Poster: Diseases of Plants: Disease Detection & Diagnosis
Recombinase polymerase-based diagnostics for in-field detection of Pseudomonas syringae pv. actinidiae.
J. STACK (1), M. Arif (2), J. Rascoe (3), M. Nakhla (3), G. Busot (1) (1) Kansas State University, U.S.A.; (2) Kansas State Univesrity, U.S.A.; (3) USDA APHIS PPQ CPHST, U.S.A.
In-field detection of the kiwifruit pathogen, P. syringae pv. actinidiae (Psa), will support plant biosecurity decisions regarding trade and production. To detect and discriminate among Psa strains (including the 2008 global outbreak strain, Psa-V), a Recombinase Polymerase Amplification (RPA) assay was developed for laboratory and field use. Primer sets targeting two effector genes (hopZ3 and hopZ5) were designed and screened against isolates of highly virulent Psa and closely related P. syringae pathovars. All Psa strains can be detected by the presence of hopZ3 amplicons but only the virulent type, Psa-V or biovar 3, is detectable by hopZ5 amplicons. Because RPA assays were coupled to lateral flow device (LFD), reverse primer were labeled with either biotin or digoxigenin and the reaction was developed in the presence of a carboxyfluorescein (FAM) labeled probe. RPA assays had an amplification time of 10 minutes. Sensitivity determinations indicated a detection limit of 1 pg or 100 fg depending of the targeted gene. Isothermal assays are a rapid diagnostic assay that enables the early detection of pathogens and a valuable tool for in-field pathogen detection to support plant biosecurity decisions.