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Iillf .Me Clue to a Speci8llmllllitmty Ofcertain Orglals, Tissues Or Organeues to Die Cocoa Virus

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Iillf .Me Clue to a Speci8llmllllitmty Ofcertain Orglals, Tissues Or Organeues to Die Cocoa Virus UNIVEIlSITY OF CAPE COAST IIIOCHIiMlCAL STUDIES OF RESISTANCE LEVELS OF COCOA VABJBTIES AT COCOA RESEARCH INSTITUTE OF GHANA, TAFO-AKIM TO COCOA SWOLLEN SHOOT VIRUS. CLMS ...0. BY .A(:CESSION NO. 28711 I DANIEL KOJO MANU CIIf''t .QlEro(f FINAL CHeeK \ ~ . I ~.,.... ._._~--- 11IESIS SUBMITTED TO THE DEPARTMENT OF CROP SCIENCE OF THE SCHOOL OF AGRICULTURE, UNIVERSITY OF CAPE COAST IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF MASTER OF PHILOSOPHY DEGREE IN CROP SCIENCE. MARCH, 2006. _ ll ."-iiE. fillIn ClAn .. DECLARATION CANDIDATE'S DECLARATION I bereby declare that this thesis is the result ofmy own original researc~ and that DO part ofit has been presented for another degree in this university orl elsewhere. r Candidate's Signature..~ Date .. ?-::f7.1. ?~ ~~l Name: Daniel Kojo Manu - SUPERVISORS' DECLARATION We hereby declare that the preparation and presentation of the thesis were supervised in accordance with the guidelines on supervision of thesis laid dowil by the University ofCape Coast. ~y(~ Prin,'poI Sup="",', S''''''ore.. .f1f.'.. .'" O'k.... Name: Mr. Yaw Opoku-Asiama C~S"P='~'" S,,,,,m,, .. ~,,)c:h.1J:67'. Name: Mr. Patrick Constantine Aculey ii ~·'i • ,. ABSTRACf BiodIemic:alllvdies were carried out on ten cocoa types to find out their 4iffeJelit levels of resistance to Cocoa Swollen Shoot Virus. The beans were iDoc:ula&ed via viruliferous nymphs ofmealy bugs, and observed after planting for 3 months. Patches of barks of severe symptom bearing cocoa types were also tested through grafting on healthy Amelonado plants and observed for 3 months. Significant differences were observed in the cocoa types in terms of symptoms appearance and in severity. PAI50 X NA33, T63/967 X T65/326 and T85/199 X PA7 were observed to be the best resistant to the CSSVD whilst T851799 X T65/238. Amelonado X Amelonado, T851799 X Amelonado, T851799 XT65/326, and T851799 X T79/501 were observed to be poor in resistance. In the investigations, there was weak correlation (0.333) between total polyphenol content and susceptibility of cocoa types to the CSSV, whereas a significant correlation (-0.58) existed between total nitrogen content and susceptibility to the Cocoa Swollen Shoot Virus. PAI50 X NA33 had the highest nitrogen content, and statistically different. There were no differences between T851799 X Amelonado. T63/967 XIMC60 and Amelonado X Amelonado. T851799 X T65/238 had the lowest and different from the rest. From the polyacrylamide gel electrophoresis, protein with molecular weights ranged from 12.0 to 62.6 kDa. A pathogenic related protein of size 23.9 kDa was detected in PAI50 X NA33. T851799 X Amelonado and on Amelonado X Amelonado. Another pathogenic related protein ofsize 46.6 kDa and 23.9 kDa were detected in T851799 X T791501. iii .' ,b,""o;: ACXNOLEDGEMENTS ,, Manypeople haYe c:ontributed to this work from its proposal to the present tbrm but mespecitieity is only fiIir. Mr. Yaw Opoku-Asiama, my Principal Supervisor, having taken me to Cocoa Res .eb institute of Ghana, Tafo several times for my practical work actually supported me to get the funding and a place for my project work. He also offiaed invaluable, unparalleled and brilliant suggestions and painstakingly read my project write up to make the necessary corrections and insertions, His special saying "we are not joking here" really spurred me on. I wish therefore to acknowledge with great sincerity my profound gratitude to him, My Patrick Constantine Aculey, my supervisor, who also became my teacher taught me some basic principles in biochemistry, laboratory instrumentation and above all found time in spite of his busy schedule to read through my work. I wish to say thank you, "jamjam". The Director, Dr. Michael Roy Appiah and Management of Cocoa Research Institute placed their facilities at my disposal free of charge. They provided me with free accommodation, free access to the laboratories at any time, free medical care, free security, and free recreational facilities, which is very, very rare to find. I therefore thank them for all the things they have done for me, I am especially indebted to Dr. Yaw Adu-Ampumah, the Deputy Director ofCocoa Research Institute and the coordinator ofCommon Fund for Commodity (CFC) projects for sponsoring my project. I wish to emphasis that without his IV .. '':M.... 'MJd1d IIIilItftate seen die light of day. I thank him very ·~tIIIiIi..itl.'liddotimIiDg time to read through my work. " ,:;V' ',' , I wiIlh to tI8It the Heads of Pathology and PhysiologylBiochemistry '.', "~ Pep.dmeDt8 tbr aUowing me to use their laboratories and for their assistance during die period of my work. I wish to thank Dr. Jimmy. F. Takrama, Dr. SIUJIUe1 T. Lowor, Dr. Alex Oppong Dwapanyin, Mr. Henry Ozahini-Obiatey, Mr. Michael Asuah; Mr. Joseph Nawo Daafo; Mr. Francis Awuku Osae, Mr. Mawuli Adoblanui, Mr. George Kwaku Oanchie; Ms. Gifty Asior Oede, Mr.Denjamin Owusu, Ms. Ophelia Kafui Torsah, Mr. Samuel Ackom, and Mr. Peter Adonteng Doamah-Dankyi, all ofCRIG for their support Ms. Korameng Ellen Oye and Mr. Gyamfi Larbi Samue~ both of CRIG library deserve my sincere thanks for getting me all the reference materials from the library and for directing me to the appropriate shelves. I acknowledge with deep appreciation the financial and moral support of my wife, Livesy. In mentioning names one may overlook the obvious so I thank all those whose contribution have not been acknowledged. Any credit is theirs. v ~/'1)t u· (CATION ·'·'.1l.a••'··._V8...· ltt GfO':'.....,~iVesy,My Children, Daniel and Persis. Q t· , ,I VI TABLE 01' CONTENTS PAGE (JUfU_rs tmBPAGE ii DECLARATION iii ABSTRACT IV ACKNOWLODGEMENT vi DEDICATION vii I TABlE OF CONTENTS .I, XI Ii,,~ LIST OF TABlES , XII LIST OF FIGURES xiii LIST OF PLATES xv LIST OF APPENDICES CHAPTER ONE INTRODUCTION 1 CHAPTERlWO LITERATURE REVIEW 7 Viruses ofcocoa 7 Cocoa Swollen Shoot Virus 7 Cocoa Swollen Shoot Virus strains 8 Physical and Biochemical Properties ofCocoa Swollen Shoot Virus 9 Mode oftransmission ofCocoa Swollen Virus in nature 9 Cocoa Swollen Shoot Virus and vector relationship 12 The use ofgraft transmission 14 Cytopathology 14 Diagnostic cocoa species and symptoms 14 vii 15 16 17 Svd. 19 Em=ct ufOocoa Swollen Shoot VINS on cocoa plant 20 Cocoa Swollen Shoot resistant progenies 24 ClIJbohycIrate content in infested plant 25 Mineral content in infested plant 25 The effect ofvirus on polyphenol content r I','.' I!' 26 CHAPTER THREE MATERIALS AND GENERAL METHODS IIIi! !, 29 i! Cocoa varieties Strains ofvirus used for the inoculation ofcocoa types 31 Experiment 1: Assessment ofcocoa types for Cocoa Swollen Shoot Virus resistance I tolerance through symptoms expressions. 31 Preparation ofCocoa Swollen Shoot Virus source plants 31 Inoculations 33 Inoculation ofcocoa types with Cocoa Swollen Shoot Virus strains using mealy bugs vector 33 The use ofpatcb-grafting in Cocoa Swollen Shoot Virus 1A strain inoculation 36 Soil media preparation, planting and care ofseedlings 36 Cocoa Swollen Shoot symptoms scoring and recording 37 Experimental Design 37 Experiment 2: Determination oftotal phenolics in Cocoa Swollen VIII .' 38 "'¥illutilfldel~ ." I Ueu'. 38 D m r IGfe-- /1•• by SoKhIet Extraction 39 TCIlII .....pMaol oonMat ....millllCion (tp) by Folin-Lowry Method 40 Blpsia:. 3: DdaildtPJerion ofuitroeen in leaves ofcocoa types 40 DigeslioD of1IIDP1es ofcocoa leaves E1ip&ia:ent 4: Dlta'minatioo ofmoleculer weight ofproteins syid t ._in infected and uninfected coooa varieties by ~ 42 Ii polyacrylamide gel electrophoresis (pAGE) \''., 43 H Extraction ofproteins samples ! ' 43 The sepII'Iling gel preparation 45 The stlddng gel preparation 48 Proteins samples preparation and loading Running gel and processing ofprotein samples 49 Disassembling and staining ofprotein gel SO Data analysis ofprotein gel electrophoresis SO CIIAP1'ER FOUR RESULTS 52 General observation ofstudies on the Cocoa Swollen Shoot Virus Disease symptoms Mean Number ofCocoa Swollen Shoot Virus Disease symptoms 57 IDcicIeoce ofCocoa Swollen Shoot Virus Disease in seedlings inoaa1ated with mealy bugs and by Patch-grafting 58 TotII poIyphenolics contents ofcocoa types 62 N"1trogen content ofcocoa \eaves 65 IX · ..., "',, ", 1r.IRt, • .,..tJf,eee- Swollen Shoot Vuus ,.·,u,,-· "<". ,,-,-'" I) { 69 _'1111'1' 5......­vr-- 80 ~.\"""" .,,~.CONCLUSIONAND UCOMMENDAll0NS 85 88 104 , I x .. lid' PAGE ~~'~n ....1: ....,.....ni. iSUudd OIl cocoa in Ghana II t'tIIW2: ....lIn' • .r......iItieI used for the experiments 30 Table J: .,..up ofCocoa Swollen Shoot Vuus Disease and their score 37 Tillie 4: ........weill' oflllndanls 51 TtIlrle 5: Number ofCocoa Swollen Shoot symptoms 58 TIbIe 6: MelD total poIypbenol content ofcocoa types 63 Tible 7: Total pheaolics content ofcocoa variety/CSSV strain interaction 64 Table 8: MelD nitlogen content ofcocoa leaves 66 Table 9: Man nitrogen content ofcocoa typelCSSV strain interaction 67 Table 10: Mo\ealI... weights ofproteins (ltDa) ofcocoa types. 79 LIST OF FIGURES PAGE 1IG1JB Figure 1: The standard curve from which the concentration of 40 absorbance was calculated 45 Figure 2: A single gel cassette properly assembled 46 Figure 3: Empty casting stands showing stacked cassettes Figure 4: A complete bio rad Protean II casting stand ready for casting 47 gels and loading ofsamples j 47 I: Figure 5: A set up showing the casting ofgel I ,I i Figure 6: A set up showing the creation ofwells for loading 48 ofprotein samples Figure 7: Histogram ofincidence ofCocoa Swollen Shoot Virus disease in mealy bug inoculated seedlings
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