2006 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Commercial interest in using living microbial agents for controlling agricultural pests such as weeds and insects is driven by an increasing public demand for environmentally-friendly, sustainable agricultural practices. The widespread use of chemical pesticides is often viewed as non-sustainable agricultural practices and has heightened interest in developing environmentally friendly pest control measures. An integrated pest management (IPM) approach using microbial biological control agents (biopesticides) in conjunction with chemical pesticides is recognized as a sustainable pest management system, which reduces chemical pesticide usage and deters the development of pest resistance to the chemical control. A significant constraint to the commercial use of living microbial agents in agriculture is the lack of low-cost methods for producing and formulating stable, effective microbial products. While plant and insect pathologists have identified hundreds of microbial agents over the past 20 years that show good potential for use as biopesticides, few living microbes have been commercially developed as biopesticides. In large part, the limited success in commercializing microbial agents is due to a lack of economical production and formulation processes that deliver microbial products with a reasonable shelf-life and that provide consistent pest control under field conditions. While living microbial agents can be produced using solid-substrate or liquid culture methods, liquid culture fermentation processes are generally the most cost-effective. Optimized deep-tank, liquid fermentation processes for the production of living microbial agents must take into account not only product yield, but also microbial stability and product efficacy. Likewise, commercially viable microbial products require formulations that provide stabilized, effective biopesticides in a form that is conducive to application technology commonly used in agriculture. Our two major research goals will be to (1) develop low-cost, deep-tank fermentation methods for the production of stable, effective living microbial propagules for use as biocontrol agents and (2) develop methods for drying and formulating fungal and bacterial microbial biopesticides that enhance the viability and efficacy of these agents during storage and application, respectively. The development of cost-effective methods for producing, stabilizing, and formulating microbial biocontrol agents will lead to the commercialization of non-chemical, microbial biocontrol agents for use in insect and weed control. Greenhouse operators, the horticulture industry, organic farmers, state, federal and local land management agencies, the fermentation and formulation industries, and producers of microbial inocula for crop protection are all customers of research directed at developing non-chemical, microbial biocontrol agents for use in insect and weed control. In addition, technology for the production and stabilization of living microbial agents will not only benefit the biological control industry but also will find application in industries such as probiotic feed additives or bioremediation where the production and stabilization of living microbial agents is required. This CRIS research project is aligned with National Program 304 "Crop and Commodity Pest Biology, Control and Quarantine," Component 5: Pest Control Technologies and Component 9: Biological Control of Weeds. 2.List by year the currently approved milestones (indicators of research progress) FY2005 Objective 1. Production of Microbial Biocontrol Agent. A.1. Screen for appropriate propagules of Metarhizium anisopliae. B. Optimize Paecilomyces fumosoroseus blastospore production. C. Optimize Mycoleptodiscus terrestris microsclerotia production. Objective 2. Formulation of Microbial Biocontrol Agents and Semiochemicals. A.1. Microsclerotia of M. terrestris--Processing techniques. A.2. Adhesive formulations of M. terrestris microsclerotia. B.1. Foam-based formulations of P. fumosoroseus. C.1. Optimize spray drying Beauveria bassiana conidia. D.1. Insect attractants--Collection of saltcedar volatiles. D.2. Insect attractants--Leaf beetle response to odors. FY2006 Objective 1. Production of Microbial Biocontrol Agent. A.1. Screen for appropriate propagules of M. anisopliae. A.2. Optimize production and fitness of M. anisopliae. C. Optimize M. terrestris microsclerotia production. Objective 2. Formulation of Microbial Biocontrol Agents and Semiochemicals. A.2. Adhesive formulations of M. terrestris microsclerotia. A.3. Field testing & refinement of adhesive formulation of M. terrestris. B.1. Foam-based formulations of P. fumosoroseus. C.2. Develop lignin formulations of B. bassiana conidia. C.3. Field test lignin formulations of B. bassiana conidia. D.2. Insect attractants – Leaf beetle response to odors. D.3. Formulation of leaf beetle odor attractants. FY2007 Objective 1. Production of Microbial Biocontrol Agent. A.2. Optimize production and fitness of M. anisopliae. C. Optimize M. terrestris microsclerotia production. Objective 2. Formulation of Microbial Biocontrol Agents and Semiochemicals A.2. Adhesive formulations of M. terrestris microsclerotia. A.3. Field testing and refinement of adhesive formulation of M. terrestris. B.2. Develop dry formulations of P. fumosoroseus. C.2. Develop lignin formulations of B. bassiana conidia. C.3. Field test lignin formulations of B. bassiana conidia. D.3. Formulation of leaf beetle odor attractants. D.4. Develop monitoring tool with odor attractants. FY2008 Objective 1. Production of Microbial Biocontrol Agent. A.2. Optimize production and fitness of M. anisopliae. C. Optimize Mycoleptodiscus terrestris microsclerotia production. Objective 2. Formulation of Microbial Biocontrol Agents and Semiochemicals. A.3. Field testing and refinement of adhesive formulation of M. terrestris. B.2. Develop dry formulations of P. fumosoroseus. C.2. Develop lignin formulations of B. bassiana conidia. C.3. Field test lignin formulations of B. bassiana conidia. D.3. Formulation of leaf beetle odor attractants. D.4. Develop monitoring tool with odor attractants. FY2009 Objective 1. Production of Microbial Biocontrol Agent. A.2. Optimize production and fitness of M. anisopliae. C. Optimize M. terrestris microsclerotia production. Objective 2. Formulation of Microbial Biocontrol Agents and Semiochemicals. A.3. Field testing & refinement of adhesive formulation of M. terrestris. B.2. Develop dry formulations of P. fumosoroseus. C.2. Develop lignin formulations of B. bassiana conidia. C.3. Field test lignin formulations of B. bassiana conidia. D.4. Develop monitoring tool with odor attractants. 4a.List the single most significant research accomplishment during FY 2006. Soy wax–insect pheromone controlled-release formulation (NP 304, Crop and Commodity Pest Biology, Control, and Quarantine, Component 5: Pest Control Technologies and Component). Granules consisting of soy wax and insect attractant pheromone were shown to be an effective pheromone release system. Mating disruption is a pesticide-free method of controlling insect pests by inhibiting the ability of insects to locate potential mates with signaling pheromones. The ideal pheromone release system for mating disruption should provide consistent, prolonged release of the active pheromone, be inexpensive to produce and apply, and be non-toxic. Consistent pheromone release rates were characterized for soy wax-pheromone formulations in a laboratory environment, and promising results were obtained in preliminary field trials that tested the formulations effectiveness for disrupting mating by the Oriental beetle. Soy wax is a renewable resource and is readily biodegradable. These studies suggest that soy wax will be a viable pheromone release system for insect control through mating disruption. 4b.List other significant research accomplishment(s), if any. None. 4c.List significant activities that support special target populations. None. 4d.Progress report. None. 5.Describe the major accomplishments to date and their predicted or actual impact. NP 304, Crop and Commodity Pest Biology, Control, and Quarantine, Component 5 - Pest Control Technologies: The fungus Paecilomyces fumosoroseus is being developed as a biological control agent for whiteflies, aphids, and subterranean termites. Refinements in large-scale production and processing technology for blastospores of P. fumosoroseus resulted in the development of a rapid 2-day fermentation process that yielded high concentration of desiccation-tolerant blastospores. Using 100-L fermentations, harvesting and drying techniques were refined and produced dry, free-flowing, blastospore powders containing high concentrations of viable blastospores with up to 2 years of shelf-life when stored at 4C. The development of this patented production and stabilization process provides industry with a cost-effective method for producing this effective bioinsecticide. In order to develop a basic approach to blastospore formulation, the physicochemical surface properties of blastospores of P. fumosoroseus were characterized. Blastospores of P. fumosoroseus were shown to have a monopolar basic surface, which is characterized as hydrophilic. The blastospores were shown to be negatively charged under neutral conditions with an isoelectric point of 3.4. The surface properties of microbes are known to control the interactions between the microorganism and the environment. Understanding these properties in biocontrol systems can guide us in formulation and application decisions, which are important in developing a successful biocontrol product. Currently, chemical pesticides are distributed as foams to treat difficult to reach insect environments. The foaming agents typically used in chemical pesticide foams are detrimental to most microbial agents. Our research has identified suitable natural foaming agents which are compatible with microbial agents including P. fumosoroseus. The use of this foaming agent in combination with P. fumosoroseus blastospores has been demonstrated to be effective in controlling Formosan termite infestations in trees. These discoveries will provide termite control operators a non-chemical option for controlling Formosan termite infestations in trees. NP 304, Crop and Commodity Pest Biology, Control, and Quarantine, Component 5 - Pest Control Technologies: Production and stabilization studies were initiated to evaluate the potential for producing a stable, infective form of the bioinsecticidal fungus Metarhizium anisopliae for use in controlling the sugar beet root maggot. These studies identified production media and drying processes that supported high yields of a desiccation tolerant form of M. anisopliae. This production and stabilization process has been successfully scaled to 100-L fermentors. In collaboration with an ARS scientist at Sydney, MT, bioassays showed that soil incorporated, dried preparations of M. anisopliae provided excellent control of the sugar beet root maggot; and field trials are currently underway to test these M. anisopliae preparations. The development of a fungus-based bioinsecticide for sugar beet root maggot control will provide farmers with another tool for controlling this troublesome pest. NP 304, Crop and Commodity Pest Biology, Control, and Quarantine, Component 5 - Pest Control Technologies; and Component 9 - Biological Control of Weeds: The fungus Mycoleptodiscus terrestris is being developed as a biological control agent for the invasive aquatic weed hydrilla. Large-scale fermentation studies were conducted with M. terrestris. Production parameters were identified that supported optimal yields of microsclerotia of M. terrestris using a 4-day fermentation process. In addition, harvesting, drying and size-reduction processes were optimized for microsclerotia of M. terrestris that resulted in the production of a stable, dry, infective form of the fungus. Significant progress has also been made in the identification of biocompatible formulations for M. terrestris that may improve the adherence of microsclerotia to hydrilla and their efficacy in infecting and killing hydrilla. Collaborative studies with another Government agency and an industrial partner continue on testing various formulations of microsclerotia of M. terrestris. These studies suggest that adhesive-based formulations may enhance the bioefficacy of M. terrestris microsclerotia formulations. Collaborative studies continue on the development of optimal formulations for microsclerotia of M. terrestris. The development of an effective fungus-based biocontrol agent for control of the aquatic weed control will aid state agencies, municipalities, water control agencies, and homeowners by providing a safe, non-chemical tool for controlling the invasive, aquatic weed hydrilla. NP 304, Crop and Commodity Pest Biology, Control, and Quarantine, Component 5 - Pest Control Technologies: Experiments were conducted to determine acceptable processing conditions for spray-drying suspensions of conidia of Beauveria bassiana with the goal of formulating this agent as a dry, stable, effective microbial insecticide. Critical to the development of this technique was the ability to formulate conidia with various coatings without compromising the viability of the spore. Processing procedures, temperature limits, and essential ingredients were identified as being critical for maintaining the viability of conidia during spray-drying. These spray-drying techniques are currently being used to formulate conidia of B. bassiana with various UV protectants such as lignin, starches, and Soyscreen based oils that protect the conidia from degradation by sunlight after application in the field. In addition, laboratory and field bioassays were conducted to evaluate the biocontrol efficacy of UV-protected granuloviruses for codling moth control in Washington apples. The development of UV protective coatings for fungal and viral bioinsecticides will benefit farmers and biocontrol producers by enhancing or prolonging the biocontrol efficacy of these bioinsecticides. NP 304, Crop and Commodity Pest Biology, Control, and Quarantine, Component 5 - Pest Control Technologies; and Component 9 - Biological Control of Weeds: Advances were made in developing baits for attracting Diorhabda elongata, which is the leaf beetle species previously introduced into the U.S. for biological control of saltcedar. Saltcedar is an environmentally and economically devastating invasive weedy tree, and the beetles are now having enormous success in combating these plants in numerous Western states. The attractant will be formulated based on the quantitation of volatile chemicals from saltcedar with feeding beetles. Further advances were made towards combining the previously described pheromone for these beetles and the volatile chemicals from saltcedar. These attractants will be useful to land managers for monitoring population parameters such as beetle dispersal and spring emergence from overwintering, and they may prove valuable for trapping live beetles for redistribution to other areas or to prevent dispersal of the beetles from the site where they are released. NP 304, Crop and Commodity Pest Biology, Control, and Quarantine, Component 5 - Pest Control Technologies; and Component 9 - Biological Control of Weeds: Granules consisting of soy wax and insect attractant pheromone were shown to be an effective pheromone release system. Mating disruption is a pesticide free method of controlling insect pests by inhibiting the ability of insects to locate potential mates with signaling pheromones. The ideal pheromone release system for mating disruption should provide consistent, prolonged release of the active pheromone, be inexpensive to produce and apply, and be non-toxic. Consistent pheromone release rates were characterized for soy wax-pheromone formulations in a laboratory environment, and promising results were obtained in preliminary field trials that tested the formulations effectiveness for disrupting mating by the Oriental beetle. Soy wax is a renewable resource and is readily biodegradable. These studies suggest that soy wax will be a viable pheromone release system for insect control through mating disruption. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The development of methods for producing and formulating the biopesticide Mycoleptodiscus terrestris for control of hydrilla continues under a new Cooperative Research and Development Agreement (CRADA) with a leading aquatic weed control company. Previous collaborations with this industrial partner and the United States Army Corps of Engineers resulted in a patent (Mycoherbicidal Compositions and Methods of Preparing and Using the Same, U.S. Patent #6,569,807), jointly developed by ARS and the U.S. Army Corps of Engineers, on the production and use of microsclerotia of M. terrestris for hydrilla control. Our CRADA partner has negotiated an exclusive license for this patent along with application for foreign patent rights in select countries. The successful development of this technology will provide a non-chemical control measure for the hydrilla and other aquatic weeds within the next 5 years. The best leaf beetle attractants were synthesized, formulated, and distributed to Animal and Plant Health Inspection Service (APHIS) for a state-wide monitoring survey of the spread and dispersal of the salt cedar beetle in the state of Nevada. In addition, attractant baits were distributed to those scientists working for the ARS-led Saltcedar Biological Control Consortium, who are currently trying to establish new populations of saltcedar beetles in Texas and Colorado. Further technology transfers were primarily of information with respect to pheromone chemistry and biology and were primarily to other scientists. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Jackson, M.A. 2006. Biocontrol of hydrilla aquatic weed. Industrial Bioprocessing. 28(5):4-5. Jackson, M.A. 2006. Mycoherbicide Hope for Hydrilla Biocontrol. Biocontol News and Information. 27(20):41N-42N. Suszkiw. J. 2006. Scientists mobilize fungus to fight hydrilla. ARS Press Release, www.ars.usda.gov/is/pr/2006/060413.htm. Leach, S. 2006. ERDC's environmental lab signs patent license ageement. The Corps Environment. 7(2):7, 16. Review Publications Dunlap, C.A., Biresaw, G., Jackson, M.A. 2005. Hydrophobic and electrostatic cell surface properties of blastospores of the entomopathogenic fungus Paecilomyces fumosoroseus. Colloids and Surfaces B: Biointerfaces. 46(4):261-266. Jackson, M.A., Erhan, S., Poprawski, T.J. 2006. Influence of formulation additives on the desiccation tolerance and storage stability of blastospores of the entomopathogenic fungus Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes). Biocontrol Science and Technology. 16(1/2):61-75. Jackson, M.A., Cliquet, S., Erhan, S.M., Connick Jr, W.J. 2005. Factors that influence the desiccation tolerance and storage stability of blastospores of the entomopathogenic fungus Paecilomyces fumosoroseus [abstract]. Society for Invertebrate Pathology Annual Meeting. Paper No. MC3, p. 79. Natwick, E.T., Behle, R.W., Cardoza, R.E., Lopez, M.I. 2005. Evaluation of insecticide bait formulations and various insecticides for control of darkling beetles, Blapstinus spp. (Coleoptera: Tenebrionidae) [abstract]. Entomological Society of America. Paper No. 21653. Behle, R.W., Compton, D.L., Laszlo, J.A., Shapiro Ilan, D.I. 2006. Potential use of SoyScreen in formulations of Beauveria bassiana for UV protection. Entomological Society of America Annual Meeting North Central Branch. Paper No. D-215. Behle, R.W., Isbell, T. 2005. Evaluation of cuphea as a rotation crop for control of Western corn rootworm (Coleoptera: Chrysomelidae). Journal of Economic Entomology. 98(6):1984-1991. Natwick, E., Cardoza, R., Lopez, M., Behle, R.W. 2006. Insecticide bait formulations evaluated for darkling ground beetles, Blapstinus spp. (Coleoptera: Tenebrionidae) control. Entomological Society of America Regional Meetings. p. 75-76. Compton, D.L., Laszlo, J.A., Isbell, T., Behle, R.W., Kurth, T.L. 2005. Synthesis and applications of biocatalytically derived UV absorbing vegetable oils [abstract]. Royal Society of Chemistry Meetings. p. 026.