We now have an efficient transformation system and are able to routinely produce transgenic plants of several grape cultivars. We have performed numerous transformation experiments with marker genes carry regulatory sequences that might be expected to cause the gene products to be secreted from the cell. We have obtained a xylem-specific protein gene from a research group in Japan in order to use its xylem-specific regulatory sequences. We have introduced a PGIP gene that has previously been shown to have disease reducing effects in other plants.

Significant progress was made on nearly all of the seven diverse objectives that comprise this long term research project. Microscopy and plant xylem chemistry studies have been initiated to understand why Xylella fastidiosa (Xf) systemically colonizes and multiplies to high concentrations in some plant hosts such as V. vinifera while only establishing “microsite” infections in other plant hosts. Field plots were established in Napa to evaluate the utility of insecticide-treated “trap” crops to reduce the numbers of blue green sharpshooter vectors that enter vineyards from adjacent riparian habitats. Experiments were conducted to better understand how cold temperatures influence the overwintering of Xf in grapevines and how severe pruning may have some promise as a management tool for PD. In vitro studies determined the concentrations of plant micronutirents such as zinc, manganese and copper that are toxic to Xf and several large field trials using these materials were established in Napa and Temecula commercial vineyards. Several methods were evaluated to introduce bactericides in grapevines. Electroporation conditions were identified that allow exogeneous DNA to be introduced into Xf cells. Numerous broad-host ranges were electroporated into Xf, however none of these vectors were stable in Xf Native Xf plasmids have been cloned, sequenced and engineered as potential shuttle vectors. Numerous crosses were made between resistant and susceptible Vitis spp. to facilitate the mapping of PD-resistance genes.

Studies on understanding how Xylella fastidiosa (Xf) moves in grape xylem were initiated using fluorescent antibodies and confocal microscopy. Xf bacteria were specifically labeled and identified in fixed grape tissues. A series of time course experiments to track Xf movement after insect or mechanical inoculation are underway. Field and greenhouse studies examining the effect of freezing temperatures on Xf survival are progressing. Pure cultures of Xf can be frozen without significant decreases in viability, however recovery of viable Xf from cold-treated canes was much lower than from non-treated canes. Experimental plots were established in several Napa vineyards to determine whether insecticide treated “trap crops” or vegetation barriers could reduce PD movement into vineyards. Vector abundance and movement is being monitored in these sites and PD incidence opposite tree barriers is being compared to that from sites without tree barriers. Four new sites were identified for the trap crop study, and St. George rootstock was planted in preparation of both foliar and soil applications of imidocloprid next spring. St. George has earlier budbreak than V. vinifera cultivars and may be used to trap early movements of sharpshooters. Extensive evaluations of this pesticide are being conducted to fine-tune its application rate and timing. In vitro Xf studies have established what concentrations of the micronutrients Zn, Cu, Mn, and Fe are toxic to Xf. Means of applying these micronutrients to grape are being evaluated, and the extraction xylem sap from grape canes has been optimized. An antibiotic produced by a Bacillus bacteria strain was shown to be very toxic to Xf on petri plates; studies for its use as a biocontrol agent for PD are initiated. Studies on the characterization of bacterial and fungal endophytes in grape xylem are also underway in vines from Davis and Napa. Work towards mapping resistance to Xf is progressing. Forty V. rupestris X M. rotundifolia selections have been tested and three have high levels of resistance (lack of symptoms and limited Xf movement based on IC-PCR results). Unfortunately, the mapping populations that were made previously, 8913-02 and 8913-21 crossed to Chardonnay, cannot be used because neither of these selections is adequately resistant. One of three highly resistant selections is 8909-15. A genetic map was created for Xiphinema index resistance using the 8909-15 X 8909-17 seedling population. This population may also be ideal to map Xf resistance since 8909-17 is susceptible. However, we also plan to produce other seedling mapping populations by crossing 8909-15 and the other resistant selections with Chardonnay. Crosses with Chardonnay will give a more clear range of responses from very resistant to very susceptible and should therefore be easier to map in. These crosses will also begin the introgression of Xf resistance fromM rotundifolia into V. vinifera. A vineyard of 10 Xf resistant / susceptible variety pairs is establishing in Florida were the effect of xylem chemistry on PD will be studied. A screenhouse was also completed so that V. vinifera varieties can be studied. Technicians and cooperators in Florida have been coordinating and testing equipment for xylem fluid analysis, Xf culture and PCR detection of Xf. Examinations into the possible role of peroxidases (known plant resistance compounds that exist at low concentrations in xylem) in Xf resistance are underway. Many recent resistance studies in other plants have identified peroxidases as important resistance compounds. Other organic and inorganic compounds, found at higher concentrations, will also be examined.

This study was initiated in cooperation with Professors Purcell and McBride to evaluate the effects on a variety of vertebrates of the removal of vegetation as well as planting of non-disease reservoir plants in riparian zones. Studies were initiated in 1997 with the establishment of eight plots, three on Conn Creek (open, managed, and not managed), and three on the Napa River (Ecological Reserve, managed, and not managed) in Napa County, and two (managed and not managed) on Maacama Creek in Sonoma County. Five sampling procedures are used per plot as follows: a.) Two Trailmaster bait stations once per month for 7 days, b.) 15 Sherman live traps for small mammals one night per month, c.) Three point census plots for birds once per week during the breeding season (approx. March 1, 1999 to June 15, 1999), d.) Eight nesting boxes for birds checked weekly during the breeding season and e.) Six 4-square foot reptile boards checked approximately twice per month. Data are tabulated through late fall, 1998. The bait stations showed the most common mammal to be the opossum, followed by squirrels, raccoons, rats, and foxes. Larger mammal activity was lowest in the open grassy plot, and highest in the Maacama Creek managed plot and the Ecological Reserve unmanaged plot. There were no significant differences in totals of all species between the managed and unmanaged pairs of plots. The most common mammal in the live traps was the deer mouse, Peromyscus spp. Activity was highest in the open unmanaged plot and lowest in the Ecological Reserve unmanaged plot, with no significant differences between the managed and unmanaged paired plots. 66 species of birds were identified on the study plots during the 1998 breeding census, and this survey will be repeated during the spring of 1999. Eight species of birds used the nest boxes and the highest occupancy rate was in the unmanaged area on the Napa River, followed by the managed area at Conn Creek. The Reserve area had the lowest occupancy rate. No patterns were evident in use between pairs of managed and unmanaged plots. The reptile boards were used by four species of lizards, two species of snakes, the Pacific tree frog, western toad, and a salamander. The most common organism was the western fence lizard. The highest use was in the managed plot on Conn Creek, with the other plots being similar to each other, with no significant differences in total numbers between managed and unmanaged plot pairs. The results to date are encouraging and with more data we should be able to determine if management of riparian zones has an impact on a variety of vertebrates.

In 1997, Pierce’s disease (PD) was detected in the winegrape-growing area of Temecula for the first time. PD is now scattered throughout Temecula with a few vineyards showing a substantial proportion of infected plants. These outbreaks are concurrent with the appearance of the Glassy-winged Sharpshooter (GWSS), Homalodisca coagulata, a PD vector new to California. PD is the principle factor limiting grape production in the southeastern Unites States where it is spread by the GWSS. Since it was first discovered in Orange and Ventura counties in 1990, numbers of the GWSS have continued to increase as it spreads throughout southern California. In the summer of 1998 the GWSS was observed for the first time in southern Kern County. Our trapping studies indicate that the main PD vector in Temecula is the GWSS, which is generated in many acres of citrus there. Early in the season (Apr-June), greater numbers of GWSS were trapped in vineyards near citrus than in vineyards near other vegetation types. At the peak of trap catches in the season (July-Sept), similar numbers of GWSS were trapped near all vegetation types demonstrating the GWSS’s ability to disperse widely from citrus. The Smoke Tree and Willow sharpshooters were also found but are relatively uncommon native insects; we do not consider them important to the PD epidemic in Temecula. Trapping studies and sweep net sampling did not indicate the presence of other PD vectors including the blue-green, the Green, and the Red-headed sharpshooters. Field studies in Temecula did not reveal any plant hosts that are likely point sources from which the PD bacterium is acquired by the GWSS. For one vineyard we documented a strong correlation between PD symptoms and the proximity of grapevines to citrus, even though we could not detect the PD bacterium in samples collected from this orchard. We are currently conducting greenhouse experiments to examine the potential of citrus to support the PD bacterium. Field experiments with grapevines showed that Admire (Bayer Corp.), a soil-applied formulation of imidacloprid, has a potential to reduce spread of the PD bacterium by killing GWSS and by inhibiting feeding. Admire showed efficacy at least 8 weeks after application. Due in part to our support, Admire was granted a section 24C registration by the California Department of Pesticide Regulation for use in grapevines against leafhoppers, including sharpshooters. In experiments that are underway we will ascertain the potential of Admire to reduce transmission of the PD bacterium by the GWSS. One of the attractions of Admire as part of a management program for PD is that it does not have a negative impact on natural enemies as with most foliar-applied insecticides. For that reason, we examined the effect of Admire in citrus to control GWSS. Citrus management relies on natural enemies to keep pest populations in check, therefore foliar-applied insecticides are frequently avoided. Unfortunately, Admire did not control GWSS in citrus to the degree desired. We suspect that its efficacy may be increased by improving application methods and timing.