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Biocontrol of postharvest gray and blue mold decay of apples with Rhodotorula mucilaginosa and possible mechanisms of action
International Journal of Food Microbiology 146 (2011) 151–156
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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o
Biocontrol of postharvest gray and blue mold decay of apples with Rhodotorula mucilaginosa and possible mechanisms of action Renping Li a, Hongyin Zhang a,⁎, Weimin Liu a, Xiaodong Zheng b,⁎⁎ a b
College of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, Jiangsu, People's Republic of China Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310029, Zhejiang, People's Republic of China
a r t i c l e
i n f o
Article history: Received 20 October 2010 Received in revised form 14 February 2011 Accepted 16 February 2011 Keywords: Apples Gray mold decay Blue mold decay Biocontrol Rhodotorula mucilaginosa Possible mechanisms
a b s t r a c t The efficacy of Rhodotorula mucilaginosa against postharvest gray mold, blue mold and natural decay development of apples and the possible mechanisms involved were investigated. The decay incidence and lesion diameter of gray mold and blue mold of apples treated by R. mucilaginosa were significantly reduced compared with the control fruits, and the higher concentration of R. mucilaginosa, the better the efficacy of the biocontrol. R. mucilaginosa also significantly reduced the natural decay development of apples following storage at 20 °C for 35 days or at 4 °C for 45 days followed by 20 °C for 15 days. Germination and survival of spores of Penicillium expansum and Botrytis cinerea were markedly inhibited by R. mucilaginosa in an in vitro test. Rapid colonization of the yeast in apple wounds was observed whether stored at 20 °C or 4 °C. In apples, the activities of peroxidase (POD) and polyphenoloxidase (PPO) were significantly induced and lipid peroxidation (malondialdehyde (MDA) content) was highly inhibited by R. mucilaginosa treatment compared with those of the control fruits. All these results indicated that R. mucilaginosa has great potential for development of commercial formulations to control postharvest pathogens on fruits. Its modes of action were based on competition for space and nutrients with pathogens, inducement of activities of defense-related enzymes such as POD, PPO and inhibition of lipid peroxidation (MDA content) of apples, so as to enhance the resistance and delay the ripening and senescence of apples. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Postharvest losses of fruits and vegetables are high, ranging from 10 and 40% depending on the species and technologies used in the packinghouses (Arras and Arru, 1999; Wilson and Wisniewski, 1994). Apple fruit (Malus domestica Borkh) is one of the most important fruits produced in China. To provide fruit throughout the year, fresh apples are stored after harvest. Postharvest losses caused by fungal diseases are the major factor limiting the storage life of apples (He et al., 2003). More than 90 fungal species may cause decay of apples during storage (Leibinger et al., 1997). Gray mold decay and blue mold decay caused by Botrytis cinerea Pers.: Fr. and Penicillium expansum Link, respectively, are two important postharvest diseases of apples (Saravanakumar et al., 2008). Traditionally, the control of postharvest diseases of fruits relies mainly on the use of synthetic fungicides. However, several fungicides are not used for postharvest treatment or have been removed from the market due to possible toxicological risks (Calvo et al., 2007). Alternative methods of control are needed because of the negative public ⁎ Corresponding author. Tel.: + 86 511 88780174; fax: +86 511 88780201. ⁎⁎ Corresponding author. Tel.: +86 571 86098861; fax: +86 571 86971139. E-mail addresses: [email protected] (H. Zhang), [email protected] (X. Zheng). 0168-1605/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2011.02.015
perceptions about the use of pesticides, development of resistance to fungicides among fungal pathogens, and high development costs of new chemicals (Calvo et al., 2007). Therefore, developing non-chemical control methods to reduce postharvest decay of fruits is becoming more important. Biological control with microbial antagonists has emerged as a promising alternative, with lower environmental impact, either alone or as part of integrated pest management to reduce synthetic fungicide usage (Wilson and Wisniewski, 1994). Currently, at least three formulated microbial antagonists Aspire, Biosave-100, and Biosave110 are now commercially available for use as a postharvest treatment (Ippolito et al., 2000). Decay on apple fruits caused by B. cinerea and P. expansum has been controlled by bacterial and yeast antagonists in laboratory studies (Fan and Tian, 2001; Ippolito et al., 2000; Mikani et al., 2008; Teixidó et al., 1999; Vero et al., 2002; Zhang et al., 2009, 2010). The phylloplane yeast Rhodotorula mucilaginosa has been reported to control B. cinerea on geranium seedlings in combination with fungicides (Buck, 2004). However, to our knowledge there is no information concerning biocontrol of postharvest diseases of fruits by R. mucilaginosa. The physiological mechanisms of control of postharvest diseases of fruits by antagonistic yeasts are not yet clear. In this paper, a yeast antagonist R. mucilaginosa is presented, which was isolated in our laboratory from the surfaces of peach blossom picked in unsprayed orchards. The primary object of this study was to evaluate
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the effectiveness of R. mucilaginosa against B. cinerea and P. expansum infection in apple fruits and determine: (1) the efficacy of R. mucilaginosa in controlling of blue mold and gray mold on apples; (2) the efficacy of R. mucilaginosa on natural decay development of apples; (3) the population dynamics of R. mucilaginosa in apple wounds; (4) the efficacy of R. mucilaginosa against pathogens in vitro; and (5) the effect of R. mucilaginosa on activities of defense-related enzymes and lipid peroxidation of apples.
used as the control. Two hours later, 30 μL of suspension of P. expansum (5× 104 spores/mL) or B. cinerea (1× 105 spores/mL) was inoculated into each wound, respectively. After air drying, the apples were stored in enclosed plastic trays to maintain a high relative humidity (about 95%) at 20 °C. Infection rate and lesion diameter of the treated fruits were measured with a slide caliper rule after 8 days (for P. expansum) or 5 days (for B. cinerea) inoculation. There were three replicates of ten fruits for each treatment, and the experiment was conducted twice.
2. Materials and methods
2.5. Efficacy of R. mucilaginosa for reducing natural decay development of apples
2.1. Pathogen inoculum The pathogens P. expansum and B. cinerea were isolated from infected apple fruits. These cultures were maintained on potato dextrose agar (PDA: extract of boiled potatoes, 200 mL; dextrose (Sangon Co., Shanghai, China), 20 g; agar (Sangon Co., Shanghai, China), 20 g and deionized water, 800 mL) at 4 °C, and fresh cultures were grown on PDA plates before use. 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. Spore concentrations were adjusted as required in sterile distilled water assisted by a hemocytometer (XB-K-250, Jianling Medical Device Co., Danyang, China). 2.2. Antagonist The yeast antagonist R. mucilaginosa was isolated from the surfaces of peach blossom picked in unsprayed orchards. Classical methods based on colony and cell morphologies were used for a preliminary characterization of the yeast (Kurtzman and Fell, 1998). Subsequently, sequence analysis of the 5.8S internal transcribed spacer (ITS) ribosomal DNA (rDNA) region was used to identify the yeast (Li et al., 2010). R. mucilaginosa has been shown to be safe in animal testing, including physiology experiments, acute toxicity studies, and the Ames test (our unpublished data). R. mucilaginosa isolates were maintained at 4 °C on Nutrient Yeast Dextrose Agar (NYDA) medium containing 8 g nutrient broth, 5 g yeast extract, 10 g glucose and 20 g agar (Sangon Co., Shanghai, China), in 1 L of distilled water. Liquid cultures of the yeast were grown in 250 mL Erlenmeyer flasks containing 50 mL of NYD Broth (NYDB) which had been inoculated with a loop of the culture. Flasks were incubated on a rotary shaker at 28 °C for 20 h. Following incubation, cells were centrifuged (TGL-16M Centrifuge, Xiangyi Co., Changsha, China) at 5000 ×g for 10 min and washed twice with sterile distilled water in order to remove the growth medium. Yeast cell pellets were re-suspended in sterile distilled water and adjusted to an initial concentration of 2–5 × 109 cells/mL before being adjusted to the concentrations required for the different experiments. 2.3. Fruits Apples (M. domestica Borkh, cv. Fuji) were harvested at commercial maturity from an orchard in Yantai of Shan dong province, and selected for uniformity of size, ripeness and absence of apparent injury or infection. Fruits were selected randomly and disinfected with 0.1% sodium hypochlorite for 1 min, washed with tap water, and allowed to air dry at room temperature (20 °C). 2.4. Efficacy of R. mucilaginosa in inhibiting blue mold decay and gray mold decay of apples A uniform wound (5 mm diameter and approximately 3 mm deep) was made at the equator of each apple fruit using the tip of a sterile dissecting needle. An aliquot (30 μL) of cell suspensions of R. mucilaginosa at 1 ×105, 1 ×106, 1 ×107, 1 ×108, or 1× 109 cells/mL was respectively pipetted into each wound site, and 30 μL of sterile distilled water was
To evaluate the effect of antagonistic yeast on development of natural decay of apples, intact fruit were inoculated by dipping them into a suspension of R. mucilaginosa (1× 108 cells/mL) for 30 s with sterile distilled water as the control, then air dried. Treated fruits were stored in enclosed plastic trays to retain high humidity (about 95%) and incubated at 20 °C for 35 days or at 4 °C for 45 days followed by 20 °C for 15 days in order to determine disease development under normal shelflife conditions, after which infection rate was measured. There were three replicate trials of 10 fruits with a complete randomization in each test and experiments were repeated three times. 2.6. Population studies of R. mucilaginosa in apple wounds Apples were prepared and wounded as described above. Each wound on fruit was inoculated with 30 μL of 1 × 108 cells/mL washed cell suspension of R. mucilaginosa. Fruits were incubated at 20 °C or at 4 °C. R. mucilaginosa was recovered from the wounds after incubation at 20 °C for 0 (1 h after treatment), 1, 2, 3, 4, 5, 6, 7 and 8 days, and at 4 °C for 0 (1 h after treatment), 3, 6, 9, 12, 15, 18, 23 and 28 days, respectively. The wounded tissue was removed with a sterile cork borer (9 mm diameter) to a depth of 10 mm and macerated in 50 mL of sterile 0.85% sodium chloride solution in a 150 mL Erlenmeyer flask using a glass rod. Serial 10-fold dilutions were made and 0.1 mL of each dilution was spread in NYDA. The plates were incubated at 28 °C for 2 days and the colonies were counted. Population densities of R. mucilaginosa were expressed as log10 CFU per wound (CFU: colonyforming units). There were three single fruit replicates per treatment, and the experiments were repeated twice (Viñas et al., 1998). 2.7. Effects of R. mucilaginosa on germination and survival of spores of P. expansum and B. cinerea in vitro The effects of R. mucilaginosa on spore germination of pathogens were tested in potato dextrose broth (PDB). A one mL aliquot of washed cell suspension of R. mucilaginosa (1× 108 cells/mL) or sterile distilled water as a control was added to 250 mL Erlenmeyer flasks containing 50 mL PDB. At the same time, aliquots (1 mL) of spore suspensions (5 × 107 spores/mL) of P. expansum or B. cinerea were added to respective flasks. After 20 h incubation at 28 °C on a rotary shaker (75 rpm), at least 100 spores per replicate were observed microscopically to determine germination rate (Droby et al., 1997). All treatments consisted of three replicates, and the experiments were repeated twice. The interactions between the antagonist and the pathogens in culture were evaluated on potato-dextrose agar (PDA) plates. Agar disks, 5 mm diameter, were cut from PDA plates and a 300 μL quantity of 1 × 108 cells/mL washed cell suspension of R. mucilaginosa or sterile distilled water as a control was added into each well site of the PDA plates. After 3 h, 100 μL of 5 × 104 spores/mL suspension of P. expansum or 100 μL of 1 × 105 spores/mL suspension of B. cinerea was inoculated into each well site, respectively. One hour later, the plates were sealed with polyethylene film in order to retain high humidity and to avoid cross contamination. The plates were incubated at 28 °C for 7 days after which the lesion diameter (total diameter minus 5 mm) of P. expansum
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2.8.1. Fruit treatment Fruit samples were treated as described above to test the efficacy of R. mucilaginosa in inhibiting blue mold and gray mold decay in vivo. The wounds were then treated with 30 μL of cell suspension of R. mucilaginosa (1 × 108 cells/mL) or sterile distilled water (control). After air drying, the apple fruits were stored in enclosed plastic trays to maintain a high relative humidity (above 95%) and incubated at 20 °C. In order to measure the elicitation effect, the tissue surrounding each wound of fruit was collected at 0 (1 h after treatment), 1, 2, 3, 4, 5 and 6 days after treatment. There were three replicates per treatment and the experiment was conducted three times. 2.8.2. Supernatant extract Extraction procedures were conducted at 4 °C. Two grams of tissue sample was ground with 10 mL of cold (4 °C) sodium phosphate buffer (50 mmol/L, pH 7.8) containing 1.33 mmol/L EDTA and 1% polyvinyl pyrrolidone (PVP) for peroxidase (POD), polyphenoloxidase (PPO) and lipid peroxidation (MDA) respectively. The homogenates were then centrifuged at 12,000 g for 15 min at 4 °C and the supernatants were assayed. 2.8.3. Analysis of enzyme activities and lipid peroxidation of apple fruits POD activity was assayed according to the method described by Lurie et al. (1997) with some modifications, using guaiacol as a substrate. The reaction mixture contained 0.2 mL of supernatant (crude enzyme extract), 2.2 mL of 0.3% guaiacol (prepared in 50 mM sodium phosphate buffer, pH 6.4) and was incubated for 5 min at 30 °C. The reaction was then initiated immediately by adding 0.6 mL of 0.3% H2O2 (prepared in 50 mM sodium phosphate, pH 6.4, and incubated at 30 °C for 5 min) and the activity was determined by measuring absorbance at 470 nm once every 30 s for 3 min. As a control, a cuvette containing all components except 0.6 mL of distilled water instead of supernatant was used. The POD activity was expressed as U per g fresh tissue weight (U/g FW). One unit was defined as an increase in A470 of 0.01 per minute. PPO activity was measured following the method described by Mohammadi and Kazemi (2002) with some modifications. The reaction mixtures contained 2.8 mL buffered substrate (50 mmol/L sodium phosphate, pH 6.4 and 100 μmol/L catechol) and 200 μL of enzyme extract. After the mixture was incubated at 30 °C for 1 min, the change in absorbance at 398 nm was measured once every 30 s for 3 min. One unit of the PPO activity is defined as the amount of the enzyme extracts producing an increase of A398 by 0.01 in 1 min and the activity is expressed as U per g fresh tissue weight (U/g FW). Lipid peroxidation was determined in terms of malondialdehyde (MDA) content by thiobarbituric acid (TBA) reaction following the method described by Du and Bramlage (1992) with slight modifications. Simply, 2 mL of 0.6% TBA in 10% trichloroacetic acid was added into 2 mL crude enzyme sample. The solution was heated at 100 °C for 10 min, quickly cooled in an ice-bath, and then centrifuged (4 °C) for 15 min at 10,000 g and the supernatants were assayed. The absorbance of supernatants were recorded at A532, A600, and A450, and MDA content was calculated according to the following formula: C (μmol/L) MDA = 6.45×(A532 −A600)−0.56×A450, and expressed as nmol MDA/g FW. 2.9. Statistical analyses The data were analyzed by the analysis of variance (ANOVA) using the statistical program SPSS/PC version II.x, (SPSS Inc. Chicago, Illinois, USA) and the Duncan's multiple range test was used for separation of means. The statistical significance was applied at the level p b 0.05.
3.1. Effects of R. mucilaginosa in inhibiting blue mold decay and gray mold decay of apples The disease incidence of both blue mold and gray mold decay of the fruits treated with R. mucilaginosa at all tested concentrations were significantly reduced compared with those of the control fruits (p b 0.05) (Fig. 1a). The lesion diameter of blue mold decay of the fruits treated with R. mucilaginosa at all tested concentrations except 1 × 105 and 1 × 106 CFU/mL were significantly reduced compared with those of the control fruits (p b 0.05) (Fig. 1b). The higher the concentrations of the antagonist, the lower the disease incidence and the smaller the lesion diameter. When the concentration of R. mucilaginosa was 1 × 109 cells/mL, blue mold decay was completely inhibited whereas there was a high disease incidence (97.2%) and large lesion diameter (2.44 cm) in the control fruit. Gray mold decay and lesion diameter were inhibited by 97.1% and 56.1% respectively compared to the control. 3.2. Effects of R. mucilaginosa on natural decay development of apples Our experiments evaluated the efficacy of yeast antagonist in reducing the natural development of decay. The results presented in Fig. 2 indicated that the application of R. mucilaginosa produced significant control of
(a) 120 100
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2.8. Effects of R. mucilaginosa on activities of defense-related enzymes and lipid peroxidation of apples
3. Results
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Treatments Fig. 1. Efficacy of R. mucilaginosa in inhibiting blue mold decay and gray mold decay of apples. Disease incidence (a) and lesion diameter (b) were measured after 8 days (P. expansum) or 5 days (B. cinerea) incubation at 20 °C. The letters of A, B, C, D, and E represent the concentrations of R. mucilaginosa at 1 × 105, 1 × 106, 1 × 107, 1 × 108 and 1 × 109 CFU/mL respectively. Each value is the mean of three experiments. Bars represent the standard error of the mean. Data in columns with the different letters are significantly different according to Duncan's multiple range test at p = 0.05.
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3.3. Population studies of R. mucilaginosa in apple wounds
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The population of R. mucilaginosa in apple wounds grew rapidly at 20 °C especially in the first day (Fig. 3a). The population reached the maximum at 3 days (6.05 × 105 CFU/mL) from an inoculum of 7.04×104 CFU/mL, after which it decreased then stabilized at a high level. Similarly, in apple wounds kept at 4 °C, rapid colonization of R. mucilaginosa was observed during the first 3 days, and then the population slowly increased to the maximum at 15 days (6.20×105 CFU/ mL) from an inoculum of 2.29×104 CFU/mL (Fig. 3b). The population then decreased, but did not fall below 1.23×105 CFU/mL.
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3.4. Effects of R. mucilaginosa on survival of P. expansum and B. cinerea in vitro
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Fig. 2. Effects of R. mucilaginosa on natural decay development of apples. Each value is the mean of three experiments. Bars represent the standard error of the mean. Data in columns with the different letters are significantly different according to Duncan's multiple range test at p = 0.05.
natural infections of apple fruits (pb 0.05). After storage at 20 °C for 35 days, the decay incidence of apples treated by R. mucilaginosa was 20% compared with 44.4% in the control fruits. Similarly, after storage at 4 °C for 45 days followed by 20 °C for 15 days, the decay incidence of apples treated with R. mucilaginosa was 22.2% compared with 53.3% in the control fruits.
After incubation with R. mucilaginosa at 28 °C in PDB for 20 h, the percentage germination of spores of P. expansum and B. cinerea was significantly reduced, by 81.87% and 82.78% respectively, compared with the control (Fig. 4a, p b 0.05). The growth on PDA of the two pathogens was also markedly controlled by R. mucilaginosa (Fig. 4b, p b 0.05), with colony diameters of P. expansum and B. cinerea reduced by 61.41% and 62.91% respectively after 7 days incubation at 28 °C, compared with the control.
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Days of incubation at 4 °C Fig. 3. Population dynamics of R. mucilaginosa in apple wounds at 20 °C (a) and 4 °C (b). Bars represented standard errors.
Fig. 4. Effects of R. mucilaginosa on survival of P. expansum and B. cinerea in vitro as measured by (a) spore germination and (b) lesion diameter. Spore germination (percentage of germinated spores from at least 100 spores) and lesion diameter (total diameter minus 5 mm) were recorded. Each value is the mean of three experiments. Bars represent the standard error of the mean. Data in columns with the different letters are significantly different according to Duncan's multiple range test at p = 0.05.
R. Li et al. / International Journal of Food Microbiology 146 (2011) 151–156
As shown in Fig. 5a, treatment with R. mucilaginosa resulted in a significant increase in the activity of POD of apples compared with the control over the entire storage time. At 4 days after inoculation, POD activity of apples peaked at 1.68 times of that of the control. 3.6. Effects of R. mucilaginosa on PPO activity of apples The PPO activity in the tissue of both the apples treated with R. mucilaginosa and the control apples increased gradually in the first 4 days after inoculation and reached a peak at the 4 days. The PPO activities decreased at 5 days then significantly increased at 6 days. However, PPO activity in the tissue treated with R. mucilaginosa was still higher than that of the control over the entire storage time. The PPO activities in treated fruit were approximately 1.20 fold and 1.18 fold of those of the control at 4 and 6 days respectively (Fig. 5b).
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3.5. Effects of R. mucilaginosa on POD activity of apples
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3.7. Effects of R. mucilaginosa on MDA content of apples
The use of biocontrol agents to manage postharvest decay of fruit has been explored as an alternative to the use of synthetic fungicides (Wilson and Wisniewski, 1989). Further identification of new antagonists is desirable because antagonists identified in specific geographic areas may be more effective against the pathogen strains present in that locale (Vero et al., 2002). In our laboratory, we have identified a strain of the yeast R. mucilaginosa which has biocontrol efficacy against blue mold and gray mold of apples caused by P. expansum and B. cinerea respectively. This is the first report of R. mucilaginosa as an antagonist of postharvest diseases of fruits. Data in this present study show that application of R. mucilaginosa significantly reduced the postharvest disease incidence and lesion diameter of apples caused by P. expansum and B. cinerea. Furthermore, in the experiments on efficacy of R. mucilaginosa for reducing natural decay development, natural disease incidence of apples was significantly reduced by R. mucilaginosa following storage at 20 °C for 35 days or 4 °C for 45 days followed by 20 °C for 15 days. This implies that R. mucilaginosa has potential to control a wide range of pathogens. Fruits and vegetables are often kept at low temperature, the biocontrol ability of antagonist under normal shelf-life conditions (including both low temperature and ambient storage) suggest that R. mucilaginosa can be used in combination with cold storage to enhance disease control. Our research also showed that R. mucilaginosa treatment did not impair quality parameters of apples during storage, including firmness, total soluble solids (TSS), ascorbic acid, and titratable acidity (our unpublished data). The results of effects of R. mucilaginosa in inhibiting blue mold decay and gray mold decay of apples indicated that biocontrol efficacy was a function of concentration: the higher the concentration of R. mucilaginosa, the better the biocontrol activity of the antagonist. Furthermore, growth studies demonstrated that R. mucilaginosa could rapidly colonize and grow in apple wounds whether at 20 °C or 4 °C. Such rapid growth in wounds indicated that R. mucilaginosa is well adapted to the wound
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4. Discussion
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The MDA content of the control apples increased in the first 4 days after inoculation and reached a peak at the fourth day (Fig. 5c). The MDA content decreased at 5 days then significantly increased at 6 days. However, the MDA content of the apples treated with R. mucilaginosa increased at 1 days after inoculation, decreased at 2 days, then increased at 3 days, and reached a peak. Thereafter, the MDA content of the apples treated by R. mucilaginosa decreased. The MDA content of apples treated with R. mucilaginosa was lower than that of the control over the duration of storage. At 2 days, 4 days and 6 days after inoculation, the MDA content of the control apples were 1.14, 1.16 and 1.17 fold higher than those of the antagonistic yeast treated apples respectively.
0
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Days after inoculation Fig. 5. Effects of R. mucilaginosa on peroxidase (POD) activity (a), polyphenoloxidase (PPO) activity (b) and lipid peroxidation as measured by malondialdehyde (MDA) content (c) of apples. Each value is the mean of two experiments. Bars represent standard errors.
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environment in fruit. These results point to the main mode of action of R. mucilaginosa being competition for space and nutrient with pathogens. Similar results for other antagonist yeasts have been reported by Bencheqroun et al. (2007), Zhang et al. (2005), Mclaughlin et al. (1992), and Spadaro et al. (2002). To further understand the mechanisms by which R. mucilaginosa controlled postharvest diseases of apples, we investigated the effects of R. mucilaginosa on spore germination and growth of the two pathogens in vitro. R. mucilaginosa significantly inhibited spore germination in PDB media and inhibited growth of P. expansum and B. cinerea on PDA. Furthermore, the percentage of disease incidence and lesion diameter of blue mold and gray mold in fruits treated with washed cell suspension of R. mucilaginosa were significantly lower than those of the control fruits. These results suggested that limiting spore germination and growth of pathogens is another mechanism of action of R. mucilaginosa in control of postharvest diseases of apples. There were reports that increased POD and PPO activities may strengthen defense systems in plants, by biosynthesizing metabolites such as phytoalexins and phenols to form lignin or toxic quinines directed against pathogen infection (Milosevic and Slusarenko, 1996; Mohammadi and Kazemi, 2002). Mittler (2002) has reported that POD and catalase are important active free-radical scavenging enzymes, and decrease in these enzymes may lead to high levels of reactive oxygen intermediates (ROI). Sustained accumulation of ROI may cause lipid peroxidation, aggravate oxidative damage, and accelerate senescence (Hariyadi and Parkin, 1991). Furthermore, in postharvest systems, increased lipid peroxidation has been regarded as a major characteristic of harvested fruits undergoing senescence and membrane injury (Deighton et al., 1999). In our research, apple fruits inoculated with R. mucilaginosa showed higher levels of POD and PPO activities and less lipid peroxidation (MDA content) than those of the control fruits over the whole storage period. These results imply that induced activity of defense-related enzymes and inhibition of ripening and senescence may be part of the mechanism of R. mucilaginosa in controlling postharvest diseases of apple fruits. In conclusion, the results reported here showed that R. mucilaginosa has great potential as a biocontrol agent for the control of postharvest blue mold decay and gray mold decay of apples caused by P. expansum and B. cinerea and is capable of reducing natural decay development of apples. Potential modes of action include competition for space and nutrient with pathogens, direct inhibition of spore germination and growth of pathogens and ability to induce defense-related enzymes such as POD, PPO and reduce lipid peroxidation of apples, enhancing resistance and delaying senescence of apples. More research into the mode of action of R. mucilaginosa to control postharvest diseases of fruits is needed, particularly to elucidate mechanisms at the molecular and proteomic level. Acknowledgements This research was supported by the Natural Science Foundation of Jiangsu Province (BK2009214), the National Natural Science Foundation of China (NNSFC-30771514) and the Foundation for the Eminent Talent of Jiangsu University. References Arras, G., Arru, S., 1999. Integrated control of postharvest citrus decay and induction of phytoalexins by Debaryomyces hansenii. Advances in Horticultural Science 13, 76–81. Bencheqroun, S.K., Bajji, M., Massart, S., Labhilili, M., El-jafari, S., Jijakli, M.H., 2007. In vitro and in situ study of postharvest apple blue mold biocontrol by Aureobasidium pullulans: evidence for the involvement of competition for nutrients. Postharvest Biology and Technology 46, 128–135.
Buck, J.W., 2004. Combinations of fungicides with phylloplane yeasts for improved control of Botrytis cinerea on geranium seedlings. Phytopathology 94, 196–202. Calvo, J., Calvente, V., Orellano, M.E., Benuzzi, D., Tosetti, M.I.S., 2007. Biological control of postharvest spoilage caused by Penicillium expansum and Botrytis cinerea in apple by using the bacterium Rahnella aquatilis. International Journal of Food Microbiology 113, 251–257. Deighton, N., Muckenschnabel, I., Goodman, B.A., Williamson, B., 1999. Lipid peroxidation and the oxidative burst associated with infection of Capsicum annuum by Botrytis cinerea. Plant Journal 20, 485–492. Droby, S., Wisniewski, M.E., Cohen, L., Weiss, B., Touitou, D., Eilam, Y., Chalutz, E., 1997. Influence of CaCl2 on Penicillium digitatum, grape fruit peel tissue, and biocontrol activity of Pichia guilliermondii. Phytopathology 87, 310–315. Du, D., Bramlage, W.J., 1992. Modified thiobarbituric acid assay for measuring lipid oxidation in sugar-rich plant tissue extracts. Journal of Agricultural and Food Chemistry 40, 1566–1570. Fan, Q., Tian, S.P., 2001. Postharvest biological control of grey mold and blue mold on apple by Cryptococcus albidus (Saito) Skinner. Postharvest Biology and Technology 21, 341–350. Hariyadi, P., Parkin, K.L., 1991. Chilling-induced oxidative stress in cucumber fruits. Postharvest Biology and Technology 1, 33–45. He, D., Zheng, X.D., Yin, Y.M., Sun, P., Zhang, H.Y., 2003. Yeast application for controlling apple postharvest diseases associated with Penicillium expansum. Botanical Studies 44, 211–216. Ippolito, A., Ghaouth, A.E., Wilson, C.L., Wisniewski, M., 2000. Control of postharvest decay of apple fruit by Aureobasidium pullulans and induction of defense responses. Postharvest Biology and Technology 19, 265–272. Kurtzman, C.P., Fell, J.W., 1998. The Yeasts: A Taxonomic Study, 4th Ed. Elsevier, Amsterdam. Leibinger, W., Breuker, B., Hahn, M., Mendgen, K., 1997. Control of postharvest pathogens and colonization of the apple surface by antagonistic microorganisms in the field. Phytopathology 87, 1103–1110. Li, S.S., Cheng, C., Li, Z., Chen, J.Y., Yan, B., Han, B.Z., Reeves, M., 2010. Yeast species associated with wine grapes in China. International Journal of Food Microbiology 138, 85–90. Lurie, S., Fallik, E., Handros, A., Shapira, R., 1997. The possible involvement of peroxidase in resistance to Botrytis cinerea in heat treated tomato fruit. Physiological and Molecular Plant Pathology 50, 141–149. Mclaughlin, R.J., Wilson, C.L., Droby, S., Ben-Arie, R., Chalutz, E., 1992. Biological control of postharvest diseases of grape, peach, and apple with the yeasts Kloeckera apiculata and Candida guilliermondii. Plant Disease 76, 470–473. Mikani, A., Etebarian, H.R., Sholberg, P.L., O'Gorman, D.T., Stokes, S., Alizadeh, A., 2008. Biological control of apple gray mold caused by Botrytis mali with Pseudomonas fluorescens strains. Postharvest Biology and Technology 48, 107–112. Milosevic, N., Slusarenko, A.J., 1996. Active oxygen metabolism and lignification in the hypersensitive response in bean. Physiological and Molecular Plant Pathology 49, 143–158. Mittler, R., 2002. Oxidative stress, antioxidants and stress tolerance. Trends in Plant Science. 7, 5–410. Mohammadi, M., Kazemi, H., 2002. Changes in peroxidase and polyphenol oxidase activities in susceptible and resistant wheat heads inoculated with Fusarium graminearum and induced resistance. Plant Sciences 162, 491–498. Saravanakumar, D., Ciavorella, A., Spadaro, D., Garibaldi, A., Gullino, M.L., 2008. Metschnikowia pulcherrima strain MACH1 outcompetes Botrytis cinerea, Alternaria alternata and Penicillium expansum in apples through iron depletion. Postharvest Biology and Technology 49, 121–128. Spadaro, D., Vola, R., Piano, S., Gullino, M.L., 2002. Mechanisms of action and efficacy of four isolates of the yeast Metschnikowia pulcherrima active against postharvest pathogens on apples. Postharvest Biology and Technology 24, 123–134. Teixidó, N., Usall, J., Viñas, I., 1999. Efficacy of preharvest and postharvest Candida sake biocontrol treatments to prevent blue mould on apples during cold storage. International Journal of Food Microbiology 50, 203–210. Vero, S., Mondino, P., Burgueño, J., Soubes, M., Wisniewski, M., 2002. Characterization of biocontrol activity of two yeast strains from Uruguay against blue mold of apple. Postharvest Biology and Technology 26, 91–98. Viñas, I., Usall, J., Teixidó, N., Sanchis, V., 1998. Biological control of major postharvest pathogens on apple with Candida sake. International Journal of Food Microbiology 40, 9–16. Wilson, C.L., Wisniewski, M.E., 1989. Biological control of postharvest diseases of fruits and vegetables: an emerging technology. Annual Review of Phytopathology 27, 425–441. Wilson, C.L., Wisniewski, M.E. (Eds.), 1994. Biological Control of Postharvest Diseases: Theory and Practice. CRC Press, Boca Raton, FL. Zhang, H.Y., Zheng, X.D., Fu, C.X., Xi, Y.F., 2005. Postharvest biological control of gray mold rot of pear with Cryptococcus laurentii. Postharvest Biology and Technology 35, 79–86. Zhang, H.Y., Wang, L., Ma, L.C., Dong, Y., Jiang, S., Xu, B., Zheng, X.D., 2009. Biocontrol of major postharvest pathogens on apple using Rhodotorula glutinis and its effects on postharvest quality parameters. Biological Control 48, 79–83. Zhang, D.P., Spadaro, D., Garibaldi, A., Gullino, M.L., 2010. Efficacy of the antagonist Aureobasidium pullulans PL5 against postharvest pathogens of peach, apple and plum and its modes of action. Biological Control 54, 172–180.
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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o
Biocontrol of postharvest gray and blue mold decay of apples with Rhodotorula mucilaginosa and possible mechanisms of action Renping Li a, Hongyin Zhang a,⁎, Weimin Liu a, Xiaodong Zheng b,⁎⁎ a b
College of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, Jiangsu, People's Republic of China Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310029, Zhejiang, People's Republic of China
a r t i c l e
i n f o
Article history: Received 20 October 2010 Received in revised form 14 February 2011 Accepted 16 February 2011 Keywords: Apples Gray mold decay Blue mold decay Biocontrol Rhodotorula mucilaginosa Possible mechanisms
a b s t r a c t The efficacy of Rhodotorula mucilaginosa against postharvest gray mold, blue mold and natural decay development of apples and the possible mechanisms involved were investigated. The decay incidence and lesion diameter of gray mold and blue mold of apples treated by R. mucilaginosa were significantly reduced compared with the control fruits, and the higher concentration of R. mucilaginosa, the better the efficacy of the biocontrol. R. mucilaginosa also significantly reduced the natural decay development of apples following storage at 20 °C for 35 days or at 4 °C for 45 days followed by 20 °C for 15 days. Germination and survival of spores of Penicillium expansum and Botrytis cinerea were markedly inhibited by R. mucilaginosa in an in vitro test. Rapid colonization of the yeast in apple wounds was observed whether stored at 20 °C or 4 °C. In apples, the activities of peroxidase (POD) and polyphenoloxidase (PPO) were significantly induced and lipid peroxidation (malondialdehyde (MDA) content) was highly inhibited by R. mucilaginosa treatment compared with those of the control fruits. All these results indicated that R. mucilaginosa has great potential for development of commercial formulations to control postharvest pathogens on fruits. Its modes of action were based on competition for space and nutrients with pathogens, inducement of activities of defense-related enzymes such as POD, PPO and inhibition of lipid peroxidation (MDA content) of apples, so as to enhance the resistance and delay the ripening and senescence of apples. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Postharvest losses of fruits and vegetables are high, ranging from 10 and 40% depending on the species and technologies used in the packinghouses (Arras and Arru, 1999; Wilson and Wisniewski, 1994). Apple fruit (Malus domestica Borkh) is one of the most important fruits produced in China. To provide fruit throughout the year, fresh apples are stored after harvest. Postharvest losses caused by fungal diseases are the major factor limiting the storage life of apples (He et al., 2003). More than 90 fungal species may cause decay of apples during storage (Leibinger et al., 1997). Gray mold decay and blue mold decay caused by Botrytis cinerea Pers.: Fr. and Penicillium expansum Link, respectively, are two important postharvest diseases of apples (Saravanakumar et al., 2008). Traditionally, the control of postharvest diseases of fruits relies mainly on the use of synthetic fungicides. However, several fungicides are not used for postharvest treatment or have been removed from the market due to possible toxicological risks (Calvo et al., 2007). Alternative methods of control are needed because of the negative public ⁎ Corresponding author. Tel.: + 86 511 88780174; fax: +86 511 88780201. ⁎⁎ Corresponding author. Tel.: +86 571 86098861; fax: +86 571 86971139. E-mail addresses: [email protected] (H. Zhang), [email protected] (X. Zheng). 0168-1605/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2011.02.015
perceptions about the use of pesticides, development of resistance to fungicides among fungal pathogens, and high development costs of new chemicals (Calvo et al., 2007). Therefore, developing non-chemical control methods to reduce postharvest decay of fruits is becoming more important. Biological control with microbial antagonists has emerged as a promising alternative, with lower environmental impact, either alone or as part of integrated pest management to reduce synthetic fungicide usage (Wilson and Wisniewski, 1994). Currently, at least three formulated microbial antagonists Aspire, Biosave-100, and Biosave110 are now commercially available for use as a postharvest treatment (Ippolito et al., 2000). Decay on apple fruits caused by B. cinerea and P. expansum has been controlled by bacterial and yeast antagonists in laboratory studies (Fan and Tian, 2001; Ippolito et al., 2000; Mikani et al., 2008; Teixidó et al., 1999; Vero et al., 2002; Zhang et al., 2009, 2010). The phylloplane yeast Rhodotorula mucilaginosa has been reported to control B. cinerea on geranium seedlings in combination with fungicides (Buck, 2004). However, to our knowledge there is no information concerning biocontrol of postharvest diseases of fruits by R. mucilaginosa. The physiological mechanisms of control of postharvest diseases of fruits by antagonistic yeasts are not yet clear. In this paper, a yeast antagonist R. mucilaginosa is presented, which was isolated in our laboratory from the surfaces of peach blossom picked in unsprayed orchards. The primary object of this study was to evaluate
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the effectiveness of R. mucilaginosa against B. cinerea and P. expansum infection in apple fruits and determine: (1) the efficacy of R. mucilaginosa in controlling of blue mold and gray mold on apples; (2) the efficacy of R. mucilaginosa on natural decay development of apples; (3) the population dynamics of R. mucilaginosa in apple wounds; (4) the efficacy of R. mucilaginosa against pathogens in vitro; and (5) the effect of R. mucilaginosa on activities of defense-related enzymes and lipid peroxidation of apples.
used as the control. Two hours later, 30 μL of suspension of P. expansum (5× 104 spores/mL) or B. cinerea (1× 105 spores/mL) was inoculated into each wound, respectively. After air drying, the apples were stored in enclosed plastic trays to maintain a high relative humidity (about 95%) at 20 °C. Infection rate and lesion diameter of the treated fruits were measured with a slide caliper rule after 8 days (for P. expansum) or 5 days (for B. cinerea) inoculation. There were three replicates of ten fruits for each treatment, and the experiment was conducted twice.
2. Materials and methods
2.5. Efficacy of R. mucilaginosa for reducing natural decay development of apples
2.1. Pathogen inoculum The pathogens P. expansum and B. cinerea were isolated from infected apple fruits. These cultures were maintained on potato dextrose agar (PDA: extract of boiled potatoes, 200 mL; dextrose (Sangon Co., Shanghai, China), 20 g; agar (Sangon Co., Shanghai, China), 20 g and deionized water, 800 mL) at 4 °C, and fresh cultures were grown on PDA plates before use. 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. Spore concentrations were adjusted as required in sterile distilled water assisted by a hemocytometer (XB-K-250, Jianling Medical Device Co., Danyang, China). 2.2. Antagonist The yeast antagonist R. mucilaginosa was isolated from the surfaces of peach blossom picked in unsprayed orchards. Classical methods based on colony and cell morphologies were used for a preliminary characterization of the yeast (Kurtzman and Fell, 1998). Subsequently, sequence analysis of the 5.8S internal transcribed spacer (ITS) ribosomal DNA (rDNA) region was used to identify the yeast (Li et al., 2010). R. mucilaginosa has been shown to be safe in animal testing, including physiology experiments, acute toxicity studies, and the Ames test (our unpublished data). R. mucilaginosa isolates were maintained at 4 °C on Nutrient Yeast Dextrose Agar (NYDA) medium containing 8 g nutrient broth, 5 g yeast extract, 10 g glucose and 20 g agar (Sangon Co., Shanghai, China), in 1 L of distilled water. Liquid cultures of the yeast were grown in 250 mL Erlenmeyer flasks containing 50 mL of NYD Broth (NYDB) which had been inoculated with a loop of the culture. Flasks were incubated on a rotary shaker at 28 °C for 20 h. Following incubation, cells were centrifuged (TGL-16M Centrifuge, Xiangyi Co., Changsha, China) at 5000 ×g for 10 min and washed twice with sterile distilled water in order to remove the growth medium. Yeast cell pellets were re-suspended in sterile distilled water and adjusted to an initial concentration of 2–5 × 109 cells/mL before being adjusted to the concentrations required for the different experiments. 2.3. Fruits Apples (M. domestica Borkh, cv. Fuji) were harvested at commercial maturity from an orchard in Yantai of Shan dong province, and selected for uniformity of size, ripeness and absence of apparent injury or infection. Fruits were selected randomly and disinfected with 0.1% sodium hypochlorite for 1 min, washed with tap water, and allowed to air dry at room temperature (20 °C). 2.4. Efficacy of R. mucilaginosa in inhibiting blue mold decay and gray mold decay of apples A uniform wound (5 mm diameter and approximately 3 mm deep) was made at the equator of each apple fruit using the tip of a sterile dissecting needle. An aliquot (30 μL) of cell suspensions of R. mucilaginosa at 1 ×105, 1 ×106, 1 ×107, 1 ×108, or 1× 109 cells/mL was respectively pipetted into each wound site, and 30 μL of sterile distilled water was
To evaluate the effect of antagonistic yeast on development of natural decay of apples, intact fruit were inoculated by dipping them into a suspension of R. mucilaginosa (1× 108 cells/mL) for 30 s with sterile distilled water as the control, then air dried. Treated fruits were stored in enclosed plastic trays to retain high humidity (about 95%) and incubated at 20 °C for 35 days or at 4 °C for 45 days followed by 20 °C for 15 days in order to determine disease development under normal shelflife conditions, after which infection rate was measured. There were three replicate trials of 10 fruits with a complete randomization in each test and experiments were repeated three times. 2.6. Population studies of R. mucilaginosa in apple wounds Apples were prepared and wounded as described above. Each wound on fruit was inoculated with 30 μL of 1 × 108 cells/mL washed cell suspension of R. mucilaginosa. Fruits were incubated at 20 °C or at 4 °C. R. mucilaginosa was recovered from the wounds after incubation at 20 °C for 0 (1 h after treatment), 1, 2, 3, 4, 5, 6, 7 and 8 days, and at 4 °C for 0 (1 h after treatment), 3, 6, 9, 12, 15, 18, 23 and 28 days, respectively. The wounded tissue was removed with a sterile cork borer (9 mm diameter) to a depth of 10 mm and macerated in 50 mL of sterile 0.85% sodium chloride solution in a 150 mL Erlenmeyer flask using a glass rod. Serial 10-fold dilutions were made and 0.1 mL of each dilution was spread in NYDA. The plates were incubated at 28 °C for 2 days and the colonies were counted. Population densities of R. mucilaginosa were expressed as log10 CFU per wound (CFU: colonyforming units). There were three single fruit replicates per treatment, and the experiments were repeated twice (Viñas et al., 1998). 2.7. Effects of R. mucilaginosa on germination and survival of spores of P. expansum and B. cinerea in vitro The effects of R. mucilaginosa on spore germination of pathogens were tested in potato dextrose broth (PDB). A one mL aliquot of washed cell suspension of R. mucilaginosa (1× 108 cells/mL) or sterile distilled water as a control was added to 250 mL Erlenmeyer flasks containing 50 mL PDB. At the same time, aliquots (1 mL) of spore suspensions (5 × 107 spores/mL) of P. expansum or B. cinerea were added to respective flasks. After 20 h incubation at 28 °C on a rotary shaker (75 rpm), at least 100 spores per replicate were observed microscopically to determine germination rate (Droby et al., 1997). All treatments consisted of three replicates, and the experiments were repeated twice. The interactions between the antagonist and the pathogens in culture were evaluated on potato-dextrose agar (PDA) plates. Agar disks, 5 mm diameter, were cut from PDA plates and a 300 μL quantity of 1 × 108 cells/mL washed cell suspension of R. mucilaginosa or sterile distilled water as a control was added into each well site of the PDA plates. After 3 h, 100 μL of 5 × 104 spores/mL suspension of P. expansum or 100 μL of 1 × 105 spores/mL suspension of B. cinerea was inoculated into each well site, respectively. One hour later, the plates were sealed with polyethylene film in order to retain high humidity and to avoid cross contamination. The plates were incubated at 28 °C for 7 days after which the lesion diameter (total diameter minus 5 mm) of P. expansum
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2.8.1. Fruit treatment Fruit samples were treated as described above to test the efficacy of R. mucilaginosa in inhibiting blue mold and gray mold decay in vivo. The wounds were then treated with 30 μL of cell suspension of R. mucilaginosa (1 × 108 cells/mL) or sterile distilled water (control). After air drying, the apple fruits were stored in enclosed plastic trays to maintain a high relative humidity (above 95%) and incubated at 20 °C. In order to measure the elicitation effect, the tissue surrounding each wound of fruit was collected at 0 (1 h after treatment), 1, 2, 3, 4, 5 and 6 days after treatment. There were three replicates per treatment and the experiment was conducted three times. 2.8.2. Supernatant extract Extraction procedures were conducted at 4 °C. Two grams of tissue sample was ground with 10 mL of cold (4 °C) sodium phosphate buffer (50 mmol/L, pH 7.8) containing 1.33 mmol/L EDTA and 1% polyvinyl pyrrolidone (PVP) for peroxidase (POD), polyphenoloxidase (PPO) and lipid peroxidation (MDA) respectively. The homogenates were then centrifuged at 12,000 g for 15 min at 4 °C and the supernatants were assayed. 2.8.3. Analysis of enzyme activities and lipid peroxidation of apple fruits POD activity was assayed according to the method described by Lurie et al. (1997) with some modifications, using guaiacol as a substrate. The reaction mixture contained 0.2 mL of supernatant (crude enzyme extract), 2.2 mL of 0.3% guaiacol (prepared in 50 mM sodium phosphate buffer, pH 6.4) and was incubated for 5 min at 30 °C. The reaction was then initiated immediately by adding 0.6 mL of 0.3% H2O2 (prepared in 50 mM sodium phosphate, pH 6.4, and incubated at 30 °C for 5 min) and the activity was determined by measuring absorbance at 470 nm once every 30 s for 3 min. As a control, a cuvette containing all components except 0.6 mL of distilled water instead of supernatant was used. The POD activity was expressed as U per g fresh tissue weight (U/g FW). One unit was defined as an increase in A470 of 0.01 per minute. PPO activity was measured following the method described by Mohammadi and Kazemi (2002) with some modifications. The reaction mixtures contained 2.8 mL buffered substrate (50 mmol/L sodium phosphate, pH 6.4 and 100 μmol/L catechol) and 200 μL of enzyme extract. After the mixture was incubated at 30 °C for 1 min, the change in absorbance at 398 nm was measured once every 30 s for 3 min. One unit of the PPO activity is defined as the amount of the enzyme extracts producing an increase of A398 by 0.01 in 1 min and the activity is expressed as U per g fresh tissue weight (U/g FW). Lipid peroxidation was determined in terms of malondialdehyde (MDA) content by thiobarbituric acid (TBA) reaction following the method described by Du and Bramlage (1992) with slight modifications. Simply, 2 mL of 0.6% TBA in 10% trichloroacetic acid was added into 2 mL crude enzyme sample. The solution was heated at 100 °C for 10 min, quickly cooled in an ice-bath, and then centrifuged (4 °C) for 15 min at 10,000 g and the supernatants were assayed. The absorbance of supernatants were recorded at A532, A600, and A450, and MDA content was calculated according to the following formula: C (μmol/L) MDA = 6.45×(A532 −A600)−0.56×A450, and expressed as nmol MDA/g FW. 2.9. Statistical analyses The data were analyzed by the analysis of variance (ANOVA) using the statistical program SPSS/PC version II.x, (SPSS Inc. Chicago, Illinois, USA) and the Duncan's multiple range test was used for separation of means. The statistical significance was applied at the level p b 0.05.
3.1. Effects of R. mucilaginosa in inhibiting blue mold decay and gray mold decay of apples The disease incidence of both blue mold and gray mold decay of the fruits treated with R. mucilaginosa at all tested concentrations were significantly reduced compared with those of the control fruits (p b 0.05) (Fig. 1a). The lesion diameter of blue mold decay of the fruits treated with R. mucilaginosa at all tested concentrations except 1 × 105 and 1 × 106 CFU/mL were significantly reduced compared with those of the control fruits (p b 0.05) (Fig. 1b). The higher the concentrations of the antagonist, the lower the disease incidence and the smaller the lesion diameter. When the concentration of R. mucilaginosa was 1 × 109 cells/mL, blue mold decay was completely inhibited whereas there was a high disease incidence (97.2%) and large lesion diameter (2.44 cm) in the control fruit. Gray mold decay and lesion diameter were inhibited by 97.1% and 56.1% respectively compared to the control. 3.2. Effects of R. mucilaginosa on natural decay development of apples Our experiments evaluated the efficacy of yeast antagonist in reducing the natural development of decay. The results presented in Fig. 2 indicated that the application of R. mucilaginosa produced significant control of
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3. Results
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Treatments Fig. 1. Efficacy of R. mucilaginosa in inhibiting blue mold decay and gray mold decay of apples. Disease incidence (a) and lesion diameter (b) were measured after 8 days (P. expansum) or 5 days (B. cinerea) incubation at 20 °C. The letters of A, B, C, D, and E represent the concentrations of R. mucilaginosa at 1 × 105, 1 × 106, 1 × 107, 1 × 108 and 1 × 109 CFU/mL respectively. Each value is the mean of three experiments. Bars represent the standard error of the mean. Data in columns with the different letters are significantly different according to Duncan's multiple range test at p = 0.05.
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The population of R. mucilaginosa in apple wounds grew rapidly at 20 °C especially in the first day (Fig. 3a). The population reached the maximum at 3 days (6.05 × 105 CFU/mL) from an inoculum of 7.04×104 CFU/mL, after which it decreased then stabilized at a high level. Similarly, in apple wounds kept at 4 °C, rapid colonization of R. mucilaginosa was observed during the first 3 days, and then the population slowly increased to the maximum at 15 days (6.20×105 CFU/ mL) from an inoculum of 2.29×104 CFU/mL (Fig. 3b). The population then decreased, but did not fall below 1.23×105 CFU/mL.
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natural infections of apple fruits (pb 0.05). After storage at 20 °C for 35 days, the decay incidence of apples treated by R. mucilaginosa was 20% compared with 44.4% in the control fruits. Similarly, after storage at 4 °C for 45 days followed by 20 °C for 15 days, the decay incidence of apples treated with R. mucilaginosa was 22.2% compared with 53.3% in the control fruits.
After incubation with R. mucilaginosa at 28 °C in PDB for 20 h, the percentage germination of spores of P. expansum and B. cinerea was significantly reduced, by 81.87% and 82.78% respectively, compared with the control (Fig. 4a, p b 0.05). The growth on PDA of the two pathogens was also markedly controlled by R. mucilaginosa (Fig. 4b, p b 0.05), with colony diameters of P. expansum and B. cinerea reduced by 61.41% and 62.91% respectively after 7 days incubation at 28 °C, compared with the control.
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Days of incubation at 4 °C Fig. 3. Population dynamics of R. mucilaginosa in apple wounds at 20 °C (a) and 4 °C (b). Bars represented standard errors.
Fig. 4. Effects of R. mucilaginosa on survival of P. expansum and B. cinerea in vitro as measured by (a) spore germination and (b) lesion diameter. Spore germination (percentage of germinated spores from at least 100 spores) and lesion diameter (total diameter minus 5 mm) were recorded. Each value is the mean of three experiments. Bars represent the standard error of the mean. Data in columns with the different letters are significantly different according to Duncan's multiple range test at p = 0.05.
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As shown in Fig. 5a, treatment with R. mucilaginosa resulted in a significant increase in the activity of POD of apples compared with the control over the entire storage time. At 4 days after inoculation, POD activity of apples peaked at 1.68 times of that of the control. 3.6. Effects of R. mucilaginosa on PPO activity of apples The PPO activity in the tissue of both the apples treated with R. mucilaginosa and the control apples increased gradually in the first 4 days after inoculation and reached a peak at the 4 days. The PPO activities decreased at 5 days then significantly increased at 6 days. However, PPO activity in the tissue treated with R. mucilaginosa was still higher than that of the control over the entire storage time. The PPO activities in treated fruit were approximately 1.20 fold and 1.18 fold of those of the control at 4 and 6 days respectively (Fig. 5b).
(a) 0.5 0.45
POD activity(U/gFW)
3.5. Effects of R. mucilaginosa on POD activity of apples
155
0.4 0.35 0.3 0.25 0.2
Control R.mucilaginosa
0.15 0.1
3.7. Effects of R. mucilaginosa on MDA content of apples
The use of biocontrol agents to manage postharvest decay of fruit has been explored as an alternative to the use of synthetic fungicides (Wilson and Wisniewski, 1989). Further identification of new antagonists is desirable because antagonists identified in specific geographic areas may be more effective against the pathogen strains present in that locale (Vero et al., 2002). In our laboratory, we have identified a strain of the yeast R. mucilaginosa which has biocontrol efficacy against blue mold and gray mold of apples caused by P. expansum and B. cinerea respectively. This is the first report of R. mucilaginosa as an antagonist of postharvest diseases of fruits. Data in this present study show that application of R. mucilaginosa significantly reduced the postharvest disease incidence and lesion diameter of apples caused by P. expansum and B. cinerea. Furthermore, in the experiments on efficacy of R. mucilaginosa for reducing natural decay development, natural disease incidence of apples was significantly reduced by R. mucilaginosa following storage at 20 °C for 35 days or 4 °C for 45 days followed by 20 °C for 15 days. This implies that R. mucilaginosa has potential to control a wide range of pathogens. Fruits and vegetables are often kept at low temperature, the biocontrol ability of antagonist under normal shelf-life conditions (including both low temperature and ambient storage) suggest that R. mucilaginosa can be used in combination with cold storage to enhance disease control. Our research also showed that R. mucilaginosa treatment did not impair quality parameters of apples during storage, including firmness, total soluble solids (TSS), ascorbic acid, and titratable acidity (our unpublished data). The results of effects of R. mucilaginosa in inhibiting blue mold decay and gray mold decay of apples indicated that biocontrol efficacy was a function of concentration: the higher the concentration of R. mucilaginosa, the better the biocontrol activity of the antagonist. Furthermore, growth studies demonstrated that R. mucilaginosa could rapidly colonize and grow in apple wounds whether at 20 °C or 4 °C. Such rapid growth in wounds indicated that R. mucilaginosa is well adapted to the wound
2
3
4
5
6
5
6
5
6
(b)
0.55 0.5
Control R.mucilaginosa
0.45
PPO activity(U/gFW)
4. Discussion
1
Days after inoculation
0.4 0.35 0.3 0.25 0.2 0.15 0.1
0
1
2
3
4
Days after inoculation
(c)
9.5 Control
9
R.mucilaginosa
MDA content(nmol/gFW)
The MDA content of the control apples increased in the first 4 days after inoculation and reached a peak at the fourth day (Fig. 5c). The MDA content decreased at 5 days then significantly increased at 6 days. However, the MDA content of the apples treated with R. mucilaginosa increased at 1 days after inoculation, decreased at 2 days, then increased at 3 days, and reached a peak. Thereafter, the MDA content of the apples treated by R. mucilaginosa decreased. The MDA content of apples treated with R. mucilaginosa was lower than that of the control over the duration of storage. At 2 days, 4 days and 6 days after inoculation, the MDA content of the control apples were 1.14, 1.16 and 1.17 fold higher than those of the antagonistic yeast treated apples respectively.
0
8.5 8 7.5 7 6.5 6 0
1
2
3
4
Days after inoculation Fig. 5. Effects of R. mucilaginosa on peroxidase (POD) activity (a), polyphenoloxidase (PPO) activity (b) and lipid peroxidation as measured by malondialdehyde (MDA) content (c) of apples. Each value is the mean of two experiments. Bars represent standard errors.
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environment in fruit. These results point to the main mode of action of R. mucilaginosa being competition for space and nutrient with pathogens. Similar results for other antagonist yeasts have been reported by Bencheqroun et al. (2007), Zhang et al. (2005), Mclaughlin et al. (1992), and Spadaro et al. (2002). To further understand the mechanisms by which R. mucilaginosa controlled postharvest diseases of apples, we investigated the effects of R. mucilaginosa on spore germination and growth of the two pathogens in vitro. R. mucilaginosa significantly inhibited spore germination in PDB media and inhibited growth of P. expansum and B. cinerea on PDA. Furthermore, the percentage of disease incidence and lesion diameter of blue mold and gray mold in fruits treated with washed cell suspension of R. mucilaginosa were significantly lower than those of the control fruits. These results suggested that limiting spore germination and growth of pathogens is another mechanism of action of R. mucilaginosa in control of postharvest diseases of apples. There were reports that increased POD and PPO activities may strengthen defense systems in plants, by biosynthesizing metabolites such as phytoalexins and phenols to form lignin or toxic quinines directed against pathogen infection (Milosevic and Slusarenko, 1996; Mohammadi and Kazemi, 2002). Mittler (2002) has reported that POD and catalase are important active free-radical scavenging enzymes, and decrease in these enzymes may lead to high levels of reactive oxygen intermediates (ROI). Sustained accumulation of ROI may cause lipid peroxidation, aggravate oxidative damage, and accelerate senescence (Hariyadi and Parkin, 1991). Furthermore, in postharvest systems, increased lipid peroxidation has been regarded as a major characteristic of harvested fruits undergoing senescence and membrane injury (Deighton et al., 1999). In our research, apple fruits inoculated with R. mucilaginosa showed higher levels of POD and PPO activities and less lipid peroxidation (MDA content) than those of the control fruits over the whole storage period. These results imply that induced activity of defense-related enzymes and inhibition of ripening and senescence may be part of the mechanism of R. mucilaginosa in controlling postharvest diseases of apple fruits. In conclusion, the results reported here showed that R. mucilaginosa has great potential as a biocontrol agent for the control of postharvest blue mold decay and gray mold decay of apples caused by P. expansum and B. cinerea and is capable of reducing natural decay development of apples. Potential modes of action include competition for space and nutrient with pathogens, direct inhibition of spore germination and growth of pathogens and ability to induce defense-related enzymes such as POD, PPO and reduce lipid peroxidation of apples, enhancing resistance and delaying senescence of apples. More research into the mode of action of R. mucilaginosa to control postharvest diseases of fruits is needed, particularly to elucidate mechanisms at the molecular and proteomic level. Acknowledgements This research was supported by the Natural Science Foundation of Jiangsu Province (BK2009214), the National Natural Science Foundation of China (NNSFC-30771514) and the Foundation for the Eminent Talent of Jiangsu University. References Arras, G., Arru, S., 1999. Integrated control of postharvest citrus decay and induction of phytoalexins by Debaryomyces hansenii. Advances in Horticultural Science 13, 76–81. Bencheqroun, S.K., Bajji, M., Massart, S., Labhilili, M., El-jafari, S., Jijakli, M.H., 2007. In vitro and in situ study of postharvest apple blue mold biocontrol by Aureobasidium pullulans: evidence for the involvement of competition for nutrients. Postharvest Biology and Technology 46, 128–135.
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