Xylem structure of four grape varieties and 12 alternative hosts to the xylem-limited bacterium Xylella fastidious.
ABSTRACT The bacterium Xylella fastidiosa (Xf), responsible for Pierce's disease (PD) of grapevine, colonizes the xylem conduits of vines, ultimately killing the plant. However, Vitis vinifera grapevine varieties differ in their susceptibility to Xf and numerous other plant species tolerate Xf populations without showing symptoms. The aim of this study was to examine the xylem structure of grapevines with different susceptibilities to Xf infection, as well as the xylem structure of non-grape plant species that support or limit movement of Xf to determine if anatomical differences might explain some of the differences in susceptibility to Xf.
Air and paint were introduced into leaves and stems to examine the connectivity between stem and leaves and the length distribution of their vessels. Leaf petiole and stem anatomies were studied to determine the basis for the free or restricted movement of Xf into the plant.
There were no obvious differences in stem or petiole vascular anatomy among the grape varieties examined, nor among the other plant species that would explain differences in resistance to Xf. Among grape varieties, the more tolerant 'Sylvaner' had smaller stem vessel diameters and 20 % more parenchyma rays than the other three varieties. Alternative hosts supporting Xf movement had slightly longer open xylem conduits within leaves, and more connection between stem and leaves, when compared with alternative hosts that limit Xf movement.
Stem--leaf connectivity via open xylem conduits and vessel length is not responsible for differences in PD tolerance among grape varieties, or for limiting bacterial movement in the tolerant plant species. However, it was found that tolerant host plants had narrower vessels and more parenchyma rays, possibly restricting bacterial movement at the level of the vessels. The implications of xylem structure and connectivity for the means and regulation of bacterial movement are discussed.
- SourceAvailable from: usda.gov[show abstract] [hide abstract]
ABSTRACT: The ultrastructural morphology of the mouthparts of the glassy-winged sharpshooter, Homalodisca coagulata, and method of plant penetration was examined using light microscopy, scanning electron microscopy, and transmission electron microscopy methods. The gross morphology of the labrum, labium, and stylet fascicle was consistent with what has been described for other plant-sucking homopterans. The ultrastructural examination of the mouthparts revealed unique details that have previously gone unreported. Several types of sensilla-like structures having the form of pegs and multi-lobed objects were identified on the outer surfaces of the labrum and within the labial groove. Dendritic canals terminated in an extensive network of smaller canals at the distal tip of the maxillary stylets below a series of surface denticles suggesting that this area may have a sensory function associated with locating xylem elements of host plants. Examination of salivary sheath pathways established that 65% of the plant penetrations by this insect terminated in the xylem vessels of the host plant. Probing by the insect was largely intracellular and terminal branching of a single probe site was common. Plant surface feeding sites varied with the stage of development which correlates with the depth of the xylem vessels and the length of the maxillary stylets of the various instars.Arthropod structure & development 11/2003; 32(2-3):189-99. · 1.11 Impact Factor
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ABSTRACT: A biofilm is a community of microorganisms attached to a solid surface. Cells within biofilms differ from planktonic cells, showing higher resistance to biocides, detergent, antibiotic treatments and host defense responses. Even though there are a number of gene expression studies in bacterial biofilm formation, limited information is available concerning plant pathogen. It was previously demonstrated that the plant pathogen Xylella fastidiosa could grow as a biofilm, a possibly important factor for its pathogenicity. In this study we utilized analysis of microarrays to specifically identify genes expressed in X. fastidiosa cells growing in a biofilm, when compared to planktonic cells. About half of the differentially expressed genes encode hypothetical proteins, reflecting the large number of ORFs with unknown functions in bacterial genomes. However, under the biofilm condition we observed an increase in the expression of some housekeeping genes responsible for metabolic functions. We also found a large number of genes from the pXF51 plasmid being differentially expressed. Some of the overexpressed genes in the biofilm condition encode proteins involved in attachment to surfaces. Other genes possibly confer advantages to the bacterium in the environment that it colonizes. This study demonstrates that the gene expression in the biofilm growth condition of the plant pathogen X. fastidiosa is quite similar to other characterized systems.FEMS Microbiology Letters 09/2004; 237(2):341-53. · 2.05 Impact Factor
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ABSTRACT: Xylella fastidiosa (Xf) is a xylem-limited bacterium that lives as a harmless endophyte in most plant species but is pathogenic in several agriculturally important crops such as coffee, citrus, and grapevine (Vitis vinifera L.). In susceptible cultivars of grapevine, Xf infection results in leaf scorch, premature leaf senescence, and eventually vine death; a suite of symptoms collectively referred to as Pierce's disease. A qPCR assay was developed to determine bacterial concentrations in planta and these concentrations were related to the development of leaf-scorch symptoms. The concentration of Xf in leaves of experimental grapevines grown in the greenhouse was similar to the concentration of Xf in leaves of naturally infected plants in the field. The distribution of Xf was patchy within and among leaves. Some whole leaves exhibited severe leaf-scorch symptoms in the absence of high concentrations of Xf. Despite a highly sensitive assay and a range of Xf concentrations from 10(2) to 10(9) cells g(-1) fresh weight, no clear relationship between bacterial population and symptom development during Pierce's disease was revealed. Thus, high and localized concentrations of Xf are not necessary for the formation of leaf-scorch symptoms. The results are interpreted as being consistent with an atiology that involves a systemic plant response.Journal of Experimental Botany 02/2007; 58(15-16):4037-46. · 5.24 Impact Factor
Xylem structure of four grape varieties and 12 alternative hosts
to the xylem-limited bacterium Xylella fastidious
David S.Chatelet1, Christina M.Wistrom2, Alexander H.Purcell2, Thomas L.Rost3and Mark A.Matthews4,*
1Department of Ecology and Evolutionary Biology, Brown University, Providence, RI 02906, USA,2Department
of Environmental Science, Policy and Management, University of California, Berkeley, CA 94720-3114, USA,3Department
of Plant Biology, University of California, Davis, CA 95616, USA and4Department of Viticulture and Enology, University
of California, Davis, CA 95616, USA
*For correspondence. E-mail: firstname.lastname@example.org
Received: 17 February 2011 Returned for revision: 14 March 2011Accepted: 21 March 2011 Published electronically: 5 May 2011
†Background and Aims The bacterium Xylella fastidiosa (Xf), responsible for Pierce’s disease (PD) of grapevine,
colonizes the xylem conduits of vines, ultimately killing the plant. However, Vitis vinifera grapevine varieties
differ in their susceptibility to Xf and numerous other plant species tolerate Xf populations without showing
symptoms. The aim of this study was to examine the xylem structure of grapevines with different susceptibilities
to Xf infection, as well as the xylem structure of non-grape plant species that support or limit movement of Xf to
determine if anatomical differences might explain some of the differences in susceptibility to Xf.
†Methods Air and paint were introduced into leaves and stems to examine the connectivity between stem and
leaves and the length distribution of their vessels. Leaf petiole and stem anatomies were studied to determine
the basis for the free or restricted movement of Xf into the plant.
†Key Results There were no obvious differences in stem or petiole vascular anatomy among the grape varieties
examined, nor among the other plant species that would explain differences in resistance to Xf. Among grape
varieties, the more tolerant ‘Sylvaner’ had smaller stem vessel diameters and 20% more parenchyma rays
than the other three varieties. Alternative hosts supporting Xf movement had slightly longer open xylem conduits
within leaves, and more connection between stem and leaves, when compared with alternative hosts that limit Xf
†Conclusions Stem–leaf connectivity via open xylem conduits and vessel length is not responsible for differ-
ences in PD tolerance among grape varieties, or for limiting bacterial movement in the tolerant plant species.
However, it was found that tolerant host plants had narrower vessels and more parenchyma rays, possibly restrict-
ing bacterial movement at the level of the vessels. The implications of xylem structure and connectivity for the
means and regulation of bacterial movement are discussed.
Key words: Grape, grapevine, Vitis vinifera, host, leaf, stem, xylem, Pierce’s disease, Xylella fastidiosa.
Xylella fastidiosa (Xf) is a xylem-limited bacterium that lives
as a harmless endophyte in most plants species, but various
subspecies and strains of Xf are differentially pathogenic in
several agriculturally important crops such as coffee, citrus
and grapevine (Hopkins and Purcell, 2002). The bacterium is
transmitted by xylem sap-feeding sharpshooter leafhoppers
(Redak et al., 2004), which acquire Xf while feeding on the
xylem of infected plants (Houston et al., 1947). In susceptible
cultivars of grapevine, Xylella fastidiosa subsp. Piercei infec-
tion results in leaf scorch, premature leaf senescence, petiole
‘matchsticks’, incomplete periderm development and even-
tually death (Stevenson et al., 2005); a suite of symptoms col-
lectively referred to as Pierce’s disease (PD).
PD symptoms were traditionally thought to result from the
accumulation of bacteria and its associated gum within the
xylem vessels, causing vascular occlusions and water deficit
(Hopkins, 1989; Purcell and Hopkins, 1996). This vascular
occlusion hypothesis implies a positive correlation between
symptom severity and pathogen concentration. Indeed, some
studies showed correlations between high Xf populations and
the apparent susceptibilities of grapevine genotypes (Raju
and Goheen, 1981; Hopkins and Thompson, 1984; Fry and
Milholland, 1990; Krivanek and Walker, 2005). However,
the relationship between PD symptoms and bacterial popu-
lations is more complex. First, Thorne et al. (2006a) showed
that visual symptoms of PD were qualitatively and quantitat-
ively different from those of various water deficits, although
water deficits exacerbate the development of PD symptoms
and, at times, localized water deficits are possible (Gambetta
et al., 2007; Choat et al., 2009). Secondly, multiple studies
showed that the overall proportion of vessels occluded by Xf
and associated gums was very low (Hopkins, 1989; Newman
et al., 2003; Alves et al., 2004; Krell et al., 2006), and was
unlikely to induce water deficit. Finally, more recently,
Gambetta et al. (2007) demonstrated, via a novel, robust quan-
titative PCR (qPCR) assay to quantify Xf in planta, that there
was very little correlation between Xf concentrations in leaves
and symptom severity. That study showed that the Xf popu-
lations were patchily distributed across whole leaves, and
that leaves could exhibit severe leaf scorch symptoms with
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Annals of Botany 108: 73–85, 2011
doi:10.1093/aob/mcr106, available online at www.aob.oxfordjournals.org
low bacterial concentrations. An alternative to the occlusion
hypothesis is that disease symptoms result from a systemic
plant response to the presence of the gums and tyloses
(Stevenson et al., 2004), via a higher rate of ethylene pro-
duction in the infected leaves (Sun et al., 2006, 2007;
Perez-Donoso et al., 2007). Phytotoxins and programmed
cell death are also considered in the induction of PD symptoms
(Gilchrist and Lincoln, 2006; Reddy et al., 2007).
Pierce’s disease is presently controlled in California by redu-
cing vector populations through habitat management (Purcell
of Vitis vinifera cultivars resistant or tolerant to Xf remains an
active area of research. Vitis vinifera cultivars vary in their sus-
ceptibilities to PD, while other Vitis species are tolerant of Xf
colonization (Purcell, 1981; Raju and Goheen, 1981; Hopkins
and Thompson, 1984; Fry and Milholland, 1990; Krivanek
lessly within numerous plant species (Freitag, 1951). Most of
those alternative hosts of Xf do not show PD symptoms,
despite allowing bacterial proliferation and movements
beyond the inoculation point, although at much lower levels
than in grapevine (Hill and Purcell, 1995; Purcell and
Saunders, 1999; Costa et al., 2004; Baumgartner et al., 2005;
Wistrom and Purcell, 2005). However, most of these studies
recorded plant longevity, the appearance and severity of the
symptoms, and the Xf populations in the plant. More detailed
investigations of the nature of the plant–pathogen interaction
are necessary to identify possible reasons for differences in
plant susceptibility to Xf colonization. Since Xf is a xylem-
limited bacterium, further investigation of the xylem structure
in resistant and susceptible plants is needed. For example,
since Xf must digest the pit membrane that separates vessels
(Newman et al., 2003; Scarpari et al., 2003), shorter and nar-
rower vessels, and internal separation of xylem tissue by rays
Conversely, the presence of long open conduits would allow
fairly quick movement of Xf over long distances (Chatelet
et al., 2006; Thorne et al., 2006b).
There are three known xylem-limited bacterial species:
Xylella fastidiosa, Clavibacter xyli and Pseudomonas syzygii.
Xylella fastidiosa is the most economically important and the
most studied; C. xyli subsp. xyli is the agent of ratoon stunting
disease of sugar cane (Davis et al., 1980); C. xyli subsp. cyno-
dontis causes stunting disease of Bermuda grass (Davis and
Augustin, 1984); and P. syzygii causes Sumatra disease of
cloves (Bennett et al., 1985; Roberts et al., 1990). Similar to
Xf, the other two xylem-limited bacteria are found in a multi-
tude of plant hosts (Kamiunten and Wakimoto, 1976; Davis
et al., 1983; Roberts et al., 1990). However, it is not known
how these bacteria spread within the xylem system, and how
the plants respond to this invasion. The same applies for
exogenous bacteria that can also invade xylem tissues, such
as Erwinia stewartii, the agent of corn wilt (Pepper, 1967),
Xylophilus ampelinus (ex Xanthomonas ampelina), the agent
of grapevine bacterial necrosis (Panagopoulos, 1969; Grall
and Manceau, 2003), or Ralstonia solanacearum, responsible
for bacterial wilt of solanaceous crop plants (Hayward,
1991). A better understanding of the propagation of these bac-
teria within the xylem may reveal multiple methods of bac-
terial movement and of plant defences.
General grapevine anatomy and its vascular tissue have been
investigated by various researchers (Pratt, 1974; Mullins et al.,
1992; Gerrath et al., 2001). Several anatomical aspects related
to PD development have also been documented (Esau, 1948;
Hopkins, 1976; Mollenhauer and Hopkins, 1976; Milholland
et al., 1981; Stevenson et al., 2004, 2005; Chatelet et al.
2006; Thorne et al., 2006b). However, generally these
studies were limited to a few plants and/or a few xylemic char-
acters. The objective of this study was to expand the scope of
these studies by examining the xylem structure of grapevines
with different susceptibilities to Xf infection, as well as
the xylem structure of non-grape plant species that do and
do not support the movement of Xf to determine if
anatomical differences might explain some of the differences
in susceptibility to Xf.
MATERIALS AND METHODS
Grapevines (Vitis vinifera, varieties ‘Sylvaner’, ‘Cabernet
Sauvignon’, ‘Pinot Noir’ and ‘Chardonnay’) were propagated
from seed or cuttings. These four cultivars are tolerant
(‘Sylvaner’), susceptible (‘Cabernet Sauvignon’) and highly
susceptible (‘Pinot Noir’ and ‘Chardonnay’) to PD (Purcell,
1977, 1980; Raju and Goheen, 1981; Hopkins and Purcell,
2002; Krivanek et al., 2005). Twelve plant species that varied
in their ability to support Xf were chosen for anatomical charac-
and Purcell, 2005). Species supporting Xf movement included
(Apocynaceae), Citrus sinensis (Rutaceae), Prunus amygdalus
derae (Solanaceae). Species that supported limited Xf move-
ment included Umbellularia californica (Lauraceae), Alnus
rhombifolia (Betulaceae), Datura meteloides (Solanaceae),
Eucalyptus globulus (Myrtaceae), Artemisia douglasiana
(Asteraceae) and Chenopodium quinoa (Amaranthaceae).
Ipomoea purpurea, H. annuus, D. meteloides, C. quinoa and
N. sanderae were grown from seeds (Lake Valley Seed,
Boulder, CO, and Botanical Interests, Inc., Broomfield, CO,
USA), while cuttings of other plants were collected on the UC
Davis campus. All greenhouse-grown plants were grown in
3.78 L pots containing U.C. Davis Mix (equal parts peat moss,
coarse sand and nitrolysed redwood sawdust) in the greenhouse
(30/20+3 8C; 40/70+10 % relative humidity; natural light),
and watered daily with modified Hoagland’s nutrient solution
(Wada et al., 2008). Grapevines, A. douglasiana, I. purpurea,
H. annuus, D. meteloides, C. quinoa and N. sanderae were
trained vertically as a single cane or stem, with lateral branches
removed. Grape canes and other plants were approx. 2–4
months old when sampled, and had intact terminal tips.
Air movement in petioles and stems
One petiole and attached leaf each from nodes 3, 7, 12, 16
and 20 were carefully removed from stems at the base of the
petioles under water. Petioles were attached to a rubber tube
under water, and air was infused into the cut ends at a pressure
of 35 kPa controlled by a pressure regulator. This pressure is 7
Chatelet et al. — Xylem structure survey and passive bacterial spread 74
% that of the lowest air-seeding threshold reported for grape-
vine (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 seconds under
pressure, the major veins and their secondary veins were cut
with a razor blade every few millimetres starting from the
margin, and moving toward 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 and the distance from the leaf margin to the
loading point were measured. Leaf lengths were variable
between species, ranging from 20 to 360 mm. In order to
compare plant species, the farthest position of bubble appear-
ance was reported as a percentage of the total length of their
leaves. Air injection in leaves was replicated five times for
each selected node for both grapes and alternative hosts.
Data are expressed as calculated means and their standard
error (n ¼ 5). Statistical analysis was performed using
analyis of variance (ANOVA). P, 0.05 was considered stat-
For stems, plants were brought to the lab and the stems were
cut under water, connected to a plastic tube and pressurized air
was pushed into the stem base at 35 kPa. Starting from the
stem apex, the primary veins of each leaf were cut as pre-
viously described until a stream of air bubbles appeared. If
air did not appear in the vein, the leaf lamina was separated
from the petiole below the lamina/petiole junction. Air
exiting at the apical end of the petiole signified that the
lamina/petiole junction was blocking the air. 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. The distance
from the loading point in the stem to the node where air
appeared in leaves was measured, and was calculated as a per-
centage of the total length of the stem. Finally the stem was cut
every few millimetres starting from the apex toward the base
until a stream of air bubble appeared at the cut, and the dis-
tance from the loading point to the appearance of the bubble
was measured. The stem lengths were very variable, ranging
from 6 to 240 cm. Therefore, for comparison, the farthest pos-
ition attained by the air in the stem of every species was cal-
culated as a percentage of the total length of their stem. Air
was injected in five different stems for both grapes and alterna-
tive hosts. Data are expressed as calculated means and their
standard error (n ¼ 5) and the statistical analysis was per-
formed using ANOVA.
Vessel length distribution in stems and petioles
Grape canes and other plants were approx. 2 months old
when sampled, and had intact terminal tips. Plants were kept
in a cool, dark location overnight prior to measurement, and
thoroughly watered to decrease transpiration and xylem
tension. The length, diameter and number of nodes were
measured for each stem or cane. Vessel length was measured
according to the technique of Ewers and Fisher (1989), modi-
fied to infuse paint under pressure. Stems were cut underwater,
and submerged until attached to the paint infusion apparatus.
Latex paint (ACE Royal High Gloss Clean Red Enamel;
ACE Hardware, Oak Brook, IL, USA) was diluted 1:300 in
deionized water, filtered through Whatman #1 filter paper,
and degassed prior to use. A modified stainless steel sprayer
(B and G Equipment Co., Jackson, GA, USA) was filled
with 3.5 L of diluted paint and pressurized with compressed
air to 100 kPa, so the paint solution flowed out of the
sprayer and into clear plastic tubing. Air was removed from
the system prior to attachment of the stem to the tube. Stems
were forced into tubing underwater and secured with wire.
All leaves were removed and the stem was placed inside a
plastic bag. Paint was infused into the stem for approx. 96 h,
until liquid ceased emerging from the distal end. Paint infusion
in leaves and stems was replicated five times for both grapes
and alternative hosts. Vessel length distribution was calculated
using Excel (Microsoft, Redmond, WA, USA), and statistical
analysis was performed using ANOVA.
Paint particles filled vessels, but were stopped by pit mem-
branes, indicating one continuous xylem vessel element.
Particles in the latex paint solution were larger than 0.22 mm
in diameter, since they did not pass through a sterilizing
filter (Millipore Corporation, Billerica, MA, USA). Pigment
and additive particles in latex paint measured between 0.3
and 7.5 mm in diameter (Croll, 2002), but pores in vessel pit
membranes measured between 0.005 and 0.17 mm, depending
on the plant species (Siau, 1984).
In a first experiment, vessel lengths were calculated for
stems of each plant species or grapevine cultivar. The paint
solution was loaded into stems as described above. Thin
(?1 mm) cross-sections were cut by hand with a Platinum
Injector razor blade (Longs Corporation, Walnut Creek, CA,
USA) every 1 cm and placed on a glass slide in 50 % glycerol.
Sections were photographed with an Olympus Vanox-AHBT
(Olympus America, Melville, NY, USA) compound light
microscope linked to a Pixera 600ES digital camera. Vessels
with paint were counted from the digital image of each section.
In a second experiment, vessel length distribution was cal-
culated for leaves from node 3, 7, 12, 16 and 20 from the
stem apex with the same technique. The leaves were excised
under water and their petiole was connected to silicone
tubing linked to the reservoir filled with the paint solution.
The leaves were kept under water and were infused with the
paint suspension at a pressure of 35 kPa until the paint solution
stopped moving. At the end of the infusion time, thin freehand
cross-sections were cut every 5 mm with a razor blade. The
petiole and major veins were sectioned every 5 mm starting
from the paint infusion point, and progressing toward the
margin of the leaf. The vessels with paint were counted in
each section and vessel length distribution was calculated as
described for stems.
Dental paste and a 0.40 mm hypodermic needle were used
to grossly imitate the wounds left by a feeding sharpshooter
(Leopold et al., 2003). Five stems of similar age from each
species mentioned above were selected to evaluate the tylose
formation after wounding. A drop of dental paste (CutterSil
Mucosa, Heraeus Kulzer, Inc., Armonk, NY, USA) was
placed above the first mature leaf proximal to the shoot
apex. The hypodermic needle filled with dental paste was
driven four times through the dental paste drop, a few milli-
metres into the stem xylem. The dental paste sealed the
Chatelet et al. — Xylem structure survey and passive bacterial spread75
wound upon withdrawing the needle. Stem segments were col-
lected at 0, 1, 3 and 6 d after wounding, and the presence of
tyloses in the vessels was observed in cross-sections made
within the wounded area, 5, 10 and 100 mm above the
wound. For each distance, the proportion of vessels with
tyloses was calculated.
Vessel diameter distribution at the base of the stem and petiole
Five stems from each species were cross-sectioned at the
base. For each stem, five zones of the xylem were randomly
selected and the number and diameter of the vessels within
each zone were counted and measured. From the same
plants, five mature leaves were collected and cross-sectioned
at the base of the petiole. For each section, all the vessels
were counted and their diameter was measured. The diameter
distributions of the vessels, at the base of the stem, and in the
petiole of a mature leaf, were calculated for each species.
Anatomical comparisons among grape cultivars and other plant
Segments of 1 cm from the base of the stem and petiole
were sectioned with a sliding microtome (AO-860, American
Optical, Buffalo, NY, USA) in transverse, tangential and
radial planes with a section thickness of 25 mm. The sections
were dehydrated through an ethanol series (Ruzin, 1999). Each
step lasted 1 h, except for the 4 h step in 50 % ethanol with 1%
safranin O, and the 1 min 95 % ethanol step with 0.5% fast
green FCF. Sections were further dehydrated and cleared in
an ethanol–xylene series (2:1, 1:1, 1:2) followed by two
xylene rinses of 10 min each. Sections were mounted with
coverslips in Permount (Fisher Scientific, Fair Lawn, NJ,
USA), and photographed with a Olympus Vanox-AHBT
(Olympus America, Melville, NY, USA) compound light
microscope linked to a Pixera 600ES digital camera. The
total numbers of vessels, bundles, rays and paratracheal par-
enchyma cells were counted. Data are expressed as calculated
means and their standard error (n ¼ 5). Statistical analysis was
performed using ANOVA (P , 0.05 was considered statisti-
Air movement in the leaves
The farthest distance travelled by air in the plant species tested
ranged from 20 to 86 % of the total length of the vascular path
from the petiole base to individual leaf vein endings (Table 1).
This range indicated that the lengths of open, continuous
xylem vessels (conduits) are highly variable, and that it is
possible for bacteria to move passively from the base of the
petiole toward the tip of the leaves.
In grapevine, air travelled up to 70 % of the leaf length in
Sauvignon’ and highly susceptible ‘Pinot Noir’. In highly sus-
ceptible ‘Chardonnay’, air only travelled 47–60 % of the leaf
length. Overall, air travelled .50 % of the total leaf length in
species allowing Xf movement, compared with 30–40% of the
leaf length in plants limiting Xf movement. Although V. major
is able to support extensive Xf movement, air only moved into
the firstthirdof itsleaves.
movement-limiting hosts D. meteloides and C. quinoa, air
moved as far as in leaves of non-limiting species. In sum,
the lengths of the open xylem conduits did not correspond
with the tolerant/susceptible category in grapevine and the
observed ability of alternative hosts to limit Xf movement.
TABLE 1. Farthest position reached by air in leaf primary veins expressed as a percentage of the total distance from the beginning
of the petiole to the margin of the leaf
Node 3Node 7 Node 12 Node 16Node 20
V. vinifera ‘Sylvaner’
V. vinifera ‘Cabernet Sauvignon’
V. vinifera ‘Pinot Noir’
V. vinifera ‘Chardonnay’
Measurements were made in leaves of four grapevine varieties as well as in plant species that do and do not support the movement of Xylella fastidiosa
beyond the inoculation site.
Data are average means (s.e.) of five leaves for each node. Values in columns with different letters are significantly different at the 95% confidence level
according to ANOVA (Turkey–Kramer test).
Chatelet et al. — Xylem structure survey and passive bacterial spread 76
Air movement within stems and from stem to leaves
The farthest position travelled by air within the stem of the
different species was also highly variable, ranging from 30 %
to almost 100 % of the stem length (Fig. 1). The farthest pos-
ition travelled by air in the stem before it branched off into a
leaf was also very variable, ranging from 10 % to almost 100
% of the stem length. In all species, air travelled up the stem
beyond the last detection point in the leaves. In the four grape-
vine varieties tested, air travelled 25–30 % of the length of the
stem before going into a leaf, but was found up to about 60 %
of the total length of the stems. In alternative hosts, the farthest
position that air travelled in the stem before entering a leaf
depended upon the plant species tested, ranging from 10–15
% in I. purpurea and C. quinoa to almost 100 % of the total
length of the stem in C. sinensis, P. amygdalus and
C. quinoa. Likewise, the farthest position attained by air in
the stem alone was very variable, ranging from 30 % in
I. purpurea and A. douglasiana to almost 100 % of the total
length of the stem in V. major, C. sinensis, P. amygdalus,
U. californica and C. quinoa. Again, the length of the open
xylem conduits and the connections between stem and leaves
did not correspond to any significant difference between toler-
ant/susceptible grapevine and between alternative host groups.
Leaf and stem vessel length distribution
Vessel length distributions were similar in leaves of the four
grapevine varieties tested. Most of the vessels were ,18 cm
and roughly 50 % of them were shorter than 3 cm (Fig. 2A).
These are similar to vessel lengths in alternative hosts
(Fig. 2B, C), where at least 40 % of the vessels in alternative
Xf hosts were ,3 cm, except for I. purpurea and C. quinoa.
The longest vessels, 24–27 cm, were found in species support-
ing Xf movement: I. purpurea, H. annuus and N. sanderae.
In grapevine stems, most of the vessels were ,50 cm, with
55 % of them being ,6 cm long and the longest measuring
about 1 m (Fig. 2D). In contrast, in all alternative hosts,
except C. sinensis, most of the vessels of the stems were
shorter than 27 cm, with 30–80 % of them being ,3 cm
long (Fig. 2E, F). The vessel length distributions in stems of
alternative hosts were similar. The longest vessel measured
in alternative hosts was about 30 cm (E. globulus).
Petiole and stem vessel diameter distribution
Vessel diameters were similar in petioles of the four grape-
vine varieties tested, with about 70 % of the vessels ranging
from 10 to 45 mm (Fig. 3A). Alternative hosts (Fig. 3B, C)
had smaller vessel diameters, mostly ,25 mm, with the excep-
tion of H. annuus, whose vessels were between 30 and 55 mm,
and D. meteloides, whose vessels were between 15 and 45 mm
Grapevine stem vessel diameters ranged from 150 to
400 mm, except for ‘Sylvaner’, which had slightly smaller
vessels, between 80 and 250 mm (Fig. 3D). In contrast, the
vessel diameters at the base of the stem of the alternative
hosts were similar to vessel diameters in petioles (Fig. 3E,
F), ranging mostly from 10 to 35 mm, except for the vessels
from H. annuus and D. meteloides whose diameter was
between 40 and 65 mm.
There were no major differences in tylose production
between grapevine varieties and between alternative hosts
(Fig. 4A–C) except for P. amygdalus, V. major and
A. rhombifolia that did not produce any tyloses in response
to wounding. In all species, the production of tyloses was
greatest near the wound. Six days after wounding, between 5
and 15 % of the vessels had tyloses at the wounding site and
the amount of tyloses decreased as the distance from the
wound increased, until the vessels became eventually free of
U. californica past 10 mm from the wound and, in all grape-
vine varieties and A. douglasiana, the vessels became free of
tylose at 100 mm. In all remaining species, some tyloses
were still present at 100 mm.
Anatomical comparison of stem and leaf cross-sections
There were no significant differences in the total vessel
number, the proportion of short vessels or the longest vessels
greenhouse-grown canes of similar length, age and diameter
(Table 2). The average grape cane measured 240 cm, with
24–36 nodes. The only significant xylem anatomy difference
noted among grape cultivars was the number of rays in
‘Sylvaner’, with about 20 % more rays compared with the
other grapevine varieties (Table 2). The average longest
Percentage of total length of stem
in a leaf
SpeciesGrapevine Limited movement
FIG. 1. Farthest position attained by air infused into stems (white) and into
stems with adjacent leaves (black), expressed as a percentage of the total
length of the stem. Data are the mean + s.e., n ¼ 5 stems.
Chatelet et al. — Xylem structure survey and passive bacterial spread77
vessel measured by paint and air infusion was 72 cm, but most
of the vessels were ,15 cm long in all cultivars. Stems from
the alternative host plants were between 6 and 150 cm long,
depending on the species. The longest vessel measured in
any alternative host was 28 cm long, in E. globulus, and the
percentage of vessels ,3 cm long ranged from 21 to 84 %.
We observed no discernible differences in vessel density,
vessel length or number of rays between alternative hosts limit-
ing and allowing Xf movement. Likewise, when the xylem
structure of the petiole was compared, there were no significant
differences among the four grapevine varieties or between the
alternative hosts (Table 3).
This was also true for the paratracheal parenchyma cells
(Table 4). With the exception of V. major, P. amygdalus and
A. rhombifolia, where they were absent, paratracheal parench-
yma cells were scanty to vasicentric. In the four grapevine cul-
tivars, vessels had 6–7 paratracheal parenchyma cells, whereas
2–3 cells were present in most of the alternative hosts.
Helianthus annuus and E. globulus were the exceptions, with
13.5 and ten cells, respectively. In longitudinal section,
% of paint-filled vessels
% of paint-filled vessels
% of paint-filled vessels
Length class (cm)
Length class (cm)
FIG. 2. Vessel length distribution in mature leaves (A–C) and stems (D–F) of grapevines and alternative hosts of Xylella fastidiosa. For each length class, the
number of paint-infused vessels was calculated as a percentage of the total number of painted vessels at the base of the petiole or stem. n ¼ 5 leaves, 5 stems.
Chatelet et al. — Xylem structure survey and passive bacterial spread 78
strands of paratracheal parenchyma cells had up to ten cells for
grapevines, while it was slightly less for the alternative
species, mostly 1–4 cells.
This study examined four varieties of grapes with different
susceptibilities to Xf infection, and 12 alternative host plant
species categorized into two groups: those that allow Xf move-
ment, and those that limit Xf movement, to determine whether
gross xylem physical characteristics had a role in Xf coloniza-
tion. The results showed that there were few or minor differ-
ences among the grapevine
alternative hosts. There was only one grape varietal difference:
the stem of the tolerant variety ‘Sylvaner’ had smaller vessel
diameters and 20 % more parenchyma rays than the other
three varieties. Among the alternative hosts, the xylem in
leaves, and the xylem connecting the stem to the leaves was
slightly more open in the hosts, allowing more bacterial move-
ment as compared with hosts in which bacterial movement was
% of vessel at the petiole base
% of vessel at the petiole base
% of vessel at the petiole base
Diameter class (mm)Diameter class (mm)
FIG. 3. Vessel diameter distribution at the base of the petiole of mature leaves (A–C) and stems (A–F) from grapevines and alternative host species of Xylella
fastidiosa. n ¼ 5 leaves, 5 stems.
Chatelet et al. — Xylem structure survey and passive bacterial spread79
limited. Therefore, the results show that gross xylem structure
and organization are not responsible for genotypic differences
in susceptibility to Xf infection and PD.
Earlier studies on different plant species showed that par-
ticle movement was limited by the frequency of vessel
endings, especially at the stem–leaf junction, where most
vessels were thought to end, except for the few vessels cross-
ing the junction (Larson and Isebrands, 1978; Wiebe et al.,
1984; Andre ´, 1999, 2002; Martre et al., 2000; Tyree and
thought to act as a safety mechanism against embolism
(Zimmermann, 1983; Aloni and Griffith, 1991; Tyree and
Ewers, 1991; Choat et al., 2005) and bacterial movement
(Zimmermann, 1983; Tarbah and Goodman, 1987; Bove ´ and
Garnier, 2002). Indeed the tracheids and short vessels in
these junctions would stop the propagation of air from the
leaf to the stem, or vice versa, and are thought to facilitate
the shedding of embolized leaves (Tyree et al., 1993; Rood
et al., 2000). Although the long open conduits described
here would allow free movement of air into the leaf blade if
they became embolized, this 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
unimpeded from stem to leaves or leaves to stems via these
open conduits could have a considerably greater impact on
pathogenesis by allowing the bacteria to move rapidly through-
out the infected plant, particularly because Xf can degrade and
traverse pit membranes (Roper et al., 2007; Pe ´rez-Donoso
et al., 2010) unlike inert gas embolisms.
The presence of long xylem vessel conduits connecting
leaves to the stem several internodes below the leaf in all the
species we examined (Chatelet et al. 2006; Thorne et al.
2006b; this study) suggests that most plant species with
vessels could have such characteristics. Since bacterial infec-
tion affects many different species, an interesting question
arises as to how such paths might be exploited by invading
bacteria. Thorne et al. (2006b) and Chatelet et al. (2006)
speculated on the importance of these conduits for the
0 1020 304050 60
Distance from the wound (mm)
70 8090 100 110
Percentage of vessels partially
filled with tyloses
Percentage of vessels partially
filled with tyloses
Percentage of vessels partially
filled with tyloses
FIG. 4. Percentage of stem vessel with tyloses distal from a simulated sharp-
shooter feeding site, 6 d after wounding with a needle. n ¼ 5 stems.
TABLE 2. Anatomical comparisons of stems of similar age from
four grape cultivars and the alternative host species of Xylella
Data are average means (s.e.) of five stems. Values in columns with
different letters are significantly different at the 95% confidence level
according to ANOVA (Turkey–Kramer test).
Chatelet et al. — Xylem structure survey and passive bacterial spread80
passive systemic spread of pathogens via the xylem and their
role in the development of diseases such as PD in grapevine.
Similarly, it was suggested that the inability to restrict bacterial
movement by the xylem was a key determinant of the appear-
ance of symptoms (Fry and Milholland, 1990; Krivanek and
Walker, 2005). However, in this study, the interconnectedness
of the organs and the vessel length distribution profiles were
similar between plant varieties and species that have been
reported to differ in susceptibility to PD, and to limit and
not limit Xf movement. These observations indicate that sus-
ceptibility to PD is not controlled by physical limitations in
xylem organization to bacterial movement and imply that
factors other than maximum open conduit length and vessel
length distribution determine the extent of bacterial movement.
The lack of differences in vessel length distributions or open
paths among the alternative hosts characterized in the literature
as ‘systemic’ and ‘non-systemic’ is interesting. There are at
least two possible implications. First, the characteristics
measured here are not the pertinent ones to ascertain the poten-
tial for Xf movement. However, in our previous studies, exper-
iments tested for open pathways in several ways including
measurements of the movement of light-emitting bacteria,
green fluorescent protein (GFP)–Xf, air and fluorescent
beads, and all produced similar results quantifying long,
open pathways in shoots, petioles and leaf lamina (Chatelet
et al., 2006; Thorne et al., 2006b). Thus, the potential for
passive movement of Xf is probably reflected in the measure-
ments reported here. However, it is clear that the bacterium
is motile (Meng et al., 2005; De La Fuente et al., 2007), and
it may well be that the environment within the xylem of differ-
ent genotypes is important to that motility. Secondly, this
classification did not account for the difference in Xf strain
specificity with the plant host. Strains of Xf differ greatly in
their abilities to move and colonize various host plant
TABLE 3. Anatomical comparisons of petioles of mature leaves from four grape cultivars and 12 alternative host plant species of
No. of vessels at cane/petiole baseVessel density% vessel ≤1 cm Longest vessel No. of rays/petiole base
V. vinifera ‘Sylvaner’
V. vinifera ‘Cabernet Sauvignon’
V. vinifera ‘Pinot Noir’
V. vinifera ‘Chardonnay’
Data are average means (s.e.) of five stems. Values in columns with different letters are significantly different at the 9 5 % confidence level according to
ANOVA (Turkey–Kramer test).
TABLE 4. Paratracheal parenchyma cells in the stem of the
grape cultivars and the alternative host species of Xylella
Up to 146.5 (0.5) Up to 10
Up to 186.7 (0.6) Up to 8
Up to 156.8 (0.4) Up to 9
Up to 13 6.4 (0.5)Up to 8
Up to 5 2.9 (0.5) 2–3
Up to 53.0 (0.2)1–2
Up to 3
Up to 52.9 (0.2)2–4
Up to 5 2.7 (0.2)2–4
Up to 15 10.0 (0.4)Up to 8
A. douglasinaUp to 4 2.6 (0.1)1–2
C. quinoaUp to 42.6 (0.2) 1–2
Data are mean (s.e.), n ¼ 5 stems.
Chatelet et al. — Xylem structure survey and passive bacterial spread81
species systemically. For example, oleander leaf scorch strains
of Xf colonized and caused disease in oleander (Neerium
oleander) but not in grape, whereas the reverse was true for
grape strains (Purcell et al., 1999). Almond strains of Xf
were weakly systemic and non-pathogenic in grape, but
grape strains were systemic and pathogenic to grape and
almond (Almeida et al., 2003). However, all the alternative
hosts in this study were chosen from previous work that had
used the Xf strain specific to PD, thereby eliminating genetic
differences in virulence and intra-plant movements. Thirdly,
there may have been false negatives in earlier work using
theless sensitive enzyme-linked
(ELISA) compared with a more sensitive qPCR assay,
leading to ‘non-systemic’ interpretation for some species
(Gambetta et al., 2007). This possibility raises the further
question of the role of bacterial distribution and population
in the development of PD.
Although high bacterial populations were previously
thought necessary for symptom development (Hopkins,
1985; Fry and Milholland, 1990; Hill and Purcell, 1995;
Krivanek and Walker, 2005), that is evidently not the case
for Xf in leaves (Alves et al., 2004; Krell et al., 2006;
Gambetta et al., 2007). Thus, the movement of small
amounts of bacteria through a limited number of open conduits
may be important in disease development. The presence of Xf
is patchy in infected susceptible plants (Newman et al., 2003;
Krell et al., 2006; Gambetta et al., 2007), and not correlated
with symptoms (Krell et al., 2006; Gambetta et al., 2007).
In the present study, there were no significant differences in
open pathways between susceptible and tolerant species.
These results point toward a systemic response of susceptible
grapevines to the presence of Xf in its xylem sap, possibly
involving ethylene (Hopkins 1985; Thorne et al., 2006a;
Pe ´rez-Donoso et al., 2007) and programmed cell death
(Gilchrist and Lincoln, 2006).
Sun et al. (2007) demonstrated that ethylene is necessary for
tylose development in wounded grapevine stems. Tyloses are
produced by paratracheal parenchyma cells, outgrowing into
the vessel lumen via vessel–parenchyma pits and eventually
occluding the vessel (Esau, 1977). They occur naturally in a
wide range of species and can be induced by wounding and
pathogen infection (Wallis and Truter, 1978; Beckman and
Talboys, 1981; Biggs, 1987; Bonsen and Kuc ˇera, 1990;
Cochard and Tyree, 1990; Pearce, 1991; Saitoh et al., 1993;
Schmitt and Liese, 1993; Clerivet et al., 2000; Salleo et al.,
2002; Sun et al., 2006). These vascular occlusions that
develop in response to infection are often thought to be
involved in the isolation of pathogens for disease defence. In
grapevines infected with Xf, tyloses are observed in primary
Mollenhauer, 1975; Stevenson et al., 2004). However, their
role in PD of grapevine is not clear as some studies reported
their frequency in PD-infected grapevines to be greater in
greater in susceptible genotypes (Krivanek et al., 2005) and
unrelated to the susceptibility of the grape genotype (Fry and
Milholland, 1990). Our results showed that the amount of
tylose produced in response to needle wounding as well as
the type and number of paratracheal parenchyma cells was
similar among the grapevine varieties and among the
alternative hosts, regardless of their ability to limit Xf move-
ment. This is in accordance with the low fraction of vessels
being occluded observed in PD studies (Hopkins, 1989;
Newman et al., 2003; Alves et al., 2004; Krell et al., 2006)
and the wounding study by Sun et al. (2006). These results
provide further evidence that tyloses may be unable to limit
Xf movement and spread in tolerant plants. The mechanisms
for Xf tolerance observed in many species are still not resolved.
The only significant differences between the tolerant and
susceptible grapevines in this study were the smaller vessel
diameters and the higher number of rays in the stem of the tol-
erant ‘Sylvaner’ cultivar compared with the more susceptible
grapevine varieties. A smaller vessel diameter suggests that
Xf would self-aggregate more easily, possibly leading to a
more rapid maturation of biofilms. However, the role of
(Hopkins, 1989; De Souza et al., 2003, 2004; Koide et al.,
2004; Newman et al., 2004; Guilhabert and Kirkpatrick,
2005; Feil et al., 2007; Chatterjee et al., 2008a, b). In addition
to narrower vessels, the higher number of rays in the tolerant
cultivar could also slow down the bacterial infection through
active secretion of defence chemical compounds. Several
studies suggested that the xylem sap composition influences
the expression of genes involved in growth, aggregation,
attachment and virulence of Xf (Bi et al., 2007; Reddy et al.,
2007; Zaini et al., 2009; Shi et al., 2010). Recently, Basha
et al. (2010) showed differences in the xylem sap composition
(amino acids, sugars and proteins) of susceptible and tolerant
Vitis species. Although more work is needed to clarify those
differences, a higher number of parenchyma cells around the
xylem could certainly facilitate the secretion of antimicrobial
compounds in the stem xylem of the tolerant Vitis cultivar
as a response to infection by Xf. In addition, the higher
number of rays in the tolerant cultivar suggests that rays
could also play a role in limiting the lateral spread of the bac-
teria. Rays are composed of one to several layers of dense
living ray parenchyma cells, without tracheids or vessel
elements (Esau, 1977), and they separate the water-conducting
xylem into longitudinal zones. These rays would present a
barrier to bacteria moving exclusively in the xylem. The
lateral spread of bacteria from one zone to another can only
occur when vessels bridge the two zones, either by crossing
the ray or where a ray ends and two contiguous zones
merge. In any case, lateral movement would be limited by
the degree of connectivity of the vessels within a zone as bac-
teria move from vessel to vessel by digesting the pit
The work by Zimmermann and Brown (1971) and
Newbanks et al. (1983) demonstrated that there are many
lateral pit field connections between vessels along their
length. The connectivity of vessels is presumed to be a key
determinant in the vulnerability of plants to the spread of
embolisms between vessels (Wheeler et al., 2005). Indeed
the more surface area of pit membrane existing between
vessels, the greater the chance of having a large pore that
will allow the movement of gas to the next vessel. A similar
logic could be applied to the movement of Xf in the xylem
of grapevine genotypes. The xylem of susceptible grapevines
could have greater intervessel pitting, thus allowing Xf
access to adjacent vessels via pit membranes, while the
development is unclear
Chatelet et al. — Xylem structure survey and passive bacterial spread82
xylem of tolerant grapevine would have more isolated vessels,
preventing bacterial movement to other vessels. Differences in
pit structure could also produce a difference between tolerance
and susceptibility by presenting pit membranes that are suscep-
tible to different degrees to digestion and breaching.
Our investigation of the xylem structure of PD-susceptible
and tolerant grapevines, and of alternative host species that
allow or restrict Xf movement produced no evidence that the
gross xylem anatomy (vessel length or number, or organ con-
nectivity) was responsible for tolerance or for limiting bac-
terial movement. However, the narrower vessels and higher
numbers of parenchyma rays found in tolerant compared
with susceptible plants imply that bacterial movement might
be restricted by more subtle differences in vessel morphology.
More subtle differences in the vessel network, such as the
degree of intervessel pitting or the thickness of the pit mem-
brane, coupled with the secretion of defensive chemical com-
pounds may play a role in limiting Xf movement and disease
development. Another important result of this study is the
observation of long, open conduits in all examined species.
The role of such conduits in bacterial colonization of plants
is largely unstudied. Future analyses of the role of the xylem
in bacterial movement should examine vessel overlapping,
spatial organization of the pit fields, and pit membrane thick-
ness and porosity.
We thank U.C. Davis Foundation Plant Materials Service for
grape cuttings; and D. Chiniquy, S. Ching, S. Hernandez,
R. Zintzun and J. Fei for technical assistance. This project
was funded by a grant from the California Department of
Food and Agriculture (Contract 01-0712).
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