A unique DNA repair and recombination gene (recN) sequence for identification and intraspecific molecular typing of bacterial wilt pathogen Ralstonia solanacearum and its comparative analysis with ribosomal DNA sequences

Abstract

Ribosomal gene sequences are a popular choice for identification of bacterial species and, often, for making phylogenetic interpretations. Although very popular, the sequences of 16S rDNA and 16-23S intergenic sequences often fail to differentiate closely related species of bacteria. The availability of complete genome sequences of bacteria, in the recent years, has accelerated the search for new genome targets for phylogenetic interpretations. The recently published full genome data of nine strains of R. solanacearum, which causes bacterial wilt of crop plants, has provided enormous genomic choices for phylogenetic analysis in this globally important plant pathogen. We have compared a gene candidate recN, which codes for DNA repair and recombination function, with 16S rDNA/16-23S intergenic ribosomal gene sequences for identification and intraspecific phylogenetic interpretations in R. solanacearum. recN gene sequence analysis of R. solanacearum revealed subgroups within phylotypes (or newly proposed species within plant pathogenic genus, Ralstonia), indicating its usefulness for intraspecific genotyping. The taxonomic discriminatory power of recN gene sequence was found to be superior to ribosomal DNA sequences. In all, the recN-sequence-based phylogenetic tree generated with the Bayesian model depicted 21 haplotypes against 15 and 13 haplotypes obtained with 16S rDNA and 16-23S rDNA intergenic sequences, respectively. Besides this, we have observed high percentage of polymorphic sites (S 23.04%), high rate of mutations (Eta 276) and high codon bias index (CBI 0.60), which makes the recN an ideal gene candidate for intraspecific molecular typing of this important plant pathogen.

Evaluation of Trichoderma spp., Pseudomonas fluorescens and Bacillus subtilis for biological control of Ralstonia wilt of tomato

“…Infected plant saps from six xylems showed similar bacterial colonies on CPG medium. All the colonies were similar to a virulent type, as the appearance was white or cream-colored, irregularly-round, fluidal, and opaque on CPG medium 34 , 35 . Gram staining and observation using a microscope showed that the bacteria were gram negative, rod-shaped and non-spore forming, which further confirmed that the bacteria was Ralstonia spp.…”

Evaluation of Trichoderma spp., Pseudomonas fluorescens and Bacillus subtilis for biological control of Ralstonia wilt of tomato

“…All the colonies were similar to avirulent type, as the appearance was white or cream-colored, irregularly-round, fluidal, and opaque on CPG medium 34, 35 . Gram staining and observation using a microscope showed that the bacteria were gram negative, rod-shaped and non-spore forming, which further confirmed that the bacteria was Ralstonia spp.…”

Host specificity and genetic diversity of race 4 strains of Ralstonia solanacearum

“…The gene for the DNA repair protein ( recN ) was amplified and sequenced according to Kumar et al . (). A 1300 bp recN fragment was amplified using denaturation at 96°C for 9 min, followed 30 cycles at 95°C for 60 s, annealing at 60°C for 60 s and extension at 72°C for 120 s, with a final extension of 72°C for 10 min.…”