Grafting, an Alternative Method for Control of Fusarium Wilt Disease in Watermelon

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2 The Islamic University-Gaza الجامعة اإلسالمية- غزة Deanery of Higher Education عمادة الد ارسات العليا Faculty of Science كلية العلوم Department of Biological Science قسم العلوم الحياتية Botany & Mycology نبات و تخصص فطريات Grafting, an Alternative Method for Control of Fusarium Wilt Disease in Watermelon Proposed by: Mohammed Kh. Al Massri Supervisor: Dr. Abboud Y. El Kichaoui Ph.D. Botany Islamic University, Faculty of Science, Biology Dept. "A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master in Biological Science, Botany & Mycology" November, 2014

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5 Dedication: I would like to dedicate this research to my university "IUG" To the committed researcher, To my parents, To my wife, To my son, Ahmed, To my daughters, To my brother, Ahmed, To all my family members, To good Friends, To all of them I dedicate this work. Mohammed Almassri I

6 Acknowledgement: First and foremost I would like to thank Allah (S. W.T) for giving me talents and showing me how to use them for honor and glory. I thank all the farmers of Rafah and Beit Lahia from Gaza Strip for their cooperation in survey and sampling from infected watermelon farms. Also, my gratitude extend to Fungal Identification Service in The Ministry of Agriculture for their services. I wish to express my deepest gratitude to the supervisor Dr. Abboud El Kichaoui, who has encouraged me along the way and has helped make this research come true. Also, thanks are extended to biological science department staff for their help during the laboratory work especially Mr. Ashraf Alshafei and Nidal Fayyad. Also, I would like to thank agricultural engineer Ali Adher who helped me in the grafting process. Addition my thanks are extended to the farmer Hani Hamodh who helped me in transplanting process. Moreover, I would like to thank my dear friends of English teachers language, Dr. Jihad AL Msalami for his revision of the research and Dr. Islam Abu Sharbain who helped me in statistical analysis. Finally, I would like to express my deepest gratitude to my wife who helped me to accomplish this thesis. The researcher: Mohammed Kh. Almassri II

7 Grafting, an Alternative Method for Control of Fusarium Wilt Disease in Watermelon Abstract: The aim of this research is to control the pathogenic fungus which causes Fusarium wilt disease in watermelon, Citrullus lanatus var lanatus cv. (Crimson Sweet 0015) by grafting it onto pumpkin, Cucurbita moschata cv. (Tetsukabuto). Pathogenic fungus was isolated from the infected roots of watermelon seedlings through growing it into PDA culture. In this study, Tongue approach grafting method was applied. Plants have been divided into four groups. Only two groups have been injected with spores of Fusarium oxysporum 1x10 6 conidia / ml, then plants were planted in industrial sterile soil contains peatmoss and vermiculite (1:1 vol/vol) in pots at a completely randomized design. Finally, results of observations were collected after the disease symptoms appeared on the plants. The results and statistical analysis for data showed that grafting had a positive effect, where the grafted plants were better than non-grafted in all the measurements in terms of the weight of the shoot and root, the length of stems and roots, as well as the number and weight of fruits, the number of lateral stems (branches) and in the number of leaves per plants. These results indicated that grafting of watermelon onto specific rootstock (pumpkin) has left a positive impact on the rate of plant growth and production of the fruit as well as disease resistance. Moreover, the results of this study reported that the use of grafting could be an advantageous alternative method to the control of Fusarium wilt disease in watermelon. Key words: Grafting, Fusarium wilt disease, Watermelon, Pumpkin, Plant growth, Infection, Grafted plants. III

8 التطعيم طريقة بديلة للتحكم في مرض الذبول الفيو ازرمي في البطيخ الملخص: التحكم في هو البحث هذا من الهدف الذبول لمرض المسبب الفطر البطيخ األحمر)كرمسون في الفيو ازرمي سويت 0015( بتطعيمه على نبات القرع البري )تيتسكابوتا( فقد تم عزل الفطر المسبب لمرض الذبول الفيو ازرمي من جذور أشتال نبات البطيخ المصابة والتي ز ر ع ت على )أجار( مستخلص البطاطا الدكستروز. است خ د مت في هذه الد ارسة طريقة التطعيم اللساني ث م ق س م ت النباتات ألربع مجموعات وتم حقن مجموعتين فقط بج ارثيم فطر فيو ازريوم أوكسيسبو ارم 10 6 x1 بتركيز كونيديا/ مل ثم ز ر ع ت النباتات في إصيصات تحتوي على تربة معقمة من صناعية البيتموس والفيرموكاليت بمعدل 1:1 في نظام عشوائي متكامل أخي ار تم جمع المالحظات ظهور المرض بعد على النباتات. وقد أظهرت نتائج الد ارسة والتحليل اإلحصائي للبيانات أن التطعيم أثر إيجابيا حيث كانت النباتات المطعمة أفضل من مثيالتها غير المطعمة في كل القياسات من حيث: وزن المجموع الخضري والمجموع الجذري طول السيقان والجذور وكذلك من حيث عدد الثمار ووزنها وعدد السيقان الجانبية )التفرعات( وأخي ار في عدد األو ارق على النبات. أوضحت هذه النتائج أن تطعيم البطيخ على أصل القرع أث ر إيجابيا معدل نمو النبات على وانتاج الثمار عالوة على مقاومته لمرض الذبول الفيو ازرمي فقد تحقق ما هدفت إليه الد ارسة وتوصل الباحث إلى إمكانية استخدام التطعيم كطريقة بديلة للتحكم في مرض الذبول الفيو ازرمي في البطيخ. الكلمات الدالة: التطعيم مرض الذبول الفيو ازرمي البطيخ القرع نمو النبات العدوى نباتات مطعمة. IV

9 Table of Contents Content Page Dedication... I Acknowledgement... II English Abstract.. III Arabic Abstract... IV List of Tables VIII List of Figures IX Abbreviations XI 1. Chapter One: Introduction 1.1 Introduction Objectives General objective Specific objectives Significance Chapter Two: Literature Review 2.1 Fusarium Wilt on Watermelon Characteristics of the pathogen Symptoms on plants Disease cycle Disease control Control management of soil borne disease Cucurbit Grafting History Watermelon s rootstocks Current Grafting Methods in Watermelon Tongue Approach Graft One Cotyledon Graft Hole Insertion Graft Side Insertion Graft Watermelon Grafting Advantages and Disadvantages Advantages Disadvantages 17 V

10 [ Content Page 3. Chapter Three: Materials and Methods 3.1 Materials Methods Isolation and Identification of Fusarium Oxysporum Cultivation and Spore Production The Grafting Process Tongue Approach Graft (TAG) method Watermelon Infection and Transplanting Fusarium spore suspensions Plant infection Watermelon transplanting Cultural practices Data collection Statistical analysis Chapter Four: Results 4.1 Isolation of suspected pathogen from diseased plants roots Characteristics of F. oxysporum on PDA culture Microscopic identification of Fusarium oxysporum Effect of grafting on plant growth Effect of grafting on dry and fresh weight of shoot and root (growth) Effect of grafting on total dry and fresh weight of the plant (whole weight) Effect of grafting on length of stems, roots and on number and weight of watermelon fruit Effect of grafting on the number of branches and leaves Effect of grafting on infected plants as control of Fusarium wilt in case of GI and NGI Effect of grafting on non-infected plants in case of GNI and NGNI Effect of grafting on infected or non-infected plants in case of GI and GNI Effect of infection or non-infection on non-grafting plants in case of NGI and NGNI Observation on plants. 50 VI

11 Content Page 5. Chapter Five: Discussion 5.1 Characteristics of Fusarium oxysporum On PDA culture Identification of the Pathogen Effect of grafting on plant growth Effect of grafting on infected plants as control of Fusarium wilt in case of GI and NGI Effect of grafting on non-infected plants in case of GNI and NGNI Effect of grafting on infected or non-infected plants in case of GI and GNI Effect of infection or non-infection on non-grafting plants in case of NGI and NGNI Analysis of observations on plants Chapter Six: conclusions & Recommendation 6.1 conclusions Recommended 65 References Appendices.. 77 VII

12 List of Tables Content Page Table (3.1) A list of the main equipments used in this study Table (3.2) A list of chemicals and culture media used in this study.. 21 Table (3.3) A list of plants and pathogen used in this study. 21 Table (3.4) Cultural practices performed 27 Table (3.5) Pesticide and fungicide application 28 Table (3.6) List of collection data 28 Table (4.1) Effect of grafting on dry and fresh weight of shoot and root of plants 32 Table (4.2) Effect of grafting on dry and fresh weight of the whole plant Table (4.3) Effect of grafting on stem, root length and on the number and the weight of watermelon fruit Table (4.4) Effect of grafting on the number of branches, live leaves dead leaves and total leaves of watermelon Table (5.1) Types of comparisons between plant groups. 52 Table (I) Data Observation 95 Table (II) Production quantities of watermelon in Gaza Strip Table (III) Comparison between quantities of pesticides, fungicides, herbicides and fumigations that input during 2013 and last years 98 VIII

13 . List of Figures Content Page Figure (2.1) Tongue approach graft.. 13 Figure (2.2) One cotyledon graft Figure (2.3) Hole insertion graft Figure (2.4) Side insertion graft 16 Figure (3.1) Isolation of Fusarium Oxysporum from infected roots as pathogen Figure (3.2) Steps of tongue approach grafting of watermelon Figure (3.3) Time schedule grafting technology by tongue approach method for watermelon during the experiment.. 25 Figure (3.4) A completely randomized design of watermelon transplanting 27 Figure (4.1) Characteristics of Fusarium oxysporum as pathogen isolated from diseased plants roots grown on PDA culture Figure (4.2) Characteristics of Fusarium oxysporum spores: (a) microconidia, (b) macroconidia and (c) chlamydospores Figure (4.3) Comparison of the effect of grafting on the dry and fresh weight of shoot and root of infected plant. 34 Figure (4.4) Comparison of the effect of grafting on the total dry and fresh weight of the whole infected plant Figure (4.5) Comparison of the effect of grafting on the length of stem, root and on the number and the weight of infected watermelon fruit Figure (4.6) Comparison of the effect of grafting on the number of branches, live leaves, dead leaves and on total leaves of infected watermelon. 37 Figure (4.7) Comparison of the effect of grafting on dry and fresh weight of shoot and root of non infected plant 38 Figure (4.8) Comparison of the effect of grafting on total dry and fresh weight of the whole non infected plant.. 39 Figure (4.9) Comparison of effect of grafting on the length of stem, root and on the number and the weight of non infected watermelon fruit Figure (4.10) Comparison of the effect of grafting on the number of branches, live leaves, dead leaves and on total leaves of non infected plant Figure (4.11) Comparison of the effect of grafting on dry and fresh weight of shoot and root of infected or non infected plant IX

14 [ Content Page Figure (4.12) Comparison of the effect of grafting on the total dry and fresh weight of the whole infected or non infected plant.. 43 Figure (4.13) Comparison of the effect of grafting on the length of stem, root and on the number and the weight of infected or non infected watermelon fruit 44 Figure (4.14) Comparison of the effect of grafting on the number of branches, live leaves, dead leaves and on total leaves of the whole infected or non infected plant. 45 Figure (4.15) Comparison of the effect of infection or non infection on dry and fresh weight of shoot and root of non grafted plants Figure (4.16) Comparison of the effect of infection or non infection on total dry and fresh weight of non grafting plants.. 47 Figure (4.17) Comparison of the effect of infection or non infection on the length of stem, root and on the number and the weight of non grafting watermelon fruit. 48 Figure (4.18) Comparison of the effect of infection or non infection on the number of branches, live leaves, dead leaves and on total leaves of non grafting plants 49 Figure (I) Stages of experiment study 77 Figure (II) Design of a randomized replications of watermelon transplanting.. 77 Figure (III) Comparison of fruit production of the four different groups of watermelon. 78 Figure (IV) Comparison of plant biomass of the four different groups of watermelon. 78 Figure (V) Comparison of the stem diameter of the four different groups of watermelon 79 Figure (VI) Comparison of the root length of the four different groups of watermelon 79 Figure (VII) Stages of plants growths 80 X

15 Abbreviations: FON MOA JICA USDA PDA LCY RLNS TAG GI NGI GNI NGNI PNK LSD SPSS Fusarium oxysporum f. sp. niveum. Ministry of Agriculture. Japan International Cooperation Agency. Union Solidarity and Development Association. Potato Dextrose Agar. Lagenaria cylindrical. The Rootstock Leaf Number Stage. Tongue Approach Graft method. Grafted & Infected Watermelon. Non Grafted & Infected Watermelon. Grafted & Non Infected Watermelon. Non Grafted & Non Infected Watermelon. Phosphorus, Nitrogen and Potassium. Least Significant Difference. Statistical Package for Social Sciences. XI

16 CHAPTER ONE INTRODUCTION 1

17 1.1. Introduction: Watermelon, Citrullus lanatus, family Cucurbitaceae, is an important vegetable, it has been widely cultivated all over the world. Its fruit has been accepted as a delicious fruit. It is a source of liquid containing over 90% water and 10 to 12% total solids including vitamin A, C, B6 and potassium (USDA, 2001). Watermelon fruits have been basic food for people and animals in different countries. Recently, watermelon fruits have become recognized more for their nutritional qualities. It has been reported that the red pigment of watermelon, lycopene, has inhibited qualities of some forms of cancer (USDA, 2007) as well as cardiovascular disease (Sesso et al. 2003). Watermelon constituted 5.7 % of total cultivated area in the Gaza Strip in During the last 3 years, the total area cultivated with watermelon in the Gaza Strip had been reduced from more than 6000 to less than 4612 dunums. Among the main reasons for such reduction was the infection with soil borne diseases. These diseases cause a decrease in yield and quality (MOA, 2014). Watermelon and pumpkin, Cucurbita moschata are belong to the same family Cucurbits (Cucurbitaceae); they are deep-rooted plants and have similar soil, light and water requirements, where they need regular irrigation (Marr, 2004). Adequate soil moisture in the early growth stages will help ensure vigorous growth. Soil moisture also is critical during blossoming and fruit development (Directorate Plant Production 2011). The Cucurbitaceae plant family is affected by several vascular wilt diseases caused by different pathogenic stains of the fungus Fusarium oxysporum, which are morphologically similar, but generally it is host-specific. This infection of watermelon and other cucurbits can cause an economic loss for farmers. The cucurbit wilt fusaria are similar in terms of their biology, epidemiology and management. Consequently, Fusarium wilt of watermelon has been caused by Fusarium oxysporum f. sp. Niveum (FON) and became one of the oldest described Fusarium wilt diseases (Egel and Martyn, 2007). Fusarium fungi live in the soil and attack plants at all stages of growth. If growers continually plant watermelon in the same soil eventually, they will wind up with a disease problem called Fusarium wilt. Symptoms of Fusarium wilt are similar on all cucurbits and dependent on several interacting factors including the amount of inoculum in the soil, environmental conditions, nutrients particularly nitrogen, and susceptibility of the host. Fusarium wilt is characterized by wilting of the vines, which at first may recover during the evening, but eventually wilt permanently. Initial symptoms often include a dull, gray green appearance of leaves that precedes a loss of turgor pressure and wilting. Wilting is followed by a yellowing of the leaves and finally necrosis. The wilting generally starts with the older leaves and progresses to the younger foliage. Under high inoculum pressure, seedlings may damp off as they emerge from the soil. Affected plants that do not die are often stunted and have considerably reduced yields (Egel and Martyn, 2007). 2

18 The primary diagnostic symptom of Fusarium wilt is a discoloration of the vascular system (xylem), which can be observed readily in longitudinal or cross section of roots or stems. In watermelon and melon, a brown necrotic streak may be visible externally on the lower stem and extending along the length of the vine. Since the pathogen is soil borne, symptomatic plants often occur in clusters corresponding to the distribution of the inoculum in the soil. Fusarium wilts are generally most severe in light, sandy, slightly acidic soils when temperatures are between C. Higher temperatures appear to impede infection often resulting in plants that are yellowed and stunted but not wilted. However, plants infected earlier in the season may display more severe symptoms later in the season, as the temperature increases, and there is more transpiration demand on the plant (Zitter et al., 1996). Until now, there are three ways of avoiding the infection of watermelon by Fusarium oxysporum f. sp. niveum: rotate the fields, treat with methyl bromide to kill the fungus, or grow resistant cultivars. The first two solutions are becoming less workable; land is becoming less available for rotations, and methyl bromide is being discontinued due to environmental concerns, while watermelon cultivars are available with varying resistance to only two races of Fusarium. Although alternative crop rotation fields and other fumigants are being tested and developed, grafting with resistant rootstocks offers one of the best methods to avoid soil-borne diseases. Thus, using grafted rootstocks seems to be an effective solution (Egel and Martyn, 2007). Grafting is an alternative approach to reduce crop damage resulting from soil-borne pathogens and increases plant a biotic stress tolerance, which in turn increases crop production. Grafting is a method of asexual plant propagation that joins plant parts for them to live together. So they will grow as one plant (Lee, 2003). Grafting is the union of two or more plant tissues that subsequently grow as a single plant (Andrews and Marquez, 1993). Now the use of grafted seedlings has been increasingly popular in the production of many fruit-bearing vegetables such as watermelon, cucumber, oriental melon, muskmelon, tomato, eggplant, and red pepper. The main purpose of grafted seedlings is to increase the yield and quality of fruits by combining a disease resistant rootstock with a genetically superior scion (Lee, 2003). Rootstocks-scion interaction showed tolerance against soil borne pathogens (Cohen et al., 2007), maybe Fusarium wilt. Fusarium wilt caused by Fusarium oxysporum that seriously damage watermelon roots and may have little or no effect on the roots of gourds, squash, or pumpkins. This selectivity among species has enabled grafting to be used to limit soil-borne diseases among cucurbit crops and used as rootstocks for watermelon (Yetisir, 2001). 3

19 Mechanisms of disease tolerance in grafted plant of the rootstocks as it is accepted that the root system synthesizes substances resistant to pathogen attack and these are transported to the shoot through the xylem (Biles et al., 1989). Grafting of watermelon scions on squash, pumpkin, or bottle gourd (Lagernaria spp) rootstocks are practiced in many of the major watermelon production regions of the world (Choi et al. 2002; Lee 1994, 2003). There are several methods for watermelon grafting techniques. The tongue approach method: is the famous grafting technique, which is used for watermelon. The tongue approach method became widespread in Asia because of its simplicity, high success rate, and little care since it does not require healing chambers (Lee and Oda, 2003). So it was the best grafting method for watermelon as reported by Hussien (2011). In Palestine, watermelon grafting has started in 2007 in coordination between Ministry of Agriculture (MOA) and Japan International Cooperation Agency (JICA) (MOA, 2008). However, with the restriction of using methyl bromide for soil fumigation, growers became aware of using grafted transplants to manage soil borne diseases. There are only few reports on vegetable grafting under the Palestinian conditions due to the limited use of this technique in commercial production as a result of the high cost of rootstocks and facilities. Several studies on grafting effects on watermelon plant growth and fruiting characteristics under normal as well as stress conditions have been conducted (Mohammed et al., 2011). However, in Palestine, a limited research has been done on the effects of grafting on increasing resistance to soil borne disease as described by Sawalha (2012) Objectives: General objective: The main objective of our study is trying to prove whether the tongue approach grafting method in local conditions can be achieve great success in controlling of Fusarium oxysporum in watermelon Citrullus lanatus var lanatus cv. (Crimson Sweet) as an alternative method Specific objectives: Isolating, identifying, cultivation and spore production of Fusarium oxysporum. Use grafting process by the tongue approach as grafting method whereas, watermelon grafted on the roots of pumpkin as resistant rootstocks to the pathogen. Infecting watermelon by identify Fusarium spore and transplanting. Evaluating the effectiveness of grafting methods by measuring the growth parameters of the grafted and non grafted plants. 4

20 1.3. Significance: Our research has been conducted for the first time in the Gaza Strip. The Gaza Strip is a small area with intensive agriculture activities, high population and a shortage of water resources. So, farmers use excessive of chemical pesticide and fungicides to increase production, this leads to pollution of the ground water, if grafting method successful this will encourage farmers to using it widespread, reducing of chemical material percentage and water consumption in agriculture this lead to improving the environment, farmer health and healthy of consumer. Cucurbits is used widely in the food system for the residents of the Gaza Strip. So, finding a practical method to save those cucurbits is essential. If this method proves to be effective, this will offer substantial production and high quality of watermelon. Compensation the relative failure in the old traditional method use to eliminate Fusarium oxysporum, Thus if this grafting method is effective in the local conditions, it will stand out as a great substitution. 5

21 CHAPTER TWO LITERATURE REVIEW 6

22 2. Literature Review: 2.1. Fusarium Wilt on Watermelon: Fusarium wilt is one of the economically most important diseases on watermelon (Walker, 1952). It is caused by Fusarium oxysporum f. sp. niveum. This disease was first described by Erwin F. Smith in 1894 in the Southeastern region of the United States. Currently, it is severe in California, mid-western states, central Wisconsin, and lower Ontario. However, the disease is now widespread throughout most regions in the world whereas, watermelon is grown (Egel and Martyn, 2007) Characteristics of the pathogen: Fusarium wilt pathogen is very host specific: It only infects watermelon. There are three pathogenic races of Fusarium oxysporum f. sp. niveum. A race is a subgroup within a species that may be capable of attacking only particular cultivars of a host. The fungus can live for many years in the soil in the absence of a susceptible watermelon crop. It survives in crop debris and on old diseased vines. Morphologically, this species produces three types of spores: macroconidia, microconidia, and chlamydospores. Chlamydospores are the dormant structure that helps this species survive for a long time without a host or in a harsh environment. The spores in the soil begin to germinate when roots of susceptible host plants are available. The fungus enters through the root tips or any openings in the root tissue (Leslie and Summerell, 2006) Symptoms on plants: Fungal infection may occur at any age of the plant. For young seedlings, dampingoff may occur and rot in the soil, where the hypocotyls are surrounded by a watery and soft rot causing the plants to become stunted. Later, wilting occurs in more mature plants causing the plant to die. A one-sided wilt is a common symptom, with one or more shoots wilting and others remaining healthy. Flaccid, withered, and brown leaves, as well as vascular discoloration are common disease symptoms (Larkin et al., 1996). The roots of infected plants may be healthy, but the vascular tissue is brown and discolored. Thus, rotten roots are not necessarily associated with Fusarium wilt disease. For observation of the vascular discoloration, we cut the vine near the soil surface and look for yellow-brown colored tissue just beneath the outer layer. On dead stems, white and cottony mycelium may be visible. Symptoms and disease incidence are expected to be most severe at temperatures between (25 27 C) (Zitter et al., 1998). 7

23 Disease cycle: This disease is not spread above-ground from plant to plant but rather from fungal spores in the soil. The fungus can be introduced to the soil through contaminated seeds, in compost, from farming tools and vehicles, on the feet of humans and animals, and with drainage water. If the seeds are infected, the fungus can be transported directly into the xylem from infected inner tissues. If the fungus is in the soil, it germinates and penetrates the fibrous root system at the root tips or at any openings. From inside the root tissue, the fungus enters the xylem and produces conidia that block water flow in the host. Three to four weeks after planting, symptoms of a one-sided wilt will appear. In the field, this symptom often occurs in patches. When the plants are dead, the fungus remains on the dead vines or in the soil in the form of white mycelia, macroconidia, and chlamydospores until a new susceptible host is available. This disease can be quite severe in fields with light, sandy soils with ph , less than 25% soil moisture, and high nitrogen content (Zitter et al., 1998) Disease control: There are no fungicides available for this disease (Wu et al., 2009). Management can be achieved through crop rotation, use of resistant varieties, and other cultural practices: 1. Crop Rotation: Controlling Fusarium disease is very difficult once the soil is infested. Crop rotation might reduce for 4 10 years the density of the fungus in the soil and with it disease incidence. It should be done with non-cucurbit vegetation. Thus watermelon planting must be avoiding in the same field for a minimum of 5-7 years (Egel and Martyn, 2007). 2. Resistant varieties: Growers should use resistant varieties available in the market such as Afternoon Delight, Crimson Sweet, Indiana, etc. Fusarium wilt resistance ratings are available in The Midwest Vegetable Commercial Guide, which is produced yearly, the Southeastern US Vegetable Crop Handbook (2009). 3. Use disease-free transplants and seeds: When transplanting in the green house, the soil used must be pathogen-free, or disinfected by steam or soil fumigation. The seed also must be pathogen free so that the fungus is not introduced into the field. Seed from infested fields should not be saved (Larkin et al., 1996). 4. Biocontrol options: Biocontrol strategies for this disease are not commercially available at this time but may become a management option in the future. Greenhouse studies of organic fertilizers in combination with different antagonistic strains of nonpathogenic Fusarium oxysporum and other microorganisms have shown promise, but are insufficient for recommendations at this time (Wu et al., 2009). 8

24 2.2. Control management of soil borne disease: Soil borne pathogens considered as a serious problem in many cultivated area of watermelons in the world, many areas with intense watermelon production as Turkey, China, Korea and Japan have suffered from soil borne pathogens (Yetisir and Sari, 2003; Cohen et al., 2005). Continuous cropping is inevitable in vegetable production in indoor areas, and therefore, reduces the yield and quality of produce. Most of the damage from continuous cropping is caused by soil-borne diseases and nematodes. It was reported that soil-borne pathogens and pests such as Verticillium, Fusarium and Meloidogyne spp. may cause yield losses of up to 78% in production (Bletsos et al., 2003). Growers in many regions including Palestine have used fumigants such as methyl bromide, to overcome soil borne diseases. Since the ongoing limiting use of fumigants, other techniques were used, watermelons were produced on crop rotation fields once every 5-6 years (Bruton, 1998; Yetisir and Sari, 2003). However crop rotation is not feasible, in addition, inadequate rotation has perhaps contributed the greatest to increased incidence and severity of soil borne diseases. Moreover, soil sterilization can never be completed. Grafting has become an essential technique for the production of repeated crops of fruit-bearing vegetables grown in indoor areas. Grafting was also combined with other alternatives such as; chemicals, biofumigation, and integration of soil solarization (Morra et al., 2007). The damages brought by Fusarium can be reduced with the use of various techniques: grafting of watermelon onto rootstocks of the genera Cucurbita or Lagenaria (Mondal et al. 1994; Qian et al. 1995; D amorer et al. 1996), applying substrate with higher ph and Fe and Cu contents, mulching the surface of the ground with plastic foil (Sun and Huang, 1985) and by biological control with bacteria or Aspergillus niger (Lin et al. 1990; Mukherjee and Sen, 1998). This technique has been also applied in the production of many vegetables. An increase in the yield of tomato, pepper, eggplant, melon and cucumber protected production has been achieved when combining grafting with other protection measures over 30 years (Yilmaz et al., 2008). Recently, vegetable production by grafting on resistant rootstocks has become a common practice to control soil-borne pathogens, especially for the cultivation of cucumber, melon, watermelon, tomato, pepper and eggplant in greenhouses in many countries (Lee, 1994; King and Davis, 2006). Grafting vegetable crops have been used extensively in greenhouse and tunnel production as a way to decrease reliance on chemical fumigants (Oda, 1999). The primary reason for grafting of vine crops is to provide protection against soil-borne diseases (Edelstein et al. 1999; Paplomatas et al. 2002), but some rootstocks have the added advantage of being resistant to nematodes, especially root-knot nematode Meloidogyne spp. 9

25 Additional benefits of grafting include increased yield, increased fruit quality especially flesh firmness, and more vigorous plants that can be grown using lower plant populations (Core, 2005; Yetisir, 2003). A grafting of watermelon onto other Cucurbitaceae rootstocks to provide soilborne disease resistance has been highly successful. With this success and with more discriminating consumers of watermelon fruit comes a second challenge: to produce a high quality fruit from grafted plants that is equal to or better than that of the non-grafted plant (Lee et al., 1998 and Rivero et al., 2004). Grafted watermelon plants onto wild watermelon rootstocks C. lanatus var. citroides were resistant or moderately resistant to the southern root knot nematode, M. incognita. (Thies and Levi, 2007) Cucurbit Grafting History: The first vegetable crops to be grafted date back to the seventeenth century; however, it did not become popular until the late 1920 s. Farmers in Korea and Japan grafted watermelon plant onto a gourd rootstock Lagenaria siceraria to provide resistance to soil borne diseases caused by successive cropping (Ashita, 1927). Research on cucurbit grafting began in the 1920s with the use of Cucurbita moschata as a rootstock for watermelon. Initial reports by Tateishi (1927) and Sato and Takamatsu (1930) described several studies of grafting. Tateishi (1931) reported grafting of watermelon onto Cucurbita moschata rootstocks, a technique that was well known at the time. In France, research on cucurbit grafting started in the 1950s, with the grafting of cucumber and melon scion onto figleaf gourd to control Fusarium wilt. During the 1960s melon plants were grafted onto Benincasa spp (Alabouvette et al., 1974). Cucumber grafting in Spain began in the mid- 1990s, but is increasing in importance (Hoyos, 2001). In 1998, approximately 95% of watermelon and oriental melons were grafted onto squash or gourd rootstocks in Japan, Korea, and Taiwan (Lee and Oda, 2003). According to Oda (2004), inter-generic grafting is used in the production of many fruit-bearing vegetables; i.e. cucumber Cucumis sativus L. grafted on pumpkin Cucurbita spp., watermelon Citrullus lanatus Matsum et. Nakai, on bottle gourd Lagenaria siceraria Standl., melon Cucumis melo L. on white gourd Benincasa hispida Cogn., are popular rootstock for watermelon production. Pumpkins and squash Cucurbita spp. are important crops and are grown in almost all arable regions of the world. 10

26 2.4. Watermelon s rootstocks: There are three economically important Cucurbita species, namely: C. pepo, C. maxima and C. moschata, which have different climatic adaptations and are widely distributed in agricultural regions worldwide (Robinson and Decker-Walters 1997; Paris and Brown 2005; Wu et al., 2007). In watermelons, there are at least three species available as plausible rootstocks suited for grafting and disease resistance. Watermelon is currently grafted on Lagenaria siceraria (bottle gourd), Citrullus lanatus (wild watermelon), Cucurbita moschata x Cucurbita maxima (interspecific squash hybrid), squash hybrids, (Cucurbita moschata x Cucurbita maxima). Lagenaria siceraria can be used to control Fusarium wilt (Yetisir and Sari, 2003). Over 95% of the commercial watermelon seedlings are grafted in Japan and Korea where farming areas are small, very intensive and crop rotation is an uncommon practice to overcome soil borne pathogens (Kurata, 1994; Lee, 1994; Traka-Mavrona et al., 2000). In Japan, watermelon plants are often grafted to some Cucurbitaceae rootstocks which are resistant to soil-borne Fusarium disease and tolerant to low temperature. Bottle gourd Lagenaria siceraria Stand1. has been mainly used as a rootstock for watermelon; but, recently, bottle gourd rootstock has become susceptible to soil-borne Fusarium disease and physiological wilt in some production sites problems, squash or pumpkin (Cucurbita moschata Duch., C. maxima C. moschata, or C. pepo L.), respectively, are sometimes used as rootstocks for watermelon instead of bottle gourd. However, squash or pumpkin rootstocks often lower fruit quality. The deterioration of melon quality on watermelon Cucurbita rootstocks is attributed to their vigorous nutritional uptake, these findings suggest that the fruit quality is related to the functions of rootstocks (Masuda et al., 1986). Grafting of watermelon scions on squash, pumpkin, or bottle gourd (Lagernaria spp.) rootstocks is practiced in all the major watermelon production regions of the world (Choi et al., 2002; Lee 1994, 2003). In a another study, watermelons are grafted on Cucurbita maxima, C. moschata, and Lagenaria siceraria rootstocks. These rootstocks influenced resistance to soil borne diseases, plant growth, yield, and fruit quality. Graft incompatibility and decrease in the fruit quality appeared depending on the scion-rootstock combination (Lee and Oda, 2003). Thirteen commercial melon rootstock and various Cucurbitaceae spp. were evaluated for potential grafting for resistance to pathogen and determined productivity and fruit quality characteristics of grafting on resistant rootstocks. Following inoculation, P360 and PGM commercial rootstocks, as well as Benincasa hispida, Cucumis metuliferus, Cucumis ficifolius, Cucurbita maxima, Cucurbita moschata, and Lagenaria siceraria were resistant to the race 1, 2 of Fusarium. Yield and quality attributes of scion cultivars (Supermarket and Proteo) grafted on P360 and PGM rootstocks were not improved relative to ungrafted controls. Grafts onto B. hispida negatively influenced both 11

27 yield and fruit quality, while C. metuliferus, and C. zeyheri had negative impacts on productivity and fruit quality (Trionfetti Nisin et al., 2002). Recently, bottle gourds Lagenaria siceraria and interspecific squash hybrids (Cucurbita moschata Cucurbita maxima) were evaluated as rootstocks for commercial watermelon production in the southeastern United States (Hassell and Memmott, 2008). In North Florida field trials, a severe infestation of root-knot nematodes unexpectedly occurred on all bottle gourd and Cucurbita rootstocks (unpublished data). In China, where farm land is limited and farmers are being forced to grow the same crop in successive years, root-knot nematodes have become a serious limitation for grafting to bottle gourd and Cucurbita rootstocks (Yong Xu, National Engineering Research Center for Vegetables, Beijing, China). Identifying root-knot-resistant germplasm and developing resistant rootstocks would provide an economical and environmentally friendly method for managing root-knot nematodes in watermelon and melon. Although BH and LCY showed high compatibility with watermelon (cv. Crimson Tide), they were not suitable rootstocks for early watermelon production, but they can be considered for late watermelon production because they were resistant to 3 known races of Fusarium oxysporum f. sp. niveum (Yetisir et al., 2003) Current Grafting Methods in Watermelon: Many different watermelon grafting techniques are available today namely the tongue approach graft, one cotyledon graft, hole insertion graft, and the side insertion graft (Cushman, 2006; Hassell and Memmott, 2008; Lee, 1994; Lee and Oda, 2003; Oda, 1995). The approach grafting method is one of the original grafting methods performed (Lee and Oda, 2003); however, the one cotyledon and hole insertion grafts are most commonly used today in commercial production. Preferences to grafting techniques are a compromise among a number of influential factors to maximize the benefit to fit the individual s needs and available resources. These contributing factors include the ease and technicality of grafting, success rate, and overall cost (Davis et al., 2008; Hassell and Memmott, 2008; Lee, 1994) Tongue Approach Graft: The tongue approach graft, or simply known as the approach graft, is relatively simple to graft as in (Fig 2.1) (Hassell and Memmott, 2008). It is the oldest grafting technique, which became widely used in the 1920 s in Asia due to its higher success rate (Lee and Oda, 2003) and the growth uniformity (Hassell and Memmott, 2008). This method continues to be preferred by inexperienced growers because of its simplicity, high success rate, and little care since it does not require healing chambers (Lee and Oda, 2003). Referring to figure one at the first true RLNS and older RLNS a diagonal 12

28 slice is made below the cotyledons, in both hypocotyls of the scion and rootstock; slices should be opposite to one another, upward and downward, respectively (Cushman, 2006; Oda, 1995). Each cut should be comparable in length so they can match up together. Each slit acts like a tongue and both are fitted together and sealed with an aluminum wrap to allow healing to take place. The rootstock meristem and cotyledons are completely removed after three days of grafting and the scion rootstock is removed at seven days after grafting. The scion is now solely dependent on the new rootstock (Oda, 1995). The plants must be individually handled manually at the time of grafting, again at three days after grafting to remove the meristem from the rootstock, and once more at day seven to remove the root portion from the scion. This makes it a very labor intensive and time consuming grafting method. Both rootstocks are then replanted together during the grafting procedure to increase the proximity during the healing time. This is a significant drawback if it s being done in a greenhouse as it occupies twice the amount of space and is costly to maintain (Cushman, 2006). Because all meristematic tissue from the rootstock is removed during the grafting procedure, rootstock re-growth can no longer occur. Figure 2.1. Tongue approach graft 1) the rootstock; 2) scion being cut; 3) union of scion and rootstock; 4) complete removal of rootstock meristem; and 5) complete removal of scion root. Picture provided by (Hassell and Memmott, 2008) One Cotyledon Graft: The one cotyledon graft is also known as splice, slant or tube graft. This graft is moderately simple being less labor intensive than the approach graft (Fig. 2.2) (Hassell and Memmott, 2008). The one cotyledon graft can be completed at one time and minimizes greenhouse occupancy making this method the most popular grafts among experienced growers and commercial nurseries in Korea. It is performed by either by hand, semi-automatic, and with automatic robots (Kurata, 1994; Lee and Oda, 2003). Plants are ready for grafting when the first true leaf is present on the rootstock or as young as the scion cotyledon stage (Cushman, 2006; Oda, 1995). The procedure is as follows: The scion is cut at an opposing 45º to 65º angle to the rootstock, approximately one inch below the 13

29 cotyledons to facilitate clamping. The rootstock meristem and one of the cotyledons are cut simultaneously from the plant at a 45º to 65º angle to maximize the grafting surface area. The sliced portion of the scion and rootstock hypocotyl is then joined together to ensure the vascular tissues are contacting each other. The graft secured with a spring clamp that is placed around the outside region of the splice. Immediately following grafting, plants require special environmental conditions for healing. This includes: high levels of dark and humidity, and healed at approximately 23ºC in a healing chamber. The healing chamber minimizes environmental stresses to allow newly grafted plants to heal without undue environmental stress rather than continue with photosynthetic activity until healing is complete. Under low light conditions, the stomata on the leaf close forcing gas exchange and photosynthetic activity to cease which slow wilting to maintain the plant vascular system at optimal survivability. The high humidity prevents the plant from excessive wilting and assists in maintain high tugor pressure which aids in graft healing. Newly grafted seedlings should be kept in the healing chamber for the duration of the graft healing lasting approximately seven days. Three days into graft healing, light intensity is increased, and humidity is gradually decreased in the healing chamber to prepare the seedlings for ambient environmental conditions outside the chamber. Figure 2.2. One cotyledon graft 1) cut scion at an approximate 65 o angle; 2) remove apical meristem and one cotyledon; 3) cut off cotyledon at an approximate 65 o angle; 4) attach scion onto rootstock; and 5) secure the graft with a clip. Picture provided by (Hassell and Memmott, 2008) Hole Insertion Graft: The hole insertion graft, which is also called terminal, cut or top insertion graft (Fig. 2.3) (Hassell and Memmott, 2008), is favored by watermelon growers in Japan because of the shorter growing time required for scion material compared to the rootstock (Lee and Oda, 2003). Grafting can begin once the first leaf emerges from the rootstock. The scion is ready for grafting during the cotyledon stage and up to the first true leaf. Some experts report that it can be used even as soon as the shoot emerges from the soil (Lee and Oda, 2003). 14

30 The procedure for this method is outlined as follows: the scion hypocotyl is cut 2 cm below the cotyledons at a slant on opposing sides to expose the vascular tissue; During this step as much of the meristematic tissue should be removed as possible. A specialized tool, such as a bamboo stick or small drill bit, is used to make a hole that is slant to the longitudinal direction between the cotyledons and into the hypocotyls which should slightly pass through the hypocotyl on one side for the scion hypocotyl to be inserted allowing the vascular system of both hypocotyls to come into contact with each other. The pointed region on the scion is then snuggly inserted through the slanted hole in the hypocotyl to complete the graft. This method does not require the same scion/rootstock hypocotyls slant cut matchup, does not require clips, and the newly grafted plant is then placed inside a healing chamber for seven days as described previously. There is a high success rate on rootstocks that are compatible with Lagenaria; however, a great concern lies within the high rate of remaining meristematic tissue since which will necessitate future re-growth removal and increasing grafting cost. Rootstock plants that have a pronounced hollow stem, such as inter-specific squash hybrids, are less likely to work because of hollow stem creates a gap which prevents the scion from adhering to the rootstock and/or inserting the seedling into the pith cavity of the rootstock. By doing so allows adventitious roots from the scion to elongate downward through the pith center and into the soil which will void the resistance and lead to complete rootstock decline (Lee and Oda, 2003). This technique has not been successfully automated because of the technicalities of performing this graft. Figure 2.3. Hole insertion grafting method 1) the scion is cut at approximately 65º on two sides forming apoint; 2) meristematic tissue is removed; 3) a hole for the scion to be fitted in is drilled at a slant between the cotyledons and just through the hypocotyl of rootstock; 4) the scion is aligned to fit snugly in the rootstock; and 5) it is then securely inserted into the rootstock. Picture provided by (Hassell and Memmott, 2008). 15

31 Side Insertion Graft: The side insertion graft, also known as the cleft or splice graft (Hassell and Memmott, 2008), is a modified whole insertion graft (Fig. 2.4) (Lee and Oda, 2003). Seedlings are ready to be grafted at the first true RLNS. The graft is as follows: Using a sharp blade, the scion is cut at an angle on both sides of the hypocotyl below the cotyledons to form a vshape. Cut a small vertical slit through the middle of the rootstock stem instead of at the top of the meristem. The slit is propped open with a toothpick.the scion is then inserted into the slit at an approximate 30º to the rootstock tip and a clip is placed over the union to secure the graft during the healing process, but its removal will be required once healing is complete. Three days after grafting carefully cut off the rootstock vegetative tissue just below its cotyledons. This grafting technique seems very simple, but inserting the scion into the rootstock can be somewhat difficult. The involvment of toothpick, makes it more time consuming and cumbersome. Once grafting is complete, the seedlings must be placed inside a healing chamber for three days after grafting, but an intense amount of labor is required to remove the rootstock shoot above the graft once the embedded scion has healed. Because of this step, this procedure cannot be automated; however, meristematic re-growth is no longer a problem. A further reason why this grafting technique is unpopular is the failure of vascular bundles to align sufficiently for a strong healing to take place to secure the graft. Figure 2.4. Side graft 1) the scion is cut at approximately 65º on two sides forming a point; 2) a simple slice is made through the rootstock hypocotyl; 3) the splice is then prop open using a toothpick or stick; 4) the scion is inserted into the rootstock, and secured with a graft clip; and 5) the vegetative portion from the rootstock is cut just below the cotyledons. Picture provided by (Hassell and Memmott, 2008). 16

32 2.6. Watermelon Grafting Advantages and Disadvantages: Advantages: Valuable benefits can also be introduced from grafting watermelons on intra- and inter specific rootstocks (Cohen et al., 2007). Resistant rootstocks can be alternated to overcome disease to maintain high watermelon production yields (Edelstein, 2004). Fusarium oxysporum f. sp. melonis can be avoided by using interspecific rootstocks (Cohen et al., 2007). Some rootstocks from Lagenaria are able to confer resistance in Cucurbitaceae against carmine spider mite, Tetranychus cinnabarinus, (Edelstein et al., 2000). Other rootstocks display tolerance for other soil borne pathogens such as Monoaporascus and Macrophomia (Koren and Edelstein, 2004). Another highly positive benefit is that some rootstocks have been known to effect fruit quality (Core, 2005; Davis and Perkins- Veazie, ). By grafting watermelons on to different rootstocks, the quality of the fruit has been known to increase fruit firmness and thus increase shelf life. These results have added to the quality of the fruit, in other countries, when shipping to foreign lands. This is a valuable potential preservation characteristic for this country in the fact that this may extend fruit longevity for both a harvest window for growers and on the shelf storage for produce buyers. It could also open new markets for the fresh cut industry. One benefit is that some grafts increase nutrient and water uptake due to a higher capacity for nitrogen uptake and transport to the scion, which greatly increases its growth (Pulgar et al., 2000). This advantage allows the plants to better use fertilizers and other nutrients that would have been left in the soil. The absorption efficiency of water is increased by vigorous rootstocks (Lee, 1994). These benefits have the potential to lower nutrient costs and amount of required water per plant to harvest the same yield. Grafted plants show a greater cold tolerance which is a great benefit since nongrafted watermelon plants have such little tolerance for low temperatures (Oda, 1995; Venema et al., 2008). Water logging is another watermelon production problem which causes the root to suffocate and crop production to halt. Studies show an increase in water logging tolerance with grafted plants (Yetisir and Sari, 2003). In another study, grafted watermelons had a greater tolerance when watered with saline water than did the nongrafted plants (Cohen et al., 2007) which implied the increase in drought tolerance in grafted plants as well (Koren and Edelstein, 2004) Disadvantages: Although there are many impressive advantages to grafting, some disadvantages have discouraged this technology from use in the U.S. These disadvantages are distributed between incompatibility, fruit quality, and cost. Incompatibility is the failure of the scion to unite and adhere to the rootstock. Lesser but still problematic incompatibilities occur when the plant is unable to grow in a healthy manner, or exhibits premature death (Garner, 17

33 1979). Other incompatibilities can cause poor fruit quality, yield reduction, and possibly plant collapse. This may be due to the reduction in or blocking of photosynthate transport. Vascular bundles must come in contact with each other in order for grafting to be successful and to avoid incompatibility (Oda et al., 1993). In order for healing to take place, vascular bundles from the scion and rootstock, severed during grafting, must come into intimate contact with one another for correct healing to take place. Vascular tissue differentiation from the callusing cells occurs in compatible grafts only (Andrews and Marquez, 1993). Grafting success can be increased by increasing the surface area and contact region between the scion and rootstock by increasing the sliced region allowing the vascular bundle on the whole to increase contact. Different plant species have a varying number of vascular bundles. This may increase the difficulty to adequately align vascular bundles from the rootstock and scion if they are unequal to achieve a successful graft (Oda et al., 1993). Some studies also shown that rootstocks can adversely affect the taste and shape of watermelon fruits (Edelstein, 2004). Plant proteins, either structural or nonstructural that are synthesized in the root, are translocated to the scion can give the fruit an off flavor that has been reported. These discrepancies are not reported in all rootstocks and can be overcome through screening procedures to evaluate for rootstock performance. Overall cost versus benefit becomes the bottom line when growers think about production within the United States: A grafted seedling in the U.S. is estimated to cost more than $ 0.75, as suggested by Taylor et al. (2008) being far more than $ 0.28 for a non-grafted seedling. There is an additional cost for growing the rootstock seedlings in comparison with a non-graft seedling transplant. This cost can be broken down into twice the amount of growing material, space, and time. Additionally equipment is needed for grafting such as a sharp blade, clips and a healing chamber. Labor is necessary to carefully handle the seedlings while performing the grafting procedure and with removing rootstock re-growth and this removal can be very expensive and of major concern due to overall cost. Rootstock re-growth occurs at the base of the rootstock cotyledons where meristematic tissue is present. Current grafting techniques attempt to remove all meristematic tissue during the grafting procedure. When the meristematic tissue is not removed, re-growth occurs at high rates. Even when grafting experience is increased and rootstock re-growth minimized, the remaining re-growth is yet too costly to remove at a reasonable cost. Overall cost must be decreased in order for grafting technology to be considered for commercial practice. This problem can be reduced by completely removing the cotyledon during grafting which eliminates the meristematic region; however, some attempts to successfully graft by removing both rootstock cotyledons in a one step fashion has not been successful (Oda, 2002a). 18

34 The overhead cost of the humidity chamber increases the overall cost to produce a quality grafted transplant. The unique spring loaded clips which are used require labor costs for placement and removal. Finally, removal of meristematic re-growth which occurs using this graft method increases overall cost. Costs can be further increased using this method if grafting is performed on older plants. Rootstock re-growth occurs at even higher rates because it is more difficult to remove all meristematic tissue during grafting which adds to the cost of labor even once the seedlings are planted in the field (Oda, 2002b). 19

35 CHAPTER THREE MATERIALS AND METHODS 20

36 3.1. Materials: Table 3.1. A list of the main equipments used in this study. Instrument Manufactures / country Incubator Heraeus (Germany) Microscope LW. Scientific (USA) Refrigerator Selecta (Spain) Spectrophotometer Chromatic (India) Autoclave Boxun (China) Balance Napco (China) Oven N-Bioteck (Korea) Hotplate Biomega (USA) Thermometer Hauhai China Ph Meter Selecta (Spain) Safety Cabinet N-Bioteck (Korea) Table 3.2. A list of chemicals and culture media used in this study. chemicals Culture media Sterile distilled water Potato Dextrose Agar Medium Normal saline Peatmoss and vermiculite Sterile Tartaric acid Solution 10% Sodium hypochlorite 2 % - 4 % 70 % Ethyl alcohol (Ethanol) Streptomycin sulfate 0.3g Chemical fertilizer Table 3.3. A list of plants and pathogen used in this study. Scion Common name Scientific name Cultivar Watermelon Citrullus lanatus var. lanatus cv.(crimson Sweet 0015). Rootstock Pumpkin Cucurbita moschata cv. (Tetsukabuto) Fusarium oxysporum f. sp. Pathogen Fusarium niveum 21

37 3.2. Methods: This experimental research was conducted during the two successive springsummer season of 2013 from 17th April to 7th July. The experiment was applied on several stages that will be explained in this chapter Isolation and Identification of the Fusarium oxysporum: Fusarium oxysporum was isolated from infected roots of watermelon plants which were collected from watermelon plot in plastic greenhouse in Rafah and Beit Lahia in Gaza Strip. The plants had a typical symptom of Fusarium wilt, and the fungus was identified as Fusarium oxysporum f. sp. niveum based on The Genus Fusarium (Booth, 1971), The Genus Fusarium- A Pictorial Atlas (Gerald & Nirenberg, 1982) and The Fusarium Laboratory Manual (Leslie and Summerell, 2006), where the pure culture of the fungi grew into potato dextrose agar (PDA) medium. Isolation Procedures of Fusarium Oxysporum: The isolation of Fusarium Oxysporum from infected watermelon is done as follows according to Burgess et al., (1988): 1. Selecting a 4 cm piece of basal stems at 1 st least 20 cm above the soil surface. 2. Washing the stem in tap water for several times, then immersion them into a 70% ethyl alcohol for 1 min, then pass it in 2 % - 4 % sodium hypochlorite solution NaOCl for 1 min as a surface sterilized and rinse it thoroughly with sterile distilled water. 3. Dry it between two sterile tissues paper or flame dry if the stem is thick. 4. Remove outer tissues by a sterile scalpel. 5. Aseptically cut the stem piece into 1 2 mm thick sections. 6. Place it into Petri dishes (9 cm) containing (PDA) medium. 7. Incubate plates at room temperature (25-28 C) for (3-7) days until suitable fungal growth appeared till identification. 8. Examining the isolated fungal under the microscope then identified it according to the description of Booth (1971), Barnett and Hunter (1986), Drenth and Sendall(2001). Figure 3.1: Isolation of Fusarium Oxysporum from infected roots as pathogen. 22

38 Cultivation and Spore Production: The developing fungal colonies from the plated seedling segments were purified by hyphal tip technique. Purring cultures of all isolated fungi were cultivated into PDA medium. Fungal colonies were identified by microscopic observation again. After suitable growth and sporulation, the plates were maintained at 4-5 C in refrigerator of the Department of Botany (Farias, 1987) The Grafting Process: The seeds of the rootstocks of pumpkin Cucurbita moschata cv. (Tetsukabuto) were sown 20 days later than the seeds of the scions of watermelon Citrullus lanatus var lanatus cv. (Crimson Sweet 0015) to ensure similar stem diameters at the grafting time due to the differences in growth vigor. Seeds were sown in 150-cell styrophoam trays under greenhouse conditions. The trays were filled with soil mixture (peatmoss and vermiculite in 1:1v/v). The environmental conditions for germination were C and 85-90% relative humidity. Tongue approach grafting method was used in our study because of its higher success rate, the uniform growth of grafted seedlings and it does not need healing chambers (Lee and Oda, 2003) The procedures of Tongue Approach Grafting (TAG) method: 1. Use a sharp knife or razor blade, to cut an angled slit halfway through the stem of the rootstock and an oppositely angled slit halfway through the stem of the scion. 2. Match the slits so that they overlap and then, seal with aluminum foil or specialty materials available for this purpose (clips). 3. Place the grafted seedlings in a seedling tray with larger cell size than what they were grown in. 4. Place root balls of both rootstock and scion together in the same cell and add potting media if needed to fill the larger cell. Return to greenhouse or other growing area. 5. High humidity and low light is not necessary to ensure success with this type of graft. 6. After healing of graft union, requires removal of top portion of rootstock about nine days after making graft. Also, requires severing of scion roots after an additional two or three days. 7. Transplanted to the field after three to four leaves have developed in about three more days. The grafting process takes about days. The success rate is depending on the combination of scion, rootstocks being used and experience (Cushman, 2006). 23

39 Some points should be taken into consideration during grafting process: The growing point of the rootstocks should be carefully removed before grafting. One cotyledon may also be removed to ensure complete removal of the growing point and to avoid overcrowding in the healing chamber. The grafting cut for rootstock should be made in a downward direction and the scion cut in an upward direction at an angle, usually C to the perpendicular axis. Grafting clips are placed to fix the graft position at the graft union site. Grafted plants are then planted together in pots. After healing the rootstock top is removed and the scion roots are cut (Lee et al., 2010). rootstock scion a b c d Figure 3.2: Steps of tongue approach grafting of watermelon a) the rootstock and scion; b) rootstock and scion being cut; c) union of scion and rootstock; d) secure the graft with a clip. 24

40 Figure 3.3: Time schedule of grafting technology by tongue approach method for watermelon during the experiment Watermelon Infection and Transplanting: Fusarium spore suspensions: After the isolation of the fungus from infected watermelon and provided by the Laboratory of plant. F. oxysporum f. sp. niveum conidia were prepared by growing plate cultures into PDA at 28 C for 10 days in the dark to induce sporulation as previously described. Plates were drenched with sterile distilled water, and spores were carefully freed from the culture surface with a fine artist s brush. Afterward, the suspension was filtered through three layers of sterile cheesecloth to eliminate mycelial fragments (Ling et al., 2012).The density of the spore suspension of each isolate was determined and adjusted to 1x10 6 conidia/ml (Boughalleb et al., 2007). 25

41 Plant infection: F. oxysporum f. sp. niveum were injected in watermelon plant by using sterile glass beakers 250 ml contain spore suspensions were prepared from cultures grown on PDA at room temperature for 10 days at a concentration of 1x10 6 conidia/ml. Seedlings of watermelon were uprooted, soil particles were removed from the roots by washing with distilled water, then cut part of it. 128 seedlings were selected and separated to four groups of 32 seedlings in each group. Two seedling groups of each grafted and non grafted watermelon were inoculated with the fungus by dipping the roots into the inoculums for seconds swirled several times to enable conidia to adhere to the roots. While others two groups of grafted and non grafted (control) were prepared by dipping their roots into sterile distilled water (Boughalleb et al., 2007) Watermelon transplanting according to Boughalleb et al., (2007): 1. Pots were arranged into 8 rows, each row had 6 m in length and contained 16 Pots. 2. Pots were filled with 12 liters of sterile peatmoss and vermiculite (1:1 vol/vol). 3. After inoculation, the grafted and non-grafted seedlings were then hand planted (one seedling per pot) according to a completely randomized block design with 4 replications, each one is consisting of 32 plants. 4. Plants were irrigated with small amounts of water so that the fungus would not leave the plants. 5. Drip irrigations system was used so that the plants would be irrigated systematically with 1-4 L / day gradually according to the age and size of plant. 6. The planting was conducted in suitable climate for watermelon growth where average air temperature during the experiment was about 27 C. 7. The observations of fungi effect on the growth were collected after 30 days after inoculating the seedlings to estimate the success of the experiment. 8. The experiment was conducted from 17April to 7 July, Types of the plant groups as the following: 1. Grafted & Infected Watermelon (GI). 2. Grafted & Non Infected Watermelon(GNI). 3. Non Grafted & Infected Watermelon (NGI). 4. Non Grafted & Non Infected Watermelon (NGNI) (Control). 26

42 N Figure 3.4: A completely randomized design of watermelon transplanting Cultural Practices: Irrigation, fertilization and pesticide were applied as recommended by the Palestinian Ministry of Agriculture. Table 3.3. Cultural practices performed. Activity Date Details Fusarium spore production 7/4/2013 Preparation of soil 14/4/2013 Plant infection 17/4/2013 Fusarium oxysporum was isolated from infected roots of watermelon on PDA. soil composed of industrial peatmoss and vermiculite by (1:1v/v) which put in containers. Watermelon infected by Fusarium spore suspensions. Transplanting 17/4/2013 Grafted and non-grafted seedling. Drip irrigations 24/4/2013 Using emitter 1-4 L/day. Fertilization 30/4/2013 Observations 17/5/2013 Pesticide and fungicide application Chemical fertilizers including: PNK, Cekresteen and Sheavah. Collected data and measurements of grafted and non grafted watermelon. 18/5/2013 Using Marshal, Vertamic, Stroby and Escoor. Evaluation of production 30/6/2013 Count of the fruits for every plant groups. Fruit harvested 5/7/2013 Off-season crop 7/7/2013 Total number of fruits harvested per plant groups and weighted (g). plant length measured, branches, live and dead leaves number counted and shoot fresh weight recorded. Roots up taken then their lengths measured and weighted. Shoot and roots dried then their dry weight recorded. 27

43 Table 3.4. Pesticide and fungicide application. pesticide Date of spraying Target pest Marshal 18/5/2013 Aphids Escoor 18/5/2013 Powdery mildew Vertimk 20/5/2013 Fly tunnels Marshal 16/6/2013 Aphids Stroby 16/6/2013 Powdery mildew Vertimk 20/6/2013 Fly tunnels 3.5. Data collection: Table 3.5. list of measuring data. No. During the cultivation period The termination data 1- Plant length(cm). Fresh and dry weight of shoot (g). 2- Number of branch per plant. Fresh and dry weight of root (g). 3- Number of leaves per branch. Length of stem and root (cm). 4- Number of male and female flowers. Number of branch, live leaves and dead leaves. 5- Number of fruits appearance in all plant groups. Total number and weight of fruits Statistical analysis: Data were analyzed using IMB SPSS statistics Version 20. Analysis of variance (ANOVA) was performed. Significance of differences among groups was tested using the least significant difference (LSD) method. LSD values were calculated at P value =

44 CHAPTER FOUR RESULTS 29

45 4. RESULTS In this chapter we will elucidate the important results which are obtained in our study and the analysis of variance for the different collected data Isolation of Suspected Pathogen from Diseased Plants Roots: The diseased watermelon plant samples were externally examined for characteristic symptoms and subjected to pathogen isolation procedure. Diseased roots showed emergence of fungi which were isolated and maintained on PDA medium. Especially, the lesion areas of roots with characteristic brown rot showed emergence of fungi which were later identified to be Fusarium Characteristics of Fusarium oxysporum On PDA culture: Fusarium species grow rapidly on various media producing white, lavender, pink, salmon, or gray colonies on potato dextrose agar. Colonies have velvety or cottony surfaces and some species change color during incubation. Conidia formed on PDA are usually variable in shape and size and so are less reliable for use in identification. However colony morphology, pigmentation and growth rates of Fusarium species on PDA are reasonably consistent if the medium is prepared carefully and if the cultures are initiated from standard inocula and incubated under standard conditions. These colony characteristics are useful secondary criteria for identification. Figure 4.1: Characteristics of Fusarium oxysporum as pathogen isolated from diseased plants roots grown on PDA culture. 30

46 4.3. Microscopic identification of Fusarium oxysporum: Definitive diagnosis requires the isolation of Fusarium species from diseased watermelon. Microscopically, Fusarium species characteristically produce microconidia, macroconidia and Chlamydoconidia. Microconidia are unicellular and ovoid with zero to three septae forming false heads at the ends of conidiophores as shown in (Figure 4.2a). Macroconidia are multicellular sickle-shaped clusters with three to five septae and footshaped basal cells as shown in (Figure 4.2b). Chlamydoconidia are sometimes present, occurring singly or in groups, with rough or smooth walls as shown in (Figure 4.2c). (a) (b) (c) Figure 4.2. Characteristics of Fusarium oxysporum spores: (a) microconidia, (b) macroconidia and (c) chlamydospores 4.4.Effect of grafting on plant growth: After 11 weeks, the fruit was harvested, counted and weighted, the aerial plant part was cut at the basal stem then the plant length measured, branches number, live and dead leaves number were counted and the vegetative fresh weight was recorded as well. The roots were up taken then washed to remove soil; their lengths were measured and fresh weight were recorded. Thereafter aerial parts and roots were placed in oven with circulating air and dried at 95 0 c for 24 hours until the constant weight was achieved; dry weight was recorded and the material was finely ground and stored in plastic bags (Karagiannidis et al., 2002). In the current study, the analysis of data concentrated on four comparisons as the following: - The first comparison: is between GI and NGI in order to understand the effect of grafting as control of Fusarium wilt. - The second comparison: is between GNI and NGNI in order to measure the effect of grafting on growth of watermelon plants in the absence of infection by Fusarium. - The third comparison: is between GI and GNI. This comparison aims to measure the effect of the existence of Fusarium on the grafted watermelon compared with grafted watermelon on the absence of Fusarium. 31

47 - The fourth comparison: is between NGI and NGNI. We aim by this comparison to show how the growth of non grafted and infected plants is influenced. The four comparisons are the main interest of us as they will elucidate the effect of grafting on plant growth and controlling of Fusarium wilt disease Effect of grafting on dry and fresh weight of shoot and root (growth): Table 4.1 illustrates the significance in the mean values of dry and fresh weight for shoot and root; that makes a comparison between of GI, NGI, GNI groups compared to NGNI watermelon as control. Table 4.1: Effect of grafting on dry and fresh weight of shoot and root of plants. Group N Shoot DW Root DW Shoot FW Root FW (g/plant) (g/plant) (g/plant) (g/plant) GI (a) 2.93 (a) (a) (a) NGI (b) 1.80 (b) (b) (b) GNI (c) 1.95 (b) (a) (b) NGNI (b) 1.96 (b) (b) 8.25 (b) * Means of similar letters in each column are not significantly different at P = 0.05 according to LSD test Effect of grafting on total dry and fresh weight of the plant (whole plant): Table 4.2 illustrates the significance in the mean values of dry and fresh weight of the whole plant; that makes comparison between of GI, NGI, GNI groups compared with NGNI watermelon as control. Table 4.2: Effect of grafting on dry and fresh weight of the whole plant. Group N Total DW Total FW (g/plant) (g/plant) GI (a) (a) NGI (b) (b) GNI (c) (a) NGNI (b) (b) * Means of similar letters in each column are not significantly different at P = 0.05 according to LSD test. 32

48 Effect of grafting on length of stems, roots and on the number and the weight of watermelon fruit: Table 4. 3 illustrates the significance in the mean values of the effect of grafting on stem, root length and number and weight of fruit, between GI, NGI, GNI groups compared with NGNI watermelon as control. Table 4.3: Effect of grafting on stem and root length and number and weight of watermelon Fruit. Group N Stem length Root length Fruit number Fruit weight (cm) (cm) (fruits /plant) (g / fruit) GI (a) (a) 1.38 (a) (a) NGI (b) (b) 0.88 (b) (b) GNI (a) (c) 1.06 (b) (a) NGNI (b) (b) 1.00 (b) (b) * Means of similar letters in each column are not significantly different at P = 0.05 according to LSD test Effect of grafting on the number of branches and leaves: Table 4.4 illustrates the significance in the mean values of the effect of grafting on number of branches, live leaves, dead leaves and total leaves between GI, NGI, GNI groups compared with NGNI watermelon as control. Table 4.4: Effect of grafting on number of branches, live leaves, dead leaves and total leaves of watermelon. Group N No. of No. of No. of No. of branch live leaves dead leaves total leaves GI (a) (a) (a) (a) NGI (b) (b) (b) (b) GNI (a) (a) (a) (a) NGNI (b) (c) (b) (b) * Means of similar letters in each column are not significantly different at P = 0.05 according to LSD test. 33

49 Effect of grafting on infected plants as control of Fusarium wilt in case of GI and NGI: Growth of infected plants. a P = b P > c d P > P > Figure 4.3: Comparison of the effect of grafting on the dry and fresh weight of shoot and root of infected plants. The results of all measurements in Table (4.1) and Figure (4.3) illustrate that the mean of grafted and infected plants is higher than in non-grafted and infected plants, and there is statistically significant difference between them at (P value = 0.05); in case of dry and fresh weight of shoot and root. These results signify that the growth of GI is better than in NGI. This implies that grafting controlled Fusarium. 34

50 The whole weight of infected plant: a P = b P > Figure 4.4: Comparison of the effect of grafting on the total dry and fresh weight of the whole infected plant. The results in two cases as in Table (4.2) and Figures (4.4) show that the growth of grafted and infected plants is greater than in non- grafted and infected ones with statistical significant difference at (P value = 0.05). These results indicate the importance of grafting to overcome the infection and to give the best growth. 35

51 b P > Length of main stem, root and the number and the weight of infected watermelon fruit. a P > b P > c P > d P > Figure 4.5: Comparison of the effect of grafting on the length of stem, root and on the number and the weight of infected watermelon fruit. The results in all situations as in Table (4.3) and Figure (4.5) show that the growth of infected and grafted plants are statistically better than the growth of infected and non grafted plants that represented by length of stem and root and number and weight of watermelon fruit and have statistical significant difference at (P value = 0.05). 36

52 Number of branches, live leaves, dead leaves and total leaves of infected watermelon: a P > b P > c P > d P > Figure 4.6: Comparison of the effect of grafting on the number of branches, live leaves, dead leaves and on total leaves of infected watermelon. The results in all cases as in Table (4.4) and Figure (4.6) show that the growth of grafted and infected plants are statistically greater than in non- grafted and infected plants as shown in branches number, live leaves and total leaves of watermelon except the dead leaves and have statistical significant difference at (P value = 0.05). 37

53 Effect of grafting on non-infected plants in case of GNI and NGNI: Growth of non-infected plants: a P > b P = c P > d P = Figure 4.7: Comparison of the effect of grafting on dry and fresh weight of shoot and root of non infected plant. The results in Table (4.1) and Figure (4.7) show that the growth of grafted and non infected plants are greater than in non-grafted and non infected ones as control, except root dry weight. There is a statistical significant difference between GNI and NGNI in case of fresh and dry weight of shoot at (P value = 0.05); but have not a significant difference in case of fresh and dry weight of root. 38

54 The whole weight of non-infected plant: a P > b P > Figure 4.8: Comparison of the effect of grafting on total dry and fresh weight of the whole non infected plant. Table (4.2) and Figures (4.8) show the mean of dry and fresh weight of the whole plant: The results of the comparison between grafted and non infected to non grafted and non infected watermelon as control show that the vegetative growth of (GNI) is larger than the control (NGNI) and have a statistical significant difference at (P value = 0.05). 39

55 Length of main stem, root and the number and weight of non infected watermelon fruit: a P > b P > c P > d P > Figure 4.9: Comparison of effect of grafting on the length of stem, root and on the number and the weight of non infected watermelon fruit. The results in all the measurements as in Table (4.3) and Figure (4.9) confirm that the growth of grafted and non infected plants are better than the non-grafted and noninfected ones as the control and have statistical significant difference at (P value = 0.05) except the number of fruit, where GNI have a little greater than NGNI. 40

56 Number of branches, live leaves, dead leaves and total leaves of non infected watermelon: a P > b P > c P > d P > Figure 4.10: Comparison of the effect of grafting on the number of branches, live leaves, dead leaves and on total leaves of non infected plant. Table (4.4) and Figure (4.10) show the mean of the number of branches, live leaves, dead leaves and total leaves of watermelon. The results in all cases show that the growth of grafted and non infected plants are statistically greater than the non-grafted and non-infected ones as shown in branches number, live leaves and total leaves of watermelon except the dead leaves where (GNI) have less than (NGNI) control. 41

57 Effect of grafting on infected or non-infected plants in case of GI and GNI: Plant growth characteristics: a P > b P > c P=0.553 d P > Figure 4.11: Comparison of the effect of grafting on dry and fresh weight of shoot and root of infected or non infected plant. All the results in Table (4.1) and Figures (4.11) illustrate that grafting has a positive effect on the growth of plants and has resistance to infection in a clear statistically relationship in the case of making a comparison between grafted and non-infected plants with grafted and infected ones, in terms of the shoot, root dry and fresh weight of plants, it is noticeable that the GI plants have root dry and fresh weight greater than the GNI which we will explain later. 42

58 The whole weight of plant: a P > b P=0.697 Figure 4.12: Comparison of the effect of grafting on the total dry and fresh weight of the whole infected or non infected plant. The results in Table (4.2) and Figures (4.12) with making the comparison between grafted and infected plants to grafted and non-infected plants support the separated results which give priority to the vegetative more than the roots. Also the dry weight for GNI plants are statistically better than GI which means that the grafting has been a positive effect on whole plant growth if it was infected by fungus or not. 43

59 Length of main stem, root and the number and the weight of watermelon fruit: a P = b P = c P > d P = Figure 4.13: Comparison of the effect of grafting on the length of stem, root and on the number and the weight of infected or non infected watermelon fruit. The results in Table 4.3 and Figure (4.13) indicate that the mean of the length of stem and root and number and weight of fruit which statistically proved the role of grafting in overcoming the infection, where GI plants have given better growth than GNI plants except the length of stem. This once again confirms the positive impact of grafting on the growth of plants. 44

60 Number of branches, live leaves, dead leaves and total leaves of watermelon. a P = b P = c P = d P = Figure 4.14: Comparison of the effect of grafting on the number of branches, live leaves, dead leaves and on total leaves of the whole infected or non infected plant. All results in Table (4.4) and Figure (4.14) show that there are similarities in the number of branches, live leaves, dead leaves and total leaves of watermelon in the case of comparison between GI with GNI. This indicates that there is a positive impact of grafting on plant growth, especially if it is infected by the fungus where the previous results were similar. 45

61 Effect of infection or non-infection on non-grafting plants in case of NGI and NGNI: Plant growth characteristics: a P = b P = c P = d P = Figure 4.15: Comparison of the effect of infection or non infection on dry and fresh weight of shoot and root of non grafted plants. The results in Table (4.1) and Figure (4.15) show that the existence of infection by the fungus has a negative effect on the vegetative growth, where the growth of non-grafted and infected plants is less than non-grafted and non-infected ones, while the growth of root growth of NGI is better than in the non- NGNI as we will explain later. All the results do not have statistical significant differences. 46

62 The whole weight of non-grafted plant: a P=0.715 b P = Figure 4.16: Comparison of the effect of infection or non infection on total dry and fresh weight of whole non grafting plants. The results in Table (4.2) and Figures (4.16) show that the total dry and fresh weight of whole plant of non grafted and non-infected are better than non-grafted and infected ones that is reaffirm the negative impact of the fungus on the total weight of the plant. 47

63 Length of main stem, root and the number and the weight of watermelon fruit: a P = b P = c P = d P = Figure 4.17: Comparison of the effect of infection or non infection on the length of stem, root and on the number and the weight of non grafting watermelon fruit. The results in Table (4.3) and Figure (4.17) show the negative impact of the presence of infection on the length of the roots, numbers and weights of the fruit, where all results in case of non-grafted and infected plants are less than non-grafted and non-infected ones without statistical significant differences except the stem length which we will explain later. 48

64 Number of branches, live leaves, dead leaves and total leaves of watermelon: a P = b P = c P = d P = Figure 4.18: Comparison of the effect of infection or non infection on the number of branches, live leaves, dead leaves and on total leaves of non grafting plants. All the results in Table (4.4) and Figure (4.18) show again the negative impact of the fungus on the number of branches, live leaves, dead leaves and total leaves of watermelon, where NGI has less growth than in the case of NGNI without statistical significant differences except dead leaves. 49

65 4.5. Observations on plants: During the study in the growing season, The following characteristics of grafted and non-grafted plants were recorded: main stem length and number of lateral stems, leaves, male and female flowers and number of fruits are recorded after 30 days from cultivation in the field as found in the table in the appendix. After a deep study to these measurements and making the comparisons among the groups, the results showed that the greatest parameters growth was observed in grafted and non-infected watermelon (GNI) and the least was in non-grafted and infected watermelon(ngi). Thus these results represent a successful indication of our experiment and the plants which were grafted on pumpkin rootstock showed the ability to resist Fusarium disease at the end. 50

66 CHAPTER FIVE DISCUSSION 51

67 Discussion: In this chapter, we will highlight on the most important results and will indicate the compatibility of the results of our study in relation to the previous studies. 5.1.Characteristics of Fusarium oxysporum on PDA culture: Fusarium oxysporum usually produces a pale to dark violet or dark magenta pigment on the agar if incubated in dark place. PDA is used by some researchers for the isolation of Fusarium species. We do not recommend this medium for this purpose as many saprophytic fungi and bacteria also can grow on the medium and interfere with the recovery of the Fusarium present. If PDA is used for the recovery of fungi from plant material, then the concentration of potato and dextrose should be reduced by %, and broad spectrum antibiotics should be included in the medium to inhibit bacterial growth (Rothrock C. and Gottlies D., 1981) Identification of the Pathogen: Pathogen was identified by studying cultural characteristics and microscopic features (Leslie and Summerell., 2006; Watanabe, 2002; Summerell et al., 2003; Gagkaeva, 2008). The pathogen sample was also sent to Fungal Identification Service of Ministry of Agriculture, for confirmation of its identity and deposition Effect of grafting on plant growth: Grafting has been proposed as a major component of an integrated management strategy for protecting plants from soil borne diseases in areas where farm land is limited and farmers are forced to grow the same crop over successive years (Ling et al., 2012). Watermelon, Citrullus lanatus is one of the most important economic vegetable crops damaged by Fusarium wilt which is caused by Fusarium oxysporum f. sp. Niveum. This is economically important wilting pathogen of watermelon because it causes significant yield losses in watermelon (Egel and Martyn, 2007). In this study, our attention is to evaluate the effect of grafting as alternative method for getting rid of Fusarium and in consequence amelioration of watermelon plant growth. So we focus to analyze four comparisons as in a table (5.1). Table 5.1: Types of comparisons between plant groups. First comparison GI and NGI Second comparison GNI and NGNI Third comparison GI and GNI Fourth comparison NGI and NGNI 52

68 Effect of grafting on infected plants as control of Fusarium wilt in case of GI and NGI: Growth of infected plants. The results in all analysis showed that the vegetative growth of grafted and infected plants (GI) was better than non grafted and infected plants (NGI) and had a significant difference at P value = In addition, regarding the growth of roots, the results are similar to the result of vegetative growth and had a clear significant difference as shown in Table (4.1) and Figure (4.3). These results indicated that the growth of pumpkin roots as rootstocks were better than the self-rooted of watermelon in presence of Fusarium; this lead to more vegetative growth. Meanwhile, the results showed that pumpkin roots had higher resistance to Fusarium wilt compared to watermelon roots. These results are a satisfied answer for the main goal of our study. In accordance with these results, Lee (1994) and Ioannou et al., (2001) found that grafted plants were more vigorous than self-rooted ones and had a larger central stem diameter. In addition, the increase in growth of grafted plants relates to stronger root growth of the rootstock (Yetisir and Sari, 2004). The vigorous rootstocks were reported to exhibit excellent tolerance to the most of serious soil borne diseases (Liu et al., 2009) The whole weight of infected plant. In case of the comparison between GI and NGI regarding on total dry and fresh weight for root and shoot, the results supported GI rather than NGI and confirmed the previous findings. The variation between the dry and wet weight demonstrates that the presence of fungus prevents the flow of water in non-grafted plant stem, where making the wet weight between grafted and non-grafted plants is too big. This means that grafting has a positive effect on plant growth if it is infected by the fungus as shown in Table (4.2) and Figure (4.4). This result is supported by the finding of Rouphael et al., (2010) who reported that the grafting improves water use efficiency. Owing to their utilization of the vigorous root system of the rootstocks, grafted plants usually show increased uptake of water and minerals when compared with self-rooted plants (Lee, Oda 2003) Length of main stem, root and the number and weight of watermelon fruit. On comparing the results of the growth based on the length of stem and root and the number and weight of watermelon fruit, the results supported clearly the previous out comes, where grafting had a significant positive impact on growth in the case of grafted and infected plants as shown in Table (4.3) and Figure (4.5). This agrees with Mohamed et al., (2012) results which indicated that pumpkin rootstock increased the yield per plant. 53

69 Number of branches, live leaves, dead leaves and total leaves of watermelon. All the results about the lateral stems, fresh leaves and total leaves of watermelon of GI compared with NGI supported what have been mentioned above, where the greatest number of them and the lowest number of dead leaves were observed in grafted and infected watermelon with a clear significant difference as shown Table (4.3) and Figure (4.6). Based on the above results, grafting had a positive impact on the growth of the infected plant. In the current study, grafted plants had much greater foliage growth and fruit yield in comparison to non-grafted plants and this is in accordance with Lee's results (1994) Effect of grafting on non-infected plants in case of GNI and NGNI: Growth of non-infected plants. A comparison between grafted and non-grafted watermelon (control) in absence of infection, showed that grafting gave a clear preference for vegetative growth compared with the control and had a clear statistical significance difference. These results supported clearly the above results, whereas the growth of the pumpkin roots gave preference vegetative growth for watermelon scion even if compared with the self-rooted of watermelon. The comparison between GNI and NGNI based on the growth of the roots, the results showed that the growth of roots of GNI which was represented by dry weight was slightly smaller than NGNI control and had no significant difference as shown in Table (4.1) and Figure(4.7). This result illustrates the agreement between root and shoot in case of NGNI which had a little bit better effect on the root growth than the growth of the GNI, where the roots are not from the same origin. Wherever the shoot growth of GNI was better than the shoot growth of NGNI with a clear significant difference, while the root growth of NGNI was slightly better than the root growth of GNI without significant difference. The nutrition that resulted from the root in grafted plants caused vegetative growth without being affected by the root-shoot variation and cause normal growth of roots. So the strength of rootstocks supports the stability of the plant; this indicates that the roots here have a clear impact on supporting the plant growth. But in case of roots growth itself, the congruence between shoot and root had a stronger impact on supporting and increasing the growth of roots. These results indicated that the flow of nutrients (mineral and water) from roots to shoot was not affected regardless of rootstock-scion, but the flow of the carbohydrates from shoot to roots seem to be affected slightly because of variation of the scion-rootstock between the two groups. If that is proved true, this result can be regarded as an important 54

70 discovery of this important research. This effect is mainly attributed to a scion-rootstock interaction which influences various plant physiological processes such as nutrient and water uptake and translocation, hormone synthesis, photosynthesis and other metabolic processes (Rouphael et al., 2010). According to Rouphael et al., (2008), rootstock-scion combination may affect the mineral concentration of aerial plant parts due to differences of physical characteristics of the root system of grafted plants, which allow for better water and mineral nutrients uptake, compared to ungrafted plants. In case of the comparison between GNI and NGNI overall results were going in the same direction. That means grafting had a positive impact on the growth of the plant especially in both cases of infected or non infected plants with the fungus. The results are in accordance and support that the dry weight of roots is one of the evidences that measures root vigor (Alan et al., 2007). Plants with vigorous root systems release more cytokinins into the ascending xylem sap resulting in increased yield due to growth promotion (Aloni et al., 2010). Moreover, physiologically King et. al.(2010) explained that the use of pumpkin and bottle gourds as watermelon rootstock encourages all vital stress factors of the plant in a way that improves the health status of the plant by increasing absorption of nutrients and plant tolerance to low soil temperature, salinity and excess humidity. The results have shown that the roots of pumpkin have the ability to absorb more water compared with the roots of watermelon and this is confirmed by the dry and wet weight of roots as shown in figure (4.7). In the case of wet and dry weight of shoot, the results showed that the dry and wet weight in grafted plants was larger than the non-grafted plants and clearly supported by the absence of infection. This demonstrates that the absorption of water and minerals was better than in the roots of pumpkin The whole weight of non-infected plant. In state of the comparison between GNI and NGNI related to the total dry and fresh weight of root and shoot together, the results were going to the same previous direction, where the grafted plants(gni) had vegetative growth greater than the non-grafted ones (NGNI) and had a clear significant difference. That means grafting had a positive impact on the growth of the plants as in Table (4.2) and Figure(4.8). Concerning the fresh and dry weight of vegetative growth, the results showed that the grafted plants onto Tetsukabuto rootstock had significantly highest values comparing with non-grafted plants (Mohammed et al., 2011). 55

71 Length of stem, root and the number and weight of watermelon fruit. Regarding to the length of stem and root, the results confirmed the above analysis that the grafting had a significant positive effect on the growth. The results clarified that there is variation between the dry weight and length of roots between GNI and NGNI, where the grafted plants had the longest root with a clear significant difference as in Table (4.3) and Figure (4.9), but had a little lower dry weight than non-grafted plants without a significant difference as in Figure (4.7). This variation is a result of cutting in roots of the grafted plants or because pumpkin roots have node which filled with water so when it is dried the weight will decrease a lot. This result agree with Alan et al., (2007), where control plants had short main stem, less number of lateral vine and low root dry weight. Furthermore, The difference in root depth between pumpkin(180cm) compared with watermelon(45cm) is perhaps the main reason for protecting those crops from the soil pathogens. In this respect, Sawalha study showed that soil fungi are present mainly in the cm depth, while their count decreased progressively below 40 cm depth of the soil horizon (Sawalha, 2012). In addition, the number of fruit in grafted plants was a little better than in the non grafted plants and had no significant difference at 5% level. This results supported by Mohammed et al., (2011) results, which elucidated that the fruit yield and average fruit weight did not significantly differ when watermelon cv. was grafted onto (Tetsukabuto). This result is relatively different from Khankahdani's et al., (2012) and Alan et al., (2007) results, which explained that fruit yield was positively influenced by grafting when compared with the control. Our result can elucidated by the damage and death of some flower. Regarding to flowering characters, the grafted and non-grafted plants did not show any significant differences (Mohammed et al., 2011) Number of branches, live leaves, dead leaves and total leaves of watermelon. Also, the results showed that the grafted plants significantly gave the highest number of lateral stems, live leaves and total leaves of watermelon except the number of dead leaves showed a positive result in case of grafting, where the low number of dead leaves in GI compared with NGNI and have a clear significant difference at 5% level as in Table (4.4) and Figure (4.10). Contrary to obtained results in the present study, Khankahdani et al. (2012) observed seedy watermelons had greater lateral stem number than in grafted watermelons but had no significant difference. Our results may be because using chemical fertilizers which causing high growth or cutting in the main stem of plants. 56

72 Effect of grafting on infected or non-infected plants in case of GI and GNI: Plant growth characteristics. The effect of grafting on watermelon growth indicated that grafting affected positively on vegetative and root development of watermelon plants. These results illustrate that plant with vigorous root systems enhanced uptake of water and minerals and increased photosynthesis which lead to increase of vegetative growth then yield as shown in Table (4.1) and Figure (4.11). Although the plants were grafted and infected, the growth of their roots had better growth than the grafted and non-infected plants with a clear statistical relationship. Root dry weight is one of the indexes that measures root vigor. This shows the importance of grafting for plants especially in the case of infection with the fungus. So there is a positive correlation between the vigorous of roots and the growth or resistance of F. oxysporum. This result can be explained as the irrigation input is removed or reduced for specific periods during the growth cycle of crops as described in eggplant (Solanum melongena L.; Kirnak et al., 2002) or watermelon (Simsek et al., 2004) or it perhaps due to a defect in weights because the process of irrigation and different date of measurement. According to Alan et al. (2007), the effect of the grafting on watermelon growth characteristics indicated that grafting affected root and vegetative development of watermelon plants but there is difference between rootstocks in root dry weight The whole weight of grafted plant. These results are identical with the explanations of the previous cases. So, the growth of roots in the infected cases supported the vegetative growth. This means plant vigour was closely related to root systems, which supply enough water and minerals nutrients to the scion which the plant needs in photosynthesis, which leads to increase in the plant growth as shown in Table (4.2) and Figure (4.12). This result was confirmed by a report of Wu et al. (2010), where reported that root exudates from resistant watermelon rootstocks suppressed Fusarium oxysporum growth while exudates from susceptible rootstocks favored the growth. According to our results, there is a positive effect of grafting on plant growth, clearly appeared only in case of infection and this is what does the study aims to Length of stems, root and the number and weight of watermelon fruit. The results showed that the root length and number and weight of fruit were significantly influenced by grafting in case of infection as compared with non-infected plants except main stem length. These results were in the same line with previous states, 57

73 confirmed the positive effect of grafting on the growth of plants and their overcoming to infection. This result is shown in Table (4.3) and Figure (4.13). In accordance with our results Alan et al. (2007) study, whereas the root length, main stem length, number of lateral vine and fruit yield were significantly influenced by grafting. It is clear that grafting increases yield since plants are resistant to soil borne disease, have strong root system and increase photosynthesis and this agrees with Mohamed et al., findings (2012) Number of branches, live leaves, dead leaves and total leaves of watermelon. The results were close together; this indicates that roots of pumpkin have the ability to maintain the growth of the plant as if it was not infected. In watermelons plants, growth is positively affected by grafting due to the increase in lateral stem and leaves number. The previous results mean that the positive impact of grafting in the case of infection without significant difference at 5% level. as in Table (4.4) and Figure (4.14). Our results agreed with the results of other researchers stated that grafting affected growth stem length, root length, number of lateral branches, number of leaves and fruit yield (Chouka and Jebari,1999; Salam et al., 2002; Yetisir et al., 2003; Yetisir and Sar,2004; Miguel, et al, 2004) Effect of infection or non-infection on non-grafting plants in case of NGI and NGNI: Effect of infection on growth of non-grafting plants. The results have shown that presence of fungus has a negative effect on the plant growth. This is the problem that our experimental study has solved by grafting which is considered as the main purpose of our study. In the same direction, the growth of non-grafted and infected plants was the least compared with the control. This result indicated that there was a negative effect on root growth followed by the same effect on the vegetative growth. This result illustrated the importance of the grafting process as Mohammed et al., (2011) and Alan et al., (2007) reported that grafting significantly affected plant growth. Despite of the plant is non-grafted and infected with the fungus, they gave the best growth of root compared with the control group. That maybe because of the physiological reaction to infection by the fungus, which stimulates the plant to produce the largest total root to give greater absorption space to increase water pressure to pump it for all vegetative plants through vascular wood. This result is supported by the results of Atkinson et al., (1999); Chaves et al., (2003) where they indicated that when water supply is limited, plant structure is modified by increasing the root: shoot ratio. 58

74 The whole weight of non-grafted plant. The results showed that there was a negative effect of the infection on the plant growth, where the growth of the NGI plants was less than the whose of NGNI. So the total dry and fresh weight of the control was better than NGI. This result indicated the importance of the grafting process as in Table (4.2) and Figure(4.16). Non grafted plants showed that severe infection with Fusarium wilt (Liu et al. 2009). One solution is the use of resistant species, such as the pumpkin, as rootstock for other curcubitaceae species (Ruiz et al. 1997) Length of stem, root and the number and the weight of non grafted watermelon fruit. All results in case of non-grafted and infected plants were less than in the controlled ones. These results indicated clearly the negative impact of infection on the growth which represented by the length of the roots, numbers and weights of the fruit, except stem length. On the other hand, infected plants had a positive effect on the length of the stem in the sense stimulate the growth of the stem more than non-infected plants. That possibly because of the watermelon is a creeping plant in research for water. This result confirms the previous findings that are related to wet weight of the roots. Hence this thesis gives attention to the importance of grafting and its impact on the growth of plants and overcomes the fungal diseases as in Table (4.3) and Figure (4.17). According to Lee (2003) grafting is an important technique for vegetable production and has become a common practice in many parts of the world where sustainable production is performed Number of branches, live leaves, dead leaves and total leaves of non grafted watermelon. All the results confirmed the above analysis; the growth of the infected plants regarding the number of branches, live leaves, dead leaves and total leaves was low. As a result towards the negative effect of infection on watermelon plants. This result again emphasizes the importance of the grafting process on preventing wilt particularly on the leaves of the plant to increase the vegetative growth that is followed by increasing of the number and weight of the fruit (Cohen et al., 2007)as in Table (4.4) and Figure (4.18). Hence, the idea of grafting in watermelon has come under the spot light as a possible alternative for controlling Fusarium wilt. In additional, grafting has great potential to have a positive effect for commercial production by overcoming soil-borne pathogen impediments (Cohen et al., 2007; Kurata, 1994; Lee, 1994; Lee and Oda, 2003; Oda, 1995; Yetisir et al., 2003), increasing fruit quality (Cohen et al., 2007; Core, 2005; Davis and Perkins- Veazie, ), and improving the plants overall environmental 59

75 efficiency (Cohen et al., 2007; Koren and Edelstein, 2004; Lee, 1994; Oda, 1995; Pulgar et al., 2000; Venema et al., 2008; Yetisir and Sari, 2003). Through-out our study, it was observed that the increased production of leaves in grafted plants was as a result of an increased uptake of water and nutrients (Pulgar et al. 2000). It is well known that the root system of the plants affects the vegetative growth and yield. So, the effects of grafting were recorded clearly in most research as a result of the differences in the root system between grafted and non-grafted plants, and the efficiency of water and nutrient uptake by the roots, or even to the distribution of growth regulators (Oda, 1995; Bletsos et al., 2003; Yetisir et al., 2003). All the measured variables supported the result that the grafted plants have higher root and shoot dry weight, longer main stem and more lateral vine than the non grafted control plants, and fruit yield of grafted plants is superior to the control. These results are supported by Lee (1994), Yetisir & Sari (2003), Yetisir et al. (2007), Han et al. (2009) whose revealed that grafted watermelon, using specific rootstocks, increased plant growth, fruit yield, enhanced water transport and plant nutrition. The results of our research were in a symmetry with the results of research conducted in the West Bank, Palestine. The results showed that grafting on bottle gourd and pumpkin rootstocks were effective in reducing the number of soil fungi and the success of the growth and production of watermelon in the soil of Sanoor in Palestine. This is due to the ability of these crops to avoid pathogens living in the soil, as well as the bottle gourd and pumpkin rootstocks are not a sensitive host to soil fungi spread in Sanoor plain, compared with watermelon, which is very sensitive to these pathogens (Sawalha, 2012). Also these results coincided with the results of other research conducted in the nearby Arab countries. The results of grafting of watermelon on bottle gourd and pumpkin rootstocks in the Lebanese, western Beqaa, showed increase in the resistance of diseases that affect non grafted watermelon seedlings and increase in production and profits significantly. The grafting process has allowed to harvest the crop three times in the season while, land cultivated with a regular seed watermelon has harvest once in a season. The roots of bottle gourd and pumpkin plants give the grafted plant large resistance in tolerance of diseases and it also gives more economical production through the number and size of a big fruits (Core 2005, Yetisir 2003). Mechanisms of diseases tolerance in grafted plants may be due to the resistance of the rootstocks as it is accepted that the root system synthesizes substances resistant to pathogen attack and these are transported to the shoot through the xylem (Biles et al., 1989). The activity of these substances, related to disease resistance can vary during the development stages of grafted plants (Heo, 1991). So watermelon grafting onto cucurbit rootstocks is one of the best alternatives to control soil-borne diseases and an agronomic interest for plant vigour and production (Blancard et al., 1991; Messiaen et al., 1991; 60

76 Gignoux, 1993; Jebari, 1994; Aounallah-Chouka and Jebari, 1999; Tarchoun et al., 2005). Therefore, our study suggests that grafting can be used for control of Fusarium wilt disease in agriculture, in particular watermelon Analysis of observations on the plants: The plants pass through significant stages during their life cycle. These stages of plant growth are (vegetative growth, flowering and fruiting). During this experiment, we recorded stem length and number of lateral stems, leaves, male and female flowers and number of fruits. The results showed that all measured variables were consistently affected by grafting watermelon onto Pumpkin and by infection with the fungus, where cultivars were ranked for Fusarium wilt disease resistance based on the amount of wilt expressed after 7 weeks. These results confirmed with the final results at the end of the study after 11 weeks. This results supported by Zitter et al., (1996), where symptoms of Fusarium wilt of watermelon vary, depending on environmental factors, the age of the infected plant, the aggressiveness and density of the pathogen population. When the pathogen population is very high, damping-off of young seedlings may occur. 61

77 CHAPTER SIX CONCLUSIONS AND RECOMMENDATION 62

78 6.1. Conclusion: Fusarium wilt diseases remains a problem in modern agriculture. Excessive use of chemical fungicides may apparently lower the density of pathogens in soil in short time duration but might give rise to mutant strains of pathogens with altered pathogenicity making the previously resistant varieties of crops susceptible. Use of biocontrol methods, crop rotations and genetically modified crops are some of the methods of disease management to curb down extreme use of chemicals on field to control pathogens and this agrees with the claims of several scientists. The objective of our study was to determine whether grafting could improve plant growth and control Fusarium wilt disease of watermelon or not. Plant grafting is a widely used means of plant propagation and growth control that is of considerable importance in the adaptation of interesting cultivars in the appropriate areas. It is also evident from our study that wilt resistance rankings are significantly affected by grafting. According to the obtained results in the present study and the groups comparison results of all variables of plants growth which were analyzed with LSD test, the concluded results from our study are: 1. The highest vegetative growth was significantly obtained by grafted and non infected plants when compared to the control plants, while the lowest was observed in nongrafted and infected plants. 2. The largest shoot dry weight was observed in grafted and non infected plants and the smallest shoot dry weight in non grafted and infected plants with a significant difference at 5% level. 3. The root growth of grafted and infected plants was significantly higher compared with the control plants, while the root growth of the control was slightly higher than NGI and had no significant difference. 4. The highest mean of root dry weight was significantly observed in grafted and infected plants, while the lowest root dry weight was in non grafted and infected plants. 5. The greatest total fresh weight was noted in grafted and non infected plants and the least was in non grafted and infected plants with significant difference. 6. The greatest total dry weight was noted in grafted and non infected plants, while the least was in non grafted and infected plants and had significant difference. 7. Grafting increased significantly the length of main stem where the longest stem was in grafted and non infected plants compared with the control plants. 8. Main root length was more in the grafted and infected plants than in non grafted and infected plants and had significant difference. 9. Fruit yield was significantly affected by grafting and infection compared to fruit number in the non-grafted and infected plants. 63

79 10. The mean of fruit weight was significantly affected by grafting, where fruit weight in grafted and infected plants was more than other groups and had significant difference together. 11. The greatest lateral stem number was significantly counted in grafted and non infected plants and the least in non grafted and infected plants but (GNI) had no significant difference to (GI). 12. Increased production of leaves was recorded in grafted plants, where the greatest number of live leaves were observed in grafted and non infected watermelon (GNI) and the least in non grafted and infected plants (NGI). 13. Grafting reduced significantly the dead leaves in grafted plants, where the least of number of dead leaves were counted in grafted and infected plants but the greatest in non grafted and infected ones. 14. The leaf number significantly differed between grafted and control plants, where the grafted plants produced the highest number of leaves per plant, while the lowest number was in non grafted ones. Our study demonstrates that all measured variables were consistently affected by grafting watermelon scion onto Pumpkin rootstock. It can be concluded that in watermelon plants, growth is positively affected by grafting due to the increase in root length, main stem length, number of lateral branches, number of leaves, number of fruit, fruit weights and vegetative fresh weights and dry weights of shoot and root were significantly affected by grafting. These results indicate that grafting watermelon onto specific rootstock influences growth, as well as disease resistance. Finally, the results of our study suggest that grafting in local conditions can be an alternative method to control Fusarium wilt disease in watermelon production. 64

80 6.2. Recommendations: Our recommends to Ministry of Agriculture: 1. We advise to reduce the use of chemical soil disinfectants and fungicides because of their negative impact on the environment, underground water and human health. 2. Train some farmers on the grafting process and give licenses to the owners of agricultural farms to apply grafting. 3. Provide the materials and tools needed for the process of grafting with the lowest prices, this will leads to reduce the cost of grafted seedlings. 4. Implement projects of grafting in cooperation with local and International Cooperation Agencies. 5. Encouraging the farmers to use grafted plants in agriculture through raising awareness programs to explain the importance of usage grafted seedlings. We advise researchers to: 1. Repeat the same method with other cucurbits to get more reliable results. 2. Use this method with other types of vegetables. 3. Perform this method in the field rather than in pots to provide an appropriate environment to the fungus. 4. Prove that the flow mineral and water from roots to shoot was not affected depending on their rootstock, but the flow of the carbohydrates from shoot to roots is affected by the variation of the rootstock which our study reached to. 65

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92 Appendices: Figure I: Stages of experimental study Figure II: Design of a randomized replications of Watermelon transplanting. 77

93 GI NGI GNI NGNI Figure III: Comparison of fruit production of the four different groups: a) grafted and infected, b) non grafted and infected, c) grafted and non infected, d) non grafted and non infected (control) of watermelon. GI NGI GNI NGNI Figure IV: Comparison of plant biomass of the four different groups: a) grafted and infected, b) non grafted and infected, c) grafted and non infected, d) non grafted and non infected (control) of watermelon. 78

94 GI NGI GNI NGNI Figure V: Comparison of the stem diameter of the four different groups: a) grafted and infected, b) non grafted and infected, c) grafted and non infected, d) non grafted and non infected (control) of watermelon. GI NGI GNI NGNI Figure VI: Comparison of the root length of the four different groups: a) grafted and infected, b) non grafted and infected, c) grafted and non infected, d) non grafted and non infected (control) of watermelon. 79

95 Figure VII: Stages of plants growth. 80