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ARS Home » Southeast Area » Stoneville, Mississippi » Biological Control of Pests Research » Research » Research Project #432515

Research Project: Biocontrol of Aflatoxin and Other Mycotoxins in Maize Using Non-toxigenic Strains of Aspergillus flavus

Location: Biological Control of Pests Research

2022 Annual Report

The overall objective of this project is the improved biological control of aflatoxin in corn through a more complete ecological understanding of the pathogen and the agroecosystem through applied investigation of biocontrol agent delivery systems. Over the next 5 years, our research will focus on the following objectives: Objective 1: Enhance shelf life, survival growth, germination, and host colonization of biocontrol agents for aflatoxin management through formulation improvements. Objective 2: Refine aerial, foliar and seed treatment application strategies of biocontrol agents for aflatoxin management. Objective 3: Develop and implement molecular markers for post-release tracking of foliar and seed treatment biocontrol applications. Objective 4: Determine population size, chemotype and mating type frequency in soil-borne Aspergillus (A.) flavus populations and subsequent infections in corn to improve predictions of aflatoxin risk and enhance biocontrol measures. Sub-Objective 4a.Correlation of aflatoxin contamination of corn with the population distribution of A. flavus in soil. Sub-Objective 4b. Correlating A. flavus mating type with corn infection. Objective 5: Apply novel formulation and application technology to other pathogen biocontrol systems.

Biological control technology is an effective method for reducing aflatoxin contamination in corn; however, present formulation and application strategies are still rudimentary and fundamental gaps remain in our knowledge of the population structure of Aspergillus (A.) flavus. Improved application technology will be developed aimed at increasing adoption of biocontrol measures by increasing efficacy and convenience of the application systems. Although previous efforts using a water dispersible formulation did not meet industry standards, they provided avenues for further research using bioplastic as a vector for application of biocontrol agents. Corn starch-based bioplastics are naturally-derived, biodegradable, recyclable, inexpensive, and provide nutrition favoring fungal growth after application. Bioplastics are easily prepared by heating commercial bioplastic for 2-3 hours at 80-90°C and applying (i.e., aerial, foliar, and seed treatment) after cooling. Bioplastic vectors promise to provide effective, efficient delivery of biocontrol agents. We also plan to develop and implement molecular markers for post-release monitoring of biological control agents. The resulting data will enable optimizing delivery tools to meet industry standards. Knowledge of local A. flavus population size, chemotype and mating type frequency will also lead to a better understanding of aflatoxin risk and permit more sound decisions of when fields warrant application of commercial biocontrol products. Domestic and international industrial companies have already inquired and invested in the development of this technology for control of various agricultural pests beyond just A. flavus. Optimization of these techniques during the next five years will improve the efficiency and practicality of biocontrol agents used in agriculture.

Progress Report
This project focused on reduction of aflatoxin and other mycotoxins in maize relevant plant health management and biologically-based and integrated disease management. This is the final year of this five-year plan and the replacement project is pending completion of reserach review. In fiscal year 2016 to 2021, this project had contributed to over 15 peer-reviewed scientific publications, had several accomplishments, several agreements, and technology transfers. All the milestones were Fully Met. Research was conducted by ARS scientists with collaborators from other institutions. Our research project was negatively impacted by the maximized teleworking requirement. Some experiments, including fungal isolation from soil and corn samples, soybean toxin tolerance bioassays, and chemical analysis of field samples for toxins, were severely delayed due to the restrictions imposed by the maximized teleworking requirement. Scheduled local, national, and international travel was restricted. So, attendance to major conferences and visiting sites of collaborative studies were impossible. The process for recruiting permanent and seasonal employees was extremely impaired due to the restrictions. Outreach activities, such mentoring, shadowing, etc., were restricted. Despite these restrictions, significant efforts were made to analyze experiments from home, coordinate with essential employees to continue laboratory experiments, write manuscripts for publication, provide progress reports to grant institutions and collaborators, plan field studies, and prepare seed treatments for the 2021 growing season. Any progress made has been due to the collaborative efforts with scientists from inside and outside the ARS. In Spring 2020, experiments for conventional corn seed treatment formulation of non-toxigenic Aspergillus (A.) flavus (Strains K49 and Afla-Guard®) formulated in starch-based bioplastic have continued to be conducted in the field and growth chamber. In Fall 2020, 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). Approximately 500 isolates of A. flavus were collected from soil, corn (silk, leaves, tassels, and seed), and rice (husk, bran, brown rice, white rice, and unfinished rice); and, the isolates were sequenced for genetic characterization. In Spring 2021, field studies were repeated in Stoneville, Mississippi, for managing levels of aflatoxin in corn by applying biocontrol strains of Aspergillus (A.) flavus (Strains K49 and Afla-Guard®) formulated with bioplastic as seed treatment. Field studies in Dawson, Georgia, were initialized for managing levels of aflatoxins in peanut using seed treated with biocontrol strains of A. flavus (Afla-Guard®) formulated with bioplastic. Meanwhile, 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. A series of bioplastic formulations have been prepared from pregelatinized cornstarch with known, defined (i.e., not proprietary) compositions. The formulations are currently undergoing chemical composition analysis prior to testing for effectiveness as soybean seed coating vehicles for A. flavus biocontrol strains and biochar. 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 1,000 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. 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. Additionally, a prototype seed coating formulated with biochar and bioplastic was developed for initial testing. Corn and soybean seeds were coated with the new formulation, and field trials in three locations (two in Washington County near Stoneville, and one in Jackson, Tennessee) were initiated in the 2021 growing season. Seed germination, stand count, and plant health of treated seeds were evaluated under laboratory, nursery, and field conditions. 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. DNA extractions are underway for genetic characterization. During the pandemic we made progress on DNA analysis with just one worker in the lab at a time by completing extractions and identifying low-quality samples for re-extraction.

1. Updating toxin roles in the pathogenicity of soybean charcoal rot disease. The fungus Macrophomina phaseolina causes charcoal rot disease in soybean and causes various other diseases in more than 500 other commercially important plants, particularly during hot, dry growing conditions. In many years, charcoal rot is a major cause of yield losses in Arkansas, Louisiana, and Mississippi. ARS researchers in Stoneville, Mississippi, conducted a study to measure the production in culture of known mycotoxins and other secondary metabolites by 89 isolates of M. phaseolina from soybean plants symptomatic for charcoal rot. Six mycotoxins and other metabolites were observed at relatively high frequencies (19.1 to 84.3% of cultures), including the previously reported mycotoxins, botryodiplodin and mellein, as well as four previously unreported substances, namely kojic acid, moniliformin, orsellinic acid, and cyclo[L-proline-L-tyrosine]. In addition, the study identified an additional nine previously unreported metabolites that were observed at relatively low frequency (<5% of cultures), including cordycepin, emodin, endocrocin, citrinin, gliocladic acid, infectopyron, methylorsellinic acid, monocerin, and N-benzoyl-L-phenylalanine. Further studies are needed to investigate possible effects of these mycotoxins and metabolites on pathogenesis by M. phaseolina and on food and feed safety, if any of them are found to contaminate the seeds of infected soybean plants at toxic levels.

2. Reducing abrasive dust by strengthening seed coating treatments using bioplastic. Seed coating treatments are commonly applied directly to seeds as part of a film-coating formulation that incorporate chemical pesticides can help seed germination or prevent early infection of the seedling by fungi and other diseases. However, many coatings lack durability and can be easily damaged during the transportation, storage, and planting processes, resulting in decreased effectiveness and environmental damage to other species such as bees and other pollinators. Strengthening the integrity of the seed coatings prevents or reduces these problems. Several new coating agent formulations were evaluated by ARS researchers in Stoneville, Mississippi, for resistance to abrasive removal before seed germination. The seed coating process was improved by first removing the outer wax layer of corn seed and then applying the protective coating. This modification reduced coating fragment loss from seeds by over 95%. Ultimately, these findings should help to protect the environment, improve plant growth, and provide better products for farmers and industry professionals.