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Hot air treatment induces resistance against blue mold decay caused by Penicillium expansum in sweet cherry (Prunus cerasus L.) fruit
Scientia Horticulturae 189 (2015) 74–80
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Hot air treatment induces resistance against blue mold decay caused by Penicillium expansum in sweet cherry (Prunus cerasus L.) fruit Lei Wang a , Peng Jin a , Jing Wang a , Hansheng Gong b , Shurong Zhang b , Yonghua Zheng a,∗ a b
College of Food Science and Technology, Nanjing Agricultural University, Nanjing, 210095, People’s Republic of China College of Food Engineering, Ludong University, Yantai, 264025, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 28 November 2014 Received in revised form 24 March 2015 Accepted 31 March 2015 Keywords: Prunus avium Hot air Induced resistance Postharvest disease Defense related genes
a b s t r a c t Effect of hot air (HA) treatment (44 ◦ C, 114 min) on reducing blue mold decay caused by Penicillium expansum in sweet cherry fruit was investigated. The results indicated that fruit treated with HA had significantly lower disease incidence and smaller lesion diameter than the control fruit did. HA treatment significantly enhanced activities of chitinase (CHI) and -1, 3-glucanase (GLU). The activities of polygalacturonase (PG) and pectinmethylesterase (PME) were significantly inhibited by HA treatment. Fruit treated with HA exhibited remarkably higher activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and polyphenoloxidase (PPO) and lower activity of ascorbate peroxidase (APX) than control. Expression of defense related genes such as PaGLU, PaCAT, and PaNPR1-like was greatly induced in HA-treated fruit during storage, while the expression of expansins (EXP) was down-regulated by HA treatment. These results suggest that HA can effectively inhibit blue mold decay caused by P. expansum in harvested cherry fruit possibly by directly inducing disease resistance and delaying fruit tissue softening that helps to ward off the spread of pathogens. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Sweet cherry (Prunus avium L.) is highly susceptible to pathogenic infection causing decay and large postharvest loss (AlHaq et al., 2002; Feliziani et al., 2013). Blue mold decay caused by Penicillium expansum Link is one of the most important postharvest diseases in sweet cherry fruit (Ceponis et al., 1987). Traditionally, the control of postharvest diseases of fruits relies mainly on the use of synthetic fungicides. However, the intense use of synthetic fungicides has led to the increasing resistance of fungal pathogens and growing concern of consumers over chemical residues (Janisiewicz and Korsten, 2002; Yu et al., 2014). Therefore, it is crucial to study eco-friendly alternative strategies to inhibit postharvest fungal decay (Droby et al., 2009). Heat treatments, including hot air (HA) treatment, hot water dipping and hot water vapor, which are more feasible for commercial application to reduce fruit decay, delay ripening and maintain quality (Jin et al., 2014; Lurie, 1998). Among the three methods, hot air treatment has received much attention and has been widely used to control fruit decay and maintain quality. It has been
∗ Corresponding author. Tel.:+86 25 8439 9080; fax: +86 25 8439 5618. E-mail address: [email protected] (Y. Zheng). http://dx.doi.org/10.1016/j.scienta.2015.03.039 0304-4238/© 2015 Elsevier B.V. All rights reserved.
reported that hot air treatment was applied effectively to reduce postharvest decay in various fruits including apple, peach and Chinese bayberry fruit (Conway et al., 2004; Wang et al., 2010; Zhang et al., 2007). In addition, HA treatment has been shown not only to limit spore germination of pathogens in vitro (Schirra et al., 2002; Zhang et al., 2007), but also induce resistance of fruit against fungal infection (Liu et al., 2010; Wang et al., 2010), thereby inhibiting decay incidence in postharvest fruit. However, there is no information concerning the effect of HA on controlling blue mold decay in sweet cherry fruit. The objective of this study was to evaluate the effect of HA treatment on controlling blue mold decay caused by P. expansum in sweet cherry fruit and to explore the possible mechanisms involved. 2. Materials and methods 2.1. Fungal pathogen The pathogen P. expansum was isolated from infected sweet cherry fruit and cultured on potato dextrose agar (PDA) medium (containing the extract of 200 g boiled potatoes, 20 g dextrose and 20 g agar in 1000 mL of distilled water). Spore suspensions were prepared from 2-week-old PDA cultures. The spores were removed
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from the surface of the cultures and suspended in 5 mL of sterile distilled water containing 0.05% (v/v) Tween 80. The number of spores was determined with a hemocytometer counting chamber, and then the spore concentration was adjusted to 5 × 104 spores mL−1 with sterile distilled water. 2.2. Fruit material Sweet cherry (Prunus avium L. cv. Hongdeng) fruits were handharvested at commercial maturity stage with healthy greenish stems. The fruits were selected for uniformity of size, ripeness, and absence of physical injuries or infections. The fruits were randomly divided into two groups of 300 fruit each. The first group was treated with HA at 44 ◦ C for 114 min (80–90% relative humidity). The second group stored at 20 ◦ C did not receive any treatment and served as control. The treatment condition in present study was chosen on the basis of our preliminary research. All fruits were airdried for approximately 1 h and surface-sterilized with 75% ethanol prior to wounding. 2.3. Pathogen inoculation and fruit tissue sampling Sweet cherries were wounded (3 mm deep and 3 mm wide) with a sterile nail at the equator of each fruit. Aliquots (15 L) of a suspension of 5 × 104 spores mL−1 P. expansum or distilled water (as control) was pipetted into each wound. The fruit were incubated at 20 ◦ C and 95% relative humidity for five days. Disease incidence and lesion diameter on each fruit wound were recorded at 1, 3 and 5 days after inoculation. There were three replicate trials of 30 fruits per treatment with complete randomization. The experiment was conducted twice. Fruit samples were taken before inoculation (time 0) and at 1, 3 and 5 days after inoculation for enzyme assays and expression of defense-related genes. Each treatment was replicated three times, and the experiment was conducted twice. 2.4. Assay of enzyme activity Chitinase (CHI, EC 3.2.1.14) and -1, 3-glucanase (GLU, EC 3.2.1.58) were extracted from 1 g of tissue sample with 5 mL of 50 mM sodium acetate buffer (pH 5.0). The homogenate was centrifugated at 12,000g for 20 min at 4 ◦ C and the supernatant was used to determine the CHI and GLU activity. CHI activity was determined according to the method of Abeles et al. (1971). CHI activity was measured by the release of N-acetylD-glucosamine (NAG) from colloidal chitin (Sigma Chemical Co., Ltd) with 4-dimethylaminobenzaldehyde method. 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 (GLU) (EC 3.2.1.58) activity was measured according to the method of Abeles et al. (1971). Aliquots (1 mL) of enzyme preparation were incubated for 1 h at 37 ◦ C with 1 mL of 4% laminarin (Aldrich, Chemical Co., Milwaukee, WI, USA). The reaction was terminated by heating the sample in boiling water for 5 min and the amount of reducing sugar was measured spectrophotometrically at 540 nm after reaction with 250 L 3, 5-dinitrosalicylic reagent. One unit is defined as the amount of enzyme catalyzing the formation of 1 mol glucose equivalents in 1 h. PG and PME were extracted from 1 g of tissue sample at 4 ◦ C with 5 mL of 40 mM sodium acetate buffer (pH 5.2) containing 100 mM NaCl, 2% (v/v) mercaptoethanol, and 5% (w/v) polyvinyl polypyrrolidone. The extracts were then homogenised and centrifuged at 25,000g for 20 min at 4 ◦ C. The supernatants were used to determine the PG and PME activity.
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PG (EC 3.2.1.15) activity was determined by the method of Zhou et al. (2011). The reaction mixture contained 40 mM sodium acetate buffer (pH 5.2), 0.1% (w/v) polygalacturonic acid and 100 L enzyme extract. One unit of activity was defined as 1 M galacturonic acid released per mg of protein per hour. PME (EC 3.1.1.11) activity was evaluated by the method of Cao et al. (2010). The reaction mixture consisted of 1 mL of enzyme extract and 10 mL 1% (w/v) citrus pectin (Sigma Chemical Co., Ltd). One unit of enzyme activity was calculated as 1 mM NaOH consumed per mg of protein per hour. SOD (EC 1.15.1.1) was extracted from1 g of flesh tissue with 5 mL of 50 mM sodium phosphate buffer (pH 7.8) at 4 ◦ C. The homogenate was centrifuged at 10,000g for 20 min at 4 ◦ C. The supernatant was used to estimate SOD activity by the method of Rao et al. (1996) in a final volume of 3 mL, which contained 0.1 mL of crude enzyme extract. One unit of SOD activity is defined as the amount of enzyme that causes 50% inhibition of nitroblue tetrazolium. CAT (EC 1.11.1.6) and APX (EC 1.11.1.11) were extracted from 1 g of tissue sample with 5 mL of 50 mM sodium phosphate buffer (pH 7.0). The homogenate was centrifugated at 12,000g for 20 min at 4 ◦ C and the supernatant was used to determine the CAT and APX activity. CAT (EC 1.11.1.6) activity was determined by the method of Chance and Maehly (1955). CAT activity was determined by adding 0.2 mL of the enzyme preparation to 3 mL of sodium phosphate buffer containing 0.2 mL H2 O2 as a substrate. One unit of CAT activity was designated as the amount of enzyme that decomposes 1 mol of H2 O2 per minute at 30 ◦ C. APX (EC 1.11.1.11) activity was measured according to the method of Amako et al. (1994). One unit of APX was defined as the amount of enzyme that oxidised 1 mol ascorbate per min. POD (EC 1.11.1.7) activity was assayed according to the method of Kochba et al. (1977). POD was extracted from 1 g of tissue sample with 5 mL of 50 mM sodium phosphate buffer (pH 8.7). The reaction mixture contained 50 mM sodium phosphate buffer (pH 8.7), 0.75% H2 O2 , 20 mM guaiacol and 0.2 mL crude enzyme extract. One unit of POD activity is defined as an increase of 0.01 in absorbance per minute at 460 nm. PPO (EC 1.10.3.1) activity was determined using the method of Tian et al. (2002). Flesh tissue (1 g) was homogenised with 5 mL of 0.2 M sodium phosphate buffer (pH 6.5). The homogenate was centrifugated at 10,000g for 20 min at 4 ◦ C. The reaction mixture contained 0.1 mL of enzyme extract and 2 mL of catechol. The increase in absorbance at 420 nm was recorded. One unit of PPO activity is defined as the amount of enzyme that caused an increase of 0.01 at 420 nm per minute. Protein content in the enzyme extracts was determined by the Bradford (1976) method, using bovine serum albumin as a standard. Specific activity of all the enzymes was expressed as units per milligram of protein. 2.5. Analysis of defense-related gene expression by RT-PCR In order to investigate the molecular mechanism of HA treatment induced disease resistance against blue mold decay in sweet cherry fruit, RT-PCR (Reverse transcription-polymerase chain reaction) was used to analyze the expression patterns of the defense-related genes (PaEXP, PaGLU, PaCAT, PaNPR1-like) in cherry fruit. Tissue samples from the HA or control fruit, with or without inoculation of P. expansum were collected at the same intervals (0, 1, 3 and 5d after treatment). Total RNA was extracted from tissue samples of cherry fruits according to the method of Chang et al. (1993). RT-PCR was performed using the PrimeScriptTM 16 1st Strand cDNA Synthesis Kit (TaKaRa, Japan). Short and conserved segments of PaEXP (GenBank ID: AF297522), PaGLU
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A
B
Fig. 1. Changes in disease incidence (A) and lesion diameter (B) of blue mold in sweet cherry fruit treated with HA and inoculated with P. expansum and incubated at 20 ◦ C. Each column represents the mean of triplicate samples. Vertical bars represent the standard errors of the means. Different letters above the bars indicate statistically significant differences at p < 0.05.
(GenBank ID: EF177488), PaCAT (GenBank ID: EF165590), PaNPR1like (GenBank ID: DQ146459) were cloned by degenerate primers. Independent PCR with 30 cycles was performed using aliquots (1 L) of cDNA samples, and a constitutively expressed gene Actin (GenBank ID: FJ560908) was used as a quantitative control in the RT-PCR analysis. The sequences of primers used for RT-PCR analysis were as follows: PaEXP forward: 5 - CTGGTGTTGTGCCTGTTG-3 , PaEXP reverse: 5 -CTGTAGGTCTGCCCGAAG3 ; PaGLU forward: 5 -GAAACGAAGTCAAGCCCTCA-3 , PaGLU reverse: 5 -GCGGCATAAACAGCATCCAA-3 ; PaCAT forward: 5 CACAAGATTACAGGCACAT-3 , PaCAT reverse: 5 -GAATAGTAGATACCAGGGACA-3 ; PaNPR1-like forward: 5 -TTTCGTTGTGGAGTTGAT-3 , PaNPR1-like reverse: 5 -TATTCGGTCTTTGTTTGC-3 ; PaACTIN forward: 5 -CAATGTGCCTGCCATGTATG-3 , PaACTIN reverse: 5 -CCAGCAGCTTCCATTCCAAT-3 . Relative mRNA levels of genes were analyzed based on densitometry values obtained using the Quality One software of Bio-Rad.
3. Results 3.1. Effect of HA treatment on disease incidence and lesion diameter of sweet cherry fruit inoculated with P. expansum As shown in Fig. 1, HA treatment significantly reduced disease incidence and lesion diameter in sweet cherry fruit inoculated with P. expansum. HA treatment reduced disease incidence and lesion diameter, respectively, by 11.0% and 32.9% on the 3rd day of inoculation compared with the control. Although all the inoculated wounds in both HA-treated and control fruit developed decay symptoms after five days of inoculation at 20 ◦ C, the lesion diameter in HA-treated fruit was still significantly (p < 0.05) smaller than that in control fruit (Fig. 1B).
3.2. Effect of HA treatment on CHI and GLU activities in sweet cherry fruit
2.6. Statistical analysis Experiments were performed using a completely randomized design. All statistical analyses were performed with SPSS 13.0 (SPSS Inc., Chicago, IL, USA). The data were analyzed by one-way analysis of variance (ANOVA), and mean separations were performed using Duncan’s multiple range tests. Differences at p < 0.05 were considered significant.
A
The activity of CHI increased gradually during storage and maintained at significantly (p < 0.05) higher levels in HA-treated fruit compared with the control (Fig. 2A). GLU activity increased sharply in both HA-treated and control fruit during the first three days of inoculation (Fig. 2B). The fruit treated with HA showed 19.6% higher activity of CHI and 26.6% higher activity of GLU than the control fruit after five days of inoculation.
B
Fig. 2. Effects of HA treatment on CHI (A) and GLU (B) activities in sweet cherry fruit inoculated with P. expansum and incubated at 20 ◦ C. Data are expressed as the mean ± SE of triplicate assays. Vertical bars represent the standard errors of the means. Asterisk (* ) indicates a significant difference at the level p < 0.05 between CK and HA.
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A
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B
Fig. 3. Effects of HA treatment on PG (A) and PME (B) activities in sweet cherry fruit inoculated with P. expansum and incubated at 20 ◦ C. Data are expressed as the mean ± SE of triplicate assays. Vertical bars represent the standard errors of the means. Asterisk (* ) indicates a significant difference at the level p < 0.05 between CK and HA.
3.3. Effect of HA treatment on PG and PME activities in sweet cherry fruit As shown in Fig. 3, the activities of PG and PME in both control and HA-treated cherries increased gradually with storage time. The increase in the activities of PG and PME was inhibited by HA treatment during the whole storage time. At the end of the storage time, the fruit treated with HA showed 14.5% lower activity of PG and 29.5% lower activity of PME than the control fruit. 3.4. Effect of HA treatment on activities of SOD, CAT and APX in sweet cherry fruit As shown in Fig. 4A, SOD activity increased sharply in the first day of incubation and decreased gradually afterwards. HA treatment induced significantly (p < 0.05) higher SOD activity compared with the control during the whole storage time. CAT activity in both control and HA-treated fruit decreased gradually with the storage
time. HA treatment retarded the decline of activity of CAT (Fig. 4B). APX activity decreased in both control and HA-treated fruit during the incubation time. HA treatment remarkably (p < 0.05) inhibited the activity of APX during the whole storage time (Fig. 4C).
3.5. Effect of HA treatment on POD and PPO activities in sweet cherry fruit POD activity in control fruit decreased gradually with the storage time. POD activity reached a peak value in HA-treated fruit on the 1st day and decreased during the remainder of incubation (Fig. 5A). PPO activity in both control and HA-treated fruit increased gradually with incubation time. HA treatment significantly (p < 0.05) enhanced the increase of PPO activity during the whole storage (Fig. 5B). The activities of POD and PPO were 23.8% and 20.5% higher in HA-treated fruit than those in control fruit after incubation at 20 ◦ C for five days, respectively.
A
B
C
Fig. 4. Effects of HA treatment on SOD (A), CAT (B) and APX (C) activities in sweet cherry fruit inoculated with P. expansum and incubated at 20 ◦ C. Data are expressed as the mean ± SE of triplicate assays. Vertical bars represent the standard errors of the means. Asterisk (* ) indicates a significant difference at the level p < 0.05 between CK and HA.
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A
B
Fig. 5. Effects of HA treatment on POD (A) and PPO (B) activities in sweet cherry fruit inoculated with P. expansum and incubated at 20 ◦ C. Data are expressed as the mean ± SE of triplicate assays. Vertical bars represent the standard errors of the means. Asterisk (* ) indicates a significant difference at the level p < 0.05 between CK and HA.
3.6. Effect of HA treatment and P. expansum inoculation on defense-related genes expression in sweet cherry fruit As shown in Fig. 6, the expression of PaEXP was up-regulated in fruit inoculated with P. expansum and down-regulated in HA-treated fruit. Expressions of PaGLU, PaCAT, PaNPR1-like were retained at very low level in the fruit treated only with distilled water (Mock) or P. expansum, however, their expression was significantly enhanced in HA-treated fruit. In fruit treated with HA and inoculated with P. expansum, transcripts of PaGLU, PaCAT and PaNPR1-like were significantly enhanced and attained at higher level compared with those treated with HA alone, suggesting that HA treatment induced stronger expression of the defensive genes in cherry fruit challenged by P. expansum. 4. Discussion Heat treatment has been widely used to prevent fungal decay, delay ripening, increase tolerance to chilling injury and extend postharvest life of fruits and vegetables (Jin et al., 2014; Paull and
A
C
Chen, 2000). In the present study, we found that HA treatment significantly reduced blue mold decay incidence and lesion diameter of cherry fruit inoculated with P. expansum. These results suggest that HA treatment, as a physical method, enhanced disease resistance of sweet cherry fruit. Induced disease resistance in harvested horticultural crops using physical, biological and/or chemical elicitors is considered as a preferred strategy for disease management (Durrant and Dong, 2004; Feliziani et al., 2013; Wang et al., 2013a,b; Yu et al., 2014). Induced resistance has been inferred to be one of the major mechanisms of heat treatment in inhibiting postharvest diseases of horticultural crops (Fallik, 2004). The disease resistance in postharvest fruit is associated with some inducible compounds including pathogenesis-related (PR) proteins (Sels et al., 2008). Among PR proteins, CHI and GLU play an important role in disease resistance of fruits. CHI has been proved to degrade chitin, which is the major component of pathogen cell walls. GLU can act indirectly by releasing oligosaccharide and eliciting defense reactions and then act synergistically with CHI to inhibit fungal growth (Sels et al., 2008). Induction of these two defensive enzymes by HA treatment was
B
D
Fig. 6. Effect of HA treatment and P. expansum on expression of representative defense related genes in sweet cherry fruit and incubated at 20 ◦ C. RT-PCR was carried out using actin as a standard. Data are expressed as the mean ± SE of triplicate assays. Vertical bars represent the standard errors of the means.
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observed in grapefruit and Chinese bayberry, which was correlated to the enhanced disease resistance and reduced disease severity (Pavoncello et al., 2001; Wang et al., 2010). In the present study, it was found that HA treatment significantly induced activities of CHI and GLU and inhibited fruit decay in sweet cherry fruit. These results imply that the induced disease resistance is involved in the mechanisms by which HA treatment reduced blue mold decay in cherry fruit. The softening of fruit will facilitate the infection of pathogen and increase postharvest decay (Wei et al., 2010). It has been reported that suppression of tomato fruit softening reduced susceptibility to Botrytis cinerea infection (Cantu et al., 2008). The softening process is correlated with the action of a number of cell wall hydrolytic enzymes. PG and PME are two major enzymes that act on the pectin fraction of the cell wall (Li et al., 2010). The decrease of softening rate after heat treatment may be due to inhibition of the synthesis of cell wall hydrolytic enzymes in various fruits (Lurie, 1998). Martínez and Civello (2008) found that PG activity was inhibited by HA treatment at 45 ◦ C for 3 h in strawberry fruit. In the present work, activities of PG and PME were significantly lower in HAtreated fruit than those in control fruit. This indicates that reduced activities of PG and PME helped to ward off the spread of pathogens, thus playing positive roles in enhancing disease resistance. The accumulation of reactive oxygen species (ROS) has the potential to serve not only as barriers against invading pathogen, but as important physiological regulators of intracellular signaling pathways that activate further plant defense reactions (Finkel, 2011; Lamb and Dixon, 1997). The metabolism of ROS is controlled by an array of enzymes including SOD, CAT and APX. SOD catalyzes the dismutation reaction of O2 − into H2 O2 and O2 . CAT and APX convert H2 O2 to O2 and H2 O and, therefore, limit the potential for further free radical production from H2 O2 (Mittler, 2002). In loquat fruit inoculated with Colletotrichum acutatum, methyl jasmonate treatment enhanced the activities of SOD, CAT and APX, which are considered to play important roles in enhancing disease resistance (Cao et al., 2008). The previous studies provided evidence that ROS accumulation had a close relationship with decreased fruit susceptibility to decay after harvest (Cao et al., 2008; Wang et al., 2013a). Wang et al. (2013b) also reported that increased activities of SOD and CAT resulted in decreased Rhizopus rot in peach fruit treated with the biocontrol agent Bacillus cereus AR156. In this work, HA treatment maintained higher activities of SOD and CAT, but lower APX activity, thus resulting in reduced blue mold decay in sweet cherry fruit. This is consistent with our previous findings in Chinese bayberry (Jin et al., 2012). POD and PPO are considered key enzymes in host defense reactions against pathogen infections. POD participates in numerous physiological processes such as lignification and suberization against attempted fungal invasion (Passardi et al., 2004). PPO has been shown to be responsible for the oxidation of phenolics into anti-microbial quinones in plant cells attacked by phytopathogens (Mohammadi and Kazemi, 2002). Liu et al. (2005) reported that BTH treatment significantly increased the activities of PPO and POD, which might account for higher disease resistance in peach fruit. Our data showed that HA treatment significantly increased the activities of POD and PPO. Similar results had been found in HA-treated Chinese bayberry (Wang et al., 2010). These results suggested that the enhanced activities of POD and PPO could be one part of mechanism of HA treatment in inducing disease resistance in sweet cherry fruit. NPR1 is a key regulator in the signal transduction pathway that leads to induced resistance and increased the expression of PR genes (Kinkema et al., 2000). Expansins (EXP) are cell wall proteins that have been proposed to play an important role in fruit softening (Brummell et al., 1999). An up-regulated expression of PpCAT, PpGLU and PpNPR1-like was observed in peach fruit treated
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with Bacillus subtilis, which resulted in enhanced level of induced resistance of peach fruit against Rhizopus rot caused by Rhizopus stolonifer (Wang et al., 2013b). In strawberry fruit, HA treatment down-regulated the expression of FaEXP and delayed the fruit softening (Dotto et al., 2011). As an indicator of defense expression, the transcription of defense related genes measured in this study showed that the expression of defensive genes was markedly increased after HA treatment. This clearly suggests that HA treatment induced disease resistance against blue mold decay in cherry fruit is associated with direct induction of defense responses, no matter whether the fruit were inoculated with P. expansum or not. Moreover, when fruit were inoculated with P. expansum, HA treatment induced stronger expression of the defensive genes in cherry fruit. In conclusion, the present study demonstrates that HA treatment at 44 ◦ C for 114 min is effective in inducing disease resistance and suppressing blue mold decay caused by P. expansum in harvested sweet cherry fruit. Moreover, this HA-induced disease resistance against P. expansum is associated with the increase of defense-related enzymes activities and defense-related genes expression. The results of this study revealed that HA treatment directly induced the expression of defensive genes, even though the fruit is not infected by phytopathogen. Meanwhile, HA treatment was effective in delaying fruit tissue softening that helps to ward off the spread of pathogens. HA treatment is a promising method for controlling blue mold decay in sweet cherry during postharvest storage and transportation. Acknowledgments This study was supported by the National Natural Science Foundation of China (no. 31172003 and 31301565), the Natural Science Foundation of Shandong Province (ZR2012CQ009) and Postgraduate Innovation Project of Jiangsu Province (CXLX13 266). References Abeles, F.B., Bosshart, R.P., Forrence, L.E., Habig, W.H., 1971. Preparation and purification of glucanase and chitinase from bean leaves. Plant Physiol. 47, 129–134. Al-Haq, M.I., Seo, Y., Oshita, S., Kawagoe, Y., 2002. Disinfection effects of electrolyzed oxidizing water on suppressing fruit rot of pear caused by Botryosphaeria berengeriana. Food Res. Int. 35, 657–664. Amako, K., Chen, G.X., Asada, K., 1994. Separate assay specific for ascorbate peroxidase and guaiacol peroxidase and for the chloroplastic and cytosolic isozymes of ascorbate peroxidase in plants. Plant Cell Physiol. 35, 497–504. 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. Brummell, D.A., Harpster, M.H., Civello, P.M., Palys, J.M., Bennett, A.B., Dunsmuir, P., 1999. Modification of expansin protein abundance in tomato fruit alters softening and cell wall polymer metabolism during ripening. Plant Cell 11, 2203–2216. Cantu, D., Vicente, A.R., Greve, L.C., Dewey, F.M., Bennett, A.B., Labavitch, J.M., Powell, A.L.T., 2008. The intersection between cell wall disassembly, ripening, and fruit susceptibility to Botrytis cinerea. Proc. Natl. Acad. Sci. U.S.A. 105, 859–864. Cao, S.F., Zheng, Y.H., Yang, Z.F., Tang, S.S., Jin, P., Wang, K.T., Wang, X.M., 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. Cao, S.F., Zheng, Y.H., Wang, K.T., Rui, H.J., Tang, S.S., 2010. Effect of methyl jasmonate on cell wall modification of loquat fruit in relation to chilling injury after harvest. Food Chem. 118, 641–647. Ceponis, M.J., Cappellini, R.A., Lightner, G.W., 1987. Disorders in sweet cherry and strawberry shipments to the New York market 1972–1984. Plant Dis. 71, 472–475. Chance, B., Maehly, A.C., 1955. Assay of catalases and peroxidase. Method Enzymol. 2, 764–775. Chang, S.J., Puryear, J., John, C., 1993. A simple and efficient method for isolating RNA from pine trees. Plant Mol. Biol. Rep. 11, 113–116. Conway, W.S., Leverentz, B., Janisiewicz, W.J., Blodgett, A.B., Saftner, R.A., Camp, M.J., 2004. Integrating heat treatment, biocontrol and sodium bicarbonate to reduce postharvest decay of apple caused by Colletotrichum acutatum and Penicillium expansum. Postharvest Biol. Technol. 34, 11–20. Dotto, M.C., Pombo, M.A., Martínez, G.A., Civello, P.M., 2011. Heat treatments and expansin gene expression in strawberry fruit. Sci. Hortic. 130, 775–780.
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Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Hot air treatment induces resistance against blue mold decay caused by Penicillium expansum in sweet cherry (Prunus cerasus L.) fruit Lei Wang a , Peng Jin a , Jing Wang a , Hansheng Gong b , Shurong Zhang b , Yonghua Zheng a,∗ a b
College of Food Science and Technology, Nanjing Agricultural University, Nanjing, 210095, People’s Republic of China College of Food Engineering, Ludong University, Yantai, 264025, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 28 November 2014 Received in revised form 24 March 2015 Accepted 31 March 2015 Keywords: Prunus avium Hot air Induced resistance Postharvest disease Defense related genes
a b s t r a c t Effect of hot air (HA) treatment (44 ◦ C, 114 min) on reducing blue mold decay caused by Penicillium expansum in sweet cherry fruit was investigated. The results indicated that fruit treated with HA had significantly lower disease incidence and smaller lesion diameter than the control fruit did. HA treatment significantly enhanced activities of chitinase (CHI) and -1, 3-glucanase (GLU). The activities of polygalacturonase (PG) and pectinmethylesterase (PME) were significantly inhibited by HA treatment. Fruit treated with HA exhibited remarkably higher activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and polyphenoloxidase (PPO) and lower activity of ascorbate peroxidase (APX) than control. Expression of defense related genes such as PaGLU, PaCAT, and PaNPR1-like was greatly induced in HA-treated fruit during storage, while the expression of expansins (EXP) was down-regulated by HA treatment. These results suggest that HA can effectively inhibit blue mold decay caused by P. expansum in harvested cherry fruit possibly by directly inducing disease resistance and delaying fruit tissue softening that helps to ward off the spread of pathogens. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Sweet cherry (Prunus avium L.) is highly susceptible to pathogenic infection causing decay and large postharvest loss (AlHaq et al., 2002; Feliziani et al., 2013). Blue mold decay caused by Penicillium expansum Link is one of the most important postharvest diseases in sweet cherry fruit (Ceponis et al., 1987). Traditionally, the control of postharvest diseases of fruits relies mainly on the use of synthetic fungicides. However, the intense use of synthetic fungicides has led to the increasing resistance of fungal pathogens and growing concern of consumers over chemical residues (Janisiewicz and Korsten, 2002; Yu et al., 2014). Therefore, it is crucial to study eco-friendly alternative strategies to inhibit postharvest fungal decay (Droby et al., 2009). Heat treatments, including hot air (HA) treatment, hot water dipping and hot water vapor, which are more feasible for commercial application to reduce fruit decay, delay ripening and maintain quality (Jin et al., 2014; Lurie, 1998). Among the three methods, hot air treatment has received much attention and has been widely used to control fruit decay and maintain quality. It has been
∗ Corresponding author. Tel.:+86 25 8439 9080; fax: +86 25 8439 5618. E-mail address: [email protected] (Y. Zheng). http://dx.doi.org/10.1016/j.scienta.2015.03.039 0304-4238/© 2015 Elsevier B.V. All rights reserved.
reported that hot air treatment was applied effectively to reduce postharvest decay in various fruits including apple, peach and Chinese bayberry fruit (Conway et al., 2004; Wang et al., 2010; Zhang et al., 2007). In addition, HA treatment has been shown not only to limit spore germination of pathogens in vitro (Schirra et al., 2002; Zhang et al., 2007), but also induce resistance of fruit against fungal infection (Liu et al., 2010; Wang et al., 2010), thereby inhibiting decay incidence in postharvest fruit. However, there is no information concerning the effect of HA on controlling blue mold decay in sweet cherry fruit. The objective of this study was to evaluate the effect of HA treatment on controlling blue mold decay caused by P. expansum in sweet cherry fruit and to explore the possible mechanisms involved. 2. Materials and methods 2.1. Fungal pathogen The pathogen P. expansum was isolated from infected sweet cherry fruit and cultured on potato dextrose agar (PDA) medium (containing the extract of 200 g boiled potatoes, 20 g dextrose and 20 g agar in 1000 mL of distilled water). Spore suspensions were prepared from 2-week-old PDA cultures. The spores were removed
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from the surface of the cultures and suspended in 5 mL of sterile distilled water containing 0.05% (v/v) Tween 80. The number of spores was determined with a hemocytometer counting chamber, and then the spore concentration was adjusted to 5 × 104 spores mL−1 with sterile distilled water. 2.2. Fruit material Sweet cherry (Prunus avium L. cv. Hongdeng) fruits were handharvested at commercial maturity stage with healthy greenish stems. The fruits were selected for uniformity of size, ripeness, and absence of physical injuries or infections. The fruits were randomly divided into two groups of 300 fruit each. The first group was treated with HA at 44 ◦ C for 114 min (80–90% relative humidity). The second group stored at 20 ◦ C did not receive any treatment and served as control. The treatment condition in present study was chosen on the basis of our preliminary research. All fruits were airdried for approximately 1 h and surface-sterilized with 75% ethanol prior to wounding. 2.3. Pathogen inoculation and fruit tissue sampling Sweet cherries were wounded (3 mm deep and 3 mm wide) with a sterile nail at the equator of each fruit. Aliquots (15 L) of a suspension of 5 × 104 spores mL−1 P. expansum or distilled water (as control) was pipetted into each wound. The fruit were incubated at 20 ◦ C and 95% relative humidity for five days. Disease incidence and lesion diameter on each fruit wound were recorded at 1, 3 and 5 days after inoculation. There were three replicate trials of 30 fruits per treatment with complete randomization. The experiment was conducted twice. Fruit samples were taken before inoculation (time 0) and at 1, 3 and 5 days after inoculation for enzyme assays and expression of defense-related genes. Each treatment was replicated three times, and the experiment was conducted twice. 2.4. Assay of enzyme activity Chitinase (CHI, EC 3.2.1.14) and -1, 3-glucanase (GLU, EC 3.2.1.58) were extracted from 1 g of tissue sample with 5 mL of 50 mM sodium acetate buffer (pH 5.0). The homogenate was centrifugated at 12,000g for 20 min at 4 ◦ C and the supernatant was used to determine the CHI and GLU activity. CHI activity was determined according to the method of Abeles et al. (1971). CHI activity was measured by the release of N-acetylD-glucosamine (NAG) from colloidal chitin (Sigma Chemical Co., Ltd) with 4-dimethylaminobenzaldehyde method. 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 (GLU) (EC 3.2.1.58) activity was measured according to the method of Abeles et al. (1971). Aliquots (1 mL) of enzyme preparation were incubated for 1 h at 37 ◦ C with 1 mL of 4% laminarin (Aldrich, Chemical Co., Milwaukee, WI, USA). The reaction was terminated by heating the sample in boiling water for 5 min and the amount of reducing sugar was measured spectrophotometrically at 540 nm after reaction with 250 L 3, 5-dinitrosalicylic reagent. One unit is defined as the amount of enzyme catalyzing the formation of 1 mol glucose equivalents in 1 h. PG and PME were extracted from 1 g of tissue sample at 4 ◦ C with 5 mL of 40 mM sodium acetate buffer (pH 5.2) containing 100 mM NaCl, 2% (v/v) mercaptoethanol, and 5% (w/v) polyvinyl polypyrrolidone. The extracts were then homogenised and centrifuged at 25,000g for 20 min at 4 ◦ C. The supernatants were used to determine the PG and PME activity.
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PG (EC 3.2.1.15) activity was determined by the method of Zhou et al. (2011). The reaction mixture contained 40 mM sodium acetate buffer (pH 5.2), 0.1% (w/v) polygalacturonic acid and 100 L enzyme extract. One unit of activity was defined as 1 M galacturonic acid released per mg of protein per hour. PME (EC 3.1.1.11) activity was evaluated by the method of Cao et al. (2010). The reaction mixture consisted of 1 mL of enzyme extract and 10 mL 1% (w/v) citrus pectin (Sigma Chemical Co., Ltd). One unit of enzyme activity was calculated as 1 mM NaOH consumed per mg of protein per hour. SOD (EC 1.15.1.1) was extracted from1 g of flesh tissue with 5 mL of 50 mM sodium phosphate buffer (pH 7.8) at 4 ◦ C. The homogenate was centrifuged at 10,000g for 20 min at 4 ◦ C. The supernatant was used to estimate SOD activity by the method of Rao et al. (1996) in a final volume of 3 mL, which contained 0.1 mL of crude enzyme extract. One unit of SOD activity is defined as the amount of enzyme that causes 50% inhibition of nitroblue tetrazolium. CAT (EC 1.11.1.6) and APX (EC 1.11.1.11) were extracted from 1 g of tissue sample with 5 mL of 50 mM sodium phosphate buffer (pH 7.0). The homogenate was centrifugated at 12,000g for 20 min at 4 ◦ C and the supernatant was used to determine the CAT and APX activity. CAT (EC 1.11.1.6) activity was determined by the method of Chance and Maehly (1955). CAT activity was determined by adding 0.2 mL of the enzyme preparation to 3 mL of sodium phosphate buffer containing 0.2 mL H2 O2 as a substrate. One unit of CAT activity was designated as the amount of enzyme that decomposes 1 mol of H2 O2 per minute at 30 ◦ C. APX (EC 1.11.1.11) activity was measured according to the method of Amako et al. (1994). One unit of APX was defined as the amount of enzyme that oxidised 1 mol ascorbate per min. POD (EC 1.11.1.7) activity was assayed according to the method of Kochba et al. (1977). POD was extracted from 1 g of tissue sample with 5 mL of 50 mM sodium phosphate buffer (pH 8.7). The reaction mixture contained 50 mM sodium phosphate buffer (pH 8.7), 0.75% H2 O2 , 20 mM guaiacol and 0.2 mL crude enzyme extract. One unit of POD activity is defined as an increase of 0.01 in absorbance per minute at 460 nm. PPO (EC 1.10.3.1) activity was determined using the method of Tian et al. (2002). Flesh tissue (1 g) was homogenised with 5 mL of 0.2 M sodium phosphate buffer (pH 6.5). The homogenate was centrifugated at 10,000g for 20 min at 4 ◦ C. The reaction mixture contained 0.1 mL of enzyme extract and 2 mL of catechol. The increase in absorbance at 420 nm was recorded. One unit of PPO activity is defined as the amount of enzyme that caused an increase of 0.01 at 420 nm per minute. Protein content in the enzyme extracts was determined by the Bradford (1976) method, using bovine serum albumin as a standard. Specific activity of all the enzymes was expressed as units per milligram of protein. 2.5. Analysis of defense-related gene expression by RT-PCR In order to investigate the molecular mechanism of HA treatment induced disease resistance against blue mold decay in sweet cherry fruit, RT-PCR (Reverse transcription-polymerase chain reaction) was used to analyze the expression patterns of the defense-related genes (PaEXP, PaGLU, PaCAT, PaNPR1-like) in cherry fruit. Tissue samples from the HA or control fruit, with or without inoculation of P. expansum were collected at the same intervals (0, 1, 3 and 5d after treatment). Total RNA was extracted from tissue samples of cherry fruits according to the method of Chang et al. (1993). RT-PCR was performed using the PrimeScriptTM 16 1st Strand cDNA Synthesis Kit (TaKaRa, Japan). Short and conserved segments of PaEXP (GenBank ID: AF297522), PaGLU
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A
B
Fig. 1. Changes in disease incidence (A) and lesion diameter (B) of blue mold in sweet cherry fruit treated with HA and inoculated with P. expansum and incubated at 20 ◦ C. Each column represents the mean of triplicate samples. Vertical bars represent the standard errors of the means. Different letters above the bars indicate statistically significant differences at p < 0.05.
(GenBank ID: EF177488), PaCAT (GenBank ID: EF165590), PaNPR1like (GenBank ID: DQ146459) were cloned by degenerate primers. Independent PCR with 30 cycles was performed using aliquots (1 L) of cDNA samples, and a constitutively expressed gene Actin (GenBank ID: FJ560908) was used as a quantitative control in the RT-PCR analysis. The sequences of primers used for RT-PCR analysis were as follows: PaEXP forward: 5 - CTGGTGTTGTGCCTGTTG-3 , PaEXP reverse: 5 -CTGTAGGTCTGCCCGAAG3 ; PaGLU forward: 5 -GAAACGAAGTCAAGCCCTCA-3 , PaGLU reverse: 5 -GCGGCATAAACAGCATCCAA-3 ; PaCAT forward: 5 CACAAGATTACAGGCACAT-3 , PaCAT reverse: 5 -GAATAGTAGATACCAGGGACA-3 ; PaNPR1-like forward: 5 -TTTCGTTGTGGAGTTGAT-3 , PaNPR1-like reverse: 5 -TATTCGGTCTTTGTTTGC-3 ; PaACTIN forward: 5 -CAATGTGCCTGCCATGTATG-3 , PaACTIN reverse: 5 -CCAGCAGCTTCCATTCCAAT-3 . Relative mRNA levels of genes were analyzed based on densitometry values obtained using the Quality One software of Bio-Rad.
3. Results 3.1. Effect of HA treatment on disease incidence and lesion diameter of sweet cherry fruit inoculated with P. expansum As shown in Fig. 1, HA treatment significantly reduced disease incidence and lesion diameter in sweet cherry fruit inoculated with P. expansum. HA treatment reduced disease incidence and lesion diameter, respectively, by 11.0% and 32.9% on the 3rd day of inoculation compared with the control. Although all the inoculated wounds in both HA-treated and control fruit developed decay symptoms after five days of inoculation at 20 ◦ C, the lesion diameter in HA-treated fruit was still significantly (p < 0.05) smaller than that in control fruit (Fig. 1B).
3.2. Effect of HA treatment on CHI and GLU activities in sweet cherry fruit
2.6. Statistical analysis Experiments were performed using a completely randomized design. All statistical analyses were performed with SPSS 13.0 (SPSS Inc., Chicago, IL, USA). The data were analyzed by one-way analysis of variance (ANOVA), and mean separations were performed using Duncan’s multiple range tests. Differences at p < 0.05 were considered significant.
A
The activity of CHI increased gradually during storage and maintained at significantly (p < 0.05) higher levels in HA-treated fruit compared with the control (Fig. 2A). GLU activity increased sharply in both HA-treated and control fruit during the first three days of inoculation (Fig. 2B). The fruit treated with HA showed 19.6% higher activity of CHI and 26.6% higher activity of GLU than the control fruit after five days of inoculation.
B
Fig. 2. Effects of HA treatment on CHI (A) and GLU (B) activities in sweet cherry fruit inoculated with P. expansum and incubated at 20 ◦ C. Data are expressed as the mean ± SE of triplicate assays. Vertical bars represent the standard errors of the means. Asterisk (* ) indicates a significant difference at the level p < 0.05 between CK and HA.
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A
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B
Fig. 3. Effects of HA treatment on PG (A) and PME (B) activities in sweet cherry fruit inoculated with P. expansum and incubated at 20 ◦ C. Data are expressed as the mean ± SE of triplicate assays. Vertical bars represent the standard errors of the means. Asterisk (* ) indicates a significant difference at the level p < 0.05 between CK and HA.
3.3. Effect of HA treatment on PG and PME activities in sweet cherry fruit As shown in Fig. 3, the activities of PG and PME in both control and HA-treated cherries increased gradually with storage time. The increase in the activities of PG and PME was inhibited by HA treatment during the whole storage time. At the end of the storage time, the fruit treated with HA showed 14.5% lower activity of PG and 29.5% lower activity of PME than the control fruit. 3.4. Effect of HA treatment on activities of SOD, CAT and APX in sweet cherry fruit As shown in Fig. 4A, SOD activity increased sharply in the first day of incubation and decreased gradually afterwards. HA treatment induced significantly (p < 0.05) higher SOD activity compared with the control during the whole storage time. CAT activity in both control and HA-treated fruit decreased gradually with the storage
time. HA treatment retarded the decline of activity of CAT (Fig. 4B). APX activity decreased in both control and HA-treated fruit during the incubation time. HA treatment remarkably (p < 0.05) inhibited the activity of APX during the whole storage time (Fig. 4C).
3.5. Effect of HA treatment on POD and PPO activities in sweet cherry fruit POD activity in control fruit decreased gradually with the storage time. POD activity reached a peak value in HA-treated fruit on the 1st day and decreased during the remainder of incubation (Fig. 5A). PPO activity in both control and HA-treated fruit increased gradually with incubation time. HA treatment significantly (p < 0.05) enhanced the increase of PPO activity during the whole storage (Fig. 5B). The activities of POD and PPO were 23.8% and 20.5% higher in HA-treated fruit than those in control fruit after incubation at 20 ◦ C for five days, respectively.
A
B
C
Fig. 4. Effects of HA treatment on SOD (A), CAT (B) and APX (C) activities in sweet cherry fruit inoculated with P. expansum and incubated at 20 ◦ C. Data are expressed as the mean ± SE of triplicate assays. Vertical bars represent the standard errors of the means. Asterisk (* ) indicates a significant difference at the level p < 0.05 between CK and HA.
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A
B
Fig. 5. Effects of HA treatment on POD (A) and PPO (B) activities in sweet cherry fruit inoculated with P. expansum and incubated at 20 ◦ C. Data are expressed as the mean ± SE of triplicate assays. Vertical bars represent the standard errors of the means. Asterisk (* ) indicates a significant difference at the level p < 0.05 between CK and HA.
3.6. Effect of HA treatment and P. expansum inoculation on defense-related genes expression in sweet cherry fruit As shown in Fig. 6, the expression of PaEXP was up-regulated in fruit inoculated with P. expansum and down-regulated in HA-treated fruit. Expressions of PaGLU, PaCAT, PaNPR1-like were retained at very low level in the fruit treated only with distilled water (Mock) or P. expansum, however, their expression was significantly enhanced in HA-treated fruit. In fruit treated with HA and inoculated with P. expansum, transcripts of PaGLU, PaCAT and PaNPR1-like were significantly enhanced and attained at higher level compared with those treated with HA alone, suggesting that HA treatment induced stronger expression of the defensive genes in cherry fruit challenged by P. expansum. 4. Discussion Heat treatment has been widely used to prevent fungal decay, delay ripening, increase tolerance to chilling injury and extend postharvest life of fruits and vegetables (Jin et al., 2014; Paull and
A
C
Chen, 2000). In the present study, we found that HA treatment significantly reduced blue mold decay incidence and lesion diameter of cherry fruit inoculated with P. expansum. These results suggest that HA treatment, as a physical method, enhanced disease resistance of sweet cherry fruit. Induced disease resistance in harvested horticultural crops using physical, biological and/or chemical elicitors is considered as a preferred strategy for disease management (Durrant and Dong, 2004; Feliziani et al., 2013; Wang et al., 2013a,b; Yu et al., 2014). Induced resistance has been inferred to be one of the major mechanisms of heat treatment in inhibiting postharvest diseases of horticultural crops (Fallik, 2004). The disease resistance in postharvest fruit is associated with some inducible compounds including pathogenesis-related (PR) proteins (Sels et al., 2008). Among PR proteins, CHI and GLU play an important role in disease resistance of fruits. CHI has been proved to degrade chitin, which is the major component of pathogen cell walls. GLU can act indirectly by releasing oligosaccharide and eliciting defense reactions and then act synergistically with CHI to inhibit fungal growth (Sels et al., 2008). Induction of these two defensive enzymes by HA treatment was
B
D
Fig. 6. Effect of HA treatment and P. expansum on expression of representative defense related genes in sweet cherry fruit and incubated at 20 ◦ C. RT-PCR was carried out using actin as a standard. Data are expressed as the mean ± SE of triplicate assays. Vertical bars represent the standard errors of the means.
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observed in grapefruit and Chinese bayberry, which was correlated to the enhanced disease resistance and reduced disease severity (Pavoncello et al., 2001; Wang et al., 2010). In the present study, it was found that HA treatment significantly induced activities of CHI and GLU and inhibited fruit decay in sweet cherry fruit. These results imply that the induced disease resistance is involved in the mechanisms by which HA treatment reduced blue mold decay in cherry fruit. The softening of fruit will facilitate the infection of pathogen and increase postharvest decay (Wei et al., 2010). It has been reported that suppression of tomato fruit softening reduced susceptibility to Botrytis cinerea infection (Cantu et al., 2008). The softening process is correlated with the action of a number of cell wall hydrolytic enzymes. PG and PME are two major enzymes that act on the pectin fraction of the cell wall (Li et al., 2010). The decrease of softening rate after heat treatment may be due to inhibition of the synthesis of cell wall hydrolytic enzymes in various fruits (Lurie, 1998). Martínez and Civello (2008) found that PG activity was inhibited by HA treatment at 45 ◦ C for 3 h in strawberry fruit. In the present work, activities of PG and PME were significantly lower in HAtreated fruit than those in control fruit. This indicates that reduced activities of PG and PME helped to ward off the spread of pathogens, thus playing positive roles in enhancing disease resistance. The accumulation of reactive oxygen species (ROS) has the potential to serve not only as barriers against invading pathogen, but as important physiological regulators of intracellular signaling pathways that activate further plant defense reactions (Finkel, 2011; Lamb and Dixon, 1997). The metabolism of ROS is controlled by an array of enzymes including SOD, CAT and APX. SOD catalyzes the dismutation reaction of O2 − into H2 O2 and O2 . CAT and APX convert H2 O2 to O2 and H2 O and, therefore, limit the potential for further free radical production from H2 O2 (Mittler, 2002). In loquat fruit inoculated with Colletotrichum acutatum, methyl jasmonate treatment enhanced the activities of SOD, CAT and APX, which are considered to play important roles in enhancing disease resistance (Cao et al., 2008). The previous studies provided evidence that ROS accumulation had a close relationship with decreased fruit susceptibility to decay after harvest (Cao et al., 2008; Wang et al., 2013a). Wang et al. (2013b) also reported that increased activities of SOD and CAT resulted in decreased Rhizopus rot in peach fruit treated with the biocontrol agent Bacillus cereus AR156. In this work, HA treatment maintained higher activities of SOD and CAT, but lower APX activity, thus resulting in reduced blue mold decay in sweet cherry fruit. This is consistent with our previous findings in Chinese bayberry (Jin et al., 2012). POD and PPO are considered key enzymes in host defense reactions against pathogen infections. POD participates in numerous physiological processes such as lignification and suberization against attempted fungal invasion (Passardi et al., 2004). PPO has been shown to be responsible for the oxidation of phenolics into anti-microbial quinones in plant cells attacked by phytopathogens (Mohammadi and Kazemi, 2002). Liu et al. (2005) reported that BTH treatment significantly increased the activities of PPO and POD, which might account for higher disease resistance in peach fruit. Our data showed that HA treatment significantly increased the activities of POD and PPO. Similar results had been found in HA-treated Chinese bayberry (Wang et al., 2010). These results suggested that the enhanced activities of POD and PPO could be one part of mechanism of HA treatment in inducing disease resistance in sweet cherry fruit. NPR1 is a key regulator in the signal transduction pathway that leads to induced resistance and increased the expression of PR genes (Kinkema et al., 2000). Expansins (EXP) are cell wall proteins that have been proposed to play an important role in fruit softening (Brummell et al., 1999). An up-regulated expression of PpCAT, PpGLU and PpNPR1-like was observed in peach fruit treated
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with Bacillus subtilis, which resulted in enhanced level of induced resistance of peach fruit against Rhizopus rot caused by Rhizopus stolonifer (Wang et al., 2013b). In strawberry fruit, HA treatment down-regulated the expression of FaEXP and delayed the fruit softening (Dotto et al., 2011). As an indicator of defense expression, the transcription of defense related genes measured in this study showed that the expression of defensive genes was markedly increased after HA treatment. This clearly suggests that HA treatment induced disease resistance against blue mold decay in cherry fruit is associated with direct induction of defense responses, no matter whether the fruit were inoculated with P. expansum or not. Moreover, when fruit were inoculated with P. expansum, HA treatment induced stronger expression of the defensive genes in cherry fruit. In conclusion, the present study demonstrates that HA treatment at 44 ◦ C for 114 min is effective in inducing disease resistance and suppressing blue mold decay caused by P. expansum in harvested sweet cherry fruit. Moreover, this HA-induced disease resistance against P. expansum is associated with the increase of defense-related enzymes activities and defense-related genes expression. The results of this study revealed that HA treatment directly induced the expression of defensive genes, even though the fruit is not infected by phytopathogen. Meanwhile, HA treatment was effective in delaying fruit tissue softening that helps to ward off the spread of pathogens. HA treatment is a promising method for controlling blue mold decay in sweet cherry during postharvest storage and transportation. Acknowledgments This study was supported by the National Natural Science Foundation of China (no. 31172003 and 31301565), the Natural Science Foundation of Shandong Province (ZR2012CQ009) and Postgraduate Innovation Project of Jiangsu Province (CXLX13 266). References Abeles, F.B., Bosshart, R.P., Forrence, L.E., Habig, W.H., 1971. Preparation and purification of glucanase and chitinase from bean leaves. Plant Physiol. 47, 129–134. Al-Haq, M.I., Seo, Y., Oshita, S., Kawagoe, Y., 2002. Disinfection effects of electrolyzed oxidizing water on suppressing fruit rot of pear caused by Botryosphaeria berengeriana. Food Res. Int. 35, 657–664. Amako, K., Chen, G.X., Asada, K., 1994. Separate assay specific for ascorbate peroxidase and guaiacol peroxidase and for the chloroplastic and cytosolic isozymes of ascorbate peroxidase in plants. Plant Cell Physiol. 35, 497–504. 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. Brummell, D.A., Harpster, M.H., Civello, P.M., Palys, J.M., Bennett, A.B., Dunsmuir, P., 1999. Modification of expansin protein abundance in tomato fruit alters softening and cell wall polymer metabolism during ripening. Plant Cell 11, 2203–2216. Cantu, D., Vicente, A.R., Greve, L.C., Dewey, F.M., Bennett, A.B., Labavitch, J.M., Powell, A.L.T., 2008. The intersection between cell wall disassembly, ripening, and fruit susceptibility to Botrytis cinerea. Proc. Natl. Acad. Sci. U.S.A. 105, 859–864. Cao, S.F., Zheng, Y.H., Yang, Z.F., Tang, S.S., Jin, P., Wang, K.T., Wang, X.M., 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. Cao, S.F., Zheng, Y.H., Wang, K.T., Rui, H.J., Tang, S.S., 2010. Effect of methyl jasmonate on cell wall modification of loquat fruit in relation to chilling injury after harvest. Food Chem. 118, 641–647. Ceponis, M.J., Cappellini, R.A., Lightner, G.W., 1987. Disorders in sweet cherry and strawberry shipments to the New York market 1972–1984. Plant Dis. 71, 472–475. Chance, B., Maehly, A.C., 1955. Assay of catalases and peroxidase. Method Enzymol. 2, 764–775. Chang, S.J., Puryear, J., John, C., 1993. A simple and efficient method for isolating RNA from pine trees. Plant Mol. Biol. Rep. 11, 113–116. Conway, W.S., Leverentz, B., Janisiewicz, W.J., Blodgett, A.B., Saftner, R.A., Camp, M.J., 2004. Integrating heat treatment, biocontrol and sodium bicarbonate to reduce postharvest decay of apple caused by Colletotrichum acutatum and Penicillium expansum. Postharvest Biol. Technol. 34, 11–20. Dotto, M.C., Pombo, M.A., Martínez, G.A., Civello, P.M., 2011. Heat treatments and expansin gene expression in strawberry fruit. Sci. Hortic. 130, 775–780.
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