Sustainable Production Technique of Satsuma

Mandarin using Plant Growth Regulators under

Climate Change

January 2020

Keiko SATO

Sustainable Production Technique of Satsuma

Mandarin using Plant Growth Regulators under

Climate Change

A Dissertation Submitted to

the Graduate School of Life and Environmental Sciences,

the University of Tsukuba

in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Agricultural Science

Keiko SATO Contents

Summary 1

Abbreviations 5

Chapter 1

General introduction 6

Chapter 2

Effects of elevated temperatures on physiological fruit drop, peel puffing and coloring of satsuma mandarin

Section 1 Effects on physiological fruit drop

Introduction 17

Materials and Methods 18

Results 19

Discussion 21

Tables and Figures 24

Section 2 Effects on peel puffing and coloring

Introduction 32

Materials and Methods 33

Results 35

Discussion 38

Tables and Figures 42

Chapter 3

Development of techniques to cope with elevated temperature by use of PGRs of satsuma mandarin

Section 1 Development of techniques to reduce peel puffing

Introduction 50

Materials and Methods 52

Results 56

Discussion 59

Tables and Figures 65

Section2 Development of handpicking techniques

Introduction 74

Materials and Methods 75

Results 80

Discussion 82

Tables and Figures 88

Section 3 Development of enriched vegetative shoots and stable flowering technique in greenhouse

Introduction 100

Materials and Methods 101

Results 104

Discussion 106

Tables and Figures 109

Chapter 4

General discussion 117

Acknowledgements 131

References 132

Summary

Cultivation areas suitable for satsuma mandarin (Citrus unshiu Marc.) have average

annual temperatures of 15–18°C and minimum winter temperatures of more than −5°C.

5 In Japan, the satsuma mandarin is cultivated mainly in the southwestern area of the Pacific

Ocean. Since global warming has progressed in recent years, high temperatures have had

various adverse effects on satsuma mandarin production. In citrus, in addition to the

advancement of the sprouting and flowering period, adverse effects include delayed

flower bud differentiation and an increase in the number of leafless flowers in greenhouse

10 cultivation. Additional adverse effects include increased frequency of physiological fruit

drop, increased fruit size, an increase in sun scalded or peel puffing fruit, low juice acidity

and low storability, and delay or decrease in peel coloring. By the end of this century,

temperatures in Japan will increase by 2.0°C (estimated using a representative

concentration pathway for greenhouse gases of 4.5) or 4.4°C (estimated using

15 representative concentration pathway 8.5), relative to the mean temperatures for 1984–

2004. Thus, global warming countermeasures will become increasingly important in the

near future.

In order to overcome the problems caused by climate change, the following research

was conducted to maintain the sustainable and stable production of satsuma mandarin.

20 First, we examined the extent of the effects of increasing temperature on physiological

fruit drop, peel puffing, and peel coloring in satsuma mandarin. Next, we developed three

techniques that utilize plant growth regulators to reduce the damage that is currently

prevalent due to global warming. The first of these techniques reduces peel puffing, the

second is a technique to improve upon the handpicking method to shorten the time

1 required for harvesting, and the third technique increases flowering, especially leafy

flowers, in greenhouse cultivation.

We first examined the effect of elevated temperature on physiological fruit drop in

satsuma mandarin cultivation. The cumulative fruit drop rate was high under temperatures

5 elevated by 2°C from about 10 to 20 days after bloom for early, medium, and late ripening

satsuma mandarins. The final drop rate increased by about 5% under temperatures

elevated by 2°C for early and medium ripening satsuma mandarins. The diameter of early

and medium ripening satsuma mandarin fruit increased when the fruit was treated with

temperature elevated by 2°C. Our results indicate that an average temperature increase of

10 2°C for about two months after bloom promoted fruit enlargement and increased the

number of physiological dropped fruit.

Next, we examined the effects of elevated temperature from the flowering to the

physiological fruit drop period and the maturity period on the peel puffing and coloration

of satsuma mandarin fruit. Peel puffing increased from the flowering to the physiological

15 fruit drop stage at temperatures elevated by 4°C and increased during the maturity stage

at temperatures elevated by 2 or 4°C. Peel coloring was promoted from the flowering to

the physiological fruit drop period at temperatures elevated by 2 or 4°C, while peel

coloring deteriorated during the maturity stage at temperatures elevated by 2°C. It was

clearly demonstrated that not only the increase in temperature during the maturity period

20 but also the increase in temperature from the flowering to the physiological fruit drop

period affected the peel puffing and coloring of satsuma mandarin fruit.

To reduce the various kinds of damage caused by global warming, we developed three

techniques using plant growth regulators. First, we developed a technique to reduce peel

−1 −1 puffing. Combinations of 1 mg∙L gibberellin A3 and 50 mg∙L prohydrojasmon, 3.3

2 −1 −1 −1 mg∙L gibberellin A3 and 25 mg∙L prohydrojasmon, and 3.3 mg∙L gibberellin A3 and

50 mg∙L−1 prohydrojasmon markedly inhibited peel puffing when compared with no

treatment. The coloring was delayed more than one week by these combined sprayings.

−1 −1 The combination of 1 mg∙L gibberellin A3 and 25 mg∙L prohydrojasmon also

5 inhibited peel puffing. The coloring was delayed just under one week by the combined

spraying. Combined sprayings of gibberellin A3 and prohydrojasmon for extremely early,

early, and medium ripening satsuma mandarin cultivars were also conducted at different

spray times from Aug. to Oct. The effects of the spraying on peel puffing and coloring

delay for the extremely early ripening cultivar were the highest from the middle of Aug.

10 to late Sep. In the early and medium ripening cultivars, the effects of spraying on peel

puffing and coloring delay were the highest in Sep.

Secondly, we examined the efficiency of handpicking fruit (and consequently the

shortening of harvest time) for satsuma mandarin treated with plant growth regulators

such as gibberellin A3, prohydrojasmon, and ethephon. Our results indicate that treatment

−1 −1 15 with 5 mg∙L gibberellin A3 plus 50 mg∙L prohydrojasmon in late Sep. combined with

ethephon (200 mg∙L−1 < ethephon < 300 mg∙L−1) from late Oct. to early Nov. was suitable

for increasing the success rate of handpicking. The success rate of handpicking increased

30–85% relative to no treatment, and the combination spraying did not impair the internal

fruit quality or cause serious leaf abscission.

20 Finally, we developed a technique for increasing the number of leafy flowers in

greenhouse cultivation. Leafy flowers may decrease, and leafless flowers may increase

under global warming conditions in heated greenhouse cultivation. In satsuma mandarin,

the quality of fruit from leafy inflorescences with around four leaves is higher than that

from leafless flowers. Enriched vegetative shoots can generate leafy flowers, which

3 produce good-quality fruit. In early-heating greenhouses heated from Nov., summer

shoots flushed by pruning after harvest were used as fruiting mother shoots (vegetative

shoots). However, sprouting some weak shoots from these vegetative shoots degraded the

enrichment of the vegetative shoots. Therefore, development of fall shoots from the

5 summer shoots was inhibited by means of ethychlozate. However, ethychlozate-induced

decline of tree vigor is a problem if ethychlozate is used every year. When 1-

naphtaleneacetic acid is used to inhibit shoot development, it is reported to enhance the

shape of vegetative shoots and improve flowering. Thus, the effects of 1-naphtaleneacetic

acid on the flowering ability and concentration of endogenous nutritional elements of the

10 vegetative shoots were compared to those of ethychlozate. Regarding flowering ability, it

was evident that physiological flower bud differentiation proceeded at a more rapid pace

and was completed earlier, and the number of leafy flowers was greater with 1-

naphtaleneacetic acid treatment. With 1-naphtaleneacetic acid treatment, the branches and

leaves contained more starch and less nitrogen relative to those under ethychlozate

15 treatment. These findings suggest that plants treated with 1-naphtaleneacetic acid will

have better floral evocation and thus bear more leafy flowers than those treated with

ethychlozate.

In this study, we used plant growth techniques to develop some techniques to

counteract the effects of global warming on satsuma mandarin cultivation. These

20 techniques will greatly contribute to sustainable satsuma mandarin production under

global warming.

4 Abbreviations

AMeDAS automated meteorological data acquisition system

ANOVA analysis of variance

ET ethephon

GA gibberellin

GA3 gibberellin A3

HLB huanglongbing

JA jasmonate

MJ methyl jasmonate

NAA 1-naphtaleneacetic acid

1-MCP 1-methylcyclopropene

PDJ prohydrojasmon

PGR plant growth regulator

RCP representative concentration pathway

SRH success rate of handpicking

SSC soluble solids content

5 Chapter 1

General introduction

5 Citrus fruits are among the most cultivated worldwide, the total citrus fruit production

for 2017 has been estimated at 146.6 million tons (9275.9 kha) (FAO, 2019), which was

16.9% of total fruit production. Among citrus fruit production, sweet oranges account for

50.0%, followed by mandarins (22.8%), lemons and limes (11.7%), and pomelos and

grapefruits (6.2%). The genus Citrus including sweet oranges and mandarins, and related

10 genera (Fortunella, Poncirus, Eremocitrus and Microcitrus) belong to the angiosperm

subfamily Aurantioideae of the Rutaceae family (Wu et al., 2018).

The satsuma mandarin (Citrus unshiu Marc.) is one of many types of mandarins, and

is the fruit with the highest production volume in Japan (Japan Fruit Growers Cooperative

Association, 2018). Satsuma mandarins accounted for 71.6% (805.1 kt) of all citrus

15 production (1124.1 kt) in 2016 in Japan, followed by ‘Shiranui’ (42.4 kt), hassaku (34.9

kt), natsudaidai (34.0 kt), iyo (32.9 kt), yuzu (26.8 kt), ponkan (23.4 kt), and ‘Kiyomi’

(14.0 kton) [Ministry of Agriculture, Forestry and Fisheries (MAFF), 2017]. Satsuma

mandarin can be divided into four groups depending on fruit maturation: extremely early,

early, medium and late ripening cultivars (Mukai and Kadoya, 1994). The times of harvest

20 are from mid Sep. to early Nov. for extremely early ripening cultivars, from mid Oct. to

late Nov. for early ripening cultivars, during Nov. for medium ripening cultivars, and from

late Nov. to late Jan. for late ripening cultivars. Satsuma mandarins are also cultivated in

heated greenhouses, and are mainly cultivated in early ripening cultivars (Mukai and

6 Kadoya, 1994). The production of greenhouses growing early ripening satsuma mandarin

was 20 kt in 2018 (MAFF, 2019).

Satsuma mandarin originated in Japan before the Edo period, but was spread

nationwide after the Meiji era and has a history of cultivation for over 100 years (Mukai

5 and Kadoya, 1994; Omura and Shimada, 2016). The fruit has many favorable quality

advantages, including juiciness, sweetness, low acidity, seedlessness, and easy peeling. It

is also rich in β-cryptoxanthin, a functional component that is good for health (Sugiura et

al., 2015). It features cultivation advantages such as high productivity, disease resistance,

parthenocarpy, and male sterility (Omura and Shimada, 2016). In addition, satsuma

10 mandarin is used for breeding and is the parent of cultivars and lines of major citrus

cultivars in Japan, such as ‘Kiyomi’ and ‘Shiranui’. In recent years, the whole genome of

citrus has been deciphered, and genome information has been published on satsuma

mandarin (Kawahara et al., 2020; Shimizu et al., 2017), clementine (Wu et al., 2014),

sweet orange (Xu et al., 2013; Wu et al., 2014), pomelo (Wang et al., 2017), ponkan

15 (Wang et al., 2018), and trifoliate orange (Kawahara et al., 2020). Genomic analysis

revealed that the parents of satsuma mandarin are ‘Kishuu mikan’ (Citrus kinokuni hort.

ex Tanaka) and ‘Kunenbo’ (Citrus nobilis Lour. var. kunip Tanaka) (Fujii et al., 2016;

Shimizu et al., 2017).

Satsuma mandarin plants grafted onto trifoliate orange trees are the most cold-

20 resistant citrus plants on the market (Yelenosly, 1985). Areas suitable for satsuma

mandarin cultivation have average annual temperatures of 15–18°C, and minimum winter

temperatures of more than −5°C (MAFF, 2015). In Japan, satsuma mandarins are

cultivated in about 20 prefectures, mainly in the southwestern area of the Pacific Ocean

(Landaniya, 2010). The prefecture with the highest production of satsuma mandarin in

7 Japan in 2018 was Wakamaya Prefecture (155.6 kt), followed by Shizuoka Prefecture

(114.5 kt), Ehime Prefecture (113.5 kt), Kumamoto Prefecture (94 kt), Nagasaki

Prefecture (49.7 kt), Saga Prefecture (46.5 kt) (MAFF, 2019). Outside Japan, satsuma

mandarin are produced in Jiangxi, Hubei, and Hunan provinces, and the Guangxi

5 Autonomous Region in China, as well as in Spain, Italy, Israel, Australia, New Zealand,

Argentina, Uruguay, Peru and South Africa (Landaniya, 2010). It is also produced on Jeju

Island, Korea (Choi, 2004), as well as in Turkey (Aksoy et al., 1998), the Gulf of the

United States (Ebel et al., 2004; Ebel et al., 2005).

In Japan, the annual average temperature is rising at rates of 1.21°C per century [Japan

10 Meteorological Agency (JMA), 2019]. The Ministry of the Environment (MOE) and JMA

(2015) estimated that temperatures in Japan would increase by 2.0°C using the

representative concentration pathway (RCP) for greenhouse gases of 4.5, or by 4.4°C

using RCP8.5 by the end of this century, relative to the mean temperatures for 1984–2004.

Therefore, measures to mitigate global warming are becoming increasingly important.

15 In recent years, because of the progress of global warming, various adverse effects on

fruit tree production are occurring in Japan. In fruit trees, the acceleration of budding and

flowering, acceleration of harvesting, delayed fruit coloring, reduction of acidity and

astringency, increase or decrease in freezing injury, increase in frost damage, and change

in disease and pest statuses have been reported (Sugiura et al., 2012).

20 The recent progression of global warming has caused several effects on satsuma

mandarin cultivation owing to high temperatures. Increased physiological fruit drop not

only reduces the annual yield for satsuma mandarin production, but also creates the

serious problem of promoting alternate bearing. In Wakayama Prefecture, there is

intensified alternate bearing due to physiological fruit drop in both the ‘Okitsu wase’ and

8 ‘Hayashi unshiu’ cultivars (Hagihara, 2011). Various factors affect physiological fruit

drop. Among these, fruit drop is promoted when there are continuous days where the

maximum daytime temperature is 25–30°C or higher during the fruit drop period. In 2011,

there were successive days with high temperatures from late Jun. to Jul., which

5 significantly promoted secondary physiological fruit drop, leading to a significant

decrease in production (Takatsuji, 2015).

Peel puffing is also a major problem for satsuma mandarin production. This is a

physiological disorder where the fruit rind tissues are separated from the flesh, which

causes problems such as reduced taste and shelf stability (Fig. 1–1). Peel puffing is closely

10 linked to high temperatures and heavy rainfall in late fall, and high humid conditions are

the primary cause; high temperature further promotes this effect (Yokoo et al., 1963;

Kawase, 1984). Therefore, if there is an increase in temperature around the time the fruit

is ripening, and there is persistent rainfall, there is an increased incidence of fruit with

peel puffing. Many prefectures have reported the effects of peel puffing (MAFF, 2018a

15 and 2018b; Table 1–1). The susceptibility to peel puffing of satsuma mandarin varies

depending on the cultivar (Kawase, 1984). In extremely early ripening satsuma mandarins,

‘Iwasaki wase’ tends to develop peel puffing while ‘Yura wase’ has minimum peel puffing.

In early ripening satsuma mandarins, peel puffing is a problem in the cultivar ‘Kitahara

wase’. In medium and late ripening satsuma mandarins, peel puffing is a problem in the

20 major cultivars: ‘Nankan No. 20’, ‘Mukaiyama unshiu’ and ‘Aoshima unshiu’, while

‘Ishiji’, ‘Sasebo unshiu’, ‘Juuman unshiu’ and ‘Imamura unshiu’ have minimum peel

puffing. However, cultivars with less peel puffing tend to require skill in cultivation

compared to the major cultivars that easily develop peel puffing.

9 While warming delays the start of harvesting due to delayed peel coloring, delayed

harvesting increases physiological disorders such as peel puffing and granulation. Thus,

since global warming is expected to cause the shortening of the suitable harvest period, it

is vital to shorten the working time needed for harvesting as much as possible. The

5 working hours needed to produce satsuma mandarins are concentrated in harvesting and

adjustment work. Since the mean number of hours spent on such work is 74.6 h · 10 a−1

(MAFF, 2010), it is difficult to shorten the harvest period. Satsuma mandarin harvesting

is generally performed with scissors, but handpicking has been investigated as a technique

that would significantly reduce harvesting time (Noro et al., 1965; Yamada et al., 1982;

10 Fig. 1–2). Handpicking does not use scissors; instead, the fruit are pulled from the trees

by hand, which approximately doubles the harvesting efficiency over harvesting with

scissors (Noro et al., 1965; Yamada et al., 1982). Handpicking has been implemented with

hassaku (Citrus hassaku), but it has not been implemented with satsuma mandarins as

handpicking breaks the soft peel of this fruit.

15 The increase in mean temperatures caused by global warming also affects flowering,

and reports were shown that the percentage of leafless flowers increases and that of leafy

flowers decreases as the temperature increases (Iwasaki et al., 2018; Ono and Iwasaki,

2010; Fig. 1–3). Fruit quality on leafy inflorescences with around four leaves are higher

than those on leafless flowers or leafy flowers with more than six leaves (Suzuki, 1973).

20 Thus, an increase in the percentage of leafless flowers due to global warming may lead

to a decline in fruit quality. This effect may be even more notable in greenhouse-cultivated

satsuma mandarin.

Plant growth regulator (PGRs) are used in a variety of applications in fruit tree

cultivation, such as chemical thinning and the reduction of fruit disorders, and are very

10 useful techniques for labor saving and stable production. Using PGRs like 1-

naphtaleneacetic acid (NAA), ethychlozate, gibberellin A3 (GA3), prohydrojasmon (PDJ),

and ethephon (ET) is also considered effective for global warming countermeasures.

Their characteristics and purposes for use in fruit trees are described below. Auxin is a

5 plant hormone that is involved in plant growth and in organizing many growth processes

(Woodward and Bartel, 2005). NAA, which is one of the major synthetic auxins, has been

recognized as a fruit abscission agent in citrus (Guardiola and Garcia-Luis, 2000), apple

(Dennis, 2000; Wertheim, 2000), and olive (Dag et al., 2009). It has been observed that

fruit growth is promoted when treated with a fruit abscission agent in citrus (Guardiola

10 and Garcia-Luis, 2000), apples (Dennis, 2000; Wertheim, 2000), and olives (Dag et al.,

2009). In addition, NAA suppress preharvest fruit drop in apples (Yuan and Carbaugh,

2007). NAA also has a sprouting inhibitory effect on citrus (Nauer and Boswell, 1978;

Phillips and Tucker, 1974), apple (Miller and Ware, 1980; Raese, 1975), pear (Keever et

al., 1998; Raese, 1975), and peach (Aldrich and Arnold, 1982). Ethychlozate, a synthetic

15 auxin, was developed as a fruit abscission agent for satsuma mandarin (Hirose, 1981;

Kamuro and Hirai, 1981). Fruit thinning effects have been observed in grapes (Ozaki and

Ichii, 1986). This chemical also promoted coloration and increased sugar content in

mature fruit of satsuma mandarin (Hirose, 1981; Tominaga and Daito, 1981). A coloring

promoting effect has been recognized in persimmons (Chijiwa et al., 2000). In addition,

20 ethychlozate has a reducing effect on peel puffing (Kawase et al., 1985). Gibberellin (GA)

is a plant hormone that plays important roles in various aspects of plant growth and

development, including stem elongation, seed germination, flower development, fruit set,

and leaf and fruit senescence (Sponsel, 2016). Exogenous GAs are widely used in

agriculture and horticulture (Rademacher, 2016). GA3, GA4 and GA7 are mainly used as

11 PGRs. In citrus, exogenous GA is mainly used to promote fruit set and to suppress

flowering. GA treatment also reduces rind disorders such as peel puffing, water spotting,

and creasing. PDJ is an analog of jasmonate (JA) (Koshiyama et al., 2006). JA is a plant

hormone, that plays an important role in inducing responses of plants to biotic or abiotic

5 stress (Browse, 2009; Campos et al., 2014; Wasternack and Hause, 2013). Preharvest PDJ

spraying promotes fruit coloring in apples, grape (Koshiyama et al., 2006), and mangoes

(Sudheeran et al., 2019). Ethylene plays an important role in fruit ripening in a wide range

of plant species (Abeles et al., 1992). ET is a chemical that generates ethylene and is used

in fruit tree cultivation for the purpose of promoting fruit maturation, fruit dropping, leaf

10 fall, and flowering. In addition, since ethylene promotes the abscission layer of fruit and

promotes fruit dropping, ET has been used as a fruit thinning agent and researched as an

abscission agent.

This study has undertaken the following research with the aim of maintaining the

stable production of satsuma mandarin plants despite the various problems caused by

15 climatic changes. First, we verified the effect of high temperature on satsuma mandarin

cultivation, and in particular, examined the extent the effect of increased temperature on

physiological fruit drop, peel puffing, and peel coloring in the future. Next, we developed

three techniques to mitigate the damaging effects that have been already occurred due to

global warming using PGRs: the first is a technique to reduce peel puffing, the second is

20 a technique to develop handpicking techniques to shorten the time needed for harvesting,

and the third is a technique to increase flowering using greenhouse cultivation in global

warming conditions.

12 Table 1–1. Prefectures with the occurrence of physiological disorders of satsuma mandarin in Japan from 2008 to 2017. Number of prefuctures Mainly effectsx 2008z 2009 2011y 2012 2013 2014 2015 2016 2017

Peel puffing 7 9 12 6 5 8 11 14 13 Poor coloring /coloring 10 7 5 4 7 1 2 6 7 delay Sunscald 9 6 5 5 6 4 2 5 5 Increase of physiolosical 6 1 fruit drop 13 zEffect of climate change on citrus (including satsuma mandarin) in Japan from 2008 to 2009. yEffect of climate change on satsuma mandarin in Japan from 2011 to 2017. xMain effects include, in addition to the above, advancement of sprouting and flowering period, poor sprouting, poor fruit growth after 2011. This table was made by processing MAFF (2018a, 2018b). (a) Puffing fruit Left: puffing fruit; right: normal fruit.

(b) Poor coloring

(c) Sunscald

Fig. 1–1. Main effects by global warming on fruits of satsuma mandarin. (a) puffing fruit, (b) poor coloring, (c) suncald.

14 (a) Harvest with scissors (general method)

(b) Handpicking

Fig. 1–2. Harvesting methods of satsuma mandarin. (a) harvest with scissors (general method), (b) handpicking.

15 (a) Leafless flowers

(b) Leafy flowers

Fig. 1–3. Flower types. (a) leafless flowers (one apical flower and no leaves on a single shoot), (b) leafy flowers (one apical flower with one or more leaves on a single shoot) . Photos by Mr. H. Fukamachi.

16 Chapter 2

Effects of elevated temperatures on physiological fruit drop, peel puffing and coloring of

satsuma mandarin

5 Section 1 Effects of elevated temperatures on physiological fruit drop

Introduction

There have been many reports on the relationship between physiological fruit drop

10 and temperature in satsuma mandarin (Inoue and Chien, 1987; Iwagaki et al., 1977; Ogata

et al., 2002; Taniguchi, 1983; Yamamoto et al., 1973). Regarding the relationship between

daily maximum temperature and the fruit drop rate during the physiological fruit drop

period, fruit drop was increased by temperatures over 30°C (Iwagaki et al., 1977;

Yamamoto et al., 1973) or over 25°C (Taniguchi, 1983). In these reports, the amount of

15 fruit drop was evaluated at different temperatures. However, the differences among the

temperature treatments were greater than 5°C; there were few reports to investigate

temperature differences smaller than 5°C, which was the difference among our

temperature treatments. MOE and JMA (2015) estimated that by the end of this century,

temperatures in Japan would increase by 2.0°C, based on the RCP for greenhouse gases

20 of 4.5 relative to the mean temperatures for 1984–2004.

The goal of this study was to clarify the effects of temperatures elevated by 2°C during

the physiological fruit drop period on physiological fruit drop using five cultivars of

satsuma mandarin.

17 Materials and Methods

Plant materials

The study was conducted during 2003–2005, 2007, at the NARO Institute of the

5 Kyushu Okinawa Agricultural Research Center, the Kuchinotsu Citrus Research Station,

(Kuchinotsu, Minamishimabara, Nagasaki, Japan; 32.60°N, 130.18°E). Satsuma

mandarin (Citrus unshiu Marc., ‘Miyagawa wase’, ‘Okitsu wase’, ‘Ishiji’, ‘Sasebo unshiu’

and ‘Shirakawa’) trees had been grafted onto the rootstocks of trifoliate orange (Poncirus

trifoliate (L.) Raf.) trees. ‘Miyagawa wase’ and ‘Okitsu wase’ were early ripening

10 cultivars. ‘Ishiji’ and ‘Sasebo unshiu’ were middle ripening cultivars. ‘Shirakawa’ was a

late ripening cultivar. The grafted trees were planted in polypropylene pots or unglazed

ceramic pots and grown in the field. Experiment years, ages of trees, full bloom date,

beginning day of treatment (days after full bloom), leaf flower (fruit) ratios at beginning

of treatment, and repetitions are shown in Table 2–1.

15

Temperature treatment using phytotron

Temperature treatments were conducted for almost two months from 0–13 days after

full bloom using two phytotrons (1,300 mm × 1,300 mm × 1,800 mm or 2,500 mm ×

2,000 mm × 2,000 mm, Koito Ind. Ltd., Tokyo, Japan). One phytotron room was set at

20 the mean temperatures for Kuchinotsu (control), based on the 1990–2000 climate normal

for temperature for 2003 and 2004 experiments, the 1978–2004 climate normal for

temperature for the 2005 experiment, and the 1978–2006 climate normal for temperature

for the 2007 experiment. The temperature was obtained from a weather observation

system located in our experimental field. The ten-day means of daily maximum,

18 minimum, and average temperatures during the treatment periods are shown in Tables 2–

2, 2–3, and 2–4. The other phytotron room was set to + 2°C relative to the temperatures

in the control. The temperatures were set to vary depending on the time of day, with

minimum temperatures from 20:00 to 6:00 h and maximum temperatures from 8:00 to

5 18:00 h in 2003 and 2004 experiments, as shown in Fig. 2–1. The temperature rose or

descended linearly for two hours from 6:00 to 8:00 h or from 18:00 to 20:00 h. In 2005

and 2007, the transition of temperature was drawn with a smooth curve, with the

maximum temperature at 12:30, minimum temperature at 0:30, and mean temperature at

19:30 and 5:30 (Fig. 2–1). The daytime length was set at 14 h, and the nighttime length

10 was set at ten hours because the average daytime length from May to Jul. is 14 h and the

nighttime length is ten hours in Nagasaki. Humidity was not controlled. The average

humidity in 2007 was 94%. Humidity were measured using data loggers (RTR-53A, T&D

Corporation, Nagano, Japan).

15 Fruit drop and diameter examination method

The fruit drop rate was measured by counting the fruit on trees every three to seven

days in 2003–2005. The fruit drop rate was measured by counting the fruit that fell from

trees every three to four days in 2007. The leaf-fruit (flower) ratio was obtained by

dividing the total number of leaves, including the old and new leaves, by the total number

20 of fruits in 2003–2005. The leaf-fruit ratio was obtained by dividing the number of old

leaves by the total number of fruits in 2007. The fruit diameters of ten fruits per tree were

measured during treatment in 2005.

Results

19 Effect of 2°C elevated temperature on physiological fruit drop

The effects of temperature elevated by 2°C on physiological fruit drop in satsuma

manrain trees are shown in Fig. 2–2. In ‘Okitsu wase’, the increase in the cumulative fruit

5 drop rate was rapid until about 40 days after full bloom both in the control and at + 2°C,

but then became more gradual. The cumulative fruit drop rate at + 2°C was always higher

than that in the control from 17 days after full bloom until the end of treatment. The final

drop rate was 95.6% in the control and 100% at + 2°C. In ‘Ishiji’, the increase in the

cumulative fruit drop rate in 2003 was rapid until 30 days after full bloom both in the

10 control and at + 2°C, and then slowed. After 51 days in bloom, fruit drop was almost

finished. The cumulative fruit drop rate at + 2°C was always higher than that of the control

from 16 days after full bloom until the end of treatment. The final fruit drop rate was

94.8% in the control and 100% at + 2°C. The increase in the cumulative fruit fall rate in

‘Ishiji’ in 2007 was rapid until 30 to 40 days after full bloom both in the control and at +

15 2°C, and then slowed. The cumulative fruit drop rate at + 2°C was always higher than

that of the control from 12 days after full bloom until the end of the survey. The final fruit

drop rate was 89.9% in the control and 93.8% at + 2°C. In ‘Shirakawa’, the increase in

the cumulative fruit drop rate was rapid until 26 days after full bloom both in the control

and at + 2°C, and then became almost constant. The final fruit drop rate was 99.1% in the

20 control and 98.7% at + 2°C.

The effects of temperature elevated by 2°C on the cumulative fruit drop rate for

‘Miyagawa wase’ and ‘Sasebo unshiu’, where the number of fruits was controlled by

thinning, are shown in Fig. 2–3. In ‘Miyagawa wase’, the increase in the cumulative fruit

drop rate was rapid until 27 days after full bloom both in the control and at + 2°C, and

20 then gradually decreased and became almost constant 45 to 48 days after full bloom. The

final fruit drop rate was 69.9% in the control and 74.2% at + 2°C. In ‘Sasebo unshiu’, the

increase in the cumulative fruit drop rate was rapid until 27 days after full bloom both in

the control and at + 2°C, and then became almost constant until 40 days after full bloom,

5 when it increased again. After 48 days in full bloom, physiological fruit drop was almost

finished. The cumulative fruit fall rate at + 2°C was always higher than that of the control

from 19 days after full bloom until the end of treatment. The final fruit drop rate was

59.4% in the control and 85.4% at + 2°C.

10 Effect of 2°C elevated temperature on fruit growth

In both ‘Miyagawa wase’ and ‘Sasebo unshiu’, fruit growth was faster at + 2°C than

in the control (Fig. 2–4). In ‘Miyagawa wase’, after 20 days of full bloom, the fruit grew

at + 2°C more vigorously than in the control, and the difference gradually increased. The

difference in the final fruit diameter 58 days after full bloom was completed was about

15 3.0 mm. In ‘Sasebo unshiu’, the fruit diameter at + 2°C was about 0.5–2.0 mm larger than

that in the control.

Discussion

20 Inoue and Chien (1987) investigated the effects of temperature on early fruit drop for

around two months after full bloom in satsuma mandarin using phytotron rooms that were

held at various daytime and nighttime temperatures in combination with 15, 20, and 25°C.

A relatively high temperature of 25/25°C (day/night) caused an earlier peak of fruit drop,

a shorter fruit drop period, and severer fruit drop (99.1%), while below 20/20°C caused a

21 later peak of fruit drop, a longer fruit drop period, and lower fruit drop (71.4–92.9%) than

those grown at other temperatures. In this study, the cumulative fruit drop rate has

remained high at + 2°C from about ten to 20 days after full bloom (Figs. 2–2, 2–3). The

final drop rate increased by about 5% for ‘Okitsu wase’ and ‘Ishiji’, and by 26% for

5 ‘Sasebo unshiu’. The average temperature was 20.6°C in the control and 22.6°C at + 2°C

for both 2005 and 2007 in this study. The highest temperature exceeded 25°C in the

control in early Jun. and at + 2°C in late May. The time when the maximum temperature

rises above 25°C, which is the temperature that promotes physiological fruit drop, was

about ten days earlier at + 2°C than in the control. It was clarified that even such a slight

10 temperature increase of 2°C increased the final physiological fruit drop rate.

High temperatures during the young fruit (cell division) stage promoted fruit

enlargement in Japanese pears and peaches (Hayama et al., 2007; Sugiura et al., 1995).

Inoue and Chien (1987) reported that in satsuma mandarin, a relatively high temperature

of 25/25°C during around one month after full bloom caused the growth of larger fruit

15 than those grown at 15/15°C during the same period. In this study, fruit diameter was

increased in fruit treated with + 2°C (Fig. 2–4). The average temperature was 20.6°C in

the control and 22.6°C at + 2°C in both 2005 and 2007. The highest temperature exceeded

25°C in the control in early Jun. and at + 2°C in late May. Thus, since the temperature at

+ 2°C was more suitable for fruit enlargement than that of the control, fruit enlargement

20 would be promoted. The final fruit drop rate was high at + 2°C because fruit enlargement

was promoted at + 2°C, and the nutrient competition between the fruits became stronger.

In heated-cultured satsuma mandarin, there is a technique to raise the temperature from

ten days after full bloom in the physiological fruit drop period in order to promote the

enlargement of the remaining fruit by dropping immature flowers and fruit (Shiraishi,

22 1989). Thus, temperature elevated by 2°C in the early period of physiological fruit drop

would be highly effective in promoting the enlargement of remaining fruit, which should

work positively for cultivation.

The experimental scheme for temperature changes throughout the day was greatly

5 different between the 2003 and 2004 experiments and the 2005 and 2007 experiments

(Fig. 2–1). However, there was no significant difference in the timing or degree of the

physiological fruit drop rate between the 2003 and 2004 trials and the 2005 and 2007

trials. Thus, it was speculated that the temperature changes had no effect on the

physiological fruit drop, although there was a difference of about 8°C between the highest

10 and lowest temperatures.

In ‘Sasebo unshiu’, the difference in final fruit yield between + 2°C and the control

was 26% (Fig. 2–3), which was significantly larger than the difference in the final fruit

yield of other varieties (5%; Figs. 2–2, 2–3). It is a problem that there are many

physiological fruit drops, leading to decrease in setting fruit in ‘Sasebo unshiu’ (Furukawa,

15 2007). It is thought that there are many sprouting shoots, leading to many fruit drop in

‘Sasebo unshiu’. The translocation of photosynthate to new shoots increases as

temperature rises (Ono et al., 1987); therefore, fruit drop would be severer in higher

temperatures.

23 Table 2–1. Condition of trees at the beggining of the tests in each year.

Beginning day of Leaf-flower (fruit) Experiment Cutivar Age of trees Full bloom date treatment ratio at beginning of Repetitions year (days after full bloom) treatment Okitsu wase 2007 Two-year-old Apr. 30th 7 0.7 5 Ishiji 2003 Two-year-old May 3rd 9 0.8 3 Ishiji 2007 Five-year-old Apr. 28th 0 1.5 3 Shirakawa 2004 Five-year-old Apr. 30th 6 1.2 2 Miyagawa wase 2005 Two-year-old May 3rd 13 5 (after thinning) 2 2 for the Control Sasebo unshiu 2005 Three-year-old May 3rd 13 10 (after thinning) 3 for + 2°C 24 Table 2–2. Daily average, mean daily minimum, and mean daily maximum temperatures (°C) during physiolosical fruit drop periods in Kuchinotsu in 2003 and 2004.

Period of Temperature (°C) temperature treatment Daily average Mean minimum Mean maximum early May 17.6 13.6 21.6 mid May 18.5 14.8 22.4 late May 19.5 15.6 23.5 early Jun. 20.8 17.5 24.6 mid Jun. 22.1 19.2 25.6 late Jun. 23.2 20.9 26.2

25 Table 2–3. Daily average, mean daily minimum, and mean daily maximum temperatures (°C) during physiolosical fruit drop periods in Kuchinotsu in 2005.

Period of Temperature (°C) temperature treatment Daily average Mean minimum Mean maximum early May 17.7 13.9 21.6 mid May 18.6 14.7 22.6 late May 19.9 16.0 24.1 early Jun. 21.2 17.5 25.2 mid Jun. 22.4 19.4 25.7 late Jun. 23.6 21.0 26.5

26 Table 2–4. Daily average, mean daily minimum, and mean daily maximum temperatures (°C) during physiolosical fruit drop periods in Kuchinotsu in 2007.

Period of Temperature (°C) temperature treatment Daily average Mean minimum Mean maximum early May 17.8 14.0 21.8 mid May 18.6 14.8 22.6 late May 19.9 16.0 24.0 early Jun. 21.3 17.6 25.4 mid Jun. 22.4 19.4 25.8 late Jun. 23.7 21.1 26.6

27 (a)

2003，2004 Daily Max. ) ℃

( Control

+ 2℃ Temeperature Temeperature

Daily Min.

6:00 8:00 18:00 20:00 6:00 Time

(b)

Daily 2005，2007 ) Max. ℃ (

Daily Mean Temeperature Temeperature

Daily Min.

5:30 12:30 19:30 0:30 5:30 Time

Fig. 2–1. Experimental scheme for temperature changes throughout the day. (a) in 2003 and 2004 experiments, (b) in 2005 and 2007 experiments.

28 100 100 (a) (b)

％ ) 80 ％ ) 80 ( (

60 60

40 Control 40

+ 2 ℃ Cumulative fruit drop fruit Cumulative 20 drop fruit Cumulative 20 Okitsu wase (2007) Ishiji (2003)

0 0 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 Days after full bloom Days after full bloom

100 100 (c) (d) ％ ) ％ )

( 80 ( 80

60 60

40 40 Cumulative fruit drop fruit Cumulative drop fruit Cumulative 20 20 Ishiji (2007) Shirakawa (2004)

0 0 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70

Days after full bloom Days after full bloom

Fig. 2–2. Effect of temperature elevated by 2°C on physiological fruit drop in satsuma mandarin trees. (a) cumulative fruit drop of ‘Okitsu wase’ in 2007 (n = 5), (b)‘Ishiji’ in 2005 (n = 3), (c) ‘Ishiji’ in 2007 (n = 3), (d)‘Shirakawa’ in 2004 (n = 2). Closed symbol: Control, opend symbol: + 2°C.

29 100 Control 100

+ 2 ℃ ％ ) 80 ％ ) 80 ( (

60 60

40 40 (a) (b) Cumulative fruit drop fruit Cumulative 20 drop fruit Cumulative 20 Miyagawa wase Sasebo unshiu (2005) (2005) 0 0 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 Days after full bloom Days after full bloom

Fig. 2–3. Effect of temperature elevated by 2°C on physiological fruit drop in satsuma mandarin trees with regulated leaf-fruit ratio. (a) cumulative fruit drop of ‘Miyagawa wase’ in 2005 (n = 2), (b)‘Sasebo unshiu’ in 2005 (n = 2 for the Control, n = 3 for + 2°C ). Closed symbol: Control, opend symbol: + 2°C.

30 30 30 Control

25 + 2 ℃ 25

20 20 (mm) (mm)

15 15

10 (a) 10 (b) Fruit diameter Fruit diameter 5 Miyagawa wase 5 Sasebo unshiu （2005） （2005） 0 0 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70

Days after full bloom Days after full bloom

Fig. 2–4. Effect of temperature elevated 2°C on fruit diameter in satsuma mandarin with regulated leaf-fruit ratio. (a) fruit diameter of ‘Miyagawa wase’ in 2005 (n = 2), (b)‘Sasebo unshiu’ in 2005 (n = 2 for the Control, n = 3 for + 2°C ). Closed symbol: Control, opend symbol: + 2°C.

31 Section 2 Effects of elevated temperatures on peel puffing and coloring

Introduction

5 In Japan, increased peel puffing and reduced peel pigmentation have already become

problems in satsuma mandarin production due to recent global warming (Sugiura et al.,

2007). Puffy mandarins are easily damaged during picking, transporting, sorting, and

packing, and their commercial value is dramatically reduced compared with normal fruit

(Fig. 1–1). These fruits are susceptible to earlier postharvest decay and taste deterioration

10 when compared with normal fruits, and therefore, puffy mandarins cannot be stored for

long periods. Climate conditions at the fruit maturation stage affect peel puffing; the

incidence of peel puffing increases with elevated relative humidity and/or elevated

temperature (Kawase, 1984; Yokoo et al., 1963). There also have been many studies on

the effects of air temperature during the maturation stage on the levels of peel

15 pigmentation (Kurihara, 1968; Nii et al., 1970; Reuther, 1973; Utsunomiya et al.,1982).

Both the decomposition of chlorophyll and the biosynthesis of carotenoids in the peel are

suppressed at 30°C or higher during this stage (Nii et al., 1970; Utsunomiya et al., 1982).

By contrast, there have been few studies on the effects of temperature during the

flowering to physiological fruit drop stage on fruit quality, including peel puffing and peel

20 pigmentation.

MOE and JMA (2015) estimated that by the end of this century, temperatures in Japan

would increases by 2.0°C using the RCP for greenhouse gases of 4.5, or by 4.4°C using

RCP8.5, relative to the mean temperatures for 1984–2004.

The goal of this study was to clarify the effects of higher temperatures on aspects of

32 fruit quality, including peel puffing and peel pigmentation, during two fruit-development

periods: from flowering to the physiological fruit drop stage and at the fruit maturation

stage.

5 Materials and Methods

Plant materials

The study was conducted during 2013–2015 at the NARO Institute of Fruit Tree and

Tea Science, Citrus Research Station, (Okitsu, Shimizu, Shizuoka, Japan; 35.05°N,

10 138.52°E). The climate normal for temperature during the 30 years between 1981 and

2010 in our experimental field was 16.3°C. The satsuma mandarin (Citrus unshiu Marc.,

‘Nankan No. 20’) trees had been grafted onto the rootstocks of trifoliate orange (Poncirus

trifoliate (L.) Raf.) trees. The grafted trees were planted in 25 L polypropylene pots and

grown in the field. We used three-year-old trees in 2013 and 2014 and five-year-old trees

15 in 2015. Trees with leaf-fruit ratios greater than 20 were thinned out to 20 in Jul., and

trees with leaf-fruit ratios of 20 or less were not thinned out.

Temperature treatment using phytotron

The temperature treatments were conducted during the flowering to physiological

20 fruit drop stage (Period A) and the maturation stage (Period B) using two phytotrons

(TPE-6S, ESPEC MIC Corp. Osaka, Japan). Since there have been few studies of the

effects on fruit quality of temperatures during the flowering to physiological fruit drop

stage (Period A), we laid out an experimental design with temperature rises during spring

and early summer in this study. The dates set for each period in each year are shown in

33 Table 2–5. One of the phytotrons was set at the recent mean temperatures for Okitsu (±

0°C), based on the 1981–2010 climate normal for temperature obtained with the

AMeDAS (automated meteorological data acquisition system) at Shimizu, located in our

experimental field. The ten-day means of daily maximum, minimum, and average

5 temperatures during Periods A and B obtained from the climate normal data are shown in

Table 2–6. The other phytotron was set to + 2°C (2013) and + 4°C (2014 and 2015)

relative to the temperatures in the ± 0°C phytotron. The temperatures were set to vary

depending on the time of day, with minimum temperatures from 0:00 to 4:00 h, maximum

temperatures from 12:00 to 16:00 h, and daily average temperatures from 6:00 to10:00 h

10 and from 18:00 to 22:00 h, as shown in Fig. 2–5. The temperatures also varied according

to the time of year (i.e., late Apr. to mid Dec.), as shown in Table 2–6. Air temperature

and humidity were measured using data loggers (RTR-53A, T&D Corporation, Nagano,

Japan) with their sensors covered by radiation shields (41304PY, Fieldpro, Inc., Tokyo,

Japan). The temperatures on Nov.17th, 2013 and Nov. 19th, 2015 are shown in Fig. 2–6.

15 The average humidity during the treatments in each year are shown in Table 2–5. During

the treatment period, appropriate watering was performed to prevent water stress on the

trees. The trees were kept in the field between Periods A and B.

Fruit quality examination method

20 Fruit measurements were conducted on the day the fruit subjected to the ± 0°C

treatment were almost fully colored. For each treatment, fruit quality analyses were

performed using 25 fruits (five from each of five potted trees) in 2013, nine fruits (three

from each of tree trees) in 2014, and 15 fruits (five from each of three trees) in 2015.

Different sets of trees were used each year. Fruit weight, peel percentage, peel

34 pigmentation, and peel puffing were assessed. Measurements of the soluble solids content

(SSC) and acidity were made using the juices of five fruits from each of five potted trees

(five biological replications) in 2013, three fruits from each of three trees (three biological

replications) in 2014, and five fruits from each of three trees (three biological replications)

5 in 2015. Fruits were randomly collected for these assessments. The measurements were

taken on Dec. 20th in 2013, Nov. 24th and Dec. 2nd in 2014, and Dec. 1st in 2015. The

level of peel puffing was assessed by finger touch and rated as 0, 1, 2, or 3, indicating a

range from firm (0) to marked puffing (3) (Kawase and Suzuki, 1981; Kawase, 1987; Fig.

2–7). Peel pigmentation was measured using a colorimeter (NF-333, Nippon Denshoku

10 Industries Co., Ltd. Tokyo, Japan), and a*/b* values were calculated. This ratio increases

as the color progresses from yellow through orange to red. We used a*/b* values from the

CIELAB scale because they are highly correlated with a/b values from the Hunter L, a, b

scale. The a/b values are highly correlated with the observed color of the fruit (Stewart

and Wheaton, 1971). Peel percentage was defined by dividing peel weight by fruit weight.

15 Peel percentage is closely correlated with peel puffing (Inoue, 1980). The SSC and acidity

of the juice samples were measured using an NH-2000 meter (HORIBA, Kyoto, Japan).

Statistical analysis

Data were subjected to a two-way analysis of variance (ANOVA), and the means were

20 separated using Tukey’s test or t-test at P < 0.05 using JMP13 (SAS Institute, Inc., NC,

USA). The percentage data were transformed using arcsine before statistical analysis.

Results

35 Effect of 2°C elevated temperature on fruit quality

The results from 2013 are shown in Table 2–7. When trees were kept at + 2°C during

Period A, the fruit weight was increased. The peel percentages for fruit kept at + 2°C

during Period A or Period B were increased. Peel puffing was accelerated in fruit kept at

5 + 2°C during Period B. The peel pigmentation levels were enhanced in fruit kept at + 2°C

during Period A, and deteriorated in fruit kept at + 2°C during Period B. The SSC in fruit

kept at + 2°C during Period A was decreased. There were no significant differences in

acidity among the treatments. Thus, when temperatures were elevated by 2°C above

normal during Period A, the fruit weight, peel percentage, peel pigmentation, and SSC

10 were affected. Temperatures elevated by 2°C during Period B also affected peel

percentage, peel puffing, and peel pigmentation.

Effect of 4°C elevated temperature on fruit quality

Although the elevated temperature during Period A tended to increase peel puffing,

15 the results were not statistically significant (Table 2–7), perhaps due to the slight

temperature difference (+ 2°C). Therefore, we investigated the effects on fruit quality of

4°C above recent means. In 2014, we compared treatments of ± 0°C and + 4°C during

Period A, with all trees at ± 0°C during Period B. Considering the possibility of advancing

the harvest of the fruit treated with + 4°C during Period A, we conducted fruit

20 measurements both one week before harvesting and at the time of harvesting. Fruit

weights were significantly higher in fruit treated with + 4°C during Period A and

measured one week before harvesting, when compared with those treated with ± 0°C. The

fruit measured at harvesting also had higher weights after treatment with + 4°C, but the

difference was not statistically significant (Table 2–8). The peel percentages and peel

36 puffing were also increased in fruit treated with + 4°C during Period A, both when

measured at harvesting and one week before. Peel pigmentation was promoted by the +

4°C treatment, with significant differences in the fruit measured at harvesting. There were

no significant differences in the SSC, but the acidity was decreased in fruit treated with +

5 4°C during Period A. Peel puffing in the fruit treated with + 4°C was significantly

increased at one week before harvesting and at harvesting. The level of peel puffing in

the fruit treated with + 4°C at one week before harvesting was higher than that in the fruit

treated with ± 0°C at harvesting. These results in 2014 demonstrated that the 4°C elevated

temperature during Period A significantly affected peel percentage, peel puffing, and

10 acidity. Fruit weight and peel pigmentation also tended to be influenced by the higher

temperature.

In 2015, we investigated the effects on fruit quality of elevated temperatures (+ 4°C)

during both Periods A and B. We laid out an experimental design with treatments of ±

0°C or + 4°C during Period A and ± 0°C or + 4°C during Period B. As in 2014, fruit

15 weight was increased in fruit treated with + 4°C during Period A (Table 2–9). There was

interaction between Periods A and B for peel percentage, with the highest percentages in

fruit treated with + 4°C during Period A and ± 0°C during Period B. Peel puffing was

increased in fruit treated with + 4°C during Period A and/or Period B. The peel

pigmentation was enhanced in fruit treated with + 4°C during Period A. There were no

20 significant differences in SSC or acidity among the treatments. These results in 2015

demonstrated that the temperature increase (4°C) during Period A affected the fruit weight,

peel puffing, and peel pigmentation. Temperature increases of 4°C during Period B also

accelerated peel puffing.

Collectively, the results in 2014 and 2015 showed that fruit weight, peel puffing, and

37 peel pigmentation were increased by treatment with + 4°C during Period A. The peel

percentage also tended to be higher in fruit treated with the higher temperature. Peel

puffing was also enhanced by the higher temperature during Period B.

5 Discussion

It is clear that peel puffing is promoted when fruit at the maturation stage are subjected

to high temperatures and high humidity or heavy rain (Kawase, 1984; Yokoo et al., 1963).

In this study, peel puffing was increased by treatment with + 2°C or + 4°C above recent

10 mean temperatures during Period B (Tables 2–7, 2–9). Peel puffing was also increased by

treatment with + 4°C during Period A (Tables 2–8, 2–9). Kawase (1984) reported that

peel puffing was increased in fruit treated with temperatures set at 5°C above the mean

and humidity maintained at almost 75% during the maturation stage, when compared with

fruit kept at the mean temperature. The mean temperatures used by Kawase (1984) were

15 almost the same as the temperatures set at ± 0°C in our study. Therefore, our results were

consistent with those of Kawase (1984), and we further showed that temperature increases

of only + 2°C can also cause increases in peel puffing.

High temperatures during the young fruit (cell division) stage promoted fruit

enlargement in Japanese pear and peach (Hayama et al., 2007; Sugiura et al., 1995). Inoue

20 and Chien (1987) reported that in satsuma mandarin, a relatively high temperature of

25°C during around one month after full bloom caused the growth of larger fruit than

those grown at 15°C during the same period. In this study, fruit weight was increased in

fruit treated with + 2°C or + 4°C during Period A (Tables 2–7, 2–8, 2–9). In 2013, the

average temperature at + 2°C during Period A was 20.5°C; in 2014, the average

38 temperature at + 4°C during Period A was 23.6°C; and in 2015, the average temperature

at + 4°C during Period A was 23.1°C. Therefore, our results for temperature and fruit

weight are in line with those of Inoue and Chien (1987), and we further showed that only

small temperature increases (+ 2°C or + 4°C) could cause increases in fruit weight. Peel

5 percentages were also increased in fruit treated with + 2°C or + 4°C during Period A

(Tables 2–7, 2–8, 2–9). The increases in fruit weight seemed to be attributable to the

increases in peel weight. In satsuma mandarin, peel thickness peaks near the end of Jun.

and beginning of Jul., when the fruit begin to grow rapidly (Kuraoka and Kikuchi, 1961).

The juice sacs begin to grow rapidly in late Jun., after the peel has grown. Thus, high

10 temperatures during Period A seemed to enhance peel growth because that is the period

when peel growth occurs. Large fruit with high peel percentages tended to have higher

degrees of peel puffing (Kawase, 1984). Therefore, the treatment of + 4°C during Period

A in our 2014 and 2015 experiments promoted fruit and peel growth, and it appears that

this led to increases in peel puffing. The reasons for the correlation between Periods A

15 and B in peel percentages are unknown.

Peel pigmentation was enhanced in fruit treated with + 2°C or + 4°C during Period A

(Tables 2–7, 2–8, 2–9). In Japanese pear and peach, high temperatures during the young

fruit stage shortened the time needed for fruit development and led to earlier harvest times

(Hayama et al., 2007; Sugiura et al., 1995). The improved peel pigmentation in this study

20 may indicate a similar effect in satsuma mandarin, with high temperatures during the

young fruit stage shortening the time needed for early fruit development. It is possible

that the optimal harvest time for fruit treated with higher temperatures during Period A

was earlier than the actual day of harvest, which was the optimal harvest time for the fruit

treated with ± 0°C during Period A.

39 In 2013, we found that pigmentation levels were reduced in fruits subjected to the +

2°C treatment during Period B (fruit maturation, Table 2–7). Studies by Nii et al. (1970),

with one month of preharvest treatment and constant day/night temperatures, showed that

carotenoid levels in the peels of satsuma mandarins at harvest were greatest after

5 treatment with 20°C, slightly lower with 15°C, and appreciably lower with treatments of

25°C or 30°C. Utsunomiya et al. (1982) reported that the chlorophyll degraded and the

carotenoids accumulated to greater extents with treatment at 15°C than with treatment at

23°C, when the fruit were treated for almost six weeks preharvest with constant day/night

temperatures. They found that chlorophyll degradation and carotenoid accumulation were

10 inhibited at 30°C. Kawase (1984) reported that peel pigmentation was delayed in fruit

treated with temperatures set at + 5°C above the mean, when compared with fruit treated

with mean temperatures. In our study, the + 2°C temperatures in late Oct., when the Period

B treatment began, had a mean of 18.9°C, a maximum of 23.5°C, and a minimum of

15.2°C. The inferior peel pigmentation may have been because the maximum temperature

15 was higher than the optimal range of 15–20°C for peel coloring. On the other hand, in

2015 the temperature during Period B had no significant effect on pigmentation levels,

even though the maximum temperature at the beginning of Period B for the + 4°C

treatment was 25.5°C (Table 2–9). This difference between 2013 and 2015 might be

explained by differences in the fruit coloration stage at the beginning of Period B. In 2013,

20 Period B was started at the beginning of the peel coloring stage in fruit subjected to the ±

0°C treatments, while in in 2015, Period B was started when 70% of the fruit were

coloring. It may be that the differences in peel coloring level at the start of treatment

affected the results. We surmised that the suppression of coloring by high temperature

was unlikely to occur if the peel coloring was advanced in more than half of the fruit.

40 We found that elevated temperatures during Period A tended to decrease the SSC and

acidity levels, although not all of these results were statistically significant (Tables 2–7,

2–8, 2–9). As we discussed above, high temperatures during the early fruit development

stages accelerated development and led to earlier harvest times in Japanese pear and peach

5 (Hayama et al., 2007; Sugiura et al., 1995). It is likely that this also occurred in the

satsuma mandarins in this study, with the fruits treated with higher temperatures

developing more rapidly than the control fruits, leading to lower SSC and acidity levels

at harvest. Our results might have been statistically significant with higher numbers of

biological replications.

41 Table 2–5. The dates set for each period and the avarage humidity during the treatment in each year.

Treatment The datas set for each Average humidity Year Fruit developmental stages in each period Days period period ± 0 °C + 2 or 4 °C

2013 Period A Apr. 30th – Jun. 4th Beginning of bloom – physiological fruit drop 36 69.2% 70.7%

Period B Oct. 28th – Dec. 20th Beginning of peel coloring stage – maturation 54 73.7% 76.1%

2014 Period A May 12nd – Jun. 9th Full bloom – physiological fruit drop 29 71.2% 67.0%

Period B Oct. 31st – Dec. 2nd Half of fruit were coloring – maturation 33 78.7% ー 42

2015 Period A May 7th – Jun. 9th Full bloom – physiological fruit drop 34 72.3% N.D.z

Period B Oct. 30th – Dec. 1st 70% of fruit were coloring – maturation 33 77.6% 77.4%

zData were not obtained. Table 2–6. Daily average, mean daily minimum, and mean daily maximum temperatures (°C) during satsuma mandarin fruit development periods in Okitsu.

Period of Temperature (°C) temperature treatment Daily average Mean minimum Mean maximum Period A late Apr. 16.2 11.9 21.1 early May 17.6 13.5 22.3 mid May 18.4 14.5 22.9 late May 19.5 15.5 24.0 early Jun. 20.8 17.2 25.1

Period B late Oct. 16.9 13.2 21.5 early Nov. 15.5 11.6 20.1 mid Nov. 13.6 9.8 18.2 late Nov. 12.0 8.1 16.8 early Dec. 10.2 6.1 15.2 mid Dec. 8.8 4.6 13.8

43 Table 2–7. Effect of high temperature on fruit quality in 2013.

Peel Peel Treatment Fruit weight Peel puffing SSC Acidity percentage pigmentation (g) (0–3) (˚Brix) (%) Period A Period B (%) (a*/b*) ± 0°C ± 0°C 129.5 19.7 0.0 0.46 10.0 0.64 ± 0°C + 2°C 147.3 20.9 1.0 0.42 9.9 0.61 + 2°C ± 0°C 159.6 20.4 0.2 0.47 9.4 0.58 + 2°C + 2°C 158.3 23.1 0.8 0.45 9.5 0.60 ANOVA Temp. of period A * * NS * * NS Temp. of period B NS * * * NS NS A×B 44 NS NS NS NS NS NS

NS and * indicate not significant and significant differences of P < 0.05 respectively, by ANOVA (n = 25 for fruit weight, peel percentage, peel pigmentation, and peel puffing; n = 5 for SSC and acidity). Table 2–8. Effect of high temperature on fruit quality in 2014.

Peel Peel Fruit measurement Treatment Fruit weight Peel puffing SSC Acidity percentage pigmentation date (g) (0–3) (˚Brix) (%) Period A Period B (%) (a*/b*) Nov. 24th ± 0°C ± 0°C 116.6 22.7 0.1 0.40 11.5 0.84 (the day one week + 4°C ± 0°C 139.1 26.6 2.2 0.40 10.9 0.67 before harvesting) t -test * * * NS NS *

± 0°C ± 0°C 118.4 23.7 0.1 0.40 11.6 0.88 Dec. 2nd + 4°C ± 0°C 133.2 27.1 2.3 0.45 11.1 0.72 (harvesting date) t -test NS * * * NS * 45 NS and * indicate not significant and significant differences, respectively, at P < 0.05 using a t-test (n = 9 for fruit weight, peel percentage, peel pigmentation, and peel puffing; n = 3 for SSC and acidity). Table 2–9. Effect of high temperature on fruit quality in 2015.

Peel Peel Treatment Fruit weight Peel puffing SSC Acidity percentage pigmentation (g) (0–3) (˚Brix) (%) Period A Period B (%) (a*/b*) ± 0°C ± 0°C 82.9 22.4 b 0.5 0.43 14.3 1.17 ± 0°C + 4°C 89.4 23.7 ab 1.5 0.44 13.6 0.99 + 4°C ± 0°C 117.1 24.6 a 1.4 0.49 14.2 0.91 + 4°C + 4°C 122.5 23.7 ab 2.5 0.48 13.9 0.85 ANOVA Temp. of period A * NS * * NS NS Temp. of period B NS NS * NS NS NS A×B NS * NS NS NS NS 46

NS and * indicate not significant and significant differences, respectively, at P < 0.05 by ANOVA . Different letters indicate significant differences among the treatments at P < 0.05 using Tukey's test (n = 15 for fruit weight, peel percentage, peel pigmentation, and peel puffing; n = 3 for SSC and acidity). 4C° + 4°C 2C° C)

° Daily + 2°C Max. ± 0°C

Daily Mean Temperature(

47 Daily Min.

0:00 4:00 6:00 10:00 12:00 16:00 18:00 22:00 0:00

Time

Fig. 2–5. Experimental scheme for temperature changes throughout the day. 25.0 25.0 a) b) 20.0 20.0 C) C) ° °

15.0 15.0

10.0 10.0 Temperature( Temperature( + 2°C 5.0 5.0 + 4°C ± 0°C ± 0°C 48 0.0 0.0 4:00 6:00 8:00 0:00 0:00 2:00 4:00 6:00 8:00 0:00 0:00 2:00 10:00 12:00 14:00 18:00 20:00 22:00 10:00 12:00 14:00 16:00 18:00 20:00 22:00 16:00

Time Time

Fig. 2–6. Temperature changes throughout the day. a) November 17, 2013. b) November 19, 2015. 0 1 2 3

Fig. 2–7. Evaluation criteria for peel puffing. The level of peel puffing was assessed by finger touch and rated as 0, 1, 2, or 3, indicating a range from firm (0) to marked puffing (3) . This figure was taken from Kawase (1987).

49 Chapter 3

Development of techniques to cope with elevated temperature by use of PGRs of

satsuma mandarin

5 Section 1 Development of techniques to reduce peel puffing

Introduction

The techniques for reducing the occurrence of peel puffing include methods using

10 fertilizer (Kawase, 1984; Torikata et al., 1955) and methods using thinning (Inoue et al.,

2003; Kitazono et al., 2008; Takagi et al., 2009). Methods using agrochemicals, such as

calcium carbonate, calcium chloride and calcium sulfate, and ethychlozate are also

employed. Recently, technique to reduce peel puffing has been developed which involves

spraying satsuma mandarin with a mixture of GA3 and PDJ. GA3 inhibition of satsuma

15 mandarin peel puffing was reported by Kuraoka et al. (1966), but treating the plants is

associated with delayed coloring and rind injury, so it was never implemented (Hirose,

1973). However, Makita and Yamaga (2004) demonstrated that spraying a mixture of

GA3 and PDJ in Sep. can effectively inhibit peel puffing and avoid causing rind injury,

and this finding served as the impetus for the progression of research into practical

20 application of this technique. As a result of these studies, GA3 and PDJ were registered

and put into use as agrochemicals to reduce satsuma mandarin peel puffing in 2010. This

technique is labor-saving because it is effective with a single spray, unlike many other

chemicals, which require multiple applications. Additionally, it is effective when sprayed

50 on the fruit, so there is no need to spray the entire tree, which also reduces the amount of

chemical required.

However, there is notable delayed coloring with this method (2010 registration

−1 −1 details: 3.3–5 mg∙L GA3 and 25–50 mg∙L PDJ solution; the spray timing is three

5 months before the scheduled harvest date, and it must be done by 45 days before

harvesting). This posed difficulties, as this method could only be used with storage

mandarin types, such as the late ripening ‘Aoshima unshiu’, which can recover the

delayed coloring after storage, or on fully mature cropping types which have significantly

later harvesting times than the customary harvesting period, and in which the fruit is

10 allowed to color completely on the tree. On the other hand, peel puffing is also a problem

with satsuma mandarin cultivars such as ‘Nankan No. 20’ and ‘Mukaiyama unshiu’, the

main medium ripening satsuma mandarins. These are harvested at the customary

harvesting time and shipped without being stored, so it is essential to investigate methods

of using GA3 and PDJ for these cropping types.

15 Research to date using the GA3 and PDJ mixture has not examined the peel puffing

and coloring of the fruit on the tree over time, so it is unclear how long the delayed

coloring occurs on the tree. This has made it difficult to investigate use of the mixture on

cropping types that are shipped at the customary times without storing. Therefore, in this

study, we aimed to examine the peel puffing and coloring of the fruit on the tree over

20 time and ascertain the timing of delayed coloring, while clarifying the effect of different

concentrations of the GA3 and PDJ mixture on reducing peel puffing. Based on the results

of this investigation, we aimed to clarify the spraying conditions depending on the

purpose of use, such as harvesting at the customary time, or intentionally delaying the

harvesting period by a few weeks, based on the reduction in peel puffing and the extent

51 of delayed coloring in medium ripening satsuma mandarins. Moreover, we also aimed to

clarify the peel puffing reduction effect and the effect on delayed coloring elicited by

different spraying times, using extremely early ripening, early ripening, and medium

ripening satsuma mandarins.

5

Materials and Methods

Plant materials

Fifteen-year-old ‘Iwasaki wase’, 15-year-old ‘Okitsu wase’, and 36, 38 to 41-year-

10 old ‘Silverhill’ satsuma mandarin (Citrus unshiu Marc.) trees, grafted onto trifoliate

orange (Poncirus trifoliate (L.) Raf.) trees were used at an orchard of the NARO Institute

of Fruit Tree and Tea Science, Citrus Research Station (Okitsu, Shimizu, Shizuoka,

Japan). ‘Iwasaki wase’ is extremely early ripening variety of satsuma mandarin. ‘Okitsu

wase’ is early ripening variety of satsuma mandarin. ‘Silverhill’ is medium ripening

15 variety of satsuma mandarin. ‘Iwasaki wase’ and ‘Silverhill’ are cultivars these are prone

to peel puffing. ‘Okitsu wase’ fruit occur peel puffing when weather condition is suitable

for outbreak of peel puffing.

The effect of GA3 concentration on peel puffing and coloring (Experiment 1)

20 Thirty-eight-year-old and 40-year-old ‘Silverhill’ satsuma mandarin were used.

Experiments were conducted in 2010 and 2012. In 2010, the PDJ concentration was set

−1 −1 −1 at 50 mg∙L , and two levels were set for the GA3 concentration: 1 mg∙L and 3.3 mg∙L .

The mixtures were sprayed on different branches on Sep. 6th (112 days after full bloom).

Five trees were used for the test; five branches were used per treatment, and three fruit

52 were collected per branch for each survey. Untreated branches were assigned as the

control. In 2012, the PDJ concentration was set at 25 mg∙L−1, and three levels were set

−1 −1 −1 for the GA3 concentration: 0 mg∙L , 1 mg∙L and 3.3 mg∙L . The mixtures were

sprayed on different branches on Aug. 31st (109 days after full bloom). Six trees were

5 used for the test, six branches were used per treatment, and six fruit were collected per

branch for each survey. Untreated branches were assigned as the control.

The fruit quality was surveyed three times: two weeks before the harvest date, one

week before the harvest date, and on the day of harvest. The harvest date in this study

was specified as the time when the untreated fruit had approximately 90% of its coloring.

10 In 2010, the SSC and acidity of the juice was measured on the day of harvest. In 2010,

there was no rain on the day the mixture was sprayed. In 2012, there was rain early in the

morning, but the mixture was sprayed after the branches, leaves and fruit had fully dried.

The effect of PDJ concentration on peel puffing and coloring (Experiment 2)

15 Thirty-nine-year-old and 41-year-old ‘Silverhill’ satsuma mandarin trees were used

−1 for this experiment. In both 2011 and 2013, the GA3 concentration was set at 1 mg∙L ,

and three levels were set for the PDJ concentration; 0, 25 and 50 mg∙L−1. Tests were

conducted in 2011 and 2013.

In 2011, the mixtures were sprayed on different branches on Aug. 29th (108 days after

20 full bloom). Eight trees were used for the test, eight branches were used per treatment,

and five fruit were collected per branch for each survey. Untreated branches were

assigned as the control. In 2013, the mixtures were sprayed on different branches on Sep.

3rd (118 days after full bloom). Six trees were used for the test, six branches were used

per treatment, and six fruit were collected per branch for each survey. Untreated branches

53 were assigned as the control. The fruit quality was surveyed three times: two weeks

before the scheduled harvest date, one week before the scheduled harvest date, and on

the scheduled day of harvest. In both years, the SSC and acidity of the juice was measured

on the scheduled day of harvest. In these years, there was no rain on the day the mixture

5 was sprayed.

The effect of spray timing on peel puffing and coloring (Experiment 3)

‘Iwasaki wase’, ‘Okitsu wase’, and ‘Silverhill’ (36 years old) satsuma mandarin were

−1 used. Test was conducted in 2008. The mixture concentration for GA3 was set at 5 mg∙L

10 with 50 mg∙L−1 for PDJ for all varieties. The mixtures were sprayed on different ‘Iwasaki

wase’ and ‘Okitsu wase’ satsuma mandarin trees on Aug. 15th (94 days after full bloom),

Sep. 5th (115 days after full bloom), Sep. 25th (135 days after full bloom) and Oct. 15th

(155 days after full bloom). Twelve trees of each variety were used for the test; three trees

were used per treatment, and ten fruit were collected per tree for each survey. Three

15 untreated trees were assigned as the control. The mixtures were sprayed on different

branches of the ‘Silverhill’ satsuma mandarin on Aug. 15th (94 days after full bloom),

Sep. 5th (115 days after full bloom), Sep. 25th (135 days after full bloom), Oct. 15th (155

days after full bloom), and Nov. 5th (176 days after full bloom). Six trees were used for

the test, six branches were used per treatment, and five fruit were collected per branch

20 for each survey. Untreated branches were assigned as the control. The fruit quality was

surveyed on the scheduled day of harvest for each variety. There was no rain on the

mixture spray dates of Aug. 15th, Sep. 5th, Sep. 25th, and Nov. 5th. On Oct. 15th, there

was light rain early in the morning, but the mixture was sprayed after the branches, leaves,

and fruit had fully dried.

54 Solution preparation of PGRs and sampling

Agricultural chemicals of GA3 (3.1% GA3 solution, Meiji Seika Pharma Co., Ltd.,

Tokyo, Japan), PDJ (5.0% PDJ solution, Meiji Seika Pharma Co., Ltd., Tokyo, Japan)

5 were used in this study. The solution was adjusted by diluting with tap water, and

immediately after preparation. The applicable solution was sprayed onto the plant with a

hand-sprayer. When treating different branches, the treatment consisted of a secondary

scaffold branch or large side branch between chest height and knee height. The fruit

sample for the fruit quality survey was an average medium to large sized fruit, collected

10 from a treated branch; the extent of peel puffing, fruit specific gravity, peel color score,

and peel pigmentation following different treatments were compared.

Fruit quality examination method

The extent of peel puffing was evaluated based on the level of peel puffing or the fruit

15 specific gravity. The level of peel puffing was assessed as Chapter 2 Section 2. The fruit

specific gravity was determined by dividing its weight by its volume. Fruit volume was

obtained by inserting a fine needle into the peduncle and measuring the volume of water

displaced when the fruit was fully immersed (Kawase, 1987). The higher the fruit specific

gravity of the fruit, the lower was the extent of peel puffing, and the lower the fruit

20 specific gravity, the greater the extent of peel puffing. The peel color score was set as 0

for fruit with no areas of orange coloring and as 10 when the fruit surface was completely

colored orange. The peel color score was evaluated objectively based on the area ratio of

the fruit surface with orange coloring. Peel pigmentation was measured as Chapter 2

Section 2. The SSC and acidity of the juice was measured with a digital refractometer

55 (PAL-1; Atago Co., Ltd., Tokyo, Japan) and titration with 0.1 N NaOH, or using an NH-

2000 meter (HORIBA, Japan).

Weather conditions

5 The rainfall and mean temperature for Nov. were obtained using data from the

AMeDAS at Shimizu installed in the Citrus Research Station (Table 3–1).

Statistical analysis

Data were subjected to ANOVA, and the means were separated by Tukey’s test at P <

10 0.05 in the experiments using JMP 7.0 (SAS Institute Inc., USA).

Results

The effect of GA3 concentration on peel puffing and coloring (Experiment 1)

15 In 2010, the extent of peel puffing was lower for the fruit in all treatments on Nov.

24th and Dec. 1st compared to the control (Fig. 3–1). The fruit specific gravity was higher

for the fruit in all treatments on all survey dates compared to the control. The peel color

score and peel pigmentation were lower for the fruit in all treatment on all survey dates

compared to the control. There was no difference in the SSC and acidity of the juice

20 between the treatments (Table 3–2).

−1 In 2012, the extent of peel puffing was lower in the GA3 3.3 mg∙L treatment than in

−1 −1 the control on Nov. 13rd, and lower in the GA3 1 mg∙L treatment and 3.3 mg∙L

treatment on Nov. 27th (Fig. 3–2). The fruit specific gravity was higher in the GA3 3.3

mg∙L−1 treatment compared with the control on Nov. 13rd and Nov. 20th, while it was

56 −1 −1 higher in the GA3 1 mg∙L treatment and the 3.3 mg∙L treatment when compared with

the control on Nov. 27th. There was no significant difference in the extent of peel puffing

−1 and the fruit specific gravity between the GA3 0 mg∙L treatment and the control. The

−1 peel color score and peel pigmentation were lower in the GA3 3.3 mg∙L treatment than

5 in the control on all survey dates, while there was no significant difference between the

−1 −1 GA3 0 mg∙L treatment and the 1 mg∙L treatment and the control on Nov. 20th and

Nov. 27th.

The effect of PDJ concentration on peel puffing and coloring (Experiment 2)

10 In 2011, the extent of peel puffing was lower in the PDJ 25 mg∙L−1 treatment and the

50 mg∙L−1 treatment than in the control on Nov. 17th and Dec. 1st (Fig. 3–3). The fruit

specific gravity was higher in the PDJ 25 mg∙L−1 treatment and the 50 mg∙L−1 treatment

than in the control on all survey dates. The peel color score was lower in the PDJ 25

mg∙L−1 treatment and the 50 mg∙L−1 treatment than in the control on Nov. 17th, and the

15 PDJ 50 mg∙L−1 treatment had lower peel color score than the control on Nov. 24th and

Dec. 1st. Peel pigmentation was lower in the PDJ 50 mg∙L−1 treatment than in the control

on all survey dates. There were no differences in the SSC and acidity of the juice (Table

3–3).

In 2013, the extent of peel puffing was lower in the PDJ 0 mg∙L−1 treatment on Nov.

20 19th, in the PDJ 25 mg∙L−1 treatment on Nov. 19th and Nov. 26th, and in the PDJ 50

mg∙L−1 treatment on all survey dates (Fig. 3–4). The fruit specific gravity was higher in

the PDJ 0 mg∙L−1 treatment on Nov. 19th, in the PDJ 25 mg∙L−1 treatment on Nov. 19th

and Dec. 3rd, and in the PDJ 50 mg∙L−1 treatment on all survey dates. The peel color score

was lower than that in the control in the PDJ 25 mg∙L−1 treatment on Nov. 19th and Dec.

57 3rd, and in the PDJ 50 mg∙L−1 treatment on all survey dates. The peel color score was

lower than the control in the PDJ 25 mg∙L−1 treatment on Nov. 19th, and in the PDJ 50

mg∙L−1 treatment on Nov. 19th and Nov. 26th. There was no difference in the SSC and

acidity of the juice between treatments (Table 3–4).

5

The effect of spray timing on peel puffing and coloring (Experiment 3)

The fruit specific gravity for ‘Iwasaki wase’ (extremely early ripening) was higher in

the treatments than in the control for Aug. 15th, Sep. 5th and Sep. 25th spray dates (Fig.

3–5). There was no significant difference between the treatment and the control for the

10 Oct. 15th spray date. Peel pigmentation was lower in the treatments than in the control

for the Aug. 15th, Sep. 5th and Sep. 25th spray dates. There was no significant difference

between treatment and control for the Oct. 15th spray.

The fruit specific gravity for ‘Okitsu wase’ (early ripening) was higher in the

treatments than in the control for the Aug. 15th, Sep. 5th, Sep. 25th and Oct. 15th spray

15 dates, and the treatment on Sep. 25th had the highest value (Fig. 3–5). Peel pigmentation

was lower in the treatment for the Aug. 15th, Sep. 5th, Sep. 25th and Oct. 15th spray dates,

and the treatment on Sep. 5th had the lowest value.

The fruit specific gravity for ‘Silverhill’ satsuma mandarin (medium ripening) was

higher in the treatment than in the control for the Aug. 15th, Sep. 5th, Sep. 25th and Oct.

20 15th spray dates, and the treatment on Sep. 25th had the highest value (Fig. 3–5). There

was no significant difference between the treatments and the control for the Nov. 5th spray.

The peel pigmentation was lower in the treatments than in the control for the Aug. 15th,

Sep. 5th, Sep. 25th and Oct. 15th spray dates, and the treatment on Sep. 25th had the

58 lowest value. There was no significant difference between the treatments and the control

for the spraying on Nov. 5th.

Discussion

5

Peel puffing becomes problem in satsuma mandarin which are harvested at the

customary harvesting time and shipped without storing. Therefore, it was considered

necessary to investigate usage methods of GA3 and PDJ for these cropping types. Thus,

in this study, we aimed to identify optimal spray concentration and spray timing of the

10 GA3 and PDJ mixture for elicitation of a reduction in peel puffing and minimization of

delayed coloring. This study formed part of the basic data when the GA3 registration

concentration was expanded to 1–5 mg∙L−1 in 2014.

When we sprayed the trees from late Aug. to early Sep. and examined the effect of

GA3 and PDJ concentration on peel puffing and coloring (Experiment 1 and 2), peel

−1 −1 15 puffing was reduced in the 1 mg∙L GA3 and 50 mg∙L PDJ mixture treatment, 3.3

−1 −1 −1 −1 mg∙L GA3 and 50 mg∙L PDJ mixture treatment, 1 mg∙L GA3 and 25 mg∙L PDJ

−1 −1 mixture treatment, and 3.3 mg∙L GA3 and 25 mg∙L PDJ mixture treatment (Figs. 3–1,

−1 −1 3–2, 3–3, 3–4). The peel color score in the 1 mg∙L GA3 and 50 mg∙L PDJ mixture

treatment was equal to or greater than the peel color score of the fruit in the control two

20 weeks before harvesting on all harvest dates in 2010 (Fig. 3–1), 2011 (Fig. 3–3), and 2013

(Fig. 3–4). Therefore, there was a one to two weeks delay in peel coloring in the 1 mg∙L−1

−1 GA3 and the 50 mg∙L PDJ mixture treatment. Given that the peel color score of the fruit

−1 −1 on the harvest date in the 3.3 mg∙L GA3 and 50 mg∙L PDJ mixture treatment was

inferior to the peel color score of the fruit in the control two weeks before harvesting, peel

59 −1 −1 coloring in the 3.3 mg∙L GA3 and 50 mg∙L PDJ mixture treatment was delayed by

−1 −1 more than two weeks (Fig. 3–1). The peel color score in the 1 mg∙L GA3 and 25 mg∙L

PDJ mixture treatment was equal to or greater than the peel color score of the fruit in the

control two weeks before harvesting at one week before harvesting in 2011 (Fig. 3–3),

5 2012 (Fig. 3–2), and 2013 (Fig. 3–4). Therefore, delay in peel coloring in the 1 mg∙L−1

−1 GA3 and 25 mg∙L PDJ mixture treatment was less than one week. Given that the peel

−1 −1 color score of the fruit on the harvest date in the 3.3 mg∙L GA3 and 25 mg∙L PDJ

mixture treatment was equal to or greater than the peel color score of the fruit in the

control two weeks before harvesting, there was a peel coloring delay of one to two weeks

10 (Fig. 3–2).

Based on the above results, an effective method for satsuma mandarins that are

harvested at the customary harvesting time and are shipped without being stored would

−1 −1 be to spray the fruit with the 1 mg∙L GA3 and 25 mg∙L PDJ mixture from late Aug. to

early Sep., as the extent of delayed peel coloring is estimated to be less than one week

−1 −1 15 with this mixture. On the contrary, the 1 mg∙L GA3 and 50 mg∙L PDJ mixture spray,

−1 −1 −1 the 3.3 mg∙L GA3 and 50 mg∙L PDJ mixture spray, or the 3.3 mg∙L GA3 and 25

mg∙L−1 PDJ mixture spray could be used to intentionally delay harvesting and disperse

the harvest workload. When there is a markedly large number of satsuma mandarins

during the same harvesting period, such as on-year, and if it is anticipated that the

20 harvesting work will not be completed within the scheduled harvesting period, then part

−1 −1 of the orchard can be sprayed with a GA3 and PDJ mixture (1 mg∙L GA3 and 50 mg∙L

−1 −1 −1 PDJ mixture, 3.3 mg∙L GA3 and 50 mg∙L PDJ mixture, or 3.3 mg∙L GA3 and 25

mg∙L−1 PDJ mixture) to intentionally delay the scheduled harvesting time, thereby

preventing notable peel puffing in the fruit and dispersing the harvesting workload.

60 −1 −1 In this study, it was clarified that spraying with 1 mg∙L GA3, and either 25 mg∙L

or 50 mg∙L−1 PDJ, concentrations lower than those at the time of agrochemical

registration in 2010, is effective for reducing peel puffing. The effect of the GA3 (1

mg∙L−1 to 3.3 mg∙L−1) and PDJ (25 mg∙L−1 to 50 mg∙L−1) mixture spray is concentration-

5 dependent; therefore, it was found that during simultaneous spraying, increasing the

concentration of one of the components increases the peel puffing reduction effect and

delays peel coloring.

This experiment provides the results in medium ripening satsuma mandarins which

the scheduled harvest date is around late Nov., but other satsuma mandarin varieties with

10 different harvest times have different varietal characteristics, therefore, the extent of

reduction in peel puffing and the extent of delayed coloring may differ from the extent

seen in this experiment. Thus, we investigated the effect of the timing of spray on peel

puffing and coloring by use of three varieties with different maturation times (Experiment

3). Peel puffing reduction and delayed coloring effects were intensified in ‘Iwasaki wase’

15 (extremely early ripening) by spraying from mid Aug. to late Sep. (Fig. 3–5). Peel puffing

reduction and delayed coloring were seen in ‘Okitsu wase’ (early ripening) following

spraying from mid Aug. to mid Oct.; the peel puffing reduction effect was strongest

following spraying in late Sep., while the delayed coloring effect was strongest following

spraying in early Sep. In ‘Silverhill’ satsuma mandarin (medium ripening), peel puffing

20 reduction and delayed coloring were seen following spraying from mid Aug. to mid Oct.,

and both outcomes were strongest following spraying in late Sep. As stated above, in

‘Okitsu wase’ and ‘Silverhill’ satsuma mandarin, the peel puffing reduction and delayed

coloring effects induced by the GA3 and PDJ mixture spray are substantial following

spraying from early to late Sep., which suggests the effect would be weaker if the timing

61 of spraying were shifted earlier or later. In ‘Iwasaki wase’, the peel puffing reduction and

delayed coloring effects induced by the GA3 and PDJ mixture spray are also intensified

by spraying from mid Aug. to late Sep., but weaker when spraying time was delayed until

mid Oct. However, we were unable to confirm in this experiment whether the peel puffing

5 reduction effect is weakened by spraying earlier than mid Aug.

This experiment has thus clarified that the strength of the peel puffing reduction and

delayed coloring effects can be adjusted by changing the timing of spraying. In

Experiments 1 and 2, relating to the spray concentration, the experiment concerned with

timing of GA3 and PDJ spraying was conducted between late Aug. and early Sep. (three

10 months before the scheduled harvest date). However, based on the results of the spray

timing experiment (Experiment 3) in the medium ripening ‘Silverhill’ satsuma mandarin,

the timing of spraying that elicited the strongest peel puffing reduction effect and delayed

coloring was in late Sep. We therefore assume that the timing of spraying used in this

experiment may not be the timing with the strongest effect. If the fruit is sprayed in late

15 Sep., when the effect tends to be more apparent, it may be possible to obtain peel puffing

reduction by using lower concentrations than were used in this experiment (minimum

−1 −1 GA3 of 1 mg∙L , and PDJ of 25 mg∙L ). We experimented using high concentration of

−1 GA3 (5 mg∙L ) in order to clearly observe the effect of spray timing on the peel puffing

reduction and delayed coloring effects, but in the future, it will be necessary to investigate

20 the effect of the timing of spraying with low concentration GA3, which was shown to be

effective in this experiment.

As part of Experiments 1 and 2, we conducted a three-year survey on the SSC and

acidity of the juice, but there was no significant difference between any of the treatments

and the control (Tables 3–2, 3–3, 3–4). Sawano (2010) reported that when late ripening

62 −1 −1 ‘Aoshima unshiu’ plants were sprayed with 5 mg∙L GA3 and 50 mg∙L PDJ mixture

−1 −1 spray or 3.3 mg∙L GA3 and 25 mg∙L PDJ mixture spray in early Sep., all treatments

had lower sugar content and higher acidity than the control on the Dec. 2nd harvest date.

However, in this experiment, there was no difference between the treatments sprayed with

5 GA3 and PDJ mixture and the control, so it is assumed that GA3 and PDJ mixture spray

used to reduce peel puffing has little effect on the SSC and acidity of the juice in medium

ripening satsuma mandarins.

In terms of the relationship between peel puffing and weather conditions, rainfall from

Nov. to the time the fruit matures promotes peel puffing (Yokoo et al., 1963), and it has

10 been reported that if the humidity is constant during this period, higher temperatures cause

more peel puffing (Kawase, 1984). In this study, 2011 had the highest rainfall and high

average temperature and the extent of peel puffing in the control was more notable than

in other experiment years (Table 3–1, Figs. 3–1, 3–2, 3–3, 3–4). There were thus annual

differences in the peel puffing due to the weather, but even in 2011, when there was

15 significant onset of peel puffing, the GA3 and PDJ mixture spray had a peel puffing

reduction effect (Fig. 3–3). Therefore, it is assumed that the peel puffing reduction effect

produced by the GA3 and PDJ mixture spray is stable regardless of annual weather

variations.

Based on the results of this study and Wakayama Prefecture’s study (Nakatani et al.,

20 2014), a manual for mitigating peel puffing for early and medium ripening satsuma

mandarin was prepared (NARO, 2014). We proposed a method of use that aims to

“reducing peel puffing and harvesting at customary times” or “controlling peel puffing

strongly and delaying harvesting time one or two weeks systematically”. The former

method is suitable for early and medium ripening satsuma mandarin that are harvested at

63 the customary time because the peel coloring delay can be suppressed within one week.

−1 −1 Specifically, a mixture of 1 mg∙L GA3 and 25 mg∙L PDJ is sprayed in early Sep.

−1 −1 (around 120 days after full bloom), or a mixture of 3.3 mg∙L GA3 and 25 mg∙L PDJ

is sprayed in late Aug. (around 100 days after full bloom). In the latter method, peel

5 coloring is delayed by one to two weeks, but it is also possible to disperse labor when the

harvest is not in time, such as on-year. It is assumed to be used in medium ripening

−1 −1 satsuma mandarin. Specifically, a mixture of 1 mg∙L GA3 and 50 mg∙L PDJ is sprayed

−1 in early Sep. (around 120 days after full bloom), or a mixture of 3.3 mg∙L GA3 and 25

mg∙L−1 PDJ is sprayed in early Sep. (around 120 days after full bloom).

10 In 2014, a registration of GA3 concentration for reducing satsuma mandarin peel

−1 −1 puffing updated from 3.3–5 mg∙L GA3 to 1–5 mg∙L GA3, while that of PDJ

−1 concentration did not changed (25–50 mg∙L PDJ). After renewal GA3 concentration for

reducing satsuma mandarin peel puffing, the technique prevails in satsuma mandarin

production area (MAFF, 2018a).

15 In the future, it will be necessary to examine the spraying concentrations and timing

of GA3 and PDJ that can effectively reduce peel puffing in the extremely early ripening

satsuma mandarins.

64 Table 3–1. Precipitation and mean temperature on November in each experiment year.

Precipitation Mean temperature Year (mm) (°C) 2010 111.5 (− 31.9)z 13.6 (− 0.1) 2011 304.5 (+ 161.1) 15.4 (+ 1.7) 2012 209.0 (+ 65.6) 13.2 (− 0.5) 2013 112.0 (− 31.4) 12.9 (− 0.8) Climate normal value 143.4 13.7

zThe value was indicated the difference from climate normal value.

65 Table 3–2. Effect of GA3 concentration on SSC and acidity of juice in 2010.

Treatment SSC (˚Brix) Acidity (%) Control 10.4 az 0.95 a −1 −1 GA3 1 mg∙L + PDJ 50 mg∙L 10.1 a 0.91 a −1 −1 GA3 3.3 mg∙L + PDJ 50 mg∙L 9.8 a 1.06 a zDifferent letters indicate significant differences among the treatments at P < 0.05 by Tukey's test (n = 5).

66 Table 3–3. Effect of PDJ concentration on SSC and acidity of juice in 2011.

Treatment SSC (˚Brix) Acidity (%) Control 10.2 az 0.80 a −1 −1 GA3 1 mg∙L + PDJ 0 mg∙L 10.1 a 0.79 a −1 −1 GA3 1 mg∙L + PDJ 25 mg∙L 10.1 a 0.83 a −1 −1 GA3 1 mg∙L + PDJ 50 mg∙L 10.1 a 0.86 a zDifferent letters indicate significant differences among the treatments at P < 0.05 by Tukey's test (n = 8).

67 Table 3–4. Effect of PDJ concentration on SSC and acidity of juice in 2013.

Treatment SSC (˚Brix) Acidity (%) Control 9.3 az 0.66 a −1 −1 GA3 1 mg∙L + PDJ 0 mg∙L 9.4 a 0.64 a −1 −1 GA3 1 mg∙L + PDJ 25 mg∙L 9.7 a 0.66 a −1 −1 GA3 1 mg∙L + PDJ 50 mg∙L 9.4 a 0.68 a zDifferent letters indicate significant differences among the treatments at P < 0.05 by Tukey's test (n = 6).

68 (a) 1.6 Control 1.4 a a −1 −1 1.2 GA3 1 mg∙L + PDJ 50mg∙L 1.0 −1 −1 GA3 3.3 mg∙L + PDJ 50mg∙L 0.8

puffing b 0.6 az b b

Peel 0.4 b 0.2 a a 0.0 Nov. 17th Nov. 24th Dec. 1st (b) 0.96 0.94 a a 0.92 a a a 0.90 b a b 0.88 b

specific gravity 0.86

Fruit 0.84

0.82 Nov. 17th Nov. 24th Dec. 1st

(c) 10 a a 8 a b c b b 6 b b 4 colorscore

Peel 2

0 Nov. 17th Nov. 24th Dec. 1st (d) 0.35 a 0.30 b )

* a b

/b 0.25 *

a a 0.20 b b 0.15 b 0.10 0.05

pigmentation ( b 0.00 Peel -0.05 Nov. 17th Nov. 24th Dec. 1st

Fig. 3–1. Effect of GA3 concentration on peel puffing and peel coloring in 2010. The diluted solutions were sprayed on Sep. 6th, 2010. (a) The level of peel puffing was assessed by finger touch and rated as 0, 1, 2, or 3, indicating a range from firm (0) to marked puffing (3). (b) The fruit specific gravity was determined by dividing its weight by its volume. The higher the fruit specific gravity of the fruit, the lower was the extent of peel puffing, and the lower the fruit specific gravity, the greater the extent of peel puffing. (c) The peel color score was set as 0 for fruit with no areas of orange coloring and as 10 when the fruit surface was completely colored orange. (d) Peel pigmentation was measured using a colorimeter and a*/b* values were calculated. This ratio increases as the color progresses from yellow through orange to red. zDifferent letters indicate significant differences among the treatments at P < 0.05 by Tukey’s test (n = 5).

69 (a) 1.6 Control 1.4 a ab −1 −1 1.2 GA3 0 mg∙L + PDJ 25 mg∙L −1 −1 1.0 b b GA3 1 mg∙L + PDJ 25 mg∙L −1 −1 0.8 GA3 3.3 mg∙L + PDJ 25 mg∙L

puffing z a 0.6 a a a ab a Peel 0.4 a b 0.2 0.0 Nov. 13rd Nov. 20th Nov. 27th (b) 0.96 0.94 0.92 0.90 a b b b a 0.88 b b b 0.86 ab a a b 0.84 0.82 0.80 Fruit Fruit specific gravity 0.78 0.76 Nov. 13rd Nov. 20th Nov. 27th (c) 10 a a a 8 a a ab a a b b b 6 c

4 colorscore

Peel 2

0 Nov. 13rd Nov. 20th Nov. 27th (d) a 0.35 a a 0.30 b a a 0.25 a 0.20 b a 0.15 ab bc 0.10 c 0.05

Peel pigmentation (a*/b*) pigmentation Peel 0.00 Nov. 13rd Nov. 20th Nov. 27th

Fig. 3–2. Effect of GA3 concentration on peel puffing and peel coloring in 2012. The diluted solutions were sprayed on Aug. 31st, 2012. (a), (b), (c), (d) are the same as in Fig. 3–1. zDifferent letters indicate significant differences among the treatments at P < 0.05 by Tukey’s test (n = 6).

70 (a) 1.6 a Control 1.4 ab −1 −1 GA3 1 mg∙L + PDJ 0 mg∙L 1.2 a a −1 −1 a bc GA3 1 mg∙L + PDJ 25 mg∙L 1.0 az a −1 −1 0.8 a c GA3 1 mg∙L + PDJ 50 mg∙L

0.6 b

Peel puffing 0.4 b 0.2 0.0 Nov. 17th Nov. 24th Dec. 1st (b) 0.88 a a 0.86 b b 0.84 a a 0.82 a b b ab bc 0.80 c 0.78

Fruit Fruit specific gravity 0.76 0.74 Nov. 17th Nov. 24th Dec. 1st

10 a (c) a a a a a b 8 a a b b b 6

4

Peel colorscore 2

0 Nov. 17th Nov. 24th Dec. 1st (d) 0.40 a a 0.35 a a b 0.30 a a 0.25 b 0.20 ab a 0.15 bc 0.10 c 0.05

Peel pigmentation (a*/b*) pigmentation Peel 0.00 Nov. 17th Nov. 24th Dec. 1st

Fig. 3–3. Effect of PDJ concentration on peel puffing and peel coloring in 2011. The diluted solutions were sprayed on Aug. 29th, 2011. (a), (b), (c), (d) are the same as in Fig. 3–1. zDifferent letters indicate significant differences among the treatments at P < 0.05 by Tukey’s test (n = 8).

71 (a) 1.6 1.4 Control −1 −1 1.2 GA3 1 mg∙L + PDJ 0 mg∙L a a az a −1 −1 1.0 ab GA3 1 mg∙L + PDJ 25 mg∙L 0.8 ab −1 −1 b bc b GA3 1 mg∙L + PDJ 50 mg∙L 0.6 bc c

Peel puffing 0.4 c 0.2 0.0 Nov. 19th Nov. 26th Dec. 3rd

(b) 0.92 a 0.90 ab b a 0.88 b c b a b a 0.86 ab b Fruit Fruit specific gravity 0.84

0.82 Nov. 19th Nov. 26th Dec. 3rd

(c) 10 a ab b a a c a ab a 8 b b c

6

4 Peel colorscore

2

0 Nov. 19th Nov. 26th Dec. 3rd (d) 0.35 a a a a a 0.30 a ab a b 0.25 ab 0.20 b c 0.15 0.10 0.05 Peel pigmentation (a*/b*) pigmentation Peel 0.00 Nov. 19th Nov. 26th Dec. 3rd

Fig. 3–4. Effect of PDJ concentration on peel puffing and peel coloring in 2013. The diluted solutions were sprayed on Sep. 3rd, 2013. (a), (b), (c), (d) are the same as in Fig. 3–1. zDifferent letters indicate significant differences among the treatments at P < 0.05 by Tukey’s test (n = 6).

72 Iwasaki wasez Okitsu wasey Silverhillx 0.92 0.95 0.90 a a a ab a 0.94 0.89 0.91 aw 0.93 bc b 0.88 bc 0.90 c 0.92 0.87 cd d 0.89 b b 0.91 0.86 d 0.90 0.85 e 0.88 0.89 0.84 0.87 0.88 0.83 0.87 0.82 Fruit specific gravity Fruit specific gravity Fruit specific gravity 0.86 0.86 0.81 0.85 0.85 0.80

Spray date Spray date Spray date

0.20 0.45 a 0.55 a a 0.40 a 0.15 0.50 a 0.35 0.10 b 0.45 0.30 b 0.05 b b 0.25 0.40 c 0.00 b b 0.20 cd b c 0.35 d -0.05 0.15 0.30 -0.10 0.10 -0.15 0.05 0.25 Peel pigmentation (a*/b*) Peel pigmentation (a*/b*) -0.20 Peel pigmentation (a*/b*) 0.00 0.20

Spray date Spray date Spray date

Fig. 3–5. Effect of the timing of GA3 plus PDJ on peel puffing and peel coloring for three satsuma −1 mandarin varieties with defferent ripening. A diluted solution containing 5 mg∙L GA3 and 50 mg∙L−1 PDJ was sprayed on Aug. 15th, Sep. 5th, Sep. 25th and Oct. 15th, respectively. The solution was also sprayed on Nov. 5th in ‘Silverhill’. zFruit specific gravity and peel pigmentation were measured on Oct. 16th, 2008. yFruit specific gravity and peel pigmentation were measured on Nov. 11st, 2008. xFruit specific gravity and peel pigmentation were measured on Dec. 1st, 2008. wDifferent letters indicate significant differences among the treatments at P < 0.05 by Tukey’s test (n = 3 in ‘Iwasaki wase’ and ‘Okitsu wase’, n = 6 in ‘Silverhill’).

73 Section 2 Development of handpicking techniques

Introduction

5 As shown Chapter 2 Section 2, suitable harvesting period is predicted to delay due to

increase fruit which are reduced peel pigmentation by elevated temperature. Suitable

harvesting period is also predicted to be short due to increase peel puffing. Thus, it is

necessary to develop the technique of harvesting fruit quickly.

The most time-consuming work for satsuma mandarin growers is harvesting, so the

10 development of a handpicking method without a negative influence on fruit quality and

tree health would be labor saving. Previous reports showed that the time required for

harvesting by handpicking is less than half that of the usual harvesting method with

scissors (Noro et al., 1965; Yamada et al., 1982). However, handpicking is not a practical

method for satsuma mandarin production at present because the fruit peel near the

15 peduncle is easily ripped by this method.

Previously, Kozaki et al. (1984) investigated fruit adhesion force and peel firmness

in relation to the efficiency of handpicking using some citrus fruit cultivars. The results

indicated that handpicking efficiency was improved by two physical factors: a decrease

in fruit adhesion force and an increase in peel firmness. Thus, we assumed that not only

20 induction of abscission layer formation between the calyx and fruit, but also enhancement

of peel firmness, are necessary to improve handpicking of satsuma mandarins.

It was reported that an abscission agent, ET, was effective for the induction of

abscission layer formation and an improvement in handpicking efficiency (Ikeda et al.,

1984; Rasmussen, 1976), although spontaneous abscission layer formation between the

74 calyx and fruit was not easy in the mature period in the satsuma mandarin, suggesting the

importance of the application period for exerting the maximum effectiveness of ET. Ikeda

et al. (1984) found that a 200 mg∙L−1 ET application improved the efficiency of satsuma

mandarin handpicking. Rasmussen (1976) also reported that 400 mg∙L−1 ET had a

5 satisfactory effect on loosening between the calyx and fruit of the satsuma mandarin fruit.

In contrast, there are no reports about improving handpicking by artificially controlling

peel firmness. Considering the peel puffing caused by reduced peel firmness in the

satsuma mandarin, spraying of GA3 and PDJ can strengthen peel firmness, which in turn

could be a potential technique to improve handpicking efficiency (Makita and Yamaga,

10 2004). Thus, we assumed that the balance between the treatments for the induction of

abscission layer formation between the calyx and fruit by ET and for strengthening peel

firmness by GA3 plus PDJ, could be key points to improve handpicking.

In the present study, we investigated the effect of combined spraying of GA3 and PDJ

−1 on handpicking efficiency, and we set the concentrations of GA3 and PDJ to 5 mg∙L

15 and 50 mg∙L−1, respectively, according to the maximum permitted concentration of

registration for reducing fruit puffing in satsuma mandarins in Japan. We then examined

the effect of the combined spraying of GA3, PDJ, and ET on handpicking efficiency.

Materials and Methods

20

Plant materials

‘Silverhill’ satsuma mandarin (Citrus unshiu Marc.) trees, 38 to 41-year-old, grafted

onto trifoliate orange (Poncirus trifoliate (L.) Raf.) trees were used at an orchard of the

NARO Institute of Fruit Tree and Tea Science, Citrus Research Station (Okitsu, Shimizu,

75 Shizuoka, Japan). The mature season of ‘Silverhill’ on this experimental site is late Nov.

to early Dec.

Solution preparation of plant growth regulators

5 Agricultural chemicals of GA3 (3.1% GA3 solution, Meiji Seika Pharma Co., Ltd.,

Tokyo, Japan), PDJ (5.0% PDJ solution, Meiji Seika Pharma Co., Ltd., Tokyo, Japan),

and ET (10.0% ET solution, Nissan Chemical Industries, Ltd., Tokyo, Japan) were used

in this study. These chemicals were diluted with tap water to prepare their spray solutions

(Table 3–5) just before spraying. These spray solutions were applied with a pressurized

10 one L capacity hand sprayer (Dia Sprayer No. 4130; Furupla, Tokyo, Japan).

Experiment 1: Effect of plant growth regulators on handpicking efficiency in 2010

Six satsuma mandarin trees (38 years old) were used. In each tree, four lateral

branches were selected and three treatments (3GP, ET, and 3GP + ET) and the control

15 were randomly assigned to each branch. For the 3GP treatment, a diluted solution

−1 −1 containing 5 mg∙L GA3 and 50 mg∙L PDJ was sprayed three times (Aug. 27th, Sep.

21st, and Oct. 27th). For the ET treatment, a diluted solution containing 200 mg∙L−1 ET

was sprayed on Nov. 9th. For the 3GP + ET treatment, both the 3GP treatment and the ET

treatment were conducted according to their respective methods. Untreated branches were

20 assigned as the control. Ten fruits from a branch in each tree were handpicked on Nov.

30th and Dec. 6th. On each handpicking day, the percentage of the fruit that did not have

the peel ripped during handpicking was calculated and designated as the success rate of

handpicking (SRH). To measure the SSC and acidity, five fruits from a branch of each

tree were sampled on Nov. 30th.

76 Experiment 2: Effect of the timing of GA3 plus PDJ on handpicking efficiency in 2011

Eight satsuma mandarin trees (39 years old) were used. For each tree, five lateral

branches were selected and four treatments [GP (Aug. 24th) + ET, GP (Sep. 12nd) + ET,

5 GP (Sep. 30th) + ET, and ET] and the control were randomly assigned to each branch.

−1 For the GP (Aug. 24th) + ET treatment, a diluted solution containing 5 mg∙L GA3 and

50 mg∙L−1 PDJ was sprayed on Aug. 24th. Additionally, a diluted solution containing 200

mg∙L−1 ET was sprayed on Oct. 26th. For the GP (Sep. 12nd) + ET treatment, a diluted

−1 −1 solution containing 5 mg∙L GA3 and 50 mg∙L PDJ was sprayed on Sep. 12nd.

10 Additionally, a diluted solution containing 200 mg∙L−1 ET was sprayed on Oct. 26th. For

−1 the GP (Sep. 30th) + ET treatment, a diluted solution containing 5 mg∙L GA3 and 50

mg∙L−1 PDJ was sprayed on Sep. 30th. Additionally, a diluted solution containing 200

mg∙L−1 ET was sprayed on Oct. 26th. For the ET treatment, a diluted solution containing

200 mg∙L−1 ET was sprayed on Oct. 26th. Untreated branches were assigned as the

15 control. Ten fruits from a branch of each tree were handpicked and measured for SRH on

Dec. 2nd and Dec. 13rd. To measure the peel puncture force, peel tensile strength, peel

color score, SSC, and acidity, five fruits from a branch of each tree were sampled on Dec.

14th.

20 Experiment 3: Effect of the concentration of ET on handpicking efficiency in 2012

Eight satsuma mandarin trees (40 years old) were used. In each tree, five lateral

branches were selected and four treatments (GP + lowET, GP + hiET, lowET, and hiET)

and the control were randomly assigned to each branch. For the GP + lowET treatment,

−1 −1 a diluted solution containing 5 mg∙L GA3 and 50 mg∙L PDJ was sprayed on Oct. 2nd.

77 Additionally, a diluted solution containing 200 mg∙L−1 ET was sprayed on Oct. 15th. For

−1 −1 the GP + hiET treatment, both diluted solutions containing 5 mg∙L GA3 and 50 mg∙L

PDJ were sprayed on Oct. 2nd. Additionally, a diluted solution containing 400 mg∙L−1

ET was sprayed on Oct. 26th. For the lowET treatment, a diluted solution containing 200

5 mg∙L−1 ET was sprayed on Oct. 15th. For the hiET treatment, a diluted solution

containing 400 mg∙L−1 ET was sprayed on Oct. 26th. Untreated branches were assigned

as the control. Ten fruits from a branch of each tree were handpicked and measured for

SRH on Dec. 3rd and Dec. 14th. To measure the peel puncture force, peel tensile strength,

and peel color score, five fruits from a branch of each tree were sampled on Dec. 4th and

10 Dec. 17th. On Dec. 17th, the SSC and acidity of juice in the sampled fruits were also

measured. For examination of ethylene production of fruit, fruit was sampled from each

treatment one week after spraying with 400 mg∙L−1 ET at Nov. 2nd. Ethylene production

was measured only with the GP + hiET treatment, the hiET treatment and the control.

15 Experiment 4: Effect of the 300 mg·L−1 ET on handpicking efficiency in 2013

Eight satsuma mandarin trees (41 years old) were used. From each tree, four lateral

branches were selected and three treatments (GP, ET, and GP + ET) and the control were

randomly assigned to each branch. For the GP treatment, a diluted solution containing 5

−1 −1 mg∙L GA3 and 50 mg∙L PDJ was sprayed on Sep. 26th. For the ET treatment, a diluted

20 solution containing 300 mg∙L−1 ET was sprayed on Nov. 11st. For the GP + ET treatment,

both a GP treatment and an ET treatment were conducted according to their respective

methods. Untreated branches were assigned as the control. Ten fruits from a section of

each tree were handpicked and measured for SRH on Dec. 2nd and Dec. 13rd. To measure

the peel puncture force, peel tensile strength, and peel color score, five fruits from a

78 section of each tree were sampled on Dec. 4th and Dec. 16th. On Dec. 16th, the SSC and

acidity of juice in the sampled fruits were also measured. For examination of ethylene

production of fruit, fruit was sampled from each treatment on 0 (Nov. 11st), 2 (Nov. 13rd),

4 (Nov. 15th), 7 (Nov. 18th), 9 (Nov. 20th), 11 (Nov. 22nd), 16 (Nov. 27th), 22 (Dec. 3rd)

5 and 30 (Dec. 11st) days after spraying with ET. Leaf abscission was measured on a branch

with almost 100 leaves before treatment by counting the leaves before (Sep. 24th, 2013)

and after (Jan. 14th, 2014) treatment.

Measurement method of the peel puncture force, peel tensile strength, peel color score,

10 SSC, and acidity

Peel puncture force and peel strength were measured in each fruit using a texture

analyzer (TA-XT plus; Microstable Systems, Surrey, UK). The maximum resistance force

when a cylinder probe (2 mm in diameter) penetrated the peel in the equatorial region of

the fruit was regarded as the peel puncture force. The maximum resistance force when a

15 tensile grip probe pulled the ends of a peel piece 3 cm long and 1.5 cm wide, which was

cut off along the longitudinal direction was regarded as the peel tensile strength. Peel

color score was assessed as Chapter 3 Section 1. To measure SSC and acidity, the juice

of five fruits was extracted, mixed. The SSC of the juice was measured with a digital

refractometer (PAL-1; Atago Co., Ltd., Tokyo, Japan). The acidity of the juice was

20 measured by titration with 0.1 N NaOH.

Measurement method of ethylene production

A fruit was placed in a 600-mL plastic container for 1 h at 20°C. A 1-mL gas was

sampled from the internal atmosphere with a plastic hypodermic syringe and injected into

79 a gas chromatograph (GC-8A; Shimadzu Co., Kyoto, Japan) fitted with an alumina

column at 70°C and a flame ionization detector to assay the ethylene concentration. The

rate of ethylene production was expressed as nL∙h −1∙g−1 FW.

5 Statistical analysis

Data were subjected to ANOVA, and the means were separated by Tukey’s test at P

< 0.05 in the experiments using JMP 7.0 or JMP13.0 (SAS Institute Inc., USA). The

percentage data of SRH, leaf abscission, and acidity were transformed using arcsine

before statistical analysis.

10

Results

Experiment 1: Effect of plant growth regulators on handpicking efficiency in 2010

The SRHs for 3GP and 3GP + ET were higher than that for the control, although there

15 was no significant difference in ET on Nov. 30th (Fig. 3–6). The SRHs for ET and 3GP

+ ET were higher than that for the control, although there was no significant difference

in 3GP on Dec. 6th. There was no difference in SSC (Table 3–6). The acidity in 3GP was

higher than that in the control. These results suggested a possible SRH improvement

because of 3GP + ET, but three treatments on different dates were too costly and labor-

20 intensive considering practical use; therefore, we investigated the effect of a one-time

treatment (Aug. 24th, Sep. 12nd, or Sep. 30th) on SRH.

Experiment 2: Effect of the timing of GA3 and PDJ on handpicking efficiency in 2011

80 The SRHs for GP (Sep. 12nd) + ET and GP (Sep. 30th) + ET were higher than that

for the control, although there was no significant difference in GP (Aug. 24th) + ET or

ET on Dec. 2nd (Fig. 3–7). The SRHs for GP (Sep. 12nd) + ET, GP (Sep. 30th) + ET, and

ET were higher than that for the control, although there was no significant difference in

5 GP (Aug. 24th) + ET on Dec. 13rd. Each treatment of GP (Sep. 12nd) + ET and GP (Sep.

30th) + ET showed a higher peel puncture force and tensile strength, but a lower color

score (Table 3–7). There was no difference in SSC. The acidity levels in GP (Aug. 24th)

+ ET and GP (Sep. 12nd) + ET were higher than that in the control. Consequently,

spraying in mid to late Sep. would be a suitable period to increase SRH, but 200 mg∙L−1

10 ET seemed to be insufficient for peel coloration. As a result, we studied the effect of

increased ET (400 mg∙L−1) on the parameters by comparison with 200 mg∙L−1 ET.

Experiment 3: Effect of the ET concentration on handpicking efficiency in 2012

The SRHs for GP + lowET, GP + hiET, and hiET were significantly higher than that

15 for the control, regardless of handpicking time (Fig. 3–8). The treatment with GP +

lowET resulted in the highest peel puncture force, while treatment with hiET resulted in

the lowest. Treatment with GP + lowET produced a higher peel tensile strength. The color

scores with GP + lowET and GP + hiET were lower than those with other treatments,

possibly due the strength of peel firmness due to GP. The SSC in hiET was lower than

20 that in the control (Table 3–8). There was no difference in acidity among the treatments.

These results demonstrated the importance of balance in concentrations between

chemicals with opposite effects, GA3 plus PDJ and ET. Ethylene production of fruit was

the highest in the GP + hiET treatment, followed by the hiET treatment among the GP +

81 hiET treatment, the hiET treatment and the control (Fig. 3–9). In addition, we observed

massive leaf abscission at 400 mg∙L−1 ET (hiET) (data not available).

Experiment 4: Effect of the 300 mg·L−1 ET on handpicking efficiency in 2013

5 Since 400 mg∙L−1 ET resulted in serious leaf defoliation, we investigated the effect

−1 of 300 mg∙L ET with a combination of GA3 plus PDJ on SRH. The SRHs for GP and

GP + ET were significantly higher than that for the control, regardless of handpicking

time, while that for ET was higher than that for the control on Dec. 13rd (Fig. 3–10).

Each treatment of GP and GP + ET resulted in a higher peel puncture force and tensile

10 strength, but lower color scores. There was no difference in SSC or acidity among any

treatments (Table 3–9). Ethylene production of fruit was always higher in the GP + ET

and the ET treatments than in the control and the GP treatments during from two days

after ET spraying to 30 days after ET spraying (Table 3–10). At four days after ET

spraying, ethylene production from fruit of GP + ET treatment was higher than that of

15 ET treatment. Leaf abscission for ET and GP + ET was unexpectedly higher than that for

the control (Fig. 3–11), suggesting that 300 mg∙L−1 ET may still be too high.

Discussion

20 Improving the efficiency of handpicking fruits using the plant growth regulators GA3,

PDJ, and ET was examined using satsuma mandarins. We intended to improve SRH by

balancing concentrations of chemicals with opposite effects on peel firmness. Triplicate

spraying of a GA3 plus PDJ solution improved SRH as compared with the control (Fig.

3–6). Furthermore, triplicate spraying of a combination of GA3 plus PDJ and ET

82 increased SRH as compared with the control. To assess the practicality of this method,

we carried out a one-time treatment. One-time treatment of GA3 plus PDJ also showed a

significant positive effect on handpicking efficiency when applied in mid to late Sep. (Fig.

3–7). However, it seemed that 200 mg∙L−1 ET may be insufficient for peel coloration

5 (Table 3–7). As for the concentration of ET, 400 mg∙L−1 ET clearly improved SRH

compared with 200 mg∙L−1 ET, but 400 mg∙L−1 ET was not enough to improve the peel

coloration (Fig. 3–8). The above results are summarized as follows: (1) The combination

treatment of GA3 plus PDJ and ET improved SRH. (2) The optimum timing of GA3 plus

PDJ treatment is mid to late Sep. (3) The optimum concentration of ET was between 200

10 mg∙L−1 and 400 mg∙L−1. (4) 400 mg∙L−1 ET caused massive leaf abscission, but this is

only our observation.

As mentioned earlier, the SRH after spraying with 400 mg∙L−1 ET was much

improved, but the treatment was accompanied by massive leaf abscission (data not

available). On the other hand, there was no noticeable defoliation in the 200 mg∙L−1 ET

15 (data not available). Therefore, we studied the effect of spraying of 300 mg∙L−1 ET: the

result on SRH was acceptable, but this concentration still resulted in 37.1% leaf

abscission (Fig. 3–11). Hirose et al. (1970) and Manago and Ogaki (1971) reported that

treatment with more than 500 mg∙L−1 ET to degreen fruit in wase unshiu caused

defoliation. Rasmussen (1976) reported that ET of 400 mg∙L−1 caused excessive

20 defoliation with satisfactory loosening of the satsuma fruit. Therefore, considering

handpicking and defoliation, the optimal concentration of ET is in the range of 200–300

mg∙L−1 ET to increase handpicking efficiency without causing massive defoliation and

poor coloration. Kender et al. (2000) reported that calcium hydroxide applied with ET

reduced ET-induced defoliation in ‘Robinson’ and ‘Sunburst’ tangerines. Pozo et al.

83 (2004) showed applications of 1-metyheycyclopropene (1-MCP) with ET reduced ET-

associated leaf abscission in orange fruits. In order to prevent massive defoliation with

400 mg∙L−1 ET and improve handpicking efficiency in the satsuma mandarin, calcium

hydroxide and 1-MCP could be investigated as protective agents.

5 Next, we discuss the SRH in terms of the optimal treatment period. The increase in

SRH with treatment by GA3 plus PDJ in late Sep. caused an increase in peel firmness

and peel tensile strength (Fig. 3–7, Table 3–7). The SRH with a combination treatment

−1 of GA3 plus PDJ (from Sep.12nd to Oct. 2nd) and 200–300 mg∙L ET (from Oct.15th to

Nov.11st) also increased peel firmness and peel tensile strength (Figs. 3–7, 3–8, 3–10).

−1 10 These results suggested that a GA3 plus PDJ treatment with 200–300 mg∙L ET spraying

increased peel firmness. Kozaki et al. (1984) observed that the efficiency of handpicking

is enhanced by fruit adhesion force and peel firmness, especially peel firmness. Our

results are the first to show the effect of combined spraying of GA3 and PDJ on

handpicking efficiency for the satsuma mandarin. This study is also the first to provide

15 information on the improvement of handpicking efficiency by the combination treatment

of GA3 plus PDJ (from Sep.12nd to Oct. 2nd) and ET (from Oct.15th to Nov.11st). In

Chapter 3 Section 1, the effect of combination spraying of GA3 plus PDJ on preventing

peel puffing was the highest for a Sep. treatment from tested spraying times from middle

Aug. to early Nov., so to increase peel firmness, the most efficient time for GA3 plus PDJ

20 treatment is mid to late Sep. In this study, the GA3 and PDJ concentrations were constant

−1 −1 at 5 mg∙L for GA3 and 50 mg∙L for PDJ, and other concentrations were not examined.

If the concentration is higher than this, it is considered that the hardness of the peel is

−1 increased, but the coloring delay is further remarkable. On the other hand, GA3 1 mg∙L

+ PDJ25 mg∙L−1 with little effect on coloring shown in Chapter 3 Section 1 might have

84 a lower effect of increasing the peel hardness. Regarding to the timing of ET spraying,

when compared at the same concentration (200 mg∙L−1), early Nov. treatment tended to

have a higher SRH than mid and late Oct. treatment (Figs. 3–6, 3–7, 3–8, 3–10).

Taniguchi (1975) reported that the longer the period from the treatment of ET to the

5 harvest date, the more abscission layer formation of fruit occurs at the time of harvest.

On the other hand, in this study, the SRH was higher when the period from the treatment

of ET to the harvest date was short.

−1 In Experiment 3, at seven days after 400 mg∙L ET spraying, fruits treated with GA3,

PDJ and 400 mg∙L−1 ET produced more ethylene than those treated with 400 mg∙L−1 ET

−1 10 (Fig. 3–9). In addition, in experiment 4, fruits treater with GA3, PDJ and 300 mg∙L ET

produced more ethylene than those treated with 300 mg∙L−1 ET spraying at four days

−1 after 300 mg∙L ET (Table 3–10). Thus, it is thought the GA3 and PDJ treatments

increased the peel hardness and the formation of abscission layer was enhanced by ET.

The mechanism that GA3 and PDJ treatment increase ethylene production by ET in fruit

15 is unclear.

Regarding fruit quality, especially SSC and acidity, the acidity with triplicate GA3

plus PDJ spraying was higher than that in the control (Table 3–6). The SSC in 400 mg∙L−1

ET treatment was lower than that in the control (Table 3–8). Sawano (2010) reported that

−1 −1 5 mg∙L GA3 plus 50 mg∙L PDJ sprayed in early Sep. to reduce fruit puffing in the

20 satsuma mandarin resulted in decreasing SSC and increasing acidity in mature fruit.

Hirose et al. (1970) and Manago and Ogaki (1971) reported that the SSC in juice

decreased with ET treatment. In our study, one-time spraying of GA3 plus PDJ in late

Sep. had no influence on SSC or acidity (Table 3–9). Furthermore, the combined spraying

−1 of GA3 plus PDJ and less than 300 mg∙L ET had less influence on SSC and the acidity

85 −1 of fruit (Tables 3–7, 3–8, 3–9). Thus, the combination treatment of 5 mg∙L GA3 plus

50 mg∙L−1 PDJ in late Sep. and less than 300 mg∙L−1 ET from late Oct. to early Nov. was

suitable to increase SRH without impairing internal fruit quality. Conclusively, we found

that the optimal conditions for achieving acceptable handpicking without impairing

5 internal fruit quality and serious leaf abscission were as follows: treatment of 5 mg∙L−1

−1 −1 −1 GA3 plus 50 mg∙L PDJ in late Sep. combined with ET (200 mg∙L < ET < 300 mg∙L )

from late Oct. to early Nov.

For practical use, we should mention two demerits derived from handpicking that

need to be addressed. First, because most handpicked fruits lose their calyces, it is

10 difficult to sell them as fresh fruit. However, as handpicked satsuma mandarin fruits

without calyces can be stored for about a month under low temperature conditions

(Yamada, 1985), handpicked fruit could also be used as cut fruit and processed products.

Second, Jozukuri and Yukinari (1976) reported that the number and length of vegetative

shoots and the number of flowers in the sprouting shoots from bearing shoots harvested

15 by clipping were more than those by handpicking in Hassaku, which indicates the

possibility that handpicking may reduce vegetative shoots and flowers in the spring. To

overcome this difficulty, a treatment such as heavy pruning of the branches is needed.

Another concern is Melanose. After handpicking, shoots or peduncles with calyx suffer

dieback, leading to Melanose. However, the number of dieback branches is limited as

20 handpicking has little effect compared to the total number of branches, so handpicking

has little influence on Melanoses (Jozukuri and Yukinari, 1976).

In addition, it is thought that this technique is difficult to use as practical harvest

because SRH don’t reach to 100%. As a method for improving the SRH, it is considered

to examine whether the GA3 and PDJ concentrations can be further increased in the peel

86 hardness. In that case, it is necessary to clarify the effects of coloring and SSC and acidity

of juice. If there is little influence on SSC and acidity of juice and only coloring is delayed,

it does not become a problem for making processed products. Moreover, it is conceivable

to promote the abscission layer formation by increasing the concentration of ET. In that

5 case, defoliation increases, so as described above, it is necessary to consider using

calcium hydroxide or 1-MCP together as a fallen leaf prevention agent. Since methyl

jasmonate (MJ) has been reported to promote orange fruit abscission (Hartmond et al.,

2000), other fruit abscission agents may be considered. We also need to explore more

suitable cultivars for using this technique than satsuma mandarin. ‘Kankitsu

10 Chukanbohon Nou 6 Gou’ mature fruit can be harvested by handpicking (Hasegawa,

2013).

Currently, the yield per 10a of satsuma mandarin is 1950 kg in Japan (MAFF, 2019).

Noro et al. (1965) reported that almost 1100 kg by handpicking, 487 kg by clipping per

a day (eight hours) of satsuma mandarin could harvested. Thus, it takes 1.8 days by

15 handpicking, four days by clipping per 10a. If this technique reached to practical use level,

harvesting time can be shortened by 2.2 days compared with clipping using this technique.

87 Table 3–5. Concentration and timing of plant growth regulators sprayed in the experiments.

Experiment Concentration and timing of Treatment Concentration and timing of GA + PDJ (year) 3 ET Experiment 1 Control - - - - −1 −1 (2010) 3GP GA3 5mg∙L + PDJ 50mg∙L Aug. 27th, Sep. 21st, Oct. 27th - - ET - - ET 200mg∙L−1 Nov. 9th −1 −1 −1 3GP / ET GA3 5mg∙L + PDJ 50mg∙L Aug. 27th, Sep. 21st, Oct. 27th ET 200mg∙L Nov. 9th Experiment 2 Control - - - - −1 −1 −1 (2011) GP (Aug. 24th) / ET GA3 5mg∙L + PDJ 50mg∙L Aug. 24th ET 200mg∙L Oct. 26th −1 −1 −1 GP (Sep. 12nd) / ET GA3 5mg∙L + PDJ 50mg∙L Sep. 12nd ET 200mg∙L Oct. 26th −1 −1 −1 88 GP (Sep. 30th) / ET GA3 5mg∙L + PDJ 50mg∙L Sep. 30th ET 200mg∙L Oct. 26th ET - - ET 200mg∙L−1 Oct. 26th Experiment 3 Control - - - - −1 −1 −1 (2012) GP / lowET GA3 5mg∙L + PDJ 50mg∙L Oct. 2nd ET 200mg∙L Oct. 15th −1 −1 −1 GP / hiET GA3 5mg∙L + PDJ 50mg∙L Oct. 2nd ET 400mg∙L Oct. 26th lowET - - ET 200mg∙L−1 Oct. 15th hiET - - ET 400mg∙L−1 Oct. 26th Experiment 4 Control - - - - −1 −1 (2013) GP GA3 5mg∙L + PDJ 50mg∙L Sep. 26th - - ET - - ET 300mg∙L−1 Nov. 11st −1 −1 −1 GP / ET GA3 5mg∙L + PDJ 50mg∙L Sep. 26th ET 300mg∙L Nov. 11st Table 3–6. Effect of the combination treatment of GA3 plus PDJ and ET on fruit quality in 2010.

Treatment SSC (˚Brix) Acidity (%)

Control 10.0 az 0.82 b 3GP 9.7 a 0.95 a ET 9.8 a 0.78 b 3GP + ET 9.5 a 0.83 ab

−1 For the 3GP treatment, 5 mg∙L GA3 plus 50 mg∙L−1 PDJ was sprayed three times (Aug. 27th, Sep. 21st, and Oct. 27th). For the ET treatment, 200 mg∙L−1 ET was sprayed on Nov. 9th. For the 3GP + ET treatment, both the 3GP treatment and the ET treatment were performed. Measurements of SSC and titratable acidity on Nov. 30th, 2010. zDifferent letters indicate significant differences among the treatments at P < 0.05 by Tukey's test (n = 6).

89 Table 3–7. Effect of timing of GA3 plus PDJ treatment on fruit firmness, peel color, and fruit quality in 2011.

Peel puncture Peel tensile Peel color Treatment SSC (˚Brix) Acidity (%) force (kg) strength (kg) score Control 0.38 bz 0.82 b 10.0 a 10.8 a 0.59 b GP (Aug. 24th) + ET 0.38 b 0.90 b 9.8 a 10.7 a 0.71 a GP (Sep. 12nd) + ET 0.49 a 1.04 a 7.9 b 10.4 a 0.71 a GP (Sep. 30th) + ET 0.45 a 1.02 a 8.3 b 10.6 a 0.63 ab ET 0.37 b 0.84 b 10.0 a 10.8 a 0.66 ab

−1 −1 For the GP (Aug. 24th) + ET treatment, both 5 mg∙L GA3 plus 50 mg∙L PDJ on Aug. 24th −1

90 and 200 mg∙L ET on Oct. 26th were sprayed. For the GP (Sep. 12nd) + ET treatment, both 5 −1 −1 −1 mg∙L GA3 plus 50 mg∙L PDJ on Sep. 12nd and 200 mg∙L ET on Oct. 26th were sprayed. −1 −1 For the GP (Sep. 30th) + ET treatment, both 5 mg∙L GA3 plus 50 mg∙L PDJ on Sep. 30th and 200 mg∙L−1 ET on Oct. 26th were sprayed. For the ET treatment, 200 mg∙L−1 ET was sprayed on Oct. 26th. Measurements on Dec. 14th, 2011. zDifferent letters indicate significant differences among the treatments at P < 0.05 by Tukey's test (n = 40 for peel puncture force, peel tensile strength, and peel color score, and n = 8 for SSC and acidity). Table 3–8. Effect of the ET concentration on fruit quality in 2012.

Treatment SSC (˚Brix) Acidity (%)

Control 11.4 az 0.56 a GP + lowET 11.4 a 0.59 a GP + hiET 10.7 ab 0.63 a lowET 11.1 ab 0.58 a hiET 10.5 b 0.64 a

−1 For the GP + lowET treatment, both 5 mg∙L GA3 plus 50 mg∙L−1 PDJ on Oct. 2nd and 200 mg∙L−1 ET on Oct. 15th were sprayed. For the GP + hiET −1 −1 treatment, both 5 mg∙L GA3 plus 50 mg∙L PDJ on Oct. 2nd and 400 mg∙L−1 ET on Oct. 26th were sprayed. For the lowET treatment, 200 mg∙L−1 ET was sprayed on Oct. 15th. For the hiET treatment, 400 mg∙L−1 ET was sprayed on Oct. 26th. Measurements on Dec. 17th, 2012. zDifferent letters indicate significant differences among the treatments at P < 0.05 by Tukey's test (n = 8).

91 Table 3–9. Effect of the 300 mg∙L-1 ET on fruit quality in 2013.

Treatment SSC (˚Brix) Acidity (%) Control 10.1 az 0.49 a GP 9.7 a 0.51 a ET 10.3 a 0.47 a GP + ET 10.0 a 0.50 a

−1 For the GP treatment, 5 mg∙L GA3 plus 50 mg∙L−1 PDJ was sprayed on Sep. 26th. For the ET treatment, 300 mg∙L−1 ET was sprayed on Nov. 11st. For the GP + ET treatment, both a GP treatment and an ET treatment were performed. Measurements of SSC and titratable acidity on Dec. 4th, 2013. zDifferent letters indicate significant differences among the treatments at P < 0.05 by Tukey's test (n = 8).

92 Table 3–10. Ethylene production of fruit after spraying with 300 mg∙L–1 ET. Ethylene production (nL∙h–1∙g–1) Treatment Days after ET spraying 0 2 4 7 9 11 16 22 30 Control 0.00 az 0.00 b 0.00 c 0.01 b 0.00 b 0.00 b 0.00 b 0.00 b 0.00 b GP 0.01 a 0.00 b 0.00 c 0.00 b 0.00 b 0.00 b 0.01 b 0.00 b 0.00 b ET 0.00 a 0.71 a 0.62 b 0.92 a 0.64 a 0.40 a 0.35 a 0.22 a 0.16 a GP + ET 0.01 a 0.90 a 0.91 a 0.98 a 0.60 a 0.49 a 0.42 a 0.24 a 0.16 a

zDifferent letters indicate significant differences among the treatments at P < 0.05 by Tukey's test (n = 8). 93 100 100 Nov. 30th Dec. 6th 90 90 80 a 80 a

picking (%) 70 70 - 60 60 a 50 50 40 a ab 40 ab 30 30 20 20 bz 10 10 b Success hand of ratio Success 0 0

Fig. 3–6. Effect of combination treatment of GA3 plus PDJ and ET on −1 handpicking in 2010. For the 3GP treatment, 5 mg∙L GA3 plus 50 mg∙L−1 PDJ was sprayed three times (Aug. 27th, Sep. 21st, and Oct. 27th). For the ET treatment, 200 mg∙L−1 ET was sprayed on Nov. 9th. For the 3GP + ET treatment, both the 3GP treatment and the ET treatment were performed. z Different letters indicate significant differences among the treatments at P < 0.05 by Tukey’s test (n = 6).

94 100 100 Dec. 2nd Dec. 13rd 90 90 80 80

picking (%) 70 70 - 60 60 a ab 50 50 a a 40 40 ab 30 30 ab bc 20 b 20 10 b 10 c Success hand of ratio Success 0 0

Fig. 3–7. Effect of timing of GA3 plus PDJ treatment on handpicking in −1 2011. For the GP (Aug. 24th) + ET treatment, both 5 mg∙L GA3 plus 50 mg∙L−1 PDJ on Aug. 24th and 200 mg∙L−1 ET on Oct. 26th were −1 sprayed. For the GP (Sep. 12nd) + ET treatment, both 5 mg∙L GA3 plus 50 mg∙L−1 PDJ on Sep. 12nd and 200 mg∙L−1 ET on Oct. 26th were −1 sprayed. For the GP (Sep. 30th) + ET treatment, both 5 mg∙L GA3 plus 50 mg∙L−1 PDJ on Sep. 30th and 200 mg∙L−1 ET on Oct. 26th were sprayed. For the ET treatment, 200 mg∙L−1 ET was sprayed on Oct. 26th. z Different letters indicate significant differences among the treatments at P < 0.05 by Tukey’s test (n = 8).

95 100 100 a Dec. 3rd a Dec. 14th 90 90 80 ab 80 ab 70 70 b picking (%) - 60 60 bc 50 50 c c 40 40 cd 30 30 20 dz 20 Success hand of ratio Success 10 10 0 0

0.60 a Dec. 4th 0.60 Dec. 17th 0.55 b 0.55 a 0.50 bc 0.50 cd d 0.45 0.45 b b 0.40 0.40 bc 0.35 0.35 c

Peel punctureforce (kg) 0.30 0.30

Dec. 4th a Dec. 17th 1.30 a 1.30 ab 1.20 1.20 ab

1.10 bc 1.10 bc bc c c 1.00 1.00 c Peel tensile strength (kg) 0.90 0.90

Dec. 4th Dec. 17th a a a 10 a a 10 a b 9 9 b b 8 b 8 7 7 Peel Peel color score 6 6

Fig. 3–8. Effect of the ET concentration on handpicking, peel puncture force, peel tensile strength, and −1 −1 peel color in 2012. For the GP + lowET treatment, both 5 mg∙L GA3 plus 50 mg∙L PDJ on Oct. 2nd −1 −1 and 200 mg∙L ET on Oct. 15th were sprayed. For the GP + hiET treatment, both 5 mg∙L GA3 plus 50 mg∙L−1 PDJ on Oct. 2nd and 400 mg∙L−1 ET on Oct. 26th were sprayed. For the lowET treatment, 200 mg∙L−1 ET was sprayed on Oct. 15th. For the hiET treatment, 400 mg∙L−1 ET was sprayed on Oct. 26th. z Different letters indicate significant differences among the treatments at P < 0.05 by Tukey’s test (n = 8 for the success ratio of handpicking, or n = 40 for peel puncture force, peel tensile strength, and color score).

96 1.80

1.60 ) 1 − a ∙g

1 1.40 − ∙h L 1.20

1.00

0.80 b 0.60

0.40

Ethylene production (n production Ethylene 0.20 cz 0.00 Control GP + hiET hiET

Fig. 3–9. Ethylene production of fruit a week after spraying with 400 mg∙L−1 ET. z Different letters indicate significant differences among the treatments at P < 0.05 by Tukey’s test (n = 8).

97 100 100 Dec. 2nd Dec. 13rd a 90 a 90 80 80 70 70 a picking (%) - 60 60 b 50 bc 50 40 40 b 30 30 cz 20 20 c Success hand of ratio Success 10 10 0 0

0.60 Dec. 4th 0.60 Dec. 16th 0.55 0.55 a 0.50 a 0.50 0.45 0.45 a ab 0.40 b 0.40 bc b 0.35 0.35 c Peel Punctureforce (kg) 0.30 0.30

Dec. 4th Dec. 16th 1.10 a a 1.10 a a 1.00 1.00 a 0.90 0.90 b 0.80 b 0.80 b Peel tensile strength (kg) 0.70 0.70

Dec. 4th Dec. 16th a a a 10 a 10 b 9 b 9 8 8 c 7 c 7 Peel Peel color score 6 6

Fig. 3–10. Effect of the 300 mg∙L−1 ET on handpicking , peel puncture force, peel tensile strength and fruit −1 −1 color in 2013. For the GP treatment, 5 mg∙L GA3 plus 50 mg∙L PDJ was sprayed on Sep. 26th. For the ET treatment, 300 mg∙L−1 ET was sprayed on Nov. 11st. For the GP + ET treatment, both a GP treatment and an ET treatment were performed. z Different letters indicate significant differences among the treatments at P < 0.05 by Tukey’s test (n = 8 for the success ratio of handpicking, or n = 40 for peel puncture force, peel tensile strength, and color score).

98 40 a a 35 30 25 20 15 b Leaf abscission (%) 10 bz 5 0 Cont. GP ET GP + ET

Fig. 3–11. Effect of the 300 mg∙L−1 ET on leaf abscission in 2013. Leaf abscission was measured on a branch with almost 100 leaves before treatment by counting the leaves before (Sep. 24th, 2013) and after (Jan. 14th, 2014) treatment. z Different letters indicate significant differences among the treatments at P < 0.05 by Tukey’s test (n = 8).

99 Section 3 Development of enriched vegetative shoots and stable flowering technique in

greenhouse

Introduction

5

The increase in mean temperatures due to global warming also affects flowering, and

it has been reported that increased temperature results in a higher percentage of leafless

flower and a lower percentage of leafy flowers (Iwasaki et al., 2018; Ono and Iwasaki,

2010). Greenhouse cultivation may be a higher temperature environment than outdoor

10 cultivation, so technique to increase the percentage of leafy flowers is indispensable.

In early-heated greenhouse mandarin cultivation, the summer shoots that form

following post-harvesting pruning are used as the fruiting mother shoots (vegetative

shoots), but when fall shoots are formed from summer shoots, these are poorly enriched

for vegetative shoots. When satsuma mandarin vegetative shoots are fully enriched, leafy

15 flowers occurs, but when vegetative shoots are not adequately enriched, leafless flowers

tend to occur (Kadoya, 2000). Therefore, development of fall shoots from the summer

shoots is inhibited by means of underground cooling (Inoue, 1994) or ethychlozate

(Hirose, 2003). However, installation of underground cooling is costly and requires effort,

and ethychlozate has a large impact on the trees, including reducing the respiratory

20 activity of the roots, thus decline of tree vigor is a problem if ethychlozate is used every

year (Manago and Hirobe, 1984).

NAA is a type of synthetic auxin, which has been found to prevent thinning of fruit

trees and fruit drop, and to inhibit shoot development (Hirose, 1976; Keever et al., 1998;

Miller and Ware, 1980; Miyagawa, 1958; Nauer and Boswell, 1978; Phillips and Tucker,

100 1974; Raese, 1975). When NAA is used to inhibit shoot development in greenhouse

mandarins, it not only performs this function, but is also reported to enhance the shape of

vegetative shoots and improve flowering. It has also been reported to have a smaller

adverse effect on the roots than ethychlozate (Manago and Hirobe, 1987).

5 In this study we investigated the effect of NAA on flowering and the components of

the shoots of greenhouse mandarin trees, with ethychlozate, normally used as a shoot

development inhibitor for summer branches on greenhouse mandarin trees, as the control

treatment.

10 Materials and Methods

Plant materials, chemical application and sampling

Ten 34-year-old ‘Miyagawa wase’ satsuma mandarin (early ripening) trees cultivated

in a greenhouse for 21 years at the Aichi Agricultural Research Center were used for the

15 experiment. This is a cropping type where the summer shoots are used as fruiting mother

shoots (vegetative shoots), so the trees were pruned immediately after harvesting on Jun.

22nd, 2005. Heating was started on Nov. 22nd, 2005 and full bloom occurred on Jan. 1st,

2006. The NAA treatment, sprayed with NAA 220 mg∙L−1; and the ethychlozate treatment,

sprayed with ethychlozate 200 mg∙L−1 as the control were set in this experiment. Five

20 trees were used each treatment. The trees were sprayed twice on day 50 and day 72 after

pruning, and were sprayed with a power sprayer until the chemical runoff from the leaves.

Five to seven average vegetative shoots at the height of the crown were randomly collected

three times from each treatment comprised of five trees, on days 49, 71, 97, 120 and 140

after pruning. The shoots were separated into leaves and branches, freeze-dried, powdered

101 and stored until analyzed. On days after 140 after pruning, shoot length and proximal

diameter of shoot were measured before the shoots were separated into leaves and

branches. On days 49, 71, 97, 120 and 140 after pruning, dry weight of separated leaves

and branches were measured.

5

Analysis of carbohydrate and nitrogen components in the shoots

For carbohydrate analysis, using the powderd freeze-dried samples (leaves and

branches, 250 mg each), sugar was extracted three times with 10 mL 80% hot ethanol for

30 min each time, and after concentrating the aqueous phase supernatant component to a

10 constant volume with an evaporator (40°C), the sugar content (glucose and sucrose) was

determined with a YSI 2700 Select Biochemistry Analyzer (Yellow Springs Incorporated,

Yellow Springs, OH, USA). The residue was then freeze-dried and suspended in 5 mL of

distilled water, to which 0.5 mL glucoamylase (1 mg∙mL−1) was added. The sample was

incubated at 55°C for 2 h. After hydrolyzing the starch in glucose, the sugar content was

15 measured using the same biochemical analyzer. The starch content was identified using a

calibration curve determined from standard starches analyzed with the same procedure.

For nitrogen analysis, analysis of nitrogen content, excluding nitrate, used the

Kjeldahl method (Miyama et al., 1973). The content of nitrogen within nitrate was

determined by suspending the dry matter in 0.2 g distilled water, and extracting at 70°C

20 for 20 minutes, then quantifying with RQflex reflectometer (Merck, Darmstadt,

Germany). The total amount obtained with the above two methods defined the total

nitrogen content.

Sprouting and flowering survey

102 On Sep. 30th, 100 days after pruning, 20 vegetative shoots with 15 to 20 cm that were

growing at the height of the crown and slightly upwards were selected from each tree. On

Dec. 20th, 181 days after pruning (29 days after heating), number of sprouting nodes,

sprouting percentage, and number of vegetative shoots were measured.

5 For forcing method of flowering survey, seven vegetative shoots were randomly

collected three times from each treatment comprised of five trees on days 97, 120 and 141

after pruning. After spraying with benzylaminopurine 100 mg∙L−1, the samples were

immediately sealed in plastic bags and sent by refrigerated transport to the Kuchinotsu

Citrus Research Station (Kuchinotsu, Minamishimabara, Nagasaki, Japan). The following

10 day, the branch base of each delivered sample was cut and the leaves were removed,

leaving the petioles. Each branch was then placed in tall beaker containing 50 mL ion

exchange water and kept in an incubator maintained at 32°C with approximately 80%

humidity. The floral percentage (number of nodes with flowering/total number of nodes

of vegetative shoot × 100) was surveyed at one to two days intervals. Timing of floral

15 evocation was defined as the time when flower buds were first seen (Okuda et al., 2004).

For conventional method of flowering survey, the numbers of leafless flowers and

leafy flowers on vegetative shoots were surveyed in the same trees as for the flowering

survey on Dec. 20th, 181 days after pruning (29 days after heating).

20 Statistical analysis

Data were subjected to the means were separated using t-test at P < 0.05 using FREE

JSTAT for Windows (Vector, Tokyo, Japan). The percentage data were transformed using

arcsine before statistical analysis.

103 Results

Effect of chemicals for sprouting and flowering on vegetative shoots

There was no sprouting at all in either treatment on 100 days after pruning (Table 3–

5 11). There was no difference between treatments in the inhibition of sprouting.

Regarding to flowering, in forcing method, timing of floral evocation was confirmed

120 days after pruning in the NAA treatment and 141 days after pruning in the

ethychlozate treatment (Fig. 3–12). The floral percentage on day 141 after pruning was

13.5% in the NAA treatment, which was 7.5% higher than in the ethychlozate treatment.

10 In conventional method, there were 3.2 leafy flowers per a vegetative shoot in the NAA

treatment, which was significantly more than in the ethychlozate treatment (Table 3–12).

The total number of flowers was also higher in the NAA treatment, albeit not significantly

higher. The sprouting ratio of the NAA treatment also tended to be higher than that of the

ethychlozate treatment.

15

Effect of chemicals for the morphology on vegetative shoots

The length of the vegetative shoots 140 days after pruning was around 15 cm in both

treatments, and there was no difference between the treatments (Table 3–13). However,

the proximal diameter (thickness) in the NAA treatment was 3.8 mm, which was

20 significantly thicker than the 3.4 mm thickness in the ethychlozate treatment. The dry

weight per a vegetative shoot varied from 1.5 g to 2.7 g for the leaves, and 0.4 g to 0.9 g

for the branches (Fig. 3–13). On day 49 after pruning, dry weight of the leaves were

approximately 1.5 g and dry weight of the branches were approximately 0.4 g in both

treatments, but dry weight of both leaves and branches continued to increase up to day

104 140. However, from day 71 onwards, both the leaves and the branches transitioned

significantly higher in the NAA treatment compared to the ethychlozate treatment.

Although the length of the vegetative shoots in the NAA treatment was almost the same

as those in the ethychlozate treatment, the proximal diameter was thicker, and the dry

5 weight was larger, suggesting that the vegetative shoots were more enriched than those in

the ethychlozate treatment.

Effect of chemicals for the nutrition on vegetative shoots

The glucose content declined in both the leaves and the branches up to 97 days after

10 pruning, and then increased thereafter (Fig. 3–14). The glucose content of the leaves was

always higher than that of the branches, up to 120 days after pruning. There was no

significant difference in glucose content between the NAA treatment and ethychlozate

treatment in either the leaves or the branches. The sucrose content increased up to 97 days

after pruning, and tending to decrease thereafter (Fig. 3–14). The content of the branches

15 was higher than that of the leaves. The sucrose content was higher in the NAA treatment

in both the leaves and branches between 97 and 120 days after pruning, compared to the

ethychlozate treatment. The starch content differed significantly between the leaves and

the branches and between treatments (Fig. 3–14). In the leaves the starch content peaked

at day 71 after pruning in both treatments, declining thereafter until day 97, followed by

20 another increase until day 140. The starch content in the leaves was always at least 2%

higher in the NAA treatment, from day 71 after pruning, compared to the ethychlozate

treatment. The starch content of the branches was lower than that of the leaves; it

remained at a low concentration from beginning to end in the ethychlozate treatment,

while in the NAA treatment it increased between day 71 and day 140 after pruning.

105 The total nitrogen content was 2.3% to 3.0% in the leaves, and 1.5% to 2.1% in the

branches, and there was no significant change during the survey period (Fig. 3–15). The

nitrogen content of the ethychlozate treatment tended to be higher than that of the NAA

treatment in both the leaves and branches.

5 C/N ratio is defined that the carbohydrate content (C), consisting of the total glucose,

sucrose and starch content, is divided by the total nitrogen content (N). The C/N ratio was

always higher in both the leaves and branches in the NAA treatment from day 71 after

pruning onwards, compared to the ethychlozate treatment (Fig. 3–16). The C/N ratio

shifted in the same way as the starch content which is the largest carbohydrate component

10 because there was little change in total nitrogen.

Discussion

Low winter temperatures promote differentiation of flower buds in satsuma mandarin

15 plants, which increases the number of flowers the following year, but in early heated

greenhouses, the plants are warmed without ever encountering low enough temperatures,

and this often creates the problem of non-sprouting buds or insufficient flowering (Hirose,

2003; Inoue, 1989). In this study, it was demonstrated that NAA treatment had an

equivalent inhibiting effect on vegetative shoot sprouting, compared to ethychlozate

20 treatment, which is currently customarily used for this purpose. It was found that NAA

treatment also enriched vegetative shoots, accelerated physiological flower bud

differentiation, and increased the number of flowers, which were mainly leafy flowers. In

the NAA treatment, which had superior flowering, the starch content in both the leaves

and branches was always higher than in the ethychlozate treatment. The total nitrogen

106 content of the leaves was always lower in the NAA treatment than in the ethychlozate

treatment, and consequently, the C/N ratio was always higher in the NAA treatment.

Reports have shown that the greater the amount of carbohydrate in the vegetative

shoots of greenhouse mandarin plants, the more flowers will be produced (Kawano, 1987;

5 Suzaki et al., 1993; Takase and Mizuno, 1997; Yahata et al., 1990). Generally, the higher

C/N ratios tend to result in increased flowering (Kawano, 1987; Okada, 2004). These

findings are consistent with the results of this study. That is, the nutritional status of the

vegetative shoots in the NAA treatment was in the preferred state for flower bud

differentiation, and as a result, there was increased flowering compared to the

10 ethychlozate treatment. Regarding the relationship between the timing of physiological

flower bud differentiation and component changes, there was almost no difference in the

fluctuations between the NAA treatment, which had early physiological flower bud

differentiation, and the ethychlozate treatment, which had delayed physiological flower

bud differentiation, so there was no correlation.

15 Manago and Hirobe (1987) examined the fresh weight of trees treated in successive

years with NAA and ethychlozate, and untreated trees, and found that the ethychlozate-

treated trees had lower fresh weight than the untreated trees, and the reduction in weight

was particularly notable for the underground part of the tree. The fresh weight of NAA-

treated trees was almost the same or slightly higher than that of the non-treated trees.

20 Tominaga et al. (1976) reported that spraying trees with NAA for successive years

inhibited the growth of the tree, resulting in a more compact tree, and although the young

shoots were shorter, the root system volume was comparatively larger than in the non-

sprayed trees, and the tree’s vigor tended to be directed towards reproductive growth.

That is, based on the findings to date, it is assumed that NAA places a smaller load on the

107 tree roots than ethychlozate and maintains tree vigor at an equivalent level to that of

untreated trees. This is thought to be one of the reasons for the increase in the total number

of flowers, which were mainly leafy flowers, and the high node sprouting rate in the NAA

treatment in this study.

5 The above findings clarify that NAA spraying after pruning of early heated

greenhouse mandarin trees enriches vegetative shoots and improves flowering compared

to ethychlozate spraying; our study data also suggest that it improves flowering in terms

of carbohydrate and nitrogen nutrition.

This study formed part of the basic data when NAA was re-registered in 2009. The

10 aim of the revisions to the registration was to inhibit the growth of summer and fall shoots,

and use is not limited to greenhouse cultivation. Young shoots grow three times in

subtropical regions, in the spring, summer and fall, but node sprouting occurs all year

round in tropical regions (Agustí et al., 2014). As global warming progresses in the future,

node sprouting patterns may appear that differ from the conventional cycles. Fall branches

15 have particularly poor flowering, so growth of these branches is not desirable. In the

future, proactive use of NAA in outdoor cultivation could be considered as a means of

inhibiting the growth of fall shoots.

108 Table 3–11. Effect of NAA and ethychlozate treatments on sprouting from vegetative shoot on 100 days after pruning.

Treatment Number of nodes Number of sprouting NAA 8.7 0.0 Ethychlozate 8.6 0.0 t -test NS NS

NS indidates non-significance at the 0.05 levels by t-test (n = 3). NAA and ethychlozate treatments were conducted on 50 and 72 days after pruning.

109 Table 3–12. Effect of NAA and ethychlozate treatments on sprouting and flowering after heating.

Number of Sprouting Number of Number of leafless Number of leafy Total number Treatment sprouting nodes percentage (%) shoots flowers flowers of flowers NAA 5.6 64.6 0.9 3.8 3.2 7.0 Ethychlozate 5.0 58.1 0.9 3.2 1.7 4.9 t -test NS NS NS NS * NS

NS and * indidate non-significance or significance at the 0.05 levels by t-test, respectively (n = 3). These numbers were per a fruitng mother shoot. The survey was conducted on 28 days after heating (181 days after pruning). 110 Table 3–13. Effect of NAA and ethychlozate treatments on length and proximal diameter of vegetative shoot on 140 days after pruning.

Length of vegetative Proximal diameter of Treatment shoot (cm) vegetative shoot (mm) NAA 15.8 3.8 Ethychlozate 15.3 3.4 t -test NS *

NS and * indidate non-significance or significance at the 0.05 levels by t-test, respectively (n = 3).

111 20 ●NAA ○Ethychlozate

15

10

Floral percentage (%) percentage Floral 5

0 90 100 110 120 130 140 150 Days after pruning

Fig. 3–12. Effect of NAA and ethychlozate treatments on floral percentage (forcing method). Floral percentage = (Number of nodes with flowering/Total number of nodes of vegetative shoots)*100. Vertical bars show standard errors of three replicates. NAA and ethychlozate treatments were conducted on 50 and 72 days after pruning. Closed symbol: NAA, opend symbol: ethychlozate.

112 ●NAA ○Ethychlozate 3.5

3

2.5 Leaf 2

1.5

1 Dryweight (g) Branch 0.5

0 40 50 60 70 80 90 100 110 120 130 140 150 Days after pruning

Fig. 3–13. Effect of NAA and ethychlozate treatments on dry weight of leaf and branch. Vertical bars show standard errors of three replicates. NAA and ethychlozate treatments were conducted on 50 and 72 days after pruning. Closed symbol: NAA, opend symbol: ethychlozate. Solid line: branch, dashed line: leaf.

113 (a) ●NAA ○Ethychlozate 3.5 ） 3

2.5

2 （ %dry weight Leaf 1.5

1

0.5 Branch

Glucose contents Glucose 0 40 50 60 70 80 90 100 110 120 130 140 150 Days after pruning

3.5 ） (b) 3

2.5

（ %dry weight 2 Branch

1.5

1

0.5 Leaf

Sucrose contents contents Sucrose 0 40 50 60 70 80 90 100 110 120 130 140 150 Days after pruning 14 (c) 12 10

8 Leaf （ %dry weight ） 6 4 2 Branch

Starch contents 0 40 50 60 70 80 90 100 110 120 130 140 150 Days after pruning

Fig. 3–14. Effect of NAA and ethychlozate treatments on glucose, sucrose and starch contents of leaf and branch. Vertical bars show standard errors of three replicates. NAA and ethychlozate treatments were conducted on 50 and 72 days after pruning. Closed symbol: NAA, opend symbol: ethychlozate. Solid line: branch, dashed line: leaf.

114 3.5 ●NAA ○Ethychlozate

） 3.0 Leaf 2.5

（ %dry weight 2.0

1.5 Branch

1.0

0.5

0.0 Total nitrogencontents 40 50 60 70 80 90 100 110 120 130 140 150 Days after pruning

Fig. 3–15. Effect of NAA and ethychlozate treatments on total nitrogen contents of leaf and branch. Vertical bars show standard errors of three replicates. NAA and ethychlozate treatments were conducted on 50 and 72 days after pruning. Closed symbol: NAA, opend symbol: ethychlozate. Solid line: branch, dashed line: leaf.

115 6.0 ●NAA ○Ethychlozate

5.0

4.0

3.0 C/Nratio Leaf 2.0

1.0 Branch 0.0 40 50 60 70 80 90 100 110 120 130 140 150 Days after pruning

Fig. 3–16. Effect of NAA and ethychlozate treatments on C/N ratio of leaf and branch. Vertical bars show standard errors of three replicates. NAA and ethychlozate treatments were conducted on 50 and 72 days after pruning. Closed symbol: NAA, opend symbol: ethychlozate. Solid line: branch, dashed line: leaf.

116 Chapter 4

General discussion

In fruit tree cultivation, it has been pointed out that the effects of recent increases in

5 temperature in Japan include the acceleration of budding and flowering, acceleration or

delay of harvesting, delayed fruit coloring, delay of endodormancy breaking or budding,

and flower bud initiation in forcing culture (Sugiura et al., 2012). The effects of recent

warming trends on fruits were enlarged fruit size, reduced acidity and astringency,

softened flesh, rapid spoiling, and increased in sun scalding. In addition, increased or

10 decreased freezing injury and late frost damage, and changes in disease and pest statuses

were pointed out. The most common effect was delayed fruit coloring. In citrus, delayed

fruit coloring was a problem, especially in extremely early and early ripening satsuma

mandarin, which were harvested in relatively high temperatures. Mulching cultivation

using a white vapor-permeable sheet is one of effective countermeasures against delayed

15 fruit coloring (Shimazaki and Nesumi, 2016). In grapes and apples, delayed fruit coloring

has also become a problem due to warming (Azuma, 2018; Honda and Moriya, 2018;

Sugiura et al., 2018, 2019); anthocyanins, the main pigments of the skin color of grapes

and apples, are suppressed by warming temperatures. In grapes, it has been shown that

the skin color is improved by LED irradiation at night (Azuma, 2018). In addition, new

20 grape and apple cultivars with better coloring are being developed (Azuma, 2018; Honda

and Moriya, 2018; Sugiura et al., 2018, 2019).

Sugiura et al. (2007) revealed the positive and negative effects of global warming on

citrus. In citrus, in addition to the advancement of the sprouting and flowering period,

adverse effects include the delayed flower bud differentiation in greenhouse cultivation,

117 increased physiological fruit drop, increased fruit size, low juice acidity, delayed and

decreased peel coloring, increased sun scalding, increased peel puffing, and low

storability. An increased delay in peel coloring also delays the harvest, which causes a

notable increase in the incidence of peel puffing and the reduction of storability, so the

5 available time for fruit harvesting tends to shorten significantly. In addition, in satsuma

mandarin greenhouse cultivation, plants have fewer flowers in high temperature years, so

this may have an adverse effect on fruit production.

The current warming countermeasure techniques that have been implemented for the

above-mentioned problems in citrus are described. Regarding peel puffing, it is a problem

10 in some major satsuma mandarin cultivars that are prone to this problem. Peel puffing

also sometimes occurred in ponkan and ‘Harumi’. As mentioned in Chapter 3 Section 1,

the techniques for reducing the occurrence of peel puffing include methods using fertilizer

(Kawase, 1984; Torikata et al., 1955) and thinning (Inoue et al., 2003; Kitazono et al.,

2008; Takagi et al., 2009). Methods using agrochemicals, such as calcium carbonate,

15 calcium chloride and calcium sulfate, and ethychlozate, are also employed for satsuma

mandarin. A mixture of GA3 and PDJ is an effective method for reducing peel puffing, as

shown Chapter 3 Section 1. This peel puffing suppression technique using the mixed

spraying of GA3 and PDJ, is superior to conventional chemicals in that the effect is stable

and it is not necessary to spray multiple times. Calcium carbonate reduces peel puffing

20 with a single treatment, but there is a problem that a thin layer of calcium residues on the

peel surface which makes the fruit unappealing to consumers remains at harvest (Shiraishi

et al., 1999).

As mentioned above, poor and delayed coloring is one of the biggest problems due

to global warming in satsuma mandarin (Table 1–1, Fig. 1–1). In order to improve peel

118 coloring, there are techniques using fertilizer and others using agrochemicals, such as

ethychlozate and calcium carbonate. Mulching cultivation is also effective for improved

peel coloring. Poor and delayed coloring is a particular problem in extremely early

ripening satsuma mandarin, which is harvested while the temperature is still high.

5 Degreening is a common commercial practice in many parts of the world used to remove

the green color from the peel of orange fruit; this is performed through exposure of the

fruit immediately after harvest to ethylene gas, especially in early harvested cultivars in

which the peel is green (Shimokawa et al., 1978; Smilanick et al., 2006). However, the

method has a risk that customers could buy fruit without acceptable internal maturity.

10 Thus, an alternative method for improving peel coloring was developed in Japan.

Fujisawa (2018) reported that peel coloring was accelerated by spraying with

ethychlozate twice at around 50 and 70 days after full bloom, and by spraying phosphoric

acid/methionine-containing fertilizer every seven days from color break.

Sunscald is also often occurred by climate change (Table 1–1, Fig. 1–1). Sunscald

15 symptoms in citrus are determined irregular yellow or brown blotches on the peel (Tsai

et al., 2013). Severe sun scalded fruits are accompanied by granulation (Chikaizumi,

2000). Sunscald tend to occur in extremely early, early ripening satsuma mandarins and

‘Setoka’. In early ripening satsuma mandarin, sun scalded fruit occurred from Aug. to Oct.

(Chikaizumi, 2007; Ogaki and Tomita, 1960). Sunscald is closely related to fruit aging as

20 well as strong solar radiation (Chikaizumi, 2007). There are some techniques that can be

used to alleviate the occurrence of sunscald, such as thinning (Kitazono et al., 2008),

white shrinkable fruit bags (Suzaki and Ichinokiyama, 2013), and covering the surface of

the fruit which exposed to direct sunlight with a white and thick tape (Katagiri, 2013).

119 The current countermeasure technique for physiological fruit drop is described.

Physiological fruit drop is a problem in all citrus. An increase in physiological fruit drop

not only reduces the annual yield but also enhances alternate bearing. The exogenous

applications of GA3 enhance fruit set of certain citrus species and cultivars (Soost and

−1 −1 5 Burnett, 1961). In Japan, 25–50 mg∙L GA3 or a mixture of 10 mg∙L GA3 and 25–50

mg∙L−1 PDJ spray on 0–10 days after full bloom were reported to reduce physiological

fruit drop in satsuma mandarin. In trees with a lot of new shoots, it is effective to remove

new some shoots around flowers that compete for nutrients with the fruit during setting.

Deterioration during storage is becoming a problem in late ripening satsuma

10 mandarins and other stored citrus cultivars. Recently, due to the effects of global warming,

the long-term storage of satsuma mandarin has become difficult because of increased peel

puffing and temperature rise during storage (MAFF, 2007). In order to increase the

storage stability of satsuma mandarin, it is necessary to suppress peel puffing. In addition,

there is a method of preventing decay during storage by treating with a fungicide

15 (iminoctadine triacetate, benomyl, thiophanate methyl, etc.) before harvesting. In recent

years, it has been shown that low-intensity blue LED irradiation of satsuma mandarin

fruit after harvesting increases the suppression of blue mold disease and the disease

resistance of the pericarp (Yamaga and Furuya, 2017; Yamaga et al., 2015).

Enlarged fruit size and reduced juice acidity are problems in all citrus species.

20 Appropriate moisture stress for satsuma mandarin trees by the plastic sheet mulching and

drip irrigation are effective as a countermeasure for increased fruit size and reduced juice

acidity. The combination of plastic sheet mulching and drip irrigation can control tree

moisture adequately (Iwasaki et al., 2012; Shimazaki and Nesumi, 2016).

120 Among the problems of diseases and pests caused by global warming, the most

threatening in the citrus industry is huanglongbing (HLB) (= citrus greening) (Gottwald,

2010; Iwanami, 2012). The disease is associated with three bacteria (Candidatus

Liberibacter spp.). A diseased tree will gradually fade and eventually die. Outbreaks of

5 HLB have been reported in Asia, Africa, Oceania, South America, and North America.

The disease is mediated by Diaphorina citri Kuwayama. There is no effective

countermeasure against this disease. In Japan, infected trees have been confirmed in

Okinawa Prefecture and on the southern islands of Kagoshima Prefecture (Iwanami,

2012). In the future, HBL will move north with Diaphorina citri Kuwayama because of

10 global warming, and there is a possibility that it will devastate satsuma mandarin

production. In Florida, HLB-resistant citrus cultivars and rootstocks have been developed

following the devastating damage of HBL (Deng et al., 2019).

In order to drastically solve the various problems caused by global warming, it is

possible to use cultivars that are resistant to each problem. There are already several

15 cultivars of satsuma mandarin that are resistant to be peel puffing. There are some

medium ripening satsuma mandarin cultivars that are resistant to peel puffing: ‘Ishiji’

(Hamana et al., 2013), ‘Kawada Unshiu’ (Ochi et al., 2018) and ‘Sasebo Unshiu’

(Furukawa et al., 2011). However, each of the cultivars requires skill in cultivation

compared to the current major medium ripening satsuma mandarin cultivars. There are

20 many cultivars of satsuma mandarin, most of which are found by bud mutation, and the

cultivars are distinguished by phenotypic differences. It is not possible to discriminate

satsuma mandarin cultivars using DNA markers (Fujii et al., 2019). There is a possibility

that the difference in the cultivars of satsuma mandarin can be distinguished by high-

precision whole genome information or epigenetic factors. In recent years, a new

121 mandarin-type cultivar, ‘Mihaya’, which matures and can be harvested at the same time

as the major medium ripening satsuma mandarins and had minimum peel puffing, has

been bred (Nonaka et al., 2012). ‘Mihaya’ is relatively easy to peel, has good flavor, and

is suitable for growing in most Japanese citrus growing areas. Switching to citrus cultivars

5 that prefer higher temperatures than satsuma mandarin is another effective

countermeasure against global warming. Sweet oranges have a good acclimation capacity

to climate change conditions because orange trees grown under high CO2 and high

temperature conditions have shown improved photosynthetic rates (Vu et al., 2002). In

fact, in Ehime Prefecture, the cultivation area for and production of blood orange is

10 increasing steadily as a countermeasure against global warming (USDA, 2018).

This study undertook the following research with the aim of maintaining the stable

production of satsuma mandarin plants despite the various problems caused by climate

change. First, we verified the effect of high temperature on satsuma mandarin cultivation,

and in particular, examined the extent of the effects of increased temperature on

15 physiological fruit drop, peel puffing, and peel coloring. Regarding physiological fruit

drop, it was verified that it was accelerated and increased by a temperature increase of

2°C. In addition, fruit enlargement was promoted by an increase in temperature of 2°C,

suggesting that the competition for nutrients between fruit became stronger, and

physiological fruit fall was accelerated and increased. As for peel puffing, it was shown

20 that it was increased by a 2°C increase in temperature from the flowering period to the

physiological fruit drop period or a 2°C or 4°C increase in the mature period. Regarding

the peel coloring, it was suggested that it was promoted by a temperature increase of 2°C

or 4°C from the flowering period to the physiological fruit drop period. It was also

clarified that an increase in temperature of 2°C during maturity suppresses peel coloring.

122 Although it was expected from previous reports that an increase of 4°C during maturity

was expected to suppress the peel coloring, this study did not suppress peel coloring, so

further study is needed. Next, we developed three techniques to mitigate the damaging

effects of global warming using PGRs. One of them is a technique to reduce peel puffing.

5 We verified the concentration and treatment timing of GA3 and PDJ, which are suitable

for the types of satsuma mandarin, that are not stored, and developed a peel puffing

mitigation technique. We have developed a method to suppress peel puffing with almost

no delay in peel coloring, as well as a method to suppress peel puffing strongly, although

peel coloring is delayed for one to two weeks. The second technique involved the

10 development of handpicking techniques to shorten the time needed for harvesting. It has

been clarified that the combination of GA3 plus PDJ and ET can improve the success rate

by 30–85%. However, since it was not 100%, it was thought that the practical level has

not been reached; further studies are needed to reach the practical level. The third

technique can be used to increase flowering using greenhouse cultivation under global

15 warming conditions. It was shown that vegetative shoots treated by NAA were enriched

nutritionally and had many leafy flowers. This suggests the possibility of promoting the

growth of leafy flowers, which produce good-quality fruit even under elevated

temperatures in greenhouse cultivation.

In this study, we developed a technique to cope with global warming using PGRs.

20 GA3 and PDJ mixed spraying was used to mitigate peel puffing. It has already been

clarified that GA3 reduces peel puffing (Kuraoka et al., 1966). Exogenous GA3 treatment

suppresses peel puffing by preventing the aging of the peel. Fruit treated with exogenous

GA3 shown reduced peel puffing, and endogenous GA-like substances in the peel were

maintain at a higher level than those in untreated fruit (Garcia-Luis et al. 1985; Kuraoka

123 et al. 1977). In addition, it has been observed that endogenous abscisic acid is suppressed

in fruit treated with exogenous GA3 (Kuraoka et al., 1977). Exogenous GA3 treatment

has been shown to increase ovary endogenous GA20 and GA1 contents and promote cell

division and hypertrophy, leading to fruit set in clementines (Mesejo et al., 2016). Thus,

5 exogenous GA3 treatment is thought to increase the endogenous GA content, thereby

reducing peel puffing and promoting fruit set. From the results of this study, it was

clarified that peel puffing and delayed coloring were affected by the concentration of PDJ,

as well as that of GA3 (Figs. 3–1, 3–2, 3–3, 3–4).

One study reported that when peach was treated with MJ at the early stage of fruit

10 growth, fruit maturation was delayed, and the expression level of the GA2ox gene in the

flesh during the maturation period decreased (Ruiz et al., 2013). GA2ox is an enzyme that

metabolizes active GA. In flesh treated with MJ, the decrease in GA2ox gene expression

is thought to be due to the suppression of GA metabolism and the maintenance of GA

activity to suppress maturation. Similarly, there is a possibility that endogenous GA

15 activity is maintained in the fruit of satsuma mandarin by PDJ treatment. In recent years,

transcriptome and metabolome analyses have been carried out to clarify the metabolism

and physiological processes of peel puffing using navel orange (Ibánez et al. 2014).

Glycolysis was severely affected in fruit with peel puffing, based on both the

transcriptomic and metabolomic results. The citric acid content of fruit with peel puffing

20 was significantly low, and the tricarboxylic acid cycle was severely affected. In addition,

this study also suggested that cytokinins and GAs would repress the disorder.

In the development of the handpicking harvesting technique, the conflicting effects of

GA3, PDJ, and ET were used. The peel is hardened by the GA3 and PDJ treatments, but

the ET treatment softens it. In addition, the GA3 and PDJ treatments delay the coloring

124 of the peel, while the ET treatment promotes coloring. In this study, we clarified the

appropriate balance of each chemical that maximizes the harvest efficiency without

compromising fruit quality as much as possible. In addition, fruits treated with GA3, PDJ,

and ET (300 or 400 mg∙L−1) produced more ethylene than those treated with ET (300 or

−1 5 400 mg∙L ) (Table 3–10, Fig. 3–9). Thus, it is inferred that the GA3 and PDJ treatments

increased the peel hardness and the formation of abscission layer was enhanced by ET.

Ethylene and JA have been reported to act synergistically and antagonistically on plant

development and disease response (Zhu et al., 2011). However, there is no report from

plant hormone perspective between GA3, PDJ and ET. It is unclear why GA3 and PDJ

10 treatment increased ethylene production of fruit by ET.

It was shown that the vegetative shoots treated with NAA were enriched nutritionally

and had many leafy flowers. NAA and ethychlozate are both synthetic auxins, but have

different migration characteristics in the tree after application. NAA has very little

migration in the tree after spraying (Hirose, 1976), while ethychlozate is rapidly

15 translocated into the roots, where it is metabolized (Kamuro and Hirai, 1981). The

metabolites from ethychlozate enhance the uptake of water and minerals. Kamuro and

Hirai (1981) stated that the metabolites promote the absorption of water and minerals, but

Manago and Hirobe (1984) reported that one to three weeks after treatment with

ethychlozate, the tree was deficient in water, N, P, Ca and Mg. Since ethychlozate is

20 sprayed for successive years, the vigor of the tree decreases, and it is likely that

ethychlozate temporarily prevents root activity and the absorption of nutrients. It is

speculated that ethychlozate weakened the tree vigor because of hindered root activity

and water absorption, resulting in poor branching and poorer flowering than those in trees

treated with NAA.

125 Comprehensive global warming countermeasures that combine the countermeasure

techniques for each of the problems described above and the techniques developed in this

study are described (Fig. 4–1). If physiological fruit drop is promoted in an off year, a

combination of GA3 treatment (or GA3 and PDJ treatment) and the removal of some

5 shoots around the flowers will be performed to ensure fruit yield. In an on-year, if the

amount of fruit is too large to be fully removed by hand, NAA and ethychlozate, which

are fruit thinning agents, are used. NAA has the effect of promoting fruit enlargement, so

it is not used in cultivars where large size is a problem. In addition, the plastic sheet

mulching and drip irrigation are performed from the time according to the cultivars. By

10 carrying out the plastic sheet mulching and drip irrigation, excessive water is prevented

from being absorbed into the tree because of heavy rain, and moderate water stress is

applied to the tree to prevent peel puffing, poor coloring/coloring delay, fruit enlargement,

and reduced juice acidity. Sunscald is a problem in the summer and autumn, especially in

extremely early and early ripening satsuma mandarins. In orchards with a lot of sun

15 scalded fruit, these symptoms can be suppressed by using white shrinkable fruit bags or

covering the surface of the fruit which exposed to direct sunlight with a white and thick

tape. In order to prevent peel puffing, it is effective to use not only the plastic sheet

mulching and drip irrigation, but also the application of a GA3 and PDJ mixed solution.

In order to increase the storage stability of satsuma mandarin, it is necessary to treat with

20 fungicide before harvesting. In addition, NAA spraying will effectively enhance the

growth of vegetative shoots and promote flowering in heated greenhouse cultivation.

Further, when warming progresses, autumn shoots are likely to occur because of an

increase in the soil temperature, and therefore, it may be possible to prevent the

enrichment of spring and summer shoots as fruiting mother shoots. In such cases, NAA

126 is sprayed when autumn buds occur, leading to enrichment of the fruit mother shoots.

When the appropriate time for harvesting is short, it is conceivable to use a handpicking

technique using PGRs.

The suitable location for satsuma mandarin production are predicted to move from

5 the southern coastal sites to inland areas of western Japan, the plains of Kanto, littoral

zones of the Sea of Japan in central and western Japan, and southern Tohoku (Sugiura et

al., 2014; Sugiura and Yokozawa, 2004). Therefore, further countermeasures against

global warming would be required in existing satsuma mandarin production area.

The effect of elevated temperature on peel puffing in Chapter 2 Section 2 and the

10 technique of reducing peel puffing by spraying a mixture of GA3 and PDJ in Chapter 3

Section 1 were investigated. As seen in Chapter 2 Section 2, when the temperature rose

by 2°C from the flowering stage to the physiology fruit drop stage and during the maturity

stage, the extent of peel puffing was increased by 0.8 (Table 2–7). The extent of peel

puffing was also increased by 2.0 when the temperature rose by 4°C from the flowering

15 stage to the physiology fruit drop stage and during the maturity stage (Table 2–9). In

Chapter 3 Section 1, the extent of peel puffing reduction with GA3 and PDJ mixed

treatment was 0.2 to 0.8 (Figs. 3–1, 3–2, 3–3, 3–4). This suggests that GA3 and PDJ

mixed treatment can offset the increase in peel puffing caused by a temperature rise of

2°C during the flowering period to the physiological fruiting period and during the

20 maturity period. However, it was thought that this technique could not suppress the

increase in peel puffing caused by temperatures elevated by 4°C from the flowering

period to the fruit drop period and during the maturity period. At temperatures elevated

by 4°C, peel puffing was alleviated not only by spraying a mix of GA3 and PDJ, but also

by a combination of other techniques for alleviating peel puffing, such as other PGRs,

127 fruit thinning methods, and fertilization management. For example, a combination of

mulching cultivation and GA3 and PDJ spraying not only reduces peel puffing, but also

alleviates peel coloring delay (Araki and Makita, 2014). In addition, if the SRH has been

improved to a practical level in Chapter 3 Section 2, it can be used as a technique for

5 rapid harvesting at a suitable time for harvesting.

In the future, if global warming has advanced greatly, it is thought that there will be

situations that cannot be controlled by current countermeasure techniques. In order to

drastically solve the various problems caused by global warming, it is necessary to select

and produce cultivars suitable for the environments where global warming is expected.

10 In Florida, HLB-tolerant new mandarin-type cultivar ‘Bingo’ was released (Gmitter,

2017). This cultivar is not only early ripening (in October) but also has excellent

coloration even at high temperatures. However, citrus breeding took a long time with the

conventional method, and it took about 20 years from the time a new cultivar was

developed and cultivated for commercial shipment (Omura and Shimada, 2016). A long-

15 juvenile period is one of the biggest problems in citrus breeding, with a juvenile period

of approximately five to ten years for orange, lemon, and mandarins (Furr et al., 1947).

However, recent advances in biotechnologies and genomics have the potential to

accelerate cultivar development (Omura and Shimada, 2016; van Nocker and Gardiner,

2014). One of the methods of improving the efficiency of the breeding process is the use

20 of marker-assisted selection. DNA markers for Citrus tristeza virus resistance from

trifoliate orange (Ohta et al., 2011), male sterility (Goto et al., 2018),

monoembryonic/polyembryonic citrus species (Shimada et al., 2018) have been

developed. Nowadays, genome-wide association studies and genomic selection, which

predict characteristics from a large amount of DNA marker information, proved useful

128 for the improvement of citrus cultivars (Imai et al., 2018; Minamikawa et al., 2017).

Using these methods, important characteristics, such as fruit weight and color and ease

of peeling, which are difficult to select by conventional DNA marker selection, can be

predicted with high accuracy at the seedling stage. Recently, integrative studies among

5 transcriptomic, proteomic and metabolomic profiles have been focused upon. Several

transcriptomics of citrus focusing on HLB have been reported together with combinations

of proteomics (Shiratake and Suzuki, 2016). Recently, some databases collecting the

transcriptome, proteome, metabolome, hormonome, and ionome data of fruit have been

published. The Mikan Genome Database (https://mikan.dna.affrc.go.jp) (Kawahara et al.,

10 2020), which provides citrus genome polymorphism information centered on satsuma

mandarin, has also been released. Variety breeding is becoming possible by efficiently

selecting new cultivars with suitable phenotypes because of the development of new

technologies, such as -omics and phenotyping (De Ollas et al., 2019). The development

of cultivars that have good flavor and are suitable for a warming environment is expected

15 to be promoted in the future.

129 Fig. 4–1. Comprehensive global warming countermeasures of satsuma mandarin.

Jan. Feb. Mar. Apr. May Jun. Jul. Aug. Sep. Oct. Nov. Dec.

Fruit growth Fruit enlargement stage Sprouting Flowering Physiological fruit drop Fruit ripening

Pruning Disbudding Fruit thinning Harvesting Mainly /Disblossoming management

130 Fertilizing Fertilizing Fertilizing

Deterioration during storage Fungicide spray

Increase peel puffing, poor and delayed coloring, fruit enlargement, reduced juice acidity Plastic sheet mulching and drip irrigation Problems of global warming Increase fruit drop (off year) Increase peel puffing Countermeasures GA3 (GA3+PDJ) spray/removal GA3+PDJ spray of some shoots around flowers Shorten appropriate Increase sunscald time for harvesting Decrease leafy flowers (green house) White shrinkable bags Handpicking NAA spray White and thick tape Acknowledgements

I wish to express my grateful thanks to Dr. T. Sugiura (Professor of Graduate School of

Life and Environmental Science, University of Tsukuba) for his continuous encouragement, persistent guidance, and invaluable suggestion.

Thanks are also due to Professors of Graduate School of Life and Environmental Science

(University of Tsukuba) Dr. T. Yamamoto, Dr. M. Kunihisa and Dr. S. Sugaya for their continuous supervision, invaluable comments, and critical reviews to further improve this dissertation.

I express my sincere gratitude to Dr. Y. Ikoma and Dr. H. Okuda for their continuous supervision, invaluable suggestion, and technical advice. I am grateful to Dr. T. Moriguchi and Dr. M. Yamada for their kindly advise.

I deeply thank Mr. H. Fukamachi, Dr. M. Iwasaki, Mr. T. Takahara, Dr. Y. Yonemoto, Mr.

K. Sugihara, Dr. H. Matsumoto, Dr. N. Nakajima for their valuable suggestions and discussions. I also thank Ms. K. Fushimi and Ms. S. Fukuda for their technical assistance.

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