Forest Ecology and Management 261 (2011) 416–426

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Forest Ecology and Management

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Application of salicylic acid induces antioxidant defense responses in the phloem

of Picea abies and inhibits colonization by Ips typographus
Andreja Urbanek Krajnc ∗ , Janja Kristl, Anton Ivancic
University of Maribor, Faculty of Agriculture and Life Sciences, Pivola 10, SI-2311 Hoče, Slovenia

a r t i c l e i n f o a b s t r a c t

Article history: The adaptive plasticity of Norway spruce (Picea abies) against attack by Ips typographus depends on sys-
Received 3 June 2010 temic acquired resistance which involves salicylic acid (SA), and an antioxidant system both recognized
Received in revised form 21 October 2010 as valuable stress markers in ecophysiological studies. In the presented field experiment, 100 mM SA
Accepted 24 October 2010
was applied to the bark sections of Norway spruce prior to being attacked by bark beetles, in order
Available online 26 November 2010
to study interactions with antioxidants and its significance for mediating stress-tolerance under natural
conditions. SA-treatments significantly elevated the total SA levels over the whole sampling period. Total
Keywords:
glutathione (tGSH) and total cysteine (tCys) increased by 167% and 80%, respectively, two weeks after
Antioxidant defence system
Ascorbic acid
treatment, in comparison with controls. In contrast, SA-treatment caused an initial deterioration in total
Glutathione ascorbic acid (tASC) and enhanced the percentage of dehydroascorbic acid (DHA), but activated tASC
Ips typographus levels over later sampling dates. The initial bark beetle attack was characterized by a significant decline
Picea abies in total SA levels, which was accompanied by a transient degradation and oxidation of their ascorbate-
Phenolics glutathione system. This initial reaction was significantly alleviated by SA-application and characterized
Salicylic acid by 175% higher tGSH contents, when compared to moderately-affected untreated trees. One month after
pheromone dispensers were placed on trees, an intensification of ascorbate-glutathione system occurred
within moderately-affected bark, but to a greater extent after SA-treatment. Total SA levels within SA-
treated moderately-affected trees remained at the control level until June. In contrast, strong attack was
characterized by a successive increase in total SA up to 252% following SA-treatment in June, whereas
a 110% increase of SA was determined within severely affected control-bark. A strong attack was fur-
ther characterized by a degradation of tGSH and total phenolics (tPH), a moderate increase in tASC and
an oxidation of the ascorbate-glutathione pool within untreated bark. In the SA-treated trees the redox
state was unaffected by severe colonization and the degradation of antioxidants was significantly allevi-
ated. In addition, SA-treated bark had significantly less entrance holes and exhibited fewer and shorter
maternal galleries than control-bark. From this perspective, exogenous SA was successfully implicated as
an activator of systemic acquired resistance in Norway spruce, providing tolerance against the complex
interactive effects of bark beetle attack and environmental factors.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction
Bark anatomy and the physiological condition of a potential host
tree are crucial for the success of bark beetles, which are the most
frequent pest of Norway spruce [Picea abies (L.) H. Karst.] in Europe.
Conifer stem pest resistance includes constitutive and inducible
defences, which have attracted much attention over recent years
Abbreviations: DW, dry weight; DHA, dehydroascorbic acid; GSSG, glutathione
disulfide; MJ, methyl jasmonate; MS, methyl salicylate; ROS, reactive oxygen
species; SA, salicylic acid; SAR, systemic acquired resistance; tASC, total ascorbic
acid; tCys, total cysteine; tGSH, total glutathione; tPH, total phenolics.
∗ Corresponding author. Tel.: +386 2 320 90 53; fax: +386 2 616 91 00.
E-mail addresses: andreja.urbanek1@uni-mb.si, andreja.urbanek@uni-mb.si
(A. Urbanek Krajnc).
(Nagy et al., 2004; Franceschi et al., 2005; Bonello et al., 2006). Dur-
ing a successful bark beetle attack, systemic acquired resistance
(SAR) becomes effective and represents a third defence strategy
for the attacked tree. It gradually develops throughout the tree
and provides a systemic change to the whole tree’s metabolism
(Christiansen et al., 1999; Evensen et al., 2000; Percival, 2001; Nagy
et al., 2004; Wermelinger, 2004; Franceschi et al., 2005). SAR relies
on a subsequent signal-transduction cascade, which involves sal-
icylic acid (SA), jasmonic acid, ethylene, hydrogen peroxide, and
superoxide radicals as major signalling compounds able to induce
the expressions of many defence-related genes through differ-
ent pathways (Shah, 2003; Durrant and Dong, 2004; Hayat et al.,
2007, 2010). General knowledge about SA metabolism, interac-
tions with other compounds involved in the SAR and its significance
for mediating stress-tolerance, has been mainly obtained through
investigations on economically important crop plants in relation

0378-1127/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.foreco.2010.10.027
 A. Urbanek Krajnc et al. / Forest Ecology and Management 261 (2011) 416–426
to increasing resistance to fungal, bacterial, and viral pathogens
(Jameson and Clarke, 2002; Vallad and Goodman, 2004; Radwan
et al., 2007; Urbanek Krajnc et al., 2008; Hayat et al., 2010). SA
has an affinity to binding with enzymes such as catalase, ascor-
bate peroxidase, aconitase, and carbonic anhydrase, and some
of these enzymes are involved in reactive oxygen species’ (ROS)
metabolisms and in redox homeostasis. Any alteration in this
homeostasis leads to the induction of a defense response in plants
(Chen et al., 2001; Mittler, 2002; Slaymaker et al., 2002; Hayat
et al., 2010). Little is known about SA accumulation in response to
the pathogen challenge in conifers. Kozlowski and Metraux (1998)
reported that SA levels increased in the roots of spruce seedlings
during infection with Pythium irregulare. Information concerning
the effects of exogenous SA-treatment on conifer defence responses
is still limited (Percival, 2001; Davis et al., 2002; Martin et al., 2003;
Hudgins and Franceschi, 2004; Rodrigues and Fett-Neto, 2009). Fol-
lowing SA-treatment, Pinus species were induced to express three
chitinase homologues, which are part of protein-based chemical
defenses, and are presumed to be effective against the cell walls
of fungal pathogens (Davis et al., 2002). Rodrigues and Fett-Neto
(2009) reported that 10 mM and 100 mM SA concentrations were
capable of significant induction of resin production in Pinus elliottii.
In contrast, Hudgins and Franceschi (2004) reported that exoge-
nous methyl salicylate (10, 25, 50, or 100 mM MS) had no apparent
effect on resin accumulation, phenolic synthesis in polyphenolic
parenchyma cells or fibre lignification in Pseudotsuga menziesii and
Sequoiadendron giganteum.
Within the broad range of defence mechanisms, the activation
of antioxidants significantly contributes to the appearance of SAR.
An antioxidant defense system is generally linked to the actions
of reactive oxygen species and is determined by the pool size of
the antioxidants (Noctor et al., 2002; Barna et al., 2003; Foyer and
Noctor, 2005; Noctor, 2006). Among antioxidants, glutathione is
a low-molecular sulfur metabolite, which performs multiple roles
in tree-environment interactions and defense (Grill et al., 2001;
Tausz et al., 2004; Tausz, 2007; Zhao et al., 2008). It functions as a
reductant in the enzymatic detoxification of ROS in the glutathione-
ascorbate cycle, and as a thiol buffer in the protection of proteins
via direct reaction with ROS or by the formation of mixed disulfides.
In this role, it has been suggested as a general redox sensor and sig-
nalling agent in plant cells (Noctor, 2006; Meyer and Rausch, 2008;
Zhao et al., 2008). Trees under stress seem to generally require and
synthesize higher concentrations of glutathione. Glutathione syn-
thesis depends on the distribution and cycling of sulfur in trees
(Herschbach and Rennenberg, 2001a,b; Tausz et al., 2001; Tausz,
2007; Bloem et al., 2005, 2007; Struis et al., 2008; Zhao et al., 2008).
Norway spruce takes up sulfate, transports it into the canopy, where
it is reduced mainly in older needles. The reduced sulfur require-
ments of the organs below the canopy may be met by root sulfur
reduction (Kostner et al., 1998; Tausz, 2007). The sulfur amino acid
cysteine is essential for most herbivores and, hence, a potential
determinant regarding the quality of the feed. Highly reduced sulfur
contents in acorns are translated directly to a high cysteine status of
feeding mites (Grill et al., 2003). Furthermore, it has been hypothe-
sized that reduced sulfur compounds in the bark are a determinant
for the breeding success of bark beetles, but a clear connection has
not been established, as yet (Mattanovich et al., 2001).
Glutathione is central to the regeneration of ascorbate within
the ascorbate-glutathione-cycle (Foyer and Noctor, 2005; Tausz
et al., 2004; Tausz, 2007). In addition to being the most abundant
water-soluble antioxidant in plant cells (Smirnoff and Wheeler,
2000), ascorbate is also required for the re-conversion of SA• to
SA yielding monodehydroascorbate, since ascorbate is highly reac-
tive against phenoxyl radicals generated by peroxidases during
oxidative stress (Kawano and Muto, 2000). Until now only a few
investigations have dealt with antioxidative systems in bark bee-
417
tle affected Norway spruce (Mattanovich et al., 2001; Pučko et al.,
2005), although antioxidative defence systems have often been
used as stress indicators for the diagnosis of disturbances in for-
est trees (Tausz et al., 2001, 2004; Tausz, 2007; Herbinger et al.,
2005; Hofer et al., 2008). In previous work (Urbanek Krajnc, 2009),
a temporal analysis of antioxidant contents in the phloem tissue
of Norway spruce was performed when the bark was exposed to
increasing levels of bark beetle attack. It has been demonstrated
that, the sequence of events in the antioxidative responses during
bark beetle attack strengthens the general well-accepted ecophysi-
ological stress-response concept (Larcher, 2003; Tausz et al., 2004).
Many studies on agricultural plants demonstrated that SA-
treatment strongly induces the synthesis of antioxidants (ascorbate
and glutathione), antioxidant enzymes (glutathione transferase,
glutathione peroxidase, ascorbate peroxidase), and provides
increased tolerance against biotic and abiotic stress factors
(Radwan et al., 2007; Urbanek Krajnc et al., 2008; Hayat et al., 2010).
To our knowledge, the role of exogenous SA in the induction of
antioxidant defence responses in conifers, has not been part of any
study, as yet. Therefore, the aim of the presented field study is to test
(1) whether SA-treatment cause an artificial elevation of antioxi-
dants in the bark of Norway spruce, (2) how long it is effective and
(3) whether its effect is systemic. Futhermore, we want to clarify (4)
whether a successful defense of Norway spruce can be induced by
SA-treatment and (5) whether SA-treatment under natural condi-
tions is sufficient to protect trees from mass-attacks by bark beetles.
In the presented field experiment, stem sections were treated with
100 mM SA prior to the induction of the bark beetle attack. Antiox-
idant levels (ascorbic acid, cysteine, glutathione, phenolics) and SA
levels were monitored (a) 72 h after SA-treatment, in order to ana-
lyze the extent to which SA increases antioxidant levels in different
stem sections; and (b) four times after bark beetle attraction during
a three-month period until the first generation of bark beetles com-
pleted their life-cycles in most of the affected trees. This approach
enabled the establishment of correlations between quantitative
changes in SA and the antioxidant system, and gave an insight into
the interactions among these defense molecules during bark beetle
colonization, ranging from successful defense to tree death.
2. Material and methods
2.1. Study site and technical preparation
The investigation took place in a Norway spruce monocul-
ture at Meranovo, Spodnji vrhov dol, Pohorje, Slovenia (latitude
46◦ 32 17,25 –46◦ 32 23,36 , longitude 15◦ 33 12,53 –15◦ 33 17,30 ,
and altitude: 474–493 m; exposition: N–NW; inclination: 5◦ ).
The studied area could be characterized as a second growth
even-aged stand with dense patches of small diameter trees (35-
year-old Norway spruce trees, tree height: 20 ± 2 m; crown length:
8 ± 0.7 m; DBH: 25 ± 2 cm). Trees, obtained from the Slovenian
Forest Service, represented a genetically variable population origi-
nating from open pollination among local genotypes.
The experimental area involved two 600 m2 plots approxi-
mately 50 m apart (bark beetle colonized, and control). The 5 m
border around the infested plot was treated twice with insecti-
cide (Fastac® , 0.3%, BASF) during maximum beetle-flight activity,
in order to protect single susceptible trees and prevent the migra-
tion of bark beetles. Both pure spruce plots were surrounded and
separated by a mixed mature species stand. The test trees (n = 16
for control and n = 16 for colonized trees) were randomly selected
within both plots. Test trees were divided into four groups:
(1) The first group of eight individuals was selected within the
control plot, the trees were pre-treated with SA and remained
unaffected (SA treatment, no attack).
418 A. Urbanek Krajnc et al. / Forest Ecology and Management 261 (2011) 416–426
(2) The second group of test trees was pre-treated with SA within
bark beetle-affected plot and three days later subjected to bark
beetles (SA treatment, attack).
(3) The third group represented a control group (untreated and
healthy trees), in order to study seasonal (environmental) vari-
ations in antioxidant contents (control, no attack).
(4) The fourth group of test trees was left untreated but was sub-
jected to bark beetles (control, attack).
The distances between the test trees ranged from 13 m to 15 m.
SA-treatment date (10 April 2007) and sampling dates were chosen
according to the weather conditions, and the activity period of the
bark beetles (Urbanek Krajnc, 2009).
2.2. Salicylic acid treatment experiment
SA treatment of test trees (group 1, 2 within control and bark
beetle-affected plots) took place on 10 April 2007. A stem-section
of each tree between 0.1 and 5 m above the ground was divided by
two vertical lines into east- and west-facing halves. The east half of
each tree was treated with 500 mL of 100 mM SA (Sigma–Aldrich)
in water with 0.1% (v/v) Tween-20 (pH 6.8), while the west half
was left untreated. Control trees were treated with 500 mL of 0.1%
Tween-20 solution (adapted from Hudgins and Franceschi, 2004).
Tween 20 is a biologically nonactive detergent used to emulsify SA
and to act as a surfactant to evenly spread the solution on the bark
surface (Franceschi et al., 2002; Hudgins and Franceschi, 2004). SA
was applied onto the stem using a paint roller. This procedure was
repeated after 5 min to ensure a uniform coating (adapted from
Erbilgin et al., 2006; Zeneli et al., 2006; Rodrigues and Fett-Neto,
2009).
2.3. Pheromone-induced attraction of bark beetles
On the 13 April 2007 (three days after SA-treatment), a
pheromone dispenser (Pheroprax® , Cyanamid Agrar, Germany)
was placed on the north side of each tree (groups 2 and 4) within
the bark beetle-affected plot, 2 m above the ground, in order to
induce bark beetle attack. At this time, the average night tem-
perature was approx. 10 ◦ C over three days. At the beginning of
the experiment, the beetle population in this area was moderate
(Urbanek Krajnc, 2009).
2.4. Assessment of the beetle attack
At the end of experiment, the trees were felled, and the outer
cork bark was carefully shaved away on both sides of the trees
(east/west sides) between 1.5 and 2.5 m above ground. The number
of entrance holes and galleries (tunnel length > 10 mm), the mean
and total lengths of all maternal galleries, and the number of larval
galleries, were recorded according to Erbilgin et al. (2006).
At the end of June, the first young beetles emerged from the
barks of severely affected trees. Two trees (control/strong attack)
that had been mass-attacked and killed were excluded from the
experiment at the end of June, since their bark was crowded with
well-developed beetle galleries. The experiment was finished in
mid-July, when the first generation of bark beetles completed their
life-cycles in most of the affected trees. To prevent the emergence
and migration of a beetles’ second generation, the trees were felled,
the logs were debarked, and the bark burned.
2.5. Sample preparation for biochemical analysis
In order to obtain a temporal sequence of defensive chemicals,
the test trees were sampled five times from 13 April to 15 July 2007.
Two samples containing bark and secondary phloem (6 cm × 6 cm)
were collected on east side of test tree at 1.3 m and 3.3 m above the
ground. The bark was removed and the total phloem immediately
frozen in liquid N2 , and transferred to the freezer (−80 ◦ C). The sam-
ples were taken on a clear day between 11.00 and 14.00 solar time.
Further processing (freeze-drying, pulverization) was done accord-
ing to Tausz et al. (2003). For biochemical analysis, both samples
of each tree were mixed together in equal volume proportions and
used as one sample.
2.6. Determination of salicylic acid
SA (free and conjugated) was determined in methanol extracts
using an isocratic HPLC technique modified according to Pasqualini
et al. (2002) and Verberne et al. (2002). SA was extracted from
bark samples (500 mg FW) by homogenization in mortar with 2 mL
30% (v/v) methanol in water. The homogenate was mixed by vor-
tex, sonicated for 5 min and centrifuged at 6000 × g for 5 min.
The supernatant was collected, the pellet was re-suspended twice
with 100% methanol, and the sonication and centrifugation steps
were repeated. After these three extraction steps the supernatans
were pooled and 10 ␮L of 0.2 M NaOH was added, in order to
prevent the sublimation of SA. The methanol:water mixture was
concentrated at ambient temperature using a Eppendorf concen-
trator. The concentrated extracts were divided into two aliquots
each. One aliquot was used for the analysis of free SA in HPLC.
In order to determine the ␤-glucosylsalicylic acid content, the
other aliquot of the methanolic extract (1 mL) was re-suspended
in 2 mL of 8N HCl and 1 mL of 3.7 M NaCl, and hydrolyzed for
1 h at 80 ◦ C. The cooled mixture was then purified through a Sep-
Pak C18 column. The samples were eluted by 1 mL 100% methanol
and then analyzed using Waters HPLC system (Waters 600E Con-
troller Pump, Waters 2475 Multi Fluorescence Detector, Waters
Software/Hardware package, cooled Waters autosampler) at exci-
tation: 315 nm wavelength and emission: 405 nm wavelength).
Column Sphericorb S5 ODS2 25 × 4.6 ␮m. The samples were frac-
tioned isocratically with 45% (v/v) methanol in water (2% (v/v)
acetic acid); the flow-rate was 0.8 mL min−1 . Recovery analysis for
determining the total SA content showed 75 ± 15% recovery; cor-
rections were made for recovery rates.
2.7. Determination of antioxidants
Total ascorbic acid (tASC) and dehydroascorbic acid (DHA) were
analyzed by an isocratic reversed-phase chromatography method
according to Tausz et al. (2003) and Herbinger et al. (2005).
Thiols (total cysteine, tCys; total glutathione, tGSH; oxidized glu-
tathione, GSSG) were determined by gradient high-pressure liquid
chromatography (HPLC), after the labelling of thiol groups with
monobromobimane, as described by Tausz et al. (2003). Total phe-
nolic compounds (tPH) were determined spectrophotometrically,
according to Ainsworth and Gillespie (2007).
2.8. Statistics
The results of biochemical analyses represented the mean and
standard deviations (S.D.) of eight replicate samples. They were
statistically evaluated by Kruskal-Wallis test, followed by post hoc
comparisons according to Conover (Bortz et al., 2000). Significant
differences were indicated by different letters (a–e). Decision rule:
P < 0.05 was regarded as significant.
The assessment of the beetle attack was evaluated by two-
way analysis of variance (ANOVA). The homogeneity of variance
was tested using Levene’s test. Post hoc comparisons among the
treatments were conducted with the help of the Fischer LSD test.
Decision rule: P < 0.05 was regarded as significant. Calculations
were performed on the Statistica 6.0 software package (StatSoft
Europe GmbH, Hamburg, Germany, www.statsoft.de).
 A. Urbanek Krajnc et al. / Forest Ecology and Management 261 (2011) 416–426 419

Fig. 1. Effect of the salicylic acid (SA)-treatment of Norway spruce on Ips typographus colonization, assessed on east-facing stem sections between 1.5 and 2.5 m above ground.
Each value is the mean of eight independent tree samples ± S.D. Different letters (a and b) indicate significant differences (P < 0.05) between the control and affected trees.

3. Results
3.1. I. typographus colonization after salicylic acid-treatment
The population level of I. typographus in the area was moderate
at the beginning of the experiment (Urbanek Krajnc, 2009). The
first entrance holes were observed on all test trees three days after
placing pheromone dispensers on the trees, but a higher number
was characterized for untreated trees.
SA treatment reduced bark beetle colonization, since it inhibited
both the entry into the bark, and sustained tunnelling activity. At
the end of the experiment, the SA-treated bark had fewer entrance
holes, although the effect was not quantified by significant interac-
tions. The differences between the SA-treated and untreated barks
were significantly greater regarding the number of galleries and
gallery lengths. The treated bark had fewer galleries, and their
lengths were shorter than in the barks of the untreated Norway
spruce trees (Fig. 1).
3.2. Salicylic acid concentrations
During the sampling period, the control/unattacked trees
showed a gradual increase in total SA levels from 7 ␮mol/g FW in
April to 13 ␮mol/g FW in July. Three days after the stem sections
were treated with 100 mM SA, a significant increase was deter-
mined in both free SA (50%) and total SA (35%). Both free and total SA
contents remained significantly higher by approximately 40% in the
SA-treated healthy trees, over the whole sampling period (Fig. 2).
Two weeks after pheromone dispensers were placed on the
trees, the initial response to bark beetle attack was characterized
by a significant decline in total SA contents (−55% above control)
in untreated trees. Within the affected SA-treated bark the total
SA concentrations also diminished but remained at the levels of
the control healthy barks. Free SA contents were lower than on the
first sampling date and remained unaltered by SA-treatment.
On 18 May 2007, both the control and SA-treated barks showed
similar patterns in response to progressive bark beetle attack but
the SA-treated barks had, in general, significantly higher SA levels
in comparison with the affected control-bark. During a moderate
attack, the total SA levels within the SA-treated barks were simi-
lar to those of the healthy controls, and remained almost constant
until July. In the untreated moderately-affected barks, the total SA
contents were 35% lower. In the severely affected SA-treated trees,
free and total SA contents were markedly raised by approximately
80%, whereas in the untreated barks only a slight increase (20%)
was determined (Fig. 2).

Fig. 2. Concentrations of free SA and total SA in the phloem tissues of SA-treated and control-trees attacked to varying degrees by Ips typographus during the sampling period.
Mean (S.D.) n = 8 (SA/no attack, control/no attack) and n = 3–5 (SA/colonized trees; control/colonized trees). Different letters (a–c) indicate significant differences (P < 0.05)
between the control and affected trees.
420 A. Urbanek Krajnc et al. / Forest Ecology and Management 261 (2011) 416–426
Fig. 3. Total Cys concentrations in the phloem tissues of SA-treated trees attacked
to varying degrees by Ips typographus during the sampling period. Mean (S.D.) n = 8
(SA/no attack) and n = 3–5 (SA/colonized trees). Different letters (a–d) indicate sig-
nificant differences (P < 0.05) between the control and affected trees.
On 15 June 2007, the total SA contents within the SA-treated
moderately-affected barks remained equal to those values deter-
mined on earlier sampling dates. Severely affected SA-treated
tissue was characterized by dramatically-enhanced total SA con-
tents (252%) but only slightly enhanced free SA levels. In severely
affected untreated barks, both free and total SA increased by 110%.
On 11 July 2007, an obvious depletion in total SA was deter-
mined in comparison with the mid-June sampling date, depending
on the intensity of the attack. In moderately-affected barks, free
SA declined by approximately 70% and total SA by 40%, in both
SA-treated and control-barks. Considerable changes were noticed
between the SA-treated and control-barks after massive bark bee-
tle colonization. In the SA-treated barks, the total SA levels stayed
as increased by 24% above the levels of the healthier barks, whereas
a 68% degradation of total SA was determined in severely-affected
control-barks (Fig. 2).
3.3. Thiol concentrations and redox state
SA-treatment, prior to a bark beetle attack had positive impacts
on the antioxidant defense response in the barks. Within the unaf-
fected barks, tGSH increased significantly by 167% and tCys by
80% two weeks after SA-treatment (Figs. 3 and 4; Supplementary
Figures 1, 2).
After initial bark beetle attack, the concentration of tGSH and
tCys dropped, and the glutathione redox state transiently shifted
towards a slightly more oxidized value (Figs. 3 and 4). In spite
of these initial events, the affected SA-treated barks had 175%
more tGSH, in comparison with the moderately-affected control-
barks (Supplementary Figure 2). Total Cys concentrations declined
more strongly within the SA-treated moderately-affected barks
but remained significantly increased by 34% over the levels of the
moderately-affected control trees (Supplementary Figure 1).
On 18 May 2007, the antioxidative shifts, after a moderate
attack, differed significantly from the reaction after massive col-
onization. After moderate bark beetle colonization, the SA-treated
barks contained 75% higher levels of total GSH when compared to
the unaffected SA-treated trees (Fig. 4), but the SA-induced increase
was insignificant when compared to the moderately-affected
control-trees (Supplementary Figure 2). Total Cys also accumu-
lated by moderate bark beetle attack (Fig. 3), but tCys contents
were found to be significantly higher in the moderately-affected
untreated barks (Supplemetary Figure 1). In the strongly-affected
SA-treated trees, tCys concentrations remained at a control
level (Fig. 3, Supplementary Figure 1). Total GSH concentrations
decreased in severely affected SA-treated trees (Fig. 4). How-
ever, the tGSH levels were 110% higher when compared to the
massively-affected control trees, which were characterized by a
strong decrease in tGSH concentrations (Supplementary Figure 2).
On 15 June 2007, the levels of tGSH and tCys decreased and
the percentages of GSSG increased in both the moderately and
strongly-affected trees (Figs. 3 and 4), but the degradation was sig-
nificantly less marked when trees were pretreated with SA. Total
Cys contents were 80% higher in the SA-treated, severely-affected
trees when compared to the untreated trees, after strong attack
(Supplementary Figure 1). Moreover, total GSH contents were 550%
higher in the SA-treated severely affected trees when compared to
the severely-affected control trees, where a massive deterioration
of tGSH was determined (Supplementary Figure 2).
On 11 July 2007, the concentration levels of tGSH within the
moderately-affected bark were slightly lower (Fig. 4), but the
SA-treated and untreated samples were not quantified by signifi-
cant interactions (Supplementary Figure 2). The strong decline in
tCys was less pronounced in the SA-treated moderately-affected
trees, which contained 72% more tCys when compared to the
moderately-affected control trees (Supplementary Figure 1). In
the severely-affected SA-treated trees, tGSH and tCys decreased
by 72% and 45%, respectively, when compared to the SA-treated
unaffected trees (Figs. 3 and 4), but both molecules remained
increased over the levels of the severely-affected control trees
(Supplementary Figures 1, 2).
3.4. Ascorbate concentrations and its redox state
Three days after the SA-application, tASC decreased by 32% on
the treated side of the investigated tree, whereas the percentage of
DHA increased by up to 45% (Fig. 5, Supplementary Figure 3). The
seasonal pattern of tASC was significantly affected by SA-treatment.
The treated trees showed the highest values two weeks after the
treatment and then one month later, and the lowest levels in mid-
June (−28%), which coincided with the tASC contents of the first
sampling date (Fig. 5, Supplementary Figure 3).
On 26 April 2007, tASC and the percentage of DHA increased
in both SA-treated and untreated barks, in comparison with those
concentrations determined on the first sampling date, probably
as a reaction to sampling wounds. The initial reaction of tASC
to bark beetle attack underwent similar changes to tGSH. In
the moderately-affected SA-treated trees the tASC concentrations
were lower (Fig. 5), however, the SA-treated trees contained sig-
nificantly higher tASC concentrations (26%) when compared to
the moderately-affected controls (Supplementary Figure 3). On
18 May 2007, the tASC contents were higher in the SA-treated
moderately-affected trees, in comparison with the moderately-
affected control-trees, although the effect of SA treatment was
insignificant (Supplementary Figure 3). The SA-treated trees, which
were strongly affected by bark-beetles, showed lower tASC levels
but a smaller percentage of DHA, in comparison with the strongly-
affected control trees, although the difference was insignificant
(Fig. 5, Supplementary Figure 3). On 15 June 2007, tASC contents
within the SA-treated unaffected samples dropped significantly
below the control levels (Supplementary Figure 3). Bark beetle
accumulation caused a significant increase in tASC contents, which
remained below the levels of bark beetle affected controls (Fig. 5,
Supplementary Figure 3). On 11 July 2007, a weak bark beetle attack
was characterized by a considerable accumulation of tASC (48%),
whereas an advanced bark beetle attack caused a moderate increase
in tASC concentrations, when compared to SA-treated unaffected
trees (Fig. 5). Any significant effect of SA-treatment was not found
(Supplementary Figure 3).
 A. Urbanek Krajnc et al. / Forest Ecology and Management 261 (2011) 416–426 421

Fig. 4. Concentrations of tGSH and GSSG (% of total) in the phloem tissues of SA-treated trees attacked to varying degrees by Ips typographus during the sampling period.
Mean (S.D.) n = 8 (SA/no attack) and n = 3–5 (SA/colonized trees). Different letters (a–e) indicate significant differences (P < 0.05) between the control and affected trees.

3.5. Total phenolic concentrations
The concentrations of tPH within the control/unattacked trees
increased by 30% on the second sampling date as a response to
wounding, and remained almost constant until the end of the sam-
pling period. SA-treated trees did not respond to elevated tPH levels
on the second sampling date and the levels remained stable during
the whole sampling period (Fig. 6, Supplementary Figure 4).
On 26 April 2007, an initial bark beetle attack caused an increase
in tPH contents within the untreated trees. Interestingly, tPH did
not respond to bark beetle attack, when these trees were treated
with SA. The tPH levels within the SA-treated moderately-affected
trees were thus significantly lower (23%) when compared to the
moderately-affected controls (Fig. 6, Supplementary Figure 4). On
18 May 2007, tPH increased similarly to tGSH in the moderately-
affected barks, but to a significantly higher level in the untreated
trees. The degradation of tPH within the strongly-affected barks
was less expressed when the bark was pre-treated with SA (Fig. 6,
Supplementary Figure 4). On 15 June 2007, the concentrations of
tPH were similar in the SA-treated trees to those in the untreated
trees, moderately-affected by bark beetles. Any advanced disease-
stage was characterized by a significant degradation of tPH, which
was more pronounced in the untreated trees. Moreover, the SA-
treated severely-affected trees had 215% higher concentrations
of tPH when compared to the severely-affected control trees
(Supplementary Figure 4).
On 11 July 2007, tPH within the moderately-affected SA-treated
samples remained at control levels. In contrast, a significant
increase in tPH contents was measured within the untreated barks.
The moderate decline in tPH after massive bark beetle colonization
within the SA-treated samples remained almost constant in com-
parison with earlier sampling dates (Fig. 6, Supplementary Figure
4).
4. Discussion
In the presented study, the effect of SA-treatment on bark bee-
tle colonization and antioxidant defense system has been studied
under natural field conditions, ranging from successful defense to
tree death. The sequence of events in antioxidative response dur-
ing bark beetle attack showed significant quantitative differences
and time-shifts in antioxidant contents between SA-treated and
control-trees. Furthermore, SA-application inhibited entry into the
bark and arrested the beetle’s gallery construction, leading to sig-
nificantly lower I. typographus colonization on SA-treated tree. A
shorter gallery length could be associated with SA-induced qualita-
tive and quantitative differences in host chemistry, and the internal
physiology of Norway spruce bark, which is less suitable for bee-
422 A. Urbanek Krajnc et al. / Forest Ecology and Management 261 (2011) 416–426

Fig. 5. Concentrations of tASC and DHA (% of total) in the phloem tissues of SA-treated trees attacked to varying degrees by Ips typographus during the sampling period. Mean
(S.D.) n = 8 (SA/no attack) and n = 3–5 (SA/colonized trees). Different letters (a–e) indicate significant differences (P < 0.05) between the control and affected trees.

Fig. 6. Total PH contents in the phloem tissues of SA-treated trees attacked to varying degrees by Ips typographus during the sampling period. Mean (S.D.) n = 8 (SA/no attack)
and n = 3–5 (SA/colonized trees). Different letters (a–c) indicate significant differences (P < 0.05) between the control and affected trees.
 A. Urbanek Krajnc et al. / Forest Ecology and Management 261 (2011) 416–426
tles. Similarly, 100 mM MJ applied to spruce bark has been reported
to inhibit both the entrance into the bark, as well as the extent of
beetle tunneling (Erbilgin et al., 2006).
In order to evaluate the effect of exogenous SA on antioxidant
defense system the preliminary objective of the presented study
was to clarify as to which level total SA contents increase in phloem
after exogenous application of SA to bark sections. This informa-
tion is of particular importance, since the SA-treatment experiment
in vivo was significantly influenced by weather conditions. Further-
more, outer bark is known as an anatomical or physical barrier to
substance exchange (Franceschi et al., 2005), and SA is supposed
to translocate inefficiently throughout the plants when applied
exogenously (Percival, 2001; Ohashi et al., 2004). The results of
the presented study demonstrated that within secondary phloem
both free and total SA contents remained significantly increased by
approximately 40% in SA-treated unaffected trees over the whole
sampling period. These preliminary results stressed the long-term
effect of exogenously applied SA in the persistence of SAR and are
in agreement with the hypothesis that, once SAR is induced it can
lead to long-lasting, broad-spectrum disease control (Kozlowski
and Metraux, 1998; Percival, 2001). It was suggested that free SA as
an active form is transported across plasma membranes (Chen et al.,
2001). Another study suggested that SA can pass through a tough
cuticular layer, in its methylated form (Ross et al., 1999). Methyl
salicylate is known as a volatile long-distance signalling molecule
that moves through phloem and can be converted to SA whenever
required (Hayat et al., 2010). Furthermore, the conversion of SA
to glycosides may prevent the phytotoxic accumulation of free SA
during the treatment (Jameson and Clarke, 2002).
In the presented experiment, the sequence of changes in SA
contents during bark beetle attack was pointed out as a dynamic
process, which depends on the severity of bark beetle colonization,
and environmental factors such as high temperatures and short-
term drought-stress in June (Urbanek Krajnc, 2009). The initial bark
beetle attack caused a significant decline in total SA levels within
untreated bark two weeks after pheromone dispensers were placed
on the trees. The initial depletion of total SA was followed by a
successive intensification of total SA contents within moderately-
affected untreated bark up to the levels of healthy control, in June.
In SA-treated moderately-affected bark, the total SA concentrations
were significantly higher when compared to bark beetle attack
alone. Later on, during a moderate bark beetle attack, the total SA
concentrations of SA-treated trees remained stable until July. Mas-
sive bark beetle attack provided a strong upward-regulation of SA,
which developed gradually until mid-June and became much more
manifested in severely affected trees after SA-treatment. These
results clearly demonstrated that the observed retention of stable
levels of SA during moderate attack, and especially high levels of
SA during massive bark beetle attack, were essential for a success-
ful defense reaction following SA-treatment. The mechanism for
SA action was previously suggested by Kawano and Muto (2000).
SA and H2 O2 are needed for an SA-generated peroxidase reaction.
Then the resulting SA• reacts with O2 to produce O2 •− , that triggers
an increase in Ca2+ . The increased Ca2+ may induce further physi-
ological responses, including the induction of defense protein and
antioxidant genes (Kawano and Muto, 2000; Kawano et al., 2004;
Hayat et al., 2010).
It is well-known that the exogenous application of SA activates
the synthesis of antioxidants and antioxidant enzymes, and also
confers resistance against various pathogens in a variety of dicot
and monocot species (Fodor et al., 1997; Urbanek Krajnc et al.,
2008; Hayat et al., 2007, 2010). The biochemistry of antioxidant
metabolism in tree cells is fundamentally similar to plant cells in
general, in modifications of the whole plant’s metabolism relat-
ing to the typical biology of trees, for example long-life spans,
long internal transport distances, and large volumes of woody
423
tissues, are significant (Tausz, 2007). The presented experiment
clearly demonstrated that SA-application to bark sections had pos-
itive effects on the accumulation of thiols (tGSH and tCys). These
results, in agreement with previous studies, suggested a close
relationship between SA and glutathione pathways (Fodor et al.,
1997; Chen et al., 2001; Freemann et al., 2005; Urbanek Krajnc
et al., 2008; Khan et al., 2010). These authors demonstrated that
the glutathione-mediated tolerance mechanism is signalled by the
constitutively-elevated levels of SA. They proposed that elevated SA
post-translationally up-regulated serin acetyltransferase activity,
causing constitutively elevated glutathione. Serin acetyltransferase
catalyzes the acetylation of L-serin to produce O-acetyl-L-serine,
which acts as a positive key regulator of sulfur assimilation and
forms a carbon skeleton for cysteine biosynthesis (Freemann et al.,
2004, 2005; Hell and Wirtz, 2008).
Glutathione is essential for the regeneration of ascorbate within
the ascorbate-glutathione-cycle (Tausz et al., 2001, 2004, 2005;
Gullner and Kömives, 2001). In addition to being the most abundant
water-soluble antioxidant in plant cells (Smirnoff and Wheeler,
2000), ascorbate is also required for the re-conversion of SA• to
SA yielding monodehydroascorbate, since ASC is highly reactive
against phenoxyl radicals generated by peroxidases during oxida-
tive stress (Kawano and Muto, 2000). However, contrary to thiols,
decreased tASC contents and a higher percentage of DHA were
measured three days after the bark was treated with SA. It can
be suggested that the exogenous application of SA is accompa-
nied by increased rates of ROS, which leads to an increased load on
the ascorbate-glutathione cycle. The results are supported by the
observation of Shi and Zhu (2008), where SA-treatment enhanced
the activities of dehydroascorbate reductase. A hypothesis has been
produced stating that any increased DHA production in SA-treated
bark could be related to the generation of SA• (Kawano and Muto,
2000). DHA is re-converted to ASC via an ascorbate-glutathione
pathway, which uses reduced glutathione as an electron donor
to regenerate ascorbate from its oxidized form (Noctor and Foyer,
1998; Noctor et al., 2002; Noctor, 2006).
Our research priority was to determine how the time-course
of antioxidant response to bark beetle attack is altered by SA-
treatment. This approach was important to clarify any cross-link
between SA pathway and ascorbate-glutathione cycle, and to define
the efficiency of antioxidant-mediated tolerance mechanism in
Norway spruce against bark beetle attack. Similarly to untreated
bark, the initial response of ascorbate-glutathione system to bark
beetle attack was also lowered in SA-treated bark, but both thiols
and tASC were significantly higher in SA treated samples than in
untreated samples after initial attack.
Relatively high temperatures in mid-May contributed to
increased flight activity. Consequently, fresh attacks on test trees
and light drought stimulated the antioxidant defense system of the
Norway spruce trees. Thus, in mid-May, obvious accumulations of
tCys and tGSH, as well as a moderate accumulation of tASC, were
detected in untreated bark after a moderate attack (Urbanek Krajnc,
2009). The increase in tGSH and tASC during moderate attack was
more pronounced after SA treatment, whereas tCys increased to a
higher percentage in untreated trees in response to moderate bark
beetle attack. Cysteine is a direct precursor of glutathione (Hell
and Wirtz, 2008), which was previously reported to increase lin-
early with higher concentrations of exogenous SA (Urbanek Krajnc
et al., 2008). Disproportionate differences in tCys and tGSH con-
tents between SA-treated and control samples during moderate
bark beetle attack reflect that cysteine was further transported,
degraded or incorporated into other synthetic pathways. The posi-
tion of cysteine biosynthesis between the assimilation of inorganic
sulfate and the metabolization of organic sulfide makes it a prime
target for the coordination of both complex processes and, thus,
plays an integral role in the regulation of primary sulfur metabolism
424 A. Urbanek Krajnc et al. / Forest Ecology and Management 261 (2011) 416–426
(Tausz, 2007; Hell and Wirtz, 2008). Moreover, the intermediate
reaction of cysteine synthesis, O-acetylserine, forms a direct con-
nection between sulfate assimilation with nitrate assimilation and
carbon metabolism (Hell and Wirtz, 2008; Urbanek Krajnc et al.,
2008; Zhao et al., 2008).
In mid-May the antioxidative shifts after massive colonization
differed quantitatively from the above discussed antioxidant reac-
tion after moderate attack. In untreated bark, massive bark beetle
colonization was characterized by strongly increasing SA levels,
whereas the tGSH contents strongly decreased and the percent-
age of GSSG rose to nearly 60% (Urbanek Krajnc, 2009). It can be
assumed, that the increase in SA contents followed by a higher
generation of SA• (Kawano and Muto, 2000) led to increase load
on ascorbate-glutathione cycle, which was characterized by more
oxidized glutathione pool. Recent studies on the ozone response of
beech suggested that the regeneration of glutathione is the limiting
step in antioxidative defence (Tausz et al., 2001; Herbinger et al.,
2005). It has been proposed that such a response can lead to an
acclimation of glutathione in the longer term or after an unsuccess-
ful defense degradation of the glutathione system was followed by
tissue dead (Tausz et al., 2004; Tausz, 2007). In the presented exper-
iment, SA treatment at this stage of defense reaction alleviated the
degradation of thiols, thus providing more efficient glutathione-
mediated tolerance mechanisms. A more reduced redox state
manifested the effective regeneration of glutathione within the
SA-treated bark.
On later sampling dates, the degradation of thiols was signif-
icantly delayed and not as obvious, when trees were pre-treated
with SA. The results reflect the importance of elevated thiol lev-
els in any initial reaction against bark beetle attack. Higher thiol
levels in mid-April seemed to be crucial for successful glutathione-
mediated defense reaction in the bark later on. They may also be
responsable for acclimatory responses of glutathione after success-
ful defense and delayed and alleviated deterioration processes in
case of unsuccessful defense. As glutathione levels have been found
to protect plants against pathogen attack and are involved in the
development of resistance against various pests, it can be assumed
that elevated glutathione contents indirectly suppress bark beetle
colonization by detoxifying pathogen-induced ROS, and by activat-
ing defense genes (Gullner and Kömives, 2001; Maughan and Foyer,
2006; Noctor, 2006).
Among antioxidants, phenolics represent a more important
component of the inducible defense strategy regarding conifer
bark. For the synthesis and accumulation of phenolic compounds,
the barks of all conifer families have polyphenolic parenchyma cells
(Krekling et al., 2000; Franceschi et al., 2000, 2005), providing phys-
ical and chemical resistance to penetration of the bark (Franceschi
et al., 2000, 2005; Schmidt et al., 2005). In our previous study, the
increase in tPH concentration two weeks after bark beetle attack
was recognized as an immediate inducible response to a bark beetle
attack (Urbanek Krajnc, 2009). Interestingly, the initial bark beetle
colonization did not cause any changes in tPH levels in SA-treated
trees, although it was reported that, after SA-treatment, the con-
tents of soluble phenolics in Fusarium oxysporum inoculated date
palm seedlings was about 4 times to 6 times higher than that in
untreated plants showing disease symptoms (Dihazi et al., 2003).
In the presented experiment, it seems that SA-treatment alleviated
the immediate response of PH compounds to bark beetle attack
and wounding observed in untreated tissues. A similar effect of
SA treatment on tPH was observed in mid-July, when moderately-
affected untreated trees responded with increased levels of tPH
due to the attack of the second generation of bark beetles (Urbanek
Krajnc, 2009). Besides these results, SA-dependent differences in
tPH concentration were observed at advanced disease stages. A
76% decline in tPH was measured in untreated trees (Urbanek
Krajnc, 2009), reflecting that the synthesis of phenolics is lacking,
when the phloem is damaged by the establishment of a complete
brood system (Franceschi et al., 2000, 2002, 2005). In contrast to
untreated trees, only a moderate decline in tPH concentration was
measured in SA-treated trees. These results provide evidence, that
SA-treatment inhibited the degradation of phenolics and activated
defense responses via a shikimic acid pathway.
In the presented experiment, the changes in the antioxidative
system during bark beetle attack are pointed out as a dynamic pro-
cess which is significantly triggered by SA-treatment. Furthermore,
SA-treatment results in a surprisingly long-term inhibition of bark
beetle colonization. From this perspective, the use of SAR against
insects and pathogens in trees would have a positive impact on
forestry management and would provide a potentially stable and
ecologically acceptable solution for minimizing attacks on living
trees.
Acknowledgments
This research was funded by Slovenian Research Agency (ARRS,
Z1-9602). The author thanks Prof. Dr. Božidar Krajnčič for valu-
able suggestions during the research. Mag. Ignac Vučko and Peter
Kramer from Maribor University Agriculture Centre, as well as Alojz
Pucko from the Slovenian Forest Service, are acknowledged for
permission to use the experimental field, and support during the
experimental work. The author also thanks Katja Urbanek, Robi
Gjergjek, Jožica Korez, Boris Sapač and Metka Visočnik for field
assistance. Prof. Dr. Maria Müller, Prof. Dr. Günther Zellnig, Dr.
Bernd Zechmann, Dr. Edith Stabentheiner and Dr. Astrid Wonisch
from the Institute of Plant Sciences, University of Graz, are also
acknowledged for effective collaboration. The authors also thank
the reviewers for valuable comments and suggestions.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.foreco.2010.10.027.
References
Ainsworth, E.A., Gillespie, K.M., 2007. Estimation of total phenolic content and other
oxidation substrates in plant tissues using Folin–Ciocalteu reagent. Nat. Protoc.
2, 875–877.
Barna, B., Fodor, J., Pogány, M., Király, Z., 2003. Role of reactive oxygen species and
antioxidants in plant disease resistance. Pest Man. Sci. 59, 459–464.
Bloem, E., Haneklaus, S., Schnug, E., 2005. Significance of sulfur compounds in the
protection of plants against pests and diseases. J. Plant Nut. 28, 763–784.
Bloem, E., Haneklaus, S., Salac, I., Wickenhäuser, P., Schnug, E., 2007. Facts and fiction
about sulphur metabolism in relation to plant–pathogen interactions. J. Plant
Biol. 9, 596–607.
Bonello, P., Gordon, T.R., Herms, D.A., Wood, D.L., Erbilgin, N., 2006. Nature and eco-
logical implications of pathogen-induced systemic resistance in conifers: a novel
hypothesis. Physiol. Molec. Plant Path. 68, 95–104.
Bortz, J., Lienert, G.A., Boenke, K., 2000. Verteilungsfreie Methoden in der Biostatistik.
Springer Verlag, Berlin, Heidelberg, New York, Tokyo.
Chen, H.-J., Hou, W.-C., Kuć, J., Lin, Y.-H., 2001. Ca2+ -dependent and Ca2+ -
independent excretion modes of salicylic acid in tobacco cell suspension culture.
J. Exp. Bot. 52, 1219–1226.
Christiansen, E., Krokene, P., Berryman, A.A., Franceschi, V.R., Krekling, T., Lieutier,
F., Lonneborg, A., Solheim, H., 1999. Mechanical injury and fungal infec-
tion induce acquired resistance in Norway spruce. Tree Physiol. 19, 399–
403.
Davis, J.M., Wu, H.G., Cooke, J.E.K., Reed, J.M., Luce, K.S., Michler, C.H., 2002.
Pathogen challenge, salicylic acid, and jasmonic acid regulate expression
of chitinase gene homologs in pine. Mol. Plant Microbe In. 15 (4), 380–
387.
Dihazi, A., Jaiti, F., Zouine, J., El Hassniand, M., El Hadrami, I., 2003. Effect of sali-
cylic acid on phenolic compounds related to date palm resistance to Fusarium
oxysporum f.sp. albedinis. Phytopath. Mediterr. 42, 9–16.
Durrant, W.E., Dong, X., 2004. Systemic acquired resistance. Annu. Rev. Phytopathol.
42, 185–209.
Erbilgin, N., Krokene, P., Christiansen, E., Zeneli, G., Gershenzon, J., 2006. Exogenous
application of methyl jasmonate elicits defenses in Norway spruce (Picea abies)
and reduces host colonization by the bark beetle Ips typographus. Oecologia 148,
426–436.
 A. Urbanek Krajnc et al. / Forest Ecology and Management 261 (2011) 416–426
Evensen, P.C., Solheim, H., Hoiland, K., Stenersen, J., 2000. Induced resistance of
Norway spruce, variation of phenolic compounds and their effects on fungal
pathogens. Forest Pathol. 30, 97–108.
Fodor, J., Gullner, G., Adam, A.L., Barna, B., Kömives, T., Kiraly, Z., 1997. Local and
systemic response of antioxidants to tobacco mosaic virus infection and to sal-
icylic acid in tobacco. Role in systemic acquired resistance. Plant Physiol. 114,
1443–1451.
Foyer, C.H., Noctor, G., 2005. Redox homeostasis and antioxidant signaling: a
metabolic inter-face between stress perception and physiological responses.
Plant Cell 17, 1866–1875.
Franceschi, V.R., Krokene, P., Krekling, T., Christiansen, E., 2000. Phloem parenchyma
cells are involved in local and distant defense responses to fungal inocula-
tion or bark-beetle attack in Norway spruce (Pinaceae). Am. J. Bot. 89, 602–
610.
Franceschi, V.R., Krokene, P., Christiansen, E., Krekling, T., 2005. Anatomical and
chemical defenses of conifer bark against bark beetles and other pests. New
Phytol. 167 (2), 353–375.
Franceschi, V.R., Krekling, T., Christiansen, E., 2002. Application of methyl jasmonate
on Picea abies (Pinaceae) stems induces defense-related responses in phloem
and xylem. Am. J. Bot. 89 (4), 578–586.
Freemann, J.L., Garcia, D., Kim, D., Hopf, A., Salt, D.E., 2005. Constitutively elevated
salicylic acid signals glutathione-mediated nickel tolerance in Thlaspi nickel
hyperaccumulators. Plant Physiol. 137, 1082–1091.
Freemann, J.L., Persans, M.W., Nieman, K., Albrecht, C., Peer, W., Pickering, I.J., Salt,
D.E., 2004. Increased glutathione biosynthesis plays a role in nickel tolerance in
Thlaspi nickel hyperaccumulators. The Plant Cell 16, 2176–2191.
Grill, D., Tausz, M., De Kok, L.J., 2001. In Significance of glutathione in plant adaptation
to the environment. In: De Kok, L.J. (Ed.), Handbook of Plant Ecophysiology, vol.
2. Kluwer, Dordrecht.
Grill, D., Tausz, M., Strnad, B., Wonisch, A., Müller, M., Raschi, A., 2003. Thiols in acorns
and feeding mites collected at sites with naturally elevated atmospheric sulphur
concentrations. In: Davidian, J.-C., Grill, D., De Kok, L.J., Stulen, I., Hawkesford,
M.J., Schnug, E., Rennenberg, H. (Eds.), Sulfur Transport and Assimilation in
Plants: Regulation, Interaction, Signaling. Backhuys, Leiden, The Netherlands,
pp. 213–215.
Gullner, G., Kömives, T., 2001. The role of glutathione and glutathione-related
enzymes in plant-pathogen interaction. In: Grill, D., Tausz, M., De Kok,
L.J. (Eds.), Significance of Glutathione to Plant Adaptation to the Environ-
ment. Kluwer Academic Publishers, Dordrecht, Boston, London, pp. 207–
239.
Hayat, Q., Hayat, S., Irfan, M., Ahmad, A., 2010. Effect of exogenous salicylic acid
under changing environment: a review. Environ. Exp. Bot. 68, 14–25.
Hayat, S., Ali, B., Ahmad, A., 2007. Salicylic acid: biosynthesis, metabolism and phys-
iological role in plants. In: Hayat, S., Ahmad, A. (Eds.), Salicylic Acid—A Plant
Hormone. Springer Verlag, Dordrecht, Boston, London, pp. 1–14.
Hell, R., Wirtz, M., 2008. Metabolism of cysteine in plants and phototrophic
bacteria. In: Hell, R., Dahl, C., Knaff, D., Leustek, T. (Eds.), Advances
in Photosynthesis and Respiration. Springer, Dordrecht, pp. 417–
435.
Herbinger, K., Then, Ch., Löw, M., Haberer, K., Alexous, M., Koch, N., Remele, K.,
Heerdt, C., Grill, D., Rennenberg, H., Häberle, K.H., Matyssek, R., Tausz, M., Wieser,
G., 2005. Tree age dependence and within-canopy variation of leaf gas exchange
and antioxidative defence in Fagus sylvatica under experimental free-air ozone
exposure. Environ. Pollut. 137, 476–482.
Herschbach, C., Rennenberg, H., 2001a. Significance of phloem-translocated organic
sulphur compounds for the regulation of sulphur nutrition. Progr. Bot. 62,
177–193.
Herschbach, C., Rennenberg, H., 2001b. Sulfur nutrition of deciduous trees. Natur-
wissenschaften 88, 25–36.
Hofer, N., Alexou, M., Heerdt, C., Löw, M., Werner, H., Matyssek, R., Rennenberg, H.,
Haberer, K., 2008. Seasonal differences and within-canopy variations of antiox-
idants in mature spruce (Picea abies) trees under elevated ozone in a free-air
exposure system. Environ. Poll. 154, 241–253.
Hudgins, J.W., Franceschi, V.R., 2004. Methyl jasmonate-induced ethylene produc-
tion is responsible for conifer phloem defense responses and reprogramming
of stem cambial zone for traumatic resin duct formation. Plant Physiol. 135 (4),
2134–2149.
Jameson, P.E., Clarke, S.F., 2002. Hormone-virus interactions in plants. Crit. Rev. Plant
Sci. 21, 205–228.
Khan, N.A., Syeed, S., Masood, A., Nazar, R., Iqbal, N., 2010. Application of salicylic
acid increases contents of nutrients and antioxidative metabolism in mungbean
and alleviates adverse effects of salinity stress. Int. J. Plant Biol. 1, 1–8.
Kawano, T., Furuichi, T., Muto, S., 2004. Controlled salicylic acid levels and corre-
sponding signaling mechanisms in plants. Plant Biotechnol. 21 (5), 319–335.
Kawano, T., Muto, S., 2000. Mechanism of peroxidase actions for salicylic acid
induced generation of active oxygen species and an increase in cytosolic calcium
in tobacco cell suspension culture. J. Exp. Bot. 345 (51), 685–693.
Kostner, B., Schnupp, R., Schulze, E.D., Rennenberg, H., 1998. Organic and inorganic
sulfur transport in the xylem sap and the sulfur budget of Picea abies trees. Tree
Physiol. 18, 1–9.
Kozlowski, G., Metraux, J.-P., 1998. Infection of Norway spruce (Picea abies (L.) Karst.)
seedling with Pythium irregular Buism. and Pythium ultimum Trow.: histolog-
ical and biochemical responses. Eurp. J. Plant Path. 104, 225–234.
Krekling, T., Franceschi, V.R., Berryman, A.A., Christiansen, E., 2000. The structure
and development of polyphenolic parenchyma cells in Norway spruce (Picea
abies) bark. Flora 195, 354–369.
425
Larcher, W., 2003. Physiological Plant Ecology. Springer Verlag, Berlin.
Martin, D., Gershenzon, J., Bohlmann, J., 2003. Induction of volatile terpene biosyn-
thesis and diurnal emission by methyl jasmonate in foliage of Norway spruce.
Plant Physiol. 132 (3), 1586–1599.
Mattanovich, J., Ehrenhöfer, M., Schafellner, C., Tausz, M., Führer, E., 2001. The role of
sulphur compounds for breeding success of Ips typographus L. (Col., Scolytidae)
on Norway Spruce (Picea abies [L.] Karst.). J. Appl. Ent. 125, 425–431.
Maughan, S., Foyer, C.H., 2006. Engineering and genetic approaches to modulating
the glutathione network in plants. Physiol. Plantarum 126, 382–397.
Meyer, A.J., Rausch, T., 2008. Biosynthesis, compartmentation and cellular functions
of glutathione in plant cells. In: Hell, R., Dahl, C., Knaff, D., Leustek, T. (Eds.),
Advances in Photosynthesis and Respiration. Springer, Dordrecht, pp. 417–435.
Mittler, R., 2002. Oxidative stress, antioxidants and stress tolerance. Trend. Plant Sci.
7 (9), 405–410.
Nagy, N.E., Fossdal, C.G., Krokene, P., Krekling, T., Lonneborg, A., Solheim, H., 2004.
Induced responses to pathogen infection in Norway spruce phloem: changes in
polyphenolic parenchyma cells, chalcone synthase transcript levels and perox-
idase activity. Tree Physiol. 24 (5), 505–515.
Noctor, G., 2006. Metabolic signalling in defence and stress: the central roles of
soluble redox couples. Plant Cell Environ. 29, 409–425.
Noctor, G., Foyer, C.H., 1998. Ascorbate and glutathione: keeping active oxygen under
control. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 249–279.
Noctor, G., Gomez, L., Vanacker, H., Foyer, C.H., 2002. Interactions between
biosynthesis, compartmentation and transport in the control of glutathione
homeostasis and sigmalling. J. Exp. Bot. 53, 1283–1304.
Ohashi, Y., Murakami, T., Mitsuhara, I., Seo, S., 2004. Rapid down and upward translo-
cation of salicylic acid in tobacco plants. Plant Biotechnol. 21, 95–101.
Pasqualini, S., Della Torre, G., Ferranti, F., Ederli, L., Piccioni, C., Reale, L., Antonielli,
M., 2002. Salicylic acid modulates ozone-induced hypersensitive cell death in
tobacco plants. Physiol. Plantarum 115, 204–212.
Percival, G.C., 2001. Induction of systemic acquired disease resistance in plants:
potential implications for disease management in urban forestry. J. Arboric. 27
(4), 181–192.
Pučko, M., Grebenc, T., Ribarič Lasnik, C., Božič, G., Konnert, M., Kraigher, H., 2005.
Effects of biotic stress on seed quality and antioxidant content in Norway
spruce trees from approved seed stands in Slovenia. Phyton-Ann. Rei Bot. 45
(3), 331–339.
Radwan, D.E.M., Fayez, K.A., Mahmound, S.Y., Hamad, A., Lu, G., 2007. Physiological
and metabolic changes of Cucurbita pepo leaves in response to zucchini yel-
low mosaic virus (ZYMV) infection and salicylic acid treatments. Plant Phys.
Biochem. 45, 480–489.
Rodrigues, K.C.S., Fett-Neto, A.G., 2009. Oleoresin yield of Pinus elliottii in a sub-
tropical climate: Seasonal variation and effect of auxin and salicylic acid-based
stimulant paste. Ind. Crops Prod. 30, 316–320.
Ross, J.R., Nam, K.H., John, C., Auria, D., Pichersky, E., 1999. S-adenosyl-L-
methionine:salicylic acid carboxyl methyltransferase, an enzyme involved in
floral scent production and plant defense, represents a new class of plant methyl-
transferases. Arch. Biochem. Biophys. 367, 9–16.
Schmidt, A., Zeneli, G., Hietala, A.M., Fossdal, C.G., Krokene, P., Christiansen, E.,
Gershenzon, J., 2005. Induced chemical defences in conifers: biochemical and
molecular approaches to studying their function. In: Schmidt, A., Zeneli, G.,
Hietala, A.M., Fossdal, C.G., Krokene, P., Christiansen, E., Gershenzon, J., Romeo,
J.T. (Eds.), Chemical Ecology and Phytochemistry in Forest Ecosystems, vol. 39.
Elsevier, Amsterdam, pp. 1–28.
Shah, J., 2003. The salicylic acid loop in plant defense. Curr. Opin. Plant Biol. 6,
365–371.
Shi, Q., Zhu, Z., 2008. Effects of exogenous salicylic acid on manganese toxicity, ele-
ment contents and antioxidative system in cucumber. Environ. Exp. Bot. 63,
317–326.
Slaymaker, D.H., Navarre, D.A., Clark, D., Del-Pozo, O., Martin, G.B., Klessig, D.F.,
2002. The tobacco salicylic acid-binding protein 3 (SABP3) is the chloroplast
carbonic anhydrase, which exhibits antioxidant activity and plays a role in the
hypersensitive defense response. Proc. Natl. Acad. Sci. U.S.A. 99, 11640–11645.
Smirnoff, N., Wheeler, G.L., 2000. Ascorbic acid in plants: biosynthesis and function.
Crit. Rev. Plant Sci. 19, 267–290.
Struis, R.P.W.J., Ludwig, C., Barrelet, T., Krähenbühl, U., Rennenberg, H., 2008. Study-
ing sulfur functional groups in Norway spruce year rings using S L-edge total
electron yield spectroscopy. Sci. Total Environ. 403, 196–206.
Tausz, M., 2007. Sulfur in forest ecosystems. In: Hawkesford, M.J., De Kok, L.J. (Eds.),
Sulfur in Plants—An Ecological Perspective. Series: Plant Ecophysiology. Springer
Verlag, Dordrecht, Boston, London, pp. 59–75.
Tausz, M., Wonisch, A., Grill, D., Morales, D., Jimenez, M.S., 2003. Measuring antiox-
idants in tree species in the natural environment: from sampling to data
evaluation. J. Exp. Bot. 54 (387), 1505–1510.
Tausz, M., Wonisch, A., Peters, J., Jimenez, M.S., Morales, D., Grill, D., 2001. Short-term
changes in free-radical scavengers and chloroplast pigments in Pinus canariensis
needles as affected by mild drought stress. J. Plant Physiol. 158, 213–219.
Tausz, M., Šircelj, H., Grill, D., 2004. The glutathione system as a stress marker in
plant ecophysiology: is a stress-response concept valid? J. Exp. Bot. 55 (404),
1955–1962.
Tausz, M., Löffler, S., Posch, S., Monschein, S., Grill, D., Kätzel, R., 2005. Do photopro-
tective pigments and antioxidants in needles of Pinus sylvestris relate to high N or
water availability at field plots in a dry year? Phyton Ann. Rei Bot. 45, 107–116.
Urbanek Krajnc, A., Zechmann, B., Stabentheiner, E., Müller, M., 2008. Artificial eleva-
tion of salicylic acid affects thiol contents in symptom development in Cucurbita
pepo during Zucchini yellow mosaic virus (ZYMV) infection. Phyton 48, 13–35.
426 A. Urbanek Krajnc et al. / Forest Ecology and Management 261 (2011) 416–426
Urbanek Krajnc, A., 2009. A temporal analysis of antioxidative defense response
in the phloem of Picea abies after attack by Ips typographus. Tree Physiol. 29,
1059–1068.
Vallad, G.E., Goodman, R.M., 2004. Systemic acquired resistance and induced sys-
temic resistance in conventional agriculture. Crop Sci. 44, 1920–1934.
Verberne, M.C., Brouwer, N., Delbianco, F., Linthorst, H.J.M., Bol, J.F., Verpoorte, R.,
2002. Method for the extraction of the volatile compound salicylic acid from
tobacco leaf material. Phytochem. Anal. 13, 45–50.
Wermelinger, B., 2004. Ecology and management of the spruce bark beetle Ips
typographus—a review of recent research. Forest Ecol. Manag. 202 (1–3), 67–82.
Zeneli, G., Krokene, P., Christiansen, E., Krekling, T., Gershenzon, J., 2006. Methyl
jasmonate treatment of mature Norway spruce (Picea abies) trees increases
the accumulation of terpenoid resin components and protects against infec-
tion by Ceratocystis polonica, a bark beetle-associated fungus. Tree Physiol. 26,
988–997.
Zhao, F.-J., Tausz, M., De Kok, L.J., 2008. Role of sulfur for plant production in agri-
cultural and natural ecosystems. In: Hell, R., Dahl, C., Knaff, D., Leustek, T. (Eds.),
Advances in Photosynthesis and Respiration. Springer, Dordrecht, pp. 417–
435.