Curr Microbiol (2014) 69:866–873 DOI 10.1007/s00284-014-0660-2
Effect of Oxygen on the Growth and Biofilm Formation of Xylella fastidiosa in Liquid Media Anthony D. Shriner • Peter C. Andersen
Received: 18 March 2014 / Accepted: 15 June 2014 / Published online: 7 August 2014 Ó Springer Science+Business Media New York 2014
Abstract Xylella fastidiosa is a xylem-limited bacterial pathogen, and is the causative agent of Pierce’s disease of grapevines and scorch diseases of many other plant species. The disease symptoms are putatively due to blocking of the transpiration stream by bacterial-induced biofilm formation and/or by the formation of plant-generated tylosis. Xylella fastidiosa has been classified as an obligate aerobe, which appears unusual given that dissolved O2 levels in the xylem during the growing season are often hypoxic (20–60 lmol L-1). We examined the growth and biofilm formation of three strains of X. fastidiosa under variable O2 conditions (21, 2.1, 0.21 and 0 % O2), in comparison to that of Pseudomonas syringae (obligate aerobe) and Erwinia carotovora (facultative anaerobe) under similar conditions. The growth of X. fastidiosa more closely resembled that of the facultative anaerobe, and not the obligate aerobe. Xanthomonas campestris, the closest genetic relative of X. fastidiosa, exhibited no growth in an N2 environment, whereas X. fastidiosa was capable of growing in an N2 environment in PW?, CHARDS, and XDM2-PR media. The magnitude of growth and biofilm formation in the N2 (0 % O2) treatment was dependent on the specific medium. Additional studies involving the metabolism of X. fastidiosa in response to low O2 are warranted. Whether X. fastidiosa is classified as an obligate aerobe or a facultative anaerobe should be confirmed by gene
A. D. Shriner USDA ARS Coastal Plains Soil, Water and Plant Research Center, 2611 W Lucas St., Florence, SC 29501, USA e-mail: [email protected] P. C. Andersen (&) University of Florida North Florida Research and Education Center, 155 Research Rd., Quincy, FL 32351, USA e-mail: [email protected]
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activation and/or the quantification of the metabolic profiles under hypoxic conditions.
Introduction Xylella fastidiosa is a gram-negative bacillus bacterium which resides in the xylem vessels of many plant species. The bacterium is transmitted to host plants by xylophagous insects such as the glassy-winged sharpshooter [Homalodisca vitripennis (Germar)] [3, 36]. Xylella fastidiosa is believed to cause diseases in its plant host by restricting the transpiration stream by clogging the xylem elements [20], by the synthesis of biofilms (a bacteria exopolysaccharides matrix) [8, 10] and/or by stimulating plant-generated tyloses [14, 16, 23, 40]. The mechanism of host plant resistance may be complex since most plant species tolerate X. fastidiosa in a benign presence, and there is not always a good correlation between bacterial titers and symptom severity [5, 17]. Xylella fastidiosa has been described as being an obligate aerobe [43]. However, recent publications describe the dissolved O2 content at hypoxic levels in xylem vessels (Table 1) during the growing season, when the metabolic demand for O2 is highest. The amount of dissolved O2 found in the xylem fluid can range from 20 to 30 lmol L-1 (0.43 mg L-1) [32], to 280 lmol L-1 (8.8 mg L-1) at 20 °C [19]. Ostensively, the O2 concentration in occluded and dysfunctional xylem vessels, or within the interior of biofilms, could be potentially lower. The xylem elements are constructed predominantly of lignin and cellulose. Xylem fluid consists of 95–99 % water, with the remaining constituents mainly consisting of amino acids, organic acids, monosaccharides, and inorganic ions [1–3]. Another constituent of xylem fluid that is
A. D. Shriner, P. C. Andersen: Growth and Biofilm Formation of Xylella fastidiosa Table 1 Oxygen concentrations found in xylem fluid from various plant species Species (conditions)
Dissolved O2 (lmol L-1)
Dissolved O2 ppm (mg L-1)
Reference
Ricinus communis
95.8
3.1
[12]
Betula pendula
42–230a
1.3–7.4
[18]
Olea europea (day) O. europea (night)
80–90a 20–30a
2.6–2.9 0.64–0.96
[32] [32]
Picea abies (irrigation)
68.5
2.2
[13]
P. abies (no irrigation)
13.7–41.1
0.43–1.3
[13]
P. abies (drought)
\13.7
\0.43
[13]
P. abies (winter)
273.8
8.8
[13]
Fagus orientalis (day)
58.36
1.87
[22]
F. orientalis (night)
50.31
1.61
[22]
Laurus nobilis
51.75
1.66
[22]
L. nobilis (flooding)
37.38
1.20
[22]
a
The data as it was presented in the primary literature. 100 % airsaturated solution (21 % O2) in water = 288 lmol L-1; 9.2 ppm
important to the survival capabilities of X. fastidiosa is the concentration of dissolved O2. The dissolved O2 enters predominantly through the root zone [31], and a small percentage enters through radial diffusion via the trunk and stems [18, 32]. The O2 concentration decreases as the transpiration stream moves away from the roots [13]. The levels of O2 are low enough to induce anaerobic fermentation, as evidenced by the generation of fermentation byproducts such as ethanol and aldehydes in the interior wood of trees [24, 29, 30]. This is especially true for the xylem environment of large trees. The terminal ends of the xylem system, (i.e., leaves), are capable of metabolizing these fermentation products [29]. The O2 concentration within the interior of biofilms is ostensibly very low, but has not been measured. There are three physical components which regulate the ability for O2 to become dissolved in water, and potentially enter into plant tissues. An increase in temperature and osmolarity reduces the solubility of O2 in water. A decrease in pressure (i.e., fluid in xylem under tension) will decrease the solubility of O2 in water. The contributions of these factors underscore the hypoxic nature of the xylem environment of many plant species. Under conditions in Florida, the air and plant temperature can exceed 35 °C, the tension of the transpiration stream can get as low as -1.6 MPa (or -230 psi), and the osmolarity of the xylem fluid is approximately 20 mM [1–3]. Other factors which affect the dissolved O2 concentration are flooding-induced anaerobiosis and extreme drought. Xylella fastidiosa strains are all classified as a single species [43], although different groupings or taxons have been proposed [34, 35, 37]. Schaad et al. [37] denoted Pierce’s disease strains of cultivated grapevine and two of
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three almond leaf scorch strains tested as X. fastidiosa subsp. piercei, subsp. nov. The genomes of several strains of X. fastidiosa have been sequenced. The first was the citrus variegated chlorosis strain [39]. The second strain to be sequenced was the Pierce’s disease strain Temecula [41], which infects and kills Vitis vinifera L. [25]. There are at least four other strains which have been sequenced: three almond leaf scorch strains (Dixon, M12 and M23) [9], and an oleander leaf scorch strain (Ann) [41]. Xylella fastidiosa can exist in a planktonic state or in the form of a biofilm, and the transition to either form can be coordinated, at least in part, by small fatty acid signaling molecule(s) [8]. A biofilm is a structured community of sessile microbial aggregates within a self-produced polymeric matrix (exopolysaccharides, proteins, DNA, and bacteria) attached to a surface [10]. Xylella fastidiosa strains from different hosts have been separated by biofilm morphology, aggregation in liquid culture, and attachment to glass surfaces [33]. Xylella fastidiosa fimbrial proteins, which are components of type I pili, are expressed in biofilm formation [7, 8, 28, 42]. The purpose of this study was to assess the in vitro growth and biofilm formation of three strains of X. fastidiosa under varying O2 concentrations. Growth and biofilm formation were then compared to Pseudomonas syringae (obligate aerobe) and Erwinia carotovora (facultative anaerobe) at various O2 concentration. Growth and biofilm of X. fastidiosa were also compared to Xanthomonas campestris pv. campestris (obligate aerobe) in air and under N2 conditions. Data concerning the growth and biofilm formation of X. fastidiosa in response to O2 concentrations may help explain the behavior of this bacterium as it experiences hypoxic conditions in planta. If X. fastidiosa is an obligate aerobe, the growth and movement of this bacterium could be influenced by plant anatomy, plant physiology, or environmental conditions that impact xylem O2 concentrations. By contrast, if X. fastidiosa is a facultative anaerobe, it is adapted to a wider range of O2 habitats, and variations in plant factors or environmental conditions that influence O2 would be expected to have a lesser impact on this bacterium.
Materials and Methods Bacteria The X. fastidiosa strains were obtained from Dr. A. Purcell and C. Wistrom (UC-Berkeley). The X. fastidiosa Pierce’s disease strains were Temecula and UCLA. The almond leaf scorch strain used was Tulare. The other bacterial cultures used were: P. syringae and E. carotovora (both obtained
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A. D. Shriner, P. C. Andersen: Growth and Biofilm Formation of Xylella fastidiosa
from Dr. T. Momol, UF) and X. campestris pv. campestris (obtained from Dr. J. Jones, UF). Cultivation Protocol Bacterial suspensions in PW? [11] and 10 % glycerol were stored in a -80 °C freezer (Baxtor Scientific Products, Cryo-Fridge). Bacterial cultures were initiated by scraping a sterile wooden applicator over the frozen stock culture and streaking onto a Buffered Charcoal Yeast Extract (BCYE) plate [43]. After 10 days, bacterial growth was scraped with an inoculation loop and placed into 5 mL of PW? broth. Bacterial cultures were incubated between 10 and 14 days until a visible turbid solution was formed before being used to make working cultures. These 5 mL cultures were transferred to 50 mL of PW? medium in 250 mL Erlenmeyer flasks with cotton plugs or silicone rubber plugs with a filter to allow gas exchange. All incubations were done in a New Brunswick incubator (model# G25-R) set at 28 °C, and all liquid cultures were placed on an orbital shaker at 150 rpm. Gram staining showed the presence of only gram-negative rods of uniform distribution. Samples that were plated on PW? medium revealed no contamination. Experiments were carried out in 15 mL polypropylene Falcon tubes with rubber septa or screw caps and 16 9 150 mm glass test tubes with slip cap closures. The media used included: (1) an enrichment medium (PW?) [11]; (2) a chemically-defined medium (CHARDS) which is a variant of CHARD2 [26], and; (3) the defined medium XDM2-PR which is a variant of XDM2 [27]. The media are described below. Optical density was measured using a Genosys 8 spectrophotometer at a wavelength of 600 nm. The machine was zeroed using an aliquot of sterile media or suspension buffer. Next, an aliquot of the bacterial suspension was placed into the cuvette and optical density was measured. PW? Medium PW? medium [11] was made in two solutions which were then combined. Solution A consisted of (for 1 L) 900 mL dH2O to which these components were added: 1.0 g soluble starch, 4.0 g soytone, 1.0 g tryptone, 1.2 g K2HPO4, 1.0 g KH2PO4, 0.4 g MgSO47H2O, 0.85 g (NH4)2HPO4, 1.0 g histidine, 25.0 mg cyclohexamide, 10.0 mL (0.1 %) hemin-Cl, and 10.0 mL (0.2 %) phenol red. Media were sterilized by autoclaving. Solution B was made by dissolving 4.0 g of glutamine into 50 mL dH2O at *60 °C, and 6.0 g bovine serum albumin (fraction V) into 30 mL of dH2O, which were then combined. Next, media were sterilized through a 0.22-lm-filter. The two solutions were then combined to form the completed broth medium. PW?
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agar plates were made by adding 12.0 g agar to solution A before sterilization in the autoclave. Solution B was then combined when solution A was cooled down to 55 °C in a hot water bath. Then, PW? medium was aseptically aliquoted into Petri dishes. CHARDS Medium CHARDS is a modified version of CHARD2 which was a chemically-defined medium based on the xylem fluid chemistry of Vitis vinifera ‘Chardonnay’ [26]. Solution A consisted of (for 1 L) 819 mL of dH2O to which these components were added: 1.0 g KH2PO4, 1.5 g K2HPO4, 0.2 g MgSO47H2O, 0.17 g alanine, 0.58 g aspartic acid, 1.8 g glutamine, 1.05 g arginine, 0.01 g cysteine-HCl, 1 mL (1 mg L-1) biotin, and 1.0 g of soluble starch. The pH was then adjusted to 6.6 using 0.1 M KOH and then sterilized by autoclaving. Solution B consisted of 0.25 g of ferric pyrophosphate dissolved in 60 mL dH2O and 4.0 g glutamine dissolved in 50 mL dH2O at *60 °C which was then combined, and was sterilized through a 0.22-lm-filter. The two solutions were then combined, aseptically, to form the completed media. XDM2-PR Medium XDM2-PR is a modified version of XDM2 [27], but lacks phenol red. XDM2 is a chemically-defined medium based on metabolic processes suggested by genome of X. fastidiosa subsp. pauca. For 1 L, 980 mL of dH2O the following components were added: 10.0 g glucose, 2.1 g K2HPO4, 0.8 g KH2PO4, 0.4 g MgSO47H2O, 0.125 g ferric pyrophosphate, 4.0 g glutamine, 1.0 g asparagine, 0.4 g serine, 0.4 g methionine, and 10 mL of vitamin solution (described below). The pH was then adjusted to 6.6 using 0.1 M KOH and then sterilized through a 0.22lm-filter. The vitamin solution (for 100 mL) consisted of 350.0 mg myo-inositol, 10.0 mg pyridoxine–HCl, 10.0 g thiamine, 5.0 mg nicotinic acid, and 0.2 mg of D-biotin. The vitamin solution was sterilized through a .22-lm-filter and stored at 4 °C in a light-protected glass bottle. PCR Protocol The polymerase chain reaction (PCR) was used to confirm the identity of the cultures. DNA extractions were conducted using the Quiagen DNeasy mini prep. The PCR protocol used a nested primer technique [35]. Oxygen Levels The LaMotte’s Dissolved Oxygen Test Kit (Model EDOCode 7414) was used to determine the treatment effects
A. D. Shriner, P. C. Andersen: Growth and Biofilm Formation of Xylella fastidiosa
over time in the aqueous environments of distilled water (dH2O) and CHARDS medium. Measurements were made just prior to daily flushing for the 21, 10, 2.1, and 0 % O2 treatments. The assays were conducted within a rubber septa closed tube by injecting 100 ll of the manganous sulfate mixed well, then 100 lL of the alkaline potassium iodide azide was added when the precipitant started to settle. A 100 lL of 50 % H2SO4 was mixed well until all the precipitants were dissolved. The tubes were opened and the solution was transferred to a beaker with a lid designed to fit around the tip of the graduated titration syringe. Next, thiosulfate was titrated into the sample until the color became clear against a white background, and the volume used represented the amount of dissolved oxygen in the sample. The O2 treatments used to assess growth and biofilm formation for all bacteria were 21, 2.1, 0.21, and 0 % O 2.
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removed and air dried. The vessels were de-stained using 70 % isopropanol. An aliquot was placed into a cuvette and the optical density was measured at a wavelength of 600 nm [15]. Statistical Analysis All experiments were designed with three replicates (three plates). The bacteria were divided and washed in order to ensure that each response observed was dependent on each treatment (and not the three measurements of the initial bacteria treatment interaction). The data were analyzed with ANOVA, t tests, means, and standard errors using the SAS V9.0 software package.
Results and Discussion Experimental Growth Conditions Mature bacterial cultures, in 50 mL batches, were removed from the incubator and pooled together into a sterilized media flask. The optical density of the culture was obtained with a spectrophotometer set at a wavelength of 600 nm. Aliquots were placed into sterile polypropylene/glass tubes and centrifuged at 2,3809g for 15 min. The supernatant was decanted and the pellet was resuspended into 3 mL of PBS (phosphate buffered saline, 0.1 M phosphate, 0.8 % NaCl, and pH 6.8). The suspension was centrifuged again. The supernatant was discarded and the pellet was resuspended into the appropriate media. The pellet was centrifuged again and the supernatant was discarded. The pellet was resuspended into 5 mL of the appropriate media. The tubes were capped with red-rubber septa and secured with parafilm. Gas treatments were done each day. The tops of the septa were sterilized by rinsing with 70 % isopropyl alcohol and dabbed dry with a Kimwipe. The gas was passed through a 0.22 lm syringe filter then injected through a 1 in., sterilized 22-gauge needle, and another sterilized 22-gauge needle was placed into the septa to act as an exhaust port to ensure gas exchange. The tubes were placed in the New Brunswick incubator set at 28 °C on an orbital shaker set at 150 rpm. Biofilm Quantification Biofilm formation on the surface walls of conical polypropylene tubes was assayed by the crystal violet method [15]. After various intervals of incubation, the supernatant was decanted and the tube was rinsed with dH2O and air dried. The vessels were filled with 0.1 % crystal violet and placed on a shaker for 1 h. The crystal violet was decanted and the tubes were rinsed until all the excess dye has been
The O2 concentrations in xylem fluid reported in the literature usually ranged from 0.43 to 3.1 mg L-1, although there are two reports of relatively high concentrations (7.4 and 8.8 mg L-1) (Table 1). The LaMotte’s method to create consistently different and stable O2 concentration was effective. The concentration of O2 in CHARDS medium measured after flushing with 21, 10, 2.1, and 0 % O2 daily for 1, 3, or 6 days was 4.5–5.0, 2.4–2.9, 0.8–1.4, and 0.4–0.7 mg L-1, respectively. The dissolved O2 content in CHARDS medium was very similar to that in distilled water (data not shown). In air, the solubility of O2 in water at 0.1 MPa pressure and 28 °C is about 8 mg L-1, and the LaMotte’s measurement of O2 in water under aerobic conditions was about 6.2 mg L-1. Also, the N2 treatment consistently resulted in a steady state concentration of about 0.5 mg L-1 O2 which may reflect a limitation of the LaMotte’s method to measure extremely low O2 concentrations. The growth of X. fastidiosa Pierce’s disease strains UCLA, STL, and almond leaf scorch strain Tulare in PW? medium (Fig. 1a) was compared to P. syringae (an obligate aerobe), and E. carotovora (a facultative anaerobe) (Fig. 1b) at four O2 concentrations. The growth of X. fastidiosa UCLA and STL was higher than the almond leaf scorch strain Tulare (Fig. 1a) and was highest in 21 % O2, and declined gradually at lower O2 concentrations. The growth of P. syringae was only substantial in the aerobic (21 % O2) treatment, and was drastically curtailed in reduced O2 environments (Fig. 1b). There was more than a sevenfold decrease in growth of P. syringae at lower O2 environments. By contrast, the growth pattern of X. fastidiosa was more analogous to the facultative anaerobe, E. carotovora (Fig. 1b), which also had an incremental growth reduction down the O2 gradient. For X. fastidiosa,
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870
A. D. Shriner, P. C. Andersen: Growth and Biofilm Formation of Xylella fastidiosa 0.6
0.50
A
A
Growth Biofilm
0.4 0.3 0.2 0.1
Xylella fastidiosa Growth Air
0.30 0.20 0.10
0.00%
0.21%
2.10%
21.00%
0.00%
0.21%
STL
1
B Pseudomonas syringae
Erwinia carotovora
1.2 1.0
Growth
0.8
Biofilm
0.6
1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0
B
7
9
11
13
Xanthomonas campestris Growth Nitrogen
1
0.4
2
3
4
5
Days
0.2 0.00%
0.21%
2.10%
21.00%
0.00%
0.21%
2.10%
21.00%
0.0
Fig. 1 The growth and biofilm formation of a three Xylella fastidiosa strains (Pierce’s disease strains STL and UCLA, and Almond Leaf Scorch (ALS) strain Tulare) in PW? media, b P. syringae (an obligate aerobe) and to E. carotovora (a facultative anaerobe) under various O2 concentrations. Initial optical density of X. fastidiosa was 0.05 and initial optical density of P. syringae and E. carotovora was 0.10. Significant differences between the optical density bars within each strain are separated by Duncan’s new multiple range test. Error bars represent standard error of the mean
the production of biofilm was numerically higher in 21 % O2 than in the other gas treatments, although significant differences did not occur. Biofilm production was negligible for P. syringae and E. carotovora. The comparison of these growth patterns raised the question as to the respiration classification of X. fastidiosa as an obligate aerobe [43]. The classification of X. fastidiosa as an obligate aerobe was based on growth responses in 1 or 5 % O2 (balance N2), 2.5 or 5.0 % CO2 with 21 % O2 (balance N2), or air [43]. The growth of X. fastidiosa (Pierce’s disease Temecula strain) in PW? medium increased eightfold and threefold
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5
Xanthomonas campestris Growth Air
ALS
1.6 1.4
3
Days
Optical Density (600nm)
1.8
2.10%
21.00%
0.00%
0.21%
2.10%
UCLA
Optical Density (600 nm)
Xylella fastidiosa Growth Nitrogen
0.40
0.00
0 21.00%
Optical Density (600nm)
0.5
Optical Density (600nm)
Xylella fastidiosa
Fig. 2 a Xylella fastidiosa strain Temecula in PW? growth over time under aerobic (21 % O2) and N2 conditions. Each point is the average of three replicates. b Growth of X. campestris in PW? under aerobic (21 % O2) and N2 conditions. Initial OD is 0.05 for X. fastidiosa and 0.10 for X. campestris. Each point is the average of three replicates. Error bars represent standard errors of the mean
compared to initial levels under 21 % O2 and N2 conditions, respectively for up to 13 days (Fig. 2a). Growth rates were linear over time. (Similar growth rates were obtained with the Pierce’s disease strain UCLA and the ALS strain Tulare, data not shown). There was a gradual reduction in biofilm formation for all three strains of X. fastidiosa, and the ratio of growth to biofilm formation was approximately the same for all gas treatments. There was a striking difference between the growth of X. fastidiosa (Fig. 2a), and a close relative, X. campestris pv. campestris (Fig. 2b), also deemed to be an obligate aerobe. For X. campestris there was a tenfold increase in growth in the aerobic treatment, and virtually no growth in the N2 treatment as indicated by the negligible slope of the line (Fig. 2b). (The same result occurred for experiments with X. campestris pv. vitians, data not shown). The designation of X. fastidiosa as a facultative anaerobe should await the analysis of fermentation profiles and gene activation consistent with anaerobiosis. An alternative
Xylella fastidiosa CHARDS Growth Air Xylella fastidiosa CHARDS Growth Nitrogen
0.25 0.2
A
0.15 0.1 0.05 0
5
10 Days
15
Xyella fastidiosa XDM2 Growth Nitrogen
A 0.20
0.15
0.10
0
5
10
15
30
Days
Xylella fastidiosa CHARDS Biofilm Air Xylella fastidiosa CHARDS Biofilm Nitrogen
0.4
0.25
0.05
30
Optical Density (600nm)
Optical Density (600 nm)
0
871
Xylella fastidiosa XDM2 Growth Air Optical Density (600nm)
Optical Density (600nm)
A. D. Shriner, P. C. Andersen: Growth and Biofilm Formation of Xylella fastidiosa
B 0.3
0.2
0.1
Xylella fastidiosa XDM2 Biofilm Air Xylella fastidiosa XDM2 Biofilm Nitrogen
0.16
B 0.12
0.08
0.04
0
0
0
5
10 Days
15
30
0
5
10
15
30
Days
Fig. 3 The comparison of a growth and b biofilm formation of Xylella fastidiosa (Pierce’s disease strain Temecula) under aerobic (21 % O2) (open squares) and N2 (closed squares) gas treatments in the CHARDS media. Initial OD was 0.10. Each point is the average of three replicates. Error bars represent standard errors of the mean
Fig. 4 The comparison of a growth and b biofilm formation of Xylella fastidiosa (Pierce’s disease strain Temecula) under aerobic (21 %) (open squares) and N2 (closed squares) gas treatments in the XDM2-PR media. Initial OD was 0.10. Each point is the average of three replicates. Error bars represent standard errors of the mean
and viable explanation is that cytochrome oxidase of X. fastidiosa has an extremely high affinity for O2, and metabolic by-products of hypoxia can be detoxified [21]. Xylella fastidiosa has the metabolic capability for sulfate reduction [21, 39, 41]; however, metabolic by-products such as hydrogen sulfide, elemental sulfur, or iron sulfide were not quantified in the current study. A biofilm growth associated repressor (BigR) controls the transcription of an operon involved in biofilm formation in X. fastidiosa [6]. Guimaraes et al. [21] showed that the BigR operon is also required for hydrogen sulfide detoxification and the continued function of cytochrome c oxidase under hypoxic conditions. Growth and biofilm formation of X. fastidiosa strain Temecula was examined from 0 to 30 days treatment under 21 % O2 and N2 conditions in chemically-defined media CHARDS (Fig. 3), and XDM2-PR (Fig. 4). The growth and biofilm formation in the CHARDS medium was not significantly different when incubation was in the 21 % O2 treatment compared to the N2 treatment; however, it should
be noticed that growth was steady and continuous. There was a twofold increase in growth and a 5- to 7-fold increase in biofilm after 30 days of culture. By contrast, the N2 treatment using XDM2-PR medium resulted in significantly more growth and biofilm formation compared to the 21 % O2 treatment. The response of X. fastidiosa to O2 treatments compared to the N2 treatment was dependent on the growth medium used. The planktonic growth, biofilm formation, and aggregation of X. fastidiosa depend on the chemistry of xylem fluid [4, 33, 38]. Monomeric compound (amino acids and organic acids) and inorganic ions were shown to greatly alter the behavior of this bacterium [4, 26]. Wulff et al. [44], working with PW and BCYE media, showed that the pH of the culture media can influence growth, aggregation, and biofilm formation; however in our study pH of all media was adjusted to 6.6. During the growing season, the dissolved O2 in the xylem fluid becomes low enough to induce fermentation within the plant tissues [24]. A relevant issue would be the
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concentration of O2 in vessels inhabited by X. fastidiosa, or within the biofilm matrix. Goodwin et al. [20] investigated the presence of possible toxic compounds generated by X. fastidiosa; however, the results were based on the washes of aerobically-grown cultures on solid media. The results presented in our study indicate that X. fastidiosa was capable of continued growth and biofilm formation under N2 conditions. The potential ability of X. fastidiosa to modify its respiration pathways during the growing season when the O2 levels drop and the metabolic demand becomes greater, offers a reasonable explanation of how X. fastidiosa is capable of continued growth and biofilm development under hypoxic conditions in-planta. In conclusion, the continued growth and biofilm formation of X. fastidiosa under N2 conditions, the great differences in growth characteristics to two obligate aerobes (P. syringae and X. campestris), and the similarity to a facultative anaerobe (E. carotovora) support the possibility that X. fastidiosa may not be an obligate aerobe [43], but rather a facultative anaerobe. During the growing season, the dissolved O2 in the xylem fluid often becomes low enough to induce fermentation within the plant tissues [24]. Xylella fastidiosa was capable of sustained growth and biofilm production in a wide range of hypoxic environments. Thus, it appears that X. fastidiosa would be adapted to variations in O2 concentrations that would result from differences in plant anatomy, plant physiology, or environmental conditions.
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8. Chatterjee S, Almeida RPP, Lindow S (2008) Living in two worlds: the plant and insect lifestyles of Xylella fastidiosa. Ann Rev Phytopathol 46:243–271 9. Chen PC, Xie G, Han S, Chertkov O, Sims D, Civerolo EL (2010) Whole genome sequences of two Xylella fastidiosa strains (M12 and M23) causing almond leaf scorch disease in California. J Bacteriol 192:4534 10. Costerton JW, Lewandowski Z, Calswell DE, Korber DR, Lappin-Scott HM (1995) Microbial biofilms. Ann Rev Microbiol 49:711–745 11. Davis MJ, French WJ, Schaad NW (1981) Axenic culture of the bacteria associated with phony disease of peach and plum leaf scald. Curr Microbiol 6:309–314 12. Dongen JTV, Schurr U, Pfister M, Geigenberger P (2003) Phloem metabolism and function have to cope with low internal oxygen. Plant Physiol 1331:1529–1543 13. Eklund L (2000) Internal oxygen levels decrease during the growing season and with increasing stem height. Trees 14:177– 180 14. Esau K (1948) Anatomic effects of the viruses of Pierce’s disease and phony peach. Hilgardia 18:423–482 15. Espinosa-Urgel M, Salido A, Ramos J-L (2000) Genetic analysis of functions involved in adhesion of Pseudomonas putida to seeds. J Bacteriol 182:2363–2369 16. Fritschi FB, Lin H, Walker MA (2008) Scanning electron microscopy reveals different response pattern of four Vitis genotypes to Xylella fastidiosa infection. Plant Dis 92:276–286 17. Gambetta GA, Fei J, Rost TL, Matthews MA (2007) Leaf scorch symptoms are not correlated with bacterial populations during Pierce’s disease. J Exp Bot 58:4037–4046 18. Gansert D, Burgdorf M, Losch R (2001) A novel approach to the in situ measurement of oxygen concentrations in the sapwood of woody plants. Plant Cell Environ 24:1055–1064 19. Gansert D (2003) Xylem sap flow as a major pathway for oxygen supply to the sapwood of birch (Betula pubescens Ehr.). Plant Cell Environ 26:803–1814 20. Goodwin PH, DeVay JE, Meredith CP (1988) Roles of water stress and phytotoxins in the development of Pierce’s disease of the grapevine. Physiol Mol Plant Pathol 32:1–15 21. Guimareaes BG, Barbosa RL, Soprano AS, Campos BM, de Souza TA, Tonoli CCC, Leme AFP, Murakami T, Benedetti CE (2011) Plant pathogenic bacteria utilize biofilm growth-associated repressor (BigR), a novel winged-helix redox switch to control hydrogen sulfide detoxification under hypoxia. J Biol Chem 286:26148–26157 22. Hierro AMD, Kronberger W, Heitz P, Offenthaler I, Richter H (2002) A new method to determine the oxygen concentration inside the sapwood of trees. J Exp Bot 53:559–563 23. Killiny N, Hernando Martinez R, Korsi Dumenyo C, Cooksey DA, Almeida RPP (2013) The exopolysaccharide of Xylella fastidiosa is essential for biofilm formation, plant virulence and vector transmission. Mol Plant Microb Interact 26:1044–1053 24. Kimmerer TW, Stringer MA (1988) Alcohol dehydrogenase and ethanol in the stems of trees: evidence for the anaerobic metabolism in the vascular cambium. Plant Physiol 87:693–697 25. Krivanek AF, Stevenson JF, Walker MA (2005) Development and comparison of symptom indices for quantifying grapevine resistance to Pierce’s disease. Phytopathology 95:36–43 26. Leite B, Andersen PC, Ishida ML (2004) Colony aggregation and biofilm formation in xylem chemistry-based media for Xylella fastidiosa. FEMS Microbiol Lett 230:283–290 27. Lemos EG, Alves LM, Campanharo JC (2003) Genomics-based design of defined growth media for the plant pathogen Xylella fastidiosa. FEMS Microbiol Lett 219:39–45 28. Lorite GS, Rodrigues CM, de Souza AA, Kranz C, Mizaikoff Cotta MA (2011) The role conditioning film formation and
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surface chemical changes on Xylella fastidiosa adhesion and biofilm evolution. J Colloid Interface Sci 359:289–295 MacDonald RC, Kimmerer TW (1991) Ethanol in the stems of trees. Plant Physiol 82:582–588 MacDonald RC, Kimmerer TW (1993) Metabolism of transpired ethanol by eastern cottonwood (Populus deltoides Bartr.). Plant Physiol 102:173–179 Mancuso S, Boselli M (2002) Characterisation of the oxygen fluxes in the division, elongation, and mature zones of Vitis roots: influence of oxygen availability. Planta 214:767–774 Mancuso S, Marras AM (2003) Different pathways of the oxygen supply in the sapwood of young Olea europaea trees. Planta 216:1028–1033 Marques LLR, Ceri H, Manfio GP, Reid DM, Olson ME (2002) Characterization of biofilm formation by Xylella fastidiosa in vitro. Plant Dis 86:633–638 Nunney L, Vickerman DB, Bromlet RE, Russell SA, Hartman JR, Morano LD, Stouthamer R (2013) Recent evolutionary radiation and host plant specialization in the Xylella fastidiosa subspecies native to the United States. Appl Environ Microbiol 79:2189– 2200 Pooler MR, Hartung JS (1995) Genetic relationships among strains of Xylella fastidiosa from RAPD-PCR data. Curr Microbiol 31:134–137 Purcell AH, Hopkins DL (1996) Fastidious xylem-limited bacterial plant pathogens. Ann Rev Phytopathol 34:131–151 Schaad NW, Postnikova E, Lacy G, Fatmi M’B, Chang C-J (2004) Xylella fastidiosa subspecies: X. fastidiosa subsp. piercei,
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subsp. nov., X. fastidiosa subsp. multiplex subsp. nov., and X. fastidiosa subsp. pauca subsp. nov. Syst Appl Microbiol 27:290– 300 Shi X, Bi J, Morse JG, Toscano NC, Cooksey DA (2013) Effect of xylem fluid from susceptible and resistant grapevines on developmental biology of Xylella fastidiosa. Eur J Plant Pathol 135:127–135 Simpson AJ, Reinach FC, Arruda P et al (2000) The genome sequence of the plant pathogen Xylella fastidiosa. Nature 406:151– 159 Sun Q, Sun Y, Walker MA, Labavitch JM (2013) Vascular occlusions in grapevines with Pierce’s disease make disease symptom development worse. Plant Physiol 161:1529–1541 Van Sluys MA, de Oliveira MC, Monteiro-Vitorello CB et al (2003) Comparative analysis of the complete genome sequences of Pierce’s disease and citrus variegated chlorosis strains of Xylella fastidiosa. J Bacteriol 185:1018–1026 Voegel TM, Warren JG, Matsumoto A, Igo MM, Kirkpatrick BC (2010) Localization and characterization of Xylella fastidiosa haemagglutinin adhesions. Microbiology 156:2172–2179 Wells JM, Raju BC, Hung H-Y, Weisberg WG, Mandeico-Paul L, Brenner DJ (1987) Xylella fastidiosa gen. Nov., sp. Nov: Gram-negative, xylem limited, fastidious plant bacteria related to Xanthomonas spp. Int J Sys Bacteriol 37:136–143 Wulff NA, Mariano AG, Gaurivaud P, de Almeida Souza LC, Virgilio ACD, Monteiro PB (2008) Influence of culture medium pH on growth, aggregation and biofilm formation of Xylella fastidiosa. Curr Microbiol 57:127–132
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Effect of Oxygen on the Growth and Biofilm Formation of Xylella fastidiosa in Liquid Media Anthony D. Shriner • Peter C. Andersen
Received: 18 March 2014 / Accepted: 15 June 2014 / Published online: 7 August 2014 Ó Springer Science+Business Media New York 2014
Abstract Xylella fastidiosa is a xylem-limited bacterial pathogen, and is the causative agent of Pierce’s disease of grapevines and scorch diseases of many other plant species. The disease symptoms are putatively due to blocking of the transpiration stream by bacterial-induced biofilm formation and/or by the formation of plant-generated tylosis. Xylella fastidiosa has been classified as an obligate aerobe, which appears unusual given that dissolved O2 levels in the xylem during the growing season are often hypoxic (20–60 lmol L-1). We examined the growth and biofilm formation of three strains of X. fastidiosa under variable O2 conditions (21, 2.1, 0.21 and 0 % O2), in comparison to that of Pseudomonas syringae (obligate aerobe) and Erwinia carotovora (facultative anaerobe) under similar conditions. The growth of X. fastidiosa more closely resembled that of the facultative anaerobe, and not the obligate aerobe. Xanthomonas campestris, the closest genetic relative of X. fastidiosa, exhibited no growth in an N2 environment, whereas X. fastidiosa was capable of growing in an N2 environment in PW?, CHARDS, and XDM2-PR media. The magnitude of growth and biofilm formation in the N2 (0 % O2) treatment was dependent on the specific medium. Additional studies involving the metabolism of X. fastidiosa in response to low O2 are warranted. Whether X. fastidiosa is classified as an obligate aerobe or a facultative anaerobe should be confirmed by gene
A. D. Shriner USDA ARS Coastal Plains Soil, Water and Plant Research Center, 2611 W Lucas St., Florence, SC 29501, USA e-mail: [email protected] P. C. Andersen (&) University of Florida North Florida Research and Education Center, 155 Research Rd., Quincy, FL 32351, USA e-mail: [email protected]
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activation and/or the quantification of the metabolic profiles under hypoxic conditions.
Introduction Xylella fastidiosa is a gram-negative bacillus bacterium which resides in the xylem vessels of many plant species. The bacterium is transmitted to host plants by xylophagous insects such as the glassy-winged sharpshooter [Homalodisca vitripennis (Germar)] [3, 36]. Xylella fastidiosa is believed to cause diseases in its plant host by restricting the transpiration stream by clogging the xylem elements [20], by the synthesis of biofilms (a bacteria exopolysaccharides matrix) [8, 10] and/or by stimulating plant-generated tyloses [14, 16, 23, 40]. The mechanism of host plant resistance may be complex since most plant species tolerate X. fastidiosa in a benign presence, and there is not always a good correlation between bacterial titers and symptom severity [5, 17]. Xylella fastidiosa has been described as being an obligate aerobe [43]. However, recent publications describe the dissolved O2 content at hypoxic levels in xylem vessels (Table 1) during the growing season, when the metabolic demand for O2 is highest. The amount of dissolved O2 found in the xylem fluid can range from 20 to 30 lmol L-1 (0.43 mg L-1) [32], to 280 lmol L-1 (8.8 mg L-1) at 20 °C [19]. Ostensively, the O2 concentration in occluded and dysfunctional xylem vessels, or within the interior of biofilms, could be potentially lower. The xylem elements are constructed predominantly of lignin and cellulose. Xylem fluid consists of 95–99 % water, with the remaining constituents mainly consisting of amino acids, organic acids, monosaccharides, and inorganic ions [1–3]. Another constituent of xylem fluid that is
A. D. Shriner, P. C. Andersen: Growth and Biofilm Formation of Xylella fastidiosa Table 1 Oxygen concentrations found in xylem fluid from various plant species Species (conditions)
Dissolved O2 (lmol L-1)
Dissolved O2 ppm (mg L-1)
Reference
Ricinus communis
95.8
3.1
[12]
Betula pendula
42–230a
1.3–7.4
[18]
Olea europea (day) O. europea (night)
80–90a 20–30a
2.6–2.9 0.64–0.96
[32] [32]
Picea abies (irrigation)
68.5
2.2
[13]
P. abies (no irrigation)
13.7–41.1
0.43–1.3
[13]
P. abies (drought)
\13.7
\0.43
[13]
P. abies (winter)
273.8
8.8
[13]
Fagus orientalis (day)
58.36
1.87
[22]
F. orientalis (night)
50.31
1.61
[22]
Laurus nobilis
51.75
1.66
[22]
L. nobilis (flooding)
37.38
1.20
[22]
a
The data as it was presented in the primary literature. 100 % airsaturated solution (21 % O2) in water = 288 lmol L-1; 9.2 ppm
important to the survival capabilities of X. fastidiosa is the concentration of dissolved O2. The dissolved O2 enters predominantly through the root zone [31], and a small percentage enters through radial diffusion via the trunk and stems [18, 32]. The O2 concentration decreases as the transpiration stream moves away from the roots [13]. The levels of O2 are low enough to induce anaerobic fermentation, as evidenced by the generation of fermentation byproducts such as ethanol and aldehydes in the interior wood of trees [24, 29, 30]. This is especially true for the xylem environment of large trees. The terminal ends of the xylem system, (i.e., leaves), are capable of metabolizing these fermentation products [29]. The O2 concentration within the interior of biofilms is ostensibly very low, but has not been measured. There are three physical components which regulate the ability for O2 to become dissolved in water, and potentially enter into plant tissues. An increase in temperature and osmolarity reduces the solubility of O2 in water. A decrease in pressure (i.e., fluid in xylem under tension) will decrease the solubility of O2 in water. The contributions of these factors underscore the hypoxic nature of the xylem environment of many plant species. Under conditions in Florida, the air and plant temperature can exceed 35 °C, the tension of the transpiration stream can get as low as -1.6 MPa (or -230 psi), and the osmolarity of the xylem fluid is approximately 20 mM [1–3]. Other factors which affect the dissolved O2 concentration are flooding-induced anaerobiosis and extreme drought. Xylella fastidiosa strains are all classified as a single species [43], although different groupings or taxons have been proposed [34, 35, 37]. Schaad et al. [37] denoted Pierce’s disease strains of cultivated grapevine and two of
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three almond leaf scorch strains tested as X. fastidiosa subsp. piercei, subsp. nov. The genomes of several strains of X. fastidiosa have been sequenced. The first was the citrus variegated chlorosis strain [39]. The second strain to be sequenced was the Pierce’s disease strain Temecula [41], which infects and kills Vitis vinifera L. [25]. There are at least four other strains which have been sequenced: three almond leaf scorch strains (Dixon, M12 and M23) [9], and an oleander leaf scorch strain (Ann) [41]. Xylella fastidiosa can exist in a planktonic state or in the form of a biofilm, and the transition to either form can be coordinated, at least in part, by small fatty acid signaling molecule(s) [8]. A biofilm is a structured community of sessile microbial aggregates within a self-produced polymeric matrix (exopolysaccharides, proteins, DNA, and bacteria) attached to a surface [10]. Xylella fastidiosa strains from different hosts have been separated by biofilm morphology, aggregation in liquid culture, and attachment to glass surfaces [33]. Xylella fastidiosa fimbrial proteins, which are components of type I pili, are expressed in biofilm formation [7, 8, 28, 42]. The purpose of this study was to assess the in vitro growth and biofilm formation of three strains of X. fastidiosa under varying O2 concentrations. Growth and biofilm formation were then compared to Pseudomonas syringae (obligate aerobe) and Erwinia carotovora (facultative anaerobe) at various O2 concentration. Growth and biofilm of X. fastidiosa were also compared to Xanthomonas campestris pv. campestris (obligate aerobe) in air and under N2 conditions. Data concerning the growth and biofilm formation of X. fastidiosa in response to O2 concentrations may help explain the behavior of this bacterium as it experiences hypoxic conditions in planta. If X. fastidiosa is an obligate aerobe, the growth and movement of this bacterium could be influenced by plant anatomy, plant physiology, or environmental conditions that impact xylem O2 concentrations. By contrast, if X. fastidiosa is a facultative anaerobe, it is adapted to a wider range of O2 habitats, and variations in plant factors or environmental conditions that influence O2 would be expected to have a lesser impact on this bacterium.
Materials and Methods Bacteria The X. fastidiosa strains were obtained from Dr. A. Purcell and C. Wistrom (UC-Berkeley). The X. fastidiosa Pierce’s disease strains were Temecula and UCLA. The almond leaf scorch strain used was Tulare. The other bacterial cultures used were: P. syringae and E. carotovora (both obtained
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A. D. Shriner, P. C. Andersen: Growth and Biofilm Formation of Xylella fastidiosa
from Dr. T. Momol, UF) and X. campestris pv. campestris (obtained from Dr. J. Jones, UF). Cultivation Protocol Bacterial suspensions in PW? [11] and 10 % glycerol were stored in a -80 °C freezer (Baxtor Scientific Products, Cryo-Fridge). Bacterial cultures were initiated by scraping a sterile wooden applicator over the frozen stock culture and streaking onto a Buffered Charcoal Yeast Extract (BCYE) plate [43]. After 10 days, bacterial growth was scraped with an inoculation loop and placed into 5 mL of PW? broth. Bacterial cultures were incubated between 10 and 14 days until a visible turbid solution was formed before being used to make working cultures. These 5 mL cultures were transferred to 50 mL of PW? medium in 250 mL Erlenmeyer flasks with cotton plugs or silicone rubber plugs with a filter to allow gas exchange. All incubations were done in a New Brunswick incubator (model# G25-R) set at 28 °C, and all liquid cultures were placed on an orbital shaker at 150 rpm. Gram staining showed the presence of only gram-negative rods of uniform distribution. Samples that were plated on PW? medium revealed no contamination. Experiments were carried out in 15 mL polypropylene Falcon tubes with rubber septa or screw caps and 16 9 150 mm glass test tubes with slip cap closures. The media used included: (1) an enrichment medium (PW?) [11]; (2) a chemically-defined medium (CHARDS) which is a variant of CHARD2 [26], and; (3) the defined medium XDM2-PR which is a variant of XDM2 [27]. The media are described below. Optical density was measured using a Genosys 8 spectrophotometer at a wavelength of 600 nm. The machine was zeroed using an aliquot of sterile media or suspension buffer. Next, an aliquot of the bacterial suspension was placed into the cuvette and optical density was measured. PW? Medium PW? medium [11] was made in two solutions which were then combined. Solution A consisted of (for 1 L) 900 mL dH2O to which these components were added: 1.0 g soluble starch, 4.0 g soytone, 1.0 g tryptone, 1.2 g K2HPO4, 1.0 g KH2PO4, 0.4 g MgSO47H2O, 0.85 g (NH4)2HPO4, 1.0 g histidine, 25.0 mg cyclohexamide, 10.0 mL (0.1 %) hemin-Cl, and 10.0 mL (0.2 %) phenol red. Media were sterilized by autoclaving. Solution B was made by dissolving 4.0 g of glutamine into 50 mL dH2O at *60 °C, and 6.0 g bovine serum albumin (fraction V) into 30 mL of dH2O, which were then combined. Next, media were sterilized through a 0.22-lm-filter. The two solutions were then combined to form the completed broth medium. PW?
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agar plates were made by adding 12.0 g agar to solution A before sterilization in the autoclave. Solution B was then combined when solution A was cooled down to 55 °C in a hot water bath. Then, PW? medium was aseptically aliquoted into Petri dishes. CHARDS Medium CHARDS is a modified version of CHARD2 which was a chemically-defined medium based on the xylem fluid chemistry of Vitis vinifera ‘Chardonnay’ [26]. Solution A consisted of (for 1 L) 819 mL of dH2O to which these components were added: 1.0 g KH2PO4, 1.5 g K2HPO4, 0.2 g MgSO47H2O, 0.17 g alanine, 0.58 g aspartic acid, 1.8 g glutamine, 1.05 g arginine, 0.01 g cysteine-HCl, 1 mL (1 mg L-1) biotin, and 1.0 g of soluble starch. The pH was then adjusted to 6.6 using 0.1 M KOH and then sterilized by autoclaving. Solution B consisted of 0.25 g of ferric pyrophosphate dissolved in 60 mL dH2O and 4.0 g glutamine dissolved in 50 mL dH2O at *60 °C which was then combined, and was sterilized through a 0.22-lm-filter. The two solutions were then combined, aseptically, to form the completed media. XDM2-PR Medium XDM2-PR is a modified version of XDM2 [27], but lacks phenol red. XDM2 is a chemically-defined medium based on metabolic processes suggested by genome of X. fastidiosa subsp. pauca. For 1 L, 980 mL of dH2O the following components were added: 10.0 g glucose, 2.1 g K2HPO4, 0.8 g KH2PO4, 0.4 g MgSO47H2O, 0.125 g ferric pyrophosphate, 4.0 g glutamine, 1.0 g asparagine, 0.4 g serine, 0.4 g methionine, and 10 mL of vitamin solution (described below). The pH was then adjusted to 6.6 using 0.1 M KOH and then sterilized through a 0.22lm-filter. The vitamin solution (for 100 mL) consisted of 350.0 mg myo-inositol, 10.0 mg pyridoxine–HCl, 10.0 g thiamine, 5.0 mg nicotinic acid, and 0.2 mg of D-biotin. The vitamin solution was sterilized through a .22-lm-filter and stored at 4 °C in a light-protected glass bottle. PCR Protocol The polymerase chain reaction (PCR) was used to confirm the identity of the cultures. DNA extractions were conducted using the Quiagen DNeasy mini prep. The PCR protocol used a nested primer technique [35]. Oxygen Levels The LaMotte’s Dissolved Oxygen Test Kit (Model EDOCode 7414) was used to determine the treatment effects
A. D. Shriner, P. C. Andersen: Growth and Biofilm Formation of Xylella fastidiosa
over time in the aqueous environments of distilled water (dH2O) and CHARDS medium. Measurements were made just prior to daily flushing for the 21, 10, 2.1, and 0 % O2 treatments. The assays were conducted within a rubber septa closed tube by injecting 100 ll of the manganous sulfate mixed well, then 100 lL of the alkaline potassium iodide azide was added when the precipitant started to settle. A 100 lL of 50 % H2SO4 was mixed well until all the precipitants were dissolved. The tubes were opened and the solution was transferred to a beaker with a lid designed to fit around the tip of the graduated titration syringe. Next, thiosulfate was titrated into the sample until the color became clear against a white background, and the volume used represented the amount of dissolved oxygen in the sample. The O2 treatments used to assess growth and biofilm formation for all bacteria were 21, 2.1, 0.21, and 0 % O 2.
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removed and air dried. The vessels were de-stained using 70 % isopropanol. An aliquot was placed into a cuvette and the optical density was measured at a wavelength of 600 nm [15]. Statistical Analysis All experiments were designed with three replicates (three plates). The bacteria were divided and washed in order to ensure that each response observed was dependent on each treatment (and not the three measurements of the initial bacteria treatment interaction). The data were analyzed with ANOVA, t tests, means, and standard errors using the SAS V9.0 software package.
Results and Discussion Experimental Growth Conditions Mature bacterial cultures, in 50 mL batches, were removed from the incubator and pooled together into a sterilized media flask. The optical density of the culture was obtained with a spectrophotometer set at a wavelength of 600 nm. Aliquots were placed into sterile polypropylene/glass tubes and centrifuged at 2,3809g for 15 min. The supernatant was decanted and the pellet was resuspended into 3 mL of PBS (phosphate buffered saline, 0.1 M phosphate, 0.8 % NaCl, and pH 6.8). The suspension was centrifuged again. The supernatant was discarded and the pellet was resuspended into the appropriate media. The pellet was centrifuged again and the supernatant was discarded. The pellet was resuspended into 5 mL of the appropriate media. The tubes were capped with red-rubber septa and secured with parafilm. Gas treatments were done each day. The tops of the septa were sterilized by rinsing with 70 % isopropyl alcohol and dabbed dry with a Kimwipe. The gas was passed through a 0.22 lm syringe filter then injected through a 1 in., sterilized 22-gauge needle, and another sterilized 22-gauge needle was placed into the septa to act as an exhaust port to ensure gas exchange. The tubes were placed in the New Brunswick incubator set at 28 °C on an orbital shaker set at 150 rpm. Biofilm Quantification Biofilm formation on the surface walls of conical polypropylene tubes was assayed by the crystal violet method [15]. After various intervals of incubation, the supernatant was decanted and the tube was rinsed with dH2O and air dried. The vessels were filled with 0.1 % crystal violet and placed on a shaker for 1 h. The crystal violet was decanted and the tubes were rinsed until all the excess dye has been
The O2 concentrations in xylem fluid reported in the literature usually ranged from 0.43 to 3.1 mg L-1, although there are two reports of relatively high concentrations (7.4 and 8.8 mg L-1) (Table 1). The LaMotte’s method to create consistently different and stable O2 concentration was effective. The concentration of O2 in CHARDS medium measured after flushing with 21, 10, 2.1, and 0 % O2 daily for 1, 3, or 6 days was 4.5–5.0, 2.4–2.9, 0.8–1.4, and 0.4–0.7 mg L-1, respectively. The dissolved O2 content in CHARDS medium was very similar to that in distilled water (data not shown). In air, the solubility of O2 in water at 0.1 MPa pressure and 28 °C is about 8 mg L-1, and the LaMotte’s measurement of O2 in water under aerobic conditions was about 6.2 mg L-1. Also, the N2 treatment consistently resulted in a steady state concentration of about 0.5 mg L-1 O2 which may reflect a limitation of the LaMotte’s method to measure extremely low O2 concentrations. The growth of X. fastidiosa Pierce’s disease strains UCLA, STL, and almond leaf scorch strain Tulare in PW? medium (Fig. 1a) was compared to P. syringae (an obligate aerobe), and E. carotovora (a facultative anaerobe) (Fig. 1b) at four O2 concentrations. The growth of X. fastidiosa UCLA and STL was higher than the almond leaf scorch strain Tulare (Fig. 1a) and was highest in 21 % O2, and declined gradually at lower O2 concentrations. The growth of P. syringae was only substantial in the aerobic (21 % O2) treatment, and was drastically curtailed in reduced O2 environments (Fig. 1b). There was more than a sevenfold decrease in growth of P. syringae at lower O2 environments. By contrast, the growth pattern of X. fastidiosa was more analogous to the facultative anaerobe, E. carotovora (Fig. 1b), which also had an incremental growth reduction down the O2 gradient. For X. fastidiosa,
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870
A. D. Shriner, P. C. Andersen: Growth and Biofilm Formation of Xylella fastidiosa 0.6
0.50
A
A
Growth Biofilm
0.4 0.3 0.2 0.1
Xylella fastidiosa Growth Air
0.30 0.20 0.10
0.00%
0.21%
2.10%
21.00%
0.00%
0.21%
STL
1
B Pseudomonas syringae
Erwinia carotovora
1.2 1.0
Growth
0.8
Biofilm
0.6
1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0
B
7
9
11
13
Xanthomonas campestris Growth Nitrogen
1
0.4
2
3
4
5
Days
0.2 0.00%
0.21%
2.10%
21.00%
0.00%
0.21%
2.10%
21.00%
0.0
Fig. 1 The growth and biofilm formation of a three Xylella fastidiosa strains (Pierce’s disease strains STL and UCLA, and Almond Leaf Scorch (ALS) strain Tulare) in PW? media, b P. syringae (an obligate aerobe) and to E. carotovora (a facultative anaerobe) under various O2 concentrations. Initial optical density of X. fastidiosa was 0.05 and initial optical density of P. syringae and E. carotovora was 0.10. Significant differences between the optical density bars within each strain are separated by Duncan’s new multiple range test. Error bars represent standard error of the mean
the production of biofilm was numerically higher in 21 % O2 than in the other gas treatments, although significant differences did not occur. Biofilm production was negligible for P. syringae and E. carotovora. The comparison of these growth patterns raised the question as to the respiration classification of X. fastidiosa as an obligate aerobe [43]. The classification of X. fastidiosa as an obligate aerobe was based on growth responses in 1 or 5 % O2 (balance N2), 2.5 or 5.0 % CO2 with 21 % O2 (balance N2), or air [43]. The growth of X. fastidiosa (Pierce’s disease Temecula strain) in PW? medium increased eightfold and threefold
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5
Xanthomonas campestris Growth Air
ALS
1.6 1.4
3
Days
Optical Density (600nm)
1.8
2.10%
21.00%
0.00%
0.21%
2.10%
UCLA
Optical Density (600 nm)
Xylella fastidiosa Growth Nitrogen
0.40
0.00
0 21.00%
Optical Density (600nm)
0.5
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Fig. 2 a Xylella fastidiosa strain Temecula in PW? growth over time under aerobic (21 % O2) and N2 conditions. Each point is the average of three replicates. b Growth of X. campestris in PW? under aerobic (21 % O2) and N2 conditions. Initial OD is 0.05 for X. fastidiosa and 0.10 for X. campestris. Each point is the average of three replicates. Error bars represent standard errors of the mean
compared to initial levels under 21 % O2 and N2 conditions, respectively for up to 13 days (Fig. 2a). Growth rates were linear over time. (Similar growth rates were obtained with the Pierce’s disease strain UCLA and the ALS strain Tulare, data not shown). There was a gradual reduction in biofilm formation for all three strains of X. fastidiosa, and the ratio of growth to biofilm formation was approximately the same for all gas treatments. There was a striking difference between the growth of X. fastidiosa (Fig. 2a), and a close relative, X. campestris pv. campestris (Fig. 2b), also deemed to be an obligate aerobe. For X. campestris there was a tenfold increase in growth in the aerobic treatment, and virtually no growth in the N2 treatment as indicated by the negligible slope of the line (Fig. 2b). (The same result occurred for experiments with X. campestris pv. vitians, data not shown). The designation of X. fastidiosa as a facultative anaerobe should await the analysis of fermentation profiles and gene activation consistent with anaerobiosis. An alternative
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Fig. 3 The comparison of a growth and b biofilm formation of Xylella fastidiosa (Pierce’s disease strain Temecula) under aerobic (21 % O2) (open squares) and N2 (closed squares) gas treatments in the CHARDS media. Initial OD was 0.10. Each point is the average of three replicates. Error bars represent standard errors of the mean
Fig. 4 The comparison of a growth and b biofilm formation of Xylella fastidiosa (Pierce’s disease strain Temecula) under aerobic (21 %) (open squares) and N2 (closed squares) gas treatments in the XDM2-PR media. Initial OD was 0.10. Each point is the average of three replicates. Error bars represent standard errors of the mean
and viable explanation is that cytochrome oxidase of X. fastidiosa has an extremely high affinity for O2, and metabolic by-products of hypoxia can be detoxified [21]. Xylella fastidiosa has the metabolic capability for sulfate reduction [21, 39, 41]; however, metabolic by-products such as hydrogen sulfide, elemental sulfur, or iron sulfide were not quantified in the current study. A biofilm growth associated repressor (BigR) controls the transcription of an operon involved in biofilm formation in X. fastidiosa [6]. Guimaraes et al. [21] showed that the BigR operon is also required for hydrogen sulfide detoxification and the continued function of cytochrome c oxidase under hypoxic conditions. Growth and biofilm formation of X. fastidiosa strain Temecula was examined from 0 to 30 days treatment under 21 % O2 and N2 conditions in chemically-defined media CHARDS (Fig. 3), and XDM2-PR (Fig. 4). The growth and biofilm formation in the CHARDS medium was not significantly different when incubation was in the 21 % O2 treatment compared to the N2 treatment; however, it should
be noticed that growth was steady and continuous. There was a twofold increase in growth and a 5- to 7-fold increase in biofilm after 30 days of culture. By contrast, the N2 treatment using XDM2-PR medium resulted in significantly more growth and biofilm formation compared to the 21 % O2 treatment. The response of X. fastidiosa to O2 treatments compared to the N2 treatment was dependent on the growth medium used. The planktonic growth, biofilm formation, and aggregation of X. fastidiosa depend on the chemistry of xylem fluid [4, 33, 38]. Monomeric compound (amino acids and organic acids) and inorganic ions were shown to greatly alter the behavior of this bacterium [4, 26]. Wulff et al. [44], working with PW and BCYE media, showed that the pH of the culture media can influence growth, aggregation, and biofilm formation; however in our study pH of all media was adjusted to 6.6. During the growing season, the dissolved O2 in the xylem fluid becomes low enough to induce fermentation within the plant tissues [24]. A relevant issue would be the
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concentration of O2 in vessels inhabited by X. fastidiosa, or within the biofilm matrix. Goodwin et al. [20] investigated the presence of possible toxic compounds generated by X. fastidiosa; however, the results were based on the washes of aerobically-grown cultures on solid media. The results presented in our study indicate that X. fastidiosa was capable of continued growth and biofilm formation under N2 conditions. The potential ability of X. fastidiosa to modify its respiration pathways during the growing season when the O2 levels drop and the metabolic demand becomes greater, offers a reasonable explanation of how X. fastidiosa is capable of continued growth and biofilm development under hypoxic conditions in-planta. In conclusion, the continued growth and biofilm formation of X. fastidiosa under N2 conditions, the great differences in growth characteristics to two obligate aerobes (P. syringae and X. campestris), and the similarity to a facultative anaerobe (E. carotovora) support the possibility that X. fastidiosa may not be an obligate aerobe [43], but rather a facultative anaerobe. During the growing season, the dissolved O2 in the xylem fluid often becomes low enough to induce fermentation within the plant tissues [24]. Xylella fastidiosa was capable of sustained growth and biofilm production in a wide range of hypoxic environments. Thus, it appears that X. fastidiosa would be adapted to variations in O2 concentrations that would result from differences in plant anatomy, plant physiology, or environmental conditions.
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