P-223 - Phytophthora ramorum screening with AmplifyRP® XRT and LAMP methods

Our goal was to compare limitations and advantages of AmplifyRP® XRT Pram (Agdia) and species (Hieno et al. 2021) and genus-specific (Mainello-Land et al. 2024) LAMP for Phytophthora ramorum screening from leaf, water, and soil samples. For on-site nursery survey, 41 plant samples were tested with AmplifyRP. Eight soil samples were collected from positive nursery and baited according to USDA P. ramorum manual. From each bait, 28 leaf disks were extracted and tested with AmplifyRP, and 10 disks extracted with Qiagen kit were used for LAMP. To assess the sensitivity of AmplifyRP and LAMP, serial dilutions of positive baited leaf and healthy tissue mixtures were used. Crude NaOH extractions were performed from three leaf disks for LAMP. P. ramorum positive leaf baits from forest stream survey samples were tested with LAMP using DNA from Qiagen kit. Both isothermal methods were compared to USDA approved ITS and Elicitin qPCRs. In result, Agdia detection system on site showed 38 negative and two failed test results, while all qPCRs were negative. P. ramorum was detected only from two soil samples with all species-specific methods. Both isothermal tests were successful for P. ramorum detection showing similar sensitivity. The detection limits of both isothermal protocols were comparable with Elicitin but lower than ITS qPCR. Interestingly, species-specific LAMP failed to detect P. ramorum in five out of 12 positive water samples, while genus-specific LAMP turn positive. Both isothermal methods can be used for P. ramorum surveillance, but more testing needed for validation.