2005 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? What 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), 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 foodborne pathogens (Listeria, Clostridium), 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, Listeria monocytogenes, has the highest hospitalization rate (92%) of any foodborne pathogen, is responsible for approximately one-quarter of foodborne disease-related deaths linked to known pathogens, and is the leading cause of product recalls. Field losses of crops from fungal and bacterial 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 insects include Bacillus thuringiensis and B. sphaericus, and species that control storage rots of fruits and vegetables are often yeasts in the genera Metschnikowia and Candida. Rapid, accurate identification of microorganisms is 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 and bacterial plant pathogens. Other National Programs benefiting from this research include NP-306, Quality and Utilization of Agricultural Products, NP-301, Plant Microbial, and Insect Genetic Resources, Genomics and Genetic Improvement, NP-304, Crop Protection and Quarantine, NP-305, Crop Production, NP-307, Bioenergy and Energy Alternatives, and NP-308, Methyl Bromide Alternatives. Each of these programs requires accurately identified microorganisms, which can be identified with the gene sequence databases developed in the current research project. The research directly supports NP-108 Food Safety priority objectives for microbial pathogens: 1.1.1.2. Develop methods that can be used both as survey methods and as speciation-molecular typing for epidemiological-ecological tracing. 1.3.1.1. Elucidate factors contributing to the population genetics and persistence of zoonotic pathogens. 1.3.1.2. Determine the factors allowing the pathogen to invade and become systemic. Outline the influence of host genetics, particularly resistance of the host to the pathogen. 1.3.1.3. Delineate virulence attributes leading to colonization. 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. Work on food spoilage/biocontrol yeasts and filamentous fungi is addressed as priority research under the following NP-108 and NP-303 Plant Diseases objectives: 1.1.1.1. Develop methodology to identify the fungi of concern. 1.1.1.2. Develop rapid methods to identify toxins for producers, regulatory and marketing agencies. 1.1.2.1. Determine the role and mechanism of action of the toxin in the pathogenesis of the fungus to the host plant. 1.1.2.5. Elucidate the genetic and molecular basis for toxin biosynthesis. Component I. Identification and classification of Fusarium Head Blight (FHB) pathogens of cereals. Component IV. Fusarium Head Blight pathogen biology, genetics, population dynamics, spread, and relationship with hosts and vectors (Corn Research need #1: monitoring of pathogens; Wheat Research Need #1: Fusarium Head Blight). 1.1.5.1. Develop biocontrol bacterial and fungal strains to control toxin produced by fungi on food crops. 2.4.1.2. Evaluate postharvest practices and pathogen survival relative to produce safety and quality. Develop methods to prevent spoilage and loss due to soft rot and fungal decay, to safely extend shelf life. 2.List the milestones (indicators of progress) from your Project Plan. This project was approved in 2003, but in order to be synchronized with other NP-108 projects, it was rewritten in 2005. The milestones reported here represent a 36 month period. Objective 1 – Listeria. Determine genotypes of lineages and individual strains of a lineage through comparative gene sequencing. Use sequences to develop multi-locus sequence typing (MLST) and microarrays for rapid detection of strains causing outbreaks of foodborne disease. Objective 2 – Clostridium. Determine strain polymorphisms for the cpe and plc genes through sequence analysis and analyze population structure to determine genotypes for disease outbreaks. Objective 3 – Fusarium. Develop multi-locus gene sequence database for global populations of A-trichothecene producers and use to develop molecular diagnostics for these populations. Objective 4 – Aspergillus and Penicillium. Develop multi-locus gene sequence database of ochratoxin-A (OA) producing species as the basis for developing a molecular detection system for these toxin producers. Objective 5 – Food spoilage and biocontrol yeasts. Conduct multigene sequencing of biocontrol species in the genus Metschnikowia and analyze species subtype populations of food spoilage yeasts in Torulaspora and Zygosaccharomyces. 4a.What was the single most significant accomplishment this past year? A system for rapid identification of microorganisms was developed. This system is based on the Luminex flow cytometer (Luminex Corporation, Austin, Texas), in which species-specific DNA probes are attached to small plastic spheres and reacted with the DNA of species to be identified. The technology allows up to 100 species-specific probes to be used in combination, which can be placed in a single well of a 96-well plate. Identification time, starting with a microbial culture, is 8 hours. The procedure has been successfully used to identify up to 30 different clinical yeasts (to be published in the September 2005 issue of the Journal of Clinical Microbiology), and is now being applied to detection of lineages of the foodborne pathogen Listeria monocytogenes, species of the plant pathogenic and mycotoxigenic genus Fusarium and species in the aflatoxin producing Aspergillus flavus complex. The successful application of this system is based on the large database of gene sequences determined by the staff of the Microbial Genomics and Bioprocessing Research Unit (MGB). 4b.List other significant accomplishments, if any. A single-well multilocus genotyping (MLGT) assay was developed for L. monocytogenes subtype identification based on nucleotide variation identified in 22 genes (23 Kilobase (Kb) of DNA sequence from 65 isolates) from seven regions of the Listeria chromosome. The current assay utilizes 59 SNP probes to distinguish among the closely related strains within lineage 1 of L. monocytogenes, which are responsible for the majority of human listeriosis outbreaks. Application of the assay to 175 additional lineage 1 isolates and comparison of MLGT results with strain histories demonstrated that the assay was able to uniquely identify isolates from each of the eight different listeriosis outbreaks examined, and was able to differentiate epidemiologically relevant groups of strains (serotypes 4b and 1/2b, and epidemic clones 1, 1a and 2). These results indicate that MLGT represents a significant new tool for use in epidemiological investigations as well as studies of the ecology, evolution, and population dynamics of L. monocytogenes. The goal of the research on Clostridium perfringens was to define distinct genetic lineages within this species on the basis of phylogenetic clustering patterns reconstructed from gene sequence data. Sequence data has been collected from seven genes (five housekeeping, two virulence) covering of a total of 6 Kb from each of 265 food, veterinary and human clinical isolates. Currently, the genes that we have sequenced (plc, colA, perfringolysin O regulator S (pfoS), histidine Kinase (hisK), gyrase subunit A (gyrA), rpoB, and ribosomal binding protien L7/L12 (rplL)) represent two distinct regions of the genome. The first region surrounds the origin of replication and extends from rpoB to colA and contains five of the seven genes that we have sequenced. The second region is on the opposite end of the genome (approximately 1.2 Megabase (Mb) from the other region). We plan to expand the number of genes from this region to 5 in order to obtain a total of 10 Kb of sequence data (approximately 5 Kb from each region). These additional data will be used to test for phylogenetic incongruence between genes within each genomic region and to test for incongruence between entire genomic regions. Over 100 strains representing food and beverage spoilage yeasts in the genus Zygosaccharomyces were identified to species from their unique gene sequences. Additional genes are being sequenced for each of the strains to determine population structure and genetic markers that may signal specificity for spoilage of particular food and beverage products. Three new food spoilage yeasts were described and placed in the genera Saturnispora, Tetrapisispora and Kregervanrija, the latter genus also newly described. Genetic boundaries of OA producing species in Aspergillus section Circumdati were determined through multi-locus DNA sequence analysis. Adding 200 new isolates from section Circumdati to the basic species set has revealed the presence of additional species that have been described by colleagues in Europe. OA is regulated in many countries, and American fruits and coffee are potential targets for the OA producing molds. OA is the putative cause of kidney failure of human populations chronically exposed to this mycotoxin. We have used multilocus DNA sequence data and multiplex polymerase chain reaction (PCR) assays to establish a baseline of FHB species and trichothecene toxin chemotype diversity globally. These studies have shown for the first time that the primary etiological agent of FHB, Fusarium graminearum, comprises at least 12 phylogenetically distinct and biogeographically structured species worldwide. Although F. graminearum accounts for over 99% of FHB within North America, three additional Fg clade species, including two recently introduced foreign FHB pathogens, were detected in our survey of FHB within the U.S. To facilitate communication among scientists within the FHB community and quarantine specialists, eight unnamed species within the Fusarium graminearum (Fg) clade resolved by our multilocus phylogeny were formally described (O’Donnell et al. 2004). Development of robust PCR multiplex and SNP-based molecular tools for Fg clade species identification and chemotype determination have significantly improved global monitoring efforts and disease surveillance, thereby making available for the first time detailed information on the host and geographic distributions of FHB pathogens and their trichothecene chemotypes. Such knowledge is critical for enhancing our knowledge of the ecology, epidemiology and population biology of these mycotoxigenic cereal pathogens. Our studies are the first to alert plant breeders charged with developing wheat and barley cultivars with broad-based resistance to FHB that the morphospecies Fusarium graminearum comprises at least 12 phylogenetically distinct and biogeographically structured species and that half of these species are still segregating for trichothecene toxin chemotype. Furthermore, our studies alert plant disease specialists and quarantine officials that only a fraction of the FHB pathogen and toxin chemotype diversity is currently represented within North America. Providing names for the newly discovered FHB pathogens should greatly facilitate communication among scientists within the FHB community, including quarantine specialists. Moreover, the unique multilocus DNA sequence database we have developed is currently being interrogated to develop the first SNP microsphere array for the high-throughput identification of all known B-trichothecene toxin-producing FHB species and their toxin chemotypes in order to improve disease surveillance efforts and to facilitate a greater understanding of the ecology, epidemiology and population dynamics of these FHB pathogens. This research has been funded in part by competitive research grants with the U.S. Wheat and Barley Scab Initiative (USWBSI) grant and the U.S. Department of Agriculture (USDA), Cooperative State Research Education Extension Service (CSREES), National Research Initiative (NRI), Competitive Grants Program and with the aid of an administer post-doc position. 4c.List any significant activities that support special target populations. None 5.Describe the major accomplishments over the life of the project, including 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, Listeria, Clostridium, Bacillus and related genera, 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. 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. For example, accomplishments from work on the current project’s objective for Listeria include the development of a large (> 450 isolates), diverse, and well-characterized (described above) collection of isolates from human, veterinary, food, and environmental sources. In addition, a robust virulence-gene phylogeny for L. monocytogenes was developed from a comparative DNA sequence database of the positive regu8latory factor A virulence gene cluster (pVGC) sequences (112 isolates; 6 genes; approximately 950,000 basepairs (bp) of comparative sequence data). Evolutionary analyses of these data demonstrated that pVGC alleles from L. monocytogenes lineages most frequently (lineage. 1)and least frequently (lineage. 3)associated with human listeriosis share a common ancestor exclusive of lineage 2 isolates, and revealed for the first time that the human epidemic associated serotype 4b is prevalent among strains from lineages 1 and 3 (Ward et al. 2004). This result demonstrated that serotype 4b strains did not represent a single monophyletic subtype and that serotype identification could not be used as a proxy for lineage identification in this case. In addition, a PCR-based test for lineage identification was developed and used in a survey of food products demonstrating that lineage 1 is overrepresented relative to lineage 2 in human listeriosis cases. This molecular surveillance work also indicated that the infrequent association of lineage 3 isolates with human listeriosis is likely due to rarity of exposure, not reduced virulence as has been previously suggested. Finally, analyses of pVGC sequences were used to determine that the epidemic-associated lineage 1 has a more recent coalescence and significantly less genetic variation than lineages 2 or 3. These findings are significant because they suggest that these lineages may have limited demographic exchangeability and could represent independent species under the cohesion species concept. The results from this work are significant in that they challenge previous assumptions regarding lineage prevalence and evolution, which have very different regulatory implications. 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 will be expanded for Fusarium graminearum/Gibberella zeae 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. USWBSI grant titled “Global Molecular Surveillance of FHB Species and Their Mycotoxin Potential” awarded to T. J. Ward and K. O’Donnell. C. P. Kurtzman and S.W. Peterson serve as Co-Principal Investigators on an NIH grant awarded to a cooperator at the University of Miami. This grant, which expired July 31, 2005, was to develop means for rapid detection of pathogenic molds and yeasts using the National Center of Agricultural Utilization Research (NCAUR)-developed gene sequence databases. The technology developed is being applied to detection of foodborne bacteria and mycotoxigenic fungi. 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). - “Peoria ag lab focusing on new crop fungus-fighting technologies” published in FarmWeek, September 13, 2004. - “Saving lives with snakes” published in the Peoria Journal Star, April 14, 2005. - “DNA test may zap Fusarium” published in Feedstuffs, June 6, 2005. - “Scientists develop fast bacteria detector” published in Food Productivity online, July 2005. - "It's the yeast they could do: Ag Labe develops test to check for 'bugs' that cause spoilage" published in the Peoria Journal Star, August 2, 2005. Review Publications Kurtzman, C.P., Statzell-Tallman, A., Fell, J.W. 2004. Tetrapisispora fleetii sp. nov., an ascosporogenous yeast in the saccharomycetaceae. Studies in Mycology. 50:397-400. Rooney, A.P., Ward, T.J. 2005. Evolution of a large Ribosomal RNA multigene family in filamentous fungi: Birth and death of concerted evolution paradigm. Proceedings of the National Academy of Sciences. 102(14):5084-5089. Martinelli, J.A., Bocchese, C.A., Xie, W., O Donnell, K., Kistler, H.C. 2004. Soybean Pod Blight and Root Rot Caused by Lineages of the Fusarium graminearum and the Production of Mycotoxins. Fitopatologia Brasileira. 29:492-498. Seabury, C.M., Honeycutt, R.L., Rooney, A.P., Halbert, N.D., Derr, J.N. 2004. Prion protein gene (PRNP) variants and evidence for strong purifying selection in functionally important regions of bovine exon 3. Proceedings of the National Academy of Sciences. 101(42):15142-15147. O Donnell, K., Sutton, D.A., Rinaldi, M.G., Magnon, K.C., Cox, P.A., Revankar, S.G., Sanche, S., Geiser, D.M., Juba, J.H., Van Burik, J.H., Padhye, A., Robinson, J.S. 2004. Genetic diversity of human pathogenic members of the fusarium oxysporum complex inferred from gene genalogies & aflp analyses: evidence for the recent dispersion of a geographically widespread clonal lineage & nosocomial orig. Journal of Clinical Microbiology. 42(11):5109-5120. Kurtzman, C.P., Robnett, C.J., Ward, J.N., Brayton, C., Gorelick, P., Walsh, T.J. 2005. Multigene phylogenetic analyses of pathogenic Candida species in the Kazachstania (Arxiozyma) telluris complex and the description of their ascosporic states as Kazachstania bovina sp. nov., K. heterogenica sp. nov., K. pintolopesii sp. nov., and K. slooffiae sp. nov. Journal of Clinical Microbiology. 43(1):101-111. Liu, Z., Slininger, P.J., Dien, B.S., Berhow, M.A., Kurtzman, C.P., Gorsich, S.W. 2004. Adaptive response of yeasts to furfural and 5-hydroxymethylfurfural and new chemical evidence for hmf conversion to 2,5-bis-hydroxymethylfuran. Journal of Industrial Microbiology and Biotechnology. 31:345-352. Available http://dx.doi.org/10.1007/s10295-004-0148-3. Kurtzman, C.P. 2005. Description of Komagataella phaffii sp. nov. and transfer of Pichia pseudopastoris to the methylotrophic yeast genus Komagataella. International Journal of Systematic and Evolutionary Microbiology. 55:973-976.