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May 23, 2017 - Tomato fruit is highly susceptible to postharvest rot caused by B. cinerea and regarded as a good model system to study postharvest dis...
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Synergistic Effects of L‑Arginine and Methyl Salicylate on Alleviating Postharvest Disease Caused by Botrysis cinerea in Tomato Fruit Xinhua Zhang,*,† Dedong Min,† Fujun Li,*,† Nana Ji,† Demei Meng,‡ and Ling Li§ †

School of Agricultural Engineering and Food Science, Shandong University of Technology, Zibo, Shandong 255049, People’s Republic of China ‡ Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, College of Food Engineering and Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, People’s Republic of China § Department of Food Science, Tianjin Agricultural University, Tianjin 300384, People’s Republic of China ABSTRACT: The effects of L-arginine (Arg, 1 mM) and/or methyl salicylate (MeSA, 0.05 mM) treatment on gray mold caused by Botrytis cinerea in tomato fruit were studied. Results indicated that Arg or MeSA alleviated the incidence and severity of fruit disease caused by B. cinerea, and that both Arg and MeSA (Arg + MeSA) further inhibited the development of fruit decay. Treatment with Arg + MeSA not only enhanced the activities of superoxide dismutase, catalase, and peroxidase but also promoted the expression levels of pathogenesis-related protein 1 gene and the activities of defense-related enzymes of phenylalanine ammonia-lyase, polyphenol oxidase, β-1,3-glucanase, and chitinase during most of the storage periods, which were associated with lower disease incidence and disease index. In addition, the combined treatment elevated the levels of total phenolics, polyamines, especially putrescine, and nitric oxide. These observations suggest that treatment of fruit with Arg + MeSA is an effective and promising way to alleviate postharvest decays on a commercial scale. KEYWORDS: L-arginine, methyl salicylate, tomato fruit, disease resistance, polyamines, nitric oxide

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aminotransferase (OAT).11 In addition, Arg can be decarboxyated by L-arginine decarboxylase (ADC) to produce PAs via an intermediate agmatine.12 Results of animal and human studies show that Arg metabolism has diverse effects on human physiology and that Arg supplementation is a safe and generally well-tolerated nutriceutical that may improve metabolic profiles in humans.13 More recently, the possible roles of Arg in plant stress responses have aroused great attention. Pretreatment with Arg alleviated the adverse effects of salt stress on canola through modulating activities of antioxidant enzymes.14 Arg at 1 mM effectively increased sunflower seedling catalase (CAT) and ascorbate peroxidase activities and alleviated the harmful effects of salinity.15 The browning of fresh-cut apple and lettuce16 and decays caused by Botrysis cinerea in tomato fruit were effectively controlled by Arg treatment.12 Our previous works also showed that exogenous Arg was effective in improving chilling tolerance and maintaining quality of tomato fruit.17,18 There are, however, no studies available on the effect of application of Arg + MeSA on disease resistance in fruit and vegetables. Tomato fruit is highly susceptible to postharvest rot caused by B. cinerea and regarded as a good model system to study postharvest disease in fruit. Thus, we used tomato fruit as material to investigate the effects of Arg and/or MeSA on fruit decay caused by B. cinerea, the activities of defensive enzymes, and the levels of total phenolics, PAs, and NO. Moreover, the changes of PR-1 expression levels were also detected.

INTRODUCTION Postharvest decay caused by pathogen infections is the most important factor leading to quality deterioration of fruit and vegetables.1,2 Although the pathogenic diseases can be eliminated by chemical fungicides, there is increasing worldwide attention about the possible harmful effects of these compounds on both human health and the environment as well as the fungicide resistance by pathogens.3,4 Consequently, it is very meaningful to explore safe and efficient alternatives to chemical fungicides for disease control of postharvest horticultural crops. Induction of disease resistance in fruit and vegetables by physical or chemical means has gained great attention in recent years, with it being considered a sustainable strategy to manage postharvest decay of horticultural crops.4 Salicylic acid (SA) is a small phenolic compound and serves as an endogenous signaling molecule, having key roles in inducing plant resistance against various stresses.2,5,6 Recently, several studies have confirmed that SA or methyl salicylate (MeSA) could inhibit decay and maintain quality in many postharvest horticultural crops. The decays caused by Alternaria alternata in jujube,6 Penicillium expansum in apple fruit,7 Penicillium italicum and Penicillium digitatum in citrus fruit,8 and mango anthracnose were effectively controlled by SA treatment.9 There has been considerable interest in the metabolism of L-arginine (Arg) in mammals over the last 2 decades, because it is the precursor for biosynthesis of signaling molecules nitric oxide (NO), polyamines (PAs), and proline.10,11 The formation of NO from Arg is catalyzed by nitric oxide synthase (NOS).13 Arg is also metabolized by arginase to urea and ornithine (Orn), the latter of which can be converted to PAs by ornithine decarboxylase (ODC) or to proline by ornithine © 2017 American Chemical Society

Received: Revised: Accepted: Published: 4890

January 25, 2017 May 20, 2017 May 23, 2017 May 23, 2017 DOI: 10.1021/acs.jafc.7b00395 J. Agric. Food Chem. 2017, 65, 4890−4896

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Journal of Agricultural and Food Chemistry

recorded on the basis of the severity of disease symptoms with a scale of 0−5,20 where 0 is no decay development, 1 is decay diameter of 0.5 but 1.0 but 2.0 but 3.5 cm. The disease index was counted by the following formula: disease index = ∑[(disease scale) × (number of fruit at the disease scale)]/(total number of fruit × 5). Enzyme Assays. For superoxide dismutase (SOD), 1 g of frozen tissue was homogenated in 5 mL of 0.05 M sodium phosphate buffer (SPB, pH 7.8) or in 0.05 M SPB at pH 7.0 for CAT and at pH 6.4 for peroxidase (POD). For chitinase (CHI) and β-1,3-glucanase (GLU), frozen tissue was homogenated in 5 volumes of 0.05 M sodium acetate buffer (pH 5.0) or in 0.2 M sodium borate buffer (pH 8.7) containing 20 mM β-mercaptoethanol for phenylalanine ammonia-lyase (PAL). For polyphenol oxidase (PPO), 1 g of frozen tissue was homogenated in 5 mL of 0.2 M SPB at pH 6.5 containing 1% (w/v) polyvinylpyrrolidone. The mixture was centrifuged at 4 °C for 20 min at 10000g. The supernatants were used for analyzing the enzyme activities. The activities of SOD and CAT were determined according to Gay and Tuzun.21 A total of 1 unit of SOD activity was defined as previously described.21 The amount of CAT decomposing 1 mmol of hydrogen peroxide (H2O2) per minute at 30 °C was regarded as 1 unit. POD activity was measured by the guaiacol method.22 A total of 1 unit of POD activity was defined as an increase in A470 of 0.01 per minute. The activities of CHI and GLU were assayed according to Zheng et al.12 The amount of enzyme catalyzing the production of 10 μg of N-acetyl-D-glucosamine for CHI or 1 mg of glucose for GLU per hour at 37 °C was regarded as 1 unit. PAL was assayed as described by Luo et al.,23 with minor modifications. A total of 0.5 mL of enzyme extract was reacted with 1 mL of 0.02 M L-phenylalanine and 2 mL of 0.2 M boric acid buffer (pH 8.8) for 1 h at 37 °C. The absorbance was recorded at 290 nm, and the increase in A290 of 0.01 per hour was regarded as 1 unit of enzyme activity. PPO activity was determined by mixing 0.5 mL of enzyme solution with 2 mL of 0.05 M SPB (pH 6.8), and the change in absorbance at 420 nm after adding 1 mL of 0.1 M catechol was recorded for 3 min. The increase in A420 of 0.01 per minute was regarded as 1 unit of enzyme activity.12 Specific activity of the enzymes was expressed as units g−1 of fresh weight (FW) . Quantitative Real-Time Polymerase Chain Reaction (qPCR) Assay. Total RNA was isolated from each sample using trizol reagent as described previously.17 Complementary DNA (cDNA) was synthesized with M-MLV reverse transcriptase (Promega, Madison, WI), oligo(dT)15 primer, and 2 μg of total RNA DNAfree. qPCR was performed using the SYBR Green I MasterMix (Toyobo, Osaka, Japan) on a LineGene 9600 detection system (Bioer, Hangzhou, China). Specific primers for PR1 (accession number X71592) and Ubi3 (accession number X58253, as a standard gene) were as follow: PR1-F (5′-CTGGTGCTGTGAAGATGTGG-3′) and PR1-R (5′-CCGACTTACGCCATACCACT-3′) and Ubi3-F (5′-TCCATCTCGTGCTCCGTCT-3′) and Ubi3-R (5′-CTGAACCTTTCCAGTGTCATCAA-3′). The qPCR parameters were: 95 °C for 2 min, followed by 40 cycles of 15 s at 95 °C and 20 s at 60 °C, and 45 s at 72 °C. Melting curves were assayed from 55 to 95 °C at 0.5 °C increments. The gene transcript levels were normalized against the threshold cycle (Ct) value of the Ubi3 gene and calculated according to the 2−ΔΔCt method. Determination of the Total Phenolic Content. A frozen sample (1 g) was ground in 2.5 mL of 95% ethanol and extracted for 48 h at 4 °C. After centrifugation (10000g for 10 min), the content of total phenolic was measured using the Folin−Ciocalteu method.12 The total phenolic content was expressed as catechin equivalent (mg g−1 of FW). Determination of Free PA and NO Contents. Free PAs in sample were extracted with 5% (w/v) cold perchloric acid and centrifuged at 4 °C for 30 min at 20000g. The PAs of the supernatants were derivatized with benzoyl chloride as described in our previous study.5

Figure 1. Effects of Arg and MeSA on (A) disease incidence and (B) disease index of tomato fruit during incubation at 25 °C for 12 days. Vertical bars represent the standard errors of the means. Values followed by different letters at the same time are significantly different according to Duncan’s multiple range test at the p = 0.05 level.

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MATERIALS AND METHODS

Pathogen. The pathogen B. cinerea was originally isolated from infected tomato fruit and cultured as described by Zheng et al.12 The spores of B. cinerea were obtained by washing 1-week-old cultures with aseptic distilled water, and the concentration of B. cinerea spores was adjusted to 2 × 105 spores mL−1. Fruit. Green mature tomato fruit (Solanum lycopersicum L. cv. Badun) were picked from a greenhouse in Zibo, Shangdong, China, and immediately transported to our laboratory. Fruit without visual defects and similar in size were selected and disinfected by 2% (v/v) NaClO for 2 min. The disinfected fruit were rinsed thoroughly with clean water and air-dried before treatment. Treatments. In preliminary experiments, three doses (0.5, 1, and 2 mM) for Arg and three concentrations (0.01, 0.05, and 0.1 mM) for MeSA treatment were investigated. The preliminary results showed that 1 mM Arg and 0.05 mM MeSA treatment were more effective than other concentrations to maintain quality and decrease the disease incidence and index of fruit infected with B. cinerea (data not shown). Thus, 1 mM Arg and 0.05 mM MeSA treatments were selected for this study. One part of fruit was treated with 1 mM Arg by vacuum infiltration, following the methods of our former study.19 After treatment with Arg, then half of the fruit were treated with 0.05 mM MeSA in 45 L airtight containers at 20 °C for 12 h. The other part of fruit were subjected to the same conditions with 0 mM Arg, and then half of the fruit were treated with 0.05 mM MeSA, as described above. Fruit treatment with 0 mM Arg and without MeSA was taken as the control. Each treatment had three replicates of 40 fruit each. After ventilation, fruit were then punctured with a sterile nail at two opposite points (2 mm wide and 4 mm deep) in the equator zone. After injection with 10 μL of conidial suspension, fruit were incubated at 25 ± 1 °C and 90−95% relative humidity. The mesocarp (5 mm away from the edge of the wound) from five fruit at 2 day intervals were collected for analysis of enzyme activities, PR-1 expression, and contents of PAs, total phenolics, and NO. Fruit disease was visually evaluated on each fruit at 4, 8, and 12 days of storage. Disease Evaluation. Disease incidence was assessed as the percentage of tomato fruit displaying symptoms. Disease index was 4891

DOI: 10.1021/acs.jafc.7b00395 J. Agric. Food Chem. 2017, 65, 4890−4896

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Journal of Agricultural and Food Chemistry

Figure 2. Effects of Arg and MeSA on activities of (A) SOD, (B) CAT, (C) POD, (D) PAL, (E) PPO, and (F) total phenolic content of tomato fruit during incubation at 25 °C for 12 days. Vertical bars represent the standard errors of the means. The benzoyl PAs were detected using high-performance liquid chromatography (HPLC) with a Symmetry C18 column (4.6 × 250 mm, 5 μm). Samples were eluted at a flow rate of 1 mL min−1 using 64% methanol and detected with an ultraviolet (UV) detector at 254 nm. Standard PAs were subjected to the same procedure. NO in the sample was extracted according to the method of Hao et al.,24 with minor modifications. A frozen sample (1 g) was ground with 5 mL of cold buffer (50 mM SBP, 1 mM dithiothreitol, and 1 mM MgCl2 at pH 7.4). After centrifugation, the NO content was measured indirectly by quantitation of nitrite and nitrate with NO assay kits (Nanjing Jiancheng Bioengineering Institute) according to our previous study.5 The NO content was expressed as micromolar per gram of FW. Statistical Analysis. All data analysis was carried out by analysis of variance (ANOVA) using software SPSS 16.0 (SPSS, Inc., Chicago, IL). Duncan’s multiple range tests were used to determine any significant differences between the means. Differences at p < 0.05 were regarded as significant.

The combined treatment of Arg with MeSA was much more effective to abate the incidence and index of disease than Arg or MeSA treatment alone (Figure 1). The incidence and index of disease in fruit treated with Arg + MeSA were 27.1 and 54.7% lower than those in the control, respectively, on day 12 after inoculation. Antioxidant Enzyme Activities. As shown in panels A−C of Figure 2, postharvest treatment with Arg and/or MeSA altered antioxidant metabolism in tomato fruit. Treatment with Arg or MeSA alone kept significantly higher activities of SOD and POD than that of the control fruit during the storage periods, and CAT activity was also enhanced by Arg or MeSA from days 6 to 12. Moreover, the combined treatment of Arg and MeSA further increased the three enzyme activities compared to application of Arg or MeSA alone during most of the storage periods, with the activities of them in combinationtreated fruit being 281.3, 122.4, and 285.0% higher than those in untreated fruit after 12 days of incubation, respectively. Activities of PAL and PPO and Total Phenolic Content. Application of Arg or MeSA alone significantly (p ≤ 0.05) improved PAL activity in comparison to the control fruit during 12 days of storage, except on day 2 or 8 (Figure 2D). Treatment with Arg + MeSA further enhanced PAL activity compared to Arg or MeSA alone during the entire storage period. PPO activity was also induced by Arg or MeSA

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RESULTS Disease Incidence and Index of Fruit. Disease incidence caused by B. cinerea in tomato fruit was reduced by Arg or MeSA treatment alone (Figure 1A). Although almost all of the inoculated wounds in fruit untreated or treated with Arg or MeSA alone showed disease symptoms after 12 days of incubation, the disease index in Arg- or MeSA-treated fruit was apparently lower than that in untreated fruit (Figure 1B). 4892

DOI: 10.1021/acs.jafc.7b00395 J. Agric. Food Chem. 2017, 65, 4890−4896

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Journal of Agricultural and Food Chemistry alone during 12 days of incubation, and the combined treatment caused an additional increase of PPO activity, which was 61.2, 24.4, or 100.8% higher than that in the treatment with Arg, MeSA alone, or the control, respectively (Figure 2E). Arg or MeSA treatment alone promoted the accumulation of total phenolics in tomato fruit from days 8 to 12 of inoculation, and the combined treatment was more effective in increasing the phenolic content compared to Arg or MeSA treatment alone. At the end of the storage time (day 12), the total phenolic content in combination-treated fruit was 38.0, 27.0, and 62.70% higher than that in the treatment with Arg, MeSA alone, or the control, respectively (Figure 2F). CHI and GLU Activities. CHI and GLU activities in tomato fruit increased during the first 8 day period after inoculation and then decreased (panels A and B of Figure 3). Treatment

Figure 4. Effects of Arg and MeSA on PR-1 gene expression of tomato fruit during incubation at 25 °C for 12 days. The expression levels of PR-1 were evaluated by qPCR, normalized to the host Ubi3 gene, and set relative to the control sample from day 0 according to the 2−ΔΔCt method. Vertical bars represent the standard errors of the means. Values followed by different letters at the same time are significantly different according to Duncan’s multiple range test at the p = 0.05 level.

Free PA Levels and NO Content. As shown in Table 1, among the three PAs, putrescine (Put) was the dominant amine, followed by spermidine (Spd) and spermine (Spm). Arg or MeSA treatment alone significantly (p ≤ 0.05) increased the Put content from days 6 to 12, with a value of about 64.1 or 83.3% higher than that of the control at day 12, respectively. However, the contents of Spd and Spm changed little following Arg or MeSA treatment alone during most of the storage period. The combined treatment resulted in a further accumulation of Put from days 6 to 10 and also increased the Spd content on days 8 and 10 as well as the Spm content on days 6 and 12. The NO content also increased following treatment with Arg or MeSA alone during the storage time. Furthermore, treatment with Arg and MeSA in combination induced a higher NO content than that of Arg or MeSA treatment separately during the days after incubation, with a maximum of nearly 30.4, 48.7, and 74.3% higher than that of Arg or MeSA treatment alone and the control fruit on day 6, respectively (Table 1).

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DISCUSSION In recent years, many studies have found that treatment with exogenous Arg has a specific role in plant responses to stresses. Nasibi et al.14 found that pretreatment with Arg alleviated the oxidative damage and improved the growth of canola seedlings. Wills and Li16 reported that Arg effectively inhibited the browning of fresh-cut lettuce and apple and had no effect on taste. In addition, preharvest treatment with Arg can also promote tomato fruit resistance against fungal infection.6 SA is a signaling component and has important roles in regulating pathogen resistance in postharvest horticultural crops.1,7 Similar to the previous studies, we found that Arg or MeSA treatment alone inhibited fruit decay caused by B. cinerea. Moreover, our present study also showed that Arg + MeSA was more effective in inducing the disease resistance of tomato fruit than the application of each treatment alone, which indicated that the combination treatment induced more defensive capacity against B. cinerea infection in tomato fruit. It has been reported that fruit resistance to pathogens is related to the maturity stages, with fruit at an earlier maturity stage showing stronger resistance against pathogen invasion.25−27 Active oxygen species (AOS) are accumulated in plants under pathological and senescence conditions, leading to

Figure 3. Effects of Arg and MeSA on activities of (A) CHI and (B) GLU of tomato fruit during incubation at 25 °C for 12 days. Vertical bars represent the standard errors of the means.

with Arg or MeSA led to significantly (p ≤ 0.05) higher activities of CHI and GLU than that of the control fruit during most of the days after inoculation. Moreover, the activities of these two enzymes in fruit were further improved by Arg combined MeSA treatment from days 6 to 12, and the activities of both enzymes in Arg + MeSA-treated fruit were 72.9 and 50.9% higher than those in the control on day 12, respectively. PR-1 Expression. The data from the tests showed that PR-1 expression in Arg- or MeSA-treated fruit increased rapidly within 2 days and maintained a high level for 6 days after inoculation (Figure 4). Moreover, treatment with Arg + MeSA resulted in a further induction of PR-1 expression compared to Arg or MeSA treatment alone during 12 days of incubation, except on days 6 and 10. In combination-treated fruit, the PR-1 expression level peaked on day 2, with a value of about 87.8, 49.8, and 206.1% higher than that of Arg or MeSA treatment alone and the control fruit, respectively. 4893

DOI: 10.1021/acs.jafc.7b00395 J. Agric. Food Chem. 2017, 65, 4890−4896

a

PAs and NO

treatment

control MeSA Arg Arg + MeSA control MeSA Arg Arg + MeSA control MeSA Arg Arg + MeSA control MeSA Arg Arg + MeSA

0

4.38 ± 0.44

71.60 ± 5.91

0.26 ± 0.03

6.69 ± 0.57 6.08 6.94 4.84 6.69 0.24 0.24 0.22 0.28 64.12 62.1 68.86 74.87 5.56 5.70 6.48 7.38

2 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.66 ab 0.74 a 0.58 b 0.61 a 0.028 a 0.027 a 0.016 b 0.022 a 6.12 ab 5.21 b 5.67 ab 5.49 a 0.46 c 0.27 c 0.28 b 0.38 a

8.18 6.99 7.44 6.47 0.31 0.28 0.37 0.25 71 82.37 87.32 73.6 5.58 6.29 8.16 7.84

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.83 a 0.70 a 0.44 a 0.58 a 0.030 ab 0.022 b 0.032 a 0.031 b 6.1 b 7.67 ab 5.32 a 7.06 ab 0.21 c 0.29 b 0.52 a 0.37 a

4 5.67 10.31 7.57 12.26 0.25 0.38 0.25 0.3 67.51 78.08 84.27 92.01 5.40 6.33 7.22 9.41

6 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.67 d 0.98 b 0.82 c 0.75 a 0.028 b 0.041 a 0.020 b 0.032 b 3.09 c 7.38 bc 8.37 ab 2.01 a 0.44 d 0.31 c 0.32 b 0.42 a

2.43 3.98 4.97 9.79 0.23 0.29 0.46 0.55 62.46 65.64 65.84 69.47 4.94 6.70 7.15 8.21

8 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.43 c 0.57 b 0.93 b 1.11 a 0.026 d 0.013 c 0.028 b 0.037 a 4.62 a 5.77 a 7.11 a 5.93 a 0.51 c 0.47 b 0.32 b 0.5 a

2.65 8.04 6.42 13.52 0.22 0.24 0.27 0.31 81.11 68.12 74.68 76.14 5.75 7.27 6.58 8.14

0.66 c 0.93 b 0.72 b 1.14 a 0.025 b 0.021 b 0.034 ab 0.024 a 7.12 a 7.02 a 8.01 a 8.61 a 0.34 c 0.37 b 0.26 b 0.31 a

10 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

Means with different letters in a column for each parameter differ significantly at p = 0.05 according to Duncan’s multiple range tests. Data are the means ± standard error (SE).

NO (μmol g−1 of FW)

Spm (nmol g−1 of FW)

Spd (μmol g−1 of FW)

Put (μmol g−1 of FW)

storage time (days)

Table 1. Effects of Arg and MeSA Treatment on the Contents of Put, Spd, Spm, and NO in Tomato Fruit during Storage at 25 °C for 12 Daysa

8.49 13.93 15.65 13.24 0.13 0.13 0.16 0.16 52.36 57.49 53.15 73.84 6.11 7.25 6.72 7.92

0.78 b 0.84 a 1.24 a 1.02 a 0.011 a 0.015 a 0.011 a 0.019 a 6.24 b 4.88 b 5.92 b 6.38 a 0.38 c 0.41 ab 0.37 bc 0.32 a

12 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

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DOI: 10.1021/acs.jafc.7b00395 J. Agric. Food Chem. 2017, 65, 4890−4896

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Journal of Agricultural and Food Chemistry oxidative injury.28 It is well-known that SOD, CAT, and POD are the key enzymes scavenging AOS, and the decrease in them may lead to high levels of AOS. Therefore, high levels of antioxidant enzymes are involved in delaying the senescence process and fruit resistance against pathogen invasion.25−27 In our present study, when compared to the control fruit, the application of Arg or MeSA induced SOD, CAT, and POD activities during most of the storage period, which might be associated with the increased disease resistance in tomato fruit when treated by Arg or MeSA alone. Moreover, our results also demonstrated that Arg + MeSA treatment further induced the three antioxidant enzyme activities than application of Arg or MeSA alone (panels A−C of Figure 2), which may partly explain why application of Arg + MeSA induced fruit resistance against B. cinerea infection more effectively than each treatment alone. The increase in total phenolic compounds has been regarded as an indicator for plant defense response. Both PAL and PPO are enzymes responsible for the processes of secondary metabolism that generates various phenolics with defensive and structure-related functions in plant.29,30 Members of the PR proteins are induced in plants infected with various pathogens and play important roles in plant resistance to pathogen infection. PR-1 is a vital group of PRs and generally used as an indicator for systemic acquired resistance.31 Both GLU (PR-2 family) and CHI (PR-3 family) are the key enzymes in the degradation of fungal cell walls.6,32 Our results demonstrated that Arg or MeSA treatment significantly increased the content of total phenolics and the activities of PAL, PPO, CHI, and GLU during most of the time after inoculated with B. cinerea. Similar effects by treatment with SA or Arg were also described in many fruit, including tomato,6 mango,8 sweet cherry,33 peach,1 and jujube.7 In addition, we also found that treatment with Arg + MeSA induced higher activities of these defense enzymes and higher levels of total phenolic content and PR-1 expression than each treatment separately. These findings may be part of the reason for the synergistic effect of Arg and MeSA treatment on alleviating fruit disease caused by B. cinerea. NO and PAs are both important signaling molecules related to several physiological and biological processes during plant growth and stress response.34,35 Recently, the influence of NO on postharvest disease has also aroused wide attention. Li et al.36 reported that NO treatment enhanced peach fruit resistance to Monilinia fructicola by activation of the phenylpropanoid pathway. Treatment of mango fruit with NO resulted in enhanced resistance against Colletotrichum gloeosporioides, increased activities of defense-related enzymes, and also delayed fruit ripening.37 Furthermore, exogenous application of the NO precursor (Arg) could promote the activities of defensive enzymes and enhance resistance against B. cinerea in tomato fruit.6 However, there is little information available on the role of PAs against pathogen infection stress, despite the continued interest in the relationship between PAs and chilling injury in postharvest fruit.18,35 Recently, Cao et al.3 pointed out that the enhanced PAs might be a major factor in reduced disease susceptibility of loquat fruit treated with methyl jasmonate. Moreover, an early work by Wang and Galletta38 showed that PAs were higher in strawberry fruit of resistant genotypes compared to the susceptible genotypes in response to anthracnose stress. Walters et al.39 also showed that MeJA treatment, which showed beneficial effects in inducing disease resistance, was accompanied by an increase in PAs in barley seedlings. Thus, a role for PAs in improving fruit resistance to

fungal infection was also proposed. In our present study, treatment with Arg (the precursor of NO and PAs), which showed beneficial effects in inducing disease resistance (Figure 1), significantly increased NO and Put contents in tomato fruit during most of the storage periods (Table 1), whereas MeSA exerted similar effects on these indexes, despite not being a precursor for NO and PAs. These effects suggest that maintained and enhanced NO or PAs may be the factors in reduced disease susceptibility of Arg- or MeSA-treated tomato fruit. Therefore, the additional induction of PAs and NO in combination treatment fruit may contribute at least in part to the increased fruit disease resistance compared to Arg and MeSA treatment alone. In conclusion, the present results support application of Arg or MeSA as a possible strategy to alleviate decay caused by B. cinerea in tomato fruit. The combination of Arg and MeSA treatment enhanced the benefits of applying each treatment separately. Furthermore, it was reported that Arg supplementation has beneficial effects on the regulation of problems related to development and health and provides great promise for improving human health. Thus, it is considered that Arg + MeSA treatment may be an effective and promising way to control postharvest decays on a commercial scale.

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AUTHOR INFORMATION

Corresponding Authors

*Telephone: +86-533-2786398. E-mail: [email protected]. *Telephone: +86-533-2786398. E-mail: [email protected]. ORCID

Xinhua Zhang: 0000-0003-4963-7443 Funding

This study was funded by the National Natural Science Foundation of China (31201432 and 31101587). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED Arg, L-arginine; MeSA, methyl salicylate; PA, polyamine; NO, nitric oxide; SOD, superoxide dismutase; CAT, catalase; POD, peroxidase; PR, pathogenesis-related protein; PAL, phenylalanine ammonia-lyase; PPO, polyphenol oxidase; CHI, chitinase; GLU, β-1,3-glucanase; FW, fresh weight; qPCR, quantitative real-time polymerase chain reaction; Put, putrescine; Spd, spermidine; Spm, spermine; AOS, active oxygen species

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REFERENCES

(1) Yang, Z. F.; Cao, S. F.; Cai, Y. T.; Zheng, Y. H. Combination of salicylic acid and ultrasound to control postharvest blue mold caused by Penicillium expansum in peach fruit. Innovative Food Sci. Emerging Technol. 2011, 12, 310−314. (2) Tian, S. P.; Qin, G. Z.; Li, B. Q.; Wang, Q.; Meng, X. H. Effects of salicylic acid on disease resistance and postharvest decay control of fruits. Stewart Postharvest Rev. 2007, 3, 1−7. (3) Cao, S. F.; Cai, Y. T.; Yang, Z. F.; Joyce, D. C.; Zheng, Y. H. Effect of MeJA treatment on polyamine, energy status and anthracnose rot of loquat fruit. Food Chem. 2014, 145, 86−89. (4) Romanazzi, G.; Sanzani, S. M.; Bi, Y.; Tian, S. P.; Gutiérrez Martínez, P.; Alkan, N. Induced resistance to control postharvest decay of fruit and vegetables. Postharvest Biol. Technol. 2016, 122, 82−94. (5) Zhang, X. H.; Shen, L.; Li, F. J.; Meng, D. M.; Sheng, J. P. Methyl salicylate-induced arginine catabolism is associated with up-regulation

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DOI: 10.1021/acs.jafc.7b00395 J. Agric. Food Chem. 2017, 65, 4890−4896

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DOI: 10.1021/acs.jafc.7b00395 J. Agric. Food Chem. 2017, 65, 4890−4896