Location: Pest Management and Biocontrol Research
2024 Annual Report
Objectives
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.
Approach
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 FY 2024 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 Sub-objective 1A, ARS researchers in Maricopa, Arizona, used simple sequence repeat (SSR) markers to characterize over 2800 A. flavus isolates from the U.S. (n = 1,014), Bangladesh (n = 279), Nigeria (n = 770), and Pakistan (n = 767). Additionally, ARS researchers sequenced the genomes of 39 Aspergillus section Flavi isolates, including 22 biocontrol isolates from Africa, 10 isolates from California tree nuts, five isolates from Bangladesh, and two isolates from a species closely related to A. flavus. SSR typing profiles the genetic diversity within target agroecosystems, allowing the fate of biocontrol agents to be tracked in the field or to identify frequent non-aflatoxigenic isolates that may be useful in the target area as biocontrol. Genomic sequencing facilitates the development of molecular assays to track relevant genetic types in the field and to profile genetic differences among isolates. This information can be used to predict features useful for biocontrol selection, such as competitive ability.
In support of Sub-objective 1B, ARS researchers continued work on a series of laboratory soil microcosm experiments aimed at determining the influence of soil biotic and abiotic factors on the competition between the non-aflatoxigenic A. flavus biocontrol strain AF36 and aflatoxin-producing A. flavus S strain. Competition between the two A. flavus isolates was evaluated in ten soils collected from fields varying in soil type and cropping history. Ground corn was incorporated into sterile (autoclaved) and non-sterile soil from each field, and soil-corn mixtures were co-inoculated with AF36 and AF70. In the non-sterile soil, growth of the two isolates was similar, but in the sterile soil AF36 growth was 10 times greater than that of AF70. AF36 comprised a greater proportion of the total A. flavus in sterile (87%) versus non-sterile (65%) soils, but outcomes of competition between AF36 and AF70 were similar among the 10 soils evaluated. Overall, A. flavus growth was greater in sterile compared to non-sterile soil with a 100-fold and 24-fold increase in growth of AF36 and AF70, respectively. Aspergillus flavus growth varied among soils with a 40-fold (AF36) and 18-fold (AF70) difference between soils supporting the greatest and least growth. Microbial communities were characterized from A. flavus inoculated and non-inoculated soils using metabarcoding. Although soil microbial community composition varied among the 10 soils, it was not influenced by the presence of A. flavus. Results suggest that both abiotic and biotic soil factors influence A. flavus growth, the presence of soil microbes had a greater influence than soil type. AF36 was more competitive in sterile compared to non-sterile soils, indicating that soil microbes modulate A. flavus competition. Though overall growth varied among soils, outcomes of competition between AF36 and AF70 were not influenced by soil type. Additional experiments will be conducted to determine the influence of soils on competition between different non-aflatoxigenic and aflatoxigenic A. flavus genotypes. Results of these experiments will aid in predicting growth and outcomes of competition between applied biocontrol and resident A. flavus in field soils. This will inform management recommendations for mitigating aflatoxin contamination through competitive displacement of aflatoxin-producing fungi.
In support of Objective 2, ARS researchers performed laboratory experiments to understand the breakdown of aflatoxin by the non-aflatoxin producing A. flavus biocontrol isolate AF36. Isolates were grown in four experiments with and without aflatoxins, with two replicates containing micronutrients and two lacking micronutrients. RNA was isolated from these cultures at two time points (four and seven days), and the growth medium was freeze-dried and sent to New Orleans for metabolomic analysis. RNA was sequenced from all four experiments, and the data from the cultures lacking micronutrients have been analyzed. Between the two experiments lacking micronutrients, there were two genes significantly upregulated on day four in flasks containing aflatoxins. While both were annotated as “uncharacterized proteins”, gene ontology indicated potential function. The first gene contained a cytochrome P450 superfamily conserved protein domain and with potential reductase and aromatase activity, while the second was similar to ABC-type membrane transporters/permeases. Additionally, there were 272 genes with significantly differential expression that were largely associated with lipid metabolism or mycotoxin biosynthesis. These genes present potential markers of aflatoxin degradation ability in non-aflatoxigenic fungi, which in turn may help select better biocontrol isolates.
In support of Sub-objective 3A, ARS researchers in cooperation with Arizona growers, conducted field studies to evaluate the impact of co-applied agrochemicals on the aflatoxin biocontrol strain A. flavus AF36. Results from the field were consistent with laboratory studies that indicated mixing the formulated biocontrol product AF36 Prevail with urea fertilizer does not reduce sporulation of the biocontrol strain. Additional field studies will be conducted to assess the impact of co-applied fungicides, insecticides, herbicides, and/or soil amendments on the efficacy of aflatoxin biocontrol products. Results will provide a basis for management recommendations regarding compatibility of aflatoxin biocontrol products and agrochemical applications.
In support of Sub-objective 3B, soils were collected pre- and post-biocontrol treatment in tree nut growing areas of Arizona for a third year to evaluate area-wide, long-term impacts of applying a non-aflatoxigenic biocontrol strain. To assess area-wide displacement of aflatoxigenic A. flavus by the applied biocontrol strain, soils were collected from treated pistachio orchards and non-treated pecan orchards, and A. flavus were isolated and genetically characterized. Prior to biocontrol application in the spring, soil populations of A. flavus were dominated by the biocontrol strain genotype in both previously treated and untreated orchards. Post-application, the biocontrol strain comprised 77% of the A. flavus soil population in treated orchards versus 22% in non-treated orchards. Frequency of biocontrol strain recovery from soils was similar across years. Results indicate the biocontrol strain is able to effectively disperse from treated to non-treated areas and survive in soils across growing seasons. However, to ensure the biocontrol strain remains a dominant component of the population within orchards when conditions are conducive for A. flavus growth and dispersal onto the crop, biocontrol products should be consistently applied though it may not be necessary to treat every orchard every year. To better understand overwintering survival of the biocontrol strain in treated pistachio orchards, a study was initiated in which soils and crop residues are collected on a monthly basis. Viable propagules of the biocontrol strain genotype and other A. flavus in soils and different types of crop residue are being quantified over time. Results will provide insight into where and to what extent the biocontrol strain and aflatoxigenic A. flavus are surviving and proliferating within orchards over time and in response to different environmental conditions.
In support of Sub-objective 3C, ARS researchers conducted a third year of field experiments evaluating the efficacy of aflatoxin biocontrol in corn grown for silage. Corn silage produced in hot, dry areas of the southwestern U.S. is frequently contaminated with aflatoxins. Because silage is primarily used as feed for dairy cows, aflatoxin concentrations must be maintained below 20 parts per billion (ppb). In 2021-2023, experiments were conducted in corn fields in Arizona. Soil was sampled both prior to application of a biocontrol product and following harvest. Biocontrol products (AF36 Prevail or Afla-Guard) were applied prior to tasseling of the corn crop at the labeled rate. Following irrigation, non-aflatoxigenic A. flavus biocontrol strains sporulated and moved onto the crop, displacing aflatoxigenic fungi. At harvest, crop sub-samples were collected from silage piles and aflatoxin concentrations were measured. Aspergillus flavus was isolated from soil and crop samples and frequencies of applied biocontrol strains were determined using DNA-based methods. Displacement of aflatoxin producing fungi in soils and on the crop varied across years. Whereas displacement by AF36 was greater in 2021 and 2022 compared to 2023, displacement by Afla-Guard was greater in 2023 compared to the other two years suggesting the two biocontrol strains may be adapted to different environmental conditions. Future studies will elucidate specific environmental factors that favor different biocontrol strain genotypes. Overall, the two biocontrol strains were similarly effective at displacing aflatoxin-producing fungi on the crop (AF36 = 81%; Afla-Guard = 64%) and reducing aflatoxin concentrations below 10 ppb, but displacement in the soil was significantly greater for AF36 (88%) compared to Afla-Guard (42%). In addition, one year following biocontrol treatment, an average of 56% of the A. flavus soil population was AF36 versus 20% for Afla-Guard. Results indicate both biocontrol strains effectively displace aflatoxin-producing fungi on corn grown for silage in Arizona and reduce aflatoxin concentrations, but AF36 may be better adapted to competition and persistence in soils compared to Afla-Guard.
Accomplishments
1. Registration of a new aflatoxin biocontrol product for Texas corn. ARS scientists in Maricopa, Arizona, assisted Texas Corn Producers with development and registration of a new four-strain aflatoxin biocontrol product (FourSure). Following over a decade of research and development, full EPA registration (EPA Reg. No. 91163-2) was granted in September 2023. This will provide Texas corn growers with an additional product for mitigation of aflatoxin contamination.
2. Biotic and abiotic factors influencing growth, competition, and survival of biocontrol and aflatoxigenic Aspergillus flavus strains in soil. The extent to which applied A. flavus biocontrol strains persist in soils is variable, and biotic and abiotic factors that impact success of specific A. flavus genotypes in soils have not been well characterized. ARS scientists in Maricopa, Arizona, conducted laboratory and field studies that demonstrated fields with different assemblages of microbes, crop histories, and soil types support different levels of overall A. flavus growth. Some soils support greater biocontrol strain growth whereas others are more favorable for aflatoxigenic A. flavus. Understanding the causes of these differences will allow identification of fields that are at greater risk of aflatoxin contamination in the absence of biocontrol applications and fields in which it may be possible to apply the biocontrol less frequently, thereby reducing grower input costs.
3. Area-wide aflatoxin management with non-aflatoxigenic-based biocontrol products in tree crops. Aflatoxin biocontrol products have been registered for tree nut crops, including pistachios, for nearly a decade, but the long-term, area-wide effects of biocontrol applications has not been well studied. ARS scientists in Maricopa, Arizona, analyzed soil and crop samples from biocontrol treated and untreated fields in tree nut growing areas of Arizona and California. Biocontrol strains were most prevalent in treated areas and frequencies decreased with distance from treatment. However, results indicated that biocontrol strains move long distances throughout the landscape, resulting in area-wide displacement of aflatoxin-producing fungi. Since biocontrol applications can reduce aflatoxin contamination risk in non-treated crops, it may not be necessary to annually treat every orchard to achieve adequate aflatoxin control in tree nuts which will reduce the overall cost of aflatoxin mitigation.
4. Efficacy of aflatoxin biocontrol and aflatoxin management recommendations for silage corn. Though efficacy of aflatoxin biocontrol using non-aflatoxigenic strains of A. flavus has been demonstrated for corn grain, the extent to which biocontrol strains can competitively displace aflatoxigenic A. flavus from pre-harvest through post-ensiling stages of corn silage has not been previously quantified. ARS researchers in Maricopa, Arizona, conducted studies in Arizona producer fields, and biocontrol products effectively displaced aflatoxin-producing fungi in the soil and on the crop, reducing aflatoxin concentrations in the pre-and post-ensiled crop below 20 ppb. This demonstrates the effectiveness of biocontrol products for mitigating aflatoxin contamination in corn silage and provides a basis for recommending biocontrol applications when corn silage is produced in regions with high risk of aflatoxin contamination.
Review Publications
Ching'anda, C., Bandyopadhyay, R., Callicott, K.A., Orbach, M.J., Cotty, P.J., Mehl, H.L. 2023. Crop host influences on outcomes of competition between Aspergillus section Flavi species co-infecting maize and groundnuts. PhytoFrontiers. 3(2):369-381. https://doi.org/10.1094/PHYTOFR-07-22-0076-R.
Kaur, N., Mehl, H.L., Langston, D., Haak, D. 2024. Evaluation of Stagonospora nodorum blotch severity and Parastagonospora nodorum population structure and genetic diversity across multiple locations and wheat varieties in Virginia. Phytopathology. 114(1):258-268. https://doi.org/10.1094/PHYTO-10-22-0392-R.
Luis, J., Mehl, H.L., Plewa, D., Kleczewski, N.M. 2023. Is Microdochium maydis associated with necrotic lesions in the tar spot disease complex? A culture-based survey of maize in Mexico and the midwestern United States. Phytopathology. 113(10):1890-1897. https://doi.org/10.1094/PHYTO-04-23-0109-R.
Ouadhene, M.A., Callicott, K.A., Ortega-Beltran, A., Mehl, H.L., Cotty, P.J., Battilani, P. 2024. Structure of Aspergillus flavus populations associated with maize in Greece, Spain, and Serbia: Implications for aflatoxin biocontrol on a regional scale. Environmental Microbiology Reports. 16(2). Article e13249. https://doi.org/10.1111/1758-2229.13249.
Legan, A.W., Mehl, H.L., Varaksa Jr., A.A., Callicott, K.A. 2023. Nanopore PCR-cDNA sequencing of the biocontrol isolate Aspergillus flavus AF36 (NRRL 18543) informs gene annotation. Microbiology Resource Announcements. 12. Article e00527-23. https://doi.org/10.1128/MRA.00527-23.
Legan, A.W., Mehl, H.L., Wissotski, M., Adhikari, B.N., Callicott, K.A. 2024. Telomere-to-telomere genome assembly of the aflatoxin biocontrol agent Aspergillus flavus isolate La3279 isolated from maize in Nigeria. Microbiology Resource Announcements. 13. Article e00696-23. https://doi.org/10.1128/mra.00696-23.
Legan, A.W., Mack, B.M., Mehl, H.L., Wissotski, M., Ching'anda, C., Maxwell, L.A., Callicott, K.A. 2023. Complete genome of the toxic mold Aspergillus pseudotamarii isolate NRRL 25517 reveals genomic instability of the aflatoxin biosynthesis cluster. G3, Genes/Genomes/Genetics. 13(9). Article jkad150. https://doi.org/10.1093/g3journal/jkad150.