Bacterial extraction from grapevine and detection ofXylophilus ampelinusby a PCR and Microwell plate detection system*.

Xylophilus ampelinusis listed as a quarantine pest in Annex II/A2 of EU Directive 2000/29. Grapevine cuttings used either as rooting or grafting material represent a primary source of inoculum because of the systemic nature of bacterial colonization. We describe a PCR-based method for the specific detection ofX. ampelinusfrom various plant materials (trunks, woody branches, growing shoots, leaves and bleeding sap), making the detection ofX. ampelinusreliable throughout the year. Bacterial cells were extracted by crushing the plant tissues and soaking them in sterile distilled water for 30–120 min at 4 °C. Bacterial cells were harvested by centrifugation at 13 000gfor 10 min through sterile paper discs, and lysed in SDS buffer. DNA was extracted by a silica-based procedure. Specific DNA amplifications were performed with a pair of primers, of which one was labelled with biotin. A digoxygenin-labelled oligonucleotide with a sequence specific for the amplicon was used as the probe. Finally, amplicons were immobilized in microplate wells coated with streptavidine, and hybridization of the digoxygenin-labelled probe was detected with alkaline phosphatase-conjugated antibiodies in a colorimetric assay. This method was sensitive, specific and allowed experiments with high throughput. [ABSTRACT FROM AUTHOR]