Application of salicylic acid induces antioxidant defense responses in the phloem of Picea abies and inhibits colonization by Ips typographus

Forest Ecology and Management 261 (2011) 416–426

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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 Hoˇce, Slovenia

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Article history: Received 3 June 2010 Received in revised form 21 October 2010 Accepted 24 October 2010 Available online 26 November 2010 Keywords: Antioxidant defence system Ascorbic acid Glutathione Ips typographus Picea abies Phenolics Salicylic acid

a b s t r a c t The adaptive plasticity of Norway spruce (Picea abies) against attack by Ips typographus depends on systemic acquired resistance which involves salicylic acid (SA), and an antioxidant system both recognized as valuable stress markers in ecophysiological studies. In the presented field experiment, 100 mM SA was applied to the bark sections of Norway spruce prior to being attacked by bark beetles, in order 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 glutathione (tGSH) and total cysteine (tCys) increased by 167% and 80%, respectively, two weeks after treatment, in comparison with controls. In contrast, SA-treatment caused an initial deterioration in total ascorbic acid (tASC) and enhanced the percentage of dehydroascorbic acid (DHA), but activated tASC levels over later sampling dates. The initial bark beetle attack was characterized by a significant decline in total SA levels, which was accompanied by a transient degradation and oxidation of their ascorbateglutathione system. This initial reaction was significantly alleviated by SA-application and characterized 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 SAtreated 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 further 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 alleviated. 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: [email protected], [email protected] (A. Urbanek Krajnc). 0378-1127/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.foreco.2010.10.027

(Nagy et al., 2004; Franceschi et al., 2005; Bonello et al., 2006). During 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 salicylic 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 different pathways (Shah, 2003; Durrant and Dong, 2004; Hayat et al., 2007, 2010). General knowledge about SA metabolism, interactions 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

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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, ascorbate 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). Following 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 exogenous 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 glutathioneascorbate 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 signalling 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 synthesis 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 requirements 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 hypothesized 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 reactive 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-

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tle affected Norway spruce (Mattanovich et al., 2001; Puˇcko et al., 2005), although antioxidative defence systems have often been used as stress indicators for the diagnosis of disturbances in forest 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 ecophysiological stress-response concept (Larcher, 2003; Tausz et al., 2004). Many studies on agricultural plants demonstrated that SAtreatment 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 antioxidants 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 conditions 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. Antioxidant levels (ascorbic acid, cysteine, glutathione, phenolics) and SA levels were monitored (a) 72 h after SA-treatment, in order to analyze 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 completed 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 monoculture 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 (35year-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 originating from open pollination among local genotypes. The experimental area involved two 600 m2 plots approximately 50 m apart (bark beetle colonized, and control). The 5 m border around the infested plot was treated twice with insecticide (Fastac® , 0.3%, BASF) during maximum beetle-flight activity, in order to protect single susceptible trees and prevent the migration 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).

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(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) variations in antioxidant contents (control, no attack). (4) The fourth group of test trees was left untreated but was subjected to bark beetles (control, attack).

ground. The bark was removed and the total phloem immediately frozen in liquid N2 , and transferred to the freezer (−80 ◦ C). The samples were taken on a clear day between 11.00 and 14.00 solar time. Further processing (freeze-drying, pulverization) was done according 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

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 temperature 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

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 vortex, 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 concentrator. 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 SepPak C18 column. The samples were eluted by 1 mL 100% methanol and then analyzed using Waters HPLC system (Waters 600E Controller Pump, Waters 2475 Multi Fluorescence Detector, Waters Software/Hardware package, cooled Waters autosampler) at excitation: 315 nm wavelength and emission: 405 nm wavelength). Column Sphericorb S5 ODS2 25 × 4.6 ␮m. The samples were fractioned 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; corrections 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 glutathione, 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 phenolic 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 twoway 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).

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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 interactions. 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 determined 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 similar 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.

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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 significant 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 determined on earlier sampling dates. Severely affected SA-treated tissue was characterized by dramatically-enhanced total SA contents (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 determined 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 beetle 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 unaffected 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 controlbarks (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 colonization. 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 accumulated 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). However, 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 significantly 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 significant 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 midJune (−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 significantly 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 moderatelyaffected 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 stronglyaffected 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).

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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 sampling 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 moderatelyaffected 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 diseasestage was characterized by a significant degradation of tPH, which was more pronounced in the untreated trees. Moreover, the SAtreated 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 comparison with earlier sampling dates (Fig. 6, Supplementary Figure 4). 4. Discussion In the presented study, the effect of SA-treatment on bark beetle 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 during 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 significantly lower I. typographus colonization on SA-treated tree. A shorter gallery length could be associated with SA-induced qualitative and quantitative differences in host chemistry, and the internal physiology of Norway spruce bark, which is less suitable for bee-

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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.

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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 information is of particular importance, since the SA-treatment experiment in vivo was significantly influenced by weather conditions. Furthermore, 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 shortterm 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 moderatelyaffected 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. Massive 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 successful 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 physiological 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 relating to the typical biology of trees, for example long-life spans, long internal transport distances, and large volumes of woody

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tissues, are significant (Tausz, 2007). The presented experiment clearly demonstrated that SA-application to bark sections had positive 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 oxidative 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 accompanied 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 SAtreatment. 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 linearly with higher concentrations of exogenous SA (Urbanek Krajnc et al., 2008). Disproportionate differences in tCys and tGSH contents 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 position 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

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(Tausz, 2007; Hell and Wirtz, 2008). Moreover, the intermediate reaction of cysteine synthesis, O-acetylserine, forms a direct connection 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 reaction 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 percentage 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 unsuccessful defense degradation of the glutathione system was followed by tissue dead (Tausz et al., 2004; Tausz, 2007). In the presented experiment, SA treatment at this stage of defense reaction alleviated the degradation of thiols, thus providing more efficient glutathionemediated 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 significantly delayed and not as obvious, when trees were pre-treated with SA. The results reflect the importance of elevated thiol levels in any initial reaction against bark beetle attack. Higher thiol levels in mid-April seemed to be crucial for successful glutathionemediated defense reaction in the bark later on. They may also be responsable for acclimatory responses of glutathione after successful 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 activating 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 physical 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 contents 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 moderatelyaffected 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 process 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. Boˇzidar Krajnˇciˇc for valuable suggestions during the research. Mag. Ignac Vuˇcko 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, Joˇzica Korez, Boris Sapaˇc and Metka Visoˇcnik 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 ecological 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. ´ J., Lin, Y.-H., 2001. Ca2+ -dependent and Ca2+ Chen, H.-J., Hou, W.-C., Kuc, 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 infection 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 salicylic 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.

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