2006 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Aflatoxins are carcinogenic toxins produced by members of the Aspergillus flavus group of fungi. Contamination with aflatoxins is the most important problem associated with crop infection by the fungus, A. flavus. Many countries have regulations prohibiting contamination above specified limits. In the U.S., contamination of foods and many feeds may not exceed 20 parts per billion (ppb). Agricultural products that contain excess aflatoxins have greatly reduced value. Aflatoxin contamination of cottonseed and corn cost agriculture in the U.S. millions of dollars each year. In Arizona alone, aflatoxin contamination of cottonseed costs the cotton industry $3 to $8 million per year. Currently, agricultural profits are severely curtailed and the burden of aflatoxin contamination has been increasingly difficult to bear. Aflatoxins are a major factor limiting international trade of several U.S. commodities. Economically viable aflatoxin management is needed to ensure the stability of agriculture in several regions of the U.S. There are no methods growers can apply to reliably and economically prevent aflatoxin contamination. Growers are at the mercy of the environment; heat and drought combined with humidity as the crop reaches maturity frequently result in disastrous levels of aflatoxins. The project seeks to develop management methods from improved understanding of both the ecology of the fungi causing contamination and the characteristics of crops that favor contamination. Strains of A. flavus that do not produce aflatoxins (atoxigenic strains) are potentially useful tools for limiting contamination. By competitively excluding aflatoxin producers, atoxigenic strains reduce the aflatoxin producing potential of fungi resident in agricultural areas and thus reduce aflatoxin contamination of the crops produced. Although a conceptual framework for the use of atoxigenic strains is well developed, knowledge of effects of agronomic practices and environmental variability on atoxigenic strain behavior is needed to develop the technology to practical use. To date, only single atoxigenic strains have been used. However, ecological theory suggests use of multiple atoxigenic strains may allow both more efficient and longer term reductions in aflatoxin content. Questions involving biopesticide registration; fungal ecology, stability, and movement of fungal communities, and practical manufacture and on farm use of this potentially useful biopesticide, need to be addressed. Furthermore, improved methods to differentiate and to monitor fungal strains in the environment are needed both to provide oversight of biocontrol practices and to facilitate continued development of these biological control agents. In collaboration with grower organizations, farm- and gin-based studies on impacts of agronomic practices on contamination are combined with large commercial field tests of atoxigenic strain technology in order to develop procedures that may be useful in area-wide aflatoxin management programs. Through detailed laboratory and field studies, the project seeks improved understanding of the population biology, ecology, evolution and physiology of causative agents of aflatoxin contamination and of biochemical aspects of crop susceptibility and contamination. 2.List by year the currently approved milestones (indicators of research progress) Milestones, Objective 1: Improved formulations of atoxigenic strains have been developed and tested in the laboratory; elite formulations have been field tested; scale-up processes for new formulations have been developed. Vegetative compatibility groups (VCGs) of A. flavus differing in incidence on crops have been compared for competitiveness on crops; data has been developed on use of single versus multiple VCGs for biological control of aflatoxin contamination. Milestones, Objective 2: Retrospective analysis of influences of agronomic practices is completed; geostatistical analysis of displacement has been completed; field tests on optimal application timing and release of atoxigenic strains in cotton has been completed; the nature of and characteristics of displacement of aflatoxin producers and movement of atoxigenic strains has been completed; field tests of influences of agronomic practices identified by the retrospective analyses have been performed. Milestones, Objective 3: Collection of data for model development from industry is complete; a model on S strain behavior in response to environmental factors is developed and tested in the lab; a model for toxin formation in the field is developed; the toxin model is tested with field experiments; the influence of modified environment field plots (misting) on toxin formation has been field tested. Milestones, Objective 4: The most common VCGs of A. flavus associated with cotton have been characterized; distribution of the common VCGs in other niches has been tested; a database of single nucleotide polymorphisms that characterize A. flavus VCGs has been intitiated; a pyrosequencing assay for AF36 on crops has been developed; a pyrosequencing assay for quantifying incidences of both the S strain and AF36 on crops has been developed; a pyrosequencing assay for multiple VCGs has been developed; variability among VCGs at known VC loci has been tested; knock-out mutants in known VC loci have developed; VC genes of interest are identified from mutants in screened mutant library. Milestones, Objective 5: Xylanases and lipases of A. flavus have been identified and characterized; XlnR homologs and XlnR regulated genes have been identified; gossypol interactions with A. flavus have been tested; genes involved in gossypol use are known; variation among VCGs in genes of putative adaptive value has been examined; an insertional knockout library of A. flavus has been developed. 4a.List the single most significant research accomplishment during FY 2006. ASSAY TO MONITOR INCIDENCE OF BIOCONTROL AGENT DEVELOPED An assay was developed based on pyrosequencing that allows for quantification of the incidence of a biological control agent used to prevent aflatoxin contamination of cottonseed on the crop after harvest. To accurately assess efficacy of biological control agents, it is frequently necessary to monitor the incidence of the biological control agent on the crop and in the environment. For atoxigenic strains of A. flavus, this is a time consuming task requiring extensive culturing. An assay to monitor an atoxigenic strain that does not require culturing was developed and proofed against cottonseed samples from commercial fields. The assay uses pyrosequencing to directly quantify the incidence of the atoxigenic strain based on variability in the fungal deoxyribonucleic acid (DNA) present on crop surfaces. The pyrosequencing assay will reduce the labor associated with evaluating the efficacy of biocontrol agents and thus facilitate development of aflatoxin management strategies based on biological control IMPACT: The developed assay for monitoring a biocontrol agent used to prevent aflatoxin contamination will aid further optimization and discovery of aflatoxin management tools. This accomplishment impacts National Program 108 Performance Goal 1.1.5 Biocontrol technologies by providing advances towards that development of a practical biological control method for reducing aflatoxin contamination. 4b.List other significant research accomplishment(s), if any. None. 4c.List significant activities that support special target populations. None. 4d.Progress report. This project is the parent research project of the following cooperative agreements involved in like research - Cotton Incorporated (Texas)(expired); Cotton Incorporated (Texas)(new); Cotton Foundation (Arizona); Antilla/Arizona Cotton Research & Protection Council (ACRPC); Olsen/University of Arizona; Van Eten/University of Arizona; and Jaime-Garcia/University of Arizona. The research relates to National Program 108, Food Safety (100%). This research is directed at understanding both the process of aflatoxin contamination and the ecology and biology of aflatoxin producing fungi. The development of procedures to limit aflatoxin contamination is a primary component of the work. This research also contributes to National Programs seeking to develop biological controls by developing concepts and technologies for production and use of fungal biocontrol. These include National Programs 104, 207, 303, 304, 305, and 308. Our laboratory continued to develop atoxigenic strain technology in collaboration with industry partners, including numerous farmers and gins, as well as, the Arizona Cotton Research and Protection Council (ACRPC), Cotton Incorporated, the National Cotton Council, South Texas Cotton and Grain Association and the Valley Coop Oil Mill. In addition to this, collaborations were expanded with the California Pistachio Commission and the University of California for work on pistachios and with the Texas Corn Growers Association for work on corn. We assisted both these latter two organizations with applications to the Environmental Protection Agency (EPA) for experimental use permits for aflatoxin management on their respective crops over the past year. Collaborations were also expanded with Mexico on aflatoxin contamination of maize in collaboration with the University of Arizona and collaborators at the National Institute of Forestry, Agricultural and Animal Research. These studies were funded by a grant from the Foreign Agricultural Service (FAS) of USDA. Collaborations also are being pursued with research in Thailand (on maize) and in Turkey (on pepper). Test acreage in Texas was expanded to include central and north central Texas in collaboration with the Texas Corn Growers Association, which funded material and application costs in the experimental areas. We continued to make improvements in product quality from improved inoculation procedures and better packaging and alterations in formulation. Efforts to further improve product formulation and application timing to improve in field performance continue in collaboration with industry partners. Cotton production in central Arizona (west of Phoenix) has considerably declined due to housing expansion. This has impacted participation in our research by gins from that region. Western Arizona remains a vital agricultural region. 5.Describe the major accomplishments to date and their predicted or actual impact. This project was certified November 5, 2005. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Technologies associated with utilizing atoxigenic strains of A. flavus to prevent aflatoxin contamination are in the process of being transferred to the Arizona and Texas cotton-producing community. As improvements are developed, transfer to producers and a producer organization is rapid. In the process of adapting these technologies for use by commercial entities, ARS scientists are attempting to solve difficulties as they are encountered in order to speed adaptation. Technologies transferred include simple strain identification, starter culture procedures, scale up procedures, quality control procedures and methods to assess efficacy. Technology transfer with industries in California, Arizona and Texas were expanded during FY 2006. These included cotton, corn and pistachio crops. During FY 2006, we continued to assist in the development and improvement of a prototype facility using ARS technology for producing pilot-scale quantities of a fungus (atoxigenic strain of A. flavus) used to prevent aflatoxin contamination is up and functioning. Scientists transfer improvements to technology directly to the organization running this facility and others as improvements are made. The greatest obstacle to implementation currently is the pesticide registration process in which ARS is engaged in order to assist industry partners in overcoming this difficult burden. During FY 2006, cottonseed, pistachio, and corn industries were assisted in pesticide registration aspects. Data on the ecology of aflatoxin producing fungi and biological control were provided to the EPA during the summer of 2006. Consultations on management of contamination of other crops (peanuts, corn, tree crops) also occur on a regular basis. This has included consultations with growers, corporations and state and federal agencies. More fundamental information on molecular relationships, physiology and ecology are transferred to the scientific community. The scientific community broadly utilizes techniques developed previously by this group. Scientists also continue to transfer techniques and knowledge useful in studying the population biology and ecology of A. flavus. A major technology transfer tool has been the inclusion of numerous farmers, gins and oil mills in the research process. During FY 2006, farmers, gins, oil mills and cooperatives throughout Central and South Texas and Arizona were included in research projects. ARS provides feedback on data and advancement; the industry provides experience and first-hand observations that often make the difference in making rapid advances. When advances are made, the relationships make transferring the information seamless and transparent. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Cotty, P. J. 2005. Update on aflatoxin management in Texas. State Support Program of Cotton Incorporated Producer. Research Review, Lubbock, TX. Cotty, P. J. 2006. Aflatoxins in Cottonseed. Annual Convention National Cottonseed Products Association, Tucson, AZ. Cotty, P. J. 2005. Biocompetitive exclusion of toxigenic fungi. World Mycotoxin Forum, Nordwijk Aan Zee, The Netherlands. Cotty, P. J. 2005. Managing Aflatoxins in Cotton-Corn Rotations. 17th Aflatoxin Elimination Workshop, Raleigh, NC. Cotty, P. J. 2006. Biocontrol of aflatoxin producing fungi in cotton-corn rotations in south Texas. Beltwide Cotton Conferences. San Antonio, TX. Review Publications Jaime-Garcia, J., Cotty, P.J. 2006. Crop rotation influences aflatoxin producing potential of Aspergillus communities in South Texas. National Cotton Council Beltwide Cotton Conference. San Antonio, TX. p. 60-63. Garber, N.P., Cotty, P.J. 2006. Timing of herbicide applications may influence efficacy of aflatoxin biocontrol. National Cotton Council Beltwide Cotton Conference. San Antonio, TX. p. 1-6. Jaime-Garcia, R., Cotty, P.J. 2006. Spatial relationships of soil texture and crop rotation to Aspergillus flavus community structure in South Texas. Phytopathology. 96(6):599-607. Jaime-Garcia, R., Cotty, P.J. 2006. Spatial relationships of soil texture and crop rotation to Aspergillus flavus community structure in South Texas. Phytopathology. 96:599-607. Bock, C.H., Cotty, P.J. 2006. Methods to sample air borne propagules of Aspergillus flavus. European Journal of Plant Pathology. 14:357-362.