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.

This continuing project seeks to evaluate a new way to reduce Pierce’s disease (PD) of grapevines by managing stream bank woodlands in north coastal California to reduce populations of the blue-green sharpshooter (BGSS) and their level of infectivity with the bacterium, Xylella fastidiosa. By replacing wild grape, blackberry, and other plants used by BGSS for breeding with plants that are not favored by the BGSS for reproduction or feeding, the numbers of BGSS that we detected in yellow sticky traps were again reduced to very low levels compared to undisturbed controls. Fir trees in the buffer remained too small to have any expected effects on BGSS movements. Winter flooding and a dry spring prevented planting along the Napa River site. Seed for replants was collected and sown and seedlings for bare-root transplants are being grown in a commercial nursery for transplanting in fall, 1997 and winter, 1998. We received provisional approval from the California Dept. of Fish and Game to conduct riparian woodland management experiments at a new site in Sonoma County along Maacama Creek. Data on the infectivity of BGSS with Xylellafastidiosa showed that in one site (Conn Creek) infectivity was about 50%, compared to a range of about 5-15% the previous year and in the two other sites this year. This high level of infectivity at one location may simply be within the range of annual fluctuations in vector infectivity.

The primary objective of this research project was to develop and evaluate a more sensitive diagnostic assay for detecting Xylella fastidiosa (X.f), the bacterium that causes Pierce’s Disease (PD) of grapevines. The development of more sensitive diagnostic assays would greatly facilitate the identification of alternate plant reservoirs of PD and grape germplasm that is more resistant or tolerant of X.f. In 1995 we evaluated the utility of a new and very sensitive diagnostic test, called the polymerase chain reaction (PCR), for detecting X.f. in grapevines and other woody plants. PCR uses a special thermostable enzyme called Taq polymerase to amplify a specific fragment of X.f. DNA. The presence of the amplified DNA indicates that the sample was infected with the X.f. bacterium. We began our evaluation by testing the specificity and sensitivity of two different sets of PCR primers which direct the Taq enzyme to amplify the X.f DNA. Results showed that both sets of PCR primers, which amplify different fragments of X.f DNA, worked equally well in detecting X.f. in grapevines. PCR detected the X.f bacterium in dormant canes in March, however the percentage of positive detections was low. The frequency of positive detections increased in diseased canes as the season progressed and the ability to consistently detect X.f. by PCR, ELISA and cultural methods directly correlated with the development of leaf symptoms. PCR consistently provided the most sensitive detection of X.f in diseased grapevines, however a low percentage of false-positive test results occasionally occurred when testing cane scrapings. PCR also detected X.f. in other woody plants, such as red willow, maple, elderberry and wild grape, woody plants that can be difficult to analyze by ELISA. The most surprising, and potentially significant, result of testing diseased grapevines over the growing season was that no X.f bacteria were detected in emerging green shoots until the middle of June. This result indicates that Xf colonizes growing tissues quite slowly and that infected grapevines are probably not a significant reservoir of the pathogen until late July or August. This observation also suggests, but doesn’t prove, that dormant buds on diseased vines probably contain few viable bacteria and, theoretically, the disease should be difficult to transmit from dormant buds collected in late winter. Experiments are now in progress to verify this hypothesis. It was also determined, using a DNA fingerprinting technique, that X.f. strains collected from diseased grapevines located throughout California were genetically quite similar to eachother and to wild grape infected with X.f. More importantly, all of these geographically diverse strains were consistently detected by both sets of PCR primers. We believe that PCR could provide more reliable pathogen detection if methods were developed to sample larger portions of the vine, without having to do multiple PCR tests on the same vine. We are now evaluating the usefulness of a technique called immunocapture PCR, which uses X.f. -specific antibodies to trap X.f. bacteria, to test large-size samples. The relative sensitivity and reliability of immunocapture PCR, standard PCR, ELISA and cultural methods will be evaluated during 1996.