| Background
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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.
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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.
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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.
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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.
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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.
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Figure 13. Number of accessions in the USDA-ARS
National Rhizobium Germplasm Collection
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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.
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Figure 14. Number of cultures dispersed from the
collection each year since 1968
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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.
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The objectives of the program include the following:
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1. Preserve the nitrogen-fixing symbionts of legumes with the goal of
maintaining wide genetic diversity.
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2. Maintain quality control of existing and new germplasm.
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3. Maintain records of accessions.
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4. Distribute cultures without charge.
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5. Provide technical information about cultures, their preservation,
and their characteristics.
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6. Acquire, maintain, and distribute type strains for all of the
different rhizobial taxa.
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7. Train scientists under the UNESCO Fellowship in Biotechnology
Program.
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| Database
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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.
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| Research
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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.
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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.
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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.
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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.
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| Selected
Achievements
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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.
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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.
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One trainee from Brazil who had independent support
was accommodated for 24 months.
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UNESCO supported one fellowship for a scientist from Spain.
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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.
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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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