Location: Crop Bioprotection Research2017 Annual Report
1a. Objectives (from AD-416):
Objective 1: Optimize fermentation and formulation protocols for selected biocontrol strains to produce products with enhanced field efficacy, consistency, and compatibility with integrated pest management methodologies. Objective 2: Elucidate selected interactions among host, biocontrol agent, and pathogen on plant surfaces using physiochemical, proteomic, and/or genomic characterization of the host or agent to guide development of effective agent production and formulation technologies. Objective 3: Develop methods and technologies to isolate and identify fastidious microorganisms that threaten U.S. agriculture and determine characteristics that contribute to pathogenicity. The encompassing goal of this project is to improve biocontrol agent product performance by both optimizing agent fermentation, formulation, and deployment protocols; and by elucidating agent, pathogen, and host interactions on plant surfaces in order to optimize antagonist colonization of host surfaces, efficacy in reducing disease and utility in integrated pest management environments. Results from research conducted in each of these key areas will be of value in meeting each individual objective as well as providing data that reciprocally provides leads and concepts that enhance the advancement of the science conducted under the other individual objective. Our Agricultural Research Service (ARS) research team possesses a unique blend of expertise and interactive research experience in plant pathology, biochemistry, and molecular biology to draw on in completing this research. Additionally, collaborators within ARS, at Universities, and within the agricultural business community have committed to providing critical skill-sets that will be instrumental in meeting our overall research goal.
1b. Approach (from AD-416):
Objective 1: Optimize fermentation and formulation protocols for selected biocontrol strains to produce products with enhanced field efficacy, consistency, and compatibility with integrated pest management methodologies. Objective 2: Elucidate selected interactions among host, biocontrol agent, and pathogen on plant surfaces using physiochemical, proteomic, and/or genomic characterization of the host or agent to guide development of effective agent production and formulation technologies. Objective 3: Develop methods and technologies to isolate and identify fastidious microorganisms that threaten U.S. agriculture and determine characteristics that contribute to pathogenicity.
3. Progress Report:
This is the final report for project 5010-22410-015-00D that terminated on May 8, 2017. For the remainder of FY 17, research commenced on newly approved research project 5010-22410-019-00D. Highlights of five years of research progress include important discoveries in each of the three research objectives of the project. Objective 1 was to optimize fermentation and formulation protocols for selected biocontrol strains to produce effective products compatible with integrated pest management methodologies. In research aligned with this objective, ARS researchers at Peoria, Illinois, worked independently and with university and industry collaborators in advancing the development of microbial biocontrol products active against Fusarium head blight (FHB) of wheat and post-harvest maladies of potato tubers in storage. In research with scientists at a university, a yeast antagonist (Cryptococcus flavescens strain 3C) of FHB disease reduced disease and the level of mycotoxin produced by the pathogen in multi-year greenhouse and field experiments conducted in Illinois and Ohio. Reductions were obtained with the yeast alone or when integrated with fungicides demonstrating the feasibility of reducing disease by incorporating biocontrol with reduced levels of more traditional chemical control measures. Multiple studies were conducted at ARS in Peoria, Illinois, on developing large-scale production methodologies in liquid culture and stabilization methods for frozen yeast biomass concentrates. New candidate biocontrol strains of the yeast were identified from multiple fields in Eastern and Midwestern states by using newly developed genetic techniques. Growing two strains of a variety of isolates of C. flavescens together (co-cultures) resulted in products that were occasionally more effective than component strains tested individually in greenhouse trials. Collectively, these results demonstrated the likelihood of successfully employing a commercial product composed of C. flavescens cells in an integrated control program with fungicides to control FHB. In work towards developing biocontrol products against maladies of potatoes in storage, improvements in the efficacy and viability of Gram negative bacterial biocontrol agents after drying and rehydration were achieved during investigations conducted under a Non Assistance Cooperative Agreement with researchers at a university in Idaho. Multiple carriers, compounds that help cells survive drying (osmoprotectants) and combinations thereof useful in preserving biocontrol agent cell viability and efficacy in reducing dry rot and Pythium leak on stored potato tubers were discovered. Peoria-based ARS scientists made important advances in preserving the viability and efficacy of Gram negative bacterial biocontrol agents. A dried product reduced post-harvest diseases of potatoes in small-pilot scale tests conducted in collaboration with a university in Idaho. These results enhance the likelihood of commercially developing effective Gram negative bacterial biocontrol agents into plant disease control products that benefit producers and consumers of agricultural crops. And in studies with a visiting Pakistani Ph.D. student, which were conducted under a Trust Fund Cooperative Agreement, ARS scientists demonstrated that hot water extraction of several plant products can provide essential nutrients and minerals for an inexpensive growth medium that produces effective biocontrol inoculum. Research progress in support of Objective 2, the characterization of interactions among host, biocontrol agent, and pathogen on host surfaces, was also significant. ARS researchers in Peoria, Illinois and scientists at a university used a deoxyribonucleic acid (DNA)-based assay they previously developed for monitoring populations of yeast antagonist Cryptococcus flavescens OH 182.9 in situ to characterize the population dynamics and spread of the yeast antagonist on field-grown wheat over time. This breakthrough provided a new tool for understanding the environmental fate of this biocontrol agent and how its population on wheat relates to biocontrol effectiveness. A new DNA-based method for rapidly classifying strains of C. flavescens for their relative potential as FHB biocontrol agents without having to conduct labor and time intensive greenhouse bioassays on wheat plants was developed and patent protection applied for. ARS researchers in Peoria, Illinois, determined what changes in gene regulation are associated with culturing FHB antagonist C. flavescens under cold adaptation conditions that enhance the stress tolerance and efficacy of cells. ARS researchers also discovered that the genome of a bacterial FHB biocontrol agent resolved to a single chromosome and then functionally annotated the genome to describe the genes identified. In other studies, ARS researchers characterized metabolite production by this organism when co-cultured with a yeast antagonist to determine if co-culturing has the potential to be managed to improve the quantity and quality of beneficial metabolites a biocontrol agent produces. Results from all of these studies will be used to optimize the production of effective biocontrol inoculum. Because surface properties play an important role in plant-microbe interactions, ARS researchers characterized the surface properties of wheat (Triticum aestivum L.) spikelet tissues under different environmental growing regimes and of FHB biocontrol yeast C. flavescens OH 182.9. Results from this work suggest that changing surface properties during crop development should be considered in spray formulations of agents to enhance colonization success. Assays were completed with wheat and yeast OH 182.9 to determine if the yeast altered wheat defense responses normally associated with infection by plant pathogens and results indicated the biocontrol organism did not modify the defense response of wheat. This suggests that the primary mode of action of the biocontrol organism is to compete for resources with the pathogen on the plant surface and limit its ability to become established. ARS researchers in Peoria, Illinois, sequenced more than twenty genomes of agriculturally important strains. This research identified gene clusters of novel, putatively bioactive metabolites including that of an ARS-discovered FHB bacterial antagonist. In additional studies, ARS researchers collaborated with university scientists in Argentina on characterizing the genomes of Bacillus species with biocontrol potential. This work also enabled Peoria researchers to significantly improve Bacillus taxonomy of this important group of agricultural and biocontrol species and settled what had been widespread confusion in interpreting previous investigations on Bacillus. A visiting scientist from Korea worked with ARS researchers to characterize the diversity of Bacillus strains that ferment soybeans. Studies continued on developing biocontrol agents that target the fungal pathogens that cause laurel wilt on avocado in Florida and Fusarium dieback on avocado in California with the discovery of bacterial strains that were effective against both pathogens in vitro. Peoria, Illinois researchers also characterized the phytobiome of raspberry plants under differing soil fertility regimes. Research objective 3, to develop new methods and technologies for isolating and identifying fastidious and/or uncultivable plant pathogens, was added midway into this five year project. The causal agents of downy mildews were chosen as the pathogens to target due to their inability to be cultured on artificial media and their economic importance worldwide. To date, studies have focused primarily on Peronospora belbahrii the causal agent of a devastating downy mildew disease on basil. ARS researchers in Peoria, Illinois, used light and scanning electron microscopy to determine that sporangia, infection causing spores of the pathogen, germinate as early as three days after host inoculation under conditions of high relative humidity. Infection resulted from direct penetration of basil leaves rather than through natural openings. Spore bearing structures emerged from stomata on both the upper and lower surfaces of leaves. Researchers then developed a first generation tissue culture medium for producing callus of basil, a medium for growing microbe-free basil seedlings, and a method of producing microbe-free sporangia of the pathogen. Studies to produce basil and downy mildew binary systems in otherwise sterile environments can now be initiated which should enable genomic characterization of the pathogen and, ultimately, the discovery of improved methods for controlling this and other downy mildew pathogens. The impact of research completed under this research project is also evident in the publishing of 32 manuscripts in peer reviewed journals and the issuance of 12 national and international patents and 16 pending national and international patents. Formal collaborations with other scientists and agricultural companies took the form of 1 Non-Assistance Cooperative Agreement (with a university); 2 Specific Cooperative Agreements (with 2 universities), 2 Material Transfer Agreements (agricultural company and a university), 1 Trust Fund Cooperative Agreement, and 1 CRADA (agricultural company). International collaborations also were established or maintained with researchers in Argentina, Turkey, France, Australia, Korea, and Pakistan.
1. Enhanced control of Fusarium head blight (FHB) of wheat using tank mixes of a yeast biocontrol agent and fungicides. Obtaining acceptable levels of reduction of FHB disease of wheat remains elusive despite advances in slowing disease using individual disease control measures that include pesticides, biological control, cultural control, disease forecasting, and resistant varieties. ARS researchers at Peoria, Illinois, in collaboration with scientists at a university, tested a fungicide tolerant variant of their patented yeast biocontrol strain Cryptococcus flavescens OH 182.9 in combination with a variety of fungicides approved for use against FHB and discovered a combination that reduced FHB symptoms in greenhouse and field studies to a greater extent than the control or the fungicide alone. Tank mixing the yeast antagonist with fungicide did not reduce the viability of antagonist cells in most cases. Collectively, these results demonstrate the possibility of reducing the impact of FHB on U.S. wheat growers by employing a yeast antagonist that has potential for commercial development in an integrated pest management program that includes reduced levels of fungicides.
2. Commercialized strains of a biocontrol bacterium differ in patterns of antimicrobial compounds they potentially produce. Bacillus velezensis is one of the most important groups of bacterial biocontrol species with activity against plant pathogens in the world yet little is known regarding how strains differ in their potential to produce antimicrobial compounds. ARS-Peoria, Illinois scientists in collaboration with scientists from Argentina, sequenced the genome of a strain of this bacterium that can limit Fusarium head blight (FHB) disease in wheat. The scientists discovered the strain contained gene clusters that code for novel potentially bioactive metabolites and that the strain produced an uncommon secondary metabolite. Using comparative genomics, scientists discovered that this strain and commercialized Bacillus velezensis strains differed in important classes of secondary metabolites they could produce. By characterizing how these metabolites impact strain field performance and overall efficacy, scientists should be able to rapidly select strains with enhance potential for reducing plant diseases for the producers of agricultural products.
3. Biocontrol potential of new bacterial agents active against avocado disease characterized. ARS-Peoria, Illinois scientists completed additional characterization of five bacterial strains that target the fungal pathogens that cause laurel wilt on avocado in Florida and Fusarium dieback on avocado in California. Genome sequencing allowed the taxonomy of the strains to be definitively determined and demonstrated that two of the strains were species that have not been previously identified as potential biocontrol agents. The study was the first to report the effectiveness of Paenibacillus thiaminolyticus and Paenibacillus apiarius against a phytopathogen. Genome mining also identified several secondary metabolites that are not known in the scientific literature. Upon the determination of the secondary metabolites these novel strains produce and of the mode of action these strains use to inhibit plant pathogens, a biocontrol product to aid U.S. producers of avocados can be realized.
Dunlap, C.A., Bowman, M.J., Schisler, D.A., Rooney, A.P. 2016. Genome analysis shows Bacillus axarquiensis is not a later heterotypic synonym of Bacillus mojavensis; Reclassification of Bacillus malacitensis and Brevibacterium halotolerans as heterotypic synonyms of Bacillus axarquiensis. International Journal of Systematic and Evolutionary Microbiology. 66:2438-2443. doi: 10.1099/ijsem.0.001048.
Dunlap, C.A., Saunders, L.P., Schisler, D.A., Leathers, T.D., Naeem, N., Cohan, F.M., Rooney, A.P. 2016. Bacillus nakamurai sp. nov., a black pigment producing strain. International Journal of Systematic and Evolutionary Microbiology. 66(8):2987-2991. doi: 10.1099/ijsem.0.001135.
Ling-Yun, G., Dunlap, C.A., Weiler, L., Rooney, A.P., Guan-Jun, C., Zong-Jun, D. 2016. Wenzhouxiangella sediminis sp. nov. isolated from coastal sediment. International Journal of Systematic and Evolutionary Microbiology. 66:4575-4579. doi: 10.1099/ijsem.0.001393.
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Muturi, E.J., Ramirez, J.L., Rooney, A.P., Dunlap, C.A. 2016. Association between fertilizer-mediated changes in microbial communities and Aedes albopictus growth and survival. Acta Tropica. 164(2016):54-63.
Palazzini, J.M., Dunlap, C.A., Bowman, M.J., Chulze, S.N. 2016. Bacillus velezensis RC 218 as a biocontrol agent to reduce Fusarium head blight and deoxynivalenol accumulation: Genome sequencing and secondary metabolite cluster profiles. Microbiological Research. 192:30-36. https://doi.org/10.1016/j.micres.2016.06.002.
Rooney, A.P., Dunlap, C.A., Weiler, L. 2016. Acinetobacter lactucae sp. nov., isolated from iceberg lettuce (Asteraceae: Lactuca sativa). International Journal of Systematic and Evolutionary Microbiology. 66:3566-3572. doi:10.1099/ijsem.0.001234.
Jin, S., Jun, X., Dunlap, C.A., Rooney, A.P., Zong-Jun, D. 2016. Psychroflexus saliphilus sp. nov., isolated from a marine solar saltern. International Journal of Systematic and Evolutionary Microbiology. 66:5124-5128. doi: 10.1099/ijsem.0.001482.
Xia, J., Dunlap, C.A., Weiler, L., Rooney, A.P., Chen, G., Du, Z. 2016. Longibacter salinarum gen. nov., sp. nov., isolated from a marine solar saltern. International Journal of Systematic and Evolutionary Microbiology. 66:3287-3292. doi:10.1099/ijsem.0.001190.
Labeda, D.P., Dunlap, C.A., Rong, X., Huang, Y., Doroghazi, J.R., Ju, K.-S., Metcalf, W.W. 2016. Phylogenetic relationships in the family Streptomycetaceae using multi-locus sequence analysis. Antonie Van Leeuwenhoek. 110(4):1-21.
Mascarin, G.M., Dunlap, C.A., Barrigossi, J.D., Quintela, E.D., De Noronha, N.C. 2016. First record of epizootics in the ocola skipper, Panoquina sp. (Lepidopera: Hesperiidae), caused by Isaria tenuipes in flooded rice fields of Central Brazil. Journal of Applied Microbiology. 122:1020-1028. doi: 10.1111/jam.13390.