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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

2019 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
A copious amount of non-toxigenic strains of Aspergillus (A.) flavus (strains K49 and Afla-Guard) spores were produced. Laboratory evaluations of spores, including viability, germination, concentration, and quality control, continue to be conducted to determine efficacy of the spores. Experiments for conventional corn seed treatments and sprayable formulation of non-toxigenic A. flavus formulated in starch-based material (bioplastic) continue to be conducted in the field and greenhouse. A collection of several thousand geo-referenced isolates of A. flavus has been made and is undergoing phenotypic and genotypic characterization. This collection, covering the Mississippi Delta, includes aflatoxigenic and non-aflatoxigenic isolates from soil and from grain. The complete genome has been sequenced and published for three biocontrol strains to facilitate comparisons with the field isolates. These experiments are proceeding in spite of limited availability of field equipment, personnel, and support needed to accomplish the goals of this objective. Some of the research in this project plan is being conducted in collaboration with scientists outside our unit and the ARS, nationally and internationally.

1. Impact of mycotoxins on yield of sesame produced in the Mississippi Delta. Four sesame varieties were planted by ARS scientists in Stoneville, Mississippi, in the Mississippi Delta at four nitrogen fertilizer application rates and evaluated for grain yield and contamination by mycotoxins and toxigenic fungi. There was no significant effect of nitrogen fertilizer application rate, variety, crop year or interaction between them for contamination of harvested seed by aflatoxins, fumonisins, Aspergillus flavus or Fusarium verticillioides in cleaned and uncleaned harvested sesame seed. In general, sesame seed is not susceptible to aflatoxin and fumonisin contamination. None of the mycotoxin levels observed in this study were significant in regard to human or animal health, but further testing is needed. This is the first report of fumonisin found in sesame seed. The results of this study indicate that sesame seed is a safe crop for growers and consumers. The information of this study will be useful to public and private sector entities including ARS, academia, industrial grain producers, and especially growers currently producing the sesame crop.

2. Toxin role in pathogenicity of soybean charcoal rot disease. Substantial research progress has been made in the development of analytical methodology for Macrophomina (M.) phaseolina mycotoxins that have already been identified. A liquid chromatographic/mass spectroscopy (LC/MS) method was developed by ARS scientists in Stoneville, Mississippi, for the detection of (-)-botryodiplodin, which plays a major role in the pathogenicity of M. phaseolina – the cause of charcoal rot disease. The limit of detection for (-)-botryodiplodin in fungal cultures was determined be 0.1 ppm. An analysis was performed on fungal culture extracts for the presence of (-)-botryodiplodin, and this toxin was detected in culture extracts at varying levels. The LC/MS method confirmed the validity of the in-culture color bioassay for the presence of (-)-botryodiplodin (i.e., red pigment formation in the presence of glycine). These results could help uncover more information on the mechanism which leads to breeders discovering variants that suppress toxin release during the infection process and develop management strategies of the disease by selecting for soybean variety resistance against the toxin. The results from this study will provide useful information to the public and private sector research units including ARS, academia, and industrial grain producers.

Review Publications
Portilla, M., Abbas, H.K., Accinelli, C., Luttrell, R.G. 2018. Laboratory and field investigations on compatibility of Beauveria bassiana spores with a sprayable bioplastic formulation for application in the biocontrol of tarnished plant bug in cotton. Journal of Economic Entomology. 112(2):549-557.
Bellaloui, N., Abbas, H.K., Ebelhar, W.M., Mengistu, A., Mulvaney, M.J., Accinelli, C., Shier, T.W. 2018. Effect of increased nitrogen application rates and environment on protein, oil, fatty acids, and minerals in sesame (Sesamum indicum) seed grown under Mississippi Delta conditions. Food and Nutrition Sciences. 9:1112-1135.
Damianidis, D., Ortiz, B.V., Bowen, K.L., Windham, G.L., Hoogenboom, G., Hagan, A., Knappenberger, T., Abbas, H.K., Scully, B.T., Mourtzinis, S. 2018. Minimum temperature, rainfall and agronomic management impacts on corn grain aflatoxin contamination. Agronomy Journal. 110:1697-1708.
Abbas, H.K., Ebelhar, W.M., Bellaloui, N., Mulvancy, M.J., Stoner, G., Kotowicz, J.K., Little, N., Accinelli, C., Shier, W. 2019. Contamination of sesame seed grown in Mississippi with aflatoxin, fumonisin, and mycotoxin-producing fungi. World Mycotoxin Journal. 12(2):123-132.
Accinelli, C., Abbas, H.K., Shier, W., Vicari, A., Little, N. 2019. Degradation of microplastic seed film-coating fragments in soil. Chemosphere. 226:645-650.
Okunowo, W.O., Osuntoki, A.A., Adekunle, A.A., Gbenle, G.O., Abbas, H.K., Shier, T.W. 2019. Optimization of Myrothecium roridum tode: Fries phytotoxin production and bioactivity on water hyacinth (Eichhornia crassipes). Toxin Reviews. pp.1-15.
Weaver, M.A., Mack, B.M., Gilbert, M.K. 2019. Genome sequences for 20 georeferenced Aspergillus flavus isolates. Microbiology Resource Announcements. 8(11):01718-18.
Fountain, J.C., Abbas, H.K., Ni, X., Scully, B.T., Lee, R.D., Kemerait, R.C., Guo, B. 2018. Evaluation of maize inbred lines and topcross progeny for resistance to pre-harvest aflatoxin contamination in the field. The Crop Journal. 7:118-125.