2010 Annual Report
1a.Objectives (from AD-416)
Manage the genetic resources of the National Rhizobium Germplasm Resource Collection. This is a service component of this project that is part of "The Germplasm Resources Information Network (GRIN)" within the National Microbial Germplasm Program (NMGP). Use genomic and phylogenetic analyses to characterize microbes that interact symbiotically with higher plants, such as strains of Bradyrhizobium, that form symbioses with soybean, and the genetic interrelations of the rhizobia that infect Medicago species. Some parts of the technologies that have been developed will be applied to the genetic and phylogenetic analysis of phytopathogenic fungi with another laboratory at Beltsville.
1b.Approach (from AD-416)
Rhizobial cultures will be managed by their preservation, quality control, and disbursement to ARS customers upon request. In some cases inoculum will be manufactured by special request for research purposes. Technical information about rhizobia, their isolation, their culturing, the symbiosis, data analysis and research advice will be given. New rhizobial cultures will be isolated from soil samples collected within the USA and from abroad. Characterization of microbes that interact symbiotically with higher plants and crops will use approaches based on genomic and phylogenetic analyses, predominantly achieved through sequence and analyses of ribosomal and housekeeping gene loci. This will include the development of several new technologies, and analysis of strains of Bradyrhizobium that form symbioses with soybean, and two projects examining the genetic interrelationships of the rhizobia that infect Medicago species.
One component of this project is responsibility for curation of the USDA ARS National Rhizobium Germplasm Resource Collection, to provide rhizobial resources and technical guidance to stakeholders and users who depend upon this program for their business and research. Progress was made in providing cultures and technical assistance to support African American institutions of higher education, 1890 Institutions and other State and Private Universities. Technological support and cultures were provided to small companies specializing in inoculant production. Our staff spent many hours in telephone and email discussions with these companies, dispensing advice on recommended strains for agronomically important legumes, protocols on media preparation and the proper growth of cultures, methods for scaling-up inoculant production, and the necessary customs requirements for certification to conduct international sales of inoculants. The program also has acclaim in the international community.
Progress was also made in the research component of this project, the goals of which are to develop and use approaches in genomics to identify rhizobial genotypes that nodulate the two most important legume crops in the USA, soybean and alfalfa. Neither legume is native to the USA and no systematic collection of rhizobia has been made in the countries of origin. Also, different nodules on the same plant harbor a range of different rhizobial genotypes and their effectiveness for nitrogen fixation varies mostly from ineffective to a few that are fully capable at nitrogen feeding of the host legume. The technology for determining bacterial genotypes is known as MultiLocus Sequence Typing (MLST) and involves the sequence analysis of 7 to 10 chromosomal loci to reveal the allele of each locus. When the alleles of all the loci are combined an allelic profile or Sequence Type (ST) of each specific bacterial genome is generated. MLST provides information about bacterial population genetic structures useful in epidemiology and should not be confused with systematics or molecular systematics that provide keys from data of type strains for species identification. Technology for MLST analysis of rhizobia nodulating soybean and alfalfa has been developed using 7 and 10 chromosomal loci, respectively and is in use in the laboratory.
Application of the Multi-locus Sequence Typing (MLST) approach for the analysis of rhizobia nodulating different legume species. The problem is that there is very little information about the variability of rhizobia in soils where important U.S. legume crops originated and how this lack of information potentially impacts U.S. production. This problem was addressed by the analysis of closely related rhizobia that originated from Spain and that were cultured from four different legume genera. There was a difference in the distribution of two rhizobial species that depended on the legume used for isolation of the cultures. This difference possibly reflects variability in compatibility of the two rhizobial species with the legume host species that were analyzed. This knowledge potentially impacts management of the symbiosis for production fields of alfalfa and alfalfa-like crops especially in organic farming.
Analysis of genes affecting the symbiosis with Medicago truncatula Jemalong A17. In the case of the alfalfa species Medicago truncatula, Jemalong A17 is the model legume undergoing full genome sequence analysis and Rm1021 is a fully sequenced strain of Rhizobium. The problem is that there is no nitrogen fixation in the symbiosis between the two and, therefore, research on the genomics of plant nitrogen nutrition has a limitation. As part of the Multilocus Sequence Typing project a specific gene region, thought to be important for nitrogen fixation, on one of the plasmids within several rhizobia was compared. The cultures were chosen based on their ability or inability to initiate nitrogen fixation with this legume species. From the analysis, no differences were observed in the gene that is thought to control nitrogen fixation (called exoH). Based on these results scientists will be able to formulate additional hypotheses and justify complete genome sequence analysis of other rhizobial strains, especially those that do form a fully functional symbiosis with the model legume. The impact will be on the acquisition of knowledge for management of the symbiosis for production fields of alfalfa and alfalfa-like crops especially in organic farming.
Bromfield, E.S., Tambong, J.T., Cloutier, S., Prevost, D., Lagurerre, G., Van Berkum, P.B., Tran Thi, T.V., Assabgui, R., Barran, L.R. 2010. Ensifer, Phyllobacterium and Rhizobium species occupy nodules of Medicago sativa (alfalfa) and Melilotus alba (sweet clover) grown at a Canadian site without a history of cultivation. Microbiology. 156:505-520.
Van Berkum, P.B., Elia, P.E., Eardly, B.D. 2010. Application of Multilocus Sequence Typing To Study the Genetic Structure of Megaplasmids in Medicago-Nodulating Rhizobia. Applied and Environmental Microbiology. 76:3967-3977.