Introduction

Pathogen attacks are responsible for losses in agricultural yield, and the use of toxic chemicals remains the general means of control. Extensive use of chemical phytosanitary products has become a major environmental concern over the last few decades. In this scenario, significant research efforts have been expended for the identification and development of newer and safer compounds, which are capable of triggering plant immune responses (Goupil and others 2012). These compounds (elicitors) are molecules that are capable of mimicking the perception of a pathogen by a plant, thereby triggering induction of a sophisticated defense response in plants. Plants are equipped with an array of defense mechanisms to protect themselves against attack by herbivorous insects and microbial pathogen. These inducers include avirulent pathogens (Hammerschmidt 1999), plant growth promoting rhizobacteria (Vivekananthan and others 2004; Acharya and others 2011a), and biotic and abiotic elicitors (Michael and others 2001; Acharya and others 2011b). Elicitation of plants with elicitor molecules results in the activation of a series of defense responses, including cell wall reinforcement by deposition of lignin and induction of an array of defense enzymes (Desender and others 2007). The corresponding plant defense responses following treatment include an oxidative burst leading to cell death, changes in cell wall composition, synthesis of antimicrobial compounds such as phytoalexins, activation of defense genes, and priming of host cells (Kuć 2006). So, these non-chemical disease control strategies are gaining importance in conventional agricultural practices diminishing negative side effects on both the environment and human health (Walling 2001; Ajay and Baby 2010; Harm and others 2011).

Induced resistance in plants can be achieved with applications of various elicitors like salicylic acid (SA) and its related compounds, such as benzo (1,2,3) thiadiazole-7- carbothioic acid S-methyl ester, 2,6-dichloro-isonicotinic acid, and dl-3-amino-n-butyric acid (Hong and others 1999; Walters and others 2005); jasmonic acid (JA) and its related compounds like methyl jasmonate (Moreno and others 2010; Yang and others 2011); benzothiadiazole (BTH) (Anttonen and others 2003; Lin and others 2011); chitosan and its derivatives (Dos Santos and others 2012; Yan and others 2012); arachidonic acid, cupric chloride (CuCl2), copper sulfate (CuSO4), isonicotinic acid, and oxalic acid (Coquoz and others 1995; Aziz and others 2006; Tian and others 2006; Acharya and others 2011b). The induction of resistance was achieved by the over expression of different defense gene products including PO, PPO, β-1,3-glucanase, and PAL (Maxson-Stein and others 2002; Pal and others 2011), along with several other enzymes and by higher accumulation of total phenolic content (Anand and others 2009; Acharya and others 2011a, b).

Tian and others (2006) used calcium chloride (CaCl2) as an elicitor for the post-harvest treatment of pear fruits and they found the induction of an increased level of defense enzyme activity. Calcium plays a fundamental role in plant growth and development. Many extracellular signals and environmental cues including hormones, light, biotic and abiotic stress factors, and elicit change in cellular calcium levels, termed as calcium signatures (Reddy 2001; Rudd and Franklin-Tong 2001; White and Broadley 2003). Plant cells also utilize a Ca2+ signal as a pivotal early signaling event in response to pathogen perception. In plant cells, the calcium ion is a ubiquitous intracellular second messenger involved in numerous signaling pathways. Cytosolic Ca2+ elevation is an important event in pathogen signaling that triggers plant innate immune responses (Dangl and others 1996).

Tea, the oldest known beverage, yields many health benefits to humans is made from the tender leaves of the tea plant (Camellia sinensis). This important horticulture crop has a key role in the economy in terms of export earnings of the country. Like other crops, tea plants also suffer from various biotic and abiotic stresses. Among the different pathogens that effect tea production, the fungal disease, blister blight, caused by Exobasidium vexans is by far the most serious disease of tea in Asia (Arulpragasam 1992; Sowndhararajan and others 2012). For the past five decades or so, several toxic chemicals have been used continuously as a general means for controlling the blister blight disease (Saravanakumara and others 2007). Around 24–28 rounds of sprays are required to keep this disease under control (Ajay and Baby 2010). The main aims of this study were (i) to evaluate the ability of calcium chloride to induce resistance at the field level against blister blight disease in tea; (ii) to determine its relation with defense enzyme activity and polyphenol content; and also (iii) to provide possible defense mechanism involved.

Materials and Methods

Plant Material

All the experiments were conducted with healthy C. sinensis (L) Kuntze plants (cultiver AV-2) raised in the experimental tea garden of Darjeeling Tea Research and Development Centre (DTR&DC), Tea Board of India, Kurseong.

Treatment

Healthy 25-year-old bushes of tea, cultiver AV-2, were treated with calcium chloride (1 % foliar application) at an interval of 15 days from May to October. Similar bushes treated with sterile distilled water were served as controls. The experiment was conducted for two seasons. There were three replications with each replicate consisting of 50 bushes. The experiment was laid out at the same elevation.

Field Trial and Percent Disease Index (PDI)

The incidence of disease in tender leaves was recorded at 30-day intervals for a period of 5 months (May–September). Three samples of fifty shoots of the same age (two leaves and a bud) and of uniform size were collected from individual plots, and they were assessed for the presence of blister spots. A shoot was considered as infected if an active blister lesion of any developmental stage was present. Disease was scored by following the method of Saravanakumara and others (2007) on a six-point scale (where 0 = no disease, 1 = 1 % leaf area affected, 3 = 2–10 % leaf area affected, 5 = 11–25 % leaf area affected, 7 = 26–50 % leaf area affected, 9 ≥ 50 % leaf area affected), and the PDI was calculated using the following formula:

$$ {\text{PDI = }}\frac{\text{Sum of all individual rating}}{\text{Total number of leaves}} \times \frac{ 1 0 0}{\text{Maxium disease grade}}. $$

Assay of the Defense-Related Enzymes

Enzyme Extraction

Tender leaves from treated and control bushes were plucked for assay during the peak time of blister blight severity at Darjeeling (late July to end of August) 24 h after the last treatment cycle. The leaf tissues collected from control and treated sets were homogenized with liquid nitrogen. Five hundred mg of powdered sample was extracted with 2 ml of different buffers containing 0.1 % polyvinylpyrrolidone and 20 µl of 1 mM phenylmethane sulphonyl fluoride to assay different enzymes: 0.1 M sodium acetate buffer (pH 5.0) for β-1,3 glucanase; 0.1 M sodium phosphate buffer (pH 7.0) for POD and PPO; and 0.1 M borate buffer (pH 8.7)for PAL. All the extraction procedures were conducted at 4 °C. The homogenate was centrifuged at 10,500×g for 20 min at 4 °C. The supernatants were used as the crude enzyme source for the enzymatic assay. Then, it was transferred to a 2-ml centrifuge tube and stored at −80 °C for further use.

Peroxidase (PO) Assay

Peroxidase activity was determined spectrophotometrically, as described (Hammerschmidt and others 1982), with some modifications. The reaction mixture consisted of 1.5 ml of pyrogallol, 0.05 ml of enzyme extract, and 0.5 ml of 1 % hydrogen peroxide in a total volume of 2.5 ml. The change in absorbance at 420 nm was recorded at each 30 s interval for 3 min. The enzyme activity was expressed as changes of absorbance of reaction mixture (∆ OD change) min−1 g−1 protein.

Polyphenol Oxidase (PPO) Assay

PPO activity was estimated as previously described (Mayer and others 1965). Two hundred μl of 0.01 M catechol was added to the reaction mixture containing 200 μl of enzyme extract and 1.5 ml of 0.1 M sodium phosphate buffer (pH 6.5). Enzyme activity was expressed as change in absorbance at 495 nm (∆ OD change) min−1 g−1 protein.

Phenylalanine Ammonia Lyase (PAL) Assay

PAL was assayed following the method of Dickerson and others (1984) determining the conversion of l-phenylalanine to transcinnamic acid spectrophotometrically at 290 nm. Enzyme extract (0.4 ml) was incubated with 0.5 ml of 0.1 M borate buffer (pH 8) and 12 mM l-phenylalanine in the same buffer for 30 min at 30 °C. Enzyme activity was expressed as synthesis of transcinnamic acid (n mol) min−1 g−1 protein.

β-1,3 Glucanase assay

β-1,3 glucanase activity was estimated according to the method of Pan and others (1991) with slight modifications. Crude enzyme extract of 50 µl was added to 50 µl of 1 % laminarin and incubated at room temperature for 30 min. The reaction was stopped by adding 300 µl of dinitrosalicylic acid and heated for 10 min in a boiling water bath. The resulting colored solution was diluted with distilled water, and the absorbance was recorded at 520 nm. The enzyme activity was expressed as µmol glucose equivalent produced min−1 g−1 protein.

Total Protein Estimation

The standard Bradford assay (1976) was employed, using bovine serum albumin as a standard, to test the protein concentration of each extract.

Analysis of Defense-Related Gene Expression by Semi-quantitative RT-PCR

Expression of the genes was analyzed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was extracted with TRIzol Reagent (Invitrogen, USA) from the control and treated sets of tea leaves. The cDNA was synthesized from the total RNA using RevertAid M-MuLV Reverse Transcriptase (Fermentas, USA) according to the manufacturer protocol. Twenty µl reaction volume contained 1 µg of RNA, 0.5 µg of Oligo(dT), 20 units of RiboLock RNase Inhibitor (Fermentas, USA), 4 µl of 5× reaction buffer (250 mM Tris–HCl (pH 8.3), 250 mM KCl, 20 mM MgCl2, 50 mM DTT), 2 µl of 10 mM each deoxynucleoside triphosphates (dNTP Mix; Fermentas, USA), and 200 units of RevertAid M-MuLV Reverse Transcriptase. The reaction was carried out at 45 °C for 60 min followed by 70 °C for 10 min. To analyze the expression of a specific gene, 1 µl of the cDNA was taken in a 50 µl PCR mixture containing 1× DreamTaq PCR buffer, 0.2 mM of each dNTPs, 1 µM of each gene specific primer, and 1.25 units of DreamTaq DNA polymerase (Fermentas, USA). Thaumatin, catalase (CAT), PAL, cinnamate 4-hydroxylase (C4H), and flavonoid 3′-hydroxylase (F3H) genes were amplified individually (Table 1). Actin gene primers were used as internal controls for expression studies (Table 1). Linearity between the amount of input RNA and the final RT-PCR products was verified and confirmed. After standardizing the optimal amplification at the exponential phase, PCR cycles were carried out under the following conditions: 94 °C for 4 min, then 30 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 60 s with a final extension step of 7 min at 72 °C in a thermal cycler (Applied BioSystem, USA). The PCR products were electrophoresed in agarose gel, stained with ethidium bromide, visualized in UV transilluminator, and then photographed.

Table 1 Primer sequences used in RT-PCR analysis
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Estimation of Total Phenol Content

Leaf samples (1 g) from the control and CaCl2-treated sets were homogenized in 10 ml of 80 % methanol and agitated for 15 min at 70 °C (Zieslin and Ben Zaken 1993). One ml of the methanolic extract was added to 5 ml of distilled water and 0.25 ml of 1 N Folin–Ciocalteau reagent, and the solution was kept at 25 °C. Phenolic content was measured spectrophotometrically at 725 nm using gallic acid as a standard. The amount of phenolics was expressed as μg gallic acid g−1 fresh weight.

Quantification of Phenolic Compounds by HPLC

Samples prepared from tea leaves for phenolic estimation were analyzed with an HPLC system (Agilant, USA) equipped with an Agilent DAAD detector and an Agilent Eclipse plus C18 column (100 mm × 4.6 mm, 3.5 μm). The mobile phases were (A) acetonitrile and (B) 0.1 % phosphoric acid. The linear gradient conditions were as follows: 0–5 min, 10 % A in B; 5–15 min, 10–20 % A in B; 15–25 min, 20-90 % A in B with a flow rate of 0.8 ml min−1; and 20 μl of injection volume. UV–Vis absorption spectra were recorded on-line from 190 to 600 nm during the HPLC analysis (Yao and others 2004). Samples were injected three times into the sample loop, and the mean of the peak areas of individual compounds was taken for quantification. Solutions of each standard, at various concentration levels, were injected into the HPLC system. The peak areas and thus the calibration curves and response factors were recorded under the same conditions as for the samples. Gallic acid (GA), caffeine, epicatechin (EC), epigallocatechin (EGC), epigallocatechin gallate (EGCG), epicatechin gallate (ECG), gallocatechin gallate (GCG), and catechin gallate (CG) (M.P. Biomedicals. USA) were used as standards. The DAAD detection was conducted at 278 nm for the quantification (Seeram and others 2006). Concentrations were calculated by comparing peak areas of reference compounds with those in the samples run under the same elution conditions.

Nitric Oxide Estimation

Production of NO was estimated by hemoglobin assay (Delledonne and others 2001) during the peak time of blister blight severity period 24 h after the treatment cycle. Leaf tissues of control and treated sets were incubated in a reaction mixture containing 10 mM l-arginine, 10 mM hemoglobin, in a total volume of 5 ml of 0.1 M phosphate buffer (pH 7.4). Production of NO was measured spectrophotometrically at 401 nm, and NO levels were calculated using an extinction coefficient of 38,600 M−1 cm−1(Salter and Knowles 1998). After 2 h of incubation, NO content in the reaction mixture was measured as nmol of NO produced mg−1 fresh weight h−1 and compared with the water control.

Real-time NO production was visualized using membrane permieant fluorochrome 4-5-diaminofluorescein diacetate (DAF-2DA) dye (Bartha and others 2005). The lower epidermis of the control and treated sets of leaves was peeled off and placed in a brown bottle containing 1 ml of loading buffer (10 mM KCl, 10 mM Tris HCl, pH 7.2) with DAF-2DA at a final concentration of 10 μM for 20 min in dark. Fluorescence was observed with a Leica DMLS microscope at an excitation wavelength of 480 nm and emission wavelength of 500–600 nm.

Statistical Analysis

Data from two consecutive seasons were analyzed by student’s t test using Microsoft® Office Excel (Microsoft®, USA), and in all the cases, results are mean ± SD (standard deviation) of at least three individual experimental data, each in triplicate. Values of P < 0.05 were considered statistically significant. The relation between changes in enzyme activity, NO level, and corresponding PDI of the treated set has been evaluated by correlation analysis using Microsoft® Office Excel (Microsoft®, USA).

Results

Under field conditions, calcium chloride-treated C. sinensis plants (cultivar AV-2) recorded a significantly lower incidence of disease than the untreated plants. Blister blight incidences were observed at an interval of 30 days from May to September (150 days) after the first spray, and PDI was calculated accordingly. During the peak time of blister blight incidence at Darjeeling tea garden in the month of July, disease incidence of blister blight was significantly reduced about 80 % when compared to untreated controls (Fig. 1).

Fig. 1
figure 1

Effects of foliar application of elicitors on blister blight disease index. Field photograph of tea plants (cultivar AV-2): a control set; b CaCl2-treated set; and c graphical presentation of disease index. Arrows indicate presence of symptoms on tender leaves. Results are mean ± SD of three separate experiments, each in triplicate

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Simultaneously, defense-related enzyme activity and polyphenol accumulation levels were also compared between the CaCl2 treated and the untreated control plants. Elicitor treatment showed higher induction of PO, PPO, PAL, and β-1,3 glucanase level along with higher polyphenol accumulation. The induction of the PO enzyme and β-1,3 glucanase enzyme was significantly higher, and about 61 % increase in enzyme accumulation was observed in tea plants treated with CaCl2 over the control set (Table 2). Likewise, higher production of PPO activity was noted in tea plants, followed by induction of PAL enzyme activity (Table 2). Correlation analysis (Table 3) between the defense enzymes and corresponding PDI is an indication for an inverse relationship. Accumulation of phenols in tea plants sprayed with CaCl2 was 7.5 % higher compared to untreated controls (Table 2).

Table 2 Effects of foliar application of elicitor, CaCl2 on tea plants on the production of defense enzymes, and total phenols
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Table 3 Correlation between the defense enzyme activity, NO production, and the corresponding PDI of the treated set
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To elucidate the effect of CaCl2 treatment on defense-related genes, expression in the transcript level was determined by semi-quantitative RT-PCR analysis. Figure 2 reveals that differential alteration of defense-related genes like PAL, thaumatin, and CAT genes occurred on treatment. Expression of the two phenylpropanoid biosynthetic pathway genes C4H and F3H increased markedly indicating a higher accumulation of flavan-3-ol compounds (Table 4).

Fig. 2
figure 2

Semi-quantitative RT-PCR of thaumatin, CAT, PAL, C4H, and F3H gene expressions as represented in lane C: control; lane T: treated. Actin band represents equal loading

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Table 4 Quantitative changes in the phenolic acid content due to the application of elicitor, CaCl2 on tea plants
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HPLC analysis showed interesting results in the total phenolic extracts from control and elicitor-treated plants. Phenolic compounds present in the samples were identified by comparing both retention times and UV–Vis spectra with those of pure standards (Fig. 3). GA, EC, EGC, and EGCG were produced in significantly higher amounts in the elicitor-treated plants, whereas caffeine and CG content were higher in the control plant sets. No significant change in ECG content was observed between the control and treated plants. GCG content was only detected in the elicitor-treated set of plants (Table 4).

Fig. 3
figure 3

HPLC chromatograms of the standard phenolic acids. Numbers above the peaks indicates retention time (min). Standard peaks represent a GA, b EGC, c caffeine, d EC, e EGCG, f GCG, g ECG, h CG

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Furthermore, an attempt has been made to evaluate the status of NO during the same period in the leaves of treated and untreated tea plants (Fig. 4). CaCl2 treatment stimulated NO production about 2.35 fold over the controls (Fig. 4). To gain a precise view of NO production during the interaction between the elicitor and tea plants, a NO-specific fluorophore DAF-2DA on leaf peals, which converts fluorescent triazol derivative upon reaction with NO, showed increased fluoresce under a fluorescent microscope (Leica DMLS) in the elicitor-treated set (Fig. 4).

Fig. 4
figure 4

Calcium chloride treatment and nitric oxide status in C. sinensis leaves. Real-time determination of NO in leaf epidermal cells by DAF-2DA staining. NO generation was detected by green fluorescence. a Control; b CaCl2-treated set; c spectrophotometric analysis of NO production in control and treated plants of C. sinensis. Results are mean ± SD of three separate experiments, each in triplicate

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Discussion

Protecting plants with the application of elicitor are so far a better alternative to the wide application of pesticides in crop protection (Sticher and others 1997). To defend themselves against attack from various pathogens present in their surrounding environment, plants are equipped both with pre-formed, constitutive chemical, and mechanical barriers as well as with inducible defense systems (Montesano and others 2003), which need appropriate stimuli or signals to activate them. Elicitors are usually capable of triggering various modes of the plant defense system resulting in the activation of a series of defense responses including cell wall reinforcement by deposition of lignin, an oxidative burst leading to a hypersensitive response, synthesis of antimicrobial compounds, and activation of defense genes. In this study, the role of defense-related gene products such as PO, PPO, PAL, β-1,3 glucanase, CAT, thaumatin, C4H, F3H, and phenolics during the CaCl2-mediated elicitation of resistance in tea plants against blister blight disease was evaluated.

This study demonstrated that CaCl2 treatment might have caused the significant reduction in disease incidence in the treated set compared to the water-treated controls. The analysis of defense-related enzymes revealed a higher accumulation of PO, PPO, PAL, and β-1,3-glucanases in the CaCl2-treated sets. This observation supports the findings of Tian and others (2006) and Acharya and others (2011b), whereas pear fruits and Raphanus leaves showed higher induction of PO, PPO, PAL, and β-1,3-glucanase enzymes upon respective treatment with several elicitors. Ajay and Baby (2010) showed that foliar application of salicylic acid (SA) and acibenzolar-S-methyl benzo-(1,2,3)-thiadiazole-7-carboxylic acid S-methyl ester (ASM) in tea plants induced higher PO, PAL, and β-1,3-glucanases activity over the untreated control and reduced disease incidence significantly. Similarly, higher accumulation of all the four defense enzymes along with reduced disease severity was observed when carrot plants were treated with different elicitors to fight against the pathogen Alternaria radicinia (Jayaraj and others 2009).

Furthermore, transcript analysis showed over expression of thaumatin, catalase, PAL, C4H, and F3H genes, which strengthens our earlier observations. Elicitor treatment enhancement of transcript levels of various plants was observed by several researchers. Over expression of thaumatin genes from rice has been demonstrated to reduce infection of rice by Rhizoctonia solani (Grover and Gowthaman 2003), of wheat by Fusarium graminearum (Chen and others 1999), of tobacco by Alternaria alternata (Velazhahan and Muthukrishnan 2003), and of carrot by Alternaria dauci, Alternaria petroselini, A. radicina, Botrytis cinerea, R. solani, and Sclerotinia sclerotiorum (Punja 2005). Increased CAT activity might have reduced the harmful effect of H2O2 accumulation. Our observations also coincide with the findings of Tasgin and others (2006) and Pal and others (2011), whereas CAT activity in wheat plants and rice plants was found to be increased due to treatment with salicylic acid and Cymbopogan citrus leaf extract respectively. PAL activity could be induced by elicitor treatment (Ajay and Baby 2010). Suspension culture of Oryza sativa cv BL-1 treated with the elicitor N-acetylchitooligosaccharide showed over expression of several defense-related genes including PAL (Yamaguchi and others 2005). Catalysis of PAL is the first step of the phenylpropanoid biosynthesis pathway, and the expression of C4H and F3H genes indicates a higher accumulation of catechin compounds. Catechin compounds are reported to act as growth and defense inducing agents (Prithiviraj and others 2007). F3H catalyzes one of the key steps of the flavonoid biosynthesis pathway yielding a large family of flavonoid compounds which are mainly involved in various biological activities (Khlestkina and others 2011). A direct relationship between the F3H expression level and disease resistance in different plants like chickpea (Cho and others 2005), avocado fruits (Ardi and others 1998), and wheat (Giovanini and others 2006) against different pathogens has already been reported earlier.

Phenolics are involved in phytolaxin accumulation, biosynthesis of lignin, and the formation of structural barriers, and play a major role in resistance against pathogens. Greater accumulation of phenolics due to elicitor-treated plants reduces pathogen attack and makes the plants more resistant to pathogen attack. The role of phenolic substances in disease resistance (Nicholson and Hammerschmidt 1992) and their accumulation by the phenyl propanoid pathway due to various elicitor treatments have already been documented earlier (Sánchez-Estrada and others 2009; Dong and others 2010; El Modafar and others 2012). Previously, Jayaraj and others (2009) and Acharya and others (2011b) showed that carrot and R. sativus leaves, respectively, upon treatment with several elicitors, accumulated higher amount of phenolics over controls. In the present study, tea plants when sprayed with CaCl2 induced higher accumulation of polyphenols. HPLC analysis of phenolic compounds revealed higher accumulation of GA, caffeine, several catechhins (GCG and CG), and epicatechins (EC, EGC, EGCG). It has also been previously reported that flavonoids are the most abundant chemical group in tea leaves and among them, flavan-3-ol compounds contribute almost 20–25 % of the dry weight of the leaves (Punyasiri and others 2004). These compounds are important for plants to adapt to various environmental conditions and also involved in the resistance to pathogens by acting as feeding deterrents (Gould and Lister 2006). The resistance of apple cultivars (Treutter and Feucht 1990) to Venturia inaequalis and avocado (Prusky and others 1996; Prusky 1996) to anthracnose has been attributed to high levels of EC, suggesting that induced epicatechin may be directly or indirectly involved in the resistance mechanism of tea against blister blight. The catechins and epicatechins are converted to proanthocyanidins via lucoanthocyanidin reductase (Tanner and others 2003) and the anthocyanidin (Xie and others 2003) reductase pathways, respectively. These proanthocyanidins are widely distributed plant defense compounds (Treutter and Feucht 1999) and have a general toxicity toward fungi, yeast, and bacteria (Scalbert 1991). In the present investigation, the enhanced accumulation of catechins and epicatechins might have induced the accumulation of proanthocyanidins, which in turn could have resulted in the observed defense enhancement. Thus, the high level of flavonoid accumulation in tea plants might be an indication of enhanced resistance against blister blight. From this observation, it can also be inferred that CaCl2 treatment might reduce the need for pesticide treatments.

Ca2+ is a well-established important intracellular messenger in plant defense signaling, which is relayed by the calcium sensor that quickly converts the signal to second messengers like NO and cyclic nucleotides. Furthermore, several recent reports have painted a picture of the signaling role of NO, which is associated with numerous physiological roles (Baudouin 2011) including defense responses in plants (Acharya and others 2005; Acharya and Acharya 2007; Hong and others 2008; Acharya and others 2011a, b; Gupta and others 2013). Lum and others (2002) showed that Ca2+ is required for H2O2-induced NO production in guard cells of mung bean. Several scientists reported that in plants Ca2+ or Ca2+- bound CaM might directly interact with the plant NOS-like enzyme (Delledonne and others 1998; Modolo and others 2005; del Rίo and others 2004). It has also been reported that H2O2 acts together with NO during programmed cell death (Zago and others 2006). Thus, NO appears as an early signaling component, possibly orchestrating a number of downstream signaling pathways (Perchepied and others 2010). In this study, CaCl2-treated tea plants showed greater NO production than the control set. Table 3 indicates that the production of NO might have a relationship with defense enzyme accumulation and PDI. This result signifies that higher accumulation of NO might have played a role in the up-regulation of defense enzyme activity, which in turn could have resulted in the reduced disease incidence. Recently, Gupta and others (2013) have also indicated the relationship between the elevated level of defense enzymes and phenols with the higher production of NO in the toxin-treated callus tissue. They also showed that co-treatment of A. alternata toxin with an NOS inhibitor (L-NAME) or NO scavenger (cPTIO) reduced the increased NO production to basal levels in R. serpentina callus showing a strong unidirectional correlation with plant defense regulators. A similar observation was recorded by Zhao and others (2007), whereas co-treatment of tobacco cells with cPTIO and oligochitosan did not increase PAL activity.

Taken together, our results demonstrate that the elicitor CaCl2 has a greater induction effect on the production of defense gene products and molecules in tea plants. This elicitor capacity of CaCl2 appeared to be associated with the higher induction of NO and defense molecules, which might have induced protection against the blister blight causing pathogen E. vexans. The increased level of NO in the treated plants suggests that NO might be the signal molecule for the stimulatory effect on induced resistance in higher accumulation of defense-related gene products and total phenolics.

Conclusion

In conclusion, the defense chemicals induced upon treatment with CaCl2 have reduced disease incidence, and thereby simultaneously increased yield would have been recorded. Our results also suggest that NO acts as a key signaling molecule in elicitor-mediated transduction cascades. NO might also be associated with the higher accumulation of defense enzymes and total phenolics. Thus, the use of elicitors can be an invaluable tool for the stimulation of defense mechanisms in plants and may open a new horizon in eco-friendly disease control and sustainable organic agriculture practices.