Rapid Detection of <i>Xanthomonas fragariae</i> in Strawberry Using Species-Specific Primers Based on Comparative Genomics

<i>Xanthomonas</i> <i>fragariae</i> poses a significant emerging threat to global strawberry production. The success of surveillance strategies and quarantine measures in controlling its international spread depends heavily on the availability of rapid and reliable in-planta detection tools. Polymerase chain reaction (PCR) is the preferred method for detecting systemic infections because of its high sensitivity, specificity, and ease of use. However, despite the availability of several PCR and real-time quantitative PCR (qPCR) methods, many face issues with analytical specificity. Given the ongoing global evolution of <i>X</i>. <i>fragariae</i> and the emergence of new variants, there is a critical need for adaptable detection methods. In this study, we designed a specific primer pair (XfOG4-F/XfOG4-R) through comparative genomic analysis of 660 genomes from the <i>Xanthomonas</i> genus. This primer set targets a region uniquely and consistently present in all 81 <i>X</i>. <i>fragariae</i> genomes available on NCBI. We validated the primer pair's specificity using colony PCR with both target <i>X</i>. <i>fragariae</i> strains and nontarget <i>Xanthomonas</i> strains. Detection sensitivity was assessed using PCR and qPCR on isolated DNA and bacterial cell suspensions, both in vitro and in artificially inoculated strawberry leaves. The qPCR method demonstrated sensitivity 100 times higher than standard PCR. Additionally, the PCR test successfully detected the pathogen in extracts from naturally infected strawberry crown samples collected on farms. The new primer set showed improved analytical specificity over previously reported primers, offering a valuable tool for detecting <i>X</i>. <i>fragariae</i>-infected plants in future field surveys.