2023 Volume 92 Issue 4 Pages 439-450
To elucidate the inhibitory effect of ethephon on the occurrence of water-soaked disorder in Japanese pear ‘Akizuki’ (Pyrus pyrifolia Nakai) fruit, we sprayed fruits with 100 mg·L−1 ethephon solution approximately 120 DAFB in the 2012, 2014, 2015, 2016, and 2017 seasons. Ethephon treatment reduced the incidence and degree of water-soaked disorder at commercial harvest time. There was no significant difference in fruit size or soluble solids content between the ethephon-treated fruits and the control fruits, except for fruit size in the 2012 season. During the nine-day storage period after harvest, water-soaked disorder did not develop in either the ethephon-treated fruits or the control fruits. In addition, flesh firmness and soluble solids content did not differ between the ethephon-treated fruits and the control fruits during the storage period. Ethylene production increased sharply in the ethephon-treated fruits a few days after the ethephon treatment, in contrast to the control fruits. Sucrose content in the ethephon-treated fruits was higher than that in the control fruits on day 10 after treatment. Sorbitol content in the ethephon-treated fruits was lower than that in the control fruits on days 10 and 20 after treatment. Water-soluble, Na2CO3-soluble, and total pectin contents were not affected by the ethephon treatment. Hemicellulose, cellulose, and starch contents in the ethephon-treated fruits decreased earlier than those in the control fruits. Sucrose content was lower, but sorbitol and glucose contents were higher, in water-soaked tissue than in sound tissue. These results suggest that ethephon treatment rapidly induces changes in the contents of fruit’s internal components and accelerates fruit maturation, thereby alleviating factors that cause water-soaked disorder in ‘Akizuki’.
‘Akizuki’, a cultivar of Japanese pear (Pyrus pyrifolia Nakai), is known for its excellent fruit quality including large size, high level of sweetness, good shape, and high yield. However, the development of physiological flesh disorders such as water-soaked disorder (Fig. 1) and cork spot like disorder in ‘Akizuki’ fruit has been reported (Hayama et al., 2017; Matsuda et al., 2006; Nakamura, 2011). A physiological disorder characterized by a watery translucent area is called “watercore in Japanese pear” (Kajiura et al., 1976). Watercore has been observed in some cultivars such as ‘Housui’ (Kajiura et al., 1976), ‘Niitaka’ (Kimura et al., 1993), and ‘Akibae’ (Tanabe et al., 2001). The water-soaked disorder in ‘Akizuki’ seems to be analogous to the watercore in Japanese pear in that their symptoms are characterized by a watery translucent area. However, water-soaked disorder is also characterized by vascular bundle browning and brown flesh in water-soaked tissue. Therefore, the water-soaked disorder in ‘Akizuki’ is considered to be different from watercore (Nakamura, 2011).
Water-soaked disorder in the Japanese pear ‘Akizuki’.
Gibberellin (GA) treatment increased the incidence of watercore in Japanese pear ‘Housui’ (Inomata et al., 1993; Sakuma et al., 1995; Kato and Kitaguchi, 2014), ‘Niitaka’ (Kimura et al., 1993), and ‘Akibae’ (Tamura et al., 2003). Conversely, forchlorfenuron (CPPU) treatment decreased the incidence of watercore in ‘Housui’ (Ohara et al., 2001). Meanwhile, GA and CPPU treatment had no effect on the development of water-soaked disorder in ‘Akizuki’ (Ohkawa et al., 2009).
Ethephon (2-chloroethylphosphonic acid), an ethylene-releasing agent, accelerates fruit maturation and ripening in a number of horticultural crops including rabbiteye blueberry (Ban et al., 2007), kiwifruit (Ampa et al., 2017; Ohara et al., 1997), yellow passionfruit (Dozier et al., 1991), fig (Crane et al., 1970), and Japanese pear (Aoki and Okada, 1976; Kimura et al., 1977; Mitani et al., 2017; Nishimoto, 1983). Ethephon treatment increased the incidence of watercore in Japanese pear ‘Housui’ (Inomata et al., 1993). Therefore, expecting that ethephon would promote the development of water-soaked disorder in ‘Akizuki’, we treated ‘Akizuki’ fruit with ethephon in a preliminary experiment and found that it tended to suppress the occurrence of this flesh disorder.
In this study, we investigated the inhibitory effect of ethephon on the occurrence of water-soaked disorder in ‘Akizuki’. In addition, changes in ethylene production and the contents of starch, sugar, and cell wall components after ethephon treatment were investigated to clarify the mechanism of action underlying the effect of ethephon on this flesh disorder.
Four-year-old (as of 2012) Japanese pear ‘Akizuki’ (Pyrus pyrifolia Nakai) trees on seedling rootstocks of P. pyrifolia planted in 60-L plastic pots filled with Andosol and planted in the ground in an open field at Chiba University (35.8°N, 139.9°E) were used. The trees planted in plastic pots were watered with 400 to 800 mL of water per tree every hour from 5:00 am to 6:00 pm, and chemical fertilizer containing N:P:K = 10:10:10 was applied monthly at a rate of 30 g per tree from April to June. As for the trees planted in the ground, organic and chemical fertilizer containing N:P:K = 6:8:2 was applied at a rate of 170 g·m−2 in December every year.
In the 2012 season, ethephon solution was applied to fruits of six trees planted in plastic pots 124 days after full bloom (DAFB) (Aug. 16). In the 2014 season, ethephon solution was applied to fruits of nine trees planted in plastic pots 120 DAFB (Aug. 7). In the 2015 season, ethephon solution was applied to fruits of 36 trees planted in plastic pots 122 DAFB (Aug. 9) and fruits of nine trees planted in the ground 120 DAFB (Aug. 6). In the 2016 season, ethephon solution was applied to fruits of nine trees planted in the ground 121 DAFB (Aug. 9). In the 2017 season, ethephon solution was applied to fruits of nine trees planted in the ground 123 DAFB (Aug. 15). In all experiments, ethephon solution was prepared from a stock solution of 100 g·L−1 (w/v) ethephon (Ethrel 10; Nissan Chemical, Corp., Japan) diluted with deionized water to 100 mg·L−1 and applied to approximately half of the fruits on the tree using a hand-held sprayer. Other fruits on the trees were untreated and used as controls.
2. Effects of ethephon treatment on the development of water-soaked disorder, harvest time, and fruit quality parametersExperiments were conducted in the 2012, 2014, 2015, 2016, and 2017 seasons. Approximately 40 fruits in the 2012 and 2014 seasons and 30 fruits in the 2015 season were harvested from trees planted in plastic pots that received the different treatments. In addition, approximately 30 fruits in the 2016 and 2017 seasons were harvested from trees planted in the ground that received the different treatments. Individual fruit tagged at treatment was harvested when its skin color reached color chart index (CCI) 3 (commercial harvest time), using the color chart for ‘Akizuki’ developed by a research group in Ibaraki Prefecture (Tahira, 2007). The weight, transverse diameter, longitudinal length, soluble solids content, and degree of water-soaked disorder were measured. Harvested fruits were sliced lengthwise into 2- to 3-mm-thick sections, and water-soaked disorder on the sections was examined. The water-soaked disorder index (WSDI) was determined on the basis of the number and size of symptoms in each fruit as follows: 0, none; 1, one to two symptoms measuring less than 1 cm2; 2, three to five symptoms measuring less than 1 cm2; 3, six or more symptoms measuring less than 1 cm2 or one or more symptoms measuring more than 1 cm2. Soluble solids content in juice was determined by a refractometer (ATC-1E; Atago Co., Ltd., Japan).
3. Changes in the occurrence of water-soaked disorderExperiments were conducted in the 2017 season. Thirty untreated fruits were harvested from trees planted in plastic pots at different maturation stages: 131 DAFB, 141 DAFB, and 151 DAFB, and at commercial harvest time (CCI 3). The WSDIs of these fruits were determined in the same manner as that mentioned in section 2.
4. Effects of ethephon treatment on fruit quality and the development of water-soaked disorder after harvestExperiments were conducted in the 2015 season. Ethephon-treated fruits (CCI 3) and control fruits (CCI 3) were harvested 155 DAFB and 162 DAFB, respectively, from trees planted on the ground and immediately stored in a room at 25°C. After storage for 0, 3, 6, and 9 days, 18 or 19 fruits were selected from the harvested ethephon-treated fruits and control fruits to measure flesh firmness, soluble solids content, and WSDI. Flesh firmness was measured using a penetrometer (FT011; Facchini, Italy) with an 11 mm plunger after removing a small disk of skin from each side of the fruit. Soluble solids content and WSDI were determined in the same manner as that mentioned in section 2.
5. Effects of ethephon treatment on fruit internal components and ethylene production 1) Sugar contents and cell wall componentsExperiments were performed in the 2012 season. Ten control fruits and 10 ethephon-treated fruits were collected on days 0, 10, and 20 after ethephon treatment and at commercial harvest time (CCI 3). Commercial harvest times of the ethephon-treated fruits and the control fruits were 28 days after treatment (152 DAFB) and 33 days after treatment (157 DAFB), respectively. Collected fruits were stored at −30°C until use for analysis of sugar contents and cell wall components.
For the analysis of sugar content, a frozen flesh sample (10 g) of sound tissue was homogenized in 80% (v/v) ethanol. The homogenate was boiled for 15 min and subjected to vacuum filtration, and the residue was washed with 80% (v/v) ethanol. After ethanol was evaporated in vacuo, the residue was dissolved in 50 mL of deionized water and passed through a 0.45 μm cellulose acetate filter unit (DISMIC-13; Toyo Roshi Kaisha, Japan). Sugar content in the filtrate was analyzed with a high-performance liquid chromatograph equipped with a refractive index detector (L-3300; Hitachi, Japan) and a Shodex Asahipak NH2P-50 column (Showa Denko Co., Ltd., Japan) that was eluted with 75% (v/v) acetonitrile. The results are presented as means of three biological replicates. Each replicate was prepared from three to four fruits, that is, a total of 10 fruits per treatment.
For the analysis of cell wall components, ethanol insoluble solids (EIS) were prepared by the method of Chun et al. (2003). A frozen flesh sample (10 g) of sound tissue was homogenized at full speed for 1 min using a homogenizer (NS-52; Microtec Co., Ltd., Japan) in ethanol to a final concentration of 80% (v/v). The homogenate was boiled for 20 min to inactivate enzymes and filtered through Miracloth (Calbiochem, USA). The residue was sequentially washed with 100% ethanol and acetone, and then dried at 37°C for 3 h.
Pectin and hemicellulose fractions were isolated according to Chun et al. (2003). The EIS (0.1 g) were continually extracted once with 25 mL of deionized water (water-soluble pectin fraction), twice with 25 mL of 50 mM trans-1,2-cyclohexanediaminetetraacetic acid (CDTA) in 50 mM sodium acetate (pH 6.5) (CDTA-soluble pectin fraction) for 12 h at room temperature, and once with 25 mL of 50 mM Na2CO3 containing 20 mM sodium borohydride (Na2CO3-soluble pectin fraction) for 6 h at 1°C. The Na2CO3 extract was neutralized with acetic acid and the residue was washed with 200 mL of 80% (v/v) ethanol and acetone for hemicellulose extraction. At the end of each extraction, the mixture was centrifuged (18,000 × g 10 min) and the supernatant was filtered through a glass microfiber filter (GC-50; Toyo Roshi Kaisha). After extraction with Na2CO3, the residue was used for the extraction of hemicellulose and cellulose. Uronic acid content in each pectin fraction was detected by the m-hydroxydiphenyl method (Blumenkrantz and Asboe-Hansen, 1973).
For hemicellulose extraction, starch was removed from the residue by incubating it in 25 mL of 100 mM Hepes solution (pH 6.9) containing α-amylase (30 units·mg−1) (Wako, Japan), 0.02% (w/v) NaN3 at 37°C for 20 h. Polysaccharide that precipitated by adding 80 mL of 100% ethanol was kept at −20°C for at least 4 h and then filtered through GC-50. The residue was washed with 80% (v/v) ethanol and acetone and then extracted sequentially with 25 mL each of 4% (w/v) KOH (4% KOH hemicellulose fraction) and 24% (w/v) KOH (24% KOH hemicellulose fraction), containing 0.1% (w/v) sodium borohydride, and continuously stirred for 24 h at room temperature. At the end of each extraction, the mixture was filtered through GC-50. Both KOH extracts were neutralized with acetic acid and used to analyze total sugar content. After extraction with 24% KOH, the residue was used for cellulose extraction.
Cellulose was determined by the method of Tabuchi and Matsumoto (2001). The residue was washed with 80% (v/v) ethanol and acetone, 0.1 M acetic acid, and deionized water, and then extracted with 4 mL of 72% (v/v) sulfuric acid for 1 h. To the mixture was added 60 mL of deionized water, and incubation was carried out at 100°C for 3 h (cellulose fraction).
Total sugar contents in hemicellulose and cellulose fractions were detected by the phenol-sulfuric acid method (Dubois et al., 1956).
The results are presented as means of three biological replicates. Each replicate was prepared from three to four fruits, that is, a total of 10 fruits per cultivar.
2) Microscopic observation of starch grains in vascular bundles and flesh cells after ethephon treatmentThe experiment was conducted in the 2012 season. Ethephon-treated fruits and control fruits were collected on day 10 after ethephon treatment and at commercial harvest time (CCI 3). The harvest time was the same as that mentioned in subsection 4. 1). After collection, a small block of tissue was excised from sound flesh and fixed in fixative solution (80% ethanol:acetic acid = 95:5, v/v). After fixation, the tissue block was dehydrated with an ethanol-butyl alcohol series and embedded in paraffin. The sample block was sliced into 20-μm-thick sections using a microtome and a section was fixed on a slide and deparaffinized with xylene. The slide was observed under a scanning electron microscope (SEM) (TM-1000; Hitachi High-Technologies Co., Ltd., Japan).
3) Ethylene productionThe experiment was conducted in the 2014 season. Five ethephon-treated fruits and 5 control fruits were collected at 2- to 10-day intervals from 0 to 36 days after ethephon treatment. Each fruit was enclosed in a jar (1.7 L) and kept at 25°C for 2 h. After this time, a 1-mL headspace gas sample was taken from the jar and injected into a gas chromatograph (GC-2014; Shimadzu, Japan) fitted with a flame ionization detector (150°C) and a glass column (Porapak Q; GL Sciences Inc., Japan) at 50°C. The results are shown as means of five biological replicates (five fruits).
4) Starch contentThe experiment was conducted in the 2014 season. Ten ethephon-treated fruits and 10 control fruits were collected on days 0, 5, 10, 15, and 20 after ethephon treatment and at commercial harvest time (CCI 3). The commercial harvest times of the ethephon-treated fruits and the control fruits were 28 days after treatment (148 DAFB) and 36 days after treatment (156 DAFB), respectively. Collected fruits were immediately stored at −30°C until analysis. A frozen flesh sample (10 g) of sound tissue was homogenized in 80% (v/v) ethanol. The homogenate was boiled for 20 min and subjected to vacuum filtration. The residue was washed with 80% (v/v) ethanol and dried at 80°C. The residue (0.03 g) was boiled for 10 min in 2 mL of deionized water and then cooled rapidly in ice. To the mixture was added 2.5 mL of 9.2 M perchloric acid. The mixture was stirred for 20 min and centrifuged at 10,000 × g for 10 min. The supernatant was collected and transferred to a 50 mL volumetric flask. The above process was repeated and the supernatant was combined with the first supernatant. The combined supernatant was made up to 50 mL with deionized water and used to determine the starch content. A 1-mL sample was added to a test tube containing 1 mL of 5% (w/v) phenol solution and 5 mL of concentrated sulfuric acid. Sugar content in the sample was quantified by a UV/VIS spectrophotometer (U-2920; Hitachi, Japan) at 490 nm wavelength. The results are presented as means of three biological replicates. Each replicate was prepared from three to four fruits, that is, a total of 10 fruits per cultivar.
6. Analysis of sugar and starch contents in water-soaked tissue and sound tissueThe experiment was conducted in the 2014 season. Ten control fruits with severe water-soaked disorder were collected at commercial harvest time (CCI 3). Water-soaked tissue and sound tissue were excised from the same fruit and stored separately at −30°C until use for analysis of sugar and starch contents. Sugar and starch extraction and analysis were conducted in the same manner as that mentioned in subsections 4. 1) and 4. 4), respectively. The results are presented as means of three biological replicates. Each replicate was prepared from three to four fruits, that is, a total of 10 fruits per cultivar.
Ethephon treatment significantly reduced WSDI in the 2012, 2014, 2015, 2016, and 2017 seasons (Table 1). In these seasons, 63 to 95% of the ethephon-treated fruits showed no signs or symptoms of water-soaked disorder. On the other hand, 63 to 82% of the control fruits showed symptoms of water-soaked disorder. Ethephon treatment in the 2012, 2014, 2015, 2016, and 2017 seasons advanced harvest time by 5 to 8 days compared with the control (data not shown). In the 2012 season, the ethephon-treated fruits were smaller than the control fruits, but their soluble solids contents did not differ (Table 2). In the 2014, 2015, 2016, and 2017 seasons, there was no significant difference in fruit size or soluble solids content between the ethephon-treated fruits and the control fruits. In both ethephon-treated fruits and control fruits, fruit size was small and weight was low in the 2012 and 2014 seasons, with larger and heavier fruit in the 2016 season compared with the other seasons. On the other hand, soluble solids content was high in the 2012 and 2014 seasons and low in the 2016 season compared with the other seasons.
Effects of 100 mg·L−1 ethephon treatment on development of water-soaked disorder in the Japanese pear ‘Akizuki’.
Effects of 100 mg·L−1 ethephon treatment on the Japanese pear ‘Akizuki’ fruit quality parameters at commercial harvest time.
Water-soaked disorder was not observed 131 DAFB in untreated fruits harvested from trees planted in plastic pots in the 2017 season (Fig. 2). This flesh disorder was occasionally observed in the untreated fruits 141 DAFB. WSDI and the incidence of water-soaked disorder increased dramatically from 141 DAFB to commercial harvest time.
Changes in the occurrence of water-soaked disorder accompanying fruit maturation in the Japanese pear ‘Akizuki’. Different letters indicate significant differences among water-soaked disorder indexes at four fruit maturation stages as determined by the Steel-Dwass multiple comparison test (P < 0.01).
WSDI was significantly lower in the ethephon-treated fruits than in the control fruits from 0 to 9 days after storage, and did not significantly change during the experimental period in either the ethephon-treated fruits or control fruits (Fig. 3). Flesh firmness and soluble solids content were not significantly different between the ethephon-treated fruits and the control fruits during the 9-day storage period.
Effects of 100 mg·L−1 ethephon treatment 120 DAFB on the development of water-soaked disorder (A), soluble solids content (B), and flesh firmness (C) after harvest in the 2015 season. Ethephon-treated fruits and control fruits were harvested 155 DAFB and 162 DAFB, respectively. Different letters (uppercase: control, lowercase: ethephon treatment) indicate significant differences among four storage days for each treatment as determined by Tukey’s multiple range test (P < 0.05). NS and * indicate no significant difference and significant difference between control and ethephon treatments for each day after storage at P < 0.05, respectively, by t-test.
Sucrose content of the ethephon-treated fruits was higher than that of the control fruits on day 10 after treatment (Fig. 4A). Glucose and fructose contents of the ethephon-treated fruits were higher than those of the control fruits on day 20 after treatment (Fig. 4B, C). Sorbitol content of the ethephon-treated fruits was lower than that of the control fruits on days 10 and 20 after treatment (Fig. 4D). Sucrose, glucose, fructose, and sorbitol contents were not significantly different between the ethephon-treated fruit and the control fruit at their respective harvest times.
Effects of 100 mg·L−1 ethephon treatment 124 DAFB on sucrose (A), glucose (B), fructose (C), and sorbitol (D) contents in ‘Akizuki’ fruit in the 2012 season. The ethephon-treated fruits and the control fruits were harvested 155 DAFB and 162 DAFB, respectively. NS and * indicate no significant difference and significant difference at P < 0.05, respectively, by t-test.
The water-soluble, Na2CO3-soluble, and total (total amount of water-soluble, CDTA-soluble, and Na2CO3-soluble pectin) pectin contents were not influenced by ethephon treatment throughout the experimental period (Fig. 5A, C, D). On the other hand, the CDTA-soluble pectin content was higher in the ethephon-treated fruits than in the control fruits at commercial harvest time (Fig. 5B). The 4% KOH-soluble and total (total amount of 4% KOH-soluble and 24% KOH-soluble hemicellulose) hemicellulose contents in the ethephon-treated fruits decreased sharply immediately after ethephon treatment and were significantly lower than those of the control fruits on days 10 and 20 after ethephon treatment (Fig. 5E, G). Although the 24% KOH-soluble hemicellulose content did not change dramatically, it was significantly lower in the ethephon-treated fruits than in the control fruits on days 10 and 20 after treatment (Fig. 5F). Cellulose content was significantly lower in the ethephon-treated fruits than in the control fruits on day 10 after treatment (Fig. 5H). All pectin contents, except for CDTA-soluble pectin content, and hemicellulose and cellulose contents did not differ between the ethephon-treated fruits and the control fruits at harvest time.
Effects of 100 mg·L−1 ethephon treatment 124 DAFB on pectin (water-soluble: A, CDTA-soluble: B, Na2CO3-soluble: C, total: D), hemicellulose (4% KOH-soluble: E, 24% KOH-soluble: F, total: G), and cellulose (H) contents in ‘Akizuki’ fruit in the 2012 season. The ethephon-treated fruits and the control fruits were harvested 155 DAFB and 162 DAFB, respectively. NS indicates no significant difference. * and ** indicate significant difference at P < 0.05 and P < 0.01, respectively, by t-test.
In the control fruits, many starch grains were present in the vascular bundles and the flesh cells on day 10 after treatment, whereas there were only a few starch grains remaining in the vascular bundles and the flesh cells at harvest time (Fig. 6A, C). On the other hand, in the ethephon-treated fruits, many starch grains disappeared on day 10 after treatment, and very few starch grains were observed in the vascular bundles and the flesh cells around vascular bundle at harvest time (Fig. 6B, D).
SEM micrographs of vascular bundle sections and flesh tissues on day 10 after treatment (A: control, B: ethephon treatment) and at harvest time (C: control, D: ethephon treatment) in the 2012 season. Ethephon treatment was performed 124 DAFB. The ethephon-treated fruits and the control fruits were harvested 155 DAFB and 162 DAFB, respectively. VB indicates vascular bundle. Arrows indicate starch grains.
Ethylene production by the control fruits remained low (less than 0.1 nL·g−1FW·h−1) until harvest time (Fig. 7). On the other hand, ethylene production of the ethephon-treated fruits increased sharply immediately after ethephon treatment, peaking on day 5 after treatment and decreasing to near-zero levels on day 15 after treatment. Ethylene production of the ethephon-treated fruits was higher than that of the control fruits on days 5 and 10 after treatment, and but decreased to levels matching those of the control fruits from day 15 after treatment onward.
Effects of 100 mg·L−1 ethephon treatment 120 DAFB on ethylene production of ‘Akizuki’ fruit in the 2014 season. NS indicates no significant difference. ** indicates significant difference at P < 0.01 by t-test.
The starch content of the control fruits decreased slowly from day 0 after treatment to harvest time (Fig. 8). On the other hand, the starch content of the ethephon-treated fruits decreased dramatically from day 0 to day 5 after ethephon treatment and leveled off thereafter. The starch contents on days 5, 10, and 20 after treatment were lower in the ethephon-treated fruits than in the control fruits. The starch content at harvest time was not significantly different between the ethephon-treated fruits and the control fruits.
Effects of 100 mg·L−1 ethephon treatment 120 DAFB on starch content in ‘Akizuki’ fruit in the 2014 season. The ethephon-treated fruits and the control fruits were harvested 148 DAFB and 156 DAFB, respectively. NS indicates no significant difference. * and ** indicate significant difference at P < 0.05 and P < 0.01, respectively, by t-test.
Total sugar, fructose, and starch contents did not differ between sound tissue and water-soaked tissue (Table 3). On the other hand, sucrose content was lower in water-soaked tissue than in sound tissue. Conversely, sorbitol and glucose contents were higher in water-soaked tissue than in sound tissue.
Sugar and starch contents in sound tissue and water-soaked tissue at harvest time in the 2014 season.
In this study, the incidence and degree of water-soaked disorder in ‘Akizuki’ were decreased by 100 mg·L−1 ethephon treatment approximately 120 DAFB in all experimental seasons (Table 1). This suggests that ethephon stably inhibits the occurrence and worsening of water-soaked disorder. On the other hand, Mitani et al. (2017) treated ‘Akizuki’ and ‘Oushuu’ fruit with ethephon approximately 100 DAFB, and observed insufficient inhibition of water-soaked disorder in ‘Akizuki’ and acceleration of this disorder in ‘Oushuu’. These facts indicate that the effects of ethephon on water-soaked disorder are dependent on the timing of treatment. In the experiment of Mitani et al. (2017), 100 DAFB corresponded to approximately 60 days before commercial harvest for ‘Akizuki’ and approximately 90 days before commercial harvest for ‘Oushuu’. In our experiment, ‘Akizuki’ was treated with ethephon approximately 40 days before commercial harvest of control fruit. The response of the fruit to ethylene application depends on its maturation stage (Adato and Gazit, 1974). Therefore, the effect of ethephon on water-soaked disorder may be influenced by the fruit maturation stage and sensitivity to ethylene during ethephon treatment. On the other hand, Mitani et al. (2017) and Hiramoto et al. (2020) reported that ethephon treatment of the Japanese pear ‘Akizuki’ approximately 100 DAFB inhibited the occurrence of cork spot like disorder.
The incidence of water-soaked disorder in ‘Akizuki’ control fruit was reported to range from 5 to 46% (Matsuda et al., 2006; Mitani et al., 2017; Shimada et al., 2014; Uemura et al., 2009). In this study, the incidence of water-soaked disorder in the control fruits ranged from 63 to 82% and was higher than that reported previously. In addition, the decrease in nitrogen fertilization increased the incidence of water-soaked disorder in ‘Akizuki’ (Shimada et al., 2013, 2014). In this study, the amount of nitrogen fertilizer applied to the field and the pots was lower than the amount applied in general Japanese pear cultivation. This may be one reason for the high incidence of water-soaked disorder in the control fruits.
In Japanese pear, several reports have shown that ethephon treatment enhanced fruit maturation, but suppressed fruit enlargement (Aoki and Okada, 1976; Hiramoto et al., 2020; Nishimoto, 1983). In this study, ethephon treatment advanced the harvest time of ‘Akizuki’ by 5 to 8 days compared with the control, but suppressed fruit enlargement in the 2012 season only (Table 2). The conditions in which ethephon suppresses fruit enlargement remain unclear and require further investigation.
Ethephon treatment reduced the shelf life of persimmon fruit (Yasunobu, 1976). In the present study, we showed that 100 mg·L−1 ethephon treatment 120 DAFB had no effect on flesh firmness or the soluble solids content in ‘Akizuki’ fruit over a 9-day storage period (Fig. 3B, C). This suggests that a shelf life up to 9 days after harvest of ‘Akizuki’ fruit is not affected by ethephon treatment before harvest.
Little information has been gathered about the development of water-soaked disorder in ‘Akizuki’ after harvest. In both the ethephon-treated fruits and the control fruits, WSDI did not significantly change from day 0 to day 9 after harvest (Fig. 3A). Therefore, water-soaked disorder may develop only in fruit on trees.
The results indicate that ethephon treatment may be a practical method to suppress the development of water-soaked disorder in ‘Akizuki’. For practical use, it is necessary to study the treatment timing and treatment dosage of ethephon.
In order to clarify how ethephon suppresses the development of water-soaked disorder in ‘Akizuki’, we investigated ethylene production and the internal components of the fruit after ethephon treatment. The control fruits produced very little ethylene (less than 0.1 nL·g−1FW·h−1) during maturation on trees (Fig. 7). ‘Akizuki’ is a low ethylene producing variety (Itai and Fujita, 2008). On the other hand, ethylene production of the ethephon-treated fruits increased sharply immediately after ethephon treatment, but reverted back to levels that matched that of the control fruits from day 15 after treatment (Fig. 7). Hence, it is conceivable that the increase in ethylene production in ‘Akizuki’ fruit following ethephon treatment is temporary. Ethylene production in ethephon-treated plant organs increases immediately after ethephon treatment (McArtney, 2002; Ohkawa et al., 2006; Torres et al., 2021). The ethylene production patterns in those reports agree with our results.
The water-soaked disorder in ‘Akizuki’ seems to be analogous to the watercore in the Japanese pear ‘Housui’ (Kajiura et al., 1976) and ‘Akibae’ (Tanabe et al., 2001) in that their symptoms are characterized by a watery translucent area. In the watercore disorder in Japanese pear, sorbitol content is high in the watercored tissue compared with that in the healthy tissue (Yamaki et al., 1976). Watercored pear fruit accumulates a large amount of sorbitol in the intercellular spaces (Liu et al., 2022). In apple, elevated sorbitol levels in intercellular spaces play an important role as an osmotic solute by attracting water through vascular bundles, resulting in watercore (Yamada et al., 2006). In this study, we found that sorbitol content in water-soaked tissue was higher than that in sound tissue (Table 3). On the other hand, sorbitol content was significantly reduced on days 10 and 20 after ethephon treatment compared with the control (Fig. 4D). However, the extent of the decrease in sorbitol content after ethephon treatment was small. Therefore, it is difficult to assume that the decrease in sorbitol content is associated with a reduction in the incidence and degree of water-soaked disorder. These should be investigated further. The conversion of sorbitol into fructose and glucose is catalyzed by NAD-sorbitol dehydrogenase (SDH) (Negm and Loescher, 1979), NADP-SDH (Yamaki, 1984), and sorbitol oxidase (SOX) (Yamaki, 1980). On day 20 after ethephon treatment, fructose and glucose contents in the ethephon-treated fruits were higher than those in the control fruits (Fig. 4B, C). These results may indicate that the increased activities of NAD-SDH and NADP-SDH promote the conversion of sorbitol into fructose and glucose.
The cell wall components, pectin, hemicellulose, and cellulose are closely related to watercore development in Japanese pear fruit. In ‘Housui’, pectin (Yamaki et al., 1976), hemicellulose (Chun et al., 2003), and cellulose (Yamaki and Kajiura, 1983) contents were lower in watercored tissue than in sound tissue. In ‘Akibae’, Na2CO3-soluble pectin, hemicellulose, and cellulose contents were lower in watercored tissue than in sound tissue (Chun et al., 2003). Yamaki and Kajiura (1983) found that the decrease in cell wall components in watercored tissue indicates partial overripeness of flesh tissue, which induces the watercore occurrence. On the other hand, ethylene and ethephon treatment affects cell wall components and the activities of associated enzymes. Exogenous ethylene treatment decreased the hemicellulose content in banana fruit (Kojima et al., 1994). Ethephon treatment enhanced water-soluble pectin content and cellulase activity in satsuma mandarin fruit after harvest (Deng et al., 2016). In this study, although pectin contents, except for CDTA-soluble pectin, were not affected by ethephon treatment, hemicellulose and cellulose contents decreased earlier in the ethephon-treated fruits than in the control fruits (Fig. 5). These results indicate that ethylene produced following ethephon treatment rapidly reduced hemicellulose and cellulose contents in ‘Akizuki’. In ‘Akibae’, CDTA-soluble pectin contents were higher in watercored tissue than in sound tissue (Chun et al., 2003). In this study, CDTA-soluble pectin content was higher in the ethephon-treated fruits than in the control fruits at commercial harvest time (Fig. 5B). Nevertheless, ethephon treatment reduced the development of water-soaked disorder (Table 1). The results suggest that the inhibition of water-soaked disorder by ethephon treatment is not related to the content of cell wall components.
Several reports have suggested a relationship between watercore disorder and starch in fruit. Gemma et al. (2002) reported that starch grains remain to some extent in the cytoplasm in matured Japanese pear ‘Housui’ grown in volcanic soil, in which watercore disorder tends to occur. In apple, starch content increases as the degree of watercore increases (Bowen and Watkins, 1997). Ohkawa et al. (2011) found many starch grains in the vascular bundles and the cells around the vascular bundles in both water-soaked tissue and sound tissue of ‘Akizuki’ at commercial harvest time. On the other hand, ethephon treatment decreased starch content in kiwifruit (Bowen et al., 1988; Ohara et al., 1997), and apple (Stover and Fargione, 2003) fruit. Ethylene is involved in the degradation of starch in ‘Tsugaru’ apple fruit (Thammawong and Arakawa, 2007). In this study, changes in starch content in ‘Akizuki’ during fruit maturation were investigated by two methods, chemical analysis and histological observation. Both investigations revealed that starch content decreased sharply after ethephon treatment (Figs. 6 and 8). These results indicate that ethylene produced by ethephon treatment induces the rapid degradation of starch in ‘Akizuki’ fruit. In addition, we found that starch grains remained in the vascular bundles and the flesh cells around the vascular bundles in the control fruits (Fig. 6C), whereas most of the starch grains disappeared in flesh tissue of the ethephon-treated fruit (Fig. 6D). There are few reports about the considerable accumulation of starch grains in vascular bundles. Ong et al. (2021) reported the accumulation of starch grains in the vascular bundles of rice leaves infected with rice orange leaf phytoplasma. The accumulation of starch grains in the vascular bundles is considered an abnormal condition. Therefore, the inhibition of water-soaked disorder following ethephon treatment in ‘Akizuki’ may be due to the degradation of starch grains in the vascular bundles and the flesh cells around the vascular bundles. However, more evidence is needed to explain the relationship between this disorder and starch content.
In most cases, vascular bundle browning was observed in the water-soaked disorder of ‘Akizuki’. Vascular bundle browning observed in Japanese pear was noted in ‘Rinka’ after low-temperature storage (Hayama et al., 2022). In addition, Hayama et al. (2022) reported that ethephon treatment 100 DAFB promoted fruit maturation and reduced the occurrence of vascular bundle browning in ‘Rinka’, and suggested that the shortened maturation period is related to resistance to the disorder because late-harvested fruit has an increased incidence of vascular bundle browning. We found that ethephon treatment shortened the maturation period and decreased the incidence of vascular bundle browning in ‘Akizuki’. The mechanism of the inhibitory effect of ethephon on vascular bundle browning may be similar in ‘Akizuki’ and ‘Rinka’.
Ethephon treatment induced very marked changes in the contents of ‘Akizuki’ fruit’s internal components such as sugar, starch, hemicellulose, and cellulose, and shortened the maturation period. However, none of the internal components at commercial harvest time were different between the ethephon-treated fruits and the control fruits. This may be due to the fact that sound flesh tissue was used for the analysis of sugars, starches, and cell wall polysaccharides, even at harvest time, when several instances of water-soaked disorder were noted.
In conclusion, we found that the incidence and degree of water-soaked disorder in ‘Akizuki’ fruits were decreased by 100 mg·L−1 ethephon treatment approximately 120 DAFB. Ethylene production increased sharply a few days after the ethephon treatment, inducing drastic changes in the contents of sugar, starch, hemicellulose, and cellulose in the ethephon-treated fruits. On the other hand, the occurrence of water-soaked disorder in the control fruits was first observed 141 DAFB. These results indicate that changes in the contents of the fruit’s internal components due to ethephon treatment from the ethephon treatment time (120 DAFB) to the onset of water-soaked disorder may contribute to the inhibition of the occurrence of this flesh disorder by ethephon. However, the results of this study could not clarify which fruit internal components are affected by the ethephon treatment leading to suppression of water-soaked disorder. Changes in cell wall components due to ethephon treatment were thought to have little relevance to the suppression of water-soaked disorder. On the other hand, starch content, which decreased sharply after ethephon treatment, and sorbitol content, which is closely related to watercore disorder in Japanese pear, should be investigated further in order to clarify the mechanism underlying the effects of ethephon treatment on this flesh disorder. It is necessary to investigate not only changes in the contents of fruit internal components, but also the activities of related enzymes and gene expressions after ethephon treatment.
