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United States Department of Agriculture

Agricultural Research Service

USDA-ARS National Rhizobium Germplasm Collection

Fungi and Bacteria: USDA-ARS National Rhizobium Germplasm Collection


USDA-ARS National Rhizobium Germplasm Collection


By Peter B. van Berkum

Location

U.S. Department of Agriculture, Agricultural Research Service, Soybean and Alfalfa Research Laboratory, BARC-West, 10300 Baltimore Boulevard, Building 011, Room 19-9, Beltsville, MD 20705

Loans

Cultures are distributed free of charge to recognized institutions and scientists at home and abroad.

Number of
accessions

4,000

Number of
additional
cultures

3,000

Types

The germplasm resource maintains all the type strains of the genera and species of legume symbionts and is the source for the type strains recognized by the International Subcommittee on the Taxonomy of Agrobacterium and Rhizobium.

Curator

Peter B. van Berkum
Phone: (301) 504-7280, fax: (301) 504-5728
e-mail

Home page



Background

The Agricultural Research Service (ARS) of the U.S. Department of Agriculture (USDA) has maintained a collection of nitrogen-fixing legume symbionts for most of the 20th century. However, in the past the activities of the collection were not directly funded by ARS. The collection of strains grew in response to the requirements of the research programs during the decades prior to the 1970's. During this time, the scientists interacted with the public and provided mainly inoculants to individuals who had legitimate needs for research purposes or for the production of specific crops.

During the 1970's, an awareness of the critical importance of biological nitrogen fixation in agricultural production ensued because of the energy crisis. It was quite evident that the limitation of nitrogen in agriculture was a worldwide phenomenon and that poor nations suffered most because of rising energy costs. The result was that it became increasingly necessary to rely on the production of leguminous crops and management of the Rhizobium-legume symbiosis to provide the world's population with food, raw materials, and alternative energy sources. Within this climate, the critical need for a culture collection of characterized Rhizobium was identified during the first three North American Rhizobium conferences. USDA was recognized as possessing the most comprehensive collection, and the established links with the public and private sectors were considered advantageous for technology transfer.

Because the goals of the culture collection were relevant at the international level, funding for its activities was at first provided by the Agency for International Development (USAID). This support was responsible for the establishment of the basic infrastructure in the laboratory to preserve and keep records of the accessions in the collection. One of the objectives of USAID's funding was to provide a means by which scientists of lesser developed countries could preserve their cultures when they lacked the technology for doing so in their laboratories. Ironically, LiphaTech took advantage of the objectives of the culture collection during this time to streamline its own collection, sending less commercially important accessions to USDA for preservation. The international status of the collection was further enhanced during the 1980's by its designation as a Microbiological Resource Center (MIRCEN) by the United Nations Educational and Cultural Organization (UNESCO) and the United Nations Environmental Program.

ARS, in recognition of the importance of the collection, initiated funding when USAID support ended in 1990. Although international funding from USAID ceased, the collection continues to participate in the UNESCO program, and the activities associated with the objectives of MIRCEN remain active. The program gained further recognition when it became the repository for type strains of genera and species, as well as for reference strains representing genetic diversity. The Rhizobium collection initiated this program in 1993 upon the request of the International Subcommittee on Agrobacterium and Rhizobium, which is a subcommittee of the International Committee on Systematic Bacteriology of the International Union of Microbiological Societies.

During the time that the collection has been funded (1975-present), the number of accessions has increased by approximately fivefold (fig. 13). The number of strains in the collection has increased over this time period because of incorporation of collections from LiphaTech, the Boyce Thompson Institute, USDA-ARS in Minnesota, the University of Sao Paulo, and Centro Internacional de Agricultura Tropical (CIAT). These collections represented significant numbers of characterized strains that could no longer be maintained or were in danger of being lost. Approximately 3,000 cultures representing the annual medic strains from the International Center for Agricultural Research in Dry Areas, the Thai soybean strains, the bean strains of CIAT, and the core collection of Nitrogen Fixation in Tropical Agricultural Legumes have been received and need to be incorporated into the ARS collection. It is estimated that over the next 5-year period the number of accessions in the Beltsville collection will double.


 Figure 13. Chart showing the number of accessions in the USDA-ARS National Rhizobium Germplasm Collection

Figure 13. Number of accessions in the USDA-ARS National Rhizobium Germplasm Collection


Over the past quarter century, the number of cultures disbursed to fill requests has increased from approximately 200 to 800 per year (fig. 14). Although some strains are more popular than others, the number of requests for any one specific strain is less than 5 percent of the total. The higher volume has increased expenditures for postage and consumed more resources for quality control and culture preservation.


Figure 14. Chart showing the number of cultures dispersed from the collection each year since 1968

Figure 14. Number of cultures dispersed from the collection each year since 1968


Since 1975, the accessions have been maintained as suspension cultures in 20 percent glycerol stored at -70 °C, as well as in lyophilized form. Also, a separate set is stored in lyophilized form for disbursement in response to requests for cultures. Newly acquired cultures are checked for contamination upon their receipt and are verified as legume symbionts by plant testing for nodulation on the original host of isolation. Soil samples are collected from centers of origin of legume hosts for the isolation and acquisition of additional biodiversity. Currently, the resources include four freezers maintained at -70 ° C and three maintained at -20 °C, a walk-in growth room for quality control, a walk-in cold room for storage of cultures and soil samples, a laminar-flow hood, a lyophilizer, and an incubator for culturing the bacteria.

The objectives of the program include the following:

1. Preserve the nitrogen-fixing symbionts of legumes with the goal of maintaining wide genetic diversity.

2. Maintain quality control of existing and new germplasm.

3. Maintain records of accessions.

4. Distribute cultures without charge.

5. Provide technical information about cultures, their preservation, and their characteristics.

6. Acquire, maintain, and distribute type strains for all of the different rhizobial taxa.

7. Train scientists under the UNESCO Fellowship in Biotechnology Program.

Database

Currently no database of the rhizobial germplasm resource is available. However, a database is being constructed. Relevant portions of holdings should eventually be available to the public through the GRIN system.

Research

The focus of the current research is on systematics of the legume symbionts for identification and classification purposes. The approach taken is predominantly molecular systematics.

Biodiversity within species is determined using protocols for the polymerase chain reaction (PCR), with primers for repeat sequences present in bacterial genomes. Repeat sequences targeted for amplification are DNA regions between repetitive extragenic palindromic (REP) sequences, enterobacterial repetitive intergenic consensus (ERIC) sequences, and BOX sequences. The amplification products are separated according to molecular size by using horizontal agarose gel electrophoresis. The gels are photographed for quantitative comparisons of the lanes according to the presence of products or their absence across lanes. This is achieved by scanning the image into a computer and by using specialized software packages. The resultant binary matrix is used to produce a dendrogram and to measure the fit of the dendrogram to the data.

DNA relatedness among strains is estimated with DNA reassociation analysis. This relies upon the double-stranded (native) nature of DNA and the property that the complementary strands dissociate at high temperature and reassociate at lower temperature. DNA homology is an attempt to answer whether the DNA of two organisms have a base sequence sufficiently similar to allow the formation of DNA heteroduplexes. DNA homology values are measurements of similarity in which the entire genome of one organism is compared with that of another. DNA homology values are used to detect similarities between closely related organisms and to conclude whether two strains belong to the same species.

Microbial phylogeny is estimated from sequencing analysis of the ribosomal genes. In particular, sequencing the 16S or small-subunit (SSU) ribosomal RNA (rRNA) gene and the 23S or large-subunit (LSU) rRNA gene. The Rhizobiaceae do not form a branch, which is distinctly different than the other genera in the alpha subdivision of the Purple Bacteria, and therefore they constitute several lineages evident as separate genera. The effort with ribosomal gene sequencing is focused on identification of the genera of accessions, preliminary estimation of speciation, and the uncovering of novel nucleotide sequences for use in strain and species identification.



Selected
Achievements

Eleven scientists were supported by the USAID program during the period 1975-87. The training period was usually 12 months. These visitors were from Iraq, Spain, Thailand, Tanzania, the People's Republic of China, Mali, Egypt, Kenya, Zambia, and Pakistan.

Three visitors were accommodated who received Fulbright Scholarships and who chose to participate in the activities associated with the collection. These visitors were scientists from Ethiopia, India, and Brazil.

One trainee from Brazil who had independent support was accommodated for 24 months.

UNESCO supported one fellowship for a scientist from Spain.

The U.S. Department of Agriculture, Foreign Agricultural Service, International Cooperation and Development, Research and Exchanges Division, supported two projects within the germplasm resource program under the Scientific Cooperation Program. Both projects supported scientific collaboration and germplasm exchange with the People's Replublic of China.

In 1993, the International Subcommittee on Agrobacterium and Rhizobium, which is a subcommittee of the International Committee on Systematic Bacteriology of the International Union of Microbiological Societies, requested that staff of the USDA-ARS National Rhizobium Culture Collection maintain and distribute the type strains of the taxa of legume symbionts. The objective was to facilitate the distribution of the type strains as reference cultures and to have this function performed by an organization capable of maintaining superior quality control of these cultures.


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Last Modified: 2/6/2002