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Arbutin- and benzotiadiazole-mediated cucumber response to Pseudomonas syringae pv. lachrymans infection in carbohydrate metabolism
Scientia Horticulturae 192 (2015) 200–210
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Arbutin- and benzotiadiazole-mediated cucumber response to Pseudomonas syringae pv. lachrymans infection in carbohydrate metabolism Maria Skłodowska, Marcin Naliwajski, Marzena Wielanek, Ewa Gajewska, ∗ ˙ ´ Elzbieta Kuzniak Department of Plant Physiology and Biochemistry, Faculty of Biology and Environmental Protection, University of Łód´z, Banacha 12/16, 90-237 Łód´z, Poland
a r t i c l e
i n f o
Article history: Received 10 December 2014 Received in revised form 17 May 2015 Accepted 4 June 2015 Keywords: Pseudomonas syringae pv. lachrymans Cucumber Arbutin Carbohydrates Invertases Lactate dehydrogenase
a b s t r a c t The effect of exogenous arbutin (ARB) or benzotiadiazole (BTH) on carbohydrate metabolism in cucumber infected with Pseudomonas syringae pv. lachrymans was studied. Glucose, sucrose, chlorophyll and carotenoid levels, acid and alkaline invertase and lactate dehydrogenase activities at the local (3rd, inoculated leaf) and systemic (5th, non-inoculated leaf) levels were determined. ARB limited disease symptom development to an extent similar to BTH. It was shown that both compounds effectively protected cucumber plants against this hemibiotrophic pathogen, however, the mechanisms of their activity are different. In the ARB-treated plants following the infection younger leaves were in better condition than BTHtreated ones due to more effective carbohydrate metabolism. Moreover, as opposed to BTH, ARB was not cytotoxic for plant tissues. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Pseudomonas syringae pv. lachrymans (Psl) is a hemibiotrophic bacterial pathogen that causes angular leaf spot disease, the second, following downy mildew, most severe cucumber (Cucumis sativus L.) disease which leads to significant economic loss. The disease is characterized by foliar and fruit brown spotting surrounded by a yellowish halo (Olczak-Woltman et al., 2009). P. syringae pathovars are adapted to the plant environment such as the leaf apoplast. They are able to secrete protein effectors through the TTSS (type III secretion system) and to produce a variety of virulence-associated systems such as toxins, ice-nucleation proteins, enzymes, phytohormones and extracellular polysaccharides. Their production by Psl has been observed (Olczak-Woltman et al., 2009). P. syringae produces effectors and toxins modifying host metabolism to its benefit, particularly by interfering with chloroplast function, which can result in nitrogen mobilization and suppression of plant defence (Rico et al., 2011; Selvaraj and Fofana, 2012). Truman et al. (2006) identified suppression of genes associated with photosynthesis, consistent with the loss of chlorophyll fluorescence, which could have an impact on primary carbon
∗ Corresponding author. Fax: +48 42 635 44 23. ´ E-mail address: [email protected] (E.. Kuzniak). http://dx.doi.org/10.1016/j.scienta.2015.06.007 0304-4238/© 2015 Elsevier B.V. All rights reserved.
metabolism, pigment biosynthesis and secondary metabolic pathways during the P. syringae infection. Glucose (Glc) and sucrose (Suc) play an important role in the regulation of developmental processes in higher plants (Gibson, 2000). Besides being a source of carbon and energy in plant metabolism, hexoses are commonly assumed to act as signals triggering up-regulation of defence related genes and down regulation of photosynthetic gene expression (Berger et al., 2004). However, carbohydrates are natural nutrients for plant pathogens so they also contribute to colonization of host plants (Peil et al., 2009). During biotic stress, including bacterial infection, these metabolites often accumulate in plant tissues and induce their senescence (Robinson et al., 2004). Plants are exposed to many different pathogens in the course of their life. Plant defence against pathogenic organisms is based on constitutive barriers and inducible mechanisms enhancing their resistance to a broad spectrum of pathogens. It was shown that induced resistance occurred after infection with necrotizing pathogens, root colonization with beneficial microorganisms and also after treatment with some natural or synthetic compounds. The utilization these inducers leads to mobilization of defence responses and establishment of systemic acquired resistance (SAR) (Conrath, 2009). SAR is a well-studied form of induced resistance and this mechanism is activated locally and systemically upon infection. However, the actual induced resistance status entails direct and indirect fitness costs (Vos et al., 2013). In general,
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Fig. 1. Symptoms of angular leaf spot disease on the 3rd cucumber leaves recorded 7 days post inoculation. A–C – general overview, D–F – close-up. The representative leaf images with symptoms routinely observed on cucumber leaves are shown. Macro photographs were taken with Panasonic DMC-FZ50 camera equipped with a close-up lens. Bar represents 0.5 cm. Severe disease symptoms in the form of irregular necrotic areas were visible on Pseudomonas syringae pv. lachrymans (Psl) infected leaves. BTH and ARB attenuated disease symptom severity, and on the leaves of BTH- or ARB-treated and subsequently Psl-inoculated plants (BTH + Psl and ARB + Psl, respectively) predominantly chlorotic lesions which covered less leaf area than in the non-treated ones were observed.
more resistant plants are less fit when the pathogen is not present (Canet et al., 2010). Benzothiadiazole (benzo(1,2,3) thiadiazole7-carbotionic acid-S-methyl ester, BTH), a functional analogue of salicylic acid (SA) is among effective inducers of SAR (Walters et al., 2005). However, BTH not only triggers plant defence but also biomass loss in a dose dependent manner (Canet et al., 2010). Cucumber is a common vegetable in which stachyose, not Suc, is the main translocated sugar. After long-distance transport to peduncles stachyose is converted to Suc (Miao et al., 2007). Utilization of Suc in cell metabolism depends on its cleavage into hexoses catalyzed, except sucrose synthase (EC 2.4.1.13), by invertase (EC 3.2.1.26). Plants possess two main isoforms of invertase (i) acid invertase (AcIn, -fructo-furanosidase) in vacuoles and apoplast which attacks Suc molecule from fructose residue and also hydrolyzes other -fructose-containing oligosaccharides such as raffinose and stachyose and (ii) Suc specific alkaline invertase (AlIn) in cytoplasm (Sturm, 1999). Changes in expression and activity of acid invertase could be used as a potential molecular marker of changes in plant primary metabolism (Biemelt and Sonnewald, 2006). Green leaves of many plant species contain low levels of lactate dehydrogenase (LDH, EC 1.1.1.27) under aerobic conditions (Sugiyama and Taniguchi, 1997). This enzyme catalyses the reversible reaction of lactate oxidation and pyruvate reduction (O’Carra and Mulcahy, 1996) and under specific conditions its higher activity may be used as a marker of cytotoxicity (Legrand et al., 1992). Arbutin (hydroquinone--d-glucopyranoside, ARB) is a natural soluble phenolic glucoside synthesised by plants of the families Ericaceae, Rosaceae, Saxifragaceae (Petkou et al., 2002). This compound can inhibit membrane lysis both free radical-mediated and enzymatic in nature (Frías et al., 2008; Oliver et al., 1996). It has been suggested that ARB contributes to membrane stabilization resulting from its interaction with membrane lipids. This phenomenon is connected with high concentration of arbutin in membranes (Frías et al., 2008). In cells ARB might be metabolized to hydroquinone
(HQ) and next to benzoquinone (2,5-cyclohexadiene-1,4-dione) which has direct antimicrobial activity (Jin and Sato, 2003). HQ is a reactive molecule capable of producing reactive oxygen species (ROS), including the superoxide anion, hydrogen peroxide and hydroxyl radical, and causing oxidative stress (Khanal et al., 2011; Xu et al., 2002). HQ also acts as a pro-oxidant that causes cytotoxicity and induces apoptosis (Xu et al., 2002). On the other hand, the studies of Casano et al. (2000) and Xu et al. (2002) showed, that the presence of additionally formed HQ molecules might enhance efficiency of photosynthesis. ARB is a well-known substrate for -glucosidase, but it may be also oxidized directly by polyphenoloxidase and by soluble and ionically bound to cell wall peroxidase (EC 1.11.1.7). (Hadˇzi-Taˇskovic´ ˇ Sukalovi c´ et al., 2007; Petkou et al., 2002). It has been suggested that the oxidation pathway of arbutin is involved in fire blight resistance of some pear varieties (Jin and Sato, 2003). On the other hand, Mo et al. (1995) reported that arbutin could serve as the signal molecule that induces production of syringomycin and other phytotoxins produced by P. syringae. To assess the ability of ARB to protect cucumber plants against angular leaf spot disease, we studied the effects of exogenous ARB in comparison to BTH on changes of carbohydrate metabolism in plants infected with Psl. We determined Glc and Suc levels as well as AcIn, AlIn, and LDH (as a marker of cytotoxity) activities both locally (in the infected leaves) and systemically (in the non-infected upper leaves). Moreover, we examined chlorophyll a (chla), b (chlb) and carotenoids (car) concentrations. 2. Material and methods 2.1. Biological material Cucumber (C. sativus L. cv. Polan) seeds were germinated in the dark at 24 ◦ C for 3 days. Then the seedlings were transferred into individual pots and grown hydroponically in the medium consisting of (mg/dm3 ): N – 224; P–39; K–312; Ca–160; Mg–33; S–44;
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Fe–0.84; Mo–0.98; Cu–0.84; Mn–8.4; B–4.2; Zn–4.2. Three-weekold plants were divided into three groups – control (non-treated) and plants treated once with 2.0 mM water solution of ARB or a commercial preparation BION 50 WG (Novartis Protection AG, Bazylea, Switzerland) containing 0.1 mM BTH as an active ingredient. The non-treated plants were sprayed with distilled water. One week later the third true leaves from the bottom were inoculated with Psl or treated with sterile distilled water (mock inoculation) using a needle-less hypodermic 1 cm3 syringe. Bacterial suspension was infiltrated at about 10 sites (per leaf) uniformly distributed on the abaxial side of the leaf blade. Bacteria for inoculation, pro´ Poland), were cultured vided by Bank of Plant Pathogens (Poznan, for 24 h on King B medium at 28 ◦ C with vigorous shaking and centrifuged at 3500 × g for 10 min. The bacterial pellet was washed twice, resuspended in sterile water, and adjusted to 107 cfu cm−3 . For biochemical analysis leaf samples without main veins (0.5 g FW) were immediately homogenized in a mortar in 5 cm3 icecold 0.05 M sodium phosphate buffer pH 7.0 containing 1 mM EDTA and 1% polyvinylpyrrolidone. Immediately after centrifugation (20,000 × g, 20 min, 4 ◦ C) the supernatant was used for measurement of enzyme (LDH, AlIn and AcIn) activities as well as protein content. For chlorophyll and carotenoids determination the tissues were stored at −20 ◦ C. To study local and systemic response of cucumber plant to Psl inoculation, the 3rd (inoculated) and the 5th (non-inoculated, upper) leaves were taken for analyses on 0, 2 and 7 days post inoculation (dpi). 2.2. Determination of leaf area and disease symptom development For determination of leaf area in control and ARB-treated plants, the leaves were taken 7 and 14 days after ARB application (time points corresponding to 0 and 7 dpi). The leaf area was assessed by a computer method of image analysis. Disease symptom development on the 3rd leaves was quantified 2 and 7 dpi and was given as percentage of the symptom-covered leaf area in relation of the total leaf area. The leaf images scanned to JPEG files were subjected to the automatic analysis and classification run in MATLAB aimed to extract discoloured regions corresponding to the pathogen-induced disease symptoms (Gocławski et al., 2012). 2.3. Determination of photosynthetic pigments To determine the contents of chlorophyll a (chla), chlorophyll b (chlb) and carotenoids (car) the frozen leaves were extracted three times with 80% acetone (1:20 w/v) and centrifuged (33,000 × g, 15 min). After measurement of absorbance of the supernatants at 470 nm, 663.2 nm and 646.8 nm, car, chla and chlb contents were calculated according to the method of Wellburn (1994). 2.4. Determination of glucose and sucrose Glc and Suc contents were determined in the extracts obtained from leaf samples (0.5 g FW) after triple 80% ethanol extraction. The ethanolic extracts were evaporated to dryness at 50 ◦ C and the residue was resolubilized with distilled water. The concentrations of both sugars were assayed using the commercial enzymatic test (Boehring Mannheim) according to the manufacturer’s instruction and expressed in g per mg protein determined in the extract used for enzyme activity analyses. 2.5. Enzyme assays Invertase activity was determined by the method described by Miller and Ranwala (1994). The reaction mixture consisted of
40 mM Suc, 50 mM Na-acetate buffer (pH 5.0 for AcIn and pH 7.5 for AlIn). The buffer and substrate were heated in water bath at 37 ◦ C for 5 min prior to substrate addition. When Suc was added, the reaction mixture was incubated at 37 ◦ C. Exactly after 30 min the reaction was stopped in a 90 ◦ C bath for 5 min. The amount of Glc released was determined as described above. One unit of invertase activity was defined as the amount of enzyme that catalysed the production of 1 mol of Glc per minute at 37 ◦ C per mg protein. LDH activity was measured by the decrease in absorbance at 340 nm resulting from oxidation of NADH ( = 6.22/mM/cm) according to Sugiyama and Taniguchi (1997) method. The assay mixture consisted of 50 mM K-phosphate buffer (pH 7.25), 0.5 mM NADH2 , 4 mM sodium pyruvate, 2.3 mM 4-methypyranozole and 2.3 mM NaCN. The activity was expressed in terms of nmol NADH oxidised per minute per mg protein. 2.6. Protein content determination The protein content was determined according to Bradford (1976), with a standard curve prepared using bovine serum albumin. 2.7. Statistical analysis The results presented are the means from 8 to 10 independent experiments. Sample variability is given as the standard deviation of the mean. The significance of differences between control and Psl or BTH and BTH + Psl or ARB and ARB + Psl as well as between Psl and BTH + Psl or ARB + Psl was determined by a non-parametric MannWhitney Rank Sum Test. Differences at P < 0.05 were considered significant. 3. Results 3.1. Infection development, vegetative growth and photosynthetic pigment levels Small chlorotic spots being the macroscopic symptoms of angular leaf spot of cucumber were observed about 2 dpi. Five days later (7 dpi) they became larger and darker, and necroses with a chlorotic halo were formed (Fig. 1). The adjacent interveinal leaf area turned yellow and about 14 dpi the affected leaf part shriveled and died. Foliar application of ARB 7 days before Psl inoculation significantly reduced angular leaf spot disease severity in comparison to the nonpretreated inoculated plants (Psl). ARB caused about 48% disease suppression in the third leaf on the 7th dpi (Figs. 1 and 2). The similar effect was observed after pretreatment of cucumber plants with ´ BTH (Kuzniak et al., 2014). ´ Contrary to BTH (Kuzniak et al., 2014), ARB treatment positively influenced growth of the 5th leaves whose area was more than 40% bigger than the control (Table 1). Moreover, the ARB-treated plants were more fit than the BTH-treated or control ones (data not shown). A few changes were observed in the levels of chla, chlb and carotenoids in all examined plant groups (Tables 2–4). In the older (3rd) infected leaves of the non-pretreated plants the changes were limited to chla concentration on the 2nd dpi (decrease by 15% in comparison to the non-infected ones), while in the ARB-treated plants following infection the lower chla levels were observed on the 2nd and 7th dpi, 75% and 79% of the non-infected ARB-treated material, respectively (Table 2). Pretreatment of plants with BTH augmented the negative effect of infection on chlorophyll content. Comparing to the non-pretreated infected plants chla and chlb contents in the 3rd leaf of the BTH-treated infected plants were 32% and 48% lower, respectively (Table 2). Similar effect was also observed in the 5th leaves. Different changes of the chlb level in
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Table 1 Influence of arbutin on the area of the 3rd and the 5th leaves of cucumber plants. Leaf area (mm2 )
Treatment
3rd leaf
Control ARB
5th leaf
7 days
14 days
7 days
14 days
9753.9 ± 1755.7 12434.5 ± 1492.1
15124.6 ± 5750.1 17055.8 ± 3752.3
2419.9 ± 362.9 3503.6 ± 595.6*
11613.5 ± 1277.5 16610.9 ± 2657.6*
Leaf area was measured 7 and 14 days after ARB treatment (which corresponds to 0 and 7 dpi). Data are means ± SD. Five plants were used for each treatment (n = 5). * indicates a significant difference between the control and ARB-treated plants at P < 0.05. Table 2 Chlorophyll a (chla) and chlorophyll b (chlb) contents (mg/g FW) in the 3rd cucumber leaf 0, 2 and 7 days post inoculation (dpi) with P. syringae pv. lachrymans. Variant
Noninfected Infected
Time (dpi)
0 2 7 2 7
Control
BTH
ARB
Chla
Chlb
Chla
Chlb
Chla
Chlb
0.778 ± 0.052 0.735 ± 0.041 0.704 ± 0.064 0.627 ± 0.051* 0.694 ± 0.078
0.315 ± 0.098 0.316 ± 0.052 0.360 ± 0.082 0.299 ± 0.103 0.389 ± 0.121
0.593 ± 0.053 0.476 ± 0.046 0.395 ± 0.118 0.429 ± 0.044# 0.518 ± 0.101
0.217 ± 0.021 0.173 ± 0.023 0.147 ± 0.030 0.157 ± 0.029# 0.215 ± 0.035* ,#
0.798 ± 0.131 0.819 ± 0.050 0.851 ± 0.072 0.611 ± 0.065* 0.677 ± 0.047*
0.345 ± 0.120 0.342 ± 0.029 0.399 ± 0.038 0.381 ± 0.068 0.289 ± 0.058*
Data are means ± SD. * indicates a significant difference between control and Psl or BTH and BTH + Psl or ARB and ARB + Psl at P < 0.05. # indicates a significant difference between Psl and BTH + Psl or ARB and ARB + Psl at P < 0.05. Table 3 Chlorophyll a (chla) and chlorophyll b (chlb) contents (mg/g FW) in the 5th cucumber leaf 0, 2 and 7 days post inoculation (dpi) with P. syringae pv. lachrymans. Variant
Time (dpi)
Noninfected
0 2 7 2 7
Infected
Control
BTH
ARB
Chla
Chlb
Chla
Chlb
Chla
Chlb
0.911 ± 0.012 0.896 ± 0.079 0.804 ± 0.158 1.123 ± 0.080* 1.023 ± 0.188*
0.424 ± 0.035 0.403 ± 0.057 0.429 ± 0.113 0.471 ± 0.042 0.443 ± 0.107
0.868 ± 0.036 0.626 ± 0.134 0.653 ± 0.136 0.576 ± 0.125# 0.595 ± 0.105#
0.405 ± 0.010 0.275 ± 0.051 0.288 ± 0.052 0.230 ± 0.030# 0.230 ± 0.048#
1.049 ± 0.080 0.859 ± 0.108 0.783 ± 0.088 1.020 ± 0.139 1.027 ± 0.139*
0.459 ± 0.024 0.392 ± 0.084 0.434 ± 0.074 0.433 ± 0.084 0.438 ± 0.044
Data are means ± SD. * indicates a significant difference between control and Psl or BTH and BTH+Psl or ARB and ARB + Psl at P < 0.05. # indicates a significant difference between Psl and BTH + Psl or ARB and ARB + Psl at P < 0.05.
the infected 3rd leaves were noted (Table 2). On the 7th dpi in the BTH-treated plants its level increased by 46% but in the ARBtreated ones decreased by 28% in comparison to the respective non-infected plants. In all other cases chla, chlb as well as car concentrations did not show significant differences between the infected and non-infected 3rd leaves. In the 5th leaves Psl infection changed these parameters only in the non-treated and ARB-treated plants (Table 3). In the non-treated plants chla levels were higher by 25% and 27% on the 2nd dpi and 7th dpi, respectively and car level was increased by 41% on the 2nd dpi in comparison to the non-infected plants (Table 4). In the 5th leaves of the BTH-treated infected plants car concentration was about 44% lower comparing to the non-pretreated infected plants. In the ARB-treated plants following infection 31% increase in chla level was observed at the end of experiment (7th dpi).
3.2. Glucose and sucrose content The obtained data indicated that after Psl inoculation in the directly inoculated (3rd) leaves on the 2nd dpi only in the BTHtreated group Glc concentration was lower (47% of the value observed in the non-infected ones) (Fig. 3). The enhancement of Glc level in the inoculated 3rd leaves in comparison to the noninoculated ones was found in the control group on the 7th dpi (213%) (Fig. 3). The younger (5th) leaves on the 2nd dpi in all infected plant groups showed higher Glc levels than the noninfected ones (Fig. 3). They were 152%, 125% and 191% for the control, the BTH-treated and the ARB-treated ones, respectively. On the 7th dpi the higher Glc level was found only in the infected ARBtreated leaves (216% of the value determined in the non-infected
Table 4 Carotenoids contents (mg/g FW) in the 3rd and 5th cucumber leaf 0, 2 and 7 days post inoculation (dpi) with P. syringae pv. lachrymans. Variant
Time (dpi)
Non-infected
0 2 7 2 7
Infected
Control
BTH
ARB
3rd leaf
5th leaf
3rd leaf
5th leaf
3rd leaf
5th leaf
0.189 ± 0.016 0.157 ± 0.028 0.165 ± 0.022 0.137 ± 0.016 0.157 ± 0.019
0.239 ± 0.016 0.221 ± 0.019 0.200 ± 0.070 0.312 ± 0.037* 0.249 ± 0.065
0.154 ± 0.023 0.155 ± 0.035 0.118 ± 0.032 0.179 ± 0.035 0.140 ± 0.025
0.205 ± 0.007 0.140 ± 0.034 0.113 ± 0.022 0.174 ± 0.020# 0.140 ± 0.029#
0.187 ± 0.021 0.183 ± 0.022 0.191 ± 0.016 0.143 ± 0.031 0.173 ± 0.028
0.257 ± 0.030 0.209 ± 0.035 0.183 ± 0.021 0.235 ± 0.049# 0.237 ± 0.059
Data are means ± SD. * indicates a significant difference between control and Psl or BTH and BTH + Psl or ARB and ARB + Psl at P < 0.05. # indicates a significant difference between Psl and BTH + Psl or Psl and ARB + Psl at P < 0.05.
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Symptom-covered leaf area (%)
16 14
Psl
ARB + Psl
12
*
10 8 6 4 2 0 2
7
Time post inoculation (days) Fig. 2. Effect of ARB treatment on angular leaf spot disease development in the 3rd leaves of cucumber plants expressed as percentage of symptom-covered area in relation to the total leaf area. * indicates a significant difference between Psl and ARB + Psl at P < 0.05 (n = 5).
ARB-treated ones), while in the control the lower level (67%) of Glc in comparison to the non-infected control plants was detected.
Pretreatment with ARB significantly influenced Glc content in the 5th leaf of the infected plants comparing to the non-pretreated infected ones. On the 2nd dpi and on the 7th dpi concentration of Glc in the ARB+Psl group exceeded the values found in the non-treated infected plants by 67% and 129%, respectively (Fig. 3). In the case of Suc levels on the 2nd dpi significant diminution was observed in the 3rd leaves in all tested groups (Fig. 4). In the infected plants as compared to the non-inoculated ones Suc levels constituted 41% of that in the BTH-treated material, 48% of that in the control and 61% of that in the ARB-treated plants. On the 7th dpi the decreased level of Suc was found in the 3rd leaves from both treated groups. It was 57% and 43% in the BTH-treated and the ARB-treated plants in comparison to the non-inoculated ones, respectively. In the 5th leaves of Psl inoculated plants Suc levels were diminished in the control and BTH-treated plants in both examined times (Fig. 4). They were 45% and 77% of the non-infected ones on the 2nd dpi and 75% and 70% on the 7th dpi, respectively. ARB impact depended on the time. In the 5th leaves of the infected plants Suc level was 29% higher on the 2nd dpi and 67% lower
Fig. 3. Effect of BTH and ARB treatments on glucose (Glc) concentration in the 3rd and the 5th leaves of cucumber plants 0, 2 and 7 days post inoculation with P. syringae pv. lachrymans (Psl). Bars represent SD of means. * indicates a significant difference between control and Psl or BTH and BTH + Psl or ARB and ARB + Psl at P < 0.05. # indicates a significant difference between Psl and BTH + Psl or ARB + Psl at P < 0.05.
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Fig. 4. Effect of BTH and ARB treatments on sucrose (Suc) concentration in the 3rd and the 5th leaves of cucumber plants 0, 2 and 7 days post inoculation with P. syringae pv. lachrymans (Psl). Bars represent SD of means. * indicates a significant difference between control and Psl or BTH and BTH + Psl or ARB and ARB + Psl at P < 0.05. # indicates a significant difference between Psl and BTH + Psl or ARB + Psl at P < 0.05.
on the 7th dpi than the respective values in the non-infected material. On the 2nd dpi both in the 3rd and the 5th leaf Suc content in BTH-treated and ARB-treated infected plants was higher comparing to the infected non-pretreated ones. Comparing to plants only inoculated, in the BTH-treated and ARB-treated and inoculated plants Suc content in the 3rd leaf was increased by 54% and 45%, respectively and in the 5th leaf by 45% and 164%, respectively. However, on the 7th dpi a significant decrease in Suc content in the ARB-treated and infected plants was found, by 48% and 26% comparing to the non-treated infected ones, in the 3rd and the 5th leaf, respectively (Fig. 4). 3.3. AlIn, AcIn and LDH activities Infection enhanced AlIn activity in the directly inoculated (3rd) leaves only in the non-treated plants at the end of experiment (7th dpi) (Fig. 5). It was 187% of the value detected in the non-inoculated control. At the same time decrease in this enzyme activity was observed in the infected BTH-treated plants (40% of the value deter-
mined in the non-infected BTH-treated ones) (Fig. 5). On the 2nd dpi, in the BTH-treated group the higher level of AlIn activity in the 5th leaves was found in the infected plants (158% of the value determined in the non-infected ones) (Fig. 5). AlIn activity was higher in the infected non-treated and ARB-treated material in the 5th leaves on the 7th dpi (308% and 242% in comparison to the noninfected group, respectively), while at this time the decrease in AlIn activity was strongly manifested in the BTH-treated plants where the difference between the infected and non-infected plants was 41%. Pretreatment with BTH and ARB decreased AlIn activity in the infected group of plants compared to the non-pretreated infected ones on the 7th dpi. In the 3rd leaf AlIn activity in the BTH + Psl and ARB + Psl groups was 53% and 38% lower than in the non-treated infected plants. Similar effect was observed in the 5th leaf on the 7th dpi. However, on the 2nd dpi AlIn activity in BTH-treated and subsequently infected plants was 107% higher comparing to plants only infected (Fig. 5). AcIn activity in the Psl inoculated 3rd leaves demonstrated a similar tendency as AlIn (Fig. 6). The increase in the infected control to 140% and decrease in the infected BTH-treated to 47% in
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Fig. 5. Effect of BTH and ARB treatments on alkaline invertase (AlIn) activity in the 3rd and the 5th leaves of cucumber plants 0, 2 and 7 days post inoculation with P. syringae pv. lachrymans (Psl). Bars represent SD of means. * indicates a significant difference between control and Psl or BTH and BTH + Psl or ARB and ARB + Psl at P < 0.05. # indicates a significant difference between Psl and BTH + Psl or ARB+Psl at P < 0.05.
comparison to the respective non-infected plants on the 7th dpi, were observed (Fig. 6). The younger leaves (5th) showed enhanced AcIn activity in the non-treated and BTH-treated groups 2 days after inoculation (Fig. 6). Its activity was by 43% and 50% higher when compared to the values observed in the respective non-infected tissues. On the 7th dpi in the 5th leaves from the infected control plants AcIn activity was as high as on the 2nd dpi (143%). On the 7th dpi in the ARB-treated ones infection enhanced this activity to 199% in comparison to the respective non-infected group (Fig. 6). On the 7th dpi AcIn activity in the 3rd leaf of the pretreated and subsequently infected plants was decreased comparing to the nonpretreated infected group, by about 40% and 30% in BTH + Psl and ARB + Psl plants, respectively. At the same time in the 5th leaf of the ARB-treated infected plants AcIn activity was 47% higher than in the infected non-treated plants (Fig. 6). Psl inoculation markedly changed the LDH activity only in the control and the BTH-treated plants in both examined leaves (Fig. 7). However, in the case of this enzyme the changes appeared earlier
in the younger leaves than in the older ones. On the 2nd dpi in the 5th leaves the respective values were 273% and 135% of the non-infected control and BTH-treated plants. Five days later (7th dpi) enhanced activity (by 50%) was observed only in the infected control (Fig. 7). The directly inoculated leaves (3rd) showed significant increase in LDH activity in comparison to the non-inoculated ones in the control and BTH-treated plants but only on the 7th dpi, the values were 127% and 143%, respectively (Fig. 7). In the 5th leaf a significant decrease in LDH activity was observed in ARBtreated infected plants comparing to infected non-treated ones, by 67% and 51% on the 2nd dpi and on the 7th dpi, respectively (Fig. 7). 4. Discussion Infection with bacterial pathogens strongly influenced photosynthesis whose efficiency is correlated with vigour and fitness of plants (Li et al., 2013). Reduced expression of genes involved in
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Fig. 6. Effect of BTH and ARB treatments on acid invertase (AcIn) activity in the 3rd and the 5th leaves of cucumber plants 0, 2 and 7 days post inoculation with P. syringae pv. lachrymans. Bars represent SD of means. * indicates a significant difference between control and Psl or BTH and BTH + Psl or ARB and ARB + Psl at P < 0.05. # indicates a significant difference between Psl and BTH + Psl or ARB + Psl at P < 0.05.
photosynthesis in Arabidopsis infected with P. syringae (Thilmony et al., 2006; Truman et al., 2006) and inhibition of tomato chloroplast function by P. syringae pv. tomato DC3000 were shown (Rico et al., 2011). In our study, in the control and ARB-treated plants following Psl infection in the directly inoculated leaves (3rd) the chlorophyll levels decreased while in the systemic leaves (5th) they increased. It might indicate that the control and ARB-treated plants switched on a compensatory mechanism connected with earlier metabolic maturation of the 5th leaves. The chlorophyll and carotenoid levels as well as our results concerning carbohydrate metabolism showed that assimilate production was more effective in these leaves than in the BTH-treated ones. However, in the ARB-treated plants this led to better plant development while in the control this promoted disease development. No significant changes in chlorophyll pigments were observed after inoculation in leaves of the BTH-treated plants, it may have been due to the fact that chl levels were initially by 20–30% lower in these plants then in the other ones. The decrease in photosynthetic pigment levels in the inoculated leaves might result from the direct influence of the pathogen on the expression of photosynthetic genes
but also from the development of chlorotic and necrotic areas on the leaves as the result of plant-pathogenic bacteria interaction (Berber et al., 2010). The latter possibility rather refers to the control plants because in the ARB-treated ones these symptoms were limited. Robinson et al. (2004) found in P. syringae pv. tagetis infected sunflower leaves that the bacterial toxins caused inhibition of photosynthetic carbon assimilation, reflected by the significant decrease in daily production of glucose, sucrose and starch, thus they postulated that infection by bacterial pathogens might influence leaf senescence via modulation of sugar status. Earlier it was suggested that leaf senescence was correlated with an increased sugar level (Yoshida, 2003), but Bonfig et al. (2006) demonstrated that sugar levels in Arabidopsis were not altered by infection with P. syringae. Here, we showed that in the ARB-treated 3rd leaves the Glc levels and AcIn activities were similar in the infected and non-infected plants, but Suc levels were lower in the infected ones. These observations suggest that pretreatment of plants with ARB might induce reactions resembling those accompanying the senescence process.
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Fig. 7. Effect of BTH and ARB treatments on lactate dehydrogenase (LDH) activity in the 3rd and the 5th cucumber leaves 0, 2 and 7 days post inoculation with P. syringae pv. lachrymans. Bars represent SD of means. * indicates a significant difference between control and Psl or BTH and BTH + Psl or ARB and ARB + Psl at P < 0.05. # indicates a significant difference between Psl and BTH + Psl or ARB + Psl at P <0.05.
On the other hand, bacterial pathogens may interfere with the source-sink balance leading to the reallocation of photoassimilates to sufficiently supply the pathogen with nutrients (Berger et al., 2004; Biemelt and Sonnewald, 2006). It is evidenced by stimulation of acid invertase that mobilizes hexoses at the infection site and a decreased rate of photosynthesis (Kocal et al., 2008). Growing bacterial population needs more nutritional compounds thus pathogen-induced modification of plant metabolism to its own advantage can be a good strategy as was shown for P. syringae by Rico et al. (2011) and Berger et al. (2004) who reported that sugars were often accumulated in plants during plant-pathogen interactions. Our results are to some extent in agreement with the finding of Berber et al. (2010) showing in P. syringae-infected tomato plants induction of cell wall invertase activities and decrease in Glc and Suc contents between the first and tenth study days, however it is worth noting that the crucial drop of Suc level appeared on the first day of experiment. In our study in the infected 3rd leaves increased Glc concentration together with enhanced AcIn activity was observed only in the control, because it took place on the 7th
dpi it probably resulted from the pathogen action. On the 2nd dpi in the BTH-treated plants Glc concentration decreased in the 3rd leaf but increased in the 5th leaf. It should be noted that in BTH-treated plants before inoculation Glc level was much higher than in the others, thus the decrease in the 3rd leaf was more pronounced. It was mainly caused by intensified synthesis of compounds involved in defence while direct pathogen impact was smaller because the activities of both invertases were decreased and the observed morphological changes connected with infection were limited. Due to ARB and BTH pretreatment decrease in Suc concentration occurred after Psl infection in directly inoculated tissues in both examined times. Taking into consideration the infected and noninfected plants, no difference for the ARB-treated or decrease for the BTH-treated ones in AlIn and AcIn activities were observed. Together with the fact that in both infected groups Glc levels were not changed a hypothesis can be put forward that pathogen access to carbohydrates was limited. The obtained results indicate that, the basic difference between BTH and ARB action after Psl infection concerned invertase activity in the directly inoculated leaves. In the BTH-treated plants it was
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decreased while in the ARB-treated ones no changes were observed. This indicates that BTH limited the rate of carbohydrate metabolism while ARB did not. The analysis of changes observed in these parameters in the systemic (5th) leaves indicate that after Psl inoculation decrease in Suc level took place in all infected groups. However, on the basis of the observed Glc level, invertase activities and morphological picture we suggest different mechanisms underlying these changes. The infected control plants showed decrease in Suc level accompanied with elevated activity of both invertases and significant decrease in Glc level between the 2nd and 7th dpi. In the BTH-treated plants, Glc level and activites of both invertases were elevated only on the 2nd dai. This might have caused diminution of Suc on the 2nd dpi which persisted till the 7th dpi, when all other parameters showed the same or lowered values in comparison to the non-inoculated plants. It may indicate that an infection-induced signal(s) was transferred from the infected area to younger tissues and mobilized BTH-mediated pathways involved in defence processes leading to inhibition of pathogen spread because the disease symptoms were not visible. In cucumber infected with Psl, SAR is induced by systemically accumulated SA (Meuwly et al., 1995). It was reported that single application of BTH at high dose increased SA content and triggered adequate defence responses after pathogen attack (Rad et al., 2005) but under these conditions the plant growth was limited (Canet et al., 2010). The reduction of plant growth may result from BTH-mediated changes in carbohydrate metabolism. The systemic ARB-treated leaves following infection showed high level of Glc and enhanced activity of both invertases accompanied with decrease in Suc level on the 7th dpi. It may be supposed that in these tissues intensification of assimilate production and export limited the losses resulting from the pathogen impact. Its action led among others to changed carbohydrate metabolism and decreased chlorophyll levels in the directly inoculated leaves. This suggestion seems correct because similarly as in the BTH-treated plants the disease symptoms were not visible. Herbers et al. (2000) proposed that sugar acted as an amplifier of plant defence responses during plant-pathogen interaction. This hypothesis was modified by Biemelt and Sonnewald (2006). They suggested that suppression or no changes of AcIn activity and hexose-mediated defence during the early phase (first 24 h) of bacterial infection allowed the bacteria to settle, but in later phase (following 48 h) the AcIn was induced to meet the nutrient demand of multiplying bacteria, thus AcIn regulation during the early and late infection phases differed. In our research this correlation was found only in the control directly inoculated leaf tissues. It is well known that pathogen infections induce rapid accumulation of reactive oxygen species (ROS) in plant tissues and their early accumulation was the key role in inhibiting penetration of a biotrophic pathogen (Barna et al., 2012). The increased carbohydrate concentrations may subsequently result in the production of ROS and activation of ROS-downstream responses (Couée et al., 2006). Arbutin presence might increase ROS level but this process requires its metabolic activation to HQ (Khanal et al., 2011). HQ causes concentration-dependent decrease in cell viability and/or increase in LDH activity, a marker of cytotoxicity (Legrand et al., 1992) but, as shown by Khanal et al. (2011), exogenous ARB did not cause a cytotoxic effect and LDH release up to 2 mM concentration in hepatoma HepG2 cell cultures. The ARB concentration used in this study was not toxic to plant cells because in the ARB-treated plants the LDH activity did not increase in any of the experimental variants. Arbutin can be metabolized by plant as well as by bacterial enzymes. Khanal et al. (2011) showed that ARB was degraded to HQ by human intestinal microflora. Earlier, Siegers et al. (2003)
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observed that Escherichia coli bacteria might liberate free HQ from its conjugates thereby killing themselves. HQ is quickly oxidized by a variety of oxidants and molecular oxygen and its oxidized derivatives are toxic for cells because they react with a large spectrum of cellular macromolecules and low molecular weight nucleophiles resulting in inhibition of some enzymes and disturbance of redox status (Pandey et al., 2005). It is probable that the plant and the pathogen used in this study showed different levels of ARB tolerance. Moreover, as it was found by Hadˇziˇ Taˇskovic´ Sukalovi c´ et al. (2007) and Casano et al. (2000) enhanced formation of HQ positively influenced plant mitochondrial and thylakoid membrane-bound hydroquinone peroxidase specific for H2 O2 and this increased the efficiency of antioxidative protection. The presence of elevated HQ level may also enhance productivity of photosynthesis (Casano et al., 2000; Xu et al., 2002). Accordingly, we observed that the ARB-treated plants exhibited better vigour and fitness and were characterised with higher level of chlorophyll than the control and BTH-treated ones. Following Psl infection LDH activity increased in the control and BTH-treated plants. It should be noted that younger leaves were affected earlier and to a greater extent than the older ones. Our results indicate that the enhanced LDH activity in the infected control and BTH-treated plants may be connected with insufficient supply of ATP and Glc. In all experimental variants in both these groups higher LDH activity was accompanied with higher Glc level. Moreover, when in the BTH-treated 3rd leaves on the 2nd dpi the significant decrease in Glc concentration took place the LDH activity was decreased, too. However, in the control plants carbon metabolism was mainly disturbed by direct pathogen action and these disturbances were observed in the inoculated and systemic leaves at all examined times, whereas in the BTH-treated ones it was connected with defence e.g. with increased synthesis of secondary metabolites. 5. Conclusions The severity of angular leaf spot disease caused by Psl in cucumber was reduced by both BTH and ARB indicating that both compounds were effective against this hemibiotrophic pathogen, however, in our opinion the mechanisms of their activity were different. BTH acts as an effective inducer of SAR (Walters et al., 2005) while ARB is a biostimulating and antimicrobial agent. Higher level of chlorophyll and more effective carbohydrate metabolism as well as less severe disease symptoms in the ARBtreated and inoculated plants may suggest that they were better fortified against the pathogen attack. A hypothesis may be put forward that ARB induced the senescence-like program in the injured leaves which restricted spread of the infection. In our opinion the use of ARB as a natural compound which may protect cucumber plants against Psl infection and simultaneously improve their growth and vigour should be taken into consideration in future studies. Acknowledgements This work was supported by National Science Centre Grant No N N310 302339. The authors are very grateful to Ms M. Fronczak for her linguistic correction of the manuscript. References Barna, B., Fodor, J., Harrach, B.D., Pogány, M., Király, Z., 2012. The Janus face of reactive oxygen species in resistance and susceptibility of plants to necrotrophic and biotrophic pathogens. Plant Physiol. Biochem. 59, 37–43, http://dx.doi.org/10.1016/j.plaphy.2012.01.014
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Arbutin- and benzotiadiazole-mediated cucumber response to Pseudomonas syringae pv. lachrymans infection in carbohydrate metabolism Maria Skłodowska, Marcin Naliwajski, Marzena Wielanek, Ewa Gajewska, ∗ ˙ ´ Elzbieta Kuzniak Department of Plant Physiology and Biochemistry, Faculty of Biology and Environmental Protection, University of Łód´z, Banacha 12/16, 90-237 Łód´z, Poland
a r t i c l e
i n f o
Article history: Received 10 December 2014 Received in revised form 17 May 2015 Accepted 4 June 2015 Keywords: Pseudomonas syringae pv. lachrymans Cucumber Arbutin Carbohydrates Invertases Lactate dehydrogenase
a b s t r a c t The effect of exogenous arbutin (ARB) or benzotiadiazole (BTH) on carbohydrate metabolism in cucumber infected with Pseudomonas syringae pv. lachrymans was studied. Glucose, sucrose, chlorophyll and carotenoid levels, acid and alkaline invertase and lactate dehydrogenase activities at the local (3rd, inoculated leaf) and systemic (5th, non-inoculated leaf) levels were determined. ARB limited disease symptom development to an extent similar to BTH. It was shown that both compounds effectively protected cucumber plants against this hemibiotrophic pathogen, however, the mechanisms of their activity are different. In the ARB-treated plants following the infection younger leaves were in better condition than BTHtreated ones due to more effective carbohydrate metabolism. Moreover, as opposed to BTH, ARB was not cytotoxic for plant tissues. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Pseudomonas syringae pv. lachrymans (Psl) is a hemibiotrophic bacterial pathogen that causes angular leaf spot disease, the second, following downy mildew, most severe cucumber (Cucumis sativus L.) disease which leads to significant economic loss. The disease is characterized by foliar and fruit brown spotting surrounded by a yellowish halo (Olczak-Woltman et al., 2009). P. syringae pathovars are adapted to the plant environment such as the leaf apoplast. They are able to secrete protein effectors through the TTSS (type III secretion system) and to produce a variety of virulence-associated systems such as toxins, ice-nucleation proteins, enzymes, phytohormones and extracellular polysaccharides. Their production by Psl has been observed (Olczak-Woltman et al., 2009). P. syringae produces effectors and toxins modifying host metabolism to its benefit, particularly by interfering with chloroplast function, which can result in nitrogen mobilization and suppression of plant defence (Rico et al., 2011; Selvaraj and Fofana, 2012). Truman et al. (2006) identified suppression of genes associated with photosynthesis, consistent with the loss of chlorophyll fluorescence, which could have an impact on primary carbon
∗ Corresponding author. Fax: +48 42 635 44 23. ´ E-mail address: [email protected] (E.. Kuzniak). http://dx.doi.org/10.1016/j.scienta.2015.06.007 0304-4238/© 2015 Elsevier B.V. All rights reserved.
metabolism, pigment biosynthesis and secondary metabolic pathways during the P. syringae infection. Glucose (Glc) and sucrose (Suc) play an important role in the regulation of developmental processes in higher plants (Gibson, 2000). Besides being a source of carbon and energy in plant metabolism, hexoses are commonly assumed to act as signals triggering up-regulation of defence related genes and down regulation of photosynthetic gene expression (Berger et al., 2004). However, carbohydrates are natural nutrients for plant pathogens so they also contribute to colonization of host plants (Peil et al., 2009). During biotic stress, including bacterial infection, these metabolites often accumulate in plant tissues and induce their senescence (Robinson et al., 2004). Plants are exposed to many different pathogens in the course of their life. Plant defence against pathogenic organisms is based on constitutive barriers and inducible mechanisms enhancing their resistance to a broad spectrum of pathogens. It was shown that induced resistance occurred after infection with necrotizing pathogens, root colonization with beneficial microorganisms and also after treatment with some natural or synthetic compounds. The utilization these inducers leads to mobilization of defence responses and establishment of systemic acquired resistance (SAR) (Conrath, 2009). SAR is a well-studied form of induced resistance and this mechanism is activated locally and systemically upon infection. However, the actual induced resistance status entails direct and indirect fitness costs (Vos et al., 2013). In general,
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Fig. 1. Symptoms of angular leaf spot disease on the 3rd cucumber leaves recorded 7 days post inoculation. A–C – general overview, D–F – close-up. The representative leaf images with symptoms routinely observed on cucumber leaves are shown. Macro photographs were taken with Panasonic DMC-FZ50 camera equipped with a close-up lens. Bar represents 0.5 cm. Severe disease symptoms in the form of irregular necrotic areas were visible on Pseudomonas syringae pv. lachrymans (Psl) infected leaves. BTH and ARB attenuated disease symptom severity, and on the leaves of BTH- or ARB-treated and subsequently Psl-inoculated plants (BTH + Psl and ARB + Psl, respectively) predominantly chlorotic lesions which covered less leaf area than in the non-treated ones were observed.
more resistant plants are less fit when the pathogen is not present (Canet et al., 2010). Benzothiadiazole (benzo(1,2,3) thiadiazole7-carbotionic acid-S-methyl ester, BTH), a functional analogue of salicylic acid (SA) is among effective inducers of SAR (Walters et al., 2005). However, BTH not only triggers plant defence but also biomass loss in a dose dependent manner (Canet et al., 2010). Cucumber is a common vegetable in which stachyose, not Suc, is the main translocated sugar. After long-distance transport to peduncles stachyose is converted to Suc (Miao et al., 2007). Utilization of Suc in cell metabolism depends on its cleavage into hexoses catalyzed, except sucrose synthase (EC 2.4.1.13), by invertase (EC 3.2.1.26). Plants possess two main isoforms of invertase (i) acid invertase (AcIn, -fructo-furanosidase) in vacuoles and apoplast which attacks Suc molecule from fructose residue and also hydrolyzes other -fructose-containing oligosaccharides such as raffinose and stachyose and (ii) Suc specific alkaline invertase (AlIn) in cytoplasm (Sturm, 1999). Changes in expression and activity of acid invertase could be used as a potential molecular marker of changes in plant primary metabolism (Biemelt and Sonnewald, 2006). Green leaves of many plant species contain low levels of lactate dehydrogenase (LDH, EC 1.1.1.27) under aerobic conditions (Sugiyama and Taniguchi, 1997). This enzyme catalyses the reversible reaction of lactate oxidation and pyruvate reduction (O’Carra and Mulcahy, 1996) and under specific conditions its higher activity may be used as a marker of cytotoxicity (Legrand et al., 1992). Arbutin (hydroquinone--d-glucopyranoside, ARB) is a natural soluble phenolic glucoside synthesised by plants of the families Ericaceae, Rosaceae, Saxifragaceae (Petkou et al., 2002). This compound can inhibit membrane lysis both free radical-mediated and enzymatic in nature (Frías et al., 2008; Oliver et al., 1996). It has been suggested that ARB contributes to membrane stabilization resulting from its interaction with membrane lipids. This phenomenon is connected with high concentration of arbutin in membranes (Frías et al., 2008). In cells ARB might be metabolized to hydroquinone
(HQ) and next to benzoquinone (2,5-cyclohexadiene-1,4-dione) which has direct antimicrobial activity (Jin and Sato, 2003). HQ is a reactive molecule capable of producing reactive oxygen species (ROS), including the superoxide anion, hydrogen peroxide and hydroxyl radical, and causing oxidative stress (Khanal et al., 2011; Xu et al., 2002). HQ also acts as a pro-oxidant that causes cytotoxicity and induces apoptosis (Xu et al., 2002). On the other hand, the studies of Casano et al. (2000) and Xu et al. (2002) showed, that the presence of additionally formed HQ molecules might enhance efficiency of photosynthesis. ARB is a well-known substrate for -glucosidase, but it may be also oxidized directly by polyphenoloxidase and by soluble and ionically bound to cell wall peroxidase (EC 1.11.1.7). (Hadˇzi-Taˇskovic´ ˇ Sukalovi c´ et al., 2007; Petkou et al., 2002). It has been suggested that the oxidation pathway of arbutin is involved in fire blight resistance of some pear varieties (Jin and Sato, 2003). On the other hand, Mo et al. (1995) reported that arbutin could serve as the signal molecule that induces production of syringomycin and other phytotoxins produced by P. syringae. To assess the ability of ARB to protect cucumber plants against angular leaf spot disease, we studied the effects of exogenous ARB in comparison to BTH on changes of carbohydrate metabolism in plants infected with Psl. We determined Glc and Suc levels as well as AcIn, AlIn, and LDH (as a marker of cytotoxity) activities both locally (in the infected leaves) and systemically (in the non-infected upper leaves). Moreover, we examined chlorophyll a (chla), b (chlb) and carotenoids (car) concentrations. 2. Material and methods 2.1. Biological material Cucumber (C. sativus L. cv. Polan) seeds were germinated in the dark at 24 ◦ C for 3 days. Then the seedlings were transferred into individual pots and grown hydroponically in the medium consisting of (mg/dm3 ): N – 224; P–39; K–312; Ca–160; Mg–33; S–44;
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Fe–0.84; Mo–0.98; Cu–0.84; Mn–8.4; B–4.2; Zn–4.2. Three-weekold plants were divided into three groups – control (non-treated) and plants treated once with 2.0 mM water solution of ARB or a commercial preparation BION 50 WG (Novartis Protection AG, Bazylea, Switzerland) containing 0.1 mM BTH as an active ingredient. The non-treated plants were sprayed with distilled water. One week later the third true leaves from the bottom were inoculated with Psl or treated with sterile distilled water (mock inoculation) using a needle-less hypodermic 1 cm3 syringe. Bacterial suspension was infiltrated at about 10 sites (per leaf) uniformly distributed on the abaxial side of the leaf blade. Bacteria for inoculation, pro´ Poland), were cultured vided by Bank of Plant Pathogens (Poznan, for 24 h on King B medium at 28 ◦ C with vigorous shaking and centrifuged at 3500 × g for 10 min. The bacterial pellet was washed twice, resuspended in sterile water, and adjusted to 107 cfu cm−3 . For biochemical analysis leaf samples without main veins (0.5 g FW) were immediately homogenized in a mortar in 5 cm3 icecold 0.05 M sodium phosphate buffer pH 7.0 containing 1 mM EDTA and 1% polyvinylpyrrolidone. Immediately after centrifugation (20,000 × g, 20 min, 4 ◦ C) the supernatant was used for measurement of enzyme (LDH, AlIn and AcIn) activities as well as protein content. For chlorophyll and carotenoids determination the tissues were stored at −20 ◦ C. To study local and systemic response of cucumber plant to Psl inoculation, the 3rd (inoculated) and the 5th (non-inoculated, upper) leaves were taken for analyses on 0, 2 and 7 days post inoculation (dpi). 2.2. Determination of leaf area and disease symptom development For determination of leaf area in control and ARB-treated plants, the leaves were taken 7 and 14 days after ARB application (time points corresponding to 0 and 7 dpi). The leaf area was assessed by a computer method of image analysis. Disease symptom development on the 3rd leaves was quantified 2 and 7 dpi and was given as percentage of the symptom-covered leaf area in relation of the total leaf area. The leaf images scanned to JPEG files were subjected to the automatic analysis and classification run in MATLAB aimed to extract discoloured regions corresponding to the pathogen-induced disease symptoms (Gocławski et al., 2012). 2.3. Determination of photosynthetic pigments To determine the contents of chlorophyll a (chla), chlorophyll b (chlb) and carotenoids (car) the frozen leaves were extracted three times with 80% acetone (1:20 w/v) and centrifuged (33,000 × g, 15 min). After measurement of absorbance of the supernatants at 470 nm, 663.2 nm and 646.8 nm, car, chla and chlb contents were calculated according to the method of Wellburn (1994). 2.4. Determination of glucose and sucrose Glc and Suc contents were determined in the extracts obtained from leaf samples (0.5 g FW) after triple 80% ethanol extraction. The ethanolic extracts were evaporated to dryness at 50 ◦ C and the residue was resolubilized with distilled water. The concentrations of both sugars were assayed using the commercial enzymatic test (Boehring Mannheim) according to the manufacturer’s instruction and expressed in g per mg protein determined in the extract used for enzyme activity analyses. 2.5. Enzyme assays Invertase activity was determined by the method described by Miller and Ranwala (1994). The reaction mixture consisted of
40 mM Suc, 50 mM Na-acetate buffer (pH 5.0 for AcIn and pH 7.5 for AlIn). The buffer and substrate were heated in water bath at 37 ◦ C for 5 min prior to substrate addition. When Suc was added, the reaction mixture was incubated at 37 ◦ C. Exactly after 30 min the reaction was stopped in a 90 ◦ C bath for 5 min. The amount of Glc released was determined as described above. One unit of invertase activity was defined as the amount of enzyme that catalysed the production of 1 mol of Glc per minute at 37 ◦ C per mg protein. LDH activity was measured by the decrease in absorbance at 340 nm resulting from oxidation of NADH ( = 6.22/mM/cm) according to Sugiyama and Taniguchi (1997) method. The assay mixture consisted of 50 mM K-phosphate buffer (pH 7.25), 0.5 mM NADH2 , 4 mM sodium pyruvate, 2.3 mM 4-methypyranozole and 2.3 mM NaCN. The activity was expressed in terms of nmol NADH oxidised per minute per mg protein. 2.6. Protein content determination The protein content was determined according to Bradford (1976), with a standard curve prepared using bovine serum albumin. 2.7. Statistical analysis The results presented are the means from 8 to 10 independent experiments. Sample variability is given as the standard deviation of the mean. The significance of differences between control and Psl or BTH and BTH + Psl or ARB and ARB + Psl as well as between Psl and BTH + Psl or ARB + Psl was determined by a non-parametric MannWhitney Rank Sum Test. Differences at P < 0.05 were considered significant. 3. Results 3.1. Infection development, vegetative growth and photosynthetic pigment levels Small chlorotic spots being the macroscopic symptoms of angular leaf spot of cucumber were observed about 2 dpi. Five days later (7 dpi) they became larger and darker, and necroses with a chlorotic halo were formed (Fig. 1). The adjacent interveinal leaf area turned yellow and about 14 dpi the affected leaf part shriveled and died. Foliar application of ARB 7 days before Psl inoculation significantly reduced angular leaf spot disease severity in comparison to the nonpretreated inoculated plants (Psl). ARB caused about 48% disease suppression in the third leaf on the 7th dpi (Figs. 1 and 2). The similar effect was observed after pretreatment of cucumber plants with ´ BTH (Kuzniak et al., 2014). ´ Contrary to BTH (Kuzniak et al., 2014), ARB treatment positively influenced growth of the 5th leaves whose area was more than 40% bigger than the control (Table 1). Moreover, the ARB-treated plants were more fit than the BTH-treated or control ones (data not shown). A few changes were observed in the levels of chla, chlb and carotenoids in all examined plant groups (Tables 2–4). In the older (3rd) infected leaves of the non-pretreated plants the changes were limited to chla concentration on the 2nd dpi (decrease by 15% in comparison to the non-infected ones), while in the ARB-treated plants following infection the lower chla levels were observed on the 2nd and 7th dpi, 75% and 79% of the non-infected ARB-treated material, respectively (Table 2). Pretreatment of plants with BTH augmented the negative effect of infection on chlorophyll content. Comparing to the non-pretreated infected plants chla and chlb contents in the 3rd leaf of the BTH-treated infected plants were 32% and 48% lower, respectively (Table 2). Similar effect was also observed in the 5th leaves. Different changes of the chlb level in
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Table 1 Influence of arbutin on the area of the 3rd and the 5th leaves of cucumber plants. Leaf area (mm2 )
Treatment
3rd leaf
Control ARB
5th leaf
7 days
14 days
7 days
14 days
9753.9 ± 1755.7 12434.5 ± 1492.1
15124.6 ± 5750.1 17055.8 ± 3752.3
2419.9 ± 362.9 3503.6 ± 595.6*
11613.5 ± 1277.5 16610.9 ± 2657.6*
Leaf area was measured 7 and 14 days after ARB treatment (which corresponds to 0 and 7 dpi). Data are means ± SD. Five plants were used for each treatment (n = 5). * indicates a significant difference between the control and ARB-treated plants at P < 0.05. Table 2 Chlorophyll a (chla) and chlorophyll b (chlb) contents (mg/g FW) in the 3rd cucumber leaf 0, 2 and 7 days post inoculation (dpi) with P. syringae pv. lachrymans. Variant
Noninfected Infected
Time (dpi)
0 2 7 2 7
Control
BTH
ARB
Chla
Chlb
Chla
Chlb
Chla
Chlb
0.778 ± 0.052 0.735 ± 0.041 0.704 ± 0.064 0.627 ± 0.051* 0.694 ± 0.078
0.315 ± 0.098 0.316 ± 0.052 0.360 ± 0.082 0.299 ± 0.103 0.389 ± 0.121
0.593 ± 0.053 0.476 ± 0.046 0.395 ± 0.118 0.429 ± 0.044# 0.518 ± 0.101
0.217 ± 0.021 0.173 ± 0.023 0.147 ± 0.030 0.157 ± 0.029# 0.215 ± 0.035* ,#
0.798 ± 0.131 0.819 ± 0.050 0.851 ± 0.072 0.611 ± 0.065* 0.677 ± 0.047*
0.345 ± 0.120 0.342 ± 0.029 0.399 ± 0.038 0.381 ± 0.068 0.289 ± 0.058*
Data are means ± SD. * indicates a significant difference between control and Psl or BTH and BTH + Psl or ARB and ARB + Psl at P < 0.05. # indicates a significant difference between Psl and BTH + Psl or ARB and ARB + Psl at P < 0.05. Table 3 Chlorophyll a (chla) and chlorophyll b (chlb) contents (mg/g FW) in the 5th cucumber leaf 0, 2 and 7 days post inoculation (dpi) with P. syringae pv. lachrymans. Variant
Time (dpi)
Noninfected
0 2 7 2 7
Infected
Control
BTH
ARB
Chla
Chlb
Chla
Chlb
Chla
Chlb
0.911 ± 0.012 0.896 ± 0.079 0.804 ± 0.158 1.123 ± 0.080* 1.023 ± 0.188*
0.424 ± 0.035 0.403 ± 0.057 0.429 ± 0.113 0.471 ± 0.042 0.443 ± 0.107
0.868 ± 0.036 0.626 ± 0.134 0.653 ± 0.136 0.576 ± 0.125# 0.595 ± 0.105#
0.405 ± 0.010 0.275 ± 0.051 0.288 ± 0.052 0.230 ± 0.030# 0.230 ± 0.048#
1.049 ± 0.080 0.859 ± 0.108 0.783 ± 0.088 1.020 ± 0.139 1.027 ± 0.139*
0.459 ± 0.024 0.392 ± 0.084 0.434 ± 0.074 0.433 ± 0.084 0.438 ± 0.044
Data are means ± SD. * indicates a significant difference between control and Psl or BTH and BTH+Psl or ARB and ARB + Psl at P < 0.05. # indicates a significant difference between Psl and BTH + Psl or ARB and ARB + Psl at P < 0.05.
the infected 3rd leaves were noted (Table 2). On the 7th dpi in the BTH-treated plants its level increased by 46% but in the ARBtreated ones decreased by 28% in comparison to the respective non-infected plants. In all other cases chla, chlb as well as car concentrations did not show significant differences between the infected and non-infected 3rd leaves. In the 5th leaves Psl infection changed these parameters only in the non-treated and ARB-treated plants (Table 3). In the non-treated plants chla levels were higher by 25% and 27% on the 2nd dpi and 7th dpi, respectively and car level was increased by 41% on the 2nd dpi in comparison to the non-infected plants (Table 4). In the 5th leaves of the BTH-treated infected plants car concentration was about 44% lower comparing to the non-pretreated infected plants. In the ARB-treated plants following infection 31% increase in chla level was observed at the end of experiment (7th dpi).
3.2. Glucose and sucrose content The obtained data indicated that after Psl inoculation in the directly inoculated (3rd) leaves on the 2nd dpi only in the BTHtreated group Glc concentration was lower (47% of the value observed in the non-infected ones) (Fig. 3). The enhancement of Glc level in the inoculated 3rd leaves in comparison to the noninoculated ones was found in the control group on the 7th dpi (213%) (Fig. 3). The younger (5th) leaves on the 2nd dpi in all infected plant groups showed higher Glc levels than the noninfected ones (Fig. 3). They were 152%, 125% and 191% for the control, the BTH-treated and the ARB-treated ones, respectively. On the 7th dpi the higher Glc level was found only in the infected ARBtreated leaves (216% of the value determined in the non-infected
Table 4 Carotenoids contents (mg/g FW) in the 3rd and 5th cucumber leaf 0, 2 and 7 days post inoculation (dpi) with P. syringae pv. lachrymans. Variant
Time (dpi)
Non-infected
0 2 7 2 7
Infected
Control
BTH
ARB
3rd leaf
5th leaf
3rd leaf
5th leaf
3rd leaf
5th leaf
0.189 ± 0.016 0.157 ± 0.028 0.165 ± 0.022 0.137 ± 0.016 0.157 ± 0.019
0.239 ± 0.016 0.221 ± 0.019 0.200 ± 0.070 0.312 ± 0.037* 0.249 ± 0.065
0.154 ± 0.023 0.155 ± 0.035 0.118 ± 0.032 0.179 ± 0.035 0.140 ± 0.025
0.205 ± 0.007 0.140 ± 0.034 0.113 ± 0.022 0.174 ± 0.020# 0.140 ± 0.029#
0.187 ± 0.021 0.183 ± 0.022 0.191 ± 0.016 0.143 ± 0.031 0.173 ± 0.028
0.257 ± 0.030 0.209 ± 0.035 0.183 ± 0.021 0.235 ± 0.049# 0.237 ± 0.059
Data are means ± SD. * indicates a significant difference between control and Psl or BTH and BTH + Psl or ARB and ARB + Psl at P < 0.05. # indicates a significant difference between Psl and BTH + Psl or Psl and ARB + Psl at P < 0.05.
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Symptom-covered leaf area (%)
16 14
Psl
ARB + Psl
12
*
10 8 6 4 2 0 2
7
Time post inoculation (days) Fig. 2. Effect of ARB treatment on angular leaf spot disease development in the 3rd leaves of cucumber plants expressed as percentage of symptom-covered area in relation to the total leaf area. * indicates a significant difference between Psl and ARB + Psl at P < 0.05 (n = 5).
ARB-treated ones), while in the control the lower level (67%) of Glc in comparison to the non-infected control plants was detected.
Pretreatment with ARB significantly influenced Glc content in the 5th leaf of the infected plants comparing to the non-pretreated infected ones. On the 2nd dpi and on the 7th dpi concentration of Glc in the ARB+Psl group exceeded the values found in the non-treated infected plants by 67% and 129%, respectively (Fig. 3). In the case of Suc levels on the 2nd dpi significant diminution was observed in the 3rd leaves in all tested groups (Fig. 4). In the infected plants as compared to the non-inoculated ones Suc levels constituted 41% of that in the BTH-treated material, 48% of that in the control and 61% of that in the ARB-treated plants. On the 7th dpi the decreased level of Suc was found in the 3rd leaves from both treated groups. It was 57% and 43% in the BTH-treated and the ARB-treated plants in comparison to the non-inoculated ones, respectively. In the 5th leaves of Psl inoculated plants Suc levels were diminished in the control and BTH-treated plants in both examined times (Fig. 4). They were 45% and 77% of the non-infected ones on the 2nd dpi and 75% and 70% on the 7th dpi, respectively. ARB impact depended on the time. In the 5th leaves of the infected plants Suc level was 29% higher on the 2nd dpi and 67% lower
Fig. 3. Effect of BTH and ARB treatments on glucose (Glc) concentration in the 3rd and the 5th leaves of cucumber plants 0, 2 and 7 days post inoculation with P. syringae pv. lachrymans (Psl). Bars represent SD of means. * indicates a significant difference between control and Psl or BTH and BTH + Psl or ARB and ARB + Psl at P < 0.05. # indicates a significant difference between Psl and BTH + Psl or ARB + Psl at P < 0.05.
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Fig. 4. Effect of BTH and ARB treatments on sucrose (Suc) concentration in the 3rd and the 5th leaves of cucumber plants 0, 2 and 7 days post inoculation with P. syringae pv. lachrymans (Psl). Bars represent SD of means. * indicates a significant difference between control and Psl or BTH and BTH + Psl or ARB and ARB + Psl at P < 0.05. # indicates a significant difference between Psl and BTH + Psl or ARB + Psl at P < 0.05.
on the 7th dpi than the respective values in the non-infected material. On the 2nd dpi both in the 3rd and the 5th leaf Suc content in BTH-treated and ARB-treated infected plants was higher comparing to the infected non-pretreated ones. Comparing to plants only inoculated, in the BTH-treated and ARB-treated and inoculated plants Suc content in the 3rd leaf was increased by 54% and 45%, respectively and in the 5th leaf by 45% and 164%, respectively. However, on the 7th dpi a significant decrease in Suc content in the ARB-treated and infected plants was found, by 48% and 26% comparing to the non-treated infected ones, in the 3rd and the 5th leaf, respectively (Fig. 4). 3.3. AlIn, AcIn and LDH activities Infection enhanced AlIn activity in the directly inoculated (3rd) leaves only in the non-treated plants at the end of experiment (7th dpi) (Fig. 5). It was 187% of the value detected in the non-inoculated control. At the same time decrease in this enzyme activity was observed in the infected BTH-treated plants (40% of the value deter-
mined in the non-infected BTH-treated ones) (Fig. 5). On the 2nd dpi, in the BTH-treated group the higher level of AlIn activity in the 5th leaves was found in the infected plants (158% of the value determined in the non-infected ones) (Fig. 5). AlIn activity was higher in the infected non-treated and ARB-treated material in the 5th leaves on the 7th dpi (308% and 242% in comparison to the noninfected group, respectively), while at this time the decrease in AlIn activity was strongly manifested in the BTH-treated plants where the difference between the infected and non-infected plants was 41%. Pretreatment with BTH and ARB decreased AlIn activity in the infected group of plants compared to the non-pretreated infected ones on the 7th dpi. In the 3rd leaf AlIn activity in the BTH + Psl and ARB + Psl groups was 53% and 38% lower than in the non-treated infected plants. Similar effect was observed in the 5th leaf on the 7th dpi. However, on the 2nd dpi AlIn activity in BTH-treated and subsequently infected plants was 107% higher comparing to plants only infected (Fig. 5). AcIn activity in the Psl inoculated 3rd leaves demonstrated a similar tendency as AlIn (Fig. 6). The increase in the infected control to 140% and decrease in the infected BTH-treated to 47% in
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Fig. 5. Effect of BTH and ARB treatments on alkaline invertase (AlIn) activity in the 3rd and the 5th leaves of cucumber plants 0, 2 and 7 days post inoculation with P. syringae pv. lachrymans (Psl). Bars represent SD of means. * indicates a significant difference between control and Psl or BTH and BTH + Psl or ARB and ARB + Psl at P < 0.05. # indicates a significant difference between Psl and BTH + Psl or ARB+Psl at P < 0.05.
comparison to the respective non-infected plants on the 7th dpi, were observed (Fig. 6). The younger leaves (5th) showed enhanced AcIn activity in the non-treated and BTH-treated groups 2 days after inoculation (Fig. 6). Its activity was by 43% and 50% higher when compared to the values observed in the respective non-infected tissues. On the 7th dpi in the 5th leaves from the infected control plants AcIn activity was as high as on the 2nd dpi (143%). On the 7th dpi in the ARB-treated ones infection enhanced this activity to 199% in comparison to the respective non-infected group (Fig. 6). On the 7th dpi AcIn activity in the 3rd leaf of the pretreated and subsequently infected plants was decreased comparing to the nonpretreated infected group, by about 40% and 30% in BTH + Psl and ARB + Psl plants, respectively. At the same time in the 5th leaf of the ARB-treated infected plants AcIn activity was 47% higher than in the infected non-treated plants (Fig. 6). Psl inoculation markedly changed the LDH activity only in the control and the BTH-treated plants in both examined leaves (Fig. 7). However, in the case of this enzyme the changes appeared earlier
in the younger leaves than in the older ones. On the 2nd dpi in the 5th leaves the respective values were 273% and 135% of the non-infected control and BTH-treated plants. Five days later (7th dpi) enhanced activity (by 50%) was observed only in the infected control (Fig. 7). The directly inoculated leaves (3rd) showed significant increase in LDH activity in comparison to the non-inoculated ones in the control and BTH-treated plants but only on the 7th dpi, the values were 127% and 143%, respectively (Fig. 7). In the 5th leaf a significant decrease in LDH activity was observed in ARBtreated infected plants comparing to infected non-treated ones, by 67% and 51% on the 2nd dpi and on the 7th dpi, respectively (Fig. 7). 4. Discussion Infection with bacterial pathogens strongly influenced photosynthesis whose efficiency is correlated with vigour and fitness of plants (Li et al., 2013). Reduced expression of genes involved in
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Fig. 6. Effect of BTH and ARB treatments on acid invertase (AcIn) activity in the 3rd and the 5th leaves of cucumber plants 0, 2 and 7 days post inoculation with P. syringae pv. lachrymans. Bars represent SD of means. * indicates a significant difference between control and Psl or BTH and BTH + Psl or ARB and ARB + Psl at P < 0.05. # indicates a significant difference between Psl and BTH + Psl or ARB + Psl at P < 0.05.
photosynthesis in Arabidopsis infected with P. syringae (Thilmony et al., 2006; Truman et al., 2006) and inhibition of tomato chloroplast function by P. syringae pv. tomato DC3000 were shown (Rico et al., 2011). In our study, in the control and ARB-treated plants following Psl infection in the directly inoculated leaves (3rd) the chlorophyll levels decreased while in the systemic leaves (5th) they increased. It might indicate that the control and ARB-treated plants switched on a compensatory mechanism connected with earlier metabolic maturation of the 5th leaves. The chlorophyll and carotenoid levels as well as our results concerning carbohydrate metabolism showed that assimilate production was more effective in these leaves than in the BTH-treated ones. However, in the ARB-treated plants this led to better plant development while in the control this promoted disease development. No significant changes in chlorophyll pigments were observed after inoculation in leaves of the BTH-treated plants, it may have been due to the fact that chl levels were initially by 20–30% lower in these plants then in the other ones. The decrease in photosynthetic pigment levels in the inoculated leaves might result from the direct influence of the pathogen on the expression of photosynthetic genes
but also from the development of chlorotic and necrotic areas on the leaves as the result of plant-pathogenic bacteria interaction (Berber et al., 2010). The latter possibility rather refers to the control plants because in the ARB-treated ones these symptoms were limited. Robinson et al. (2004) found in P. syringae pv. tagetis infected sunflower leaves that the bacterial toxins caused inhibition of photosynthetic carbon assimilation, reflected by the significant decrease in daily production of glucose, sucrose and starch, thus they postulated that infection by bacterial pathogens might influence leaf senescence via modulation of sugar status. Earlier it was suggested that leaf senescence was correlated with an increased sugar level (Yoshida, 2003), but Bonfig et al. (2006) demonstrated that sugar levels in Arabidopsis were not altered by infection with P. syringae. Here, we showed that in the ARB-treated 3rd leaves the Glc levels and AcIn activities were similar in the infected and non-infected plants, but Suc levels were lower in the infected ones. These observations suggest that pretreatment of plants with ARB might induce reactions resembling those accompanying the senescence process.
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Fig. 7. Effect of BTH and ARB treatments on lactate dehydrogenase (LDH) activity in the 3rd and the 5th cucumber leaves 0, 2 and 7 days post inoculation with P. syringae pv. lachrymans. Bars represent SD of means. * indicates a significant difference between control and Psl or BTH and BTH + Psl or ARB and ARB + Psl at P < 0.05. # indicates a significant difference between Psl and BTH + Psl or ARB + Psl at P <0.05.
On the other hand, bacterial pathogens may interfere with the source-sink balance leading to the reallocation of photoassimilates to sufficiently supply the pathogen with nutrients (Berger et al., 2004; Biemelt and Sonnewald, 2006). It is evidenced by stimulation of acid invertase that mobilizes hexoses at the infection site and a decreased rate of photosynthesis (Kocal et al., 2008). Growing bacterial population needs more nutritional compounds thus pathogen-induced modification of plant metabolism to its own advantage can be a good strategy as was shown for P. syringae by Rico et al. (2011) and Berger et al. (2004) who reported that sugars were often accumulated in plants during plant-pathogen interactions. Our results are to some extent in agreement with the finding of Berber et al. (2010) showing in P. syringae-infected tomato plants induction of cell wall invertase activities and decrease in Glc and Suc contents between the first and tenth study days, however it is worth noting that the crucial drop of Suc level appeared on the first day of experiment. In our study in the infected 3rd leaves increased Glc concentration together with enhanced AcIn activity was observed only in the control, because it took place on the 7th
dpi it probably resulted from the pathogen action. On the 2nd dpi in the BTH-treated plants Glc concentration decreased in the 3rd leaf but increased in the 5th leaf. It should be noted that in BTH-treated plants before inoculation Glc level was much higher than in the others, thus the decrease in the 3rd leaf was more pronounced. It was mainly caused by intensified synthesis of compounds involved in defence while direct pathogen impact was smaller because the activities of both invertases were decreased and the observed morphological changes connected with infection were limited. Due to ARB and BTH pretreatment decrease in Suc concentration occurred after Psl infection in directly inoculated tissues in both examined times. Taking into consideration the infected and noninfected plants, no difference for the ARB-treated or decrease for the BTH-treated ones in AlIn and AcIn activities were observed. Together with the fact that in both infected groups Glc levels were not changed a hypothesis can be put forward that pathogen access to carbohydrates was limited. The obtained results indicate that, the basic difference between BTH and ARB action after Psl infection concerned invertase activity in the directly inoculated leaves. In the BTH-treated plants it was
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decreased while in the ARB-treated ones no changes were observed. This indicates that BTH limited the rate of carbohydrate metabolism while ARB did not. The analysis of changes observed in these parameters in the systemic (5th) leaves indicate that after Psl inoculation decrease in Suc level took place in all infected groups. However, on the basis of the observed Glc level, invertase activities and morphological picture we suggest different mechanisms underlying these changes. The infected control plants showed decrease in Suc level accompanied with elevated activity of both invertases and significant decrease in Glc level between the 2nd and 7th dpi. In the BTH-treated plants, Glc level and activites of both invertases were elevated only on the 2nd dai. This might have caused diminution of Suc on the 2nd dpi which persisted till the 7th dpi, when all other parameters showed the same or lowered values in comparison to the non-inoculated plants. It may indicate that an infection-induced signal(s) was transferred from the infected area to younger tissues and mobilized BTH-mediated pathways involved in defence processes leading to inhibition of pathogen spread because the disease symptoms were not visible. In cucumber infected with Psl, SAR is induced by systemically accumulated SA (Meuwly et al., 1995). It was reported that single application of BTH at high dose increased SA content and triggered adequate defence responses after pathogen attack (Rad et al., 2005) but under these conditions the plant growth was limited (Canet et al., 2010). The reduction of plant growth may result from BTH-mediated changes in carbohydrate metabolism. The systemic ARB-treated leaves following infection showed high level of Glc and enhanced activity of both invertases accompanied with decrease in Suc level on the 7th dpi. It may be supposed that in these tissues intensification of assimilate production and export limited the losses resulting from the pathogen impact. Its action led among others to changed carbohydrate metabolism and decreased chlorophyll levels in the directly inoculated leaves. This suggestion seems correct because similarly as in the BTH-treated plants the disease symptoms were not visible. Herbers et al. (2000) proposed that sugar acted as an amplifier of plant defence responses during plant-pathogen interaction. This hypothesis was modified by Biemelt and Sonnewald (2006). They suggested that suppression or no changes of AcIn activity and hexose-mediated defence during the early phase (first 24 h) of bacterial infection allowed the bacteria to settle, but in later phase (following 48 h) the AcIn was induced to meet the nutrient demand of multiplying bacteria, thus AcIn regulation during the early and late infection phases differed. In our research this correlation was found only in the control directly inoculated leaf tissues. It is well known that pathogen infections induce rapid accumulation of reactive oxygen species (ROS) in plant tissues and their early accumulation was the key role in inhibiting penetration of a biotrophic pathogen (Barna et al., 2012). The increased carbohydrate concentrations may subsequently result in the production of ROS and activation of ROS-downstream responses (Couée et al., 2006). Arbutin presence might increase ROS level but this process requires its metabolic activation to HQ (Khanal et al., 2011). HQ causes concentration-dependent decrease in cell viability and/or increase in LDH activity, a marker of cytotoxicity (Legrand et al., 1992) but, as shown by Khanal et al. (2011), exogenous ARB did not cause a cytotoxic effect and LDH release up to 2 mM concentration in hepatoma HepG2 cell cultures. The ARB concentration used in this study was not toxic to plant cells because in the ARB-treated plants the LDH activity did not increase in any of the experimental variants. Arbutin can be metabolized by plant as well as by bacterial enzymes. Khanal et al. (2011) showed that ARB was degraded to HQ by human intestinal microflora. Earlier, Siegers et al. (2003)
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observed that Escherichia coli bacteria might liberate free HQ from its conjugates thereby killing themselves. HQ is quickly oxidized by a variety of oxidants and molecular oxygen and its oxidized derivatives are toxic for cells because they react with a large spectrum of cellular macromolecules and low molecular weight nucleophiles resulting in inhibition of some enzymes and disturbance of redox status (Pandey et al., 2005). It is probable that the plant and the pathogen used in this study showed different levels of ARB tolerance. Moreover, as it was found by Hadˇziˇ Taˇskovic´ Sukalovi c´ et al. (2007) and Casano et al. (2000) enhanced formation of HQ positively influenced plant mitochondrial and thylakoid membrane-bound hydroquinone peroxidase specific for H2 O2 and this increased the efficiency of antioxidative protection. The presence of elevated HQ level may also enhance productivity of photosynthesis (Casano et al., 2000; Xu et al., 2002). Accordingly, we observed that the ARB-treated plants exhibited better vigour and fitness and were characterised with higher level of chlorophyll than the control and BTH-treated ones. Following Psl infection LDH activity increased in the control and BTH-treated plants. It should be noted that younger leaves were affected earlier and to a greater extent than the older ones. Our results indicate that the enhanced LDH activity in the infected control and BTH-treated plants may be connected with insufficient supply of ATP and Glc. In all experimental variants in both these groups higher LDH activity was accompanied with higher Glc level. Moreover, when in the BTH-treated 3rd leaves on the 2nd dpi the significant decrease in Glc concentration took place the LDH activity was decreased, too. However, in the control plants carbon metabolism was mainly disturbed by direct pathogen action and these disturbances were observed in the inoculated and systemic leaves at all examined times, whereas in the BTH-treated ones it was connected with defence e.g. with increased synthesis of secondary metabolites. 5. Conclusions The severity of angular leaf spot disease caused by Psl in cucumber was reduced by both BTH and ARB indicating that both compounds were effective against this hemibiotrophic pathogen, however, in our opinion the mechanisms of their activity were different. BTH acts as an effective inducer of SAR (Walters et al., 2005) while ARB is a biostimulating and antimicrobial agent. Higher level of chlorophyll and more effective carbohydrate metabolism as well as less severe disease symptoms in the ARBtreated and inoculated plants may suggest that they were better fortified against the pathogen attack. A hypothesis may be put forward that ARB induced the senescence-like program in the injured leaves which restricted spread of the infection. In our opinion the use of ARB as a natural compound which may protect cucumber plants against Psl infection and simultaneously improve their growth and vigour should be taken into consideration in future studies. Acknowledgements This work was supported by National Science Centre Grant No N N310 302339. The authors are very grateful to Ms M. Fronczak for her linguistic correction of the manuscript. References Barna, B., Fodor, J., Harrach, B.D., Pogány, M., Király, Z., 2012. The Janus face of reactive oxygen species in resistance and susceptibility of plants to necrotrophic and biotrophic pathogens. Plant Physiol. Biochem. 59, 37–43, http://dx.doi.org/10.1016/j.plaphy.2012.01.014
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