Ascorbic Acid Enhances Oxidative Stress Tolerance and Biological

Article pubs.acs.org/JAFC

Ascorbic Acid Enhances Oxidative Stress Tolerance and Biological Control Efficacy of Pichia caribbica against Postharvest Blue Mold Decay of Apples Chaolan Li, Hongyin Zhang,* Qiya Yang, Mahunu Gustav Komla, Xiaoyun Zhang, and Shuyun Zhu School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, Jiangsu, People’s Republic of China ABSTRACT: The effect of ascorbic acid (VC) on improving oxidative stress tolerance of Pichia caribbica and biocontrol efficacy against blue mold caused by Penicillium expansum on apples was investigated. P. caribbica showed susceptibility to the oxidative stress in vitro test, and 250 μg/mL VC treatment improved its oxidative stress tolerance. The higher viability exhibited by VCtreated yeast was associated with a lower intracellular ROS level. The activities of antioxidant enzymes of P. caribbica were improved by VC treatment, including catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX). Additionally, VC-treated yeast exhibited greater biocontrol activity against P. expansum and faster growth when stored at 25 and 4 °C, respectively, compared to the performance of the non-VC-treated yeast. In response to the VC treatment under oxidative stress, several differentially expressed proteins were identified in P. caribbica, and most of the poteins were confirmed to be related to basic metabolism. Therefore, the application of ascorbic acid is a useful approach to improve oxidative stress tolerance of P. caribbica and its biocontrol efficacy on apples. KEYWORDS: oxidative stress, antioxidant enzymes, Pichia caribbica, ascorbic acid, Penicillium expansum, biocontrol, differentially expressed proteins

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INTRODUCTION For the past few years, use of antagonistic microbial agents to control postharvest disease has spread greatly.1 Similar to other nonfungicide resources, biocontrol yeasts cannot achieve the same effect compared with synthetic fungicides.2 Due to all kinds of environmental stresses imposed by a variety of biotic and abiotic stresses, which have major impacts on yeast growth, the biocontrol efficacy of yeasts decreased because of the lower viability.3 Oxidative stress, one of the most common environmental stresses, plays a pivotal role in biocontrol systems.4 The ability of antagonistic agents to survive and proliferate in wounds or on fruit surfaces when applied in the orchard is a key factor for antagonists.5 When fruits are exposed to the practical environment, the fruit tissue is associated with the accumulation of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and superoxide anions (O2• −) that can influence host response,4 pathogen virulence,6 yeast efficacy,7 and vital cellular macromolecules, via denaturation of proteins, mutations of DNA, and peroxidation of lipids. Thus, enhancing the oxidative stress tolerance of biocontrol yeasts may be a useful approach to improve the controlling activity. Many yeast species have been confirmed to contol postharvest diseases effectivly.8 Among these yeast antagonists, Pichia caribbica has exhibited control efficacy against postharvest diseases of strawberries.2 To keep survival viability under oxidative stress, appropriate methods adjusting biochemical and physiological states have to be applied with P. caribbica. Once exposed to oxidaitve stress induced by H2O2, yeasts activate a number of antioxidative stress-related defense mechanisms. These include multiple isoforms of superoxide dismutase (SOD) converting O2• − into H2O2, and then catalases and peroxidases further metabolize H2O2 to H2O.9,10 © 2014 American Chemical Society

Correlation between increased levels of antioxidative compounds and enzymes with adaptation to oxidative stress conditions has become a hot topic. Ascorbic acid (VC), an important water-soluble antioxidant, is one of the most common food additives and endogenous substances that plays a crucial role in protecting plants, animals, microorganisms, and humans from oxidative stress.11 As an important antioxidant compound, cellular ascorbic acid can protect fruit membranes from lipid peroxidation.12 Additionally, Pinto et al.13 reported that browning happened to the fruits during storage under various CO2 concentrations because of the VC decrease in pears. Moreover, it was reported that exogenous application of VC improved antioxidant enzyme activity and reduced MDA in the eyes of rats, so as to increase its effect on oxidative stress.14 However, to our best knowledge, there is little information about the effect of VC on antioxidant systems in microorganisms, especially in postharvest biocontrol area. Furthermore, there is no information concerning the differentially expressed proteins between non-VC-treated yeast and VCtreated yeast under oxidative stress. The purpose of the present study was to determine the effect of VC on oxidative stress tolerance and biocontrol efficacy of the yeast antagonist P. caribbica. Specifically, this study investigated (i) the effect of VC on the activity of P. caribbica in controlling blue mold decay of apples; (ii) the viability of P. caribbica under a range of oxidative stress conditions induced by different concentrations of H2O2; (iii) the effect of VC on Received: Revised: Accepted: Published: 7612

April 25, 2014 July 3, 2014 July 3, 2014 July 3, 2014 dx.doi.org/10.1021/jf501984n | J. Agric. Food Chem. 2014, 62, 7612−7621

Journal of Agricultural and Food Chemistry

Article

concentration of 1 × 108 cells/mL, and the non-VC-treated (NVCtreated) and 250 μg/mL VC-treated yeast suspensions were respectively exposed to 10 mM H2O2 according to the previous study and incubated at 28 °C at 190 rpm. After 20 min, colonies were evaluated with the same method as described above to test the survival of P. caribbica under oxidative stress. There were three replicates of each treatment, and the experiment was conducted three times. Determination of Intracellular Reactive Oxygen Species. 2,7Dichlorodihydrofluorescein diacetate (H2DCFDA), the oxidantsensitive probe, was used to determine the intracellular ROS production of P. caribbica according to the procedure of Liu et al.,3 with a slight modification. Non-VC-treated and VC-treated yeast cells (the final concentration of VC was 250 μg/mL) were collected and then exposed to 10 mM H2O2 at 0, 20, 40, and 60 min, and cells without H2O2 treatment was considered as control. After exposure, cells were harvested quickly by centrifugation and washed twice with phosphate-buffered saline (PBS) buffer (pH 7.0) and resuspended in the same buffer containing 25 μM H2DCFDA (dissolved in dimethyl sulfoxide). The suspension was incubated in the dark at 30 °C for 1 h. After washing twice with PBS buffer, intracellular ROS were examined with a fluorospectrophotometer using λexcitation = 504 nm and λemission = 522 nm. The higher relative intensity indicates a higher ROS level. There were three replicates of each treatment, and the entire experiment was repeated three times. Determination of the Enzyme Activities of CAT, SOD, and GPX. Yeasts were prepared as described and then supplemented with VC at the final concentration of 250 μg/mL. The yeast cells without VC treatment were considered as control. Cells were exposed to 10 mM H2O2 at 28 °C at 190 rpm for 1 h, then harvested by centrifugation at 10000 rpm for 10 min, and washed three times with sterile distilled water to remove residual VC and H2O2. The yeast samples at each time point (before H2O2 treatment and 0, 20, 40 and 60 min after exposure to H2O2) were harvested by centrifugation at 10000 rpm for 10 min and washed three times with sterile distilled water. Cells were disintegrated in liquid nitrogen and suspended in chilled potassium phosphate buffer (50 mM, pH 7.8, containing 5 mM DL-dithiothreitol and 5% PVPP). The cell suspension was centrifuged at 10000 rpm for 20 min at 4 °C, and the supernatant was used for enzyme assays. SOD activity was evaluated by spectrophotometric assay according to the method of Bernstein et al. 18 A reaction mixture containing no protein extract was used as the control. Test tubes with the reaction mixture were irradiated under fluorescent lights at 4000 lx for 20 min. Then the absorbance of each solution was measured at 560 nm. One unit of enzyme activity was defined as the amount of enzyme inhibiting 50% of NBT photoreduction. Results were analyzaed from three independent replicates. CAT activity was measured by monitoring at 240 nm with the decrease in absorbance due to the decomposition of H2O2 according to the method of Reverberi et al.19 The reaction solution (3 mL) contained 2.8 mL of 30 mM H2O2 (dissolved in phosphate buffer (pH 7.8)) and 200 μL of extracted solution. After addition of the enzyme solution, the reaction was initiated, and one unit was defined as a decrease in A240 of 0.01 per minute. Results were analyzed from three independent replicates. GPX was assayed by monitoring at 422 nm with the decrease in absorbance due to the reduction of GSH at 37 °C. The reaction mixture (1 mL) contained 0.4 mM GSH, 0.3 mM H2O2, and 0.3 mL of enzyme solution. The mixture was placed in an electrically heated thermostatic water bath at 37 °C for 5 min. Subsequently, 4 mL of 1.67% (w/v) meta-phosphoric acid (containing 0.05% (w/v) EDTA; 28% (w/v) NaCl) was quickly added to terminate the reaction followed by centrifugation for 10 min at 3000 rpm. Two milliliters of supernatant was added with 0.32 M Na2HPO4 (2.5 mL) and 0.5 mL of DTNB for 5 min. One unit was defined as a decrease of 1 μM GSH in 37 °C per minute. Results were analyzed from three independent replicates The activities of SOD, CAT, and GPX were expressed as units per milligram of protein. Protein content was measured as described by Bradford,20 bovine serum albumin (BSA) being used as standard.

the viability of P. caribbica under oxidative stress; (iv) the effect of VC on ROS accumulation; (v) the effect of VC on induction of antioxidant enzymes, including catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX) in P. caribbica; (vi) the effect of VC on population dynamics of P. caribbica both in wounds and on the surface of apple fruit; and (vii) the biological function of the differentially expressed proteins of P. caribbica combined with VC and P. caribbica as sole treatment.

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MATERIALS AND METHODS

Yeast. P. caribbica was obtained from the culture collection maintained by our laboratory at 4 °C on nutrient yeast dextrose agar (NYDA; 8 g of nutrient broth, 5 g of yeast extract, 10 g of glucose, and 20 g of agar, 1 L of distilled water; Sangon, Shanghai, China). A loop of the culture was inoculated in nutrient yeast dextrose broth (NYDB) and incubated on a rotary shaker at 180 rpm at 28 °C for 24 h. After incubation, cells were centrifuged at 5000 rpm for 10 min and washed twice with sterile distilled water to remove the residual medium.15 Cell pellets were then resuspended in sterile distilled water and adjusted to an appropriate concentration with a hemocytometer for experiments. Pathogen. Penicillium expansum was isolated from infected apples and maintained on potato dextrose agar (PDA; extract of boiled potatoes, 200 mL; dextrose, 20 g; agar, 20 g; and distilled water, 800 mL) at 4 °C. Spore suspensions were harvested by removing the spores from a 7-day-old culture and suspending them in sterile distilled water. With a hemocytometer, spore concentrations were determined and adjusted as required. Fruits. Apple (Malus domestica Borkh.) fruits at commercial maturity without wounds or rot were selected on the basis of uniformity of size and color, disinfected with 1% (w/v) sodium hypochlorite for 1−2 min, rinsed with tap water, air-dried, and prepared for subsequent experiments. Efficacy of P. caribbica in Controlling Blue Mold Decay of Apples. Three wounds were made on the equator of each apple with a sterile cork borer (approximately 5 mm diameter and 3 mm deep) and treated with 30 μL of different treated solutions as follows: (1) cell suspensions of P. caribbica (1 × 108 cells/mL); (2−6) cell suspensions of P. caribbica alone (1 × 108 cells/mL) supplemented with different concentrations of VC at 150, 200, 250, 300, or 350 μg/mL separately; and (7) sterile distilled water as the control. Biocontrol activity of P. caribbica was tested according to the procedure of Cao et al. 16 with some modifications. Three hours later, 30 μL of P. expansum suspension (5 × 104 spores/mL) was inoculated into each wound. After being air-dried, fruits were placed in enclosed plastic trays to maintain a high relative humidity (about 95%) and incubated at 25 °C. Disease incidences of apple fruits caused by P. expansum were determined after 15 days. There were three replications of each treatment that consisted of 12 fruits. The entire experiment was repeated three times, and the overall percentage of infection was calculated. Evaluation of Survival of P. caribbica under Oxidative Stress. The survival of P. caribbica under oxidative stress was determined according to the method of Liu et al.,3 with a slight modification. Yeasts was prepared as described, and 4 mL of yeast cell suspension at a final concentration of 1 × 108 cells/mL was placed in a 20 mL of test tube. Then the same volume of H2O2 was added at different concentrations (0, 5, 10 and 20 mM) into the test tube, exposing the yeast cells to oxidative stress at 28 °C on a shaker at 190 rpm for 0, 20, 40, or 60 min. At the described time points, 100 μL of serial 10-fold dilutions (from 5 × 107 to 5 × 103 cells/mL) of the samples were spread on NYDA. Colonies were counted after incubation at 28 °C for 3 days and expressed as the log10 colon-forming units per milliliter (CFU/mL) of each wound. There were three replicates of each treatment, and the entire experiment was conducted three times. Effect of VC on Oxidative Stress Tolerance of P. caribbica. The effect of VC on survival of P. caribbica after H2O2 treatment was determined according to the method of Deveau et al.17 with some modifications. Yeasts was prepared as described and adjusted to a final 7613

dx.doi.org/10.1021/jf501984n | J. Agric. Food Chem. 2014, 62, 7612−7621


Article

Population Dynamics of P. caribbica in Apple Fruits. Population Dynamics of P. caribbica on the Surface of Apple Fruits. Four circles (approximately 14 mm diameter) were made on the upper part of each apple fruit with a marker pen. Two treatment solutions were used, VC-treated and non-VC-treated suspensions of P. caribbica. Three drops of 10 μL of these solutions were added evenly in each circle. Treated fruits in plastic trays were stored at 20 and 4 °C, respectively. Fruit samples were collected at different time points after treatment. At each time point, yeasts were harvested by removing the circled tissues with a sterilized knife. Samples were then ground with a mortar and pestle in 50 mL of sterile distilled water. Then, 100 μL of serial 10-fold dilutions were spread on NYDA plates. One hour after samples had been taken served as time 0. Fruits stored at 20 °C were assessed at 0 (1 h after the apples were inoculated with P. caribbica), 12, 24, 36, 48, 60, 72, 84, and 96 h, whereas those stored at 4 °C were assessed at 0 (1 h after the apples were inoculated with P. caribbica), 4, 5, 6, 7, 8, 9, 10, and 11 days. Colonies were counted after incubation at 28 °C for 48 h and expressed as the log10 CFU per wound. Each treatment contains three replications that consisted of 24 fruits, and the experiment was repeated three times. Population Dynamics of P. caribbica in Wounds of Apple Fruits. Apples were prepared and wounded as described above. A 30 μL suspension of P. caribbica (1 × 108 cells/mL) alone (NVC) or P. caribbica (1 × 108 cells/mL) added with VC at a final concentration of 250 μg/mL (VC) was applied to each wound. Treated fruits were placed in enclosed plastic trays and stored at 20 and 4 °C, respectively. Fruit samples were collected with a sterile cork borer (9 mm diameter) to a depth of 10 mm21 at different time points after treatment as described above. Fruits stored at 20 °C were assessed each day for 10 days, whereas those stored at 4 °C were assessed every 3 days for 33 days (1 h after the apples were inoculated with P. caribbica served as time 0). Colonies were counted after incubation at 28 °C for 48 h and expressed as the log10 CFU per wound. There were three replications of each treatment, and the entire experiment was repeated three times. Biological Function of the Differentially Expressed Proteins of P. carbbica Combined with VC and P. caribbica as Sole Treatment. Protein Sample Preparation. Non-VC-treated and VCtreated yeast cells were collected and then exposed to 10 mM H2O2 for 60 min. The yeast cells were harvested by centrifuging at 10000g for 10 min (4 °C) and washed three times with cold double-distilled water to remove residual VC and H2O2. Protein sample preparation was performed as described by Li et al.22 Two-Dimensional Electrophoresis (2-DE) and Image Analysis. 2DE and image analysis were performed as described by Wang et al.23 using the vertical electrophoresis systems, Image Scanner III and Image Master 2D Platinum 7.0 software (GE Healthcare Bio-Science, Uppsala, Sweden), respectivly. Protein In-Gel Digestion. Differentially expressed protein spots were excised from the gel and prepared for MS analysis according to the procedure of Zhang et al.24 Protein Identification by MALDI-TOF/TOF and Database Query. The peptide solution was analyzed using MALDI TOF/TOF mass spectrometer (Bruker, Germany). The resulting monoisotopic peptide masses were queried against protein database in NCBInr using MASCOT software according to the procedure of Majoul et al.25 Data Analysis. All statistical analyses were performed by analysis of variance (ANOVA) using the statistical program SPSS/PC version 16.0 (SPSS Inc., Chicago, IL, USA), and Duncan’s multiple-range test was used for means separation. Differences at p < 0.05 were considered significant.

Figure 1. Efficacy of P. caribbica in controlling blue mold decay of apples stored at 25 °C. Sterilized water was used as control. Each value is the mean of three experiments. Bars represent the standard error of the mean. Data in columns with different letters are significantly different according to Duncan’s multiple-range test at p < 0.05.

yeast combined with 250 μg/mL VC were 38.9 and 19.4%, respectively, whereas disease incidence in the control fruits (inoculated with water followed by the pathogen) reached 94.4%. Evaluation of Survival of P. caribbica under Oxidative Stress. The viability of P. caribbica cells decreased with increasing H2O2 concentration and exposure time within each concentration (Figure 2). At 0 min, the yeast with all

Figure 2. Population of P. caribbica under oxidative stress induced by H2O2. Each value is the mean of three experiments. Bars represent the standard error of the mean. Data in columns with different letters at the same time point are significantly different according to Duncan’s multiple-range test at p < 0.05

treatments had the same population level, whereas quite different levels were recorded at other time points. Furthermore, the population of P. caribbica cells reduced dramatically with a higher H2O2 concentration, particularly under the oxidative stress condition induced by 20 mM H2O2 for 60 min, in which yeast cells were nearly all dead. On the basis of the result of the viability assay, 10 mM H2O2 was chosen to be the appropriate stress condition for the following experiments. Effect of VC on Oxidative Stress Tolerance of P. caribbica. As indicated in Figure 3, VC treatment had a significant effect on P. caribbica viability (p < 0.05); supplementation of VC improved the yeast viability from 2.73 × 104 cells/mL to 4.15 × 104 cells/mL. ROS Accumulation of P. caribbica under Oxidative Stress. As illustrated in Figure 4, the ROS accumulation of

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RESULTS Efficacy of P. caribbica in Controlling Blue Mold Decay of Apples. As shown in Figure 1, P. caribbica effectively controlled postharvest blue mold decay of apples caused by P. expansum after 15 days of storage at 25 °C compared with the control fruits (p < 0.05). However, VC-treated yeast exhibited a better biocontrol efficacy compared to non-VC-treated yeast. Disease incidences in apple fruits treated with yeast alone and 7614



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Figure 3. Effect of VC on oxidative stress tolerance of P. caribbica. Each value is the mean of three experiments. Bars represent the standard error of the mean. Data in columns with different letters are significantly different according to Duncan’s multiple-range test at p < 0.05.

Figure 4. ROS accumulation in non-VC-treated and VC-treated cells of P. caribbica exposed to oxidative stress (10 mM H2O2) for various periods of time. Sterilized water was used as control. Each value is the mean of three experiments. Bars represent the standard error of the mean. Data in columns with different letters at the same time point are significantly different according to Duncan’s multiple-range test at p < 0.05.

both treatments reached the highest level at 40 min. VC-treated cells showed significantly lower reactive oxidative stress (ROS) level compared to non-VC-treated cells at all tested time points except 60 min. Activities of Antioxidant Enzymes. Adding exogenous VC enhanced the enzyme activities of SOD, CAT, and GPX of P. caribbica at some time point under oxidative stress. H2O2 induced the SOD activity, and VC improved its activity after exposure (Figure 5A). At 20 min, VC had the most obvious efficacy, and at 40 min, both treatments had the highest level of SOD activity, but all decreased at 60 min. VC did not increase CAT activity significantly until 40 min. However, CAT activity in non-VC-treated cells decreased greatly, whereas the CAT activity in VC-treated cells increased markedly at 40 min, and both of them exhibited a decreasing trend at 60 min (Figure 5B). When exposed to H2O2, GPX decreased tremendously before 20 min and then increased (Figure 5C). GPX activity of VC-treated P. caribbica was significantly higher than that of non-VC-treated P. caribbica at all tested times. Population Dynamics of P. caribbica in Apple Fruits. Population Dynamics of P. caribbica on the Surface of Apple Fruits. The population of P. caribbica on the surface of apple fruits decreased quickly at the first stage at both temperatures (20 and 4 °C) and then increased (Figure 6A,B). However, irrespective of the storage temperature, the population of VC-

Figure 5. Change of SOD (A), CAT (B), and GPX (C) activities in control and VC-treated cells of P. caribbica. Cells without VC treatment served as control. Sterilized water was used as control. Data in columns with different letters are significantly different according to Duncan’s multiple-range test at p < 0.05.

treated cells was significantly higher than that of non-VCtreated cells at all tested times. Population Dynamics of P. caribbica in Wounds of Apple Fruits. Population dynamics of cells in the wounds compared with those on the fruit surfaces indicated a quite different trend (Figure 6). P. caribbica multiplied quickly in apple fruit wounds, and the number of yeast cells increased almost 10-fold at the first day (Figure 6C). The population of P. caribbica reached the maximum at days 7 and 8 for non-VC-treated cells and VCtreated cells, respectively. Although the yeast in wounds of apple fruits stored at 4 °C grew more slowly than that stored at 20 °C, rapid colonization of P. caribbica was observed during the first 3 days (Figure 6D). Although during all time points this population trend showed little fluctuation, it remained at a high level and did not fall below 1 × 106 CFU/wound at 4 °C. 7615



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Furthermore, irrespective of the temperature, the population of VC-treated P. caribbica was higher than the population of nonVC-treated P. caribbica at all tested times. Identification of Differentially Expressed Proteins. Two aspects, changes in abundance of common spots between the two treatments (non-VC-treated and VC-treated) and appearance or absence of spots specifically, were considered when the differentially expressed proteins were evaluated. More than 200 protein spots were detected in each gel using IMP7.0 (Figure 7). Among them, 61 protein spots showing statistically significant (p < 0.05) changes of >1.5-fold in relative abundance between the two treatments were selected. Of the 61 proteins, 41 proteins were up-regulated and 20 proteins were downregulated. All 61 proteins were focused on differences in degree of peak protein (Table 1). Most of the proteins were related to basic metabolism. Besides, there are proteins that correlate with protein synthesis, transportation, and stress response.

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DISCUSSION New methods to control postharvest decay have been found as an alternative to the use of synthetic fungicides; biocontrol agents were considered to be one of the most effective means.26 In the present study, the results indicated that P. caribbica has showed control efficacy against postharvest blue mold decay of apples. This observation is in agreement with the one reported by Lahlali et al.,27 but there are many differences between laboratory tests with host/parasite systems and performance of biocontrol agents/products under practical conditions,28,29 which have great impact on the efficacy of controlling the postharvest disease of fruits. Oxidative stress, a crucial environmental factor, influenced the survival and efficacy of biocontrol yeasts.30,31 Consequently, to maintain or increase the biocontrol activity of antagonists under commercial conditions, improving its stress tolerance is a useful strategy.32,33 P. caribbica at 1 × 108 cells/mL combined with 250 μg/mL VC was demonstrated to be more effective in reducing P. expansum infection in apple fruit wounds than treatment with P. caribbica alone in this present study. Combining antagonists with some food additives has drawn researchers’ attention recently. 34 Previous studies have demonstrated the efficacy of NH4Mo on improving the biocontrol ability of the yeast Pichia membranifaciens in fruit.34 Ascorbic acid (VC) has been reported in various fruits and vegetables,35 is an essential nutrient for physiological function of most animal species,36 and plays an important role as a water-soluble antioxidant. From our experiments, we have got a similar result that VC is a potential approach to enhance the biocontrol efficacy of the antagonistic yeast P. caribbica. Specifically, the biological control activity of P. caribbica was greatly enhanced when the yeast was amended with VC at a final concentration range from 150 to 300 μg/mL, compared with the performance of P. caribbica used alone (Figure 1). It was found that 250 μg/mL VC showed the greatest enhancement for the biocontrol efficacy of P. caribbica (Figure 1). With regard to oxidative stress, yeasts subjected to oxidative stress produce ROS. ROS can initiate cataract reactions leading to the production of OH• and other destructive species such as lipid peroxides, which do serious harm to the cells.37 Hydroxyl radicals are known to be very reactive and can damage vital cellular macromolecules, resulting in a decrease in cell viability.18 As a consequence, in Figure 2 it was observed that P. caribbica was susceptible to oxidative stress, which led to a

Figure 6. Population dynamics of P. caribbica both on the surface and in wounds of apple fruits stored at 20 and 4 °C, respectively. Each value is the mean of three experiments. Bars represent the standard error of the mean. Data in columns with different letters are significantly different according to Duncan’s multiple-range test at p < 0.05. (A, B) Fruits were circled and inoculated with three 10 μL drops of non-VC-treated (NVC) or VC-treated (VC) cell suspension of P. caribbica at 1 × 108 cells/mL. Yeast colonies were counted after incubation at 28 °C for 36−48 h and expressed as the log10 CFU per circle. (C, D) Fruits were wounded and inoculated with 30 μL of nonVC-treated (NVC) or 250 μg/mL VC-treated (VC) cell suspension of P. caribbica at 1 × 108 cells/mL. Population levels at 20 °C were assessed each day for 10 days; meanwhile, the levels at 4 °C were assessed every 2 days for 33 days. Yeast colonies were counted after incubation at 28 °C for 36−48 h and expressed as the log10 CFU per wound. 7616



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Figure 7. Two-dimensional pattern of intracellular proteins of non-VC-treated (NVC) or 250 μg/mL VC-treated (VC) cell under oxidative stress. Proteins were separated by 2D gel electrophoresis using 24 cm IPG strips with a pH 3−10 nonlinear gradient. After electrophoresis, proteins were visualized by Coomassie blue staining.

negatively correlated with cell viability. Thus, the application of VC was capable of reducing the intracellular ROS accumulation, which could improve the population level of antagonistic yeast. To reduce the excess of ROS, yeasts have developed an antioxidant defense system during evolution, which comprises various enzymatic and nonenzymatic components. The antioxidant enzymes that can directly react with ROS and scavenge ROS include SOD, CAT, and various peroxidases. In the present study, it was found that the activities of the three enzymes (SOD, CAT, and GPX) of P. caribbica cells were increased by VC treatment when exposed to H2O2 (Figure 5) at all tested time points. The results suggested that the induction of antioxidant enzyme activity in P. caribbica by VC treatment may be a key factor in lowering ROS levels, thus improving the viability of yeast cells exposed to oxidative stress. It has been confirmed that there is a direct relationship between the population and the biocontrol efficacy of the antagonists.5 The expected biocontrol efficacy will be achieved if antagonistic yeasts are able to survive and proliferate on the surface of the fruits.5 There are many differences between laboratory tests and practical conditions,29 which have great impact on the efficacy of controlling postharvest diseases of fruits. Oxidative stress, a crucial environmental factor,

low viability that influenced its biocontrol efficacy. This observation is in agreement with that reported by Liu et al.,3 who indicated that with the increasing H2O2 concentration and exposure time, the viability of Cystofilobasidium infirmominiatum decreased rapidly.3 At the same time, the integration with VC is an effective measurement to improve the oxidative tolerance of the antagonistic yeast (Figure 3) . Further studies showed that lower ROS accumulation was exhibited in VC-treated cells compared to non-VC-treated cells (Figure 4). Compared with the yeast viability presented under oxidative stress in Figure 2, the decreasing survival ability of the yeast with high concentrations of H2O2 might be caused by the higher accumulation of intracellular ROS. However, at 60 min, the ROS accumulation decreased tremendously compared with 40 min. This result is different from that achieved by Liu et al.,3 who observed much higher ROS accumulation at 60 min. This may be attributed to cell death including cell apoptosis and necrocytosis. ROS produced under stress will induce cell apoptosis and lead to necrocytosis. Besides, a high concentration of ROS will cause necrocytosis directly. Cells release the cell content such as mitochondria and cytoplasm due to necrocytosis. This will be a possible mechanism leading to the decrease of ROS level. From the results of this present study, it is possible to conclude that the trend of ROS accumulation is 7617



Article

Table 1. . Identification of Cellular Proteins of P. caribbica Showing Differential Expression under Oxidative Stress Using MS/ MS Analysis NCBI accession no.

mass

PI

species

score

SC (%)

matches

guanyl-nucleotide exchange factor

gi|528061614

168581

5.51

71

8

10

hypothetical protein BC1G_10068 small GTPase Rac1 GTP-binding nuclear protein GSP1/Ran

gi|154301833 gi|299755668 gi|146421317

73084 21794 24413

4.32 8.22 6.52

69 75 97

11 35 45

8 5 8

polyketide synthase hypothetical protein SMAC_00264 hypothetical protein PGUG_05024

gi|296808807 gi|336273934 gi|146414197

41053 16556 32118

9.41 5.55 7.77

61 68 124

14 43 34

4 4 10

conserved hypothetical protein hypothetical protein TRIATDRAFT_51448 phosphoglycerate mutase-like protein short-chain dehydrogenase reductase malate dehydrogenase [Aspergillus oryzae RIB40] CYP52A16 hypothetical protein CaO19.11314 glyceraldehyde-3-phosphate dehydrogenase

gi|115384822 gi|358400748 gi|512200498 gi|320585844 gi|317148785 gi|29469871 gi|68477373 gi|146419367

140175 110004 37855 39123 32665 62054 77594 35831

5.51 4.99 5.06 5.36 6.85 6.88 8.63 6.6

75 70 70 65 63 46 47 111

11 8 17 25 14 11 9 44

12 7 8 7 5 4 5 10

KLTH0E13530p hypothetical protein BN14_11504 hypothetical protein PGUG_05024

gi|255715569 gi|471868712 gi|146414197

76761 55505 32118

8.9 8.73 7.77

77 68 95

19 33 31

11 14 7

hypothetical protein PGUG_04204

gi|190347768

37468

5.42

86

25

8

RNA helicase

gi|405118839

75720

9.83

63

13

8


gi|146418176

33223

5.22

60

31

4

hypothetical protein F503_06451 xylose reductase inorganic pyrophosphatase malate dehydrogenase conserved hypothetical protein 40S ribosomal protein S0

gi|512187970 gi|4103055 gi|344232670 gi|317148785 gi|189191866 gi|146417344

85551 36247 31666 32665 71626 28665

8.21 5.58 5.18 6.85 6.57 4.77

64 129 69 63 67 70

12 41 25 14 11 31

9 12 6 5 6 6

conserved hypothetical protein

gi|146415847

42169

6.08

89

36

12

hypothetical protein TPHA_0N00980 nicotinate phosphoribosyltransferase putative high-affinity nicotinic acid transporter protein 50S ribosomal protein l19

gi|367007166 gi|344230982 gi|500252021

96418 47536 64344

5.8 5.51 9.51

Schizosaccharomyces octosporus yFS286 Botryotinia f uckeliana B05.10 Coprinopsis cinerea okayama 7#130 Meyerozyma guilliermondii ATCC 6260 Arthroderma otae CBS 113480 Sordaria macrospora k-hell Meyerozyma guilliermondii ATCC 6260 Aspergillus terreus NIH2624 Trichoderma atroviride IMI 206040 Glarea lozoyensis ATCC 20868 Grosmannia clavigera kw1407 Aspergillus oryzae RIB40 Candida tropicalis Candida albicans SC5314 Meyerozyma guilliermondii ATCC 6260 Lachancea thermotolerans Rhizoctonia solani AG-1 IB Meyerozyma guilliermondii ATCC 6260 Meyerozyma guilliermondii ATCC 6260 Cryptococcus neoformans var. grubii H99 Meyerozyma guilliermondii ATCC 6260 Ophiostoma piceae UAMH 11346 Meyerozyma guilliermondii Candida tenuis ATCC 10573 Aspergillus oryzae RIB40 Pyrenophora tritici-repentis Pt-1C-BFP Meyerozyma guilliermondii ATCC 6260 Meyerozyma guilliermondii ATCC 6260 Tetrapisispora phaf f ii CBS 4417 Candida tenuis ATCC 10573 Togninia minima UCRPA7

60 42 69

14 15 24

6 4 7

gi|405124304

21403

65

27

5

putative oligopeptide transporter enolase 1

gi|328863365 gi|146415384

79177 46951

8.41 5.42

70 147

20 34

8 13

hypothetical protein COCHEDRAFT_1157214 enolase 1

gi|451997726 gi|146415384

55174 46915

10.22 5.42

59 154

18 34

6 13

hypothetical protein BN14_07940 MCM7 hypothetical protein CANTEDRAFT_128012 hypothetical protein ASPNIDRAFT_44377 phosphoglycerate kinase

gi|471884071 gi|359294723 gi|344228724 gi|350632783 gi|146416915

21986 22843 51735 30733 44452

9.82 5.97 6.53 6.15 5.9

53 74 82 69 71

34 32 20 24 24

4 5 9 6 7

T-complex protein 1 subunit α Short=TCP-1-α homogentisate 1,2-dioxygenase DEHA2F09570p conserved hypothetical protein

gi|471909326 gi|494828579 gi|50424381 gi|146414762

38733 51230 44438 35538

7.62 5.56 6.3 4.54

71 65 135 81

22 20 36 20

8 5 13 8


gi|146421948

35991

5.22

60

15

5

protein name

7618

11.6

Cryptococcus neoformans var. grubii H99 Melampsora larici-populina 98AG31 Meyerozyma guilliermondii ATCC 6260 Bipolaris maydis C5 Meyerozyma guilliermondii ATCC 6260 Rhizoctonia solani AG-1 IB Trichosporon laibachii Candida tenuis ATCC 10573 Aspergillus niger ATCC 1015 Meyerozyma guilliermondii ATCC 6260 Rhizoctonia solani AG-1 IB Coniosporium apollinis CBS 100218 Debaryomyces hansenii CBS767 Meyerozyma guilliermondii ATCC 6260 Meyerozyma guilliermondii ATCC 6260



Article

Table 1. continued NCBI accession no.

mass

PI

isocitrate lyase

gi|146413757

61937

6.31

pyruvate kinase

gi|146422809

55672

6.62


gi|190345229

53074

7.23


gi|146423756

53100

7.23

acetyl-CoA hydrolase

gi|146417797

58385

5.96


gi|146419296

73680

5.91


gi|146420955

85853

5.68


gi|146421560

84975

5.92


gi|146418675

69418

8.31

heat shock protein 60, mitochondrial precursor

gi|190348913

60586

5.17

hypothetical protein SEPMUDRAFT_147302 hypothetical protein PGUG_00667

gi|453087363 gi|146422710

83575 56048

5.94 5.44

heat shock protein 70 2

gi|146413777

70177

5.04


gi|190347716

81682

6.69


gi|190347716

81682

6.69

protein name

influenced the survival and efficacy of biocontrol yeasts.31 Hence, the improvement of oxidative tolerance has an important significance for adequate biological effectiveness.33 It was observed that VC improved the oxidative stress tolerance of P. caribbica and so did the population dynamics both on the surface and in the wounds of apples. The biocontrol activity of the antagonist is based on its capacity for rapid colonization and competition for space and nutrients.5 In this study, 250 μg/mL VC improved the oxidative stress tolerance of P. caribbica and the population of P. caribbica both in the wounds and on the surface of the apple fruit (Figure 6), which probably explains the enhancement of the efficacy of P. caribbica exhibited in a previous assay. Similar results for other antagonist yeasts have been reported by Bencheqroun et al.38 and Zhang et al.39 In addition, the first 2 days after inoculation into apple wounds are quite important for P. expansum germination and infection.40 As shown in Figure 6, it is notable that VC-treated cells of P. caribbica had a relatively higher population level than non-VC-treated cells both in wounds and on the surface of apple fruits during the first 2 days after inoculation. Thus, the higher population of VC-treated yeast cells compared to non-VC-treated cells in the early days may have produced the better biocontrol ability. As to the molecular mechanism, most differentially expressed proteins in response to oxidative stress by the addition of VC were related to basic metabolism, which indicated that the basic metabolism of P. caribbica was improved by application of VC, so as to improve its biocontrol efficacy. Enolase 1, which can catalyze 2-phosphoglyceric acid to phosphoenolpyruvic acid, is a key enzyme in the glycolytic pathway; polyketide synthase is the key enzyme mediating polyketide formation; and malate dehydrogenase corresponded to the tricarboxylic acid cycle.

species Meyerozyma guilliermondii ATCC 6260 Meyerozyma guilliermondii ATCC 6260 Meyerozyma guilliermondii ATCC 6260 Meyerozyma guilliermondii ATCC 6260 Meyerozyma guilliermondii ATCC 6260 Meyerozyma guilliermondii ATCC 6260 Meyerozyma guilliermondii ATCC 6260 Meyerozyma guilliermondii ATCC 6260 Meyerozyma guilliermondii ATCC 6260 Meyerozyma guilliermondii ATCC 6260 Sphaerulina musiva SO2202 Meyerozyma guilliermondii ATCC 6260 Meyerozyma guilliermondii ATCC 6260 Meyerozyma guilliermondii ATCC 6260 Meyerozyma guilliermondii ATCC 6260

score

SC (%)

matches

172

35

14

154

30

17

90

27

11

67

20

7

107

28

13

80

21

9

253

47

27

173

32

19

117

31

18

258

45

26

80 101

23 21

11 13

213

43

22

247

45

28

92

24

13

According to these, we can conclude that the higher basic metabolic activity can induce higher oxidative tolerance. In conclusion, the results of the present study showed that the oxidative stress tolerance of the antagonist can be improved markedly by the application of VC. Application of VC was also efficient in reducing the ROS level and inducing the activities of antioxidant enzymes. Consequently, VC can increase the population dynamics and the biocontrol efficacy of P. caribbica against postharvest blue mold decay of apples. This may offer great practical potential in reducing postharvest diseases of apples. However, according to results reported by Macarisin et al.,4 the appropriate concentration of ROS production stimulated by these biomicrobial biocontrol agents is important for fruits to initiate their protective effect, so the interation among the ROS production, antagonist, pathogen, and host fruits/vegetables should be investigated in the future.

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AUTHOR INFORMATION

Corresponding Author

*(H.Z.) Phone: +86-511-88780174. Fax: +86-511-88780201. E-mail: [email protected]. Funding

This research was supported by the National Natural Science Foundation of China (31271967), the Technology Support Plan of Jiangsu Province (BE2014372), the Research Fund for the Doctoral Program of Higher Education of China (20123227110015), and the Technology Support Plan of Zhenjiang (NY2013004). Notes

The authors declare no competing financial interest. 7619



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Article

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ACKNOWLEDGMENTS We thank Qingzhi Ding for technical assistance. We also acknowledge Dr. Yanhua Yang for help with mass spectrometry analysis.

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Ascorbic Acid Enhances Oxidative Stress Tolerance and Biological

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