Ralstonia solanacearum race 4 isolates were obtained from Zingiberaceae plants in India during bacterial wilt outbreaks. Polyphasic phenotypic and genotypic analysis revealed intraracial diversity and dominance of biovar 3 over biovar 4. Biovar 3 strains were isolated from very severely wilted Zingiberaceae plants in the field and found to be present across diverse geographical, host and seasonal boundaries. It was hypothesized that these isolates belong to a single, 'fast wilting', lineage. Using one 'fast wilting' isolate in controlled inoculations, rapid wilt was observed in ginger within 5-7 days. Wilting was also observed in several other closely and distantly related hosts such as turmeric (Curcuma longa), aromatic turmeric (Curcuma aromatica), black turmeric (Curcuma caesia), sand ginger (Kaempferia galanga), white turmeric (Curcuma zeodaria), awapuhi (Zingiber zerumbet), greater galangal (Alpinia galanga), globba (Globba sp.), small cardamom (Elettaria cardamomum) and large cardamom (Ammomum subulatum) of the Zingiberaceae family, and in tomato (Solanum lycopersicum). Molecular analysis, including multiplex PCR-based phylotyping, sequence analysis of 16S rDNA, 16-23S intergenic spacer and the recN gene, and multilocus sequence typing, revealed minimal differences between fast wilting isolates, confirming that almost all belong to the same lineage. Biovar 4 was isolated from plants showing slow wilt progression and self-limiting wilting in restricted geographical locations instead, and was identified to be genetically distinct from the fast wilting biovar 3 isolates. To the authors' knowledge, this is the first report of host range and genetic analysis of R. solanacearum race 4 in India.

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Small cardamom (Elettaria cardamomum Maton.) and ginger (Zingiber officinale Roxb) bacterial wilt is caused by same strain of Ralstonia solanacearum: a result revealed by multilocus sequence typing (MLST) Abstract Bacterial wilt in cardamom (Elettaria cardamomum Maton) was observed in Kerala state of India. Infected plants showed wilting wherein all leaves roll or curl upward towards the midrib centre, turn yellow, and the whole plant finally dies; the collar region shows water-soaked lesions initially and turns dark brown eventually; copious quantity of bacterial exudate is observed on the cut end of the pseudostem. The bacterium was identified as Ralstonia solanacearum based on a panel of phenotypic characters such as fluidal white colony on Kelman's medium, biovar assay and biolog assay (BiologGN), and genotypic characters such as Multiplex-PCR based phylotyping, sequences of 16S rDNA, 16-23S intergenic region, and recN gene. Collectively these tests revealed that the R. solanacearum infecting cardamom belong to biovar 3 and phylotype 1 confirming its Asian origin. Upon soil inoculation, the bacterium caused typical wilting of the cardamom plants in three weeks and ginger plantlets in two weeks. Cross transmissibility of the bacterium was observed in cardamom and ginger wherein the plants succumbed to wilt when R. solanacearum from either of the host was inoculated. BOX-PCR fingerprinting revealed that the strain is identical (100%) to a ginger strain of R. solanacearum, which is widely prevalent in the Indian subcontinent. Furthermore, Multilocus Sequence Typing (MLST) based strain comparison confirmed that cardamom and ginger strain were identical to each other at 11 loci. Apart from striking phenotypic and genotypic (allelic) similarities, geographical origin, and cross transmissibility of the cardamom strain of R. solanacearum strongly suggest that the new occurrence of wilt of cardamom in India could have an origin in bacterial wilt of ginger. Perusal of records on Ralstoniainduced bacterial wilt in crop plants, particularly among the Zingiberaceae family, reveals that this is a new report of bacterial wilt disease in small cardamom.

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.

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