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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

2021 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
This is the final report for the project 2020-42000-022-00D which terminated in May 2021. Ongoing projects related to this project’s objectives are being completed as part of the bridging project 2020-42000-023-00-D which will end in December 2021. The new project is currently going through NP108 OSQR review. A summary of results for all the expiring project objectives are described below. In support of Objective 1, research continued to 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 with the development and deployment of new aflatoxin biocontrol products and formulations. A method for monitoring frequencies of biocontrol strains in tree canopies was developed, and this method was used to initiate long-term monitoring of the presence of the biocontrol strain AF36 in tree nut (e.g. pistachio and almond) production areas in Arizona and California. Results demonstrated the ability of biocontrol applications to increase the frequency of non-aflatoxin producing fungi and decrease the aflatoxin-producing potential of fungal populations associated with tree nuts. Furthermore, area-wide benefits resulting from movement of biocontrol strains to non-treated areas and long-term increases in frequencies of the non-aflatoxigenic strain resulting from annual applications of the biocontrol product have been documented. Thus, widespread use of aflatoxin biocontrol products by tree nut growers will result in long-term control of aflatoxin-producing fungi in production areas and minimize the incidence and severity of aflatoxin contamination in high value crops including pistachio and almonds. In collaboration with the Arizona Cotton Research and Protection Council (ACRPC), ARS researchers evaluated the effectiveness of field perimeter biocontrol treatments in cotton. The applied biocontrol strain was detected in the center of fields at similar frequencies compared to those on the crop from the treated perimeter of the field. This suggests that adequate displacement of aflatoxin-producing fungi can be achieved without treating the entire field, thus reducing the time and cost of biocontrol product application. In addition to optimizing aflatoxin biocontrol recommendations for currently registered products, research contributed to the development and registration of new aflatoxin biocontrol products and formulations. There is a need for aflatoxin biocontrol worldwide, and optimal biocontrol strains for target regions are selected based on their natural occurrence in association with crops. In collaboration with the International Institute of Tropical Agriculture (IITA), ARS scientists conducted research that led to the selection of regionally adapted biocontrol strains, formulation of strains into granular products, development of biocontrol product manufacturing infrastructure, and registration of aflatoxin biocontrol products for countries throughout Sub-Saharan Africa including Senegal, The Gambia, Burkina Faso, Ghana, Tanzania, Mozambique, Malawi, and Zambia. Substantial contributions were also made to the development of a biocontrol product for Pakistan, and the registration application for the product is currently under review by the Pakistan Department of Plant Protection. Dissemination of the aflatoxin biocontrol technology internationally has led to improved food safety and increased the ability of developing nations to trade with countries that strictly enforce aflatoxin limits in commodities. A major focus of the expiring project was to work with the Texas Corn Producers to develop a multi-strain biocontrol product for use in Texas corn. This built upon work conducted in previous projects that identified atoxigenic strains associated with Texas corn that were able to effectively displace aflatoxin-producing fungi on the crop across a range of environments in the state. Large scale field trials were conducted over several years in commercial fields to generate efficacy data, and the registration package for the product (FourSure) is currently under the final stages of review by the Environmental Protection Agency. This will be the third commercial aflatoxin biocontrol product registered for use in the United States, and it will provide Texas corn growers with an additional tool to mitigate crop aflatoxin contamination. In support of Objective 2, research continued to develop an understanding of the distribution of Aspergillus flavus genetic types and vegetative compatibility groups worldwide in order to improve selection of biological control agents. This resulted in selection of strains used to develop the aflatoxin biocontrol products. Approximately 29,000 isolates originating from 35 countries in North America, Central America, Africa, Europe, Asia, and Australia were genotyped using DNA-based markers, and whole genomes were sequenced for the most common genetic types. An online database was developed that includes the genotype data for these isolates, and the database will become publicly available by the end of 2021. The database has been used to identify the most common atoxigenic genotypes associated with target regions and crops. These genotypes were selected as candidate biocontrol strains and evaluated for aflatoxin biocontrol efficacy in lab and field studies. Population genetic analyses of A. flavus isolates at regional, continental, and inter-continental scales indicated there is long-distance dispersal and broad distribution of individual A. flavus genotypes. In support of Objective 3, research focused on understanding the development, evolution, and stability of populations of A. flavus. Isolates originating from Africa and North America but belonging to a single genetic type were sequenced and compared. In addition, selected isolates grown in culture over multiple generations were sequenced to determine types and rates of genetic change. Frequent, small changes were observed in genomes, but changes in markers used for genotyping isolates were not observed. In addition, the genetic stability of genotypes that are used as biocontrol strains were assessed before and after their application to crops. 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.

1. Development of a multi-strain aflatoxin biocontrol product for Texas corn. Aflatoxin contamination of corn grain causes significant economic losses. Two aflatoxin biocontrol products are commercially available in the United States, but neither one was developed specifically for corn, and both are based on a single non-aflatoxigenic strain. At the request of the Texas Corn Producers, ARS researchers in Maricopa, Arizona, identified four biocontrol strains that are well adapted to corn in different regions of Texas, and these were developed into a formulated biocontrol product. Field studies demonstrated the efficacy and safety of the product, which is now under the final stages of review by the Environmental Protection Agency for full registration. This will be the third commercial aflatoxin biocontrol product registered for use in the United States, and it will provide Texas corn growers with an additional tool to mitigate crop aflatoxin contamination.

2. Development of an aflatoxin biocontrol product for Pakistan. Aflatoxin biocontrol is one of the most effective approaches to minimize the incidence and severity of crop contamination, however, developing countries do not always have access to the technology. In collaboration with industry and government partners in both the United States and Pakistan, ARS researchers from Maricopa, Arizona, developed an aflatoxin biocontrol product based on a non-aflatoxigenic strain of Aspergillus flavus that is endemic to the corn production region of Pakistan, and provided guidance for the commercial manufacturing and distribution of the biocontrol product in Pakistan. This is the first aflatoxin biocontrol product to be developed for South Asia, and its availability in the region will improve food/feed safety and the ability of Pakistan to meet international trade standards.

3. AflaSat: A database of global genetic diversity of Aspergillus flavus. Global populations of Aspergillus flavus are genetically diverse. Understanding distributions of aflatoxigenic and non-aflatoxigenic genotypes is important for developing effective aflatoxin control strategies for crops. ARS researchers in Maricopa, Arizona, utilized DNA-based markers to genotype nearly 29,000 isolates originating from 35 countries in North America, Central America, Africa, Europe, Asia, and Australia. An online database called “AflaSat” was developed, and standardized genotype data for each of these isolates was uploaded and made accessible to collaborators. AflaSat will be publicly available in Fall 2021, serving as a valuable resource for aflatoxin researchers worldwide, especially for identification of non-aflatoxigenic genotypes common to target areas that have potential as biocontrol strains.

4. Identification of widely distributed naturally occurring non-aflatoxigenic genotypes of Aspergillus flavus. A goal of aflatoxin biocontrol research is to identify non-aflatoxigenic strains of Aspergillus flavus with superior ability to displace aflatoxin-producing fungi in crops and soils. Even though A. flavus populations are genetically diverse, certain genotypes are widely distributed within and across regions. An ideal biocontrol strain is one that persists in a wide range of cropping systems over multiple years. ARS researchers in Maricopa, Arizona, quantified the distributions and frequencies of strains that are active ingredients in two commercial aflatoxin biocontrol products available in the United States in soil and crop samples collected from regions where the biocontrol products had not been previously applied. One strain was common across multiple Kenyan agroecosystems, and the other strain was detected throughout the southern United States. Results indicate these atoxigenic strains are broadly adapted to a range of agroecosystems, and biocontrol products based on these strains are likely to be effective on a regional basis.

5. Identification of aflatoxin degradation as a mechanism of aflatoxin biocontrol by non-aflatoxigenic strains of Aspergillus flavus. Displacement of aflatoxin-producing fungi on crops by applied biocontrol strains is considered the primary mechanism of aflatoxin biocontrol, but some strains reduce aflatoxin more than can be explained by competitive displacement alone. ARS researchers in Maricopa, Arizona, tested strains that are active ingredients in commercially available aflatoxin biocontrol products for their ability to degrade aflatoxin. All strains tested were able to degrade aflatoxin in corn grain by over 40% after seven days, with some doing better than others. The results demonstrate that in addition to displacement capacity, screening atoxigenic strains for a superior ability to degrade aflatoxin may also help identify new or superior strains for biocontrol products.

6. A simple, cost-effective method for differentiating between two morphologically similar aflatoxin-producing species from West Africa. Aflatoxins can result from crop infection by a variety of Aspergillus species, and identifying the causal agent is important for developing strategies to mitigate aflatoxin contamination. There are several species of highly aflatoxigenic Aspergillus in West Africa that are morphologically indistinguishable. ARS researchers in Maricopa, Arizona, developed a culture-based method utilizing defined microbiological media that can distinguish between A. aflatoxiformans and A. minisclerotigenes based on their production of aflatoxins in media with different sources of nitrogen. This method will be useful for distinguishing these two species in a region where aflatoxin researchers frequently do not have access to the equipment and supplies necessary for DNA-based identification of fungal species.

7. A new method for monitoring dispersal of an aflatoxin biocontrol strain in tree nut cropping systems. The use of the aflatoxin biocontrol technology in tree nuts is relatively new, and the extent to which the applied biocontrol strain moves within tree canopies has not been documented. Efficient movement of the soil-applied biocontrol strain into the tree canopy is necessary for the biocontrol strain to displace aflatoxin-producing fungi on the nut crop and prevent aflatoxin contamination. ARS researchers in Maricopa, Arizona, developed a method for monitoring populations of Aspergillus flavus throughout the entire tree canopy during the growing season. This method is currently being utilized to monitor the effectiveness of aflatoxin biocontrol in tree nut production areas and to document the long-term, area-wide effects of biocontrol applications.

Review Publications
Adhikari, B.N., Callicott, K.A., Cotty, P.J. 2021. Conservation and loss of a putative iron utilization gene cluster among genotypes of aspergillus flavus. Microorganisms. 9(1). Article 137.
Singh, P., Callicott, K.A., Orbach, M., Cotty, P.J., Mehl, H.L. 2020. Phenotypic differentiation of two morphologically similar aflatoxin producers from West Africa. Toxins. 12(10). Article 656.
Islam, M.S., Callicott, K.A., Mutegi, C., Bandyopadhyay, R., Cotty, P.J. 2020. Distribution of active ingredients of a commercial aflatoxin biocontrol product in naturally occurring fungal communities across Kenya. Microbial Biotechnology. 14(4):1331-1342.