2007 Annual Report 1a.Objectives (from AD-416) (1) Improve manufacture and formulation of ATOX for management of aflatoxin contamination. Specifically address problems in applying atoxigenic strain technology to commercial practice and optimize formulation for successful delivery of biocontrol agents. (2) Characterize influences of agronomic practices (tillage, irrigation, crop rotation, application strategy) on biocontrol of aflatoxin contamination and optimize field use of ATOX on a commercial scale. (3) Develop an epidemiological model to explain formation of aflatoxins in Texas and Arizona, with emphasis on the contamination after crop maturation (the second phase of contamination). (4) Characterize the major vegetative compatibility groups (VCGs) associated with Arizona and Texas agriculture and associated niches, and test geographical and niche specialization in order to improve understanding of the etiology of contamination and selection of ATOX with improved competitive ability in target crops/areas. Develop both a single nucleotide polymorphism (SNP) database and molecular tracking techniques useful in characterizing the composition of A. flavus communities, at both the strain and VCG level on crops and in the environment. (5) Characterize A. flavus responses to varying environments and ecological niches (including animal and plant hosts) to assess fungal adaptations leading to niche competence and to facilitate selection of elite biocontrol strains and development of management practices. Data will be used to contribute to select elite biocontrol strains with improved efficacy and safety. Objectives 1-5 address development of a knowledge base and technologies that could have generic value in protection of all the crops vulnerable to aflatoxin contamination. 1b.Approach (from AD-416) Develop improved formulations and production techniques to address problems in commercial practice of biological control and to increase efficacy against aflatoxin producing fungi. Through field tests and retrospective analyses, characterize influences of agronomic practices on biological control and use recommendations. Apply geostatistical and epidemiological tools to development of a model to predict aflatoxin contamination after crop maturation. Collect representative A. flavus from crops in Texas and Arizona and characterize strain specialization, adaptive traits, and optimal atoxigenic strains for distinct cropping systems. Develop both a SNP database for differentiating distinct A. flavus strains and a molecular technique for quantifying strain incidence in environmental samples. Select elite biocontrol strains based on improved knowledge of A. flavus adaptations and responses to relevant environments and ecological niches. 3.Progress Report This ARS research project is also the parent project of the following cooperative agreements involved in like research: Cotton Foundation/6435-42000-020-02T; Arizona Cotton Research & Protection Council/6435-42000-020-03S; University of Arizona/6435-42000-020-04S; University of Arizona/6435-42000-020-06S; and Cotton Incorporated/6435-42000-020-07R. 4.Accomplishments Identification of the causal agent responsible for poisonous maize in Kenya. -- Maize may become highly contaminated with aflatoxins both in the U.S. and Africa. In Kenya, hundreds of people died from acute aflatoxin poisoning from highly contaminated maize. The specific fungus causing the contamination episodes was identified to be the S strain of Aspergillus flavus, a fungus that also infects maize in central Texas. This is a rare case of actually identifying the specific fungus causing an aflatoxin outbreak. Identification of the causal agent is an important first step in developing rational approaches to management. This research is covered under National Program 108 Component 2, Mycotoxins and Plant Toxins, Problem Statement 2.1.2: Crop/Fungal/Insect/Toxin Relationships, Problem Statement 2.1.3: Production Practices and Expert Systems and Problem Statement 2.1.5: Biocontrol Technologies. Vegetative compatibility groups of A. flavus behave as clones. -- A. flavus is divided into many different genetic groups known as vegetative compatibility groups. There has been controversy about potential genetic exchange among these vegetative compatibility groups VCGs) in recent years. Deoxyribonucleic acid (DNA) sequences of multiple A. flavus isolates belonging to each of 5 vegetative compatibility groups collected from several states were compared. The resulting DNA finger prints indicate that, at least in the examined group of isolates, genetic exchange among groups did not occur. The results suggest that A. flavus reproduces and disperses clonally in U.S. agriculture and that biocontrol strains of A. flavus are unlikely to obtain the ability to produce aflatoxins from aflatoxin producers. Management needs to be directed at specific clones that have abilities both to prosper in crop environments and to produce large quantities of aflatoxins. This research is covered under National Program 108 Component 2, Mycotoxins and Plant Toxins, Problem Statement 2.1.2: Crop/Fungal/Insect/Toxin Relationships, Problem Statement 2.1.3: Production Practices and Expert Systems and Problem Statement 2.1.5: Biocontrol Technologies. Resolution of A. flavus community structure. -- Quantities and distributions of vegetative compatibility groups (VCGs), clonal genetic groups, of Aspergillus flavus in agricultural fields are highly complex. Such observations in earlier studies indicate a lack of host specialization among the many A. flavus vegetative compatibility groups. Commercial cottonseed crops in south Texas and southern Arizona were sampled to represent the crop across vast areas over a four-year period. A randomized representative sample of A. flavus associated with the crop was subjected to vegetative compatibility analyses. The results indicated that certain VCGs frequently infect cottonseed in both Texas and Arizona and suggest these strains are adapted to success in the cotton crop environment. The most common atoxigenic VCG was found to be the VCG to which the biocontrol strain AF36 belongs. This suggests that AF36 is the atoxigenic strain best adapted to cotton production in Arizona and Texas and may be the best biocontrol agent for contamination of cottonseed in those states. Several other relatively common atoxigenics were also identified that might be useful in improving biocontrol by use with AF36. In addition, several aflatoxin-producing VCGs were found to be prevalent and possibly important etiologic (causal) agents of contamination and, as such, important targets for management efforts and good agents to include in screens seeking germplasm resistant to aflatoxin contamination. The A. flavus population structure detected by vegetative compatibility analyses also suggests that recombination (i.e. a sexual growth phase) is uncommon or nonexistent among A. flavus associated with cotton. This research is covered under National Program 108 Component 2, Mycotoxins and Plant Toxins, Problem Statement 2.1.2: Crop/Fungal/Insect/Toxin Relationships, Problem Statement 2.1.3: Production Practices and Expert Systems and Problem Statement 2.1.5: Biocontrol Technologies. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of web sites managed 1 Number of non-peer reviewed presentations and proceedings 15 Number of newspaper articles and other presentations for non-science audiences 25 Review Publications Ehrlich, K., Kobbeman, K., Montalbano, B.G., Cotty, P.J. 2006. Aflatoxin-Producing Aspergillus Species from Thailand. International Journal of Food Microbiology. 114:153-159. Ehrlich, K., Montalbano, B.G., Cotty, P.J. 2007. Analysis of Single Nucleotide Polymorphisms in Three Genes Shows Evidence for Genetic Isolation of Certain Aspergillus flavus Vegetative Compatibility Groups. Federation of European Microbiological Societies Microbiology Letters. 268(2):231-236. Probst, C., Njapau, H., Cotty, P.J. 2007. Outbreak of an Acute Aflatoxicosis in Kenya in 2004: Identification of the Causal Agent. Applied and Environmental Microbiology. 73(8):2762-2764. Cotty, P.J., Mellon, J.E. 2006. Ecology of aflatoxin producing fungi and biocontrol of aflatoxin contamination. Mycotoxin Research. 22(2):110-117. Cotty, P.J. 2006. Biocompetitive Exclusion of Toxigenic Fungi. In: Barug, D., Bhatnagar, D., van Egmond, H.P., van der Kamp, J.W., van Osenbruggen, W.A., Visconti, A., editors. The Mycotoxin Factbook. The Netherlands: Wageningen Academic Publishers. 400 p.