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? Insects are major pests of food and fiber, contributing to billions of dollars in direct losses to American agriculture and further reductions in agricultural product quality. While chemical insecticides often provide adequate control of insect pest populations, they may be detrimental to product quality and cause adverse effects in humans and non-target organisms. Microbial entomopathogens are natural biological agents that may be effective for control of pest insects and are safer for non-target organisms. However, their production and application is generally more expensive and they are often difficult to use effectively. Thousands of microbes harmful to insects have been discovered, but few have been successfully deployed to control pest insects. Some reasons for this lack of success are incomplete knowledge of their biological properties, lack of understanding of how they impair or kill insects, and the factors they produce for biocontrol. The research to be undertaken falls under National Program 304 - Crop Protection and Quarantine. It addresses the Biology of Pests and Natural Enemies (including microbes) for Insects and Mites; Basic Biology (Component IIA) and Pest Control Technologies; Traditional Biological Control (Component VA). Under National Program 301, a component of objective A of this project addresses Genomic Characterization and Genetic Improvement. This project was initiated in December, 2005. It encompasses basic and applied research aimed at developing effective, environmentally sound microbial (fungal, viral, and bacterial) pesticides, through natural and biotechnological methods, for use against agricultural and forest pests. The project has three specific goals:. 1)to characterize microbial entomopathogens;. 2)to evaluate molecular and cellular mechanisms of pathogenesis and virulence utilized by microbial entomopathogens; and. 3)to develop applied technologies for use of microbial entomopathogens as biopesticides. This project aims to enable development of improved microbial biocontrol agents (fungi, bacteria, and viruses) through natural selection of strains with better properties or through genetic engineering. It will analyze biological and molecular traits of a broad range of microbes harmful to insects; discover and describe genetic components that may be conserved among similar species to facilitate their use in biological control; and contribute to taxonomic characterization and identification of genetic components relating to success as control agents. This project also aims to determine how microbes harmful to insects disable pest insects and facilitate their use in biocontrol through understanding how they impair or kill insects. It will provide improved production technologies and utilization practices for insect pathogens in insect pest management. The resulting reduced use of chemical insecticides in important agricultural systems will lower health risks for humans while decreasing crop losses. Better pest control based on natural microbial biopesticides will yield safer agricultural products and less environmental damage. 2.List by year the currently approved milestones (indicators of research progress) Year 1 (FY 2006) No milestones indicated for this year (new OSQR project implemented December 2005). Year 2 (FY 2007) • Beauveria multilocus phylogeny completed. • Entomopathogenic bacteria toxic to one or more pest insects identified. • P. luminescens JHE-like toxin characterized. • Impact of insect transient toxin exposure determined. • Analysis of PDV gene transcription and expression over the course of gypsy moth parasitization completed. • Two new Lepidopteran cell lines established and characterized. Analysis of virus susceptibility to new lines complete. Year 3 (FY 2008) • Metarhizium multilocus phylogeny completed. • Genetic profiles of both IBL collection and transconjugant Bt strains obtained. • PDV full genome obtained and validated. • EST libraries and identification of genes expressed during fungal pathogenesis completed. • Bt, Chromobacterium, and Photorhabdus toxins identified. • Characterization of bacterial strain(s) by phenotypic microarray completed. • Lymantria dispar cDNA libraries generated. • Glyptapanteles indiensis cell culture initiated. Year 4 (FY 2009) • M. anisopliae multilocus phylogeny completed. • Stem cell mitogens produced in response to bacterial toxins characterized and identified. • New baculovirus virulence/host range genes identified and characterized. • L. dispar ESTs sequence analysis completed. • Functional identity of PDV pathogenic genes obtained. • Non-lepidopteran cell lines established and characterized with emphasis on Apis mellifera and Hypothenemus hampei lines. • Virus production procedures for large scale cultures developed. • Analysis of B. bassiana GHA in field populations completed. • Chromobacterium efficacy established through field trials. Year 5 (FY 2010) • Four novel baculovirus genomes sequenced and analyzed. • B. bassiana gene expression studies during insect infection cycle completed. • Novel Bt, P. luminescens, or Chromobacterium, toxins chacterized. • L. dispar genome arrays developed and screened for pest response to PDV. • Analysis of new lines for pathogen studies completed. • Baculovirus efficacy in lepidopteran hosts established. 3a. List the milestones that were scheduled to be addressed in FY 2006. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. No milestones were scheduled to be addressed this year (new OSQR project implemented December 2005). 4a.List the single most significant research accomplishment during FY 2006. Molecular markers developed for Beauveria. PCR and sequencing primers to four nuclear genomic regions were developed for multilocus phylogenetic analysis of the entomopathogenic fungus Beauveria. Substantial progress was made through development of a multilocus data set that will form the basis for a comprehensive phylogenetic analysis and taxonomic revision of the genus. The phylogeny and molecular markers will be used by scientists to improve the accuracy of identifications, strain verification and ecological monitoring strains used in biological control of insects. (NP304 component IIA, VA, and NP 301 objective A) 4b.List other significant research accomplishment(s), if any. New cell lines developed from two lepidopteran species. Cell lines were established from pickleworm (Diaphania nitidalis) and black cutworm (Agrotis ipsilon). These are the first lines from the pickleworm and are part of a cooperative effort with Clemson University and the USDA/ARS Vegetable Laboratory in Charleston, SC, with an effort to develop an effective pest control based on insect viruses for this pest of cucumbers and cucurbit crops. These new lines will provide a means to screen in vitro for appropriate viruses for their biocontrol. The new black cutworm cell line shows improved susceptibility to the virus from this insect. This line will allow more extensive study of virus for improved biological control of this pest of many agricultural crops, including corn, soybeans and many vegetables. (NP304 component IIA) Time- and tissue-specific expression of 10 G. indiensis polydnavirus (GiBV) parasitism genes analyzed in the larval gypsy moth host. Expression of protein products of PDV genes within the pest host results in inhibition of immune response, development, and alteration of physiology, enabling successful development of the wasp. These GiBV genes (protein tyrosine phosphatase and ankyrin repeat genes) have importance as regulators of signal transduction and inhibition of signaling in the parasitized host. All were expressed early in parasitization and most were sustained at least 7 days, suggesting their importance in early virus-induced immunosuppression and alteration of physiology. This information helps explain how the virus assists the wasp’s survival, which may lead to new biocontrol strategies based on wasp disruption of insect pest immune systems. (NP304 component IIA and VA) Genome sequence of a baculovirus from diamondback moth, Plutella xylostella, determined and analyzed. This virus, P. xylostella multiple nucleopolyhedrovirus (PxMNPV), is very similar to the A. californica MNPV, but can kill diamondback moth larvae at much lower doses. The PxMNPV genome sequence was determined and compared to the sequence of AcMNPV, and genetic differences between the two viruses that could account for the difference in their activities against P. xylostella were identified. This information adds to our knowledge of the molecular genetic determinants of baculovirus virulence and host range, and may facilitate the engineering of baculoviruses with enhanced efficacy against P. xylostella and other lepidopteran pests. (NP304 component IIA and VA and NP 301 objective A) Novel method developed to isolate bacterial pathogens from the environment. Most environmental samples contain insect bacterial pathogens but they are usually in too low number to be effective biocontrol agents. Merging concepts from chemical combinatorics (testing of compounds in mixtures) and insect pathology (isolating pathogens from dead insects), a method was developed for amplifying bacteria, increasing the numbers that were effective at killing insect larvae. This method could allow rapid discovery of new and effective biocontrol microbes. (NP304 component IIA and VA) Toxin complex a (Tca) from P. luminescens was shown to be highly toxic to both the Colorado potato beetle and the sweet potato whitefly. The activity of Tca against the Colorado potato beetle is comparable to the Cry III proteins of B. thuringiensis. The breadth of activity of Tca contrasts starkly with the generally narrow selectivity of B. thuringiensis d-endotoxins; the expression of Tcs in transgenic plants may allow development of crops that are broadly resistant to insect pests, allowing reduction of insecticide applications to control secondary pests. (NP304 component IIA and VA) 4c.List significant activities that support special target populations. This project does not support special target populations 4d.Progress report. NONE. 5.Describe the major accomplishments to date and their predicted or actual impact. Developed a series of molecular markers for the entomopathogenic fungus Beauveria bassiana for phylogenetic and population genetic investigations. These markers were made available to other scientists for characterizing natural Beauveria assemblages, identifying strains released as biocontrol agents, and for use in culture collections to genetically characterize accessioned isolates (NP304 component IIA and VA, NP 301 objective A). Developed new insect cell lines and made available to the scientific community. These have unique properties and are useful in a wide variety of scientific disciplines, especially for study and production of insect viruses as pest management tools (NP304 component IIA and VA). Discovered new bacterial pathogens with insecticidal properties in Colorado potato beetle and lepidopteran pest insects and assessed their toxic properties. These could provide new and environmentally sound tools for the control of insect pests (NP304 component IIA and VA). Discovered several viral genes and gene families and examined their time and tissue specificity during lepidopteran pest infection- including protein tyrosine phosphatase (PTPs) and others that may disrupt insect pest immune systems, growth, and development. These viral genes will be useful for deployment in pest biocontrol (NP304 component IIA and VA). Determined the genome sequence of a baculovirus highly virulent to diamondback moth and assessed virulence profile by comparison to a closely related virus of different host range. This improved our knowledge of baculovirus evolution, host and range and virulence and may facilitate the construction of improved baculovirus-based biopesticides for control of lepidopteran pests (NP304 component IIA and VA). Numerous material transfer agreements (MTAs) were established with scientists, universities, and industry for transfer of cell lines, research tools, and microbials developed or discovered in this project that could lead to their commercialization. The primary customers for these research accomplishments are scientists, University researchers, companies, grower groups, and corporations involved and/or interested in a variety of ecologically sound and biologically important factors relating to both insect biology/pathology and pest insect control. 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? MTAs were processed for providing cell lines to two foreign research institutes, one U.S. Corporation and two universities. These material transfers as well as more than 30 in the past five years, show that certain aspects of this project's technology are regularly transferred to other scientists and industry for use in their research and development programs. Ultimately, the use of these cell culture/virus systems needs to be optimized to make them economically competitive with other pest control methods. MTAs were processed with a University that will examine environmental strains to eliminate cross reactive background when tracking Bacillus strains after release; with a Medical Center that is are looking at background of environmental Bacillus as a source of pathogenic genes; and, with a State University that will examine Bt strains with attached crystals to understand the mechanism for crystal attachment. A novel Chromobacterium strain with insecticidal properties remains under patent review. Toxins from this strain, Chromobacterium subtsugae, were provided under MTAs to two major agricultural companies for evaluation of toxic properties with pest insects. These transfers could facilitate commercialization of a new ARS-discovered biopesticide. This strain will be available to the end user as soon as patent issues resolve. A series of molecular markers for B. bassiana, targeting intergenic regions in the nuclear genome was developed encompassing regions rich in nucleotide polymorphisms appropriate for phylogenetic and population genetic investigations. These markers were made available to other scientists for characterizing natural assemblages of Beauveria entomopathogens, to identify and track Beauveria strains released as biocontrol agents, and for use in culture collections to genetically characterize accessioned isolates. The PxMNPV genome sequence was submitted to GenBank sequence database, where it is available for use by other insect virologists and molecular geneticists studying insect pathogens. Variant sequences for AcMNPV identified in the course of this study were also submitted to GenBank. Review Publications Blackburn, M.B., Domek, J.M., Gelman, D.B., Hu, J.S. 2005. the broadly insecticidal photorhabdus luminescens toxin complex a (tca): activity against colorado potato beetle and sweet potato whitefly.. Journal of Insect Science. 5:32. Farrar, R.R., Shapiro, M., Shepard, M.B. 2005. Enhanced activity of the nucleopolyhedrovirus of the fall armyworm (lepidoptera:noctuidae) on bt- transgenic and nontransgenic sweet corn through the use of a fluorescent brightener and a feeding stimulant. Environmental Entomology. 34:825-832. Farrar, R.R., Shapiro, M. 2005. Control of artifical populations of the diamondback moth (lepidopetera: plutellidae) on collard by a nucleopplythedrovirus with a stillbene-based enhancer and an ultraviolet light protectant. Journal of Entomological Science. 40:280-290. Ferkovich, S.M., Lynn, D.E. 2005. Enhanced egg laying in adult predators fed artificial diet supplemented with an embyonic cell derived from eggs of Ephestia kueniella Zeller (Lepidoptera: Pyralidae). Florida Entomologist. 88:329-331. Lynn, D.E. 2006. Lepidopteran cell lines after long-term culture in alternative media: comparison of growth rates and baculovirus replication. In Vitro Cellular and Developmental Biology. 42:149-152. Gundersen, D.E., Pedroni, M.J. 2006. Characterization and transcritptional analysis of protein tyrosine phosphatase (ptp) genes of the parasitoid glyptapanteles indiensis polydnavirus (gibv) in the parasitized host.. Journal of General Virology. 87(2):311-322. Dougherty, E.M., Narang, N., Loeb, M.J., Lynn, D.E., Shapiro, M. 2005. Fluorescent brightener inhibits apoptosis in baculovirus-infected gypsy moth larval midgut cells. Biocontrol Science and Technology. 16:157-168.