Rhodosporidium paludigenum induces resistance and defense-related responses against Penicillium digitatum in citrus fruit

Postharvest Biology and Technology 85 (2013) 196–202

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Rhodosporidium paludigenum induces resistance and defense-related responses against Penicillium digitatum in citrus fruit Laifeng Lu a,b , Huangping Lu a,b , Changqing Wu c , Weiwen Fang d , Chen Yu a,b , Changzhou Ye a , Yibing Shi a , Ting Yu a,b,∗ , Xiaodong Zheng a,b,∗∗ a

Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310058, People’s Republic of China Fuli Institute of Food Science, Zhejiang University, Hangzhou 310058, People’s Republic of China c Department of Animal and Food Sciences, University of Delaware, Newark, DE 19716-2150, USA d Chun’an County Agriculture Bureau, Chun’an 311700, People’s Republic of China b

a r t i c l e

i n f o

Article history: Received 19 July 2012 Accepted 11 June 2013 Keywords: Rhodosporidium paludigenum Penicillium digitatum Citrus fruit Induced resistance Postharvest biological control

a b s t r a c t Induced disease resistance against plant pathogens is a promising non-fungicidal decay control strategy. In this study, a potential biocontrol yeast, Rhodosporidium paludigenum, was investigated for its induction of disease resistance against Penicillium digitatum in citrus fruit. The results showed that R. paludigenum is the most effective yeast among three selected yeasts in stimulating the resistance of citrus fruit to green mold. When R. paludigenum was applied 48–72 h before inoculation with P. digitatum, disease incidence and disease severity in citrus fruit significantly decreased. Application of R. paludigenum at concentrations of 1 × 108 and 1 × 109 cells mL−1 respectively resulted in 49.6% and 52.5% reductions in the percentage of infections. Induction of resistance to P. digitatum by R. paludigenum treatment significantly enhanced the activities of defense-related enzymes, including ˇ-1,3-glucanase, phenylalanine ammonia-lyase, peroxidase, and polyphenoloxidase, which may be an important mechanism by which the biocontrol yeast reduces the fungal disease of citrus fruit caused by P. digitatum. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Citrus reticulata Blanco cv. Ponkan is a well-known citrus fruit grown in China and Japan. Losses caused by various postharvest pathogens account for nearly 50% of the total wastage in citrus fruit, which occur at different stages in the farm and after harvest during marketing (Ladaniya, 2008). Penicillium digitatum (Pers.:Fr.) Sacc., the causal agent of green mold, is responsible for major postharvest losses of citrus fruit and accounts for up to 60–80% of the total fungal decay during fruit storage (Ballester et al., 2011). Fungicides such as thiabendazole, imazalil, and sodium o-phenulphenate are used worldwide to minimize the postharvest decay of citrus fruits (Schirra et al., 2011). However, with increasing concerns regarding the dietary and environmental safety of fungicides and the occurrence of fungicide resistance in pathogens, developing safer and

∗ Corresponding author at: Zhejiang University, No. 866 Yuhangtang Road, Hangzhou 310058, People’s Republic of China. Tel.: +86 571 88982398; fax: +86 571 88982191. ∗∗ Corresponding author at: Zhejiang University, No. 866 Yuhangtang Road, Hangzhou 310058, People’s Republic of China. Tel.: +86 571 88982861; fax: +86 571 88982191. E-mail addresses: [email protected] (T. Yu), [email protected] (X. Zheng). 0925-5214/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.postharvbio.2013.06.014

more eco-friendly alternatives are necessary to control postharvest decay (Sharma et al., 2009; Smilanick et al., 2005). Induced resistance against plant pathogens is a very promising non-fungicidal decay control strategy, providing long-term systemic resistance to a broad spectrum of pathogens and pests (Walling, 2001). Induced resistance can be achieved by the application of physical (Arcas et al., 2000; Liu et al., 2010), chemical (Venditti et al., 2005; Zhang et al., 2011), and biological treatments (Droby et al., 2002; El Ghaouth et al., 2003; Nantawanit et al., 2010). In the postharvest fruit-yeast biocontrol system, disease resistance can be stimulated by Candida famata and Candida oleophila in citrus fruit (Krihak et al., 1996; Droby et al., 2002), by Aureobasidium pullulans and Candida saitoana in apple fruit (Ippolito et al., 2000; El Ghaouth et al., 2003), by Cryptococcus laurentii in jujube fruit (Tian et al., 2007), and by Pichia guilliermondii in chili fruit (Nantawanit et al., 2010). However, the mechanism by which yeasts induce resistance remains largely unknown because of the complex interactions among host, pathogen, and antagonist (Droby et al., 2009). Previous studies indicated that Rhodosporidium paludigenum Fell & Tallman can significantly inhibit various fungal diseases of harvested fruits (Wang et al., 2008, 2010a,b). However, further study on the use of this biocontrol yeast to induce fruit resistance has yet to be conducted, and the mechanism by which R. paludigenum suppresses diseases is incompletely understood. Thus, this study was undertaken to determine whether or not R. paludigenum

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induces resistance against P. digitatum in citrus fruit and investigate the potential defense mechanism involved in such resistance. The specific aims are (1) to compare the efficacy of biocontrol and induced resistance activated by different yeasts to inhibit green mold in citrus fruit; (2) to determine the treatment time and inoculation level of R. paludigenum required to induce resistance against P. digitatum in citrus fruit; and (3) to investigate the influence of R. paludigenum on the activity of defense-related enzymes of citrus fruit. 2. Materials and methods 2.1. Citrus fruit and pretreatment Citrus (C. reticulata Blanco cv. Ponkan) fruit without physical injuries or infections by observation were hand-harvested from an orchard located in Chun’an City, Zhejiang Province, China. Citrus fruit with similar size and color were selected for the experiments. Fruit were surface-sterilized in a water solution of 0.1% sodium hypochlorite for 2 min, rinsed thoroughly with tap water, and allowed to air dry at room temperature (20 ◦ C).

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group (n = 12) was used as an experimental unit. The percentages of infected wounds and average lesion diameters of twelve fruit in each treatment were computed as one observation value for statistical analysis, respectively. 2.3.2. Induction of disease resistance to P. digitatum by different yeasts Two wound sites were made around the blossom end in each fruit. Each wound was treated with 50 ␮L of sterile distilled water as the control or with 50 ␮L of one of the following agents: (1) cell suspension of S. cerevisiae at 1 × 108 cells mL−1 as another control; (2) cell suspension of C. laurentii at 1 × 108 cells mL−1 , or (3) cell suspension of R. paludigenum at 1 × 108 cells mL−1 . After incubation at 25 ◦ C for 48 h, a second wound (2 mm deep and 5 mm diameter) was made approximately 6 mm away from the initial wound and inoculated with 30 ␮L of a spore suspension of P. digitatum (1 × 104 spores mL−1 ). The fruit were stored in enclosed plastic trays to maintain high RH (90–95%) at 25 ◦ C. The percentages of infected wounds and average lesion diameters were determined 60 h after inoculation. A total of 36 citrus fruit were randomly separated into three groups, and each group of twelve fruit was treated as an experimental unit.

2.2. Biocontrol agent and fungal cultures Yeast antagonist R. paludigenum Fell & Tallman was originally isolated from the South China Sea and identified by CABI Bioscience Identification Services (IMI 394084) (Wang et al., 2008). Yeast antagonist C. laurentii (Kufferath) Skinner (strain zju 10), which shows biocontrol potential (Yu et al., 2012), was originally isolated from pear fruit. Baker’s yeast (Saccharomyces cerevisiae) was obtained from the Institute of Microbiology, Chinese Academy of Sciences, China, and used as a yeast control. Yeast cells were cultured in a 250 mL flask in 50 mL of nutrient yeast dextrose broth (NYDB) medium on a gyratory shaker at 3.3 s−1 for 36 h at 28 ◦ C. The NYDB medium consisted of 8 g of nutrient broth, 5 g of yeast extract, and 10 g of glucose in 1 L of distilled water. Yeast cultures were then centrifuged (KA1000, Shanghai Anke, Shanghai, China) at 50 s−1 for 15 min. Yeast cells were washed twice with sterile distilled water and re-suspended with sterile distilled water. The cell concentration was counted with a hemocytometer. The pathogen P. digitatum (Pers.:Fr.) Sacc. was isolated from a decayed citrus fruit and maintained on potato dextrose agar (PDA) at 25 ◦ C in the dark. The PDA medium consisted of 200 g of potato extract, 20 g of glucose, and 20 g of agar in 1 L of distilled water. The spore suspension was prepared by flooding 7-day-old sporulating cultures of P. digitatum with sterile distilled water containing 0.05% Tween-20. The spore concentration of the pathogen was adjusted to 1 × 104 spores mL−1 by a hemocytometer. 2.3. Treatment with antagonistic yeasts 2.3.1. Preventive activity of different yeasts in inhibiting green mold Each citrus fruit was gently wounded with a sterile borer around the blossom end to form four wounds of 2 mm depth and 5 mm diameter. Each wound was treated with 50 ␮L of one of four following agents: (1) sterile distilled water as the control; (2) cell suspension of S. cerevisiae at 1 × 108 cells mL−1 as another control; (3) cell suspension of C. laurentii at 1 × 108 cells mL−1 ; or (4) cell suspension of R. paludigenum at 1 × 108 cells mL−1 . After 2 h, 30 ␮L of a spore suspension of P. digitatum (1 × 104 spores mL−1 ) was inoculated into each wound. The fruit were then stored in enclosed plastic trays to maintain high (90–95%) relative humidity (RH) at 25 ◦ C. The number of infected wounds and lesion diameters of wounds were recorded 72 h after inoculation. A total of 36 citrus fruit were randomly separated into three groups. Each

2.3.3. Effect of the time between biocontrol treatment and pathogen inoculation on induced disease resistance by R. paludigenum Each citrus fruit was gently wounded as described in Section 2.3.2. Each wound was treated with 50 ␮L of sterile distilled water as the control or R. paludigenum at 1 × 108 cells mL−1 . At various time intervals (0, 24, 48, and 72 h) after treatment, a second wound (2 mm deep and 5 mm diameter) was made approximately 6 mm away from the initial wound and inoculated with 30 ␮L of spore suspension of P. digitatum (1 × 104 spores mL−1 ). The fruit were then stored as described in Section 2.3.2. The percentages of infected wounds and average lesion diameters were determined 60 h after inoculation. A total of 36 citrus fruit were randomly separated into three groups, and each group of twelve fruit was treated as an experimental unit. 2.3.4. Effect of biocontrol agent concentration on induced disease resistance by R. paludigenum Each citrus fruit was gently wounded as described in Section 2.3.2. Each wound was treated with 50 ␮L of sterile distilled water as the control or with 50 ␮L of one of the following: (1) suspension of R. paludigenum at 1 × 107 cells mL−1 , (2) suspension of R. paludigenum at 1 × 108 cells mL−1 , or (3) suspension of R. paludigenum at 1 × 109 cells mL−1 and then incubated at 25 ◦ C for 48 h. After inoculation, all fruit were stored in enclosed plastic trays to maintain high RH (90–95%). Then, a second wound (2 mm deep and 5 mm diameter) was made approximately 6 mm away from the initial wound and inoculated with 30 ␮L of a spore suspension of P. digitatum (1 × 104 spores mL−1 ). The fruit were stored as described in Section 2.3.2. The percentages of infected wounds and average lesion diameters were determined 60 h after inoculation. A total of 36 citrus fruit were randomly separated into three groups, and each group of twelve fruit was treated as an experimental unit. 2.4. Effect of R. paludigenum on induction of defense-related enzyme activities of citrus For enzyme extraction, each citrus fruit was gently wounded as described above, and each of the two wounds was treated with 50 ␮L of sterile distilled water (control) and R. paludigenum at 1 × 108 cells mL−1 , respectively. Wound sites were removed using a sterile borer at various time intervals (0, 24, 48, and 72 h) after treatment and frozen in liquid nitrogen. Each sample consisted of

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fruit tissues pooled from six wounds that were collected from three fruit. For the extraction of ˇ-1,3-glucanase (GLU), 100 mg of frozen fresh tissue was ground with 4.5 mL of cold (4 ◦ C) 50 mmol L−1 sodium acetate buffer (pH 5.0) containing 1% (w/v) polyvinylpolypyrrolidone (PVPP) in a mortar and pestle. For phenylalanine ammonia-lyase (PAL) extraction, 300 mg of frozen fresh tissue was ground with 4.5 mL of cold (4 ◦ C) borate buffer (pH 8.8) containing 5 mmol L−1 ˇ-mercaptoethanol and 0.1% (w/v) PVPP in a mortar and pestle. For peroxidase (POD) and polyphenoloxidase (PPO) extraction, 300 mg of frozen fresh tissue was ground with 4.5 mL of cold (4 ◦ C) 50 mmol L−1 sodium phosphate buffer (pH 7.8) containing 1.33 mmol L−1 ethylenediamenetetraacetic acid and 1% (w/v) PVPP in a mortar and pestle. The homogenates were centrifuged for 15 min at 133 s−1 and 4 ◦ C. The supernatants were used as crude enzyme extracts to assay the enzyme activities and protein contents. The soluble protein content was assayed according to the Bradford method (1976) using bovine serum albumin as a standard protein. GLU activity was determined by measuring the amount of reducing sugar released from larminarin following the method of Abeles and Forrence (1970) and using glucose as the standard. GLU activity was assayed by incubating 62.5 ␮L of enzyme in 62.5 ␮L of a 5% (w/v) larminarin (L9634, Sigma) preparation for at least 2 h at 40 ◦ C. Then, 50 ␮L of the reaction mixture was collected and diluted to 1:4 with sterile distilled water. The reaction was terminated by heating the sample in boiling water for 5 min. The amount of reducing sugars was measured spectrophotometrically at 492 nm after reaction with 372 ␮L of a dinitrosalicyclic reagent using SpectraMax Plus 384 (MD, USA). Final activity values were expressed in millimoles of glucose per kilogram of total protein and per second. PAL activity was determined based on the production of transcinnamic acid following the method of Yao and Tian (2005). A reaction mixture of 1 mL of enzyme extract, 2 mL of 50 mmol L−1 borate buffer (pH 8.8), and 1 mL of 20 mmol L−1 l-phenylalanine (107256, Merck) was incubated for 2 h at 37 ◦ C. The reaction was terminated by the addition of 1 mL of 1 mol L−1 HCl, and the absorbance of the supernatant was measured at 290 nm using a SpectraMax Plus 384 (MD, USA). The amount of trans-cinnamic acid produced was determined based on its standard curve (C80857, Sigma). The specific activity of PAL was defined as the amount of enzyme extract that produces an increase of trans-cinnamic acid per kilogram of total protein and per second.

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All treatments were arranged in a randomized complete block design and conducted at least twice. The data were obtained from one individual experiment and representative of two independent experiments with similar results. All statistical analyses were done using Statistical Program SPSS/PC ver. II.x (SPSS Inc., Chicago, IL, USA) with two-way analysis of variance and Duncan’s multiple range tests. Mean differences were considered to be significant at P < 0.05. 3. Results 3.1. Preventive activity of different yeasts in inhibiting green mold in citrus fruit As shown in Fig. 1, S. cerevisiae did not show a reduction in disease incidence and treated fruit showed disease incidence similar to control (water-treated) fruit. Compared with the control or S. cerevisiae treatments, treatment with antagonistic yeasts (C. laurentii and R. paludigenum) caused a significant reduction in disease incidence (Fig. 1A) and disease severity (Fig. 1B). The percentages of infection in the C. laurentii and R. paludigenum treatment groups were 11.1% and 16.7%, or lower by 81.8% and 72.7%, respectively, that in the control group. The average lesion diameters of wounds in the C. laurentii and R. paludigenum treatments were 2.6 and 2.8 mm, respectively, compared with 18.9 mm in the control treatment.

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POD activity was measured using guaiacol as a substrate following the method described by Lurie et al. (1997) with some modifications. The reaction mixtures contained 220 ␮L of 0.3% guaiacol, 60 ␮L of 0.3% (w/v) H2 O2 , and 20 ␮L of crude enzyme extracts. The reaction was allowed to proceed for 5 min by measurement at 470 nm once every 30 s after the mixture was incubated for 1 min at 30 ◦ C. One unit of POD activity was defined as the amount of enzyme extract that produces an increase of Abs470 by 1.0 per second. PPO activity was measured using catechol as a substrate fol˜ and Mercado-Silva (2004) lowing the method of Aquino-Bolanos with some modifications. The reaction mixture contained 290 ␮L of 10 mmol L−1 catechol and 10 ␮L of crude enzyme extracts. The reaction was allowed to proceed for 5 min by measurement at 398 nm once every 30 s after the mixture was incubated for 1 min at 30 ◦ C. One unit of PPO activity was defined as the amount of enzyme extract that produces an increase of Abs398 by 1.0 per second.

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Fig. 1. Preventive activity of different yeasts against Penicillium digitatum on citrus fruit stored at 25 ◦ C. Fruit were wounded and treated with 50 ␮L of sterile water (control), Saccharomyces cerevisiae, Cryptococus laurentii, or Rhodosporidium paludigenums, after which wounds were inoculated with 30 ␮L of a P. digitatum suspension at 1 × 104 spores mL−1 . Disease incidence (A) and lesion diameter (B) in citrus fruit were measured 72 h after pathogen inoculation at 25 ◦ C and 90–95% RH. Bars represent standard errors of three replicates. Different letters indicate significant differences (P < 0.05) according to the Duncan’s multiple range tests.

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Fig. 2. Induced resistance of different yeasts against Penicillium digitatum on citrus fruit stored at 25 ◦ C. Fruit were wounded and inoculated with 50 ␮L of sterile water (control), Saccharomyces cerevisiae, Cryptococus laurentii, or Rhodosporidium paludigenums. After 48 h incubation, wounds were inoculated with 30 ␮L of a P. digitatum suspension at 1 × 104 spores mL−1 . Disease incidence (A) and lesion diameter (B) in citrus fruit were measured 60 h after pathogen inoculation at 25 ◦ C and 90% to 95% RH. Bars represent standard errors of three replicates. Different letters indicate significant differences (P < 0.05) according to the Duncan’s multiple range tests.

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Fig. 3. Effect of time and concentration of Rhodosporidium paludigenum on induced resistance of citrus fruit against Penicillium digitatum. The disease incidence (A, C) and lesion diameter (B, D) of time and yeast cell concentration that exhibited disease symptoms were measured 60 h after pathogen inoculation at 25 ◦ C and 90% to 95% RH. Bars represent standard errors of three replicates. Different letters indicate significant differences (P < 0.05) according to Duncan’s multiple range tests. Data of the variable disease incidence (%) were arcsine-transformed before statistical analysis and non-transformed data are shown.

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Fig. 4. Effect of Rhodosporidium paludigenum on the activities of (A) ˇ-1,3-glucanase, (B) phenylalanine ammonia-lyase, (C) peroxidase, and (D) polyphenoloxidase in citrus fruit. The fruit were kept for various time intervals (0, 24, 48, and 72 h) after antagonistic yeast treatment at 25 ◦ C and 90–95% RH, before crude extracts were obtained. Short bars represent standard errors of three replicates. Asterisks indicate significant differences (P < 0.05) according to the Duncan’s multiple range tests.

3.2. Induction of disease resistance by different yeasts Inoculation of antagonists C. laurentii and R. paludigenum significantly affected the induction of resistance in citrus fruit (Fig. 2). Disease incidences in these treatments were 60.0% and 50.8%, or lower by 17.3% and 30.0%, respectively, that of the control (Fig. 2A). The lesion diameters of wounds in the C. laurentii and R. paludigenum treatments were 9.5 and 7.7 mm, or lower by 19.2% and 34.4%, respectively, compared with 11.7 mm in the control group (Fig. 2B). Both the lesion diameter and disease incidence in the R. paludigenum treatment were significantly lower than those in the C. laurentii treatment. 3.3. Effect of time between biocontrol treatment and pathogen inoculation and the agent concentration on induced disease resistance by R. paludigenum The percentage of infection significantly decreased compared with that of the 0 h treatment when the time interval between treatment with R. paludigenum and inoculation of P. digitatum was 24 h or longer (Fig. 3A). The development of average rot diameter was significantly inhibited after 48 and 72 h of incubation and resulted in 55.8% and 45.8% decay reductions, respectively, compared with the 0 h treatment (Fig. 3B). No significant difference in the percentage of infections and average lesion diameter was observed between 48 and 72 h of incubation.

Induction of resistance in citrus fruit depended on the concentration of R. paludigenum treatment. The ability of antagonistic yeast to induce fruit disease resistance increased as the concentration of R. paludigenum increased (Fig. 3C and D). Inoculation with R. paludigenum at 108 and 109 cells mL−1 significantly decreased disease incidence by 39.2% and 42.4%, respectively, compared with that induced by the 107 cells mL−1 treatment (Fig. 3C). However, no significant differences in percentage of infections and average lesion diameter between the treatments with inoculation concentrations of 108 and 109 cells mL−1 were observed. 3.4. Effect of R. paludigenum on induction of defense-related enzyme activities Changes in GLU, PAL, POD, and PPO activities in response to R. paludigenum are shown in Fig. 4. Treatment with R. paludigenum rapidly induced GLU activity in the initial 48 h of incubation. GLU activity levels then remained stable with increasing incubation time. After 48, 72, or 96 h, the GLU activity in yeast-treated tissues was 1.25, 1.23, and 1.20 times higher, respectively, than that of the control and changed only slightly throughout the incubation period (Fig. 4A). Changes in PAL activity in response to wounding or antagonistic yeast showed a pattern similar to that in GLU. PAL activity in yeast-treated tissues increased within 24 h after treatment and remained considerably higher (1.51-fold) than that of the control at

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48 h (Fig. 4B). Although a decline in activity was observed after 48 h, the PAL activity induced by yeast treatment was still significantly higher than that of the water control at 72 h. The same trends were also observed in POD and PPO activities. Application of R. paludigenum in citrus fruit wounds significantly increased POD and PPO activities throughout the incubation period compared with those induced by the water treatment. In yeasttreated tissues, POD and PPO activities rapidly increased within 48 h and respectively reached maxima of 1.77 and 1.48 times higher than that of the water treatment (Fig. 4C and D) at 48 h.

4. Discussion In recent years, yeast-induced resistance in plants has become an increasingly attractive option for suppressing plant pathogens (Raacke et al., 2006). However, induced resistance is not usually a major direct mechanism of postharvest biocontrol agents, most likely because this event is difficult to monitor since both yeast and pathogen are applied at the same site (Castoria and Wright, 2010). In the present study, the antagonistic activity of R. paludigenum was investigated separately from its ability to induce resistance. The antagonistic yeast R. paludigenum and the pathogen P. digitatum were applied in spatially separated wounds on the citrus fruit surface. The results of this study demonstrate that R. paludigenum is more efficient than any other yeast used in this study as a postharvest biocontrol yeast for inducing resistance. Results of this study show that the ability of R. paludigenum to stimulate disease resistance depends on the time of yeast inoculation. The percentage of infection and disease severity in citrus fruit significantly decreased with increasing incubation time. This result agrees with previous reports that show that the ability to induce chili fruit resistance is positively correlated with increased incubation time after application of the yeast P. guilliermondii strain R13 (Nantawanit et al., 2010). However, both disease incidence and disease severity after 72 h incubation were slightly higher than those at 48 h, which may be related to over-maturity of the fruit under high storage temperatures. These results indicate that resistance induced by R. paludigenum can only protect fruit from further infection after treatment. Other antifungal treatments showing curative activities must be combined with the antagonistic yeast to yield the best results. Another contributing factor to the level of resistance of the fruit is the inoculation concentration of R. paludigenum. The positive relationship between inoculation concentration of R. paludigenum and the resistance level indicates that higher inoculation concentrations are effective in inhibiting pathogen invasion. However, no significant difference was observed between the resistances induced by inoculation concentrations of 108 and 109 cells mL−1 . Thus, 108 cells mL−1 was considered the optimum yeast incubation concentration for eliciting resistance to P. digitatum. This finding is similar to that reported by Droby et al. (2002), who found that the development of induced resistance in harvested grapefruit after C. oleophila application is related to the yeast concentration. Host responses in stored fruits induced by biocontrol agents share many features with the defense mechanisms that are induced in actively growing plant tissues (Castoria and Wright, 2010). GLU, PAL, POD, and PPO are commonly studied in the postharvest biocontrol area and known to be involved in plant disease resistance (Zhao et al., 2008). Thus, induction of enhanced activities of GLU, PAL, POD, and PPO by R. paludigenum may be closely correlated with the mechanism by which R. paludigenum induces resistance against P. digitatum in citrus fruit. As one of the most important pathogen-related proteins and hydrolytic enzymes, GLU can directly inhibit fungal growth by

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decomposing ˇ-1,3-glucan in the fungal cell wall (Edreva, 2005). In the present study, compared with the control treatment (water), GLU activity was significantly induced by R. paludigenum 48–96 h after inoculation, especially at 48 and 72 h (Fig. 4A). This result is supported by previous studies that report that GLU accumulation in apple (Ippolito et al., 2000; El Ghaouth et al., 2003) and jujube fruit (Tian et al., 2007) is correlated with increased levels of induced resistance elicited by various antagonistic yeasts. The first enzyme in the phenylpropanoid metabolism pathway is PAL, which is responsible for the biosynthesis of p-coumaric acid derivatives, phytoalexin, and lignins that contribute to plant defense systems (Nicholson and Hammerschmidt, 1992). PAL also participates in the biosynthesis of the defense hormone salicylic acid, which is required for both local and systemic acquired resistance in plants (Dixon and Paiva, 1995). Induction of PAL activities increased significantly in fruit surface-wounds following treatment with the antagonist cells. This result is in agreement with previous findings that PAL is involved in increasing resistance and significantly increases in response to the stimulation of different resistance elicitors in citrus fruit (Droby et al., 2002; Ballester et al., 2010; Nantawanit et al., 2010; Sánchez-Estrada et al., 2009). POD controls the availability of H2 O2 in the cell wall, which is a prerequisite for the cross-linking of phenolic groups in response to various external stress, such as wounds, pathogen interactions, and environmental constraints, through the formation of a physical barrier of lignin or suberin (Passardi et al., 2004). High concentrations of phenolic compounds around wounds or pathogen-infected areas can restrict or weaken pathogen growth (Reimers and Leach, 1991). Ballester et al. (2010) found that soluble POD contributes to the beneficial effect of pathogen infection treatment in reducing disease incidence. Thus, the increase in POD is one of the markers of induced resistance. Besides POD, rapidly elevated levels of PPO are also important for resistant genotypes following infection (Mayer, 2006). PPO can produce antimicrobial phenolic substances through oxidizing phenolic compounds (Mayer and Harel, 1979). Compared with the control treatment, which showed slight changes with increasing incubation time, the activities of POD and PPO in R. paludigenum-treated citrus fruit were markedly enhanced in this study. Similarly, Tian et al. (2006) documented that C. laurentii reduces the disease incidence caused by Alternaria alternata in pear fruit, and the increased activities of defensive enzymes such as GLU, PAL, PPO, and POD are correlated with the onset of induced resistance. In conclusion, the results from this study indicate that postharvest treatment of citrus fruit with R. paludigenum induces higher levels of disease resistance against P. digitatum and significantly enhances the activities of defense-related enzymes, which is an important mechanism by which this biocontrol yeast reduces disease incidence in citrus fruit. However, to understand the mechanism underlying such induced resistance completely, global gene expression changes and the functions of antifungal products obtained after incubation with R. paludigenum require further study.

Acknowledgements This research was partially supported by the National Natural Science Foundation of China (31271962) and the Foundation for the Author of National Excellent Doctoral Dissertation of PR China (201061) and the Ph.D. Programs Foundation of Ministry of Education of China (20100101110087) and the National Public Benefit (Agricultural) Research Foundation of China (200903044) and the Program for Key Innovative Research Team of Zhejiang Province (2009R50036).

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