Novel Detection Marker of Xanthomonas fragariae from Strawberry Plants Using SYBR Green Real-Time PCR

Angular leaf spot, caused by the phytopathogenic bacterium <i>Xanthomonas fragariae</i>, results in considerable yield losses in strawberry production. This pathogen is designated as a quarantine pest in several countries, including South Korea. Therefore, early identification of this pathogen in young plants is essential for preventing disease spread and ensuring rapid eradication. However, intraspecific genetic variation among isolates from different geographic origins imposes challenges for the accurate diagnosis of <i>X. fragariae</i>. In this study, we developed a novel gene marker and primer set (XF-212F/R) through comparative genomic analysis of <i>X. fragariae</i> strains and related bacterial species for pathogenspecific detection. As a result, the XF-212F/R primer set amplified only <i>X. fragariae</i> strains without cross-reactivity to other <i>Xanthomonas</i> spp. or related bacteria. The detection limits of the SYBR Green real-time PCR assay were 1.41 × 10² plasmid copies/μL, 500 fg of genomic DNA, and 1.52 × 10³ CFU/mL of bacterial cells. The assay also accurately detected the pathogen from cells directly extracted from infected strawberry leaves, enabling rapid detection without the need for DNA extraction. This diagnostic method showed improved coverage for <i>X. fragariae</i> strains and is suitable for early detection and disease surveillance in strawberry plants.