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 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. A total of 3,518 strains have been accessioned into the general collection and 89 deposits made into the Patent Collection. The Patent Culture Collection has distributed 415 strains (since June 1, 2009), including 272 to scientists in the United States, and 183 to foreign scientists. Strain distributions from the general collection totaled approximately 15,368 (since June 1, 2009), including 6,507 strains to the Institute of Genomic Biology (IGB), University of Illinois as the ARS contribution to a collaborative research project, 2,547 to ARS scientists, 3,747 to non-ARS scientists in the United States, and 2,567 to foreign scientists. The ARS Culture Collection website now has 11,843 strain records available in the public access catalog, had over 21,600 visitors from 124 different countries and has been indexed by 82 different search engines. The online strain request module for the Culture Collection database system/website went live on January 1, 2010, greatly facilitating strain requests by customers and simplifying request fulfillment and tracking by the collection staff. 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. Collaboration with scientists at the IGB, University of Illinois, has been leveraged to identify 190 uncharacterized strains from the ARS Actinobacterial Culture Collection through the 16S ribosomal DNA (rRNA) gene sequences provided by the IGB high-throughput sequencing facility. This collaboration is also facilitating the development and testing of a multi-gene sequence database by providing sequence for more than 900 known and unidentified Streptomyces strains. In addition, phylogenetic analyses to study the genetic relatedness of the genus Bacillus has resulted in the description of a new subspecies. Lastly, 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.
1. MOLECULAR CHARACTERIZATION OF PRIORITY MICROBIAL GENETIC RESOURCES. The biodiversity of much of the microbial germplasm held in the Agricultural Research Service (ARS) Culture Collection located at the National Center for Agricultural Utilization Research Center in Peoria, Illinois, is not known because of limited characterization for many strains, making it difficult to assess its real or potential value by customers involved in agricultural and biotechnology research. Molecular diversity was estimated, particularly for poorly characterized isolates, based on the sequences of various genes. The partial sequences of four different protein-coding genes for plant pathogenic Streptomyces species and several thought to be closely related to them demonstrated that the 10 different pathogenic species can be discriminated and identified based on these gene sequences. Additionally, similar sequences are being collected for all streptomycetes in the ARS Actinobacterial Culture Collection by leveraging a National Institutes of Health (NIH)-funded collaboration with the Institute of Genomic Biology (IGB), University of Illinois. A similar study of the fungal genus Hamigera and the genera of all ascomycete yeasts was also completed, providing an overview of genetic relationships that are useful for prediction of utility of species agricultural, biotechnological and medical applications. The knowledge obtained from these research accomplishments provides improved characterization of the germplasm held in the Collection and discernment of potential uses in biotechnology as well as insights for disease control, toxin reduction, and prevention of food spoilage.
2. CHARACTERIZATION OF AN INTRODUCED CEREAL PATHOGEN POPULATION. Fusarium head blight [FHB] is a disease of cereal crops, caused by a fungal pathogen, which results in significant economic and health problems throughout the world. A novel population of FHB pathogen was found in Louisiana, including a fungal species previously reported only from Asia and South America. In addition, FHB pathogens producing the mycotoxin nivalenol, considered to be rare in the United States, were found at high frequencies. These results are critical to promoting food safety and cereal production through improved detection of mycotoxin-contaminated grain and through variety improvement efforts that account for the entire spectrum of pathogen and toxin types. Field tests for differences in toxin production and aggressiveness on wheat in Germany and Canada using strains representing the previously dominant (15-acetyldeoxynivalenol (15ADON) population) and novel (3-acetyldeoxynivalenol (3ADON)) populations indicated that 3ADON isolates produced more toxin and could pose a greater risk to food safety, but aggressiveness and deoxynivalenol (DON) production of 3ADON and 15ADON chemotypes was quite similar on highly resistant lines of wheat, indicating that breeding and use of highly resistant lines should be an effective means of minimizing the threat to food safety posed by the novel 3ADON population. Infection of a susceptible wheat variety by members of the novel 3ADON population results in grain that is more heavily contaminated with trichothecene toxins, while the 3ADON and 15ADON isolates produced essentially the same amount of toxin when infecting moderately resistant wheat varieties, suggesting that as the percentage of 3ADON strains increases, DON levels in cereals are likely to increase in epidemic years. The wider use of moderately resistant cultivars, however could help to mitigate the food safety impact of changes in the pathogen population.
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