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Presymptomatic multiplication of Xanthomonas campestris pv. vesicatoria on the surface
of pepper leaves
EDNASHARON,
YOAVBASHAN,'YAACOV
OKON,A N D YIGALHENIS
Department of Plant Pathology and Microbiology, Faculty of Agricrtlture, The Hebrew University of Jerusalem,
P.O. B0.x 12, Rehovot 76 100, Israel
Received August 26, 1981
SHARON,
E., Y. BASHAN,
Y. OKON,and Y. HENIS.1982. Presymptomatic multiplication of Xanthornonas campestris pv.
vesicatoria on the surface of pepper leaves. Can. J. Bot. 60: 1041- 1045.
Xanthomonas vesicatoria infection of pepper leaves was monitored by scanning electron and light microscopy. During
incubation the bacteria became located in the intercellular spaces between the mesophyll cells. The primary infection sites were
the vein areas. Xanthomonas vesicatoria reach high popula60n levels on the leaf surface but usually not in the stomata. Necrosis
was first observed microscopically 120 h after inoculation near the leaf veins. Bacteria were detected in infected tissue but not in
necrotic tissue.
SHARON,
E., Y. BASHAN,
Y. OKONet Y. HENIS.1982. Presymptomatic multiplication of Xanthomonas campestris pv.
vesicatoria on the surface of pepper leaves. Can. J. Bot. 60: 1041- 1045.
L'infection des feuilles de piment par le Xanthomonas vesicatoria a CtC suivie par la microscopie photonique et Clectronique i
balayage. Au cours de l'incubation, les bactiries viennent se longer dans les espaces intercellulaires du mCsophylle. Les regions
des nervures sont les sites d'infection primaires. Le X. vesicatoria peut former de fortes populations sur la surface de la feuille,
mais gCnCralement pas dans les stomates. La nCcrose est d'abord observke microscopiquement 120 h aprks l'inoculation prks des
nervures de la feuille. Les bactCries sont dCtectCes dans les tissus infect&, mais non dans les tissus nCcrosCs.
[Traduit par le journal]
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Introduction
~anthomonasvesicatoria (Doidge) Dowson causes
severe damage to crops of pepper in Israel (18). The
pathogen attacks both pepper and tomato and produces
typical leaf symptoms on pepper leaves, e.g., small (3 to
5 mm diameter) pale brown spots with a distinct yellow
halo (6). In artificial inoculation there is a period of 5 to 9
days between inoculation and the appearance of disease
symptoms (5).
Many ultrastructural studies of the interaction between phytopathogenic bacteria and plant cells have
been reported using transmission electron microscopy
(9, 15, 16, 17). Most investigators have suggested that
the compatible bacterial pathogen multiplied freely in
the intercellular spaces of host tissue (3, 7 , 8 , 9 , 15, 17).
There are only a few reports, however, that desci-ibe
events occumng on the surface of the infected plant
leaves (3,7,11,14). However, multiplication and tissue
necrosis caused by X. vesicatoria have as yet not been
monitored at the ultrastructural level.
The purpose of this study was to follow the multiplication of X. vesicatoria and the development of
lesions in susceptible pepper plants.
'present address: Division of Plant Pathology, Agriculture
Research Organization, Volcani Centre, Bet Dagan, P.O. Box
6, 50250 Israel.
Materials and methods
Organisms and growth conditions
Xanthomonas vesicatoria (R-3) isolated from infected
pepper plants in Israel were kept at room temperature on
nutrient agar slants (Difco) and transferred weekly to a fresh
medium. To avoid loss of pathogenicity, pepper leaves were
inoculated monthly with isolate R-3 using the enrichment
method of Henis et al. (10) and the pathogen was reisolated
from the leaves and again stored on agar slants. Inocula were
grown on nutrient broth (Difco) in a shaker bath (100
strokes/min) at 30°C for 24 h, centrifuged at 10 000 x g
for 10 min, and resuspended in 0.06 M potassium phosphate
buffer (pH 6.8) to give 0.1 absorbance units at 540 nm in a
Junior I1 Coleman spectrophotometer which corresponded to
10' cells/mL.
Pepper plants (Capsicum annuum) of the susceptible cultivar Mabor were grown in vermiculite and peat (50:50, v/v) in
a phytotron at 27OC (day) and 21°C (night) under a 14-h day
(day light) and were irrigated with Hoagland's solution. Plants
with four to six true leaves were inoculated.
Bacterial inoculation
Inoculation was made using one of the two following
methods: ( a ) eight plants were kept under periodic mist (5 s
mist every 30 min) (4) for 24 h, sprayed with lo4 cells/ml
until runoff and incubated in a mist chamber of approximately
100% relative humidity at 25OC (2); ( b ) 20 detached leaves
were surface-sterilized with commercial 0.5% NaOCl for 3
min. The samples were washed five times with sterile phosphate buffer to remove traces of hypochlorite and then placed
0008-4026/82/07 1041-05$01 .OO/O
01982 National Research Council of Canada/Conseil national de recherches du Canada
CAN. J. BOT. VOL. 60. 1982
ABBREVIATIONS:
B, bacteria; V, vein; S, stomata; N, necrotic tissue; H, apparently healthy tissue.
F I G . 1. SEM of the surface of pepper leaf before inoculation. x 1000. F I G . 2. SEM of X. vesicatoria cells on the surface of
pepper leaf immediately after inoculation. x 2000. F I G . 3. SEM of X . vesicatoria cells in an open stoma. x 4300. F I G . 4. SEM of
X . vesicatoria 48 h after inoculation. X 2000. FIG. 5. Multiplication of X . vesicatoria 48 h after inoculation over the lower areas
of the epidermal cell surface. x 2000.
SHARON ET AL.
1043
FIG. 6. Multiplication of X. vesicatoria 72 h after inoculation along the leaf veins. X 2700. FIG.7. Typical microcolonies of X.
vesicatoria on the leaf surface 120 h after inoculation. X 1600. FIG. 8. First necrosis near a vein 120 h after inoculation. x 180.
FIG. 9. Border line between necrotic area and apparently healthy tissue. x 1000. Insert in Fig. 8 shows the location of Fig. 9.
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CAN. I. BOT. VOL. 60, 1982
(three leaves per Petri dish) on 0.5% water agar (10). Four
areas of the lower surface of each leaf were inoculated with a
) incu2mL suspension of X . vesicatoria cells ( 1 0 4 / m ~ and
bated in a growth chamber under constant light (12 000 lx) at
25 2 2°C.
bacteria seen in the open stomata (Fig. 3). These
observations on the early stages of infection are in
accordance with the hypothesis that upon reaching the
infection site even very small numbers of phytopathogenic bacteria are capable of inducing primary infection
Determination of bacterial population from leaves
(8).
Incubated leaves were placed in 3% sodium hypochlorite for
Between 48 and 100 h following infection, bacteria
5 min, washed three times with 3 L sterile distilled water and were observed randomly dispersed over the leaf surface
homogenized in 20 mL saline solution (8.5 g NaCl/L) in a (Fig. 4), in particular over the lower areas of the
sterile Omnimixer (Sorvall). Suspensions (0.1 mL) were epidermal cell surfaces (Fig. 5). In specific areas,
spread on solid nutrient agar plates supplemented with
such as along the leaf veins, extensive multiplication
150mg/L sodium deoxycholate (NS) to prevent growth of
Gram-positive bacteria. Colonies (CFU) were counted after occurred and the tissue began to collapse (Fig. 6 ) .
After 120 h many microcolonies appeared in isolated
48 h incubation at 30°C. For bacterial counts of the surface
population, leaves were suspended in saline, vigorously patches but the stomata were usually free of bacterial
shaked for 30 min, and counted on the same media as described cells (Fig. 7). A similar pattern of bacterial development
above. Counts of bacteria from leaves represent trends of was observed in both inoculation methods. This finding
populations. No attempt was made to measure absolute is in agreement with that of Leben (12, 13) who regarded
numbers.
X . vesicatoria as an epiphytic bacterium. It seems that
the leaf population of the pathogen can multiply and
Light microscopy and scanning electron tnicroscopy (SEM)
Leaves from differently inoculated plants were examined by survive on the leaf surface without further supply of
SEM at 24-h intervals during the first 6 days after inocula- bacterial cells from the inside of the infected leaf, in
tion. Light microscopy was carried out only 24 h after spite of the fact that the pathogen has the ability to
inoculation. From each treatment 10 samples were prepared multiply inside the leaf tissue (Fig. IOB). Therefore, this
for observation as previously described (1, 2, 4).
bacterium differs from Pseudomonas tomato, another
leaf
pathogen of tomato plants (3), and from XanthoResults and discussion
monas pruni, a leaf pathogen of peach trees (14).
To date, it has generally been recognized that the However, there is still a possibility that bacteria,
natural openings of plants (e.g., stomata, lenticels, observed on the surface of leaves, come from the
hydathodes, trichomes) and wounds are sites of entry internal tissues through the same openings that were
into the leaf by pathogenic bacteria. After penetrating originally used for tissue invasion.
the tissue, rapid multiplication occurs in the intercellular
One hundred and fifty hours after inoculation bacteria
spaces which is followed by the development of disease were concentrated mainly on the leaf veins and necrotic
symptoms (8). Therefore, most studies using different areas (spots) became microscopically apparent at these
ultrastructural methods have concentrated on the events
taking place inside the tissue, rather than on the leaf
I
I
I
I
I
I_
surface.
@
In our study we found bacterial cells neither on
uninoculated leaves nor inside their tissue using either lL 7light microscopy or SEM. Bacterial counts for X .
6vesicatoria from samples inside and on the surface of
pepper leaves or from washings from these were
5negative using a diagnostic medium (NS) (Fig. 1). Ten E'
minutes after inoculating the leaves with a suspension 2 b containing lo4 cells/mL of X . vesicatoria, bacteria 8
were sparsely covering the leaf surface (lo3 CFU/g
g3
leaf) (Figs. 2, 10). It was not possible to determine a 7whether any cell of X . vesicatoria penetrated the tissue.
S
However, 24 h following inoculation aggregates of the
1pathogen, lo4 CFU/g leaf, could already be observed
under a light microscope in the intercellular spaces
i0 ,d,
TIME AFTER INOCULATION (h)
between the mesophyll cells.
the leaf tissue
and
FIG. 10. Multiplication of Xanthomonas vesicatoria in
under a light microscope of stomata and substomata1 pepper leaves. (A) On the leaf surface. (B) Within the leaf
chambers it was found that in most cases bacteria were tissue. Counts of bacteria from leaves represent trends of
not located in these areas. Only in very few cases were populations.
;
.
d5
SHARON ET
sites (Fig. 8). This event was subsequently supported by
the appearance of visual symptoms at these sites 9 days
after inoculation.
The necrotic sites were free of bacterial cells. The
border between the necrotic area and apparently healthy
tissues was sharp (Fig. 8) and bacteria were found only
on the surface of cells far from necrosis (Fig. 9). This
suggests that the cells surrounding the necrotic area
inhibited the multiplication of X. vesicatoria in a yet
unknown way. A similar phenomenon was first observed in tomato leaves infected with P. tomato (3).
These findings fill the gap in understanding the events
taking place o n the pepper leaf surface between inoculation by X. vesicatoria and the appearance of disease
symptoms.
Acknowledgements
W e thank Mrs. Naomi Bahat, Electron Microscopy
Department, for excellent technical assistance. This
research was partially supported by grant No. 8231026
from the Agricultural Research Organization, Ministry
of Agriculture, Israel, and by grant No. 1-214-80 from
the United States - Israel Agricultural Research and
Development fund (BARD).
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