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Postharvest Biology and Technology 77 (2013) 94101

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Postharvest Biology and Technology


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Effects and possible mechanisms of tea tree oil vapor treatment on the main disease in postharvest strawberry fruit
Xingfeng Shao , Hongfei Wang, Feng Xu, Sai Cheng
Department of Food Science and Engineering, Ningbo University, Ningbo 315211, Zhejiang, PR China

a r t i c l e

i n f o

a b s t r a c t
The purpose of this study was to investigate the effect of tee tree oil (TTO) against the main fungal disease in strawberries and a possible mechanism for the effects. TTO vapor exhibited a higher activity against spore germination and mycelial growth of Botrytis cinerea and Rhizopus stolonifer under in vitro conditions. TTO vapors at 0.9 g/L signicantly reduced articially inoculated gray mold and soft rot in vivo, and treated strawberries maintained a fresher quality than untreated strawberries during storage. In addition, this treatment also enhanced the resistance of strawberries against B. cinerea, which caused a higher hydrogen peroxide (H2 O2 ) level and activities of superoxide dismutase (SOD), phenylalanine ammonia-lyase (PAL), peroxidase (POD) and -1,3-glucanase during the rst period of incubation. These results indicate that TTO can reduce fruit decay, possibly by inhibiting pathogen growth directly and inducing disease resistance indirectly, and TTO vapor may provide an alternative means of controlling disease in strawberries. 2012 Elsevier B.V. All rights reserved.

Article history: Received 29 September 2012 Accepted 24 November 2012 Keywords: Melaleuca alternifolia Fragaria ananassa Fungus Disease resistance

1. Introduction Strawberries (Fragaria ananassa Duch.) are an especially perishable fruit that are susceptible to mechanical injury and decay during storage. Botrytis cinerea and Rhizopus stolonifer are the two major pathogens affecting strawberries during storage (Reddy et al., 2000). Application of synthetic fungicides for the control of these diseases is a standard commercial practice worldwide; however, interest is increasing in physical or biological methods to maintain postharvest quality and control disease in fruit because of the increasing awareness of chemical compounds that are potentially harmful to human health and the environment. In recent years, essential oils (EOs) have gained particular interest to control postharvest disease due to their volatility, relatively safe status, wide acceptance by consumers, and eco-friendly and biodegradable properties (Tzortzakis and Economakis, 2007). An important property of EOs is their bioactivity in the vapor phase and the limitation of aqueous sanitation for many commodities such as strawberries and grapefruit, making them possible fumigants (Tzortzakis, 2009). For example, in the vapor phase, Lippia scaberrima Sond EO controlled two mango postharvest spoilage pathogens (Regnier et al., 2008), origanum oil inhibited the growth of B. cinerea and gray mold in tomato fruit (Soylu et al., 2010), and EO from dill (Anethum graveolens L.) reduced fungal spoilage of cherry

Corresponding author. Tel.: +86 574 87609573; fax: +86 574 87609573. E-mail address: shaoxingfeng@nbu.edu.cn (X. Shao). 0925-5214/$ see front matter 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.postharvbio.2012.11.010

tomatoes (Tian et al., 2011). Furthermore, the volatile phases of the EOs are found to be more toxic to fungi than the contact phase (Regnier et al., 2008; Chutia et al., 2009; Soylu et al., 2010). Tea tree oil (TTO) is an EO steam-distilled from Melaleuca alternifolia L., a species native to northern New South Wales, Australia (Swords and Hunter, 1978). The oil is considered nontoxic, with a pleasant odor, and has been used as an alternative antimicrobial, antioxidant and anti-cancer agent (Kim et al., 2004; Giordani et al., 2006; Hammer et al., 2006). To date, less attention has been paid to TTO application against plant pathogens. Szczerbanik et al. (2007) suggested that the TTO vapor may provide an alternative means of controlling postharvest pathogens. Our previous studies revealed that the antifungal activity of TTO against B. cinerea was better than clove oil, garlic oil, peppermint oil and eucalyptus leaf oil (Cheng and Shao, 2011), which may be used to control disease in fresh fruit. Control of postharvest diseases by treatments such as heat, UV-C and antagonists seems to occur through two different mechanisms: a direct germicide effect on pathogens and an indirect effect by inducing defense mechanisms in the fruit tissue (Schirra et al., 2000; Zhao et al., 2008; Liu et al., 2010; Pombo et al., 2011). Previous studies have demonstrated that EOs exhibit a dose-dependent activity against spore germination and mycelial growth in vitro, which is associated with the degeneration of fungal hypha, the alternations of ultrastructure, and the disruption of the cell membrane (Soylu et al., 2006, 2010; Liu et al., 2009; Tian et al., 2011). However, little information is available on the effect of EOs on disease resistance. Induced host resistance always relates to the changes of defense-related enzymes, including peroxidase (POD),

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polyphenoloxidase (PPO), superoxide dismutase (SOD), catalase (CAT), phenylalanine ammonia-lyase (PAL), chitinase (CHI) and 1,3-glucanase (Cao et al., 2008; Zhao et al., 2008; Liu et al., 2010; Shao et al., 2010; Pombo et al., 2011). Arrebola et al. (2010) found that lemongrass oil treatment failed to stimulate the total phenolic content, PAL, -1,3-glucanase and CHI activities on naturally infected peach fruit. In addition, some studies suggest that EOs can enhance the resistance of strawberries or sunowers, but no detailed information about defense-related enzymes was men tioned in these reports (Fernndez-Ocana et al., 2004; Wang et al., 2007). To our knowledge, the effect of TTO on the disease resistance ability in strawberries has not been studied. The objectives of this study were (1) to investigate the antifungal activity of TTO against B. cinerea and R. stolonifer in vitro, (2) to examine the effects of TTO on articially inoculated infection in vivo, (3) to study the changes of disease resistance ability by TTO treatment and (4) to assess the quality change of strawberries by TTO vapor treatment.

2.3. Measurement of the effects of TTO on the articially inoculated infection in strawberries Strawberry fruit were surfaced-disinfected with ethanol and air-dried. Each fruit was articial wounded once to a depth of 2 mm and 3 mm in diameter by pressing them down on the head of a nail. A 20 L aliquot of a conidial suspension B. cinerea or R. stolonifer at 104 conidia/mL was inoculated into each wound. After drying, the inoculated fruit were randomly distributed into two groups. For the treated group, 15 fruit were placed into 1 L polystyrene containers with snap-on lids (the residual air spaces of the containers was approximate to 670 mL). Based on our preliminary experiments, an aliquot of 0.6 g TTO was put into a small beaker and placed in the sealed fruit container. TTO were spontaneously volatilized inside the residual air space of the containers at 25 C for 3 h (TTO concentration was recorded as 0.9 g/L air). Non-treated fruit were used as a control group. After treatment, the wounded fruit were released from the TTO vapor and stored at 20 C for 3 d. At the end of storage, the incidence and severity of the decay were measured. The incidence of decay was expressed as the percentage of infected wounds. Lesion diameter was expressed as the mean of the width and the length of each area of decay. Each treatment was replicated three times, and 15 fruit were used in each replicate. The entire experiment was conducted in triplicate. 2.4. Measurement of the effects of TTO on the induction disease resistance ability of strawberries Strawberries were wounded and inoculated with B. cinerea and then randomly distributed into two groups (a control and a TTO treatment). The treatment and storage methods were the same as the detailed information in Section 2.3. To evaluate the elicitation of active defense responses by TTO treatment, tissue samples surrounding each wound of the fruit were collected at 0, 12, 24, 36, 48, 60 and 72 h in each group. Each treatment involved three replications, and 70 fruit were used in each replicate. Ten fruit each per replication were evaluated at each sampling time. The entire experiment was conducted in triplicate. 2.4.1. Measurement of enzyme activity All enzyme extraction procedures were conducted at 4 C. For SOD, 1 g of the fresh sample was ground with 5 mL of 50 mM sodium phosphate buffer (pH 7.8). Fresh sample (1 g) was ground with 5 mL of 100 mM sodium phosphate buffer (pH 7.0) for ascorbate peroxidase (APX) and CAT. PAL was extracted with 100 mM sodium borate buffer at a pH of 8.7, containing 3% PVPP (m/v) and 0.14% -mercaptoethanol (v/v). For the measurement of POD and PPO, 1 g of the fresh sample was ground with 5 mL of 200 mM sodium phosphate buffer (pH 6.4) containing 3% PVPP (m/v). For chitinase and -1,3-glucanase, a fresh sample (10 g) was mixed with 10 mL of citric acidphosphate buffer (50 mM, pH 5.0). The extracts were then homogenized and centrifuged at 10,000 g for 20 min at 4 C. The supernatant was used for the enzyme assay. SOD activity was determined by the assay kits purchased from Nanjing Jiancheng Institute of Bioengineering (Nanjing, Jiangsu, China) according to the instructions of the manufacturer. APX activity was determined by adding 0.1 mL of the enzyme preparation to 3 mL of sodium phosphate buffer containing 0.1 mL of 9 mmol AsA and 0.1 mL of hydrogen peroxide (H2 O2 ), the substrates (Cao et al., 2008). Enzyme activity was dened as the decrease of absorbance, where one unit was dened as the change of 0.001 absorbance unit per minute at 400 nm. CAT activity was determined by adding 0.1 mL of the enzyme preparation to 3 mL of sodium phosphate buffer containing 0.2 mL of 75% H2 O2 , which was the substrate. One unit was dened as the change in 0.01 absorbance units per minute at 240 nm (Cao et al., 2008). PAL activity was analyzed using the method of Assis et al. (2001). One unit was dened as the change in 0.01 absorbance units per hour at 290 nm. POD activity was determined by the method of Kochba et al. (1977). Enzyme activity was dened as the increase in absorbance, where one unit was dened as the change in 0.01 absorbance units per minute at 460 nm. PPO activity was determined using the method of Tian et al. (2002). Enzyme activity was dened as the increase of absorbance, where one unit was dened as the change of 0.1 absorbance unit per minute at 400 nm. -1,3-Glucanase activity was determined using the method of Abeles et al. (1970); one unit was dened as the creation of 1 mg of glucose per hour. Chitinase activity was measured using the method of Boller et al. (1983); one unit was dened as the creation of 1 mol N-acethl-d-glucosamine per hour. The specic activity of all the enzymes was expressed as units per gram fresh weight. 2.4.2. Measurement of H2 O2 content For the determination of H2 O2 content, 2 g of fresh tissue was homogenized with 5 mL of chilled 100% acetone and centrifuged at 10,000 g at 4 C for 20 min. The supernatant was immediately analyzed using assay kits purchased from Nanjing Jiancheng Institute of Bioengineering (Nanjing, Jiangsu, China) according to the instructions of the manufacturer. H2 O2 content was expressed as mmol g1 FW. 2.5. The effects of TTO on fruit quality Un-inoculated strawberries were randomly distributed into a control and a TTO treatment group. Each group contained 60 fruit. The method of TTO vapor treatment

2. Materials and methods 2.1. Essential oil, pathogen and fruit TTO was obtained from the Agricultural Products Processing Research Institute at the Chinese Academy of Tropical Agricultural Sciences. It was analyzed by gas chromatographymass spectrometry (GCMS) prior to experimentation. The TTO contained 50.28% terpinen-4-ol and 4.22% 1,8-cineole, which was adjusted to comply with the international standard (Hammer et al., 2006). The highly virulent B. cinerea and R. stolonifer were isolated from spoiled strawberry fruit in a greenhouse and then identied by morphological and molecular biology methods. They were grown for 7 d on potato dextrose agar (PDA: 1 L of an infusion from potatoes containing 20 g/L glucose and 15 g/L agar) at 25 C. Spores from a 7-day-old culture were suspended in 0.05% Tween-20 and adjusted to 104 conidia/mL using a hemacytometer. Strawberries (Fragaria ananassa Duch. cv. Hongyan) were harvested by hand at the mature red stage from a commercial greenhouse around Ningbo University, PR China. The fruit were transferred to the laboratory within 2 h. All fruit samples were uniform in size (approximately 25 g per fruit) and color and free of physical injury or signs of infection.

2.2. Measurement of the antifungal ability of TTO on B. cinera and R. stolonifer under in vitro conditions 2.2.1. Spore germination The inhibition of spore germination and mycelial growth was analyzed by using the modied method of Soylu et al. (2010). Glass Petri dishes (90 mm 20 mm, which offer 80 mL air spaces after addition of 20 mL of agar media) were lled with 20 ml of PDA, and 50 L aliquots of the spore suspension (104 conidia/mL) were spread onto the surface of PDA medium. Sterile lter paper discs (10 mm diameter) were attached to the inner surface of each Petri dish lid. The appropriate amount of oil (40, 80 and 120 mg) was added onto the lter paper, and the dishes were quickly covered. The petri dishes were wrapped with paralm along the rim to inhibit the release of volatile components. The compounds were allowed to volatilize inside the Petri dishes spontaneously at 25 C for 3 h. The TTO concentrations were recorded as 0.5, 1.0 and 1.5 g/L air. The controls were prepared similarly with the exception of the volatile treatment. After the vapor treatment, the lter paper was taken out of the dishes and the TTO vapor was replaced with normal air. All dishes were incubated at 20 C for 3 d, and fungal colonies were marked and counted. The percent inhibition = [(Gc Gt)/Gc] 100, where Gc and Gt represent the mean number of germinated conidia in control and treated dishes, respectively. The experiments were conducted nine times.

2.2.2. Mycelial growth Glass petri dishes were lled with 20 mL of PDA and one disc (5 mm diameter) of a mycelial plug that was taken from the edge of a 7-day-old fungal culture and was placed onto PDA in the Petri dishes. The method of TTO vapor treatment has been described in Section 2.2.1. After the vapor treatment, all plates were incubated at 20 C for 7 d. The efcacy of the treatment was evaluated by measuring the average of two perpendicular diameters of each colony. Percentage mycelial inhibition = [(dc dt)/dc] 100, where dc is the mean colony diameter for the control sets and dt is the mean colony diameter for the treatment sets. All tests were repeated nine times.

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Fig. 1. The effects of different tea tree oil (TTO) vapor concentrations on spore germination and mycelial growth of B. cinerea and R. stolonifer. Vertical bars represent the standard error of the mean. has been described in Section 2.3. After treatment, these fruit were released from the TTO vapor and stored at 20 C for 3 d to investigate the changes in quality parameters and natural disease development. Each day, 15 fruit per group were evaluated. Three replicates for each parameter were measured, and the entire experiment was conducted three times. Weight loss was expressed as the reduction in weight as a percentage of the total weight. Fruit rmness (N) was measured on two opposite sides of 5 fruit from each replicate with a TA-XTplus texture analyzer (Stable Micro System Ltd., Haslemere, UK) equipped with a 5 mm diameter probe at a speed of 1 mm/s. The soluble solid content (SSC) and titrated acid (TA) were measured by the method of Shao et al. (2012). Anthocyanin in fruit extracts was determined using the pH differential method (Esti et al., 2002). 2.6. Statistical analyses SAS Software (Version 8.2; SAS Institute, Cary, NC, USA) was used to conduct statistical analyses. Data from all the triplicate experiments were analyzed by oneway analysis of variance (ANOVA). Comparison of means was performed by Duncans multiple range tests. A value of P < 0.05 was considered statistically signicant. Fig. 2. The effects of tea tree oil (TTO) vapor treatment on the articially inoculated infection in strawberries. Vertical bars represent the standard error of the mean.

spore inhibition rate reached 60 and 75% and the mycelial inhibition rate reached 87.3% and 40%, respectively. Compared with the lower concentration, TTO at 1.0 and 1.5 g/L showed a higher antifungal effect on B. cinerea or R. stolonife (Fig. 1). However, the difference between 1.0 and 1.5 g/L was not signicant.

3.2. Effects of TTO vapor on articially inoculated infection in strawberries Based on our pre-test, the TTO treatment at 0.9 g/L air for 3 h is suitable for strawberry preservation. The TTO treatment (0.9 g/L air) can reduce the gray mold caused by B. cinerea and soft rot caused by R. stolonifer (Fig. 2). After 3 days of storage at 20 C, the articial decay incidence of the control groups for both fungi was 100%, and the lesion diameter was 11.5 and 12 mm, respectively. The decay incidence and diameter of gray mold were reduced to 55% and 5.0 mm by the TTO vapor treatment, respectively. The soft rot was reduced to 80% and 7.5 mm, respectively. TTO showed a more effective alleviation of the infection caused by B. cinerea than that caused by R. stolonifer in articially inoculated strawberries.

3. Results 3.1. Effects of TTO vapor on spore germination and mycelial growth The different concentrations of TTO were found to inhibit the spore germination and mycelial growth of B. cinerea and R. stolonifer (Fig. 1). Even at the lower concentration (0.5 g/L air), the

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Fig. 3. The effects of tea tree oil (TTO) vapor on the superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) activity and hydrogen peroxide (H2 O2 ) content of strawberries inoculated with B. cinerea. Vertical bars represent the standard error of the mean.

3.3. Effects of TTO vapor on the SOD, APX and CAT activity and H2 O2 content SOD activity of control and TTO-treated groups generally increased during incubation (Fig. 3A). The TTO treatment improved the SOD activity, which resulted in higher SOD activities in the treatment than in the control across the entire incubation period. The signicant (P < 0.05) difference was especially apparent in the rst 24 h of incubation. APX activity in both the control and treatment declined at the rst 36 h, increased to a peak value at 48 h and then decreased sharply (Fig. 3B). The fruit treated with TTO showed signicantly (P < 0.05) lower APX activity in comparison with the control fruit during incubation. CAT activity of both groups showed a similar pattern (Fig. 3C), with TTO-treated fruit showing a higher level of activity in the rst 24 h compared with the control. The H2 O2 content in TTO-treated fruit was signicantly (P < 0.05) higher than that in control fruit except at 72 h (Fig. 3D). 3.4. Effects of TTO vapor on PAL, POD and PPO activity PAL activity of both groups initially increased, followed by a decrease, and a peak value occurred at 24 h. PAL in the TTO-treated group exhibited a signicantly (P < 0.05) higher value than that of the control during the rst 48 h, followed by lower values after this time-point. POD activity in both control and treatment groups showed a similar pattern (Fig. 4B), with a peak value found at 48 h. Prior to the rst 36 h, the TTO treated group showed a statistically (P < 0.05) higher POD value than that of the control. PPO activity of both groups also showed a similar pattern (Fig. 4C). The PPO

activity was signicantly lower in TTO-treated fruit than in control fruit (Fig. 4C). 3.5. Effects of TTO vapor on the activity of -1,3-glucanase and CHI The changes of -1,3 glucanase and CHI are erratic (Fig. 5). The TTO treatment increased the activity of -1,3 glucanase during the rst 24 h (Fig. 5A), but a signicant (P < 0.05) difference was only observed in the rst 12 h. The activity of CHI in the TTO-treated fruit showed no signicant (P > 0.05) change during storage (Fig. 5B). 3.6. Effects of TTO vapor on the fresh quality in un-inoculated strawberry fruit The TTO treated group showed in a decline in fruit rmness (Fig. 6A). A signicant difference (P < 0.05) occurred after 24 h of storage. For the SSC and TA contents (Fig. 6B and C), the TTO treatment maintained signicantly (P < 0.05) higher values than the control at the end of storage. Furthermore, this treatment markedly (P < 0.05) decreased weight losses (Fig. 6D). The TTO treated group showed higher anthocyanin content; however, the difference was not signicant (P > 0.05) during the 72 h of storage. The TTO treatment did not affect the avor of strawberries after 24 h of storage. 4. Discussion TTO vapor treatment can signicantly reduce the spore germination and mycelial growth of B. cinerea and R. stolonifer in vitro

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Fig. 4. The effects of tea tree oil (TTO) vapor on the phenylalanine ammonia-lyase (PAL), peroxidase (POD) and polyphenoloxidase (PPO) activity of strawberries inoculated with B. cinerea. Vertical bars represent the standard error of the mean.

(Fig. 1). Chutia et al. (2009) suggested that EOs in the volatile phase are effective at a very low concentration and are advantageous in limiting the spread of the pathogen by lowering the spore load. For the in vivo experiment, the TTO vapor treatments also alleviated the gray mold and soft rot in articially infected strawberries (Fig. 2). This treatment also maintained the fresh quality of

strawberries and allowed for higher rmness, SSC and TA content and lower weight loss (Fig. 6). TTO is classied as generally regarded as safe (GRAS) by the United State Food and Drug Administration (Hammer et al., 2006). One of the earliest and most prominent fruit defense responses is an oxidative burst, which generates reactive oxygen species

Fig. 5. The effects of tea tree oil (TTO) vapor on the activity of standard error of the mean.

-1,3-glucanase and chitinase (CHI) of strawberries inoculated with B. cinerea. Vertical bars represent the

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Fig. 6. The effects of tea tree oil (TTO) vapor on the quality changes of un-inoculated strawberries. Vertical bars represent the standard error of the mean.

(ROS) (Lamb and Dixon, 1997). The accumulation of ROS serves not only as a toxin to pathogens, but as a signal that activates additional fruit defense reactions (Lamb and Dixon, 1997). This is especially true for H2 O2 , which can be induced by different postharvest treatments (including heat treatment, methyl jasmonate and antagonist) to improve disease resistance (Cao et al., 2008; Liu et al., 2010, 2012; Shao et al., 2010). Generally, the metabolism of ROS is controlled by an array of enzymes including SOD, CAT and APX. O2 is efciently converted to H2 O2 by the action of SOD, while H2 O2 is predominantly destroyed by APX and CAT (Nishikawa et al., 2003). Zhang et al. (2012) demonstrated that 1-methylcyclopropene treatment induced activities of CAT and SOD in jujube fruit inoculated with Penicillium expansum. Cao et al. (2008) found that methyl jasmonate treatment signicantly inhibits the activities of CAT and APX, while SOD activity was not signicantly affected in loquat fruit, resulting in a higher level of H2 O2 in the earlier period of incubation. In this study, the TTO treatment signicantly improved SOD activity during the initial period of incubation and reduced APX activity during storage, which may have caused higher levels of H2 O2 in the rst 60 h of incubation. This indicated that TTO may affect the

accumulation of H2 O2 in the rst period of incubation, resulting in enhanced pathogen resistance in strawberries. This might be due to the mechanism developed by fruit that uses secondary metabolites in TTO as a signal to induce defense. The phenylpropanoid pathway is important in secondary plant metabolism and produces a variety of phenolics with structural and defense-related functions including lignins, phenolic acids, stilbenes, and avonoids. PAL is a key enzyme in the rst step of the phenylpropanoid pathway, which is related to the synthesis of phenolic acid and lignin (Dixon et al., 2002). In addition to PAL, POD is involved in the last step of the polymerization of cinnamyl alcohols to form lignin and is directly involved in the induction of defense mechanisms (Passardi et al., 2004). Heat treatment was previously found to induce the expression and activity of PAL in infected peaches (Liu et al., 2012). Pombo et al. (2011) demonstrated that UV-C treatment improved the expression and activities of PAL and POD in strawberries inoculated with B. cinerea during the rst 24 h of storage. However, Arrebola et al. (2010) suggested that EO treatment cannot stimulate the levels of total phenolic content and PAL activities in naturally infected peaches. In this study, we

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observed that TTO treatment enhanced the PAL and POD activities during the initial period of incubation. Hence, POD and PAL could be implicated in the formation of a mechanical barrier preventing pathogen penetration at the wound site (Pombo et al., 2011). PPO is a copper enzyme that can catalyze several reactions leading to the formation of quinones. Quinone synthesis is one of the rst symptoms in response to fungal attack or wounding, and these compounds have effective antimicrobial properties (Yoruk and Marshall, 2003). Zhang et al. (2012) demonstrated that 1methylcyclopropene treatment induces activities of PAL and PPO in jujube fruit inoculated with P. expansum. Heat treatment significantly enhanced the activities of POD and PPO activity in Chinese bayberries to control disease (Wang et al., 2010). However, some researchers suggested that PPO may be not involved in disease resistance in apples and loquat fruit (Liu et al., 2010; Shao et al., 2010). In this study, PPO activity in strawberries was reduced by TTO treatment during the incubation. Pathogenesis-related (PR) proteins are a class of defense molecules that are induced by pathogen attack, wounding, or environmental stressors. CHI, which belong to the PR-2 family, and -1,3-glucanase, which belong to the PR-8 family, are the most fully characterized PR proteins associated with defense responses (Van Loon et al., 2006). There is compelling evidence that -1,3glucanase can act directly by degrading the pathogen cell wall, or indirectly by releasing oligosaccharide elicitors of defense reactions, both of which are potential defense mechanisms against fungal infection (Liu et al., 2010; Zhao et al., 2008). CHI can also degrade chitin, one component of the pathogen cell wall. Researches demonstrated that heat treatment can induce the activities of CHI and -1,3-glucanase in infected peaches (Liu et al., 2012) and Chinese bayberries (Wang et al., 2010). For strawberries treated with UV-C, CHI activity increased at 10 h post-treatment, while -1,3-glucanase was enhanced after 4 and 24 h of storage. Our data indicated that TTO treatment can signicantly increase -1,3-glucanase activity during the rst 12 h of incubation, but did not signicantly enhance the CHI activity in strawberries. However, lemongrass oil treatment cannot affect the activities of both enzymes in peaches (Arrebola et al., 2010). This discrepancy could be due to the different fruit examined and EOs used. Further studies are needed to explore the mechanism of PR proteins in strawberries treated by TTO. In conclusion, TTO vapor signicantly reduced B. cinerea and R. stolonifer in vitro, the main pathogens effecting postharvest strawberries. TTO vapor at 0.9 g/L also inhibited articially inoculated gray mold caused by B. cinerea and soft rot caused by R. stolonifer and maintained the fresh quality of strawberries during storage. This treatment may enhance the resistance of strawberries against B. cinerea through induction of H2 O2 levels and several defenserelated enzymes in the rst period of incubation. These results suggest that the mode of action of TTO appears to be both via direct interaction with the fungus itself and via defensive responses of fruit tissue. TTO vapor treatment is a promising method for controlling pathogen effects on strawberries during postharvest storage. Acknowledgements This study was sponsored by the National Science Foundation of China (No. 31271943), Natural Science Foundation of Zhejiang Province (No. Y12C200009) and Ningbo City (No. 2012A610143) and the K.C. Wong Magna Fund at Ningbo University. References
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