Rapid identification of Clavibacter michiganensis subspecies sepedonicus using two primers random amplified polymorphic DNA (TP-RAPD) fingerprints

Twenty strains of Clavibacter michiganensis subsp. sepedonicus from different geographic origins and other reference strains of the same and different species, including other potato pathogens, were analysed with a new procedure named TP-RAPD that originates fingerprints of bacterial species. This procedure uses two primers to amplify the 16S rDNA gene. At 45 degreesC of annealing, the PCR product electrophoresed in agarose gels produced a band pattern that was different in all bacterial species studied as well as in the subspecies of C. michiganensis. All strains of C. michiganensis subsp. sepedonicus displayed the same TP-RAPD number of pattern. Unlike Gram negative bacteria, Gram positives of high G + C content, such as Clavibacter, produced low bands in TP-RAPD. By using a different set of two primers also based in the 16S rDNA sequence from Escherichia coli a more adequate amplification of Gram positives of high G + C including a greater number of bands was obtained. TP-RAPD patterns using the new set of primers described in this work is a reliable and fast method to identify C. michiganensis subsp. sepedonicus.