New taxonomic markers for identification of Rhizobium leguminosarum and discrimination between closely related species
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ORIGINAL PAPER
New taxonomic markers for identiWcation of Rhizobium leguminosarum and discrimination between closely related species Monika Janczarek · Michai Kalita · Anna Maria Skorupska
Received: 18 June 2008 / Revised: 16 September 2008 / Accepted: 3 November 2008 / Published online: 20 November 2008 © Springer-Verlag 2008
Abstract Rhizobia, producing species-speciWc exopolysaccharides (EPSs), comprise a very diverse group of soil bacteria that are able to establish nitrogen-Wxing symbioses with legumes. Based on the sequences of R. leguminosarum EPS synthesis genes, a sensitive and reliable PCR-based method for identiWcation and subsequent discrimination between Rhizobium species has been developed and tested. For identiWcation of R. leguminosarum, primer sets I–III complementary to sequences of rosR, pssA and pssY genes were proposed. Further sets of primers (IV–VII) were designed for discrimination between R. leguminosarum biovars. The usefulness of the method was examined using a wide range of R. leguminosarum strains isolated from diVerent host plants nodules originating from diVerent regions of Poland. We demonstrate a high discriminating power of primer sets I–III that allow distinguishing R. leguminosarum and two closely related species, R. etli and R. gallicum. This new approach is applicable to identiWcation of R. leguminosarum strains, originating from nodules or soil, where many other closely related bacteria are expected to be present. Based on the nucleotide sequence of rosR and pssA genes, phylogenetic relationships of selected R. leguminosarum isolates were determined. Our results indicate that both rosR and pssA might be useful markers to diVerentiate and deWne relationships within a group of R. leguminosarum strains.
Communicated by Ursula Priefer. M. Janczarek · M. Kalita · A. M. Skorupska (&) Department of Genetics and Microbiology, Institute of Microbiology and Biotechnology, University of M. Curie-Skiodowska, 19 Akademicka, 20-033 Lublin, Poland e-mail: [email protected]; [email protected]
Keywords Diversity · IdentiWcation of R. leguminosarum · PCR detection · Pss genes Abbrevations EPS Exopolysaccharide ITS Intergenic transcription sequence RFLP Restriction fragment length polymorphism IP Isoprenylphosphate
Introduction Rhizobia, currently comprising the genera Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium and Azorhizobium, are the widely occuring soil bacteria belonging to the Proteobacteria that are able to establish nitrogenWxing symbioses with legumes. Establishment of symbiosis is a complex process involving an exchange of a series of signals between the plant and the bacterium. Plant Xavonoids secreted from roots and bacterial Nod factors are the best characterised signals involved in the infection process (Perret et al. 2000). Moreover, acidic exopolysaccharide (EPS) secreted in large amounts by Rhizobium species plays an important role in the establishment of an eVective symbiosis with host plants that form indeterminate (i.e., with pe
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