Project : USDA ARS

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Research Project: Development of New Production Methodologies for Biocontrol Agents and Fastidious Microbes to Improve Plant Disease Management

Location: Crop Bioprotection Research

2021 Annual Report

Objectives
Objective 1: Develop new microbial culturing and mass production technologies for biocontrol agents and nutritionally fastidious plant pathogens. Subobjective 1a: Develop new microbial culturing technologies for biocontrol agents. Subojective 1b: Develop new methodologies for culturing nutritionally fastidious plant pathogens. Objective 2: Define interactions between biocontrol agents, hosts, and pathogens using traditional and genomic approaches to increase disease management success.

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
In year four of the project, Objective 1 studies completed in collaboration with the University of Hawaii and the Agricultural Research Organization of Israel identified several thousand genes that are differentially expressed in basil leaves (from a susceptible variety) in response to downy mildew infection. Additional analyses indicated the downy mildew induced a number of potential basil disease resistance genes, as well as, genes that might contribute to disease susceptibility. The gene expression of downy mildew was also analyzed and identified genes that likely degrade plant tissues during infection. Additional experiments were conducted to identify a sterile two-organism culturing system for basil downy mildew, which could potentially identify new information in how to grow this pathogen in pure culture. It was determined that basil downy mildew spores were not able to infect cultured basil cells that were growing on artificial growth medium in plastic dishes. This finding indicates that growth of basil downy mildew from spores require nutrients present in basil leaves, but which are not present in cell cultures. Under Objective 2, progress was made in understanding how biocontrol agents interact with each other. In collaboration with the Technological University of Mixtec in Mexico, we evaluated the combination of two biocontrol agents in controlling fungal diseases in tomatoes. In this study, we combined a bacterial and fungal biocontrol agent and tested their effectiveness against vascular wilt and early blight in tomatoes. The results showed the combination performed the same as using the two biocontrol agents independently. The work also established that this bacterial biocontrol agent does not directly inhibit the plant pathogen in confrontation assays, and its activity may result from inducing plant resistance to the pathogens or through competition for resources with the pathogen. In a second collaboration, with the National University of Rosario in Argentina, we completed a large genomic study of strains that comprise the Bacillus cereus group. The Bacillus cereus group is made up of species that are important as biological control agents, plant growth promoters, probiotics, sources of food processing enzymes, food fermentation inoculants, food poisoning agents, human and livestock pathogens. This study describes the latest methods for correctly identifying these organisms and exploring how important phenotypes (e.g. toxin production) are distributed in the genus. We were also able to establish how phenotypes are correlated to a given species. Correct taxonomic identification and phenotype predication are very important for regulatory concerns, intellectual property, and communication of ideas. In a subordinate project on controlling laurel wilt in avocadoes, progress has been made in developing new methods of crop protection. Laurel wilt is spread by ambrosia beetles, which bore galleries into the trees and grow the plant pathogenic fungus to feed their offspring. We have developed methods on the use of beneficial microbes for controlling both the ambrosia beetle vector and the disease-causing fungus. We initiated an experiment to evaluate two new biocontrol strains as potential antagonists of the disease. These were compared with established methods for treating a local area during phyto-sanitation after a diseased tree is identified.

Accomplishments
1. Identification and characterization of a novel plant-associated bacterium. Microorganisms can be a source of valuable chemicals and enzymes useful in solving a variety of problems. Useful products from microorganisms include antibiotics and enzymes used in detergents. Examining microorganisms from unusual sources can potentially lead to such products. A bacterium found growing in association with corn cell cultures was characterized and found to be a new species. Genome sequencing indicated the presence of genes coding for enzymes and proteins involved with beneficial plant associations, antibiotic compounds including a novel peptide, and the breakdown of complex carbohydrates. Further investigation may lead to the development of products, such as effective and safe antibiotics produced by the bacterium could be utilized to protect crops from plant-pathogenic fungi and bacteria.

2. Identification of novel antifungal compounds. Agricultural products can be contaminated with fungi at harvest. These fungi can grow during post-harvest storage and cause losses of the commodity. In addition, some contaminating fungi can produce mycotoxins which are harmful to livestock and humans. It is imperative to identify safe and effective technologies that reduce fungal contamination of food. An ARS scientist at Peoria, Illinois, and a colleague from Henan University of Technology (China) utilized two naturally occurring chemicals produced by plants to completely inhibit growth of fungi that were present on the surface of peanuts. The two chemicals are utilized in the food industry as flavoring substances, and generally recognized as safe, but they have not previously been utilized for control of fungi infecting peanuts during post-harvest storage. This research is an important demonstration that these safe, volatile chemicals can be used to reduce fungal spoilage of stored peanuts and reduce dependence on synthetic pesticides, which may cause adverse effects on humans.

3. Genomic and taxonomic analysis of the Bacillus cereus group. The Bacillus cereus group is made up of agriculturally important bacteria that have a wide range of biological activities. They have been used to control insect pests in crops, identified as food and human pathogens, and found closely associated with plants. Our research analyzed over 2000 genomes from this group to clarify the taxonomic relationships and identify the prevalence of traits associated with each species. This research allows us to understand how these bacteria evolve to serve different functions in agricultural systems and will allow us to better predict their behavior. This helps us design strategies to limit the bacteria with negative traits and better exploit the bacteria with positive traits.

U.S. DEPARTMENT OF AGRICULTURE

Crop Bioprotection Research: Peoria, IL