Location: Crop Bioprotection Research
2023 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 Objective 1, we 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 and extracted DNA from more than 2,500 strains. We have modified and developed methods for greatly reducing the cost of library preparation for these strains. We completed our first round of sequencing and data analysis. We accessioned and publicly released data for more than 600 genomes. 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). A Bacillus subtilis host strain was identified that had optimal competence for DNA transformation. A novel shuttle vector, with both positive and negative selectable markers for yeast transformation, was constructed for recombination of large biosynthetic gene clusters at the Bacillus subtilis amyE locus. The iturin A biosynthetic gene cluster from Bacillus velezensis was cloned into the shuttle vector and successfully propagated in yeast. Optimization of transformation of the large vector into E. coli cells is in progress. The B. subtilis host strain was successfully modified to produce three bioactive compounds, surfactin, fengycin and subtilosin. Experiments are underway to mutate the surfactin and subtilosin biosynthetic gene clusters in the B. subtilis hosts strain.
Under Objective 2, we made significant progress in evaluating Bacillus velezensis as a seed coating for Midwest row crops. We completed producing seeds and testing three wheat lines in germination assays with different genotypes of B. velezensis. Assays to evaluate the microbiome of germinating seedlings are in progress. In a second experiment, we evaluated the compatibility of three common groups of Bacillus biocontrol and biostimulant strains against essential oils commonly used as crop protection products. The results showed that the Bacillus strains were compatible with most essential oils at anticipated usage rates. In collaboration with a researcher in Brazil, we evaluated the biocontrol and biostimulant activity of two novel Trichoderma sp. strains. The isolates were tested for their ability to control white rot in cotton plants and for their ability to act as a biostimulant to promote plant growth. The results showed that one of these strains was effective at controlling white rot in cotton, while the second strain was better adapted to serve as a biostimulant. The results suggest that Trichoderma strains may be useful in providing multiple benefits for cotton plants. Progress was made in evaluating a new technique to control Phytophthora blight using gene silencing. In this technique, the expression of genes is blocked using compounds known as dsRNA that are specific for a given gene. The potential genes to be targeted were identified and their dsRNAs produced. Experiments are under way to evaluate nanoparticles as a method to deliver the dsRNAs inside the plant pathogen. If successful, this approach could be a novel method of controlling the disease in crops.
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 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 to the tunnels and galleries inside of trees where they reproduce. 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 pest-killing fungi into the galleries of this pest. The results obtained so far provide evidence that the mites can carry and spread spores of beneficial pest-killing fungal species and kill the beetles. However, additional research is needed to optimize the production and inoculation of mites with entomopathogenic fungi.
Accomplishments
1. Lysinibacillus, a new group of plant biostimulants, increases corn growth and produces plant hormones. The goal of this research was to identify microbes that stimulate the growth of plants. Biostimulants are an important class of microbial products that stimulate the growth of plants providing an agronomic benefit. ARS researchers in Peoria, Illinois, in collaboration with Colombian researchers reported the discovery and characterization of Lysinibacillus species as biostimulants. They characterized this group of bacteria for their ability to promote plant growth in corn and to produce auxin family plant hormones. The study demonstrated that most of the bacteria from the genus Lysinibacillus could promote plant growth. This research expands our knowledge of how microbes interact with plants. This research benefits corn and other row crops growers in the United States by providing information that can be used to develop new products to enhance crop growth and yields.
Review Publications
Silva, L.G., Camargo, R.C., Mascarin, G.M., De Oliveira Nunes, P., Dunlap, C.A., Bettiol, W. 2022. Dual functionality of Trichoderma: Biocontrol of Sclerotinia sclerotiorum and biostimulant of cotton plants. Frontiers in Plant Science. 13. Article 983127. https://doi.org/10.3389/fpls.2022.983127.
Baati, H., Siala, M., Azri, C., Ammar, E., Dunlap, C.A., Trigui, M. 2022. Genomic analysis of heavy metal-resistant Halobacterium salinarum isolated from Sfax solar saltern sediments. Extremophiles. 26. Article 25. https://doi.org/10.1007/s00792-022-01273-0.
Pantoja-Guerra, M., Burkett-Cadena, M., Cadena, J., Dunlap, C.A., Ramirez, C.A. 2023. Lysinibacillus spp.: An IAA-producing endospore forming-bacteria that promotes plant growth. Antonie Van Leeuwenhoek. 116: 615-630. https://doi.org/10.1007/s10482-023-01828-x.
Ramirez-Carino, H.F., Morales, I., Guadarrama-Mendoza, P.C., Gonzalez-Terreros, E., Martinez-Gutierrez, G.A., Dunlap, C.A., Valadez-Blanco, R. 2023. Biofertilizing effect of putative plant growth promoting rhizobacteria in vitro and in tomatillo seedlings (Physalis ixocarpa Brot.). Scientia Horticulturae. 308. Article 111567. https://doi.org/10.1016/j.scienta.2022.111567.
Dowd, P.F., Naumann, T.A., Johnson, E.T. 2023. Potential role of a maize metallothionein gene in pest resistance. Plant Gene. 34. Article 100409. https://doi.org/10.1016/j.plgene.2023.100409.
