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

Abstract

Angular leaf spot, caused by the phytopathogenic bacterium Xanthomonas fragariae, 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 X. fragariae. In this study, we developed a novel gene marker and primer set (XF-212F/R) through comparative genomic analysis of X. fragariae strains and related bacterial species for pathogen-specific detection. As a result, the XF-212F/R primer set amplified only X. fragariae strains without cross-reactivity to other Xanthomonas 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 X. fragariae strains and is suitable for early detection and disease surveillance in strawberry plants.