioxidants of the fruit (Sun et al., 2011). But each indicator was given the equal weight, which can't explain the degree of importance of indicators. A comprehensive evaluation model of coal mine safety, established by the entropy weights and TOPSIS, was applied to evaluate safety conditions of production in four coal mines (Li et al., 2011).
In this study, we used Principal Component Analysis to eliminate the correlation between indicators. And then the wine samples were clustered by Weighted Cluster Analysis, where weights were determined by information entropy. In addition, in order to verify the accuracy of the weights, we used Back Propagation (BP) Neural Network to update weights. Finally, we used weighted TOPSIS method to evaluate the quality of various types of wine and determine the grade of wine. It is worth mentioning that the weights were respectively determined by information entropy method for red wine of the first category and the second category. Likewise, BP neural network was used to test the accuracy of the weights.
Data sources and original indicators:
Research data is quoted from the 2012 China Undergraduate Mathematical Contest in Modeling, with 27 kinds of red samples monitoring 12 parameters as a case study (http://www.mcm.edu.cn/problem/2012/2012.html).
PCA of indicators:
A widely used multivariate analytical statistical technique, Principle Component Analysis can simplify a set of dependent texture variables to a smaller set of underlying variables based on patterns of correlation among the original variables (Lawless and Heymann, 1999). PCA can use fewer new variables instead of the original variables with the largest variability (He et al., 2007).
Information entropy weighted clustering: Cluster
Analysis is a tool of exploratory data analysis to solve classification problems. The degree of association is strong between members of the same cluster and weak between members of different clusters (Burin et al., 2011). Cluster quality is largely under the influence of the weights of features. Shannon Entropy is used to defines indexes weights (Zhang et al., 2009).
Below are steps for weighted information entropy cluster: • Normalize the original data matrix. Let m stands for wine samples, n is located as physicochemical ( 1) Under the j-th index, value of i-th sample valuation is p ij : (2) • Calculate weights of the properties. Information entropy of j-th index is: (3) Below is the formula of j-th index of entropy weights w j : (4) • Use weights to calculate the squared Euclidean distance • Do clustering analysis using ward method with squared Euclidean distance • Analyze evaluation results We applied a BP neural network model in iterating weight calculated by entropy method for weights accuracy inspection. BP neural network, the most widely used neural network model, is a multi-layer network model of one-way communication (Xie et al., . Normalized data of the red wine's main constituent was regarded as input and weight determined by information entropy was regarded as output. Component weight calculated by information entropy is definitely accurate if there is little difference between iterative weights and initial weights. Comprehensive evaluation based on TOPSIS method: TOPSIS, developed by Hwang and Yoon (1981), is a simple ranking method in conception and application (Hwang and Yoon, 1981). The standard TOPSIS method attempts to choose alternatives that simultaneously have the shortest distance from the positive ideal solution and the farthest distance from the negative-ideal solution. Making full use of attribute information, TOPSIS provides a cardinal ranking of alternatives and does not require attribute preferences to be independent (Chen and Hwang, 1992;Yoon and Hwang, 1995). The evaluation object is ranked in accordance with the value of the relative degree of approximation. The bigger the value, the better the evaluation object.
RESULTS AND DISCUSSION
Analysis of the outcome of PCA: As is shown in Table 2, a total 89.06% of data information was explained by four principal components. So it was reasonable to take the principal components F 1 , F 2 , F 3 , F 4 to represent the original 12 targets to conduct the cluster analysis. The matrix of the red wine component score coefficients are represented in Table 3.
From Table 3, we knew that component 1 of the red wine contained information of anthocyanin, Tannins, total phenols, Flavonoids, DPPH Semiinhibition volume, which could be accordingly named taste factors; Component 2 of the red wine contained information of a* (D65), C (D65), which could be named chromaticity factors; Component 3 of the red wine contained information of H (D65), b* (D65), which could be named cool tone factors; Component 4 contained information of aromatic, L* (D65), resveratrol, which could be named incense factors.
Analysis of information entropy weighted cluster:
We calculated the entropy weights of four principal components of the red wine. The results of our calculation are shown in Table 4. The weights of principal components will be greater if more information is contained in the main ingredient. It indicates that the principal components are very important when they have high weights. As was shown in Table 4, we knew that the principal component 1 had the greatest impact on wines clustering.
In Table 4, we can see that iterative weights calculated by the BP neural network had a small difference from weights before iterating, which proved that weights determined by information entropy had a high accuracy.
We divided samples into different categories, based on the standard that the distance between the two classes was greater than 10 and the within-class distance was about 5.
Results for the red wine classification are shown in clustering tree (Fig. 1). According to the standard, we , 5, 8, 9, 14, 17, 22, 23, 24 and 27 and the second category contained samples 1, 3, 4, 6, 7, 10, 11, 12, 13, 15, 16, 18, 19, 20, 21, 25 and 26, respectively. Values of all the physicochemical indicators but the color ones of the first category were greater than those of the second category. It showed that the first class of the red wine was relatively dark and the tone was darker. It had sour taste and rich aroma. However, the second class was lighter in color and partially brick red. It tasted thin and had less odor than the first class.
Result of TOPSIS comprehensive evaluation:
We calculated the entropy weights of four principal components of the red wine. As shown in Table 5, iterative weights calculated by BP neural network had a small difference from weights before iterating, proving that weights determined by information entropy had a high accuracy. We conducted TOPSIS comprehensive evaluation regarding the two categories of red wine samples. The optimal value stood for the relative closeness to the ideal solution can be calculated from negative and positive ideal solution. To be exact, the optimal value stood for the quality of wine. The higher the score was, the better the wine was. The wine grading standard can be determined according to the optimal values and their distribution. According to the distribution of optimal values of wine samples, we divided values into three intervals. Meantime, every category of wine was divided into three levels. Each interval corresponded to a particular grade of wine quality. Grade I indicated the worst quality, while Grade III stood for the best quality. The grading standards of red wine are shown in Table 6. Table 7 shows scores of wine samples. If optimal value was less than 0.5, then the solution of corresponding wine samples approached the negative solution. On contrast, if optimal value was more than 0.5, the solution of corresponding wine samples approached the positive solution. From Table 7, we found that most optimal values were less than 0.5. So we concluded that the whole qualities of most wine samples were generally not high. And the distance between the value of the best wine and that of the worst wine in three categories were bigger than 0.5, which showed good discrimination of using TOPSIS method. Based on optimal values, we graded the red wine according to the standard shown in Table 6. Table 8 shows wine classification results. In the first category of red wine, wine of grade I accounted for 50%, wine of grade II accounted for 30%, wine of grade III accounted for 20%; In the second category of red wine, wine of grade I accounted for 17.65%, grade II accounted for 52.94%, grade III accounted for 29.41%. On the whole, with red wine, the quality of the lighter category was slightly higher than the darker category.
CONCLUSION
We grasped the principal components of the physicochemical indicators using Principle component analysis. And then we calculated each main component weight based on the method of entropy weights. To verify the accuracy of the weights calculation, we used BP neural network model to iterate weights. The results of BP neural network showed that there were narrow difference between iterative weights and initial weights, which proved that weights determined by information entropy had a high accuracy. After weights accuracy was verified, we clustered red wine samples into two categories. Weighted cluster analysis worked well in clustering. We applied the weighted TOPSIS method to objectively evaluating the quality of various types of wine, which showed good discrimination in assessment of wine quality. The method has displayed good practicality and can be used in cases where there are no other objective criteria available. It steers clear of the thorny problem of determining subjective weights in general evaluation and conducts a comprehensive evaluation of the quality of the wine, playing an important role in the promotion of scientific, standardized and institutionalized evaluation of the wine quality. What's more, the model can be widely used in food and other quality evaluation. | v3-fos |
2016-05-12T22:15:10.714Z | {
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} | s2 | Chemical, Thermal and Spectroscopic Methods to Assess Biodegradation of Winery-Distillery Wastes during Composting
The objective of this work was to study the co-composting process of wastes from the winery and distillery industry with animal manures, using the classical chemical methods traditionally used in composting studies together with advanced instrumental methods (thermal analysis, FT-IR and CPMAS 13C NMR techniques), to evaluate the development of the process and the quality of the end-products obtained. For this, three piles were elaborated by the turning composting system, using as raw materials winery-distillery wastes (grape marc and exhausted grape marc) and animal manures (cattle manure and poultry manure). The classical analytical methods showed a suitable development of the process in all the piles, but these techniques were ineffective to study the humification process during the composting of this type of materials. However, their combination with the advanced instrumental techniques clearly provided more information regarding the turnover of the organic matter pools during the composting process of these materials. Thermal analysis allowed to estimate the degradability of the remaining material and to assess qualitatively the rate of OM stabilization and recalcitrant C in the compost samples, based on the energy required to achieve the same mass losses. FT-IR spectra mainly showed variations between piles and time of sampling in the bands associated to complex organic compounds (mainly at 1420 and 1540 cm-1) and to nitrate and inorganic components (at 875 and 1384 cm-1, respectively), indicating composted material stability and maturity; while CPMAS 13C NMR provided semi-quantitatively partition of C compounds and structures during the process, being especially interesting their variation to evaluate the biotransformation of each C pool, especially in the comparison of recalcitrant C vs labile C pools, such as Alkyl /O-Alkyl ratio.
Introduction
Currently, the European Union (EU-27) is the world's leader in the wine sector, with 55% of the global vineyard and around 60% of wine production, France, Italy and Spain being the largest world wine producing countries [1], which implies a great generation of wastes, whose structures [16]. Moreover, correlation of DTG profiles and time variation in samples can help to identify specific types and organic matter transformations [17,19].
Fourier transform infrared spectroscopy (FT-IR) constitutes an useful technique to study the development of the composting process, since it can help in the qualitative characterization of the principal chemical groups of the raw organic matter [20,21,22]. With FT-IR spectra, the relative intensity of the typical bands of components can be selected and used to follow the composting process, monitoring the transformation of the organic and inorganic materials.
Solid-state 13 C magnetic resonance spectroscopy (solid-state C 13 NMR) constitutes one of the most powerful tools for studying the carbon composition of organic matter [21], since this technique allows to investigate samples without any need of extraction and fractionation and thus, to collect direct information on the structural characteristic of whole organic matter during the composting process [23]. So, several studies have proposed the cross-polarization and magic angle spinning (CPMAS) 13 C NMR technique for monitoring the stabilization process during composting, analyzing the complete sample and the extracted humic acids [13,19,23].
Therefore, the main aim of this work was to study the development of the co-composting process of winery-distillery wastes with animal manures using the traditional analytical methods together with advanced instrumental methods (thermal analysis, FT-IR and CPMAS 13 C NMR techniques) to ascertain organic matter changes during the process. The quality assessment of the final composts obtained was also carried out.
Compost procedure
Three different piles (A, B and C) were prepared by mixing wastes from the winery and distillery industry (grape marc (GM) and exhausted grape marc (EGM)), with two different animal manures (cattle manure (CM) and poultry manure (PM)). GM was obtained from a winery placed in Bullas (Murcia, Spain), EGM was collected from an alcohol distillery placed in Villarrobledo (Albacete, Spain); CM was obtained from a cattle farm in Santomera (Murcia, Spain) and PM was collected from a poultry farm of laying hens located in Orihuela (Alicante, Spain). The main characteristics of the raw materials are shown in Table 1.
Then, the raw materials were thoroughly mixed, and each mixture obtained (about 150 kg) was separately placed in thermo-composters with and efficient volume of 350 L. The thermocomposters, 70 cm x 70 cm x 85 cm, were made of high-density polyethylene (HDPE) and have a lateral system of natural ventilation to guarantee aerobic conditions. The moisture of the piles was controlled weekly by adding the necessary amount of water to obtain a moisture content not less than 40%.
In order to compost the maximum weight of winery-distillery wastes with a sufficient amount of animal waste, as nitrogen and micro-organisms source, the composting mixtures were prepared on a dry weight basis (fresh weight basis in brackets) in the following proportions: temperature was close to the ambient and re-heating did not occur. Then, composts were left to mature over a period of two months, approximately.
Samples were taken at seven sites of the pile from the whole profile (from the top to bottom). Composite representative samples were obtained after mixing and homogenizing thoroughly the previous seven sub-samples.
Each sample was divided into two fractions: one of them was dried in a drying-oven at 105°C for 24 h to determine the moisture content and the second was air-dried and ground to less than 0.5 mm for the rest of the classical analytical determinations. For the thermal and spectroscopic analyses, composting samples were air-dried, ground in an agate mill, then sieved through a 0.125 mm mesh, and milled again with an agate mortar.
Classical determinations: chemical analyses
In the raw materials and in the composting samples, EC and pH were analyzed in a 1:10 (w/v) water-soluble extract. Organic matter (OM) was assessed by determining the loss-on ignition at 500°C for 24 h; water-soluble organic carbon (WSC) was determined in a 1:10 (w/v) water extract by using an automatic carbon analyzer for liquid samples (TOC-V CSN Analyzer, Shimadzu).
Total organic C (TOC) and total N (TN) were determined were determined by dry combustion at 950°C using a Leco TruSpec C-N Elemental Analyzer (Leco Corp., St. Joseph, MI, USA). The humic-like fractions (extractable organic carbon (Cext), fulvic acid-like carbon (Cfa), and humic acid-like carbon (Cha)) were also determined using an automatic carbon analyzer for liquid samples (TOC-V CSN Analyzer, Shimadzu); these extractions were carried out according to the methods used by Bustamante et al. [8]. After HNO 3 /HClO 4 digestion, P was assessed colorimetrically as molybdovanadate phosphoric acid, Na and K were determined by flame photometry (Jenway PFP7 Flame Photometer, Jenway Ltd., Felsted, UK) and Ca, Mg, Fe, Cu, Mn, Zn by inductively coupled plasma-optical emission spectrometry (ICP-OES, Thermo Elemental Co. Iris Intrepid II XDL, USA). All the analyses were made in triplicate. The germination index (GI) was calculated using seeds of Lepidium sativum L. according to the method of Zucconi et al. [24]. The humification indexes (HR, HI, Pha, Cha/Cfa) and the losses of OM (from the initial (X 1 ) and final (X 2 ) ash contents) were calculated according to the equations described by Bustamante et al. [8].
Advanced instrumental determinations: thermal and spectroscopic analyses
Thermal analyses were performed with a Mettler Toledo (TGA/SDTA851e/LF/1600) and Pfeiffer Vacuum (Thermostar GSD301T) mass spectrometer that enables the recording of thermograms and mass spectra of combustion gases simultaneously. All samples were combusted with a mixing stream of oxygen/He (20/80%), a gas flow 100 ml min −1 within a temperature range from 25 to 650°C, a heating rate 10°C min −1 , a sample weight around 5 mg, Al 2 O 3 pan, and self-controlled calibration. The FT-IR spectra were collected on a Bruker IFS 66 spectrometer. The resolution was set to 4 cm −1, and the operating range was 400−4000 cm −1 . The analytical technic used was FT-IR attenuated total reflection spectroscopy (ATR). Samples (7−10 mg) were mixed with 100 mg of dried KBr, and then the mixture was pressed into pellets. In all cases, 20 scans per sample were recorded, averaged for each spectrum, and corrected against the spectrum with ambient air as background.
CPMAS 13 C NMR experiments were performed on a Bruker Advance DRX500 operating at 125.75 MHz for 13 C. Samples were packed into a 4 mm diameter cylindrical zirconia rotor with Kel-F end-caps and spun at 10000 ± 100 Hz. A conventional CPMAS pulse sequence [25] was used with a 1.0 ms contact time. Between 2000 and 5000 scans were accumulated with a pulse delay of 1.5 s. Line broadening was adjusted to 50 Hz. Spectral distributions (the distribution of total signal intensity among various chemical shift ranges) were calculated by integrating the signal intensity in seven chemical [26]. The labels only indicate major types of C found in each region. Spin counting calculation were performed using the method of Smernik and Oades [27]. Glycine (analytical reagent grade; SIGMA) was used as an external intensity standard. The proton spin-lattice relaxation time (T 1 H) and the proton spin-lattice relaxation rate in the rotating frame (T 1ρ H) were determined as described in Smernik and Oades [27]. These parameters are very important in order to avoid signal loss in the CP-1 MAS spectra, since it is possible to choose the best conditions of the CP-MAS pulse sequence (these experimental conditions have been previously described). The percentage of potential 13 C NMR signal, which was actually observed (Cobs), was in the range 60-66% for the CP-MAS technique [27]. The main source of error was uncertainty in the integrated NMR intensities. Two replicate measurements were carried out for all samples.
Statistical analysis
Data corresponding to OM losses (OM degradation) produced throughout the composting process were fitted to a kinetic function by the Marquardt-Levenberg algorithm to minimize the sum of the squared differences between the observed and predicted values of the dependent variable, using the Sigmaplot 11.0 computer program. A first-order kinetic model was used for OM degradation during composting [28]. This model was chosen as the best fit because it gave a randomized distribution of the residuals together with the lowest residual mean square (RMS) value and a highly-significant F-value (data not shown). The model was: where A is the maximum degradation of OM (% C T ), k the rate constant (d -1 ) and t the composting time (d). The RMS and F-values were calculated to compare the fittings of different functions and the statistical significance of curve fitting. MATLAB version 6.5 from Math-Works was used for the calculations, as well as the iToolbox (including methods for iPLS, biPLS and dynamic biPLS), EMSC Toolbox (for pre-processing methods), and GA-PLS Toolbox, available from http://www.models.kvl.dk.
Temperature evolution of the composting piles
The temperature profile in the composting process determines the rate at which many of the biological reactions take place, being a signalling parameter about nutrient bioavailability and the presence of potential limiting factors (salinity, polyphenols, etc.); also, it is associated to the capacity of the process to reduce the pathogen contents [8]. At the beginning of the process, the temperature increased very slowly in all the mixtures except for pile B (Fig 1), probably as a consequence of its higher contents in water-soluble easy-degradable compounds ( Table 2), due to the use of GM, with greater amounts of these compounds than EGM [2]. The initial inhibition of the thermophilic phase during composting of winery and distillery wastes has been reported by other authors [7,10,29]. The presence of compounds with antimicrobial effect, such as polyphenols, and the acidic character of this type of wastes could be responsible for this temperature profile [10,29,30]. The use of the manures as co-composting ingredients, in general, induced better conditions in the initial composting mixtures, reducing some of the limiting factors linked to winery-distillery wastes, such as the acidic character, since the pH of the initial mixtures (pH = 7.9 for pile A; pH = 7.1 for piles B and C) (S1 Table) was in the range 6-8, suggested as suitable for composting [8]. However, in pile C, the use of PM seemed not to have the same positive effect, showing this pile the lowest thermophilic temperature values at the beginning of the process. This initial inhibition could be due to the higher concentrations of polyphenolic compounds of this waste (Table 1), which induced the highest contents of these compounds in the initial mixture of pile C (2568 mg/kg for pile C compared to 1125 mg/ kg for pile A and 1446 mg/kg for pile B) (S1 Table). The turnings carried out throughout composting, especially the second one, reactivated the process in all the mixtures by the increase in the microbial activity, producing the maximum temperature rise in all the composting mixtures. This reactivation of the composting process with turnings was also reported by Bustamante et al. [29] during an experiment of composting of anaerobic digestate using different bulking agents. According to the EXothermic Index, EXI (calculated as the summation of the daily value obtained by subtracting the ambient temperature from the temperature value in the composting pile during the bio-oxidative phase of composting, and expressed as cumulated°C), pile B had the most exothermic behavior (1741 cumulated°C), while piles A and C were quite similar (1344 and 1317, respectively), probably due to the previously commented different content in labile compounds in GM and EGM, the last one obtained after grape marc-washing processes carried out in the distillery.
Organic matter evolution during composting: classical analytical approach The organic matter degradation profile during composting, as determined by OM losses (data not shown), followed a first-order kinetic equation where RMS is the residual mean square. All equations were significant at P < 0.001. The OM degradation kinetics of all the piles fitted satisfactorily this equation. The A and k values obtained were lower than those reported by different authors in other composting experiments using livestock and agroindustrial wastes [8,31,32], probably due to the composting scale used, with a pile weight much lower than those of the commented experiments. The maximum degradation of OM (A) was observed in pile C, but the greatest OM degradation rate was observed in pile B (in accordance with the highest previously commented EXI), since this pile showed the highest values of k and the product of A x k, showing the highest degradation rate the mixture with GM compared to those with EGM.
All the piles showed a decrease in the TOC/TN ratio values (Table 2), especially at the beginning of the composting process and in piles A and B, corresponding to the highest OM degradation rates observed in these piles, showing pile C the lowest decrease in the TOC/TN ratio. At the end of the composting process, the TOC/TN ratio reached values <20 in all piles, suggesting that all composts had reached an acceptable degree of maturation [4]. However, in pile C, the initial TOC/TN ratio value was lower than the reference value for mature compost and therefore, this maturity parameter cannot be used as the only maturity indicator for this pile. Thus, this fact evidences the previously commented need of using different parameters to estimate compost maturity.
The water-soluble organic C represents the most active fraction of carbon and is indicative of compost stability, since it is constituted by sugars, hemicellulose, phenolic substances, amino acids, peptides and other easily biodegradable substances [33]. Therefore, the study of the transformations occurring in the soluble OM can be useful for assessing compost maturity. In this sense, the evolution of the water-soluble organic C and of the water-soluble organic C to the total organic N ratio (WSC/TN) can be considered as suitable parameters for assessing compost maturity [4]. The contents of water-soluble C decreased in all the piles throughout the composting process (Table 2), due to the degradation of simple, water-soluble organic compounds [8]. At the end of the process, in general, all the piles had values lower or close to 1.7%, the maximum value suggested for a compost to be considered mature [4]. The WSC/TN also decreased in all the piles throughout the composting process (Table 2), with decreases in relation to the initial value of 70%, 62% and 73% for the piles A, B and C, respectively, due to the degradation of simple water-soluble organic compounds, such as sugars, amino acids, and peptides [33]. The final values of this ratio ranged between 0.31 and 0.69, within the limit value established for a mature compost [4].
Regarding the humification parameters, such as the humification indexes (HI, HR, Pha, Cha/Cfa) and/or the contents in humic and fulvic acid-like C (Cha and Cfa), their increases during composting have been reported in different studies as indicative of OM humification [4]. However, in this experiment, these parameters have not shown the classical trend of increasing throughout the composting process, showing, in general, all the piles a decrease from the initial values (Table 2), this fact being also observed by other authors [8,34]. This could be attributed to the alkaline co-extraction and partial acid co-precipitation of incompletely or not humified components of organic matter, such as the polyphenols present in the winery and distillery wastes, which hid the real evolution of the humic fraction. This fact was reported by Marhuenda-Egea et al. [18] that confirmed this interference using fluorescence excitation-emission matrix to determine humic-fulvic evolution during composting of winery and distillery residues. Moreover, this shows the high dependence of these parameters on the origin of the raw materials used, being not useful to evaluate the humification in all types of compost, highlighting the need of using other analytical and/or instrumental techniques to evaluate compost maturity.
Organic matter evolution during composting: advanced instrumental approach Thermal tools. Thermogravimetric analysis (TG, DTG and DTA) were carried out to assess the changes in organic matter during the composting process. The thermograms of the compost samples for the piles A, B and C, corresponding to the initial and maturity stage of composting are shown in Fig 2. These thermograms displayed different steps-regions in the thermal analysis, linked to the complexity of the organic matter present in the compost samples. In the presence of atmospheric oxygen, two exothermic phenomena may occur in compost characterization, such as volatilization of aliphatic compounds or carbohydrates and the oxidation of high molecular weight compounds [35]. A clear change within the range of 250°C to 550°C is shown, which corresponds to the combustion of carbohydrates, aromatic compounds and other substances [35]. The three piles showed a similar behavior, the amount of matter that was burned being higher in the initial sample than in the corresponding mature compost sample at the same temperature. This trend suggests a progressive transformation of the biomass in the polyelectrolyte macromolecules known as humified matter and thus, the increase in the molecular weight, stability, and aromatization degree during the co-composting process [19,36]. The heat-labile material was decomposed in the early stages of composting, producing that with time the most recalcitrant material become the material predominant in the compost piles. In the DTG profiles, three peaks can be distinguished between 250 and 530°C (Fig 3), associated to the degradation of organic matter [35]. The first peaks were obtained in the range between 250 and 400°C and the third one appeared between 450 and 500°C. The two peaks within the range of 250-420°C could be attributed to the combustion of carbohydrates, such as cellulose and lignocellulosic [35], which are main components of the plant material present in the winery and distillery wastes. The first peak decreased considerably with time in the piles B and C; however, in the samples of pile A only a slight change was observed (Fig 3). This fact could be due to a higher availability of easily degradable compounds, such as carbohydrates and proteins, in these piles. This confirms the previous results concerning the higher degradation rate observed in these piles, especially in pile B, using the classical analytical approach. Moreover, the contents in carbohydrates in the piles decreased throughout the composting process due to the microbial degradation processes, implying an enrichment in recalcitrant material due to the concentration effect. These recalcitrant compounds constitute the fraction of material that combusted in the range of 450 and 500°C. Different authors [35,37] have also attributed the range between 350-500°C to the degradation of complex aromatic structures, such as the humified organic matter. Therefore, more stabilized samples take more energy for decomposition, e.g. require higher temperatures to achieve the same mass losses, due to these samples are richer in highly complex aromatic compounds compared to the initial ones, which indicates the OM stabilization during the composting process.
The DTA profiles of the organic matter displayed two distinct exothermic peaks (between 250-400°C and between 400-580°C) (Fig 4), which indicated the thermal reactions of organic components characterized by different thermal stability [19,37]. A decrease in the relative intensity of the first exothermic peak was observed in DTA profiles. This fact indicates the progressive degradation of the carbohydrates and aliphatic compounds [19,38] and of some easily biodegradable aromatic structures [39]. At the same time, an increase in the second peak intensity was observed in sequential samplings of the composting piles. This peak is associated to more complex aromatic structures with high molecular weights, such as lignin [39,40]. The increase in the intensity of this peak can be related to the release of the aromatic structures after deterioration of the lignocellulose complex and thus, the condensation of these structures [40]. Therefore, this peak can be attributed to the increase in the aromatic fraction in the mature samples. These exothermic reactions were associated with the peaks in the TG and DTG curves, linked with loss weight, but not with energy. The indexes R1 and R2 can Chemical and Instrumental Tools to Study Winery-Distillery Composting summarize the observed organic matter transformations [41]. R1 is the ratio between the mass loss associated with the two main exothermic reactions at TG (Table 3), and R2 is the ratio of the peak areas in the DTA analysis. In all the piles, the R1 ratio increased during composting, thus revealing a high sensitivity of this parameter to the chemical changes induced by the biotransformation of organic materials. This shows the relative amount of the most thermally-stable fraction of the organic matter with respect to the less stable one. R2 behavior was similar to the R1, but the difference associated between the initial and mature samples was higher than in R1. R2 evolution showed that the most labile organic matter (i.e., cellulose), which requires less energy for its combustion, disappeared during the first stages of the composting process, remaining an organic matter more complex and therefore, with higher thermal stability (Table 3). These results suggested an enhancement of the number of stable compounds through two ways, either by a concentration effect due to the loss of the most labile material during composting and/or by the novo synthesis of more stable and complex compounds, such as the humic-like substances. Presumably, both processes occur simultaneously due to the microbial activity during the composting process.
Spectroscopic tools: FT-IR and CP-MAS 13 C-NMR techniques FT-IR spectra from the most representative samples (initial and mature samples of pile C) are displayed in Fig 5. Some of the most significant peaks are pointed. The variations in the FT-IR spectra during composting time were minor. The region between 2930-2880 cm -1 could be attributed to aliphatic groups in fatty acids [22] and its stationary behavior during the composting indicated the presence of vegetable material highly resistant to microbiological degradation, probably associated to molecules of vegetable origin like cutines, suberines or lignins [18]. Variations between piles and time of sampling in the FT-IR spectra were appreciated between 1800 and 600 cm -1 region. The band around 1540 cm -1 can be assigned to amide II and components containing lignin. These bands were identified in biowastes due to their content of wood and plants, which are rich in lignin [20]. The peak at 1420 cm -1 was due to the Chemical and Instrumental Tools to Study Winery-Distillery Composting OH in-plane bend of carboxylic acids, the CO 2 stretch of carboxylates and the aliphatic CH 2 group of alkanes. The band at 1384 cm −1 was assigned to nitrate and inorganic components as carbonates absorb at 875 cm -1 . However, a visual inspection of the FT-IR spectra bands of the samples did not show valuable differences among the samples during the composting process. In order to detect possible variations of the FT-IR bands during composting, the relative absorbance (rA) of certain signals was used [11,20]. The relative absorbance is the height of one distinct band multiplied by 100 and divided by the sum of all compared band heights [42]. Bands heights were measured and corrected referring to the chosen baseline by OMNIC 5.1b software. Eight bands were used to calculate relative absorbances (rA): 2927, 2854, 1640, 1548, 1428, 1384, 1037, and 875 cm -1 (Table 4). In this experiment, only the rA ratio between 1037 and 1384 cm -1 signal showed changes with time and type of pile, associated to the infrared (IR) spectra bands assigned to the C/N ratio (1037 and 1384 cm -1 ). Piles B and C showed increases of 17 and 9% in this ratio during the composting that were negatively correlated to the observed increase of the TOC/TN ratio using the classical analytical approach, which could be used as indicator of compost stability. In addition, there was an increase in the values of rA of signals attributed to C aliphatic (2927 cm -1 band); however, the latest aspect was only clear for pile C. These results could be explained by the fact that these signals are generated by molecules of vegetable origin, very resistant to degradation, which could induce a concentration effect, since other labile compounds are degraded during the composting process.
The CP-MAS 13 C-NMR spectra show several principal peaks corresponding to the samples collected at the beginning of the composting process at 204, 175, 142, 130, 105, 72, 62, 55, 33, 30 and 24 ppm (Fig 6). An increase of alkyl C in the 0-50 ppm region, associated to the degradation of aliphatic components was measured for the sample of pile B. This result could be due to the breakdown of polysaccharides, presumably in higher amount in pile B due to the use of GM as component, yielding simple alkyl chains [21,23]. A shoulder appears around 25 ppm in Table 4. Relative absorbance in % of the sum of all the peak heights of the FT-IR spectra of the composting samples. CP-MAS 13 C-NMR spectra, indicating the presence of methyl groups in alkyl chains. The methyl group presents an elevated mobility and, therefore, a weak coupling [11]. At 30 and 33 ppm, appear the methyl and methylen groups, respectively. The main difference between methyl and methylene groups is related to carbon dipolar interactions with linked protons.
The peak that appears around 55 ppm in CP-MAS 13 C-NMR spectra supports the assignment to Cα of polypeptides [43]. However, the signal at 55 ppm in CP-MAS 13 C-NMR spectra can be also assigned to O-CH3 groups in lignin (phenolmethoxyl of coniferyl and sinapyl moieties) and in hemicellulose (glucoronic acid in xylan) [19]. These composts principally derived from plant and organic remains, and thus, contain different biomolecules, such as proteins and peptides, as well as lignin and hemicellulose. So, the signal around 55 ppm has contribution from Cα of polypeptides and O-CH3 groups in lignin and in hemicellulose [44]. Also, the spectra were dominated by a strong signal around 72 ppm, assigned to cellulose and hemicellulose in CP-MAS spectra. The chemical shift at 105 ppm has been assigned to different carbons of lignin-type moieties in CP-MAS spectra: The C2 carbons of both guaiacyl and syringyl lignin structures and the C6 carbon of syringyl units. This peak could be also attributed to quaternary aromatic carbons in tannins [11]. However, the low signal to noise (S/N) ratio in these spectra made difficult the assignment. In the CPMAS 13 C-NMR spectra, the region between 110 ppm and 170 ppm can be divided in two sub-regions: the first one, between 110-140 ppm is assigned to non-substituted aromatic C and C-substituted aromatic carbons [45]. The second region, between 140-160 ppm is attributed to aromatic carbons linked to O or N. The first region only shows peaks at 112 and 130 ppm. The second sub-region, between 140-160 ppm, exhibits peaks usually attributed to lignin or tannins [11]. The peak centered at 142 ppm is attributed to methoxy-substituted or hydroxy-substituted phenyl C [11]. The peak at 153 ppm is assigned to oxygen-substituted aromatic C, including both C-OCH3 and C-OH groups [46]. Also, the region between 170-210 ppm is assigned to carbonyl/carboxyl carbons of ester and amide groups [46]. The contribution of amides at the signals in this region should be important, because the nitrogen content is high ( Table 5).
The area under the curves in the CP-MAS 13 C-NMR spectra was calculated for the different regions (the regions are detailed in Materials and Methods section) ( Table 5). The main C types are O-alkyl (95-60 ppm) and alkyl (45 to -10 ppm). These two regions experimented opposite evolutions with time. The O-alkyl (from cellulose and hemicellulose) signals decreases with the composting time, especially in piles A and C (Fig 6 and Table 5). Moreover, in all the piles, the intensity of the alkyl signals (from aliphatic chains, such as lipids, cutin or suberin) increases in the mature composts. Aliphatic structures of cutin and suberin molecules are resistant to biodegradation and thus, these molecules could have been accumulated throughout the composting process [36]. This shows a concentration effect over the C-alkyl by a preferential Table 5. Relative intensity (%) of each C type by integration of the solid-state 13 degradation of sugar polymer, as cellulose and hemicellulose, during the composting process (Fig 6) [36]. This preferential degradation pathway of cellulose and hemicellulose observed in the CP-MAS 13 C-NMR data agreed with the previous thermal analysis data. Minor components in the CP-MAS 13 C-NMR spectra of the compost samples were carbonyl (210-165 ppm), O-aromatic (165-145 ppm), aromatic (145-110 ppm), O2-alkyl (110-95 ppm), and N-alkyl/methoxy (60-45 ppm) [37]. The scatter of the data (Fig 6) could reflect the non-quantitative characteristic of the CP-MAS 13 C-NMR technique, as the errors show (the observable carbon contents for this type of compost samples were generally around 60-66%).
Quality of the composts obtained
The main physico-chemical, chemical and biological properties of the final composts obtained are shown in Table 6. The composts obtained had final pH values close to neutrality and within the range (6.0-8.5) suggested as suitable for compost [47]. The EC values in all the composts were close to 2 dS/m, probably due to the presence of manure in the formulation of all the piles, showing the composts elaborated with CM (A and B), the greatest EC values. However, these values were similar or lower to those reported in composts with similar origin [7,8,10]. Organic matter (OM) concentrations were similar in all the composts and higher than the minimum values established by the Spanish and the European legislation (35% and 30%, respectively for OM) [48,49]. TN contents were greater than 2% in all the composts, which were affected by the manure nature, showing the compost elaborated with PM (compost C) the highest TN concentrations. These contents were similar to those reported in composts elaborated using agroindustrial wastes and manures [8,10,12,28,29,32]. The concentrations in other macronutrients, such as P and K, were higher or similar to those found in other materials usually considered as organic amendments, such as composts from agroindustrial wastes and manures [12] K contents in the compost B, elaborated using GM, were significantly higher than in the rest, due to the higher contents of water-soluble elements in this material [2], while P contents were similar in the composts A and B, elaborated using CM, and lower than in compost C, due to the use of PM. Contents of Ca ranged between 34-66 g/kg, showing compost C higher levels, and Na contents were significantly lower than those reported for manurederived composts [29], this aspect being an additional added value of these composts. The micronutrient concentrations were similar or lower than those observed in composts from agroindustrial wastes and manures, previously mentioned, the values of Cu and Zn being lower than the limit values established for compost by the Spanish legislation and the European guidelines [48,49]. Regarding other parameters related to the compost maturity and/or stability, the contents in water-soluble polyphenols, compounds that in high levels inhibit the germination [8], were quite lower than those observed in the raw materials. All the composts also showed absence of phytotoxicity, with values of the germination index > 50%, the minimum value established by Zucconi et al. [24].
Conclusions
In conclusion, the classical analytical methods have shown a suitable development of the composting process in all the piles, with a more significant effect of the winery-distillery waste used than of the type of manure, but obtaining in all the scenarios end-products with suitable properties and a good degree of maturity for their agricultural use. However, the classical analytical approach has also been shown to be ineffective to study the humification process during composting of this type of materials. Therefore, thermal analysis techniques has been useful to elucidate the degradability of the remaining material and additionally, to assess qualitatively the rate of OM stabilization and recalcitrant C in the compost samples; FT-IR has allowed to identify functional groups in composting, which indicates composted material stability and maturity; while CPMAS 13 C-NMR has provided semi-quantitatively partition of C compounds and structures during the process, being especially interesting their variation to evaluate the biotransformation of each C pool, especially in the comparison of recalcitrant C vs labile C pools, e.g. Alkyl /O-Alkyl ratio. Therefore, the combination of instrumental techniques clearly complemented the existing classical analytical tools, especially those based on non-specific chemical determinations. Also, the knowledge of the structural and functional nature of mature compost will help to predict the behavior of compost in agronomic uses. | v3-fos |
2017-06-06T04:46:51.268Z | {
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} | s2 | Global Transcriptome Profiles of 'Meyer' Zoysiagrass in Response to Cold Stress
A long green period is essential for a turfgrass species with high ornamental value and a wide area of use. Zoysiagrasses (Zoysia spp. Willd.) are perennial turfgrass species popular in tropical, subtropical and temperate zones, possessing many properties necessary to be economically useful turfgrass. They do not have a long green period because of cold sensitivity. A main focus in zoysiagrass research is to develop cold tolerant cultivars. Understanding the cold response in zoysiagrass is a fundamental area of research. In the present study, ‘Meyer’ zoysiagrass (Zoysia japonica), a widely cultivated variety in the genus, is used. We employed RNA-Seq to investigate genome-wide gene expression profiles in leaves under cold stress (4°C). Using the Illumina sequencing platform, we obtained approximately 206 million high-quality paired-end reads from three libraries (0 h, 2 h, and 72 h cold treatment at 4°C). After de novo assembly and quantitative assessment, 46,412 unigenes were generated with an average length of 998 bp and an N50 of 1,522 bp. A total of 25,644 (55.2%) unigenes were annotated by alignment with public protein databases including NR, SwissProt, KEGG and KOG. Differentially expressed genes (DEGs) were investigated using the RPKM method. A total of 756 DEGs were identified between 0h and 2h-cold treatment, with 522 up-regulated and 234 down-regulated; and 5327 DEGs were identified between 0h and 72h-cold treatment, with 2453 up-regulated and 2874 down-regulated. The expression profile of 15 DEGs selected randomly was confirmed with qRT-PCR. The results suggest that cold stress can induce desiccation and oxidative stress, inhibit photosynthesis and substance transport. In response to the stress, genes involved in proline synthesis, in starch hydrolysis, in methionine and ascorbic acid metabolism, in SOD activity, and in DREBs response pathway were up-regulated. GA metabolism, ABA and JA stimulus response were affected under cold exposure. This is the first transcriptome sequencing of Z. japonica, providing a large set of sequence data as well as gene expression profiles under cold stress. It will improve our current understanding of the cold response of zoysiagrass and be beneficial in breeding research.
Introduction
Material and Methods Plant material and cold treatment 'Meyer' zoysiagrass (Zoysia japonica) was collected from the nursery garden of turfgrass resources, Jiangsu Province and the Chinese Academy of Sciences. Dr Jianxiu Liu, one of the corresponding authors of this study, is in charge of the nursery garden and provided the plant material. This collection does not involve endangered or protected species. Grass patches of 10cm×10cm×10cm in size were grown in pots (20cm) in a greenhouse for 6 months, with a 14h light period and 20°C/25°C (Light/Dark) temperature conditions. For cold treatment, grass pots were transferred to a growth chamber set at 4°C for 2h (CT_2h) and 72h (CT_72h). Grass pots transferred to a chamber set at 25°C were used as the control (CK). The light period was 14h, and light intensity was 100 μmol m -2 s -1 . The second expanded leaf from the top was collected and stored at -80°C after freezing in liquid nitrogen.
Physiological response assay
Relative water content [17], levels of total soluble carbohydrate, proline [8], and malondialdehyde (MDA) [18] were measured using previously described protocols. Activities of detoxification enzymes including superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) were determined according to Li &Yi (2012) [19]. One unit of SOD activity was defined as the amount of enzyme inhibiting NBT reduction by 50%. One unit of POD and CAT activity was defined as the amount of enzyme causing a change of 0.01 in absorbance per minute. Experiments were conducted with five biological replicates, and results were expressed as the mean ±standard error (SE). All data were subjected to one-way analysis of variance (ANOVA) and the LSD multiple comparison test (p < 0.05) using the SPSS statistical package.
Total RNA extraction, RNA-seq library construction and sequencing Total RNA was extracted using the Universal Plant Total RNA Extraction Kit RP3301 (Bio Take Corporation, Beijing, China) following the manufacturer's instructions, with three biological replicates. RNA integrity was confirmed using an Agilent 2100 Bio Analyzer. Samples with RIN 7 and 28S:18S ratio 1.5:1, total amount 0.1∽4 ug, were considered acceptable. RNA samples from the same treatments were mixed and used for the determinations. Pair-end libraries were constructed as previously described [20] and sequenced on a HiSeq2000 system according to the manufacturer's instructions (Capitalbio Company, Beijing, China). The raw sequence reads were deposited in the NCBI (National Center for Biotechnology Information) SRA database.
RNA-seq data processing, de novo assembly and annotation RNA-seq reads of each sample were first processed by removing the adapter sequence, then merged and assembled to transcripts using Trinity with default parameters (v.r2013-08-14) [21]. Alternative splicing transcripts were assigned to a unigene cluster. The longest transcript in a cluster was assigned as a unigene to form a set of non-redundant transcript sequences. Unigene sequences were aligned to a series of protein databases by BLAST (blastx, E-value < = 1e-5) (v2.2.6) following a priority order of Uniprot (Swiss-Prot/TrEMBL), Nr (non-redundant protein sequences in NCBI), KEGG (Kyoto Encyclopedia of Genes and Genomes database) and KOG (eukaryotic orthologous groups). The best hit of alignment was used to infer biological function of a unigene. The representative Gene Ontology annotation was performed by the software Blast2GO [22] which assigned homologous sequences aligned by BLAST with Uniprot and NCBI nr database to GO terms. We predicted transcription factors from protein sequences obtained by CDS predictions using hmmsearch to search the domains of plant transcription factors (http://plntfdb.bio.uni-potsdam.de/v3.0/). The transcription factor-related unigenes (E-value < = 1e-5) were classified according to the gene family information.
Gene expression quantification and differential expression analysis
We used the Trinity platform for gene expression quantification and differential expression analysis, which included RSEM (v1.2.6) [23] for transcript abundance estimation, and normalization of expression values as FPKM (Fragments per kilobase of transcript permillion fragments mapped), and edgeR [24] for identifying differentially expressed genes (absolute value of log 2 (ratio) 2and adjusted p-value < 0.001). GO terms and pathways enriched in the set of differentially expressed genes were calculated by the hypergeometrictest [25].
Quantitative real-time PCR analysis
For quantitative real-time PCR (qRT-PCR) analysis of transcripts, 1 μg DNase I-treated total RNA was used to synthesize cDNA by M-MLV (Promega) using poly(dT) oligonucleotides. Actin-1 was used as an internal control. qRT-PCR was performed using One Step SYBR Prime-Script RT-PCR Kit (TAKARA, Dalian, China) according to the manufacturer's instructions. Products were verified by melting curve analysis. Quantification was achieved by normalizing the number of target transcript copies to the reference Actin-1 gene using the comparative ΔΔCt method [26]. All reactions were performed with at least three biological replicates. Primers used in all quantitative RT-PCR experiments are listed in S1 Table. Results
Physiological changes induced by low temperature
To investigate physiological responses in leaves, we measured relative water content (RWC) and levels of total soluble carbohydrate, proline and malondialdehyde (MDA). The activities of antioxidant enzymes, including SOD, CAT and POD, were also determined. After 2h at 4°C, RWC declined significantly, and the content of total soluble carbohydrate increased (P < 0.05). After 72h at 4°C, RWC remained constant, whereas soluble carbohydrate, proline and MDA increased (P < 0.05) considerably (Fig 1A-1D). The activities of SOD and CAT in leaves exposed to 2h did not change significantly (P 0.05), while the activity of POD increased by 14.67% (P < 0.05). After a 72h-cold exposure, SOD activity increased by 10.78%, while activities of CAT and POD decreased by 35.99% (P <0.05) and 13.85% (P <0.05), respectively ( Fig 1E-1G).
Illumina sequencing and reads assembly
To measure the transcriptome in the cold, we selected 'Meyer' leaves from normal condition (CK), 2h cold treatment (CT_2h), and 72h treatment (CT_72h) for RNA-Seq analysis. Three gene pair-end libraries, constructed from cDNA synthesized from leaf RNA, were sequenced with the Illumina HiSeq2000. Total clean nucleotides generated from each sample exceeded 5.5 Gb. We obtained 206,232,142 raw reads in total. Low-quality reads which contained adapter and unknown or low quality bases were discarded, and a total of 206,164,694 clean reads were obtained. Of these clean reads, the average Q30 percentage (sequencing error rate < 1%) was 88.47%. All clean reads were deposited in the NCBI and can be accessed in the Short Read Archive (SRA) under the study accession number SRP051412 (PRJNA270938).
A total of 89,868 transcripts with length 300 nt were generated using Trinity software ( Table 1). The mean transcript size was 1262 nt, and N50 is 1854 nt. Clustering analysis was The second expanded leaf from the top was used for determinations. Experiments were conducted with five biological replicates, and results were expressed as mean ± standard error (SE). *P < 0.05. doi:10.1371/journal.pone.0131153.g001 Transcriptome Profiles of 'Meyer' Zoysiagrass used to assign all transcripts to 46,412 unigene clusters. A set of non-redundant unigenes was obtained by keeping the longest transcript in a cluster as representative. Among these unigenes, the mean length was 998 nt, with N50 being 1522 nt. A total of 28103 (60.55%) unigenes were longer than 500 nt, and 5818 (12.53%) unigenes were longer than 2000 nt (Fig 2), implying that a batch of transcripts with complete CDS were obtained. Reads were then mapped back to unigenes, 82.76%, 85.68%, and 83.60% of reads in libraries of CK, CT_2h, and CT_72h respectively, were mapped successfully, indicating that the transcript data was suitable for further analysis.
Among the unigene clusters, 33,837 only contained one transcript sequence, while 12575 clusters contained more than one sequence (Fig 3). These multiple transcript unigene clusters can represent transcription variants, allelic variants, closely related paralogues, misassembled transcripts, or transcripts that were fragmented due to low coverage. The largest unigene cluster contains 188 transcript sequences and was blasted to a probable trehalose-phosphate phosphatase (TPP) gene family in Setaria italic. TPP removes the phosphate from trehalose-6-phosphate to produce free trehalose. There is also a large TPP gene family in the genome of rice and Arabidopsis, some of which are involved in the response to stress [27][28].
Functional annotation and classification
We first annotated the unigenes by homologous searches against public databases in the priority order of Uniprot, Nr, KEGG and KOG. Using a cutoff value of E<10 −5 , a total of 25,644 (55.2%) unigenes had significant BLASTx hits in at least one of the four databases. Among them, 25438(54.8%), 25468(54.9%), 19031(43.1%), 13187(28.6%) unigenes were blasted to Uniprot, Nr, KEGG and KOG, respectively. A total of 12162 (26.2%) unigenes had hit in all four databases, while 20777(44.8%) unigene were not identified ( Table 2). To study the sequence conservation of 'Meyer' in other plant species, we analyzed the species distribution of unigene datasets by aligning sequences against the Uniprot database. The species distributions for the best match from each sequence are shown in Fig 4. Specifically, 98.0% of these unigenes had the greatest number of hits to monocotyledonous species. The majority of unigenes had a significant level of sequence identity to foxtail millet (Setariaitalica) genes (52.07%). The second closest reference species was Sorghum bicolor (21.92%). Oryza sativa (9.99%), Zea mays (6.30%), Oryza brachyantha (3.99%), Brachypodium distachyon (3.62%), and Hordeum vulgare (0.15%) are reference species in decreasing order (Fig 4). We used the Gene Ontology (GO) classification system to assess the possible functions of the unigenes. A total of 16,206 (16206/46421) unigenes were successfully assigned to at least one GO term (Fig 5). The unigenes were then classified into three categories: biological processes, cellular component, and molecular function. The category of biological processes consisted of 2021 GO terms, which were assigned to 82.94% of the unigenes; "metabolic process", "cellular process" and "single-organism process" were the top three. The cellular component category consisted of 391 GO terms, which were assigned to 93.99% of the unigenes; "cell", "cell part" and "organelle" were the major components. The category of molecular function Transcriptome Profiles of 'Meyer' Zoysiagrass consisted of 1359 GO terms, which were assigned to 73.66% of the unigenes; "catalytic activity", "binding" and "transporter activity" were the top three functions.
We then aligned all the unigenes to the KOG database and 11,308 unigenes were identified. These unigenes were grouped into 25 functional categories (Fig 6). The top three categories were "signal transduction mechanism", "general function prediction only", and "posttranslational modification, protein turnover, chaperone"; the smallest category was "cell motility".
Transcription factors are important upstream regulatory proteins and play critical roles in plant responses to abiotic stresses. In the present study we identified a total of 1979 transcription factors from the 'Meyer' unigenes that were classified into 55 different families ( Table 3). The largest group of transcription factors was bHLH (203, 10.26%), followed by MYB-related
Identification of differentially expressed unigenes
To identify a significant change in gene expression induced by low temperature, we used the FPKM method (Fragments per kilobase of transcript permillion fragments mapped) allowing us to calculate the expression levels of genes. Compared with CK, unigenes with absolute values of log 2 (ratio) 2 and p-value 0.001 were considered as DEGs (differentially expressed genes). There were 756 DEGs between CK and CT_2h, including 522 up-regulated unigenes and 234 down-regulated ones (Fig 7, S2 Table). There were 5327 DEGs between CK and CT_72h, including 2453 up-regulated unigenes and 2874 down-regulated ones (Fig 7, S3 Table). To further validate our RNA-seq expression profile data, we performed qRT-PCR assays with primers designed according to fifteen randomly-selected unigenes (S1 Table). qRT-PCR data were normalized using the 'housekeeping' gene Actin-1.The results showed that gene expression profiles were consistent using either RNA-seq or qRT-PCR analysis, although the exact fold change varied between the two techniques ( Table 4). The correlation coefficient between RNA-Seq data and qRT-PCR data is 0.837 (p < 0.01). In addition, PCR products of these primers showed the expected fragment size by agarose gel electrophoresis, which was consistent with the reliability of the assembled sequences.
GO and KEGG enrichment analysis of DEGs
To investigate the function of the DEGs, we then mapped all DEGs to the Gene Ontology database (http://www.genontology.org/) and calculated the gene numbers from each GO term. Using a hypergeometric test, we identified significantly enriched GO terms (p-value < 0.05) in DEGs compared to normal condition. After 2h at low temperature, 39 GO terms were enriched and can be categorized into 6 types (S4 Table): (1) Cell wall and external encapsulating structure organization; (2) Water and carbohydrate transport; (3) Electron carrier activity; (4) Tetrapyrrole binding; (5) Sequence-specific DNA binding transcription factor activity; (6) Auxin mediated signaling pathway. After a 72h-cold exposure, 112 GO terms were enriched (S5 Table). GO term types listed in (2) to (4) above were enriched more intensively. Other enriched GO terms in the CT_72h treatment were mainly related to functions such as: (1) Photosystem and photosynthesis activity; (2) Oxidation-reduction process; (3) Carbohydrate metabolism; (4) Nitrogenous compound metabolism and transport; (5) Lipid metabolism; (6) GA catabolism; (7) Response to ABA and JA stimulus.
KEGG enrichment analysis identified some biochemical pathways and signal transduction pathways. Compared with CK, 5 pathways showed significant change (p-value < 0.05) in the 2h-cold treatment, including biosynthesis of unsaturated fatty acids, biosynthesis of flavonoid and isoflavonoid, MAPK signaling pathway, and terpenoid backbone biosynthesis (S6 Table).
In the 72h-cold treatment, 32 pathways showed significant changes (p-value < 0.05) (S7 Table). These pathways included: (1) Photosynthesis, including "antenna proteins", "chlorophyll metabolism", and "carbon fixation"; (2) Carbohydrate metabolism, including "starch and sucrose metabolism" and "pentose and glucuronate interconversions"; (3) Lipid metabolism, including "Linoleic acid metabolism" and "alpha-Linolenic acid metabolism"; (4) Antioxidants metabolism, including "ascorbate and aldarate metabolism" and "cysteine and methionine metabolism"; (5) Biosynthesis of secondary metabolites, including "phenylpropanoid biosynthesis", "isoquinoline alkaloid biosynthesis", and "flavonoid biosynthesis"; (6) Plant hormone signal transduction. The results of KEGG enrichment and GO term enrichment in the 72hcold stress were in general similar. Taken together, after exposure to 4°C for 2h, electron carrier activity and the biological processes of cell wall organization, water homeostasis and carbohydrate transport were affected. After 72 h of cold exposure electron carrier activity, redox balance, photosynthetic activity, carbohydrate and lipid metabolism, biosynthesis of some secondary metabolite, and organic substance transport were affected significantly. The metabolism of GA and responses to ABA and JA stimulus were also involved in cold stress response at the 72 h time point.
Discussion
Temperature is an important environmental factor that affects the distribution, growth and development of plants. Cold stress, including chilling (<20°C) and/or freezing (<0°C) temperatures, prevents the expression of full genetic potential of plants owing to its direct inhibition of metabolic reactions and, indirectly, through cold-induced osmotic, oxidative and other stresses [6][7]29]. To understand the cold response in 'Meyer' zoysiagrass at the molecular level, we compared the transcriptomes at normal growth temperature and at 4°C. The characteristics of the cold response in 'Meyer' are discussed below.
Photosynthesis and nitrogen assimilation
Photosynthesis is greatly inhibited by low temperature in a number of plant species, of which the main impact is photosystem and its action [29]. Similar inhibition occurred in 'Meyer' under cold stress, indicated by the down-regulated expression of unigenes involved in porphyrin and chlorophyll synthesis, photosystem structure, photosynthetic electronic transport, and carbon dioxide fixation (S8 Table).
Chlorophylls are complex molecules exquisitely suited to the light absorption, energy transfer, and electron transfer functions carried out in photosynthesis. During the 72h-4°C stress, most of the DEGs involved in chlorophyll synthesis were down-regulated (Fig 8). At the same time, one unigene of ferrochelatase (Hem H), and one of pheophorbide a oxygenase (PAO) were up-regulated. Hem H catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme, which departs from the chlorophyll biosynthetic pathway. PAO, the key enzyme in chlorophyll catabolism, catalyzes porphyrinmacrocycle cleavage of pheophorbide a (pheide a) to a primary fluorescent catabolite (pFCC) [30]; its expression was up-regulated during senescence [31]. Taken together, these gene expression changes suggest that chlorophyll biosynthesis decreases and chlorophyll catabolism increases during low temperature treatment. An intact photosystem is necessary for effective light-absorption and energy transformation. In this study, 10 unigenes encoding chlorophyll a-b binding proteins, 8 unigenes encoding proteins in the reaction center of photosystem Ⅰ (PSⅠ), and 3 unigenes encoding oxygen evolving complex (PsbQ, PsbO and PsbR) were down-regulated. It can be inferred that light harvesting, the water-splitting reaction, and electron transport in PSⅠdecrease in leaves under low temperature exposure. The photosynthetic electron transport activity was also affected, as demonstrated by the down-regulated unigenes of plastocyanin and ferredoxins-1. These changes might lead to abnormal photosynthetic electron transport.
In the carbon fixation process, gene expressions of four enzymes, including carbonic anhydrases (CA), phosphoenolpyruvate carboxylase (PEPC), ribulosebisphosphate carboxylase (Rubisco) and fructose-1,6-bisphosphatase (FBP) were altered considerably during cold treatment. CA catalyzes the reaction of carbon dioxide with water to form bicarbonate, which is then transported to photosynthesizing cells. In this study, the expression of three unigenes encoding CA was down-regulated, suggesting that the exchange rate of carbon dioxide decrease during the 72h cold treatment. This result was consistent with previous research demonstrating that the exchange rate of carbon dioxide in zoysiagrass fell by 80-95% when canopy air temperature was 10°C-12°C, compared to the control (optimal temperature) [32]. PEPC, a key enzyme during C 4 photosynthesis, has been suggested to be related to the cold sensitivity of C 4 plants [33]. Findings in Amaranthus hypochondriacus (a NAD-ME type C4 species) have shown that PEPC activity fell to 70% on exposure to a low temperature of 15°C, but the protein content and phosphorylation of the enzyme did not change significantly [34] compared to the control. The present study showed that the expression of six PEPC unigenes changed significantly at 4°C, three were up-regulated and three were down-regulated. A previous study indicated that the activity of PEPC in zoysiagrass decreased drastically under 10°C/7°C (L/D) chilling stress [35]. The depressed enzyme activity might be related to the expression changes of PEPC isogenes under cold stress. In addition, one unigene of Rubisco small chain and one unigene of chloroplast located FBP were down-regulated. The expression changes of these carbon-fixation related unigenes support previous data showing that photosynthesis in zoysiagrass decreases greatly under chilling stress compared with that at 25°C [32].
In contrast to carbon fixation, nitrogen assimilation appears to be stimulated under cold stress. The expression changes of related unigenes are shown in S9 Table. First, the expressions of three nitrate reductase (NR) unigenes were up-regulated with fold changes of 6 to 11. Second, two unigenes, homologous with ferredoxin-6 and FNR root isozyme genes in Zea mays, respectively, were up-regulated. In Zea mays the two genes are nitrate-inducible and might function to transfer electrons from the NADPH to Fd-dependent enzymes such as nitrite reductase (NiR) and glutamine synthetase in root plastids [36][37]. Third, eight DEGs with GO term "glutamine family amino acid biosynthetic process" (GO:0009084), including two glutamine synthetase unigenes, were all up-regulated. NR catalyzes the reaction: NO 3 -+ NAD(P)H + H + + 2 e -!NO 2 -+ NAD(P) + + H 2 O. Nitrite can be converted to ammonia nitrogen by NiR, and the latter can then be assimilated as amide nitrogen through the glutamine synthetase pathway. It has been shown that in wheat seedlings the activity of glutamine synthetase and nitrate reductase increases during cold acclimation [38]. The overexpression of a glutamine synthetase gene in rice was found to enhance salt and chilling tolerance and increase photorespiration [39]. The up-regulated expression of these genes might increase nitrogen assimilation and photorespiration. It could be inferred that, owing to the decrease of carbon fixation under cold stress, nitrogen assimilation and photorespiration activities were enhanced so as to divert surplus assimilatory power-ATP and NADPH.
Oxidative stress
Reactive oxygen species (ROS) are produced in plants as by products during many metabolic reactions, such as photosynthesis and respiration. Oxidative stress occurs when there is a serious imbalance between the production of ROS and antioxidant defense, causing rapid cell damage by triggering a chain reaction. In the present study, after a 72h-4°C exposure, a total of 270 DEGs were assigned to the GO term "oxidation-reduction process" (GO:0055114), including 177 down-regulated and 93 up-regulated with the fold changes of -10.54 to 13.16, compared to the control. The expression changes of these genes could affect some oxidationreduction activities and trigger oxidative stress. GO enrichment analysis of DEGs indicated that GO terms "cellular response to reactive oxygen species" (GO:0034614) and "cellular response to oxidative stress" (GO:0034599) were enriched in the 72h-cold treatment. Oxidative stress can dramatically promote nonenzymatic lipid peroxidation [40], of which malondialdehyde (MDA) is one of the important end products. Physiological measurement showed that MDA levels in leaves increased significantly during the 72h treatment.
Desiccation stress and inhibition in substance transport
Chilling stress can inhibit water uptake [41]. This study showed that the relative water content in leaves of 'Meyer' declined significantly (p < 0.05) after being exposed to 4°C for 2h, and remained at this level during the 72h exposure. Genes characterized by the GO term "response to desiccation" (GO:0009269) were enriched under cold stress for 2h and 72h, demonstrated by the down-regulated expression of two and four putative aquaporin unigenes, respectively (S10 Table). These results indicated that 'Meyer' became water stressed after cold treatment. Aquaporins are intrinsic membrane proteins that mediate the transport of water, small neutral solutes and CO 2 . It is well documented that regulation of aquaporins plays a role in early responses and acclimation to water stress [42]. In Arabidopsis, 12 aquaporin genes are downregulated during cold acclimation and recover to original expression levels during de-acclimation [43]. The over-expression of aquaporin genes of Rhododendron catawbiense in Arabidopsis plants leads to a significant decrease in cold tolerance and the increase of dehydration rate of leaves [43]. The down-regulation of aquaporin unigenes thus might help to prevent water loss in 'Meyer' leaves under cold stress.
Desiccation stress might affect the activities of transporters. GO enrichment analysis of DEGs indicated that genes with GO terms "dicarboxylic acid transport" (GO:0006835) and "carbohydrate transport" (GO:0008643) were enriched during 2h-cold stress. After 72h-cold stress, genes with GO terms including "nucleobase transmembrane transporter activity" (GO:0015205), "nitrogen compound transport" (GO:0071705), "amino acid transmembrane transport" (GO:0003333), "peptide transport" (GO:0015833), and "carboxylic acid transport" (GO:0046942), were enriched. A total of49 DEGs, 37 of which were down-regulated, were found to have these GO terms, suggesting that transporter activities are inhibited during cold stress.
The affect of cold stress on "Carbohydrate transport" genes was the most noteworthy. After a 2h-cold exposure, three DEGs with this GO term, one encoding a bidirectional sugar transporter (SWEET1a) and two encoding monosaccharide transporter 3 (MST3) respectively, were up-regulated. SWEET1a mediates both low-affinity uptake and efflux of sugar across the plasma membrane. MST3 in rice is involved in the accumulation of monosaccharides required for cell wall synthesis during cell thickening [44]. It is possible that early sugar transport adjustment is involved in cell wall thickening, which is supported by the enrichment of DEGs with the GO term "cell wall organization or biogenesis" in the 2h-cold treatment. During the 72hcold exposure, 14 DEG sassigned to the GO term "carbohydrate transport" were differentially regulated (S11 Table), including the nine down-regulated and the five up-regulated genes. Unigenes encoding sugar transporter proteins (STP), sucrose transport protein (SUT), hexose carrier, and polyol transporter were down-regulated. SUTs are responsible for the transport of sucrose into the cell with the concomitant uptake of protons (symport system) [45]. STPs have been shown to catalyze proton symport of hexoses across the plasma membrane into the cell, as demonstrated by heterologous expression in yeast [46]. The down-regulated expression of SUT and STP will restrict the phloem loading activity and, thus can affect the long distance transport of sugar. Unigenes encoding putative isoform plastidic glucose transporter 2, vacuolar monosaccharide symporter, and CMP-sialic acid transporter 3-like isoform X1 were upregulated in the cold. In Arabidopsis thaliana, plastidic glucose transporter 4 is located in the chloroplast inner membrane and may be involved in the efflux of glucose into the cytosol [47]; the tonoplast monosaccharide transporter 1 has been shown to be involved in the transport of monosaccharides into the vacuole during cold stress [48]; and a CMP-sialic acid transporter is responsible for transporting CMP-sialic acid to the Golgi lumen [49]. It could be inferred that intracellular sugar transport was stimulated in 'Meyer' during the exposure to low temperature. Abnormal sugar transport in the cold could possibly lead to abnormal distribution of photosynthetic products in cells. Rogers et al [32] found that, when zoysiagrass was chilled at 10°C the chloroplasts accumulated starch and were substantially inflated.
Resistance to desiccation stress
In response to drought stress brought about by low temperature treatment, osmotic adjustment occurs in 'Meyer', including the adjustment of potassium ion homeostasis and the accumulation of proline and soluble sugars. GO and KEGG enrichment analysis indicated that genes with the GO terms "potassium ion homeostasis" (GO:0055075), "proline biosynthetic process" (GO:0006561), and "starch and sucrose metabolic" KEGG pathway (ko00500) were enriched in 'Meyer' during the 72h-4°C stress.
Potassium (K + ) is the most important and abundant cation in living plant cells and plays crucial roles in osmoregulation. In the enriched GO term "potassium ion homeostasis" two unigenes encoding calcineurin B-likes (CBLs) and ABC transporter C family member 5-like (abcC5-like) respectively were up-regulated, while a unigene encoding calcium-activated outward-rectifying potassium channel 1(KCa1) was down-regulated significantly (S10 Table). CBLs are commonly related to abiotic stress tolerance in plants [50], participating in the regulation of K + uptake [51]; abcC5 is involved in regulation of K + and Na + cell content, and is required for regulation of stomatal opening by auxin, abscisic acid (ABA) and external Ca 2+ ; KCa1, also known as two-pore potassium channel 1(TPK1), is apotassium-selective tonoplast ion channel functioning in K + transport across the vacuolar membrane and plays a role in intracellular K + homeostasis. The up-regulation of CBL and abcC5 accompanied with the down-regulation of KCa1 might change the distribution of K + in apoplast, cytoplast and vacuolar to adapt to cold stress.
Proline is an important osmolyte in plant cells. It is also considered to be a scavenger of free radicals, an energy sink and a stress-related signal. The synthesis of proline is stimulated under multiple abiotic stresses [52]. In the present study, 6 DEGs with the GO term "proline biosynthetic process" (GO:0006561) were found to be up-regulated during the 72h-cold (S10 Table) but not during the 2h-cold exposure, which corresponded to the accumulation of proline in leaves.
Soluble sugars play an important role in osmotic adjustment. Sucrose and glucose accumulate and comprise the majority of the total soluble sugar in the stolon and rhizome of zoysiagrass during cold acclimation [8]. This study showed that the level of total soluble sugar went up significantly at the 72h-cold time point. At the transcriptional level, 102 DEGs were assigned to the "starch and sucrose metabolism" KEGG pathway (ko00500), including 34 upregulated and 68 down-regulated. Among these, unigenes encoding starch branching enzyme (SBE), starch phosphorylase and α-amylase were up-regulated with fold changes of 3 to 12 in the 72h-cold treatment. SBE influence the structure of starch by catalyzing the formation of a-1,6-branch points with varied frequency and branch chain length. Overexpression of SBEIIb in an SBEIIb-deficient mutant of rice resulted in the accumulation of excessive branched, watersoluble polysaccharides instead of amylopectin [53]. The changes of these genes suggest that amylohydrolysis and starch-branching may contribute to the accumulation of soluble sugars in leaves. Sucrose synthase (SUS), a key enzyme involved in sucrose metabolism, catalyzes the reversible conversion of sucrose and UDP to UDP-glucose and fructose. Induced expression and increased activity of SUSs occurs in several species, such as Arabidopsis [54], wheat [55] and chickpea [56]. In the present study, three of the four SUS unigenes identified were induced with fold changes of 2.50, 2.35 and 1.29, respectively. During the reproductive stage of chickpea, a significantly higher specific activity of SUS was observed in seeds of most of the cold-tolerant lines but not in the cold susceptible lines [56]. It may be worthwhile to determine whether SUSs can be used as candidate genes to evaluate cold tolerance of different germplasm in Zoysia genus.
Resistance to oxidative stress
Antioxidants play an important role in combating oxidative stress. Ascorbic acid (AsA) functions as a major redox buffer and as a cofactor for enzymes involved in regenerating other antioxidants. In the present study, genes with "ascorbate and aldarate metabolism" KEGG pathway were enriched at the 72h time point, in which two encoding GME (GDP-Mannose 3',5'-epimerase) were induced 2.3-fold and 6.2-fold, respectively. GME catalyzes an important step in the AsA biosynthetic pathway in higher plants. Overexpression of GMEs in tomato leads to AsA accumulation and enhanced tolerance to oxidative, cold, and salt stress [57]. The up-regulated expression of GME may enhance tolerance to oxidative stress in 'Meyer' exposed to low temperature.
Methionine (Met), a sulfur-containing amino acid, is highly susceptible to oxidation [58]. In arsenic-exposed rats methionine can result in the reversal of oxidative stress with a significant (P < 0.05) decline in tissue arsenic burden [59]. In the present study, genes with GO terms "Cysteine and methionine metabolism" KEGG pathway (ko00270) were enriched in 'Meyer' after a 72h-cold exposure, among which four encoding cobalamin-independent methionine synthase (MetE), homocysteine S-methyltransferase (HMT), S-adenosyl-l-methionine synthase (SAMS), and methionine γ-lyase (MGL) respectively, were up-regulated. Both MetE and HMT catalyze the synthesis of methionine, whose role in the stress response has been largely unreported. Methionine is converted to S-adenosylmethionine (SAM) which serves as a precursor for metabolites including glycinebetaine, methylated polyols, polyamines and ethylene. MGL is involved in cellular Met homeostasis and Ile synthesis. Transgenic Arabidopsis lines overexpressing a SAMS gene from potato exhibited higher salt and drought stress tolerance compared to controls [60]. MGL of Arabidopsis was up-regulated by simultaneous water deficit and nematode stress in root tissue, and conferred resistance to nematodes when overexpressed [61]. The up-regulation of the four genes could increase the level of Met and its derivatives, which may alleviate oxidative damage and enhance tolerance to cold in 'Meyer'. The induced expression of MetE and HMT genes by low temperature provides a new insight into the cold response in plants.
Antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) are important in reducing levels of ROS. Several of these enzymes have been shown to be induced under cold stress at either the transcript or protein level [62]. In the present study, the activity of POD increased during the 2h-cold stress, but declined significantly at the 72h-cold time point; the activity of SOD increased significantly during the 72h-cold stress; the activity of CAT declined during the cold stress. Compared with the control, two POD unigenes were transcriptionally up-regulated in the 2h-cold treatment, one SOD unigene was up-regulated but 12 POD unigenes were down-regulated in the 72h-cold treatment. These findings were consistent with the changes of POD and SOD activity. Three unigenes for CAT were identified and designated as comp35050_c4, comp30190_c0 and comp30190_c4. Under cold stress, the expression of comp35050_c4 increased, while that of the other two decreased. The decrease of CAT activity under cold stress suggests that the protein product of comp30190_c0 and comp30190_c4 could have great activity than that of comp35050_c4. The lower activities of CAT and POD under cold stress may be one of reasons why zoysiagrasses are cold sensitive. Increasing antioxidant enzymes activities should be considered in cold tolerance breeding research.
Expression changes of DREBs and COR/dehydrin genes
The DREBs (Dehydration Responsive Element Binding proteins/C-repeat Binding Factors) proteins namely, DREB1 and DREB2, are thought to be the major transcription factors (TFs) that control stress-inducible gene expression in the ABA-independent pathway [63]. Overexpressing DREB genes in plants can significantly enhance plant tolerance to multiple abiotic stresses, including drought, freezing and high salinity [63]. Moreover, findings in some plants indicate that the different stress tolerance ability is related to diverse gene structures and expression patterns of DREB1 and DREB2. For example, in winter cultivars of wheat (Tritic eaeaestivum and T. monococcum) the constitutive and inducible expression of DREB1/CBFs within five pooideae-specific groups may play a predominant role in their superior freezing tolerance ability [64][65]; Among Arabidopsis natural populations, the relaxed selection on the CBF/DREB1s in some accessions from the southern part of Europe has compromised the ability of these genes to act as efficient transcriptional activators during the cold acclimation process [66]; In Zea mays, it was reported that natural variation in drought tolerance at the seedling stage was related to the variation of ZmDREB2.7 [67]. In this study, eight and five unigenes assigned to DREB1 and DREB2 subgroups, respectively, were identified (Table 5). Compared with the control, six DREB1 and two DREB2 unigenes were up-regulated significantly at the 2h-cold time point, and one DREB2 unigene was up-regulated at the 72h-cold time point. The induced expression of these DREB1 and DREB2 genes will be involved in increasing cold resistance in zoysiagrass. The relationship between the DREBs and cold tolerance in zoysiagrass is worthy of further research.
A number of cold-regulated (COR) genes have been characterized in plants and some are identified as regulons of DREBs [7]. The dehydrins, belonging to the late-embryogenesis abundant (LEA) class of proteins [68], are among the COR genes that have been extensively studied [69] and shown to have major roles in conferring low temperature tolerance. Studies in wheat (T.aestivum L.) have shown that the expression of the COR genes Wcs120, Wcor410, and Wcor14 was highest for low temperature-tolerant and lowest for more cold sensitive genotypes [70]. In Arabidopsis, the expression levels of COR genes are higher in more cold-tolerant leaves than in cold-sensitive pollen [71]. In this study, ten unigenes annotated as dehydrin/COR were detected, six of which were up-regulated several fold during 72h-cold stress, indicating a potential relationship to the cold tolerance ability in 'Meyer'.
The relationship of the COR413 family to cold tolerance is of interest. The COR413 family consists of two distinct groups: plasma membrane targeted (COR413-PM) and thylakoid membrane targeted (COR413-TM), respectively [72]. The expressions of one gene of group COR413-PM and one gene of group COR413-TM are closely associated with the acquisition of freezing tolerance in several plant species such as wheat, rye, and Arabidopsis. On the other hand, group Cor413-pm and-tm transcripts are not induced in the low temperature-sensitive species rice and maize [72]. In the present study, a unigene encoding COR413-pm 1 was induced by low temperature in 'Meyer', which is the reverse of the expression profiles in the low temperature-sensitive species mentioned above. Determining whether this induced expression is related to cold tolerance in zoysiagrass requires further research.
Adjustment in phytohormone metabolism and signaling pathway
Phytohormones play important roles in the response of plants to stress. In this study, genes with GO terms "gibberellin metabolic process" (GO:0009685), "response to abscisic acid stimulus"(GO:0009737), and "response to jasmonic acid stimulus" (GO:0009753) were enriched after the 72h-cold exposure. The expression changes of DEGs in these GO terms are shown in S12 Table. Gibberellins (GAs) play a central role in plant development and the metabolism of GAs in plants is highly regulated [73]. From a regulatory standpoint, two biosynthetic enzymes-GA 20-oxidase (GA20ox) and GA 3-oxidase (GA3ox)-and an enzyme involved in gibberellin catabolism, GA 2-oxidase (GA2ox), are most notable. The gene transcription of these enzymes is highly regulated. In this study, three unigenes of GA20ox were down-regulated; three of GA2ox were up-regulated (Fig 9). These changes suggest that gibberellin catabolic pathways were activated and lead to the decreased endogenous level of bioactive GA just like other plant species [74]. ABA serves as an endogenous messenger in abiotic stress responses in plants, and has been called a 'stress hormone'. Genes with the GO term of "response to abscisic acid stimulus" was enriched in 'Meyer' during the 72h-4°C exposure. Three regulators of ABA signal transduction pathways, including type 2C protein phosphatases (PP2C), sucrose nonfermenting 1-related protein kinase2 (SnRK2/ SAPK4), and proline-rich receptor-like protein kinase (PERK4), were up-regulated significantly in the cold. Without ABA, PP2C dephosphorylates SnRK2 kinases and blocks signal transduction. The ABA-bound receptor inactivates PP2C, which causes the SnRK2 kinases to be in a phosphorylated state, allowing them to activate bZIP group transcription factors (TFs) that then promote downstream ABA-responsive gene transcription [75][76][77]. PERK4, localized to the plasma membrane, has been shown to be an ABA-and Ca 2+ -activated protein kinase, is required for abscisic acid responses in Arabidopsis [78]. In this study, the expression of three PP2C unigenes, one SnRK2/ SAPK4 unigene and one PERK4 unigene were induced with fold changes of 2.53 to 7.10, suggesting that the ABA signaling pathway was stimulated in 'Meyer' exposed to cold.
Jasmonates, including jasmonic acid (JA) and its related metabolites, are essential components of the signaling pathway triggering the expression of plant defense genes in response to various sources of environmental stress [79]. Levels of endogenous jasmonates have been reported to increase upon pathogen infection, insect wounding, and various abiotic stresses [80][81]. In this study, genes with the GO term "response to jasmonic acid stimulus" (GO:0009753) was enriched during 72h cold stress, among which the regulation of allene oxide synthase (AOS) unigenes and indole-3-acetic acid-amidosynthetase GH3.12 unigenes were noteworthy. AOS is a major control point in Arabidopsis thaliana octadecanoid signaling. This enzyme acts on a number of unsaturated fatty-acid hydroperoxides forming the corresponding allene oxides. Several AOS enzymes have been cloned from various plant species. Most of them prefer 13-hydroperoxides as substrates and are involved in JA production [82][83]. JA can also induce the expression of AOS [84]. OsGH3.12 belongs to group I of GH3 genes in plants [85], which can conjugate JA with isoleucine to produce bioactive JA-Ile [86]. In 'Meyer' under 4°C stress for72h, two AOS genes were down-regulated with fold changes of -2 to -5, while AOS2-like, AOS3-like and AOS4-like were up-regulated 4 to 8-fold, and three OsGH3.12 homologs were down-regulated-7 to -9-fold. The expression changes of these unigenes suggest that metabolism and signaling pathways of JA are significantly regulated by cold stress in 'Meyer', which has also been reported in rice [81].
Cross-talk between the different plant hormones results in synergistic or antagonistic interactions that play crucial roles in the response to abiotic stress in many plants [87] As shown in this study, the expression changes of multiple unigenes that are involved in metabolism and signaling pathway of different plant hormones might indicate a coordination of the regulatory network of stress signaling and cold response in zoysiagrass.
Conclusion
In conclusion, this study provided the first large-scale transcriptome data set in warm-season turfgrass in response to low-temperature stress. More than 206 million high-quality 100-bp paired-end reads were generated and assembled into 46,412 unigenes in 'Meyer' zoysiagrass. Furthermore, 756 and 5327 unigenes were identified as DEGs in 2h-and 72h-4°C treatments, respectively; in addition, their classification, functions, and metabolic pathways are shown here for the first time. The down-regulated expression of genes involved in electron carrier activity, chlorophyll biosynthesis and carbon-fixation activity, water transport, organic substance transport, and antioxidant enzyme activity are consistent with the cold sensitivity of 'Meyer'. The induced expression of genes involved in osmotic adjustment, antioxidants, nitrogen assimilation, phytohormone metabolism and signaling, and the DREB response pathway may play a vital role in cold resistance. The high-resolution expression patterns presented here further our understanding of the molecular response to low temperature in zoysiagrass, and would facilitate the breeding of cold tolerant varieties.
Supporting Information S1 Table. The expression changes of DEGs related to nitrate assimilation. (XLSX) S10 Table. The expression changes of DEGs involved in desiccation stress and osmotic adjustment. (XLSX) S11 Table. The expression changes of DEGs in GO term of carbohydrate transport. (XLSX) S12 Table. The expression changes of DEGs related to plant hormone metabolism or stimulus. (XLSX) | v3-fos |
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"year": 2015
} | s2 | Uncovering the genetic architecture of Colletotrichum lindemuthianum resistance through QTL mapping and epistatic interaction analysis in common bean
Colletotrichum lindemuthianum is a hemibiotrophic fungal pathogen that causes anthracnose disease in common bean. Despite the genetics of anthracnose resistance has been studied for a long time, few quantitative trait loci (QTLs) studies have been conducted on this species. The present work examines the genetic basis of quantitative resistance to races 23 and 1545 of C. lindemuthianum in different organs (stem, leaf and petiole). A population of 185 recombinant inbred lines (RIL) derived from the cross PMB0225 × PHA1037 was evaluated for anthracnose resistance under natural and artificial photoperiod growth conditions. Using multi-environment QTL mapping approach, 10 and 16 main effect QTLs were identified for resistance to anthracnose races 23 and 1545, respectively. The homologous genomic regions corresponding to 17 of the 26 main effect QTLs detected were positive for the presence of resistance-associated gene cluster encoding nucleotide-binding and leucine-rich repeat (NL) proteins. Among them, it is worth noting that the main effect QTLs detected on linkage group 05 for resistance to race 1545 in stem, petiole and leaf were located within a 1.2 Mb region. The NL gene Phvul.005G117900 is located in this region, which can be considered an important candidate gene for the non-organ-specific QTL identified here. Furthermore, a total of 39 epistatic QTL (E-QTLs) (21 for resistance to race 23 and 18 for resistance to race 1545) involved in 20 epistatic interactions (eleven and nine interactions for resistance to races 23 and 1545, respectively) were identified. None of the main and epistatic QTLs detected displayed significant environment interaction effects. The present research provides essential information not only for the better understanding of the plant-pathogen interaction but also for the application of genomic assisted breeding for anthracnose resistance improvement in common bean through application of marker-assisted selection (MAS).
Introduction
Members of the ascomycete genus Colletotrichum cause devastating anthracnose diseases in many agronomically important crops in temperate, tropical and subtropical regions (Bailey and Jeger, 1992). The specialized hemibiotrophic fungus C. lindemuthianum [(Sacc. & Magnus) Lams. -Scrib]. has a reduced number of plant hosts, mainly common bean (Phaseolus vulgaris L.), although in less extent and severity, it can also colonize P. acutifolius var. lactifolius, P. coccineus, P. aureus, P. lunatus, P. limensis, Medicago sativa, and Vicia faba (Sicard et al., 1997;Mahuku et al., 2002). The pathogen has a sequential biotrophicand necrotrophic-infection process to invade and colonize the plant hosts, that involves the transition from an asymptomatic biotrophic phase (characterized by intracellular thick primary hyphae) to a destructive necrotrophic phase (characterized by thin filamentous secondary hyphae) referred to as the biotrophynecrotrophy switch, which is essential for anthracnose disease development (Bhadauria et al., 2011). The remarkable resistance of C. lindemuthianum and its capacity for survivability in any environmental condition renders its presence responsible for losses in crops. In fact, the damage caused by this fungus in bean crops is so great that it has produced an economical loss in productive countries (Vigidal-Filho et al., 2007). Besides, C. lindemuthianum causes a hypersensitive response in bean resistant plants -groups of red-brownish wounds of different sizes that are produced by the plant to delimit the spread of the pathogenic fungus (Martínez-Pacheco et al., 2009). The process of co-evolution between the fungus and bean resistant plants has led this fungal species to produce new pathogenic variants, which can be detected on the basis of the phenotypic response to anthracnose infection shown by different varieties of common bean Rodríguez-Guerra et al., 2003). Thus, more than 100 races have been described for C. lindemuthianum (Rodríguez-Guerra et al., 2003) and new pathotypes are reported every day, indicating a large pathogenic variability of this fungus.
Resistance to anthracnose in common bean generally follows a qualitative mode of inheritance where resistant and susceptible reactions are clearly differentiated. The specific resistance genes follow the classic gene-for-gene model (Flor, 1955), and the qualitative resistance provided by them is often less durable than quantitative resistance, since pathogens can more easily adapt to single gene-mediated resistance (St. Clair, 2010). In contrast, quantitative resistance usually confers broad-spectrum protection toward different races of biotrophic or necrotrophic pathogens (Oliver and Ipcho, 2004). The genetic regulation of quantitative traits is often complex due to their polygenic nature. However, trait dissection through Quantitative Trait Loci (QTL) analysis is a useful approach to identify chromosomal regions harboring genes that control these quantitative traits. Yet, in addition to mapping main effect QTLs, epistatic interactions between QTLs are important. Identification of quantitative disease resistance main and epistatic effects from multiple environments does not only help to extend the applicability of results, but is also essential for the development of an efficient markerassisted selection (MAS) program aimed at improving breeding efficiency.
Despite the fact that genetics of anthracnose resistance in common bean has been studied for a long time, few QTL studies have been conducted on this species. The present work studies the genetic basis of quantitative resistance to two races of C. lindemuthianum in different organs of a segregating common bean recombinant inbred line population (RIL) from the cross PMB0225 × PHA1037. Using multi-environment QTL mapping approach, race specific anthracnose resistance QTLs were identified showing significant main additive effects in stem, petiole and leaf organs, which were co-localized with NL genes. In addition to identifying main effect QTLs, this analysis revealed epistatic interactions that explained phenotypic variation beyond those controlled by main effects of individual loci. Thus, markers associated with QTLs reported here constitute useful tools for MAS breeding programs directed toward improved anthracnose resistance.
Biological Material
A RIL population consisting of 185 F 7 lines was developed by single-seed descent from an F 2 population from the cross between PMB0225 (a common bean line as P1) and PHA1037 (nuña bean line abbreviated as P2) accessions belonging to the Andean gene pool. Mesoamerican (17, 73, 448, and 1545) and Andean (7,23,39,55,and 102) anthracnose races were inoculated to the two parents. The twelve differential cultivars (Michelite, MDRK, Perry Marrow, Cornell 49242, Widusa, Kaboon, Mexico 222, PI207262, TO, TU, AB136, and G2333) were used to confirm the identity of the C. lindemuthianum races. Only races 23 and 1545 were pathogenic on PMB0225 parent and chosen for the present study.
Plant Growth Conditions, Inoculation, and Disease Evaluation
Plants were grown in plastic pots containing a mixture of clay soil and organic compound (1:1; v/v), under natural and artificial (12-h photoperiod, 166 µE s −1 m −2 ) photoperiod growth conditions with average day and night temperatures of 25 and 20 • C, respectively. Plants were irrigated according to water needs. The anthracnose races were kept on potato-dextrose agar (PDA) at 19-21 • C in darkness. To obtain conidia, fungus was grown for sporulation for about 15 days, and medium plates were flooded with 10 mL of 0.01% Tween 80 in distilled water. The conidial suspension was collected and filtered twice to remove mycelial fragments. The number of conidia was estimated using a haemocytometer and inoculum concentration was adjusted to 3 × 10 6 conidia mL −1 with distilled water. Spore suspension was sprayedinoculated onto 2-week-old bean plants showing the fully expanded primary leaves using an atomizer. Inoculated plants were sealed in order to increase 95-100% humidity for 48 h.
The infected phenotypes were assessed on the basis of symptom severity on the primary leaves (L), stems (S), and petioles (P) at intervals of 7, 14, and 21 days post-inoculation (dpi). Numerical disease scores (DC) were assigned based on visual appreciation of the percentage of the organ presenting symptoms. A score of 1 represented no observed symptoms, while 9 corresponded to 100% of the organ covered by brown typical lesions of anthracnose (Figure 1). The Area Under the Disease Progress Curve (AUDPC) was calculated according to Shaner and Finney (1977) as: AUDPC = n i = 1 [x i + x i + 1 /2] t, where x i is the disease score on date i, n the number of evaluations made and the time in days between evaluations x i and x i + 1 . The use of AUDPC is an effective method to take both duration and severity of disease into account.
Experimental Design and Statistical Data Analysis
The experiment was set up as a randomized complete block design with four replicates in artificial (named A-Ppd) and natural (named N-Ppd) photoperiod conditions, respectively. Each RIL genotype was represented by one plant in each block. Independent four-block experiments were carried out for each race, and the parental lines PMB0225 and PHA1037 were included.
Descriptive statistical parameters (mean value, standard deviation and range of variation) and normality (Kolmogorov-Smirnov test) were obtained for each quantitative trait and environment. Variation in the expression of traits through the environments was analyzed using PROC MIXED (SAS Institute Inc V. 9.04, Cary, NC, USA). Variance components and broadsense heritabilities with their standard errors were estimated by restricted maximum likelihood (REML) option of the PROC MIXED and IML (SAS Institute Inc. v. 9.04, Cary, NC, USA) for the phenotypic traits (Holland et al., 2003;Holland, 2006). Phenotypic Pearson correlation coefficients among traits were implemented using PROC CORR across the environments (SAS Institute Inc. v. 9.04, Cary, NC, USA).
QTL Analysis
The genetic linkage map described by Yuste-Lisbona et al. (2012) was used for QTL analysis. The SCAR SW13 and SW12 (Fourie et al., 2004;Rodríguez-Suárez et al., 2008), 1 AFLP, 3 SSR, 29 SNP, and the seed coat color gene (P) were added to this map, which finally consisted of 229 loci (86 AFLP, 98 SSR, 42 SNP, 2 SCAR, and P locus) distributed on 11 LGs. The map spanned 858.4 cM, with an average distance of 3.7 cM between adjacent markers. Marker data were analyzed by JoinMap R 4.0 software (Van Ooijen, 2006). A minimum logarithm of odds ratio (LOD) score of 6.0 and a recombination frequency value of 0.3 were set as the linkage threshold for grouping markers. The Kosambi map function (Kosambi, 1944) was used to calculate the genetic distance between markers. The LGs were designated according to Pedrosa-Harand et al. (2008). QTLNetwork 2.0 software (Yang et al., 2008) was used to identify single-locus QTL, epistatic QTL (E-QTL) and their environment interaction effects (QTL × Environment, QE; and E-QTL × Environment, E-QE). The mixedmodel based on composite interval mapping method (MCIM) was carried out for one-dimensional genome scan to detect putative single-locus QTL (defined as those showing significant main additive effects) and their environment interactions. In addition, a two-dimensional genome scan was carried out to identify epistatic interaction effects. An experimental-wise significance level of 0.05 was designated for candidate interval selection, putative QTL detection, and QTL effect. Both testing and filtration window size were set at 10 cM, with a walk speed of 1 cM. The critical F-value to declare putative QTLs was determined by a 1000 permutation test at 95% confidence level. The effects of QTL and environment interactions were estimated by the Markov Chain Monte Carlo method (Wang et al., 1994). QTL with only genetic effects indicated that these were expressed in the same way across environments. In addition, QTL with environment interaction effects suggested that their expressions were environmentally dependent. The detected QTLs were designated as recommended by Miklas and Porch (2010). The genetic map and the QTL detected were drawn using the MapChart 2.2 software (Voorrips, 2002).
Identifying Location of QTL in Common Bean Genome
Nucleotide sequences of the markers flanking the main effect QTLs were used as queries for BLASTN search (Altschul et al., 1997) against the first chromosome scale version of P. vulgaris genome (Schmutz et al., 2014) available in the Phytozome database (http://www.phytozome.net/).
Resistance Variation in the RIL Population
The bean accession PMB0225 was fully susceptible to anthracnose infection to race 1545 in all tested organs, and displayed susceptibility in leaf, intermediate resistance in stem, and full resistance in petiole to race 23. The PHA-1037 accession was fully resistant to both races in all organs. Table 1 shows the mean values and standard errors of the parental genotypes and the RIL population, as well as the ranges of variation of the RIL population for the resistance traits for each environment. In the RIL population, a continuous but bimodal distribution skewed toward the resistant parent PHA1037 was found regardless of the organ and race tested ( Figure S1). The relative skewedness toward the resistant PHA1037 parent would imply that multiple genes with complementary additive effects are conferring resistance to anthracnose. Variance analysis was conducted for each environment and difference between blocks was not significant for most of the environments and resistant traits ( Table 1). PMB0225 and PHA1037 parents and RIL progeny were significantly different for resistance traits in each environment (P = 0.001), demonstrating a genetic origin for the different levels of resistance in the RIL population.
The estimated broad-sense heritability estimates for resistance traits between organs for a given race were high, with values ≥0.70 ( Table 2). These values are in accordance with those reported by Geffroy et al. (2000) for anthracnose resistance between aerial organs of the plant to isolates 45 and A7, with heritability values ranging from 0.90 to 0.98. There was a strong correlation (P ≤ 0.001) for disease resistance scores between the different organs for a given race ( Table 2). This was in sharp contrast to the absence of correlation when the data regarding both races were compared. These findings suggest that different genes could be involved in resistance for a given race, while identical genes condition resistance against the same race in different organs. In addition and in order to determine if the same set of genes encode resistance against both races, co-segregations for resistance to races 23 and 1545 were considered. Twentynine and thirty-four RILs were resistant to each race 23 and 1545, respectively, while evidence of co-segregation of disease response to both races was observed in 114 RILs (14 and 74 RILs were susceptible and resistant to both races in all organs, and 26 RILs presented resistance to both races but not in all organs). Accordingly, these results suggest that different genes determined specific resistance to races 23 and 1545.
Mapping of Main Effect QTL
The evaluation of the RIL population developed from the cross PMB0225 x PHA1037 under different environments has led to the identification of 10 and 16 main effect QTLs involved in resistance against anthracnose races 23 and 1545, respectively. These QTLs were mapped on eight LGs, with the exception of LGs 02, 10, and 11 (Figure 2). All QTLs detected had significant additive effects and did not display significant additive-by-environment interaction effects. A complete report of the single-locus QTL detected for anthracnose resistance traits is given in Table 3.
Ten main effect QTLs were identified for resistance to race 23: one on each LGs 01 and 03, four on LG04, and two on each LGs 05 and 09 (Figure 2). Three of them had significant effects on stem resistance (SDC and SAUDPC traits), positioned on LGs 01 and 04, and explaining a phenotypic variance from 2.64 to 7.90%. The remaining QTLs were involved in leaf resistance (LDC and LAUDPC traits) on LGs 03, 04, 05, and 09, explaining a phenotypic variance from 3.06 to 11.43%. Two QTLs for resistance 1 | Estimates of means, standard errors, range of variation, and variance analysis results for anthracnose resistance to races 23 and 1545 of the two common bean parents, PMB0225 and PHA1037, and the RIL population, grown in two environments (Env).
LAUDPC RACE 23
For resistance to race 1545, 16 main effect QTLs were found: two on each LG 01, 03, and 07, six on LG05, and four on LG08 (Figure 2). Five of them had significant effects on stem resistance (SDC and SAUDPC traits), positioned on LGs 03, 05, and 08, and explaining a phenotypic variance from 4.33 to 14.24%. For petiole resistance (PDC and PAUDPC traits), five QTLs were detected on LGs 01, 03, and 05, which explain a phenotypic variance from 4.08 to 10.22%. The remaining six QTLs were involved in leaf resistance (LDC and LAUDPC traits) on LGs 05, 07, and 08, with phenotypic variance explained ranging from 1.49 to 16.75%. Organ-specific QTLs were identified for petiole resistance on LG01 (PDC 1545 -1 and PAUDPC 1545 -1) and for leaf resistance on LG07 (LDC 1545 -7 and LAUDPC 1545 -7). Six main effect QTLs (SDC 1545 -5, SAUDPC 1545 -5, PDC 1545 -5, PAUDPC 1545 -5, LDC 1545 -5, and LAUDPC 1545 -5) were co-localized or nearly The location of the anthracnose resistance main effect QTLs to races 23 and 1545 was different in most cases, which is in agreement with the absence of correlation between the resistance reactions against both races. Only the QTLs LDC 23 -3, SDC 1545 -3.1, and PAUDPC 1545 -3 were co-localized or nearly co-localized on LG03 for resistance to race 23 in leaf, and to race 1545 in stem and petiole, although with opposite additive values ( Table 3).
Detection of Epistatic QTL
A total of 39 E-QTLs (twenty-one for resistance to race 23 and 18 for resistance to race 1545) involved in 20 epistatic interactions (eleven and nine interactions for resistance to races 23 and 1545, respectively) were detected by the combined analysis of the multi-environment phenotypic values. None of the epistatic interactions detected displayed significant environment interaction effects. These E-QTLs were mapped on seven LGs, with the exception of LGs 02, 07, 10, and 11. The positive and negative additive-by-additive epistatic effect values obtained for these epistatic interactions indicate that both parent alleles could contribute to increasing the resistance to anthracnose races 23 and 1545. A complete description of digenic epistatic interaction analysis for anthracnose resistance traits toward both races is shown in Table 4.
For resistance to race 23, 6 of the 21 E-QTLs identified were previously detected as main effect QTLs. Thus, not only did the E-QTLs E-SDC 23 -1, E-SDC 23 -4, E-LDC 23 -3, E-LAUDPC 23 -4, E-LAUDPC 23 -5.1, and E-LAUDPC 23 -9 participate in epistatic interactions, but they also had an individual genetic effect. The analysis revealed novel loci on LGs 01, 03, 05, and 08 interacting so as to influence resistance to race 23. The percentage of phenotypic variance explained by the interaction of the E-QTLs ranged from 1.13 to 7.92%. Among the E-QTLs detected, it is noteworthy that the genomic region located between markers BMc316 and E45M50-69 on LG08 bears E-QTLs (E-SAUDPC 23 -8, E-LDC 23 -8, and E-LAUDPC 23 -8) involved in epistatic interactions for resistance in stem and leaf, which indicates that this region could participate in non-organ-specific resistance to anthracnose race 23. Collectively, the percentage of phenotypic variance explained by the interaction of the E-QTLs varied from 6.85 to 16.06% for stem traits (SDC and SAUDPC, respectively), and from 7.92 to 15.29% for leaf traits (LAUDPC and LDC, respectively).
Novel loci on LGs 01, 03, 04, 06, 08, and 09 were detected as involved in resistance to race 1545. Among the 18 E-QTLs identified, two E-QTLs were previously identified as main effect QTLs (PAUDPC 1545 -1 and PAUDPC 1545 -5). The percentage of phenotypic variance explained by the interaction of the E-QTLs varied from 2.25 to 7.05%. All the E-QTLs detected for resistance to race 1545 were organ-specific. The total phenotypic variation explained by the additive-by-additive epistatic effects of all E-QTLs detected ranged from 6.38 to 7.05% QTLs are depicted as vertical bars to the right of the LG. QTL detected for resistance to race 23 are indicated in black. QTLs identified for resistance to race 1545 are shown in gray. Co-location of resistance-associated genes encoding NB-LRR (NL) proteins with anthracnose QTLs are represented to the right of the QTLs.
Identifiying Location of QTL in Common Bean Genome
BLASTN analysis of the nucleotide sequences of the markers flanking the main effect QTLs with common bean genome revealed that the homologous regions spanning 17 of the 26 QTLs identified were positive for the presence of NL genes (Figure 2). Thus, the main effect QTLs SDC 23 -1, PDC 1545 -1,
Discussion
To gain insight in basic knowledge dealing with resistance, a pathosystem that involves C. Lindemuthianum and P. vulgaris model legume has been characterized. The gene action governing anthracnose resistance was studied in a broad set of RILs generated from a cross between susceptible and resistant Andean accessions. Thus, insights into the number of quantitative resistance loci involved in anthracnose resistance to races 23 and 1545 were provided, as well as their epistatic interactions. The pathogen infected and colonized PMB0225 line, although symptoms and pathogen development were significantly reduced in race 23 as compared to race 1545. Genetic and molecular analysis revealed different features associated with the resistance of PHA1037.
Genetic Architecture of Resistance
The phenotypic dissection of anthracnose resistance carried out in the RIL population has led to the detection of different kinds of resistance components. The pathogen resistance response was consistent between the testing environments, which evidenced that anthracnose resistance is mostly influenced by genes rather than environmental conditions. The occurrence of major resistance factors was found across both races and in the different organs tested. Moreover, genomic regions controlling anthracnose resistance displayed additive main effects, epistatic effects or both. This architecture has been frequently reported for other quantitative resistances (Young, 1996). Hence, in addition to main effect QTLs, significant epistatic interactions between QTLs have previously been reported in quantitative resistance against other fungus, such as Phytophthora capsici in pepper (Lefebvre and Palloix, 1996;Thabuis et al., 2003), Rhizoctonia solani in rice (Liu et al., 2014) or Puccinia triticina in wheat (Singh et al., 2014). However, genetic mapping studies considering epistatic interaction effects have not been performed so far in common bean. In this work, depending on the race and organ tested, the total phenotypic variation explained by main effect QTLs ranged from 2.64 to 23.89% (SAUDPC and SDC traits, respectively), whereas epistatic interactions explained a total phenotypic variation from 3.34 to 15.29% (PDC and LDC traits, respectively). Most of the epistatic interactions detected were due to loci without detectable QTL additive main effects, which show the importance of the epistatic effects in genetic resistance to anthracnose. Furthermore, resistant alleles came from the resistant parent PHA1037 more frequently, but they occasionally originated from the susceptible parent PMB0225, as observed in petiole resistance to race 1545. This result suggests that the susceptible parent also develops defense mechanisms, even though their activity could be insufficient to stop fungal progression. The dissection of resistance into distinct phenotypic resistance components allows for a more precise QTL detection and facilitates the exhaustive selection of resistance factors in breeding programs. This type of approach has led to the identification of organ-specific defense mechanisms for resistance to Phytophthora infestans in potato (Gao et al., 2013) and Colletotrichum graminicola in maize (Balmer et al., 2013). This study has identified QTLs located in the same genomic region for resistance to different organs (SDC 23 -4, SAUDPC 23 -4, LDC 23 -4.1, LAUDPC 23 -4, SDC 1545-5, SAUDPC 1545-5, PDC 1545-5, PAUDPC 1545-5, LDC 1545-5, LAUDPC 1545-5, SDC 1545-8, SAUDPC 1545-8, LDC 1545-8, and LAUDPC 1545 or for different races of infection (LDC 23 -3, SDC 1545 -3.1, and PAUDPC 1545 -3), which are usually described as "generalist QTLs" (Lefebvre and Palloix, 1996;Thabuis et al., 2003). In addition, "specialist QTLs" have also been identified, which were involved in organ-or race-specific resistance (SDC 23 -1, PDC 1545 -1, PAUDPC 1545 -1, LDC 23 -5.1, LAUDPC 23 -5.1, LDC 1545 -7, LAUDPC 1545 -7, LDC 23 -9 and LAUDPC 23 -9). However, it is not possible to conclude whether those genomic regions containing "generalist QTLs" resulted from the clustering of "specialist QTLs" or from the pleiotropic effect of a single gene. The presence gene clusters acting on the same trait is widespread among higher plants, and it has also been described in the common bean genome (Schmutz et al., 2014). Geffroy et al. (1999) pointed out that the origin of these clusters preceded the geographic separation of the wild common bean gene pools, as well as the role of selection in the emergence of such clusters, which might confer a selective advantage to the genotype that possesses them.
Co-Localization of QTL with Resistance Genes
The association between NL genes and QTLs conferring resistance to Colletotrichum species has been reported in several plant species. In maize, a gene conferring resistance to anthracnose stalk rot, caused by C. graminicola, encoded a CC-NB-LRR protein (Abad et al., 2006). The physical mapping of RCT1, a host resistance gene against C. trifolii in M. truncatula showed that RCT1 was part of a complex locus containing numerous genes homologous to previously characterized TIR-NB-LRR resistance genes (Yang et al., 2007). In common bean, QTLs associated with anthracnose resistance were mapped in a cluster on the LG04, which was composed by CC-NB-LRR genes (Ferrier-Cana et al., 2003;Geffroy et al., 2009). Therefore, there is strong evidence that NB-LRR genes confer gene-for-gene resistance to Colletotrichum species in diverse plant hosts. Schmutz et al. (2014) stated that the majority of NL genes were physically organized in complex clusters in the common bean genome. In the present study, the homologous regions spanning 17 of the 26 main effect QTLs detected were positive for the presence of NL genes. The main effect QTLs detected on LG01 were co-localized with a cluster of 17 NL genes at the bottom of Chr1. The Co-1 anthracnose resistance cluster is also located in this position of the Chr1, which includes Co-1, Co-58 kb region that comprises eight genes: three phosphoinositidespecific phospholipases C (PI-PlC), one zinc finger protein, and four kinases, which suggests that Co-x is not a classical NL gene. Moreover, genes for resistance to angular leaf spot, common bacterial blight, Fusarium root rot, and white mold have been located at the bottom of Chr1 (Miklas and Singh, 2007), as well as Phg-1 and Ur-9 genes, which confer resistance against Pseudocercospora griseola and Uromyces appendiculatus, respectively (Kelly and Vallejo, 2004;Gonçalves-Vidigal et al., 2011).
Resistance main effect QTLs to race 23 acting in stem and leaf organs were positioned in an important cluster of 41 NL genes on Chr4. Geffroy et al. (1999) stated that three specific resistant genes for anthracnose were clustered in this region, which originated either from the Mesoamerican BAT93 parent (Co-9) or the Andean Jalo-EEP558 parent (Co-x, Co-y). Furthermore, a major-effect QTL for resistance to isolate 45 (for leaf, stem, and petiole resistance) and a reverse-effect QTL (for leaf resistance) for resistance to isolate A7 (Geffroy et al., 2000), as well as the Co-3 anthracnose resistance cluster (Ferreira et al., 2013;Campa et al., 2014) were also located in this genomic region of Chr4. On the other hand, the main effect QTLs detected at the top of LG08 were placed within a cluster of 17 NL, where the Co-4 anthracnose resistance cluster is located (Melotto et al., 2004;Rodríguez-Suárez et al., 2007;Campa et al., 2014).
In addition, it is worth noting that the main effect QTLs detected on LG05 for resistance to race 1545 in stem, petiole and leaf were positioned within a 1.2 Mb region where the LRR gene Phvul.005G117900 is located. Based on genome sequence analysis, it can be considered an important candidate gene for the non-organ-specific QTL identified here. However, given that regions containing fast evolving genes, such NL genes, that are susceptible to chromosomal rearrangement and transposition or genomic duplication (Meyers et al., 2005), it is not possible to determine if the non-organspecific resistance resulted from the pleiotropic effect of the Phvul.005G117900 gene or from the clustering of different genes, which could not be present in the reference genome sequence. Thereby, further studies on fine mapping of the target genomic regions would be necessary to draw definitive conclusions.
Concluding Remarks
The results stated herein provide essential information not only for a better understanding of the plant-pathogen interaction but also for the application of genomic assisted breeding for anthracnose resistance improvement in common bean. This research has also shown the importance of the epistatic effects in genetic resistance to anthracnose, which has never been studied so far. Thereby, both main and epistatic interaction effects of genes or QTLs should be considered for a successful application of MAS, which provides an opportunity to use a pyramiding strategy for durable resistance. As well as providing useful tools for MAS of anthracnose resistance in common bean, this work also offers valuable clues for further study on cloning the candidate gene corresponding to the non-organ-specific QTL for resistance to race 1545 located on Chr5. | v3-fos |
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} | s2 | Controlling Smart Green House Using Fuzzy Logic Method
To increase agricultural output it is needed a system that can help the environmental conditions for optimum plant growth. Smart greenhouse allows for plants to grow optimally, because the temperature and humidity can be controlled so that no drastic changes. It is necessary for optimal smart greenhouse needed a system to manipulate the environment in accordance with the needs of the plant. In this case the setting temperature and humidity in the greenhouse according to the needs of the plant. So using an automated system for keeping such environmental condition is important. In this study, the authors use fuzzy logic to make the duration of watering the plants more dynamic in accordance with the input temperature and humidity so that the temperature and humidity in the green house plants maintained in accordance to the reference condition. Based on the experimental results using fuzzy logic method is effective to control the duration of watering and to maintain the optimum temperature and humidity inside the greenhouse
I. INRODUCTION
The development of agriculture industry in Indonesia increased along with increasing knowledge of agricultural techniques. From traditional farming systems evolved into a modern agricultural system where agricultural product is increased, but by using dangerous pesticides in an effort to increase agricultural output. In other hand will destroy the environment. Then developing agricultural technology by manipulating the climate around the plant called micro-climate.
Climate change is happening today has made farmers difficulties for increasing agricultural product. Erratic weather conditions led to the planting and harvesting cannot be determined and estimated. Farmers are difficult to predict the weather in the growing season. Mark and Davel [1] describe an annotated bibliography on issues relating to changes in the concentrations of Earth's greenhouse gases. The areas covered include theory and numerical modelling of climate change; cycles involving carbon dioxide and other radiatively important trace gases; observations of climate change and the problems associated with those observations; paleoclimatology as it relates to previous changes in the greenhouse gases; the impacts on and interactions with managed and natural ecosystems from climate change; policy issues related to climate change and to the limitation of climate change; history of the study of the greenhouse effect; and some other causes of climate change. Then, in India Sahay and Ghosh [4] explain cities across the globe are considered as major anthropogenic sources of greenhouse gases (GHG), yet very few efforts has been made to monitor ambient concentration of GHG in cities, especially in a developing country like India.
Andrasko [2] mention that measurement and monitoring issues are emerging at the intcrsections of the project and national scales, referred to here as monitoring-domain edge effects. Adrasko would like to show following actions are necessary to improve existing monitoring capabilities and to help resolve project/national edge effects.
Smart greenhouse technology is an alternative solution to control the micro-climatic conditions in the plant. The use of smart greenhouse in the cultivation of plants is one way to provide an environment that is closer to better conditions for plant growth. The use of smart greenhouse so that plants can obtain air temperature and humidity is optimum.
David and Murat [3] describe an autonomous computer vision-guided plant sensing and monitoring systemwas designed and constructed to continuously monitor temporal, morphological, and spectral features of lettuce crop growing in a nutrient film technique (NFT) hydroponics system.
In other hand, to control the temperature and humidity inside the smart green house would require an automatic controller that can read existing activities in the smart green house. Park et al [5] show the system collected temperature of leaves and humidity on leaves of crop. As well as greenhouse environmental information such as temperature, humidity, etc. In this study the temperature and humidity in the greenhouse can be regulated by watering when the value of high temperatures and low humidity. It is necessary for setting the watering time duration for controlling the temperature and humidity values. The author uses fuzzy logic to produce more crop watering duration dynamic where the temperature and humidity in accordance with the desired plants.
Compared with conventional systems, the system is capable of processing input of real values (exact) into a fuzzy scale and process them using the rule base to make the decision which is the output fuzzy system very quickly and accurately.
II. CONTROL SYSTEM OF SMART GREENHOUSE
Plants can grow properly if schedule for watering is done every day. In the conventional greenhouse, watering is done manually by the owner to perform continuous scheduling watering during the day.
This being smart greenhouse that plants watering process is performed automatically in accordance with the desired watering schedule. Schedule watering is done at 07.00 in the morning and at 17.00 in the afternoon. In addition to watering schedules, the greenhouse smart also uses temperature and humidity sensors to maintain the condition of the plants in the green house.
Conditioning system for temperature and humidity inside the greenhouse smart refers to climatic factors that affect plant growth. In this case, the parameter is temperature and humidity controlled.
In this study, samples were taken where the tomato plants good growing condition is at a temperature of 24o -32o C. Under the terms of the growth made control systems using temperature and humidity sensors (DHT11) where the sensor detects changes in temperature and humidity that occur in green house.
III. CONTROL DURATION OF GREENHOUSE SPRAYING PUMP SYSTEM USING FUZZY LOGIC METHOD a. Design Fuzzy Logic System for Smart Greenhouse
Smart greenhouse has a system of spraying plants that use water pumps. Working of water pump is programmed at microcontoller i.e., Arduino-Uno for controlling an electronic device as a link between the power source to the water pump.
Watering duration is controlled by fuzzy logic system. Fuzzy logic method begins by receiving input from a temperature sensor and humidity sensor then their value will be calculated to produce the output as the duration of watering in ths smart green house.
In this system the temperature sensors and humidity sensors as inputs, where the sensor will detect temperature and humidity in the smart green house. Then the sensor will give a signal to the microcontroller Arduino-uno which will give the decision of the time duration to electronic devices that connected to a power source of the water pump. Flow diagram for determining the duration of the water pump settings by using fuzzy logic as shown in Figure 1. In regulating the duration of the water pump using fuzzy These are the reference conditions of temperature and humidity conditions in fact, was to get the output of the temperature and humidity sensors in the form of value for the duration of the water pump used mamdani and defuzzification method used is minimized. The output value of the duration of this water pump will then be sent to the microcontroller types arduino uno.
By using Fuzzy Inference System (FIS) on software matlab, created a program file in matlab toolbox. Engine FIS will be saved by clicking: File -export. There are two options available i.e., Workspace and File. When selected to Workspace then will appear in Matlab active.
At the FIS Editor at setting the two inputs namely Temperature Sensor with conditions of low, medium and high and humidity sensor input with the condition of low, medium and high, as seen in Figure 2.
At the FIS Editor at setting the two inputs namely Temperature Sensor with conditions of low, medium and high and humidity sensor input with the condition of low, medium and high, as seen in Figure 2. The first input is a temperature sensor with conditions of low, medium and high use trimf using the membership function types can be seen in Figure 3. The range values from 0 to 40 express the magnitude of the temperature in the green house.
. Figure 3. Temperature as an input of Fuzzy system A second input of humidity with level of membership function low, medium and high, here, it is using the sigmoid function type for representative of membership function, can be seen in Figure 4. Here, it was chosen three membership function using sigmoid formula. Decisions given by the fuzzy controller is derived from the rules which exist in the database. These decisions are stored as a set rule. Rule-the rule is an if-then statement is intuitive and easy to understand, because only a linguistic statement.
There are 9 rules used in setting the duration of the water pump at this Greenhouse. These rules are inserted into the "rule editor" as in Figure 6.
Rule which has been included in the rule editor can be seen in the rule option viewer as shown in Figure 7. Rule viewer is used to set the duration of the likelihood of the water pump, the condition is a short, medium or long, such condition depends on the value of the temperature on the output. To set the trends can be done by shifting the center red line for each membership function. Figure 7 shows the value when the temperature of 34 0 C and humidity indicate 31%, the output obtained output watering for 90.4 seconds. The value of this watering output varies depending on the value of the input temperature and humidity in the green house.
Results of rule viewer can be seen in the form of threedimensional surface viewer as shown in Figure 8. Surface viewer is plot a graph of the data released in the process of defuzzification. The temperature and the humidity data has been obtained used as input variables in this system to get the duration of watering plants as the output variable. The data has been obtained and then inserted into the rule to get the duration of the pump. For the duration of the pump that produced less than 20 seconds otherwise the pump is not running and if the duration of the pump is greater than 20 seconds otherwise the pump running so at 07:00 and 08:00 pump is not running. The duration of the pump obtained at 12.00 where the temperature is 35 ° C and humidity of 35% can be seen in Table 1. Table 1. Result of greenhouse system using Fuzzy Logic Simulation of fuzzy logic control shows the results performed on variable temperature and humidity as input variables obtained hour duration of the pump taken at 7:00 to 17:00 as in the table above and can be seen in Figure 9 below. From the results of the duration of watering with fuzzy logic is done on tomato plants in the green house can keep the temperature and humidity are optimal temperature of 24 ° C -32 ° C and humidity 40% -80%. It is effective for the duration of watering with fuzzy logic obtained in accordance with the temperature and humidity in the green house.
IV. CONCLUSIONS 1. Simulation of watering duration was success using Matlab.
The result was duration of watering which inputs of temperature and humidity in smart green house. 2. Watering duration system using fuzzy logic method was effective to keep the temperature on 24 °C -32 °C dan humidity on 40 % -80 %. 3. The fuzzy logic method is available to implement at smart greenhouse, which has two or more inputs and single output. | v3-fos |
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} | s2 | Feed Conversion, Survival and Development, and Composition of Four Insect Species on Diets Composed of Food By-Products
A large part of the environmental impact of animal production systems is due to the production of feed. Insects are suggested to efficiently convert feed to body mass and might therefore form a more sustainable food and/or feed source. Four diets were composed from by-products of food manufacturing and formulated such as to vary in protein and fat content. These were offered to newly hatched Argentinean cockroaches, black soldier flies, yellow mealworms, and house crickets. The first two species are potentially interesting as a feed ingredient, while the latter two are considered edible for humans. Feed conversion efficiency, survival, development time, as well as chemical composition (nitrogen, phosphorus, and fatty acids), were determined. The Argentinean cockroaches and the black soldier flies converted feed more efficiently than yellow mealworms, and house crickets. The first two were also more efficient than conventional production animals. On three of the four diets yellow mealworms and house crickets had a feed conversion efficiency similar to pigs. Furthermore, on the most suitable diet, they converted their feed as efficiently as poultry, when corrected for edible portion. All four species had a higher nitrogen-efficiency than conventional production animals, when corrected for edible portion. Offering carrots to yellow mealworms increased dry matter- and nitrogen-efficiency and decreased development time. Diet affected survival in all species but black soldier flies, and development time was strongly influenced in all four species. The chemical composition of Argentinean cockroaches was highly variable between diets, for black soldier flies it remained similar. The investigated species can be considered efficient production animals when suitable diets are provided. Hence, they could form a sustainable alternative to conventional production animals as a source of feed or food.
Introduction
Several insect species can be produced for food and/or feed, for instance house crickets are produced for food in Thailand and Laos, and black soldier flies are used as fish feed [1][2][3][4]. Conventional animal production systems contribute greatly to anthropogenic greenhouse gas production and use vast amounts of fossil energy and arable land [5,6]. For a large part, these indicators of environmental impact are determined by the amount and type of feed used during animal production [7,8]. Another important factor of environmental impact is how efficiently this feed is converted into body mass. One of the suggested advantages of insects over conventional production animals such as chickens, pigs and cattle, is a higher feed conversion efficiency, due to insects being poikilothermic [2,9,10]. However, feed conversion efficiency depends on a variety of factors, such as the species and the diet consumed. Due to differences in digestive systems and nutrient requirements the same diet may result in other feed conversion efficiencies in different species [11]. Furthermore, diet composition affects development rate and the chemical composition of the insect body [12][13][14][15]. To quantify these variables, four insect species, two edible for humans and two suitable as animal feed, were selected.
In our experiment we used several by-products to compose four diets, differing in protein and fat content. The objectives of this experiment were 1) to compare the feed conversion efficiency of several insect species to be used as production animals, and 2) to determine the effects of diet composition on survival, development time, and chemical composition of these species.
Insects
Four insect species were selected: Argentinean cockroach (Blaptica dubia (Serville); Dictyoptera: Blaberidae), black soldier fly (Hermetia illucens (L.); Diptera: Stratiomyidae), yellow mealworm (Tenebrio molitor (L.); Coleoptera: Tenebrionidae), and house cricket (Acheta domesticus (L.); Orthoptera: Gryllidae). Adult Argentinean cockroaches were provided by a private Dutch insect breeder and were checked daily for new-born offspring. Newly hatched nymphs of the house cricket and larvae of the black soldier fly were taken from colonies maintained at the Laboratory of Entomology, Wageningen University. These two species had been reared on chicken feed for over four years (Opfokmeel farmfood, Agruniek Rijnvallei Voer BV, Wageningen, The Netherlands). Mealworm eggs were provided by Kreca V.O.F. (Ermelo, The Netherlands). For all species, larvae or nymphs were randomly allocated to control and experimental groups within 24 hours of egg hatch (black soldier flies, yellow mealworms, and house crickets) or birth (Argentinean cockroaches).
production, were different in colour and structure suggesting differences in composition; hence, we tested both. For black soldier flies the chicken feed used for the black soldier fly colony at the Laboratory of Entomology of Wageningen University (Wageningen, The Netherlands) served as a control diet (Opfokmeel farmfood; Agruniek Rijnvallei Voer B.V., Wageningen, the Netherlands). Diets were stored at-20°C.
Experimental setup
Argentinean cockroach: Ten nymphs were placed in a plastic container (17.5 x 9.3 x 6.3 cm) with aeration slits on the sides. A piece of egg carton was placed in the container to provide a hiding place for the nymphs. Nymphs were provided with four grams of either an experimental diet, or control diet. Moisture was provided three times per week by applying a few drops of tap water in a corner of the container.
Black soldier fly: One hundred larvae were placed in a plastic container (17.8 x 11.4 x 6.5 cm) of which the sides were manually perforated to allow air flow. Larvae were provided with four grams of either an experimental diet, or control diet. For each gram of diet, approximately two ml of water was added by means of a syringe.
Yellow mealworm: Fifty larvae were placed in a plastic container (17.5 x 9.3 x 6.3 cm) with aeration slits in the sides. Larvae were provided with one gram of either an experimental diet or one of the two control diets. In some rearing facilities, this species is provided with carrot as a source of water [8]. Therefore, the effect of providing carrot was tested for each experimental and control diet. Larvae allocated to a treatment with carrot, were provided with 0.30 g, three times per week.
House cricket: Fifty nymphs were housed in a plastic cage (35.6 x 23.4 x 22.8 cm; Faunarium type pt2665, Hagen, Holm, Germany), with aeration slits in the lid. To increase surface area, two layers of hollow plastic tubes (20 cm long and 3 cm in diameter) were placed in each cage. Nymphs were provided with one gram of either an experimental diet or control diet. Furthermore, a water dispenser (Gebroeders de Boon, Gorinchem, The Netherlands), with a piece of tissue paper placed in the opening to prevent drowning, was placed in each cage.
For each species, six replicate containers per dietary treatment were set up, after which the containers were placed in a climate chamber at 28°C with a relative humidity of 70% and a photoperiod of 12 hours.
Three times per week, all containers were checked visually. If the feed provided was almost depleted, as indicated by changes in colour and particle size, more was added to ensure ad libitum feeding. Water for the house crickets was replenished when required.
All insects were harvested per container when the first prepupa (black soldier fly), pupa (yellow mealworm), or adult (Argentinean cockroaches and house crickets) was observed. Most specimens per container would be expected to be in their last larval or nymphal stage at that moment. This is when yellow mealworms are normally sold, and when house crickets have the highest edible portion [9]. Furthermore, black soldier flies have a higher digestibility in their last larval stage than in their prepupal stage [16]. For Argentinean cockroaches the same moment was chosen. Development time was considered to be the number of days between the start of the experiment and the day a container was harvested. After harvesting, animals were killed by freezing and then all animals were dried per container at 70°C until a constant weight. Subsequently these were ground with a batch mill (Ika Labortechnik, Staufen, Germany) and stored at -20°C until further analysis.
Chemical analysis
Nitrogen (N) and phosphorus (P) content of the diets, insects and faeces were determined according to Novozamsky, Houba [17]. When insufficient samples were available (four samples), N content was determined according to Patton and Kryskalla [18], and P content according to Rowland and Haygarth [19]. Fatty acids were extracted according to Folch, Lees [20] and the fatty acid profiles were determined according to Raes, De Smet [21].
Calculations and statistics
Feed conversion efficiency can be expressed in different ways. The most common measure in animal production systems is the Feed Conversion Ratio (FCR), which is the amount of feed needed (in kg) to obtain one kg of weight increase of the production animal. Entomologists, however, commonly use Efficiency of Conversion of Ingested food (ECI) as a measure for feed conversion efficiency on a dry matter (DM) basis. ECI is calculated as: (weight gained / weight of ingested food) à 100% [22]. For FCR and ECI calculations it was assumed that all provided feed had been consumed by all species. This assumption was supported by changes in colour and structure of the residual material; however, it cannot be excluded that a small amount of feed was left unconsumed. Both the FCR and the ECI can be calculated on a fresh and a DM basis, and can also be used for specific nutrient conversion efficiencies. In this paper, FCR is expressed on a fresh weight basis, whereas ECI is expressed on a DM basis. FCRs for concentrates (feeds with a high nutrient density) exclude the weight of provided carrots. Crude protein content was calculated by multiplying the nitrogen content by 6.25. Total fatty acid content (TFA) was calculated by summing the contents of individual fatty acids. Nitrogen conversion efficiency (N-ECI) was calculated as: (Insect N-content à insect weight at harvest) / (Dietary N-content à feed provided).
Significant differences between treatments (P < 0.05) were determined by means of a Kruskall-Wallis test followed by a Scheffé test for post-hoc testing. The degrees of freedom equalled the number of compared treatments minus one. If only one sample was analysed for a dietary treatment, it was excluded from the dataset for post-hoc testing. Correlations between parameters were determined by Pearson correlation tests. Statistical analysis for all data was performed using SPSS 19.0.
Results and Discussion
This study compares the effects of different diets composed of food by-products on four insect species. Effects on feed conversion efficiency, survival and development, as well as insect chemical composition are reported.
Diet composition
Dry matter percentage and nutrient composition of diets are listed in Table 2. The experimental and the control diets used in this study had a high DM content (88-95%) whereas the carrots, provided to the yellow mealworms in the respective treatments, had a low DM content (9%), making them a suitable source of water. High protein, and high fat diets contained more crude protein and TFA, respectively, than control diets. The opposite was true for the low protein and the low fat diets.
In the high protein diets the phosphorus content was more than double that of the low protein diets (Table 2), preventing an accurate distinction between the effects of protein and phosphorus. Increased levels of dietary P are reported to have positive effects on life history traits of certain insect species including house crickets [23][24][25][26] The dietary fat composition varied between diets. The most prevalent fatty acids were tridecylic acid (C13:0), palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1n9c), and linoleic acid (C18:2 n6c). The latter was especially abundant in control diets (30-48% of TFA). In the high protein, low fat diet the main fatty acid was C13:0 (62% of TFA), whereas in the control diets it accounted for 12-22% of TFA, and only 4-6% of TFA in the high fat diets. In the high fat diets, myristic acid (C14:0) was present in larger concentration (5-9% of TFA) than in the other diets (< 1%).
Feed conversion efficiency
When using economic allocation, by-products with a lower monetary value than the main product are considered to have a lower environmental impact [7]. These by-products are becoming increasingly important feed ingredients [27]. Their usefulness depends, among others, on how efficiently these are converted to body mass by the production animal. Combinations of by-products could make suitable insect diets. Diet composition is the main variable determining feed conversion efficiency for a given insect species [28]. The two species suitable as animal feed, the Argentinean cockroaches and black soldier flies, used their food more efficiently than the species suitable for human consumption, the yellow Mealworms and house Table 2. Dry matter (DM) percentage, crude protein (CP), phosphorus, and total fatty acid (TFA) percentages on a DM basis, and fatty acid composition (as % of total fatty acids*) of diets provided to Argentinean cockroach, black soldier fly, yellow mealworm, and house cricket (results based on single analysis). crickets (Table 3). This was apparent for both the FCR (feed conversion ratio on a fresh matter basis) and the ECI (feed conversion efficiency on a dry matter basis). Whether these more favourable FCRs would lead to economic and environmental benefits when these insects are used as a feed ingredient depends on whether their diets could also be used directly and efficiently by the consuming production animal.
For conventional production animals, FCRs for concentrates to edible product are reported to be 2.3 for poultry meat, 4.0 for pork, and 8.8 for cereal beef [27]. The yellow mealworms had high FCRs (> 3.8) on all diets. However, when only the concentrate feed is used for FCR calculations (carrots are excluded), the FCRs for carrot-supplemented diets is between 1.8 (high protein, high fat diet), and 3.1 (high protein, low fat diet). The lower values are similar to the FCR for concentrates of commercially produced mealworms (2.2) provided with carrots [8]. Also the house crickets, on their control diet, had a similar FCR (2.3). This indicates that the two species suitable for human consumption were as efficient as poultry in converting their feed to food for humans.
The Argentinean cockroaches had a higher FCR on their control diet than on the low protein, low fat and the two high fat diets (1.5-2.7; Table 3). On the low protein, high fat diet this species had the highest ECI of all species-diet combinations (5-30%).
For the black soldier flies the FCRs and ECIs were similar over dietary treatments, although they tended to use the low protein diets less efficiently than the other diets (P = 0.051).
In conventional production animals the energy content of the feed determines growth rates and efficiencies [29]. In insects the protein density and composition seem to be more important [30][31][32] because they do not use energy to maintain a constant body temperature. Indeed, high protein diets resulted in lower FCRs and higher ECIs for most species. Furthermore, for all species-diet combinations in our experiment N-ECI > ECI, indicating that N was more efficiently converted to body mass than other diet components. N-ECI in yellow mealworms and house crickets ranged between 22 and 58%. This can be considered high compared to the conversion of dietary protein to edible protein in conventional production animals (12% for cereal beef, 23% for pork and 33% for chicken) [27]. This can only in part be explained by the higher edible portion for the insects, compared to the conventional production animals. With a 50% increase in demand of animal based protein expected by 2050, this high N-ECI may be the most relevant benefit of insects over conventional production animals [33]. When compared over the four insect species studied, large differences in N-ECI between species and between dietary treatments were apparent. Argentinean cockroaches provided with the low protein high fat diet had the highest N-ECI (87%). On the other diets, N-ECI for this species was also high (51-66%). Both Argentinean and American cockroaches (Periplaneta americana) harbor endosymbionts that produce methane [34,35]. The cockroach endosymbiont Blattabacterium enables American cockroaches to convert nitrogenous waste products, such as uric acid, to amino acids, and vitamins [36]. This explains their high N-ECI (51-83%), and possibly the high N-ECI of Argentinean cockroaches in this study. N-ECIs calculated for the black soldier fly larvae were slightly lower (43-55%) and these were not affected by diet.
For the yellow mealworms, the high water content of carrots might be expected to increase the FCR for all carrot-supplemented diets, because of the high water content of the carrots. However, this was only evident for the low protein diets. These diets resulted in longer development times, and thereby longer periods of carrot provision, and hence larger amounts of carrot being provided. On most other diets, carrot provision resulted in similar FCRs, and greatly increased N-ECI (22-31% to 35-58%) and ECI (7-12% to 13-21%). The latter values seem low compared to previously reported ECIs (17-29%), in a study where yellow mealworms were also provided with diets varying in protein content and supplemented with carrot [37]. However, the higher values in that study (28-29%) were found on diets with an extremely high protein content (33-39% DM). This indicates that dietary protein content is a primary determinant in feed conversion efficiency. However, these extremely high protein diets resulted in a higher excretion of uric acid, and N-ECIs calculated from that study (14-23%) were low compared to our study.
For house crickets, no differences in FCR were found between dietary treatments (2.3-6.1). Published FCR values for this species (1.5-2.8) indicate that feed conversion was inefficient on most of our diets [2,10,32]. Furthermore, house cricket ECI (5-12%) was the lowest of the four species investigated. Similarly to the FCR, reported ECI values for last stage nymphs and adults (20-38%), were more favorable than in our experiment [38][39][40]. House cricket N-ECI (23-41%) was similar to yellow mealworm not provided with carrot, but lower than the other species-treatment combinations. The N-ECI observed in our study was similar to the N-ECI reported by Lundy & Parrella (25%) for house crickets on grain-based diets [32]. In our study water provision might have been suboptimal, affecting the ECI similarly as observed for yellow Table 3. Survival rate (%), development time (days), Feed Conversion Ratio (FCR), Dry matter conversion of ingested food (ECI;%), and nitrogen efficiency (N-ECI; %), of Argentinean cockroach, black soldier fly, yellow mealworm without and with carrot, and house cricket on different diets (Mean ± SD). Different superscripts in a column, per species, denote significant differences (Kruskal Wallis followed by Scheffé's post-hoc test; P < 0.05). mealworms. Another explanation might be that a densovirus (AdDNV), present in most European and North-American house cricket production facilities, interfered with nutrient absorption, increased mortality and decreased growth rates [41][42][43].
Survival rates and development time
Whereas all diets were accepted by the four species, development times were strongly affected by dietary treatment ( Table 3). The same was true for survival rate except for the black soldier flies, in which survival was high on all treatments (72-86%). Higher survival rates were correlated with shorter development times in the other species; for the Argentinean cockroaches (R = -0.708; P < 0.001), yellow mealworms (R = -0.524; P < 0.001), and house crickets (-0.718; P = 0.001). As both parameters are considered indicators of dietary quality, strong correlation can be expected [11].
In the Argentinean cockroaches the survival rate was 47% on the high protein, low fat diet, whereas it was 80% on the high protein, high fat diet. Similarly, development took ca. ten months on the high protein, low fat diet while this was only seven months on the high protein, high fat and their control diet.
The black soldier flies developed fastest on the high protein, high fat, and their control diet (three weeks), whereas on the low protein diets this took over five weeks. On a high quality diet such as chicken feed, or a diet designed for houseflies, black soldier fly larvae develop in two to three weeks [44,45]. The extended development on the low protein diets indicates these had a lower dietary quality for black soldier flies [11,12].
The yellow mealworms in our study developed in 12 to 32 weeks. Similar to the black soldier flies, development times were extended in yellow mealworms on low protein diets, compared to high protein diets or their control1 diet. Furthermore, survival was higher on high protein diets than on low protein diets, while their control diets were intermediary. Except for the low protein diets, development time and survival of yellow mealworms in our study were similar to published values (10.5-24 weeks and 69-92%, respectively) [37,46,47]. It appears that in this species dietary protein content is a determining factor for development and survival. The inclusion of beer yeast in the high protein diets, which works as a feeding stimulant [48], and contains important growth factors for yellow mealworms [49] might also have contributed to a shorter development time and higher survival. Carrot provision decreased development time and resulted in uniform survival rates (! 80%) except for the low protein, high fat diet (< 20%). Irrespective of carrot provision, survival was low on this diet, which might well have been caused by the presence of cinnamon in the cookie remains (which comprised 50% of that diet), as was suggested by van Broekhoven, Oonincx (37). The increased survival and shortened development time could be due to the carrot functioning as a water supply to the yellow mealworms [47,49]. However, other nutrients, for instance β-carotene, could also have been of influence [11,37].
The development time of house crickets in our study varied substantially between the control diet (7 weeks) and the low protein, high fat diet (24 weeks). On the high protein, high fat and their control diet these values were similar as reported in literature (4.5-11.5 weeks), but development was strongly prolonged on the other diets [2,40,[50][51][52]. Furthermore, survival rates can be considered low in this species on all diets, with the possible exception of their control diet (55%). Older studies report that house crickets do well on most animal feeds or poultry mashes and that a 20% crude protein content is sufficient to support growth [38,40]. Furthermore, survival can be up to 80% [51]. A more recent study reports survival rates similarly low (24-47.5%) as our study [2]. These differences might be attributed to the previously mentioned densovirus.
Insect body composition
Considerable differences in dry matter content were found for the four different species (Table 4). House crickets had the lowest DM content (~25%), whereas the highest was observed in yellow mealworms (up to 42%). Compositional differences between the four species were apparent (Table 4, Fig 1). Crude protein content was the lowest in black soldier flies, followed by yellow mealworms, whereas Argentinean cockroaches and house crickets had higher crude protein contents (P < 0.001). Crude protein and TFA contents were within published values for black soldier flies, yellow mealworms, and house crickets (Table 5). Argentinean cockroaches had a lower P content than the three other species (P<0.001). Phosphorus and crude protein content were strongly correlated in the Argentinean cockroach (R = 0.776; P = 0.001), black soldier fly (R = 0.827; P < 0.001), and yellow mealworm (R = 0.546; P = 0.001), but not in house crickets (P = 0.11). When analysed for the four species together, no such correlation was found (P = 0.572), which could suggest species-specific crude protein and P ratios. Because in most diets higher crude protein contents coincided with higher P contents this could also have been a dietary effect, as was seen in black soldier flies produced on manure [53].
Within species, dietary treatment had the largest effect on composition for the Argentinean cockroaches. Considerable plasticity was observed regarding DM (28-39%), crude protein (38-73% DM), and TFA content (15-40% DM) in our study. Whereas Yi, Lakemond [54] reported a lower DM content (24%), their crude protein and TFA content were within the range found in our study (Table 5). On both the high protein, low fat and their control diet, Argentinean cockroaches contained almost twice as much crude protein, compared to the low protein, high fat diet. In American cockroaches uric acid (which contains N) is stored when they are provided with high protein diets [36]. If this is also the case for Argentinean cockroaches, on such high protein diets N-based crude protein determinations would overestimate true protein content.
The black soldier flies in our study showed little compositional variation. Their DM content was between 33 and 36%, whereas a far larger range (17-40%) has been reported [1,44,53,55]. Crude protein contents in our study had the same range as reported in other studies with black soldier flies (38-46% DM). This was elevated on the high protein, high fat diet compared to the low protein diets. Phosphorus content of black soldier flies on their control diet was higher than those on low protein diets, but low on all diets compared to results from other studies ( Table 5). Their TFA content was not affected by dietary treatments, and similar to published values (Table 5).
In the yellow mealworms carrot supplementation decreased DM content but it did not affect crude protein, P, or TFA content. This is in contrast with the findings of Urs and Hopkins [46] who reported an increase in fat content when water was provided. When comparing diets supplemented with carrots, the highest TFA content was found on the control2 diet, although the diet itself had an intermediate TFA content. TFA content was more variable (23-35% of DM) than crude protein and P content, which were similar on most diets. Whereas crude protein and TFA content were within the range published for yellow mealworms, the P content of our yellow mealworms was lower (Table 5).
House crickets had a high crude protein (58-59% DM) and a low TFA content (17-21% DM) on the diets on which sufficient material for chemical analysis could be collected (the control and the high protein diets). Although our diets had a large variation in fat content, this was not reflected in the TFA content of the crickets, whereas other studies indicate that large variability is possible (Table 5).
Fatty acids
No butyric acid (C4:0), caproic acid (C6:0), caprylic acid (C8:0), undecylic acid (C11:0), or erucic acid (C 22:1n9) was detected in any of the insect species. Capric acid (C10:0) was detected only in black soldier flies (0.8-1.3% of TFA; Table 6). Fig 2 illustrates that fatty acid profiles were determined not only by diet, but were in part species-specific. The clearest example of this was the high lauric acid (C12:0) concentration in the black soldier flies, which contributed between a third to half to TFA, while this was 0.5% in the other species. In dipterans C16 fatty acids are suggested to predominate [56]; however, for black soldier flies this seems to be C12:0 (Table 5). Also C14:0 was present in higher levels in black soldier flies than in the other species investigated. While C16:0 concentrations were affected by diet, all species had similar ranges. The contribution of C18:0 to TFA was highest in house crickets, followed by Argentinean cockroaches and yellow mealworms, while for black soldier flies this was lowest (P < 0.001). The main fatty acid in Argentinean cockroaches and yellow mealworms was C18:1n9c, while the Table 4. Dry matter (DM), crude protein (CP; %DM), phosphorus (P; g/kg DM) content, and total fatty acids (TFA, %DM), of Argentinean cockroach, black soldier fly, yellow mealworm without and with carrot, and house cricket on different diets (Mean ± SD). Different superscripts in a column, per species, denote significant differences (Kruskal Wallis followed by Scheffé's post-hoc test; P < 0.05). Experimental diet abbreviations: HPHF = high protein, high fat; HPLF = high protein, low fat; LPHF = low protein, high fat; LPLF = low protein, low fat, C indicates carrot supplementation. For DM% n = 6, for CP, P & TFA n = 3 unless indicated otherwise.
Large variation due to dietary treatment was apparent for C18:2n6c in all species, as indicated by the error bars in Fig 2. A small proportion of the house cricket fatty acids consisted of eicosatrienoic acid (C20:3n3; 0.4% of TFA), and docosahexaenoic acid (C22:6n3;~0.1% of TFA). These were not detected in the other species analysed, or in any of the diets. Furthermore, both house crickets on the high protein, high fat diet, and black soldier flies on the low protein, high fat diet contained 0.1% eicosapentaenoic acid (C20:5 n3), while in the Argentinean cockroaches and yellow mealworms this fatty acid was not detected. In all species investigated, n3 fatty acids were present in low concentrations ( 1.5%), while n6 fatty acids were present in higher concentrations ( Table 6). The content of n3 fatty acids, as well as the n6/n3 ratio is relevant for human and animal health. A n6/n3 ratio < 5 is considered optimal for human health [57]. In most animal products this ratio is between 10 and 15, but in Table 5. Comparative data on crude protein (CP; %DM), fat (%DM), and phosphorus content (P; %DM), and the main fatty acids (as a % of total fatty acids) for black soldier fly larvae, yellow mealworms and house crickets. [57]. Similarly, the fatty acid profile of insects is considered to reflect the fatty acid profile of the diet [11], although this is not true for all species, for instance yellow mealworms [58]. Our experimental diets differed in their n6/n3 ratio (4.9-13.5), and affected insect n6/n3 ratios (5.8-102.1; Table 7). However, none of the insectdiet combinations resulted in a n6/n3 ratio < 5. The lowest n6/n3ratios were present in the Argentinean cockroaches and black soldier flies, followed by house crickets, whereas yellow mealworms had high n6/n3 ratios (>20) on all diets tested. In order to optimize n6/n3 ratios in insect-derived food or feed further experiments on the plasticity of fat content and fatty acid profile are needed.
The main fatty acid profile in Argentinean cockroaches followed a pattern similar to an earlier study with this species; the main fatty acid was C18:1n9c, followed by C16:0, and C18:2n6c ( Table 5). The concentration of the latter fatty acid showed considerable variation due to dietary treatment (1.7-19.5% of TFA). The fatty acid profile of the Argentinean cockroaches partially followed the dietary fatty acid profile. However, it seems that C18:1n9c was selectively accumulated, especially on the high protein, low fat diet. In a study with several cockroach species this fatty acid accounted for 30-55% of TFA [56], which might indicate that cockroaches are especially rich in C18:1n9c.
Overall, fatty acid profiles of black soldier flies in this study were similar to published values (Table 5), although C12:0 concentrations were higher and C18:1n9c concentrations were lower. The fatty acid profiles of the black soldier flies did not follow the dietary fatty acid pattern in general. black soldier flies on the high fat diets, rich in C18:1n9c, retained more of this fatty acid than on the other diets, but the low protein, low fat diet, resulted in relatively low concentrations of C18:1n9c. On all diets this species had a high concentration of C12:0. It appears black soldier flies metabolize a large proportion of fatty acids to C12:0 when lower levels of fat are provided, whereas these are stored in their dietary form when higher amounts are Table 7. Ratios between n6 and n3 fatty acids in experimental and control diets provided to Argentinean cockroach, black soldier fly, yellow mealworm, and house cricket. provided, indicating limited possibilities to tailor the fatty acid profile of black soldier flies. On most diets black soldier flies did, however, have a relatively low n6/n3 ratio. Yellow mealworms were rich in C18:1n9c,C18:2n6c, and C16:0 on all diets, which corresponds with published fatty acid profiles for this species (Table 5). Whereas carrot provision strongly influenced N-ECI in yellow mealworms, it did not influence their general fatty acid profile, which agrees with the results of Urs and Hopkins [46] on water provision to yellow mealworms. The fat composition of yellow mealworm seems to be fairly constant. Whereas the n6/n3ratio is flexible, yellow mealworms accumulate n6 fatty acids more efficiently than n3 fatty acids, resulting in a higher n6/n3ratio in the yellow mealworm compared to their diet [37]. Yellow mealworm had the highest n6/n3 ratio on all diets tested, although carrot provision resulted in a decrease.
Fatty acid data on house crickets are available for only three diets, due to a limited amount of sample. The main fatty acid in this species was C18:2 n6, although C16:0 and C18: 1n9 were also present in high concentrations. Together these made up ! 75% of TFA. Large differences in C18:2n6 and α-linolenic acid (C18:3n3) concentrations were found due to dietary treatment. This suggests large plasticity in the content of these fatty acids, similar to the study of Collavo, Glew [2]. House crickets can convert C18:1n9 into C18:2n6 [59,60], but probably require one of these in their diet. The low concentrations of these fatty acids in the high protein, low fat diet might have prolonged development and lowered survival. Because C20:3n3 and C22:6n3 were not detected in the diet, but was present in the house crickets, this could suggest de novo synthesis. House crickets can elongate C18:3n3 to C20:5n3 [61], however, formation of C20:3n3 and C22:6n3 has not previously been described. In contrast to our findings, no C20:3n3 or C22:6n3 was detected by Tzompa-Sosa, Yi [62], whereas they reported a higher concentration of C20:5n3 (0.6 vs. 0.1% of TFA). The latter fatty acid was, however, detected in the diet used in that study, and might therefore have been selectively accumulated.
Conclusions
This study shows that 1) insects can be produced on diets composed of food by-products, 2) Argentinean cockroaches and black soldier flies use feed more efficiently than yellow mealworms and house crickets, 3) yellow mealworms and house crickets were equally efficient in converting feed to edible body mass as poultry, 4) on suitable diets the insects utilized protein more efficiently than conventional production animals, and 5) the composition of insect species can be altered through their diet.
Further studies should focus on finding optimised combinations of insect species and diet composition, in order to efficiently produce insects that meet the nutritional requirements of humans and other animals. | v3-fos |
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} | s2 | Differential response to sulfur nutrition of two common bean genotypes differing in storage protein composition
It has been hypothesized that the relatively low concentration of sulfur amino acids in legume seeds might be an ecological adaptation to nutrient poor, marginal soils. SARC1 and SMARC1N-PN1 are genetically related lines of common bean (dry bean, Phaseolus vulgaris) differing in seed storage protein composition. In SMARC1N-PN1, the lack of phaseolin and major lectins is compensated by increased levels of sulfur-rich proteins, resulting in an enhanced concentration of cysteine and methionine, mostly at the expense of the abundant non-protein amino acid, S-methylcysteine. To identify potential effects associated with an increased concentration of sulfur amino acids in the protein pool, the response of the two genotypes to low and high sulfur nutrition was evaluated under controlled conditions. Seed yield was increased by the high sulfate treatment in SMARC1N-PN1. The seed concentrations of sulfur, sulfate, and S-methylcysteine were altered by the sulfur treatment in both genotypes. The concentration of total cysteine and extractible globulins was increased specifically in SMARC1N-PN1. Proteomic analysis identified arcelin-like protein 4, lipoxygenase-3, albumin-2, and alpha amylase inhibitor beta chain as having increased levels under high sulfur conditions. Lipoxygenase-3 accumulation was sensitive to sulfur nutrition only in SMARC1N-PN1. Under field conditions, both SARC1 and SMARC1N-PN1 exhibited a slight increase in yield in response to sulfur treatment, typical for common bean.
INTRODUCTION
Common bean (dry bean, Phaseolus vulgaris) is an important source of protein and fiber in human diets. Like other grain legumes, its protein quality is sub-optimal, being limited by the levels of the essential sulfur amino acids, methionine, and cysteine. During the past decades, a lot of effort has been dedicated to improving protein quality in grain legumes, primarily using transgenic approaches (Krishnan, 2005;Ufaz and Galili, 2008;Amir et al., 2012;Galili and Amir, 2013). Transgenic expression of foreign proteins can be limited by the supply of sulfur, and often results in a shift of sulfur away from endogenous, sulfur-rich proteins (Streit et al., 2001;Tabe and Droux, 2002;Chiaiese et al., 2004). Table 1 lists the different experiments that were performed involving transgenic expression of sulfur-rich proteins in legumes and their outcomes. In Vicia narbonensis, co-expression of Brazil nut 2S albumin and a feedback-insensitive, bacterial aspartate kinase was associated with increased sulfur concentration in seed (Demidov et al., 2003). A common concern with these approaches is the potential allergenicity of the foreign proteins (Nordlee et al., 1996;Kelly and Hefle, 2000;Krishnan et al., 2010). A possible solution to this problem is the expression of a de novo synthetic protein, MB-16. An alternative approach involves the transgenic manipulation of sulfur amino acid pathways. Overexpression of cytosolic serine acetyltransferase in developing soybean seed led to a 70% increase in total cysteine concentration (Kim et al., 2012). Expression of a feedback-insensitive Arabidopsis cystathionine γsynthase (AtD-CGS), encoding a protein lacking 30 amino acids in the N-terminal domain, raised total methionine concentration by 1.8 to 2.3-fold, with an overall increase in seed protein concentration (Song et al., 2013). By contrast, expression of the feedback-insensitive mto1-1 allele, harboring a point mutation, led to elevated levels of free methionine, but not total methionine in soybean, whereas in azuki bean, the levels of cystathionine were raised while total methionine concentration was actually decreased (Hanafy et al., 2013a,b). A completely different approach proposed to improve protein quality in common bean involves the introduction of highly digestible phaseolin types from wild accessions by conventional breeding (Montoya et al., 2010). Based on in vitro protein digestibility corrected amino acid score, genotypes having highly digestible phaseolin types could increase bioavailable sulfur amino acids by approximately 30% as compared with S type phaseolin present in Mesoamerican cultivars.
Grain yield in legumes has a low heritability due to environmental variables. Consequently, agronomic practices combined with proper fertilizer management heavily influence yield. Sulfur, which has long been known to play a major role in plant metabolism (Takahashi et al., 2011), increases yield in common bean (Malavolta et al., 1987) and influences seed quality via the 1 | Attempts to improve sulfur amino acid concentration in legumes by transgenic expression of sulfur-rich proteins.
Crop plant Foreign protein Increase in sulfur amino acids Reference
Soybean Brazil nut 2S albumin Methionine 26% Townsend and Thomas (1994) Soybean 15 kDa δ-zein Methionine by 20% and cysteine by 35% Dinkins et al. (2001) Soybean 27 kDa γ-zein Methionine by 19% and cysteine by 30% Li et al. (2005) Soybean 11 kDa δ-zein Methionine (alcohol soluble fraction) Kim and Krishnan (2004) Common bean Brazil nut 2S albumin Methionine by 20% Aragão et al. (1999) Lupin Sunflower seed albumin Methionine by 90% Molvig et al. (1997), Tabe and Droux (2002) Chickpea Sunflower seed albumin Methionine by 90% Chiaiese et al. (2004) Vicia narbonensis Brazil nut 2S albumin and feedbackinsensitive aspartate kinase Methionine by 100% and cysteine by 20% Demidov et al. (2003) Soybean MB-16 Methionine by 16% and cysteine by 66% Zhang et al. (2014) proportion of sulfur containing amino acids, cysteine, and methionine. Sulfate is the most significant and readily mobilized form of sulfur. Sulfate taken up by the roots is reduced to sulfide and further incorporated into cysteine. Cysteine is converted to methionine or incorporated into glutathione and proteins. Sulfate, and/or organic forms of sulfur, such as glutathione (Anderson and Fitzgerald, 2001) or S-methylmethionine (Bourgis et al., 1999;Lee et al., 2008;Tan et al., 2010), is transported through the phloem, followed by uptake by transporters into the developing embryo and translocation between seed tissues (Zuber et al., 2010). Delivery of adequate sulfur to seed tissues is needed for maximizing production and to improve protein quality (Hawkesford and De Kok, 2006). Nutrient status of the plant regulates the uptake and assimilation of sulfate (Smith et al., 1995;Buchner et al., 2004). Studies have shown that a decrease in sulfate availability results in a several-fold enhanced expression of sulfate transporter genes, which enhances the capacity for sulfate uptake (Hawkesford, 2000(Hawkesford, , 2003. Sulfur fertilization favorably affects protein quality by increasing the expression of proteins rich in sulfur amino acids. Control of seed protein accumulation by the sulfur status has been well documented in several legumes, including globulins in soybean and lupine (Blagrove et al., 1976;Gayler and Sykes, 1985), and globulins, and albumins in pea (Chandler et al., 1983(Chandler et al., , 1984Higgins et al., 1987). Reduced expression of pea legumin and albumin 1 genes in response to sulfur deficiency was further confirmed in transgenic tobacco (Rerie et al., 1991;Morton et al., 1998). In general, high sulfur stimulates the expression of sulfurrich globulins and albumins while sulfur deficiency increases the expression of sulfur-poor globulins. In soybean, the accumulation of the sulfur-poor β-subunit of β-conglycinin is repressed by exogenous methionine (Holowach et al., 1984(Holowach et al., , 1986. This was confirmed in transgenic Arabidopsis (Naito et al., 1995). The immediate metabolic precursor of cysteine, O-acetylserine, seems involved in the up-regulation of the β-subunit of β-conglycinin under conditions of sulfur deficiency (Kim et al., 1999). A high nitrogen to sulfur ratio not only increases the accumulation of the β-subunit of β-conglycinin, but also reduces the levels of sulfur-rich Bowman-Birk inhibitor . Recent research has focused on adaptation of legumes to sulfur deficiency, highlighting the possible role of a vacuolar sulfate transporter in Medicago truncatula (Zuber et al., 2013). This research is relevant to improvement of sulfur use efficiency (De Kok et al., 2011). Crop plants mitigate the effect of silencing or deficiency in storage proteins through rebalancing of the seed proteome (Marsolais et al., 2010;Herman, 2014;Wu and Messing, 2014). SARC1 and SMARC1N-PN1 are related genotypes of common bean differing in seed protein composition (Osborn et al., 2003). They share 87.5 and 83.6% of the recurrent, Sanilac parental background, respectively. SARC1 integrates the lectin arcelin-1 from a wild accession. SMARC1N-PN1 lacks phaseolin and major lectins, through introgressions from a P. coccineus accession and Great Northern 1140, respectively. These changes are associated with an increased concentration of methionine and cysteine, by 10 and 70%, respectively, concomitant with 70% decrease in Smethylcysteine concentration (Taylor et al., 2008). Proteomic and transcript profiling indicated that several sulfur-rich proteins have increased levels in SMARC1N-PN1, including the 11S globulin legumin, albumin-2, defensin D1, Bowman-Birk type proteinase inhibitor 2, albumin-1, basic 7S globulin, and Kunitz trypsin protease inhibitor (Marsolais et al., 2010;Yin et al., 2011;Liao et al., 2012). SARC1 and SMARC1N-PN1 offer a unique system to investigate how related legume genotypes, harboring natural genetic variation in storage protein composition, respond to sulfur deficiency. The presence of an endogenous sink for sulfur in SMARC1N-PN1 is associated with an increased plasticity of the seed composition in response to sulfur nutrition.
PLANT MATERIALS AND GROWTH CONDITIONS
SARC1 and SMARC1N-PN1 were evaluated for their response to sulfur nutrition by fertilizing with a nutrient solution containing low sulfur (LS) or high sulfur (HS) as described in previous work with common bean (Sánchez et al., 2002) and chickpea (Chiaiese et al., 2004) (Pandurangan et al., 2012).
FIELD TRIAL
The response of SARC1 and SMARC1N-PN1 genotypes to sulfur fertilization was assessed in a field trial conducted at the Cereal Research Centre Morden, MB, Canada, in 2012. Soil was sampled in the fall 2011 and analyzed at Exova, Calgary, AB, Canada, to determine the amount of nutrients needed for the treatment. Nutrient analysis found 24:129:1345 kg ha −1 as nitrogen, phosphorus, potassium, and 47 kg ha −1 as sulfur. Crops were either grown with or without applied sulfur (30 kg ha −1 ) as gypsum (CaSO 4 ·2H 2 O). Recommended seed rate (250,000 seeds ha −1 = 25 seed m −2 ) and cultural practices were used at all plots. Plot size was 1 m × 5.5 m trimmed to 5.0 m 2 with spacing of two rows at 0.5 m between plots. All plots were planted in a randomized complete block design with four replications for each treatment, each replication consisting of two rows of 5.5 m long accounting for 550 seeds per treatment. Two adjacent rows represented one replicate. A post emergent herbicide, Basagran (BASF Canada, Mississauga, ON, Canada) was applied at the rate of 2.2 l ha −1 . Fertilizer added in all the treatment plots was 120 kg N ha −1 . Dry mature seeds from the net area of each plot were harvested separately, weighed, and recorded as seed yield (kg ha −1 ).
AMINO ACID ANALYSIS
Extraction and quantification of sulfur amino acids from mature seed tissue was performed as previously described, using HPLC after derivatization with phenylisothiocyanate (Hernández-Sebastià et al., 2005;Taylor et al., 2008). Cysteine was quantified separately as cysteic acid after oxidation with performic acid.
ALBUMIN AND GLOBULIN EXTRACTION AND QUANTIFICATION
Albumin and globulin fractions were extracted from mature seed as described by Rolletschek et al. (2005). Protein in the extracts was quantified using the Bio-Rad Protein Assay reagent (Mississauga, ON, Canada) with bovine serum albumin as standard. Protein concentration was normalized according to the volume of extract recovered. A volume of sample equivalent to the same weight of tissue extracted was subjected to SDS-PAGE on a 12% polyacrylamide gel. Following staining with Coomassie R-250, band intensities in globulin extracts were measured with Quantity One 4.2.1 (Bio-Rad). Quantity One is very tolerant of an assortment of electrophoretic artifacts, and can measure total and average quantities, determine relative and actual amounts of protein. Prior to quantification the image acquired from scanning the gels was optimized by the software by performing lane background subtraction to reduce any noise or background density while maintaining image quality followed by filtering to remove small noise features while leaving larger features relatively unaffected. The software was used for identifying lanes and defining, quantitating, and matching bands. Lane-based quantitation used to calculate intensity of similar bands across lanes involves calculating the average intensity of pixels across the band width and integrating over the band height. The quantity of a band as measured by the area under its intensity profile curve is expressed as intensity × mm. Apparent molecular mass was calculated based on standards using the same software.
SAMPLE PREPARATION AND MASS SPECTROMETRY
Proteomic experiments were performed at the London Regional Proteomics Centre of the University of Western Ontario. Sample preparation was carried out at the Functional Proteomics Facility. Protein bands of interest identified by band intensities in the globulin extracts were excised by the robotic Ettan Spot Picker (GE Healthcare Life Sciences, Baie d'Urfé, QC, Canada) and suspended in 50% methanol and 5% acetic acid for digestion. Trypsin digestion was performed using the MassPREP automated digester (Waters, Mississauga, ON, Canada). Gel pieces were destained using 50 mM ammonium bicarbonate and 50% acetonitrile followed by protein reduction with 10 mM dithiothreitol, alkylation with 55 mM iodoacetamide and tryptic digestion. Peptides were extracted using a solution of 1% formic acid and 2% acetonitrile and lyophilized. Peptides were dissolved in a solution of 30% acetonitrile and 0.1% trifluoroacetic acid mixed with α-cyano-4hydroxycinnamic acid in 50% acetonitrile, 12.5 mM ammonium citrate, 0.1% trifluoroacetic acid, and analyzed on a 4700 Proteomics Discovery System (Life Technologies, Burlington, ON, Canada) at the MALDI-MS facility. MS analysis was carried out in an m/z range of 500-3500 and mass tolerance of 50 ppm. Data acquisition and processing were done using 4000 Series Explorer and Data Explorer (Life Technologies). The instrument was equipped with a 355 nm Nd:YAG laser and the laser rate was 200 Hz. Reflectron and linear positive ion modes were used. Each mass spectrum was collected as a sum of 1000 shots. Samples from protein bands no. 1, 4, and 5 were further analyzed by LC-MS-MS at the Biological Mass Spectrometry Laboratory. They were reconstituted in 18 μl of 0.1% formic acid in water and 10 μl was injected into the UPLC-MS/MS system. The system was comprised of a Waters nanoAcquity UPLC with a Waters C18 trapping and Waters 25 cm analytical column coupled to a Waters QToF Ultima Global Mass Spectrometer. The sample was run at a flow rate of 0.3 μl/min. Solvent A was water:formic acid 0.1% and solvent B was acetonitrile:formic acid 0.1%. Solvent B was set to go from 5% to 60% in 40 min and then reach 95% by 42.5 min. B was kept at 95% for 5 min and brought back to 5% at 50 min. The column was re-equilibrated for 25 min prior to the following injection. Sample loading took 3 min with a flow rate of 10 μl/min at 99% A and 1% B. MS survey scan was performed at a cone voltage of 35 V and set to 1.4 s with 0.1 s interscan and recorded from 300 to 1800 m/z. In a given survey scan, all doubly and triply charged www.frontiersin.org ions with intensities greater than 40 counts were considered candidate to undergo MS/MS fragmentation. MS/MS acquisition would stop as soon as the total ion current would reach 25000 counts per second or after a maximum time of 6 s. MS/MS scan was acquired from 50 to 1800 m/z for 1.4 s with an interscan time of 0.1 s. Selected ions were fragmented with a collision energy of 30 eV.
Peptide mass fingerprint data were analyzed by searching peptide mass values against a translated version of the preliminary release of the common bean genome (June 26, 2012;Schmutz et al., 2014) using MASCOT (Matrix Science, Boston, MA, USA). The following parameters were used: 1 missed cleavage; fixed carbamidomethyl alkylation of cysteine; variable oxidation of methionine; peptide mass tolerance: ± 1.2 Da; peptide charge state: +1, significant threshold: p < 0.05. For MS-MS, raw data were converted to mgf files using PEAKS 5.3 (Bioinformatics Solutions Inc., Waterloo, ON, Canada). MS/MS ion search was performed with MASCOT against the same database, as well the Mascot database (MSDB, August 31, 2006) using similar cleavage and post-translational modification parameters.
SULFATE ANALYSIS
Replicate samples (∼50 seeds) were ground to a fine powder in a Kleco Ball Mill (Visalia, CA, USA) and lyophilized. Approximately 100 mg of ground tissue was used for sulfate analysis by chemical suppression ion chromatography and conductivity detection using a Dionex DX-600 Ion Chromatograph (Thermo Fisher Scientific, Sunnyvale, CA, USA), as described in Herschbach et al. (2000) with modifications. Approximately 100 mg of tissue was extracted in 0.5 ml of deionized water. The suspension was centrifuged at 16,000 × g for 10 min at 4 • C. A 300 μl aliquot of the cleared supernatant was transferred to an ion chromatography vial for testing using an IonPac anion-exchange column (AS14A, 4 mm; Thermo Fisher Scientific) and eluted with a mixture of 3.5 mM sodium hydrogen carbonate, and 1.0 mM sodium carbonate. A 10 μl aliquot of the solution contained in vials was injected into the eluent stream and background conductivity of eluents reduced by a suppressor (Anion Self-Regenerating Suppressor Ultra, 4 mm). An AS50 auto sampler equipped with a refrigerated chamber was used to house the vials and Dionex Peaknet 6.0 software was employed to track and analyze data.
ELEMENTAL ANALYSIS
Approximately 500 mg of ground seed tissue was submitted to elemental analysis which was performed by dry combustion with a CNS-2000 Elemental Analyzer (LECO Instruments ULC, Mississauga, ON, Canada) as described by Taylor et al. (2008).
STATISTICAL ANALYSIS
Analysis of variance was performed using SAS version 9.2 (Toronto, ON, Canada). Homogeneity of the variances was inspected by residual graphic analysis.
INCREASED YIELD OF SMARC1N-PN1 IN RESPONSE TO HIGH SULFUR UNDER CONTROLLED CONDITIONS
To determine whether differences in response to sulfur nutrition are associated with the presence of an additional, endogenous sink for sulfur in SMARC1N-PN1, an experiment was performed under controlled conditions with two levels of sulfate fertilization. Treatment conditions were designed so that the LS conditions correspond to a sulfur deficiency at the reproductive stage. The LS condition was found to be non-limiting for vegetative growth (Figure 1). The nitrogen levels selected are non-limiting (Sánchez et al., 2002;Chiaiese et al., 2004). The two genotypes were compared for their agronomic parameters. The following variables were evaluated: number of seeds, seed weight, and seed yield ( Table 2). The fact that SARC1 and SMARC1N-PN1 are not completely isogenic explains the occurrence of genotypic differences for some of these characteristics. There were significant interactions between factors for seed weight and yield. Whereas the average seed weight decreased under HS for SARC1, it actually increased for SMARC1N-PN1 (G × T; p ≤ 0.01). This was associated with increased yield, specifically in SMARC1N-PN1, by 8% (G × T; p ≤ 0.05). A trial was performed to determine if the differences observed under controlled conditions would be replicated in the field. The large difference in yield between genotypes indicates that SMARC1N-PN1 is not well adapted to agronomic conditions in Manitoba. Both genotypes exhibited a limited yield response to sulfate fertilization, by 3-15% (Table 3). This response is typical for common bean and other legume crops.
INCREASED SEED CONCENTRATION OF SULFUR AND SULFATE IN RESPONSE TO HIGH SULFATE TREATMENT
To determine if the sulfur treatment effectively altered seed composition and particularly the concentration of sulfur and its metabolites, mature seeds were analyzed for total carbon, nitrogen, and sulfur by elemental analysis and for sulfate concentration by ion analysis. Previously, SARC1 and SMARC1N-PN1 were shown to have similar nitrogen concentration in seed (Hartweck and Osborn, 1997), and Taylor et al. (2008) reported a similar seed concentration of carbon, nitrogen, and sulfur. The sulfur treatment did not change carbon and nitrogen concentration, but had a significant effect on sulfur and sulfate concentration ( Table 4). Sulfur concentration was raised by the HS treatment by approximately 15 to 20% in both genotypes. Sulfate concentration was increased by 17% in SARC1 and 38% in SMARC1N-PN1. The differences in sulfur and sulfate concentrations indicate that treatment conditions are suitable to investigate whether the two genotypes respond differently to sulfur nutrition.
INCREASED CONCENTRATION OF CYSTEINE AND GLOBULINS IN SMARC1N-PN1 UNDER HIGH SULFATE CONDITIONS
To evaluate whether sulfur nutrition has an effect on the total concentration of sulfur amino acids, methionine, cysteine, and the non-protein amino acid, S-methylcysteine were quantified after acid hydrolysis of ground seed tissue. As expected, the concentration of these three amino acids was different between genotypes, methionine, and cysteine being higher, and S-methylcysteine lower in SMARC1N-PN1 than in SARC1 (Table 5), as previously reported (Taylor et al., 2008). HS increased the levels of S-methylcysteine by approximately 40% in both genotypes. Cysteine concentration was raised in response to the HS treatment specifically in SMARC1N-PN1, by 16% (G × T; p ≤ 0.03). On average, the combined levels of methionine and cysteine were elevated by 13% in SMARC1N-PN1, while they were decreased by 2% in SARC1 in response to the HS treatment.
To investigate whether the differences in sulfur nutrient allocation influenced seed storage protein composition, an important determinant of seed quality, albumins, and globulins were sequentially extracted and their concentration quantified ( Table 6). The concentration of extractible albumins was unchanged by the treatment. However, the concentration of extractible globulins increased specifically in SMARC1N-PN1, by 24% (G × T; p ≤ 0.008).
The globulin extracts were analyzed by SDS-PAGE. A volume of sample equal to a similar weight of seed tissue extracted was separated by electrophoresis (Figure 2). The volume of five protein bands appeared to be increased by the sulfur treatment. This was confirmed by image analysis with Quantity One ( Table 7). The fold change and statistical significance of the changes was confirmed by analyzing replicate extracts of each genotype on separate gels (Supplementary Figure S1; Supplementary Tables S1 and S2). The protein bands were excised, digested with trypsin and analyzed by MALDI-MS or LC-MS-MS and identified by MASCOT search against the MASCOT database, or a translated database of the common bean genome (Schmutz et al., 2014). Protein bands no. 2 and 3 could be identified by MALDI-MS, whereas protein bands no. 1, 4, and 5 required more sensitive LC-MS-MS analysis. Tables 8 and 9 provide information about protein identifications and list the number of methionine, cysteine, and total residues in each protein. In all cases, the apparent molecular mass measured by electrophoresis matched the predicted molecular mass relatively closely.
Protein band no. 2 was identified as a lipoxygenase, which was named lipoxygenase-3, based on its similarity to the corresponding soybean and pea proteins. Its baseline levels were higher in SMARC1N-PN1 than in SARC1 by approximately twofold. These results are consistent with the prior identification of this lipoxygenase as elevated in SMARC1N-PN1 as compared with SARC1, both by spectral counting (1.6-fold) and immunoblotting (2.5-fold; Marsolais et al., 2010). This protein had been identified on the basis of the soybean lipoxygenase-3 sequence (Yenofsky et al., 1988). The common bean accession shares 88% identity with soybean lipoxygenase-3, and 84% identity with pea lipoxygenase-3 (Ealing and Casey, 1988). Lipoxygenase-3 levels were increased by the sulfur treatment by approximately twofold. This was observed exclusively in SMARC1N-PN1. The apparent molecular mass determined for lipoxygenase-3 is consistent with the fact that the pea lipoxygenase A1 polypeptide, whose N-terminal sequence matched the deduced amino acid sequence of the lipoxygenase-3 cDNA, had an apparent molecular mass greater than 97.4 kDa (Domoney et al., 1990). Lipoxygenase-3 is particularly rich in sulfur amino acids with 1.7% of its residues as methionine and 0.5% as cysteine.
Protein band no. 3 was identified as albumin-2. Its baseline levels were sixfold higher in SMARC1N-PN1 than in SARC1. These results are consistent with the prior identification of albumin-2 www.frontiersin.org
Error 15
Values are the average ± SD; n = 5.
as being elevated by about 10-fold in SMARC1N-PN1 as compared with SARC1 according to two-dimensional electrophoresis based proteomics (Marsolais et al., 2010). This protein had been identified by de novo sequencing, on the basis of its similarity with pea albumin-2 (Higgins et al., 1987) and mung bean seed albumin [Uniprot:Q43680], and its full-length cDNA had been subsequently cloned (Yin et al., 2011). Its transcript levels were elevated in developing seeds of SMARC1N-PN1 as compared with SARC1 (Liao et al., 2012). Albumin-2 levels were increased by the HS treatment in both genotypes, by approximately 20-30%. The sequence of albumin-2 is relatively rich in sulfur amino acids, with 0.9% of its residues as methionine and 1.3% as cysteine. Protein band no. 1 was identified as arcelin-like protein 4 (Lioi et al., 2003). It was only present in the SARC1 extracts. This is consistent with previous spectral counting and two-dimensional electrophoresis based proteomic data (Marsolais et al., 2010). Arcelin-like protein 4 contains 0.4% its residues as methionine and 0.8% as cysteine. Protein bands no. 4 and 5 were only observed in SMARC1N-PN1. They were identified as α-amylase inhibitor β subunit (Kasahara et al., 1996). The levels of this protein had been shown to be elevated by 20-fold in SMARC1N-PN1 as compared with SARC1 by spectral counting, and this had been validated by two-dimensional electrophoresis based proteomics (Marsolais et al., 2010). The apparent molecular mass measured for protein band no. 4 is in agreement with the results obtained with αamylase inhibitor purified from Great Northern beans (Furuichi et al., 1993). A protein band of 15.5 kDa had been identified as the β subunit by N-terminal sequencing and appeared not to be glycosylated. Protein band no. 5 is likely be a minor form of the α-amylase inhibitor β subunit lacking one or more residues at the C-terminal. Indeed, a peptide containing the sequence of the Nterminus was detected for this band. In the work by Furuichi et al. (1993) a protein band of 13.5 kDa band was tentatively assigned as the α subunit and appeared to be glycosylated. Here, the proteomic data were unambiguous and did not identify a match to the α subunit. Interestingly, the α subunit was conspicuously absent from the SMARC1N-PN1 proteome determined by spectral counting (Marsolais et al., 2010). Purification of the β subunit yielded a complex with the α-amylase inhibitor like protein, with a predicted molecular mass of 25 kDa, in which the α subunit was Frontiers in Plant Science | Plant Physiology Values are the average ± SD; n = 5.
absent (Yin and Marsolais, unpublished results). The apparent molecular mass of this complex measured by size exclusion chromatography was equal to 41.2 kDa. In SDS-PAGE, the purified fraction contained a minor band of approximately 13.8 kDa, similar to the results in Figure 2. The α-amylase inhibitor β chain is devoid of sulfur amino acids. However, the corresponding αamylase-1 precursor contains 1.6% of its residues as methionine without any cysteine (Prescott et al., 2005). The four methionines are located at the N-terminus of the polypeptide precursor. Sulfur amino acid residues are neither present in the α subunit (Kasahara et al., 1996).
DISCUSSION
The most important finding reported from this study is that the protein pool of SMARC1N-PN1 was able to accommodate an increase in sulfur amino acids, particularly cysteine, in response to enhanced sulfate nutrition, whereas this was not the case in SARC1. This property is associated with the presence of an endogenous sink for sulfur in SMARC1N-PN1. The increase in cysteine concentration was associated with a specific increase in the concentration of extractible globulins and seed yield in SMARC1N-PN1. It has been reported that sulfur nutrition influences the response of chickpea plants to the transgenic expression of sunflower seed albumin (Chiaiese et al., 2004). An increase in the seed concentration of reduced sulfur was more pronounced in the transgenic line than in wild-type under conditions of low nitrogen and high sulfur nutrition. Further results were suggestive of a higher methionine concentration in the transgenic line in response to high sulfur and high nitrogen, although this could not be analyzed statistically due to a lack of replication. Recently, Kim et al. (2014) reported that adequate sulfate nutrition is required to maximize the accumulation of maize δ-zein in transgenic soybean lines where expression of the endogenous β-conglycinin was suppressed by RNA interference. The seed concentration of sulfate reported here for common bean is much lower than the concentration of oxidized sulfur in lupin or pea, by approximately 100-fold (Tabe and Droux, 2002;Chiaiese et al., 2004). Whereas in lupin or pea, sulfate represents a reserve of sulfur, whose levels can be significantly reduced upon transgenic expression of a sulfur-rich protein, Smethylcysteine plays a similar role in common bean. This is supported by the fact that the S-methylcysteine concentration was reduced by approximately twofold in SMARC1N-PN1 as compared with SARC1. However, the effect of sulfur nutrition on the concentration of S-methylcysteine was similar between genotypes. Likewise in chickpea, nitrogen and sulfur treatments had a similar effect on oxidized sulfur concentration in wild-type and a transgenic line expressing sunflower seed albumin (Chiaiese et al., 2004).
The increase in the extractible globulin fraction associated with enhanced levels of cysteine stimulated an analysis of sulfurresponsive proteins in the extracts. Two lectins, uniquely present in either genotype, arcelin-like protein 4, and the β subunit of the α-amylase inhibitor, were identified as sulfur-responsive. In the case of the α-amylase inhibitor, sulfur is needed for the accumulation of the polypeptide precursor but not the mature β subunit. Albumin-2 was found to be increased by a similar fold change in response to HS in both genotypes, although its baseline levels were higher in SMARC1N-PN1. In pea, albumin-2 levels were initially found to be reduced in response to severe sulfur deficiency (Randall et al., 1979). In later work, protein levels were found to be relatively unchanged in response to moderate sulfur deficiency (Higgins et al., 1987). Albumin-2 is characterized by the presence of four repeats of the hemopexin domain. The results www.frontiersin.org of crystallization studies have revealed that these proteins bind spermine (Gaur et al., 2010(Gaur et al., , 2011. Binding of heme and spermine was found to be mutually exclusive in grasspea hemopexin. A pea mutant lacking albumin-2 had altered levels of polyamines, and this was associated with increased seed protein concentration (Vigeolas et al., 2008). Whether SARC1 and SMARC1N-PN1 differ in their level of polyamines could be the subject of future investigation. The identification of lipoxygenase-3 as a sulfur-responsive protein is particularly interesting, because this response was observed in only one of the genotypes. The present result strongly suggests that the sulfur-responsive albumin protein of 95 kDa identified in pea is actually lipoxygenase-3 (Higgins et al., 1987). We speculate that differences in sulfur-responsiveness between the two common bean genotypes are probably determined by polymorphisms in the promoter or 3 -untranslated region of the lipoxygenase-3 gene. These polymorphisms must arise from recombination at the lipoxygenase-3 locus between the different parents. If this is true, sequence comparisons between the two genotypes might lead to the identification of a much sought after cis-acting regulatory element determining a positive response to sulfur nutrition in higher plants. To date, the only known cis-acting regulatory motif determining a transcriptional response to sulfur, the SURE motif, is involved in the up-regulation of sulfate transporter and assimilatory genes in response to sulfur deficiency (Maruyama-Nakashita et al., 2005).
The present results have special implications for the agronomic management of common bean, if storage protein deficiency is used as a trait for the improvement of protein quality through conventional breeding. Although SARC1 and SMARC1N-PN1 responded equally to sulfate fertilization under field conditions, the results obtained under controlled conditions suggest that adequate sulfur Frontiers in Plant Science | Plant Physiology nutrition is required to maximize the concentration of sulfur amino acids and therefore protein quality in genotypes lacking phaseolin and major lectins like SMARC1N-PN1. As deposition of sulfur due to atmospheric pollution decreases, sulfate fertilization might become necessary for common bean production in Southwestern Ontario. It is currently an integral part of agronomic production in the plains of Manitoba.
ACKNOWLEDGMENTS
We thank staff at the Southern Crop Protection and Food Research Centre, Igor Lalin for sulfate analysis, Tim McDowell for elemental analysis, Patrick Chapman for bioinformatics, and Alex Molnar for graphics. We are grateful to staff at the London Regional Proteomics Centre, Paula Pittock and Kristina Jurcic. We acknowledge Rey Interior, at the SPARC BioCentre, Hospital for Sick Children, for help with amino acid analyses. We thank Larry Stitt of LW Stitt Statistical Services for help with statistical interpretations. We are indebted to Cynthia Grant at the Brandon Research Centre of Agriculture and Agri-Food Canada, for advice on field sulfur trials, and to Derek Lydiate at the Saskatoon Research Centre of Agriculture and Agri-Food Canada, for discussions that stimulated this work. Steven Karcz of the Saskatoon Research Centre, who passed away in 2012, helped with MASCOT. This work was supported in part by a grant from the Ontario Research Fund for Research Excellence, Round 4 to FM (project 043), and grants from the Manitoba Pulse Growers Association, and Pulse Science Cluster of the AgriInnovation Program of Agriculture and Agri-Food Canada to AH and FM. | v3-fos |
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} | s2 | Response of wheat growth, grain yield and water use to elevated CO 2 under a Free‐Air CO 2 Enrichment (FACE) experiment and modelling in a semi‐arid environment
Abstract The response of wheat crops to elevated CO 2 (eCO 2) was measured and modelled with the Australian Grains Free‐Air CO 2 Enrichment experiment, located at Horsham, Australia. Treatments included CO 2 by water, N and temperature. The location represents a semi‐arid environment with a seasonal VPD of around 0.5 kPa. Over 3 years, the observed mean biomass at anthesis and grain yield ranged from 4200 to 10 200 kg ha−1 and 1600 to 3900 kg ha−1, respectively, over various sowing times and irrigation regimes. The mean observed response to daytime eCO 2 (from 365 to 550 μmol mol−1 CO 2) was relatively consistent for biomass at stem elongation and at anthesis and LAI at anthesis and grain yield with 21%, 23%, 21% and 26%, respectively. Seasonal water use was decreased from 320 to 301 mm (P = 0.10) by eCO 2, increasing water use efficiency for biomass and yield, 36% and 31%, respectively. The performance of six models (APSIM‐Wheat, APSIM‐Nwheat, CAT‐Wheat, CROPSYST, OLEARY‐CONNOR and SALUS) in simulating crop responses to eCO 2 was similar and within or close to the experimental error for accumulated biomass, yield and water use response, despite some variations in early growth and LAI. The primary mechanism of biomass accumulation via radiation use efficiency (RUE) or transpiration efficiency (TE) was not critical to define the overall response to eCO 2. However, under irrigation, the effect of late sowing on response to eCO 2 to biomass accumulation at DC65 was substantial in the observed data (~40%), but the simulated response was smaller, ranging from 17% to 28%. Simulated response from all six models under no water or nitrogen stress showed similar response to eCO 2 under irrigation, but the differences compared to the dryland treatment were small. Further experimental work on the interactive effects of eCO 2, water and temperature is required to resolve these model discrepancies.
Introduction
Crop simulation models are robust tools to extrapolate likely behaviour under circumstances that are difficult or impossible to experimentally test in the field, such as a changing climate. In agriculture, many studies have employed crop modelling with various future climatic scenarios (e.g. Hoogenboom et al., 1995;Boote et al., 1996;Semenov et al., 1996;Howden & O'Leary, 1997;Amthor, 2001;Howden et al., 2003;Asseng et al., 2004Asseng et al., , 2013aPower et al., 2004;Ludwig & Asseng, 2006;Anwar et al., 2007;Lobell & Ortiz-Monasterio, 2007;O'Leary et al., 2011). The range of crop models available for use in such studies is large because of differences in complexity and function. Models that include some realistic simulation of the effects of elevated atmospheric CO 2 (eCO 2 ), temperature and water stress on crop growth and development are considered a prerequisite in terms of model function for future climate change studies. Other subcomponents involving mineral uptake may also be critical for some applications (e.g. nitrogen in soil C sequestration studies).
Comparison against measured field data is fundamental to improving crop models (Boote et al., 1996). Such improvement offers assurance that subsequent simulation scenario studies are the best available. This study tests the performance of six wheat crop models against 3 years of measured crop growth and water use data from the Australian Grains Free-Air CO 2 Enrichment (AGFACE) experiment, located in a semiarid (seasonal VPD ranging from 0.2 to 1.1 kPa with a mean around 0.5 kPa) environment in southern Australia. Previous studies from other environments that have compared simulations with FACE results have reported response to eCO 2 to be close to the observed response in biomass, yield and water use (Tubiello et al., 1999;Jamieson et al., 2000;Asseng et al., 2004;Ko et al., 2010). Seasonal growth responses to eCO 2 for wheat have been in the order of 5-40% (mean~18%) (Ainsworth & Long, 2005;Sun et al., 2009) depending on other growth limiting factors. In general, C 3 cereal crops under water stress responded proportionally more to eCO 2 (~22%) than unstressed crops (~16%), showing the importance of water supply (Kimball, 2011). The lack of observed and consistent high responses (>25%) in field crops is puzzling (Long et al., 2006), but one reason proposed might be artefacts of the experimental environment (Long et al., 2006;Bunce, 2013). Another aspect needing consideration is the relative dryness of the atmosphere during the season that will affect transpiration efficiency. Previous FACE experiments are not considered representative of semiarid regions because their annual potential evapotranspiration/rainfall (PET/R) ratio is either less than or close to unity or very high. The PET/R ratio at the AG-FACE site in southern Australia is around 3 compared to around 10 in Arizona. Similarly, from a seasonal perspective and despite crops being irrigated in Arizona, the seasonal VPD also remains about three times the Horsham AGFACE site (1.4 kPa cf. 0.5 kPa). Because of the intermediate dryness of the Australian AGFACE experiment and the present view that the relative response to eCO 2 is much higher under conditions of water stress (Leakey et al., 2009;Sun et al., 2009;Kimball, 2011;Dias De Oliveira et al., 2012), this study offers a new opportunity to test the performance of a range of crop models under eCO 2 in a new unirrigated environment. In addition, to testing models in terms of the accumulation of crop biomass, grain yield and water use and subsequent water use efficiency (WUE) in response to eCO 2 , we explore whether model components used to calculate these responses need improvement.
FACE experiment
The AGFACE experiment at Horsham, Australia (36°45 0 S, 142°07 0 E, 128 m elev.) (Mollah et al., 2009), was used to measure the growth, yield and water use of wheat under various controlled conditions. The experiment included multiple cultivars, but we restricted our study to one popular local cultivar (cv. Yitpi) that was grown under two water regimes (rain-fed and supplemental irrigation), two nitrogen fertilisation regimes (0 and 53-138 kg N ha À1 ) and two sowing dates (to create two growing season temperature environments) for both daytime ambient (365 lmol mol À1 ) and elevated (550 lmol mol À1 ) CO 2 atmospheric conditions. Injection of CO 2 was controlled by wind speed and direction whereby it was injected approximately 150 mm above the crop canopy upwind when wind speed was between 0.3 and 10 m s À1 between 2 h after sunrise and 2 h before sunset. The primary design criteria of daytime 550 lmol mol À1 CO 2 were achieved 86-94% of time ranging from median 495-605 lmol mol À1 (within AE10%) in the centre of 12-m (2007 and 2008) and 16-m (2009) diameter octagonal rings (Mollah et al., 2009). Within 1-m downwind of the rings, CO 2 levels ranged from 500 to 1700 lmol mol À1 . The agronomic design with the two times of sowing (TOS) over 3 years (2007)(2008)(2009)) comprised a complete randomised block experimental design of four replicates. This equates to 48 crops over the 3 years (2CO 2 9 2TOS 9 2 irrigation 9 2 nitrogen 9 3 years). In 2007, plots were split for TOS, where half of each plot was sown to each TOS. In subsequent years, plots were split for irrigation, in which a 0.8-m-deep buried plastic barrier separated the two rain-fed and supplemental irrigated half-plots. Each replication was one 'bay' with two aCO 2 experimental areas and two eCO 2 rings per bay. Sowing time alters biomass partitioning including yield as crops are forced to develop into warmer, less efficient conditions approaching summer (O'Leary et al., 1985).
Gravimetric soil water content was measured at sowing and harvest using a hydraulically operated soil sampler where sampling was in increments of 0-0.1, 0.1-0.2 and 0.2 m increments thereafter to 2 m from one core per plot (42 mm diameter). Soil mineral nitrogen (NO 3 and NH 4 ) was also measured from an additional core taken close to the sampling time of the soil water measurements. Site bulk density was measured from 70 mm diameter 9 75 mm deep sampling rings from each octagonal area. We defined seasonal crop water use as the change in soil water content (mm) during the growing season (sowing to harvest) plus rainfall and irrigation between sowing and harvest. This therefore includes losses from soil evaporation, crop transpiration, deep drainage and run-off, with the latter two considered insignificant from an agronomic perspective at this location (i.e. within the error of measurement, after O' Leary & Connor, 1997). For example, seasonal drainage is known to be around 20 mm with typical errors of water use for these soils around 50 mm. We defined WUE as grain yield divided by water used (soil evaporation plus transpiration plus deep drainage plus run-off). Large soil mineral nitrogen content (~300 kg N ha À1 ) at the site precluded any significant effects of applied N, so soil analyses were pooled across the N treatments.
There were 12 subplots per ring in 2007 and 2008, each 1.4 by 4 m. There were 24 subplots in 2009, the additional plots containing cultivars not analysed here. Each subplot was 8 plant rows wide. Row spacing was 0.217 m in 2007 and 0.19 m in 2008 and 2009. Within each subplot, there were three destructive harvests; at maximum tillering/initiation of stem elongation (DC31), anthesis (DC65) and maturity (DC90) (Zadoks et al., 1974). The centre rows were sampled, leaving the outer rows on each side as buffers. In 2007, destructive samples for the DC31 and DC65 samplings were randomly distributed within each subplot as 0.5-m and 1.0-m row segments at maturity (DC90). Beginning in 2008, all samples were collected as quadrats of four rows by 0.5 m (DC31 and DC65) and four rows by 1-m areas at DC90. The DC31 and DC65 samples were collected within a reserved area termed 'growth' subplot, and the DC90 sample was collected from a designated area termed 'maturity' subplot. Biomass samples were oven-dried at 70°C, and leaf and stem area measurements were made from using an electronic planimeter from subsamples comprising approximately 25% of the collected fresh biomass. Mean sowing plant density measured by plant counts about 3 weeks after the emergence was 120 plants m À2 and ranged from 60 to 175 plants m À2 . Agronomic management at both sites was according to local practices, including spraying fungicides and herbicides, as needed. Granular phosphorus and sulphur (as 'superphosphate') were incorporated into the soil at sowing at rates between 7-9 kg P ha À1 and 8-11 kg S ha À1 depending on year.
The experimental site had previously been laser-graded to assist in flood irrigation and had been irrigated with sewage water for over 20 years to assist bulking seed in various plant breeding programmes. This history resulted in extreme variability of critical soil properties at the site, and as a consequence for modelling the response to eCO 2 , we pooled the initial soil water content at sowing across the ambient and eCO 2 treatments to be consistent with single soil type parameters for the site (Table S1). This option, as opposed to modelling each ring, provided an effective and simple solution to compare the experimental data with all its site variance, to the modelled data.
Simulation models
Six crop simulation models were tested against the AGFACE data (Table 1). They represent two primary groups of popular models dealing with the effects of eCO 2 and final crop yield by the primary method of daily biomass accumulation: (1) those that primarily use radiation use efficiency (RUE) calculations and (2) those that primarily use transpiration efficiency (TE) calculations. Both approaches are approximations to the real photosynthetic mechanisms, but their utility across diverse environments is well known. Each model was run by the respective modelling groups/authors without adjusting parameters to fit the data apart from the data to define the cultivar phenology. In addition to the selected models, we analysed the median of all the models as an ensemble to explore whether an ensemble result was more accurate than any individual model.
The experimental site is defined by its soil type (Table S1) and daily weather conditions that were measured mostly on site. When on-site measurements were unavailable, locally calibrated calculated values for temperature were made from two nearby meteorological stations approximately 10 km away. Other generic site parameters needed by some of the models are listed in Table S2.
Simulation modelsthe APSIM-Wheat model. The Agricultural Production Systems Simulator (APSIM) is a farming systems simulation framework that has been designed to allow evaluation of farm decision-making in the face of climatic risk, climate change or changes in policy (Keating et al., 2003). The Wheat model within APSIM has been broadly tested across Australia and internationally in a range of experimental (Holzworth et al., 2011;Zhang et al., 2012) and farm (Hochman et al., 2009;Carberry et al., 2013) conditions. Testing of simulated responses to increasing atmospheric CO 2 has been published for predecessors of the current APSIM-Wheat model (Reyenga et al., 1999;Asseng et al., 2004). Increased atmospheric CO 2 concentration impacts upon simulated crop growth and resource use via changes to RUE, TE and the critical nitrogen concentration (CNC) for crop growth (Reyenga et al., 1999). To capture CO 2 effects on RUE, and interactions with temperature, AP-SIM-Wheat (v 7.4) scales RUE using the ratio (/ p ) of the These models have all been used in various climate change applications.
light-limited photosynthetic response at the enhanced CO 2 level to that at 350 lmol mol À1 : where the temperature-dependent CO 2 compensation point (Г) is calculated as: where t is the temperature (°C). The responses of transpiration efficiency (g m À2 mm À1 ) and leaf CNC (%) to increased CO 2 are assumed to be linear with changes of +37% and À7%, respectively, for a doubling of CO 2 concentration to 700 lmol mol À1 (Fig. 1). Actual transpiration is indirectly reduced under eCO 2 through the gain in TE. Simulation modelsthe APSIM-Nwheat model. The APSIM-Nwheat model (v 1.55) was developed from an earlier version of APSIM. This model has been tested and applied in Australia (Asseng et al., 1998a(Asseng et al., ,b, 2003Asseng & Van Herwaarden, 2003) and many other growing environments in the world (Asseng et al., 2000(Asseng et al., , 2002(Asseng et al., , 2004Wessolek & Asseng, 2006;Bassu et al., 2009). It uses the same CO 2 functions by Reyenga et al. (1999) as described above for APSIM v7.4 but has accelerated senescence functions and a physiological approach for water-soluble carbohydrate remobilisation, which is critical for terminal drought situations (Asseng & Van Herwaarden, 2003). Table S4 lists the salient cultivar parameters for APSIM-Nwheat used to simulate the AGFACE experiments.
Simulation modelsthe CAT-Wheat model. .5) (Weeks et al., 2008) was selected as a simple model with respect to biomass accumulation (see below) but has high utility in spatial analyses across landscapes (O'Leary et al., 2011;Christy et al., 2013). It includes modules for phenological development, crop growth and yield, together with dynamics of water and nitrogen in the crop and soil. Photosynthetic area (includes leaf and stem) is determined by above-ground biomass. The model was based on a generic annual crop model to enable the simulation of any crop and is based on extensively used contemporary models (Williams et al., 1989;Littleboy et al., 1992). Biomass accumulation is determined from intercepted radiation, RUE, water and nutrient stress factors and a photoperiod factor (Williams et al., 1989). The minimum value of these factors (i.e. the most limiting) is always used to reduce RUE. The fertilisation effect of eCO 2 is achieved by multiplication of RUE by a factor (RUECC) from (St€ ockle et al., 1992).
where CO 2 is the atmospheric CO 2 concentration (lmol mol À1 ) such that RUECC = 1, 1.18 and 1.23 at 350, 550 and 650 lmol mol À1 , respectively ( Fig. 1). Potential or actual crop transpiration is not reduced by eCO 2 . Grain yield is determined using the harvest index approach that is modified by water and nitrogen stress. Components of the water balance include crop interception, soil evaporation, transpiration, run-off and drainage that provide daily soil water content. Sadras, 2002;D ıaz-Ambrona et al., 2005). We used CROPSYST (v 4.18.27) which calculates dry matter accumulation as a function of daily intercepted solar radiation and daily crop transpiration, using constant coefficients of RUE (Monteith 1981), and variable coefficients of TE (Kremer et al., 2008). The minimum of the two calculations each day of the growing season determines the dry matter production for the day. For unstressed wheat, a ratio (r) of 1.134 for a CO 2 The factors for atmospheric CO 2 of 550 lmol mol À1 vary slightly between the models. Note: the various models apply RUE and TE in different ways, and as TE is related to RUE by growth, both correction factors are applied in all these models such that double accounting is avoided (Table 1). change between 360 and 560 lmol mol À1 was used in this study. The value of this ratio as a function of CO 2 is given by a nonrectangular hyperbola (NRH) fitted to three points: r = 1 at CO 2 = 360 lmol mol À1 , r = 1.134 at CO 2 = 560 lmol mol À1 and r = 0.95 9 NRH maximum ratio at CO 2 = 900 lmol mol À1 (CO 2 approaching saturation for C3 species). The ratio for any given CO 2 of interest is applied directly to RUE. In the case of TE, the effect of CO 2 on canopy conductance for water vapour (and therefore on crop transpiration) is accounted for following Allen (1990). Thus, both TE (increase) and crop transpiration (reduction) are adjusted to obtain the expected value of r. Table S6 lists the salient cultivar parameters for CROP-SYST used to simulate the AGFACE experiments.
Simulation modelsthe SALUS model. The Systems Approach to Land Use Sustainability (SALUS) (Basso et al., 2006(Basso et al., , 2010 is an evolution of the CERES models (Ritchie et al., 1988). When perceived improvements in CERES models became available after 2002, those were included in SALUS. The primary purpose of SALUS is to do long-term simulation cropping systems with emphasis on simulating yields along with environmental consequences of various the management strategies. SALUS has been tested for crop yield (e.g. Basso et al., 2007Basso et al., , 2009Basso et al., , 2010Basso et al., , 2012Asseng et al., 2013a;Rosenzweig et al., 2013;Dzotsi et al., 2013), soil C dynamics (e.g. Senthilkumar et al., 2009), plant N uptake and phenology (e.g. Basso et al., 2010Basso et al., , 2011a, nitrate leaching (e.g. Giola et al., 2012;Syswerda et al., 2012), and tillage effects on soil properties (Basso et al., 2006(Basso et al., , 2011b. Growth is primarily determined by the RUE approach and is then reduced based on the transpiration and nitrogen limitations. The effects of eCO 2 are simulated by adjusting RUE as a function of CO 2 with a corresponding increase in the amount of transpiration use efficiency (Fig. 1). The effect of temperature on RUE and transpiration is made by applying a multiplier to both whereby the factor varies nonlinearly from 1.25 to unity below or above the optimum temperature for photosynthesis. The advantage of this model is its utility in soil management across diverse landscapes. Table S8 lists the salient cultivar parameters for SALUS used to simulate the AGFACE experiments.
Statistical analyses
Comparisons of simulated data against the observed data were made by simple regression of treatment means (arithmetic) of the form y = a + bx where y is the observed and x is the simulated data (n = 48). The regression coefficients (a, b), their standard errors (SE), 95% lower and upper confidence intervals of b, residual degrees of freedom (df), coefficient of determination (R 2 ), significance of regression (P) and root mean square error (RMSE) are reported and listed in Table S9. The response of a number of variables to eCO 2 was analysed by simple regression of treatment means (arithmetic) of the eCO 2 treatments against the ambient treatments, forced through zero. The classical method of determining the response to eCO 2 by dividing the elevated measurement (e.g. grain yield) by the ambient measurement is, by default, a fit forced through the origin with the slope (eCO 2 /aCO 2 ratio) above unity indicating positive response to the level of CO 2 elevation. One of the problems of measuring response to eCO 2 for each treatment is the problem of increasing or decreasing response as the absolute growth or yield approaches zero (Amthor, 2001). The method of fitting a single slope that we employed weights the response against such sensitivity because the mean slope is forced through zero over the wide range of absolute growth or yield. This relative approach provided a robust and simple way to reduce experimental noise and obtain gross estimates of the effects of eCO 2 on observed and simulated yield, water use and WUE rather than by differences through classical ANOVAs. This method then allows both graphical and statistical comparison of the experimental data against the simulated data in an efficient way. For this comparison, the resultant degrees of freedom are halved (n = 24).
FACE experiment
FACE experimentwheat response to elevated CO 2 . The AGFACE field data have shown that early growth, yield and WUE of wheat increased under eCO 2 . At stem elongation (DC31) and anthesis (DC65), biomass under ambient CO 2 was 1200 and 6200 kg ha À1 , respectively, but increased by 21% and 23% under eCO 2 (Table 2). There was also a corresponding increase in crop leaf area index of 21% and photosynthetic area index of 25% due to eCO 2 . The increases in vegetative growth under eCO 2 resulted in a 26% increase in grain yield. For these crops, the WUE of producing biomass increased 36% (21.3-28.9 kg ha À1 mm À1 ) and for grain increased 31% (7.2-9.6 kg ha À1 mm À1 ) under eCO 2 with a corresponding small reduction (6%, P = 0.1) in total accumulated water use from 320 to 301 mm during the growing season.
FACE experimentwheat response to water availability. Early growth of wheat up to stem elongation was not affected by irrigation, but by anthesis, supplemental irrigation increased biomass and LAI by 10% and 37%, respectively, compared with the rain-fed crops (Table 3) showing the more sensitive nature of LAI to water stress. This translated through to a 19% increase in yield and 17% greater water use under irrigation treatments. There was no effect of water regime on wheat WUE for biomass or grain.
FACE experimentwheat response to time of sowing. Early growth up to stem elongation was greater (50%) with late sowing, but this pattern reversed at anthesis where late sowing caused a 42% and 60% reduction in biomass and leaf area compared with early-sown wheat (Table 3). This translated to a 43% reduction in yield where crops were sown late due to crop exposure to increased temperature. Additionally, late sowing reduced water use and WUE of grain production by 10% and 37%, respectively, compared with early-sown crops.
FACE experimentcombined effect of elevated CO 2 , water and time of sowing. For early growth of wheat up to stem elongation, the increase in growth (relative and absolute) due to eCO 2 was greatest (30%) for crops sown late under rain-fed conditions (Table 3). At anthesis, this pattern changed to the greatest growth response due to eCO 2 . Late-sown crops under supplemental irrigation had the greatest absolute increases in growth due to eCO 2 (40%) ( Table 4). For grain yield, both least and greatest response to eCO 2 occurred for rain-fed and irrigated crops, respectively, when crops were late sown, although the greatest absolute increases in yield due to eCO 2 occurred in combination when wheat was sown early under supplemental irrigation. The WUE of grain production was reduced under eCO 2 (À50%) when crops were sown late under rain-fed condition; however, when these crops were irrigated, there was a 34% increase in WUE due to eCO 2 . Increases in WUE due to eCO 2 of early-sown wheat were 41% and 27% for rain-fed and irrigated crops, respectively. There were no effects of applied N on growth due to the high levels of soil mineral nitrogen content at the site (see Materials and methods).
The timing of emergence, anthesis and maturity were not affected by eCO 2 (Table 5) despite the expectation of accelerated development through raised canopy temperatures under eCO 2 (Kimball, 2011). In simulating phenological development, there were, however, larger than expected unexplained errors (6À8 days) in all of the models in simulating time to anthesis and maturity (Table 5). Despite this variation in phenological performance, all the models were able to simulate the response of biomass, yield and water use to eCO 2 . Table 2 Observed gross response to elevated carbon dioxide (b) of wheat (cv. Yitpi) biomass at stem elongation (DC31) and anthesis (DC65), leaf area index at DC65 (LAI65), leaf + stem area index at DC65 (PAI65), yield, water use and water use efficiency for biomass (WUEb) and grain (WUEg) under FACE (free-air carbon dioxide enrichment) conditions The fitted slope (b) of the model y = bx where y is the parameter under eCO 2 and x is the parameter under ambient CO 2 is shown together with its standard error (SE) and 95% lower and upper confidence intervals of b and its coefficient of determination (R 2 ). The total residual degree of freedom was 23.
Simulation performance
Simulation performancebiomass and yield. Despite large variation in the data, the simulated biomass and grain yield showed no serious bias, and all models provided a similar mean error (Table S9). There were, however, some differences between the models in the absolute simulation of early biomass and LAI. Comparison of modelled growth over time at the early growth stage of DC31 is somewhat difficult without observations from multiple sampling dates, because the cause of mismatch may not be clear from a single DC31 measurement. Nevertheless, the overall response to eCO 2 was similar among the models (Table S10). For crop growth up to anthesis, RMSE between observed and simulated biomass ranged from 1400 to Table 3 The observed treatment-mean performance of wheat (cv. Yitpi) growth at stem elongation (DC31) and anthesis (DC65), leaf area index at DC65 (LAI), yield, water use and water use efficiency for biomass (WUEb) and grain (WUEg) under ambient (aCO 2 ) and elevated carbon dioxide (eCO 2 ) 1500 kg ha À1 across the six models (Table S9). All the models tended to simulate biomass until anthesis reasonably well, despite over prediction of LAI. Both AP-SIM-Wheat (R 2 = 0.24 and RMSE = 0.70 m 2 m À2 ) and APSIM-Nwheat (R 2 = 0.39 and RMSE = 1.18 m 2 m À2 ) simulated LAI closer to the observed data than the other models simulating PAI. No differences in model performance in simulating biomass to anthesis occurred between the ambient and eCO 2 treatments. For crop yield (Table S9), comparison of RMSE between observed and predicted ranged from 600 to 1300 kg ha À1 . The SALUS model had the highest R 2 (0.69) and the lowest RMSE (600 kg ha À1 ) with CAT-Wheat and OLEARY-CONNOR models exhibiting typical means errors around 750 kg ha À1 and the APSIM-Wheat and APSIM-Nwheat >1000 kg ha À1 . This variation among the models highlights the extent of accuracy that can be expected using such models without fitting the models to the data and is considered sufficiently accurate to test the relative response of eCO 2 .
Simulation performancewater use and water use efficiency. The average water use across all treatments was 310 mm during the growing season and ranged from 272 to 357 mm (Table 3). All the models simulated water use with a relatively high degree of accuracy and consistency with R 2 ranging from 0.58 to 0.31 and RMSE from 31.3 to 67.6 mm (Table S9). No differences in model performance in simulating water use occurred between the ambient and eCO 2 treatments. There was some variation between the models in simulating WUE for biomass and grain yield. For the accumulation of biomass, the water use efficiency (WUEb) was simulated with varied accuracy by AP-SIM-Wheat, APSIM-Nwheat, CAT-Wheat, CROPSYST and SALUS with R 2 ranging from 0.18 to 0.84 and RMSE from 6.63 to 9.69 kg ha À1 mm À1 ). For grain yield, the water use efficiency (WUEg) R 2 ranged from 0.03 to 0.51 and RMSE from 2.48 to 4.12 kg ha À1 mm À1 (Table S9).
Biomass and leaf area response to eCO 2
The observed increase in growth of wheat, up to stem elongation, due to eCO 2 was 21% (using regression) (Table S10). This early growth response was not captured well by all the models where AP-SIM-Wheat, APSIM-Nwheat, CAT-Wheat, CROPSYST, OLEARY-CONNOR and SALUS produced a predicted response to eCO 2 of 29%, 18%, 25%, 40%, 45% and 9%, respectively ( Fig. 2 and Table S10). The models: APSIM-Wheat, CAT-Wheat and SALUS substantially oversimulated early biomass (stem elongation), whereas the OLEARY-CONNOR model undersimulated the amount of biomass but oversimulated the response to eCO 2 for the same period. Wheat growth up to anthesis increased on average (using regression) by 23% due to eCO 2 ( Fig. 3 and Table S10). In comparison, simulated response was 22%, 21%, 20%, 28%, 25% and 16% for the APSIM-Wheat, APSIM-Nwheat, CAT-Wheat, CROPSYST, OLEARY-CONNOR and SALUS models, respectively. All the six models performed within or close to the 95% confidence limits of observed slope. However, in contrast to the oversimulation of early growth, SALUS was not able to capture the observed range of biomass. The simulated increase in LAI at DC65 from eCO 2 was 19% for both APSIM-Wheat and AP-SIM-Nwheat and 13%, 7%, 24% and 8% for PAI for CAT-Wheat, CROPSYST, OLEARY-CONNOR and SALUS models, respectively ( Fig. 4 and Table S10). For both APSIM models, the range of both observed and simulated data matched, whereas the CAT-Wheat, CROPSYST, OLEARY-CONNOR and SALUS did not match well the observed range of PAI. The observed and simulated biomass and LAI response to eCO 2 at DC65 (50% anthesis) under irrigation was generally increased despite irrigation not fully preventing stress (Table S10). Under irrigation, the effect of late sowing on response to eCO 2 to biomass accumulation at DC65 was substantial in the observed data (~40%), but the simulated response ranged from 17% from SALUS to 28% from CROPSYST (Table 4). Simulated response from all the six models under no water or nitrogen stress showed similar response to eCO 2 under irrigation, but the differences compared to the dryland treatment were small.
Yield response to eCO 2
The observed mean wheat yield response to eCO 2 was 26% (using regression) ( Fig. 5 and Table S10). Similarly, simulations match well the response with simulated The simulated response to elevated CO 2 (• and solid fitted lines) compared to the observed response to elevated CO 2 (○ and dotted fitted lines slope = 1.21). The 1 : 1 unity dashed line is the line of zero response to elevated CO 2 . CROPSYST does not simulate stage DC31, but simulated biomass was outputted on the observed date of DC31 for comparison.
Water use and water use efficiency response to eCO 2 The water use during the growing season of wheat was slightly reduced by eCO 2 , but simulated water use from all the models showed no consistent reduction (Table S10). Some models actively reduce daily transpiration under eCO 2 conditions (e.g. CROPSYST and OLEARY-CONNOR), but the accumulated WU (Table S10) and T (data not shown) were near 1 : 1 with respect to eCO 2 . Of all the parameters, simulated water use gave the most consistent nonresponse to eCO 2 (Table S10). The observed water use efficiency of biomass (WUEb) and wheat yield (WUEg) increased by 36% and 31% (using regression) under eCO 2 , respectively (Tables 2 and 4). In comparison, simulations tended to underestimate the effect of eCO 2 on WUEb and WUEg but were closest for APSIM-Nwheat (22% and 27%) and CROPSYST (29% and 29%), respectively. Given the large variance in WUEb and WUEg data, differences between the models are not considered significant (Tables 4 and S9).
Discussion
The measured response of wheat cv. Yitpi to eCO 2 over the 3 years was strong and consistent for all above-ground biomass at DC31 and DC65, and LAI/ PAI at DC65 and grain yield. The response is considered large but not necessarily indicative of some future climate because the experimental conditions evaluated CO 2 response under ambient temperature and VPD conditions and did not consider likely future elevated temperature or increased VPD. Nevertheless, an interactive effect of temperature was seen in the observed data where the late-sown crops consistently responded proportionally greater to eCO 2 than the normal time of sowing (Table 4). All the six models did not match this observed interaction, and a closer focus on such response is needed in future experiments. The overall observed response to eCO 2 (~550 lmol mol À1 ) of around 21-23% in biomass, LAI and yield was slightly higher than reported meta-analysis means around~18% (Ainsworth & Long, 2005;Sun et al., 2009), but near identical expected for dry environ-ments (cf.~22% in Arizona, USA, Kimball, 2011). The assumption that there is a larger response under waterstressed conditions of the experiment is, however, not supported by the experimental data nor modelling data. Indeed, the measured response to eCO 2 of biomass accumulation by anthesis was higher under irrigation (28% cf. 18%). This response was contrary to that observed in Arizona whereby unstressed conditions had a lower response to eCO 2 (cf. ~16% Kimball, 2011). The reasons are unclear and warrant further investigation. This magnitude of response to eCO 2 was also close to the findings of Yang et al. (2007a) that reported an average increase of 24.8% in wheat grain yield across various N levels over 2 years in Nanjing, China. These two FACE experiments were conducted under near-identical CO 2 concentrations (365 vs. 550 lmol mol À1 for AG-FACE; 350 vs. 550 lmol mol À1 for China FACE). The response in biomass to eCO 2 was reported to be an average of 13.6% across key growth stages (Yang et al., 2007b). The extent of the increase in biomass from China FACE is relatively small compared with our findings. This was partly due to their reported negative impact of eCO 2 on biomass accumulation from heading to maturity (À5.5%) even though positive effects were found in the pre-anthesis growth phases ranging from 11% to 41% (Yang et al., 2007b). It is possible that excess pre-anthesis storage and translocation resulted in greater yield gain.
The modelled gross response to eCO 2 was similar among all models despite their different primary mechanism of computing daily growth. None of the models were specifically fitted to the data and thus show a robustness that should allow confident application to climate change scenarios involving eCO 2 in combination with varying water and N supply and growing season temperatures. Despite the good overall performance of each model with respect to response to eCO 2 against the measured data at Horsham, differences were evident. This was particularly noted by growth stage DC31 where without temperature corrections to the TE-RUE functions OLEARY-CONNOR and CROP-SYST appear to overpredict the response to eCO 2 (45% and 40%, respectively, Fig. 2 and Table S10), but this is a relatively small error occurring so early in the season that this apparent discrepancy did not affect later growth. APSIM-Nwheat followed the early observed response data more closely than all the other models. We concur with Ewert (2004) regarding the need to improve LAI simulation to achieve the correct CO 2 response in all the models except the APSIM-Nwheat model. Nevertheless, despite the poorly modelled early growth by anthesis, all the models matched the seasonal biomass production quite well. Because the grain yield response to eCO 2 was simulated well by all models, the consequences of poor early simulation were not detectable by maturity at this location. This is a particularly critical finding for the applicability of these models to other similar dryland areas where incomplete cover is a dominant feature of the growing season.
Another potential uncertainty is the artefacts of high frequency changes in CO 2 concentration in FACE raised by Bunce (2013). As the modelled response does not contain any physiological mechanisms that might mimic such processes, and the modelled crop response at 550 lmol mol À1 was within the experimental error for all models for biomass at DC65, we consider the pulse CO 2 injection artefact to likely be small and within the experimental error of our experiment. However, whilst the mean response to eCO 2 was consistent across the range of treatments and years, results from individual crops did exhibit large variability. The effect of such variability was reduced by the forcing of the regression through the origin (Table S10). Similarly, the 6-model ensemble generally reduced the modelled variability, but some models matched the observed data better than the ensemble (e.g. SALUS in grain yield). This is not unexpected because the ensemble represents a statistical outcome rather than an explanatory outcome represented by each model. Careful application of such ensemble data is therefore needed.
Despite the modelled response being within or close to the experimental error, an obvious issue of FACE experiments is the spatial and temporal variation in CO 2 concentrations. The destructive samples for the three growth stages were collected at least 1 m away from CO 2 delivery rings, thus avoiding the areas of highest CO 2 concentrations, at times over 1200 lmol mol À1 within 1 m of the ring (Mollah et al., 2012). Median plot CO 2 concentrations varied in 2009 from 524 to 640 lmol mol À1 , depending on prevailing winds, and were as high as 722 lmol mol À1 in one plot in 2007. Whilst the equipment maintained its designed 550 lmol mol À1 in the centre, no crops were grown at the centre and we suggest that a 650-lmol mol À1 level is probably more realistic for majority of the area within the rings for the measured crops, as noted in the standard deviation ring map in Mollah et al. (2012).
The lack of a phenological response to eCO 2 is an interesting result because of the strong growth response; however, the differences of <2 days may not have been noted in the AGFACE experiment. At Nanjing in China, eCO 2 -accelerated phenological development was reported by 1.3 days to heading and another 1.3 days between heading and maturity (Yang et al., 2007a). Such very small changes in phenology are unlikely to gain any large benefit in terms of water savings that might ultimately occur in high-VPD environments. Future FACE experiments should consider measuring canopy temperatures throughout the day and night to better explain water fluxes with canopy temperature and crop development. The performance of all the models in terms of predicting anthesis date with RMSE's over 6 days provides room for improvement; however, notably only one crop in 2008 was responsible for the large error. Interestingly, uncertainties in simulating the impact of increasing global temperature and temperature by eCO 2 interactions have been recently pointed out as the largest shortcomings in wheat models for simulating future climate change impacts, with additional experimental research required for targeted model improvements (Asseng et al., 2013a). Our analyses support that view.
The simulated WUE was quite poor particularly for grain yield despite significant performance in WU and biomass and yield (Table S9). This probably reflects the problem of compounding of errors in both the numerator and denominator in calculating WUE. A way to dampen such effects would be to fit regressions through the prime data with various sources of variance reduced such as removing soil evaporation and examining the TE dynamics. Nevertheless, the modelled response matches the observed first-order processes sufficiently well with acceptable absolute errors (Table S10).
The gross response to eCO 2 was simulated satisfactorily; however, on close examination, there are aspects requiring model improvement. For APSIM-Wheat, CAT-Wheat, OLEARY-CONNOR and SALUS models, the primary focus should be on early growth in terms of biomass and leaf and stem components and water relations under drought and unstressed conditions. Despite simple temperature reduction via RUE in CROPSYST and OLEARY-CONNOR, a temperature interactive term with eCO 2 might be needed. This would allow very high responses to eCO 2 to be reduced early in the season when mean temperatures are typically below 10°C and TE is high because of the low VPD. Similarly, the failure of all models to simulate the eCO 2 response under late sowing might be indicative of a poorly understood eCO 2 response under such conditions. For CAT-Wheat and SALUS, the simple RUE approach with no TE or temperature reduction and a simple harvest index method simulated the gross response reasonably well, but its interaction of eCO 2 and water supply conditions warrants further experimentation because of the relatively high early growth. New field experiments are needed focusing on early growth and water relations. The phenological development should also be scrutinised in future experiments with a focus on canopy heating and cooling as water fluxes change under FACE conditions. The FACE experiment was conducted under ambient temperature and VPD conditions that are unlikely to be directly representative of future climates. It remains to be seen what the effect of high temperatures and VPD when combined with eCO 2 will be on leaf area development, tiller number, above-ground biomass and grain yield. Certainly, the large response to eCO 2 under late sowing shows that the interactive effects will be of continuing interest. It was recently shown that biomass and particularly grain yield of vigorous and nonvigorous wheat genotypes increased when grown under eCO 2 and a mean temperature of 1.0°C above the mean ambient temperature (Dias De Oliveira et al., 2012). However, when grown under eCO 2 and mean temperatures of 2.0 and 3.0°C above the mean ambient temperature, grain yields tended to decrease linearly, mainly because the combination of eCO 2 and high temperature reduced the time between anthesis and physiological maturity (Dias De Oliveira et al., 2012). Consequently, it is expected that the predicted increments in the temperature for the Mediterranean climatic region of Australia (Christensen et al., 2007) will reduce the grain yield response of wheat to eCO 2 unless wheat cultivars with high tolerance to temperature are developed. We suggest field experimentation using open air heating for heating the air around field-grown crops be explored (Kimball & Conley, 2009).
This experiment at Horsham has allowed for the first time the testing of several crop models to eCO 2 under semi-arid unirrigated conditions. At this Mediterranean-type climatic site, the response to eCO 2 was higher under irrigation. However, no elevated temperature treatments were explicitly applied in our FACE experiment, and the models did not fully match the observed interaction of the late-sown crops; therefore, some caution is required when applying the models to some future climate. These findings can therefore be cautiously used as representative of semi-arid environments, and the models used here can equally be applied in eCO 2 impact and adaptation studies for other similar semi-arid regions of the world.
Overall, without any amendment, six different crop models were able to simulate gross response to eCO 2 at Horsham, Australianotwithstanding some secondorder deviations at the early growth stage for APSIM-Wheat, CAT-Wheat, CROPSYST, OLEARY-CONNOR and SALUS models. Whilst APSIM-Nwheat performed well across many variables, it was not the better performer for all of them. General improvements in all the models are expected with future local calibration, but the RUE and TE methods of biomass accumulation represented in the models were sufficient in reproducing the main responses in the FACE experiment and therefore applicable for future climate change analyses in similar dry environments where increased water supply increases the response to eCO 2 at around 550 lmol mol À1 . Department of Agriculture, Fisheries and Forestry. Technical and engineering support was provided by Mahabubur Mollah, Russel Argall, Peter Howie, Janine Fitzpatrick, Justine Ellis, Jennifer Briggs and Jason Ellifson. At the outset of the project, we invited guest collaborators to join the team (O'Leary & Anwar, 2008;O'Leary et al., 2010). Participation of CS and CROPSYST simulations was supported by the project of Regional Approaches to Climate Change for Pacific Northwest Agriculture (REACCH-PNA) which is funded through award #2011-68002-30191 from the US National Institute for Food and Agriculture. We acknowledge helpful discussions during the project from Doug Arbrect, Jeanphilippe Aurambout, Craig Beverly, Hamish Brown, Deli Chen, Kithsiri Bandara Dassanayake, Andrew Fletcher, Peter Grace, Bruce Kimball, Robert Norton, Eduardo Dias de Oliveira, David Stephens, Humaira Sultana, Michael Tausz, Gerard Wall and Jeff White. The experimental data set comprising meteorological observations and treatment crop and soil data from the 48 crops presented here are available upon request from the senior author. Helpful comments were made by an anonymous referee on an earlier manuscript.
Author contribution
The various contributions from the authors were as follows: concept, primary author: GOL; modelling data contributions: GOL, BC, JN, NH, DC, CS, BB, IS and SA; and discussion contributions with respect to other published data: GF, QL, IFC and JP.
Supporting Information
Additional Supporting Information may be found in the online version of this article: Table S1. Soil profile data describing the mean air dry, crop lower limit (LL), drained upper limit (DUL), saturation (SAT), bulk density (BD), pH, organic carbon (OC) and organic nitrogen (ON) content of the experimental site used by all models. APSIM-Wheat used additional water availability coefficients (KL and XF). Table S2. Additional generic site specific and soil type parameters defining the experimental site including some APSIM-Wheat specific parameters (APSIM). Table S3. Key parameters used to define wheat growth in APSIM-Wheat for cultivar Yitpi. Table S4. Key parameters used to define wheat growth in APSIM-Nwheat for cultivar Yitpi. Table S5. Key parameters used to define wheat growth in CAT-Wheat for cultivar Yitpi. Table S6. Key parameters used to define wheat growth in CROPSYST for cultivar Yitpi. Table S7. Key parameters used to define wheat growth in the OLEARY-CONNOR model for cultivar Yitpi. Table S8. Key parameters used to define wheat growth in SALUS for cultivar Yitpi. Table S9. Comparison of simulated and observed data showing regression coefficients (a, b), their standard errors (SE), 95% lower and upper confidence intervals of b, coefficient of determination (R 2 ), significance of regression (P) and root mean square error (RMSE) for the linear model y = a + bx where y is the observed and x is the simulated data of biomass at DC31 (BIO31 kg ha À1 ), biomass at DC65 (BIO65 kg ha À1 ), leaf area index (LAI at DC65 m 2 m À2 ), leaf and stem area index (PAI at DC65 m 2 m À2 ), grain yield (GY kg ha À1 ), water use (WU mm), water use efficiency for biomass (WUEb kg ha À1 mm À1 ) and water use efficiency for grain (WUEg kg ha À1 mm À1 ) for the APSIM-Wheat, APSIM-Nwheat, CAT-Wheat, CROPSYST, OLEARY-CONNOR, SALUS wheat models and the 6-model ENSEMBLE. The residual degrees of freedom was 46. a CROPSYST does not simulate stage DC31 but simulated biomass was output on the observed date of DC31 for comparison. Table S10. Regression coefficients of the mean response to elevated CO 2 on biomass at DC31, biomass at DC65, leaf area index at DC65 (LAI at DC65), leaf + stem area index (PAI at DC65), grain yield, water use and water use efficiency for biomass and grain for the observed experimental data (OBS) and APSIM-Wheat, APSIM-Nwheat, CAT-Wheat, CROPSYST, OLEARY-CONNOR, SALUS models and the 6-model ENSEMBLE. The response under dryland and irrigated conditions and simulated unstressed conditions showing the varied general effect of added water to the response to eCO 2 . The fitted slope (b) of the model y = bx where y is the parameter under eCO 2 (550 lmol mol À1 ) and x is the parameter under ambient CO 2 (365 lmol mol À1 ) is shown together with its standard error (SE) and 95% lower and upper confidence intervals of b for the observed data. The residual degrees of freedom was 23. a CROPSYST does not simulate stage DC31 but simulated biomass was output on the observed date of DC31 for comparison. | v3-fos |
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} | s2 | Socio-Economic and Technical Efficiency Level of Organic Rice Farming with System of Rice Intensification: A Case Study in Morowali Regency Indonesia
E-mail: [email protected] Abstract: This research aimed to (1) analyze technical efficiency level of rice farming with System of Rice Intensification and non-System of Rice Intensification and (2) analyze the factors that affected technical efficiency of rice farming. The research was conducted in Morowali Regency Indonesia. The populations in this research were farmer associations of water users, who membered as many as 104 household heads. Sampling was done by census. The number of samples that were used as many as 104 household heads, 47 household heads of rice farmers with System of Rice Intensification and 57 household heads of farmers with non-System of Rice Intensification. The result showed rice farming with System of Rice Intensification had a higher technical efficiency level when compared with rice farming with non-System of Rice Intensification. This was due to rice farming with System of Rice Intensification had more farmers who used organic fertilizers. The usage of organic fertilizers, age of farmers, education of farmers, frequency of following extension and System of Rice Intensification affected toward the technical efficiency of rice farming. Farmers could increase organic rice production with System of Rice Intensification.
Introduction
Rice is one of the important food crops in Indonesia. The dependence on food crops, especially rice is still very large. Total calories consumed by the community in Indonesia are almost 60% that satisfied by rice (Pirngadi et al., 2002;Makarim and Suhartatik, 2006).
The achievement of rice self-sufficiency in Indonesia in 1984 was the transition of traditional farming technique (organic farming) to non-organic farming. Rice produced is the result of modern agriculture by using fertilizers and chemical pesticides. The techniques create negative impact of excessive chemicals usage. Excessive chemicals will affect the health of farmers as farming actors and farmers will have difficulty in fulfilling the input.
One of alternatives farming systems that can resolve the problem is organic farming (Sumardi et al., 2007). This alternative is very important in facing the issue of food safety. When the community became increasingly aware of the dangers of fertilizers usage and pesticides made by chemical then farming that produced healthy organic food was expected could be a solution.
Farming system that used organic materials is the System of Rice Intensification (SRI). In 1991, SRI began to be tested and applied to Asia region, this method was used to improve the state of soil fertility and rice productivity due to the saturation of the usage of fertilizers and chemical pesticides. This was proved by the results that were enough positive, namely rice that produced were about 8 tons/hectare were higher when compared with the results of national average (Pirngadi, 2009). SRI is method of rice crops cultivation by intensively, efficient and environmental friendly. SRI method is water-saving and do not use an-organic fertilizers (Ferdinan and Harmailis, 2007;Sumardi et al., 2007).
SRI technology is one of rice crops cultivation of water-saving, high yield and cost efficiency. Rice crops cultivation by organically with the usage of SRI on rice fields, will be obtained some advantages such as the usage of more efficient seeds per hectare and productions achieved will increase by 50% (Marlina et al., 2012).
SRI had been tested in more than 18 countries in the world, such as China, Cambodia, Philippine, Myanmar, Bangladesh, India, Nepal, Cuba, Indonesia and the others with results that were achieved between 7-12 tons/ha. This system had been developed in a number of regions in Indonesia and was proved could increase rice production (Pirngadi et al., 2002;Anugrah et al., 2008).
The application of rice crop cultivation by using SRI is basically intended to obtain high production. SRI also includes cultivation system of water-saving, so it is very suitable to be applied in the irrigation area which always has a shortage of water, especially during the dry season. Rice cultivation method on SRI was expected to give a significant contribution toward production increasing and profits.
Besides the method of cultivation, the efficient of production input usage also very determines the production of rice farming (Hormozi et al., 2013;Ojogho and Alufohai, 2010;Wen et al., 2011;Maruyama et al., 2014;Watkins et al., 2014). The effort of rice production increasing through the efficiency of input usage became the right choice. Rice production is affected by agricultural land, labor, capital and management, but in practice, the factors that affect rice production can be divided into two groups, namely biological factors, such as agricultural land and fertilizers and socio-economic factors such as labor. This research aimed to (1) analyze technical efficiency level of rice farming with SRI and non-SRI and (2) analyze the socio-economic factors that affect technical efficiency of rice farming.
Research Methods
The research was conducted in Wita Ponda Sub-Regency Morowali Regency Indonesia. It was done because Wita Ponda Sub-Regency had applied SRI pattern in organic rice farming. The populations in this research were farmer associations of water users in Wita Ponda Sub-Regency. Associations of water users had members as many as 104 Household Heads (HH), but from all of associations of water users members were not entirely applied SRI pattern in their farming. Considered the condition, then the determination of respondent in this research was done by census method. Farmers of associations of water users members amounted 104 HH that consisted of 47 HH who applied SRI and 57 HH who applied non-SRI.
The stochastic frontier production function was used to answer the first objective. This function is based on the model that developed by Coelli et al. (2005): The production function was assumed to have a Cobb-Douglas form who transformed into natural logarithm form: Where : Y = Rice production (kg) BNH = Seed (kg) PUK = Fertilizers (kg) Pest = Pesticides (liter) TK = Labor (HOK) V i = Error of random model U i = Random variable that presented the inefficiency of i sample technical Technical efficiency level of rice farming was estimated by the formula of (Coelli et al., 2005): Where: y i = Actual production * i y = Alleged of frontier production Multiple linear regression model was used to answer the second objective: Where: TE i = Technical efficiency that was estimated; δ 1 -δ 5 = Regression coefficient X 1 = Age of respondent X 2 = Frequency of following agricultural extension; D 1 = Dummy education D = 0 for farmers who did not graduate from elementary school D = 1 for other farmers D 2 = Dummy organic D = 0 for farmers without organic D = 1 for farmers used organic D 3 = dummy SRI D = 0 for non-SRI farmers D = 1 for SRI farmers µ i = error term
Technical Efficiency Level
Technical efficiency of rice farming in this research used the stochastic frontier production function. This analysis was used to measure the technical efficiency level of rice farming with SRI and non-SRI. The form of production function that used was Cobb Douglas Stochastic Frontier. This production function is taken on the grounds: (1) homogeneous, (2) a simpler form, (3) could be made in form of additive linear and (4) rarely caused problems (Debertin, 1986).
The analysis results of technical efficiency level of organic rice farming are seen on Table 1.
Technical efficiency level of rice farming with SRI ranged from 0.4804 to 0.9604 with average value of 0.8432 and rice farming with non-SRI ranged from 0.3819 to 0.9390 with average value of 0.7463.
The Effect of Farmers Socio-Economic toward Technical Efficiency Level
Multiple regression analysis was used to analyze the effect of farmers socio-economic toward technical efficiency level of rice farming. Farmers socio-economic in this research were age of farmers, education of farmers, frequency of following extension, usage of organic fertilizers and SRI cropping. The results of multiple regression analysis of farmers socio-economic effect toward technical efficiency level of rice farming are seen on Table 2.
Adjusted R Square 0.677 shows that variation of technical efficiency level of rice farming could be explained by the independent variables of age of farmers, education of farmers, frequency of following extension, usage of organic fertilizers and SRI cropping simultaneously amounted 67.70%, while its remaining 32.30% were explained by the other factors. Table 1 shows SRI organic rice farming had a higher technical efficiency level when compared with non-SRI organic rice farming. This was due to SRI rice farming had more farmers who used organic fertilizers. SRI farming in the research area was semi-organic agriculture, where farmers used organic materials as one of the inputs that served as soil and supplement of synthetic chemical fertilizers (Las et al., 2006). Pesticides were used selectively and limited. The main cornerstone was a modern agricultural system Good Agricultural Practices (GAP) that prioritized productivity, efficiency of production system, security and preservation of natural resources and environmental. Table 2 shows F statistic = 44.185 significant at α 1%, it meant simultaneously independent variables of age of farmers, education of farmers, frequency of following extension, usage of organic fertilizers and SRI cropping affected toward technical efficiency of rice farming. Partially can be explained as following.
Discussion
Age of farmers affected positive and significant toward technical efficiency level of rice farming at confidence level 99%. Age of farmers could reduce inefficiencies of farmers in rice farming. This was consistent with the research of (Islam et al., 2012;Balde et al., 2014;Ogundari et al., 2010;Piya et al., 2012). Age of farmers correlate with farming experience if farming was done continuously. The older age of farmers then tended the more experiences they had, in this case knowledge and ability in rice farming. Farming experience could encourage farmers to do actions that could advance their farming.
Education of farmers affected positive and significant toward technical efficiency level of rice farming at confidence level 95%. Education of farmers could reduce inefficiencies of farmers in rice farming. This was supported by research of (Ogisi et al., 2012;Ogundari et al., 2010;Donkoh et al., 2013;Piya et al., 2012;Mailena et al., 2014a;2014b). The level of person education could affect the decision making process, which was related to the maturity of thinking that owned to manage farming activities that more effective and efficient as well as easier in receiving information and new technology. The level of education was one of factors that supported the production process of a farming. Education is the main modal in analyzing a job, so that farmers can streamline production factors used. The usage efficiency of production factors can reduce the cost of production so that income of farmers can increase.
Frequency of following extension affected positive and significant toward technical efficiency level of rice farming at confidence level 99%. Following extension could reduce inefficiencies of farmers in rice farming. It was strengthened by research of (Lawal et al., 2014;Islam et al., 2012;Ogisi et al., 2012). Extension is one of innovation diffusion ways, so that farmers who were often followed the extension been expected could adopt the innovation which recommended in order of increasing their farming production.
The usage of organic fertilizers affected positive and significant toward technical efficiency level of rice farming at confidence level 99%. The usage of organic fertilizers could reduce inefficiencies of farmers in rice farming. This is due to the organic materials serve as soil and supplement of synthetic chemical fertilizers (Las et al., 2006). The Sustainability of high efficiency would depend on the continued support which farmers received in the field of inputs providing (Donkoh et al., 2013).
Cropping rice with SRI affected positive and significant toward technical efficiency level of rice farming at confidence level 99%. SRI could reduce inefficiencies of farmers in rice farming. This was due to most of SRI farmers used semi-organic input. Organic SRI can restore the state of soil fertility and rice productivity due to the saturation of fertilizers usage and chemical pesticides (Pirngadi, 2009).
Conclusion
SRI rice farming had a higher technical efficiency level when compared with non-SRI rice farming. This was due to SRI rice farming had more farmers who used organic fertilizers. Partially age of farmers, education of farmers, frequency of following extension, usage of organic fertilizers and SRI affected toward technical efficiency of rice farming. Farmers could increase rice production with organic SRI.
Acknowledgement
The author would like to thank Prof. Dr. Ir. Muhammad Basir, SE., MS and Prof. Dr. Ir. Made Antara, MP who had guided and directed the author. The author also liked to thank the reviewers so this paper could be published.
Funding Information
This article was funded by the University of Tadulako. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Ethics
This article is original and contains unpublished material. | v3-fos |
2019-03-30T13:12:29.394Z | {
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} | s2 | Fungi Associated with the Spoilage of Post-harvest Tomato Fruits Sold in Major Markets in Awka, Nigeria
Tomato contains large amount of water which makes it more susceptible to spoilage by fungi. These fungi that spoil tomatoes produce mycotoxins that are detrimental to human health. This study was therefore carried out to isolate, characterize and identify the fungi associated with the spoilage of tomato fruits sold in Okpuno, Amaudo, EkeAwka, Nodu and Amaikwo Markets in Awka, Nigeria. The average fungal counts ranged between 1.3 x 10 and 2.0 x 10 cfu/ml, while the fungal isolates were Aspergillus niger, Rhizopus stolonifer, Fusarium oxysporum, Saccharomyces cerevisiae, Alternaria alternata, Penicillium digitatum and Geotrichum candidum. The percentage occurrence of the isolates in the fruits from Eke Awka was the highest (32.73%) while that of the fruits from Nodu market was the least (12.73%). Aspergillus niger had the highest percentage occurrence (47.27%) in the fruits studied, with Saccharomyces cerevisiae and Geotrichum candidum having the lowest percentage occurrence (3.64%). Pathogenicity test result showed that Aspergillus niger also had the highest decay diameter of 30mm in the healthy tomato fruits while Geotrichum candidum had the lowest decay diameter. Proper handling and adequate storage facilities must therefore be employed to prolong the shelf life of tomato fruits.
Introduction
Tomato is a widely consumed fruit eaten in both raw and processed forms [1]. It has the botanical name Lycopersicum Esculentum and belongs to the plant family solanaceae. It is rich in vitamins including vitamin A and vitamin C, carbohydrates, proteins, fats, fibres and potassium [2]. It is rich in lycopene which has many beneficial health effects. It contains large amount of water which makes it more susceptible to spoilage by the action of microorganisms [3]. Tomato has a much lower sugar content than other fruits and is therefore not as sweet.
Tomatoes may be pear-shaped, elongated, flattened and heart shaped. They are edible, fleshy and reddish when ripe and vary in their acid composition, with white and yellow ones being less acidic. Tomatoes can be used as savoury or flavouring in soups and cooked foods or can be eaten as fruits. It is used in many dishes, salads, sauces and drinks and can also be dried and ground into pancakes [4].
The consumption of tomatoes throughout the world is believed to benefit the heart and other organs. The richest source of lycopene is tomato and tomato-based products [5]. Lycopene has been found to prevent prostate cancer, improve the skin's ability to protect itself against the harmful ultra violet rays, decrease the risk of breast, lung, stomach, bladder, uterine, head and neck cancers, protect against neurodegenerative diseases, lower urinary tract infections and reduce the cardiovascular risk associated with type 2 diabetes [6][7][8][9][10]. Tomatoes have serious challenges to their existence. These include changes in climate conditions, pests, inadequate rainfall and microorganisms particularly fungi. One of the limiting factors that influence tomato economical value is its relatively short shelf life caused by pathogen attack.
Spoilage of tomatoes are those adverse changes in the quality of tomatoes that are brought about by the action of predominantly biological and physical factors. These may be changes in taste, smell, appearance or texture of the fruits. Ghosh [11] reported that fungi were the source of spoilage of most of the tomato samples accessed than bacteria. Fungi affecting tomatoes include Aspergillus phoenicis, Absidia spp, Trichoderma spp, Alternaria alternata, Fusarium oxysporum, Fusarium moniliformis, Aspergilli us niger, Mucor spp, Rhizopus stolonifer, Penicillium spp, Geotrichum spp and Phytophthora spp [12][13][14][15][16][17][18][19].
Fungal spoilage of tomatoes has been recognized as a source of potential health hazard to humans and animals due to the fact that they produce mycotoxins which are capable of causing mycotoxicoses in man following ingestion or inhalation [18]. The mycotoxins are not limited to their areas of infections. Since tomatoes contain large amount of fluid, these mycotoxins diffuse rapidly throughout them, contaminating all parts and making the fruits unfit for consumption. The importance of tomato in the food industry and its nutritional benefits cannot be overemphasized. Routine microbiological examination of tomatoes is very crucial as it contributes to a large extent to economic development.
Tomatoes are consumed either raw or cooked worldwide. Numerous reports on their spoilage organisms in the developed countries are available but there is dearth of information on the spoilage organisms of tomatoes in a developing city such as Awka, Nigeria. There is therefore a need to evaluate the fungi associated with their spoilage, hence in this work, the fungi associated with the spoilage of post-harvest tomatoes sold in major markets in Awka, Nigeria were isolated, characterized and identified.
Samples Collection
Thirty tomato samples were purchased from Okpuno, Amaudo, Eke Awka, Nodu and Amaikwo markets in Awka, Anambra State, Nigeria. They were transported to the microbiology laboratory of Nnamdi Azikiwe University Awka in sterile polythene bags for fungal isolation. The samples were left for one week for spoilage to occur. The thirty spoilt tomatoes were used for the study.
Materials Sterilization
All the glass wares were properly washed, dried and sterilized in the oven at 160 0 C for one hour. The entire working surfaces were also disinfected with ethanol to reduce contaminants.
Samples Processing
One gram of each of the spoilt tomatoes was carefully cut with the aid of a sterile scalpel and enriched in sterile sabouraud dextrose broth for twenty four hours. Ten fold serial dilutions of the samples were thereafter carried out.
Isolation of Fungi
The pour plate method was used. One milliliter of the serially-diluted sample (10 3 ) was dispensed into a conical flask containing sterile sabouraud dextrose agar (SDA) and two percent chloramphenicol to inhibit bacterial growth. The contents were properly mixed and dispensed aseptically into sterile petri-dishes. Incubation was carried out in an inverted position at 28 0 C for five days. The colonies that developed were counted and subcultured repeatedly on sabouraud dextrose agar plates to obtain pure cultures. They were later stored on SDA slants for characterization and identification.
Characterization and Identification of the Isolates
The pure cultures of the moulds were identified on the basis of their colony growth pattern, conidial morphology and pigmentation using the slide culture technique and microscopic examination. The yeasts were characterized and identified using the Gram stain, Chlamydospores formation, Germ tube, sugar assimilation and motility tests. The identity of each fungus was confirmed with the aid of a mycological atlas.
Slide Culture Technique
A fragment of the aerial mycelia was picked with a sterile inoculating needle and inoculated on a slide containing prepared sabouraud dextrose agar. The slide was thereafter incubated at room temperature for twenty four hours after which it was examined under the microscope.
Microscopic Examination
Lactophenol cotton blue solution was used. A drop of the solution was placed on a clean grease-free slide. A fragment of the mould was emulsified in the solution after which the slide was covered with a cover slip, avoiding bubbles. The slide was thereafter viewed under the microscope.
Gram Stain
A smear of the test yeast cell was made on a slide and stained, using the Gram stain procedure. The slide was thereafter viewed under the microscope for the presence of gram positive yeast cells.
Chlamydospore Formation Test
The procedure employed by Menza et al [20] was used. The isolate was inoculated on corn meal agar containing tween 80 and incubated at 25℃ for seventy two hours. The isolate was thereafter introduced on a slide and stained with lactophenol cotton blue solution. The slide was thereafter viewed under the microscope for chlamydospore formation.
Germ Tube Test
The procedure of Menza et al [20] was employed in the test. A colony of the test yeast was inoculated into human serum and incubated at 37℃ for three hours. A drop of the incubated serum was placed on a microscope slide and covered with a coverslip. The slide was thereafter examined under the microscope for the presence of germ tube.
Sugar Assimilation Test
The test was performed using the procedure of Menza et al [20]. Carbohydrate-impregnanted filter paper discs were placed on carbohydrate-free yeast nitrogen base agar contained in petri-dishes and incubated at 30℃ for eighteen hours Growth around the paper discs was thereafter observed. Growth in the medium indicated the ability of the isolate to assimilate a sugar. The sugars used were glucose, sucrose, lactose, raffinose and galactose. Hanging drop slides were used. Vaseline was spread with a sterile toothpick on the corners of a clean coverslip. A drop of the yeast suspension was aseptically placed on the centre of a coverslip using a sterile inoculating loop. The depression slide was lowered on the coverslip so that the drop protruded into the centre of the concavity of the slide. The hanging drop slide was turned over and placed on the stage of the microscope so that the drop was on the light hole. The drop was thereafter examined under the microscope.
Pathogenicity Test of the Isolates
The procedures of Chukwuka et al [21], Baiyewu et al [22] and Onuorah et al [23] were used. Fifteen healthy tomatoes were properly washed with tap water, rinsed with distilled water and surface-disinfected with ethanol. Sterile cork borers were used to bore holes in each of the tomato fruits. Each of the isolated fungi was thereafter inoculated into the fruits after which the cores of the fruits were replaced. Sterile petroleum jelly was used to seal the holes of the fruits to prevent contamination. Fifteen tomato fruits wounded with the cork borers but were not inoculated with the fungi served as controls.
The inoculated tomato fruits and the controls were placed in sterile polythene bags (one fruit per bag). Each of the fruits was moistened with wet balls of absorbent cotton wool to create a humid condition. The fruits were thereafter incubated at 28℃ for five days and observed for spoilage. The fungi were re-isolated from the fruits and compared with the original isolates. The decay rate of each fungus in the healthy fruits was also determined by measuring its rot diameter after two weeks of its inoculation into the healthy tomato fruit.
Results
The average fungal counts of the spoilt tomato fruits are presented in Table 1. The fruits from Eke Awka market had the highest count of 2.0 x 10 3 cfu/ml while those from Nodu market had the lowest count of 1.3 x 10 3 cfu/ml. The fungal isolates from the spoilt tomato fruits are shown in Table 2. They were Aspergillus niger, Rhizopus stolonifer, Fusarium oxysporum, Saccharomyces cerevisiae, Alternaria alternata, Penicillium digitatum and Geotrichum candidum. The occurrence of the fungi in relation to the markets is shown in Table 3. The fungi occurred most in the samples from Eke Awka market (32.73%) while they had the lowest occurrence in the fruits from Nodu market (12.73%). The percentage occurrence of the fungi from the spoilt tomato fruits is presented in Table 4. Aspergillus niger occurred most frequently (47.27%) while Saccharomyces cerevisiae and Geotrichum candidum had the lowest percentage occurrence (3.64% each). The decay diameter of the fungi in the healthy tomato fruits is presented in Table 5. Aspergillus niger had the highest decay diameter of 30mm while Geotrichum candidum had the least decay diameter of 10mm.
Discussion
The fungi associated with the spoilage of post-harvest tomato fruits sold in major markets in Awka, Nigeria were studied and the result revealed the presence of a teeming population of fungi. The average fungal counts ranged from 1.3 x 10 3 to 2.0 x 10 3 cfu/ml. The fruits from Eke-Awka market had the highest count of 2.0 x 10 3 cfu/ml while those from Nodu market had the lowest count of 1.3 x 10 3 cfu/ml ( Table 1).
The fungal isolates from the fruits were Aspergillus niger, Rhizopus stolonifer, Fusarium oxysporum, Saccharomyces cerevisiae, Alternaria alternata, Penicillium digitatum and Geotrichum candidum ( Table 2). Ibrahim et al [15] isolated Aspergillus niger as one of the major fungi responsible for the production of volatile compounds in spoilt tomatoes. Baker [18] also isolated Aspergillus niger from rotten tomato fruits and reported that they are pathogenic on tomato fruits. Akinmusire [13] reported that Rhizopus spp were associated with the spoilage of tomatoes. Wogu and Ofuase [24] isolated Aspegillius spp, Penicillum spp, Fusarium spp and Saccharomyces spp from spoilt tomato fruits. Mbajiuka and Enya [25] also isolated Aspergillius spp, Penicillum spp and Saccharomyces cerevisiae from spoilt tomatoes while Fatih et al [19] reported the presence of Alternaria alternata and Fusarium oxysporum in the spoilt tomato fruits they studied. Ghosh [11] also isolated Fusarium oxysporum, Aspergillius niger and Rhizopus stolonifer from the spoilt tomato fruits studied.
The percentage occurrence of the fungi in relation to the markets showed that the fungi had the highest percentage occurrence of 32.73% in the samples from Eke Awka market while their percentage occurrence was lowest (12.73%) in the fruits from Nodu market ( Table 3). The detection of more fungi in the fruits from Eke-Awka market could be as a result of poor sanitation, overcrowding, poor storage and unhygienic practices by the fruits handlers.
Aspergillus niger had the highest percentage occurrence of 47.27% in the spoilt tomato fruits examined while Saccharomyces cerevisiae and Geotrichum candidum each had the lowest percentage occurrence of 3.64% in the fruits studied in (Table 4). The result agreed with the work of Akinmusire [13] and Ibrahim et al [15] They reported that Aspergillus niger had the highest rate of occurrence in the tomato fruits they studied and concluded that the fungus may be the major organism responsible for the spoilage of tomato fruits.
The result of the pathogenicity test showed that the fungi inoculated into the healthy tomato fruits had the same features as the ones re-isolated from them, indicating that the fungi were responsible for the spoilage of the tomato fruits. Aspergillus niger produced the highest rot in the tomato fruits, with a rot diameter of 30mm while Geotrichum candidum produced the lowest rot diameter of 10mm in the tomato fruits (Table 5).
Fungal spoilage of tomatoes is attributable to the high water content, environmental conditions, state of handling, state of storage facilities, the fungal load of the handlers and the quality of the tomatoes. These fungi isolated in this study are sources of potent mycotoxins which are detrimental to health. Aspergillus niger is a source of Ochratoxin which is considered to be a potent Carcinogen, therefore spoilt tomatoes must not be consumed but disposed off, since such consumption could be detrimental to health. Farmers and marketers of the produce are also advised to take appropriate precautions during the harvesting, transportation, storage and sale of tomatoes to reduce the risk of these toxins and other metabolites that are deleterious to health.
Conclusions
Tomato fruits have high dietary and nutritional qualities. Their spoilage by fungi results in loss of economic resources as well as food poisoning. The fruits are usually transported from areas of production to areas of consumption in locally woven baskets and sacks under conditions that encourage the growth of fungi. Good quality control measures must therefore be employed by the farmers, marketers and consumers during the harvesting, transportation, handling and processing of the fruits. Frequent inspection of the fruits for sale by food inspectors is also recommended. These will go a long way in preventing the consumption of contaminated tomato fruits thereby reducing the health hazards posed by the mycotoxins produced by these fungi isolated in this study. | v3-fos |
2016-03-14T22:51:50.573Z | {
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} | s2 | Effects of Wheat Naturally Contaminated with Fusarium Mycotoxins on Growth Performance and Selected Health Indices of Red Tilapia (Oreochromis niloticus × O. mossambicus)
An 8-week feeding trial was conducted to examine effects of wheat naturally contaminated with Fusarium mycotoxins (deoxynivalenol, DON 41 mg·kg−1) on growth performance and selected health indices of red tilapia (Oreochromis niloticus × O. mossambicus; initial weight = 4.3 g/fish). Five experimental diets were formulated by replacement of clean wheat with naturally contaminated wheat resulting in graded levels of DON and zearalenone (ZEN) (Diet 1 0.07/0.01, Diet 2 0.31/0.09, Diet 3 0.50/0.21, Diet 4 0.92/0.37 and Diet 5 1.15/0.98 mg·kg−1). Groups of 50 fish were randomly allocated into each of 20 aquaria and fed to near-satiety for eight weeks. Growth rate, feed intake and feed efficiency of fish fed the experimental diets decreased linearly with increasing levels of Fusarium mycotoxins (p < 0.05). Although growth depression was associated with feeding diets naturally contaminated with Fusarium mycotoxins, especially DON, no biochemical and histopathological parameters measured in blood and liver appeared affected by Fusarium mycotoxin concentrations of diets (p > 0.05). Though there was no clear evidence of overt DON toxicity to red tilapia, it is recommended that feed ingredients should be screened for Fusarium mycotoxin contamination to ensure optimal growth performance.
Introduction
Feed represents over 60% of operating costs in aquaculture [1]. Efforts to reduce the cost of feed have led to a reduction in the level of costly fishmeal and increased levels of plant ingredients in the feed of most fish species [2,3]. Feeds for tilapia and other omnivorous fish species are currently formulated to contain high levels of plant feedstuffs. Several surveys have found extensive mycotoxin contamination of both plant feedstuffs [4][5][6][7][8]. As a result, fish have a high risk of exposure to feed that might contain significant levels of mycotoxins and this may potentially lead to significant economic losses [8][9][10].
Mycotoxins are secondary toxic metabolites, produced by filamentous fungi, such as Aspergillus, Penicillium and Fusarium fungi. Though numerous mycotoxins commonly occur in feed and feedstuffs, some toxins, namely aflatoxin B1 (AFB1), zearalenone (ZEN), deoxynivalenol (DON), fumonisin B1 (FB1) and ochratoxin A (OTA), draw the most scientific attention due to their toxic potentially adverse impacts on animal health [4,5,7]. Mycotoxin contamination is largely dependent on temperature and moisture conditions and may occur at various stages of production (e.g., in the field, during harvest, transport or storage) [9,11]. Fusarium mycotoxins, particularly DON, ZEN, and FB1, are ubiquitous in cereal grains worldwide [7,8,12]. Prevalence of these mycotoxins was found in different geographic regions according to a mycotoxin survey program between 2012 and 2013 in Central Europe, North Europe, North Asia, Africa and South Africa [13]. Notably, in a recent annual survey, 50% to 65% of samples were detected to contain DON concentrations ranging from 0.3 to 1.7 mg·kg −1 depending on commodity type and geographical region of origin [14].
DON is trichothecene mycotoxins produced mainly by Fusarium graminearum and F. culmorumthat is frequently found in feeds and feedstuffs [5,7]. It acts as a potent inhibitor of protein synthesis via binding to the 60S ribosomal subunit and interfering with peptidyl transferase activity. The 9,10 double bond and the C-12,13 epoxide are involved in inhibition of protein synthesis during the elongation and termination steps [15,16]. DON causes deleterious effects in animals. Clinical symptoms of acute toxicity (e.g., vomiting and diarrhea) and symptoms of sub-chronic and chronic toxicity e.g., decreased growth rate, feed intake, feed efficiency and histopathological alteration in organs of animals exposed to DON have been reported [17,18]. Influence on serotoninergic activity is one mechanism of action that leads to vomiting and feed refusal after exposure to high and low levels of DON, respectively [19,20]. Immunosuppression resulting in decreased disease resistance is also believed to be associated with DON [21,22].
The effects of DON on animals vary depending on the nutritional and health status of animals prior to exposure, environmental conditions, and forms of DON (e.g., purified and natural forms) as well as dose and duration of exposure [23,24]. Pigs are considered as most sensitive animal species to DON; concentrations as low as 1 to 2 mg·kg −1 caused decreases in growth and feed intake following oral exposure. Pigs exhibit feed refusal and vomiting when fed diets containing 12 and 20 mg DON kg −1 , respectively [25,26]. In contrast, the growth performance of poultry and ruminants were not found to be significantly affected by consumption of diets containing 9.7 and 8.5 mg DON kg −1 , respectively [27,28]. Only few studies have reported adverse impacts of dietary DON on aquatic species, e.g., rainbow trout (Oncorhynchus mykiss) and channel catfish (Ictalurus punctatus) [24,29]. Significant reduction in growth, feed efficiency and feed intake were observed in rainbow trout fed diets containing corn artificially contaminated with graded levels of DON ranging from 1 to 12.9 mg·kg −1 for eight weeks [29]. More recently, Hooft, et al. [24] demonstrated that rainbow trout are extremely sensitive to low dietary levels of DON (<1 mg·kg −1 ). These authors observed significant linear or quadratic decreases in weight gain, feed intake, feed efficiency, retained nitrogen, recovered energy, energy retention efficiency and nitrogen retention efficiency of rainbow trout fed diets containing low levels of DON ranging from 0.3 to 2.6 mg·kg −1 from naturally contaminated corn. In addition, livers of rainbow trout fed dietary DON exhibited fatty infiltration and pyknosis and karyolysis were observed in hepatocytes [24]. Conversely, channel catfish appeared to be more resistant, as diets with high levels of dietary DON (10 mg·kg −1 ) did not affect performance [21,30].
It is apparent that adverse impacts on growth performance and histopathological alteration of organs associated with feeding dietary DON vary considerably among fish species. Additionally, some evidence has shown co-occurrence of several Fusarium metabolites in naturally contaminated feeds and grains [31][32][33][34]. To our knowledge, the effects of feeding diets containing DON on tilapia, an economically important hybrid fish in South East Asia [1], have not been investigated. In the present study, wheat naturally contaminated with Fusarium mycotoxins (DON 41 mg·kg −1 ) was used as a source of DON to examine effects of diets containing graded levels of DON from naturally contaminated wheat on growth performance, selected health parameters and histopathological alteration of red tilapia.
Chemical Composition of Experimental Diets
All experimental diets were formulated to be isonitrogenous and isoenergetic and to meet all nutrient requirements of tilapia as estimated by the NRC [35]. There was no significant difference (p > 0.05) nutritional composition among diets.
Determination of Mycotoxins and Fungal Metabolites in Diets
The replacement of clean wheat with naturally contaminated wheat resulted in five diets, containing graded levels of DON and ZEN. The concentrations of DON and ZEN as analyzed by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in Diets 1-5 were 0.07/0.01, 0.31/0.09, 0.50/0.21, 0.92/0.37 and 1.15/0.98 mg·kg −1 , respectively. A total of 50 secondary fungal metabolites were determined by LC-MS/MS. Surprisingly, some Fusarium metabolites, including aurofusarin (0.01 to 2.46 mg·kg −1 ), culmorin (0.20 to 1.39 mg·kg −1 ) and 15-hydroxyculmorin (0.06 to 1.83 mg·kg −1 ), were detected in all diets at higher levels than DON in this study.
The co-occurrence of fungal metabolites in feed and feed ingredients naturally contaminated with mycotoxins has been shown in several reports. The findings in the present study are in agreement with previous reports [4,5,8,[31][32][33] indicating that use of grains naturally contaminated with Fusarium mycotoxins results in variations in the concentrations of Fusarium mycotoxins (e.g., DON and ZEN), as well as in inescapable fungal metabolites, namely Fusarium metabolites and Alternaria metabolites (e.g., alternariol, AOH; alternariolmonomethyl ether, AME).
Growth Performance
Growth performance (e.g., weight gain, thermal-unit growth coefficient, feed intake and feed efficiency) decreased linearly with increasing levels of Fusarium metabolites ingested (Table 1). Some evidence suggests that intake of a combination of mycotoxins could result in a synergistically/additively adverse impact on animals than that of a single mycotoxin [36][37][38]. The depression of growth in red tilapia in the present study may be related to presence of any one of these Fusarium metabolites, or a possible interaction among them. This remains to be investigated. However, the current data on toxic effects of these fungal metabolites on animals is insufficient to decisively clarify whether they could impair growth of red tilapia in the present study. Sauer, et al. [39] demonstrated that a diet containing high levels of AME 24 (mg·kg −1 ) and AOH (39 mg·kg −1 ) for 21 days did not reduce weight gain or alter tissues on gross and microscopic examinations. In addition, Alexander, et al. [40] suggested that AOH and AME are less toxic to animals than any other in the Alternaria group. Rotter, et al. [41] reported that diets containing 2 mg culmorin kg −1 alone or in combination with 6 mg DON for 21 days did not reduce weight gain and feed intake of pigs (average weight = 22.8 kg/each). Based on these findings, trace amounts of AOH (0.12 mg·kg −1 ), AME (0.15 mg·kg −1 ) and culmorin (1.39 mg·kg −1 ) in the present study likely did not cause any deleterious effects on the growth of red tilapia. Furthermore, a small amount of ZEN (up to 0.4 mg·kg −1 ) was detected in the experimental diets in the study of Hooft, et al. [24] and 4 mg·kg −1 was detected in corn used as a source of dietary DON in the study of Woodward, et al. [29]. Although there was evidence suggesting growth promoting action in ruminants and assessment of oestrogenic potency in vitro [42,43], possible additive or synergistic effects on fish growth resulting from a combination of DON and ZEN require further investigation [5,44,45].
In red tilapia, weight gain, growth rate (expressed as thermal-unit growth coefficient, TGC), feed intake and feed efficiency decreased linearly (p < 0.01) with increasing levels of DON ranging from 0.07 to 1.15 mg·kg −1 . Additionally, diets containing increasing levels of DON were associated with linear and quadratic decreases in mortality (p < 0.05). Growth performance of red tilapia in the present study was in agreement with previous studies on rainbow trout in which growth rate and feed intake were linearly depressed by increasing levels of dietary DON [24,29]. Table 1. Effects of diets naturally contaminated with Fusarium mycotoxins on growth performance and mortality rate of red tilapia (initial average weight = 4.3 g/fish) over the 8-week trial. Mortality is based on number of deaths after the 8-week trial relative to initial number. In rainbow trout, feeding dietary DON as low as 1 mg·kg −1 caused a reduction in feed intake, feed efficiency and growth rate [24,29]. Woodward, et al. [29] reported that significant reductions in feed intake, feed efficiency and weight gain were associated with increasing levels of DON ranging from 1 to 12.9 mg·kg −1 diet. Hooft, et al. [24] found that feeding naturally contaminated diets containing DON ranging from 0.3 to 2.6 mg·kg −1 diet resulted in significant linear or quadratic reductions in feed intake feed efficiency and growth rate (TGC) of rainbow trout. Observed differences in sensitivity of rainbow trout to DON in these two studies could be accounted for, in part, by the use of different sources of DON. Woodward, et al. [29] used artificially infected corn while Hooft, et al. [24] used naturally contaminated corn. Additionally, the initial weight of fish used in the study of Hooft, et al. [24] was half that in the study by Woodward et al. [29] (23 and 50 g/fish). In contrast, feed intake and weight gain in channel catfish were not affected by graded levels of DON in either naturally contaminated wheat and corn, or purified DON ranging from 0 to 10 mg DON kg −1 diet [21,30]. However, catfish fed diet containing >15.0 mg DON kg −1 diet from naturally contaminated sources of wheat exhibited reduced growth and poor feed conversion ratio [30]. Clearly, channel catfish are more tolerant to DON than rainbow trout and red tilapia demonstrating differences among species.
Fishes vary in their sensitivity to DON which may be associated with differences inthe detoxification capacity of microorganisms in the digestive tract and liver to transform DON to the less toxic metabolites (e.g., de-epoxy deoxynivalenol, DOM-1; DON-3-glucuronide) [46][47][48]. DON is transformed to de-epoxy deoxynivalenol (DOM-1) via deepoxidation and deacetylation by microorganisms from the digestive tract [46,47]. In common carp, Cyprinus carpio, hepatic microsomes in the liver have been shown to transform DON to DON-3-glucuronide [46]. Microbes from the digestive tract of brown bullhead (Ameiurus nebulosus) were more capable of transforming DON to DOM-1 than brown trout (Salmo trutta), pink salmon (Oncorhynchus gorbuscha) and other fishes [48]. The significant reduction in feed intake by red tilapia in the present study is in agreement with previous findings in rainbow trout and pigs. Reduced intake of feed contaminated with Fusarium mycotoxins (e.g., DON, T-2 toxin, fusaric acid) might be a response to gastrointestinal tract irritation by DON and co-occurring mycotoxins [44,45] inhibition of gastric emptying [49] and enhanced production serotonin (5-hydroxytryptamine, 5-HT), a brain neurotransmitter [20], linked to behavioral changes such as loss of appetite, vomiting, and emesis [49]. Nevertheless, Hooft, et al. [24] showed that rainbow trout pair-fed a diet containing 0.3 mg DON kg −1 had higher growth rates, feed efficiency, and carcass crude protein content than those fed with a higher concentration of DON (2.6 mg·kg −1 ). This suggests dietary DON does not simply result in a reduction in feed intake of rainbow trout but also negatively affects nutrient metabolism [24]. Accordingly, Lun, et al. [50] reported a depression of growth of pig associated with dietary DON may be related to the disturbance of minerals absorption and/or metabolism.
Significant linear and quadratic decreases (p < 0.05) in the mortality of red tilapia were associated with feeding diets containing increasing levels of DON in the present study. Manning, et al. [21] did not observe mortality of channel catfish fed diets containing high levels of DON (10 mg·kg −1 ) from naturally contaminated corn. Likewise, no significant difference in mortality of rainbow trout fed a diet containing 6.4 mg·kg −1 purified DON or fish pair-fed the control diet was observed by Ryerse, et al. [51]. Interestingly, Manning, et al. [21] demonstrated that feeding diets containing 5 and 10 mg DON kg −1 significantly decreased mortality of channel catfish during a trial of 21-day post-challenge with the pathogenic bacterium (Edwardsiella ictaluri). This finding was supported by Ryerse, et al. [52], who observed a significant reductions in the cumulative mortality of rainbow trout fed diets containing 4.1 or 5.9 mg·kg −1 DON from naturally contaminated corn compared to control and pair-fed groups following infection with Flavobacterium psychrophilum. Likewise, feeding a diet containing 6.4 mg·kg −1 purified DON significantly reduced mortality of rainbow trout after experimental F. psychrophilum infection compared to the control group (<0.1 mg·kg −1 DON) [51]. The authors suggest that reduced feed intake associated with DON may positively influence survival during disease outbreaks [51]. The exact cause of the mortalities observed in the present study is unclear.
Hematological and Biochemical Parameters
Hematocrit (Hct), plasma alanine aminotransferase (ALT), plasma aspartate aminotransferase (AST) and hepatosomatic index (HSI) of fish were not significantly affected by the experimental diets ( Table 2). Hepatosomatic index (HSI) in tilapia fed the control and DON-treated diets were similar which suggests that dietary DON did not cause extensive changes to their liver.
However, there were trends of the decreases in both Hct and HSI with increased ingestion of dietary DON. In accord, Hct in channel catfish and rainbow trout did not exhibit a significant change when feeding 17.5 and 1.96 mg DON kg −1 diet, respectively [30,53]. Further, Matejova, et al. [53] reported that AST and ALT in rainbow trout did not differ significantly from those for a control group when fed diets artificially contaminated with 1.96 mg DON kg −1 .
Histopathological alterations in livers
Although histopathological change is not a toxicity-specific response, hepatocytes are known to respond to a variety of toxicants [54]. Most red tilapia fed the control and other diets had healthy livers (n = 12), only few livers appeared to have changes upon gross and microscopic examinations (Figure 1 (a)). Areas of focal necrosis were observed in red tilapia fed diet 3 (Figure 1 (b)) and 5. Cytoplasmic vacuolation was observed in each liver from fish fed diet 2 (Figure 1 (c)) and 3. Seven samples of fish fed diets 2 ( Figure 1 (d)), 3 and 4 showed subcapsular edema in both of the gross and microscopic examinations.
Histopathological alterationsin red tilapia were not associated with DON dose, based on the scoring system by Bernet et al. [55], although lesions were observed in hepatic tissue in some fish. Histopathological alterations in livers and kidneys of rainbow trout have been reported and related to dietary DON [24,53]. Subcapsular edema were observed in livers of rainbow trout fed 1.4 mg DON·kg −1 along with fatty infiltration and phonotypical alteration of rainbow trout fed 2.6 mg DON kg −1 [24]. Kidneys of rainbow trout displayed severe hyaline droplet denegation in tubular epithelial cells when fed 1.96 mg DON·kg −1 [53]. Focal necrosis and subcapsular edema observed in hepatic tissues of red tilapia were similar to the findings of Hooft et al. [24], who suggested edema may be associated with the inhibition of protein synthesis by DON resulting in a lack of plasma protein. Furthermore, cytoplasmic vacuolation was noticed in some livers of red tilapia fed dietary DON, perhaps a result of lipid accumulation or lack of glycogen in hepatocytes. Liver is considered as a good indicator of nutritional and toxic pathology because of its function in metabolizing substances from the digestive tract [56].
Experimental Condition
This experiment was conducted at Applied Centre of Aquaculture Nutrition (ACAN), Biomin (Thailand) Co., Ltd. (Bangkok, Thailand). Twenty of 120-L aquaria in a recirculation indoor system with a flow rate of approximately 1.5 cm 3 per second flow rate per tank were used. Each tank was continuously aerated to assure ambient dissolved concentrations near air saturation. The average values of each water quality parameter over eight weeks were: dissolved oxygen = 4.44 ± 0.59 mg·L −1 , temperature = 30.50 ± 0.6 °C, pH = 6.95 ± 0.16, ammonia = 0.42 ± 0.12 mg·L −1 , nitrate = 20 mg·L −1 , and nitrite = 0.40 ± 0.13 mg·L −1 . Feces and sediment were removed every morning before feeding. Water volume (50%) in the recirculation system was replaced with dechlorinated tap water weekly.
Experimental Animal
Two thousand juvenile red tilapia (O. niloticus × O. mossambicus; initial average weight = 4.3 g/fish) obtained from a commercial farm (Bangkok, Thailand) were held in aquaria for three weeks. Prior to the beginning of an experiment, 50 fish of similar body weight were weighed at the onset of an experiment and randomly distributed into each of 20 aquaria. Fish were fed to near-satiety with the experimental diets three times daily for eight weeks (900, 1300 and 1600 h on the workday, and once per day at 900 h on the weekend). Mortality was recorded daily and feed intake was recorded weekly.
Experimental Diets
Naturally contaminated wheat (41 mg DON kg −1 ) and clean wheat flour (0.23 mg DON kg −1 ) were used in the dietary preparation and referred to as "contaminated wheat" and "clean wheat", respectively. Five isonitrogenous and isoenergetic diets containing graded levels of DON were formulated by replacement of clean wheat with contaminated wheat (Table 3). Spaghetti-like pellets, 2 mm diameter, were made using a meat grinder and were broken by hand to the appropriate size for fish. The pellets were dried in an oven at 70 °C for two hours, left to cool overnight and kept in plastic bags until used. Five hundred grams of each experimental diet were sampled and ground for proximate analysis according to AOAC [57] and mycotoxin analysis by LC-MS/MS. Table 3. Ingredient composition of the five isonitrogenous and isoenergetic diets and levels of detected Fusarium mycotoxins.
Detection and quantification were performed with a liquid chromatography/tandem mass spectrometry (LC-MS/MS) system (QTrap5500, Applied Biosystems, Foster City, CA, USA) equipped with an electrospray ionisation (ESI) source (TurboIonSpray) and an ultra-high performance liquid chromatography system (1290 Series, Agilent Technologies, Waldbronn, Germany). Chromatographic separation was performed at 25 °C on a Gemini C18-column, 150 × 4.6 mm inner diameter, (5 μm particle size), equipped with a C18 security guard cartridge, 4 × 3 mm i.d. (all from Phenomenex, Torrance, CA, USA). Elution was carried out in binary gradient mode. Both mobile phases contained 5 mM ammonium acetate and were composed of methanol/water/acetic acid 10:89:1 (v/v/v; eluent A) and 97:2:1 (v/v/v; eluent B), respectively. After an initial time of 2 min at 100% A, the proportion of B was increased linearly to 50% within 3 min. Further linear increase of B to 100% within 9 min was followed by a hold-time of 4 min at 100% B and 2.5 min column re-equilibration at 100% A. The flow rate was 1000 μL/min. Data acquisition was performed in the scheduled multiple reaction monitoring (sMRM) mode both in positive and negative polarities in two separate chromatographic runs. The sMRM detection window of each analyte was set to the respective retention time ±27 s and ±42 s in positive and negative modes, respectively. The target scan time was set to 1 s. Confirmation of positive analyte identification was obtained by the acquisition of two sMRMs per analyte (with the exception of moniliformin and 3-nitropropionic acid that each exhibited only one fragment ion), which yields 4.0 identification points according to commission decision 2002/657/EC (EU 2002). Analyst ® software version 1.5.1 (AB Sciex, Foster City, CA, USA) was used to control the LC-MS/MS instrument, as well as for automatic and manual integration of the peak. Quantification of the >300 metabolites included in the method was done based on linear, 1/x weighed calibration curves derived from the analysis of serial dilutions of a multi-analyte stock solution. Results were not corrected for apparent recoveries due to lack of a suitable blank sample. The accuracy of the method was verified on a routine basis by participation in a proficiency testing scheme organized by BIPEA (Gennevilliers, France), which included samples of animal feed. The following analytes were positively identified in one or more of the samples: DON, DON-3-Glucoside, ZEN, zearalenon-4-Sulfat, alpha zearalenol, beta zearalenol, sterigmatocystin, averufin, 3-nitropropionic acid, moniliformin, siccanol, equisetin, apicidin, enniatin B, enniatin B1, enniatin A1, enniatin A, beauvericin, monocerin, aurofusarin, rubrofusarin, culmorin, 15-hydroxyculmorin, tenuazonic acid, alternariol, alternariolmethylether, macrosporin, tentoxin, secalonic acid D, chanoclavin, curvularin, tryptophol, brevinamid F, emodin, chrysophanol, ergometrin, ergometrin, ergosinin, ergosin, ergocornin, ergocorninin, ergotamines, ergocryptin, ergocryptinin, ergocristin, ergocristinin, linamarin, and lotaustralin, while all other investigated metabolites were below their respective limits of detection.
Sample Collection
All fish from each aquarium were weighed for initial and final total body weight as well as at the end of 4th and 8th-week. Feed intake was recorded weekly. At the end of the experiment, three fish from each aquarium were anaesthetized with eugenol (Sigma-Aldrich Ptd., Ltd., Singapore, Singapore) prior to blood collection. Blood samples were analyzed for Hct, ALT and AST. Livers were dissected for HSI, and histological examination were calculated and conducted, respectively.
Laboratory Analysis
AST and ALT in plasma were examined using Architect c 16000Analyzer (Abbott Laboratories, Abbott Park, IL, USA). Hct was estimated using a micro HT centrifuge (DSC-100MH-2 Digisystem Laboratory Instrument Inc., Taipei, Taiwan). Crude protein, ether extract (lipid), ash, moisture, dry matter and gross energy of diets were analyzed. Samples of experimental diets were analyzed for crude protein (CP), ether extract (EE, lipid), ash, moisture, dry matter (DM) and gross energy (GE) according to [57]. Tecator Kjeltec digestion and distillation units were used for CP analysis and the percentage of total nitrogen was determined based on a dry matter basis (%N × 6.25). Lipid was extracted from samples using petroleum ether by Soxhlet apparatus and GE was analyzed using automated bomb calorimeter (AC500 Isoperibol bomb calorimeter, LECO Corporation, St. Joseph, MI, USA).
Histopathological Examination
Sample tissues were processed according to Clark [59]. In brief, tissues were processed using a Shandon citadel 2000 tissue processor (International medical equipment Inc., San Marcos, CA, USA). Then they were dehydrated, cleared, infiltrated with liquid paraffin, and finally embedded in paraffin blocks.
Tissues were sectioned at 4 µm thick using a Leitz 1512 rotary microtome and stained with Hematoxylin and Eosin (H & E stain). Mounted slides were observed under a light microscope. Five liver specimens of fish from the lowest and highest DON level treatments were inspected. Images were captured by a digital camera (Micropublisher 5.0, Qimage, BC, Canada) coupled with a light microscope (Openlab) at the UG/OMNR Fish Nutrition Research Laboratory, Department of Animal and Poultry Science, University of Guelph (Guelph, ON, Canada).
All parameters, such as body weight gain, TGC, feed intake, feed efficiency, mortality rate, HSI, Hct, AST and ALT are presented as treatment mean and standard error of the mean (SEM). The GLM procedure of SAS for Windows (SAS version 9.2, SAS Institute Inc., Cary, NC, USA) was used to perform linear and quadratic orthogonal polynomial contrasts. Statistical significance was declared at p < 0.05.
Conclusions
Impairment of growth performance of red tilapia found in the present study is associated with diets contaminated with Fusarium mycotoxins, particularly DON. The adverse impacts observed in the present study may be a possible result of DON contamination alone or due to synergistic effects resulting from co-occurrence of DON and other Fusarium metabolites, which are commonly found in naturally contaminated feed. The effects of these Fusarium metabolites on the growth of animals are still largely unknown. Hence, the effects of consumption of multiple mycotoxins on animals and fish require further investigation. Inclusion of plant ingredients naturally contaminated with Fusarium mycotoxins in fish feed not only increases the risk of exposing farmed fish to DON but may also result in contamination of fish feeds with unidentified Fusarium metabolites. Consequently, it is necessary to screen for Fusarium mycotoxins and other fungal metabolites in feed and feed ingredients before use in order to ensure good growth performance. | v3-fos |
2019-04-24T13:13:07.142Z | {
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} | s2 | Macrofungi in Some Forests of Telangana State, India
The fruiting bodies of macrofungi were collected from some forests, fences, waste fields, timber depots of Telangana state during rainy season. This is an attempt to give a broad picture of diversity of macrofungi belonging to the class Basidiomycetes in some forest areas of Telangana region. A total number of 50 fruiting bodies were collected and cultured and among them only ten were identified based on their macroscopic features and molecular identification since they showed good lignolytic activity.
Introduction
The total forest cover in India according to the latest Forest Report 2013 is 69.8 million hectares and this constitutes 20.64% of the geographic area. The state of Telangana is one of the progressive states in the southern part of India with an area of 114,840 sq.km, while the recorded forest area is 29242.08 sq.km. The annual rainfall is between 900 to 1500 mm in northern Telangana and 700 to 900 mm in southern Telangana, from the southwest monsoons and it is a semiarid area and has a predominantly hot and dry climate. The Basidiomycota is the second largest phylum of kingdom Fungi, with approximately 23,000 species [1] including many of the common macroscopic forest fungi (e.g., mushrooms, shelf fungi). Mushroom is a general term utilised mostly for the fruiting body of the macrofungi, belongs to Ascomycota and Basidiomycota, and represents only a short reproductive stage in their life cycle. Investigations on the taxonomy and diversity of macrofungi are gaining importance, as many macrofungi are facing a threat of extinction due to environment destruction. Wild mushrooms have deep biological and economical impact. Information on mushroom diversity is important because of its major functions in natural and organised eco-schemes as ectomycorrhizal fungi. The fungal group becomes a significant component for reforestation programs. Furthermore, they are significant as nourishment source for human beings and animals [2]. The macrofungi were also used as a bioindicator of environmental quality [3]. Most of the fleshy and gilled macrofungi were prevalent in the rainy times of the year as this time is favourable for their output, since there is ample moisture, favourable warmth, relative humidity, and sunshine, which furthermore aids the macrofungi in the decomposition of dead organic tissue. The early dry time of the year collection was predominated by the polypores since there is decline in rainfall and relative humidity, boost in warmth, and sunshine and most of the fleshy macrofungi will not withstand these conditions. During rainy season, there is abundant growth of several kinds of Basidiomycetes. Many fungal species groups do not produce visible fruit bodies or other species-specific characteristics, or these characteristics are extremely rare and cannot be detected in traditional surveys; hence these can be studied using molecular methods [4][5][6]. Further, Basidiomycetes are considered to be a very interesting group of fungi with their exceptional adjustment abilities and as natural lignocellulose degraders with different ecological groups such as white rot, brown rot, and leaf litter fungi [7]. Many kinds of macrofungi are not edible but possess variety of pharmaceutical and medicinal qualities. A fraction of complete fungal wealth has been subjected to scientific scrutiny and mycologists continue to unravel the unexplored and hidden wealth; one-third of fungal diversity of the globe exists in India and of this only 50% is characterized until now [8]. About 10,000 species within the overall fungal estimates of 1.5 million belong to 2 Journal of Mycology this group. Mushrooms alone are represented by about 41,000 species, of which approximately 850 species are recorded from India [9]. Some of the wild edible mushrooms have been reported from South West India [10]. The first listed Indian Fungi was published [11] and that was revised by Vasudev [12] and extra additional lists appeared in between culminating with the fungi of India (Bilgrami et al. [13]).
The present communication is the collection, culture, identification, and preservation of the macrofungi in some forests of Telangana region. This study adds extra information to the present knowledge on the data of diversity of fungi in Telangana region and also to understand their bioprospects.
Study Area.
The fruiting bodies of white rot fungi were collected in rainy season from the forest of Etunagaram, Bhadrachalam, Kothagudem, Narsampet, Ilandhu, Khammam, Bodhan, and Nirmal of Telangana region.
Culturing and Preservation.
The collected fruit bodies were cultured for their mycelial growth as per the method suggested [14]. A small piece of fruiting body was dipped in 0.01% mercuric chloride to remove surface contamination and washed several times with distilled water to remove the traces of mercuric chloride and transferred aseptically on to 3% malt extract agar [15] slants and was incubated for 5-7 days. The mycelium collected from the growing edge of those slants was transferred on to new malt extract agar slants and incubated further 5-7 days to obtain pure culture. Pure cultures were subcultured on malt extract agar slants and plates every fortnight.
Characterization of Macrofungi.
Based on macroscopic features, namely, size, shape, sporocarp nature, color, spore print, margin of pileus, characters of lamella, spacing of gills, characters of the stipe, and the presence or absence of veils on stipes, the fungi were identified, http://en.wikipedia.org/ wiki/Template:Mycomorphbox.
Macroscopic Identification.
Seven mycological characters useful in tentative identification of mushrooms are hymenium type, cap shape, gills, stipe character, color of the spore print, ecological type, and edibility.
Molecular Identification.
Based on their lignolytic activity (data not shown here) ten species of wild macrofungi were selected for confirmation of their identities using molecular methods (see Figure 1). Collected fruiting bodies are initially cultured on malt agar (MEA), and the genomic DNA of the macrofungi was extracted using modified CTAB method [16]. The genomic DNA of the macrofungi was then subjected to PCR to amplify the ITS regions of the nuclear ribosomal DNA using two primers: ITS 1 (5 TCCGTAGGTGAA CCTTGCGG3 ) and ITS 4 (5 TCCTCCGCTTATTGATATGC3 ) [17]. The PCR products were then purified using QIAGEN purification kit following the manufacturer's instructions and the purified PCR products were sent to SciGenom, Cochin, for outdoor DNA sequencing. Related gene sequences for each of the macrofungal specimens were obtained from NCBI GenBank and then automatically aligned using ClustalW program incorporated in BioEdit v. 7.1.9 [18]. Manual sequence alignments were then performed using Bioedit to allow maximum sequence similarity. Finally, phylogenetic tree was constructed based on maximum parsimony using PAUP v. 4.0 b10 [19].
Results
The fruiting bodies of white rot fungi were collected in rainy season from few forest areas of Telangana state ( Table 1). The collected fruit bodies were cultured until the pure cultures were obtained. The lists of identified wild mushrooms were depicted in Tables 2 and 3. The fungi that belong to Polyporales are more in number than other orders. Schizophyllum radiatum belong to the order Agaricales. All the collected fungi were saprotrophic and were not edible. Based on seven mycological characters they were characterized. The climatic conditions in Telangana are congenial for prevalence of macrofungi and hence recorded maximum genera. During the systematic surveys at different forests of Telangana, total 50 mushroom samples were collected and these samples belong to different genera out of which only 10 mushroom samples were identified based on their lignolytic activity (data not shown here) and were identified basing on the molecular identification and macroscopic characters. All collected mushrooms have been deposited in the department herbarium with accession numbers. The ten identified mushrooms by molecular level are Trametes versicolor (GenBank Accession number HF953984), Pycnoporus cinnabarinus strain SYBC-L14, Daedaleopsis flavida strain 5A, Trametes elegans voucher, Trametes hirsuta, Fomitopsis feei (GenBank Accession number AY 515327.1), Trametes gibbosa (GenBank Accession number AY351924.1), Trametes elegans GenBank (Accession number AY351925.1), Ganoderma lucidum, and Schizophyllum radiatum (GenBank Accession number HE863742.1).
Discussion
In Basidiomycotina more than 2000 species of edible mushrooms are reported in the publications from different components of the world. People all over Asian countries in the twentieth century know that mushrooms are important biosource of novel secondary metabolites. In India, the alternative systems of medicine utilize the curative properties of mushrooms. Secondary metabolites of these mushrooms are chemically diverse and possess a broad spectrum of biological activities, which are explored in traditional medicines [20]. In India, several mushrooms have been reported as medicinal mushrooms which have antioxidant, antimicrobial, and anti-inflammatory activity with antitumor and other properties [21]. Study on mushrooms in South India such as Tamil Nadu, Kerala, Karnataka, and Andhra Pradesh was neglected as regards to studies on agarics until 1975 [22]. All these collected white rot fungi were rich in medical importance. Ganoderma lucidum is well known to promote health and longevity, lowers the risk of cancer and heart disease, and boosts the immune system [23]. Polysaccharides from mushrooms as anticancer agents, other constituents exhibiting antioxidants and antihypertensive, cholesterol-lowering, liver protection, antifibrotic, anti-inflammatory, antidiabetic, antiviral, and antimicrobiallike activities, have overtly primed its potential as dietary supplements [24]. Some of the Indian mushrooms which are having the medicinal properties are Agaricus bisporus [25], Astraeus hygrometricus [26], Volvariella bombycina [27], Ramaria formosa [28], Pleurotus sajor-caju [29], Pleurotus pulmonarius [30], Pleurotus ostreatus [31], Phellinus rimosus [32,33], Lycoperdon perlatum [34], Lentinus tuber-regium [35], and Lentinus squarrosulus [36]. One of the authors [37] worked on Schizophyllum radiatum and reported the potentials of extracellular biosynthesis of silver nanoparticles, their characterization, and antimicrobial activity on grampositive and gram-negative bacteria. The S. radiatum was also studied for its antimicrobial and antioxidative properties under submerged fermentation. The supernatant of the seed media obtained after separating the mycelia has been used for the synthesis of silver nanoparticles, whereas the species of Fomitopsis feei were recorded in decolorization of triphenyl methane dyes, namely, bromophenol blue, basic fuchsin, methyl violet, methyl green, ethyl violet, and malachite green [38]. This report clearly explains that the bioremediation by utilising fungal organisms was advised to be the costeffective and ecofriendly method of decolorization of effluents released from the dye industries. The genus Trametes and its species have many immunomodulatory and anticancer effects [39]. The genus of Daedaleopsis has antibiotic, antihypersensitive, and antitumour properties [40]. A constant monitoring and collection, identification, and preservation of the wild mushrooms are the need of the hour to explore and make use of the bioprospects of the diversified macrofungal species. This is a preliminary and basic work carried out to locate and identify the bioprospects of the existing macrofungi which will pave the way for understanding an elaborate study on this aspect.
Conclusion
Amongst the vast number of living forms very little amount of attention has been paid to conservation of fungal biodiversity. Due to loss of natural habitats, soil and air pollution expansion of monocropping and loss of genetic diversity many fungal species are on threat. For the smooth working of this terrestrial environment, the preservation of mushroom differences is discriminating. Keeping in view this gigantic mushroom treasure it is the high time to completely save this biodiversity. A few mushrooms are known to be the wellsprings of different bioactive substances like antibacterial, antifungal, antiviral, antiparasitic, antioxidant, antiinflammatory, antiproliferative, anticancer, antitumour, cytotoxic, anti-HIV, hypocholesterolemic, antidiabetic, anticoagulant, and hepatoprotective substances, among others. These mushrooms have been utilized as ethnomedicines by tribals for treatment of different sicknesses. Numerous mushrooms still stay unreported and their healthful and in addition medical advantages are obscure to us. Henceforth, an opportune examination in regard to isolation, identification, and characterization of the current mushroom vegetation is vital. Biotechnological devices can be utilized with a specific end goal to accomplish the in situ and ex situ preservation of huge numbers of the mushroom species. The outcome of the present study elaborates the information on the facts and figures of diversity of fungi of the study area. | v3-fos |
2019-03-21T13:04:47.629Z | {
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} | s2 | Length – weight relationship and prediction equations of body composition for growing-finishing cage-farmed Nile tilapia
The objective of the present study was to develop models for predicting live weight from the lengthweight relationship and body composition of Nile tilapia. A total of 3,000 juvenile fish (initial weight = 28.6±4.16 g and standard length = 13.8±0.16 cm) were distributed into three circular cages (12 m3 each). The fish were hand-fed extruded diets containing 332 g kg–1 of crude protein and 3,230 kcal kg–1 of digestible energy, until apparent satiety, twice daily, for 100 days. Twelve fish were collected from each cage every 20 days for measurements of body weight and length, and proximate composition analysis; statistical analysis was conducted using linear regression. The value of the slope b and the intercept for the length-weight relationship were 3.0604 and 0.0203, respectively. The prediction equations obtained for body moisture (MO), crude protein (CP), crude lipid (CL), and ash against body weight (BW) in g/100 g of fish were as follows: MO = 70.0090 – 0.0071BW; CP = 13.7550 + 0.0037BW; CL = 9.2636 + 0.0057BW; and ash = 4.2392 – 0.0024BW. It is possible to develop equations to predict body weight and composition, which can be used to control the production of Nile tilapia and improve its commercial value.
Introduction
Mathematical models of fish growth offer an objective and practical method for describing patterns of growth data and estimating fish weight at times between sampling intervals. Accurate estimations of standing biomass, and therefore, of the amount of feed that must be provided, are vital to aquaculture management. Additionally, knowledge of the relationships between body weight and composition supports selection during efforts for the improvement of aquaculture genetics. An accurate length-weight relationship equation allows for the conversion of growth-in-length to growth-in-weight in stock assessment models, as well as the estimation of biomass from the length frequency distribution, condition (Petrakis and Stergiou, 1995), and morphological characteristics of fish populations (Stergiou and Moutopolous, 2001); the relationship equation, thus, is an important aquacultural management tool.
Growth, which is defined as a change in magnitude, can be measured in size and tissue composition and represents one of the most significant parameters in aquaculture. The body composition of fish has recently received attention in studies on nutrition, genetics, and health (Tobin et al., 2006) because of the increasing interest in the quality and safety of fish products (Dumas et al., 2010). Body composition is an important aspect of nutritional quality (Kamal et al., 2007;Breck, 2014) and affects the nutritional value and consumption quality of fish (Azam et al., 2004). Despite the usefulness of the length-weight relationship and body composition, as well as the great economic importance of Nile tilapia (Oreochromis niloticus) to global aquaculture, little information on these factors is available in cagefarmed growing-finishing populations of the species. The present study was conducted to determine the lengthweight relationship and develop prediction equations of body composition for growing-finishing cage-farmed Nile tilapia by using regression analysis.
Material and Methods
The management was in accordance with the guidelines of the Animal Care Committee of Universidade Estadual de Maringá, Paraná, Brazil.
A total of 3,000 masculinized juvenile Nile tilapia (initial weight = 28.6±4.16 g; standard length = 13.8±0.16 cm; age, 60 days) were randomly distributed into three hexagonal cages (11 m 3 each) placed at a local mean depth of 6.0±1.3 m in the Paranapanema River (22°34'07"S; 52°33'34"W). A mix of vegetable and animal protein sources was used to formulate an extruded diet (Table 1) containing 332 g kg −1 of crude protein and 3,230 kcal kg −1 of digestible energy (Table 2). Crystalline amino acids were added to maintain the quantitative and essential amino acid profile recommended for Nile tilapia as described in NRC (2011). Fish were hand-fed to apparent satiety twice daily for 100 days. Water quality parameters were monitored daily during the feeding trial. The average water temperature was 28.5±1.3 °C; pH, 7.34±0.21; and dissolved oxygen varied from 6.2 to 6.6 mg L −1 .
At the beginning of the experiment and every 20 days afterwards, 12 fish were randomly selected from each cage, starved for 24 h and collected to determine individual weight, standard length, and whole-body composition. Whole-body samples of fish were pooled, ground, and stored at −20 °C for proximate analysis.
The proximate composition analyses of the fish samples were performed following the procedures of the AOAC (1990). Water content was determined by placing the fish in a previously weighed aluminum foil tray, drying in an electric oven at 55 °C until constant weight was achieved, and then oven-drying at 105 °C for 24 h. Crude protein (nitrogen × 6.25) was determined using the micro-Kjeldahl method after acid hydrolysis. Lipid was extracted using petroleum ether in a Soxhlet apparatus and determined gravimetrically. Ash was determined by combustion at 550 °C in a muffle furnace for 6 h.
Gross energy was determined using an adiabatic bomb calorimeter. Dietary amino acids were analyzed by hydrolyzing 0.3 mg fish sample in 1 mL of 6N HCl for 22 h. The obtained sample was diluted in 0.02N HCl and injected in an automatic Amino Acid Analyzer. Recovery hydrolysis was performed in 4N methanesulfonic acid for the analysis of tryptophan and in performic acid for the recovery of sulfur amino acids.
Data on the total body length (L) and body weight were recorded from each fish. The parameters a and b of the length-weight relationship were estimated by logarithmic transformation of the equation W = a × L b , in which W is the body weight (g); L is the standard body length (cm); a is the intercept; and b is the slope. Length-weight relationships were used to provide the condition of fish and determine whether growth is isometric (b = 3) or allometric (negative allometric: b<3, or positive allometric: b>3) (Ricker, 1973). To check whether the average b value was significantly different from 3.0, the t-test was conducted at 0.05 significance.All statistical analyses were performed using the statistical package SPSS 14.0. The body composition data were analyzed by oneway analysis of variance (ANOVA) at a significance level of p<0.05. The prediction equations for body composition were developed using linear (ŷ i = ß 0 + ß 1 x i ) or second-order (ŷ i = ß 0 + ß 1 x i + ß 1 x i 2 ) polynomial regression analysis (Zeiton et al., 1976). The degree of association between the variables was analyzed according to the determination coefficient (r 2 ) for each variable.
Results and Discussion
At the end of the experiment, a high daily weight gain (7.5 g) was observed (Table 3). Although indoor experiments offer uniform chemical and physical waterquality parameters that can be controlled, the advantage of conducting experiments in rivers is the high rates of water turnover, resulting in high and constant levels of dissolved oxygen and water temperature, which allow for a high daily weight gain in fish. The daily weight gain of fish in the present study was higher than that reported by Moraes et al. (2009) for Nile tilapia reared in indoor cages at the same density. Factors such as diet composition (Moraes et al., 2009), stocking density (Araujo et al., 2010), and chemical and physical parameters of the water may affect the fish growth; however, the higher weight gain of fish in this study was because the fish were fed until apparent satiation. Feed intake is a major factor for tilapia growth (Tran-Duy et al., 2012), and in our study, fish preferred to be fed to apparent satiation because of the constant and high dissolved oxygen concentration during trial studies. Besides this, all essential amino acids were supplied to meet the dietary requirement of Nile tilapia, as described in NRC (2011) based on the ideal-protein concept, to optimize protein (amino acids) utilization for fish growth and health (Li et al., 2009). The length-weight relationship was established using the equation W = 0.0203 × L 3.0604 (R² = 0.9914) (Figure 1). The mean value of b (3.0604) did not significantly differ (P<0.05) from the standard value of 3.0, implying that the "cube law" could be applied for this species (Ricker, 1973). When the weigh-length exponent b is equal to 3.0, the body form maintains a constant proportion to the length and the fish grows isometrically, resulting in an ideal shape (Pauly, 1983). However, when b is less than 3.0, the fish shows negative allometric growth, and when the b value is greater than 3.0, the fish shows positive allometric growth (Weatherley and Gill, 1987). Similar results were obtained for Nile tilapia collected from the Atbara River and Khashm El-Girba reservoir by Ahmed et al. (2011), who found a b value higher than 3.0 (3.415) for this species. Thus, the fish are expected to grow proportionally in all directions.
Changes in fish weight are generally greater than those in fish length (Ahmed et al., 2011). In general, when the value of b exceeds 3.0, fish become fatter, and when the value falls below 3.0, fish become leaner. The value of b found in the present study is within the interval of 2.5 to 3.5 recorded for many fish species by Froese (2006), as well as between the values of 2.299 and 3.684 recorded for Nile tilapia in the Atbara River and Khashm El-Girba reservoir, respectively (Ahmed et al., 2011). However, this value is slightly higher than the mean value (b = 2.908) described by Britton and Harper (2008) for Nile tilapia with 4.0-23.1 cm in length.
All linear regressions were significant, with coefficients of determination (r 2 ) ranging from 0.7743 to 0.8628. The relationships between body weight and body moisture, crude protein, and crude lipid were best expressed using linear regression analysis (Table 4). As shown in several studies, when the size of fish increase, more fat is deposited than the formation of other tissues (Salam and Davies, 1994;Salam et al., 2001). The compositions of various organs and bodily tissues may also show considerable differences (Weatherly and Gill, 1987). However, whole-body composition follows a similar pattern among different species as fish size increases (Lupatsch et al., 2001;Dumas et al., 2010), and the live weight of the majority of fishes consists of approximately 700-800 g kg −1 of water, 200-300 g kg −1 of protein, and 20-120 g kg −1 of lipid (Love, 1980). The whole-body composition of fish is affected by species (Ali et al., 2005), environmental parameters , nutrition, and body size (Ebrahimi and Ouraji, 2012). Information on body composition related to fish size can be used to select fish with higher protein contents at a specific size, for human consumption (Ali et al., 2005). Fish represents one of the main sources of protein in developing countries (Louka et al., 2004). The moisture content is a good indicator of body protein and lipid contents (Ali et al., 2005), as observed in this study ( Figure 2); lower moisture percentages are associated with higher lipid contents in fish (Dempson et al., 2004). Hartman and Margraf (2008) reported a significant fat-water relationship and developed models to predict the proximate composition for chum salmon (Oncorhynchus keta), rainbow trout (Oncorhynchus tshawytscha), brook trout (Salvelinus fontinalis), and striped bass (Morone saxatilis).
In the present study, moisture and ash contents decreased linearly with the increase in body weight. However, the body fat content increased linearly as a function of body weight. This result is in agreement with that described by Salam et al. (2001), who observed an increase in fat content with increase in the size of Nile tilapia. Memid et al. (2006) reported that the body constituents of Russian sturgeon (Acipenser gueldenstaedtii) could be estimated with reasonable accuracy from the weight of the fish by using a predictive regression model. Pangle and Sutton (2005) described a linear regression model for estimating temporal changes in the proximate composition of juvenile lake herring (Coregonus artedi) during winter periods. Breck (2014) reviewed the close relationship of body size to body composition for many freshwater and marine fish species. Understanding the relationship between body weight and length, as well as developing prediction equations for body composition, provide important support for genetic improvement, fish management, feeding strategies, and marketing.
Conclusions
The body composition of Nile tilapia varies according to its body weight and can be estimated using the lengthweight relationship. Prediction equations of body composition derived from linear regression analysis can be employed to address the requirements of specific consumer markets.
Acknowledgments
Financial support: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Ajinomoto do Brasil Indústria de Comércio de Alimentos -Animal Nutrition Division for the amino acids donation and analysis. We wish to thank the staff of the "São João" fish farm group for allowing us the use the Fish Farm base, and Mr. Manuel Silva and Mrs. Rosa Silva for their support during this study. | v3-fos |
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} | s2 | Garlic (Allium sativum): diet based therapy of 21st century–a review
Hafiz Ansar Rasul Suleria, Masood Sadiq Butt, Nauman Khalid, Saira Sultan, Ali Raza, Muhammad Aleem, Munawar Abbas School of Medicine, The University of Queensland, Australia National Institute of Food Science and Technology, University of Agriculture Faisalabad, Pakistan Department of Global Agriculture, Graduate School of Agriculture and Life Sciences, University of Tokyo, Japan Faculty of Agriculture, Universitat Hohenheim, Stuttgart, Germany Department of Food Science, Government College University Faisalabad, Pakistan Asian Pac J Trop Dis 2015; 5(4): 271-278
Introduction
Functional and nutraceutical foods are becoming popular all over the world owing to their health promoting perspectives. There are several traditional plants, used as a therapeutic carrier for different physiological threats. Investigation through diet based therapies, elucidate the significance of these traditional plants including garlic, onion, black cumin, green tea, ginger, peanut etc., however, several avenues are yet to be explored for researchers [1]. Pakistan being diverse in indigenous phphytocoenosium has such types of traditional plants that are contributing to health.
Functional foods are significant in this milieu owing to easy accessibility, low price and allied health promoting perspectives. In this circumstance, many fruit and vegetables, cereals, nuts and pulses etc. are vital because they possess therapeutic potential due to certain arrangement of biologically active chemical substances known as phytochemicals. These are the biologically important chemical molecules in plant food that are mainly concerned with modulation of various metabolic pathways, like free radical scavenging, antimicrobial properties and providing protection against diseases [2].
The diversity in human diet cannot be understood properly as socioeconomic and environmental factors lead to wide variations in the P E E R R E V I E W A B S T R AC T K E Y WO R D S Garlic, Functional food, Nutraceuticals food, Diet based therapy Functional and nutraceutical foods provide an opportunity to improve one's health by reducing health care costs and to support economic development in rural communities. For this reason, various phyto-based functional foods are becoming popular worldwide owing to number of evidences for their safer therapeutic applications. Garlic (Allium sativum L.,) is an essential vegetable that has been widely utilized as seasoning, flavoring, culinary and in herbal remedies. The consumption of traditional plants especially garlic has progressively increased worldwide because of their great effectiveness, fewer side effects and relatively low cost. Garlic is well known to contain an array of phytochemicals. These bioactive molecules are playing pivotal role in maintaining human health and having potential to reduce various ailments. It has distinct nutritional profile with special reference to its various bioactive components that can be used in different diet based therapies to cure various life-style related disorders. The present review is an attempt to explore the functional/nutraceutical role of garlic against various threats including dyslipidemia and hyperglycemia, cardiovascular disorders, antioxidant capacity and carcinogenic perspectives.
dietary pattern of the global population. The abrupt increase in medical care costs and human's desire for maintenance of one's health result in greater attention for nutritionists and public health scientists to elucidate the diet-health linkages. Diet and health linkages are correlating as consumer thread is varied and now they are more conscious toward nutritional and functional attributes of food [3]. However, a number of strategies are needed, to explore nutrients dense sources, their potential utilization and applications, and especially their mode of actions [4]. Nevertheless, vegetables are one of the important segments of human diet since ancient times and hold unique position in all dietary guidance systems. The American Dietician Association recommended daily 4-5 servings of vegetables to meet the requirements of desired nutrients. Vegetables are rich sources of bioactive molecules such as carotenoids, chlorophylls, anthocyanins and flavonoids [5]. These bioactive molecules have the ability to target at molecular level effectively by modulating enzyme kinetics, release of cytokines, and signal transduction [6]. Among the functional foods, use of garlic is mounting day by day because of its health promoting potential besides basic nutrition. The medicinal uses of various herbs have been proven experimentally as lifesaving entities, associated with health claims owing to their rich phytochemicals profile [7]. Health promoting aspects of garlic are mainly accredited to its sulfur containing compounds mainly allicin and S-allylcysteine ( (SAC). Among organosulfur compounds, thiosulfinates, ajoenes and allicin (diallyl thiosulphate) are the chief bioactive components in garlic responsible for curing various ailments.
Links of crucial importance have been settled between dietetic components and human health safety [8]. There are lot of evidences that consumption of different vegetables are important for human health as they are very good sources of dietary fibers, antioxidants, carotenoids, sulfurcontaining compounds, vitamins, and minerals [9]. Epidemiological studies conducted worldwide have brought to light that liberal consumption of vegetables especially rich in nutraceuticals is associated with a guarantee of prevention and reduction of health related chronic disorders [10].
Garlic, an overview
Garlic (Allium sativum L.) is one of the most commonly used plants utilized both for medicinal purposes and culinary practices as providing flavor and taste to the final product. It is believed to be originated from Central Asia over 6 000 years ago and has been extended towards West, South and East. However, intact garlic as well as its components/ fractions is used since long time ago in Chinese medicines even 3 000 years while Egyptians fed garlic to pyramid crews to boost their immunity thereby render safe from various maladies and improve their performance [11].
Garlic health promoting perspectives have been proven and it is recommended worldwide as a dietary supplement. It is also cultivated for its medicinal value owing to an increase in its consumption both in cooking and curing. Lately, a wide range of its cooking related and medicinal preparations show their availability in market. In many countries, health potentials of garlic and its various products have been recognized and are useable as dietary supplementation. Dehydrated garlic as well as its extracted oil is gaining popularity and are being sold as dietary supplements [12].
Garlic is a rich source of sulfurous compounds, for example, diallyl sulfide, allicin (diallyl thiosulphate), -glutamyl-S-allyl-L-cysteines, and S-allyl-L-cysteine sulfoxides (alliin) and altogether they are called allium compounds. They are found efficacious in reduction of certain types of cancer as well as in management of cholesterol and eventually blood pressure [13,14]. The garlic organosulfur components are mainly responsible for its health promoting properties. Major research interventions have focused on its potency in prevention of ailments related to lifestyle related disorders for instance in case of sedentary lifestyle like hypercholesterolemia, dyslipidemia, and high blood pressure.
The nutritional composition of garlic bulb contains approximately 65% water, 28% carbohydrates 2.3% organosulfur compounds, 2% protein, 1.2% free amino acids and 1.5% fiber. Briefly, raw garlic contains water, carbohydrate, and proteins in the amounts of 58.58, 33.06, and 6.36 g/100 g, respectively. The consumption of 100 grams of garlic provides 149 kilocalories. Carbohydrates and proteins are the major components of garlic powder accounting for more than 80% while among vitamins, thiamin have special consideration with high bioavailability owing to some specific sulfur containing components. Regarding minerals, selenium needs special interest as its level is at least 9 ppm higher than almost all other plants. Main pharmacological effects of garlic are due to its featured organo-sulphur compounds [15]. Allicin (diallyl thiosulphate), which is the most important bioactive component in garlic holds the responsibility for its distinctive pungent smell and other remedial properties [16].
Chemistry of garlic
Garlic bioactive compounds are placed into categories as: sulfurcontaining and non-sulfur compounds. Sulphur is chiefly found as alkylcysteine sulphoxides and gamma-glutamyl peptides which in combination constitute more than 70% of the total sulphur content in garlic. The degradation of alkyl cysteine sulphoxides, alliin, methiin, and isoalliin is brought about by alliinase enzyme and result in a release of powerful smelling volatiles, provide particular odor and flavor to the Allium species. Alliin is converted into allicin by crushing the garlic clove within seconds. Garlic remains odorless unless it is mashed. Cross-section investigation has exhibited that location of the substrate alliin and the enzyme alliinase are separate compartments. In garlic the flavor precursor showing dominant characteristic is alliin having less concentrations of isoalliin, methiin and trace amounts of propiin [17]. The therapeutic effects of garlic are mainly attributed to a sulfur-containing compound known as allicin [18]. The garlic clove lack allicin content but contains its precursor, the non-protein amino acid alliin. Garlic bulbs contain alliin and alliinase at about 1.7 and 2.8% of their dry weight, respectively. By the crushing of garlic bulbs variable amounts of allicin is produced ranging from 1.6-13.0% of their dry mass [19].
The distinguishing flavor of fresh garlic is associated with thiosulfinates and various other volatile substances produced by alliinase (EC4.4.1.4) like S-alkyl-substituted cysteine sulfoxide derivatives, alkyl alkane thiosulfinates, pyruvic acid, and ammonia [20]. The enzyme action starts as soon as garlic tissues are disrupted. The major substrate in garlic is non protein amino acid alliin (S-allyl-L-cysteine sulfoxide). Allicin (diallylthiosulfinate) formed by enzymatic activity constitutes about 60 to 80% of total garlic thiosulfinates. Gene coding for the enzyme alliinase has disclosed that it is constituted of 448 amino acids having a molecular mass of 51.45 kDa and carbohydrate amount of 5.5-6.0% with 55 000 kDa. Alliinase enzyme contain 10 cysteine residues, all exist in S-S bridges which make the enzyme inactive by their reduction, or by removing pyridoxal coenzyme factor. Indeed, none other compound outside the thiosulfinates has been discovered to contribute for a fairly large portion of the pharmacological preparations of mashed garlic at similar levels. Allicin amount in fresh garlic varies to a large extent. According to British pharmacopoeia 1998, the minimum allicin content ensuring pharmaceutical and economic viability of garlic powder products is 4.5 mg/g. It is well observed that garlic allicin content varies with various geographical regions. Difference in allicin content among various ecotypes is directly related to genetic variations [19].
Characterization of garlic preparations
Estimation of garlic flavor components is important to the food industry since the quality of garlic and garlic products affects overall food quality. Similarly, chromatographic methods have been utilized by the dietary supplements industry to investigate garlic volatiles that may affect the overall acceptability of supplements to the consumer. Several sulfur components were investigated including allyl methyl sulfide, allyl mercaptan, 3,3'-thiobis-1-propene and diallyl disulfide. The diallyl disulfide appeared to be the more prominent component for all garlic preparations. The biological potentials of different components of garlic for example lectins (abundantly found proteins in garlic), prostaglandins, pectin, adenosine, fructan, vitamins B1, B2, B6, C and E, biotin, nicotinic acid, glycolipids, phospholipids, fatty acids and essential amino acids, have been investigated for years. Recently, the importance of pharmacological activities, including antifungal, antitumor, antithrombotic and hypocholesterolemic attributes [21] of definite sapogenins and steroid saponins, such as beta-chlorogenin has been discovered [22]. Some other distinctive chemical components include allicin and organo-selenium which show many biological influences, like lowering cholesterol level, preventing cancer etc. and most likely act synergistically. Flavonoids, abundant in many fruits especially onion, are almost not found in garlic, while a minimal quantity of non-volatile water-soluble sulphur containing compounds exist in garlic. These are SAC (enzyme induced transformation of gamma-glutamylcysteines during aqueous solution) which are also useful in the betterment of many health disorders [23]. The most important garlic preparations used as dietary supplements are the one containing the most safe and effective, high stability and odorless components. In accordance with various researches, aged garlic extract by dint of its extraction method, has more effectiveness and safety as compared to fresh garlic, garlic powder and related formulations [24].
Garlic oil mainly contains diallyl disulphide, diallyl trisulphide, allyl propyl disulphide, and disulphide, also containing a small amount of diallyl polysulphide [25]. It is considered to be more effective than aqueous extracts and reveals a wide range of pharmacological effects including antidiabetic and anticarcinogenic [26]. Conversely, its application in the food industry is very restricted due to its strong flavor and odor, water insolubility, and low physicochemical stability.
Life style related disorders
Poor dietary habits and changing lifestyle often lead to various physiological threats such as cardiovascular complications, immune dysfunctions and cancer [27][28][29]. It is estimated that 30-40% of different diseases are curable with a vigorous lifestyle and dietary modules [30][31][32]. Disease prevention stratagem should include nutritional and exercise guidelines; particular targeted dietary components could be a part of this approach. Diet and healthy foods have a significant impact on the antioxidant potential of the body and combat deficiency of nutrients, like tocopherols, polyphenols that reduce the risk of various maladies like diabetes mellitus, and atherosclerosis. Plants rich in phytochemicals profile have the ability to scavenge free radicals and ameliorate oxidative stress [33]. Among these phytochemicals, phytosterols, antioxidants and flavonoids have shown hypoglycemic and hypocholesterolemic potential [13]. Functional foods and their bioactive moieties have shown therapeutic potential against various biological threats including antioxidant, anti-cancer and immunomodulation [34]. Free radicals being reactive in nature, damage to macromolecules. Imbalance of these reactive oxygen species result in oxidative stress on different cells and tissues. Oxidative stress is a disturbance between the production of reactive oxygen species and antioxidant defense, resulting in tissue injury. Oxidative stress also leads to DNA damaging, cardiovascular and neuropathy disorders and cancers [35]. It is urged to utilize herbal products having better efficiency and safety, as a substitute for chemical therapy. Various herbal products that may be comprised of one or blend of different antioxidants have been investigated to protect against hepatic injury, having immune modulatory or antiviral properties [36].
Diabetes mellitus
Diabetes is a metabolic syndrome that steadily affects different physiological systems of the human body. It is one of the leading causes of mortality worldwide and, if uncontrolled, can threat multiorgan systems [37]. Uncontrollable blood glucose is known to be the core feature in the inception of diabetes difficulties of Type 1 as well as Type 2 (American Association of Diabetes Educators, 2002). Most common type is Type 2 category, while Type-1 diabetes develops mostly in early childhood. Kidneys play a significant role and become the target for investigation in studies of diabetic complications as many other factors involved in the development of diabetic nephropathy are the same as in other common diabetic complications, like retinopathy and microvascular diseases.
There is an estimate that in 2030, 376 million peoples worldwide will be affected with diabetes [38]. It is mainly characterized by relative deficiency in insulin secretion or insulin action associated with hyperglycemia and malfunctioning in the metabolism of carbohydrate, lipid, and protein. Diabetes may also lead to various other complications like, cardiovascular disorders, oxidative stress and immune dysfunction [39]. Cardiovascular diseases are the main cause of illness and death all over the world. By increasing the level of cholesterol and low density lipoprotein (LDL) oxidation triggers events that initiate atherosclerosis [40]. As a result of various health disparities there is an onset of autoimmune disorders and immune dysfunction. According to Nogichi [41], 33% of all the diabetic patients take various medications that may be considered effective, among them garlic is one of the most frequently used. Garlic and its preparations have shown varied biological activities, including antidiabetic, antiatherosclerotic, anticarcinogenic and antithrombotic actions [42]. However, these biological activities are well famous, only certain complications, like cardiovascular diseases and tumor development, have been widely studied [43]. It is also predicted that the number of studies related to garlic hypoglycemic perspectives is limited and the outcomes are also inconsistent.
It is investigated that the effect of garlic extract prepared with water or combination of other organic solvents are studied on normal as well as alloxan-induced diabetic rabbits. It is inferred that all those garlic preparations possess an intense hypoglycemia action, which was accomplished by employing ethyl ether extract with that of tolbutamide. Later on, the influence of garlic oil was studied on diabetic animals by Anwar [44], in order to verify the hypoglycemic perspectives of garlic. Lately, garlic juice was determined to cure hyperglycemia in diabetic rats, and it also eased out the oxidative pressure and harm in their liver and kidney. In addition to garlic extract and garlic oil, allyl sulfide, S-allyl-cysteine sulfoxide (alliin), was also found to be possessed with the same hypoglycemic effect as was in case of glibenclamide [45].
Recently, it is accounted that the hypoglycemic action of garlic, garlic oil and its organosulfur constituents especially di-allyl-trisulfide, result in higher insulin secretion and insulin sensitivity in streptozotocin-induced diabetic rats. However, there was not an appropriate hypoglycemic activity of garlic powder in diabetic animals induced with streptozotocin. It is concluded that incoherent results are partly due to different garlic preparations used in the different experimental observations. The chemical profile of garlic products mainly depend upon the processing and working conditions, like temperature, preparation time, and the type of solvents used for extraction [46].
Cholesterol lowering potential
Cardiovascular disorder is one of the leading causes of both mortality and morbidity all over the globe. Oxidation of cholesterol fractions, especially of LDLs, is playing a cardinal role in atherosclerosis development. Lipid peroxidation by toxic radicals increases the concentration of free radicals and causes certain diseases including atherosclerosis in humans. Management of plasma cholesterol level is a keystone element to protect cardiovascular disease disorders. Various medications have been applied in treatment but due to the long time therapy and their side effects, natural products may be considered as a suitable remedy. Garlic has been being used in traditional medicine to improve the lipid profile and its different supplementations hold remarkable effect on cholesterol level, LDL-cholesterol, and high density lipoprotein-cholesterol. Consumption of garlic and garlic preparations are very useful in regulating plasma lipid levels [47], plasma anticoagulant activity [48] and also contributed toward the prevention of the atherosclerosis process [49]. Garlic preparations have been reported owing to numerous remedial benefits, including decreasing plasma cholesterol levels and blood pressure, decreasing platelet aggregation, protecting LDL from oxidation. Various garlic preparations like garlic oil, garlic extract etc. are also effective in lowering blood pressure and oxidative stress in hypertensive persons. Ackermann et al. [50] estimated that garlic preparations have comparatively lower declines (1.2-17.3 and 12.4-25.4 mg/dL) in total cholesterol level as compared to whole garlic after 1 and 3 months correspondingly. Effect of garlic on lipid profile was elaborated that garlic significantly reduced total cholesterol and triglycerides level [51].
Aqueous garlic extracts has the ability to decrease cholesterol synthesis up to 75% without cellular toxicity mediated by sterol 4-alpha-methyl oxidase. It is also effective in reducing coronary calcium progression [52]. It was elaborated in one study that S-allyl cysteine sulfoxide decreased the activity of 3-hydroxy-3-methylglutaryl-CoA reductase enzyme while significantly increased the activity of lecithin acyl transferase in isoproterenol-induced myocardial ischaemia [49]. According to Yeh and Liu [52] observations, SAC, S-propylcysteine and S-ethylcysteine inhibited cholesterol synthesis up to 40-60% as compared to gammaglutamyl-S-methylcysteine, gamma-glutamyl-S-allylcysteine and gamma-glutamyl-S-propylcysteine that contributed 20-35%. Moreover, only SAC appears to decrease the activity of 3-hydroxy-3-methylglutaryl-CoA reductase by enhancing sulfhydryl oxidation. There is no doubt that garlic and its various preparations possess anticoagulant perspectives [48], but still there is a lot of controversy with respect to the garlic lipid profile and its antioxidant status. As there is not an exact molecular mechanisms identified for garlic perspectives in the atherosclerosis, further establishment should be made to develop a relation among garlic extract, antioxidant status, and blood lipid profile.
Cardiovascular complications
Epidemiological studies have investigated various risk factors associated with atherosclerosis development such as elevation in serum lipid level (cholesterol and triglycerides), increase in plasma fibrinogen level, elevation in platelet activation, modification in metabolism of glucose and smoking [53]. One of the important cause in the atherosclerosis formation is oxidation of LDL [54]. It is revealed through various epidemiological studies that atherosclerosis-related conditions like coronary artery disease, ischemic stroke and peripheral artery disease increase with age. It usually happens in the muscular arteries such as aorta, coronary, carotid, femur, and ilium arteries.
Aging influences structural and functional attributes of the vessels that leads to various cardiovascular complications. By virtue of that the morbidity and mortality increase especially in hypertension conditions. Aging induces alleviation in endothelium-dependent, alteration in endothelial cells and vascular tone regulating, it also decreases endothelial synthesis and eventually releases nitric oxide. During aging increased collagen-elastin ratio as well as sedimentation and sticking of calcium along with lipids inside the blood vessels leads to enhancement of the hardness of vascular wall [55]. In old age groups increase in oxidative pressure with respect to aging is significant factor in the occurrence of atherosclerosis. Both in vitro and in vivo studies exposed that an important stimulator of early atherosclerotic lesion is oxidative modification of LDL that contributes to continual rise in amount of LDL hydroxy fatty acids with the increasing age in healthy individuals. "Aging" may also occur in a condition when there is a delay in the removal of lipoprotein from the plasma, leads to a gradual "senescent" type of passing on lipoprotein that is particularly more exposed to oxidation as in oxygen radical-stimulated modification. Oxidized LDL have pro-atherogenic perspectives including increase in smooth muscle proliferation, smooth muscle cells and fibroblasts and cytotoxicity to vascular endothelial cells as well as the induction of apoptotic activity in vascular endothelial cells [56].
Garlic and its various preparations have been used to assess and cure various cardiovascular disorders for a long time. Studies on both rats and rabbits have shown that garlic extract has been used to alleviate dietinduced hypercholesterolemia [57]. It is clarified that garlic extract and aged garlic extract are efficacious in reduction of plasma cholesterol, triglyceride level and LDL-cholesterol in hyper-lipidemic persons however, no any effect in normolipidemic ones [49]. Preparations utilizing garlic powder have also been studied for reduction of plasma cholesterol levels in high cholesterol people [58], while a few objections have also been observed by Gardner et al [59]. It is clearly predicted that garlic and its various components have a potential to decrease plasma cholesterol and triglyceride level by inhabitation of important enzymes of cholesterol and fatty acid synthesis mechanism [52].
Platelet aggregation and gradual thrombus formation is reduced competently by utilizing garlic and its components whilst firbrinolysis increases as a result of dissolution of clot and thrombi. Daily ingestion of 2.4 g of garlic supplemented with aged garlic extract has high threshold level of collagen, epinephrine-induced platelet assemblage as well as platelet adherence to fibrinogen among healthy subjects [60]. It is also observed in one case study that high intake of age garlic extract (7.2-8.0 g per day) enhanced threshold values of ADP-stimulated platelet accumulation, platelet adhesiveness to collagen as well as von Willebrand factor. In addition suppression of the ADP-stimulated platelet aggregation has been observed in normolipidemic individuals with dietary intervention of aged garlic extract at the rate of 5 mL per day (equivalent to 1.5 g per day) [49]. Aged garlic extract prohibited both percentage as well as staring rate of platelet congregation up to 10 mol/ L at concentrations of ADP. The Km value for ADP-induced platelet aggregation was nearly doubled after dietary modification with age garlic extract whilst the maximum rate of aggregation was unchanged. Garlic oil is found to limit platelet accumulation particularly caused by adrenaline; arachidonic acid, collagen and calcium that alleviate the constitution of pro-aggregatory prostaglandin thromboxane-A2 known as thromboxane-B2 in heart patients. In animals, studies have revealed that fresh garlic extracts are effective against cyclooxygenase activity in cell culture [43]. In vitro studies have pointed out that use of aged garlic extract improved both blood properties and microcirculation controlling hemolysis in oxidized erythrocytes cells as well as lipid peroxidation rate [61]. This confirmation was made by another subsequent research in which chronic garlic powder ingestion inclines to rarefy aging in aortic hardness. This contributes in protecting the aorta elasticity in term of correlate to aging among humans. Studies on utilization of aqueous garlic extracts has confirmed that it not only assist in the constricting factor in pulmonary arteries as well as in producing and functioning of endothelial-derived relaxing factor (NO) [62], but could also decrease blood pressure in high cholesterolemic individuals. Oxidative pressure was related to preservation of enzymatic activities like glutathione peroxidase and superoxide dismutase [63]. Animal modeling has been utilized to describe the influence of garlic oil preparation on rats with nicotine-caused lipid peroxidation. Supplementation of garlic oil removes lipid peroxidation activity in all major organs of the rodents like heart, lung, liver and kidney. This reducing of lipid peroxidation activity was related to a rising antioxidant property of enzymes (super oxide dismutase, catalase, and glutathione peroxidase) as well as higher level of glutathione. If garlic and its preparations are recommended as an antioxidant tools, it is very important to prove its effectiveness in human clinical trials. However, the data revealed that garlic consumption may reduce oxidative stress and aging related diseases [18].
This was determined that the antioxidant condition in non-smoking people is twofold higher than smokers. Introduction of age garlic extract for nearly 14 days tend to reduce the plasma and urine concentration in non-smoking people by 29% and 37% respectively, whereas by 35 and 48% in smokers respectively. After fortnight in both groups plasma and urine concentrations becomes nearly equal to values as before dietary supplementation of age garlic extract while changes other than these were not noticed in other bio-chemical parameters [64]. Garlic consists of a complicated concoction of pytochemicals outlook that act in a synergistic way in order to provide beneficial effects and particularly its antioxidant potential. Flavonoid is high potency antioxidants and their utilization causes significant reduction in LDL to lipid peroxidation [65]. Ingestion of phytochemical especially flavonoids, for instance apigenin, myricetin, quercetin, is related to mortality in an inverse ratio because of cardiovascular disorders [66].
Antioxidant activity
Oxidation of DNA, lipid and protein by ROS plays an important role in wide range of diseases, including aging, cancer and cardiovascular disorders, inflammatory and neurodegenerative diseases. Research studies elucidated phytotherapy, potent source of antioxidant phytochemicals, like vitamins C and E, flavonoids, pigments and glutathione, protect against various cellular degradations [67]. Among garlic preparations, age garlic extracts have higher antioxidant potential than fresh garlic and commercial garlic supplementations. Water-soluble organo-sulphur compounds, such as SAC and S-allylmercaptocysteine (SAMC), have a great antioxidant potential [68]. The two constituents SAC and SAMC are the prime organo-sulphur constituents that are found in age garlic extract whereas some other components which possess an antioxidant potency include stable fat-soluble allyl sulphides as diallyl sulfide, diallyl disulfide, diallyl trisulfide and diallyl polisulphides [23]; some tetrahydro-b-carboline derivatives that formed during the natural aging process; flavonoids, saponins and some essential micronutrients like selenium and macronutrients, as lectins with antiperoxide perspectives are prominent in the kidney, liver and heart of rats. Phytochemical profile of age garlic extract may act as synergistically that exerts their antioxidant potential by scavenging ROS [69]. Age garlic extract owing to its antioxidant potential, reduces the risk of cardiovascular and cerebrovascular syndromes by preventing LDL oxidation as well as lipid peroxidation [47].
Cancer perspectives
Garlic is incepted to be utilized as an anticancer agent in 1950's and the findings depicted that thiosulfinate constituents of garlic had a subduing effect on the growth of tumor cells [70]. Based on these findings, many corresponding epidemiological and laboratory scale researches were conducted to assure the presence of chemo-preventive or anti-cancer effects in garlic and its closely related allium species like garlic and onion [71]. These research trails have been confirmed that intake of both garlic and onion reduces carcinoma as well as sarcoma risk in different tissues and body organs, like bladder, colon, prostate, lungs, oesophagus, stomach, skin, brain and liver [72]. These effects are not fully understood and their mode of action is not yet completely known. It is stated that garlic and onion exhibit indirect way of anti-carcinogen action by different mechanisms that inhibit carcinogen metabolism, increase the detoxification and facilitate its excretion from the body. Garlic may also effect on cytochrome P450 and inhibit the procarcinogens' activation, alter oxidative damage by their antioxidant action and further stop cell proliferation by inducing apoptosis that inhibit unnecessary cell division. It also helps in chromosomal abbreviation prevention (anticlastogenic effect) [73] and alteration of the cyclooxygenase as well as lipoxygenase activities (anti-inflammatory effects) [74].
It is also investigated from modern research that both water and fat-soluble sulphur containing compounds from garlic supply anticarcinogenic advantages like diallyl sulfide, diallyl sulphoxide, diallyl disulfide, diallyl sulfone, diallyl trisulfide, and SAC. Other sulphur containing compounds, such as SAMC, methiin, ajoene, along with some diallyl disulfide and diallyl trisulfide, can change the mechanism of cell increase by stimulating apoptosis in various human cells like human leukaemic cells. Apart from some organo-sulphur containing compounds, eruboside-B is a crucial steroid saponin which is extracted from garlic bulb as well as some organo-Se containing compounds, and gives the anti-carcinogenic potency to garlic as well as onion. The pure Se containing compounds have been proved as superior anticancer agents than S-analogues, like diallyl selenide that has activity of about 300 times more as compared to diallyl sulfide for the treatment of mammal cancer [13].
The two major Se containing compounds having potential to cure cancer activity in garlic and onion are Se-methyl selenocysteine and g-glutamyl-Se-methyl selenocysteine, but Se-methyl selenocysteine and Se-allyl selenocysteine are the most promising chemopreventive [75]. For quantification of Se-containing compounds, new analytical techniques are required [76]. Advancement in analysis of gene expression along with various functional assays elaborates the significant information on anti-carcinogenic capabilities of all active components. Facts from cDNA array studies explicate the anti-proliferative perspectives of diallyl disulfide that may be due to variation in gene expression of aggrecan 1, cadherin 5, tenascin R and vitronectin [77]. Similarly, it is reported that garlic and its active components response mainly depends on the consumer's genetic backgrounds (nutrigenetic effects), changes in cellular molecular weight within respect to dose (metabolomics effects), DNA methylation, and histone regulation (nutritional epigenetic effects). Identification biomarkers can also be used to determine the response of garlic or other allium foods on reducing cancer burden that is important to develop strategies for the functional food [78].
In addition, in recent days researchers have focused on anti-mutagenic view of garlic, which is an estimate that particular suphur containing compounds like diallyl sulfide, have a positive impact on DNA repair mechanisms [73]. It is concluded that daily recommended intake of garlic and onion provides protection against cancer insurgence. The United States National Cancer Institute accounted garlic is a potential food because of its various functional and nutraceutical aspects with special reference to cancer prevention.
Conclusions
Garlic (Allium sativum L.) is one of the most essential herbaceous vegetables that have been probed against various life sight related disorders. It is helpful in various biological functions due to array of phytochemicals. Garlic nutrition includes manganese, selenium, calcium, vitamins B1 and B6, tryptophan and protein. These components provide synergistic effect against various threats but still need further attention of the researchers. It is assumed that health promoting potential of garlic is mainly attributed to sulfur containing compounds like allicin, citral, geraniol, linalool, phellandrene, s-methyl-1-cysteine sulfoxide. However, pharmacological effectiveness is linked to organosulphur compounds, particularly cysteine sulfoxides and thiosulfinates. Globally, garlic and its various preparations like fresh garlic juice, garlic powder, garlic extract, and garlic oil are becoming popular against numerous physiological menaces. With their unique combination of bioactive moieties, allium vegetables should be in our diet on a regular basis. Both in vivo and in vitro studies have elucidated the potential of garlic against variety of metabolic syndromes. Garlic has wide applications as antioxidant, antifungal, antithrombotic, hypocholestrolemic and hypoglycemic agent. It reduces glucose metabolism in diabetics, slows the development of arteriosclerosis and lowers the risk of various cancers. Furthermore, it reduces the chance of heart attacks in myocardial infarct patients and could also improve the immune dysfunction.
Conflict of interest statement
We declare that we have no conflict of interest.
Background
Functional and nutraceutical foods are becoming popular in all over the world owing to their health promoting perspectives. There are various traditional plants that used as a therapeutic carrier for different physiological threats. Vegetables are enriched sources of these bioactive substances, such as carotenoids, chlorophylls, anthocyanins, and flavonoids. These bioactive molecules have the ability to target at molecular level effectively and their molecular aspects include enzyme kinetics, release of cytokines, and signal transduction. Among the functional foods, use of garlic is mounting day by day because of its health promoting potential besides basic nutrition. The medicinal uses of various herbs have been proven experimentally as lifesaving entities, associated with health claims owing to their rich phytochemicals profile. Health promoting aspects of garlic are mainly accredited to its sulfur containing compounds mainly allicin and SAC. Among organosulfur compounds, thiosulfinates, ajoenes and allicin (diallyl thiosulphate) are the chief bioactive components in garlic.
Applications
This review is helpful for further researcher related to garlic and health perspectives.
Peer review
The present review explored the functional/nutraceutical role of garlic against various threats including dyslipidemia and hyperglycemia, cardiovascular disorders, antioxidant capacity and carcinogenic perspectives. It's looking good and comprehensive. | v3-fos |
2016-05-15T20:03:17.014Z | {
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} | s2 | Tryptophan-Ethylester, the False (Unveiled) Melatonin Isomer in Red Wine
Among the food plants, the presence of melatonin in grapes (Vitis vinifera L.) deserves particular attention because of the production of wine, an alcoholic beverage of economic relevance and with putative healthy effects. Furthermore, melatonin isomers have been detected in wine too. Recently, one of these isomers has been identified as tryptophan-ethylester, a compound with the same molecular weight of melatonin. In this Commentary, we briefly comment the source(s) of tryptophan-ethylester in wine and the putative nutritional role(s).
Introduction
The topic of melatonin in grape products began less than a decade ago, when it was detected, for the first time, in berry skin of Italian and France grapevine varieties (Vitis vinifera L. cv. Barbera, Cabernet Franc, Cabernet Sauvignon, Croatina, Marzemino, Merlot, Nebbiolo, and Sangiovese) grown in north-western Italy. 1 Since then, indoleamine was reported in other grape tissues with varying levels according to both endogenous and exogenous factors, such as genetic traits, phenological stages, environmental and climatic conditions, and agricultural practices ( Table 1). The presence of melatonin was also ascertained in red and white wine produced in different geographical areas (Table 1).
In the field of melatonin research, the occurrence of melatonin isomers in nature represents an emerging topic. 2 Isomers can be classified according to the position of the two side chains present in the indole ring of melatonin, the methoxy (M) group at position 5 and the N-acetylaminoethyl (A) group at position 3. Hypothetically, either one of these two side chains can be relocated to any one of the seven positions in the indole nucleus of melatonin to form isomers. 3 In particular, different isomers were found in grape products, including red wine, even if their chemical structure has not been identified yet. [4][5][6][7] Very recently, in the attempt to determine the conformation of the most abundant (putative) melatonin isomer detected in red wine, we have identified it as tryptophan-ethylester, a compound with the same molecular weight of melatonin paper subject to independent expert blind peer review by minimum of two reviewers. all editorial decisions made by independent academic editor. upon submission manuscript was subject to anti-plagiarism scanning. prior to publication all authors have given signed confirmation of agreement to article publication and compliance with all applicable ethical and legal requirements, including the accuracy of author and contributor information, disclosure of competing interests and funding sources, compliance with ethical requirements relating to human and animal study participants, and compliance with any copyright requirements of third parties. this journal is a member of the committee on publication ethics (cope).
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( Fig. 1). 8 In particular, the concentrations of tryptophanethylester and melatonin in wine were 84 and 3 ng mL -1 , respectively. 8 However, to date, the relationship between concentrations of melatonin and tryptophan-ethylester in grape products is still unknown: it seems that tryptophan-ethylester may arise from a pathway different from the melatonin biosynthetic route, possibly directly from tryptophan.
Amino acid esters readily cross cell membranes because of their lipophilicity and are subject to intracellular enzymatic hydrolysis, thus regenerating the native amino acids. 9 Therefore, dietary tryptophan-ethylester, a lipid-soluble tryptophan derivative, may bypass defective gastrointestinal neutral amino acid transport and be metabolized to melatonin in enteroendocrine cells of the gastrointestinal mucosa. In a child with Hartnup disease (an autosomic recessive metabolic disorder affecting the absorption of nonpolar amino acids, particularly tryptophan), tryptophan-ethylester administration successfully corrected tryptophan deficiency state, and in vitro experiments demonstrated that ester was hydrolyzed by intestinal mucosa, liver, and kidney to provide tryptophan. 10 More recently, in rats, tryptophan-ethylester, but not tryptophan, evoked a rapid and transient dose-dependent decrease in mean arterial pressure and heart rate and significantly promoted vasodilatation in small mesenteric arteries by blocking voltage-operated calcium channels on vascular smooth muscle cells. 11 Noteworthy, to the best of our knowledge, it seems that gut microflora has not the capacity to synthesize tryptophanethylester or melatonin isomers. However, the exact contribution of grapes to melatonin, melatonin isomers, and tryptophan-ethylester in wine has not entirely been elucidated yet, and a pivotal role of yeasts and, possibly, of bacteria in the production of these metabolites in wine has been suggested. In a pioneering paper, Sprenger and colleagues demonstrated that, in Saccharomyces cerevisiae, melatonin is synthesized and metabolized to other 5-methoxylated indoles (5-methoxytryptamine and 5-methoxytryptophol). 12 In other yeast species, ie, Saccharomyces uvarum and S. cerevisiae var. bayanus, melatonin production in synthetic grapes must depend on growth conditions and medium, including tryptophan concentration. 13 Though no information is available on tryptophan-ethylester, the ability of yeasts to enrich indoleamine-fermented foods and beverages different from wine is corroborated by a number of studies. [14][15][16][17][18][19] In this view, these microorganisms may also contribute to the biosyn thesis of tryptophan-ethylester in wine. Our preliminary results showed that high levels of tryptophan-ethylester are produced by yeasts in enological conditions. 20 In conclusion, as the source(s) of tryptophan-ethylester in wine is(are) still unknown, we can only speculate on the putative nutritional role(s) of this compound: it may provide a pool of tryptophan able to cross the gastrointestinal tract and, possibly, the blood-brain barrier; then, de-esterified tryptophan-ethylester may be rapidly metabolized in target cells like enteroendocrine cells, which produce serotonin and melatonin, two paracrine and endocrine factors. 21 However, we have to take into account that pharmacologically active tryptophan-ethylester concentrations (5-20 mg/kg and higher) are unlikely to be reached in wines, at least based on current knowledge; therefore, we solicit further studies to quantify tryptophan-ethylester in a range of wines and grapes. | v3-fos |
2018-04-03T02:32:36.234Z | {
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} | s2 | Soil Fertility, Phosphorus Fractions, and Maize Yield as Affected by Poultry Manure and Single Superphosphate
A field experiment was conducted in 2007 and 2008 on a slightly acidic alfisol. Poultry manure (PM) was applied at 0, 5 t ha−1, 10 t ha−1, 15 t ha−1, and 20 t ha−1 in combination with SSP at 0, 15 kg P ha−1, 30 kg P ha−1, 45 kg P ha−1, and 60 kg P ha−1, which was replicated three times. The pH and organic C were significantly increased by the application of PM alone while available P was highly increased by the sole application of SSP. Plant tissue P was significantly increased with the application of 30 kg P ha−1 while the largest grain yield was obtained when PM at 20 t ha−1 was combined with SSP at 60 kg P ha−1. The buildup of organic P was observed when PM was applied at 15 t ha−1 while the combination of the two treatments increased residual P and Fe-P. However, P occlusion was effectively reduced with the sole application of PM. Organic P and residual P however had a strong positive relationship with the grain yield. Comparing the sole and combined application of the treatments, the combined application was more effective for most of the parameters observed.
Introduction
Phosphorus (P) is one of the essential elements for plant growth and the number of plants available P in the soil is often inadequate to meet plant requirements. Phosphorus is a macronutrient that plays a number of important roles in plant. Adequate phosphorus results in higher grain production, improved crop quality, greater stalk strength, increased root growth, and earlier crop maturity. Mokwunye et al. [1] and Warren [2] stated that phosphorus deficiency is one of the largest constraints to food production in tropical African soils due to low native P and high fixation by iron and aluminum oxides. It has also been established that phosphorus is relatively unavailable for plant uptake in highly weathered soils like the Ultisols [3]. In order to meet the need of phosphorus in these soils, farmers make use of inorganic fertilizers for their crops. However, P fertilizer is not readily available to these farmers because of scarcity in some cases and also due to the high cost of these fertilizers. Recently, farmers especially in the developed countries make use of organic manure which has been found to be effective like inorganic fertilizers for the release of some nutrients especially phosphorus but the resultant yield has been found not to be as high as when inorganic fertilizer is combined with the organic manure. Combining organic manure with P fertilizers has been found to not only increase yield but also reduce P fixation and subsequently increase phosphorus availability and uptake [3]. The study however aims at studying the effect of combined application of poultry manure and single superphosphate on phosphorus forms, soil fertility, and the yield maize.
Materials and Methods
The field experiment was conducted at I.A.R&T experimental field at Ibadan in 2007 and 2008 early planting season (see Table 1). The experimental design was a 5 × 5 factorial experiment in a RCBD replicated three times. The plot size was 5 m × 4 m (20 m 2 ) with spacing of 75 cm × 50 cm to give a total plant population of 106,666 plants ha −1 . Three seeds of maize (TZB-SR) were planted after 2 weeks of treatment incorporation. The seedlings were thinned to two per stand 2 International Scholarly Research Notices Organically stable organic and inorganic P two weeks after planting. The plots were not tilled in the early season (May) for the second year (2008) in order to avoid mixing up of treatments. Gramoxone and primextra were used as herbicides. Weeding was done at the 4th and 8th week after planting.
Leaf sampling of maize was done at eight weeks after planting. Leaf just below and opposite the ear was sampled randomly per plot. Ears of maize were allowed to dry on the stalk. Ears in the four middle rows of each plot were harvested (making allowance for two guard rows) on each side of the plot. The cobs were dehusked and weighed. Ten cobs per plots were randomly selected for cob weight measurement. The cobs were sun dried and the grains were weighed. Yield per hectare was computed at 12% moisture content for maize. Soil samples were analyzed before and after for phosphorus fractions and soil nutrient composition.
Soil Physical and Chemical Analysis.
Particle size distribution was determined by the hydrometer method [4] using sodium hexametaphosphate as the dispersing agent. Soil pH was determined in distilled water (1 : 1 soil water ratio) with a pH meter. Exchangeable cations (K, Na, Ca, and Mg) by extraction, with ammonium acetate (pH = 7). Available P was extracted with 0.03 N NH 4 F in 0.025 N HCl solution [5] and P in the extract was analysed colorimetrically by the molybdenum blue method at 660 nm. Soil organic carbon was determined by wet oxidation with sulphuric acid [6]. Total P was determined by wet digestion using perchloric acid and nitric acid while organic P was by ignition method. Phosphorus concentration in the digests was determined using the ascorbic acid method.
Phosphorus Fractionation Study.
Fractionation procedure by Chang and Jackson [7] was used to quantify different forms of inorganic P in the studied soils (Table 2). Fractionation was done on the soil before and after amendment for incubation, greenhouse, and field experiment. The sequence of the chemical extraction and a brief description of the targeted P forms are shown in Table 2.
Determination of Phosphorus in the Extracts.
The phosphorus in the extracts was determined by the methyl blue colour method of Murphy and Riley [8]. An aliquot of 10 mL was used in developing the colour. The determination of P in NH 4 F, NaOH, and DCB extracts however requires the following pretreatment before the development of the colour.
(a) NH 4 F extract: 0.05 g of H 3 BO 3 per 50 mL of the extract was added before taking the aliquot.
(b) NaOH extract: after precipitating the organic matter with conc. H 2 SO 4 , the pH of the extract was adjusted with NaOH using 2,4-dinitrophenol indicator. An aliquot of the clear solution was then taken into a 50 mL volumetric flask.
(c) DCB extract (reductant soluble P): 10 mL of the extract was pipetted into a test tube and 1 mL of HClO 4 was added to it and boiled to destroy the dithionite in the extract. It was then transferred into a 50 mL volumetric flask and 3 mL of 5% ammonium molybdate solution was added while distilled water was used to make up to mark. Absorbance was read after 30 minutes.
International Scholarly Research Notices 3
Statistical Analysis.
The data collected were subjected to analysis of variance using the statistical analysis system (SAS)-general linear model [10]. Means were separated by Duncan multiple range test.
Initial Chemical Properties of the Soil Used.
The pH of the soil was slightly acidic and therefore a need for the application of organic manure. The soil was low in N, Na, and organic carbon while P was moderately available in it. It was however adequate with cations like Ca, Mg, and K (Table 3).
Characterization of the Poultry Manure Used and the Initial Phosphorus Fractions of the Soil.
Nitrogen in the poultry manure used was 5.82% which was similar to the result observed for phosphorus (5.54%) while potassium was low in the soil (0.94%). Organic C had a high percentage of 13.38% which led to the resultant C/N ratio of 2.3 (Table 4). Also notable was the result obtained for Fe (1555 mg/kg). The order of abundance of the phosphorus fractions before the application of treatments was organic P > Fe-P ≡ occluded P > residual P (Table 5).
Effect of Combined Application of Poultry Manure and SSP on the Soil Chemical Properties.
There was a decrease in pH as a result of the sole application of SSP in 2007, which further decreased in 2008 ( Table 6). Application of poultry manure alone increased the pH of the soil significantly when compared to the other treatments. However, the combined application of poultry manure and SSP increased the pH of the soil in 2007 with a further increase in 2008. In other words, poultry manure alone can be compared favorably with the combined application of poultry manure and SSP in increasing soil pH especially in slightly acidic soil. Effect of SSP on organic C was not significant and decreased when compared to the initial value. However, the application of poultry manure increased organic C significantly when poultry manure was applied alone in 2007. In 2008, organic C further increased with the sole application of poultry manure. The effect of the combined application of poultry manure and SSP was not significant on organic carbon in 2007 but significant in 2008 with the combined application of poultry manure and SSP. Available P increased over the control either with the sole application of poultry manure or SSP or when the two treatments were combined. However, in 2007 the sole application of SSP was more effective in increasing available P than the sole application of poultry manure but was the most effective with the combined application of poultry manure and SSP, while, in 2008, the combined treatments were not significant but the sole application was.
Effect of the Combined Application of Poultry Manure and SSP on Plant Tissue P Concentration and Grain Yield of
Maize. Effect of poultry manure and SSP alone with their combinations was significant in both years (Table 7). In 2007, plant tissue P was high when the treatments were applied solely but combined application was highly significant. Plant tissue P continued to increase in 2008 with the sole and combined application but with the most significant increase with the sole application of SSP. The sole application of SSP had no significant effect on the grain yield of maize in 2007 while the sole application of poultry manure and the combined application of the two treatments were significant in the same year. However, in 2007, the highest grain yield of maize was obtained by the sole application of poultry manure. In 2008, effect of the sole application of poultry manure and SSP on maize grain yield was significant while the combined application was not. The combined application of the two treatments gave the highest grain yield in 2008. (Table 8). In 2007, organic P was significantly increased over the initial value when poultry manure was applied solely than when SSP was applied alone. Combining the two treatments also increased organic P significantly in 2007. The application of poultry manure alone however gave the highest increase in organic P in 2007. In 2008, organic P reduced with the sole application of SSP but increased when poultry manure was applied alone and with some combined treatments. The sole application of poultry manure was again effective in 2008 in increasing organic P. Residual P increased over the control when poultry manure and SSP were applied alone and when combined in both years. Residual P increased in 2007 with the combined application of the two treatments but reduced in 2008. A similar trend to that of residual P was observed for Fe-P but with few exceptions when the 2 treatments were combined. In 2008, the largest buildup of Fe-P was observed with the combined application of poultry manure and SSP. In both years of cropping, sole application of poultry manure was effective in reducing P occlusion more than the combined application.
Effect of the Combined Application of Poultry
International Scholarly Research Notices 5
Relationship among the P Fractions, Soil Chemical Properties, and Maize Grain Yield.
In 2007, the pH of the soil had a strong negative relationship with Al-P and occluded P but positive relationship with residual P (Table 9), while in 2008, increase in the pH of the soil favored the buildup of Fe-P (Table 10). Available P in 2007 had a positive correlation with organic P, Ca-P, and Fe-P but a negative correlation with occluded P. Organic C had no significant relationship with the P fractions in 2007 but a positive correlation with organic P and a negative correlation with occluded P in 2008. Organic P and residual P contributed positively to the maize grain yield in both 2007 and 2008.
Initial Chemical Properties of the Soil Used.
The pH of the soil was slightly acidic and this will make Fe-P be more available than the other inorganic P fractions. Although phosphorus was moderately available, poultry manure and SSP were further applied in order to study the effect of continuous application of organic and inorganic fertilizer on such soils yearly as has been practiced by many farmers in Nigeria.
Characterization of the Poultry Manure Used and Initial
Phosphorus Fractions of the Soil. A common and traditional practice has utilized manure as an organic fertilizer or soil improver due to its contribution of organic matter and nutrients, mainly N and P [11]. The high percentage of nitrogen in the poultry manure used could have been due to the decomposition of poultry manure during the curing stage before application to the soil with a subsequent release of N which was detected during the analysis. Manure in past studies has been applied according to the nitrogen needs of the soil [12]. Macronutrients such as phosphorus are part of the advantage of using organic waste in agriculture, which may be recovered and incorporated into natural cycle and this was evident in the percentage of phosphorus in the poultry manure used. It also contains a high level of organic matter SSP * * * * which generates changes in chemical, physical, and biological soil amendment properties [12]. Organic P was the highest P fraction at the initial stage of this study. About two-thirds of the phosphorus in fresh manure is in organic form [12]. The organic P pool is usually considered a major source of organic phosphorus and can be converted to inorganic P by ignition or wet digestion. Initial abundance of Fe-P could be attributed to the acidic nature of the soil; however occluded P also had the same value which could mean there is an equilibrium reaction between the labile and the nonlabile P fractions.
Effect of Combined Application of Poultry Manure and SSP on the Soil Chemical
Properties. Acidity in sols if not properly managed will eventually make the soil be low or not yielding and therefore a need for amendment. In this study, application of poultry manure compared favourably with the combined application of poultry manure and SSP in increasing the pH of the soil while applying inorganic fertilizer alone decreased the soil pH. Soil pH of an acidic Ultisol in the eastern Nigeria was observed to increase soil pH [13]. Sole and combined organic and inorganic fertilizer has been found to increase soil nutrients favorably. The combined application of the two treatments was highly effective in increasing soil organic C in both 2007 and 2008. Earlier studies on poultry manure ability to increase soil organic C have been reported [13,14], likewise when it is combined with SSP [15]. Single superphosphate was however more effective than the sole application of poultry manure for increasing the available P content of the soil although the combined application of the two treatments still gave the highest value [16]. The effectiveness of SSP for available P over poultry manure could be due to the immediate release of phosphate into the soil when SSP is being applied.
Effect of the Combined Application of Poultry Manure and SSP on Plant Tissue P Concentration and Grain Yield of
Maize. High plant tissue P was obtained in 2007 and 2008 with the sole and combined application of poultry manure and SSP. However, the sole application of SSP was however sustainable in the 2nd year to cause a further increase in the concentration of P in the shoot. A similar result has been observed by Adeniyan and Ojeniyi, [17]. The increase in the plant tissue P in 2008 was as a result of P accumulation observed in both years, which would have resulted in the transfer to the shoot of the plant instead of being washed into nearby river and hereby preventing eutrophication.
When the treatments were applied solely, application of poultry manure alone was more effective than SSP, that is, considering the high grain yield. Although this was in the first year of planting, the combined application of poultry manure and SSP was again sustainable in the second year. The combined application of organic and inorganic resources has been found to favor maize production [14].
Effect of the Combined Application of Poultry Manure and SSP on Phosphorus
Fractions. The buildup of organic P was observed in both years of this study with the sole application of poultry manure and this signifies the importance of organic manure when considering amendments for an acidic soil. Addition of soybean residue and wheat residue or in combination with fertilizer P favored the buildup of organic P in a study [18]. The inorganic P fractions considered, that is, residual P and Fe-P, was significantly increased when poultry manure was combined with SSP. This indicates the ability of the combined application of poultry manure and SSP to maintain the fertility of the soil through increase in the labile P fraction [19]. Residual P however reduced in 2008 and this was also observed by Agbenin and Goladi, [20] in an experiment involving the addition of P fertilizers or in combination with cow manure. Significant was the ability of poultry manure alone to reduce P occlusion in both years, that is, occluded P, which further confirms an earlier study which states that addition of organic materials to soil decreases P fixation [3]. Inorganic and organic products are generated during the partial decomposition of organic waste and organic acids can be adsorbed into the soil surface thereby decreasing the potential P adsorption by blocking the formation of complexes [21]. 4.6. Relationship among the P Fractions, Soil Chemical Properties, and Maize Grain Yield. Organic P and Fe-P contributed significantly to the increase observed in available P in 2007 while occluded P decreased with an increase in available P. This further confirms the importance of poultry manure while considering soil amelioration. In both years of cropping, organic P was sustainable in increasing grain yield but Fe-P was only effective in 2007. Residual P (a nonlabile P fraction) equally contributed to the increase observed in grain yield in both years. In other words, there would have been an equilibrium reaction between the nonlabile P (residual P) and labile P (Fe-P) fraction which would have subsequently resulted in an increase in available P.
Conclusion
Application of poultry manure alone was effective in increasing the pH, organic C, and available P content of the soil. However, application of SSP was highly significant in increasing the available P content of the soil and this was evident in the increase obtained for the plant tissue P in 2008, while, for grain yield, the most effective was the combined application of poultry manure and SSP. Increase in organic P fraction was as a result of the application of poultry manure while the combined application of poultry manure and SSP increased the Fe-P fraction. Poultry manure alone was also the most effective treatment for the reduction of P occlusion in the soil. Significant were also the importance of poultry manure and the combined application of poultry manure and SSP exhibited through the strong relationship that existed among available P, Fe-P, and organic P. Although poultry manure was compared favorably with the combined application of poultry manure and SSP, the combination of the treatments was more suitable for the sustainability of the fertility of the slightly acidic soil. | v3-fos |
2016-05-12T22:15:10.714Z | {
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} | s2 | Distillation Time as Tool for Improved Antimalarial Activity and Differential Oil Composition of Cumin Seed Oil
A steam distillation extraction kinetics experiment was conducted to estimate essential oil yield, composition, antimalarial, and antioxidant capacity of cumin (Cuminum cyminum L.) seed (fruits). Furthermore, regression models were developed to predict essential oil yield and composition for a given duration of the steam distillation time (DT). Ten DT durations were tested in this study: 5, 7.5, 15, 30, 60, 120, 240, 360, 480, and 600 min. Oil yields increased with an increase in the DT. Maximum oil yield (content, 2.3 g/100 seed), was achieved at 480 min; longer DT did not increase oil yields. The concentrations of the major oil constituents α-pinene (0.14–0.5% concentration range), β-pinene (3.7–10.3% range), γ-cymene (5–7.3% range), γ-terpinene (1.8–7.2% range), cumin aldehyde (50–66% range), α-terpinen-7-al (3.8–16% range), and β-terpinen-7-al (12–20% range) varied as a function of the DT. The concentrations of α-pinene, β-pinene, γ-cymene, γ-terpinene in the oil increased with the increase of the duration of the DT; α-pinene was highest in the oil obtained at 600 min DT, β-pinene and γ-terpinene reached maximum concentrations in the oil at 360 min DT; γ-cymene reached a maximum in the oil at 60 min DT, cumin aldehyde was high in the oils obtained at 5–60 min DT, and low in the oils obtained at 240–600 min DT, α-terpinen-7-al reached maximum in the oils obtained at 480 or 600 min DT, whereas β-terpinen-7-al reached a maximum concentration in the oil at 60 min DT. The yield of individual oil constituents (calculated from the oil yields and the concentration of a given compound at a particular DT) increased and reached a maximum at 480 or 600 min DT. The antimalarial activity of the cumin seed oil obtained during the 0–5 and at 5–7.5 min DT timeframes was twice higher than the antimalarial activity of the oils obtained at the other DT. This study opens the possibility for distinct marketing and utilization for these improved oils. The antioxidant capacity of the oil was highest in the oil obtained at 30 min DT and lowest in the oil from 360 min DT. The Michaelis-Menton and the Power nonlinear regression models developed in this study can be utilized to predict essential oil yield and composition of cumin seed at any given duration of DT and may also be useful to compare previous reports on cumin oil yield and composition. DT can be utilized to obtain cumin seed oil with improved antimalarial activity, improved antioxidant capacity, and with various compositions.
Introduction
Cumin (Cuminum cyminum L.) is an annual herbaceous plant belonging to family Apiaceae. The plant originates from the Mediterranean region; however, it is presently grown in many regions across the world as a spice and essential oil crop [1,2]. Due to its unique, pleasant warm and spicy aroma, cumin has been used as spice since ancient times; ancient Greek and Roman cultures utilized it as frequently as pepper and salt are used today. Cumin seed is an essential and main ingredient in many spice mixes or cooking powders, including curry powder, chilli powder, sambar powder, and others [1]. Currently, cumin seed is produced in India, Pakistan, and in most Mediterranean, Eastern European, and in Latin American countries [1,3]. Cumin seed contains both essential and fatty oil [1,4]. Cumin essential oil (Oleum cumini) is a yellowish fluid with a strong sweet-spicy aroma and is utilized as an aromatic ingredient mainly in the food and liquor industry and to a limited extent in perfumery, cosmetics and pharmaceuticals. Both cumin seed and cumin essential oil have medicinal properties such as antioxidant, cholesterol lowering, and antimicrobial properties [5,6]. The essential oil of cumin was also reported to have fumigant toxicity against pulse beetle, Callosobruchus chinensis [7]. Consequently, cumin seed and cumin essential oil have been utilized in various homebased remedies, especially in the Mediterranean and Asian cultures [1,3,5,8,9].
The essential oil of cumin is traditionally extracted from cumin seed via steam distillation [2]. However, various authors reported cumin oil composition following different durations of the steam distillation time (DT). There is no agreement in the literature regarding the optimum duration of the DT for maximum oil yield. In addition, it is not clear if the duration of the distillation time would affect cumin essential oil bioactivity or composition. Recently, [10] reported that DT had a significant effect on oil yield and composition of anise seed (Pimpinella anisum L.), a plant from the same family. The seeds (fruits) of the two plant species (cumin and anise) has very similar essential oil secretory ducts (vittae). Therefore, our working hypothesis was that the duration of the steam DT would affect essential oil yield, composition, and the bioactivity of cumin seed essential oil.
Steam distillation and the duration of the steam distillation times (DT)
The cumin seed essential oil was extracted via steam distillation of whole seed using 2-L steam hydrodistillation units (Heartmagic, Rancho Santa Fe, CA) as described previously for anise [10]. The experiment was conducted at the University of Wyoming Sheridan Research and Extension Center in 2013, whereas the essential oil analyses were performed at the USDA-ARS, Natural Product Utilization Research Unit located at the University of Mississippi. The certified bulk cumin seed used in this study originated from India, and was purchased from Starwest Botanicals (Rancho Cordova, CA, U.S.A.).
Ten different distillation times (DT) were performed in this study: 5, 7.5, 15, 30, 60, 120, 240, 360, 480, and 600 min. These DT were based on preliminary studies conducted by the authors, and on literature reports [2]. All DT were performed in 3 replicates, resulting in 30 separate distillations. Each individual sample extracted consisted of 900 g of cumin seed, in order to provide sufficient oil for analyses and for biological evaluations.
At the end of each DT, the cumin oils were collected in glass vials, separated from water, measured on an analytical scale and kept in a freezer until the gas chromatography analyses could be performed. Cumin oil yield (content) was expressed as g of oil per 100 g of cumin seed.
Gas Chromatography Flame Ionization Detection (GC-FID) Essential Oil Quantitative Analysis
Oil samples were analyzed by GC-FID on a Varian CP-3800 GC equipped with a DB-5 fused silica capillary column (30 m × 0.25 mm, with a film thickness of 0.25 μm) operated using the following conditions: injector temperature, 240°C; column temperature, 60-120 at 3°C/min, then held at 240°C at 20°C/min for 5 min; carrier gas, He; injection volume, 1 μL (split on FID, split ratio 50:1); FID temperature was 300°C.
Commercial standards (-)-β-pinene, (-)-α-pinene, γ-terpinene and cuminaldehyde were obtained from Aldrich (Sigma-Aldrich, Spain) and p-cymene was obtained from Fluka (Buchs, Switzerland). With seven concentration points, a least squares regression for quantification was used. Each specific analyte was used to formulate a separate calibration curve using FID response data except for α-terpinene-7-al and β-terpinene-7-al, which were quantitated using the response factor for cumin aldehyde. Linearity was imposed by using response factors (RF) and regression coefficients independently. Response factors (RF) were calculated using the equation RF = DR/C, where DR was the detector response in peak area (PA) and C was the analyte concentration. The chromatograms of each of the essential oil samples from the field experiments were compared to the chromatograms from standards. Target analytes were confirmed by retention time. Confirmed integrated peaks were used to determine percentage of each chemical constituent in the essential oil itself. The RF of the target chemical constituent was used to determine the individual analyte percentage for each sample using the equation PA/RF/C × 100 = % analyte in the oil on a wt (analyte)/wt (oil) basis.
Antioxidant capacity of the oils from different hydrodistillation times (HDT)
The antioxidant capacity of cumin seed oil extracts from 15, 30, 60, 120, 240, 360, 480, and 600 min were measured at the University of Nebraska-Lincoln, Small Molecule Analysis Laboratory, by the oxygen radical absorbance capacity (ORAC oil ) method [11,12]. The specifics of the method utilized at the University of Nebraska-Lincoln were detailed previously [10]. Trolox, (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a polar derivative of Vitamin E, was used as a standard, therefore the results are reported as μmole Trolox g -1 . The 5 and 7.5 min DT did not yield sufficient oil quantity for antioxidant activity. Each of the oils from the above 8 DT was analyzed in triplicate, hence generating 9 readings for each treatment.
Antimalarial activity testing of the cumin oils from various DT
The antimalarial activity of the cumin essential oils from the 5, 7.5, 15, 30, 60, 120, 240, 360, 480 and the 600 min DT treatments (all in two replicates) was tested using a method described previously [13] at the National Center for Natural Product Research (NCNPR), The University of Mississippi, University, MS. Additionally, in a secondary late assay, we subjected the pure compounds p-cymene, cuminaldehyde, (-)-beta-pinene, (-)-alpha-pinene, and gamma-terpinene to the same antimalarial assay.
Antimicrobial activity testing
The antimicrobial testing of the cumin oils was also performed at the NCNPR, University, MS. The primary screening for antimicrobial activity of cumin essential oils obtained from the 5, 7.5, 15, 30, 60, 120, 240, 360, 480 and the 600 min DT treatments (all in two replicates) were tested for antifungal activity against Candida albicans, C. glabrata, C. krusei, Aspergillus fumigatus, Cryptococcus neoformans and antibacterial potential against gram +ve bacteria Staphylococus aureus, methicillin-resistant S. aureus and Mycobacterium intracellulare and gram-ve bacteria Escherichia coli and Pseudomonas aerogenosa at a concentration of 50 μg/ml, and % inhibition was calculated following the method published previously [13]. The antifungal activity was tested using ampothericin B, the antibacterial drug control was ciprofloxacin.
Statistical analyses
The effect of distillation time on oil content (%), and the concentration (%) and yield (mg/100 seed) of α-pinene, β-pinene, γ-cymene, γ-terpinene, cumin aldehyde, α-terpinen-7-al, and βterpinen-7-al, as well as ORAC (uM Trolox equiv./g) was determined using a one-way analysis of variance. For each response, the validity of model assumptions was verified by examining the residuals as described in literature [14]. The oil content and all yield responses required a transformation to achieve normality assumption, however, the averages shown in the tables are back-transformed to the original scale. Since the effect of distillation time was significant (pvalue < 0.05) on all responses, multiple means comparison was completed using Duncan's multiple range test at the 5% level of significance, and letter groupings were generated. The analysis was completed using the GLM Procedure of SAS [15].
The relationships between distillation time (DT) and oil content (%), and between DT and the yield of β-terpinen-7-al were adequately modelled by the Michaelis-Menten model (Eq 1); and the relationships between DT and the concentrations of α-pinene, β-pinene, γ-terpinene, cumin aldehyde, and α-terpinen-7-al, as well as the relationships between DT and the yields of α-pinene, β-pinene, γ-cymene, γ-terpinene, cumin aldehyde, and α-terpinen-7-al were adequately modelled by the Power model (Eq 2). The relationships between DT and the concentration of γ-cymene and ORAC were very week to describe them by a regression model. The parameters of these nonlinear models (Michaelis-Menten and Power) were estimated iteratively using the NLIN Procedure of SAS [15] and the fitted models met all adequacy requirements [16].
Where Y is the dependent (response) variable, X is the independent (DT) variable, and the error term ε is assumed to have normal distribution with constant variance. Validity of the normality, constant variance and independence assumptions on the error terms were verified by examining the residuals [16].
Effect of distillation time (DT) on oil profile
DT had significant effects on all of the measured responses, and especially on oil profile (Fig 1). Maximum oil yields were obtained at 480 min DT; further increase of DT to 600 min did not result in significant increase of oil yields (Table 1, Fig 2). Generally, the concentrations of α- pinene, β-pinene, γ-cymene, γ-terpinene in the oil increased with the increase in the duration of the DT. The concentration of α-pinene was highest in the oil obtained at 600 min DT (Table 1). Maximum concentrations of β-pinene and γ-terpinene were reached in the oil at 360 min DT; further increase of DT did not significantly change the concentration of these two compounds in the oil (Table 1, Fig 2). The concentration of γ-cymene reached its maximum in the oil at 60 min DT; further increase in the duration of the DT did not bring a corresponding increase of γ-cymene in the oil. The concentration of cumin aldehyde (major cumin oil oil constituent, 50-66% concentration range) was high in the oils obtained at 5-60 min DT, and low in the oils obtained at 240-600 min DT (Table 1, Fig 2). The concentration of α-terpinen-7-al (3.8-15.8% range) was low at shorter DT, increased with the increase of DT and reached maximum in the oils obtained at 480 or 600 min DT. The concentration of β-terpinen-7-al (11.6-20.4% range) was low in 5-15 min DT, reached maximum concentration in the oil at 60 min DT, and decreased after 240 min DT (Table 1, Fig 2).
Effect of distillation time (DT) on the yield of individual oil compounds
The yield of individual oil compounds was calculated from the oil yield and the concentration of given compound in the oil at each DT. Overall, the yield of all essential oil compounds increased with an increase of the duration of the DT and reached maximum at either 600 min DT (α-pinene, β-pinene, γ-cymene, γ-terpinene, and α-terpinen-7-al), or at 480 min DT (cumin aldehyde), or at 360 min DT (β-terpinen-7-al) ( Table 2, Fig 3).
Effect of distillation time (DT) on antioxidant capacity and on antimicrobial activities
The antioxidant activity of the oils from this study varied from 202 uM Trolox equiv./g (in the oil from 360 min DT) to 338 uM Trolox equiv./g (in the oil from the 30 min DT) ( Table 2). Cumin oils obtained during the 0-5 and 5-7.5 min DT timeframes showed high antimalarial activity; 77 and 75% respectively, suppression of Plasmodium falciparum ( Table 2). The antimalarial activity of these oils was significantly higher (twice as much) compared to the antimalarial activity of the oils from the other DT (Table 2). However, in a later assay, the pure commercially available compounds did not show antimalarial activity against P. falciparum up Table 1. Mean oil content (%), and the concentration (%) of α-pinene, β-pinene, γ-cymene, γ-terpinene, cumin aldehyde, α-terpinen-7-al, and β-terpinen-7-al obtained from the 10 distillation times (DT).
DT (min)
Oil content α-pinene β-pinene γ-cymene γ-terpinene cumin aldehyde α-terpinen-7-al β-terpinen-7-al to a concentration of 12 μg/ml (which was 2.5 times higher than our routine testing concentration for pure compounds). The antimicrobial activity of the cumin oils from various DT was not significant against most of the organisms with the exception of Cryptococcus neformans. The cumin oils obtained at 0-5 and at 0-7.5 min showed above 50% activity against Cryptococcus neformans in the primary screening. The activity of the cumin oils obtained from the later DT showed between 20 and 50% activity against Cryptococcus neformans.
Nonlinear regression analysis of the data revealed that the relationship between DT and oil content can be described by the Michaelis-Menton model (Fig 2). The relationship between DT and β-terpinene-7-al yield was also very well described by the Michaelis-Menton model (Fig 3). The parameters shown in the last plot of Fig 3 suggest that the maximum β-terpinene-7-al yield that can be achieved is 412 mg, and the distillation time required to achieve half of this maximum yield is 202 min. As shown in Figs 2 and 3, the other relationships were described by the Power model.
Discussion
This is the first report on the effect of DT on cumin whole seed oil yield, oil composition, on cumin oil antimalarial activity, and on cumin oil antioxidant activity. DT has been recently shown to significantly change essential oil yield and composition of oil from anise seed [10], from biomass of crops from other families such as peppermint (Mentha ×piperita L.), lemongrass (Cymbopogon flexuosus Steud.), palmarosa (Cymbopogon martinii Roxb.) [17] and from lavender [18], among other crops.
The concentration of the seven major oil constituents (α-pinene, β-pinene, γ-cymene, γ-terpinene, cumin aldehyde, α-terpinen-7-al, and β-terpinen-7-al) varied significantly as a function of the DT, instead of their boiling points. Although it may seem logical that concentrations of constituents would vary as a function of their boiling points, there are many additional factors that have a greater influence on final concentration and yield. This is not a true distillation of the oil but rather steam at 100 C is passed through the plant material volatilizing these constituents. Furthermore, the constituents in the tissue may be available for extraction by the steam to varying degrees creating these differences in concentration unrelated to boiling point. This is a distillation process that mimic the process employed by the commercial oil extraction facilities. Table 2. Mean yield (mg/100 seed) of α-pinene, β-pinene, γ-cymene, γ-terpinene, cumin aldehyde, α-terpinen-7-al, and β-terpinen-7-al, antioxidant capacity, ORAC (uM Trolox equiv./g), and antimalarial activity (% inhibition) of the cumin seed oils obtained from the 10 distillation times (DT).
DT (min) α-pinene β-pinene γ-cymene γ-terpinene cumin aldehyde α-terpinen-7-al β-terpinen-7-al ORAC Antimalarial activity In this study, the oil from 600 min DT had the highest concentration of α-pinene compared to the oils from the other DT. The oils obtained at 360 min were characterized with the highest concentrations of β-pinene and γ-terpinene. The oil at 60 min had the highest concentration of γ-cymene compared to the other oils. The concentration of cumin aldehyde (the main oil constituent) was high in the oils obtained at 5-60 min DT, and low in the oils obtained at 240-600 min DT. Furthermore, α-terpinen-7-al reached maximum in the oils obtained at 480 or 600 min DT, whereas β-terpinen-7-al reached maximum concentration in the oil at 60 min DT. Generally, the yields of all oil constituents increased with the increase in DT and reached their respective maximums at 480 or 600 min DT, which was due to increase in essential oil yield and the increased concentration of some individual compounds in the oil.
The cumin seed oils in the 5 and in the 7.5 min DT had twice the antimalarial activity of the oils from the other DT. This opens the possibility for distinct marketing and utilization for these improved oils. Currently, the plant that is used for malaria treatment is Artemisia annua L. (Asteraceae), because it contains the active ingredient artemisinin, which has proven antimalarial activity. Artemisia annua is currently the only commercial source for production of artemisinin-based combination therapies (ACT) [19,20]. It is estimated that around 2 million doses of artemisinin from A. annua are needed on yearly basis to be used as the first line of defense against multi-drug-resistant Plasmodium falciparum malaria, as recommended by the World Health Organization [20]. Cumin seed oil with high antimalarial activity may be a welcoming new antimalarial agent. Indeed, there is a pending patent on the use of cumin seed powder and cumin oil as capsules for malaria treatment [21]. However, in this study we describe a method that can produce cumin oil with twice as much inhibition power than the common cumin oil. The cumin oil with improved antimalarial activity may render greater acceptance and utilization as it comes from a spice, edible plant.
In a follow up later assay, the pure commercially available compounds (p-cymene, cuminaldehyde, (-)-β-pinene, (-)-α-pinene, and γ-terpinene) did not show antimalarial activity against P. falciparum. Therefore, minor constituents are likely responsible for the antimalarial activity. A full bioassay-directed fractionation will be performed but it is the subject of a more intensive study and not part of this study.
DT in this study affected the antioxidant activity of cumin seed oil, with the highest antioxidant capacity of the oil obtained at 30 min DT and the lowest in the oil from 360 min DT. Recent studies found that DT affected antioxidant activity of oils of other crops such as wormwood, Artemisia annua [22], fennel, Foeniculum vulgare Mill. biomass [23], Rocky mountain juniper, Juniperus scopulorum Sarg. [24]. Hence, when antioxidant activity of a certain oil is reported, DT must also be reported.
The regression models developed in this study can be utilized to predict essential oil yield and composition of cumin seed at any given duration of DT. The regression models could also be useful to compare data from reports on cumin oil yield and composition. This study demonstrated DT can be utilized to obtain cumin seed oil with various compositions and bioactivity from the same batch of seed.
University of Wyoming Outreach School and the Department of Plant Science allocations to Valtcho D. Jeliazkov (V.D. Zheljazkov).
Author Contributions
Conceived and designed the experiments: VZ. Performed the experiments: VZ. Analyzed the data: TA. Contributed reagents/materials/analysis tools: AG CC TA VZ VS. Wrote the paper: VZ AG CLC TA VS. Writing and editing of the manuscript: VZ AG CLC TA VS. | v3-fos |
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