P-161 - Development and validation of LAMP assays for rapid detection of Phacidiopycnis spp. and Sphaeropsis sp. on pome fruit

Phacidiopycnis washingtonensis, Phacidiopycnis pyri, and Sphaeropsis pyriputrescens represent emerging pathogens of concern to pome fruit growers in the U.S. Pacific Northwest. Their detection through conventional and morphological methods presents challenges due to their phenotypic similarities and to their latency before harvest. To facilitate early detection, a molecular diagnostic assay based on loop-mediated isothermal amplification (LAMP) was developed for each pathogen, by targeting the beta-tubulin gene. The assays have been validated on artificially inoculated symptomatic and asymptomatic fruits samples, eliminating the need for DNA extraction. The detection limits for P. washingtonensis, P. pyri, and S. pyriputrescens were established at 0.001, 0.01, and 0.01 ng/μl, respectively. The LAMP primer sets designed for each pathogen demonstrated high specificity, effectively amplifying their respective species but not the other closely or distantly related fungal pathogens of pome fruits, as verified through quantitative real-time LAMP and qualitative colorimetric assays. Consequently, the developed methods demonstrated high sensitivity, specificity, reliability, and rapid performance in detecting the three pathogens in both symptomatic and asymptomatic pome fruits. These point-of-care detection assays are anticipated to facilitate future epidemiological studies, enable timely disease management, and enhance the safety of fruit exports.