Abstract: To induce resistance of tomato plants against Tetranychus urticae Koch nine different compounds were used. The effect of these compounds on T. urticae, the essential oil components and defense enzymes activity in tomato leaves were determined. The highest reduction percentages of T. urticae movable stages recorded with potassium humates (55.38%) followed by salicylic acid, (Potassium humates+Salicylic acid), methyl jasmonate, potassium silicate, propolis and vital power calcio cao, (50.76, 47.34, 46.37, 37.81, 25.15 and 23.44%), respectively. The major components of essential oils in treated tomato leaves were Caryophyllene, Humulene, β-phellandrene, d-Limonene, cis-α-Copaene-8-ol, β-Spathulenol, Eugenol, 8-Cedren-13-ol, Spathulenol, Geraniol, Humulene epoxide II, Caryophyllene Oxide, Delta-elemene, Linalool, β-Elemene and Methyl salicylate. The levels of defense enzymes such as Catalase (CAT), Peroxidases (POD), Polyphenol oxidase (PPO), Phenylalanine ammonia lyase (PAL, β-glycosidase and inhibition (%) of the Proteinase inhibitors (PIs) were increased with most of treatments compared with untreated plant (control).While the levels of superoxide dismutase (SOD, glutathione-S-transferase (GST), acrobat peroxidase (APX) and lipoxygenase (LOXs) were increased with untreated plant (control). Potassium humates and Salicylic acid given highest anti-Tetranychus activity, enzymes one of the important aspects of Host Plant Resistance (HPR) against T. urticae. The treated tomato plants by different compounds showed defense response against herbivores and enhanced resistance against T. urticae and these results correlate with chemically determined such as essential oil components and defense enzymes.
INTRODUCTION
The two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), is become one of the important pests of many vegetable corps, especially Solanaceae and Cucurbitaceae. Use of acaricides is the main strategy to control T. urticae in field crops and development of resistance in mite populations is common (Cranham and Helle, 1985; Devine et al., 2001). The elicitors of induced responses can be sprayed on crop plants to build up the natural defense system against damage caused by herbivores. Induced resistance could be exploited as an important tool for the pest management to minimize the use insecticides in pest control. Plants respond to herbivory through various morphological, biochemicals and molecular mechanisms to counter/offset the effects of herbivore attack (War et al., 2012).
Depending upon the modes of feeding of insect pests, different defense signaling pathways are activated, which induce the production of specific volatile compounds (Walling, 2000). The Herbivore-Induced Plant Volatiles (HIPVs) include terpenes, green leafy volatiles (GLVs), GLVs consist of C6-aldehydes [(Z)-3-hexenal, n-hexanal] and their respective derivatives such as (Z)-3-hexenol, (Z)-3-hexen-1-yl acetate and the corresponding E-isomers (War et al., 2012; Engelberth et al., 2004). The HIPVs defend the plants either directly by repelling, deterring and toxicity to the herbivore or indirectly by attracting the natural enemies of the attackers and thus, protect the plants from further damage (Dudareva et al., 2006; Maffei, 2010).
The enzymes that impair the nutrient uptake by insects through the formation of electrophiles includes peroxidases (PODs), polyphenol oxidases (PPOs), ascorbate peroxidases and other peroxidases by oxidizing mono-or dihydroxyphenols, that lead to the formation of reactive o-quinones, which in turn polymerize or form covalent adducts with the nucleophilic groups of proteins due to their electrophilic nature (e.g.,-SH or e-NH2 of Lys) (Bhonwong et al., 2009; Gulsen et al., 2010; Gill et al., 2010). Other important antioxidative enzymes include lipoxygenases, phenylalanine ammonia lyase, superoxide dismutase, etc. Induction of antioxidative enzymes in plants following herbivory has received considerable attention in recent years (Gill et al., 2010; Usha Rani and Jyothsna, 2010; War et al., 2011a, b; Chen et al., 2005, 2009; Chakraborti et al., 2009; Vandenborre et al., 2011). Most of the plant defense responses against insects are activated by signal transduction pathways mediated by jasmonic acid, salicylic acid and ethylene (Gill et al., 2010; Shivaji et al., 2010). A broad spectrum of defensive responses are induced by jasmonates that include antioxidative enzymes, proteinase inhibitors (PIs), volatile organic compounds (VOCs), alkaloid production, trichome formation and secretion of extrafloral nectar (EFN) plays an important role as plant indirect defence (Mao et al., 2007; Dickens, 2006; Pauwels et al., 2009). A large numbers of genes involved in defense against herbivores are regulated by JA. Concentration of indole glucosinolate, an important defensive compound, is induced by jasmonates (Shivaji et al., 2010). Also in strawberry plants fertilized by CaSO4 and K2SO4, the mite, T. urticae infestation was lower than on unfertilized plants, these treatment lead to an increase in total phenols and amino acid in plants. Increased potassium levels in strawberry plants lead to an increase in plant resistance to T. urticae infestation (Afifi et al., 2010).
The present study was conducted to evaluate the efficiency of nine different compounds against the two-spotted spider mites Tetranychus urticae Koch in tomato plants and the role of these compounds in enhancement of resistance and raise the plant defense mechanism.
MATERIALS AND METHODS
Tomato hybrid: Hybrid tomato super strain B was chosen for this study. It was obtained from Agrimatco For Agriculture Company, Egypt.
Tested compounds: Nine different compounds were used in this study. These compounds with their concentration and produced company were tabulated in Table 1.
| Table 1: | Treatments of nine different compounds used against Tetranychus urticae on tomato plants |
Experimental procedure: The experiment was conducted in Acarology Greenhouse, Faculty of Agriculture, Cairo University, Giza Egypt, during season 2013. The experimental area was divided into 11 treatments according to complete randomized block design including three replicates for each treatment. Soil was well prepared before the tomato seedlings planted; tomato plants received all normal agricultural processes without any pesticides application. Tomato hybrid seeds were sown at the first week of January in the nursery then seedlings were planted in the greenhouse at the first week of February. The infestation occurred naturally by T. urticae on tomato hybrid. The application of spray was started after one weak of cultivation. Check treatment was sprayed with water only. A compressor spryer (5 L capacity) was used. Samples (30 leaves) were randomly collected from treatments (10/each replicate), just before spraying then weekly afterwards. Additional sprays were conducted in the fourth, seventh and ninth weeks of seedling cultivation. Leaf samples were kept into perforated polyethylene bags, closed with rubber bands and kept in an ice box then transferred to the laboratory for examination using a stereomicroscope. Different stages of T. urticae were counted and recorded.
Essential oils extraction and GC/MS analysis: Samples of tomato fresh leaves (100 g) were collected form each treatment. Samples were hydro-distilled in Clevenger-type apparatus according to Council of Europe (1997). The amount of oil obtained from each sample was calculated as:
The essential oil samples were stored in the dark at 4°C and analyzed by GC-MS according to Adam et al. (1998) method. GC/MS analysis was performed on a Thermoquest-Finnigan Trace GC-MS equipped with a DB-5 (5% phenyl) methylpolysiloxane column (60 m-0.25 mm i.d., film thickness 0.25 μm). The injection temperature was 220°C and the oven temperature was raised from 40°C (3 min hold) to 250°C at a rate of 5°C min-1, then held at 250°C for 2 min; transfer line temperature was 250 °C. The 1 μL of sample was injected and helium was used as the carrier gas at a flow rate of 1.0 mL min-1. The mass spectrometer was scanned over the 40-500 m/z with an ionizing voltage of 70 eV and identification was based on standard mass library that National Institute of Standards and Technology (NIST Version 2.0) to detect the possibilities of essential oil components.
Enzyme extractions and assays: The enzyme analysis in tomato leaves were carried out after the final spray. Tomato fresh leaf samples were collected from each replicate and placed into polyethylene bags and kept in an ice box with liquid nitrogen then transferred to Central Agriculture Pesticide Laboratory (CAPL) for chemical analysis. Fresh leaf samples were submersed for 5 min in liquid nitrogen. The frozen leaves were kept at -80°C for further analyses. Enzymes were extracted from 0.5 g leaf tissue using a mortar and pestle with 5 mL extraction buffer containing 50 mM potassium phosphate buffer, pH 7.6 and 0.1 mM Na-EDTA. The homogenate was centrifuged at 15,000×g for 15 min and the supernatant fraction was used to assay for the various enzymes. All steps in the preparation of enzyme extracts were performed at 4°C. Superoxide dismutase (SOD) is estimated by the method of Marklund and Marklund (1974). The activity of catalase (CAT) was assayed by the method of Nakano and Asada (1981). Peroxidase (POD) activity was assayed spectrophotometrically at 470 nm using guaiacol as a phenolic substrate with hydrogen peroxide (Diaz et al., 2001). Polyphenol oxidase (PPO) activity was determined using a spectrophotometric method based on an initial rate of increase in absorbance at 410 nm (Soliva et al., 2000). Glutathion-S-transferase (GST) was assayed by the method of Habig et al. (1974). Glutathione peroxidase was assayed by the method of Paglia and Valentine (1967). APX activity was determined by Cakmak and Marschner (1992). Extraction of protease inhibitor by Pichare and Kachole (1996) and activity of protease inhibitor against protease was assayed according the procedure described by Kunitz (1945) with slight modifications. Determination of lipoxygenase was prepared by a modification of the method of Surrey (1964) and was assayed activity according to the method of Zimmerman and Vick (1970). β-glucosidase activity was determined according to Bhat et al. (1993). Determination of phenylalanine ammonia-lyase activity was determined according to Lamb et al. (1979).
Statistical analysis: Data was analyzed by one-way analysis of variance and mean comparison at 5% level of significance using Fisher's Least Significant Difference (LSD). Using the Statistical Analysis System (SAS., 1988) and ASSISTAT according to Silva and de Azevedo (2009). Reduction percentages of T. urticae were calculated according to Henderson and Tilton (1955).
RESULTS AND DISCUSSION
Effect of different compounds on Tetranychus urticae: Data in Table 2 indicated the efficacy of nine different compounds against T. urticae movable stage in tomato plants. At the end of the experiment, the numbers of movable stages/leaflet in sprayed treatments averaged 16.00, 14.24, 12.11, 11.56, 10.05, 8.72, 7.50, 7.31, 6.76 and 6.01 individuals/leaflet for Berelex (Gibberellic acid), Universal ergofito, Lemongrass oil, Propolis, Vital Power Calcio Cao (Potassium humates+ Salicylic acid), Methyl jasmonate, Potassium silicate, Salicylic acid and Potassium humates, respectively, compared with the check (13.47 individuals/leaflet).
The highest reduction percentages of T. urticae movable stages occurred after nine weeks of spraying, which observed with potassium humates, (55.38%) followed by salicylic acid, (Potassium humates+Salicylic acid), methyl jasmonate, potassium silicate, propolis and vital power calcio cao, (50.76, 47.34, 46.37, 37.81, 25.15 and 23.44%, respectively), while the lower reduction percentages observed with Lemongrass oil and Universal ergofito (11.78 and 4.06%, respectively) no reduction percentage was recorded with Berelex (Gibberellic acid).
Data in Table 3 showed the effect of these compounds, on the number of T. urticae eggs.
| Table 2: | Mean numbers and reduction percentages of Tetranychus urticae movable stages in tomato plants after treated with nine compounds under greenhouse condition |
| *Pre-treatment and the first spray, **Additional sprays | |
| Table 3: | Mean number and reduction percentages of Tetranychus urticae eggs in tomato plants after treated with nine compounds under greenhouse condition |
| *Pre-treatment and the first spray, **Additional sprays | |
At the end of the experiment, the numbers of eggs/leaflet in sprayed treatments averaged 13.16, 12.00, 10.05, 9.24, 8.21, 6.88, 6.08, 5.42, 5.01 and 4.57 for Berelex (Gibbereillc acid), Universal ergofito, Lemongrass oil, Propolis, Vital Power Calcio Cao (Potassium humates+Salicylic acid), Methyl jasmonate, Potassium silicate, Salicylic acid and Potassium humates respectively, compared with the check (11.23 eggs/leaflet). The highest reduction percentages of T. urticae eggs after nine weeks of spraying observed with Potassium humates, (57.24%), followed by (Potassium humates+Salicylic acid), Potassium silicate, Methyl jasmonate, Lemongrass oil and Salicylic acid, which averaged 49.78, 46.66, 35.64, 26.78 and 14.83, respectively, while the lower reduction percentages recorded with Universal ergofito and Vital Power Calcio Cao (3.83 and 2.00%, respectively). No reduction percentages were recorded with Berelex (Gibbereillc acid) and Propolis.
The statistical analysis show that there were significant differences between treatments; Berelex (Gibbereillc acid), Control, (Potassium humates+Salicylic acid) and Potassium humates, while no significant differences appeared among Salicylic acid, Methyl jasmonate and Potassium silicate.
Chemical composition of essential oils in tomato leaves: The data present in Table 4 showed that, the essential oil components and its concentrations in treated tomato leaves extract (using steam distillation apparatus) were depend on treatments type compared with untreated leaves (control). Some of essential components were detected in all treatments in addition to control group; such as Caryophyllene oxide at 25.27 Rt and 2, 7 Di-t-butyl-p-cresol (20.21 Rt), Humulene epoxide II (26.41 Rt), 2-Pentadecanone 6, 10, 14-tri methyl (26.80 Rt), Cis-α-copaene-8-al (27.65 Rt). However, some essential oil components present only in control group and disappeared in all treatment groups such as; 1,3,5 Heptatriene (E,E) at (0.82%), P-Menth-8-ene, 3-methylene (0.48%), 4,6,6 tri methyl-bicyclo (3.1.1) hepta-3-en-2-one (0.61%), alpha-acorenol (2.04%), 6-tetradecyne (0.78%). Moreover, as shown in Table 4 some components found only in treatments group and disappeared in control group such as; 2-carene, di-limonene, B-Phellondrene, Terpilene, isolimonene, B-spathulenol-etc. These phenomena may be due to the effect of different organic treatments on the gene expression of tomato plants and formation of new components or alteration in the concentration of components present in untreated sample (control).
Also, the highest essential oil component numbers were found in tomato plants treated with Potassium silicate (T8) followed by untreated samples (T11), Berelex (Gibbereillc acid) (T3), Potassium humates (T4), Propolis (T5), Vital Power Calcio Cao (T6), Universal ergofito (T7), Salicylic acid (T2) and methyl jasmonic acid (T1). These numbers were 39, 37, 34, 29, 28, 27, 24, 23, 23 and 19, respectively (Table 4).
The major components found in most treatments were Caryophyllene, Humulene, β-phellandrene, d-Limonene, cis-α-Copaene-8-ol, β-Spathulenol, Eugenol, 8-Cedren-13-ol, Spathulenol, Geraniol, Humulene epoxide II, Caryophyllene Oxide, Delta-elemene, Linalool, β-Elemene and Methyl salicylate. The highest concentrations of Caryophyllene were recorded with treatment; Berelex (Gibbereillc acid) (21.43) followed by Potassium humates and Methyl jasmonate (17.85 and 13.9%, respectively) compared with the untreated plants (control) (12.43%). In case salicylic acid, propolis and potassium silicate the concentrations recorded ratios close to the control (11.85, 11.75 and 10.92% respectively). Also, caryophyllene oxide recorded highest concentrations in treatment; (Potassium humates+Salicylic acid) (3.95%) followed by treatments; Potassium silicate, Vital Power Calcio Cao, Lemongrass oil, Universal ergofito, Propolis, Potassium humates, Berelex (Gibbereillc acid), Salicylic acid and Methyl jasmonate (3.75, 3.53, 2.71, 2.70, 2.53, 2.03, 2.00, 1.72 and 1.72%), respectively compared with control (0.61%).
| Table 4: | Relative concentration of essential oil components in tomato leaves after spraying with nine different compounds against Tetranychus urticae Koch |
Also, data in Table 4 indicated that, the highest concentrations of Humulene were observed with treatment; Berelex (Gibbereillc acid) (6.11%) followed by treatments; Potassium humates and Salicylic acid (4.97 and 4.56%, respectively), compared with the control (4.44%). In case of Propolis, Potassium silicate and Methyl jasmonate treatments, the concentrations recorded ratios close to control (4.01, 3.83 and 3.76%, respectively). While Humulene epoxide II the highest concentration was observed with treatment; (Potassium humates+ Salicylic acid) (3.03%), followed by treatments; Universal ergofito, Vital Power Calcio Cao and Lemongrass oil (2.99, 2.47 and 2.36%), respectively compared with the control (2.30%). The highest concentrations of Delta-elemene founded in treatment; salicylic acid (12.31%) followed by treatments; Berelex (Gibbereillc acid), Methyl jasmonate, Potassium humates, Propolis and (Potassium humates+ Salicylic acid) (11.25, 10.54, 9.81, 7.89 and 7.28%), respectively compared with the control (6.63%). Also The highest concentrations of β-Elemene recorded in treatment; Berelex (Gibbereillc acid) (2.95%) followed by treatments; Methyl jasmonate, Potassium humates, Salicylic acid, Potassium silicate and Propolis (2.45, 2.43, 2.26, 2.11 and 2.2%, respectively) compared with the control (1.96%). The β-phellandrene, d-Limonene and Linalool were found with some plant treated without the control; Potassium humates and Berelex (Gibbereillc acid) (16.08 and 11.85%); Propolis, Potassium humates and Berelex (Gibbereillc acid) (12.5, 2.90 and 2.56%); Methyl jasmonate, Potassium humates, Propolis, Potassium silicate and Berelex (Gibbereillc acid) (3.36, 1.97, 1.97, 1.77 and 1.49%), respectively. The highest concentrations of Methyl salicylate recorded in plant treated with Potassium humates (1.66%), followed by; Berelex (Gibbereillc acid) and Potassium silicate (1.37 and 1.19%) respectively, compared with the control (0.99%).
The highest concentrations of Geraniol were found in treatment; Methyl jasmonate (1.7%), followed by treatments; Propolis and Potassium silicate (0.97 and 0.82%, respectively), compared with control (0.51%). Also, the highest concentrations of Eugenol was recorded with treatment; Methyl jasmonate (8.75%) followed by treatments; Salicylic acid, Potassium silicate, Vital Power Calcio Cao, Propolis and (Potassium humates+Salicylic acid) (7.74, 4.21, 4.14, 3.11 and 2.91%, respectively) compared with control (2.91%). Spathulenol and β-Spathulenol, highest concentrations recorded in treatments; (Potassium humates+ Salicylic acid) (8.12%) and Lemongrass oil (8.28%), respectively, followed by treatments; Potassium silicate (4.85%) and Propolis and Methyl jasmonate (4.09 and 2.92%), respectively, compared with the control (4.47 and 0.00%), respectively. The highest concentrations of cis-α-Copaene-8-ol was observed with treatment; Universal ergofito (10.88%) followed by treatments; (Potassium humates+ Salicylic acid), Salicylic acid, Lemongrass oil and Vital Power Calcio Cao (8.38, 7.15, 6.97 and 6.96%), respectively, compared with the control (5.86%). While in case, 8-Cedren-13-ol recorded highest concentrations in treatment Universal ergofito (4.26%) than other treatments.
These results may be due to, the relation between the response of plant to biotic stress caused by the two spotted spider mites T. urticae and secretion of essential oil compounds as one of most defense mechanism against the pest. These results were in agreement with the results obtained by Verhage et al. (2010) who stated that, the plant hormones play a critical role in regulating plant growth, development and defense mechanisms. War et al. (2012) who found that, the defensive (Secondary) metabolites can be either constitutive stored as inactive forms or induced in response to the insect or microbe attack, Also, these phenomena confirmed by Kanchiswamy et al. (2010) who reported that, extension rearrangement in gene expression occur in plants in response to herbivory with hundreds and even up to several thousands of genes getting up or down regulated. More studies had indicated the role of jasmonic acid, salicylic acid and gibberellin and other components in plant induced resistance (Omer et al., 2000; Traw and Bergelson, 2003; Ament et al., 2004; Van Schie et al., 2007; Kappers et al., 2010; Farouk and Osman, 2012; Sarmento et al., 2011; Rahimi et al., 2013; Miyazaki et al., 2014).
Defense enzyme activities in tomato leaves: Enzymes are one of the important aspects of Host Plant Resistance (HPR) against mites is the disruption of mite’s nutrition. The defense enzyme, catalase (CAT), peroxidases (POD), superoxide dismutase (SOD), polyphenol oxidase (PPO), glutathione peroxidase (GPO), glutathione-S-transferase (GST), Ascorbate peroxidase (APX), phenylalanine ammonia lyase (PAL), lipoxygenase (LOXs), proteinase inhibitors (PIs) and β-glycosidase were determined in leaves of infested tomato plants which were treated with different compounds against T. urticae. Results in Table 5 indicated that the levels of defense enzymes CAT, POD, SOD, PPO, glutathione peroxidase and GST were increased in the treatments (4, 2 and 8) and given highest anti-Tetranychus activity compared with untreated plants (control) and these results may be due to the chemical nature of spraying compounds, because all of the previous mentioned compounds are phenolic compounds.
The highest activity of enzyme catalase (CAT) increased in treated treatments; Gibbereillc acid to 120.31 U g-1 tissues, followed by treatments; Salicylic acid and Potassium humates to 107.64 and 90.01 U g-1 tissues, respectively, compared with untreated one (72.82 U g-1 tissues). While this enzymes activity was reduced in other treatments, recorded the lowest rate was with application by Lemongrass oil (20.8182 U g-1 tissues). The results emphasized the role of Potassium humates in improving plant growth and inducing resistance against T. urticae which resulted in lower infestation, followed by Salicylic acid. The highest rate activity of enzyme peroxidases (POD) was achieved by Salicylic acid as 9399.67 U g-1 tissues followed by treatments; Potassium humates, Methyl jasmonate and Vital Power Calcio Cao to 9188.69, 9121.99 and 8259.97 U g-1 tissues, respectively compared with control treatment (7071.02 U g-1 tissues), while the lowest rate was implemented by the pesticide, Potassium silicate(4828.05 U g-1 tissues). Oxidative state of the host plants has been associated with HPR to insects (He et al., 2011; Zhao et al., 2009) which results in production of ROS, that are subsequently eliminated by antioxidative enzymes. POD constitutes one such group of enzymes, which scavenges the ROS besides having other defensive roles. PODs are an important component of the immediate response of plants to insect damage (Usha Rani and Jyothsna, 2010; War et al., 2011a; Gulsen et al., 2010). A number of process are regulated by PODs that have direct or indirect role in plant defense, including lignification, suberization, somatic embryogenesis, auxin metabolism and wound healing (He et al., 2011; Heng-Moss et al., 2004; Sethi et al., 2009).
The treatments; Potassium humates, Potassium silicate, Lemongrass oil, Universal ergofito, (Potassium humates+Salicylic acid), Gibbereillc acid and Salicylic acid application boosted polyphenol oxidase (PPO) by 491.57, 461.47, 370.70, 362.17, 354.57, 306.57 and 299.80 mg g-1 tissues, respectively, over that in infested plants (control) 292.43 mg g-1 tissues, while Methyl jasmonate, Vital Power Calcio Cao and Propolis by 257.97, 238.80 and 236.97 mg g-1 tissues, respectively. PPO highly reactive quinones autopolymerize to form brown polyphenolic catechol melanins, a process thought to protect damage to plants from pathogens and insects (Deverall, 1961; Kessler and Baldwin, 2002; Pierpoint, 1969). The PPOs are important enzymes in plants that regulate feeding, growth and development of insect pests and play a leading role in plant defense against the biotic and abiotic stresses (He et al., 2011; Bhonwong et al., 2009).
| Table 5: | Defense enzymes concentration in tomato leaves after spraying with different compounds against the two-spotted spider mite Tetranychus urticae Koch |
*Values are mean of three replicates, number in the same column followed by the same letter are not significantly different at p<0.05 |
|
In plants treated with Vital Power Calcio Cao, (Potassium humates+Salicylic acid), Propolis, Potassium humates, Gibbereillc acid, Salicylic acid, Methyl jasmonate and Universal ergofito Glutathione peroxidase (GPO) activity was increased in leaves of tomato plant to 9113.91, 8332.53, 8066.67, 7959.67, 7794.32, 7674.36, 7703.54 and 7632.21 U g-1 tissue, respectively over untreated plants (7577.09 U g-1 tissue), while Lemongrass oil and Potassium silicate droppings (5631.76 and 4188.96 U g-1 tissue), respectively. Tremendous increase in superoxide dismutase (SOD), glutathione-S-transferase (GST), ascorbate peroxidase (APX) and lipoxygenase (LOXs) were provoked by T. urticae infestation. In Table 5, induction of LOXs activity in response to herbivory has been studied in many plants such as soybean in response to two-spotted spider mite (T. urticae) (Hildebrand et al., 1986).
Enormous increase in Phenylalanine Ammonia Lyase (PAL) activity was observed in tomato leaves treated with Gibbereillc acid, Vital Power Calcio Cao and Potassium humates to 55.53, 23.93 and 22.33 nmol/min/g tissues, respectively compared with the control (13.96 nmol/min/g tissues), while the low activity was found in leaves of other treatments. Increased the highest activity levels of β-glycosidase recorded in leaves treated with Propolis, followed by Potassium humates and Gibbereillc acid compared with the control (Table 5). Greater increasing inhibition (%) of the Proteinase inhibitors (PIs) activities in tomato leaves were observed in treatments; Gibbereillc acid, (Potassium humates+Salicylic acid), Universal Ergofito, Potassium humates, Vital Power Calcio Cao, Methyl jasmonate and Lemongrass oil to 79.99, 57.87, 56.84, 47.92, 32.61, 16.03 and 14.62%, respectively compared with control (13.75%), while in treatments; Salicylic acid, Propolis and Potassium silicate the inhibition (%) was reduced to 9.24, 8.73 and 13.34%, respectively. The defensive function of many PIs against insect pests, directly or by expression in transgenic plants to improve plant resistance against insects has been studied against many lepidopteran (Dunse et al., 2010) and hemipteran insects (Azzouz et al., 2005).
The statistical analysis show that in Table 5 there were significant differences between treatments; Control, Potassium humates, Salicylic acid, Potassium silicate and Potassium humates+Salicylic acid with most of defense enzymes such as Catalases, Peroxidases, Superoxide dismutase, Glutathione-s-transferees, Lipoxygenase, Phenylalanine ammonia-lyase and Proteinase inhibitors.
CONCLUSION
In conclusion, foliar application of different elicitors, particularly Potassium humates, Salicylic acid, (Potassium humates+Salicylic acid), Methyl jasmonate and Potassium silicate enhanced concentrations of essential oil components and enzymatic and non-enzymatic antioxidants in tomato leaves infested by T. urticae, thus suppressing invasion-induced oxidative damage and enhancing tolerance. Current knowledge limits the complete description of elicitor signal-transduction pathways in plants: Future studies are needed to dissect the complex network of elicitors and its involvement in plant defence against biotic and abiotic stresses, using genetic, genomic and biochemical approaches.
ACKNOWLEDGMENT
This study was supported in part by the Faculty of Agriculture, Cairo University, Giza, Egypt.