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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 #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

2023 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 fiscal year (FY) 2023 progress for project 2020-4200-023-000D, Improvement of the Aflatoxin Biocontrol Technology Based on Aspergillus flavus Population Biology, Genetics, and Crop Management Practices, which began in May 2021. 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, over 3,800 A. flavus isolates were genetically characterized from Cameroon (1049 isolates), Greece (780 isolates), Serbia (687 isolates), Spain (624), and the United States (Arizona, California, Texas, and Illinois; 613 isolates). These A. flavus were isolated from corn grain, corn silage, peanut, pistachio, almonds, walnuts, and crop-associated soil. The genetic characterization primarily used simple sequence (SSR) repeat markers, and additional molecular assays were used to identify single nucleotide polymorphisms (SNPs) or deletions in the aflatoxin biosynthesis gene cluster. The SSR data were used to identify frequent non-aflatoxigenic genotypes that may be useful for future biocontrol products or to find isolates genetically similar to registered biocontrol products. In support of Sub-objective 1B, ARS researchers in Maricopa, Arizona, continued to conduct 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 A. flavus S strain. Crop debris (corn or cotton) was inoculated with an aflatoxigenic strain and/or biocontrol strain AF36 then either placed on top of soil or mixed into the soil. Inoculated crop/soil samples were grown in a temperature-cycling growth chamber simulating conditions in southern Arizona from October through March, when overwintering occurs. Growth of the two strains in soil varied over time and burying crop debris impacted competition between the aflatoxin-producing and biocontrol strain, likely because of increased interactions with other soil microbes. Ability of crop debris to support sporulation by the two strains after different periods of overwintering was also assessed. Burying crop debris reduced sporulation potential to a greater extent than exposure to typical winter temperatures, and sporulation by aflatoxigenic S strain on crop debris was more impacted by exposure to soil microbes than was sporulation by the biocontrol strain. Similar experiments are being conducted with a variety of field soils to elucidate how different soil biological and physical properties influence growth, survival, and competition of A. flavus strains in the soil environment. Results of these experiments will aid in predicting the persistence of biocontrol strains over time and help inform better management practices for mitigating aflatoxin contamination. In support of Objective 2, ARS researchers performed laboratory experiments to understand the breakdown of aflatoxin by the non-aflatoxin producing biocontrol isolate AF36. Following growth in flasks of liquid media with and without aflatoxin, RNA was isolated from mycelia of AF36, and media were frozen for future metabolomic analysis to quantify aflatoxin degradation and identify potential degradation products. RNA transcripts are currently being sequenced, and a transcriptomic analysis will be conducted to identify changes in gene expression that occur in association with aflatoxin degradation. In support of Sub-objective 3A, ARS researchers conducted laboratory and field experiments to evaluate the impact of co-applied agrochemicals on the aflatoxin biocontrol strain A. flavus AF36. Though mixing with other products is not recommended, some farmers mix and co-apply aflatoxin biocontrol products and granular fertilizers in order to reduce time and equipment costs associated with treating fields. The formulated biocontrol product AF36 Prevail (spores coated on sorghum grain) was mixed with granular urea fertilizer and incubated at either 22°C or 31°C under moderate (approximately 50%) or high (greater than 85%) relative humidity for 0, 1, 4, 8, 24, or 168 hours. Granules of the biocontrol product were then incubated in a 24-well plate under high humidity at 31°C to evaluate sporulation of the active ingredient A. flavus AF36. Exposure of the biocontrol product to urea fertilizer for up to one week did not reduce sporulation of AF36 compared to the non-exposed control unless the biocontrol-fertilizer mixture was incubated under warm, humid conditions which resulted in a 100% reduction in sporulation. Applied mixtures of AF36 and urea fertilizer were also evaluated in the field, and there was no evidence that co-applications reduced the viability or efficacy of the biocontrol product. Furthermore, mixtures of AF36 and fertilizer provided by farmers were evaluated, and sporulation of the biocontrol product was not compromised even several weeks after mixing. In support of Sub-objective 3B, ARS researchers in Maricopa, Arizona, sampled soils and crops from tree nut growing areas of Arizona and California for a second year to evaluate the area-wide impacts of aflatoxin biocontrol product application in pistachios and almonds. Soil and leaves from upper and lower canopies were collected from biocontrol-treated and non-treated nut orchards, and soil was collected from non-treated fields and non-cultivated areas at varying distances from treated areas. Aspergillus flavus was isolated from soil and leaves, DNA was extracted, and isolates were genotyped using molecular markers. Biocontrol strain genotypes were detected in soil and tree canopies at similar frequencies. Biocontrol strains were most prevalent in treated areas and frequencies decreased with increasing distance from treated orchards. However, results suggest that biocontrol strains move long distances (greater than 10 km) throughout the landscape, resulting in area-wide displacement of aflatoxin-producing fungi. Thus, biocontrol applications may reduce aflatoxin contamination risk in non-treated crops, and it may not be necessary to treat every orchard every year to achieve adequate aflatoxin control in tree nuts. Additional years of sampling will provide insight into both dispersal and persistence of biocontrol strains and allow for improved recommendations for timing and frequency of biocontrol application for mitigation of aflatoxins. In support of Sub-objective 3C, ARS researchers conducted a second year of field experiments evaluating the efficacy of aflatoxin biocontrol in silage corn. Corn silage produced in hot, dry areas of the southwestern U.S. is frequently contaminated with aflatoxins, and since silage is primarily used as feed for dairy cows, aflatoxin concentrations must be maintained below 20 parts per billion (ppb). Samples were collected from five Arizona corn fields in which AF36 Prevail was applied and three fields in which Afla-Guard was applied. All fields had received biocontrol applications in previous years. Aspergillus flavus isolates belonging to the same genetic types as the biocontrol strains were recovered from all soils prior to biocontrol application at frequencies ranging from 20-92%, suggesting there is substantial carryover of biocontrol strains between growing seasons. Following biocontrol application and harvest of the crop, biocontrol strains made up 36-100% and 50-100% of the A. flavus population in soil and on the crop, respectively. Whereas a single isolate of Afla-Guard was recovered from fields that were not treated with Afla-Guard, AF36 made up an average of 32% and 18% of A. flavus in soil and on the crop, respectively, in fields that did not receive an AF36 application. Relatively high frequencies of AF36 in non-treated fields may be due to widespread application of AF36 in Arizona in multiple crops over several decades. Aflatoxin concentrations in the pre-and post-ensiled crop from all treated fields were below 10 ppb, demonstrating the effectiveness of biocontrol products for mitigating aflatoxin contamination in corn silage.

1. Identification of potential biocontrol isolates for mitigation of aflatoxin contamination in chilies. Dried red chili is frequently contaminated with aflatoxins. Although aflatoxin biocontrol products have been developed specifically for mitigation of aflatoxin contamination in oil seed crops, biocontrol strains with superior ability to reduce aflatoxin contamination in chilies have not been previously identified. Non-aflatoxigenic Aspergillus flavus genotypes associated with chilies collected in Nigeria were isolated and characterized by ARS researchers in Maricopa, Arizona, and in laboratory tests they reduced aflatoxins in chilies significantly more than biocontrol strains that are active ingredients in currently registered biocontrol products. These isolates are a potential resource for developing new biocontrol products specifically for control of aflatoxins in red chilies.

2. An analysis of natural Aspergillus flavus populations from Greece, Spain, and Serbia is a close relative of a registered biocontrol product present across southern Europe. To date, there is one aflatoxin biocontrol product in Europe, AF-X1, and it is currently registered for use only in Italy. Aspergillus flavus populations were examined on corn from Spain, Greece, and Serbia, all countries with an aflatoxin problem, to determine whether use of AF-X1 would require the introduction of novel genetic types. Using simple sequence repeat (SSR) markers, ARS researchers in Maricopa, Arizona, discovered that close relatives of AF-X1 were found in all three countries, suggesting that AF-X1 can be safely and effectively used throughout southern Europe where aflatoxin mitigation is needed.

3. Characterization of crop host influences on competition between species of aflatoxin-producing fungi. Maize and groundnuts, two crop hosts that differ in nutrient composition and physiology, are staple crops that are highly susceptible to aflatoxin contamination. Aflatoxins are produced by several Aspergillus species that co-occur and compete on crops. Results of a series of co-inoculation experiments on maize and groundnut kernels by ARS researchers in Maricopa, Arizona, indicated that complex interactions between crop hosts and different Aspergillus species modulate Aspergillus community structure and subsequent aflatoxin contamination. Understanding factors that impact sporulation, dispersal, crop infection, and aflatoxin production by different Aspergillus species is critical for developing crop management practices that will minimize aflatoxin contamination.

4. A new chromosome-level genome assembly of Aspergillus pseudotamarii reveals extensive genomic rearrangements relative to A. flavus. Aspergillus pseudotamarii is an understudied aflatoxin-producing fungus related to A. flavus. A highly contiguous genome of this species was generated by ARS researchers in Maricopa, Arizona, using a combination of short read and long read sequencing technologies. This contiguous genome showed numerous large-scale genomic rearrangements relative to A. flavus, including the region containing the aflatoxin biosynthesis cluster. Better quality genomes of Aspergillus species will be useful for developing genetic screens to identify opportunistic pathogens in crops and stored food products and for understanding the molecular evolution of secondary metabolite biosynthesis gene clusters in molds.

Review Publications
Garcia-Gonzalez, J., Mehl, H.L., Langston, D.B., Rideout, S.L. 2022. Planting date and cultivar selection to manage southern blight in potatoes in the mid-Atlantic United States. Crop Protection. 162. Article 106077.
Kaur, N., Mehl, H.L. 2022. Distribution of G143A mutations conferring fungicide resistance in Virginia populations of Parastagonospora nodorum infecting wheat. Plant Health Progress. 23(1):28-32.
Singh, P., Mehl, H.L., Orbach, M.J., Callicott, K.A., Cotty, P.J. 2022. Genetic diversity of Aspergillus flavus associated with chili in Nigeria and identification of haplotypes with potential in aflatoxin mitigation. Plant Disease. 106(7):1818-1825.
Weaver, M.A., Callicott, K.A., Mehl, H.L., Opoku, J., Park, L.C., Fields, K., Mandel, J.R. 2022. Characterization of the Aspergillus flavus population from highly aflatoxin-contaminated corn in the United States. Toxins. 14(11). Article 755.
Hagler, J.R., Casey, M.T., Hull, J.J., Machtley, S.A. 2022. A labor-saving marking and sampling technique for mark-release-recapture research. Entomologia Experimentalis et Applicata. 171(2):138-145.
Brent, C.S., Heu, C., Gross, R.J., Fan, B., Langhorst, D.R., Hull, J.J. 2022. RNAi-mediated manipulation of cuticle coloration genes in Lygus hesperus Knight (Hemiptera: Miridae). Insects. 13(11). Article 986.