The Horticulture Journal
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Print ISSN : 2189-0102
ISSN-L : 2189-0102
ORIGINAL ARTICLES
Induction of Parthenocarpy by Bordeaux Mixture and Its Use for Cultivation in Japanese Pear ‘Kosui’
Taishi HayashidaKazuyoshi NadaShin Hiratsuka
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2016 Volume 85 Issue 3 Pages 193-200

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Abstract

Since both artificial pollination and fruit thinning are necessary for marketable fruit production of Japanese pears (Pyrus pyrifolia (burm. f.) Nakai) in Japan, about a 20% fruit set is ideal without pollination because only 5% of flowers are actually subjected to fruit production. In this study, copper (Cu2+) and ferrous (Fe2+) ions were shown to be effective for inducing a nearly-ideal fruit set of the Japanese pear ‘Kosui’. Fruit induced by ferrous sulfate (FeSO4) solution or a Bordeaux mixture, which is a combination of copper sulfate (CuSO4), lime, and water, were parthenocarpic, because 1) self-pollen tube growth was not promoted by Cu2+ and Fe2+, 2) almost no perfect seeds were observed at harvest, and 3) Cu2+ and Fe2+ acted as strong inhibitors of pollen tube growth in vitro. The effective stage for inducing parthenocarpy was sprouting time to 4 days after anthesis in the Bordeaux mixture, and sprouting time to 4 days before anthesis in the FeSO4 solution. Annual changes in the effectiveness were found in both chemicals, and the Bordeaux mixture showed no inhibitory effect on the fruit set of cross-pollinated flowers. The growth of Bordeaux mixture-induced fruit was improved by gibberellin (GA) paste or GA paste mixed with N-(N-(2-chloro-4-pyridy1)-N'-phenylurea (CPPU) treatment of the fruit stalk; the treated fruit was about 100 g heavier than the untreated fruit. The GA paste treatment is currently conducted to promote fruit growth and maturation in ‘Kosui’ fruit production in Japan, and the Bordeaux mixture can be substituted for chemical fungicide generally used before anthesis to control scab and black spot disease. Accordingly, the Bordeaux mixture is a promising chemical for great labor-saving in ‘Kosui’ cultivation.

Introduction

In Japanese pear cultivation in Japan, artificial pollination and fruit thinning practices are necessary, and account for about 30% of total cultivation labor during the restricted season (Kumashiro, 2000). Artificial pollination is conducted because the Japanese pear has S-RNase-based self-incompatibility, and does not have the economic parthenocarpy found in the European pear (Nyéki et al., 1998). Although compatible pollinizers are interplanted in the orchards, “The Positive List System” introduced recently in Japan by the Ministry of Health, Labour and Welfare of Japan (2006, http://www.mhlw.go.jp/english/topics/foodsafety/positivelist060228/introduction.html, June 11, 2015), where restricts pesticide application, excludes pollinizers with different harvest seasons from the growing cultivar. Therefore, monoculture is strongly desired. After confirming the fruit set, fruit thinning is needed to promote fruit growth and quality; about a 20% fruit set is ideal because only 5% of flowers are actually used for fruit production (Hiratsuka et al., 2009).

The Japanese pear ‘Kosui’ is the most popular cultivar, accounting for 40% of total pear production in Japan (Ministry of Agriculture, Forestry and Fisheries, 2011, http://www.maff.go.jp/j/tokei/kikaku/nenji/88nenji/other/n555.xls, June 11, 2015). Recently, we found that some heavy metal ions such as Cu2+, Fe2+, and Zn2+ inhibit the stylar RNase activity of ‘Kosui’, and give a 15%–40% fruit set by application before anthesis (Hayashida et al., 2013), indicating that a cultivation system without both artificial pollination and fruit thinning practices can be established (Hayashida et al., 2015). However, the mechanism regarding fruit set by the ions was not clear, and the induced fruit were sometimes small (Hayashida et al., 2013).

Bordeaux mixture containing Cu2+ can be substituted for chemical pesticides usually used by farmers before anthesis to control scab and black spot disease in Japanese pears. If Bordeaux mixture has fruit-inducing ability similar to CuSO4 solution, great labor saving will be accomplished. In this study, we first aimed to clarify the promotive effect of the Bordeaux mixture on the fruit set of ‘Kosui’ compared with the effects of CuSO4 and FeSO4 solutions. Then, whether the fruit set by the Bordeaux mixture is due to incompatibility breakdown was investigated by observing pollen tube growth and seed formation. Finally, the promotive effects of gibberellin (GA) and CPPU on Bordeaux mixture-induced fruit growth were determined.

Materials and Methods

Plant materials

The present study was conducted for 6 years from 2008 to 2013. During the experiment, 42–47 year-old Japanese pear trees (Pyrus pyrifolia (Burm. f.) Nakai) ‘Kosui’ were used at the Experimental Farm of Mie University, Tsu, Mie, Japan. For the pollination experiments, pollen grains were gathered from ‘Kosui’ (S4S5) (self-pollen source) and ‘Chojuro’ (S2S3) (cross-pollen source) flowers, and stored at −30°C until use in respective years. Self-incompatibility of Japanese pears is controlled by the S-haplotype (S1, S2, S3, …), and the style rejects the pollen with the same S-haplotype (Hiratsuka, 2004).

Influence of the Bordeaux mixture and CuSO4 solution application on the fruit set

In 2008, to confirm whether Bordeaux mixture containing CuSO4 induces the fruit set, IC Bordeaux 48QTM containing 2.5% Cu2+ (Inoue Calcium Corporation Co., Ltd., Kochi, Japan) was diluted 30-fold, and sprayed on ‘Kosui’ flowers 8 days before anthesis, and they self-pollinated at anthesis. Pollinated flowers were covered with paper bags immediately after pollination, and the percentage of fruit set was checked 4 weeks after pollination. As a control, flowers sprayed with 2 mM CuSO4 (Nacalai Tesque Co., Ltd., Kyoto, Japan) solution were used. Both the Bordeaux mixture and CuSO4 solution contained 0.1% Tween-20 as a wetting agent. In both treatments, 20 flower clusters (a cluster consisted of 3–4 developmentally uniform flowers) were employed.

Pollen tube growth the Bordeaux mixture- and FeSO4 solution-treated style and seed formation in the fruit

In the above experiments, since the Bordeaux mixture was proven to have similar effect on fruit sets with CuSO4 solution, the Bordeaux mixture was used hereafter instead of CuSO4 except for in vitro experiments. In 2009, the Bordeaux mixture or 2 mM FeSO4 (Nacalai Tesque Co., Ltd., Kyoto, Japan) solution containing 0.1% Tween-20 was sprayed on ‘Kosui’ flowers 8 days before anthesis, and they were pollinated either by self or cross (‘Chojuro’) pollen at anthesis; the treatment 8 days before anthesis was carried out because the stage was relatively effective for inducing fruit in the preliminary experiments. Flowers sprayed with distilled water containing 0.1% Tween-20 were used as a control. Then, the flowers were covered with paper bags, and sampled 72 hours after pollination when cross-pollen tubes have already passed the base of the style but self-pollen tubes have not under field conditions (Hiratsuka and Tezuka, 1980). The flowers were fixed immediately with FAA solution (formaldehyde:acetic acid:70% ethanol = 1:1:18), and paraffin sections were made at the base of styles. After removing paraffin through xylene-ethanol series, each section was stained with 0.1% aniline blue dye (Wako Pure Chemicals Industries Co., Ltd., Osaka, Japan) dissolved in 30 mM K3PO4 for two hours, and observed under a fluorescent microscope (BH-2 RFL-T2; Olympus, Tokyo, Japan). More than 25 styles sampled from 5 different flower clusters were used for each treatment. In addition, the Bordeaux mixture- and FeSO4 solution-induced fruit were kept on trees, and their seed number and appearance were checked at harvest. As a control, fruit sprayed with 0.1% Tween-20 was used. Six fruit in the Bordeaux mixture treatment, 5 fruit in the FeSO4 solution and 5 fruit in the control were used, respectively.

Influence of various concentrations of CuSO4 and FeSO4 on pollen germination in vitro

Into an agar media consisting of 10% sucrose, 1% agar, and 0.01% H3BO3, various concentrations of CuSO4 or FeSO4, 0.5 to 5 mM, were added. The pollen grains of ‘Kosui’ were scattered onto the medium, and cultured under darkness at 25°C for 24 hours. After culture, the medium was stained with cotton blue dye (1% cotton blue dye (Wako Pure Chemicals Industries Co., Ltd., Osaka, Japan) dissolved in a solution of glycerin:phenol:lactic acid:H2O = 1:1:1:1), and pollen germination was observed under an optical microscope (BH-2; Olympus). More than 50 pollen grains were observed in each treatment and experiments were carried out 3 times. The control did not contain FeSO4 or CuSO4. The influence of FeSO4 was investigated in 2009, and that of CuSO4 was in 2013.

Determination of the optimum stage for inducing a fruit set by the Bordeaux mixture and FeSO4 solution

FeSO4 solution treatment was conducted in 2010 and the Bordeaux mixture in 2013. FeSO4 solution treatment 4 days after anthesis (DAA) was not done since no promotive effect was observed in the preliminary experiments. Self-pollination was not done hereafter unless otherwise indicated because Cu2+ and Fe2+ were confirmed to cause parthenocarpy in the above experiments. The Bordeaux mixture or 2 mM FeSO4 solution containing 0.1% Tween-20 was sprayed on ‘Kosui’ flowers at various developmental stages, sprouting times, 8 and 4 days before anthesis, and 0 and 4 DAA. Flower clusters with developmentally uniform 3–4 flowers were used in all experiments except for sprouting time treatment. Application of both chemicals before anthesis was carried out at respective developmental stages of the flower determined by their appearance according to Hiratsuka et al. (1985). Flowers just before opening were used for the treatment at anthesis. For the treatment at 4 DAA, flowers had been covered with paper bags 0 DAA, and had Bordeaux mixture 4 DAA applied. In all treatments, flowers were covered with paper bags until 30 DAA to avoid contamination. The flowers sprayed with 0.1% Tween-20 at 8 days before anthesis were used as a control. At least 20 flower clusters were used for each treatment.

Annual changes in induced fruit sets by the Bordeaux mixture and FeSO4 solution

To confirm the annual changes in the effectiveness of the Bordeaux mixture and 2 mM FeSO4 solution, both chemicals were sprayed on ‘Kosui’ flowers 8 days before anthesis, from 2008 to 2013. The Bordeaux mixture, and FeSO4 solution contained 0.1% Tween-20, and the flowers treated with 0.1% Tween-20 were used as a control. The treated flowers were covered with paper bags, and the percentages of fruit sets were checked 4 weeks after anthesis. More than 20 flower clusters (a cluster consisted of 3–4 developmentally uniform flowers) were used in each year.

Influence of Bordeaux mixture application on fruit sets of compatibly pollinated flowers

To clarify whether Bordeaux mixture application inhibits the fruit set of compatibly pollinated ‘Kosui’ flowers, Bordeaux mixture containing 0.1% Tween-20 was used 8 days before anthesis, and the flowers were pollinated by ‘Chojuro’ pollen at anthesis in 2010. As a control, flowers sprayed with 0.1% Tween-20 were used. At least 20 flower clusters (a cluster consisted of 3–4 developmentally uniform flowers) were employed, and the percentages of fruit sets were checked 4 weeks after pollination.

Influence of GA and CPPU on the growth of Bordeaux mixture-induced fruit

Since the fruit induced by the Bordeaux mixture was often smaller than that by artificial pollination, GA and CPPU were tested to improve the fruit growth in 2010. After Bordeaux mixture-induced fruit were thinned at a leaf-to-fruit ratio of 25:1 at 30 DAA, the following 4 treatments were conducted.

  1. 1)   CPPU paste: 15 ppm CPPU (Kyowa Hakko Bio Co., Ltd., Tokyo, Japan) mixed in lanoline (Wako Pure Chemicals Industries Co., Ltd., Osaka, Japan) was applied to fruit stalks of pre-thinned fruit (about 20 mg per fruit) 20 DAA, and fruit thinning was carried out 30 DAA.
  2. 2)   GA paste: a commercial GA lanolin paste (containing 2.7% active ingredient GAs) purchased from Kyowa Hakko Bio Co., Ltd. was applied to fruit stalks (about 20 mg per fruit) 30 DAA.
  3. 3)   CPPU solution + GA paste: 15 ppm CPPU solution was sprayed on pre-thinned fruit (about 3 mL per fruit) 20 DAA first, then GA paste was applied to fruit stalks 10 days after CPPU application.
  4. 4)   CPPU + GA paste: 15 ppm CPPU mixed in GA paste was applied to fruit stalks (about 20 mg per fruit) 30 DAA.

As a control, untreated fruit induced by the Bordeaux mixture was used. At harvest, fruit weight was determined. More than 9 fruit were employed for each treatment.

Statistical analysis

Data were calculated using Microsoft Excel (ver. 15.0.4615.1000), and standard errors were set at each data point. The significance between the data was analyzed by the Tukey-Kramer test at a 5% level. In fruit set experiments (Figs. 4, 5, and 6), statistical analyses were not conducted because no replication was made.

Results

Influence of Bordeaux mixture application on fruit sets

Whether the Bordeaux mixture could be substituted for CuSO4 solution was tested in terms of inducing fruit in 2008 because the Bordeaux mixture can also be used for disease control such against as rust and black spot disease. Fruit sets induced by CuSO4 solution were 32%, and those by the Bordeaux mixture were 15%. Although the Bordeaux mixture effect seemed to be somewhat weaker than the CuSO4 solution, we concluded that the Bordeaux mixture can be substituted for CuSO4 solution. Bordeaux mixture application on open flowers, however, sometimes turned the petals brown, although the fruit surface showed no harmful effect.

Pollen tube growth in Bordeaux mixture- and FeSO4 solution-treated styles and seed formation in the fruit

In FeSO4 solution-treated flowers, self-pollen tubes were not observed at the base of styles 72 hours after pollination (Fig. 1A), and almost no tubes were detected in Bordeaux mixture-treated ones (Fig. 1B), although a large number of pollen tubes was present beneath the stigma (data not shown). The growth of cross-pollen tubes was not affected by either treatment (data not shown). In controls, many cross-pollen tubes were present (Fig. 1C), but no self-pollen tubes were observed (data not shown). When the seed number was checked in the fruit at harvest, almost no fully developed seeds were formed in either treatment, which was similar to the control (Fig. 2A). The empty seeds, however, were often black and large (Fig. 2B).

Fig. 1

Horizontal sections at the stylar base in the Japanese pear ‘Kosui’. (A): self style 72 hours after pollination and treated with 2 mM FeSO4 solution, (B): self style 72 hours after pollination and treated with the Bordeaux mixture, (C): crossed style 72 hours after pollination. Abbreviations: cor., cortex; tt, transmitting tract; pt, pollen tube.

Fig. 2

Seed formation in ‘Kosui’ fruit induced by the Bordeaux mixture or 2 mM FeSO4 solution. (A): Seed number in the fruit. Vertical bars indicate SE (n ≥ 5). Different letters indicate significance at P < 0.05 by the Tukey-Kramer test. (B): Appearance of seeds in the fruit. (a): fully developed seeds, (b): large empty seeds, (c): small empty seeds.

Influence of various concentrations of CuSO4 and FeSO4 on pollen germination in vitro

Pollen germination on the media containing 0, 0.5, 2, and 5 mM CuSO4 was 70%, 3%, 0.9%, and 0.3%, respectively (Fig. 3). FeSO4 also inhibited the pollen germination dose-dependently; germination percentages on 0, 0.5, 2, and 5 mM FeSO4 media were 39%, 17%, 4%, and 2%, respectively.

Fig. 3

Influence of various concentrations of CuSO4 or FeSO4 on ‘Kosui’ pollen germination 24 hours after incubation in vitro. Vertical bars indicate SE (n = 3). Different letters indicate significance at P < 0.05 by the Tukey-Kramer test.

Determination of optimum stage for inducing fruit sets by the Bordeaux mixture and FeSO4 solution

The effective stages were determined for inducing fruit sets by the Bordeaux mixture and FeSO4 solution treatments, respectively (Fig. 4). When they were applied to young flowers, sprouting time to 4 days before anthesis, 23%–56% fruit sets were achieved by the treatments. Although the Bordeaux mixture treatment was also effective for more developed flowers, 0 to 4 DAA, FeSO4 was not at 0 DAA. Since both treatments showed almost 2 times higher fruit sets than controls, it is clear that the Bordeaux mixture and FeSO4 solution have an ability to induce fruit sets of ‘Kosui’. The optimum period was considered between the sprouting time and 4 DAA in Bordeaux mixture application, and at sprouting time to 4 days before anthesis in the FeSO4 solution.

Fig. 4

Timing effect of the Bordeaux mixture or 2 mM FeSO4 solution treatment on fruit sets of ‘Kosui’. FeSO4 solution and Bordeaux mixture treatments were conducted in 2010 and 2013, respectively. FeSO4 solution treatment 4 DAA was not tested. Fruit sets were checked 30 DAA.

Annual changes in inducing fruit sets by the Bordeaux mixture and FeSO4 solution

Annual changes in the effectiveness were found in the Bordeaux mixture and FeSO4 solution; fruit sets by the Bordeaux mixture changed from 15% to 56% over the 4 years tested and those by FeSO4 solution from 17% to 40% over 5 years (Fig. 5). Although the promotive effect was different year by year, both chemicals induced higher fruit sets compared with controls.

Fig. 5

Annual changes in the fruit set of ‘Kosui’ treated with Bordeaux mixture or 2 mM FeSO4 solution 8 days before anthesis. Fruit sets were checked 30 DAA. Bordeaux mixture in 2010 and 2011, and FeSO4 solution treatment in 2013 were not tested, respectively.

Influence of Bordeaux mixture application on fruit sets of compatibly pollinated flowers

Bordeaux mixture-treated flowers set 70% fruit following cross-pollination, and cross-pollination without Bordeaux mixture application also induced 70% fruit sets (Fig. 6). All fruit resulted from pollination; small parthenocarpic fruit were clearly discriminated from cross-pollinated fruit. Thus, the Bordeaux mixture had no inhibitory effect on fruit sets in cross-pollinated flowers.

Fig. 6

Influence of the Bordeaux mixture on fruit sets of cross-pollinated ‘Kosui’ flowers. Bordeaux + Cross: Bordeaux mixture was applied 8 days before anthesis and plants cross-pollinated at anthesis. Cross: 0.1% Tween-20 was used 8 days before anthesis and plants cross-pollinated at anthesis. Fruit sets were checked 30 DAA.

Influence of GA and CPPU on the growth of Bordeaux mixture-induced fruit

To improve the growth of Bordeaux mixture-induced fruit, GA and CPPU were treated by several application methods (Fig. 7). The mean weight of the fruit was 250 g in an untreated control at harvest. In contrast, fruit grew to 336 g when CPPU paste was applied to the fruit stalks 20 DAA, and to 363 g when GA paste was applied to fruit stalks 30 DAA. Thus, both treatments showed a tendency to improve fruit growth. The combined effect of CPPU solution with GA paste was the strongest, when CPPU solution was sprayed on fruit 20 DAA and GA paste was subsequently applied to fruit stalks 30 DAA, fruit reached 431 g, the largest among the treatments. Interestingly, the fruit size was almost the same with GA paste only when CPPU mixed in GA paste was applied 30 DAA.

Fig. 7

Influence of GA and/or CPPU treatments on the growth of ‘Kosui’ fruit induced by the Bordeaux mixture. CPPU paste: CPPU mixed in lanoline was applied to fruit stalks 20 DAA. GA paste: a commercial GA paste was applied to fruit stalks 30 DAA. CPPU solution + GA paste: CPPU solution was sprayed on fruit 20 DAA first, then GA paste was applied to fruit stalks 10 days after CPPU application. CPPU + GA paste: CPPU mixed in GA paste was applied to fruit stalks 30 DAA. Cont.: untreated control. Vertical bars indicate SE (n ≥ 9). Different letters indicate significance at P < 0.05 by the Tukey-Kramer test.

Discussion

The Bordeaux mixture showed similar effect on inducing parthenocarpy to ‘Kosui’ with 2 mM CuSO4 solution, where Cu2+ in the Bordeaux mixture may function. This fact is desirable because fruit set induction and disease control can be fulfilled simultaneously in the ‘Kosui’ orchard. The fruit set induction effect of the Bordeaux mixture, however, was somewhat lower than that of CuSO4 solution, although Bordeaux mixture contains almost a 6 times higher concentration of Cu2+ (13 mM) than the 2 mM CuSO4 solution. This may be explained by the fact that not enough Cu2+ was absorbed into the flower cluster after Bordeaux mixture application (unpublished data), which may be due to the complex conjugation manner of Cu2+ with Ca(OH)2; Cu2+ in the Bordeaux mixture is present as CuSO4·xCu(OH)2·yCa(OH)2·zH2O on flower tissue (Instruction leaflet for IC Bordeaux mixture 2015; Inoue Calcium Corporation Co., Ltd.). Therefore, when unseasonable weather is expected during anthesis, the Bordeaux mixture should be sprayed twice or used as a supplementary practice to artificial pollination.

Cu2+ strongly inhibited stylar RNase activity and led to ‘Kosui’ fruit sets as reported earlier (Hayashida et al., 2013). First, we considered that Cu2+ causes breakdown of self-incompatibility. In this study, however, it was proved that self-pollen tube growth was not promoted in the styles and almost no fully developed seeds were formed in the fruit by Bordeaux mixture and FeSO4 solution treatments. Thus, Cu2+ and Fe2+ induce parthenocarpy, but not breakdown, of self-incompatibility. This well matches the fact that FeSO4 solution lead to fruit sets of ‘Kosui’ despite little inactivation of stylar RNase (Hayashida et al., 2013). Why self-incompatibility was not defeated is unclear. Nevertheless a considerable decrease occurred in stylar RNase activity by Cu2+. There seem to be several reasons as follows; 1) since ‘Kosui’ styles possess strong non-S-RNase together with S4- and S5-RNase (Norioka et al., 2007), Cu2+ may inhibit predominantly the non-S-RNase activity, 2) pear style does not absorb enough amount of Cu2+ after treatment to inactivate S-RNase, because estimated increase of Cu2+ in the style is 0.05 mM after Bordeaux mixture application (unpublished data). Investigations are in progress to clarify this point.

No report is available on relation of Cu2+ and Fe2+ to parthenocarpy of the pear. Usually, plant hormones are involved in the fruit set; ethylene induces fruit abscission in apples (Dal et al., 2005; Fukui et al., 1984), Citrus fruit (Gomez et al., 2000; Iglesias et al., 2006), and the Japanese persimmon (Suzuki et al., 1988), and ethylene seems to act as a fatal factor in abscission under various stresses (Tudela and Primo, 1992). There is no report on ethylene biosynthetic inhibition by heavy metal ions. On the inhibition of ethylene action, Ag+ and Cu2+ are reported to block the ethylene effects (Beyer, 1976), but Cu2+ may promote not only ethylene perception but also its signaling (Hirayama and Alonso, 2000). Accordingly, the relation of heavy metal ions to ethylene action is not clear, so fruit sets by Cu2+ are still unclear in the Japanese pear ‘Kosui’. On the other hand, gibberellin can induce parthenocarpy in various fruit tree species such as grapes (Fellman et al., 1991) and the Japanese pear (Nakagawa et al., 1968; Pharis and King, 1985; Vivian and Koltunow, 1999; Zhang et al., 2010). Cytokinins also induce parthenocarpy in some fruit trees (Bangerth and Schroder, 1994; Hayata et al., 1995; Iwahori et al., 1988; Lewis et al., 1996; Ogata et al., 1989; Takagi et al., 1994; Yu, 1999). Accordingly, Cu2+ and Fe2+ may stimulate gibberellin and/or cytokinin production in young flowers and bourses.

Cu2+ and Fe2+ seemed to be effective during any bud stages of the flower to induce parthenocarpy, from sprouting time to 4 days before anthesis. In more developed flowers, the effectiveness was different between the Bordeaux mixture and FeSO4 solution; the former also induced fruit at and after anthesis, but the latter did not. Little information is available on heavy metal absorption by flower organs. Absorption of Cu2+ into the roots is influenced by organic substance concentration, pH, and concentration of heavy metal ions in the root (Hasegawa et al., 2010). Ferrous chelate can be available to the root under several conditions (Hasegawa et al., 2010) so that chelation of Fe2+ may be difficult in older flower tissues of the Japanese pear. Alternatively, the mode of action of Fe2+ may be different from that of Cu2+ on inducing parthenocarpy in the pear.

Although annual changes in the effect of inducing fruit sets were found in Bordeaux mixture and FeSO4 solution treatments, percentages of fruit sets in both treatments were higher than those in control in all years tested. The changes may be due to differences in the environmental conditions; in 2008 and 2012, for example, the effectiveness was low and we had a lot of rainfall during the flowering period (Ministry of Meteorological Agency of Japan, 2015, http://ds.data.jma.go.jp/gmd/tcc/tcc/products/climate/climatview/graph_mkhtml.php?&n=47651&p=24&s=1&r=1&y=2009&m=4&e=0&k=0, June 11, 2015). On the other hand, in 2009, 2011, and 2013, we confirmed an adequate effect and had enough sunshine during flowering seasons. Absorption and/or action of Cu2+ and Fe2+ may be different under different environmental conditions.

The weight of the Bordeaux mixture-induced fruit was significantly lower than that of cross-pollinated fruit; the fruit was often about 250 g, which may be due to seedlessness. Since GA and/or CPPU are known to stimulate fruit growth in the pear, they were tested in this study. When GA paste was applied to fruit stalks 30 DAA, fruit weight became almost the same as cross-pollinated fruit. Fruit growth was much greater when CPPU solution was sprayed 20 DAA first, and then GA paste applied 30 DAA. The sugar content in the fruit was almost the same among all treatments (data not shown). However, the promotion effect of CPPU on fruit growth tended to disappear when the same concentration of CPPU was mixed with GA paste and applied 30 DAA, which may save a lot of labor. Usually, fruit growth is classified into two stages in the pear; 1) the cell division stage from about 10 to 30 DAA, and 2) the cell enlargement stage from about 30 DAA to maturation. Since CPPU enlarges fruit by stimulating cell division, application at 30 DAA may be too late to exhibit its function. Otherwise, the promotive effect of CPPU may be nullified by GA if they are used simultaneously. Investigation is underway to determine the optimum stage of CPPU application and its concentration to improve Bordeaux mixture-induced fruit. CPPU is reported to delay the harvest time of Japanese pear fruit (Zhang et al., 2008), but it may be useful for improving Bordeaux mixture-induced fruit because the maturation delay by CPPU was not observed compared to conventionally cultivated fruit without GA paste (data not shown). At present, GA treatment 30 DAA appears to be useful for producing marketable Bordeaux mixture-induced fruit.

Since the artificial pollination and fruit thinning practices account for about 30% of the total labor in ‘Kosui’ cultivation (Kumashiro et al., 2000), a cultivation system using the Bordeaux mixture is extremely attractive; this system needs no labor for pollination and much less labor for fruit thinning (Hayashida et al., 2015) because the Bordeaux mixture induces far lower fruit sets than with cross-pollination. In addition, we consider that ‘Kosui’ monoculture is possible, which may lead to an increase in fruit yield and a lot of labor saving. Meanwhile, since the Bordeaux mixture had no inhibitory effect on fruit sets in cross-pollinated flowers, the Bordeaux mixture application can also be useful as a supplementary practice to artificial pollination when unseasonable weather is expected such as spring frost or rainfall during anthesis.

In conclusion, the Bordeaux mixture can be substituted for CuSO4 solution to induce parthenocarpy and can achieve an almost ideal fruit set in ‘Kosui’. The Bordeaux mixture-induced fruit can be provided to market by treating with GA paste 30 DAA. Since the Bordeaux mixture is a fungicide used for controlling scab and black spot disease, and GA paste treatment is currently used to promote fruit growth and maturation in ‘Kosui’ fruit production in Japan, these practices do not always require additional labor. The cultivation method proposed here may provide a new cultivation system for ‘Kosui’ fruit production without artificial pollination and much less labor for fruit thinning practice.

Literature Cited
 
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