2005 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? What does it matter? There is both an unmet need and a great potential to develop biological control agents and to find new products for agriculture and industry from microbes and other natural sources. We use the ARS Collection of Entomopathogenic Fungal Cultures (ARSEF) as a major focal point for research activities. One thrust of the project is the curation of a large culture collection of fungal pathogens of invertebrates; activities include the distribution, permanent storage, characterization, and identification of these fungi, together with research on their isolation, maintenance and preservation. Research is directed toward the taxonomy, systematics, and organismal biology of these fungi. The other major thrust of this project is to use these fungi as well as other selected organisms as source materials in our search for natural products with biological activities that might be useful in agricultural, veterinary, pharmaceutical, or other value-added contexts. Our research efforts are directed toward the successful development of effective biological control agents, biopesticides, and high value products, with a major emphasis on utilizing fungi from the ARSEF collection. The project has three specific goals:. 1)Conserve, characterize, and exchange insect pathogenic fungi, genetic resources and associated information from the ARS Collection of Entomopathogenic Fungal Cultures (ARSEF),. 2)Develop molecular tools of functional genomics for use in systematics and in defining secondary metabolite potential of entomopathogenic fungi, and. 3)Identify, characterize, and develop natural products from entomopathogenic fungi and selected organisms for agricultural and other high-value uses. The germplasm activities of this CRIS project fall within ARS National Program 301, Component I - Genetic Resource Management, and the activities in problem areas 1a-1e. (expand diversity of germplasm collections, maintain living genetic resources and information, document and characterize genetic resources, expand germplasm evaluation and characterization, make available these resources and information), as they relate to fungal insect pathogens of invertebrates. We are also expanding the use of these fungi and other microbes by evaluating their potential to produce biopesticides or other high-value products, conducted under National Program 306, Quality and Utilization of Agricultural Products, Component 2. New Product Technology. As part of the ARS effort to safeguard genetic resources, we maintain and expand the ARSEF Collection to foster efforts for their use in biological control agents and biopesticide discovery. Research on classifying, preserving, and supplying fungal germplasm will be useful for potential development as biological control agents. Our research to understand the role of fungal secondary metabolites in the infection process may be of particular relevance in addressing the benefits and risks of fungal biological controls. The discovery of novel, biobased products may offer new opportunities for effective alternatives to chemical insecticides or other high value products. Fungal biological control agents or the biopesticide products derived from them will provide safe, effective biological alternatives to synthetic chemical pesticides. 2.List the milestones (indicators of progress) from your Project Plan. NOTE: many of the milestones are multi-year and ongoing activities throughout the life of the project. Year 1 (FY 2004) 1.1. Conserve germplasm in culture collection to supply needs for these fungi. We will incorporate orphaned collections, exchange germplasm, and participate in Russian/U.S. germplasm exchange program. 1.2. Characterize morphology of germplasm in the ARSEF collection. 2.1. Begin systematic studies of entomopathogenic fungi, specifically to revise Beauveria taxonomy. 2.2. Assess gene expression as a function of media and environment. 3.1 Prepare and characterize materials in testing program. 3.2. Evaluate microbial compounds such as thaxtomin analogs and Cordyceps products. Year 2 (FY 2005) 1.1. Conserve germplasm in culture collection to supply needs for these fungi. We will incorporate orphaned collections, exchange germplasm, and participate in Russian/U.S. germplasm exchange program. 1.2. Characterize morphology of germplasm in the ARSEF collection. 2.1. Complete systematic studies of entomopathogenic fungi, specifically to revise Beauveria taxonomy. 2.2. Begin to assess gene expression as a function of media and environment 3.1 Prepare and characterize materials in testing program. 3.2. Evaluate microbial compounds such as Cordyceps and Drechmeria products. Year 3 (FY 2006) 1.1. Conserve germplasm in culture collection to supply needs for these fungi. We will incorporate orphaned collections and exchange germplasm. 1.2. Characterize morphology of germplasm in the ARSEF collection. 2.1. Begin systematic studies of entomopathogenic fungi to refine classification of Metarhizium. 2.2. Complete gene expression as a function of media and environment. 3.1 Prepare and characterize materials in testing program. 3.2. Evaluate microbial compounds such as thaxtomin analogs, Cordyceps and Drechmeria products and other promising extracts identified in testing program. Year 4 (FY 2007) 1.1. Conserve germplasm in culture collection to supply needs for these fungi. We will incorporate orphaned collections and exchange germplasm. 1.2. Characterize morphology of germplasm in the ARSEF collection. 2.1. Complete systematic studies of entomopathogenic fungi, specifically to refine classification of Metarhizium and to re-identify Verticillium isolates. 2.2. Begin to assess gene expression as a function of asexual and sexual states of the fungus. 3.1 Prepare and characterize materials in testing program. 3.2. Evaluate microbial compounds such as other promising extracts identified in testing program. Year 5 (FY 2008) 1.1. Conserve germplasm in culture collection to supply needs for these fungi. We will exchange germplasm. 1.2. Characterize morphology of germplasm in the ARSEF collection. 2.1. Complete systematic studies of entomopathogenic fungi, specifically to re-identify Verticillium isolates. 2.2. Complete assessment of gene expression as a function of asexual and sexual states of the fungus. 3.1 Prepare and characterize materials in testing program 3.2. Evaluate microbial compounds such as promising extracts identified in testing program. 4a.What was the single most significant accomplishment this past year? Putative virulence factors of fungus not needed for successful insect infection. A major impediment in the development of biocontrol agents has been an incomplete understanding of the factors that make them efficacious, including the role of secondary metabolites. Both a genetic and chemical approach was used to address the role of destruxins, a group of small peptides, in pathogenicity of Metarhizium anisopliae to insect pests, in work conducted collaboratively and described more fully under Projects 1907-22410-003-05T and 1907-22410-003-06S. Two important points can be made from this study:. 1)destruxins were found in only a subset of insects killed by the fungus, suggesting that they may not be important virulence factors for the fungus. If they were critical, we would expect to detect them invariably in all cadavers.. 2)A previously unreported group of mycotoxins, closely related to the mutagenic fusarins produced by Fusarium, was discovered in M. anisopliae. Overproduction of these mycotoxins by two knockout mutants did not contribute to increased virulence against insect hosts, suggesting that they may be dispensable for these insect-fungus interactions. Although further genetic analyses are required to confirm this hypothesis, this information is relevant to strain selection and development since toxin-deficient strains will be perceived as safer for insect biological control than those with the genetic potential to make such compounds. This research supports the development of alternatives to chemical pest controls by contributing to the knowledge base that defines a safe and effective biological control agent. 4b.List other significant accomplishments, if any. None. 4c.List any significant activities that support special target populations. None. 4d.Progress report. Lyophilization of ARSEF germplasm resumed in fall 2004 upon the hire of a technician to conduct this important aspect of collection operation. Three shipments comprising 103 critically important isolates of Metarhizium species from the orphaned collection of entomopathogenic fungal cultures at the Division of Entomology, CSIRO (Canberra, Australia) were received. Thirty-nine isolates from Russian sites in the Siberian Far East and near the Black Sea (adjacent to the Georgian border) were shipped to ARSEF under terms of a joint US/Russian research program in fall 2004. About 175 isolates of Aschersonia and Hypocrella were characterized in conjunction with NSF-funded studies directed by K. Hodge (Cornell Univ.) on the systematics and phylogeny of these genera pathogenic for scale insects; many of these isolates have been included in a detailed PCR-based phylogenetic and taxonomic analysis. Nearly 130 isolates of Metarhizium and allied genera were examined prior to shipment to Dr. S. Rehner (ARS, Beltsville) for preliminary multigenic molecular taxonomic analyses similar to those done in the previous two years for species of Beauveria. Collaborative work with S. Rehner (ARS Insect Biocontrol, Beltsville) on Beauveria systematics on a study of a block of Danish isolates from a single small farm that suggests that (1) there may be a much greater diversity of Beauveria species present in a small area than ever suspected, and (2) despite the lack of obvious production of sexual stages in nature, (para)sexual recombination may still be actively occurring in field-based populations of Beauveria. The background molecular studies on more than 700 ARSEF isolates of Beauveria have been completed, and the anticipated taxonomic evaluation of the tree derived from these multigenic studies will be completed in FY06. Reidentification of entomopathogenic isolates formerly classified in Verticillium is proceeding on an ad hoc basis as these isolates are pulled from cryogenic storage for lyophilization and reidentification; more than 60 isolates have already been processed, and the remaining isolates should be completed by the end of FY06. Studies of Metarhizium studies are being conducted in collaboration with S. Rehner (Beltsville) for the reidentification of ARSEF isolates under the current molecular systematics and for the verification and expansion of the molecularly based systematics of this genus (originally based on the isolates now being transferred to ARSEF from the CSIRO collection noted above in 1.1). This report documents research conducted under a Specific Cooperative Agreement between ARS and the Entomology Research Laboratory at the University of Vermont entitled "Entomopathogenic Fungi for IPM of Pear Thrips and Western Flower Thrips in Forests and Greenhouses" CRIS 1907-22410-003-01S. Pear thrips (PT; Taeniothrips inconsequens) populations are building again throughout Vermont, and a model developed to predict risk of damage to maples is being used to provide warnings to maple growers likely to be affected. The management of western flower thrips (WFT; Frankliniella occidentalis) in greenhouses by either of two protocols - ‘Better Management Practices’ (BMP) and ‘Conventional’ (CON) - were monitored, and BMP was found to be superior to CON in total costs, labor and time required, and resulted in higher quality and value of the resulting crops. Predatory mites were found to be the most effective natural enemy for use in the BMP greenhouse; the use of the mites was compatible with fungal sprays that provided a slower although sustained suppression of pest WFT populations. The protocols for greenhouse pest control are all being developed in cooperation with growers operating in real-world conditions rather than based on laboratory studies only, so that the end-users of the results have a direct stake in the development, improvement and adoption of new IPM recommendations. New production and formulations technologies have been developed and continue to be refined to provide improved granular whey-based protein matrix formulations for increasingly effective and economical applications of entomopathogenic fungi against soil-borne stages of WFT. Terminated and replaced by 1907-22410-003-10S. This report serves to document research conducted under a 2 1/2-year Trust Fund Cooperative Agreement with the National Research Initiative, USDA, established in FY 2002 entitled "Are destruxins virulence factors for the fungus Metarhizium anisopliae?" CRIS 1907-22410-003-05T. This project was directed toward defining the role of destruxins, a family of biologically active peptides, in the insect disease process of Metarhizium anisopliae. Destruxins are cyclic depsipeptides with chemical features suggesting synthesis by a nonribosomal peptide synthetase (NRPS). Knockout (KO) mutants were created by disrupting a NRPS gene fragment, ma267 predicted to be involved in destruxin production via Agrobacterium tumefaciens-mediated transformation. All KO strains were mitotically stable and phenotypically similar to wild type, but all strains produced comparable levels of destruxins. Based on these results, we concluded that gene ma267 does not encode the destruxin NRPS. There were no detectable differences in pathogenicity among the KO mutants and wild type strains in multiple bioassays with beet armyworm and Colorado potato beetle. We conclude that the unknown NRPS gene ma267 does not encode a virulence factor for insect disease. Efforts are underway to identify the natural product synthesized by ma267 since it is appears to be expressed early in the infection cycle. Quantitative estimates of destruxin levels in mycosed larvae from HPLC MS/MS analyses ranged from 1 microgram-10 picograms/insect. Destruxin was not detectable in all cadavers and destruxin estimates for insects killed by KO strains did not differ from those for insects killed by WT strains. A fourth KO transformant has an additional uncharacterized mutation evidenced by a marked increase in the production of yellow pigment. We identified two major components, NG-391 and NG-393, previously reported from Fusarium sp. as stimulants of nerve-cell growth; these compounds have not been previously reported from a Metarhizium. These compounds are closely related to the fusarins, mutagenic Fusarium mycotoxins that contaminate corn. Detectable levels of these compounds were also found in control strains; levels in two KO mutants were 60- and 20-fold higher, respectively, than the other strains analyzed. Several new NRPS gene fragments cloned from cDNA of DTX-producing cultures will serve as targets for the next gene disruption studies. Neither destruxins nor the fusarin-like compounds appear to be required for virulence of the fungus, suggesting that they are dispensable for infection. Although further proof will be needed, this information is relevant to strain selection and development since toxin-deficient strains will be perceived as safer for insect biological control than those with the genetic potential to make such compounds. Terminated 2/28/05. This report serves to document research conducted under a Specific Cooperative Agreement with A.C.L. Churchill at Boyce Thompson Institute, a co-investigator on a grant from the National Research Initiative, CSREES, USDA, entitled "Genetic manipulation of destruxins in Metarhizium anisopliae" CRIS 1907-22410-003-06S. This project was used to conduct the molecular analyses of a peptide synthetase gene predicted to play a role in the biosynthesis of destruxins. Enhancing the efficacy and safety of fungal biological control agents requires an understanding of the genetics and chemistry of disease processes used by these organisms. Agrobacterium tumefaciens-mediated transformation was used to generate knockout mutants of the peptide synthetase gene ma267, based on strong correlative evidence of its role in destruxin biosynthesis. Few detectable differences in phenotype were observed in the mutants compared to control strains, and destruxin production was comparable to the wild type control, leading to the conclusion that gene ma267 does not encode the destruxins peptide synthetase. A fourth transformant has an additional uncharacterized mutation correlated with overproduction of metabolites not previously reported from a Metarhizium. Several new NRPS gene fragments have been cloned from cDNA of DTX-producing cultures in order to aid in studies to identify the putative destruxin peptide synthetase. Terminated 2/28/05. This report serves to document research conducted under a Non-funded Cooperative Agreement with A.C.L. Churchill, Boyce Thompson Institute, a co-investigator with J. Clardy, on a grant funded through the National Cancer Institute, entitled "Screening of fungal cultures for anti-cancer compounds" CRIS 1907-22410-003-07N. Over 1000 isolates from the ARS Collection of Entomopathogenic Fungi (ARSEF) have been provided to the industrial partner for culturing, extraction, and screening over a 4 year period. Extracts are also provided to ARS for in-house screening purposes for potential utility as biopesticides, in addition to information concerning antimicrobial activities. From a core collection of insect pathogenic fungi, over 53% of the fungi screened were shown to contain genes for peptide synthetase. Of those gene fragments found in the molecular screening program, over 70% appeared to be unique gene fragments in comparison to those reported in Genbank. These fragments are being used to explore means of following gene expression in vitro by varying fermentation conditions in order to optimize conditions for product discovery. Terminated 4/30/2005. This report documents research conducted under a Specific Cooperative Agreement between ARS and the Department of Plant Pathology at Cornell University entitled "Identifying the components for scab resistance in potato" CRIS 1907-22410-003-08S. We evaluated chemically fractionated oat bran for the elicitation of thaxtomin synthesis since oat-based media is the only known liquid medium that supports thaxtomin production. The ability to support thaxtomin production was found almost exclusively in the insoluble cellulosic material. Starch, cellulose, and simple sugars did not support production, but xylan and glucan, two carbohydrates present in the hemicellulose fraction in oat bran, supported elevated levels of thaxtomin production, comparable to that found in oatmeal and oat bran broth. Limited enzymatic digestion of xylan to produce smaller xylose oligomers resulted in higher production of thaxtomin. Cellobiose, a building block of cellulose, also supported thaxtomin production. These carbohydrates appear to be signal molecules that turn on metabolic pathways required for pathogenicity in Streptomyces. However, the techniques we have used to date do not provide the resolution to discriminate among the various cell wall components in terms of their ability to produce thaxtomin. Future work will concentrate on the specific signal receptors and putative regulatory genes in the vicinity of the thaxtomin biosynthetic pathway. This report serves to document research conducted under Specific Cooperative Agreement with ARS and Gary Bergstrom at the Department of Plant Pathology at Cornell University entitled "Isolation and characterization of microbial metabolites from biological biocontrol agents" CRIS 1907-22410-003-09S. Effective and economic means of controlling Fusarium diseases are needed and new chemical or biological control strategies have yet to meet the immediate and long-term needs of growers. In some cooperative tests, the Bergstrom strain of Bacillus subtilis designated TrigoCor 1448 has reduced infection and mycotoxin contamination comparable to synthetic fungicides. This success, however, is tempered by the fact that the biology of the microbial antagonist and its interaction with Fusarium within the plant environment is poorly understood, and biocontrol efficacy is not consistent across environments. A U.S. patent covering this proprietary strain was awarded to Cornell Research Foundation and Embrapa Trigo in Brazil in 2005 (U.S. patent 6,896,883 B2- Biocontrol for plants with Bacillus subtilis, Inventors: Gary C. Bergstrom and Wilmar C da Luz). To order to develop microbial production protocols that maximize the active chemical components in the microbial product, chemical profiling of TrigoCor 1448 broth cultures was used to identify active components as a function of days of bacterial growth. In collaboration with L.Walker (Dept. of Biological and Environmental Engineering, Cornell Univ.), fermentation trials identified the critical factors necessary to produce a quality biocontrol product. Surface response modeling was used to predict the effect of temperature, aeration, and time of culture on component production, while principal component analysis was used to delineate which components correlated with activity. We found an optimal time window of 5-6 days for production of active chemistries in shake broth culture that correlated with greater biocontrol activity. The active antifungal components included iturins, fengycins, and surfactins; mass analysis was used to confirm active chemistries. In greenhouse experiments, the application of Bacillus subtilis strain TrigoCor 1448 to wheat spikes, prior to inoculation with conidial suspensions of Fusarium graminearum, consistently reduced the incidence and severity of head blight symptoms and produced dramatic (5- to 10-fold) reductions in contamination of grain by deoxynivalenol. This protection occurred as inoculation times varied from peak anthesis through soft dough stages. Large, viable populations of spore-forming bacteria were shown to persist on sprayed wheat spikes more than a week following the application of TrigoCor 1448, while no spore-forming bacteria were found on the surface of wheat spikes sprayed with water. This is a striking demonstration of the potential for TrigoCor 1448 to persist during grain filling on spikes of wheat. Further studies are directed at developing a formulation for use in greenhouse conditions and in understanding the mode of action involved in toxin reduction. Another research component of the work is the use of Cornell facilities and analytical equipment to chemically characterize various microbial metabolites for structural determination. This report documents research conducted under a Specific Cooperative Agreement between ARS and the Plant and Soil Sciences at the University of Vermont entitled "Entomopathogenic Fungi for Biological Control of Pear Thrips and Western Flower Thrips in Forests and Greenhouses" CRIS 1907-22410-003-10S. This new agreement was established in May 2005, so no significant results are yet available to report. However, the work plan for the remainder of FY-05 and FY-06 includes further work on testing the persistence and efficacy of granular formulations of fungal isolates active against pear thrips (PT; Taeniothrips inconsequens) in maple forest loam soils or against western flower thrips (WFT; Frankliniella occidentalis) in standard soil and soil-free potting media in greenhouses. Particular attention will be given to determining the most effective timing for and the impacts of applications of fungi against WFT in greenhouses. Research on improving formulations for thrips-active fungal isolates will continue. Long-term cryopreservation and cataloging of an extensive collection of fungal cultures isolated from thrips will continue, as will testing of >250 recent isolates of Metarhizium anisopliae and Beauveria bassiana against WFT to find optimal isolates for further development against this major pest insect. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. ARSEF increases holdings and diversity of culture collection. The ARSEF culture collection continues to expand in size and diversity. It now encompasses more than 7800 isolates from more than 480 fungal taxa isolated from nearly 1100 hosts and other substrates from 1620 collection sites around the world. The use of lyophilized isolates greatly facilitates delivery times for many of the most requested isolates and increases the stocks for isolates ready for an off-site back-up of the ARSEF collection's holdings [Milestone 1.1; Objective 1.1 and 1.2 of NP301, Component 1]. ARSEF supplies customer needs for fungal germplasm. Over the past two years of the project, the ARS Collection of Entomopathogenic Fungal Cultures (ARSEF) sent out more than 1400 cultures in response to over 200 requests from an international group of academic and industrial customers. Major recipients included an ARS laboratory in Beltsville and 2 large pharmaceutical firms; significant numbers of isolates to laboratories at Cornell University and the University of Maryland, and large numbers of isolates to a foreign client at Zhejiang University (PRChina), with small numbers of isolates sent too many other labs throughout the world. During this same period of time, 700 isolates were formally accessioned, and 1800 isolates (approximately 20% of the current collection) were processed for long-term storage either cryogenically in liquid nitrogen dewars and/or by freeze-drying [Milestone 1.1.and 1.2; Objective 1.1 and 1.2; NP301, Component 1]. Molecular analysis of Beaveria completed and Metarhzium analysis underway. Molecular multigenic studies conducted collaboratively with S. Rehner (ARS Insect Biocontrol, Beltsville) on Beauveria systematics using more than 700 ARSEF isolates of Beauveria have been completed. Systematics of Metarhizium in collaboration with S. Rehner (Beltsville) are underway with the receipt of the Australian isolates from the CSIRO collection. [Milestone 2.1.; Objective 1.1 and 1.2; NP301, Component 1]. Over 1800 extracts submitted for high throughput screening. Over 1800 extracts representing 1000 isolates from the ARS Collection for Entomopathogenic Fungi were submitted to the Chemical Screening Program, Institute of Chemistry and Cell Biology, Harvard Medical School (221907-22410-003-07N), including one extract identified as having potent antibacterial activity. Participation in this screening program should broaden the knowledge base of assays in which these materials display biological activity, in our efforts to discover compounds having value-added properties (Milestone 3.1.; Objective 3.1; NP306, Component 2). Novel peptides discovered in fungus infecting cicadas. Microbes, and especially those organisms found in close association with other organisms as pathogens or endophytes, are known to be prolific producers of structurally diverse, biologically active compounds, that can be used directly or as lead chemistries for new biological pesticides. Novel peptides from a fungus originally isolated as a pathogen of locusts (Cordyceps heteropoda) have been chemically characterized as belonging to the peptaibol family of compounds. These compounds were active against bacteria, fungi, and mosquito larvae in laboratory testing. Myriocin, a known antifungal and potent immunosuppressant was also purified from this isolate, previously unknown as a source of this compound [Milestone 3.2; Objective 3.2.; NP306, Component 2). Phytotoxic derivatives used to probe structural components needed for biological activity. Plant pathogenic organisms can be sources for novel chemistries, especially in the search for phytotoxic compounds potentially useful as herbicides. Streptomyces species that cause “scab” diseases on potato tubers, sweet potato storage roots and expanded tap roots produce a family of phytotoxins known as thaxtomins that are critical for pathogenicity. A series of thaxtomin derivatives were generated in order to investigate the key structural components required for activity. Only a specific configuration of the compound was active, and additional groups added at carbon position 14 had reduced activity. These studies will be useful for defining the chemical requirements necessary for herbicidal activity [Milestone 3.3; Objective 3.2.; NP306, Component 2). 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? Humber provided training in diverse aspects of the biology and systematics of fungal pathogens affecting insects and in maintaining culture collections in a workshop for scientists, graduate students, and technical staff, and delivered a departmental seminar on the biology and changing systematics of Beauveria bassiana at the University of Sao Paulo’s agricultural campus (ESALQ) in Piracicaba, Brazil. Humber delivered a departmental seminar at Fordham University on the natural occurrence and importance of wall-less stages of entomopathogenic fungi in the development of these organisms, and provided 2 days of consultation at Fordham’s field station, the Louis Calder Center (Armonk, NY), for various projects dealing with the biology, ecology, and evolution of fungal pathogens affecting insects. Provided on-site consultation on appropriate doctoral research program for new PhD student in the Department of Agronomy at the Universidade Federal Rural de Pernambuco, Recife. Formulations and data concerning the efficacy of Bacillus subtilis under greenhouse and field conditions have been discussed with a commercial biological pesticide company potentially interested in product development. 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). None. Review Publications Humber, R.A. 2005. Insect-fungus associations: ecology and evolution. Mycological Research. 109(2):383-384. Humber, R.A. 2005. Fungi associated with insects or used for applied biocontrol. Mycological Research. 109(3):258. Benjamin, R.K., Blackwell, M., Chapela, I.H., Humber, R.A., Jones, K.G., Klepzig, K.D., Lichtwardt, R.W., Malloch, D., Spatafora, J.W., Weir, A. 2004. Insect- and other arthropod-associated fungi. In: Mueller, G.M., Bills, G.F., Foster, M. S., editors. Biodiversity of Fungi: Inventory and Monitoring Methods. Burlington, MA: Elsevier Academic Press. Chapter 18, pp. 395-434. Ireland, C.M., Andersen, R.J., Clardy, J., Churchill, A.C., Frost, P., Gibson, D.M., Greenberger, L., Greenstein, M. 2004. Anticancer agents from unique natural products sources. Pharmaceutical Biology. 41:S15-38. Wach, M., Kers, J., Krasnoff, S.B., Loria, R., Gibson, D.M. 2005. Nitric oxide synthase inhibitors and nitric oxide donors modulate the biosynthesis of thaxtomin a, a nitrated phytotoxin produced by streptomyces spp. Nitric Oxide. 12:46-53. Wach, M.J., Johnson, E.G., Gibson, D.M., Loria, R. 2004. Nitric oxide's role in common scab of potato: phytotoxin biosynthesis and what else? [abstract]. American Phytopathological Society Annual Meeting. 94:S106 Kim, B., Gibson, D.M., Shuler, M.L. 2005. Relationship of viability and apoptosis to taxol production in taxus sp. suspension cultures elicited with methyl jasmonate. Biotechnology Progress. 21:700-707. Kers, J.A., Cameron, K.D., Joshi, M.V., Bukhalid, R.A., Morello, J.E., Wach, M.J., Gibson, D.M., Loria, R. 2005. A large, mobile pathogenicity island confers plant pathogenicity on streptomyces species. Molecular Microbiology. 55(4):1025-1033. Krasnoff, S.B., Reategui, R., Wagenaar, M.M., Gloer, J.B., Gibson, D.M. 2004. Cicadapeptins i and ii: new aib-containing peptides from the entomopathogenic fungus cordyceps heteropoda. Journal of Natural Products. 68(1):50-55. Gibson, D.M., Krasnoff, S.B., Churchill, A. 2005. Exploring polyketides of insect pathogenic fungi. Proceedings of American Chemical Society National Meeting. 229(1-2):AGRO 10. Kim, B., Gibson, D.M., Shuler, M.L. 2004. Effect of subculture and elicitation on instability of taxol production in taxus sp. suspension cultures. Biotechnology Progress. 20:1666-1673. Kanga, L.H., Jones, W.A., Humber, R.A., Boyd, Jr., D.W. 2004. Fungal pathogens of the glassy-winged sharpshooter, Homalodisca coagulata (Say) (Homoptera: Cicadellidae). Florida Entomologist. 87:225-228. Tosi, S., Caretta, G., Humber, R.A. 2004. Conidiobolus antarcticus, a new species from continental antarctica. Mycotaxon. 90:343-347. Posada, F.J., Vega, F.E., Rehner, S.A., Blackwell, M., Weber, D.C., Suh, S., Humber, R.A. 2004. Syspastospora parasitica on beauveria bassiana attacking the colorado potato beetle.. Journal of Insect Science. Vol.#4, Pg. 24 Mineiro, J.L., Raga, A., Leite, L.G., Humber, R.A., Sato, M.E., Nicastro, R.L. 2004. Pathogenicity in the laboratory of the fungus lecanicillium muscarium against phytophagous mites. Brazilian Entomological Congress Abstracts and Proceedings, Sept. 2004, Gramado, Brazil. p.176. Mineiro, J.L., Raga, A., Leite, L.G., Humber, R.A., Sato, M.E., Nicastro, R.L. 2004. First report of lecanicillium muscarium attacking mites on coffee in brazil. In: Brazilian Entomological Congress Abstracts and Proceedings. Proc. 20th Brazilian Entomological Congress, Sept. 2004, Gramado, Brazil. p.175. Krasnoff, S.B., Gibson, D.M., Wagenaar, M., Reategui, R., Gloer, J.B. 2004. Cicadapeptins, new aib-containing peptides, from an entomopathogenic cordyceps sp [abstract]. Society for Invertebrate Pathology Annual Meeting Proceedings. p. 66.