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.
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.
Margaria P;Turina M;Palmano;
2009
Abstract
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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
