INTE GRATED US E OF GRAFTING TECHNOLOGY IN SPECIALTY MELON PRODUCTION: GRAFTING TECHNIQUES , ROOT KNOT NEMATODE MANAGEMENT AND FRUIT QUALITY By 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
Â© 2014 Wenjing Guan
To my husband, Yihai, for his unconditionally love and support
4 ACKNOWLEDGMENTS I sincerely thank my major advisor, Dr. Xin Zhao, for financial support, and y ears of guidance on my research, and being a supportive mentor in the four year s journey. My appreciation also goes to my committee members, Dr s . Donald W. Dic kson, Donald J. Huber, and Nicholas S. Dufault for the ir excellent suggestions that help ed me continuously moving forward. I thank my husband, Yihai Wang , for his unconditional ly love and support. I also feel very lucky to have a family w ho always stoo d 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 w here I made man y 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 M aria Mendes, Michael Alligo od, 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 department al staff for the administrative support, especially to Curtis Smyder for his co nstant and patient assistances in all the aspects.
5 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 Me thods ................................ ................................ ............................ 61 Transplant Production ................................ ................................ ...................... 61 Field Planting and Harvest ................................ ................................ ............... 62 In sect Pests Management ................................ ................................ ................ 64 Disease Evaluations ................................ ................................ ......................... 64 Evaluations of Fruit Characteristics ................................ ................................ .. 65
6 Statistical Analyses ................................ ................................ .......................... 65 Results and Discussion ................................ ................................ ........................... 65 Anthesis and Harvest Dates ................................ ................................ ............. 65 Fruit Yields ................................ ................................ ................................ ....... 66 Fruit Characteristics ................................ ................................ ......................... 69 Disease Observations ................................ ................................ ...................... 71 Conclusi ons ................................ ................................ ................................ ............ 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 Me thods ................................ ................................ .......................... 100 Melon Production ................................ ................................ ........................... 100 Sample Preparation ................................ ................................ ........................ 102 Consumer Sensory Analyses ................................ ................................ ......... 103 Instrumental Measurements ................................ ................................ ........... 104 Statistical Analyses ................................ ................................ ........................ 104 Results and Discussion ................................ ................................ ......................... 104 .......... 104 C. metulifer Rootstock ................. 106 Off flavor of ................................ ................................ .... 107 ................................ ................... 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
7 Conclusions ................................ ................................ ................................ .......... 124 7 SUMMARY AND FUTURE PROSPECT ................................ ............................... 129 Summary ................................ ................................ ................................ .............. 129 Future Prospect ................................ ................................ ................................ .... 132 LIST OF REFERENCES ................................ ................................ ............................. 135 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 154
8 LIST OF TABLES Table page 1 1. The major events in the history of implementing grafting technology in cucurbit c rop 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 a nd 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 a nd 46 days after grafting in the first and second experiments, respectively. ................................ ................................ ................................ ........ 56 3 cantaloupe grown in organic and conventional fields during Spring 2011 at Citra, FL ................................ ................................ ................................ .............. 74 3 cantaloupe under organic and conventional production during Spring 2011 at Citra, FL. ................................ ................................ ................................ ............. 75 3 organic (Org) and conventional (Con) production during Spring 2011 at Citra, FL. ................................ ................................ ................................ ...................... 76 3 4. Fruit weight, length, shape, soluble solids concentration (SSC), and f lesh and conventional (Con) production during Spring 2011 at Citra, FL ................... 77 3 5. Aboveground diseases severity and root knot nematode (RKN) gall ratings of production during Spring 2011 at Citra, FL. ................................ ........................ 78
9 4 1. Root gall index (GI), egg mass index (EMI), egg recovery, and reproduction factor (Rf) of grafted and non the greenhouse study (2011). ................................ ................................ ................... 93 4 2. Root gall index (GI) and numbers of Meloidogyne javanica second stage juveniles (J2) in so il of grafted and non ...................... 94 4 3. Total and marketable fruit yields (kg/plant) of grafted and non grafted conventional field studies (2012). ................................ ................................ ....... 95 4 4. Marketable fruit number per plant in different fruit size categories of grafted and non the organic and conventional field studies (2012). ................................ .............. 96 5 1. Grafting treatments included in certified organic field, non fumigated conventional field, and fumigated convent ional field in Spring 2012. ................ 112 5 2. Consumer sensory evaluation and instrumental measurements of grafted g 2012. ................................ ................................ ......... 113 5 3. Percent distribution of panelists in the consumer sensory evaluations of sweetness and firmness level, and the p ercentage of panelists who detected off ................................ ................................ ... 114 5 4. Consumer sensory evaluation and instrumental measurements of grafted ............. 115 5 5. Percent distribution of pa nelists in the consumer sensory evaluations of sweetness and firmness level, and the percentage of panelists who detected off ................................ ................................ ... 116 5 6. Consumer sensory evaluation and instrumental measurements of grafted ................................ ............................ 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 ................................ ...................... 118 6 1. Early and total yields of grafted and non ................................ .... 125 6 2. Duration of fruit development from anthesis to harvest of grafted and non ................................ ................................ ................................ . 126 6 3. Fruit quality attributes of grafted and non ................................ ... 127
10 6 4. Length (cm) of the longest vine of grafted and non days after transplanting (DAT) and 46 DAT. ................................ ..................... 127
11 LIST OF FIGURES Figure page 1 1. Hole insertion grafting. A) Remove true leaves from rootstock. B) Make a slit with a 45Âº angle at the growing tip. C) Keep toothpick inserted while preparing scion.. ................................ ................................ ................................ . 37 1 2. One cotyledon grafting. A) r emove one cotyledon and true leaf, cut rootstock at a slant, with a 45Âº angle. B) Cut scion 1.5 to 2 cm below cotyledon, also a 45Âº 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 scio n 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 f irst experiment. RC: rootstock control, SC: scion control, HI: hole insertion, OC: one cotyledon, NC: non cotyledon. ................................ ................................ ............ 57 2 2. Ro ot 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 (ananas melon) ................................ ................................ ................................ ... 79 6 1. Cumulative numbers of female flowers per plant in grafted and non grafted .................... 128 6 root system; B) Cell death initiated in th e roots, as indicated by the arrow. ...... 128
12 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 INTEGRATE D USE OF GRAFTING TECHNOLOGY IN SPECIALTY MELON PRODUCTION: GRAFTING TECHNIQUE S, ROOT KNOT NEMATODE MANAGEMENT AND FRUI T QUALITY By 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. E ffects of grafting methods and root excisi on were evaluated in greenhouse experiments with regard to plant quality and growth characteristics ( Cucumis melo ) grafted onto hybrid squash ( Cucurbita maxima Ã— 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 wer e observed in grafting success and plant g rowth among one cotyledon, hole insertion, and tongu e approach methods . Interest in producing specialty melon is increasing in Florida ; however, the lack of disease resistance is an obstacle limiting specialty mel on production. Honeydew melon better performances in specialty melon cultivar evaluation trials. However, they are susceptible to root knot nematode s (RKN) damage.
13 G rafting was proposed to take a dvantage of RKN resistance in C ucumis metulifer 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 C. metulifer s. The grafted plants showed promising results in RKN management ; however, the yields were not improved in either organic or conventional field s . Rootstock effects on melon fruit quality deserve more attention in specialty melon production as they are ofte n marketed for outstanding taste and unique flavor. hybrid squash rootstock soluble solids concentration (SSC ). Moreover, more c onsumers dete cted off flavor in the 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 ibit significant effect s on SSC , flesh firmness, and consumer
14 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 tolerance s (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 root stocks ( Cucurbita moschata ) to overcome yield loss caused by f usarium wilt ( caused by Fusarium oxysporum ) (Sato and Takamatsu, 1930). Since then, grafting has been widely used in Asia n and European countries for controlling soil borne 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 e t al., 2008). Exploring the histor y of implementing grafting technology in cucurbit crop production in other countries, and understanding rootstock and scion interaction s will not only benefit the integration of grafting technique s 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 anc ient literatures in Asia, but there w ere no large scale grafting practices recorded before 1920 .
15 From 1920 to 1940. Re search in cucurbit grafting initiated in Japan in 1920s as a respon se to the increasing interest in grafting watermelon onto squash ( Cucurbita moschata ) for f usarium wilt control . Evaluation of different cucurbit species le d 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 watermel on, melon ( Cucumis melo ) and cucumber ( Cucumis sativus ) . In the early years, c left 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 n ot only provided cold tolerance, but also showed early ripening, increased flow er ing, higher yield, prolonged production season , and increase d sugar content in fruit (Lebedeva, 1937 ). From 1940 to 1950. L. siceraria wa s 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 m any local cultivars (Jurina, 1949; Pajacyk , 1948). Cucurbit grafting was introduced to Europe right after World War II for the purpose of controlling f usarium 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 f usarium wilt
16 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 go urd 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 sp p . rootstocks were evaluated in the USSR (Jur ina, 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 t he Netherlands and German y . As grafted plants exhibited fusariu m wilt tolerance , good fruit quality and early harvest, t he 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 war m conditions inside greenhouses . Moreover, due to the physical injury caused by grafting process, grafted cucumber s were mor e susceptible to C u cumis virus es (Groenewegen, 1953). In this period, m elon 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. pep o were found to delay harvest (Groenewegen, 1953; Naaldwijk, 1952). From 1960 to 1970. Extensively using bo ttle gourd rootstock s resulted in losi ng resistance to f usarium wilt of grafted watermelons in Japan . A new strain, F. oxysporum f.sp. Lagenariae , to gether with Pythium s pp. and physiological disorder s resulted in a
17 watermelon sudden wilt that spread across Japan in the 1950s and 1960s (Sakata et al., 2007). Re evaluation of Cucurbita sp p . and B. hispida species identified C. pepo and C. maxima Ã— C. moschata rootstocks that were resistant to the new F. oxysporum strain, and gr aft compatible with watermelon (Marukawa and Yamamuro , 1964; Marukawa and Yamamuro , 1967). As oriental melons were generally less su sceptible to f usarium wilt, m elon grafting wa s less common in Japan than watermelon grafting. However, s ome of the new ly introduced melon cultivars were susceptible to the disease and le d to the evaluation of Cucurbita spp . as melon rootstocks (Marukawa, 1969). In Europe in this period, the most com m on ly 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 on to C. ficifolia were widely ad o pted . 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 t hat result ed in losing f usarium 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 disease s . From 1970 to 1980. In Japa n, growing grafted cucurbit crops has bec ome a routine practice that led to systemic evaluation s of grafting with other agricultural practices . Grafted cucumbers were found to absorb pesticide s at higher rates , which le d to the readjustment of pesticide application rate s for grafted plants (Suda et al. , 1976).
18 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 wh o reported that mineral nutrient uptake of cucumber s grafted onto C. ficifolia was different from non grafted cucumber plants. In Europe, interest in melon grafting for f usarium wilt management was gradually declining. One of the reason s was the emergence of a new Fusarium races, F. oxysporum f.sp. melonis , which compromised root stock 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 pressure s (Buitelaar, 1987 ). Furthermore, melons were susceptible to Phomopsis sclerotioides and Verticillium dahlia . U nfortunately, 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 evaluation s of wild Cucumis species le d to the identification of C ucumis metulifer , which was resistant to multiple diseases (f usarium wilt, gummy stem blight, and root knot nematode) and compatible with cucumber s and melon s (Igarashi et al. , 1987). Cucumber s grafted onto C. moschata almost completely supp ressed wax, as wax free cucumber has a di stinct appearance and a longer shelf life, the popularity of
19 grafted cucumbers was increased (Sakata et al. , 200 8). With the increased dem and for grafted cucumber plant s , cucumber grafting robots w ere 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 wa s developed in China (Zhang and Wei , 1999). Response of grafted cucumber to low temperature was a research fo cu s 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 Ve rticillium dahlia , led the work of reevaluating rootstocks. However, few promising rootstocks were identified ( Nisini et al., 2000 ; Paplomatas et a l., 2000 ). From 2000 to present. Phas e out of the broad spectru m soil fumigant methyl bromide stimulated the introduction of vegetable grafting into the U.S. and Mediterranean area s. 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).
20 Understanding Rootstock and Scion Interactions During US SR (Mudge, et al., 2009). Researchers believed that two far distant species could derive intermediate characteristics through vegetative hybridization (grafting), and characteris tics fro m both rootstock and scio n 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 s cion ( Ludilov , 1964; Ludilov , 1969; Parhomenko, 1941 ). M any 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 b ioc hemical studies were conducted in this period. Catalase, peroxidase, and polyphenol oxidase activit ies were found increased in watermelon and melon plant s grafted onto gourd. Grafting also improved c hlorophyll content in scion leaves (Merkis, 1955). Rad iographic techniques were used to study translocation of substance between r ootstock and scion. The study confirmed that C14 labeled photoassimilate can transport from scion to rootstock (D e Stigter, 1961). Despite t he enthusiasm towards grafting hybrid , different opinions were expressed . By comparing f usarium wilt symptoms among cantaloupe scion grafted onto resistant cucumber rootstock, reciprocal grafting, self grafted canta loupe , and self grafted cucumber, Cox (1944) concluded that scion did not affec t resistance of rootstock while rootstock did not affect susceptibility of scio n against the pathogen. Mustafin
21 (1962) reported that even though xylem vessels of cucumbers g rafted onto squash rootstock were almost twice as large as non grafted cucumbers, t hey were caused by the increased nutrient supply but not the genetic modification s . 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 g rafting did not change heritable characteristics of both rootstock and scion was commonly accepted (Mockaiti s, 1976), although morphological alterations of the grafted plants were observed. T he observed changes were often attributed to one of the following physiological mechanisms. Firstly, the overall improved p lant health. Due to disease resistance or stress tolerance provided by rootstocks, grafted plants were generally healthier compared with non grafted plants, particu larly 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 characteriz ed 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 deve lop 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 CO 2 , and transpiration rate (Liu, et al., 2011). Fourthly, hormonal balance s . Plant hormones are importan t factors that regulate all aspects of plant development.
22 Cytokinins are known to be produce d 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 translocate d 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 cytoki nin 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 a nthracnos e after grafting onto an infected resistant rootstock with the same pathogen . D isease resistance signals were suspected transmitting across grafting union ( ,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 t hese characteristics were less likely to be affe cted by physiological mechanisms , grafting induced alternations at molecular level became the study focus. Grafting increased the activities of several important enzyme s (Guan et al., 2012). C ucumber s grafted onto C. moschata induced expression of resistance rel ated 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 transcription al profil e, the relative expression of Mn Superoxide dismutase (SOD) and Cu/Zn SOD mRNA was
23 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). A pples grafted onto roots tock 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 level s . Cucurbits were model plant s to study phloem exudates, in which large amount of proteins with diverse functions have been identified (Lough and Lucas, 2006). Phloem proteins we re confirmed transmissible across grafting union (Golecki et al., 1998). Studies on flower l ocus (FL) proteins indicated that FL proteins may act as the long dist ance florigenic signals in the c ucurbits (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 i n grafted plants. In addition to proteins, long distance trafficking of RNA s was d emonstrated (Kehr and B uhtz 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 integrat ing it into genome was demonstrated. However, little is known about the involvement of this mechanism in grafting induced genetic modification (Adler, 2001). Studies in epigenetic s unraveled a new mechanism in gene expression regulation that described heritable changes were induced by mechan isms 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
24 grafting transmissible signals participated in any of the gene expression regulation mechanisms in grafted plants . The m ost direct evidence of gra fting induced genetic changes ca me from genetic information exchange via plastid genomes at grafting site ( Liu et al., 2010a; Stegemann and Bock , 2009). However, curr ent evidence s 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 Im proved 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., 20 10) . Diseases controlled by grafting in different cucurbit crops are listed in Table 1 2. Soil borne fungal and oomycete diseases. The earliest reported use of vegetable grafting for disease control was for management of fusarium wilt in cucurbits (Sakata e t 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, primaril y 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
25 and cucumbers (Paplomatas et al., 2002) . Monosporascus sudden wilt, caused by Monosporascus cannonballus , is an important soil borne disease of melon and watermelon in hot and semiarid areas. Grafting scions of susceptible melon cultivars onto C. maxima and C. maxima Ã— C. moschata rootstocks improved resistance of melon (Edelstein et al., 1999) despite the fact that Cucurbita is n ormally 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 growi ng 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. moschat a , 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, 2 010) . 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 metulife r , Cucumis ficifolius , and bur cucumber ( Sicyos angulatus ) (Fassuliotis, 1970; Gu et al., 2006) . Using C. metulifer as a rootstoc k to graft RKN susceptible melons led to lower levels of root galling and nematode numbers at harvest (Sig Ã¼ enza 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
26 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 root stocks for controlling the soil borne 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, s ome 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 includ e 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 (Lou ws et al., 2010; Sakata et al., 2006) . Challenges and future prospective . E xtensively 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
27 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 1930 s, 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 hig h 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 t hat could not be controlled by rootstocks, such as viral diseases and foliar fungal diseases (Guan et al., 2012). H owever, in depth study to prove the assumption is challengeable as m any external factors could affect these disease performances . Establishin g 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 he l p 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., 2012 a ; 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
28 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 you ng 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 gr eater 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 inter specific and can have varied hypocotyl diameters. Grafting methods then need to be adjusted base d 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 eff ects 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
29 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 grafti ng 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 wh en 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 gr afting technique will depend on Hole Insertion Graft ing Procedure of the hole insertion graft ing was demonstrated in Fig ure 1 1 . First, leaves and meristem tissue are removed at the growing tip of the rootstock. Next, a slit is made across the growi ng 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 while the insertion stick is removed. Hole insertion graft ing produces high quality grafted transpla nts 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 graft ing cost, as well as the labor involved in collecting clips after healing.
30 Hole insertion graft ing 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 tr ue leaves should be avoided. Hole insertion graft ing 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 thi s stage, scion hypocotyls are strong enough to be inserted while still easy to fit into the slits on the rootstock. Si nce the ideal plant size for hole insertion graft ing 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 Graft ing O ne cotyledon gra ft ing is easier to conduct than hole insertion graft ing . Procedure of this method was shown in Fig ure 1 2. First, the rootstock growing tip is cut at a 45 degree angle. The cut removes true leaves, meristem tis sue , and one of the cotyledons. N ext, 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 hypoc otyl 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 meth od 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
31 scion seeds. Similar to hole insertion graft i ng s, 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 roo tstocks 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 ÂºC and relatively humidity 95%. Our experiments indicated that temperatures lower than 21 Âº 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 requir ed. Placing plants in complete darkness for longer than 48 hours, however, could result in unfavo rable stem elongation and weak seedlings. After the critical 48 hours, exp osing 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 (Fig ure 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, hum idity is gradually reduced by partially opening the container. For larger scale grafting, a healing chamber that can accommodate several seedling trays can
32 easily be constructed inside of a greenhouse. More information regarding the construction of a heali ng 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 incomp lete remov al of meristem tissue during hole insertio n 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 m ajor 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 gr owth points of rootstocks. Tongue Approach Graft ing Tongue approach graft ing 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 graft ing can achieve a high survival rate without high humidity healing conditions. Direct sunlight shou ld be avoided on newly grafte d plants, but healing is often achieved in a normal greenhouse environment. Some rootstock and
33 scion cultivars have very short hypocotyls, and they are not suitable for this method. Tongue approach graft ing requires rootstock a nd 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 Graft ing Non cotyledon graft ing is very similar to one cotyledon graft, but the rootstocks are cut at the hypocotyls, a nd 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.
34 Table 1 1 . The major eve nts in the history of implementing grafting technology in cucurbit crop production . Time Japan USSR z Europe China U.S. 1920 1930 Watermelon was grafted onto Cucurbita moschata for f usarium wilt control 1930 1940 Selected f usarium wilt rootstocks: Lagenaria siceraria , Benincasa hispida , Luffa cylindrical , Cucurbita maxima , C. pepo , C. ficifolia . W atermelon and melon grafting were introduced for cold tolerance 1940 1950 L. siceraria beca me the most popular rootstock for watermelon grafting. Evaluation of grafting local watermelon and melon cultivars onto C. maxima rootstock s Cucumbers were grafted onto C. ficifolia for f usarium wilt management 1950 1960 Polyploidy watermelons were grafted Eco nomic analyses were condu cted on watermelon and melon grafting. Cucumbers were grafted Economic analyses were conducted on cucumber grafting. Leaf chlorosis and virus problems were found associated with grafted cucumber s . Melons were grafted 1960 1970 L. siceraria lost resistance to f usarium wilt . C. pepo and C. maxima Ã— C. moschata were proved resistance to the new F. oxysporum strain. Melon and cucumber grafting became popular. B. hispida was regarded as the most suitable melon rootstock. Emerging diseases wi th cucumber rootstock C. ficifolia . Combining g rafting with other disease management approaches achieve d better results.
35 Table 1 1 Continued . Time Japan USSR z Europe China U.S. 1970 1980 Systematical evaluation of grafting with other production practices. Mineral nutrient uptake was found differed between grafted and non grafted plants . Melon gra fting was declining because 1. The e volution of new pathogens that broke rootstock resistance; 2. n ew int roduced resistant melon cultivars; 3. g rafting unable to control new emerging diseases. 1980 1990 Resistance to F. oxysporum f.sp. Lagenariae was found in L. siceraria . I t again became the mo st popular watermelon rootstock. C. metulifer was evaluated as cucumber and melon rootstock s . became popular . Grafting robot was developed for cucumber grafting Sicyos angulatus was evaluated as cucumber rootstock for root knot nematode management 1990 2000 Cucurbit grafting increased tremendously . Other important cucurbit crops were also grafted 2000 prese nt Cucurbit grafting was introduced . z USSR: Union of Soviet Socialist Republics
36 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 rot Fusarium oxysporum; Fusarium solani watermelon Verticillium wilt Verticillium dahliae watermelon, melon, cucumber Monosporascus sudden wilt Monosporascus cannonballus w atermelon, melon Phytophthora blight Phytophthora capsici watermelon, cucumber Target leaf spot Corynespora cassiicola c ucumber Black root rot Phomopsis sclerotioides c ucumber, melon Gummy stem blight Didymella bryoniae m elon Powdery mildew Podosphaera xanthii c ucumber Downy mildew Pseudoperonospora cubensis c ucumber Nematodes Root knot Meloidogyne spp. c ucumber, melon, watermelon Viral diseases Melon necrotic spot virus Melon necrotic spot virus (MNSV) w atermelon Information was adapted from published reviews (King et al., 2008; Louws et al., 2010).
37 Figure 1 1 . Hole insertion graft ing . A ) Remov e true leaves from rootstock. B) Make a slit with a 45Âº angle at the growing tip. C) Keep toothpick in serted while preparing scion. D) Cut scion hypocotyls at insert scion into the slit. F) Bamboo toothpick used for insertion.
38 Figure 1 2 . One cotyledon graft ing . A) remove one cotyledon and true leaf, cut rootstock at a slant, with a 45Âº angle. B) Cut scion 1.5 to 2 cm below cotyl edon, also a 45Âº angle. C) Attach the rootstock and scion cut surfaces and hold them t ogether with a grafting clip. D) A newly grafted melon plant with one cotyledon graft.
39 Figure 1 3 . A storage container ( 63 cm long, 46 cm wi de 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 .
40 Figure 1 5 . Tongue approach graft ing . A) Rootstock and scion have similar stem diameters. B) Rootstock is cut at a downward a ngle and scion is cut u pward. 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.
41 Figure 1 6 . Non cotyledon graft ing . A) C ut rootstock at hypocotyl. B) Attach rootstock and scion cut surfaces with a grafting clip
42 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 adapta tion 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 geogra phic 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 method s 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 bein g easily performed by hand, one cotyledon is th e only method which can also be performed by machine (Hassell et al., 2008). A grafting machine can produce 60 0 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
43 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 pr oblem 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 graft ing. As the name indicates, non cotyledon method excises both rootstock cotyledon s, 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) repo rted 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 an d 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 preventin g growing media
44 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 en ergy 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 consider able 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 ÂºC to 30 ÂºC, and average relative Cucumis melo ) was grafted onto hybrid squash ro C. maxima Ã— 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 f irst and second experiments, respectively, when both rootstock and scion developed first true leaves. Plants were grafted with four methods, n amely, hole insertio n (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
45 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 c ell 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 hum idifiers 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 Â± 3 ÂºC 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 d ay by adjusting the humidifier and partially opening the clear plastic. The air conditioner was turned off unless the temperature was above 35 ÂºC. After seven days, plants were moved out from the healing chamber and grown under natural greenhouse condition s. 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).
46 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 we re 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 fert ilizer (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 expe riments, respectively, when at least two female flowers bloomed on each of the melon plants.
47 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 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 roo t 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 v ascular 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 t o 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 area s 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 with out 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
48 develop ment 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 (Nguyen, et al., 2013). Survival Rates a nd Quality o f 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 regen eration 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 pot ential of grafting survival . Hole insertion method works best with small
49 scions that could be easily inserted int o the holes of rootstocks, whereas TA method works best when scion and rootstock plants have similar stem diameter. Rootstock a nd 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 grafte d 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 gr afting 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 graf ted 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 H I and OC grafted plants, even though the majority of the plants were alive at 16 DAG. This was d ifferent from other methods in which graft failure was attributed to the lack of union between scions and rootstocks, which resulted in scion death. The majorit y of NC grafted plants he aled 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
50 NC grafted pla nts 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 hy pocotyls. 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) reporte d that root excision significantly 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 con trols were observed in this experiment. Grafting methods and root treatment also did not affect flowering date in this study.
51 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 w as 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 surfa ce 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 did exhib it 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 co ntent, 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 g rowth characteristics was observed among HI, OC and TA grafted plants, suggesting the three grafting methods did not differ in their effects on plant growth.
52 Conclusion s The experiment demonstrated that without root excision, plants grafted with hole ins ertion, one cotyledon and tongue approach methods performed similarly regarding quality and growt h characteristics, however, non cotyledon method resulted in quality reduction of the grafted plants. Root excision was unsuccessful with tongue approach metho d, 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.
53 Table 2 1. The charact eristics of hole insertion, one cotyledon and tongue approach grafting methods . Character istics Grafting methods Hole insertion One cotyledon Tongue approach Rootstock regrowth Yes Yes No Location of graft union High High Low Requirements for post grafting healing High High Low Requirement of grafting clips No Yes Yes Grafter training Moderate to high Low Moderate to 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 95.8 z 91.6 One cotyledon 100 100 Non cotyledon 91.6 87.5 Tongue approach 45.8 83.3 z Average of the three replications
54 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 9.08 y aA x 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 = mod erately stunted; 6 = somewhat stunted; 7 = fair but not acceptable; 8 = borderline acceptable; 9 = good and acceptable but not the best ac ceptable; 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 LSD ) Table 2 4 . Aboveground biomass at 16 days after grafting . Treatment z First experiment Second experiment Fresh weight (g) Dry weight (g) Fresh weight (g) Dry weight (g) Rootstock c ontrol 2.72 a y 0.28 a 2.02 a 0.25 a Scion c ontrol 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 Ã— Grafting interaction effects were observed. y Means followed by the same letter were not significantly different according to least significant difference ( LSD ) at P
55 Table 2 5 . P value s 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 . Facto r z Root growth characteristics Aboveground growth characteristics Length surfac e area Fresh weight Dry weight Leaf area Stem diameter Chlorophyll content Stomatal 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 Ã— 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 Ã— 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.
56 Table 2 6 . Root and aboveground growth characteristics of plants without root excision at 44 and 46 days a fter grafting in the first and second experiments, respectively. Treatment Root growth characteristics Aboveground growth characteristics Length (cm) surface area (cm 2 ) Fresh weight (g) Dry weight (g) Leaf area (cm 2 ) Stem diameter z (mm) Chlorophyll Content y (CCI x ) Stomatal conductance (mmol/m 2 s) First experiment Rootstock control 2301.799 a w 714.741 a 191.07 a 22.60 a 3471.66 a 6.66 a 18.03 a 2078.43 a 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 approach 2368.076 a 817.823 a 174.03 a 17.26 bc 3444.63 a 7.46 a 18.03 a 1522.52 ab Second experiment Rootstock control 4008.548 a 846.982 a 284.40 a 41.17 a 4126.06 a 9.63 a 27.27 a 1445.6 a 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 approach 4560.601 a 833.892 a 213.98 b 25.36 b 2965.71 a 8.29 a 22.23 ab 2145.2 a 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 w
57 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 withou t root excision. C) Root surface area of plants with root excision. D) Root surface area of plants without root excision.
58 Figure 2 2. Root growth during the 16 days after grafting (DAG) in the second experiment. RC: rootstock control, SC: scion contr ol, HI: hole insertion, OC: one cotyledon, NC: non cotyledon. A) Root length of plants with root excision. B) Root length of plants withou t root excision. C) Root surface area of plants with root excision. D) Root surface area of plants without root excision.
59 CHAPTER 3 SPECIALTY MELO N CULTIVAR EVALUATION UNDER ORGANIC AND CONVENTIONAL PRODUCTION IN FLORIDA Introduction Melon is a c rop 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 ty pe 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. Amo ng 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 me lon 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
60 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 addit ion, 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 hea lthy, 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 speci alty melon types were not provided. The major production barriers are lack of dise ase resistant cultivars and low marketable yield (Maynard, 1989). The challenge is more pronounced in Florida, since the humid subtropical conditions often result in high lev els 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). Therefo re, 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
61 problems in melon production. Some specialty melon cul tivars 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 Flor ida 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 p roduction 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 convenie ntly 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, t hese 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
62 cantaloupe, one o f 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 2011. Untreated or org anic 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. Conve ntional 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 Scien ce 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 (50:5 0, by weight) at the rate of 448 kgÂ·ha 1 . Both the organic and conventional field experiments were arran ged in a randomized complete block design with four blocks and 10 plants per cultivar in each block. Plan ts 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 irriga tion. The bed s pacing and in row spacing
63 we re 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 a l., 2011). The 10N 4.4P 8.3K fertilizer (Premium Vegetable Grower Fertilizer; Southern States, Lebanon, KY) was applied preplant at 84 kgÂ·ha 1 nitrogen (N) to the conventional field. Plants were fertigated 2 weeks after transplanting with 6N 0P 6.6K fertil izer (Dyna Flo; Chemical Dynamics, Plant city, FL) at a weekly rate of 8.4 kgÂ·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 kgÂ·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 recorde d. 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. external color changi ng from green to golden yellow or creamy white. Canary melon is less aromatic than honeydew melon. Hence, the change of fruit external color from tudy.
64 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 la bels. 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 m elon 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 necr osis 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 = pl ant totally wilts and cannot recover; 5 = plant is dead. After
65 the final harvest, all 10 plants i n 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 = numero us 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 ro tting 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 record ed. 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 Tes ter; 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) . difference 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
66 type melon were also early ma turing cultivars. The first harvest date of these four cultivars was 52 DAT under both organic and conventional production. By contrast, the occur until 62 DAT (Tabl e 3 2). Although the two canary melon cultivars in this study earlier harvest time than honeydew melon cultivars in Delaware (Johnson and Ernest, 2010). As the rainy season norma lly 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 syste ms 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 the h ighest marketable yield under conventional production (10.7 kg/plant). However, wi 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, ca organic and conventional fields (8.3 and 8.9 kg/plant, respectively). The high marketable
67 culls. High yi elds and excellent fruit quality of canary melon cultivars were also observed by Strang et al. (2007) in Kentucky. per plant compared with other specialty melon cultivars in b oth fields (Table 3 3). higher than other types of specialty melon cultivars in the organic field, but interestingly, such difference appeared to diminish in the convention al field. Other than the asian melon cultivars, no significant differences in the marketable fruit number were observed conventional field. alia 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 Galia 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 harves ted the melon fruit every 4 5 d instead of a shorter ripe, resulting in an increased percentage of culls.
68 Differential performance of the two cultivars within the same specialty melon type was an excellent performance in open field conditions, with marketable yields of 7.2 and 7.6 high yie lding 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) conclude d 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 enviro nmental 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 increase in the conventional field compared with the organic field. Asian melon increase in marketable fruit weight by over 35% compared with melons in the organic marketable fruit weight in the organic and conventional fields.
69 Fruit Characteristics Fruit weight and size varied significantly among specialty melon c ultivars (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 the ir elongated fold greater than the fruit width. Oval ced the largest fruit, with average fruit weight of 2.7 and 2.3 kg in organic and conventional fields, respectively. 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, S SC, 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 15.3 and 15.6% in the organic and conventional fields, respectively (Table 3 melon reported in a specialty melon trial conducted in Kentucky (Strang, et al. 2007). Ernest (2010) reported that SSC of some canary melon cultivars may reach 16%.
70 tivar grown in the southeastern United harvested at half to 3/4 slip maturity stage. However, with respect to consumer 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 Spa in, 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 qua lity 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 s helf life. In this study, the flesh firmness of 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, ana nas 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 a ssociated with the distinct ripening patterns of climacteric and non climacteric melons (Pech et al., 2008). Compared with melons in
71 Reticulatus and Inodorus groups, little information is available about biochemical and physiological characteristics of mel ons 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 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 independe nt 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 th e 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 compared with other melon cultivars (Table 3 N galling, it might be possible that they carry some exhibited less galling than the oth er specialty melon cultivars evaluated, except for
72 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, ars, asian assessments over multiple seasons which include examination of individual diseases are needed to fully evaluate disease resistance and tolerance of these melon cul tivars. 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 thi s cultivar evaluation trial w here no fungicides or nematicides were disease performance, and produced high quality fruit, although the yield of hon eydew 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 c ultivars 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 deman d, 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.
73 Conclusions While high temperatures and humidity pre sent 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 thi s cultivar evaluation trial where no fungicides or nematicides were used, can 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 warra nt 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.
74 Table 3 1. taloupe grown in organic and conventional fields during Spring 2011 at Citra, FL Melon type Cultivar name Seed source z Fruit characteristics y Ananas ( C. melo var. reticulatus ) Creme de la Creme W. Atlee Burpee & Co. Orange yellow and lightly netted skin; fruit is creamy white, marbled with pale orange; fragrant, very sweet and slightly spicy San Juan Seeds Orange yellow rind and heavily netted; ivory colored flesh; pear like, sweet flavor Canary ( C . melo var. inodorus ) Brilliant Seeds Dark yellow and lightly wrinkled skin; white and juicy flesh, sweet and nutty in flavor Camposol Seedway, LLC Bright yellow rind and lightly wrinkled skin; white and juicy flesh; honey dew like taste; large fruit Asian ( C. melo var. makuwa ) Ginkaku Kitazawa Seed Co. Small, oval shaped; deep golden color with white stripes; white flesh, is quite thick, crisp, smooth and remarkably sweet Sun Jewel Seeds Oblong fruit, lemon yellow with shallow white stripes; white flesh, crisp when ripe, moderately sweet Galia type Arava Seeds Golden yellow and lightly netted rind; lime green and juicy flesh; extra sweet flavor with tropical and perfumed aromatics ( C. melo var. reticulatus ) Diplomat Seeds Golden yellow and lightly netted rind; lime green and juicy flesh; extra sweet flavor with tropical and perfumed aromatics Honeydew ( C . melo var. inodorus ) Honey Pearl Seeds White skinned fruit with white flesh; sweet flavor and grainy texture, like asian pears, round, uniform and middle sized fruit Honey Yellow Seeds Yellow skinned fruit with orange flesh; juicy and very sweet; smooth skin, round, uniform and medium sized fruit Cantaloupe ( C. melo var. reticulatus ) Athena Seedway, LLC Well netted, sutureless; ripe fruit seldom crack and have a tough rind; good shelf life even when harvested ripe; thick, sweet, orange flesh
75 Table 3 2. conventional production during Spring 2011 at Citra, FL. Cultivar Anthesis z (DAT y ) First harvest (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.
76 Table 3 3. under organic (Org) and conventional (Con) production during Spring 2011 at Citra, FL. Cultivar Marketable fruit wt (kg/plant) z Marketable fruit number (no./plant) Culls (%) y Org Con Org Con Org Con Creme de la Creme 7.3 ab x 7.4 bcd 4 cd 5 bcd 7.0 bc 12.0 de 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 difference (LSD) test at P
77 Table 3 4. Fruit weight, length, shape, soluble solids concentration (SSC), and flesh firmness o f 10 specialty melons and Cultivar Fruit wt (kg/fruit) z Fruit length (cm) Fruit shape y SSC (%) Flesh firmness (N) Org Con Org Con Org Con Org Con Org Con Creme de la creme 1.8 c x 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 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 Yellow 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 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 difference (LSD) test at P
78 Table 3 5. Aboveground diseases severity and root knot nematode (RKN) gall ratings conventional production during Spring 2011 at Citra, FL. Cultivar Organic field Conventional field Disease severity rating (0 5 scale) z RKN gall rating (0 10 scale) y Defoliation (%) x Creme de la creme 2.9 bc w 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 a nd 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 ga lls, 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 healt hy 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 P
79 Fig ure 3 1. Pictures of melons in the field and harvested fruit of 10 specialty melon cultiva 1) a 2) (ananas melon); B 1) 2) type melon); E 2)
80 CHAPTER 4 ROOT KNOT NEMATODE RESISTANCE AND YIEL D 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 specialt y 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 hortic ultural crops is largely dependent on soil fumigation (Duniway, 2002). Given the Reprinted with permission from ASHS journal
81 increasing costs and rising environmental concerns associated with soil fumigation, alternative approaches are needed for integrated management to improve sustainable melon pr oduction (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 natu rally 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 co nsumed as food supplements by loca l 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.,201 1). 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 f usarium wilt (caused by Fusarium oxysporum ), g ummy stem blight (caused by Didymella
82 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, v egetable grafting was proposed as an alternative approach to take advantage of RKN resist ance in wild Cucumis species (Kokalis Burelle and Rosskopf, 2011; SigÃ¼enza, 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 des irable 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 cur rently 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 (K okalis Burelle and Rosskopf, 2011; Lee and Oda, 2003; SigÃ¼enza, et al., 2005; Thies et al., 2013). However, information is limited regarding its grafting compatibility with other melon types, an d the rootstock effects on RKN management and yield of the gra fted 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
83 determine the effects of C. metulifer on RKN management, yield performance, and fruit quality of grafted plants. Materials and Methods Plant Materials C. melo var. inodorus ) C. melo var. reticulatus 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 Intr oduction (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 th e C. metulifer rootstocks in the present greenhouse and field studies, respectively. Greenhouse RKN Study The greenhouse RKN inoculation study was conducted during Fall 2011. C. metulifer rootstock. N on grafted and self 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 originat ed from tobacco ( Solanum lycopersicum ) in a greenhouse ) . The RKN species and race were identified by their isozyme patterns resolved by polyacryalimide electrophoresis and differ ential host tests (Taylor and Sasser, 1978).
84 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 ve rmiculite, 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 blo cking healing chamber in the greenhouse where temperature was maintained at 28 Â± 3 ÂºC 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 aft er 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 inocul ated 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 arr anged 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 ÂºC. Fertilized female flowers were removed to pre vent fruit formation. The experiment was terminated on 15 Oct. Roots were washed gently and
85 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 ne matode chamber counting slide under a 40Ã— 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 were grafted onto C. metulifer rootstock for the field trials. the conventional and organic trials, respectively. Seeds of C. metulifer and melon cultivars were sown on 20 and 22 Feb., respectively. A p eat b ased 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.
86 The field experime nts were conducted at the University of Florida Plant Science Research and Education Unit (PSREU), Citra, FL. The organic site was naturally Abelmoschus esculentus ) was grown in the plots fro m 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 pla nt growth and need throughout the season. In both fields, the treatments were: non C. metulifer rootstock. Self ontrols 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 fiel ds 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 Harvest; Gloucester, MA) at a rate of 2.3 kg N/ha. In the conventional field, 10N 4.4P -
87 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 Fl o; 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 recom mendations (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, respective harvested at full external fruit 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 final harvest and separated from ripened fruit. Fruit were weighe d 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 o rganic 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
88 (1.75 cm diam. Ã— 20 cm depth) were collected in the root zone of six plants in the center of each plot. Each soil samp le was thoroughly mixed and second stage juveniles (J2) were extracted from 100 cm 3 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 orga nic 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 g alling. 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, s 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 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 u nspecified race of M. incognita (Dalmasso et al., 1981; Fassuliotis, 1967; Nugent and Dukes, 1997; SigÃ¼enza 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
89 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 findin g 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 ha s 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 . onto C. metulifer showed significantly lower root gall index (GI), egg mass index (EMI), and Rf than non grafted and self indicating grafting RKN susceptible melon onto C. metulifer was effective in reducing RKN reproduction. No sig nificant differences in EMI and Rf were observed between non grafted C. metulifer C. metulifer . Field Study of RKN Management 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 4 2). Root galling was significantly reduced on C. metulifer (GI < 1). Grafting with C. metulifer rootstock also reduced J2 numbers in the
90 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 do uble 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 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 cr opping 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 C. metulifer with non grafted and self grafted melon plants (Table 4 3). Similar results were also reported on C. metulifer grafted muskmelons t hat 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 press ure was at a relatively high level. According to Barrett et al. (2012 b
91 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 lower range of RKN damage compared to what was previously reported (Barrett et al., 2012 b ). In the conventional field, no significant differences in total and marketable yields were observed between non C. metulifer rootstock. However, total yield (but not marketable yield) C. metulifer was significantly lower than that of non conventional field (Tab le 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 C. metulifer rootsto ck affected fruit size and uniformity (Table 4 4). Although some studies showed that grafting melons onto Cucurbita interspecific hybrid rootstock increased single fruit weight (CrinÃ² et al., 2007), it may not be the case for melons grafted onto C. metulif er 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
92 melons. Although yield was not improved by grafting, incorporati ng 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 ef forts may be directed to develop more vigorous C. metulifer rootstocks with greater potential for yield enhancement in grafted melon production.
93 Table 4 1. Root gall index (GI), egg mass index (EMI), egg recovery, and reproduction factor (Rf) of grafted and non greenhouse study (2011). Treatment z GI y EMI Egg recovery x Rf w NGHY 5.0 a u 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 C. metulifer ; HY/Cm = C. metulifer ; HY/HY = self 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 significant difference test at P
94 Table 4 2. Root gall index (GI) and numbers of Meloidogyne javanica second stage juveniles (J2) in soil of grafted and non (2012). Treatment z GI y Meloidogyne density (J2/100 cm 3 of soil) Honey Yellow NGHY 7.14 a x 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 Ar/Ar = self C. metulifer , respectively. y GI was rated on a 0 10 s cale: 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 sys tem 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 interrup ted, 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 P
95 Table 4 3. Total and marketable fruit yields (kg/plant) of grafted and non grafted honeydew m conventional field studies (2012). Treatment z Organic Conventional Total yield Marketable yield Total yield Marketable yield Honey Yellow NGHY 4.93 a x 3.85 a 10.20 a 9.06 a HY/Cm 3.81 a 3.26 a 8.53 b 7.44 a HY/HY y 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 Ar/Ar = self C. metulifer , respectively. y Self x Means within a column followed by the same letter were not significantly different P
96 Table 4 4 . Marketable fruit numbe r per plant in different fruit size categories of grafted and non the organic and conventional field studies (2012). Treatment z Organic Conventional 0.45 1.35 kg 1.36 1.80 kg 1.81 2.25 kg >2.25 kg 0.45 1.35 kg 1.36 1.80 kg 1.81 2.25 kg >2.25 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/HY y 1.62 0.96 0.51 0.05 P value NS x 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 Ar/Ar = self C. metulifer , respectively. y Self x NS = Non significant, i.e., P > 0.05.
97 CHAPTER 5 STUDYING QUALITY ATTRIBUTES O F GRAFTED SPECIATLY MELONS USING BOTH CONSUMER SENSORY ANALYSIS AND INSTRUMENTAL MEASUREMENTS Introduction Melon ( Cucumis melo L.) is an important component of fresh consumed vegetable s and fruit s 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 ad dition 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 (Chapt er 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 conside rable 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 plant s 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 soil borne diseases were managed through grafting, such as fusarium wilt (caused by Fusarium oxysporum ), monospor ascus sudden wilt (caused by Monosporascus cannonballus ), gummy stem blight (caused by Didymella bryoniae ), verticillium wilt (caused by Verticillium dahliae ), and root knot nematode
98 (caused by Meloidogyne spp.) (Guan et al., 2012) . In addition to the dise ase 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 resu lts 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 Ã— C. moschata mpact on the total Cucumis melo inodorus ) (CrinÃ² et al., 2007) Cucumis melo inodorus ) (Fita et al., 2007). With the same scion cultivars, different roots tocks might exhibit different year study, while C. maxima Ã— 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).
99 Fruit quality is a multivariate characteristic, and SSC and flesh firmness were among the most important melon quality attributes that were widely u sed 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, a bnormal 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 compou nds in the determination of melon fruit quality are increasingly recognized , grafting effe cts on these characteristics were also studied . In add ition, reduced fruit sh elf life following 1 C. maxima Ã— 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 impact s 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 t he overall
100 evaluation of melon fruit quality need to combine both instrumental measurement and sensory analysis (Aulenbach and Worthington, 1974) . Melon q uality attributes as influenced by cultivars, ripening stages, and postharvest treatments were evaluated by combining consumer sensory analysis and instr umental 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 fr uit quality of grafted melons. A reliable consumer sensory a nalysis normally involve s 75 to 150 consumers (Lawless, 1998) . A 9 point hedonic is commonly used , which was regarded as one of the most valid and reliable scale s used in consumer sensory analysis. In this study, grafted specialty melons with different ro otstock 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 o ur 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 C. melo L. var. reticulatus Ser.) and the honeydew melon C. melo L. var. inodorus Naud.) were grafted onto each of the two Cucurbita max ima Duchesne Ã— Cucurbita moschata Duchesne), and Cucumis metulifer E. Mey. ex Naud. Seeds of the two melon
101 rootstock seeds were donated by Syngenta Seeds (Research Triangle Par k, 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 C. maxima Duchesne Ã— 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 cot yledon 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
102 design with five replications in 2012 and four replica tions 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 based on fruit color and total soluble solids content. At the beginning of the first harvest, Among them, the highest SSC was 16%. The color that corresponded to the highest SSC was then Sample Preparation Analyses of the two melon cultivars were conducted separately on different days. a melons were harvested and stored at 10 ÂºC overnight. Ten fully ripe melons were chosen from each treatment based on fruit size, i.e., approximately 1.5 kg per fruit, and absenc e 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 melo n 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 C. metulifer rootstock grafted melon plants). In 2013, only one sensory analysis with three treatments, i.e., non on June 14.
103 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 c ounterparts 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 3Ã—3 cm cubes. Fruit cubes from ten halves of each t reatment were well mixed and stored in a plastic container at 4 ÂºC 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 proce dures of the sensory evolution and layout of sensory analysis lab were describ ed previously (Barrett et al., 2012 c ) . 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 samp le, 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 descr ibe 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 aske d to indicate whether they exp erienced off flavor in the sample, and to describe the off flavor they detected.
104 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) . Statistic al Analyses Analysis of variance was performed using the Proc Glimmix procedure of SAS program (Version 9.2 C for Windows; SAS Institute, Cary, NC) . measurement s among treatments. Results and Discussion Consistent among all the evaluated production conditions in 2012 and 2013, the uced consumer rated overall acceptability and flavor liking compared with non 5 2 ). Averaged among production conditions in 2012, consumers who rated the hile 36% more 5 3 ). As Just about right scale provided directional information for the hedonic questions (Van Trijp et al., 2007) , the result s suggested that consumers like d the Ar/ST melons less because they were not sweet enough. This was
105 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 s grafted onto compared with NGAr (Table 5 4 ). Hybrid squash rootstock is well kn own 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 g rowths were commonly observ ed in plants grafted onto vigor ous 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 promotin g light infiltration and improving fruit quality (Cohen et al., 1999; Yang et al., 20 07 ) compared with non grafted cont rols, 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 factor s that deserve more attention are effects of grafting on fruit scion grafted onto hybrid squash rootstocks delayed anthesis of female flowers, but did not aff ect early harvest (Chapter 6 ). Considering fruit sugar content is correlated with the length of the fruit development period (Burger and
106 Schaffer, 2007) , stimula ted 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 squas h 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 product s as whole pattern s, they lack the capability to se parate individual product attributes (Lawless and Heymann, 2010) . As a result, they may est ablish a positive correlation between two unrelated attributes, such as sweetness and firmness (Clark and Lawless, 1994) . Therefore, the higher rating on firmness li king of non sweetness more than gra fted melons. Another potential explanation could be that texture attributes other than firmness were affected by grafting, such as juiciness and adhesiveness (Krame r and Szczesniak, 1973) . Because consumers were not asked to rate these attributes, they may express their feeling on other fruit texture at tributes as firmness liking (Lawless and Heymann, 2010) . A more accurate sensory specification on melon firmness may be provided by trained panelists. C. metulifer Rootstock 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 f irmness between NGAr and Ar/Cm were detected under either of the production conditions (Table 5 2 ).
107 Known for root knot nematode and fusarium wilt resistances, C. metulifer was tested as a rootstock for grafting melons (SigÃ¼ enza et al., 2005) . However, unl ike 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 t he 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 field s yielded different results in consumer sensory properties. This may suggest the rootstock effects on melon fru it quality were subjected to influences of environmental conditions . C. metulifer rootstock did not affect SSC and flesh firmness, indicating that its effects on sensory p roperties might be attributed to quality attributes other than sweetness and firmness . Off 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 o f melon consumption. Although lacking capability of defining off flavor, it was consistent among the tests that a greater percentage of panelists reported off 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 fiel d. 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.
108 f lavor 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 . 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 th e 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 Gri mm, 2001) , and some alcohols n oted for fermented flavor and are . 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 Regardless of the producti on conditions and the rootstock selections , grafted t any significant differences in sensory properties (overall acceptabilit y, flavor liking, and firmness liking), SSC, and flesh firmness compared with non 5 6 ). In addition, the percentages of consumers
109 who detected off flavor were similar between non 5 7 ). Our findings were in accordance with previous reports of insignificant influence of grafting on quality attributes of honeydew melons ( Traka (CrinÃ² et al., 2007) , However, it was also demonstrated that unsuitable rootstock might cause quality (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 climacteric fruit that exhibit an autocatalytic ethylene production peak during fruit ripening, while the ethylene production peak was not observed in non climact er ic honeydew m elons (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 g alia 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 honeyde period of fruit growth and the initial stage of sugar accumulation (Pratt et al., 1977), the enti re fruit development stages (Bianco and Pratt 1977). Considering the dry weight accumulation would require an efficient water removal mechanism (Schaffer et al.,
110 1996), the enhanced water status in the grafted melon plants might affect the efficiency of su gar 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 Ã— scion int e raction all contribute to the ultimate fruit quality, the present study indicated that the scion might play a more critical r ole in the process. A study using three cherry cultivars grafted onto five rootstocks with varied siz e controlling potential found that the scion accounted for the highes t percentage of variation in quality modification, and concluded that fruit quality was more of a scion dependent character (GonÃ§alves 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 cultivar s (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 off flavor that might be contributed by volatile compounds could also explain the Co mpared with non instrumental measurements. The differential responses of the two specialty melon types to grafti ng practice in terms of fruit quality deserve further investigations. More in depth
111 studies especially those involving flavor related volatile compounds and profiles are also warranted to better understand the rootstock and scion interaction effects on fru it quality and consumer perceived sensory properties of grafted melon fruit. .
112 Table 5 1 . Gra f ting treatments included in certified organic field, non fumigated conventional field, and fumigated conventional field in Spring 2012. Certified organic field Non fumigated conventional field Fumigated conventional field grafted onto C. metulifer rootstock grafted onto C. metulifer rootstock r ootstock None rootstock Non Self None Self and
113 Table 5 2 . Consumer sensory evaluation and instrumental measurements of grafted Treatment z Consumer sensory evaluation y Instrumental measurements Overall acceptability Flavor liking Firmness liking SSC (ÂºBrix) Flesh firmness (kg) Test 1 x F NGAr 6.51 a w 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 C. metulifer rootstock, respectively. F, NF, and O indicated melons produced from fumigated conventional fi eld, non fumigated conven tional 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 June 1 and June 12, 2012, respectively. Test 1 included 96 panelists, and Test 2 included 100 panelists. w Means within a column follower by the same letter were no t significantly different by P
114 Table 5 3 . Percent age distribution of panelists in the consumer sensory evaluations of sweetness and firmness level, and the percentage of panelists who detected off Treatment z Sweetness level (%) y Firmness level (%) Off flavor (%) 1 2 3 4 5 1 2 3 4 5 Test 1 x 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 C. metulifer rootstock , respectively . F, NF , and O indicated melons produced from fumigated conventional field, non fumigated conventio nal 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 to o 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 pan elists.
115 Table 5 4 . conventional field in Spring 2013. Treatme nt z Sensory evaluation y Instrumental measurements Overall acceptability Flavor liking Firmness liking TSS (ÂºBrix) Flesh firmness (kg) Titratable acidity pH NGAr 6.89 a w 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 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 difference test at P
116 Table 5 5 . Percent age distribution of panelists in the consumer sensory evaluations of sweetness and firmness level, and the percentage of panelists who detected off Treatment z 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 z NGAr, Ar/Ca, Ar/ST indicated non 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/s omewhat too sweet, 5= too firm/m uch too sweet. This test included 105 pa nelists.
117 Table 5 6 . Consumer sensory evaluation and instrumental measurements of grafted Treatment z Consumer sensory evaluation y Instrumental measurements Overall acceptability Flavor liking Firmness liking SSC (ÂºBrix) Flesh firmness (kg) Test 1 x 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 NS w 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 grafted onto C. metulifer rootstock, respectively. F, NF and O indicated melons produced fr om fumigated conventional field, non fumigated conven tional 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.
118 Table 5 7 . Percent age distribution of panelists in the consumer sensory evaluations of sweetness and firmness level, and the percentage of panelists who detected off Treatment z Sweetness level y Firmne ss level Off flavor (%) 1 2 3 4 5 1 2 3 4 5 Test 1 x 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 grafted onto C. metulifer rootstock , respectively . F, NF and O indicated melons produced from fumigated conventional field, non fumigated conven tional 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.
119 CHAPTER 6 PHYSIOLOGICAL CHANGES IN GRAFTED MELON PLANTS WITH A HYBRID SQUA S H 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., 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 fundamen tally 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 modifi cations 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 Univers ity of Florida Plant Science Research and Education Unit in Citra, FL, USA. The soil texture is loamy sand. , Inc. Boise, ID, USA). Non and self 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 m ixture of vermiculite,
120 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 g rafted 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 i nto 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 supplemen ted 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 treatmen t 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 3 6 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 develop ed 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. (2012 c ). The day before the
121 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 ÂºC 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 firmn ess 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 Ã— 3 cm melon cubes. All the fruit cubes were then well mixed and stored in a plastic container at 4 ÂºC 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 perf ormed using the Proc Glimmix procedure of SAS program ( significant difference (HSD) test ( measurements between different treatments. Results and Discussion The first female flower of the self and non and self grafted plant s had about 12 blooming ure 6 1). The delayed bloom of female flowers was also observed
122 on cucumbers ( Cucumis sativus ) (Satoh, 1996; Yilmaz et al., 2011) and watermelons ( Citrullus lanatus ) (Sakata et al., 2007) grafted onto hybrid squash rootstocks. Although 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 BalÃ¡zs (2010). On average, the length of fruit development period from anthe sis to full slip for and self 6 2). The result 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 wi acceptability, flavor liking, and firmness liking by consumers in the sensory analysis. Interestingly, self of overall acceptability and flavor liking than non instrumental measurements did not show any significant differences (Table 6 3). As fruit sugar content is correlated with the length of frui t 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, a pplication of ethephon, an ethylene releasing compound, reduced
123 melon SSC ( Ouzounidou et al., 2008 ). The role of ethylene in the regulation of fruit ripening and quality of grafted plants deserves further studies. 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 (Fig ure 6 2). Similarly, Minuto et al. (2010) reported the sudden collapse of melon plants grafted onto h ybrid squash rootstocks, and the reduction in final fruit yield per ha as a result of the high incidence of plant sensitive scion rootstock combination to this physiological disorder, and plant collapse became more severe with higher temperature (30 Â°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 degenera tion 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. compared with non and self 6 4). The enhanced vegetative growth is generally ass 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 im provement. Further studies are warranted to evaluate the
124 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 d eserve 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
125 Table 6 1. Early and total yields of graft ed and non Treatment Early harvest Total harvest Total yield (kg/plant) Marketable yield (kg/plant) Total number (no./plant) Marketable number (no./plant) Total yield (kg/plant) Marketable yield (kg/plant) Total number (no./plant) Marketable number (no./plant) NA r z 0.58 a y 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 y difference test at P
126 Table 6 2. Duration of fruit development from anthesis to harvest of grafted and non Treatment z Days from anthesis to harvest NAr 38 a y Ar/Ar 39 a Ar/St 34 b z NAr = non y Means within a column followed by the same letter were not significantly different P
127 Table 6 3. Fruit quality attributes of grafted and non Treatment y Instrumental measurement Consumer sensory analysis z SSC (ÂºBrix) Flesh firmness (kgf) Titratable acidity (%) pH Overall acceptabilit y Flavor liking Firmnes s liking NAr 7.0 a x 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 z 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 Means wit hin a column followed by the same letter were not significantly different P Table 6 4. Length (cm) of the longest vine of grafted and non 36 days after transplanting (DAT) and 46 DAT. Treatment z 36 DAT 46 DAT NAr 72.4 b y 127.2 b Ar/Ar 71.3 b 130.4 b Ar/St 83.0 a 148.2 a z NAr = non y Means within a column followed by the same letter were not significantly different P
128 Figure 6 1. Cumulative numbers of female flowers per plant in grafted and non grafted Figure 6 healthy root system; B) Cell death initiated in the root s, as indicated by the arrow; C) Cell death spread to the stem of rootstock, necrosis of vascular tissue develop ed as indicated by the arrow; D) T he entire rootstock was dead; E) dead rootstock and healthy scion stem separated by the graft union.
129 CHAPTER 7 SUMMARY AND FUTURE PROSPECT Summary Melon grafting is used for managing soilborne diseases, and improving abiotic stress tolerance s . The h igh cost of producing graf ted seedling s is an obstacle preventing wide adap tion of this technology in the U.S. Optimizing grafting procedure and producing high quality grafted seedling s is important in reducing the cost. Four grafting method s including hole insertion, one coty ledon, to ngue approach, and non cotyledon were examined for their impacts on seedling growth and root characteristics The grafted plants were examined with or without root excision (Chapter 2 ) . Except the non cotyledon grafting method , root excised plants initiated roo t 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 n on cotyledon method resulted in a shorter root length and less surface area compared with hole insertion and on e cotyledon grafted pla nts at 16 days after grafting . Non cotyledon method significantly decreased quality of grafted plants . Root excision d id not exhibit significa nt impacts on either root growth or above ground growth characteristics at the flowering stage . Without roo t excision, hole insertion, one cotyledon , and tongue approach methods did not show consistent differences in above ground growth characteristics com pared with non grafted scion controls at about six weeks after grafting . Growing special ty melons is challenging in Florida as they often lack disease resistance s . Cultivar evaluation trials including ten specialty melons and a commonly grown cantaloupe m rn U.S were conducted (Chapter 3) .
130 Compare specialty melons are generally more susceptible to root knot nematode s (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 hone 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 Cucumi s metulifer was further tested as a rootstock in conventional and organic field (Chapter 4) . C. metulifer exhibited significantly lower gal ling 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 enhanceme nts, 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, w hich was C. metulifer rootstock had a significantly lower total fruit yield than non C. metulifer rootstock and no n plants.
131 P revious 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 outst anding taste and unique fruit flavor. and root knot nematode resistant C. metulifer (Chapter 5) . Regardless of the production systems, received significantly lower scores in consumer overall acceptability and flavor liking comp ared to non . Reduced SSC were detected in the graft combination . Grafting with C. metulifer significantly decreas ed consumer overall between grafted and non grafted treatments was not detected in melons produced from the non fumigated conventional filed. More consumers detected of f flavor in the grafted , which may partially explain the decreased preference for the fruit from grafted plants. TSS, flesh firmness, and consumer perceived sensory att melons. Plant phy siological changes were explored to understand the rootstock effects on fruit quality modifications in the (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 were not significantly different between grafted and non celerated fruit development as the duration between anthesis and harvest was about 4 and 5 days shorter than that of non and
132 self grafted plants, respectively. Vegetative growth was greater in the grafted plants with rvest period, about 27% of grafted plants with pathogenic. The wilt plants eventually died at the end of the season. Analysis of the collapsed grafted plants indicated that the cell death initiate d 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 P rospect 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 studie s 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 melons onto C . metulifer . T he 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 devel op more vigor ous 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 t o enhance the production of high value vegetables in RKN infested fields. Moreover, the grafting practice involving diverse types of specialty melon
133 cultivars will need to be tested at locations with differ ent disease pressures and varying climate conditio ns. 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 chang es that might be related to fruit quality determination have been investigated in this project . More in depth studies are warranted to understand the cause s of these changes, such as water uptake and transport, plant hormones, and nutritional status . I n addition, future research needs to elucidate rootstock and scion interactions at the cellular and molecular level. More research is warranted to conduct b ioc hemical analysis of 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. B reakthroughs 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 mechanism s of rootstock scion interactions , it wil l 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 a nd
134 scion plants before grafting. While grafting technology has been used in vegetable production for decades, many underlying mechanisms are still poorly understood . Further ing our s to grafting will not only improve ou r fundamental knowledge in plant science but also benefit sustainable vegetable production through more targeted breeding and use of rootstocks.
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154 BIOGRAPHICAL SKETCH the Southwest the Chinese Aca demy 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 part icular 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.