Location: Mycotoxin Prevention and Applied Microbiology Research
2022 Annual Report
Objectives
The major goals of this project are to maintain and enhance the quality, diversity, and utility of the ARS Culture Collection holdings, including strains held in the ARS Patent Culture Collection, and to conduct and support microbiological research that advances agricultural production, food safety, public health, and economic development. Objective 1 provides for the curation and management of the ARS Culture Collection, including the acquisition and distribution of microbial strains and associated information. This objective provides scientists worldwide with access to high quality microbial germplasm through culture deposit and distribution services that facilitate research, publication, and technology transfer. Objective 2 is designed to improve understanding and utilization of microbial diversity through genetic and phenotypic characterization of strains in the ARS Culture Collection, and through the development of improved tools for microbial identification and characterization. Successful completion of goals under Objective 1 will provide scientists working on Objective 2 with access to microbial cultures, associated metadata, and tools to transfer research findings to the scientific community. Research conducted under Objective 2 will provide novel germplasm, microbiological expertise, and strain characterization data that enhances the value of the ARS Culture Collection and improves its curation.
Objective 1: Efficiently and effectively acquire, distribute, back-up, and maintain the safety, genetic integrity, health, and viability of priority microbial genetic resources and associated information in the ARS Culture Collection.
Objective 2: Enhance the value of priority microbial genetic resources in the ARS Culture Collection by conducting comparative phenotypic, phylogenetic and genomic analyses. Record and disseminate characterization data via the ARS Culture Collection database and other data sources.
Approach
More than 98,000 accessions consisting of microbial cultures of agriculturally, industrially, and medically important bacteria and fungi will be maintained in a secure centralized limited-access facility. These microbial genetic resources will be effectively preserved and secured, characterized to improve their utility, and made available to support agricultural and other research projects worldwide. In addition to maintaining and distributing currently held strains, high priority microbial genetic resources will be acquired and safeguarded so that these critical resources are widely available for microbial research and development. Microbial cultures will be characterized with a combination of genetic, genomic, and phenotypic approaches and analyses to elucidate their phylogenetic relationships, ensure accurate species identification, and enhance their utility for research and development.
Progress Report
Objective 1. This year, in response to ARS research program needs and customer requests, 154 new strains were accessioned into the open collection and 75 isolates were deposited in the Patent Culture Collection. In addition, 4,738 strains were sent to 550 scientists from across the United States and 67 other countries. These strain distributions conservatively represent an in-kind contribution of $1.4 million in support of microbiological research and biotechnological innovation. The utility and security of the collection was improved by adding or updating 5,692 strain history or inventory records.
Research under the first objective is focused on maintaining the integrity of the existing collection and acquiring material to improve the taxonomic representation and utility of the collection. To this end the ARS Culture Collection acquired and accessioned type strains of nine species of Sarocladium, 12 species of Parastagonospora, and 19 species of Colletotrichum from The Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre – an institute of the Royal Netherlands Academy of Arts and Sciences Culture collection in the Netherlands. These strains will fill important taxonomic gaps in the ARS Culture Collection (which goes by the initials NRRL for the previous name of the researcher center, the Northern Regional Research laboratory) and represent agricultural important pathogens and potential biological control organisms that are important to the ARS mission. These strains are currently being used to determine the genetic diversity and species identity of strains of Colletotrichum, Parastagonospora, and Sarocladium already in the ARS Culture Collection that have not been identified using DNA sequence-based phylogenetic methods. We sequenced multiple genes that are used for the accurate identification of species in these genera. These data are currently being analyzed to assign proper taxonomic names to these strains. All sequence data will be made publicly available through depositing in GenBank. This work contributed to milestone three (acquiring 300 high priority strains), and to meeting milestone four (DNA based characterization of strains in the ARS Culture Collection).
The lyophilization system was previously inoperable from July 2018 to August 2021 due to the system needing repairs and upgrades followed by staffing challenges. This resulted in a sizable backlog of strains that needed new lyophilized preps for distribution and prevented us from completing milestone two: Replicate and deliver (semi-annually) new accession to the backup collection at the National Laboratory for Genetic Resources Preservation (NLGRP), in previous years. This year, we hired a new Biological Science Technician to address this backlog. We have lyophilized over 500 strains, which includes 12 ampoules per strain for a total of 6,000 ampoules. During fiscal year 2022 (FY22), with the system repairs and hiring of a new technician, we were able to lyophilize all strains accessioned over the last three years of the project. This allowed us to meet milestone two for FY22, and lyophilize all strains deposited in FY21, FY20, and FY19 that could not be lyophilized due to systems repairs and staffing challenges.
Objective 2. Research under the second objective is focused on generating and utilizing DNA-sequence based analyses of genetic variation and phenotypic data to characterize agronomically important microbes in the NRRL and to make these strains and associated metadata publicly available to promote agricultural production, biotechnological development, and food safety. This year, approximately 1,500 agronomically important toxin producing and plant pathogenic fungal strains in the ARS Culture Collection or from ongoing research studies within the ARS location in Peoria, Illinois, were typed using single or multilocus DNA sequence data. A specific project completed during FY 2022 focused on the assessment of all 79 strains listed as Colletotrichum in the ARS Culture Collection using a DNA-based approach. We chose Colletotrichum as it is an important plant pathogenic genus and only nine of the 79 strains of Colletotrichum in the NRRL database are included in the public catalog. Also, to our knowledge no DNA sequence data has been generated for any of these strains, so the morphology-based species level identifications require molecular validation employing phylogenetically informative sequence data. The collection also houses many strains that were deposited in the past by ARS researchers in Peoria, Illinois, that have limited strain data, unknown viability tests and lack a DNA sequence-based identification. These strains are referred to as “A-strains,” as the accession number begins with an “A” and are not listed in the public catalog due to this lack of proper identification. Our study revealed that of the 24 A-strains listed as Colletotrichum in the NRRL database, 18 were not viable, three were not Colletotrichum, and only three strains were determined to be species of Colletotrichum. Our study also revealed that of the 55 Colletotrichum strains with NRRL numbers, 25 were misidentified. In many cases this was the result of a single morphospecies of Colletotrichum, such as C. acutatum being resolved as a species complex based on published multigene molecular phylogenetic analyses by other researchers. With the acquisition of strains from the CBS Culture Collection, and molecular identification of strains already in the collection, NRRL now has 70 strains of Colletotrichum representing 32 species, all with DNA-based molecular identifications. We also identified two novel species among the strains present in the collection and we are working on the formal description. This work also contributed to meeting milestone four’s characterization of 1,000 strains in the ARS Culture Collection. This work also contributed to milestone one as we work to improve the accuracy of strain identifications in the NRRL database.
Accomplishments
1. Investigated the origin and diversity of fungi causing tar spot disease of corn. The fungal pathogen Phyllachora maydis causes the disease tar spot on corn, which has recently emerged as a major threat to corn production throughout North America. The origin of this disease is still unclear, and there may be multiple pathogens causing the disease. An ARS researcher in Peoria, Illinois, collaborated with researchers at the University of Illinois and several other universities in the United States to sample tar spot disease from across South, Central, and North America to understand the origin and diversity of the pathogen causing tar spot disease. Analyses of DNA data determined there are three species of Phyllachora that cause tar spot of corn in the United States. It is likely these three species originated in Mexico, Central America, and the Caribbean. These results indicate the pathogens are native to North America and the emergence of the disease is likely due to a combination of change in climate making the corn growing region of the United States more hospitable for the fungus and a shift in corn genetics that resulted in a lack of resistance to the three Phyllachora species that cause this disease. These results will be of interest to corn breeders, pathologists, agronomists, and corn growers as they try to better understand the biology of this fungus and develop resistant varieties and crop management strategies to reduce yield loss due to this disease.
2. Updated the FUSARIUM-ID v.3.0 online database. Members of the mold genus Fusarium are the most important group of toxin-producing plant pathogens. They are responsible for multibillion U.S. dollar annual losses to the world’s agricultural economy due to reduction in yields, price discounts, and toxin contaminated food and feed that is unsuitable for consumption by humans and animals. Accurate species-level identification of these pathogens is crucial for disease diagnosis and management. This can be a daunting task because this agriculturally important genus is currently estimated to comprise well over 400 genetically distinct species and because morphology cannot be used to accurately identify most of these species. To address the critical need for accurate pathogen identification, a team of ARS researchers in Peoria, Illinois, along with collaborators at Penn State University updated FUSARIUM-ID v.3.0 creating a comprehensive database enabling scientists worldwide to identify Fusarium species using DNA sequence data. This database is being used by a wide range of scientists and quarantine officials charged with minimizing the threat these pathogens pose to global agricultural biosecurity and food safety.
3. Building more resilient culture collections. Bacterial pathogens are a major threat to food and fiber production worldwide. Microbial culture collections play an important role in monitoring and surveying bacterial pathogens by housing important reference strains that can be used to identify and characterize these pathogens. However, there are many gaps in these collections. An ARS researcher in Peoria, Illinois, worked with scientists and curators at culture collections in Australia, France, New Zealand, and the United Kingdom to review the role of culture collections in monitoring emerging pathogens and described areas where the scientific community can contribute by depositing more plant pathogenic bacteria. This study also provides guidelines on the best practices to improve microbial preservation and alerted the global scientific community that plant bacterial culture collections must increase deposits to be prepared for new races or introduced bacterial pathogens.
Review Publications
Wyka, S., Mondo, S., Liu, M., Nalam, V., Broders, K. 2022. A large accessory genome and high recombination rates may influence global distribution and broad host range of the fungal plant pathogen Claviceps purpurea. PLoS Pathogens. 17(2). Article e0263496. https://doi.org/10.1371/journal.pone.0263496.
Liu, M., Findlay, W., Dettman, J., Wyka, S.A., Broders, K., Shoukouhi, P., Dadej, K., Kolarik, M., Basnyat, A., Menzies, J.G. 2021. Mining indole alkaloid synthesis gene clusters from genomes of 53 Claviceps strains revealed redundant gene copies and an approximate evolutionary hourglass model. Toxins. 13(11). Article 799. https://doi.org/10.3390/toxins13110799.
Broders, K., Aspin, A., Bailey, J., Chapman, T., Portier, P., Weir, B.S. 2022. Building more resilient culture collections: A call for increased deposits of plant-associated bacteria. Microorganisms. 10(4). Article 741. https://doi.org/10.3390/microorganisms10040741.
Broders, K., Iriarte-Broders, G., Bergstrom, G.C., Byamukama, E., Chilvers, M., Cruz, C., Dalla-Lana, F., Duray, Z., Malvick, D., Mueller, D., Paul, P., Plewa, D., Raid, R., Robertson, A.E., Salgado, C., Smith, D., Telenko, D., VanEtten, K., Kleczewski, N.M. 2022. Phyllachora species infecting maize and other grass species in the Americas represents a complex of closely related species. Ecology and Evolution. 12(4): Article e8832. https://doi.org/10.1002/ece3.8832.
Munkvold, G.P., Weieneth, L., Proctor, R.H., Busman, M., Blandino, M., Susca, A., Logrieco, A., Moretti, A. 2018. Pathogenicity of fumonisin-producing and nonproducing strains of Aspergillus species in section Nigri to maize ears and seedlings. Plant Disease. 102(2):282-291. https://doi.org/10.1094/PDIS-01-17-0103-RE.
