Submitted to: Plant Pathology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 12/29/2004
Publication Date: 7/1/2005
Citation: Fatmi, M., Damsteegt, V.D., Schaad, N.W. 2005. A combined agar absorbent and bio-pcr assay for sensitive detection of xylella fastidiosa in grape and citrus. Plant Pathology. 54:1-7 Interpretive Summary: Pierce's disease of grape (PD) and citrus variegated chlorosis (CVC) are serious diseases caused by a very small, slow-growing bacterium. Because 10-14 days are needed to culture the bacterium, disease diagnosis is very difficult. CVC causes severe losses in Brazil and the pathogen is on the APHIS List of plant pathogens; the presence of CVC in the USA would result in severe losses to the citrus industry. To prevent possible introduction, methods for less time consuming detection of the pathogen are needed. Rapid molecular-based techniques such as polymerase chain reaction (PCR) are available for routine detection of bacterial pathogens, however, use has been limited by the presence of materials in plant tissues that inhibit PCR. We describe a simple agar absorbent-PCR assay to overcome PCR inhibitors. By using the new agar absorbent assay, 97% of infected grape samples were positive whereas only 13% were positive by a traditional PCR assay. With citrus 100% of samples were positive by the new assay, but only 33% by a traditional PCR assay. This simple PCR-based assay should prove useful for routine detection of slow-growing, difficult to detect pathogens.
Technical Abstract: Application of PCR for disease diagnosis has been limited in part by the presence of PCR inhibitors. Inhibition can be overcome and sensitivity increased with BIO-PCR by enriching bacteria on agar media, however, Xylella fastidiosa (Xf) grows slowly. We have developed an agar absorbent BIO-PCR method for detecting Xf in grape and citrus plants. Optimum lengths of time for absorption of inhibitors by the agar medium or enrichment of bacteria on the medium were determined for grapes infected by Pierce's disease and citrus variegated chlorosis. Direct PCR assays of grape and citrus samples showed that 13% (4/32) and 33% (2/6) were positive, respectively. In contrast, with agar absorbent-PCR, 97% (31/32) and 100% (6/6) were positive after 2 d and 4 h for grape and citrus, respectively. In a separate experiment, petioles of symptomatic grape and citrus leaves were spotted onto agar media and the spots washed, after various intervals, and assayed for bacteria by real-time PCR. By agar-absorption, 50% (14/28) of the grape spots were positive after 1h or 4h, and 89% (25/28) after 5d. All six citrus samples were positive after only 4h. Viable Xf were recovered from all samples after 14 days. This simple technique should be useful for routine detection of Xf and other slow-growing bacteria in the presence of PCR inhibitors.