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Synergistic effect of the combined treatment with gamma irradiation and sodium dichloroisocyanurate to control gray mold (Botrytis cinerea) on paprika
Radiation Physics and Chemistry 98 (2014) 103–108
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Synergistic effect of the combined treatment with gamma irradiation and sodium dichloroisocyanurate to control gray mold (Botrytis cinerea) on paprika Minchul Yoon, Koo Jung, Kwang-Youll Lee, Je-Yong Jeong, Ju-Woon Lee, Hae-Jun Park n RT Practical Application Division, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeong-up 580-185, Republic of Korea
H I G H L I G H T S
Paprikas were treated with irradiation and NaDCC to control gray mold. We confirmed that the combined treatment was synergistically affected. The treatment can contribute to a reduction of postharvest losses caused by fungi. This combined treatment can also reduce the doses of irradiation.
art ic l e i nf o
a b s t r a c t
Article history: Received 1 September 2013 Accepted 30 December 2013 Available online 17 January 2014
Gray mold (Botrytis cinerea) is one of the most major fungal pathogens in paprika. Generally, gamma irradiation over 1 kGy is effective for the control of fungal pathogens; however, a significant change in fruit quality (physical properties) on paprika was shown from gamma irradiation at over 0.6 kGy (po 0.05). Therefore, in this study, the synergistic disinfection effect of the combined treatment with gamma irradiation and sodium dichloroisocyanurate (NaDCC) was investigated to reduce the gamma irradiation dose. In an artificial inoculation experiment of B. cinerea isolated from naturally-infected postharvest paprika, fungal symptoms were observed in the stem and exocarp of paprika after conidial inoculation. From the sensitivity of gamma irradiation and NaDCC, B. cinerea conidia were fully inactivated by 4 kGy of gamma irradiation (D10 value 0.99 kGy), and were fully inactivated by 50 ppm NaDCC treatment. The fungal symptoms were not detected by the dose-dependent gamma irradiation ( 44 kGy) and NaDCC ( 450 ppm). As a result of the combined treatment of gamma irradiation and NaDCC, the D10 value was significantly reduced by 1.06, 0.88, 0.77, and 0.58 kGy (po 0.05). Moreover, fungal symptoms were more significantly reduced in combined treatment groups (gamma irradiation and NaDCC) than single treatment groups (gamma irradiation or NaDCC). These results suggest that combined treatment with irradiation and NaDCC treatment can be applied to preserve quality of postharvest paprika or other fruits. & 2014 Elsevier Ltd. All rights reserved.
Keywords: Botrytis cinerea Gamma irradiation Sodium dichloroisocyanurate Synergistic effect Postharvest paprika
1. Introduction Paprika (spice red pepper; Capsicum annuum L.) is one of the most economically important fruit crops. It is an excellent vitamin source which has been confirmed by various epidemiological studies (Gey and Puska, 1989; Gerster, 1991). In spite of this economic importance, the productivity decreases and quality losses occur during the preharvest and postharvest management of this fruit crop because of a number of diseases. The gray mold
n
Corresponding author. Tel.: þ 82 63 570 3190; fax: þ82 63 570 3195. E-mail address: [email protected] (H.-J. Park).
0969-806X/$ - see front matter & 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.radphyschem.2013.12.039
caused by Botrytis cinerea develops mainly on fruits, leaves, or flowers (Dik and Elad, 1999) in a greenhouse, causing a decrease in the productivity of paprika throughout the world, and is usually managed by the frequent use of prophylactic fungicides (Bulger et al., 1987; de Visser, 1996; Raposo et al., 1996) in fruit crops, including pepper crops during preharvest management. Furthermore, B. cinerea causes an important postharvest decay of pepper (Pernezny et al., 2003), and disinfection technologies of this pathogen are needed for a postharvest management strategy. Most postharvest management technologies have applied chemical fumigation for the preservation of fruit quality from fungi mediated diseases, but alternative methods are needed because of
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growing public concerns over the human health and environmental risks (Spotts and Cervantes, 1986). Therefore, many researchers have focused on eco-friendly postharvest technologies that can contribute to replacing the use of chemical fumigation techniques for crop quality preservation. Alternative treatments have been reported: heat treatments (Lurie, 1998), photochemical treatments (Baka et al., 1999), pulsed light treatments (GómezLópez et al., 2005b), microwave drying (Orsat et al., 2006), UV treatments (Vicente et al., 2005), and ozone treatments (Bialka and Demirci, 2007). Although these alternative treatments show promise, individual treatments are not effective for fungicides. Therefore, it is necessary to develop a technology that combines other disinfection treatments for synergistic effects (Conway et al., 2005). Although fresh fruits and vegetables can be irradiated at a gamma ray dose of up to 1 kGy (US FDA, 2004), this is not possible to fully control postharvest fungi (Blank and Corrigan, 1995). Moreover, the radiation dose required to control fungi has a negative effect on the skin color and texture of stored fruits and vegetables (Beraha et al., 1960; Tiryaki et al., 1994). Therefore, it is important to reduce the irradiation dose for an inhibition of postharvest photogenes through a combined treatment. One promising treatment is the use of gamma irradiation with a combination of other treatments. Consequently, this combined treatment can contribute to a reduction of postharvest losses caused by fungi and reduce the use or doses of fungicides for disease control (Cia et al., 2007). Chlorination is applicable to specific fruits and vegetables, including paprika, using a postharvest process with flumes, water dump tanks, and spray washers. This postharvest treatment was applied to various fruits and vegetables such as tomatoes (Goodin, 1977; Showalter, 1993), citrus (Hough and Kellerman, 1971), apples (Hendrix, 1991), pears (Sanderson and Spotts, 1995), and peppers (Sherman and Allen, 1983). In this study, the effect of gamma irradiation on the physical property of postharvest paprika was evaluated using a texture analyzer. The sensitivity of gamma irradiation and dichloroisocyanurate (NaDCC) was evaluated through in vivo and in vitro tests. The objective of the present study is to evaluate the combination effect of gamma irradiation and NaDCC for disease control and shelf-life of the paprika.
2. Materials and methods
2.2. Preparation of fungal conidia For harvesting the conidia, about 10 mL DW was added to a culture dish and the conidia were gently harvested by filtration through four sterile layers of gauze. The conidial suspensions were collected in sterile screw-cap test tubes (16 100 mm2) containing 15 mL of sterile distilled water and filtered twice using sterile Pasteur pipettes (4.62 mm) packed with glass wool. The procedure was used to remove mycelial fragments and conidial clumps (Saleh et al., 1988). The concentration of conidia was measured using a hemacytometer (Warner-Lambert Technologies Inc., Buffalo, N.Y.). The final conidial concentration was adjusted to 105 conidia mL 1 with DW for further study (Slade et al., 1987). 2.3. Gamma irradiation and NaDCC treatment The samples were irradiated in a cobalt-60 irradiator (point source, AECL, IR-79, Nordion, Canada) with various absorbed doses (from 0.2 to 4 kGy). Dosimetry was performed using 5 mm diameter alanine dosimeters (Bruker Instruments, Rheinstetten, Germany). For chlorination, the samples were immersed in sodium dichloroisocyanurate (NaDCC; Sigma-Aldrich, Poole, UK) at various concentrations (from 5 to 50 ppm). Following treatment, the suspensions were serially diluted in DW and plated on PDA. Incubation was carried out at 25 1C for 5 days. Survival curves were constructed by plotting the survivor CFU/mL versus the actual radiation doses and NaDCC concentrations. The curves were fitted by linear regression, and the radiation sensitivity was expressed in terms of the D10 values. A D10 value is defined as the dose required for reducing the given population by 90% of its initial value. The D10 value was determined from the reciprocal of the slope for the straight-line portion of the survival curve (Ley, 1983). 2.4. Artificial inoculation on paprika Pre-sterilized paprika fruits were used in an artificial inoculation test, and was wounded to a 1.5 mm thickness by puncturing them with the point of a sterile 0.5 mm diameter needle. Each wound site was artificially inoculated with 10 μL of suspension containing 105 conidia mL 1 on the paprika. To protect secondary contamination, the inoculated paprika was transferred into a sterilized oxygen-impermeable plastic container, and was kept at 25 1C and a relative humidity of 490% for 1 week.
2.1. Preparation of paprika and fungus Paprika was purchased from an agricultural expert company (Rosepia Co., Ltd., Jeollabuk-do, Korea). The paprika was selected for a uniform size and freedom from defects, surface sterilized with 2% sodium hypochlorite for 1 min, rinsed in sterile distilled water (DW), and dried on a clean bench. The fungus was isolated from a naturally-infected paprika stem. For a pure culture, isolated individual conidia were transferred to potato dextrose agar (PDA, Difco Laboratories, Detroit, MI). All cultures were grown on PDA at 25 1C for 14 days. Cultures were transferred to a fresh PDA every 30 days. For fungal identification, the genomic DNA was isolated from the fungus using a DNA extraction kit (Bioneer, Korea) according to the manufacturer0 s protocols. The extracted DNA was purified using a QIAquicks DNA purification kit (Qiagen, Valencia, CA, USA). PCR amplification of 18s rRNA genes was carried out in 50 μL PCR reactors using the primers ITS1 and ITS4 (White et al., 1990). PCR products were purified using the QIAquicksGel Extraction Kit (Qiagen), and gene sequencing and a blast search were requested from a commercial analysis service (Solgent Inc., Daejeon, Korea).
2.5. Physical properties of irradiated paprika Following gamma irradiation, the physical properties were monitored from a tissue of rectangular shaped irradiated and control paprika. Tissues (3 cm 1 cm) were trimmed from the exocarp to endocarp tissue with razor blades. Thirty tissues from 10 samples were tested using a textural measurement instrument (model TA-XT2i, stable Micro System, Surrey, UK) equipped with a 5 kg mechanical load cell, and were compressed 3 times per sample with a 2-mm-diameter puncture probe. Data are presented as the maximum force of hardness and fracturability recorded during tissue compression. 2.6. Statistical analysis All experiments were carried out in triplicate with three observations. A one-way analysis of variance was performed using the SPSS software system, and Duncan0 s multiple range tests were used to compare the differences among the mean values.
M. Yoon et al. / Radiation Physics and Chemistry 98 (2014) 103–108
0 day
105
7 days
Stem
Exocarp and calyx
Fig. 1. Artificial inoculation experiments of Botrytis cinerea on paprika. Three different anatomic positions (stem, calyx, and exocarp) were inoculated with 10 μL of suspension containing 105 conidia mL 1. After 1 week, two positions (stem and exocarp) on paprika were successfully developed.
3. Results and discussion 3.1. Artificial inoculation on paprika According to the results of the fungal identification, isolated fungus from paprika stem was identified as B cinerea in this study. To confirm the symptoms caused by this fungus, an artificial inoculation test was carried out at different anatomic positions (Fig. 1). After 1 week, the fungal symptoms were observed on the stem and exocarp, whereas artificially inoculated fungal conidia on calyx were not developed. In the results of the artificial inoculation test, isolated B. cinerea was kept for fungal pathogenicity, and the disease development on the paprika stem and exocarp except calyx was observed using a suspension containing 105 conidia mL 1. According to these results, further artificial inoculation was carried out on the paprika exocarp because of high reproducibility. 3.2. Changes in physical properties following gamma irradiation Following gamma irradiation, the irradiated paprika was kept in a cooling chamber at 7 1C for 1 week. No significant changes in color of any samples compared with the controls were observed (data not shown). However, after storage, the physical properties were significantly decreased by the dose-dependent irradiation (Fig. 2). The firmness of the 0.6 kGy-irradiated samples was significantly decreased (po 0.05). Continuously, a firmness reduction of about 28% was evaluated by irradiation at a dose of 1 kGy (Fig. 2A). In addition, fracturability of endocarp was also significantly decreased by 0.6 kGy gamma irradiation (p o0.05). About 33% fracturability reduction was observed at a dose of 1 kGy (Fig. 2B). In previous literature, Mitchell et al. (1992) reported that
irradiation at low doses of 0.3–0.75 kGy leads to a reduction of up to 11% of vitamin C in fruits before storage, and up to 79% of vitamin C after 3 weeks of storage. Therefore, combined treatment is necessary to reduce the use or doses of irradiation for fruit quality preservation. 3.3. Sensitivity of gamma irradiation and NaDCC on B. cinerea All survival curves were obtained after 3 days of incubation. The survival curve of B. cinerea conidia following gamma irradiation and chlorination is shown in Fig. 3. The initial values of fungal suspension following gamma irradiation and NaDCC treatment were observed to be 4.23 and 4 log10 CFU/mL, respectively. The sensitivities of both treatments were significantly increased by dose-dependent gamma irradiation and NaDCC treatment (po0.01). Continuously, B. cinerea conidia did not survive irradiation at a dose of 4 kGy (Fig. 3A), and did not survive after 50 ppm NaDCC treatment (Fig. 3B). In particular, a reduction profile of the survival curve by NaDCC treatment showed a gradual decrease at below 20 ppm NaDCC, and a sharp decrease at over 20 ppm NaDCC. In addition, to confirm the fungal pathogenicity for the following treatments, the irradiated and NaDCC treated conidial suspensions were artificially inoculated on paprika exocarp (Fig. 3C). In the case of gamma irradiation, the fungal symptoms of irradiated conidia gradually disappeared by the dose-dependent gamma irradiation. At over 2 kGy irradiation, the conidia were not developed. Moreover, the effect of NaDCC treatment on B. cinerea conidia showed that the treatment of 50 ppm NaDCC leads to fungal disinfection. Various treatments and technologies have been evaluated to aid in the management of postharvest pathogens. Among them, numerous reports have introduced the irradiation of fruits and vegetables (Kader, 1986). These reports suggest that irradiation may
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Firmness (g)
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a
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de
e
200 100 0
1.2
0
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0.2
0.4
0.6
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1
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Gamma irradiation (kGy)
Fig. 2. Physical properties of irradiated paprika with various doses of gamma radiation (0–1.2 kGy). Different letters are significantly different (po 0.05) by Duncan0 s multiple range test.
4.5
4
4
3.5
3.5
Log10 CFU/mL
Log10 CFU/mL
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3 2.5 2 1.5
3 2.5 2 1.5 1
1
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30
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0 kGy
1 kGy
2 kGy
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Ir
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Fig. 3. Sensitivity of gamma irradiation and NaDCC on B. cinerea. (A). Radiation sensitivity of B. cinerea with various doses of gamma radiation (0, 1, 2, and 4 kGy ). (B). NaDCC sensitivity of B. cinerea with various doses of NaDCC (0, 10, 20, 30, and 50 ppm). (C). The fungal pathogenicity following treatment at over 2 kGy irradiation; conidia were not developed. In addition, the effect of chlorination on B. cinerea conidia showed that the treatment of 50 ppm NaDDC leads to fungal disinfection.
have some applications for the preservation of fruits and vegetables. Consequently, the United States Food and Drug Administration allows the irradiation of fresh fruits and vegetables at up to 1 kGy (US FDA, 2004). However, single treatment by gamma irradiation will not resolve most microbiological populations because of their different radiation sensitivities. Additionally, a few fruits and vegetables will receive a negative effect in physio-chemical properties or other quality parameters at up to 1 kGy (Beraha et al., 1960; Tiryaki and Maden, 1991), whereas, many fruits and vestibules can tolerate 1 kGy
irradiation without significant sensory impact by proper controlling of temperature during irradiation and by combination of other sanitizers or techniques (Herdt and Feng, 2009; Fan, 2013). NaDCC, also known as sodium dichloro-s-triazinetrione, chlorine donor, has been approved by the United States Environmental Protection Agency and the World Health Organization for the routine treatment of drinking water (Clasen and Edmondson, 2006). Bloomfield and Miles (1979) reported the antimicrobial properties of NaDCC and sodium hypochlorite (NaOCl). A 30 min
M. Yoon et al. / Radiation Physics and Chemistry 98 (2014) 103–108
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y = -0.9433x + 4.1419 R² = 0.9899
3
y = -1.1405x + 3.9207 R² = 0.9883
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y = -1.7127x + 2.2216 R² = 0.9649
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Fig. 4. Synergistic effect of combined treatment on B. cinerea. (A). Synergistic effect of combined treatment with gamma irradiation and chlorination on B. cinerea conidia. (B). The combined treatment of 0.4 kGy irradiation and 30 ppm NaDCC fully inhibited the fungal development.
soak in NaDCC reduced the initial microbial load in various vegetables by 1.69–2.42 log compared to water washed controls (Nicholl and Prendergast, 1998). Moreover, raw salads are frequently subjected to chlorine wash to reduce microbial numbers, inhibit enzymatic activity, and improve the sensory quality shelflife (Lee and Frank, 1991). Beuchat and Brackett (1990) found that a chlorine dip system significantly reduced the initial population of natural contaminants on lettuce. However, with 4 days of storage at 5 1C, no significant difference in the populations of aerobic microbes was found on the lettuce. From the previous literature, although NaDCC treatment shows a reasonable antibiotic activity, continuous efficiency is not entirely maintained during storage. Therefore, a combined disinfection method of NaDCC and another efficient treatment should be considered. 3.4. Synergistic effect of combined treatment on B. cinerea Synergistic effect of combined treatment with gamma irradiation and NaDCC treatment on the disinfection of B. cinerea conidia is shown in Fig. 4. A synergistic effect was observed for combined treatment against B. cinerea conidia. The sensitivity was markedly increased with combined treatment compared with the gamma irradiation was used alone (Fig. 4A). As a result of artificial inoculation, the combined treatment with 0.4 kGy of irradiation and 30 ppm of NaDCC fully inhibited the fungal development (Fig. 4B). The synergistic effect of combined treatment is summarized in Table 1. In the comparison of single and combined treatments, the initial value of the conidial number by dosedependent chlorine was significantly reduced by 4.15, 4, 3.5, and
Table 1 Synergistic effect of combined treatments for control of gray mold (B. cinerea) on paprika. Treatments
Initial value (log10 CFU/mL)
D10 value (kGy)
r2
Ir Only NaDCC (10 ppm) þIr NaDCC (20 ppm) þ Ir NaDCC (30 ppm) þ Ir
4.15a 4.00a 3.50b 2.15c
1.06a 0.88b 0.77c 0.58d
0.989 0.988 0.993 0.964
Ir, gamma irradiation. NaDCC, sodium dichloroisocyanurate. Within the same column, different letters indicate significant difference (p o 0.05).
2.15 log10 CFU/mL (p o0.05). In addition, the D10 value following combined treatment was also significantly reduced by 1.06, 0.88, 0.77, and 0.58 kGy, correspondingly (po 0.05). Recently, the combined treatments of gamma irradiation and chlorination were attempted in seafood for a reduction of natural microflora (Kim et al., 2012; Park et al., 2012). The authors reported that the combined chlorine–ionizing radiation disinfection treatments result in synergistic benefits in regard to reducing the numbers of natural microflora. Similarly, in this study, the combined methods result in a greater reduction than when the single methods are used alone for the control of gray mold. Moreover, the minimum dose of the developmental inhibition on B. cinerea was markedly reduced from 2 kGy to 0.4 kGy irradiation by the combined treatment with 30 ppm NaDCC. This decreased
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dose contributed to the preservation of the physical properties of post-harvested paprika. Especially, in the postharvest management of fruits, the application of chlorination has various advantages. The postharvest handling of many fruits usually involves the use of flumes, water dump tanks, spray washers, or hydrocoolers. Most chlorination processes recirculate used water to conserve water and energy, and are easy to apply to fruits by adjusting the optimal chlorine concentration. Therefore, this postharvest treatment was applied to various fruits (Goodin, 1977; Showalter, 1993; Hough and Kellerman, 1971; Hendrix, 1991; Sanderson and Spotts, 1995; Sherman and Allen, 1983). Furthermore, gamma irradiation is not only a promising disinfectant method of pathogens but also has high penetration power (Postma et al., 2001). During the postharvest processes, gamma irradiation can be applied to disinfect pathogens following the commercial packing process during the previous transportation and distribution. Thus, a chlorination process is not necessary in the new postharvest process, and gamma irradiation can rapidly handle a large quantity at once. 4. Conclusion The complete prevention of pathogens in fruits requires relatively high doses of gamma irradiation, which cause reduction of physical properties and consequent loss of fruits quality. On the other hand, the use of combined treatment with irradiation and NaDCC treatment has a synergistic effect on reducing the fungi load and the irradiation dose required to eliminate pathogens was also reduced. Our results suggest that industrial application with combined treatment (NaDCCþgamma irradiation) is possible for quality preservation in paprika. Furthermore, the application requirement profile of a combination of irradiation with NaDCC treatment must be justified in future.
Acknowledgments This work was supported by the Export Promotion Technology Development Project from the Korea Ministry of Agriculture, Food and Rural affairs and the Nuclear Research Foundation of Korea funded by Ministry of Science, ICT and Future Planning. The authors thank Ho-Je Kwon, Don-Sun Im, Byoung Hun Lee, Jae-Ho Kim and Tai-Jin Kang for support of gamma irradiation in KAERI (Korea Atomic Energy Research Institute). References Baka, M., Mercier, J., Corcuff, F., Castaigne, F., Arul, J., 1999. Photochemical treatment to improve storability of fresh strawberries. J. Food Sci. 64, 1068–1072. Beraha, L., Ramsey, G.B., Smith, M.A., Wright, W.R., 1960. Gamma radiation dose response of some decay pathogens. Phytopathology 50, 474–476. Beuchat, L.R., Brackett, R.E., 1990. Survival and growth of Listeria monocytogenes on lettuce as influenced by shredding, chlorine treatment, modified atmosphere packaging and temperature. J. Food Sci. 55, 755–758. Bialka, K.L., Demirci, A., 2007. Decontamination of Escherichia coli O157:H7 and Salmonella enterica on blueberries using ozone and pulsed UV-light. J. Food Sci. 72 (9), 391–396. Blank, G., Corrigan, D., 1995. Comparison of resistance of fungal spores to gamma and electron beam radiation. Int. J. Food Microbiol. 26, 269–277. Bloomfield, S.F., Miles, G.A., 1979. The antibacterial properties of sodium dichloroisocyanurate and sodium hypochlorite formulations. J. App. Bacteriol. 46, 65–73. Bulger, M.A., Ellis, M.A., Madden, L.V., 1987. Influence of temperature and wetness duration on infection of strawberry flowers by Botrytis cinerea and disease incidence of fruit originating from infected flowers. Phytopathology 77, 1225–1230. Cia, P., Pascholati, S.F., Benato, E.A., Camili, E.C., Santos, C.A., 2007. Effects of gamma and UV-C irradiation on the postharvest control of papaya anthracnose. Postharvest Biol. Technol. 43, 366–373. Clasen, T., Edmondson, P., 2006. Sodium dichloroisocyanurate (NaDCC) tablets as an alternative to sodium hypochlorite for the routine treatment of drinking water at the household level. Int. J. Hyg. Environ. Health 209, 173–181.
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Synergistic effect of the combined treatment with gamma irradiation and sodium dichloroisocyanurate to control gray mold (Botrytis cinerea) on paprika Minchul Yoon, Koo Jung, Kwang-Youll Lee, Je-Yong Jeong, Ju-Woon Lee, Hae-Jun Park n RT Practical Application Division, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeong-up 580-185, Republic of Korea
H I G H L I G H T S
Paprikas were treated with irradiation and NaDCC to control gray mold. We confirmed that the combined treatment was synergistically affected. The treatment can contribute to a reduction of postharvest losses caused by fungi. This combined treatment can also reduce the doses of irradiation.
art ic l e i nf o
a b s t r a c t
Article history: Received 1 September 2013 Accepted 30 December 2013 Available online 17 January 2014
Gray mold (Botrytis cinerea) is one of the most major fungal pathogens in paprika. Generally, gamma irradiation over 1 kGy is effective for the control of fungal pathogens; however, a significant change in fruit quality (physical properties) on paprika was shown from gamma irradiation at over 0.6 kGy (po 0.05). Therefore, in this study, the synergistic disinfection effect of the combined treatment with gamma irradiation and sodium dichloroisocyanurate (NaDCC) was investigated to reduce the gamma irradiation dose. In an artificial inoculation experiment of B. cinerea isolated from naturally-infected postharvest paprika, fungal symptoms were observed in the stem and exocarp of paprika after conidial inoculation. From the sensitivity of gamma irradiation and NaDCC, B. cinerea conidia were fully inactivated by 4 kGy of gamma irradiation (D10 value 0.99 kGy), and were fully inactivated by 50 ppm NaDCC treatment. The fungal symptoms were not detected by the dose-dependent gamma irradiation ( 44 kGy) and NaDCC ( 450 ppm). As a result of the combined treatment of gamma irradiation and NaDCC, the D10 value was significantly reduced by 1.06, 0.88, 0.77, and 0.58 kGy (po 0.05). Moreover, fungal symptoms were more significantly reduced in combined treatment groups (gamma irradiation and NaDCC) than single treatment groups (gamma irradiation or NaDCC). These results suggest that combined treatment with irradiation and NaDCC treatment can be applied to preserve quality of postharvest paprika or other fruits. & 2014 Elsevier Ltd. All rights reserved.
Keywords: Botrytis cinerea Gamma irradiation Sodium dichloroisocyanurate Synergistic effect Postharvest paprika
1. Introduction Paprika (spice red pepper; Capsicum annuum L.) is one of the most economically important fruit crops. It is an excellent vitamin source which has been confirmed by various epidemiological studies (Gey and Puska, 1989; Gerster, 1991). In spite of this economic importance, the productivity decreases and quality losses occur during the preharvest and postharvest management of this fruit crop because of a number of diseases. The gray mold
n
Corresponding author. Tel.: þ 82 63 570 3190; fax: þ82 63 570 3195. E-mail address: [email protected] (H.-J. Park).
0969-806X/$ - see front matter & 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.radphyschem.2013.12.039
caused by Botrytis cinerea develops mainly on fruits, leaves, or flowers (Dik and Elad, 1999) in a greenhouse, causing a decrease in the productivity of paprika throughout the world, and is usually managed by the frequent use of prophylactic fungicides (Bulger et al., 1987; de Visser, 1996; Raposo et al., 1996) in fruit crops, including pepper crops during preharvest management. Furthermore, B. cinerea causes an important postharvest decay of pepper (Pernezny et al., 2003), and disinfection technologies of this pathogen are needed for a postharvest management strategy. Most postharvest management technologies have applied chemical fumigation for the preservation of fruit quality from fungi mediated diseases, but alternative methods are needed because of
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growing public concerns over the human health and environmental risks (Spotts and Cervantes, 1986). Therefore, many researchers have focused on eco-friendly postharvest technologies that can contribute to replacing the use of chemical fumigation techniques for crop quality preservation. Alternative treatments have been reported: heat treatments (Lurie, 1998), photochemical treatments (Baka et al., 1999), pulsed light treatments (GómezLópez et al., 2005b), microwave drying (Orsat et al., 2006), UV treatments (Vicente et al., 2005), and ozone treatments (Bialka and Demirci, 2007). Although these alternative treatments show promise, individual treatments are not effective for fungicides. Therefore, it is necessary to develop a technology that combines other disinfection treatments for synergistic effects (Conway et al., 2005). Although fresh fruits and vegetables can be irradiated at a gamma ray dose of up to 1 kGy (US FDA, 2004), this is not possible to fully control postharvest fungi (Blank and Corrigan, 1995). Moreover, the radiation dose required to control fungi has a negative effect on the skin color and texture of stored fruits and vegetables (Beraha et al., 1960; Tiryaki et al., 1994). Therefore, it is important to reduce the irradiation dose for an inhibition of postharvest photogenes through a combined treatment. One promising treatment is the use of gamma irradiation with a combination of other treatments. Consequently, this combined treatment can contribute to a reduction of postharvest losses caused by fungi and reduce the use or doses of fungicides for disease control (Cia et al., 2007). Chlorination is applicable to specific fruits and vegetables, including paprika, using a postharvest process with flumes, water dump tanks, and spray washers. This postharvest treatment was applied to various fruits and vegetables such as tomatoes (Goodin, 1977; Showalter, 1993), citrus (Hough and Kellerman, 1971), apples (Hendrix, 1991), pears (Sanderson and Spotts, 1995), and peppers (Sherman and Allen, 1983). In this study, the effect of gamma irradiation on the physical property of postharvest paprika was evaluated using a texture analyzer. The sensitivity of gamma irradiation and dichloroisocyanurate (NaDCC) was evaluated through in vivo and in vitro tests. The objective of the present study is to evaluate the combination effect of gamma irradiation and NaDCC for disease control and shelf-life of the paprika.
2. Materials and methods
2.2. Preparation of fungal conidia For harvesting the conidia, about 10 mL DW was added to a culture dish and the conidia were gently harvested by filtration through four sterile layers of gauze. The conidial suspensions were collected in sterile screw-cap test tubes (16 100 mm2) containing 15 mL of sterile distilled water and filtered twice using sterile Pasteur pipettes (4.62 mm) packed with glass wool. The procedure was used to remove mycelial fragments and conidial clumps (Saleh et al., 1988). The concentration of conidia was measured using a hemacytometer (Warner-Lambert Technologies Inc., Buffalo, N.Y.). The final conidial concentration was adjusted to 105 conidia mL 1 with DW for further study (Slade et al., 1987). 2.3. Gamma irradiation and NaDCC treatment The samples were irradiated in a cobalt-60 irradiator (point source, AECL, IR-79, Nordion, Canada) with various absorbed doses (from 0.2 to 4 kGy). Dosimetry was performed using 5 mm diameter alanine dosimeters (Bruker Instruments, Rheinstetten, Germany). For chlorination, the samples were immersed in sodium dichloroisocyanurate (NaDCC; Sigma-Aldrich, Poole, UK) at various concentrations (from 5 to 50 ppm). Following treatment, the suspensions were serially diluted in DW and plated on PDA. Incubation was carried out at 25 1C for 5 days. Survival curves were constructed by plotting the survivor CFU/mL versus the actual radiation doses and NaDCC concentrations. The curves were fitted by linear regression, and the radiation sensitivity was expressed in terms of the D10 values. A D10 value is defined as the dose required for reducing the given population by 90% of its initial value. The D10 value was determined from the reciprocal of the slope for the straight-line portion of the survival curve (Ley, 1983). 2.4. Artificial inoculation on paprika Pre-sterilized paprika fruits were used in an artificial inoculation test, and was wounded to a 1.5 mm thickness by puncturing them with the point of a sterile 0.5 mm diameter needle. Each wound site was artificially inoculated with 10 μL of suspension containing 105 conidia mL 1 on the paprika. To protect secondary contamination, the inoculated paprika was transferred into a sterilized oxygen-impermeable plastic container, and was kept at 25 1C and a relative humidity of 490% for 1 week.
2.1. Preparation of paprika and fungus Paprika was purchased from an agricultural expert company (Rosepia Co., Ltd., Jeollabuk-do, Korea). The paprika was selected for a uniform size and freedom from defects, surface sterilized with 2% sodium hypochlorite for 1 min, rinsed in sterile distilled water (DW), and dried on a clean bench. The fungus was isolated from a naturally-infected paprika stem. For a pure culture, isolated individual conidia were transferred to potato dextrose agar (PDA, Difco Laboratories, Detroit, MI). All cultures were grown on PDA at 25 1C for 14 days. Cultures were transferred to a fresh PDA every 30 days. For fungal identification, the genomic DNA was isolated from the fungus using a DNA extraction kit (Bioneer, Korea) according to the manufacturer0 s protocols. The extracted DNA was purified using a QIAquicks DNA purification kit (Qiagen, Valencia, CA, USA). PCR amplification of 18s rRNA genes was carried out in 50 μL PCR reactors using the primers ITS1 and ITS4 (White et al., 1990). PCR products were purified using the QIAquicksGel Extraction Kit (Qiagen), and gene sequencing and a blast search were requested from a commercial analysis service (Solgent Inc., Daejeon, Korea).
2.5. Physical properties of irradiated paprika Following gamma irradiation, the physical properties were monitored from a tissue of rectangular shaped irradiated and control paprika. Tissues (3 cm 1 cm) were trimmed from the exocarp to endocarp tissue with razor blades. Thirty tissues from 10 samples were tested using a textural measurement instrument (model TA-XT2i, stable Micro System, Surrey, UK) equipped with a 5 kg mechanical load cell, and were compressed 3 times per sample with a 2-mm-diameter puncture probe. Data are presented as the maximum force of hardness and fracturability recorded during tissue compression. 2.6. Statistical analysis All experiments were carried out in triplicate with three observations. A one-way analysis of variance was performed using the SPSS software system, and Duncan0 s multiple range tests were used to compare the differences among the mean values.
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0 day
105
7 days
Stem
Exocarp and calyx
Fig. 1. Artificial inoculation experiments of Botrytis cinerea on paprika. Three different anatomic positions (stem, calyx, and exocarp) were inoculated with 10 μL of suspension containing 105 conidia mL 1. After 1 week, two positions (stem and exocarp) on paprika were successfully developed.
3. Results and discussion 3.1. Artificial inoculation on paprika According to the results of the fungal identification, isolated fungus from paprika stem was identified as B cinerea in this study. To confirm the symptoms caused by this fungus, an artificial inoculation test was carried out at different anatomic positions (Fig. 1). After 1 week, the fungal symptoms were observed on the stem and exocarp, whereas artificially inoculated fungal conidia on calyx were not developed. In the results of the artificial inoculation test, isolated B. cinerea was kept for fungal pathogenicity, and the disease development on the paprika stem and exocarp except calyx was observed using a suspension containing 105 conidia mL 1. According to these results, further artificial inoculation was carried out on the paprika exocarp because of high reproducibility. 3.2. Changes in physical properties following gamma irradiation Following gamma irradiation, the irradiated paprika was kept in a cooling chamber at 7 1C for 1 week. No significant changes in color of any samples compared with the controls were observed (data not shown). However, after storage, the physical properties were significantly decreased by the dose-dependent irradiation (Fig. 2). The firmness of the 0.6 kGy-irradiated samples was significantly decreased (po 0.05). Continuously, a firmness reduction of about 28% was evaluated by irradiation at a dose of 1 kGy (Fig. 2A). In addition, fracturability of endocarp was also significantly decreased by 0.6 kGy gamma irradiation (p o0.05). About 33% fracturability reduction was observed at a dose of 1 kGy (Fig. 2B). In previous literature, Mitchell et al. (1992) reported that
irradiation at low doses of 0.3–0.75 kGy leads to a reduction of up to 11% of vitamin C in fruits before storage, and up to 79% of vitamin C after 3 weeks of storage. Therefore, combined treatment is necessary to reduce the use or doses of irradiation for fruit quality preservation. 3.3. Sensitivity of gamma irradiation and NaDCC on B. cinerea All survival curves were obtained after 3 days of incubation. The survival curve of B. cinerea conidia following gamma irradiation and chlorination is shown in Fig. 3. The initial values of fungal suspension following gamma irradiation and NaDCC treatment were observed to be 4.23 and 4 log10 CFU/mL, respectively. The sensitivities of both treatments were significantly increased by dose-dependent gamma irradiation and NaDCC treatment (po0.01). Continuously, B. cinerea conidia did not survive irradiation at a dose of 4 kGy (Fig. 3A), and did not survive after 50 ppm NaDCC treatment (Fig. 3B). In particular, a reduction profile of the survival curve by NaDCC treatment showed a gradual decrease at below 20 ppm NaDCC, and a sharp decrease at over 20 ppm NaDCC. In addition, to confirm the fungal pathogenicity for the following treatments, the irradiated and NaDCC treated conidial suspensions were artificially inoculated on paprika exocarp (Fig. 3C). In the case of gamma irradiation, the fungal symptoms of irradiated conidia gradually disappeared by the dose-dependent gamma irradiation. At over 2 kGy irradiation, the conidia were not developed. Moreover, the effect of NaDCC treatment on B. cinerea conidia showed that the treatment of 50 ppm NaDCC leads to fungal disinfection. Various treatments and technologies have been evaluated to aid in the management of postharvest pathogens. Among them, numerous reports have introduced the irradiation of fruits and vegetables (Kader, 1986). These reports suggest that irradiation may
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Firmness (g)
1600
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a
1200
bc
c
d
d
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b
Fracturability (g)
106
0.4
0.6
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1
ab
ab
bc
cd
300
de
e
200 100 0
1.2
0
Gamma irradiation (kGy)
0.2
0.4
0.6
0.8
1
1.2
Gamma irradiation (kGy)
Fig. 2. Physical properties of irradiated paprika with various doses of gamma radiation (0–1.2 kGy). Different letters are significantly different (po 0.05) by Duncan0 s multiple range test.
4.5
4
4
3.5
3.5
Log10 CFU/mL
Log10 CFU/mL
4.5
3 2.5 2 1.5
3 2.5 2 1.5 1
1
y = -1.054x + 4.3049 R² = 0.9963
0.5 0 0
1
2
3
y = -0.079x + 4.4686 R² = 0.9048
0.5 0 4
0
10
Irradiation dose (kGy)
20
30
40
50
NaDCC (ppm)
0 kGy
1 kGy
2 kGy
0 ppm
10 ppm
20 ppm
4 kGy
Ir
50 ppm
NaDCC
Fig. 3. Sensitivity of gamma irradiation and NaDCC on B. cinerea. (A). Radiation sensitivity of B. cinerea with various doses of gamma radiation (0, 1, 2, and 4 kGy ). (B). NaDCC sensitivity of B. cinerea with various doses of NaDCC (0, 10, 20, 30, and 50 ppm). (C). The fungal pathogenicity following treatment at over 2 kGy irradiation; conidia were not developed. In addition, the effect of chlorination on B. cinerea conidia showed that the treatment of 50 ppm NaDDC leads to fungal disinfection.
have some applications for the preservation of fruits and vegetables. Consequently, the United States Food and Drug Administration allows the irradiation of fresh fruits and vegetables at up to 1 kGy (US FDA, 2004). However, single treatment by gamma irradiation will not resolve most microbiological populations because of their different radiation sensitivities. Additionally, a few fruits and vegetables will receive a negative effect in physio-chemical properties or other quality parameters at up to 1 kGy (Beraha et al., 1960; Tiryaki and Maden, 1991), whereas, many fruits and vestibules can tolerate 1 kGy
irradiation without significant sensory impact by proper controlling of temperature during irradiation and by combination of other sanitizers or techniques (Herdt and Feng, 2009; Fan, 2013). NaDCC, also known as sodium dichloro-s-triazinetrione, chlorine donor, has been approved by the United States Environmental Protection Agency and the World Health Organization for the routine treatment of drinking water (Clasen and Edmondson, 2006). Bloomfield and Miles (1979) reported the antimicrobial properties of NaDCC and sodium hypochlorite (NaOCl). A 30 min
M. Yoon et al. / Radiation Physics and Chemistry 98 (2014) 103–108
107
4.5 4
Log10 CFU/mL
3.5
y = -0.9433x + 4.1419 R² = 0.9899
3
y = -1.1405x + 3.9207 R² = 0.9883
2.5 2 1.5
y = -1.2974x + 3.4793 R² = 0.9939
Ir Only
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NaDCC (10ppm) + Ir
0.5
NaDCC (20ppm) + Ir NaDCC (30ppm) + Ir
y = -1.7127x + 2.2216 R² = 0.9649
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0.4
0.6
0.8
1
1.2
Gamma irradiation (Gy) 0 kGy
0.2 kGy
0.4 kGy
0.8 kGy
NaDCC (30 ppm) + Ir
Fig. 4. Synergistic effect of combined treatment on B. cinerea. (A). Synergistic effect of combined treatment with gamma irradiation and chlorination on B. cinerea conidia. (B). The combined treatment of 0.4 kGy irradiation and 30 ppm NaDCC fully inhibited the fungal development.
soak in NaDCC reduced the initial microbial load in various vegetables by 1.69–2.42 log compared to water washed controls (Nicholl and Prendergast, 1998). Moreover, raw salads are frequently subjected to chlorine wash to reduce microbial numbers, inhibit enzymatic activity, and improve the sensory quality shelflife (Lee and Frank, 1991). Beuchat and Brackett (1990) found that a chlorine dip system significantly reduced the initial population of natural contaminants on lettuce. However, with 4 days of storage at 5 1C, no significant difference in the populations of aerobic microbes was found on the lettuce. From the previous literature, although NaDCC treatment shows a reasonable antibiotic activity, continuous efficiency is not entirely maintained during storage. Therefore, a combined disinfection method of NaDCC and another efficient treatment should be considered. 3.4. Synergistic effect of combined treatment on B. cinerea Synergistic effect of combined treatment with gamma irradiation and NaDCC treatment on the disinfection of B. cinerea conidia is shown in Fig. 4. A synergistic effect was observed for combined treatment against B. cinerea conidia. The sensitivity was markedly increased with combined treatment compared with the gamma irradiation was used alone (Fig. 4A). As a result of artificial inoculation, the combined treatment with 0.4 kGy of irradiation and 30 ppm of NaDCC fully inhibited the fungal development (Fig. 4B). The synergistic effect of combined treatment is summarized in Table 1. In the comparison of single and combined treatments, the initial value of the conidial number by dosedependent chlorine was significantly reduced by 4.15, 4, 3.5, and
Table 1 Synergistic effect of combined treatments for control of gray mold (B. cinerea) on paprika. Treatments
Initial value (log10 CFU/mL)
D10 value (kGy)
r2
Ir Only NaDCC (10 ppm) þIr NaDCC (20 ppm) þ Ir NaDCC (30 ppm) þ Ir
4.15a 4.00a 3.50b 2.15c
1.06a 0.88b 0.77c 0.58d
0.989 0.988 0.993 0.964
Ir, gamma irradiation. NaDCC, sodium dichloroisocyanurate. Within the same column, different letters indicate significant difference (p o 0.05).
2.15 log10 CFU/mL (p o0.05). In addition, the D10 value following combined treatment was also significantly reduced by 1.06, 0.88, 0.77, and 0.58 kGy, correspondingly (po 0.05). Recently, the combined treatments of gamma irradiation and chlorination were attempted in seafood for a reduction of natural microflora (Kim et al., 2012; Park et al., 2012). The authors reported that the combined chlorine–ionizing radiation disinfection treatments result in synergistic benefits in regard to reducing the numbers of natural microflora. Similarly, in this study, the combined methods result in a greater reduction than when the single methods are used alone for the control of gray mold. Moreover, the minimum dose of the developmental inhibition on B. cinerea was markedly reduced from 2 kGy to 0.4 kGy irradiation by the combined treatment with 30 ppm NaDCC. This decreased
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dose contributed to the preservation of the physical properties of post-harvested paprika. Especially, in the postharvest management of fruits, the application of chlorination has various advantages. The postharvest handling of many fruits usually involves the use of flumes, water dump tanks, spray washers, or hydrocoolers. Most chlorination processes recirculate used water to conserve water and energy, and are easy to apply to fruits by adjusting the optimal chlorine concentration. Therefore, this postharvest treatment was applied to various fruits (Goodin, 1977; Showalter, 1993; Hough and Kellerman, 1971; Hendrix, 1991; Sanderson and Spotts, 1995; Sherman and Allen, 1983). Furthermore, gamma irradiation is not only a promising disinfectant method of pathogens but also has high penetration power (Postma et al., 2001). During the postharvest processes, gamma irradiation can be applied to disinfect pathogens following the commercial packing process during the previous transportation and distribution. Thus, a chlorination process is not necessary in the new postharvest process, and gamma irradiation can rapidly handle a large quantity at once. 4. Conclusion The complete prevention of pathogens in fruits requires relatively high doses of gamma irradiation, which cause reduction of physical properties and consequent loss of fruits quality. On the other hand, the use of combined treatment with irradiation and NaDCC treatment has a synergistic effect on reducing the fungi load and the irradiation dose required to eliminate pathogens was also reduced. Our results suggest that industrial application with combined treatment (NaDCCþgamma irradiation) is possible for quality preservation in paprika. Furthermore, the application requirement profile of a combination of irradiation with NaDCC treatment must be justified in future.
Acknowledgments This work was supported by the Export Promotion Technology Development Project from the Korea Ministry of Agriculture, Food and Rural affairs and the Nuclear Research Foundation of Korea funded by Ministry of Science, ICT and Future Planning. The authors thank Ho-Je Kwon, Don-Sun Im, Byoung Hun Lee, Jae-Ho Kim and Tai-Jin Kang for support of gamma irradiation in KAERI (Korea Atomic Energy Research Institute). References Baka, M., Mercier, J., Corcuff, F., Castaigne, F., Arul, J., 1999. Photochemical treatment to improve storability of fresh strawberries. J. Food Sci. 64, 1068–1072. Beraha, L., Ramsey, G.B., Smith, M.A., Wright, W.R., 1960. Gamma radiation dose response of some decay pathogens. Phytopathology 50, 474–476. Beuchat, L.R., Brackett, R.E., 1990. Survival and growth of Listeria monocytogenes on lettuce as influenced by shredding, chlorine treatment, modified atmosphere packaging and temperature. J. Food Sci. 55, 755–758. Bialka, K.L., Demirci, A., 2007. Decontamination of Escherichia coli O157:H7 and Salmonella enterica on blueberries using ozone and pulsed UV-light. J. Food Sci. 72 (9), 391–396. Blank, G., Corrigan, D., 1995. Comparison of resistance of fungal spores to gamma and electron beam radiation. Int. J. Food Microbiol. 26, 269–277. Bloomfield, S.F., Miles, G.A., 1979. The antibacterial properties of sodium dichloroisocyanurate and sodium hypochlorite formulations. J. App. Bacteriol. 46, 65–73. Bulger, M.A., Ellis, M.A., Madden, L.V., 1987. Influence of temperature and wetness duration on infection of strawberry flowers by Botrytis cinerea and disease incidence of fruit originating from infected flowers. Phytopathology 77, 1225–1230. Cia, P., Pascholati, S.F., Benato, E.A., Camili, E.C., Santos, C.A., 2007. Effects of gamma and UV-C irradiation on the postharvest control of papaya anthracnose. Postharvest Biol. Technol. 43, 366–373. Clasen, T., Edmondson, P., 2006. Sodium dichloroisocyanurate (NaDCC) tablets as an alternative to sodium hypochlorite for the routine treatment of drinking water at the household level. Int. J. Hyg. Environ. Health 209, 173–181.
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