Methyl jasmonate primes defense responses against Botrytis cinerea and reduces disease development in harvested table grapes

Scientia Horticulturae 192 (2015) 218–223

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Methyl jasmonate primes defense responses against Botrytis cinerea and reduces disease development in harvested table grapes Lulu Jiang, Peng Jin, Lei Wang, Xuan Yu, Huanyu Wang, Yonghua Zheng ∗ College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, PR China

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Article history: Received 22 March 2015 Received in revised form 7 June 2015 Accepted 10 June 2015 Keywords: Methyl jasmonate Table grapes Botrytis cinerea Defense priming Induced disease resistance

a b s t r a c t The effect of a postharvest treatment with 10 ␮mol/L of methyl jasmonate (MeJA) on controlling gray mold decay caused by Botrytis cinerea in table grapes and the possible action mechanisms were investigated. The results indicated that MeJA treatment significantly reduced the incidence and development of gray mold decay in grapes. Meanwhile, MeJA treatment markedly enhanced activities of superoxide dismutase (SOD) and catalase (CAT), decreased ascorbate peroxidase activity, promoted the accumulation of H2 O2 and improved the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. MeJA treatment also significantly enhanced activities of defense related enzymes including chitinase (CHI), ␤-1,3-glucanase (GNS), phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO) and peroxidase (POD). Moreover, transcripts of defense related genes encoding CHI, GNS, PAL, SOD and CAT were only significantly enhanced in fruit pre-treated with MeJA and then inoculated with B. cinerea compared with those that were only treated with MeJA or inoculated with B. cinerea. These results suggested that 10 ␮mol/L of MeJA treatment primes defense responses for enhanced disease resistance against Botrytis cinerea and thus reducing gray mold decay in harvested table grapes. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Table grape (Vitis vinifera L.) is a non-climacteric fruit that is susceptible to fungal infection. Gray mold decay caused by Botrytis cinerea is the major postharvest disease that limits the storage of grapes. Currently, the most common method to control post-harvest diseases of grapes is the use of SO2 gas, either by repeated fumigation in storage rooms, or by packing the grapes in polyethylene-lined boxes with SO2 generators. However, SO2 can damage grapes manifested as bleaching of the berries and discolo-ration of the rachis, and induce allergy phenomena in sensitive individuals, which limit the commercial application of SO2 in many countries (Sanzani et al., 2012). Therefore, many alternative approaches including chemical treatment with ethanol (Lurie et al., 2006), chitosan (Meng et al., 2008) or salt solutions (Youssef and Roberto, 2014), physical treatment with hot water (Karabulut et al., 2004), and biological treatment with Cryptococcus laurentii (Meng et al., 2010) have been explored to control postharvest decay of grapes in the last decade.

∗ Corresponding author. Fax: +86 25 8439 5618. E-mail address: [email protected] (Y. Zheng). http://dx.doi.org/10.1016/j.scienta.2015.06.015 0304-4238/© 2015 Elsevier B.V. All rights reserved.

Methyl jasmonate (MeJA), a naturally occurring plant growth regulator, mediates diverse developmental processes and defense responses against biotic and abiotic stresses in plants (Creeman and Mullet, 1997). Moreover, MeJA has been shown effective to enhance disease resistance against fungal infection in various harvested fruits such as tomatoes (Ding et al., 2002), sweet cherry (Yao and Tian, 2005), loquat (Cao et al., 2008), peaches (Jin et al., 2009) and Chinese bayberries (Wang et al., 2009). These results suggest that MeJA treatment could be a useful approach to reduce postharvest diseases in horticultural produce. However, the specific defense mechanisms involved in MeJA-induced resistance in harvested fruits are still not well understood. Recently, the MeJAinduced disease resistance against Penicillium citrinum infection in harvested Chinese bayberries was found to be associated with an enhanced capacity to mobilize infection-induced cellular defense responses (Wang et al., 2014), a phenomenon called ‘priming’ (Conrath et al., 2002). However, whether defense priming is a common phenomenon of MeJA-induced disease resistance in harvested fruits is unknown. The objectives of this study were first to evaluate the efficacy of MeJA on controlling gray mold decay caused by Botrytis cinerea in harvested table grapes, and then to investigate if the MeJA-induced disease resistance against gray mold decay is associated with priming of defense responses in the fruit.

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2. Materials and methods 2.1. Plant material ‘Kyoho’ table grapes (Vitis vinifera×V. labrusca) were handharvested from a commercial vineyard in Nanjing, Jiangsu province, and transported to the laboratory within 2 h. Grapes were selected for uniform size, color and absence of blemishes or disease prior to MeJA treatment and pathogen inoculation. 2.2. Pathogen Botrytis cinerea was isolated from infected grape berries and maintained on potato dextrose agar medium (PDA: extract of boiled potatoes, 200 mL; dextrose, 20 g; agar, 20 g in 800 mL sterile distilled water). Spores of B. cinerea were harvested with sterile distilled water after 2 weeks of incubation on PDA petri dishes at 25 ◦ C, and removed from the surface of PDA petri dishes and suspended in 5 mL of sterile distilled water. The concentration of spores was adjusted to 1 × 105 conidia mL −1 using a haemocytometer. 2.3. Efficacy of MeJA for control of gray mold decay caused by B. cinerea In a preliminary in vivo study, we found that 10 ␮mol/L was the lowest concentration for MeJA to effectively induce disease resistance against B. cinerea and reduce disease incidence in grapes (data not shown), thus this specific concentration was used for MeJA treatment in this work. The selected grapes with pedicel attached were randomly divided into two lots of 300 berries each, and then placed in 40-L airtight containers for MeJA treatment. An appropriate amount of MeJA liquid (Aldrich Chemical Co., Milwaukee, WI) was spotted onto filter paper that was placed inside the containers with the berries and incubated at 25 ◦ C for 12 h. MeJA concentrations of 0 (control) and 10 ␮mol/L were calculated on the basis of the assumption that MeJA evaporated completely. After MeJA treatment, the containers were opened and ventilated at 25 ◦ C for 3 h before pathogen inoculation. For pathogen inoculation, both MeJA-treated and the control grapes were first surface-sterilized with 75% ethanol, then wounded (2 mm diameter × 2 mm deep) at 2 points on the equatorial zone with a sterilized nail and inoculated with 15 ␮L of a 1.0 × 105 spores/mL suspension of B. cinerea in each wound. Afterwards, all the grapes were incubated at 25 ◦ C with high relative humidity (approx. 95%) for 60 h. Disease incidence and lesion diameter on each fruit wound were observed at 24, 36, 48, 60 h post inoculation. Meanwhile, tissue samples of healthy pulp were taken for measurements of enzyme activity, protein, total phenolic and H2 O2 contents, and DPPH radical-scavenging activity. 2.4. Measurement of enzyme activity Chitinase (CHI, EC 3.2.1.14) was extracted from 5 g of tissue sample and homogenized with 5 mL of 50 mM sodium acetate buffer (pH 5.2), containing 5 mM ␤-mercaptoethanol and 1 mM ethylenediaminetetraacetic acid (EDTA), and centrifuged at 12000 × g at 4 ◦ C for 30 min. The supernatant was collected for the enzymatic assay. CHI activity was assayed by the amount of N-acetyl-D-glucosamine (NAG) released from colloidal chitin (Ippolito et al., 2000). One unit of CHI activity is defined as the amount of enzyme required to catalyze the production of 1 ␮g NAG per hour at 37 ◦ C. ␤-1,3-glucanase (GNS, EC 3.2.1.58) was assayed by measuring the amount of reducing sugar released from the substrate according to the method of Ippolito et al. (2000). The enzyme preparation was extracted from 1 g of tissue sample and homogenized with 5 mL of sodium acetate buffer (50 mM, pH 5.2). Then the homogenate was centrifuged at 12000 × g for 30 min at 4 ◦ C. Enzyme prepara-

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tion (1 mL) and 4% laminarin (1 mL) reacted for 1 h at 37 ◦ C. The sample was incubated with 2 mL 3, 5-dinitrosalicyclic reagent, and heated in boiling water for 5 min. The blank was the crude enzyme preparation heated for 5 min. After that the amount of reducing sugars was measured by spectrophotometric methods at 540 nm. One unit is defined as the amount of enzyme required to catalyze the production of 1 ␮mol glucose equivalent h−1 at 37 ◦ C. Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) was assayed by the method described by Assis et al. (2001). One gram of tissue sample was ground with 5 mL of sodium borate (0.2 M, pH 8.7). PAL activity was assayed by incubating 1 mL enzyme preparation in 1 mL L-phenylalanine at 37 ◦ C for 1 h. The reaction was stopped by addition of 10 ␮L HCl. One unit of PAL activity is defined as the amount of enzyme caused the increasing absorbance of 0.01 at 290 nm h−1 . Superoxide dismutase (SOD, EC 1.15.1.1) activity was assayed according to the method of Rao et al. (1996). Enzyme preparation was extracted from 3 g of tissue samples and homogenized with 5 mL sodium phosphate buffer (50 mM, pH 7.8) at 4 ◦ C. The reaction mixture contained 0.1 mL the enzyme preparation, 50 mM sodium phosphate buffer (pH 7.8), 14 mM methionine, 3 ␮M EDTA, 1 mL Nitro-Blue-Tetrazolium (NBT) and 60 ␮M riboflavin. One unit of SOD activity is defined as the volume of enzyme corresponding to 50% inhibition of NBT reduction. Catalase (CAT, EC 1.11.1.6) was extracted from 3 g of tissue sample and homogenized with 5 mL sodium phosphate buffer (5 mM, pH 7.0) at 4 ◦ C according to the method of Chance and Maehly (1955). The reaction mixture contained 50 mM sodium phosphate buffer (pH 7.0), 12.5 mM H2 O2 and 200 ␮L enzyme preparation. The increase in absorbance at 240 nm was recorded for 6 min at 25 ◦ C. One unit of CAT activity is defined as the amount of enzyme that decomposed 1 ␮moL H2 O2 per min at 30 ◦ C. Ascorbate peroxidase (APX, EC 1.11.1.11) was determined according to the method of Vicente et al. (2006). The enzyme was extracted from 3 g of tissue sample and homogenized with 5 mL sodium phosphate buffer (50 mM, pH 7.0). The homogenate was centrifuged at 12000 × g for 20 min at 4 ◦ C. The reaction mixture contained sodium phosphate buffer (50 mM, pH 7.0), 9 mM ascorbic acid and 30% H2 O2 . APX activity was determined by monitoring the decrease in absorbance at 290 nm as ascorbate was oxidized. One unit of APX activity is defined as the amount of enzyme oxidizing 1 ␮moL ascorbate per min at 30 ◦ C. Peroxidase (POD, EC 1.11.1.7) was determined by the method of Kochba et al. (1977). Enzyme preparation was extracted from 5 g of tissue sample and homogenized with 5 mL sodium phosphate buffer (50 mM, pH 8.7) at 4 ◦ C.The trails use 0.3% guaiacol as donor and 0.75% H2 O2 as substrate. One unit of POD activity is defined as the amount of enzyme that caused an increase in absorbance of 0.01 at 470 nm per minute. Polyphenol oxidase (PPO, EC 1.10.3.1) was extracted from 3 g of tissue sample and homogenized with 5 mL sodium phosphate buffer (0.2 M, pH 6.5) at 4 ◦ C according to the method of Gonzalez et al. (1999). The reaction mixture contained 1 mL enzyme extract and 2 mL catechol. One unit of PPO activity is defined as the amount of enzyme caused an increase in absorbance of 0.01 at 410 nm in 1 min under the specified conditions. Protein content in the enzyme extracts was estimated using the Bradford (1976) method, using bovine serum albumin as a standard. Specific activity of all the enzymes was expressed as units per milligram protein. 2.5. Measurement of H2 O2 content and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity H2 O2 content was determined by the method of Patterson et al. (1984) based on titanium oxidation. H2 O2 was extracted from 3 g of

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2.6. Defense-related gene expression analysis In order to investigate the mechanism of MeJA-induced disease resistance against B. cinerea in grape fruit, tissue samples were collected from 10 berries at 6, 12, 18, 24 and 36 h after inoculation to analyze the expression of several defense-related genes in grapes only treated with distilled water (Mock), inoculated with B. cinerea or treated with MeJA, and in those pre-treated with MeJA and then inoculated with B. cinerea. Total RNA was extracted from tissue sample of grape fruit according to the method of Chang et al. (1993). RT-PCR was performed using the PrimeScriptTM 16 1st Strand cDNA Synthesis Kit (TaKaRa, Japan). Specific primers were designed from their nucleotide sequences by referring to National Center for Biotechnology Information (NCBI) and synthesized by SBS Genetech Company (Shanghai, China). Short and conserved segments of CHI (GenBank ID: Z54234.1), GNS (GenBank ID: U73709), PAL (GenBank ID: U73709), SOD (GenBank ID: JQ692111.2) and CAT (GenBank ID: AF236127.1) were cloned by degenerate primers. 18S-rRNA (GenBank ID: AJ421474.1) was used as a quantitative control in the RT-PCR analysis. The sequences of primers used for RT-PCR analysis were as follows: CHI forward: 5’-GGGTTGTGGGCATTGGTA-3’, CHI reverse: 5’-GCTGCGATTTCCCTTTTA-3’; GNS forward: 5’-AATGTCAGATTCCGATACG-3’, GNS reverse: 5’-AAGTGCCGAGTAAACAGC-3’; PAL forward: 5’-TCATCCGAGCATCAACTA-3’, PAL reverse: 5’-GAGGAGATTAAGCCCAAG-3’; SOD forward: 5’-GTAATGAGGGTGTTTGTGG-3’, SOD reverse: 5’-TTCTCGTCTTCAGGAGCA-3’. CAT forward: 5’-CCCAGTCTTCTTTATTCG-3’, CAT reverse: 5’-TCCAACTCTTATGGCTTC -3’. 18S-rRNA forward: 5’-TGACCAAGCCAATGATGC-3’, 18S-rRNA reverse: 5’-CGACTTTCACTTTCAACCC-3’. 2.7. Statistical analysis The experiment was conducted twice using completely randomized design and each treatment was replicated three times. The data were expressed as means ± standard error (SE) and subjected to statistical analysis using the SPSS version 16.0 (SPSS Inc., Chicago, IL, USA). Data were analyzed by one-way analysis of variance (ANOVA), and means were compared by Duncan’s multiple range test at a significance level of P < 0.05.

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tissue sample and homogenized with 5 mL of chilled 100% acetone. Then the homogenate was centrifuged at 12000 × g for 20 min at 4 ◦ C. Absorbance of the supernatant was measured at 412 nm. The H2 O2 content is expressed as ␮moL g−1 fresh weight (FW). The DPPH radical scavenging activity was assayed by the method of Larrauri et al. (1998) with some modifications. One gram of tissue sample was homogenized with 5 mL 50% ethanol, then centrifuged at 12000 × g for 20 min at 4 ◦ C. The reaction mixture contained 0.5 mL extracting solution and 1.5 mL DPPH. An ethanol solution of DPPH served as control. The DPPH radical-scavenging activity was calculated by the following formula: DPPH radical scavenging activity (%) = [1 −(absorbance of sample/absorbance of control)] × 100%.

disease incidence (%)

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0

hours after inoculation (hour) Fig. 1. Changes in disease incidence (A) and lesion diameter (B) in table grapes treated with MeJA and inoculated with B. cinerea during incubation at 25 ◦ C. Each column represents the mean of triplicate samples. Vertical bars represent the standard errors of the means. Different letters above the bars for each time point indicate statistically significant differences according to Duncan’s multiple range test at P < 0.05 level.

41.7%, 60.6% and 86.5%, respectively, of that in the control on the 24th, 36th and 48th hour of inoculation (Fig. 1A). Meanwhile, as shown in Fig. 1B, the lesion diameter in grape fruit treated with MeJA was only 31.3%, 50.9% and 60.4%, respectively, of that in the control on the 24th, 36th and 48th hour of inoculation. Although all the inoculated wounds in both MeJA treated and control fruit developed decay symptoms after 60 h of inoculation, the lesion diameter in fruit treated with MeJA was still significantly (P < 0.05) smaller than that in the control (Fig. 1B). 3.2. Effect of MeJA treatment on SOD, CAT, APX activities and H2 O2 content in grape fruit inoculated with B. cinerea SOD activity in both MeJA-treated and control fruit increased sharply at the first 24 h of incubation and then decreased gradually. MeJA treatment maintained significantly (P < 0.05) higher SOD activity throughout the incubation compared with the control (Fig. 2A). CAT and APX activities declined gradually in grape fruit during the whole incubation period. MeJA treatment maintained significantly (P < 0.05) higher CAT activity but lower APX activity (Fig. 2B and C). The level of H2 O2 in both MeJA-treated and control fruit increased during incubation, but significant (P < 0.05) higher H2 O2 concentrations were detected in MeJA-treated fruit throughout the incubation period (Fig. 2D). 3.3. Effect of MeJA treatment on CHI and GNS activities in grape fruit inoculated with B. cinerea CHI activity in both MeJA-treated and control fruit increased gradually with incubation time. MeJA treatment maintained significantly (P < 0.05) higher CHI activity during the whole incubation period compared with the control. The activity of CHI was 44.0% higher in MeJA-treated fruit than the control after 60 h of incubation (Fig. 3A). As shown in Fig. 3B, GNS activity increased rapidly and reached a peak value on the 36th hour of the inoculation, thereafter it declined gradually. Fruit treated with MeJA showed significantly (P < 0.05) higher GNS activity during the whole incubation time except on the 24th hour (Fig. 3B).

3. Results 3.1. Efficacy of MeJA for control of gray mold decay caused by B. cinerea

3.4. Effect of MeJA treatment on PAL, POD, PPO activities and DPPH radical scavenging activity in grape fruit inoculated with B. cinerea

As shown in Fig. 1, MeJA treatment significantly (P < 0.05) reduced disease incidence and lesion diameter in grape fruit inoculated with B. cinerea during the first 48 h of incubation at 25 ◦ C. The disease incidence in grape fruit treated with MeJA was only

PAL activity in control fruit increased slightly at the first 48 h of incubation and then decreased. MeJA treatment induced significantly (P < 0.05) higher PAL activity during the entire incubation period (Fig. 4A). POD and PPO activities in both control and MeJA

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Fig. 2. Effect of MeJA treatment on SOD (A), CAT (B), APX (C) activities and H2 O2 content (D) in table grapes inoculated with B. cinerea during incubation at 25 ◦ C. Data are expressed as the mean of triplicate samples. Vertical bars represent the standard errors of the means.

inoculated with B. cinerea or treated with MeJA. However, in fruit pre-treated with MeJA and then inoculated with B. cinerea, transcripts of all tested genes were significantly enhanced and attained at higher levels at all the sampling points compared with the other three treatments (Fig. 5). Therefore, it is clear that MeJA induced stronger expression of the five defense related genes in grape fruit upon challenged with the pathogen B. cinerea.

4. Discussion

Fig. 3. Effect of MeJA treatment on CHI (A) and GNS (B) activities in table grapes inoculated with B. cinerea during incubation at 25 ◦ C. Data are expressed as the mean of triplicate samples. Vertical bars represent the standard errors of the means.

treated fruit increased steadily during incubation. MeJA treatment maintained significantly (P < 0.05) higher activities of POD and PPO than the control (Fig. 4B and C). DPPH radical-scavenging activity in control fruit showed little change during incubation. However, MeJA treatment significantly (P < 0.05) enhanced the increase in DPPH radical-scavenging activity (Fig. 4D). 3.5. Effect of MeJA treatment and B. cinerea inoculation on defense-related genes expression in grape fruit As shown in Fig. 5, the transcript levels of five defense-related genes encoding CHI, GNS, CAT, SOD and PAL were analyzed by using RT-PCR. Transcripts of all these genes were retained at very low level in grape berries only treated with distilled water (Mock),

In last decade, MeJA has been shown effective to suppress postharvest diseases of various fruits including tomatoes (Ding et al., 2002), sweet cherry (Yao and Tian, 2005), loquat (Cao et al., 2008) and peaches (Jin et al., 2009) and Chinese bayberries (Wang et al., 2009), possibly by directly inhibiting pathogen growth, and/or indirectly inducing disease resistance. In our study, we found that treatment with 10 ␮mol L−1 of MeJA significantly reduced the incidence and development of gray mold decay in grapes inoculated with B. cinerea (Fig. 1). These results suggest that the disease resistance of grape fruit was enhanced by postharvest MeJA treatment. The protection of plant from invading fungal pathogens is largely connected with induction of reactive oxygen species (ROS), and H2 O2 as one kind of ROS has been reported to exert various effects on plant defense responses (Bolwell et al., 2001; Torres, 2010). Generally, the metabolism of ROS is controlled by an array of enzymes including SOD, CAT, and APX. And H2 O2 is destroyed predominantly by APX and CAT. Recently, there is increasing evidence showing that a close relationship exists between ROS accumulation and decreased susceptibility to fungal infection in harvested fruits treated with different elicitors. For examples, higher levels of H2 O2 were correlated with lower susceptibility of MeJA-treated loquat fruit to Colletotrichum acutatum, MeJA-treated peach fruit to Penicillium expansum, BTH-treated muskmelon fruit to Trichothecium roseum, and MeJA-treated Chinese bayberries to Penicillium citrinum infections (Cao et al., 2008; Jin et al., 2009; Ren et al., 2012; Wang et al., 2014). These findings suggest that H2 O2 may act as a signaling molecule for the induction of disease resistance. In this study, MeJA treatment significantly enhanced activities of SOD and CAT (Fig. 2A and B), but inhibited the activity of APX (Fig. 2C), thus resulting in higher level of H2 O2 in grape fruit (Fig. 2D). These results suggest that enhanced H2 O2 generation may play crucial

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Fig. 4. Effect of MeJA treatment on PAL (A), POD (B), PPO (C) activities and DPPH radical scavenging activity(D) in table grapes inoculated with B. cinerea during incubation at 25 ◦ C. Data are expressed as the mean of triplicate samples. Vertical bars represent the standard errors of the means.

role for MeJA-induced disease resistance against B. cinerea infection in grape fruit. In previous studies, MeJA-induced resistance in postharvest fruits has been found to involve the activation of pathogenesisrelated proteins such as CHI and GNS and stimulation of phenylpropanoid pathway (Ding et al., 2002; Yao and Tian, 2005). CHI and GNS break down the cell wall components of pathogens, and play a crucial role in resistance of plants against pathogen infection (Ferreira et al., 2007). PAL is the key enzyme of the phenylpropanoid pathway and associated with the enhancement of physical barriers in plant against the attack by pathogens (Shadle et al., 2003). POD and PPO are both involved in lignification of host plant cells and considered as key enzymes related to defense reaction against pathogen infections (Mohammadi and Kazemi, 2002). The induction of these defense related enzymes by different elicitors has been reported in various harvested fruits including apple, Chinese bayberry, loquat, mango and tomato, which is correlated to increased disease resistance and reduced disease severity (Ippolito et al., 2000; Zeng et al., 2006; Cao et al., 2008; Charles et al., 2009; Wang et al., 2010). In line with these results, our study showed that MeJA treatment enhanced activities of CHI, GNS, PAL, POD and PPO (Figs. 3 and 4) and reduced gray mold decay in grapes inoculated with B. cinerea (Fig. 1). These results suggest that the induction of these defense related enzymes may be one part of the mechanism by which MeJA suppressed B. cinerea infection in grape fruit.

Induced resistance protects plants from attacks of a wide spectrum of pathogens. For a long time, it was assumed that protection of plants by induced resistance is based on direct activation of defense responses. However, there is increasing evidence showing that after treatment with resistance inducing agent, plants exhibit a faster and stronger activation of specific defense responses only after they have been infected by a pathogen (Kohler et al., 2002; Verhagen et al., 2010; Ahn et al., 2011). This phenomenon of plant-pathogen interaction is called priming, and is considered as a mechanism that is common to different types of induced disease resistance in plants (Conrath et al., 2002). However, studies on defense priming have been exclusively focused on field crops and model plants (Conrath, 2011). More recently, we demonstrated the B. cereus AR156-induced disease resistance against Rhizopus rot in peach fruit and the MeJA-induced disease resistance against Penicillium citrinum in Chinese bayberries were associated with priming of defense responses (Wang et al., 2013; Wang et al., 2014). Yu et al. (2014) also reported that treatment with ␥-aminobutyric acid could induce disease resistance against Penicillium expansum in pear fruit by priming of defense responses. Very recently, Wang et al. (2015) found that 10 ␮mol/L of MeJA treatment primed defense responses against B. cinerea in Kyoto grapes through inducing higher expression of the defense-related gene VvNPR1.1. In this study, we found that transcripts of several defense related genes encoding CHI, GNS, PAL, SOD and CAT were only significantly

Fig. 5. Expression of representative defense related genes in table grapes only inoculated with distilled water (Mock), B. cinerea (control/B. cinerea) or treated with MeJA, and in those pre-treated with MeJA and then inoculated with B. cinerea (MeJA/B. cinerea). RT-PCR was carried out using 18S-rRNA as an internal reference.

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enhanced in fruit pre-treated with MeJA and then inoculated with B. cinerea compared with those that were only treated with MeJA or inoculated with B. cinerea (Fig. 5). This result further confirmed that the MeJA-induced disease resistance against gray mold decay in grapes is also associated with priming of defense responses. Taken together, we suggest that defense priming might also be a common phenomenon of induced disease resistance in postharvest fruits. However, further investigations are needed to elucidate the molecular mechanisms of defense priming in harvested fruits. In conclusion, our results demonstrated that 10 ␮mol/L of MeJA can effectively induce disease resistance and suppress gray mold caused by B. cinerea in harvested table grapes. Moreover, this MeJAinduced disease resistance against B. cinerea is associated with priming of defense responses through enhancing defense-related enzymes activity and genes expression. Defense priming might be a common phenomenon of MeJA-induced disease resistance in harvested fruits. Acknowledgement This study was supported by National Natural Science Foundation of China (No. 31172003) and the Priority Academic Program Development of Jiangsu Higher Education Institutions. References Ahn, I.P., Lee, S.W., Kim, M.G., Park, S.R., Hwang, D.J., Bae, S.C., 2011. Priming by rhizobacterium protects tomato plants from biotrophic and necrotrophic pathogen infections through multiple defense mechanisms. Mol. Cells 32, 7–14. Assis, J.S., Maldonado, R., Munoz, T., Escribano, M.I., Merodio, C., 2001. Effect of high carbon dioxide concentration on PAL activity and phenolic contents in ripening cherimoya fruit. Postharvest Biol. Technol. 23, 33–39. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle-dye binding. Anal. Biochem. 72, 248–254. Bolwell, P.P., Page, A., Pislewska, M., Wojtaszek, P., 2001. Pathogenic infection and the oxidative defences in plant apoplast. Protoplasma 217, 20–32. Cao, S., Zheng, Y., Yang, Z., Tang, S., Jin, P., Wang, K., Wang, X., 2008. Effect of methyl jasmonate on the inhibition of Colletotrichum acutatum infection in loquat fruit and the possible mechanisms. Postharvest Biol. Technol. 49, 301–307. Chance, B., Maehly, A.C., 1955. Assay of catalases and peroxidase. Methods Enzymol. 2, 764–775. Chang, S., Puryear, J., Cairney, J., 1993. A simple and efficient method for isolating RNA from pine trees. Plant Mol. Biol. Rep. 11, 113–116. Charles, M.T., Tano, K., Asselin, A., Arul, J., 2009. Physiological basis of UV-C induced resistance to Botrytis cinerea in tomato fruit. V. Constitutive defence enzymes and inducible pathogenesis-related proteins. Postharvest Biol. Technol. 51, 414–424. Kochba, J., Lavee, S., Spiege, R.P., 1977. Difference in peroxidase activity and isoenzymes in embryogenic and non-embryogenic ‘Shamouti’ orange ovular callus lines. Plant Cell Physiol. 18, 463–467. Conrath, U., Pieterse, C.M.J., Mauch-Mani, B., 2002. Priming in plant-pathogen interactions. Trends Plant Sci. 7, 210–216. Conrath, U., 2011. Molecular aspects of defence priming. Trends Plant Sci. 16, 524–531. Creeman, R.A., Mullet, J.E., 1997. Biosynthesis and action of jasmonate in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48, 355–381. Ding, C.K., Wang, C.Y., Gross, K.C., Smith, D.L., 2002. Jasmonate and salicylate induce the expression of pathogenesis-related-protein genes and increase resistance to chilling injury in tomato fruit. Planta 214, 895–901. Ferreira, R.B., Monteiro, S., Freitas, R., Santos, C.N., Chen, Z., Batista, L.M., Duarte, J., Borges, A., Teixeira, A.R., 2007. The role of plant defence proteins in fungal pathogenesis. Mol. Plant Pathol. 8, 677–700. Gonzalez, E.M., de Ancos, B., Cano, M.P., 1999. Partial characterization of polyphenol oxidase activity in raspberry fruits. J. Agric. Food Chem. 47, 4068–4072. Ippolito, A., El Ghaouth, A., Wilson, C.L., Wisniewski, M., 2000. Control of postharvest decay of apple fruit by Aureobasidium pullulans and induction of defense responses. Postharvest Biol. Technol. 19, 265–272. Jin, P., Zheng, Y., Tang, S., Rui, H., Wang, C.Y., 2009. Enhancing disease resistance in peach fruit with methyl jasmonate. J. Sci. Food Agric. 89, 802–808.

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