References

Debelle F et al. (1996) Mol. Microbiol. 22, 303-314

Moulin L et al. (2001) Nature 411, 948-950

Quinto C et al. (1997) Proc. Natl. Acad. Sei. USA 94, 4336-4341

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DIVERSITY OF SINORHIZOBIUM MELILOTI ISOLATES NATIVE TO CENTRAL ASIAN AND SIBERIAN GENE CENTERS OF ALFALFA

M.L. Roumiantseva, E.E. Andronov, B.V. Simarov

Research Institute for Agricultural Microbiology, Ch. Podbelskogo 3, 196608 St.-Petersburg-Pushkin, Russia

Central Asian and Siberian centers of diversity located on the territory of Russia and Tadjikistan according to discovery of N.I. Vavilov (1926) and A.I. Ivanov (1988) comprise perennial species of Medicago, Melilotus and Trigonella (alfalfa cross-inoculation group) and were never studied for diversity of indigenous rhizobia. Twenty-seven isolates of Sinorhizobium meliloti were recovered from nodules of host plants belonged to 7 species from alfalfa cross-inoculation group and from soil samples by using trap host plants at 8 sites/15 locations in the Central Asian center of diversity. Fifty-six S. meliloti isolates were obtained from soil samples at 2 sites/18 locations in Siberian centre of diversity by using trap host plants. Two megaplasmids bands with a size of more than 1400 kb were revealed in all isolates with a surprising variety of sizes. From 8 to 11 plasmid profiling groups containing from 0 to 3 plasmids in addition to megaplasmids were identified in Central Asian and Siberian populations. Plasmids with a size at about 200 kb represented by similar percentage of isolates in both populations occurred to be more specific for nodule than for soil Central Asian isolates. The number of ISiim2011-2 elements per genome was ranged up to 23 copies among tested isolates, while three soil isolates native to both populations were free from this IS element. Both populations did not show significant difference in average copies of lSRm2Q\\-2 per genome, while a sharp contrast was revealed between nodule and soil Central Asian isolates, as the latter possessed half as many ISi?»i2011-2 copies per genome. RFLP analysis revealed from 7 to 8 distinct chromosomal types generated by probes on leu and recA loci in each population, but only two types were shared by both populations. One of these two types was the type similar to that of Rm2011, which was more frequently detected among both populations. Among p<Sy?nB types developed by probes on exo/exs and exp gene clusters (Becker et al. 1997) type Rm2011 was strongly dominant in both populations. Other types were highly specific for each population. pSymA types were generated by PCR amplified 224 bp inner fragment of nodDl of Rm2011 and by w//KDH probe (Roumiantseva et al. 1999). Among 14 or 19 pSymA types identified among isolates of both populations type Rm2011 was predominant among Central Asian population, but that was not the case in Siberian population. The majority of the remaining types were highly specific for each population. Mean genetic diversity (H) confirms a slight difference between tested populations and between nodule and soil Central Asian isolates at chromosomal loci, and significant difference at pSymB loci. A highest value of diversity was observed at pSymA loci among all groups of isolates. Applied disequlibrium statistics for all pairs of studied loci showed a strict link between chromosomal and pSymA pairs of loci in isolates native to both gene centers of alfalfa.

References

Becker A (1997) J. Bacteriol. 179, 1375-1384

Ivanov Al (1988) In Brezhnev DD (ed), Alfalfa (Lyutserna), pp. 1-318, Amerind Publishing Co. Pvt.

Ltd., New Delhi Roumiantseva ML (1999) Russian J. Genetics 35, 159-135

Vavilov NI (1926) Trends in Practical Botany, Genetics and Selection (in Russian) 16, 3-248

Acknowledgements

This work was financed by INTAS694.

USE OF AUTOMATED SYSTEMS TO STUDY THE BIODIVERSITY OF RHIZOBIA

K. Lindstrom, A. Dresler-Nurmi, L.A. Rasanen, Z. Terefework

Dept Appl. Chem. Microbiol., Viikki Biocenter, University of Helsinki, Finland

To evaluate the biodiversity of 320 rhizobial strains isolated from the nodules of Calliandra calothyrsus growing in humid tropics in Central America, Africa and New Caledonia, PCR-RFLP of 16S rDNA was used. RFLP of 150 strains with two enzymes revealed that the diversity of the strains was very large. A direct PCR reaction to amplify ribosomal genes was set up using intact cells without prior DNA isolation. The process was automated by using 96 Deep Well Plates for growing the bacteria and 0.2 mL Skirted 96 Tube Plates for PCR reactions. To distinguish bacterial isolates, amplified 16S rDNA genes were digested with three different enzymes and run in a 96-sample tank. 16S rDNA was successfully amplified from all tested strains without prior isolation of the DNA. Different, distinct restriction patterns were detected with each of endonucleases used. Next, the IGS regions will be analyzed by a traditional RFLP approach (agarose gel electrophoresis) as well as by basepair-accurate fluorescence based T-RFLP analysis (capillary electrophoresis). The latter will also be tried for 16S rDNA to create a reliable database of patterns representing both recognized species and Calliandra rhizobia.

The genetic diversity of Rhizobium galegae strains and taxonomically diverse rhizobia representing the recognized species was assessed using a fluorescent AFLP method. Unlike other PCR-based fingerprinting techniques such as RAPD, the AFLP reactions are not as much dependent on minor changes in PCR conditions. The AFLP fingerprints are highly reproducible, and changing the selective bases of the primers can regulate the number of bands obtained. The detection methods, fluorescent based reactions on ABI 310 and ABI 377 genetic analyzers and silver staining gave consistent fingerprint patterns from all the strains studied. However, a relatively lower number of bands were observed on the polyacrylamide gels due to their smaller resolution power and due to the size of the gel when compared with the capillary and slab gel electrophoresis separation of fragments from ABB 10 and ABI377. Using capillary electrophoresis with a proper polymer and standard conditions it is possible to obtain data within the range of 50-2000 bp. Pairwise comparison of the fluorescent samples from the ABB 10 and ABI377 runs revealed that more distinguished peak are resolved by capillary electrophoresis (CE) than the slab gel electrophoresis. We used the recognized species of rhizobia in this study to demonstrate the broad use and advantages of the AFLP method over other fingerprinting techniques, and show the applicability of the technique in rhizobial diversity studies. The groupings in the clusters, however, do not accord with the anticipated relatedness of most of the species supported by other rhizobial systematic data. Rhizobium galegae, a species that makes successful symbiosis only with Galega officinalis and Galega orientalis plants, shows an interesting host-microsymbiont specificity. The Rhizobium galegae strains are able to infect both plants, however, strains isolated from Galega officinalis form effective nodules on the respective plant, but ineffective nodules on Galega orientalis and vice versa. All methods delineated the G. orientalis strains from G. officinalis strains, the G. orientalis strains formed a tight cluster whereas the G. officinalis strains seem to show a greater level of genetic diversity. The AFLP results warrant a subspecies status to the two biovars that are previously delineated by their symbiotic genotypic and phenotypic characters.

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