Multiple detection of several grapevine pathogens by taqman® real-time rt-pcr assays from crude-sap extracts.

Grapevine is one of the most important cultivars worldwide. Among grapevinepathogens, phytoplasma-associated with grapevine yellows Flavescence dorée (FD) and Bois noir (BN), and viruses are serious threat in Europe, causing heavy economic losses. Constant monitoring is fundamental for limiting the spread of infections. Osman and Rowhani (2006) described a high-throughput method to screen grape samples for virus infection by RT-PCR of the genomic RNA directly from crude extract, and Osman et al., (2007, 2008) improved this system using Real Time RT-PCR. Margaria et al. (2007)developed a rapid RT-PCR protocol for Flavescence dorée detection from leaf-extracts based on 16SrRNA). For phytoplasma detection, using RNA as template instead of DNA can take advantage of the high ribosomal-RNA copy number compared to the two copies of the 16SrRNA gene present in the phytoplasma genome (Schneider and Seemüller, 1994). Moreover, RNA detection allows indirect evaluation of metabolically active cells. We now report improvement of the methodof Margaria et al. (2007) using Taqman® Real-Time RT-PCR to detect the 16SrRNA of FD and BN phytoplasmas. We have hnow used the same crude-extract of grapevine to detect, on the same plate, Fd, BN and viruses.Grapevine Leafroll associated Virus-1, and -3, and Grapevine Virus A, which are the major viruses infecting grapevines in Piedmont, Italy (F. Mannini, personal communication). In 2007, over 200 samples with suspicious symptoms were collected. Single and mixed infections of both the phytoplasmas and the viruses were found.