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 the responsibility to curate the USDA, ARS National Rhizobium Germplasm Resource Collection and to provide rhizobial resources and technical guidance to stakeholders and users who depend upon this program for their business and research. Rhizobia is the common name for numerous bacterial species that form associations (known as symbiosis) with plants of the legume family (peas, beans, soybeans and alfalfa, for example) to “fix” gaseous nitrogen from the atmosphere that is then used for plant growth. Progress was made in providing rhizobial 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 the production of Rhizobium cultures to inoculate legume crops and to non-profit organizations. Assistance was given to stakeholders who are developing bio-energy production resources through the cultivation of specific leguminous trees. Our staff spent many hours in telephone and email discussions with these stakeholders, dispensing advice on recommended strains for agronomically important legumes, protocols on media preparation on which to grow rhizobia cultures and the proper growth of cultures, and methods for scaling-up rhizobial inoculant production. Progress was made on analyses to characterize rhizobia that interact symbiotically with higher plants, including strains of Bradyrhizobium that form symbioses with soybean and the genetic interrelations of the rhizobia that infect Medicago species such as alfalfa. One of the technologies is the determination of variation in the bacteria by measuring differences in the DNA of specific genes and is known as MultiLocus Sequence Typing (MLST). When this methodology was applied to bacteria forming a symbiosis with alfalfa we were able to identify differences in the DNA sequence of a number of genes. When these differences in DNA sequence were combined, they allowed us to define the “genotype” of each individual that was analyzed. By then combining the different genotypes, it was possible to describe the genetic structure of the bacterial population that infects the roots of plants and then forms a beneficial symbiosis. The method proved to be far superior in providing information about the genetic structure of the bacteria than the conventional techniques that are based on DNA fingerprinting or serology. When the method was used with collections of isolates obtained from Western Asia and North Africa it was possible to identify the genotypes of bacteria that normally form a symbiosis with alfalfa in its natural environment. Progress was also made in the design of a database with the information obtained using the MLST technique. The database can be used as a reference for subsequent projects that determine how biotic and abiotic factors in the environment affect the population and symbiosis of these bacteria.
1. Development of MultiLocus Sequence Typing (MLST) for the rhizobial genus Bradyrhizobium and analysis of nodule occupancy of soybean grown at the USDA, ARS, Beltsville South Farm. Soybean is a very important crop for U.S. agriculture. Soybean is a legume and may benefit from a symbiosis with bacteria referred to as rhizobia that establishes in the roots. The benefit to the plant and in turn to the farmer is that crop production is possible without the need for nitrogen fertilizer applications. However, one of the problems is that different genetic types or “genotypes” of rhizobia vary substantially in their ability to provide the plant with the nitrogen required for full production. Another problem is that if different soybean rhizobia are present in the soil then different locations on the soybean roots usually are occupied by different genotypes of rhizobia, which vary greatly in their efficiency. Therefore, it is necessary to be able to identify the different genotypes of soybean rhizobia that have been preserved in Rhizobium collections as well as those that are in symbiosis with the roots of the soybean plant. ARS researchers at Beltsville, MD successfully developed the MLST method to be able to describe soybean rhizobia genotypes. This was accomplished by the DNA sequence analysis of seven genes in the chromosome of the rhizobia and then comparing the variation of these seven genes across different cultures. A method also was developed to extract rhizobia DNA directly from the locations on the soybean roots where the symbiosis is established. In this way it is now possible to describe the genotypes of the rhizobia in each of the locations of the symbiosis on the roots in order to identify soybean genetic types that form symbiosis with the most efficient rhizobia.
Van Berkum, P.B., Elia, P.E., Song, Q., Eardly, B. 2012. Development and application of a multilocus sequence analysis method for the identification of genotypes within genus Bradyrhizobium and for establishing nodule occupancy of soybean (Glycine max L. Merr). Molecular Plant-Microbe Interactions. 25(3):321-330.