2006 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Crop production is an energy-intensive process that is dependent on the availability and low cost of fossil fuels to manufacture fertilizer. Problem one is that fossil fuel resources are dwindling, their cost is increasing substantially with the result that agriculture will become more expensive and the prices of agricultural products will rise. Problem two is that domestic agricultural production is dependent upon the importation of foreign petroleum products. Problem three is that chemical fertilizer use causes widespread pollution, which may impact the food supply and has clear and direct consequences for human health. The potential health effects of high nitrate levels are diverse, including reproductive problems, methemoglobinemia, and cancer. These three problems can be addressed by the use of legumes, which are an essential component of low-input sustainable agriculture because of a nitrogen-fixing symbiosis with rhizobia that has minimal impact on the environment. However, improvement in the agricultural use of the symbiosis is needed to enhance efficiency of legume production and for use in sustainable agriculture. The research to be undertaken falls under National Program 301 - Plant, Microbial, and Insect Genetic Resources, Genomics and Genetic Improvement and addresses goals 1, objectives 1.2 described in the National Program Action Plan. Specifically these are: 1.2.1.1 Develop molecular genetics and novel approaches in genomics to investigate new rhizobial Germplasm that is being isolated. 1.2.1.2 as methods become available apply them to the USDA-ARS National Rhizobium Germplasm Resource Collection 1.2.1.3. provide ARS customers and end-users with rhizobial products and technical information about the symbiosis for use in the manufacture of inocula and individual research programs. 2.List by year the currently approved milestones (indicators of research progress) This is a new project that was submitted to OSQR for review on 12-22-05. FY2005: I. Culture Collection 1. Fill all requests and replenish dispensary 2. Commence with the isolation of rhizobia for Trifolium thompsonii (Thompson clover) from eight soil samples collected along the Columbia River in Washington State. 3. Provide quality control data based upon plant tests of 161 annual medic rhizobia that are part of the Rhizobium Germplasm Resource Collection. II. Genetic characterization of soybean rhizobia Nothing planned in FY2005 and FY 2006 III. Medicago edgeworthii rhizobia 4. Sequence analysis of 16S rRNA genes VI. MultiLocus Sequence Typing (MLST)of Medicago-nodulating rhizobia 5. Conduct purity tests for 231 strains used in MLST analysis. FY2006: I. Culture Collection 1. Fill all requests and replenish dispensary 2. Thompson Clover isolates purified and authenticity verified 3. Preserve annual medic rhizobia to replenish deficiencies in the collection II. Genetic characterization of soybean rhizobia Nothing planned in FY2005 and FY2006 III. Medicago edgeworthii rhizobia 4. Sequence analysis of 23S rRNA genes in the representative strains VI. MultiLocus Sequence Typing (MLST)of Medicago-nodulating rhizobia 5. Design and test PCR primers for chromosomal genetic loci FY2007: I. Culture Collection 1. Fill all requests and replenish dispensary 2. Thompson Clover isolates incorporated into the collection 3. Recover 200 ICARDA strains from preserved stocks that were received. II. Genetic characterization of soybean rhizobia 4. Sequence analysis of ITS regions of a select number of strains belonging to different serogroups III. Medicago edgeworthii rhizobia 5. Develop and apply Ligation-mediated suppression PCR for application in rhizobia research VI. MultiLocus Sequence Typing (MLST)of Medicago-nodulating rhizobia 6. Optimize PCR reactions of chromosomal loci FY2008: I. Culture Collection 1. Fill all requests and replenish dispensary 2. Isolate rhizobia from nodules of soybeans grown in 19 soils 3. Test viable ICARDA strains for symbiotic reaction on plants II. Genetic characterization of soybean rhizobia 4. Complete sequence analysis of ITS regions of a select number of non-soybean bradyrhizobial strains III. Medicago edgeworthii rhizobia 5. Complete sequence analysis of nodABC of Rhizobium mongolense, Sinorhhizobium medicae and two representative M. edgeworthii-nodulating isolates. VI. MultiLocus Sequence Typing (MLST)of Medicago-nodulating rhizobia 6. Complete sequence analysis of 5 chromosomal loci of 231 strains FY2009: I. Culture Collection 1. Fill all requests and replenish dispensary 2. Soybean isolates purified and authenticity verified 3. Incorporate ICARDA strains that pass quality test into collection II. Genetic characterization of soybean rhizobia 4. Complete AFLP analysis of soybean strains III. Medicago edgeworthii rhizobia Nothing planned VI. MultiLocus Sequence Typing (MLST)of Medicago-nodulating rhizobia 5. Complete sequence analysis of additional 5 chromosomal loci of 231 strains 4a.List the single most significant research accomplishment during FY 2006. Analysis of the Genomes of Rhizobia that Fix Nitrogen in Symbiosis with Two Legume Species Closely Related to Alfalfa: The genomes of 148 rhizobia, the nitrogen fixing bacteria that nodulate Medicago laciniata and M. truncatula (relatives of alfalfa) were precisely characterized by DNA sequence analysis of 10 genes on their chromosomes. This is an accomplishment in a subordinate project 1275-21000-220-02S in collaboration with the Institut National de Recherche Scientifique et Technique (INRST), BP95, 2050 Hammam Lif, Tunisia that addresses the need to develop molecular genetic and novel approaches in genomics to investigate new rhizobial germplasm that is being isolated. The DNA sequence analysis approach is referred to as Multi-Locus Sequence Typing (MLST). Before the development of MLST such a precise analysis of agriculturally important bacteria was not possible. Although there was more DNA sequence variation among the M. truncatula rhizobia, 4 rhizobial chromosomes of the 26 identified were the same for rhizobia of both host legumes species. Since specificity of nodulation in this case is regulated by genes on an extra-chromosomal element known as a plasmid, it was demonstrated that genes on these plasmids have a history of transfer and recombination among Medicago-nodulating rhizobia. The impact is the demonstration that DNA is shared among members of the microbial community in soil and that agriculture has been influenced by the release of DNA originating from crops, their pests and nitrogen fixing symbionts. This accomplishment aligns with National Program 301, Plant Genetic Resources, Genomics and Genetic Improvement, Program Component 2, Genomic Characterization and Genetic Improvement. 4b.List other significant research accomplishment(s), if any. MLST was developed ahead of schedule with the objective of applying the approach to the Subordinate Project. 4c.List significant activities that support special target populations. None 4d.Progress report. None 5.Describe the major accomplishments to date and their predicted or actual impact. This research project has been in place for 12 months. The activities of the USDA ARS National Rhizobium Resource Collection have made and continue to make significant impact on industry and research both at home and abroad. Strains have been made available to inoculum companies for the manufacture of legume inocula. An example for this year would be Rizobacter Argentina S.A., Pergamino, Argentina, who sought permission to use USDA 110, USDA 122 and USDA 422 in the manufacture of soybean inoculants to supply their market, which is all of Latin America. Also, this category includes U.S. small business and start-up companies that otherwise would not have this resource available and would not come into existence and subsequently flourish. These smaller businesses tend to have more specialized needs since they have to find a business niche that is not covered by the larger companies. An example for this year would be a cooperator in Indiana, who runs a small business manufacturing specialized formulas of inocula. This in turn means that the Resource Collection has to acquire, preserve and maintain more unusual and exotic rhizobial germplasm. This activity is almost exclusively accomplished by this ARS program. Besides having importance to industry, the Resource also is the backbone of National and International research programs. Numerous USDA cultures are used in rhizobial research. The importance of the collection in this manner is exemplified by the soybean strain USDA 110 (A USDA isolate from soybean), which was chosen as the candidate for the complete genome sequencing of Bradyrhizobium japonicum. Significant impact of this program is also felt in our Nation’s schools and colleges. Many Masters and Doctoral theses have depended both on strains and technical advice from the program. Many High School students have used rhizobia from the collection in Science Fair projects. Scientific impact has been the development of expertise in Multi-Locus Sequence Typing (MLST). Although the approach that has been developed is specific for one group of rhizobia, those forming symbioses with Medicago spp., the experience gained will aid in the development of MLST for other groups. Further impact has been evident by the external funding of a small collaborative research project with a scientist from Tunisia that will lead to two manuscripts for publication in scientific journals over the next six months. Also, the development of MLST led to the invitation to give an oral presentation at the 14th Australian Nitrogen Fixation Conference in November 2005. These accomplishments align with National Program 301, Plant Genetic Resources, Genomics and Genetic Improvement, Program Component 2, Genomic Characterization and Genetic Improvement. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The importance of the USDA ARS National Rhizobium Resource Collection Program for ARS customers and end-users was described earlier. The products, Germplasm and knowledge, are already available for use by industry and by scientists. The constraints are that the collection is dynamic and is a live resource that requires day-to-day attention otherwise stability of the resource would not be guaranteed. The attention required includes lengthy procedures of isolation, preservation, quality control and restocking. In many cases specific inquiries demand some research or prior knowledge of culture characteristics. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). None Review Publications Van Berkum, P.B., Bernal, G.R., Tlusty, B., Esteves De Jensen, C., Graham, P.H. 2004. Characteristics of rhizobia nodulating beans in the central region of minnesota. Canadian Journal of Microbiology 50:1023-1031. Van Berkum, P.B., Tlusty, B., Grahma, P.H. 2004. Characteristics of rhizobia associated with dalea spp. in three prairie areas of minnesota.. Canadian Journal of Microbiology 51:15-23. Van Berkum, P.B., Eardly, B.D., Leibold, J.M. 2006. Proposal for combining bradyrhizobium spp. (aeschynomene indica) with blastobacter denitrificans and to transfer blastobacter denitrificans (hirsh and muller 1985) to the genus bradyrhizobium as bradyrhizobium denitrificans. Systematic and Applied Microbiology 29:207-213.