2007 Annual Report 1a.Objectives (from AD-416) Conserve, characterize, and exchange insect pathogenic fungi, genetic resources and associated information from the ARS Collection of Entomopathogenic Fungal Cultures (ARSEF). Develop molecular tools of functional genomics for use in systematics and in defining secondary metabolite potential of entomopathogenic fungi. Identify, characterize, and develop natural products from entomopathogenic fungi and selected organisms for agricultural and other high-value uses. Develop, preserve, and evaluate fungal pathogens as microbial control agents in IPM systems for thrips in maple and greenhouse crops as a component of a comprehensive IPM strategy. 1b.Approach (from AD-416) Develop preservation conditions, including lyophil cultures, to maintain cultures for customer base. Develop backup culture collection off-site. Expand culture collection via orphaned collections and additional germplasm from collaborators. Improve public access to and information content of ARSEF website. Conduct systematic studies of insect pathogenic fungi. Utilize molecular tools to characterize virulence and pathogenicity factors of insect pathogenic fungi. Develop chemical fingerprinting for promising biocontrol isolates. Characterize biological activity and chemistries of compounds produced by entomopathogenic fungi and other control agents. Develop collaborations to foster screening and utilization of bioproducts. 3.Progress Report Molecular and traditional taxonomic approaches are being used to reconcile species relatedness within Beauveria and Metarhizium via collaboration between the ARS Insect Biocontrol Laboratory, Beltsville, and ARSEF staff. This effort is establishing a multi-gene DNA-based study to evaluate evolutionary relationships and documenting the extent to which traditional morphological characters correlate with the genetically defined groups that may be recognized as separate species. Collaboration with staff at the Center for Studies of Parasites and Vectors, National University of La Plata, Argentina occurred during a 2-week visit to Argentina as part of the Fulbright Senior Specialist program. Research on Entomophthorales, conidial fungi affecting a range of agricultural hosts, and the mosquito pathogen Leptolegnia chapmanii were conducted during the visit. These studies improve knowledge of the fungal flora of South America and directly support US efforts to use some of these same fungi as potential biocontrol agents against major insect pests. New regulations have enabled shipments of Brazilian microbial germplasm for the sole purposes of taxonomic identification and ex situ preservation. Approximately 30 isolates have been sent to ARSEF from the Federal University of Goias for cooperative evaluation. Under this new ruling, ARSEF can receive Brazilian germplasm for documentation, identification, and preservation. Fungi and bacteria are prolific producers of natural products, but only a fraction of their productive capabilities have been discovered. To establish a more systematic means of evaluating factors that affect natural product synthesis and more easily detect novel chemistries, we have employed the use of genetic expression analysis, genetic knockouts, and multiple chemical analysis tools (mass infusion analysis, 2D nuclear magnetic resonance spectroscopy) to identify unique chemical signatures. SCA 1907-22410-00003-10S (Entomopathogenic fungi for biological control of pear thrips and western flower thrips in forests and greenhouses (see separate annual report for further details) with University of Vermont continues refining means to produce, apply, monitor and integrate fungal pathogens into models to manage thrips in greenhouses and forests. SCA 1907-22410-003-09S (Isolation and characterization of microbial metabolites from biological biocontrol agents) with Cornell University terminated in 2007 (see separate annual report for further details) focused on developing technologies to evaluate the biological control potential of TrigoCor 1448, a Bacillus subtilis strain. Chemical profiling and response surface modeling was used to identify active components as a function of culture time, media formulation, and temperature, in order to develop an optimized product. SCA 1907-22410-003-011S (Molecular systematics, functional genomics, and chemistries of entomopathogenic fungi) with Cornell University (see separate annual report for further details) is evaluating the effect of molecular expression patterns and specific developmental stages as a function of media formulation in an effort to identify novel metabolites. 4.Accomplishments ARSEF supplies customer needs for fungal germplasm. The ARS entomopathogenic fungal collection (ARSEF) accessioned >370 new isolates, prepared >800 isolates for cryogenic or lyophilized preservation, and distributed >500 isolates to 52 recipients in response to 99 requests from ARS, other state, academic, and commercial labs in the US and abroad. ARSEF currently comprises more than 8500 isolates of more than 580 fungal taxa from 1150 different hosts and 1780 locations throughout the world. ARSEF continues to fulfill its mission to supply customers’ needs as the world’s largest collection of insect pathogenic fungi. NP 304, Component I- Identification and Classification of Insects and Mites (microbes), Problem 1D- Systematic Studies of Emerging Pests and New Beneficials (microbes) Developing novel systems for identification of novel metabolites. Genome-wide searches of microbial systems have revealed a number of genes involved in secondary metabolites for which no products are known; one such gene family are nonribosomally derived peptide synthetases (NRPSs). We used a combined genetic and chemical search strategy in a pathogenic fungus to identify gene products of two specific NRPSs as several highly conserved iron-binding peptides (ferrocrocin, coprogen, and triacetylfusarinine) involved in regulating iron metabolism. These peptide products were found to play significant roles in the fungus by affecting its ability to be pathogenic and to produce spores, thus illustrating their role in microbial virulence, morphology, and development. NP304, Component II- Biology of Pests and Natural Enemies (microbes), Problem 2A- Basic biology Thaxtomin is a potent phytotoxin produced by plant pathogenic Streptomyces species that cause scab diseases of multiple root crops, including potato. The complete complement of genes and the regulatory signals for production of the peptide family of thaxtomins have been delineated, leading to the discovery that the pathway is controlled by cellobiose, the smallest component of plant cell wall biosynthesis. These studies have provided a model for plant pathogenic Streptomyces-plant host interactions where the cell wall components are key to the regulation of this pathway. This knowledge may aid in developing control strategies for this disease. NP304, Component II- Biology of Pests and Natural Enemies (microbes), Problem 2A- Basic biology 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of active CRADAs and MTAs 1 Number of U.S. patents granted 1 Number of web sites managed 1 Number of non-peer reviewed presentations and proceedings 9 Number of newspaper articles and other presentations for non-science audiences 2 Review Publications Gibson, D.M., Krasnoff, S.B., Churchill, A.C. 2006. Searching for polyketides in insect pathogenic fungi. In: Rimando, A., Bauer, S., editors. Polyketides: Biosynthesis, Genetics, and Biological Activity. ACS Book Series 955. p. 48-67. Wach, M.J., Loria, R., Krasnoff, S.B., Gibson, D.M. 2007. Effect of carbohydrates on the production of thaxtomin a by streptomyces acidiscabies. Archives Of Microbiology. 188:81-88 Bischoff, J., Humber, R.A., Rehner, S.A. 2007. Metarhizium frigidum sp. nov.: a cryptic species of m. anisopliae and a member of the m. flavoviride complex. Mycologia 98:737-745. James, T.Y., Kauff, F., Schoch, C., Matheny, B., Hofstetter, V., Cox, C.J., Celio, G., Guiedan, C., Fraker, E., Miadlikowska, J., Lumbsh, T., Rauhut, A., Reeb, V., Arnold, A., Amtoft, A., Stajich, J.E., Hosaka, K., Sung, G., Johnson, D., O'Rourke, B., Crockett, M., Binder, M., Curtis, J.M., Slot, J.C., Wang, Z., Wilson, A.W., Schueller, A., Longcore, J.E., O Donnell, K., Mozley-Standridge, S., Porter, D., Letcher, P.M., Powell, M.J., Taylor, J.W., White, M.M., Griffith, G.W., Davies, D.R., Humber, R.A., Morton, J.B., Sugiyama, J., Rossman, A.Y., Rogers, J.D., Pfister, D.H., Hewitt, D., Hansen, K., Hambleton, S., Shoemaker, R.A., Kohlmeyer, J., Volkmann-Kohlmeyer, B., Spotts, R.A., Serdani, M., Crous, P.W., Hughes, K.W., Matsuura, K., Langer, E., Langer, G., Untereiner, W.A., Lucking, R., Budel, B., Geiser, D.M., Aptroot, D.M., Diederich, P., Schmitt, I., Schultz, M., Yahr, R., Hibbett, D.S., Lutzoni, F., Mclaughlin, D.J., Spatafora, J.W., Vilgalys, R. 2006. Reconstructing the early evolution of Fungi using a six-gene phylogeny. Nature. 443:818-822. Pryor, S.W., Gibson, D.M., Krasnoff, S.B., Walker, L.P. 2006. Identification of antifungal compounds in a biological control product using a microplate inhibition bioassay and ms analysis. Proceedings of the American Society of Agricultural and Biological Engineers International (ASABE). 49:1643-1649. Oide, S., Moeder, W., Krasnoff, S., Gibson, D.M., Haas, H., Yoshioka, K., Turgeon, G. 2006. Nps6, encoding a non-ribosomal peptide synthetase involved in siderophore-mediated iron metabolism, is a conserved virulence determinant of plant pathogenic ascomycetes. The Plant Cell. 18:2836-2853. Mendez Sanchez, S., Humber, R.A., Roberts, D., Freitas, A., Lima, L., Silva, G., De Almeida, C., Nunes, E. 2002. Prospección de hongos Entomophthorales para el control natural de insectos en Bahía, Brasil [Prospecting Entomophthoralean fungi for the natural control of insects in Bahia, Brazil]. Revista Manejo Integrado de Plagas. 66:20-30. Schroeder, F.C., Gibson, D.M., Churchill, A.C., Punchapat, S., Wursthorn, E.J., Krasnoff, S.B., Clardy, J. 2007. Differential Analysis of 2D NMR Spectra: New Natural Products from a Pilot-Scale Fungal Extract Library. Angewandte Chemie International Edition. 46:901-904. Pryor, S.W., Gibson, D.M., Bergstrom, G.C., Walker, L.P. 2007. Minimization of Between-well Sample Variance of Antifungal Activity Measurements Using a High-Throughput Screening Microplate Bioassay. Biotechniques. 42(2):168-172. Kornfeld, A., Kaufman, P.B., Lu, C.R., Gibson, D.M., Bolling, S.F., Warber, S.L., Chang, S., Kirakosyan, A. 2007. The production of hypericins in two selected Hypericum perforatum shoot cultures is related to differences in black gland structure. Plant Physiology and Biochemistry. 45:24-32. Hibbett, S.A., Binder, M., Bischoff, J. F., Blackwell, M., Cannon, P. F., Eriksson, O. E., Huhndorf, S., James, T., Kirk, P. M., Lücking, R., H. Lumbsch, T., Lutzoni , F., Matheny, P. B., Mclaughlin, D. J., Powell, M. J., Redhead , S., Schoch, C. L., Spatafora, J. W., Stalpers, J. A., Vilgalys, R., Aime, M. C., Aptroot, A., Bauer, R., Begerow, D., Benny, G. L., Castlebury, L. A., Crous, P. W., Dai, Y.-C., Gams, W., Geiser, D. M., Griffith, G. W., Gueidan, C., Hawksworth, D. L., Hestmark, G., Hosaka, K., Humber, R. A. , Hyde, K. D., Ironside, J. E., KLõljalg, U., Kurtzman, C. P., Larsson, K.-H., Lichtwardt, R., Longcore, J., Mi¿dlikowska, J., Miller, A. Moncalvo, J.-M., Mozley-Standridge, S., Oberwinkler, F., Parmasto, E., Reeb, V., Rogers, J. D., Roux, C., Ryvarden, L., Sampaio, J. P., Schüßler, A., Sugiyama, J., Thorn, R. G., Tibell, L., Untereiner, W. A., Walker, C., Wang, Z., Weir, A., Weiss, M., White, M. M., Winka, K., Yao, Y.-J., and Zhang, N. 2007. A higher-level phylogenetic classification of the Fungi. Mycological Research. 111:509-547.