A diagnostic tool for improved detection of Xanthomonas fragariae using a rapid and highly specific LAMP assay designed with comparative genomics

摘要

Molecular diagnostics of plant pathogens are crucial to prevent disease spread and to enhance food quality and security. A comparative genomics approach using genomes of different Xanthomonas species and pathovars was applied to identify highly specific targets in the genome of Xanthomonas fragariae, the causal agent of angular leaf spot of strawberry, listed under quarantine regulations in Europe. A reliable and sensitive loop-mediated isothermal amplification (LAMP) assay was designed using a unique marker, providing a highly specific and rapid detection technique, convenient for on-site detection. Specificity of the designed assay was tested on 37 strains from a culture collection of X. fragariae, 82 strains of other Xanthomonas species and pathovars and 11 strains of other bacterial genera isolated from strawberry leaves. A detection limit of 10(2) fg was achieved, approximating to 20 genome copies per reaction. When performing analyses with crude plant material, a consistent lower detection efficiency of 10(2) CFU mL(-1) was achieved. The LAMP assay designed in this study was adapted to work on crude plant material without any prior extensive extraction steps or incubation period; moreover, it does not require advanced analytical knowledge or a fully equipped laboratory. Results were produced within 7-20 min, depending on the pathogen concentration, thus providing a high-throughput and user-friendly method for detection and screening of plant material in support of quarantine regulations.