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. 2006 Sep;98(3):483–494. doi: 10.1093/aob/mcl124

Xylem Structure and Connectivity in Grapevine (Vitis vinifera) Shoots Provides a Passive Mechanism for the Spread of Bacteria in Grape Plants

DAVID S CHATELET 1,*, MARK A MATTHEWS 2, THOMAS L ROST 1
PMCID: PMC2803575  PMID: 16790469

Abstract

• Background and Aims Bacterial leaf scorch occurring in a number of economically important plants is caused by the xylem-limited bacterium Xylella fastidiosa (Xf). In grapevine, Xf systemic infection causes Pierce's disease and is lethal. Traditional dogma is that Xf movement between vessels requires the digestion of inter-vessel pit membranes. However, Yersinia enterocolitica (Ye) (a bacterium found in animals) and fluorescent beads moved rapidly within grapevine xylem from stem into leaf lamina, suggesting open conduits consisting of long, branched xylem vessels for passive movement. This study builds on and expands previous observations on the nature of these conduits and how they affect Xf movement.

• Methods Air, latex paint and green fluorescence protein (GFP)-Xf were loaded into leaves and followed to confirm and identify these conduits. Leaf xylem anatomy was studied to determine the basis for the free and sometimes restricted movement of Ye, beads, air, paint and GFP-Xf into the lamina.

• Key Results Reverse loading experiments demonstrated that long, branched xylem vessels occurred exclusively in primary xylem. They were observed in the stem for three internodes before diverging into mature leaves. However, this stem—leaf connection was an age-dependent character and was absent for the first 10–12 leaves basal to the apical meristem. Free movement in leaf blade xylem was cell-type specific with vessels facilitating movement in the body of the blade and tracheids near the leaf margin. Air, latex paint and GFP-Xf all moved about 50–60 % of the leaf length. GFP-Xf was never observed close to the leaf margin.

• Conclusions The open vessels of the primary xylem offered unimpeded long distance pathways bridging stem to leaves, possibly facilitating the spread of bacterial pathogens in planta. GFP-Xf never reached the leaf margins where scorching appeared, suggesting a signal targeting specific cells or a toxic build-up at hydathodes.

Keywords: Xylella fastidiosa, pathogens, xylem, stem—leaf connection, grapevine, passive movement, systemic

INTRODUCTION

Recent studies on xylem connections between organs have focused on water transport (Martre et al., 2000; Tyree and Zimmermann, 2002; Zwieniecki et al., 2003; Orians et al., 2005). The movement of water in xylem, however, and the movement of bacteria and particulate matter suspended in the water may encounter different constraints. While there is a water continuum from roots to leaves (Honert, 1948; Slatyer and Taylor, 1960), several studies show that particle movement is limited by the frequency of vessel endings, especially at the stem—leaf junction, where most vessels have been thought to end, except for the few vessels crossing the junction (Larson and Isebrands, 1978; Wiebe et al., 1984; Andre, 2002; Tyree and Zimmermann, 2002). Such vascular organization is thought to act as a protection against the movement of potential air embolisms (Zimmermann, 1983; Aloni and Griffith, 1991; Tyree and Ewers, 1991) or pathogens (Zimmermann, 1983). A vessel end signifies that water and everything that flows passively with it (minerals, particles, bacteria) must cross the inter-vessel pit membrane through pores that are smaller than 0·2 µm in diameter (Siau, 1984).

Three pathogenic endophytic bacteria, Xylella fastidiosa, Clavibacter xyli and Pseudomonas syzygii live exclusively within xylem vessels when in planta (Bové and Garnier, 2002). Pierce's disease of grapevine is caused by the xylem-limited bacterium, Xylella fastidiosa (Hopkins and Mollenhauer, 1973; Wells et al., 1987). The bacterium multiplies and spreads within the xylem, reportedly plugging the xylem vessels (Esau, 1948; Hopkins, 1981; Tyson et al., 1985). In addition, xylem vessels become occluded with pectins, tyloses, and gums produced by the plant in response to invasion by the bacterium (Teackle et al., 1975; Hopkins, 1981; Huang et al., 1986; Fry and Milholland, 1990; Stevenson et al., 2004a) or in response to phytotoxins produced by the bacteria (Mircetich et al., 1976; Lee et al., 1982). Symptoms produced include marginal leaf necrosis, chlorosis, leaf matchsticks (petioles that remain attached to the stem after the lamina have fallen off) and green islands (incomplete initiation of the phellogen of the stem) (Stevenson et al., 2005), wilting of the fruit and death of the plant. The colonization of the plant requires that the bacteria move within the xylem from one organ to another (Stevenson et al., 2004b), implying that the bacteria must move from one vessel to another vessel across pit membranes. However, Xylella fastidiosa (Xf) is too big to move passively from one intact vessel to another with water flow (0·25–0·35 × 0·9–3·5 µm; Nyland et al., 1973; Holt et al., 1994). A favoured hypothesis to explain how bacteria become systemic is that the bacteria digest the pit membrane cell wall (Roper et al., 2002; Stevenson et al., 2004b). Another more recent twist in the mechanism is that bacteria might also move through torn or remnant pit pore membranes (Carlquist and Schneider, 2004; Stevenson et al., 2004b). This propagation by digestion could be rather slow if vessels are short and if numerous membranes have to be crossed. Bacterial movement in the stem of Vitis is relatively easy because vessels are plentiful and can be up to 1 m long (Sperry et al., 1987; Thorne et al., 2006b, A. Perez and J. M. Labavitch, Plant Sciences Department, University of California, Davis, unpubl. res.). However, there is still the problem of bacterial passage into leaves if most of the vessels end at the stem—petiole and petiole—lamina junctions.

Recent experiments by Thorne et al. (2006b) showed that there are open, continuous vessels from the stem to the leaf lamina of grapevine. They analysed bacterial movement using Yersinia enterocolitica (Ye) containing the lux operon. With this construct, the bacteria give off light through the luciferin/luciferase reaction which allows its movement to be followed using X-ray film. Ye is a good surrogate to Xf as it has a similar shape and size (0·5–0·8 µm × 1–3 µm; Bercovier and Mollaret, 1984), but it does not secrete any wall-digesting enzyme. Hence, any movement of Ye in the xylem is purely passive and not a consequence of pit membrane digestion. In addition, Thorne et al. (2006b) tested xylem translocation of 1-µm-diameter fluorescent beads. They found that both Ye and the beads moved freely from the stem into primary and secondary veins of the leaf blade for up to three nodes distal to the loading point. The Ye and beads travelled 60–80 % of the total length of the potential vascular path from petiole base towards the leaf margins. This shows an open xylem conduit all the way from the stem through the petiole and into the leaf blade without the need to digest a pit membrane, but also suggests that some features of the vascular structure preclude the movement of the bacteria all the way to the leaf blade periphery.

In the present study, the open conduits shown by Thorne et al. (2006b) were expanded on by using two additional methods to study xylem vessel lengths, air and latex paint movement. Using air and paint confirmed their existence and allowed their length to be measured. The paint also permitted the identification of these conduits in the stem and the leaf. The open vessel network was observed at different stages of leaf development and they were identified along the xylem pathway by using latex paint (particle movement) and toluidine blue (water movement). The xylem anatomy of the leaf blade was also studied to identify the change in the vascular structure that might restrict movement of Ye, beads, air and paint to within 50–60 % of the leaf length. Leaves were also inoculated with Xf-expressing green fluorescent protein (GFP) to check bacterial movement within the leaf at different times after inoculation. Since Xf has the ability to digest cell walls, the bacteria would theoretically be able to move farther than 50–60 % of the leaf blade length and eventually be found at the leaf margin. This work provides a partial explanation for how xylem-dwelling pathogenic bacteria, such as Xf, move within Vitis and can become systemic.

MATERIALS AND METHODS

Plant material

Grapevines (Vitis vinifera L. ‘Chardonnay’) were grown in a greenhouse at the University of California, Davis. Plants were pruned to two-bud spurs resulting in a Y-shaped plant with two shoots emerging from a common subtending trunk. Green shoots with about 20–30 nodes and without a periderm were used for the different experiments. Each node and their subtending leaf were numbered starting from the first visible leaf closest to the apex (number one) increasing in basipetal order down the stem.

Air movement

Five leaves from nodes 5–26 were cut off at the base of the petiole under water and brought to the laboratory, care being taken not to allow any air to enter the cut end of the petiole. Leaves from node 26 were about 3 months older than node 5 and the leaves above node five were immature, too small and fragile to be used. The petioles of the selected leaves were attached under water to a rubber tube and household air was pushed through at a pressure of 35 kPa controlled by a pressure regulator. This pressure is 7 % that of the lowest air seeding threshold reported for grapevine (Sperry et al., 1987) and about 20 % that of the lowest value reported for trees (Choat et al., 2003; Hacke et al., 2004; Sperry and Hacke, 2004). After a few minutes under pressure, the five major veins and their secondary veins were cut with a razor blade every few millimetres starting from the margin towards the base of the leaf. The incisions were made until a stream of air bubbles appeared at the cut. The distance from where the bubbles first appeared to the air loading point was measured and the distance from the leaf margin to the loading point was also recorded.

Green stems with about 25 nodes were cut under water and brought to the laboratory. The stem was re-cut at different internodes, connected to a plastic tube and household air was pushed into the stem base with a pressure of 35 kPa. Starting from the stem apex, the veins of each leaf were cut as previously described to observe the first appearance of a stream of air bubbles. For each vein, the distance from the loading point to the first appearance of a stream of bubbles was recorded along with the total possible length of the vascular pathway. Once the veins of all the leaves were cut and appearance of air and distances were recorded, starting from the stem apex, the blades of leaves showing no air were separated from their petiole below the blade/petiole junction. If air exited at the apical end of the petiole that signified that the junction at the blade/petiole was preventing the air from going further. If no air exited the apical end of the petiole, it was cut at its base to verify whether air was able to travel within the petiole. Finally, the stem was cut every few millimeters starting from the apex towards the base until a stream of air bubble appeared at the cut. The closest node position was noted and the remaining length of the stem was measured.

Paint movement and leaf vessel length distribution

Two experiments were conducted to study paint movement between stem and leaf, and leaf vessel length distribution was investigated in a third experiment. A 1 : 300 red latex paint : water solution was prepared and left to stand for 24 h while the larger particles settled. The supernatant was first prefiltered through Whatman no. 1 filter paper (Whatman Laboratory Division, Springfield Mill, Maidstone, Kent, UK) to remove all particles greater than 11 µm in diameter, thus allowing the paint solution to travel within the vessels without clogging them. Since the pit membrane pores of dicotyledons range in size from about 0·005 to 0·17 µm in diameter (Siau, 1984), paint particles larger than 0·17 µm need to be removed from the remaining solution. A sample of the solution run through a MF-Millipore membrane filter with a pore size of 0·22 µm (Millipore Corporation, Massachusetts, USA) showed that most of the particles were stopped by the membrane filter and thus that the paint particles were larger than 0·22 µm and could not pass through pit membrane pores. As a result, the accumulation of paint particles in the xylem revealed the end of the vessels. The paint solution was loaded at different internodes of stems bearing 20–25 leaves under a pressure of 35 kPa for several days. When the paint solution stopped moving into the stem after several days, the presence of paint in the leaves above the loading point was checked microscopically from free-hand stem cross-sections. The petiole, the five major veins and their secondary veins were free-hand sectioned every 5 mm and observed with a Zeiss SV 8 stereomicroscope (Carl Zeiss, Oberkochen, West Germany) linked to a Pixera 600ES digital camera (Pixera Corporation, Los Gatos, USA).

The second experiment consisted of observing the movement of paint from the leaf to the stem at different nodes and its propagation in the stem to visualize eventually the leaf traces. The leaf blade was removed and the paint was loaded (35 kPa) into the apical end of the petiole still attached to the stem for 48 h. The leaves tested this way ranged from node 4 to node 20 counting from the apex towards the shoot base. The presence of paint in the stem was studied by observing cross-sections of the stem with a stereomicroscope connected to a digital camera. A section was cut every centimetre above and below the infused leaf until no paint was visible in the xylem. The same experiment was repeated with a filtered (Whatman no. 1 filter paper) 0·1 % Toluidine Blue O (TBO) (w/v) solution loaded for 24 h. TBO is able to cross the pit membrane between tracheary elements (Shane et al., 2000) and its movement should reflect the movement of water from the petiole to the stem and in the stem, which will show differences with the movement of paint which cannot cross a pit membrane.

In a final experiment, vessel lengths were measured using the paint perfusion technique (Zimmermann and Jeje, 1981; Ewers and Fisher, 1989). For each of ten stems, the leaf from node 15, counting from the apex, was excised under water. The petiole was connected to silicone tubing linked to a reservoir filled with the paint solution. The leaves were kept under water and were infused with the latex suspension at a pressure of 35 kPa for 48 h. At the end of the infusion time, thin free-hand cross-sections were cut every 5 mm with a razor blade. Starting from the loading point and progressing towards the margin of the leaf, the petiole was first sectioned every 5 mm, followed by the five major veins and secondary veins. The paint-filled or partially filled vessels were counted in the petiole, in the five major veins and in the secondary veins. Vessel length distribution was calculated according to Zimmermann and Jeje (1981), as modified by Ewers and Fisher (1989). The distance travelled by the paint from the loading point was calculated and the distance from the leaf margin to the loading point was also recorded.

Tracheary elements of the leaf blade

Leaf blade xylem structure was studied by paraffin, plastic embedding and maceration. Leaf punches were made with a cork borer, and vein segments 5 mm long were separated from surrounding leaf blade tissues with a razor blade. One sample was collected from each major vein at the base of the blade and at mid-distance. Two more samples were taken close to the margin of the leaf. Six mature leaves from node 15 were used for each method. For the paraffin method, the segments were fixed in formalin—acetic acid—alcohol (FAA) fixative and dehydrated through an ethanol series (Ruzin, 1999). The samples were then infiltrated with Hemo-De (Fisher Scientific, Hampton, NH, USA) followed by Paraplast Extra paraffin using a Leica TP 1020 automatic tissue processor. Then the samples were embedded with a Leica EG 1160 paraffin embedding centre (Leica Microsystems, Bannockburn, IL, USA), and sectioned at 10 µm with a Microm HM 304E rotary microtome (Microm, Walldorf, Germany). Sections were stained following the Johansen's safranin (1 % w/v, 2 h) and fast green (0·05 % w/v, 15 s) protocol (Ruzin, 1999).

For plastic embedding, the segments were fixed in FAA fixative and dehydrated through an ethanol series (Ruzin, 1999). The samples were then infiltrated and embedded in glycol methacrylate (JB-4 Plus embedding kit; Polysciences, Inc., USA). First the tissues were infiltrated with JB-4 solution A mixed with benzoyl peroxide (catalyst) for several hours, then the specimens were transferred to a mold holder and embedded with an embedding solution made from JB-4 Plus solution B added to the infiltration solution. The mold holder was then transferred into a chamber where the air was replaced by N2 gas in order for the embedding solution to polymerize. The resulting plastic blocks with the specimen were then fixed onto a block holder with epoxy glue and prepared for sectioning. The segments were sectioned at 5 µm with a Microm HM 304E rotary microtome. Sections were stained with 0·1 % TBO (aqueous, w/v).

For maceration, the segments were immersed separately in capped vials containing a maceration solution (1 : 4 : 5 30 % hydrogen peroxide : distilled water : glacial acetic acid) and placed in the oven at 57 °F until the tissues became translucent (Ruzin, 1999). After washing with water several times, the tissues were stained with 0·1 % aqueous Safranin O (w/v) for several hours and then transferred to a microscope slide. Using a binocular microscope, xylem strands were gently extracted and other tissues were removed to have a clear view of the xylem. The tracheary elements were then carefully separated with forceps and needles and spread on the slide.

All material was examined with an Olympus Vanox-AHBT (Olympus America, Melville, New York, USA) compound light microscope linked to a Pixera 600ES digital camera.

Movement of GFP-Xylella fastidiosa in the leaf

A set of grapevines was inoculated with Xylella fastidiosa (Xf) strain Temecula that constitutively expresses a green fluorescent protein (GFP) (Newman et al., 2003). Ready-to-inoculate GFP-Xf was obtained from Dr Bruce Kirkpatrick, Department of Plant Pathology, UC, Davis. Leaves between nodes 5 and 15 were inoculated with GFP-Xf in the middle of the petiole, at mid-distance of the main vein in the middle of the lamina (central vein) or in the central vein near the tip of the leaf. Inoculation consisted of placing a drop of bacterial culture (20 μL, 108 CFU) on the adaxial surface of the petiole or vein and pushing a 27-gauge hypodermic needle through the drop and into the leaf vascular structure, trying to pierce the xylem vessels. Because the water column inside the xylem is under tension and the liquid drop under atmospheric pressure, the liquid is sucked into the xylem upon removal of the needle when the xylem has been pierced. An additional set of plants was similarly inoculated with fluorescent polystyrene beads suspended in TYE at a concentration of 1·0 × 108 microspheres mL−1 [FluoSpheres polystyrene microspheres, 1·0 µm, blue-green fluorescent (430/465 nm), Molecular Probes, Eugene, OR, USA] to serve as negative controls. Previous studies showed that the leaf symptoms of Pierce's disease usually appear in greenhouse-grown grape in 5–12 weeks (Hill and Purcell, 1995; Purcell and Saunders, 1999). Consequently bacteria-inoculated leaves and control leaves were sampled 5 weeks after inoculation before Pierce's disease symptoms appeared and at about 11 weeks when the symptoms appeared. Infected leaves were cut from plants immediately prior to sectioning. All leaves were cross-sectioned every 5 mm with a thin razor blade, starting from the base of the petiole and continuing in the veins of the blade to the margins of the leaf. The presence of the fluorescent Xf cells was visualized with an Olympus Vanox-AHBT epifluorescent microscope, using filters that permitted an excitation wavelength between 380 nm and 490 nm (Blue light) and emission wavelength above 515 nm, and photographed as described above.

RESULTS

Air and latex paint movement in leaves

The distance travelled by air and paint ranged from 40 % to 60 % of the total length of the vascular path from petiole base to individual vein endings (Fig. 1). Neither air nor paint can move from one vessel to another across intact inter-vessel pit membranes. Consequently, the similar results obtained with air and paint suggest that both moved through open, continuous xylem conduits until they reach a location in the leaf blade where a change occurs in the xylem vascular structure that precludes further movement (Fig. 2).

Fig. 1.

Fig. 1.

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Air and paint position in the leaf veins calculated as percentage of the total length of the vascular pathway from petiole to leaf margin. Total length ranged from 8 cm to 25 cm. Air and paint was loaded at the base of the petiole of leaves from different nodes. The farthest position of the air or paint inside the leaf veins was recorded. For each node, the presence of air or paint was observed in the five major veins and ten secondary veins of five leaves. Data are mean ± standard error, n = 5.

Fig. 2.

Fig. 2.

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Map of a leaf showing the longest distance (dashed line) and the averaged distance travelled by air (blue dots) and paint (red dots) in the primary and secondary veins when loaded into the petiole base. Images on the right are cross-sections of the petiole (A) and of the midvein (B) showing paint-filled vessels. Scale bars = 1 mm.

Air and paint movement from stem to leaves

About 75 % of the time, air and paint loaded in the stem were found in primary and secondary veins of the three leaves immediately apical to the loading point (Fig. 3). No air and paint was observed leaving the stem and moving into petioles beyond these three leaves, although air or paint could be observed in the stem axis up to 1 m past the loading point. The progression of air and paint inside the three leaves was comparable to that seen when air and paint were loaded into an individual isolated leaf via the base of the petiole (Fig. 2). This suggests that the open, continuous vessels present from petiole to leaf blade are also continuous from the stem axis into the leaf blade for three internodes (about 20–30 cm). However, these vessels connecting stem to leaves were not present at all stages of vine development. Air and paint loaded in the stem above nodes 10–12 (younger leaves) could not be found in any leaves, although they did move up the stem. The open vessels connecting the stem axis and leaves seem to form at nodes 10–12 (about 1 m behind the apex).

Fig. 3.

Fig. 3.

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Diagram representing the movement of air or paint within the stem and into the leaves when loaded at different internodes. Below nodes 10-12, air and paint moved into the three leaves above the loading point (average = 3·06 petioles, s.d. = 0·47, n = 5 shoots) and into the stem. From nodes 10–12, air and paint moved only in the stem. A pressure of 35 kPa was used and the presence of air and paint was first confirmed into the leaves then into the stem starting from the apex of the stem towards its base.

Paint and water movement from leaf to stem

Two other experiments were conducted to examine the xylem connections between the stem and the leaf by removing the leaf blade and back loading paint or a TBO solution into the apical end of the petiole (Fig. 4). Similarly to the previous experiment that studied the movement of paint from stem to leaves, no paint could be observed moving into the stem from leaves above nodes 10–12. However, paint did move into the stem from older leaves. After 48 h, paint loaded in a petiole at node 15 was observed in the stem at its attached node (Fig. 4A). Paint was also observed 1 cm below the node (Fig. 4B) and as far as three internodes below the loaded petiole without moving into the leaves (Fig. 4C). This agrees with the previous experiment showing the paint moving for three internodes before branching off the stem to go into the leaves. Below the loaded node, paint was observed only near the stem pith where the primary xylem is located (Fig. 4B). No paint was observed in the stem secondary xylem.

Fig. 4.

Fig. 4.

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Downward movement of red latex paint (A–C) and Toluidine Blue O (TBO) (D–H) in stems when loaded in the petiole. Paint and TBO were loaded under a pressure of 35 kPa in nodes ranging from 4 to 20. (A–C) Cross-sections of a stem with paint loaded at node 15; (D–F) cross-sections of a stem with TBO loaded at node 15; (G, H) cross-sections of a stem with TBO loaded at node 6. Hand sections of the stem were made after 48 h for the paint and after 24 h for TBO. (A, D, G) Sections made within the loaded node, a few millimetres below the stem-petiole junction; (B, E, H) sections made 1 cm below the node; (C) a section made above the third node below the loaded petiole; (F) a section made five nodes below the loaded petiole. (I) Primary vessels are shown containing red paint compared with secondary xylem which is without paint. Scale bars: A–H = 1 mm; I = 200 μm.

Results were different when the petiole was loaded with TBO. First, the dye was able to move into the stem from mature leaves (node 15; Fig. 4D–F) and, contrary to the paint, it also moved into the stem from young leaves (node 6; Fig. 4G, H). However, the dye loaded in young leaves was not found past the first node below the loading site. The second difference is that after only 24 h, TBO can be already observed five nodes below the loading point (Fig. 4F). The third difference is that TBO was not restricted to the primary xylem and could be seen in the stem secondary xylem. This dye movement was mostly radial over a distance of five nodes with little tangential propagation.

Leaf vessel length distribution

The frequency distribution of vessel lengths passing into leaves produced a highly skewed, non-normal distribution with a high frequency of short vessels (Fig. 5). About 70 % of the vessels of grapevine leaves were <3 cm long. The remaining vessels were equally distributed among the length classes, each class representing about 5 %. However, the longest vessels (11–14 cm) represented only 1–2 % of the paint-filled vessels. This means that there are few vessels through which the paint is able to access the middle of the leaf blade from the stem, but these long vessels were always present.

Fig. 5.

Fig. 5.

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Vessel length distribution in mature leaves of greenhouse-grown Chardonnay. For each length class, the number of painted-vessels was calculated as a percentage of the total number of painted vessels at the petiole base. The maximum vein length ranged from 15 to 25 cm (average ± standard error, n = 50 veins).

Tracheary elements of the leaf blade

Studies of air and paint movement in the xylem showed that the average distance travelled is about 50–60 % of the leaf length, with maximum distance reaching about 80 % of the leaf length (Fig. 2). Since air and paint cannot cross intact cell walls or pit membranes between vessels, this means that the open, continuous tracheary elements conducting air or paint must end in this area. Consequently, the type of tracheary element (vessel member or tracheid) was studied before this limit, at this limit and past this limit.

Vessel members with helical secondary walls and simple perforation plates were predominant in the basal 50–60 % of the lamina (Fig. 6). Past the limit of air and paint mobility, closer to the leaf margins, no perforation plates were observed in any tracheary elements. Macerations revealed that the xylem near the leaf margin was composed of close-ended tracheids (Fig. 6). Clearings of the areoles of the leaf blade showed that the endings of the minor veins were formed of tracheids with unusual shapes (Fig. 7). They may be clavate, gnarled or ramified with short knobs.

Fig. 6.

Fig. 6.

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Map of a leaf showing the change in the xylem structure in relation to the limit set by air and paint movement. Images on the right show the presence of simple perforation plates (opp) within the xylem of the midvein, obtained with paraffin embedding (A), plastic embedding (B) and by maceration (C). (D, E) Tracheids are shown in the midvein near the tip of the lamina. Six leaves were used for each method. For each leaf, ten samples were taken before the limit and ten samples were taken after the limit. A total of 120 samples were examined for each method. Scale bars = 25 μm.

Fig. 7.

Fig. 7.

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Vein-ending of a minor vein from an areole within the leaf blade showing tracheids after clearing, and observed with a confocal microscope. Scale bar = 10 μm.

Movement of GFP-Xf in leaves

Open, continuous vessels were shown to be present from the stem to 50–60 % of the leaf length. Vessels and vessel members terminate in this area and are replaced by tracheids towards the leaf margin. The working hypothesis for the present paper was that bacterial movement would be affected by the change of tracheary element and would be stopped or slowed because further travel would involve passage across a primary cell wall or pit membrane. GFP-Xf was needle-inoculated into leaves, and there was no difference in Xf movement between leaves collected at 5 weeks and around 11 weeks (Fig. 8). In all the leaves examined, bacteria were observed, on average, in 50–60 % of the leaf lamina, but never reached the leaf margin. Bacteria had already reached the end of the vessels at 5 weeks and did not move closer after 11 weeks. Curiously, the first symptoms of leaf necrosis in Pierce's disease occur at the leaf blade margin, some distance past the location of the maximal distance of Xf travel in the blade.

Fig. 8.

Fig. 8.

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Map of a leaf showing the average (green spots) and the longest (dashed line) distance travelled by GFP-Xf in the xylem vessels when loaded into the petiole base. Images on the right are cross-sectional views of the infected petiole (A) and the midvein (B). Scale bars = 0.5 mm.

Bacteria were loaded at different places on leaves to determine if the loading site influenced the distance the bacteria could travel or the outer limit of its travel. The results showed that GFP-Xf could not be found past the previously seen limit in leaves infected in the petiole or at mid-distance of the primary central vein, regardless of the presence or absence of symptoms. Green fluorescence was observed in the petiole of leaves infected in the midvein, signifying that the bacteria were able to move against the transpiration stream. In leaves infected in the petiole, the bacteria could be detected in most of the vessels of the petiole while only a few reached the lamina of the same leaf. When leaves were inoculated near the leaf margin, GFP-Xf could not be observed at 5 weeks, nor at 11 weeks. The bacteria may not have survived in the tracheids, or possibly no tracheids were actually infected during the needle inoculation as there are few of them near the margin.

Fluorescent beads, loaded similarly to GFP-Xf, in the petiole or at mid-distance of the central vein, were often found a few millimeters proximal to the inoculation site, most of them upstream and sometimes downstream. Since the beads move passively (no cell wall enzymatic digestion possible) with the transpiration stream and cannot move from one vessel to another because of their size (1 µm), they were stopped by vessel endings. In some leaves, however, they were found up to 60 % of the lamina length but not farther, which is similar to GFP-Xf movement. This means that some of the beads were loaded in the open vessels and moved with the water flow until they reach the end, where they accumulated.

DISCUSSION

Using several different techniques, this study confirmed the existence of open and continuous xylem vessels from the stem to the leaf blade in grapevine (Thorne et al., 2006b). It was not clear if the observed movement of paint, air, fluorescent beads or Ye in the xylem was restricted to a single xylem vessel or whether more than one vessel was seen joined to another through broken primary cell walls or broken pit membranes. This led to the use of the term ‘conduit’ to mean one or more xylem vessels joined to allow free movement of particles ∼1 µm in diameter. Additionally, it was shown that the longest open conduits consistently extend, on average, to approx. 60 % of the leaf length. At the end of these conduits in the leaf blade, the xylem vessels are replaced by tracheids. These observations have important implications for xylem development and bacterial migration through xylem.

Vascular structure of the leaf

The diameter of xylem conduits decreased towards the margin in leaves of grape, similar to sunflower (Wang, 1985; Canny, 1993). At about 50–60 % of the leaf length, movement of paint particles and low-pressure air halted, indicating that the open conduits ended before the leaf margins. No perforation plates were found in the tracheary elements of the veins past this limit; vessels and vessel members were replaced by short and small annular or spirally thickened tracheids. This transition can be understood from the description of the primary vascularization of grapevine leaves by Fournioux and Bessis (1977). According to them, the differentiation of the xylem does not progress continuously from the base of the leaf lamina towards its margins, but occur at several points near the leaf margin along an axis that will become a vein (Fig. 9). These points are called nodal points. At each ‘nodal point’, the differentiation of vascular xylem occurs in three directions: (1) acropetal towards the leaf margin along the axis; (2) as a bifurcation to form a secondary vein branching from the axis; and (3) basipetal to meet the xylem forming towards the nodal point. It is suggested that these nodal points produce only tracheids. Accordingly, acropetal xylem differentiation from the lamina base may form vessels, and the transition from vessels to tracheids is at the interface of xylem originating from the lamina base and from a nodal point at about 50–60 % of the leaf length.

Fig. 9.

Fig. 9.

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Diagram showing changes in tracheary structure along the leaf axis in grapevine.

Movement of GFP-Xf in vessels and tracheids

As with paint and air, GFP-Xf was restricted to 50–60 % of the leaf length when loaded in the petiole or in the middle of the lamina. There are several possible explanations for this. The limit of GFP-Xf movement coincides with the location of the transition from vessel to tracheid. Bacterial accumulation at the end of the vessels and possibly in the first tracheids could form biofilms, therefore reducing bacterial movement towards the leaf margin. Tracheid shortness would also require repeated breaching of inter-tracheid primary walls in order for bacteria to move towards the leaf margin. Thus, movement via wall degradation would be slow in tracheids compared with vessels. Surprisingly, the bacteria failed to advance through the leaf tracheids even over an 11-week period. A structural difference in the tracheid primary wall that averts its digestion is implicated because the primary cell wall structure could interfere with adhesion (de Souza et al., 2003) or present a poor substrate for the enzymes that are effective for breaching the pit membrane in vessels.

The limit of GFP-Xf movement was the same in infected leaves without symptoms as in leaves with scorching symptoms at their margin. Thus, the presence of the bacteria right at the leaf margin is not necessary to produce Pierce's disease symptoms. This is consistent with the hypothesis of a signal or toxin traveling from the bacterial location to the margin of the leaf and inducing the marginal leaf necrosis (Thorne et al., 2006a). Alternatively, a toxin might be swept up the transpiration stream and concentrate in the margins.

Continuity between stem and leaf lamina

Anatomical studies of the stem—petiole and petiole—lamina junctions in different plant habit types—trees, shrubs, vines and grasses—indicate that most vessels end near these junctions in alfalfa (Wiebe et al., 1984), tall fescue (Martre et al., 2000), trees (sycamore maple, walnut, London plane, oak), shrubs (rose, American poke) and vine (clematis) (Andre, 2002), and that vascular continuity is maintained by a bridge of tracheids (Meyer, 1928) or by few, short and narrow vessels as shown by Larson and Isebrands (1978) and Larson and Fisher (1983) in eastern cottonwood or by André et al. (1999) and André (2002) in the species mentioned above. This arrangement is thought to allow the unimpeded movement of water, but not particles that may be found in the transpiration stream. André (2002) used a microcasting technique that can be compared with air and paint movement, because the silicone used for infiltration is able to move through open perforation plates but not through intact pit membranes and primary walls. André investigated the stem—petiole junction for a number of species, trees, shrubs and one vine, Clematis vitalba. He showed that, for all of them, some vessels were continuous from stem to petiole. As such, finding this connection in Vitis was expected. However, his study was limited to the stem—petiole abscission zone, and he did not investigate whether these vessels terminated in the leaf blade. In their study of grapevine, Fournioux and Bessis (1974, 1977) observed that most of the vessels appeared at the junctions and formed acropetally or basipetally on either side, except for a few xylem elements that are continuous between stem and petiole and between petiole and lamina. However, they did not investigate the functionality of these vessels or whether they formed an open pathway between stem and leaf lamina. Here, the findings of Fournioux and Bessis (1974, 1977) are extended by demonstrating functionally open conduits from stem to leaf lamina in Vitis.

The presence of these open conduits is surprising because the structures of the stem—petiole and petiole—lamina junctions are thought to be a safety mechanism against embolism (Zimmermann, 1983; Aloni and Griffith, 1991; Tyree and Ewers, 1991) and particle movement (Zimmermann, 1983; Thorne et al., 2006b). Indeed the tracheids and short vessels in these junctions would stop the propagation of air from the leaf to the stem, and are thought to facilitate the shedding of the embolized leaves (Tyree et al., 1993; Rood et al., 2000). Although the open conduits would allow free movement of air into the leaf blade, their embolism can be expected to have a limited impact on overall leaf hydraulic conductance because they are so few in number (1–2 % of all vessels) and because water can bypass the obstruction easily through the finely reticulate vein network of a leaf (Wylie, 1938; Roth-Nebelsick et al., 2001; Salleo et al., 2001; Cochard et al., 2004). However, allowing a few bacterial cells to pass may have a considerably greater impact on pathogenesis.

The present results with paint and air indicate a differential time course for establishing hydraulic and open conduit connections between stem and leaves. Although the young leaves already had open conduits from petiole to lamina and the leaf traces could be distinguished in the stem (Fournioux and Bessis, 1973), the open conduits from stem to leaf lamina were not found near the shoot apex but appeared in leaves at about 12 nodes below the shoot apex and older. The hydraulic connection between leaves and stem is present in young stems as indicated by the movement of TBO from petiole to stem at node 6. Paint and air could not move across the petiole—stem junction at node 6, signifying that the conduits between stem and leaf were not yet open, although they were already open between petiole and lamina. Beyond nodes 10–12, both air and paint were able to move through the stem—petiole junction and into the lamina, meaning that conduits opened and connected the stem to the already open conduits of the leaf.

Martre et al. (2000) suggested that when monocotyledonous leaves unfold and become functional, their base may still be meristematic and the xylem in the basal part may not yet have reached its full water-conducting capacity. The xylem tracks of the leaf lamina become fully connected to the stem only after the maturation of the leaf-base. In grapevine, a similar maturation seems to occur around nodes 10–12, and this is approximately the position where leaf growth is complete (Schultz and Matthews, 1988). As the leaf matures, these open conduits in the lamina extend through the petiole towards the stem and eventually connect with the stem. Paint movement from petiole to stem showed that these open conduits belong to the primary xylem, probably metaxylem. The protoxylem is usually damaged by tissue expansion, and metaxylem is the main pathway for conduction until secondary xylem is formed (Esau, 1965). The temporal formation of these conduits and the connectivity between leaf and stem needs to be investigated more closely.

Continuity for three internodes

The present study also showed that these same conduits were open and continuous for typically only three internodes in the stem. This essentially agrees with Fournioux and Bessis (1973) who reported that the traces of each leaf separate from the axial xylem of the stem four (rather than three) internodes before they leave the stele. Stevenson et al. (2004a) could not see such early separation of traces in their stem serial cross-sections because the traces that were continuous from the leaf were indistinguishable from stem secondary xylem using their technique. The results presented here are compelling because of the open connection between leaf and stem established by paint that was introduced in the primary xylem of the leaf.

CONCLUSIONS

An important aspect in the colonization of hosts by xylem-dwelling bacterial pathogens such as Xf is the systemic movement of the bacteria within the vascular system. The present results indicate that, at least in grapevine, rapid passive bacterial colonization is possible via the primary xylem. This could explain the rapid colonization observed in several studies. Agrobacterium tumefasciens inoculated at the base of the stem reached the top of the grapevine stem (about 30 cm) within 24 h using the transpiration flow (Tarbah and Goodman, 1987). Burkholderia sp. appeared in the fifth leaves of grapevine only 72 h after the roots were put in contact with the bacteria (Compant et al., 2005). Thorne et al. (2006b) found Ye at 50–60 % of the length of the leaf within 30 min after being loaded at the petiole base and moved by transpiration. Since bacterial infection affects many different species, an interesting question arises as to whether such paths are unique to grapevine or whether they can be found in other species susceptible to bacterial infection.

Another result from this study is the absence of the bacteria at the margin of the leaves where symptoms of Pierce's disease first appear. Thus, marginal necrosis from Pierce's disease must be due to soluble signals or toxins transported from infection sites in the leaves of Vitis.

Acknowledgments

The authors thank Eleanor Thorne, Qiang Sun, Gregory Gambetta and Brendan Choat for helpful discussions and technical assistance. We also thank Caroline Roper from Dr Kirpatrick's laboratory at UC Davis for providing GFP-Xf, and Christina Wistrom from Dr Purcell's laboratory at UC, Berkeley for sharing her experience with the paint infusion technique. We also thank David Gilchrist for allowing us to use his confocal microscope and Barney Ward for technical assistance. Cal-Western Nurseries, Visalia, CA, is acknowledged for providing Chardonnay grapevines. This work was funded by the California Department of Food and Agriculture, Agreement No. 01-0712.

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