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ARS Home » Pacific West Area » Maricopa, Arizona » U.S. Arid Land Agricultural Research Center » Pest Management and Biocontrol Research » Research » Research Project #430864

Research Project: Improved Environmental and Crop Safety by Modification of the Aspergillus flavus Population Structure

Location: Pest Management and Biocontrol Research

2017 Annual Report

1a. Objectives (from AD-416):
1. Optimize and expand use of biological control of aflatoxins based on atoxigenic strains of Aspergillus flavus in order to improve access, affordability, and area-wide management. Subobjective 1.1. Evaluate area-wide influences where atoxigenic biopesticides are widely used and develop strategies to increase cost-savings and efficacy based on area-wide effects. Subobjective 1.2. Evaluate the potential to adapt hydropriming from seed technology to use with atoxigenic strain products to increase atoxigenic strain release under low humidity. Subobjective 1.3. Advance biological control products based on atoxigenic strains of A. flavus with commercial field testing. Subobjective 1.4. Improve access to atoxigenic strain biopesticides by assisting stakeholders to reduce costs of manufacture and distribution and expand biopesticide products while engaging USEPA in dialogue on biocontrol regulatory issues and public sector roles. 2. Develop an understanding of the distribution of Aspergillus flavus genetic haplotypes and vegetative compatibility groups worldwide in order to improve selection of biological control agents. Subobjective 2.1. Identify A. flavus endemic in and adapted to target agroecosystems. Subobjective 2.2. Determine utility of SSRs in tracking mechanisms and histories of divergences within A. flavus. Subobjective 2.3. Develop an SSR database to support global efforts to delineate distributions of A. flavus genotypes and relationships among strains under investigation in diverse locations. 3. Improve understanding of development, evolution, and stability of populations of Aspergillus flavus, including phenomena occurring both within and between VCGs, in order to inform to inform optimization of long-term beneficial effects of atoxigenic strain biocontrol. Subobjective 3.1. Determine the nature of clonal evolution in A. flavus with genomic analyses. Subobjective 3.2. Assess mutation rate in an A. flavus genome during asexual reproduction in controlled laboratory evolution studies.

1b. Approach (from AD-416):
Biological control products, developed during previous projects, with atoxigenic strains of Aspergillus flavus as active ingredients have been successful at greatly reducing aflatoxin contamination of corn and peanut in commercial fields in the US and in thousands of farmer’s fields across Nigeria, Kenya, Senegal, Burkina Faso, Zambia, the Gambia, and Ghana. The current project seeks to improve biological control to increase both single-season and long-term aflatoxin management to provide a context for both efficient area-wide aflatoxin management and reductions in cost of biological control programs. Area-wide influences of current commercial practices utilizing atoxigenic strain biocontrol agents will be quantified with culture and DNA based techniques. Diversity among and distributions of naturally occurring atoxigenic strains of potential use in biological control products will be determined and atoxigenics will be selected and field tested for the next generation of aflatoxin prevention biocontrol products. Simple Sequence Repeat (SSR) analyses will be expanded to allow better understanding of strain distribution and divergence. A worldwide SSR database for A. flavus will be developed to allow the global scientific community to identify genotypes reported in the literature and/or incorporated into biocontrol products under development around the world. Comparative genomic analysis will be performed to characterize adaptation, divergence, and the relative contributions of recombination and clonality to A. flavus community structure. The resulting information will provide improved cost effective tools for production of safe foods and feeds.

3. Progress Report:
Efforts to optimize and expand use of biological control of aflatoxins based on atoxigenic strains of Aspergillus flavus included field work on pistachios and corn, interactions with regulatory authorities, and interactions with public sector manufacturers of biocontrol products. Field studies in 6 commercial orchards in Eastern Arizona on optimal timing of AF36 Prevail on pistachios were initiated during 2016 and analyses will be completed in 2017. Results suggest some commercial applications are made too early in the season and that tree crop producers should be more concerned with long-term multi-season influences of applications and emphasize less influences during the year of application. Area-wide influences of atoxigenic strain applications in a region of North Central Texas, where the two registered atoxigenic active ingredients have been widely used, suggest excellent residual and cumulative influences and the potential for reduced applications where use patterns are high. This is clear evidence that area-wide management was occurring with normal intensive use in this area that has been severely hurt economically by aflatoxin contamination in the past. The Environmental Protection Agrency (EPA) approved an Experimental Use Registration for FourSure™ an atoxigenic strain based biopesticide being developed with the Texas Corn Producers Board. FourSure™ contains four active ingredients each of which is an atoxigenic genotype of A. flavus not previously registered for use in a biopesticide. Registration of FourSure™ will increase the number of atoxigenic strain genotypes registered in the U.S. by 300% and facilitate long-term, area-wide management. Fifty thousand pounds of FourSure™ were produced by staff in Tucson in a temporary manufacturing facility and shipped to Texas where farmers treated commercial fields in compliance with the EPA approved Experimental Use Program. Crops will be harvested and analyzed for required data over the next year to determine efficacy and the relative presence of the four genotypes on treated crops and in soils a year after treatment. A program to evaluate use of the atoxigenic strain based AF36 Prevail™ in a two crop (cotton and corn silage) area-wide management program was developed with the Arizona Cotton Research and Protection Council (ACRPC) and is being initiated in 2017. ACRPC has taken the lead role in this project which extends over two Townships. Results to date on treatment of only single boarders with biocontrol agents suggest this might be an inexpensive way to treat low value crops in area-wide aflatoxin management programs. ACRPC is applying these results with a novel pneumatic applicator to treat parameters of cotton fields, over 3,000 acres, in the area-wide assessment area. Dairies are solid treating the silage fields. As a result of ARS activity and collaboration with stakeholders and colleagues, several successes in registrations of atoxigenic strain products were achieved including improved and expanded labels from EPA for AF36 Prevail™ that include improved use and less constrained storage requirements, and expansion of permitted crops to include almonds and figs. This is the first pesticide directed at preventing pre-harvest aflatoxin contamination registered for use on either almonds or figs. The registration allows for future area-wide management of contamination in important U.S. productions regions where these two crops are produced in close proximity to pistachios, for which a registration was already available, and, as such, will facilitate aflatoxin prevention on all three crops. Registrations were also expanded in Africa including AflaSafe SN01 (contains four genotypes of A. flavus from West Africa) directed at maize and peanut in the Gambia and Senegal and AflaSafe BF01 (contains four different genotypes of A. flavus native to Burkina Faso) directed at maize and peanut in Burkina Faso. Both the AflaSafe registrations are approved across the The Permanent Interstate Committee for Drought Control in the Sahel (French: Comité permanent inter-État de lutte contre la sécheresse au Sahel (CILSS) nations which means that once the active ingredients are found in additional francophone West African countries, the products will be already approved for use in those locations. Collections of A. flavus were constructed with the assistance of collaborators across five continents including Asia (Bangladesh, Pakistan, and China), Europe (Italy, Serbia, and Greece), Australia, North America (U.S., Haiti, and Costa Rica), and Africa (including nations in East, West, and Southern Africa). The isolates are being DNA profiled based on 17 simple sequence repeat loci. This data will be combined with future data and assembled into a global database. Whole genomes of several A. flavus genotypes have been sequenced, assembled and annotated. Several of these are atoxigenic strains for which exact mechanisms of atoxigenicity were needed for regulatory consideration. In addition, several isolates belonging to the same vegetative compatibility group were included to allow improved understanding of development, evolution, and stability within and between VCGs. This latter approach will allow insight into use of atoxigenic genotypes in a manner that encourages long-term benefit to agricultural systems.

4. Accomplishments
1. Expansion of Environmental Protection Agency (EPA) registered biopesticides for aflatoxin management. Biopesticides based on atoxigenic strains of Aspergillus flavus have become the most widely used intervention for preventing aflatoxin contamination. However, these biopesticides must be approved by and registered with regulatory authorities and each target crop and each atoxigenic genotype require additional regulatory action. Thousands of atoxigenic genotypes of A. flavus exist with broad adaptation but regulatory approval for use in commercial products has only been granted for a few genotypes. The ARS aflatoxin laboratory in Tucson, Arizona, addressed this through direct interactions with regulatory authorities, field and laboratory experimentation, and collaborations with the Arizona Cotton Research and Protection Council, the University of California, the International Institute of Tropical Agriculture, commodity groups, and several national governments. The result is new and expanded registrations of biopesticides directed at preventing aflatoxin contamination including approval for new target crops (figs and almonds), additional A. flavus genotypes, and less restrictive handling requirements. Full registrations in the U.S., Senegal, and Burkina Faso were added to existing registrations in the United States, Kenya, and Nigeria.

2. Iron utilization gene cluster variation within Aspergillus flavus provides new molecular tools for atoxigenic genotype selection. Certain genotypes of Aspergillus flavus, produce aflatoxins that contaminate food crops, including maize, peanuts, and tree nuts. Aspergillus flavus genotypes that do not produce toxin are widely used as biological control agents to reduce aflatoxin contamination of food and feed. Filamentous fungi like A. flavus have diverse mechanisms to uptake and use iron. ARS researchers in Tucson, Arizona, have identified and characterized a cluster of genes involved in iron uptake and utilization. Variation among A. flavus genotypes in iron utilization genes provides greater understanding of iron utilization process in the aflatoxin producing fungi. The genes identified in this study can potentially be used as genetic markers for use in profiling atoxigenic genotypes for biological control product development.

Review Publications
Adhikari, B.N., Bandhyopadhyay, R., Cotty, P.J. 2016. Degeneration of aflatoxin gene cluster in Aspergillus flavus from Africa and North America. Applied Microbiology and Biotechnology Express (AMB Express). 6:62. doi: 10.1186/s13568-016-0228-6.

Kachapulula, P.W., Akello, J., Bandyopadhyay, R., Cotty, P.J. 2017. Aflatoxin contamination of groundnut and maize in Zambia: observed and potential concentrations. Journal of Applied Microbiology. 122:1471-1482.

Thakare, D., Zhang, J., Wing, R.A., Cotty, P.J., Schmidt, M.A. 2017. Aflatoxin-free transgenic maize using host-induced gene silencing. Science Advances. 3(3):e1602382.

Singh, P., Cotty, P.J. 2017. Aflatoxin contamination of dried red chilies: Contrasts between the United States and Nigeria, two markets differing in regulation enforcement. Food Control. 80:374-379.

Bandyopadhyay, R., Ortega-Beltran, A., Akande, A., Mutegi, C., Atechnkeng, J., Kaptoge, L., Senghor, A.L., Adhikari, B.N., Cotty, P.J. 2016. Biological control of aflatoxins in Africa: current status and potential challenges in the face of climate change. World Mycotoxin Journal. 9(5):771-789.