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United States Department of Agriculture

Agricultural Research Service

Research Project: MANAGEMENT AND GENETIC CHARACTERIZATION OF AGRICULTURAL AND BIOTECHNOLOGICAL MICROBIAL RESOURCES
2013 Annual Report


1a.Objectives (from AD-416):
Objective 1: Strategically expand the genetic diversity in the ARS Culture Collection and improve associated information for priority microbial genetic resources. Sub-objective 1.A. Acquire from diverse sources samples of food-borne pathogenic bacteria, actinobacteria from equine sources, basidiomycetous yeasts, plant pathogenic fungi, and grain storage molds to fill current gaps in the ARS Culture Collection for these priority microbial strains. Sub-objective 1.B. In consultation with the microbial research community, identify microbial genetic resources associated with discontinued research programs, or held by researchers who are nearing retirement, and attempt to acquire those of strategic importance to current or future agricultural research programs. Objective 2: Conserve priority microbial genetic resources efficiently and effectively, and distribute them and associated information worldwide. Sub-objective 2.A. Conserve more than 90,000 accessions of priority microbial genetic resources and associated information, emphasizing food-borne pathogenic bacteria, actinobacteria from equine sources, basidiomycetous yeasts, plant pathogenic fungi, and grain storage molds, as well as microbes of biomedical and biotechnological importance. Sub-objective 2.B. Back-up at the National Center for Genetic Resources Preservation (NCGRP) approximately 15,000 strains (emphasizing Fusarium) that are currently preserved under liquid nitrogen vapor only at the National Center for Agricultural Utilization Research (NCAUR). Sub-objective 2.C. Continue to improve data management and technology transfer procedures, emphasizing improvements in user interface for the public access catalog system. Sub-Objective 2.D. Distribute on request microbial accessions and information that meet the specific needs of agricultural, biomedical, and biotechnological researchers. Objective 3: Strategically characterize (“genotype”) and evaluate (“phenotype”) priority microbial genetic resources through multigene analyses, and with key morphological, physiological, and biochemical descriptors. Sub-objective 3.A. Develop and apply multigene markers for phylogenetic and genetic diversity analyses of priority microbial genetic resources. Incorporate characterization data into GRIN and/or other databases, and apply the data to providing accurate taxonomic identifications, as well as to predicting the agricultural and biotechnological utility of newly discovered taxa. Sub-objective 3.B. Determine the phenotypic diversity and elucidate the population genetic structure for the Fusarium Head Blight (FHB) species Fusarium graminearum and F. asiacticum. Map their worldwide distribution, as a first step of establishing a molecular surveillance system for the early detection of Fusarium populations introduced to North America.


1b.Approach (from AD-416):
New species and novel strains of known species of plant pathogens and mycotoxigenic fungi, food-borne pathogens, actinobacteria important to animal health and biotechnology, and yeasts will be isolated from nature or acquired from reports in the literature and from cooperators worldwide. New strain accessions will be cataloged in the collection database, preserved by lyophilization and/or freezing in liquid nitrogen vapor where appropriate, and information related to well characterized strains will be made publicly available on the Collection website. Information provided by ARS Program leadership, national and international microbiology societies and culture collection organizations will identify microbial collections in danger of being lost and important collections will be acquired and accessioned where existing resources permit. The entire collection is secured in a limited access room and records for strain inventory and distribution are maintained on the collection database system. High priority microbial strains held only as frozen preparations under liquid nitrogen vapor phase will be duplicated and shipped to NCGRP for backup in a liquid nitrogen freezer dedicated for microbial germplasm. Strains will be freely distributed to the scientific community worldwide but requestors must provide required documentation or permits before animal or plant pathogenic strains or those requiring Biosafety Level II confinement are distributed. Through phylogenetic analysis of sequences from multiple gene loci, evaluate the diversity and systematics of actinomycetes, Bacillus, Aspergillus, and yeasts of importance to agriculture, food safety, and biotechnology. A multilocus genotyping assay previously developed for identification of FHB species and trichothecene chemotypes will be applied to a global collection of FHB isolates to determine the current distribution and trichothecene chemotype diversity of Fusarium graminearum and F. asiacticum populations. Population diversity and relatedness will then be assessed using a published panel of variable number tandem repeat markers. Differences in pathogen fitness and aggressiveness in individual populations will be evaluated by determining a range of phenotypic characteristics, such as growth, reproduction, and toxin production.


3.Progress Report:
The objectives of this project were designed to enhance the quality, diversity, and utility of the ARS Culture Collection holdings, and to produce and support microbiological research that advances agricultural production, food safety, public health, and economic development. Over the course of the project, we accessioned 10,118 isolates into the general collection and 667 isolates into the ARS Patent Culture Collection. The ARS Patent Culture Collection distributed 1,945 isolates, including 1,140 domestically and 845 to foreign scientists. More than 39,700 isolates were distributed from the general collection, including 6,422 isolates sent to ARS scientists, 22,965 isolates sent to other scientists within the United States, and 10,313 isolates sent to scientists in 76 different countries. Using a conservative estimate of monetary value based on fees charged by other major culture collections, strain distributions from the general collection during this project cycle represent an $8 million in-kind contribution in support of microbiological research and biotechnological innovation. The impact of this contribution is evidenced through the citation of strains from the ARS Culture Collection in 3,515 publications and 2,350 patent applications during this period. The addition of an online strain request module for the ARS Culture Collection database greatly facilitated public access to strains and request processing. A full time collection staff was developed, improving operations and significantly advancing efforts to generate a comprehensive electronic inventory. Approximately 48,230 inventory records have been added or updated and 3,544 previously uncataloged strains have been added to the permanent collection. These activities provide for continued preservation of agriculturally and biotechnologically significant microbial germplasm and distribution to researchers in ARS and throughout the world.

Accurate identification of microbial isolates is a critical requirement for agricultural, medical, and industrial science. Development and testing of novel DNA sequence databases for rapid, accurate identification of microbial groups represented in the ARS Culture Collection provided tools to make accurate identification of microbes accessible to a wider variety of scientists, including non-specialists. Gene sequence databases were compiled and evaluated for identification of several microbial groups and provide an invaluable resource for strain identification and pathogen surveillance by researchers worldwide. Original research detailing microbial diversity is represented in 61 peer-reviewed papers and 42 authoritative book chapters including the discovery and description of more than 14 new species of filamentous fungi important in food safety, plant pathology, environmental, veterinary, or human health, as well as numerous new yeast species and new or revised species of Streptomyces important in biotechnology or animal health. These research activities and accomplishments underscore the importance of microbial germplasm collections as an invaluable resource and driving force for innovative research.


4.Accomplishments
1. Maintenance and distribution of strains in the ARS Culture Collection. Access to accurately identified microbial isolates is critical to microbiological research that advances agricultural production, food safety, public health, and economic development. ARS scientists in the Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agricultural Utilization Research, Peoria, Illinois, have supported microbiological research by operating and improving the ARS Culture Collection. In the previous 12 months, nearly 400 microbial isolates were accessioned into the collection and nearly 6,000 microbial isolates were distributed to scientists in the United States and 51 other countries. The utility and availability of this collection was improved by updating 48,000 inventory records and adding 3,500 previously uncataloged microbial isolates to an electronic database of available cultures. Using a conservative estimate of monetary value, strain distributions from the ARS Culture Collection during the last 12 months represent an $1.2 million in-kind contribution in support of microbiological research and biotechnological innovation. The broad impact of the ARS Culture Collection in advancing agricultural science and biotechnology is evidenced by the fact that microbial strains from the ARS Culture Collection have been cited in more than 1,200 scientific publications and 100 patents during the last year.

2. Molecular characterization of priority actinobacterial genetic resources. The range of biodiversity represented by the microbial strains held in the ARS Culture Collection in Peoria, Illinois, is not known because of the limited or incorrect characterization of many strains, making it difficult to assess its real or potential value by customers involved in agricultural and biotechnology research. The diversity within the ARS Culture Collection of strains of the genus Streptomyces, whose species produce many commercially important antibiotics, was estimated by ARS scientists in the Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agricultural Utilization Research, Peoria, Illinois, based on the sequences of five protein-coding genes. A prototype database containing sequences of the five genes includes 87% of described species and analysis of the dataset confirmed the taxonomic diversity of this genus and also identified a number of described species that are equivalent as well as several strains that have published genome sequences that are misidentified. These studies demonstrated the value of the ARS Culture Collection as a source of reference germplasm for gene sequencing studies to address practical problems of strain identification.

3. Yeast species could provide a tool for control of codling moths. Codling moths are important pests in apple production that reduce the marketable yield of orchards. ARS scientists in the Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agricultural Utilization Research, Peoria, Illinois, in collaboration with scientists in Sweden and Brazil, discovered that species of the yeast genus Metschnikowia appear essential for development of codling moth larvae in apple fruits. This knowledge will be useful in developing new strategies for the control of this important apple pathogen.

4. Protecting future commercial harvests of true morels. Commercial harvesting of true morels has grown into highly lucrative cottage industries in several morel-rich countries, including China, Turkey, and the United States. To protect this renewable resource, ARS scientists in the Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agricultural Utilization Research, Peoria, Illinois, in collaboration with scientists in China, Europe, and North America, determined the utility of the fungal barcode gene for identifying morels so that previously unknown species can be detected and identified for further genetic evaluation. Towards this end, we developed a dedicated, web-accessible informational database called Morchella MLST (http://www.cbs.knaw.nl/morchella/) that facilitates DNA sequence-based identifications of true morels via the web. Moreover, pure cultures of diverse morel species were made and are stored in the ARS Culture Collection to benefit biotechnologists interested in cultivating morels commercially. Finally, the results of this study provide invaluable information needed to develop appropriate practices for the conservation and sustainable harvest of morels.

5. Novel azuki bean pathogen discovered in Hokkaido, Japan. Production of azuki beans in Japan, which are used in numerous traditional foods in Asia, is threatened by a novel root rot disease. The present study was initiated to characterize the azuki bean pathogen in Japan, as part of a broad research program to establish a global picture of bean pathogen diversity, host range, and geographic distribution. ARS scientists in the Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agricultural Utilization Research, Peoria, Illinois, in collaboration with scientists in Japan and Argentina, discovered that the novel pathogen of azuki bean was also pathogenic to mung bean, kidney bean, and soybean, and theorize that the azuki bean pathogen was introduced into Japan from South America. These research findings promote agricultural biosecurity by providing plant pathologists and quarantine officials with tools for pathogen detection and identification and they should assist bean breeders in their efforts to develop cultivars with broad-based resistance to the diverse bean root rot pathogens.

6. Protecting avocado production worldwide from invasive beetles and pathogenic fungi. ARS scientists in the Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agricultural Utilization Research, Peoria, Illinois, discovered at least eight closely related pathogenic fungi that are being cultivated for food by invasive wood boring beetles in the genus Euwallacea, an exotic beetle native to Asia. These beetles and the Fusarium fungi that they farm currently pose a significant threat to avocado production worldwide. In addition, these wood infesting beetles are economically destructive pests of cacao, rubber trees, and tea in Asia, and literally hundreds of urban landscape trees in Los Angeles and surrounding counties in southern California. Research reported in this study promotes agricultural biosecurity by providing plant pathologists, insect biologists, and quarantine officers with robust tools for the early detection and identification of these economically destructive plant pathogens.


Review Publications
Sugui, J.A., Peterson, S.W., Clark, L.P., Nardone, G., Folio, L., Riedlinger, G., Zelazny, A.M., Holland, S.M., Kwon-Chung, K.J. 2012. Aspergillus tanneri sp. nov., a new pathogen that causes invasive disease refractory to antifungal therapy. Journal of Clinical Microbiology. 50(10):3309-3317.

Du, X., Zhao, Q., Yang, Z.L., Hansen, K., Taskin, H., Buyukalaca, S., Dewsbury, D., Moncalvo, J., Douhan, G.Q., Robert, V.A., Crous, P.W., Rehner, S.A., Rooney, A.P., Sink, S.L., O'Donnell, K. 2012. How well do ITS rDNA sequences differentiate species of true morels (Morchella)? Mycologia. 104(6):1351-1368.

Salter, C.E., O'Donnell, K., Sutton, D.A., Marancik, D.P., Knowles, S., Clauss, T.M., Berliner, A.L., Camus, A.C. 2012. Dermatitis and systemic mycosis in lined seahorses Hippocampus erectus associated with a marine-adapted Fusarium solani species complex pathogen. Diseases of Aquatic Organisms. 101(1):23-31.

Peter, G., Dlauchy, D., Price, N.P., Kurtzman, C.P. 2012. Diddensiella caesifluorescens gen. nov., sp. nov., a riboflavin-producing yeast species of the family Trichomonascaceae. International Journal of Systematic and Evolutionary Microbiology. 62(12):3081-3087.

Kurtzman, C.P., Robnett, C.J. 2013. Relationships among genera of the Saccharomycotina (Ascomycota) from multigene phylogenetic analysis of type species. Federation Of European Microbiological Societies Yeast Research. 13(1):23-33.

Labeda, D.P., Goodfellow, M., Brown, R., Ward, A.C., Lanoot, B., Vanncanneyt, M., Swings, J., Kim, S.B., Liu, Z., Chun, J., Tamura, T., Oguchi, A., Kikuchi, T., Kikuchi, H., Nishii, T., Tsuji, K., Yamaguchi, Y., Tase, A., Takahashi, M., Sakane, T., Suzuki, K.I., Hatano, K. 2012. Phylogenetic study of the species within the family Streptomycetaceae. Antonie Van Leeuwenhoek. 101(1):73-104.

Aoki, T., Tanaka, F., Suga, H., Hyakumachi, M., Scandiani, M.M., O'Donnell, K. 2012. Fusarium azukicola sp. nov., an exotic azuki bean root-rot pathogen in Hokkaido, Japan. Mycologia. 104(5):1068-1084.

Chen, Y., Ntai, I., Ju, K., Unger, M., Zamdborg, L., Robinson, S.J., Doroghazi, J.R., Labeda, D.P., Metcalf, W.W., Kelleher, N.L. 2012. A proteomic survey of nonribosomal peptide and polyketide biosynthesis in actinobacteria. Journal of Proteome Research. 11(1):85–94.

Xess, I., Mohapatra, S., Shivaprakash, M.R., Chakrabarti, A., Benny, G.L., O'Donnell, K., Padhye, A.A. 2012. Evidence implicating Thamnostylum lucknowense as an etiological agent of Rhino-orbital Mucormycosis. Journal of Clinical Microbiology. 50(4):1491-1494.

Mendel, Z., Protasov, A., Sharon, M., Ben-Yehuda, S., O'Donnell, K., Rabaglia, R., Wysoki, M., Freeman, S. 2012. An Asian ambrosia beetle Euwallacea fornicatus and its novel symbiotic fungus Fusarium sp. pose a serious threat to the Israeli avocado industry. Phytoparasitica. 40(3):235-238.

Kurtzman, C.P. 2012. Candida kuoi sp. nov., a new anamorphic species of the Starmerella yeast clade that synthesizes sophorolipids. International Journal of Systematic and Evolutionary Microbiology. 62(9):2307-2311.

Kuo, M., Dewsbury, D.R., O'Donnell, K., Carter, M., Rehner, S.A., Moore, J., Moncalvo, J., Canfield, S.A., Stephenson, S.L., Methven, A.S., Volk, T.J. 2012. Taxonomic revision of true morels (Morchella) in Canada and the United States. Mycologia. 104(5):1159-1177.

Jurjevic, Z., Peterson, S.W., Horn, B.W. 2012. Aspergillus section Versicolores, nine new species and multilocus DNA sequence based phylogeny. IMA Fungus. 3(1):59-79.

Peterson, S.W. 2012. Aspergillus and Penicillium identification using DNA sequences: Barcode or MLST? Applied Microbiology and Biotechnology. 95(2):339-344.

Witzgall, P., Proffit, M., Rozpedowska, E., Becher, P., Andreadis, S., Coracini, M., Lindblom, T., Bengtsson, M., Ream, L.J., Kurtzman, C.P., Piskur, J., Knight, A.L. 2012. “This is not an apple”–yeast mutualism in codling moth. Journal of Chemical Ecology. 38(8):949-957.

Horn, B.W., Olarte, R.A., Peterson, S.W., Carbone, I. 2013. Sexual reproduction in Aspergillus tubingensis from section Nigri. Mycologia 105(5): 1153-1163.

Last Modified: 12/18/2014
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