| Summary: | <i>Xanthomonas euroxanthea</i> is a bacterial species encompassing both pathogenic and non-pathogenic strains and is frequently found colonizing the same host plants as <i>X. arboricola</i>. This presents the need to develop a detection and genotyping assay able to track these bacteria in microbial consortia with other xanthomonads. Eight <i>X. euroxanthea</i>-specific DNA markers (XEA1-XEA8) were selected by comparative genomics and validated in silico regarding their specificity and consistency using BLASTn, synteny analysis, CG content, codon usage (CAI/eCAI values) and genomic proximity to plasticity determinants. In silico, the selected eight DNA markers were found to be specific and conserved across the genomes of 11 <i>X. euroxanthea</i> strains, and in particular, five DNA markers (XEA4, XEA5, XEA6, XEA7 and XEA8) were unfailingly found in these genomes. A multiplex of PCR targeting markers XEA1 (819 bp), XEA8 (648 bp) and XEA5 (295 bp) was shown to successfully detect <i>X. euroxanthea</i> down to 1 ng of DNA (per PCR reaction). The topology of trees generated with the concatenated sequences of three markers (XEA5, XEA6 and XEA8) and four housekeeping genes (<i>gyrB</i>, <i>rpoD</i>, <i>fyuA</i> and <i>acnB</i>) underlined the equal discriminatory power of these features and thus the suitability of the DNA markers to discriminate <i>X. euroxanthea</i> lineages. Overall, this study displays a DNA-marker-based method for the detection and genotyping of <i>X. euroxanthea</i> strains, contributing to monitoring for its presence in <i>X. arboricola</i>-colonizing habitats. The present study proposes a workflow for the selection of species-specific detection markers. Prospectively, this assay could contribute to unveil alternative host species of <i>Xanthomonas euroxanthea</i>; and improve the control of phytopathogenic strains.
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