Location: Crop Bioprotection Research
2022 Annual Report
Objectives
Objective 1: Discover and optimize the use of bioactive metabolites associated with beneficial microbes.
Sub-objective 1A: Genome sequencing of Bacillus microbial resources.
Sub-objective 1B: Heterologous expression of biosynthetic gene clusters.
Sub-objective 1C: Creation of lipopeptide producer strains and evaluation of synergy and efficacy.
Objective 2:Evaluate the application of microbes, such as seed coatings, for their interaction with plant pathogens and their role in biocontrol efficacy.
Sub-objecitve 2A: Evaluation of seed coatings and biocontrol agent genotype.
Sub-objective 2B: Development of genetic modification protocols and functional genomics to understand the determinants of biocontrol efficacy.
Approach
Our approach will be to apply technologies allied with the fields of fermentation science, microbial physiology, metabolomics, genomics, and proteomics for two purposes: to enhance the efficacy and shelf-life of the antagonist biomass manufactured and to produce gnotobiotic (i.e., all of a limited number of organisms in a culture are known) or axenic cultures of nutritionally fastidious plant pathogens. More specifically, the shelf-life and efficacy of biocontrol strains will be improved by isolating efficacious stress tolerant variants of a yeast biocontrol agent and then testing the more promising strains isolated in small pilot tests against Fusarium head blight of wheat. Other studies will strive to discover cell production methodologies that promote the production of compounds that enhance cell stress tolerance. Strain transcriptional response to culture conditions will be determined to facilitate optimizing these cell production studies. This will include studies to elucidate the transcriptional response of a yeast biocontrol strain to cold-adaptation that improves cell survival and biocontrol efficacy. Gnotobiotic culturing studies will include establishing a selection of host plants in sterile tissue culture boxes or as callus cell cultures and evaluating methods for infecting these host tissues with axenic propagules of an obligate pathogen. The transcriptional response of gnotobiotic host cell tissue to infection by an obligate plant pathogen will then be determined as a prelude to attempting to grow one or more obligate plant pathogens in axenic culture.
Progress Report
Under Objective 1, we evaluated several DNA extraction methods and identified a method that provided high quality DNA and a high yield. We evaluated a few culturing methods and labware to determine the best method to grow more than 4500 Bacillus strains. We identified labware and strategies to manage the high number of samples. We have grown and extracted the DNA out of more than 800 samples. The second aspect of this project is to identify novel metabolites that the beneficial microbes can produce. This is accomplished by identifying unique genes in their genomes and expressing them under laboratory conditions.
Under Objective 2, we have completed a study of our collection of selected Bacillus strains and have selected seven strains to evaluate as seed coatings in wheat and soybeans. We anticipate conducting the seed coating experiments in the fall. We have made progress in inserting a fluorescent marker in these strains. This will allow us to easily see the bacterium with a fluorescent microscope when it is on the surface or inside the tissues of a plant.
Accomplishments
