2011 Annual Report 1a.Objectives (from AD-416) 1. Understand the phylogenetics of aflatoxin production through an examination of the population dynamics of aflatoxigenic and atoxigenic Aspergillus (A.) species in mixed cultures. Determine the potential for restoration of toxigenicity in toxigenic strains of A. flavus and development of toxicity in other species or genera which contain partial aflatoxin gene clusters. 2. Identify A. flavus pathogenicity factors required during invasion of oilseed crops and link these factors to aflatoxin contamination. Identify the critical enzymes that will allow the formulation of viable biological control strategies for a given crop situation. 1b.Approach (from AD-416) Aflatoxins (AFs) are polyketide-derived, toxic and carcinogenic secondary metabolites produced mainly by Aspergillus (A.) flavus on cotton, corn, peanuts, and tree nuts. One of the main strategies for prevention of AF contamination in these crops involves introducing a non-aflatoxigenic competitor Aspergillus to the soil of the plants during the growing season. Recent work revealed that A. flavus has a complicated evolutionary history that includes a history of recombination. It is the purpose of this proposal to better understand A. flavus population dynamics in agricultural environments in order to more safely use biocontrol as a strategy to reduce crop contamination with AFs. To do this we will develop new competitor strains with improved properties for tracking their dispersal after introduction onto crops and with improved ability to over-winter in order to decrease the need for annual reapplication. Optimal candidates should be unable to produce the neurotoxin, cyclopiazonic acid, without altering their competitive ability. The potential for restoration of both AF- and CPA-producing ability of the atoxigenic strain in the laboratory or the field will be assessed. We will determine if the ability to produce hydrolases by the biocontrol strain is important for its competitive ability. With these studies, we expect to be able to either develop new biocontrol strains or improve the design of currently used biocontrol strains to reduce preharvest AF contamination. We also expect to provide additional insight into the evolution of diversity within A. flavus. 3.Progress Report This project started in January 2011 and replaced project 6435-42000-020-00D "Identification and Enhancement of Seed-Based Biochemical Resistance in Crops to Aflatoxin Producing Pathogens" which terminated in December 2010. We are in the process of preparing a green-fluorescent protein-labeled fungus that can be tracked while in the field. Using this marked fungus we will assess fungal population dynamics of natural non-aflatoxigenic strains in corn and cotton fields. We are identifying specific genes that are turned on in fungal (Aspergillus (A.) flavus) cells when this fungus infects corn and cottonseed. We are trying to find specific enzymes that the fungus uses to invade the crop tissues. Once we identify these enzymes we will examine different A. flavus strains to see if there are significant differences between these strains in their ability to produce these enzymes, and consequently their ability to invade the crops, and produce aflatoxins.