Location: Crop Bioprotection Research2014 Annual Report
1a. Objectives (from AD-416):
Objective 1: Optimize fermentation and formulation protocols for selected biocontrol strains to produce products with enhanced field efficacy, consistency, and compatibility with integrated pest management methodologies. Objective 2: Elucidate selected interactions among host, biocontrol agent, and pathogen on plant surfaces using physiochemical, proteomic, and/or genomic characterization of the host or agent to guide development of effective agent production and formulation technologies. Objective 3: Develop methods and technologies to isolate and identify fastidious microorganisms that threaten U.S. agriculture and determine characteristics that contribute to pathogenicity. The encompassing goal of this project is to improve biocontrol agent product performance by both optimizing agent fermentation, formulation, and deployment protocols; and by elucidating agent, pathogen, and host interactions on plant surfaces in order to optimize antagonist colonization of host surfaces, efficacy in reducing disease and utility in integrated pest management environments. Results from research conducted in each of these key areas will be of value in meeting each individual objective as well as providing data that reciprocally provides leads and concepts that enhance the advancement of the science conducted under the other individual objective. Our Agricultural Research Service (ARS) research team possesses a unique blend of expertise and interactive research experience in plant pathology, biochemistry, and molecular biology to draw on in completing this research. Additionally, collaborators within ARS, at Universities, and within the agricultural business community have committed to providing critical skill-sets that will be instrumental in meeting our overall research goal.
1b. Approach (from AD-416):
Objective 1: Optimize fermentation and formulation protocols for selected biocontrol strains to produce products with enhanced field efficacy, consistency, and compatibility with integrated pest management methodologies. Objective 2: Elucidate selected interactions among host, biocontrol agent, and pathogen on plant surfaces using physiochemical, proteomic, and/or genomic characterization of the host or agent to guide development of effective agent production and formulation technologies.
3. Progress Report:
Significant progress in support of both objectives of this research project was made in FY14 by ARS scientists in the Crop Bioprotection Research Unit (CBP) at the USDA-ARS, National Center for Agricultural Utilization Research (NCAUR), Peoria, Illinois, in collaboration with universities and industry. In studies conducted under Objective 1, important steps towards maintaining the efficacy and viability of Gram negative bacterial biocontrol agents during drying and rehydration were made in investigations conducted under a Specific Cooperative Agreement (SCA) with researchers at the University of Idaho. ARS identified multiple carriers useful in preserving cell viability during drying and biocontrol efficacy in reducing dry rot on stored potato tubers. We then investigated combining the best osmoprotectants and carriers identified to produce dried Gram negative bacteria products. We discovered that strains differed in which osmoprotectant/carrier combination was superior in enhancing survival during drying and efficacy against post harvest dry rot of potato tubers in laboratory bioassays. Small pilot scale studies of the best single strain biocontrol products developed are ongoing. Considerable progress also was made in support of Objective 2 in FY14. ARS scientists in the Crop Bioprotection Research Unit (CBP) unit at USDA-ARS, National Center for Agricultural Utilization Research (NCAUR) in Peoria, Illinois, completed experiments and data acquisition for studies that determined what changes in gene regulation are associated with culturing fusarium head blight (FHB) antagonist C. flavescens under cold adaptation conditions that enhance the stress tolerance and efficacy of cells. Bacterial FHB biocontrol agent Bacillus subtilis OH 131.1 was cultivated under iron starvation conditions and evaluated for metabolite production including iron-binding siderophores. In other studies, ARS characterized metabolite production by B. subtilis OH 131.1 when co-cultured with a yeast antagonist to determine if co-culturing has the potential to be managed to improve the quantity and quality of production of beneficial metabolites by a biocontrol agent. FY14 research progress was also evident in ARS scientists extending an existing Cooperative Research and Development Agreement (CRADA), extending a Specific Cooperative Agreement (SCA), maintaining a Non-Funded Cooperative Agreement (NFCA) for a second year, and receiving two patent issuances. These results will enhance the development of biological control products active against plant disease, as well as basic knowledge on how to produce even more effective biocontrol products by understanding gene expression and interactions of antagonists with the host and pathogen.
1. Sequencing and annotation of the genome of biocontrol agent Bacillus subtilis OH 131.1. Though strain B. subtilis OH 131.1 has demonstrated biocontrol activity against Fusarium Head Blight (FHB) of wheat, the genomic potential of the strain to produce bioactive metabolites was not known. ARS scientists in the Crop Bioprotection Research Unit at the USDA-ARS, National Center for Agricultural Utilization Research (NCAUR), Peoria, Illinois, discovered that the genome resolved to a single chromosome and then functionally annotated the genome to describe the genes identified. After the genome was analyzed to determine the secondary metabolite synthetic clusters present, ARS scientists demonstrated that the strain produced several of the predicted bioactive metabolites. Additionally, they used comparative genomics to identify genes unique to the strain, accurately determined the phylogeny of the strain for the first time, and identified a core genome possessed by closely related biocontrol strains. The completed genome was made publically available by accession into a national repository. This work provides insight into the modes of action the strain may utilize in its biological activity and to identify related strains that may also benefit wheat producers and consumers by reducing the impact of FHB disease.
2. Characterization of surface properties of wheat heads and a biocontrol agent. Surface properties play an important role in plant-microbe interactions including determining if microbial agents adhere to plant surfaces, yet the surface properties of wheat heads and biocontrol agents had not been determined. To better understand how surface properties of wheat may affect Fusarium Head Blight (FHB) disease development and spray application effectiveness, ARS scientists in the Crop Bioprotection Research Unit at the USDA-ARS, National Center for Agricultural Utilization Research (NCAUR), in Peoria, Illinois, characterized the surface properties of wheat (Triticum aestivum L.) spikelet tissues under different environmental growing regimes as were the surface properties of FHB biocontrol yeast Cryptococcus flavescens OH 182.9. When two wheat cultivars were grown in greenhouse versus field environments, changes occurred in surface energy parameters and estimates of roughness over time on heads of the two cultivars. Additionally, tissues from greenhouse grown plants tended to be more hydrophobic than when field grown and the surface properties of lawns of yeast OH 182.9 were hydrophobic in water. This work suggests that changing surface properties during crop development could influence colonization success by biocontrol agents and should be considered in spray formulations of agents to enhance colonization success.
Schisler, D.A., Core, A.B., Boehm, M.J., Horst, L., Krause, C.R., Dunlap, C.A., Rooney, A.P. 2014. Population dynamics of the Fusarium head blight biocontrol agent Cryptococcus flavescens OH182.9 on wheat anthers and heads. Biological Control. 70(1):17-27.
Venkateswaran, K., Vaishampayan, P., Benardini III, J.N., Rooney, A.P., Spry, J.A. 2014. Deposition of extreme-tolerant bacterial strains isolated during different phases of Phoenix spacecraft assembly in a public culture collection. Astrobiology. 14(1):24-26.
Slininger, P.J., Schisler, D.A. 2013. High-throughput assay for optimizing microbial biological control agent production and delivery. Biocontrol Science and Technology. 23(8):920-943.
Dunlap, C.A., Schisler, D.A. 2014. Characterization of the surface properties of wheat spikelet components grown under different regimes and the biocontrol yeast Cryptococcus flavescens. Journal of Agricultural and Food Chemistry. 62(1):809-815.