Plant Pathology (2013) 62, 11141122 Doi: 10.1111/ppa.

12013

Effects of plant age on disease development and virulence of

Clavibacter michiganensis subsp. michiganensis on tomato

G. Sharabania, D. Shtienberga, M. Borensteina, R. Shulhania, M. Lofthouseb, M. Soferb,

L. Chalupowicza, V. Barela and S. Manulis-Sassona*
a
Department of Plant Pathology and Weed Research, ARO, Volcani Center, Bet Dagan,50250; and bNegev R & D Center, D.N. Negev,
85400, Israel

The effect of plant age at the time of inoculation on the severity of bacterial wilt and canker disease caused by Clavib-
acter michiganensis subsp. michiganensis (Cmm) was examined in six greenhouse experiments. The period during
which inoculations led to wilt and death of tomato plants was defined. This period, designated window of vulnerabil-
ity, ranged from transplanting to the 17- to 18-leaf stage. Plants inoculated after this period expressed disease symp-
toms but did not wilt or die. No significant changes in disease incidence were observed when leaves of different ages
were inoculated. Yield accumulation was significantly reduced in plants inoculated within the window of vulnerability
compared with those inoculated after this period. Expression of virulence genes, viz. celA, encoding a secreted cellulase,
and the serine protease-encoding pat-1, chpC and ppaA, was induced during the early stages after inoculation in plants
inoculated within the window of vulnerability. Differences in Cmm population between plants inoculated within and
outside of this period were insignificant after the first week post-inoculation, indicating that differences in disease sever-
ity, yield loss and expression of virulence determinants are not correlated with Cmm population level. Results suggest
that implementation of precautionary measures during the window of vulnerability to avoid secondary spread of Cmm
will have a season-long effect on plant mortality and may minimize, or even prevent, yield losses.

Keywords: bacterial canker, epidemiology, Solanum lycopersicum

challenged by the pathogen (Whalen, 2005). The change

Introduction
Bacterial wilt and canker caused by Clavibacter michi-
ganensis subsp. michiganensis (Cmm) is one of the most
important diseases of tomato (Solanum lycopersicum)
worldwide (Strider, 1969; Davis et al., 1984). The patho-
gen causes extensive damage and substantial economic
losses by reducing tomato yield quantity and quality
(Gartemann et al., 2003; Eichenlaub & Gartemann,
2011). Disease prevention is mainly achieved through the
use of certified pathogen-free seeds (de Leon et al.,
2011), but new outbreaks and first reports of its causal
agent are still periodically noted in different regions (de
Leon et al., 2011). At present, neither commercial resis-
tant plants nor effective chemical controls are available
(de Leon et al., 2008). Other measures, such as biologi-
cal control (Yogev et al., 2009; Amkraz et al., 2010),
have been investigated, but they are still far from provid-
ing successful means to cope with the disease. Thus, its
successful suppression remains a serious challenge for the
tomato industry worldwide.
The response of host plants to pathogens often
depends on the developmental stage of the host when
*E-mail: shulam@volcani.agri.gov.il
Published online 16 November 2012
in host-plant resistance as a function of developmental
stage at the time of infection has been given several
names: ontogenic resistance, developmental resistance,
mature-seedling resistance, adult-seedling resistance,
adult-plant resistance and, occasionally, age-related
resistance (Kus et al., 2002; Whalen, 2005). Changes in
tomato response to Cmm with age have been reported
in a few studies (Kendrick & Walker, 1948; Chang
et al., 1992). In general, incubation period increases and
severity decreases with plant age (Kendrick & Walker,
1948). For example, under favourable environmental
conditions, symptoms of systemic infection developed
faster on 2- to 4-week-old seedlings than on 5- to 6-
week-old seedlings (Chang et al., 1992). Kendrick &
Walker (1948) reported that wilted leaves appeared ear-
lier in younger plants, but the rate of symptoms devel-
opment was the same. Although the conclusion from
these studies is that young plants are more susceptible
than mature plants, the ages at which tomato plants are
most vulnerable to infection have not been determined.
Furthermore, in those studies, plant age was determined
chronologically (weeks or days). As plant growth and
development are affected by environmental conditions
(e.g. temperature and radiation intensity) as well as cul-
tural practices (e.g. growth media, fertilization, irriga-
tion), chronological age does not necessarily reflect the
plants physiological age.

1114 2012 British Society for Plant Pathology

 Plant age and disease development by Cmm
Thyr (1968) reported that the appearance of bacterial
canker symptoms is closely related to the population of
the pathogen colonizing the host. The pathogen can
survive as an effective endophyte in tomato but appar-
ently has to establish an endophytic population of
>108 CFU g 1 plant tissue to induce disease symptoms
(Gartemann et al., 2003). The effect of plant age at the
time of inoculation on population dynamics of Cmm in
planta has not been reported.
Cmm pathogenicity is dependent on virulence genes
located on plasmids and the chp/tomA pathogenicity
island (PAI) located on the chromosome (Eichenlaub &
Gartemann, 2011). In the wildtype strain NCPPB382,
two plasmids, pCM1 and pCM2, are essential for viru-
lence (Meletzus et al., 1993), as loss of either one
reduces it. pCM1 encodes celA, a secreted cellulase
with endo-b-1,4-glucanase activity (Jahr et al., 2000),
and pCM2 encodes Pat-1, a putative serine protease
(Dreier et al., 1997). The chp/tomA PAI, a genomic
island of about 129 kb with significantly lower GC con-
tent than in the genome, encodes several serine prote-
ases that are important for colonization (Gartemann
et al., 2008). A mutant lacking the complete PAI region
exhibits impaired virulence and is unable to effectively
colonize tomato. The virulence factors located on the
chp/tomA PAI or on pCM1 and pCM2 are required for
effective movement of the pathogen in tomato and for
the formation of cellular aggregates (Chalupowicz et al.,
2011). The effect of plant age on the expression of vir-
ulence genes has not been reported and it is not known
whether that expression is correlated with Cmm coloni-
zation at different plant ages.
The aim of the present study was to elucidate the sig-
nificance of tomato age at the time of inoculation on
bacterial canker development. Plant age was defined in
terms of number of mature leaves. The specific objec-
tives were: (i) to define the range during which plant
age affects disease severity; and (ii) to determine the
effect of plant age at the time of inoculation on coloni-
zation and expression of bacterial virulence genes in
planta.
(a)
1 2 3 4
Figure 1 Diagrammatic schemes of inoculation procedures for experiments studying the effects of tomato plant age (a) and leaf age (b) at the time
of inoculation on bacterial canker development. (a) Experiments V4, B1 and B2. Numbers 1, 2, 3 and 4 correspond to plants with 78, 1213, 1617
and 1920 leaves, respectively. Plants were inoculated by cutting the petioles of the two youngest leaves close to the stem. (b) Experiments V4, B1
and B2, in which plants with 1920 leaves were used. Numbers 1, 2, 3 and 4 correspond to leaf numbers 78, 1213, 1617 and 1920,
respectively. Inoculated leaves are shown in black, blossoms clusters are marked with asterisks and circles represent small fruits.
Plant Pathology (2013) 62, 11141122
1115
Materials and methods
Bacterial strains and inoculation procedures
Strain Cmm32, which was used in this study, was originally iso-
lated in 2001 from Talmi Eliyahu, Israel and belongs to group
B, according to pulsed field gel electrophoresis analysis (Kleit-
man et al., 2008). Bacteria were cultured on nutrient agar (NA)
(Difco) plates at 28C for 72 h. Bacterial cells were collected in
2 mL distilled water, and the resulting suspension was adjusted
to an OD595 of 0
5, which corresponds to c. 109 cells mL 1.
This suspension was diluted to a final concentration of c. 108
cells mL 1 and used for tomato inoculation. Tomato cv. 1125
(Top Seeds) plants were inoculated by cutting the petioles close
to the stem with scissors which had been dipped into the bacte-
rial suspension (contaminated-scissors method; Thyr, 1968). All
experiments included a non-inoculated control treatment in
which the petioles were cut with scissors dipped in water.
Experimental design
The effect of plant age at the time of inoculation on disease
development was determined in six experiments in 2009 and
2010. The results obtained in the six experiments were validated
in an additional experiment, carried out in 2011. Plants were
grown under commercial-like conditions at the Volcani Center
(experiments V1, V2, V3 and V4) and at the experimental sta-
tion of the Negev R&D Center, located in the southwest of
Israel (experiments B1, B2 and B3). Each experiment consisted
of three to four treatments that differed in the age of the plants
at the time of inoculation (Fig. 1a). The ages were: 45, 78, 12
13 leaves (experiments V1, V2 and V3); 78, 1213, 1617, 19
20 leaves (experiments V4, B1 and B2) and 78, 1617, 1920,
2930 leaves (experiment B3). The two upper open leaves of
each plant were inoculated using the contaminated-scissors
method, as described above.
The effect of leaf age on disease development was determined
by inoculating leaf numbers 78, 1213, 1617 and 1920 of
plants at the 19- to 20-leaf stage of experiments V4, B1 and B2
(Fig. 1b). Each experimental plot consisted of 510 plants in
one row. All experiments included a non-inoculated control
treatment using water only.
Seedlings (at the 3- to 4-true-leaf stage) were obtained from
commercial nurseries. Before transplanting, to confirm the
(b)
1 2 3 4
1116 G. Sharabani et al.
absence of Cmm infection, seedlings were observed visually for
detection of symptoms, then 10% of the seedlings were sampled
randomly, macerated in sterile distilled water and subjected to
PCR analysis (Kleitman et al., 2008). None of the seedlings
exhibited any bacterial canker symptoms and all attempts to iso-
late Cmm were negative. Experiments V1 to V4 were carried
out in large net-houses. Tomato seedlings (cv. 1125) were trans-
planted on 23 March 2009 (experiment V1), 10 August 2009
(experiment V2), 23 March 2010 (experiment V3) and 6 Sep-
tember 2010 (experiment V4). Seedlings were transplanted into
grow bags (Pelemix Ltd) in beds, with two rows per bed and 15
plants per row. The distances between beds, between rows in a
bed and between plants within a row were 2
2, 0
3 and 0
3 m,
respectively. The plants were irrigated daily and fertilized three
times a week with liquid fertilizer containing N:P:K at 5:3:8 and
3% of a 0
2% solution of microelements. In all experiments
plants were cultivated according to the practices used by Israeli
tomato growers in commercial greenhouses for irrigation, fertil-
ization, disbudding, removal of lower leaves, trellising, harvest-
ing, pest management and discarding the plants after c.
45 months. These experiments were laid out in a complete block
design with three to four rows (replicates) for each treatment.
Experiments B1 and B2 were performed in large walk-in tun-
nels (6 9 6 9 2
5 m) covered with polyethylene. Tomato cv.
1125 seedlings were transplanted into grow bags, with four
rows per tunnel, on 15 April 2010 and 2 August 2010. The dis-
tances between rows and between plants within a row were 1
2
and 0
3 m, respectively. These experiments were laid out in a
complete block design with four replicates. Within each tunnel
(replicate), each row was designated as a treatment.
Experiment B3 was performed to validate the results obtained
in the previous six experiments. Tomato seedlings were trans-
planted directly into the soil on 13 April 2011 in two commer-
cial-like greenhouses (9 9 10 9 5 m) covered with
polyethylene. In each greenhouse there were 15 experimental
plots, each containing two rows of 10 plants each. The distances
between beds, between rows within a bed, and between plants
within a row were 1
6, 0
5 and 0
3 m, respectively. This experi-
ment was laid out in a complete block design with six repli-
cates.
The effect of plant age at the time of inoculation on yield
accumulation was determined in experiments V4 and B3. At
weekly intervals, all edible fruits were collected from each plant
and weighed. The cumulative yield (kg per plant) was calculated
at the end of the experiments, and the significance of the differ-
ences in yield between treatments determined by ANOVA with the
TukeyKramer HSD test at P  0
05.
Disease assessments
All plants were inspected visually at weekly intervals from
1 week after planting, and the appearance of typical bacterial
canker symptoms was recorded on an individual-plant basis.
Disease severity was determined using a four-grade scale as fol-
lows: 0 = symptomless plant, typical bacterial canker symptoms
not observed on any of the leaves; 1 = low severity, disease
symptoms observed on a few leaflets or leaves, with less than
5% of the plants leaf area exhibiting disease symptoms;
2 = intermittent severity, disease symptoms observed on many
leaves, with 1040% of the plants leaf area exhibiting disease
symptoms; and 3 = high severity or wilting and dying plants,
disease symptoms observed on most leaves and the plant in
advanced stages of wilting or already dead. Data were used to
calculate the changes in the incidence of plants with symptoms
(grades 1 to 3, as a percentage) and the incidence of wilting and
dying plants (grade 3, as a percentage) over time. Data were
analysed using regression analysis in which the dependent vari-
able was disease incidence (Y, expressed as percentage) and the
independent variable was time from inoculation (X, in days).
The results were fitted to the regression equation Y = a/(1 + e^
(X X0)/b)) in which a, b and X0 are the regression coefficients:
a = the asymptotic percentage; b = the value of X at the inflex-
ion point; and X0 = the time at which Y is equal to 50% of the
asymptote.
Determination of Cmm population size
The effect of plant age at the time of inoculation on the dynam-
ics of the Cmm population in planta was determined in plants
inoculated at the 7- to 8- and 19- to 20-leaf stages, at different
time points after inoculation. Seedlings of tomato cv. 1125, at
the 3- to 4-true-leaf stage) were transplanted into 13-cm plastic
pots. Potted plants were placed in a growth chamber and main-
tained at 27C with a 12 h photoperiod until use. The plants
were irrigated daily and fertilized three times weekly with liquid
fertilizer containing N:P:K at 5:3:8 and 3% of a 0
2% solution
of microelements.
Samples were taken 1, 2, 4 and 8 weeks after inoculation
from the lower part of the stem close to the ground, from the
inoculation site and from the apex. Samples were weighed and
macerated in sterile distilled water. Serial tenfold dilutions in
distilled water were prepared and 100 lL of each dilution was
plated on three mCNS plates (Sharabani et al., 2012). Plates
were incubated at 28C and the resulting colonies were counted
5 days later. The samples were taken from six plants (replicates)
per treatment and the data were used to calculate the average
and SE of Cmm population size. The experiment was repeated
once.
Relative expression of virulence genes
The effect of plant age at the time of inoculation on expression
of virulence genes was determined in plants inoculated at the 7-
to 8- and 19- to 20-leaf stages, at different time points after
inoculation, as described above for determination of Cmm pop-
ulation size. Tissue samples were taken from the inoculation
site. For each time point, two separate pools of two inoculated
stem junction parts (c. 1 cm long) were cut from the infected
plants and used for isolation of RNA with a MasterPure RNA
Purification Kit (Epicentre Biotechnologies). RNA quantification
was performed with a NanoDrop 1000 spectrophotometer
(Thermo Fisher Scientific). To remove residual genomic DNA,
the isolated RNA was treated with DNase (Turbo DNA-free,
Ambion Inc.). Reverse transcription was carried out from 1 lg
total RNA using the Verso cDNA Kit (Thermo Scientific) with
random hexamer primers, according to the manufacturers
instructions. Triplicates of cDNA synthesis were performed for
45 min at 42C. The resulting cDNA was subjected to PCR
amplification with Power SYBR Green PCR Master Mix
(Applied Biosystems) in a final volume of 20 lL. Specific prim-
ers for real-time PCR for the virulence genes pat-1, celA, ppaA
and chpC were used as described by Chalupowicz et al. (2010).
Real-time detection was performed with a 7300 Real-Time PCR
System (Applied Biosystems). Taq DNA polymerase was acti-
vated at 95C for 10 min, followed by a thermal cycling pro-
gramme consisting of 40 cycles of 15 s at 95C and 1 min at
60C. Relative expression was normalized with gyrA and bipA
as internal references and inoculated plants at time of inocula-
Plant Pathology (2013) 62, 11141122
 Plant age and disease development by Cmm 1117

tion (time 0) were used as controls. Results represent the means

(a)
of two different experiments with three samples for each time
100
point. Relative quantification and statistical analysis were per-
formed with the data analysis program of REAL-TIME PCR soft-
80
ware (Applied Biosystems).
60
Results
40

Final disease incidence (%)

Effect of plant and leaf age at the time of inoculation
on disease development
Data recorded in the six experiments were used to calcu-
late the relationship between plant age at the time of
inoculation and final incidence of plants with symptoms
(grades 13) or of wilting and dying plants (grade 3). By
the end of the experiments disease symptoms were
observed in all plants inoculated from the 4- to 5-leaf
stage to the 12- to 13-leaf stage (n = 334) and on >85%
of the plants inoculated at the 16- to 17- (n = 95) and
19- to 20-leaf stages (n = 95) (Fig. 2a). The results
observed in the seventh experiment carried out for vali-
dation of the window of vulnerability, corroborated
these findings (Fig. 2a). In this experiment, plants were
also inoculated at the 29- to 30-leaf stage; 40% of these
exhibited disease symptoms, but none had wilted or died
by the end of the experiments (Fig. 2a). On average, 65
72% of the plants inoculated from the 4- to 5-leaf stage
to the 13- to 14-leaf stage (n = 334) eventually wilted or
died. About 30% of the plants inoculated at the 16- to
17-leaf stage (n = 95) and none of the plants inoculated
at the 19- to 20-leaf stage (n = 95) wilted or died by the
end of the experiments (Fig. 2b). None of the plants
inoculated at the 29- to 30-leaf stage wilted or died. No
disease symptoms were observed in any of the experi-
ments on the non-inoculated, control plants. Based on
these results, a period of time called the window of vul-
nerability was defined as the period during which inocu-
lation led to wilting and dying of the plants. The
window of vulnerability ranged from transplanting to the
16- to 17-leaf stage (Fig. 2b).
In all experiments, disease symptoms were observed
up to 50 days post-inoculation in plants inoculated from
the 4- to 5-leaf stage to the 16- to 17-leaf stage (Fig.
S1a,c; Table S1). In plants inoculated at the 19- to 20-
leaf stage disease symptoms were observed up to 75 days
post-inoculation. Wilting and dying were first observed
25105 days post-inoculation in plants inoculated
between the 4- to 5- and 16- to 17-leaf stages. In plants
inoculated at the 19- to 20- and 29- to 30-leaf stage,
wilting and dying were not observed until the end of the
experiments (Fig. S1b,c; Table S2).
Differences in susceptibility resulting from leaf age
were determined by inoculating leaves of different ages
on plants with 1920 leaves. First disease symptoms
were observed 5465 days post-inoculation, and within
24 weeks >90% of the inoculated plants exhibited typi-
cal bacterial canker symptoms (data not shown). None
of the plants showing symptoms wilted or died until the
end of the experiment (100130 days post-inoculation).
20
0
4 8 12 16 20 30
100
(b) y = 669/(1+272^((x169)/0136))
R2 = 0976;P = 00237
80
60
40
20
0
4 8 12 16 20 30
Plant age at the time of inoculation
(number of leaves)
Figure 2 Relationship between tomato plant age at time of inoculation
with Clavibacter michiganensis subsp. michiganensis and final disease
incidence of (a) plants with symptoms and (b) wilting and dying plants.
Data were taken at the end of the experiments, 100130 days post-
inoculation. Open squares represent the means of six experiments: V1,
V2, V3, V4, B1 and B2. Open circles represent results of the additional
validation experiment B3. Vertical bars represent the standard error.
None of the control plants showed any symptoms until
the end of the experiments.
Effect of plant age at the time of inoculation on yield
accumulation
Effects of bacterial canker on yield accumulation, as
related to the age of the plants at time of inoculation,
were examined in two experiments. Results of one repre-
sentative experiment are presented in Figure 3. The dis-
ease did not affect yield accumulation rate at any
inoculation time. However, in plants inoculated at the 7-
to 8-leaf stage (within the window of vulnerability), yield
accumulation was arrested c. 110 days from transplant-
ing, resulting in 40
5% yield reduction compared with
the non-inoculated control plants. In plants inoculated at
the 16- to 17-leaf stage (close to the end of the window
of vulnerability), yield accumulation continued until
125 days after transplanting, with a 21
5% yield
reduction. Yield was not significantly reduced in plants
inoculated at the 19- to 20- or 29- to 30-leaf stage
(Fig. 3); these plants were inoculated after the window
of vulnerability. Similar trends were observed in the
repeated experiment (data not shown).

Plant Pathology (2013) 62, 11141122

1118 G. Sharabani et al.

10 10

Cumulative yield (kg per plant)

Plant age Plant age

Population size (log CFU)

7/8
Non-inoculated a
a 19/20
8 7/8 a 8
16/17
ab
6 19/20 6
29/30 b

4 4

2 2

0 0
0 50 100 150 200 LS IS Apex LS IS Apex
Time (days from transplanting) One week Two weeks
Time (weeks after inoculation)
Figure 3 Effect of tomato plant age at time of inoculation with
Clavibacter michiganensis subsp. michiganensis on yield Figure 4 Effect of tomato plant age at time of inoculation on the
accumulation. The two youngest fully expanded leaves were inoculated population dynamics of Clavibacter michiganensis subsp.
by cutting the petioles close to the stem on plants bearing 78, 1617, michiganensis in planta. Plants with 78 or 1920 leaves were
1920 or 2930 leaves. Fruits were harvested from 18 plants (three inoculated with a bacterial suspension of 108 cells mL 1 by cutting the
repeats of six plants). Arrows indicate the time of inoculation in each petioles of the two youngest open leaves close to the stem. Samples
treatment. At the end of the experiment, values followed by different were taken from the lower part of the stem close to the ground (LS), at
letters are significantly different (P  0
05) according to the Tukey the inoculation site (IS) or from the apex, 1 and 2 weeks post-
Kramer HSD test. Statistics of regression equations are presented in inoculation. Results are means of six plants for each time point and
Table 1. vertical bars represent the standard error.

19- to 20-leaf stage was significantly lower than in

Effect of plant age at the time of inoculation on Cmm
those inoculated at the 7- to 8-leaf stage. Transcript
population dynamics in planta
levels of pat-1 reached a peak at 48 hpi of 10-fold
The population size of Cmm at various locations on the induction and then declined to four- and twofold induc-
plants was determined 18 weeks after inoculation on tion at 96 and 168 hpi, respectively. Relative expression
plants inoculated at the 7- to 8- or 19- to 20-leaf stage. of celA in plants inoculated at the 7- to 8-leaf stage
One week post-inoculation, the population size of Cmm was significantly higher than in those inoculated at the
in the lower stem and apex was significantly higher in 19- to 20-leaf stage. In plants bearing 78 leaves, three-
plants inoculated at the 7- to 8-leaf stage than in plants and twofold induction was measured at 12 and 24 hpi,
inoculated at the 19- to 20-leaf stage; differences respectively, reaching a peak of 11-fold at 48 hpi and
between the two treatments at the inoculation site were then declining to six- and onefold induction after 96
insignificant (Fig. 4). However, 2 weeks post-inoculation and 169 hpi, respectively (Fig. 5). In contrast, celA was
(Fig. 4) and up to 8 weeks post-inoculation (data not barely expressed in plants inoculated at the 19- to 20-
shown), differences in population size were insignificant leaf stage throughout the tested period. Similarly,
in the lower stem, inoculation site and upper stem increased transcript levels were also observed in the
between plants inoculated at the 7- to 8- and 19- to 20- case of the chromosomal serine proteases, ppaA and
leaf stages. The pathogen was not isolated from the non- chpC, in plants inoculated at the 7- to 8-leaf stage
inoculated, control plants. Similar trends were observed compared with those inoculated at the 19- to 20-leaf
in the repeated experiment (data not shown). stage. Transcription of ppaA in plants inoculated at the
7- to 8-leaf stage was induced 1
5- and 3
8-fold at 12
and 24 hpi, respectively, reaching a peak of 7
8-fold at
Effect of plant age at the time of inoculation on
48 hpi and decreasing to 6
1- and threefold at 96 and
expression of Cmm virulence genes
168 hpi, respectively (Fig. 5). In plants inoculated at
The transcript levels of the virulence genes pat-1, celA, the 19- to 20-leaf stage, expression of ppaA was
ppaA and chpC were measured by quantitative RT- induced 2
1-fold at 48 hpi, reached a peak of fivefold
PCR in the inoculation site of plants inoculated at the induction at 96 hpi and then decreased to threefold at
7- to 8- and 19- to 20-leaf stages (within and outside 168 hpi. A similar induction pattern was observed for
of the vulnerability period, respectively) at various times chpC in infected tomato plants inoculated at the 7- to
post-inoculation (Fig. 5). Transcript level of the serine 8-leaf stage; chpC expression was induced by factors of
protease pat-1 in plants inoculated at the 7- to 8-leaf 2
5 and 2
1 at 12 and 24 hpi, respectively, reaching a
stage increased threefold in the initial 12 h post-inocu- peak of sevenfold at 48 hpi and then decreasing to
lation (hpi), reached a peak of 14-fold induction at 2
1- and 2-fold at 96 and 168 hpi, respectively. In con-
24 hpi and then declined to eight-, six- and threefold trast, expression of chpC in plants inoculated at the
induction after 48, 96 and 168 hpi, respectively 19- to 20-leaf stage was barely induced during the
(Fig. 5). Expression of pat-1 in plants inoculated at the experimental period (Fig. 5).

Plant Pathology (2013) 62, 11141122

 Plant age and disease development by Cmm 1119

20 14
(a) pat-1 Plant age (b) celA
15 7/8 12
19/20

12 10

8
9
6
6
4
3
Relative expression
2

0 0
12 24 48 96 168 12 24 48 96 168
10 10
(c) ppaA (d) chpC
8 8

6 6

4 4

2 2

0 0
12 24 48 96 168 12 24 48 96 168
Time (hours post-inoculation)

Figure 5 Effect of tomato plant age at time of inoculation with Clavibacter michiganensis subsp. michiganensis on relative expression of virulence
genes. Transcript levels of pat-1 (a), celA (b), ppaA (c) and chpC (d) were determined by quantitative RT-PCR at various times after inoculation.
Plants with 78 or 1920 leaves were inoculated with a bacterial suspension of 108 cells mL 1 by cutting the two youngest open leaf petioles close
to the stem. Relative expression was normalized with gyrA and bipA as internal references and inoculated plants at time 0 were used as controls.
Results represent the mean of two different experiments with three samples for each time point. Vertical bars represent the standard error.

of them died and only 40% exhibited disease symptoms,

Discussion
Changes in host response to a pathogen over time,
expressed as plant age, leaf age and leaf position, have
been reported for several pathogens (Whalen, 2005;
Develey-Riviere & Galiana, 2007). As plants mature,
they often become increasingly resistant to normally vir-
ulent pathogens (Kus et al., 2002). In young tomato
plants, disease symptoms caused by Cmm appeared ear-
lier than in older plants (Kendrick & Walker, 1948;
Chang et al., 1992). In those studies, inoculations were
performed on the first true leaves of plants at ages rang-
ing from 2 to 6 weeks. Obviously, the ages of these
leaves were dissimilar in terms of the effect of leaf age
on disease expression and development (Chang et al.,
1992). In the present study, plant age was determined as
number of leaves developed, corresponding to the plants
physiological age.
Results of six experiments enabled the identification of
a period during which inoculation led to wilting and
dying of plants; this period was designated the window
of vulnerability and ranged from transplanting to the 17-
to 18-leaf stage. Plants inoculated outside that window of
vulnerability expressed disease symptoms but did not wilt
or die until the end of the experiment (Fig. 2). These
observations were confirmed in a validation experiment
(Fig. 2), which strengthened the determination of the
window of vulnerability. In the validation experiment,
plants bearing 2930 leaves were inoculated as well; none
supporting the hypothesis that plants inoculated at an
age beyond the window of vulnerability are less suscepti-
ble to Cmm infection. It should be noted that the actual
time passed from the 16- to 17-leaf stage to the 19- to
20-leaf stage is relatively short, 13 weeks depending on
temperature. The reason for the sharp difference in
response to Cmm inoculation by plants with 1617
leaves compared to those with 1920 leaves is not
known. It is not likely that this difference was related to
Cmm population size colonizing the infected tissues
because only minor differences in colonization were
found (Fig. 4). It might have been related to differences
in the developmental stage of the plants with 1617 com-
pared to 1920 leaves. Although plants in both groups
were already bearing flower clusters, plants at the 19- to
20-leaf stage had already developed small fruits. Fruit
bearing has previously been associated with changes in
host responses to pathogens (Develey-Riviere & Galiana,
2007). In some cases, fruit bearing increases host resis-
tance, whereas in others it decreases it. For example,
potatoes plants are relatively resistant to Alternaria solani
and cotton plants are relatively resistant to Alternaria
macrospora during the vegetative stages of plant growth.
However, when tuberization (in potatoes) or boll
development (in cotton) is initiated, the plants start to
become susceptible, and susceptibility increases during
development of the reproductive stage (Shtienberg et al.,
1995).

Plant Pathology (2013) 62, 11141122

1120 G. Sharabani et al.
Table 1 Coefficients and parameters for the regression equations
describing the effect of tomato plant age at the time of inoculation with
Clavibacter michiganensis subsp. michiganensis on yield accumulation a
Regression
coefficientsb
Plant age (no. leaves)
at inoculation a b X0 R2 P tissue. Some of the Cmm proteins which may be respon-
Non-inoculated 8
4 14
7 97
0 0
961 <0
0001
78 5
0 8
4 90
4 0
946 <0
0001
1617 6
6 9
6 90
5 0
954 <0
0001
1920 7
6 9
6 91
7 0
978 <0
0001
2930 8
4 11
4 96
0 0
971 <0
0001
a chpC) were induced during the early stages of infection
Disease progress curve presented in Figure 3.
b
The equation Y = a/(1 + e^ (X X0)/b)) was fitted to the data.
Y = cumulative yield (kg/plant); X = time (in days) from transplanting.
a, b and X0 are regression coefficients: a = asymptote; b = value of X
at inflexion point; X0 = time when Y = 50% of asymptotic value.
Effects of leaf age on disease development have been
described in several studies. In some cases, mature leaves
were more resistant to infection (Kus et al., 2002; Kurt
& Tok, 2006) whereas in others, younger leaves were
less susceptible (Heilbronn & Harrison, 1989; Bouhassan
et al., 2004). In the present study, inoculation of leaves
of different ages on plants of the same age did not result
in any significant change in disease incidence. First dis-
ease symptoms developed 8495 days after transplanting
and, within 24 weeks, more than 90% of the plants
(bearing 1920 leaves) exhibited typical disease symp-
toms. Nevertheless, none of the plants wilted or died
until the end of the experiment. It could be argued that
this lack of difference in the early response results from
the fact that the inoculated leaves were on plants past
the window of vulnerability. However, similar results
were obtained when inoculating leaves 45, 78 or 12
13 on plants bearing 1213 leaves (within the window of
vulnerability; data not shown). These results imply that
young tomato leaf tissue is as sensitive as older leaf tis-
sue to Cmm infection, and that the window of vulnera-
bility is directly related to plant age rather than leaf age.
Yield loss in tomato as a result of bacterial canker
disease has been estimated in several studies (Strider,
1969; Gleason et al., 1991). Ricker & Riedel (1993)
reported that late infections of mature processing toma-
toes appeared to be less detrimental to yield. Emmatty
& John (1973) showed that early Cmm inoculation at
biweekly intervals resulted in a greater decrease in
yield. A study examining the effect of Cmm inoculation
at different phenological stages of tomato showed sig-
nificantly reduced yield when inoculation was per-
formed on seeds and plants during transplanting, first
pruning and first flowering, but not when inoculation
was performed during first harvest (Dullahide et al.,
1983). The results of the present study confine the cru-
cial phenological period when yield loss can be pre-
vented to the period between transplanting and fruit
formation (Fig. 3). This time corresponds to the win-
dow of vulnerability.
The difference between susceptible and resistant
response outcomes may be related to the speed at which
those responses occur (Yang et al., 1997). During com-
patible Cmmtomato interactions, basal defence is
induced (Savidor et al., 2011). However, Cmm is able to
invade the plant, colonize the xylem and damage plant
sible for colonization and induction of disease symptoms
are members of the serine protease families which are
secreted during infection along with cell-wall-degrading
enzymes (Savidor et al., 2011). The genes encoding endo-
glucanase (celA) and serine proteases (pat-1, ppaA and
of young plants bearing 34 leaves (Chalupowicz et al.,
2010). In the present study, experiments conducted with
plants bearing 78 leaves corroborated those results,
demonstrating highly induced expression of virulence
genes in plants during the window of vulnerability. In
contrast, in plants bearing 1920 leaves, expression of
pat-1 and ppaA was significantly reduced and delayed
relative to plants bearing 78 leaves (Fig. 5). celA and
chpC were not expressed at all in plants bearing 1920
leaves. The delayed and lower level of expression of
these, and probably other virulence genes, can be
explained by the enhanced resistance in mature tomato
plants, which allows the host defence mechanisms to
overcome Cmm virulence and thus reduce development
of severe disease symptoms. Age-related resistance (ARR)
is associated with the accumulation of secondary metab-
olites or defence proteins. In Arabidopsis thaliana, the
ARR response to Pseudomonas syringae pv. tomato is
correlated with the intercellular level of salicylic acid
(Carviel et al., 2009). Transcriptional analysis of young
tomato plants in response to Cmm infection revealed
that a subset of host genes is highly induced, including
defence-related genes, production and scavenging of free
oxygen radicals and hormone synthesis (Balaji et al.,
2008). It might be hypothesized that in older tomato
plants, enhanced host defence responses lead to an
increased ARR response to Cmm. This hypothesis war-
rants further investigation in Cmmtomato interaction
studies.
Differences in Cmm population between plants within
and outside the window of vulnerability were found only
in the first week post-inoculation in the lower stem and
apex. However, from 2 weeks on, no differences in colo-
nization were found between plants bearing 78 and 19
20 leaves. These results indicate that the differences in
disease incidence, yield loss and expression of virulence
determinants found between plants within and outside
the window of vulnerability are not correlated with level
of Cmm multiplication. Cmm is known to be an effective
endophyte of tomato and related plants (Eichenlaub
et al., 2006). However, the ability to effectively multiply
in the plant is a prerequisite, but not sufficient for trig-
gering symptom induction. It might be speculated that in
mature plants, Cmm cannot overcome the ARR response
and thus virulence determinants are less expressed or
retarded.
Plant Pathology (2013) 62, 11141122
 Plant age and disease development by Cmm
One of the primary sources of inoculum is bacteria
colonizing plant debris remaining from the previous
crops in or on the soil. Although endophytic populations
of Cmm colonizing the plants infected beyond the win-
dow of vulnerability do not cause wilting, death and
yield loss, they may be a source of inoculum for subse-
quent plantings. Accordingly, by the end of each produc-
tion cycle, growers should invest all efforts possible to
remove the infected tissues from greenhouses.
Age-related resistance can have implications for disease
management strategies. The main mechanisms of second-
ary spread of Cmm are believed to be disbudding and
removal of the lower leaves, workers hands and pesti-
cide spraying (Strider, 1967). These procedures facilitate
pathogen invasion of the vascular tissues of target plants
through the newly opened wounds, leading to systemic
infection (Gleason et al., 1991; Kawaguchi et al., 2010).
Moreover, it has recently been shown that touching
symptomless infected plants bearing guttation droplets
prior to touching nearby plants transfers the pathogen,
which is exuded in the guttation fluid, within rows
(Sharabani et al., 2012). The results presented here
demonstrate the importance of defining the window of
vulnerability for effective disease control. By avoiding
the secondary spread of Cmm during this period, plant
mortality and yield losses can be prevented.
Acknowledgements
This study is part of the Khosen Clavibacter project,
financed by the Israeli Ministry of Agriculture and Rural
Development and by the Israeli Council of Vegetable
Growers. We would like to acknowledge the assistance of
H. Yechezkel, L. Gadot, B. Oren, E. Blu and G. Fridman
of the Negev R & D Center for their technical assistance.
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Supporting Information
Additional Supporting Information may be found in the online version of
this article.
Table S1. Coefficients and parameters for the regression equations
describing the effect of plant age at the time of inoculation on incidence
of symptomatic plants in experiments V1, V2, V3, V4, B1, B2 and B3.
Table S2. Coefficients and parameters for the regression equations
describing the effect of plant age at the time of inoculation on incidence
of wilting and dying plants in experiments V1, V2, V3, V4, B1, B2 and
B3.
Figure S1. The effect of plant age at the time of inoculation on bacte-
rial canker development. (a) Disease incidence over time of symptomatic
plants as recorded in experiments V1, V2, V3, V4, B1 and B2. (b) Dis-
ease incidence of wilting and dying plants recoded in experiments V1,
V2, V3, V4, B1 and B2. (c) Changes in disease incidence of symptomatic
and of wilting and dying plants in the validation experiment, B3. Plants
at the 45, 78, 1213, 1617, 1920 or 2930 leaf stage were inocu-
lated by cutting the petioles of the two youngest leaves close to the stem.
Statistics of regression equations are presented in Tables S1 and S2.
Plant Pathology (2013) 62, 11141122