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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

2019 Annual Report

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.

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.

Progress Report
Related to Objective 1, Aspergillus (A.) flavus colonization of corn and cotton is well described, but the occurrence of A. flavus in the canopy of tree crops is not documented or understood. ARS researchers in Tucson, Arizona, are monitoring the year-round diversity and abundance of the A. flavus community in the phylosphere of a commercial pistachio orchard. Experimental releases of biocontrol formulations have been conducted in this orchard with the goal of identifying better application methods. This information is being shared with cooperators and stakeholders. ARS researchers have contributed to the registration of FourSure™, a biopesticide end-use product, that includes four strains of A. flavus (TC16F, TC35C, TC38B, and TC46G) to control aflatoxin. An ARS-developed, DNA-based fingerprinting method was used to identify locally adapted biocontrol strains. FourSure™ was initially produced by ARS, and production has been transferred to a commodity stakeholder organization. The increased commercial production has facilitated approximately 5,000 acres of field trials in 2018 and 2019. Treated fields are being monitored by ARS researchers to characterize the A. flavus soil population before and after treatment, the level of colonization of the grain, and the subsequent level of aflatoxin contamination. Preliminary results have been very encouraging, with efficient displacement of the initial A. flavus community by the four FourSure™ genotypes. Several farms with four to seven treated fields averaged over 95 percent displacement with a single 10 pound per acre treatment. Texas corn producers have provided excellent support in conducting these field trials to support the ongoing registration process. Practical, affordable biological control of aflatoxin requires well-adapted, atoxigenic A. flavus isolates and efficient application methods. Work with the Arizona Cotton Research and Protection Council and grower-cooperators are evaluating novel application technologies. Because A. flavus sporulates abundantly on the applied inoculum and the spores are readily dispersed, it is possible to provide protection for an entire field when the biocontrol product is only applied on the field perimeter. This experiment has been conducted for two years. These data could be used to validate the feasibility of area-wide suppression of aflatoxigenic A. flavus. In support of Objective 2, 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 beginning in Mali and Ethiopia. Continued collaborations are occurring to advance atoxigenic strain-based products in Serbia and Italy. In support of Objective 3, a novel Aspergillus species was discovered in grain samples from Arkansas, Louisiana, and Texas in addition to soil samples from Texas. These isolates had a colony morphology similar to A. flavus S-type biotypes, but with a unique aflatoxin production profile. Sequence analysis of this newly-described species revealed that they are genetically distinct from A. flavus, clustering in a monophyletic clade.

1. Molecular methods for precise identification of Aspergillus flavus isolates. Aspergillus flavus, the principal cause of aflatoxin contamination of food and feed, is globally distributed. This fungus is common in the agroecosystem, but tools for strain-specific identification have been imprecise or very labor-intensive. ARS scientists in Tucson, Arizona, have assembled a global collection of over 40,000 A. flavus isolates and developed a SSR (simple sequence repeat) based method to characterize isolates at 17 loci with exceptional precision. Screening of the first approximately 29,000 isolates has revealed a remarkable degree of diversity, with over 13,000 unique haplotypes discovered to date. This methodology and the resulting dataset is being shared with other researchers to document the geographic structure of the A. flavus population and explore the interactions between environment and genotype.

Review Publications
Ortega-Beltran, A., Moral, J., Picot, A., Puckett, R.D., Cotty, P.J., Michaildes, T.J. 2019. Atoxigenic Aspergillus flavus isolates endemic to almond, fig, and pistachio orchards in California with potential to reduce aflatoxin contamination in these crops. Plant Disease. 103(5):905-912.
Singh, P., Cotty, P.J. 2019. Characterization of Aspergillis from dried red chilies (Capsicum spp.): Insights into the etiology of aflatoxin contamination. International Journal of Food Microbiology. 289:145-153.
Singh, P., Orbach, M.J., Cotty, P.J. 2018. Aspergillus texensis: A novel aflatoxin producer with S morphology from the United States. Toxins. 10(12):1-15.
Ohkura, M., Cotty, P.J., Orbach, M.J. 2018. Comparative genomics of aspergillus flavus s and l morphotypes yield insights into niche adaptation. G3, Genes/Genomes/Genetics. 8(12):3915-3930.