Kobe University Repository : Thesis

Kobe University Repository : Thesis Development of a novel high-value added hydroponic 学位論文題目 technique using dry-fog spray fertigation(ドライフォグ Title を用いた新しい高付加価値水耕栽培技術の開発に関する研究) 氏名 Hikosaka, Yosuke Author 専攻分野博士（農学） Degree 学位授与の日付 2016-03-07 Date of Degree 公開日 2017-03-01 Date of Publication 資源タイプ Thesis or Dissertation / 学位論文 Resource Type 報告番号乙第3304号 Report Number 権利 Rights URL http://www.lib.kobe-u.ac.jp/handle_kernel/D2003304 ※当コンテンツは神戸大学の学術成果です。無断複製・不正使用等を禁じます。著作権法で認められている範囲内で、適切にご利用ください。

Create Date: 2017-12-18

Doctoral Dissertation

Development of a novel high-value added hydroponic technique

using dry-fog spray fertigation

January 2016

Graduate School of Agricultural Science, Kobe University

Yosuke Hikosaka

Table of contents

Summary ------1

Chapter 1. General introduction ------5

Chapter 2 ------9 A novel aeroponics technique using dry-fog spray fertigation to grow leaf lettuce (Lactuca sativa L. var. crispa) with water saving hydroponics

2-2. Materials and methods ------12 2-3. Results and discussion ------15

Chapter 3 ------25 Growth and physiological characteristics of leaf lettuce grown in an environment controlled room using a new aeroponic system with dry-fog spray fertigation

3-2. Materials and methods ------28 Dry-fog aeroponic system Plants and culture conditions Measurements of growth and photosynthesis Measurements of root activity Measurement of leaf constituents Data analysis

3-3. Results and discussion ------33 Flux density of dry-fog particles and lettuce growth Physiological activity of the leaves and roots Leaf constituents

Chapter 4 ------46 Growth and physiological characteristics of leaf lettuce grown in a greenhouse using a new aeroponic system with dry-fog spray fertigation

4-2. Materials and methods ------48

4-3. Results and discussion ------50 Growth of Leaves and Roots Physiological Activity of the Leaves and Roots Leaf Constituents

i Chapter 5 ------58 Ever-bearing strawberry culture using a new aeroponic system with dry-fog spray fertigation during the summer

5-2. Materials and methods ------60 5-3. Results and discussion ------63 Plant temperature under the mid-summer climate conditions Flowering and Fruiting Photosynthetic Ability

Chapter 6. Concluding remarks ------70

References ------73

Acknowledgment ------81

ii Abbreviation

A: CO2 assimilation rates

ABA: abscisic acid

BSA: bovine serum albumin

Ci: intercellular CO2 concentrations

DF: dry-fog

DFT: deep flow techniques

DTT: dithiothreitol

DW: dry weight

EC: electrical conductivity

EDTA: ethylenediaminetetraacetate

FW: fresh weight gs: stomatal conductance

LED: light emitting diode

HPLC: high-performance liquid chromatography

PPFD: photosynthetic photon flux density

SDF: semi-dry-fog

SE: standard error

SLA: specific leaf area

TPF: triphenylformazan

TSP: total soluble protein

TTC: triphenyltetrazolium chloride

TSS: total soluble solids

iii T/R: top/root fresh weight ratios

φ: apparent CO2 fixation efficiency

iv Summary

The Hydroponic culture is one of the most important plants production techniques to address the anticipated hood shortage in the near future. The dry-fog aeroponics cultivating the plants with extremely fine fog of nutrient solution is a novel hydroponic technique expected for improving the yields and water resource efficiency. In this study, the author investigated the growth characteristics cultivated with the dry-fog aeroponics and optimum environment of foggy rhizosphere to increase the yields.

In chapter 2, growth characteristics of lettuce cultivated using a dry-fog hydroponic technique were investigated and compared to lettuce cultivated using deep flow technique

(DFT) as the prevailing hydroponic technique. Dry-fog hydroponics is an aeroponic technique that sprays a very fine foggy nutrient solution with an average droplet diameter of less than 10 µm into the root zone. The roots extend into the chamber filled with dry-fog of the liquid fertilizer and absorb water and nutrients from the dry-fog that fills the rhizosphere. This soilless culture system needs less water than any other hydroponic technique, and no differences were found in growth and harvest quality of plants between the two tested systems. For dry-fog culture, root growth was encouraged and root hair significantly developed to catch the foggy nutrient solution efficiently. The contents of ascorbic acid, nitrate nitrogen, Ca2+ and chlorophyll of leaves were not significantly different between the two hydroponic cultures. However, respiration rate of roots and photosynthetic rate of leaves significantly increased with dry-fog culture. Because the amount of water around the roots is less with dry-fog, horticultural crops are expected to grow well with this novel hydroponic technique, which optimizes the growth and quality of plants with water-saving hydroponics.

1 In chapter 3, The growth characteristics and physiological activities of leaves and roots of lettuce cultivated in dry-fog aeroponics with different flow rates of nutrient dry-fog (FL, 1.0 m s-1; NF, 0.2 m s-1) were investigated under a controlled environment for two weeks and compared to lettuce cultivated using deep-flow technique (DFT). The growth of leaves of FL and DFT was not different and was significantly higher than that of NF. The amount of dry-fog particles adhering to the objects was higher in FL than in

NF, so that the root growth in NF was significantly higher than that of FL. The respiration rate of roots was significantly higher in dry-fog aeroponics, but the dehydrogenase activity in the roots was significantly higher in DFT. There were no differences in the contents of chlorophyll and total soluble protein in the leaves or the specific leaf area.

Photosynthetic rate and stomatal conductance were higher in dry-fog aeroponics. The contents of nitrate nitrogen, phosphate and potassium ions in the leaves were significantly higher in DFT, but the content of calcium ions was significantly higher in FL. Thus, changing the flow rate of the dry-fog in the rhizosphere can affect the growth and physiological activities of leaves and roots.

In chapter 4, the author investigated the growth characteristics and physiological activity of leaf lettuce cultivated using different sizes of liquid fertilizer droplets and different rates of fog flow through the rhizosphere using a dry-fog hydroponics technique in a greenhouse. These characteristics were subsequently compared to lettuce cultivated using DFT. The cultivation chambers were filled with dry-fog (DF) containing droplets that average less than 10 µm in diameter or semi-dry-fog (SDF) containing droplets that average 20 µm in diameter. The plants were grown for three weeks following transplantation, and the author measured the growth, respiration rate and triphenyltetrazolium chloride (TTC) reduction ability of the roots in addition to the

2 photosynthesis rate and the contents of total soluble protein, nitrate nitrogen and calcium ions. The amount of leaf growth increased with a slower flow rate under both DF and

SDF conditions and was increased in both dry-fog aeroponic conditions compared to leaves grown in DFT. SDF with a slow flow rate increased root growth and activity and resulted in an increase in stomatal conductance and photosynthesis rate. DF with a slow flow rate also significantly increased the growth of leaves and roots, but the physiological activities were not affected. Thus, the growth and physiological characteristics of leaf lettuce cultivated using dry-fog aeroponics were improved in different ways by changing both the atomized droplet size and flow rate in the root zoon.

In chapter 5, the author developed the dry-fog hydroponics for establishing ever-bearing strawberryFragaria x ananassa Duch. var. Summer ruby production during the summer. This aeroponic system keeps rhizosphere temperature lower than air temperature owing to the release of latent heat by dry-fog evaporation and adiabatic expansion of solution from the exhaust nozzle. Runner seedlings of strawberry plants were cultivated with dry-fog aeroponic system from early June to December in a greenhouse, the growth, flower bud formation, fruit quality, photosynthetic rates and the contents of chlorophyll and total soluble protein in leaves were compared with them of plants cultivated by drip fertigation with palm shell medium as a control. Rhizosphere and the crown temperatures of dry-fog aeroponics were reduced by approximately 3-5˚C throughout the day compared with control and were maintained below 25˚C during the late summer. Although ever-bearing strawberry plants are insensitive to high temperature under long day conditions from a viewpoint of flower initiation, low temperature, especially the crown part of shoot base, is considered important for inducing flowering.

For dry-fog aeroponics, the number of inflorescence and ˚Brix of fruits significantly

3 increased but the number of runner and vegetative growth significantly decreased compared with control. Photosynthetic rates of leaves grown with dry-fog aeroponics

increased than those of control under both light and CO2 limited and saturated measurement conditions. In addition, leaf chlorophyll and total soluble protein contents significantly increased in dry-fog aeroponics compared with control. From these findings, the author suggested that dry-fog aeroponics is an usable new hydroponics for raising ever-bearing strawberry plants during the summer by maintaining rhizosphere temperature low suitable for shifting their growth phase from vegetative to reproductive.

In conclusion, dry-fog aeroponics can improve the growth of plants and yields, and this new hydroponic technique will contribute to food production.

4 Chapter 1. General introduction

To address the food shortages in the near future caused by a decrease in arable land or increase in the world population, greenhouse horticultures, especially hydroponic cultures without soil, are becoming more important. While one advantage of a hydroponic culture is that plants can be cultivated under optimally controlled conditions for nutrients regardless of the soil conditions, a large amount of water is required to grow crops with nutrient solution. New hydroponic systems that enable stable crop production while saving water have been anticipated in recent years. Aeroponics is a water-saving hydroponic technique without rooting media (Weathers and Zobel, 1992; Ritter et al.,

2001; Farren and Mingo-Castel, 2006). In this culture, liquid fertilizer is periodically sprayed on the roots from nozzles with pipes in the rooting zone. The sprayed nutrient solution that is not absorbed by the roots is usually re-sprayed using a re-circulation system or it is discarded directly. Tomato, one of the most important crops world-wide, has recently been cultivated using aeroponics (Biddinger et al., 1998; Zhao et al., 2010).

One of the features of this culture system is nutrient accessibility in which nutrients are absorbed efficiently by the roots. Furthermore, root growth in most vegetable crops is expected to increase under the aerobic conditions of the rhizosphere environment because there is no solid phase and less liquid phase compared to the physical composition of soil or any other rooting media (Hillel and David, 1988; Cherif et al., 1997).

Dry-fog aeroponics is a novel hydroponic technique that sprays a very fine fog of atomized liquid fertilizer using a specialized nozzle as a double-fluid atomizer with nutrient solution and compressed air (developed and patented by Ikeuchi Co., Ltd., Osaka,

Japan). Dry-fog is the finest fog and is less than 10 µm in diameter on average per droplet.

5 The droplets are too small to wet objects, such as roots. Currently, dry-fog is mainly used to humidify factories producing electronic components or printing to keep the environment static-free and has not yet been applied to crop cultivation in commercial horticulture. In dry-fog aeroponics, dry-fog fertilizer is atomized using a specialized nozzle in the rhizosphere where the plant roots directly absorb water and nutrients from the dry-fog (Fig. 1-1), like an aerial root of epiphytic orchids. Because the fine, foggy, nutrient solution is atomized, only one specialized nozzle can occupy the large space for the rooting zone in a chamber (1000 × 660 × 300 mm), with a smaller amount of water compared to the other types of predominant spray hydroponic techniques; therefore, dry-fog aeroponics can reduce water usage and the number of nozzles that are needed, resulting in a lower running cost and greater environmental conservation. The dry-fog nutrients that are not being absorbed by the roots accumulate as condensation on the inner wall of the cultivation chamber, and the collected condensation in the nutrient reservoir is atomized again. Thus, there is little puddle of the fertilizer solution in the chamber, and the roots hang in the dry fog.

Generally, the spray nozzles especially atomizing fine fog have risks for clogging.

In agriculture, sometimes it had been big problems that the failure of fertilization or environmental control by the clogged nozzles with traditional aeroponics or the mist cooling systems and it has taken large costs to the measures. But the double-fluid nozzles used in the dry-fog aeroponics have the structure that is hard to clogging. An ultrasonic humidifier also can atomize the very fine fog, and has been used for many bioreactors to generate the nutrient fog (Mohammad et al., 2000; Liu et al., 2003; Jing and Yunwei,

2013). In contrast, it is believed that the nozzle used for dry-fog aeroponics atomizes a larger amount of fine fog without problems.

6 As above, Dry-fog aeroponics is expected to increase the yields and improve the water resource efficiency, but has not yet been investigated or established as a commercial cultivation system for horticultural crop production. This study is intended to establish the dry-fog aeroponics as a new hydroponic technique. In chapter 2, the author evaluated the production capacity based on the novelty of the dry-fog aeroponics by comparison with deep flow technique as the conventional hydroponics. In chapter 3 and 4, the author developed the cultivation conditions that focused to the rhizosphere dry-fog environment under the controlled environment and in a greenhouse. Finally, in chapter 5, the author introduced about an effect of the rhizosphere cooling ability to the strawberry cultivation in summer glasshouse as one of the applications of the dry-fog aeroponics.

Spray unit

Fig. 1-1. a) Cutaway drawing of the dry-fog hydroponic chamber. b) System chart of the dry-fog hydroponic technique.

Chapter 2

A novel aeroponics technique using dry-fog spray fertigation to grow leaf lettuce (Lactuca sativa L. var. crispa) with water saving hydroponics

9 2-1. Introduction

In dry-fog aeroponics, the fog of atomized liquid fertilizer that consists of nutrient solution and compressed air passed through a specialized nozzle functioning as a double-fluid atomizer in a chamber made of lightweight polystyrene is less than 10 µm in diameter on average per droplet, so less nutrient solution exists in the hydroponic system than in the general aeroponics system that is used for research and commercial cultivation

(Biddinger et al., 1998; Farren and Mingo-Castel, 2006; He et al., 2013). Dry-fog nutrients that are not absorbed by the roots condense and fall down the inner wall to accumulate at the bottom of the chamber, and the collected solution is atomized again.

Thus, the nutrient solution is circulated in the system, and there is no wastewater outside of the system. Because of these features, dry-fog aeroponics is expected to be an effective technique for protected cultivation to address the food shortage in the future caused by decreases in both arable land and water resources and cultural eutrophication caused by nutrient wastewater. Spray culture using a fog box with nozzles as double-fluid atomizers

(Ehara et al., 1966) or with an ultrasonic humidifier, which can atomize the very fine fog, has been used for aeroponic systems, and many bioreactors have been developed to generate nutrient fog (Mohammad et al., 2000; Chun-Zhao et al., 2003), but the advantage of these methods for practical cultivation is not well understood.

In the present study, leaf lettuces were cultivated using dry-fog aeroponics in a controlled environment. The growth and leaf constituents were measured and compared to plants cultivated with traditional hydroponic techniques (DFT) to evaluate the efficacy of dry-fog aeroponics. Root respiration and leaf photosynthetic rate were also investigated and compared in order to evaluate affects on water and nutrient absorption resulting from

10 differences in the rhizosphere environment in the two hydroponic systems.

11 2-2. Materials and methods

A plastic chamber (1000 × 660 × 300 mm) was fitted with a dry-fog atomizing nozzle to establish a dry-fog cultivation system (Fig. 2-1). A double-fluid atomizer nozzle was connected to the nutrient solution reservoir with a plastic tube (6 mm across) and a compressor with Teflon tube (8 mm across), with an air pressure of 0.3 MPa. Size fractionation of atomized droplets was measured using LDSA-SPR (Tohnichi computer applications Co. Ltd., Tokyo, Japan), which analyzes the droplets by Fraunhofer diffraction and calculates the size distribution of the droplets by Rosin-Rammler distribution. A commercial liquid fertilizer (Otsuka Chemical Co. Ltd., Tokyo, Japan, EC

-1 1.2 mS cm , pH 6.0, N: 130 ppm, P: 60 ppm, K: 200 ppm, Ca: 115 ppm, Mg: 30 ppm,

Fe: 1.4 ppm, Mn: 0.8 ppm, Zn: 0.05 ppm, Cu: 0.02 ppm, B: 0.75 ppm, Mo: 0.02 ppm) was added to the nozzle from a nutrient solution reservoir equipped at the end of an aeroponic chamber. The nozzle atomized fine foggy nutrients that continuously filled the chamber. Each chamber had 48 holes (20 mm across) across the top to hold plants and only the roots remained in the chamber. As a control experiment, a DFT hydroponic chamber (800 × 665 × 310 mm) was filled with 30 L of the same liquid fertilizer. Leaf lettuce (Lactuca sativa L. cv. “Okayama Saradana”, Takii Co. Ltd., Kyoto, Japan) was sown on sponge blocks (10 × 10 × 20 mm) supplied with enough pure water. The air temperature and relative humidity throughout the cultivation were maintained at 26 ˚C

-2 -1 and 60 % respectively. The photosynthetic photon flux density was 250 µmol m s supplied by cool white tubular fluorescent lamps with a 16-h day length. After

-1 germination, the liquid fertilizer (EC 0.6 mS cm , pH 6.0) was supplied by bottom irrigation for two weeks, and 48 seedlings that had grown uniformly were transplanted

12 into each hydroponic system and grown for four weeks. Every week, plants were sampled from both hydroponic systems and the fresh weight, leaf length, and fresh weights of roots were recorded. After sampling, the leaves and roots were dried in an oven at 80 ˚C for two days and the dry weights were recorded. At three and four weeks after transplantation, intact root samples (1 g FW) were taken from a growing plant that developed new roots under hydroponic conditions, and the rate of root respiration was measured polarographically at 26˚C using a Clark-type gas-phase oxygen electrode

(CB1D, Hansatech, Norfolk, UK) with incoming humidified air and 21% O2 under dark conditions.

The contents of nitrate nitrogen, calcium ion, ascorbic acid and chlorophyll in the leaves were measured at three and four weeks. Leaves (10 g FW) were sampled to determine nitrate nitrogen and calcium contents. The leaves were homogenized in de-ionized water and the nitrate nitrogen and calcium contents in the filtrated fraction were measured using RQ-FLEX (Merck Millipore, Darmstadt, Germany). The other leaves (10 g FW) were also sampled and homogenized in a 5% metaphosphoric acid solution, and the content of ascorbic acid in the filtrated fraction was measured using

RQ-FLEX. The content of leaf chlorophyll was measured using SPAD meter (SPAD-502,

KONICA MINOLTA, INC., Tokyo, Japan). At four weeks after planting, CO2 assimilation rates (A) of fully expanded mature leaves were measured under different

-1 CO2 concentrations (50-1500 µmol mol ) using an LI-6400 portable photosynthesis system (Li-Cor, Lincoln, NE, USA). The measurement conditions were leaf temperature,

20 ˚C, and photosynthetic photon flux densities, 1200 µmol m-2 s-1. The maximum rate

(Max) and apparent CO2 fixation efficiency (φ) were estimated as parameters of the best-fitted non-rectangular hyperbola for the photosynthetic responses to intercellular

13 CO2 concentrations (Ci). All data were subjected to one-way analysis of variance

(ANOVA), and the mean differences were compared using the Tukey HSD test when the

F-test indicated a significant difference at P0.01. Each data point was the mean of six replicates and a comparison with P0.05 was considered significantly different.

Fig. 2-1. Dry-fog aeroponic systems (A) and DFT (B) settled in a temperature-controlled room.

14 2-3. Results and discussion

In order to characterize dry-fog spray fertigation conditions, the fog was measured using LDSA-SPR of droplets of various sizes (Fig. 2-2). The minimum and maximum diameter of droplets were 1.64 µm and 149 µm, respectively. LDSA-SPR analyzed the size distribution of droplets, and the Sauter mean diameter, which commonly means the average particle size of powders and liquid droplets, was found to be 8.85 µm by calculation. Therefore, the fog in the chamber was certainly dry-fog.

3 Content percentage ( % ) percentage Content 2

1 0 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 Diameter of droplet(µ

Fig. 2-2. The particle size distribution of droplets using a double-fluid atomizer nozzle to establish a dry-fog hydroponic technique measured by LDSA-SPR. The mean diameter of droplets was analyzed and calculated based on this distribution.

15 There was no significant difference in the fresh and dry weight of leaves between the dry-fog and DFT hydroponic cultures (Fig. 2-3). Root growth was not significantly different at three weeks after planting, but during the fourth week of dry-fog culture the root fresh weight increased by 29 % compared to roots grown using DFT. The roots from the dry-fog culture had many lateral roots, and many root hairs developed on the surface.

This development was not observed in the roots grown using DFT (Fig. 2-4). The root respiration rate at four weeks after planting was significantly high in the dry-fog culture, but there was no significant difference at three weeks between the two hydroponic systems (Fig. 2-5). The photosynthetic rates of the mature leaves grown for four weeks dry-fog culture significantly increased in both limited and saturated Ci compared to those in DFT (Fig. 2-6). In both hydroponic cultures, the photosynthetic rate increased linearly

-1 -1 up to approximately 300 µmol mol Ci and saturated at approximately 600 µmol mol Ci.

Leaf nutrient contents were compared between the two hydroponic cultures at three and four weeks after transplanting (Fig. 2-7). The nitrate nitrogen content in the leaves grown using dry-fog culture significantly increased by 16 % compared to those grown using

DFT at three weeks after planting, but there was no significant difference at four weeks.

The content of ascorbic acid in the leaves grown using dry-fog culture increased by 9 % at three weeks and 4 % at four weeks, compared to the leaves grown using DFT, however there were no significant differences. The calcium content in the leaves grown using dry-fog culture significantly increased at three weeks after planting, but there was no significant difference at four weeks. The chlorophyll content of mature leaves significantly increased with dry-fog culture only at four weeks.

16 120 12 a) Dry-fog FW A DFT FW A 100 Dry-fog DW 10 DFT DW 80 8

a 60 a 6

FW of leaves ( g ) of leaves FW 40 4 DW of leaves ( g ) of leaves DW

20 2

0 0 0 1 2 3 4 Weeks after settling b) 25 2.5 A Dry-fog FW DFT FW

20 Dry-fog DW B 2.0 DFT DW

15 1.5

10 1.0 a FW of roots ( g ) FW

of roots ( g ) DW b 5 0.5

0 0.0 0 1 2 3 4 Weeks after settling

Fig. 2-3. Changes in fresh and dry weights of lettuce leaves and roots grown using dry-fog or DFT hydroponics during the four weeks after transplanting. Different uppercase and lowercase letters show a significant difference (P0.05), bars represent

±se (n=6).

Dry-fog DFT

5cm

5mm

Fig. 2-4. Leaves and roots of the lettuce grown using dry-fog or DFT hydroponics at four weeks after transplanting.

120 Dry-Fog DFT a 100 s ) 80

60 A b / Root gFW gFW Root /

2 A 40 The root repiration rete rete repiration root The O (µmol 20

3 4 Weeks after transplanting

Fig. 2-5. Respiration rates of lettuce roots grown using dry-fog or DFT hydroponics for three and four weeks after transplanting. Different letters show significant difference

(P0.05), bars represent ±se (n=6).

20 A (Crt)(DFT) 18 É A (Dryfog) ÉÉ

É ÉÉ É É 14 É É •s ) É 2 É É É É É 12 ÉÉ mol/m É µ É É 10 ÉÉ É

É É 8 É ÉÉ

É É Photosynthetic rate ( rate Photosynthetic 6 É É É É ÉÉ 4 É ø Max a a DFTCrt: 0.034 11.2 É b b 2 Dryfog: 0.054 16.6 É É 0

0 100 200 300 400 500 600 700 800 900 1100 1000 1200 1300 1400 1500

Ci ( µmol/mol )

Fig. 2-6. Relationships between CO2 assimilation rates (A) and intercellular CO2 concentration (Ci) measured for the attached mature leaves of lettuce grown using dry-fog or DFT hydroponics for four weeks after transplanting. The saturated rate (Max) and the apparent CO2 fixation efficiency (φ) were estimated as parameters of the best-fitted non-rectangular hyperbola response curve to Ci. Different letters show significant difference (P0.05).

1200 1200 a Dry-fog DFT a Dry-Fog DFT

1000 1000

A A a 800 800 a B B 600 600 contents (mg/l) contents 2+ 400 400 Ca

200 200 Nitrate nitrogen contents ( mg/l ) ( mg/l contents nitrogen Nitrate 0 0 1400 3 4 50 3 4 Weeks after settling Dry-fog DFT a Dry-Fog DFT 1200 a 40 1000 A a A A A 30 b 800

600 SPAD 20 400

Ascorbic acid contents ( mg/l ) ( mg/l contents acid Ascorbic 10 200 0 0 3 4 3 4 Weeks after settling Weeks after settling

Fig. 2-7. Nitrate nitrogen, ascorbic acid, calcium ion and chlorophyll content of mature lettuce leaves grown using dry-fog or DFT hydroponics for three and four weeks after transplanting. Different letters show significant difference (P0.05), bars represent ±se

(n=6).

To evaluate the efficacy of dry-fog culture as a novel aeroponic system, the growth of lettuce was investigated and compared to DFT hydroponic culture. One of the features of dry-fog culture is water conservation. For the systems used in this study, the DFT always needed at least 30 L of liquid fertilizer, while dry-fog culture could fill a chamber with only 1 L of solution because the droplets of dry fog are very fine. In addition, the total amount of liquid fertilizer in a whole dry-fog culture system was less than 10 L. This aspect is an advantage not only in terms of water resource depletion but also because the

Styrofoam chamber culturing unit remains light, making it possible to install the system almost anywhere. Chamber units can also be multistaged easily with low costs, meaning this system is suitable for a plant factory or urban farming. Furthermore, because the efforts to maintain the chamber units and irrigate plants are also greatly reduced, dry-fog aeroponics is expected to be usable not only for commercial crop production but also for welfare and food education.

Despite using less liquid fertilizer in the rhizosphere for the dry-fog culture, there was no difference in leaf growth in the two different hydroponic techniques. It was thought that the increase of growth in the dry-fog culture was due to changes in root morphology and physiology. Root hairs that developed only in the dry-fog culture had a normal increase in the efficiency of nutrient absorption from heterogeneous soil due to a significant increase in the root surface area (Bates and Lynch, 1996; Ma et al., 2001).

Root hairs are not needed in DFT hydroponic technique because there is adequate water and liquid nutrients in the root zone. They are not formed in traditional aeroponics because nutrient solutions are sprayed on roots that are always kept wet. In dry-fog culture, because the rhizosphere is almost an air phase, roots have to catch the very fine

22 droplets of nutrient solutions floating in the air in a chamber. Importantly, as an adaptation mechanism, root hairs develop to catch more droplets of fertilizer. In the present study, the significant increase in root growth was observed only at four weeks after planting. Also the respiration rate of the roots was significantly higher in the dry-fog cultures than in the DFT hydroponic system at four weeks (Fig. 2-4). The respiration rate of roots has a close relationship with water and nutrient absorption in the roots (Hansen,

1980). Indeed, concomitant increases in both root growth and respiration rate in the dry-fog culture were observed, which might result in promotion of water and nutrient absorption and increases of growth and yield. However, no significant increase in leaf growth was observed in dry-fog culture as compared with DFT. Because the growth and respiration activity of roots significantly increased at four weeks and the morphology of new roots developed in the dry-fog culture changed, it may take longer for plant roots to adapt to the dry-fog culture environment as compared with other hydroponic systems.

The ascorbic acid content in the leaves also tended to be slightly higher in the dry-fog culture, but there were no significant differences. Leaf ascorbic acid is a kind of secondary metabolite, and its content in leaves increases under environmental stress conditions (Robinson and Bunce, 2000; Reddy et al., 2004, Koyama et al., 2012).

Because there is nearly no extra water in the rhizosphere in dry-fog culture, roots might suffer from slightly drought conditions. The increase in secondary metabolites caused by environmental stresses generally comes with a decrease in growth and harvest

(Champolivier and Merrien, 1996; Kirda et al., 2004, Koyama et al., 2012). However in the present study, such decreases were not observed. Under drought conditions, the development of root hairs and increase in root surface area are countermeasures to growth depression due to a water shortage. In dry-fog cultures, plants develop root hairs to catch

23 the very fine droplets of nutrient solution, and this development is thought to effect secondary metabolism. Dry-fog culture might be useful to adjust mild drought conditions in rhizosphere by changing the airflow speed or the frequency of atomization, which affect harvest quality (Brix or secondary metabolite content). Furthermore, the atomized dry-fog from the nutrient solution in a chamber fills the rhizosphere space immediately, meaning the rhizosphere environment is changed quickly and drastically. This aspect is expected to lead to new cultivation methods that can increase the content of secondary metabolites by controlling environmental stresses without decreasing growth or harvest.

With regard to the photosynthetic rates of mature leaves grown in the dry-fog culture, significant increases were shown in both φ and Max compared with DFT culture. This indicates that the rhizosphere environment of dry-fog culture may affect the photosynthetic ability of lettuce leaves by improving CO2 assimilation efficiency of mesophyll cells. The content of leaf chlorophyll was significantly increased in the dry-fog culture at four weeks, and this may be considered to be due to improvement in the activity or quantity of photosynthetic light or dark reaction components. The relationship between enhancement of photosynthetic activity and rhizosphere environment in the dry-fog culture remains unclear.

Chapter 3

Growth and physiological characteristics of leaf lettuce grown in an environment

controlled room using a new aeroponic system with dry-fog spray fertigation

25 3-1. Introduction

Specific features of dry-fog aeroponics are the aerobic environment in the rhizosphere and flexible control of the flow rate and particle size of atomizing foggy solution using a specialty nozzle in the root zone in a chamber. In dry-fog aeroponics, plant roots are hanged in the foggy nutrient solution and absorb water and nutrients directly from the fog particles. No part of the roots is soaked in the nutrient solution, and fresh airflow is always supplied to the root surface. Because the flow rate of dry-fog sprayed by a nozzle can be changed easily using a fan set in a chamber, it is possible to control the density and speed of the particles of nutrients and water adhering to the roots.

Root growth in most vegetable crops is expected to increase under the aerobic conditions of the rhizosphere environment because there is no solid phase and less liquid phase compared to the physical composition of soil or any other rooting media (Hillel and

David, 1988; Cherif et al., 1997). It has been predicted that the completely aerobic rhizosphere environment would improve the absorption of water and nutrients and the growth of leaves (Changoo and Takakura, 1994; Yoshida et al., 1997), but studies to date have not been sufficient to determine the optimal rhizosphere environment for dry-fog aeroponics.

The growth promotion and increased viability of roots in an aerobic rhizosphere filled with foggy nutrient solution and an increase in the water absorption efficiency by continuous flow of dry-fog on the surface of roots are expected in dry-fog aeroponics.

There has been no report indicating a relationship between the flow rate and particle size of foggy nutrient solution sprayed in the rhizosphere and plant growth with aeroponics, therefore, the author cultivated lettuce plants with the dry-fog aeroponic technique and

26 investigated the effect of the flow rate and particle size by changing them with a controlled fan and nozzles set in the rhizosphere on the growth characteristics and physiological activities of plants.

27 3-2. Materials and methods

Dry-fog aeroponic system

Two dry-fog aeroponics systems, as it has been described in chapter 2, consisting of a rhizosphere chamber made of polystyrene (1000 × 660 × 300 mm) and a dry-fog atomizing nozzle (developed and patented by Ikeuchi Co., Ltd., Osaka, Japan) were settled in a temperature-controlled room. Each chamber had 22 holes (20 mm across) for setting plants on the top, and only the roots remained in the chamber. An axial flow fan was equipped in one chamber, and the dry-fog flow rate was adjusted to 1.0 m s-1 (FL).

The other chamber was not equipped with a fan, and the flow of the dry-fog did not occur in the chamber except for the spraying from the nozzle (NF). The flow rate in the chamber was measured at a depth position of 70 mm from the planting hole using an omnidirectional anemometer (testo445; TESTO, Lenzkirch, Germany) (Fig. 3-1). The nozzle continuously atomized fine foggy nutrients of a commercial liquid fertilizer (OAT

Agrio Co., Ltd., Tokyo, Japan, EC 1.2 mS cm-1, pH 6.0, N: 130 ppm, P: 60 ppm, K: 200 ppm, Ca: 115 ppm, Mg: 30 ppm, Fe: 1.4 ppm, Mn: 0.8 ppm, Zn: 0.05 ppm, Cu: 0.02 ppm,

B: 0.75 ppm, Mo: 0.02 ppm) that always filled the chamber. As a control experiment, a deep-flow technique (DFT) hydroponic chamber (480 × 380 × 114 mm) was filled with

30 L of the same liquid fertilizer. To confirm the amount of dry-fog particles flowing through the rhizosphere, a slide glass that was painted with thin silicon oil (1000 cs) was exposed perpendicularly upstream of the dry-fog for one second. Then, the number and size of particles that were trapped in the silicon oil layer on a slide glass were observed and recorded with a microscope (Fig. 3-1, Photos).

28 Plants and culture conditions

Commercial leaf lettuce seeds (Lactuca sativa L. cv. ‘Greenspan’, Kaneko Seeds

Co. Ltd., Tokyo, Japan) were sown on sponge blocks (20 × 20 × 30 mm) and were supplied with enough pure water. The air temperature and relative humidity in the cultivation room were maintained at 25˚C and 60%, respectively. The photosynthetic photon flux density (PPFD) was 120 µmol m-2 s-1 supplied by red (660±10 nm, 96 µmol m-2 s-1) and blue (455±10 nm, 24 µmol m-2 s-1) light-emitting diodes (LEDs) lamps (Legu

LED, HRD Co., Ltd. Tootori, Japan) with a 16-h day length. After germination, half-strength liquid fertilizer (EC 0.6 mS cm-1, pH 6.0) was supplied by bottom irrigation for two weeks, and 20 seedlings that grew uniformly were transplanted in each hydroponics system and were grown for two weeks.

Measurements of growth and photosynthesis

Every week after transplanting, six plants were sampled from each hydroponic system, and the fresh weight, the number of leaves and the fresh weight of roots were recorded. On the 2nd week after transplantation, a leaf disk (10 cm2) was sampled from each plant and dried in an oven at 80°C for two days and its dry weight was measured to estimate the specific leaf area. Transpiration and the CO2 assimilation rates of a fully expanded mature single attached leaf were measured under controlled environmental conditions using an LI-6400 portable photosynthesis system (Li-Cor, Lincoln, NE, USA) on the 2nd week after transplanting. The measurement conditions were as follows: leaf temperature, 25°C; PPFD supplied by a red-blue LED light source (10% blue based on

-2 -1 PPFD), 150 µmol m s ; leaf vapor pressure deficit, 1.0 kPa; and ambient CO2 concentration, 500 µmol mol-1.

Measurements of root activity

On the 2nd week after transplanting, intact root samples were taken from six growing plants that developed new roots under each hydroponic condition, and the rates of root respiration were measured polarographically at 25˚C using a Clark-type gas-phase oxygen electrode (CB1D; Hansatech, Norfolk, UK) with incoming humidified ambient air

(21% O2) under dark conditions. After measuring the respiration rate, the root was blotted by pressing slightly for 10 seconds between two sheets of filter paper (No. 1,

ADVANTEC, Tokyo, Japan), then fresh weight was measured and the dehydrogenase activity of the roots was tested according to the triphenyltetrazolium chloride (TTC) method. Root samples (1.0-1.5 g FW) were cut into small pieces of 10 mm and placed in a tube containing 5 ml of equally mixed solution of 0.1% TTC and 0.1 M phosphate buffer (pH 7.0) for a reduction reaction for 2 h at 37°C. Then, 2 ml of 1 M H2SO4 was added to the tube to stop the reaction. The root was blotted with paper towel and homogenized in ethyl acetate. The extract was transferred to a tube and was quantified to

6 ml by adding ethyl acetate. The absorbance of the extract at 485 nm was recorded using a spectrophotometer, and the concentration of triphenylformazan (TPF) as a reaction product was calculated by comparing to a standard curve with diluting TPF after complete reduction of 0.1% TTC (Wako Pure Chemical Industries, Ltd., Osaka, Japan) with Na2S2O4.

Measurement of leaf constituents

The contents of nitrate nitrogen, calcium ions, phosphate ions and potassium ions in the leaves were measured every week, and the total soluble protein (TSP) and chlorophyll

30 were measured on the 2nd week after transplantation. The leaves (3 g FW) were homogenized in deionized water. The homogenate solution was passed through a filter paper (No. 1, ADVANTEC, Tokyo, Japan) and was measured using RQflex (Merck

Millipore, Darmstadt, Germany). Because RQflex has previously used for measuring the ion contents (Kintzios et al., 2004) and it was reported that the measurements were closely correlated with HPLC analysis (Ito et al., 2013). In present study, accurate quantitative correction was not carried out because of comparative experiments. Other leaves (2 g FW), which were sampled for measuring the concentration of TSP, were homogenized in 50 mM Tris-HCl buffer (pH 8.0) containing 10 mM MgCl2, 0.2 mM ethylenediamine tetra-acetic acid and 5 mM dithiothreitol on ice, and 1 ml of the extract was transferred to a tube. Then, 1 ml of 20% (w/v) trichloroacetic acid was added to the tube and left for more than 15 min. The supernatant was removed, and the pellet was resolved in 1 ml of 1 M NaOH for 24 h. The TSP concentration of the samples was measured by the Coomassie Brilliant Blue protein assay (Nacalai tesque, Kyoto, Japan) and calculated by comparing to a standard curve with diluted BSA (Nacalai tesque, Kyoto,

Japan). The chlorophyll content of the leaves (1 g FW) was analyzed after extraction by grinding the leaves in pre-cooled 80% (v/v) acetone and quantified spectrophotometrically according to the method of Arnon (1949).

Data analysis

All of data were subjected to a one-way analysis of variance (ANOVA) and the mean differences were compared using the Tukey’s HSD test when the F-test indicated a significant difference at P≤0.01. The hydroponic culture experiment was repeated twice under the same conditions; each data point was the mean of 12 replicates, and a

31 comparison with P≤0.05 was considered significantly different.

32 3-3. Results and discussion

Flux density of dry-fog particles and lettuce growth

The number of dry-fog particles trapped in the silicon oil layer on a slide glass was apparently superior in FL than in NF (Fig. 3-1 Photos), meaning that the rhizosphere with fast flow rate was filled with much of foggy water and nutrient solution compared to that with low flow rate. In FL, approximately three times the nutrient droplets were collected on the silicon oil layer compared to that in NF (Table 3-1). There are direct observation showing a relationship between the flow rate of dry-fog spray and the amount of dry-fog particles in the root zone. The average diameter of the droplets was less than 10 µm in both FL and NF and showed no significant difference. There was no difference other than in the flow rate for the supply of nutrient solution. Because a nozzle continuously sprayed dry-fog and saturated relative humidity was maintained in a chamber filled with dry-fog nutrients, more foggy nutrient droplets can contact the root surface under active flow conditions than under non-flow conditions. So, it was thought that the roots in FL could absorb water and nutrients easier than in NF when the roots had same volume and same surface area.

A ) A 1.0 1.1 1.1 1.0 1.0 1.3 1.0 0.8 1.0 0.8 1.1 A

0.7 0.8 0.8 1.0 0.8 1.0 1.0 1.2 1.5 1.5 1.0 ( m s-1, flow rate ) B B ) B 0.1 0.2 0.1 0.1 0.1 0.1 0.1 0.2 0.1 0.2 0.1

0.1 0.1 0.2 0.2 0.1 0.2 0.2 0.1 0.1 0.1 0.1 ( m s-1, flow rate ) C )

Air flow slide glass Axial fan Fog nozzle

Fig. 3-1. Flow rate (m s-1) in the rhizosphere at each planting hole (filled circles in A, FL and in B, NF) and cutaway drawing of the dry-fog aeroponic chamber (C). Photographs show the droplets of dry-fog trapped in the thin silicon oil layer on a slide glass exposed perpendicularly upstream of the dry-fog in the rhizosphere (dotted line position) in FL

(A) and NF (B). Dotted line shows the position of slide glass in the chamber (A, B: horizontal; C: vertical).

Table 3-1. Average diameter and density of droplets trapped in the silicon oil layer.

Average number Distribution of the droplet diameter (%) Average diameter of droplets of droplets -2 ( mm ) 10 µm 1020 µm 2030 µm 30 µm ( µm ) FL 1905 ± 179 15.1 8.3 1.4 0.1 9.5 ± 0.25

NF 867 ± 143 15.1 9.0 0.5 0.4 9.2 ± 0.43 Means ± standard deviations.

The growth of leaves at each week after transplantation was significantly higher in

FL than in NF but showed no difference between FL and DFT (Fig. 3-2). The growth of roots on the 1st week in both dry-fog cultures was significantly higher than that in DFT but showed no difference between the two dry-fog cultures. The root fresh weight in NF on the 2nd week was significantly higher than those in other two by more than double of

DFT. As a result, the top/root fresh weight ratios (T/R) in dry-fog aeroponics were significantly lower at each week compared to that of DFT. The T/R of tobacco plants was affected by various environmental factors in especially rhizosphere (Andrews et al., 2006).

These findings suggested that lettuce plant growth, especially the root growth respond sensitively to rhizosphere in aeroponics, where environment conditions might be less stable compared to those in DFT and soil. There was no change in the T/R between the

1st and 2nd week after transplantation in FL, but it decreased significantly in the 2nd week compared to that in the 1st week in NF. In addition, well-developed lateral roots and root hairs on its surface were observed in dry-fog aeroponics and these root traits were especially pronounced in NF (Fig. 3-3).

The author considered that, in the dry-fog culture, lettuce roots in the aerobic

35 rhizosphere absorbed enough water and nutrients from the foggy particles flowing in the root zone by altering root morphogenesis resulted from developing branched roots with root hairs and by enlarging root biomass resulted from distributing new assimilates preferentially into the growth of roots rather than leaves, and these growth and developmental changes are adaptations to the aerobic rhizosphere environment by increasing root surface area to improve the efficiency of catching dry-fog particles and absorbing water and nutrients. The author considered that the roots that grew in FL quickly adapted to aerobic rhizosphere conditions within one week after transplantation and that shoot growth increased in the 2nd week, but the roots that grew in NF could not completely adapt within one week and maintained assimilation partitioning to increase root growth in the 2nd week. Although root growth had priority over shoot growth for catching dry-fog nutrients efficiently, the shoot fresh weight showed no significant difference in either week after transplantation between FL and DFT, whose roots grew without any morphological changes. It is expected that well-developed roots in dry-fog aeroponics promote the absorption of water and nutrients and subsequent shoot growth.

Therefore, the author concluded that dry-fog aeroponics increases shoot growth after the adaptation of roots to the aerobic conditions in the rhizosphere, and further investigations are needed for the growth characteristics after three weeks.

36 35 a 30 FL a 25 NF b 20 DFT

FW of leaves ( g ) of leaves FW 10 a a

5 b 0

4 a

3 b 2 c of roots ( g ) FW 1 a a b 0 0 1 2 Weeks after transplantinga 20

15 a

T/R ratio T/R b b

5 b c

0 0 1 2 Weeks after transplanting

Fig. 3-2. Changes in the fresh weight of lettuce leaves and roots and the T/R ratio when grown using dry-fog aeroponics with different flow speeds (FL: 1.0 m s-1, NF: 0.1 m s-1) or DFT hydroponics in the two weeks after transplanting. Different lowercase letters show a significant difference (P≤0.05); bars represent ± SE (n=12).

FL NF DFT

50 mm

FL NF DFT

5 mm

Fig. 3-3. Leaves and roots of the lettuce grown using dry-fog aeroponics with different flow speeds (FL: 1.0 m s-1, NF: 0.1 m s-1) or DFT hydroponics in the two weeks after transplanting. The enlargement photos show the typical root hairs on lateral roots of each root system.

38 Physiological activity of the leaves and roots

The photosynthetic rate of the attached mature leaves in the 2nd week after transplanting increased significantly by dry-fog aeroponics and was significantly higher in NF than FL (Table 3-2). The stomatal conductance was also higher in dry-fog aeroponics, especially in NF, whose leaves absorbed more CO2 into the intercellular space than did those that were grown in DFT under the same atmospheric CO2 concentration. There were no differences in the contents of chlorophyll and TSP and SLA, indicating the thickness of a leaf, among all of the hydroponic systems, so it was thought that the photosynthetic rate of leaves grown under dry-fog aeroponics increased due to the elevated CO2 concentration in leaves with opening stomata and not due to an increased photosynthetic ability.

In terms of the root morphogenesis in sprayponics, it is particularly worth nothing that root hairs formed on the surface of roots under the dry-fog aerobic conditions because of no related reports found so far. Bibikova and Gilroy (2003) reported that the development of the root hair was influenced by abscisic acid (ABA) production under water stress. Furthermore, the availability of phosphorus or nitrate in the rhizosphere affects the length, number and density of root hair (Bates and Lynchand, 1996; Ma et al.,

2001; Hammac et al., 2011). Thus, the development of root hair in some kinds of plant species may be related to water stress or nutrient stress as a survival adaptation under the unhealthy rhizosphere environment. However, in this study, lettuce plants had never experienced both water and nutrient shortages because of the continuous spraying with nutrient solution at proper concentration all the day. So the development of root hair in dry-fog aeroponics was not affected by the water stress related ABA biosynthesis, because the stomatal conductance of dry-fog aeroponics, especially in NF with

39 significantly developed root hair, was higher than DFT where plants were never exposed to water stress. This increased stomatal conductance at the 2nd week after transplanting to dry-fog aeroponics showed that the evapotranspiration demand was filled much more in dry-fog aeroponics than in DFT by the enough absorption of water resulted from increased surface area of roots and decreased T/R.

The respiration rate of roots is an important physiological parameter for evaluating root activity for absorbing water and nutrients. There was no significant difference in the root respiration rate within dry-fog aeroponics (FL vs. NF) and that of the DFT was significantly lower compared to those of dry-fog aeroponics in the 2nd week after transpiration (Table 3-2). These results indicate that the roots increased not only the surface area of developing root hairs to improve the dry-fog trapping efficiency but also the root absorption activity of water and nutrients adhering to the root surface.

On the other hand, the TTC reducing activity was significantly higher in DFT, but there was no difference between FL and NF (Table 3-2). TTC is absorbed into tissue cells and reduced to red-colored TPF by H+ generated by succinate dehydrogenase in the

TCA-cycle within the mitochondria. So the quantity of TPF has been used as an indicator of succinate dehydrogenase activity in many studies of root viability. At the same time,

TTC reduction activity is also used as a parameter to evaluate uptake of water and nutrients (Wang et al., 2006). It has been considered that good growth of shoots needs both a high TTC reduction activity and a large root surface area (Wen-Zeng et al., 2011).

The TTC reduction activities of the roots in FL and NF were approximately 60% and

70% of those in DFT (Table 3-2), but the root fresh weights were 144% and 233% of those in DFT in the 2nd week after transplantation (Fig. 3-2). Considering total root activity as a product of TTC reduction activities per FW and total FW of whole roots per

40 plant, NF was expected to have the highest absorbing activity in the total absorption activity per plant, and there was no marked difference between DFT and FL. Because the

TTC absorbed by roots is reduced mainly by dehydrogenase in mitochondria, the relationship between the TTC reduction activity and respiration rate of roots has been investigated (Louise et al., 2000). Furthermore, considering that the root morphology changed drastically in dry-fog aeroponics, the physiological activities of roots are expected to adapt to the dry-fog rhizosphere environment. Increases in both stomatal conductance and photosynthetic rate of the plants grown in dry-fog rhizosphere might be caused by improving root morphology and physiological activity to enhance absorption of water and nutrient solution. These were results of root growth adaptation to the dry-fog environment at two weeks after transplanting, however, their influences to the leaf growth could be reflected in a short while later. Additional researches are necessary to explain the changes in root growth adaptation and growth of plants cultivated in dry-fog aeroponics for a longer period.

41 )

-1 min

-1 g 2 Root

0.04 a 0.35 ± 0.04 a 0.42 ± 0.19 ± 0.02 b 0.19 ± molO respiration rate respiration µ (

) -1 fog or DFT - FW h FW -1 TPF production

0.03 b 0.31 ± 0.02 b 0.32 ± 0.04 a 0.46 ± (mg g (mg

DW) -1 g 2 Specific area leaf (cm 0.125 ± 0.009 a 0.125 ± 0.002 b 0.107 ± 0.012 a 0.132 ±

FW) -1

protein (mg g (mg 5.58 ± 0.48 a 5.58 ± 0.35 a 5.89 ± 0.33 a 5.47 ± Total soluble Total

)

-2 a+b (g m Chlorophyll 1.52 ± 0.09 a 1.52 ± 0.04 a 1.44 ± 0.02 a 1.51 ±

) -1 s -2

O m 2 Stomatal

conductance 0.10 ± 0.002 b 0.10 ± 0.002 a ± 0.11 0.003 c 0.09 ± (molH )

-1 s -2 m

2 rate

7.11 ± 0.06 b ± 7.11 0.05 a 7.93 ± 0.07 c 6.95 ± molCO Photosynthetic µ (

2. Leaf physiological and morphological traits and root physiological activities when grown using dry - 0.05, n=12). ≤

FL NF DFT P Means ± standard deviations. Different lowercase letters in each column denote significant differences among three treatments according to a Tukey's HSD Tukey's a to according treatments three among differences significant denote column each in letters lowercase Different deviations. standard Means ± test ( test

Table 3 transplanting. two weeks after the in hydroponics

42 Leaf constituents

The contents of nitrate nitrogen and phosphate ions of mature leaves were the highest in

DFT in both weeks (Fig. 3-4). FL had significantly higher values than those of NF in the

1st week, but there was no significant difference between these values in the 2nd week due to the increased surface area of the roots in NF. Because the roots in NF had almost the same absorption activity of nutrients to FL, increases in both the biomass and the absorbing activity of the roots could promote the shoot growth after the 3rd week after transplanting. Although the roots in dry-fog aeroponics were less exposed to nutrient solution in the rhizosphere than in DFT and the leaf contents of nitrate nitrogen and phosphate were lower in both FL and NF than those in DFT with unchanged chlorophyll content, no nutrient deficiencies were observed in the leaves in dry-fog aeroponics. The content of potassium ions in mature leaves was also significantly higher in DFT in both weeks compared to that in dry-fog aeroponics. NF had a significantly higher value than

FL in the 2nd week. The content showed no significant changes between the 1st and 2nd weeks in DFT and FL, but NF increased this content by 30%. The content of calcium ions in mature leaves significantly increased in the FL than DFT in both weeks, and the FL was higher than the NF in the 2nd week. Because calcium and potassium have a competitive relationship, enhancing potassium absorption in NF in the 2nd week inhibited an increase in calcium. There was no significant difference in the calcium content between NF and DFT in the 2nd week. The insufficient absorption and transportation of calcium causes the physiological disorder “tip-burn” in the shoots of lettuce (Bangerth,

1979; Barta and Tibbitts, 2000) and decreases the production of lettuce. Many studies have investigated the improvement of calcium absorption during the cultivation of leafy vegetables (Shibata et al., 1995; Goto and Takakura, 2003; Takahashi et al., 2012).

43 Although tip-burn did not occur in lettuce cultivation with dry-fog aeroponics, lettuce plants can grow well with healthy consumption values. The author suggest that dry-fog ae1roponics inhibits the occurrence of tip-burn in lettuce production with minimum amounts of nutrient solution and water.

44 2.5

2 a

FW)

-1 1.5 a b b

( mg g ( mg b - 1 3

NO c 0.5

0 1 2 1.5 Weeks after transplanting a 1.2 b FW) a

-1 b 0.9 b ( mg g ( mg

2- 0.6

4 c

PO 0.3

0 1 2 4 Weeks after transplanting a a b 3 b FW) -1 b c 2

g ( mg + K 1

0 1 2 0.9 Weeks after transplanting a 0.9 ab b b FL b FW) FW) bc -1 -1 0.60.6 NF DFT ( mg g ( mg ( mg g ( mg + 2+ FL Ca Ca 0.30.3 NF DFT 0 0 1 2 1 2 WeeksWeeks after after transplanting transplanting Fig. 3-4. Changes in the ion content of lettuce leaves when grown using dry-fog aeroponics with different flow speeds (FL: 1.0 m s-1, NF: 0.1 m s-1) or DFT hydroponics in the two weeks after transplanting. Different lowercase letters show a significant difference (P≤0.05); bars represent ± SE (n=12).

Chapter 4

Growth and physiological characteristics of leaf lettuce grown in a greenhouse

using a new aeroponic system with dry-fog spray fertigation

46 4-1. Introduction

In chapter 3, the growth characteristics and physiological activities of leaf lettuce grown with dry-fog aeroponics under the controlled environment were investigated. The plant cultivation under controlled environment has received attention as ‘plant factory’. In plant factories the plants like leaf lettuce can be cultivated all around year regardless of climates, but there are many problems such as initial and running costs to popularization.

So it is need to assume that the dry-fog aeroponics systems are used in both plant factories and traditional greenhouse. In a greenhouse, the plants can receive much stronger lights from the sun than that from LEDs, so the growth is expected to increase more than that under the LEDs. But, because the increase of the evaporation demands of leaves is also expected, it is thought that the different rhizosphere environment to archive the improvement of the growth. In this chapter, the author investigated and evaluated the growth and physiological characteristics of leaf lettuce cultivated in a greenhouse using a dry-fog aeroponics system with dry-fog (less than 10 µm in diameter on average per droplet) and semi dry-fog (20-30 µm in diameter on average per droplet) and two different flow rates.

47 4-2. Materials and methods

Leaf lettuce (Lactuca sativa L. ‘Greenspan’, Kaneko Seeds Co. Ltd., Tokyo, Japan) was grown in a greenhouse with a dry-fog (DF) or semi dry-fog (SDF) aeroponics system consisting of a rhizosphere chamber made of polystyrene (6000 × 700 × 300 mm) and a dry-fog or semi dry-fog atomizing nozzle (developed and patented by Ikeuchi Co., Ltd.,

Osaka, Japan). The chambers were equipped with an axial flow fan, and the flow rate of each fog was adjusted to 1.0 m s-1 (1 m) or 0.1 m s-1 (0 m). The nozzle continuously atomized fine particles of nutrients from a commercial liquid fertilizer that always filled the chamber. As a control experiment, deep-flow technique (DFT) hydroponic chambers

(480 × 380 × 114 mm) were filled with 30 L of the same liquid fertilizer.

Plants were transplanted in each hydroponic system at two weeks post-germination and were grown for three weeks. Each week, the growth of the leaves and roots and the dehydrogenase activity of the roots were measured. On the third week, the CO2 assimilation rates of fully expanded, mature leaves were measured using an LI-6400 portable photosynthesis system (Li-Cor, Lincoln, NE, USA); the rates of respiration of the roots were measured polarographically at 25˚C using a Clark-type gas-phase oxygen electrode (CB1D; Hansatech, Norfolk, UK) with incoming humidified ambient air (21%

O2) under dark conditions; and the dehydrogenase activity of the roots were measured according to the triphenyltetrazolium chloride (TTC) method. In addition, the nitrate nitrogen, calcium ion and total soluble protein (TSP) contents were extracted from mature leaves and measured on the third week. All of data were subjected to a one-way analysis of variance (ANOVA), and the mean differences were compared using Turkey’s HSD test when the F-test indicated a significant difference of P≤0.01. Each data point was the

48 mean of 10 replicates, and a comparison with a P value ≤ 0.05 was considered to be significantly different.

49 4-3. Results and discussion

Growth of Leaves and Roots

The growth of leaves and roots at three weeks post-transplantation was significantly greater under DF and SDF conditions compared to the DFT condition (Fig.

4-1). There were no differences in the growth of the leaves or roots in either the DF or

SDF condition with a faster flow rate, but the leaves and roots in the DF condition showed significantly more growth with the slower flow rate compared to the SDF condition. In addition, there were no significant changes in the T/R from the first to third week post-transplantation when grown in the faster flow rate, but the T/R decreased significantly and well-developed lateral roots were observed under the slower flow rate condition (Fig. 4-2). The decrease in T/R ratio shows an increased growth of early stage roots, which is thought to be due to an adaptation to the aerobic rhizosphere environment by increasing the root surface area to improve the efficiency of catching fog particles and absorbing water and nutrients (Terence and Jonathan, 2000). Because the amount of fog particles that can contact the root surface is affected by the flow rate, it was considered that the growth of roots increased under slower flow rates to improve the contact efficiency with the fog. Furthermore, because the DF particle size was smaller than the

SDF particle size and the total amount of water and nutrients on the root surface was thus decreased in DF vs. SDF conditions, it was considered that the growth of roots would be increased in DF conditions compared to SDF conditions. This increase in early stage root growth could serve as an adaptation to the rhizosphere environment and result in a greater efficiency in water and nutrient absorption compared to plants grown under DFT conditions, thus significantly increasing the growth of leaves.

70 DFT 60 A SDF 0m A 50 SDF 1m B 40 DF 0m B C 30 DF 1m

( g ) of leaves FW 20 10

0 1 2 3 DFT 20 Weeks after transplanting A SDF 0m SDF 1m B 15 B DF 0m B DF 1m 10

of roots ( g ) FW 5 C

0 20 1 2 3 DFT DF 0m 18 Weeks after transplanting SDF 0m DF 1m 16 SDF 1m 14

12 10 A

T/R ratio T/R 8 6 B 4 B B 2 B 0 1 2 3 Weeks after transplanting

Fig. 4-1. Changes in the fresh weight of lettuce leaves and roots and the T/R ratio when grown using dry-fog aeroponics with different flow rates or DFT hydroponics in the three weeks after transplanting. Different lowercase letters show a significant difference

(P≤0.05); bars represent ± SE (n=10).

51 70 mm

DFT SDF 0 m DF 0 m SDF 1 m DF 1 m

10 mm

Fig. 4-2. Leaves and roots of the lettuce grown using dry-fog aeroponics with different flow speeds or DFT hydroponics in the three weeks after transplanting. The enlargement photos show the typical root hairs on lateral roots of each root system.

Physiological Activity of the Leaves and Roots

The photosynthesis rate of mature leaves grown in DFT and SDF conditions with a slower flow rate was significantly increased, as shown by both saturated intercellular CO2 concentrations (Ci) and the apparent CO2 fixation efficiency (Ф) (Fig. 4-3). The photosynthetic rate measured in ambient air CO2 concentration (400 ppm) was significantly increased under SDF conditions with a slower flow rate and DF conditions with faster flow rate. This is likely because their stomatal conductance was high enough

52 that their Ci was also increased compared to the other hydroponic conditions (Table 4-1).

The specific leaf area, which is an indicator of the thickness of a leaf, was significantly smaller under DFT and SDF conditions with a slower flow rate, which could be the cause of the increase in photosynthetic rate under saturated Ci and Ф conditions. There were no differences in the total soluble protein content in plants grown under DF or SDF conditions, while it was decreased in plants grown under DFT conditions. Furthermore, there were no differences in protein content between plants grown under DFT or DF conditions with a faster flow rate, which suggests that the increase in photosynthetic rate was not due to an increased photosynthetic ability.

The respiration rate of roots, which indicates their ability to absorb water and nutrients, was significantly higher in plants grown under SDF conditions with a slower flow rate compared to those grown in DFT and DF with a slower flow rate (Fig. 4-4). The dehydrogenase activity of roots grown in aeroponic conditions was significantly decreased compared to DFT in the first and second weeks, but there were no differences in the third week expected in plants grown in DF with a slower flow rate (Fig. 4-5). These results suggest that the root activity was decreased by the drastic change in rhizosphere environment but that the roots adapted to the environment by altering their physiology in addition to increasing their surface area such that sufficient water and nutrients could be absorbed (Wen-Zeng et al., 2011).

40 ) -1

s -2 30 mol m mol µ ø Max 0.087b 32.4b 20 SDF 1m a a SDF 0m 0.094 35.4 DF 1m 0.086b 33.0b 10 DF 0m 0.086b 32.5b

Photosynthetic rate ( rate Photosynthetic DFT 0.094a 36.1a

0 0 200 400 600 800 1000 1200 Ci ( µmol mol-1)

Fig. 4-3. Relationships between CO2 assimilation rates and intercellular CO2 concentration (Ci) measured for the attached mature leaves of lettuce grown using dry-fog aeroponics with different flow speeds or DFT hydroponics in the three weeks after transplanting. The saturated rate (Max) and the apparent CO2 fixation efficiency (ø) were estimated as parameters of the best-fitted non-rectangular hyperbola response curve to Ci.

Different letter shows significant difference (P≤0.05).

Table 4-1. Leaf physiological and morphological traits when grown using dry-fog with different Table 1. Leaf physiological and morphological traits when grown using dry-fog with flow different rates or flow DFT rates hydroponics or DFT inhydroponics the three weeks in the after three transplanting. weeks after transplanting.

Photosynthetic Stomatal Specific Total soluble rate conductance leaf area protain -2 -1 -2 -1 2 -1 -1 (µmolCO2 m s ) (molH2O m s ) (cm mg DW) (mg g FW) DFT 15.75 ± 0.25 c 0.21 ± 0.005 b 0.65 ± 0.05 d 6.16 ± 0.31

SDF 0 m 18.06 ± 0.24 a 0.24 ± 0.004 a 0.82 ± 0.04 c 7.24 ± 0.32 1 m 16.19 ± 0.18 b 0.18 ± 0.001 d 0.98 ± 0.03 a 7.14 ± 0.32 DF 0 m 15.93 ± 0.27 c 0.19 ± 0.006 c 0.91 ± 0.03 b 7.04 ± 0.42 1 m 17.84 ± 0.24 a 0.23 ± 0.003 a 1.01 ± 0.06 a 6.60 ± 0.43 Means ± standard deviations. Different lowercase letters in each column denote significant differences among three treatments according to a Tukey's HSD test (P≤0.05, n=10).

0.7 A

) 0.6 B -1 B 0.5 C C

FW min FW 0.4 -1 g 2 0.3

molO 0.2 µ Roots respiration rate ( 0.1

0 DFT SDF 0m SDF 1m DF 0m DF 1m

Fig. 4-4. Respiration rate of lettuce roots grown using dry-fog aeroponics with different flow speeds or DFT hydroponics in the three weeks after transplanting. Different lowercase letters show a significant difference (P≤0.05); bars represent ± SE (n=10).

2.5 )

-1 A A A 2 A FW h FW -1

1.5 B

DFT DF 0m 0.5 SDF 0m DF 1m TPF production ( mg g ( mg TPF production

SDF 1m 0 1 2 3 Weeks after transplanting

Fig. 4-5. Changes in the dehydrogenase activity of lettuce roots grown using dry-fog aeroponics with different flow speeds or DFT hydroponics in the three weeks after transplanting. Different lowercase letters show a significant difference (P≤0.05); bars represent ± SE (n=10).

Leaf Constituents

The nitrate nitrogen content of mature leaves was significantly increased by a slower flow rate, especially in SDF conditions, and was caused by the increase in both root surface area and activity; those plants grown in DFT or DF with faster flow rate showed no statistically significant differences (Table 4-2). The calcium ion content was also significantly increased in aeroponic-grown plants, especially those grown in SDF

56 conditions with a slower flow rate. Although tip-burn, which is caused by the insufficient absorption and transport of calcium ions (Bangerth, 1979; Barta and Tibbitts, 2000), did not occur in this study, dry-fog or semi-dry-fog aeroponics are expected to inhibit the occurrence of tip-burn during lettuce production with minimum amounts of nutrient solution and water, thereby increasing yield.

Table 4-2. The ion contents of lettuce leaves when grown using dry-fog with different flow rates or DFT hydroponics in the three weeks after transplanting.

Chapter 5

Ever-bearing strawberry culture using a new aeroponic system with dry-fog spray

fertigation during the summer

58 5-1. Introduction

In Japan, strawberry is one of most popular fresh fruits supplied by year-round production in a greenhouse except for mid-summer as an off-crop season because of its flowering habit, in spite of much demand for fresh fruits by most of cake shop. A large amount of supply during summer was imported from oversea with very high cost. Most varieties are June bearers defined as facultative short-day plants, however, ever-bearing varieties, which are classified as long-day plants (Durner et al., 1984) or as day-neutral plants (Bringhurst et al., 1988), have been breeding for suitable for summer production.

Even in ever-bearing varieties, their flower bud initiation is restricted and unstable at high temperature over 25 ºC (Nishiyama and Kanahama, 2002; Sonsteby and Heide, 2007), so they are cultivated within a confined cool region. In late years, there were several researches to attempt to cool runner seedlings (Kumakura and Shishido, 1995), the rooting medium (Ikeda et al., 2006, 2007) and the crowns (Dan et al., 2005) of strawberry grown for high-bench culture in summer culture.

The objectives of this study were to determine the effects of cooling root zone and crowns of ever-bearing strawberry plants cultured using a dry-fog sprayponic system in summer and to confirm the effects of temperatures of root zone and crowns on flower bud initiation and fruit setting. The results will encourage strawberry grower to attempt very early forcing culture even in lowland and warm areas where summer temperature is too high to initiate flowering.

59 5-2. Materials and methods

Commercial ever-bearing strawberry seedlings (Fragaria x ananassa Duch. var.

Summer ruby, Mikamo-Fletec Co., Ltd, Tokushima, Japan) were transplanted in late June

2012. The air temperature and solar radiation in a greenhouse were recorded through the experiment.

A dry-fog aeroponics system consisting of a rhizosphere chamber made of polystyrene with 6.0 m in length, 0.3 m in width and 0.7 m in height was settled in a greenhouse (Fig. 5-1). Each chamber had 16 holes (0.2 m across) for setting plants on the top, and only the roots remained in the chamber. A dry-fog atomizing nozzle (developed and patented by Ikeuchi Co., Ltd, Osaka, Japan) was equipped at one end and an axial flow fan with the flow rate of 1 m s-1 at the other end in a chamber. The nozzle continuously atomized fine foggy nutrients of a commercial liquid fertilizer (Tank Mix A

& F, OAT Agrio Co., Ltd, Tokyo, Japan, EC 1.0 mS cm-1, pH 6.0, N:54 ppm, P:37 ppm,

K:78 ppm, Ca:44 ppm, Mg:14 ppm, Fe:1.0 ppm, Mn:0.7 ppm, Zn:0.03 ppm, B:0.34 ppm,

Mo:0.02 ppm) that always filled the chamber. Dry-fog aeroponics was divided into two culture treatments with (DF,Crown cooling) or without (DF) mist cooling crowns of the rosette shoot base by withdrawing fog from the chamber (Fig. 5-2). As a control experiment, plants were cultured in a container (0.4×0.7×0.3 m in height, planting space of 0.2 m) filled with palm shell fiber substrate (Cocoblock, Kaneko Seeds Co., Ltd,

Tokyo, Japan) and were irrigated sufficiently with the same liquid fertilizer.

Fig. 5-1. Strawberries cultivated with dry-fog aeroponic systems in a greenhouse.

Mist cooling device

Nozzle

Axial fan for circulating fog

Fig. 5-2. Cutaway drawing of a dry-fog hydroponic chamber. Crowns were continuously misted by dry-fog stream withdrawing from the root zone in a chamber

61 Measurements of root zone and crown temperatures

Temperatures of the root zone and the crown surface of rosette shoot were recorded continuously throughout the experiments using fine thermocouples (ø 0.5 mm, copper-constantan) located in rhizosphere and on the crown. The numbers of the flower truss and total flowering were recorded every week after plant establishment in each culture conditions from the early summer to fall. The CO2 assimilation rates of a fully expanded mature single attached leaf in the mid-summer was measured in the end of

August under varied ambient CO2 concentration and PPFD at constant leaf temperature

(25°C) and leaf vapor pressure deficit (1.0 kPa) using an LI-6400 portable photosynthesis system (Li-Cor, Lincoln, NE, USA). Marketable valued fruits were harvested, weighed and the total soluble solids (TSS) of the fruit were measured using hand refractometer

(PAL-J, ATAGO Co., Ltd, Tokyo, Japan). All plants were harvested in the mid-December and the contents of chlorophyll and total soluble protein (TSP) in mature leaves were extracted with 80% acetone and 50mM Tris-HCl buffer (pH 7.9, 10 mM

MgCl2, 0.2 mM EDTA, 5 mM DTT) then measured spectrophotometrically after the

Coomassie brilliant blue dye-binding assay.

Statistical analysis

All of the data (n=12) were subjected to a one-way analysis of variance (ANOVA), and the mean differences were compared using the Tukey HSD test and t-test indicated a significant difference at P ≤0.05.

62 5-3. Results and discussion

Plant temperature under the mid-summer climate conditions

The weather parameters were recorded during the growth period of strawberry

(Fig.5-3). Daily mean air temperature varied over 30ºC between late July and mid-

September with the maximum temperature of 35ºC, which was frequently happen during the seating summer with strong solar radiation. After early October, it usually decreased below 25ºC in fall. Diurnal crown temperature of plants was 3 to 5ºC lower in DF culture than in palm shell culture during most of growing period. Although both crowns of DF and control plants were exposed to warm air and occasional sunshine because of its above ground part, the crown of DF plants might be cooled by contacting leaked fog through the planting hole. In the same way, diurnal root zone temperature also always ca. 3 to 4ºC lower in DF plants than in control plants. Owing to the release of latent heat by dry-fog evaporation and adiabatic expansion of solution from the atomizing nozzle, the fog temperature in rhizosphere was kept cool compared to the temperature of air surrounding the well-insulated chamber. Both temperatures of crown and root zone of DF plants began to decrease below 25ºC, which might be close to the upper limit temperature for flower bud initiation of ever-bearing strawberry even under long day conditions (Nishiyama and

Kanahama, 2002), ca. 20 days earlier than in control. After runner plants were transplanted in late summer, cooling medium accelerated flower bud emergence almost

10 days compared with plants grown in uncooled medium (Ikeda et al., 2007). They mentioned that the decrease in temperature in rooting medium enough to accelerate flower-bud emergence and fruit set on the primary axillary branch. There was non-significant difference in both temperatures of crown and root zone of DF plants

63 between with and without forced-mist cooling.

Fig. 5-3. Changes in daily solar radiation and average air temperature in a greenhouse in

2012 (above) and average temperatures of the crown surface (middle) and the rooting zone (below) of strawberry plants grown with dry-fog aeroponics (DF) or with palm shell culture. Crowns were continuously misted by dry-fog stream withdrawing from the root zone in a chamber (DF, Crown cooling).

64 Flowering and Fruiting

The numbers of flower truss (inflorescence) and flower of DF and control plants

were shown in Fig.5-4. The crown temperatures were too high to develop the flower truss

even in ever-bearing strawberry grown in both cultures during early to mid-August. After

late of August, the number of flower truss start to increase significantly for DF plants

compared to control plants which flower truss rarely developed until late September.

Flower bud initiation of DF plants was promoting in September, lead to significantly

higher number of flowering than control plants from September to November.

On the contrary, the emergence number of runner as vegetative growth was

extremely high for control plants (7.2), while DF plants developed few runners (0.8-1.3)

in summer from August to September. The interactive effects of temperature and

photoperiod on the flowering habit have been reported for ever-bearing strawberry (var.

Summerberry) plants, responding as qualitative long-day plants with the critical

photoperiod between 13 and 14 hours at high temperature (>25 ºC) and quantitative

long-day plants at lower temperature (10 to 25 ºC) (Nishiyama et al., 2006). When

ever-bearing strawberry plants were transplanted in early summer, daily high temperature

above 25 ºC with nearly critical photoperiod in subsequent growing period strongly

inhibit inflorescence development. The forced-mist cooling had no effect on the flower

truss development, however, it affected significantly the number of flowering in

November with more flower settings by cooling crowns directly.

Axial fan for circulating fog

Fig. 5-4. Changing in the number of flower truss (above) and flowering (below). [Value] shows the number of runner grown from August to September.

Photosynthetic Ability

The photosynthetic rates of mature leaves expanded in summer showed no significant difference at both rate-limited CO2 concentration and PPFD between DF and control plants (Fig.5-5). However, the CO2 or PPFD saturated rate seemed to be higher in

DF plants than in control plants. These increases in the maximum photosynthetic ability in DF plants could be caused by significantly increased contents of chlorophyll and TSP in their leaves compared to them in control plants (Table 5-1). Furthermore, high photosynthetic ability of DF plants measured at saturated PPFD was owing to an increase of stomatal conductance (gs) resulted in high intercellular CO2 concentrations (Ci)

(Fig.5-6) because of a close link between gs and Ci. The photosynthetic rate was light saturated at 400 and 800 µmolPPFD/m2/s for control and DF plants, respectively. These values are almost in the same range of 400-600 µmolPPFD/m2/s obtained in Fragaria vasca (Chabot, 1978).

DF plants had fewer leaves than control plants because of their growth tendency toward vegetative growth phase, most assimilates by photosynthesis might be distributed into the flower truss development and fruit setting, reading to increases in marketable fruit yield and TSS in fruits. The total number of marketable fruits per plant was significantly high by ca. three times in DF plants compared with it in control plants. For

Fragaria vasca, temperature during growth period shifted reproductive allocation from flowering at low temperature, to vegetative propagation at higher temperature (Chabot,

1978). Kumakura and Shishido (1995) also found that fruit production with marketable value was not satisfactory at diurnal mean temperature above 20ºC, and the yield was reduced when the day temperature exceeded 25ºC. Although a high temperature (23ºC) of

67 solution in nutrient film technique decreased photosynthesis rate of single attached leaf in ever-bearing strawberry (Udagawa et al., 1989), the ambient photosynthetic rates measured under growth conditions did not decreased for both DF and control plants even their root zone temperatures exceeded over 23ºC in the present study.

Fig. 5-5. Relationships between photosynthetic rates and intercellular CO2 concentrations

(Ci at 1500 µmol/m2/s saturated PPFD) or light intensities (PPFD at 350 µmol/mol ambient CO2) measured for the attached mature leaves of strawberry plants grown in the summer.

68 Table 5-1. Leaf physiological traits and harvested fruit quality of strawberry plants grown

using dry-fog hydroponics in the summer.

LeavesY Economically harvested fruitsX

Total number Chl. a+b TSP Total number FW TSS per plantZ (mg g-1 FW) (mg g-1FW) per plant (g fruit-1) (ºBrix) DF 15.2 ± 2.1 b 2.78 ± 0.22 a 3.54 ± 0.21 a 18.2 ± 2.8 a 10.5 ± 1.2 a 10.4 ± 0.15 a

Palm shell 21.5 ± 1.8 a 2.17 ± 0.14 b 2.86 ± 0.23 b 6.8 ± 1.6 b 9.8 ± 1.8 a 8.2 ± 0.31 b

Means ± standard deviations. Different lowercase letters in each column denote significant differences by t-test (P≤0.05, n=12). ZAll plants were harvested and measured in mid-December. YLeaf constituents were determined for the leaves measured their photosynthesis in mid-August. XMature fruits were harvested from November to December.

Fig. 5-6. A relationship between Ci and stomatal conductance (gs) at the measurements shown in Fig. 4. A value in parenthesis shows PPFD 50 and 1500 µmol/m2/s at each measurement, respectively.

69 Chapter 6. Concluding remarks

In this study, the growth characteristics and physiological activities of plants cultivated with the dry-fog aeroponics were investigated and evaluated to establish the dry-fog aeroponics as a new hydroponic technique.

In chapter 2, it was showed that the dry-fog aeroponics enables lettuce plants to grow with less water without sacrificing yield and leaf quality as compared to traditional hydroponics. The lightweight cultivation chamber can decrease the workload for growers and is easy for new growers to maintain. Dry-fog aeroponics posses potential as a novel hydroponic system to promote high-value crop production and yield more than other hydroponic systems by controlling the rhizosphere environment while saving water and nutrients. It is worth noting that the nozzle used in this study can atomize enough dry-fog to fill a much larger chamber (6000 × 700 × 450 mm).

In chapter 3, in the dry-fog aeroponics, the flow rate of dry-fog through the rhizosphere can be controlled easily by using a fan. The shoot and root growth of leaf lettuce plants was altered depend on the flow rate, which affected on the amount of dry-fog particles that adhere to the surface of the roots. The roots grew and developed adaptively to dry-fog aerobic rhizosphere with well branching, root hairs and high respiration activity. These roots might be prefer to increase ability of water and nutrient absorption, lead to fill the evapotranspiration demand sufficiently and enhance the photosynthetic rate and stomatal conductance. Accordingly, dry-fog aeroponics is thought to be an effective cultivation system to promote plant growth.

As the flow rate of dry-fog aeroponics indirectly affects on the plant growth by the root development, optimally-controlled flow rate might make it possible to accelerate the

70 root growth adaptation, thereby leading to improved growth in the early stages after planting. Therefore, further investigations are necessary to better understand the optimal rhizosphere environment for root growth that makes the production yield maximum in dry-fog aeroponics.

In chapter 4, the growth of leaves and overall yield were increased by a slower flow rate of fog though the rhizosphere compared to DFT; the root growth was greatly increased under DF conditions. SDF with a slower flow rate increased the root biomass and surface area and also increased the absorption activity of roots. Because sufficient water was absorbed from the adapted root, the evaporation demand was met, and the photosynthetic rate was increased in response to the increase in stomatal conductance.

Therefore, aeroponics using semi-dry-fog with a 0.1 m s-1 flow rate is expected to improve the productivity and yield of greenhouse lettuce production. Under DF conditions with a 0.1 m s-1 flow rate, the significant increase in the root system affected the growth of leaves, although the author did not observe a significant increase in the physiological activity of the leaves and roots. Thus, the yield for lettuce cultivated using dry-fog aeroponics was improved in different ways by changing the droplet size. Further studies are warranted to elucidate the optimal rhizosphere environment for dry-fog aeroponics.

In chapter 5, Cooling the plant parts (crown and root) of ever-bearing strawberry affected the growth behavior with shifting from vegetative to reproductive phase, resulting in early emergence of inflorescence and flowering in late summer. For this purpose, dry-fog aeroponics is seemed to be an effective hydroponic system, where the root zone and crowns are kept cooler than surrounding air temperature by maximum 5ºC.

It is important to lower and save the cost for forcing culture of ever-bearing strawberry in

71 summer without extra-electricity cost for cooling, for example, using a chiller for running a cold water to cool crowns and rooting medium. Dry-fog aeroponics has double operations of both supplying nutrient solution and cooling root zone at the same time without any other specific devices. Furthermore, strawberry plants grown with dry-fog aeroponics have a high ability of photosynthesis that contributes to fast growth, produce more inflorescences and fruiting. The author considered that dry-fog aeroponics could be helpful and suitable new hydroponic system for growers of ever-bearing strawberry production in warm and lowland horticulture in summer.

As above, improvement of the plants yields and the creation of the new cropping type by the dry-fog aeroponics were anticipated, and possibility of popularization as a new hydroponic technique was shown enoughly. Further research would be helpful to determine rhizosphere environmental conditions (density of dry-fog, frequency of atomizing) to maximize plant growth and the amount of secondary metabolites produced.

In addition, optimization of the composition and strength of the nutrient solution, especially for dry-fog culture, is needed and could be determined through investigation of the absorption nutrients in the plants.

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80 Acknowledgment

I am most grateful to my supervisor, Dr. Michio Kanechi, Associate Professor of the

Horticultural Crop Production and Development Laboratory, Graduate School of

Agricultural Science, Kobe University, for his courteous instruction and encouragement throughout the research and writing of papers.

I am also grateful to Dr. Yuichi Uno, Associate Professor of the Horticultural Crop

Production and Development Laboratory, Graduate School of Agricultural Science, Kobe

University, for all his kind support, and helpful advice and discussions.

I sincerely thank Mr. Norio Onishi and Mr. Daisuke Kataoka for their fruitful discussions and help to balance my commute to college with my assignment at the Ikeuchi Co. Ltd. I am grateful to Ikeuchi Co.Ltd. for supporting my postgraduate studies.

I would like to thank all members of the Horticultural Crop Production and Development

Laboratory, Graduate School of Agricultural Science, Kobe University, especially Ms.

Chiyo Fukuda, Mr. Daiki Takegami, Ms. Mizuki Sato and Ms. Tan Oh, for their support with my studies and campus activities.