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

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.

Phylogenetic Analysis and Polyphasic Characterization of Clavibacter michiganensis Strains Isolated from Tomato Seeds Reveal that Nonpathogenic Strains Are Distinct from C. michiganensis subsp. michiganensis

“…These genes did not display any signs of evolution involving recombination. The analysis confirmed the results of previous studies reporting a robust use of gyrB as a phylogenetic marker in the genus Clavibacter (83) and the use of recA for differentiation of the five subspecies of C. michiganensis (78). Both the gyrB and recA loci were sufficiently discriminating to identify infraspecies sequence variation.…”

Characterization of phenotypic variants of Clavibacter michiganensis subsp. michiganensis isolated from Capsicum annuum

“…More recently, genetic diversity related to geographical area was observed within the Cmm population (De Leon et al 2009;Ignatov et al 2004;Kaneshiro et al 2006;Nazari et al 2007). Among the five Cm subspecies, the relatively high genetic diversity of Cmm strains was confirmed by PCR-RFLP and PFGE analysis (Kleitman et al 2008;Waleron et al 2011).…”

Diversity of Plant Associated Actinobacteria

“…Housekeeping Genes Another valuable method is the genetic analyses of the housekeeping genes such as rpoB (the RNA polymerase β subunit-encoding gene), rpoD, gyrB (gyrase subunit β gene), recA (encoding a protein involved in repairing damaged DNA in the SOS regulon) and multilocus sequence analysis MLSA (Dahllöf et al 2000;Kasai et al 2000;Maréchal et al 2000;St-Onge et al 2008;Bernèche-D'Amours et al 2011;Waleron et al 2011;Jacques et al 2012;Carro et al 2012;Xu et al 2012b). …”

“…michiganensis in pure cultures with a detection threshold of 10 3 cfu ml −1 (Zhao et al 2007;Luo et al 2008). Also, attempts aiming to differentiate Clavibacter subspecies as well as to differentiate between saprophytes, non virulent and virulent Clavibacter strains have been performed by many researchers (Dreier et al 1995;Dreier et al 1997;Louws et al 1998;Pastrik and Rainey 1999;Smith et al 2001;Bach et al 2003;Waleron et al 2011;Jacques et al 2012;Zaluga et al 2013). Molecular methods include qualitative gene PCR (Dreier et al 1995;Li and de Boer 1995;Slack et al 1996;Waleron et al 2011;Jacques et al 2012) rep-PCR (Louws et al 1998;Smith et al 2001), Nested PCR (Lee et al 1997b), RAPD-PCR (de Leon et al 2009), SSR-PCR , RT-PCR (Bach et al 2003), rDNA sequencing (Kaneshiro et al 2006) and DNA hybridization (Slack et al 1996).…”

“…subsp. michiganensis strains (Waleron et al 2011). More recent work by Jacques et al (2012) used polyphasic approach to study diversity among a worldwide collection of pathogenic and non pathogenic C. michiganensis subsp.…”