Use of Microorganisms to Manage Weeds and Insect Pests in Turf and Agricultural Systems

Goals / Objectives
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.

Project Methods
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.

Progress 09/14/15 to 09/13/20

Outputs
Progress Report Objectives (from AD-416): 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. Approach (from AD-416): 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. Accomplishments 01 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.

Impacts
(N/A)

Publications

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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.

Progress 10/01/18 to 09/30/19

Outputs
Progress Report Objectives (from AD-416): 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. Approach (from AD-416): 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. Significant progress has been made in meeting the project objectives through four years of research. For Objective 1, the goal was to develop baculoviruses for insect control in turf and other major crops. In year four of the project, the baculovirus that specifically infects the black cutworm (AgipMNPV) was shown to provide effective control of this pest of bentgrass turf. Field experiments demonstrated that virus treatments were as effective as a standard chemical insecticide for controlling small and medium-sized larvae, resulting in obviously less insect feeding damage to golf course greens managed turfgrass. The baculovirus treatment was more effective when compared with other biological microbes (fungus and bacteria) applied alone or in combinations with the baculovirus demonstrating that the baculovirus can serve as a stand-alone biological replacement for chemical insecticides for control of the black cutworm in turf. Additional efforts to improve the insecticidal activity of the virus were unsuccessful in that plant-based compounds shown to improve the activity of other baculoviruses provided no beneficial effect for this virus. For Objective 2, the goal was to determine the relationship between microbial communities and the characteristics of invasive weeds. In year four of the project, microbial communities of various plant parts were determined for samples of bindweed, medusahead grass, and quack grass. The research shows the microbial communities vary significantly across the samples, with the time of season providing the biggest effect. The research has highlighted the difficulty of conducting comparisons between locations when the locations and the ability to sample the locations occur at different times. This knowledge provides valuable information on how to design future experiments on microbial communities in plants. For Objective 3, the milestone was to identify viruses that can target key endophytes that contribute to the negative characteristics of weeds. This milestone was modified due to the inability to identify key endophytes that meet these requirements. The milestone was broadened to include characterizing microbes isolated from these weeds species to include microbes that may also provide beneficial traits to agriculturally important plants. This work has identified several new species 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 interest from commercial entities interested in exploring these microbes for novel traits. Accomplishments 01 Identifying microbes transmitted in the seeds of invasive weeds. The goal of this research was to identify novel methods of controlling the invasive weed, medusahead. Medusahead is an important weed in the western U.S. and represents a serious threat to ranchers, since it can greatly reduce the grazing capacity of pastures. The current research provides the first characterization of the microbes the weeds pass to other generations through their seeds. Microbes are known to provide plants with beneficial properties that help their survival in stressful environments and may help weeds outcompete beneficial plants. In this research, ARS researchers at Peoria, Illinois, compared microbes from the seeds of plants in their native range (Europe) with seeds of plants in the U.S. and identified significant differences between the plants. The research shows the seeds of introduced plants contain several families of fungi not seen in the European strains. This research allows us to understand how these microbes are vertically transmitted by the weeds and which of these microbes may provide the weed with a competitive advantage. This research provides knowledge that may be useful in developing novel strategies to control invasive weeds by targeting the microbes that provide beneficial traits to weeds, which would directly benefit ranchers and nature ecosystems impacted by these weeds.

Impacts
(N/A)

Publications

Knight, C., Bowman, M.J., Frederick, L., Day, A., Lee, C., Dunlap, C.A. 2018. The first report of antifungal lipopeptide production by a Bacillus subtilis subsp inaquosorum strain. Microbiological Research. 216:40-46.
Johnson, E.T., Dunlap, C.A. 2019. Phylogenomic analysis of the Brevibacillus brevis clade: a proposal for three new Brevibacillus species, Brevibacillus fortis sp. nov., Brevibacillus porteri sp. nov. and Brevibacillus schisleri sp. nov. Antonie Van Leeuwenhoek.
Mascarin, G.M., Junior, R.P., Fernandes, E.K., Quintela, E.D., Dunlap, C.A. , Arthurs, S.P. 2018. Phenotype responses to abiotic stresses, asexual reproduction and virulence against whiteflies among strains of the entomopathogenic fungus Cordyceps javanica (Hypocreales: Cordycipitaceae). Microbiological Research. 216:12-22.
Dunlap, C.A. 2019. Taxonomy of registered Bacillus spp. strains used as plant pathogen antagonists. Biological Control. 134:82-86.
Martins, S.J., Rocha, G.A., De Melo, H.C., Georg, R.D., Ulhoa, C.J., Dianese, E.C., Oshiquiri, L.H., Da Cunha, M.G., Da Rocha, M.R., De Araujo, L.G., Vaz, K.S., Dunlap, C.A. 2019. Plant-beneficial bacteria mitigate drought stress in soybean. Plant Physiology and Biochemistry. 25:13676⿿13686.
Wang, X., Li, C., Dunlap, C.A., Rooney, A.P., Du, Z. 2018. Marinicella sediminis sp. nov., isolated from marine sediment. International Journal of Systematic and Evolutionary Microbiology. 68:2335-2339.
Schisler, D.A., Yoshioka, M., Vaughan, M.M., Dunlap, C.A., Rooney, A.P. 2018. Nonviable biomass of biocontrol agent Papiliotrema flavescens OH 182. 9 3C enhances growth of Fusarium graminearum and counteracts viable biomass reduction of Fusarium head blight. Biological Control. 128:48-55.

Progress 10/01/17 to 09/30/18

Outputs
Progress Report Objectives (from AD-416): 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. Approach (from AD-416): 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. Substantial progress has been made in the third year of this research project. For Objective 1, In vivo production of the baculovirus of the black cutworm remains the most plausible approach for commercialization of a biopesticide product. Optimization of the incubation temperature increased production of wild-type virus about three fold. Laboratory evaluations demonstrated that newly hatched larvae remain highly susceptible to virus infection for about 5 days. When applied to field grown grass, the virus applications had good residual activity for several days, which (when combined with larval susceptibility) suggest that once weekly applications may provide control of this pest in the field. ARS scientists in Peoria, Illinois made significant progress in Objective 2. ARS scientists in Peoria, Illinois extracted DNA from the invasive weed, medusahead (Taeniatherum caput-medusae) to determine what microbes reside inside the plant. The plants were sampled from their invaded range in the western United States and from their native range in Europe. These DNA samples were used to determine the microbial communities living inside the plant, based on high throughput DNA sequencing techniques. ARS scientists in Peoria, Illinois updated the collection and analysis protocols, to reflect knowledge gained during the course of the project. ARS scientists in Peoria, Illinois targeted analysis to seeds, since they can transmit these microbes from plant to offspring. These studies will allow us to determine how microbes that live inside the plant affect these weeds. ARS scientists in Peoria, Illinois made significant progress in Objective 3. ARS scientists in Peoria, Illinois extracted nucleotide samples from individual cultures of fungal endophytes of the invasive weed, medusahead (Taeniatherum caput-medusae). A subset of the most interesting samples were used and subjected to a process to isolate potential mycoviruses. Additional studies are needed and under way to confirm and characterize these samples. Mycoviruses have the potential to alter the properties of fungi that live inside of plants, which can confer unique properties to the plant. These properties can provide increased stress tolerance to plants allowing them to out-compete other plants. ARS scientists in Peoria, Illinois successfully completed all elements of Objective 4, which was taxonomic characterization of a collection of fungal endophytes isolated from medusahead. These strains were characterized with DNA sequencing to identify them to the species level. The strains have been prepared for long-term preservation and storage, which will allow them to be evaluated for potential commercial properties at a later date. Accomplishments 01 Extending storage stability of biological insecticides. Maintaining viability of beneficial microbes during storage continues to be a problem for biological insecticides. Temperature and moisture availability limit the survival of microbial propagules and shorten the shelf-life of biopesticides. ARS scientists in Peoria, Illinois have demonstrated that maintaining dry conditions during storage is beneficial to the stability of the virus that infects the black cutworm. After one year at room temperature, dry formulations do not lose insecticidal activity, whereas samples at 33 degrees C and 75% relative humidity displayed decreased activity (i.e., showing half-lives of 8 months and 2 months, respectively). This information has practical importance for developing commercial products by providing product parameters necessary to achieve industry standards, which in turn increases the efficiency with which private companies could commercially produce this biopesticide. In addition, this research provides new pest control solutions for turf insects.

Impacts
(N/A)

Publications

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.
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.
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., 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.
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.

Progress 10/01/16 to 09/30/17

Outputs
Progress Report Objectives (from AD-416): 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. Approach (from AD-416): 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. Substantial progress has been made in the second year of this research project. For Objective 1, In vivo production of the baculovirus (AgipMNPV) of the black cutworm remains the most attractive method for production and commercialization of this biopesticide. Our laboratory was successful in optimizing variables to increase production of wild-type virus about three fold. Laboratory evaluations demonstrated that newly hatched larvae remain highly susceptible to virus infection for about 5 days. When applied to field grown grass, the virus applications had good residual activity for several days, which (when combined with larval susceptibility) suggest that once weekly applications may provide continued control of this pest in the field. We have made significant progress in Objective 2, for a second year we have isolated fungal strains from the invasive weed, medusahead (Taeniatherum caput-medusae). The plants were sampled from their invaded range in the western United States and from their native range in Europe. For this year, plants were sampled from Bulgaria and Serbia. We successfully obtained fungal isolates from the roots, seeds, stem and leaves from these plant samples. In addition, DNA samples have been extracted from all of the plant tissues for future analysis of the microbial communities based on high throughput DNA sequencing techniques. These studies will allow us to determine how endophytes impact the success of invasive weeds and may offer potential approaches to limit their invasions. We have made significant progress in Objective 4. All of the strains isolated from this year�s field work have been accessioned into the database and are available for future research needs. We have preliminary identification on most samples using two separate genes. Accomplishments 01 Optimized production of baculovirus effective against black cutworm. ARS researchers at Peoria, Illinois, were successful in improving the production yield through optimizing the production conditions of the baculovirus AgipMNPV (a pathogen specifically infects cutworm larvae). This accomplishment is in support of developing the baculovirus AgipMNPV as a biopesticide for control of the black cutworm infesting turf grass grown for golf tees and greens as a replacement for chemical insecticide applications. Defining parameters for economical production of baculoviruses to be used as biological insecticides remains a major factor limiting successful commercialization. Evaluating virus products, for both the number of particles produced and the insecticidal activity of those particles, insures the quality of the active agent for subsequent research for development of this agent as an ecologically benign pest control technology. Successful adoption for highly managed turf grass, will support expanded use of this baculovirus for other crops and vegetables where the cutworm is a pest. In addition, successful development of biological pest controls will prevent plant damage without adverse effects on beneficial insects or non-target animals, contamination of environmental resources such as surface water, or posing a toxicity hazard to pesticide applicators or others in the treated area. 02 Completed fungal endophyte survey of the invasive weed, medusahead. ARS researchers at Peoria, Illinois, were successful in completing the first survey of the fungal endophytes from the invasive weed, medusahead (Taeniatherum caput-medusae). Endophytes are microorganisms that live within plants and are thought to help the plant tolerate different environmental stresses. In this emerging scientific field, it is unclear of the role these endophytes play in helping invasive weeds out compete the native plants. This initial dataset provides the foundation to start understanding these complex interactions and to determine if these interactions can be exploited to control the spread of this weed. In addition, we have identified and described a variety of new bacteria species that are new to science. These novel species were accessioned in to our culture collection to be evaluated in the future for commercial potential. Understanding the relationship between these microbes and the weed will allow us to develop new strategies to selectively target and control the weed for the benefit of ranchers and the natural ecosystems.

Impacts
(N/A)

Publications

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.
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.
Chow, A., Dunlap, C.A., Jackson, M.A., Flores, D., Patt, J.M., Setamou, M. 2016. Oviposition behavior and survival of Tamarixia radiata (Hymenoptera: Eulophidae), an ectoparasitoid of the Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae), on hosts exposed to an entomopathogenic fungus. Journal of Economic Entomology. 109:1995-2005. doi: 10.1093/jee/ tow164.
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.
Hamm, P.S., Caimi, N.A., Northup, D.E., Valdez, E.W., Buecher, D.C., Dunlap, C.A., Labeda, D.P., Lueschow, S.R., Porras-Alfaro, A. 2017. Western bats as a reservoir of novel Streptomyces species with antifungal activity. Applied and Environmental Microbiology. 83:e03057-03016. doi: 10. 1128/AEM.03057-16.
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. doi: 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.

Progress 10/01/15 to 09/30/16

Outputs
Progress Report Objectives (from AD-416): 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. Approach (from AD-416): 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. Substantial progress has been made in the first year of this research project. For Objective 1, important factors for in vivo production of the black cutworm baculovirus has been evaluated in order to optimize yield of this beneficial microbe. Larval age, exposure dosage, larval diet, and incubation temperature all impact the amount of wild-type virus produced by infected larvae. In addition, optimal conditions for production of selected virus isolates were also determined. Isolates with a specific gene deletion are known to provide faster kill of the pest insect, but tend to have lower levels of production relative to wild-type virus. Characterizing the impact of higher production on kill-rate will guide the selection of the isolates for the most effective pest control application. We have made significant progress in Objective 2 by isolating more than 350 fungal strains from the invasive weed, medusahead (Taeniatherum caput- medusae). The plants were sampled from their invaded range in the western United States (U.S.) and from their native range in Europe. We successfully obtained fungal isolates from the roots, seeds, stems, and leaves from both invaded (U.S.) and native (Europe) plant samples. In addition, DNA samples have been extracted from all of the plant tissues for future analysis of the microbial communities based on high throughput DNA sequencing techniques. We have made significant progress in Objective 4 by establishing an inventory system and strain database to manage newly isolated fungal strains. All of the strains isolated from this year�s field work have been accessioned into the database and are available for future research needs. Accomplishments 01 Research on the benefits provided to plants by fungi that live symbiotically within living plant tissue (endophytes) is a new and emerging science that generally focuses on improving crop plants. It is likely that the endophytes provide an ecological advantage to invasive weeds, although these mechanisms have yet to be discovered. As the initial phase of this research on weed endophytes, we have successfully collected, identified, cultured, and stored over 350 fungal isolates from the invasive noxious weed medusahead to establish a culture collection as a specific resource for studying symbiotic interactions. This collection will support the ultimate research goal of developing weed control by selective manipulation of the endophytic fauna. Defining parameters for economical production of entomopathogens to be used as biological insecticides remains a major factor limiting successful commercialization. In support of developing the baculovirus AgipMNPV as a biopesticide for control of the black cutworm infesting turf grass grown for golf tees and greens, the impact of important in vivo production factors have been characterized to maximize production of virus particles including: selection of the artificial diet, age (size) of the exposed larvae, exposure dosage, and incubation temperature. Evaluating virus products, for both the number of particles produced and the insecticidal activity of those particles, insures the quality of the active agent for subsequent research for development of this agent as an ecologically benign pest control technology for use on highly managed turf grass.

Impacts
(N/A)

Publications

Behle, R.W., Richmond, D.S., Jackson, M.A., Dunlap, C.A. 2015. Evaluation of Metarhizium brunneum F52 (Hypocreales: Clavicipitaceae) for control of Japanese beetle larvae in turfgrass. Journal of Economic Entomology. 108(4) :1587-1595. doi:
Dunlap, C.A. 2015. Phylogenomic analysis shows that �Bacillus vanillea� is a later heterotypic synonym of Bacillus siamensis. International Journal of Systematic and Evolutionary Microbiology. 65(10):3507-3510.
Dunlap, C.A., Kwon, S.W., Rooney, A.P., Kim, S.J. 2015. Bacillus paralicheniformis sp. nov., isolated from fermented soybean paste. International Journal of Systematic and Evolutionary Microbiology. 65(10) :3487-3492.
Kim, S.J., Dunlap, C.A., Kwon, S.W., Rooney, A.P. 2015. Bacillus glycinifermentans sp. nov., isolated from fermented soybean paste. International Journal of Systematic and Evolutionary Microbiology. 65(10) :3586-3590.
Mascarin, G.M., Jackson, M.A., Kobori, N.N., Behle, R.W., Dunlap, C.A., Delaibera, I. 2015. Glucose concentration alters dissolved oxygen levels in liquid cultures of Beauveria bassiana and affects formation and bioefficacy of blastospores. Applied Microbiology and Biotechnology. 99(16) :6653-6665. doi: 10.1007/s00253-015-6620-3.
Qayyum, M.A., Wakil, W., Arif, M.J., Sahi, S.T., Dunlap, C.A. 2015. Infection of Helicoverpa armigera by endophytic Beauveria bassiana colonizing tomato plants. Biological Control. 90:200-207.
Dito, D.F., Shapiro-Ilan, D.I., Dunlap, C.A., Behle, R.W., Lewis, E.E. 2016. Enhanced biological control potential of the entomopathogenic nematode, Steinernema carpocapsae, applied with a protective gel formulation. Biocontrol Science and Technology. 26(6):835�848.
Dunlap, C.A., Kim, S.J., Kwon, S.W., Rooney, A.P. 2016. Bacillus velezensis is not a later heterotypic synonym of Bacillus amyloliquefaciens; Bacillus methylotrophicus, Bacillus amyloliquefaciens subsp plantarum and �Bacillus oryzicola� are later heterotypic synonyms of Bacillus velezensis based on phylogenomics. International Journal of Systematic and Evolutionary Microbiology. 66(3):1212-1217. doi: 10.1099/ ijsem.0.000858.
Behle, R.W., Goett, E.J. 2016. Dosage response mortality of Japanese beetle, masked chafer, and June beetle (Coleoptera: Scarabaeidae) adults when exposed to experimental and commercially available granules containing Metarhizium brunneum. Journal of Economic Entomology. 103(3) :1109-1115. doi: 10.1093/jee/tow080.