Effects of thiamine on Trichothecium and Alternaria rots of muskmelon fruit and the possible mechanisms involved

Journal of Integrative Agriculture 2017, 16(11): 2623–2631 Available online at www.sciencedirect.com

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RESEARCH ARTICLE

Effects of thiamine on Trichothecium and Alternaria rots of muskmelon fruit and the possible mechanisms involved GE Yong-hong, LI Can-ying, LÜ Jing-yi, ZHU Dan-shi College of Food Science and Engineering, Bohai University/Food Safety Key Laboratory of Liaoning Province/National & Local Joint Engineering Research Center of Storage, Processing and Safety Control Technology for Fresh Agricultural and Aquatic Products, Jinzhou 121013, P.R.China

Abstract The effects of thiamine against pink and black spot rots caused by Trichothecium roseum and Alternaria alternata and modulation on the metabolism of reactive oxygen species (ROS) and phenylpropanoid pathway were investigated in this paper. In vitro test indicated that thiamine significantly inhibited mycelia growth and spore germination of T. roseum and A. alternata. Thiamine at 100 mmol L–1 effectively inhibited lesion development of muskmelon fruit inoculated with T. roseum or A. alternata, enhanced production rate of O2-. and H2O2 content, activities of catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) in muskmelon fruit. Thiamine also affect phenylpropanoid pathway in muskmelon fruit by increasing the activities of phenylalanine ammonia lyase (PAL) and peroxidase (POD), the content of total phenolic compounds, flavonoids and lignin. These results suggest that the effects of thiamine on pink and black spot rots in muskmelon fruits are associated with its direct fungitoxic against the pathogens and the modulation of O2-. and H2O2 production, eliminating enzymes and phenylpropanoid pathway. Keywords: induced resistance, thiamine, muskmelon fruit, antimicrobial activity, phenylpropanoid pathway

synthetic fungicides, such as imazalil, iprodione, azoxystrobin,

1. Introduction Muskmelon (Cucumis melo L.) fruit is one of the most important horticultural crops cultivated in the Northwest China (Ren et al. 2012). However, fruit are easily infected by Trichothecium roseum and Alternaria alternata which cause pink and black spot rots in the fruit and lead to considerable postharvest losses in China (Ge et al. 2008). Applications of

effectively control postharvest diseases of muskmelon (Aharoni et al. 1992; Ma et al. 2004). However, fungicide usage has become limited due to the development of fungicide resistant pathogens, potential effects on human health and environment, and the need for new alternative methods have been proposed (Tripathi and Dubey 2004; Bi et al. 2009). One of the potential methods of controlling disease is the induced resistance in plants and fruit (Kuc 2001). Several literatures have showed that vitamins, such as riboflavin (vitamin B2), menadione sodium bisulphite (vitamin K3)

Received 10 September, 2016 Accepted 25 February, 2017 Correspondence GE Yong-hong, E-mail: [email protected] © 2017 CAAS. Publishing services by Elsevier B.V. All rights reserved. doi: 10.1016/S2095-3119(16)61584-8

and thiamine (vitamin B1) can induce disease resistance in plants or fruit (Dong and Beer 2000; Borges et al. 2004; Ahn et al. 2005; Yin et al. 2012). Preharvest application of thiamine induced resistance in pearl millet, grapevine, rice and barley against Sclerospora graminicola, Plasmo-

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para viticola, Rhizoctonia solani and Rhopalosiphum padi (Pushpalatha et al. 2011; Bahuguna et al. 2012; Boubakri et al. 2012, 2013; Hamada and Jonsson 2013). In vitro test indicated that thiamine can inhibit spore germination and mycelia growth of A. alternata (Yin et al. 2012). Postharvest thiamine dipping treatment effectively inhibited lesion development in Asian pear fruit inculated with A. alternata (Yin et al. 2012). These results suggested that decreased disease development by thiamine was closely related to modulation of various physiological, chemical and molecular host-defense responses including generation of reactive oxygen species (ROS), pathogenesis-related (PR) genes expression, phenolics and lignin accumulation, callose deposition, hypersensitive responses and direct inhibition the pathogen growth ( Ahn et al. 2007; Bahuguna et al. 2012; Boubakri et al. 2012, 2013; Yin et al. 2012). However, the thiamine efficiency in disease control and the physiological and chemical changes induced in muskmelon fruit are still unknown. Therefore, the objectives of this study were to investigate the effects of thiamine on the control of the postharvest diseases caused by T. roseum and A. alternata in muskmelon fruit, to evaluate the antifungal activity of thiamine against T. roseum and A. alternata in vitro, to determine the production rate of O2-. and H2O2, eliminating enzymes activities, and to investigate the enzymes activities and secondary metabolites of phenylpropanoid pathway in the muskmelon fruit.

2. Materials and methods 2.1. Fruit and treatment Muskmelon fruits (Cucumis melo L. cv. Jinhongbao) were harvested at 40 d after full blossom in the field in Liaoning Province, China. Fruit without mechanical injuries or diseases were cleaned, disinfected, and subsequently immersed in 0, 25, 50, and 100 mmol L–1 thiamine solutions for 10 min. After air-drying, fruits were kept in cartons and stored at (22±2)°C, RH=70–80% conditions for the following experiments. Three replicates per treatment were made, each replicate containing 15 fruits, and the entire experiment was performed twice.

2.2. Pathogen and inoculation T. roseum and A. alternata were originally isolated from decay fruit and cultured on potato dextrose agar (PDA) at 25°C. Spore suspensions were prepared by flooding the 7-dold culture plates with 4–5 mL of sterile distilled water. The inoculums were diluted to 1×105 spores mL–1 and confirmed using a haemocytometer (XB-K-25, Solarbio, China). Inoculations were carried out 24 h after thiamine treat-

ment according to Ren et al. (2012). All fruits were sterilized with 70% ethanol around the equator of each fruit, and then four wounds were made with a dissecting needle (3-mm deep×3-mm diameter). A volume of 20 μL of spore suspensions was injected into each wound. Fruits were air-dried, then kept in cartons and incubated at room temperature. The lesion diameter was recorded 7 d after inoculation.

2.3. Effects of thiamine on spore germination and mycelia growth of T. roseum and A. alternata in vitro To assess the effects of thiamine on spore germination of T. roseum and A. alternata, 20 µL aliquots of spore suspensions (1×105 spores mL–1) were added into 5 mL potato dextrose blended with 0, 25, 50, and 100 mmol L–1 thiamine, and then incubated in Petri dishes at 25°C. After 4, 6, and 8 h of incubation, the germination rates were assessed by observing 100 spores per treatment replicate under a light microscope. Three replicates per treatment were made, and the experiment was performed twice. Effects of thiamine on mycelia growth of T. roseum and A. alternata were assessed according to the method of Li et al. (2009). Mycelia disks (5-mm in diameter) from 7-d-old cultures were placed in the centre of Petri dishes (90-mm in diameter) with 15 mL of PDA containing different concentrations of thiamine (0, 25, 50, 100 mmol L-1), and then incubated at 25°C. The mycelia growth was determined by measuring the colony diameter 7 d after inoculation. Three replicates per treatment were made, and the experiment was performed twice.

2.4. Samples collection Approximately 3.0 g of flesh tissue was taken from 3–8 mm below the skin around the equator of fruit at 0, 2, 4, 6, and 8 d after 100 mmol L-1 thiamine and distilled water treatment. Each sample was packed in aluminum foil individually and frozen in liquid nitrogen immediately, and kept at –80°C until the biochemical determination. There were three replicates for enzyme assays in each treatment, and the experiment was repeated twice.

2.5. Production rate of O2-. and H2O2 content assay Production rate of O2-. was determined according to the method of Ren et al. (2012). The reaction mixture contained 2 mL of the supernatant, 1 mL 50 mmol L-1 phosphate buffer (pH 7.8), and 0.5 mL hydroxylamine hydrochloride (10 mmol L-1) solution for 30 min at 25°C. Then 1 mL 4-aminobenzene sulfonic acid (17 mmol L-1) and 7 mmol L-1 α-naphthylamine were added for a further 40 min. After that 4 μL of n-butanol was added into the reaction mixture, and n-butanol phase

GE Yong-hong et al. Journal of Integrative Agriculture 2017, 16(11): 2623–2631

was used for the determination of O2-.. The production rate of O2-. was expressed as ΔOD530 min-1 g-1 FW. H2O2 content was assayed by the method of Ren et al. (2012). And 1 mL of the supernatant was re-centrifuged followed by the addition of 200 μL, 20% titanium tetrachloride, and 200 μL of concentrated ammonia solution to precipitate the titanium-hydro peroxide complex. Precipitate was washed repeatedly by cold acetone and then dissolved in 3 mL of 1 mol L-1 H2SO4 and then re-centrifuged. H2O2 content was monitored by taking the absorbance at 410 nm and expressed as µmol H2O2 g-1 FW.

2.6. Crude enzymes extraction All enzyme extracts were conducted at 4°C. Frozen samples (3.0 g) were ground in a mortar on ice, using the following extraction solutions: 5 mL of 50 mmol L-1 phosphate buffer (pH 7.5) containing 10 g L-1 of polyvinylpyrrolidone (PVP), 0.01% Triton X-100 (v/v) for peroxidase (POD); 5 mL phosphate buffer (pH 7.0, 50 mmol L-1) containing 5 mmol L-1 dithiothreitol (DTT) and 8 g L-1 PVP for catalase (CAT); 4 mL phosphate buffer (pH 7.5, 50 mmol L-1) containing 1 mmol L-1 ethylenediaminetetraacetic acid (EDTA) for ascorbate peroxidase (APX) and glutathione reductase (GR); 5 mL, 0.2 mol L-1 sodium borate buffer (pH 8.7) containing 50 mmol L-1 β-mercaptoethanol, 2 mmol L-1 EDTA and 8 g L-1 of PVP for phenylalanine ammonia lyase (PAL). The homogenates were centrifuged at 15 000×g for 20 min, and the supernatants were assayed for enzymatic activities.

2.7. Enzyme activity assays CAT activity was assayed according to Ren et al. (2012) following the disappearance of H2O2 at 240 nm. CAT activity was expressed as ΔOD240 g-1 FW. APX activity was determined as described by Nakano and Asada (1987) with slight modifications. The reaction mixture included 2 mL phosphate buffer (0.1 mol L-1, pH 7.5), 150 µL, 5 mmol L-1 ascorbic acid, 100 µL crude enzymes and 200 µL H2O2 (10 mmol L-1). Absorbance of the solution was measured at 290 nm. APX activity was expressed as µmol ascorbic acid (AsA) g-1 FW. GR activity was assayed by the method of Foyer and Halliwell (1976) with minor modifications. The reaction mixture included 2 mL phosphate buffer (0.1 mol L-1, pH 7.5), 200 µL 5 mmol L-1 reduced glutathione (GSH), 100 µL crude enzymes and 30 µL 4 mmol L-1 NADPH. Absorbance of the solution was measured at 340 nm. GR activity was expressed as µmol NADPH g-1 FW. PAL activity was assayed as described by Kozukue et al. (1979) with some modifications. The reaction solution contained 3 mL of L-phenylalanine and 500 µL of crude

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enzyme extract. The mixture was incubated at 40°C for 1 h and stopped by the addition of 0.2 mL of 6 mol L-1 HCl. Absorbance at 290 nm was determined for PAL. PAL activity was expressed as U g-1 FW, where, U=0.01 ΔOD290 min-1. POD activity was assayed according to the method of Liu et al. (2005). The reaction mixture contained 200 µL of crude enzyme extract, 2.5 mL guaiacol (25 mmol L-1), and 200 µL of 250 mmol L-1 H2O2. The oxidation rate of guaiacol was monitored at 470 nm for 2 min. POD activity was expressed as U g-1 FW, where U=0.01 ΔOD470 min-1.

2.8. Total phenolic compounds, flavonoids and lignin contents assay Total phenolic compounds and flavonoids contents were determined according to the method of Wang et al. (2011). The contents of total phenolic compounds and flavonoids were expressed as OD325 g-1 FW and OD280 g-1 FW, respectively. Lignin content was determined as described by Wang et al. (2011). The lignin content was expressed as OD280 g-1 FW.

2.9. Statistical analysis All statistical analyses were performed using SPSS ver. 17.0 (SPSS Inc., Chicago, IL, USA). Fisher’s least significant differences (LSD) at P<0.05 were determined to compare differences between means. Data are presented as the standard error of means.

3. Results 3.1. Effects of thiamine on spore germination and mycelia growth of T. roseum and A. alternata Thiamine at 25, 50, and 100 mmol L-1 significantly inhibited mycelia growth of T. roseum and A. alternata in vitro, with the highest concentration have a greater inhibition (Fig. 1- A and B). The inhibitory rate of T. roseum and A. alternata treated with 100 mmol L-1 thiamine was 55.5 and 56.7%, respectively. All concentrations of thiamine significantly inhibited spore germination of T. roseum and A. alternata, with higher concentrations having a greater inhibition (Fig. 1-C and D). After 8 h incubation, thiamine at 100 mmol L-1 significantly inhibited spore germination of T. roseum and A. alternata to a rate of 41 and 56%, respectively.

3.2. Effects of thiamine on pink and black spot rots development in muskmelon fruit Postharvest application of thiamine significantly decreased lesion diameter in muskmelon fruit inoculated with T. roseum or A. alternata compared to the control fruit (Fig. 2-A and

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B 10 a

6 4

b

b

b

2 0

Control

25 50 Concentration (mmol L–1) Control

C 100 Spore germination (%)

Colony diameter (cm)

8

25 mmol L–1

80

40

c

b a

b

c

20 0

b

a

4

c

b

6

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4

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2 Control

50 mmol L–1

25 50 Concentration (mmol L–1)

100

100 mmol L–1

D 100

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60

a

8

0

100

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d

c 6 Incubation time (h)

8

Spore germination (%)

Colony diameter (cm)

A

a

80

b b

60

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a b

40 a b c

20 0

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Fig. 1 Effects of different concentrations of thiamine treatment on mycelia growth and spore germination of Trichothecium roseum (A and C) and Alternaria alternata (B and D), respectively, at 25°C for 7 d incubation. Bars indicate the standard error, and bars with different letters are significantly (P<0.05) different according to the least significant differences (LSD).

concentrations. After incubation at (22±2)°C for 7 d, fruits treated with thiamine at 100 mmol L-1 showed the least lesion diameter which was 32.1 and 38.7% lower than the control fruit inoculated with T. roseum or A. alternata, respectively.

A 3.0 Lesion diameter (cm)

B). The reduction was enhanced by increasing thiamine

2.5

3.3. Effects of thiamine treatment on O2 and H2O2 production and antioxidative enzymes activity

a

b

2.0

c

1.5 1.0 0.5 0.0

-.

a

Control 25 50 100 Concentration (mmol L–1)

in the control fruit, and was significantly lower than that

in thiamine-treated fruit (Fig. 3-A). The highest level of -.

O2 production rate was determined at the 2nd day after treatment which was 42.6% higher than that in the control

fruit. H2O2 content was increased in the former 4 d after treatment, and peaked at the 4th day, then decreased. But

thiamine treatment significantly increased the content of H2O2 in muskmelon fruit (Fig. 3-B).

The activity of CAT in the control fruit kept at a low level

throughout the assay time. Thiamine treatment enhanced CAT activity, and peaked at 2 d after treatment which was 65.5% higher than that in the control fruit (Fig. 4-A). APX activity was induced by thiamine treatment in

Lesion diameter (cm)

B 1.5

Production rate of O2-. maintained at a relatively low level

1.2 0.9

a

b

b c

0.6 0.3 0

Control 25 50 100 Concentration (mmol L–1)

Fig. 2 Effects of different concentrations of thiamine treatment on lesion development in muskmelon fruit inoculated with Trichothecium roseum (A) and Alternaria alternata (B) storage at room temperature 7 d after treatment. Bars indicate the standard error, and bars with different letters are significantly (P<0.05) different according to the least significant differences (LSD).

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Control

Thiamine

CAT activity (U g–1 FW)

4 2

B

15

H2O2 content (µmol H2O2 g–1 FW)

12

0

2 4 6 Days after treatment (d)

B

9 6 3 0 0

2 4 6 Days after treatment (d) -

Fig. 3 Effects of thiamine treatment on O2 . production rate (A) and H2O2 content (B) in muskmelon fruit. Each value is the mean for three replicates and the vertical bars indicate the standard error.

muskmelon fruit (Fig. 4-B). The highest activity of APX was determined at 6 d after treatment, which was 40.9% higher than that in the control fruit. Thiamine treatment also significantly increased GR activity in muskmelon fruit throughout the assay time, and peaked at 6 d after treatment that was

6 4 2

0

2 4 6 Days after treatment (d)

8

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2 4 6 Days after treatment (d)

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8

35 30 25 20 15 10

8

C 15 12 9 6 3

28.8% higher than control fruit (Fig. 4-C).

3.4. Thiamine elicited the key enzymes activities and antifungal compounds contents in phenylpropanoid pathway PAL activity significantly increased in muskmelon fruit after thiamine treatment, and peaked at 6 d after treatment (Fig. 5-A). POD activity was also induced by thiamine treatment, and significant differences were determined at 4, 6 and 8 d after treatment, which was 45.5, 56.5 and 21.4% higher than that in the control fruit (Fig. 5-B).

Thiamine

8

0

8

APX activity (U g–1 FW)

Production rate of O2–. (ΔOD530 min–1 g–1 FW)

6

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Control

A 10

8

GR activity (U g–1 FW)

A

Fig. 4 Changes in activity of catalase (CAT) (A), ascorbate peroxidase (APX) (B) and glutathione reductase (GR) (C) in muskmelon fruit treated with thiamine. Each value is the mean for three replicates and the vertical bars indicate the standard error.

lignin content in thiamine-treated fruit reached the maximum level at 6 d after treatment, which was 82% higher than that in the control fruit (Fig. 5-E).

4. Discussion

Content of total phenolic compounds increased by thiamine treatment, and peaked at 4 d after treatment which was

Several chemicals have been reported to induce disease

significantly higher than that in the control fruit (Fig. 5-C).

resistance in plants or fruit by either direct inhibition patho-

Postharvest thiamine treatment significantly increased the

gen growth or/and induction of disease to pathogen invasion

content of flavonoids and lignin (Fig. 5-D). The highest level

(Wang et al. 2010; Li W H et al. 2012; Li Y C et al. 2012;

of flavonoids was observed at 4 d after treatment, which was

Yin et al. 2012). Thiamine has been recently reported to

54.2% higher than that in the control fruit (Fig. 5-D). The

induce resistance in Asian pear fruit through activating

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Control 25 20 15 10 5 0

0

C

2 4 6 Days after treatment (d)

8

20 15 10 5 0

0

2 4 6 Days after treatment (d)

8

0

2 4 6 Days after treatment (d)

8

D 0.30

1.5 Flavonoids content (OD280 g–1 FW)

Total phenolic compounds content (OD325 g–1 FW)

Thiamine B 25 POD activity (U g–1 FW)

PAL activity (U g–1 FW)

A

1.2 0.9 0.6 0.3

0

2 4 6 Days after treatment (d) E

0.24 0.18 0.12 0.06 0

8

2.0

Lignin content (OD280 g–1 FW)

1.6 1.2 0.8 0.4 0

0

2 4 6 Days after treatment (d)

8

Fig. 5 Effects of thiamine treatment on the activity of phenylalanine ammonia lyase (PAL) (A) and peroxidase (POD) (B), the content of total phenolic compounds (C), flavonoids (D) and lignin (E) in muskmelon fruit. Each value is the mean for three replicates and the vertical bars indicate the standard error.

defense-related enzymes and accumulation of O2-. and H2O2 (Yin et al. 2012). The present study indicated that 25, 50, and 100 mmol L-1 thiamine significantly inhibited spore germination and mycelia growth of T. roseum and A. alternata. Our results were agreed with that of Yin et al. (2012) who found that 25–200 mmol L-1 thiamine inhibited spore germination and mycelia growth of A. alternata which caused black spot of pear fruit. However, Ahn et al. (2005) reported that thiamine at low concentrations (<50 mmol L-1) did not inhibit the growth of Magnaporthe grisea or Xanthomonas oryzae pv. oryzae. The differences could be explained that the antifungal activity of thiamine depends on using concentrations or vary among pathogen and host

species. We also found that postharvest application of thiamine significantly decreased lesion diameter in muskmelon fruit inoculated with T. roseum or A. alternata, and inhibition efficacy showed dose-dependent. Similar results were reported in Asian pear, rice, tobacco, tomato, cucumber, pearl millet, and Arabidopsis against semi-biotrophic and biotrophic pathogens (Ahn et al. 2005; Ahn et al. 2007; Liu et al. 2008; Pushpalatha et al. 2011; Yin et al. 2012). Ahn et al. (2005) have firstly reported that thiamine induced resistance in rice and Arabidopsis through activation of host defense responses. Our findings indicated that thiamine function as elicitor agent in muskmelon fruit because of inducing host defense responses without any

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pathogen infection. Thiamine induced the accumulation of reactive oxygen species at the early stage after treatment, and reactive oxygen species served as signal to activate the production of pathogenesis-related proteins and phenylpropanoid pathway to enhance the resistance of muskmelon fruit to pathogen invasion. The present results indicated that application of thiamine after harvest induced O2 . production and H2O2 accumulation in muskmelon fruit. Boubakri et al. (2012) found that accumulation of H2O2 in grapevine was closely related with resistance against Plasmopara viticola. Study in pear fruit also showed that thiamine induced H2O2 production was beneficial to disease resistance against A. alternata (Yin et al. 2012). Generation of ROS is one of the earliest cellular events in plants or fruit after pathogen invasion or following elicitor treatment (Boubakri et al. 2012). H2O2 has several functions in host against pathogen infection including direct inhibition pathogen growth, activation PR genes expression, cross-linking cell wall, triggering phytoalexins accumulation and hypersensitive responses (Torres et al. 2006; Shetty et al. 2008). The present study also indicated that thiamine increased the activities of CAT, APX and GR in muskmelon fruit. Study has shown that application of ascorbic acid improved antioxidant enzymes including CAT, and SOD of Pichia caribbica to enhance its biocontrol efficacy on apple fruits (Li et al. 2014). CAT and APX can scavenge excess H2O2 to eliminate the damage to host tissues. GR keeps the balance between reduced glutathione (GSH) and glutathione disulfide (GSSH) through ascorbate-glutathione (AsA-GSH) cycle. Different results were found in pear fruit that CAT activity was inhibited by thiamine treatment (Yin et al. 2012). This difference suggested that the H2O2 scavenging system in muskmelon fruit maybe depend on both CAT and AsA-GSH cycle. Similar inducing effects in fruit have been reported after treatment with other elicitors, including acibenzolar-S-methyl (ASM) (Ren et al. 2012), sodium silicate (Li et al. 2012). In addition, POD can also eliminate H2O2 to avoid harmful damage to cells. The present study showed that thiamine treatment significantly increased PAL activity in muskmelon fruit. As a rate-limiting enzyme in the phenylpropanoid pathway, the increasing activity of PAL is associated with the biosynthesis of antifungal compounds including phenolics, flavonoids and lignin (Vogt 2010). Application of thiamine after harvest also induced the increase of total phenolic compounds, flavonoids and lignin in the muskmelon fruit. In addition, the accumulation of phenolics may provide adequate substrate to produce fungitoxic quinines at the presence of polyphenol oxidase (PPO) and/or POD to directly inhibit pathogen growth (Lattanzio et al. 2006). Phenolics have also been reported to be involved in the restriction of A. alternata growth in pear fruit (Yin et al. 2012). Similar results have been found in muskmelon fruit pre-harvest or postharvest

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treated with ASM (Zhang et al. 2011; Liu et al. 2014). Higher levels of POD were found in muskmelon fruit after treating with thiamine. POD is also involved in lignin accumulation together with cinnamate-4-hydroxylase and laccase (Liu et al. 2014). The accumulation of lignin could strengthen the cell wall and act as an effective barrier against pathogen penetration and spread (Hematy et al. 2009; Kärkönen and Köutaniemi 2010). Induction of lignin accumulation was also reported in muskmelon fruit after dipping with ASM (Liu et al. 2014). Flavonoids are phenylpropanoid metabolites, which are synthesized from one of the branches of the phenylpropanoid pathway and significantly contributed to anthocyanin formation (Allan et al. 2008). Flavonoids contribute to resistance against pathogens commonly found during the storage of fruit and vegetables, and also have antimicrobial bioactivity (Pan and Liu 2011). In our experiment, thiamine induced the deposition of flavonoids in muskmelon fruit. Studies in grapevine, Asian pear fruit demonstrated that thiamine treatment increased flavonoids content by increasing the activities of key enzymes in phenylpropanoid pathway (Yin et al. 2012; Boubakri et al. 2013). These results suggest that increases in activities of PAL, POD are closely related to phenylpropanoid metabolites accumulation in fruit tissues.

5. Conclusion Our data demonstrate that thiamine could directly inhibit the growth of T. roseum and A. alternata and activate the phenylpropanoid pathway, production of ROS and enhanced antioxidative enzymes activities in muskmelon fruit. This suggests that postharvest application of thiamine could be promising in reducing decay and utilization of synthetic fungicides to control postharvest diseases in fruit and vegetables. However, further studies at the molecular level are needed to investigate the action of thiamine.

Acknowledgements This study was supported by the National Natural Science Foundation of China (31401554), and the Doctoral Initial Funding of Bohai University, China (bsqd201405).

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