Influence of Spraying Various Agricultural Compounds Containing Bioactive Substances on the Skin Color and Wine Hue of ‘Muscat Bailey A’ Grapes

Abstract

The cultivation of ‘Muscat Bailey A’ for fresh consumption is popular in western Japan, but growers are facing issues with poor coloration of grape berries partly due to insufficient cooling of nighttime temperatures. To ameliorate the coloration defects in ‘Muscat Bailey A’ grapes grown with gibberellin treatment for seedlessness under high nighttime temperatures, and to improve the color characteristics of red wine made from these grapes, the effectiveness of agricultural materials aimed at correcting poor coloration was tested on grape berries intentionally subjected to conditions that induce poor coloration in 2017 and 2018. Inactivated yeast derivatives, shochu lees filtrate, and proteolytic enzymes were sprayed on leaves, and fertilizer containing optically active abscisic acid (S-ABA) was sprayed on clusters during veraison as agricultural materials that could potentially improve fruit characteristics such as grape skin color and the hue of wine. After veraison, the grapes were grown under conditions in which the nighttime temperature was kept above 25°C. The L*, a*, and b* values of these grape skins were determined using colorimetry. Low L* values, indicating darker berry coloration, and notable decreases in hue angle, suggesting a shift towards red and purple hues, were observed after treatment with S-ABA-containing fertilizer. The absorbance of wine made from the grapes described above was measured spectrophotometrically. A significant difference in the color characteristics of the wine was observed for various absorbance indices (A₄₂₀ representing yellow, A₅₂₀ representing red-purple, and A₅₂₀, at pH 1.0 as an indicator of total anthocyanins). The tannin content in the wine from S-ABA-treated grapes was significantly higher compared to the control. However, no differences were observed in taste among these wines. Comprehensive analysis by LC-MS confirmed that mainly levels of malvidin, petunidin (both purple-colored anthocyanins), and peonidin (a red-colored anthocyanin) compounds had increased. These results suggest that S-ABA-containing fertilizer works effectively in alleviating the poor coloration of ‘Muscat Bailey A’ clusters grown under high nighttime temperature conditions and in improving coloration of the wine made from these grapes.

Introduction

‘Muscat Bailey A’ [‘Bailey’ (Vitis labruscana) × ‘Muscat Hamburg’ (V. vinifera)] was bred to be suitable as both a table grape and a wine grape (Kawakami, 1940). It was registered as a variety in the list of International Organisation of Vine and Wine in 2013 and is the leading red wine grape in Japan (Takata et al., 2014). It is widely cultivated nationwide, with a cultivation area of 386 ha for table grapes in 2018, the eighth largest in Japan (Tokusan kaju seisan doutai tou chousa (kazyu), Ministry of Agriculture, Forestry and Fisheries of Japan, https://www.maff.go.jp/j/tokei/kouhyou/sakumotu/sakkyou_kazyu/index.html, March 20, 2022). In western Japan, seedless cultivation of ‘Muscat Bailey A’ for fresh consumption is particularly popular and widely known under the name ‘New Bailey A’.

Under unsuitable conditions, the surfaces of berries become poorly colored and they do not retain the original color of the variety. Poor coloration has been attributed to various physiological factors, such as over-flowering, early defoliation, excess or lack of inorganic nutrients, pathological factors such as viral diseases and environmental factors such as high temperatures and low solar radiation (Kataoka, 1996). The relationships between temperature and grape growth have been studied, including the relationship between temperature and cumulative temperature (Winkler, 1962), the suppression of coloration by high daytime temperatures (Naito and Ueda, 1964), and the effect of nocturnal berry temperature on coloration in ‘Kyoho’ (Tomana et al., 1979). Therefore, both daytime and nighttime temperatures can affect grape skin coloration. The annual average temperature in Japan is rising, albeit with various fluctuations, but is expected to increase at a rate of 1.26°C per 100 years over the long term (Climate Change Monitoring Report 2020, Japan Meteorological Agency, 2021), indicating that the warming trend is expected to accelerate. With studies predicting the skin color of grapes from air temperature (Sugiura et al., 2018) and making recommendations for adaptation measures (Sugiura et al., 2019b), the development of viticulture and winemaking techniques that can accommodate the changes due to global warming is an urgent issue. In addition to poor coloration arising in part from insufficient cooling of nighttime temperatures, gibberellin-induced defects in coloration have also been observed in black or red colored grapes such as ‘Muscat Bailey A’ (Kabumoto et al., 1983), ‘Delaware’ (Takeshita et al., 1984), ‘Kyoho’ and ‘Aki Queen’ (Gamo and Fumuro, 2000). Poor coloration is more serious in areas where table grapes are cultivated mainly by seedless treatment with gibberellin, in addition to the effects of global warming.

Various methods have been proposed as countermeasures to poor coloration. Girdling was reported to be effective for ‘Pione’ (Fujishima et al., 2005) and for ‘Aki Queen’ (Yamane and Shibayama, 2006), but can cause tree weakness (Yamane, 2008), and its effectiveness varies depending on treatment conditions such as the timing and number of grape clusters (Yamane and Shibayama, 2007; Yamane et al., 2007). Low nighttime temperature treatment of grape clusters (Koshita et al., 2007), light treatment of grape berries at night (Azuma et al., 2012), and light intensity control around clusters by defoliation (Matsuyama et al., 2014) have also been reported, but the costs and labor requirements must be considered. Wine with a high anthocyanin content and high color density was obtained using lateral shoot grapes (Kishimoto et al., 2017). A relationship between abscisic acid (ABA) and berry ripening has been suggested by the rapid increase in endogenous ABA content during the ripening period (Coombe and Hale, 1973) and the coloration effect of ABA in ‘Delaware’ (Matsui et al., 1980) and in ‘Kyoho’ and ‘Pione’ (Kataoka, 1986). In 2003, a fertilizer containing 10% natural, optically active abscisic acid (hereinafter S-ABA) was registered in Japan under the trade name ‘Miyobi Gold’ (Kamuro, 2004). The improvement effect of S-ABA treatment on coloration was reported in ‘Pione’ (Kawase, 2005), ‘Flame seedless’ (Peppi et al., 2006), ‘Malbec’ (Sadamatsu et al., 2007), and ‘Benitaka’ (Shahab et al., 2019). In ‘Gorby’, however, there were differences in the effect depending on the time of treatment (Habu et al., 2008). From previous reports, it is clear that S-ABA, unlike ABA, has a stable effect on improving the coloration of berry skin. A foliar spray fertilizer, LalVigne^® MATURE (Lallemand Inc., ON, Canada, hereinafter Mature), consisting of 100% natural, inactivated wine yeast components, including 7.0% nitrogen, was reported to increase berry anthocyanin content after maceration (Segade et al., 2016; Villangó et al., 2015). It is considered to accelerate the secondary metabolism of grapevines and the ripening of phenols in red wines (Kogkou et al., 2017). Shochu lees filtrate contains extracts of inactivated yeast, rice koji (cultured Aspergillus luchuensis on steamed rice) and components derived from sweet potatoes. Arazyme is a proteolytic enzyme preparation. We expect that these treatments will improve the coloration of grapes by metabolizing phenols and producing anthocyanins through a mechanism similar to that of Mature. Applying these agricultural materials to the vine may induce the production of secondary metabolites, which is a plant biological defense mechanism, and we expected that the same mechanism as in Mature would also be effective for improvements in grape coloration. Establishing new grape cultivation techniques that can be used for table and wine grapes is beneficial for growers facing grapes with coloration defects that are likely to be induced by global warming and gibberellin treatment.

In this study, we investigated the effects of the four agricultural materials described above on coloration and their effects on the characterization of wine from ‘Muscat Bailey A’ grapes that were treated with gibberellin and exposed to high nighttime temperatures after veraison.

Materials and Methods

Experimental area and plant materials

Experiments were conducted in 2017 and 2018 in an unheated plastic greenhouse in Numakuma-cho, Fukuyama City, Hiroshima (34°25′N, 133°19′E, elevation 85 m). The four-roofed greenhouse, 6 m wide, 25 m deep, and 4 m high, was covered with agricultural polyethylene film (0.15 mm thick).

One 15-year-old (2017) ‘Muscat Bailey A’ grapevine (10 m between vines, 4 m in a row) using H-shaped training and spur-pruning culture, was used. The grape samples used in this study were cultivated using an H-shaped training system, a common method in Japan. This spur pruning technique shapes the main branches into an H pattern when viewed from above, with the branches trained at a manageable height. It optimizes sunlight exposure and air circulation, which are crucial in Japan’s humid climate. To induce poor grape coloration, the top and sides of the entire grapevine were covered with agricultural polyethylene film (0.1 mm thick), and the nighttime temperature was maintained above 25°C using a heater (KA321 and four-stage thermos; NEPON Inc., Tokyo, Japan) after veraison. The clusters were dipped in 100 ppm gibberellin solution (GA₃; Kyowa Hakko Bio Co., Ltd., Tokyo, Japan) supplemented with 1 ppm 1-(2-chloro-4-pyridyl)-3-phenylurea (CPPU, Fulmet; Kyowa Hakko Bio Co., Ltd.) to induce seedless berries 10 days before full bloom, and then dipped again in 100 ppm gibberellin 10 days after full bloom to stimulate berry growth. The final number of berries was adjusted to approximately 60 per cluster, with one cluster per shoot. The clusters were covered with white fruit bags 10 days before the treatment, which were removed just before the treatment, and were replaced after the clusters dried.

Treatments

Four agricultural materials were used as treatments during veraison (Table 1). Foliar fertilizer, Mature (inactivated wine yeast), was diluted 200 times with distilled water. Fertilizer containing 10% S-ABA (Miyobi Gold; BAL Planning Co., Ltd., Aichi, Japan) was diluted 100 times (equivalent to 1,000 ppm of S-ABA) with distilled water. Shochu lees (Satsuma Muso Co., Ltd., Kagoshima, Japan) was filtered through filter paper (Whatman [3030-909]) and used as a shochu lees filtrate. The proteolytic enzyme Arazyme (Power Cell C; Biomio Inc., Kumamoto, Japan) was diluted 1,000 times with distilled water.

Table 1

Treatment of the grape cultivar ‘Muscat Bailey A’ with various agricultural materials.

The solutions (except that of S-ABA-containing fertilizer) were hand-sprayed on the leaves from the surface and the underside of the leaf. The S-ABA-containing fertilizer solution was hand-sprayed on the clusters from six directions (top, bottom, left, right, front, and rear). For each experimental group, three clusters were placed on each of the four main branches. The number of samples in each plot was n = 12.

Sampling and measurement of berry characteristics

Grape clusters were collected at approximately 7:00 a.m. on the 40^th day after treatment and the values were measured on the same day. The clusters were weighed and the average weight of three randomly sampled berries from the top, middle, and bottom of a cluster (nine berries per cluster) was used as the berry weight. After removing the bloom and dirt from the surface of berries, the X, Y, and Z values were analyzed using a colorimeter (CR-400; Konica Minolta Inc., Tokyo, Japan) to measure the L*, a*, and b* values with the formula given by the manufacturer. The value of tan⁻¹ (b*/a*) was used as the hue angle. After freezing and storage, the pulp of berries was squeezed, and the total soluble solids (TSS, %) and acidity (%) of juice converted to tartaric acid were measured with a brix and acidity meter (PAL-BX | ACID2; Atago Co., Ltd., Tokyo, Japan).

Winemaking

For each of the treatments, except for grapes treated with shochu lees filtrate, 500 g of grapes was used to make experimental wine. The shochu lees filtrate was excluded because its berry skin color measurement results were similar to those of the Mature treatment. The samples (n = 4 in all cases) were grouped in each of the four H-shaped branches. After thawing, destemming, and crushing the cryopreserved samples, 0.15 g·L⁻¹ of potassium pyrosulfite was added to the crushed samples. The pH, titratable acidity, and formol nitrogen of the juice were measured using a pH meter (F-52; Horiba Ltd., Kyoto, Japan). The specific gravity of the juice (15°C) was determined with a specific gravity hydrometer, and the soluble solids content was calculated. These measurements were conducted in accordance with the Official Analysis Method (National Tax Agency of Japan, 2017). Sucrose was added to 23% (w/v) of the final sugar concentration in the juice.

Wine yeast OC-2 after culture with shaking in YPD liquid medium (1% yeast extract, 2% Bacto peptone, and 2% glucose) was inoculated with a yeast cell concentration of 3 × 10⁶ cells·mL⁻¹. Yeast nutrient Fermaid^® K (Lallemand Inc., ON, Canada) was added as directed by the manufacturer. The experimental wines were stirred twice a day. After five days of maceration at 25°C, the wines were pressed. After fermentation was complete, 0.10 g·L⁻¹ of potassium pyrosulfite was added to the wines, and they were stored at 15°C for several days. Then, the supernatants were collected and stored at 4°C until use. The alcohol content and extract content were determined by the Official Analysis Method (National Tax Agency of Japan, 2017).

Spectrophotometric determination of wines

Sample absorbance was measured using a NovaspecPlus spectrophotometer (Amersham Biosciences, Amersham, UK) in accordance with the methods of Somers and Evans (1977) and Boulton (2001). A₄₂₀ and A₅₂₀ were measured as intensity of yellow and red-purple in wine, respectively. The A₅₂₀ at pH 1.0, indicating the total anthocyanin content, was measured 45 minutes after mixing 100 μL of wine with 900 μL of 0.2 M acetic acid/hydrochloric acid buffer (pH 1.0) and 20 μL of 10% acetaldehyde. The A₅₂₀ (SO₂)/A₅₂₀ (DW) ratio, indicating the ratio of stable red pigments, was determined by measuring A₅₂₀ one minute after mixing 160 μL of 5% potassium pyrosulfite or distilled water (DW) with 2 ml of wine.

Analysis of wine tannins

Tannin concentration was analyzed using the methyl cellulose precipitation method following Bindon et al. (2014). Briefly, wine samples were mixed with methyl cellulose solution, followed by the addition of saturated ammonium sulfate and water. After mixing and centrifugation, tannin levels were quantified by measuring the absorbance difference at 280 nm between treated and control samples, with tannic acid as the standard.

Metabolome analysis of wine using LC-MS

The wine samples were frozen until analysis. The wine samples made from S-ABA-treated grapes were mixed by the year of harvest, and submitted to the Kazusa DNA Research Institute metabolome for contract analysis. LC-MS analysis of the metabolome was performed using an HPLC system, Ultimate 3000 RSLC (Thermo Fisher Scientific Inc., San Jose, CA, USA), coupled with a high-resolution mass spectrometer, Q Exactive (Thermo Fisher Scientific). The identification of the compounds detected was performed based on the exact mass and compositional formula of the fragment ions and the exact compositional formula deduced from the results of precise measurements obtained from LC-MS using PowerGetBatch software (Sakurai and Shibata, 2017). MF searcher (Sakurai et al., 2012) was used to search for the compounds in the UC2 database (Sakurai et al., 2018), which integrates the KNApSAcK family database (http://kanaya.naist.jp/KNApSAcK/) (Afendi et al., 2012) and Human Metabolome Database (http://www.hmdb.ca), the theoretical compositional database and the in-house database. From the obtained data, focusing on anthocyanins, compounds were listed by peak area in order of the S-ABA-treated wine samples and control samples.

Statistical analysis

Data were compared to the controls by one-way analysis of variance and Dunnett’s multiple comparison test using GraphPad Prism version 8.0.0 (GraphPad Software, Inc., San Diego, CA, USA).

Results

Effects of treatments on TSS, acidity, berry weight, and cluster weight

The results of TSS (%), acidity of juice (%), berry weight, and cluster weight are shown in Table 2. No significant differences were observed for TSS in the juice or for berry weight in any experiments. In 2017, the acidity of juice (%) was higher in all experiments than in the control, with a significant difference in the Arazyme sample (P = 0.0093). In 2017, cluster weight was higher in all experiments than in the control, with significant differences in the Mature (P = 0.0086) and Arazyme (P = 0.0475) samples. No significant differences were observed in other experiments.

Table 2

Effects of various treatments on ‘Muscat Bailey A’ grape quality in 2017 and 2018, presented in two separate tables.

Effects of treatments on berry coloration

The results for skin color measured by a colorimeter are shown in Fig. 1. The hue angle calculated from a* and b* values, indicating color tone of skin (90, yellow: 0, red: −90, blue), was significantly lower in the S-ABA-treated sample than in the control (P < 0.0001 in 2017, P = 0.0021 in 2018). The L* value indicating lightness was significantly lower in the S-ABA-treated sample than in control (P < 0.0001 in 2017, P = 0.0012 in 2018). Additionally, both the hue angle and L* value, which indicate skin color, were lower in 2018 than in 2017. The nighttime temperatures in 2018 were slightly lower than in 2017 (data not shown), resulting in a greater cooling effect at night and, consequently, greater coloration of the grapes in 2018. Figure 2 shows photographs of clusters and individual berries, illustrating significant differences in appearance between the control group and the S-ABA-treated group. Although the hue angle in the Arazyme-treated sample was significantly lower in 2017 (P = 0.0059), the L* value was higher than in the control (P = 0.0192) in 2018 (Fig. 1). These results indicated that among the agricultural materials used in this study, S-ABA had a marked color-enhancing effect on berry skin.

Fig. 1

Effect of each treatment on the skin color of grapes exposed to high nighttime temperature (≥ 25°C) after veraison. Data are expressed as the mean (SE) (2017: n = 99–108, 2018: n = 108) *P < 0.05 vs. control. **P < 0.01 vs. control. ****P < 0.0001 vs. control.

Fig. 2

The photograph illustrates the effect of a treatment on the skin color of grapes exposed to high nighttime temperatures (≥ 25°C) after veraison, with S-ABA-treated clusters (left) and sampled berries (right) showing significant differences from the control.

Effects of treatments on wine characteristics

We conducted a small-scale vinification test to compare the color of wine made from grapes treated with various agricultural materials, as the colors of these skins were different. No significant differences compared with the control were observed in pH, titratable acidity, formol nitrogen in the juice, alcohol content, or wine extracts (Table 3) in any treatments, although some differences in extracts were observed for the 2017 S-ABA and 2018 Mature treatments. The absorbance indices obtained by spectrophotometer and tannin concentrations are shown (Fig. 3). The yellow color (A₄₂₀) was significantly higher in the S-ABA-treated sample than in the control in 2018 (P = 0.0273) (Fig. 3F). The red color (A₅₂₀) was significantly higher in the S-ABA-treated sample than in the control, with significant differences in 2017 (P = 0.0271) and 2018 (P < 0.0001) (Fig. 3B and G). Tannin concentration was significantly higher in the S-ABA-treated samples (P < 0.0001) compared to the control in 2017, as well as in the S-ABA-treated samples (P < 0.0001) in 2018 (Fig. 3C and H). The total anthocyanin content (A₅₂₀ at pH 1.0) was significantly higher in the S-ABA-treated samples than in the control in 2017 (P = 0.0011) and 2018 (P < 0.0001) (Fig. 3D and I). No specific trends were observed in other absorbance values and treatments. In addition, as a result of sensory evaluation conducted by a panel of four authors, no taste differences were noted (data not shown). These results indicated that among the agricultural materials used in this study, S-ABA increased the anthocyanin and tannin content in red wine, resulting in enhanced color, but had no effect on the taste of the wine.

Table 3

The analysis values for grape juice and red wine made from grapes treated with various agricultural materials in 2017 and 2018, presented in two separate tables.

Fig. 3

Comparison of wine color at A₄₂₀ (A, F), A₅₂₀ (B, G), tannin (C, H), total anthocyanin content at pH 1.0 indicated by A₅₂₀ (D, I), and the ratio of stable red pigment indicated by A₅₂₀ (SO₂)/A₅₂₀ (DW) (E, J) of wines made from grapes treated with Mature, S-ABA, and Arazyme in 2017 (upper panel) and 2018 (lower panel). Data are expressed as mean (SE) (n = 4). *P < 0.05 vs. control. **P < 0.01 vs. control. ****P < 0.0001 vs. control.

Metabolome analysis by LC–MS

The S-ABA-treated wine samples, which were notably different from the control samples as shown by the absorbance analysis (Fig. 3), were further compared with the control samples using LC-MS metabolome analysis. Focusing on anthocyanins, the compounds are listed in Table 4 for 2017 and Table 5 for 2018, arranged by the largest differences in peak area between the control and S-ABA-treated samples. Malvidins, petunidins, and peonidins showed a tendency to increase with S-ABA treatment across both years. Based on the differences in peak area sizes, malvidins appear to have a particularly large impact among these compounds. This aligns with the findings of Koyama et al. (2017), who demonstrated that malvidin is the predominant anthocyanin in ‘Muscat Bailey A’. However, for compounds with multiple candidates that have the same estimated molecular formula, further investigation is required to identify them. These findings suggested that S-ABA induces these anthocyanins, influencing the color of the grape skins and the intensity of wine color.

Table 4

Analytical comparison of the metabolite profiles using LC-MS between wines made from S-ABA treated grapes and control grapes, highlighting the identified anthocyanin compounds in 2017.

Table 5

Analytical comparison of the metabolite profiles using LC-MS between wines made from S-ABA treated grapes and control grapes, highlighting the identified anthocyanin compounds in 2018.

Discussion

The poor coloration of grapes is a major problem in western Japan; specifically, the Fukuyama area, where Fukuyama University is located, in part owing to the insufficient cooling of nighttime temperatures caused by global warming. Various methods specific to grape variety, such as girdling, ABA treatment, and the regulation of temperature or/and light environment around the grapevine, have been reported to improve grape coloration. A limited number of techniques have been put into practice in the field of cultivation. In this study, a practical method to improve the coloration of ‘Muscat Bailey A’ grape was investigated for use with table grapes, which can be used for red wine grapes as long as the skin color is good.

S-ABA-containing fertilizer (product containing 10% S-ABA) was officially registered as a foliar application fertilizer in Japan in 2003 (Kamuro, 2004). Kataoka (1986) examined the differences in the effect of ABA treatment on grape berry coloration by variety, with a focus on the control of coloration by ABA. The anthocyanin content in the control was higher than that of ABA-treated grapes in both the seeded and seedless berries of ‘Muscat Bailey A’ and Super Hamburg, and there was no effect on the appearance of coloration. However, in the present study, S-ABA-treated grapes had a clearly deeper skin color (Fig. 2) while the hue angle and L* value of S-ABA-treated grapes were significantly lower than those of the control (Fig. 1). The main cause of the differences between them may have been the type of ABA (S-ABA contains only the left-handed form, while synthetic ABA contains both left-handed and right-handed forms), although treatment conditions, anthocyanin content, and temperature conditions are interconnected (Kataoka, 1986). A coloration-enhancing effect of S-ABA treatment was reported for ‘Kyoho’ (Habu et al., 2008), ‘Gorby’ (Habu et al., 2009), ‘Aki Queen’ (Katayama-Ikegami et al., 2016, 2017) and ‘Pione’ (Sugiura et al., 2019a) grapes. These results were consistent with our results. In this study, there was no significant difference in TSS (%), acidity of juice (%), berry weight, cluster weight, pH, titratable acidity, or formol nitrogen in the juice (Tables 2 and 3). In contrast, Peppi and Fidelibus (2008) reported that a reduction in titratable acidity was caused by the effect of S-ABA on components of the red grape Flame Seedless. This occurrence may have been due to the difference between cultivars.

There are five major types of anthocyanin pigments in grapes and they can be classified into two groups: red-colored anthocyanins, namely cyanidin and peonidin, and purple-colored anthocyanins, namely delphinidin, petunidin, and malvidin (Azuma, 2016). In the wine made from grapes treated with S-ABA in this study, the metabolomic analysis of mid-polar compounds using LC-MS indicated that the increased levels of derivatives of purple pigments, such as malvidin and petunidin, and red pigments such as peonidin, were the main cause of the improvement in coloration of the wine (Tables 4 and 5). Most of the differences in the color and taste of wine are derived from phenolic compounds, among which flavonoids confer astringency and bitterness (Yokotsuka, 1995). There are some reports indicating an increase in total phenolic content in addition to anthocyanins with S-ABA treatment (Deis et al., 2011; Pessenti et al., 2019). In this study, tannin content was significantly increased (Fig. 3C and H). These data indicate that S-ABA treatment improved berry coloration of ‘Muscat Bailey A’ grapes. However, it did not appear to have any effect on most grape qualities, except for the coloration of the wine.

Regarding other agricultural materials used in this study, it has been reported that spraying of Mature increased anthocyanin levels in table grapes (Crupi et al., 2020) and wine (Villangó et al., 2015; Segade et al., 2016), affecting the berry skin color. Foliar spraying of this material was expected to improve coloration by inducing anthocyanin synthesis. However, in our study, little coloration effect was observed (Fig. 1). The shochu lees filtrate used in this study contained inactivated yeast and malted rice; thus, we expected it to induce anthocyanin synthesis in grape skins and to improve coloration by the same mechanism as Mature. The results showed that in some cases there were significant differences in a* and b* values, but as with Mature, there appeared to be no effect (Fig. 1). Arazyme consists of highly active proteases (Park et al., 2008). We considered that the foliar application of this enzyme may induce a secondary metabolic reaction similar to that during pest infestation of the grapevine, and we expected it to function as an elicitor. In the present study, the application of Arazyme had some or no effect on color parameters, but there were no differences in appearance (data not shown) and no effect on the quality of wine vinified with it. We consider that agricultural materials other than S-ABA used in this study, which were not intended for use to address coloring deficiencies, did not yield any effects under the conditions applied in this study.

As described above, this study concluded that the application of S-ABA-containing fertilizer on grape clusters was extremely effective in improving the coloration of berry skin and wine, although the use of this fertilizer presents some issues. The first issue is in the timing of treatment. Fertilizer containing S-ABA was applied during the veraison period, which is approximately 10 days after bagging for pest prevention. In this study, the bag was removed and fertilizer containing S-ABA was applied. This is inefficient in terms of cultivation management. Kugiyama et al. (2011) also made this point and judged the procedure to be complicated and impractical. However, a detailed study of the allowable ranges for the timing of bagging and treatment with fertilizer containing S-ABA would be useful. The second issue is how to apply S-ABA treatment. In this study, cluster spraying was performed by hand. Spraying of material on grape clusters is time consuming, and leaves marks on the clusters. If the grapes are to be used for wine making, it is not a problem if traces of material remain, but if they are for table grapes, this causes a decrease in market valuation. It may be worthwhile to consider spraying the entire vineyard or spraying at the same time as gibberellin treatment is applied, as described by Kamuro (2011).

As noted in Introduction, several methods and their combinations have been studied to improve grape coloration. This study aimed to address the poor coloration of ‘Muscat Bailey A’ in a region affected partly by insufficient cooling of nighttime temperatures. In this experiment, an artificially elevated nighttime temperature was used to induce poorly colored clusters of ‘Muscat Bailey A’. As anticipated, these conditions resulted in poor coloration with low anthocyanin content. It has been reported that S-ABA-containing fertilizers are effective for enhancing grape coloration when applied at optimal harvest timing (Kamuro, 2011). Recently, another S-ABA formulation, ABSUP^® Liquid (Sumitomo Chemical Co., Ltd., known as ProTone^® in other countries), has been registered as a plant growth regulator by Japan’s Ministry of Agriculture, Forestry and Fisheries. Although similar effects are expected, it is currently approved only for Kyoho and Pione grapes. Our results suggest that improving the coloration of ‘Muscat Bailey A’ could prevent the need to discard these grapes, thereby reducing waste. Furthermore, the grapes could be used as raw material for winemaking, offering a potential solution to the challenges faced by grape growers in western Japan, including the Fukuyama area. ‘Muscat Bailey A’ was originally bred to be suitable for both table and wine grapes (Kawakami, 1940). On the other hand, Okuda et al. (2014) showed that while the seeds of ‘Muscat Bailey A’ contain a significant amount of proanthocyanidins, these polyphenols are difficult to extract during winemaking. For ‘Muscat Bailey A’ wines, the contribution of seed-derived tannins to the flavor is minimal. As a result, using seedless ‘New Bailey A’, which is simply ‘Muscat Bailey A’ treated with gibberellin to produce seedless grapes, does not pose a problem. In fact, there is a winery in Hiroshima that uses seedless ‘New Bailey A’ for vinification, and the wines have been well received. Thus, wines made from ‘New Bailey A’ could offer a viable variation of ‘Muscat Bailey A’ wines. It is hoped that the findings of this study can be applied in regions facing similar challenges, leading to the development of a highly profitable viticultural model.

Acknowledgements

The authors thank Dr. Y. Kamuro for providing the fertilizer containing S-ABA (Miyobi Gold), and BIOMIO Co., Ltd. and Satsuma Muso Co., Ltd. for providing the experimental materials. This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI (grant numbers JP18KT0047 and JP22K05532).

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