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
Collections of well characterized microbial germplasm are an invaluable resource for agricultural and biotechnology research. The overall goals of this project are to enhance the Agricultural Research Service (ARS) Culture Collection through acquisition of novel microbial germplasm and to characterize this germplasm genetically through gene sequence analysis. In FY 2008, a total of 3,133 strains have been accessioned into the general collection, including large collections of Listeria and Salmonella strains and 137 deposits made into the Patent Collection. The Patent Culture Collection has distributed 468 strains (since July 1, 2008), including 41 to ARS scientists, 239 to non-ARS scientists in the United States, and 188 to foreign scientists. Strain distributions from the general collection totaled 4,124 (since July 1, 2007), including 719 to ARS scientists, 1,838 to non-ARS scientists in the United States, and 2,039 to foreign scientists. The ARS Culture Collection website now has 11,217 strain records available in the public access catalog, had over 21,000 visitors from 129 different countries and has been indexed by 69 different search engines. The development of an online, automated system for client requests for microbial germplasm from the collection is in progress. These activities provide for continued preservation of agriculturally and biotechnologically significant microbial germplasm and distribution to researchers in ARS as well as throughout the world. Phylogenetic analyses of the genus Bacillus has resulted in the description of a new subspecies. A phylogenetic study of plant pathogenic Streptomyces based on the sequences of 4 house-keeping genes is almost completed and will be expanded to encompass species representing the entire genus. It has been determined that ochratoxigenic Aspergillus niger strains can be disinguished from non-toxigenic A. niger strains on the basis of calmodulin gene sequences, providing a basis for the development of gene probes for rapid detection of toxin producers. Multigene sequence analysis resolved genus assignments of the biotechnologically important yeast species that can grow on methanol as a sole source of carbon. The majority of the species were placed in the genus Ogataea, but three of the species were recognized as separate from Ogataea and their earlier placement in the genus Komagataella was confirmed. Progress achieved in FY2008 has potential scientific impact for academic, government,and industrial researchers by providing new and better characterized microbial germplasm that can be exploited in many areas of research such as those related to energy production disease control in plants and animals and food safety.
1. MOLECULAR CHARACTERIZATION OF PRIORITY MICROBIAL GENETIC RESOURCES. The biodiversity of much of the microbial germplasm held in the Agriculture Research Service (ARS) Culture Collection is not known. Molecular diversity was estimated, particularly for poorly characterized isolates, based on the sequences of various gene loci. A retrospective phylogenetic analysis of 16S rRNA gene sequences for unidentified strains of Streptomyces isolated from equine placentas by collaborators at University of Kentucky demonstrated that the emerging pathogen Streptomyces atriruber has been present in horses since the mid-1990's, as well as the presence of several undescribed species. The phylogenetic mapping of morphological character distribution onto the 16S rRNA gene phylogenetic tree demonstrated that there should not have been distinction between spore chains in tight spirals or open loops. It has been determined that ochratoxigenic Aspergillus niger strains can be distinguished from non-toxigenic A. niger strains on the basis of calmodulin gene sequences, providing a basis for the development of gene probes for rapid detection of toxin producers. The information gained from this research demonstrated the value of molecular sequence databases based on well characterized collections of microorganisms in providing new insights for disease control in animals and rapid detection and reduction of ochratoxin producing fungi in commodities.
2. CHARACTERIZATON OF AN INTRODUCED CEREAL PATHOGEN POPULATION. We completed a multi-year, multi-agency, integrated analysis of a highly toxigenic Fusarium head blight pathogen population identified from Canada and the northern U.S. Our analyses demonstrated that this pathogen population was likely introduced into North America from eastern Asia during the last 30 years and has rapidly spread across the northern portions of North America by competitively excluding the resident pathogen population. These pathogens produce more toxin and appear to be more aggressive toward susceptible wheat varieties. This research was supported in part by a grant from the Western Grains Research Foundation, a producer group, and provides critical information for disease control and toxin reduction efforts.
3. MOLECULAR CHARACTERIZATION OF BIOTECHNOLOGY YEASTS. The yeast species Pichia pastoris is commonly used in molecular biology for expression of foreign genes. Based on gene sequence analyses, P. pastoris was transferred to the genus Komagataella. Our multigene analysis showed that the strain of K. pastoris used for gene expression is actually another species, which is K. phaffii. This work has provided genetic clarification concerning the species widely used in molecular biology and will be useful in further applications of the species for biotechnological advances.Balajee, A., Baddley, J.W., Peterson, S.W., Nickle, D., Varga, J., Boey, A., Lass-Florl, C., Samson, R.A. 2009. Aspergillus alabamensis, a New Clinically Relevant Species in the Section Terrei. Eukaryotic Cell. 8(5):713-722.