We are investigating the response of Pierce’s diseased grapevines in the field and laboratory-greenhouse to cold – especially freezing – temperatures. The geographical distribution of PD, its therapy by freezing treatments, and the failures of most late season infections to survive the following winter all suggest that cold temperatures play a key role it the spread of PD in temperate climates, but more information is needed to determine the underlying cause of freezing mortality of the causal bacterium, Xylella fastidiosa. In field studies, we used infective insect vectors to inoculate X. fastidiosa into 12 vines each month at University viticultural plots in Oakville and Davis from April through August, 1997. We recorded symptoms and attempted to culture X. fastidiosa from inoculated vines at both sites in the fall of 1997 to determine infection rates. April and August inoculations at Oakville were lower than planned. We will record symptoms and diagnose inoculated vines for infection with X. fastidiosa in the fall of 1998 to reconfirm the recovery of most late season infections of X. fastidiosa that have been previously reported (in 1979) by observing symptoms. During January and February, we took cuttings from inoculated canes and rooted them in the greenhouse to assess the rate of spread of X. fastidiosa from the point of insect infection, based on the date of inoculation. We inoculated an additional set of Ruby cabernet vines in the Central Valley (Kearney Agricultural Station) and Cabernet Sauvignon vines at UC Davis in April, with additional inoculations planned for June and July, 1998. Some vines were inoculated with infective insect vectors and other vines with needle inoculations of cultured X. fastidiosa. These vines will be assessed in fall, 1998 and again in the fall of 1999 for continued survival of X. fastidiosa. We prepared pot-grown grapevines with Pierce’s disease, allowed them to become dormant outdoors, and tested the effects of various laboratory freezing treatments on the survival of X. fastidiosa. We completed freezing treatments over a range of temperatures in March, 1998. Preliminarily, we have not detected bacterial growth in about 70%of the plants, but we will assess the plants again in July. In addition, we are recording the effects of temperature on the survival or cultured cells of X. fastidiosa in various solutions to compare to mortality rates in dormant grapevines.

Major Objectives:

1. To screen the following commonly used rootstocks for the ability to be symptomless carriers of, or non-hosts to, Pierce’s Disease Bacteria (PDB): St. George, 5C, 5BB, 161-49, 3309C, 101-14, Schwarzmann, 44-53 Mgt, 11 OR, 1103P, Ramsey, Freedom, Harmony, 039-16.
2. To screen selections from rupestris X rotundifolia seedling populations for resistance/non-host status to PDB.
3. To screen 4 accessions of each of a range southern Vitis species for susceptibility to PD; the ability to act as non-disease expressing carriers of PDB; and the ability to be non-hosts to PDB.
4. To test the possibilities of grafting vinifera scions onto an infected stock/trunk combination, and grafting to non-hosts identified above under greenhouse and Davis field conditions, in preparation for future field studies.

Summary:

We have prepared plant materials for PD screening, developed the inoculation system, have optimized the PD – PCR detection system, and have seedlings from a cross of Chardonnay X (rupestris X rotundifolia). These plant materials will be inoculated with PD during summer 1998 and results should be ready near the end of 1998.

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 is the final year of a continuing project on the fate of the Pierce’s disease (PD) bacterium (Xylella fastidiosa) in various plant species. The selected plants are preferred by the principal insect vector of PD in coastal California, the blue-green sharpshooter (BGSS) or are common perennials in riparian vegetation. We inoculated plants in the lab with X. fastidiosa using infective BGSSs and inoculated plants in the field mechanically with cultured bacteria. After keeping the plants in a greenhouse or waiting 2-4 months for mechanically inoculated plants in the field to develop infections, we attempted to isolate the bacterium from the plants. We previously were not able to recover by culture X. fastidiosa from arroyo willow, red willow, sandbar willow, or yellow willow that had been inoculated by either method, but polymerase chain reaction assays indicated the presence of X. fastidiosa in willows in the field. After inoculating red or yellow willows with X. fastidiosa using BGSS, we recovered the bacterium in culture after less than 1 week (8 of 15 positive) and after 3 weeks (8 of 13 positive) but made only one recovery in 16 attempts after 12 weeks. We concluded that the bacterium undergoes a short period of growth in willow, but does not survive well beyond 2-3 months. This would explain previous reports that willow is a host of the bacterium, and might have caused the positive PCR results in willows from which we did not culture X. fastidiosa. To examine the effects of overwintering, we are maintaining greenhouse-infected plants outdoors at Oakville during the winter of 1996-97. These will be returned to the greenhouse in March, 1997 to hold for retesting (culture) later this spring. We have further refined and expanded our analysis of strains of X. fastidiosa. Pierce’s disease strains from Georgia and Florida are closely related to California strains, especially north coastal California strains. Strains from peach (phony disease), oak leaf scorch, and oleander leaf scorch are all quite distinct from grape and almond strains. Two of the almond strains overlapped slightly with grape strains.

This is a continuing project to develop new methods of managing Pierce’s disease (PD) of grapevines by managing the plant habitats of the principal insect vector of PD, the blue-green sharpshooter (BGSS). Populations of BGSS would be reduced by replacing plants such as wild grape, blackberry, and others with plants that are not favored by the BGSS for reproduction or feeding. We are also testing buffer strip plantings of redwood and Douglas fir between vineyards and riparian woodlands as a barrier to reduce the influx of BGSS into vineyards during spring. The second goal is to reduce the percentage of BGSSs that are infective with the Pierce’s disease bacterium {Xylella fastidiosa) by replacing plants that support the multiplication, within-plant movements, and year-round survival of A”. Fastidiosa with plants that do not. Managing riparian woodlands to reduce the threat of PD also must meet requirements to maintain good habitats for fish and wildlife, prevent water contamination, and not degrade other public uses or values of this ecosystem. During 1995-96 we monitored populations of BGSS before and after the management treatments. We also estimated that about 6%of BGSS in the study site were infected with Z Fastidiosa in September, 1995. Sticky trap catches of BGSS along both sides of the study site along Conn Creek, Napa County, verified that BGSS was common along the entire study site. Trap catches in vineyards were much lower than in adjacent riparian vegetation (10%or less of riparian catches). During the fall, selected plants were removed from treatment plots by cooperating growers using California Conservation Corps work crews. Replantings and buffer strip plantings were made during February-March. Additional plantings to replace trees that died will be made after September, 1996, depending on rainfall. As expected, trap catches of BGSS were drastically reduced in plots where riparian vegetation was removed. Two additional experimental sites should be added in 1996. Approval from California Dept. Of Fish and Game for these experiments was given for a Napa River site. Permission will be sought to establish a Sonoma County site on Maacama Creek if BGSS activity proves to be sufficient based on our sampling of BGSS populations. Future replications of this experiment on other sites will give more realistic short term estimates of the effectiveness of the vegetation management approach because not as many large trees will be removed. A mix of University and AVF funding is planned to support this complex, multi-year project. This year funding (>$20,000/year) for 2 or 3 years shared funding has been awarded through a competitive grant from the California Department of Pesticide Regulation.