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ARS Home » Midwest Area » Peoria, Illinois » National Center for Agricultural Utilization Research » Crop Bioprotection Research » Research » Research Project #432596

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

Location: Crop Bioprotection Research

2020 Annual Report

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.

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 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 three of the project, Objective 1 studies completed by ARS scientists at Peoria, Illinois, and collaborators at the University of Hawaii and North Carolina State University identified several genes likely involved in nutrient acquisition in the pathogen that causes downy mildew in basil. A number of genes coding for sugar transporters, which are critical for pathogen growth, were discovered to be highly expressed by the pathogen when infecting basil leaves in Hawaii, Illinois, and North Carolina. The research found that basil downy mildew utilizes multiple different transporters to obtain sugars from its host or to move sugars from cell to cell throughout the plant. Similar studies identified basil downy mildew genes that are differentially expressed during disease progression in infected leaves; though their function is not yet known. Understanding the resources used by the pathogen during the infection process will allow us to develop strategies to propagate the fungus outside of the plant, which is the main goal of the project. We identified a suitable medium to grow callus derived from basil leaves, which are undifferentiated plant cells that are used to grow the fungus in the lab. Successfully growing callus is the first step in developing culture conditions to grow fastidious plant pathogens, such as downy mildew. During this research, a novel bacterium species was discovered that accidentally contaminated callus in the laboratory; substantial characterization of this new species was completed. For project Objective 2, significant progress was made in understanding the interactions between microbes and their plant hosts. We completed two significant studies of comparative genomics and metabolomics on a group of Bacillus species widely developed as biocontrol agents against plant pathogens. The first study reports the diversity in a class of antifungals known as iturinic lipopeptides. Comparative genomic analysis followed by mass spectroscopy of culture media allowed us to assign the biosynthetic clusters with the antifungal iturinic compounds they produce. This research identified compounds that have not yet been commercialized and are likely targets for the development of future crop protection products. In addition, this research also identified the role of these metabolites in the speciation of this genus. 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 in the use of beneficial microbes for controlling both the ambrosia beetle vector and the disease-causing fungus. Treating ambrosia beetles with antagonistic bacteria in a greenhouse assay resulted in significant control in the form of beetle death and reduction in fecundity of the surviving vectors. The beneficial Bacillus strain utilized in this study possesses strong antifungal activity and is phylogenetically close to commercial Bacillus strains that are used as plant pathogen antagonists. Treatment with both the bacteria and production supernatant was more effective than the supernatant alone and the beetles can vector the bacteria to the insect galleries. The bacteria then kill the fungus (plant pathogen) that is grown by the insect to feed the larvae. These findings supported scaling up testing to a limited field trial this year with a commercial orchard partner.

1. Importance of antifungal metabolites Bacillus crop protection agents. Species belonging to the genus Bacillus are the most successful bacteria developed as biocontrol agents for control of plant pathogens and also serve as important human, livestock, and aquaculture probiotics. ARS scientists in Peoria, Illinois, discovered that distribution of antifungal compounds produced by these bacteria can be used to predict their taxonomy. It was also discovered this class of antifungal compounds evolved from one compound into six related antifungal compounds. By understanding how these antifungal molecules originated and evolved, we have gained insights into the interactions between crop protection bacteria and the plant pathogenic fungi they control, which should allow us to better utilize these beneficial bacteria as crop protection products.

2. Identification of antifungal compounds from bacteria to control onion rot. Many bacteria naturally produce biochemicals that inhibit the growth of fungal plant pathogens and ARS scientists in Peoria, Illinois, identified a bacterium from the ARS Culture Collection with this ability. In liquid culture, the bacterium secreted biochemicals that inhibited growth of many fungal plant pathogens and was effective against a fungal pathogen of onion grown in greenhouse assays. These metabolites were identified as edeines, a class of antibiotic metabolites that exhibit broad antibacterial, antifungal and bioherbicidial activity. This work contributes to the broader scientific community through increasing our knowledge of the biological activity of this important soil microbe. In addition, comparative genomics research determined that gaining the ability to produce edeines played an important role in the evolution of the bacteria in this genus (Brevibacillus). Therefore, these compounds may be more useful in post-harvest or other unique applications rather than traditional crop protection treatments.

Review Publications
Dowd, P.F., Naumann, T.A., Johnson, E.T., Price, N.P.J. 2020. A maize hydrolase with activity against maize insect and fungal pests. Plant Gene. 21:100214.
Araujo, R., Dunlap, C.A., Barnett, S., Franco, C. 2019. Decoding wheat endosphere-rhizosphere microbiomes in Rhizoctonia solani-infested soils challenged by Streptomyces biocontrol agents. Environmental Microbiology. 10:1038. https://doi:10.3389/fpls.2019.01038.
Dunlap, C.A., Bowman, M.J., Rooney, A.P. 2019. Iturinic lipopeptide diversity in the Bacillus subtilis species group – important antifungals for plant disease biocontrol applications. Frontiers in Microbiology. 10:1794.
Hamm, P.S., Caimi, N.A., Northup, D.E., Valdez, E.W., Buecher, D.C., Dunlap, C.A., Labeda, D.P., Porras-Alfaro, A. 2019. Streptomyces corynorhini sp. nov., isolated from Townsend's big-eared bats (Corynorhinus townsendii). Antonie Van Leeuwenhoek. 112:1297-1305.
Johnson, E.T., Bowman, M.J., Dunlap, C.A. 2020. Brevibacillus fortis NRS-1210 produces edeines that inhibit the in vitro growth of conidia and chlamydospores of the onion pathogen Fusarium oxysporum f. sp. cepae. Antonie van Leeuwenhoek. 113:973-987.
Masmoudi, F., Abdelmalek, N., Tounsi, S., Dunlap, C.A., Trigui, M. 2019. Abiotic stress resistance, plant growth promotion and antifungal potential of halotolerant bacteria from a Tunisian solar saltern. Microbiological Research. 229:126331.
Araujo, R., Dunlap, C., Franco, C.M.M. 2020. Analogous wheat root rhizosphere microbial successions in field and greenhouse trials in the presence of biocontrol agents Paenibacillus peoriae SP9 and Streptomyces fulvissimus FU14. Molecular Plant Pathology. 21(5):622-635.
Dunlap, C.A., Bowman, M.J., Zeigler, D.R. 2020. Promotion of Bacillus subtilis subsp. inaquosorum, Bacillus subtilis subsp. spizizenii and Bacillus subtilis subsp. stercoris to species status. Antonie Van Leeuwenhoek. 113:1-12.
Burkett-Cadena, M., Sastoque, L., Cadena, J., Dunlap, C.A. 2019. Lysinibacillus capsici sp. nov, isolated from the rhizosphere of a pepper plant. Antonie van Leeuwenhoek. 112:1161-1167.
Dunlap, C.A. 2019. Lysinibacillus mangiferihumi, Lysinibacillus tabacifolii and Lysinibacillus varians are later heterotypic synonyms of Lysinibacillus sphaericus. International Journal of Systematic and Evolutionary Microbiology. 69(9):2958-2962.
Dowd, P.F., Johnson, E.T. 2020. Transgenic expression of a previously uncharacterized maize AIL1 gene in maize callus increases resistance to multiple maize fungal and insect pests. Plant Gene. 23:100235.
Juma, E.0., Allan, B.F., Kim, C., Stone, C., Dunlap, C.A., Muturi, E.J. 2020. Effect of life stage and pesticide exposure on the gut microbiota of Aedes albopictus and Culex pipiens L. Scientific Reports. 10. Article 9489.