Location: Crop Bioprotection Research2020 Annual Report
1. Demonstrate production potential for baculovirus for insect control (such as black cutworm MNPV), evaluate formulations for storage stability and residual efficacy, and identify and evaluate insect semiochemicals such as attractants or feeding stimulants that can be integrated into formulations to improve control of major insect pests of turf or other crops. 2. Determine the relationship between microbial communities and the characteristics of weeds (such as bindweed, medusahead grass, or quack grass) that make them harmful to turf, natural ecosystems, and agricultural commodities. 3. Identify viruses that can target potential key endophytes or microorganisms that contribute negative characteristics of weeds. 4. Identify, describe, and preserve microorganisms isolated from weeds as part of the characterization of microbial communities associated with important weeds.
Grasses planted as turf and pasture represent a commodity that has been underserved when considering the use of biological control based on microbial agents. For urban and athletic turf grasses, a newly discovered baculovirus offers the opportunity to develop a biological pesticide for control of the black cutworm. Research will focus on basic and applied aspects of production, formulation, and efficacy of this baculovirus for development as a biological insecticide. Invasive weed species among range grasses such as Medusahead may obtain enhanced fitness as a result of associations with endophytic microbes. Research will utilize classic microbial and newly developed molecular techniques to characterize endophytic microbes of the weedy plants and identify those providing competitive advantages to the weeds. Subsequent research will strive to discover mycoviruses to attach the endophytes of the weedy plant, to convert the competitive advantage back to the desired crop plant.
This is the final report for Project 5010-22000-011-00D. Significant progress has been made in meeting the project objectives through the final year of the project. For Objective 1, the goal was to develop baculoviruses for insect control in turf and other major crops. In year five of the project, we addressed the milestone to promote technology transfer of research to potential commercial partners by engaging in communication with industry representatives to discuss the benefits of applying ARS baculovirus biopesticide production and formulation technologies to commercial baculovirus biopesticides. Research throughout this project on AgipMNPV, the baculovirus that specifically infects the black cutworm, demonstrated selection of the artificial diet, age (size) of the exposed larvae, exposure dosage, and incubation temperature are important factors in vivo production of virus particles (occlusion bodies). When evaluating in vivo virus products, both the number of occlusion bodies and the insecticidal activity of those occlusion bodies needed to be evaluated to ensure the quality of the active agent. Further, we demonstrated higher temperature, and moisture levels, limited the survival of microbial propagules, and shortened the shelf-life of prototype biopesticide formulations. Yet, properly prepared dry formulations did not lose insecticidal activity when stored for one year at room temperature. Under laboratory and field conditions in collaboration with Purdue University, AgipMNPV treatments effectively controlled cutworm larvae when applied alone, but showed no synergistic activity when applied in combination with other biological control agents. For Objective 2, the goal was to determine the relationship between microbial communities and the characteristics of invasive weeds. In the final year of the project, microbial communities of various plant parts from different locations were selected for sampling of bindweed. Over the course of the five year project, microbial communities were selected in the invasive range of the United States and from their native ranges in Europe for bindweed, medusahead grass, and quack grass. Direct comparisons of the endophytic communities of each plant species between the locations were more difficult than anticipated, due to the strong effect of life stage on the microbiota. The microbiota of plants changes rapidly during the late spring and early summer. To overcome some of these limitations, we focused on the microbiota vertically transmitted by seeds. These results showed significant differences between the locations. It also identified the U.S. seeds of medusahead grass contained the entomopathogen Beauveria bassiana. Additional research is needed to identify how these seed transmitted endophytes effect the insect resistance of the plants. For Objective 3, the milestone was to identify viruses that can target key endophytes contributing to the negative characteristics of weeds. The original goal was to identify endophytes that may provide weeds with a competitive advantage and target those microbes with viruses. We were unsuccessful in identifying microbes that provided the weeds with a competitive advantage. We were able to identify isolates containing mycoviruses, but experiments to generate mycovirus free isolates were unsuccessful. For Objective 4, the goal was to create a microbial collection of endophytic fungi isolated from bindweed, medusahead grass, and quack grass weed species. We created a collection of more than 350 fungal isolates that may provide beneficial traits to agriculturally important plants. These strains were characterized with DNA sequencing to identify them to the species level using two different genes. The strains collected over the course of this project have been prepared for long-term preservation and storage, which will allow them to be evaluated for potential commercial properties at a later date. This work has identified several new species of microbes previously unknown to science, which are currently being evaluated for their impact on agronomic traits. In addition, the unique nature of this collection of microbes, isolated from weeds, has attracted commercial entities interested in exploring these microbes for novel traits. For the next project cycle, this project will be merged with related project 5010-22410-017-00D, "Development of Production and Formulation Technologies for Microbial Biopesticides in Conjunction with the Development of Attractants and Repellents for Invasive Insect Pests." Isolates obtained from this study will be included in a large genome sequencing proposal in the next project cycle.
1. Identifying plant compounds that synergize a baculovirus for better insect control. Beneficial baculoviruses are highly specific microorganisms able to infect and kill targeted pests in a field environment. Yet, widespread commercialization of virus based biological insecticides lags because of high production costs. When comparing baculovirus applications to several crop plants, ARS scientists in Peoria, Illinois, observed that applications to soybeans were more effective than the same applications to green bean, cabbage, or cotton. Follow-up analysis identified three compounds that were uniquely found in only the soybean leaves and laboratory experiments using artificial insect diet confirmed the synergy of these compounds with the virus, resulting in higher toxicity to the pest caterpillars. Successful incorporation of natural plant-based synergistic compounds provides the opportunity to reduce application rates needed for pest control, which will effectively lower treatment costs to the grower.
Hay, W.T., Behle, R.W., Berhow, M.A., Miller, A.C., Selling, G.W. 2020. Biopesticide synergy when combining plant flavonoids and entomopathogenic baculovirus. Nature. 10:6806. https://doi.org/10.1038/s41598-020-63746-6.
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. https://doi.org/10.3389/fmicb.2019.01794.
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.
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. https://doi.org/10.1007/s10482-020-01404-7.
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. https://doi.org/10.1007/s10482-019-01261-z.
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. https://doi.org/10.1016/j.micres.2019.126331.
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. https://doi.org/10.1007/s10482-019-01248-w.
Hay, W.T., Behle, R.W., Ruiz-Vera, U.M., Fanta, G.F., Selling, G.W. 2020. Use of novel film forming starch complexes to directly and indirectly reduce insect damage to plants. Crop Protection. 130:105048. https://doi.org/10.1016/j.cropro.2019.105048.
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. https://doi.org/10.1099/ijsem.0.003577.
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. https://doi.org/10.1007/s10482-019-01354-9.
Sayed, A.M.M., Dunlap, C.A. 2019. Virulence of some entomopathogenic fungi isolates of Beauveria bassiana (Hypocreales:Cordycipitaceae) and Metarhizium anisopliae (Hypocreales:Clavicipitaceae) to Aulacaspis tubercularis (Hemiptera:Diaspididae) and Icerya seychellarum (Hemiptera:Monophlebidae) on mango crop. Journal of Economic Entomology. 112(6):2584-2596. https://doi.org/10.1093/jee/toz187.
Buhl, M., Dunlap, C., Marschal, M. 2019. Prevotella brunnea sp. nov., isolated from a wound of a patient. International Journal of Systematic and Evolutionary Microbiology. 69(12):3933-3938. https://doi.org/10.1099/ijsem.0.003715.
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. https://doi.org/10.1111/mpp.12918.