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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

2020 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
In Spring 2019, a field study was conducted to manage levels of aflatoxin in corn by applying biocontrol strains of Aspergillus (A.) flavus (Strains K49 and Afla-Guard®) formulated with bioplastic. In Fall 2019, field samples were collected and processed by counting and isolating colonies of A. flavus and determining the toxigenicity of the colonies. Meanwhile, lab evaluations, including host viability, germination, concentration, and quantity control, of K49 and Afla-Guard spores were conducted to determine the efficacy of the spores. In Spring 2020, further experiments for conventional corn seed treatment formulation of non-toxigenic A. flavus formulated in starch-based material (bioplastic) have continued to be conducted in the field and growth chamber. Additionally, experiments were conducted in Spring 2020 for biochar seed coating formulated in bioplastic with soybean and corn seeds to investigate the ability of seed-zone biochar to protecting seedlings from infection by undesirable pathogens. Field experiments were started in Stoneville, Mississippi, and Jackson, Tennessee, to study the effect of biochar on plant health, yield, and seed germination. 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. 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. Fortunately, the above mentioned experiments are currently in progress despite major obstacles, such as lack of personnel and field equipment, caused by the COVID-19 pandemic. Any progress made has been due to the collaborative efforts with scientists from inside and outside the ARS. In spite of this, significant efforts were made to continue work on the project by teleworking, conducting experiments from home, writing manuscripts for publication and progress reports for Mississippi Soybean Promotion Board and coordinating with essential employees to continue laboratory experiments.

1. Updating the impact of mycotoxins on yield of sesame produced in the Mississippi Delta. Four sesame varieties were planted 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 (A.) flavus or Fusarium verticillioides in cleaned and uncleaned harvested sesame seed. Close to 500 A. flavus isolates were analyzed using a rapid molecular approach for ability to cause low level aflatoxin contamination in the four varieties of sesame seeds. ARS researchers in Stoneville, Mississippi, studied Aspergillus flavus isolates, nearly all lacked the biosynthetic gene clusters that produce toxins (aflatoxins and CPA), and the cause is suspected to be due to prior applications of Afla-Guard® in fields near the experiment and collection sites. The results of this study indicate that sesame may be a safe crop for growers and consumers in relation to crop contamination by pathogenic fungi and mycotoxins. 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. Updating toxin roles in the pathogenicity of soybean charcoal rot disease. Substantial research progress has been made in the development of analytical methodology for known Macrophomina (M.) phaseolina mycotoxins. A liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the detection of (-)-botryodiplodin, which plays a major role in the pathogenicity of Macrophomina phaseolina – the causal agent of charcoal rot disease. The limit of detection for (-)-botryodiplodin in fungal cultures was determined be 0.1 ppm. ARS researchers in Stoneville, Mississippi, analyzed a collection of fungal culture extracts for the presence of (-)-botryodiplodin, and the toxin was detected in most culture extracts at varying levels. The LC-MS/MS method helped confirmed the validity of a previously developed in-culture colorimetric bioassay for the presence of (-)-botryodiplodin (i.e., red pigment formation in the presence of glycine. Approximately, 101 cell-free culture filtrates were prepared from cultures isolated from soybean plants with charcoal rot disease. The results of the analysis showed that 78% of these isolates produced (-)-botryodiplodin, and isolates that produced the toxin correlated well with the results of the in-culture assay, helping validate the in-culture assay method. Laboratory studies have been demonstrated that reports in the literature of pigment production by isolates of M. phaseolina are an artifact based on the same color-formation reaction underlying the in- culture assay method. The results of this experiment could help uncover more information on the pathogenicity of charcoal rot disease which could help breeders discover variants that suppress toxin release or interaction 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
Abbas, H.K., Bellaloui, N., Accinelli, C., Smith, J.R., Shier, W.T. 2019. Toxin production in soybean (Glycine max L.) plants with charcoal rot disease and by Macrophomina phaseolina, the fungus that causes the disease. Toxins. 11(11):645.
Abbas, H.K., Bellaloui, N., Butler, A.M., Nelson, J.L., Abou-Karam, M., Shier, T.W. 2020. Phytotoxic responses of soybean (Glycine max L.) to Botryodiplodin, a toxin produced by the charcoal rot disease fungus, Macrophomina phaseolina. Toxins. 12(1):25.
Accinelli, C., Abbas, H.K., Bruno, V., Nissen, L., Vicari, A., Bellaloui, N., Little, N., Shier, W.T. 2020. Persistence in soil of microplastic films from ultra-thin compostable plastic bags and implications on Aspergillus flavus population. Waste Management. 113: 312-318.
Chang, P.-K., Scharfenstein, L.L., Abbas, H.K., Bellaloui, N., Accinelli, C., Ebelhar, M.W. 2020. Prevalence of NRRL21882-like (Afla-Guard®) Aspergillus flavus on sesame seeds grown in research fields in the Mississippi Delta. Biocontrol Science and Technology. 30:1090-1099.
Bellaloui, N., McClure, A.M., Mengistu, A., Abbas, H.K. 2020. Influences of agricultural practices, environment, and cultivar differences on soybean seed protein, oil, sugars, and amino acids. Plants. 9(3),378.
Pruter, L.S., Brewer, M.J., Weaver, M.A. 2020. Overview of risk factors and strategies for management of insect-derived ear injury and Aflatoxin accumulation for Maize grown in subtropical. Journal of Integrated Pest Management. 11:1-8.