Location: Crop Bioprotection Research
2022 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
This is the final report for this project which terminated in March 2022. See the report for the replacement project, 5010-22410-024-000D, “Develop an Improved Understanding of Microbe-pathogen Interactions for Biological Control” for additional information.
Objective 1: Develop new microbial culturing and mass production technologies for biocontrol agents and nutritionally fastidious plant pathogens. Under Objective 1 of this project, studies were completed to evaluate potential methods of culturing downy mildews, which currently cannot be grown outside of plants. Limited information is known about how downy mildew pathogens extract nutrients from the leaves of their plant host. Expression of genes encoding transporters of metabolites were measured in downy mildew infecting tobacco leaves. This study identifies potential suitable targets for controlling downy mildews that cause economic losses in the production of basil, spinach, and tobacco.
A second study determined that basil downy mildew turned on genes involved in carbohydrate degradation during leaf infection. Research with pathogens related to downy mildews found that these enzymes are important for pathogen virulence, but little is known about these processes in downy mildews. Three basil downy mildew genes were transferred into laboratory strains of bacteria or yeast to study them more closely.
Objective 2: Charactierize the molecular basis of interactions to improve the bioefficacy and consistency of biological control agents. Under Objective 2, studies were completed to identify the modes of action of biocontrol agents and their metabolites for reducing Fusarium head blight in wheat. In collaboration with researchers from Argentina, we evaluated ten strains of Bacillus inaquosorum as a potential antagonist of the causal agent of Fusarium head blight. We previously identified B. inaquosorum as a strong producer of antifungal metabolites, but this species has rarely been reported as a potential biocontrol agent. The strains were all successful in inhibiting the growth of Fusarium graminearum. In addition, the strains were tested in combination with two Bacillus velezensis strains known to be effective biocontrol agents. The preliminary results suggest that there is no direct synergism or antagonism between the Bacillus strains. Greenhouse and field assays are currently underway for the best performing strains and will continue as part of the next project plan.
In collaboration with researchers from Tunisia, we evaluated a B. velezensis and Bacillus spizizenii strains with exceptional halotolerance for their ability to protect tomato plants from osmotic and salt stress. Both strains were successful in improving plant traits while under salt and osmotic stress and were able to reduce sodium levels in the plants while increasing potassium and calcium levels. These strains also demonstrated plant growth promoting traits under these conditions in a greenhouse assay.
In a subordinate project, our laboratory has been developing and evaluating potential microbial biological control options to manage two ambrosia beetle vectored diseases of avocado: laurel wilt and Fusarium dieback. We demonstrated entomopathogenic fungi can reduce the population of the different beetle species that are spreading laurel wilt. However, ambrosia beetle infestations remain difficult to control because of their cryptic habits and the inability to deliver pesticides inside of their galleries. Among the few organisms inhabiting the beetle’s galleries are a type of mite. These mites were found in close association with the ambrosia beetles and their fungal gardens. We evaluated the ability of this mite to spread beneficial fungi into the galleries of this pest. The results obtained so far are encouraging. There is evidence the mites can carry and spread spores of beneficial fungi species and kill the beetles. However, similar trials under semi-field and field conditions are needed to confirm the results.
Summary of research over the five-year project:
Over the course of the project, we were successful in making progress towards our objectives. Under Objective 1, we characterized the life cycle of Peronospora belbahrii, the causal agent of downy mildew in sweet basil. This knowledge of the infection process should lead to the design of better crop protection products for the control of downy mildew. We completed a study looking at the genes expressed by the pathogen during the infection process in basil. This allows us to better understand the requirements the pathogen needs to live and reproduce inside the plant. We were unsuccessful in developing a method of culturing the pathogen. However, we were successful in developing a culturing method of the closely related fungus that causes downy mildew of tobacco.
Under Objective 2, we completed several successful studies using Bacillus species as antagonists of plant pathogens. In collaboration with scientists from 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 seeds and alter the microbial community colonizing the emerging plant. Results from these studies should improve the commercial development potential of these biocontrol products.
We also completed large scale genomic analysis of several species of the bacterium genus Bacillus to understand how they have evolved and the distribution of metabolites characteristics across the species. This research identified compounds not yet 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. For the Bacillus cereus group, we identified species important for biological control agents, plant growth promoters, probiotics, sources of food processing enzymes, food fermentation inoculants, food poisoning agents, and human and livestock pathogens. This study describes the latest methods for correctly identifying these organisms and explores how key biological attributes (e.g. toxin production) are distributed in the genus.
We were also able to establish how physical traits are correlated to a given species. Correct naming and trait prediction are very important for regulatory concerns, intellectual property, and communication of ideas. In a discovery project, we screened and identified a bacterium that secreted biochemicals inhibiting 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 bioherbicidal 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 gaining the ability to produce edeines played an important role in the evolution of the bacteria in this genus (Brevibacillus). All of these studies help understand the mode of action of these important crop protection agents and provide insights to address efficacy and regulatory hurdles associated with their use. This research on the mode of action of biocontrol bacteria will be built upon in the next project as we explore the genetic determinants of biocontrol activity through sequencing a large number of bacteria and identifying the biosynthetic clusters associated with bioactive metabolites.
Accomplishments
1. Identified genes that participate during the infection process of downy mildew. Downy mildew infects susceptible basil cultivars every year in the United States and can cause significant losses in yield and crop quality. The pathogen responsible for downy mildew disease on basil has not been well studied at the molecular level, primarily because the pathogen will not grow on artificial growth medium, but only on living plants. The ability to grow it outside the plant will expedite research on this important pathogen. ARS researchers in Peoria, Illinois, collaborated with scientists from the Agricultural Research Organization of Israel and the University of Hawaii to identify the genes in basil plants that are turned on during course of the disease. We also identified the genes the pathogen uses during the infection process. Knowledge of the nutrients the pathogen is utilizing will enable us to better develop methods to cultivate it outside the plant. These results contribute important knowledge of the infection process of basil downy mildew and could aid the development of more effective measures for reducing the severity of the disease.
Review Publications
Soni, R., Keharia, H., Dunlap, C.A., Pandit, N., Doshi, J. 2022. Functional annotation unravels probiotic properties of a poultry isolate, Bacillus velezensis CGS1.1. LWT - Food Science and Technology. 153. Article 112471. https://doi.org/10.1016/j.lwt.2021.112471.
Dowd, P.F., Naumann, T.A., Johnson, E.T. 2022. A maize gene coding for a chimeric superlectin reduces growth of maize fungal pathogens and insect pests when expressed transgenically in maize callus. Plant Gene. 30. Article 100359. https://doi.org/10.1016/j.plgene.2022.100359.
Ma, W., Johnson, E.T. 2021. Natural flavour (E,E)-2,4-heptadienal as a potential fumigant for control of Aspergillus flavus in stored peanut seeds: finding new antifungal agents based on preservative sorbic acid. Food Control. 124. Article 107938. https://doi.org/10.1016/j.foodcont.2021.107938.
Dowd, P.F., Johnson, E.T. 2022. Different maize (Zea mays L.) inbreds influence the efficacy of Beaveria bassiana against major maize caterpillar pests, which is potentially affected by maize pathogen resistance. Biocontrol Science and Technology. 32(7):847-862. https://doi.org/10.1080/09583157.2022.2055745.
