Plants have evolved several layers of defense responses in the face of
attack by microbial microorganisms that threaten their survival. One of
these responses is Systemic Acquired Resistance (SAR) that can be induced
by some of pathogen avirulent genes product or by abiotic physical or
chemical agents called elicitors (Durrant and Dong, 2004). Applications
of chemicals which activate plant defense mechanisms before pathogen attack
with no environmental side effects have drawn a considerable attention
of researches in this area. Tomato (Lycopersicon esculentum Mill.)
is one of the popular cash crops, recently recognized as a model plant
for plant-pathogen interactions (Arie et al., 2007). Its cultivation,
however, has been limited by an abundance of diseases caused by fungi,
bacteria, viruses and nematodes. Alternaria leaf spot or stem canker is
a common foliar disease of tomato occurring in most regions of Iran and
causing 10-15% annual loss. Tomato stem canker is one of the most
devastating diseases worldwide, caused by Alternaria alternata
f. sp. lycopersici Grogan and is responsible for significant economic
losses sustained by tomato producers each year (Thomma, 2003).
One of the potential plant disease management strategies is the use of
Systemic Acquired Resistance (SAR) to activate host defense mechanisms
(Ryals et al., 1994). Chemical activation of disease resistance
in plants represents an additional option for growers to protect their
crops from losses due to plant diseases. One of the most commonly used
chemicals inducers is Salicylic Acid (SA), which appear to mimic the systemic
effects of localized infection (Kessmann et al., 1994; Vallad and
Goodman, 2004). SA appears to be a central signaling component in SAR
(Yalpani et al., 1991; Vallad and Goodman, 2004). The necessity
of SA for the expression of SAR in tobacco was shown in experiments with
the salicylate hydroxylase (nahG) transgene, which converts salicylate
into catechol (Gaffney et al., 1993). There are some reports regarding
pre-treatment of plants by SA can be effective against Alternaria
sp. fungi in tomato and other plants (Spletzer and Enyedi, 1999; Coquoz
et al., 1995; Weete, 1992). Systemic resistance to A. solani
in tomato using 200 μM SA feeding treatment in a hydroponically grown
system has been induced (Spletzer and Enyedi, 1999). Also, inducing systemic
resistance to A. cassiae in sicklepod (C. obtusifolia)
by using a foliar application of 1000 μM SA has been achieved (Weete,
Exogenous application of SA and some other chemicals including: polyacrylic
acid, acetyl salicylic acid, 2, 6-dichloroisonicotinic acid, methyl salicylate,
jasmonic acid and jasmonic methyl ester, benzothiadiazole derivatives,
DL-B-aminobutyric acid and oxalic acid, can induce accumulation of pathogenensis-related
proteins and lead to reduced incidence of several diseases on many crops
Since SA is an important signal molecule that plays a critical role in
plant defense against pathogen invasion (Spletzer and Enyedi, 1999; Coquoz
et al., 1995; Weete, 1992; Malamy et al., 1990) and the
potential use of that against tomato stem canker has not investigated
yet, we examined if the exogenous foliar application of SA could activate
SAR against A. alternata f. sp. lycopersici in tomato leaves.
MATERIALS AND METHODS
Plant materials: Seeds of tomato cv. Ergon were surface sterilized
with 1% Ca (OCl)2, washed three times with sterile distilled
water and dried under a laminar flow hood. The seeds were planted in 25
cm diameter pots containing 4 kg steam-sterilized sandy-loam soil and
kept in an environmentally controlled greenhouse at 24±2°C
with 16 h of light and 8 h of dark till desired stage. Young plants, approximately
3 weeks old at four-leaf stage were selected and transplanted for use
in the experiments.
Inoculum preparation: Single spore of Alternaria alternata
isolate M11 (isolated from infected tomato in Hamadan Province, Iran,
2006) was grown on Potato Carrot Agar (PCA). The incubation temperature
was 22±2°C, under a diurnal cycle of 8 h light and 16 h darkness.
Three-week-old cultures were flooded with approximately 10 mL of distilled
water and conidia were dislodged with a glass spreader. The inoculum density
was adjusted with sterile distilled water to 1x105 conidia
mL-1 using haemocytometer.
Greenhouse experiments: Greenhouse experiments were setup at College
of Agriculture, Bu Ali Sina University greenhouses in summer 2006. SA
was obtained from Sigma-Aldrich and was dissolved in sterile distilled
water to final concentration of 200 and 400 μM solution, pH 6.4.
Three days prior to conidial suspension application, three-weeks-old
at four-leaf stage tomato plants were planted in plastic pots and sprayed
by concentrations of 200 and 400 μM of SA. Forty eight hours before
inoculation, pots were kept in growth chamber with relative humidity near
85-90% and temperature between 24±2°C. After foliar inoculation
of the plants with conidial suspensions (1x105 conidia mL-1)
they were remained in a fairly dark condition for another 24 h. Plants
were evaluated daily for appearance of disease symptoms. Control plants
were sprayed with sterile distilled water. Treatments were consisted of
four pots each containing two plants and were arranged in a completely
randomized design with four replications. This experiment was conducted
three times and representative data from one of the trials are shown in
Microscopic observations of the leaf surface: Leaves of the inoculated
tomato plants were detached at 1 and 14 days after challenge-inoculation.
Five leaf disks from a newly emerged compound leaf (not SA-treated leaves)
were collected from each plant per four replications. The numbers of penetration
attempts and single cell necrosis reactions were observed using a microscope
(Zeiss, Biomed, Germany) equipped with filter set 05 (BA-420 long-pass).
The extend of disease symptoms was determined by measuring the infected
area of necrotic lesions and counting the necrosis lesions number on all
compound leaves 14 days after inoculation with the aid of low magnification
of microscope. This experiment also was conducted three times and representative
data from one of the trials are shown in the results.
HPTLC analysis: a densitometric method by USING HIGH Performance
Thin Layer Chromatography (HPTLC) which already has established for the
rapid and accurate identification and quantitative determination of acetylsalicylic
acid, ascorbic acid and salicylic acidby Krzek and Starek (1999) was performed
in this study.
Inoculated and non-inoculated leaf tissue samples from different treatments
were harvested 14 days after SA application, examined and assessed in
terms of their free salicylic acid content. Fresh tissue samples (0.5
g) were ground in 20 mL methanol and sonicated for 20 min. The extracts
were filtered and the solvent was evaporated to dryness in vacuo.
Then the extracts were resuspended in 5 mL of 3-flouro acetic acid. The
free SA was extracted by ethyl-acetate: cyclo hexan: iso-propanol (50:50:1,
v/v), two times, each with 10 mL of solvent mixture. Finally the organic
phase was collected and re-evaporated to dryness and redissolved in 1
mL of acetone.
HPTLC system include TLC Scanner 3 and Linomat 5 sprayer samples winCAST
1.2.2 software (CAMAG, Switzerland, Muttenz) was used for determination
of free SA in leaf samples. TLC was performed on silica gel plate (Merck,
60F254, 20x30 cm). The solvent system which has been used was:
Normal hexane: Di methyl ether: Formic acid (0.4: 1: 1).
Sample spraying done with Linomat 5 sprayer samples and under liquid
nitrogen atmosphere. A microliter Hamilton syringe used for sampling.
Applied 10 ppm of SA as internal standard and maximum absorption obtained
at 305 nm wavelength of spectra region (Krzek and Starek, 1999).
Statistical analysis: All studies were arranged in a completely
randomized design with four replicates. Data were subjected to analysis
of variance and mean values were separated by the Least Significant Difference
(LSD) test at p = 0.05.
RESULTS AND DISCUSSION
In recent years, there has been an increasing interest in non-chemical
control methods in plant disease management. White (1979) has earlier
shown that exogenous SA application on tobacco can activate SAR. This
raises the interesting possibility of altering the normally compatible
Alternaria-host plant interaction to an incompatible one by using
SA treatment. To this aim we analyzed the effect of exogenous applications
of SA, to clarify it`s potential ability to suppress stem canker disease
of tomato plants.
The results from greenhouse and laboratory experiments indicated that,
challenge inoculation of SA-treated tomato plants using conidia of A.
alternata f. sp. lycopersici resulted in 54.7% fewer necrosis
lesions per leaf and 87.5% reduction in blighted leaf area (mm2)
as compared with controlled plants not receiving SA (Fig.
The results obtained from foliar application of two different concentrations,
demonstrated that application of 200 μM dosage of SA was insufficient
for inducing disease resistance. But, Spletzer and Enyedi (1999), have
found that the addition of 200 μM SA to the root system significantly
increased the endogenous SA content of tomato leaves. This firstly, might
be due to the different SA application methods and secondly because of
the different fungal species which have been used in these two pathosystems.
In present study, foliar application of 400 μM SA significantly
increased the endogenous SA content of leaves. The blighted areas also
were significantly reduced in all plants treated with 400 μM SA.
However, earlier results obtained by Spletzer and Enyedi (1999) indicated
that hydroponically tomato root treated with 500 μM SA exhibited
a loss of leaf turgor as evidenced by leaf wilting. Also, Yildiz and Guven
(2007) found that application of 1000 μM SA on grapes against Uncinula
necator has induced systemic resistance, but has caused reduction
in chlorophyll content, resulted in chlorosis symptoms. But results obtained
in current study had no such side effects which already have been found.
||Protection of tomato leaves against Alternaria alternata
by a foliar spray with 400 μM salicylic acid (a) and distilled
water (b) four days after inoculation with Alternaria conidial
suspension (1x105 conidia mL-1)
When a strong/fast resistance reaction is induced, this is usually associated
with a host cell death pattern whose consequence is the isolation of the
invading pathogen from healthy host tissue. A microscope-based screening
for apparent discoloration of the host cell wall on different treatments
was conducted. No visible responses were found in the leaf tissues at
24 h after inoculation. The number of HR lesions and cell wall browning
were not detectable by the naked eye. Penetration events were not distributed
randomly on the treated leaves (Fig. 2, 3).
There was significant effect of plant cell reactions in terms of percentage
of penetration events that occurred 14 days after challenge-inoculation
in the site of attempted penetration. SA treated plants were identified
with higher penetration attempts, therefore higher numbers of single cell
HRs, but lower blighted area and discolorations as compared with non treated
plant (control), which had higher rate of blighted area (Fig.
2, 3). This is probably because of the hypersensitive
response which is a common manifestation of plant disease resistance that
is characterized by rapid cell death around the point of infection, restricting
the spread of pathogens. Grant and Mansfield (1999) have already reported
such a necrosis-inducing activity in tomato and also single cell reactions
in which failed penetration attempts in other Alternaria-host pathosystems
has been indicated (McRoberts and Lennard, 1996). Recently it has been
found that, in tomato and another necrotrophic pathogen (Botrytis cinerea),
callose deposition was present as single spots corresponding with unsuccessful
fungal penetration attempts (Asselbergh and Hofte, 2007).
||Percentage of necrosis single cell on tomato leaves
in treated leaves with Salicylic Acid (SA) and non-treated (infected
control) in compare with their endogenous free-SA level in different
||Single cell necrosis reaction against Alternaria
alternata in SA-treated tomato leaves
The results obtained in greenhouse experiments clearly indicated that
exogenous application of SA, in concentration of 400 μM on tomato
leaves have reduced the blighted leaf area (2.25 mm2) significantly
(p<0.05), as compared with infected control (10.31 mm2).
This compound has shown it`s ability to accumulate free-SA content in
leaves (10.4 μg g-1 fresh weight) which was three-fold
higher than the control plants (3.2 μg g-1 fresh weight).
It also activated Systemic Acquired Resistance(SAR) which was effective
against stem canker disease. The results obtained from HPTLC analysis
have indicated highly correlation between increasing the endogenous level
of free SA in treated plants and reduction of the blighted leaf area (Fig.
4). This is in agreement with the results of other publications which
also found that any disruption in the plant`s ability to accumulate salicylic
acid results in the loss of pathogenesis-related protein gene expression
and attenuation of the SAR response (Gaffney et al., 1993; Lawton
et al., 1996; Vernooij et al., 1994). These reports support
our finding that reduced disease incidence could be achieved by using
SA pre-treatment. Foliar application of arachidonic acid to potato has
activated SAR against A. solani (Coquoz et al., 1995). It
has been indicated that, arachidonic acid caused an increase in the endogenous,
free SA levels in potato (Coquoz et al., 1995) and this is consistent
with our observation that SAR in tomato occurs in correlated with SA levels
||Effect of foliar application of 400 μM Salicylic
Acid (SA) on endogenous free-SA content of tomato leaves and disease
index of Alternaria leaf spot in different treatments
We have also demonstrated that the ability of a compound to accumulate
endogenous free-SA correlates with its potential to induce resistance
of tomato to Alternariosis. This is in accordance with the results obtained
by Spletzer and Enyedi (1999), as found that the addition of 200 μM
SA to the root system significantly increased the endogenous SA content
of leaves, resulted in 77% reduction in blighted leaf area (caused by
Alternaria solani) as compared with control plants not receiving
SA. But, foliage application of 200 μM in our experiment was not
sufficient for inducing disease resistance. This might be due to the different
species which have been used in these two pathosystems. However, Thomma
et al. (1998, 2001) have found that neither salicylic acid nor
ethylene is directly involved in resistance against Alternaria
in Arabidopsis. This contradiction results seems to be related
with different host plant system which have been used in these different
Our data indicate that induced resistance may have potential as a disease
control method in tomato crop. Furthermore, the finding that exogenous
SA treatment increased the accumulation of free SA content of leaves suggests
that in tomato the systemic induced response is SA-dependent and therefore
may offer other means of SAR induction. This may be a particularly attractive
option for greenhouse tomato growers where elicitor application could
be easily integrated with existing management practices.
This is the first report of enhancing systemic acquired resistance against
tomato stem canker disease, induced by foliar application of salicylic
acid. Further studies are needed on different tomato cultivars to evaluate
more accurately the efficacy of this chemical inducer and duration of
the SA-induced response in tomato.
This study was supported by the Iran National Science Foundation (Grant
No. 84695/15), we also wish to express our gratitude to the Research Council
of Bu-Ali Sina University, Hamadan, Iran, for providing glasshouse facilities.
Also, our appreciation is expressed to Prof. P. Salehi, the director of
Medicinal Plant Researches Centre, Shahied Beheshti University, Iran,
for providing the necessary facilities to carry out this work.