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United States Department of Agriculture

Agricultural Research Service

Research Project: DEVELOPMENT AND APPLICATION OF MOLECULAR PHYLOGENETICS OF FUNGI TO ENHANCE FOOD SAFETY AND FOOD SECURITY
2009 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, place species into genetically related groups (genera or subgenera). Second, define the genetic lineages (species or subspecies) that make up each group. Following this, gene-based systems are developed that identify species groups as well as the species within the groups. In FY08, the last of the yeast groups was characterized in which a multigene analysis was completed for species assigned to the genus Pichia. This analysis included those Pichia species that grow on methanol and are of biotechnological importance, as well as related species. From this multigene analysis, methanol assimilating species not previously transferred to the genus Ogataea were reassigned to this genus. Additionally, the genus Peterozyma was described for the species Pichia toletana and P. xylosa. Species of the food spoilage genus Citeromyces grouped with the genera Kuraishia, Nakazawaea and Pachysolen. Species of Aspergillus and Penicillium produce toxins in grains and other food products, which can severly inpact human and animal health. For FY08, the phylogeny of the genus Aspergillus and the ochratoxin-A (OTA) producing species in it has been completed and published. The databases generated in the phylogenetic study can now be exploited to develop DNA oligonucleotide probes that accurately identify the various species of Aspergillus and Penicillium that produce OTA contamination in foods and commodities. Ochratoxin-A is a regulated toxin in the European Union and some other parts of the world. Members of the Fusarium oxysporum species complex (FOSC) are ubiquitous soil borne pathogens responsible for vascular wilts, rots, and damping-off diseases of over 80 agronomically and horticulturally important crops. We constructed a two-locus database, comprising partial translation elongation factor (EF-1a) gene sequences and nearly full-length sequences of the nuclear ribosomal intergenic spacer region (IGS rDNA) for 850 isolates spanning the phylogenetic breadth of the FOSC. This database represents a unique resource for typing plant host-specific pathogens (i.e., formae speciales) and opportunistic pathogens of humans. Of the 850 isolates typed, 101 EF-1a, 204 IGS rDNA, and 257 two-locus sequence types (STs) were differentiated. Analysis of the combined dataset suggests that two-thirds of the STs might be associated with a single host plant. This analysis also revealed that the 26 STs associated with human mycoses were genetically diverse, including several which appear to be nosocomial in origin. In addition, experiments were conducted to assess the potential of genetically diverse members of the FOSC to produce moniliformin, fumonisin and enniatin mycotoxins in vitro. The impact of this work is to develop gene probes for rapid diagnosis of human and plant pathogens.


4.Accomplishments
1. PHYLOGENETIC RECOGNITION OF ASCOMYCETOUS YEAST GENERA. Known ascomycetous yeasts have been compared from multigene phylogenetic analyses, which has resulted in the grouping of species into genera based on their genetic relatedness. Most of the Zygosaccharomyces, cause food and beverage spoilage. With the assignment of species to genetically defined genera, DNA probes can be developed that detect numerous spoilage species with a single test.

2. MOLECULAR IDENTIFICATION OF OCHRATOXIN-A PRODUCING FUNGI. To date oligonucleotide primers complementary to specific polymorphisms in the RNA polymerase beta gene have been developed and tested using manual methods for a small number of OTA producing species from section Circumdati, including Aspergillus westerdijkiae, A. robustus and A. steynii. Initial studies indicate that other species require additional oligonucleotide probe development, including diversification to additional loci that can provide unequivocal identification. The impact of this work is to rapidly detect and identify molds that produce toxins in food and feed products.

3. IDENTIFICATION OF FUSARIUM BY GENE TYPING. Fusarium is the most important group of toxin producing phytopathogens, and the species infect all major agricultural plants causing crop loss and toxic contamination of crops. A comprehensive 2-locus DNA sequence typing scheme was developed for agriculturally and medically important members of the Fusarium oxysporum species complex, economically the most important group of pathogens within the genus. This database represents a unique resource for typing plant host-specific pathogens (i.e., formae speciales) and opportunistic pathogens of humans. These data will be incorporated into the web-accessible Fusarium-ID database for rapid identification of these pathogens, which will benefit plant pathologists, grain exporters and the medical community.


6.Technology Transfer

Number of Other Technology Transfer1

Review Publications
O Donnell, K., Ward, T.J., Aberra, D., Kistler, H.C., Aoki, T., Orwig, N.G., Kimura, M., Bjornstad, A., Klemsdal, S.S. 2008. Multilocus Genotyping and Molecular Phylogenetics Resolve a Novel Head Blight Pathogen within the Fusarium graminearum Species Complex from Ethiopia. Fungal Genetics and Biology. 45(11):1514-1522.

Kurtzman, C.P., Robnett, C.J., Basehoar Powers, E.A. 2008. Phylogenetic Relationships Among Species of Pichia, Issatchenkia and Williopsis Determined from Multigene Sequence Analysis, and the Proposal of Barnettozyma gen. nov., Lindnera gen. nov. and Wickerhamomyces gen. nov. Federation of European Microbiological Societies Yeast Research. 8(6):939-954.

O Donnell, K., Sutton, D.A., Fothergill, A., Mccarthy, D., Rinaldi, M.G., Brandt, M., Zhang, N., Geiser, D.M. 2008. Molecular Phylogenetic Diversity, Multilocus Haplotype Nomenclature, and In Vitro Antifungal Resistance within the Fusarium solani Species Complex. Journal of Clinical Microbiology. 46(8):2477-2490.

Sera, R., Peterson, S.W., Venancio, A. 2008. Multilocus Sequence Identification of Penicillium Species in Cork Bark During the Manufacture of Wine Bottle Stoppers. Research in Microbiology. 159(3):178-186.

Vega, F.E., Posada, F.J., Aime, M.C., Peterson, S.W., Rehner, S.A. 2008. Fungal endophytes in green coffee seeds. Mycosystema. 27:75-84.

Peterson, S.W., Horn, B.W. 2009. Penicillium parvulum and Penicillium georgiense sp. nov. Isolated from the Conidial Heads of Aspergillus Species. Mycologia. 101(1):71-83.

Schroers, H., Lamprecht, S.C., O Donnell, K., Kammeyer, P.L., Johnson, S., Sutton, D.A., Rinaldi, M.G., Summerbell, R.C. 2009. Taxonomy and Phylogeny of the Fusarium dimerum Species Group. Mycologia. 101(1):44-70.

Last Modified: 10/23/2014
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