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Polymorphism analysis of housekeeping genes for identification and differentiation of Clavibacter michiganensis subspecies

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Abstract

The utility of polymorphism analysis was determined for differentiation of the following subspecies of the Gram-positive plant pathogenic bacterium, Clavibacter michiganensis: C. m. subsp. michiganensis, C. m. subsp. sepedonicus, C. m. subsp. insidiosus C. m. subsp. nebraskensis, and C. m. subsp. tessellarius. Specific primers designed for amplification of the housekeeping genes recA, rpoB, and rpoD generated 827-, 1037-, and 862-bp DNA fragments, respectively. PCR products obtained from 40 C. michiganensis strains were analysed using RFLP with four restriction endonucleases, and those PCR products with specific RFLP patterns were sequenced. The genotypes discriminated after PCR–RFLP were specific for each subspecies and also allowed for differentiation of C. m. subsp. michiganensis strains. Sequence analysis of the recA, rpoB, and rpoD gene fragments also distinguished C. michiganensis subspecies and was useful for phylogenetic analysis of all subspecies. For rapid, inexpensive, and effective differentiation of the five subspecies in this research, we recommend the amplification of recA and/or rpoD gene fragments and digestion of the PCR products with the restriction endonuclease FnuDII.

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Acknowledgements

The authors thank Bogumila Tyszkiewicz from the State Plant Health and Seed Inspection Service in Gdansk and Dr Joanna Pulawska from the Research Institute of Pomology and Floriculture in Skierniewice for providing Cm strains. This work was supported by the Ministry of Science and Higher Education, Project No N 303 086 32/2778.

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Authors and Affiliations

  1. Department of Biotechnology, Intercollegiate Faculty of Biotechnology of the University of Gdansk and Medical University of Gdansk, Kladki 24, 80–822, Gdansk, Poland

    Malgorzata Waleron, Krzysztof Waleron & Ewa Lojkowska

  2. Department of Virology and Bacteriology, Institute of Plant Protection, National Research Institute, Wladyslawa Wegorka 20, 60–318, Poznan, Poland

    Joanna Kamasa

  3. Department of Potato Protection and Seed Science at Bonin, Plant Breeding and Acclimatization Institute at Radzikow, 76–009, Bonin 3, Poland

    Wlodzimierz Przewodowski

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  1. Malgorzata Waleron
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  2. Krzysztof Waleron
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  5. Ewa Lojkowska
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Correspondence to Malgorzata Waleron.

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Waleron, M., Waleron, K., Kamasa, J. et al. Polymorphism analysis of housekeeping genes for identification and differentiation of Clavibacter michiganensis subspecies. Eur J Plant Pathol 131, 341–354 (2011). https://doi.org/10.1007/s10658-011-9812-4

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