QUANTITATIVE DETECTION OF PSEUDOMONAS CORRUGATA AND PSEUDOMONAS MEDITERRANEA IN TOMATO PLANTS BY DUPLEX REALTIME PCR
G. Licciardello, P. Bella, V. Catara
Pseudomonas corrugata and P. mediterranea are two closely related bacterial species both causal agents of tomato pith necrosis. To screen tomato planting material reliably, a quantitative real-time PCR assay was developed, to detect and/or discriminate both bacterial pathogens in a single tube. So, two species-specific primer/probe sets were designed on the sequences of two DNA fragments amplified by a previously reported specific PCR protocol. TaqMan real-time PCR assays were developed for individual (simplex PCR) and simultaneous (duplex PCR) amplifications. The assays were performed with the SmartCycler TD II System (Cepheid) and the fluorescence from both FAM and Texas Red channels were recorded at the annealing step. Specificity was tested with an extended range of P. corrugata and P. mediterranea strains, with other Pseudomonas spp. and with a number of tomato bacterial pathogens. The detection limit was approximately 10 cells per reaction for both bacteria, and quantification was linear over a six-log range. The duplex real-time PCR assay was validated on tomato plants artificially inoculated by pricking the stem with a strain of each species either separately or together.