Detection of Xylella fastidiosa in grapevine cuttings
The development of a detection method for Xylella fastidiosa (EPPO A1 quarantine pest) in asymptomatic grapevine propagation material was the objective of a Italian-French research project. The result of the investigations is a scheme for testing woody grapevine material and one year old cuttings is based essentially on the extraction of the bacteria from the xylem by vacuum washing, their concentration through centrifugation and their identification by immunofluorescence staining and/or ELISA using polyclonal antibodies. A final confirmation of positive results might be obtained through using gel electrophoresis (SDS-PAGE) of bacterial cells which are directly isolated from the infected tissue and cultured on a suitable medium (e.g. PD3). The scheme as proposed by the authors is as follows:
Wash with tap water, disinfect surface with
70% ethanol, wrap ends with paper tape
Vacuum flush (80kPa) with SDW or PBS
Collect and centrifuge sap extracts
(10 000 rpm for 15 min.)
Resuspend pellets in SDW or PBS
(total volume 0,5 ml), dilute ten-fold
Direct Isolation (PD3)
PD-89-1 } Polyclonal antisera produced in Italy
Zaccardelli, M.; Bazzi, C.; Chauveau, J.F.; Paillard, S. (1993) Sero-diagnosis of Xylella fastidiosa in grapevine xylem extracts.
Phytopathologia Mediterranea 32, 174-181.