Combination of UV-C Treatment and Metschnikowia pulcherrimas for Controlling Alternaria Rot in Postharvest Winter Jujube Fruit Dongqi Guo, Lixia Zhu, and Xujie Hou
Abstract: The potential of using antagonistic yeast Metschnikowia pulcherrimas alone or in combination with ultravioletC (UV-C) treatment for controlling Alternaria rot of winter jujube, and its effects on postharvest quality of fruit was investigated. The results showed that spore germination of Alternaria alternata was significantly inhibited by each of the 3 doses (1, 5, and 10 kJ m−2 ) in vitro. In vivo, UV-C treatment (5 kJ m−2 ) or antagonist yeast was capable of reducing the percentage of infected wounds and lesion diameter in artificially inoculated jujube fruits, however, in fruit treated with combination of UV-C treatment and M. pulcherrima, the percentage of infected wounds and lesion diameter was only 16.0% and 0.60 cm, respectively. The decay incidence on winter jujube fruits treated with the combination of UV-C treatment and M. pulcherrima was 23% after storage at 0 ± 1 °C for 45 d followed by 22 °C for 7 d. None of the treatments impaired quality parameters of jujube fruit. Thus, the combination of UV-C radiation and M. pulcherrima could be an alternative to synthetic fungicides for controlling postharvest Alternaria rot of winter jujube.
Introduction Jujube (Zizyphus jujuba Mill.) fruit has been commonly used as a raw material for foodstuffs as well as for traditional medicine in China. In the past decades, production of Chinese jujubes, especially fresh-eating varieties, has rapidly increased owing to the strong demand of fruit market (Cao and others 2013). However, fresh jujubes are very susceptible to diseases caused by various pathogenic fungi. Considerable losses of fresh jujubes may occur due to infections during harvest, storage, processing, and transportation, and thereby the postharvest supply period of the fruit is greatly limited. Previous studies showed that black mold rot caused by Alternaria alternata(Fr.) Keissl is responsible for most of the postharvest losses of fresh jujubes even though the fruit were stored at low temperature (Wang and others 2009;Yan and others 2011, 2012). Control of postharvest Alternaria rot of fresh jujubes has been accomplished mainly with the use of synthetic fungicides. However, the efficacy of chemical treatments has often been limited in the face of pathogen resistance to these compounds, as well as concerns about environment contamination and public health. Accordingly, alternative control strategies are needed (Droby and others 2009; Asghari and Aghdam 2010; Wu and others 2010; P´erez and others 2011). Several microorganisms, particularly yeasts occurring naturally on the surface of fruits or vegetables, have been identified for the control of postharvest diseases. Some are capable of effectively reducing the incidence of postharvest fungal pathogens on different fruits, both in small-scale experiments and at industrial scale (Lima and others 2003; Xu and others 2008; Sharma and others 2009; Zong and others 2010). The yeast antagonist M. pulcherrima has been demonstrated to be effective against postharvest diseases of MS 20140991 Submitted 6/8/2014, Accepted 10/15/2014. Authors are with College of Life Science, Tarim Univ, and Xinjiang Production & Construction Group Key Laboratory of Agricultural Products Processing in Xinjiang South, 843300, Alar, Xinjiang, People’s Republic of China. Direct inquiries to author Guo (E-mail: [email protected]).
R C 2014 Institute of Food Technologists
doi: 10.1111/1750-3841.12724 Further reproduction without permission is prohibited
several fruits (Leverentz and others 2006; Csutak and others 2007). In addition to biological control, physical control such as UV-C irradiation has been recently used as one of the promising nonpesticide technologies to control of postharvest fruit senescence and especially control decay in different fruit and vegetable species (Shama and Alderson 2005; Allende and others 2006; Cia and others 2010). UV-C illumination at the dosage of 5 kJ m−2 was shown to effectively induce resistance against Monilinia fructicola (G. Winter) Honey in harvested “Yali” pear fruits (Li and others 2010). Mustafa Erkan and others (2008) found that 3 UV-C irradiations with dosages of 0.43, 2.15, and 4.30 kJ m−2 significantly reduced the incidence of natural infection in strawberry fruits. Although several nonchemical methods including the use of yeast antagonists and UV-C treatment, when used alone, each presents limitations that could affect its commercial applicability (Xu and Du 2011). Therefore one approach to use these methods as an alternative to synthetic fungicides is the integration of different treatments, taking advantage of their additive or synergistic effects in order to overcome the performance and improve the efficacy of each method. The aim of this study was to evaluate the effectiveness of yeast antagonist, as a stand-alone treatment and in combination with the application of UV-C, for the control of Alternaria rot on winter jujube, and to investigate the effects on quality of jujube after storage, including firmness, total soluble solids, mass loss, ascorbic acid, and titratable acidity.
Materials and Methods Plant material Winter jujubes were harvested at commercial maturity from Alar of Xinjiang province and sorted based on size and the absence of physical injuries or infections. Fruits were disinfected with 2%(v/v) sodium hypochlorite for 2 min, washed with tap water and dried in air. These fruits were either used immediately or stored at 2 ± 1 °C until use. Vol. 00, Nr. 0, 2014 r Journal of Food Science M1
M: Food Microbiology & Safety
Keywords: Alternaria rot, M. pulcherrimas, quality parameters, UV-C treatment, winter jujube
Controlling Alternaria rot in jujube fruit . . . Yeast antagonist and pathogen M. pulcherrima was isolated from the surface of jujube fruit following the method of Wilson and Chalutz (1989) and identified by Sangon Biotech (Shanghai) Co., Ltd. The yeasts were cultured in nutrient yeast dextrose broth (NYDB: 8 g of nutrient broth, 5 g of yeast extract, and 10 g of dextrose in 1000 mL distilled water) for 24 h at 28 °C with a shaker at 200 rev min−1 . Yeast cells were collected by centrifugation at 5000 g for 10 min. The concentration of the yeast was determined with a hemacytometer and adjusted to 1×108 cells/mL with sterile distilled water. A. alternata was isolated from infected jujube fruits and cultured on potato dextrose agar (PDA: extract of boiled potatoes, 200 mL; dextrose, 20 g; agar powder, 20 g; and distilled water, 800 mL) for 14 d at 25 °C. The spores were removed by inoculating loop from the surface of the cultures and suspended in sterile distilled water containing 0.05% (v/v) Tween 80.The suspension was filtered through 5 layers of sterile cheesecloth to remove adhering mycelia. The spore concentration of A. alternata was determined with the aid of a hemocytometer and adjusted as required.
M: Food Microbiology & Safety
UV-C treatment The fruits were placed on trays under 2 UV-C lighting tubes (peak emission at 254 nm) at a distance of 20 cm. The radiation intensity of the lamps was measured by a UV digital radiometer (ZQJ-254, Shanghai Jinpeng Analytical Instrument Co. Ltd., China) (Xu and Du 2011). In vitro tests To determine the effects of UV-C Treatment on A. alternata in vitro, spore suspension of the pathogen prepared from the 14 d culture incubated at 25 °C was adjusted to 1×103 spores/mL with sterile distilled water. The fungal spore suspension was transferred to a petri dish and exposed to UV-C radiation for 3(1 kJ m−2 ), 15 (5 kJ m−2 ), or 30 (10 kJ m−2 ) min, and the conidial suspension without UV-C radiation was used as a control. After treatment, aliquots of 100 µL of each suspension were pour-plated onto PDA agar plates. After 72 h incubation at 25 °C, the colonies were counted and the results were expressed as the mean number of colony-forming units (cfu)/mL. Each treatment was repeated 3 times (Li and others 2010). Effects of UV-C treatment and M. pulcherrima on decay development in artificially inoculated fruits Winter jujubes were wounded with a dissecting needle (3 mm deep × 3 mm wide) and wound sites were inoculated by adding 20 µL of spore suspension of A. alternata (1×105 spores/mL) and allowed to dry for 2 h. Wound-inoculated fruits were divided into 4 groups: (1) jujube fruits which were illuminated by UV-C for 15min (5 kJ m−2 ); (2) aliquots (20 µL) of cell suspension of M. pulcherrima (1×108 cells/mL) were pipetted into wounds; (3) jujube fruits which were first treated with UV-C as described in Group 1, then inoculated with M. pulcherrima as described in Group 2; and (4) jujube fruit did not receive any of these 2 treatments, which served as control. All fruits were stored at 22 °C for 7 d. The disease incidence and lesion diameter on each jujube fruit caused by A. alternata were recorded. Each treatment consisted of 3 replicates with 400 fruits per replicate and the experiment was repeated 3 times. M2 Journal of Food Science r Vol. 00, Nr. 0, 2014
Effects of UV-C treatment and M. pulcherrima on naturally infection To evaluate the effect of M. pulcherrima with or without UV-C treatment on development of natural decay, intact winter jujube fruits were divided into 4 groups: for the 1st group, fruits which were illuminated by UV-C for 15 min (5 kJ m−2 ); for the 2nd group, fruits inoculated by dipping for 1 min into cell suspension of M. pulcherrima, the concentration of which had been adjusted to 1×108 cells/mL with sterile distilled water, and then air-dried; for the 3rd group, fruits which first received the UV-C treatment as described above, and then which received the antagonist treatment as described in Group 2; for the 4th group, fruits did not receive any of these 2 treatments, which served as control. Treated fruits were stored at 22 °C for 7 d or (0 ± 1 °C) for 45 d followed by 22 °C for 7 d in order to determine disease development under normal shelf-life conditions. The percentage of infected fruits was recorded afterwards. There were 3 replicate trials of 400 fruits per treatment with complete randomization. The experiment was repeated 3 times. Effects of UV-C treatment and M. pulcherrima on postharvest quality of jujube fruit To evaluate the effect of antagonistic yeast, UV-C treatment, or both of them to postharvest quality of jujubes, jujube fruits were treated with these treatments, and stored as described above in the experiment to evaluate the effect of antagonistic yeast, UV-C treatment, or both on development of natural decay. Quality parameters were measured after storage. Quality measurements were made on 3 replicates of 400 fruits each, and performed at ambient temperature (about 22 °C). The testing methods used are described below. Fruit firmness. Firmness values of each individual jujube were measured at 2 points of the equatorial region by using the TA-XT2i Texture Analyser (Microstable Instruments, the UK) with a 5-mm diameter flat probe. The probe descended toward the sample at 5.0 mm s−1 and the maximum force (N) was defined as firmness. The firmness of each jujube was measured 3 times on different sides (Zhang and others 2007). Total soluble solids. Total soluble solids (TSS) were determined by measuring the refractive index of the same juice with a hand refractometer and the results expressed as percentages (g per 100 g fruit weight) (Larrigaudi`ere and others 2002). Mass loss. The mass was measured by an MP2000-2 balance (±0.001 g) (Shanghai Balance, China) before treatment (A) and after storage (B), respectively, and the mass loss was calculated as (A−B)/A (Zhou and others 2002). Ascorbic acid. Ascorbic acid (ASA)was measured by titrating with 2,6-dichlorophenolindophenol according to the method of Lin and others (2008). Results were expressed as milligrams of ¨ ascorbic acid per 100 g sample (Ozden and Bayindirli 2002). Titratable acidity. Acidity was measured by titration with 0.1 N NaOH to pH 8.1; 4g of juice diluted with 20 mL of distilled water was evaluated for each replicate. Titratable acidity was calculated as percentage malic acid (Wright and Kader 1997). Statistical analysis Data from a single experiment were subjected to analysis of variance (ANOVA) with the SPSS 17.0 statistical software (IBM International Business Machines Corporation) and the mean values of the significant interactions were compared by Duncan’s multiple range test. Differences at P
Abstract: The potential of using antagonistic yeast Metschnikowia pulcherrimas alone or in combination with ultravioletC (UV-C) treatment for controlling Alternaria rot of winter jujube, and its effects on postharvest quality of fruit was investigated. The results showed that spore germination of Alternaria alternata was significantly inhibited by each of the 3 doses (1, 5, and 10 kJ m−2 ) in vitro. In vivo, UV-C treatment (5 kJ m−2 ) or antagonist yeast was capable of reducing the percentage of infected wounds and lesion diameter in artificially inoculated jujube fruits, however, in fruit treated with combination of UV-C treatment and M. pulcherrima, the percentage of infected wounds and lesion diameter was only 16.0% and 0.60 cm, respectively. The decay incidence on winter jujube fruits treated with the combination of UV-C treatment and M. pulcherrima was 23% after storage at 0 ± 1 °C for 45 d followed by 22 °C for 7 d. None of the treatments impaired quality parameters of jujube fruit. Thus, the combination of UV-C radiation and M. pulcherrima could be an alternative to synthetic fungicides for controlling postharvest Alternaria rot of winter jujube.
Introduction Jujube (Zizyphus jujuba Mill.) fruit has been commonly used as a raw material for foodstuffs as well as for traditional medicine in China. In the past decades, production of Chinese jujubes, especially fresh-eating varieties, has rapidly increased owing to the strong demand of fruit market (Cao and others 2013). However, fresh jujubes are very susceptible to diseases caused by various pathogenic fungi. Considerable losses of fresh jujubes may occur due to infections during harvest, storage, processing, and transportation, and thereby the postharvest supply period of the fruit is greatly limited. Previous studies showed that black mold rot caused by Alternaria alternata(Fr.) Keissl is responsible for most of the postharvest losses of fresh jujubes even though the fruit were stored at low temperature (Wang and others 2009;Yan and others 2011, 2012). Control of postharvest Alternaria rot of fresh jujubes has been accomplished mainly with the use of synthetic fungicides. However, the efficacy of chemical treatments has often been limited in the face of pathogen resistance to these compounds, as well as concerns about environment contamination and public health. Accordingly, alternative control strategies are needed (Droby and others 2009; Asghari and Aghdam 2010; Wu and others 2010; P´erez and others 2011). Several microorganisms, particularly yeasts occurring naturally on the surface of fruits or vegetables, have been identified for the control of postharvest diseases. Some are capable of effectively reducing the incidence of postharvest fungal pathogens on different fruits, both in small-scale experiments and at industrial scale (Lima and others 2003; Xu and others 2008; Sharma and others 2009; Zong and others 2010). The yeast antagonist M. pulcherrima has been demonstrated to be effective against postharvest diseases of MS 20140991 Submitted 6/8/2014, Accepted 10/15/2014. Authors are with College of Life Science, Tarim Univ, and Xinjiang Production & Construction Group Key Laboratory of Agricultural Products Processing in Xinjiang South, 843300, Alar, Xinjiang, People’s Republic of China. Direct inquiries to author Guo (E-mail: [email protected]).
R C 2014 Institute of Food Technologists
doi: 10.1111/1750-3841.12724 Further reproduction without permission is prohibited
several fruits (Leverentz and others 2006; Csutak and others 2007). In addition to biological control, physical control such as UV-C irradiation has been recently used as one of the promising nonpesticide technologies to control of postharvest fruit senescence and especially control decay in different fruit and vegetable species (Shama and Alderson 2005; Allende and others 2006; Cia and others 2010). UV-C illumination at the dosage of 5 kJ m−2 was shown to effectively induce resistance against Monilinia fructicola (G. Winter) Honey in harvested “Yali” pear fruits (Li and others 2010). Mustafa Erkan and others (2008) found that 3 UV-C irradiations with dosages of 0.43, 2.15, and 4.30 kJ m−2 significantly reduced the incidence of natural infection in strawberry fruits. Although several nonchemical methods including the use of yeast antagonists and UV-C treatment, when used alone, each presents limitations that could affect its commercial applicability (Xu and Du 2011). Therefore one approach to use these methods as an alternative to synthetic fungicides is the integration of different treatments, taking advantage of their additive or synergistic effects in order to overcome the performance and improve the efficacy of each method. The aim of this study was to evaluate the effectiveness of yeast antagonist, as a stand-alone treatment and in combination with the application of UV-C, for the control of Alternaria rot on winter jujube, and to investigate the effects on quality of jujube after storage, including firmness, total soluble solids, mass loss, ascorbic acid, and titratable acidity.
Materials and Methods Plant material Winter jujubes were harvested at commercial maturity from Alar of Xinjiang province and sorted based on size and the absence of physical injuries or infections. Fruits were disinfected with 2%(v/v) sodium hypochlorite for 2 min, washed with tap water and dried in air. These fruits were either used immediately or stored at 2 ± 1 °C until use. Vol. 00, Nr. 0, 2014 r Journal of Food Science M1
M: Food Microbiology & Safety
Keywords: Alternaria rot, M. pulcherrimas, quality parameters, UV-C treatment, winter jujube
Controlling Alternaria rot in jujube fruit . . . Yeast antagonist and pathogen M. pulcherrima was isolated from the surface of jujube fruit following the method of Wilson and Chalutz (1989) and identified by Sangon Biotech (Shanghai) Co., Ltd. The yeasts were cultured in nutrient yeast dextrose broth (NYDB: 8 g of nutrient broth, 5 g of yeast extract, and 10 g of dextrose in 1000 mL distilled water) for 24 h at 28 °C with a shaker at 200 rev min−1 . Yeast cells were collected by centrifugation at 5000 g for 10 min. The concentration of the yeast was determined with a hemacytometer and adjusted to 1×108 cells/mL with sterile distilled water. A. alternata was isolated from infected jujube fruits and cultured on potato dextrose agar (PDA: extract of boiled potatoes, 200 mL; dextrose, 20 g; agar powder, 20 g; and distilled water, 800 mL) for 14 d at 25 °C. The spores were removed by inoculating loop from the surface of the cultures and suspended in sterile distilled water containing 0.05% (v/v) Tween 80.The suspension was filtered through 5 layers of sterile cheesecloth to remove adhering mycelia. The spore concentration of A. alternata was determined with the aid of a hemocytometer and adjusted as required.
M: Food Microbiology & Safety
UV-C treatment The fruits were placed on trays under 2 UV-C lighting tubes (peak emission at 254 nm) at a distance of 20 cm. The radiation intensity of the lamps was measured by a UV digital radiometer (ZQJ-254, Shanghai Jinpeng Analytical Instrument Co. Ltd., China) (Xu and Du 2011). In vitro tests To determine the effects of UV-C Treatment on A. alternata in vitro, spore suspension of the pathogen prepared from the 14 d culture incubated at 25 °C was adjusted to 1×103 spores/mL with sterile distilled water. The fungal spore suspension was transferred to a petri dish and exposed to UV-C radiation for 3(1 kJ m−2 ), 15 (5 kJ m−2 ), or 30 (10 kJ m−2 ) min, and the conidial suspension without UV-C radiation was used as a control. After treatment, aliquots of 100 µL of each suspension were pour-plated onto PDA agar plates. After 72 h incubation at 25 °C, the colonies were counted and the results were expressed as the mean number of colony-forming units (cfu)/mL. Each treatment was repeated 3 times (Li and others 2010). Effects of UV-C treatment and M. pulcherrima on decay development in artificially inoculated fruits Winter jujubes were wounded with a dissecting needle (3 mm deep × 3 mm wide) and wound sites were inoculated by adding 20 µL of spore suspension of A. alternata (1×105 spores/mL) and allowed to dry for 2 h. Wound-inoculated fruits were divided into 4 groups: (1) jujube fruits which were illuminated by UV-C for 15min (5 kJ m−2 ); (2) aliquots (20 µL) of cell suspension of M. pulcherrima (1×108 cells/mL) were pipetted into wounds; (3) jujube fruits which were first treated with UV-C as described in Group 1, then inoculated with M. pulcherrima as described in Group 2; and (4) jujube fruit did not receive any of these 2 treatments, which served as control. All fruits were stored at 22 °C for 7 d. The disease incidence and lesion diameter on each jujube fruit caused by A. alternata were recorded. Each treatment consisted of 3 replicates with 400 fruits per replicate and the experiment was repeated 3 times. M2 Journal of Food Science r Vol. 00, Nr. 0, 2014
Effects of UV-C treatment and M. pulcherrima on naturally infection To evaluate the effect of M. pulcherrima with or without UV-C treatment on development of natural decay, intact winter jujube fruits were divided into 4 groups: for the 1st group, fruits which were illuminated by UV-C for 15 min (5 kJ m−2 ); for the 2nd group, fruits inoculated by dipping for 1 min into cell suspension of M. pulcherrima, the concentration of which had been adjusted to 1×108 cells/mL with sterile distilled water, and then air-dried; for the 3rd group, fruits which first received the UV-C treatment as described above, and then which received the antagonist treatment as described in Group 2; for the 4th group, fruits did not receive any of these 2 treatments, which served as control. Treated fruits were stored at 22 °C for 7 d or (0 ± 1 °C) for 45 d followed by 22 °C for 7 d in order to determine disease development under normal shelf-life conditions. The percentage of infected fruits was recorded afterwards. There were 3 replicate trials of 400 fruits per treatment with complete randomization. The experiment was repeated 3 times. Effects of UV-C treatment and M. pulcherrima on postharvest quality of jujube fruit To evaluate the effect of antagonistic yeast, UV-C treatment, or both of them to postharvest quality of jujubes, jujube fruits were treated with these treatments, and stored as described above in the experiment to evaluate the effect of antagonistic yeast, UV-C treatment, or both on development of natural decay. Quality parameters were measured after storage. Quality measurements were made on 3 replicates of 400 fruits each, and performed at ambient temperature (about 22 °C). The testing methods used are described below. Fruit firmness. Firmness values of each individual jujube were measured at 2 points of the equatorial region by using the TA-XT2i Texture Analyser (Microstable Instruments, the UK) with a 5-mm diameter flat probe. The probe descended toward the sample at 5.0 mm s−1 and the maximum force (N) was defined as firmness. The firmness of each jujube was measured 3 times on different sides (Zhang and others 2007). Total soluble solids. Total soluble solids (TSS) were determined by measuring the refractive index of the same juice with a hand refractometer and the results expressed as percentages (g per 100 g fruit weight) (Larrigaudi`ere and others 2002). Mass loss. The mass was measured by an MP2000-2 balance (±0.001 g) (Shanghai Balance, China) before treatment (A) and after storage (B), respectively, and the mass loss was calculated as (A−B)/A (Zhou and others 2002). Ascorbic acid. Ascorbic acid (ASA)was measured by titrating with 2,6-dichlorophenolindophenol according to the method of Lin and others (2008). Results were expressed as milligrams of ¨ ascorbic acid per 100 g sample (Ozden and Bayindirli 2002). Titratable acidity. Acidity was measured by titration with 0.1 N NaOH to pH 8.1; 4g of juice diluted with 20 mL of distilled water was evaluated for each replicate. Titratable acidity was calculated as percentage malic acid (Wright and Kader 1997). Statistical analysis Data from a single experiment were subjected to analysis of variance (ANOVA) with the SPSS 17.0 statistical software (IBM International Business Machines Corporation) and the mean values of the significant interactions were compared by Duncan’s multiple range test. Differences at P
