DNA Markers for Detection and Genotyping of Xanthomonas euroxanthea

Xanthomonas euroxanthea is a bacterial species encompassing both pathogenic and non-pathogenic strains and is frequently found colonizing the same host plants as X. arboricola. This presents the need to develop a detection and genotyping assay able to track these bacteria in microbial consortia with other xanthomonads. Eight X. euroxanthea-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 X. euroxanthea 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 X. euroxanthea 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 (gyrB, rpoD, fyuA and acnB) underlined the equal discriminatory power of these features and thus the suitability of the DNA markers to discriminate X. euroxanthea lineages. Overall, this study displays a DNA-marker-based method for the detection and genotyping of X. euroxanthea strains, contributing to monitoring for its presence in X. arboricola-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 Xanthomonas euroxanthea; and improve the control of phytopathogenic strains.