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United States Department of Agriculture

Agricultural Research Service

2010 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
The primary goal of this research is to develop rapid, accurate methods utilizing gene sequences for detection and identification of yeasts that cause food spoilage or are used for biocontrol, and for filamentous fungi that produce toxic compounds and/or cause plant diseases. This research has two approaches. First, genetically define the lineages (species or subspecies) of the spoilage organisms and pathogens. Following this, gene-based systems are used to place the species in genetically defined groups such as genera. Using these data, rapid DNA-based systems are developed that identify species and groups of species. Correct identification is essential for solving problems in food spoilage, plant disease and formation of toxins by fungi. Progress toward accomplishing these objectives includes the following: .
1)Multigene analysis was completed for the ascomycetous yeasts resulting in redefinition of present genera and description of nearly 20 new genera. Included in the 100 genera now recognized are many groups that cause food spoilage. Multigene analysis was applied to 400 strains assigned to the genus Metschnikowia to recognize species in this genus that are used for biocontrol and to correlate biocontrol activity with genetically defined species; .
2)A multigene-based phylogeny of the genus Aspergillus, which includes Ochratoxin-A (OTA) producing species, has been completed and published. Species of Aspergillus and Penicillium produce toxins in grains and other food products, which can severely affect human and animal health. The databases generated in the phylogenetic study are being used to develop DNA-based probes that accurately identify the species that produce OTA contamination in foods and commodities. OTA is a regulated toxin in the European Union and some other parts of the world and its presence in U.S. produced commodities can affect exports; .
3)We constructed a gene sequence database for 850 isolates spanning the phylogenetic breadth of the Fusarium oxysporum species complex (FOSC). Members of the FOSC are ubiquitous soil borne pathogens responsible for vascular wilts, rots, and damping-off diseases of over 80 agronomically and horticulturally important crops. In addition, some species of Fusarium produce potent toxins. This database represents a unique resource for typing plant host-specific pathogens and opportunistic pathogens of humans. Of the 850 isolates compared, 562 strains were genetically differentiated using two genes. This analysis also revealed that Fusarium isolates associated with human mycoses are genetically diverse. Experiments were also conducted to assess the potential of genetically diverse members of the FOSC to produce the mycotoxins moniliformin, fumonisin and enniatin. The purpose of this work is to develop gene probes for rapid detection of Fusarium plant pathogens, but because some Fusarium species are human pathogens, the gene sequence database developed has immediate use in the diagnosis of human disease.

1. PHYLOGENETIC RECOGNITION OF FUSARIUM PLANT PATHOGENS BY GENE TYPING. The fungus Fusarium is pathogenic to a large number of agricultural crops such as Fusarium head blight (FHB) of cereals, which results in large annual grain losses and accumulation of toxin in the harvested wheat. The only accurate method for identifying the species is by examination and comparison of gene sequences. Bacterial Foodborne Pathogens and Mycology Research Unit scientists at the National Center for Agricultural Utilization Research in Peoria, IL, developed gene assay probes for the detection and identification of two novel FHB pathogens. These probes will facilitate the identification of fungal strains in the field and allow early intervention for prevention of toxin contamination in food and feed.

2. IDENTIFICATION OF OCHRATOXIN A PRODUCING FUNGI FROM GENE SEQUENCES. The toxin Ochratoxin A (OTA) is often found in foods and beverages as a result of contaminating species of the fungi Aspergillus and Penicillium. Proper identification of the fungus is critical to preventing this toxin from entering the food supply. Because these fungi are so similar in appearance, accurate identification is only possible from examination of the gene sequences. Bacterial Foodborne Pathogens and Mycology Research Unit scientists at the National Center for Agricultural Utilization Research in Peoria, IL, investigated the DNA sequences from numerous fungal isolates and developed a database that can now be used to accurately identify toxin producing species as well as look-alike non-toxin producing species. This work will allow food producers to develop better strategies to identify fungal contamination and to prevent toxin production.

3. MOLECULAR GENETIC DIVERSITY OF BIOCONTROL YEASTS. Species of the yeast genus Metschnikowia are used internationally to control mold-induced rots of fruits. There are numerous species of Metschnikowia that are difficult to differentiate. To provide accurate identification, Bacterial Foodborne Pathogens and Mycology Research Unit scientists at the National Center for Agricultural Utilization Research in Peoria, IL, developed a diagnostic database of DNA sequences from four genes and tested the database on 400 strains that include all known species. From this database, diagnostic probes are being developed to identify the species used by producers for biocontrol of fruit rots and to follow survival and dispersal of the biocontrol species in the field. This work will facilitate application of biocontrol technology to prevent fruit storage rots.

Review Publications
Kurtzman, C.P., Suzuki, M. 2010. Phylogenetic analysis of ascomycete yeasts that form coenzyme Q-9 and the proposal of the new genera Babjeviella, Meyerozyma, Millerozyma, Priceomyces and Scheffersomyces. Mycoscience. 51(1):2-14.

Yli-Mattila, T., Gagkaeva, T., Ward, T.J., Aoki, T., Kistler, H.C., O Donnell, K. 2009. A Novel Asian Clade Within the Fusarium graminearum Species Complex Includes a Newly Discovered Cereal Head Blight Pathogen from the Far East of Russia. Mycologia. 101(6):841-852.

O Donnell, K., Gueidan, C., Sink, S.L., Johnston, P.R., Crous, P., Glenn, A.E., Riley, R.T., Zitomer, N.C., Colyer, P., Waalwijk, C., Van Der Lee, T., Moretti, A., Kang, S., Kim, H., Geiser, D.M., Juba, J., Baayen, R.P., Cromey, M.G., Bithel, S., Sutton, D.A., Skovgaard, K., Ploetz, R., Kistler, H.C., Elliott, M., Davis, M., Sarver, B.A. 2009. A Two-locus DNA Sequence Database for Typing Plant and Human Pathogens Within the Fusarium oxysporum Species Complex. Fungal Genetics and Biology. 46(12):936-948.

Kurtzman, C.P., Robnett, C.J. 2010. Systematics of Methanol Assimilating Yeasts and Neighboring Taxa from Multigene Sequence Analysis and the Proposal of Peterozyma gen. nov., a New Member of the Saccharomycetales. Federation Of European Microbiological Societies Yeast Research. 10(3):353-361.

O Donnell, K., Sutton, D.A., Rinaldi, M.G., Gueidan, C., Crous, P.W., Geiser, D.M. 2010. A Novel Multi-Locus Sequence Typing Scheme Reveals High Genetic Diversity of Human Pathogenic Members of the Fusarium incarnatum-F. equiseti and F. chlamydosporum Species Complexes within the U. S. Journal of Clinical Microbiology. 47(12):3851-3861.

O Donnell, K., Sink, S.L., Scandiani, M.M., Luque, A.M., Colletto, A., Biasoli, M., Lenzi, L., Salas, G., Gonzalez, V., Ploper, L.D., Formento, N., Pioli, R.N., Aoki, T., Yang, X.B., Brice, A.J. 2010. Soybean Sudden Death Syndrome Species Diversity within North and South America Revealed by Multilocus Genotyping. Phytopathology. 100(1):58-71.

Schoch, C., Blackwell, M., Bonito, G., Castlebury, L.A., Crous, P., Geiser, D., Lutzoni, F., O Donnell, K., Rossman, A.Y., Spatafora, J. 2009. The Ascomycota tree of life: A phylum wide phylogeny clarifies the origin and evolution of fundamental reproductive and ecological traits. Systematic Biology. 58(2):224-239.

Vega, F.E., Simpkins, A., Aime, M., Posada, F., Peterson, S.W., Rehner, S.A., Infante, F., Castillo, A. 2009. Fungal endophyte diversity in coffee plants from Colombia, Hawaii, Mexico and Puerto Rico. Fungal Ecology. 3:122-138.

Peterson, S.W., Jurjevic, Z., Bills, G.F., Stchigel, A.M., Vega, F.E. 2010. The Genus Hamigera, Six New Species and Multilocus DNA Sequence Based Phylogeny. Mycologia. 102(4):847-864.

Taskin, H., Buyukalaca, S., Dogan, H.H., Rehner, S.A., O Donnell, K. 2010. A multigene molecular phylogenetic assessment of true morels (Morchella) in turkey. Fungal Genetics and Biology. 47(8):672-682.

Last Modified: 11/30/2015
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