Location: Crop Bioprotection Research2013 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. 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:
ARS Crop Bioprotection Research Unit scientists at the National Center for Agricultural Utilization Research (NCAUR), Peoria, IL, worked with university and industry collaborators in FY13 to make significant progress in support of the two objectives of this project. Lipopeptide tolerant variants of the Fusarium head blight (FHB) yeast antagonist Cryptococcus flavescens OH 182.9 were developed by repeatedly coculturing the yeast with varying initial inoculum concentrations of the lipopeptide producing, FHB antagonist Bacillus amyloliquefaciens AS 43.3. Selected OH 182.9 variants grew more competitively in the presence of B. amyloliquefaciens and some cocultures of variants and strain AS 43.3 exhibited modestly increased activity against FHB in greenhouse tests. This work was conducted in support of Project Objective 1, is to optimize fermentation and formulation protocols to enhance the effectiveness of biocontrol products active against plant diseases. Also in Objective 1, an alternative co-cultured product, composed of strain OH 182.9 and a Bacillus spp. that produced greatly reduced levels of antimicrobial compounds was developed. Varying initial inoculum levels of both microbial isolates in dual cultures resulted in differing final concentrations of each strain and differing FHB biocontrol efficacy of the final products in greenhouse tests. The reproducibility of small pilot scale production of inoculum of a yeast biocontrol agent and the stability and efficacy of the product produced was determined. A concentrated yeast cell product maintained acceptable-viability and efficacy in greenhouse bioassays of the FHB antagonist. Research progress in support of Objective 2, the characterization of interactions among host, biocontrol agent, and pathogen on host surfaces, was also significant. ARS scientists in collaboration with scientists at the Ohio State University (OSU), had previously developed DNA-based assay for monitoring OH 182.9 populations and confirmed its accuracy in greenhouse bioassays. In field experiments conducted this year we confirmed the utility of the assay for characterizing the population dynamics and spread of yeast antagonist OH 182.9 on wheat. Collaborative studies were conducted with OSU that made significant progress in developing a DNA-based method for rapidly classifying strains of C. flavescens for their relative potential as FHB biocontrol agents without having to conduct labor and time intensive greenhouse bioassays on wheat plants. FY13 research progress was also demonstrated in the form of a CRADA extension, two new Specific Cooperative Agreements (SCA) with universities, a patent application, and a patent issuance. These results and agreements 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. Gene-based monitoring of a benign biocontrol agent of Fusarium head blight. A yeast previously discovered by ARS Crop Bioprotection Research Unit scientists at the National Center for Agricultural Utilization Research (NCAUR), Peoria, Illinois, reduces Fusarium Head Blight (FHB), a globally important disease of wheat that is difficult to control. Understanding parameters that support survival and efficacy of this yeast on wheat would enhance its potential as a FHB control tool, but tracking the yeast in the field is labor intensive. In this study, Ohio State University and ARS scientists developed a gene-based technique to quantify the population dynamics of this yeast and successfully employed it on field grown wheat inoculated with the biocontrol agent. We discovered that the dispersal of the yeast from inoculation sites increased with time and that the yeast could be detected on harvested grains and postharvest field residue. This breakthrough provides a new tool for understanding the environmental fate of this biocontrol agent and how its population on wheat relates to biocontrol effectiveness. Such information is crucial to the development of this environmentally benign yeast as a commercial biocontrol product to benefit wheat producers, processors, and consumers.
2. Glume blotch pathogen of wheat does not readily produce alternariol during the wheat infection process. The plant pathogen responsible for causing glume blotch in wheat, Stagonospora nodorum was recently shown to have the genetic potential to produce the mycotoxin, alternariol, which can be toxic to plant and animal cells. In a collaborative project, ARS Crop Bioprotection Research Unit scientists at the National Center for Agricultural Utilization Research (NCAUR), Peoria, Illinois, and scientists of the Australian National University tested wheat plant components (leaves, stems, and wheat heads) for this mycotoxin after infecting plants with the pathogen. Under the test conditions utilized, no alternariol mycotoxin was detected among the metabolites produced by the pathogen on infected wheat plant parts. The integration of functional genomics and the study of metabolite production profiles enabled us to specifically unravel complex interactions in a wheat-plant pathogen system and broadly contributes methodologies for understanding how to develop more effective plant disease control measures.
Dunlap, C.A., Bowman, M.J., Schisler, D.A. 2013. Genomic analysis and secondary metabolite production in Bacillus amyloliquefaciens AS 43.3: A biocontrol antagonist of Fusarium head blight. Biocontrol. 64(1):166-175.