A Loop-Mediated Isothermal Amplification Portable Assay for Visual Detection of Phacidiopycnis washingtonensis and...

Phacidiopycnis washingtonensis and Phacidiopycnis pyri are emerging postharvest pathogens affecting apple and pear fruit, respectively, in the U.S. Pacific Northwest (PNW). Both pathogens cause latent infections in the orchard and eventually manifest as spongy and water-soaked lesions later in storage. In addition to resulting in significant fruit loss, these species are classified as quarantine pathogens in some countries to which PNW fruit is exported. To facilitate the removal of export barriers, it is important to prioritize early detection of Phacidiopycnis spp. to ensure appropriate and timely disease management. A loop-mediated isothermal amplification (LAMP) assay, targeting the β-tubulin gene, was designed, tested, and validated to detect the two pathogens. The analytical sensitivity for P. washingtonensis and P. pyri was 1 pg/μl and 10 pg/μl, respectively. The LAMP primer sets designed for each pathogen demonstrated specificity to their respective Phacidiopycnis species and did not amplify other closely or distantly related pome fungal pathogens which were evaluated using real-time reactions with a portable LAMP machine. The assays were further optimized to enable the visual detection of the two species through a colorimetric method, achieving similar detection limits. The assays were validated using artificially inoculated symptomatic and asymptomatic fruits using crude samples. The developed LAMP assays demonstrated high sensitivity, specificity, reliability, and rapid performance in detecting P. washingtonensis and P. pyri in both symptomatic and asymptomatic pome fruits. These assays are anticipated to facilitate future epidemiological studies, enable timely management, and ensure safety of fruit exports.