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ARS Home » Southeast Area » Stoneville, Mississippi » Biological Control of Pests Research » Research » Research Project #423007

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

Location: Biological Control of Pests Research

2015 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: Determine the environmental fate of non-toxigenic strains of Aspergillus (A.) flavus using molecular tools. Sub-Objective 1a. Compare DNA sequence information from current biocontrol strains with indigenous strains to identify unique molecular markers. Sub-Objective 1b. Monitor the post-release spatial and temporal distribution of A. flavus biocontrol strains in corn fields. Objective 2: Determine the mechanisms of biocontrol efficacy with non-toxigenic Aspergillus flavus strains. Objective 3: Optimize a water dispersible formulation and application procedure for the use of non-toxigenic strains of Aspergillus flavus for biocontrol of mycotoxins in corn. Sub-Objective 3a. Develop and evaluate water dispersible granular (WDG) formulation of non-toxigenic Aspergillus flavus strains. Sub-Objective 3b. Evaluate the Accu-Flo™ spray nozzle for aerial application of biological materials. Sub-Objective 3c. Determine the effect of timing of the application of K49 and Afla-Guard® as a WDG formulation. Sub-Objective 3d. Compare the effect of a combined inoculum of K49 and Afla-Guard® to these inoculants used separately.

Maize (corn) production in the United States is valued at $65 billion annually. Infection of corn by some strains of Aspergillus (A.) flavus, and subsequent contamination with the mycotoxin aflatoxin, results in costs of $923 million (UN Food and Agriculture Organization) and illnesses, including cancer or death in livestock and humans. Fungicides, altered agronomic practices and breeding efforts, including the use of transgenic Bt-corn have all been insufficient in mitigating aflatoxin contamination. Presently, the most effective approach to reduce aflatoxin contamination in corn is biological control, using non-aflatoxin-producing strains of A. flavus, as developed by USDA-ARS researchers. This technology is now commercially available as Afla-Guard®. Substantial progress has been made in the implementation of this product, but important research questions remain, which are addressed in this proposal: First, the post-release environmental fate of non-toxigenic strains of A. flavus must be evaluated. This is an essential environmental stewardship issue and may yield insight into A. flavus ecology and the plant disease cycle. Another objective includes experiments to evaluate mechanisms of biological control, including a model to explain why biocontrol strains are more effective at reducing aflatoxin contamination than predicted by simple competition. Finally, improvements in formulation and application methods of A. flavus biocontrol strains are needed for better, more consistent aflatoxin control. The commercialization of Afla-Guard® was important in the effort to exclude aflatoxin from food and feed. The basic and applied research in this proposal is essential to complete the implementation of the biocontrol strategy for reducing aflatoxin contamination of corn.

Progress Report
This project is in its fourth year with two SY personnel. This project is proceeding as anticipated. The initial focus was on implementing field experiments and beginning other laboratory and greenhouse projects later in the year. In spring of 2015, field plots were planted. Fields were planted late in the season due to weather conditions. Objective 1: HiSEQ Illumina-based sequencing was performed on two commercially available and one experimental biocontrol strain and is being compared to a native toxin-producing strain. Subsequently, the sequence data was mapped to a published sequence and a de novo assembly has been performed on each strain. Additional sequencing has been performed on 150 Mississippi-native strains to evaluate indigenous diversity. The third season of the four-year study has been implemented on a private farm and biocontrol strains have been applied. Soil samples were collected and A. flavus strains were isolated as a reference for the A. flavus population prior to introduction of biocontrol strains. A PCR-independent method for amplification of A. flavus-specifc DNA has been developed. Objective 2: An assay was developed for the in vitro study of competition by measuring fungal growth and aflatoxin production. This competition assay is being applied to whole-ear assays in the field in the current growing season. Objective 3: Fungal spores of both strains (i.e., K49 and Afla-Guard) were mass produced as a corn starch (CS) formulation (liquid and granule) in 2015. Research on biological methods for controlling aflatoxin in corn is progressing, however difficulties such as weather impacts, lack of appropriate equipment and personnel availability in 2015 has hampered the effort. Field studies in several locations for optimizing CS formulation for improved delivery of both non-toxigenic Aspergillus flavus K49 and Alfa-Guard strains is in its first season. These studies included preliminary studies on rate of application, time of application, and mixed vs sole application of both strains of CS formulation. Some treatments were eliminated due to excessively wet weather and broken machinery. Another product called the Water Dispersible Granule (WDG) formulation of both strains was provided to the collaborator in objective 3 to perform preliminary evaluation of droplet characteristics in 2012 to present in order to determine useful data that could be used for adjusting droplet size in field aerial application of WDG by plane. Accu-Flo® nozzles obtained for correct droplet size range had needles too small to pass the formulation. Alternative nozzles with larger openings were procured and experiments were conducted for this nozzle using water and adjuvant, which provided baseline information on canopy penetration. Some of the research is being accomplished in cooperation with scientists at other institutions. For example, research in objective 3 is being conducted for Syngenta under a specific cooperative agreement between ARS and Syngenta.

1. Aspergillus flavus whole genome sequencing. Through collaboration with the Genomics and Bioinformatics Research Unit, Stoneville, MS, the entire genome of Aspergillus flavus strain K49, AF36 and Afla-Guard was sequenced by Illumina HiSeq sequencer, resulting in over 75 million reads, which have been assembled into about 40 million base pairs of high quality, highly overlapping sequence. These assemblies have been annotated and compared to a toxigenic strain to reveal Single Nucleotide Polymorphisms that may be useful for strain-specific identification.

2. Evaluation of starch-corn Bioplastic non-aflatoxigenic A. flavus formulation. A new formulation for applying non-toxigenic strains of A. flavis was evaluated under field conditions in Europe and the USA in an effort to control aflatoxin production in pre-harvest corn. Assessment of two formulations (bioplastic granules and grain formulation) were accomplished. These two solid formulations controlled aflatoxin by 67.2% to 95% reduction in Europe while aflatoxin contamination was reduced up to 90% in USA.

3. Evaluation of ustiloxin toxins in false smut galls in rice in U.S. In China, Japan and other Asian countries false smut galls from rice produced mycotoxin Ustiloxins. False smut from rice grown in Arkansas was tested for toxins and none were identified. Evidently rice with false smut in the Arkansas is safe for human and animal consumption.

4. Genetic variability of Aspergillus flavus isolates from a Mississippi corn field. Isolates of Aspergillus flavus from a single field were found to be genetically variable and were of two predominant types toxigenic and non-toxigenic. Toxigenic isolates were predominate (60%) in the population.

5. The effect of Bt traits of corn on mycotoxin contamination. Bt corn has been shown to reduce insect feeding on corn. This was hoped to also reduce mycotoxins because insect damage increases mycotoxin levels. This study was essentially negative in that no consistent relationship was found between Bt traits and reduction in Mycotoxins.

Review Publications
Abbas, H.K., Shier, T.W., Cartwright, R.D., Sciumbato, G.L. 2014. Ustilaginoidea virens infection of rice in Arkansas: Toxicity of false smut galls, their extracts and the Ustiloxin fraction. American Journal of Plant Sciences. 5: 3166-3176.
Weaver, M.A., Abbas, H.K., Falconer, L.L., Allen, T.W., Pringle III, H.C., Sciumbato, G.L. 2014. Biological control of aflatoxin is effective and economical in Mississippi field trials. Crop Protection Journal. 69:52-55.
Abbas, H.K., Rex, P. 2015. Presence of adhesive vesicles in the mycoherbicide Alternaria helianthi. Journal of Plant Studies. 4(2): 21-29.
Chang, P.-K., Scharfenstein, L.L., Solorzano, C.D., Abbas, H.K., Hua, S.-S. T., Jones, W.A., Zablotowicz, R.M. 2015. High sequence variations in the region containing genes encoding a cellular morphogenesis protein and the repressor of sexual development help to reveal origins of Aspergillus oryzae. International Journal of Food Microbiology. 200:66-71.
Solorzano Torres, C.D., Abbas, H.K., Zablotowicz, R.M., Chang, P., Jones, W.A. 2014. Genetic variability of Aspergillus flavus isolates from a Mississippi corn field. The Scientific World. 2014: 8 pages.
Boyette, C.D., Abbas, H.K., Johnson, B., Hoagland, R.E., Weaver, M.A. 2014. Biological Control of the weed Sesbania exaltata using a microsclerotia formulation of the bioherbicide Colletotrichum truncatum. Natural Science. 5(18):2672-2685.
Thomson, S.J. 2014. Evaluation of a solid stream radial nozzle on fixed-wing aircraft for penetration of spray within a soybean canopy. Journal of Plant Protection Research. 54(1):96-101.
Weaver, M.A., Hoagland, R.E., Boyette, C.D. 2015. Kudzu response to foliar applied herbicides. American Journal of Plant Sciences. 6(7):856-863.
Accinelli, C., Abbas, H.K., Vicari, A., Shier, W.T. 2015. Evaluation of recycled bioplastic pellets and a sprayable formulation for application of an Aspergillus flavus biocontrol strain. Crop Protection. 72: 9-15.
Bianchini, A., Horsley, R., Jack, M., Kobielush, B., Rue, D., Tittlemier, S., Wilson, W.W., Abbas, H.K., Abel, S., Harrison, G., Miller, J.D., Shier, W.T., Weaver, G. 2015. DON occurrence in cereal grains: A North American perspective. Cereal Foods World. 60: 32-56.