Abstract
A DNA marker that distinguishes plant associated bacteria at the species level and below was derived by comparing six sequenced genomes of Xanthomonas, a genus that contains many important phytopathogens. This DNA marker comprises a portion of the dnaA replication initiation factor (RIF). Unlike the rRNA genes, dnaA is a single copy gene in the vast majority of sequenced bacterial genomes, and amplification of RIF requires genus-specific primers. In silico analysis revealed that RIF has equal or greater ability to differentiate closely related species of Xanthomonas than the widely used ribosomal intergenic spacer region (ITS). Furthermore, in a set of 263 Xanthomonas, Ralstonia and Clavibacter strains, the RIF marker was directly sequenced in both directions with a success rate approximately 16% higher than that for ITS. RIF frameworks for Xanthomonas, Ralstonia and Clavibacter were constructed using 682 reference strains representing different species, subspecies, pathovars, races, hosts and geographic regions, and contain a total of 109 different RIF sequences. RIF sequences showed subspecific groupings but did not place strains of X. campestris or X. axonopodis into currently named pathovars nor R. solanacearum strains into their respective races, confirming previous conclusions that pathovar and race designations do not necessarily reflect genetic relationships. The RIF marker also was sequenced for 24 reference strains from three genera in the Enterobacteriaceae: Pectobacterium, Pantoea and Dickeya. RIF sequences of 70 previously uncharacterized strains of Ralstonia, Clavibacter, Pectobacterium and Dickeya matched, or were similar to, those of known reference strains, illustrating the utility of the frameworks to classify bacteria below the species level and rapidly match unknown isolates to reference strains. The RIF sequence frameworks are available at the online RIF database, RIFdb, and can be queried for diagnostic purposes with RIF sequences obtained from unknown strains in both chromatogram and FASTA format.
Highlights
Bacterial phytopathogens cause billions of dollars in crop losses annually
Our results indicate that replication initiation factor (RIF) is a suitable marker for the classification of strains from the six genera used in this study, complements other DNA markers in Xanthomonas MLSA studies, and may be expanded to other bacterial genera, the majority of which contain a single copy of the dnaA gene
The RIF marker is suitable for the classification and meaningful grouping of strains from the six genera examined in this study
Summary
Bacterial phytopathogens cause billions of dollars in crop losses annually (as summarized by Baker et al [1]). The bacterial genera Clavibacter, Ralstonia, Pseudomonas, Dickeya, Erwinia, Pantoea, Pectobacterium, and Xylella contain species of important plant pathogens [5,6,7,8,9,10,11,12]. The historical designations of Xanthomonas pathovars and Ralstonia solanacearum races, based on the plant host and symptoms produced, are being replaced by modern DNA based methods [17] that have been used to reclassify some pathovars of X. axonopodis into different species and subspecies [15,16] 16S rDNA sequencing, DNA-DNA hybridization and other genetic and phenotypic traits have been used to reclassify some species of Pseudomonas, X. maltophilia, Erwinia chrysanthemi, E. carotovora and E. herbicola into species of Ralstonia, Stenotrophomonas maltophilia, Dickeya, Pectobacterium and Pantoea agglomerans, respectively [9,10,11,12,28]
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