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

Location: Pest Management and Biocontrol Research

2020 Annual Report

Objectives
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.

Approach
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
In support of Objective 1, research continued on evaluating area-wide influences where atoxigenic biopesticides are heavily used and developing strategies to increase cost-savings and efficacy of aflatoxin biocontrol based on region-specific management of aflatoxin-producing fungal populations. A major activity under this objective was further development of a new, multi-strain aflatoxin biocontrol product in cooperation with the Texas Corn Producers Board. The biocontrol product FourSure was applied to over 10,000 acres of commercial corn in Texas in 2019, and efficacy of the product was assessed based on displacement of aflatoxin-producing fungi on the crop and reductions in aflatoxin concentrations in harvested grain. On average, corn grain harvested from fields treated with FourSure had 80% less aflatoxin than non-treated fields. In addition, the FourSure biocontrol strains were recovered from fields two years post-treatment, which indicates long-term reductions in aflatoxin contamination of crops in these fields may be possible when biocontrol products are applied. The U.S. Environmental Protection Agency (EPA) was provided with the 2019 data in support of the biopesticide registration package for FourSure. In addition, researchers in Maricopa (Tucson, Arizona worksite) contributed to a bridging document that will be submitted to the EPA to request waivers for toxicology and ecotoxicology studies on the FourSure active ingredients. The waiver request is based on the premise that the atoxigenic A. flavus genotypes in FourSure are genetically and physiologically similar to the active ingredients in currently registered aflatoxin biocontrol products (i.e. AF36 Prevail and Afla-Guard) for which these studies have been conducted. International activities aimed at expanding the aflatoxin biocontrol technology included collaborations with the International Institute of Tropical Agriculture (IITA) and the USDA Foreign Agricultural Service (FAS). In cooperation with IITA, ARS researchers from Maricopa (Tucson, Arizona worksite) supported work to expand biocontrol registrations and manufacturing facilities throughout Africa (e.g. Kenya, Nigeria, Senegal). In cooperation with FAS, the Tucson, Arizona lab also was involved in providing technical expertise and training to facilitate development and registration of a biocontrol product for Pakistan. Under Objective 2, additional A. flavus isolates from global collections were genetically characterized using molecular markers. Over 20,000 isolates originating from the United States and countries in Central America, Africa, Europe, Asia, and Australia have been genetically characterized, and genomes have been sequenced for the most common genetic types (Sub-objective 2.1). Population genetic analyses at regional, continental, and inter-continental scales suggest there is long-distance dispersal and broad distribution of individual A. flavus genotypes (Sub-objective 2.2). Genetic information for over 20,000 isolates has been deposited in an online database, AflaSat, which is currently in development (Sub-objective 2.3). The database has been released to selected U.S. and international collaborators who are externally verifying the functionality and utility of AflaSat. For Objective 3, studies were conducted to determine evolution and stability of A. flavus genotypes. Isolates originating from Africa and North America, but belonging to a single genetic type, were sequenced and compared (Sub-objective 3.1). In addition, selected isolates grown in culture over multiple generations were sequenced to determine types and rates of genetic change (Sub-objective 3.2). Frequent, small changes were observed in genomes, but changes in markers used for genotyping isolates were not observed. Results suggest that rates of change within A. flavus lineages are slow and A. flavus genotypes are stable over time. Genetic differences unique to A. flavus genotypes that are active ingredients in biocontrol products were used to develop molecular assays to track and quantify proportions of these genotypes in crops. These molecular assays are a rapid alternative to culture-based methods for assessing the efficacy of biocontrol products based on the ability of the active ingredient genotypes to displace aflatoxin-producing fungi on the crop.

Accomplishments
1. Development and deployment of aflatoxin biological control products in Africa. Crop aflatoxin contamination is a worldwide problem impacting food and feed safety as well as international trade. In warm regions of Africa, there is a perennial risk of aflatoxin contamination in staple crops such as maize and groundnut. Collaborative work between ARS scientists from Maricopa, Arizona, and scientists at the International Institute of Tropical Agriculture (IITA) has led to registration of new atoxigenic-strain based aflatoxin biocontrol products in Ghana and Senegal and increased adoption of the biocontrol technology by smallholder farmers in Kenya through implementation of market incentives. This work contributes to reduced aflatoxin concentrations in West African crops which will ultimately decrease incidence of negative health effects that result from consumption of aflatoxin contaminated food/feed and increase the value of crops and access to international markets. Results are of value to smallholder farmers, national agriculture and health ministries, and international development organizations interested in protecting the health of consumers and increasing the value of agricultural goods produced in these countries.

2. Discovery of new aflatoxin-producing Aspergillus species in the southern United States. Effective control of crop aflatoxin contamination and evaluation of aflatoxin contamination risk within a region requires an understanding of the causal agents. Previously it was assumed that aflatoxin contamination of U.S. crops was due solely to Aspergillus flavus and A. parasiticus. Collaborative work between ARS scientists from Maricopa, Arizona, and scientists at the University of Arizona, characterized species of high aflatoxin-producing fungi, isolated from agricultural soils in the southern U.S. In addition to A. flavus, three additional Aspergillus species, including two previously undescribed taxa, were identified. The four species differed in the types and quantities of aflatoxins produced, so the frequency of different Aspergillus species within a field will impact the relative risk of high levels of crop contamination. This work has resulted in a better understanding of the aflatoxin-producing potential of different Aspergillus species that are associated with crops in the southern United States. Results are of value to researchers, growers, and regulators who are trying to quantify risk of aflatoxin contamination events and develop aflatoxin management strategies that target specific causal agents.

Review Publications
Senghor, L., Ortega-Beltran, A., Atehnkeng, J., Callicott, K.A., Cotty, P.J., Bandyopadhyay, R. 2019. The atoxigenic biocontrol product Aflasafe SN01 is a valuable tool to mitigate aflatoxin contamination of both maize and groundnut cultivated in Senegal. Plant Disease. 104(2):510-520. https://doi.org/10.1094/PDIS-03-19-0575-RE.
Ortega-Beltran, A., Callicott, K.A., Cotty, P.J. 2020. Founder events influence structures of Aspergillus flavus populations. Environmental Microbiology. 22(8):3522-3534. https://doi.org/10.1111/1462-2920.15122.
Shenge, K.C., Adhikari, B.N., Akande, A., Callicott, K.A., Atehnkeng, J., Ortega-Beltran, A., Kumar, P., Bandyopadhyay, R., Cotty, P.J. 2019. Monitoring Aspergillus flavus genotypes in a multi-genotype aflatoxin biocontrol product with quantitative pyrosequencing. Frontiers in Microbiology. 10. https://doi.org/10.3389/fmicb.2019.02529.
Kachapulula, P.W., Bandyopadhyay, R., Cotty, P.J. 2019. Aflatoxin contamination of dried wild fruits in Zambia. Frontiers in Microbiology. 10. https://doi.org/10.3389/fmicb.2019.01840.
Ezekiel, C., Ortega-Beltran, A., Oyedeji, E., Atehnkeng, J., Kossler, P., Tairu, F., Hoeschle-Zeledon, I., Karlovsky, P., Cotty, P.J., Bandypadhyay, R. 2019. Aflatoxin in chili peppers in Nigeria: extent of contamination and control using atoxigenic Aspergillus flavus genotypes as biocontrol agents. Toxins. 11(7). https://doi.org/10.3390/toxins11070429.
Bandyopadhyay, R., Atehnkeng, J., Ortega-Beltran, A., Akande, A., Falade, T., Cotty, P.J. 2019. “Ground-truthing” efficacy of biological control for aflatoxin mitigation in farmers’ fields in Nigeria: from field trials to commercial usage, a 10-year study. Frontiers in Microbiology. 10. https://doi.org/10.3389/fmicb.2019.02528.
Agbetiameh, D., Ortega-Beltran, A., Awuah, R.T., Atenhking, J., Islam, M.S., Callicott, K.A., Cotty, P.J., Bandyopadhyay, R. 2019. Potential of atoxigenic Aspergillus flavus vegetative compatibility groups associated with maize and groundnut in Ghana as biocontrol agents for aflatoxin management. Frontiers in Microbiology. 10. https://doi.org/10.3389/fmicb.2019.02069.
Singh, P., Callicott, K.A., Orbach, M.J., Cotty, P.J. 2020. Molecular analysis of S morphology aflatoxin producers from the United States reveals previously unknown diversity and two new taxa. Frontiers in Microbiology. 11. https://doi.org/10.3389/fmicb.2020.01236.
Aikore, S., Ortega-Beltran, A., Erubetine, D., Atehnkeng, J., Falade, T., Cotty, P.J., Bandyopadhyay, R. 2019. Performance of broilers fed with maize colonized by either toxigenic or atoxigenic strains of aspergillus flavus with and without an aflatoxin-sequestering agent. Toxins. 11(10). https://doi.org/10.3390/toxins11100565.