2007 Annual Report 1a.Objectives (from AD-416) Establish a molecular evolutionary framework for understanding the genetic diversity, pathogenicity and mycotoxin potential of fusarial pathogens that cause Fusarium head blight (FHB) of wheat and barley and sudden death syndrome (SDS) of soybeans. Determine from multilocus sequence analysis the genetic diversity of Aspergillus and Pencillium species that produce ochratoxin-A, and develop rapid detection methods for these fungi using oligonucleotide probes. Develop molecular genetic methods, based on analysis of multilocus gene sequences, for rapid detection and identification of yeasts responsible for food and beverage spoilage as well as for biocontrol yeasts that are applied to fruit, vegetables and grains to ensure species safety and to determine the fate of these species in the environment. 1b.Approach (from AD-416) Multiple diagnostic gene sequences will be developed for phytopathogenic and toxigenic species for molds in the genera Aspergillus, Fusarium, Penicillium and their relatives, and for food spoilage and biocontrol yeasts. The gene sequences will be used to develop population- and species- specific molecular probes for rapid detection and for understanding the genetic diversity and relationships of each group of pathogens, food spoilage and biocontrol yeasts. 3.Progress Report All known strains of the yeast genera Torulaspora and Zygotorulaspora in the ARS Culture Collection are being characterized from gene sequences of large subunit rDNA and from translation elongation factor-1 alpha. The data indicate that species of these common food and beverage spoilage yeasts can be separated by both gene sequences, thus providing a means for rapid species detection. Certain Metschnikowia species are commonly used for biocontrol of fruit rots. Strains of all known species of Metschnikowia and those of the closely related genus Clavispora are being characterized from gene sequences of large subunit rDNA and translation elongation factor-1 alpha to provide a rapid means for detecting and identifying species and for prediction of other species that may be useful for biocontrol. In addition, species in related clades have undergone multigene sequencing resulting in expansion of the genera Trichomonascus and Trigonopsis, and the description of the new genus Sugiyamaella. Genealogy of Aspergillus and rapid identification of the species. Over 460 isolates of Aspergillus have been sequenced at each of four genetic sites. The information developed will provide the foundation for DNA-based identification techniques that are quicker and more accurate than prior techniques. Because some species of Aspergillus can produce harmful mycotoxins, while others are useful in food fermentations, in biotechnology and in medicine, the rapid and accurate identification of species is essential to medical laboratories, food processing labs and biotechnologists. 4.Accomplishments Rapid Identification of Fusarium Head Blight Pathogens. Our unique multilocus genotyping (MLGT) assay for the high-throughput discrimination of B-FHB pathogen species and B-trichothecene chemotypes was used to continue molecular surveillance of FHB species and trichothecene toxin chemotype diversity within North America. Species and chemotypes were determined for over 3,000 North American FHB isolates in collaboration with from colleagues the Canadian Grain Commission, and Connecticut Agricultural Experiment Station. The results provide the first evidence of a rapid, adaptive evolutionary shift in FHB pathogen populations and conclusively demonstrate that a highly toxigenic 3ADON F. graminearum population has been rapidly replacing the dominant 15ADON FHB pathogen in North America. These results directly contradict the hypothesis of a homogeneous F. graminearum population in North America that can be represented by local inocula in breeding, as we identified significant genetic subdivision associated with trichothecene chemotype differences (GST >0.24, P <0.001). The recent dramatic expansion of 3ADON populations observed in our analyses demonstrates the need to re-evaluate chemotype and population diversity throughout North America. Our analyses of trichothecene chemotype distributions across Canada revealed a dramatic longitudinal cline in which 3ADON producers were significantly more common in Eastern Canada than in Western provinces. A recent range expansion is consistent with the high level of population differentiation observed between 3ADON and 15ADON populations in western Canada (GST = 0.311, P <0.001). The rapid and significant increase in 3ADON frequency in Western Canada indicates that isolates from the 3ADON populations have a selective advantage over isolates from the dominant 15ADON population. Phenotypic differences that might affect 3ADON pathogen fitness, including toxin accumulation, growth, production of conidia (fecundity), and pathogenicity, were investigated. The most striking finding was that isolates from the two 3ADON populations accumulated significantly (P <0.05) more trichothecene than isolates from the 15ADON population. The fact that isolates from the rapidly spreading 3ADON populations accumulated significantly (P <0.05) more trichothecene than isolates from the 15ADON population could have a profoundly negative affect on food safety and the economics of cereal production in North America. Our results highlight the potential for the exchange of adaptations between FHB populations via recombination and stress the need to consider pathogen variation in addition to host variation in combating FHB. Specifically, our data indicate that FHB species and population-level variation require consideration in the development of cereal cultivars with broad-based resistance to FHB pathogens. Our findings also indicate the need to evaluate trichothecene contamination independent of FHB infection levels, as different pathogen populations have different toxigenic capabilities. This accomplishment fully supports NP-303 Plant Diseases component IV Wheat Research Need #1 - Fusarium Head Blight. 6.Technology Transfer Number of patent granted 1 Number of non-peer reviewed presentations and proceedings 15 Review Publications White, M.M., James, T.Y., O Donnell, K., Cafaro, M.J., Tanabe, Y., Sugiyama, J. 2006. Phylogeny of the Zygomycota based on nuclear ribosomal sequence data. Mycologia. 98:872-884. Hill, N.S., Schwarz, P., Dahleen, L.S., Neate, S., Horsley, R., Glenn, A.E., O Donnell, K. 2006. Elisa analysis for Fusarium in barley: Development of methodology and field assessment. Crop Science. 46:2636-2642. Zhang, N., O Donnell, K., Sutton, D.A., Nalim, A., Samuels, G.J., Summerbell, R.C., Padhye, A.A., Geiser, D.M. 2006. Members of the Fusarium solani species complex causing infections in both humans and plants are those most commonly encountered in the environment. Journal of Clinical Microbiology. 44(6):2186-2198. Peterson, S.W. 2006. Multilocus sequence analysis of Pencillium and Eupenicillium species. Revista Iberoamericana De Micologia. 23:134-138. Page, B.T., Shields, C.E., Merz, W.G., Kurtzman, C.P. 2006. Rapid identfication of ascomycetous yeasts from clinical specimens by a molecular-based flow cytometry method and comparision with identifications from phenotypic assays. Journal of Clinical Microbiology. 44(9):3167-3171. Chang, D.C., Grant, G.B., O Donnell, K., Wannemuehler, K., Noble-Wang, J., Rao, C.Y., Jacobson, L.M., Crowell, C.S., Sneed, R., Lewis, F.M., Kainer, M.A., Genese, C.A., Alfonso, E.C., Jones, D.B., Srinivasan, A., Fridkin, S.K., Park, B.J. 2006. An outbreak of Fusarium keratitis associated with use of a new contact lens solution. Journal of the American Medical Association. 296:953-963. Bar-Meir, M., Sutton, D.A., Wickes, B., Kurtzman, C.P., Goldman, S., Zheng, X. 2006. Catheter-related fungemia due to Candida thermophila. Journal of Clinical Microbiology. 44(8):3035-3036. 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. Kurtzman, C.P., Robnett, C.J. 2007. Multigene phylogenetic analysis of the Trichomonascus, Wickerhamiella and Zygoascus yeast clades, and the proposal of Sugiyamaella gen. nov. and fourteen new species combinations. Federation Of European Microbiological Societies Yeast Research. 7:141-151. Deyrup, S.T., Gloer, J.B., O Donnell, K., Wicklow, D.T. 2007. Kolokosides A-D: Triterpenoid glycosides from a Hawaiian isolate of Xylaria sp. Journal of Natural Products. 70(3):378-382. Serra, R., Peterson, S.W. 2007. Penicillium astrolabium and Penicillium neocrassum, two new species isolated from grapes and their phylogenetic placement in the P. olsonii and P. brevicompactum clade. Mycologia. 99(1):78-87. 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. O Donnell, K., Sarver, B., Brandt, M., Chang, D.C., Nobel-Wang, J., Park, B.J., Sutton, D., Benjamin, L., Lindsley, M., Padhye, A., Geiser, D.M., Ward, T.J. 2007. Phylogenetic diversity and microsphere array-based genotyping of human pathogenic Fusaria, including isolates from the multistate contact lens-associated U.S. keratitis outbreaks of 2005 and 2006. Journal of Clinical Microbiology. 45(7):2235-2248.