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United States Department of Agriculture

Agricultural Research Service

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

Location: Biological Control of Pests Research

2013 Annual Report

1a. Objectives (from AD-416):
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.

1b. Approach (from AD-416):
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.

3. Progress Report:
The initial focus was on planning and establishing field experiments with other lab and greenhouse projects to follow as the year progressed. In spring of 2013, field experiments were planted where indicated above. Due to weather and technical problems with spore production planting was late with some treatments missed. Objective 1: HiSEQ Illumina-based sequencing was performed on 2 commercially available and 1 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 second season of the four-year study has been planted on a private farm and biocontrol strains have been applied. Soil samples were collected and Aseprgillus (A.) flavus strains were isolated as a reference for the A. flavus population prior to introduction of biocontrol strains. Objective 2: An assay was developed for the in vitro study of competition by measuring fungal growth and aflatoxin production. Objective 3: Fungal spores of both strains K49 and Afla-Guard were mass produced as a Water Dispersible Granule (WDG) formulation in 2013. Research on biological methods for controlling aflatoxin in corn is progressing with difficulties such as weather effects, lands, and lack of equipment availability in 2013. Field studies in several locations for optimizing WDG formulation for improvement of delivery of both non-toxigenic Aspergillus flavus K49 and Alfa-Guard strains is in its second season. These studies included rate of application; time of application; and mixed vs sole application of both strains of WDG formulation. Some applications were missed as indicated above. The WDG formulation of both strains was provided to ARS scientist (collaborator) to perform preliminary evaluation of droplet characteristics in 2012 and 2013 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 have been ordered and pattern tests will be conducted using these new nozzles. A filtration scheme may also be considered as long as liquid pressure drop is not too large.

4. Accomplishments
1. Aspergillus flavus whole genome sequencing. Through collaboration with the Genomics and Bioinformatics Research Unit at 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. Evaluated the role of Bt-transformed corn in mycotoxin contamination. Field experiments evaluated contemporary genetically modified (GM) corn varieties for susceptibility to mycotoxin contamination. While these GM varieties have very high resistance to several economically important insect pests of corn, there was no clear effect on the level of aflatxoxin contamination. These results were discussed in the context of other mycotoxins and other published experiments.

3. Measurement of aflatoxin response to agronomic management practices. Aflatoxin contamination of corn was monitored in three-year, multi-site experiment to assess the role of irrigation, planting density and host-plant genetics. Corn genotypes with the "flex-ear" trait had the lowest mycotoxin levels. Planting density, in contrast, had limited effect on aflatoxin or fumonsin concentration. Aflatoxin levels were highest in the driest year, regardless of the irrigation practice, suggesting that factors other than simple water deficit are important.

4. Development of non-aflatoxigenic Aspergillus (A.) flavus strains and other biological control fungi. Field evaluations assessing the efficacy of A. flavus, strain K49, and Trichoderma spp to control damping off were studies under field conditions, and assessments of two formulations for application of non-toxigenic isolates via soil application (bioplastic granules) and directed spray application (water dispersible granule formulation). These methods controlled aflatoxin by >65% and damping off >80%.

5. Documented the role of crop selection on Aspergillus (A.) flavus soil populations. Experimental evidence was collected over 3 years that supported the conclusion that crop selection has a role in shifting the soil population of A. flavus. Because there are limited control measures for aflatoxin contamination of corn, crop rotation may be an attractive strategy to safeguard this commodity. The population of A. flavus was significantly higher in soil after corn production than after soybean production. Over time, corn produced in a corn-soybean rotation may have lower risk of aflatoxin contamination than a continuous corn system.

Review Publications
Abbas, H.K., Mascagni, Jr., H.J., Bruns, H.A., Shierd, W.T., Damanne, K.E. 2012. Effect of planting density, irrigation regimes, and maize hybrids with varying ear size on yield, and aflatoxin and fumonisin contamination levels. American Journal of Plant Sciences. 3:1341-1354.

Kebede, H.A., Abbas, H.K., Fisher, D.K., Bellaloui, N. 2012. Relationship between aflatoxoin contamination and physiological responses of corn plants under drought and heat stress. Toxins. 4:1385-1403.

Koger III, C.H., Zablotowicz, R.M., Patterson, M.R., Walker, T.W., Weaver, M.A., Street, J.E. 2013. Effect of Winter Flooding on Weeds, Soybean Yield, Straw Degradation, and Soil Microbiol Activity. American Journal of Plant Sciences. 4:10-18.

Manning, B. B., Abbas, H. K. 2012. nThe effect of Fusarium mycotoxins deoxynivalenol, Fumonisin, and Moniliformin from contaminated moldy grains on aquaculture fish. Toxin Reviews. 31:1-5.

Bellaloui, N., Mengistu, A., Fisher, D.K., Zobiole, L.H., Abbas, H.K. 2012. Irrigation management: effects of soybean diseases on seed composition in genotypes differing in their disease resistance under irrigated and nonirrigated conditions. Nova Hedwigia. 1:1-42.

Willcox, M., Davis, G., Warburton, M.L., Windham, G.L., Abbas, H.K., Betran, J., Holland, J.B., Williams, W.P. 2013. Confirming quantitative trait loci for aflatoxin resistance from Mp313E in different genetic backgrounds. Molecular Breeding. 32(1):15-26.

Abbas, H.K., Bellaloui, N., Zablotowicz, R.M., Bruns, H.A., Gillen, A.M. 2012. Corn-soybean rotation systems in the Mississippi Delta, implications on mycotoxin contamination and soil populations of Aspergillus flavus. International Journal of Agronomy. 2012, Article ID 935463, 7 pages doi:10.1155/2012/935463.

Herath, B., Jacob, M., Wilson, A., Abbas, H.K., Nanayakkara, D. 2012. New secondary metabolites from bioactive extracts of the fungus Armillaria tabescens. Natural Product Research. 27(17):1562-1568.

Manning, B.B., Abbas, H.K., Wise, D.J., Greenway, T. 2013. The effect of feeding diets containing deoxynivalenol contaminated corn on channel catfish (Ictalurus punctatus) challenged with Edwardsiella ictaluri. Aquaculture Research, 1-5; doi:10.1111/are.12123.

Last Modified: 05/28/2017
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