2011 Annual Report 1a.Objectives (from AD-416) 1. Refine aflatoxin biocontrol technology for peanuts and develop an effective system for achieving biological control of aflatoxins in corn, an important crop grown in rotation with peanuts. 2. Determine characteristics of soil populations important for invasion of peanut seeds by aflatoxigenic fungi and evaluate the competitiveness of nontoxigenic biocontrol strains of A. flavus. 3. Determine the chemical barriers of peanut to fungal challenge, particularly challenge by A. flavus. Investigate the basis for greater resistance to A. flavus invasion and aflatoxin contamination possessed by certain peanut genotypes for possible exploitation in breeding programs. 4. Conduct the necessary laboratory and field trials required by the EPA to extend the use of Aflaguard to other crops susceptible to aflatoxin, such as corn. 1b.Approach (from AD-416) Experiments to extend the shelf life of afla-guard(r) will be conducted by producing afla-guard(r) with a variety of oils covering a range of oxidative stabilities. Samples will be placed in long-term storage at 4 degrees, 23 degrees, 30 degrees, 37 degrees, and 44 degrees C and tested once a month to determine the survival and viability of conidia on the coated barley. A multi-year (at least three) study will be conducted to determine the possibility of achieving biological control of aflatoxin contamination of corn. The field tests will include two plantings (3-4 weeks apart) of four treatments in a randomized complete block design with eight replications. Corn will be ground in a Romer subsampling mill, and the quantity and toxigenicity of A. flavus in the corn will be determined. Aflatoxins will be quantified in the same samples. Native fungal populations in 20 different soils will be quantified and species will be identified either directly on the dilution plates or by subculturing to Czapek agar slants. Peanut seeds will be aseptically wounded and inoculated with 7.0 mg of soil paste using a small spatula. Forty seeds will be inoculated with each soil and incubated 14 d at 37 C. Twenty-four uninoculated wounded seeds will serve as controls in each experiment. A. flavus and A. parasiticus sporulating on seeds will be identified by subculturing to Czapek agar slants. In a related series of experiments, nontoxigenic biocontrol strains (conidial-color mutant A. parasiticus NRRL 21369 and a nitrate-nonutilizing mutant of A. flavus NRRL 21882) will be added to soils at different concentrations to examine their interactions with native aflatoxin-producing populations. Aflatoxin analyses of individual seeds will be performed by extracting overnight in methanol and quantifying with high performance liquid chromatography. A series of experiments will be conducted to. 1)isolate, identify, and quantify chemicals produced in peanuts in response to fungal invasion;. 2)characterize the chemical response of peanuts representing a range of pod/kernel maturity to fungal challenge;. 3)characterize the chemical responses of peanuts representing a genotypic range of recognized differences in susceptibility to A. flavus invasion and aflatoxin contamination;. 4)characterize peanut wax composition and evaluate different genotypes for peanut wax content and composition. 3.Progress Report The sexual stage of the aflatoxin-producing fungus Aspergillus nomius was discovered by crossing strains of the opposite mating type. The two other major aflatoxin-producing species, A. flavus and A. parasiticus, were earlier shown through this collaboration to also reproduce sexually. Progeny strains from A. nomius crosses often differed in aflatoxin production compared to parental strains and are being examined at the molecular level for genetic recombination. In addition, DNA analyses of A. flavus progeny obtained from sexual crosses were completed in a collaborative effort with North Carolina State University and data showed extensive recombination with respect to the aflatoxin gene cluster due to the independent assortment of chromosomes and crossing over within the gene cluster. These recombination events coincide with patterns of genetic variation observed in field populations of A. flavus. Evaluation of eight non-toxigenic strains of A. flavus for biological control of aflatoxin was completed and the data were analyzed statistically. The laboratory assay involved the co-inoculation of viable, artificially wounded peanut seeds with mixtures of non-toxigenic and aflatoxin-producing strains. Eight non-toxigenic strains (including the strain present in Afla-Guard®) that differed genetically in their inability to produce aflatoxin were paired with eight genetically different aflatoxin-producing strains in all combinations. Five of the eight non-toxigenic strains were superior to the Afla-Guard® strain in reducing aflatoxin in peanuts. The dynamics of phytoalexin synthesis in seeds from different peanut genotypes were investigated. Disease-resistant cultivars demonstrated a faster defensive response to fungal invasion and a higher production of stilbenoid phytoalexins compared to susceptible peanut cultivars. In addition, the first systematic study on the biological activity of all known peanut stilbenoids and their analogs was completed. New stilbenoids were discovered and examined for biological activity. These compounds possessed significantly higher activity against fungal pathogens than other major peanut stilbenoids. Furthermore, the new stilbenoids showed strong antioxidant, anticancer and anti-inflammatory properties in a panel of human cell lines. Lower hydrophobicity of stilbenoids was associated with higher biological activity in numerous fungal and human-cell assays. The position of hydroxy and prenyl groups on the carbon skeleton of stilbenoids had a significant effect on their biological activity. Molecular markers are being developed to aid in the breeding of peanuts for disease resistance. A phytoene desaturase gene is being tested as a selectable marker for herbicide resistance in peanut in the development of a method for genetic transformation. Molecular markers for Valencia peanut also are being developed in collaboration with the University of New Mexico to be used in their breeding program. In addition, molecular markers are being developed for Cercospora species that infect soybeans and peanuts. This bridged project replaces #6604-42000-008-00D through review of the National Program. 4.Accomplishments 1. Sexual reproduction is responsible for variation in aflatoxin production by Aspergillus. Collaboration between ARS researchers at Dawson, GA, and North Carolina State University is directed toward understanding the origins of genetic variation in aflatoxin-producing Aspergillus. The major aflatoxin-producing fungi, A. flavus, A. parasiticus and A. nomius, were previously considered to be strictly asexual in reproduction. In this study, mating-type genes were identified and strains of opposite mating type were crossed, resulting in the formation of the sexual stage in all three species. Approximately 3500 progeny strains from sexual crosses have been generated. Examination of progeny showed that meiosis results in genetic recombination with respect to the aflatoxin gene cluster due to the independent assortment of chromosomes and crossing over within the gene cluster. Therefore, sexual reproduction is responsible for the genetic variation in aflatoxin production by Aspergillus in crops. The discovery of sexuality redefines all that is known about the biology of aflatoxin-producing fungi and will be an important consideration in devising control measures for aflatoxin. 2. Evaluation of new strains of non-toxigenic fungi for biological control of aflatoxin in crops. Aflatoxin is a potent carcinogen produced by fungi in crops, and contamination of commodities with aflatoxin threatens the competiveness of United States agriculture in the world market. Application of non-toxigenic strains of Aspergillus flavus to crops effectively reduces aflatoxin contamination. Based on a laboratory peanut seed assay, ARS researchers at Dawson, GA, showed that five non-toxigenic strains of A. flavus exhibited a greater reduction of aflatoxin than the currently used strain that is incorporated into the biocontrol formulation Afla-Guard®. A patent has been submitted for the use of these strains in the biological control of aflatoxin. 3. Evaluation of new peanut phytoalexins for crop resistance and medical applications. Peanuts produce defensive phytoalexins as a means to resist invasion by pathogens but little is known about the specific biological activity of these compounds. New stilbenoid phytoalexins were discovered in peanuts by ARS researchers in Dawson, GA. These compounds possess significantly higher activity against fungal pathogens than other major peanut stilbenoids, which makes them key objects for consideration in peanut breeding programs. In addition, the new stilbenoids were shown to possess strong antioxidant, anticancer and anti-inflammatory properties in a panel of human cell lines; therefore, these compounds may find applications in medicine. 4. Development of genetic tools to monitor populations of fall armyworm. Fall armyworm, Spodoptera frugiperda, is an insect pest that causes large economic losses in crops such as corn, rice and cotton. In Puerto Rico, the fall armyworm has developed resistance to the typical mode of control, Bt-toxin. Concerns exist about Bt resistance spreading to the continental USA; however, molecular tools were previously inadequate for monitoring populations. Molecular markers were developed for this insect by ARS researchers at Stoneville, MS, and Dawson, GA, and were shown to be effective in identifying S. frugiperda populations. These markers are now used as tools to monitor possible invasion and cross breeding involving Bt-resistant fall armyworm in the USA and Latin American countries. 5. Development of molecular markers for charcoal-rot disease in soybean. Molecular markers were developed by ARS researchers at Stoneville, MS, and Dawson, GA, for the fungal pathogen Macrophomina phaseolina, which causes charcoal-rot disease in soybean. For years farmers needed to know whether isolates of this pathogen were host specific or differed in pathogenicity. This knowledge is necessary for making decisions on pesticide applications and crop rotations, as well as for incorporating resistance to charcoal rot in breeding programs. The molecular markers for M. phaseolina were shown to be associated with physiological functions and host specificity. These markers are being used by soybean breeders and mycologists in ARS in Mississippi and Tennessee. Review Publications Arias, R.S., Blanco, C.A., Portilla, M., Snodgrass, G.L., Scheffler, B.E. 2011. First microsatellites from Spodoptera frugiperda (Lepidoptera: Noctuidae) and their potential use for population genetics. Annals of the Entomological Society of America. 104(3):576-587. Arias, R.S., Ray, J.D., Mengistu, A., Scheffler, B.E. 2011. Discriminating microsatellites from Macrophomina phaseolina and their potential association to biological functions. Plant Pathology. 60(4):709-718 DOI:10.1111/j.1365-3059.2010.02421.x. Arias, R.S., Stetina, S.R., Scheffler, B.E. 2011. Comparison of whole-genome amplifications for microsatellite genotyping of Rotylenchulus reniformis. Electronic Journal of Biotechnology. DOI:10.2225/vol14-issue3-fulltext-13. Arias, R.S., Techen, N., Rinehart, T.A., Olsen, R.T., Kirkbride, J.H., Scheffler, B.E. 2010. Development of simple sequence repeat markers for Chionanthus retusus (Oleaceae) and effective discrimination of closely related taxa. HortScience. 46(1):23-29. Blanco, C., Portilla, M., Jurat-Fuentes, J., Sanchez, J.F., Viteri, D., Vega-Aquin, P., Teran-Vargas, A.P., Azuara-Dominguez, A., Lopez, J., Arias De Ares, R.S., Zhu, Y., Barrera, D., Jackson, R.E. 2010. Susceptibility of Spodoptera frugiperda (Lepidoptera: noctuidae) isofamilies to Cry1Ac and Cry1F proteins of Bacillus thuringiensis. Southwestern Entomologist. 35(3):409-415. Han, K.M., Dharmawardhana, P., Arias, R.S., Ma, C., Busov, V., Strauss, S.H. 2011. Gibberellin-associated cisgenes modify growth, stature and wood properties in Populus. Plant Biotechnology Journal. 9(2):162-178. Sobolev, V., Khan, S.I., Tabanca, N., Wedge, D.E., Manly, S.P., Cutler, S.J., Coy, M.R., Becnel, J.J., Neff, S.A., Gloer, J.B. 2011. Biological Activity of Peanut (Arachis hypogaea) Phytoalexins and Selected Natural and Synthetic Stilbenoids. Journal of Agricultural and Food Chemistry. 59:1673-1682. Sheppard, G.S., Berthiller, F., Dorner, J.W., Lombaert, G.A., Malone, B., Maragos, C.M., Sabino, M., Solfrizzo, M., Trucksess, M.W., Van Egmond10, H.P., Whitaker, T.B. 2010. Developments in mycotoxin analysis: an update for 2008-2009. World Mycotoxin Journal. 3(1):3-23. Horn, B.W., Moore, G.G., Carbone, I. 2011. Sexual reproduction in aflatoxin-producing Aspergillus nomius. Mycologia 103:174-183. Abbas, H.K., Zablotowicz, R.M., Horn, B.W., Phillips, N.A., Johnson, B.J., Jin, X., Abel, C.A. 2011. Comparison of major biocontrol strains of non-aflatoxigenic Aspergillus flavus for the reduction of aflatoxins and cyclopiazonic acid in maize. Journal of Food Additives & Contaminants. 28:198-208. Horn, B.W., Dorner, J.W. 2011. Evaluation of different genotypes of nontoxigenic Aspergillus flavus for their ability to reduce aflatoxin contamination in peanuts. Biocontrol Science and Technology. 21(7):865-876. Sobolev, V., Neff, S.A., Gloer, J.B., Khan, S.I., Tabanca, N., De Lucca Ii, A.J., Wedge, D.E. 2010. New pterocarpenes elicited by Aspergillus caelatus in peanut (Arachis hypogaea) seeds. Phytochemistry and Agriculture. 71:2099-2107.