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New taxonomic markers for identification of Rhizobium leguminosarum and discrimination between closely related species

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Abstract

Rhizobia, producing species-specific exopolysaccharides (EPSs), comprise a very diverse group of soil bacteria that are able to establish nitrogen-fixing symbioses with legumes. Based on the sequences of R. leguminosarum EPS synthesis genes, a sensitive and reliable PCR-based method for identification and subsequent discrimination between Rhizobium species has been developed and tested. For identification 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 different host plants nodules originating from different 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 identification 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 differentiate and define relationships within a group of R. leguminosarum strains.

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Abbreviations

EPS:

Exopolysaccharide

ITS:

Intergenic transcription sequence

RFLP:

Restriction fragment length polymorphism

IP:

Isoprenylphosphate

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Acknowledgments

We gratefully acknowledge Prof. E. Velazquez for kindly providing the R. lusitanum p1-7T strain (Universidad de Salamanca, Departamento de Microbiologia y Genetica, Edificio Departamental de Biologia, Salamanca, Spain), Dr. Cecile Revellin for providing the R. giardinii bv. giardinii H152T and R. gallicum bv. gallicum R602T strains from collection of the Laboratoire de Microbiologie des Sols, Institut National de la Recherche Agronomique, Dijon, France, Dr. R. Mhamdi (Laboratoire des Interactions Legumineuses-Microorganismes, Centre de Biotechnologie, Hammam-lif, Tunisia) for providing the R. gallicum 8a3 strain and Dr. J. Sanjuan (Grupo Interacciones Planta-Bacteria, Dpto. Microbiologia del Suelo y Sistemas Simbioticos Estacion Experimental del Zaidin, CSIC, Granada, Spain) for providing R. leguminosarum bv. phaseoli GR-84 strain. We are grateful to Prof. P. Graham (Department of Soil, Water and Climate, Universitet of Minnesota, St. Paul, USA) for providing the strains (UMR1632, UMR1899, UMR6204, UMR6358, UMR6821, UMR7370, UMR7639) representing different species of Rhizobium and Mesorhizobium genera and Prof. W. Małek (Marie Curie-Skłodowska University, Lublin, Poland) for providing the isolates of B. japonicum and Mesorhizobium spp. We thank Mrs. Maria Małek for excellent technical assistance. This work was supported by grant from the Ministry of Science and Higher Education no. N N303 092234.

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  1. Department of Genetics and Microbiology, Institute of Microbiology and Biotechnology, University of M. Curie-Skłodowska, 19 Akademicka, 20-033, Lublin, Poland

    Monika Janczarek, Michał Kalita & Anna Maria Skorupska

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  1. Monika Janczarek
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  2. Michał Kalita
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  3. Anna Maria Skorupska
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Correspondence to Anna Maria Skorupska.

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Janczarek, M., Kalita, M. & Skorupska, A.M. New taxonomic markers for identification of Rhizobium leguminosarum and discrimination between closely related species. Arch Microbiol 191, 207–219 (2009). https://doi.org/10.1007/s00203-008-0447-6

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