2008 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 A 3-locus typing scheme for members of the Fusarium solani species complex (FSSC) was developed using portions of the following three genes: the internal transcribed spacer (ITS) region plus domains D1 + D2 of the nuclear large subunit (LSU) ribosomal ribonucleic acid (rRNA), translation elongation factor (EF-1a), and the second largest subunit of RNA polymerase II (RPB2). Of the 251 isolates subjected to multilocus deoxyribonucleic acid (DNA) sequence typing, 191 sequence types (STs) were differentiated. The in vitro activity of 10 antifungals tested against 19 isolates representing 18 species that span the breadth of the FSSC phylogeny show that members of this complex are broadly resistant to these drugs. The systematics and taxonomy of the ochratoxin-A producing species from Aspergillus section Circumdati (the Aspergillus ochraceus group) was assessed using multilocus DNA sequence analysis. Additional species have been identified from section Cirdumdati that were not previously known. Some of the these species are non-ochratoxigenic, whereas others make ochratoxin-A. Having established the species that exist in section Circumdati, it is now possible to prepare oligonucleotide probes to rapidly identify each of the ochratoxigenic species. Collaborative studies are underway to determine the characters that can be used to describe and distinguish these species from others in the section. The ochratoxigenic species from section Nigri (the Aspergillus niger group) were also examined. Suprisingly, the beta-tubulin locus typically used to assess relationships and species boundaries in this group of fungi was found to occur as one of two unrelated forms (paralogs). A multigene sequence analysis of the approximately 150 known species of Pichia, Debaryomyces, Dekkera and Brettanomyces provided the basis for reassigning species to phylogenetically defined genera, resulting in the proposal of eight new genera (Babjeviella, Barnettozyma, Lindnera, Meyerozyma, Millerozyma, Priceomyces, Scheffersomyces, Wickerhamomyces), redefinition of four previously described genera (Debaryomyces, Pichia, Schwanniomyces, Yamadazyma), and the elimination of two genera (Issatchenkia, Williopsis). The taxonomic changes described provide a better understanding of species that cause food and beverage spoilage as well as those important to biotechnology and biocontrol. Species of the genus Metschnikowia, which are often used for biocontrol of fruit rots, were characterized from additional gene sequences. This research directly addresses NP 108, Component 1. 4.Accomplishments 1. IDENTIFICATION OF FUSARIUM BY GENE TYPING. Species of Fusarium are important pathogens of plants and humans. A comprehensive 3-locus deoxyribonucleic acid (DNA) sequence typing scheme was developed for medically and agriculturally important members of the species-rich Fusarium solani species complex (FSSC). This research has had a significant impact on the ability to accurately identify members of this diverse group of agriculturally and medically important species. This research addresses NP 108, Component 1, Problem Areas 1.2.1 and 1.2.5. 2. OCHRATOXIN PRODUCING SPECIES OF ASPERGILLUS. Ochratoxin-A, produced by species of Aspergillus in agricultural products, is toxic to humans. A multigene dataset was used to clarify genetic relationships among species of Aspergillus that produce the mycotoxin ochratoxin-A. This research has had a significant impact on the ability to accurately identify and rapidly detect these mycotoxigenic molds. This research addresses NP 108, Component 1, Problem Areas 1.2.1 and 1.2.5. 3. PHYLOGENY OF ASCOMYCETOUS YEASTS. A multigene analysis of ascomycetous yeasts has provided the genetic basis for defining genera. This research has had a significant impact on identification of groups of yeasts that are responsible for food and beverage spoilage. This research addresses NP 108, Component 1, Problem Area 1.2.1 and 1.2.5. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of New Commercial Licenses Executed 2 Review Publications Kurtzman, C.P. 2007. Eleven new species of Sugiyamaella and Candida from forest habitats. Federation Of European Microbiological Societies Yeast Research. 7(6):1046-1063. Elmer, W.H., Covert, S.F., O Donnell, K. 2007. Investigation of an outbreak of Fusarium foot and fruit rot of pumpkins within the United States. Plant Disease. 92:1142-1146. Imamura, Y., Chandra, J., Mukherjee, P.K., Lattif, A., Szczotka-Flynn, L.B., Pearlman, E., Lass, J.H., O Donnell, K., Ghannoum, M.A. 2007. Fusarium and Candida albicans biofilms on soft contact lenses: model development, influence of lens type and susceptibility to lens care solutions. Antimicrobial Agents and Chemotherapy. 52:171-182. Covert, S.F., Aoki, T., O Donnell, K., Starkey, D.E., Holliday, A., Geiser, D.M., Cheung, F., Town, C., Strom, A.L., Juba, A., Scandiani, M., Yang, X.B. 2007. Sexual reproduction in the soybean sudden death syndrome pathogen Fusarium tucumaniae. Fungal Genetics and Biology. 44(8):799-807. Peterson, S.W., Varga, J., Frisvad, J.C., Samson, R.A. 2008. Phylogeny and subgeneric taxonomy of Aspergillus. In: Varga, J., Samson, R.A., editors. Wageningen Academic Publishers: Wageningen, The Netherlands. p. 33-56. Torres, O.A., Palencia, E.R., Lopez De Pratdesaba, L., Grajeda, R., Fuentes, M., Speer, M.C., Merrill Jr, A.H., O Donnell, K., Bacon, C.W., Glenn, A.E., Riley, R.T. 2007. Estimated Fumonisin Exposure in Guatemala Greatest in Consumers of Lowland Maize. Journal of Nutrition. 137(12):2723-2729. Posada, F.J., Aime, M.C., Peterson, S.W., Rehner, S.A., Vega, F.E. 2007. Inoculation of coffee plants with the fungal entomopathogen Beauveria bassiana (Ascomycota: Hypocreales). Mycological Research. 111(6):748-757. Starkey, D.E., Ward, T.J., Aoki, T., Gale, L.R., Kistler, H.C., Geiser, D.M., Suga, H., Toth, B., Varga, J., O Donnell, K. 2007. Global molecular surveillance reveals novel Fusarium head blight species and trichothecene toxin diversity. Fungal Genetics and Biology. 44(11):1191-1204. Klich, M.A., Frisvad, J.C., Peterson, S.W., Varga, J., Geiser, D.M., Samson, R.A. 2007. The Current Status of Species Recognition and Identification in Aspergillus. Studies in Mycology. 59:1-10. Horn, B.W., Peterson, S.W. 2008. Host specificity of Eupenicillium ochrosalmoneum, E. cinnamopurpureum and two Penicillium species associated with the conidial heads of Aspergillus. Mycologia 100(1):12-19. Peterson, S.W. 2008. Phylogenetic analysis of Aspergillus species using DNA sequences from four loci. Mycologia. 100(2):205-226. Ward, T.J., Clear, R.M., Rooney, A.P., O Donnell, K., Gaba, D., Patrick, S., Starkey, D.E., Gilbert, J., Geiser, D.M., Nowicki, T.W. 2008. An adaptive evolutionary shift in Fusarium head blight pathogen populations is driving the rapid spread of more toxigenic Fusarium graminerarum in North America. Fungal Genetics and Biology. 45(4):473-484.