XYLEM FLUID CHEMISTRY MEDIATION OF PIERCE’S DISEASE: STIMULATION OF AGGREGATION AND BIOFILM FORMATION Project Leader:

Xylella fastidiosa (Xf) is the causal agent of Pierce’s disease in grapevines. The mechanisms of pathogenicity are largely due to occlusion of xylem vessels by aggregation of Xf and biofilm formation. Our previous work has documented the effects of xylem constituents on both Xf proliferation and biofilm formation. This current research utilizes 1) addition of xylem constituents to defined media in-vitro, and 2) exposure of Xf to xylem fluids of different Vitis germplasms to investigate effects of xylem components on Xf growth and biofilm formation. Xylem fluid is typically low in O2 and our in-vitro studies have established the capability of Xf to grow under hypoxic conditions. The growth in the defined (minimal) media is often superior or equal under the oxygen-limited conditions as compared to the air-saturated media. These effects were found to be variable with Xf strain and media formulation indicating interactive effects between O2 and specific xylem components. Short term (1 hour) to long term (12 days) exposure of Xf to Vitis xylem fluids showed highly significant differences in both Xf growth rates and biofilm formation dependent on Vitis genotype. Xf growth in Vitis xylem fluid was correlated to the concentration of the organic acid citric acid, many of the amino acids including glutamic acid, glutamine, histidine, valine, methionine, isoleucine and phenylalanine and inorganic ions including copper, magnesium, phosphorous and zinc. Biofilm formation was also correlated to specific xylem constituents. Our next experiments will test the growth and biofilm formation of Xf in response to changing the concentration of these constituents noted above in xylem fluid so that the role of each constituent can be assessed. INTRODUCTION We have previously established that a functional relationship exists between Pierce’s disease (PD) expression and Xylella fastidiosa (Xf) colony growth within Vitis germplasms. Colony growth results from proliferation of individual Xf (cfu), aggregation, and biofilm formation. Both in-vitro and in-vivo experiments have shown that both Xf proliferation and biofilm formation may be impacted by a variety of constituents including specific inorganic ions, O2, antioxidants, amino and organic acids and sulfhydryl groups. Newly developed defined media result in variable patterns of Xf colony growth (i.e. PW+ results in rapid bacterial proliferation with comparatively little biofilm formation whereas our newly defined media CHARDS provides slower bacterial growth but high biofilm formation). CHARDS media is equivalent to CHARD2 (Leite et al. 2004) except that starch at 0.2g/liter is added. These media provide an array of tools to test effects of individual compounds on Xf colony growth. Lastly, we can also examine Xf colony formation within xylem fluids of varying Vitis germplasms, and correlate patterns of growth to composition of the xylem fluid. OBJECTIVES 1. Utilize defined media to examine the effects of O2 and other xylem components on Xf growth. 2. Quantify the relationship between naturally occurring xylem constituents (inorganic ions, amino acids and organic acids) and Xf colony growth utilizing xylem fluid from a variety of Vitis germplasms. Objective 1. The effects of O2 on Xf growth and biofilm Our initial work focused on the role of O2 in Xf colony growth. The levels of oxygen found in xylem fluid are highly variable between almost atmospheric to anoxic levels (Gansert et al. 2001; Eklund, 2000). There may also be great variation within a plant (Dongen et al. 2003). Levels documented in xylem are generally well below atmospheric levels, and Xf has been defined as an obligate aerobe incapable of growth without O2 (Wells et al. 1987). We subjected Xf growing in liquid PW+ media to 5 levels of O2 ranging from atmospheric O2 (21%) to anaerobic conditions (0%). Gas treatments were applied for 5 minutes every 24 hours. Xf was cultured under these conditions for 15 days at which time Xf growth, quantified by optical density (OD), and biofilm formation were measured. OD was measured using a Genosys 8 spectrophotometer at a wavelength of 600nm. The formation of biofilm on the surface of polypropylene tubes was assayed by the crystal violet method (Espinosa-Urgel et al. 2000). The oxygen levels were determined using a LaMotte’s Dissolved Oxygen Test Kit (model EDO•code 7414) to insure the accuracy and persistence of treatment conditions. Our results established a relationship between O2 concentrations and Xf growth. For many strains tested, Xf growth rates were highest under atmospheric conditions and declined as O2 levels declined. Growth comparison of Xylella fastidiosa pv. Pierce’s disease strain ‘Temecula’ and Xanthomonas campestris under 21% oxygen and 0% oxygen headspaces, indicate that there was a discrepancy among the ability to grow under a hypoxic condition. There was no change in the optical density for Xanthomonas but there was continued growth for Xylella under the nitrogen gas treatments (0% O2; Figure 1). This was also 119 found for other strains of Xylella, such as the Pierce’s disease strain ‘UCLA’ and the almond leaf scorch strain ‘Tulare’ (data not shown). Effects of oxygen on biofilm appear more variable than effects on OD, yet in about half of the experiments conducted biofilm increased significantly under hypoxic conditions. Media formulation had more significant effects on biofilm than did O2 concentrations. The absolute rates of Xf growth and the subsequent reactions to declining O2 were strain dependent. Furthermore, the effects of oxygen varied greatly depending on media used during assays (Table 1). When grown in the defined media XDM2 effects of varying oxygen were highly significant, whereas effects were not evident for Xf grown in CHARDS. The latter is a defined media based on xylem fluid composition (Leite et al. 2004), whereas the former is based on genomic analysis (Lemos et al. 2003). Variations in effects suggest interactions between oxygen and other xylem constituents that may be important to Xf growth. Preliminary analysis of media composition, CHARDS and XDM2, after sustained Xf growth under differing oxygen levels (21% versus 0%) showed both qualitative and quantitative variations in organic acid profiles. These results, along with analysis of terminal oxidase (the high through-put, low affinity cytochrome bo) in the electron transport pathway and the lack of production of hydrogen sulfide, suggest the possibility that the bacterium may be employing an anaerobic energy production pathway. Current research is addressing if such pathways may be fermentative. We are also analyzing these metabolic products to determine if these products are related to pathogenicity. Objective 2: Xf colony growth in xylem fluid from varying Vitis germplasms. Xylem fluid was collected from Vitis cultivars ranging in PD susceptibility. These included Vitis rotundifolia cvs. Carlos and Noble, Vitis rupestris cv. St. George, Vitis simpsoni cv. Pixialla, Vitis champinii cvs. Dogridge and Ramsey and Vitis vinifera cvs. Chardonnay, Chenin blanc and Exotic all collected from cut bleeding spurs (Andersen and Brodbeck 1991) at the NFREC-Quincy Research Center in March 2005 and also from Vitis vinifera cv. Chardonnay and Vitis rotundifolia cv. Noble in California during March and April 2006. Profiles of inorganic nutrients (phosphorous, potassium, magnesium, zinc, manganese, copper, boron and sodium) amino acids (glutamine, asparagine, aspartic acid, glutamic acid, serine, glycine, histidine, arginine, threonine, alanine, proline, tyrosine, valine, methionine, cysteine, isoleucine, leucine, phenylalanine and lysine), organic acids (oxalic, citric, tartaric, malic, malonic, lactic and succinic acids), electrical conductivity and pH were established for the xylem fluid used in each assay. Cell suspensions of the Temecula strain were pelleted from PW+ medium and re-suspended in xylem fluid from the varying cultivars for periods of one hour, 5 days and 12 days. Bacterial concentration and biofilm formation were quantified for each treatment and correlated to individual chemical constituents in the xylem fluid. Tests were run in March 2006 and again in June 2006 to insure repeatability. We note that the only way to collect sufficient quantities of xylem fluid for this experiment is to use bleeding xylem fluid from cut Vitis spurs which is only available in late winter. These fluids from dormant vines may or may not be representative of xylem fluid from specific germplasms in summer when Xf is actively proliferating. Thus, this methodology was developed to provide a range of xylem profiles to assess effects of individual xylem components on Xf growth rather than to quantify characteristics of Xf growth as a function of specific Vitis germplasms. Our results emphasize how dramatically even short term exposure of Xf to xylem fluid of varying composition quickly alters Xf growth and biofilm formation (Table 2). Significant effects in biofilm (p<0.0001) were present after 1 hour and persisted throughout 12 days. Effects on OD were delayed (not apparent after 1 hour) but became highly significant with time (p<0.0001 for the 5 and 12 day intervals). Optical density varied greater than 3-fold and biofilm formation greater than 5fold in the various fluids during the experiments. In all cases both OD and biofilm formation increased with time, but rates of increase varied dramatically between xylem fluid treatments. For example, biofilm formation in Carlos fluid was significantly higher than in the other fluids when measured after one hour, was intermediate in value after five days and was lower than in all other fluids after 12 days. Exposure to specific xylem fluids often had the opposite effect on overall growth rates (OD) and biofilm formation. Optical density for Xf incubated in Ramsey fluid was consistently higher than Xf in other fluids at each time period, but also consistently lower in biofilm formation. Analyses of xylem fluids showed that many xylem constituents were highly correlated to optical density and biofilm formation. Optical density (growth) became more highly correlated with these constituents over time with only two significant correlations after 1 hour (Table 3). Glutamine, the predominant amino acid in Vitis xylem fluid, was weakly but consistently correlated with OD after 5 and 12 days. Some of the minor amino acids (histidine, valine, methionine, isoleucine and phenylalanine) were much more strongly correlated to Xf growth. The organic acid citric acid was also very highly correlated to OD after 5 and 12 days. For the inorganic ions, phosphorous, copper and zinc were well correlated with OD. For all of the constituents mentioned above, results appeared consistent over time as significant relationships apparent after 5 days also persisted through 12 days. We have previously hypothesized the importance of calcium, magnesium, phosphorous and citric acid to Xf growth (Andersen 2005). Equations utilizing these as variables yielded higher correlations than regression analyses based on any single chemical constituent (P<0.0001; R =0.90). The strength of these relationships suggest that our original hypothesis merits further investigation.