Polymorphism analysis of housekeeping genes for identification and differentiation of <Emphasis Type="Italic">Clavibacter michiganensis</Emphasis> subspecies |
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| Authors: | Malgorzata Waleron Krzysztof Waleron Joanna Kamasa Wlodzimierz Przewodowski Ewa Lojkowska |
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| Institution: | (1) Department of Biotechnology, Intercollegiate Faculty of Biotechnology of the University of Gdansk and Medical University of Gdansk, Kladki 24, 80–822 Gdansk, Poland;(2) Department of Virology and Bacteriology, Institute of Plant Protection, National Research Institute, Wladyslawa Wegorka 20, 60–318 Poznan, Poland;(3) Department of Potato Protection and Seed Science at Bonin, Plant Breeding and Acclimatization Institute at Radzikow, 76–009 Bonin 3, Poland |
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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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