Integrated Use of Grafting Technology in Specialty Melon Production: Grafting Techniques, Root-Knot Nematode Management and Fruit Quality

INTEGRATED USE OF GRAFTING TECHNOLOGY IN SPECIALTY MELON PRODUCTION: GRAFTING TECHNIQUES, ROOT-KNOT NEMATODE MANAGEMENT AND FRUIT QUALITY

WENJING GUAN

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2014

To my husband, Yihai, for his unconditionally love and support

ACKNOWLEDGMENTS

I sincerely thank my major advisor, Dr. Xin Zhao, for financial support, and years of guidance on my research, and being a supportive mentor in the four years journey.

My appreciation also goes to my committee members, Drs. Donald W. Dickson, Donald

J. Huber, and Nicholas S. Dufault for their excellent suggestions that helped me continuously moving forward. I thank my husband, Yihai Wang, for his unconditionally love and support. I also feel very lucky to have a family who always stood behind me through both good and bad times.

I am very grateful of being in the Zhao lab and in the Horticultural Sciences

Department, from where I made many lifelong friends and received kindly help from colleagues. I would like to thank Desire Djidonou, Charles Barrett, and Libby Rens for their help, not just in research, but also in how to adapt to graduate student life when I struggled at the beginning. I also thank Jason Neumann, Zack Black, and Maggie

Goldman for their kind help with my projects and dissertation writing. Thanks to Yushen

Huang for helping me when I was overwhelmed.

My experiments could not have been done without technique support from Maria

Mendes, Michael Alligood, Buck Nelson, and the crew members at the Plant Science

Research and Education Unit. It was also impossible without help from Callie Cooper,

Tarik Eluri and Isaac Vincent. I extend my gratitude to all the departmental staff for the administrative support, especially to Curtis Smyder for his constant and patient assistances in all the aspects.

TABLE OF CONTENTS page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 8

LIST OF FIGURES ...... 11

ABSTRACT ...... 12

CHAPTER

1 LITERATURE REVIEW ...... 14

An Overview of the History of Cucurbit Grafting ...... 14 Grafting in Cucurbit Crop Production ...... 14 Understanding Rootstock and Scion Interactions ...... 20 Diseases Controlled by Grafting in Cucurbit Production ...... 24 Techniques for Melon Grafting ...... 27 Hole Insertion Grafting ...... 29 One-Cotyledon Grafting ...... 30 Post-Graft Healing ...... 31 Sucker Development ...... 32 Tongue Approach Grafting ...... 32 Non-Cotyledon Grafting ...... 33

2 EFFECTS OF GRAFTING METHODS AND ROOT EXCISION ON THE GROWTH CHARACTERISTICS OF GRAFTED MUSKMELON PLANTS ...... 42

Introduction ...... 42 Materials and Methods...... 44 Results and Discussion...... 47 Root Growth ...... 47 Survival Rates and Quality of Grafted Transplants ...... 48 Plant Growth ...... 50 Conclusions ...... 52

3 SPECIALTY MELON CULTIVAR EVALUATION UNDER ORGANIC AND CONVENTIONAL PRODUCTION IN FLORIDA ...... 59

Introduction ...... 59 Materials and Methods...... 61 Transplant Production ...... 61 Field Planting and Harvest ...... 62 Insect Pests Management ...... 64 Disease Evaluations ...... 64 Evaluations of Fruit Characteristics ...... 65

Statistical Analyses ...... 65 Results and Discussion...... 65 Anthesis and Harvest Dates ...... 65 Fruit Yields ...... 66 Fruit Characteristics ...... 69 Disease Observations ...... 71 Conclusions ...... 73

4 ROOT-KNOT NEMATODE RESISTANCE AND YIELD OF SPECIALTY MELONS GRAFTED ONTO CUCUMIS METULIFER ...... 80

Introduction ...... 80 Materials and Methods...... 83 Plant Materials ...... 83 Greenhouse RKN Study ...... 83 Field Study ...... 85 Statistical Analyses ...... 88 Results and Discussion...... 88 Greenhouse RKN Study ...... 88 Field Study of RKN Management ...... 89 Fruit Yields in the Organic and Conventional Field Experiments ...... 90 Conclusions ...... 91

5 STUDYING QUALITY ATTRIBUTES OF GRAFTED SPECIATLY MELONS USING BOTH CONSUMER SENSORY ANALYSIS AND INSTRUMENTAL MEASUREMENTS ...... 97

Introduction ...... 97 Materials and Methods...... 100 Melon Production ...... 100 Sample Preparation ...... 102 Consumer Sensory Analyses ...... 103 Instrumental Measurements ...... 104 Statistical Analyses ...... 104 Results and Discussion...... 104 Fruit Quality of ‘Arava’ Scion Grafted onto Hybrid Squash Rootstocks ...... 104 Fruit Quality of ‘Arava’ Scion Grafted onto C. metulifer Rootstock ...... 106 Off-flavor of Grafted ‘Arava’ Fruit ...... 107 Fruit Quality of Grafted ‘Honey Yellow’ Fruit ...... 108 Conclusions ...... 110

6 PHYSIOLOGICAL CHANGES IN GRAFTED MELON PLANTS WITH A HYBRID SQUASH ROOTSTOCK ...... 119

Introduction ...... 119 Materials and Methods...... 119 Results and Discussion...... 121

Conclusions ...... 124

7 SUMMARY AND FUTURE PROSPECT ...... 129

Summary ...... 129 Future Prospect ...... 132

LIST OF REFERENCES ...... 135

BIOGRAPHICAL SKETCH ...... 154

LIST OF TABLES

Table page

1-1. The major events in the history of implementing grafting technology in cucurbit crop production...... 34

1-2. Diseases reported to be controlled by grafting in different cucurbit crops...... 36

2-1. The characteristics of hole insertion, one-cotyledon and tongue approach grafting methods...... 53

2-2. The percentages of alive plants at 16 days after grafting in the first experiment. .. 53

2-3. Quality of grafted plants at 16 days after grafting in the first experiment...... 54

2-4. Aboveground biomass at 16 days after grafting...... 54

2-5. P values in the analysis of variance of effects of grafting treatment and root excision on the root and aboveground growth characteristics at 44 and 46 days after grafting in the first and second experiments, respectively...... 55

2-6. Root and aboveground growth characteristics of plants without root excision at 44 and 46 days after grafting in the first and second experiments, respectively...... 56

3-1. Seed sources and descriptions of 10 specialty melon cultivars and ‘Athena’ cantaloupe grown in organic and conventional fields during Spring 2011 at Citra, FL ...... 74

3-2. Anthesis and first harvest dates of 10 specialty melon cultivars and ‘Athena’ cantaloupe under organic and conventional production during Spring 2011 at Citra, FL...... 75

3-3. Fruit yields of 10 specialty melon cultivars and ‘Athena’ cantaloupe under organic (Org) and conventional (Con) production during Spring 2011 at Citra, FL...... 76

3-4. Fruit weight, length, shape, soluble solids concentration (SSC), and flesh firmness of 10 specialty melons and ‘Athena’ cantaloupe under organic (Org) and conventional (Con) production during Spring 2011 at Citra, FL ...... 77

3-5. Aboveground diseases severity and root-knot nematode (RKN) gall ratings of 10 specialty melons and ‘Athena’ cantaloupe under organic and conventional production during Spring 2011 at Citra, FL...... 78

4-1. Root gall index (GI), egg mass index (EMI), egg recovery, and reproduction factor (Rf) of grafted and non-grafted honeydew melon ‘Honey Yellow’ in the greenhouse study (2011)...... 93

4-2. Root gall index (GI) and numbers of Meloidogyne javanica second-stage juveniles (J2) in soil of grafted and non-grafted honeydew melon ‘Honey Yellow’ and galia melon ‘Arava’ in the organic field study (2012)...... 94

4-3. Total and marketable fruit yields (kg/plant) of grafted and non-grafted honeydew melon ‘Honey Yellow’ and galia melon ‘Arava’ in the organic and conventional field studies (2012)...... 95

4-4. Marketable fruit number per plant in different fruit size categories of grafted and non-grafted honeydew melon ‘Honey Yellow’ and galia melon ‘Arava’ in the organic and conventional field studies (2012)...... 96

5-1. Grafting treatments included in certified organic field, non-fumigated conventional field, and fumigated conventional field in Spring 2012...... 112

5-2. Consumer sensory evaluation and instrumental measurements of grafted ‘Arava’ melons in Spring 2012...... 113

5-3. Percent distribution of panelists in the consumer sensory evaluations of sweetness and firmness level, and the percentage of panelists who detected off-flavor in ‘Arava’ in Spring 2012...... 114

5-4. Consumer sensory evaluation and instrumental measurements of grafted ‘Arava’ melons grown in fumigated conventional field in Spring 2013...... 115

5-5. Percent distribution of panelists in the consumer sensory evaluations of sweetness and firmness level, and the percentage of panelists who detected off-flavor in ‘Arava’ in Spring 2013...... 116

5-6. Consumer sensory evaluation and instrumental measurements of grafted ‘Honey Yellow’ melons in Spring 2012...... 117

5-7. Percent distribution of panelists in the consumer sensory evaluations of sweetness and firmness level, and the percentage of panelists who detected off-flavor in ‘Honey Yellow’ in Spring 2012...... 118

6-1. Early and total yields of grafted and non-grafted ‘Arava’...... 125

6-2. Duration of fruit development from anthesis to harvest of grafted and non- grafted ‘Arava’...... 126

6-3. Fruit quality attributes of grafted and non-grafted ‘Arava’...... 127

6-4. Length (cm) of the longest vine of grafted and non-grafted ‘Arava’ plants at 36 days after transplanting (DAT) and 46 DAT...... 127

LIST OF FIGURES

Figure page

1-1. Hole insertion grafting. A) Remove true leaves from rootstock. B) Make a slit with a 45º angle at the growing tip. C) Keep toothpick inserted while preparing scion...... 37

1-2. One-cotyledon grafting. A) remove one-cotyledon and true leaf, cut rootstock at a slant, with a 45º angle. B) Cut scion 1.5 to 2 cm below cotyledon, also a 45º angle...... 38

1-3. A storage container (63 cm long, 46 cm wide and 30 cm deep) used for post- graft healing...... 39

1-4. Rootstock re-growth as indicated by arrows. Plants were grafted with one- cotyledon method...... 39

1-5. Tongue approach grafting. A) Rootstock and scion have similar stem diameters. B) Rootstock is cut at a downward angle and scion is cut upward. C) Approach rootstock and scion together...... 40

1-6. Non-cotyledon grafting. A) Cut rootstock at hypocotyl. B) Attach rootstock and scion cut surfaces with a grafting clip ...... 41

2-1. Root growth during the 16 days after grafting (DAG) in the first experiment. RC: rootstock control, SC: scion control, HI: hole insertion, OC: one- cotyledon, NC: non-cotyledon...... 57

2-2. Root growth during the 16 days after grafting (DAG) in the second experiment. RC: rootstock control, SC: scion control, HI: hole insertion, OC: one- cotyledon, NC: non-cotyledon...... 58

3-1. Pictures of melons in the field and harvested fruit of 10 specialty melon cultivars and ‘Athena’ cantaloupe. A-1) ‘Creme de la Creme’, A-2) ‘San Juan’ (ananas melon) ...... 79

6-1. Cumulative numbers of female flowers per plant in grafted and non-grafted ‘Arava’ plants from 16 days after transplanting (DAT) to 41 DAT...... 128

6-2. Roots and stems of ‘Arava’ grafted onto ‘Strong Tosa’ rootstock. A) A healthy root system; B) Cell death initiated in the roots, as indicated by the arrow...... 128

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

INTEGRATED USE OF GRAFTING TECHNOLOGY IN SPECIALTY MELON PRODUCTION: GRAFTING TECHNIQUES, ROOT-KNOT NEMATODE MANAGEMENT AND FRUIT QUALITY

Wenjing Guan

August 2014

Chair: Xin Zhao Major: Horticultural Sciences

Grafting is an effective approach in controlling soilborne diseases and improving stresses tolerance in melon production. Effects of grafting methods and root excision were evaluated in greenhouse experiments with regard to plant quality and growth characteristics of muskmelon ‘Athena’ (Cucumis melo) grafted onto hybrid squash rootstock ‘Strong Tosa’ (Cucurbita maxima × Cucurbita moschata). Non-cotyledon method resulted in lower plant quality. Root excision was unsuccessful with tongue approach method, while it did not exhibit significant impacts in the growth of plants that were grafted with one-cotyledon and hole insertion methods. No consistent differences were observed in grafting success and plant growth among one-cotyledon, hole insertion, and tongue approach methods.

Interest in producing specialty melon is increasing in Florida; however, the lack of disease resistance is an obstacle limiting specialty melon production. Honeydew melon

‘Honey Yellow’ and galia melon ‘Arava’ exhibited relatively better performances in specialty melon cultivar evaluation trials. However, they are susceptible to root-knot nematodes (RKN) damage.

Grafting was proposed to take advantage of RKN resistance in Cucumis metulifer. In a greenhouse experiment, ‘Honey Yellow’ was grafted onto C. metulifer and inoculated with Meloidogyne incognita race 1. The grafted plants exhibited significantly lower gall and egg mass indices, and fewer eggs compared with non- and self-grafted

‘Honey Yellow’. C. metulifer was further tested as a rootstock in field trials using ‘Honey

Yellow’ and ‘Arava’ as scions. The grafted plants showed promising results in RKN management; however, the yields were not improved in either organic or conventional fields.

Rootstock effects on melon fruit quality deserve more attention in specialty melon production as they are often marketed for outstanding taste and unique flavor.

Regardless of production systems, ‘Arava’ grafted onto hybrid squash rootstock ‘Strong

Tosa’ reduced consumer sensory properties and total soluble solids concentration

(SSC). Moreover, more consumers detected off-flavor in the grafted ‘Arava’ melons.

Accelerated fruit development and the plant wilt at the end of the season may partially explain the reduced fruit quality of the graft combination. Different from the results with

‘Arava’, grafting did not exhibit significant effects on SSC, flesh firmness, and consumer perceived sensory attributes of ‘Honey Yellow’ melons.

CHAPTER 1 LITERATURE REVIEW

An Overview of the History of Cucurbit Grafting

By physically conjoining a plant with desirable fruit characteristics (called scion) onto another plant with specific disease resistances or stress tolerances (called rootstock), grafted plants combine beneficial characteristics from both rootstock and scion cultivars (Lee et al., 2010). Research on cucurbits grafting began in the early

1990s, with watermelon (Citrullus lanatus) grafted onto squash rootstocks (Cucurbita moschata) to overcome yield loss caused by fusarium wilt (caused by Fusarium oxysporum) (Sato and Takamatsu, 1930). Since then, grafting has been widely used in

Asian and European countries for controlling soilborne diseases and improving stress tolerances. Interest in cucurbit grafting has recently grown in the U.S. as an integrated pest management practice in crop production systems (Kubota et al., 2008). Exploring the history of implementing grafting technology in cucurbit crop production in other countries, and understanding rootstock and scion interactions will not only benefit the integration of grafting techniques into cucurbit production in the U.S., but also advance our knowledge in plant physiology at the whole plant level.

Grafting in Cucurbit Crop Production

The major events in the history of implementing grafting technology in cucurbit crop production are listed in Table 1-1.

Before 1920. A few descriptions about vegetables grafting can be found in ancient literatures in Asia, but there were no large-scale grafting practices recorded before 1920.

From 1920 to 1940. Research in cucurbit grafting initiated in Japan in 1920s as a response to the increasing interest in grafting watermelon onto squash (Cucurbita moschata) for fusarium wilt control. Evaluation of different cucurbit species led to the identification of bottle gourd (Lagenaria siceraria), wax gourd (Benincasa hispida), sponge gourd (Luffa cylindrical), and other species of squash (Cucurbita maxima, C. pepo, and C. ficifolia) as rootstocks for grafting watermelon, melon (Cucumis melo) and cucumber (Cucumis sativus). In the early years, cleft grafting was the grafting method used in Japan (Sakata et al., 2007; Sakata et al., 2008).

Cucurbits grafting was introduced to the former Union of Soviet Socialist

Republics (USSR) in the 1930s as cold tolerance of grafted plants made it possible to grow cucurbits in Moscow area. It was then noticed that the grafted plants not only provided cold tolerance, but also showed early ripening, increased flowering, higher yield, prolonged production season, and increased sugar content in fruit (Lebedeva,

1937).

From 1940 to 1950. L. siceraria was the most widely used watermelon rootstock in Japan (Sakata et al., 2007). In the USSR, watermelon and melon grafting became popular. Grafting with C. maxima rootstocks was evaluated on many local cultivars

(Jurina, 1949; Pajacyk, 1948).

Cucurbit grafting was introduced to Europe right after World War II for the purpose of controlling fusarium wilt (Fusarium elegans, F. martiella) of cucumbers. The early recommended rootstock was a selected strain of C. ficifolia. Early grafting methods were cleft grafting and tongue approach (Maan, 1946). Besides fusarium wilt

resistance, cold tolerance, higher yield, and drought resistance were reported in grafted cucumbers (Kostoff, 1948).

From 1950 to 1960. Polyploidy watermelons were grafted onto bottle gourd in

Japan. The grafted polyploidy watermelons also exhibited improved yield (Furusato,

1952). In the USSR, melon grafting was approved to be an economical feasible practice

(Murtazov, 1950). More Cucurbita spp. rootstocks were evaluated in the USSR (Jurina,

1957). Following melon and watermelon, cucumber was also grafted for cold tolerance and early harvest (Avdeev, 1955).

Since the 1950s, greenhouse grown grafted cucumber became popular in the

Netherlands and Germany. As grafted plants exhibited fusarium wilt tolerance, good fruit quality and early harvest, the increased income was estimated eight times more than the extra cost generated by producing grafted plants (Hoppmann and Harnisch,

1958). However, the grafted cucumber was found sensitive to leaf chlorosis since the cold tolerant rootstock may not adapt to warm conditions inside greenhouses.

Moreover, due to the physical injury caused by grafting process, grafted cucumbers were more susceptible to Cucumis viruses (Groenewegen, 1953).

In this period, melon grafting was initiated in Europe. Cucumber rootstock C. ficifolia was found incompatible to melons, while C. pepo var. ovifera performed better as melon rootstock. However, melons grafted onto C. pepo were found to delay harvest

(Groenewegen, 1953; Naaldwijk, 1952).

From 1960 to 1970. Extensively using bottle gourd rootstocks resulted in losing resistance to fusarium wilt of grafted watermelons in Japan. A new strain, F. oxysporum f.sp. Lagenariae, together with Pythium spp. and physiological disorders resulted in a

watermelon sudden wilt that spread across Japan in the 1950s and 1960s (Sakata et al., 2007). Re-evaluation of Cucurbita spp. and B. hispida species identified C. pepo and C. maxima × C. moschata rootstocks that were resistant to the new F. oxysporum strain, and graft compatible with watermelon (Marukawa and Yamamuro, 1964;

Marukawa and Yamamuro, 1967).

As oriental melons were generally less susceptible to fusarium wilt, melon grafting was less common in Japan than watermelon grafting. However, some of the newly introduced melon cultivars were susceptible to the disease and led to the evaluation of Cucurbita spp. as melon rootstocks (Marukawa, 1969).

In Europe in this period, the most commonly used melon rootstock was B. hispida, and cleft grafting was replaced by top grafting (Louvet and Peyriere, 1962).

Grafting melons onto B. hispida, and cucumbers onto C. ficifolia were widely adopted.

However, emerging diseases such as foot rot (caused by Nectria haematococca var. cucurbitae) and black root rot of cucumber (caused by Phomopsis sclerotioides) were found associated with C. ficifolia rootstock (Cruger, 1969; Kerling and Bravenboer,

1967). Moreover, Fusarium javanicum var. ensiformc that resulted in losing fusarium wilt resistance of C. ficifolia was isolated (Bravenboer, 1964). Combining grafting with other practices such as soil steaming and soil fumigation was proposed for better control of soilborne diseases.

From 1970 to 1980. In Japan, growing grafted cucurbit crops has become a routine practice that led to systemic evaluations of grafting with other agricultural practices. Grafted cucumbers were found to absorb pesticides at higher rates, which led to the readjustment of pesticide application rates for grafted plants (Suda et al., 1976).

Understanding nutrient requirement of grafted plants was a research focus since late 1970s, and continuously attracted attention afterwards in Japan. Gomi (1980) was one of the pioneers who reported that mineral nutrient uptake of cucumbers grafted onto

C. ficifolia was different from non-grafted cucumber plants.

In Europe, interest in melon grafting for fusarium wilt management was gradually declining. One of the reasons was the emergence of a new Fusarium races, F. oxysporum f.sp. melonis, which compromised rootstock resistance and resulted in reoccurrence of the disease (Benoit, 1974). It was also attributed to the newly released melon cultivars that performed better than grafted melons under disease pressures

(Buitelaar, 1987). Furthermore, melons were susceptible to Phomopsis sclerotioides and Verticillium dahlia. Unfortunately, melons grafted onto Benicasa spp. did not control these pathogens. Cucurbita spp. rootstock exhibited partial resistance to these pathogens, but their performances were not consistent (Alabouvette et al., 1974).

From 1980 to 1990. L. siceraria with resistance to F. oxysporum f.sp. Lagenariae was identified in Japan (Matsuo et al., 1985). Because of the potential negative impacts of Cucurbita spp. on watermelon fruit quality (Matsuda and Honda, 1981), L. siceraria once again became a popular watermelon rootstock.

New evaluations of wild Cucumis species led to the identification of Cucumis metulifer, which was resistant to multiple diseases (fusarium wilt, gummy stem blight, and root-knot nematode) and compatible with cucumbers and melons (Igarashi et al.,

1987).

Cucumbers grafted onto C. moschata almost completely suppressed wax, as wax-free cucumber has a distinct appearance and a longer shelf life, the popularity of

grafted cucumbers was increased (Sakata et al., 2008). With the increased demand for grafted cucumber plants, cucumber-grafting robots were developed in Japan (Suzuki,

1990).

In Europe, Sicyos angulatus was evaluated as a cucumber rootstock for root-knot nematode control (Honick, 1984; Visser and den Nijs, 1987).

From 1990 to 2000. Cucurbit grafting increased tremendously in China. Many popular cucurbit species such as squash (C. pepo) and bitter melon (Momordica charantia) were also grafted (Chen et al., 2000; Liao and Lin et al., 1996; Lin et al.,

1998). Meanwhile, grafting robot was developed in China (Zhang and Wei, 1999).

Response of grafted cucumber to low temperature was a research focus in the

1990s in China. Studies were conducted to evaluate the best suitable rootstocks (Yu et al., 1998) and their potential cold tolerance mechanisms (Li, et al., 1998; Yu et al.,

1999).

In Europe, emerging diseases caused by Didymella bryoniae and Verticillium dahlia, led the work of reevaluating rootstocks. However, few promising rootstocks were identified (Nisini et al., 2000; Paplomatas et al., 2000).

From 2000 to present. Phase-out of the broad spectrum soil fumigant methyl bromide stimulated the introduction of vegetable grafting into the U.S. and

Mediterranean areas. Many studies were conducted with their focuses on one of the four areas: 1.effects of grafting on disease management, stress tolerance, yield, fruit quality of solanaceous vegetables and cucurbits; 2.selection of new rootstocks; 3. economic feasibility of the practices; and 4. incorporation of grafting into the whole production systems (Cohen et al., 2007; Kubota et al., 2008).

Understanding Rootstock and Scion Interactions

During 1940s to 1970s, ‘grafting hybrid’ was a hot topic in genetic studies in the

USSR (Mudge, et al., 2009). Researchers believed that two far distant species could derive intermediate characteristics through vegetative hybridization (grafting), and characteristics from both rootstock and scion could be heritably affected. Continuously grafting progenies of the scion onto the same rootstock (similar to back crossing) would eventually generate new plants combining favorable characteristics of both rootstock and scion (Ludilov, 1964; Ludilov, 1969; Parhomenko, 1941).

Many studies in this period reported that a variety of plant characteristics, either in the first generation or several following generations, were changed by grafting (Galun

1958; Hohlaceva, 1955; Mustafin, 1961; Nakamura and Meakawa, 1957; Nakamuram and Meakawa, 1960; Sanaev, 1966). Exchanging substances across the graft union was assumed the reason of genetic modifications (Gaskova, 1944; Haschkova, 1944).

Early biochemical studies were conducted in this period. Catalase, peroxidase, and polyphenol-oxidase activities were found increased in watermelon and melon plants grafted onto gourd. Grafting also improved chlorophyll content in scion leaves (Merkis,

1955). Radiographic techniques were used to study translocation of substance between rootstock and scion. The study confirmed that C14 labeled photoassimilate can transport from scion to rootstock (De Stigter, 1961).

Despite the enthusiasm towards ‘grafting hybrid’, different opinions were expressed. By comparing fusarium wilt symptoms among cantaloupe scion grafted onto resistant cucumber rootstock, reciprocal grafting, self-grafted cantaloupe, and self- grafted cucumber, Cox (1944) concluded that scion did not affect resistance of rootstock while rootstock did not affect susceptibility of scion against the pathogen. Mustafin

(1962) reported that even though xylem vessels of cucumbers grafted onto squash rootstock were almost twice as large as non-grafted cucumbers, they were caused by the increased nutrient supply but not the genetic modifications. Hayase (1966) reported that grafted cucumber developed hermaphrodite flowers, but this was due to occasionally unstable flower primordial but not genetic changes.

In the late 1970s, the idea that grafting did not change heritable characteristics of both rootstock and scion was commonly accepted (Mockaitis, 1976), although morphological alterations of the grafted plants were observed. The observed changes were often attributed to one of the following physiological mechanisms. Firstly, the overall improved plant health. Due to disease resistance or stress tolerance provided by rootstocks, grafted plants were generally healthier compared with non-grafted plants, particularly under biotic- and abiotic- stress conditions. Secondly, water and mineral nutrient transport. Many rootstocks developed for vegetable grafting were selected or bred from wild genotypes. In addition to specific disease resistance, they are characterized by large and vigorous root systems (Davis et al., 2008; Lee, 1994).

Therefore, water and mineral nutrient uptake could be improved in the grafted plants

(Guan et al., 2012; Savvas et al., 2010). On the other hand, some of the graft combinations may develop hydraulic barriers at graft union, which decrease hydraulic conductivity of grafted plants (Koepke and Dhingra, 2013). Thirdly, net photosynthesis.

Corresponding to the enhanced mineral nutrient and water uptake, grafting muskmelons onto hybrid squash rootstock improved net photosynthesis rate, stomatal conductance, intercellular CO2, and transpiration rate (Liu, et al., 2011). Fourthly, hormonal balances.

Plant hormones are important factors that regulate all aspects of plant development.

Cytokinins are known to be produced in the roots and translocate to the shoot, where they affect shoot growth (Aloni et al., 2010). The production of cytokinins is under the regulation of auxin, which is produced in the shoot and translocated to the root, where it affects root development. In the grafted plants, the auxin and cytokinin balance is interrupted. The vigorous property of rootstock enhanced scion growth, which might be due to increased supply of cytokinin to the shoot and decreased supply of IAA (Lee and

Oda, 2003; Sorce et al., 2002).

Signal transmission between rootstock and scion (graft transmission) again attracted attention in the 1980s. Anthracnose (caused by Colletotrichum lagenarium) susceptible cucumber scion was resistant to anthracnose after grafting onto an infected resistant rootstock with the same pathogen. Disease resistance signals were suspected transmitting across grafting union (Dean and Kuć 1986; Jenns and Kuć,1979). Short- day plant, Sicyos angulatus L. Sicyos, was induced to flower by grafting it onto a flower- induced plant of the same species suggesting that floral stimulus might transmit from rootstock to scion (Takahashi and Saito, 1981). Since these characteristics were less likely to be affected by physiological mechanisms, grafting induced alternations at molecular level became the study focus.

Grafting increased the activities of several important enzymes (Guan et al.,

2012). Cucumbers grafted onto C. moschata induced expression of resistance-related R proteins and photosynthesis-related proteins (Li et al., 2009). Sucrose phosphate synthase and stachyose synthase activities were improved in muskmelons grafted onto hybrid squash rootstocks (Liu, et al., 2011). With regard to transcriptional profile, the relative expression of Mn-Superoxide dismutase (SOD) and Cu/Zn-SOD mRNA was

higher in grafted cucumbers under low temperature stress, corresponding to the improved activities of SOD, Mn-SOD and Cu/Zn-SOD (Gao et al., 2009). Apples grafted onto rootstock with moderate resistance to blight disease (caused by Erwinia amylovora) showed increased stress-related gene expression, while scions grafted onto susceptible rootstock did not (Jensen et al., 2003).

Identifying long-distance transmissible signals is critical in understanding grafting induced modifications at molecular levels. Cucurbits were model plants to study phloem exudates, in which large amount of proteins with diverse functions have been identified

(Lough and Lucas, 2006). Phloem proteins were confirmed transmissible across grafting union (Golecki et al., 1998). Studies on flower locus (FL) proteins indicated that FL proteins may act as the long-distance florigenic signals in the cucurbits (Lin et al.,

2007). Considering the important roles of proteins in regulation of gene expression, the transmissible proteins might be one of the signals that induce genetic modification in grafted plants. In addition to proteins, long distance trafficking of RNAs was demonstrated (Kehr and Buhtz 2008; Liang et al., 2012). siRNA mediate post- transcriptional gene silencing, a graft-transmissible silencing was confirmed in tomato

(Shaharuddin et al., 2006). Reverse transcription of mRNA into cDNA, and integrating it into genome was demonstrated. However, little is known about the involvement of this mechanism in grafting induced genetic modification (Adler, 2001). Studies in epigenetics unraveled a new mechanism in gene expression regulation that described heritable changes were induced by mechanisms other than changes in DNA sequence

(Bird, 2007). Although specific proteins and RNAs were involved in currently documented epigenetic mechanisms, it is still unclear whether these long-distance

grafting transmissible signals participated in any of the gene expression regulation mechanisms in grafted plants.

The most direct evidence of grafting induced genetic changes came from genetic information exchange via plastid genomes at grafting site (Liu et al., 2010a; Stegemann and Bock, 2009). However, current evidences only proved that such transmission was limited to cells adjacent to graft union, whether the genetic information could transmit between root and shoot is unclear.

Diseases Controlled by Grafting in Cucurbit Production

Improved resistances against many soilborne fungal, oomycete, and nematode pathogens have been reported in grafted cucurbit crops. Moreover, certain foliar fungal and viral diseases were suppressed when susceptible scions were grafted onto specific rootstocks (Louws et al., 2010). Diseases controlled by grafting in different cucurbit crops are listed in Table 1-2.

Soilborne fungal and oomycete diseases. The earliest reported use of vegetable grafting for disease control was for management of fusarium wilt in cucurbits

(Sakata et al., 2007). Commonly used cucurbitaceous rootstocks are non-hosts to most formae speciales of F. oxysporum, and thus grafting has been successfully employed to control fusarium wilt in cucurbit production (Louws et al., 2010). Verticillium wilt, primarily caused by Verticillium dahliae, is another vascular wilt disease that often affects Cucurbitaceae. Studies with plants grafted onto commercial rootstocks and subjected to infection with V. dahliae indicated that both scions and rootstocks contributed to disease resistance of the grafted combinations in watermelons, melons,

 Reprinted with permission from ASHS journal

and cucumbers (Paplomatas et al., 2002). Monosporascus sudden wilt, caused by

Monosporascus cannonballus, is an important soilborne disease of melon and watermelon in hot and semiarid areas. Grafting scions of susceptible melon cultivars onto C. maxima and C. maxima × C. moschata rootstocks improved resistance of melon

(Edelstein et al., 1999) despite the fact that Cucurbita is normally regarded as a host for

M. cannonballus (Mertely et al., 1993). However, the improved resistance and better yield with grafted plants was inconsistent. The variable results might be attributed to differences in rootstock-scion combinations and growing conditions. Phytophthora blight, caused by Phytophthora capsici, is regarded as one of the most destructive diseases in production of cucurbits. In P. capsici infested fields, yields of cucumbers grafted on bottle gourd (Lagenaria siceraria), C. moschata, and wax gourd (Benincasa hispida) rootstocks were significantly increased and vegetative growth was more vigorous (Wang et al., 2004). Watermelons grafted onto selected bottle gourd rootstocks also exhibited resistance to P. capsici (Kousik and Thies, 2010).

Root-knot nematodes (RKN). Root galling of susceptible plants is a typical response to RKN (Meloidogyne spp.) infection, resulting in poor absorption of water and nutrients. In cucurbits, resistance to M. incognita was identified in Cucumis metulifer,

Cucumis ficifolius, and bur cucumber (Sicyos angulatus) (Fassuliotis, 1970; Gu et al.,

2006). Using C. metulifer as a rootstock to graft RKN susceptible melons led to lower levels of root galling and nematode numbers at harvest (Sigüenza et al., 2005).

Moreover, C. metulifer showed high graft compatibility with several melon cultivars

(Trionfetti Nisini et al., 2002). Cucumbers grafted on the bur cucumber rootstock exhibited increased RKN resistance (Zhang et al., 2006). Promising progress has also

been made in developing M. incognita resistant germplasm lines of wild watermelon

(Citrullus lanatus) for use as rootstocks (Thies et al., 2010). However, at present cucurbit rootstocks with resistance to RKN are not commercially available (Thies et al.,

2010).

Viral diseases. Vegetable grafting research on resistance to viral diseases yielded mixed results because of the lack of systematic studies in this area. Wang et al.

(2002) reported improved antivirus performance in grafted seedless watermelon plants.

In Israel, use of resistant rootstocks for controlling the soilborne melon necrotic spot virus (MNSV) in cucurbits was a significant advantage over soil fumigation with methyl bromide, which does not control this viral disease (Cohen et al., 2007). However, some reports indicated that grafted plants were more vulnerable to viral diseases, possibly due to graft incompatibility that weakened the scion plants (Davis et al., 2008).

Other diseases. Other fungal diseases which have been controlled by grafting include target leaf spot (caused by Corynespora cassiicola) on cucumbers, black root rot (caused by Phomopsis sclerotioides) on cucumbers and melons, and gummy stem blight (caused by Didymella bryoniae) on melons (King et al., 2008; Louws et al., 2010)

(Table 1-2). Grafting has also been reported to improve crop resistance to the foliar fungal diseases such as powdery mildew (caused by Podosphaera xanthii) and downy mildew (caused by Pseudoperonospora cubensis) on cucumbers, when certain rootstocks were used (Louws et al., 2010; Sakata et al., 2006).

Challenges and future prospective. Extensively using grafting technique with limited rootstocks led to the emergence of new virulent strains, and the recurrence of suppressed diseases. As the inhibition of major diseases, some of the former minor

diseases became more important. To solve these problems, it is important to have a backup pool of cucurbit rootstocks. However, most of the currently used rootstocks were developed in the 1930s, and minimal progress has been achieved since that period.

More rootstock breeding programs equipped with up-to-date breeding technologies should be established. Development of transgenic rootstocks might be one of the directions. A cucumber green mottle mosaic virus coat protein gene (CGMMV-CP) and a cucumber fruit mottle mosaic tobamovirus (CFMMV) replicase gene have been introduced into watermelon and cucumber rootstocks, respectively. Susceptible scions grafted onto transgenic rootstocks exhibited high resistance against these viral pathogens, but the transgenic rootstocks have not been commercially available (Gal-On et al., 2005; Park et al., 2005; Yi et al., 2009).

Grafting-induced systemic defense was proposed to explain resistance to the diseases that could not be controlled by rootstocks, such as viral diseases and foliar fungal diseases (Guan et al., 2012). However, in-depth study to prove the assumption is challengeable as many external factors could affect these disease performances.

Establishing a worldwide research standard including a model scion and rootstock plants combination, a well-controlled environment condition, a standard inoculation system, and a carefully designed experimental protocol will be critical to repeat the results, and help to improve our understanding in rootstock-scion interactions in disease management.

Techniques for Melon Grafting

Grafted plants are more costly than the regular transplants (Barrett et al., 2012a;

Djidonou et al., 2013). Cost, along with the desire to customize scion cultivars, and the need to produce organic transplants, leads many small and organic growers to choose

to graft plants by themselves. However, achieving a high grafting survival rate can often be rather challenging for growers especially during their first attempt at grafting.

Although melon grafting follows the same principles as grafting other types of vegetables, it has some unique considerations. Firstly, both rootstock and scion plants develop hollow stems (hypocotyls) soon after seed germination, and this stem cavity expands as the hypocotyls become thicker. Hollow stems reduce the contact area between rootstock and scion tissues; therefore, grafting should be conducted with young seedlings when the central cavities are small. Secondly, in contrast to tomato grafting where scions can be cut above the cotyledons, melon scions should be cut at the hypocotyl area and maintain both cotyledons. The presence of cotyledons results in greater leaf surface area, and causes water loss to be more severe with newly grafted plants. Thirdly, rootstocks used for melon grafting can be intra- or interspecific and can have varied hypocotyl diameters. Grafting methods then need to be adjusted based on rootstock species and hypocotyl diameters.

Hybrid squashes are widely used melon rootstocks (King et al., 2010). They are highly resistant to fusarium wilt and tolerant to verticillium wilt, monosporascus sudden wilt, and gummy stem blight (Guan et al., 2012; Louws et al., 2010). In addition, hybrid squashes have superior tolerance to low temperatures and saline conditions (Colla et al., 2010; Davis et al., 2008). However, hybrid squashes are susceptible to root-knot nematodes, and may have adverse effects on fruit quality of some melon cultivars

(Davis et al., 2008, Sakata et al., 2008).

Melon cultivars with resistance to Fusarium oxysporum f. sp. melonis (race 0, 1,

2, and 1.2) are also used as melon rootstocks (Davis, et al., 2008). The advantages of

melons grafted onto melons are fewer incompatibility issues and less fruit quality concerns. However, insufficient disease resistance is the major obstacle of using melon as rootstocks (King et al., 2010). C. metulifer was recently tested for melon grafting as it is resistant to root-knot nematode and fusarium wilt (Sig enza et al., 2005; Trionfetti

Nisini et al., 2002). While C. metulifer is compatible with melon cultivars, it has thinner stems compared with melons, and special attention should be paid when it is used as a melon rootstock.

To help small and organic growers achieve high survival rate of melon grafts, commonly used grafting techniques and their application in specific circumstances were introduced. Decisions on choosing the most suitable grafting technique will depend on available resources, rootstock and scion cultivars, and the grafter’s experience.

Hole Insertion Grafting

Procedure of the hole insertion grafting was demonstrated in Figure 1-1. First, leaves and meristem tissue are removed at the growing tip of the rootstock. Next, a slit is made across the growing point from the bottom of one-cotyledon to the other side of the hypocotyl. A shaved stick such as a toothpick or bamboo barbecue skewer can be used as the insertion tool. Leave the stick inserted in the growing point, while cutting the scion hypocotyl at both sides into a ‘V’ shape. The scion is then inserted into the slit while the insertion stick is removed.

Hole insertion grafting produces high quality grafted transplants because it maximizes contacting surfaces between rootstock and scion, and affords protection of the grafting union with both rootstock cotyledons. Another advantage of this method is that it does not require grafting clips, which reduce grafting cost, as well as the labor involved in collecting clips after healing.

Hole insertion grafting works best for hybrid squash rootstocks as they normally have thicker hypocotyls than those of melon scions. Due to the concern of hollow hypocotyls, rootstocks with more than two expanded true leaves should be avoided.

Hole insertion grafting has a strict requirement for the grafting stages of scions. The ideal graft period for scions is when the first true leaves start to emerge but are not fully expanded. During this stage, scion hypocotyls are strong enough to be inserted while still easy to fit into the slits on the rootstock. Since the ideal plant size for hole insertion grafting is relatively narrow, timing of sowing rootstock and scion seeds is critical for this method. If a new rootstock or scion cultivar is used, it is recommended to record the relative seed germination and growth rate of both rootstock and scion plants before conducting large scale grafting with this method.

One-Cotyledon Grafting

One-cotyledon grafting is easier to conduct than hole insertion grafting.

Procedure of this method was shown in Figure 1-2. First, the rootstock growing tip is cut at a 45 degree angle. The cut removes true leaves, meristem tissue, and one of the cotyledons. Next, the hypocotyl of the scion is cut at the same angle as the rootstock.

The scion is then attached to the rootstock with a grafting clip.

This method is most suitable for melon rootstocks as it works best when the rootstock and scion have similar hypocotyl diameters. Hybrid squash rootstocks are also grafted with this method as it is simple to conduct. Since hybrid squashes generally have thicker hypocotyls compared with melon scions, scion seeds should be planted earlier than rootstock seeds. This method can also be applied to the C. metulifer rootstock. However, opposite to hybrid squash rootstock, C. metulifer has a thinner hypocotyl than melons, thus rootstock seeds should be sowed a few days earlier than

scion seeds. Similar to hole insertion graftings, scions grafted at a younger stage have higher survivability due to less water evaporation. While young plants are desirable, a compromise must be made so that scion stem diameters are similar to those of rootstocks, particularly when hybrid squash rootstocks are used. It is important to note that grafting needs to be completed soon after plants are cut to prevent the surfaces of both scion and rootstock from drying off, which will greatly reduce the survival rate of grafted plants.

Post-Graft Healing

With the hole insertion and one-cotyledon graft methods, maintaining high humidity and favorable temperature during the first 48 hours is essential for grafting success. The optimal temperature is around 28 ºC and relatively humidity 95%. Our experiments indicated that temperatures lower than 21º C and/or average RH lower than 85% in the first 48 hours result in grafting failure (unpublished data). If scions severely wilt during this period, their chance to recover is very low. To reduce water loss through evaporation and photosynthesis, heavy shade or completely dark conditions are required. Placing plants in complete darkness for longer than 48 hours, however, could result in unfavorable stem elongation and weak seedlings. After the critical 48 hours, exposing the plants to a gradual reduction of humidity and an increase in the amount light is also important. For small scale grafting, storage containers can be used for post- graft healing (Figure 1-3). The container should be placed in a dark room or covered with black cloth during the critical hours. High humidity can be achieved by putting a thin layer of water on the bottom, and spraying water inside the closed container. After the critical hours, humidity is gradually reduced by partially opening the container. For larger scale grafting, a healing chamber that can accommodate several seedling trays can

easily be constructed inside of a greenhouse. More information regarding the construction of a healing chamber can be found at (Johnson et al.,2011). For commercial post-graft healing, a storage room with humidity and temperature control is normally used.

Sucker Development

Sucker development refers to re-growth of rootstocks as a result of the incomplete removal of meristem tissue during hole insertion and one-cotyledon graft methods (Figure 1-4). Scouting and removing suckers should be conducted before transplanting. However, suckers may continuously emerge in the field, which can become one of the major problems of using grafted transplants. Rootstock regrowth compete with scion plants for water and nutrients, and reduce yield. Two graft methods, tongue approach and modified one-cotyledon graft, eliminate sucker problems by completely removing the growth points of rootstocks.

Tongue Approach Grafting

Tongue approach grafting is a popular graft method used in Spain. Rootstock and scion seeds are sowed in the same pot. After they both emerge, the rootstock hypocotyl is cut halfway through at a downward 45 degree angle. At the same height, the scion hypocotyl is cut halfway through at an upward 45 degree angle. Then, rootstocks and scions are joined together with the cut surfaces mated, and the stems are fixed with a grafting clip. Ten days after grafting, the top of the rootstocks and the roots of scions are cut off with a razor blade (Figure 1-5).

Tongue approach grafting can achieve a high survival rate without high humidity healing conditions. Direct sunlight should be avoided on newly grafted plants, but healing is often achieved in a normal greenhouse environment. Some rootstock and

scion cultivars have very short hypocotyls, and they are not suitable for this method.

Tongue approach grafting requires rootstock and scion to have similar hypocotyl diameters. Thus the timing of sowing rootstock and scion seeds should be adjusted based on their hypocotyl diameters.

Non-Cotyledon Grafting

Non-cotyledon grafting is very similar to one-cotyledon graft, but the rootstocks are cut at the hypocotyls, and removed both cotyledons (Figure 1-6). Although this method is easy to conduct, it may result in rootstock decline as cotyledons are important for early root growth.

Table 1-1. The major events in the history of implementing grafting technology in cucurbit crop production. Time Japan USSRz Europe China U.S. 1920- Watermelon was grafted onto 1930 Cucurbita moschata for fusarium wilt control

1930- Selected fusarium wilt rootstocks: Watermelon and 1940 Lagenaria siceraria, Benincasa melon grafting hispida, Luffa cylindrical, Cucurbita were introduced maxima, C. pepo, C. ficifolia. for cold tolerance

1940- L. siceraria became the most Evaluation of Cucumbers were grafted 1950 popular rootstock for watermelon grafting local onto C. ficifolia for fusarium grafting. watermelon and wilt management melon cultivars onto C. maxima rootstocks

1950- Polyploidy watermelons were Economic Economic analyses were 1960 grafted analyses were conducted on cucumber conducted on grafting. watermelon and Leaf chlorosis and virus melon grafting. problems were found Cucumbers were associated with grafted grafted cucumbers. Melons were grafted

1960- L. siceraria lost resistance to B. hispida was regarded as 1970 fusarium wilt. the most suitable melon C. pepo and C. maxima × C. rootstock. moschata were proved resistance Emerging diseases with to the new F. oxysporum strain. cucumber rootstock C. Melon and cucumber grafting ficifolia. became popular. Combining grafting with other disease management approaches achieved better results.

Table 1-1 Continued. Time Japan USSRz Europe China U.S. 1970- Systematical evaluation of grafting Melon grafting was 1980 with other production practices. declining because 1. The Mineral nutrient uptake was found evolution of new pathogens differed between grafted and non- that broke rootstock grafted plants. resistance; 2. new introduced resistant melon cultivars; 3. grafting unable to control new emerging diseases.

1980- Resistance to F. oxysporum f.sp. Sicyos angulatus was 1990 Lagenariae was found in L. evaluated as cucumber siceraria. It again became the most rootstock for root-knot popular watermelon rootstock. nematode management C. metulifer was evaluated as cucumber and melon rootstocks. Grafted ‘bloomless’ cucumber became popular. Grafting robot was developed for cucumber grafting

1990- Cucurbit grafting 2000 increased tremendously. Other important cucurbit crops were also grafted

2000- Cucurbit prese grafting nt was introduced. zUSSR: Union of Soviet Socialist Republics

Table 1-2. Diseases reported to be controlled by grafting in different cucurbit crops. Disease and Pest Pathogen Cucurbit Crops Fungal and oomycete diseases Fusarium wilt Fusarium oxysporum watermelon, melon, cucumber Fusarium crown and root Fusarium oxysporum; Fusarium solani watermelon Verticilliumrot wilt Verticillium dahliae watermelon, melon, cucumber Monosporascus sudden Monosporascus cannonballus watermelon, melon Phytophthorawilt blight Phytophthora capsici watermelon, cucumber Target leaf spot Corynespora cassiicola cucumber Black root rot Phomopsis sclerotioides cucumber, melon Gummy stem blight Didymella bryoniae melon Powdery mildew Podosphaera xanthii cucumber Downy mildew Pseudoperonospora cubensis cucumber

Nematodes Root-knot Meloidogyne spp. cucumber, melon, watermelon

Viral diseases Melon necrotic spot virus Melon necrotic spot virus (MNSV) watermelon Information was adapted from published reviews (King et al., 2008; Louws et al., 2010).

Figure 1-1. Hole insertion grafting. A) Remove true leaves from rootstock. B) Make a slit with a 45º angle at the growing tip. C) Keep toothpick inserted while preparing scion. D) Cut scion hypocotyls at both sides into a ‘V’ shape. E) insert scion into the slit. F) Bamboo toothpick used for insertion.

Figure 1-2. One-cotyledon grafting. A) remove one-cotyledon and true leaf, cut rootstock at a slant, with a 45º angle. B) Cut scion 1.5 to 2 cm below cotyledon, also a 45º angle. C) Attach the rootstock and scion cut surfaces and hold them together with a grafting clip. D) A newly grafted melon plant with one-cotyledon graft.

Figure 1-3. A storage container (63 cm long, 46 cm wide and 30 cm deep) used for post-graft healing.

Figure 1-4. Rootstock re-growth as indicated by arrows. Plants were grafted with one- cotyledon method.

Figure 1-5. Tongue approach grafting. A) Rootstock and scion have similar stem diameters. B) Rootstock is cut at a downward angle and scion is cut upward. C) Approach rootstock and scion together. D) Make sure cut surfaces make contact with each other. E) Fix with a grafting clip. F) Cut rootstock top and scion roots 10 days after grafting.

Figure 1-6. Non-cotyledon grafting. A) Cut rootstock at hypocotyl. B) Attach rootstock and scion cut surfaces with a grafting clip

CHAPTER 2 EFFECTS OF GRAFTING METHODS AND ROOT EXCISION ON THE GROWTH CHARACTERISTICS OF GRAFTED MUSKMELON PLANTS

Introduction

Grafting has proven to be an effective approach in controlling soilborne diseases and overcoming abiotic stresses in production of solanaceous and cucurbitaceous vegetables in many Asian and European countries (Lee and Oda, 2003). High cost of producing grafted seedlings is an obstacle limiting wide adaptation of this technology in the U.S (Davis, et al., 2008; Memmott and Hassell, 2010). Optimizing grafting procedures and producing high quality grafted seedlings are important in reducing the cost.

Cucurbit grafting methods differ considerably among geographic regions and nurseries, and vary by rootstock and scion combinations (Chapter 1; Lee 1994; Lee and

Oda, 2003; Lee et al., 2010). Today, the most popular methods are hole insertion, one- cotyledon and tongue approach. Characteristics of the three methods are summarized in Table 2-1.

Tongue approach grafting was initiated in the Netherlands in the 1960s, and spread to Korea, Japan, and other European countries thereafter (Davis et al., 2008).

Although the method requires additional labor to cut rootstock top and scion bottom a few days after initial grafting, it does not need high humidity post-graft healing conditions, thus the high grafting survival rates are guaranteed (Davis et al., 2008).

Aside from being easily performed by hand, one-cotyledon is the only method which can also be performed by machine (Hassell et al., 2008). A grafting machine can produce

600 grafts per hour using the one-cotyledon method as compared to about 1000 grafts per person per day (Hassell et al., 2008). Hole insertion is the most popular method

used in China (Davis et al., 2008). With this method, the grafted plants have a high graft union, and do not require grafting clips. Although relatively easy to conduct, re-growth of rootstocks, also known as rootstock suckers, is a problem of hole insertion and one- cotyledon methods. Additional efforts are required to remove suckers after planting. For large-scale production of grafted plants in the U.S., the non-cotyledon method and fatty alcohol treatment of rootstock meristems were recently proposed to eliminate the rootstock sucker problem (Daley and Hassell, 2014; Memmott, 2010; Memmott and

Hassell, 2010).

One of the major differences of the aforementioned grafting methods is the number of rootstock cotyledons remaining after grafting. As the name indicates, non- cotyledon method excises both rootstock cotyledons, while hole insertion and one- cotyledon methods maintain two and one of the root cotyledons, respectively. Tongue approach method also removes both rootstock cotyledons, but the cotyledons are excised five to ten days after grafting, when the graft union has healed.

Cucurbits have leaf-like cotyledons. Their photosynthetic activity supports early hypocotyl and root growth (Lovell and Moore 1971). Bisognin et al., (2005) reported that before the true leaf area of cucumber seedling was equivalent to cotyledon area, cotyledons were essential to maintain aerial and root growth. In addition to seedling development, cotyledon damage on cucumbers can influent plant sex expression and maturity (Omran, 1981).

In addition to the number of rootstock cotyledons removed during grafting, grafting methods are further distinguished by whether roots are excised prior to graft healing. As root excision facilitates grafting process by preventing growing media

contaminating machines, excising roots and allowing them to regenerate during the graft healing process is a method currently used by mechanical grafting machines

(Memmott, 2010). In addition, since both graft healing and root growth are energy requiring processes, after excising the roots, the energy reserved in rootstock hypocotyl could be conserved to facilitate graft healing, which might improve graft success (Lee,

1994; Memmott and Hassell, 2010; Penny, et al., 1976).

With the considerable variances existing in the methods of cucurbit grafting, greenhouse experiments were conducted to evaluate the effects of different grafting practices on seedling quality and growth characteristics of melon plants grafted onto hybrid squash rootstock.

Materials and Methods

Two experiments were conducted from Nov. 2013 to March 2014 in a greenhouse where the temperature ranged from 20 ºC to 30 ºC, and average relative humidity was 56%. Cantaloupe ‘Athena’ (Cucumis melo) was grafted onto hybrid squash rootstock ‘Strong Tosa’ (C. maxima × C. moschata). Scion and rootstock seeds were planted on the same day, 23 Nov. 2013 and 11 Jan. 2014 in the first and second experiments, respectively. Seeds were sown into 128-cell styrofoam flats (Sun City, FL).

The cells were filled with Metro-Mix 200 potting soil (Sun Gro Horticulture, Bellevue,

WA) containing a mixture of vermiculite, peat moss, perlite, and starter nutrient.

Seedlings were grafted on 5 Dec. 2013 and 23 Jan. 2014 in the first and second experiments, respectively, when both rootstock and scion developed first true leaves.

Plants were grafted with four methods, namely, hole insertion (HI), one-cotyledon

(OC), non-cotyledon (NC), and tongue approach methods (TA). Non-grafted rootstock and scion plants were included as controls. Both grafted and non-grafted plants were

examined with or without root excision. For the root excision treatment, plants were cut just above the root zone and replanted in pre-moistened soil in a 128-cell tray. A randomized complete block design with three replications and 12 plants per treatment per replication was used in the grafting experiments.

Except for the plants grafted with the TA method, all the other plants were placed in a healing chamber immediately following grafting and root excising. The chamber was built on the greenhouse bench with PVC pipes as its frame. A clear plastic, followed by a white on black plastic and a shade cloth were used to cover the chamber. An air conditioner and humidifiers were used to control temperature and humidity inside of the chamber. On the first two days, a dark environment was provided by closing the chamber completely. Temperature was maintained at 28 ± 3 ºC and relative humidity was 95% to 100% inside of the chamber. On the third day, plants were exposed to light by partially opening the black plastic and shade cloth, while the humidity and temperature were maintained at the same levels as the previous days.

Humidity was gradually reduced from the fourth day by adjusting the humidifier and partially opening the clear plastic. The air conditioner was turned off unless the temperature was above 35 ºC. After seven days, plants were moved out from the healing chamber and grown under natural greenhouse conditions.

Conforming to the standard healing conditions of plants grafted with TA method, these plants were placed in a shaded area on the bench under the natural greenhouse conditions. The top of the rootstocks and the bottom of the scions were cut 9 days after grafting (DAG).

The numbers of live plants of each grafting treatment were recorded in the first experiment; while in the second experiment, a 0-10 scale (Memmott, 2010) was used to evaluate qualities of the grafted plants at 16 DAG.

All the plants were individually transplanted into 0.4L square plastic pots filled with Metro-Mix 200 potting soil at 10 DAG. For each treatment, randomly selected three healed plants, one from each replication, were destructively measured at 4, 8, 12 and

16 DAG. Total root length and root surface area were evaluated using a root scanning apparatus (EPSON color image scanner LA1600, Toronto, Canada) and image analysis software WinRhizo 2008a (Regent Instruments, Quebec, Canada). At 16 DAG, aboveground fresh and dry weights were measured. As the graft healing process of TA grafted plants was considerably different from the other treatments, above measurements were not conducted on TA grafted plants.

Plant growths were further evaluated in the following part of the experiments. Six plants of each treatment that rated as superb (Memmott, 2010) were individually transplanted into 3.8 L pots filled with Metro-Mix 200 potting soil. Plants were arranged in a completely randomized design with six plants that served as six replications in each treatment. NC grafted plants (both with and without root excision) and TA grafted plants with root excision were not included in this part of the experiment. Plants were grown in the greenhouse, hand watered, and fertilized with 20N-8.7P-16.6K fertilizer (Peters

Professional; United Industries, St. Louis, MO) at the rate of 2.8 g N/plant/week. The blooming dates of male and female melon flowers were recorded on each plant. The experiments were terminated at 44 and 46 DAG in the first and second experiments, respectively, when at least two female flowers bloomed on each of the melon plants.

Aboveground growth characteristics including fresh weight, dry weight, leaf area, stem diameter, chlorophyll content, and stomatal conductance were measured. Root length and surface area of each plant were evaluated as previously described.

Analysis of variance was performed using the XLSTAT software (Addinsoft, New

York, NY, USA). Fisher’s least significant difference test (α = 0.05) was conducted for multiple comparisons of different measurements among treatments.

Results and Discussion

Root Growth

Using the HI, OC, and TA methods, grafted plants with intact roots (without root excision) exhibited an increase in root length and surface area in the first 4 DAG. But active root growth in the early days following grafting was not observed on root excised plants regardless of grafting methods used (Figure 2-1 and Figure 2- 2). As vascular bundles between scion and rootstock started to connect soon after grafting (Aloni et al.,

2008), the observation partially supported the previous assumption that if the roots were excised, energy reserved in rootstock hypocotyl could be conserved to initiate graft healing instead of root growth (Memmott and Hassell, 2010).

In general, root regeneration was initiated on the root excised plants at 4 DAG

(Figure 2-1 A, C and Figure 2-2 A, C), which then developed similar root lengths and surface areas as the root intact plants at 16 DAG. No significant differences in root length and surface area were observed among rootstock control, scion control, HI and

OC grafted plants at 16 DAG in the first experiment (Figure 2-1). However, OC grafted plants without root excision had shorter root length and less surface area compared with the same root treatment of HI grafted plants in the second experiment (Figure 2-2). As one of the cotyledons was removed from the rootstock hypocotyls, root formation and

development might be affected (Katsumi et al., 1969), although the remaining cotyledon could partially complement the role of the removed cotyledon (Mayoral et al., 1985).

NC grafted plants exhibited a root growth pattern different from that of the other plants. The excised root initiated new root growth 8 DAG instead of 4 DAG (Figure 2-1

A, C and Figure 2-2 A, C). In addition, the root length and surface area of the intact roots did not exhibit a significant increase during 16 DAG in the first experiment (Figure

2-1 B, D), and showed a moderate growth in the second experiment (Figure 2-2 B, D).

As a result, the root length and surface area of NC grafted plants were significantly lower than other plants at 16 DAG. Katsumi et al. (1969) reported that root formation of cucumber hypocotyl cuttings were inhibited if cotyledons were completely removed.

Lacking auxin, which is supplied by cotyledons, was suggested as the reason of the root growth inhibition (Elkinawy, 1980).

Hybrid squash rootstock was known for vigorous root systems (Davis et al.,

2008). But the rootstock control and all the grafted plants did not exhibit significantly longer root length and larger surface area compared with the scion control in the early stage of seeding development. The same observation was also reported in a germination test including both hybrid squash ‘Strong Tosa’ and melon ‘Athena’

(Nguyen, et al., 2013).

Survival Rates and Quality of Grafted Transplants

Survival rates were generally above 80 %, except plants grafted with TA method and had root excised (45.8 %) (Table 2-2). The graft healing conditions that were unsuitable for root regeneration were the main reason for the lower survival rate of those plants. The comparative size of rootstock and scion plants is important to achieve the highest potential of grafting survival. Hole insertion method works best with small

scions that could be easily inserted into the holes of rootstocks, whereas TA method works best when scion and rootstock plants have similar stem diameter. Rootstock and scion seeds are normally planted on different days (Davis, et al., 2008). However, in order to accommodate different methods, the rootstock and scion seeds were sow on the same day in the present study, which might affect survival rates of plants grafted by some of these methods. The highest survival rate (100%) was achieved in plants grafted with OC method, suggesting this method might have a low requirement for the relative size of rootstock and scion plants.

Significant interaction effects between grafting methods and root excision on quality of the grafted plants were observed in the second experiments (Table 2-3).

Similar to the results in the first experiment, grafted plants with TA method and root excision had the lowest quality. While plants grafted with the same method but with intact roots exhibited similar plant quality as plants grafted with the HI and OC methods.

Interestingly, regardless of whether roots were excised or intact, quality of the NC grafted plants was significantly lower than HI and OC grafted plants, even though the majority of the plants were alive at 16 DAG. This was different from other methods in which graft failure was attributed to the lack of union between scions and rootstocks, which resulted in scion death. The majority of NC grafted plants healed initially, whereas about one-third of plants exhibited slowed and stunted growth, and the rootstock hypocotyl declined and eventually died in the following days.

Without the phytosynthates from rootstock cotyledons, insufficient nutrient reserved for rootstock growth may be one of the reasons that lead to rootstock hypocotyl deterioration (Memmott and Hassell, 2010). On the other hand, the decline of

NC grafted plants after healing may be due to the inhibition of root growth as a result of removing rootstock cotyledons (Figure 2-1 and 2-2). However, when both rootstock cotyledons were removed, plants grafted with TA method did not exhibit the decline of rootstock hypocotyls. Since rootstock cotyledons were removed after graft healing on plants grafted with TA methods, the critical roles of rootstock cotyledons might be replaced by cotyledons or true leaves of scion plants.

Using the NC method, Memmott (2010) reported that root excision significantly enhanced grafting success when watermelon was grafted onto ‘Strong Tosa’ at the rootstock second-true-leaf-unexpanded stage of the rootstock. However, the advantage of root excision was not observed in the present study. The plant quality was evaluated at 16 DAG in this study versus 7 DAG in the previous report by Memmott (2010), which may partially explain the discrepancies. No significant difference in grafting success was observed between HI and OC grafted plants.

With the randomly selected three healed grafted plants, the aboveground fresh weight and dry weight of NC grafted plants at 16 DAG were significantly lower than that of non-grafted scion controls in the first experiment but not in the second experiment

(Table 2-4).

Plant Growth

Cucumbers and watermelons grafted onto hybrid squash rootstocks have been found to display delayed bloom of female flowers (Satoh, 1996; Yilmaz et al., 2011), but no differences on the anthesis date between grafted plants and scion controls were observed in this experiment. Grafting methods and root treatment also did not affect flowering date in this study.

A significant interaction effect between grafting methods and root excision on root length was observed in the first experiment (Table 2-5). Rootstock controls with root excision displayed 3039 cm roots, which was significant longer than those without root excision (2031 cm). However, such an effect of root excision was not observed on the scion controls and the grafted plants. It was also not observed in the second experiment. Therefore, root excision was less likely to affect root growth at this stage of plant development. Significant grafting effects were observed on fresh and dry weight, and stem diameter in both experiments. But it was not consistent for stomatal conductance and chlorophyll content (Table 2-5).

Among the grafting treatments that did not involve root excision, rootstock controls had significant higher values of aboveground dry weight, but not root length and surface area compared with scion controls (Table 2-6). Although hybrid squash rootstocks are often suggested to have vigorous root systems (Davis et al., 2008), large root biomass of ‘Strong Tosa’ was not observed in this study. However, the rootstock did exhibit thicker stem diameters compared with that of the scion.

Enhanced vegetative growth was often reported on grafted plants with hybrid squash rootstocks, however, no consistent differences in aboveground fresh weight, dry weight, leaf area, chlorophyll content, and stomatal conductance were observed between scion controls and grafted plants in the experiment. Growing plants for about six weeks in a greenhouse with 3.8 L pots might obscure differences between treatments. No significant difference in plant growth characteristics was observed among HI, OC and TA grafted plants, suggesting the three grafting methods did not differ in their effects on plant growth.

Conclusions

The experiment demonstrated that without root excision, plants grafted with hole insertion, one-cotyledon and tongue approach methods performed similarly regarding quality and growth characteristics, however, non-cotyledon method resulted in quality reduction of the grafted plants. Root excision was unsuccessful with tongue approach method, while it did not exhibit significant impacts on quality, as well as plant growth of one-cotyledon and hole insertion grafted plants. Further field studies are warranted to evaluate the yield performance of grafted melons as affected by grafting methods.

Economic analysis of different grafting methods should also be considered at selecting the most suitable methods in specific circumstances.

Table 2-1. The characteristics of hole insertion, one-cotyledon and tongue approach grafting methods. Characteristics Grafting methods Hole insertion One- Tongue cotyledon approach Rootstock regrowth Yes Yes No Location of graft union High High Low Requirements for post-grafting High High Low healing Requirement of grafting clips No Yes Yes Grafter training Moderate to Low Moderate to high high Grafting efficiency Moderate High Low Remaining rootstock cotyledons Two One None

Table 2-2. The percentages of live plants at 16 days after grafting in the first experiment. Grafting treatment Root treatment Excised root Intact root Hole insertion 95.8z 91.6 One-cotyledon 100 100 Non-cotyledon 91.6 87.5 Tongue approach 45.8 83.3 z Average of the three replications

Table 2-3. Quality of grafted plants at 16 days after grafting in the first experiment. Graft treatment z Root treatment Excised root Intact root Hole insertion 9.08y aAx 8.63 aA One-cotyledon 9.50 aA 9.42 aA Non-cotyledon 5.67 bA 5.38 bA Tongue approach 2.67 cB 7.92 aA z Two-factor factorial analysis of variance was conducted in the analysis y Quality of grafted plants were evaluated on a 0-10 scale: 0 = dead; 1 = almost dead; 2 = moderating between surviving or not; 3 = borderline but will probably die; 4 = severely stunted; 5 = moderately stunted; 6 = somewhat stunted; 7 = fair but not acceptable; 8 = borderline acceptable; 9 = good and acceptable but not the best acceptable; 10 = optimal results (Memmott and Hassell, 2010). x Means followed by the same lowercase letter within a column, and means followed by the same uppercase letters within a row were not significantly different according to Fisher’s least significant difference (LSD) at P ≤ 0.05.

Table 2-4. Aboveground biomass at 16 days after grafting. Treatmentz First experiment Second experiment Fresh weight Dry weight Fresh weight Dry weight (g) (g) (g) (g) Rootstock control 2.72 ay 0.28 a 2.02 a 0.25 a Scion control 2.07 a 0.18 b 1.23 b 0.10 b Hole insertion 2.68 a 0.23 ab 1.80 a 0.24 a One-cotyledon 2.23 a 0.18 bc 1.27 b 0.13 b Non-cotyledon 1.26 b 0.12 c 1.10 b 0.10 b z.Two-factor factorial analysis of variance was conducted in the analysis. No Root and Root × Grafting interaction effects were observed. y Means followed by the same letter were not significantly different according to Fisher’s least significant difference (LSD) at P ≤ 0.05.

Table 2-5. P values in the analysis of variance of effects of grafting treatment and root excision on the root and aboveground growth characteristics at 44 and 46 days after grafting in the first and second experiments, respectively. Factorz Root growth Aboveground growth characteristics characteristics Length surfac Fresh Dry Leaf area Stem Chlorophyll Stomatal e area weight weight diameter content conductance First experiment Grafting 0.362 0.061 0.032 0.000 0.471 0.000 0.810 0.008 Root 0.058 0.062 0.936 0.050 0.865 0.965 0.053 0.474 Grafting × Root 0.013 0.228 0.343 0.433 0.179 0.176 0.801 0.816 Second experiment Grafting 0.492 0.568 0.024 0.000 0.240 0.000 0.001 0.459 Root 0.711 0.726 0.219 0.440 0.056 0.126 0.253 0.438 Grafting × Root 0.897 0.946 0.534 0.668 0.508 0.322 0.740 0.552 z Two-factor factorial analysis of variance was used in the analysis.

Table 2-6. Root and aboveground growth characteristics of plants without root excision at 44 and 46 days after grafting in the first and second experiments, respectively. Treatment Root growth Aboveground growth characteristics characteristics Length (cm) surface Fresh Dry Leaf area Stem Chlorophyll Stomatal area weight (g) weight (g) (cm2) diameterz Contenty conductance (cm2) (mm) (CCIx) (mmol/m2s) First experiment Rootstock 2301.799 aw 714.741 a 191.07 a 22.60 a 3471.66 a 6.66 a 18.03 a 2078.43 a control Scion control 2688.182 a 761.273 a 161.61 a 15.39 c 3167.19 a 4.51 b 18.47 a 1333.35 ab Hole insertion 2988.523 a 669.417 a 163.31 a 17.05 bc 3197.58 a 7.19 a 19.37 a 1592.10 ab One-cotyledon 2402.692 a 732.402 a 193.62 a 18.82 b 3775.86 a 6.92 a 19.17 a 915.47 b Tongue 2368.076 a 817.823 a 174.03 a 17.26 bc 3444.63 a 7.46 a 18.03 a 1522.52 ab approach Second experiment Rootstock 4008.548 a 846.982 a 284.40 a 41.17 a 4126.06 a 9.63 a 27.27 a 1445.6 a control Scion control 3562.452 a 731.688 a 232.34 b 28.02 b 3890.30 a 6.46 b 17.47 b 1350.2 a Hole insertion 3632.287 a 692.679 a 244.59 ab 31.17 b 3668.99 a 8.43 a 19.50 b 1382.8 a One-cotyledon 3493.939 a 787.102 a 225.52 b 27.66 b 3591.10 a 8.72 a 20.13 b 1264.6 a Tongue 4560.601 a 833.892 a 213.98 b 25.36 b 2965.71a 8.29 a 22.23 ab 2145.2 a approach z Stem diameter was measured at 2cm above soil surface. y Chlorophyll content and stomatal conductance were measured on the newly expanded leaves at the longest stem x Chlorophyll concentration index. w Means followed by the same letter within a column were not significantly different according to Fisher’s least significant difference (LSD) test at P ≤ 0.05.

Figure 2-1. Root growth during the 16 days after grafting (DAG) in the first experiment. RC: rootstock control, SC: scion control, HI: hole insertion, OC: one- cotyledon, NC: non-cotyledon. A) Root length of plants with root excision. B) Root length of plants without root excision. C) Root surface area of plants with root excision. D) Root surface area of plants without root excision.

Figure 2-2. Root growth during the 16 days after grafting (DAG) in the second experiment. RC: rootstock control, SC: scion control, HI: hole insertion, OC: one-cotyledon, NC: non-cotyledon. A) Root length of plants with root excision. B) Root length of plants without root excision. C) Root surface area of plants with root excision. D) Root surface area of plants without root excision.

CHAPTER 3 SPECIALTY MELON CULTIVAR EVALUATION UNDER ORGANIC AND CONVENTIONAL PRODUCTION IN FLORIDA 

Introduction

Melon is a crop with diverse fruit characteristics. According to the International

Code of Nomenclature for Cultivated Plants, C. melo is divided into 16 groups within two subspecies: C. melo ssp. melo and C. melo ssp. agrestis (Burger et al., 2010). Sweet melons are mainly in the groups of Cantalupensis, Reticulatus, and Inodorus that are in the subspecies of C. melo ssp. melo, as well as the group of Makuwa that is in the subspecies of C. melo ssp. agrestis (Burger et al., 2010). The most commonly cultivated melon type in the United States is cantaloupe (Reticulatus group) (Sargent and

Maynard, 2009). In 2011, 72,590 acres of cantaloupe were planted in the United States with a production value of $349 million (U.S. Department of Agriculture, 2011). Besides cantaloupe, other melon types with distinctive fruit attributes are generally referred to as specialty melon in the United States.

Commonly known specialty melon include charentais, galia-type, ananas, persian, honeydew, casaba, crenshaw, canary, and asian melon. Among them, galia- type, ananas and persian melon are in the same Reticulatus group as cantaloupe

(Shellie and Lester, 2004). They produce aromatic, climacteric fruit that slip from vines with an identifiable abscission zone during ripening. Other specialty melon such as honeydew, casaba, crenshaw, and canary melon are in the Inodorus group. Fruit in the

Inodorus group generally lack aromatic flavor and do not slip from vines when ripening

(Lester and Shellie, 2004). Sweet asian melon is primarily in the Makuwa group. Their

 Reprinted with permission from ASHS journal

fruit are oblate, oval or pyriform shaped, and have white flesh with light aroma (Akashi et al., 2002).

With unique flavor, shape, and color, specialty melon generally command a higher price than ordinary muskmelon (Bachmann, 2002). In addition, demand for specialty melon is increasing in the United States because of the burgeoning ethnic diversity in the population, for whom specialty melon is staple fruit (Walters et al., 2008).

The expanding market also reflects consumer preference for healthy, new, unusual produce and cuisines (Greene, 1992).

Despite the increase in their popularity, according to a report by U.S. Department of Agriculture (2009), only 2 acres of honeydew melon were harvested in Florida in

2007, whereas data on other specialty melon types were not provided. The major production barriers are lack of disease resistant cultivars and low marketable yield

(Maynard, 1989). The challenge is more pronounced in Florida, since the humid subtropical conditions often result in high levels of disease pressures on melon production (Elmstrom and Maynard, 1992). Moreover, root-knot nematodes (RKN) thrive in Florida sandy soils, causing root galling and interfering with water and nutrient uptake of melon plants (Zitter et al., 1996). Therefore, specialty melon cultivars with disease resistance or tolerance would be valued by producers and create novel markets.

Breeding for disease resistance is one of the major goals in developing new melon cultivars. Combining high levels disease resistance with excellent horticultural characteristics, however, is often a challenging task in vegetable breeding (Guan et al.,

2012). Damages caused by several soilborne and foliar diseases are still the major

problems in melon production. Some specialty melon cultivars released in recent years had resistance to powdery mildew, downy mildew, and certain races of Fusarium oxysporum (Cornell University, 2011). However, limited information is available regarding their performance in Florida. Hence, research under Florida production conditions is needed to evaluate yield, disease performance, and fruit quality of these specialty melon cultivars in order to provide updated recommendations to Florida growers.

Consumer demand for organic produce, and interest in organic production among producers have continued to increase in recent years (U.S. Department of

Agriculture, 2010). Although the need for developing cultivars specifically suitable for organic crop production has been recognized (Adam, 2005), limited information is available regarding cultivar selection for organic melon production. While performance of cultivars may differ significantly between organic and conventional systems (Murphy et al., 2007), it is also suggested that conventional cultivars can be conveniently adapted to organic conditions (Lorenzana and Bernardo, 2008). The objective of this study was to evaluate the performance of different specialty melon cultivars in terms of yield potential, disease resistance, and fruit characteristics. In addition, these cultivars were assessed under both organic and conventional production to determine varietal response to the different systems.

Materials and Methods

Transplant Production

Specialty melon evaluated in this study consisted of 10 cultivars from five different types including ananas melon Creme de la Creme and San Juan, canary melon Brilliant and Camposol, asian melon Ginkaku and Sun Jewel, galia-type melon

Arava and Diplomat, and honeydew melon Honey Pearl and Honey Yellow. ‘Athena’ cantaloupe, one of the most popular muskmelon cultivars in the southeastern United

States, was included as a control (Table 3-1 and Figure 3-1). Seeds (Table 3-1) were sown into Pro-Tray 72-cell flats (Johnny’s Selected Seeds, Winslow, ME) on 20 Feb.

2011. Untreated or organic seeds were used for producing organic transplants. Peat- based medium (Natural & Organic 10; Fafard, Agawam, MA) and 2N-1.3P-0.8K fertilizer

(Organic fish and seaweed; Neptune’s Harvest, Gloucester, MA) was used for organic transplant production. Conventional potting soil with a mixture of vermiculite, bark, peat moss, and perlite (Metro-Mix 200; Sun Gro Horticulture, Bellevue, WA) and 20N-8.7P-

16.6K fertilizer (Peters Professional; United Industries, St. Louis, MO) were used for conventional transplant production.

Field Planting and Harvest

Melon plants with three true leaves were transplanted on 28 Mar. 2011 to the certified organic (Quality Certification Services, Gainesville, FL) and conventional field plots at the University of Florida Plant Science Research and Education Unit in Citra, FL

(USDA Hardiness Zone 9a). The soil texture at both sites is loamy sands. The organic field was used for conducting organic vegetable production research from 2006 to 2008.

Then bahiagrass (Paspalum notatum) and bermudagrass (Cynodon spp.) were grown in the field until melon experiment. The conventional field was fumigated using methyl bromide chloropicrin (50:50, by weight) at the rate of 448 kg·ha-1. Both the organic and conventional field experiments were arranged in a randomized complete block design with four blocks and 10 plants per cultivar in each block. Plants were grown in raised beds (75 cm wide and 23 cm high) covered with black plastic mulch. Drip tapes with a

30 cm emitter spacing were used for irrigation. The bed spacing and in-row spacing

were 183 and 91 cm, respectively. Fertilization program was based on the soil test conducted prior to bed preparation and the University of Florida recommendations for muskmelon production in sandy soils (Olson et al., 2011). The 10N-4.4P-8.3K fertilizer

(Premium Vegetable Grower Fertilizer; Southern States, Lebanon, KY) was applied preplant at 84 kg·ha-1 nitrogen (N) to the conventional field. Plants were fertigated 2 weeks after transplanting with 6N-0P-6.6K fertilizer (Dyna Flo; Chemical Dynamics,

Plant city, FL) at a weekly rate of 8.4 kg·ha-1 N for 10 weeks. In the organic field, 10N-

0.9P-6.6K fertilizer (All Season Fertilizer; Nature Safe, Cold Spring, KY) was applied preplant at 224 kg·ha-1 N.

Anthesis dates (9 out of 10 plants in each plot showed at least one open male flower) of each cultivar were recorded as days after transplanting (DAT). Melon fruit were harvested five times from 14 May to 2 June 2011. The first harvest dates of each cultivar were recorded. Cantaloupe was harvested at 3/4 slip, i.e., abscission zone between fruit and stem is 3/4 separated (Beaulieu et al., 2004). Galia-type and ananas melons were harvested when rinds turned color from green to light yellow, and burnt orange, respectively. The fruit of asian melon ‘Sun Jewel’ slipped off the vines when ripe, while ‘Ginkaku’ was harvested when rind turned yellow while still attached to vines.

The fruit of ‘Honey Yellow’ and ‘Honey Pearl’ were harvested based on aroma and external color changing from green to golden yellow or creamy white. Canary melon is less aromatic than honeydew melon. Hence, the change of fruit external color from green to golden yellow, was the primary index for harvesting canary melons ‘Brilliant’ and ‘Camposol’ in our study.

Marketable fruit weight and number were recorded on 10 plants for each cultivar per block. Percentage of cull fruit (immature, misshapen, or defective fruit with cracking, sunburn, or disease and insect damages) was calculated by dividing the number of unmarketable fruit by total fruit number.

Insect Pests Management

In the conventional field, esfenvalerate (Asana; DuPont, Wilmington, DE), methoxyfenozide (Intrepid; Dow AgroSciences, Indianapolis, IN), cyfluthrin (Baythroid;

Bayer CropScience, Research Triangle Park, NC), dimethylcyclopropane carboxylate

(Mustang Max; FMC Corporation, Philadelphia, PA), and carbaryl (Sevin XLR; Bayer

CropScience, Research Triangle Park, NC) were applied in a rotational scheme at the rates based on the product labels. OMRI (Organic Materials Review Institute) listed pesticides spinosad (Entrust; Dow Agrosciences, Indianapolis, IN) and pyrethrins

(Pyganic; McLaughlin Gormley King Company, Minneapolis, MN) were used in the organic field plot mainly for controlling melon aphids (Aphis gossypii).

Disease Evaluations

Disease severities were evaluated at the end of harvest. In the conventional field, severity of the combination of powdery mildew and downy mildew was evaluated based on visual estimation of the percentage of defoliated leaves to total canopy coverage of each plot. In the organic field, plant wilting was visible, thus a rating with 0-5 scale was developed to measure above-ground disease severity: 0 = no symptoms on leaves, stems, and crown; 1 = moderate necrosis on leaves, no symptoms on stems and crown;

2 = severe necrosis on leaves, water-soaked symptom, and some lesions on stems and crown, plants wilt in full sun; 3 = severe lesions on stems, large lesions girdle vines, part of the plant is wilting; 4 = plant totally wilts and cannot recover; 5 = plant is dead. After

the final harvest, all 10 plants in each organic plot were dug, and RKN galling was evaluated based on a 0-10 scale: 0 = no galls, 1 = very few small galls, 2 = numerous small galls, 3 = numerous small galls, some of which are grown together, 4 = numerous small galls and some big galls, 5 = 25% of roots severely galled, 6 = 50% of roots severely galled, 7 = 75% of roots severely galled, 8 = no healthy roots but plant is still green, 9 = roots rotting and plant dying, and 10 = plant and roots dead (Zeck, 1971).

Evaluations of Fruit Characteristics

At the third harvest, four typical marketable ripe melon fruit of each cultivar from each block were randomly selected for evaluations of fruit characteristics. Average fruit weight, fruit length (from stem-end to blossom-end), and fruit width (measured halfway between stem-end and blossom-end) were recorded. Soluble solids concentration

(SSC) and flesh firmness were also measured within 24 h following fruit harvest. SSC in fruit juice was determined by a refractometer (Ni; Atago, Bellevue, WA). Mesocarp firmness was measured using a penetrometer (Fruit Tester; Wagner Instruments,

Greenwich, CT) with an 8-mm plunger.

Statistical Analyses

Analysis of variance was performed using the Proc Mixed procedure of SAS program (version 9.2C for Windows; SAS Institute, Cary, NC). Fisher’s least significant difference test (α = 0.05) was conducted for multiple comparisons of different measurements among melon cultivars.

Results and Discussion

Anthesis and Harvest Dates

Anthesis dates ranged from 12 to 18 DAT for all the evaluated melon cultivars

(Table 3-2). ‘Honey Yellow’ has been reported as an early maturing melon cultivar in

Kentucky (Strang et al., 2007). In our study, in addition to ‘Honey Yellow’, the other honeydew melon ‘Honey Pearl’, asian melon ‘Sun Jewel’, and galia-type melon

‘Diplomat’ were also early-maturing cultivars. The first harvest date of these four cultivars was 52 DAT under both organic and conventional production. By contrast, the first harvest of canary melons ‘Brilliant’ and ‘Camposol’, and cantaloupe ‘Athena’ did not occur until 62 DAT (Table 3-2). Although the two canary melon cultivars in this study matured late, canary melon ‘SEM 6798’, ‘ACR 2056CN’, and ‘HSR 4325’ showed an earlier harvest time than honeydew melon cultivars in Delaware (Johnson and Ernest,

2010). As the rainy season normally starts in May in north and central Florida, using early maturing cultivars may help to alleviate negative impacts caused by warm and wet conditions.

Fruit Yields

Differential performance of crop cultivars between organic and conventional farming systems has been reported. Murphy et al. (2007) found that the highest yielding wheat (Triticum aestivum) cultivar in a conventional system did not exhibit the highest yielding potential in the organic system. In the present study, ‘Athena’ cantaloupe had the highest marketable yield under conventional production (10.7 kg/plant). However, marketable yield of ‘Athena’ ranked fifth in the organic field (6.8 kg/plant). Honeydew melon ‘Honey Yellow’ had significantly lower marketable yield (6.5 kg/plant) compared with canary melon ‘Camposol’ (8.3 kg/plant) under organic production but their yields were similar in the conventional field (8.9 kg/plant) (Table 3-3).

Although some specialty melon cultivars performed differently in the contrasting production systems, canary melon ‘Camposol’ consistently exhibited high yields in both organic and conventional fields (8.3 and 8.9 kg/plant, respectively). The high marketable

yield of ‘Camposol’ was primarily attributed to large fruit size and low percentage of culls. High yields and excellent fruit quality of canary melon cultivars were also observed by Strang et al. (2007) in Kentucky.

Asian melon ‘Ginkaku’ produced significantly greater numbers of marketable fruit per plant compared with other specialty melon cultivars in both fields (Table 3-3).

Marketable fruit number of the other asian melon ‘Sun Jewel’ was also significantly higher than other types of specialty melon cultivars in the organic field, but interestingly, such difference appeared to diminish in the conventional field. Other than the asian melon cultivars, no significant differences in the marketable fruit number were observed between ‘Athena’ cantaloupe and other specialty melon cultivars in either the organic or conventional field.

Asian melon ‘Sun Jewel’, galia-type melon ‘Diplomat’, and ananas melon ‘San

Juan’ had relatively high percentages of cull fruit under both organic and conventional production, resulting in lower marketable yields compared with other specialty melon cultivars. As a result of having a thin rind, premature cracking was the main issue with

‘Sun Jewel’ (Liu et al., 2012), particularly following heavy rainfall (Strang et al., 2007).

Galia-type melon ‘Diplomat’ also cracked prematurely at the stem end. Growing melons in greenhouse or high tunnels may overcome this problem (Cantliffe et al., 2002).

Ananas melon has a rapid ripening process (Schultheis et al., 2002). Strang et al.

(2007) suggested that these types of melons should be harvested daily when skin begins to change color. We harvested the melon fruit every 4-5 d instead of a shorter interval. As a result, some of the ‘San Juan’ melons were over-ripe, resulting in an increased percentage of culls.

Differential performance of the two cultivars within the same specialty melon type was also observed. By contrast with ‘Diplomat’, galia-type melon ‘Arava’ had an excellent performance in open-field conditions, with marketable yields of 7.2 and 7.6 kg/plant under organic and conventional production, respectively. ‘Arava’ was also a high-yielding galia-type melon in Delaware (Johnson and Ernest, 2010).

The majority of melon cultivars tended to have higher yields in the conventional than organic field. By conducting a comprehensive meta-analysis on various crops,

Seufer et al. (2012) concluded that organic crop yields were generally lower than conventional yields although the differences could vary considerably with production sites and cultivation systems. As organic matter decomposition and nitrogen mineralization rates are subject to environmental conditions, nitrogen availability was found to be the main yield-limiting factor in organic systems (Pang and Letey, 2000).

This was reflected by smaller-sized canopies of organically grown plants compared to conventional ones observed in our study. Nevertheless, yield differences between conventional vs. organic production varied among melon cultivars. The largest difference was observed for cantaloupe ‘Athena’, with a 58% marketable fruit weight increase in the conventional field compared with the organic field. Asian melon

‘Ginkaku’ and honeydew melon ‘Honey Yellow’ in the conventional field also showed an increase in marketable fruit weight by over 35% compared with melons in the organic field. However, ananas melon ‘Creme de la Creme’ and ‘San Juan’, canary melon

‘Camposol’, asian melon ‘Sun Jewel’, and galia-type melon ‘Arava’ exhibited similar marketable fruit weight in the organic and conventional fields.

Fruit Characteristics

Fruit weight and size varied significantly among specialty melon cultivars (Table

3-4). Averaged across the two production systems, asian melon produced the smallest fruit, with an average fruit weight of 0.7 and 0.8 kg for Ginkaku and Sun Jewel, respectively. One of the distinctive characteristics of asian melon was their elongated fruit shape. In particular, the fruit length of ‘Sun Jewel’ was almost 2-fold greater than the fruit width. Oval-shaped canary melon ‘Camposol’ produced the largest fruit, with average fruit weight of 2.7 and 2.3 kg in organic and conventional fields, respectively.

‘Athena’ and specialty melons other than the canary and asian melons were close to round-shaped (fruit length to maximum width ratio close to 1), with their fruit length ranging from 11.7 to 19.2 cm.

Of all the quality attributes, SSC, which reflects the level of sugar accumulation during fruit ripening, is used as a primary index in melon grading and marketing (Burger et al., 2000). According to the U.S. muskmelon grade standard, minimum values of SSC are 11 and 9% for Fancy and No.1 fruit, respectively (Shellie and Lester, 2004). For honeydew melon, the SSC of marketable fruit needs to reach at least 8% (Lester and

Shellie, 2004). Among all the evaluated specialty melon cultivars, honeydew melon

‘Honey Yellow’ had the highest SSC of 15.3 and 15.6% in the organic and conventional fields, respectively (Table 3-4), although it was not significantly different from ‘Honey

Pearl’ (14.8%) under conventional production. ‘Honey Yellow’ was also the sweetest melon reported in a specialty melon trial conducted in Kentucky (Strang, et al. 2007).

Although in the present study, the SSC of canary melons was ≤ 14%, Johnson and

Ernest (2010) reported that SSC of some canary melon cultivars may reach 16%.

Honeydew melon ‘Honey Pearl’ and asian melon ‘Sun Jewel’ also exhibited higher SSC

than ‘Athena’ in both fields, and canary melon ‘Brilliant’, ananas melon ‘San Juan’, and asian melon ‘Ginkaku’ also had higher SSC than ‘Athena’ under organic production.

‘Athena’ is the most popular cantaloupe melon cultivar grown in the southeastern United

States because of its disease resistance and shipping quality. Commercially, ‘Athena’ is harvested at half to 3/4 slip maturity stage. However, with respect to consumer perceived eating quality, ‘Athena’ did not show the best performance compared with other melon cultivars that were harvested at the same maturity stage (Simonne et al.,

2003).

Fruit firmness is an important component in fruit quality preference by consumers and postharvest shelf-life assessment. In Spain, consumers indicated a preference for tender melon fruit (Pardo et al., 2000), while in a sensory study performed in the United

States, honeydew melon breeding lines with firmer flesh exhibited a higher consumer rating on textural and overall eating quality compared with cantaloupe (Saftner, et al.,

2006). Thus, melons with both soft and firm flesh may be differentially favored by specific consumer groups. However, melons with low external firmness may reduce shipping capability and shorten postharvest shelf-life. In this study, the flesh firmness of

‘Athena’ cantaloupe, ananas, and galia-type melons ranged from 5.1 to 10.0 N, significantly lower than that of full-ripe canary and honeydew melons (flesh firmness ranged from 15.8 to 28.8 N). Cantaloupe, ananas and galia melons often exhibit climacteric fruit characteristics, while honeydew and canary melons are categorized as non-climacteric fruit (Burger et al., 2006). As a result of ethylene-dependent softening, the difference in flesh firmness is likely associated with the distinct ripening patterns of climacteric and non-climacteric melons (Pech et al., 2008). Compared with melons in

Reticulatus and Inodorus groups, little information is available about biochemical and physiological characteristics of melons in the Makuwa group (e.g., asian melon). Liu et al. (2004) reported that Golden No. 9, a melon cultivar in the Makuwa group, had a climacteric pattern of ethylene production. However, in the present study, ‘Ginkaku’ and

‘Sun Jewel’ from the same Makuwa group exhibited significantly higher flesh firmness compared with other climacteric melons. It is likely that melons in the Makuwa group might include both climacteric and non-climacteric fruit, or their softening could be controlled by ethylene-independent mechanisms.

Disease Observations

Plants grown in the organic field showed wilt symptoms during the fruit expansion stage. Stem canker and brown, gummy exudates were observed in the cortical tissues, with black specks (pycnidia) appearing on the cankers. Root galls were also observed on the plants. The diseases identified as causing these symptoms were gummy stem blight (caused by Didymella bryoniae, UF Plant Disease Clinic) (Zitter et al., 1996) and

RKN (a mixed population of M. javanica and M. incognita, UF Nematode Lab). Overall, canary melon ‘Camposol’ and galia-type melon ‘Arava’ and ‘Diplomat’, and honeydew melon ‘Honey Pearl’ and ‘Honey Yellow’ showed less aboveground symptoms compared with other melon cultivars (Table 3-5). Given that ‘Honey Yellow’ and

‘Diplomat’ exhibited severe RKN galling, it might be possible that they carry some potential tolerance to RKN infestation. Asian melon ‘Ginkaku’ was also heavily infested by RKN, which may be reflected by severe aboveground disease symptoms. ‘Athena’ exhibited less galling than the other specialty melon cultivars evaluated, except for

‘Camposol’ which showed a similar level of galling as ‘Athena’.

Powdery mildew and downy mildew occurred simultaneously at the end of the season, and spread to the whole field within a week. Some specialty melon cultivars evaluated in this study exhibited good foliar disease performances. For example, honeydew melon ‘Honey Yellow’, canary melon ‘Brilliant’ and ‘Camposol’, and asian melon ‘Sun Jewel’, had less than 40% defoliation. Among all the melon cultivars, asian melon ‘Ginkaku’ had the highest foliar disease severity rating. However, cultivar assessments over multiple seasons which include examination of individual diseases are needed to fully evaluate disease resistance and tolerance of these melon cultivars.

While high temperatures and humidity present unique challenges for high quality specialty melon production in Florida, interest in growing specialty melon as a high- value crop is increasing among local producers. In this cultivar evaluation trial where no fungicides or nematicides were used, canary melon ‘Camposol’, and honeydew melon

‘Honey Yellow’ and ‘Honey Pearl’ showed high yield potential and relatively good foliar disease performance, and produced high quality fruit, although the yield of honeydew melon cultivars appeared to be lower under organic production as compared with conventional production. Given the differential yield performance of some cultivars in organic and conventionally managed fields, selections of promising specialty melon cultivars for different cultivation systems warrant further study. For melons with higher percentages of cull fruit, protected culture may be a beneficial alternative to enhance marketable yield. Taking into consideration the grower needs and consumer demand, further research involving multiple years and locations are also expected to assess yield performance and fruit quality of different melon types and cultivars in different farming systems in Florida.

Conclusions

Table 3-1. Seed sources and descriptions of 10 specialty melon cultivars and ‘Athena’ cantaloupe grown in organic and conventional fields during Spring 2011 at Citra, FL Melon type Cultivar name Seed sourcez Fruit characteristicsy Ananas Creme de la W. Atlee Burpee & Co. Orange-yellow and lightly netted skin; fruit is creamy white, marbled (C. melo var. reticulatus) Creme with pale orange; fragrant, very sweet and slightly spicy San Juan Johnny’s Selected Orange-yellow rind and heavily netted; ivory colored flesh; pear-like, Seeds sweet flavor

Canary Brilliant Johnny’s Selected Dark-yellow and lightly wrinkled skin; white and juicy flesh, sweet and (C. melo var. inodorus) Seeds nutty in flavor Camposol Seedway, LLC Bright yellow rind and lightly wrinkled skin; white and juicy flesh; honey-dew like taste; large fruit

Asian Ginkaku Kitazawa Seed Co. Small, oval shaped; deep golden color with white stripes; white flesh, is (C. melo var. makuwa) quite thick, crisp, smooth and remarkably sweet Sun Jewel Johnny’s Selected Oblong fruit, lemon yellow with shallow white stripes; white flesh, crisp Seeds when ripe, moderately sweet

Galia-type Arava Johnny’s Selected Golden yellow and lightly netted rind; lime-green and juicy flesh; extra- Seeds sweet flavor with tropical and perfumed aromatics (C. melo var. reticulatus) Diplomat Johnny’s Selected Golden yellow and lightly netted rind; lime-green and juicy flesh; extra- Seeds sweet flavor with tropical and perfumed aromatics

Honeydew Honey Pearl Johnny’s Selected White-skinned fruit with white flesh; sweet flavor and grainy texture, (C. melo var. inodorus) Seeds like asian pears, round, uniform and middle-sized fruit Honey Yellow Johnny’s Selected Yellow-skinned fruit with orange flesh; juicy and very sweet; smooth Seeds skin, round, uniform and medium-sized fruit

Cantaloupe Athena Seedway, LLC Well-netted, sutureless; ripe fruit seldom crack and have a tough rind; (C. melo var. reticulatus) good shelf life even when harvested ripe; thick, sweet, orange flesh

Table 3-2. Anthesis and first harvest dates of 10 specialty melon cultivars and ‘Athena’ cantaloupe under organic and conventional production during Spring 2011 at Citra, FL. Cultivar Anthesisz First harvest (DATy) (DAT) Organic Conventional Organic Conventional Creme de la Creme 14 14 62 57 San Juan 14 14 57 57 Brilliant 18 16 62 62 Camposol 18 16 62 62 Ginkaku 18 18 57 62 Sun Jewel 16 16 52 52 Arava 14 14 62 57 Diplomat 14 14 52 52 Honey Pearl 16 14 52 52 Honey Yellow 14 12 52 52 Athena 14 12 62 62 z 9 out of 10 plants in each plot showed at least one open male flower. y DAT: Days after transplanting.

Table 3-3. Fruit yields of 10 specialty melon cultivars and ‘Athena’ cantaloupe under organic (Org) and conventional (Con) production during Spring 2011 at Citra, FL. Cultivar Marketable fruit wt Marketable fruit number Culls (%)y (kg/plant)z (no./plant) Org Con Org Con Org Con Creme de la 7.3 abx 7.4 bcd 4 cd 5 bcd 7.0 bc 12.0 de Creme San Juan 5.9 bc 5.6 cd 3 cd 4 cd 17.8 b 23.0 bc Brilliant 6.4 bc 7.5 bcd 4 cd 5 bcd 0 c 4.9 ef Camposol 8.3 a 8.9 ab 4 cd 5 bcd 0 c 0.9 f Ginkaku 5.6 c 8.0 abc 10 a 12 a 11.1 bc 17.0 cd Sun Jewel 5.6 c 5.5 cd 8 b 7 b 38.6 a 41.1 a Arava 7.2 ab 7.6 bcd 4 cd 6 bc 4.6 c 0.5 f Diplomat 4.0 d 5.0 d 3 d 3 d 36.9 a 31.6 ab Honey Pearl 7.2 ab 8.9 ab 5 c 6 bc 1.4 c 3.7 ef Honey Yellow 6.5 bc 8.9 ab 4 cd 5 bcd 0 c 3.3 ef Athena 6.8 bc 10.7 a 3 cd 5 bcd 1.5 c 1.0 ef P value 0.0002 0.0096 <0.0001 <0.0001 <0.0001 <0.0001 z wt: weight, 1 kg = 2.2046 lb y Percentage of unmarketable fruit for the season. x Means within a column followed by the same letter are not significantly different according to Fisher’s least significant difference (LSD) test at P≤0.05.

Table 3-4. Fruit weight, length, shape, soluble solids concentration (SSC), and flesh firmness of 10 specialty melons and ‘Athena’ cantaloupe under organic (Org) and conventional (Con) production during Spring 2011 at Citra, FL Cultivar Fruit wt (kg/fruit)z Fruit length (cm) Fruit shapey SSC (%) Flesh firmness (N) Org Con Org Con Org Con Org Con Org Con Creme de la 1.8 cx 2.0 b 15.9 ef 16.1 cd 1.0 ef 1.1 e 10.3 de 10.3 e 5.1 f 6.5 f creme San Juan 2.3 b 1.5 cd 17.3 cd 14.2 e 1.0 ef 1.0 e 11.7 c 11.9 cd 8.4 e 7.9 ef Brilliant 1.8 cd 1.8 bc 18.3 bc 17.7 b 1.2 c 1.2 d 13.9 b 12.4 c 23.4 c 28.8 b Camposol 2.7 a 2.3 a 20.4 a 20.1 a 1.2 c 1.3 c 10.4 de 11.0 de 24.4 c 21.3 c Ginkaku 0.7 g 0.7 f 14.7 gh 15.2 de 1.6 b 1.6 b 11.2 cd 10.5 e 38.3 a 35.3 a Sun Jewel 0.9 f 0.7 f 18.7 b 17.2 bc 1.9 a 1.9 a 13.1 b 14.2 b 19.9 d 21.7 c Arava 1.3 e 1.1 e 13.7 h 11.7 e 1.0 f 0.9 f 10.2 e 10.8 de 8.4 e 10.0 e Diplomat 1.7 cd 1.4 d 15.6 efg 14.2 e 1.1 ef 1.0 e 9.9 e 11.4 cd 6.7 ef 6.2 f Honey Pearl 1.7 cd 1.6 cd 16.6 de 15.0 de 1.1 de 1.0 e 11.9 c 14.8 ab 19.1 d 15.8 d Honey 1.6 d 1.6 cd 15.4 fg 14.8 de 1.0 ef 1.0 e 15.3 a 15.6 a 26.9 b 23.8 c Yellow Athena 2.3 b 1.6 cd 19.2 b 15.3 de 1.2 cd 1.0 e 9.8 e 11.7 cd 7.4 ef 7.7 ef P value <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 z wt: weight y Ratio of fruit length to maximum width. x Means within a column followed by the same letter are not significantly different according to Fisher’s least significant difference (LSD) test at P≤0.05.

Table 3-5. Aboveground diseases severity and root-knot nematode (RKN) gall ratings of 10 specialty melons and ‘Athena’ cantaloupe under organic and conventional production during Spring 2011 at Citra, FL. Cultivar Organic field Conventional field Disease severity RKN gall rating (0- Defoliation (%)x rating (0-5 scale)z 10 scale)y Creme de la creme 2.9 bcw 2.6 d 57.5 bc San Juan 3.4 ab 4.0 c 70.0 ab Brilliant 2.8 dc 3.6 c 37.5 cd Camposol 2.0 e 1.7 de 37.5 cd Ginkaku 3.7 a 5.0 ab 80.0 a Sun Jewel 2.9 c 1.9 d 37.5 cd Arava 2.2 e 4.4 bc 42.5 cd Diplomat 2.3 de 4.5 abc 70.0 ab Honey Pearl 2.3 de 3.7 c 75.0 ab Honey Yellow 2.3 de 5.4 a 27.5 d Athena 3.2 bc 0.9 e 57.5 bc P value <0.0001 <0.0001 <0.0001 z Disease symptoms were caused by gummy stem blight and RKN. The severity was rated using a 0-5 scale: 0 = no symptoms on leaves, stems, and crown; 1 = moderate necrosis on leaves, but no symptoms on stems and crown; 2 = severe necrosis on leaves, water-soak and some lesions on stems and crown, plants wilte in full sun; 3 = severe lesions on stems, large lesions girdle vines, part of plants is wilting; 4 = plants totally wilt and cannot recover; 5 = plants are dead. y Roots were rated on a 0-10 scale: 0 = no galls, 1 = very few small galls, 2 = numerous small galls, 3 = numerous small galls, some of which are grown together, 4 = numerous small and some big galls, 5 = 25% of roots are severely galled, 6 = 50% of roots are severely galled, 7 = 75% of roots are severely galled, 8 = no healthy roots but plant is still green, 9 = roots rotting and plant is dying, and 10 = plant and roots are dead. x Percentage of defoliation of whole plot (10 plants), attributed by powdery and downy mildews. w Means within a column followed by the same letter are not significantly different according to Fisher’s least significant difference (LSD) test at P≤0.05.

Figure 3-1. Pictures of melons in the field and harvested fruit of 10 specialty melon cultivars and ‘Athena’ cantaloupe. A-1) ‘Creme de la Creme’, A-2) ‘San Juan’ (ananas melon); B-1) ‘Brilliant’, B-2) ‘Camposol’ (canary melon); C-1) ‘Ginkaku’, C-2) ‘Sun Jewel’ (asian melon); D-1) ‘Arava’, D-2) ‘Diplomat’ (galia- type melon); E-1) ‘Honey Pearl’, E-2) ‘Honey Yellow’ (honeydew melon); F) ‘Athena’ cantaloupe.

CHAPTER 4 ROOT-KNOT NEMATODE RESISTANCE AND YIELD OF SPECIALTY MELONS GRAFTED ONTO CUCUMIS METULIFER 

Introduction

Specialty melons generally refer to melon types other than cantaloupe, which is the most widely grown melon type in the United States. Galia, ananas, persian, honeydew, casaba, crenshaw, canary, and asian melons are among the major specialty melons in the market (Strang, et al., 2007). Driven by the consumer preference for healthy and specialty produce, demand for specialty melons has been increasing in recent years (Guan et al., 2013).

Root-knot nematodes (RKN) are a major limiting factor for melon production in the southern United States, and in other semitropical and tropical regions of the world.

Meloidogyne incognita, M. javanica, and M. arenaria are the most widespread RKN species in southeastern United States (Zitter et al., 1996), and all of them cause root galling on specialty melons. More recently, two other species, namely M. enterolobii and

M. floridensis, also have been found in Florida infecting plants of the Cucurbitaceae family (Brito et al., 2004; Handoo et al., 2004). Within M. incognita, four races (races 1-

4) that are morphologically similar but different in their ability to reproduce on host plants were reported (Taylor and Sasser, 1978; Nickle, 1991). M. incognita race 1 was reported as the most common race, accounting for 60% in a worldwide collection

(Taylor and Sasser, 1978).

In conventional production systems, management of RKN in high value horticultural crops is largely dependent on soil fumigation (Duniway, 2002). Given the

 Reprinted with permission from ASHS journal

increasing costs and rising environmental concerns associated with soil fumigation, alternative approaches are needed for integrated management to improve sustainable melon production (Martin, 2003). Under organic production, RKN management is more challenging because of their wide host range and the lack of highly effective management strategies. The use of soil solarization, cover crops, soil amendment, and biological control for disease and nematode management often yields only moderate results (Oka et al., 2007).

Resistant cultivars are a cornerstone of RKN management in both vegetable and agronomic crops (Williamson and Roberts, 2010). Resistances in plants are either naturally occurring, or transferred to crop cultivars through breeding. Naturally occurring resistance was found in wild species of Cucumis, such as C. metulifer and C. anguria

(Fassuliotis, 1967; Thies and Levi, 2013; Wehner et al., 1991), and wild melon C. melo var. texanus (Faske, 2013), but it was absent in melon cultivars (Thomason and

McKinney, 1959).

Cucumis metulifer is a wild species from Africa. Its common name is African horned cucumber as the fruit has horn-like spines. In Africa, the fruit was consumed as food supplements by local people, and the fruit pulp is used for treatment of many diseases, including diabetes mellitus, hypertension, typhoid fever, malaria, and human immunodeficiency virus infections (Jimam, et al., 2010; Abubakar, et al.,2011). The medicinal properties were attributed to alkaloids, flavonoids, glycosides contained in the fruit pulp (Wannang, et al., 2008).

In addition to RNK resistance, C. metulifer also exhibited resistance to fusarium wilt (caused by Fusarium oxysporum), gummy stem blight (caused by Didymella

bryoniae), melon aphid (Aphis gossypii Glover) and melonworm (Diaphania hyalinata L.)

(Guillaume and Boissot, 2001; Igarashi et al., 1987; MacCarter and Habeck, 1974;

Trionfetti Nisini et al., 2002).

Incorporating RKN resistance from C. metulifer into C. melo was unsuccessful, as the interspecific hybrid failed to set fruit or produce viable seeds (Deakin et al.,

1971). Therefore, vegetable grafting was proposed as an alternative approach to take advantage of RKN resistance in wild Cucumis species (Kokalis-Burelle and Rosskopf,

2011; Sigüenza, et al., 2005; Thies et al., 2013). As grafted plants combine desirable characteristics from both rootstock and scion plants, RKN can be managed by grafting melon cultivars with desirable horticultural traits onto rootstocks containing RKN resistance. Grafting has been shown to be an effective tool for combating several soilborne diseases in cucurbit production worldwide (Davis et al., 2008; Guan et al.,

2012). Unfortunately, all currently available commercial melon rootstocks are susceptible to many of the RKN species, thereby making development of resistant rootstocks a pressing need in melon production.

Cucumis metulifer can be used as a rootstock for grafting cantaloupe melons

(Kokalis-Burelle and Rosskopf, 2011; Lee and Oda, 2003; Sigüenza, et al., 2005; Thies et al., 2013). However, information is limited regarding its grafting compatibility with other melon types, and the rootstock effects on RKN management and yield of the grafted plants. In this study, C. metulifer was used as a rootstock for grafting honeydew and galia melons. We conducted a RKN inoculation study in the greenhouse, and two field trials under conventional and organic production systems. The objectives were to

determine the effects of C. metulifer on RKN management, yield performance, and fruit quality of grafted plants.

Materials and Methods

Plant Materials

Two specialty melons, honeydew melon ‘Honey Yellow’ (C. melo var. inodorus) and galia melon ‘Arava’ (C. melo var. reticulatus) (Johnny’s Selected Seeds, Winslow,

ME) were evaluated as scions grafted onto C. metulifer rootstock. A C. metulifer line

(USVL-M0046) was selected for RKN resistance and grafting compatibility with cantaloupe melons from Plant Introduction (PI) 526242 (U.S. PI C. metulifer Collection)

(J.A. Thies, U.S. Vegetable Laboratory, USDA, ARS, Charleston, SC, unpublished).

USVL-M0046 was self-pollinated to produce S2 and S3 seeds (provided by USDA,

ARS, Charleston, SC), which were used as the C. metulifer rootstocks in the present greenhouse and field studies, respectively.

Greenhouse RKN Study

The greenhouse RKN inoculation study was conducted during Fall 2011.

Honeydew melon ‘Honey Yellow’ scions were grafted onto C. metulifer rootstock. Non- grafted and self-grafted ‘Honey Yellow’, as well as non-grafted C. metulifer were included as controls.

The RKN (M. incognita race 1) inoculum used in this experiment was provided by

Dr. Donald W. Dickson ( It was obtained from an isolate that originated from tobacco grown in north Florida, and was maintained and multiplied on tomato ‘Rutgers’

(Solanum lycopersicum) in a greenhouse). The RKN species and race were identified by their isozyme patterns resolved by polyacryalimide electrophoresis and differential host tests (Taylor and Sasser, 1978).

Rootstock and scion seeds were planted on 8 and 10 July 2011, respectively.

Seeds were sown into 128-cell styrofoam flats (Sun City, FL). The cells were filled with conventional potting soil with a mixture of vermiculite, peat moss, perlite, and starter nutrient (Metro-Mix 200; Sun Gro Horticulture, Bellevue, WA). Seedlings were grafted on July 22, using the one-cotyledon method (Davis et al., 2008). After grafting, plants were immediately placed into a light-blocking healing chamber in the greenhouse where temperature was maintained at 28 ± 3 ºC and relative humidity was 90% to 95%. After three days, humidity in the healing chamber was gradually reduced, and grafted plants were exposed to light. At seven days after grafting, plants were removed from the healing chamber and grown in the greenhouse for another seven days. Completely healed grafted plants and non-grafted plants were then transplanted individually into 15- cm-diam. clay pots containing pasteurized soil (89% sand, 3% silt, 5% clay; pH 6.1,

1.1% organic matter) and a slow release fertilizer 18N-2.6P-9.9K (Osmocote, The

Scotts Company, Marysville, OH) added at 6 g N/pot. Seven days after transplanting, the plants were then moved to a shade house and inoculated with 5,000 eggs or juveniles of M. incognita race 1. 10 mL (500 eggs or juveniles/mL) of nematode inoculum were evenly dispensed into three 6-cm-deep holes around the plants, and topped with moist soil to protect eggs from desiccation. Plants were arranged in a completely randomized design with five plants that served as five replications in each treatment.

Plants were grown in the shade house for eight weeks with air temperatures ranging from 24 to 39 ºC. Fertilized female flowers were removed to prevent fruit formation. The experiment was terminated on 15 Oct. Roots were washed gently and

allowed to air dry. Then the roots were stained with 10% (v:v) solution of red food coloring (Thies et al., 2002) to aid with an estimation of the amount of galling and egg masses based on a 0-5 scale (Taylor and Sasser, 1978). The number of eggs per root system was counted after their extraction using a 0.25% sodium hypochlorite solution and blender technique (Hussey and Barker, 1973). Eggs were quantified with a nematode chamber counting slide under a 40× magnification microscope. Nematode reproduction factor (Rf) was calculated as the ratio of final eggs recovered to the initial inoculum number.

Field Study

Two field experiments were conducted during Spring 2012, one in a certified organic field (Quality Certification Services, Gainesville, FL) and another in a fumigated conventional field. Honeydew melon ‘Honey Yellow’ and galia melon ‘Arava’ scions were grafted onto C. metulifer rootstock for the field trials. Untreated ‘Honey Yellow’ seeds were used for both trials, and untreated and organic ‘Arava’ seeds were used for the conventional and organic trials, respectively. Seeds of C. metulifer and melon cultivars were sown on 20 and 22 Feb., respectively. A peat-based medium (Natural &

Organic 10; Fafard, Agawam, MA) was used for organic transplants. 2N-1.3P-0.8K fertilizer (Organic fish and seaweed; Neptune’s Harvest, Gloucester, MA) was applied three times a week after seed germination at a concentration of 90 mg/L (based on N) for organic transplants. Conventional potting soil Metro-Mix 200 was used for conventional seedling production. Two weeks after seed germination, conventional plants were fertilized twice a week with 20N-8.7P-16.6K (Peters Professional; United

Industries, St. Louis, MO) at a concentration of 120 mg/L (based on N). Plants were grafted on 8 Mar. as previously described for the greenhouse study.

The field experiments were conducted at the University of Florida Plant Science

Research and Education Unit (PSREU), Citra, FL. The organic site was naturally infested with RKN. The RKN susceptible okra ‘Clemson Spineless’ (Abelmoschus esculentus) was grown in the plots from Aug. to Dec. 2011 to increase the RKN population density. The beds in both organic and conventional fields were 76 cm wide x

23 cm high and row centers were spaced at 1.8 m. Beds in the conventionally managed field were prepared and fumigated with methyl bromide-chloropicrin (50:50, by weight) mixture at a dosage of 454 kg/ha three weeks before transplanting. Fumigant was applied 25 cm deep in bed, and the beds were immediately covered with low-density black polyethylene film (Intergro, Safety Harbor, FL). A double-wall single drip tube

(Chapin, Watertown, NY) with emitters spaced 30.5 cm apart and a flow rate of 1.9

L/min/30.5 m was placed under the polyethylene mulch near the row center. Drip irrigation was applied twice or three times daily based on plant growth and need throughout the season. In both fields, the treatments were: non-grafted ‘Honey Yellow’ and ‘Arava’, and ‘Honey Yellow’ and ‘Arava’ scions grafted onto C. metulifer rootstock.

Self-grafted ‘Honey Yellow’ and ‘Arava’ were also planted as controls in the organic field. The experimental design for both field trials was a randomized complete block design with five replications and eight plants per treatment per replication.

Melon plants were transplanted into the organic and conventional fields on 29

Mar. 2012. Plant in-row spacing was 0.9 m. In the organic field, 10N-0.9P-6.6K fertilizer

(All Season Fertilizer; Nature Safe, Cold Spring, KY) was applied preplant at 227 kg

N/ha and supplemented by weekly injection of liquid fertilizer 2N-1.3P-0.8K (Neptune’s

Harvest; Gloucester, MA) at a rate of 2.3 kg N/ha. In the conventional field, 10N-4.4P-

8.3K fertilizer (Premium Vegetable Grower Fertilizer; Southern States, Lebanon, KY) was applied preplant at 85 kg N/ha, and 6N-0P-6.6K fertilizer (Dyna Flo; Chemical

Dynamics, Plant City, FL) was injected weekly at a rate of 14.3 kg N/ha. Cucurbit disease and insect controls for both conventional and organic crops followed the

University of Florida Institute of Food and Agricultural Sciences Extension recommendations (Olson and Santos, 2010). All pesticides used in the organic field were approved by the Organic Material Review Institute (OMRI). Plots were hand weeded in the organic field. In the conventional field, glyphosate (Roundup; Monsanto

Company, St. Louis, MO) and halosulfuron-methyl (Sandea; Gowan Company, Yuma,

AZ) were applied preplant for weed control.

Melons were harvested seven times from 21 May to 10 June, and nine times from 21 May to 22 June in the organic and conventional fields, respectively. ‘Arava’ was harvested at full-slip stage, and ‘Honey Yellow’ was harvested based on external fruit color. At the first harvest, soluble solids contents (SSC) of 10 ‘Honey Yellow’ melons with slight variations in fruit color were tested. Among them, the highest SSC was 16%, and the color that corresponded to the highest SSC was then used as the harvest index for ‘Honey Yellow’ (Guan et al., 2013). Immature melons were also harvested at the final harvest and separated from ripened fruit. Fruit were weighed individually. Small fruit (weighing less than 0.45 kg), immature, and misshapen fruit, and fruit with cracking and sunburn, as well as defective fruit with insect or disease damage were categorized as unmarketable fruit.

After the final harvest of the organic field, the root systems of eight plants were dug and rated for root-knot nematode galling on a 0-10 scale (Zeck, 1971). Soil cores

(1.75-cm diam. × 20-cm depth) were collected in the root zone of six plants in the center of each plot. Each soil sample was thoroughly mixed and second-stage juveniles (J2) were extracted from 100-cm3 soil of each sample using a centrifugal-flotation method

(Jenkins, 1964). In addition, two RKN females were excised from each of five randomly selected plants from the organic field, and were identified to species using specific polymerase chain reaction primers in the lab of Dr. Donald W. Dickson (Dong et al.,

2001). In the conventional field plot, 20 randomly selected plants for each melon cultivar were dug and rated for galling.

Statistical Analyses

Analysis of variance was performed using the Proc Glimmix program of SAS statistical software package for Windows (Version 9.2C for Windows, SAS Institute,

Cary, NC). Tukey’s honestly significant difference test (α = 0.05) was conducted for multiple comparisons of different measurements among treatments.

Results and Discussion

Greenhouse RKN Study

The reproduction factor (Rf) values were used to determine RKN resistance.

Plants with Rf > 1 were considered susceptible (Sasser et al., 1984). C. metulifer exhibited resistance (Rf = 0.48) and ‘Honey Yellow’ was susceptible (Rf = 22.0) to M. incognita race 1 (Table 4-1).

Most of the previous studies on RKN resistance of C. metulifer were conducted with M. incognita race 3 or an unspecified race of M. incognita (Dalmasso et al., 1981;

Fassuliotis, 1967; Nugent and Dukes, 1997; Sigüenza et al., 2005; Thies and Levi,

2013; Walters et al., 2006). In the present experiment, M. incognita race 1was used as the inoculum to assess the RKN resistance of C. metulifer. We recovered 2,395 RKN

eggs per plant from an initial inoculum level of 5,000 eggs per plant, which indicated that C. metulifer has resistance to M. incognita race 1. This finding was similar to that reported by Nugent and Dukes (1997) in which 2,900 eggs per plant were extracted from roots of C. metulifer plants that had been inoculated with 5,000 eggs per plant of

M. incognita race 3. In addition to M. incognita, C. metulifer has also been reported resistant to M. arenaria and M. javanica (Walters et al., 1993). Fassuliotis (1970) found no hypersensitive reaction to infection by M. incognita, but the development of J2 to adult was delayed in C. metulifer.

‘Honey Yellow’ grafted onto C. metulifer showed significantly lower root gall index

(GI), egg mass index (EMI), and Rf than non-grafted and self-grafted ‘Honey Yellow’, indicating grafting RKN susceptible melon onto C. metulifer was effective in reducing

RKN reproduction. No significant differences in EMI and Rf were observed between non-grafted C. metulifer and ‘Honey Yellow’ grafted onto C. metulifer.

Field Study of RKN Management

Recorded eight days after grafting, the survival rates of ‘Honey Yellow’ and

‘Arava’ grafted onto C. metulifer were both above 80%, which were similar to that of self-grafted melons. Graft incompatibility was not observed between the two specialty melon cultivars and the C. metulifer rootstock.

No root galling was observed on plants in the conventional field, indicating RKN infestation was reduced to an undetectable level in the fumigated soil. The RKN species in the naturally infested organic field was identified as M. javanica. Both non-grafted and self-grafted ‘Honey Yellow’ and ‘Arava’ plants exhibited severe galling (GI > 4) (Table 4-

2). Root galling was significantly reduced on ‘Honey Yellow’ and ‘Arava’ grafted onto C. metulifer (GI < 1). Grafting with C. metulifer rootstock also reduced J2 numbers in the

soil compared with non-grafted melon plants, suggesting that grafting RKN susceptible specialty melon cultivars onto C. metulifer could be an effective approach in reducing

RKN damage and decreasing nematode population densities in the soil.

Preplant RKN density can have an important impact in plant growth and yield

(Barker and Olthof, 1976). Crop rotation is commonly suggested as a management tactic for reducing RKN population densities in the soil. Thies et al. (2003) demonstrated that double-cropping cucumber and squash following RKN resistant bell pepper increased the yields of cucumber and squash. Yield improvement was also observed on muskmelons that were double-cropped after RKN resistant tomato (Hanna, 2000). RKN population densities for ‘Honey Yellow’ and ‘Arava’ scions grafted onto C. metulifer were about 99.7% and 97.6% lower, respectively, than non-grafted plants of the same melon cultivars in the organic field. Thus, incorporating melons grafted onto C. metulifer into a double-cropping system with RKN susceptible vegetables may be an effective approach to improve overall crop yields.

Fruit Yields in the Organic and Conventional Field Experiments

In the organic field experiment, despite the improved RKN management, no significant differences in total and marketable yields were observed when comparing

‘Honey Yellow’ and ‘Arava’ grafted onto C. metulifer with non-grafted and self-grafted melon plants (Table 4-3). Similar results were also reported on C. metulifer grafted muskmelons that exhibited resistance to Fusarium wilt, but did not show yield improvement as opposed to the non-grafted muskmelons (Trionfetti Nisini et al., 2002).

Yield improvement by grafting with disease resistant rootstocks was more pronounced when disease pressure was at a relatively high level. According to Barrett et al. (2012b), tomato grafted with RKN resistant rootstock ‘Multifort’ had significantly

higher yield when almost the entire roots of the non-grafted plants were severely damaged by RKN (GI > 8). However, yield improvement was not observed when gall index of non-grafted plants were between 6 and 7. Using the same 0-10 gall index scale

(Zeck, 1971) in the present experiment, the average gall index of non-grafted and self- grafted ‘Honey Yellow’ and ‘Arava’ were 6.92 and 4.82, respectively. This was in the lower range of RKN damage compared to what was previously reported (Barrett et al.,

2012b).

In the conventional field, no significant differences in total and marketable yields were observed between non-grafted ‘Arava’ and ‘Arava’ grafted onto C. metulifer rootstock. However, total yield (but not marketable yield) of ‘Honey Yellow’ grafted onto

C. metulifer was significantly lower than that of non-grafted ‘Honey Yellow’ in the conventional field (Table 4-3). The smaller stem diameter below the graft union was observed in contrast to that above the graft union. The growth vigor of C. metulifer as a potential rootstock deserves more investigations with respect to its impact on growth and development of the grafted melon plants.

Within each melon weight class, no significant differences were observed for numbers of marketable fruit among grafting treatments. There was no indication that grafting ‘Honey Yellow’ and ‘Arava’ scions onto C. metulifer rootstock affected fruit size and uniformity (Table 4-4). Although some studies showed that grafting melons onto

Cucurbita interspecific hybrid rootstock increased single fruit weight (Crinò et al., 2007), it may not be the case for melons grafted onto C. metulifer rootstock.

Conclusions

Specialty melons grafted onto C. metulifer exhibited less root galling, and RKN population densities in the rhizosphere than those for non-grafted and self-grafted

melons. Although yield was not improved by grafting, incorporating specialty melons grafted onto C. metulifer into a double-cropping system with RKN susceptible vegetables may be an alternative approach to manage Meloidogyne spp. and enhance production of high-value vegetables in RKN infested fields. Future breeding efforts may be directed to develop more vigorous C. metulifer rootstocks with greater potential for yield enhancement in grafted melon production.

Table 4-1. Root gall index (GI), egg mass index (EMI), egg recovery, and reproduction factor (Rf) of grafted and non-grafted honeydew melon ‘Honey Yellow’ in the greenhouse study (2011). Treatmentz GIy EMI Egg recoveryx Rfw NGHY 5.0 au 5.0 a 110,027 a 22.0 a NGCm 0.8 c 0.4 b 2,395 b 0.48 b HY/Cm 1.6 b 0.4 b 3,339 b 0.68 b HY/HY 5.0 a 4.6 a 141,500 a 28.3 a z NGHY = non-grafted ‘Honey Yellow’; NGCm = non-grafted C. metulifer; HY/Cm = ‘Honey Yellow’ grafted onto C. metulifer; HY/HY = self-grafted ‘Honey Yellow’. Plants were inoculated with 5,000 eggs or juveniles of M. incognita race 1. y GI and EMI were rated on a 0-5 scale: 0 = no galls or egg masses; 1 = 1-2 galls or egg masses; 2 = 3-10 galls or egg masses; 3 = 11-30 galls or egg masses; 4 = 31-100 galls or egg masses; 5 = more than 100 galls or egg masses (Taylor and Sasser, 1978). x Egg recovery = Number of extracted root-knot nematode eggs from the entire root system of each plant. w Rf = egg recovery / initial inoculum. u Means within a column followed by the same letter were not significantly different according to Tukey’s honestly significant difference test at P ≤ 0.05.

Table 4-2. Root gall index (GI) and numbers of Meloidogyne javanica second-stage juveniles (J2) in soil of grafted and non-grafted honeydew melon ‘Honey Yellow’ and galia melon ‘Arava’ in the organic field study (2012). Treatmentz GIy Meloidogyne density (J2/100 cm3 of soil)

Honey Yellow NGHY 7.14 ax 378.2 a HY/HY 6.70 a 515.6 a HY/Cm 0.08 b 1.2 b Arava NGAr 5.20 a 200.2 a Ar/Ar 4.45 a 140.2 ab Ar/Cm 0.15 b 4.8 b z NGHY and NGAr = non-grafted ‘Honey Yellow’ and ‘Arava’, respectively; HY/HY and Ar/Ar = self-grafted ‘Honey Yellow’ and ‘Arava’, respectively; HY/Cm and Ar/Cm = ‘Honey Yellow’ and ‘Arava’ grafted onto C. metulifer, respectively. y GI was rated on a 0-10 scale: 0 = no gall; 1 = very few small galls; 2 = small galls, more numerous and easy to detect; 3 = numerous small galls, some may grow together; 4 = numerous small galls, some big galls are present, but roots are still functioning; 5 = 25% of the root system is out of function due to severe galling; 6 = 50% of the root system is out of function due to severe galling; 7 = 75% of the root system is heavily galled and lost for production; 8 = no healthy roots are left, the nourishment of the plant is interrupted, but the plant is still green; 9 = the completely galled root system is rotting, the plant is dying; 10 = plant and roots are dead (Zeck, 1971). x Means within a column followed by the same letter were not significantly different according to Tukey’s honestly significant difference test at P ≤ 0.05.

Table 4-3. Total and marketable fruit yields (kg/plant) of grafted and non-grafted honeydew melon ‘Honey Yellow’ and galia melon ‘Arava’ in the organic and conventional field studies (2012). Treatmentz Organic Conventional Total yield Marketable yield Total yield Marketable yield Honey Yellow NGHY 4.93 ax 3.85 a 10.20 a 9.06 a HY/Cm 3.81 a 3.26 a 8.53 b 7.44 a HY/HYy 4.30 a 3.58 a Arava NGAr 5.85 a 4.21 a 10.06 a 9.59 a Ar/Cm 5.20 a 2.95 a 9.62 a 8.54 a Ar/Ar 5.68 a 3.60 a z NGHY and NGAr = non-grafted ‘Honey Yellow’ and ‘Arava’, respectively; HY/HY and Ar/Ar = self-grafted ‘Honey Yellow’ and ‘Arava’, respectively; HY/Cm and Ar/Cm = ‘Honey Yellow’ and ‘Arava’ grafted onto C. metulifer, respectively. y Self-grafted ‘Honey Yellow’ and ‘Arava’ were only evaluated in the organic field study. x Means within a column followed by the same letter were not significantly different according to Tukey’s honestly significant difference test at P≤ 0.05.

Table 4-4. Marketable fruit number per plant in different fruit size categories of grafted and non-grafted honeydew melon ‘Honey Yellow’ and galia melon ‘Arava’ in the organic and conventional field studies (2012). Treatmentz Organic Conventional 0.45- 1.36 - 1.81 - >2.25 0.45- 1.36 - 1.81 - >2.25 1.35 1.80 2.25 kg 1.35 1.80 2.25 kg kg kg kg kg kg kg Honey Yellow NGHY 1.52 1.00 0.52 0.10 0.77 2.10 1.80 0.50 HY/Cm 1.27 0.75 0.47 0.05 1.30 1.57 1.32 0.35 HY/HYy 1.62 0.96 0.51 0.05 P-value NSx NS NS NS NS NS NS NS Arava NGAr 2.07 1.26 0.46 0.05 1.02 2.12 1.77 0.65 Ar/Cm 1.49 1.03 0.30 0.07 1.00 1.62 1.72 0.60 Ar/Ar 1.98 1.10 0.36 0.22 P-value NS NS NS NS NS NS NS NS z NGHY and NGAr = non-grafted ‘Honey Yellow’ and ‘Arava’, respectively; HY/HY and Ar/Ar = self-grafted ‘Honey Yellow’ and ‘Arava’, respectively; HY/Cm and Ar/Cm = ‘Honey Yellow’ and ‘Arava’ grafted onto C. metulifer, respectively. y Self-grafted ‘Honey Yellow’ and ‘Arava’ were only evaluated in the organic field study. x NS = Non-significant, i.e., P > 0.05.

CHAPTER 5 STUDYING QUALITY ATTRIBUTES OF GRAFTED SPECIATLY MELONS USING BOTH CONSUMER SENSORY ANALYSIS AND INSTRUMENTAL MEASUREMENTS

Introduction

Melon (Cucumis melo L.) is an important component of fresh consumed vegetables and fruits in the U.S. In 2011, consumptions of cantaloupe and honeydew melons were 4.5 kg per capita, ranked six among fresh vegetables (U.S. Department of

Agriculture, 2011). In addition to the most consumed melon types, demand for specialty melons (charentais, galia, ananas, persian, orange-fleshed honeydew, casaba, crenshaw, canary, and asian melons) has grown dramatically as more consumers prefer unique and healthy produce (Chapter 3). From 2001 to 2010, the yield of cantaloupe increased from 26656 to 28224 kg per hectare (U.S. Department of

Agriculture, 2011). However, loss of methyl bromide as a broad spectrum soil fumigant in the same period brought vegetable production considerable challenges (Noling and

Becker, 1994). In order to maintain the high yield and meet the domestic demand, innovative practices for controlling soilborne pests were needed.

Although relatively new in the U.S., vegetable grafting has been used in Asian and European countries for decades mainly for soilborne disease management in cucurbit and solanaceous crops (Guan et al., 2012; Louws et al., 2010). Similar to tree grafting, it unites two plants as a single plant. The grafted plant combines a scion plant with desirable horticultural characteristics and a rootstock plant that is resistant to target diseases (Lee et al., 2010). Several soilborne diseases were managed through grafting, such as fusarium wilt (caused by Fusarium oxysporum), monosporascus sudden wilt

(caused by Monosporascus cannonballus), gummy stem blight (caused by Didymella bryoniae), verticillium wilt (caused by Verticillium dahliae), and root-knot nematode

(caused by Meloidogyne spp.) (Guan et al., 2012). In addition to the disease management, grafted plants have also shown improved abiotic stress tolerance, and enhanced water and nutrient uptake (Schwarz et al., 2010).

As interest in melon grafting is growing in the U.S., its impacts on fruit quality are gaining more attention. This is particularly true for specialty melons, as they are usually marketed for unique fruit flavor and outstanding eating quality.

Previous studies in melon grafting generated contradictory results with respect to fruit quality attributes. The rootstock and scion combination is one of the major factors that contribute to the mixed results. For example, hybrid squash rootstock Cucurbita maxima Duchesne × C. moschata Duchesne ‘RS841’ exhibited no impact on the total soluble solids content (SSC) of melon fruit ‘Incas’ (Cucumis melo inodorus) (Crinò et al.,

2007), but reduced the SSC of ‘Piñonet Torpedo’ melons (Cucumis melo inodorus) (Fita et al., 2007). With the same scion cultivars, different rootstocks might exhibit different impacts. ‘Supermarket’ and ‘Proteo’ melons were grafted onto eight rootstocks in a two- year study, while C. maxima × C. moschata rootstock had no impact on SSC of the two melon cultivars, Benincasa hispida reduced SSC (Trionfetti Nisini et al., 2002).

Production conditions may also affect the quality attributes of fruit from grafted melon plants (Traka-Mavrona et al., 2000). Moreover, grafting might influence fruit ripening behavior. If fruit from grafted and non-grafted control plants were harvested simultaneously, it is likely that the differences in quality assesment could simply be a reflection of harvest maturity(Soteriou et al., 2014).

Fruit quality is a multivariate characteristic, and SSC and flesh firmness were among the most important melon quality attributes that were widely used for assessing grafted melon quality (Lester and Shellie, 2004; Shellie and Lester, 2004). Other quality related observations, such as pre-harvest internal decay, internal breakdown, abnormal fruit fermentation, fibrous flesh, and poor netting of grafted melons were found associated with fruit of grafted melons in the early Japanese and Korean literatures

(Rouphael et al., 2010). As the roles of aroma volatiles and health-related compounds in the determination of melon fruit quality are increasingly recognized, grafting effects on these characteristics were also studied (Condurso et al., 2012; Kolayli et al., 2010; iao et al., 2010; Yars and Sar , 2012). In addition, reduced fruit shelf-life following 1- methylcyclopropene treatment was found in cantaloupe ‘Athena’ grafted onto

‘Tetsukabuto’ rootstocks (C. maxima × C. moschata) (Zhao et al., 2011). Although a variety of quality attributes have been evaluated in grafted melons, only one study included a sensory analysis (Kolayli et al., 2010).

Sensory evaluation involves the interpretation of sensory experiences by human brains, and thus it can provide the most accurate prediction of how humans are likely to react to the fruit (Lawless and Heymann, 2010). Meanwhile, sensory evaluation provides an approach to study different quality variables and their impacts on the sensory attributes in an integrated manner, thus it is impossible to be replaced by individual instrumental measurement (Heintz and Kader, 1983). While substitution of instrumental measurements for sensory tests did not always work well (Aulenbach and

Worthington, 1974; Yamaguchi et al., 1977), it has been suggested that the overall

evaluation of melon fruit quality need to combine both instrumental measurement and sensory analysis (Aulenbach and Worthington, 1974).

Melon quality attributes as influenced by cultivars, ripening stages, and postharvest treatments were evaluated by combining consumer sensory analysis and instrumental measurements (Saftner and Lester, 2009; Senesi et al., 2002; Senesi et al., 2005; Vallone et al., 2013). However, well-designed consumer sensory analysis was seldom applied in assessing fruit quality of grafted melons. A reliable consumer sensory analysis normally involves 75 to 150 consumers (Lawless, 1998). A 9-point hedonic is commonly used, which was regarded as one of the most valid and reliable scales used in consumer sensory analysis.

In this study, grafted specialty melons with different rootstock and scion combinations were grown under three different production systems. Using both consumer sensory evaluation and instrumental measurement, this study assessed quality attributes of grafted specialty melons with the overall goal of enhancing our comprehensive understanding of rootstock induced grafting impacts on melon quality.

Materials and Methods

Melon Production

The field experiments were conducted in the Spring seasons of 2012 and 2013 at the University of Florida Plant Science Research and Education Unit in Citra, FL. In

2012, galia melon ‘Arava’ (C. melo L. var. reticulatus Ser.) and the honeydew melon

‘Honey Yellow’ (C. melo L. var. inodorus Naud.) were grafted onto each of the two rootstocks: hybrid squash ‘Strong Tosa’ (Cucurbita maxima Duchesne × Cucurbita moschata Duchesne), and Cucumis metulifer E. Mey. ex Naud. Seeds of the two melon scions were purchased from Johnny’s Selected Seeds (Winslow, ME). Hybrid squash

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rootstock seeds were donated by Syngenta Seeds (Research Triangle Park, NC), and

C. metulifer rootstock seeds were provided by the USDA ARS Vegetable Laboratory

(Charleston, SC). Non-grafted and self-grafted melon plants were included as controls.

Grafted plants and the controls were evaluated in three production systems, i.e., certified organic production (Quality Certification Services, Gainesville, FL), conventional production with soil fumigation, and conventional production without soil fumigation. Specific treatments included in each of the three production systems are listed in Table 5-1. Based on the findings from the 2012 studies, only ‘Arava’ melon scion was evaluated in the 2013 study. ‘Arava’ was grafted onto the ‘Strong Tosa’ rootstock and another hybrid squash rootstock ‘Carnivor’ (C. maxima Duchesne × C. moschata Duchesne). Grafted plants as well as non-grafted controls were planted in the fumigated conventional field in 2013.

Conventional and organic transplants were produced as previously described

(Chapter 4) Grafting was conducted by using the one-cotyledon method (Davis et al.,

2008). Plants were transplanted in the field at the three-true-leaf stage, on March 29 and April 10 in 2012 and 2013, respectively. The soil fumigant, methyl bromide: chloropicrin (50:50, by weight), was applied at the rate of 448 kg/ha three weeks before transplanting in the conventional field. Non-fumigated conventional field was treated with halosulfuron-methyl (Sandea; Gowan Company, Yuma, Arizona) for nutsedge control one week before transplanting at the rate according to product label. Production practices in both organic and conventional fields were followed as described previously

(Chapter 3). Experiments in all the fields were arranged in a randomized complete block

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design with five replications in 2012 and four replications in 2013. Eight plants were included per treatment per replication.

Harvests lasted from May 21 to June 14 in 2012, and from June 6 to June 20 in

2013. ‘Arava’ was harvested at the full-slip stage and ‘Honey Yellow’ was harvested based on fruit color and total soluble solids content. At the beginning of the first harvest,

SSC values of 10 ‘Honey Yellow’ melons with slight variations in fruit color were tested.

Among them, the highest SSC was 16%. The color that corresponded to the highest

SSC was then used as the harvest index for ‘Honey Yellow’ fruit (Chapter 4).

Sample Preparation

Analyses of the two melon cultivars were conducted separately on different days.

Each cultivar was analyzed twice in the 2012 harvest season (June 1 and 12 for ‘Arava’, and May 25 and June 13 for ‘Honey Yellow’). The day before the sensory analysis, melons were harvested and stored at 10 ºC overnight. Ten fully ripe melons were chosen from each treatment based on fruit size, i.e., approximately 1.5 kg per fruit, and absence of defects. Six treatments were included in each of the analyses. The first analysis of each melon cultivar included melons produced from the fumigated conventional field (i.e., fruit of non-grafted, self-grafted, and ‘Strong Tosa’ rootstock grafted melon plants) and the organic field (i.e., fruit of non-grafted, self-grafted, and C. metulifer rootstock grafted melon plants). The second analysis assessed melons from the fumigated and the non-fumigated conventional fields (i.e., fruit of non-grafted, and

‘Strong Tosa’ and C. metulifer rootstock grafted melon plants). In 2013, only one sensory analysis with three treatments, i.e., non-grafted ‘Arava’, ‘Arava’ grafted onto

‘Strong Tosa’ rootstock, and ‘Arava’ grafted onto ‘Carnivor’ rootstock, were conducted on June 14.

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On the day of consumer sensory analysis, each of the whole melon fruit were washed in tap water and dried with paper towel. They were then cut longitudinally into halves. Half of the samples were used for sensory analysis while the remaining counterparts were kept for instrumental measurements.

Consumer Sensory Analyses

Rinds and contiguous flesh (about 1.5 cm) of the melon halves were discarded.

The rest of the melon flesh was cut to roughly 3×3 cm cubes. Fruit cubes from ten halves of each treatment were well mixed and stored in a plastic container at 4 ºC during consumer sensory tests that typically lasted for five hours.

Consumer sensory analyses were conducted at the University of Florida Sensory

Analysis Lab in Gainesville, FL. The procedures of the sensory evolution and layout of sensory analysis lab were described previously (Barrett et al., 2012c). Each of the consumer sensory tests had 96 to 100 panelists. They were mostly students, and faculty and staff members on campus who reported they had eaten melons before. Six samples with 2 melon cubes of each sample were randomly arranged and presented to consumers. Consumers were first asked to answer demographic questions including gender, age, and melon consumption frequency. For each sample, consumers were asked to score overall acceptability, firmness liking and flavor liking using a 1-9 hedonic scale (9 = like extremely, 5 = neither like nor dislike, 1 = dislike extremely). Following hedonic scale questions, consumers were asked to describe firmness and sweetness levels using a 1-5 Just-about-right scale, e.g. 1= too soft, 2 = slightly too soft, 3 = just about right, 4 = slightly too firm, 5 = too firm (Lawless and Heymann, 2010). At last, consumers were asked to indicate whether they experienced off-flavor in the sample, and to describe the off-flavor they detected.

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Instrumental Measurements

Flesh firmness and SSC were measured in the 2012 samples, while flesh firmness, SSC, titratable acidity, and pH were measured in the 2013 samples. Flesh firmness was measured twice in the middle of the mesocarp of each melon half using a penetrometer (Fruit Tester; Wagner Instruments, Greenwich, CT) with an 8-mm plunger.

SSC was determined by a refractometer (AR200; Reichert Technologies, Depew, NY), while titratable acidity and pH were measured using Titrino (791S; Metrohm USA Inc.

Riverview, FL, USA) following the methods described previously (Zhao et al., 2011).

Statistical Analyses

Analysis of variance was performed using the Proc Glimmix procedure of SAS program (Version 9.2 C for Windows; SAS Institute, Cary, NC). Tukey’s honestly significant difference test (α = 0.05) was conducted for multiple comparisons of different measurements among treatments.

Results and Discussion

Fruit Quality of ‘Arava’ Scion Grafted onto Hybrid Squash Rootstocks

Consistent among all the evaluated production conditions in 2012 and 2013, the

‘Arava’ scion grafted onto ‘Strong Tosa’ rootstock (Ar/ST) reduced consumer rated overall acceptability and flavor liking compared with non-grafted ‘Arava’ (NGAr) (Table

5-2). Averaged among production conditions in 2012, consumers who rated the sweetness level as ‘Just about right’ of Ar/ST was 20% less than NGAr, while 36% more consumers considered the sweetness level of Ar/ST was either ‘Not sweet at all’ or ‘Not quite sweet enough’ (Table 5-3). As Just-about-right scale provided directional information for the hedonic questions (Van Trijp et al., 2007), the results suggested that consumers liked the Ar/ST melons less because they were not sweet enough. This was

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further confirmed by instrumental measurement. SSC that reflects the sugar content showed a significantly lower value in the melon flesh of Ar/ST than NGAr (Table 5-2).

The similar rootstock effects were also observed in the ‘Arava’ scions grafted onto

‘Carnival’ rootstock in 2013, which reduced overall acceptability, flavor liking, and SSC compared with NGAr (Table 5-4).

Hybrid squash rootstock is well known for the vigorous root system and large vessel elements. Water status of the grafted melon plants was enhanced as indicated by improved leaf water potential, leaf stomata conductance, transpiration rate, and the amount of xylem saps (Agele and Cohen, 2009; Jifon, 2010). It was suggested that the reduced SSC of fruit from grafted plants may be due to a water dilution effect (Albrigo,

1977). However, there was no direct evidence to support such assumption. Improved vegetative growths were commonly observed in plants grafted onto vigorous rootstocks

(Bie et al., 2010; Leoni et al., 1990). Whether the large leaf areas contribute to the modification of fruit quality is unclear. SSC of melons was reduced when 50% of the leaves were removed (Long et al., 2005), even though pruning is a common practice in melon production for promoting light infiltration and improving fruit quality (Cohen et al.,

1999; Yang et al., 2007). Heavier canopies were observed on the grafted ‘Arava’ plants compared with non-grafted controls, but pruning was not conducted in the present study. Further study was warranted to investigate the effects of pruning on melon quality of grafted plants. Other factors that deserve more attention are effects of grafting on fruit development. ‘Arava’ scion grafted onto hybrid squash rootstocks delayed anthesis of female flowers, but did not affect early harvest (Chapter 6). Considering fruit sugar content is correlated with the length of the fruit development period (Burger and

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Schaffer, 2007), stimulated fruit development of grafted melons may partially explain the reduced SSC.

Except for the late harvested melons in the fumigated conventional field, firmness liking of non-grafted ‘Arava’ was significantly higher than ‘Arava’ grafted onto hybrid squash rootstocks in all the other production conditions, whereas no significant differences in flesh firmness were detected by firmness measurement. Since consumers are untrained panelists who evaluate products as whole patterns, they lack the capability to separate individual product attributes (Lawless and Heymann, 2010). As a result, they may establish a positive correlation between two unrelated attributes, such as sweetness and firmness (Clark and Lawless, 1994). Therefore, the higher rating on firmness liking of non-grafted ‘Arava’ may be simply because consumers like its sweetness more than grafted melons. Another potential explanation could be that texture attributes other than firmness were affected by grafting, such as juiciness and adhesiveness (Kramer and Szczesniak, 1973). Because consumers were not asked to rate these attributes, they may express their feeling on other fruit texture attributes as firmness liking (Lawless and Heymann, 2010). A more accurate sensory specification on melon firmness may be provided by trained panelists.

Fruit Quality of ‘Arava’ Scion Grafted onto C. metulifer Rootstock

Organically produced ‘Arava’ grafted onto C. metulifer (Ar/Cm) decreased overall acceptability and flavor liking compared with NGAr. However, the negative rootstock effects on sensory properties were not detected when plants were grown in the non- fumigated conventional field. No differences in SSC and flesh firmness between NGAr and Ar/Cm were detected under either of the production conditions (Table 5-2).

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Known for root-knot nematode and fusarium wilt resistances, C. metulifer was tested as a rootstock for grafting melons (Sigüenza et al., 2005). However, unlike the hybrid squash rootstock, the improvement of soilborne disease resistance did not translate into yield enhancements (Trionfetti Nisini et al., 2002). Lacking rootstock vigor may be part of the reason, so the mechanisms by which C. metulifer affected melon quality were unlikely the same ones utilized by hybrid squash rootstocks. Melons grown in the organic and non-fumigated conventional fields yielded different results in consumer sensory properties. This may suggest the rootstock effects on melon fruit quality were subjected to influences of environmental conditions. C. metulifer rootstock did not affect SSC and flesh firmness, indicating that its effects on sensory properties might be attributed to quality attributes other than sweetness and firmness.

Off-flavor of Grafted ‘Arava’ Fruit

As the definition of off-flavor was not provided to the consumer panelists, their report of off-flavor in this study more likely reflected an expression of unpleasant flavor detected based on their prior experience of melon consumption. Although lacking capability of defining off-flavor, it was consistent among the tests that a greater percentage of panelists reported off-flavor in fruit from grafted ‘Arava’ than non- and self-grafted controls.

Panelists who found off-flavor in the organically produced Ar/Cm, NGAr, and

Ar/Ar were 32.6%, 17.9% and 25.3%, respectively (Table 5-3). The greater percentage of panelists finding off-flavor may partly explain the deterioration of sensory properties of Ar/Cm in the organic field. The same trend, albeit with a smaller difference (26.3% and 18.2% for Ar/Cm and NGAr, respectively) was observed for melons grown in the non-fumigated conventional field.

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‘Arava’ melons grafted onto hybrid squash rootstocks also exhibited greater off- flavor compared with NGAr grown under the same production conditions. The biggest difference was observed in the 2013 study. The percentages of panelists who detected off-flavor were 37.3% and 10.7% for Ar/ST and NGAr, respectively (Table 5-5).

Differences in the presence of off-flavors detected by the consumer panelists implied that the rootstock might affect melon volatile compositions, which was demonstrated by previous studies with various rootstock and scion combinations

(Condurso et al., 2012; Xiao et al., 2010; Yars and Sar , 2012). More than 240 volatile compounds were identified in muskmelon fruit (Kourkoutas et al., 2006). These volatiles and their unique combinations contribute to melon flavors and off-flavors. Identification and isolation of the compounds causing melon off-flavors are one of the focuses in sensory and volatile studies. Previous studies have identified some of the volatiles, such as hexanal and nonanal, which may lead to development of off-flavors during storage

(Beaulieu and Grimm, 2001), and some alcohols noted for fermented flavor and are regarded as “negative” compounds in melon flavor (Yars and Sar , 2012). Interestingly, contents of some alcohol compounds were found to be higher in grafted melon (Xiao et al., 2010). This may explain the higher number of reports of off-flavor in grafted melon fruits. However, due to the complicated nature of volatile compounds, how their synthesis and metabolism may be influenced by grafting is still largely unknown.

Fruit Quality of Grafted ‘Honey Yellow’ Fruit

Regardless of the production conditions and the rootstock selections, grafted

‘Honey Yellow’ did not exhibit any significant differences in sensory properties (overall acceptability, flavor liking, and firmness liking), SSC, and flesh firmness compared with non-grafted ‘Honey Yellow’ fruit (Table 5-6). In addition, the percentages of consumers

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who detected off-flavor were similar between non-grafted and ‘Strong Tosa’ grafted

‘Honey Yellow’ melons (Table 5-7). Our findings were in accordance with previous reports of insignificant influence of grafting on quality attributes of honeydew melons

‘Lefko Amynteou’ (Traka-Mavrona et al., 2000) and ‘Incas’ (Crinò et al., 2007),

However, it was also demonstrated that unsuitable rootstock might cause quality deterioration of the ‘Incas’ fruit (Verzera et al., 2014).

A distinct difference in grafting effect on fruit quality was observed in the two specialty melon cultivars. One of the fundamental differences between the two melon cultivars is the fruit ripening pattern (Paul et al., 2012). Galia melon ‘Arava’ produce climacteric fruit that exhibit an autocatalytic ethylene production peak during fruit ripening, while the ethylene production peak was not observed in non-climacteric honeydew melons (Guan et al., 2013; Pech et al., 2008). As many aroma compounds are only produced through ethylene-dependent pathways, climacteric melons generally have higher aroma levels than non-climacteric fruit (Obando-Ulloa et al., 2008).

Because climacteric galia melons are rich in aroma components, its sensory properties perceived by consumers might be more likely to be affected by grafting practice than honeydew melons.

Another difference between the two melon groups that deserve future study is that honeydew fruit (‘Honey Yellow’) showed a rapid dry weight gain during the early period of fruit growth and the initial stage of sugar accumulation (Pratt et al., 1977), while muskmelon (‘Arava’) exhibited an increase in the dry matter accumulation during the entire fruit development stages (Bianco and Pratt 1977). Considering the dry weight accumulation would require an efficient water removal mechanism (Schaffer et al.,

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1996), the enhanced water status in the grafted melon plants might affect the efficiency of sugar accumulation, which may have a more pronounced effect on fruit quality of muskmelons than honeydew melons, as it exhibited a continuous dry weight accumulation toward the end of fruit development.

Although rootstock, scion, and rootstock × scion interaction all contribute to the ultimate fruit quality, the present study indicated that the scion might play a more critical role in the process. A study using three cherry cultivars grafted onto five rootstocks with varied size-controlling potential found that the scion accounted for the highest percentage of variation in quality modification, and concluded that fruit quality was more of a scion-dependent character (Gonçalves et al., 2006). In citrus, it was also observed that some citrus cultivars may be naturally high in quality, thus grafting practice as well as rootstock choice may be less influential in these cultivars compared with low-quality cultivars (Castle, 1995).

Conclusions

Combining consumer sensory analysis and instrumental measurements provided a more thorough evaluation of the grafting effects on the quality attributes of specialty melons. Reduced SSC may be the primary reason for deteriorated sensory properties of galia melon ‘Arava’ fruit when plants were grafted with the hybrid squash rootstocks.

Melon off-flavor that might be contributed by volatile compounds could also explain the reduced flavor liking and overall acceptability of fruit from grafted ‘Arava’ plants.

Compared with non-grafted honeydew melon ‘Honey Yellow’ fruit, grafted ‘Honey

Yellow’ did not exhibit any significant differences in fruit sensory properties and instrumental measurements. The differential responses of the two specialty melon types to grafting practice in terms of fruit quality deserve further investigations. More in-depth

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studies especially those involving flavor-related volatile compounds and profiles are also warranted to better understand the rootstock and scion interaction effects on fruit quality and consumer perceived sensory properties of grafted melon fruit.

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Table 5-1. Grafting treatments included in certified organic field, non-fumigated conventional field, and fumigated conventional field in Spring 2012. Certified organic field Non-fumigated Fumigated conventional conventional field field ‘Arava’ and ‘Honey Yellow’ ‘Arava’ and ‘Honey Yellow’ ‘Arava’ and ‘Honey Yellow’ grafted onto C. metulifer grafted onto C. metulifer grafted onto ‘Strong Tosa’ rootstock rootstock rootstock None-grafted ‘Arava’ and ‘Arava’ and ‘Honey Yellow’ Non-grafted ‘Arava’ and ‘Honey Yellow’ grafted onto ‘Strong Tosa’ ‘Honey Yellow’ rootstock Self-grafted ‘Arava’ and None-grafted ‘Arava’ and Self-grafted ‘Arava’ and ‘Honey Yellow’ ‘Honey Yellow’ ‘Honey Yellow’

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Table 5-2. Consumer sensory evaluation and instrumental measurements of grafted ‘Arava’ melons in Spring 2012. Treatmentz Consumer sensory evaluationy Instrumental measurements Overall Flavor Firmness SSC Flesh firmness acceptability liking liking (ºBrix) (kg) Test 1x F NGAr 6.51 aw 6.32 a 6.46 a 11.43 a 1.49 a F Ar/Ar 6.17 a 6.07 a 6.17 ab 11.51 a 1.25 ab F Ar/ST 5.39 bc 5.20 bc 5.79 b 9.61 b 1.20 ab O NGAr 5.97 ab 5.62 ab 5.95 ab 7.62 c 0.95 b O Ar/Ar 5.04 c 4.72 c 5.80 b 7.32 c 1.02 b O Ar/Cm 5.04 c 4.78 c 5.73 b 7.74 c 0.93 b p-value <0.0001 <0.0001 0.0059 <0.0001 <0.0001 Test 2 F NGAr 6.36 a 6.36 a 5.88 ab 10.07 a 1.17 F Ar/Ar 5.74 bc 5.47 bc 5.77 ab 9.03 ab 1 31 F Ar/ST 5.55 cd 5.24 cd 5.59 b 8.72 b 1.29 NF NAr 6.18 ab 5.95 ab 6.21 a 9.81 a 1.55 NF Ar/Cm 5.72 bc 5.42 bc 5.82 ab 9.60 ab 1.18 NF Ar/ST 5.12 d 4.81 d 5.39 b 8.82 b 1.28 P-value <0.0001 <0.0001 0.0012 0.0008 0.3308 z NGAr, Ar/Ar, Ar/ST, Ar/Cm indicated non-grafted ‘Arava’, self-grafted ‘Arava’, ‘Arava’ grafted onto ‘Strong Tosa’ rootstock and ‘Arava’ grafted onto C. metulifer rootstock, respectively. F, NF, and O indicated melons produced from fumigated conventional field, non-fumigated conventional field, and organic field, respectively. yAttributes were evaluated on a 9-point hedonic scale: 1=Dislike extremely, 2=Dislike very much, 3=Dislike moderately, 4=Dislike slightly, 5=Neither like nor dislike, 6=Like slightly, 7=Like moderately, 8=Like very much, 9=Like extremely. xTest 1 and Test 2 were conducted on June 1 and June 12, 2012, respectively. Test 1 included 96 panelists, and Test 2 included 100 panelists. wMeans within a column follower by the same letter were not significantly different by Tukey’s honest significant difference test at P ≤ 0.05.

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Table 5-3. Percentage distribution of panelists in the consumer sensory evaluations of sweetness and firmness level, and the percentage of panelists who detected off-flavor in ‘Arava’ in Spring 2012. Treatmentz Sweetness level (%)y Firmness level (%) Off-flavor 1 2 3 4 5 1 2 3 4 5 (%) Test 1x F NGAr 2.1 16.8 63.2 13.7 4.2 2.1 13.7 68.4 13.7 2.1 25.3 F Ar/Ar 2.1 25.3 52.6 15.8 4.2 4.2 29.5 55.8 9.5 1.1 29.5 F Ar/ST 7.4 37.9 44.2 10.5 4.2 40.0 48.4 6.3 1.1 36.8 O NAr 13.7 42.1 42.1 2.1 5.3 34.7 51.6 8.4 17.9 O Ar/Ar 30.5 46.3 23.2 7.4 29.5 53.7 8.4 1.1 25.3 O Ar/Cm 21.1 51.6 25.3 2.1 5.3 28.4 50.5 13.7 2.1 32.6 Test 2 F NGAr 3.0 24.2 61.6 8.1 3.0 10.1 22.2 57.6 9.1 1.0 17.2 F Ar/Ar 13.1 41.4 39.4 5.1 1.0 9.1 19.2 51.5 19.2 1.0 29.3 F Ar/ST 8.1 43.4 42.4 6.1 3.0 29.3 49.5 15.2 3.0 30.3 NF NAr 5.1 38.4 46.5 8.1 2.0 2.0 20.2 58.6 15.2 4.0 18.2 NF Ar/Cm 5.1 50.5 38.4 6.1 6.1 28.3 54.6 8.1 3.0 26.3 NF Ar/ST 16.2 54.6 27.3 2.0 4.0 18.2 48.5 23.2 6.1 36.4 z NGAr, Ar/Ar, Ar/ST, Ar/Cm indicated non-grafted ‘Arava’, self-grafted ‘Arava’, ‘Arava’ grafted onto ‘Strong Tosa’ rootstock, and ‘Arava’ grafted onto C. metulifer rootstock, respectively. F, NF, and O indicated melons produced from fumigated conventional field, non-fumigated conventional field, and organic field, respectively. y Firmness and sweetness level were evaluated with a Just-About-Right scale: 1= too soft/not sweet at all, 2= slightly too soft/not quite sweet enough, 3= just about right, 4= slightly too firm/somewhat too sweet, 5= too firm/Much too sweet. x Test 1 and Test 2 were conducted on June 1 and June 12, 2012, respectively. Test 1 included 96 panelists, and Test 2 included 100 panelists.

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Table 5-4. Consumer sensory evaluation and instrumental measurements of grafted ‘Arava’ melons grown in fumigated conventional field in Spring 2013. Treatme Sensory evaluationy Instrumental measurements z nt Overall Flavor liking Firmness TSS (ºBrix) Flesh firmness Titratable pH acceptability liking (kg) acidity NGAr 6.89 aw 6.87 a 6.57 a 9.56 a 1.02 b 0.052 6.85 Ar/Ca 5.12 b 4.69 b 6.01b 5.77 b 1.22 a 0.046 6.78 Ar/ST 4.83 b 4.33 b 5.69 b 5.72 b 1.03 b 0.052 6.72 P-value <0.0001 <0.0001 0.0001 <0.0001 0.0446 0.2386 0.1903 z NGAr, Ar/Ca, Ar/ST indicated non-grafted ‘Arava’, ‘Arava’ grafted onto ‘Carnival’ and ‘Arava’ grafted onto ‘Strong Tosa’. y Attributes were evaluated on a 9-point hedonic scale: 1=Dislike extremely, 2=Dislike very much, 3=Dislike moderately, 4=Dislike slightly, 5=Neither like nor dislike, 6=Like slightly, 7=Like moderately, 8=Like very much, 9=Like extremely. This test included 105 panelists. w Means within a column follower by the same letter were not significantly different by Tukey’s honest significant difference test at P ≤ 0.05.

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Table 5-5. Percentage distribution of panelists in the consumer sensory evaluations of sweetness and firmness level, and the percentage of panelists who detected off-flavor in ‘Arava’ in Spring 2013. Treatmentz Sweetness level (%)y Firmness level (%) Off-flavor 1 2 3 4 5 1 2 3 4 5 (%) NGAr 1.3 22.7 68.0 6.7 1.3 18.7 76.0 5.3 10.7 Ar/Ca 26.7 49.3 21.3 2.7 22.7 57.3 18.7 1.3 28.0 Ar/ST 32.0 57.3 9.3 1.3 2.7 32.0 61.3 4.0 37.3 zNGAr, Ar/Ca, Ar/ST indicated non-grafted ‘Arava’, ‘Arava’ grafted onto ‘Carnival’ rootstock and ‘Arava’ grafted onto ‘Strong Tosa’ rootstock y Firmness and sweetness level were evaluated with a Just-About-Right scale: 1= too soft/not sweet at all, 2= slightly too soft/not quite sweet enough, 3= just about right, 4= slightly too firm/somewhat too sweet, 5= too firm/much too sweet. This test included 105 panelists.

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Table 5-6. Consumer sensory evaluation and instrumental measurements of grafted ‘Honey Yellow’ melons in Spring 2012. Treatmentz Consumer sensory evaluationy Instrumental measurements Overall Flavor Firmness SSC (ºBrix) Flesh acceptability liking liking firmness (kg) Test 1x F NGHY 6.00 5.74 5.85 15.07 2.83 F HY/HY 6.10 5.96 6.09 14.22 2.71 F HY/ST 5.98 5.81 6.08 14.20 2.59 O NGHY 6.18 6.14 5.92 14.80 2.87 O HY/HY 6.04 6.02 5.74 14.12 2.87 O HY/Cm 5.95 5.77 5.95 14.47 3.19 P-value NSw NS NS NS NS Test 2 F NGHY 6.28 6.41 5.94 14.48 2.82 F HY/HY 6.06 6.05 5.62 15.42 2.82 F HY/ST 5.91 5.88 5.92 13.65 3.06 NF NGHY 6.05 6.06 6.04 14.73 3.06 NF HY/Cm 6.11 6.03 6.05 14.70 3.19 NF HY/ST 6.03 5.98 6.13 14.01 3.06 P-value NS NS NS NS NS z NGHY, HY/HY, HY/ST, HY/Cm indicated non-grafted ‘Honey Yellow’, self-grafted ‘Honey Yellow’, ‘Honey Yellow’ grafted onto ‘Strong Tosa’ rootstock, and ‘Honey Yellow’ grafted onto C. metulifer rootstock, respectively. F, NF and O indicated melons produced from fumigated conventional field, non-fumigated conventional field, and organic field, respectively. y Attributes were evaluated on a 9-point hedonic scale: 1=Dislike extremely, 2=Dislike very much, 3=Dislike moderately, 4=Dislike slightly, 5=Neither like nor dislike, 6=Like slightly, 7=Like moderately, 8=Like very much, 9=Like extremely. x Test 1 and Test 2 were conducted on May 25 and June 13 2012, respectively. Test 1 included 97 panelists and Test 2 included 98 panelists. w NS = Non-significant, i.e., P > 0.05.

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Table 5-7. Percentage distribution of panelists in the consumer sensory evaluations of sweetness and firmness level, and the percentage of panelists who detected off-flavor in ‘Honey Yellow’ in Spring 2012. Treatmentz Sweetness levely Firmness level Off-flavor 1 2 3 4 5 1 2 3 4 5 (%) Test 1x F NHY 9.3 23.7 49.5 15.5 2.1 2.1 15.5 51.6 21.7 9.3 26.8 F HY/HY 4.1 29.9 54.6 11.3 13.4 50.5 33.0 3.1 23.7 F HY/ST 5.2 30.9 53.6 10.3 1.0 16.5 53.6 20.6 8.3 26.8 O NHY 2.1 26.8 54.6 15.5 1.0 1.0 9.3 50.5 29.9 9.3 16.5 O HY/HY 5.2 25.8 60.8 8.3 2.1 18.6 43.3 26.8 9.3 23.7 O HY/Cm 3.1 34.0 50.5 11.3 1.0 3.1 9.3 57.7 24.7 5.2 30.9 Test 2 F NHY 2.1 22.7 63.9 11.3 1.0 7.2 54.6 24.7 12.4 15.5 F HY/HY 4.1 22.7 61.9 10.3 1.0 1.0 9.3 37.1 38.1 14.4 16.5 F HY/ST 11.3 25.8 53.6 8.3 1.0 1.0 13.4 49.5 28.9 7.2 16.5 NF NHY 2.1 33.0 54.6 6.2 4.1 5.2 44.3 32.0 18.6 26.8 NF HY/Cm 7.2 25.8 55.7 8.3 3.1 1.0 9.3 50.5 27.8 11.3 14.4 NF HY/ST 7.2 28.9 55.7 8.3 1.0 5.2 56.7 24.7 12.4 22.7 z NGHY, HY/HY, HY/ST, HY/Cm indicated non-grafted ‘Honey Yellow’, self-grafted ‘Honey Yellow’, ‘Honey Yellow’ grafted onto ‘Strong Tosa’ rootstock, and ‘Honey Yellow’ grafted onto C. metulifer rootstock, respectively. F, NF and O indicated melons produced from fumigated conventional field, non-fumigated conventional field, and organic field, respectively. y Firmness and sweetness level were evaluated with a Just-About-Right scale: 1= too soft/not sweet at all, 2= slightly too soft/not quite sweet enough, 3= just about right, 4= slightly too firm/somewhat too sweet, 5= too firm/Much too sweet. x Test 1 and Test 2 were conducted on May 25 and June 13 2012, respectively. Test 1 included 97 panelists and Test 2 included 98 panelists.

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CHAPTER 6 PHYSIOLOGICAL CHANGES IN GRAFTED MELON PLANTS WITH A HYBRID SQUASH ROOTSTOCK

Introduction

Hybrid squash rootstocks are highly resistant to fusarium wilt (Fusarium oxysporum) and have good tolerance to cold and saline conditions (Davis et al., 2008).

However, the use of hybrid squash rootstocks for melon grafting has not been adopted as they could impair fruit quality attributes of certain melon cultivars (Sakata et al.,

2008). Galia melon ‘Arava’ grafted onto hybrid squash rootstock ‘Strong Tosa’ reduced fruit SSC when they were grown in fumigated fields (Guan et al., 2013). Although fruit quality is usually considered an inherent characteristic of the melon scion cultivars, which may not be fundamentally changed without genetic modifications, plant physiological changes caused by grafting with specific rootstocks may result in certain adverse effects on melon fruit quality attributes. The objective of this field experiment was to determine the modifications of plant growth and development of the galia melon grafted onto the interspecific hybrid squash rootstock, and their potential impacts on fruit quality modifications.

Materials and Methods

The field trial was conducted in Spring 2013 at the University of Florida Plant

Science Research and Education Unit in Citra, FL, USA. The soil texture is loamy sand.

Galia melon ‘Arava’ (Johnny’s Selected Seeds, Winslow, ME, USA) was grafted onto interspecific hybrid squash rootstock ‘Strong Tosa’ (Syngenta seeds, Inc. Boise, ID,

USA). Non- and self- grafted ‘Arava’ plants were included as controls. The scion and rootstock seeds were planted on 10 Feb. and 14 Feb., respectively, into the 128-cell

Speedling flats (Sun City, FL, USA) containing potting soil with a mixture of vermiculite,

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peat moss, perlite, and starter nutrient (Metro-Mix 200; Sun Gro Horticulture, Bellevue,

WA, USA) in the research greenhouse on campus in Gainesville, FL. Seedlings were grafted on 23 Feb. using the one-cotyledon method (Davis et al., 2008). The experimental field was fumigated with methyl bromide and chloropicrin (50:50, w/w) at the rate of 454 kg/ha three weeks before transplanting. Completely healed grafted plants with three true leaves as well as control plants were transplanted into the field on

19 Mar. The bed spacing was 1.8 m from center to center, and plant in-row spacing was

0.9 m. 10N-4.4P-8.3K fertilizer (Premium Vegetable Grower Fertilizer; Southern States,

Lebanon, KY, USA) was applied preplant at 85 kg/ha N and supplemented by weekly injection of liquid fertilizer 6N-0P-6.6K fertilizer (Dyna Flo; Chemical Dynamics, Plant

City, FL, USA) at a rate of 14.3 kg/ha N. The field was arranged in a randomized complete block design with four replications and ten plants per treatment per replication.

The anthesis dates of each female flower on each plant were recorded and labeled every day from 16 days after transplanting (DAT) to 41 DAT. The length of the longest vine was measured on three plants per treatment per replication at 36 DAT and

46 DAT. Melons were harvested seven times from 25 May to 10 June at the full-slip stage. The early (fruit from the first two harvests that occurred on 25 and 28 May, respectively) and total yields were recorded. The harvest dates of fruit developed from the labeled female flowers that were successfully fertilized were recorded, and the fruit development durations were calculated.

Consumer sensory analysis was conducted on 31 May at the University of

Florida Sensory Analysis Lab in Gainesville, FL. The sensory evaluation procedures were similar to those described previously by Barrett et al. (2012c). The day before the

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analysis, ten fully ripe marketable melons of similar size (about 1.5 kg per fruit) and without any defects were selected from each treatment. The fruit were stored at 10 ºC overnight. In the next morning, melons were washed in tap water and dried with paper towel. They were cut longitudinally into halves. Instrumental measurements were conducted on one half of each fruit. Flesh firmness was measured twice in the middle of mesocarp using a penetrometer (Fruit Tester; Wagner Instruments, Greenwich, CT,

USA) with an 8-mm plunger. Soluble solids content (SSC), titratable acidity, and pH were measured as previously described (Zhao et al., 2011). The other half of each fruit were cut into roughly 3 × 3 cm melon cubes. All the fruit cubes were then well mixed and stored in a plastic container at 4 ºC during the consumer sensory test that lasted for approximately five hours. In this study, 106 consumer panelists who had eaten melons before were asked to rate overall acceptability, firmness liking, and flavor liking using a

1-9 hedonic scale (1 = dislike extremely, 5 = neither like nor dislike, 9 = like extremely).

Statistical analysis was performed using the Proc Glimmix procedure of SAS program (version 9.2C for Windows; SAS Institute, Cary, NC, USA). Tukey’s honest significant difference (HSD) test (α = 0.05) was conducted for multiple comparisons of measurements between different treatments.

Results and Discussion

The first female flower of the self- and non- grafted ‘Arava’ plants bloomed around 29 and 30 DAT, respectively, 8 to 9 days earlier than that of ‘Arava’ grafted onto

‘Strong Tosa’ rootstock. At 41 DAT, non- and self-grafted plants had about 12 blooming female flowers per plant, while only 7 were observed on the grafted plants with ‘Strong

Tosa’ rootstock (Figure 6-1). The delayed bloom of female flowers was also observed

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on cucumbers (Cucumis sativus) (Satoh, 1996; Yilmaz et al., 2011) and watermelons

(Citrullus lanatus) (Sakata et al., 2007) grafted onto hybrid squash rootstocks. Although grafting with ‘Strong Tosa’ delayed the appearance of female flowers, the first harvest dates were not affected in our study, and the early and total yields did not differ significantly between the grafted and non-grafted plants (Table 6-1). The unaffected early and total yields of grafted muskmelon were also reported by Balázs (2010).

On average, the length of fruit development period from anthesis to full-slip for

‘Arava’ grafted with ‘Strong Tosa’ was 34 days, significantly shorter than those of non- and self-grafted ‘Arava’, which were 38 and 39 days, respectively (Table 6-2). The result suggested an accelerated fruit development of ‘Arava’ grafted onto ‘Strong Tosa’.

Fruit quality assessment showed similar levels of fruit flesh firmness, titratable acidity, and pH between treatments (Table 6-3). However, the present study confirmed our previous observations (Guan et al., 2013) that grafting with ‘Strong Tosa’ affected the fruit quality of ‘Arava’ melon by decreasing fruit SSC, and overall ratings of acceptability, flavor liking, and firmness liking by consumers in the sensory analysis.

Interestingly, self-grafted ‘Arava’ exhibited higher scores of overall acceptability and flavor liking than non-grafted ‘Arava’ in the consumer sensory test, even though the instrumental measurements did not show any significant differences (Table 6-3).

As fruit sugar content is correlated with the length of fruit development (Burger and Schaffer, 2007), the shorter fruit development period observed in the grafted plants may partially explain the reduced fruit SSC. Ethylene is known to accelerate the natural process of fruit development, ripening, and senescence (Saltveit, 1999). During fruit development period, application of ethephon, an ethylene releasing compound, reduced

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melon SSC (Ouzounidou et al., 2008). The role of ethylene in the regulation of fruit ripening and quality of grafted plants deserves further studies.

About 27% of grafted plants with ‘Strong Tosa’ wilted during the late fruit development stage, and eventually died at the end of the season. Analysis of these plants indicated that it was not caused by pathogens. Cell death initiated in the roots and later expanded to the entire rootstock (Figure 6-2). Similarly, Minuto et al. (2010) reported the sudden collapse of melon plants grafted onto hybrid squash rootstocks, and the reduction in final fruit yield per ha as a result of the high incidence of plant collapse. They found ‘Fiola’ melon grafted onto ‘Shintosa’ rootstock was the most sensitive scion-rootstock combination to this physiological disorder, and plant collapse became more severe with higher temperature (30 °C) and exogenous application of auxins. It was suspected that auxins transported from the scion to the root triggered production of reactive oxygen species, which lead to degeneration of roots in the grafted plants (Aloni et al., 2008). The declined root systems may cause a decrease of plant photosynthesis. Xu et al. (2005) reported that grafted melons exhibited significantly lower photosynthetic rates in the late fruit development stage, which might contribute to the reduced fruit quality.

The vine length was significantly greater in ‘Arava’ grafted onto ‘Strong Tosa’ as compared with non- and self-grafted ‘Arava’ plants (Table 6-4). The enhanced vegetative growth is generally associated with the rootstock’s vigorous root systems that help improve water and mineral nutrient uptake during plant growth and development

(Davis, et al., 2008). This is one of the reasons that grafting with selected rootstocks may result in fruit yield improvement. Further studies are warranted to evaluate the

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relationship between fruit quality modification and the increased plant vigor in grafted plants.

Conclusions

By replacing the root systems to achieve targeted goals, grafting also creates a long-distance signaling network between rootstocks and scions. Understanding physiological changes that take place during plant growth and development and underlying mechanisms is essential for improving both fruit yield and quality of grafted plants. This study showed that grafting with the interspecific hybrid squash rootstock enhanced vegetative growth, delayed bloom of female flowers, and accelerated fruit development in galia melon plans. The impacts of these modifications on fruit quality attributes deserve further investigations. The scion-rootstock interactions can be rather complex given the diverse range of melon types. Analyzing the performance of various scions grafted onto the interspecific hybrid squash rootstock may help systematically examine the scion-rootstock interactions, which could offer more insights into identifying contributing factors to the physiological changes in grafted plants and their influence on fruit development and ripening

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Table 6-1. Early and total yields of grafted and non-grafted ‘Arava’. Treatment Early harvest Total harvest Total yield Marketable Total Marketable Total yield Marketable Total Marketable (kg/plant) yield number number (kg/plant) yield number number (kg/plant) (no./plant) (no./plant) (kg/plant) (no./plant) (no./plant) NArz 0.58 ay 0.56 a 0.67 a 0.62 a 7.0 a 5.1 a 7.4 a 4.9 a Ar/Ar 0.68 a 0.66 a 0.74 a 0.70 a 7.3 a 4.1 a 7.2 a 4.0 a Ar/St 0.36 a 0.28 a 0.47 a 0.32 a 6.5 a 4.2 a 7.2 a 3.9 a z NAr = non-grafted ‘Arava’; Ar/Ar = self-grafted ‘Arava’; Ar/St = ‘Arava’ grafted onto ‘Strong Tosa’. y Means within a column followed by the same letter were not significantly different according to Tukey’s honest significant difference test at P ≤ 0.05.

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Table 6-2. Duration of fruit development from anthesis to harvest of grafted and non- grafted ‘Arava’. Treatmentz Days from anthesis to harvest NAr 38 ay Ar/Ar 39 a Ar/St 34 b z NAr = non-grafted ‘Arava’; Ar/Ar = self-grafted ‘Arava’; Ar/St = ‘Arava’ grafted onto ‘Strong Tosa’. y Means within a column followed by the same letter were not significantly different according to Tukey’s honest significant difference test at P ≤ 0.05.

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Table 6-3. Fruit quality attributes of grafted and non-grafted ‘Arava’. Treatmenty Instrumental measurement Consumer sensory analysisz SSC Flesh Titratable pH Overall Flavor Firmnes (ºBrix) firmness acidity acceptabilit liking s liking (kgf) (%) y NAr 7.0 ax 1.2 a 0.070 a 6.6 a 5.7 b 5.3 b 6.1 a Ar/Ar 7.8 a 1.3 a 0.069 a 6.7 a 6.3 a 6.2 a 6.2 a Ar/St 5.4 b 1.2 a 0.067 a 6.6 a 4.8 c 4.4 c 5.4 b y NAr = non-grafted ‘Arava’; Ar/Ar = self-grafted ‘Arava’; Ar/St = ‘Arava’ grafted onto ‘Strong Tosa’. zAttributes were evaluated on a 9-point hedonic scale: 1 = Dislike extremely, 2 = Dislike very much, 3 = Dislike moderately, 4 = Dislike slightly, 5 = Neither like nor dislike, 6 = Like slightly, 7 = Like moderately, 8 = Like very much, 9 = Like extremely. x Means within a column followed by the same letter were not significantly different according to Tukey’s honest significant difference test at P ≤ 0.05.

Table 6-4. Length (cm) of the longest vine of grafted and non-grafted ‘Arava’ plants at 36 days after transplanting (DAT) and 46 DAT. Treatmentz 36 DAT 46 DAT NAr 72.4 by 127.2 b Ar/Ar 71.3 b 130.4 b Ar/St 83.0 a 148.2 a z NAr = non-grafted ‘Arava’; Ar/Ar = self-grafted ‘Arava’; Ar/St = ‘Arava’ grafted onto ‘Strong Tosa’. y Means within a column followed by the same letter were not significantly different according to Tukey’s honest significant difference test at P ≤ 0.05.

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Figure 6-1. Cumulative numbers of female flowers per plant in grafted and non-grafted ‘Arava’ plants from 16 days after transplanting (DAT) to 41 DAT.

Figure 6-2. Roots and stems of ‘Arava’ grafted onto ‘Strong Tosa’ rootstock. A) A healthy root system; B) Cell death initiated in the roots, as indicated by the arrow; C) Cell death spread to the stem of rootstock, necrosis of vascular tissue developed as indicated by the arrow; D) The entire rootstock was dead; E) dead rootstock and healthy scion stem separated by the graft union.

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CHAPTER 7 SUMMARY AND FUTURE PROSPECT

Summary

Melon grafting is used for managing soilborne diseases, and improving abiotic stress tolerances. The high cost of producing grafted seedlings is an obstacle preventing wide adaption of this technology in the U.S. Optimizing grafting procedure and producing high quality grafted seedlings is important in reducing the cost. Four grafting methods including hole insertion, one-cotyledon, tongue approach, and non- cotyledon were examined for their impacts on seedling growth and root characteristics of muskmelon ‘Athena’ grafted onto hybrid squash rootstock ‘Strong Tosa’. The grafted plants were examined with or without root excision (Chapter 2). Except the non- cotyledon grafting method, root excised plants initiated root growth at 4 days after grafting, and developed similar root length and surface area as root intact plants at 16 days after grafting. Plants grafted with non-cotyledon method resulted in a shorter root length and less surface area compared with hole insertion and one-cotyledon grafted plants at 16 days after grafting. Non-cotyledon method significantly decreased quality of grafted plants. Root excision did not exhibit significant impacts on either root growth or above ground growth characteristics at the flowering stage. Without root excision, hole insertion, one-cotyledon, and tongue approach methods did not show consistent differences in above ground growth characteristics compared with non-grafted scion controls at about six weeks after grafting.

Growing specialty melons is challenging in Florida as they often lack disease resistances. Cultivar evaluation trials including ten specialty melons and a commonly grown cantaloupe melon ‘Athena’ in the eastern U.S were conducted (Chapter 3).

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Compare with ‘Athena’, specialty melons are generally more susceptible to root-knot nematodes (RKN) damage, which is one of the major obstacles in melon production.

Sustainable management approaches are demanded by both conventional and organic growers.

Melon grafting was proposed as an alternative approach to take advantage of

RKN resistance in Cucumis metulifer. A greenhouse experiment was conducted with the honeydew melon ‘Honey Yellow’ grafted onto C. metulifer, and inoculated with M. incognita race 1 (Chapter 4). The grafted plants exhibited significantly lower gall and egg mass indices, and fewer eggs compared with non- and self- grafted ‘Honey Yellow’.

Cucumis metulifer was further tested as a rootstock in conventional and organic field trials using honeydew melon ‘Honey Yellow’ and galia melon ‘Arava’ as scions (Chapter

4). ‘Honey Yellow’ and ‘Arava’ grafted onto C. metulifer exhibited significantly lower galling and reduced RKN population densities in the organic field; however, total and marketable fruit yields were not significantly different from non- and self-grafted plants.

Although the improvement of RKN resistance did not translate into yield enhancements, incorporating grafted specialty melons with C. metulifer rootstock into double-cropping systems with RKN susceptible vegetables may benefit the overall crop production by reducing RKN population densities in the soil. At the conventional field site, which was not infested with RKN, ‘Honey Yellow’ grafted onto C. metulifer rootstock had a significantly lower total fruit yield than non-grafted ‘Honey Yellow’ plants; however, fruit yields were similar for ‘Arava’ grafted onto C. metulifer rootstock and non-grafted ‘Arava’ plants.

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Previous research revealed mixed results regarding rootstock impacts on fruit quality. In grafted specialty melon production, the rootstock effect on fruit quality deserves more attention as specialty melons are marketed for outstanding taste and unique fruit flavor. Galia melon ‘Arava’ and honeydew melon ‘Honey Yellow’ were grafted onto commercial hybrid squash rootstock ‘Strong Tosa’ and root-knot nematode resistant C. metulifer (Chapter 5). Regardless of the production systems, ‘Arava’ grafted onto ‘Strong Tosa’ received significantly lower scores in consumer overall acceptability and flavor liking compared to non-grafted ‘Arava’. Reduced SSC were detected in the graft combination. Grafting with C. metulifer significantly decreased consumer overall acceptability and flavor liking for organically grown ‘Arava’ fruit, but the difference between grafted and non-grafted treatments was not detected in melons produced from the non-fumigated conventional filed. More consumers detected off-flavor in the grafted

‘Arava’ fruit, which may partially explain the decreased preference for the fruit from grafted plants.Different from ‘Arava’, grafting did not exhibit any significant effect on

TSS, flesh firmness, and consumer perceived sensory attributes of ‘Honey Yellow’ melons.

Plant physiological changes were explored to understand the rootstock effects on fruit quality modifications in the grafted ‘Arava’ melons (Chapter 6). The grafted plants delayed the anthesis of female flowers by about 8-9 days but did not affect the harvest dates compared with the non-and self- grafted ‘Arava’ plants. The early and total yields were not significantly different between grafted and non-grafted plants. ‘Arava’ plants grafted onto ‘Strong Tosa’ demonstrated accelerated fruit development as the duration between anthesis and harvest was about 4 and 5 days shorter than that of non- and

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self-grafted plants, respectively. Vegetative growth was greater in the grafted plants with

‘Strong Tosa’ rootstock. During the harvest period, about 27% of grafted plants with

‘Strong Tosa’ rootstock wilted, which was identified as non-pathogenic. The wilt plants eventually died at the end of the season. Analysis of the collapsed grafted plants indicated that the cell death initiated in the roots and expanded to the entire rootstock.

Underlying mechanisms that cause these physiological changes as related to fruit quality deserve further research.

Future Prospect

Optimizing grafting methods to produce high quality grafted plants is essential in implementing grafting technology in specialty melon production. In this project, the effects of grafting methods and root excision on the growth characteristics of grafted melons were evaluated under greenhouse conditions. Future field studies are needed to assess yield performance of grafted melons as affected by different grafting methods taking into consideration of the economic analysis.

This research demonstrated the effectiveness of RKN management through grafting ‘Honey Yellow’ and ‘Arava’ melons onto C. metulifer. The improvement of RKN resistance did not translate into yield enhancements, which might relate to the thinner stem diameter of the rootstock compared with that of the scions. Future breeding efforts could be directed to develop more vigorous C. metulifer rootstocks to better match the scion stem diameter. Future studies are also warranted to incorporate the grafting practice into a double-cropping system with RKN susceptible vegetables, which might be an alternative approach to enhance the production of high-value vegetables in RKN infested fields. Moreover, the grafting practice involving diverse types of specialty melon

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cultivars will need to be tested at locations with different disease pressures and varying climate conditions.

In addition to C. metulifer, other wild Cucumis species, such as C. anguria, C. ficifolius and C. melo var. texanus, which have shown disease resistances, should also be evaluated for their potential use as rootstocks for melon grafting. Having a pool of rootstocks to cope with a variety of soilborne diseases has been and will continuously be an important task. More efforts should be devoted to developing new rootstocks, either from selection of wild species, recombination of current plant materials, or application of new technologies like gene editing or transgenic approaches.

The physiological changes that might be related to fruit quality determination have been investigated in this project. More in-depth studies are warranted to understand the causes of these changes, such as water uptake and transport, plant hormones, and nutritional status. In addition, future research needs to elucidate rootstock and scion interactions at the cellular and molecular level. More research is warranted to conduct biochemical analysis of important enzymes’ activity and expression analysis of genes with specific functions in the grafted plants. Identification of long-distance signals and genetic modification of the grafted plants are hot topics.

Breakthroughs in these areas will greatly advance our understandings in the rootstock and scion interactions, leading to more effective and efficient use of the grafting technology. To better understand the mechanisms of rootstock-scion interactions, it will be important to establish a widely accepted research protocol that allows for better synthesis and analysis of findings across studies. This will include but not limited to rootstock and scion genotypes, age of grafted plants, and maturity of rootstock and

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scion plants before grafting. While grafting technology has been used in vegetable production for decades, many underlying mechanisms are still poorly understood.

Furthering our understanding of the plants’ responses to grafting will not only improve our fundamental knowledge in plant science but also benefit sustainable vegetable production through more targeted breeding and use of rootstocks.

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BIOGRAPHICAL SKETCH

Wenjing Guan earned her bachelor’s degree in Plant Protection at the Southwest

Agricultural University, Chongqing, China, and master’s degree in Plant Pathology at the Chinese Academy of Agricultural Sciences. She worked as a research assistant under the direction of Dr. Xin Zhao in the Horticultural Sciences Department at the

University of Florida since 2010. Her research interest is in sustainable and organic vegetable production, with the particular focus on integrated use of grafting technology in specialty melon production. Wenjing received her Ph.D. degree from the University of

Florida in the summer of 2014.

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