Mechanisms of glycine betaine enhancing oxidative stress tolerance and biocontrol efficacy of Pichia caribbica against blue mold on apples

Biological Control 108 (2017) 55–63

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Mechanisms of glycine betaine enhancing oxidative stress tolerance and biocontrol efficacy of Pichia caribbica against blue mold on apples Xiaoyun Zhang a, Guochao Zhang b, Pengxia Li c, Qiya Yang a, Keping Chen b, Lina Zhao a, Maurice Tibiru Apaliya a, Xiangyu Gu d, Hongyin Zhang a,⇑ a

School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, Jiangsu, People’s Republic of China Institute of Life Sciences, Jiangsu University, Zhenjiang 212013, Jiangsu, People’s Republic of China Institute of Agricultural Products Processing, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, Jiangsu, People’s Republic of China d School of Grain Science and Technology, Jiangsu University of Science and Technology, Zhenjiang 212003, Jiangsu, People’s Republic of China b c

h i g h l i g h t s
GB enhanced biocontrol efficacy of P. caribbica to blue mold decay of apples.
P. caribbica treated with GB exhibited faster growth in wounds of apples.
GB increased oxidative stress tolerance of P. caribbica.
GB reduced ROS accumulation, protein carbonylation, lipid oxidation of P. caribbica.
GB induced differential expression of some proteins of P. caribbica.

a r t i c l e

i n f o

Article history: Received 23 December 2016 Revised 25 February 2017 Accepted 25 February 2017 Available online 2 March 2017 Keywords: Glycine betaine Oxidative stress tolerance Biocontrol efficacy Pichia caribbica Blue mold ROS

a b s t r a c t The effects of glycine betaine (GB) treatment on biocontrol efficacy of Pichia caribbica against blue mold decay of apples and oxidative stress tolerance were investigated. To reveal the mechanism of GB enhancing oxidative stress tolerance and biological control efficacy of P. caribbica, the accumulation of intracellular reactive oxygen species (ROS), protein carbonylation, lipid oxidation and differentially expressed proteins were analyzed. Compared with P. caribbica treated without GB, P. caribbica treated with GB exhibited enhanced biocontrol activity (disease incidence decreased from 48.81% to 32.14%) and oxidative stress tolerance when exposed to oxidative stress (survival ability increased from 60.1% to 77.7%), as well as improved growth in wounds of apples. GB treatment reduced accumulation of ROS, levels of oxidative damage to cellular proteins and lipids in P. caribbica. The proteomic analysis showed that 51 proteins were differentially expressed in P. caribbica after GB treatment, 33 proteins of which were successfully identified by MALDI-TOF-MS and database Queries. The up-regulation of some identified proteins related to carbohydrate transport and metabolism (enolase, pyruvate kinase and isocitrate lyase), stress response and regulation (peroxisomal catalase and Serine/threonine protein kinase) were involved in the enhancement of oxidative stress tolerance and biocontrol efficacy of P. caribbica. Ó 2017 Elsevier Inc. All rights reserved.

1. Introduction Postharvest losses of fruits and vegetables caused by fungal pathogens are very serious throughout the world. Furthermore, some fungi could produce mycotoxins that is a major health hazard for consumers (Mahunu et al., 2016; Liu et al., 2017). Blue mold decay caused by Penicillium expansum is one of the most important postharvest rots of apples, which affects fruit quality and causes ⇑ Corresponding author. E-mail address: [email protected] (H. Zhang). http://dx.doi.org/10.1016/j.biocontrol.2017.02.011 1049-9644/Ó 2017 Elsevier Inc. All rights reserved.

significant economic losses (Fan and Tian, 2000; Zhang et al., 2007; Calvo et al., 2007). P. expansum is also regarded as a major producer of patulin (PAT), a mycotoxin which has cytotoxic, genotoxic and immunosuppressive activity (Wouters and Speijers, 1996). Traditionally, the control of fungal diseases is mainly based on chemical treatments (Salomao et al., 2008; Sansone et al., 2005). However, the use of chemical fungicides is restricted due to the concerns on the resistance of spoilage fungi to fungicides, toxicity of residual fungicides and the environmental pollution (Ragsdale and Sisler, 1994; Sharma et al., 2009; Förster et al., 2007).

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Biological control with microbial antagonists alone or integrated with other measures is considered as a promising alternative strategy, which could reduce synthetic fungicide usage (Wilson and Wisniewski, 1994; Wisniewski et al., 2007; Sui et al., 2015). A number of antagonistic yeasts have been used to control postharvest decay of fruits and vegetables. The antagonistic yeasts have showed significant biocontrol activity against pathogenic fungi strains, such as P. expansum, which lead to the decay of apples fruits(Janisiewicz and Korsten, 2002; Fan and Tian, 2001; Droby et al., 2003; Droby, 2006; Ippolito et al., 2000; Zhang et al., 2009; Sharma et al., 2009). Recent reports have concerned the important role of antagonistic yeasts not only in the control of fungal contamination but also mycotoxin produced by fungi in fruits, such as patulin by P. expansum in apple fruits (Castoria et al., 2002, 2005). However, some studies reported that antagonistic yeasts used alone cannot provide as satisfactory biocontrol efficacy against decay of fruits as chemical fungicide. Therefore, some compounds, such as indole-3-acetic acid (IAA), chitosan，glycine betaine or antioxidant compounds, were used to enhance the biocontrol activity of antagonistic yeasts (Yu et al., 2007, 2009; Sharma et al., 2009; Liu et al., 2011; Zhao et al., 2012). Oxidative stress, a potential threat for most aerobic organisms, has a pivotal role in biocontrol systems (Castoria et al., 2003; Macarisin et al., 2010). The presence of oxidative stress (i.e. hydrogen peroxide) in the environment of wounds in fruits represents part of the plant defense response to microbial attack. The previous study demonstrated that antagonistic yeasts could rapidly colonize wounds of fruits and then limit the growth of pathogenic fungi even in this oxidative stressful environment (Castoria et al., 2003). The tolerate ability of antagonistic yeasts to oxidative stress effected their biocontrol ability against fungal pathogens greatly. Therefore, increasing survival ability of antagonistic yeasts under oxidative stress conditions would be a useful strategy for enhancing biocontrol efficacy (Liu et al., 2011; Sui et al., 2015). Glycine betaine (GB, N, N, N-trimethylglycine), one of the most common compatible solutes, plays a vital role in osmotic regulation of bacteria, fungi, and plants (Ashraf and Harris, 2004; De Zwart et al., 2003; Rhodes and Hanson, 1993). GB can protect enzymes by stabilizing the protein structure and maintains the integrity of membranes against osmotic stress (Ashraf and Foolad, 2007; Boncompagni et al., 1999; Wang et al., 2007). GB induces antioxidant defense responses in plant species such as tea (Kumar and Yadav, 2009), wheat (Raza et al., 2007), and rice (Farooq et al., 2008). However, there are few reports about the effect of GB on antioxidant systems and biocontrol efficacy of antagonistic yeast, not to mention the mechanism involved. Liu et al. (2011) report that GB-treated Cystofilobasidium infirmominiatum exhibited greater biocontrol activity against P. expansum. The accumulation of reactive oxygen species (ROS) and protein oxidation in GB-treated cells decreased. While the activities of antioxidant enzymes, including catalase, superoxide dismutase and glutathione peroxidase of GB-treated cells increased. The results by Sui et al. (2012) also showed that GB-treated Candida oleophila exhibited greater biocontrol activity against P. expansum and Botrytis cinerea. GB-treated cells exhibited less accumulation of ROS and lower levels of oxidative damage to cellular proteins and lipids. Additionally, the expression of major antioxidant genes, including peroxisomal catalase, peroxiredoxin TSA1, and glutathione peroxidase was elevated in the yeast by GB treatment. The objective of the present study was to determine the effect of GB on oxidative stress tolerance and biocontrol efficacy of antagonistic yeast Pichia caribbica, and explore the mechanism of GB enhancing oxidative stress tolerance and biocontrol efficacy of P. caribbica. We investigated (1) the effects of GB on biocontrol efficacy of P. caribbica against blue mold decay of apples caused by

P. Expansum, population dynamics of P. caribbica in the wounds of apples, viability of P. caribbica under oxidative stress; (2) the effects of GB on reactive oxygen species (ROS) accumulation in P. caribbica, protein carbonylation and lipid oxidation; (3) the effects of GB on proteome of P. caribbica. 2. Material and methods 2.1. Yeast P. caribbica preserved in the China General Microbiological Collection Center (CGMCC, No. 3616) was stored at 4 °C on nutrient yeast dextrose agar (NYDA) in our laboratory. NYDA medium contained (per liter): 8 g nutrient broth, 5 g yeast extract, 10 g glucose, and 20 g agar. Liquid cultures in nutrient yeast dextrose broth (NYDB) inoculated with a loop of the above culture were incubated at 28 °C for 24 h. And then, cultures were centrifuged at 7000g for 10 min and precipitates were washed three times with sterile distilled water to remove residual growth medium. The cells was resuspended and adjusted to the concentration of 5  108 cells/ ml with sterile distilled water. 2.2. Pathogen P. expansum isolated from infected apples was stored at 4 °C on potato dextrose agar (PDA). PDA medium contained (per liter): extract of boiled potato (200 g), 20 g dextrose, 20 g agar. Spore suspensions of P. Expansum were prepared by collecting spores from the sporulating edges of a 7-day-old culture into sterile distilled water and adjusted to the appropriate concentration. 2.3. Fruits Apples (Malus  domestica Borkh, cv. Fuji) were harvested at commercial maturity from a orchard in Yantai, Shangdong Province. Fruits without apparent injury or infection were selected based on uniformity of size, ripeness and color, and disinfected with 1% (w/v) sodium hypochlorite for 1–2 min, then washed with tap water and air-dried. These fruits were used in subsequent biocontrol assays. 2.4. GB treatment of P. caribbica P. caribbica at concentration of 5  108 cells/ml was prepared according to the method described as 2.1. Afterward, 1 ml of the above-mentioned P. caribbica suspension was cultivated in NYDB (not-GB-treated) or NYDB amended with GB (GB-treated) at different concentrations (0.5 mM, 1 mM, 2 mM and 3 mM) in a rotary shaker at 180 rpm at 28 °C for 24 h. Afterwards, cultures were centrifuged at 7000g for 10 min and washed three times with sterile distilled water to remove residual GB and medium. Cell pellets were re-suspended and adjusted to a concentration of 1  108 cells/ml with sterile distilled water for subsequent experiments. 2.5. Biocontrol assay of P. caribbica against P. expansum on apples Three wounds were made on the equator of each apple using a sterile cork borer (approximately 5 mm in diameter, and 3 mm in depth). Each wound was inoculated with the following solutions: (1) suspension of not-GB-treated P. caribbica (1  108 cells/ml) prepared according to the method described as 2.4; (2) suspension of GB-treated P. caribbica (1  108 cells/ml) prepared according to the method described as 2.4; (3) sterile distilled water as the control. After three hours, 30 ml of P. expansum suspension (5  104 spores/ml) prepared according to the method described as 2.2

X. Zhang et al. / Biological Control 108 (2017) 55–63

was inoculated into each wound. Treated apples were then placed in plastic trays and enclosed with polyethylene bags to maintain relative humidity (approximately 95%), and stored at 20 °C. Disease incidences were determined after 15 days. Each treatment was performed in triplicate and each replicate consisted of 12 apples and three wounds per apple. The average percentage of infection for each replicate was calculated (numbers of wounds with infection/36  100). The entire experiment was repeated in triplicate and the percentage of infection was the mean with triplicate.

2.6. Population dynamics of P. caribbica in wounds of apples Apples were prepared and wounded as described above. P. caribbica (1  108 cells/ml) was prepared according to the method described as 2.4. Each wound was inoculated with a 30 ml suspension of not-GB-treated or GB-treated P. caribbica (1  108 cells/ml). Fruits were incubated at 20 °C, and then population dynamics was assessed daily for 8 days. The wounded tissue was removed with a sterile cork borer (9 mm diameter and 10 mm depth) and ground completely in a sterile mortar with 50 ml of sterile 0.85% sodium chloride solution. Serial 10-fold dilutions (100 ml) were made and then spread on NYDA plates. Samples taken 1 h after treatment were served as time 0. Colonies were counted after incubation at 28 °C for 2 days and expressed as log10CFU/wound. Each treatment was performed in triplicate and experiment was repeated three times.

2.7. Effect of GB on oxidative stress tolerance of P. caribbica The effect of GB on survival of P. caribbica after H2O2 treatment was determined according to the method described as Deveau et al. (2010) with some modifications. P. caribbica (1  108 cells/ ml) was prepared according to the method described as 2.4 and then exposed to 10 mM H2O2 (based on our previous study, data were not shown) at 28 °C and 180 rpm for 20 min. Thereafter, 100 ml of serial 10-fold diluted samples (from 1  103 to 1  107 cells/ml) were spread on NYDA plates. Colonies were counted after incubation at 28 °C for 2 days. The oxidative stress tolerance was denoted as survival (the survival numbers of P. caribbica treated with H2O2 / the initial numbers of P. caribbica  100). Each treatment was replicated in triplicate and the experiment was performed three times.

2.8. Determination of intracellular ROS P. caribbica (1  108 cells/ml) was prepared according to the method described as 2.4. And then the oxidant-sensitive probe, 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA) was used to assess intracellular ROS production in P. caribbica according to the method of Li et al. (2014). The ROS level was proportional to the relative intensity. Each treatment was performed in triplicate and the entire experiment was repeated three times.

2.9. Determination of protein carbonylation P. caribbica (1  108 cells/ml) was prepared according to the method described as 2.4 and exposed to 10 mM H2O2 for 0, 20, 40 and 60 min. Then protein carbonylation was measured by determining carbonyl content (Liu et al., 2011). The carbonyl content was expressed as nmol/mg protein. Protein content was determined according to the method described by Bradford (Bradford, 1976). There were three replicates in each treatment, and the experiment was repeated three times.

57

2.10. Assay of lipid peroxidation To assay lipid peroxidation, a method based on the reaction of thiobarbituric acid (TBA) with malondialdehyde (MDA) was used (Du and Bramlage, 1992). Briefly, 2 ml of 0.6% TBA in 20% trichloroacetic acid was added into 2 ml disrupted P. caribbica. Heating the solution at 100 °C for 10 min, then quickly cooled in an ice-bath, and centrifuged (4 °C) for 15 min at 10000g, afterwards the supernatants were harvested and assayed. The absorbance of supernatants was recorded at 532, 600, and 450 nm. MDA was calculated based on the formula: C (lM)MDA = 6.45  (A532  A600)  0.56  A450, expressed as nmol/mg protein. Protein content was determined according to the method described by Bradford (Bradford, 1976). There were three replicates and each repeated three times. 2.11. Proteomic analysis Protein Samples Preparation. Not-GB-treated and GB-treated P. caribbica were collected and then exposed to 10 mM H2O2 for 60 min. Then the cells were harvested by centrifuging at 10000g for 10 min (4 °C) and washed three times with cold doubledistilled water to remove residual H2O2. Protein samples preparation was performed as described by Li et al. (2009). Two-Dimensional Electrophoresis (2-DE) and Image Analysis. 2DE and image analysis were performed as described by Wang et al. (2005) using the vertical electrophoresis systems, Image Scanner III and Image Master 2D Platinum 7.0 software (GE Healthcare BioScience, Uppsala, Sweden), respectively. Three biological replicates were performed for each treatment. 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. (2011). Protein Identification by MALDI-TOF-MS (Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry) and Database Query. The peptide solution was analyzed using MALDI-TOFmass 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. (2004). 2.12. Statistical analysis The data were analyzed by analysis of variance (ANOVA) using the statistical program SPSS/PC version II.x, (SPSS Inc., Chicago, Illinois, USA) and Duncan’s multiple range test used for separation of means. Statistical significance was applied at the level p < 0.05. 3. Results 3.1. Assay of biocontrol efficacy of P. caribbica against P. expansum on apples The disease incidence of blue mold decay of apples treated with P. caribbica and the control was shown in Fig. 1. It was shown that P. caribbica could effectively controlled postharvest disease caused by P. expansum on apples. The disease incidence of the control fruits (inoculated with water followed by the pathogen) was 91.66%, while the disease incidence of apples treated with notGB-treated P. caribbica was 48.81%. However, the disease incidence of blue mold decay of apples treated with P. caribbica treated with GB was significantly lower than that of not-GB-treated P. caribbica, especially when the treated concentration of GB was 1 mM (32.14%). The result showed that GB treatment enhanced the biocontrol efficacy of P. caribbica against blue mold decay of apples.

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Fig. 1. The effect of GB on the biocontrol efficacy of P. caribbica against P. expansum on apples. Sterilized water as control (CK). Each value is the mean of three experiments. Bars represent 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.

3.2. Population dynamics of P. caribbica in wounds of apples The population of P. caribbica in apple wounds increased quickly at 20 °C, especially on the first day, the numbers of P. caribbica increased almost 12-fold (Fig. 2), and reached its maximum at the third day. After that the population decreased and stabilized at a high level. As shown in Fig. 2, GB-treated P. caribbica multiplied more quickly in wounds of apples than the untreated cells, especially at the first three days. It showed that GB treatment is beneficial to the growth of P. caribbica in apple wounds. 3.3. Effect of GB on oxidative stress tolerance of P. caribbica The oxidative stress tolerance of P. caribbica treated with GB or not was shown in Fig. 3. Under the condition of H2O2-induced oxidative stress, GB treatment had a significant (p < 0.05) effect on survival of P. caribbica. The survival of not-GB-treated P. caribbica (60.1%) was lower than that of GB-treated P. caribbica (77.7%). It suggested that GB-treated P. caribbica showed higher

oxidative stress tolerance and viability compared with untreated P. caribbica. 3.4. ROS accumulation of P. caribbica under oxidative stress As shown in Fig. 4, large amounts of ROS accumulated in both GB-treated and not-GB-treated P. caribbica under the condition of H2O2-induced oxidative stress, and ROS accumulation reached the peak at 40 min, and then decreased at 60 min. While the ROS accumulation in GB-treated P. caribbica was significantly lower compared with that in not-GB-treated P. caribbica, indicating that GB treatment is an effective method to reduce ROS accumulation of P. caribbica exposed to oxidative stress. 3.5. Assay of protein carbonylation To investigate the protective effect of GB on cellular proteins under the condition of H2O2-induced oxidative stress, protein carbonylation of P. caribbica was measured. As shown in Fig. 5, the carbonyl content of proteins in P. caribbica treated with GB or not were both low before H2O2 treatment at 0 min. Oxidative stress (10 mM H2O2) resulted in a marked increase of carbonyl content in P. caribbica with the extension of exposure time. However, GBtreatment significantly (p < 0.05) reduced carbonyl content of P. caribbica exposed to H2O2-induced oxidative stress for same time. 3.6. Assay of lipid oxidation MDA content was an indicator of lipid peroxidation. The MDA content of P. caribbica exposed to H2O2-induced oxidative stress were determined and shown in Fig. 6. The effects of oxidative stress and GB on the MDA content was similar to that on protein carbonylation. That is oxidative stress significantly (p < 0.05) increased MDA content of P. caribbica with the extension of exposure time, but lower MDA content was observed in P. caribbica treated with GB at all of the exposure time. 3.7. Differentially expressed proteins of P. carbbica treated with GB

Fig. 2. Population dynamics of P. caribbica in apple wounds at 20 °C. Each value is the mean of three experiments. Bars represent the standard error of the mean. NGB: P. caribbica was not treated by glycine betaine. GB: P. caribbica was treated by glycine betaine.

More than 200 protein spots were detected in each gel after ignoring very faint spots and spots with undefined shapes and areas using IMP7.0 software (Fig. 7). When P. caribbica was exposed to H2O2, 51 differential proteins (>1.5-fold) of GB-treated cells

X. Zhang et al. / Biological Control 108 (2017) 55–63

59

Fig. 3. The effect of GB 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. NGB: P. caribbica was not treated by glycine betaine. GB: P. caribbica was treated by glycine betaine.

Fig. 4. ROS accumulation in GB-treated and not-GB-treated P. caribbica exposed to oxidative stress. 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. NGB: P. caribbica was not treated by glycine betaine. GB: P. caribbica was treated by glycine betaine.

were determined compared with untreated cells, in which 19 proteins were down-regulated and 32 proteins were up-regulated. Thirty-three proteins were successfully identified by MALDI-TOFMS and Database Queries, and the results were showed in Table 1. Besides the unnamed and hypothetical proteins, most of which (Table 1) were related to carbohydrate, amino acid, nucleotide transport and metabolism. For example, enolase (7), pyruvate kinase (12), isocitrate lyase (13), glyceraldehyde-3-phosphate dehydrogenase (22) were all up-regulated by GB treatment. In

Fig. 5. Protein carbonylation in GB-treated and not-GB-treated P. caribbica exposed to oxidative stress. 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. NGB: P. caribbica was not treated by glycine betaine. GB: P. caribbica was treated by glycine betaine.

addition, the expression of some stress response and regulatory proteins also changed. For instance, serine/threonine protein kinase (14) and peroxisomal catalase (11) were up-regulated. 4. Discussion P. caribbica, an antagonistic yeast, significantly reduced postharvest blue mold decay of apples caused by P. expansum (Cao et al., 2013). However, the biocontrol efficacy of P. caribbica needs to be improved for commercial applications.

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Fig. 6. MDA contents in GB-treated and not-GB-treated P. caribbica exposed to oxidative stress. 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. NGB: P. caribbica was not treated by glycine betaine. GB: P. caribbica was treated by glycine betaine.

Fig. 1 showed that the biocontrol efficacy of GB-treated P. caribbica against the blue mold decay of apples was enhanced compared

Fig. 7. Two-dimensional pattern of intracellular proteins in GB-treated and not-GBtreated P. caribbica exposed to oxidative stress. A: not-GB-treated P. caribbica; B: GB-treated P. caribbica.

to not-GB-treated cells, and when the concentration of GB was 1 mM, the biocontrol efficacy was best. As shown in Fig. 2, GBtreated P. caribbica multiplied more quickly in wounds of apples than not-GB-treated cells, especially at the first three days. A direct relationship exists between the population of the antagonist in fruit wounds and biocontrol efficacy, a high population density is advantageous for obtaining nutrients and space when competing with pathogen, both of which play important role in biocontrol efficacy (Wisniewski et al., 2007; Janisiewicz and Korsten, 2002; Droby et al., 2009). Furthermore, the quicker propagation of P. caribbica treated with GB at the first three days is an advantage in the control of P. expansum for the reason that the early stage after inoculation in apple wounds is important for the germination and infection of P. expansum (Li et al., 2008). So the higher population of GB-treated P. caribbica may lead to the improved biocontrol efficacy to postharvest blue mold decay of apples. Oxidative stress plays an important role in biocontrol systems (Castoria et al., 2003; Macarisin et al., 2010), and thus enhancing oxidative stress tolerance may be a useful approach for improving the biocontrol efficacy of P. caribbica. Fig. 3 showed that P. caribbica untreated with GB was more susceptible to oxidative stress, which led to a lower survival ability of P. caribbica. Conversely, GB treatment improved tolerance of P. caribbica to oxidative stress, an important factor related to biocontrol efficacy of antagonistic yeasts (Castoria et al., 2003; Macarisin et al., 2010). Under the condition of oxidative stress, cells are able to generate a large amounts of ROS including hydroxyl radicals and other destructive species which do harm to cells and tissues (Castoria et al., 2003; Madeo et al., 1999). Hydroxyl radicals are known to be very reactive and can cause oxidative damage to cell components including proteins, lipids, and nucleic acids, resulting in a decrease in cell survival ability (Dat et al., 2000; Bernstein et al., 2010; Branduardi et al., 2007; Reverter-Branchat et al., 2004). As shown in Fig. 4, when exposed to H2O2, the accumulation of ROS in P. caribbica untreated with GB was higher than that in the GBtreated cells. Higher concentration of ROS in not-GB-treated P. caribbica would result in more serious necrocytosis and then lower survival ability (Fig. 3). Therefore, the increased survival ability of GB-treated P. caribbica exposed to H2O2 (Fig. 3) might be contributed to the lower accumulation of intracellular ROS. And the survival ability of P. caribbica treated with GB has a positive influence on the population dynamics in fruit wounds, and then on the biocontrol efficacy. Protein oxidation and lipid oxidation occurs frequently in cells under oxidative stress and has a deleterious effect on the structure and function. The level of carbonyl groups and MDA content are widely used as indicators of oxidative damage to proteins and lipids, respectively (Abegg et al., 2010; Li et al., 2010). As shown in Figs. 5 and 6, GB-treated P. caribbica showed lower carbonyl and MDA levels compared with cells untreated with GB. These results indicated that GB treatment reduced oxidative damage to protein and lipid to enhance the oxidative stress tolerance and increased the survival ability of P. caribbica exposed to H2O2 as well. When P. caribbica was exposed to H2O2-induced oxidative stress, the differentially expressed proteins of P. caribbica showed that enolase (7), pyruvate kinase (12), and isocitrate lyase (13) related to carbohydrate transport and metabolism were upregulated by GB treatment. Enolase (7), one of the enzymes involved in glycolysis and fermentation, is virtually common to all living cells. It catalyzes the dehydration of glycerate-2phosphate to enolpyruvate phosphate. Enolase was detected in tomato cells inoculated with protective Fusarium Oxysporum, but not in the control (tomato cells inoculated with non-protective F. Oxysporum) (L’Haridon et al., 2011). It indicated that enolase was involved in the biocontrol activity of protective F. Oxysporum. Pyru-

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Table 1 Identification of cellular proteins of P. caribbica showing differential expression under GB using MS/MS analysis. Proteins were excised from the 2D PAGE gel and digested with trypsin. The peptides were extracted and their masses were measured by MS/MS. Protein name, Accession number, Mass, isoelectric point (PI), Taxonomy, Database, Score, sequence coverage SC (%) and matches are listed. Function

Spot

Protein name

NCBI accession

Mass

PI

Score

SC (%)

Matches

Up-regulation (") or downregulation (;)

Carbohydrate transport and metabolism

7

enolase

46951

5.42

98

20

7

"1.5

12

pyruvate kinase

55672

6.62

148

29

15

"2.5

13

isocitrate lyase

61937

6.31

115

2.4

10

"4.0

16

acylphosphatase

10121

4.72

77

42

5

"2.8

22

glyceraldehyde-3-phosphate dehydrogenase xylose reductase purine biosynthesis protein purH

gi| 146415384 gi| 146422809 gi| 146413757 gi| 493628159 gi| 146419367 gi|4103055 gi| 491530247 gi| 495712653 gi| 493747471 gi| 492690463 gi| 494992005 gi| 397312433 gi| 146420447 gi| 515601576 gi| 497382957 gi| 119720498 gi| 551219704 gi| 194380858 gi| 47214952 gi| 503075714 gi| 146417687 gi| 146418431 gi| 224066993 gi| 595841877 gi| 146417697 gi| 516341928 gi| 146417697 gi| 515791360 gi| 255088982 gi| 146421560 gi| 504413047 gi| 308273298 gi| 575067628

35831

6.60

113

41

11

"1.5

36247 55169

5.58 5.84

173 87

46 26

13 7

"1.7 "1.5

122442

6.01

70

9

7

"1.8

76461

5.17

92

19

8

;1.5

31667

5.51

84

15

10

;1.5

35614

5.17

80

30

7

;1.5

24897

8.2

68

18

4

"1.5

55227

6.26

106

34

10

"2.2

48991

7.60

86

30

7

"2.2

56849

5.77

80

19

8

;2.1

17992

9.27

88

43

4

;1.5

47804

9.13

29

88

8

;1.5

51592

5.9

73

20

7

;1.5

46901

9.30

87

28

6

"2.0

8032

5.69

95

71

6

"1.6

36156

6.15

94

21

7

;1.6

39463

7.02

119

38

9

;1.5

10270

8.98

77

45

5

"2.4

65012

5.43

84

16

8

"6.2

39440

10.72

108

40

10

"3.3

115639

5.81

82

33

14

"2.8

39440

5.42

114

37

9

"4.6

14315

9.10

86

46

5

;1.5

146546

9.29

90

12

16

"1.5

84975

5.92

117

20

11

"1.5

117356

8.37

91

13

12

"2.0

47248

9.52

89

27

9

;2.3

104262

8.56

83

12

8

;1.5

Nucleotide transport and metabolism

28 8 10 24 31

Amion acid transport and metabolism Stress response and regulatory protein

Energy production and conversion hypothetical protein

ribonucleoside-diphosphate reductase DNA gyrase subunit B

25

nicotinate phosphoribosyltransferase ABC transporter

4

integrase, partial

11

peroxisomal catalase

14

serine/threonine protein kinase

32

transcriptional regulator

33 30

PadR-like family transcriptional regulator ATPase AAA

1

unnamed protein product

2

unnamed protein product

3

hypothetical protein

5

conserved hypothetical protein

6

hypothetical protein PGUG_02910 Hypothetical protein POPTR_0002s10120 g hypothetical protein PRUPE_ppa003246 mg hypothetical protein PGUG_02545 hypothetical protein

9 15 17 18 19 20

hypothetical protein PGUG_02545 hypothetical protein

21

predicted protein

23

conserved hypothetical protein

27

hypothetical protein

26

hypothetical protein N47_F15960 hypothetical protein HETIRDRAFT_383311

29

vate kinase (12) is involved in ATP generating by glycolysis pathway for cells. It was inferred that the enhancement of pyruvate kinase activity was connected with the increased stress tolerence of cells. The results by Benjaphokee et al. (2012) suggested that

the increase in pyruvate kinase activity of the high-temperature (41 °C) growth Saccharomyces cerevisiae caused the alteration in glycolytic flux of stressed cells to optimize ATP generation for maintenance of homeostasis. Isocitrate lyase (13) is a signature

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X. Zhang et al. / Biological Control 108 (2017) 55–63

enzyme of the glyoxylate cycle, involved in the metabolic adaptation in response to environmental changes. ICL is essential for abiotic stress tolerance of the fungal biocontrol agent Trichoderma atroviride (Dubey et al., 2013). The icl deletion mutant showed decreased antagonism on B. cinerea in plate confrontation assays and a reduced systemic defense in A. thaliana leaves, and then a reduced protection against B. cinerea. These data showed that ICL is important for biocontrol effect in plants. Accordingly, the upregulation of enolase (7), pyruvate kinase (12), and isocitrate lyase (13) by GB treatment may contribute to higher oxidative tolerance and enhanced biocontrol efficacy of P. caribbica. Serine/threonine protein kinase (14) can catalyze many functional protein phosphorylation, which regulates various cell life activities (Alderwick et al., 2006; Faucher et al., 2008). It is also involved in signal transduction of environmental stress, and signal can induce specific function gene expression through a series of transmission (Schenk and Snaar-Jagalska, 1999; Stone and Walker, 1995). Under the condition of oxidative stress, the increased level of serine/threonine protein kinase expression of P. caribbica may induce specific function gene expression for resisting oxidative damage. Peroxisomal catalase (11) is a major antioxidant gene. The enzymatic detoxification of ROS rely on the upregulation of antioxidant genes such as peroxisomal catalase (Nakagawa et al., 2010). GB treatment enhanced the expression of peroxisomal catalase in the C. oleophila (Sui et al., 2012). Banu et al. (2009) also reported that the expression of peroxisomal catalase in tobacco cells was enhanced by GB treatment under salt stress. It suggested that the up-regulated expression of antioxidant gene such as peroxisomal catalase in C. oleophila by GB treatment may be a key factor in reducing ROS accumulation and oxidative damage, and then improving the viability of yeast cells in fruit wounds and biocontrol efficacy. Therefore, the up-regulation of Serine/threonine protein kinase and peroxisomal catalase in P. caribbica by GB treatment also made a contribution to the higher oxidative tolerance and enhanced biocontrol efficacy. In conclusion, reduced intracellular ROS accumulation, damage to cellular proteins and lipids, up-regulated expression of some proteins participating in carbohydrate transport and metabolism, stress response and regulation by GB treatment increased the oxidative stress tolerance of P. caribbica. While increased oxidative stress tolerance, and improved growth by GB treatment contributed directly to enhanced biocontrol efficacy of P. caribbica. Therefore, GB treatment may be a potential strategy for enhancing the biocontrol efficacy of antagonistic yeasts to postharvest decay of fruits. Acknowledgments This work was supported by Jiangsu Agriculture Science and Technology Innovation Found (JASTIF, CX(15)1048), the National Key Research and Development Program of China (2016YFD0400902), the National Natural Science Foundation of China (NNSFC-31571899), the Technology Support Plan of Jiangsu Province (BE2014372), the Startup Foundation of Jiangsu University (08JDG004). References Abegg, M.A., Paulo, V.G., Casanova, A., Hoscheid, J., Salomon, T.B., Hackenhaar, F.S., Medeiros, T.M., Benfato, M.S., 2010. Response to oxidative stress in eight pathogenic yeast species of the genus Candida. Mycopathologia 170, 11–20. Alderwick, L.J., Molle, V., Kremer, L., Cozzone, A.J., Dafforn, T.R., Besra, G.S., Fütterer, K., 2006. Molecular structure of EmbR a response element of Ser/Thr kinase signaling in Mycobacterium tuberculosis. Proceedings of the Natl. Acad. Sci. U.S. A. 103, 2558–2563. Ashraf, M., Foolad, M., 2007. Roles of glycine betaine and proline in improving plant abiotic stress resistance. Environ. Exp. Botany 59, 206–216.

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