Alleviation of Vascular Bundle Browning in the Japanese Pear ‘Rinka’ by Preharvest Application of Ethephon

Abstract

Vascular bundle browning (VBB) of the flesh tissue of the Japanese pear ‘Rinka’ was observed when the fruit was stored at 4°C. This symptom was not observed in mature fruit at harvest nor after storage at 25°C for one week following harvest; however, VBB frequently appeared when fruit was stored at 4°C. More than 50% of the fruit had the VBB symptom after storage at 4°C for one week. Further incidences and degrees of the symptoms were observed when the fruit was stored at 4°C for longer periods. No other cultivars have shown this kind of disorder induced by a short period of cold storage, indicating that VBB is a novel type of chilling injury (CI) in pears. The application of a 100 ppm ethephon (2-chloroethylphosphonic acid) solution at approximately 100 days after full flowering (DAF) significantly reduced the occurrence of VBB. Ethephon advances maturation and leads to an early harvest, causing a significant reduction in VBB, but ethephon-treated fruit harvested at the same harvest time as untreated fruit showed a reduced incidence of VBB. Treatment with 1-methylcyclopropene (1-MCP), an inhibitor of ethylene perception, increased the VBB development during cold storage and a subsequent storage period at 25°C. These results suggest that ethylene has a positive effect on alleviating the VBB development in the Japanese pear ‘Rinka’.

Introduction

Japanese pear (Pyrus pyrifolia (Burm.f.) Nakai), also called Asian pear or Nashi pear, is cultivated throughout most East Asian countries, including Japan, China, and Korea, as well as other countries, such as Australia, New Zealand, and the United States. The fruit is very juicy, has a crisp texture, and does not need to ripen after harvest, unlike European pears (Pyrus communis L.). ‘Rinka’ is a new cultivar released in 2013 by the National Agriculture and Food Research Organization (NARO), Ibaraki, Japan. ‘Rinka’ is an early-maturing cultivar, and the harvest time and fruit quality are almost comparable with those of ‘Kosui’, the principal cultivar in Japan (Saito et al., 2020). Recently, ‘Kosui’ trees cultivated in the southwestern regions (warmer climate regions) of Japan have been observed to have dead flower buds in the spring (Ito et al., 2018; Sakamoto et al., 2017; Tominaga et al., 2019), whereas the rate of dead flower bud occurrence in ‘Rinka’ is much lower than that in ‘Kosui’ (Saito et al., 2020; Sugiura, 2019). Therefore, ‘Rinka’ is expected to be a suitable cultivar for adapting to a warmer climate; however, vascular bundle browning (VBB) of the fruit’s flesh in cold storage has presented a problem (Saito et al., 2020).

Low temperatures are usually effective for maintaining postharvest fruit quality and extended shelf life (Wills and Golding, 2016). However, depending on the chilling tolerance of the fruit, chilling injury (CI) occurs in some fruits (Lurie and Crisosto, 2005; Patel et al., 2016) including pears (Franck et al., 2007; Nath and Panwar, 2018). On the other hand, Japanese pear can typically be stored for more than one month at 0–5°C without any CI symptoms or low temperature-associated disorders (Bal, 2020; Choi et al., 2015; Itai and Tanahashi, 2008; Li and Wang, 2009). To our knowledge, no other Japanese pear cultivars have been reported to exhibit VBB in cold storage, and little is known about the symptom.

Ethylene plays a predominant role during fruit maturation and ripening (Barry and Giovannoni, 2007). Moreover, ethylene is also involved in regulating cold tolerance in postharvest fruit. Ethephon (2-chloroethylphosphonic acid), a plant hormone regulator that is converted to ethylene by plants, is registered as a maturity accelerator for agricultural use on Japanese pears in Japan, and it has been reported that its application at approximately 100 days after full flowering (DAF) accelerates the maturity and advances the harvest date of Japanese pears (Aoki and Okada, 1976; Hiramoto et al., 2020; Kimura et al., 1977; Takase et al., 1982). On the other hand, the effects of ethylene on cold tolerance vary in different species. It was shown that ethylene increases CI symptoms in avocado (Pesis et al., 2002) and plum (Candan et al., 2008), whereas ethylene alleviates CI symptoms in peach (Zhou et al., 2001), and Chinese pears (Pyrus bretschneideri Rehder) (Wei et al., 2019). Similarly, it was also shown that 1-methylcyclopropene (1-MCP), an ethylene action inhibitor, had either a positive or a negative effect on CI symptoms (Cheng et al., 2015; Fan et al., 2002; Jiang et al., 2004; Salvador et al., 2004).

In this study, we observed the VBB disorder that developed during storage at 4°C in ‘Rinka’ fruit. In addition, we studied the efficacy of ethephon application or 1-MCP treatment on VBB occurrence to clarify the relationship between ethylene and VBB.

Materials and Methods

Plant materials and treatments

The experiments were conducted using the Japanese pear ‘Rinka’ planted in an experimental orchard (andosols) at the Institute of Fruit Tree and Tea Science, NARO (Ibaraki, Japan). For ethephon treatment, two trees on to which ‘Rinka’ was top grafted onto ‘Kosui’ grafted onto P. pyrifolia seedlings in 2012, were used. These trees were 44 years old in 2019. Trees were grown using a pergola training system with horizontal wire trellises. Each tree had three main branches. One branch per tree was treated with ethephon, and the other two branches were left untreated as controls. An ethephon solution (100 ppm) was prepared from a stock solution of 100 g·L⁻¹ ethephon (Ethrel 10; Nissan Chemical, Corp., Japan) that was dissolved in distilled water and sprayed at approximately 100 days after full flowering (DAF). For 1-MCP treatment, mature fruit harvested from 8-year-old ‘Rinka’ trees grafted onto Pyrus betulaefolia Bunge seedlings were used. Fruit were placed in a 56-L plastic container and treated with 1 ppm 1-MCP (SmartFresh^TM; AgroFresh Inc., USA) for 18 h at 23°C. For the control, fruit were left in air for 18 h at 23°C.

Trial establishment

Experiment 1: Effect of ethephon treatment, cold storage periods, and shelf-life following cold storage on the incidence of VBB (2019)

Ethephon-treated fruit were harvested on 1 August 2019 (106 DAF). Control fruit were harvested on 15 August 2019 (121 DAF). Harvest timing was determined based on the ground color of fruit with a color chart score of between “2” to “3” (Yamazaki and Suzuki, 1980) using a color chart for ground color of Japanese pear (Fujihira Industry Co., Ltd., Japan). Harvested fruit from the treatments and controls were uniformly divided into six groups by their skin ground colors. There were 13 control samples and 10 ethephon treatment samples in each group. In each treatment, the fruit of one group was analyzed on the harvest day, and the remaining groups were packed into corrugated fiberboard boxes. One group per treatment was transferred to a 25°C storage room with 80–85% relative humidity (RH) for one week immediately after harvest to evaluate shelf life. The other four groups per treatment were placed in a storage room at 4°C with 85–95% RH. Two groups per treatment were removed from the 4°C storage room after either two or three weeks. One group was analyzed immediately after cold storage, whereas the other group was examined after a stay of one week at 25°C with 80–85% RH following the cold storage period.

Experiment 2: Effects of ethephon treatment, harvest date, and storage period on the incidence of VBB (2020)

To examine the effect of ethephon treatment on fruit growth during maturation, twenty-four fruit per treatment were randomly selected to measure fruit width after the ethephon treatment. Fruit width was measured with digital calipers (Mitsutoyo Corp., Japan) at intervals of 4 to 12 days until the commercial harvest time for the control fruit (134 DAF).

To analyze the effect of harvest date, ethephon-treated fruit were harvested twice on 7 August 2020 (123 DAF) (Ethephon) at the commercial harvest time and on 18 August 2020 (134 DAF) at the commercial harvest time for untreated fruit (Ethephon-late harvest). Untreated fruit were harvested on 18 August 2020 (134 DAF) at the commercial harvest time (Control). Commercial harvest time was determined when the ground color of fruit achieved a color chart score of “3”. Fruit from each treatment/harvest time were divided into three groups. One group was analyzed on the harvest day. The other two groups were packed as described for Experiment 1 and stored at 4°C for 7 or 15 days.

Experiment 3: Effect of 1-MCP treatment on the incidence of VBB (2021)

Mature fruit were harvested on 11 August 2021 (132 DAF) and divided into five groups. One group was analyzed on the harvested day. Two groups were used for 1-MCP treatments and the other two groups were used as controls. After 1-MCP treatment, fruit were packed as described for Experiment 1 and stored at 4°C with 85–95% RH for two weeks. One group for each treatment was analyzed just after the cold storage, and another group was analyzed after a stay of one week at 25°C with 80–85% RH following the cold storage period.

Fruit quality analysis

Fruit was individually weighed. Flesh firmness was measured using a penetrometer (FT011; Fujihira Industry) with an 8 mm plunger after removing a small disk of skin from each side of the fruit. Firmness values were recorded as the force in Newtons (N) required to insert the penetrometer. A juice squeezer (RE-29301; Atago Co., Ltd., Japan) was used to extract juice from paired flesh samples taken from opposite sides of the fruit. The soluble solids content (SSC) and pH of each fruit juice sample were determined using a refractometer (PR-101; Atago) and pH meter (C-73, Phasion; Az One Corp., Japan), respectively. Extracted juice was also collected in a 1.5 mL-micro tube and stored at −20°C for analysis of sugar composition. Juice samples were dissolved, centrifuged at 15,000 rpm for 10 min, and the supernatant liquid was diluted with water. Mannitol was added as an internal standard. The diluted mixture was passed through a StrataSax cartridge (Shimadzu GLC Ltd., Japan). Aliquots of the eluate were separated by high-performance liquid chromatography (HPLC, Prominence; Shimadzu Corp., Japan) equipped with a refractive index detector (RID-10A; Shimadzu) and a Rezex^TM RCM-Monosaccharide Ca column (300 mm × 7.8 mm, Phenomenex, USA). The column was kept at 80°C and eluted with water at a flow rate of 0.8 mL·min⁻¹. Sugars were identified by their retention times and quantified from a standard curve using authentic standards.

Ethylene production was determined by withdrawing a 1-mL gas sample from the headspace of a 1.25-L air-tight glass chamber into which fruit had been incubated for 1 h at 25°C. Ethylene concentration in the gas sample was determined using a gas chromatograph (GC-2014A; Shimadzu) equipped with an activated alumina column and a flame ionization detector. The injection, column, and detector temperatures were 120, 80, and 120°C, respectively. The rate of ethylene production was expressed as μL·kg⁻¹·h⁻¹.

Measurement of VBB severity

After collecting the juice samples, the remaining fruit were cut transversely at the equatorial plane, and the severity of the VBB symptoms was assessed visually according to the following four-stage scale: 0, absent; 1, slight (less than five scattered browning spots), 2, moderate (spread on less than 50% of the plane) 3, severe (spread on more than 50% of the plane). Since VBB symptoms were scattered evenly in the middle of the flesh in preliminary tests, VBB severity was evaluated at the equatorial aspect.

Statistical analysis

Statistical analysis was performed using JMP^® ver. 14 software (SAS Institute Inc., USA). Data were subjected to a one-way analysis of variance (ANOVA) to determine the significance of differences. The means were evaluated using Tukey-Kramer’s multiple comparison test (pairwise comparisons) or Dunnett’s multiple comparison test (comparisons to harvest day) at P < 0.05.

Results

Experiment 1: Effects of ethephon treatment, cold storage periods, and shelf-life following cold storage on the incidence of VBB

In the ethephon-treated and control groups, no VBB symptoms were detected at harvest or after 25°C storage for a week after harvest (Fig. 1). VBB was found in fruit exposed to cold temperature, and the ethephon treatment significantly reduced the VBB incidence in this group. More than 50% of control fruit showed VBB symptoms, including severe symptoms, after two weeks in cold storage. On the other hand, 70% of the ethephon-treated fruit were healthy, and the remaining affected fruit had symptoms corresponding to the slight level (Fig. 1). The exposure of control fruit to 25°C for a week after cold storage made the symptoms slightly more severe. Even in fruit with severe symptoms of VBB, browning appeared only in the vascular strands and not in the mesocarp cells adjacent to the brown vascular bundles when the fruit was stored at 4°C and 25°C.

Fig. 1

The percentage of fruit exhibiting none to severe vascular bundle browning (VBB) symptoms in control fruit (C) and ethephon-treated fruit (E) (Experiment 1). The VBB index: 0, absent; 1, slight (less than five scattered browning areas); 2, moderate (spread to less than 50% of the plane); 3, severe (spread to more than 50%). There were 13 control samples and 10 ethephon samples for each sampling date.

VBB was observed in the peripheral vascular bundles scattered in the middle of the flesh tissue (Fig. 2), and not in the main ventral and dorsal bundles near the core or the flesh close to the skin. Furthermore, flesh close to the VBBs was water-soaked in fruit with severe symptoms, but it did not discolor even when the fruit was kept at 25°C after cold storage.

Fig. 2

Vascular bundle browning symptom appearing in ‘Rinka’ pear during cold storage.

Fruit quality is described in Table 1. Ethephon-treated fruit were relatively small and had significantly lower SSC values than the control. The pH tended to increase after harvest in the control, but did not change or tended to decrease in the ethephon-treated samples. Thus, the pH in ethephon-treated fruit was lower than the control after harvest.

Table 1

Fruit quality of control fruit and ethephon-treated fruit in cold storage and subsequent storage at 25°C (Experiment 1).

Ethylene production in both treatments was very low or at an undetectable level at harvest (< 0.05 μL·kg⁻¹·h⁻¹). Ethylene levels remained very low in fruit kept either in cold storage or stored at 25°C (data not shown).

Experiment 2: Effects of ethephon treatment, harvest date, and storage period on the incidence of VBB

In the control and ethephon-treated fruit, VBB was detected after cold storage for one week and became more severe after longer storage periods (Fig. 3). This result indicated that VBB appeared after a short period in cold storage and ethephon alleviated the injury. Comparing the same harvest time for the ethephon-late group and the control, the ethephon-late group had a lower incidence of VBB than the control (Fig. 3). Comparing the two harvest times for ethephon-treated fruit, a commercial harvest and a late harvest, VBB was slightly more severe in fruit from the late harvest group than the commercial harvest group.

Fig. 3

The percentage of fruit exhibiting none to severe vascular bundle browning (VBB) symptoms in control fruit (C), ethephon-treated fruit (E), and ethephon-treated fruit at late harvest (E-LH) (Experiment 2). The VBB index: 0, absent; 1, slight (less than five scattered browning areas); 2, moderate (spread to less than 50% of the plane); 3, severe (spread to more than 50%). Numbers in parentheses indicate the number of samples tested.

A summary of fruit quality is shown in Table 2. Ethephon treatment reduced fruit size, SSC, and pH when fruit were harvested at a commercial harvest time (Table 2). When ethephon-treated fruit remained on trees until the control harvest time, fruit quality parameters, including fruit size, SSC, and pH, were identical to the control. The size of ethephon-treated fruit ranged from almost the same size to slightly larger than the untreated control; however, the differences were not significant (data not shown). Early harvest resulted in smaller ethylene-treated fruit at the commercial harvest time. Ethephon-treated fruit contained significantly less sucrose and sorbitol, but had more fructose and glucose at commercial harvest (Fig. 4). Notably, the sucrose content of ethephon-treated fruit was less than one-third of the control; however, the sucrose content of ethephon-treated and control fruit was almost equal when the ethephon-treated fruit was left on the trees until the control fruit harvest time. The sugar content and composition did not change during cold storage for 15 days (data not shown).

Table 2

Fruit quality of control and ethephon-treated fruit at commercial harvest (Ethephon) and late harvest (Ethephon-late harvest) (Experiment 2).

Fig. 4

Total and specific sugar contents of control fruit (Control), ethephon-treated fruit (Ethephon), and ethephon-treated fruit at late harvest (Ethephon-LH) on harvest day (Experiment 2). Control and Ethephon fruit were harvested at the commercial harvest time. Ethephon-LH fruit were harvested at the same time as Control fruit. Values with the same letters on the same label are not significantly different by Tukey-Kramer’s test (P < 0.05).

Experiment 3: Effect of 1-MCP treatment on the incidence of VBB

To clarify the role of ethylene on the incidence of VBB, 1-MCP treatment was given to the fruit after harvest. 1-MCP treatment promoted VBB incidence during cold storage for two weeks (Fig. 5). Furthermore, all the fruit tested showed severe symptoms when kept at 25°C for one week after 1-MCP treatment. 1-MCP treatment did not affect any fruit quality parameters (fruit weight, firmness, SSC, pH) after the cold storage and the subsequent storage at 25°C for one week (data not shown).

Fig. 5

The percentage of fruit exhibiting none to severe vascular bundle browning (VBB) disorders in control fruit and 1-MCP treated fruit after two weeks’ storage at 4°C and subsequent storage at 25°C for one week (Experiment 3). The VBB index: 0, absent; 1, slight (less than five scattered browning areas); 2, moderate (spread to less than 50% of the plane); 3, severe (spread to more than 50%). Numbers in parentheses indicate the number of samples tested.

Discussion

Pears are known to be susceptible to various physiological disorders after harvest, including superficial scald, bitter pit, internal breakdown, and black end (Franck et al., 2007; Li and Wang, 2009; Nath and Panwar, 2018); however, VBB has not been reported previously. Similar VBB symptoms have been observed in avocado (Woolf et al., 2005) and pineapple (Luengwilai et al., 2016) during cold storage; however, these symptoms were accompanied by flesh browning that was not observed in ‘Rinka’. As VBB did not discolor flesh around the vascular bundles or cause outer flesh browning in this study, it was impossible to identify fruit with the disorder, even in fruit with severe symptoms, from the external appearance. In addition, VBB did not affect the taste or odor of ‘Rinka’ either, although a lack of appetite often resulted from the visual effects of the symptom.

In ‘Rinka’, VBB symptoms did not appear in fruit at harvest or after 7-day storage at 25°C following harvest (Fig. 1). VBB was frequently found in fruit after cold storage for more than a week, and longer periods of cold storage resulted in higher rates and more severe incidences (Figs. 1 and 3). These results indicated that VBB appeared in response to cold temperature and was a type of chilling injury. Although differences between cultivars were not compared in this study, no other pears have been shown to have such a cold-associated disorder after short-term cold exposure of less than a week. This cultivar’s response to cold temperature may be a unique characteristic of ‘Rinka’. The degree of VBB symptoms slightly increased at 25°C after a week following cold storage, although VBB was not observed at 25°C following harvest (Figs. 1 and 5). This result indicates that VBB symptoms may develop slowly even at room temperature after the initial appearance of VBB.

The ethephon treatment at around 100 DAF significantly reduced VBB incidence (Figs. 1 and 3). Ethephon advanced the fruit to maturity and led to an earlier harvest time than the control. The shortened maturation period was related to resistance to the disorder because late-harvested fruit had an increased VBB incidence compared to fruit harvested at two different harvest times (commercial harvest and late harvest) in the ethephon-treated fruit (Fig. 3). These results suggested that shortening the maturation period contributes to a reduction in the incidence of VBB; however, the decrease in VBB cannot be explained only by a shorter maturation period based on the lower incidence in ethephon-treated fruit than control fruit harvested on the same day (Fig. 3). 1-MCP treated fruit showed more severe symptoms and higher incidences than control fruit during the cold storage and subsequent storage at 25°C (Fig. 5). Therefore, we propose that other factors, directly or indirectly related to ethylene, affect the incidence of VBB induced by cold temperature.

Ethylene is known to regulate fruit maturation in Japanese pear. However, the amount of ethylene production in Japanese pear fruit varies depending on the cultivar (Itai et al., 1999; Itai and Fujita, 2008). ‘Rinka’ fruit produced very little ethylene (0.1 < μL·kg⁻¹·h⁻¹) during cold storage and storage at 25°C following harvest or cold storage and is judged to be a low-level type, the same as ‘Hosui’, ‘Akizuki’, and ‘Nijisseiki’ (Itai and Fujita, 2008). In these cultivars with very low ethylene production, changes in fruit skin color, flesh firmness, and sugar accumulation during fruit maturation occur naturally, and the fruit has been shown to respond to exogeneous ethylene or 1-MCP (Hiramoto et al., 2020; Itai and Tanahashi, 2008; Kimura et al., 1977; Ma et al., 2016; Mitani et al., 2017; Wang et al., 2018). Thus, a low level of ethylene contributes to regulation of the fruit maturation and ripening process in Japanese pear. However, the degrees of regulation for each maturation and ripening process including skin color change, firmness loss, and sugar accumulation may be different. Ethephon treatment at 110DAF has been shown to increase the SSC and affect skin color, but not fruit enlargement in ‘Hosui’ (Kimura et al., 1977) and ‘Akizuki’ (Hiramoto et al., 2020). Ma et al. (2016) noted that sugar accumulation and acidity were likely ethylene-independent, while fruit firmness and chlorophyll degradation in the fruit skin were ethylene dependent in ‘Niitaka’. In our study, ethephon treatment resulted in advancing the harvest time of ‘Rinka’ by about two weeks, a result consistent with previous studies; however, the treated fruit tended to be smaller in size and have a lower SSC than the control (Tables 1 and 2). Furthermore, our study also demonstrated that the sugar composition was significantly different between the control and ethephon-treated fruit. Ethephon-treated fruit had more glucose and fructose and less sucrose and sorbitol than untreated fruit (Fig. 4). Sucrose accumulates rapidly during the late-maturation stage of Japanese pear concomitant with increasing activities of sucrose synthase (SS; EC 2.4.1.13) and sucrose-phosphate synthase (SPS; EC 2.4.1.14) (Moriguchi et al., 1992; Tanase and Yamaki, 2000; Zhang et al., 2014). These results suggested that ethephon treatment at around 100 DAF in ‘Rinka’ can be more effective in promoting the changes in fruit skin color than increases in fruit size, SSC or sucrose accumulation.

In conclusion, this study showed that Japanese ‘Rinka’ pear fruit exhibited VBB symptoms after cold temperature storage at 4°C for at least one week. The results indicate that VBB appeared after fruit exposure to cold temperature and is, thus, a cold-associated disorder. As no other cultivars have shown this kind of disorder in pear, ‘Rinka’ is probably very sensitive to cold temperature. Ethephon treatment at around 100 DAF effectively reduced the occurrence of VBB disorder, when the fruit were harvested at the same stage determined by the ground color. Therefore, early harvest may reduce the incidence. However, ethephon alleviated the incidence of VBB even when the fruit was harvested at the same time as the control. In addition, 1-MCP promoted the VBB incidence during the storage at 4°C and the subsequent storage at 25°C. Our study suggests that other factors related to ethylene besides a shorter maturation period contribute to reducing VBB incidence in ‘Rinka’.

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