Methyl jasmonate induced responses in four plant species and its effect on Spodoptera litura Fab. performance

Journal of Asia-Pacific Entomology 14 (2011) 263–269

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Journal of Asia-Pacific Entomology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j a p e

Methyl jasmonate induced responses in four plant species and its effect on Spodoptera litura Fab. performance Ching-Wen Tan, Ju-Che Lo, Jitendra Yadav, Kaliova Tavou Ravuiwasa, Shaw-Yhi Hwang ⁎ Department of Entomology, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung 402, Taiwan

a r t i c l e

i n f o

Article history: Received 13 August 2010 Revised 17 March 2011 Accepted 19 March 2011 Available online 24 March 2011 Keywords: Induced defense Polyphenol oxidase Trypsin inhibitor Tobacco cutworm Relative growth rate

a b s t r a c t Defensive proteins, such as polyphenol oxidase (PPO) and trypsin inhibitor (TI), are induced by herbivore wounding and exogenous methyl jasmonate application in various plant species. This study was conducted to measure induction of PPO and TI in radish, sweet pepper, tomato, and water spinach plants following herbivore wounding (I), methyl jasmonate application (M), and a combination of the two treatments (M + I). The effect of induced responses was also examined against third instar Spodoptera litura Fab. PPO activity was induced in radish by treatment I only; in sweet pepper, by treatments I and M; in tomato, by treatments I, M, and M + I; and in water spinach, by treatments M and M + I. The activity of TI was enhanced 1.2–1.4-fold in radish, sweet pepper, and tomato by M and M + I treatments, whereas in water spinach, it was enhanced 1.2-fold by all 3 treatments. The relative growth rate (RGR) of S. litura was reduced by 53% on radish plants following M treatment only. It was reduced by 37% and 42% on sweet paper plants following M and M + I treatment, respectively. RGR was significantly reduced on test tomato plants following I, M, and M + I treatments. The RGR of S. litura was unaffected on water spinach plants following any treatment. Collectively, the results of this study indicated that induction of plant defensive proteins in response to S. litura feeding or exogenous methyl jasmonate application varied among plant species, which further affected the induced plant resistance to the caterpillars. © Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society, 2011. Published by Elsevier B.V. All rights reserved.

Introduction Plants face diverse levels of herbivory during their life span. They protect themselves against herbivory with a suite of morphological and chemical defense strategies (Karban and Baldwin, 1997; Dicke and Hilker, 2003; Harrison, 2005; Johnson and Agrawal, 2005). Chemical defense strategies involve secondary metabolites and proteins which may be present constitutively or induced by challenges such as herbivore wounding (Ryan, 1990; Bennett and Wallsgrove, 1994; Duffey and Stout, 1996; Zhu-Salzman et al., 2008). Such induced plant responses include indirect, volatile-mediated defenses, and direct elevation in concentrations of plant chemical defensive substances. Indirect induced defenses attract natural enemies of herbivores (De Moraes et al., 1998; Thaler, 1999), whereas direct induced defenses directly affect the performance and preference of the attacking herbivore, as well as other species that colonize the plant (Agrawal, 2000; Traw and Dawson, 2002; Poelman et al., 2008). In addition to secondary metabolites, which have been traditionally perceived as the major components of chemical defense strategies that regulate host plant utilization by insects (Berenbaum and Zangerl,

⁎ Corresponding author. Tel.: +886 422840363; fax: + 886 4 22875024. E-mail address: [email protected] (S.-Y. Hwang).

2008), proteins are also an important contributor of the plant's chemical defense mechanism (Felton, 2005; Browse and Howe, 2008). Proteins are a major and the most common limiting nutrient for insect growth (Mattson, 1980; Bernays and Chapman, 1994). Past studies have indicated that dietary protein quality has a significant impact on outcomes of plant–insect relations (Broadway and Duffey, 1988; Felton, 1996). Protein based chemical defense compounds, which include different kinds of anti-nutritive compounds such as proteinase inhibitors (PIs) and oxidative enzymes, may have negative effects on insect herbivores (Felton, 2005; Bhonwong et al., 2009). PIs impair insect digestive proteases in the insect gut and ultimately reduce growth rate and increase mortality (Chen et al., 2005; Felton, 2005). PIs are generally categorized according to the kind of protease they inhibit (Ryan, 1990; Koiwa et al., 1997). Trypsin inhibitor (TI) is a protease inhibitor in plant species which have the ability to reduce the performance of insects (Ryan, 1990; Koiwa et al., 1997). Oxidative enzymes destroy or modify dietary amino acids and fatty acids and include polyphenol oxidase (PPO), peroxidase, and lipoxygenase (Duffey and Felton, 1991), as well as the essential amino acid-degrading enzymes arginase and Thr deaminase (Chen et al., 2005, 2007). Among these, PPO is the most widespread oxidative enzyme in plants that may act as an anti-herbivore protein (Constabel and Ryan, 1998; Bhonwong et al., 2009). Therefore, protein based defense chemicals may also play an important role in plant defense.

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A variety of signaling compounds that are involved in the induction of proteinous defense chemicals in plant tissues have been identified, including systemin peptides (Pearce et al., 1991; Ryan and Pearce, 2003), oligogalacturonides (Bergey et al., 1999), and jasmonates (Farmer and Ryan, 1990; Farmer et al., 1992). Among these, jasmonate and jasmonic acid (JA) are master signals that elicit synthesis of proteinous defensive compounds such as PIs and PPO (Farmer and Ryan, 1990; Steppuhn and Baldwin, 2007) and may increase host resistance to insect herbivores (Bodenhausen and Reymond, 2007; Koornneef and Pieterse, 2008; van Dam and Oomen, 2008; Zheng and Dicke, 2008). Herbivory can trigger endogenous accumulation of JA, which may act as a signaling molecule to stimulate the expression of induced defensive chemicals such as PIs and PPO (Farmer and Ryan, 1990; Farmer et al., 1992; Steppuhn and Baldwin, 2007). In addition, JA signaling may also induce plant volatiles emission (Dicke et al., 1998; Ament et al., 2004; van Schie et al., 2007). These emitted volatiles include terpenoids and short chain aldehydes which enable parasitic wasps to locate leaf-eating caterpillars. Exogenous application of JA or its volatile derivative methyl jasmonate (MeJA) on plants may also induce PPO, PIs, and volatile blends (Orozco-Cardenas et al., 1993; Thaler et al., 1996, 2001; Cipollini and Sipe, 2001). Past studies have shown that the exogenous application of JA and MeJA on plants results in higher plant fitness, increased parasitism, and reduced abundance and performance of herbivores (Baldwin, 1998; Thaler, 1999, 2001; Kessler and Baldwin, 2001). Hence, endogenous accumulation and exogenous application of JA can play important roles in a plant's induced defense response. However, such induced responses may vary across plant species and may be influenced differentially by different elicitors. Most past studies regarding such induced plant responses have been confined to single plant species and lack a comparative study on the ability of different elicitors, separately and in combination, to induce defensive responses in different plant species. Therefore, this study was carried out with the aims to (1) compare the induction of two protein-based defenses, polyphenol oxidase and trypsin inhibitors, among tomato (Lycopersicon esculentum Mill.), sweet pepper (Capsicum annuum L.), water spinach (Ipomoea aquatica Forsk.), and radish (Raphanus sativus L.) in response to insect wounding, methyl jasmonate, and the two treatments combined; and (2) to examine the effects of the above treatments on relative growth rates of Spodoptera litura caterpillars. Materials and methods

(25 ± 2 °C, 16:8 (L/D) h). Seeds were purchased from a local seed company (Known-You Seed Company, Kaohsiung, Taiwan). They were soaked in warm water (45 °C) for 30 min and rinsed with distilled water 3 times to eliminate contamination before sowing. Seeds were then sown in standard potting soil (MOS-010; Known-You Seed Company, Kaohsiung, Taiwan) in 104-well plates and were watered daily. Seedlings with 1–2 true leaves were transplanted into plastic pots (10.5 cm height× 12 cm diameter) filled with standard soil. The plants were watered daily and thirty five to forty day old plants were used in this study. Induction treatments Five different treatments were used on the four different plant species in this study. Insect wounding (I), exogenous application of MeJA (M), combination of both (M + I), and two kinds of controls, mock (MC) and negative (XC), were applied to the test plants. Methyl-jasmonate (BioWorld, USA) was first dissolved in 95% alcohol (1:10) then diluted in water to the final concentration of 1.5 mM. The surfactant Break-Thru S-240 (Evonik Goldschmidt GmbH, Germany) was added to the spraying solution (1/10,000). The exogenous application treatment (M) was performed by spraying 1.5 mM MeJA solution on the whole above-ground part of test plants 3–5 times until the MeJA solution started to drip from the plant. Fortyeight hours after spraying, the fifth leaf (from bottom) samples were used for insect bioassays and were chemically analyzed. The insect wounding treatment (I) was performed by allowing one fourth instar S. litura to feed on the second leaf of radish, sweet pepper, and water spinach plants and on the last leaflet of the second leaf of tomato test plants for 16 h (causing damage to approximately 30% of the leaf area). Mesh bags were used to enclose the larvae on leaves. After 16 h of caterpillar feeding, leaf samples were used for insect bioassays and chemical analysis. Combination M and I treatments (M + I) were performed by first treating the test plants with MeJA for 32 h. Each plant was then fed to one fourth instar S. litura for 16 h as described in insect wounding treatment. Forty-eight hours after MeJA spray and 16 h after feeding, leaf samples were used for insect bioassays and chemical analysis. Mock control (MC) treatment was similar to that of the M treatment, except that no MeJA was added in the spraying solution. Sample plants without any treatment were used as the negative control (XC) group.

Insects Relative growth rate (RGR) bioassay S. litura eggs were collected from the field in Taichung County, Taiwan, and kept in a plastic rearing cup (250 ml; Alpha Plus Scientific Corporation, Taoyuan, Taiwan) containing a small moistened cotton ball. The rearing cup was placed in a growth chamber (Model A 414931206; Yuh Chuen Chiou Industries Ltd., Taiwan) at 25 ± 2 °C, 70% ± 3% RH, 16:8 (L/D) h until caterpillar hatch. Hatched larvae were reared under the same controlled conditions in the growth chamber and were fed artificial diet. Artificial diet was prepared following a procedure developed by Gupta et al. (2005). Pupae were collected, sexed, and males and females were kept separately in plastic rearing cups (250 ml) until adult emergence. After emergence, males and females were paired (10 pairs) in a glass cylinder (22 cm height× 14.5 cm diameter) lined with tissue paper for egg collection. The glass cylinder was kept at room temperature and paired adult insects were fed sugar solution. A colony was maintained throughout this study. Plants Tomato (L. esculentum Mill.; Known-You 301), sweet pepper (C. annuum L.; Known-You Green Star), water spinach (I. aquatica Forsk.; Known-You Tauyang 1), and radish (R. sativus L.; Known-You New 6) plants were grown in a greenhouse under controlled conditions

A bioassay was conducted to assess the effect of the above treatments on insect performance on the 4 species of plants. The growth rate (RGR) of third instar S. litura was calculated as mean dry weight gain per hour. In this bioassay, the fifth leaf (from bottom) of a plant after various treatments was used to rear the caterpillars. The petiole of each leaf was placed in an Eppendorf microcentrifuge tube (2 ml; Bioscience Inc., UT, USA) filled with water to maintain leaf turgor. Each assay consisted of a newly molted and weighed larva placed into a Petri dish (1 cm height × 5.5 cm diameter) containing a leaf from a plant that had received one of the five different treatments (N = 10 replicates per plant treatment). At the same time, 10 newly emerged third instar larvae were weighed then frozen at −20 °C for 24 h. They were then oven dried at 45 °C for 1 week to estimate the percent water content. Larval water content was estimated as the difference between the wet and dry weights of these 10 larvae. Larvae feeding on leaves were examined regularly 6–7 times daily until molting. At the molting stage, larvae were transferred individually to another Petri dish (1 cm × 5.5 cm diameter; Alpha Plus Scientific Co., Taoyuan, Taiwan). As soon as the fourth instar emerged, it was immediately frozen and oven dried to calculate the RGR following the formula developed by Waldbauer (1968).

Foliar chemical analyses Polyphenol oxidase (PPO) and trypsin inhibitor (TI) activities were measured by spectrophotometric assays (Thaler et al., 1996; Moran, 1998; Stout et al., 1999). Foliar samples were collected for chemical analysis simultaneously with the RGR bioassays. The leaflets of the fifth leaf of tomato plants and the fifth leaf of the other plant species were harvested with surgical scissors. Whole leaves were ground in liquid nitrogen then homogenized in pH 7 phosphate buffer containing 7% (wt./ vol.) polyvinylpolypyrrolidone. A 1 ml volume of homogenate was removed and placed in a 1.7 ml centrifuge tube. Then, 100 μl of a 10% Triton X-100 solution was added to the homogenate. This mixture was then centrifuged at 10,000 rpm at 4 °C for 15 min. The resulting supernatant was used to determine enzyme activity. Total protein was first assessed with bovine serum albumin as the standard (Bradford, 1976). PPO activity was measured as the rate of formation of melanin-like materials from phenolic substrates (Stout et al., 1999). For this assay, 10 to 100 μl of enzyme extract was added to 500 μl of 10 mM catechol in pH 8 potassium phosphate buffer (0.1 M), and the change in absorbance of the mixture at 470 nm was recorded for 30 s. PPO activities were reported as ΔOD470 min− 1 mg fresh weight− 1 (Ryan et al., 1982). The levels of TI were determined using trypsin with protocols modified from the method of Lee and Lin (1995). TI activity assay was based on the ability of plant extracts containing trypsin inhibitors to inhibit the catabolism of casein by trypsin. Leaf tissues were homogenized in liquid nitrogen, then protein was extracted in 10 mM pH 7.8 phosphate buffer (1% polyvinylpolypyrrolidone (PVPP; Sigma), 1% ascorbic acid (Sigma), 1 mM potassium chloride (KCl; Sigma), 10 mM magnesium chloride (MgCl2; Sigma), and 50 mM ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA– Na2; Sigma)). The homogenate was centrifuged at 12,000 rpm at 4 °C for 15 min. There were three sets of groups: sample, blank, and standard. The standard group was prepared with 125 μl double-distilled water (DDW), 250 μl trypsin solution (0.8 mg/ml, 0.25 mM HCl) (trypsin; Sigma), and 250 μl of heated 2% casein solution (100 °C, 15 min, 10 mM pH 7.8 phosphate buffer) in a 2 ml centrifuge tube and was incubated at 37 °C for 20 min. The sample group was prepared with 50 μl DDW, 250 μl of heated 2% casein solution, and 75 μl clarified leaf extract in a centrifuge tube and was incubated at 37 °C for 20 min. After adding 250 μl trypsin solution, the sample was incubated at 37 °C again for 20 min. The blank group was prepared with 50 μl DDW, 250 μl of heated 2% casein solution, and 75 μl of clarified leaf extract in a centrifuge tube and was incubated at 37 °C for 20 min. An additional 250 μl DDW was added and the blank was incubated again at 37 °C. The reactions were stopped with 750 μl trichloroacetic acid (TCA; 10% wt./vol. H2O; Sigma) and placed at room temperature for 2 h. Finally, the tubes were centrifuged at 10,000 rpm at 4 °C for 15 min and the absorbance values of supernatants were detected by spectrophotometer at 280 nm. The activity of TI was expressed in TI units, which was defined as the amount of TI activity that inhibits 1 mg of trypsin within 20 min. TI activity was calculated by the following equation: ((OD280 of standard+OD280 of blank−OD280 of sample)/ OD280 of standard)×100%. Statistical analysis All statistical analyses were conducted using the one-way ANOVA and Tukey multiple range test (Version 8; SAS Institute Inc., Cary, NC, USA, 1999). The effect of different treatments were analyzed by analysis of variance (ANOVA; PROC GLM), followed by comparisons of means by the Tukey multiple range test (significance level, P b 0.05). Results Foliar chemistry PPO induction differed across experimental plant species and treatments (Fig. 1). In radish plants, it was only induced significantly

Polyphenol oxidase activity (ΔOD/mg/min) Polyphenol oxidase activity (Δ OD/mg/min) Polyphenol oxidase activity (ΔOD/mg/min) Polyphenol oxidase activity (Δ OD/mg/min)

C.-W. Tan et al. / Journal of Asia-Pacific Entomology 14 (2011) 263–269

265

25

Radish

P =0.0003 a

20

15

a a

a

b

10

5

0 XC

Sweet pepper

MC

30

I

PMI=0.0002

Treatments

a

20

M

Col 3 Col 3: -Col 3: -Col b 3: -Col 3: --

b

XC

MC

a b

10

0 50

I

M

Tomato

MI

P< 0.0001

Treatments 40

30

20

a Col 7 Col 7: -Col 7: -Col 7: -Col 7: --

a

a

b

c

10

0 XC

MC

Water spinach

I

M

Treatments

40

a 30

Col 11 Col 11: -Col 11: -b -Col 11: Col 11: --

b

XC

MC

MI P<0.0001

a

b

20

10

0 I

M

MI

Treatments Fig. 1. Polyphenol oxidase activity of radish, sweet pepper, tomato, and water spinach with five different induced treatments (XC, clear and healthy plant; MC, sprayed with water, EtOH, and spreader surfactant; I, damaged by larva; M, treated with MeJA; MI, treated with both I and M treatments). Data are expressed as mean ± SE (n = 5); bars with different letter(s) are significantly different (P b 0.05, Tukey's test).

The effect of an induced plant response on the relative growth rate (RGR) of third instar S. litura was inconsistent across test plant species and induction treatments (Fig. 3). The relative growth rate of third instar S. litura was significantly reduced on radish plants induced by MeJA only (P = 0.0007) as compared with third instar S. litura grown on control foliage. RGR of S. litura was significantly reduced on sweet pepper plants induced by both MeJA and the combination treatment (P = 0.0027). It was significantly reduced on the tomato plant following all treatments (insect wounding, MeJA application, and combination of both these treatments) (P b 0.0001). Finally, none of the induced responses of water spinach affected the RGR of the third instar S. litura (P = 0.2828). Discussion Our study confirmed that all the tested plant species used specific elicitors to induce PPO and TI activities. The different eliciting treatments show different effects on each of the plant species, thus

Table 1 Summary of the induced polyphenol oxidase (PPO) and trypsin inhibitor (TI) activities (data presented as fold relative to the control treatments; I compared with XC; M and MI compared with MC) in four plant species following different treatments and the percent reduced relative growth rate (RGR) of the third instar S. litura.

PPO activity Insect wounding Exogenous MeJA M+I TI activity Insect wounding Exogenous MeJA M+I RGR of S. litura Insect wounding Exogenous MeJA M+I

Radish

Sweet pepper

Tomato

Water spinach

(I) (M)

1.5 NS NS

1.7 1.2 NS

1.9 1.6 1.5

NS 1.4 1.4

(I) (M)

NS 1.2 1.2

NS 1.2 1.4

NS 1.3 1.2

1.2 1.2 1.2

(I) (M)

NS 53 NS

NS 37 42

43 74 89

NS NS NS

NS indicates no significant difference compared to the control.

Trypsin inhibitor activity (unit/g FW)

Larval performance

Radish

P<0.0001

100

80

b

b

a

a

M

MI P=0.0001

b

60

40

20

0 100

Sweet XC pepper

MC

I

Treatments 80

60

a Col 3

Col 3: -bc Col 3: -Col 3: -Col 3: --

ab

a

I

M

c

40

20

0

Trypsin inhibitor activity (unit/g FW)

by insect wounding compared with the control treatment (XC) (P = 0.0003). In sweet pepper plants, it was induced significantly by both insect wounding and MeJA application (P = 0.0002). In tomato plants, it was induced significantly by all the treatments (I, M, M + 1) (P b 0001). In water spinach, it was induced significantly by MeJA application and the combination of the two treatments (P b 0.0001). Insect wounding induced PPO activity 1.5-, 1.7-, and 1.9-fold in radish, sweet pepper, and tomato plants, respectively, as compared with the control treatments (Table 1). MeJA application induced PPO activity 1.2, 1.6, and 1.4 times higher in sweet pepper, tomato, and water spinach plants, respectively, than in their respective control treatments. The combination of the 2 treatments induced PPO activity 1.5- and 1.4-fold in tomato and water spinach plants, respectively, as compared with their control treatments. Induction of TI also varied across plant species and treatments (Fig. 2). Insect wounding significantly induced TI activity in water spinach only (P b 0.0001), whereas it was induced significantly by MeJA application and by the combination of insect damage and MeJA treatment in all test plant species (radish (P b 0.0001), sweet pepper (P = 0.0001), tomato (P b 0.0001), and water spinach (P b 0.0001)) as compared with controls. Insect wounding induced TI activity 1.2-fold in water spinach. MeJA induced TI activity 1.2-fold in radish, sweet pepper, and water spinach, and 1.3-fold in tomato. The combined treatment of MeJA and insect wounding induced TI activity 1.2-fold in radish, tomato, and water spinach, and 1.4-fold in sweet pepper.

Trypsin inhibitor activity (unit/g FW)

C.-W. Tan et al. / Journal of Asia-Pacific Entomology 14 (2011) 263–269

XC Tomato

MC

a 80

60

MI P <0.0001

Treatments

100 Col 7

Col 7: -b 7: -Col Col 7: -Col 7: --

b

a

b

40

20

0

Trypsin inhibitor activity (unit/g FW)

266

WaterXC spinach MC

I

a

60

MI P<0.0001

Treatments

100

80

M

Col 11

Col b 11: -Col 11: -Col 11: -Col 11: --

a

a

b

40

20

0 XC

MC

I

M

MI

Treatments Fig. 2. Trypsin inhibitor activity of radish, sweet pepper, tomato, and water spinach with five different induced treatments (XC, clear and healthy plant; MC, sprayed with water, EtOH, and spreader surfactant; I, damaged by larva; M, treated with MeJA; MI, treated with both I and M treatments). Data are expressed as mean ± SE (n = 5); bars with different letter(s) are significantly different (P b 0.05, Tukey's test).

C.-W. Tan et al. / Journal of Asia-Pacific Entomology 14 (2011) 263–269

exhibiting diverse effects on the induction of plant resistance against insect herbivores. Contents of the protein-based defense chemicals, PPO and TI, vary in the constitutive and induced levels within and among plant families

Relative growth rate (mg/mg/h)

0.08

P=0.0007

Radish a

a

a

0.06

a

0.04

b

0.02

0.00

Relative growth rate (mg/mg/h)

SweetXCpepperMC a

0.06

ab Col 3 0.04

I

M

MI P=0.0027

Treatments

ab

Col 3: -Col 3: -Col 3: -Col 3: --

b b

0.02

0.00

Relative growth rate (mg/mg/h)

XC Tomato

0.06

a

MC

I

a

X Data

M

MI P<0.0001

Col 7

0.04

Col 7: -Col 7: -Col 7: -Col 7: --

b

c

0.02

c 0.00

Relative growth rate (mg/mg/h)

MC WaterXCspinach

I

M

MI P=0.2828

Treatments 0.06 Col 11

0.04

Col 11: -Col 11: -Col 11: -Col 11: --

267

(Farmer and Ryan, 1990; Constabel and Ryan, 1998; Constabel and Barbehenn, 2008). Moreover, induced responses of plants are related to the type of stimulations (e.g., category of herbivory, concentration of chemical elicitor, and strength of damage) (Thaler et al., 1996; Constabel and Ryan, 1998; Stout et al., 1998b; van Dam et al., 2001). Induction of PPO by MeJA treatment or by insect wounding has been confirmed in several plants, including both herbaceous crops and trees (Felton et al., 1994; Constabel and Ryan, 1998; Stout et al., 1998b). Our results clearly demonstrated that insect wounding, MeJA, and combined treatments are all specific elicitors that could induce PPO activities in the tested plants. Our study also shows that the levels of induction vary significantly (from 1.2- to 1.9-fold) (Table 1). Constabel and Ryan (1998) also indicated that plant species vary in their capacity to induce PPO activity, which ranges from 0- to 46-fold. Earlier study shows that MeJA (0.45 mM) application could not induce PPO activity in sweet pepper (C. annuum) (Constabel and Ryan, 1998); however, in our study, we observed a significant increase of PPO and TI activities in sweet pepper responding to MeJA (1.5 mM) treatment. This contrasting result could be attributed to the higher concentration of MeJA that was used in our experiment. In addition to MeJA, we also found that mock (MC) treatment could induce PPO activity in some plant species. Therefore, we suggest that the application of MeJA solvent to plants should be thoroughly considered for effective results in further studies. Trypsin inhibitors (TIs) are present in all forms of life (Fritz, 2000) and have been reported to be induced in many plant species by various stress conditions (Koiwa et al., 1997; van Dam et al., 2001). TI activities may also be affected by various factors, such as plant species, concentration of MeJA, and duration of stimulation (Farmer and Ryan, 1990). Our results revealed a clear variation in TI activities among different plant species for the different treatments. They show that insect wounding caused significant elevation of TI in water spinach only, which is similar to a previous study (Walker-Simmons and Ryan, 1977) where only 10 of the 23 species had induced TI activities in their tested plants. The degree of induction for TI activity, however, was similar in all plant species, ranging from 1.2- to 1.4-fold for the different treatments. Our results indicated that both insect wounding and exogenous application of MeJA resulted in various PPO and TI activities in each of the plant species. Moreover, exogenous MeJA increased the activity of PPO and TI in most of the tested plant species. The reasons behind why insect wounding and exogenous MeJA treatment differed in their capacity to stimulate activity of protein-based defense chemicals (PPO and TI) in our study are not clear. The concentration of MeJA (Thaler et al., 1996), degree of damage by insects (Felton et al., 1994), and duration of insect wounding are possible factors that could affect induction responses. JA and MeJA application reduced growth performance, survival rate, and oviposition rate of insects and increased the parasitism rate by natural enemies (Thaler, 1999; Kessler and Baldwin, 2001). This finding suggested that MeJA is a useful tool to induce plant resistance in some agricultural crops. Damage by herbivores may not be totally avoidable in the field. Therefore, exogenous application of MeJA and insect wounding should be considered in field conditions. Our study revealed that no additive effect of these elicitors was found on expression of either PPO or TI. The reason for this insignificant interaction is not clear and further studies are needed to clarify the causes. Previous laboratory and field studies showed that induced defense may affect herbivore survivor rate, relative growth rate, and population size (Karban and Myers, 1989; Stout and Duffey, 1996; Karban and

0.02

0.00 XC

MC

I

Treatments

M

MI

Fig. 3. Effect of different induced treatments (XC, clear and healthy plant; MC, sprayed with water, EtOH, and spreader surfactant; I, damaged by larva; M, treated with MeJA; MI, treated with both I and M treatments) of radish, sweet pepper, tomato, and water spinach on growth rate of S. litura. Data are expressed as mean ± SE (n = 10); bars with different letter(s) are significantly different (P b 0.05, Tukey's test).

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Baldwin, 1997; Thaler et al., 2001). Herbivore performance could be affected by a wide variety of defense compounds and by the distribution of nutrition in plants (Schoonhoven et al., 2005; Soler et al., 2005; Kaplan et al., 2008). The chemical defensive compounds PPO and TI reduce the host plant foliar nutritive value, particularly for noctuid larvae. Our study also shows that the RGR of third instar S. litura was significantly reduced in tomato plants following each of the treatments. This indicates that tomato plants could express significant resistance after insect wounding and that the growth performance of insects was greatly reduced (Thaler et al., 2001; Joem and Muthukumaran, 2008). Previous studies also found that tomato plants showed the most dramatic induction responses in PIs (proteinase inhibitors) and PPO activities after treatment with MeJA, JA, and insect wounding stimulations (Farmer and Ryan, 1990; Orozco-Cardenas et al., 1993; Constabel et al., 1995; Thaler et al., 1996; Constabel and Ryan, 1998; Stout et al., 1998b). The relative growth rate of S. litura third instars significantly reduced in radish, sweet pepper, and tomato plants for each specific treatment (Table 1). Moreover, the reduced relative growth rate is related to the total consumption of leaf area. Insects fed with radish and tomato plants consumed less, while those fed with sweet pepper consumed more in comparison to their respective control plants (data are not shown). Earlier studies (Stout and Duffey, 1996; Stout et al., 1998a; Joem and Muthukumaran, 2008) also suggested that both PPO and PI are important protein based chemical defenses in plants and that they can be induced systemically. Therefore, our and other studies reveal that PPO and TI are important factors that could be induced to affect insect performance. However, TI activity shows higher inducibility within different elicitors and plant species in our study. This suggests that TI and PPO act differently in plant species when exposed to threats. In summary, exogenous methyl jasmonate can be a useful chemical elicitor to strengthen the resistance of radish, tomato, and sweet pepper against S. litura. More research is needed to further support and clarify the mechanism of higher inducibility of TI when plants are exposed to various elicitors. Acknowledgment We thank two anonymous reviewers for their comments on the manuscript. References Agrawal, A.A., 2000. Specificity of induced resistance in wild radish: causes and consequences for two specialist and two generalist caterpillars. Oikos 89, 493–500. Ament, K., Kant, M.R., Sabelis, M.W., Haring, M.A., Schuurink, R.C., 2004. Jasmonic acid is a key regulator of spider mite-induced volatile terpenoid and methyl salicylate emission in tomato. Plant Physiol. 135, 2025–2037. Baldwin, I.T., 1998. Jasmonate-induced responses are costly but benefit plants under attack in native populations. Proc. Natl Acad. Sci. U.S.A. 95, 8113–8118. Bennett, R.N., Wallsgrove, R.M., 1994. Secondary metabolites in plant defence mechanisms. New Phytol. 127, 617–633. Berenbaum, M.R., Zangerl, A.R., 2008. Facing the future of plant–insect interaction research: le retour à la “raison d'Être”. Plant Physiol. 146, 804–811. Bergey, D.R., Orozco-Cardenas, M., de Moura, D.S., Ryan, C.A., 1999. A wound- and systemin-inducible polygalacturonase in tomato leaves. Proc. Natl Acad. Sci. U.S.A. 96, 1756–1760. Bernays, E.A., Chapman, R.F., 1994. Host–Plant Selection by Phytophagous Insects. Chapman and Hall, New York. Bhonwong, A., Stout, M.J., Attajarusit, J., Tantasawat, P., 2009. Defensive role of tomato polyphenol oxidases against cotton bollworm (Helicoverpa armigera) and beet armyworm (Spodoptera exigua). J. Chem. Ecol. 35, 28–38. Bodenhausen, N., Reymond, P., 2007. Signaling pathways controlling induced resistance to insect herbivores in Arabidopsis. Mol. Plant. Microbe. Interact. 20, 1406–1420. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Broadway, R.M., Duffey, S.S., 1988. The effect of plant protein quality on insect digestive physiology and the toxicity of plant proteinase inhibitors. J. Insect Physiol. 34, 1111–1117. Browse, J.B., Howe, G.A., 2008. New weapons and a rapid response against insect attack. Plant Physiol. 146, 832–838.

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