2010 Annual Report 1a.Objectives (from AD-416) Optimize agronomic systems and environmental practices, including fertilization and rotation that minimize inoculum potential of Aspergillus flavus and other mycotoxin-producing fungi while maximizing corn yield and profits in the Mid-south USA. Gain an understanding of the role of crop management practices on the ecology of Aspergillus flavus and aflatoxin contamination in order to optimize the application of competitive exclusion products. Develop economical biologically-based strategies, including antagonists from soil fungi and bacteria, microbial competitors and antagonists, and natural compounds from biological sources, to minimize mycotoxins and their respective fungi in corn, and continue ecological studies on Aspergillus populations under various management strategies, e.g., rotations, tillage, and cover crops, and herbicide-resistant crops. Evaluate insect-resistant and susceptible maize lines for insect damage and aflatoxin and fumonisin contamination. Evaluate corn accession for aflatoxin resistance. Develop isolines with stable resistance and susceptibility. Cooperate with industry in using molecular markers to develop resistent inbreds/hybrids. 1b.Approach (from AD-416) Evaluate potassium fertility as a means to decrease mycotoxins. Evlaluate corn maturity as a management practice to avoid heat and/or drought stress and mycotoxins. Evaluate double-cropped corn for mycotoxin contamination. Evaluate corn-soybean rotation to reduce fungal inoculum. Characterize population dynamics of Aspergillus propagules in soil, air, insects and corn at various ontogeny in BT and conventional corn. Compare efficacy of non-toxigenic A. flavus strains as biocontrol agents. Optimize surfactant concentration and formulation for improvement of efficacy of non-toxigenic strains. Determine efficacy of Pichia anomala to control aflatoxin and fumonisin. Identify and characterize bacteria from corn field soils as biocontrol agents of Aspergillus and Fusarium. Isolate and identify factors in corn-earn-worm-resistant corn silks for control of insects and mycotoxins. Cooperate with breeders to develop isolines and inbreds/hybrids. 3.Progress Report The project is proceeding as anticipated. The initial focus was on planning and establishing field experiments with other lab and greenhouse projects to follow as the year progressed. Research in corn physiology for the FY 2010 mainly focuses on determining how much aflatoxin contamination can be reduced by (a) combining biological control method with new stress tolerant commercial hybrids and (b) using new drought and heat stress tolerant commercial hybrids, as compared to aflatoxin resistant corn. The experiment in (a) is planted and being evaluated in two locations in Stoneville and (b) is being evaluated under irrigated and non-irrigated conditions for the second year by monitoring stress levels using physiological and ecological parameters. A complementary greenhouse physiological experiment is being conducted for these field studies. A third study on evaluation of dry grind ethanol commercial hybrids for aflatoxin contamination is also being conducted in two locations in Stoneville. Plots have been inoculated with toxigenic Aspergillus (A.) flavus strain K54 and at harvest the corn samples will be evaluated for aflatoxin and fumonisin contaminations. Research on biological methods for controlling aflatoxin in corn is progressing in 2010. Field studies are continuing using the pin-bar inoculation technique to characterize the colonization patterns of corn by various strains of A. flavus including K49 as a biocontrol agent. Field studies, in two locations, for optimizing formulation for improved delivery of non-toxigenic A. flavus strains is in its first season. Field trials are going on in 7 states for evaluating this new technology, “Development and Evaluation of Water Dispersible Granular Formulations of Non-toxigenic Aspergillus flavus Strains”. This work established in a “CRADA” which was completed on May 25, 2010, and it is progressing well by both teams. Some of the research on this project is being done in cooperation with scientists at other institutions. Research in objective 1 is being conducted by Louisiana State University (LSU) under a Specific Cooperative Agreement between ARS and LSU. Separate report summarize work completed under this agreement (subordinate project 6402-42000-003-01S). Research in objective 1 is being conducted by Mississippi State University (MSU) under a Non-funded Specific Cooperative Agreement between ARS and MSU. A separate report summarize work completed under this agreement (subordinate project 6402-42000-003-06N). A scientist at University of Bologna, Italy, is cooperating on research listed in objective 2 under a Non Funded Cooperative Agreement between ARS and University of Bologna. A separate report summarizes work completed under this agreement (subordinate project 6402-42000-003-04N). Research in objective 2 is being conducted by Mississippi State University under a Funded Specific Cooperative Agreement between ARS and MSU. A separate report summarizes work completed under this agreement (subordinate project 6402-42000-003-05T). 4.Accomplishments 1. Development of Non-aflatoxigenic Aspergillus (A.) flavus Strains. Field evaluations assessing the efficacy of A. flavus, K49 have been focused on characterization of competitive displacement of toxigenic isolates under field conditions, and assessments of two novel formulations for application of non-toxigenic isolates via soil application (bioplastic granules) and directed spray application (water dispersible granule formulation). Limited incidence of aflatoxin contamination in research plots the previous year provided limited data on efficacy of control, although information on colonization potential was developed. The Patent Provisional Application was converted to a full patent in February 3 and filed on February 10, 2010. 2. Development of a Quantitative Pin–bar and Side Needle Inoculation Techniques. These techniques were developed for use in field studies to characterize the dynamic and optimum colonization of non-toxigenic Aspergillus (A.) flavus as biological control strains. These were used to compare and to evaluate colonization by non-formulated spores of A. flavus. Using these two technique, Side needle injection and pin-bar inoculation techniques were found better than spray, tooth pick inoculation, and sprinkling wheat infected with Aspergillus flavus on the ground. The development of these techniques led to many benefits, including the accurately and economically screening of large numbers of Aspergillus strains. Also, these two techniques can be used in a screening study for resistance and in a fungal ecology study, especially biocontrol of aflatoxin in corn. 3. Comparisons of Herbicide Treated and Cultivated Herbicide-Resistant Hybrids. No differences in yields or mycotoxin contamination occurred in herbicide-resistant corn hybrids when treated with their respective herbicide when compared to receiving no herbicide applications and only cultivated for weed control. 4. Applying Additional Potassium Fertilizer as a Method of Reducing Aflatoxin in Corn. Applying extra levels of potassium had no effect in reducing aflatoxin contamination in pre-harvest corn. Yields did not increase due to the added potassium and in fact declined on a sandy loam soil at the higher rates of fertilizer application due to probably interference in the uptake of other nutrients. Review Publications Bruns, H.A., Abbas, H.K. 2010. Comparisons of Herbicide Treated and Cultivated Herbicide-Resistant Corn. Field Crops Research. doi:10.1155/2010/798127. Baird, R., Mcneil, D., Wadl, P.A., Trigiano, R.N., Allen, T., Shier, T., Wang, X., Rinehart, T.A., Abbas, H.K., Moulton, J.K. 2010. Variability of United States isolates of Macrophomina phaseolina based on simple sequence repeats and cross genus transferability to related Botryosphaeraceae. Mycopathologia. 170(3):169-80. Abbas, H.K. 2008. Introduction to the Special Issues on Emerging Issues in Mycotoxin Research. Journal of Toxicology Toxins Reviews. 28(2&3):61-62. Bruns, H.A., Abbas, H.K. Aflatoxin Contamination in Corn Differs Among Inoculation Techniques. Plant Health Progress, Published on-line - DOI:10.1094/PHP-2010-0601-01-RS. Palumbo, J.D., O Keeffe, T.L., Kattan, A., Abbas, H.K., Johnson, B.J. 2010. Inhibition of Aspergillus flavus in soil by antagonistic Pseudomonas strains reduces the potential for airborne spore dispersal. Phytopathology. 100:532-538.