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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

2018 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:
There is a lack of sound techniques for assessing the distribution of Aspergillus flavus in the canopy of tree crops and particularly pistachios. Studies to develop useful methods to monitor the dynamics of A. flavus communities in the canopies of pistachio trees in Bowie, Arizona, were initiated. Early results are promising and may help fine tune application strategies. Small trials to assess the potential of novel formulations of biocontrol projects were undertaken in pistachios with the help of commercial collaborators. During 2018, ARS researchers in Tucson, Arizona, produced fifty-thousand pounds of FourSure™, a biopesticide end-use product, containing as active ingredients four strains of A. Flavus (TC16F, TC35C, TC38B, and TC46G) to control aflatoxin. It was produced in a temporary manufacturing facility and shipped to several locations in Texas where farmers treated commercial fields in compliance with an Environmental Protection Agency (EPA) approved Experimental Use Program. Samples from approximately 5,000 acres of treated maize crops and untreated maize were collected by the participating farms and shipped to Tucson, Arizona, where the aflatoxin content was determined. Composition of A. flavus on the crops was examined with deoxyribonucleic acid (DNA) fingerprinting using simple sequence repeats (SSR). In addition, communities of fungi resident in soils of treatment areas prior to treatment were analyzed using similar fingerprinting. As of August 2018, the DNA fingerprinting of the 2017 trials is nearing completion with excellent results showing very high efficacy to alter the A. flavus community and displace aflatoxin producers with the four FourSure™ genotypes. Several farms with 4 to 7 treated field’s averaged over 95 percent displacement with a single 10 pound per acre treatment. Small plots (replicates were 0.5 to 1.0 acre) also provided evidence that all the FourSure™ genotypes and the combined FourSure™ product were highly effective. The Arizona Cotton Research and Protection Council in Phoenix, Arizona agreed to produce the FourSure™ for the 2018 trials which included over 5,000 acres of maize. The 2018 commercial Texas maize trials are currently being harvested. Soils across the treatment areas have been taken to assess the long-term benefits of the area-wide program. Cottonseed from modules both at the interior and the center of fields indicate that the perimeter treatments provide some level of efficacy for farmers without incentive to treat entire cotton fields. The distribution pattern of the pneumatic applicator is being determined along with time required to treat various sized fields. Efforts to fill the void of techniques to assess distribution of atoxigenic strain active ingredients in harvested silage were initiated in 2018. Progress continued to be made across Africa with the launch of the now registered AflaSafe GH01, a natural biocontrol product in Ghana. Also, advanced trials of AflaSafe products with specific fungi for the target regions were continued in Tanzania, Malawi, and Mozambique. The Tucson, Arizona, laboratory assisted with expanded efforts by using molecular tools to select well adapted fungi for Uganda and Rwanda. Work is initiating in Mali and Ethiopia. Work advancing atoxigenic strain-based products in Serbia and Italy has continued with collaborators. Data for the database of SSR Haplotypes was collected during 2018, expanding beyond 8,000 ascensions, including fungi from Australia, North America, Central America, Africa, Europe, and Asia. Collections of fungi were expanded in China, Pakistan, and Guatemala. Each of these are in support of collaborators interested in utilizing atoxigenic strain-based biocontrol.

4. Accomplishments
1. Routine use of atoxigenic strains of Aspergillus flavus for biological control of aflatoxins. Utilizing atoxigenic strains of A. flavus has become routine in the U.S. and several portions of Africa, including Kenya for control of aflatoxins. An ARS researcher at Tucson, Arizona, performed a large population genetic analysis on A. flavus isolated from soil obtained from Kenyan maize fields. The study determined that A. flavus populations are very large, ancient, and evolving through mutation-driven, clonal reproduction. Under all conditions, all genetic loci were in linkage disequilibrium suggesting that atoxigenic A. flavus biocontrol agents used to modify fungal communities to produce fewer toxins, and thus be less dangerous, have remained genetically stable over thousands of years and should remain stable without sexual reproduction that would introduce new gene combinations. This novel and very effective biocontrol technology provides a simple inexpensive tool for improving the value and safety of crops and the environment.

2. Biocontrol manufacturing facility using modular design approach. A modular manufacturing facility to produce atoxigenic strain-based biocontrol products to prevent aflatoxin contamination in Kenya was completed at the Katumani Station of the Kenya Agriculture and Livestock Research Organization in Machakos, Kenya. Machakos is in the area with the greatest frequency of lethal aflatoxicosis resulting from aflatoxin-contaminated maize. An ARS scientist in Tucson, Arizona, made the initial facility design, developed the process utilized by the facility and worked with scientists and engineers at the International Institute of Tropical Agriculture to implement the design. The facility's processing capabilities are flexible, allowing for expansion of production capacity by addition of modules and for construction of similar sister facilities with different numbers of modules. The manufacturing capacity allows Kenya to produce commercial quantities of a biocontrol effective at preventing aflatoxin contamination of maize and allows for production of increased safe food during periods of food insufficiency.

Review Publications
Picot, A., Doster, M., Islam, M.S., Callicott, K.A., Cotty, P.J., Michalides, T., Ortega-Beltran, A. 2017. Distribution and incidence of atoxigenic Aspergillus flavus VCG in tree crop orchards in California: A strategy for identifying potential antagonists, the example of almonds. International Journal of Food Microbiology. 265:55-64.

Mauro, A., Garcia Cela, E., Cotty, P.J., Battilani, P. 2018. Biological control products for aflatoxin prevention in Italy: Commercial field evaluation of atoxigenic A.flavus active ingredients. Toxins. 10:1-14.

Ayalew, A., Kimanya, M., Matumba, L., Bandyopadhayay, R., Menkir, A., Cotty, P.J. 2017. Controlling aflatoxins in maize in Africa: strategies, challenges and opportunities for improvement. In: Watson, D., editor. Achieving Sustainable Cultivation of Maize Cultivation Techniques, Pest, and Disease Control. Volume 2. Cambridge, UK: Burleigh Dodds Science. p. 1-24.

Kachapulula, P.W., Akello, J., Bandyopadhyay, R., Cotty, P.J. 2017. Aspergillus section Flavi community structure in Zambia influences aflatoxin contamination of maize and groundnut. International Journal of Food Microbiology. 261:49-56.

Ortega-Beltran, A., Cotty, P.J. 2018. Frequent shifts in aspergillus flavus populations associated with maize production in Sonora, Mexico. American Phytopathological Society. 108(3):412-420.

Agbetiameh, D., Ortega-Beltran, A., Awuah, R.T., Atehnkeng, J., Cotty, P.J., Bandyopadhyay, R. 2018. Prevalance of aflatoxin contamination in maize and groundnut in Ghana: Population structure, distribution, and toxigenicity of the causal agents. Plant Disease. 102(4):764-772.

Ortega-Beltran, A., Moral, J., Puckett, R.D., Morgan, D.P., Cotty, P.J., Michailides, T.J. 2018. Fungal communities associated with almond throughout crop development: implications for aflatoxin biocontrol management in California. PLoS One. 13(6):e0199127.

Islam, M.S., Callicott, K.A., Mutegi, C., Bandyopadhyay, R., Cotty, P.J. 2018. Aspergillus flavus resident in Kenya: High genetic diversity in an ancient population primarily shaped by clonal reproduction and mutation-driven evolution. Fungal Ecology. 35:20-33.

Kachapulula, P.W., Akello, J., Bandyopadhyay, R., Cotty, P.J. 2018. Aflatoxin in dried insects and fish in Zambia. Journal of Food Protection. 81(9):1508-1518.