Detection of phloem restricted bacteria responsible for strawberry marginal chlorosis (SMC) by real-time PCR in a single assay

Two uncultured phloem restricted plant pathogens, the γ3 proteobacterium «Candidatus Phlomobacter fragariae » and the stolbur phytoplasma (group 16SrXII-A) are associated with strawberry marginal chlorosis (SMC) in France. As “Ca. P. fragariae” and stolbur phytoplasma induce identical symptoms, the only way to identify the pathogen infecting a given diseased plant is to perform conventional PCR assays. Because using two PCR techniques for detecting separately each of the two bacteria is time consuming and because specificity and sensitivity of the detection test needed to be improved, a new approach using triplex real time PCR was developed for the routine detection of “Ca. P. fragariae “ and stolbur phytoplasma. The real time PCR has the advantage of being faster reduces the risks of producing false positives. Furthermore, real-time PCR techniques provide the possibility of multiplexing by using probes with different compatible fluorescent dyes. Here, we present a new sensitive Taqman® method which permits the simultaneous amplification of three DNA targets in one test: the map gene of stolbur phytoplasma, the spoT gene of “Ca. P. fragariae” and the cox gene of strawberry chloroplast taken as an internal control. The specificity and the efficiency of this method were determined.