2009 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 This project officially began on March 29, 2007, replacing project 6402-42000-002-00D. Progress and accomplishments related to the previous project were reported separately in 2006. To date, this 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. In spring of 2009, Field experiments were planted successfully as indicated above. Research directed toward environmental manipulation to minimize aflatoxin and optimize yield in corn is proceeding. Experiment is being conducted to evaluate corn hybrids under drought stress and irrigated conditions. Some physiological data related to drought stress are being collected. Plots have been inoculated with toxigenic A. flavus strain K54 and at harvest the corn samples will be evaluated for aflatoxin and fumonisin contaminations. Data collection on corn-soybean crop rotation experiment was completed in 2009. The impact on fungal inoculum source of Aspergillus contamination in non-Bt and Bt corn is planted in 2009 for the fourth season and will be harvested shortly. Research on biological and chemical methods for controlling aflatoxin in corn is progressing well in 2009. 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 biocontrol agent. Field studies in two locations are optimizing formulation for improvement of delivery of non-toxigenic A. flavus strains is in its second season. An experiment in one location was destroyed by severe winds and storms on June 23, 2009. The potential for using two bacteria isolated from corn field soil, to control mycotoxins on corn have been discontinued in field studies. The mycotoxin contamination in corn for the past three seasons 2006 to 2008 was not reduced. This was effective under controlled conditions but not in the field. Inbred lines of maize were developed with a new source of resistance to corn earworm. Closely related susceptible inbred lines were also developed. These lines will enhance a new series of studies to develop genetic markers for the new trait, understand the mechanisms of resistance and determine genetic control. The corn earworm resistance lines are not resistant to aflatoxin contamination per se; however, they are highly resistant to corn earworm whose feeding can worsen infection of the corn ear by A. flavus. The new trait will be useful for developing insect resistant corn hybrids that may reduce the incidence of mycotoxins through reduced insect feeding. Some of the research on this project is being done in cooperation with scientists at other institutions; according the following agreements 6402-42000-003-01S, 6402-42000-003-04N, 6402-42000-003-05T, 6402-42000-003-06N. Work completed on these agreements will be submitted in separate reports. 4.Accomplishments 1. Aspergillus flavus and Aflatoxin in Soil. Soil in agricultural fields plays a major role in the life cycle of A. flavus, and is used in providing the primary inoculum of fungus during the growing season. Little is known about the saprophytic activities of these fungi in soil. A two year survey of Aspergillus populations was conducted generating a culture collection of over 1,200 isolates, including several strains that were later demonstrated to be useful for biocontrol. This information is useful in assessing the risks of soil conservation practices in relation to potential problems of food quality for corn growers and to the corn processing industry. 2. Effects of Planting Date on Mycotoxins in Bt- corn Hybrids in Arkansas. Farmers are interested in determining optimum planting dates for corn in Arkansas to avoid mycotoxin contamination. Mid-April was identified as the optimal planting date for growing short-season cultivars to reduce mycotoxin risk. Three years of field studies using several Bt- and non- Bt corn hybrids in mid-April showed a reduction in mycotoxin levels compared to mid- May plantings. Bt- corn hybrids showed more resistance to mycotoxin than non-Bt corn hybrids. These studies have provided information to growers regarding what hybrids to use to decrease mycotoxin contamination and have assisted breeders in developing strategies for the next generation of hybrids. 6.Technology Transfer Number of Invention Disclosures Submitted 1 Number of Other Technology Transfer 3 Review Publications Myung, K., Li, S., Butchko, R.A., Busman, M., Proctor, R., Abbas, H.K., Calvo, A.M. 2009. FvVE1 Regulates Biosynthesis of the Mycotixins and Fumonisins and Fusarims verticillioides. Journal of Agriculture and Food Chemistry. 57(11):5089-94 Wilkinson, J.R., Abbas, H.K. 2008. Aflatoxin, Aspergillus, Maize, and the Relevance to Alternative Fuels (or Aflatoxin: What is It, Can We Get Rid of It, and Should the Ethanol Industry Care?). Journal of Toxicology Toxins Reviews. 27:227-260 Abbas, H.K., Accinelli, C., Zablotowicz, R.M., Abel, C.A., Bruns, H.A., Dong, Y., Shier, W.T. 2008. Dynamics of Mycotoxin Concentrations in Aging Corn Residues Under Mississippi No-Till Conditions. Journal of Agricultural and Food Chemistry. 56:7578-7585 Palumbo, J.D., O Keeffe, T.L., Abbas, H.K. 2008. Microbial interactions with mycotoxigenic fungi and mycotoxins. Journal of Toxicology Toxins Reviews.27(3):261-285. Lyn, M.E., Abbas, H.K., Zablotowicz, R.M., Johnson, B.J. 2009. Delivery systems for biological control agents to manage aflatoxin contamination of pre-harvest maize. 2009. Food Additives & Contaminants. Part A(26):381-387. Accinelli, C., Sacca, M.L., Abbas, H.K., Zablotowicz, R.M., Wilkinson, J.R. 2009. Use of a Granular Bioplastic Formulation for Carrying Conidia of a Non-aflatoxigenic Strain of Aspergillus flavus. Biological Control. 100:3997-4004, 2009. Baird, R.E., Wadl, P.A., Wang, X.W., Hadziabdic, D., Rinehart, T.A., Abbas, H.K., Shier, T., Trigiano, R.N. 2009. Microsatellites from the Charcoal Rot Fungus (Macrophomina phaseolina). Molecular Ecology Resourcespgs 946-948.