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

2019 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
Significant progress has already been made in meeting the five-year project objectives which are to 1) Develop new microbial culturing and mass production technologies for biocontrol agents and nutritionally fastidious plant pathogens, and 2) Define interactions between biocontrol agents, hosts, and pathogens using traditional and genomic approaches to increase disease management success. In year two of the project, first objective studies completed by ARS scientists at Peoria, Illinois, include work that identified several new species of bacteria from the ARS culture collection that can inhibit a fungal pathogen of onion. The most promising one of these new species was evaluated to determine the nature of the antifungal compound. This research identified the active component as edeine, a broad-spectrum antibiotic. Greenhouse assays are being conducted to evaluate the potential of the strain to control fungal pathogens in onion. In research on culturing nutritionally fastidious plant pathogens, studies continued to determine the ability of several different culture media to produce callus of basil plant cells as a prelude to infecting callus with Peronospora belbahrii, the causal agent of the economically important downy mildew disease of basil. Plant callus are a type of cells that are used to grow and maintain fungal plant pathogens that require active plant tissue for growth. Tissue culture experiments determined that sterile basil leaves were not suitable for producing plant callus. It was however discovered that a system based on excised surface-sterilized basil leaves was found to support consistent infection of the downy mildew after inoculation. In addition, downy mildew of corn and turfgrass were obtained from the field but were not amenable to propagation in growth chambers. A downy mildew of cultivated impatiens collected by an ARS collaborator was easily maintained in our laboratory. Callus from impatiens leaves is currently being generated to test as a suitable host for the downy mildew. In the research conducted in support of project Objective 2, significant progress was made in understanding the interactions between microbes and their plant hosts. This excised leaf plate assay was used for experiments to conduct gene expression studies of the pathogen infecting the plant to understand the infection process. ARS researchers in Peoria, Illinois, continue to make significant progress on a project on peanut kernel shrivel disease with collaborators from a university at Southern Queensland. The goal of the research is to identify the causal agent of this important disease of peanuts. Our previous research identified a fungus as the potential agent of the disease, and we are currently validating this observation. Greenhouse testing was conducted this year and the harvested peanuts are currently being evaluated for the disease. Finally, additional progress has been made on research to understand the effect on biocontrol seed coatings on soil diseases of wheat. In collaboration with scientists from a university at Adelaide, Australia, we completed microbiome testing and analysis of wheat seeds treated with biocontrol agents and exposed to disease stress. The results showed the biocontrol agents persist for at least 8 weeks after planting treated seed and alter the microbial community that colonizes the emerging plant. Results from these studies should improve the commercial development potential of these biocontrol products. In a subordinate project on controlling laurel wilt in avocadoes, progress has been made in controlling the insect and the disease-causing fungus they vector with beneficial microbes. Recent research by our team has found that the species of ambrosia beetles found in infested avocado fields can use both plant-pathogenic fungi or non-pathogenic fungi as symbionts. These fungi are harbored by the insect in special structures in their mouths and used to inoculate galleries inside of trees. The fungi are then used as food for the beetle larvae. During the current project period, we were able to demonstrate that the antagonistic bacteria we previously isolated with activity against the plant pathogen strains have selective activity against pathogenic fungal symbionts. The results showed the strains are much more antagonistic against the plant pathogenic symbionts than the non-pathogenic symbionts. These results suggest it may be possible to push the insect population to utilize the non-pathogenic symbionts with this selective pressure, which would be a novel control strategy.

Accomplishments
1. Microscopy studies determine basil downy mildew rapidly germinates on leaves. Downy mildew caused by Peronospora belbahrii is a devastating disease of sweet basil (Ocimum basilicum) production worldwide. Traditional methods for reducing plant disease, such as the spraying of fungicides, requires using a variety of chemicals to limit the possibility of the pathogen developing resistance. Organic basil growers have very limited means to control this disease. Documenting how the pathogen infects basil plants and how quickly it produces spores would help scientists to know when in the pathogen’s life cycle, it is most vulnerable to control measures. ARS scientists in Peoria, Illinois, utilized a scanning electron microscope to determine that spores of the pathogen on basil leaves germinated in 3 days and the mildew directly penetrated leaves soon after. After 7 more days, spore producing reproductive structures of the pathogen formed on both the bottom and top of young leaves which enhances dispersal to other plants. These results contribute to more knowledge of the infection process and indicates that downy mildew control products should be present on all leaf surfaces or introduced into the plant systemically, which should lead to the design of better crop protection products for the control of downy mildew.

U.S. DEPARTMENT OF AGRICULTURE

Crop Bioprotection Research: Peoria, IL