Development of Trapping Systems to Trap the Glassy-Winged Sharpshooter Homalodisca Coagulata Adults and Nymphs in Grape

Four trap types in addition to Pherocon AM traps and clones were field tested in 2001 for the ability to catch the glassy-winged sharpshooter Homalodisca coagulata (GWSS). Traps were deployed in citrus groves and grape vineyards with known high populations in addition to groves and vineyards with low populations. Trap types tested included flight intercept traps (5 colors), plates (11 colors), colored discs (12 colors), and nymph traps (3 colors). Traps were checked weekly and visual count of egg masses, nymphs, and adults were made. Trapped GWSS were sexed, and females with forewing spots of brochosomes or residue were noted. The data from the intercept traps and colored plates clearly indicated that GWSS are attracted to yellow as well as orange. Attraction to these colors was statistically significant and demonstrated that even though the AM type trap may have reliability issues, it is clearly not a ?blunder trap.? Traps reliability stems from three key issues. First, the glue is not sticky enough on the AM trap (and clones) especially above 90E F. Second, the reflected wavelengths (8) below 500 nanometers are somewhat repellent to GWSS. Finally, dusty conditions can shorten the AM traps effectiveness from two weeks to 1 week or less, and temperatures above increased the probability of escape.

The yellow and orange colored plates with Stickem Special Holdfast formulation were very successful in catching adult GWSS. Yellow plates caught statistically more GWSS than AM traps while orange traps usually caught more than the AM traps. The interesting thing is that the yellow plates were more reliable at catching GWSS at low population levels than the AM traps. The nymph traps reliably caught 1st through 5th instar nymphs in moderate to low populations. These traps are easy to deploy in grape canes in situations where it could take hours of searching to locate nymphs. Low populations of GWSS nymphs in a vineyard may pose threats of moving X. fastidiosa from vine to vine within trellises. Flourescent yellow and canary yellow intercept traps attracted large numbers but the collection mechanism only caught about 15%of the bugs that encountered the panels, which made the traps unreliable. However, intercept traps were capable of catching live insects and may have utility when a better capture devise is incorporated.
Additional progress was made in determining that adult and nymphs are attracted to upper UV, and certain wavelengths in the yellow and orange ranges. The preliminary data indicated relationships between the number of ovipositional females trapped and oviposition in associated vegetation.

Female GWSS secrete and deposit brochosomes on the forewings just prior to oviposition. These spots are then scraped off during oviposition. Furthermore, white spots are secreted before each egg mass is laid, and female GWSS can only produce rod shaped brochosomes after mating. It is therefore feasible to relate preovipositional females with white spots and residues to egg masses in associated vegetation analysis. The white spots are very visible on females caught in traps suggesting they are looking for a place to lay eggs.

PDF: Development of Trapping Systems to Trap the Glassy-Winged Sharpshooter Homalodisca Coagulata Adults and Nymphs in Grape

Management of Pierce’s Disease of Grape by Interfering with Cell-Cell Communication in Xylella Fastidiosa

Xylella fastidiosa is an “endophytic” bacterium?one that colonizes the internal tissues of the host plant. A key determinant of success for an endophyte (and symptom formation in the plant) is the ability to spread within the plant from the initial site of infection because the bacteria use up local resources as their population grows. As a large group of bacteria accumulates, they start behaving differently. There is strong evidence that Xylella, like its related plant pathogenic bacteria such as Xanthomonas campestris, secretes a gum that protects it, and produces degradative enzymes such as cellulases that break down plant tissues, but only after a large number of cells have developed at a site. These virulence traits, which allow them to expand into new vessels in a plant, would be ineffective for solitary cells. Hence the pathogen must repress the production of such traits at low cell numbers when it would be futile, and express such traits only when it has reached higher cell density, basically “ganging-up” on the plant. The bacteria keep tabs on their population size by communicating via a small diffusible signal molecule. Many different types of signals are employed by different species of bacteria, however more than one species often employ the same signal. In addition, some bacteria are capable of degrading the signal of another species, or blocking it with a “look-alike” analog. Our research involves finding other bacteria that can block or confuse the signals Xylella uses to spread in the plant. The first step in this research is to identify the signal used by Xylella. We have found that Xylella employs the same signal molecule, DSF, as the related plant-pathogen Xanthomonas. We have engineered a collection of DSF “signal-sensing” strains of Xylella to be used as molecular tools that will allow us to monitor signaling in Xylella itself. These bacteria glow when they sense DSF and will indicate which genes in Xylella are being expressed in such a cell-density-dependent fashion. We are fortunate in finding that Xylella shares the same cell signaling system as Xanthomonas, since we can use a fluorescent “signal-sensing” strain of Xanthomonas to help us find other bacteria that grow with Xylella in the plant that can disrupt the signal. We have engineered a mutant strain of Xylella that should be unable to make the signal, but are still characterizing it. When finished, we will use this strain to verify the importance of signaling for survival and spread of the bacteria in the plant. We expect that the mutant will have a smaller population size and will be less efficient at spreading throughout the plant. We have collected potential DSF degrader and DSF analog producer strains of bacteria from infected grapevines and are preparing to test them with our signal-sensing strains to see if they can disrupt Xylella signaling. Such strains should be effective in reducing disease when introduced into plants as endophytes or if the genetic ability to produce these compounds or degradative enzymes is introduced into grape.

PDF: Management of Pierce’s Disease of Grape by Interfering with Cell-Cell Communication in Xylella Fastidiosa

Host Selection Behavior and Improved Detection For Glassy-Winged Sharpshooter, Homalodisca coagulate (Say)

Leafhopper vectors of Xylella fastidiosa (including GWSS) behave very differently than most herbivorous insects. GWSS has evolved many unusual adaptations that enable subsistence on xylem fluid. Adult GWSS may feed on hundreds of different host species, are long lived and exceptionally mobile and fecund. Nutrition affects GWSS female fecundity and longevity and malnutrition is a primary source of mortality of immatures. We have established that adults prefer to feed on xylem fluid with specific chemical characteristics (high amide concentrations). Nymphs develop more successfully on xylem fluid with low amide concentrations and proportionally higher concentrations of many of the more dilute amino acids that are deemed essential for the development of most insects. We have also established the physiological basis for this phenomenon: adults can efficiently use nitrogen and carbon from high amide concentrations, whereas young developing nymphs cannot. Given the pivotal status of host plant nutrition on GWSS behavior we examined GWSS behavior and that of its parasitoids in field and laboratory experiments to elucidate the underlying mechanisms. We determined GWSS response in feeding and oviposition to host plant species of different quality in the field by varying plant spatial patterns. We determined that previous GWSS host use affects present host use and future longevity. We found that the relative quantity/frequency of a plant species in the landscape does not influence GWSS oviposition preference in the field. We found that GWSS feed in differential numbers on Vitis spp. and selected cultivars. New predators of eggs (tree crickets) were identified. The behavior of parasites in parasitism of GWSS eggs was found not to be related to host plant species or frequency of availability. We determined that parasites can overwinter in GWSS eggs and that they can successfully parasitize eggs up to 1-2 days before nymphal emergence.

PDF:  Host Selection Behavior and Improved Detection For Glassy-Winged Sharpshooter, Homalodisca coagulate (Say)

Bacterial Polysaccharides Expressed by Infective Xylella Fastidiosa During Pierce’s Disease

Xylella fastidiosa are gram-negative bacteria that occur in the xylem of PD-infected grape plant, and are transmitted by xylem-feeding insects; leafhoppers and sharpshooters. Our hypothesis is that bacterial polysaccharides (EPS and LPS) produced by X. fastidiosa may be causative agents of Pierce’s disease on grapes.

We have chemically analyzed isolated PD-infected grape xylem sap and Xylella cultures for Xylella EPS and LPS. We have found no evidence for the production of a “xanthan-like” EPS gum. We concluded that the symptoms of Pierce’s disease are not due to a buildup of a Xylella ESP gum in infected vines. Physical occlusion of infected xylem tissue is therefore more probably due to adhering and clumping bacteria cells, and/or to the production of an abundant LPS.

Our research has identified a Xylella cell-wall LPS (lipopolysaccharide), composed of polymeric D-galactose and L-rhamnose sugars. Significantly, Xylella LPS from the grape strain is structurally different to LPS isolated from orleander-specific Xylella.

Perhaps most important, we have shown than an antibody raised against the Xylella grape-pathogen recognizes the purified LPS from this strain, but does not detect LPS from the orleander Xylella or from control bacteria. This antibody also detects the Xylella LPS in PD-infected grape sap isolated from plants in the field.

Our results suggested that an Xylella LPS antibody kit could be developed as a early diagnostic for Pierce’s disease infected vines, that could be used by the non-specialist in the field. With this goal in mind, we are presently developing a quantitative, color-based LPS-antibody dot blot assay for the early detection of PD.

PDF: Bacterial Polysaccharides Expressed by Infective Xylella Fastidiosa During Pierce’s Disease

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.

Key to Management of Glassy-winged Sharpshooter: Interactions Between Host Plants, Malnutrition and Natural Enemies

The goal of the program is to determine the relationship of host plant xylem chemistry, and leaf morphology on host selection, feeding and ovipositional behavior of GWSS and its parasites.

GWSS oviposits in many plant species, yet the majority of GWSS egg masses tend to be concentrated on a few select host species that apparently offer the quality of xylem fluid (food) required for survival of nymphs. Food quality for nymphs appears to be an important factor affecting the population increase of GWSS. We concentrated on developing field methods and data towards determining the host selection behavior and use for oviposition of known host plants preferred for feeding by the adult GWSS. Experimental sites that offered mixed host plants were established in isolated islands in an open field and a large planting of crape myrtle. Plants on the islands and the surrounding crape myrtle were examined for the presence of GWSS life stages weekly. Results from both sites indicated a statistically significant preference of GWSS for ovipositing on holly plants over all other plant species. The second host most frequently used by GWSS for oviposition was Bradford pear. A few egg masses were also found on other hosts. Pyracantha was chosen as oviposition host only very early in the summer and in the fall. Parasites were able to utilize the GWSS eggs on all hosts. The data suggests that proximity of adult and ovipositional hosts may greatly increase exposure of adult hosts plants to GWSS. We also worked on standardizing and quantifying methodology for rearing GWSS. Strict attention must be given to lighting conditions especially from late to early season. When diapause occurs, diapause can be terminated if three weeks of short daylength are followed by long daylength (we employ 16:8 light:dark regime) so long as the proper ovipositional hosts are present.

We have now begun the second phase of this experiment, which is to assess the suitability to GWSS based on xylem chemistry of California-relevant host species. We have collected xylem chemistry data on the host plants used in the field plots and these are ready for analyses. We are currently investigating Chardonnay grapes, Navel oranges, Spanish Pink Lemon and Crape Myrtle. Data from these hosts will be compared to rates of development on soybean in order to assess the value of each of these species as developmental hosts for GWSS. Quantitative analysis is needed to prioritize the role of each potential host for implementation of GWSS control measures.

By exposing GWSS eggs to parasite adults, we determined the duration of the susceptibility of GWSS eggs to the parasites Gonatocerus ashmeadi and Gonatocerus morrilli. Parasitoids can successfully parasitize 100%of GWSS eggs for at least 7 days after oviposition. We also investigated the overwintering behavior of Gonatocerus sp. and GWSS. We determined for the first time in the U.S. that GWSS as eggs and Gonatocerus sp. within parasitized egg masses can overwinter at north Florida winter temperatures. Laboratory experiments showed that parasitoids fed honeydew provided from excised leaves with live whiteflies lived twice as long as those fed simple honey solution. Perhaps parasitoid abundance could be enhanced by providing alternative food sources.

PDF: Key to Management of Glassy-winged Sharpshooter: Interactions Between Host Plants, Malnutrition and Natural Enemies

Developing an Integrated Pest Management Solution for Pierce’s Disease Spread by the Glassy-Winged Sharpshooters in Temecula

Monitoring of non-agricultural areas demonstrated that glassy-winged sharpshooters occur at significant densities within the landscaped, riparian, and natural areas surrounding and interspersed within the agricultural areas of the Temecula valley. As it will be both politically and biologically impossible to eradicate sharpshooters from these non-agricultural areas associated with the Temecula vineyards, these areas will have to be considered to be permanent and constant sources of glassy-winged sharpshooters.
Insecticide treatments of citrus had a dramatic and significant effect on parasitoids of glassy-winged sharpshooters in the Temecula region. With insecticide treatments few sharpshooter eggs were found in treated areas and only a minute proportion of these were parasitized. At the beginning of April, 2000, glassy-winged sharpshooter parasitoids were recorded in 30%of all sharpshooter eggs collected. By June the parasitism rate had risen to 90%. Parasitism rates continued in excess of 90%for the remainder of the year. The primary parasitoid encountered was Gonatocerus ashmeadi (Hymenoptera: Mymaridae).
Results from the first six months of sampling have yielded positive detection of X. fastidiosa in grapes at many sites throughout the Temecula valley. Xylella fastidiosa was also consistently detected in almond and oleander in the Temecula region. Three other plant species (coyote brush, mustard, and elderberry) showed weak positive results for the presence of X. fastidiosa when tested by ELISA; however, neither culturing or PCR techniques supported these results. We expect that more plants will yield positive results as we begin to routinely use the more sensitive immunocapture PCR method. Thus far, no obvious source of inoculum of PD, other than grape and almond, was detected in the valley using these methods.

To date, five parasitoid species have been investigated as potential biological control agents of GWSS. Two were found to lack the host plant range required of an effective biological control agent and the remaining three were found suitable for further research. Rearing methodologies for sharpshooters and parasitoids have been significantly improved. We are now providing sharpshooter egg masses to two laboratories for biocontrol research, one laboratory for behavior research and three laboratories involved in insecticide research. The work determining host plant preference of sharpshooters was largely abandoned as it was deemed no longer necessary with the successful development of rearing methodologies. The construction of the phenological degree-day model is continuing. To date we have taken the insect through a single generation at several temperatures; this research will require additional funding and at least another 18 months of effort before any significant conclusions are made.

Field-trials conducted at U.C. Riverside in 2000 showed that two soil-applied and one foliar-applied neonicotinoid induced high sharpshooter mortality through an 8 week period. Most striking is our observation that imidacloprid applied to grapevines in September 1999 had a substantial and disruptive impact on sharpshooter feeding almost a year later. This may, in fact, be more important to protecting plants from Xylella fastidiosa-carrying sharpshooters then inducing mortality as disease acquisition and transmission will likely be disrupted as well.

Plant microelements such as zinc, copper, manganese and iron, as well as three antibiotics were tested for inhibition against Xylella fastidiosa in vitro. Tetracycline was the most effective antibiotic and zinc was the most toxic microelement. Prophylactic materials being evaluated included 3 inducers of systemic acquired resistance (SAR) and 4 microelement formulations. Therapeutic materials include several formulations of microelements and 2 antibiotics. All prophylactic field plots were mapped for PD each fall, however no new infections were found in either the treated or control vines in 2000. Bactericides were applied as foliar sprays, as materials packed into hollow nylon (DP) screws or in drilled holes packed with bactericides that were suspended in agarose and the ends of the holes were sealed with DP screws. Several of the drilled through/DP screw treated vines did not show any PD symptoms following treatment, however these vines were also severely pruned following treatment. A final assessment of the efficacy of these bactericide treated vines will be made in the summer of 2001. An injection machine that is widely used for injecting avocado trees was found to work well for injecting vines in the spring but less effective in the fall. Several potted plant experiments were performed using soil drenches of microelements as therapeutic or prophylactic bactericides. Although significant phytotoxicity occurred with some of the materials, managanese and zinc treatments may have some potential. A custom-made pressure bomb was purchased and used to express xylem sap from 1 meter long grapevine canes that were treated with various microelements. Surprisingly high concentrations of zinc and managanese were found in the xylem sap of grapevines treated with amino acid chelates of these elements. Additional experiments are now being done to determine whether the xylem sap is actually toxic to Xylella fastidiosa or if the ions are too tightly bound.

PDF: Developing an Integrated Pest Management Solution for Pierce’s Disease Spread by the Glassy-Winged Sharpshooters in Temecula

Sequence of Xylella fastidiosa Strain Causing Pierce’s Disease of California Grapevine

The annotation software will be based on the one being used in the Xanthomonas axonopodis project. Whose bioinformatics is also carried partially by LBI. The annotation environment was developed internally by LBI and is composed basically of software that interfaces typical annotation oriented programs (e.g. BLAST). The annotation Web interface was also developed by LBI. All annotation data are managed by MySQL, a relational database manager system (DBMS).

Many different shotgun libraries were prepared and distributed for sequencing. About 690 plates of 96 clones each have been distributed to the Sls and Sus for DNA preparation and sequencing. A cosmid library was constructed and a total of 2,717 cosmid ends were sequenced.
The submitted shotgun sequences are only accepted as 96 sample plates, with at least 65%of the sequences with 400 bases with phred quality above 20. When the plates did not meet this criterion they were sequenced again.

Accepted reads deposited (28/02/01): We have a total of 84,167 accepted reads covering about 19 fold of the genome.

We have achieved around 19-fold coverage of the genome with the shotgun sequences alone. The cosmid library end sequencing was done to help in the assembly of the genome. At this point we have identified 17 gaps of which 14 should be closed by cosmid sequencing. We are going to use the PCR strategy to address the other three gaps. The cosmids to close the gaps were distributed in the last week of February to the SLs interested.

The genome assembly, as mentioned above, is done using Phil Green’s phrap. At this point of the project, assemblies are done only to solve genome closure problems (varying stringency and including complete cosmid and plasmid sequences). Scaffolding is done by a program developed at LBI. Genome closure is done based on cosmid and plasmid ends anchoring and ORF colinearity with Xylella fastidiosa-CVC genes when it applied.

While the sequencing to close the gaps is going on, the genome annotation interface is being built in order to undertake detailed comparison to the CVC strain.

PDF: Sequence of Xylella fastidiosa Strain Causing Pierce’s Disease of California Grapevine

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

Objective 1: Understand how Xf moves, and the patterns of its movement, in systemic (grape, blackberry) and non-systemic (willow) plant hosts using microscopy. (Purcell)

Confocal microscopy of red willow inoculated with Xf showed that Xf can multiply to very high levels within individual xylem elements but does not move to adjacent cells in non-systemic hosts. Thus, the low populations of Xf recovered from willow by culturing represent many Xf cells within a very few colonized cells rather than fewer numbers of Xf in a larger number of cells. High magnification scanning electron microscope and modified staining methods of Xf in grape revealed “tentacle-like” structures (fimbriae) at the narrow ends and division plane of dividing Xf cells collected from expressed xylem sap of infected grape but not when collected from cultured Xf cells from PW medium. Xf aggregated on the surface of xylem vessels in PD-infected grape petioles as well as dormant infected grape tissue (green and woody stem pieces) early or late in colonization of grape xylem. The bacteria were embedded in a matrix of fibers that covered them in a net-like fashion. This matrix of Xf cells and extracellular materials (a biofilm) adhered to the xylem cell walls. The fimbriae of attached Xf connected only in contact with plant tissues. Biofilm formation appears to be a consistent feature of colonization of grape by Xf. Cells of Xf grown in a new culture provided by Brazilian collaborators produced abundant extracellular fibrils but not fimbriae, and these cells attached strongly to glass, unlike cells grown in traditional Xf media (PW, etc). Our findings suggest that Xf produces attachment structures only under specific environmental cues.

Objective 2. Understand how temperature influences the movement and survival of Xf and the incidence and/or severity of PD. (Purcell)

Field inoculations of grape at different times of the year at Oakville (1997), Davis (1997-98), Fresno (1998-99) and Hopland (1999) consistently confirmed that infections during April through May and to a much lesser in June resulted in persistent infections of Xf the following year; whereas most June, July or August inoculations resulted in non-persistent infections unless the base of canes were inoculated. At the Hopland site, recovery of early-season infected vines was the first evidence of possible climate-mediated recovery of PD-infected vines that was not explainable by pruning eliminating the Xf infections.

Inoculations of vines during April 2000 in San Luis Obispo Co. and Santa Barbara Co. suggest that cool temperatures (not exceeding 16 C) following inoculation may not allow systemic infection by Xf. The highest populations of Xf occurred at the bases of symptomatic canes and decreased towards the tip of sampled canes in experimentally inoculated or naturally infected vines.

Experimental freezing of dormant, PD-infected potted vines cured the plants of PD at rates of 0 to 70%after exposures to temperatures ranging from -2 to -10 C for 3 successive freezing exposures. Cane segments treated identically at the same time never recovered, suggesting that some aspect of plant physiology, rather than the direct action of freezing alone, kills Xf by freezing in grape xylem. The survival of Xf after freezing in various liquid media further supports this view.

Objective 3: Determine whether vegetation barriers or trap crops can reduce the incidence of PD in riparian areas. (Weber)

Two trap crop trials have been established on either side of a large vineyard in Napa. One trial borders the Napa River, the other Milliken Creek. Vines are spaced 9 feet (rows) by 5 feet (in-row). In each trial, St. George rootstock is planted at the ends of adjacent rows to create the trap crop treatments. Trap crop treatments include the first 6 vines in 12 adjacent rows. In each trial, there are three replicate trap crop planting and three control plantings where Chardonnay or Pinot Noir are planted to the end of the row.

The vineyard was planted in May 1999. By October 2000, trap crop vines along the Napa River had reached the upper trellis wire and had filled in well. They were pruned in December along with the rest of the vineyard leaving wood on the ?fruiting? wire. On the Milliken Creek side, deer did considerable grazing damage throughout the year and the vines did not develop as well. Admire (soil-applied imidacloprid) was applied to trap crop vines in November 1999. Due to concerns about its efficacy, this will be replaced with a spring 2001 treatment of Provado (foliar-applied imidacloprid). BGSS were monitored in 2000 in both trials using yellow sticky cards placed at the ends of rows. BGSS were detected in all treatments, although counts were relatively low. Per-trap catch totals ranged from 2 to 23 BGSS for the trapping period March-October. A PD disease survey was conducted in both trials in Sept. 2000 extending approximately 40 vines into the vineyard. Only 5 vines were found showing PD symptoms.

Monitoring and mapping will continue in 2001.

Objective 4: Develop transformation and transposon mutagenesis for Xylella fastidiosa. (Kirkpatrick)
Procedures were developed to successfully introduce the transposon, Tn5, into two different strains of Xylella fastidiosa (Xf). Electroporation conditions that were similar to those that have been used to electroporate DNA into several Xanthomonas species were found to be efficacious for electroporating DNA into Xf. Several attempts to transpose Xf with two different Tn5 and two Tn10 suicide constructs failed to produce transposed Xf cells. However, using identical electroporation conditions, we obtained several hundred Tn5 mutants of both the Fetzer and Temecula Xf strains using a ?transposome? complex composed of a transposase protein/DNA complex containing Tn5. Southern blot analysis showed these were random, single, Tn5 inserts throughout the Xf genome. Sequence analysis of Xf genomic DNA that flanked the Tn5 insertion identified several genes that were found in the CVC strain of Xf. We now have approximately 500 Tn5 mutants stored at ?80; inoculation of grapevines and other analyses of the mutants will begin in a few months after several thousand mutants have been obtained.

Three, 1.8 kb Xf plasmids were cloned and sequenced from the UCLA Xf strain. The largest open reading frame (ORF) on these small Xf plasmids had significant homology with another phage replicase gene, suggesting this Xf ORF encodes a plasmid replicase gene. Initial cloning of these plasmids probably interrupted the promoter for this ORF and we have since recloned these plasmids at another location on the plasmid. We are now introducing the KanR gene from the Tn5 construct described above, which we know is expressed in Xf, into the cloned Xf plasmids. In this manner we hope to construct an Xf/E. coli shuttle vector that will greatly facilitate molecular genetic analyses of Xf.

Objective 5: Isolate and identify endophytic bacteria that systemically colonize grapevine xylem. Identify natural, or genetically engineer, endophytes to be antagonist to Xf. (Kirkpatrick)

Several hundred bacterial isolates were obtained from both healthy and Xf-infected grapevines located in Napa and Yolo counties. Specific grapevines of two cultivars were sampled bimonthly during 1999 and 2000. In addition, healthy appearing grapevines that were growing in the middle of vineyards that were decimated by PD were also randomly sampled. Four different media were used to cultivate the bacteria, however no significant differences in the types or numbers of bacteria were observed using the different media. Gram stains were performed on all of the isolates so that the appropriate Biolog plates can be used to initially identify the isolates, at least to the genus level. Beginning in the spring these isolates will be pin-prick inoculated into grapevines growing in the greenhouse. After several weeks, attempts will be made to recover the bacteria from sections of xylem that are 2 or 3cm from the point of inoculation. Any bacteria that are found to systemically colonize grapevine xylem in reasonable concentrations will be tested for natural antagonism towards Xf. We are now screening a random peptide library for synthetic peptides that are inhibitory to Xf. If these peptides are identified, attempts will be made to genetically engineer grape endophytes to express these peptides within grapevine xylem.

Objective 6: Genetics of Resistance to Xf. (Walker)
A Design II mating design with a set of 6 females by a set of 6 male parents, from which we will select sets of seedlings to study the inheritance of Xf resistance, is completed. The mean expression of resistance within a given female across all males and similar comparisons of males across females will allow us to draw conclusions about the inheritance of Xf resistance.

We define Xf resistance as the ability of a genotype to limit the movement of Xf, particularly in a downward direction, and have tested a set of resistant and susceptible individuals to determine whether this definition is valid. The known susceptible genotypes were V. rupestris ? A. de Serres? (the female parent of the 89 population), Chardonnay and the V. rupestris x M rotundifolia genotype 8909-19; potentially resistant genotypes were 8909-04 and 8909-11; and the resistant genotypes were 890915 and 890917. After 4 weeks, Xf was easily detected in the three susceptible genotypes. By 16 weeks, the differences among resistant and susceptible genotypes are very clear in terms of both symptom expression in leaves and unevenly lignified stems and ELISA readings. ELISA is cheaper, faster, quantifiable and more simple than PCR detection of Xf, and can reliably detect 10,000 cfu/ml of Xf in ground plant sap.

150 samples with duplicate readings can be processed in a day at a material cost of $0.28 per plant sample. IC-PCR detection is 10 more sensitive, but is not quantifiable and costs about $1.44 per sample. Nested PCR gave very good results and is 100 times more sensitive than IC-PCR, but few samples can be run per day, and the cost of materials and labor is high ($2.88 per sample). We also examined spot-PCR which would allow several hundred samples to be run per day, but the sensitivity is equivalent to ELISA and cost $2.70 per sample. (All costs exclude labor).

We are mapping Xf resistance in the V. rupestris X M. rotundifolia 9621 population (previously used for Xiphinema index resistance). We have AFLP marker data on about 70 of the 150 individuals and are testing 4 replicates from each of these 150. Xf resistance data from 70 individuals are complete and the others are due for analysis in about 4 months. We plan to place Xf resistance on our existing AFLP based map. If the resistance trait does not place on the existing map, we have seeds for a second mapping population based on a cross of the 8909-15 X 8909-19; a resistant by susceptible genotype which will allow greater power in mapping the resistance genes. However, this seedling population will have to be grown out from seed, propagated and screened for resistance and marker information. We also have (8909-08 and 8909-23) to Chardonnay, which should also segregate widely.

We bench-grafted Chardonnay on each of the following rootstocks: AXR#1, St. George, 3309C, 101-14Mgt, Schwarzmann, 44-53 Malegue, Riparia Gloire, 1616C, Lenoir, 5BB, 5C, Börner, 110R, 1103P, Harmony, Freedom, and Ramsey, and Chardonnay (control), Riesling, Sylvaner, Chenin blanc, and Colombard.

Own-rooted Chardonnay, Riesling, Sylvaner, Chenin blanc and Colombard were also inoculated. We have evaluated all three reps for PD expression and ELISA sampling to determine the extent of downward movement is continuing. The vines were cut back to basal buds, are now re-growing and will once again be evaluated for symptoms and by ELISA.

Objective 7: Determine whether xylem chemical composition is involved with PD resistance or susceptiblity in grape varieties and common Xf plant hosts. (Andersen)

In an effort to determine whether xylem fluid chemistry is related to resistance to Xylella we investigated the chemistry of Vitis genotypes covering a wide range of resistance/tolerance. Xylem fluid chemistry of 10 grape genotypes belonging to 5 species has been characterized, although only the amino acid data was presented. Total amino acids varied over 4-fold, and many amino acids only occurred in less than 10uM or trace quantities. A complete correlation with resistance will await compilation of organic acid and sugar data. Total amino acids in xylem fluid collected from Chardonnay grafted on 4 different rootstocks showed an effect of rootstock and of infection with Xylella fastidiosa. Xylem fluid of Chardonnay on 3309 rootstock tended to be most dilute of the rootstocks and XF infection increased total amino acids in xylem fluid of Chardonnay on all rootstocks except SO4. Arginine was the amino acid that showed the biggest increase in amino acids with XF infection. A more complete analysis of the effect of xylem chemistry of grape genotypes on resistance to Xylella fastidiosa and the change in chemistry with Xylella fastidiosa infection awaits further research.

An investigation is underway to determine whether resistance to Xylella fastidiosa is influenced by xylem fluid chemistry across plant species common to both Florida and California. Xylem fluid chemistry of 32 host plants species/cultivars was analyzed and natural X. fastidiosa (resistant/susceptible) was noted by PCR analysis. About 50%of the plant species/cultivars were Xylella negative. Those that were Xylella-negative were mechanically inoculated with X. fastidiosa during the fall of 1999. After 4 weeks only one species was positive, Vitis rotundifolia (wild grape). Each of the Xylella negative species/cultivars have also been sampled fall 2000 and are currently being analyzed via PCR. Those species that still do not harbor Xylella may be inoculated with different strains for confirmation of resistance to multiple Xylella strains. In vitro nutrient requirements of Xylella have been studied for 3 months without much success. A UCLA strain of X. fastidiosa was subjected to 3 months of experimentation using Chang and Donaldson?s chemically defined media. This UCLA strain grew well on PD or PW+ but did not grow on the chemically defined media. We switched strains to ATCC 35881 and tested this strain on all the media. The result was that it grew well on all the media and it even grew slowly on a media consisting of glutamine as the only amino acid source. Thus, nutritional fastidiousness is extremely strain dependent. Additional experiments will be performed with a different strain (probably the Temecula strain) after consultation with B. Kirkpatrick, S. Purcell and A. Walker.

Lytic peptides were extremely effective against Xylella. They act by disrupting cell membrane integrity of bacteria but not eucaryotic cells of higher animals. Cecropins were one to three orders more effective than tetracycline. Xylella fastidiosa incubated with Cecropin A and B at 1 uM resulted in 100%inhibition of growth; incubation with Xylella at 0.1 uM resulted in 95%inhibition. Indolicidin was a fairly strong inhibitor of Xylella fastidiosa with 100%inhibition at 9.5 uM. Magainin II was followed by Magainin I in potency. For tetracycline 100%inhibition was achieved at 112 uM. These compounds may serve as a potent naturally occurring pesticide against Xylella.

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

Genetic Transformation to Improve the Pierce’s Disease Resistance of Existing Grape Varieties

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.

PDF: Genetic Transformation to Improve the Pierce’s Disease Resistance of Existing Grape Varieties