Development and Evaluation of a Polymerase Chain Reaction (PCR) Assay for Detecting Xylella Fastidiosa in Plants

This was the second and final year of a project whose goal was to develop and evaluate a polymerase chain reaction (PCR) assay to detect Xylella fastidiosa (X.f), the bacterium which causes Pierce’s disease (PD) of grapevines. PCR results obtained in 1996/97 using chronically infected PD-grapevines located in the Napa Valley yielded results that were similar to those obtained in 1995-6. PCR and direct culture methods detected X.f. in woody cane samples collected during fall, winter and spring months during 1996-97. PCR consistently provided more sensitive detection of X.f compared to direct culture or ELISA, results which suggest that PCR will be a valuable research tool for screening grape germplasm accession for resistance to X.f Surprisingly, as in 1996, we did not detect the bacterium using PCR or culture until May, 1997 in leaves or stems of newly developed canes. Detection of the bacterium in the oldest leaves and shoots which developed from chronically infected spurs rose from 8%of the samples being positive on May 9 to 42%positive on June 9 and 75%positive on July 9, 1997. We will continue sampling these vines again in August and in the fall to complete this time course study. However, these results indicate that under Napa Valley conditions, X.f. multiplies to significant populations only after mid to late June. These results indicate that insect vectors are probably unlikely to acquire the bacterium from PD-infected grapevines until late June, which is consistent with the lack of evidence for vine to vine transmission of X.f. by sharpshooter vectors. In a previous study, we found that the X.f. bacterium was unlikely to survive the following winter in vines that were newly infected after May-June. The pattern of negative PCR reactions interspersed over the season with positive reactions from grapevines that were known to have Pierce’s disease indicates that the bacterium is irregularly distributed within infected grapevines. Because PCR normally uses only a very small (less than 0.1 gram) piece of tissue, we believe that some of our pathogen detection “misses” were the result of testing a small sample that did not happen to contain detectable numbers of X.f. In order to increase the size of the sample from a particular vine, we are now evaluating a technique called immunocapture PCR. This technique uses PD-specific antibodies to capture and immobilize X.f. bacteria on small beads that can be recovered with a magnet. The beads are then tested for the presence of bound X.f. using PCR. In this manner we can test several samples collected from various positions on the vine to increase the likelihood of detecting X.f. in the vine. Initial results using immunocapture PCR have been encouraging. We will continue this work and compare the relative sensitivities and reliability of direct versus immunocapture PCR.