Multiple detection of the phytoplasmas associated with Flavescence dorée (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.