2006 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Human and animal health and well-being are markedly impacted by agricultural products contaminated with pathogens and microbially produced toxins, and the agricultural economy can be devastated by product losses caused by plant pathogens, fungal toxins, and foodborne pathogens. We are producing genetic information and molecular tools required to enhance the safety and quality of the U.S. food supply and the productivity of U.S. agriculture by resolving outstanding taxonomic issues that impact regulatory policy [e.g. Food Safety Inspection Service (FSIS), Food and Drug Administration (FDA), U.S. Centers for Disease Control and Prevention (CDC)], developing molecular identification and subtyping methods for use in pathogen surveillance and outbreak detection, and characterizing the population genetics and ecology of food and beverage spoilage yeasts (Zygosaccharomyces, Torulaspora, Candida, Pichia) and mycotoxigenic and plant pathogenic fungi (Fusarium, Aspergillus, Penicillium). These organisms have a significant deleterious impact on human and animal health and the agricultural economy. For example, field losses of crops from fungal diseases and storage losses from toxin producing molds such as Fusarium, Aspergillus and Penicillium cost the U.S. billions of dollars annually. Furthermore, wheat scab fusaria, most notable the Fusarium graminearum species complex, caused over $2.6 billion in losses to American farmers between the years 1991 and 1997. In addition, food and feed contaminated by toxins and pathogens are a serious threat to human and animal health worldwide. Chemical pesticides are widely used in agriculture, but some pose a serious threat to the environment. Development of biological control agents for pests and the inhibition of spoilage organisms can represent a safe alternative to chemical pesticides. Commonly used biocontrol species for storage rots of fruits and vegetables are often yeasts in the genera Metschnikowia and Candida. Rapid, accurate identification of microorganisms and their tracking in the environment are essential for solution to the preceding problems. This can only come from development and widespread use of molecular genetic diagnostic technologies such as those being developed by our scientists. The main focus of this work is on National Program (NP) 108, Food Safety, but NP-303, Plant Diseases, benefits from the molecular databases being developed that allow detection of fungal plant pathogens. The research directly supports NP-108 Food Safety and Security priority objectives as well as those of NP-303 Plant Diseases. The Project Plan focuses on the use of genomics information for the development and construction of detection technologies for fungal pathogens and their products as well as food spoilage and biocontrol yeasts of critical importance in food safety and food security under Sections 1.2.1 Detection and Validation; 1.2.3 Production and Processing Ecology; 1.2.5 Omics; 1.2.7 Risk Assessment; 1.2.8 Pathogenicity; 1.2.9 Food Security; 2.2 Crop/Fungal/Toxin Relationships; and 2.5 Biocontrol Strategies in the 2006-2010 NP-108 Action Plan. The research addresses Agency Performance Measure 3.1.2: Develop and transfer to Federal agencies and the private sector, systems that rapidly and accurately detect, identify, and differentiate the most critical and economically important foodborne microbial pathogens. 2.List by year the currently approved milestones (indicators of research progress) This project was rewritten and approved in 2005, in order to be synchronized with other NP-108 projects, and replaces and extends the yeast and mold component of 3620-42000-031-00D. FY2006: - Conduct Surveys for Fusarium Head Blight (FHB) and soybean sudden death syndrome (SDS) pathogens. - Conduct miltilocus DNA sequence analyses of FHB and SDS pathogens. - Initiate mycotoxin analyses and pathogenicity testing. - Conduct multilocus sequencing and analysis of the Aspergillus ochraceus group. - Develop multilocus DNA sequence database for Zygosaccharomyces. - Develop multilocus DNA sequence database for Metschnikowia and related species. FY2007: - Continue surveys and identify novel FHB and SDS pathogens through analyses of multilocus DNA sequence data. - Identify single nucelotide polymorphisms (SNPs) from comparative DNA sequence data for species-specific detection of FHB and SDS pathogens. - Continue mycotoxin analyses and pathogenicity testing. - Conduct multilocus sequencing and analysis of the terverticillate Penicillium species producing ochratoxin-A (OTA). - Develop multilocus DNA sequence database for Torulaspora and Zygotorulaspora. - Continue development of multilocus DNA sequence database for Metschnikowia and related species. FY2008: - Validate SNP microsphere arrays for FHB and SDS pathogens. - Conduct global molecular surveillance of FHB and SDS pathogens using validated SNP microsphere array. - Continue mycotoxin analyses and pathogenicity testing. - Develop probes for identification of OTA producing species in the Aspergillus ochraceus group. - Develop multilocus database for Dekkera and Brettanomyces. - Determine population structure of species in the Metschnikowia clade. FY2009: - Expand SNP microsphere arrays to accommodate newly discovered FHB and SDS pathogens. - Continue global molecular surveillance of FHB and SDS pathogens using validated SNP microsphere array. - Continue mycotoxin analyses and pathogenicity testing. - Conduct multilocus sequencing and analysis of the Aspergillus niger group. - Determine population structure of species in preceding genera and test lineages for resistance to preservatives. Develop microsphere assay for detection of lineages and species of food and beverage spoilage yeasts. - Develop a robust phylogeny of the Metschnikowia clade. Test lineages for efficiency of biocontrol. Develop microsphere assay for detection of lineages and species of the Metschnikowia clade. FY2010: - Expand microsphere assay for newly discovered FHB and SDS pathogens to continue global surveillance. Complete mycotoxin and pathogenicity analyses. - Develop DNA probes for all OTA producing species. - Develop robust species phylogeny for food spoilage and biocontrol yeasts and complete testing of a rapid detection assay. 4a.List the single most significant research accomplishment during FY 2006. Identify Human Pathogenic Species of Fusarium. Species of Fusarium associated with a worldwide outbreak of human ocular keratitis were identified from multigene DNA sequence analysis. The study was initiated at the request of the CDC and showed that several species of Fusarium were the cause of this outbreak of keratitis. Accurate identification was possible only because of the extensive Fusarium gene sequence database that was developed at the National Center for Agricultural Research (NCAUR). This study will be reported in a 2006 issues of the Journal of American Medical Association. This fully supports NP-108 Food Safety (animal and plant products) Component 1.1.1.2 Develop methods that can be used both as survey methods and as speciation-molecular typing for epidemiological-ecological tracing. 4b.List other significant research accomplishment(s), if any. Molecular Identification of Aspergillus Mycotoxin Producers. Ochratoxin-A (OTA) producing species in section Circumdati have been well characterized by multilocus DNA sequence phylogenetic analysis. Oligonucleotide markers are being developed and tested for the rapid identification of the OTA producing species. Penicillium species living endophytically in coffee plants have been characterized taxonomically and tested for OTA production. Very low levels of OTA have been found in a few isolates. Aspergillus species in coffee have also been characterized taxonomically and from the viewpoint of OTA production. We have found that parasites that have been introduced into coffee growing regions to control the "broca" beetle are themselves vectors of Aspergillus westerdijkiae, a major producer of OTA. A more thorough study of the ecology of coffee and its parasites and pathogens is needed to establish relative risks from different methods used to improve coffee quality. This accomplishment supports NP-108 Food Safety (animal and plant products) component 1.1.1.2 Develop methods that can be used both as survey methods and as speciation-molecualr typing for epidemiological-ecological tracing. Molecular Identification of Food Spoilage and Biocontrol Yeasts. Food spoilage species of Zygosaccharomyces have undergone gene sequencing (26S ribosomal DNA and elongation factor (EF) 1-apha) to determine species and subspecies. These genetic lineages will be correlated with types of food spoilage and natural habitats. Similarly, biocontrol species of Metschnikowia and related genera (Clavispora, some species of Candida) were compared from 26S rDNA and EF-1-alpha to provide a genetic framework of species relationships. This accomplishment supports NP-108 Food Safety (animal and plant products) component 1.1.5.1 Develop biocontrol bacterial and fungal strains to control toxin produced by fungi on food crops; and 2.1.1 Agriculture Research Service (ARS) should take the lead to develop methodologies that have both regulatory, industry and research use: a commonality of interests between government and stakeholders. Include survey methods and molecular typing methods for trace back. Molecular Diversity of Fusarium Head Blight Pathogens. Through our ongoing survey of genetic diversity within the B-trichothecene lineage of FHB pathogens (B-FHB) we identified two novel species (Fusarium gerlachii and Fusarium vorosii) within the Fusarium graminearum (Fg) species complex. One of these is the first novel FHB species identified from the U.S., and our phylogenetic analyses demonstrate that it is the sister group to F. graminearum. In addition, we determined that isolates from this previously unrecognized species have a nivalenol (NIV) chemotype that may present a greater threat to food safety than the 15ADON-producing F. graminearum isolates that predominate in the U.S. The other new species was originally isolated from FHB-infected wheat in Europe, but additional isolates have been identified from Japan. This species is most closely related to F. asiaticum, and isolates tested to date have a 15ADON chemotype. In addition, our molecular surveillance identified a novel, and highly divergent, evolutionary lineage of F. graminearum from the Gulf Coast of the U.S. While these isolates appear to be distinct from other F. graminearum, some gene-genealogies suggest gene flow between these groups. Alternatively, the Gulf Coast isolates may represent a novel species that has retained limited ancestral polymorphism shared with F. graminearum. Population genetic analyses and specific tests of gene flow between the Gulf Coast isolates and other F. graminearum are proposed to address this question and to determine the extent to which this previously unrecognized group of isolates contributes to FHB in the U.S. This accomplishment fully supports NP-303 Plant Diseases component IV Wheat Research Need #1 - Fusarium Head Blight. Rapid Identification of Fusarium Head Blight Pathogens. We have completed the development and validation of a high-throughput single tube assay for the simultaneous identification of all B-FHB species and chemotypes via flow cytometry. This multilocus genotyping (MLGT) assay consists of 44 probes targeting single nucleotide differences unique to individual species or chemotypes, and was validated using a panel of isolates with known chemotype and species identity. To date, the assay has been used to determine species and chemotype for 1,000 isolates from North America, Asia, Africa and Europe. As a result, we have identified a trichothecene chemotype cline across Canada and yearly increases of the 3ADON chemotype within North America, suggesting that 3ADON isolates have a selective advantage in the Northeast U.S. and Canada. In addition, through collaborations with Randy Clear (Canadian Grain Commission), we have determined that Canadian F. graminearum with a 3ADON chemotype produce significantly (P < 0.001) higher levels of trichothecenes than those with a 15ADON chemotype, suggesting a possible explanation for the increasing frequency of 3ADON isolates. Our active surveillance using the MLGT assay, conducted in collaboration with the Cereal Disease Lab, St. Paul, Minnesota, also revealed for the first time the presence of F. asiaticum (f = 30%) and NIV-producing F. graminearum (f = 33%) in the Southern U.S. Taken together, the results of our molecular surveillance suggest large-scale changes in B-FHB pathogen composition are taking place in North America. This finding has significant implications for disease control efforts and regulatory policy regarding trichothecene-contaminated grain. The MLGT assay provides significant new capabilities for pathogen surveillance that will further our understanding of the ecology and epidemiology of FHB pathogens. This accomplishment fully supports NP-303 Plant Diseases component IV Wheat Research Need #1 - Fusarium Head Blight. 4c.List significant activities that support special target populations. None. 5.Describe the major accomplishments to date and their predicted or actual impact. The major accomplishments of this project are the development of molecular diagnostic tools and databases for rapid detection and accurate identification of species in the following microbial groups: Fusarium, Aspergillus, Penicillium, and ascomycetous yeasts. These databases allow, for the first time, rapid, accurate identification of species, and this is essential to progress in food safety, plant pathology, plant breeding, mycotoxin research, biocontrol, and bioconversion, the focal areas of our work. These databases are being used by clinicians because human and animal pathogenic species are found within the groups studied, and they have the potential for use by Animal, Plant Health Inspection Service (APHIS), FSIS and other Federal agencies concerned with the import and movement of hazardous microorganisms. The databases are also being used at NCAUR and elsewhere to develop rapid tests based on DNA probes for use in clinical and food quality and safety laboratories. In addition, the preceding work has led to the discovery of over 100 new microbial species, which are being formally described. Many of the species have already been shown to be important to agriculture. This accomplishment supports NP-108 Food Safety (animal and plant products) component 1.1.5.1 Develop biocontrol bacterial and fungal strains to control toxin produced by fungi on food crops; and 2.1.1 Agriculture Research Service (ARS) should take the lead to develop methodologies that have both regulatory, industry and research use: a commonality of interests between government and stakeholders. Include survey methods and molecular typing methods for trace back. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The species-specific rDNA sequence database for ascomycetous yeasts is now available to scientists and industry through GenBank, the National Institutes of Health (NIH) genetic sequence database, and is being used commercially by two companies in the U.S. and one in Japan. Electronically portable multigene DNA sequence databases are being expanded for Fusarium graminearum/Gibberella zeae and other Fusarium species to rapidly and accurately identify these pathogens for the first time. USDA-CSREES-NRI-Competitive Grants Program grant titled "A Molecular Database of Fusarium mycotoxicology" awarded to a cooperator at Pennsylvania State University with K. O'Donnell as Co-Principal Investigator. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). "Eye Specialists See More Cases of Fungal Disease," published in the Wall Street Journal, April 6, 2006. This is a newly initiated project. Publications are listed on the report for project 3620-42000-031-00D, which has now been replaced.