Progress 10/01/23 to 09/30/24
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. In October of 2023, recently harvested field samples of corn, soybean, and soil have continued to be collected for processing by drying, weighing, and grinding (grain only). In continuation of decades long monitoring, samples have been sporulated in agar media coated petri dishes. Evaluation of pathogenic fungi, Aspergillus flavus, has been performed by counting colony forming units (CFU) and determining the percentage of toxigenic isolates using phenotypic characterization methods (UV, pigmentation, and ammonia vapor). Phenotypic variation is being documented and explored for causes and effects and atoxigenic strains of A. flavus continue to be identified. For the third year, field studies were harvested in Stoneville and Leland, Mississippi, to determine the effects of different formulations of various biochars and bioplastic on mycotoxins and the health of the corn and soybean plants. This research was conducted with other ARS researchers at Stoneville, Mississippi, and their teams. Samples have been shared with an ARS researcher in the Crop Genetics Researcher Unit at Stoneville, Mississippi, to analyze for harvested seed quality and composition. Field studies in Dawson, Georgia, in a continued collaboration with the ARS researchers at the National Peanut Laboratory, Dawson, Georgia, were conducted for managing levels of aflatoxins in peanut using seed treated at Stoneville facilities with biocontrol strains of A. flavus (Afla- Guard® and K49), with and without biochar formulated with bioplastic. Samples from 2023,422 from peanut and 157 from soil, were received from Dawson, Georgia, to analyze for mycotoxins and isolate Aspergillus isolates. Samples were shared with an ARS researcher at 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 out-competing and suppressing populations of toxigenic Aspergillus flavus when combined with biochar and bioplastic seed treatments. New field studies at Stoneville, Mississippi, Starkville, Mississippi, and Reform, Alabama, to study the effects of biochar seed coatings of corn on Aspergillus and Fusarium growth and mycotoxin production were harvested in Fall of 2023. We are continuing to analyze results and samples from this collaboration with researchers at Unted States universities, Toxigenicity has been analyzed for 240 corn samples, while isolations of Aspergillus and Fusarium CFUs for the corn samples and an additional 480 soil samples are ongoing. Laboratory studies were conducted with ARS researchers at Stoneville, Mississippi, and Peoria, Illinois, along with university faculty from Minnesota and Mississippi analyze mycotoxin metabolism in crickets and yellow meal worms. Two new trials were begun incorporating naturally contaminated corn to explore additional factors of fungal contamination and their effects on their complete lifecycles. Continued collaboration with researchers in the United States and Nigeria, have investigated the possible role of mycotoxins in the pathological mechanism of the fungus, Coniothyrium glycines. Results from Spring of 2024 have thus far have indicated two known phototoxic mycotoxins produced by the fungus, cercosporin and elsinochrome A, may be responsible for red leaf blotch in soybeans in Africa. Both molecular tools and chemical analyses are being performed to determine the exact roles of these and possibly other mycotoxins in the pathology of the disease. A Mississippi Delta indigenous, genetically distinct, non-aflatoxigenic isolate was used in testing commercially acceptable, low-cost seed treatments for suppressing aflatoxin levels. Planting this seed resulted in a population shift of the soil and rhizosphere communities towards a less aflatoxigenic state. The second year of field trials included alternative seed-applied pesticides and continued with locations in Corpus Christ, Texas, where there is more aflatoxin pressure and Stoneville, Mississippi, where there is less. To monitor the Aspergillus flavus population we are collecting isolates from grain and soil as well as airborne spores and spores that are associated with insects. The genetic composition of this population is being compared to the genetic profiles of over 20,000 isolates in the AflaSat database as well as our own growing collection of A. flavus isolates. Field surveys of A. flavus isolates from Mississippi Delta corn and soil samples continued for the fifth year to detect year-to-year genetic shifts. This collection has been supplemented with over 300 isolates from Guatemala and 300 from Texas and a smaller number from highly contaminated corn collected by the United States Federal Grain Inspection Service. This collection now stands at over 4,000 isolates in storage that have been georeferenced and scored for morphotype and aflatoxin production. Over 3,000 DNA extractions are complete and genetic characterization is underway. Genetic analysis of A. flavus isolates from Guatemala identified several potential biocontrol genotypes, which have been characterized in lab experiments for aflatoxin suppressive activity. Three of the most competitive genotypes were selected for field trials, now underway, in Guatemala. ACCOMPLISHMENTS 01 Discovery that house crickets, as an emerging livestock feed supplement, can convert mycotoxin contaminated grain into a safer product. A team of ARS researchers at Stoneville, Mississippi, and academia evaluated the role of the house cricket to remediate mycotoxin contaminated grains, specifically, fumonisin B1 in corn. Its qualities as a beneficial insect were tested to see if they would retain their economic value as an alternative source of protein for animal feed after being fed a corn-based diet contaminated with different levels of fumonisin B1. Crickets can consume fumonisin contaminated corn and convert that into an insect meal with nearly three times the protein content of high protein corn while reducing the available fumonisin content by 96% in the insect meal. These findings resulted in a peer reviewed article published in Journal of Economic Entomology in 2024. During this research, the ARS scientists were invited to be a part of a USDA-ARS collaborative collective that is working to research and promote the use of beneficial insects in a circular economy. Two livestock feed magazines have reached out for details about the potential for crickets to improve feed quality and reduce the waste of contaminated corn. Stakeholders and lobbyists in cricket farming have reached out for our expertise and advice to improve cricket safety and increase their value. If adopted, corn stakeholders will have their contaminated corn retain a higher value while insect-livestock stakeholders will be able to safely access cheaper and previously undesirable agricultural waste streams for cricket rearing substrate. Livestock stakeholders will know that their use of cricket as feed supplements is safe. 02 Development, transfer, and release of two improved soybean germplasm lines in the United States with tolerance to mature seed damage (MSD). Soybean is a global crop and a major source of plant protein for both human and animal nutrition. Mature seed diseases and abiotic factors, such as high heat and wet weather, promote mature seed damage caused by mold, insects, heat, sprouting seed, and mycotoxins. Profits are lost to growers when damaged grain is sold at grain elevators and again by domestic buyers who cannot sell damaged grain to international markets. ARS researchers at Stoneville, Mississippi, developed two improved soybean germplasms (DS1260-2, and DS49-142) from two exotic, damage- resistant sources (PI 587982A and PI 603756, respectively) and made them available to soybean scientists for research and breeding. These lines, which have less damage from mold, stinkbugs, heat, and mycotoxins, were publicly released by ARS on 5 September 2023 and 16 March 2024, respectively. The improved lines were transferred through four material transfer agreements to multiple research partners. University partners have developed multiple new breeding lines from DS49-142 and DS1260-2. These universities will release high-yielding cultivars with improved tolerance to mature seed damage that is derived from DS49-142 and DS1260-2. The improved cultivars will be utilized by soybean producers to increase United States producer and seller profits by decreasing the amount of damaged grain produced and sold.
Impacts
(N/A)
Publications
- Paulk, R.T., Abbas, H.K., Rojas, M.G., Morales Ramos, J.A., Busman, M., Little, N., Shier, T.W. 2024. Evaluating Acheta domesticus (Orthoptera: Gryllidae) for the reduction of fumonisin B1 levels in livestock feed. Journal of Economic Entomology. https://doi.org/10.1093/jee/toae025.
- Garg, V., Dudchenko, O., Wang, J., Khan, A.W., Gupta, S., Kaur, P., Han, K. , Saxena, R.K., Kale, S.M., Pham, M., Yu, J., Chitikineni, A., Zhang, Z., Fan, G., Lui, C., Valluri, V., Meng, F., Bhandari, A., Liu, X., Yang, T., Chen, H., Valliyodan, B., Roorkiwal, M., Shi, C., Yang, H., Durand, N.C., Pandey, M.K., Li, G., Barmukh, R., Wang, X., Chen, X., Lam, H., Jiang, H., Zong, X., Liang, X., Liu, X., Liao, B., Guo, B., Jackson, S., Nguyen, H.T., Zhuang, W., Wan, S., Wang, X., Aiden, E.L., Bennetzen, J.L., Varshney, R. K. 2021. Chromosome-length genome assemblies of six legume species provide insights into genome organization, evolution, and agronomic traits for crop improvement. Journal of Advanced Research. 42:315-329. https://doi. org/10.1016/j.jare.2021.10.009.
- Weaver, M.A., Bowen, C., Bastidas, A., Park, L.C., Drewry, S.G., Mandel, J. R. 2023. Genetic Diversity of Aspergillus flavus on Maize in Guatemala. Foods. https://doi.org/10.3390/foods12203864.
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Progress 10/01/22 to 09/30/23
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. 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 01 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. 02 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. 03 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. 04 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.
Impacts
(N/A)
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.
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Progress 10/01/21 to 09/30/22
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. 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 first year of this five-year plan. In fiscal year 2022 (to date) has contributed to 3 peer-reviewed scientific publications. Research was conducted by ARS researcher in Stoneville, Mississippi, with collaborators from other institutions. Objective 1 Progress: In Spring 2022, 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 2021, 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 2022, 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 Researcher Unit in Stoneville, Mississippi, to analyze for seed quality and composition. We also conducted studies in Stoneville with using 80 germplasms of corn to screen them 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, (156) peanut and (180) soil samples were received back from Dawson, Georgia to analyze for mycotoxins and isolate Aspergillus isolates from soil. Samples were also given to ARS researcher in the Crop Genetics Researcher 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 in Tifton, Georgia, with corn and peanuts to look for resistant lines for aflatoxin and heat stress and sends samples (848 corn and 40 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 2021 to present, for other people inside and outside the ARS were analyzed for mycotoxins and some were analyzed for the presence of fungi. 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 2022 has included alternative seed-applied pesticides to evaluate the compatibility with other products in case neonicotinoidinsecticides are not available. 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 01 Development and release of improved MG IV Soybean Germplasm Line with tolerance to mature seed damage and free of mycotoxins. A new soybean germplasm (DS31-243) released in 2022 by ARS researchers in Stoneville, Mississippi, to have less mature seed damage (mold, insect damage, wrinkled green seed) was tested by the incumbent for its ability to resist toxin contamination produced by Fusarium species (spp.). Fusarium spp. are well known to produce toxins such as the group of toxins called trichothecenes, including T-2 and nivalenol, in soybean and other crops. ARS researchers showed that seed from DS31-243 produced less of these toxins than current commercial cultivars, such as Progeny 4211. DS31-243 had less seed damage, higher seed germination, and competitive yield and could save growers financial losses from the toxins and damage that develop prior to harvest. Seed was provided to Southern soybean breeders through MTAs to develop improved cultivars and will be deposited in the USDA Soybean Germplasm Collection for use by scientists around the world. 02 In culture and instrumental (liquid chromatography tandem mass spectrometry; LC-MS/MS) methods developed that measure (-)- Botryodiplodin Produced by Macrophomina phaseolina. An ARS researcher in Stoneville, Mississippi, established and lead a multi-institutional team studying the mycotoxins produced by M. phaseolina, the cause of charcoal rot in soybean and many other economically important crops. Charcoal rot is the major cause of economic losses in the Mid-South and an increasing problem world-wide due to climate change. The team developed an in-culture assay that detects production of the mycotoxin (-)-botryodiplodin by reaction with glycine in the medium to form a red pigment. This simple method does not require specialized technical expertise or equipment and can be used by laboratories with limited resources. Results with this method were confirmed by LC-MS/MS. (-)- Botryodiplodin is believed to play an important role in the root infection process of the fungus.The in-culture assay is being used by the team to study the mechanisms by which M. phaseolina in the soil reservoir finds soybean roots, enters them and spreads to adjacent plants. These results of these studies will be useful for growers, industry and scientists working in this area of research. 03 Bioplastic as a binder, a sprayable carrier and a seed coating for the delivery of agricultural chemicals and biocontrol agents. Bioplastic made from cornstarch has properties that give it many advantages over previous delivery systems for agricultural agents, including both biocontrol agents and conventional agrochemicals, such as fungicides, insecticides, and nematicides, either individually or together. Studies by a multi-institutional team established by an ARS researcher in Stoneville, Mississippi, have shown that bioplastic has a variety of valuable properties that enable it to be used as granules, sprayable liquid suspensions and seed coatings. It is sufficiently hydrophilic to absorb water to form a hydrogel, particles of which deform sufficiently to pass through a sprayer head. It is sufficiently lipophilic so that particles in sprayable formulations bind agrochemicals and biocontrol microorganisms as well as stick to the cuticle on leaf surfaces after spraying. It provides nutrition for the survival and proliferation of biocontrol agents after application to leaf surfaces. It is inert, stable and compatible with most agrochemicals and biocontrol microorganisms. Suspensions of bioplastic particles dry to form hard, durable coatings on seeds that can incorporate agrochemicals and biocontrol microorganisms in a form the resists dust-off of incorporated agents. It is completely biodegradable and environmentally friendly. This research has had major impacts in agricultural biotechnology nationally and internationally.
Impacts
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Publications
- Alam, S., Abbas, H.K., Sulyok, M., Khambhati, V.H., Okunowo, W.O., Shier, T.W. 2022. Pigment produced by glycine-stimulated macrophomina phaseolina is a (-)-botryodiplodin reaction product and the basis for an in-culture assay for (-)-botryodiplodin production. Pathogens. 11(3):280. https://doi. org/10.3390/pathogens11030280.
- Bellaloui, N., Mengistu, A., Smith, J.R., Abbas, H.K., Accinelli, C., Shier, T.W. 2021. Effects of charcoal rot on soybean seed composition in soybean genotypes that differ in charcoal rot resistance under irrigated and non-irrigated conditions. Plants. 10(9):1801. https://doi.org/10.3390/ plants10091801.
- Accinell, C., Abbas, H.K., Bruno, V., Khambhati, V.H., Little, N., Bellaloui, N., Shier, T.W. 2022. Field studies on the deterioration of microplastic films from ultra-thin 1 compostable bags in soil. Journal of Environmental Management. 305:114407. https://doi.org/10.1016/j.jenvman. 2021.114407.
- Weaver, M.A., Mizra, N., Boyette, C.D., Brown, S.P., Mandel, J.R. 2022. Whole genome sequence and draft assembly of the biocontrol fungal pathogen Albifimbria verrucaria CABI-IMI 368023. Microbiology Resource Announcements. 11:1. https://doi.org/10.1128/MRA.00909-21.
- Weaver, M.A., Hoagland, R.E., Boyette, C.D., Brown, S.9. 2021. Taxonomic evaluation of a bioherbicidal isolate albifimbria verrucaria, formerly myrothecium verrucaria. The Journal of Fungi. 7(9):694. https://doi.org/10. 3390/jof7090694.
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