2012 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.
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 2012, field experiments were planted successfully where indicated above.
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. Additional sequencing has been performed on 150 Mississippi-native strains to evaluate indigenous diversity. The first season of the four-year study has been planted 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 Aspergillus (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 2012. Research on biological methods for controlling aflatoxin in corn is progressing with minimal difficulties such as weather effects, lands, and lack of equipment availability in 2012. 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 first season. These studies included rate of application; time of application; and mixed vs sole application of both strains of WDG formulation. The WDG formulation of both strains was provided to collaborator to perform preliminary evaluation of droplet characteristics in 2012 in order to determine useful data that could be used for adjusting droplet size in field aerial application of WDG by plane.
Some of the research on this project is being done in cooperation with scientists at other institutions. Research in objective 3 is being conducted by Syngenta under a specific cooperative agreement and a CRADA between ARS and Syngenta. Separate reports summarize work completed under these agreements (Subordinates 6402-42000-005-02T and 6402-42000-005-04T, respectively). Research in objective 3 is being conducted by Mississippi State University scientists and ARS scientists under a Specific Cooperative Agreement with Mississippi Corn Promotion Board (MCPB). A separate report summarizes work completed under this agreement (Subordinates 6402-42000-005-05T and 6402-42000-005-03T).
Development of non-aflatoxigenic Aspergillus flavus strains. Field evaluations assessing the efficacy of Aspergillus flavus, strain K49, have been focused on characterization of competitive displacement of toxigenic isolates under field conditions, and assessments of two novel formulations for application of non-toxigenic isolates via soil application (bioplastic granules) and directed spray application (water dispersible granule formulation). Limited incidence of aflatoxin contamination in research plots the previous year provided limited data on efficacy of control, although information on colonization potential was developed. A U.S. Patent was granted on 05/08/2012: “Granular bioplastic biocontrol composition”, Docket no. 0013.09, and U.S. Serial No. 8,173,179.
Aspergillus flavus whole genome sequencing. Through collaboration with ARS scientist in the Genomics and Bioinformatics Research Unit in 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 are being annotated and compared to a toxigenic strain to reveal Single Nucleotide Polymorphisms that may be useful for strain-specific identification.
Abbas, H.K., Accinelli, C. 2011. New perspectives for the application of bioplastic materials in the biocontrol of Aspergillus flavus in corn. Toxin Reviews. 30(2-3):71-78.
Trucksess, M.W., Abbas, H.K., Weaver, C.M., and Shier, W.T. 2011. Distribution of aflatoxins in shelling and milling fractions of naturally contaminated rice. Journal of Food Additives & Contaminants. 28:1076-1082.
Manning, B.B., Abbas, H.K., Wise, D.J., Peterson, B.C. 2011. Channel catfish, Ictalurus punctatus, fed diets containing aflatoxin from moldy corn do not experience increased mortality after challenge with Edwardsiella ictaluri. Journal of the World Aquaculture Society. 42(4):598-602.
Jiang, T., Zhou, B., Luo, M., Abbas, H.K., Kemerait, R., Lee, R.D., Scully, B.T., Guo, B. 2011. Expression analysis of stress-related genes in kernels of different maize (Zea mays L.) inbred lines with different resistance to aflatoxin contamination. Toxins. 3(6):538-550.
Bruns, H.A., Ebelhar, M.W., Abbas, H.K. 2012. Comparing single-row and twin-row corn production in the mid south. Crop Management. 1:1-48, doi:10.1094/CM-20120404-01-RS.
Weaver, M.A., Zablotowicz, R.M., Krutz, L.J., Locke, M.A., Bryson, C.T. 2012. Microbial and vegetative changes associated with development of a constructed wetland. Ecological Indicators. 13:37-45.
Chang, P., Abbas, H.K., Weaver, M.A., Ehrlich, K., Scharfenstein, L.L., Cotty, P.J. 2012. Identification of genetic defects in the atoxigenic biocontrol strain Aspergillus flavus K49 reveals the presence of a competitive recombinant group in field populations. International Journal of Food Microbiology. 154:192-196.