Location: Crop Bioprotection Research
2024 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 Sub-objective 1.A, ARS researchers in Peoria, Illinois, made significant progress in sequencing the genomes of bacteria in the family Bacillaceae in collaboration with the ARS culture collection in Peoria, Illinois. Sequencing the genomes of microbes associated with plants will allow us to better understand how plants and microbes interact with each other. This will facilitate identification of novel compounds from the microbes that may be useful in crop protection applications. We have successfully cultured, extracted DNA, sequenced, and assembled the genomes of more than 2,000 bacterial strains this year. We accessioned and publicly released data for more than 2,000 genomes from the previous year. We screened more than 5,000 available bacterial genomes previously produced by our laboratory for proteins with pesticidal properties. We have identified 12,607 putative bacterial pesticidal proteins, that have potential antifungal, insecticidal and/or nematocidal activity. Further research is needed to confirm these potential beneficial activities against agricultural pests.
Under Sub-objective 1.B and 1.C, we made significant progress optimizing procedures for the cloning and expression of large secondary metabolite clusters (> 40 kb). We constructed strains expressing the lipopetides bacillomycin D, bacillomycin F, bacillomycin L, mojavensin, and mycosubtilin. The expression of these compounds is currently being optimized. Having access to milligram quantities of purified lipopeptides will allow us to test the efficacy of these compounds against a variety of plant pathogens as pure chemicals or combination of pure chemicals. In addition, two unknown biosynthetic gene clusters from Bacillus sonorensis and Bacillus velezensis were transformed into the optimized Bacillus subtilis host strain; characterization of the novel metabolite(s) is ongoing. Identifying new metabolites with biological activity will allow us to better manage fungicide resistance and provide new crop protection products.
Under Sub-objective 2.A, we made significant progress in evaluating Bacillus velezensis as a seed coating for Midwest row crops. We completed testing of two soybean lines in germination assays and plant growth promoting assays with nine genotypes of Bacillus velezensis. Assays to evaluate the effect of these treatments on the microbiome of germinating seedlings are in progress. In addition, we evaluated the production of auxin, a plant growth regulator from these genotypes of Bacillus velezensis. In a different experiment, during genome mining of a strain of Chromobacterium vaccinii, we discovered it can produce a broad-spectrum antibiotic (2,4-diacetylphloroglucinol), which has previously been shown to be useful in controlling plant diseases. We also discovered that this bacterium produces two other compounds with known antimicrobial and insecticidal activity. These findings suggest that strains of this bacterial species should be explored for potential applications in biocontrol of plant diseases and pest insects.
Under Sub-objective 2.B, we made substantial progress in evaluating the ability of two transformation methods to introduce foreign DNA plasmids harboring a gene encoding a green fluorescent protein into 10 strains of Bacillus velezensis. Electroporation was unsuccessful in transforming all 10 strains of Bacillus velezensis while natural competence induced by nitrogen starvation was successful for 6 out of 10 strains. Additional methods are being evaluated to increase the success rate and efficiency of transforming these strains.
Tar spot of corn caused by the fungus Phyllachora maydis is a major disease of corn that is rapidly spreading in the continental U.S. and Canada. Tar spot can cause significant loss in grain yield and in 2021, this fungus caused grain yield loss with an economic impact of $1.25 billion. The use of fungicides has not been very effective because once the disease is noticed in the field, subsequent infection becomes difficult to control. As part of a new initiative, we conducted research to discover biocontrol agents for the tar spot of corn pathogen. Bacterial and fungal communities were isolated from tar spots occurring in corn and alternate weedy grass hosts collected from multiple sites and evaluated for effectiveness in controlling tar spot in overwintering corn debris relative to commercial strains. The most effective of those tested was a laboratory isolate that is also not pathogenic to corn leaves. Identification and commercialization of biological control organisms effective on corn debris, should add an important component to an integrated control program for tar spot of corn.
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. However, ambrosia beetle infestations remain difficult to control because of their cryptic habitats and the inability to deliver pesticides or biopesticides to the tunnels and galleries inside of trees where they reproduce. Among the few organisms inhabiting the beetle’s galleries is a type of mite. These mites were found in close association with ambrosia beetles and their fungal gardens. We previously showed that this mite could spread beneficial pest-killing fungi into the galleries of these beetles. The current research was focused on optimizing the production of these mites and their inoculation with beneficial fungi. These mites cultivate and consume a variety of fungi to feed their offspring, and little is known about the range of fungi they can utilize for this purpose. We assessed the potential of nine fungal species to serve as diet for the mites and evaluated methods for inoculating the mites with beneficial fungi. We have identified conditions in which we can produce the mites and inoculate them with beneficial fungi. Additional research is needed to identify the optimal level of inoculation of the beneficial fungi without inhibiting mites’ movement.
Accomplishments
1. New weapons to fight tar spot disease in corn. Tar spot disease of corn can cause significant yield losses when environmental conditions are conducive to spread of this fungus in the Midwestern United States. Biological control is an approach that uses natural microbes to inhibit or kill plant pathogens, which provides a more sustainable method for farmers to protect their crops. In spring of 2022, ARS researchers in Peoria, Illinois, isolated two strains of bacteria, Priestia megaterium, from corn that has potential biological control properties. These bacteria were grown in large quantities in the laboratory, and a portion of each strain was applied to corn seeds that were then sown in field plots outside of the laboratory. This year we completed our second year of field trials, marking the second year of successfully reducing disease incidence when the two bacterial strains are applied as a seed coating. These bacteria could be utilized in pest management strategies to reduce the impact of tar spot disease in corn.
2. Identifying broken genes in corn to improve pest resistance. Corn is one of the most important grain crops in the world and its production can be severely limited by fungal pathogens and insect pests. Breeding corn plants with improved resistance to pests and pathogens has been helpful, and multiple genes appear to be involved in both insect and pathogen resistance. During the breeding process, mutations can unintentionally be introduced into these resistance genes and reduce their effectiveness. ARS researchers in Peoria, Illinois, identified mutations in a group of pest resistance genes that make them less effective. Identifying these problematic genes allows them to be removed from breeding stock or have them repaired. This discovery will allow corn breeders to produce plants with better pest resistance and on farm performance.
Review Publications
Muturi, E.J., Doll, K.M., Dunlap, C.A. 2023. Non-target effects of essential oils on selected beneficial bacteria. Journal of Plant Diseases and Protection. https://doi.org/10.1007/s41348-023-00825-6.
Lyon, R.M., Johnson, E.T., Dowd, P.F. 2024. Undesirable protein sequence variations in maize genes that confer resistance to fungal pathogens and insect pests. Plant Gene. 37. Article 100441. https://doi.org/10.1016/j.plgene.2023.100441.
Surovy, M., Dutta, S., Mahmud, N., Gupta, D., Farhana, T., Paul, S., Win, J., Dunlap, C.A., Oliva, R., Rahman, M., Sharpe, A.G., Islam, T. 2024. Biological control potential of worrisome wheat blast disease by the seed endophytic bacilli. Frontiers in Microbiology. 15-2024. https://doi.org/10.3389/fmicb.2024.1336515.
Johnson, E.T., Bowman, M.J., Gomes, R.P., Carneiro, L.C., Dunlap, C.A. 2023. Identification of 2,4-diacetylphloroglucinol production in the genus Chromobacterium. Scientific Reports. 13. Article 14292. https://doi.org/10.1038/s41598-023-41277-0.
Johnson, E.T., Dowd, P.F., Ramirez, J.L., Behle, R.W. 2023. Potential biocontrol agents of corn tar spot disease isolated from overwintered Phyllachora maydis stromata. Microorganisms. 11(6). Article 1550. https://doi.org/10.3390/microorganisms11061550.
Dowd, P.F., Johnson, E.T. 2024. Appropriate selection of organic hybrid sweet corn varieties can positively influence both the effectiveness of the insect biological control agent Beauveria bassiana and fungal disease resistance. Organic Agriculture. https://doi.org/10.1007/s13165-024-00453-w.