Location: Biological Control of Pests Research
2023 Annual Report
Objectives
Objective 1: Optimize aerial and seed treatment application strategies for biological and chemical pest control agents for row crops such as corn, peanut, and soybean.
Sub-objective 1.A: Managing aflatoxin in corn by biochar and non-toxigenic Aspergillus flavus.
Sub-objective 1.B: Managing aflatoxins in peanut by biochar and non-toxigenic Aspergillus flavus.
Sub-objective 1.C: Highly economical deployment of non-aflatoxigenic A. flavus for corn production in Guatemala.
Sub-objective 1.D: Managing mycotoxin in soybean infected with charcoal rot disease caused by Macrophomina phaseolina.
Objective 2: Utilize Aspergillus genomics for population studies, and improve risk models.
Sub-objective 2.A: Genetic characterization of the A. flavus population within niches of the corn production ecosystem.
Sub-objective 2.B: Improve emerging weather-based aflatoxin risk models with field level data of pathogen genotypes.
Approach
Mycotoxin contamination in row crops like corn, peanut and soybean contributes to significant economic losses for farmers and industry. Mycotoxin contamination (e.g., aflatoxins in corn and peanuts) poses substantial food and feed safety risks. The project goal is to continue work on reducing and managing major toxins in row crops by improving biological control and other methods using more effective formulations incorporating bioplastic and biochar. Bioplastic formulations for delivering biological agents will be refined to improve plant health by increasing fungal control and broadening to other plant diseases. Biochar seed coating formulations will also be developed with bioplastic that provide nutrients for plant seedlings, improve nutrient-holding capacity of soil, and prevent fungal pathogens from infecting seeds by binding substances needed for infection. Research will be expanded to Macrophomina phaseolina, a fungus which produces multiple mycotoxins (e.g., botryodiplodin, moniliformin, others) and causes significant yield losses in soybean and other row crops. Assays for routine laboratory use have been developed for M. phaseolina mycotoxins, which will be used to test soybean seeds harvested from infected plants to determine their impact on food and feed quality and safety. Knowledge of Aspergillus flavus population biology and aflatoxin risk models will be improved by exploring agroecosystems with high aflatoxin occurrence, including isolating non-aflatoxigenic A. flavus and field testing as regionally adapted biocontrol products for high stress environments. Atoxigenic fungi will also be evaluated for compatibility with seed-applied pesticides needed for improved yield with more economical deployment strategies. New formulations will be evaluated by mycotoxin analyses (ELISA, HPLC, LC-MS/MS) on plant samples with and without treatment. Mycotoxin control allows US farmers to grow non-toxic grains with improved quality, safety, and value, benefiting farmers and the public. Data obtained will enable optimization of industrial quality biological control delivery tools during the next five years, improving the efficiency and practicality of bioagent products in agriculture.
Progress Report
The project falls under National Program 303 and will be centered on reduction of harmful mycotoxins and fungal diseases in row crops such as maize, peanut and soybean. The proposed research has two research objectives and six research support objectives and is relevant to Component 2 - Biology, Ecology, and Epidemiology of Plant Pathogens; Problem Statement 2A: Advance an Understanding of Fundamental Pathogen Biology; Problem Statement 2C: Characterize Microbial Ecology and Epidemiology of Plant Diseases; Component 3– Plant Health Management; Problem Statement 3B: Advance Biologically-based and Integrated Disease Management Strategies; and Problem Statement 3C: Develop Pre-plant Approaches to Reduce Pathogen Pressure for Commercial Crop Production Systems.
This is the second year of this five-year plan. In spite of several obstacles and restriction during FY23, in fiscal year 2023 (to date) the incumbent has contributed to 4 peer-reviewed scientific publications and 2 abstracts. Research was conducted by ARS researchers in Stoneville, Mississippi, and with collaborators from other institutions in Mississippi and different states.
Objective 1 Progress: In Spring 2023, experiments for conventional corn seed treatment formulation of non-toxigenic Aspergillus flavus (Strains K49 and Afla-Guard®) formulated in starch-based bioplastic have continued to be conducted in the field. In Fall 2022, field samples including corn seeds and soil were collected, processed by drying, weighing, and grinding (seed only), and evaluated by counting the number of A. flavus colony forming units (CFU) and determining the percentage of toxigenic isolates using cultural methods (UV, pigmentation, and ammonia vapor). More than 1000 isolates of A. flavus were collected from soil, corn (soil and seed), and peanuts (soil and seed), and the isolates were sequenced for genetic characterization. In Spring 2023, field studies were done in Stoneville and Leland, Mississippi, for the effects of biochar and bioplastic on mycotoxins and the health of the plant on corn and soybean. This research is conducted with other ARS researchers in Stoneville, Mississippi, and their teams. Samples were given to an ARS researcher in the Crop Genetics Research Unit in Stoneville, Mississippi, to analyze for seed quality and composition. We also conducted studies in Stoneville using 80 germplasms of corn to screen for resistance to mycotoxins (aflatoxin and fumonisin) by artificial injection with toxigenic isolates of Aspergillus and Fusarium under field condition. This study is conducted in cooperation with ARS researcher in Starkville, Mississippi. Field studies in Dawson, Georgia, in collaboration with the ARS researchers at the National Peanut Laboratory, were conducted for managing levels of aflatoxins in peanut using seed treated in our laboratory with biocontrol strains of A. flavus (Afla-Guard®) formulated with bioplastic and biochar. Later, 120 peanut and 180 soil samples were received from Dawson, Georgia, to analyze for mycotoxins and isolate Aspergillus isolates from soil. Samples were also given to an ARS researcher in the Crop Genetics Research Unit in Stoneville, Mississippi, to analyze nutritional components. Lab evaluations, including host viability, germination, concentration, and quality control, of K49 and Afla-Guard spores were conducted to determine the efficacy of the spores. Another study is conducted annually with ARS researchers with corn and peanuts to look for resistant lines for aflatoxin and heat stress and send samples (over 500 corn and over 50 peanut) to our laboratory for chemical analysis for the presence of aflatoxins. We also collected over 1500 isolates of Aspergillus flavus from peanut, corn and soil and evaluated them for toxigenicity. These isolates were sent to other ARS researchers for DNA sequencing evaluation. All samples received in fall 2022 to present, for other people inside and outside the ARS were analyzed for mycotoxins and some were analyzed for the presence of fungi. We are collaborating with researchers from Mississippi State and University of Minnesota to conduct field studies in Starkville, Mississippi and Reform, Alabama to study the effects of biochar seed coatings of corn on Aspergillus and Fusarium growth and mycotoxin production. Laboratory studies were conducted with ARS researchers from Stoneville, Mississippi and Peoria, Illinois along with university faculty from Minnesota and Mississippi to analyze mycotoxin metabolism in crickets and yellow meal worms. Analyzed 269 isolates for aflatoxin and microbial analyses for Baozhu Guo in FY23 at ARS in Tifton, Georgia.
A locally indigenous, genetically distinct, non-aflatoxigenic isolate was used in testing commercially acceptable, low-cost seed treatments. Planting this seed resulted in a population shift of the soil and rhizosphere communities towards a less aflatoxigenic state. Follow-up work in 2023 has included alternative seed-applied pesticides and testing in South Texas, where there is more aflatoxin pressure. Objective 2 Progress: To monitor the Aspergillus flavus population in the corn-producing agroecosystem we have collected A. flavus isolates from a field near Stoneville, Mississippi, and Corpus Christi, Texas. We are collecting isolates from grain and soil as well as airborne spores and spores that are insect associated. The genetic composition of this population is being contextualized by comparing the SSR haplotypes with over 20,000 isolates in the AflaSat database as well as our own growing collection of A. flavus isolates. In 2021 the survey of A. flavus isolates from Mississippi Delta corn and soil continued. This collection has been supplemented with over 100 isolates from Guatemala and 300 from Texas and a smaller number from highly contaminated corn collected by the Federal Grain Inspection Service. This collection now stands at over 3,000 isolates in storage that have been georeferenced and scored for morphotype and aflatoxin production. Over 2,500 DNA extractions are complete and genetic characterization is underway.
Accomplishments
1. Production of mellein by Macrophomina phaseolina and the role of mellein in the pathogenicity of in charcoal rot. Mellein is one of many toxins produced by M. phaseolina, the fungus that causes charcoal rot of soybean and numerous other plant diseases. Many fungi that infect crop plants through the roots from a soil reservoir appear to use toxins to facilitate infection by causing a readily penetrated necrotic area in root tissue. Thus, ARS researchers in Stoneville, Mississippi, have evaluated root toxicity of mellein in soybean seedlings, examining treated seedlings to determine if mellein causes the symptoms of charcoal rot. Results showed that mellein can cause wilting in the soybean host but only at much higher concentrations than found in nature. These results suggest that more research is needed to evaluate other, unknown mycotoxins produced by isolates of this fungus to determine their role in root infection and in causing charcoal rot symptoms in soybean.
2. Determination of the impact of microplastic field contamination on birds. Microplastic consists of small pieces of plastic found in the environment that are produced by the natural breakdown of plastic materials (e.g., biodegradable plastic mulch) in soil. Foraging birds, such as crows, pigeons, and other wild birds, consume microplastics that are associated with crop seeds left in the field after harvest, such as in adherent soil or seed coatings that contain them. This study by ARS researchers in Stoneville, Mississippi, investigates methods for repelling birds from agricultural seeds. Coating seed corn with a slurry containing organic chemicals that can repel birds from consuming recently planted seeds should both increase stand and reduce microplastic contamination in wild bird diets. It was found in field studies that adding feathers or hairy cotton fibers to seed coatings was effective at reducing predation, presumably by triggering the so-called “feather-fear” reaction in birds. Also, birds won’t eat seed when it is covered with a black coating. These methods are relatively simple and may be useful to decrease bird predation of seed corn, increase stand and thereby increase yields for growers. It may also reduce ingestion of microplastic pollutants by wild birds, which may improve their health.
3. Evaluation of biochar seed coatings in crop production. Mycotoxins are common crop contaminants. Aflatoxin, produced by Aspergillus flavus, is the primary determinant of crop quality and a leading cause of economic loss in corn production. Biochar is reported to increase crop performance, encourage growth, increase seedling vigor, and may prevent fungi from infecting seedling roots. This study by ARS researchers in Stoneville, Mississippi, investigated the utility of biochar-containing seed coatings under laboratory and field conditions. In this study, it was demonstrated that biochar seed coatings reduced germination time, reduced bird predation, did not negatively affect yield, and did not promote A. flavus growth in field soil. These results create a better understanding of the positive effects of biochar as a seed coating component that can be used to influence future research and improve seed treatment technology. Reduced germination time and reduced bird predation should result in reduced production costs for growers.
4. Evaluation of insect feeding as a bioremediation strategy for reduction of mycotoxins in animal feed. In principle, feeding mycotoxin-contaminated grain to insects instead of discarding or burning it, may allow nutrient components to remain in the food-cycle as insect-based protein supplements in animal feed. In this study ARS researchers in Stoneville, Mississippi, reared on a corn-based diet spiked with fumonisin and evaluation of their potential as an animal feed supplement is ongoing. When completed, this research will enable an evaluation of insect feeding as a bioremediation strategy for fumonisin-contaminated grain. Application of this research may lead to healthier livestock and reduce agricultural losses for the farmers and feed lot operators.
Review Publications
Khambhati, V.H., Abbas, H.K., Sulyok, M., Tomaso-Peterson, M., Chen, J., Shier, W.T. 2023. Mellein: Production in culture by macrophomina phaseolina isolates from soybean plants exhibiting symptoms of charcoal rot and its role in pathology. Frontiers in Plant Science. 14-2023. https://doi.org/10.3389/fpls.2023.1105590.
Bellaloui, N., Mengistu, A., Smith, J.R., Abbas, H.K., Accinelli, C., Shier, W.T. 2023. Soybean seed sugars: A role in the mechanism of resistance to charcoal rot and potential use as biomarkers in selection. Plants. 12:1-14. https://doi.org/10.3390/plants12020392.
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. https://doi.org/10.3390/toxins14110755.
