SEROLOGICAL DIAGNOSTIC SURVEY AND FARMER PERCEPTION OF CUCUMBER MOSAIC VIRUS DISEASE IN THE GREATER ACCRA REGION OF GHANA This thesis is submitted to the: UNIVERSITY OF GHANA, LEGON GRADUATE SCHOOL OF NUCLEAR AND ALLIED SCIENCES (COLLEGE OF BASIC AND APPLIED SCIENCES) DEPARTMENT OF NUCLEAR AGRICULTURE AND RADIATION PROCESSING BY ASEM WISDOM (ID: 10806431) B.Sc Agriculture Technology (2012) (University for Development Studies) IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF MASTER OF PHILOSOPHY IN NUCLEAR AGRICULTURE DEGREE (MUTATION BREEDING AND PLANT BIOTECHNOLOGY OPTION) SEPTEMBER, 2023 University of Ghana http://ugspace.ug.edu.gh i DECLARATION This thesis is the results of research work undertaken by Asem Wisdom, at the school of Nuclear and Allied Sciences (Department of Nuclear Agriculture and Radiation Processing), University of Ghana under the supervision of DR. ANDREW SARKODIE APPIAH and DR. SAMUEL AMITEYE Signature Date 25- 01- 2023 ASEM WISDOM (Candidate) Signature Date: 25 – 01 - 2023 Dr. Andrew Sarkodie Appiah (PRINCIPAL SUPERVISOR) Signature Date 25-01-2023 Dr. Samuel Amiteye (Co-SUPERVISOR) University of Ghana http://ugspace.ug.edu.gh i ACKNOWLEDGEMENTS This thesis could not have been produced without the help of the God Almighty, many individuals, groups and friends who supported me in diverse ways especially in prayers. I wish to express my sincere thanks to my supervisors, Dr. Andrew Sarkodie-Appiah and Dr. Samuel Amiteye for their critical suggestions, immense support and analysis at the various stages of the work. I wish to thank all the staff of the Molecular Biology Laboratory (Ghana Atomic Energy Commission / Biotechnology and Nuclear Agriculture Research Institute (BNARI) especially Mr. Robert Appiah for their immense help all throughout this study, Doris Mensah-Wonkyi and Rosemary Kusi-Adjei thank you, for you were very instrumental during the mechanical sap inoculation stage of this work. And to all the farm hands at the Institute for their immense assistance. I wish to thank BNARI for granting me access to their laboratory and facilities. I am also grateful to all the lecturers of the Department of Nuclear Agriculture and Radiation Processing for the training and knowledge they imparted onto me throughout the duration of the course. My special thanks go to Rev. Fr. Moses Huadji, the Parish Priest of Holy Trinity Catholic Church, Agomenya and Mr. Reuben Boafo for your spiritual, moral and physical support. Lastly to my uncles and aunties for their support both in cash and in kind. Thumps up for my cousins for their words encouragement, especially Irene Agbo, Benjamin Oduro-Kissi, Grace Akorfa, University of Ghana http://ugspace.ug.edu.gh ii Alhassan Mohammed, Ebenezer Akomea, Kinsley Obeng, Baffour-Awuah Dennis and Obed Koduah. University of Ghana http://ugspace.ug.edu.gh iii DEDICATION I dedicate this work to Almighty God and to my late parents. This would not have been achieved without you. May God gives you eternal rest forever. University of Ghana http://ugspace.ug.edu.gh iv TABLE OF CONTENTS DECLARATION…………………………………………………………………………………………..i ACKNOWLEDGEMENTS ........................................................................................................................ ii DEDICATION .......................................................................................................................................... iiii LIST OF TABLES ..................................................................................................................................... xi LIST OF FIGURES .................................................................................................................................. xii LIST OF ABBREVIATIONS .................................................................................................................. xiv ABSTRACT ........................................................................................................................................... xviii CHAPTER ONE ..........................................................................................................................................1 1.0 INTRODUCTION .................................................................................................................................1 1.1 Background of study ..............................................................................................................................1 1.2 Statement of problem and justification of study ....................................................................................3 1.3 Objectives ..............................................................................................................................................4 CHAPTER TWO .........................................................................................................................................5 2.0 LITERATURE REVIEW ......................................................................................................................5 2.1 Types of vegetables and importance in human and animal health ........................................................5 University of Ghana http://ugspace.ug.edu.gh v 2.2 Production levels and economic value of vegetables ............................................................................7 2.3 Constrains to vegetable production in Ghana ........................................................................................9 2.3.1 Insect Pest of Vegetable grown in Ghana ...........................................................................................9 2.3.2 Diseases of vegetables ......................................................................................................................11 2.3.3 Bacterial Disease of Vegetables........................................................................................................12 2.3.4 Fungal Diseases of Vegetables .........................................................................................................13 2.3.5 Viral Diseases of Vegetables ............................................................................................................14 2.4 Cucumber mosaic virus (CMV) ...........................................................................................................14 2.4.1 Origin and Geographical Distribution of CMV ................................................................................14 2.4.2 Transmission of Cucumber mosaic virus ..........................................................................................16 2.4.2.1 Natural transmission ......................................................................................................................16 2.4.2.2 Experimental transmission .............................................................................................................17 2.4.3 Host Range of CMV .........................................................................................................................18 2.4.4 Alternative Hosts of CMV ................................................................................................................18 2.4.5 Symptoms of CMV ...........................................................................................................................19 2.4.6 Genome Organization of CMV .........................................................................................................22 University of Ghana http://ugspace.ug.edu.gh vi 2.4.7 Detection of CMV.............................................................................................................................24 2.4.7.2 Nucleic Acid based Test ................................................................................................................26 2.4.7.3 Electron Microscopy (EM) ............................................................................................................28 2.4.7.4 Immuno-electron microscopy ........................................................................................................29 2.4.7.5 Biological Test Using Indicator Plants ..........................................................................................30 2.4.8 Economic Importance of CMV .........................................................................................................30 2.4.9 Control and Management of CMV ...................................................................................................31 REFERENCES ..........................................................................................................................................33 CHAPTER THREE ...................................................................................................................................56 3.2 INTRODUCTION ...............................................................................................................................56 3.3.1 Study area and location .....................................................................................................................58 3.3.2 Ayawaso West Municipal District ....................................................................................................59 3.3.3 Ga East Municipal District................................................................................................................59 3.3.4 Tema West Municipal District ..........................................................................................................60 3.4 SELECTION OF FARMERS ..............................................................................................................60 3.5 SURVEY AND DATA COLLECTION ..............................................................................................60 University of Ghana http://ugspace.ug.edu.gh vii 3.6 RESULTS AND DISCUSSION ..........................................................................................................61 3.6.1 Demographic Characteristics of respondents....................................................................................61 3.6.2 Cropping systems and agronomic practices of respondents .............................................................65 3.5.4 Cucumber mosaic virus awareness and management by farmers .....................................................68 3.7 CONCLUSION ....................................................................................................................................69 REFERENCES ..........................................................................................................................................71 CHAPTER FOUR ......................................................................................................................................74 4.1 NATURAL HOST RANGE AND INCIDENCE OF CUCUMBER MOSAIC VIRUS IN SOME SELECTED VEGETABLE FIELDS IN THE GREATER ACCRA REGION OF GHANA...........74 4.2 INTRODUCTION ...............................................................................................................................74 4.3 PRINCIPAL OBJECTIVE OF THE STUDY .....................................................................................75 4.4 SPECIFIC OBJECTIVES OF THE STUDY .......................................................................................76 4.5 MATERIALS AND METHODS .........................................................................................................76 4.5.1 Study area and location ..................................................................................................................76 4.5.2 Materials and Methods. .....................................................................................................................76 4.5.3. Data collection .................................................................................................................................77 University of Ghana http://ugspace.ug.edu.gh viii 4.5.4 Sample collection during survey .......................................................................................................79 4.5.5.2 Preparation of samples ...................................................................................................................79 4.6 RESULTS AND DISCUSSION ..........................................................................................................81 4.6.1 Symptoms of CMV observed on the field ........................................................................................81 4.6.2 Viral disease incidence and distribution across the three districts ....................................................82 4.6.2.1 Disease incidence ...........................................................................................................................82 4.6.3 Severity of viral disease symptom among vegetables crops .............................................................86 4.6.4 ELISA detection of CMV in leaf samples collected from the field ..................................................89 REFERENCES ..........................................................................................................................................95 CHAPTER FIVE .....................................................................................................................................101 5.1 DETERMINATION OF HOST RANGE OF CUCUMBER MOSAIC VIRUS AMONG SOME SELECTED VEGETABLES IN GHANA ..............................................................................................101 5.2 INTRODUCTION .............................................................................................................................101 5.3 MAIN OBJECTIVE OF THE EXPERIMENT .................................................................................103 5.4 SPECIFIC OBJECTIVES OF THE EXPERIMENT .........................................................................104 5.5 MATERIALS AND METHODS .......................................................................................................104 University of Ghana http://ugspace.ug.edu.gh ix 5.5.1 SELECTION OF POTENTIAL HOST CROP ...............................................................................104 5.5.2 Mechanical sap inoculation of CMV ..............................................................................................105 5.5.3 Confirmation of CMV transmission by ELISA test .......................................................................106 5.6 RESULTS AND DISCUSSION5.6.1 Mechanical sap inoculation of Cucumber mosaic virus ........106 Table 5.1 Mechanical sap inoculation of selected vegetable crops with CMV .......................................107 REFERENCES ............................................................................................................. 112_Toc147291635 CHAPTER SIX ........................................................................................................................................115 6.1 GENERAL CONCLUSIONS AND RECOMMENDATIONS ........................................................115 6.2 GENERAL CONCLUSIONS ............................................................................................................115 6.3 RECOMMENDATIONS ...................................................................................................................116 APPENDICES .........................................................................................................................................117 Coating buffer (pH 9.6)...........................................................................................................................118 PBS (pH 7.4) phosphate buffered saline ................................................................................................118 PBS-Tween (PBST) .................................................................................................................................118 Sample extraction buffer (pH 7.4) ..........................................................................................................119 Sample extraction buffer (pH 8.5) for Begomoviruses ..........................................................................119 University of Ghana http://ugspace.ug.edu.gh x Conjugate buffer .....................................................................................................................................119 Substrate buffer .......................................................................................................................................119 University of Ghana http://ugspace.ug.edu.gh xi LIST OF TABLES Table 4.1 Five-point scoring scale for determination of severity index of Cucumber Mosaic virus disease……………………………………………………………………………………………………77 Table 4.2 ELISA detection of Cucumber mosaic virus in different vegetable crops in Dzorwulu and Haatso……………………………………………………………………………………………………89 Table 4.3 ELISA detection of Cucumber mosaic virus in different vegetable crops in Tema motorway area………………………………………………………………………………………………………91 Table 5.1 Mechanical sap inoculation of selected vegetable crops with CMV……………………………………………………………………………………………………103 University of Ghana http://ugspace.ug.edu.gh xii LIST OF FIGURES Figure 2.1 Insect pests affecting vegetable production (A) Colorado potato beetle larvae on the leaves of potatoes (B) The woolly bear caterpillar on a spinach plant (D) Aphids on broccoli (E) Squash vine borer adults (F) whitefly on a leaf………………………………………………………………………………14 Figure 1.2 Some symptoms of Cucumber mosaic virus; A- leaf yellowing in lettuce, B- Leaf blistering in okra, C-mosaic leaf deformation in pepper, D-leaf deformation in tomato, E- leaf curl upwards in Radish, F- Yellow patches (mosaic) in cucumber………………………………………………………………..24 Figure 2.3 Genome organization of CMV……………………………………………………………….26 Figure 3.1 Educational level of respondent farmers in the study areas……………………………………62 Figure 3.2The number of years the respondent farmers have practiced vegetable farming…………………………………………………………………………………………………..63 Figure 3.3 Size of farmland cultivated by respondent farmers in the study area………………………………………………………………………………………………………64 Figure 3.4 Cropping systems practiced by respondents…………………………………………………66 Figure 3.5 Type of seeds planted by the respondent farmers………………………………………… 67 Figure 3.6 Source of planting materials used by the respondents……………………………………….67 Figure 3.7 Cucumber mosaic virus symptoms observed on the farmers’ fields………………………...68 Figure 3.8 Respondents’ perception on causes of abnormalities on their fields………………………...69 Figure 4.1 Symptoms of CMV observed on the vegetable farms……………………………………….81 Figure 4.2The map showing the districts within Greater Accra region as well as sample collection sites………………………………………………………………………………………………………82 Figure 4.3 Incidence of viral diseases on vegetable farms at Haatso……………………………………84 Figure 4.4 Incidence of viral diseases on vegetable farms at Dzorwulu…………………………………84 Figure 4.5 Incidence of viral diseases on vegetable farms along the Tema motorway…………………..85 Figure 4.6 Index of severity symptoms for diseased plants only assessed within the three districts of Greater Accra Region……………………………………………………………………………………86 University of Ghana http://ugspace.ug.edu.gh xiii Figure 4.7 Index of severity of symptoms for all plants assessed within the three districts of the Greater Accra Region……………………………………………………………………………………………………87 Figure 4.8 Microliter plate showing positive CMV samples…………………………………………….89 Figure 5.1 (A) Inoculated pepper plant showing symptoms of mosaic and (B) uninoculated healthy pepper plant…………………………………………………………………………………………………… 104 Figure 5.2 (A) Inoculated cabbage plant showing yellow patches of mosaic and (B) uninoculated healthy cabbage plant…………………………………………………………………………………………..105 Figure 5.3 (A) Healthy uninoculated okro plant and (B) Inoculated okro plant showing leaf curl and blistering………………………………………………………………………………………………105 Figure 5.4 (A) Asymptomatic inoculated eggplant and (B) uninoculated eggplant…………………..105 University of Ghana http://ugspace.ug.edu.gh xiv LIST OF ABBREVIATIONS Abbreviation Definition ADRRI Africa Development and Resources Research Institute AVRDC Asians Vegetable Research Development Center BBWV Broad Bean Wilt Virus BNARI Biotechnology and Nuclear Agriculture Research Institute CMV Cucumber Mosaic Virus DAS-ELISA Double Antibody Sandwich Enzyme Linked Immunosorbent Assay DNA Deoxyribonucleic Acid DSMZ Deutsche Sammlung von Milkroorganismen und Zellkulturen ELISA Enzyme-Linked Immunosorbent Assay University of Ghana http://ugspace.ug.edu.gh xv EM Electron Microscopy FAO Food and Agriculture Organization FAOSTAT Food and Agriculture Organization’s Statistical Database GAEC Ghana Atomic Energy Commission GDP Gross Domestic Product GLD Grapevine Leafroll Disease GLSS Ghana Living Standards Survey GSS Ghana Statistical Service ICTV International Committee on Taxonomy of Viruses IDM Integrated Disease Management IEM Immuno-Electron Microscopy University of Ghana http://ugspace.ug.edu.gh xvi IFPRI International Food Policy Research Institute IOSR International Organization of Scientific Research ISSAP Index of Symptom Severity of all plants ISSER Institute of Statistical, Social and Economic Research. IWMI International Water Management Institute. MOFA Ministry of Food and Agriculture MP Movement Protein ORF Open Reading Frame PBS Phosphate Buffered Saline PCR Polymerase Chain Reaction PVMV Pepper Veinal Mottle Virus University of Ghana http://ugspace.ug.edu.gh xvii PVP Polyvinylpyrrolidone RFLP Restriction Fragment Length Polymorphism RNA Ribose Nucleic Acid RT-PCR Reverse Transcription Polymerase Chain Reaction SPSVV Sweet Potato Sunken Vein Virus TEM Transmission Electron Microscopy TMV Tobacco Mosaic Virus UNICEF United Nation International Children’s Emergency Fund UV-ray Ultraviolet ray WEDC Water Engineering and Development Centre. WHO World Health Organization University of Ghana http://ugspace.ug.edu.gh xviii ABSTRACT Vegetables are important in diets of practically every household in Ghana. Vegetables are essential dietary portion that provide important vitamins and minerals. In addition to providing farmers with a source of income, vegetable cultivation helps the economy of the country to grow by creating jobs and bringing in substantial amount of foreign currency. Viral diseases are one of the largest obstacles to vegetable production, and in Ghana they are regarded to be a significant factor limiting the output of vegetable production. The Cucumber Mosaic Virus (CMV), one of these viruses, is extremely devastating and infects more plant families than any other plant virus. Unfortunately, since its discovery in 1974, the host range of CMV among important vegetable crops in Ghana has gotten comparatively little research attention. Therefore, the aim of this study was to identify the prevalence, host range, and severity of CMV among the main vegetables grown in Ghana's Greater Accra region, namely in the districts of Tema West, Ga East, and Ayawaso West. A standardized questionnaire was used to conduct a survey involving 120 farmers in these districts to assess the perception of the importance of CMV in vegetable production. It was discovered that the majority of farmers had little to no knowledge about viruses and instead implicated abiotic and biotic factors for their problems. After making extensive visual observations, it was discovered that CMV symptoms were present in every farm that was visited. The presence of CMV in tomato, broccoli, lettuce, spinach, cucumber, radish, and cabbage was confirmed by Enzyme-Linked Immunosorbent Assay (ELISA) on samples taken from symptomatic plants. This is the first account of broccoli, cabbage, lettuce, spinach, and radish in Ghana testing positive for CMV. The disease is spread mechanically through sap inoculation from an infected plant to a healthy plant, according to an University of Ghana http://ugspace.ug.edu.gh xix ELISA confirmation test. The findings of this study will contribute to the development of efficient control methods that would help manage the disease, particularly given the new host range of CMV discovered in Ghana. University of Ghana http://ugspace.ug.edu.gh 1 CHAPTER ONE 1.0 INTRODUCTION 1.1 Background of study In Ghana, vegetables play a significant role in almost every household's diet. They are crucial dietary additives that supply critical vitamins and minerals (Graham et al., 2007). Almost all of Ghana's ecological zones are used for vegetable farming, which is done either as a monoculture or in intercropping arrangements with other crops including cereals, legumes, root and tuber crops, plantation etc. The most extensively grown and economically significant vegetables are tomato, pepper, eggplant, and okra. Ghana is able to produce yields of 15,000 kg/ha for tomatoes, 30,000 kg/ha for eggplant, and 20,000 kg/ha for peppers. However, it was anticipated that the comparable national mean yields in 2016 would only be around half of these yields (MOFA, 2017). Vegetables are extremely susceptible to biotic and abiotic stressors, which contribute to low yields, as does the lack of governmental and private investment in productivity enhancing technology. Vegetable crops are frequently susceptible to a variety of diseases, which can affect both the quality and quantity of harvests. Infected plants frequently experience stunting, reduced leaf size, and leaf mottling and deformation. For instance, some tomato mosaic virus strains might result in brown dead streaks along the stem or the death of a portion of the plant, notably the tip (Lamptey et al., 2005) Numerous bacterial, viral, and fungal infections result in financial losses for vegetable farming. Among them, viral infections stand out because they significantly lower quality and yield while University of Ghana http://ugspace.ug.edu.gh 2 having a wide range of symptoms that differ greatly from host to host. Because most farmers are unaware of this issue, specialized methods for locating and managing virus infected plants under field conditions, including identification and control methods are poorly understood. According to Biswas et al. (2016), one of the best ways to stop the spread of viral infections in the field is to identify the causal agent and practice good hygiene in accordance with the most common diseases. Although many plant viruses have been reported to affect vegetables, Cucumber mosaic virus (CMV), one of the oldest viruses identified, is still being studied on the basis of epidemiological reviews of viral diseases of vegetables (Gallitelli, 2000). One of the most ubiquitous plant viruses, having a very diverse host range, the virus is a member of the family Bromoviridae and the genus Cucumovirus. (Meena et al., 2022). It infects over 1000 species of plants, including grains, fruits, vegetables, and ornamentals. More than 75 species of aphids can non-persistently spread the virus from infected to healthy plants (Krenz et al., 2015). The virus’s wide range of natural hosts, non-persistent transmission by numerous aphid species, and seed transmission in some hosts may be the causes of its widespread distribution (Biswas et al., 2016). The management and monitoring of CMV infestations is challenging due to the virus's spreading properties. Thus, viral disease management and precise strategies to recognize CMV in vegetables are greatly needed. The availability of multiple CMV variants makes it challenging to recognize the isolate from the symptoms alone, which is another problem with CMV in terms of identification. Various plant viruses may be identified, according to Katoch et al. (2003), using techniques such as bioassays, indicator plants, symptom observation, host range estimation, viral particle morphology, biochemistry, and immunology. However, it is necessary to develop methods for simultaneously detecting many plant species and multiple University of Ghana http://ugspace.ug.edu.gh 3 viral diseases (James et al., 2006). The most effective strategy to tackle the cucumber mosaic virus disease would be to do an extensive study on the distribution, diagnosis, and currently popular and advanced disease management techniques. CMV is one of the most prevalent viral infections in the globe due to its extensive dispersion. (ICTV, 2018) resulting in decreased in production (Rivera-Toro et al., 2020). Three positive sense single-stranded RNA segments make up the CMV genome (RNA1, RNA2 and RNA3). Each piece of viral RNA is packaged into an isometric particle, where it is translated into vital proteins that control viral encapsidation, replication, and transportation throughout the plant (ICTV, 2018). Two subgroups of CMV isolates, subgroup I and II, have been identified based on serology, nucleic acid hybridization, and gene sequencing (Dubey and Singh, 2008). 1.2 Statement of problem and justification of study In Ghana, vegetable production generates income for farmers and contributes to the development of the country’s economy through creation of employment and earning of foreign revenue (Kofi and Torvikey, 2018). For instance, in 2017 hot pepper contributed 4.5% of the foreign revenue generated from the sale of vegetables (Abdulai et al., 2017). One of the biggest challenges associated with vegetable production is viral diseases (Ogundeji, et al., 2012) and in Ghana viral diseases are thought to be an important factor restricting the output of vegetable production (Appiah et al., 2014). Vegetables can be infected by over 40 University of Ghana http://ugspace.ug.edu.gh 4 viruses (Lee et al., 2018) with numerous symptoms, including mosaic, mottle, leaf deformation, vein chlorosis, and stunting which considerably affect plant vigor and yield. Plant viruses are known to reduce crop quality and yield, cause horrific, enormous losses, and other adverse effects (Rao and Reddy, 2020). One of these viruses, the Cucumber mosaic virus (CMV), is devastating and it infects more plant families than any other plant virus. The host range of CMV among Ghana's significant vegetable crops has received relatively little research since its detection in 1974. This research therefore, sought to determine the prevalence, host range and severity of CMV among the major Ghanaian vegetables. The outcome of this research will aid in the management of the disease through the formulation of effective control measures. 1.3 Objectives The principal objective of this research was to assess the prevalence, severity and host range of Cucumber mosaic virus among vegetable crops in the Greater Accra region of Ghana Specific objectives were to: 1. Assess farmer awareness on virus disease incidence within Greater Accra region. 2. Conduct sero-diagnostic survey for CMV in the different vegetable crops. 3. Determine the experimental host range of CMV among Ghanaian vegetables using mechanical sap inoculation. University of Ghana http://ugspace.ug.edu.gh 5 CHAPTER TWO 2.0 LITERATURE REVIEW 2.1 Types of vegetables and importance in human and animal health Vegetables are horticultural crops that may be cultivated either annually or perennially. They have parts that may be consumed raw or cooked, including their roots, stalks, flowers, fruits, and leaves (Welbaum, 2015). In consideration of the plant component utilized as food and the specific nutritional value, vegetables can be categorized Khashroom et al., 2019). They can be classified as leaf (for instance, spinach, lettuce, curly lettuce, chard, purslane, and chicory), stalk (for instance, celery and asparagus), fruit, and flower vegetables (example are Broccoli, cauliflower, artichoke, tomatoes, peppers, cucumber and eggplant), Vegetables with root, bulb, and tuber (such as carrot, beet, turnip, fennel, onion, radish, and potato) and Legumes (example are peas, and soya beans) (Ülger et al., 2018). Vegetables are essential for human nutrition because they include bioactive nutritional molecules such as dietary fiber, vitamins, and minerals, as well as non- nutritive phytochemicals (phenolic compounds, flavonoids, bioactive peptides, etc.). These nutrients and non-nutrient molecules lower the risk of chronic illnesses like obesity, diabetes, some malignancies, and cardiovascular disorders (Pennington et al., 2009; September-Malaterrer et al., 2018). Consuming a variety of food groups in the proper amounts is necessary for a healthy, high-quality diet. Globally, the number of people who experienced hunger decreased to 795 million in 2015 (Pelletier et al., 2016), demonstrating improvement in guaranteeing appropriate access to staple University of Ghana http://ugspace.ug.edu.gh 6 foods as assessed by caloric intake. However, according to McLean et al. (2009), 2.1 billion more individuals are either overweight or obese, and micronutrient deficiencies affect an estimated 2 billion individuals worldwide (Ng et al., 2014). The important sources of the micronutrients required for healthful diets are fruits and vegetables. Vegetables provide dietary fiber, which reduces cholesterol levels in the blood, which may lessen the risk of heart disease. Maintaining a healthy blood pressure requires potassium whilst folate (folic acid) reduces the likelihood of cardiac disease and the chance of birth abnormalities. The health of the eyes and skin is maintained by vitamin A, while the teeth and gums are safeguarded by vitamin C, which also aids in the body's absorption of iron. In order to prevent chronic diseases (especially heart diseases, cancers, and diabetes) and provide essential micronutrients (especially calcium, iron, iodine, vitamin A, and zinc), the World Health Organization (WHO) recommends a minimum intake of 400 g of fruits and vegetables per day (WHO and UNICEF, 2003; WHO/FAO, 2003) . Consumers today, including those with more income, are thought to be falling short of this goal. In order to narrow this dietary gap and provide people with access to vegetables' nutritional worth, greater effort must be made. With a rising interest in foods that encourage healthy living and the preservation of excellent health, many people have started to adapt their eating habits in recent years. Vegetables are therefore increasingly being included in diets to support the maintenance of a healthy lifestyle and boost disease resistance (Afari-Sefa et al., 2015). University of Ghana http://ugspace.ug.edu.gh 7 2.2 Production levels and economic value of vegetables Vegetable cultivation takes place in close to 200 countries, and in many parts of the world, vegetables make up a significant portion of the average person's diet. Three hundred and ninety- two (392) vegetable crops comprising 70 families and 225 genera were reported in a global assessment of vegetables (Silva Dias, 2010). The most often consumed category of vegetables were the leaves or young green shoots (53% of the total), followed by vegetable fruits (15%). Roots, tubers, rhizomes, corms, and stolons were the most often used below ground crop vegetable organs, making up 17% of the total (Silva Dias, 2011). Vegetables are cultivated everywhere, by both large commercial producers and small holder farmers, on good and marginal soils, in urban and rural locations, and on large and small farms. A major portion of vegetables grown globally are produced on small plots and have short production cycles, which makes it difficult to gather reliable production numbers thus hindering the understanding of the importance of these crops to the global food supply. Even with the poor dependability of FAO figures about vegetables grown in impoverished countries, global production of vegetables in 2007 was about 900 million tonnes (FAO, 2009). Asia produced 74.7% of the world's vegetables on 72.8% of the global vegetable producing land (671 million tonnes; 52.7 million ha) with China contributing significantly to global vegetable accounting for almost 50% of global vegetable production (Silva Dias 2011) Similar to other emerging economies in Sub-Saharan Africa, the agriculture industry contributes significantly to Ghana's GDP and provides the majority of the country's jobs (ISSER, 2013). For instance, a record-high of GH¢ 8441 million (18.3%) of the nation's GDP was provided by the agriculture industry in 2017 (Ghana Statistical Service, 2018) University of Ghana http://ugspace.ug.edu.gh 8 In Ghana, the agricultural industry and the economy as a whole continue to be dominated by the crops sub-sector in terms of GDP contribution. In 2016 and 2017, it contributed 14.6% and 14.2% of nominal GDP, respectively (GSS, 2018). Vegetable cultivation, especially non-traditional or exotic vegetables, is a significant production area within Ghana's agricultural industry's crop sub- sector, which has significant potential for both domestic as well as global markets. Vegetable production levels have expanded dramatically over the previous years, rising from 682.43 million metric tons in 2000 to about 1.09 tonnes in 2017 (Statista, 2019), and as a result, they have greatly influenced global consumption, employment, and income demands. Growing vegetables in Ghana has the potential to reduce poverty and increase food security. Asians Vegetable Research Development Center (AVRDC) report in 2006 indicates that vegetable farming offers smallholder farmers substantially better revenue and more jobs per hectare than growing basic crops (Asravor, 2016). According to the most recent consumption figures from the Ghana Living Standards Survey (GLSS), in homes, vegetables accounted for 12.8% of the overall food budget in 2012 to2013, with the most common vegetables being tomatoes, onions, carrots, and chillies. For example, tomatoes accounted for 35.2% of all vegetable spending, followed by onions (19.0%), chilies (9.7%), and carrots (1.3 %) (Asselt et al., 2018). According to Badmus and Yekinni (2011), growing exotic vegetables is a lucrative industry that takes little start-up money and has turned into a source of income for the farmers involved. Urban vegetable cultivation provides people with a nutritious diet and a source of income, ensuring food security, nutrition, and better livelihoods (Hoornweg and Munro-Faure, 2008). Cabbage (Brassica University of Ghana http://ugspace.ug.edu.gh 9 oleracea var. capitata L.), lettuce (Lactuca sativa L.), spring onions (Allium fistulosum L.), bell peppers (Capsicum annuum L.), and cucumber (Cucumis sativus L.) are common urban vegetables that are not indigenous to Ghana (Cofie et al., 2003). but are cultivated by peri-urban vegetable growers. 2.3 Constrains to vegetable production in Ghana In Ghana, Vegetable production is hindered by a variety of variables that impact negatively on production, processing, marketing, and the entire exploitation of the potential prospects that vegetables may generate (Djokoto et al., 2015; Obuobie et al., 2006; Afari-Sefa et al., 2015). The land tenure structure, limited access to water for irrigation, and crop destruction by pests and diseases are the key factors limiting productivity. The types of crop that can be cultivated, as well as the overall cost involved in producing vegetables, are determined by the land's availability, size, and location, while the water source for irrigation dictates whether the farmer can cultivate throughout both the dry and rainy seasons. 2.3.1 Insect Pest of Vegetable grown in Ghana The primary biotic barrier to the production of vegetables in Ghana is insect infestations. Many of them not only cause direct harm but also serve as carriers of many viral infections. Vegetable crop losses of between 30 and 40 percent have been reported (Rai et al., 2014). Pre-harvest pests University of Ghana http://ugspace.ug.edu.gh 10 globally cause the loss of 35% of potential crop output, around 45% of total food production losses of the world's total food are caused by insect pests, diseases and weeds (Oerke, 2006). Pests on crop plants have an agricultural relevance because of the harm they do, which lowers the yield's quality or quantity (or both). The appearance of the crop, which may display certain forms of pest damage or disease signs, is frequently the first sign of the existence of a pest or disease (Hein, 2003). According to their feeding behavior, insect pests create two basic forms of agricultural damage (Imam et al., 2010). The first is harm caused by the chewing of plant materials by insects such as grasshoppers, locusts, and crickets (Orthoptera), caterpillar (Lepidoptera) and beetles (Coleoptera) (Khan et al., 2020). The second is harm caused by sucking plant sap from tissues of leaves, roots, or fruits, as well as from the phloem (or xylem) system (Khan et al., 2020). The bugs (Hemiptera) and thrips (Thysanoptera) are the two primary groups of sucking insects (Khan et al., 2020). Crop damage caused by insect pests includes decreased yield, disease transmission, decreased market value of crops, and increased agricultural costs. The Chenopodiaceae and Solanaceae groups of plants contain both succulent and leafy plants, such as spinach and tomatoes, which are attacked by pest insects with a broad host range. Monophagous insects are only found on a small number of the host plants in the system, notably the leafy vegetables like spinach, lettuce, and kale. If all or most of the plants in a mixed system are edible to a polyphagous pest, it is likely that the pest will stay longer because of its capacity to expand its menu of food sources and subsequently multiply, causing more harm (Imam et al., 2010) University of Ghana http://ugspace.ug.edu.gh 11 Figure 2.1 Insect pests affecting vegetable production :(A) Colorado potato beetle larvae on the leaves of potatoes (B) The woolly bear caterpillar on a spinach plant, (C) Thrips feeding causes silvering on underside of a potato leaf, (D) Aphids on broccoli (E) Squash vine borer adults (F) whitefly on a leaf. 2.3.2 Diseases of vegetables Plants infected with bacteria, fungi, parasites, and viruses exhibit certain symptoms that are similar to those caused by insect attack. Finding the pest close to the damage on the plant typically makes it easier to identify the destructive organisms while studying crop damage and agricultural pest (Hein, 2003). A B B C D E F University of Ghana http://ugspace.ug.edu.gh 12 The many vegetable varieties that are often produced in Ghana are vulnerable to a variety of diseases. Both abiotic and biotic factors may contribute to the development of these disorders. The abiotic influences may include some climatic and cultural circumstances including high temperatures, nutritional deficiencies, and excessive sunshine (Roberts et al., 2004). The Food and Agriculture Organization (FAO) generally classifies the biotic disease causing organisms into three groups: bacteria, fungus, and viruses (FAO, 2004). These diseases drastically lower crop yields, raise production costs, and thus have a negative impact on farmer revenue (Manneh et al., 2016). 2.3.3 Bacterial Disease of Vegetables Many vegetable crops are susceptible to leaf blights and spots caused by plant pathogenic bacteria. These diseases often start off as little, water-soaked patches that gradually turn brown, and they are most frequently brought on by Pseudomonas and Xanthomonas species. The bacterial diseases include bacterial canker (Clavibacter michiganensis subsp. michiganensis), bacterial spot (Xanthomonas axonopodis pv. vesicatoria), bacterial wilt (Ralstonia solanacearum), bacterial speck (Pseudomonas syringae pv. tomato) and bacterial stem rot (Erwinia spp, Pectobacterium carotovorum subsp. carotovorum and Pectobacterium carotovorum subsp. atrosepticum, Pectobacterium chrysanthemi). (Bastas, 2013). Vegetable leaves, fruit, and stems develop lesions from the bacterial spot disease (Jones et al., 2000). The lesions prevent the leaves from performing photosynthetic processes, which ultimately University of Ghana http://ugspace.ug.edu.gh 13 results in leaf death. The fruit may also be impacted, rendering it unfit for processing or export. Although the bacteria are spread over a field by wind-driven raindrops, wounds from field work (grafting, cutting, tying, harvesting, pesticide spraying), and aerosols (Lindemann and Upper, 1985; McInnes et al., 1988), seeds are the main source of inoculum (Obradovich et al., 2004; Jones et al., 1991) 2.3.4 Fungal Diseases of Vegetables A variety of dangerous plant diseases are brought on by fungi, which make up the majority of plant pathogens. Generally, fungi are responsible for vegetable diseases. Fungal infections are spread through infected soil, weeds, infected seed, agricultural waste, and nearby crops. They are disseminated by the movement of contaminated soil, animals, humans, tools, equipment, seedlings, and other plant material, as well as by wind and water splash. They penetrate plants through stomata, which are naturally occurring openings, as well as wounds caused by cutting, picking, hail, insects, other diseases, and mechanical damage (Tournas, 2005). Various fungi are the cause of foliar diseases. The following are some of the most prevalent foliar diseases: White blister, Powdery mildew, and Downy mildew. Other soil-borne fungus includes Club root, Sclerotinia species, Sclerotium species, Pythium species, Fusarium species, and Rhizoctonia species. Numerous vegetables are susceptible to some fungi-related infections. Anthracnose, Botrytis rots, Downy mildews, Fusarium rots, Powdery mildews, Rusts, Rhizoctonia rots, Sclerotinia rots, and University of Ghana http://ugspace.ug.edu.gh 14 Sclerotium rots are some of the fungal diseases. Other examples are Red root complex in beans, Leaf blight (Alternaria dauci) in carrots, and Club root in brassicas (Plasmodiophora brassicae). Punja and Utkhede (2004). 2.3.5 Viral Diseases of Vegetables Viral infections are emerging as a significant limitation to agricultural production in the context of changing climatic conditions. Worldwide, virus infections drastically reduce the productivity and quality of agricultural produce and are responsible for significant yield losses of up to 100%. Viral infections are often confused with other irregularities, making it difficult to select the best management approach in order to produce crops free of viruses. (Nagendran et al., 2017). Some important viral diseases in vegetables are; Tobacco mosaic virus disease, Tomato spotted wilt virus disease, Pepper veinal mottle virus disease, Cucumber mosaic virus disease, Potato virus Y disease, Cauliflower mosaic virus disease, Tomato mosaic virus disease, Tomato yellow leaf curl virus disease, Plum pox virus, and Potato virus X. (Arogundade et al., 2014). 2.4 Cucumber mosaic virus (CMV) 2.4.1 Origin and Geographical Distribution of CMV University of Ghana http://ugspace.ug.edu.gh 15 In addition to its importance as a model for studying plant viral interactions, Cucumber MosaiVirus (CMV), has a significant effect on agricultural production all over the world. Among plant viruses, it is one of the most common (Palukaitis and Garca-Arena, 2003). The first cases of CMV infections on cucumber and other Cucurbitaceae plants were reported concurrently by Doolittle and Jagger in Michigan and New York respectively (Garcıa-Arenal and Palukaitis, 2008). Since then, approximately 1200 species in over 100 groups of monocots and dicots, including agricultural plants, ornamental plants, and woody plants have been documented to suffer CMV infections, particularly in temperate to tropical regions. There are two subgroups of CMV isolates that have recently been identified, namely I and II. While isolates from different subgroups have only 69–77% sequence identity, the nucleotide sequences of CP genes from CMV isolates within each subgroup can share up to 95% nucleotide sequence identity (Palukaitis and Garca-Arena, 2003). Isolates of subgroup I are able to tolerate high-temperatures compared to those in subgroup II, while symptoms in subgroup II are less severe. In terms of the country's warm climate, subgroup I isolates are therefore more prone to infect Ghana's vegetable crops. Subgroup I can be further divided into subgroups IA and IB, and populations of subgroup IB is very common in East Asia (Palukaitis et al., 1992; Roossinck et al., 1999). Worldwide dissemination of CMV isolates is evident in open-field crop cultivation and greenhouses. Isolates from subgroup I are more prevalent than those from subgroup II, with the latter being mostly restricted to colder regions or times of year in temperate zones (Garca-Arenal and Palukaitis, 2008). The majority of the subgroup IB isolates found in the Mediterranean region may have been transported there from East Asia, the subgroup's supposed place of origin (Garca-Arenal and Palukaitis, 2008). In Greece, tomato plants were used to identify a severe, satellite-free CMV University of Ghana http://ugspace.ug.edu.gh 16 isolate known as CMV-G that belonged to the subgroup IB. Through further inoculation on a host with a local lesion, two distinct genotypes that infected tobacco with either moderate symptom (green mosaic) or more severe yellow mosaic were isolated (Sclavounos et al., 2006). According to Lavina et al. (1996), CMV causes significant viral reservoirs in native weed populations by infecting a variety of weed species close to horticulture crops. CMV was discovered, CMV strains from subgroup II were discovered in France (Gallitelli et al., 2004). and southern Italy (Palukaitis and Garcia-Arenal, 2003; Roossinck, 2005; Paradies et al., 2000). There are further credible reports of CMV infection from Spain (Gallitelli et al., 2012) and Tunisia (Chabbouh and Cherif, 1990). Uganda, Zambia, Tanzania, Ethiopia, Zimbabwe, Kenya, Malawi, Madagascar, Sudan, Rwanda, Ghana, and Nigeria are among the African countries where the virus has been reported. (Skelton et al., 2018; Appiah et al., 2014). 2.4.2 Transmission of Cucumber mosaic virus 2.4.2.1 Natural transmission Aphids and mechanical inoculation of plant sap are the most important natural transmission of cucumber mosaic virus, and they are also the most effective (Li et al., 2020). In a non-persistent, stylet-borne method, CMV has been discovered to be transmitted from plant to plant by at least 75 species of aphids (Palukaitis et al., 1992). CMV is not passed to offspring aphids and does not multiply in its aphid carrier. According to Li et al. (2020), Myzus persicae and Aphis gossypii are the most efficient vectors for CMV transmission in vegetables. One of the main characteristics of University of Ghana http://ugspace.ug.edu.gh 17 non-persistent viral transmission is that aphids only pick up the virus for brief periods of time (often a few seconds) before losing it after their usual feeding on plants. Additionally, CMV can spread through pollen, contaminated soil particles, infected seed, infected agricultural debris, and other means (Pares and Gunn, 1989; Alvarez et al., 2003; Chen et al., 2000). Additionally, the virus can spread by the parasitic plant dodder; Cuscuta spp. (Palukaitis and Garca-Arenal, 2003). 2.4.2.2 Experimental transmission CMV infection has frequently been produced in experimental settings by mechanical inoculation using sap, pure virions, or viral RNA. In addition to many other plant species, CMV principally causes mosaic and stunt diseases in the families; Cucurbitaceae, Solanaceae, Leguminosae, Brassicaceae, and Gramineae. Symptoms of the disease can vary based on the host species and CMV strain, and may include leaf deformity, systemic necrosis, stunting, and chlorosis. Normally utilized for CMV replication, Nicotiana glutinosa and N. tabacum are well recognized for their aggressive CMV reproduction. The mechanical inoculation of CMV causes necrotic local lesions in the test plants Vigna unguiculata, Chenopodium amaranticolor, and C. quinoa. Garcıa-Arenal & Palukaitis (2009) Ipomoea setifera has been the source of CMV isolation Loebenstein (2012) was able to mechanically inoculate Ipomoea nil, Ipomoea purpurea, Ipomoea lacunosa, and Ipomoea trichocarpa with CMV but not Ipomoea batatas cv. Porto Rico. CMV transmission to healthy sweet potato plants was unsuccessful, according to Cohen and Loebenstein et al. (2009). However, aphid, or graft inoculations can quickly transmit CMV to sweet potatoes in the presence University of Ghana http://ugspace.ug.edu.gh 18 of Sweet Potato Sunken Vein Virus (SPSVV); whitefly-transmitted virus (Loebenstein et al., 2003). 2.4.3 Host Range of CMV Cucumber mosaic virus (CMV) has a very wide host range of wild and cultivated plants with more than 1,200 known hosts, comprising some monocotyledons and a large number of dicotyledonous plants (Chen, 2003). CMV has also been reported to infect cowpea, celery, cucurbits, pepper, lettuce, tomato, chilies, banana, pasture legumes, and some ornamental plants (Palukaitis et al., 1992); Flasinski et al., 1995; Davis et al., 1996; Gafny et al., 1996; Latham et al., 1999; Iqbal et al., 2011; Ashfaq et al., 2014b). 2.4.4 Alternative Hosts of CMV Weeds or other alternate natural hosts serve as the primary hosts for the majority of plant viruses from which the economically important crop plants may contract viruses (Neeraj and Zaidi, 2008; Mathews and Dodds, 2008). Due to the biotrophic nature of plant viruses, which obviously needs alternate hosts to survive if the original crop host is unavailable, in close proximity to agricultural fields, weeds, ornamental plants, and wild plants appear to be infected with plant cultivable species' viruses. (Sivalingam and Varma, 2007). These plants also play a significant role in the University of Ghana http://ugspace.ug.edu.gh 19 development of virus disease epidemics. As a result, the epidemiology of viral infections is more complex than that of other plant diseases Budnik (1995). Many weeds have the potential to serve as CMV reservoirs, which can aid in the virus' early- season transmission to crops. This is due to the virus's wide variety of potential hosts. Common milkweed (Asclepias syriaca), yellow rocket (Barbarea vulgaris), marsh yellowcress (Rorippa islandica), and yellow toadflax (also known as butter-and-eggs, Linaria vulgaris) are a few examples of perennial, biennial, and winter annual plants that harbor CMV in their roots, tubers, and underground organs all winter long. They were shown to be significant causes of infection in lettuce (Zitter and Murphy, 2009). 2.4.5 Symptoms of CMV Numerous vegetable and horticultural crops are susceptible to CMV, which affects 1200 species in more than 100 plant families and can lead to severe economic losses. In most hosts, the virus induces systemic infection; but, in particular crops, like alfalfa, it may go undetected. Depending on the crop affected and the age of the plant when infection occurs, cucumber mosaic symptoms can vary widely (Zitter and Murphy, 2009) CMV infected cucurbits, particularly zucchini, squash, melons and cucumber, exhibit severe mosaic symptoms as well as leaf deformation and constriction. Infected plants also exhibit stunting, decreased fruit output with pitted and deformed fruits which are unmarketable. Mixed infections with different viruses are typical, which makes the situation worse. The virus, and the University of Ghana http://ugspace.ug.edu.gh 20 usual mosaic signs on and melon leaves. On fruits, it is also common to see mottling or mosaic. A quick and total wilt is seen on mature plants of several cucumber cultivars a few days following CMV infection. The symptoms of CMV in zucchini and squash are quite severe and include mosaic, yellow spots, and leaf deformities. Infected plants continue to be stunted, and fruit setting is typically significantly decreased or even stopped. Deformed fruits have little depressions all over them. Watermelon reacts with black necrotic lesions when it has CMV infection, which is an uncommon occurance (Garcia-Arnal and Palukaitis (2009). Chlorotic local lesions between the secondary leaf veins are one of the early CMV infection symptoms in crops like tomato plants. Necrosis advances toward the base of the plant, which might die a few weeks after infection, starting with brown spots on the leaves and progressing to brown lines along petioles and stems. Fruits are misshaped, sunburned, and necrotic if they are present at all. Infected plants typically exhibit reduced growth, a bushy look, and deformed leaves when they are grown (shoestring-like leaves). Fruits shrink in size and do not ripen. Depending on a variety of variables, including the existence of satellite RNAs, plants may have normal- appearing leaves but exhibit fruit necrosis (losses are much decreased in this instance), or they may exhibit usual symptoms but with more pronounced manifestations (in this case, losses can be very high) (Bragard et al., 2013). The viral strain and the age of the plant at the time of infection affect the CMV infection symptoms in pepper plants. CMV induces symptoms of necrosis on both the fruit and foliage of young pepper plants, including leaf yellowing, leaf constriction, and fruit necrosis. If older plants are infected, University of Ghana http://ugspace.ug.edu.gh 21 CMV signs can be subtle and show up on lower, yellowing leaves as green ring spots or oak-leaf patterns, as well as a subtle mosaic. CMV infected celery and parsley exhibit yellowing and necrosis of the leaves. When the temperature falls below 13 °C, CMV infected lettuce exhibits strong mosaic, vein chlorosis, and commonly vein browning and necrosis. Stunting, yellowing, and mottling of the older leaves as well as deformation of the younger leaves are some of the indications of CMV infection in spinach (Bragard et al., 2013). A B C D E F University of Ghana http://ugspace.ug.edu.gh 22 Figure 2.2 Some symptoms of Cucumber mosaic virus; A- leaf yellowing in lettuce, B- Leaf blistering in okra, C-mosaic leaf deformation in pepper, D-leaf deformation in tomato, E- leaf curl upwards in Radish, F- Yellow patches (mosaic) in cucumber. 2.4.6 Genome Organization of CMV Three positive-sense, single-stranded RNAs packed in distinct particles may be found in the CMV genome (Palukaitis et al., 1992). Additionally, two subgenomic RNAs are present in virus particles (Palukaitis, 2003). The 1a and 2a proteins, which make up the two components of the viral replication complex, are encoded by RNAs 1 and 2, respectively (Hayes and Buck, 1990). Additionally, RNA 2 encodes the 2b protein, a multifunctional protein implicated in host-specific, long-distance migration, symptom development, and virulence determination via inhibiting gene silencing (Brigneti et al., 2015). Additionally, a recent study has shown that the 2b gene controls the choice of inter-viral recombination (Shi et al., 2008). Cell-to-cell movement protein (MP) 3a and capsid protein (CP), which is translated from a subgenomic RNA 4, are both proteins that are encoded by the CMV RNA 3. Cell-to-cell communication, virion assembly, and aphid-mediated transmission are all facilitated by CP (Perry et al., 1998). Based on serology, nucleic acid hybridization, RT-PCR followed by RFLP, and nucleotide sequence identity, CMV strains have been divided into two major subgroups known as subgroups I and II (Roossinck, 2002). The nucleotide identity between these two groupings is 75% (Roossinck, 2002), with subgroup I being more diverse than subgroup II (Palukaitis, 2003). Subgroup I has been further divided into IA and IB, with 92–95 percent nucleotide similarity between these two A phylogenetic examination of a few CMV strains revealed that the University of Ghana http://ugspace.ug.edu.gh 23 subgrouping from CP ORF analysis is not entirely supported by the estimated trees for the distinct open reading frames (ORFs) found on the different RNAs. As a result, several RNAs may have independent evolutionary histories (Roossinck, 2002). In agricultural regions, the subgroups are not spread equally. Subgroups IA and II are distributed globally, although subgroup IB is reportedly confined mostly to Asia (Roossinck et al., 1999) Random mutation, recombination, and re-assortment are the most frequent causes of RNA virus evolution and variety, and RNA viruses are capable of undergoing fast genetic change (Garca-Arenal et al., 2001). All three CMV subgroups have been observed to exhibit RNA re-assortment (Bonnet et al., 2005; Chen et al., 2007). Recombination in the 5′ and 3′ NTR, between ORFs 3a and 3b, and in naturally occurring populations of CMV harboring satellite RNA have also been demonstrated to be additional sources of heterogeneity in the viral population (Bonnet et al., 2005; Chen et al., 2002; Pierrugues et al., 2007). As a result, CMV is a heterogenic species with a wide range of isolates. This enables the virus to quickly adapt in distinctive habitats with fluctuating selection pressures (Roossinck, 2002; Lin et al., 2004; Escriu et al., 2000; Koundal and Praveen, 2011). For a better understanding of the evolutionary mechanisms that produce variety, studies concentrating on this genetic diversity and the causes of variation in viral populations are crucial. For many years, CMV has been prevalent in several regions of the United States. But in the 2000s, a number of viral outbreaks impacted the processing snap bean (Phaseolus vulgaris L.) in crucial agricultural areas in the upper Midwest and northeast of the US (German et al., 2004; Nault et al., 2006). The introduction of new variations in viral populations can help to partially explain the origin of such plant virus outbreaks. University of Ghana http://ugspace.ug.edu.gh 24 Figure 2.3 Genome organization of CMV. Figure 2.4 Genome organization of CMV. The nucleotide and amino acid numbers are for the Fny strain. RNAs 1, 2, and 3 are genomic, and required for infection. RNAs 4 and 4 A are subgenomic. RNA 4 is packaged in virions of all strains; RNA4A is only packaged in subgroup II strains. The satRNA is a molecular parasite sometimes associated with the virus. It is packaged in the virions of the virus. 2.4.7 Detection of CMV A plant that has a viral infection may show a variety of indications indicating the virus is active. However, since viruses can induce symptoms that are identical to those caused by abiotic stressors, using symptoms alone to diagnose the presence of viruses may be misleading (Gao, 2020). University of Ghana http://ugspace.ug.edu.gh 25 Additionally, it is uncommon for plant viruses of the cryptic viral class to ever cause any visible symptoms (Marwal et al., 2021; Lukács, 1994; Valverde and Gutierrez, 2008). Therefore, rapid diagnostic approaches must be used to enable accurate, early diagnosis and disease control due to the catastrophic impact of viral diseases on economically important plants. In situations where symptoms may be similar to those of more than one virus, molecular and serological diagnostic approaches have shown to be very helpful (Shukla, 2015). The cucumber mosaic virus has been identified using these methods, which include the Enzyme Linked Immunosorbent Assay (ELISA), Reverse-Transcription PCR, and Electron Microscopy.2.4.7.1 Serological Detection (ELISA) One of the most commonly used immunodiagnostic techniques is the Enzyme Linked Immunosorbent Assay (ELISA), which is popular due to its simplicity, accuracy, and relatively low cost. The use of ELISA for the detection of plant viruses is widely known and has proven to be one of the most effective technologies for the identification of plant viruses The ELISA technique relies on the sensitive detection of non-precipitates reaction, made possible by the use of enzyme-labeled antibodies. González-Garza (2017), discovered that ELISA differs from nearly all other serological techniques in plant virology that were based on the formation and detection of immune precipitates. For practical purposes, the ratio of antibodies to antigen has little effect on the effectiveness of ELISA technique. Thus, once the proper concentrations had been established, they could be used in subsequent tests to detect the virus at all concentrations, and since the reaction of enzyme-labeled antibodies was dependent on the virus concentration, the method has a high quantitative potential for figuring out how sensitive ELISA is. University of Ghana http://ugspace.ug.edu.gh 26 The ELISA test enables the researcher to do both a quantitative and qualitative analysis and the test has been used extensively by researchers studying the Cucumber mosaic virus due to its usefulness and simplicity of use (Gan and Patel, 2013). 2.4.7.2 Nucleic Acid based Test Due to its capacity to identify viruses at low titer levels or concentrations. Reverse Transcription Polymerase Chain Reaction (RT-PCR) is the most widely used technique for diagnosing plant viruses (Scagliusi et al., 2009; Mekuria et al., 2003). Several investigations have demonstrated that the RT-PCR is more accurate at detecting plant viruses than ELISA, with fewer false-negative findings (Mekuria et al., 2003; McGavin et al., 2011). By calculating the DNA concentration following each amplification step in the PCR process, the qPCR, also known as real-time PCR, can calculate the viral titer level in the samples (Taylor et al., 2010; Deepak et al., 2007). The PCR technique employs a pair of artificial oligonucleotides or primers, each of which hybridizes to one strand of a double-stranded DNA (dsDNA) target and which spans an area that will be exponentially replicated. The DNA Taq polymerase uses the hybridized primer as a substrate, and the hybridized primer is used to sequentially add deoxynucleotides to the substrate to form a complementary strand. The Taq polymerase is most frequently produced from the thermophilic bacteria Thermus aquaticus. Three phases make up the process: (i) dsDNA separation at temperatures over 90 °C, (ii) primer annealing at temperatures between 50 and 75 °C, and (iii) optimum extension at temperatures between 72 and 78 °C. A programmable thermal cycler University of Ghana http://ugspace.ug.edu.gh 27 regulates the rate of temperature change, often known as the ramp rate, the duration of incubation at each temperature, and the number of times each set of temperatures is repeated. With the use of fan-forced warm air flows or electrically controlled heating blocks, modern technologies have drastically decreased ramp times. As a result, several of the best cell culture, antigenaemia, and serological techniques are being replaced by PCR (Niubo et al., 1994). In order to gather quantitative data, it has been possible to employ existing PCR and detection assay combinations (referred to as "conventional PCR"), with encouraging outcomes. However, these methods have been hampered by the time-consuming post-PCR processing procedures needed to assess the amplicon (Guatelli et al., 1989). The conventional method for detecting amplified DNA involves electrophoresis of the nucleic acids in the presence of ethidium bromide and visual or densitometric examination of the bands that emerge after exposure to UV light (Kidd et al., 2000). Additionally, PCR-ELISA may be used to capture amplicon onto a solid phase using biotin or digoxigenin-labelled primers, oligonucleotide probes (oligoprobes), or just after the incorporation of the digoxigenin into the amplicon (Watzinger et al., 2001). An enzyme-labeled avidin or anti-digoxigenin reporter molecule can be used to detect the amplicon once it has been collected, much like a conventional ELISA format. Real-time PCR has demonstrated its worth in labs all over the world, adding to the vast quantity of data produced by traditional PCR tests. In contrast to the traditional assays, real time PCR allows the detection of the amplicon to be seen as the amplification developed (Mackay, 2004). University of Ghana http://ugspace.ug.edu.gh 28 The use of fluorogenic molecules to label primers, probes, or amplicon has made it possible to monitor accumulating amplicon in real time. The technique has become faster in large part as a result of shorter cycle durations, the elimination of post-PCR detection steps, the use of fluorogenic markers, and sensitive methods for detecting their emissions (Wittwer et al., 1990; Wittwer et al., 1997). Comparatively to conventional PCR, real-time PCR has significant drawbacks, such as the inability to monitor amplicon size without opening the device, the incompatibility of some platforms with certain fluorogenic chemicals, and the relatively limited multiplex capabilities of present applications. When utilized in low-throughput facilities, real-time PCR's startup costs might also be too high. The hardware of the system or the fluorogenic dyes or fluorophores available both have certain restrictions that contribute to these flaws. 2.4.7.3 Electron Microscopy (EM) Scientists have worked to understand the structure of viruses ever since they were identified as the disease-causing agents in the last decades of the nineteenth century (reviewed in Mettenleiter, 2017). In order to conduct investigations at the nanoscale and obtain direct pictures of viruses for use in research and diagnostics, electron microscopy (EM) has a high resolving capacity. Due to its capacity to identify contaminations, the use of TEM is crucial for quality control of reference material, viral preparations used for antibody generation, or directly for vaccination. During an unexpected regional viral outbreaks or epidemics, prompt generation of consistent and comparable University of Ghana http://ugspace.ug.edu.gh 29 data is crucial for effective diagnosis, making TEM a vital component of the procedures followed by national reference laboratories. EM image databases have just been created and made accessible by Laue and Möller (2016). The expansion of these archives will offer the ease of identification of unexpected as well as newly developing infections. For instance, plant viruses can act as markers to forecast the presence of harmful human viruses in irrigation water (Shrestha et al., 2018) 2.4.7.4 Immuno-electron microscopy The same serological concepts that underlie ELISA are also the foundation of immuno-electron microscopy (IEM), which may be used to further identify viruses during regular Transmission electron microscopy (TEM) testing. IEM has the benefit of not requiring further immunoglobulin purification or conjugation procedures because it operates directly with raw serum. Antibody usage is minimal since only modest reaction volumes are needed. Most TEM labs maintain extensive antisera collections tailored to a variety of viral types and isolates. Antisera may be kept for a long time at 4°C with 0.05% sodium-azide without suffering a substantial loss of activity. Polyclonal antisera may respond in a heterogeneous manner depending on the antigen composition and the epitopes found in the initial viral purification and therefore can be used for a variety of purposes during regular diagnostics (Griffiths and Lucocq, 2014). While serological associations will become apparent by the intensity of antibody attachment during the decorating process, some antisera are adequate for IEM capture of several viruses within a genus. In a homologous response, the virion is shown to be firmly packed with antibodies, whereas emerging isolates or heterogonous viruses would only have a thin antibody coating (Richert-Pöggeler et al., 2019). When present, University of Ghana http://ugspace.ug.edu.gh 30 monoclonal antibodies that target single epitopes offer the best specificity and repeatability for differentiating between viral isolates (Griffiths and Lucocq, 2014). 2.4.7.5 Biological Test Using Indicator Plants Accurate disease identification is difficult due to the fact that viral infections do not always result in immediately noticeable visual signs in the host plants. With the use of biological indexing, it is possible to validate the existence of a virus that could otherwise go undetected in some plants, identify an uncommon host plant for the virus, and quantify the virus (Smith, 1977). The sensitive plant species or kinds that often exhibit typical symptoms after exposure to the pathogenic viruses are the indicator plants (Smith, 1977). Biological indexing is still employed in conjunction with lab-based testing techniques to effective disease diagnosis (Wolfenden et al., 2018). The main drawback in the utilization of indicator plants for virus detection is the lengthy time (weeks or months) it takes between inoculation and the appearance of disease symptoms (Legrand, 2015). Furthermore, indicator plant symptoms may change depending on the surrounding environment. Constable et al. (2013) discovered that rugose wood symptoms on Rupestris St. George indicator plants could not be seen in a cold climate but symptoms were evident in hot climate. Contrarily, Cabernet Franc in a cool location exhibited Grapevine Leafroll Disease (GLD) symptoms, but no symptom was observed in the hot climate site for the identical treatments, limiting the use of this variety as an indication (Wang et al., 2022) 2.4.8 Economic Importance of CMV University of Ghana http://ugspace.ug.edu.gh 31 Viruses are to blame for significant quality and production losses in crops all around the world. Cucumber mosaic virus (CMV), one of the most significant viruses affecting vegetable crops globally, transmitted by polyphagous aphid vectors (Montasser et al., 2006; Palukaitis and Garcia- Arenal, 2003; Pakdeevaraporn et al. 2005; Biswas et al. 2013). CMV is particularly severe on chilli (Green, 1993) and up to 41% yield reductions in vegetable has been reported (Selvarajan et al., 2023). Additionally, more than 60% yield losses resulting from CMV infection in peppers has been documented (Rahman et al., 2015). If an infection occurs early, yield losses might be as high as 100% (Rahman et al., 2015). However, the quantity of crop losses is influenced by the virus strain, vegetable type, plant age and, environmental variables, viral disease during disease development, etc (Thackray et al., 2004). One of the about 80 species of aphids that can spread CMV is the green peach aphid, Myzus persicae, Cerkauskas et al. (2004). CMV is not spread by pepper seed. Although CMV may be mechanically transferred, workers handling infected pepper plants do not readily spread it because it is less stable than TMV. Weeds serve as hosts for both the virus and the aphid vectors. The high prevalence of CMV in field plants is caused by the abundance of aphid vector species and natural host reservoirs (Rahman et al., 2015). 2.4.9 Control and Management of CMV Several strategies are often used to reduce the prevalence and spread of CMV. The devastation brought on by viral infections has been fought against with intensive efforts that include University of Ghana http://ugspace.ug.edu.gh 32 interdisciplinary approaches to viral disease control. Aphid vector management with chemicals, cultural techniques, and the adoption of virus-resistant cultivars are some of the strategies that are commonly used. 2.4.9.1 Integrated Management Practices Worldwide, large agricultural losses are caused by plant diseases, which are regarded as a serious biotic constraint. Particularly in the tropics and semi-tropics, where viruses and their vectors thrive, viral infections of plants result in significant economic losses. Furthermore, viral diseases have been made worse by intensive agricultural techniques that have been made necessary by the population's expanding needs and the introduction of novel genotypes and cropping patterns (Varma, 2007). To reduce the losses brought on by these diseases, a variety of innovative strategies have been tested. The main strategies involve avoidance of potential infection sources, avoiding or controlling vectors, altering cultural practices, using resistant varieties developed through conventional breeding techniques, cross-protection, systemic acquired resistance, and using transgenic plants that have been genetically modified to express alien genes that confer viral resistance (Varma, 2007). In contrast to adopting a single component method of control, integrated disease management (IDM), which integrates biological, cultural, physical, and chemical control measures, has been shown to be more successful and long-lasting (Ownley and Trigiano, 2016). Chemical pesticides are viewed as being both uneconomical and environmentally unfriendly. Despite the fact that several studies have been done to identify non-chemical alternatives. Pesticide- University of Ghana http://ugspace.ug.edu.gh 33 related issues including pest resistance, dangers to non-target organisms, pest comeback, and pesticide residue have gotten much worse as a result of overuse of pesticides. Thus, the creation of disease-resistant cultivars or hybrids is the most efficient, long-lasting, and sustainable method of CMV management (Yao et al., 2013). Although it has been shown that using resistant cultivars is the most practical and inexpensive option, sustainable agriculture must adopt an integrated strategy for the viral infections to be effectively managed. Therefore, in order to create efficient integrated management strategies, it is essential to have a complete grasp of the ecology of viruses and their vectors (Wightman 2007). Several methods have been used to combat CMV, including as cultural practices like weeding, eradicating infected plants, using disease-free seeds, and using chemical and biological pesticides like pyrethroids, abamectin, and carbamate to control aphids (insect vectors). Disease management is highly challenging due to the wide host range and many insect vectors. (Palukaitis et al., 1992). It is possible to increase yield by using methods to postpone early infection. Use of certified seeds and plants, screening and disinfection of infected mother stock, washing and disinfecting of hands and tools, and planting resistant cultivars are the most effective ways to manage CMV. Vegetables should also be kept out of weedy border areas and planted next to taller border plants like maize, which can serve as a non-susceptible host. (Arogundade et al., 2019). University of Ghana http://ugspace.ug.edu.gh 34 REFERENCES References for chapter one and chapter two are merged as directed by the University of Ghana thesis guidelines. Abdulai, J., Nimoh, F., Darko-Koomson, S., & Kassoh, K. F. S. (2017). Performance of vegetable production and marketing in peri-urban Kumasi, Ghana. Afari-Sefa, V., Asare-Bediako, E., Kenyon, L., & Micah, J. A. (2015). Pesticide use practices and perceptions of vegetable farmers in the cocoa belts of the Ashanti and Western Regions of Ghana. Agriculture Organization. (2004). The State of Food and Agriculture 2003-04 (No. 35). 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