Changes in Antioxidative Enzyme Activities in ...

Article

J. Korean Soc. Appl. Biol. Chem. 54(4), 507-514 (2011)

Changes in Antioxidative Enzyme Activities in Cucumber Plants with Regard to Biological Control of Root-knot Nematode, Meloidogyne incognita with Cinnamomum cassia Crude Extracts Dang-Minh-Chanh Nguyen1,2, Dong-Jun Seo2, Ro-Dong Park2, Bok-Rye Lee3, and Woo-Jin Jung2* 1

Western Highlands Agro-Forestry Scientific and Technical Institute, 53 Nguyen Luong Bang Street, Buon Ma Thuot City, DakLak Province, Vietnam 2 Division of Applied Bioscience and Biotechnology, Environment-Friendly Agriculture Research Center (EFARC), Institute of Agricultural Science and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea 3 Department of Animal Sciences, Institute of Agricultural Science and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea Received March 8, 2011; Accepted April 11, 2011 Cucumber plants were infected with the root-knot nematode Meloidogyne incognita and were then treated with Cinnamomum cassia crude extracts (CCE). The activities of antioxidative enzymes such as superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), and ascorbate peroxidase (APX, EC 1.11.1.11, class I peroxidase) were detected in the cucumber leaves. On day 28 after treatment of C. cassia (0, 1, 5, and 10 mg/mL), the relative galling formation decreased significantly in a dose-dependent manner. The C. cassia crude extracts also effectively inhibited the growth of M. incognita in soil and roots. The number of second-stage juveniles was significantly decreased as the concentration of the C. cassia crude extracts increased. Twenty-eight days after treatment with CCE, the SOD activities of plant leaves treated with 5 and 10 mg/mL CCE were greater than that of leaves treated with nematode (Ne) only. In addition, the CAT and ascorbate peroxidase (APX) activities increased as the concentration of CCE increased. The relative activities of the antioxidative enzymes decreased as the galling formation in the roots increased in response to M. incognita infection. Polyacrylamide gel electrophoresis (PAGE) with activity staining revealed active bands of SOD corresponding to at least five isozymes (93, 79, 71, 66, and 41 kDa), including three Fe-SOD bands (71, 66, and 41 kDa) and two Mn-SOD bands (93 and 79 kDa). Analysis of the cucumber leaves revealed one active band corresponding to the CAT isozyme (>97 kDa) on 10% native gels. At least four bands corresponding to APX were observed in 10% polyacrylamide, including one major band (>97 kDa) and three minor bands (79, 71, and 66 kDa). Compared with the leaves of cucumber plants that received other treatments, SOD (41 kDa) and APX (70 kDa) isozymes in the leaves of plants treated with Ne only showed intensified bands in PAGE gels. Key words: ascorbate peroxidase, catalase, Cinnamomum cassia, Meloidogyne incognita superoxide dismutase

Systemic acquired resistance (SAR) is a mechanism of induced defense that confers long-lasting protection against a broad spectrum of microorganisms. SAR requires the signal molecule salicylic acid (SA) and is associated with *Corresponding author Phone: +82-62-530-3960; Fax: +82-62-530-2129 Email: [email protected] http://dx.doi.org/10.3839/jksabc.2011.078

accumulation of pathogenesis-related proteins, which are thought to contribute to resistance [Durrant and Dong, 2004]. Such acquired and induced resistance has been reported in many plant species challenged with different pathogens and chemicals [Ryals et al., 1996; Sticher et al., 1997]. In many cases, SAR involves expression of a set of genes, including those encoding pathogenesisrelated (PR) proteins and enzymes [Ryals et al., 1996; Sticher et al., 1997]. SA is thought to be essential for establishment of SAR in plants [Malamy and Klessig,

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1992; Ryals et al., 1996]; exogenous SA induces resistance in many plant species. Furthermore, SAR to Meloidogyne hapla was found in tomato (Lycopersicon esculentum Mill.) and pyrethrum (Chrysanthemum cinerariaefolium) plants through prior inoculation with two other host incompatible, root-knot nematode species, Meloidogyne incognita and Meloidogyne javanica [Ogallo and McClure, 1995; 1996]. In cucumber, induction of SAR by different pathogens as well as by SA, 2,6dichloroisonicotinic acid (INA) and thiadiazole-7carbothionic acid S-methyl ester (BTH) have been reported [Feussner et al., 1996; Smith-Becker et al., 1998]. The generation of reactive oxygen species (ROS), such as the superoxide anion (O2) and hydrogen peroxide (H2O2), is a common event associated with normal plant on biochemical processes including chloroplast and mitochondrial electron transport, and oxidase in the plasma membrane [Laloi et al., 2004; Zhou et al., 2004]. Both the formation of oxidized products and the induction of antioxidant mechanisms may be signs of ROS overproduction and thus oxidative stress. ROS production has been well established in several plant tissues associated with the expression of hypersensitivity (HR) or SAR [Laloi et al., 2004]. Generally, aerobic organisms have equivalent antioxidative molecules or at least one form of superoxide dismutase (SOD, EC 1.15.1.1) to cope with oxidative stress [Dufernez et al., 2008]. SOD converts two molecules of superoxide anion (O2) into oxygen (O2) and hydrogen peroxide (H2O2) at rates limited only by diffusion, whereas catalase (CAT, EC 1.11.1.6) reduces H2O2 into H2O and O2. Their function is to maintain low steady state levels of ROS in the cell [Barbehenn, 2002]. Cinnamon extracts with compatible flavors can be used as antifungal agents in products that are easily subjected to fungal contamination, such as bakery products. Nielsen and Rios [2000] demonstrated that cinnamon extracts exerted not only antifungal but also antibacterial activities and acted as an insecticide. Nguyen et al. [2009b], in our previous work, suggested that cinnamon methanol extracts could be useful as fungicides against Rhizoctonia solani. Moreover, it was shown to exert more strong nematicidal activity against the pine wood nematode Bursaphelenchus xylophilus in vitro [Nguyen et al., 2009a]. In the present study, the effects of Cinnamomun cassia crude extracts and antioxidative enzyme activities (SOD, CAT, and ascorbate peroxidase (APX)) on cucumber plants were investigated after infection with root-knot nematode, M. incognita.

Materials and Methods Preparation and extraction of C. cassia. C. cassia samples were purchased from a traditional medicine market in Vietnam. The samples were cut into 3-cm pieces, after which they were placed in paper bags and dried by heating in an oven at 50oC for 3 days. The samples were then stored at room temperature until use. Subsequently, the samples were extracted in 99% methanol at a ratio of 1:5 (v/v, dry plant material/solvent) at 30oC with shaking at 150 rpm for 7 days. Subsequently, the extracts were vacuum-filtered through a Whatman No. 2 filter, after which the solvents were dried by vacuum evaporation at 40oC to give paste. The pastes were then weighed and re-dissolved in methanol to give a final concentration of 100 mg/mL. Finally, the C. cassia crude extracts (CCE) were diluted in water to give final concentrations of 1, 5, and, 10 mg/mL immediately before the experiment. Plant material and growth conditions. Cucumber (Cucumis sativus L. Asia Unchun F1) seeds were sterilized in 70% ethanol for 3 min, after which they were thoroughly washed with sterile distilled water. Three seeds were then sown in each pot, and the plants were thinned to a density of one per pot after 1 week. Cucumber plants were grown in seedbed soils that had been steamsterilized and mixed with sterilized quartz sand and vermiculate at a ratio of 2:2:1 (v/v/v) and then placed into pots to a volume of 1200 mL. The plant growth medium consisted of modified Johnson’s solution [Johnson, 1957]. Culture preparation of root-knot nematodes on cucumber plants. To inoculate the cucumber plants with nematodes, second stage juveniles (J2) of M. incognita were extracted from the roots of infected cucumbers using the modified Baermann’s method [Southey, 1986]. The seedlings (3-week-old) were then inoculated with 10 mL of nematode suspension (5000 of second stage juveniles) per pot at four sites around the root system. In addition, an un-inoculated pot was used as a control. One week after inoculation, the cucumber seedling (4-weekold) in each pot was separately treated with 100 mL of 1, 5, or 10 mg/mL of CCE by irrigation or with 100 mL of water as a control. Each treatment was replicated three times with fifteen pots prepared. Assessment of relative galling formation. The cucumber root was collected on day 28 after treatment with CCE, after which all root galls were washed. The relative galling formation (RGF) was then determined to assess the disease severity using the following equation:

Antioxidative Enzyme Activities in Biocontrol of Root-knot Nematode

RGF (%)=NGCCE/NGN×100; where NGCCE is the number of galls in CCE treatments and NGN is the number of galls in nematode treatment only. Preparation of crude enzymes from cucumber leaves. The fifth cucumber leaves from the top were collected on day 28 after treatment with CCE and then washed under running tap water. All samples were frozen in liquid nitrogen and stored at −80oC until used. Samples (500 mg) were powdered with liquid nitrogen and then homogenized in 1 mL of homogenization buffer (100 mM potassium phosphate buffer at pH 7.0 containing 2 mM EDTA, 1% polyvinylpyrrolidone (PVP), and 1 mM phenylmethyl sulfonyl fluoride (PMSF). The homogenates were then centrifuged at 14,000 rpm for 10 min at 4oC, and the supernatants were subsequently used for activity assays and native polyacrylamide gel electrophoresis (PAGE). The protein concentration was measured according to the method described by Bradford [1976] using bovine serum albumin (BSA) as a standard. Activity assays of antioxidative enzymes. The activity of SOD was determined according to the method described by Giannopolitis and Ries [1977], with some modifications [Chowdhury and Choudhuri, 1985; Zhang et al., 1995]. Because SOD competes with nitroblue tetrazolium (NBT) for O2, the presence of SOD inhibits the color development; therefore, the SOD activity was expressed as the % inhibition of NBT reduction. The reaction mixture contained 63 µmol NBT, 1.3 µmol riboflavin, 13 µmol methionine, 0.1 mM EDTA, 100 mM potassium phosphate buffer (pH 7.0), and 50 µL of enzyme extract in a total volume of 3.0 mL. The reaction was initiated by illuminating the reaction mixture with light at an intensity of 78 µmol photon/s/m2 for 2 min, after which the light was turned off to terminate the reaction, after which the absorbance was read at 560 nm. Control reactions, in which the enzyme extracts were replaced with an equal volume of homogenizing buffer, were also performed. The enzyme activity was expressed as the unit activity/g of fresh weight of cucumber leaves. One unit of SOD activity was defined as the amount of enzyme required to cause a 50% inhibition in the rate of reduction of NBT under specified conditions. CAT activity was determined according to method described by Chance and Maehly [1955], applying the modification developed by Racchi [2001]. Briefly, 3 mL reaction mixtures composed of 100 mM potassium phosphate buffer (pH 7.0), 15 mM H2O2, and 20 µL of enzyme extract, which was used to initiate the reaction, were prepared. The decomposition of H2O2 was measured based on the decline in absorbance at 240 nm for over 1 min after the reaction was initiated. The activity was expressed as µmol H2O2 decomposed per min. The

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activity was expressed as µmol H2O2 decomposed per min. One unit of CAT was defined as the absorbance difference value of 0.01. Ascorbate peroxidase (APX, EC 1.11.1.11, class I peroxidase) activity was determined according to the method described by Chen and Asada [1989]. Briefly, a 1-mL reaction mixture composed of 100 mM potassium phosphate buffer (pH 7.5), 0.5 mM ascorbate, 0.2 mM H2O2, and 50 µL of extraction solution was prepared. The oxidation of APX was then determined based on the decrease in the absorbance at 290 nm (2.8 mM/cm). One unit of APX activity was defined as the amount of the enzyme that oxidized 1 µmol of ascorbate per min. Activity staining of antioxidative enzymes. The isozyme patterns of SOD, CAT, and APX were identified by native PAGE on 10, 7, and 10% acrylamide gels, respectively. Twenty micrograms of C. cassia crude proteins mixed with equal volume of loading buffer was loaded directly onto the gel. For SOD activity staining, electrophoresis was conducted cold, in running buffer at 100 V in a stacking gel and 200 V in a resolving gel. Following electrophoresis, the gels were subjected to staining as follows: washing three times in distilled water, followed by soaking in 2.45 mM NBT solution (prepared in 75% N, N-dimethyl formamide) for 30 min in the dark. The samples were then soaked in 28 µM each riboflavin and tetramethylethylenediamine (TEMED) in 50 mM potassium phosphate buffer (pH 7.8) for 30 min. To distinguish the three types of SOD, inactivation of SOD substrates for both H2O2 and potassium cyanide (KCN) was conducted individually in each treatment. Upon illumination, an achromatic band indicating the zone of activity appeared in the region of the gel where the SOD protein was located. Cu/Zn-SOD was inhibited by KCN and H2O2, Fe-SOD was inactivated by H2O2, and MnSOD was resistant to both inhibitors [Fridovich, 1989]. Catalase activity staining was conducted according to the method described by Chandlee and Scandalios [1983] after pre-treating the gels with 0.01% (v/v) H2O2 for 10 min. The stain mixture contained 1% (w/v) ferric chloride (FeCl3) and 1% (w/v) potassium ferricyanate [K3Fe(CN)6] in distilled water. Subsequently, the bands were visualized in an illuminator after washing the gels with distilled water three times. APX activity staining was conducted according to the method described by Chen and Asada [1989]. Briefly, the gel was washed three times in distilled water, after which it was incubated with 4 mM ascorbate and 2 mM H2O2 for 25 min in the dark. The gel was again washed with distilled water, and dipped in 2.45 mM NBT solution and 28 mM TEMED in 50 mM potassium phosphate buffer (pH 7.8) for 1 min. Finally, the gels were washed with

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Table 1. Effect of different concentrations of CCE on the growth of second-stage juveniles at 0, 14, and 28 days after treatment Number of second stage juveniles (J2)/g root FW

Treatment

0 Control Ne Ne and CCE 1 mg/mL Ne and CCE 5 mg/mL Ne and CCE 10 mg/mL

14 b

00±0 219±2a 212±1a 216±2a 211±2a

28 e

00±0 268±8a 245±3b 133±4c 117±3d

00±0d 301±15a 236±18b 168±8c 144±7c

Values shown in each column are the means±SD of three replicates. Data followed by the same letter within columns are not significantly different (p≤0.05) as determined by Tukey’s Studentized Range (HSD) test Control: without nematode; Ne: 5000 of nematodes; CCE: C. cassia crude extracts.

distilled water and then visualized using an illuminator. Statistical analysis. Treatment effects were determined by analysis of variance (one-way ANOVA) conducted according to the general linear model procedure of the Statistical Analysis System 9.1. Means were separated with Tukey’s Studentized Range Test at p=0.05. All data are presented as mean values±standard deviation.

Results After 14 days of treatment, the number of second-stage juveniles of M. incognita in the root was significantly decreased in plants treated with 1, 5, and 10 mg/mL CCE when compared to plants that received the Ne treatment alone (Table 1). Interestingly, after 28 days of treatment with 5 and 10 mg/mL CCE, the number of second-stage juveniles (J2) was significantly lower than that of J2 observed on plants treated with 1 mg/mL CCE and those that received the Ne treatment alone (Table 1). However, the number of J2 on plants treated with 5 and 10 mg/mL CCE did not differ significantly except at 14 days after treatment of CCE. The number of second-stage juveniles in soils was significantly decreased in all CCE treatments on day 28 compared with Ne treatment alone (Fig. 1). The relative galling formation on the cucumber plants on day 28 after treatment with 1, 5, and 10 mg/mL of C. cassia crude extracts were 88.3, 73.7, and 64.2% lower than the relative galling formation observed on the control plants, respectively (Table 2). The activities of the antioxidative enzymes of the CCEtreated plants and the control plants differed significantly. In each of the treatment groups, the SOD, CAT, and APX activities tend toward a low value in the plants treated

Fig. 1. Effects of different concentrations of C. cassia crude extracts on number of second-stage juveniles in soil 0, 14, and 28 days after treatment of CCE. Each value is mean±SD for n=3. Data followed by the same letter within columns are not significantly different (p≤0.05) as determined by Tukey’s Studentized Range (HSD) test. Table 2. Effect of different concentrations of C. cassia crude extracts on relative galling formation of cucumber plants 28 days after treatment Treatment Control Ne Ne+1 mg/mL CCE Ne+5 mg/mL CCE Ne+10 mg/mL CCE

Relative galling formation (%) 000±0e 100.0±2.6a 088.3±3.1b 073.7±3.5c 064.2±2.1d

Values given in the column are the means±SD based on three replicates. In the column, data followed by the same letter are not significantly different (p≤0.05) as determined by Tukey’s Studentized Range (HSD) test Control: without nematode; Ne: 5000 of nematodes; CCE: C. cassia crude extracts.

with Ne alone. The SOD activity was significantly higher in both the 5 mg/mL CCE (30.4 U/g FW) and the control (33.6 U/g FW) treatment groups than in plants treated with Ne alone (20.0 U/g FW) and in those treated with 1 mg/mL CCE (17.0 U/g FW) (Table 3). Upon comparison, the SOD activity in the leaves of plants treated with 1 mg/ mL CCE did not differ significantly from that of the leaves of plants treated with Ne alone, whereas plants treated with 10 mg/mL CCE showed a significantly higher SOD activity than Ne-treated plants. The CAT activity in cucumber leaves increased as the CCE concentration increased, with the highest level (319.6 U/g FW) being observed in the leaves of plants that were treated with 10 mg/mL CCE, and the CAT activity was lowest (87.7 U/g FW) in the leaves of plants treated with Ne alone. In particular, the APX activities in the leaves of plants that were treated with 10 mg/mL CCE (7.8 U/g


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Table 3. SOD, CAT, and APX activities of the 5th cucumber leaves of plants treated with M. incognita and different concentrations (1, 5, and 10 mg/mL) of CCE 28 days after treatment Treatment

Antioxidative activity (U/g FW) SOD

Control Ne Ne+1 mg/mL CCE Ne+5 mg/mL CCE Ne+10 mg/mL CCE

CAT a

33.6±1.2 0 20.0±1.6bc 17.0±0.3c 30.4±1.8a 22.9±1.1b

APX b

170.2±20.9 087.7±11.7c 218.5±6.3b0 287.3±5.2a0 319.6±32.5a

7.3±0.2a 4.2±0.2c 4.5±0.3c 6.0±0.3b 7.8±0.3a

Values shown in each column are the means±SD based on three replicates. Data followed by the same letter within columns are not significantly different (p≤0.05) as determined by Tukey’s Studentized Range (HSD) test Control: without nematode; Ne: 5000 of nematodes; CCE: C. cassia crude extracts.

FW) and the leaves of the control plants (7.3 U/g FW) were significantly higher than the APX activity of plants treated with Ne alone (4.5 U/g FW). There was no significant difference in the APX activity of the leaves of plants treated with Ne alone and those treated with 1 mg/ mL CCE. Taken together, these results suggest that C. cassia crude extracts play an important role in activation of the antioxidative enzymes in cucumber leaves under experimental conditions, and the activation of enzymes depends on amount of CCE. The relationship between galling formation and the antioxidative activities are shown in Fig. 2; the relative CAT activity was negatively correlated with the relative galling formation (R2=0.7276, p≤0.01) (Fig. 2B). Similarly, the relative APX activity was negatively correlated with the relative galling formation (R2=0.9175, p≤0.001) (Fig. 2C). However, the relative galling formation was not correlated with the relative SOD activity in cucumber leaves (R2=0.1338) (Fig. 2A). Following PAGE, the activities of SOD, CAT, and APX were detected to determine if the isozyme levels in cucumber leaves were up-regulated on day 28 after treatment with CCE (Figs. 3 and 4). The electrophoresis pattern of SOD in cucumber leaves revealed at least five clear isoforms of activities (93, 79, 71, 66, and 41 kDa). These isoforms were identified as three Fe-SOD (71, 66, and 41 kDa) and two Mn-SOD (93 and 79 kDa) (Fig. 3A, B, and C). The electrophoresis pattern of CAT in cucumber leaves revealed the presence of at least one band of strong activity (>97 kDa) (Fig. 4A). Interestingly, APX activity in cucumber leaves revealed the presence of at least four bands composed of one dark band (>97 kDa)

Fig. 2. Scatter plots used to determine the correlation between the relative galling formation and the relative antioxidative enzyme activities. SOD (A), CAT (B), and APX (C) in the 5th cucumber leaves on day 28 after treatment with nematode (Ne) and/or C. cassia crude extracts (CCE). Ne (●), Ne+1 mg/mL CCE (▲), Ne+5 mg/mL CCE (■), and Ne+10 mg/mL CCE (◆).

and three minor bands (79, 71, and 66 kDa) (Fig. 4B). The antioxidative enzymes [SOD (41 kDa) and APX (79 kDa)] showed the greatest activity in the leaves of plants that were treated with Ne alone.

Discussion Onifade et al. [2008] demonstrated that carbofuran and combination (1:1) of medicinal plants, both Haplophyllum tuberculatum and Plectranthus cylindraceus, produced fewer galls on the tomato roots at 0.6~1.3 µg/mL oils than tomato plants infected with M javanica. Also, tomato plants from nematode juvenile infested soils treated with Trichoderma harzianum at 106 spore/mL exhibited a significant reduction in the number of galls per plant root [Sahebani and Hadavi, 2008]. Treatment of chitinase-producing Paenibacillus illinoisensis KJA-424 showed an inhibition of egg hatching on root-knot

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Fig. 3. Activity staining of SOD [(A) PAGE (without KCN and H2O2 treatment), (B) KCN treatment, and (C) H2O2 treatment] in 5th cucumber leaves on day 28 after treatment with C. cassia crude extracts. Twenty microgram aliquots of crude proteins extracts were loaded onto 10% native PAGE gels and then stained for activity. M: standard protein marker; Lane 1: control (without nematode); 2: with nematode (5000 J2); 3: nematode+1 mg/mL CCE; 4: nematode+5 mg/mL CCE; 5: nematode+10 mg/mL CCE.

Fig. 4. Activity staining of CAT (A) and APX (B) in the 5th cucumber leaves on day 28 after treatment with C. cassia crude extracts. Twenty microgram aliquots of crude proteins extracts were loaded onto 7 and 10% native PAGE gels. M: standard protein marker; Lane 1: control (without nematode); 2: with nematode (5000 J2); 3: nematode+1 mg/mL CCE; 4: nematode+5 mg/mL CCE; 5: nematode+10 mg/mL CCE.

nematode M. incognita in vitro [Jung et al., 2002]. In our experimental results, the numbers of second-stage juveniles per one gram of soil within the root system were 53.7, 29.3, and 24.3 on day 14 after treatment and 43.4, 26.7, and 15.1 on day 28 after treatment at different concentrations (1, 5, and 10 mg/mL) of C. cassia crude extracts, respectively (Fig. 1). Whereas in Ne treatment without C. cassia, the number of second-stage juveniles per one gram of soil showed 58.5 and 61.3 on days 14 and 28 after treatment, respectively. The second-stage juveniles in sterilized soil were not observed in the soil throughout whole growth period. According to Onifade et al. [2008], there was enhanced reduction in M. javanica juveniles when the oils from the plants of H. tuberculatum and P. cylindraceus were mixed and tested. Javed et al. [2008] also showed that the total numbers of M. javanica in treatment of cake and leaves in neem plants were significantly lower than those of the control at 7 and 14

days after inoculation. Also, the total number of M. javanica within roots decreased with increasing concentration of the neem cake. Oka et al. [1999] suggested that foliar spray or soildrenching with SA, jasmonic acid (JA) or methyl jasmonic acid (MeJA) was either phytotoxic to tomato plants or did not induce resistance to M. javanica. The mechanism governing plant resistance to plantpathogenic nematodes may be somewhat different from the defense mechanism against fungal, bacterial or viral pathogens. In our experiments, the activity of antioxidative enzymes increased in the leaves of cucumber plants treated with 5 or 10 mg/mL CCE on day 28 after treatment, whereas the antioxidative activity of the leaves of cucumber plants treated with M. incognita alone was very low (Table 3). Increased activity of oxidative metabolism was frequently found in localized infection of foliar tissues by obligate biotrophic or necrotrophic pathogens associated with a rapid development of hypersensitive response [Mehdy et al., 1996; ThordalChristensen et al., 1997]. Furthermore, induction of oxidative stress has been observed in roots exposed to stresses, such as salt, aluminum, and chill [Shalata and Tal, 1998; Yamamoto et al., 2003; Zhou et al., 2004]. The present study suggests that the activity of the antioxidative enzymes (SOD, CAT, and APX) in cucumber leaves increased as the relative galling formation decreased in cucumber roots (Fig. 2). Additionally, galling indices of tomato plants sprayed with 20 or 40 mM DL-β-amino-n-butyric acid (BABA) were lower than those of untreated plants. Reductions in the number of J2 in roots and in galling indices of plants soil drenched with 2.5 or 5 mM BABA were recorded relative to the other treatments [Oka et al., 1999]. Several defense reactions induced during SAR following inoculation with an avirulent pathogen were also induced after tunicamycin treatment. They include increased levels of free and bound SAs and expression of PR proteins.


The level of free and bound SAs increased similarly to the increase reported after pathogen inoculation in cucumber [Rasmussen et al., 1991; Molders et al., 1996]. In our experiments, after infection with the root-knot nematode, different patterns of SOD isozyme activity were observed in the leaves of cucumber plants subjected to various treatments. Specifically, the Fe-SOD isozymes were found to be strongest in Ne-treated leaves (Fig. 3A, B, lane 2). These findings indicate that the application of cinnamon crude extracts may induce SOD-specific activity in cucumber leaves as a defensive response against infection by the root knot nematode, M. incognita. In the present study, the activities of antioxidative enzymes were significantly higher in cucumber leaves on day 28 after treatment with CCE. Jung et al. [2008] demonstrated that the elicitor-active oligosaccharides, chitin hexamer (a hexamer of GlcNAc) and thuricin 17 (a small bacterial peptide), induced phenyl ammonia lyase and tyrosine ammonia lyase activities in soybean leaves. These liginification-related enzymes are associated with changes in total phenol metabolism and have also been found to trigger changes in the activity of antioxidative enzymes such as peroxidase and SOD. An increase in the activity of SOD in response to low temperature has been reported in several studies. Our results showed that the strongest intensified bands of SOD (Fig. 3) and APX were produced by the Ne-treated leaves on day 28 after treatment (Fig. 4B). Finally, the level of resistance increased with the application of increasing concentrations of C. cassia crude extracts. We, therefore, concluded that both direct and indirect effects of C. cassia crude extracts could be important mechanisms in cucumber plants infected by M. incognita. The mechanisms governing resistance to M. incognita in cucumber and the effects of C. cassia on other nematode host plants and nematode species are under investigation as next study. Acknowledgments. This study was supported by the Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

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