A multiplex Real Time Taqman RT-PCR assay for the detection of grapevine flavescence doree and bois noir phytoplasmas using crude sap extracts.

Flavescence dorée (FD) and Bois noir (BN) are the most important and spread grapevine yellows diseases in Europe and are caused by phytoplasmas of the 16SrV and 16SrXII-A groups, respectively. Detection of BN and FD phytoplasmas is particularly difficult, as they are generally present in low concentrations and unevenly distributed in the grapevine phloem. Many efforts recently focused on developing of a rapid and sensitive method for phytoplasma detection. In this study a triplex real time TaqMan RT-PCR methodology has been set up to simultaneously, but specifically, amplify a 16SrDNA gene fragment of FD and BN phytoplasmas and a 18SrDNA gene region of the host plant using a crude sap extract as template. The use of RNA as template, instead of DNA, takes advantage from the high ribosomal-RNA copy number present in pathogen cells and provides an evidence of viability and metabolic activity of phytoplasma, which is not provided by DNA-based detection. During our study an efficient and rapid method for GY phytoplasmas detection in different host plants has been obtained. Absence of nucleic acid extraction step allows to greatly decrease both costs and time necessary to perform the assay. Therefore the method presented is a rapid, specific and sensitive alternative to conventional nested-PCR. We successfully applied this technique to the detection of BN and FD phytoplasmas on more than three hundred samples of grapevine, strawberry, tomato, wild host plants and vector insects