Biological, Cultural, and Chemical Management of Pierce’s Disease

Understand how Xf moves, and the patterns of its movement in systemic (grape, blackberry) and non-systemic (willow) plant hosts using microscopy. We concentrated our studies of the patterns of colonization of plants by Xf during the past year, on using high magnification scanning electron microscopy (SEM) to document how Xf systemically colonizes the xylem of grape and symptomless plant hosts. As reported previously, platinum-gold vapor staining of freeze-dried tissues, followed by high magnification SEM (HMSEM), allowed us to visualize thread-like attachment structures on Xf cells that we tentatively have identified as bacterial fimbriae. The EM lab at Berkeley has had continuing problems with the chamber needed to coat (stain) specimens for HMSEM, which postponed and then greatly delayed our progress in further studies needed to estimate how common and under what conditions these structures occur. For example, we have not seen these fimbriae-like structures in Xf cells grown in culture. In addition to fimbriae-like structures, we noted that Xf colonization of grape resulted in interconnected networks of amorphous strands to which many of the bacteria adhered. We speculate that these structures are what results from freeze-drying (critical point fixation) the plant samples to be examined. This aspect of the project is now being further explored by a grant begun late last year to Lindow and Purcell at UC Berkeley on bacterial attachment. Under the new grant, mutants of Xf are being generated that target particular genes, such as those presumed through gene sequence homology to code for fimbrial proteins. Progress was made by Helene Feil in the Lindow lab at UC Berkeley to develop GFP-mutants by developing a method of homologous recombination to target specific genes into Xf. This also would enable the systematic production of mutants to explore gene function in Xf.We still need to further examine the behavior of Xf in symptomless, systemic and non-systemic hosts. Our results to date indicate that willow is a poor choice of a non-systemic host for experimental studies because it is very difficult and time-consuming (and thus also expensive) to find plant cells colonized by Xf. The number of colonized cells in willow was very low, even when we examined restricted insect inoculation of leaf veins to a very small region. However, our studies revealed that xylem cells in willow that are colonized by Xf are completely filled by the bacterium. This answered one question about how Xf colonizes non-systemic host plants. Other species of non-systemic host plants should be examined to test the generality of our findings, but we intend this final year of funding under the joint project to concentrate on systemic hosts such as tobacco. We can easily and reliably infect tobacco, and we so far have found no evidence for the occurrence of a visible matrix or network in tobacco that is characteristic of systemic populations of Xf in grape. One of our objectives in further examining symptomless systemic hosts is to seek further evidence that symptoms may be due to the formation of a bacterial matrix with Xf infections rather than the simple plugging of xylem vessels with bacterial cells alone. This work also may provide clues to help identify any plant signals or processes that might induce or enable matrix formation by Xf cells. § Understand how temperature influences the movement and survival of Xf and the incidence and/or severity of PD. Continuing research on temperature effects will be concentrated on how ambient temperatures affect the growth and movement of Xf in weeds, for which we have preliminary data from ongoing studies this winter. Three cool-weather alternate systemic hosts of Xf are fava bean, prickly lettuce, and poison hemlock. Summer annuals, which we propose using to examine Xf colonization at warm temperatures, are common cocklebur, common sunflower, and annual bur-sage. Prickly lettuce can also be used in the tests of high temperatures, since it persists in the field throughout the summer. Results from December 2001 and January 2002 are described and are currently underway in Bakersfield to examine the effects of winter temperatures on Xf and systemic movement in fava bean, prickly lettuce and poison hemlock. Results shown are for the first three weeks of growth in the greenhouse at an average temperature of 25°C (+/- 5°C) and field average of 9°C (range 3 to 13°C). Xylella was recovered from 40%of plants grown in the field (17 of 43 inoculation sites) and 51%of plants grown in the greenhouse (21 of 41 inoculation sites). However, initial indications are that bacterial populations were lower, and there was less systemic movement of the bacteria for field-grown plants compared to greenhouse-grown plants.§ Determine whether vegetation barriers between riparian areas and vineyards and/or insecticide-treated “trap crops” on the ends of vineyard rows can reduce the incidence of PD. Our findings that the systemic insecticide imidacloprid (Provado, Bayer Corp.) did not rapidly kill adult blue-green sharpshooters (BGSS) or GWSS required a re-evaluation of this approach. Instead of Admire, a soil-applied form of imidacloprid, we have applied the Provado formulation of imidacloprid for foliar applications at 2-week intervals during April-May. As was true of 2000, trap catches of BGSS were low in 2001, but each of 3 replicates of treated and control plots at the experimental site were mapped for PD and monitored during the season for BGSS. So far the incidence both of BGSS and PD has been very low in these young (third year) vines.§ Determine the epidemiological role of seasonal fluctuations of Xylella fastidiosa populations in riparian host plants of the North Coast. The objective of this research is to determine how populations of the Pierce’s Disease (PD) bacterium, Xylella fastidiosa (Xf), behave in five riparian hosts over time. A replicated field experiment was initiated at three commercial vineyards in Napa County, CA, to measure Xf populations in five riparian plant species: Vitis californica (California grape), Rubus ursinus (California blackberry), Rubus discolor (Himalayan blackberry), Sambucus mexicana (blue elderberry), and Vinca major (periwinkle). All five species are known hosts of Xf and its vector, Graphocephala atropunctata (blue-green sharpshooter, BGSS). All five species are listed as prime candidates for removal in the Information Manual for Riparian Vegetation Management for Pierce’s Disease in North Coast California Vineyards. In June 2001, individuals of the five plant species were inoculated with a local strain of Xf by placing a drop of a suspension of cultured Xf cells on a stem and piercing the stem with a pin beneath the suspension. In mid summer and early fall, Xf populations were estimated in petioles located immediately distal to stem inoculation sites by culturing on selective media. Populations of Xf reached detectable levels in California blackberry, blue elderberry, and California grape by mid summer and increased by early fall. Xf was not detected in periwinkle until early fall, when populations were found to be as high as that of California blackberry, blue elderberry, and California grape. Increased flight activity of young adult BGSS, which peaks in mid summer and remains high through early fall, may contribute to spread of Xf from infected California blackberry, blue elderberry, periwinkle, and California grape to adjacent vineyards. This may not contribute to increased disease incidence in adjacent vineyards within the same growing season, since canes that become infected late in the growing season are pruned out the following winter. However, adult BGSSs that acquire Xf from California blackberry, blue elderberry, periwinkle, and California grape in mid summer/early fall and survive the winter are still capable of spreading Xf to vineyards the following spring. We will continue to collect petioles from inoculated plants and monitor Xf populations until spring 2004. We are especially interested in determining if Xf overwinters in Himalayan blackberry, blue elderberry, and California grape (California blackberry and periwinkle are known overwintering hosts). Overwintering hosts likely play an important role in the epidemiology of PD in providing a source of bacteria for spring infections, especially near vineyards where infective adult BGSS do not survive the winter. Xf was not detected in inoculated Himalayan blackberry in mid summer or early fall. Based on past research, we expected to detect Xf in Himalayan blackberry at both times. Differences in our results, with respect to results of past research, may be due to differences in inoculation method, Xf strain, and/or environment.§ Evaluate plant micronutrients and antibiotics as potential bactericides for elimination of Xylella fastidiosa (Xf) in grapevines and determine the efficacy of using micronutrients and antibiotics to cure Xf-infection in field-grown grapevines.Symptom evaluation data from the 8-year-old Merlot vineyard in Napa suggests that the most effective mode of treatment delivery is to directly inject the materials in a gel-like matrix into the vines cordon. Foliar treatments must first be taken into the plant to become systemic, and the concentration of material that actually makes it into the xylem is going to be much lower than the concentration at the plant surface. D.P. screws place the treatments within the xylem, but only a small area of exposure is produced and materials must be taken up by xylem passing through the cotton pellet. Drill/injection seems to result in a more efficient uptake, possibly due to the increase in xylem surface area exposure created by drilling completely through the vine. The agarose matrix also creates a continuous bridge for xylem passing from below the application site to plant parts above. The difference in efficacy between Streptomycin applied as a DP screw and as a drill/injection further strengthens this argument.Micronutrients (zinc, manganese, and copper) applied as direct injections and at higher rates than labeled for micronutrient deficiencies appear to have the potential of creating an unfavorable environment for Xf. This is evident in the 8-year-old Merlot vineyard in Napa where these micronutrients produced disease ratings up to 50%lower than ratings recorded for untreated controls when applied as a direct injection.Severe pruning continues to give initial positive growth response in the summer after the vine is severely pruned in the preceding winter. The low disease ratings recorded in the fall 2001 for 6-year-old Merlot vines in Napa pruned in February of that year coincide with similar results recorded during the fall of 2000 for 8-year-old vines pruned early in the 2000 season (see 2000 Progress Report). Pruning a cordon to within 6 to 8-in. of the graft union allows a previously infected vine to send up a new shoot that will be trained into a cordon that is free of xylem blockage. Ideally, this new shoot can then be treated prophylactically to avoid re-infection.Prophylactic trials are located in both northern and southern California. Of trials evaluating all treatments, two are located in Temecula, CA and four are located in the Napa Valley. An additionally vineyard in Temecula has been treated with the three systemic acquired resistance (SAR) inducers included in the prophylactic treatments. Two additional trials containing all treatments except the Rezist/Stabilizer are located in the Santa Cruz Mountains.From the preliminary data obtained in this experiment, copper amino acid chelate shows the most potential in raising the level of available metallic ions that may negatively affect the growth of Xf of all the metal-amino acid chelates that were tested. This information is unfortunate due to the significant level of phytotoxicity that the copper amino acid chelate causes when applied as a foliar spray to grapevines. These results also bring into question the results obtained from nutrient analyses of xylem sap collected from Cabernet and Thompson Seedless vines treated with zinc, manganese, and copper amino acid chelates that showed that very significant increases in the concentration of these metal ions after foliar applications of zinc and manganese chelates (see 2000 Progress Report). Zinc and manganese amino acid treatments, although shown to raise microelement levels in grapevine xylem to concentrations that would be theoretically inhibitory to Xf, may not produce metallic ion concentrations that are actually toxic to Xf en planta. It is possible that the chelated ions are not free to act as bactericides against Xf cells in the xylem. Experiments designed to test bactericidal properties of xylem sap extracted from a prophylactically treated vine are now being performed in order to develop a standard protocol for screening potential bactericides en planta.§ Isolate and identify endophytic bacteria that systemically colonize grapevine. Develop methods to genetically transform grape endophytes to express anti-Xf peptides.We were very pleased that the results of grapevine inoculation showed that many of the bacterial isolates could multiply and move in grapevine xylem vessels. Additional endophytes collected from field isolations will continue to be screened for systemic movement within grapevine as well as natural antagonism to Xf. In the future, grapevines that were inoculated with endophytes that were recovered at least 6cm above or 6 cm below the point of inoculation will be re-sampled to find out how far the endophyte actually moved within the 4 week period. We find the results of this subset of data promising. Large scale processing of the more than 600 endophytes collected over the past three years is currently under way. We will also be screening non-tumorogenic strains of Agrobacterium vitis acquired from Thomas Burr at Cornell University, and a Bacillus species obtained from Ken Eastwell at Washington State, Prosser, WA for systemic movement in grapevine as well as antagonism to Xf.§ Develop transformation/transposon mutagenesis systems for Xf and use Xf mutants to identify potential pathogenicity, movement or insect transmission genes.Understanding the complex interactions between the plant, pathogen, and insect vector is imperative for the development of effective disease controls. Recently, the complete genome sequence of a citrus strain of Xylella. Fastidiosa (Xf) was determined (Simpson et al., 2000). Analysis of its genome revealed important information on potential plant pathogenicity and insect transmission genes. However, more than half (53%) of the identified ORFs in X. fastidiosa encode proteins with no assignable function. In addition, some of the putative gene functions assigned on the basis of sequence homology with other prokaryotes may be incorrect. In order to identify and understand the function of X. fastidiosa genes, it is imperative to develop techniques to knock out and complement putative Xf pathogenicity or transmission genes.§ Genetics of XF Resistance.The following objectives are currently underway and described in detail in the annual report:1. Complete analysis of a series of crosses (Design II mating scheme) allowing the quantitative inheritance of Xf resistance to be evaluated.. Complete a genetic map of a Vitis rupestris x Muscadinia rotundifolia seedling population using AFLP (amplified fragment length polymorphism) markers to allow the identification of DNA markers to Xf resistance and eventual identification of Xf resistance genes and their genetic engineering into vinifera cultivars. 200hi3. Utilize genetic markers to Xf resistance to accelerate the introgression of Xf resistance into table, raisin and wine grapes.4. Develop DNA markers from resistance sources other than M. rotundifolia for use in breeding table, raisin and wine grapes through the development of additional mapping populations and the bulk segregant analysis of DNA markers. § Xylem Fluid Chemistry Mediation of PD Resistance.Our primary objective was to establish the effects of xylem chemistry on the resistance/susceptibility of Vitis to Pierce’s Disease. In 2001, we determined seasonal changes in the xylem chemistry of a wide variety of Vitis genotypes that expressed differential rates of Xylella fastidiosa (Xf) susceptibility. Chemical differences between Vitis genotypes were pronounced, and also changed greatly throughout the year. Moreover, we established that even short-term exposure to xylem fluid caused differential growth habits and colony formation in subsequent Xylella growth. Xf exposed to the xylem fluid from susceptible genotypes of Vitis formed significantly larger colonies than bacteria exposed to resistant genotypes. Large colony formation may be critical to expression of Xf virulence, as Xf typically can survive and persist in resistant Vitis genotypes; it simply does not form large colonies that adhere to xylem walls and eventually occlude xylem vessels.We developed a simplified in vitro diet for Xf that suggests that certain strains of Xf may not be as “fastidious” in nutritional requirements as once thought. Xf developed (and flourished) with the only organic sources being a non-amino source of nitrogen and one of each of three primary organic compounds found in xylem fluid (1 amino acid, 1 organic acid and 1 carbohydrate). Xylella was able to persist with the non-amino nitrogenous compound as the sole organic source, but we found each of the main xylem constituents to play a major role in the formation of large colonies.We completed an extensive database looking at xylem chemistry throughout the year on a large variety of alternative (non-Vitis) host plants and compared these to rates of Xf infection. The statistical analysis is now being completed to determine if resistance mechanisms are the same for other host species as for Vitis, and to further our knowledge of alternative hosts that may be important in the spread of Xf. Lastly, we completed our study on naturally occurring peptides with high antimicrobial activity (lytic peptides). We identified the lytic peptides most lethal to Xf, and also examined how stable they will be in xylem fluid. These compounds may eventually be incorporated in control strategies for Xf via genetic engineering or direct application of compounds into the xylem fluid.