Skip to main content
ARS Home » Pacific West Area » Maricopa, Arizona » U.S. Arid Land Agricultural Research Center » Pest Management and Biocontrol Research » Research » Research Project #440431

Research Project: Improvement of the Aflatoxin Biocontrol Technology Based on Aspergillus flavus Population Biology, Genetics, and Crop Management Practices

Location: Pest Management and Biocontrol Research

2022 Annual Report

Objective 1: Characterize Aspergillus section Flavi diversity and population dynamics in response to biotic and abiotic factors with a focus on the soil environment. Sub-objective 1A: Characterize Aspergillus section Flavi diversity in target agroecosystems and develop/refine tools for typing and quantifying specific genotypes in the environment. Sub-objective 1B: Evaluate the survival, growth, and dispersal of biocontrol strains (atoxigenics) versus high aflatoxin producers in response to biotic and abiotic factors with a focus on the soil environment. Objective 2: Elucidate molecular mechanisms involved in aflatoxin degradation by atoxigenic Aspergillus flavus. Objective 3: Identify management practices that will increase the efficacy and reduce the cost of aflatoxin biocontrol in diverse cropping systems. Sub-objective 3A: Evaluate impact of co-applied agrochemicals on aflatoxin biocontrol efficacy. Sub-objective 3B: Optimize management recommendations for area-wide aflatoxin management with atoxigenic-based biopesticides in tree crops. Sub-objective 3C: Evaluate efficacy of aflatoxin biocontrol and develop aflatoxin management recommendations for silage corn.

Sub-objective 1A: Global populations of Aspergillus flavus and related species will be characterized to identify genotypes that are dominant in target agroecosystems and to provide genomic targets useful for typing and tracking those lineages in the environment. Isolates of A. flavus will be provided by U.S. and international collaborators. Isolates will be genotyped using simple sequence repeat (SSR) markers, and data will be added to the previously developed SSR database (AflaSat). Molecular assays that distinguish between species/genotypes will be designed based on whole genome sequencing of multiple species/isolates within Aspergillus section Flavi. Sub-objective 1B: A series of soil microcosm experiments aimed at understanding A. flavus population dynamics in agricultural soils will be conducted. The focus will be on competition between non-aflatoxigenic biocontrol strains of A. flavus and high aflatoxin-producing S strain A. flavus in soil. Experiments will be conducted in different soil types, in autoclaved versus non-autoclaved field soil, and at different soil temperatures and moisture contents. Influences of treatments on survival, growth, and sporulation of non-aflatoxigenic and S strain A. flavus will be assessed using a combination of culture- and DNA-based methods. Objective 2: The phenomenon of aflatoxin degradation by non-aflatoxigenic A. flavus isolates will be assessed using transcriptomic analysis. Changes in gene expression in the presence or absence of aflatoxin and glucose as carbon sources will be used to identify potential mechanisms of aflatoxin degradation. In addition, a metabolomic study will determine products of aflatoxin degradation by non-aflatoxigenic A. flavus. Sub-objective 3A: A combination of laboratory, small plot, and large-scale field studies will be used to assess impacts of fertilizer, herbicide, insecticide, and fungicide co-treatments on efficacy of aflatoxin biocontrol. Sporulation of biocontrol strains on formulated products and growth of active ingredient strains will be quantified with and without exposure to co-treatment agrochemicals. Sub-objective 3B: Movement and persistence of an applied non-aflatoxigenic biocontrol strain will be quantified in a tree crop production area in Arizona. Soil will be collected from biocontrol treated pistachio orchards, non-treated tree crop orchards, fields with an annual crop (e.g., corn, cotton), and crop-adjacent desert lands. Sampling will be conducted along transects with increasing distance away from biocontrol treated areas. This will allow for quantification of biocontrol strain movement across the landscape in a tree crop production area. Sub-objective 3C: Efficacy of aflatoxin biocontrol products will be assessed in commercial fields of silage corn in Arizona. Soil will be collected prior to biocontrol application and following harvest. Chopped samples of corn silage will be sampled immediately following harvest and monthly from silage piles. Percentages of A. flavus in soil and on the crop belonging to the same genotype as the applied biocontrol strain will be quantified, and aflatoxin concentrations in silage will be measured.

Progress Report
This report documents progress for project 2020-42000-023-000D, Improvement of the Aflatoxin Biocontrol Technology Based on Aspergillus flavus Population Biology, Genetics, and Crop Management Practices, which started in May 2021 and continues research from project 2020-42000-022-000D, Improved Environmental and Crop Safety by Modification of the Aspergillus flavus Population Structure. In support of Objective 1, ARS researchers in Maricopa, Arizona, characterized Aspergillus section Flavi diversity and population dynamics in response to biotic and abiotic factors with a focus on the soil environment. For Sub-objective 1A, A. flavus populations from Sudan (1,021 isolates), the Democratic Republic of the Congo (874 isolates), and Bangladesh (205 isolates) were genetically characterized using simple sequence repeat (SSR) markers. In addition, non-aflatoxigenic isolates were identified through detection of deletions in the aflatoxin biosynthesis gene cluster. The genetic profiles of these isolates have been analyzed to determine if any of these isolates are genetically similar to biocontrol isolates that are currently used in other countries and to identify additional frequently occurring non-aflatoxigenic genotypes that may be the foundation of region-specific biocontrol products. In support of Sub-objective 1B, ARS researchers in Maricopa, Arizona, conducted a series of laboratory soil microcosm experiments aimed at determining the influence of soil biotic and abiotic factors on competition between the non-aflatoxigenic A. flavus biocontrol strain AF36 and aflatoxin-producing S strain. Molecular assays (real-time polymerase chain reaction (PCR) and droplet digital (dd)PCR) aimed at quantifying specific A. flavus genotypes in soils were optimized. Soils varying in physical, chemical, and biological properties were collected from ten locations, and autoclaved and non-autoclaved soils were co-inoculated with AF36 and S strain A. flavus. Soils supported different levels of overall A. flavus growth, and the extent to which non-sterilized soils suppressed A. flavus growth compared to autoclaved soils varied among the different soils. Furthermore, some soils supported greater growth of AF36 whereas others were more favorable for growth of the S strain. In a separate series of experiments, the impacts of soil moisture and temperature on growth and competition between AF36 and S strain A. flavus were evaluated in four soils varying in texture (e.g., sand/silt/clay content). Currently, different soil types from fields with variable cropping histories are being collected throughout Arizona and California, and these will be used to conduct additional soil microcosm experiments aimed at elucidating the relative importance of soil physical, chemical, and biological properties on growth, survival, and competition between biocontrol strains and aflatoxin-producing A. flavus. For Objective 2, ARS researchers in Maricopa, Arizona, initiated joint transcriptomics and metabolomics studies in conjunction with ARS scientists at the Food and Feed Safety Research Unit in New Orleans, Louisiana, in order to understand the breakdown of aflatoxins by non-aflatoxin-producing biocontrol isolates currently registered for use in the United States. Protocols for growth of non-aflatoxigenic A. flavus isolates in liquid media with and without aflatoxin, for the extraction of RNA from these cultures, and for the extraction of metabolites from both the mycelia and liquid media have been developed in preparation for the actual experiments. Under Sub-objective 3A, ARS researchers in Maricopa, Arizona, conducted laboratory experiments to evaluate the impact of co-applied agrochemicals on the aflatoxin biocontrol strain AF36, in order to identify management practices that will increase the efficacy and reduce control costs in diverse cropping systems. The formulated biocontrol product AF36 Prevail (spores coated on sorghum grain) was mixed with granular urea fertilizer for 0, 1, 12, and 24 hours at room temperature (approximately 28°C) and then incubated in a 24-well plate under high humidity to evaluate sporulation of the fungus. Exposure of the biocontrol product to fertilizer for up to 24 hours did not reduce sporulation of AF36 compared to the non-exposed control suggesting that mixing AF36 Prevail with granular fertilizer may not reduce its efficacy. In vitro experiments were conducted to evaluate the sensitivity of AF36 and aflatoxigenic L and S strain isolates of A. flavus to fungicides (e.g., azoxystrobin). All A. flavus isolates evaluated were sensitive to azoxystrobin, but AF36 and aflatoxigenic L strain were less sensitive than S strain to the fungicide. Additional studies will evaluate the sensitivity of AF36 on the formulated biocontrol product to a variety of agrochemicals in the lab and in field experiments. In support of Sub-objective 3B, ARS researchers in Maricopa, Arizona, sampled soils and crops from tree nut growing areas of Arizona to evaluate the area-wide impacts of AF36 Prevail application in pistachios. Aspergillus flavus was isolated from the samples, and AF36 was identified based on a genotype-specific single nucleotide polymorphism (SNP). Over 50% of the A. flavus population in treated pistachio orchards was comprised of the AF36 genotype, and approximately 40% of the A. flavus population was AF36 in non-treated pecan orchards adjacent to treated pistachio. Just under 30% and 15% of the population was the AF36 genotype in pecan orchards and other crop fields, respectively, that were up to two miles from treated pistachio. Soils and crops in tree nut growing areas in Arizona and California are currently being sampled to further evaluate the extent to which biocontrol applications increase the frequency of biocontrol strains and reduce aflatoxin-producing fungi in both treated fields and surrounding non-treated areas. In support of Sub-objective 3C, ARS researchers in Maricopa, Arizona, collected soil and crop samples from six corn silage fields in Arizona where aflatoxin biocontrol products (AF36 Prevail or Afla-Guard) were applied in 2021. Four fields had received biocontrol applications in previous years, whereas two fields received their first biocontrol application in 2021. Aspergillus flavus isolates belonging to the same genetic types as the biocontrol strains were recovered from all soils prior to biocontrol application in 2021, though at relatively low frequencies. Prior to biocontrol application, 26-70% of the soil population of A. flavus was comprised of aflatoxin-producing strains. Following AF36 application and harvest of the crop, AF36 made up 95-100% of the A. flavus soil population. In fields where Afla-Guard was applied, both Afla-Guard and AF36 strains were recovered, likely because AF36 is already a natural component of field soils in Arizona. On the crop, both biocontrol products were effective in displacing aflatoxin-producing fungi by 80-100% and aflatoxin concentrations in the pre-ensiled harvested crop from all fields was below 20 parts per billion (ppb). Aspergillus flavus populations declined over time in silage piles, but the biocontrol strains remained the dominant genotypes and aflatoxin concentrations remained low post-ensiling. Similar field experiments are being conducted in 2022.


Review Publications
Faske, T.R., Kandel, Y., Allen, T.W., Grabau, Z.J., Kemerait, R.C., Lawrence, G.W., Lawrence, K.S., Mehl, H.L., Overstreet, C., Thiessen, L.D., Wheeler, T. 2022. Meta-analysis of the field efficacy of seed- and soil-applied nematicides on Meloidogyne incognita and Rotylenchulus reniformis across the U.S. Cotton Belt. Plant Disease.
Schmidt, M., Mao, Y., Opoku, J., Mehl, H.L. 2021. Enzymatic degradation is an effective means to reduce aflatoxin contamination in maize. BioMed Central (BMC)Biotechnology. 21. Article 70.
Ching'Anda, C., Atehnkeng, J., Bandyopadhyay, R., Callicott, K.A., Orbach, M., Cotty, P.J., Mehl, H.L. 2022. Spatial and temporal population dynamics of Aspergillus flavus in commercial pistachio orchards in Arizona. Plant Health Progress. 23:272-280.
Wei, X., Langston, D.B., Mehl, H.L. 2022. Comparison of current peanut fungicides against Athelia rolfsii through a laboratory bioassay of detached plant tissues. Plant Disease. 106(8):2046-2052.