Droplet Digital PCR for the Detection of pv. in Asymptomatic Olive Plant Material.

Olive knot disease, caused by pv. , severely impacts olive tree yield and oil quality. Early and accurate detection of the bacterium's presence, particularly in asymptomatic plants, is crucial for effective disease management. This study aimed to develop an improved protocol for processing plant samples and adapting quantitative PCR to droplet digital PCR (ddPCR). For this purpose, four plant preparations-EW (external washing), PELLET (bacterial concentration), and enrichment in liquid media for 24 or 48 h (24hE, 48hE)-were tested using spiked samples. The ddPCR was set up and compared with qPCR to evaluate analytical sensitivity and specificity. Additionally, field samples from symptomatic and asymptomatic olive orchards were tested to evaluate the performance of the selected methods in naturally infected plants. ddPCR showed higher sensitivity than qPCR, particularly with the PELLET and 24hE preparations. The PELLET from the spiked sample preparation achieved a limit of detection of 10 CFU/mL for both molecular tests. The ddPCR, combined with the PELLET preparation, offers a highly sensitive and reliable tool for detecting pv. in asymptomatic olive material. This protocol shows great potential for improving early bacterial detection and disease prevention, thus aiding control strategies in nurseries and olive orchards, and supporting the production of certified plant propagation material.