Improving Lettuce Productivity while Suppressing Biofilm Growth and Comparing Bacterial Profiles of Area and Nutrient Solutions in Windowfarm Systems

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

By

Seungjun Lee

Graduate Program in Food Science and Nutrition

The Ohio State University

2014

Master's Examination Committee:

Dr. Jiyoung Lee, Advisor

Dr. Ahmed Yousef

Dr. Luis Rodriguez-Saona

Copyrighted by

Seungjun Lee

2014

Abstract

Hydroponic systems have gained worldwide popularity and are increasingly used in various purposes in different geographic areas. To improve and produce more hydroponic crops, a variety of hydroponic systems have been developed, such as: wick, drip, ebb- flow, water culture, , aeroponic, and windowfarm systems.

Numerous studies show that have many advantages over culture systems; i.e., reuse of water, ease in controlling external factors, and reduction of traditional farming practices (e.g., cultivating, weeding, watering, and tilling). However, limitations include: high setup cost, rapid pathogen spread, and specialized management. The purpose of this Review (Chapter 1), ‘Introduction to Hydroponic Systems’ are to: 1) characterize the trends, advantages, and limitations of different systems; 2) introduce different types and methods of operation; and 3) discuss research being conducted in plant diseases and the role of beneficial . The Review (Chapter 1) provides a better understanding of hydroponics and newly applied systems and discusses their optimization to enhance food quality and quantity, and reduce plant diseases.

In Chapter 2, the windowfarm is described, when plants are often prone to infections by phytopathogens that slow plant growth and reduce crop productivity and excessive biofilm build-up due to high concentration of nutrients in the system. The major items ii discussed in Chapter 2 are: 1) development of a new windowfarm system that uses minimal water; 2) presentation of methods that promote plant health by making plants more resistant to phytopathogen infection and enhance growth using biosurfactant- producing around plant ; and 3) demonstration of methods that minimize biofilm build-up, such as UV irradiation, thereby extending the usable lifespan of the whole hydroponic system. To examine these items, Romaine lettuce was cultivated for 11 weeks in a windowfarm system. P. chlororaphis was inoculated near the roots, subsequently producing pyoverdine (yellow-green fluorescent pigments). For enhancing bacterial colonization, glass beads or granules were added.

The water reservoir was treated once a week for 5 min with low pressure ultraviolet (UV) irradiation to minimize biofilm build-up in the system while maintaining the colonized beneficial microbial around the roots. Food productivity was measured by the number of leaves and length and weight of the lettuce; quality was measured by lettuce color. ultimum, a plant pathogen, was inoculated on the root areas to examine the protective effects of P. chlororaphis against P. ultimum. The results show that P. chlororaphis-treated lettuce grew significantly better than non-treated lettuce as indicated by enhancement of color, weight, length, and number of leaves per head (p < 0.05). The death rate of the lettuce was also reduced by half when the lettuce was treated with P. chlororaphis. UV irradiation reduced the concentration of bacteria (4 log reduction) and algae (4 log reduction) in the water reservoirs and water tubing systems. In summary, the iii results suggest that introduction of P. chlororaphis into a windowfarm system promotes plant growth and reduces damage caused by plant pathogens. Additionally, UV irradiation of the water reservoir results in reduction of algal and biofilm growth and extends the lifespan of the system.

In Chapter 3, how the use of beneficial bacteria, addition of beads around roots and

UV treatments affect bacterial diversity and community in hydroponic systems were examined. . The objectives of this study were to: 1) examine bacterial diversity and community in hydroponic systems; 2) compare the effect of three different treatments

(beads, P. chlororaphis, and UV irradiation) in root areas using the PCR-DGGE method; and 3) identify bacteria in root areas and water in a windowfarm system. Bacterial profiles were compared under three different treatments (bead and P. chlororaphis treatments in root area, and UV irradiation of reservoir water) using PCR-DGGE and banding pattern analysis. After cultivation of lettuce in windowfarm systems, clay, bead and water samples were collected and bacterial community DNA was extracted for analysis. The DGGE bands were analyzed and interesting bands were selected for bacterial identification. The results demonstrated that 1) P. chlororaphis had positive effects on lettuce’s growth and root development; 2) the bacterial community of the root area was affected significantly by beads (clay pelltes versus glass beads with clay pelltes) and P. chlororaphis treatments; 3) Beads and P. chlororaphis treatments did not make the change of bacterial diversity around root area in the windowfarm system. Bacteria iv that are beneficial for plant growth were found in the windowfarm sytem (Variovorax paradoxus, Pseudomonas fluorescens, Pseudomonas thivervalensis, and Pseudomonas brassicacearum).

In conclusion, the results suggest that 1) P. chlororaphis-treated lettuce grew significantly better than non-treated lettuce as indicated by a 50% reduction in the death rate and enhancement of color, weight, and number of leaves per head (p < 0.05); 2) UV irradiation reduced the biofilm growth in water tubing systems and extended the lifespan of the system; 3) the bacterial profile provides a better understanding of bacterial activities in the root area and water in hydroponic systems and how plants and interact.

Keywords: hydroponic systems, windowfarm, beneficial bacteria, Pseudomonas chlororaphis, Pythium ultimum, UV irradiation, bacterial profile

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Acknowledgments

I would like to express the deepest appreciation to my advisor, Dr. Jiyoung Lee.

She continually and convincingly conveyed a spirit of adventure in regard to research and an excitement in regard to teaching. Without her guidance and persistent help this dissertation would not have been possible.

I would like to thank my committee members, Dr. Ahmed Yousef and Dr. Luis

Rodriguez-Saona for their constant interests and support during the course of my

Master’s study at The Ohio State University.

In addition, a thank you to Dr. Zuzana Bohrerova, Dr. Parwinder S. Grewal, and

Dr. Chongtao Ge have helped in enriching my research experience and manuscripts. The authors thank Dr. Brian B. McSpadden Gardener for supporting the source of the pathogen (P. ultimum), Dr. Qinghua Sun for his support in this study, Dr. Bruce Casto for editing the manuscript, Minseok Kim and Jill Stiverson for their kind support in experiments and data analysis, and Hyokyung Kim for her contribution in making figures.

This study was supported by Food Innovation Center at The Ohio State University.

I would also like to thank my lab members: Chenlin Hu, Cheonghoon Lee, Chris

Rea, Tyler Gorham, and Jessica Healy, Eunyoung Park, Feng Zhang, and Tsung-Ta Hsu, for all the help in and outside the lab.

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I wish to thank my family, friends at the Department of Food Science and

Technology for their continuous support, as well as the faculties, staff members at the

Department of Food Science and Technology.

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Vita

March 2003 ...... Kyeongbuk High School, South Korea

2011...... B.A. Applied Biology and Chemistry,

...... Kyungpook National University University,

...... South Korea

2012 to present ...... Department of Food Science and

Technology, The Ohio State University

Publications

Lee, S., Ge, C., Bohrerova, Z., Grewal, P. S., and Lee, J. (2014) Enhancing plant productivity while suppressing biofilm growth in a windowfarm system using beneficial bacteria and ultraviolet irradiation. Journal of Applied Microbiology. (submission)

Lee, S., and Lee, J. (2014) A review of hydroponic food production systems: commonly used conventional types and an emerging vertical windowfarm system. Agricultural systems. (submission)

Fields of Study

Major Field: Food Science and Nutrition

viii

Table of Contents

Abstract ...... ii

Acknowledgments...... vi

Vita ...... viii

Publications ...... viii

Fields of Study ...... viii

Table of Contents ...... ix

List of Tables ...... xiii

List of Figures ...... xv

Chapter 1: Literature Review ...... 1

1.1. Abstract ...... 1

1.2. Introduction...... 2

1.3. Advantages and limitations of hydroponics ...... 5

1.4. Common hydroponic models ...... 7

1.4.1. The wick system ...... 7

1.4.2. The drip system...... 8

ix

1.4.3. The system ...... 9

1.4.4. The (deep) water culture system ...... 9

1.4.5. The nutrient film technique system ...... 10

1.4.6. Aeroponic systems ...... 11

1.5. Hydroponic window farming: an emerging model ...... 11

1.6. Beneficial bacteria in hydroponic systems ...... 12

1.7. Conclusion ...... 14

Chapter 2: Enhancing plant productivity while suppressing biofilm growth in a windowfarm system using beneficial bacteria and ultraviolet irradiation ...... 16

2.1. Abstract ...... 16

2.2. Introduction...... 17

2.3. Materials and Methods ...... 20

2.3.1. and growing conditions ...... 20

2.3.2. Construction and optimization of the windowfarm system ...... 21

2.3.3. Pseudomonas chlororaphis inoculation ...... 23

2.3.4. Pythium ultimum inoculation ...... 24

2.3.5. UV-C irradiation ...... 25

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2.3.6. Heterotrophic plate count ...... 25

2.3.7. Food quality and productivity: Color, length, weight, number, and lettuce

leaf mortality ...... 26

2.3.8. Measurement of Chlorophyll a in the water tubing systems ...... 27

2.3.9. Statistical analysis ...... 28

2.4. Results ...... 28

2.4.1. Evaluation of food quality and productivity of P. chlororaphis treated

lettuce. 28

2.4.2. Effect of UV irradiation on the windowfarm system ...... 30

2.5. Discussion ...... 31

Chapter 3: Comparison of bacterial profiles of root area and nutrient solution in the windowfarm using DGGE and banding pattern analysis ...... 36

3.1. Abstract ...... 36

3.2. Introduction...... 37

3.3. Materials and Methods ...... 40

3.3.1. Sample preparation ...... 40

3.3.2. Bacterial Community DNA Extraction ...... 41

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3.3.3. PCR Amplification of 16S rRNA Genes ...... 42

3.3.4. Denaturing Gradient Gel Electrophoresis (DGGE) ...... 43

3.3.5. DGGE gel data analyses ...... 44

3.3.6. Sequencing Analysis of the Selected DGGE Bands ...... 45

3.4. Results ...... 46

3.4.1. Bacterial Species Diversity ...... 46

3.4.2. Bacterial Community Structure ...... 47

3.4.3. Bacterial Identification ...... 48

3.5. Discussion ...... 50

3.5.1. Bacterial Identification ...... 52

Chapter 4: Conclusions ...... 54

List of References ...... 56

Appendix A ...... 78

Appendix B ...... 85

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List of Tables

Appendix A ...... 78

Table 1. The advantages of hydroponic systems compare to culture and their

limitations...... 79

Table 2. Summary of test conditions of 8 different groups in each test ...... 80

Table 3. Color measurements (L*, a*, b* and hue angle) of the 9-week aged lettuce. 81

Table 4. Death of lettuce 2 weeks after Pythium ultimum inoculation...... 82

Table 5. Diversity indices calculated from the DGGE banding profiles (DGGE OTUs

richness (S), Shannon diversity index (H), and Evenness index (E)) generated from

DGGE fingerprints of microbial communities from bead samples. The comparison of

diversity indices between each group was performed using an independent two samples

t-test. There is no significantly difference between clay pellets and a mixture, non-P.

chlororaphis treatment and P. chlororaphis treatment, or non-UV irradiation and UV

irradiation, except for asterisk groups (*); evenness index between non-P. chlororaphis

treated groups and P. chlororaphis treated groups was significantly difference (p <

0.05)...... 83

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Table 6. Identification of bands obtained by PCR-DGGE based on the V3 region of

16S rRNA and the closest sequence match of known bacteria in other references...... 84

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List of Figures

Appendix B ...... 85

Figure 1. Trends in the total number of documents concerning hydroponic systems by

year. The figure shows a time series plot of the number of hydroponic system papers

since 1930s. The key words used for search [Scopus] were: hydroponics, hydroponic

system, hydroponic food, and soilless culture. Journal articles and books were

included...... 85

Figure 2. Proportion of documents related to hydroponic system by subject area over

76 year period from 1937 to 2013. The key words used for database search [Scopus]

were: hydroponics, hydroponic system, hydroponic food, and soilless culture. Journal

articles and books were included...... 86

Figure 3. The number of documents related to hydroponic systems by country from

1937 to 2013. The key words used for database searching [Scopus] were: hydroponics,

hydroponic system, hydroponic food, and soilless culture. Journal articles and books

were included...... 87

Figure 4. The percentage of major types of hydroponic crops found from publications.

The key words used for database searching [Scopus] were: hydroponics, hydroponic

xv system, hydroponic food, and soilless culture. Journal articles and books were included...... 88

Figure 5. Six different types of traditional hydroponic systems. (a) wick system, (b) drip system, (c) Ebb-Flow system, (d) water culture system, (e) nutrient film technique, and (f) aeroponic system...... 89

Figure 6. Schematic diagram of a single unit of a vertical windowfarm system (a) and a multiple-unit windowfarm system (b). Water in the water reservoir goes up through tubing to each pot using an air pump and watering time can be control by an electronic timer...... 90

Figure 7. . Schematic diagram of a single unit of the windowfarm system (a) and a complete windowfarm system (b). Water in the water reservoir ① goes up through a tube ④ to each pot ③ using an air pump ②...... 91

Figure 8. Number of lettuce leaves in P. chlororaphis treated (+) and non-treated (-) lettuce groups after 9-weeks of planting in the windowfarm system...... 92

Figure 9. Crop productivity as measured by thelength (a) and weight (b) in 9-week aged lettuce with (+) and without (-) P. chlororaphis. Box plots showing means and medians of lettuce length and weight. The length of each box shows the interquartile range and 50% of cases of the variable. The line and the dot in the box indicate the

xvi mean and the median, respectively, while extended lines from the box show maximum and minimum values. Comparing P. chlororaphis treated lettuce versus non-P. chlororaphis treated lettuce in their appearances of leaves and roots (c)...... 93

Figure 10. Concentration of bacteria in the water reservoir during 5-min exposure to

UV irradiation. Bacterial levels were measured using heterotrophic plate counts to determine the optimal UV dose for minimizing biofilm build-up while maintaining P. chlororaphis in the windowfarm system...... 94

Figure 11. Effect of UV irradiation during lettuce growing period on concentration of heterotrophic bacteria in the water reservoirs (a) and on the beads around the lettuce roots (b)...... 95

Figure 12. Comparison of Chlorophyll a concentration on the inner surfaces of water tubing in the UV irradiated groups and the non-UV irradiated groups after the study period (a). Measurement of Chlorophyll a on the inner surfaces of water tubing in the non-UV irradiated groups by three day interval during the lettuce growing period (b).

Photos of both tubing in the UV irradiated groups and the non-UV irradiated groups after the study period (c)...... 96

Figure 13. Schematic diagram of the windowfarm systems. Clay: clay pellets growing media, Class + glass: a mixture with clay pellets and glass beads (1:1 ratio),

xvii

Pseudomonas: P. chlororaphis inoculation around root, UV: UV irradiation in water reservoirs for 5 minutes every week...... 97

Figure 14. Comparing P. chlororaphis treated lettuce versus non-P. chlororaphis treated lettuce in their appearances of leaves and roots; (a) P. chlororaphis treated lettuce root with clay pellets, (b) non-P. chlororaphis treated lettuce root with clay pellets. (c) P. chlororaphis treated lettuce root with clay pellets; (d) P. chlororaphis treated lettuce root with a mixture of clay pellets and glass beads; (e) root of non-P. chlororaphis treated lettuce with clay pellets. Claya: clay pellets growing media,

Mixtureb: a mixture with clay pellets and glass beads (1:1 ratio)...... 98

Figure 15. Flowchart of the windowfarm experimental design. This chart describes the each step in the process of entire windowfarm experiment based on three different conditions (beads, P. chlororaphis inoculation, and UV irradiation). Each group had 24 replicates...... 99

Figure 16. 16S rRNA gene DGGE profile (bands marked with ● were selected for sequencing). The first set of 16S rRNA gene DGGE profile using 30% - 70% denaturant range (Lanes 1 to 8 are bead samples; Lanes 9 to 16 are water samples.

Each lane describes in this figure (a: clay pellets growing media, b: a mixture of clay pellets and glass beads, c: P. cholororaphis treatment, d: UV irradiation)...... 100

xviii

Figure 17. . Banding pattern analysis of bacterial community around root area by band- search algorithm and band comparison among the different groups. (a) all bead samples; (b) clay pellets versus a mixture of clay pellets and glass beads; (c) P. chlororaphis treated groups versus non-P. chlororaphis treated groups...... 101

Figure 18. Banding pattern analysis of bacterial community in water samples of the windowfarm system by band-search algorithm and band comparison among the different groups...... 102

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Chapter 1: Literature Review

1.1. Abstract

Hydroponic systems have gained worldwide popularity and are increasingly used for various purposes in different geographic areas. In order to produce more and improved hydroponic crops, a variety of modified hydroponic systems have been developed, such as: the wick, drip, ebb-flow, water culture, nutrient film technique, aeroponic, and windowfarm systems. According to numerous studies, hydroponics have many advantages over field culture systems, such as: reuse of water, ease in controlling external factors, and a reduction in traditional farming practices (e.g., cultivating, weeding, watering, and tilling). However, several limitations have also been identified in hydroponic culture systems: high setup cost, rapid pathogen spread, and a need for specialized management knowledge.

The purposes of this review are to: 1) characterize the trends, advantages, and limitations of hydroponic systems; 2) introduce different types of hydroponic systems and methods of operation; and 3) discuss research being conducted in plant diseases and

1 the role of beneficial bacteria in hydroponic systems. This review intends to provide a better understanding of hydroponic and newly applied systems and the optimization of existing systems in order to enhance food quality and quantity, and reduce plant diseases.

Keywords: hydroponic systems, vertical windowfarm system, conventional hydroponics, beneficial bacteria

1.2. Introduction

Hydroponic systems are cultivation technologies that use nutrient solutions rather than soil substrates. Sometimes they use artificial media, such as peat moss, sawdust, charcoal, sawdust, rockwool, coco , clay granule, gravel, or ceramics, to provide mechanical support (Bhattarai et al., 2008; FAO, 2014; Jones, 1983; Roberto 2004; Yu et al., 1993). Since hydroponic production techniques can offer higher yields and higher quality products, the supply of, and demand for, hydroponic systems have dramatically increased in the United States (US) (Brentlinger, 2007; Van Patten, 2008). The commercial hydroponics industry has grown by approximately fivefold in the last 10 years, and its global value is currently estimated to be about $8 billion US dollars

(Carruthers, 2002). Hydroponic research has increased steadily from 1930 to 2013

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(Figure 1) with about half of this research focused on the Agricultural and Biological

Sciences fields (Figure 2).

With the development of new materials and equipment (e.g., media, tubes, connectors, valves, pots, water reservoirs or tanks, air or water pumps and electronic timers), new hydroponic techniques have become available, notably: the static aerated, ebb and flow, deep flow, aerated flow, nutrient film, gravel flow sub irrigation, , root mist, and fog feed systems. Most hydroponic systems operate automatically to control the amount of water, nutrients, and lighting time, based on the requirements of different plants (Hochmuth and Hochmuth, 2011; Resh, 2013). Likewise, different artificial media can provide different particle sizes, shapes, and penetrability; each medium affects plants and roots differently by retaining water, supporting plants, and making pore space at different rates (Asao et al., 1999). The selection of a medium depends on the nature of the plants, cost, and the type of hydroponics that is employed

(Jones, 1997).

Although hydroponics are commonly used for personal gardening, education, and research, most systems have been used for crop and vegetable production, i.e., tomatoes, beans, spinach, , cucumbers, and lettuce (Nichols, 2006; Stajano et al., 2003).

A large amount of hydroponic crops are produced in developed countries to meet consumer demand. Among the countries found in the literature, the US and China are the top two countries generating the most publications about hydroponic plants and 3 hydroponic systems (Figure 3) (Carruthers, 2002). In order to enhance production quality and quantity, many different kinds of plants have been studied in these systems.

Tomatoes, as a representative research crop, are the most commonly studied (Figure 4).

Hydroponic systems offer a number of benefits, including: the ability to reuse water and nutrients, easy environmental control, and prevention of soil-borne diseases and pests

(Lommen, 2007; Molitor, 1990). However, waterborne diseases can contaminate and spread through the water tubing systems; therefore many studies have focused on preventing fungal infections or developing remedial agents for phytopathogens

(Chatterton et al., 2004; Itoh et al., 1998; Song et al., 2004).

Almost all hydroponic systems are indoor, located in , so they rely less on external conditions and have less impact on the environment (Sundin et al., 1995).

Because they can be used not only in urban areas, but also in non-arable lands, their current applications include supplying food for astronauts in space, growing crops in desert areas or the Polar regions, and providing food for poor or rural communities (FAO,

2001; Jones, 1997; Stajano, 2003). For instance, people living in underdeveloped and poor regions of Thailand cannot grow enough food using traditional farming practices because of high and a lack of natural nutrients in the soil, but hydroponic systems can successfully generate additional crop production and provide agricultural education for the regions’ children (Ortiz et al., 2009).

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1.3. Advantages and limitations of hydroponics

There are many advantages of hydroponic systems over soil culture systems (Table

1). Hydroponics perform well, even in areas that are otherwise unsuitable for growing crops due to soil contaminants like toxic chemicals or heavy metals (Jones, 1997).

Hydroponic systems also make it easy to control growth conditions, such as temperature, volume of water, nutrients, humidity, and duration of lighting in order to optimize crop production (Norén et al., 2004). In addition, plants in hydroponic systems are not easily influenced by climate change; therefore, plants can be cultivated year-round under a wide range of conditions (Gibeaut et al., 1997; Norström et al., 2004). Further, as the systems operate automatically, they may be expected to reduce labor and several traditional agricultural practices can be eliminated, such as cultivating, weeding, watering, and tilling (Jovicich et al., 2003).

Soil-based crops can be contaminated by many environmental agents, some of which are hard to prevent. However, using hydroponics, most media and other materials can be sterilized by ultraviolet (UV) irradiation, chemical compounds, steam, and/or high temperatures (Knutson, 2000). Furthermore, indoor hydroponics are not expected to be infected by diseases common to plants cultivated in soil (Stanghellini and Rasmussen,

1994; Zlnnen, 1988), thereby reducing or eliminating the use of pesticides and their resulting toxicity (Fu et al., 1999). Delivering recycled or used directly to the root area 5 provides a more effective utilization of resources, reduces water loss, and distributes nutrients evenly to the entire plant, requiring less fertilizer than soil systems (Güohler et al., 1989; Midmore and Deng-lin, 1999; Resh, 2013). Finally, pH can be easily controlled, according to the plant’s requirements (Rolot and Seutin, 1999). Because of these advantages, many studies report that hydroponic systems can increase the yield and quality of crops (Cornish, 1992; Resh, 2012; Rolot and Seutin, 1999; Sarooshi and

Cresswell, 1994).

However, there are also some limitations to hydroponic systems (Table 1). The main problem is the high initial setup cost, as the fundamental supplies are expensive

(Domingues et al., 2012; Resh, 2013). Hydroponic systems are also vulnerable to power outages, as the electrical-driven machines in the systems cannot supply water or nutrient solution without power (Knutson, 2000). In addition, when phytopathogens

(microorganisms such as Verticillium, Pythium, and Fusarium) contaminate solutions or crops, waterborne diseases can rapidly spread through the entire systems as water tubing systems are connected to each pot (Ikeda et al., 2002). These infectious agents may multiply and accumulate, potentially causing a severe disease outbreak in the system

(Schnitzler, 2004). Finally, hydroponic system operators need specialized skills and knowledge to produce high yields of crops; they must learn the proper amounts of nutrients and lighting, manage complex nutritional problems, maintain pest control, and

6 prevent the production of biofilms in the water tubing system (Guo et al., 2002; Sutton et al., 2000; Zekki et al., 1996).

1.4. Common hydroponic models

Hydroponic systems are highly customizable and many modified versions have been used to optimize growing conditions for particular plants. Overall, there are six common hydroponic systems described herein: the wick, drip, ebb-flow, water culture, nutrient film technique, and aeroponic systems.

1.4.1. The wick system

The wick or passive system is an excellent model for cultivating indoor plants: it is a self-feeding model and does not require a water pump (Figure 5 (a)) (Shrestha and

Dunn, 2013). Water or a nutrient solution in a reservoir is supplied through a wick

(typically nylon) that can absorb and transport water from the reservoir to the root area by capillary action. The wick system has been used in small-scale gardens, such as personal home or office gardens, to grow flowers or flowering plants. Even though it effectively inhibits the diseases common to overwatering, the wick system is not suitable for large or

7 long term plants, which need a larger amount of water than the wick can supply (Harris,

1988).

1.4.2. The drip system

The drip or drip irrigation system is a popular and widely used commercial system (Reed, 1996; Rouphael and Colla, 2005). Water or a nutrient solution in the reservoir is delivered to each plant or pot using a pump with the amount of water for each plant adjusted by an electronic timer (Figure 5 (b)) (Rouphael and Colla, 2005). The drip system is divided into two models, recovery and non-recovery, depending on the processing of the reused water or nutrient solution (Saaid et al., 2013). In the recovery system, the water or nutrient solution is collected and returned to the reservoir, and then recirculated through the system (Schröder and Lieth, 2002). This makes it more economical than the non-recovery model, but reusing the solutions may result in pH changes and growth of algae or mold in the reservoir or tubing system. The non-recovery drip system needs to have the amount of water or nutrient solution frequently monitored in the reservoir to ensure that enough water or nutrient solution reaches the roots of the plants (Santamaria et al., 2003).

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1.4.3. The ebb and flow system

The ebb and flow system uses an automatic flood and drain watering technique, in which plants are flooded temporarily and periodically (Figure 5 (c)) (Buwalda et al.,

1994). The water or nutrient solution in the reservoir ascends to a growth tray via a water pump, accumulates to a certain level, and stays in the growth tray for a set amount of time, providing water and nutrients to the plants. After a predetermined time, the solution is drained back into the reservoir through a tubing system. This multifaceted circulation system requires continual observation to control the amount of water provided to the system. Although it is possible to grow many different kinds of plants and provide them with a large amount of water, root disease and growth of algae or molds may easily occur in this system (Buttner et al., 1995; Buwalda et al., 1994); therefore, some modified ebb- flow systems include a filtration or other methods for sterilization of the water (Nielsen et al., 2006).

1.4.4. The (deep) water culture system

Most modified hydroponic systems were originally derived from the water culture system (Harris, 1988). The water culture system is a simple model, composed of a reservoir, an air stone, a tubing system, an air pump, and a floating platform (Figure 5 9

(d)) (Hoagland and Arnon, 1950). Unlike the wick system, it produces food actively: a floating platform supports plants or pots in a reservoir, where the root parts are constantly immersed in the water or nutrient solution (Saaid et al. 2003) and oxygen is supplied by an air pump and air stone. For optimization of growing conditions, it is necessary to monitor the oxygen and nutrient concentrations, salinity, and pH (Domingues et al.,

2012). Although fast-growing or water-loving plants grow well in this system, large or long-term plants do not, and algae and molds can grow rapidly in the reservoir.

1.4.5. The nutrient film technique system

The nutrient film technique (NFT) system generates oxygen rich conditions

(Figure 5 (e)) (Jones, 1997). Water or a nutrient solution in a reservoir circulates throughout the entire system; it enters the growth tray via a water pump without a time control, and then constantly flows around the roots (Domingues et al., 2012). The solution is collected and reused, and the amount of water is controlled by the slope of the tray and the power of the water pump. However, the roots are susceptible to fungal infection because they are always immersed in water or nutrient solution (Thinggaard and

Middelboe, 1989).

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1.4.6. Aeroponic systems

In an aeroponic system, water or a nutrient solution is sprayed around the roots by a water pump (Figure 5 (f)). Supports maintain the pots or plants, and the water or nutrient solution is in a mist form and supplied for a specified period using a special nozzle and an electronic timer. Customizing the misting cycles to particular plants is important, because their roots are exposed to the air and can dry rapidly. The mist can easily be affected by the outside temperature, which makes these systems difficult to operate under cold or frigid conditions.

1.5. Hydroponic window farming: an emerging model

Window farming is an emerging concept in urban agriculture, enabling residents to grow vegetables and herbs all year in urban settings with an available window (Lee et al., 2014). The window farm system is generally a vertical hydroponic growing system constructed of simple household materials, including plastic bottles, a water reservoir, and a small scale water pump with tubing (Figure 6). Water circulates through the system via an automatic drip irrigation configuration using a pump and an electronic timer. The sun supplies natural light, although artificial light may be needed on cloudy days.

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The windowfarm system requires much less space than traditional hydroponic systems and provides an alternative method for growing crops in urban environments; an innovation of special interest to people in congested cities. This trend is expected to continue, as window farming helps create sustainable agriculture, and is able to provide urban residents with fresh and healthy foods.

1.6. Beneficial bacteria in hydroponic systems

Even though hydroponics grow plants in closed systems, pathogens still threaten plants’ viability (Owen-Going et al., 2003). Many pathogens can grow under to hydroponic conditions due to the high nutrient concentration. Circulation of the water rapidly spreads pathogens throughout the system and may ruin the entire crop

(Stanghellini and Rasmussen, 1994). Pythium spp. are common fungal root pathogens that are spread through water circulation systems (Rankin and Paulitz, 1994) and cause in hydroponically grown cucumber, pepper, and lettuce (Khan et al., 2003;

Stanghellini et al., 1996; Utkhede et al., 2000). Pythium ulimum has been shown to cause root rot in tomatoes, whereas Pythium aphanidermatum and Pythium dissotocum cause root rot in spinach cultivated in temperature between 17-27°C (Gold, 1985; Gravel et al.,

2006). Fusarium spp. are ubiquitous and cause wilt and root diseases in various plants

(Fravel, 2002). Fusarium oxysporum is responsible for root rot in basil, tomatoes, and 12 bananas (de Ascensao and Dubery, 2000; Fravel, 2002; Nahalkova et al., 2008).

Consequently, managing fungal infections is a vital component of hydroponic operations.

Options for preventing pathogen contamination include physical, chemical, and biological methods (Igura et al., 2004; Song et al., 2004; Zhang and Tu, 2000). We and others have studied the effect of plant growth-promoting (PGPR) in hydroponic systems. PGPR have been introduced into both soil culture and hydroponic systems (Kıdoğlu et al., 2009; Lee et al., 2010; Lee et al., 2014) with positive effects on plant quality and quantity (Woitke and Schitzler, 2005). PGPR act through N2 fixation, control of plant stress, extracting nutrients from soil, competition with pathogens, production of various kinds of plant hormones and biological controls, and promotion of plant growth (Bull et al., 1991; Freitas et al., 1993; Gaskins et al., 1985; Kloepper, 1993;

Lugtenberg and Kamilova, 2009).

Other research using Bacillus spp. and Pseudomonas spp. has suggested that both bacteria may prevent or diminish the effect of plant pathogens or fungal pathogens

(Raaijmakers et al., 2010). When Pseudomonas chlororaphis or Bacillus cereus was applied to chrysanthemums, infection by Pythium decreased by about 20% (Liu et al.,

2007). Although the mechanism where Bacillus spp. promotes plant growth and prevents diseases is still poorly understood, they can produce against phytopathogens

(Nihorimbere et al., 2012). Bacillus subtils, a Gram-positive bacterium, is well known as a plant growth enhancer with the ability to decrease high salinity concentrations of water 13 or nutrient solutions (Böhme, 1999; Woitke et al., 2004). Bacillus amyloliquefaciens was shown to increase water use efficiency, along with the quality (higher vitamin C than control groups) and quantity (8~9%) of tomatoes (Gül et al., 2008). Bacillus licheniformis has increased the diameter and weight of tomatoes and peppers, and promoted higher yields of each crop (García et al., 2004). Pseudomonas spp. shows antagonistic and antifungal activity against Fusarium graminearum and prevents root rot

(Benizri et al., 1995), whereas treatment of hydroponically grown tomatoes, cucumbers, lettuce, and potatoes bring about increased root and shoot weight and reduce root rot

(Peer and Schippers, 1988; Rankin and Paulitz, 1994). Finally, the introduction of

Pseudomonas chlororaphis on peppers in a hydroponic system was effective in suppressing infection of Pythium aphanidermatum and Pythium dissotocum and in controlling root rot (Chatterton et al., 2004).

1.7. Conclusion

The popularity of hydroponic systems has increased significantly, both in personal gardening and agriculture, because of its notable advantages over soil cultures. Various modified hydroponic models have been developed to improve materials and tools, but some problems still exist: fungal infections, lighting systems, wastewater treatment, maintenance of the system, construction cost per acre, and education for system operators 14

(Arteca and Arteca, 2000). Further innovations will no doubt address these problems, particularly by understanding the mechanisms whereby beneficial bacteria promote plant growth and prevent phytopathogens. In the future, hydroponic systems may be widely used in underdeveloped countries to produce food in harsh climates or in areas with limited space; applications may also be developed for use in outer space. Furthermore, the demand for indoor hydroponic systems may surge in the near future, due to the effect of climate change on plant variety and yield and the need for alternate sources of high- quality vegetable products.

15

Chapter 2: Enhancing plant productivity while suppressing biofilm growth in a windowfarm system using beneficial bacteria and ultraviolet irradiation

2.1. Abstract

Aim: Common problems in a windowfarm system (a vertical and indoor hydroponic system) are phytopathogen infections to plants and excessive build-up of biofilms. The objectives were to: 1) promote plant health by making plants more resistant to infection using beneficial biosurfactant-producing Pseudomonas chlororaphis around the roots; and 2) minimize biofilm build-up employing ultraviolet (UV) irradiation, thereby extending the lifespan of the whole system with minimal maintenance.

Methods and Results: Romaine lettuce was cultivated in the windowfarm systems with

P. chlororaphis inoculated near the roots. The water reservoir was periodically treated with UV to minimize biofilm formation. For enhancing colonization, glass beads or clay granules were added. Pythium ultimum, a plant pathogen, was inoculated around the roots.

Food productivity (number of leaves, and length and weight of the lettuce) and food quality (color of the lettuce) were measured. The results show that P. chlororaphis- treated lettuce grew significantly better than non-treated lettuce as indicated by enhancement of color, weight, length, and number of leaves per head (p < 0.05). The 16 death rate of the lettuce was reduced by ~50% when the lettuce was treated with P. chlororaphis. UV irradiation reduced the growth of bacteria (4 log reduction) and algae

(4 log reduction) in the water reservoirs and water tubing systems.

Conclusion: Introduction of P. chlororaphis into the system promoted plant growth and reduced damage caused by plant pathogen, P. ultimum. UV irradiation of the water reservoir reduced algal and biofilm growth and extended the lifespan of the system.

Significance and Impact of Study: The results provide significant data about the effect of P. chlororaphis on plants and plant pathogens in a window farm. UV irradiation minimized biofilm build-up in the system, while optimizing the colonization of beneficial microbial flora.

Keywords: windowfarm, hydroponic, Pseudomonas chlororaphis, Pythium ultimum, UV irradiation, romaine lettuce, biofilm

2.2. Introduction

Hydroponic systems, including window-based hydroponic systems, are crop cultivation systems without soil that are increasing in popularity in the United States

(Brentlinger 2007). The windowfarm is a vertical and indoor hydroponic system that is an emerging concept for plant cultivation in urban areas, providing residents with high- quality organic vegetables and herbs throughout the year. Such systems could also be 17 used to provide healthy food in developing countries that are challenged with water shortages and contaminated soil. The windowfarm system has several advantages over the conventional farming systems including: minimal use of space, utilization of various types of windows, higher crop yields, control of ambient temperature and humidity, more efficient water and fertilizer use, exclusion of pests, and control of plant diseases (Jensen

1999; Manzocco et al. 2011). In addition, this system can be assembled from simple household tools and materials. The water in the windowfarm system can be fully reused by recycling the excess water dripping from a plant growing unit to the next unit, which is located under the first one, and so on. Natural sunlight can be fully utilized and supplementary electric light can be used if needed. For these reasons, the system has the potential to significantly contribute to the urban food supply, particularly where access to healthy soil is limited. Furthermore, since most hydroponic systems are installed in green houses or indoor facilities, plants can be protected from soilborne plant pathogens and infections.

However, certain pathogens, especially waterborne pathogens, may contaminate fresh produce even in this closed system (Calvo-Bado et al. 2006). Plant pathogens can easily grow in the presence of high concentrations of nutrients that are used in hydroponic systems. When the systems are infected by pathogens, they can rapidly disseminate through the water tubing systems and contaminate the entire fresh produce (Chinta et al.

2014; Stanghellini and Rasmussen 1994). So far, hydroponic culture systems have 18 significantly unresolved barriers such as root-infection by fungal and bacterial pathogens

(Nonomura et al. 2001) and . Fusarium spp. and Phythium spp. have been reported to be the common crop pathogens that cause root rot in fresh produce grown in a hydroponic system (Anderson and Guerra 1985; Utkhede et al. 2000). Pythium spp. can infect a large range of plant species (Mitchell 1986) and proliferate in nutrient solutions

(Watanabe et al. 2008). These uncertainties have raised major health concerns as many studies have reported that crops can be contaminated with human pathogens when the water is polluted (Ge et al. 2013).

Another concern found in hydroponic systems is biofilm build-up in water circulation systems that can shorten the lifespan of the system and interfere with circulation of water.

Biofilms are complex structures, which consist of aggregated cells adhering to each other that develop quickly in hydroponic systems due to available nutrients in the system

(Elasri and Miller 1999). UV-C disinfection is a commonly accepted method for drinking and wastewater disinfection, and is applied in various fields, including food industry ( et al, 2012). The effectiveness of UV against suspended and aggregated bacteria, virus and protozoa, and biofilm has been demonstrated under many conditions

(Bohrerova and Linden, 2006; Friedberg et al. 1995; Elasri and Miller 1999; Karsten et al.

2007; Vu and Kuo, 2010).

In this study, windowfarm systems were constructed in which plants were grown with minimal maintenance. The factors evaluated included: colonization of Pseudomonas 19 chlororaphis (P. chlororaphis) around the roots by adding small beads (clay granules and glass beads); and reducing the growth of biofilms, including algae (that can inhibit flow of the water in the windowfarm system) by optimizing both dose and mode of application of UV-C. In summary, this study provides data that examine ways to prevent root disease, suppress biofilm growth, improve food quality, increase in crop yields, and promote plant health by proliferation of beneficial microbial flora in a windowfarm system.

2.3. Materials and Methods

2.3.1. Germination and growing conditions

Romaine lettuce (Lactuca sativa L. var. longifolia) seeds were purchased from

Botanical Interests, Inc. (Broomfield, CO, USA). The lettuce was germinated using a heated germination station (Hydro Farm Horticultural Products, Petaluma, CA, USA) and grown for 2 weeks at 25°C - 27°C and ~50% humidity (47% ~ 51%) in the laboratory at the College of Public Health, The Ohio State University (Columbus, OH, USA). After two weeks, the lettuce was transplanted into vertical net pots of the windowfarm systems

(Figure 7). Each pot was filled with either 80g of sterile clay granules (10 mm in diameter) (Easy Green Hydroton, Georg Buchner, Eschborn, Germany) or 80g of sterile mixture (one-to-one ratio) of clay granules (10 mm in diameter) and glass beads (5 mm in 20 diameter) (Glass Beads Solid, Fisher Scientific Inc.). During the 7-week experimental period, growing conditions were 22-29 °C and 28-40% relative humidity. Nutrients (132 ml/100 L of water) (Botanicare® Pro Grow Nutrients, Chandler, AZ) were added into the water reservoir (container at the bottom of the windowfarm system, (Figure 7) which was refilled every week. The nutrient solution was circulated from top to bottom through the vertical hydroponic system to provide irrigation and fertilization using an air pump

(Figure 7).

2.3.2. Construction and optimization of the windowfarm system

As shown in Figure 7, the windowfarm system was constructed using 500 mL plastic water bottles (Nestle, Stamford, CT 06902) for use as pots, 3 L plastic water bottles served as water reservoirs (Nestle, Stamford, CT 06902), 5 cm x 200 cm PVC rods (JM eagle, Los Angeles, CA 90045), 421 cm standard 3/16" flexible airline tubes

(Penn-Plax, Hauppauge, NY 11788), tubing connectors (Como filtration system,

Janesville, WI 53546), a 25L air pump with 8 outlets (Hydrofarm, Grand Prairie, TX

75050), and black drop cloths (Warp's Jiffy-Cover, Chicago, IL 60651) over each bottle and water reservoirs to prevent exposure to sunlight and to reduce the algal boom in the water reservoir. All materials were immersed in 70% ethanol (Decon Laboratories Inc.,

King of Prussia, PA 19406) for 24 hours and sterilized using UV irradiation for 2 hours prior to use. The lettuce was exposed for 16 hours per day to artificial light (Hydrofarm 21

Fluorescent System, Fairless Hills, PA, USA). The light system was controlled using a mechanical timer (24-Hour Mechanical Timer, General Electric,

Fairfield, CT 06825). The air pump circulated 2.5L of water through each pot with a flow rate of ~2ml/minute and the water circulation interval (4 hours per day) was controlled using a mechanical timer (General Electric, Fairfield, CT 06825). There were 8 different groups (Table 2) per test condition; Group 1 (control group), lettuce with clay granules without P. chlororaphis inoculation and without UV irradiation; Group 2, lettuce with clay granules and P. chlororaphis inoculation; Group 3, lettuce with clay granules with

UV irradiation for 5 minute intervals (all UV irradiations were for 5 min a week); Group

4, lettuce with clay granules and with P. chlororaphis inoculation and with UV irradiation; Group 5, lettuce with a mixture of clay granules and glass beads; Group 6, lettuce with a mixture of clay granules and glass beads and with P. chlororaphis inoculation; Group 7, lettuce with a mixture of clay granules and glass beads and with

UV irradiation; and Group 8, lettuce with a mixture of clay granules and glass beads and with P. chlororaphis inoculation and UV irradiation. After 9 weeks, the suspension of P. ultimum was added to all groups. Each groups had 8 pots to grow lettuce and all experiments were repeated 3 times.

22

2.3.3. Pseudomonas chlororaphis inoculation

P. chlororaphis (ATCC 9446) was cultured in Tryptic Soy broth (TSB) (Becton

Dickinson, Sparks, MD, USA) for 24 hours at 28°C with shaking (180 rpm). The culture was then centrifuged at 8,000 rpm for 10 min and the bacterial pellet was re-suspended in deionized (DI) water (Milli-Q, Millipore, Billerica, MA, USA). The concentration of P. chlororaphis was measured using a cell density meter (WPA Biowave, Biochrom,

Cambridge, UK) and was confirmed using plate counts. In order to determine the optimal concentration of P. chlororaphis for inoculation of the lettuce, the bacteria suspension was diluted to the levels of 107, 108, and 109 CFU/ml using DI water and then was added near the lettuce roots and onto the clay granules and glass beads. Based on the results of color measurements (as stated in below paragraph), the optimal concentration for P. chlororaphis inoculation was 1.0 x 108 CFU/ml as there was a significant difference between the 1.0 x 107 CFU/ml and 1.0 x 108 CFU/ml treatment groups (p<0.05), but no significant difference between the 1.0 x 108 CFU/ml and 1.0 x 109 CFU/ml treatment groups (p>0.05). Therefore, in all the experiments, 1.0 x 108 CFU/ml of P. chlororaphis was added to each pot 2 weeks after germination. P. chlororaphis of the same concentration was inoculated 3 times once a week in each pot, and then the inoculated lettuce was grown in the windowfarm systems for additional 5 weeks to develop

23 colonization of P. chlororaphis near the roots and to determine the overall effects of P. chlororaphis treatment.

2.3.4. Pythium ultimum inoculation

A strain of P. ultimum Trow (ATCC 96195) was provided by the laboratory of Dr.

Brian B. McSpadden Gardener (Department of at The Ohio State

University). P. ultimum was cultured in Potato Dextrose (PD) broth or PD agar (Becton

Dickinson, Sparks, MD, USA) and grown for 1 week at 24°C with shaking (150 rpm).

The broth was centrifuged at 8,000 rpm for 10 min, and the bacterial pellet was re- suspended in DI water (Milli-Q, Millipore, Billerica, MA, USA). A propagule suspension in DI water of P. ultimum was adjusted to 105 zoospores per ml based on hemocytometer counts. The suspension of P. ultimum was added twice a week near the lettuce roots and onto the clay granules and glass beads 7 weeks afterP. chlororaphis inoculation. The P. ultimum inoculated lettuce was grown in the windowfarm systems for additional 2 weeks.

24

2.3.5. UV-C irradiation

A 254 nm UV system was purchased from Atlantic Ultraviolet Co. (Hauppauge,

NY, USA). The average intensity of UV (4800 μW/cm2) was determined using a UV-C meter (UV512C, Digital UV-C Meter, General Tools, New York, NY, USA). The UV-C system was placed into the water reservoir for 5 minutes once a week (on days 13, 20, 27) at room temperature (~25 °C) to reduce the total microorganisms in the windowfarm systems. The approximate average UV dose per treatment was 50 mJ/cm2 (Bolton and

Linden, 2003).

2.3.6. Heterotrophic plate count

To monitor the concentration of bacteria present in the hydroponic system (clay granules, glass beads, water) and the efficiency of UV treatment, the levels of total bacteria were determined using a heterotrophic plate count method (American Public

Health Association 1992). 2g of beads were collected and suspended in DI water (Milli-Q,

Millipore, Billerica, MA, USA) with vortexing for 10 minutes. The solution was serially diluted (101 - 105) with phosphate buffered saline (PBS; Fisher Scientific, Fair Lawn, NJ,

USA) and the bacterial concentrations in both beads and water in the reservoirs were

25 determined using TSA media (Tryptic Soy Agar, Becton Dickinson, Sparks, MD, USA),

PCA media (Plate Count Agar, Becton Dickinson, Sparks, MD, USA) and Pseudomonas

Isolation Agar (Becton Dickinson, Sparks, MD, USA). All plates were aerobically incubated at 28°C for 24 hours. Colonies of P. chlororaphis were verified and counted under a UV lamp because P. chlororaphis produces pyoverdines which are yellow-green fluorescent pigments (Meyer. 1998; Yamaoka et al. 2000).

2.3.7. Food quality and productivity: Color, length, weight, number, and lettuce

leaf mortality

After 9 weeks, the color of the lettuce leaves was measured using a portable tristimulus colorimeter with an 8 mm diameter measuring area in the L*a*b* mode

(Konica Minolta CR 300 series Chroma Meters, Ramsey, NJ, USA). The values of L*

(lightness, white-black), a* (greenness, green-red) and b* (blue-yellow) at three selected areas on the lettuce leaves were measured. The hue angle, which is an important indicator of discoloration, was calculated using the following equation; Hue angle = Tan-1 (b*/a*)

(Castaner et al. 1996; Hosoda et al. 2000). The length and weight of lettuce were measured and the numbers of leaves per pot were counted. Lettuce that showed signs of withering and mortality of leaves or root rot was enumerated during the final 2 weeks to

26 evaluate the protection effect by P. chlororaphis against plant disease caused by P. ultimum inoculation.

2.3.8. Measurement of Chlorophyll a in the water tubing systems

As a measurement of algal biofilm growth in the hydroponic system, the concentration of chlorophyll a was measured for 48 days using water tubing pieces that were additionally added (4 tubes per system, 1 cm each). By installing a small section of by-pass water tubing to the main water circulating tubing, it was possible to collect biofilm samples on a regular basis without changing the water circulation in the hydroponic system. The tubing samples were placed into 30 mL sterile DI water (Milli-Q,

Millipore, Billerica, MA, USA), vortexed for 10 minutes, and the level of chlorophyll a was quantified in vivo using the intact cells without filtration or extraction described in our previous study (Marion et al., 2012) using a two-channel handheld Aquaflour™ flourometer (Turner Designs®, Sunnyvale, CA). Chlorophyll a (excitation at 460 ± 20 nm, emission > 665 nm) was standardized (R2 = 99.9%) with liquid primary chlorophyll a standards (catalog number 10-850, Turner Designs®).

27

2.3.9. Statistical analysis

The experimental data including food quality and productivity after P. chlororaphis treatments (color tests, measurement of leaf length and weight, and number of leaves), and heterotrophic plate counts to observe microbial changes in the windowfarm systems were analyzed using SPSS 17.0 statistical software (SPSS Inc.,

Chicago, IL, USA). Analyses of variance (ANOVA) were performed to determine the differences between the means of the data in each independent experiment. Results were considered significant at p < 0.05.

2.4. Results

2.4.1. Evaluation of food quality and productivity of P. chlororaphis treated

lettuce.

After 9 weeks, the color (edible part), total length, weight, and number of leaves were measured to compare food quality and productivity among the 8 treatment groups.

The color parameters (L*, a*, b* and hue angle) of the lettuce with and without P. chlororaphis and UV treatments are presented in Table 3. When the lettuce was cultivated using different beads in the pots, clay granules or a mixture of clay granules 28 and glass beads, the beads did not significantly alter lettuce lightness or the greenness (- a*) (p > 0.05). The lightness of Groups 1 (clay granules) and Group 5 (a mixture of clay granules and glass beads) was 51.03±1.62 and 53.11±2.46 and ranged from 45.39 to

55.87 and 49.14 to 55.99, respectively. The greenness of the Group 1 was -25.68±0.88 and of the Group 5 was -25.44±0.58, and varied from -28.14 to -21.55 and -28.04 to -

21.96, respectively. In addition, UV irradiation of the water for 5 minutes did not affect the lightness (L*) or greenness of the lettuce (p > 0.05). However, P. chlororaphis treatment was found to significantly affect the lightness and greenness of the lettuce (p <

0.05) regardless of the kind of beads (Group 2, 4, 6, and 8 vs. Groups 1, 3, 5, and 7).

P. chlororaphis treatment significantly affected the number of edible lettuce leaves (p <

0.05). The average number of lettuce leaves in the P. chlororaphis treated groups was about 17 and ranged from 15 to 18, whereas the average number of lettuce leaves without

P. chlororaphis treatment was about 9, ranging from 8 to12 (Figure 8).

The average length and weight of the lettuce were significantly affected by P. chlororaphis treatment as well, regardless of UV irradiation (Figure 9). The average length of the P. chlororaphis treated lettuce group was 27.3 cm and ranged from 24.8 to

29.1 cm, whereas the average length of the untreated lettuce was 20.9 cm ranging from

18.1 to 24.5 cm. The average weight of the P. chlororaphis treated lettuce group was

31.86 g, and ranged from 28.50 g to 36.10 g, however the untreated lettuce weighed an average of 27.38 g (23.0 - 32.8 g). 29

In order to evaluate the ability of P. chlororaphis to protect the plant from damage by P. ultimum infection, survival of the lettuce was assessed by observing the severity of root rot and withering and mortality of leaves during the final two weeks

(Table 4). All non-treated lettuce died, but 54% of the P. chlororaphis treated lettuce survived. Those surviving lettuce did not show any symptoms of root rot, withering or mortality of leaves.

2.4.2. Effect of UV irradiation on the windowfarm system

In order to find an optimal dose UV for minimizing biofilm build up while sustaining P. chlororaphis in the system, the concentration of bacteria in the water reservoir after UV irradiation was measured (Figure 10). After 5 min of UV irradiation, an immediate 5-log reduction in the total number of CFU/ml was observed in the water reservoir, and ~ 2 log CFU/mL remained in the water reservoir.

The total effect of UV irradiation on bacteria levels in the water reservoirs and on the beads were measured using the heterotrophic plate count method every other day and

UV irradiation of the water reservoir for 5 minutes on days 13, 20, and 27 (Figure 11).

UV irradiation significantly affected the number of bacteria in the water reservoir immediately after the irradiation (p<0.05), but the number of bacteria on the beads in each pot did not change (p>0.05). The initial average concentration of bacteria in the 30 water reservoir was 1.07 x 107 CFU/ml however, after each UV treatment of the water reservoirs, a 5-log reduction was observed (Figure 11a). The bacteria in the water reservoir re-grew to the initial non-irradiated concentration within two days of the post irradiation. The level of bacteria on the beads remained at 4.20 x 107 CFU/g bead, regardless UV irradiation (Figure 11b).

During the lettuce growing period, chlorophyll a in the water tubing system was measured in the UV irradiated and non-UV irradiated groups to examine the effect of UV on biofilm build-up (Figure 12). The final average chlorophyll a concentration was 1.81

μg/L/cm2 and 0.126 ng/L/cm2 (14,000 times reduction) in the non-UV irradiated systems and UV irradiated systems (p < 0.05), respectively.

2.5. Discussion

Root rot by Pythium spp. threatens the productivity of crops in different kinds of hydroponic systems (Owen-Going et al. 2003). Various Pythium spp. are frequently found contaminating plants in hydroponic systems (Khan et al. 2003). Mild temperatures, high concentrations of nutrients in water, and high humidity make P. ultimum extremely virulent under hydroponic conditions resulting in destruction of crops in the water circulation systems within few days (Ch rif et al. 1997; Vallance et al. 2012). In order to prevent Pythium spp. infection, several methods have been used such as a diversity of 31 filtering systems, chemical treatments, or biological management tactics (Compant et al.

2005; Ellis et al. 1999; Liu et al. 2007). Plant root-colonizing Pseudomonas spp., which are plant growth-promoting rhizobacteria (PGPR), can accelerate the growth of healthy plants through the production of , antimicrobial metabolites, and plant hormones (Dowling and O'Gara 1994; Jiao et al. 2013; Utkhede et al. 2000). Most of the previous studies have employed biocontrol using Pseudomonas spp. against various plant pathogens, particularly Fusarium spp., that produce phenazine-1-carboxamide (PCN)

(Chin-A-Woeng et al. 2001). P. chlororaphis PCL1391 can reduce root rot caused by

Fusarium oxysporum (Chin-A-Woeng et al. 2000) and Khan et al. (2003) showed that P. chlororaphis can prevent root browning caused by pathogens in hydroponic systems.

Results from the current study demonstrate that P. chlororaphis will not only prevent root rot and reduce P. ultimum infection, but will also promote the color, number of leaves, height and fresh mass of the lettuce. Various treatments were examined including clay granules, glass beads, P. chlororaphis treatment, and UV irradiation to test the efficiency of different management conditions for growth of romaine lettuce at room temperature in our windowfarm system. P. chlororaphis treatment alone induced a color change (increased lightness and greenness) on the lettuce regardless of the use of different beads or UV irradiation. P. chlororaphis also enhanced the number, length, and weight of leaves vs. the non-treated lettuce. Furthermore, according to our survival data,

32

P. chlororaphis treatment reduced lettuce mortality associated with Pythium ultimum infection.

UV irradiation has been used to destroy microbes in air, water, and surfaces. It is also used for food safety following approval of its use by the Food and Drug Administration

(FDA, USA). UV light in the range of 250-260 nm is effective in reducing viability of microorganisms such as viruses, bacteria, fungi (Bintsis et al. 2000). Most of the previous studies concerning UV irradiation focused on food, water or surface disinfection to reduce the level of microorganisms (Guerrero-Beltrán and Barbosa-Cánovas, 2004). In addition, UV irradiation has been used to eliminate or reduce pathogens in order to prevent root disease, which is a major concern in closed hydroponic systems (Zhang et al.

2000).

Biofilm build-up is another major problem found in hydroponic water circulation systems. Biofilm build-up because of high nutrient concentration in the hydroponic water causes nuisance and reduces the lifespan of the systems. Therefore, it is necessary to clean or replace as biofilm can inhibit the flow of water to the plants.

In order to minimize biofilm build-up while maximizing the effect of P. chlororaphis in the windowfarm systems, the UV irradiation was limited to 5 minutes of exposure at three separate intervals. Unlike the results found in the water reservoirs (> 5 log reduction,

Figure 10), the concentration of the beneficial bacteria on the beads was unaffected after

UV irradiation. Consequently, the UV irradiation of the water reservoirs can temporarily 33 decrease the total load of the system, while maintaining the desired concentration of P. chlororaphis near the roots. Although the bacterial population in the water reservoir decreased only temporarily, we found that the concentration of chlorophyll a on the inner surface of the water tubing was greatly reduced in the UV irradiated systems. Borderie et al. (2011) demonstrated that UV-C irradiation has detrimental effects on algal growth and photosynthetic activity, but the ability of algal regrowth and repairing the UV-C damage is unknown. Although we saw fast regrowth of the heterotrophic bacterial cells post UV-C irradiation in the water reservoir due to the nutrient and carbon availability, the same phenomenon was not observed for the algae in the tubing system.

In conclusion, P. chlororaphis treatment of romaine lettuce in the windowfarm system significantly enhanced growth (the number, weight, and height of leaves) and quality (lightness and greenness) of lettuce. In addition, P. chlororaphis treatment was able to significantly reduce the consequences of P. ultimum infection in lettuce. UV-C irradiation temporarily reduced the concentration of bacteria in the water reservoir and caused reduction in the level of chlorophyll a on the inner surface of water tubing systems, but it did not affect the concentration of bacteria on the beads in the pots. As a result, it was possible to sustain the concentration of bacteria on the beads and maintain their beneficial growth promoting properties on lettuce, while minimizing algal growth in the water circulation system. 34

The windowfarm system can be made of very simple household materials such as plastic bottles, water containers, and a small scale water pump with tubing. It can grow plants efficiently with minimal space, water, and nutrient solution while controlling plant disease and pests. In addition, having these windowfarm systems can provide enjoyment and education opportunities in classrooms as well as providing healthy food. For those areas with food supply challenges, contaminated soil, and water shortages, these systems can provide healthy emergency food.

35

Chapter 3: Comparison of bacterial profiles of root area and nutrient solution in the windowfarm using DGGE and banding pattern analysis

3.1. Abstract

Microorganisms play an important role in plant growth and prevention of plant disease in cultured hydroponic systems. The objectives of this study were to: 1) examine bacterial diversity and community in hydroponic systems; 2) compare the effect of three different conditions in root areas using the PCR-DGGE method; and 3) identify bacteria in root areas and water in a windowfarm system. Bacterial profiles of three different conditions (bead treatment, P. chlororaphis treatment, and UV irradiation) in root area and reservoir water were compared using PCR-DGGE and banding pattern analysis.

After cultivation of lettuce in windowfarm systems, soil and water samples were collected and bacterial community DNA was extracted for analysis. The DGGE bands were analyzed and interesting bands were selected for bacterial identification. The results demonstrated that 1) P. chlororaphis had positive effects on lettuce’s growth and root development; 2) the bacterial community of the root area was affected significantly by beads (clay pelltes versus glass beads with clay pelltes) and P. chlororaphis treatments;

3) Beads and P. chlororaphis treatments did not make the change of bacterial diversity

36 around root area in the windowfarm system. Four bacteria (Variovorax paradoxus,

Pseudomonas fluorescens, Pseudomonas thivervalensis, and Pseudomonas brassicacearum), beneficial for plant growth, were found. These results provide a better understanding of bacterial activities in the root area and water in hydroponic culture systems and how plants and microorganisms interact.

3.2. Introduction

With the development of materials, tools, and equipment, various hydroponic systems have been expanded for use in disciplines such as biology, agriculture, , education, and personal gardening (Resh, 2013). Because of the many advantages of hydroponic systems, such as reuse of water and nutrients, ease of environmental control, and prevention of soilborne diseases and pests, these hydroponic systems can help in the cultivation of various crops (Jones, 1997). However, waterborne organisms can contaminate and rapidly spread through the water tubing of hydroponic systems (Lee et al. 2014). When the systems are infected by pathogens, they can rapidly disseminate through the water tubing systems and contaminate the entire crop of fresh produce (Koseki et al. 2011; Stanghellini et al. 1996). Plant pathogens can easily grow in the presence of the high concentrations of nutrients that are used and may cause severe

37 disease outbreaks (Khan et al. 2003). Root rot infection is a significant problem and is often caused by fungal or bacterial pathogens and oomycetes (Nonomura et al. 2003).

Consequently, many past studies have focused on preventing fungal infection or developing remedial agents under hydroponic conditions. For instance, a slow filtration system was developed to eliminate Phytophthora cinnamomi (van Os et al. 1999), while others have shown that oxygen treatment in tomato plants reduced Pythium root rot

(Chérif et al., 1997).

In a previous windowfarm study, we showed that the introduction of P. chlororaphis into a windowfarm system improved the quality and quantity of lettuce and reduced the damage caused by the plant pathogen P. ultimum (Lee et al. 2014).

Biological control with beneficial bacteria, such as Pseudomonas chlororaphis or

Bacillus polymyxa, can suppress the growth of plant disease by producing and/or substances (Walker et al. 1998), thereby creating a hostile environment for the pathogen and promoting crop growth and improving yields (Zheng et al., 2000). However, it is often difficult to determine how deliberate bacterial treatment of the soil affects existing microflora (Gul et al., 2012; Peer and Schippers, 1989;

Schönwitz and Ziegler, 1986). Despite multiple studies over several decades, there is still a dearth of information about the effect of microflora and microbial ecology in the , although microbial communities are associated with both healthy and

38 infected roots. In addition, the medium in hydroponic systems, (e.g. sawdust, charcoal, ceramics, sawdust, rockwool, coco coir, clay pellet, or gravel) can affect the population of microorganisms in the rhizosphere soil because each media has different properties and effects on roots and microflora (Resh, 2012).

In a previous study, we developed a windowfarm system and cultivated lettuce using this system (Figure 13). Although addition of beads around roots showed little effect on lettuce productivity, a P. chlororaphis inoculation increased production significantly and also reduced P. ultimum infections (Figure 14). Based on these results, we examined those mechanisms surrounding root area inoculation of P. chlororaphis, including interaction with other bacteria (e.g. change in bacterial diversity, bacterial community strtucture), using PCR-DGGE analysis. The present study investigated eight different groups of plants, based on three different conditions: 1) different types of beads (clay pellets versus clay pellets with glass beads); 2) with and without treatment with

Pseudomonas chlororaphis; and 3) with and without ultraviolet irradiation. The objectives of the current study was to examine how these different conditions affect bacterial diversity, the community of the root area, and the water in windowfarm systems.

We use PCR-DGGE and banding pattern analysis to obtain information on both culturable and nonculturable bacteria (Su et al., 2013). This research was designed to

39 demystify microbial diversity and community structure and help clarify some of the interactions between microorganisms and plants in hydroponic systems.

3.3. Materials and Methods

3.3.1. Sample preparation

The windowfarm systems were made for growing romaine lettuce (Figure 13).

Figure 15 shows a brief flowchart of the study design about the entire windowfarm experiments, including the initial lettuce production stages. The operation method of the windowfarm, the growing conditions, and the conditions of UV irradiation were described in detail previously (Lee et al. 2014). During the 11-week experimental period,

Romaine lettuce (Lactuca sativa L. var. longifolia) (Broomfield, CO, USA) was cultivated using a laboratory windowfarm system at The Ohio State University

(Columbus, OH, USA). Each pot was filled with either 80g of sterile clay pellets (10 mm in diameter) (Easy Green Hydroton, Georg Buchner, Eschborn, Germany) or 80g of a sterile 1:1 mixture of clay pellets (10 mm in diameter) and glass beads (5 mm in diameter) (Glass Beads Solid, Fisher Scientific Inc.).

There were 8 different groups per test condition: Group 1 (the control group) had lettuce with clay pellets without either P. chlororaphis inoculations or UV irradiation; 40

Group 2 had lettuce with clay pellets and P. chlororaphis inoculations; Group 3 had lettuce with clay pellets and UV irradiation at 5 minute intervals (all UV irradiations were for 5 minutes/week); Group 4 had lettuce with clay pellets, P. chlororaphis inoculations, and UV irradiation; Group 5 had lettuce with a mixture of clay pellets and glass beads;

Group 6 had lettuce with a mixture of clay pellets and glass beads and P. chlororaphis inoculations; Group 7 had lettuce with a mixture of clay pellets and glass beads and with

UV irradiation; and Group 8 had lettuce with a mixture of clay pellets and glass beads, with both P. chlororaphis inoculation and UV irradiation. After harvesting the lettuce, 16 different samples were collected, including eight bead samples from pots and eight water samples from water reservoirs using eight different windowfarm systems.

3.3.2. Bacterial Community DNA Extraction

10 grams of each bead sample were mixed with 100 ml of 1X phosphate buffered saline (PBS, Fisher Scientific, Waltham, MA). At room temperature, samples were shaken for 10 minutes using a vortex mixer (Barnstead/Thermolyne Maxi Mix II,

Dubuque, Iowa), then shacked for two hours in an incubator shaker (200 rpm) (New

Brunswick Scientific Co., Inc. Incubator Shaker I 2400, Edison, NJ), both at room temperature. After removing the beads, 50 ml PBS samples were prepared.

From all the water reservoirs, 100 ml water samples were collected. Out of the 100ml sample volume, 50 ml was prefiltered through a sterile 20 μm pore size nylon filter 41 membrane (Osmonics, Minnetonka, MN, USA) to remove large debris, and then filtered through a sterile 0.4 μm pore size polycarbonate membrane filters (Millipore, Billerica,

MA, USA). The membranes were transferred into a 2 ml sterile tube. 1.4 ml of ASL buffer in the QIAamp® DNA stool kit (Qiagen, Valencia, CA), and 0.1 mm and 0.5 mm diameter autoclaved glass beads (Biospec Products, Bartlesville, OK, USA) were added into the tubes. Bead-beating was performed using a Mini-Beadbeater-96 (Biospec

Products, Bartlesville, OK, USA) at 2,100 oscillations/min for 3 min. The supernatant was transferred and the remaining DNA extraction process was performed using a

QIAamp® DNA stool kit, according to the manufacturer’s instructions. The eluates were used immediately for the next procedure and subsequently stored at -80°C.

3.3.3. PCR Amplification of 16S rRNA Genes

The basic experimental design followed that of our previous research (Ge et al.,

2012). Two universal bacterial 16S rRNA gene primers, BA338f (5’-ACT CCT ACG

GGA GGC AGC AG-3’) and 518r (5’-ATT ACC GCG GCT GCT GG-3’), were used to amplify the 200 bp fragment of 16S rRNA genes of the V3-region. The PCR products had a DNA concentration of approximately 30 μg ml-1 DNA templates (Qubit

Fluorometer, Invitrogen, US). In addition, for the DGGE experiment, a 40 bp GC clamp was added to the 5’ end of primer BA338fG (5’-GCC CGC CGC GCG CGG CGG GCG 42

GGG CGG GGG CAC GGA CTC CTA CGG GAG GCA GCA G-3’). The total PCR reaction volume was 50 μl, which included 2.5 μl of bacterial DNA, 5 μl of 10 × PCR buffer (Qiagen, Valencia, CA), 10 μl of Q solution (PCR enhancer, Qiagen, Valencia,

CA), 0.5 μl of each forward and reverse primer from each 100 μM primer stock, 1.5 μl of

50 mM MgCl2, 1 μl of 10 mM dNTPs, 0.2 μl of Taq polymerase (Invitrogen, Carlsbad,

CA), and 28.8 μl of sterilized distilled water. PCR amplification was conducted using a

MutiGene Thermal Cycler (Labnet, Edison, NY) under the following conditions: 94°C for 3 minutes; 30 cycles of 92°C for 1 minute, 55°C for 30 s, and 72°C for 1 minute; and a final extension at 72°C for 10 minutes. The PCR product was confirmed by agarose gel

(1.0%) electrophoresis, and the gel was stained using ethidium bromide (1 μl·100·ml–1).

3.3.4. Denaturing Gradient Gel Electrophoresis (DGGE)

The amplified 16S rRNA gene PCR products were run in a DCode System (Bio-

Rad, Hercules, CA) with a density gradient gel consisting of 8% (w/v) polyacrylamide

(37.5:1), with a denaturing gradient ranging from 30% - 70%, at 60°C for 18 hours. The final gel images were captured using the Molecular Imager Gel Doc XR System (Bio-

Rad, Hercules, CA). The DGGE band profiles were compared using the BioNumerics software (version 7.10, BIOSYSTEMATICA, Tavistock, Devon, UK) as described by

Hovda et al (2007). Reference samples were loaded in the first and last lanes of the 43

DGGE gel. The obtained DGGE pattern images were subsequently normalized to correct any gel smiling or uneven band migration by comparison with two external reference lanes. The DGGE banding profiles were done with Pearson’s coefficient and the

Neighbor-Joining algorithm.

3.3.5. DGGE gel data analyses

The DGGE bands were selected using the band-searching algorithm of

BioNumerics software (version 7.10, Biosystematica, Tavistock, Devon, UK). After normalization of the gel images, only those bands with a peak height intensity exceeding

2.0% of the strongest band in each lane (lane 1 to 16) were selected for further analyses.

DGGE band data were used to examine biological diversity using two equations: the

Shannon-Wiener index, H = -ΣPi ln(Pi) (Shannon and Weaver, 1963; Yu and Morrison,

2004), and the Evenness index, E = H´/lnS (Pielou, 1966; Yu and Morrison, 2004). Each band was treated as an individual operational taxonomic unit (OTU). S (richness) is the total number of DGGE bands in each lane, and was also used to indicate the number of species. Pi is the probability of the bands in each lane, calculated from ni/N; ni is the peak height of a band and N is the sum of all peak heights in the curve of a given sample (N).

The experimental data of the microbial diversity indices (Table 5) were analyzed using SPSS 17.0 statistical software (SPSS Inc., Chicago, IL, USA). The comparison of 44 diversity indices between each group was performed using an independent two samples t- test. Results were considered significant at p < 0.05.

3.3.6. Sequencing Analysis of the Selected DGGE Bands

The unique bands were excised from the gel using a sterile blade under UV transillumination and the piece of gel was placed into 30 μl of nuclease-free water at 4°C for 24 hours. 3 μl of the supernatant were used as the template for PCR for re- amplification under the same conditions described earlier. After confirmation with agarose gel (1.0%) electrophoresis, the PCR products were purified with a QIA quick

PCR purification kit (Qiagen, Valencia, CA). They were further sequenced using an ABI

Prism 3730 DNA analyzer (Applied Biosystems, Foster City, CA) in the Plant-Microbes

Genomics Facility at The Ohio State University (http://pmgf.biosci.ohio-state.edu). The sequences were compiled with a Sequence Scanner v1.0 (Applied Biosystems, Foster

City, CA) to check for accuracy, and their taxonomic identification was determined by comparison with reference sequences in the GenBank database, using the BLAST search program.

45

3.4. Results

3.4.1. Bacterial Species Diversity

Figure 16 shows the DGGE gel pattern of the bacterial community from the beads and water samples using the 30% to 70% denaturant range and the 34 bands selected for bacterial identification. The diversity indices obtained from the PCR-DGGE banding profiles are shown in Table 5. The average amount of richness (R) of the clay pellet samples was about 24.8±2.63, while that of the mixture samples (clay pellets and glass beads) was about 24.8±2.22. The average of the Shannon-Wiener index (H) of the clay pellet groups was 3.03±0.08, and the mixture groups’ average was 3.02±0.14. The

Evenness scores (E) of the clay pellet samples was 0.95±0.02, and the E of the mixture samples was 0.94±0.02. The bacterial diversity around the root was not significantly different between the clay pellet groups and the mixture groups.

However, the Evenness index was significantly different between the non-treated and P. chlororaphis treated groups (p<0.05), even though the richness and Shanon-

Wiener index were not significantly different between the groups. The average richness

(S) of the non-treated and P. chlororaphis treated groups was 25.0±2.45 and 24.5±0.38, respectively. The average of the Shannon-Wiener index of the untreated and treated groups was 3.003±0.13 and 3.04±0.10, respectively. 46

UV-C irradiation (254nm with intensity of 4800 μW/cm2; UV dose per treatment:

50 mJ/cm2) was applied to the water reservoir of the windowfarm system for 5 minutes once a week at room temperature (~25 °C) to prevent biofilm build-up in the tubing system. The microbial diversity was not significantly different between the non-UV and the UV irradiation groups. The non-irradiated groups’ average richness (S), Shannon-

Wiener index, and Evenness index were 26.5±4.20, 3.09±0.20, and 0.95±0.02, respectively. The irradiated groups’ average richness (S), Shannon-Wiener index, and

Evenness index were 26.8±3.59, 3.16±0.15, and 0.97±0.0126, respectively.

3.4.2. Bacterial Community Structure

DGGE band pattern was analyzed for bacteria community investigation in each group using the Bionumerics software. The similarity results of the root area among different groups are summarized in Figure 17, in the form of a band-comparison dendrogram. The similarity between the different types of bead samples (clay pellet groups versus groups with a mixture of clay pellets and glass beads) was significantly different (Figure 17 (b)). Bands of each clay pellet treated group (lanes 3 & 4) and bands of each of the mixture treated groups (lanes 5 & 8) were tightly clustered; the similarity index in the bacterial DGGE gel between groups 1 (lane 4) and 3 (lane 3) was 80%, and

47 the similarity between groups 5 (lane 8) and 7 (lane 5) was 84%. P. chlororaphis treatment also affected the development of the microbial community around the root area

(Figure 17 (c)). The similarity of groups 3 (lane 3) & 7 (lane 5) was 92%, and the similarity of groups 4 (lane 1) & 8 (lane 7) was 94%.

The results of similarity in the water samples seem to follow the bead sample pattern (Figure 18). The bacterial community of each treated clay pellet group and each treated mixture group was similar. The similarity of the treated clay pellet groups was

80% and the similarity of the treated mixture groups was 83%. Also, the overall similarity of P. chlororaphis treated groups was 94%, and the similarity of non-P. chlororaphis treated groups was 97%. In contrast, the banding pattern analysis shows that

UV irradiation in the water reservoirs does not change the formation of a bacteria community around the root area and in the water (Figures 17 and 18). The banding profiles between UV irradiation groups and non-UV irradiation groups were not distinctly clustered and no significant clustering phenomenon was observed.

3.4.3. Bacterial Identification

To determine bacterial taxonomic identification, 82 bands were directly selected from the DGGE gel and were sequenced. Sequences were analyzed by comparing the samples with the most closely related bacterial 16S rRNA sequences from the National 48

Center for Biotechnology Information (NCBI) database. From the analyzed samples from the excised bands, 34 samples had sequences showing high similarity (≥95%) with the reference sequences, and the results are summarized in Table 6. From the root area, various bacteria were detected (Figure 16): Rhodanobacter spathiphylli (band 1),

Variovorax paradoxus (band 2), Simplicispira limi (band 3), Pseudomonas chlororaphis

(band 5), Variovorax boronicumulans (band 6), Giesbergeria sinuosa (band 7),

Simplicispira psychrophila (band 8), Variovorax defluvii (band 9), Acidovorax avenae

(band 10), Mycobacterium kumamotonense (band 14), Mycobacterium fortuitum (band

16), Mycobacterium senegalense (band 17), Mycobacterium abscessus (band 18),

Pseudomonas fluorescens (band 20), Pseudomonas asturiensis (band 21),

Phenylobacterium conjunctum (band 23), and Roseomonas riguiloci (band 27).

The bacteria from the water reservoirs were (Figure 16): Pseudomonas brassicacearum (band 4), Rhodoferax ferrireducens (band 11), Sulfurimonas autotrophica

(band 12), Wolinella succinogenes (band 13), Acidovorax ebreus (band 15), Sulfurimonas paralvinellae (band 19), Pseudomonas fragi (band 22), Nocardia brasiliensis (band 24),

Rhodococcus kunmingensis (band 25), Mycobacterium hiberniae (band 26), Pedobacter duraquae (band 28), Pedobacter westerhofensis (band 29), Arthrobacter phenanthrenivorans (band 30), Pedobacter metabolipauper (band 31), Arthrobacter arilaitensis (band 32), Beijerinckia indica (band 33), and Pseudomonas thivervalensis

(band 34). 49

3.5. Discussion

The PCR-DGGE analysis is a reproducible and effective method to determine microbial diversity and community from similar or different samples (Gelsomino et al.

1999). Specifically, PCR-DGGE analysis is used to examine the effect of plant growth- promoting rhizobacteria (PGPR) on microbial diversity and community (Lauber et al.

2009). Even though the mechanism of PGPR has not been fully examined, PGPR are used in various ways to promote plant growth and to protect or reduce plant pathogens

(García et al. 2004). P. chlororaphis, which is a non-pathogenic rhizobacterium, has plant growth-promoting characteristics such as inducing systemic resistance against plant disease, production of indole-3-acetic acid, siderophores, and ability for phosphate solubilization (Kim et al. 2004; Roesti et al. 2006). P. chlororaphis PCL1391 treatment in tomatoes protects against root rot caused by Fusarium oxysporum by making phenazine-1-carboxamide (PCN) that is a control agent for the disease (Chin-A-Woeng et al. 2000). In addition, P. chlororaphis treatment around the root area has been shown to promote plant growth and reduce damage from the plant pathogen, Pythium ultimum (Lee et al. 2014). However, Piromyou et al. (2011) revealed that, in the soil, dominant species of the microbial community, such as Pseudomonas spp. or Bacillus spp, were not affected by introduction of PGPR.

50

Even though the development of lettuce root between treated clay pellet groups and treated mixture groups was not different (Figure 14 (c) and (d)), the roots of the P. chlororaphis treated groups were more highly developed than the non- treated groups

(Figure 14 (c) and (e)). However, in spite of three different treatments, the bacterial diversity around the root area was not significantly different (p > 0.05), with the exception of the Evenness index (E) between P. chlororaphis non-treated and treated groups. Although these conditions may provide different environments around the root area for bacterial growth in hydroponic systems, bacterial diversity around the root area may not be significantly affected by different types of supporting materials (e.g. clay pellets vs. mixture of clay pellets and glass beads), P. chlororaphis inoculation, and/or

UV irradiation.

P. chlororaphis treatment and different bead usage affected bacterial community structure in the root area (Figure 17). The banding pattern of P. chlororaphis treated groups was clustered together, and that of non-P. chlororaphis treated groups was closely related. However, UV irradiation of the water reservoir did not affect the bacterial community around the root area.

Ultraviolet (UV) irradiation has been applied to disinfect water because it is an effective treatment to inactivate microorganisms (Li et al. 2010). In hydroponic systems, biofilm build-up, which is the product of adhesion and development of microorganisms on surfaces, is a problem awaiting a solution (Schwartz et al., 2003). Hydroponic industry 51 may have to change parts of their hydroponic systems because massive production of biofilms in the water tubing hinders operation of the system (Resh, 2012). In order to minimize biofilm build-up in our windowfarm system, 254 nm UV-C irradiation

(intensity: 4800 μW/cm2, UV dose per treatment: 50 mJ/cm2) was delivered and optimized the exposre (5 minutes per week). Consequently, UV irradiation of the water reservoirs can temporarily decrease (> 5 log reduction) the total microorganism of the system (Lee et al. 2014). However, in this study, UV irradiation did not affect the bacterial diversity of water reservoirs, or the bacterial community structure in water.

Bacterial diversity was not significantly different between the non-UV and the UV irradiation groups (p > 0.05). Use of beads treatment or P. chlororaphis treatment had a much stronger effect on bacterial community change in water than did UV irradiation.

3.5.1. Bacterial Identification

Several species found in this study were plant growth promoting rhizobacteria

(PGPR): Variovorax paradoxus, Pseudomonas fluorescens, Pseudomonas thivervalensis, and Pseudomonas brassicacearum. Variovorax paradoxus was cadmium-tolerant and culturable bacteria were associated with the roots of young seedlings. V. paradoxus has a root length-promoting effect due to their ability to produce 1-aminocyclopropane-1-

52 carboxylate (ACC) deaminase, the immediate precursor of the plant hormone ethylene

(Belimov et al. 2005). As V. paradoxus strains can reduce ACC concentration in plant roots by ACC deaminase, it can possibly promote root elongation and nodule formation

(Maimaiti et al. 2007). Pseudomonas fluorescens, which is an ubiquitous bacterium on soil, water, and plant surfaces, is an excellent biological control organism because it can produce antibiotics for suppression of plant disease and also other secondary metabolites, such as pyrrolnitrin, 2,4-diacetylphloroglucinol, hydrogen cyanide, and siderophores

(Paulsen et al. 2005). P. fluorescens produce defense enzymes and contribute to prevention of root rot caused by Fusarium oxysporum in tomato roots (Ramamoorthy et al. 2002). Pseudomonas thivervalensis and Pseudomonas brassicacearum, which are endophytic bacteria, increase germination rates and crop growth. Pseudomonas thivervalensis develops root and shoot growth, and increases total biomass in (Persello-Cartieaux et al. 2001). P. brassicacearum has both antifungal and phytotoxic activities against fungal diseases (Chung et al. 2008). P. brassicacearum

MA250, is a biocontrol agent against pink snow mold (Microdochium nivale) in wheat and shows good effects on control of seed-borne Microdochium and Fusarium spp

(Levenfors et al., 2008). Plant pathogenic bacteria were found in our samples, such as

Acidovorax avenae which is a serious seed-borne pathogen in the world and the causal agent of a watermelon and melon seedling blight and fruit blotch (Schaad et al. 2008).

53

Chapter 4: Conclusions

First, as development of the windowfarm system using simple materials, the system can provide enjoyment and education opportunities in classrooms as well as providing healthy food. For those areas with food supply challenges, contaminated soil, and water shortages, these systems can provide healthy emergency food.

Secondly, this research demonstrated that P. chlororaphis treatment of romaine lettuce in the windowfarm system significantly enhanced growth and quality of lettuce. P. chlororaphis treatment was able to significantly reduce the consequences of P. ultimum infection in lettuce. UV-C irradiation temporarily reduced the concentration of bacteria in the water reservoir and caused reduction in the level of chlorophyll a on the inner surface of water tubing systems, but it did not affect the concentration of bacteria on the beads in the pots. As a result, it was possible to sustain the concentration of bacteria on the beads and maintain their beneficial growth promoting properties on lettuce, while minimizing algal growth in the water circulation system.

Third, in spite of three different treatments, the bacterial diversity around the root area was not significantly different (p > 0.05). However, P. chlororaphis treatment and different bead usage affected bacterial community structure in the root area. The banding pattern of P. chlororaphis treated groups was clustered together, and that of non-P.

54 chlororaphis treated groups was closely related. However, UV irradiation of the water reservoir did not affect the bacterial community around the root area.

Lastly, several species found in this study were plant growth promoting rhizobacteria (PGPR): Variovorax paradoxus, Pseudomonas fluorescens, Pseudomonas thivervalensis, and Pseudomonas brassicacearum. Also, plant pathogenic bacteria were found in our samples, such as Acidovorax avenae which is a serious seed-borne pathogen in the world and the causal agent of a watermelon and melon seedling blight and fruit blotch.

55

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Appendix A

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Table 1. The advantages of hydroponic systems compare to soil culture and their limitations.

Issues Hydroponic system Soil Culture Reference Unaffected by soil and external factors Gibeaut et al. 1997; Unsuitable if soil is contaminated with heavy metal and plant Land usage and Indoor system; Easy nutrient control; supplement lighting is efficient Jones, 1997; Norén disease; Limited by nutrients in soil; installation of supplement light effect of per unit area; control of the environment such as temperature, et al. 2004; is expensive; hard to control external environments; sensitive to environment humidity and lighting time; not sensitive to climate change, Norström et al. 2004 climate change; cultivation all year round is limited in certain areas cultivation all year round everywhere Labor Jovicich et al. 2003 Traditional practices are largely eliminated; no weeds Cultivating, weeding, watering, tilling and additional practices

Sanitation Easy handling of medium and all materials and maintaining sanitary Difficult to sanitize soil and equipment; hard to maintain sanitation Knutson, 2000 conditions conditions consistently Diseases and pest No soil-borne diseases; easy to control insects and animals; no Soil-borne diseases; hard to control insects and animals (loss of crop Jones, 1997; Zlnnen, pesticide damage yield) 1988 Efficient water usage; water can be recycled or reused; no nutrient Güohler et al. 1989; Inefficient water usage; water cannot be recycled or reused; 7 Water waste due to water runoff; Water goes directly to root areas; Midmore and Deng- 9 eutrophicastion of the environment due to run-off; hard to control possibility of controlling water-holding ability by using different lin 1999 water-holding capacity kinds of medium Fertilizers and Uneven distribution to crops (partial deficiency); often use of Resh, 2013; Rolot Even distribution to crops; efficient use of fertilizers and saving the nutrient solution excessive amount of nutrient ; high variation, hard to control pH and 1999 cost; easy control of pH and amount of nutrient amount of nutrient Unstable and uneven amount of production due to pests/soilborne Cornish, 1992; Stable and even amount of production; tomato,60-300 tons per acre; Quantity and pathogens; tomato, 5-10 tons per acre; cucumber, 7,000 lb per acre; Resh, 2012; Rolot cucumber, 28,000 lb per acre; lettuce, 21,000 lb per acre; bean, 21 Quality of crop lettuce, 9,000 lb per acre; bean, 5 tons per acre; uneven quality of 1999; Sarooshi and tons per acre; even quality of production production Cresswell, 1994  High initial setup cost for supplies and continuous replacement cost for maintaining Domingues et al.  Vulnerable to power outage leading to problems in water or nutrient supply, and witheredness 2012; Guo et al.  Easy spread of phytopathogens throughout water tubing systems 2002; Knutson, Limitations  Requirement of experts to maintain the systems 2000; Resh, 2013;  Needs of nutrients background to controlling amounts of nutrients Schnitzler, 2004;  Biofilm build-up in the system interfering nutrient uptake and reducing life span of the system Sutton et al. 2000; Zekki et al. 1996

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Table 2. Summary of test conditions of 8 different groups in each test

P. chloraphis Test group Bead type UVb treatmenta Group 1 No No

Group 2 Yes No Clay granules Group 3 No Yes Group 4 Yes Yes

Group 5 No No Group 6 Yes No

c Group 7 Mixture No Yes

Group 8 Yes Yes aP. chlororaphis was inoculated in designated pots (+). bUV irradiation of the water reservoir for 5 minutes every week (days 13, 20, and 27). cA mixture of clay granules and glass beads.

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Table 3. Color measurements (L*, a*, b* and hue angle) of the 9-week aged lettuce.

Groups Color

Bead UVb L* a* b* Hue angel Pca

1 0 0 51.03±1.62 -25.68±0.88 39.22±2.22 -56.8±0.09

Clay 2 + 0 44.21±2.15 -22.82±0.55 27.42±2.15 -50.2±0.05 granules 3 0 + 55.44±1.22 -26.13±2.35 43.78±2.36 -59.2±0.10

4 + + 43.06±1.20 -21.40±0.82 27.66±1.99 -52.3±0.06

5 0 0 53.11±2.46 -25.44±0.58 38.78±2.66 -56.7±0.10

6 + 0 42.77±2.00 -22.42±0.72 27.89±0.67 -51.2±0.09 Mixturec 7 0 + 56.08±1.98 -27.55±0.71 44.47±1.57 -58.2±0.06

8 + + 43.27±2.47 -22.07±0.99 29.71±1.93 -53.4±0.07 a P. chlororaphis was inoculated in designated pots (+). bUV irradiation of the water reservoir for 5 minutes every week (days 13, 20, and 27). c A mixture of clay granules and glass beads.

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Table 4. Death of lettuce 2 weeks after Pythium ultimum inoculation.

Group Death (%)

P. chlororaphis treated lettuce 54

Non-treated lettuce 100

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Table 5. Diversity indices calculated from the DGGE banding profiles (DGGE OT s richness (S), Shannon diversity index (H), and Evenness index (E)) generated from

DGGE fingerprints of microbial communities from bead samples. The comparison of diversity indices between each group was performed using an independent two samples t- test. There is no significantly difference between clay pellets and a mixture, non-P. chlororaphis treatment and P. chlororaphis treatment, or non-UV irradiation and UV irradiation, except for asterisk groups (*); evenness index between non-P. chlororaphis treated groups and P. chlororaphis treated groups was significantly difference (p < 0.05).

Bead sample group Richness (S) Shannon-Wiener index (H) Evenness index (E)

Clay pellets 24.8±2.63 3.03±0.08 0.95±0.02

Mixturea 24.8±2.22 3.02±0.14 0.94±0.02

Non-Pcb inoculation 25.0±2.45 3.00±0.13 0.94±0.02*

Pc inoculation 24.5±2.38 3.04±0.10 0.95±0.01*

Water sample group Richness (S) Shannon-Wiener index (H) Evenness index (E)

Non-UV irradiation 26.5±4.20 3.09±0.20 0.95±0.02

UV irradiation 26.8±3.59 3.16±0.15 0.97±0.01 a: mixture of clay pellets and glass beads (1:1 ratio) b: P. chlororaphis inoculation in root area

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Table 6. Identification of bands obtained by PCR-DGGE based on the V3 region of 16S rRNA and the closest sequence match of known bacteria in other references.

AccessionNo. Similarity Banda Identityb Potential source Reference (%)c 1 Rhodanobacter spathiphylli NR042434.1 97 Soils Kostka et al. 2012 2 Variovorax paradoxus NR074654.1 97 Soil Franzetti et al. 2012 3 Simplicispira limi NR043773.1 98 Water, active sludge Liu et al. 2012 4 Pseudomonas brassicacearum NR074834.1 99 Soil Ortet et al. 2011 5 Pseudomonas chlororaphis NR042939.1 96 Soil, water Dimkpa et al. 2011 6 Variovorax boronicumulans NR041588.1 98 Soil, water Liu et al. 2013 7 Giesbergeria sinuosa NR028711.1 95 Water, wasted water Lu et al. 2007 8 Simplicispira psychrophila NR028712.1 95 Active sludge Li et al. 2009 9 Variovorax defluvii NR109102.1 96 sewage Satola et al. 2013 10 Acidovorax avenae NR102856.1 97 Plant Burdman et al. 2005 11 Rhodoferax ferrireducens NR035948.1 96 Mud Shawkey et al. 2005 12 Sulfurimonas autotrophica NR074451.1 98 Water Inagaki et al. 2003 13 Wolinella succinogenes NR025942.1 96 Human Schröder et al. 1988 14 Mycobacterium kumamotonense NR041346.1 97 Human Masaki et al. 2006 15 Acidovorax ebreus NR074591.1 97 Sludge of water Huang et al. 2012 16 Mycobacterium fortuitum NR104775.1 96 Human Silcox et al. 1981 17 Mycobacterium senegalense NR042921.1 97 feces Hamid et al. 2002 18 Mycobacterium abscessus NR074427.1 95 water Wallace et al. 1996 19 Sulfurimonas paralvinellae NR041439.1 96 Inagaki et al. 2003 20 Pseudomonas fluorescens NR102835.1 99 Soil Lindsay et al. 2002 21 Pseudomonas asturiensis NR108461.1 96 Plant, Soil Gonz lez et al. 2013 22 Pseudomonas fragi NR024946.1 95 Soil Drapeau,,1980 23 Phenylobacterium conjunctum NR041963.1 96 Fresh water Abraham et al. 2008 24 Nocardia brasiliensis NR074743.1 95 Soil Wallace et al. 1995 25 Rhodococcus kunmingensis NR044034.1 96 Soil Wang et al. 2008 26 Mycobacterium hiberniae NR026092.1 97 Sludge McNabb et al. 2004 27 Roseomonas riguiloci NR109149.1 97 Wet land Baik et al. 2012 28 Pedobacter duraquae NR042601.1 99 Fresh water Muurholm et al. 2007 29 Pedobacter westerhofensis NR042602.1 95 Fresh water Muurholm et al. 2007 30 Arthrobacter phenanthrenivorans NR074770.1 96 soil Kallimanis et al. 2009 31 Pedobacter metabolipauper NR042603.1 96 Water Muurholm et al. 2007 32 Arthrobacter arilaitensis NR074608.1 97 Food Irlinger et al. 2005 33 Beijerinckia indica NR004554.1 95 sludge Vilcheze et al. 1994 34 Pseudomonas thivervalensis NR_024951.1 98 Soil, water Ross et al. 2000 aBand number as indicated on Figure 16 bClosest match to band sequence obtained by comparison with BLAST search. Numbers in parentheses indicate the

GenBank accession number cSimilarity was the ratio of identical sequence between the closest sequence from database entry and the band sequence, which was obtained after BLAST. 84

Appendix B

Figure 1. Trends in the total number of documents concerning hydroponic systems by year. The figure shows a time series plot of the number of hydroponic system papers since 1930s. The key words used for search [Scopus] were: hydroponics, hydroponic system, hydroponic food, and soilless culture. Journal articles and books were included.

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Figure 2. Proportion of documents related to hydroponic system by subject area over 76 year period from 1937 to 2013. The key words used for database search [Scopus] were: hydroponics, hydroponic system, hydroponic food, and soilless culture. Journal articles and books were included.

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Figure 3. The number of documents related to hydroponic systems by country from 1937 to 2013. The key words used for database searching [Scopus] were: hydroponics, hydroponic system, hydroponic food, and soilless culture. Journal articles and books were included.

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Figure 4. The percentage of major types of hydroponic crops found from publications.

The key words used for database searching [Scopus] were: hydroponics, hydroponic system, hydroponic food, and soilless culture. Journal articles and books were included.

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Figure 5. Six different types of traditional hydroponic systems. (a) wick system, (b) drip system, (c) Ebb-Flow system, (d) water culture system, (e) nutrient film technique, and

(f) aeroponic system.

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Figure 6. Schematic diagram of a single unit of a vertical windowfarm system (a) and a multiple-unit windowfarm system (b). Water in the water reservoir goes up through tubing to each pot using an air pump and watering time can be control by an electronic timer.

90

Figure 7. . Schematic diagram of a single unit of the windowfarm system (a) and a complete windowfarm system (b). Water in the water reservoir ① goes up through a tube

④ to each pot ③ using an air pump ②.

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Figure 8. Number of lettuce leaves in P. chlororaphis treated (+) and non-treated (-) lettuce groups after 9-weeks of planting in the windowfarm system.

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Figure 9. Crop productivity as measured by thelength (a) and weight (b) in 9-week aged lettuce with (+) and without (-) P. chlororaphis. Box plots showing means and medians of lettuce length and weight. The length of each box shows the interquartile range and 50% of cases of the variable. The line and the dot in the box indicate the mean and the median, respectively, while extended lines from the box show maximum and minimum values.

Comparing P. chlororaphis treated lettuce versus non-P. chlororaphis treated lettuce in their appearances of leaves and roots (c). 93

Figure 10. Concentration of bacteria in the water reservoir during 5-min exposure to UV irradiation. Bacterial levels were measured using heterotrophic plate counts to determine the optimal UV dose for minimizing biofilm build-up while maintaining P. chlororaphis in the windowfarm system.

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Figure 11. Effect of UV irradiation during lettuce growing period on concentration of heterotrophic bacteria in the water reservoirs (a) and on the beads around the lettuce roots

(b).

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Figure 12. Comparison of Chlorophyll a concentration on the inner surfaces of water tubing in the UV irradiated groups and the non-UV irradiated groups after the study period (a). Measurement of Chlorophyll a on the inner surfaces of water tubing in the non-UV irradiated groups by three day interval during the lettuce growing period (b).

Photos of both tubing in the UV irradiated groups and the non-UV irradiated groups after the study period (c).

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Figure 13. Schematic diagram of the windowfarm systems. Clay: clay pellets growing media, Class + glass: a mixture with clay pellets and glass beads (1:1 ratio),

Pseudomonas: P. chlororaphis inoculation around root, UV: UV irradiation in water reservoirs for 5 minutes every week.

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Figure 14. Comparing P. chlororaphis treated lettuce versus non-P. chlororaphis treated lettuce in their appearances of leaves and roots; (a) P. chlororaphis treated lettuce root with clay pellets, (b) non-P. chlororaphis treated lettuce root with clay pellets. (c) P. chlororaphis treated lettuce root with clay pellets; (d) P. chlororaphis treated lettuce root with a mixture of clay pellets and glass beads; (e) root of non-P. chlororaphis treated lettuce with clay pellets. Claya: clay pellets growing media, Mixtureb: a mixture with clay pellets and glass beads (1:1 ratio).

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9

9

Figure 15. Flowchart of the windowfarm experimental design. This chart describes the each step in the process of entire

windowfarm experiment based on three different conditions (beads, P. chlororaphis inoculation, and UV irradiation). Each

group had 24 replicates.

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Figure 16. 16S rRNA gene DGGE profile (bands marked with ● were selected for sequencing). The first set of 16S rRNA gene DGGE profile using 30% - 70% denaturant range (Lanes 1 to 8 are bead samples; Lanes 9 to 16 are water samples. Each lane describes in this figure (a: clay pellets growing media, b: a mixture of clay pellets and glass beads, c: P. cholororaphis treatment, d: UV irradiation). 100

Figure 17. . Banding pattern analysis of bacterial community around root area by band- search algorithm and band comparison among the different groups. (a) all bead samples;

(b) clay pellets versus a mixture of clay pellets and glass beads; (c) P. chlororaphis treated groups versus non-P. chlororaphis treated groups.

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Figure 18. Banding pattern analysis of bacterial community in water samples of the windowfarm system by band-search algorithm and band comparison among the different groups.

102