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.
3. Progress Report
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 and Foundations, including Dba Plant Probotics, Strategic Enzyme Applications, Inc., MSI/Bio-Next, Inc., Rizobacter Argentina, and the Samuel Roberts Noble Foundation. 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. In the past year strains have been sent from National Rhizobium Germplasm Resource Collection to Argentina, Canada, Egypt, India, Portugal, and Tunisia. 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.
1. Development of Multi Locus Sequence Typing (MLST) for two large extra chromosomal plasmids within the rhizobia that form symbioses with various species of Medicago and Annual Medics. Approximately half the DNA content of Medicago-nodulating rhizobia is present in the form of two large plasmids. One of these plasmids carries all the genetic information necessary for the formation of the nitrogen fixation symbiosis with the legume host plant. The question was whether these two plasmids diverged in accordance with variation of the chromosome of each strain. MLST technology was developed using 6 genes to analyze each plasmid present in 230 strains that had been used for chromosomal analysis. Also, methods were developed to obtain the entire sequence for one of the nodulation and one of the nitrogen fixation genes. More diversity was present in each of the two plasmids than in the chromosome. Extensive sharing of genes during the evolution of the two plasmids was identified. Successful infection followed by the establishment of an effective symbiosis with alfalfa was related to the presence of specific alleles of the two genes for symbiosis that were analyzed. The impact will be in management of the symbiosis in production fields of alfalfa for forage production and in organic farming for successful establishment of the cover crop for green manuring.
Van Berkum, P.B., Fuhrmann, J.J. 2009. Evidence from ITS sequence analysis of 31 and 110 serogroup soybean strains that extant members of the genus Bradyrhizobium are likely the products of reticulate evolutionary events. Applied and Environmental Microbiology. 75:78-82.