# Chlorine Dioxide Treatment Modulates Ripening-Related Genes and Antioxidant System to Improve the Storability of Tomato

Authors: Me-Hea Park; Siva Kumar Malka
Journal: Journal of Food Quality
 (2022)

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## Abstract

Chlorine dioxide (ClO2) is used to maintain quality and safety of fresh produce. However, ClO2 action mechanism in fresh produce is unknown. In this study, firstly, we evaluated the efficacy of ClO2 treatment on the quality, chilling injury, and calyx molding of tomatoes stored at two different temperatures. Then, ClO2 effect on the expression of cell wall- and ripening-related genes and on the activity of antioxidant enzymes was investigated. Tomatoes were treated with gaseous ClO2 for 15 min before transferring them to 13°C for 12 days and/or 4°C for 14 days, followed by 5 days at 20°C (shelf-life conditions). ClO2 treatment marginally reduced the rate of respiration but did not affect ethylene production at 13°C and 4°C storage or at shelf-life conditions. When stored at 13°C, treatment with ClO2 reduced the loss of firmness, with concomitant repression of pectin esterase 1, a cell wall-related gene. Additionally, at 13°C storage conditions, ClO2 treatment maintained tomato quality in terms of soluble solid content, titratable acidity, and color and was associated with the downregulation of the ripening-relatedethylene response factors B3/C1/E1 and the induction of antioxidant genes encoding catalase and ascorbate peroxidase. At 4°C storage conditions, ClO2 at a concentration of 15 ppm not only maintained the firmness and quality of tomatoes but also inhibited pitting during shelf-life with a concomitant increase of catalase activity. Moreover, treatment with 15 ppm ClO2 significantly reduced the calyx molding that is generally observed in fruits stored at 13°C and under shelf-life conditions. Hence, our results indicate that ClO2 treatment effectively maintained tomato quality and inhibited calyx molding by partially regulating ripening-related genes and antioxidant systems, thereby improving the storability of postharvest tomatoes.


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## Body

## 1. Introduction

The shelf-life of tomatoes is short owing to their fast-ripening nature. During storage and distribution, ripening progresses with a color change from green to red as well as softening and compositional changes in chemicals associated with flavor and aroma, such as organic acids, sugars, and other volatiles. Cold storage is a common practice to extend the shelf-life of fresh produce. However, tomatoes are prone to developing chilling injuries (CIs) under prolonged cold storage conditions, such as pitting, the development of sunken areas on the fruit, and increased susceptibility to rotting and decay [1]. CI symptoms usually become pronounced under market shelf conditions following cold storage, thereby reducing consumer desirability [1, 2]. Storage above 10°C is recommended to avoid CI in tomatoes; however, high storage temperatures accelerate softening and promote microbial growth especially on calyces, resulting in quality deterioration [1, 3–5]. The calyx is considered an indicator of the freshness and quality of the tomato fruit. Moreover, consumers are highly attracted to the green leaf aroma provided by these green parts. However, calyces are vulnerable to microbial spoilage (epiphytic bacteria and molds) during shipping and storage and are usually the first part of tomatoes to show fungal growth. The presence of mold on the calyx influences the marketability and shelf-life of tomatoes, even if the fruit itself is not infected [5]. Thus, the maintenance of microbial safety and quality of tomatoes during storage and distribution requires investigation.Chlorine dioxide (ClO2) is a strong oxidizing gas used to disinfect fresh produce because of its antimicrobial efficacy against bacteria, fungi, and viruses [6–8]. As it is water-soluble, ClO2 can be used in both aqueous and gaseous forms. However, gaseous ClO2 is more effective for pathogen inactivation [8]. A comparison of the disinfection efficacy of various chemical and physical sanitizers revealed gaseous ClO2 to be more effective in microbial inactivation than other sanitizers [9]. The antimicrobial effect of ClO2 gas has been evaluated for a wide range of produce, such as spinach, potatoes, mung bean sprouts, lettuce, onions, cabbage, cantaloupe, and strawberries [6, 7]. The mechanism of action of ClO2 against microbes involves the destabilization of the cell membrane, alteration of membrane permeability, and interruption of protein synthesis [8]. Furthermore, ClO2 reacts with oxygenated compounds and proteins, resulting in the disruption of cellular metabolism [6].Several studies have highlighted the efficacy of ClO2 application in maintaining the quality of fresh produce. For instance, the controlled release of ClO2 gas regulates the firmness of berries during storage [10]. ClO2 exposure positively affects the composition of volatile compounds and free amino acids in citrus fruits, resulting in the retention of their distinct flavors [11]. ClO2 exposure retains the titratable acidity (TA), soluble solid content (SSC), and vitamin C content in tomato and mulberry [12, 13]. In another study, the sensory properties of plums were preserved upon ClO2 treatment [14]. However, ClO2 application may negatively affect the quality of some fresh produce. For instance, ClO2 treatment results in rapid color changes in spinach leaves and browning of grapefruit, cabbage, lettuce, peaches, and apples [15]. It is evident that different fresh produce may respond differently to ClO2 application; hence, the optimization of treatment conditions for each produce of interest is necessary. The effect of ClO2 on the respiration rate and ethylene biosynthesis plays a key role in ClO2-derived quality maintenance in fresh produce [8]. However, the molecular mechanisms by which ClO2 maintains the quality of fresh produce remain to be fully understood.In this study, we optimized the ClO2 treatment conditions and evaluated the efficacy of ClO2 application on the quality, calyx molding, and CI of tomatoes stored at two different temperatures. To understand the mechanism of action of ClO2, we assessed the impact of ClO2 treatment on the gene expression profile of ripening-related and antioxidant genes and the activity of antioxidant enzymes.


## 2. Materials and Methods

### 2.1. Plant Materials and Treatments

“Kamma” tomatoes (Solanum lycopersicum Mill.) grown in Jungyeum, South Korea, were harvested at the pink-red stage and transported to the laboratory. In our preliminary experiments, tomatoes were treated with 5, 10, and 15 ppm ClO2 for 15 min and/or 30 min. ClO2 at 5 ppm did not have any effect on CI and calyx molding (Figure S1). Additionally, ClO2 effect on CI and calyx molding was not significantly different when the treatment time was extended from 15 min to 30 min under our experimental conditions (data not shown). Hence, the fruits were treated with 10 or 15 ppm gaseous ClO2 (mixed with ambient air) using a ClO2 generator (CA300, South Korea) or left untreated (control) inside a commercial cardboard box for 15 min in a closed chamber. Twenty boxes containing 20 fruits each were used for each treatment. The ClO2 concentration in the closed chamber was verified using a built-in ClO2 meter. Following treatment, the fruit-containing boxes were covered with a plastic film and transferred to 13°C for 12 d or 4°C (cold storage) for 14 d followed by five days at 20°C (14 + 5 d; shelf-life conditions). During storage, the relative humidity was maintained at 90 ± 5%.


### 2.2. Gas Chromatography Analysis

The rate of respiration and ethylene production were analyzed using gas chromatography (Bruker 450-GC; Bruker Corp, Billerica, MA, USA). One milliliter of gas was sampled using a syringe from 2-L containers with four fruits from each treatment that had been sealed for 2 h. The injection and column temperatures were 110°C and 70°C, respectively. The thermal conductivity detector and flame ionization detector used for the ClO2 and ethylene measurements were set at 150°C and 250°C, respectively. ClO2 and ethylene measurements were obtained from three independent replicates per treatment per day.


### 2.3. Fruit Quality Evaluation

On the day of evaluation, fifteen fruits per treatment were randomly sampled to assess fruit quality. Progressive changes in skin color were monitored in a fixed set of fruits per treatment using a color difference meter (Minolta CR-400; Konica Minolta, Osaka, Japan) and reported based on Hunter’s scale. Firmness was analyzed using a texture analyzer (TA Plus Lloyd Instruments Ltd, Fareham, Hampshire, UK) at a speed of 2 mm/s with a plunger head of 5 mm in diameter. The total SSC of the samples was analyzed using a digital refractometer (PAL-1, ATAGO CO. LTD, Tokyo, Japan) and TA, expressed in grams of citric acid per 100 g of sample juice, was determined by titrating 5 mL of juice from the fruit with 0.1 N NaOH until a pH of 8.2 was reached. This procedure was performed using an auto pH titrator (TitroLine Easy; SCHOTT Instruments GmbH, Mainz, Germany). Fruit pitting was expressed as the percentage of fruits that exhibited pitting. The final reported pitting rate was obtained from three independent replicates per treatment per day.


### 2.4. Calyx Molding

Development of molding on calyx was recorded after 4, 8, and 12 d in fruits stored at 13°C; after 7 and 14 d in fruits stored at 4°C, and after 3 and 5 d at 20°C. For each treatment group, data reported are from three independent replicates (three boxes with 20 fruits each) per treatment per day.


### 2.5. RNA Isolation and cDNA Synthesis

The tomatoes stored at 13°C were sampled on days 0, 4, and 12, whereas those stored at 4°C were sampled on days 0, 7, and 14. Subsequently, five fruits were pooled from each sample, and the pericarp tissue was used for RNA isolation using the cetyltrimethylammonium bromide protocol [16]. First-strand cDNA was synthesized using a ReverTraAce kit (Toyobo, Japan).


### 2.6. Quantitative Real-Time PCR (qRT-PCR)

qRT-PCR was performed as described previously by Park et al. [17] using a CFX96 Touch™ Real-Time PCR detection system (Bio-Rad, Hercules, CA, USA). Amplification was performed using the iQ™ SYBR Green Supermix (Bio-Rad) with specific primers (Table S1). qRT-PCR was performed under the following conditions: 95°C for 30 s, followed by 40 cycles of 95°C for 10 s and 55°C or 58°C for 40 s. Relative gene expression was calculated using the 2−ΔΔCt method and normalized to the expression levels of the housekeeping genes actin and elongation factor 1 (EF1). The qRT-PCR gene expression analysis was performed using three biological replicates.


### 2.7. Enzyme Extraction

All enzyme extraction procedures were performed at 4°C. Powdered freeze-dried fruit tissues (0.1 g) were homogenized in 3 mL of extraction buffer (100 mM potassium phosphate buffer (pH 7.5), 1% polyvinylpolypyrrolidone, 2 mM EDTA-Na, and 1 mM PMSF). The slurry was centrifuged (15,000 rpm, 4°C, 30 min) in a refrigerated centrifuge (LaboGene 2236R, Gyrozen Co, Ltd, Daejeon, Korea) and filtered. Then, the activities of catalase (CAT), ascorbate peroxidase (APX), and superoxide dismutase (SOD) enzymes of the clear supernatants were immediately measured, as described below.


### 2.8. CAT Activity

CAT activity was measured using a method previously described by Beers and Sizer [18] with some modifications. The supernatant (50 µL) was mixed with 1 mL of sodium phosphate buffer (pH 7.0) and 500 µL of H2O2 (100 mM). Absorbance was recorded every 30 s for 5 min. One unit of CAT activity was defined as the change in absorbance by a factor of 0.01 at 240 nm per min. CAT activity was expressed as units per minute per gram of dry weight (U·min−1 g−1).


### 2.9. APX Activity

APX activity was measured according to Chen and Asada [19] with modifications. The supernatant (100 µL) was mixed with 1 mL of reaction mixture (100 mM potassium phosphate buffer, 0.1 mM H2O2, and 0.5 mM ascorbate). The absorbance of the mixture was measured at 290 nm every 10 s for 1 min using a spectrophotometer (Epoch 2; BioTek Industries, Highland Park, USA). One unit of APX activity was defined as a decrease in absorbance by a factor of 1 per minute under the assay conditions. APX activity was expressed as units per minute per gram of dry weight (U·min−1 g−1).


### 2.10. SOD Activity

SOD activity was assayed by measuring its ability to inhibit the photochemical reduction of nitro blue tetrazolium (NBT), as previously described by Rao et al., [20] with some modifications. The supernatant (20 µL) was mixed with 100 µL of 100 mM potassium phosphate buffer (pH 7.5) and 100 µL of the reaction mixture (13 mM methionine, 10 µM EDTA-Na2, 2 µM riboflavin, and 120 µM NBT). The absorbance of the mixture was measured at 560 nm using a spectrophotometer (Epoch 1; BioTek Industries, Highland Park, USA). One unit of SOD activity was defined as the amount of enzyme that produced the half-maximal inhibition of NBT reduction. SOD activity was expressed as units per minute per gram of dry weight (U min−1 g−1).


### 2.11. Statistical Analyses

Values are presented as the mean ± standard error. Differences between groups were evaluated by analysis of variance, and means were compared with Duncan’s multiple range test; statistical significance was set atP<0.05. Data were analyzed using SAS version 9.2 software (SAS Institute, Cary, NC, USA). For gene expression data, statistical analysis was performed using the t-test using Microsoft Excel v.2010 (Seattle, Washington). P-values <0.05 were considered statistically significant.


## 2.1. Plant Materials and Treatments

“Kamma” tomatoes (Solanum lycopersicum Mill.) grown in Jungyeum, South Korea, were harvested at the pink-red stage and transported to the laboratory. In our preliminary experiments, tomatoes were treated with 5, 10, and 15 ppm ClO2 for 15 min and/or 30 min. ClO2 at 5 ppm did not have any effect on CI and calyx molding (Figure S1). Additionally, ClO2 effect on CI and calyx molding was not significantly different when the treatment time was extended from 15 min to 30 min under our experimental conditions (data not shown). Hence, the fruits were treated with 10 or 15 ppm gaseous ClO2 (mixed with ambient air) using a ClO2 generator (CA300, South Korea) or left untreated (control) inside a commercial cardboard box for 15 min in a closed chamber. Twenty boxes containing 20 fruits each were used for each treatment. The ClO2 concentration in the closed chamber was verified using a built-in ClO2 meter. Following treatment, the fruit-containing boxes were covered with a plastic film and transferred to 13°C for 12 d or 4°C (cold storage) for 14 d followed by five days at 20°C (14 + 5 d; shelf-life conditions). During storage, the relative humidity was maintained at 90 ± 5%.


## 2.2. Gas Chromatography Analysis

The rate of respiration and ethylene production were analyzed using gas chromatography (Bruker 450-GC; Bruker Corp, Billerica, MA, USA). One milliliter of gas was sampled using a syringe from 2-L containers with four fruits from each treatment that had been sealed for 2 h. The injection and column temperatures were 110°C and 70°C, respectively. The thermal conductivity detector and flame ionization detector used for the ClO2 and ethylene measurements were set at 150°C and 250°C, respectively. ClO2 and ethylene measurements were obtained from three independent replicates per treatment per day.


## 2.3. Fruit Quality Evaluation

On the day of evaluation, fifteen fruits per treatment were randomly sampled to assess fruit quality. Progressive changes in skin color were monitored in a fixed set of fruits per treatment using a color difference meter (Minolta CR-400; Konica Minolta, Osaka, Japan) and reported based on Hunter’s scale. Firmness was analyzed using a texture analyzer (TA Plus Lloyd Instruments Ltd, Fareham, Hampshire, UK) at a speed of 2 mm/s with a plunger head of 5 mm in diameter. The total SSC of the samples was analyzed using a digital refractometer (PAL-1, ATAGO CO. LTD, Tokyo, Japan) and TA, expressed in grams of citric acid per 100 g of sample juice, was determined by titrating 5 mL of juice from the fruit with 0.1 N NaOH until a pH of 8.2 was reached. This procedure was performed using an auto pH titrator (TitroLine Easy; SCHOTT Instruments GmbH, Mainz, Germany). Fruit pitting was expressed as the percentage of fruits that exhibited pitting. The final reported pitting rate was obtained from three independent replicates per treatment per day.


## 2.4. Calyx Molding

Development of molding on calyx was recorded after 4, 8, and 12 d in fruits stored at 13°C; after 7 and 14 d in fruits stored at 4°C, and after 3 and 5 d at 20°C. For each treatment group, data reported are from three independent replicates (three boxes with 20 fruits each) per treatment per day.


## 2.5. RNA Isolation and cDNA Synthesis

The tomatoes stored at 13°C were sampled on days 0, 4, and 12, whereas those stored at 4°C were sampled on days 0, 7, and 14. Subsequently, five fruits were pooled from each sample, and the pericarp tissue was used for RNA isolation using the cetyltrimethylammonium bromide protocol [16]. First-strand cDNA was synthesized using a ReverTraAce kit (Toyobo, Japan).


## 2.6. Quantitative Real-Time PCR (qRT-PCR)

qRT-PCR was performed as described previously by Park et al. [17] using a CFX96 Touch™ Real-Time PCR detection system (Bio-Rad, Hercules, CA, USA). Amplification was performed using the iQ™ SYBR Green Supermix (Bio-Rad) with specific primers (Table S1). qRT-PCR was performed under the following conditions: 95°C for 30 s, followed by 40 cycles of 95°C for 10 s and 55°C or 58°C for 40 s. Relative gene expression was calculated using the 2−ΔΔCt method and normalized to the expression levels of the housekeeping genes actin and elongation factor 1 (EF1). The qRT-PCR gene expression analysis was performed using three biological replicates.


## 2.7. Enzyme Extraction

All enzyme extraction procedures were performed at 4°C. Powdered freeze-dried fruit tissues (0.1 g) were homogenized in 3 mL of extraction buffer (100 mM potassium phosphate buffer (pH 7.5), 1% polyvinylpolypyrrolidone, 2 mM EDTA-Na, and 1 mM PMSF). The slurry was centrifuged (15,000 rpm, 4°C, 30 min) in a refrigerated centrifuge (LaboGene 2236R, Gyrozen Co, Ltd, Daejeon, Korea) and filtered. Then, the activities of catalase (CAT), ascorbate peroxidase (APX), and superoxide dismutase (SOD) enzymes of the clear supernatants were immediately measured, as described below.


## 2.8. CAT Activity

CAT activity was measured using a method previously described by Beers and Sizer [18] with some modifications. The supernatant (50 µL) was mixed with 1 mL of sodium phosphate buffer (pH 7.0) and 500 µL of H2O2 (100 mM). Absorbance was recorded every 30 s for 5 min. One unit of CAT activity was defined as the change in absorbance by a factor of 0.01 at 240 nm per min. CAT activity was expressed as units per minute per gram of dry weight (U·min−1 g−1).


## 2.9. APX Activity

APX activity was measured according to Chen and Asada [19] with modifications. The supernatant (100 µL) was mixed with 1 mL of reaction mixture (100 mM potassium phosphate buffer, 0.1 mM H2O2, and 0.5 mM ascorbate). The absorbance of the mixture was measured at 290 nm every 10 s for 1 min using a spectrophotometer (Epoch 2; BioTek Industries, Highland Park, USA). One unit of APX activity was defined as a decrease in absorbance by a factor of 1 per minute under the assay conditions. APX activity was expressed as units per minute per gram of dry weight (U·min−1 g−1).


## 2.10. SOD Activity

SOD activity was assayed by measuring its ability to inhibit the photochemical reduction of nitro blue tetrazolium (NBT), as previously described by Rao et al., [20] with some modifications. The supernatant (20 µL) was mixed with 100 µL of 100 mM potassium phosphate buffer (pH 7.5) and 100 µL of the reaction mixture (13 mM methionine, 10 µM EDTA-Na2, 2 µM riboflavin, and 120 µM NBT). The absorbance of the mixture was measured at 560 nm using a spectrophotometer (Epoch 1; BioTek Industries, Highland Park, USA). One unit of SOD activity was defined as the amount of enzyme that produced the half-maximal inhibition of NBT reduction. SOD activity was expressed as units per minute per gram of dry weight (U min−1 g−1).


## 2.11. Statistical Analyses

Values are presented as the mean ± standard error. Differences between groups were evaluated by analysis of variance, and means were compared with Duncan’s multiple range test; statistical significance was set atP<0.05. Data were analyzed using SAS version 9.2 software (SAS Institute, Cary, NC, USA). For gene expression data, statistical analysis was performed using the t-test using Microsoft Excel v.2010 (Seattle, Washington). P-values <0.05 were considered statistically significant.


## 3. Results

### 3.1. Effect of ClO2 Treatment on Respiration Rate and Ethylene Production

Respiration is a major factor that contributes to postharvest quality losses in fruits. Treatment with ClO2 marginally reduced the rate of respiration in tomatoes at both storage temperatures (13°C and 4°C). At 13°C, the effect of ClO2 on the respiration rate was limited to 1 d during storage, and no difference between the treatments was observed thereafter (Figure 1(a)). However, ClO2-treated fruits recorded a lower respiration rate throughout the duration of storage at 4°C and at subsequent shelf-life conditions (Figure 1(b)). No significant difference in respiration rate was observed between the ClO2 treatment at concentrations 10 and 15 ppm (Figure 1). Further, ClO2 at 10 and 15 ppm concentrations did not affect ethylene production under either storage condition (Figures 1(c) and 1(d)).Figure 1
Effect of ClO2 treatment on the respiration rate of tomatoes stored at (a) 13°C for 12 days and (b) 4°C for 14 days followed by 5 days at 20°C, and ethylene production in tomatoes stored at (c) 13°C for 12 days and (d) at 4 °C for 14 days followed by 5 days at 20°C. Data are represented as the mean ± standard error (SE) of three replicates. Effect of ClO2 treatment on fruit quality.
(a)(b)(c)(d)Regardless of ClO2 treatment and storage temperature, firmness was observed to steadily decrease over time during storage. However, ClO2 treatment significantly delayed the loss of firmness in fruits stored at 13°C, as evidenced by the consistently higher firmness in ClO2-treated fruits than in control fruits (Table 1). In contrast, firmness was maintained at values similar to the control even upon ClO2 treatment during cold storage at 4°C. Further, no significant difference in firmness was observed between the treatment groups under shelf-life conditions (Table 2). ClO2 fumigation did not affect the SSC and TA of tomatoes at either storage temperature (Tables 1 and 2). Moreover, the surface color of the fruits was not altered upon ClO2 treatment, as evidenced by consistently similar Hue values among the different treatment groups (Tables 1 and 2).Table 1
Effect of ClO2 treatment on quality (firmness; SSC: soluble solid content; TA: titratable acidity) and color (Hue values) in fruits stored at 13°C for 12 days.
Storage period (days)TreatmentFirmness (N)SSC (%)TA (%)Hue value00 ppm ClO216.73 b4.0 a0.58 a63.61 a10 ppm ClO218.70 a4.1 a0.59 a63.69 a15 ppm ClO217.60 ab4.1 a0.56 a61.24 b40 ppm ClO213.59 b4.0 a0.48 a51.94 a10 ppm ClO215.16 a4.1 a0.49 a51.56 a15 ppm ClO214.05 ab4.2 a0.49 a51.13 a80 ppm ClO212.34 b4.4 a0.53 a47.56 a10 ppm ClO213.42 a4.1 b0.47 b47.53 a15 ppm ClO213.59 a4.2 ab0.49 b47.59 a120 ppm ClO211.79 a4.1 a0.43 a46.66 a10 ppm ClO212.35 a4.2 a0.46 a46.42 a15 ppm ClO211.97 a4.2 a0.45 a46.62 aTable 2
Effect of ClO2 treatment on quality (firmness; SSC: soluble solid content; TA: titratable acidity) and color (Hue values) in tomatoes stored at 4°C for 14 days followed by 5 days at 20°C.
Storage period (days)TreatmentFirmness (N)SSC (Brix)TA (%)Hue value00 ppm ClO₂16.73 b4.03 a0.58 a63.61 a10 ppm ClO₂18.70 a4.07 a0.59 a63.69 a15 ppm ClO₂16.73 b4.07 a0.56 a61.24 b70 ppm ClO₂15.10 a4.13 a0.52 a56.90 a10 ppm ClO₂15.34 a4.13 a0.53 a57.87 a15 ppm ClO₂15.19 a4.10 a0.50 a57.39 a140 ppm ClO₂15.03 a4.20 a0.50 a57.21 a10 ppm ClO₂12.82 b4.03 ab0.50 a58.24 a15 ppm ClO₂14.53 a3.97 b0.54 a57.49 a14 + 50 ppm ClO₂10.44 a3.97 a0.46 a52.02 a10 ppm ClO₂10.74 a4.03 a0.45 a52.66 a15 ppm ClO₂10.89 a3.97 a0.38 b52.27 aValues within a column with different letters are significantly different atP<0.05, as determined by Duncan’s multiple range test.Values within a column with different letters are significantly different atP<0.05, as determined by Duncan’s multiple range test.


### 3.2. Effects of ClO2 Treatment Calyx Molding

No calyx molding was observed in tomatoes after 4 days at 13°C (data not shown), whereas 35% of control fruits showed calyx molding after 8 days at 13°C; this was restricted to only ∼14% in the 15 ppm ClO2 treatment group (Figure 2(b)). ClO2 at 10 ppm showed an intermediate effect on calyx molding at 13°C (Figures 2(a) and 2(b)). No visible molding was observed on the calyces of tomatoes during cold storage (data not shown). However, upon transferring them to shelf-life conditions, molding rapidly appeared in all treatment groups. ClO2 at 15 ppm was effective in controlling the calyx molding under shelf-life conditions (Figures 2(a) and 2(c)). On day 3 at 20°C, 15 ppm ClO2-treated fruits demonstrated almost 50% less calyx molding than the control fruits (Figure 2(c)). However, the 10 ppm ClO2 treatment did not significantly affect calyx molding under shelf-life conditions. Furthermore, the impact of ClO2 treatment on calyx molding diminished on the final day of storage at both temperatures (Figures 2(b) and 2(c)).Figure 2
Effect of ClO2 treatment on calyx molding in tomato. (a) Representative images of tomatoes with differential calyx molding after various treatments and storage conditions. (b) Calyx molding in tomatoes stored at 13°C and (c) 4°C for 14 days followed by 5 days at 20°C. Graphs plotted represent mean values ± standard error (SE) of three replicated measurements. Different lowercase letters in the graph indicate statistically significant differences at P<0.05.
(a)(b)(c)


### 3.3. Effects of ClO2 on CI

Prolonged exposure to cold temperatures can cause pitting, a classical symptom of CI, in tomatoes. No pitting was observed during cold storage in our experiments. However, regardless of the treatment, pitting was observed after transferring the tomatoes from cold storage to shelf-life conditions. Tomatoes treated with 15 ppm ClO2 were less prone to pitting under shelf-life conditions than the control fruits (Figure 3). By the end of the shelf-life, more than 70% of the control fruits showed pitting, compared to only 40% of the 15 ppm ClO2-treated fruits (Figure 3). ClO2 at 10 ppm did not have any significant (P<0.05) effect on pitting in tomatoes (Figure 3).Figure 3
Effect of ClO2 treatment on chilling injury in tomatoes stored at 4°C for 14 days followed by 5 days at 20°C. Graphs plotted represent mean values ± standard error (SE) of three replicated measurements. Different lowercase letters in the graph indicate statistically significant differences at P<0.05.


### 3.4. ClO2-Induced Gene Expression Profiles

To elucidate the mechanisms of action of ClO2, we examined the expression profile of cell wall–related (pectinesterase 1 (PE1), pectin lyase (PL), and glucanases), ripening-related(ethylene response factors; ERF.B3/C1/E1), and antioxidant genes (APX, CAT, and peroxidase 42) in fruits stored at 13°C. In addition to the higher firmness observed in ClO2-treated fruits, the transcripts of the PE1 gene were reduced in response to 15 ppm ClO2 treatment on day 0; however, no difference in the expression profile between the treatment groups was observed thereafter (Figure 4(a)). Notably, the expression profiles of glucanase and PL were not altered by the ClO2 treatment (Figure 4(a)).Figure 4
Effect of ClO2 treatment on expression of genes related to (a) cell wall disassembly (PE1: pectin esterase 1; PL: pectate lyase), (b) ripening-associated ethylene response factors (ERF.B3/C1/E1), and (c) antioxidant defense (APX: ascorbate peroxidase; CAT: catalase; peroxidase 42) in fruits stored at 13°C for 12 days. The bar represents the mean ± standard error of three biological replicates. Different lowercase letters in the graph indicate statistically significant differences at P<0.05. ClO2-induced antioxidant activity in tomatoes.
(a)(b)(c)We then evaluated genes associated with ripening process (ERF.B3/C1/E1) and found ERF.B3 to be downregulated upon ClO2 treatment until day 4 at 13°C storage conditions. Additionally, ERF.C1 transcripts were also suppressed in response to ClO2 treatment on day 0 at 13°C significantly. ERF.E1 levels were reduced by ClO2 treatment but not statistically different (Figure 4(b)). This downregulation of ERF.B3 and ERF.C3 levels may influence ripening-associated changes in tomatoes. The expression levels of antioxidant genes APX, CAT, and peroxidase 42 were induced in ClO2-treated fruits when stored at 13°C. While the induction of these genes continued until day 4 at 13°C, APX was restricted to day 0. By the end of the storage period, no difference was observed in the expression levels of these genes between the treatment groups (Figure 4(c)).Chilling temperatures destroy the balance between reactive oxygen species formation and antioxidant defense mechanisms, which causes oxidatively induced CI [1]. In our experiments, ClO2 treatment resulted in significantly lower surface pitting (Figure 3). Hence, we examined the effect of ClO2 treatment on the activity of the antioxidant enzymes APX, CA, and SOD during cold storage and shelf-life conditions. During 4°C storage, the activity of APX was not altered (Figure 5(a)), whereas SOD activity in 15 ppm ClO2-treated fruits remained stable or even marginally declined, suggesting that a source of H2O2 not linked to SOD activity may exist (Figure 5(c)). CAT activity was induced upon ClO2 treatment during storage at 4°C (except on day 14) and shelf-life conditions, suggesting the potential involvement of this antioxidant enzyme in ClO2-induced CI inhibition (Figure 5(c)).Figure 5
Effect of ClO2 treatment on activity levels of antioxidant enzymes. (a) Ascorbate peroxidase (APX), (b) catalase (CAT), and (c) superoxide dismutase (SOD) in fruits stored at 4°C for 14 days followed by 5 days at 20°C. Data are shown as the mean ± standard error (SE) of three replicates. Different lowercase letters in the graph indicate statistically significant differences at P<0.05.
(a)(b)(c)


## 3.1. Effect of ClO2 Treatment on Respiration Rate and Ethylene Production

Respiration is a major factor that contributes to postharvest quality losses in fruits. Treatment with ClO2 marginally reduced the rate of respiration in tomatoes at both storage temperatures (13°C and 4°C). At 13°C, the effect of ClO2 on the respiration rate was limited to 1 d during storage, and no difference between the treatments was observed thereafter (Figure 1(a)). However, ClO2-treated fruits recorded a lower respiration rate throughout the duration of storage at 4°C and at subsequent shelf-life conditions (Figure 1(b)). No significant difference in respiration rate was observed between the ClO2 treatment at concentrations 10 and 15 ppm (Figure 1). Further, ClO2 at 10 and 15 ppm concentrations did not affect ethylene production under either storage condition (Figures 1(c) and 1(d)).Figure 1
Effect of ClO2 treatment on the respiration rate of tomatoes stored at (a) 13°C for 12 days and (b) 4°C for 14 days followed by 5 days at 20°C, and ethylene production in tomatoes stored at (c) 13°C for 12 days and (d) at 4 °C for 14 days followed by 5 days at 20°C. Data are represented as the mean ± standard error (SE) of three replicates. Effect of ClO2 treatment on fruit quality.
(a)(b)(c)(d)Regardless of ClO2 treatment and storage temperature, firmness was observed to steadily decrease over time during storage. However, ClO2 treatment significantly delayed the loss of firmness in fruits stored at 13°C, as evidenced by the consistently higher firmness in ClO2-treated fruits than in control fruits (Table 1). In contrast, firmness was maintained at values similar to the control even upon ClO2 treatment during cold storage at 4°C. Further, no significant difference in firmness was observed between the treatment groups under shelf-life conditions (Table 2). ClO2 fumigation did not affect the SSC and TA of tomatoes at either storage temperature (Tables 1 and 2). Moreover, the surface color of the fruits was not altered upon ClO2 treatment, as evidenced by consistently similar Hue values among the different treatment groups (Tables 1 and 2).Table 1
Effect of ClO2 treatment on quality (firmness; SSC: soluble solid content; TA: titratable acidity) and color (Hue values) in fruits stored at 13°C for 12 days.
Storage period (days)TreatmentFirmness (N)SSC (%)TA (%)Hue value00 ppm ClO216.73 b4.0 a0.58 a63.61 a10 ppm ClO218.70 a4.1 a0.59 a63.69 a15 ppm ClO217.60 ab4.1 a0.56 a61.24 b40 ppm ClO213.59 b4.0 a0.48 a51.94 a10 ppm ClO215.16 a4.1 a0.49 a51.56 a15 ppm ClO214.05 ab4.2 a0.49 a51.13 a80 ppm ClO212.34 b4.4 a0.53 a47.56 a10 ppm ClO213.42 a4.1 b0.47 b47.53 a15 ppm ClO213.59 a4.2 ab0.49 b47.59 a120 ppm ClO211.79 a4.1 a0.43 a46.66 a10 ppm ClO212.35 a4.2 a0.46 a46.42 a15 ppm ClO211.97 a4.2 a0.45 a46.62 aTable 2
Effect of ClO2 treatment on quality (firmness; SSC: soluble solid content; TA: titratable acidity) and color (Hue values) in tomatoes stored at 4°C for 14 days followed by 5 days at 20°C.
Storage period (days)TreatmentFirmness (N)SSC (Brix)TA (%)Hue value00 ppm ClO₂16.73 b4.03 a0.58 a63.61 a10 ppm ClO₂18.70 a4.07 a0.59 a63.69 a15 ppm ClO₂16.73 b4.07 a0.56 a61.24 b70 ppm ClO₂15.10 a4.13 a0.52 a56.90 a10 ppm ClO₂15.34 a4.13 a0.53 a57.87 a15 ppm ClO₂15.19 a4.10 a0.50 a57.39 a140 ppm ClO₂15.03 a4.20 a0.50 a57.21 a10 ppm ClO₂12.82 b4.03 ab0.50 a58.24 a15 ppm ClO₂14.53 a3.97 b0.54 a57.49 a14 + 50 ppm ClO₂10.44 a3.97 a0.46 a52.02 a10 ppm ClO₂10.74 a4.03 a0.45 a52.66 a15 ppm ClO₂10.89 a3.97 a0.38 b52.27 aValues within a column with different letters are significantly different atP<0.05, as determined by Duncan’s multiple range test.Values within a column with different letters are significantly different atP<0.05, as determined by Duncan’s multiple range test.


## 3.2. Effects of ClO2 Treatment Calyx Molding

No calyx molding was observed in tomatoes after 4 days at 13°C (data not shown), whereas 35% of control fruits showed calyx molding after 8 days at 13°C; this was restricted to only ∼14% in the 15 ppm ClO2 treatment group (Figure 2(b)). ClO2 at 10 ppm showed an intermediate effect on calyx molding at 13°C (Figures 2(a) and 2(b)). No visible molding was observed on the calyces of tomatoes during cold storage (data not shown). However, upon transferring them to shelf-life conditions, molding rapidly appeared in all treatment groups. ClO2 at 15 ppm was effective in controlling the calyx molding under shelf-life conditions (Figures 2(a) and 2(c)). On day 3 at 20°C, 15 ppm ClO2-treated fruits demonstrated almost 50% less calyx molding than the control fruits (Figure 2(c)). However, the 10 ppm ClO2 treatment did not significantly affect calyx molding under shelf-life conditions. Furthermore, the impact of ClO2 treatment on calyx molding diminished on the final day of storage at both temperatures (Figures 2(b) and 2(c)).Figure 2
Effect of ClO2 treatment on calyx molding in tomato. (a) Representative images of tomatoes with differential calyx molding after various treatments and storage conditions. (b) Calyx molding in tomatoes stored at 13°C and (c) 4°C for 14 days followed by 5 days at 20°C. Graphs plotted represent mean values ± standard error (SE) of three replicated measurements. Different lowercase letters in the graph indicate statistically significant differences at P<0.05.
(a)(b)(c)


## 3.3. Effects of ClO2 on CI

Prolonged exposure to cold temperatures can cause pitting, a classical symptom of CI, in tomatoes. No pitting was observed during cold storage in our experiments. However, regardless of the treatment, pitting was observed after transferring the tomatoes from cold storage to shelf-life conditions. Tomatoes treated with 15 ppm ClO2 were less prone to pitting under shelf-life conditions than the control fruits (Figure 3). By the end of the shelf-life, more than 70% of the control fruits showed pitting, compared to only 40% of the 15 ppm ClO2-treated fruits (Figure 3). ClO2 at 10 ppm did not have any significant (P<0.05) effect on pitting in tomatoes (Figure 3).Figure 3
Effect of ClO2 treatment on chilling injury in tomatoes stored at 4°C for 14 days followed by 5 days at 20°C. Graphs plotted represent mean values ± standard error (SE) of three replicated measurements. Different lowercase letters in the graph indicate statistically significant differences at P<0.05.


## 3.4. ClO2-Induced Gene Expression Profiles

To elucidate the mechanisms of action of ClO2, we examined the expression profile of cell wall–related (pectinesterase 1 (PE1), pectin lyase (PL), and glucanases), ripening-related(ethylene response factors; ERF.B3/C1/E1), and antioxidant genes (APX, CAT, and peroxidase 42) in fruits stored at 13°C. In addition to the higher firmness observed in ClO2-treated fruits, the transcripts of the PE1 gene were reduced in response to 15 ppm ClO2 treatment on day 0; however, no difference in the expression profile between the treatment groups was observed thereafter (Figure 4(a)). Notably, the expression profiles of glucanase and PL were not altered by the ClO2 treatment (Figure 4(a)).Figure 4
Effect of ClO2 treatment on expression of genes related to (a) cell wall disassembly (PE1: pectin esterase 1; PL: pectate lyase), (b) ripening-associated ethylene response factors (ERF.B3/C1/E1), and (c) antioxidant defense (APX: ascorbate peroxidase; CAT: catalase; peroxidase 42) in fruits stored at 13°C for 12 days. The bar represents the mean ± standard error of three biological replicates. Different lowercase letters in the graph indicate statistically significant differences at P<0.05. ClO2-induced antioxidant activity in tomatoes.
(a)(b)(c)We then evaluated genes associated with ripening process (ERF.B3/C1/E1) and found ERF.B3 to be downregulated upon ClO2 treatment until day 4 at 13°C storage conditions. Additionally, ERF.C1 transcripts were also suppressed in response to ClO2 treatment on day 0 at 13°C significantly. ERF.E1 levels were reduced by ClO2 treatment but not statistically different (Figure 4(b)). This downregulation of ERF.B3 and ERF.C3 levels may influence ripening-associated changes in tomatoes. The expression levels of antioxidant genes APX, CAT, and peroxidase 42 were induced in ClO2-treated fruits when stored at 13°C. While the induction of these genes continued until day 4 at 13°C, APX was restricted to day 0. By the end of the storage period, no difference was observed in the expression levels of these genes between the treatment groups (Figure 4(c)).Chilling temperatures destroy the balance between reactive oxygen species formation and antioxidant defense mechanisms, which causes oxidatively induced CI [1]. In our experiments, ClO2 treatment resulted in significantly lower surface pitting (Figure 3). Hence, we examined the effect of ClO2 treatment on the activity of the antioxidant enzymes APX, CA, and SOD during cold storage and shelf-life conditions. During 4°C storage, the activity of APX was not altered (Figure 5(a)), whereas SOD activity in 15 ppm ClO2-treated fruits remained stable or even marginally declined, suggesting that a source of H2O2 not linked to SOD activity may exist (Figure 5(c)). CAT activity was induced upon ClO2 treatment during storage at 4°C (except on day 14) and shelf-life conditions, suggesting the potential involvement of this antioxidant enzyme in ClO2-induced CI inhibition (Figure 5(c)).Figure 5
Effect of ClO2 treatment on activity levels of antioxidant enzymes. (a) Ascorbate peroxidase (APX), (b) catalase (CAT), and (c) superoxide dismutase (SOD) in fruits stored at 4°C for 14 days followed by 5 days at 20°C. Data are shown as the mean ± standard error (SE) of three replicates. Different lowercase letters in the graph indicate statistically significant differences at P<0.05.
(a)(b)(c)


## 4. Discussion

Respiration and ethylene biosynthesis are the key determinants of the shelf-life of tomatoes. Hence, postharvest handling methods of tomatoes primarily focus on controlling the respiratory metabolism and regulating the climacteric rise of ethylene. ClO2 treatment is known to influence the rate of respiration and ethylene production in fresh produce [8]. For instance, gaseous ClO2 application reduced the respiration rate in fresh-cut “Hami” melon and green peppers [21, 22]. The respiratory activity, ripeness, and senescence of peaches are controlled by ClO2 treatment [23]. Our results corroborate these findings as treatment with ClO2 reduced the respiration rate in tomatoes at both storage temperatures (13 and 4°C); however, the effect was more pronounced at 4°C (Figure 1). Storage of produce at low temperatures is known to inhibit respiration. Hence, the continued lower respiration rates observed here in ClO2-treated fruits under shelf-life conditions indicate a synergetic effect of ClO2 and cold storage (Figure 1). Guo et al. [24] reported that gaseous ClO2 reduced ethylene biosynthesis in mature green tomatoes. However, in the present study, ethylene production was not affected. One possible reason is that the climacteric rise of ethylene in the pink-stage tomato used in this experiment has already attained before ClO2 treatment.Higher storage temperatures favor ripening processes that result in the loss of firmness and changes in fruit quality. We found that ClO2-treated fruits were firmer than the control fruits throughout storage at 13°C (Table 1), indicating a positive effect of ClO2 treatment on the regulation of firmness. Firmness was also maintained during 4°C storage (Table 2). These results are in agreement with previous reports: Islam et al. [13] reported that 12 h of gaseous ClO2 treatment can yield tomatoes with greater firmness. Similarly, ClO2 delayed the softening of grape tomatoes [25], and a ClO2self-releasing sheet was effective in maintaining the firmness of cherry tomatoes [26]. ClO2 treatment has also been shown to maintain firmness in other produce, such as strawberries, blueberries, and litchi [10, 27–29]. Interestingly, ClO2 had no effect on firmness at 20°C, as we observed no difference in firmness between the ClO2-treated and control groups under shelf-life conditions (Table 2). This indicates a temperature-specificity in ClO2 action. Indeed, in a previous study, ClO2 was more effective in maintaining blueberry firmness at 10°C than 20°C [10]. Further, the ClO2 effect was not apparent on the firmness of strawberries at 20°C [29].Loss of firmness in fruits is related to biochemical alterations in cell wall components and/or a loss of turgor pressure in cells due to water loss.PE1, PL, and glucanase are known genes involved in cell wall–associated biochemical changes during fruit ripening [30]. PE1 is specifically expressed during the ripening process in strawberries [31]. Silencing of PE1 in tomatoes resulted in increased levels of soluble solids and decreased levels of soluble polyuronides in cell walls, which enhanced fruit rigidity [32]. Transient genetic manipulation of PE1 in strawberries by overexpression and silencing significantly influences the fruit firmness [33]. In this study, PE1 transcripts were found to be reduced in response to 15 ppm ClO2 treatment on day 0, indicating their involvement in regulating firmness (Figure 4). PLs are pectin-modifying enzymes that cleave glycosidic bonds via a β-elimination mechanism between galacturonosyl residues [34, 35]. PL is dominant during fruit maturation, and RNA interference of SlPL results in enhanced fruit firmness and changes in pericarp cells [36]. Further suppression of the PL gene in tomatoes enhances fruit firmness and extends the shelf-life without affecting fruit quality [36, 37]. Glucanase is the key enzyme involved in the hydrolytic cleavage of 1,3 beta-D glucosidic linkages in beta-1,3 glucans. Glucanases are expressed during the ripening and maturation of tomatoes [38]. Previously, the ripening-associated expression of glucanase has been shown to be cultivar-specific in three banana cultivars [39]. However, the expression of glucanase and PL was not affected by ClO2 treatment under our experimental conditions, suggesting that glucanase and PL may not be involved in the regulation of fruit firmness by ClO2 treatment (Figure 4).ClO2 treatment is known to retain color and quality in fresh produce [8]. Active packing of strawberries with ClO2-generating sachets maintained SSC, TA, and color at 4°C [28]. Further, it has been reported that color, TA, and SSC are not significantly affected in mangoes treated with ClO2 [40]. Similarly, we did not find any significant effect of ClO2 treatment on SSC and TA of tomatoes. ClO2 has previously been shown to downregulate ethylene biosynthetic pathway genes, including ACS2, ACO1, and ACO3, in mature green tomatoes and melons [22, 24]. In our experiment, the expression of ACS2 and ACS4 was decreased in ClO2-treated mature green and breaker-stage tomatoes (Figure S2) but not in the pink-stage tomatoes (Figures 1(c) and 1(d)). Hence, we checked the expression of ethylene responsive genes. ERFs are the downstream components of ethylene signaling that mediate ethylene-dependent gene transcription and are among the largest families of plant transcription factors. ERF.B3, ERF.C1, and ERF.E1are induced during tomato ripening [41]. Suppression of ERF.B3 has been shown to delay the onset of ripening in tomatoes [42]. In our study, ERF.B3, ERF.C1, and ERF E1 levels were downregulated upon ClO2 treatment at the beginning of storage (Figure 4). Therefore, the suppression of ERFs may contribute to slowing maturation processes in ClO2-treated fruits.The accumulation of reactive oxygen species is associated with fruit ripening and causes over ripening [43, 44]. Postharvest application of antioxidant compounds can effectively extend the shelf-life of tomatoes and other crops by reducing reactive oxygen species [45, 46]. ClO2 treatment altered the redox status and enhanced the antioxidant capacity in longan fruit, preventing pericarp browning [47]. Chumyam et al. [48] reported that ClO2 could restore the redox balance, leading to a reduction and delay in fruit senescence. In our experiments, ClO2 treatment upregulated the antioxidant genes APX, CAT, and peroxidase42 at 13°C storage conditions (Figure 4). An alteration in redox status in tomatoes after ClO2 treatment may induce the expression of these antioxidant genes. The stable redox balance in ClO2-treated tomatoes may contribute to delayed fruit maturation. Our results suggest that the ClO2 treatment may have the potential to slowing fruit maturation via suppressing the expression of PE1 and ERF.B3/C1 and induction of APX, CAT, and peroxidase 42 transcripts. However, further research is needed to fully discover the role of ClO2 treatment on ripening-associated genes in tomatoes.We verified the efficacy of ClO2 treatment on calyx molding, considering that lower storage temperatures inhibit microbial growth. In this study, no visible mold was observed on calyces of tomatoes stored at 4°C, whereas high storage temperatures of 13°C and 20°C (shelf-life) promoted calyx molding (Figure 3). However, ClO2 at 15 ppm effectively controlled molding on the calyces at both storage temperatures. It has been reported that ClO2 can inactivate microbial infestation during postharvest storage [7, 8]. Bhagat et al. [49] demonstrated that treating tomatoes with 0.5 ppm ClO2 gas for 12 min delayed the growth of natural microflora and extended its shelf-life by 7 days when stored at 22°C. Further, ClO2 treatment significantly delayed the development of white molds and black spots in Roma tomatoes [50]. Similarly, ClO2 treatment reduced total aerobic bacterial, yeast, and mold counts approximately by 1 log-scale in grapefruit after 6 weeks of storage at 10°C [25]. The impact of ClO2 on microbial activity diminishes over time during storage [8]. For instance, ClO2-generating pads effectively reduced the growth of yeast and molds for 8 days, but no effect was observed after a 12-day storage period at 2°C [40]. Similarly, the impact of ClO2 treatment on calyx molding diminished by the end of storage at both temperatures (Figure 3).Chilling temperatures trigger the accumulation of reactive oxygen species, which can initiate lipid peroxidation and cause oxidative damage to the cell membrane and eventual tissue deterioration [1]. Surface pitting is a visual symptom of CI in tomatoes. It is known that CI may develop during exposure to low temperatures, but the symptoms usually appear after the transfer of the produce to nonchilling temperature conditions [1]. We observed that surface pitting in tomatoes during shelf-life following cold storage was significantly inhibited by 15 ppm ClO2 treatment (Figure 3). Plants have developed antioxidant defense mechanisms to diminish the deleterious effects of reactive oxygen species on cells. These mechanisms include antioxidant enzymes SOD, APX, CAT, peroxidases, and several nonenzymatic antioxidants. Cold stress induces the activity of APX, CAT, and SOD during cold storage and shelf-life. Interestingly, our results indicate a higher activity of CAT in 15 ppm ClO2-treated fruits, suggesting that higher antioxidant activity after ClO2 treatment may contribute to the inhibition of pitting in tomatoes (Figure 5). ClO2 has been shown to increase the total antioxidant capacity of produce [51]. Wang et al. reported that ClO2 application induces peroxidase and CAT activities in barley roots and aerial parts during seed germination. ClO2 significantly enhances the activities of SOD, CAT, and APX in the longan pericarp [47]. Reduced CI and microbial incidence in tomato can retain greater fruit quality and improve storability.


## 5. Conclusions

In summary, the application of 15 ppm gaseous ClO2 for 15 min was found to be effective in controlling the loss of firmness and maintaining the quality of tomatoes stored at 13°C and 4°C by controlling their respiratory metabolism and microbial incidence. Ripening-relatedERFs were suppressed by ClO2. Furthermore, the expression and activity of antioxidant genes and enzymes were modulated upon ClO2 treatment. These findings will be useful for further understanding the molecular mechanism of action of ClO2 for quality maintenance. Taken together, short-term 15 ppm ClO2 treatment represents an efficient method to improve storability of tomatoes [53].



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*Source: 3818269-2022-12-06.xml*