2012 Annual Report
1a.Objectives (from AD-416):
1. Culture, conserve, characterize, and exchange insect pathogenic fungi, fungal 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 metabolites and products of insect pathogenic fungi that affect biological control potential.
3. Discover natural fungal metabolites and products to evaluate their roles in the survival, development, virulence, and pathogenicity of fungal biological control agents.
4. Identify and characterize novel lead chemistries for the development of new fungal metabolite-based pesticides for agricultural applications.
1b.Approach (from AD-416):
Use various preservation methods (lyophil, cryogenic, offsite backup) to maintain culture collection and to accession new germplasm for customer base. Develop new methods to isolate, culture, and preserve isolates. Conduct studies on the systematics, taxonomy, and organismal biology of these fungi. Use molecular and bioinformatic tools to evaluate insect pathogenic genomes and gene families. Use molecular tools to target secondary metabolite pathways. Characterize biological activity and chemistries of compounds produced by fungi at various developmental stages.
Holdings of ARS Collection of Entomopathogenic Fungal Cultures (ARSEF). In the last 12 months (1 Aug 2011 – 31 July 2012), the ARSEF culture collection has accessioned 360 isolates, received 400 cultures awaiting formal accessioning and long-term preservation, and distributed 609 cultures in response to 110 requests. ARSEF currently comprises 11569 accessions of 673 fungal taxa from 2312 locations worldwide, and 1259 different hosts and substrates. Accession information for ARSEF holdings continues to be revised as molecular reidentifications are received from collaborating laboratories.
Material Transfer Agreements (MTA) with EMBRAPA. MTAs to cover exchanges of germplasm between the ARSEF collection and its Brazilian counterpart, the entomopathogenic fungal collection at Cenargen (Embrapa Genetic Resources and Biotechnology; Brasilia, BR) are in final stages of preparation for signatures.
Identification of Multiple Hydrophobin Genes and their Expression during Fungal Development. At least one strain of the broad host range insect pathogen Metarhizium brunneum has been commercially developed, so it is important to evaluate components related to development and pathogenicity for further improvements. Hydrophobins are a group of small peptides that are important for fungal growth and development. ARS researchers in Ithaca, NY, together with Cornell University researchers, identified and characterized three different hydrophobin genes, and produced mutants in which each gene had been deleted. These mutants showed alterations in expression patterns during development, and in pigmentation, spore production, morphology, and hydrophobicity, as well as a marked delay in virulence against two different insects. The studies showed that that these three hydrophobins have distinct, but compensating, roles in providing functionality for their producing organism, and that their level of expression in important in enhancing virulence against insects.
Refuting role of dextruxins' importance in insect pathogenicity and virulence by Metarhizium. The small peptide family known as destruxins has been thought to be critically important for virulence against insects, but conclusive proof as to their importance has been lacking. ARS researchers at Ithaca, New York identified the gene cluster responsible for biosynthesis and generated knockout strains incapable of producing destruxins. The mutants had no significant changes in virulence levels against several different insect species and no changes in fungal morphology or development. Gene expression was detectable at low levels during early growth phase and increased with culture time, and gene transcripts were also detectable in later stages of infected insects and in conidia. That destruxins appear to be dispensable for virulence against insects casts serious doubt on their long-purported role in insect pathogenicity and virulence.
Genetic clarification and reclassification of fungi in Entomophthorales. These fungi include some of the most important fungal insect pathogens worldwide, but the relationships among these fungi and with other fungal groups have remained comparatively unstudied until now. Multigenic studies have proven that this group of fungi is a closed related taxonomic unit, and have basically supported the previous traditionally based classification in six families. Several of the genera will need more study because the existing relationships with others in this group were not supported. Several distinct lineages were recognized, and this group has been reclassified by ARS researchers at Ithaca, New York as a new fungal phylum, Entomophthoromycota, comprising three newly recognized classes (Basidiobolomycetes, Neozygitomycetes, and Entomophthoromycetes).
Sanchez, S., Humber, R.A., Freitas, A., Pinheiro, A. 2011. Batkoa apiculata (Thaxter) Humber affecting Anopheles (Diptera: Culicidae) in the municipality of Una, Southern Bahia, Brazil. Entomotropica. 25(2):63-68.
Donzelli, B., Krasnoff, S., Sun-Moon, Y., Churchill, A., Gibson, D.M. 2012. Genetic basis of destruxin production in the entomopathogen Metarhizium robertsii. Current Genetics. 58:105-116.
Sevim, A., Donzelli, B., Wu, D., Demirbag, Z., Gibson, D.M., Turgeon, B. 2012. Hydrophobin genes of the entomopathogenic fungus, Metarhizium brunneum, are differentially expressed and corresponding mutants are decreased in virulence. Current Genetics. 58:79-92.
Humber, R.A. 2012. Identification of entomopathogenic fungi. In: Lacey, L.A., editor. Manual of Techniques in Invertebrate Pathology. London,UK: Academic Press. p 151-187.
Humber, R.A. 2012. Preservation of entomopathogenic fungal cultures. In: Lacey, L.A., editor. Manual of Techniques in Invertebrate Pathology. Chapter 10. London, UK: Academic Press. p. 317-328.
Rehner, S.A., Humber, R.A., Minnis, A., Sung, Gi-Ho, Luangsa-Ard, J., Devotto, L. 2011. Phylogeny and systematics of the anamorphic, entomopathogenic genus Beauveria. Mycologia. 103(5):1055-1073.
O Donnell, K., Humber, R.A., Geiser, D.M., Kang, S., Park, B., Robert, V.A., Crous, P.W., Johnston, P.R., Aoki, T., Rooney, A.P., Rehner, S.A. 2011. Phylogenetic diversity of insecticolous fusaria inferred from multilocus DNA sequence data and their molecular identification via FUSARIUM-ID and Fusarium MLST. Mycologia. 104(2):427-445.
D'Alessandro,, W., Humber, R.A., Luz,, C. 2012. Occurrence of pathogenic fungi to Amblyomma cajennense in a rural area of central Brazil and their activities against vectors of Rocky Mountain spotted fever. Veterinary Parasitology. 188:156-159. DOI: 10.1016/j.vetpar.2012.02.016.
Gams,, W., Humber, R.A., Jaklisch,, W., Kirschner,, R., Stadler,, M. 2012. Minimizing the chaos following the loss of article 59: suggestions for a necessary discussion. Mycotaxon. 119:495-507. DOI: 10.5248/119.495.
Sung, J., Lee, J., Humber, R.A., Sung, G., Shrestha, B. 2006. Cordyceps bassiana and production of stromata in vitro showing Beauveria anamorph in Korea. Mycobiology. 34(1):1-6. DOI: 10.4489/MYCO.2006.34.1.001.
Rocha,, L., Inglis,, P., Humber, R.A., Kipnis,, A., Luz,, C. 2012. Occurrence of Metarhizium spp in central Brazilian soils. Journal of Basic Microbiology. DOI: 10.1002/jobm.201100482.