Detection of Flavescence dore´ and Bois noir phytoplasmas, grapevine leafroll associated virus-1 and-3 and grapevine virus A from the same crude extract by reverse transcription Real Time Taqman assays.

Multiple detection of the phytoplasmas associated with Flavescence dore´e (FD) and Bois noir (BN) diseases and of the
viruses Grapevine leafroll associated virus -1 and -3 (Ampelovirus) and Grapevine virus A (Vitivirus) is described, using the
same crude extract as template. Sap was prepared by semi-automatic maceration requiring minimal time and effort, consisting
of a tissue grinding step in carbonate buffer and a boiling step in glycine buffer; two microlitres were used as template in
each pathogen-specific assay. RealTime reverse transcription (RT)-PCR for FD phytoplasma detection was found to be five
orders of magnitude more sensitive than the RT-PCR method described previously. However, the RealTime RT-PCR assay
for the detection of BN phytoplasma needed a nested step to achieve high sensitivity, suggesting low concentration of
template in the host. The viruses were detected by RealTime nested-PCR, which was more sensitive than the ELISA and
RealTime RT-PCR assays previously described. The methods presented here have been successfully used to monitor
infections in field and nursery samples during the 2008 grapevine growing season.