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ARS Home » Midwest Area » Peoria, Illinois » National Center for Agricultural Utilization Research » Crop Bioprotection Research » Research » Research Project #429739

Research Project: Development of Production and Formulation Technologies for Microbial Biopesticides in Conjunction with the Development of Attractants and Repellents for Invasive Insect Pests

Location: Crop Bioprotection Research

2018 Annual Report

Objective 1. Utilizing transcriptomics to link gene function to fungal morphogenesis, develop liquid culture methods for producing propagules of fungal biocontrol agents such as Metarhizium spp., Beauveria bassiana, and Mycoleptodiscus terrestris by optimizing nutritional and environmental conditions during fungal growth for optimal biocontrol efficacy and storage stability to control pests in agricultural, urban, and natural ecosystems. Objective 2. Develop novel fungal formulation technologies through the selection and application of innovative processes and ingredients that lead to improved storage stability, product delivery, field stability, and efficacy of fungal pathogens for biocontrol of insects [Beauveria bassiana, Metarhizium spp., and Isaria fumosorosea] and weeds [Mycoleptodiscus terrestris]. Subobjective 2A: Evaluate the compatibility of improved entomopathogen formulations with representative corn pest resistance mechanisms for control of sweet corn pests. Objective 3. Identify, chemically and behaviorally characterize, and deploy natural insect semiochemicals (pheromones/kairomones and plant volatiles) with microbial biocontrol agents for management of important agricultural insect pests such as brown marmorated stink bug and coffee berry borer.

Our approach to the development of production methods for fungal biocontrol agents will focus on the use of liquid fermentation techniques. We will identify defined or semi-defined media that support the growth of our fungal biocontrol agents. Propagule form yield, storage stability, and biocontrol efficacy are critical “fitness” factors that will be considered during medium optimization. Initially, we will focus on producing propagules of fungal biocontrol agents such as Metarhizium spp, Beauveria bassiana, and Mycoleptodiscus terrestris. We also will use transcriptomics to identify pathways that may impact these factors. We will evaluate the impact of culture harvest techniques, stabilization processes, and formulation ingredients on the physical characteristics, biological activity, storage stability, and field efficacy of selected biocontrol agents. Also, we will identify and chemically characterize insect attractants and deterrents and evaluate formulations for management of important agricultural insect pests such as the brown marmorated stink bug, coffee berry borer, polyphagous shot hole borer, and tea shot hole borer.

Progress Report
Significant progress was made in FY18 on Objectives 1, 2, and 2a related to the production and formulation of microbial agents for controlling insects. Under Objective 1, ARS scientists at Peoria, Illinois, continued to develop and optimize liquid culture production processes for beneficial fungi. Liquid culture techniques focused on production of two diverse fungal structures: spores (ideal for insect control as foliar spray applications) and microsclerotia (a specialized resting state of fungi that is ideal for use in granular formulations). Production and stabilization processes for microsclerotia of the insect-killing fungus Metarhizium were refined. Development of liquid culture fermentation methods of the insect-killing fungi Beauveria bassiana focused on the rapid production of high concentrations of a type of spore known as a “blastospore”. There is significant industry interest in this ARS patent-pending production process for blastospores for the control of whiteflies, aphids, and other important insect pests worldwide due to high yields coupled with excellent desiccation-tolerance, shelf-life, and biocontrol efficacy. Successful production using a variety of Beauveria strains demonstrated a general versatility of the fermentation media and process for blastospore production. Research expanded slightly to include fermentation production of beneficial bacteria with liquid culture processes. One goal is to optimize production of newly identified strains of beneficial bacteria known to control pests of honey bees. A second goal is to develop co-fermentation techniques to produce insecticidal fungus and bacteria strains simultaneously, based on previous research that demonstrated improved pest control when certain strains are applied together. Techniques are currently under evaluation for reduced production costs and for potential benefits to product storage and efficacy. Under Objective 2, ARS scientists at Peoria, Illinois, developed drying and formulation processes that optimized the stability and efficacy of blastospores of Beauveria. The blastospore formulations are excellent for spray applications on plant surfaces for control of whiteflies, aphids and thrips. Specifically, progress continues on developing dual microbe biopesticides that have demonstrated synergistic (better than expected) control efficacy. When applied to field grown plants, a mixed microbe formulation containing both Beauveria blastospores and the bacterium Bacillus thuringiensis (Bt) was shown to provide more effective pest control than either product alone. ARS scientists at Peoria, Illinois, also made substantial progress in developing shelf-stable, granular formulations of Metarhizium microsclerotia for use in controlling soil-dwelling insect pests including white grubs in turf. Research continues on the development of specific formulations of Metarhizium microsclerotia for application to control insect pests of trees (Asian longhorned beetle) and nuts (Walnut husk maggot). Granular formulations of Metarhizium microsclerotia are ideal for use in the soil environment or in above ground applications onto trees, and new powdered formulations that were developed offer expanded opportunities for novel applications for pest control. Dried granular formulations of Metarhizium microsclerotial and powdered formulations of Beauveria blastospores were developed and show excellent storage stability at room temperature. All studies demonstrate the importance of water activity and storage atmosphere in optimizing the shelf-life of fungal microsclerotia and blastospores. Formulation processes matched pilot plant production capabilities with the ability to supply kilogram quantities of product for field testing. Under Objective 2A, ARS scientists at Peoria, Illinois, found that corn with enhanced resistance to bacterial pathogens reduced the ability of a commercially formulated bacterial biopesticide Bacillus thuringiensis to control corn earworms and fall armyworms. In addition, corn cell lines with fungal plant pathogen resistance genes were found to inhibit the growth of commercial preparations of the fungal insect pathogen Beauveria bassiana in several cases. By contrast, insects fed leaves and stalks from sorghum lines that overproduced a regulatory gene for increased resistance to sorghum pathogens were killed more quickly by applications of commercial preparations of the bacterial insect pathogen Bacillus thuringiensis and the fungal pathogen Beauveria bassiana. This information defines interactions between host plant resistance and biopesticides used for control of plant diseases and insect pests and, in turn, improves integrated pest management strategies that focus on controlling insect pests of corn.

1. Improved low cost liquid culture fermentation. High production costs continue to limit commercialization of microbial-based biological insecticides. ARS scientists in Peoria, Illinois characterized the impact of lower cost nitrogen sources on the yield and storage stability of the commercially important biopesticide, Beauveria bassiana. The study identified an inexpensive nitrogen source that improved the yield and storage stability of this biopesticide. The nitrogen component is the most expensive nutrient in liquid media composition, but this study showed that low-cost nitrogen compounds composed mainly of agro-industrial by-products can be used for liquid culture production of beneficial microbes for biological insecticides.

2. Discovery of corn genes that improve plant resistance to insects and fungi. Insects and disease greatly reduce corn yields and grain quality. Corn ear molds can produce toxins harmful to people and animals, causing hundreds of millions of dollars in losses in the U.S. ARS scientists in Peoria, Illinois identified a corn-gene that produces a protein that inhibits insect growth and enhances the effectiveness of a commercial fungicide. In addition, the researchers found a second gene that produces a protein that reduces rates of corn ear rot fungal infection and also reduces growth rates of insects that damage corn ears. Identifying and using beneficial genes for plant resistance is an economical means for growers to reduce corn ear damage caused by insects and ear rots.

Review Publications
Dowd, P.F., Zilkowski, B.W., Johnson, E.T., Berhow, M.A., Muturi, E.J. 2018. Transgenic expression of a maize geranyl geranyl transferase gene sequence in maize callus increases resistance to ear rot pathogens. AGRI GENE. 7:52-58.
Dowd, P.F., Berhow, M.A., Johnson, E.T. 2018. Enhanced pest resistance and increased phenolic production in maize callus transgenically expressing a maize chalcone isomerase -3 like gene. Plant Gene. 13:50-55.
Dowd, P.F., Johnson, E.T. 2017. Insect damage influences heat and water stress resistance gene expression in field grown popcorn: Implications in developing crop varieties adapted to climate change. Mitigation and Adaptation Strategies for Global Change. 23(7):1063-1081.
Dowd, P.F., Naumann, T.A., Price, N.P., Johnson, E.T. 2017. Identification of a maize (Zea mays) chitinase allele sequence suitable for a role in ear rot fungal resistance. AGRI GENE. 7:15-22.
Cao, W., Guo, L., Du, Z., Das, A., Saren, G., Jiang, M., Dunlap, C.A., Rooney, A.P., Yu, X., Li, T. 2017. Chengkuizengella sediminis gen. nov. sp. nov., isolated from sediment. International Journal of Systematic and Evolutionary Microbiology. 67:2672–2678. doi: 10.1099/ijsem.0.002006.
Cote, G.L., Dunlap, C.A., Vermillion, K.E., & Skory, C.D. 2017. Production of isomelezitose from sucrose by engineered glucansucrases. Amylase. 1(1):82-93. doi: 10.1515/amylase-2017-0008.
Dunlap, C.A., Lueschow, S.R., Carillo, D., Rooney, A.P. 2017. Screening of bacteria for antagonistic activity against phytopathogens of avocados. Plant Gene. 11:17-22.
Dunlap, C.A., Mascarin, G.M., Romagnoli, E.M., Jackson, M.A. 2017. Rapid discrimination of Isaria javanica and Isaria poprawskii from Isaria spp. using high resolution DNA melting assays. Journal of Invertebrate Pathology. 150:88–93.
Dunlap, C.A., Ramirez, J.L., Mascarin, G.M., Labeda, D.P. 2018. Entomopathogen ID: a curated sequence resource for entomopathogenic fungi. Mycologia. 111:897-904. doi:10.1007/s10482-017-0988-2.
Dunlap, C.A., Schisler, D.A., Perry, E.B., Connor, N., Cohan, F.M., Rooney, A.P. 2017. Bacillus swezeyi sp. nov. and Bacillus haynesii sp. nov., isolated from desert soil. International Journal of Systematic and Evolutionary Microbiology. 67:2720-2725. doi: 10.1099/ijsem.0.002007.
Johnson, E.T., Proctor, R., Dunlap, C.A., Busman, M. 2017. Reducing production of fumonisin mycotoxins in Fusarium verticillioides by RNA interference. Mycotoxin Research. 34:29-37.
Lu, D., Xia, J., Dunlap, C.A., Rooney, A.P., Du, Z. 2017. Salibacter halophilus gen. nov., sp. nov., isolated from a saltern. International Journal of Systematic and Evolutionary Microbiology. 67:1784–1788. doi: 10.1099/ijsem.0.001807.
Lu, D., Xia, J., Dunlap, C.A., Rooney, A.P., Du, Z. 2017. Gracilimonas halophila sp. nov., isolated from a marine solar saltern. International Journal of Systematic and Evolutionary Microbiology. 67:3251-3255. doi: 10.1099/ijsem.0.002093.
Torres-Crus, T.J., Billingsley Tobias, T.L., Almatruk, M., Hesse, C.N., Kuske, C.R., Desiro, A., Benucci, G., Bonito, G., Stajich, J.E., Dunlap, C.A., Arnold, A., Porras-Alfaro, A. 2017. Bifiguratus adelaidae, gen. et sp. nov., a new member of Mucoromycotina in endophytic and soil-dwelling habitats. Mycologia. 109(3):363-378.
Xia, J., Xie, Z., Dunlap, C.A., Rooney, A.P., Du, Z. 2017. Rhodohalobacter halophila gen. nov., sp. nov., a moderately halophilic member of the family Balneolaceae. International Journal of Systematic and Evolutionary Microbiology. 67:1281–1287. doi: 10.1099/ijsem.0.001806.
Xu, Z., Zhang, H., Han, J., Dunlap, C.A., Rooney, A.P., Mu, D., Du, Z. 2017. Colwellia agarivorans sp. nov., an agar-digesting marine bacterium isolated from coastal seawater. International Journal of Systematic and Evolutionary Microbiology. 67(6):1969-1974. doi: 10.1099/ijsem.0.001897.
Behle, R.W. 2017. In vivo production of Agrotis ipsilon nucleopolyhedrovirus for quantity and quality. Journal of Economic Entomology. 111(1):101-107. doi: 10.1093/jee/tox315.
Mushtaq, R., Behle, R.W., Liu, R., Niu, L., Song, P., Shakoori, A.R., Jurat-Fuentes, J. 2017. Activity of Bacillus thuringiensis Cry1Ie2, Cry2Ac7, and Cry7Ab3 proteins against Anticarsia gemmatalis, Chrysodeixis includens and Ceratoma trifurcata. Journal of Invertebrate Pathology. 150(2017):70-72.
Sayed, A.M., Behle, R.W. 2017. Comparing formulations for a mixed-microbial biopesticide with Bacillus thuringiensis var. kurstaki and Beauveria bassiana blastospores. Archives of Phytopathology and Plant Protection. 50:15-16, 745-760.
Sayed, A.M., Kim, S., Behle, R.W. 2017. Characterization of silver nanoparticles synthesized by Bacillus thuringiensis as a nanobiopesticide for insect pest control. Biocontrol Science and Technology. 27(11):1308-1326.
Sayed, A.M., Behle, R.W., Tiilikkala, K., Vaughn, S.F. 2018. Insecticidal activity of bio-oils and biochar as pyrolysis products and their combination with microbial agents against Agrotis ipsilon (Lepidoptera: Noctuidae). Pesticides and Phytomedicine. 33:39-52.
Mascarin, G.M., Kobori, N.N., Jackson, M.A., Dunlap, C.A., Delalibera, I. 2018. Nitrogen source affects productivity, desiccation tolerance, and storage stability of Beauveria bassiana blastospores. Journal of Applied Microbiology. 124:810-820. doi: 10.1111/jam.13694.
Dunlap, C.A., Rooney, A.P. 2018. Acinetobacter dijkshoorniae is a later heterotypic synonym of Acinetobacter lactucae. International Journal of Systematic and Evolutionary Microbiology. 68:131-132. doi:10.1099/ijsem.0.002470.
Santos, V., Mascarin, G.M., Da Silva Lopes, M., Fregolente Alves, M.C., Rezende, J.M., Viccari Gatti, M.S., Dunlap, C.A., Delaliber Junior, I. 2017. Identification of double-stranded RNA viruses in Brazilian strains of Metarhizium anisopliae and their effects on fungal biology and virulence. Plant Gene. 11:49-58.