A combined agar absorbent and BIO-PCR assay for rapid, sensitive detection of Xylella fastidiosa (Xf) in grape and citrus

• Author(s): Damsteegt, V.; Fatmi, M.; Schaad, Norman;
• Abstract: Application of PCR for disease diagnosis has been limited by the presence of PCR inhibitors. Inhibition can be overcome and sensitivity increased with BIO-PCR by enriching bacteria on agar media, however, X. f. grows slowly. We have developed an efficient agar absorbent BIO-PCR assay for detecting X. f. in grape and citrus plants. By spotting symptomatic grape petioles and citrus leaf veins onto PD2 agar, we found 43% of the grape and 100% of the citrus samples were positive after a 1 or 4 h absorption treatment. All grape samples were positive after 5 days with BIO-PCR. Two of 6 citrus and none of 12 grape samples were positive by direct PCR conducted at spotting. Viable X. f. were recovered from all samples after 14 d growth on PD2. For routine assays, we recommend touching tissue to the bottom of a microfuge tube for direct real-time PCR and to each of four PD2 agar microfuge tubes. After one h, add 50 ul of PCR water to two tubes, vortex, and use 5ul for real-time PCR. If negative, assay the other two tubes after 5 days enrichment.
• Publication Date: Jun 2003
• Journal: Phytopathology