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Optimal Cultivation Time for Yeast and Lactic Acid Bacteria in Fermented Milk and Effects of Fermented Soybean Meal on Rumen Degradability Using Nylon Bag Technique

The objectives of this study were to determine an optimal cultivation time for populations of yeast and lactic acid bacteria (LAB) co-cultured in fermented milk and effects of soybean meal fermented milk (SBMFM) supplementation on rumen degradability in beef cattle using nylon bag technique. The study on an optimal cultivation time for yeast and LAB growth in fermented milk was determined at 0, 4, 8, 24, 48, 72, and 96 h post-cultivation. After fermenting for 4 days, an optimal cultivation time of yeast and LAB in fermented milk was selected and used for making the SBMFM product to study nylon bag technique. Two ruminal fistulated beef cattle (410±10 kg) were used to study on the effect of SBMFM supplementation (0%, 3%, and 5% of total concentrate substrate) on rumen degradability using in situ method at incubation times of 0, 2, 4, 6, 12, 24, 48, and 72 h according to a Completely randomized design. The results revealed that the highest yeast and LAB population culture in fermented milk was found at 72 h-post cultivation. From in situ study, the soluble fractions at time zero (a), potential degradability (a+b) and effective degradability of dry matter (EDDM) linearly (p<0.01) increased with the increasing supplemental levels and the highest was in the 5% SBMFM supplemented group. However, there was no effect of SBMFM supplement on insoluble degradability fractions (b) and rate of degradation (c). In conclusion, the optimal fermented time for fermented milk with yeast and LAB was at 72 h-post cultivation and supplementation of SBMFM at 5% of total concentrate substrate could improve rumen degradability of beef cattle. However, further research on effect of SBMFM on rumen ecology and production performance in meat and milk should be conducted using in vivo both digestion and feeding trials.

INTRODUCTION
In livestock production, feed resources are very important in tropical regions, especially during the dry season. The security of feed is becoming critical in terms of quantity and quality, particularly protein sources which effect productivity performance. Researchers are attempting to find alternative protein sources which may help to increase livestock productivity (Wanapat and Rowlinson, 2007). Soya bean (Glycine max) is an economic crop used for both human and animal feeding. Soybean meal is the by-product of the extraction of soybean oil. It is the most important protein source used to feed farm animals. Soya bean meal is usually standardized commercially to contain 44% or 48% crude protein (CP) (Pond et al., 2005). Many researchers are seeking strategies to improve nutritive value of by-products and local feeds such as cassava chip, rice straw, rice bran, soybean meal, etc. Incorporation of microbial additives such as a culture of Saccharomyces cerevisiae and lactic acid bacteria (LAB) to the diet has become a common practice in ruminant nutrition (Polyorach et al., 2012). It has been widely used for protein production. Eukaryotic and prokaryotic microorganisms can be considered a suitable host for the production due to the rapid growth of microorganisms and a broader range of materials which may be considered as suitable substrates depending on the microorganism chosen.
The process of protein enrichment of animal feed using microorganisms to improve the nutritional value of animal has been evaluated. Oboh and Akindahinsi (2003) reported that Saccharomyces cerevisae could also be used for enriching cassava products. Boonnop et al. (2009) demonstrated that supplementation of cassava chips with Bakers' yeast (S. cerevisae) could increase CP from 2.0% to 32.4%. Moreover, Polyorach et al. (2012) reported that yeast fermented cassava chip protein (YEFECAP) could be prepared to increase CP level up to 47%. The effect of YEFECAP as protein source on rumen ecology and ruminant production performance has been evaluated by previous studies of Polyorach et al. (2014a) Boonnop et al. (2010) and Wanapat et al. (2011). The method of upgrading the protein content of soybean meal has been developed. Moreover, LAB are the principal organism involved in the production of fermented feed. It is well known that LAB in fermented feed can exert health benefits for animals (Canibe and Jensen, 2012;Sugiharto et al., 2015).
Fermented milk products are regarded as predominantly lactic fermentations, the frequent occurrence of yeasts and LAB has led to the suggestion that interactions may occur which could influence product characteristics and quality. However, the mechanisms of possible interaction between yeasts and the lactic flora have not been widely studied. Such interaction may produce stimulation or inhibition of growth of one, or both, of the co-cultured strains (Narvhus and Gadaga, 2003). Recently, Polyorach et al. (2014b) has reported that soybean meal nutritive value has been improved with fermented milk using yeast and LAB. Fermented soybean meal with fermented milk product could increase CP from 46.8% to 70.6% while fiber content was decreased compared with unfermented group. Therefore, soybean meal fermented milk (SBMFM) is a potential protein source for ruminant.
However, the use of SBMFM as a protein source for ruminants is still affected by lack of data. Therefore, the objectives of this study were to determine an optimal cultivation time of populations of yeast and LAB co-culture in fermented milk and the effects of SBMFM supplementation on rumen degradability in beef cattle using nylon bag technique.

Fermented milk preparation
Yeast and LAB cultivation was done according to the method of Polyorach et al. (2014b) and in brief was as follows: i) Activated yeast: weighed 20 g of Baker's yeast into a flask and mixed with 20 g of sugar plus 100 mL of distill water. The mixture was then incubated at room temperature for 1 h (A). ii) Activated LAB: mixed commercial yoghurt 50 g, molasses 25 g and adding distilled water 25 mL, and then incubated at room temperature for 2 h (B). iii) Mixed (A) and (B) with 500 g of raw milk. The fermented milk was sampled at 0, 4, 8, 24, 48, 72, and 96 h post-cultivation for determination of yeast and LAB populations using direct counting.

Soybean meal fermented milk production
The SBMFM was prepared by mixing fermented milk (72 h post-cultivation) with soybean meal at proportion of 1 mL: 1 kg (fermented milk product: soybean meal) and fermented for 3 days. The product then was sundried for 2 days and stored in plastic bag.

In situ trial
Animals and dietary treatment: Two rumen fistulated crossbred beef cattle (410±10 kg) were used to determine the effect of SBMFM supplementation on rumen degradation using nylon bag technique according to completely randomized design. The dietary treatments were different levels of SBMFM supplement at 0%, 3%, and 5% of total concentrate substrate. All animals were housed in individual pens and fed rice straw ad libitum with concentrate (14% CP) supplemented at 0.5% body weight. Animals had free access to fresh water and trace mineralized salt. The diets were offered in two equal meals at 7.00 and 16.30 hour for 10 days before the initiation of the in situ experiment.
Nylon bag techniques: Samples were collected, ovendried at 60°C for 72 h, ground to pass through a 1 mm screen and prepared for in sacco analysis. Ruminal degradation of the feed fractions was determined by the nylon bag technique (Orskov and McDonald, 1979). Bags 7×4 cm in size with a pore size of 38 μm, were used. Three grams of each sample treatments were placed into the bags and tied tightly with nylon thread. Bags were tied to a weighed chain and placed in the ventral rumen sac of steers at approximate 2 h after the morning feeding. All feeds were incubated simultaneously in both steers using duplicates bags per feed at each time point and a blank bag containing no sample for each removal time. Bags of each feed were removed after 0, 2, 4, 6, 12, 24, 48, and 72 h of incubation. Immediately after removal from the rumen, the bags were washed with cold tap water until rinsed clear water and oven-dried at 60°C for 72 h. The bags were weighed and residues were removed and then analyzed for dry matter (DM). All bags of feed samples were collected for their corresponding blank. The 0 h incubation samples were washed and dried in similar condition. During each time, ruminal pH and temperature were measured immediately in both cattle using a portable pH and temperature meter from.

Chemical composition analysis
The samples were analyzed for DM, organic matter (OM), ether extract (EE), and CP according to AOAC (1990). Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined using the method of Goering and Van Soest (1970).

Rumen degradability
The ruminal disappearance characteristics of DM was fitted to the exponential following the procedure described by Orskov and McDonald (1979) and using the NEWAY program (Chen, 1996): p = a+b(1-e -ct ); where, p = disappearance (%) of DM from the bags at the time t, a = proportion of immediately soluble faction of DM (g/kg), b = potentially but not immediately degradable fraction (g/kg), c = fractional rate of soluble degradation (%/h) of b and t is the incubation time (h). Solubility of DM were determined similarly by washing of 0 h bags.
The effective degradability (ED) of DM were calculated using the equation shown below: ED = a+b[c/(c+k)]; where k assuming the rate of particulate outflow from the rumen, k, is 0.05 h -1 by equation of Ørskov and McDonald (1979).

Statistical analysis
All data were statistically analyzed according to a completely randomized design using the analysis of variance procedure of SAS (1998). Data were analyzed using the model Y ij = μ+T i +ε ij where Y ij is the observation, μ, the overall mean; T i , effect of treatment (i = 1 to 3); and ε ij , the residual effect. Differences between treatment means were determined by Duncan's New Multiple Range Test (Steel and Torrie, 1980). Supplement level's trends were analyzed by using orthogonal polynomials. Differences between means with p<0.05 were accepted as representing statistically significant differences.

Kinetics growth of yeast and lactic acid bacteriacoculture in fermented milk
Kinetics growth of yeast and LAB co-culture in fermented milk are shown in Table 1 and Figure 1. The population of yeast was the highest (p<0.01) (6.90 log cell/mL) started from 72 h-post cultivation while LAB started from 48 h-post cultivation (9.67 log cell/mL). Under current study, optimal cultivation time was in agreement with Polyorachet al. (2013) who reported that yeast fermented liquid at 66 h post-cultivation was 3×10 11 cell/mL.  In addition, Narvhus and Gadaga (2003) also reported that the fermentation of natural fermented milk usually takes 2 to 3 days and if the product is immediately consumed, or kept under refrigeration, then any effect of interaction between yeasts and LAB in the product has to be evident within this short fermentation period. The yeast counts recorded in this study were in similar range to those reported by Mathara et al. (2008) who found yeasts counts of <1.0 to 7.4 log10 cfu/mL. The presence of yeast in traditionally fermented milk products, in varying numbers, has been reported by Kebedeet al. (2007). The frequent concurrence of LAB and yeasts has led to the suggestion that there could be interactions that may an influence the product characteristics and quality (Narvhus and Gadaga, 2003). The fermented milk pH was the highest (p<0.01) at 0 h of cultivation (pH 7.01) and the lowest (p<0.01) was at 96 h post-cultivation (pH 3.4). The decreasing of ruminal pH might be due to the milk fermentation process where LAB ferments the lactose in the milk to lactic acid. Wszoleket al. (2001) reported that fermented milk products utilize a wide variety of microorganisms to produce a wide variety of products in addition to lactic acid including ethanol, free fatty acids, and acetaldehyde. When yeasts grow with LAB in milk, they need either to be able to obtain sufficient energy-giving substrates from the milk components or to be able to avail themselves of metabolites from LAB. S. cerevisiae has been associated with the production of alcohols and other aroma compounds, stimulation of LAB, improvement of nutritional value, and inhibition of undesirable microorganisms (Jespersen, 2003). However, the yeasts present in this product need to be investigated further to establish their exact role in the fermentation process, including their interaction with LAB and their metabolic properties.

Chemical composition of feeds
The chemical compositions of concentrates, SBMFM and soybean meal (SBM) are presented in Table 2. Concentrate diets contained DM, OM, CP, EE, NDF, and ADF at 89.3%, 95.9% DM, 14.2% DM, 3.3% DM, 16.6% DM and 12.4% DM, respectively. Moreover, SBMFM diets contained DM, OM, CP, EE, NDF, and ADF at 92.0%, 94.0% DM, 58.9% DM, 6.2% DM, 11.3% DM, and 6.5% DM, respectively. The CP and EE of SBMFM in the present study were increased compared to SBM. These results were consistent with the findings reported by Polyorach et al. (2014b) who showed that when soybean meal was fermented with milk by yeast and LAB the nutritive values of soybean meal, especially CP and EE, were significantly improved. The increase of CP and EE of SBMFM could be due to the effect of microorganisms (yeast and LAB) which could utilize milk and soybean meal and also products from fermented milk. Based on the previous works of Wanapat et al. (2011) and Polyorach et al. (2012, 2014a, it was reported that yeast could be prepared to increase CP content of cassava chip up to 30% or 47% of DM. Nasseri et al. (2011) and Yamada and Sgarbieri (2005) found that yeast (S. cerevisiae) is rich in protein, soluble fiber, some minerals, and predominantly saturated fatty acids. Therefore, the product of SBMFM could be used as a source of protein for ruminant feeding which could improve productivity and this could increase the economic gains for both dairy farming and fattening cattle.

Ruminal environment
Ruminal pH and temperature in various hours post feeding are shown in Table 3. The average ruminal pH and temperature were 6.5°C and 39.1°C, respectively. Supplementation of SBMFM in the present study changed  neither ruminal pH nor temperature. Chanjula et al. (2003) reported that ruminal pH and temperature ranged from 6 to 7 and 39°C, respectively, and were considered to be an optimal range for the microbial activity in the rumen. Change of ruminal pH after one hour post feeding could be due to greater amounts of starch in concentrate diets which may yield greater lactic acid concentration (Kang et al., 2014; thus a lowered ruminal pH.

Ruminal dry matter disappearance and characteristics
The effects of SBMFM supplementation on degradability characteristics are presented in Figure 2 and Table 4. The finding of current study revealed that the soluble fractions at time zero (a) was linearly (p<0.05) increased with increased supplemental levels and the highest was at 5% of total concentrate substrate supplemented group. Moreover, the potential degradability (a+b) and effective degradability of dry matter (EDDM) were quadratically (p<0.05) increased when increasing supplemental levels while the insoluble degradability fractions (b) and the rate of degradation (c) presented did not affect by treatments. The reason could be the effect of SBMFM as a high quality protein source developed from soybean meal by fermentation with milk and microbes (yeast and LAB) (Polyorach et al., 2014b) on microbial activity and the digestion of N in the rumen. Traore et al. (2010) reported an improvement in feed digestibility and microbial activity when degradable protein was added in the rumen. Faster and more completed digestion of the feed by microbes apparently reduces the fill of the feed in the rumen. Khy et al. (2012) reported that supplementation of protein source (Leucaena leucocephala) at 450 g/h/d could improve voluntary intake, digestibility, and rumen fermentation parameters in swamp buffaloes.
Moreover, SBMFM also contained yeast and LAB which improved feed utilization by promoting ruminal microbes. Yeast can promote the growth of cellulolytic bacteria and lactate-utilizing bacteria. Yeast also stimulates bacterial growth through removal of oxygen that occurs in ruminal fluid and in that way prevents oxygen toxicity to the ruminal anaerobes (Chaucheyras-Durand et al., 2008;Doto and Liu, 2011). Jouany (2006) reported that yeast culture could consume oxygen in rumen fluid that enters the rumen by coating with feed particles. Yeast culture, in addition, could provide some of the important nutrients and co-factors (vitamins B) to ruminal microflora. Mosoni et al. (2007) found that proportions of 16S ribosomal RNA of the three main cellulolytic bacterial species (F. succinogenes, R. albus, and R. flavefaciens) increased in the rumen of sheep fed with yeast. Additional of S. cerevisiae leads to increase  germination of zoospores from a rumen fungal strain of Neocallimastix frontalis as shown in in vitro study (Chaucheyras-Durand et al., 2008). Wanapat et al. (2013) and Polyorach et al. (2013Polyorach et al. ( , 2014a used yeast to improve the nutritional value of cassava chips (YEFECAP) by increasing ruminal microorganisms and thereby improving nutrient digestibility of ruminants.
In addition, LAB could interact with rumen microorganisms in such a way that their activity is enhanced feed utilization, in a recent study (Khuntia and Chaudhary, 2002), dietary addition of a mixed culture of LAB increased DM intake, weight gain, and DM digestibility in calves. Salawu et al. (2001) found that application of Lactobacillus plantarum to pea-wheat silage increased the rate of nitrogen and NDF degradation in the rumen. Malik and Sharma (1998) inoculated rumen fluid with various microorganisms in the presence of wheat straw and concentrates, and found that Lactobacillus acidophilus improved DM and OM digestibility in vitro, compared with an untreated control.

CONCLUSION
Based on this study, it could be concluded that milk fermented to the optimal time of 72 h-post cultivation resulted in the highest yeast and LAB populations. Moreover, supplementation of SBMFM could improve degradability in the rumen. This study suggested that SBMFM could be used as a high quality protein source with the supplemental level up to 5% of total concentrate substrate. However, further research should be investigated in in vivo trial on ruminal performance and animal production.

CONFLICT OF INTEREST
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.


extfieldsofstudy: Biology Medicine

provenance: AgriculturalAndFoodSciences,1970-2015-Agricultural And Food Sciences-2015.jsonl.gz.gz

text:
Pathotypes of Bacterial Spot Pathogen Infecting Capsicum Peppers in Korea

Sixty-seven isolates of bacterial spot pathogen (Xanthomonas spp.) collected from six provinces of Korea were tested for the identification of their pathotypes and determination of their distribution throughout Korea in an effort to genetically manage the disease. Near isogenic lines of Early Calwonder (Capsicum annuum) pepper plants carrying Bs1, Bs2 and Bs3, and PI235047 (C. pubescens) were used as differential hosts. Race P1 was found to be predominant, followed by race P7, and races P3 and P8 were also observed. This is the first report of races P7 and P8 in Korea. The races P7 and P8 were differentiated from the former races P1 and P3, respectively, on the basis of their ability to elicit hypersensitive reactions to PI235047.

Bacterial spot disease is one of the major diseases damaging pepper plants in Korea. This pathogen, formerly called Xanthomonas campestris pv. vesicatoria (Doidge) Dye, is currently subdivided into Xanthomonas euvesicatoria, X. vesicatoria, X. gardneri, and X. perforans on the basis of DNA homology and phenotypes (Hamza et al., 2010;Jones et al., 2004Jones et al., , 2005. However, Korean strains of the bacterial spot pathogen have not been reclassified into these four species. Although treatment of seeds with hot water or chemicals, spraying plants with chemicals including copper and antibiotics, rotation with non-host plants, and sanitation by removing volunteer plants and diseased plant residue are recommended for control of the disease (Cox, 1982;Goode and Sasser, 1980), host resistance is considered the most economically and ecologically reliable method for control of bacterial spot disease in pepper plants (Hibberd et al., 1989). Five hypersensitive resistance (HR) genes, namely Bs 1 , Bs 2 , Bs 3 , Bs 4 and Bs 7 , have been reported in pepper plant accessions PI163192, PI260535, PI271322, PI235047, and UENF1556, respectively (Cook and Guevara, 1984;Cook and Stall, 1963;Kim and Hartmann, 1985;Potnis et al., 2012), and two recessive genes, bs 5 and bs 6 , that govern non-hypersensitive resistance and act additively with each other were also screened from PI271322 and PI163192, respectively (Jones et al., 2002;Vallejos et al., 2010). One or more of the HR genes have been transferred to the Early Calwonder (ECW) strain of the pepper plant to develop near isogenic lines for the respective genes, and the varieties developed were named ECW 10R, ECW 20R, ECW 30R and ECW 70R, depending on the respective HR gene incorporated, and as ECW 12346, with Bs 1 , Bs 2 , Bs 3 , bs 5 and bs 6 incorporated (Jones et al., 2002;Stall et al., 2009;Vallejos et al., 2010). Genetic control of bacterial spot disease by hypersensitive resistance genes has not been very successful due to the occurrence of pathogenic races that can overcome the HR genes (Cook and Stall, 1969;Hibberd et al., 1987b;Kousik and Ritchie, 1995;Sahin and Miller, 1995). The hypersensitive reaction of pepper plants is known to be induced by an interaction between the Bs genes in pepper plants and the corresponding effector (avirulence, avrBs) genes in the pathogen in a gene-for-gene model (Minsavage et al., 1990;Potnis et al., 2012). Bacterial spot pathogens of peppers are classified into 11 pathotypes on the basis of interactions between the Bs 1 , Bs 2 , Bs 3 and Bs 4 genes that confer an HR reaction in the pepper and corresponding avirulence genes (avrBs 1 , avrBs 2 , avrBs 3 and avrBs 4 ) in the pathogen (Stall et al., 2009). ECW 10R, ECW 20R, ECW 30R, and PI235047 (Capsicum pubescens) have been used as differential hosts in the classification of pathogen races (Sahin and Miller, 1998;Stall et al., 2009). In Korea, Kim et al. (2007) transferred Bs 2 and Bs 3 to a local Chilseong strain of *Corresponding author. Phone) +82-53-950-5729, FAX) +82-53-950-5722 E-mail) bskim@knu.ac.kr Capsicum annuum, a susceptible cultivar grown in Yeongyang in Gyeongbuk province, and produced Chilbok-2 and Chilbok-3 for use as differential hosts.
In Korea, only race P1 and race P3 had been found (Kim, et al., 1990;Lee and Cho, 1996;Pae et al., 1994) until the 1990s. These races have been subdivided into races P1 and P7, and races P3 and P8, respectively, based on their abilities to elicit HR reactions in pepper strain PI235047 in the USA (Sahin and Miller, 1998;Stall et al., 2009). The prevalence of many races makes breeding and controlling the disease by resistant cultivars complicated. Therefore, a better understanding of the pathological races distributed in a given area, as well as host-pathogen relationships, is important in planning a breeding program for pepper plants for durable resistance to the disease. Here, we report results from testing Korean strains of Xanthomonas spp. infecting peppers to identify their pathotypes according to the race identification system (Stall et al., 2009).
The differential host sets consisting of Early Calwonder (ECW) and its isogenic lines ECW10R, ECW20R, and ECW30R, carrying Bs 1 , Bs 2 and Bs 3 , respectively, and PI235047 with Bs 4 gene, were kindly provided by USDA while Chilseong NILs, Chilbok-2, and Chilbok-3 were bred by us. All materials were sown in 200-cell trays with Wonjo mix (Nongkyung Agroindustrial Co., Ltd., Korea) and 16 plants of each line were transferred to 32 cell-trays one month after sowing in July 2012. In September 2012, the plants were transferred to pots 13 cm in diameter, again filled with Wonjo Mix, to grow more leaves.
Isolates of Xanthomonas spp. have been collected from various locations of Korea since the 1990s. These cultures have been maintained in water and on Yeast Extract Dextrose Calcium Carbonate (YDC) slants with routine subculture. Dry specimens of the collected leaves have also been stored in the refrigerator. In 2012, additional isolates were collected in the Chungnam, Jeonbuk and Jeonnam area of Korea. Overall, 67 isolates, 42, 3, 6, 3, 8 and 5 of which were originally collected from Daegu and Gyeongbuk, Chungnam, Chungbuk, Gangwon, Jeonnam and Jeonbuk province, respectively, were tested to determine their pathological races in September 2012. The confirmation study was conducted only with ECW NILs and PI235047 after transplanting the differential host plants to 25 cm pots in January 2013.
Isolates of pathogenic Xanthomonas spp., stored as stocks strains, were subcultured on yeast extract dextrose calcium carbonate (YDC) agar plates and incubated for 2-3 days at 28-29ºC to obtain pure cultures. Two-dayold, typical round, convex, mucoid, and yellow colonies were suspended in distilled water with a sterile cotton swab to obtain a suspension. The bacterial concentration was adjusted to approximately 10 8 cfu/ml (0.2 OD at 470 nm) using a spectrophotometer (Kim, 1983). The bacterial suspension was infiltrated into the abaxial surface of the leaves on each side of the midrib using a syringe with the needle removed. Two leaves were used as replication for each isolate.
Races were determined on the basis of hypersensitive (HR) and susceptible (Sus) reactions in response to infiltration of the bacterial suspension into leaf tissues, according to the race identification system reported by Stall et al. (2009). A hypersensitive reaction was recognized by tissue collapse at the infiltration site, with the tissue turning from dark green to light black within 24-48 h post inoculation, while a susceptible or non-hypersensitive reaction by water-soaked lesions appearing 3 or 4 days post infiltration. HR lesions turned tan to papery white with light twisting while non-HR lesions spread with yellow margins with time. Infiltration sites on tender leaves of susceptible plants often turned wet clear with water-soaking and yellowing margins (Fig. 1).
The reactions of the differential hosts to the 67 isolates collected from the six provinces of Korea are given in Table 1. Chilbok-2 and Chilobok-3 carrying Bs 2 and Bs 3 , respectively, were included in the test but their reactions were the same as ECW 20R and ECW 30R, respectively, therefore, not listed here. Races P1, P3, P7, and P8 were found in the six provinces of Korea. Among the 67 isolates, 29 (43.3.0%) were race P1, 3 (4.5%) were race P3, 23 (34.3%) were race P7, and 12 (17.9%) were race P8. The races appeared to be evenly distributed in the six provinces although the number of isolates collected was limited and concentrated in Daegu and Gyeongbuk province. Races P7 and P8 have been subdivided from races P1 and P3, respectively, on the basis of their abilities to elicit an HR reaction in PI235047 carrying the Bs4 gene (Sahin and Miller, 1998;Stall et al., 2009). Thus, race P1 was found to be predominant in Korea, followed by race P7, and races P3 and P8 were also identified (Table 1). To date, races P4, P5, P6, P9 and P10 that can overcome the Bs 2 gene have not been found in Korea. ECW 20R, ECW 12346, and Chilbok-2 carrying Bs 2 gene exhibited hypersensitive resistance to all strains collected in Korea. However, ECW 10R, carrying Bs 1 , was susceptible to all isolates tested, indicating that races P0, P2 and P5 are not present in Korea, and ECW 30R and Chilbok-3 plants, carrying the Bs 3 gene only, were resistant to races P1 and P7 only. In the 1990s, only races P1 and P3 were reported in Korea (Kim et al., 1990;Lee and Cho, 1996;Pae et al., 1996). Thus, the pathotype distribution in Korea has been essentially unchanged for the last 15 years, although races P7 and P8 could be differentiated from race P1 and P3, respectively, according to their ability to induce HR reactions in PI235047 carrying the Bs4 gene. Regarding the relative frequency of pathotypes, Kim et al. (1990) and Lee and Cho (1996) reported that race P3 was detected more frequently than race P1, while Pae et al. (1994) reported that races 1 and 3 were found at a similar frequency. In the present study, however, the majority of the 67 isolates identified were race P1 (43.3%), followed by race P7 (34.3%). Race P1 and race P7 are formerly race 1. Thus, the relative frequencies of pathotypes found in this study differed from the previously reported results. The discrepancy in the frequency of pathotypes may be due to the different collection sites of the studies, since races P1 and P3 can be observed in the same field and even in the same leaf (Kim et al., 1990). Pohronezny et al. (1992) reported a sudden shift in the predominant race from P2 to P1 in Southern Florida, and Kousik and Ritchie (1996) also reported a rapid shift from race P1 to race P2 to race P3 in North Carolina, due to the inactivation of effector genes (avrBs1, avrBs2 and avrBs3) in the pathogen by various mutations, including insertion elements. Likewise, a shift from race P3 to race P1 could have happened in Korea as reported by Oh et al. (2011). However, resistant varieties of  1-2, 17, 22, 23, 25, 26, 27, 38, 39, 041, 48, 49, 51, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 74, 75, 76, 77 6, 10, 12-1, 12-2, 14, 36, 43, 46, 47, 50, 51, 52, 53-1, 53-2, 56, 58, 73, 79, 80, 81 Total 67 a HR = hypersensitive reaction characterized by rapid tissue collapse at infiltration site within 48 h post infiltration of the pathogenic bacterium; Sus = susceptible reaction characterized by water-soaked or yellow lesions appearing 72 h or later post infiltration (Fig. 1).
pepper plants carrying any of the HR genes have not been distributed for cultivation in Korea. Selection pressure for a race shift, therefore, has not been present in Korea. The difference in the frequency of the races is more likely due to a difference in collection sites, since all strains that were newly isolated for this study from the Chungnam, Jeonbuk, and Jeonnam areas in 2012 were either P1 or P7. Thus, the hypersensitive resistance gene, Bs 2 , is still functioning in Korea and may be used in breeding for resistance. The validity of Bs 1 and Bs 3 , for resistance, however, is over in Korea (Kim et al., 2009). In breeding for resistance, the exploitation of quantitative resistance not specific to races or the integration of the major HR genes with quantitative resistance would be desirable for more durable resistance (Hibberd, 1989;Jones et al., 2002;Stall et al., 2009;Vallejos et al., 2010). Substantial genetic diversity has been found among Korean strains of the bacterial spot pathogen (Chung et al., 1997). Abundant sources of non-hypersensitive resistance have also been reported (Kim et al., 2009;Sowell, 1960;Sowell and Dempsey, 1977;Tran and Kim, 2007) and are available at hand. Most of the sources of resistance are in hot chili type peppers in horticultural characters including fruit shape. Therefore, these sources of resistance may be more readily available for breeding Korean chili peppers, the most common type of pepper grown in Korea, than for breeding bell type peppers. In the current classification system of bacterial spot pathogen affecting peppers, bacterial spot disease may be caused by one of four species, including X. euvesicatoria, X. vesicatoria, X. gardneri, and X. perforans, or by any mixture of these variants (Jones et al., 2004(Jones et al., , 2005Stall et al., 2009). The association of pathotypes with the four species of Xanthomonas has not yet been reported, but this information would be useful to breed peppers resistant to bacterial spot disease.


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Asian Soybean Rust Resistance: An Overview

Soybean is one of the most important economic crops as a source of protein and oil. Biotic stresses such as pathogens, insects and weeds can cause negative impacts on its production. Among the diseases, Asian soybean rust caused by Phakopsora pachyrhizi (Sydow. & Sydow.)is the most destructive, and over 80% losses are common when environmental conditions are conducive to disease development (Figure 1). The infected plants undergo defoliation and early maturation in relation to non-infected plants, which causes reduction in weight and quality of the grains. Due to the limited availability of resistant varieties, the fungicide application is the only management tool available for farmers, which significantly raises the production cost and the risk of environmental and human contamination (Figure 2). In addition, some pathogen populations have shown increased tolerance to certain fungicides [1]. Thus, the search for resistant varieties is a critical strategy for economically and environmentally sustainable control.

Introduction
Soybean is one of the most important economic crops as a source of protein and oil. Biotic stresses such as pathogens, insects and weeds can cause negative impacts on its production. Among the diseases, Asian soybean rust caused by Phakopsora pachyrhizi (Sydow. & Sydow.)is the most destructive, and over 80% losses are common when environmental conditions are conducive to disease development ( Figure 1). The infected plants undergo defoliation and early maturation in relation to non-infected plants, which causes reduction in weight and quality of the grains. Due to the limited availability of resistant varieties, the fungicide application is the only management tool available for farmers, which significantly raises the production cost and the risk of environmental and human contamination ( Figure 2). In addition, some pathogen populations have shown increased tolerance to certain fungicides [1]. Thus, the search for resistant varieties is a critical strategy for economically and environmentally sustainable control.
Asian soybean rust is present in the most countries growing soybeans. The first report of rust epidemic was around 1914 in Southeast Asian countries. However, until the middle of last century the rust fungus reports occur only in East Asia and Australia. In 1970 it was reported in India [2], in 1976 in Puerto Rico [3] and in 1994 in Hawaii [4]. Probably the introduction of soybean rust in Africa in 1975 took place from urediniospores transported by air currents from west of India [5]. In 2001, the fungus was first detected in Brazil and Paraguay [6][7][8][9] and in 2003 in Argentina [7][8][9][10]. In the continental US, the pathogen was first reported in 2004 [11], and the rust epidemics have been limited to the southern states, with reduced movement toward producing regions of the North [12]. There are no reports of negative economic effects due to soybean rust in the United States, although there are up to 33% yield losses documented in experiments with no fungicide application [13]. In Argentina, soybean rust epidemics occur every year, usually late in the growing season and significant losses are limited to a few north locations of the country [10].
The main difference in the effect of the rust between Argentina, Brazil and the US is due to the unique environmental characteristics of Brazil that increase the rust risk. The environmental condition in Brazil is much more conducive to rust development than that in the US and Argentina. Uredinia survive only in areas of southern US and northern Argentina due to low temperatures during the winter at high latitudes [14]. The high rainfall regime in the summer in Brazil increases substantially the risk of losses caused by Asian soybean rust when compared with Argentina and the US. In Brazil, the main factors for rust epidemics are a great soybean field extension and the continued monoculture, which favors urediniospores production and dissemination, and accelerates race shifts. The favorable climate, fungicide ineffectiveness, high plant density, sowing period from September to January, pathogen survival in volunteer plants of soybean and secondary hosts, are additional factors that decrease efficiency of control measures of the disease [15].
Sources of resistance have been reported and to date six dominant genes have been identified: Rpp1 [16], Rpp2 [17], Rpp3 [18], Rpp4 [19], Rpp5 [20] and Rpp6 [21]. These genes are not effective against all populations of P. pachyrhizi [22]. The transfer of resistance genes through classical breeding or through marker-assisted selection allow to develop resistant varieties and their use as an efficient and costeffective method for soybean rust control. Therefore, studies on the inheritance of resistance in soybean to P. pachyrhizi are very important for varieties development. Many breeding programs have used some identified resistance genes with success, although the diversity of rust isolates present in the nature is a continuous challenge. The objective of this review is to provide a broad overview of the soybean rust resistance, and a useful tool to guide future researches as well.
emerged as a domesticated specie by the eleventh century BC. Soybean belongs to the Magnoliophyta Division, Magnoliopsida Class, Fabales Order, Fabaceae Family, Faboideae Subfamily, Glycine gender [23]. Glycine max is a domesticated specie over the 18 known species of the genus Glycine, and its diploid number of chromosomes is forty. Soybean is cultivated in temperate and subtropical regions as oil and protein source.
Soybean is a legume species that has a great adaptability to different latitudes, climate and soil conditions. Their grains are important source of protein and vegetable oil for human and animal diet. Soybeans contain about 20% oil and 40% protein in the form of essential amino acids. The oil is used mainly for human consumption and as an industrial raw material, and the beans after oil extraction are used for animal feed [24].
In Brazil, the introduction of soybeans in a commercial scale occurred in the 1930's, using American varieties originally adapted to cultivation in the southern US. However, the large increase in area and production of soybeans took place in the 80's, when the success in breeding allowed the expansion of culture to the Cerrado areas [25]. From an area equal to 1.29 million hectares and a production of 2.2 million tons in 1980, the region began to grow to 5.08 million hectares, with a production of 10.3 million tons in 1989. Currently soybean is one of the most important crops produced in Brazil and accounts for about 46% of the grain produced in the country. In the 2014-2015 season, the Brazilian soybean production reached 96 million tons, an increase of 11.5% over the previous season [26]. Exports by the production chain of the oilseed reached nearly US $31 billion and represented 31% of total exports from Brazil agribusiness [27]. Worldwide soy is cultivated in about 118 million hectares, with production estimated at 315 million tons [28]. Brazil is the second largest producer and accounts for about 30% of world production of oilseeds. The US is the first producer, with an output of 108 million tons in an area of 33.6 million hectares, representing 34% of total world production.

The Pathogen
The fungus that causes Asian soybean rust belongs to the Fungi Kingdom, Basidiomycetes Class, Uredinales Order, Phakopsoraceae Family, Phakopsora pachyrhizi specie. To date, the described fungus stages were uredinial, teleomorphic and basidial. The aecial stages have not been reported yet [29]. Like all fungi of the group, P. pachyrhizi is a biotrophic pathogen that requires a living host to grow and survive. Naturally, P. pachyrhizi infects 31 species in 17 genera of legumes [30]. The main hosts are Glycine max, G. soja, Pachyrhizus erosus, Pueraria lobata and Vigna unguiculata. According to Yeh et al. [31] and Bromfield [2] the fungus could attack up to 87 and 95 hosts, respectively.
Symptoms caused by soybean rust are different from those caused by other types of rusts. The uredinia are the fruiting bodies, which produce the urediniospores released through an ostiole. The symptoms of soybean rust are characterized by small brownish to dark brown lesions, with one or more uredinia mainly on the bottom side of the plant leaflets. The lesions tend to be angular with 2 to 5 mm in diameter. May also occur on petioles, pods and stems. The color of the lesions varies with age and with the interaction between the host genotype and the pathogen isolate. The new lesions are initially light brown, becoming darker with the age [32].
The urediniospores are the primary inoculum of soybean rust. These are asexual, small, lightweight spores, which are removed from uredinia when the infected leaf surface is dry. After removal, the air currents can transport the spores over long distances, which explain its spread from one field to another. In the presence of water and temperature between 21 and 25°C, the urediniospores deposited on the host leaf surface begins the germination process and infection [32]. The penetration of the parasite occurs directly through the cuticle and epidermal cell wall of the host. The direct penetration rather than through the stomata, is a characteristic that differentiates P. pachyrhizi from other rusts fungi. The colonization begins shortly after penetration, the primary branching hyphae gives rise to formation of a dense mycelium filling the intercellular spaces and inserting haustoria in the mesophyll and epidermal cells. The fungus reproduction begins approximately at eight days after infection, and its first evidence is the hyphae aggregation forming the uredinia primordium. Uredinia haves light brown to red brown appearance. In about 3 to 4 days, it starts the production of urediniospores. According Alves et al. [33], the latency period length varies with the temperature, being shorter for temperatures around 23°C, in agreement with previous studies of Melching et al. [34].

Pathogen Variability
Regarding the variability of the pathogen, the asexual production of urediniospores suggests a low genetic diversity, with a small number of clonal lineages and recurrent events [35]. In general, data from different molecular markers shows that the genetic diversity in P. pachryrhizi is low between large geographic areas and high when considering the variation within local populations. The high capacity of dispersion over long distances enables virulent genotypes of P. pachyrhizi move  quickly between different populations [36]. In Nigeria, P. pachyrhizi has approximately 90% of their genetic diversity within the soybean fields, with little diversity distributed among the fields [35,[37][38][39].
On the other hand, the presence of resistance genes in the host did not lead to reduction of frequency of virulence genes in pathogen populations. The probable explanation for this is that asexual way is not the unique reproduction mechanism of P. pachyrhizi. The anastomosis of the germ tube and hyphae, and the possibility of parasexual cycle [40,41], contribute to increase the genetic diversity of the pathogen. Thus, breeding for soybean rust resistance, should consider the likelihood of conversion of avirulent to virulent isolates. In addition, the local development of varieties containing only one resistance gene exposes it to these pathogen populations containing the full range of genetic variation present at regional level.
The size of the genome of P. pachyrhizi was estimated between 300 and 950 Mb depending on the analysis method used [42]. Igor Grigoriev provided similar information, suggesting a genome greater than 850 Mb [43]. However, due to ignorance about the degree of heterozygosity between or within the dikaryotic fungus genome, this size can be overrated. It is possible that the genome of P. pachyrhizi has sustained a high level of gene duplication during evolution with an important activity of transposable elements, which can explain the enormous size of the estimated genome for that species.

Resistance
The plants generally have two main defense mechanisms against pathogens, race-specific and race-nonspecific resistance. Race-specific resistance is controlled by single R genes and generally less durable. In contrast, race-nonspecific resistance is a polygenic trait and more durable [44].
During the early phases of infection, all pathogen classes deliver effector molecules into the plant cell to enhance microbial fitness. In response, plants have evolved a dynamic immune system to defend themselves against plant pathogens. The perception of pathogen associated molecular patterns (PAMPs) by plant extracellular pathogen recognition receptors (PRRs) is the first stage of plant basal defenses. The PAMPs are essential structures or components that are conserved throughout the whole classes of pathogens, including oligogalacturonides, ergosterol, bacterial flagelin, xylanases, cold-shock protein and lipopolysaccharides. Such response is referred to as PAMPtriggered immunity (PTI) and activates a myriad of process, including mitogen-activated protein kinase (MAPK) cascades, production of reactive oxygen species (ROS). According to the zig-zag model, proposed by Jones & Dangl [45], there are numerous PRRs in plants to recognize PAMPs and to initiate basal defense responses. However, some pathogens can secrete effectors to evade recognition by plant PRRs and to promote pathogen growth and virulence, called effectortriggered susceptibility (ETS). In response to ETS, host plants trigger R proteins to interact directly or indirectly with pathogen effectors or avirulence (Avr) proteins, and induce a stronger defense response, referred to as effector-triggered immunity (ETI) [44]. ETI triggers salicylic acid (SA) biosynthesis and signaling, leading to local and systemic acquired resistance (SAR) against biotrophic pathogens.
According to Van de Mortel [46], a biphasic gene response to P. pachyrhizi infection was seen in resistant and susceptible genotypes. The early transcriptional response observed in susceptible and resistant plants might represent a general response of soybean to the non-specific recognition of any pathogen, presumably by interaction with PAMPs. By contrast, the second likely response relates to R gene detection of P. pachyrhizi, typically mediated by ETI pathway. Typically ETI culminates in hypersensitive cell death (HR) that retard pathogen growth, particularly in interactions involving haustorial parasites.
There are three types of soybean reaction to infection by P. pachyrhizi, which are associated with qualitative resistance genes ( Figure 3). The first type is the immunity (IM) or complete resistance, without the presence of reproductive structures, such as uredinia or urediniospores. The second type is incomplete (or partial) resistance, which leads to the development of red brown (RB) lesions. According Parlevliet [47] and Ribeiro Do Vale [48], incomplete resistance allows some growth or reproduction of the pathogen in the host tissues. Finally, the tan colored lesions (TAN), indicative of susceptibility [49]. Either IM or RB reaction are initiated with the early perception of the pathogen avirulence proteins by plant R proteins, according the classical gene-for-gene resistance theory [50]. This incompatible interaction is followed by a localized programmed cell death, called hypersensitive response (HR), to limit the pathogen growth. On the other hand, TAN reaction means a compatible interaction, without the perception of the pathogen by the plant. Studies of host-pathogen interaction show that RB lesions tend to have longer latency period, smaller and fewer uredinia than the TAN lesions. Although RB type injury can also vary in color from light red to dark red, and sometimes have larger lesions than the TAN type. These observations suggest that the color of the lesions may not be a reliable indicator of susceptibility or resistance [2,51,52].
The wide variation in the type of reaction, color and intensity of sporulation observed in the field can be a difficulty factor in genotype characterization studies [53]. Although the type of lesions are widely used in detached leaves and seedlings inoculations studies, the appearance of rust lesions in adult plants is often different from that originally reported by Bromfield & Hartwig [17]. Besides the influence of the host genotype and levels of pathogen sporulation, the color of the lesions varies with their age, especially in the field where the infection events are continuous. In the work of Miles et al. [49], the IM and RB reactions were considered the unique forms of resistance expression. However, partial resistance also occurred in the interaction between P. pachyrhizi and soybeans, since differences were observed between the lines with TAN lesions. The variation in the number of uredinia is one of the parameters to be considered in the differentiation of genotypes with partial resistance and is inversely correlated with yield [2,51,54,55].
The incompatible interaction expressed as IM phenotype is mediated by Rpp1 gene [56], while the resistance conferred by the other genes is characterized by the formation of RB lesions, and limited  [51]. Soybean varieties with partial resistance allow the development of a few lesions and limited sporulation during the growing season [55]. Race-nonspecific resistance has also been observed. It acts by reducing the amount and rate of rust development, even if the type of infection is similar to that produced in highly susceptible varieties [2]. This type of resistance can be effective against most of the pathogen population, being more useful than the racespecific resistance. The difficulties associated with race-specific and race-nonspecific resistance have led to the search for new types of resistance such as tolerance [57]. Tolerance is the relative ability of soybeans to produce under the stress caused by rust. This type has been used to minimize yield losses associated with soybean rust [58,59].
The first report of race-specific resistance in soybean to P. pachyrhizi occurred in the 70's [60]. In 1975, the US Department of Agriculture (USDA), in cooperation with the Asian Vegetable Research and Development Center (AVRDC), tested approximately 16,000 genotypes against a mixture of five isolates. They found 805 accessions with low severity or RB lesions, which were considered as a potential resistance sources. However, it was not possible to determine whether these resistant genotypes represented new sources of resistance genes or just alternative sources for the same four genes already known at that time. This is still an important question to be answered about the new resistance sources identified in recent studies. Studies conducted in Australia by McLean [61] and McLean & Byth [16] found that a single dominant gene controls resistance of PI 200492 to certain races of P. pachyrhizi. At the same time, Singh & Thapliyal [62] reported that PI 462312 also carries a single dominant gene.
The first report indicating the occurrence of variability in the pathogen population was in 1980. In tests carried out by Bromfield & Hartwig [17], the PI 200492 and PI 462312 showed susceptibility to an isolate from Taiwan and resistance to an isolate from India. On the other hand, PI 230970 and PI 230971 had just RB lesions when inoculated with isolates from Australia, India, Taiwan and Philippines. With the same set of varieties, Hartwig & Bromfield [18] indicated that Rpp1, Rpp2 and Rpp3 genes not had the same spectrum of reaction against different rust isolates. While Rpp1 and Rpp3 genes conferred resistance only to an isolate from India, the Rpp2 gene conferred resistance to isolates from India and Taiwan [63].
Between 2000 and 2010, with the incorporation of Rpp1 and Rpp2 genes in the Asian breeding programs, the resistance conferred by these genes was quickly defeated [64]. Similarly, the effectiveness of these genes was lost only two years after soybean rust had been reported in Brazil [7].
According to studies from Miles et al. [49], the rust severity was not related to the type of lesions. Among the varieties with RB lesions, PI 561356 and PI 594538A showed lower levels of severity than PI 230970, PI 423972 and PI 459025B, who were the varieties with higher rates of severity. On the other hand, among the varieties with TAN lesions and low levels of severity, PI 548463, PI 548484 and PI 549017 produced similar numbers and sizes of uredinia as from the susceptible variety Williams, indicating no relationship between the severity of the disease and the reproduction of the fungus.
Walker et al. [53], studying the field reaction of reported resistance genes in the southern United States, noted that Rpp1 gene, present in PI 200492, and Rpp6 gene, in PI 567102B, still conferred high levels of resistance. The varieties carrying Rpp2, Rpp3, Rpp4 or Rpp5 showed incomplete resistance and moderate levels of rust development when compared to susceptible controls. Although Rpp2, Rpp3 and Rpp4 genes have conditioned lower levels of resistance, this moderate resistance was stable at the different sites. Regarding the resistant varieties reported in the literature, but whose genes have not been mapped yet, the resistance in PI 567104B was similar of PI 567102B (Rpp6). Coincidentally both these varieties came from the same research center in Indonesia, yet the relationship between them is unknown. PI 416826A, PI 417125, PI 567024, PI 567025A, PI 567034 and PI 605823 also showed good resistance to most field isolates of P. pachyrhizi. The authors reported no immunity among the varieties tested and a low frequency of sporulating uredinia, even in highly resistant accessions, such as PI 200492 and PI 567102B. This indicates that in some regions, the fungal population is heterogeneous and contains one or more isolates able to overcome the resistance conferred by Rpp1 and Rpp6 genes. They also observed that moderate resistance conditioned by certain genes make it difficult to distinguish between race-specific and race-nonspecific resistance. An example is the case of PI 506764 (Hyuuga), carrying resistance genes Rpp3 and Rpp5 [65], but whose classification field was the same of PI 200492 (Rpp1) and PI 462312 [Rpp3].
Recently, Chen et al. [66] identified a new resistance allele on chromosome 18, where several alleles of Rpp1 family have been identified. According to the authors, the identified locus differs from those previously identified in PI 200492 [67], PI 587886 and PI 587880A [68], PI 561356 [69] and PI 594538A [70]. According to Ray et al. [68], the resistance observed in PI 587880A and PI 587886 is controlled by an Rpp1 allele with incomplete dominance. The presence of RB lesions in heterozygous individuals (Rpp1/rpp1), and IM or TAN lesions in homozygous (Rpp1/Rpp1 or rpp1/rpp1) individuals are indicative of incomplete resistance conferred by Rpp1. Furthermore, the presence of three classes of lesions (IM, RB and TAN) in mapping populations is also an indicative of incomplete dominance. In this sense, Smith et al. [71] also conclude that the gene action of an Rpp1 allele mapped in PI 587905 can be dominant or incompletely dominant, depending on the age or development stage of plants. So far, resistance alleles to soybean rust were identified and mapped in different loci of chromosomes 3, 6, 16 and 18 (Table 1).
Resistant varieties are useful tools to reduce the economic losses caused by Asian soybean rust. However, varieties with a single resistance gene tend to be readily broken [2,64,72]. The effectiveness of the racespecific resistance is often of short duration, especially in biotrophic pathogens like P. pachyrhizi, with high virulence variability. Varieties with two pyramided resistance genes tend to express greater durability of resistance than varieties with a single gene [73,74]. Lemos et al. [75] and Yamanaka et al. [76] reported that a variety with three resistance genes (Rpp2, Rpp4 and Rpp5) showed significantly higher resistance than its ancestor, showing that pyramiding strategy is effective to increase the rust resistance.
The main factors that determine which strategies and methods should be employed in rust resistance breeding programs include the genetic distance between the varieties to be improved and the resistance donor, the screening methods, the genetics of resistance and the number of characteristics to be improved [77]. The soybean breeding for rust resistance has focused on qualitative genes, which have stability limitations in the control of diseases. Furthermore, the presence of multiple virulence genes in the pathogen population, and the lack of multiple resistance genes in the host gives a competitive advantage to rust, and makes the race-specific resistance less effective and short as compared to race-nonspecific resistance [78]. Ribeiro et al. [79] suggest that breeding for race-nonspecific resistance is more effective to achieve durable resistance. Currently, with the launch of new resistant varieties, it is expected the appearance of new rust races. Therefore, it is crucial to make continued efforts on identifying newer genes for resistance, developing and refining molecular markers for use in breeding programs for Asian soybean resistance.


extfieldsofstudy: Medicine Chemistry

text:
Anesthetic activity and bio-guided fractionation of the essential oil of Aloysia gratissima (Gillies & Hook.) Tronc. in silver catfish Rhamdia quelen

This work aimed to determine the efficacy of the essential oil of A. gratissima as anesthetic for silver catfish, and to perform the bio-guided fractionation of essential oil aiming to isolate compounds responsible for the noted effects. Fish were submitted to anesthesia bath with essential oil, its fractions and isolated compounds to determine time of anesthetic induction and recovery. Eugenol (50 mg L) was used as positive control. Essential oil of A. gratissima was effective as an anesthetic at concentrations of 300 to 900 mg L. Fish presented involuntary muscle contractions during induction and recovery. The bio-guided fractionation of essential oil furnished E-(-)-pinocamphone, (-)-caryophyllene oxide, (-)-guaiol and (+)-spathulenol. E-(-)pinocamphone caused the same side effects observed for essential oil. (-)-Caryophyllene oxide, (-)-guaiol and (+)-spathulenol showed only sedative effects at proportional concentrations to those of the constituents in essential oil. (+)-Spathulenol (51.2 mg L) promoted deep anesthesia without side effects. A higher concentration of (+)-spathulenol, and lower or absent amounts of E-(-)-pinocamphone could contribute to increase the activity and safety of the essential oil of A. gratissima. (+)-Spathulenol showed potent sedative and anesthetic activities in silver catfish, and could be considered as a viable compound for the development of a new anesthetic.

INTRODUCTION
There are different handling practices in fish farming, such as biometrics and transport. These practices can cause stress, which can be detrimental to animal performance. Anesthetics are used in this context to maintain fish welfare, minimizing stressful situations and facilitating handling (Coyle et al. 2004, Zahl et al. 2012). Natural products have been evaluated as an alternative to synthetic anesthetics, such as 2-phenoxyethanol, quinaldine sulfate and benzocaine, which may cause some side effects in fish and handlers (Inoue et al. 2003, Velisek et al. 2007. Some compounds isolated from plants, such as eugenol (Cunha et al. 2010a, Becker et al. 2012) and menthol (Simões and Gomes 2009), and also some essential oils, such as the ones obtained from Eugenia caryophyllata (Inoue et al. 2003), Lippia alba (Cunha et al. 2010b(Cunha et al. , 2011, Ocimum gratissimum (Benovit et al. 2012 and Melaleuca alternifolia (Hajek 2011) have shown anesthetic activity on different fish species.
The genus Aloysia (Verbenaceae) is originally from South America, with 34 species cataloged, among which 12 can be found in Brazil (Cordo and Deloach 1995). Certain species of Aloysia are known to exhibit central activity. A. gratissima (Gillies & Hook) Tronc. is known in Brazil as erva-santa (Souza and Wiest 20007), and has been used in folk medicine due to its sedative properties (Goleniowski et al. 2006). The essential oil of A. triphylla showed potent anesthetic activity in white shrimp (Litopenaeus vannamei)  and silver catfish (Rhamdia quelen) (Gressler et al. 2014). Benovit et al. (2012) verified anesthetic activity of essential oil of A. gratissima in Brazilian flounder (Paralichthys orbignyanus). However, there are no studies correlating its depressant effect with some chemical constituent.
This work aimed to determine the efficacy of the essential oil obtained from leaves of A. gratissima (EOA) as anesthetic for silver catfish (Rhamdia quelen), and to perform the bio-guided fractionation of EOA aiming to isolate the active compounds responsible for the noted effects. Acetylcholinesterase (AChE) activity under the influence of EOA was also investigated.

PLANT MATERIAL
Aerial parts of A. gratissima were collected in April 2010 in Santa Maria, Rio Grande do Sul, Brazil. A voucher specimen (SMDB 13.077) was kept in the Biology Department of the Universidade Federal de Santa Maria and identified by Professor Marcos Sobral, UFSJ, and Professor Solon Jonas Longhi, UFSM.
ESSENTIAL OIL EXTRACTION The essential oil was obtained from fresh leaves of A. gratissima by hydrodistillation with a Clevenger type apparatus for 3h (British Pharmacopoeia 2007). Samples of the essential oil were stored at −4°C in amber glass bottles prior to the tests or isolation.
Optical rotations were obtained on a Perkin Elmer 343 polarimeter. NMR spectra were recorded in CDCl 3 on Bruker HPX 300 FT-NMR at 400 MHz for 1 H and 100 MHz for 13 C with TMS as internal standard.
FRACTIONATION AND ISOLATION OF ESSENTIAL OIL (EOA) EOA (6.03 g) was first submitted to fractionation by column chromatography (CC, 3.5 x 52 cm) with 300 g silica gel 60 (Macherey-Nagel, 70-230 mesh) as stationary phase and hexane:acetone (99:1 v/v) as mobile phase. The fractions obtained (100 mL each) were gathered in 16 main fractions (Fractions A-P) based on TLC profile (silica gel F254; detection: anisaldehyde H 2 SO 4 ). The fractions G and J were subsequently fractionated.

EXPERIMENTAL PROCEDURE
Three groups of experiments were performed to evaluate the sedative and anesthetic potential, as well as the recovery of silver catfish exposed to EOA, fractions and isolated compounds. Animals were transferred to aquaria containing 1 L of water and the samples to be tested, these samples were previously diluted in ethanol 95% (1:10), to evaluate the time required to reach stages of anesthesia . In this method, each juvenile was used only once in order to observe deep sedation (S2), partial (S3a) and total loss (S3b) of equilibrium, anesthesia (S4) and/or medullar collapse (S5). Anesthesia was determined by loss of reflex activity and no reaction to strong external stimuli (pressure on caudal peduncle). The animals remained in the anesthetic bath until they reached S4 or for 30 min. After anesthesia induction, each fish was measured, weighed and transferred to an anesthetic-free aquarium to recover. The fish were considered to have recovered if their normal posture and behavior were restored, and the maximum time observed for this to occur was 30 min. After that, the animals were transferred to 30 L aquariums, where possible side effects (spasms, contortions, secretion of mucus, seizures) or mortality was examined after 24h of exposition. Control experiments were performed using aquarium containing ethanol at the highest concentration used to dilute the EOA. In experiment 1, the anesthetic activity of EOA was evaluated. Ten silver catfish juveniles (6.82 ± 2.04 g; 9.10 ± 0.91 cm) were used for each concentration of EOA (90, 300, 450, or 900 mg L -1 ). Experiment 2 was performed to compare the activity among 450 mg L -1 EOA and the fractions with higher chromatographic yield obtained by column chromatography (B, D, F, G and J). Six juveniles (8.66 ± 2.32 g; 9.52 ± 0.86 cm) were used for testing each fraction, at proportional concentrations to those of the major constituents in the EOA. In experiment 3, the sedative and anesthetic activities of the isolated compounds were evaluated. Six juveniles (6.99 ± 2.24 g; 9.44 ± 1.03 cm) were used for each concentration. The isolated compounds were evaluated at proportional concentrations to those of the constituents in the EOA at concentrations of 450, 675 and 900 mg L -1 , which corresponded to: 19.06, 28.60 and 38.14 mg L -1 of E-(-)-pinocamphone; 10.00, 15.00, 20.00 mg L -1 of (-)-caryophyllene oxide; 32.65, 48.96 and 65.30 mg L -1 of (-)-guaiol; and 12.80, 19.20 and 25.60 mg L -1 of (+)-spathulenol. Additional concentrations of (-)-caryophyllene oxide (40 mg L -1 ) and (+)-spathulenol (51.20 mg L -1 ) were tested. (-)-Guaiol and (+)-spathulenol were also evaluated together (32.65 and 12.80 mg L -1 , respectively). Eugenol at concentration of 50 mg L -1 was used as positive control (Cunha et al. 2010a).

LONG EXPOSURE TIME
This experiment evaluated the effects of long exposure time (24h) of silver catfish to EOA and (+)-spathulenol at sedative concentrations. Considering the sedative concentrations determined in the experiments described in the previous sections, four experimental groups were evaluated: EOA at 90 mg L -1 , (+)-spathulenol at 2.6 mg L -1 (proportional concentration of the constituent in the EOA at 90 mg L -1 ), ethanol (at the concentration used to dilute the EOA) and water.
Silver catfish (6.72 ± 2.01 g; 8.75 ± 1.02 cm) were transferred to aquaria containing 1 L of water and the sample to be tested. Six animals were used for each group to determine the time it would take to reach stage S3a. Initially, animal condition was evaluated in the first 30 min of exposure to confirm the occurrence of sedation and then after 24h, aiming to evaluate the toxicity of the substances during this time. After exposure, fish were transferred to anesthetic-free 7 L aquariums, where possible signs of intoxication or mortality were examined for an additional 24h.

ACETYLCHOLINESTERASE ASSAY
Six silver catfish juveniles (20.49 ± 6.71 g; 16.50 ± 1.33 cm) were exposed to each concentration of EOA (90, 300 or 450 mg L -1 ), firstly diluted in ethanol 95% (1:10). Control experiments were performed using water alone or ethanol at the highest concentration used to dilute the EOA.
Fish were euthanized by severing the spinal cord after they reached S4 or after 30 min of exposure (control group and concentrations that did not promote anesthesia). Whole brain and muscle samples were removed, packed in eppendorf tubes and kept at −20°C for AChE analysis. AChE assay was performed in homogenates of brain and muscle according to the method described by Ellman et al. (1961) and modified by Villescas et al. (1981). Homogenates (50-100 µL) were incubated with 0.8 mM of acetylthiocholine and 1.0 mM of 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) at 25°C for 2 min. Protein content was determined according to Bradford (1976). Enzyme activity was expressed as micromoles of acetylthiocholine hydrolyzed per minute per milligram of protein.

STATISTICAL ANALYSIS
Values are presented as mean ± SEM (standard error of the mean). A Levene test was used with all data to verify the homogeneity of variances. The results were analyzed by one-way ANOVA and Tukey test or Kruskal-Wallis and Dunn test, when appropriate. Comparison between spathulenol and eugenol was performed with t-test. The minimum significance level was set at P<0.05. Regression analyses were performed to EOA and isolated compounds.
Silver catfish reached anesthesia with EOA at concentrations ranging from 300 (approximately 18 min) to 900 mg L -1 (approximately 12 min). Fish exposed to 90 mg L -1 reached stage 3a after 8 min and they remained in this stage for 30 min of observation. There were no significant differences in the induction times to anesthesia  between the concentrations of 450 and 900 mg L -1 , but these induction times (approximately 12 min) were significantly faster than those of 300 mg L -1 . A significant relationship between the EOA concentration and the time required for anesthesia induction was noted for all stages (Table II), except for recovery time. Time of recovery was significantly faster at 900 mg L -1 (approximately 12 min) when compared to 300 and 450 mg L -1 . During this experiment, and 24h after it, no mortality occurred. However, involuntary muscle contractions were observed in the fish submitted to EOA during induction and recovery. The group exposed to ethanol showed light sedation (S2 in  approximately 7.9 min), but it was significantly different from all EOA-treated groups. AChE activity did not show any significant difference among treatments (Table III).
The fractions B, D, F, G and J showed higher yield from the EOA chromatography process. Similar chemical composition was verified for fractions F and G, while the others showed different major constituents (Table I). No sedative effect was observed for fraction D in silver catfish within 30 min of exposure. Fish exposed to fractions B and F reached only S3a (Table IV). Both fractions G and J resulted in anesthesia (S4) in 50% of fish with no significant differences between them. However, fraction G caused involuntary muscle contractions similar to those verified with the EOA. Fraction J caused no side effects, but it resulted in increased recovery time in relation to the other fractions. Since fractions G and J showed anesthetic or side effects, we chose to isolate their constituents in order to identify the possible active compounds. E-(-)-pinocamphone and (-)-caryophyllene oxide were obtained from fraction G, while purification of fraction J resulted in the isolation of (-)-guaiol and (+)-spathulenol. The isolated constituents were identified by comparing the MS and NMR data with literature reports (Coxon et al. 1984, Raharivelomana et al. 1995, Chaves and Santos 2002, Höld et al. 2002, Bolzan 2007  III Brain and muscle specific AChE activity (µmol min -1 mg protein -1 ) of Rhamdia quelen exposed to EOA. *Values are means ± SEM. Data was submitted to one-way ANOVA (P<0.05).

Juveniles submitted to E-(-)-pinocamphone
showed different behavior of sedated or anesthetized fish. In the first minutes of exposure, a period of hyperactivity could be noted. Animals tended to present faster and erratic swimming with partial loss of equilibrium, and sudden jumping towards the SIMONE C. BENOVIT et al.

Fraction
Time of induction to anesthesia (

TABLE IV Time (s) required for induction and recovery from anesthesia using EOA (at concentration of 450 mg L -1 ) and their fractions of higher yield (at proportional concentrations to those of the major constituents in the EOA) in Rhamdia quelen.
water surface. Afterwards, there was a period of total loss of equilibrium, animals tumbled on the bottom of the aquarium and remained immobile even after pressure on caudal peduncle, but they responded to external stimuli made with a glass rod in the bottom of the aquarium. Then, fish went back to swimming erratically, and, at that moment, involuntary muscle contractions were noted. Fish writhed to the right and left, or remained contracted in "C or S-shape". There was a significant relationship between the concentration of E-(-)-pinocamphone and the time required for hyperactivity stage (y = 283.198 − 15.111x + 0.228x 2 ; r 2 = 1) and tipping stage (y = − 13.117+18.650x-0.406x 2 ; r 2 = 1). Three juveniles were found dead 24h after the experiment.
The other isolated compounds reached stages S2 and S3a at proportional concentrations to EOA. Sedation (S2) obtained with (-)-caryophyllene oxide was not significantly different from EOA, except at the lowest concentration tested, or (+)-spathulenol (Fig. 1a). EOA was the most effective for loss of equilibrium (S3a), except at concentration of 675 mg L -1 , where it did not differ from (+)-spathulenol. (-)-Caryophyllene oxide and (-)-guaiol did not present statistical differences to reach stage S3a, except at the highest concentration, in which (-)-caryophyllene oxide was more effective (Fig. 1b). In addition, both compounds showed different induction times from EOA.
The association of (-)-guaiol and (+)-spathulenol resulted in decrease of induction time until stage S3a, when compared to the isolated compounds (Fig. 1b). However, animals submitted to this association showed mucus secretion and greater recovery time than with the isolated compounds (Fig. 1c). When compared with EOA, the recovery time of the association did not differ statistically. The three isolated compounds did not show statistical differences in the recovery time at all concentrations. Moreover, only one fish submitted to EOA recovered within 30 min at the concentration of 675 mg L -1 .
Regarding the long exposure time (24h), fish reached S3a at 241.50±20.28 s with EOA and 1752.50±70.94 s with (+)-spathulenol. After 24h of exposure, no mortality was observed, and the animals submitted to EOA and (+)-spathulenol were not sedated. Fish submitted to (+)-spathulenol did not present signs of intoxication or mortality after 24h in anesthetic-free aquaria. However, 33.33% of mortality was observed with EOA.

DISCUSSION
Previous studies showed quantitative differences in the chemical composition of the EO obtained from leaves of A. gratissima. Dambolena et al. (2010) identified 1,8-cineole (45.5%) and sabinene (8.3%) as major constituents. However, the proportion of these compounds found by these authors was different from the corresponding data found in the present study. Similar proportion of guaiol (6.27%) was found by Franco et al. (2007). Varied amounts of bicyclogermacrene (0 -12.8%) were described by Ricciardi et al. (2006), and this variation depended on sites and period of collection. Stages are according to Gomes et al. (2011). Maximum observation time was 30 min. N= 6 for each concentration tested. Data are expressed as mean ± SEM. Different letters indicate a significant difference between the different products at the same stage using t-test (P<0.05). Aloysia gratissima: ANESTHESIA AND FRACTIONATION Anesthesia (S4) is achieved when fish lose reflex activity and do not show a reaction to strong external stimuli . Silver catfish exposed to concentrations of 300-900 mg L −1 EOA reached this stage within 12-18 min. The concentration of 900 mg L −1 was the most effective, because it showed lower anesthetic induction and recovery time. Similar EOA concentrations (270-900 mg L −1 ) also provoked deep anesthesia in Brazilian flounder (Benovit et al. 2012). To reach the same level of anesthesia in a similar period of time (approximately 12 min) in silver catfish, 30 mg L −1 of eugenol and 100 mg L −1 of EO of Lippia alba are made necessary (Cunha et al. 2010a, b). Moreover, 200 µL L −1 of EO of Melaleuca alternifolia and 250 mg L −1 of menthol lead to S4 in common carp, Cyprinus carpio, and in Nile tilapia, Oreochromis niloticus, respectively (Simões andGomes 2009, Hajek 2011).
The fractionation process showed that compounds present in fraction D did not contribute to the depressor effects of EOA, while constituents of the fractions B and F could be involved in its sedative effect. Previous reports described central activity for some compounds present in fractions B and F, such as limonene, 1,8-cineol, α-and β-pinene (Kasanen et al. 1998, Santos and Rao 2000, Vale et al. 2002, which need to be confirmed in aquatic animals. On the other hand, fractions G and J promoted anesthesia in 50 % of fish, which indicates that the compounds present in these fractions contribute to the anesthetic effect of EOA. The involuntary muscle contractions and mortality observed in fish exposed to EOA and/ or fraction G may be explained by the presence of E-(-)-pinocamphone in these samples. Additionally, with this isolated compound the fish showed two distinct stages, hyperactivity and tipping. Similar effects were characterized as seizure in zebrafish, Danio renio, exposed to caffeine, pentylenetetrazole (PTZ) and picrotoxin (Wong et al. 2010). E-and Z-pinocamphone are known antagonists of gamma-aminobutyric acid type A (GABA A ) receptor (Höld et al. 2002). These compounds are able to promote convulsions in mouse that can be alleviated by diazepam, and their signs of intoxication are similar to picrotoxin (Höld et al. 2002). As fish submitted to E-(-)-pinocamphone showed more intense side effects than EOA, the compounds with sedative/ anesthetic activity present in EOA may have reduced their effects.
The results obtained for E-(-)-pinocamphone are not inconsistent with the results of the AChE assay of EOA. Action in AChE was researched because the muscle inhibition of this enzyme leads to increased acetylcholine levels, resulting in excessive muscular stimulation (Kirby et al. 2000). However, AChE activity was not affected by EOA at the tested concentrations and, therefore, the contractures observed on the fish were not due to alterations in the activity of this enzyme. On the other hand, this response could be attributed, as reported earlier, to the GABAergic system, and future studies should be conducted to clarify this issue.
(-)-Caryophyllene oxide and (-)-guaiol promoted sedation at all tested concentrations. Higher concentrations of these compounds were not used because they caused mucus secretion on the animals. Additionally, (-)-caryophyllene oxide showed light spasm in fish. Secretion of mucus has been observed with some synthetic anesthetics for fish, such as 2-phenoxyethanol, quinaldine sulfate and benzocaine (Inoue et al. 2003, Velisek et al. 2007); as well as 2,4-D [(2,4-dichlorophenoxy) acetic acid], a herbicide (Sarikaya and Yilmaz 2003). Caryophyllene oxide, isolated from the Psidium guajava, showed depressant effects on the central nervous system of mice, through potencialization of the pentobarbital induced-sleeping time (Meckes and Calzada 1996). However, studies on the central activity of (-)-guaiol were not found.
Sedation induced by (+)-spathulenol is in accordance with those described for the EO obtained from Baccharis uncinella, which showed sedative effects in mice and contains spathulenol as one of the major constituents (Ascari et al. 2012). There were no other studies related to the central activity of (+)-spathulenol. Since this compound did not cause any side effects at sedative concentrations, one higher concentration was tested, which resulted in anesthesia. In this concentration, fish submitted to (+)-spathulenol showed faster sedation, slower anesthesia and recovery, than eugenol. Eugenol is largely used as anesthetic for fish and its activity has been proven in different species (Vidal et al. 2007, Honczaryk and Inoue 2009, Cunha et al. 2010a, Becker et al. 2012). On the other hand, (+)-spathulenol promoted anesthesia in lower concentrations (51.2 mg L -1 ) than S-(+)-linalool (153 mg L -1 ) isolated from the EO of L. alba in silver catfish . Additionally, with S-(+)-linalool, fish did not recover within 30 min, similar to (+)-spathulenol in the present study.
(+)-Spathulenol at 2.6 mg L -1 was shown to be a safe compound to sedate silver catfish for 24h. An opposite pattern occurred with EOA, because mortality was noted after exposure for the same period of time. Spathulenol at 8 mg L -1 for 24h was classified by Ito et al. (2004) as ichthyotoxic in medaka, Oryzias latipes. It is probable that this concentration of spathulenol led to S5 of anesthesia induction (medullar collapse) and thus, caused mortality to this fish. An anesthetic may induce different results due to variations between fish species (Zahl et al. 2012).
A relationship between concentration and recovery time was noted in fish exposed to (-)-caryophyllene oxide and (-)-guaiol, while the other tested compounds did not show the same relation. Studies with eugenol and EO of L. alba in silver catfish also showed that the concentration of these substances did not influence in the recovery time (Cunha et al. 2010a, b).
The fractionation of EOA resulted in a decrease of the anesthetic potential. The isolated compounds did not promote anesthesia at proportional concentrations to those of the constituents in the EOA. Moreover, the association of (-)-guaiol and (+)-spathulenol caused a decrease in the time required to reach stage S3a. These evidences indicate that the anesthetic and sedative effects are due to the synergism of the constituents present in EOA. Similar results were observed regarding the anticonvulsive activity of the EO of Ocimum gratissimum in mice. Moreover, the EO of this plant was more effective, than their major constituents tested alone or in association, eugenol, 1,8-cineole and E-caryophyllene, which were evaluated in the same proportion found for the EO (Galindo et al. 2010).
Conversely, the hypothesis that the minor constituents are responsible for the anesthetic activity of EOA in silver catfish cannot be ruled out, since the fractions of lower yield were not tested. Essential oils contain several substances, which can show different mechanisms of action. These different mechanisms can act in synergy resulting in improved activity (Harris 2002), but antagonism can also occur (Savelev et al. 2003). Pharmacokinetic synergism can also be observed, where absorption, distribution, biotransformation or elimination of a compound can be influenced by other substance, obtaining positive results (Spinella 2002).
EOA showed anesthetic properties, however, it should be used carefully in silver catfish due to the observed side effects. Additionally, EOA had a higher induction time than ideal, of 3-5 min (Cunha et al. 2010b). The presence of E-(-)-pinocamphone and (-)-caryophyllene oxide contributed to the side effects of EOA, whereas anesthetic activity was partially related to (+)-spathulenol. Then, higher concentration of (+)-spathulenol, and lower or absent amounts of E-(-)-pinocamphone, could contribute to improve the anesthetic activity and safety of the EOA. (+)-Spathulenol was considered a promising anesthetic and sedative for aquatic animals.