Location: Crop Bioprotection Research
2025 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., ARS researchers in Peoria, Illinois, made significant progress in sequencing the genomes of bacteria from agricultural environments in collaboration with the ARS Culture Collection in Peoria, Illinois. The goal of the research is to identify new bioactive compounds for use as crop protection products in Midwest agriculture systems, with an emphasis on controlling corn, soybean and wheat plant pathogens. Sequencing the genomes of these microbes provides a method of identifying novel compounds faster than traditional methods. The genomes of these bacteria can be quickly screened to determine the likelihood they contain novel compounds. It also allows us to understand which strains make known compounds; and prioritize studies with the strains most likely to contain novel compounds. This will facilitate the identification of novel compounds from microbes that are useful for controlling plant pathogens of the major Midwest crops. New compounds are constantly needed to manage resistance in existing crop protection products and control new or emerging pests. We have sequenced hundreds of additional genomes and made them available through public repositories. Under this project, we have released 2,765 microbial genomes.
ARS researchers made substantial progress in identifying novel, helpful (“bioactive”) compounds from agriculturally important bacteria that were identified by examining their genomes. Bioactive compounds can be discovered by searching the genomes for clusters of genes that can produce these compounds (“biosynthetic genes”). ARS researchers in Peoria, Illinois, identified the product of a previously unknown biosynthetic cluster from bacterial strains that have been registered as crop protection agents. This metabolite was confirmed to have an antimicrobial activity and its role in controlling soybean diseases is currently under investigation. The US microbial seed coating market is currently valued at $1.58 billion and is projected to reach $2.00 billion by 2030. ARS researchers have also made progress in preparing a bacterial host that will be used to express the biosynthetic clusters from other bacteria. The first version of this host contained DNA sequences that could interfere with subsequent genetic transformation events. We have removed the unwanted DNA and modified our procedures to enhance the efficiency of genetic transformation. We have completed three of four gene modifications in the bacterial host that are necessary before the host can receive the large biosynthetic gene clusters cloned from other bacteria. We have cloned three large, biosynthetic gene clusters into yeast plasmids and we are working to complete modifications to the bacterial host; once the modifications to the host are finished, the yeast plasmids will be moved into the new host. The ability to produce targeted bioactive compounds from these bacterial hosts will help meet the growing demand from American farmers for new compounds to combat increased pesticide resistance, as well as emerging diseases.
ARS researchers in Peoria, Illinois, continued making progress in understanding the function and effectiveness of lipopeptides, a type of crop protection compound commonly found in biocontrol products. Lipopeptides are produced by one of the most successful biological crop protection products on the market, so improving their efficacy against plant pathogens will benefit a large class of agricultural products. They are frequently used in foliar spraying and seed coatings to control plant pathogens in corn, soybeans and wheat in the Midwest. We have successfully created microbial strains that lack the genes that produce lipopeptides. By applying these lipopeptide-lacking microbes to plants, both in isolation and with external lipopeptides, we will be able to determine the functions of these lipopeptides and establish whether they are essential to the microbes’ effectiveness. We have also tested a variety of bacteria, that make different combinations of lipopeptides, against the cause of red crown rot in soybeans. These results will help us understand what combinations of lipopeptides are most effective in controlling the pathogen. The results of these in-laboratory tests need to be confirmed in the field before guidance on the best combinations can be provided.
Under Objective 2, ARS researchers in Peoria, Illinois, evaluated the ability of essential oils and other natural products to inhibit the plant pathogen that causes red crown rot disease in soybeans. Red crown rot is an increasing problem in the Midwest soybean growing regions, and the disease can cause significant yield reductions, with estimates ranging from 15% to 70%. We identified several compounds that can inhibit the pathogen-producing fungus and confirmed that these compounds had no detrimental effect on soybean seed germination. In addition, we tested a variety of commercial biocontrol bacteria and strains from our collection for the ability to inhibit the pathogen that causes red crown rot in soybeans. We identified several strains that successfully inhibited the pathogen in the laboratory. In collaboration with researchers in Champaign, Illinois, we are conducting field trials in central Illinois this growing season to determine the effectiveness of commercial microbial products in controlling this disease. This addresses producers’ requests to better understand how these products perform under regional weather conditions.
ARS researchers in Peoria, Illinois, evaluated the ability of a popular species of microbial biocontrol agent to be genetically modified using different techniques. This species of bacteria is the most successful commercial microbial biocontrol agent for controlling plant pathogens and is commonly used in corn and soybeans as a seed coating. Bacillus velezensis strains have been very successful as biocontrol agents but have been historically difficult to genetically modify. The ability to genetically modify them will provide new opportunities to improve their effectiveness and better understand how these strains interact with the plant and the plant pathogens. We tested a variety of methods to introduce foreign DNA into these strains and identified one method that was successful for most of the strains. Introducing DNA into the cells was the first step in the process as the DNA must be integrated into the microbial genome with the ability to control when these new genes are turned on. We are currently evaluating different DNA sequences to test their ability to regulate the expression of introduced genes. This research will lead to better crop protection products that provide solutions to existing and emerging plant diseases facing farmers.
In a subordinate project, our laboratory has been developing and evaluating potential microbial biological control options to manage Laurel wilt and Fusarium dieback, two avocado diseases spread by the ambrosia beetle.
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 has 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. This year we evaluated the mite’s ability to survive off fungi associated with three different types of ambrosia beetles. The mite exhibits distinct feeding behaviors on fungal species. In addition, field trials were conducted, and it was confirmed that released mites attach themselves to Ambrosia beetles and establish themselves within their nests. Additional studies are needed to optimize the production of mites and their ability to deliver beneficial microbes to the nest of pest insect.
Tar spot is a major fungal disease of corn that is prevalent in the United States Midwest region. Tar spot disease 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 is only moderately effective at reducing the disease. As part of a continuing research initiative, ARS researchers have identified three bacterial strains that reduce tar spot disease, two of which are now in the patent process. The third bacterial strain reduces the amount of reproductive structure release after application to tar spots. The experiments utilized diseased freeze-dried corn leaves and intact corn plants infected with tar spot pathogen. An invention disclosure was filed in June for the use of the third bacterial strain as a plant protection product. This year we continued to analyze grass samples that were collected close to cornfields for the presence of tar spot pathogen. We found that reed canary grass, brome grass, Barnyard grass, sedge, tall fescue, and green bristle could serve as alternative hosts for tar spot pathogen.
Accomplishments
1. Controlling tar spot of corn by managing weedy grasses that serve as alternative hosts. Tar spot is an invasive fungal disease of corn responsible for billions in losses to U.S. farmers. Practical disease management practices are needed to help farmers control this growing threat to corn production. ARS researchers in Peoria, Illinois, discovered that the fungus that causes tar spot disease is also found in reed canary grass, brome grass, Barnyard grass, sedge, tall fescue, and green bristle grass close to corn fields. These results indicate that farmers in the U.S. corn belt can lower the incidence of tar spot disease by managing weedy grasses adjacent to their cornfields. Integrating this strategy with other management strategies will boost agricultural production allowing the U.S. to meet the domestic and global market for U.S. corn.
Review Publications
Yang, Y., Ma, W., Johnson, E.T., Xie, Y., Zhao, R. 2024. Antifungal effects of three natural branched medium-chain fatty acids and their potential as fumigants against Aspergillus flavus in stored peanut seeds. Food Control. https://doi.org/10.1016/j.foodcont.2024.110950.
Adorada, D.L., Dunlap, C.A., Ash, G.J. 2024. Scope, distribution, and cause of the peanut kernel shrivel (PKS) syndrome: An emerging threat to Australia’s Peanut Industry. Agronomy Journal. https://doi.org/10.3390/agronomy14071435.
Muturi, E.J., Dunlap, C.A., Perry, W.L., Rhykerd, R.L. 2024. Cover crop species influences soil fungal species richness and community structure. PLOS ONE. 19(9). Article e0308668. https://doi.org/10.1371/journal.pone.0308668.
Dowd, P.F., Johnson, E.T. 2024. Efficacy of the insect biocontrol fungus Beauveria bassiana towards the European corn borer feeding on treated maize leaf and stalk tissue is inbred dependent and associated with resistance to the maize pathogen Fusarium. International Journal of Plant Biology. https://doi.org/10.3390/ijpb15030049.