Article pubs.acs.org/JAFC

Enhanced Biocontrol Activity of Rhodotorula mucilaginosa Cultured in Media Containing Chitosan against Postharvest Diseases in Strawberries: Possible Mechanisms Underlying the Effect Hongyin Zhang,*,† Lingling Ge,‡ Keping Chen,‡ Lina Zhao,† and Xiaoyun Zhang† †

College 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

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ABSTRACT: The effect of Rhodotorula mucilaginosa cultured in media containing chitosan on its antogonistic activity against postharvest diseases of strawberries and the possible mechanisms involved are discussed. Two-dimensional gel electrophoresis were applied in the analysis of the proteins of R. mucilaginosa in response to chitosan. Compared with the application of R. mucilaginosa alone, the biocontrol efficacy of the yeast combined with 0.5% chitosan was enhanced greatly, with significant increase in chitinase activity of antagonistic yeast, polyphenoloxidase, peroxidase, phenylalanine ammonia lyase, chitinase and β1,3-glucanase activity, and with an inhibition of lipid peroxidation of strawberries. The population of R. mucilaginosa harvested from NYDB amended with chitosan at 0.5% increased rapidly in strawberry wounds compared with those harvested from NYDB without chitosan. In the cellular proteome, several differentially expressed proteins were identified, most of which were related to basic metabolism. KEYWORDS: chitosan, Rhodotorula mucilaginosa, antagonistic activity, strawberries, differentially expressed proteins

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INTRODUCTION Rhizopus stolonifer and Botrytis cinerea are two of the most destructive fungi of strawberries which can cause Rhizopus decay and gray mold separately.1 Due to these diseases, tremendous economic losses are caused. Traditionally, R. stolonifer and B. cinerea are controlled mainly by treatment with synthetic fungicides. However, with deeper research, gradually developed fungicide resistance in pathogens were detected and potential harmful effects on human health and the environment were perceived, which leads to an urgent demand for alternative control approaches with good efficacy, low residues, and ensured safety.2 Biological control with antagonistic yeasts has emerged as a potential method to reduce synthetic fungicide usage.2,3 Phylloplane yeast Rhodotorula mucilaginosa combined with fungicides has been reported to effectively control Botrytis cinerea on geranium seedlings.4 Our research team found that Rhodotorula mucilaginosa showed biocontrol efficacy against blue mold and gray mold of apples caused by Penicillium expansum and B. cinerea, respectively.5 However, making biological control a practical way requires the enhancement of the consistency and efficacy of antagonistic yeasts (including R. mucilaginosa) in controlling postharvest diseases.2,6 Many attempts have been proposed to improve the performance of postharvest biocontrol yeasts. Besides combining antagonists with some exogenous substances (organic and inorganic additives),2,6−9 combination with some physiological manipulation may also be an effective way.2 For example, Teixido et al. (1998) found that the cultivation of the yeast in a low water activity modified liquid media can improve the biocontrol efficacy of Candida sake.10 According to Li and Tian (2006), induced accumulation of internal trehalose could improve both the viability and biocontrol activity of © 2014 American Chemical Society

Cryptococcus laurentii under stresses of low temperature and controlled atmosphere.11 Recently, it has reported that the combination with chitin in the culture media may increase the bicontrol activity of C. laurentii significantly.12 Chitosan, a high molecular weight β-(1,4)-glucosamine polymer, produced from the chitin components by deacetylation, which is the second most abundant polysaccharide found in nature, is an important structural component of the cell wall of some plant-pathogenic fungi.13 It has been demonstrated to be nontoxic and safe to animals and hold great potential for food applications with its unique physiological and biological properties.14−16 Chitosan has been shown to form an ideal coating on fruit and also have antifungal activity against a wide range of fungal pathogens.17 Li and Yu (2001) found that chitosan could reduce the incidence of brown rot caused by Monilinia fructicola significantly and delay the development of disease compared with the control.18 Moreover, Yu et al. (2007) reported that combination of chitosan and antagonistic yeast C. laurentii resulted in a synergistic inhibition of the blue mold rot.19 However, to our knowledge, there is no report referring to enhancing the biocontrol efficacy of antagonistic yeast against postharvest disease by addition of chitosan to the growth media and the mode of action involved. The objective of this study was to determine the impact of adding chitosan in the culture media on the efficacy of the R. mucilaginosa against Rhizopus decay, gray mold decay, and natural decay of strawberries and discuss the possible physiological and molecular mechanisms involved. Received: Revised: Accepted: Published: 4214

January 7, 2014 April 13, 2014 April 13, 2014 April 14, 2014 dx.doi.org/10.1021/jf500065n | J. Agric. Food Chem. 2014, 62, 4214−4224

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Efficacy of Cell-Free Culture Filtrate of R. mucilaginosa Incubated in Different Media in Controlling Rhizopus Decay and Gray Mold Decay of Strawberries. Fresh fruits were treated as described above to evaluate the efficacy of R. mucilaginosa harvested from different media in the control of the Rhizopus decay and gray mold decay of strawberries. Then 30 μL of following solution were added in each wound of fruits: (1) a solution of cell-free filtered supernatant through a Millipore membrane (0.45 μm) from the media of NYDB or NYDB + chitosan at different concentrations (0.01%, 0.1%, 0.25%, 0.5%, 1%), (2) sterile distilled water as the control. Two hours later, 30 μL of R. stolonifer suspensions (1 × 104 spores mL−1) or B. cinerea suspensions (1 × 105 cells mL−1) were added into each wound. After air-drying, the samples were put in trays sealed with plastic film. Then the samples in the trays were incubated at 20 °C and a high relative humidity (about 95%). The number of the infected fruits and their lesion diameters were examined after 3 d inoculation. There were three replicates per treatment and 18 fruits in each replicate. All treatments were arranged in a randomized complete block design, and the experiment was conducted twice. Effects of Chitosan on Biocontrol Efficacy of R. mucilaginosa to Natural Decay Development of Strawberries. To inoculate yeast on fruits, dipping them in the cell suspensions of (1) R. mucilaginosa (1 × 108 cells mL−1) harvested from the media of NYDB, (2) R. mucilaginosa (1 × 108 cells mL−1) harvested from the media of NYDB + chitosan at different concentrations (0.01%, 0.1%, 0.25%, 0.5%, 1%), and (3) sterile distilled water as the control respectively for 30 s, then air-dried. The treated fruits were sealed separately in polyethylene-lined plastic boxes to retain high humidity. Fruits were stored at 4 °C for 20 d, after that, transferred them to the condition at 20 °C for 7 d, which can determine the disease development under normal shelf life conditions. After observation, infection severity was recorded. There were three replicate trials of 20 fruits with a complete randomization in each test, and experiments were repeated twice. Effects of Chitosan on Population Growth of R. mucilaginosa in Strawberry Wounds. Fruits were wounded as described above to evaluate the biocontrol efficacy against Rhizopus decay and gray mold decay of strawberries. Then 30 μL of 1 × 108 cells mL−1 R. mucilaginosa solution harvested either from NYDB or NYDB + 0.5% chitosan were added in each wound. At different times (0, 12, 24, 48, and 72 h) at 20 °C after the treatment, the samples were harvested. The tissue was removed with a sterile cork borer (9 mm diameter and 10 mm deep) and ground with a 150 mL Erlenmeyer flasks, grinding the sample pieces by a glass rod in 10 mL of sterile 0.85% sodium chloride solution. After that, serial 10-fold suitable dilutions were made and 0.1 mL of suitable dilution was spread in NYDA plates. The plates were incubated at 28 °C for 2 d, and the colonies were counted afterward. Population densities of R. mucilaginosa were expressed as log 10 CFU (colony-forming units) per wound. There were two replicates per treatment and 5 fruits per replicate, and the experiments were repeated twice. Assay for Chitinase Activity of the Cell-Free Culture Filtrate of R. mucilaginosa Incubated in Different Growth Media. To determine the chitinase activity, a reaction mixture was prepared by adding 0.5 mL of 5 mg mL−1 colloidal chitosan containing 1.2 μmol sodiumazide and 56 μmol sodium acetate to 0.5 mL of enzyme supernatant.21 The enzyme−substrate mixture was incubated at 37 °C for 2 h with constant shaking. After that, centrifuging at 7680g for 10 min was used to stop the reaction. Following the centrifuging, 0.5 mL of enzyme supernatant was incubated at 37 °C for 2 h with 0.1 mL of 3% (w/v) desalted snail gut enzyme (Sigma) and 0.1 mL of 1 mol L−1 potassium phosphate buffer (pH 7.1) to hydrolyze the chitosan oligomers.22 The resulting N-acetyl-D-glucosamine(GlcNAc) was determined following the method described by Reissig et al. (1955) using internal standards of GlcNAc.23 For the sample, substrate blanks and standard, each contained in a volume of 0.5 mL in a test tube, 0.1 mL of potassium tetraborate was added. The tubes were heated in a vigorously boiling water bath for exactly 3 min, and then the tubes were cooled in tap water. p-Dimethylaminobenzaldehyde (DMAB) reagent of 3 mL was added afterward, then they were mixed and placed in a bath at 37 °C. After precisely 20 min, the tubes were cooled

MATERIALS AND METHODS

Fruits Material. Strawberries (Fragaria ananassa Duch.) cultivars “fengxiang” were harvested early in the morning and rapidly transferred to the laboratory. Berries were sorted on the basis of size, ripeness, and blemishes such as injuries or infections. Chitosan. Chitosan with 90% deacetylation was bought from Sangon Company (shanghai, China) . Antagonist and Growth Conditions. The yeast antagonist Rhodotorula mucilaginosa was isolated from the surfaces of peach blossom which was picked in unsprayed orchards. The yeast was first identified by a VITEK 32 Automicrobic system (BioMérieux Company, Marcy l’Etoile, France). To identify the yeast, sequence analysis of the 5.8S internal transcribed spacer (ITS) rDNA (rDNA) region was used.20 In animal tests, including physiology experiments, acute toxicity studies, and the Ames test, R. mucilaginosa was demonstrated to be safe (our unpublished data). R. mucilaginosa isolates were maintained at 4 °C on nutrient yeast dextrose agar (NYDA) medium (8 g og nutrient broth, 5 g of yeast extract, 10 g of glucose, and 20 g of agar in 1 L of distilled water). Liquid cultures of the yeast were grown in 250 mL Erlenmeyer flasks containing 50 mL of nutrient yeast dextrose broth (NYDB) which had been inoculated with a loop of the culture. Flasks were incubated on a QYC-200 rotary shaker (FuMa, China) at 28 °C for 20 h. After incubation, cells were harvested by centrifugation at 7000g for 10 min by using a TGL-16 M centrifuge (XiangYi, China) and washed twice with sterile distilled water in order to remove the residual medium. Then the yeast cell pellets were resuspended in sterile distilled water, counted using a JSQA hemocytometer (HongTai, China), and adjusted to an initial concentration of 5 × 108 cells mL−1. Then 1 mL of the abovementioned yeast suspensions were added to nutrient yeast dextrose broth (NYDB) or NYDB amended with chitosan at different concentrations (0.01%, 0.1%, 0.25%, 0.5%, 1%) (NYDB + chitosan) on a rotary shaker at 200 rpm at 28 °C for 24 h. After incubation, centrifuged the yeast cells at 7000g for 10 min and washed twice with sterile distilled water. Cell pellets were resuspended and counted as described above and adjusted to a concentration of 1 × 108 cells mL−1. Besides, the growth media was filtered through a Millipore membrane (0.45 μm) and the supernatant was used for biocontrol and chitinase activity assay. Fungal Pathogen and Growth Conditions. The pathogen Rhizopus stolonifer (Ehrenb.: Fr) Vuill. and Botrytis cinerea (Pers.: Fr.) were isolated from infected strawberries. The organisms were identified according to their cultural and micro- and macroscopic morphological characteristics. These cultures were maintained on potato dextrose agar (PDA) media (containing the extract from 200 g of boiled potato, 20 g of glucose, and 20 g of agar in 1 L of distilled water) at 4 °C. Before use, the mold should be activated on PDA at 25 °C. Spore suspensions were prepared by removing the spores from the sporulating edges of a 7 day old culture with a bacteriological loop and suspending them in sterile distilled water. After counting with a hemocytometer, the spore concentration was adjusted to a required concentration. Effects of Chitosan on Biocontrol of R. mucilaginosa to Rhizopus Decay and Gray Mold Decay of Strawberries. The surface of strawberries was wounded using a sterile cork borer (approximately 3 mm diameter and 3 mm deep) and treated with 30 μL of following solution: (1) the cell suspensions of R. mucilaginosa (1 × 108 cells mL−1) which were harvested either from the media of NYDB or NYDB + chitosan at different concentrations (0.01%, 0.1%, 0.25%, 0.5%, 1%); (2) sterile distilled water as the control. Two hours later, 30 μL of R. stolonifer suspensions (1 × 104 spores mL−1) or B. cinerea suspensions (1 × 105 cells mL−1) were added into each wound of strawberries. After air-drying, the samples were put in trays sealed with plastic film. Then the samples in the trays were incubated at 20 °C and a high relative humidity (about 95%). The number of the infected fruits and their lesion diameters were observed after 3 d inoculation. There were three replicates per treatment and 18 fruits in each replicate. All treatments were arranged in a randomized complete block design, and the experiment was conducted twice. 4215

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(μmol L−1) = 6.45 × (A532 − A600) − 0.56 × A450 and expressed as μmol MDA g−1 FW. Assay of chitinase activity of strawberries was determined as described above to evaluate the chitinase activity of different growth media, and the specific activity was expressed as the U per gram FW, one unit was defined as microgram of GlcNAc per hour. β-1,3-Glucanase was assayed by measuring the amount of reducing sugar released from the substrate by the method reported by Ippolito et al., with some modifications.28 A crude enzyme sample of 250 μL was routinely added to 250 μL of 0.2% laminarin (w/v) in 50 mM, pH 5.0, potassium acetate buffer, and incubated at 37 °C for 1 h. For the control, the same mixture was similarly diluted at zero incubation time. Then 1.5 mL of 3,5-dinitrosalicylate was added and boiled for 5 min on a water bath to stop the reaction, and the amount of reducing sugars was measured spectrophotometrically at 500 nm using a UV1601 spectrophotometer (Shimadzu, Japan). The formation of 1 mg glucose equivalents per hour was defined as one unit of the β-1,3glucanase activity, and the specific activity was expressed as the U per gram FW. Protein Sample Preparation. Liquid cultures of the yeast were grown in NYDB or NYDB + 0.5% chitosan, respectively, as described above (Antagonist and Growth Conditions). The yeast cells were harvested from NYDB or NYDB + 0.5% chitosan by centrifuging at 10000g for 10 min (4 °C) and washed three times with cold doubledistilled water to remove residual medium. The protein sample preparation for 2-DE, and image analysis were performed as described by Zhao et al.29 Protein samples were kept at −70 °C until use. The protein concentration was determined according to Bradford’s method using bovine serum albumin as standard.30 2-DE and Image Analysis. Two-dimensional electrophoresis (2DE) and image analysis were performed as described by Wang et al.31 Protein In-Gel Digestion. Protein in-gel digestion was performed as described by Zhang et al.32 Protein Identification by MALDI-TOF/TOF and Database Query. Protein identification by MALDI-TOF/TOF and database query were performed as described by Wang et al.31 Statistical Analysis. The data were analyzed by the analysis of variance (ANOVA) using the statistical program SPSS/PC version II.x (SPSS Inc. Chicago, Illinois, USA). The Duncan’s multiple range test was used for means separation. In addition, when the group of the data was two, the independent samples t test was applied for means separation. The statistical significance was assessed at the level P = 0.05.

in tap water and read without delay at 585 nm. The specific activity was expressed as microgram of GlcNAc per milliliter. There were three replicates in each experiment. The experiment was conducted twice. Effects of R. mucilaginosa Incubated in Different Growth Media on Strawberry Enzyme Activities and Lipid Peroxidation. Fruits were wounded as described above to evaluate the yeast biocontrol activity against Rhizopus decay and gray mold decay of strawberries. Then 30 μL of a cell suspension of R. mucilaginosa at 1 × 108 cells mL−1 harvested either from the media of NYDB or NYDB + 0.5% chitosan after the incubation for 24 h were added in each wound. Treatment with sterile distilled water was served as the control. After air-drying, the strawberries were put in trays sealed with plastic film. Then the samples in the trays were incubated at 20 °C and a high relative humidity (about 95%). At 0, 24, 48, and 72 h after treatment, samples were harvested. After removing the wound tissue with a sterile borer (6 mm diameter and 5 mm deep), the fresh tissue around the wound was picked up by another sterile borer (9 mm diameter and 10 mm deep). Two grams of the fresh tissue from six fruits was grounded with 10 mL of cold (4 °C) 50 mM different buffers containing 1.33 mmol per liter EDTA and 1% polyvinyl pyrolidone (PVPP) in a mortar and pestle to assay different enzymes: sodium acetate buffer (pH 5.0) for chitosanase and β-1,3-glucanase, sodium phosphate buffer (pH 6.4) for polyphenoloxidase (PPO), peroxidase (POD), and malondialdehyde (MDA) and borate buffer (pH 8.8) for phenylalanine ammonia lyase (PAL). The homogenates were centrifuged (4 °C) for 15 min at 12000g, and the supernatants were kept for the assay. There were three replicates per treatment. The experiment was conducted twice. The testing methods of enzyme activities were described as below. The PPO activity was measured following the method described by Waite (1976) with some modifications.24 First 0.1 mL of crude enzyme extract (supernatant extract) was added to 2.9 mL of catechol (50 mM sodium phosphate, pH 6.4, and 0.1 M catechol, incubated at 30 °C for 5 min) as a substrate. Then the change in absorbance at 398 nm was recorded once every 30 s for 3 min. The amount of the enzyme extract producing an increase of A398 by 0.01 in 1 min was defined as one unit of the PPO activity. The PPO enzyme activity was expressed as U per gram fresh weight (FW). The POD activity was measured as the method described by Lurie et al. (1997) with some modifications, using guaiacol as a substrate.25 The reaction mixture should be inoculated for 5 min at 30 °C, which containing 0.2 mL of crude enzyme extract, 2.2 mL of 0.3% guaiacol (prepared by 50 mM sodium phosphate buffer, pH 6.4, incubated at 30 °C for 5 min). Then 0.6 mL of 0.3% H2O2 (prepared by 50 mM sodium phosphate, pH 6.4, and incubated at 30 °C for 5 min) was added to initiate the reaction. The POD activity was determined by measuring at A470 once every 30 s for 3 min. A cuvette containing all components except added 0.6 mL of distilled water was used as a control. The POD activity was expressed as U per gram FW. One unit was defined as ΔA470 of 0.01 per min. The PAL activity was assayed as the method described by Assis et al. (2001) with some modifications.26 Simply, after preincubation of 3 mL of borate buffer (50 mM, pH 8.8) containing 10 mM L −1 phenylalanine for 10 min at 37 °C, 1 mL of crude enzyme extract was added. After mixing, the initial absorbance of the reaction mixture was immediately recorded at 290 nm. Then the reaction mixture was incubated at 37 °C. After 60 min, A290 was measured and recorded again. Calculating the difference value between the pre- and postabsorbance. The blank was a cuvette containing all components except 1 mL of distilled water was added. One unit of the PAL was defined as the formation of 1 μg of cinnamic acid equivalents per hour, and the specific activity was expressed as the U per gram FW. The lipid peroxidation was determined in terms of MDA content by thiobarbituric acid (TBA) reaction as described by Du and Bramlage.27 Briefly, 2 mL of 0.6% TBA in 20% trichloroacetic acid was added into 2 mL of crude enzyme sample. The solution was heated at 100 °C for 10 min, then quickly cooled in an ice-bath and centrifuged (4 °C) for 15 min at 10000g, and afterward the supernatants were harvested and assayed. The absorbance of supernatants was recorded at 532, 600, and 450 nm. MDA content was calculated according to the formula CMDA

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RESULTS Effect of Chitosan on Biocontrol of R. mucilaginosa to Rhizopus Decay and Gray Mold Decay of Strawberries. Compared with the control after 3 d storage at 20 °C, treatments of R. mucilaginosa at 1 × 108 cells mL−1 effectively controlled Rhizopus decay of strawberries (Figure 1, P < 0.05), and the application of chitosan at the concentration 0.5% during cultivation significantly improved the biocontrol efficacy of R. mucilaginosa compared with the cultivation without chitosan (P < 0.05). Compared with the control after 3 d storage at 20 °C, R. mucilaginosa at 1 × 108 cells mL−1 cultivation in the NYDB had no impact on the disease incidence of gray mold decay (Figure 1, P < 0.05). However, treatments with application of R. mucilaginosa cultivation in the NYDB media amended with chitosan at all tested concentrations except 1% could reduce the disease incidence of gray mold decay of strawberries. The antagonistic activity of R. mucilaginosa was shown to be greatly enhanced through cultivation in the NYDB media amended with chitosan at the optimal concentration (0.5%) compared with that cultivated in NYDB without chitosan (P < 0.05). Efficacy of Cell-Free Culture Filtrate of R. mucilaginosa Incubated in Different Media in Controlling Rhizopus 4216

dx.doi.org/10.1021/jf500065n | J. Agric. Food Chem. 2014, 62, 4214−4224

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Effects of Chitosan on Biocontrol Efficacy of R. mucilaginosa to Natural Decay Development of Strawberries. Compared with the control after 20 d at 4 °C followed by 7 d at 20 °C, R. mucilaginosa at 1 × 108 cells mL−1 cultivated in the NYDB media had no impact on the natural decay incidence of strawberries (Figure 3). However, treat-

Figure 1. Effect of chitosan on biocontrol of R. mucilaginosa to Rhizopus decay and gray mold of strawberries. Fruit treatments are as follows: Control = sterile distilled water, 0 = R. mucilaginosa which were harvested from the media of NYDB (0.01%, 0.1%, 0.25%, 0.5%, 1%) = R. mucilaginosa which were harvested from the media of NYDB + chitosan at different concentrations (0.01%, 0.1%, 0.25%, 0.5%, 1%). Each value is the mean of two experiments. Bars represent standard deviations. Different letter indicates significant differences (P = 0.05) according to the Duncan’s multiple range test. Figure 3. Effect of chitosan on biocontrol of R. mucilaginosa to natural decay of strawberries. Fruit treatments are as follows: Control = sterile distilled water, 0 = R. mucilaginosa which were harvested from the media of NYDB (0.01%, 0.1%, 0.25%, 0.5%, 1%) = R. mucilaginosa which were harvested from the media of NYDB + chitosan at different concentrations (0.01%, 0.1%, 0.25%, 0.5%, 1%). Each value is the mean of two experiments. Bars represent standard deviations. Different letter indicates significant differences (P = 0.05) according to the Duncan’s multiple range test.

Decay and Gray Mold Decay of Strawberries. The cellfree culture filtrates of R. mucilaginosa incubated in NYDB had no significant control efficacy on the Rhizopus decay and gray mold decay of strawberries after 3 d storage at 20 °C. However, compared with the control, the cell-free culture filtrates of NYDB media amended with 0.25% 0.5%, and 1% chitosan in which R. mucilaginosa was incubated for 24 h exhibited a significant control activity against Rhizopus decay in strawberry wounds after 3 d incubation at 20 °C (Figure 2). Similarly, the cell-free culture filtrates of NYDB media added with 0.5% chitosan significantly controlled the gray mold decay in strawberry wounds (Figure 2).

ments with application of R. mucilaginosa cultivation in the NYDB media added with chitosan at all tested concentrations except 1% had a significantly biocontrol efficacy on the natural decay incidence of strawberries (P < 0.05). R. mucilaginosa cultivation in the NYDB media amended with 0.5% chitosan was the most effective in preventing natural decay development, which decreased the decay incidence of the fruit to 33.3% compared to 71.7% for the control and 58.9% for the R. mucilaginosa cultivation in the NYDB. Effects of Chitosan on Population Growth of R. mucilaginosa in Strawberry Wounds. The antagonistic yeast R. mucilaginosa, whether cultivated in the NYDB or NYDB media amended with 0.5% chitosan, proliferated rapidly in strawberry wounds, especially during the first 24 h, and stabilized relatively thereafter (Figure 4). However, the population of R. mucilaginosa cultivated in NYDB amended with chitosan at 0.5% in strawberry wounds was higher than that of R. mucilaginosa cultivated in NYDB over the whole storage time except at 72 h (P < 0.05). Particularly, after 24 h of incubation in the wounds, the amount of R. mucilaginosa grown in chitosan-supplemented NYDB media was more than 3-fold higher than that of R. mucilaginosa grown in NYDB without chitosan. Assay for Chitinase Activity of the Cell-Free Culture Filtrate of R. mucilaginosa Incubated in Different Growth Media. After 24 h incubation, the chitinase activity of the cell-free culture filtrates of R. mucilaginosa incubated in NYDB added with chitosan at 0.01, 0.5, and 1% had a higher level than that of R. mucilaginosa grown in NYDB. Especially

Figure 2. Effect of cell-free culture filtrate of R. mucilaginosa incubated in different media in controlling Rhizopus decay and gray mold decay of strawberries. Fruit treatments are as follows: Control = sterile distilled water, 0 = a solution of cell-free filtered supernatant from the media of NYDB (0.01%, 0.1%, 0.25%, 0.5%, 1%) = a solution of cellfree filtered supernatant from the media of NYDB + chitosan at different concentrations (0.01%, 0.1%, 0.25%, 0.5%, 1%). Each value is the mean of two experiments. Bars represent standard deviations. Different letter indicates significant differences (P = 0.05) according to the Duncan’s multiple range test. 4217

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Figure 4. Effects of chitosan on population growth of R. mucilaginosa in strawberry wounds. 0 = R. mucilaginosa which were harvested from the media of NYDB 0.5% = R. mucilaginosa which were harvested from the media of NYDB + chitosan at the concentration of 0.5%. Bars represent standard errors. An asterisk (*) indicates significant differences (P = 0.05) according to the independent samples t test.

Figure 6. Effects of R. mucilaginosa harvested from different media on PPO activity of strawberries. Control = sterile distilled water, NYDB = R. mucilaginosa which were harvested from the media of NYDB, chitosan (0.5%) = R. mucilaginosa which were harvested from the media of NYDB + chitosan at the concentration of 0.5%. Each value is the mean of two experiments. Bars represent standard deviations.

the chitinase activity of the cell-free culture filtrates incubated in NYDB with 0.5% chitosan had a higher level than that of the cell-free culture filtrates grown in NYDB amended with chitosan at other concentrations (Figure 5, P < 0.05). However,

NYDB without chitosan throughout the whole storage period (24, 48, and 72 h after inoculation). Effects of R. mucilaginosa Harvested from Different Media on POD Activity of Strawberries. POD activity of strawberries treated with R. mucilaginosa cultivated in the chitosan-supplemented media (0.5%) and in the media of NYDB showed an uptrend within 48 h after inoculation, and, in the former, POD activity reached the highest peak at 72 h, while in the latter, almost no increase was observed after 48 h (Figure 7). The POD activity of the strawberries treated with R.

Figure 5. Chitinase activity of the cell-free culture filtrate of R. mucilaginosa incubated in media containing chitosan. Each value is the mean of two experiments. Bars represent standard deviations. Data in columns with different letters are statistically different according to Duncan’s multiple range test at P = 0.05.

there was no significant difference of the chitinase activity between the cell-free culture filtrates of R. mucilaginosa grown in NYDB and the media added with 0.1 or 0.25% chitosan. Effects of R. mucilaginosa Harvested from Different Media on PPO Activity of Strawberries. A peak of PPO activity of strawberries was exhibited in the treatment with R. mucilaginosa cultivated in the NYDB media amended with chitosan (0.5%) at 24 h after inoculation. However, after the decrease of the PPO activity at 24 h, it showed a following significant increase again after 48 h (Figure 6). On the other hand, no evident induction of PPO activity was observed in treatment with water or R. mucilaginosa cultivated in the NYDB throughout the whole storage period except at 72 h. It was obvious that the PPO activity of strawberries treated with R. mucilaginosa cultivated in the chitosan-supplemented media (0.5%) was higher than that of the control and that of strawberries treated with R. mucilaginosa cultivated in the

Figure 7. Effects of R. mucilaginosa harvested from different media on POD activity of strawberries. Control = sterile distilled water, NYDB = R. mucilaginosa which were harvested from the media of NYDB chitosan (0.5%) = R. mucilaginosa which were harvested from the media of NYDB + chitosan at the concentration of 0.5%. Each value is the mean of two experiments. Bars represent standard deviations.

mucilaginosa cultivated in the NYDB amended with chitosan (0.5%) was significantly higher than that of the control strawberries at 48 and 72 h. Effects of R. mucilaginosa Harvested from Different Media on PAL Activity of Strawberries. Treatment with R. mucilaginosa cultivated in NYDB containing chitosan at 0.5% induced a peak of PAL activity of strawberries within 48 h after 4218

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treated with R. mucilaginosa cultivated in the NYDB at 48 h after inoculation. Effects of R. mucilaginosa Harvested from Different Media on Chitinase Activity of Strawberries. Throughout the whole storage period, the chitinase activity of strawberries treated with R. mucilaginosa cultivated both in the chitosansupplemented media and in NYDB showed the similar tendency for change, moreover, the chitinase activity in the former treatment was slightly higher than that of the latter (Figure 10). However, in the control strawberries, the chitinase

inoculation and then decreased (Figure 8). Nevertheless, in the treatment with water or R. mucilaginosa cultivated in NYDB, no

Figure 8. Effects of R. mucilaginosa harvested from different media on PAL activity of strawberries. Control = sterile distilled water, NYDB = R. mucilaginosa which were harvested from the media of NYDB, chitosan (0.5%) = R. mucilaginosa which were harvested from the media of NYDB + chitosan at the concentration of 0.5%. Each value is the mean of two experiments. Bars represent standard deviations.

evident peak of PAL activity was induced during the whole storage period. The PAL activity of strawberries treated with R. mucilaginosa incubated in chitosan-supplemented (0.5%) NYDB was significantly higher than that of strawberries treated with R. mucilaginosa incubated in NYDB and the control (P < 0.05) at 48 and 72 h after storage. Effects of R. mucilaginosa Harvested from Different Media on MDA Content of Strawberries. The MDA contents of strawberries treated with R. mucilaginosa and water were demonstrated to have gradual linear ascending tendency during the storage period (Figure 9). The MDA content of strawberries treated with R. mucilaginosa cultivated in the NYDB amended with chitosan (0.5%) was obviously lower than that of the control (P < 0.05) at the whole storage period. What’s more, it was obviously lower than that of strawberries

Figure 10. Effects of R. mucilaginosa harvested from different media on chitinase activity of strawberries. Control = sterile distilled water, NYDB = R. mucilaginosa which were harvested from the media of NYDB, chitosan (0.5%) = R. mucilaginosa which were harvested from the media of NYDB + chitosan at the concentration of 0.5%. Each value is the mean of two experiments. Bars represent standard deviations.

activity increased and reached the peak at 24 h, then decreased at 48 h, and then changed to be steady after 48 h. The chitinase activity of the control strawberries was significantly lower than those of the R. mucilaginosa cultivated in the chitosansupplemented media treated strawberries at 24 and 72 h. Effects of R. mucilaginosa Harvested from Different Media on β-1,3-Glucanase Activity of Strawberries. In the control strawberries, β-1,3-glucanase activity showed the down trend generally and only increased slightly at 48 h. A peak of β-1,3-glucanase activity at 48 h after inoculation and followed by an indistinctive decrease was observed in the treatment with R. mucilaginosa cultivated in the media of NYDB induced. However, compared with R. mucilaginosa cultivated in NYDB without chitosan and the control, the treatment with R. mucilaginosa cultivated in the NYDB media amended with chitosan (0.5%) can improve the β-1,3-glucanase activity of strawberries during the whole storage period, especially after 72 h incubation (Figure 11, P < 0.05). Identification of Differentially Expressed Proteins. For protein identification by means of peptide mass fingerprints (PMF), MASCOT was applied to search protein database of Viridiplantae. In this study, ignoring very faint spots and spots with undefined shapes and areas, more than 480 protein spots were detected in each gel with Image Master 2D Elite software (Figure 12). A total of 52 proteins were differentially expressed when the yeast antagonist R. mucilaginosa were harvested from the NYDB amended with chitosan. Among the 52 proteins, there are 26 up-regulated proteins and 26 down-regulated

Figure 9. Effects of R. mucilaginosa harvested from different media on MDA content of strawberries. Control = sterile distilled water, NYDB = R. mucilaginosa which were harvested from the media of NYDB, chitosan (0.5%) = R. mucilaginosa which were harvested from the media of NYDB + chitosan at the concentration of 0.5%. Each value is the mean of two experiments. Bars represent standard deviations. 4219

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Figure 11. Effects of R. mucilaginosa harvested from different media on β-1,3-glucanase activity of strawberries. Control = sterile distilled water, NYDB = R. mucilaginosa which were harvested from the media of NYDB, chitosan (0.5%) = R. mucilaginosa which were harvested from the media of NYDB + chitosan at the concentration of 0.5%. Each value is the mean of two experiments. Bars represent standard deviations.

proteins. Due to the under-representation of yeast proteins in the protein database, only 16 protein spots were unambiguously identified by protein database analysis (Table 1). Of all the differentially expressed proteins identified, most of them were about the basic cell metabolism, including: N-6 DNA methylase (K-6), threonine synthase (K-14), glutaminase (K4), phosphopantetheine-binding (K-15), amidohydrolase 3 (K11), and actin (K-13), which can maintain cells normal morphology, protein kinase Cdc7 (K-10), which is involved in regulating complex cellular functions, while two-component system sensor protein (K-5) is concerned about osmotic stress on cells. In addition, we also identified sulfatase (K-3), bifunctional proline dehydrogenase/pyrroline-5-carboxylate dehydrogenase (K-16), putative peptidoglycan-binding protein, lysm containing domain (K-7), and several hypothetical proteins (K-1, -2, -8, -9, -12).

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DISCUSSION The results from this study showed that when R. mucilaginosa was cultivated in the media of NYDB amended with 0.5% chitosan, its antogonistic efficacy against the Rhizopus decay, gray mold decay, and natural decay of strawberries was greatly improved. Considering that the widely available natural component, chitosan, is cheap and safe to humans and the environment, it might be an effective, safe, and economical approach to enhance the biocontrol efficacy of R. mucilaginosa to postharvest decay of fruits. In our results, treatments with application of R. mucilaginosa cultivation in the NYDB media amended with chitosan at concentrations of 1% could not reduce the disease incidence of gray mold decay of strawberries compared with the control. This should be further studied to test the reason. For harvested fruits, the diseases caused by fungi usually begin as a result of latent infections established in the field or wound infections.2,33 The wounds, which inevitably occur during harvest, transport, and handling, not only damage the quality of harvested fruit34 but also provide pathways for pathogen invasion, especially for the wound invading necrotrophic fungi such as R. stolonifer.2,35 It has reported

Figure 12. Two-dimensional pattern of intracellular proteins of R. mucilaginosa after cultivation in NYDB or NYDB amended with 0.5% chitosan powder.

that yeasts are capable of rapidly colonizing and growing in the wounds, which can provide an advantage over pathogen growth. Due to this property, antogonistic yeasts were chosen for biocontrol.36 This study showed that R. mucilaginosa harvested from the chitosan-supplemented (0.5%) media proliferated more rapidly in strawberry wounds compared with those grown in NYDB without chitosan. Competition for nutrients and space is proposed to be the principal modes of action of R. mucilaginosa in antagonism with pathogens and the better biocontrol activity of the antagonists can be obtained with a higher the concentration of R. mucilaginosa,5 we assume that the enhanced growth of the antagonistic yeast might be a major reason for adding chitosan in culture media, leading to the increased biocontrol efficacy of R. mucilaginosa against the Rhizopus decay, gray mold decay, and natural decay of strawberries. In our test, chitinase activity was detected in different cellfree culture filtrate of R. mucilaginosa cultivated in different 4220

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Table 1. Identification of Cellular Proteins of R. mucilaginosa Showing Differential Expression under Chitosan Cultivation Using MS/MS Analysis NCBI accession

mass

PI

species

score

NP/PD

K-1 K-2 K-3 K-4 K-5

spot

hypothetical protein FNP_2207 hypothetical protein sulfatase glutaminase two-component system sensor protein

gi|254302157 gi|169777975 gi|134101323 gi|51596263 gi|21229958

109274 99456 55546 35967 83320

5.07 6.18 6.43 5.01 6.06

71 88 45 68 82

7/29 6/22 3/8 3/22 7/15

K-6 K-7

N-6 DNA methylase putative peptidoglycan- binding protein, lysm containing domain hypothetical protein UM01172.1 hypothetical protein MGL_0058 protein kinase Cdc7

gi|92109757 gi|285019619

188696 41099

5.79 8.98

Fusobacterium nucleatum subsp. Aspergillus oryzae RIB40 Saccharopolyspora erythraea NRRL 2338 Yersinia pseudotuberculosis IP 32 953 Xanthomonas campestris pv campestris str. ATCC 33 913 Nitrobacter hamburgensis X14 Xanthomonas albilineans

88 84

11/29 7/21

gi|71005306 gi|164662323 gi|148228724

71955 70112 54289

6.35 5.01 9.04

Ustilago maydis 521 Malassezia globosa CBS 7966 Xenopus laevis

80 97 58

7/13 9/23 4/6

amidohydrolase 3

gi|163941091

59770

5.61

Bacillus weihenstephanensis KBAB4

79

7/18

hypothetical protein ANHYDRO_00219

gi|212695699

36793

9.18

Anaerococcus hydrogenalis DSM 7454

67

5/11

actin

gi|57157304

41945

5.29

Candida boidinii

91

8/19

threonine synthase

gi|242398391

39150

6.81

Thermococcus sibiricus MM 739

47

3/8

phosphopantetheine- binding

gi|242261690

8997

4.76

Clostridium cellulovorans 743B

53

3/6

bifunctional proline dehydrogenase/pyrroline-5-carboxylate dehydrogenase

gi|58584796

116883

5.94

Wolbachia endosymbiont strain TRS of Brugia malayi

75

10/28

K-8 K-9 K10 K11 K12 K13 K14 K15 K16

protein name

media, so R. mucilaginosa can produce chitinase in all kinds of growth media, this was similar to some other strains of postharvest biocontrol yeasts such as C. laurentii and Candida oleophila, which were reported to be able to produce chitinase.12,37,38 Besides, compared with the chitinase activity of the cell-free culture filtrates grown in NYDB media and NYDB plus chitosan at other concentrations, the activity of the cell-free culture filtrates of R. mucilaginosa grown in NYDB amended with 0.5% chitosan showed a higher level. On the test of efficacy of cell-free culture filtrate of R. mucilaginosa incubated in different media in controlling of Rhizopus decay and gray mold decay of strawberries, the filtrates incubated in NYDB had no significant control efficacy on the Rhizopus decay and gray mold decay of strawberries after 3 d storage at 20 °C. However, the cell-free culture filtrates of NYDB media added with 0.5% chitosan could effictively control the Rhizopus decay and gray mold decay in strawberry wounds compared to the control. The chitinase activity may have diverse biological roles including the antifungal activity, which is usually induced in the presence of the chitin.39 Carstens et al. (2003) demonstrated that the chitinase produced by Sacharomyces cerevisiae could inhibit spore germination and hyphal growth of B. cinerea in vitro.40 Yu et al. (2008) reported the antagonistic activity of C. laurentii combined with chitinase treatment showed a increased inhibition against blue mold decay caused by Penicillium expansum of pear fruit associated with the higher chitinase activities.12 So we propose that the enhanced chitinase activity of antagonistic yeast is one of the determining factors to improve antagonistic activity of R. mucilaginosa against postharvest decay of strawberries. In nature, plants survive the attack of many microorganisms by employing several layers of defense responses.41 Besides constitutively expressed barriers, plants can recognize the presence of pathogens and respond by activating defense reactions.42 The success of this defense response depends on

some key factors, including the speed by which the plant recognizes the attacking pathogen and the intensity by which the appropriate defense mechanism is activated. According to the research, the effectiveness of this basal resistance can be improved by elicitors including specific biotic and abiotic stimuli experienced by the plant before contact with the pathogen.43 Recent studies have revealed that the induction of disease resistance by elicitors is a sensitization process that primes the plant for more rapid deployment of the defenses.44−46 PPO catalyzes the oxidation of phenolics to quinones, which are more toxic to pathogens than the former.47 Increased PPO activity is correlated with disease resistance in plants.48,49 POD is bifunctional enzymes that can oxidize various substrates in the presence of H2O2, but it also produce reactive oxygen species (ROSs). POD catalyzes the last step of lignin biosynthesis. High POD activities are associated with the onset of induced resistance which involves several important plant defense mechanisms such as lignification and suberization.25,28,33 So enhancement of lignification and suberization related to maintenance of integrity and vital functions of the cell and increased the levels of antimicrobial phenolic compounds in plant cells attacked by phytopathogens resulted in improvement of disease resistance. PAL is the first committed enzyme of the phenylpropanoid pathway which is directly involved in the synthesis of phenolics, phytoalexins, and lignins that are associated with the localized resistance processes.50 It also has been reported that PAL is transcriptionally induced in response to development and ripening.51 In addition, previous study reported that higher disease resistance and lower decay index accompanied by significantly increased PAL activity in MeJA-treated vegetable soybean pods, sweet cherry, and peach fruit.52−54 Our results showed that PPO, POD, and PAL activity were enhanced in strawberry fruit while treated with R. mucilaginosa incubated in the NYDB media 4221

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pothogenesis-related (PR) proteins such as chitinase, β-1,3glucanase of fruits, and to reduce lipid peroxidation, so as to enhance the resistance and delay the senescence of fruits. The proteomic analysis and comparison of R. mucilaginosa in response to chitosan suggested that 0.5% chitosan improved the basic metabolism of R. mucilaginosa, and this may be one of the molecular mechanisms by which enhancement the efficacy of R. mucilaginosa by adding chitosan in the culture media. All these should be further studied.

amended with 0.5% chitosan compared with the case that without chitosan. On the basis of these, we assume that enhancement the activity of R. mucilaginosa to induce more defense enzymes such as PPO, POD, and PAL so as to enhance the resistance of strawberries may be another mechanism that chitosan enhances the antagonistic activity of R. mucilaginosa against postharvest decay of strawberries. R. mucilaginosa incubated in the NYDB media amended with 0.5% chitosan acted as biotic stimuli, which primed the strawberries for more rapid deployment of the defenses to pathogens. MDA, a decomposition product of polyunsaturated fatty acids hydroperoxides, has been utilized very often as a suitable biomarker for lipid peroxidation, which is an effect of oxidative damage.55 Increased lipid peroxidation has been regarded as a major characteristic of harvested fruits undergoing senescence and membrane injury.56 In this study, MDA contents of strawberries treated with R. mucilaginosa cultivated in the culture media of the chitosan-supplement (0.5%) showed a lower level than that of both the control during the whole storage period and the treatment with R. mucilaginosa cultivated in NYDB at some storage time. This suggests that enhancing the activity of antagonistic yeast to inhibit the ripening and senescence of fruits may be one of the action modes by adding chitosan to improve antogonistic activity of R. mucilaginosa against postharvest decay of strawberries. Our study displayed that R. mucilaginosa cultivated in the NYDB media amended with 0.5% chitosan induced more chitinase and a higher level of β-1,3-glucanase activity of strawberries compared with both the control and the R. mucilaginosa cultivated in NYDB. Previous study indicates that in plants, pathogenesis-related (PR) proteins are induced by a pathogen invasion, such as chitinases and β-1,3-glucanases, which are related to their capacity to degrade fungal cell wall in desease resistance process.57 This induction can be enhanced by some elicitors with the improvement of resistance to pathogens in plants.58,59 Therefore, we infer that improving the activity of antagonistic yeast to induce the production of pathogenesis-related (PR) proteins of strawberries may be another reason that a more remarkable upward trend was seen in the efficacy of R. mucilaginosa cultivated in the NYDB media amended with 0.5% chitosan in controlling postharvest decay of strawberries than the case in NYDB. Furthermore, the identification of differentially expressed proteins showed that, compared with R. mucilaginosa harvested from the NYDB, there are a total of 52 proteins that were differentially expressed when the yeast antagonists were harvested from the NYDB amended with chitosan. Among the 52 proteins, there are 26 up-regulated proteins and 26 down-regulated proteins, and most of them are related to basic metabolism. This indicated that the basic metabolism of R. mucilaginosa was improved by chitosan induced incubation, so as to improve it's biocontrol efficacy to postharvest diseases of strawberries. Response patterns of R. mucilaginosa to chitosan induced incubation are complex, as the differentially abundant proteins are involved in multiple metabolic pathways. In short, our results showed that chitosan induced incubation can improve the antagonistic activity of R. mucilaginosa, which may offer great practical potential in reducing the postharvest diseases of strawberry fruit. The mode of actions may include its ability to enhance competitiveness of yeast antagonist for nutrients and space with pathogens, to enhance chitinase activity of antagonistic yeast, to induce more defense enzymes such as PPO, POD, and PAL, to induce the production of

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86-511-88780201. Fax: +86-511-88780201. E-mail: [email protected] (Hy Zhang). Funding

This research was supported by the National Natural Science Foundation of China (31271967), the Research Fund for the Doctoral Program of Higher Education of China (20123227110015), and the Technology Support Plan of Zhenjiang (NY2013020). Notes

The authors declare no competing financial interest.

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