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POSTERS: Pathogen detection, quantification and diagnosis

Evaluation of isothermal recombinase polymerase amplification (RPA) to detect the four major soilborne diseases of strawberry in California nurseries
Seyed Mojtaba Mansouripour - Strawberry Center, California Polytechnic State University. Gerald Holmes- Strawberry Center, California Polytechnic State University, Natalia Peres- University of Florida - Gulf Coast Research and Education Center, Nan-Yi Wang- Gulf Coast Research and Education Center, University of Florida

The soilborne pathogens Verticillium dahliae, Macrophomina phaseolina, Fusarium oxysporum f. sp. fragariae and Phytophthora sp., causal agents of Verticillium wilt, Macrophomina crown rot, Fusarium wilt, and Phytophthora crown rot, respectively, have emerged as prevalent pathogens in California strawberry production after the phase-out of methyl bromide. There is a need to detect these pathogens in nurseries to minimize their spread on transplants. To evaluate the sensitivity and specificity of isothermal recombinase polymerase amplification (RPA) assay, 125 plant samples from 13 locations of high elevation strawberry nurseries were collected in August 2018. All crowns were plated on APDA, ACMA, NP10, and PARP media. Extracted DNA (1µl) was added to the master mix and each pathogen’s presence was determined using a TwistDx T16-ISO instrument (RPA) and High-Resolution Melting (HRM) methods. The results showed that 31(25%), 32(26%) and 43(34%) samples were positive for Phytophthora sp. using plating, RPA and HRM, respectively. The remaining pathogens were not detected by either method. The RPA diagnostic sensitivity was 74% while its specificity was 100% compared to HRM. Since the RPA instrument is easily transportable, highly sensitive with the same specificity as PCR, but faster, RPA could be used as a rapid method for nurseries to detect important soilborne pathogens of strawberry in the field before shipping plants to fruit production fields around the world.