ACKNOWLEDGEMENTS

All thanks and praises are for "Allah Almighty" who bestowed me with His countless blessings and enabled me to seek knowledge and invigorate me for this work. I am mortified from head to toe for my deeds before His unlimited bounties bestowed upon me. At this stage of submission of my dissertation of the highest degree of my study career, I am unable to express my feelings of thankfulness for "Allah almighty". No words to explain the love and gratitude for Holy Prophet Hazrat Muhammad (Peace Be Upon Him), forever a source of guidance and hope, and whose love is the only key to success in this and next world.

This thesis arose as a result of research that has been conducted in parts, one part at Lahore College for Women University (LCWU) and other at National Institute for Biotechnology and Genetic Engineering (NIBG, Faisalabad).

Special gratitude is due to Higher Education Commission Pakistan for providing financial support to my studies. I offer my special thanks to Dr. Uzma Quraishi, Vice Chancellor of LCWU, Prof. Dr. Bushra Khan, Incharge Faculty of Natural Sciences for providing me support and all available facilities.

It is my utmost pleasure to extend my heartiest gratitude to my supervisor Prof. Dr. Farah Khan Chairperson, Department of Botany, LCWU. I want to thank for her personal interest, valuable guidance, ample support, suggestion and help during research work and thesis writing. I shall always be thankful to her for her affectionate behavior towards me. She has always been a source of inspiration, guidance and encouragement for me. Beyond than the duties as a supervisor, her involvement on all life matters make Dr. Farah Khan more privileged, honored and a true spiritual parent in the eyes of her students. I wish to express my deep sense of gratitude for my co- supervisor Dr. Shahid Mansoor (Director, NIBGE) who has made a great contribution for the successful completion of this work. His skilful advices, sincere cooperation and learned guidance enabled me to complete this work. I learned a lot from him and he has been very kind teacher.

I am deeply indebted to Dr. Imran Imin (Senior Scientists, NIBGE) for his cooperation, sincere suggestions, encouragement, for his 24 hours available help, value able advices in science discussion and help in learning Molecular Biology techniques. I have respectful appreciation for Dr. Muhammad Saeed and Aliyu Ibrahim Dabai for his all time available help and sincere cooperation.

My cordial thanks to all my colleagues, lab fellows particularly Atiq-ur-Rehman (big brother), Dr. Zafar Iqbal, Huma Mumtaz, Maryam Umair, Hira Kamal, Shaista Javed, Aqsa, Ali Tahir, Hamza, Ghulam Rasool, Ishtiaq Hassan, Qamar Abbas, Zayab Khalid, Kiran Zahid, Shazia Kanwal, Sumaira Iqbal, friends Hannan Mukhtar, Maryam Fatima, Sadaf, Saima Zafar and especially the supporting staff in LCWU and NIBGE for their available help and sincere cooperation. I am also thankful to all, who directly or indirectly helped me in my work.

I have no words to express my feelings for the everlasting prayers of my mother Mrs. Muhammad Siddique, my father Mr. Muhammad Siddique and parents in law, who encouraged me in every moment of my life. I am very thankful to my dear husband Shahid Waheed Qamar, my beloved daughters Maheen Fatima and Hareem Fatima for their cooperation, support and to tolerate my unavailability particularly when they needed me more. My brother Kamran Siddique, sisters Ghazala Siddique, Saima Siddique, Mahwish Siddique and Zaib-Un-Nisa Siddique and all family members Taimoor Khalid, Salahud-Ud-Din, my brothers and sisters in law, for their affection, care and devotion during my whole study. I am also thankful to Adeel Ahmad, M. Munir, Sardar Ali Sajid and M. Amin for their all time availability to help me in accomplishment of my tasks. May God bless them all with everlasting happiness.

RIFFAT SIDDIQUE

CONTENTS

Title Page No. List of Tables i List of Figures iii List of Text Figures iv List of Abbreviations vi Abstract xii Chapter 1: Introduction 01 Chapter 2: Review of Literature 24 Chapter 3: Materials and Methods 64 3.1 Materials 64 3.1.1 Collection of infected samples 64 3.1.2 Classification of Tomato 64 3.1.3 Nutritional value of Tomato 65 3.1.4 Health benefits of tomatoes 65 3.1.4.1 Heart health 65 3.1.4.2 Prevention from cancer 65 3.1.4.3 Skin health and vision improvement 66 3.1.4.4 Pregnancy and depression 66 3.1.4.5 Reduces urinary tract infections 66 3.1.4.6 Counters the consequences of cigarette smoke 66 3.1.4.7 Good for hair and reduce migraine 66 3.1.4.8 Enhance immunity 66 3.1.4.9 Strengthen bone, neutralize acidosis and wound repair 67 3.1.4.10 Antipathy for tomato 67 3.1.4.11 Economic importance of tomato 67 3.2 Methods 68 3.2.1 Extraction of DNA 68 3.2.2 Quantification of DNA 68 3.2.3 Amplification of DNA using polymerase chain reaction (PCR)69 3.2.4 DNA amplification via rolling circle amplification (RCA) 69 3.2.5 Gel electrophoresis 70 3.2.6 Gel extraction and purification of PCR products 70 3.2.7 DNA purification by phenol-chloroform extraction 71 3.2.8 Ligation and cloning of amplified DNA 71 3.2.9 Preparation of competent Escherichia coli cells by heat shock 71 3.2.9.1 Transformation of ligated product in competent cells of E. coli 72 3.2.9.2 Isolation of plasmid DNA 72 3.2.9.3 Restriction by restriction enzymes (endonucleases) 73 3.2.9.4 Preservation of clones (glycerol stocks and plasmids)74 3.2.9.5 Renewal of clones74 3.2.10 Preparation of electro-competent cells of Agrobacterium 74

3.2.10.1 Transformation by using Agrobacterium tumefaciens 74 3.2.10.2 Transient analysis via Agrobacterium tumefaciens 75 3.2.11 Plants growth conditions 75 3.2.11.1 Transfer of plants into individual pots 76 3.2.11.2 Observations of plants 76 3.2.12 Detection of ToLCNDV in resistant and susceptible cultivars 76 3.2.13 DIG labeled probe synthesis 77 3.2.14 Southern hybridization 77 3.2.14.1 Dot-Blot hybridization 79 3.2.14.2 Quantification of bands on blots 79 3.2.15 Extraction of plant samples in n-hexane 82 3.2.16 Gas chromatography mass spectrometry 82 3.2.17 Photography and computer graphics 83 3.2.18 Sequencing and sub sequencing 83 3.2.19 Phylogenetic analysis 83 3.2.20 Sequence demarcation tool (SDT) analysis 83 3.2.21 Percentage identity and divergence 84 3.2.22 Recombination detection program (RDP) 84 Chapter 4: Results 85 4.1 Characterization of DNA-A component of begomoviruses 85 4.1.1 Observation of symptoms severity in tomato samples 85 4.1.2 Amplification of begomoviruses infecting tomato 85 4.1.3 Molecular characterization of DNA-A components of Begomoviruses 86 4.1.4 Open reading frames (ORFs) analysis 86 4.1.5 Percentage homologies with database sequences 90 4.2 Species Demarcation Tool (SDT) analysis 92 4.3 Identification of new strain of PaLCuV (PaLCuV-LHR strain 98 4.4 Phylogenetic analysis of monopartite and bipartite DNA-A components of begomoviruses 98 4.5 Phylogenetic analyses of full length sequences and proteins of present isolates 102 4.5.1 Phylogenetic comparison of full length sequences and Rep proteins of studied species 102 4.5.2 Phylogenetic analysis of AC2 encoded TrAP 102 4.5.3 Phylogenetic tree of AC3 encoded REn protein 103 4.5.4 Phylogenetic analysis of AC4 protein 103 4.5.5 Phylogenetic analysis of AV1 encoded coat protein (CP) 109 4.5.6 Phylogenetic analysis of AV2 encoded pre CP 109 4.6 Phylogeography 112 4.7 Recombination (inter and intraspecific) analysis among current isolates 112 4.8 Molecular characterization of DNA-B of begomoviruses 122 4.8.1 Amplification and cloning of DNA-B components of Begomoviruses 122 4.8.2 Molecular characterization and ORFs analysis of DNA-B

components of begomoviruses 122 4.9 Phylogenetic Analysis of DNA-B components of begomoviruses 123 4.9.1 Comparison of isolates based on full length sequences and BV1 encoded NSP 124 4.9.2 Comparison of phylogenetic tree for full length genome and BC1 encoded MP 132 4.10 Recombination analysis of DNA-B 132 4.11 Characterization of Betasatellites 138 4.11.1 Amplification of betasatellites 138 4.11.2 Molecular characterization of betasatellites 138 4.11.3 Papaya leaf curl betasatellites (PaLCuB) 141 4.11.4 Cotton leaf curl Multan betasatellites (CLCuMuB) 141 4.12 Analysis of conserve regions of betasatellites 141 4.12.1 βC1 protein 141 4.12.2 A-Rich region 142 4.12.3 Satellite conserve region (SCR) of betasatellites 142 4.13 Phylogenetic dandogram 142 4.13.1 Phylogenetic analysis of full length nucleotides sequence and βC1 protein 143 4.14 Recombination 143 4.15 Characterization of Alphasatellites 150 4.15.1 Amplification and cloning of alphasatellites 150 4.15.2 Molecular characterization of alphasatellites 150 4.15.3 Okra leaf curl alphasatellite (OLCuA) 152 4.15.4 Tomato leaf curl Pakistan alphasatellite (ToLCPKA) 152 4.16 Conserve regions of alphasatellites 152 4.16.1 Rep of all isolates 152 4.16.2 A-rich region 152 4.17 Phylogenetic analysis 153 4.17.1 Phylogenetic analysis based on full length nucleotide sequences and Rep proteins 156 4.18 Recombination among alphasatellites 156 4.19 Pathogenicity developed in Ty resistant cultivars on ToLCNDV Inoculation 160 4.20 Detection of ToLCNDV in inoculated cultivars 164 4.20.1 Confirmation of ToLCNDV in systemic leaves of inoculated plants 164 4.20.2 Southern Blot Hybridization 167 4.22 Metabolites (volatile compounds) profiling of ToLCNDV inoculated resistant and susceptible Ty tomato cultivars by GCMS 170 4.22.1 Healthy S. lycopersicum plant 170 4.23 Tolerant or resistant cultivars 170 4.23.1 Cultivar R6 of S. lycopersicum 170

4.23.2 Cultivar R10 of S. lycopersicum 171 4.23.3 Cultivar R11 of S. lycopersicum 171 4.23.4 Cultivar R5 of S. lycopersicum 171 4.23.5 Cultivar R13 of S. lycopersicum 172 4.24 Mild symptomatic cultivars 172 4.24.1 Cultivar R2 of S. lycopersicum 172 4.24.2 Cultivar R3 of S. lycopersicum 172 4.24.3 Cultivar R9 of S. lycopersicum 173 4.25 Moderate symptomatic cultivar 173 4.25.1 Cultivar R12 of S. lycopersicum 173 4.26 Severe symptomatic cultivars 174 4.26.1 Cultivar R7 of S. lycopersicum 174 4.26.2 Cultivar R8 of S. lycopersicum 174 4.27 Very severe symptomatic cultivars 174 4.27.1 R14 Cultivar of S. lycopersicum 174 4.27.2 Cultivar R15 of S. lycopersicum 175 4.27.3 Nagina variety of S. lycopersicum 175 Chapter 5: Discussion 195 References 213 Plagiarism Report List of Publications and Reprints

i

LIST OF TABLES

Table No. Title Page No. Degenerate back to back primer sets used for amplification of 3.1 begomoviruses, associated satellites and for probe synthesis with 80 respective PCR profiles. Coordinates of Open Reading Frames (ORFs) i.e. Virion Sense (AV1 & AV2) and Complementary Sense (AC1, AC2, AC3 & AC4), 4.1 89 accession numbers and origin coordinates of the DNA-A components of begomoviruses. Percentage identities for current isolates of each species and homology 4.2 sharing isolates, which were retrieved from National Center for 91 Biotechnology Information (NCBI) database. Comparison of complementary and virion sense ORFs among RS92 4.3 101 and three other isolates of PaLCuV. Recombination analysis of complete nucleotide sequences of DNA-A 4.4 120 components of begomoviruses by RDP 4.2 program Coordinates of BCI and BVI Open Reading Frames (ORFs) of DNA-B 4.5 component of bipartite begomoviruses, their accession numbers and 126 origin (Longitude & Latitude) coordinates. BLAST results of current DNA-B components of begomoviruses and 4.6 128 their percentage homology with database sequences. Recombination analyses of complete nucleotide sequences of DNA-B 4.7 137 components of begomoviruses by RDP4.2 program. Coordinates of βC1 protein of betasatellites, genome length, samples 4.8 139 origin and GeneBank accession numbers. Blast results and maximum homology values, shared by present isolates 4.9 140 with database sequences. Recombination analysis done for complete nucleotide sequence of 4.10 149 betasatellites by RDP4.2 program. Coordinates of Rep protein, genome size, samples origin and 4.11 151 GeneBank accession numbers of alphasatellites. BLAST results and maximum homology percentages, shared by present 4.12 151 isolates with database sequences. Recombination analysis done for complete nucleotide sequences of 4.13 159 alphasatellites by RDP4.2 program. Ty resistant cultivars with distribution codes and various Ty genes 4.14 161 combination. Percentages of infectivity and types of symptoms caused by ToLCNDV 4.15 166 in Ty resistant and Nagina plants. Volatile compounds analyzed by GCMS from the n-hexane extract of 4.16 176 leaves and stem of healthy S. lycopersicum plant. Volatile compounds isolated from the n-hexane extract of leaves and 4.17 stem of S. lycopersicum (R6 cultivar which showed complete resistance 177

after inoculation with ToLCNDV) by GCMS. Table continue page turn over ii

Volatile compounds isolated from the n-hexane extract of leaves and 4.18 stem of S. lycopersicum (R10 cultivar which showed complete 179 resistance after inoculation with ToLCNDV) by GCMS. Volatile compounds isolated from the n-hexane extract of leaves and 4.19 stem of S. lycopersicum (R11 cultivar which showed complete 180 resistance after inoculation with ToLCNDV) by GCMS. Volatile compounds isolated from the n-hexane extract of leaves and 4.20 stem of S. lycopersicum (R5 cultivar which showed complete resistance 181 after ToLCNDV inoculation) by GCMS. Volatile compounds isolated from the n-hexane extract of S. 4.21 182 lycopersicum (R13 cultivar after ToLCNDV inoculation) by GCMS. Volatile compounds isolated from the n-hexane extract of leaves and 4.22 stem of S. lycopersicum (R2 cultivar which showed mild symptoms 183 after ToLCNDV inoculation) by GCMS. Volatile compounds isolated from the n-hexane extract of leaves and 4.23 stem of S. lycopersicum (R3 cultivar which showed mild symptoms 184 after ToLCNDV inoculation) by GCMS. Volatile compounds isolated from the n-hexane extract of leaves and 4.24 stem of S. lycopersicum (R9 cultivar which showed mild symptoms 185 after ToLCNDV inoculation) by GCMS. Volatile compounds isolated from the n-hexane extract of leaves and 4.25 stem of S. lycopersicum (R12 cultivar which showed moderate 186 symptoms after ToLCNDV inoculation) by GCMS. Volatile compounds isolated from the n-hexane extract of leaves and 4.26 stem of S. lycopersicum (R7 cultivar which showed severe symptoms 187 after ToLCNDV inoculation) by GCMS. Volatile compounds isolated from the n-hexane extract of leaves and 4.27 stem of S. lycopersicum (R8 cultivar which showed severe symptoms 188 after ToLCNDV inoculation) by GCMS. Volatile compounds isolated from the n-hexane extract of leaves and 4.28 stem of S. lycopersicum (R14 cultivar which showed very severe 190 symptoms after ToLCNDV inoculation) by GCMS. Volatile compounds isolated from the n-hexane extract of leaves and 4. 29 stem of S. lycopersicum (R15 cultivar which showed very severe 191 symptoms after ToLCNDV inoculation) by GCMS. Volatile compounds isolated from the n-hexane extract of leaves and 4.30 stem of S. lycopersicum (Nagina variety which showed very severe 193 symptoms after ToLCNDV inoculation) by GCMS.

iii

LIST OF FIGURES

Figure No. Title Page No. 3.1 Southern hybridization assembly. 81 4.1 Symptomatic and healthy tomato plants 87 4.2 Gel picture exhibiting PCR amplified fragment of 2.8 kb 88 sized begomoviruses. 4.3 Gel picture indicating restriction pattern for full length 88 begomoviruses clones. 4.4 Phylogeography 113 4.5 Gel picture exhibiting PCR amplified fragment of ~2.7 kb 125 sized begomoviruses 4.6 Restriction of DNA-B components of bipartite 125 begomoviruses 4.7 Amplification and restriction of betasatellites. 139 4.8 Alphasatellites amplification and restriction 151 4.9 Pathogenicity of ToLCNDV in Ty resistant cultivars and 163 Nagina variety. 4.10 Detection of ToLCNDV DNA-A in systemic leaves by PCR 165 in inoculated tomato plants. 4.11 Detection of ToLCNDV DNA-B in systemic leaves by PCR 165 in inoculated tomato plants. 4.12 Southern hybridization of DNA-A component of 168 ToLCNDV in Ty inoculated plants. 4.13 Southern blot detection of DNA-B component of 169 ToLCNDV in inoculated Ty cultivars.

iv

LIST OF TEXT FIGURES

Figure No. Title Page No. 4.1 SDT analyses of present and already reported ToLCV 93 isolates. 4.2 SDT analysis for ToLCKeV isolates 94 4.3 SDT analysis for PaLCuV isolates 95 4.4 SDT analysis of ToLCPalV isolates 96 4.5 SDT analysis performed for ToLCNDV isolates 97 4.6 Phylogenetic analysis of complete nucleotide sequences of 100-101 DNA-A components of begomoviruses 4.7 Phylogenetic analysis of Rep 105 4.8 Phylogenetic analysis of TrAP 106 4.9 Multiple alignments of TrAP of PaLCuV (RS33, RS58, 107 RS60 and RS92) and ToLCV (RS96 and RS97) isolates. 4.10 Comparison of REn protein and full length sequences 107 4.11 Alignment of REn protein of all ToLCV 108 4.12 Phylogenetic dendogram of AC4 sequences 108 4.13 Phylogenetic dendogram of AV1 (CP) sequences 110 4.14 Comparison of AV2 protein and full length sequence of all 111 isolates. 4.15 RDP analysis of RS 157 (ToLCKeV) isolate 114 4.16 RDP analysis performed for RS2 ToLCKeV isolate 114 4.17 RDP analysis performed for RS 92 PaLCuV isolate 117 4.18 RDP analysis performed for RS 33 PaLCuV isolate 117 4.19 RDP analysis performed for RS 82 ToLCV isolate. 118 4.20 Recombination analysis performed by RDP4.2. 119 4.21 Phylogenetic analysis of DNA-B components 129-130 4.22 Comparison of isolates, based on full length sequences and 131 BV1 proteins 4.23 Comparison of isolates based on full length nucleotide 135 sequences and BC1 proteins 4.24 RDP analysis performed for RS156 ToLCNDV DNA-B 136 isolate 4.25 Recombination analysis performed by RDP4.2 for DNA-B 136 components of begomoviruses 4.26 Multiple alignment of βC1 of CLCuMuB and PaLCuB 144 isolates 4.27 A-rich region in PaLCuB and CLCuMuB 145 4.28 SCR region of PaLCuB and CLCuMuB 145 4.29 Phylogenetic tree of betasatellites 146 4.30 Comparison of βC1 and whole genome phylogeny of all 147 isolates v

4.31 RDP analysis of RS 107 PaLCuB isolate 148 4.32 RDP analysis of RS163-25PaLCuB isolate. 148 4.33 multiple alignments of Rep of OLCuA and ToLCPKA 154 4.34 A-rich region in ToLCPKA and OLCuA 154 4.35 Phylogenetic analyses of Alphasatellites 155 4.36 Comparison of Rep proteins and full length sequences of 157 Alphasatellites 4.37 RDP analysis of RS 5-ToLCPKA isolate 158 4.38 RDP analysis of RS69-3 (OLCuA) isolate 158 4.39 Symptoms severity shown by Ty cultivars after ToLCNDV 162 inoculation 4.40 Gradual increase in symptomatic plants number after 162 ToLCNDV inoculation

vi

ABBREVIATIONS

µg microgram ml milliliter µl microliter µM micromolar NBT 4-nitro blue tetrazolium BCIPT 5-bromro-4-chloro-3-indolyl-phosphate AVRDC Asian Vegetable Research and Development Center AMP Adenosine mono phosphate ATP Adenosine tri phosphate BLAST Basic local alignment search tool

Ca(NO3)2.4H2O calcium nitrate tetrahydrate

CaCl2 calcium chloride CTAB cetyltrimethylammonium bromide CP coat protein CR common region CSR complementary strand replication cccDNA covalently closed circular DNA

CuSO4.5H2O copper sulfate pentahydrate DNA deoxyribonucleic acid dNTPs deoxyribonucleotide triphosphate ds(RF) double stranded replicative form dsDNA double-stranded DNA dsRNA double-stranded RNA EDTA ethylene diamine tetraacetic acid FID flame ionization detector GC-TOF-MS gas chromatography-time-of-flight-mass spectrometry GC-MS gas-chromatography-mass-spectrometry GFP green fluorescent protein HR hypersensitive response IR intergenic region ICTV International Committee on Taxonomy of viruses

KH2PO4 potassium dihydrogen phosphate vii

KOH potassium hydroxide kV kilo Volt kDa kilo Dalton LIR large intergenic region SIR small intergenic region LB Lauria Bertani NaCl sodium chloride MeSA methyl salicylate

MgCl2 magnesium chloride mM millimolar MM moneymakers MP movement protein NaOH sodium hydroxide LC-MS liquid chromatography-mass-spectrometry ssRNA single-stranded RNA NIG NSP interacting GTPase NIK NSP-interacting kinase NES nuclear export signal NPC nuclear pore complex NSP nuclear shuttle protein NSP nuclear shuttle protein ORFs open reading frames PD plasmodesmata PCR polymerase chain reaction ssRNA positive sense single-stranded RNA PTGS post transcriptional gene silencing PreCP pre coat protein PCNA proliferating cell nuclear antigen PERK proline-rich extension-like receptor protein kinase PKC protein kinase C RHP random haxamer primers RDR recombination detection program REn replication enhancer protein viii

RFC replication factor C RFLP restriction fragment length polymorphism RT retention time pRBR retinoblastoma-related protein RNAi RNA interference RCR Rolling circle replication SAM S-adenosyl-methionine SCR Satellite conserve region SDT Sequence Demarcation Tool siRNAs short interfering RNAs ssDNA single-stranded DNA SSC standard sodium citrate SDW sterile distilled water SNF1 sucrose nonfermenting-1 TAE tris-acetate EDTA SEL size exclusion limit ToLCD tomato leaf curl disease TYLCD tomato yellow leaf curl disease TrAP transcriptional activator protein

ix

VIRUSES AND SATELLITES Abutilon mosaic virus (AbMV) African cassava mosaic virus (ACMV) Ageratum enation virus (AEV) Ageratum yellow vein alphasatellite (AYVA) Ageratum yellow vein virus (AYVV) Honey suckle yellow vein virus (HSYVV) Barley mild mosaic virus (BaMMV) Barley yellow mosaic virus (BaYMV) Bean dwarf mosaic virus (BDMV) Bean golden mosaic virus (BGMV) Bean golden yellow mosaic virus (BGYMV) Bean yellow dwarf virus (BeYDV) Beat curly top virus (BCTV) Beet curly top Iran virus (BCTIV) Beet severe curly top virus (BSCTV) Bendhi yellow vein mosaic alphasatellite (BYVMA) Bhindi yellow vein mosaic virus (BYVMV) Cauliflower mosaic virus (CaMV) Chilli leaf curl betasatellites (ChLCuB) Chilli leaf curl India virus (ChiLCINV) Chloris striate mosaic virus (CSMV) Cleome leaf crumple virus (ClLCRV) Cotton leaf crumple virus (CLCrV) Cotton leaf curl Burewala alphasatellite (CLCuBuA) Cotton leaf curl Khokhran virus-Burewala (CLCuKoV-Bu) Cotton leaf curl Multan alphasatellite (CLCuMuA) Cotton leaf curl Multan betasatellite (CLCuMuB) Cotton leaf curl Rajasthan virus (CLCuRV) Cotton leaf curl-1alphasatellite (CLCu-1A) Cucumber mosaic virus (CMV) Duranta leaf curl alphasatellite (DuLCA) East African cassava mosaic virus (EACMV) x

East African cassava mosaic Zanzibaar virus (EACMZV) Eragrostis curvula streak virus (ECSV) Euphorbia mosaic virus (EuMV) Gossypium darwinii symptomless alphasatellites (GDarSLA) Guar leaf curl alphasatellite (GuLCuA) Hibiscus leaf curl alphasatellite (HLCuA) Hollyhook yellow vein symptoms less alphasatellite (HoYVSLA) Honeysuckle yellow vein mosaic virus (HYVMV) Horseradish curly top virus (HCTV) Indian cassava mosaic virus (ICMV) Macroptilium yellow spot virus (MaYSC) Maize streak virus (MSV), Melon chlorotic mosaic virus (MeCMV) Mungbeen yellow mosaic India virus (MYMIV) Mungbeen yellow mosaic virus (MYMV) Okra enation leaf curl virus (OELCuV) Papaya leaf curl betasatellites (PaLCuB) Papaya leaf curl virus (PaLCuV) Papaya leaf curl virus-Lucknow ( PaLCuV-Luc) Potato virus X (PVX) Radish leaf curl virus (RaLCuV) Rice tungro bacilliform virus (RTBV) Sida golden mosaic virus (SiGMV) Soybean mosaic virus (SMV) Spinach curly top Arizona virus (SCTAV) Spinach severe curly top virus (SSCTV) Spinach severe curly top virus (SSCTV) Squash leaf curl virus (SqLCV) Sri Lankan cassava mosaic virus (SLCMV) Sweet potato leaf curl Sichuan virus1 ( SPLCSiV -1) Sweet potato leaf curl virus (SPLCV -US) Tobacco curly shoot virus (TbCSV) Tobacco leaf curl betasatellite (TbLCB) Tobacco leaf curl Japan virus (TbLCJV) xi

Tobacco mosaic virus (TMV) Tobacco necrosis satellite virus (TNSV) Tobacco ringspot virus (TRS V) Tobacco yellow crinkle virus (TbYCV) Tobacco yellow dwarf virus (ToYDV) Tobacco yellow rugose virus (TbYRV) Tomato golden mosaic virus (TGMV) Tomato leaf curl Bangalore Virus (ToLCBaV) Tomato leaf curl betasatellites (ToLCuB) Tomato leaf curl Gujrat virus (ToLCGV) Tomato leaf curl Joydebpur virus (ToLCJV) Tomato leaf curl Kerala virus (ToLCKeV) Tomato leaf curl New Delhi virus (ToLCNDV) Tomato leaf curl Palampur virus (ToLCPalV) Tomato leaf curl Patna virus (ToLCPatV) Tomato leaf curl Pune virus (ToLCPuV) Tomato leaf curl Rajasthan virus (ToLCRaV) Tomato leaf curl virus (ToLCV) Tomato leaf curl Yunnan virus (TLCYnV) Tomato leaf deformation virus (ToLDeFV) Tomato mosaic virus (ToMV) Tomato mottle virus (ToMoV) Tomato pseudo curly top virus (TSCTV) Tomato pseudo-curly top virus (TPCTV) Tomato ring spotted virus (TRSV) Tomato spotted wilt virus (TSWV) Tomato yellow leaf curl China alphasatellites (TYLCCNA) Tomato yellow leaf curl China virus (TYLCCNV) Tomato yellow leaf curl Sardinia virus (TYLCSV) Tomato yellow top virus (TYTV) Turnip curly top virus (TCTV) Vernonia yellow vein Fujian alphasatellite (VYVFA) Wheat dwarf virus (WDV) xii

ABSTRACT

Tomato, an economically important crop is heavily affected by Tomato Leaf Curl Disease (ToLCD) and Tomato Yellow Leaf Curl Disease (ToYLCD), caused by whitefly transmitting geminiviruses (family Geminiviridae; genus Begomovirus). Begomoviruses containing single stranded DNA are bipartite with two genomic segments (DNA-A and DNA-B), mostly found in both areas of biospheres i.e. the New World (NW) and Old World (OW), but thought to be originated from OW viruses. Monopartite viruses (having a single genomic component equivalent to the DNA-A of bipartite begomoviruses) are mostly restricted to the OW, usually associated with alphasatellite or betasatellite. Betasatellites are dependent on DNA-A for encapsidation, replication and are vital for infectivity of several host plants as well as encode for a pathogenicity determinant the βC1 protein. Alphasatellites are self- replicating and their function in begomoviruses infections is unclear. Begomoviruses among geminiviruses cause great economic losses by infecting various plants. Cultivated Tomato is considered the most appropriate host for the evolution and emergence of monopartite and bipartite begomoviruses as it is widespread and infected by highest number of begomoviruses than any other species. Diversity of begomoviruses associated with recent disease severity of Tomato in various regions of the Pakistan was evaluated during this study. Recent incidences and severity of ToLCD was exploited by collected infected plant samples from major Tomato cultivation areas of Punjab (Lahore and Faisalabad) and Khyber Pakhtunkhawa (KP; Swat, Dir and Malakand) during year 2013-2015. Full length 40 DNA-A, 41 DNA-B components, 19 betasatellites and 05 alphasatellites were cloned, sequenced and submitted into the International DNA databases. Phylogenetic analysis, based on complete nucleotide sequences, identified the presence of 05 begomoviruses, 02 betasatellite and 02 alphasatellite species. The identified begomoviruses species were Tomato leaf curl Kerala virus (ToLCKeV), Tomato leaf curl virus (ToLCV), Papaya leaf curl virus (PaLCuV) Tomato leaf curl New Delhi virus (ToLCNDV) and Tomato leaf curl Palampur virus (ToLCPalV). Isolated betasatellites were Papaya Leaf Curl Betasatellite (PaLCuB) and Cotton Leaf Curl Multan Betasatellite (CLCuMuB). Whereas alphasatellites identified were Tomato Leaf Curl Pakistan Alphasatellite (ToLCPKA) and Okra Leaf Curl Alphasatellite (OLCuA). We humbly claim to identify three begomoviruses (ToLCKeV, ToLCV and PaLCuV) of tomato plant for xiii the first time in Pakistan. Among different strategies being followed to control begomoviruses infection, natural resistance produced by Tomato yellow (Ty) genes has been proved useful. However, single Ty gene containing cultivars did not show broad spectrum resistance, in contrast to two or many Ty genes containing pyramided cultivars which were considered more promising under high disease pressure and were evaluated against Tomato leaf curl New Delhi virus (ToLCNDV). Thirteen Ty resistant cultivars developed by pyramiding Ty-1/Ty-3, Ty-2, Ty-3 and Ty-5 resistance genes in various combinations were analyzed. Ty cultivars and the susceptible native Nagina variety were evaluated for resistance by agro inoculation of bipartite ToLCNDV which is prevalent in Pakistan and is infecting many economically important crops. Results from this study showed that R5, R6, R10, R11 & R13 cultivars showed complete, R2, R3 & R9 moderate, R12 mild, R7 & R8 very less while R14, R15 and Nagina cultivars showed no resistance against ToLCNDV. ToLCNDV DNA-A and DNA-B titer in systemic leaves was determined by Southern hybridization which exhibited comparable virus titer to symptoms severity observed in each cultivar. These resistant and susceptible cultivars were further exploited for metabolites identification by GCMS technique. This analysis led to the identification of various compounds, mainly aliphatic hydrocarbons, diterpenes, triterpenes, phenol, alcohols, esters, fatty acids, aldehydes and ketones etc. The illustrated research was conducted for the better understanding of diversity of begomoviruses and associated satellites, resistance evaluation of Ty cultivars followed by their GCMC analysis. A more detailed understanding of begomoviruses disease complex may allow development of better control methods in future.

CHAPTER NO. 1 INTRODUCTION

1

INTRODUCTION

Tomato (Solanum lycopersicum) syn (Lycopersicon esculentum L.) belongs to family Solanaceae which has 90 genera and more than 3000 species. It is an important member of genus Solanum which includes more than 1000 species (Bohs, 2005; Särkinen et al., 2013). The fruit of tomato is edible, berry shaped and usually red in colour. It is one among most important vegetables, grown all around the world because it requires short duration to produce fruits, gives high yield and has a significant economic impact. Tomato represents well balanced and healthy diet as rich source of minerals, essential amino acids, vitamins particularly vitamin B and C, dietary fibers, sugars, iron and phosphorus (Bhowmik et al., 2012). It is consumed in various ways as uncooked fresh form like salad, in cooked form as sauces, soups and integral component of many fish or meat dishes. Tomato fruit is processed in juices, purees, ketchup form, dried and canned products are another form of processed products (Noonari et al., 2015).

Tomato is ranked as second most important vegetable after Potato in global vegetable production (Noonari et al., 2015; Bakht and Khan, 2014).Worldwide almost 163.4 million tons tomatoes are being produced annually (FAOSTAT 2015). Tomato is grown in several parts of Pakistan, since 2001 to 2012 area for tomato crop production has increased from 27.9 to 50 thousand hectares while its yield increased from 268.8 to 476.8 thousand tons (MINFAL 2015). In 2013, area under cultivation has increased more than 58.196 thousand hectares with an estimated production of around 574.052 thousand tons (Noonari et al., 2015).

In Pakistan tomato yield has reduced to great extent due to pathogens and pests as major biological constrains (Kamran et al., 2012). Bacterial pathogens pose a serious threat to many economically important plants all around the world. Solanaceous species e.g. tomato, potato, tobacco and capsicum are rigorously affected by various bacterial diseases (Allen et al., 2005). Commonly found diseases of bacteria in tomato are bacterial wilt caused by Ralstonia solanacearum (Khokhar, 2013).

Fungal diseases include fusarium wilt caused by the Fusarium oxysporum which is responsible for leaves wilting, yellowing and curling at the edge. Plants wilt from one side, leaving other half healthy for a long time. Similarly verticillium wilt caused by Verticillium albo-atrum, is common in cooler climates and symptoms of this disease 2 are similar to fusarium but slower than that. Symptoms include wilting, leaves yellowing, necrosis, vein clearing and stunting. This disease affects other solanaceous crops as well (Fradin and Thomma, 2006).

Nematodes are also important plant pathogens which cause severe economic damages to agricultural vegetables production all around the world (Anwar et al., 2007; Williamson and Hussey, 1996). These parasites during feeding induce 'gall' or 'giant cells' formation on their hostʼs roots. Meloidogyne incognita is ranked as main destructive pathogen of vegetables including tomato (Fourie and Mc Donald, 2000; Shahid et al., 2007.

Tomatoes are infected by various RNA viruses causing mosaic diseases. Several viruses have been reported to infect tomato from Pakistan, as Tomato mosaic virus (ToMV), Potato virus x (PVX), Cucumber mosaic virus (CMV), Tomato ring spotted virus (TRSV), Tomato spotted wilt virus (TSWV) and Tomato yellow top virus (TYTV; Mughal, 1985). ToMV causes mottling, alternate yellow and dark green areas sometime uneven thickenings appear in the form of blisters. ToMV being most common has distributed all over the Pakistan and has gained the status of second biggest threat infecting tomato after Tomato Yellow Leaf Curl Virus (TYLCV; Imran et al., 2013).

Among all viral diseases of tomato, begomoviruses (members of Geminiviridae) caused diseases i.e. Tomato yellow leaf curl disease (TYLCD) and Tomato leaf curl disease (ToLCD) are of immense importance. TYLCD was identified for first time in Israel in 1930 (Cohen and Lapidot, 2007). Latter TYLCD and ToLCD have been studied from different regions including Asia, Europe, South America, Africa, Australia and North America etc. (Moriones and Navas-Castillo, 2000). These are destructive agents not only for tomato and other members of family Solanaceae (pepper, petunia and tobacco) but also for other families like Euphorbiaceae, Cucurbitaceae, Malvaceae and Fabaceae (Seal et al., 2006a). However cultivated tomato is considered most appropriate for evolution and emergence of monopartite and bipartite begomoviruses (Melgarejo et al., 2013).

Word virus is derived from Latin word which means poison. Viruses are contagious microscopic entities composed of DNA or RNA encapsulated in protein coat, by using hostʼs machinery transcribe and translate inside the host and remained 3 functionally inactive outside host as they are unable to capture or accumulate free energy (Hull, 2009; Hull, 2013).

Plant viruses are very prevalent and economically imperative pathogens, nearly all plants grown by human being for food, fiber and feed are variously affected by at least one virus. Viruses of cultivated crops are well studied because of the financial impact of losses caused by them. Even many wild plants are also their host and study of these plant viruses led to the better understanding of viruses in various aspects (Hull, 2009).

A comprehensive list of standards used for the classification of viruses, are based on following 04 properties: Virion properties (shape, size and type of genome), protein properties (genome organization and replication), antigenic and biological properties (transmission, host range and pathogenicity). Modern classification has taken molecular characterization into consideration and has become more authentic and reliable (Van Regenmortel et al., 2000; Fauquet et al., 2005; King et al., 2011).

By considering organization or nature of genome, 06 main types of viruses are defined as; double-stranded DNA (dsDNA), double-stranded RNA (dsRNA), single- stranded DNA (ssDNA), positive sense single-stranded RNA (ssRNA+), negative sense single-stranded RNA (ssRNA-) and reverse-transcribing viruses. Almost more than 90 % of the viruses have RNA while less than 10 % of viruses have DNA genome (Rojas et al., 2005). Viruses taxonomy and nomenclature is now managed by the International Committee on Taxonomy of viruses (ICTV). Different groups of viruses have been developed on the basis of various kinds of genomes and hosts.

Geminiviruses belong to Geminiviridae, which consists of circular ssDNA, derived from ''geminus '' a Latin word called twin as these consist of 02 identical, incomplete T=1 icosahedra in ssDNA containing capsid which is composed of 110 coat protein (CP) subunits arranged to form 22 pentamers in protein capsid (Bottcher et al., 2004). According to three dimensional analysis of Maize streak virus (MSV), which indicates its dimensions 22 x 38 nm and similar dimensions are found for African cassava mosaic virus (ACMV; Bottcher et al., 2004), these viruses contain ssDNA genome consists of one or two components of 2.5-3.0 kb (Harrison et al., 1977; Rybicki et al., 2000; Stanley et al., 2005; Lu et al., 2015). 4

Geminiviridae is classified into seven genera named as Masterivirus, Turncurtovirus, Topocuvirus, Begomovirus, Becurtovirus, Curtovirus and Eragrovirus, based on their host range, genome organization and insect vector (Adams et al., 2014; Varsani et al., 2014a). Geminiviruses infect monocot and dicot plants all around the world and are transmitted by insects from infected to healthy plants (Stanley et al., 2005). These are considered smallest among known viruses with bidirectional transcription mode and are surrounded by overlapping genes. Virion consists of one structural protein named as CP used for encapsidation of ssDNA.

Geminiviruses those are grouped into the genus Masterivirus are consists of monopartite genome (have one genomic molecule), which is of ~2.6-2.8 kb size (Behjatnia et al., 2011). These viruses are responsible for infections both in monocot and dicot plants, transmitted and carried by leaf hoppers and found in old world (OW; Boulton, 2002). Their genome contains 04 open reading frames (ORFs), 02 in virion sense (V1 and V2) and 02 in complementary sense (C1 and C2; Wright et al., 1997; Sahu et al., 2014), large (LIR) and small intergenic region (SIR), regulatory elements containing intergenic regions (IRs; Gao et al., 2004). Complementary sense genes encode 02 kinds of replication associated proteins (Rep and Rep A). AC1 only encodes for Rep A which interact with retinoblastoma-related protein (pRBR) and responsible for cell cycle progression. AC1 and AC2 encode for full length Rep which is responsible for initiation of virion strand replication and its own expression control (Palmer and Rybicki, 1998; Gutierrez, 1999; Gutierrez et al., 2004). V1 encodes for the movement protein (MP) required for inter and intracellular movement of virus while V2 ORF encodes for CP which is used for virus encapsidation, movement and transmission by insects. MSV is well characterized and the representative member of the genus (Willment et al., 2007). Wheat dwarf virus (WDV), Bean yellow dwarf virus (BeYDV) and Tobacco yellow dwarf virus (ToYDV) are the other examples of the genus (Wright et al., 1997). Chickpea chlorotic dwarf virus (CpCDV) has recently been reported from Pakistan (Nahid et al., 2008; Kanakala et al., 2013; Manzoor et al., 2014).

5

Classification of viruses (This figure has been reproduced from http://www.studfiles.ru/preview/4491305/).

Structures of Geminivirus. Electron microscopic images of geminate particles of Maize streak virus (A). Three dimensional reconstructions for the MSV geminate particle of about 38 nm and 22 nm by the electron microscopy data (B). Image A and B were reproduced from (Shepherd et al., 2010).

6

Members of the genus Curtovirus contain ~3.0 kb sized monopartite genome, transmitted by leafhopper (Circulifer tenellus); found in NW and OW, infecting dicotyledonous (~44 families) plants (Hur et al., 2007). Beat curly top virus (BCTV) is well characterized and type member of the genus. According to recent taxonomic revision, curtoviruses has been reclassified the already known viruses into 03 species BCTV, Spinach severe curly top virus (SSCTV) and Horseradish curly top virus (HCTV; Varsani et al., 2014b).

Replication, virion development and gene expression of curtoviruses takes place in the nucleus (Esau, 1977). Curtoviruses have 07 ORFs which are transcribed in a bidirectional fashion from an IR which contains origin of replication and composed of about 450 nucleotides. Three ORFs are extremely conserved virion sense (V1, V2 and V3) while 04 ORFs are highly divergent complementary sense (C1, C2, C3 and C4; Stanley et al., 1986; Stenger, 1994; Klute et al., 1996; Briddon et al., 1998; Baliji et al., 2004; Sahu et al., 2014). V1 encodes CP as well as involved in vector specificity, virion formation and movement. V2 (Pre coat) gene products are involved in movement or replication and V3 gene encodes for MP (Soto and Gilbertson, 2003).

C1 encodes for Rep protein, product of C2 is involved for recovery phenotype in infected hosts, C3 is implicated to form product called replication enhancer protein (REn) that works by the interaction of Rep and host factors. While C4 gene is involved in the synthesis of a protein which works as pathogenicity determinant like C4 gene of monopartite begomoviruses (Stanley and Latham, 1992; Latham et al., 1997).

Genus Topocovirus has Tomato pseudo-curly top virus (TPCTV) the only member, isolated from USA, Florida (NW), has monopartite genome and it causes infections in dicots. It is being transmitted by tree hopper (Micrutalis malleiffera). Its genome contains 06 ORFs, 04 in complementary-sense (Rep, C2, C3 and C4) and 02 in virion- sense (CP and V2; Sahu et al., 2014). As the functions performed by these genes are not investigated, but it is believed that due to the similarity of these genes with other begomoviruses infecting dicots, have alike functions. TPCTV genome analysis indicates such characteristics which are typical for masteriviruses and begomoviruses, due to which topocoviruses are considered natural recombinant of both (Briddon et al., 1996). 7

Genus Eragrovirus contains Eragrostis curvula streak virus (ECSV) which causes disease in Eragrostis curvula grass and produces MSV-like symptoms (Varsani et al., 2009). Its genome is composed of 04 CP, C2, V2 and Rep genes (Sahu et al., 2014). Genes showed similar arrangements as in masteriviruses and possess 02 IRs. Virion- sense genes showed more sequence relatedness with positionally similar masteriviruses genes, but products of complementary-sense genes exhibited more sequence relatedness with that of Rep and C2 proteins of curtoviruses, begomoviruses and topocoviruses. As compare to masteriviruses, IR which contains nonanucleotide sequence is smaller than the other IR and termed as IR-1, while other IR is larger and termed as IR-2. ECSV nonanucleotide sequence is like becurtoviruses.

Genus Turncurtovirus has also been established recently, represented by Turnip curly top virus (TCTV), which is its only species (Briddon et al., 2010). Members of genus turncurtovirus have monopartite genome and are transmitted via leafhopper (Circulifer hematoceps) vector (Varsani et al., 2014a). These viruses infect several hosts including sugarbeet, turnip and cowpea (Razavinejad et al., 2013). TCTV genome contains Rep, transcriptional activator protein (TrAP), REn and C4 in complementary-sense, is similar with curtoviruses. But in contrast to curtoviruses which contains 03 genes in virion-sense, TCTV possesses 02 genes i.e. CP and V2 in virion-sense. Inspite of this, complementary-sense genes organization of TCTV is similar to curtoviruses, but product of C4 gene only showed highest identity (~70 %) with that of curtoviruses. TCTV virion-sense gene like CP showed low sequence similarity with CP of curtoviruses but product of V2 gene did not show significant identity with any protein from database (Briddon et al., 2010).

Genus Becurtovirus is represented by Beet curly top Iran virus (BCTIV) and another species i.e. Spinach curly top Arizona virus (SCTAV) has also been identified, which produces similar symptoms as by BCTV (curtoviruses; Heydarnejad et al., 2007; Soleimani et al., 2013; Hernández-Zepeda et al., 2013). BCTIV can cause infections in many plants and its vector is leaf hopper (Circulifer hematoceps; Taheri et al., 2012).

This genus shows similarity with curtoviruses in several aspects and its genome encodes merely 05 (Rep, C2, CP, V2 and V3) genes (Sahu et al., 2014). Virion-sense genes arrangement in becurtoviruses is similar to that in curtoviruses. While, 02 genes in complementary-sense strand are like to the masteriviruses and products of these 02 8 genes are also homologous to masteriviruses genes products. Genome of becurtoviruses has 02 IRs, termed SIR and a LIR. Its LIR enclose a novel nonanucleotide sequence (TAAGATTCC) containing hairpin-loop structure. So it has chimeric genome which arose by the curtoviruses and masteriviruses recombination (Yazdi et al., 2008).

Genus begomovirus is economically most imperative, geographically most wide spread and contains approximately more than 300 species (Brown et al., 2012; Varsani et al., 2014a). The genus name has been derived from earliest known and characterized type member Bean golden yellow mosaic virus (BGYMV; Stanley et al., 2005). Begomoviruses are transmitted by whitefly which causes infections in dicotyledonous (tomato, beans, cassava, cotton and squash) crops. Begomoviruses are well studied and characterized, found in NW and OW regions. Begomoviruses are monopartite and bipartite on the basis of genomic components. Monopartite begomoviruses are associated with ssDNA alphasatellites or betasatellites.

Begomoviruses who have divided their genomic component into two parts DNA A and DNA B are termed as bipartite begomoviruses. These viruses are mostly found in NW (Brown et al., 2012) but some are also present in OW (Zhang and Ling, 2011). Every genomic component has about ~2.6 to 2.8 kb size (Seal et al., 2006b). Numerous begomoviruses are bipartite such as, Tomato leaf curl new Delhi virus (ToLCNDV), Tomato leaf curl Palampur virus (ToLCPalV), Tobacco yellow crinkle virus (TbYCV) and Tobacco yellow rugose virus (TbYRV) etc (Harrison et al., 1977; Bisaro et al., 1982; Padidam et al., 1995; Dominguez et al., 2002).

Sequence analysis of both genomic components indicates very less sequence similarity that is of about ~170 nucleotides part of IR named as common region (CR; Hanley-Bowdoin et al., 1999). Within the IR or CR, all geminiviruses sequenced to date contain an inverted repeat which is inconsistent in length and sequence, separated with a nonanucleotide (TAATATT/AC) sequence. Which also represents site of initiation of virion-sense DNA replication and Rolling circle replication (RCR; Harrison and Robinson, 2002).

In majority of the cases, CRs sequences of two components are found almost similar but there are few exceptions i.e. variation in sequences of CRs among both DNA A and DNA B. As an example CR sequence of DNA A and DNA B of Tomato leaf curl 9

Gujrat virus (ToLCGV) shows 40 % difference (Chakraborty et al., 2003). Similar is the case of Cotton leaf crumple virus (CLCrV), where CRs of both components vary upto 37 % (Idris and Brown, 2004). Inspite of these differences, sequence vital for the replication are identical among both components of every individual virus.

DNA A component of bipartite begomoviruses encodes 06 ORFs, 04 (AC1 to AC4), 02 (AV1 and AV2) are present on complementary-sense and virion-sense strand respectively. AV1 ORF forms CP and AV2 responsible for movement. AC1encodes for Rep, AC2 and AC3 encode for TrAP and REn proteins respectively, AC4 gene for AC4 protein which involved in host-virus movement and symptoms development (Jupin et al., 1994; Laufs et al., 1995; Wartig et al., 1997).

DNA B component holds 02 ORFs (BV1 in virion-sense strand and BC1 in complementary-sense strand) BV1 encodes protein called nuclear shuttle protein (NSP) while BC1 encodes MP required for intercellular and intracellular movement of virus in host plants. For proficient systemic infectivity and virus propagation both components are required (Stanley, 1983).

Monopartite begomoviruses contain genome which consists of only one DNA component which is homologous to the DNA A of bipartite begomoviruses (Navot et al., 1991; Rojas et al., 2001). Monopartite contains ~2.8 kb genome (Fauquet et al., 2008) found in OW. First NW monopartite begomovirus has recently been identified signifying that this genome is not only reserved for OW begomoviruses (Melgarejo et al., 2013; Sánchez-Campos et al., 2013). Begomoviruses genome contains 06 ORFs i.e. 04 in complementary and 02 in virion-sense as in DNA-A of bipartite begomoviruses. Products of these genes are responsible for all the functions like replication, movement, transcriptional activation and encapsidation but unlike to NW viruses AV2 in OW viruses acts as pathogenicity determinant (Padidam et al., 1996).

The satellites are sub viral nucleic acid molecules that may rely on their helper viruses for the process of their replication, encapsidation, movement and share very less homology with their helper viruses (Briddon et al., 2008; Mayo et al., 2005).

The betasatellites are circular, ssDNA component of about half the size of their begomovirus genome that is ~1350 nucleotides and have single ORF in complementary-sense strand which encodes for the βC1 protein (Briddon et al., 2003; Briddon et al., 2008). This protein is composed of 118 amino acids and is considered 10 responsible for all functions performed by satellites known so far (Cui et al., 2005; Saeed et al., 2007). This protein also known to assists suppression of host mediated silencing and accumulation of helper virus (Cui et al., 2005; Leke et al., 2013). Betasatellites are necessary for the induction of specific disease symptoms (Zhang et al., 2015) as they are symptoms modulator (Briddon et al., 2003).

Sequences alignments have indicated that betasatellites have three common characters first is satellite conserve region (SCR) that is adjacent to nonanucleotide 5ʹ TAATATTAC 3ʹ sequence containing putative stem loop like structure, second βC1, third a A-rich region that is located upstream of βC1 (Saunders et al., 2000). SCR position in betasatellites is similar as common region in bipartite begomoviruses (Brown et al., 2012). Nucleotides sequence analysis of betasatellites indicates very less or no sequence identity with DNA A or DNA B genomes of begomoviruses else than a conserved hair pin like structure having ubiquitous 09 nucleotides (TAA/GTATTAC; Briddon and Stanley, 2008).

Several begomoviruses associated diseases (allied with another ssDNA molecule) are now recognized as alphasatellites and were initially named as DNA-1 (Mansoor et al., 1999; Saunders and Stanley, 1999; Nawaz-Ul-Rehman and Fauquet, 2009). However these are not typical satellites because satellites are considered those organisms which depend upon their helper viruses for the process of replication, due to this fact these are known as ''Satellite-like'' (Saunders et al., 2000).

Alphasatellites contain genomic component of ~1375 nucleotides and a single ORF which encodes for a Rep protein similar to nanoviruses and it can autonomously replicate due to this protein (Mansoor et al., 1999; Saunders and Stanley, 1999).

These satellites also contain A-rich region and an envisage hairpin structure along with TAGTATTAC sequence (Briddon et al., 2004), but for other processes like encapsidation, transmission and movement, alphasatellites rely on their helper viruses (Mansoor et al., 1999; Leke et al., 2015). Due to this reason it is expected that alphasatellites may had been confined by begomoviruses through mixed infection via insect vector as whitefly having nanoviruses like particle which consequently became a component of begomovirus/betasatellites complex (Saunders and Stanley, 1999; Mansoor et al., 2003; Briddon et al., 2004). Exact role of alphasatellites is not yet known as these are not required for disease establishment. 11

Masterivirusʼs genome organization and leaf hopper (Cicadulina mbila) vector of Maize streak virus. Genes position and orientation is represented by arrows. Rep is spliced mRNA translation product of Rep A and B ORFs, which are encoded on complementary sense strand. While MP and CP encoded in virion-sense strand, SIR and LIR are indicated. Nonanucleotide (TAATATTAC) sequence containing predicted hairpin loop structure is located within LIR.

Genome organization of Curtovirus and its leaf hopper vector (Circulifer tenellus). Arrows represent genes position and orientation. Genome encodes 07 genes, 03 in virion sense strand CP, V2 and V3 proteins, 04 in complementary sense strand C2, Rep, C4 and REn. Intergenic region has a nonanucleotide (TAATATTAC) sequence.

Genome organization of Topocovirus and its transmission vector treehopper (Micrutalis malleifera). Arrows are showing position and genes orientation. TPCTV encodes 06 genes among these 02 are in virion-sense orientation as CP and V2 proteins, but other 04, C2, Rep, REn and C4 are in complementary sense. Intergenic region contains a putative hair pin structure which is having nonanucleotide sequence (TAATATTAC). 12

Genome organization of Eragrostis curvula streak virus (ECSV), a type member of eragrovirus. Arrows are indicating genes position and orientation. Genome encodes 04 genes, 02 CP and V2 are encoded on virion while Rep and C2 genes encoded on complementary sense strand. 02 intergenic regions LIR and SIR are present however; SIR contains nonanucleotide (TAAGATTCC) sequence.

Genome organization of Turnip curly top virus (TCTV), a type member of turncurtovirus. Position and orientation of genes is represented by arrows. Genome encodes 06 genes among these 04 genes (C1, C2, C3 and C4) are on complementary, while 02 genes V2 and CP in virion-sense strand. Intergenic region contains stem loop structure having nonanucleotide (TAATATT/AC) sequence as loop part.

Genome organization of Beet curly top Iran virus (BCTIV) of becurtovirus. Arrows exhibit position and orientation of genes in genome. Genome consists of 05 ORFs among these 03 (CP, V2 and V3) encode on virion-sense strand and remaining 02 (RepA and RepB) proteins encode on complementary sense-strand. 02 small (SIR) and large (LIR) which contain hairpin structure having novel nonanucleotide (TAAGATTCC) sequence part of loop structure are present. 13

Genome organization of begomoviruses, their associated satellites and white fly. Bipartite begomoviruses contain genome comprised of DNA A and DNA B components. Arrows are exhibiting genes position and orientation. Genes encodes for Rep, TrAP, REn and C4 protein in complementary sense. 02 genes encode for V2 and CP are in virion sense-strand. DNA B in bipartite begomoviruses encodes for 02 proteins NSP, MP in virion sense and complementary sense strand respectively, these proteins are required for systemic and local movement. Both DNA-A and DNA-B genomic components in case of bipartite begomoviruses contained CR, which encompasses the hairpin loop, in their intergenic regions. Monopartite contain DNA A along with alphasatellite or betasatellite. Alphasatellites encodes Rep as well as contain A-rich sequence. Betasatellites encode βC1, SCR and A- rich sequence. All begomoviruses and their associated satellites intergenic region (IR) have a hair pin loop structure which contains nonanucleotide sequence as loop part.

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Majority of the viruses which need a transmission vector such as aphids, whitefly, leafhopper and plant hoppers are very common and transmit majority of the viruses. Begomoviruses are transmitted by whitefly (Bemisia tabaci) Gennadius (Genus Hemiptera; Family Aleyrodidae; Navas-Castillo et al., 2011) and caused great yield reduction in numerous economically significant crops (Leke et al., 2015).

B. tabaci, an insect species complex, has discrete genotypic and phenotypic variants (Bird et al., 1978; Perring et al., 1993; Bedford et al., 1994; Brown et al., 1995; Frohlich et al., 1999; Tiwari et al., 2013a). On the basis of strong geographical distribution of B. tabaci, it has genetically different biotypes (De Barro et al., 2000). All around the world most predominant and damaging whiteflies are Mediterranean (MED) which previously were called Q biotype and Middle East Asia Minor 1 (MEAM1) B biotype (Brown et al., 1995; Brown, 2007). Viral diseases those emerged during last two decades were caused by whitefly transmitted viruses mostly (Anderson et al., 2004). Whiteflies are considered highly mobile and can fly for short distance as well as can travel upto several kilometers due to wind assistance (Byrne et al., 1996; Byrne , 1999). Due to this reason whitefly vectors are called as naturally occurring ''flying syringes'' which can trial viruses over space and time (Ng et al., 2011).

About 111 viruses are transmitted by whitefly (Tiwari et al., 2013a), 90 % (more than 280 species) out of which belong to begomovirus, crinivirus are 6 % (12 species) and closterovirus, carlavirus and ipomovirus genera are 4 % (Jones, 2003; Navas- Castillo et al., 2011). Whitefly consists of not less than 37 species previously were called as biotypes, which can be differentiated mostly by DNA markers (Frohlich et al., 1999; De Barro et al., 2011; Liu et al., 2012; Firdaus et al., 2013a).

In geminiviruses, AC1 (Rep) and AC3 (REn) ORFs on A genome are essential for increased replication (Elmer et al., 1988). A variety of interactions have been evolved between host factors, viral proteins (Hanley-Bowdoin et al., 1999; Gutierrez et al., 2004), and protein kinases (Kong and Hanley-Bowdoin, 2002) to accomplish viral replication cycle.

Geminiviruses replicate through Rolling Circle Replication (RCR) mechanism, by converting ssDNA into double stranded replicative form (dsRF; Sunter and Bisaro, 1992) by Recombination Dependent Replication (RDR; Jeske et al., 2001; Hanley- 15

Bowdoin et al., 2004; Alberter et al., 2005) and by complementary strand replication (CSR; Saunders et al., 1991; Jeske et al., 2001; Preiss and Jeske, 2003; Erdmann et al., 2010). In RF, viral strand is nicked from a particular site in the replication origin due to AL1 (Rep) which was found to contain endonuclease activity (Laufs et al., 1995).

Viral ssDNA after formation of dsDNA intermediate inside the nucleus of the infected host plant cells, assembles further into the minichromosomes, which is also transcribed inside the cells as transcription and replication is based on the host enzymes. Host differentiated cells are lacking replication enzymes, to encompass this hindrance geminiviruses induce DNA replication machinery accumulation in mature cells, probably by transcriptional control and cell cycle modification (Hanley- Bowdoin et al., 2004).

Rolling Circle Replication (RCR) is an asymmetric replication mechanism meant for copying single strand genome via double stranded intermediate by Rep protein (Campos-Olivas et al., 2002a). RCR occurs typically in infected cellʼs nuclei (Davies, 1987) and occasionally in plastids (Gröning et al., 1987).

Mechanistically RCR initiation by Rep protein is involved in its binding to the replication origin and nicking of DNA, which took place in strand and site-specific manner in a conserved nonanucleotide (TAATATT↓AC) sequence (Heyraud- Nitschke et al., 1995; Choudhury et al., 2006). In this way it forms 3' primer for the process of unidirectional DNA synthesis by using DNA polymerase of host and Rep covalently linked to 5' end (Khan, 2000). After that when one polymerization round termination takes place, on reencounter of a new recognition site by the Rep. Now Rep through the cleavage and ligation of newly synthesized strand, leads to the production of a circular ssDNA. Newly formed ssDNA modify to form either dsDNA for use in RCR or virion synthesis after acquiring CP. Rep has topoisomerase activity which is Adenosine tri phosphate-dependent (ATP; Pant et al., 2001). Rep isolated from diverse geminiviruses showed ATPase activity (Bagewadi et al., 2004).

Geminiviruses Recombination Dependent Replication (RDR) is analogous to T4 bacteriophage (Jeske et al., 2001; Preiss and Jeske, 2003) also called as ''break- induced replication'', ''Join-copy pathway'' and ''bubble migration synthesis'' (George and Kreuzer, 1996; Kreuzer, 2000; Jeske et al., 2001). RDR is mainly observed in 16 begomoviruses their satellites and curtoviruses. Begomoviruses and curtoviruses during RDR use viral covalently closed circular DNA (cccDNA) as template (Jeske et al., 2001; Pilartz and Jeske, 2003; Preiss and Jeske, 2003; Alberter et al., 2005; Morilla et al., 2006; Jovel et al., 2007). RDR starts by using a 3' free end of the ss DNA or ssDNA overhang that interacts with the supercoiled dsDNA and extends followed by homologous recombination as well as loop migration. So minus strand synthesis takes place, a new ssDNA is discharged, which acts as template to continue replication or might be packaged into virions (Jeske, 2009).

Emerging viruses are major threat to plants which cause catastrophic losses. Viral emergence occurs due to several factors, for example more significant mechanism is involuntary long distance transportation of viruses due to enhanced global movement of people and plants. Ability of viruses to acquire new hosts has caused severe diseases in new hosts. Viruseʼs emergence in novel geographical regions may also has commenced via the infected plant (propagated materials, seeds or plants) materials. Afterwards, emergent viruses spread by existing vectors (Rojas and Gilbertson, 2008). Finally begomoviruses keep on evolving swiftly due to increased genetic variability, because of pseudorecombination, component capture, reassortments and mutation phenomena (Roberts and Stanley, 1994; Hou and Gilbertson, 1996; Zhou et al., 1997; Frischmuth and Stanley, 1998; Padidam et al., 1999; Saunders et al., 2001a; Rojas and Gilbertson, 2008; Singh et al., 2012) which leads to increase in diversification (Prasanna and Rai, 2007).

Due to the recombination and pseudorecombination phenomenon begomoviruses keep on evolving (Roberts and Stanley, 1994; Hou and Gilbertson, 1996; Zhou et al., 1997; Frischmuth and Stanley, 1998; Padidam et al., 1999; Saunders et al., 2001a; Singh-Pant et al., 2012). Recombination among already existing species, leads to the appearance of novel begomovirus species and it occurs frequently in nature (Zhou et al., 1997; Saunders et al., 2001a).

Breeders working for resistance induction by the integration of begomoviruses resistant genes into tomato must consider the fact of increased level of genetic variety present in tomato affecting begomoviruses. Another problem is that a particular resistance gene can be tremendously efficient against a specific begomovirus species but entirely useless against distinct or dissimilar species. Along with these facts, pace of begomoviruses evolution is exceedingly rapid which consequently leads to the fast 17 emergence of novel species and strains which may overcome the effect of resistance genes (Padidam et al., 1999).

Viral diseases cause catastrophic losses of agriculture (Agrios, 1997), similarly members of Geminiviridae are liable for distressing plant diseases particularly in tropical, sub tropical and in temperate regions of the planet due to the universal climatic changes and international trading (Mansoor et al., 2006). Since almost a century before, many diseases such as beet curly top, maize streak and cassava mosaic viruses were considered serious threat for crop production.

From the few preceding decades many new emerging geminiviruses have caused disease epidemics in tomato, cotton and grain legumes (Varma and Malathi, 2003).

These infections lead to the heavy economic losses all around the world like in Florida for tomato US $ 140 million loss (Moffat, 1999), in Pakistan during 1992- 1997 was US $ 5 billion loss for cotton (Briddon and Markham, 2000), and for cassava in Africa US $ 1300-2300 million loss (Thresh et al., 1998). African cassava mosaic disease produced by begomoviruses at pandemic level has caused 15-24 % losses (Legg and Fauquet, 2004). Ninety % losses because of cassava mosaic disease complex were reported in India for grain (Patil et al., 2004), for legumes US $ 300 million (Varma et al., 2011) loss. Masteriviruses caused maize streak disease, which is a major corn distracting disease in Africa (Palmer and Rybicki, 1998).

Similarly in Western United States of America in 1900s sugar beet production was badly affected by beet curly top disease (Stenger and Mcmahon, 1997; Soto et al., 2005). Monopartite begomoviruses causing tomato yellow leaf curl disease (ToYLCD) in various regions is a big constrain to the production of tomato (Salati et al., 2002). In America, bean production was reduced due to bean golden mosaic disease (Brown and Bird 1992). Bean yellow dwarf virus (BeYDV) in South Africa reduced dry beans (Phaseolus vulgaris) production upto 90 % (Rybicki and Pietersen, 1999).

18

Replication of geminiviruses by RCR. Viral ssDNA enters nucleus and form complementary strands by DNA polymerase of host and resultant to dsDNA. This dsDNA will act as template for early (Rep) leftward and late (coat protein) rightwards by bidirectional transcription. Proteins are translated by RNA into the cytoplasm, Rep moves to nucleus to start replication of dsDNA by RCR. Rep nicks in the origin of replication on virion strand, further extended by host DNA polymerase at 3ʹ-end of virion strand, on complementary strand template. After replication Rep nicks and religates virion strands, multiple copies of ssDNA produced are either packaged into virions or under goes replication process. This figure has been reproduced from (Pooggin, 2013).

Recombination dependent replication (RDR). Viral DNA primer attached with circular dsDNA, extended by DNA polymerase of host along circular template. During or after replication, same or another DNA polymerase complex, linear ssDNA (newly synthesized) converted into either fully or partially dsDNA. Heterogeneous linear dsDNA are generated by RDR. Those linear dsDNA which are long and carrying more than 01 origin of replication, transcribed in both orientation by PolII to produce viral mRNA. After translation Rep starts replication of long, linear and more than one replication origin carrying dsDNAs. SsDNA produced by the replication of linear multimeric dsDNA leads to the production of circular ssDNA that get packed or reenter in replication. This figure has been reproduced from (Pooggin, 2013). 19

Plant metabolism is defined as "a complex of chemical and physical events of the photosynthesis, respiration, synthesis as well as degradation of organic substances". Products of photosynthesis not only provides respiratory substrate but are also used as building blocks for the biosynthesis of aminoacids, nucleic acids, proteins, carbohydrates, lipids, organic acids and natural products.

A variety of chemical compounds are required for survival and subsistence of every organism (Bernhoft, 2010). Metabolites are produced by organisms via enzyme mediated chemical processes termed as metabolic pathways.

Primary metabolism in plants consists of all metabolic processes and pathways those are indispensable for the survival of plants.

Primary metabolites are those which are produced for basic functions required for life like cell division, reproduction, growth, respiration and storage (Bourgaud et al., 2001) these includes aminoacids, carbohydrates and lipids.

Secondary metabolism in plants produces those products which aid in the process of development and growth of plants; however are not obligatory for the plantʼs survival. It plays key role to keep plants healthy and it facilitates primary metabolism of plants.

Secondary metabolites are different from primary metabolites as they generally are not essential for basic metabolic processes in plants (Dixon, 2001), but they have role in plantʼs interaction with environment. Secondary metabolites assist plants to keep all its systems working accurately, for defense purposes and impart characteristics like color in plants. These are also utilized for regulation and signaling of primary metabolic pathways. Secondary metabolites such as plant hormones are frequently used in order to regulate metabolic activity inside the cells and to supervise the overall plant development. In different species, production of secondary metabolites is according to particular need, which is selected during the course of evolution (Hijazi et al., 2013).

There is no commonly agreed and fixed classifying system for secondary metabolites. Plants secondary metabolites based on biosynthetic origins can be divided into three major groups. First group consists of flavonoids, allied polyphenolic and phenolic compounds, their structures vary from simple phenolic compounds to polymer with high molecular weight. Structures of phenolic compounds determine its antioxidant activity particularly by substitution, nature and position of some hydroxyl groups 20

(Amarowicz et al., 2004; Balasundram et al., 2006; Das et al., 2010). Second group consists of terpenoids often described as natural products which are active against various herbivores (Litvak and Monson, 1998), bacteria (Trombetta et al., 2005; Schelz et al., 2006), viruses (Sun et al., 1996) and fungi (Rana et al., 1997). Third group is composed of nitrogen-containing alkaloids as well as sulphur containing compounds (Crozier et al., 2006). Among ~50,000 secondary metabolites found in plants, 30,000 are terpenoids, 2,500 phenylpropanoids, 12,000 alkaloids and 2,500 are other compounds (De Luca and St Pierre, 2000). Whereas phytochemicals involved in plant defense belong to three main classes: phenolics, terpenoids and alkaloids (Doshi et al., 2015).

Structurally secondary metabolites are divided into five: Isoprenoids (i.e. terpenoids), polyketides, alkaloids, flavonoids and phenylpropanoides groups. Secondary metabolites have also been classified into following more specific types as alkaloids, non protein amino acids, glucosinolates, amines, cyanogenic glycosides, alkamides, lectins, peptides and polypeptides, flavonoids, tannins, terpenes, saponins, steroids, phenylpropanoids, coumarins, lignins, polyketides, waxes, polyacetylenes and organic acids (Wink, 2009).

Secondary metabolites are important for plantsʼs survival to environment due to their antimicrobial, anti insect activities, their ability to reject competing plant species and to attract symbionts and pollinators (Dixon, 2001). Plants are continuously exposed to variety of pathogenic microorganisms below and above ground, which as a consequence leads plants to evolve different ways to recognize and then defend against such infections (Bolton, 2009). Enormous natural products are found in plants, many of which have evolved to produce selective benefits against microbial attack (Dixon, 2001).

Secondary metabolites play very important role in defense mechanisms and researchers know that this is a common trait in numerous plants, but this is still complex to determine the specific role for every secondary metabolite.

Metabolites profiling is defined as an analytical method used for relative quantification of numeral metabolites from various biological samples (Fiehn, 2002). Samples vary from single cell to specific tissues as this can be a part of tissue or a mixture of various organs (as whole shoot). 21

Metabolite profiling concept is known for several decades (Fernie et al., 2004). It has been widely used in various research areas like in comparative display for genes functional analysis and in system biology (Fiehn et al., 2000; Fernie et al., 2004).

Historically, metabolites measurements have mostly been gained by spectrophotometric assays which can detect either single metabolite, or via chromatographic separation of low complex mixtures. Over the last decade, various methods based on high sensitivity and accuracy for compounds measurements in extremely complex mixtures had been developed. Various terms have been implemented to illustrate these methods ranging from Metabolic Finger Printing, Metabolite Profiling to Metabolomics and Metabonomics.

Different mass spectrometry methods such as Gas chromatography-time-of-flight- mass spectrometry (GC-TOF-MS), Gas-chromatography-mass-spectrometry (GC- MS) and liquid chromatography-mass-spectrometry (LC-MS) are current main mass spectrometry methods used for metabolite analysis (Fernie et al., 2004).

GC-MS is used for the robust quantification and identification of some hundred metabolites in a single extract (Halket et al., 1999; Fiehn et al., 2000; Roessner et al., 2001). Main advantage for this instrumentation stem is the fact that it has been used for metabolite profiling for long, which led to the availability of stable protocols for sample preparation, chromatogram interpretation, evaluation, analysis, machine maintenance and set up (Fernie et al., 2004).

During last decades GC-MS has confidently developed as an important technological podium for profiling of both plant and non-plant metabolites (Fiehn, 2002; Sumner et al., 2003; Fernie et al., 2004; Kell et al., 2005; Robertson, 2005). With the passage of time there are many improvements in this technique which led to address many biological problems. Previously GC-MS has also been used for certain diagnostic purposes such as for the analysis and expression of metabolite reactions in different experimental variations (Sauter et al., 1991). Now the purpose of metabolite profiling varies from simple diagnostics such as for studies of herbicidal mode of action on barley seedlings (Sauter et al., 1991; Oikawa et al., 2006) to distinguish Arabidopsis, tomato and potatoʼs genotypes (Fiehn et al., 2000; Roessner et al., 2001). Study of different tissue types of plants e.g Lotus japonicus (Desbrosses et al., 2005), tomato fruit ripening steps (Roessner-Tunali et al., 2003), to study complications innate in 22 system biology and in integrative genomics (Sauter et al., 1991; Nicholson and Wilson, 2003; Carrari et al., 2006; Gibon et al., 2006) have also been done by this technique.

Ty cultivars have been developed for resistance against TYLCV by the acquisition of Ty-1, Ty2, Ty3 and Ty5 genes which produce resistance for virus, along with this, Ph2 and Ph3 genes (to impart resistance against Phytopthora infestans and to create resistance against fusarium wilt) and Sm gene (for gray leaf spots resistance). Pyramiding of resistance genes showed either broader resistance spectrum or an increased level of resistance. Resistance genes pyramiding have been affective in other plants-viruses interactions, like a study by Kelly et al. (1995) by pyramiding genes in Phaseolus vulgaris against Bean common mosaic virus was proved effective. Similar results were found in other plant-virus interactions as in Glycin max against Soybean mosaic virus (SMV; Shi et al., 2009) and in Hordeum vulgare against Barley mild mosaic virus (BaMMV) and against Barley yellow mosaic virus (BaYMV; Werner et al., 2005). However pyramiding for various plant-pathogens has also been successful for examples in rice bacterial leaf blight disease caused by Xanthomonas oryzae (Suh et al., 2013) and Bt (Bacillus thuringiensis) genes pyramiding against insects Spodoptera litura and Heliothis armigera (Li et al., 2014). Similarly in tomato, pyramiding strategy has also been used for multiple disease resistance (Hanson et al., 2016).

Although Ty cultivars are resistant for TYLCV a monopartite begomovirus, are also resistant against bipartite begomoviruses which are prevalent and infect tomatoes, economically important crops and ornamental plants. Here we have infected these Ty resistant tomato cultivars with ToLCNDV, in order to compare their resistance response against begomoviruses which are prevalent in Pakistan.

Begomoviruses are great trouble for the production of various economically important crops and are wide spread all over the world. Proposed study was also aimed to characterize the begomoviruses infecting tomato plants in different tomato cultivation of Pakistan. New begomoviruses species are still evolving due to trading, over population of whitefly and recombinations which consequently led to increase in diversity of begomoviruses (Prasanna and Rai, 2007). This work will help scientists and researchers in better understanding of tomato diseases for the improvement of programs involved in virus resistant transgenic crops production. Similarly resistant 23 cultivarʼs acquisition and their inoculation with ToLCNDV (common in Pakistan) may help us to understand the resistance level of Ty cultivars against ToLCNDV. Resistant cultivars may later be used for cultivation purpose in Pakistan. GC-MS assisted metabolite profiling of ToLCNDV inoculated thirteen Ty (according to number and combination of Ty genes) cultivars and one native variety (Nagina), showed the repeated presence of various compounds found in resistant types. It seems that these compounds may be responsible for the elevated immunity of these resistant cultivars.

Aims and Objectives

 Amplification, identification and characterization of begomoviruses affecting tomato plants.

 Quantification of metabolites particularly secondary metabolite or volatile metabolite in resistant and susceptible tomato plants.

 Correlation of plant phenotypes and their metabolites to search out the resistant tomato varieties against different begomoviruses.

CHAPTER NO. 2 REVIEW OF LITERATURE 24

REVIEW OF LITERATURE

Tomato belongs to family solanaceae (Nightshades) and its name was derived from genus Solanum "The night shade plant". It is a medium sized family consists of about 3000 to 4000 species (Knapp et al., 2004) and 98 genera (Olmstead and Bohs, 2006). Approximately half among these species belong to diverse and large genus Solanum (Knapp et al., 2004). Economically solanaceae is ranked third but as vegetable crops ranked first (Wu and Tanksley, 2010).

Solanum is the one among 10 major species rich flowering plants genera (Frodin, 2004) and economically most important genus as it includes crops e.g. potato, tomato, eggplant, pepper, petunias and tobacco (Chase et al., 1993; Kimura and Sinha, 2008; Chase and Reveal, 2009). It contains ~1500 species (Knapp et al., 2004), about more than 50 % species of Solanaceae and 13 known wild tomato species (Kimura and Sinha, 2008).

Tomato word was derived from Spanish word Tomate, which came from Nahuatl (Rick, 1978) word (tomatl) meaning “fat water” or “fat thing”. Previously in 1753 Linnaeus cited tomato in genus Solanum as S. lycopersicum. In 1754, it was placed in its own genus, calling it Lycopersicum esculentum by Philip Miller (Peralta and Spooner, 2006; Foolad, 2007). This name was widely used, but it was in breach of plant naming rules. Genetic evidence has shown now that Linnaeus was right to place tomato in Solanum genus, turning S. lycopersicum as correct name. Inspite of this, it is likely that taxonomic position of tomato will be contentious for some time to come, as both names are found in literature (Gerszberg et al., 2015).

Tomato is usually cultivated as annual, usually grows upto the height of 01-03 meters, having bipinnate and compound leaves, hairy stem, sympodial shout (Kimura and Sinha, 2008), flowers typically have 05 petals but flowers with 07 or more petals are commonly found and styles are generally inserted. It is grown due to its fleshy fruits which have great variety in color, size and shape. Commonly it is accepted that genetic diversity of tomato is very less due to certain problems in its diffusion and domestication (Miller and Tanksley, 1990; Williams and Clair, 1993; Noli et al., 1999; Park et al., 2004a; Tam et al., 2005). Tomato is a diploid species having 24 chromosome i.e. 12 pairs (2n=24; Wu and Tanksley, 2010), encoding almost 35,000 genes and small genomic size (840 Mb; Shusei et al., 2012).

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Very little information regarding origin of tomatoes is available, although it is of great economic value (Blanca et al., 2012). Its origin was in South America, while this was assumed that its domestication took place in Central America (Kimura and Sinha, 2008) but Mexico was its food origin (Smith, 1994). Some members of the cultivated tomatoes such as its wild relatives were inhabitant to western South America as of northern Ecuador in the course of Peru toward northern Chile, together with Gala pages Islands. Afterward it spread all over the world following Americas Spanish colonization, however its introduction in Asia (Burma, India, Dutch East Indies, Malaya and Indochina) took place during 18th century as Joannis de Loureiro in 1790 has written in his book entitled "Flora Cochinchinens" about tomato cultivation in fields and gardens of Cochin china. Similarly in India tomato cultivation was indicated in 19th century by William Roxburgh in 1832 in book entitled as "Flora Indica". While, its cultivation in Punjab (Lahore) Pakistan was first indicated by Bamber in his book "Plants of the Punjab" in 1916 (Mccue, 1952).

Tomato is important as a food has not only bred to improve fruit quality, productivity but to enhance resistance against biotic and abiotic stresses. It is also used as research material as model plant due to its several important features like fleshy fruit, compound leaves and sympodial shoot which other model plants (e.g. Arabidopsis and rice) are lacking. Wild tomato species are known important for evolutionary studies, contained desirable traits and exhibited great diversity in phenotypes, which can be crossed with cultivated verities indicating a significant source of breeding to get desirable traits (Kimura and Sinha, 2008).

The most primitive evidence for geminiviruses diseases was found in 752 AD in the original biggest omnibus of Japanese poetry called as ''The Man'yoshu'' its meaning is the 'collection of ten thousand leaves'. A poem indicated the autumnal or yellowish symptoms appearance in Eupatorium plants during summer, was written by Empress Koken. This surveillances was allied to the yellow vein illness of Eupatorium plant (Inouye and Osaki, 1980) which now a days is considered as caused by geminiviruses as well as their associated satellites (Saunders et al., 2003). Particular symptoms shown by geminiviruses, in plants grown in tropical and sub tropical regions were being observed since the mid 1800s (Wege et al., 2000). In 1970s, it was found that ssDNA viruses were responsible for such symptoms (Goodman, 1977a; Harrison et al., 1977) and were named as geminiviruses (Goodman, 1977b).

26

As Geminiviruses were named from ''geminus '' means twin due to twin morphology, studied for first time by Hatta and Francki (1979) after examining 1.7 nm electron- dense fissure visibly separating into 02 parts, under electron microscope in Chloris striate mosaic virus (CSMV) particles. The ssDNA associated with geminate particles of Cassava latent virus also called ACMV, BGYMV and MSV was demonstrated for first time by Harrison et al. (1977) and Goodman (1977a). Genomic DNA of BGYMV transmitted by whitefly was mechanically inoculated via sap of infected plant caused infection in plants (Phaseolus vulgaris; Goodman, 1977b). On the basis of such conclusions geminiviruses were kept separate from other all viruses known, till that time. In 1979 geminiviruses group had been established but did not acquire the status of the family (Matthews, 1979). It was observed that BGYMV as well as TGMV were few geminiviruses those have separated their genomic components into more than one molecules of nucleic acids (Bisaro et al., 1982; Hamilton et al., 1982). ACMV was first fully sequenced geminivirus, which has supported the demonstration of the bipartite genome by constructing infectious clones (Stanley, 1983). Afterwards BCTV and MSV were known as monopartite after sequencing and characterization of their genome (Howell, 1984; Mullineaux et al., 1984; Stanley et al., 1986; Grimsley et al., 1987).

In 1995 group of geminiviruses was converted to family Geminiviridae (Murphy et al., 1995). Initially geminiviruses were divided into 03 genera named as Begomovirus, Mastrevirus and Curtovirus (Briddon et al., 1996). But after the identification of TPCTV which was transmitted by specific insect, a different vector as compare to other vectors, carrier for the other geminiviruses. As a consequence of this study geminiviruses were divided into 04 genera (Begomovirus, Masterivirus, Curtovirus and Topocovirus by ICTV (Stanley et al., 2005). However due to new molecular techniques and cost effective sequencing new geminiviruses are being discovered from different cultivated and non cultivated plants. In order to accommodate recently discovered geminiviruses those are divergent and in some aspects have unique genome structure, led to the establishment of 03 new genera i.e. Becurtovirus, Turncurtovirus and Eragrovirus (Varsani et al., 2014a).

Names of the following genera were derived from the abbreviations of the type members such as MSV for mastrevirus, BCTV for curtovirus, TSCTV for

27 topocuvirus, similarly eragrovirus by ECSV, turncurtovirus by TCTV, becurtovirus from BCTIV and BGMV for begomovirus (Jeske, 2009).

Up till now about ~400 geminiviruses are known and among them more than 300 species are included into the begomovirus. Greater than 1000 complete virus sequences had been submitted in International DNA databases, which is increasing day by day, indicating the huge diversity, extensive geographic circulation, host adaptation and economic significance of these viruses (Brown et al., 2012). This number has increased as 3,123 complete begomoviruses genomes were available in public databases, in December 2012 (Brown et al., 2015).

Geminiviruses, particularly those classified in genus begomovirus were perceived as emerging viruses (Brown et al., 2015), because of their frequent occurrence and disease severity in numerous economically significant crops (Polston and Anderson, 1997; Lefeuvre and Moriones, 2015). Approximately 20 geminiviruses species, mainly those included in begomoviruses had been considered more competent for disease in tomato (Polston and Anderson, 1997). Cultivated tomatoes were considered most appropriate for evolution and emergence of monopartite and bipartite begomoviruses, because tomato was infected by greater than 67 species of begomoviruses indicating greatest digit infecting any other crop (Fauquet et al., 2008).

Begomoviruses are separated into 02 main geographically distinct groups according to phylogenetic analysis, the OW and NW viruses. OW viruses are found in areas such as Eastern Hemisphere, Africa, Europe, Asia while those belong to NW viruses are found along the Americas and Western Hemisphere (Rybicki, 1994; Padidam et al., 1999; Paximadis et al., 1999). Both OW and NW begomoviruses have enormous characteristics vary among them. OW viruses consist of both monopartite and bipartite, while NW viruses have only bipartite begomoviruses but recently ToLCD in NW countries such as Ecuador and Peru was caused by a monopartite Tomato leaf deformation virus (ToLDeFV; Melgarejo et al., 2013). Along with these differences, all OW begomoviruses contained AV2 an additional ORF in DNA A which is lacking in NW begomoviruses (Rybicki, 1994; Stanley et al., 2005). In contrast to OW begomoviruses NW begomoviruses contain N- terminal PWRSMAGT motif that is the conserved sequence of amino acids present in CP region, encoded by ORF (AV1) which is lacking in OW begomoviruses (Harrison et al., 2002). In most of the OW

28 begomoviruses 02 iterons are present in CR (first is the upstream of AC1 TATA box, second is complementary downstream), while downstream iteron is absent in NW begomoviruses (Argüello-Astorga et al., 1994). OW begomoviruses are more diverse than NW begomoviruses (Rybicki, 1994). Genomic DNA component of monopartite begomoviruses is approximately 2.9 kb which is larger than NW DNA A of bipartite begomoviruses of approximately 2.6 kb, containing additional V2 gene (however DNA-A of bipartite begomoviruses found in OW contain homologous AV2 gene).

Rybicki (1994) hypothesized that nearly all NW begomoviruses are recent than OW and may have evolved 130 million years ago, after continental partition of Americas from Gondwana. He also proposed that whiteflies may have transmitted viruses from Asia to Americas, that's why these viruses were considered ancestors of NW begomoviruses known today. These viruses consequently evolved separately under entirely various conditions which led to the gain or loss of different genomic components. Recently there is the evidence of NW begomoviruses occurrence in OW and vice versa, due to the diversity of whitefly (B biotype) and supply of contaminated propagating materials. However evidence provided by Ha et al. (2008) showed that NW begomoviruses were present in OW before continental separation. As strains of TYLCV have been recognized in NW (Florida and Caribbean Islands; Polston et al., 1999; Czosnek and Laterrot, 1997) NW virus i.e. Abutilon mosaic virus (AbMV) has been recognized in New Zealand (Lyttle and Guy, 2004) and UK (Brown et al., 2001) in Abutilon species.

Geminiviruses ssDNA encodes for a conserved (named as Rep) protein, also called (AC1, AL1, L1, C1 or C1) which can catalyzes the initiation of rolling-circle DNA replication (Laufs et al., 1995; Orozco and Hanley-Bowdoin, 1996; Nash et al., 2011). It is a virus encoded protein exclusively necessary for replication. Rep protein has homology with initiator protein of DNA replication found in prokaryotic plasmids (Laufs et al., 1995). Rep, 41 kDa protein is multi functional, but unable to work as polymerase depends totally on host machinery for their genome replication (Elmer et al., 1988; Orozco et al., 1997; Campos-Olivas et al., 2002b). It mediates recognition of virus-specific cognate origin (Fontes et al., 1994), as well as transcriptional repression (Sunter et al., 1993; Eagle et al., 1994). Rep carried initiation and termination of replication of viral genome (Heyraud-Nitschke et al., 1995; Laufs et al., 1995), and induction of accumulation of host replication factors in infected host

29 cells (Nagar et al., 1995). Rep attaches particularly to the dsDNA (Fontes et al., 1994; Singh et al., 2008), cleaving and ligating DNA within a conserved sequence located in hairpin loop of plus-strand origin (Laufs et al., 1995; Orozco and Hanley-Bowdoin, 1996). It acts as helicase in order to unwind DNA in the course of replication of plus- strand (Desbiez et al., 1995; Clerot and Bernardi, 2006; Singh et al., 2008). Its helicase activity is oligomeric conformation dependent (Choudhury et al., 2006).

Rep alone can start RCR without other accessory viral factors (Hong et al., 2003). Rep N-terminus is concerned for nicking, site-specific DNA binding and ligation, middle portion consists of oligomerization domain, while C-terminus contained ATPase activity due to ATP binding (Orozco et al., 1997). N-terminus site specificity of Rep was mapped in initial 116 amino acids in case of TYLCV Rep (Jupin et al., 1995). N-terminal sequences of Rep from geminiviruses have 03 motifs (RCRI, RCRII and RCRIII), on comparing with prokaryotic RCR (Dasgupta et al., 2004; Vadivukarasi et al., 2007).

Geminiviruses were not capable to go into meristem, but Rep-mediated initiation of the host plant cells DNA replication machinery turned geminiviruses replication to occur into differentiated cells (Kong et al., 2000; Egelkrout et al., 2002; Richter et al., 2016). Rep by binding to pRBR in cell cycle established s-phase (Collin et al., 1996; Hanley-Bowdoin et al., 2004). Experimentally it was demonstrated that a region (~80 amino acid) of Rep of TGMV, consists of 02 expected α- helices which interact with pRBR (Arguello-Astorga et al., 2004). In mature leaves transcription of host was activated via Rep by relieving the pRBR/E2F repression. Rep pRBR binding capability and its ability to prevail over the proliferating cell nuclear antigen (PCNA; processivity factor meant for δ promoter of DNA polymerase) repression mediated by E2F, was the indication of a model through which host genes expression by pRBR/E2F was mend by Rep (Castillo et al., 2003). As stated by this model, E2F binds with PCNA promoter and this binding led to chromatin remodeling acts, such as histone deacetylases (SW1/SNFI enzymes), which however in turns form a repressor complex and enroll pRBR in mature host cells. In this way, host genes activation led to the commencement of host DNA replication machinery. Geminiviruses encoded protein related to replication, interacted with several host plant replication factor C (RFC) complex, which is a clamp loader as well as shift PCNA towards replication fork (Luque et al., 2002; Castillo et al., 2003). It was expected about such interactions

30 to occur during early phase of DNA replication complex formation. ACMV Rep protein, after yeast cell inoculation led to the re-replication induction resultant into morphological variations (Kittelmann et al., 2009). These variations were elongation of yeast cells upto 03 folds in contrast to non-induced cells and these cells contained less compact but enlarged nuclei. Cells expressing Rep, presented evidence for DNA content beyond 2C, which was the indicative of uninterrupted replication, with no mitosis superseding. TGMV Rep attaches to histone H3 which is a mitotic kinesin, a novel protein kinase (GRIK; Kong and Hanley-Bowdoin, 2002) and Ubc9 are sumoylation pathway factor (Castillo et al., 2004).

TrAP also nominated as AC2, AL2 and C2, is ~15 kDa, exclusive to begomoviruses as not found in masteriviruses but curtoviruses (BCTV) L2 related protein (C2) do not contain transcriptional activity (Jackel et al., 2015). This protein encoding gene was transcribed by a promoter located within Rep gene (Shivaprasad et al., 2005). In masteriviruses role of AC2 is being played by AC1 (Liu et al., 1998a). TrAP was named because of its property to transactivate virion-sense promoter, as to activate late genes. TrAP (AL2) was considered essential for late genes transactivation (Sunter and Bisaro, 1997), gene silencing suppression (Hanley-Bowdoin et al., 1999; Voinnet et al., 1999; Vanitharani et al., 2004; Trinks et al., 2005; Raja et al., 2010), and as basal defense suppressor (Rajeswaran et al., 2007; Yang et al., 2007; Amin et al., 2011a).

More recently, some researchers had shown that AC2 of Cabbage leaf curl virus (CaLCuV) in mesophyll activated CP promoter and de repress the promoter present in vascular tissue as observed in TGMV (Lacatus and Sunter, 2008). AC2 activated TGMV CP promoter through a bipartite component located among -125 and -60 base pairs, upstream on the start site for transcription (Sunter and Bisaro, 2003). But in opposite AL2 activated CP promoter by de repression in phloem which was activated by sequences positioned in between 1.2 and 1.5 kb upstream of transcription start site of CP (Sunter and Bisaro, 1997). As a counter defensive measure, begomoviruses Rep, the TrAP of CaLCuV and TGMV, as well as related C2 protein found in curtoviruses prevent methylation and ultimately suppress transcriptional gene silencing (TGS; Buchmann et al., 2009; Zhang et al., 2011; Rodriguez-Negrete et al., 2013).

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According to previous studies conducted, AL2 function was not virus specific showed that AL2 gene products from various begomoviruses can accompaniment a TGMV al2 mutants, in protoplast of tobacco and planta (Sunter et al., 1994; Saunders and Stanley, 1995; Sung and Coutts, 1995a). However many attempts in order to discover a conserved motif present in AL2 responsive promoters, had obtained very less success (Sunter and Bisaro, 2003). Signifying it may interact with the multiple host factors, supported by the fact that AL2 feebly binds to the dsDNA, although minimum sequence was recognized as AL2 response component and that binding was not sequence specific (Noris et al., 1996; Sung and Coutts, 1996; Hartitz et al., 1999; Sunter and Bisaro, 2003).

Moreover, AC2 (1 to 100) is a successful suppressor similar to that of full-length AC2 and silencing activation suppression seems to be autonomous activity. According to Gopal et al. (2007) Bhindi yellow vein mosaic virus (BYVMV) AC2 was concerned in transactivation and just slight in gene silencing suppression of monopartite begomoviruses.

AL2 interacted with Adenosine Kinase (ADK, an enzyme significant for adenosine recovery as well as maintenance of methyl cycle) and sucrose nonfermenting-1(SNF1 a type of kinase modulating metabolism of sugar), as a part of viral reaction to host defense. It also activated CP and BR1 (recently called NSP) gene promoters late in infection (Hao et al., 2003; Wang et al., 2003; Wang et al., 2005). According to Wang et al. (2003) L2 and AL2 interrelated proteins were considered pathogenicity determinants when expressed into transgenic plants which caused increased susceptibility. L2 and AL2 In vitro and subsequent coexpression in the yeast and E. coli inactivated ADK. They showed reduction in ADK activity occurred in geminiviruses-infected host plant tissues as well as in transgenic plants expressing viral proteins. But in contrast to this, after various RNA viruses or functional L2 gene lacking geminiviruses, their inoculation into plants led to increased ADK activity. AL2 have the ability of multiple cellular kinase interaction and they demonstrated that AL2 was found in cytoplasm and nucleus of infected host plant cells. As this data indicated that ADK being targeted through viral pathogens and supported this with the evidence that, this ''housekeeping'' enzyme may be a part of host plant defense response. Previously they had shown that L2 and AL2 interacts and inactivates SNF1 Kinase. All such observations indicated that SNF1 mediated metabolic alterations are

32 a significant factor of innate antiviral defenses as well as ADK and SNF1 inactivation by means of geminiviruses proteins signifying dual strategy in order to respond to this defense. Viral proteins activities are being regulated to some extent by their interaction with itself. A zinc finger like motif (CCHC) was required but it was not enough for AL2 self interaction. Substitutions of alanine for invariable cysteine residues which comprise the motif, eliminated self-interaction or were responsible for abnormal subnuclear localization; however this did not destroy ADK and SNF1 interaction. With the aid of bimolecular fluorescence complementation, it was shown that AL2:AL2 complexes gather mainly in the nucleus while AL2:ADK as well as L2:ADK complexes gather primarily into the cytoplasm. Moreover, cysteine residue mutations damaged the coat protein promoter activation ability of AL2 but local silencing suppression remains unaffected. AL2 self-interaction is associated with the nuclear localization as well as proficient activation of transcription, while L2 and AL2 monomers by interacting with ADK can suppress local silencing (Yang et al., 2007). Sunter and Bisaro (1992) recognized that AC2-mediated AR1 i.e. now named as CP or AV1 and BR1 (termed as NSP) genes activation occurred at the transcriptional level. TrAP function of transactivation was confirmed more by Hong et al. (1997) they developed N. benthamiana transgenic plants intended for the plant ribosome- inactivating protein (RIP) transgene expression by a virus inducible promoter. PVX system was utilized in order to express ACMV-TrAP in Planta. Interruption of TrAP expression through anyway decreased RIP transcripts level, validated the need of AC2 mediated magnification of RIP act in transgenic plants.

As begomoviruses encoded TrAP was found linked with pathogenicity determinant (Chandran et al., 2013) and silencing activities. TrAP of numerous begomoviruses has been shown as PTGS suppressor (Vanitharani et al., 2004; Zrachya et al., 2007; Glick et al., 2008; Amin et al., 2011b). PTGS is RNA degradation that is sequence specific following to the targeted mRNA down-regulation which led to the decrease in protein expression (Bisaro, 2006; Raja et al., 2010; Raja et al., 2008). Related protein L2 of curtoviruses has similar pathogenicity functions (Yang et al., 2007).

AC2 and AC4 genes of East African cassava mosaic virus (EACMV) were studied to demonstrate synergistic interaction among them by means of an Agrobacterium tumefaciens infiltration assay. This interaction led to the PTGS suppression stimulated by green fluorescent protein (GFP) as discover by the reduced levels of short

33 interfering RNAs (siRNAs) and enhanced GFP mRNA accumulation level. AC2 protein of Indian cassava mosaic virus (ICMV), in addition was observed to act as PTGS suppressor (Vanitharani et al., 2004). As well as it over come the virus stimulated hypersensitive cell death (Hussain et al., 2007; Mubin et al., 2010).

It has been established that viruses cause host defense suppression (which acts against RNA and DNA viruses both), shared their capability of suppressing host stresses.

Transgenic N. tabacum and N. benthamiana containing truncated AC2 i.e. AC2 1-100 (lacking transactivation ability) and full length C2 transgenes were prepared. Inoculation of Tobacco mosaic virus (TMV; a discrete RNA virus), BCTV and TGMV resultant to enhanced susceptibility (ES) phenotype distinguished by less mean latent period (time of first symptoms appearance), although there was neither major symptoms severity nor major increase in virus titer. Less inoculum level was found enough to cause ES phenotype. It was also concluded from these results that transcription activation domain of TrAP however is not necessary for suppression (Sunter et al., 2001). Wang et al. (2005) again confirmed earlier results by creating a construct expressing an inverted repeat GFP RNA (dsGFP). Truncated AC2 1-100 (without transcriptional activation domain) and C2 of TGMV and BCTV respectively, all suppressed silencing of RNA directed against the construct. After further investigation they concluded that proteins encoded by geminiviruses acquired a novel mechanism for silencing suppression along with ADK inhibition. As many reports during 2004 to 2005, dispensed begomoviruses TrAP and BCTV C2 as RNA silencing suppressor or defense response, in plants against viruses (Baulcombe, 2004; Ding et al., 2004; Roth et al., 2004; Voinnet, 2005; Wang et al., 2005).

Increased susceptibility caused by AL2 and L2 was characterized mainly by enhancement in viral infectivity, when these genes were expressed in transgenic plants. According to Hao et al. (2003) as they provided biochemical and genetic evidences about increased susceptibility which was due to AL2 and L2 interaction with SNF1 kinase (metabolism global regulator). They showed In vivo and In vitro, that L2 and AL2 inactivate SNF1 and also demonstrated that antisense SNF1 transgene expression in N. benthamiana produced enhanced susceptibility, similar as expected by AL2, L2 transgene, but over expression of SNF1 enhanced the resistance. AL2 protein missing important portion of SNF1 interaction domain when expressed in transgenic plants do not show enhanced susceptibility. These observations

34 suggested that SNF1mediate metabolic alterations were a constituent of innate antiviral defenses as well as SNF1 inactivation via AL2, L2 was a counter defensive measure.

Trinks et al. (2005) proposed that transcriptional activation and silencing suppression via TrAP are connected functionally. conserved 03 domains were recognized in AC2 protein, a basic domain at N- terminus of which a nuclear localized signal (NLS) present, a central DNA binding (with a nonclassical Zn- finger motif) domain, an acidic activator (at the C- terminus) domain. They discovered host genes transctivation is vital for suppression of silencing activity of TrAP. Complementary results about the need of TrAP transactivation domain for silencing suppressor purpose advocate 02 mechanisms (Transcription-dependent and transcription- independent) involvement. By the work of Trinks and colleagues (2005) AC2 transcription-dependent mechanism as suppressor can be explained. For Mungbeen yellow mosaic virus (MYMV) AC2 functioning, NSL, Zinc finger and activation domain is required as explained by them. According to them ACMV and MYMV AC2 proteins, transient expression of which led to the expression of some host genes as Wermer exonuclease-like 1 (WEL 1), which was able to suppress RNA silencing in N. benthamiana. Studies conducted by Wang et al. (2003) presented an evidence for transcription-independent silencing suppression. In yeast with the aid of 02 hybrid screening, interaction of TGMV truncated TrAP lacking activation domain and L2 of BCTV with ADK (Adenosine Kinase) was observed. ADK mainly localized in cytoplasm, was a nucleoside kinase intended for catalyzing the Adenosine mono phosphate (AMP) synthesis from ATP and adenosine, as well as its activity represented a part of response to the virus infection.

Evidences to date indicated that geminiviruses target ADK in order to suppress antiviral RNA silencing, probably by inhibiting methyl cycle (Wang et al., 2005; Raja et al., 2008; Buchmann et al., 2009). As ADK is vital for supporting S-adenosyl- methionine- (SAM) as well as SAM dependent methyl transferase activity, yeast and Arabidopsis lacking ADK have reduced methylation (Lecoq et al., 2001; Weretilnyk et al., 2001; Moffatt et al., 2002; Schoor and Moffatt, 2004). As methylation of DNA and histone are known to be linked with RNA silencing, which propose a connection among AL2/C2 induced ADK inhibition and viral pathogenesis. It is suggested in recent reports that AL2 by preventing cellular transmethylation reactions, suppress

35 transcriptional gene silencing, therefore counteracting viral chromatin methylation (Buchmann et al., 2009).

REn, encoded by mostly dicot infecting geminiviruses also called as AL3/AC3, C3. AC3 is ~16 kDa protein; this protein mainly increases viral DNA accretion (as much as 50 folds) by unknown mechanism (Elmer et al., 1988; Sunter et al., 1990; Castillo et al., 2003), via Rep interaction (Settlage et al., 2005). It also caused symptoms and viral infection development in plants (Hormuzdi and Bisaro,1995; Sung and Coutts, 1995b), as well as stimulates viral DNA replication (Pasumarthy et al., 2011). This is absent in few curtoviruses and all masteroviruses, REn lacking masteroviruses use RepA to execute same functions (Settlage et al., 2001; Castillo et al., 2003). A distinctive curtovirus BCTIV in Iran consists of a novel nonanuleotide (TAAGATT/CC) sequence with an exclusive nick site, lacking REn and C4 ORFs (Yazdi et al., 2008).

Experimental interpretations proposed that REn might enhance Rep affinity for the origin (Mohr et al., 1990). Complementation studies discovered REn could proceed on heterologous viruses (Sunter et al., 1994). It is needed for optimal replication of ssDNA genomes as REn after interaction with Rep starts RCR. REn can interact with 02 host encoded proteins, PCNA and pRBR. It was proposed these proteins interact to contribute to REn function (Castillo et al., 2003). By using REn protein of TYLCV, mutations impact was examined on amino acids those were conserved, among REn protein family, upon protein interaction and replication enhancement. Replication enhancement activity of C3 was unaffected in tobacco protoplasts, by many mutations. Some mutations were detrimental for REn replication function or some had enhanced replication function. Mutated protein analysis in yeast two hybrid assays exhibited that certain mutations inactivated REn replication enhancement ability. In addition, inactivated or reduced REn oligomerization can abolish PCNA and Rep interactions (Settlage et al., 2001). But sometimes, C3 mutated proteins weakens pRBR binding. Middle of C3 protein contained hydrophobic residues which were concerned for REn interaction with Rep, itself and PCNA, but both C and N termini with polar residue were significant for the C3-pRBR interactions. This indicated the significance of interactions among C3-C3, C3-PCNA and C3-C1, in geminiviruses replication. However evidences proposed that REn performs protein-protein interaction, as no catalytic action had been established for this protein (Settlage et al.,

36

2005; Pasumarthy et al., 2010). TGMV carrying mutations in REn gene was less efficiently replicating as compare to wild-type counterpart (showing lesser viral DNA levels) along with delayed and attenuated symptoms (Sunter et al., 1990). Expression of the REn associated with Tomato leaf curl virus (ToLCV) by TMV vector, caused N. benthamiana stunting which may be credited to the cell cycle interfering proteins, probably by interaction with RBR (Selth et al., 2004). PVX-mediated expression of TYLCV REn led to the major increase in the miR159 accumulation (Settlage et al., 2005).

With the help of yeast two hybrid system and REn protein from Tomato yellow leaf curl Sardinia virus (TYLCSV) as bait, 03 clones of PCNA were obtained from Arabidopsis thaliana. REn use to interact with tomato PCNA (LePCNA) was demonstrated by pull-down assay and two-hybrid technology. LePCNA, REn binding domain was located between 132 and 187 amino acids by the truncated protein analysis, while every REn deletion used either eliminate or severely reduced its capability to interact with PCNA. Tomato PCNA also interacts with TYLCSV Rep (Castillo et al., 2003). REn interact with Rep as well as plant factors by the synthesis of multimeric complexes (Selth et al., 2004; Settlage et al., 2005).

C4/AC4, AL4, a significant protein for plant/virus interactions which displayed diverse functions (Vanitharani et al., 2004; Chellappan et al., 2005). AC4 is highly inconsistent among all begomoviruses, expressed from single ORF, embedded in Rep protein coding sequence, but its reading frame is entirely different (Hanley-Bowdoin et al., 1999). It in begomoviruses might have implicated intercellular movement and viral defense (Vanitharani et al., 2004; Gopal et al., 2007). C4 has also been concerned for monopartite geminiviruses movement (Jupin et al., 1994; Rojas et al., 2001). C4 encoded by the curtoviruses (monopartite) acts as movement proteins such as NSP and MP in bipartite geminiviruses in order to shuttle between nuclear membrane and cellular periphery/ or between cytoplasm and nucleus (Kotlizky et al., 2000; Teng et al., 2010). Although its function is still not so clear, but for several viruses act as pathogenicity determinant as well as PTGS suppressor by siRNA binding (Vanitharani et al., 2004; Bisaro, 2006; Gopal et al., 2007; Zrachya et al., 2007; Glick et al., 2008; Saeed et al., 2008; Pandey et al., 2009; Amin et al., 2011b). In a report, its involvement in gene silencing suppression synergistically with ACMV AC2 was shown (Vanitharani et al., 2004).

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In a yeast two hybrid assay C4 of BCTV showed interaction with 02 members of the shaggy-related protein kinase family (AtSKζ and AtSKη) as well as with leucine rich repeat and receptor like Kinase (LRR-RLK). TGMV AC4 although bound with alike efficiency with AtSKζ and AtSKη, but was not able to interact to LRR-RLK. BCTV C4 and TGMV AC4 interaction with AtSKη proposed, proteins interaction in brassinosteroid signaling pathway. BCTV C4 was localized in cell periphery in N. benthamiana, due to N-terminal myristoylation motif which was consistent with the plasma membrane targeting (Piroux et al., 2007).

C4 protein of ToLCV in a yeast two hybrid assay, acts as gene silencing suppressor as well as interacts with the novel shaggy-like kinase (SISK; Dogra et al., 2009). Sri Lankan cassava mosaic virus (SLCMV) and ACMV in bipartite begomoviruses, their C4 was found homologue of Beet severe curly top virus (BSCTV) C4 which by binding miRNA and siRNA can suppress PTGS (Vanitharani et al., 2004; Chellappan et al., 2005).

In BCTV previously it was studied C4 was considered liable for symptom determination, C4 mutations caused relatively different symptoms in host plants as produced by wild C4 (Frischmuth and Stanley, 1992; Iqbal et al., 2012). As Teng et al. (2010) described the functional analysis of curtovirus (BSCTV) C4 protein. DNA replication and viral infectivity analysis of BSCTV and C4 lacking BSTCV mutant upon inoculation of N. benthamiana and Arabidopsis indicated C4 was found essential for the disease symptoms development but not needed for the viral DNA replication. BSCTV C4 mutations damaged viral DNA accumulation into newly emerging host plant leaves, in contrast, wild-type virus was found in newly emerged leaves of N. benthamiana as well as Arabidopsis. BSTCV C4 mutant retained their ability to replicate, indicating this C4 ORF not required for the replication. Further they found C4 expression in plants can assists C4 lacking BSCTV mutant to move systemically. C4 localized in cytosol and nucleus in Arabidopsis protoplast and N. benthamiana leaves, seemed to bind with viral DNA as well as ds/ssDNA nonspecifically indicating novel DNA binding characteristics.

AC4 in bipartite begomoviruses had been shown its role as gene silencing suppressor (Vanitharani et al., 2004), as it obstructed the miRNA pathway and acts as symptoms determinant (Chellappan et al., 2005). Likewise in monopartite geminiviruses C4 had been found to be anticipated in symptoms determination (Latham et al., 1997; Krake

38 et al., 1998). In monopartite begomoviruses those are associated with the betasatellites; βC1 had been involved in suppression of PTGS and determination of symptoms (Bisaro, 2006; Briddon and Stanley, 2006). This indicated that C2 and C4 functions should be explained, as done by Gopal et al. (2007) they aimed to understand the role of βC1, C2 and C4 of BYVMV. They found C4 and βC1 were strong PTGS suppressors while C2 was found involved in transactivation and gene silencing suppression.

A new begomovirus species discovered from China (province Yunnan) from Malvastrum coromandelianum and tomato due to that, it was named as Tomato leaf curl Yunnan virus (TLCYnV). Whole genome of TLCYnV has great sequence similarity with Tomato yellow leaf curl China virus (TYLCCNV) except the sequence of C4 gene. Inoculation of TLCYnV by Agrobacterium showed it was highly infectious for the diverse range of plants species but less infectious for M. coromandelianum. As compare to TYLCCNV, this virus showed infection in tomato without betasatellite. By the transgenic expression it was demonstrated that C4 protein of TYLCCNV did not bring developmental disorders but C4 of TLCYnV act opposite in N. benthamiana evocative for virus symptoms. However, its genome in plants was less methylated than TYLCCNV, so its C4 protein demonstrated TGS and PTGS more efficiently than TYLCCNV C4. This indicated TYCYnV evolution from TYLCCNV by recombination which turned its C4 more virulent even without betasatellite requirement (Xie et al., 2003).

Numerous infections caused by monopartite (begomoviruses and curtoviruses) geminiviruses were considered linked with particular vein-swelling phenotype that was due to cell deregulation (hyperplasia) in phloem, as studied in BCTV (Stanley et al., 1986; Park et al., 2004b). Product of C4 of BCTV was responsible for such phenotypes which were demonstrated by genetic analysis (Stanley and Latham, 1992; Stanley et al., 1992). C4 expression in transgenic N. benthamiana also showed ectopic cell division which led to develop severe phenotype due to abnormal development of tissues (Latham et al., 1997). As C4 embedded in Rep coding sequence, showed their expression coordination which might be expected if Rep control S-phase entry and C4 elicit successive cell division. In contrast TGMV (bipartite begomovirus homologue) AC4 did not produce disease phenotype (Pooma and Petty, 1996). This might be due to second genetic component encoding proteins for inter and intracellular movement.

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However, this was not always same, as AC4 of SLCMV and ACMV (both bipartite begomoviruses) upon expression as transgene can cause developmental abnormalities, but they also have capacity to bind to siRNAs and miRNAs for PTGS suppression (Vanitharani et al., 2004; Chellappan et al., 2005).

Piroux et al. (2007) had performed experiment in order to map BCTV region significant for phenotype production (pathogenicity) by C4 and AC4 recombinants via PVX vector in N. benthamiana expression. Proteins separated into 03 regions, among those central region produced symptoms similar to wild type C4 of BCTV.

Virion-sense strand encoded protein called coat protein also termed as (CP, AV1, V1) responsible for virus encapsidation and transmission via whitefly vector (Rojas et al., 2001; Zrachya et al., 2007; Sharma and Ikegami, 2009; Amin et al., 2011a), ssDNA accumulation, inter and intracellular movement (Boulton et al., 1993). It might be involved in limiting DNA copy number of virus by Rep activity down-regulation, particularly nicking (Yadava et al., 2010).

CP binds to ssDNA and dsDNA as experimental evidences are accessible and demonstrated that binding was not in sequence specific manner (Liu et al., 1997; Lazarowitz and Beachy, 1999; Liu, 2008). DNA binding domain was located at the N terminal of this protein. Tomato leaf curl Bangalore virus (ToLCBV-[Ban5]) CP also nonspecifically binds to ssDNA via a C2H2 type conserved zinc finger motif which was considered accountable for this interaction (Kirthi and Savithri 2003). Moreover SNL sequence found in N-terminal of CP was shown concerned for viral DNA transport into the nucleus (Kunik et al., 1998; Palanichelvam et al., 1998). Studies on TYLCV CP had indicated that CP also involved in mediating ssDNA and dsDNA export (Rojas et al., 2001). Studies led to conclusion that CP is multifunctional. According to Padidam et al. (1995) truncation of ToLCNDV CP, after 65 or 172 amino acids did not change symptom development and systemic movement in N. benthamiana and tomato.

In monopartite geminiviruses CP is required for systemic infections (Noris et al., 1998), but not extremely vital for this process (Guevara-González et al., 1999). Rojas et al. (2001) explained a probable model in whitefly-transmitted monopartite begomoviruses for CP-mediated nuclear export of viral DNA as well as V1- mediated release of viral DNA, to the cell periphery. According to their study C4 remained at

40 cell periphery and had very less ability to mediate movement of viral DNA from cell to cell (Rojas et al., 2001).

Zhang et al. (2001) and Bottcher et al. (2004) explained structure of MSV geminate particle, which consists of 110 CP copies properly fit into the reconstructed density map, arranged as 22 pentameric capsomers. It is very vital for movement and systemic infection in monopartite but nonessential in bipartite begomoviruses for same activities. V1 TYLCSV mutants caused infection in N. benthamiana that was asymptomatic and contained decreased level of viral DNA, but in tomato these mutants were not able to produce infection (Wartig et al., 1997). Deletions in the nuclear targeting sequences located in the N-terminus of CP of ACMV restricted the assembly of twinned particle (Unseld et al., 2004). Evidence for CP involvement in ssDNA uptake on initial infection, obtained from microinjection experiment with ssDNA of MSV for which it was explained that in CP absence, injected DNA did not enter nucleus, but upon CP and DNA conjunction it was shown DNA swiftly transported into nucleus (Liu et al., 1999).

CP involvement in movement was confirmed by Liu et al. (2001a), according to their mutational analysis, they showed that there was a reduced level of viral DNA in infected plants, but it was unaffected in protoplast, resultant into impairment of virus movement. They also demonstrated that single amino acid alteration (lysine [K] substitution at 182 position with valine [V]), in a very conserved motif of coat protein of MSV, abolished systemic infection in maize plants indicating mutation affect on virion formation by virus particle instability or encapsidation defect.

CP of TYLCV was found responsible for DNA export and import in nucleus and anticipated for DNA shuttling into nucleoplasm and cytoplasm indicating their homology with BV1 of bipartite begomoviruses. TYLCSV, a closely related species to TYLCV, its CP gene disruption abolished its ability of symptoms development and viral DNA accumulation in tomato and N. benthamiana (Wartig et al., 1997). Moreover CP mutant of TYLCSV isolated from Sicily, proposed virions requirement for systemic infection (Noris et al., 1998). V1 mutants of TYLCSV had caused symptomless infections as well as decreased DNA level in N. benthamiana plants, while in tomato were not essentially infectious (Wartig et al., 1997). Analysis of monopartite ToLCV have shown that CP was needed for systemic infection (Rigden

41 et al., 1993; Rigden et al., 1994). These results indicated that CP was necessary for systemic infections in monopartite begomoviruses (Rojas et al., 2001).

TYLCV CP was found localized into nucleus, by fusing CP with GUC (β- glucuronidase) reporter enzyme in order to assay nuclear import into the petunia protoplast (Kunik et al., 1998). However its localization into nucleus was also seek out by observing nuclear uptake of coat protein in drosophila embryos by using purified fluorescently labeled CP via microinjection. These assays confirmed the contribution of an active process of nuclear import through a NLS specific pathway, for the transport purpose of CP among plant and insect cell-nuclei. NLS sequence was recognized in CP at amino terminus. CP of MYMV and Rice tungro bacilliform virus (RTBV) were observed to have 02 NLS at least. NLS of RTBV CP were found positioned within N- and C- terminal regions both (residues 479RPK/ 497KRK and 744KRK/758RRK) while in case of CP of MYMV located within the N-terminal region (residues 3KR and 14KRRK). CPs nuclear import in case of RTBV as well as MYMV both was being mediated via a nuclear import factor, importin α-dependent pathway (Guerra-Peraza et al., 2005).

CP C-terminal also recognized to have nuclear export signal (NES) needed for host receptors recognition. TYLCV CP was found localized in cell periphery, around the nucleus and had capability of plasmodesmata interaction which enhances their size exclusion limit (SEL) and facilitate intercellular virus movement (Rojas et al., 2001). TYLCV CP has also been recognized to have C-terminal located NES (Rhee et al., 2000).

However, the role of CP in systemic infection i.e. cell to cell and long distance movement depends on particular geminivirus-host combination. But in few bipartite begomoviruses, in several host plant species, CP was found not needed for systemic infection as well as symptoms development (Azzam et al., 1994; Ingham et al., 1995; Pooma et al., 1996; Sudarshana et al., 1998; Guevara-González et al., 1999; Wang et al., 1999). In opposite case CP was needed for systemic infection in monopartite begomoviruses (Wartig et al., 1997), curtoviruses (Briddon et al., 1989), and masteriviruses (Boulton et al., 1989; Liu et al., 1998b). Inspite of this, which type of geminiviruses was studied, CP was vital for insect transmission and as vector specificity determinant (Hofer et al., 1997; Höhnle et al., 2001; Briddon et al., 1990).

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According to Briddon et al. (1989) CP of BCTV was necessary for infectivity determinant, with the help of Agrobacterium-mediated inoculation system they expressed BCTV CP mutant were non-infectious indicating CP is vital part for infectivity. It however was complemented by another mutant (having lethal mutation in Rep) co-inoculation. When was electroporated in protoplasts of N. tabacum, CP mutant maintained replicative function for dsDNA and ssDNA both. Likewise Boulton et al. (1989) by different mutational analyses showed the infectivity role of MSV CP. ToLCV CP destruction, led to interruption of virus spread (Rigden et al., 1993).

CP is also responsible for insect vector specificity and transmission. ACMV which is a whitefly transmissible bipartite begomovirus, its CP was replaced by BCTV (curtoviruses) transmitted by leafhopper so a chimeric virus was constructed. Leafhopper vector of BCTV transmitted chimeric virus and BCTV both, but ACMV was not, indicating CP role as insect vector specificity determinant (Briddon et al., 1990). Virus transmission via insect vector required safe passage from plant to insect as well as again to plant. Endosymbionts residing in whitefly body produced GroEL protein. TYLCV CP sequences were found to interact with GroEL in vector haemolymph to make sure virus maintenance and its safe passage (Rana et al., 2012; Yaakov et al., 2011). According to Liu et al. (2001b) and Unseld et al. (2001) central domain of CP was considered essential for transmission by vector.

AV2/V2 encodes pre coat protein (Pre CP), which is responsible for viral movement inside plants (Wartig et al., 1997; Hanley-Bowdoin et al., 1999; Harrison and Robinson, 1999; Sharma and Ikegami, 2009; Ho et al., 2014). It also overcome ds RNA called RNA interference (RNAi) triggered host defenses. So V2 of numerous begomoviruses has been shown as PTGS suppressor (Rojas et al., 2001; Zrachya et al., 2007; Glick et al., 2008; Yadava et al., 2010; Amin et al., 2011a). In monopartite begomoviruses V2 is considered concerned for viral spread (Padidam et al., 1996; Wartig et al., 1997; Hanley-Bowdoin et al., 1999). In OW begomoviruses Pre CP found involved for monopartite begomoviruses movement but in bipartite its role is not very clear (Rothenstein et al., 2007). NW begomoviruses are V2 lacking, but 02 OW begomoviruses (CoYVV and CoGMV) are known without V2 and are being considered as viruses remnants those who transmitted to NW from OW (Ha et al., 2006; Ha et al., 2008).

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This protein contains 112 aminoacids, concerned for symptoms development and viral DNA accumulation (Padidam et al., 1996). V2 gene overlaps a tiny CP portion and its effect on CP gene was studied by Bull et al. (2007). As V2 mutations did not show any outstanding effect on expression of CP, and no down regulation of CP was noted, but when AV2 and AV2/CP double mutants were inoculated into plants, DNA accumulation was very less and very mild symptoms were shown. In case of bipartite begomoviruses AV2 mutational analysis showed its involvement for movement function (Padidam et al., 1996). Intercellular viral transport was examined by the use of GFP as reporter gene by Rothenstein et al. (2007) for this purpose constructs (AV2:GFP) expressed in plasmids and replicating viruses were localized into cell periphery making cytoplasmic and nuclear inclusion bodies. AV2:GFP construct transportation into adjacent cells of epidermal tissue was shown by particle bombardment. V2 in monopartite begomoviruses showed similar localization pattern as those of MP in bipartite begomoviruses. In TYLCV, V2 localizes at cell periphery, around nucleus and co-localizes with the endoplasmic reticulum. However Hak et al. (2015) described that TYLCV-Is movement in planta did not require V2, as they demonstrated that V2 mutants TYLCV-Is as well as its related viruses had tendency for induction of symptomless infections along with attenuated viral DNA level in the protoplasts, indicates V2 role as movement protein. Due to the discovery of plant silencing and viral silencing suppression mechanism, V2 role in viral movement was reconsidered. They studied two mutants, one with abolished V2 suppression activity, other with nontranslatable V2, both spread to newly emerged leaves alike to wild type, however their DNA accumulation levels as compare to wild were lower (10 folds) suggesting reduced virus proliferation, formerly ascribe to movement hindrance, is because of silencing suppression activity.

AV2 pathogenicity depends on conserved motif 40TxR42, a protein kinase C (PKC) phosphorylation motif (Rojas et al., 2001; Chowda-Reddy et al., 2008).

By the mutagenesis studies of AV2 of East African cassava mosaic Zanzibaar virus (EACMZV; Bull et al., 2007) suggested no vital role for AV2 in pathogenicity and symptom development, but in contrast symptoms attenuation was observed. Plants contain antiviral defense system which is counteracted with suppress proteins responsible for blocking RNA silencing pathway, produced by viruses (Baulcombe, 2002; Bisaro, 2006). Monopartite begomoviruses V2 by different reports have been

44 proved working as RNA silencing pathway suppressor. TYLCV V2 was shown to directly interact with host SGS3 in planta, this interaction was credited for V2 suppressor activity, for RNA silencing suppression (Glick et al., 2008). TYLCV-Is V2 targets step of RNA silencing pathway through Dicer-mediated dsRNA cleavage. GFP as transgene was used in this experiment, increased GFP transcript and protein level was observed after RNA silencing inhibition by V2 (Zrachya et al., 2007).

BC1, BL1 and MP are synonyms referred for complementary-sense gene (Gardiner et al., 1988; Pooma et al., 1996; Stanley and Townsend, 1986), MP found in NW begomoviruses (Ho et al., 2014). DNA B of bipartite begomoviruses encodes 02 i) NSP ii) MP. Those are needed for virus spread between and within cells (Rojas et al., 2005), MP shuttles among cellular periphery and nuclear envelop. By NSP interaction and cooperation MP binds with DNA, this binding is size and form specific (Rojas et al., 2005). MP mediates dsDNA movement and localized at plasmodesmata level (Noueiry et al., 1994). A ''couple-skating model'' and ''relay race model'' had been proposed to explain the bipartite begomoviruses movement (Rojas et al., 1998; Hehnle et al., 2004). In relay race model, NSP firstly binds with dsDNA and transfers dsDNA from nucleus to cytoplasm where MP takes over for crossing by plasmodesmata. In couple skating model, first of all NSP binds with ssDNA of virus in the nucleus, transfers it to cytoplasm, where MP interacts with a DNA-NSP complex in order to facilitate intracellular movement. For Bean dwarf mosaic virus (BDMV) intracellular movement, both proteins are needed, mutation in any (NSP and MP) of 02 proteins inhibited viral DNA movement from cell to cell (Sudarshana et al., 1998).

An electron microscopic study of AbMV NSP has discovered the redirection of NSP, towards plasma membrane was found promoted by MP in fission yeast (Frischmuth et al., 2007). Mungbeen yellow mosaic India virus (MYMIV) MP transgenic expression in N. benthamiana showed, that this MP was localized around host epidermal cells (Radhakrishnan et al., 2008), where as AbMV MP was localized at plasmodesmata and plasma membrane and this protein supports NSP redirection to the plasma membrane in the fission yeast (Kleinow et al., 2009).

Both domains (anchor and pilot domain) of MP are significant for intracellular movement, anchor domain was necessary for localization at cell periphery. This domain may consist of an amphiphilic helix which functions to fasten protein on the

45 membrane leaflet (Zhang et al., 2002). Plasmodesmata and MP both for the intracellular movement delimit viral genome size (Gilbertson et al., 2003). In injected cells, MP of BDMV was involved for cell to cell transport of viral DNA by increasing the SEL of plasmodesmata (Noueiry et al., 1994).

Participation of begomovirus MP for symptoms development had also been reported. Squash leaf curl virus (SqLCV) MP and Tomato mottle virus (ToMoV) MP constitutive expression in N. benthamiana and N. tabacum respectively, showed virus like symptoms demonstrating its role for pathogenicity (Duan et al., 1997). Similar study was carried out by Pascal et al. (1993) via transgenic expression of SqLCV MP and NSP in plants. Their results explained that MP expression merely is sufficient to induce particular SqLCV symptoms (mosaic and leaf curling), results indicated function of MP in bipartite geminiviruses, as symptoms determinant.

Saunders et al. (2001b) had studied discrete disease phenotypes shown by 02 TGMV [common strain (cs) and yellow vein (yv) strain] strains which were determined via nucleotide variation in MP encoding gene at 3'-teminal region. In N. benthamiana, bipartite begomoviruses csTGMV caused extensive chlorosis, yvTGMV in systemically infected leaves caused veinal chlorosis but in Datura stramonium, yvTGMV caused only tiny chlorotic lesions in systemically infected leaves, while csTGMV produce severe chlorotic mosaic and leaf distortion. Site-directed mutagenesis and genetic recombination studies, by using infectious clones of yvTGMV and csTGMV, helped to recognize the role of MP encoding gene for the production of symptom. At 272 MP amino acids, either isoleucine (yvTGMV) or valine (csTGMV), they influenced symptoms by intermediate phenotype induction in both hosts, when were exchanged among 02 strains. When an additional strain- specific amino acid of MP at 288 position was exchanged, either lysine (yvTGMV), or glutamine (csTGMV) leading to the symptoms phenotype change to that of other strain.

Nuclear shuttle protein (NSP), BR1 and BV1 are synonyms referred for virion sense gene. This protein has the capability of ssDNA and dsDNA binding and to shuttle between cytoplasm and nucleus (Hehnle et al., 2004; Zhou et al., 2011). It is considered as multifunctional protein and its synthesis is being regulated at the transcription level by TrAP (Sunter and Bisaro, 1991). As geminiviruses replication occurs in the nucleus, due to which they have to travel from cell to cell, as well as

46 from cytoplasm to nucleus. NSP supported viral DNA nucleocytoplasmic trafficking. Current model for cell to cell movement of bipartite begomoviruses proposed that BV1 coordinate viral DNA movement from nucleus to cytoplasm via nuclear pore complex (NPC), while BC1mediate movement from cell to cell by plasmodesmata across cell wall (Lazarowitz and Beachy, 1999; Gafni and Epel, 2002; Rojas et al., 2005).

NSP or MP role for the pathogenicity of a bipartite begomovirus (ToLCNDV) was also studied. Both MP and NSP genes were expressed in plants (L. esculentum, N. benthamiana and N. tabacum) by PVX or via stable transformation of the gene construct under 35S promoter control in N. tabacum. Expression of NSP by PVX in N. benthamiana showed leaf curling particular to those caused by ToLCNDV in L. esculentum and N. tabacum produced hypersensitive response (HR) indicated NSP of ToLCNDV was a target of the host defense response in following host. No phenotypic variation was seen when MP was expressed by same PVX vector in any of hosts. When NSP was expressed under 35S promoter control, as transgene led to necrotic lesions formation in expanded leaves which started from a point latter spread whole leaf. Deletion from N-terminal of NSP for 60-100 aminoacids abolished HR response, indicating need of these sequence for HR response while 100 Amino acid deletion at C-terminal, did produce HR response indicating no role of this region in HR. So it was indicated by experiment that NSP of ToLCNDV is determinant of pathogenicity and targets of host plant defense responses (Hussain et al., 2005).

NSP indicate strong and sequence-independent affinity for ssDNA and dsDNA both (Hehnle et al., 2004). NSP is highly basic protein and contained 02 NLS. Mutations in any of the 02 NLSs led to abolish viral infectivity. NSP C-terminal is considered to be involved in MP interaction (Sanderfoot and Lazarowitz, 1996). NSP interacts with different host factors in order to complete viral movement task, which include acetyl transferase from Arabidopsis thaliana, and a receptor-like protein kinase from soybean and tomato, NSP-interacting kinase (NIK), Proline-rich extension-like receptor protein kinase (PERK) and NSP interacting GTPase (NIG). Separation of ssDNA from that of dsDNA for encapsidation and movement may required acetylation, whereas NSP phosphorylation by kinase might be supportive in protein masking by triggering host resistance reaction during the viral DNA movement process, while interaction with NIG increases viral protein (nucleus to cytoplasm)

47 translocation, so redirect viral DNA towards cell surface in order to interact with viral MP (Mcgarry et al., 2003; Mariano et al., 2004; Florentino et al., 2006; Carvalho et al., 2008; Santos et al., 2010).

The term "satellites" first was used in 1962 by Kassanis describing a subviral agent associated with Necrovirus called as Tobacco necrosis satellite virus (TNSV; Kassanis, 1962). In 1969 first satellite virus was recognized and characterized called RNA-sat, found associated with nepovirus Tobacco ringspot virus (TRSV; Schneider, 1969). After that a huge number of RNA satellites were found associated with many plant viruses (Mayo et al., 2005). Majority of the satellites consists of RNA and found associated with RNA genomes containing viruses and have no distinct effects on the symptoms shown in plants by their helper viruses (Hu et al., 2009). But few such satellites were recognized for their ability to strengthen the infection and to show novel symptoms may not otherwise being produced by virus merely (Collmer and Howell, 1992; Zhang et al., 2015).

Dry et al. (1997) for first time published a report on satellites associated with begomoviruses, found with ssDNA of ToLCV isolated from Australia. ToLCV-sat was circular, very small consist of 682 nt, share very little sequence homology expect for sequences inside the apex of the 02 stem-loop structures i.e. first having nonanucleotide (TAATATTAC) while second having ToLCV Rep binging motif and encodes not even a single protein (Behjatnia et al., 1998). ToLCV-sat has all the particular characteristics of the true satellite, although it does not play any role in symptoms development but for the process of replication, encapsidation, inter and intracellular movement, it needs ToLCV. Another satellite molecule, structurally similar to that of ToLCV-sat was identified latter, associated with NW bipartite begomoviruses (Fiallo-Olivé et al., 2012). Many begomovirus infected plants also have half sized betasatellite defective mutant which resemble in structure to satellites (Briddon et al., 2003; Akhtar et al., 2014).

Betasatellite for the first time was identified from Ageratum conyzoides infected by monopartite begomovirus Ageratum yellow vein virus (AYVV); this single genomic component was considered infectious (Tan et al., 1995). Cloned genomes of this virus show symptoms after inoculation into N. benthamiana but it was non infectious or weakly infectious for the host (Ageratum plant; Saunders and Stanley, 1999). When AYVV was inoculated with DNA component, cloned genome show particular

48 symptoms in host (Saunders et al., 2000; Briddon et al., 2001). This specific component was named as DNAβ at that time but after that, it was renamed as betasatellite (Briddon and Stanley, 2008). Transmission of both of these components and disease spread in Ageratum established the etiology of disease. Upon single geminivirus component inoculation, particular symptoms were not shown which led to ambiguity in etiology for a long duration in cotton leaf curl disease. Later on an entire set of genomic components which was necessary for the particular symptoms induction in cotton leaf curl disease, was known as the betasatellite (Briddon and Markham, 2001).

However sometimes, as it was shown by studies that, for betasatellites is not compulsory to induce symptoms but it might enhance symptoms severity induced by their helper virus as studied in Tobacco curly shoot virus (TbCSV) related satellite. Due to the reason TbCSV may be considered as evolutionary intermediary among monopartite as well as betasatellite associated monopartite begomoviruses (Li et al., 2005).

Since the discovery of betasatellites about 02 decade ago, more than 400 complete betasatellites sequences had been submitted in sequence database indicating isolation easiness as well as agricultural significance (Briddon et al., 2003; Briddon et al., 2008; Sattar et al., 2013). Monopartite begomoviruses containing betasatellites were found widespread all around the OW (Andleeb et al., 2010).

Diversity in betasatellites investigated by Briddon et al. (2003) indicated diversity centre lies inIndian subcontinent (South Asia) and Southern China or in South East Asia; indicating possible centre of betasatellites origin. Numerous such new complexes had been isolated from a wide range of plants growing still, all around the Africa and Asia (Amin et al., 2002; Briddon et al., 2003; Jose and Usha, 2003; Bull et al., 2004; Cui et al., 2004a; Cui et al., 2004b; Jiang and Zhou, 2004; Jiang and Zhou, 2005; Rouhibakhsh and Malathi, 2005; Were et al., 2005; Wu and Zhou, 2005; Xiong et al., 2005). A parallel study based on larger number of sequences was conducted by Nawaz-Ul-Rehman and Fauquet (2009) set the centre of origin and diversity in South East Asia. Betasatellites had been divided into 02 groups on the basis of phylogeny. First those isolated from host plants which are members of family Malvaceae (as cotton, okra, hollyhock and hibiscus) but second consists of betasatellites isolated

49 from (non-malvaceous) plants (as tomato, chilies, Ageratum, Zinnia, honeysuckle etc.)

Betasatellites encoded βC1 protein is considered responsible for all functions performed by betasatellite, known up till now (Cui et al., 2005; Saeed et al., 2007).

In contrast to ToLCV-sat, betasatellites boost their helper begomoviruses accumulation, increase infection symptoms in a number of host plants (Saunders et al., 2000; Briddon et al., 2001; Nawaz-Ul-Rehman and Fauquet, 2009; Patil and Fauquet, 2010), virus movement (Cui et al., 2005; Kon et al., 2007; Qazi et al., 2007; Saeed et al., 2007; Amin et al., 2011b; Iqbal et al., 2012), and most probably because of the silencing suppressor action of βC1 protein (Cui et al., 2005).

DNA-A part of monopartite begomoviruses consists of C4 act as symptoms determinants. DNA-A mutants having C4 deletion can induce symptoms in the presence of betasatellites indicating that βC1 act as a strong inducer for diseases symptoms induction (Saeed et al., 2008; Zhang et al., 2015). It cause abnormal phenotypic expression in transgenic tobacco plants when it was expressed under 35S promoter of Cauliflower mosaic virus (CaMV; Cui et al., 2004a; Saunders et al., 2004; Saeed et al., 2005). Systemic expression shown by βC1 with potato virus X (PVX) vector led to the vein thickening, darkening and enation like symptoms development in Nicotiana tabacum plant (Amin et al., 2011a). Even same results found when similar studies were done for N. benthamiana (Kon et al., 2007), but it has been considered that βC1 alone was not sufficient for the development of symptoms as shown in studies carried by Ding et al. (2009). βC1 self-interaction in In vivo and In vitro forms large granular soluble bodies and this interaction is needed for induction of symptoms in host plant (Cheng et al., 2011). βC1 was localized towards the periphery of cells was studied with the aid of transient expression of the green fluorescent protein (GFP) which fused with βC1 and contained export and import signals (Kumar et al., 2006). Sequence analysis of βC1 indicated that it contained isoleucine and leucin loaded region near C-terminal end that might be accountable for peripheral localization and nuclear shuttle activity (Cheng et al., 2011).

In the case of ToLCNDV (bipartite), when its DNA B is missing, betasatellite acts as functional complement of DNA B and cause infection which indicated that betasatellites encode for movement functions (Saeed et al., 2007; Saeed et al., 2008).

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βC1 of Cotton leaf curl Multan betasatellite (CLCuMuB) when was expressed via PVX vector in N. benthamiana resultant into miRNA regulation, concerned for developmental processes. Intensity of miR164, miR165, miR166, miR169 and miR170 was less but there was prominent increase in miR159 and miR160 observed among infected plants (Amin et al., 2011a). A well-known fact about pathogenicity determination ability of βC1 was found, but more recent studies indicated that influence of A-rich region part of βC1 promoter was considered vital for symptoms development, that might be due to temporal and spatial βC1 regulation by promoter (Ding et al., 2009). According to Kon et al. (2007) βC1 was considered suppressor for PTGS as well as implicated for RNA mediated defense of host. As described by them sequences upstream of βC1 start codon and A-rich had been contributing for pathogenicity (Kumar et al., 2006). For few viruses e.g. TYLCCNV, betasatellite was found vital for typical symptoms induction in those plant hosts, they were isolated from (Cui et al., 2004a). BC1 encoded by TYLCCNB act as pathogenicity factor (Cui et al., 2004a), suppressor of both PTGS and TGS (Cui et al., 2005; Yang et al., 2011). It also interfere host auxin and jasmonate signaling pathways (Yang et al., 2008).

Alphasatellites role in begomoviruses disease etiology is ambiguous (Briddon and Stanley, 2006; Nawaz-Ul-Rehman and Fauquet, 2009). However, in one study Rep of Gossypium darwinii symptomless alphasatellites (GDarSLA) and Gossypium mustelinium symptomless alphasatellite (GMusSLA) showed to suppress RNA silencing (Nawaz-Ul-Rehman et al., 2010). It was usually considered that alphasatellites decrease virus infection impact leading to increase in host plant survival and further complex spread, by competing for the cellular resources (Mansoor et al., 1999; Saunders and Stanley, 1999). According to Idris et al. (2011) a strange group of alphasatellites had been involved to attenuate betasatellite associated begomoviruses by decreasing DNA accumulation of betasatellites. Symptoms establishment in tomato plants was studied by infecting plants with 01 or 02 betasatellite associated monopartite begomoviruses from Oman. Results showed establishment of attenuated symptoms in the presence of alphasatellites, so this study indicated the role of alphasatellites for modulating virulence of begomovirus betasatellite complex.

Alphasatellites previously had been recognized solely from monopartite begomovirus- betasatellite complexes infected plants. However, it was found that experimentally

51 alphasatellites were able to maintain in planta via a bipartite begomoviruses and curtoviruses, but it is not the case in masteriviruses. But, more recently, 02 discrete alphasatellites were found to be related to NW begomoviruses, they were linked with 02 weeds (Euphorbia mosaic virus and Cleome leaf crumple virus) infected by bipartite begomoviruses in Brazil, and they have conserved genome characteristics of alphasatellites, such as presence of Rep protein encoding gene, hair pin structure and A-rich region in both of the cases, like to those of alphasatellites group from Africa (Paprotka et al., 2010). In Venezuela alphasatellites like molecules were found associated with Melon chlorotic mosaic virus a bipartite begomovirus and even though its sequence from particular OW alphasatellites have diverged, it however had all genome characteristics of this kind of DNA satellite as above mentioned (Romay et al., 2010).

All begomoviruses are transmitted by whitefly (Brown et al., 2012; Becker et al., 2015). Whitefly represents species complex i.e. the combination of greater than 30 species which are morphologically distinguishable (De Barro et al., 2011; Liu et al., 2012; Boykin and De Barro, 2014). White fly for the first time was reported as pest in India in1919, since that time it was known, with huge vast host range of about 500 plant species and 74 families (Greathead, 1986). About 60 various geminiviruses had been reported were transmitted by whitefly (Markham et al., 1994). In 1930, transmission of ACMV and Tobacco leaf curl virus (TbLCV) geminiviruses for the first time was demonstrated by B. tabaci, which caused disease in cassava and tobacco plants respectively (Storey, 1936; Storey and Nichols, 1938).

Different ways of plant virus transmission by whiteflies are well-known. Virus transmission occurs either in non persistent (require less than few hours for acquisition, stylet born, non circulative, shed in molting), semipersistant (for acquisition require minutes to hours, in foregut, retention time is from several hours to days, non circulative, shed in molting) or persistant (for acquisition require hours, in haemolymph, retention time is from days to whole life) way. In persistent transmission, virus can either replicate in propagative transmission or in circulative transmission, inside the insect (Gray and Banerjee, 1999; Ng and Falk, 2006; Hogenhout et al., 2008). Whitefly transmit begomoviruses (monopartite and bipartite) in persistant, circulative (Rosen et al., 2015) and nonpropagative way (Duffus, 1987; Hull, 2002).

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Vector transmission involves specific interaction among virus, host and vector determinants and it is variable and complex process (Andret-Link and Fuchs, 2005; Purcell and Almeida, 2005; Martinière et al., 2013). This interaction turned more complex if plants are infected by more viruses simultaneously (Salvaudon et al., 2013). Numerous transmission events by different virus containing vectors led to the ''mixed infection'' or it can be caused by single transmission via single vector having two viruses at same time. Multiple viral illnesses are very common in crops (Syller, 2012).

In Pakistan 02 biotypes have been recognized, on geographical basis, B and non-B biotype (Haider et al., 2003), non-B biotype from Pakistan and from Spain is extra proficient for transmission than B biotype from Mexico, South Africa and USA. The prevalence of disease considerably was augmented in America because of B biotype introduction from OW (Power, 2000).

Virus particles ingested by whitefly through stylet passed into gut and entered haemolymph enclosed in haemocoel. Haemolymph carries virus to salivary glands, a site from it can be egested out via saliva. Virus translocation route was studied by immunolocalization study on TYLCV. For this purpose, TYLCV-particular antiserum was used to find, immunogold lable showed it presence in stylet, then food canal, then in descending midgut and finally in lumen. Label was moreover found from microvilli. Under the light of these interpretation, microvilli were considered long term storage and virus internalization site (Brown and Czosnek, 2002; Czosnek et al., 2002; Ghanim et al., 2009; Skaljac and Ghanim, 2010).

CP along with its derivatives and non structural proteins as helper components (HC) are main determinants for transmission specificity and many proteins act as receptor such as virion-binding vector proteins (Andret-Link and Fuchs, 2005). A functional gene encode for CP is required for BCTV for transmission (Azzam et al., 1994). In an experiment, displacement of CP of ACMV (bipartite begomovirus; transmitted by whitefly) was done with CP of BCTV (curtovirus; transmitted by leafhopper). Consequently ACMV was transmitted by leafhopper, leading to the confirmation of CP role as specificity determinant for transmission via insects (Briddon et al., 1990). In similar way a non transmissible isolate of AbMV via whitefly, by substituting its CP with CP of a closely correlated Sida golden mosaic virus (SiGMV) was made transmissible (Hofer et al., 1997). In the CP of TYLCV two amino acids were

53 replaced which led to the suppression of virus transmission by whitefly but acquisition was intact (Noris et al., 1998). So epitopes for acquisition and transmission are different. Some viruses like AbMV and Honey suckle yellow vein virus (HSYVV) drop the capacity of transmission via insect vector. In nontransmissible AbMV isolate, an exchange of aminoacids in CP region at position 124 and 149 converted it whitefly transmissible (Höhnle et al., 2001).

Virus particles taken into the insect digestive systems are vulnerable for enzymatic degradation but nature has addressed this problem by acquiring third partner involved in transmission (Symbiotic bacteria) live as endosymbionts in the body of whitefly. A variety of endosymbionts in B. tabaci have been found such as Wolbachia, Hamiltonella, Cardinium, Fritschea, Rickettsia and Arsenophonus (Gueguen et al., 2010). A protein produced by these endosymbionts is a homolog of GroEL (63 kDa) and it prevents virus from degradation. Via yeast two hybrid systems, GroEL interaction with CP of viruses was observed. In translocation assay, TYLCV was found in digestive system, salivary glands and haemocoel of B. tabacci after 8 hours of sucking, but AbMV which is nontransmissible was found nowhere else digestive tract even following 96 hours after feeding. Yeast two hybrid assay led to the results showing proficient GroEL interaction with CPs of viruses. It proposed that AbMV encounters some troubles while moving from digestive tract towards haemolymph (Morin et al., 2000; Ohnesorge and Bejarano, 2009). An interaction among CP of TYLCV and BtHSP16 was found making virus passage easy. Some viral DNA as in TYLCSV, its DNA was noticed which inherited transovarially by B. tabaci progeny and this DNA latter on was identified in eggs, in nymphs and even in adults of first generation progeny (Ghanim et al., 1998; Bosco et al., 2004). TYLCV was only found to be sexually transmitted and can move among viruliferous male to female and vice versa, but not among same sex individuals (Ghanim et al., 2001).

TYLCD/ToLCD are being caused by numerous begomoviruses species and characterized by yellowing and curling of apical leaves. These diseases led to 100 % yield losses if infection occurred at an early stage of growth (Prasanna et al., 2015a), as flowers are abscised and plant growth retarded (Cohen and Lapidot, 2007). TYLCD was first identified in about 1940 in Middle East (Cohen and Antignus, 1994). TYLCV was the cause of TYLCD known for first time in early 1960 in Middle East, this virus type usually called as "Israel" type, but now this virus has

54 spread numerous NW and OW regions and it is regularly evolving through mutation and recombination (Lefeuvre et al., 2010). In 1991 isolate of TYLCV was characterized from Israel which showed monopartite genome (Navot et al., 1991). Latter it was distributed in Egypt, Jordan, Lebanon, Cyprus and Israel. Now, after that TYLCV (TYLCV-IL considered now as a 'type' isolate) has spread in NW and emerged as main constrain for tomato crop in Dominican Republic, TYLCV-IL was introduced there in the early 1990s (Salati et al., 2002). Various anecdotal reports indicated its possible spread via tomato transplants import from Israel in early 1990s, in Dominican Republic which provide well suited environment for its establishment and its rapid spread to southern and northern regions (Salati et al., 2002). Afterwards it spread to Puerto Rico, Cuba, Jamaica and other Caribbean islands. TYLCV in 1999 was reported for first time from Florida in USA from sold tomato in retail store, high winds may cause viruliferous whiteflies blown to Florida (Polston et al., 1999). Afterwards reported from Louisiana, Georgia, North Carolina, northern Mexico (Brown and Idris, 2006), and in 2007 into California (Rojas et al., 2007). Sequence analysis of these isolates have confirmed the presence of TYLCV-IL in NW. TYLCV emergence is an excellent example of possible risks linked with global plant material movements (Rojas and Gilbertson, 2008).

TYLCV is well known and best studied being major threat for tomato crop (Butterbach et al., 2014), since then different forms of TYLCV emerged in various regions and TYLCD spread in Asian countries and Pacific regions particularly Korea, Australia, Eastern China and Japan, however numerous other begomoviruses species and strains emerged in many countries of these regions. In tomato about 70 different begomoviruses are known naturally infecting this crop in various parts of the world (Tsai et al., 2011a). In Indian subcontinent, number of species has increased to the present situation than those were first identified in early 1990 (Chowda Reddy et al., 2005; Reddy et al., 2011) where ToLCD, in tomato growing areas is a major problem.

Numerous new strains had been identified from New Delhi, Lukhnow, Mirzapur, Banglore, Vadodara and Varanasi (Chakraborty et al., 2003). Among these, a few viruses like ToLCNDV (Padidam et al., 1995), ToLCPalV (Kumar et al., 2008), ToLCV are bipartite (Chakraborty et al., 2003). While others, like Tomato leaf curl Bangalore Virus (ToLCBaV; Muniyappa et al., 2000), Tomato leaf curl Kerala virus (ToLCKeV; Pasumarthy et al., 2010) , Tomato leaf curl Joydebpur virus (ToLCJV;

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Tiwari et al., 2013b), Tomato leaf curl Patna virus (ToLCPatV; Kumari et al., 2010), Tomato leaf curl Pune virus (ToLCPuV), Tomato leaf curl Rajasthan virus (ToLCRaV) have a monopartite genome. Recently identified 03 monopartite begomoviruses infecting tomato in India were Cotton leaf curl Khokhran virus- Burewala (CLCuKoV-Bu; Kumar et al., 2013), Ageratum enation virus (AEV) and TbCSV (Swarnalatha et al., 2013). While Cotton leaf curl Rajasthan virus (CLCuRV) have been identified infecting tomato in Pakistan (Shahid et al., 2009).

ToLCD and TYLCD are rapidly emerging due to the arrival of vectoring efficient B. tabacci biotypes and increased population of whiteflies carrying viruses (Khan et al., 2015). Over the previous 20 years, B as well as other biotypes had mediated the emergence of more than 100 new begomoviruses species (Rojas and Gilbertson, 2008). Likewise begomoviruses, their vector diversity has also been reported from the Philippines (Tsai et al., 2011b), Indonesia (Tsai et al., 2006; De Barro et al., 2008), Bangladesh (Maruthi et al., 2007), Thailand (Sawangjit et al., 2005) and Pakistan (Haider et al., 2003).

Geminiviruses being foremost warning to the world agriculture have caused devastating yield losses (Sahu et al., 2014). Different strategies have been adopted for begomoviruses control. These include physical measures based on catching whitefly via hands, racquets and shovels use of plastic membrane, insect preventing net or net house and use of UV absorbing plastic sheets (Cohen and Antignus, 1994).

Extracts of plant parts and crushed plants leaves had also been used as biological insecticides. Among chemical controls used, inorganic insecticides were successful at the beginning; latter led to the development of insecticidal resistance in B. tabaci (Horowitz et al., 1998). Afterwards breeding for resistance to insect vector or virus was sought to control begomoviruses (Basak, 2016). Merely controlling whitefly population is labor intensive, expensive and sometimes proves ineffective. This situation led to the use of resistant cultivars which presents a good solution for the TYLCD control (Caro et al., 2015).

So in order to address this problem, alternate control approaches for these ever evolving pathogens should be devised. Breeding for geminiviruses resistance relies on the availability of resistant genes from the wild cultivars, development of pathogen resistance associated dominant molecular markers and quick resistance introgression

56 into susceptible cultivars (Lapidot and Friedmann, 2002). However, breeding of resistant crops to enhance resistance against these DNA viruses is a big challenge (Vanderschuren et al., 2007).

Among most prevalent viruses infecting tomato crop, TYLCV has very significant economical impact all around the world (Czosnek, 2007; Verlaan et al., 2013; Caro et al., 2015), and also being caused by more than 10 begomoviruses species (Brown et al., 2015). Genetic resistance against plant viruses, if available in the germplasm, is considered one of the most efficient ways to control viral infections. The genes conferring such resistant phenotypes can be transferred to cultivated varieties by breeding often assisted by molecular markers. Against begomoviruses, very few resistance genes are known, the most important of them are the Ty series of genes available in wild tomato (Solanum chilense, S. habrochaites, S. peruvianum, S. pimpinellifolium; Ji et al., 2007a). Ty gene and its alleles had been introgressed into commercial tomato cultivars for TYLCV resistance but convenient molecular tools for its widespread use are not yet available (Verlaan et al., 2011).

To develop efficient strategies in order to grow resistant cultivars and for pest control, extrication of tomato plant response for pathogens is very essential for the proliferation of resistant cultivars which were established by the introgression of resistant genes into domestic tomato (Vidavsky et al., 1998). For this purpose 06 genes which produced tolerance type resistance against begomoviruses (referred Ty for TYLCV) had been recognized from various wild tomato species and introgressed into the cultivated tomatoes (Prasanna et al., 2015b).

These 06 main loci ranging Ty-1 to Ty-6, associated with resistance and low viral replication, had been described and mapped in tomato, which showed resistance not only against TYLCV as well as other relevant begomoviruses species, so far identified (Zamir et al., 1994; Hanson et al., 2006; Ji et al., 2007b; Anbinder et al., 2009; Ji et al., 2009; Hutton et al., 2012; Hutton and Scott, 2013).

These genes were obtained from different origins, as Ty-1 which possibly have another linked locus for resistance, located on accession LA1963 and Ty-3 on LA2779, both being present on chromosome 06 shown to be allelic (Verlaan et al., 2011; Verlaan et al., 2013), LA1932 was reported to have an allele at this locus as Ty- 3a (Scott et al., 1996; Ji et al., 2007b), Ty-4 mapped on chromosome 03 (Ji et al.,

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2009) and Ty-6 originated from accession LA2779 carried by chromosome 06 (Hutton and Scott, 2013). These all Ty-1, Ty-3, Ty-4 and Ty-6 genes were carried by various S. chilense accessions (Zamir et al., 1994; Hanson et al., 2006; Ji et al., 2007b; Anbinder et al., 2009; Ji et al., 2009; Hutton et al., 2012). Ty-2 introgressed from S. habrochaites (accessionB6013) was located on chromosome 11 (Yang et al., 2014), Ty-5 from S. peruvianum Ty172 carried by 04 chromosome, symbol ty-5 was proposed for the genes due to its recessive nature (Hutton et al., 2012).

Various Ty genes recognized may not were equally efficient for resistance in all strains or species of begomoviruses. In order to develop tomato lines suitable for wide range of viruses in various countries, and to attain long lasting and strong resistance against numerous different species/ stains of begomoviruses, a strategy adapted was to pyramid various Ty genes in a same tomato variety. Asian vegetables research and development center (AVRDC) had produced verities combining Ty-1, Ty-2, Ty-3/Ty- 3a, ty-5, 'ty6' genes. Its initial test had indicated the effectiveness of pyramidal strategy (Hanson et al., 2011). So single or combinations of resistance genes are needed to be assessed for different begomoviruses species/strains, in different areas to determine the affectivity of single or pyramidal genes in each location (De Resende et al., 2009). But breeding programmes are developing very slowly because of TYLCV resistance genetics as well as virus-vector-host interactions complexity (Ji et al., 2007a).

Different Ty resistant cultivars had been evaluated previously, indicating various level of effectiveness against variety of begomoviruses as study conducted by Shahid et al. (2013) had indicated Ty-1 and Ty-2 homozygous hybrids of tomato challenged by monopartite begomoviruses via Agrobacterium-mediated inoculation. Ty-1 hybrids were found tolerant to Honeysuckle yellow vein mosaic virus (HYVMV) as well as with many plants being neither symptoms free, nor virus free. Ty-1 hybrid response was resistant to moderately resistant against Tobacco leaf curl Japan virus (TbLCJV), while response of Ty-2 hybrids was found resistant to extremely resistant for 03 (TbLCJV, HYVMV and TYLCV) monopartite begomoviruses as compare to susceptible plants.

Pyramidal effect of 02 (Ty-2 and Ty-3) genes was detrimental against the begomoviruses species. This assessment was made based on diagnostic features of markers linked with Ty genes as well as by using marker assisted selection. In order to

58 produce pyramidal tomato lines by crosses among Ty stocks, 05 tomato lines developed were assessed for their response by agroinoculation method, while field tests were conducted in diseases hot spots. Tomato pyramidal lines could be a significant genetic resource for adequate tomato production, particularly in those regions affected by diseases caused by tomato leaf curl viruses. Ty-3 carrying and pyramidal lines showed high resistance level to 02 bipartite (ToLCNDV and ToLCPalV) and 01 monopartite (ToLCBV), however combined results of resistance test exhibited that Ty-3 was significant and showed broad spectrum resistance (Prasanna et al., 2015a).

Similarly Ty-1 resistance gene was explored which encodes for RNA-dependent RNA polymerase and it was found allelic with Ty-3. Upon TYLCV inoculation of Ty-a and Ty-3 resistant lines, low virus titers were found with the production of siRNA, in opposite to the susceptible moneymakers (MM) having higher virus titer and less siRNA level. SiRNA circulation exhibited a steady and precise enhancement of siRNA, was derived from C3 and V1 genes in Ty-3 and Ty-1 genes upon comparative analysis. In Ty-2 resistant plants virus was barely demonstrable, but siRNA profile resemble with susceptible tomato MM after TYCLV challenge. Additionally hypermethylation was observed in V1 promoter region of TYLCV from genomic DNA isolated from Ty-1resistant plants, than the susceptible (MM). Ty-1 resistance was found effective even for bipartite Tomato severe rugose virus (ToSRV) and it also associated with hypermethylation of V1 promoter region of virus, but in case of mixed infection of Cucumber mosaic virus (CMV) and TYLCV, resistance was compromised. So, Ty-1 imparts resistance by enhancing cytosine methylation of virus genomes evocative of enhanced TGS (Butterbach et al., 2014).

Resistant plants were not only constructed for TYLCV by Ty genes, but also to control B. tabacci by breeding for the durable host plant resistance, as all tomato varieties cultivated all over the world are highly susceptible for whitefly (Lucatti et al., 2015). Many wild tomato plants being resistant to white fly (Liedl et al., 1995; Nombela et al., 2000; Muigai et al., 2002; Muigai et al., 2003; Baldin et al., 2005; Firdaus et al., 2012; Firdaus et al., 2013b; Lucatti et al., 2013), act as resistance donor against whitefly, for various resistance breeding programs. Different resistance mechanisms in wild tomato plants which are so far recognized, were based on various chemical compounds synthesized in glandular trichomes as acyl sugars, methylketons

59 and sesquiterpenes were found affecting antibiosis and antixenosis behaviour of whitefly (Freitas et al., 2002; Muigai et al., 2003; Antonious et al., 2005; Bleeker et al., 2009; De Resende et al., 2009; Bleeker et al., 2011; Lucatti et al., 2013).

Almost all plant organs as flowers, leaves or roots emit various volatile organic compound (VOCs), these VOCs includes alkanes, alkenes, ketones, aldehydes, terpenes (modified terpenes and terpenoids) and aromatic compounds (Weingart et al., 2012). Emission of VOCs by sessile plants enable them to send signals over comparatively long distances, as well as VOCs are well recognized to be involved in the defense linked processes and can also be used for signaling inside the plant (Choudhary et al., 2008). Plant VOCs generally are consists of complex mixtures, for that reason comprehensive study of metabolites is significant for better understanding of the fundamental molecular mechanism (Weingart et al., 2013).

During present study Ty resistant and susceptible tomato cultivars were studied for resistance and susceptibility response as well as were exploited for metabolites profiling i.e. bioactive compounds (or metabolites), isolated from extract by GCMS, in response to resistance or susceptibility of Ty cultivars against ToLCNDV.

During previous 15 years metabolomics has appeared as a recent of the so called "Omics" research fields and it deals with analytical characterization of the metabolome, i.e. low molecular weight having metabolite components of the biological systems under consideration. Various biological systems from simple microbes to plant and very complex multi organism systems as mammals have benefited from metabolomics studies. However general purpose of almost all metabolomics is to create a snapshot of metabolic status of a biological system as well as to characterize variations in the abundances of measured metabolites produced as a consequence of natural or external fluctuations and experimental abiotic or biotic perturbations (Schuhmacher et al., 2013). Metabolite profiling based on analytical approach implemented for relative quantification of many metabolites of samples (Fiehn, 2002).

Metabolism based on intermediates and products which are termed as metabolites, in metabolomics context metabolites are defined as, a molecule having size less than 1kDa (Samuelsson and Larsson, 2008), in plants these termed as primary and secondary metabolites (Bentle, 1999). More than 50,000 metabolites from whole

60 kingdom while many thousands metabolites from a single plant have been identified and characterized with diverse biological functions (De Luca and St Pierre, 2000; Griffin and Shockcor, 2004).

Horning and Horning (1971) introduced metabolic profiling terminology after measuring different compounds by using a technique called GC-MS (Van Der Greef and Smilde, 2005; Novotny et al., 2008). GC-MS has been used as an analysis tool for profiling of complex amalgamations of primary metabolites now-a-days (Osorio et al., 2012). GC-MS systems are regularly used for profiling as it is useful for the separation of ~400 compounds (Jonsson et al., 2006). However metabolites range identified by using such techniques is restricted to particular experimental protocol employed along with physical and chemical features of the compounds investigated (Von Roepenack-Lahaye et al., 2003; Bednarek et al., 2005; Fayos et al., 2006; Zacarés et al., 2007; Bellés et al., 2008; López-Gresa et al., 2011). Molecular plant pathology metabolites characterization involved in defense response is still a pending goal (López-Gresa et al., 2012). Numerous biomolecules having various biological functions are assumed may be involved in plant pathogens interactions. Therefore much better analytical systems are required for the accurate identification and detection of primary and secondary metabolites (Glauser et al., 2010).

Tomato is an outstanding model to scrutinize interactions among plants and pathogens, as this plant is highly susceptible for the attack of numerous microbe pathogens (viruses, bacteria, fungal members and nematodes). Plant microbial interaction includes incompatible and compatible interactions, among these incompatible had been studied extensively due to their practical applications in the field than compatible (Vlot et al., 2009), which is less studied (O'donnell et al., 2001; Huang et al., 2003). In incompatible interactions, upon pathogens perception by host led to host defense activation response, causing localized cell death and necrosis development (Ryals et al., 1996). However in compatible interactions due to gene-for- gene resistance absence, necrosis did not occur, but in such interactions microbes multiply and spread actively all over the body consequently leading to plant death (Dixon et al., 1994). Inspite of these differences, both of these interactions share some common characters such as plantsʼs signal compounds inductions such as saliaclic acid (2-hydroxybenzoic acid; Vlot et al., 2009), ethylene and pathogens defense proteins (Van Loon et al., 2006). One among well established defense responses is the

61 phenolic metabolism which led to the complex range of natural chemical products (Dixon et al., 2002; Abdel-Farid et al., 2009).

Whitefly is a serious threat to tomato cultivation all around the world but a few wild relatives of tomato were found resistant. Various studies had been conducted to observe biochemical changes among plants in response to whitefly attack. Different types of metabolites produced in resistant and susceptible plants are linked genetically. Resistant S. pennellii exhibited high level of resistance to B. tabacci, by using mapping approach genetic background of resistance linked traits in association with biochemical traits was elucidated. Bulked segregant analysis on pool of resistant and susceptible plants for whitefly, led to the identification of various resistance and susceptibility metabolites production (Lucatti et al., 2016). Concluding that there is a direct genetic correlation among reduced whitefly incidence and biochemical based resistance in S. pennellii.

Metabolomics might be an outstanding tool to determine phenotype, as a consequence of genetic manipulation like gene insertion or deletion. Another aspect was to predict about unknown genes functions, after comparing metabolic commotion at insertion or deletion of genes, this technique has also been implemented for mammalian systems (Saghatelian et al., 2004; Chiang et al., 2006). As a study conducted by Zhou et al. (2012) exhibited that cowpea d-endotoxin and trypsin inhibitor genes of Bacillus thuringiensis had been introduced into the rice genome to enhance its resistance against pest via Agrobacterium-mediated transformation. Metabolic profiling acquired from GC-MS was applied to determine capricious metabolic variations produced as a consequence of In vitro growth of the tissue as well as insertion of genes in plants. Metabolic profiles attained from GC-MS were the main component analysis which proposed that great metabolic variations occurred during In vitro growth of tissue than genes insertion or transformation.

A metabolite study was performed by López-Gresa (2012) for the compatible interactions among Tomato mosaic virus (ToMV) and plant, by comparing inoculated and systemically infected (non-inoculated) tomato leaves. It helped to understand metabolic condition of plant due to ToMV infection, this analytical approach led to the 32 metabolites (Including flavonoids, phenylpropanoids, sugars, aminoacids, organic acids and other miscellaneous compounds) identification. By multivariate data analysis, metabolites subset produced during defense response in plant was

62 identified, however a distinct variation in several metabolites contents was observed associated with asymptomatic ToMV infection, in inoculated as well as systemically infected leaves.

Deng et al. (2005) explained different plants rapidly produce and assemble plant- signaling molecule in the leaves, such as methyl salicylate (MeSA), which arouse resistance against disease caused by virus and herbivores infections. A simple, fast and sensitive protocol was developed to establish MeSA production directly by introducing samples into leaves, thermal desorption and by using GC-MS afterward. This method has also been applied to study defense against TMV in tomato, by quick exploration of volatile compounds produced in leaves, a huge quantity of MeSA in tomato plants against TMV was known to produce for resistance. MeSA can generate defense reaction, being an important plant-signaling molecule, synthesized in tomato leaves against TMV. Similarly 02 tomato lines one resistant (R), other susceptible (S) for TYLCV, showed different stress response upon TYLCV infection. S plants showed stunted growth and do not yield, while R were symptoms less as well as remain yielded. Metabolite profile comparison represented different stress response in both lines. S plants were showing high levels of ROS, wound induced and pathogenesis related proteins. In contrast R plants did not activate similar host defense mechanisms (Moshe et al., 2012).

Effects of various other pathogenic organisms on tomatoes have also been studied, indicating association of various unique compounds against pathogens. Hussaini et al. (2011) exhibited that tomato fruits produce volatile metabolites profiled by the use of GC-MS, after inoculation by three toxigenic fungus strains acquired from spoilt tomatoes. Volatile metabolites studied during this experiment vary in numbers and quantity, as 52 volatile metabolites were found. Healthy tomato fruits produced 28 metabolites; Aspergillus flavus inoculated fruit produced 10 metabolite unique for this strain while Fusarium oxysporum and A. niger each produced 04 metabolites which were unique to them. This study explained these unique compounds can be exploited to identify tomato diseases/ pathogen or infections of toxigenic fungi before disease development as biomarkers.

Another similar study by Ibrahim et al. (2011a) exhibited that different volatile metabolites produced, when tomato fruits were inoculated by three bacterium species isolated from spoilt tomatoes. Differences in quantity as well as numbers of

63 metabolites were studied, as 66 volatile compounds found accumulatively, while in totality 28 metabolites found in healthy fruits. Tomato fruits after inoculation with Listeria monocytogenes produced14 volatile metabolites same after infection of Brevibacillus laterosporus while inoculation with Bacillus megaterium produced 16 metabolites. Among all metabolites only octadecanoic acid and oleic acid found comparatively consistently. 11 metabolites present in Bacillus megaterium, 10 in Brevibacillus laterosporus and 08 were unique for Listeria monocytogenes inoculation respectively.

However inspite of various resistance strategies availability, breeders working for resistance induction, by the integration of resistant genes into tomato, must consider the fact of increased level of genetic variety present in tomato infecting begomoviruses. Another problem is that a particular resistance gene can be tremendously efficient against a specific begomovirus species but entirely useless against distinct, dissimilar species. As well as pace of begomoviruses evolution is exceedingly rapid, consequently leading to the fast emergence of novel species and strains those may prevail over resistance genes.

CHAPTER NO. 3 MATERIALS AND METHODS 64

MATERIALS AND METHODS

3.1 Materials

Begomoviruses infected plants and Ty resistant cultivarʼs seeds were used during the study

3.1.1 Collection of infected samples

Symptomatic tomato samples were collected from major tomato cultivation areas (Gogdarra, Sherpalam, Kamaala, Korigram, Piraan, Tormang, Jaban, Shamo Zai and Khadag Zai) in Swat, Dir and Malakand districts of KP, in Faisalabad (NIAB) and in Lahore (Rakh Burj), during 2013-2015. Phenotypically different symptomatic samples were collected from various fields in each area, where infected plants showed symptoms variability. Tomato plants which showed particular viral infection symptoms were captured by high resolution camera. A total of 30 samples were collected from various fields of Rukh Burj, while 21 samples from NIAB and 24 from KP fields. Newly emerging infected leaves of plants were collected and saved in zip lock polythene bags, labeled with area, date and symptoms by using permanent marker. Samples were preserved at low temperature -20 oC until utilized.

Seeds of Ty resistant cultivars were obtained from the World Vegetable Center entitled as Asian Vegetable Research and Development Center (AVRDC) Taiwan.

3.1.2 Classification of Tomato

Kingdom: Plantae

Subkingdom: Tracheobionta

Division: Magnoliophyta

Class: Magnoliopsida

Subclass: Asteridae

Order: Solanales

Family: Solanaceae

Genus: Solanum L.

Species: Solanum lycopersicum L. (Knapp and Peralta, 2016)

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3.1.3 Nutritional value of Tomato

Tomatoes contain 95 % water content and remaining 5 % are fibers and carbohydrates (3 gm sugar, 1 gm protein and 1 gm fiber; Claye et al., 1996). One medium sized i.e. about 123 gm tomato gives 22 calories, 0 gm fat, rich in vitamin A, C and K1 which are important for bone health and blood coagulation (Bolton‐Smith et al., 2007; Bügel, 2003). Potassium (essential mineral, significant for cardiovascular disease and blood pressure control; D'elia et al., 2011) and folate (B9) are also present which are vital for normal tissue growth and cell functions, particularly in pregnancy (Fekete et al., 2012). Along with these many beneficial nutrients and antioxidants as alpha lipoic acid, choline, lycopene which imparts red color in tomato and act as antioxidant (Story et al., 2010) are found in highest amount in tomato peel (Viuda-Martos et al., 2014). Lycopene is highest in processed product than fresh (Nguyen et al., 2001) as (fresh tomato contain 1-8 mg/100 g but ketchup contain 10-14 mg/100 g; Rao et al., 1998). However other tomato products (processed products) as tomato juices, ketchup and tomato based sauces are richest dietary sources (Clinton, 1998; Rao et al., 2006). Lutein, beta-carotene, a yellow antioxidant which is converted into vitamin A inside the body and naringenin is also present in tomato skin that contains antiapoptotic and anti-inflammatory activity (Bharti et al., 2014). Chlorogenic acid, a strong antioxidant compound which reduces blood pressure is also present in tomato (Kozuma et al., 2005; Watanabe et al., 2006).

3.1.4 Health benefits of tomatoes

Tomatoes health benefits to mankind are known since ancient times.

3.1.4.1 Heart health

Tomato enrich with lycopene protects from cardiovascular diseases (heart attacks and strokes). A study conducted on middle aged men indicated that less amount of beta carotene and lycopene in blood can increase the risk of heart strokes and attacks (Karppi et al., 2012a; Karppi et al., 2012b). Clinical trials showed protective effect of tomato on blood vessels against blood clotting risk (Palomo et al., 2012).

3.1.4.2 Prevention from cancer

Tomato being a rich source of many antioxidants can help to combat free radicals formation those are recognized to cause cancer. Different studies link tomatoes and

66 products of tomatoes to less occurrence of lung, prostrate, stomach (Giovannucci, 2002), pancreas, rectum, colon, oesophagus, oral cavity and cervix cancer (Giovannucci, 1999). Lycopene richness is considered as main source for its protective effects but quality human trials are needed (Chen et al., 2013; Holzapfel et al., 2013; Lin et al., 2015). High carotenoids amount can protect from breast cancer (Sato et al., 2002; Aune et al., 2012).

3.1.4.3 Skin health and vision improvement

Tomato is beneficial for skin due to its richness in lycopene and other compounds which may protect skin from sunburn (Aust et al., 2005; Stahl et al., 2006). It is rich in vitamin A which is good for vision improvement.

3.1.4.4 Pregnancy and depression

Adequate folic acid is required in pregnancy, so tomato being rich source of folic acid can protect infants from neural tube defects (El-Shabrawi et al., 2015). Folic acid by reducing excessive homocysteine formation in body also decreases depression. As surplus homocysteine interferes with feel-good hormones (dopamine, norepinephrine and serotonin) which not only regulate mood but appetite and sleep also (Permoda- Osip et al., 2013).

3.1.4.5 Reduces urinary tract infections

Tomatoes are rich in water content which stimulates urination and reduces toxins, salts, excessive water, uric acid and some fats which ultimately decreases urinary tract infections (Bhowmik et al., 2012).

3.1.4.6 Counters the consequences of cigarette smoke

Chlorogenic acid and coumaric acid are found in tomato which fight against nitrosamines produced in body as main carcinogens present in cigarettes (Bhowmik et al., 2012; Hecht et al., 2015).

3.1.4.7 Good for hair and reduce migraine Vitamin A makes hair strong and shiny, is also good for bones and teeth. Riboflavin supports migraine attacks reduction is also found in tomato (Bhowmik et al., 2012). 3.1.4.8 Enhance immunity Tomato consumption helps to protect from colds and flu. Drinking tomato juices is effective for immunity development (Bhowmik et al., 2012).

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3.1.4.9 Strengthen bone, neutralize acidosis and wound repair Vitamin K activates main non-collagen protein (osteocalcin) in bones which mineralizes calcium in bones (Bhowmik et al., 2012). Acidosis mainly is responsible for calcium loss, headache, fatigue, muscle ache, sleeplessness, acne, arteriosclerosis, eczema, degenerative condition, hormone imbalance and sexual dysfunctioning. Tomato being rich source of magnesium, calcium and potassium help in acidosis. By taking correct amount of tomato per day, vitamin C found in it can help wounds in body to get repaired (Bhowmik et al., 2012). 3.1.4.10 Antipathy for tomato

Tomatoes are usually better endured and very rarely cause allergy (Asero et al., 2009; Asero, 2013). These allergic reactions are called as pollen-food (or oral) allergy syndrome (Boccafogli et al., 1994). In this syndrome, immune system attacks against vegetables and fruits proteins resultant to scratchy throat, itching in mouth and throat swelling (Webber and England, 2010). Along with this, oxalic acid content of tomato which form an insoluble calcium oxalate (calcium salts) precipitates as kidney stones.

3.1.4.11 Economic importance of tomato

Tomato is considered economically valuable crop in Pakistan as annual export of this vegetable from Pakistan is almost 9832 tons for the duration of 2009-2013. During 2009-10 Pakistan exported 5692 tons tomatoes and got profit of 77 million rupees. Tomato is tremendously exported to the international market due to ever increasing demand in Afghanistan, Iran, UAE, Saudi Arabia, Sri Lanka and India. Present average yield, during 10 years is quite less to meet the needs, therefore different high production strategies and better yielding varieties are required to be cultivated in order to increase yield and export (Khokhar, 2013).

Growing population of Pakistan has resulted in increased demand of tomato but due to biotic and abiotic factors its production is far less than need. Consequently in order to meet the needs particularly in rainy season and Ramadan tomatoes are imported from neighboring countries especially India. India has been the major source of imported tomato in Pakistan for the last many years, with increasing vegetables imports of worth more than US $ 120 million during 2013 (Amir and Hyder, 2015). Tomato import fluctuates according to the needs in the country as Pakistan has imported 0.36 million Kg of tomatoes of worth 0.17 million US $ from India during

68 first two weeks of May, 2015 (Agricorner 2015). Due to duty free land route, tomatoes import from India has been increased over US$ 100 million. Regarding import shares, India is the major tomato exporter for Pakistan (Khan and Hussain, 2014).

3.2 Methods

Various methods used for the characterization, resistance evaluation and volatile profiling are as follows.

3.2.1 Extraction of DNA

Genomic DNA extraction from symptomatic plant samples was done by Cetyltrimethyl ammonium bromide (CTAB) protocol explained by (Doyle and Doyle, 1990). For this purpose approximately 100 mg plant sample was taken and crushed in liquid nitrogen till fine paste, by using pre sterilized autoclaved and pre-cooled mortar and pestle. CTAB used was pre heated at 65 oC, 700 µl CTAB buffer (1.4 M NaCl, 02 % (w/v) CTAB and 100 mM Tris HCl with basic pH i.e. pH=08 and 20 mM EDTA) having 0.2 % (v/v) β-mercaptoethanol was added, mixed properly and ground tissue was poured in microfuge tube which was incubated at 65 oC for half an hour. During incubations microfuge tubes were occasionally inverted to mix CTAB with ground tissues following a particular interval. After incubation samples were kept at room temperature to cool and 700 µl (equal volume to CTAB) of chloroform-isoamyl alcohol in (24:01) ratio was added. Plant tissues were mixed and spun in centrifuge machine (HETTICH, EBA, 200) at 1800xg for 05 minutes. Afterwards supernatant was taken in a new eppendorf and isopropanol, equal to the 0.6 volume of the supernatant was added to precipitate the DNA. Eppendorfs were kept at room temperature for one hour or overnight. These tubes were spun at high speed 3220xg for 15 minutes to precipitate DNA in the form of pellet. 70 % ethanol was used to wash pellet by dissolving excessive salts, pellet was then air dried. Appropriate amount of sterile distilled water (SDW) was added to dissolve the pellet.

3.2.2 Quantification of DNA

Quantification of purified DNA, by measuring the absorbance at 260 nm via spectrophotometer (Smartspec Plus, BIORAD, U.S.A.) was performed. DNA was diluted with SDW to make it 25 ng/ul. SWD was taken in cuvette for mock reading afterwards diluted DNA was added in cuvette, reading was noted and dilution factor

69 was multiplied to acquire final reading. Optical density OD260 of 01 is equivalent to 50 µg/ml of genomic DNA.

3.2.3 Amplification of DNA using polymerase chain reaction (PCR) mechanism

DNA was amplified by single PCR reaction added in PCR tubes of 0.25 ml or 0.5 ml capacity, reaction mixture consists of following components as, 05 µl (50-100 ng) of template that is genomic DNA, 05 µl dNTPs (02 mM), 10X Taq Polymerase buffer (05 µl ; Fermentas), 01 µl (0.5 µM) of each (forward and reverse) primers, MgCl2 03µl (1.5 mM) and Taq DNA polymerase 01 µl (1.25 units; Fermentas) was prepared by keeping and adding all these reagents on ice. Amplification was performed by placing reaction mixture, for incubation in thermocycler (My cyclerTM, Bio-Rad or Master cycler gradient, eppendorf). Reaction was carried out by using particular PCR profile, which consists of three following steps; denaturation, annealing and extension, these cycles were repeated 35 to 40 times to amplify DNA. First of all, preheating of genomic DNA was proceeded by heating at 94 oC (05 minutes), followed by denaturation, annealing and extension, at 94 oC (01 minute), 48 oC to 55 oC (02 minutes), 72 oC (03 minutes; vary in time according to fragment length being amplified) respectively. After this 72 oC temperature was kept for 10 minutes and PCR reaction was obtained from PCR machine as temperature had reduced due to hold at 04 oC. Different PCR profiles, varied in annealing temperatures and time, were used for amplification of begomoviruses, alphasatellites and betasatellites (Table 3.1).

3.2.4 DNA amplification via rolling circle amplification (RCA) mechanism

RCA method was utilized to amplify circular DNA (Lizardi et al., 1998). In order to prepare RCA reaction mixture following reagents were added by keeping on ice, template DNA [(100-200 ng, random haxamer primers (RHP; 50 µM), 10X ᶲ-29 DNA and 10X polymerase buffer (02 µl)]. The reaction mixture was added in pre sterilized PCR tubes and heated for 03 minutes at 94 oC to denature double strands of DNA. Then reaction mixture was allowed to cool to room temperature and 01 mM dNTPs, enzyme mix contains ᶲ-29 DNA polymerase (05-07units) and pyrophosphatase was added. Reaction mixture was incubated at 30 oC for 18-20 hours. Then ᶲ-29 DNA polymerase enzyme was inactivated via heating at 65 oC for 10 minutes. Amplified products were confirmed latter, by running amplification product (01µl), on the ethidium bromide stained agarose gel.

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3.2.5 Gel electrophoresis

DNA fragments were resolved in agarose gel to determine the size and quantity of DNA fragments. 01% (w/v) agarose gel was used which was developed by dissolving (1.3 g of agarose in 130 ml of 1X TAE buffer) in oven. After heating, gel was allowed to cool about 60 oC and 03 µl (0.5 µg/ml) ethidium bromide was mixed homogenously. Gel was poured in tray and minigel (12×9) as well as midigel (18×15) electrophoretic apparatus were used. In the molten gel, combs were inserted and left for 10 minutes to solidify. Gel tray was then dipped either in 1X TBE (89 mM boric acid, 02 mM EDTA and 89 mM Tris) or 01X TAE buffer (40 mM Tris acetate [pH 8.4], 01 mM EDTA). Combs were then removed to load DNA samples mixed with 5X loading dye. To observe the size of DNA fragment, 1kb DNA marker (Fermentas) was also run with samples. TBE and TAE gels were electrophorised at about 50 or 100 V respectively. Size of DNA fragments was studied by comparing with 1kb Ladder by using Gel Doc System (UVP-Digidoc-It 130) and photographed.

3.2.6 Gel extraction and purification of PCR products

Amplified DNA by PCR was run on 01 % agarose gel; required fragments were observed under UV transilluminator and cut by using blade. DNA purification was done by using Wizard SV Gel as well as PCR Clean-Up kit (Promega). Cut gel slice was placed in an already weighted 1.5 ml eppendorf, gel slice further was treated with 10 µl of membrane binding solution for 10 mg of gel slice was added, gel was dissolved in solution by vortex and incubated at 55-65 oC till gel slice was completely dissolved in membrane binding solution. This whole mixture (gel and PCR product) was poured into mini column assembly, kept at room temperature for 01 minute, centrifuged at 16000×g for 60 seconds. All content of gel was passed through column and discarded, DNA stacked with column which was inserted into the collection tube. 700 µl of membrane wash solution was poured on column to purify PCR product which remained stick on column and centrifuged at 16,000×g for 60 seconds. After discarding flow through, by adding 500 µl membrane wash solution was further centrifuged at 16,000×g till 05 minutes, afterwards all course through was disposed of, again minicolumn with empty collection tube and opened lid was centrifuged for 60 seconds so that remaining ethanol should be evaporated. Mini column was put in new microcentrifuge tube, 50 µl of SDW was poured on mini column, incubated at

71 room temperature for 01 minute and then centrifuged for 01 minute at 16,000×g. Then course through collected in tube was kept at -20 oC and minicolumn was discarded.

3.2.7 DNA purification by phenol-chloroform extraction

In order to remove impurities such as proteins, DNA solutions and restricted vectors were cleaned by phenol chloroform extraction. For this purpose, 18µl of DNA solution was diluted by adding 182 µl of SDW to get 200 µl final volume. This DNA solution was further vortex, after mixing equal volume (01:01) of phenol chloroform to get milky white solution, this solution was then centrifuged for 10 minutes at 16,000×g, uppermost transparent layer (phase) was then carefully collected in sterile microcentrifuge tube without mixing interface among two phases. 3M Sodium acetate at pH (5.4) added into supernatant equal to 1/10 volume, mixed properly, and chilled absolute ethanol (2.5 volumes) was added, kept at -70 oC for half an hour. DNA pellet was obtained after centrifugation at high speed for 10 minutes, 70 % ethanol added to wash then pellet was incubated at 37 oC to get dry and dissolved by adding 20 µl of SDW.

3.2.8 Ligation and cloning of amplified DNA

Amplified DNA via PCR was used to ligate in (pTZ57R/T) T/A cloning vector with insTAclone, PCR cloning kit (Fermentas), according to the information provided by manufacturers. Reaction mixture of 20 µl was prepared by adding (18-540 ng) vector, 02 µl or 04 µl of (10X ligation, 5X ligation buffer), 0.2-0.4 µl (01 to 02 units) of T4 DNA ligase and at last SDW to make 20 µl final volume. All reagents of ligation were kept and added on ice in autoclaved micocentrifuge tubes, which were then incubated in ligation chamber, over night at 16 oC. By using particular restriction enzymes RCA amplified DNA and pBlueScriptII KS/SK [+] or pTZ57R/T circular cloning vector were digested to produce blunt or sticky ends making it favorable for insertion. Due to blunt end ligations, reaction mixture had same reagents and their quantity except for 50 % PEG4000 (02 µl) solution as well as 5 units (01 µl) of ligase, was used inspite of 02 units. Ligation in (pGreen 0029) binary vector was done by same protocol.

3.2.9 Preparation of competent Escherichia coli cells by heat shock

Heat shock competent E. coli cells were made by following protocol described by (Cohen et al., 1972). A colony of E. coli (strain Top10) from a freshly grown plate was singled out by using an autoclaved toothpick and transferred into 50 ml flask

72 containing 50 ml liquid LB media. Flask was incubated for vigorous shaking at 37 oC over night to grow E. coli cells. Next day 02 ml of liquid LB media containing E. coli cells was added into 250 ml liquid LB media in 1000 ml flask and kept again at 37 oC for vigorous shaking till an OD600 of 0.5 to 01 was obtained. Culture was kept for half an hour on ice then equally poured into sterile 50 ml falcon vials to centrifuge for 10 minutes at 3220xg and 04 oC. Pellet obtained was dissolved after discarding supernatant, in 20 ml of 0.1 M MgCl2 and again centrifuged. Supernatant poured out to dissolve pellet into 20 ml of 0.1 M CaCl2, was chilled for 30 minutes and then centrifuged. After discarding supernatant pellet was again suspended into 03 to 04 ml of 0.1 M CaCl2, cold sterilized glycerol was added as 200 µl glycerol for every 01 ml o of 0.1 M CaCl2. Cells were transferred in 200 µl aliquots and stored at -70 C. All steps of the competent cell genesis were performed in laminar air flow cabinet.

3.2.9.1 Transformation of ligated product in competent cells of E. coli

Ligated DNA was transformed in E. coli competent cells and transformation was done by heat shock method explained by (Sambrook et al., 1989). A vial of competent cells (200 µl) taken from -70 oC, 05 to10 µl of ligated DNA was mixed in it and kept on ice for 30 minutes. Dry bath adjusted at 42 oC to give heat shock to E. coli containing vial for 02 minutes, then vial was immediately transferred on ice for 02 minutes, 01ml liquid LB medium was poured in vials and cells were kept at 37 oC cells to grow for 45 to 60 minutes. Vials were spun at 3220xg for 02 minutes to get pellet, only 200 µl of supernatant was kept to dissolve pellet, remaining was discarded. Plates of LB agar, having suitable antibiotic were used to spread (by glass spreader, already sterilized on flame after dipping in ethanol and cooled till room temperature) transformed cells then were incubated overnight at 37 oC. IPTG and X-Gal (130 µg/ml, 270 µg/ml respectively) were spread on LB plates before cells spreading, for blue white screening with suitable antibiotics. Vector chosen for ligation led to the selection of various antibiotics for making LB agar plates such as ampicillin 100 µg/ml for TA vector with ampicillin resistance gene while kanamycin 50 µg/ml was used for pGreen0029 with kanamycin resistance gene.

3.2.9.2 Isolation of plasmid DNA

Alkaline lysis method described by Birnboim and Doly (1979) and Ish-Horowicz and Burke (1981) was used with few modifications. A single transparent and clear colony

73 of E. coli cells was picked up from freshly grown plate by using autoclaved sterile toothpick and mixed with 05 ml LB (broth) liquid media in sterile culture tubes having appropriate antibiotics (ampicillin or kanamycin) wrapped with aluminum foil. All steps were performed in laminar air flow cabinet. Culture tubes were incubated in shaker at 37 oC over night to multiply cells. About 01 ml of culture was taken in 1.5 ml capacity microcentrifuge tube and centrifuged at 16000xg to obtain pellet of E. coli. After eliminating supernatant, pellet was resuspended in 100 µl of resuspension soln. (10 mM EDTA, 50 mM Tris-HCl [pH 8.0], 100 µg/ml RNase A). Micro centrifuged tubes containing resuspension soln. and pellet were vortex till pellet was completely dissolved. Then 150 µl of lysis solution (0.2 M NaOH, 1 % [w/v] SDS) was poured and these eppendorfs were inverted gently 05 to 06 times and left for some time until a transparent string appeared on opening lid of tubes. Then 200 µl neutralization soln. (03 M potassium acetate [pH 5.5]) was poured and mixed properly by inverting tubes 07 to 08 times, after that tubes were centrifuged at 16000xg for 15 minutes. Supernatant was accumulated in another sterile eppendorf, chilled 100 % ethanol was added (2.5 to the total supernatant volume obtained), mixed and kept at - 70 oC for an hour to get DNA fibers precipitated. Following this step, tubes were centrifuged for 12 minutes at 16000xg to acquire DNA pellet. Pellet was then washed with 70 % ethanol and incubated at 37 oC to get dry, SDW was added and plasmids stored at -20 oC.

3.2.9.3 Restriction by restriction enzymes (endonucleases)

Restriction enzymes and relevant buffers as according to the supplier's (Thermo) information were used for the restriction of plasmid DNA (for screening of clones), RCA and PCR (for cloning) products, according to the size of inserts. For plasmid screening 10 µl reaction mixture consists of 500 ng DNA, 03 units of restriction enzymes, their buffers and SDW was made for the screening of plasmid to find particular insert size, while digestion of cloning a 20 µl reaction mixture consists of 02 µg DNA, 10 units endonucleases, buffer and SDW was added. Samples were incubated at 37 oC an optimum temperature for most of the restriction enzymes. Sometimes digestion is kept overnight, while for partial digestion low efficiency buffers and less incubation is required. Agarose gel stained by ethidium bromide was used for the resolution of fragments, by comparing with DNA marker, after attaining particular sized fragments plasmids or clones were preserved.

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3.2.9.4 Preservation of clones (glycerol stocks and plasmids)

Preservation of clones was being done by different ways in the form of plasmid that is preserved as such at about -20 oC to -70 oC or bacterial culture is saved in the form of glycerol stocks at -70 oC. For glycerol stocks preparation sterilized glycerol was added in bacterial culture in laminar air flow cabinet. Bacterial culture was mixed with glycerol in the 07:03 ratios, 700 µl of bacterial culture was taken in eppendorf and 300 µl of glycerol added, mixed properly and stored at -70 oC.

3.2.9.5 Renewal of clones

Bacterial culture (clone) can be renewed by using flame sterilized wire loop, it was then cooled to room temperature, dip in glycerol stock and streak it on LB agar plate containing specific antibiotics according to resistance marker in clone. All these steps were performed under sterilized conditions in laminar air flow cabinet. Agar plate after streaking was incubated at 37 oC over night to grow cells.

3.2.10 Preparation of electro-competent cells of Agrobacterium tumefaciens

A single colony was picked with the help of autoclaved sterile toothpick from a recently grown Agrobacterium tumefaciens (strain GV3101) plate as an inoculum, added into 20 ml of LB liquid medium along with 25 µg/ml rifampicin, contained in 50 ml sterilized autoclaved flask. Culture was placed for 48 hours in incubator shaker adjusted as160 rpm at 28 oC. After cells growth, 05 ml of culture was taken for inoculation in 01 liter flask having 250 ml LB medium, along with 25 µg/ml rifampicin and kept in shaker set at 28 oC, until 0.5-1 OD600 of the cells was attained. Then culture was aseptically transferred in ice chilled 50 ml propylene tubes, which were incubated for 10 minutes on ice, and centrifuged for 10 minutes at 04 oC and 3220xg to pellet cells. Obtained pellet after dissolving in 50 ml sterile cold SDW was centrifuged, it again dissolved in SDW, centrifuged and was further dissolved in cold 10 ml SWD with 10% [v/v] cold sterilized glycerol and centrifuged. This step was repeated twice. Ultimately 03 to 04 ml of cold, filter sterilized 10 % [v/v] glycerol was taken to dissolve the cells. At the end under laminar air flow cabinet these cells were transferred in 1.5 ml microcentrifuge tubes to store at -80 oC for use in future.

3.2.10.1 Transformation by using Agrobacterium tumefaciens competent cells

An aliquot of electro competent Agrobacterium tumefaciens cells was kept on ice after taken out from -80 oC and let it to thaw for about 5 minutes. Then 02 µl ligation

75 mixture or plasmid DNA was added in this cell which was further transferred into cold electroporation cuvette. Electroporation cuvette was washed thoroughly with 100 % ethanol and it was dried completely. Cells with plasmid were then poured in cuvette and put on ice to inhibit heat shock of cells. Electroporator (ECM 600, BTX, USA) was adjusted at 1.44 KV. Cells containing cuvette was set aside in chamber for electric shock and button for start was pushed. Following electric shock, liquid LB medium (01ml) was mixed with shocked cells and transferred from cuvette to new sterilize microcentrifuge tube and it was incubated at 28 oC in a shaker for 02 to 03 hours. Cells were centrifuged for some time to obtain pellet. All the supernatant was discarded except 100 µl that was used to dissolve pellet of cells. These cells were lastly stretched on a plate (solid LB agar medium) containing antibiotics as tetracyclin (10 µg/ml) rifampicin (25 µg/ml) as well as kanamycin (50 µg/ml) and plate was incubated for 48 hours at 28 oC.

3.2.10.2 Transient analysis via Agrobacterium tumefaciens

Binary vector (pGreen0029) having clones, were transformed in cells (electro competent) of Agrobacterium tumefaciens (GV3101 strain) for inoculum preparation used for transient analysis. By using a sterile wire loop a single well developed colony was picked up and mixed in LB medium (50 ml) having kanamycin, tetracyclin and rifampicine as (50 µg/ml, 10 µg/ml, 25 µg/ml, respectively) antibiotics, this step was performed under laminar air flow cabinet and wire loop was sterilized by flame. LB medium containing flask was incubated at 28 oC and 160 rpm for 48 hours. Next step was to harvest the cells from LB medium by shifting culture into falcon tubes. Culture in falcon tubes was centrifuged at 3220xg for 15 minutes and at 04 oC. Pellet obtained was thus dissolved in 10 ml (10 mM) MgCl2 to have an OD600 ranges from 0.6 to 01. Then acetosyringone (100 µM) was mixed in the culture and kept for 03 to 04 hours at room temperature, to activate and increase virulence. Now this culture was ready for infiltration, it was infiltrated in the abaxial side of the leaves with syringe of 05 ml injection. Tomato plants were not watered before 24 hours of inoculation.

3.2.11 Plants growth conditions

Seeds of thirteen Ty resistant cultivars of tomato (from AVRDC Taiwan) and one native susceptible tomato variety, were sown in large earthen pots (about 10 inch diameter) having combination of silt, clay, sand and compost added in equal

76 proportion, pots held in relative humidity (65 %), dark (16 hrs.), light (08 hrs.) at 26 oC. Every plant was watered day by day while with Hoagland soln. (1.5 mM

Ca(NO3)2.4H2O, 0.75 mM MgSO4.7H2O, 1.25 mM KNO, 0.5 mM KH2PO4, micronutrients [15µM MnCl2.4H2O, 50 µM H3BO3, 2.0 µM ZnSO4.7H2O, 0.5µM

Na2MoO4.2H2O,1.5 µM CuSO4.5H2O] and Fe-EDTA [01 mM KOH, 30 µM

FeSO4.7H2O and 30 µM EDTA]) one time per week.

3.2.11.1 Transfer of plants into individual pots

After almost 15 days of seeds sowing, seedlings having proper height were transferred individually into new pots for further experimentation. Plastic pots with 05 inch diameter containing compost, sand, silt and clay in almost same proportion under same conditions were used. Seedlings started growing in new pots, till the size of about one and half feet (18-19 inches), were divided into three groups first (infiltrated by ToLCNDV acquired from Dr. Zafar Iqbal on personal communication), second (infiltrated with Agrobacterium tumefaciens containing pGreen0029) and third were kept uninoculated as healthy control. Infiltration was done for first and second groups of plants using syringe of 05 ml injection on lower side of leaves by injecting infectious clones or pGreen0029. While third control plants were kept under same conditions in glass house.

3.2.11.2 Observations of plants

These four groups of plants were regularly observed after interval of one week. Plants showing symptoms were observed carefully and various levels of infections were noted. Pictures were taken at every stage of infection, in order to compare the infected plants with the healthy control. Plants showing symptoms (very severe, severe, moderate, mild and non symptomatic) were harvested for every cultivar. Plant samples were collected in polythene bags for further use for PCR, southern hybridization and GCMS analysis.

3.2.12 Detection of ToLCNDV in resistant and susceptible cultivars

ToLCNDV inoculated resistant and susceptible cultivars were confirmed for the presence of ToLCNDV genome which was responsible for symptoms development in the resistant and susceptible cultivars. DNA was extracted from putative plants to confirm the presence of ToLCNDV by using specific primers (Table 3.1) which were designed for ToLCNDV detection through PCR.

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3.2.13 DIG labeled probe synthesis

For DIG labeled probe synthesis, a 50 µl PCR reaction mixture, by means of Dig Probe Synthesis Kit (Roche, Germany) was prepared. Reaction mixture contained

10X PCR buffer 05 µl with MgCl2, 0.5 µl of forward and reverse primers for each pair, PCR DIG Probe Synthesis Mix 05 µl (130 µl dTTP, 200 µM dATP, dGTP, dCTP and 70 µM DIG-dUTP), 01 µl of enzyme mix supplied with the kit as well as template DNA 05µl (10-100 pg or 01to 50 ng for plasmid or genomic DNA respectively) and PCR-grade water for volume make up to 50 µl. Latter reaction mixture was incubated in thermal cycler, according to the size of amplification profile was set. Probe used for hybridization was taken in microcentrifuge tube and was denatured via heating at 95 oC in a water bath, for 05 minutes. Immediately it was cooled on ice for 04 to 05 minutes and was poured in hybridization bottle.

3.2.14 Southern hybridization

For southern hybridization, genomic DNA extracted from samples, were subjected to quantification by nanodrop (Thermo Scientific, Wilmington, USA). After that 05 to10 µg genomic DNA of each sample was loaded in wells of 1.5 % agarose gel and its voltage was maintained at 60 volts. After appropriate gel run, it was observed under ultra violet light. It was further treated with three different solutions which were used for its washing, with light agitation at room temperature. First washing required Depurination solution (0.25 M HCl) and this washing prolonged for 30 minutes. After this washing, gel was thoroughly and carefully rinsed with SDW in order to avoid two solutions mixing. Second denaturation solution (0.5 M NaOH and 1.5 M NaCl) was used for washing and again gel was washed with SDW, to get rid of solution's residues. Final gel treatment was given for 30 minutes by Neutralization solution (1.5 M NaCl, 01 M Tris [pH=7.4]). Next step was to transfer genomic DNA from gel to the nylon membrane (Hybond, Amersham) by making assembly, in assembly DNA was transferred by using 5X SSC solution, to the membrane. Carefully gel was shifted on filter papers wedge in order to avoid any air bubble in filter papers those were dipped in 5X SSC solution from both sides. Nylon membrane was placed on the upper side of gel. Right, upper side of membrane and gel was cut in order to signify samples numbers. Layers of 5X SSC dipped filter papers were put on membrane's upper side, which was further covered by mass of paper towels in order to facilitate capillary action accountable for DNA transfer from gel to membrane, towel layers were further

78 kept in balanced by applying a light weight on the top of towels layers (Figure 3.1). To get DNA completely transferred, assembly was kept overnight at room temperature.

Next day nylon membrane was acquired back from assembly and gel was observed by gel doc, in order to ensure complete transfer of DNA from gel to membrane. Membrane after drying at room temperature was crossed linked in order to fix DNA to membrane by UV cross-linker (CL-1000 Ultraviolet Cross-linker-UVP) at 120 mjcm-2 energy. Blot was then treated with the pre-hybridization solution (10 ml 20X SSC, 05 ml 50 X Denhardt [ficoll 01 % (w/v), BSA 01 % (w/v) PVP 01 % (w/v)], 10 µl salmon sperm DNA (05 ng), 05 ml 10 % SDS and 30 ml SDW for 50 ml volume make up), for o3 hours at 42 oC in hybridizer (Hybaid, Midi-dual 14). After prehybridization, for DNA probe and blot hybridization, blot transferred into hybridization bottle, by keeping DNA carrying side of blot inside the hybridization, to make sure blotsʼs complete contact with probe. Denatured, Dig-labeled probe was inserted and incubated overnight at 42 oC.

Dig-labeled probe was removed and stored at -20 oC for future use, and blot was treated for post hybridization (stringency washings) washings. For first washing, 2X SSC, 0.1 % (w/v) SDS were used for 15 minutes at 60 oC and then solution was removed. In second washing, 01X SSC, 0.1 % (w/v) SDS was used for blot washing for 15 minutes at 60 oC and solution was discarded. For third time, blot was washed by using 0.1 % X SSC, 0.1 % (w/v) SDS for 15 minutes at 60 oC. After these washings blot was treated with the blocking solution (01X Maleic acid and 10 % Blocking reagent, for 30 minutes at 28-30 oC); this treatment was meant for blocking of non-specific binding of probe on the membrane. Following Blocking Solution washing, Antibody solution treatment was given to blot (Anti-Digoxigenin, Blocking solution [75 mµ/ml [01:10000]) by placing it in the hybridization bottle containing antibody solution, which was incubated for 30 minutes at 28 to 30 oC. Afterwards blot was washed twice by using washing solution (150 µl Tween-20, 50 ml of 01X Maleic acid) at 28-30 oC for 15 minutes. Blot washing was again repeated, after these washings, blot was treated further with detection buffer (0.1 M NaCl, 0.1 M Tris-HCl, pH=9.5) at room temperature for 03 to 05 minutes and was eliminated from hybridization bottle. All washing solutions as well as buffers, required for blot washing and various treatments were 10 to 15 ml in quantity. For chromogenic blot

79 hybridization, visualization solution was consists of 5-bromro-4-chloro-3-indolyl- phosphate and 4-nitro blue tetrazolium (BCIPT/NBT). NBT (100 mg NBT/02ml of 70 % N, N-dimethylformamide) and BCIPT (100mg BCIPT/ 02ml of 100% N, N- dimethylformamide) were added as 70µl of BCIPT and 90 µl of NBT solution into detection buffer and blot containing try. Before adding visualization solution tray was completely wrapped with aluminum foil, due to photosensitive nature of BCIPT/ NBT. Wrapped tray was left for few minutes to hour or overnight to develop blot completely. All washing solutions as well as buffers required for blot washing and various treatments were used in volume of 15 to 100 ml (Southern, 1975).

3.2.14.1 Dot-Blot hybridization

For dot blot hybridization, genomic DNA extracted from samples were subjected for quantification by nanodrop, required quantity (02 µg) was loaded for each sample in 1.5 % agarose gel and voltage was maintained at 60 volts. After appropriate gel run, it was observed by gel doc. Similarly 02 µg (~4 µl) of each sample and denaturation solutions (01 M NaOH and 200 mM EDTA, pH 8.2), in order to obtain final concentration of 0.4 M NaOH/10mM EDTA was added. Denaturation solution containing genomic DNA was heated at 100 oC for 10 minutes and was snap cooled for 10 minutes. After denaturation DNA was spotted on the positively charged nylon membrane (Amersham hybond) which was previously submerged in distilled water for 05 minutes and dried. Membrane after drying at room temperature was crossed linked in order to fix DNA to membrane by UV cross-linker at 120 mjcm-2 energy. Blot was then treated similarly as explained previously for the southern blot hybridization (section 3.14).

3.2.14.2 Quantification of bands on blots

Dot intensities were quantified with the help of ImagJ software (http://rsbweb.nih/ij/). Data acquired from this analysis was normalized, against the intensity of genomic DNA bands stained by ethidium bromide, for every sample.

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Table 3.1 Degenerate back to back primer sets used for amplification of begomoviruses, associated satellites and for probe synthesis with respective PCR profiles

Primer Pairs Sequence PCR Profile

ToLCNDV A1 5'GATATCATCATTTCAACGCCCGCATCGAA3' 95 oC for 01min

ToLCNDV A2 5'GATATCTGCTGGTCGCTTCGCCATAGTTC3' 52 oC for 02min

72 oC for 03min

ToLCNDV A3 5'GAGCTCGTGCAGTTGTCCCCATTGCCCGCGTCAC3' 95 oC for 01min

ToLCNDV A4 5'GAGCTCCATAGGGGCTGTCGAAGTTG3' 52 oC for 02min

72 oC for 03min

ToLCNDV B1 5'AAGCTTCTGCTCGAACATGGACGGAAATGAC3' 95 oC for 01min

ToLCNDV B2 5'AAGCTTAGCCAGTTGAGGAATAGATGCATG3' 52 oC for 02min

72 oC for 03min

ToLCNDV B3 5'GGTACCCGTAACGATCTTGAACTATGTCCC3' 95 oC for 01min

ToLCNDV B4 5'GGTACCCTATCTGGCTATAGGTCCGAACG3' 52 oC for 02min

72 oC for 03min

Begomo F 5'ACGCGTGCCGTGCTGCTGCCCCCA3' 94 oC for 01min

Begomo R 5'ACGCGTATGGGCTGYCGAAGTTSAGACG3' 52 oC for 02min

72 oC for 03min

DNA101 5'CTGCAGATAATGTAGCTTACCAG3' 94 oC for 30 sec

DNA102 5'CTGCAGATCCTCCACGTGTATAG3' 50 oC for 30 sec

72 oC for 45sec

β01 5'GGTACCACTACGCTACGCAGCAGCC3' 94 oC for 30 sec

β02 5'GGTACCTACCCTCCCAGGGGTACA3' 50 oC for 30 sec

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72 oC for 45sec

ND -A qPCR F 5′GTCGAAGCGACCAGCAGATAT3′ 95 oC for 30 sec

ND-A qPCRR 5′GGAACATCTGGACTTCTGTAC3′ 58 oC for 30 sec

72 oC for 45 sec

BMP qPCR F 5′GCCCATGATTCGTTCGGAC3′ 95 oC for 30 sec

BMP qPCR R 5′GAATTCCGACCACCAAAGAT3′ 60 oC for 30 sec

72 oC for 45 sec

Primers for Probe Synthesis

ND -A Probe F 5′CCTTTAATCATGACTGGCTT3′ 95 oC for 30 sec

ND-A Probe R 5′CATTTCCATCCGAACATTC3′ 58 oC for 30 sec

72 oC for 45 sec

ND -B Probe F 5′GCCCATGATTCGTTCGGAC3′ 95 oC for 30 sec

ND-B Probe R 5′CACGTGGTACTGGAATATCGCA3′ 60 oC for 30 sec

72 oC for 45 sec

Figure 3.1: Southern hybridization assembly. Apparatus used for genomic DNA shifting from agarose gel to the nylon membrane via capillary transfer mechanism.

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3.2.15 Extraction of plant samples in n-hexane

Harvested plant samples were shade dried at room temperature for about 02 to 03 weeks. Dried samples were weighted and ground fine with the help of clean dried and autoclaved pestle and mortar. 1 gm sample and 10 ml of n-hexane solvent was taken in a flask, the flask was then shaken vigorously to let all the material soaked and to get dissolved in solvent. Flasks with plant samples and solvents were kept in shaker at 37 oC for 02 to 03 days. Extracts were filtered by using Whatman filter paper and collected in a falcon tube. Plant materials were again soaked in 10 ml n-hexane and put on shaking so that remaining bioactive compounds could be isolated; extracts were again collected in falcons. Extract obtained after repeating three times, were about 10 to 15 ml, then these were allowed to evaporate. When 3 to 4 ml of extract was left it was stored at 04 oC and solvent was repeatedly added into extracts to prevent it from evaporation. Before GCMS, this extract was evaporated to 01 ml.

3.2.16 Gas chromatography mass spectrometry

Bioactive compounds were analyzed by GC-MS (QP2010 plus Shimadzu, Japan) with the flame ionization detector (FID). DB-5 capillary column (30mx0.25mm, film thickness 0.25 µm J&W scientific, Folsom C.A) was used. Helium was used as carrier gas with a constant flow rate of 0.8 ml/min. approximately 01 µl of extract was injected by micro injector into GCMS. Oven temperature was kept at 50 oC for 05 minutes, latter was programmed from 50-100 at the rate of 10 oC/min and was maintained at the final temperature for 05 minutes. Injector and detector temperatures were kept 200 oC and 250 oC respectively. Ionization voltage was 70eV with mass scan range of 55-950m/z with the rate of 0.5 scans/s and scanning was done for 40 minutes. Computer generated a graph called chromatogram which represented compounds in the form of peaks, X-axis and Y-axis on the chromatogram represents retention time (RT; The time when sample was injected to when eluted) and the abundance of the compounds respectively. For unknown compounds identification, mass spectrum comparison of the constituents with National Institute Standard and Technology Library (NIST-2005) and literature was done, which gives about 90 % identity. Then compounds molecular formulas, retention time and molecular weight were tabulated. Quantification was done by relative abundance percentage for isolated compounds individually in each sample.

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3.2.17 Photography and computer graphics

Photographs of infected plants during collection and experiment were taken via a digital camera with good resolution (Sony DSC W50). In order to edit pictures, Adobe Photoshop 7 was used while Corel DRAW 12 software was used for graphic design and figures production.

3.2.18 Sequencing and sub sequencing

Plasmids isolated by using Gene JET Plasmid Miniprep Kit (Fermentas) were sent for Sanger sequencing to Genomics and Bioinformatics Research Unit (GBRU) Stoneville MS, USA. Sequencing was done by primer walker strategy with the help of universal sequencing primers (M13 F [20] and M13 R [-20]). Completed sequences were further analyzed and those were not completely sequenced in first reaction, sent again after designing sub sequencing primers i.e. primer walking. These primers were designed by analyzing available sequences, after alignments in Clustal X2 and by selecting conserved regions. Primers designed for sub sequencing along with sequences needed to be completed, were sent. Sub sequencing was repeatedly done to complete the sequences. Laser gene package for sequence analysis (DNAStar Inc., Madison, WI, USA) software was used by assembling and then analyzing sequences, while with the help of Basic Local Alignment Search Tool (BLAST) online (www.ncbi.nlm.nih.gov/Blast.cgi) was used for the comparison of homologies with previously submitted sequences in database. Each sequence was further analyzed for ORFs by ORF Finder (www.ncbi.nlm.gov), to ensure presence of stop, start codon, its truncation or full length in particular sequence. Duly complete sequences were submitted to gene bank (www.ena.ac.uk) to get accession numbers.

3.2.19 Phylogenetic analysis

Dully complete sequences were used for phylogenetic analyses, which were done by using current and maximum homology sharing sequences retrieved from NCBI database and an outgroup. Alignments were performed using Mega6 via Muscle, by nucleotides Neighbor Joining algorithm with 1000 bootstrap value. Trees were interpreted and viewed by Mega6 and TreeView (Page 1996).

3.2.20 Sequence demarcation tool (SDT) analysis

Multiple aligned sequences by muscle Mega6 were preceded by SDT software by selecting 93 % cut off value for strain and 91 % for species demarcation with three

84 color mode selections. For SDT analysis, maximum homology sharing sequences, mentioned in current geminivirus list at international committee on taxonomy of viruses (ICTV) website (www.ictvonline.org) were retrieved from NCBI, analyzed to indicate virus species and presence of diversity among present isolates.

3.2.21 Percentage identity and divergence

By using MegAlign software, all isolates were aligned by Clustral W method. Sequence distances were observed by comparing percentage identity and divergence among isolates of same species, different strains and different species.

3.2.22 Recombination detection program (RDP)

Recombination among sequences and breakpoints of recombination were determined by RDP (RDP4.22). RDP software based on various methods like RDP, GENCONV, BootScan, MaxChi, Chimaera, SiScan, PhylPro LARD and 3Seq, having default setting with 0.05 P-value was used. Analysis was done by P-Values based on majority of parameters (more than three or four) among these, having less Av. P-values.

CHAPTER NO. 4 RESULTS 85

RESULTS

Results are divided into two sections.

SECTION I

4.1 Characterization of DNA-A component of begomoviruses

Characterization of DNA-A component of begomoviruses was done as follows.

4.1.1 Observation of symptoms severity in tomato samples

Samples were collected from different fields, among all collected samples those were collected from Rukh Burj fields showed very severe symptoms, exact location of each field with GPS coordinates has been described (Table 4.1). Different fields adjacent to tomato fields were of chilies and cotton which also showed symptoms similar to begomoviruses infections. Symptomatic plants exhibited typical symptoms, which include severe leaf curling, rolling, leaf thickening, shortening, puckering, dark margins, flower dissemination, bushy appearance due to short internodes and severe growth stunting were more common in samples of Lahore and Faisalabad (Figure 4.1) but yellowing, curling, less flowering/fruiting and stunted growth were more prominent symptoms in infected plants from KP fields. However, overall ToLCD symptom severity and its incidence was more than previously surveyed (Shahid Mansoor personal communication).

4.1.2 Amplification of begomoviruses infecting tomato

A total of 75 samples were subjected to PCR amplification by using RCA amplification product dilutions, with already reported universal primers (Akhter et al., 2009) and newly designed oligonucleotides primers, 66 samples showed 2.8 kb amplification (Figure 4.2). Among 66, 13 samples showed amplification with Begomo F/Begomo R, universal primers (Akhter et al., 2009), 19 amplifications were obtained by newly synthesized primers (designed for ToLCNDV DNA-A) A1/A2 primer pair, 25 by A3/A4 primers pair (Table 3.1, section 3.4). RCA was also employed for 09 samples and amplified products

86 were further restricted by BamHI which led to release of 2.8 kb fragments.

Restricted RCA products and PCR amplified products (from dilution of RCA product) of begomoviruses were ligated into cloning vector (pTZ57/RT) and transformed into E. coli (Top10 stain). After acquiring positive clones confirmed via restriction by using EcoRI and PstI restriction enzymes and unique restriction enzymes i.e. MluI for Begomo F/Begomo R, EcoRV for A1/A2 and SacI for A3/A4 were also used when required. Clones after confirmation by restriction fragment length polymorphism (RFLP; Figure 4.3), were sent for sequencing.

4.1.3 Molecular characterization of DNA-A components of begomoviruses

All cloned products get sequenced, their complete nucleotides sequences were determined and a total of 40 DNA-A complete sequences were obtained after sequencing. Duly complete sequences were submitted in database to obtain accession number for each clone (Table 4.1). Complete nucleotide sequences of DNA-A genome consist of 2738- 2766 nucleotides. For initial comparison nucleotides BLAST analysis was performed for DNA-A sequences (monopartite and bipartite). Complete analyses revealed that 13 clones showed homology with ToLCNDV isolates, 17 with ToLCV, 04 with PaLCuV, 04 with ToLCPalV and 02 with ToLCKeV, exhibited five species identification.

4.1.4 Open reading frames (ORFs) analysis

All full length confirmed nucleotide sequences of DNA-A of monopartite and bipartite begomoviruses were compared for protein coding ORFs by using ORF finder of NCBI (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). All begomoviruses contained the typical genome organization, 04 ORFs were found situated on complementary strand (AC1, AC2, AC3 and AC4) and 02 ORFs in virion sense (AV1 and AV2), coordinates for each ORF have given (Table 4.1). These ORFs in all DNA-A were separated by intergenic region, within intergenic region CR of DNA-A, shared homology with CR of its cognate DNA-B. A conserved, nonanucleotide sequence TAATATTAC was found in intergenic region of all begomoviruses. AC1 found in all DNA-A of present begomoviruses isolates vary from 1499-2619, AC2 1192-1631, AC3 1047-1086, AC4 2245-2467, AV1 280-1083 and AV2 120-458. 87

Figure 4.1: Symptomatic and healthy tomato plants. Reduced leaf area shown in "A" growth stunting "B" leaves thickening, leaf curling and yellowing "C" overview of healthy field "D" deformation "E" rolling of leaves "F" leaf thickening and dark coloration "G" and healthy tomato plant "H". 88

Figure 4.2: Gel picture exhibiting PCR amplified fragment of 2.8 kb sized begomoviruses. Amplified products were loaded with 1 kb DNA marker for size specification of amplified products, samples containing wells were designated as 01, 02, 03, 04, 05 and 06. Wells 07 and 08 represented negative and positive controls respectively. Well marked by "M" contained 1kb DNA marker which was also shown independently with each fragment size specification. Previously confirmed infected plant sample, gave amplification of 2.8 kb was used as positive control, while in well 7 PCR product of genomic DNA of healthy plant was run as negative control. Samples in well 01 to 06 showed amplification of 2.8 kb.

Figure 4.3: Gel picture indicating restriction pattern for full length begomoviruses clones. Restriction products were run in 01 to 07 wells gave full length (2.8 kb) fragment accumulatively, showed two types of restriction patterns. Restriction product of clone 01 and 02 have similar pattern, while sample in well 03 to 07 showed different restriction patterns indicated restriction fragment length polymorphism. "M" here was used to represent DNA marker to compare fragment length.

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Table 4.1: Coordinates of Open Reading Frames (ORFs) i.e. Virion Sense (AV1 & AV2) and Complementary Sense (AC1, AC2, AC3 & AC4), accession numbers and origin coordinates of the DNA-A components of begomoviruses. Coordinates of Virion and Complementary ORFs Clone Accession Genome Identity in Location Longitude Latitude AV1 AV2 AC1 AC2 AC3 AC4 name Numbers Size (nt) Database RS8 LN878118 2739 ToLCNDVA NIAB Fsb 31.396249 73.031816 280-1050 120-458 1499-2584 1192-1596 1047-1457 2251-2427 RS12 LN886522 2739 ToLCNDVA NIAB Fsb 31.396249 73.031816 280-1050 120-458 1499-2584 1192-1596 1047-1457 2251-2427 RS21 LN886523 2743 ToLCNDVA NIAB Fsb 31.396249 73.031816 280-1050 120-458 1499-2584 1192-1596 1047-1457 2251-2427 RS23 LN887902 2739 ToLCNDVA NIAB Fsb 31.396249 73.031816 280-1050 120-458 1499-2584 1192-1596 1047-1457 2251-2427 RS28 LN886524 2739 ToLCNDVA NIAB Fsb 31.396249 73.031816 280-1050 120-458 1499-2584 1192-1596 1047-1457 2251-2427 RS57 LN887908 2738 ToLCNDVA Rakh Burj 31.387026 74.515358 279-1049 119-457 1498-2583 1176-1595 1046-1454 2250-2426 RS99 LN887905 2740 ToLCNDVA Rakh Burj 31.387026 74.515358 281-1051 121-459 1500-2585 1178-1597 1048-1458 2252-2428 RS124 LT556071 2740 ToLCNDVA Korigram 34.76807 72.02393 280-1050 120-458 1499-2584 1177-1596 1047-1457 2251-2427 RS163 LT556076 2739 ToLCNDVA Rakh Burj 31.387026 74.515358 280-1050 120-458 1499-2584 1177-1596 1047-1457 2251-2427 RS219 LT556070 2739 ToLCNDVA Sherpalam 34.89428 72.41253 280-1050 120-458 1499-2584 1192-1596 1047-1457 2251-2427 RS220 LT556082 2739 ToLCNDVA Sherpalam 34.89428 72.41253 280-1050 120-458 1499-2584 1192-1596 1047-1457 2251-2427 RS234 LT556081 2740 ToLCNDVA Gogdarra 34.74286 72.28895 280-1050 120-458 1499-2584 1177-1596 1047-1457 2251-2427 RS235 LT556074 2740 ToLCNDVA Gogdarra 34.74286 72.28895 280-1050 120-458 1499-2584 1177-1596 1047-1457 2251-2427 RS142 LT556072 2756 ToLCPalV Kamaala 34.64119 71.79818 280-1050 120-467 1499-2584 1177-1596 1047-1457 2269-2445 RS143 Not Submitted 2756 ToLCPalV Kamaala 34.64119 71.79818 280-1026 120-467 1499-2602 1177-1596 1047-1457 2269-2445 RS145 LT556077 2756 ToLCPalV Kamaala 34.64119 71.79818 280-1050 120-467 1499-2584 1177-1596 1047-1457 2269-2445 RS147 LT556078 2756 ToLCPalV Kamaala 34.64119 71.79818 280-1050 120-467 1499-2584 1177-1596 1047-1457 2269-2445 RS71 LN878121 2757 ToLCV Rakh Burj 31.387026 74.515358 304-1074 144-500 1523-2608 1216-1620 1071-1475 2158-2451 RS72 LN878122 2759 ToLCV Rakh Burj 31.387026 74.515358 304-1074 144-500 1523-2608 1216-1620 1071-1475 2158-2451 RS79 LN878123 2761 ToLCV Rakh Burj 31.387026 74.515358 304-1074 144-500 1523-2608 1216-1620 1071-1475 2158-2451 RS80 LN887903 2761 ToLCV Rakh Burj 31.387026 74.515358 304-1017 144-500 1524-2609 1217-1621 1072-1476 2159-2452 RS82 LN878125 2758 ToLCV Rakh Burj 31.387026 74.515358 305-1075 145-501 1524-2609 1217-1621 1072-1476 2159-2452 RS86 LN887904 2760 ToLCV Rakh Burj 31.387026 74.515358 304-1074 144-500 1523-2608 1216-1620 1071-1475 2158-2451 RS88 LN887907 2760 ToLCV Rakh Burj 31.387026 74.515358 304-1074 144-500 1523-2608 1216-1620 1071-1475 2158-2451 RS90 LN878103 2760 ToLCV Rakh Burj 31.387026 74.515358 304-1074 144-500 1523-2608 1216-1620 1071-1475 2158-2451 RS91 LN878104 2761 ToLCV Rakh Burj 31.387026 74.515358 304-1074 144-500 1523-2608 1216-1620 1071-1475 2158-2451 RS 96 LN878127 2757 ToLCV Rakh Burj 31.387026 74.515358 304-1074 144-491 1523-2608 1216-1620 1071-1475 2158-2451 RS 97 LN878128 2754 ToLCV Rakh Burj 31.387026 74.515358 304-1074 144-491 1523-2608 1216-1620 1071-1475 2158-2451 RS101 LN887906 2757 ToLCV Rakh Burj 31.387026 74.515358 304-1074 144-500 1523-2608 1216-1620 1071-1475 2158-2451 RS176 LT556079 2766 ToLCV Rakh Burj 31.387026 74.515358 304-1074 144-500 1523-2608 1216-1620 1071-1475 2158-2451 RS178 LT556083 2766 ToLCV Rakh Burj 31.387026 74.515358 304-1074 144-500 1523-2608 1216-1620 1071-1475 2158-2451 RS179 LT556080 2760 ToLCV Rakh Burj 31.387026 74.515358 304-1074 144-500 1523-2608 1216-1620 1071-1475 2158-2451 RS186 LT556073 2760 ToLCV Rakh Burj 31.387026 74.515358 304-1074 144-500 1523-2608 1216-1620 1071-1475 2158-2451 RS187 Not Submitted 2759 ToLCV Rakh Burj 31.387026 74.515358 306-1076 146-502 1525-2610 1218-1622 1073-1477 2160-2453 RS33 LN878119 2764 PaLCuV Rakh Burj 31.387026 74.515358 306-1076 146-502 1531-2616 1224-1628 1079-1483 2202-2459 RS58 LN878120 2763 PaLCuV Rakh Burj 31.387026 74.515358 308-1078 148-504 1533-2618 1226-1630 1081-1485 2204-2461 RS 60 LN886525 2761 PaLCuV Rakh Burj 31.387026 74.515358 306-1076 146-502 1531-2616 1224-1628 1073-1483 2202-2459 RS 92 LN878129 2750 PaLCuV Rakh Burj 31.387026 74.515358 304-1083 144-500 1522-2607 1215-1619 1070-1474 2193-2450 RS 2 LN886521 2764 ToLCKeV NIAB Fsb 31.396249 73.031816 304-1074 144-491 1529-2614 1222-1626 1077-1481 2155-2463 RS 157 LT556075 2765 ToLCKeV NIAB Fsb 31.396249 73.031816 305-1075 145-492 1530-2615 1223-1627 1078-1482 2120-2464 90

4.1.5 Percentage homologies with database sequences

Percentage homologies of present isolates were compared with already submitted sequences in database indicated that complete nucleotide sequences of all ToLCV shared 96 to 98 % homology with ToLCV isolates from database. These maximum homology sharing sequences were found submitted from Indians. Similarly, present isolates of ToLCKeV shares 92 to 93 % homology with NCBI sequences, homology sharing NCBI sequences were also submitted by India. A maximum of 97 to 98 % homologies were shared by present PaLCuV isolates with respective virus isolates in NCBI database, interestingly three present isolates shared maximum homology with isolates submitted and reported from India. However, a new strain of PaLCuV (RS92) identified in current study showed 91 % maximum homology with isolates submitted from Pakistan. Isolates of ToLCPalV (04) on comparing with database sequences shared 97 % to 98 % homology with isolates submitted from India, although this virus has already been reported from Pakistan and India. In contrast to all, ToLCNDV herein isolates shared 91% to 98 % maximum homology with ToLCNDV isolates submitted from Pakistan. A complete detail of BLAST results for each isolate, with percentage identities has been given (Table 4.2).

All isolates of same species were also compared for percentage identities and divergence among them by using multiple alignments of complete nucleotide sequences, aligned by Clustral W method of MegAlign program of DNA Star. All isolates of ToLCV showed percentage identities which vary from 94.7 % to 99.8 %, similarly among ToLCkeV isolates 99.8 % to 99.9 %. Comparison for PaLCuV and ToLCPalV isolates showed 99.6 % to 100 % and 98.8 % to 99.7 % identities respectively, for ToLCNDV identities vary from 92.2 % to 99.7 % among all ToLCNDV isolates. Distance analysis of herein full length sequences indicated, all isolates of particular species showed greater homology percentage than threshold level for species demarcation i.e. greater than 91 %, with NCBI isolates of particular species.

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Table 4.2: Percentage identities for current isolates of each species and homology sharing isolates, which were retrieved from National Center for Biotechnology Information (NCBI) database.

Clone Accession Percentage Blast Results name Numbers Homology RS8 LN878118 ToLCNDV-[PK:Sn:PT10:04]-DQ116883 97 % RS12 LN886522 ToLCNDV-[PK:Sn:PT10:04]-DQ116883 97 % RS21 LN886523 ToLCNDV-[PK:Sn:PT10:04]-DQ116883 96 % RS28 LN886524 ToLCNDV-[PK:Sn:PT10:04]-DQ116883 97 % RS23 LN887902 ToLCNDV-[PK:Sn:PT10:04]-DQ116883 96 % RS99 LN887905 ToLCNDV-[PK:Sn:97]-AJ620187 97 % RS124 LT556071 ToLCNDV- [IN:Tom:o6]-EF068246 97 % RS57 LN887908 ToLCNDV-[PK:Sn:97]-AJ620187 97 % RS234 LT556081 ToLCNDV-[PK:Dar:T5/6:01]-AF448058 97 % RS163 LT556076 ToLCNDV-[PK:Mul:Luf:04]-AM292302 98 % RS219 LT556095 ToLCNDV-[PK:06]-EF620534 96 % RS220 LT556096 ToLCNDV-[PK:06]-EF620534 96 % RS235 LT556074 ToLCNDV-[PK:Dar:T5/6:01]-AF448058 97 % RS142 LT556072 ToLCPalV-[IN:Pal:047]-AM884015 98 % RS147 LT556078 ToLCPalV-[IN:Pal:047]-AM884015 98 % RS143 Not Submitted ToLCPalV-[IN:Pal:047]-AM884015 97 % RS145 LT556077 ToLCPalV-[IN:Pal:047]-AM884015 98 % RS72 LN878122 ToLCV-[IN:Luc:Phy:12]-JX524172 96 % RS82 LN878125 ToLCV-[IN:Luc:Phy:12]-JX524172 97 % RS178 LT556083 ToLCV-[IN:Luc:Phy:12]-JX524172 97 % RS79 LN878123 ToLCV-[IN: Ban:Chi:08]-HM007094 98 % RS90 LN878103 ToLCV-[IN: Ban:Chi:08]-HM007094 98 % RS91 LN878104 ToLCV-[IN: Ban:Chi:08]-HM007094 98 % RS71 LN878121 ToLCV-[IN:Luc:Phy:12]-JX524172 96 % RS80 LN887903 ToLCV-[IN: Ban:Chi:08]-HM007094 98 % RS86 LN887904 ToLCV-[IN: Ban:Chi:08]-HM007094 98 % RS88 LN887907 ToLCV-[IN: Ban:Chi:08]-HM007094 98 % RS101 LN887906 ToLCV-[IN:Luc:Phy:12]-JX524172 97 % RS176 LT556079 ToLCV-[IN:Luc:Phy:12]-JX524172 97 % RS179 LT556080 ToLCV-[IN: Ban:Chi:08]-HM007094 98 % RS 96 LN878127 ToLCV-[NP:Pan:00]-AY234383 99 % RS 97 LN878128 ToLCV-[NP:Pan:00]-AY234383 98 % RS186 LT556073 ToLCV-[IN:Luc:Phy:12]-JX524172 98 % RS187 Not Submitted ToLCV-[IN:Luc:Phy:12]-JX524172 97 % RS33 LN878119 PaLCuV-IN[IN:Har2:Aca:07]-FN645926 97 % RS58 LN878120 PaLCuV-IN[IN:Har2:Aca:07]-FN645926 98 % RS 60 LN886525 PaLCuV-IN[IN:Har2:Aca:07]-FN645926 97 % RS 92 LN878129 PaLCuV-PK[PK:Cot:02]-AJ436992 91 % RS 2 LN886521 ToLCKeV-[IN:Ker5:07]-EU910140 93 % RS 157 LT556075 ToLCKeV-[IN:Ker5:07]-EU910140 93 %

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4.2 Species Demarcation Tool (SDT) analysis

Following initial comparison of isolates for species identification and demarcation, SDT analysis was performed for all isolates acquired during present study, according to revised criteria of begomoviruses taxonomy by using MUSCLE alignment of MEGA6 software as described by Brown et al. in 2015. All isolates of each particular species, mentioned in current geminivirus list ICTV website (www.ictvonline.org), were retrieved in order to confirm the particular species of begomoviruses. Isolates of ToLCV showed more than 93 % homology with NCBI sequences which was greater than threshold level for strains and species demarcation, SDT analysis of ToLCV was shown (Text Figure 4.1) in the form of heat plot.

SDT analysis of present ToLCKeV and database isolates indicated present isolates showed greater than 91 % pairwise identity with already submitted sequences and were ToLCKeV isolates, shown by heat plot method (Text Figure 4.2).

Similarly PaLCuV isolates were also analyzed by retrieving maximum homology sharing isolates for species demarcation and confirmation, among 04 isolates of PaLCuV, three were found belong to same strain of PaLCuV. Other than (RS92), which represented a new strain of PaLCuV (Text Figure 4.3), as it shared less than 93 % homology was named as PaLCuV-LHR strain.

All present isolates shared maximum homology with ToLCPalV were used in SDT analysis for species demarcation. This analysis was based on ToLCPalV isolates acquired from present study, from database as well as 02 isolates of ToLCNDV were also included to indicate similarity among isolates of same species and demarcation among isolates of different species. Results here indicated all isolates (except ToLCNDV isolates) were from the single strain and single species, SDT results exhibited by heat plot (Text Figure 4.4).

Likewise all isolates of ToLCNDV were analyzed for species demarcation, indicated all isolates belong to the same strain and species. This SDT analysis was also performed by adding ToLCPalV isolates to differentiate among isolates of ToLCNDV species, and to confirm isolates under study were from same strain of ToLCNDV species (Text Figure 4.5). For this analysis an appropriate number of ToLCNDV isolates was selected from online available sequences. 93

Text Figure: 4.1 SDT analyses of present and already reported ToLCV isolates. Heat plot indicated present ToLCV were grouped together with maximum homology sharing isolates of ToLCV retrieved from database, which exhibited pairwise identity greater than 91 % and belonging to same species. Identities were compared manually with pairwise identity showing scale attached at right side of figure, herein ToLCV isolates were bold.

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Text Figure: 4.2 SDT analysis for ToLCKeV isolates. Present isolates and all homology sharing accession numbers so far present in begomoviruses list were used for analysis. Comparison with manual scale attached hereby indicated present isolates were ToLCKeV isolates and represented as bold.

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Figure Text Figure 4.3: SDT analysis for PaLCuV isolates. PaLCuV isolates in plot, clearly exhibited that three present isolates have grouped with similar isolates of PaLCuV and showed maximum similarity with Indian isolates. RS92 which was located at different position in plot indicated possibly a new strain of PaLCuV and it showed maximum pairwise identity with isolate submitted from Pakistan and India. 96

Text Figure 4.4: SDT analysis of ToLCPalV isolates. Multiple alignments performed for present ToLCPalV isolates and those retrieved from database were analyzed by pairwise percentage identity for species demarcation. Present isolates were indicated as bold which showed greater than 91 % similarity and belonging to ToLCPalV species. ToLCNDV isolates showed less than 85 % identity with ToLCPalV isolates which was a separate species. Four ToLCPalV isolates showed greater than 95 % pairwise identity with other already reported isolates of ToLCPalV.

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Text Figure 4.5: SDT analysis performed for ToLCNDV isolates. Heat plot indicated pair wise percentage identity among isolates. Present isolates showed greater than 91 % identity with already submitted ToLCNDV isolates from Pakistan, compared manually by scale attached hereby at right side and all present isolates were highlighted as bold.

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4.3 Identification of new strain of PaLCuV (PaLCuV-LHR strain) For strain demarcation, SDT analysis for RS92 (LN878129) was performed, which indicated that this was a new strain of PaLCuV as its similarity with other strains of PaLCuV was less than threshold level (Txt Figure 4.3). On comparing its homologies it showed maximum homologies 91 % with PaLCuV-PK[PK:Cot:02]-AJ436992 accession number from NCBI sequences, while minimum homology it shared with database isolates belongs to PaLCuV was 85 %. Percentage identity showed RS92 shared 86 to 86.6 % percentage identities with other isolates of PaLCuV. This analysis indicated RS92 as new strain of PaLCuV. RS92 analyzed for recombination showed two significant interspecific recombination events first with ToLCV- [IN:Sas:Tom:09]-KP178730 and second with RaLCuV-[IN:Bih:To:09]-GU732204 (Text Figure 4.17). These recombination events may lead to form RS92 a different strain. RS92 shared 79.4 to 83.5 % identities with ToLCV isolates, while its identity with ToLCKeV varies from 83.1 to 83.2 %, with ToLCPalV 68.8 to 69.4 % and 71.4 to 72.8 % for isolates of ToLCNDV. RS92 was also compared with other present PaLCuV isolates for ORFs (AC1, AC2, AC3, AC4, AV1 and AV2) and similarity index among ORFs had shown (Table 4.3).

4.4 Phylogenetic analysis of monopartite and bipartite DNA-A components of begomoviruses

Multiple alignments performed by MUSCLE algorithm of MEGA6 software for present and database sequences were used to construct phylogenetic trees of DNA-A components of monopartite and bipartite begomoviruses. Phylogenetic tree clearly exhibited 05 clades formation (Text Figure 4.6). This clade pattern obviously indicated that herein isolates belong to 05 distinct species. First clade consists of sequences of ToLCV isolates, all database sequences which grouped in this clade with our isolates were submitted from India. Second clade contained ToLCKeV isolates, sequences of ToLCKeV under present study showed maximum homology with Indian isolates and grouped also with Indian isolates and form a separate clade. Isolates of first clade (ToLCV) and second (ToLCKeV) showed percentage identity with isolates vary from 82.2 % to 88.5 %. Third clade represented PaLCuV isolates, which was further consists of two sub clades, sub clade "A" represented three PaLCuV isolates grouped with PaLCuV isolates were submitted from India, while other subclade "B" represented one isolate grouped with PaLCuV isolates from Pakistan and exhibited 99 new PaLCuV strain. Isolates of ToLCKeV and PaLCuV shared 80.8 % to 82.9 % identities. Fourth clade represented ToLCPalV in this clade all present isolates were found grouped with isolates shared maximum homology with sequences submitted from India. All isolates of ToLCPalV shared maximum homology with ToLCKeV and PaLCuV isolates vary from 69.4 % to 73.1 % (Text Figure 4.6). Fifth clade showed ToLCNDV isolates, herein sequences clustered with isolates from Pakistan. Isolates of ToLCNDV shared identities with ToLCKeV, ToLCPalV and PaLCuV that vary from 73.6 % to 85.7 %. However phylogenetic analyses along with SDT analyses revealed the presence of 05 various virus species as ToLCV, ToLCKeV, PaLCuV, ToLCPalV and ToLCNDV were found associated with present begomoviruses infected plants. Among begomoviruses, ToLCNDV and ToLCPalV were found infecting tomatoes previously in different areas of Pakistan, but ToLCKeV, ToLCV and PaLCuV species identified for first time from tomato in Pakistan, which showed maximum similarity with respective isolates from India.

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Text Figure 4.6: Phylogenetic analysis of complete nucleotide sequences of DNA-A components of begomoviruses. Multiple sequence alignments of complete nucleotides sequences of DNA A of monopartite and bipartite begomoviruses and maximum homology sharing accession numbers retrieved from NCBI database were performed to construct phylogenetic tree by neighbor joining method. Numbers at the nodes represent percentage (1000 replicates) bootstraps confidence values. Horizontal lines were proportional to the distances among species and vertical lines were arbitrary. Dandogram was rooted on DNA-A of a distantly related and similar genome size containing NW, Sida golden mosaic virus (SiGMV). Isolate descriptor and begomoviruses acronyms were as proposed by Brown et al. in 2015. Isolates during present study were clustered to form five distinct clades, first represented Tomato leaf curl virus (ToLCV) isolates, second Tomato leaf curl Kerala virus (ToLCKeV), third Papaya leaf curl virus (PaLCuV), fourth Tomato leaf curl palampur virus (ToLCPalV) and fifth Tomato leaf curl new Delhi virus (ToLCNDV). Isolates from current studies in each cluster were highlighted as bold.

Table 4.3: Comparison of complementary and virion sense ORFs among RS92 and three other isolates of PaLCuV.

Isolates AC1 AC2 AC3 AC4 AV1 AV2

RS33 92 % 92 % 98.3 % 87.5 % 68.5 % 86.9 %

RS58 69.7 % 92 % 89.3 % 87.5 % 82.8 % 86.9 %

RS60 92 % 89.9 % 85 % 87.5 % 82.4 % 86.9 % 102

4.5 Phylogenetic analyses of full length sequences and proteins of present isolates

This comparison was performed to indicate variation in proteins of different species in comparison to full length nucleotide sequences as follows.

4.5.1 Phylogenetic comparison of full length sequences and Rep proteins of studied species

Phylogenetic tree for complete nucleotide sequences clearly indicated 05 distinct species grouped into 05 distinct clades; however different strains of particular species were also discrete in clade of that particular species. Clade pattern for each species found in full length phylogenetic tree was compared with phylogenetic tree of Rep protein sequences, in order to observe Rep ORFs similarity and differences among each species. Rep protein sequences of all isolates were used to perform multiple alignments by Clustral W method of MEGA6 software, while full length genome of isolates were allowed to align by MUSCLE algorithms. By comparing clade pattern of both phylogenetic trees similar species distinction was observed (Text Figure 4.7). First clade of Rep phylogenetic tree represented ToLCV sequences, all the protein sequence grouped together to form a big clade of ToLCV, this clade was further consists of subclades among these isolates; Rep of RS82 represented a subclade. This clade pattern might be due to recombination as among theses isolates RS82 contained interspecific recombination (Text Figure 4.18). RS96 and RS97 which represent subclade of ToLCV in full length phylogenetic tree, same was observed in Rep phylogenetic tree. However for further confirmation of this clade pattern multiple alignments were considered, which clearly indicated that few amino acids in these proteins were different from other Rep sequences. Another interesting feature of this Rep phylogenetic tree was seen for Rep sequences of ToLCNDV and ToLCPalV, in full length phylogenetic tree, all ToLCNDV were grouped in a separate single clade, but in Rep tree, isolates have been divided into 02 subclades. One subclade consists of Rep of ToLCNDV isolates only while other subclade consists of Rep of ToLCPalV and ToLCNDV both. This clade pattern indicated Rep sequences of these ToLCPalV and ToLCNDV isolates have some more similarity.

4.5.2 Phylogenetic analysis of AC2 encoded TrAP

Multiple sequence alignments for TrAP sequences of all isolates were performed by Clustral W method of Mega6 and used for phylogenetic tree construction. Isolates of 103 particular species grouped to form distinct clades. In TrAP dandogram, sequences of ToLCV, ToLCKeV, ToLCPalV and ToLCNDV isolates indicated similar clade formation like in full length dandogram. On the other hand TrAP tree indicated one isolate (RS92) of PaLCuV was grouped separately from other PaLCuV isolates, like to RS96 and RS97 (ToLCV) isolates in contrast to full length phylogenetic tree (Text Figure 4.8).

So, amino acid sequence in AC2 of RS 92 was compared with other isolates of PaLCuV, RS96 and RS97 (Text Figure 4.9). It was very clear from alignment that few amino acid sequences it shares to that of RS96 and RS97 than other isolates of ToLCV and PaLCuV. Other possible reason for its grouping with ToLCV isolates may be the presence of recombination event in RS92 with ToLCV (Text Figure 4.17).

4.5.3 Phylogenetic tree of AC3 encoded REn protein

All present isolates were compared for REn protein, multiple sequence alignments of REn were used to construct phylogenetic tree, which ensured REn sequence and full length sequences of each species were clustered to form distinct clade. Isolates grouping in their respective species as well as distinction among different species was mark able in full length tree and similar pattern was found in REn phylogenetic tree. However one exception was found i.e. RS96 and RS97 were not grouped with other ToLCV isolates as in full length dendogram shown (Text Figure 4.10).

Possibly variation in amino acid sequences of REn of RS96 and RS97 from other isolates led to form separate subgroup. These isolates due to the reason were not grouped with any other isolates of other species, rather grouped separately to form a subclade and these sequences were analyzed for recombination as well, but no significant recombination was found. However protein alignments of these sequences showed significant variation than other protein sequences of same species might be the possible reason of their divergence (Text Figure 4.11). Another interesting feature of REn tree was the grouping of protein sequences of ToLCNDV and ToLCPalV as subclade, represented maximum similarity in proteins as compare to whole sequences.

4.5.4 Phylogenetic analysis of AC4 protein

AC4 protein phylogenetic tree in contrast to full length dendogram indicated AC4 proteins of ToLCNDV isolates represented first clade, and all isolates except RS124 were clustered together. Although all proteins of ToLCPalV were clustered together, 104 but formed subclade with isolates of ToLCNDV indicating AC4 similarity among both species than other species, however RS124 a ToLCNDV formed a separate subclade, than other ToLCNDV protein sequences. Few Amino acid differences in RS124, from other ToLCNDV isolates on comparing multiple alignments were found, that can be possible reason for its distinct subclade formation or for its divergence from other isolates. Protein sequences of ToLCKeV, PaLCuV, ToLCPalV and ToLCV exhibited clade formation similar to full length sequences (Text Figure 4.12).

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Text Figure 4.7: Phylogenetic analysis of Rep. Phylogenetic comparison among complete nucleotide sequences tree and Rep protein tree of herein isolates. Phylogenetic tree were constructed by MEGA 6, by Neighbor joining method and with bootstrap (1000 replicates) value, percentage bootstraps confidence values have not shown here. Horizontal lines were proportional to the distances among species and vertical lines were arbitrary, full length nucleotide based dandogram was rooted on a distantly related SIGMV-[PR:04]-AY965900 represented by pink color. Here ''A'' represented phylogenetic tree of full length and ''B'' exhibited dandogram of Rep proteins of present isolates and it was routed on Rep sequence of SiGMV-[US:Flo:Pha:06]-GQ357649. Complete and protein sequences of each species were marked by different colors. Green color represents complete genome and Rep sequence of ToLCV, purple for ToLCKeV, blue for PaLCuV, yellow for ToLCPalV and white for ToLCNDV.

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Text Figure 4.8: Phylogenetic analysis of TrAP. Phylogenetic comparison among complete nucleotide and TrAP protein sequences of herein isolates. Phylogenetic tree was constructed by MEGA 6, by Neighbor Joining method and with bootstrap (1000 replicates) value, percentage bootstraps confidence values have not shown here. Horizontal lines were proportional to the distances among species and vertical lines were arbitrary, full length nucleotide based dendogram ''A'' rooted on a distantly related SIGMV-[PR:04]-AY965900, TrAP based dendogram ''B'' was routed on TrAP sequence of Spinach severe curly top virus SSCTV-[USA: Spi:09]-NC_014631. Here "A" represents phylogenetic tree of full length and "B" dandogram of TrAP sequences of all present isolates. Complete nucleotides and protein sequences of each species were marked by different colors. Green color represented complete genome and TrAP sequence of ToLCV, purple for ToLCKeV, blue for PaLCuV, yellow for ToLCPalV and white color represents isolates of ToLCNDV. TrAP phylogenetic tree showed isolates of ToLCV, ToLCKeV, ToLCPalV and ToLCNDV showed almost similar clade formation to that of full length nucleotide sequences. RS92 in AC2 tree showed different clade formation than their respective full length sequences as it grouped with ToLCV isolates.

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Text Figure 4.9: Multiple alignments of TrAP of PaLCuV (RS33, RS58, RS60 and RS92) and ToLCV (RS96 and RS97) isolates. Alignment of TrAP of RS92, RS33, RS58, RS60, RS96 and RS97 clearly exhibited similarity among RS92, RS96 and RS97 aminoacids at certain points in sequence. Boxes outlined black indicated amino acids similarity it shared with RS96 and RS97.

Text Figure 4.10: Comparison of REn protein and full length sequences. Phylogenetic tree was constructed by MEGA 6 by using Neighbor Joining method. Horizontal lines were proportional to the distances among species and vertical lines were arbitrary, full length nucleotide based dendogram was rooted on a distantly related SIGMV-[PR:04]-AY965900. REn based dendogram was routed on REn sequence of Sweet potato leaf curl virus SPLCV-[US:MS:1b-1a:07]. Here ''A'' represented phylogenetic tree of full length and ''B'' represented dandogram of REn proteins of all present isolates. Green color represented complete genome and REn sequence of ToLCV, purple for ToLCKeV, blue for PaLCuV, yellow for ToLCPalV and white color represented isolates of ToLCNDV. REn phylogenetic tree showed isolates of ToLCKeV, ToLCPalV and ToLCNDV, PaLCuV showed almost similar clade formation to that of full length nucleotide sequences, but RS96 and RS97 in REn protein tree showed different clade formation than other isolates of ToLCV species. 108

Text Figure 4.11: Alignment of REn protein of all ToLCV. This alignment was rooted on REn protein of SPLCV-[US:MS:1b-1a:07]-HQ333139. RS96 and RS97 indicated differences in REn protein on comparison with other ToLCV isolates. Amino acid differences were indicated by black boxes. Amino acid sequences at the end of ORF was more various, consists of 01, 02 and 05 amino acids patches, however these differences were consistent in both (RS96 and RS97) isolates, turning them divergent in AC3 phylogenetic tree among ToLCV isolates.

Text Figure 4.12: Phylogenetic dendogram of AC4 sequences. ''A'' here for complete nucleotide sequences and "B" for protein based dandogram. Dendogram was rooted on AC4 of a distantly related Macroptilium yellow spot virus MaYSC-[BR:Crb2:11]-KC004116. AC4 proteins of present Isolates were clustered to form distinct clades, similar to the whole genome phylogenetic dendogram. Green color represented complete genome and AC4 sequence of ToLCV, purple for ToLCKeV, blue for PaLCuV, yellow for ToLCPalV and white for ToLCNDV. 109

4.5.5 Phylogenetic analysis of AV1 encoded coat protein (CP)

All isolates of 05 species were also compared for virion sense proteins, to analyze difference of CP among different species, phylogenetic relation among full length and CP were shown (Text Figure 4.13). In CP dendogram all isolates of ToLCV were clustered together accept RS96 and RS97 which were clustered with isolates of PaLCuV (RS33) showed more similarity with these sequences. This variation in clade formation might be due to recombination, as RS33 (PaLCuV) showed 03 recombination events with ToLCV isolate shown (Text Figure 4.18). So this can lead to RS96 and RS97 group with RS33. Similarly one PaLCuV isolate (RS92) also showed variation in clade pattern, it clustered with RS2 and RS157 which were ToLCKeV isolates. Grouping of RS92, RS2 and RS157 as subclade may be due to their recombination with RaLCuV (Text Figure 4.15; 4.16), as these events were mapped in AV1 regions along with other sites. Along with this, multiple alignments were also performed for RS92, RS2, RS157 and other ToLCV isolates, indicated RS92 shared more similarity with RS2 and RS157 protein sequence than other all isolates in analysis, results not shown here.

4.5.6 Phylogenetic analysis of AV2 encoded pre CP

Phylogenetic dendogram of pre CP of present isolates compared with full length phylogenetic dendogram (Text Figure 4.14). This analysis showed very interesting clade formation based on AV2 proteins, all isolates have grouped into 02 main clades, first clad contain ToLCV isolates, PaLCuV and ToLCKeV while second clade have ToLCNDV and ToLCPalV. All isolates of ToLCNDV, ToLCPalV and ToLCKeV were grouped in respective clades, similar to full length sequence dandogram. Isolates of ToLCV forms 03 subclades, else than RS96 and RS97 isolates, as these isolates were distinct in full length tree as well. RS92 (PaLCuV) showed grouping with RS2 and RS157 ( ToLCKeV) in AV2 protein, as previously studied these isolates showed recombination with RaLCuV, may this recombined fragment involved in this clade pattern formation, but no significant recombination was observed in AV2 region particularly. Similarly RS33, RS58 and RS60 also intermingle with ToLCV isolates, RS33 was recombinant of ToLCV it can form clade with ToLCV isolates. Conclusively, this tree consists of 02 main clades first clade represents isolates of ToLCV, ToLCKeV, PaLCuV may contain more similar AV2 protein as compare to isolates in second clade (ToLCPalV and ToLCNDV). 110

Text Figure 4.13: Phylogenetic dendogram of AV1 (CP) sequences. ''A'' represents complete nucleotide sequences and "B" protein based dandogram, constructed by Neighbor Joining method. Horizontal lines were proportional to the distances among species and vertical lines were arbitrary. CP dendogram was rooted on CP sequence of a distantly related Sweet potato leaf curl Sichuan virus 1- [CN:Sc15:12]-KC488316. CP sequences of present Isolates were clustered to form distinct clades, similar to the whole genome phylogenetic dendogram, but few exceptions as grouping of RS96 ad RS97 (ToLCV) with RS33 (PaLCuV) and grouping of RS92 (PaLCuV) with RS2 and RS157 (ToLCKeV) were found. Green color here represented complete genome and CP sequence of ToLCV, purple for ToLCKeV, blue for PaLCuV, yellow for ToLCPalV and white color represents isolates of ToLCNDV. 111

Text Figure 4.14: Comparison of AV2 protein and full length sequence of all isolates. Phylogenetic tree constructed by Neighbor Joining method with bootstrap (1000 replicates) value. Horizontal lines were proportional to the distances among species and vertical lines were arbitrary, full length nucleotide based dendogram ''A'' was rooted on a distantly related SIGMV-[PR:04]-AY965900 represented. AV2 based dendogram ''B'' was routed on AV2 sequence of Sweet potato leaf curl Sichuan virus1-[CN:Sc15:12]-KC488316. Green color represented complete genome and AC4 sequence of ToLCV, purple for ToLCKeV, blue for PaLCuV, yellow for ToLCPalV and white color represents isolates of ToLCNDV. Phylogenetic tree showed two main clades, first contained ToLCV, ToLCKeV and PaLCuV and second contained ToLCPalV and ToLCNDV.

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4.6 Phylogeography

A phylogeographic analyses was done by using complete nucleotide sequences of DNA-A components of begomoviruses from this study and database sequences. The analyses demonstrated the geographical distribution of various components of ToLCD complex. Three major centers of diversity were found including southern, eastern and northwestern Indian subcontinent. Two major viruses ToLCKeV and ToLCV were prominent in the region 01 (southern region). Region 02 includes ToLCV as major contributor, region 03 (northwestern region) indicated maximum diversity including PaLCuV, ToLCPalV and ToLCNDV. Apart from these complexes, this was observed for first time that ToLCKeV, and ToLCV had also infected tomatoes in this region. These spatial distributions indicated PaLCuV, the most prevalent virus that was found in western region of subcontinent (KP: Pakistan) moreover, this was a recent phenomena (Figure 4). These results also suggested that the movement of ToLCKeV, ToLCV and PaLCuV was probably from India to Pakistan as the center of diversity lies in the Indian regions.

4.7 Recombination (inter and intraspecific) analysis among current isolates Isolates of present study were analyzed for recombination among viruses, which indicated RS2, RS157, RS92, RS101, RS186, RS176, RS33, RS163 and RS82 have inter specific recombinations, while RS80 and RS179 contain intraspecific recombinations and RS60 contain both inter as well as intraspecific recombinations.

RS157 ToLCKeV isolate showed 03 recombination events with Radish leaf curl virus (RaLCuV-[IN:Var:03]-EF175733), RaLCuV-[IN:Bih:09]-GU732203, RaLCuV- [IN:Bih06:10]-HQ257375 isolates, but recombination with EF175733 was more significant and mapped in AC1, AC2 and AC3 regions, while GU732203 and HQ257375 showed less significant recombination in AV1 and AC1 regions respectively. Major and minor parents and P-vales for these recombination events were listed (Table 4.4; Text Figure 4.15).

Similarly RS2 which was another isolate of ToLCKeV, was the recombinant of RaLCuV-[IN:LabIso:11]-JQ411026 having significant P-values (Text Figure 4.16; Table 4.4) and it was mapped in AC1 region.

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Figure 4.4: Phylogeography. Phylogeography was based on isolates obtained by current study and related isolates retrieved from NCBI database. Phylogeography "A" represented occurrence of ToLCKeV, ToLCV, PaLCuV, ToLCPalV and ToLCNDV species in Pakistan after trade and dotted lines were indicating these viruses movement direction, and the picture herein at bottom indicate distribution of ToLCKeV and ToLCV in Pakistan and Indian subcontinent before trade. While "B" represented distribution of isolates, specifically which were used for dandogram tree construction in Pakistan and Indian regions, indicating center of origin of these viruses was in Indian subcontinent. 114

Text Figure 4.15: RDP analysis of RS 157 (ToLCKeV) isolate. It recombined with RaLCuV- [IN:Var:03]-EF175733, RaLCuV-[IN:Bih:09]-GU732203, RaLCuV-[IN:Bih06:10]-HQ257375 isolates. Recombination with EF175733 was mapped in AC1 region, indicated by GENECONV algorithm of RDP and P-value were shown on Y-axis, but particular to this event were shown over the region, where exchange of genetic material took place.

Text Figure 4.16: RDP analysis performed for RS2 ToLCKeV isolate. This isolate recombined with RaLCuV-[IN:LabIso:11]-JQ411026. Breakpoints for this recombination event were 1959-2107 where exchange of genetic material took place, and this region was mapped in AC1 region of RS2, indicated by GENECONV algorithm of RDP and P-value particular to this event have been shown over this region.

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New strain of PaLCuV (RS92) showed 02 recombination events, first with ToLCV- [IN:Sas:Tom:09]-KP178730 this event took place in AV2 and IR regions. Second event with RaLCuV-[IN:Bih:To:09]-GU732204 was mapped in AV1, AC1, AC2 and AC3 ORFs. This recombinant exhibited a particular example of 02 interspecific recombinations among begomoviruses which might lead to the evolution of new strains (Text Figure 4.17), P-values for these events (Table 4.4).

RS33 contained 03 recombinations, first with Tomato leaf curl virus-Janti (TOLCV- Jan[IN:Jan:05]-AY754812) was mapped in AV1 and AV2 regions, second with Tomato leaf curl virus-Banglore (ToLCV-Ban-[IN:ND:09]-HM851186) in AC3. Third recombination took place with AY754812, in AC1 and AC4 regions (Text Figure 4.18).

RS82 a ToLCV isolate showed 03 more significant and interspecific recombination events, first was with Chilli leaf curl India virus ChiLCINV-[IN:08]-FM877858 mapped in IR, second with Tobacco curly shoot virus (TbCSV-[BD:Tom:06]- KM383757) in AC1 region, while third event with ToLCPalV-[PK:Lah:06]- AM494776 in AC4 and AC1 regions (Text Figure 4.19).

RS163 (ToLCNDV), showed 02 interspecific recombination events. First with ToLCV-[PK:Tom:15]-LN878121, second with ToLCPalV-[Pk:Lah:06]-AM494976 were mapped in AV1, AC2 and AC3 regions respectively (Text Figure 4.20).

RS176 a ToLCV isolate exhibited 02 interspecific recombination events, one with OELCuV-[IN:Odi:Okr:14]-KT390343, this recombination event was mapped in AC1 regions, second event with ToLCPalV-[Pk:Lah:06]-AM494976, which was mapped in IR region (Text Figure 4.20), P-values for these events (Table 4.4).

RS186 a ToLCV isolate has 04 recombination events, first and forth recombination events were with ToLCPalV-[Pk:Lah:06]-AM494976, second and third events were with Papaya leaf curl virus-Lucknow (PaLCuV-Luc-[IN:Luc]-Y15934). These four events were mapped in AV1, AC1, AC1 and AC1 respectively (Text Figure 4.20), P- values for these events (Table 4.4). RS101 which was also a ToLCV isolate, showed interspecific recombination with Okra enation leaf curl virus (OELCuV- [IN:Odi:okr:14]-KT390343) and ToLCPalV-[Pk:Lah:06]-AM494976. These recombination events were mapped in AC1 and AC4 regions respectively (Text Figure 4.20). 116

RS 80 a ToLCV isolate exhibited 02 intraspecific recombination events one with ToLCV-Ban[IN:IKH12:09]-HM803118 located in AC1, other event with ToLCV- [PK:Tom:15]-LT556073 which was also located in AC1, all these recombinants showed homologies with isolates from India, proposing this recombination event may took place there, as these all viruses were prevalent there shown (Text Figure 4.20).

RS179 ToLCV which has intraspecific recombination with ToLCV-[PK:Tom:15]- LT556073, in AC1 region P-values for these events given (Table 4.4).

RS 60 a PaLCuV isolate showed interspecific and intraspecific recombination with ToLCNDV-[PK:Tom:15]-LT556076 and PaLCuV-[IN:Har2:Aca:07]-FN625926 and these recombination events were located in AV2, AC1 and IR regions respectively (Text Figure 4.20).

Significant recombination events found in present isolates, their P-values, major and minor parents, recombination events numbers, events break points and percentage similarity have been given (Table 4.4).

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Text Figure 4.17: RDP analysis performed for RS 92 PaLCuV isolate. This isolate recombined with ToLCV-[IN:Sas:Tom:09]-KP178730 and RaLCuV-[IN:Bih:To:09]-GU732204, indicated by GENECONV algorithm of RDP P-value were shown on Y-axis and P-value for the event were given over the region.

Text Figure 4.18 : RDP analysis performed for RS 33 PaLCuV isolate. This isolate recombined with TOLCV-Jan[IN:Jan:05]-AY754812 and ToLCV-Ban[IN:ND:09]-HM851186. Recombination with AY754812 was more significant indicated by GENECONV algorithm of RDP, P-value were shown on Y-axis but for highlighted event shown over the region.

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Text Figure 4.19: RDP analysis performed for RS 82 ToLCV isolate. This isolate recombined with ChiLCINV-[IN:08]-FM877858, TbCSV-[BD:Tom:06]-KM383757 and ToLCPalV-[PK:Lah:06]- AM494776 at several points. Recombination with KM383757 was more significant indicated by GENECONV algorithm of RDP, P-value were shown on Y-axis but for highlighted event shown over the region.

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Text Figure 4.20: Recombination analysis performed by RDP4.2. All recombinants and recombination events detected in DNA-A components of begomoviruses, through RDR were shown accumulatively.

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Table 4.4: Recombination analysis of complete nucleotide sequences of DNA-A components of begomoviruses by RDP 4.2 program

Recombinant $ Breakpoints No./ Intraspecific

Interspecific/ Interspecific/ Recombination parents # P-values@

Major Minor RDP GENECONV Botscan MaxChi Chimaera SiScan PhylPro LARD 3Seq

Events Events

(3) i)594- PaLCuV-IN[IN:Pra3:Cro:o8]- 812 Interspecific GQ200448 RaLCuV-[IN:Bih:09]-GU732203 3.346x10-03 NS NS 5.190x10-06 1.220x10-06 NS NS NS NS

ii) 1088- RaLCuV-[IN:Bih06:10]- RS157 1759 Interspecific HQ257375 RaLCuV-[IN:Var:03]-EF175733 7.426x10-12 3.420x10-12 2.982x10-10 1.040x10-18 2.994x10-19 3.292x10-27 NS NS 8.512x10-27

iii) 2450- 2765 Interspecific RaLCuV-[IN:Bih:09]-GU732203 RaLCuV-[IN:Bih06:10]-HQ257375 NS 2.580x10-02 NS NS NS 8.307x10-17 NS NS NS

(1) 1959- RS2 2107 Interspecific RaLCuV-[IN:Bih:09]-GU732203 RaLCuV-[IN:LabIso:11]-JQ411026 3.475x10-31 1.936x10-31 NS 5.653x10-12 3.005x10-6 NS NS NS 8.523x10-34

(4) i) 645- 880 Interspecific PaLCuV-Luc[IN:Luc]-Y15934 ToLCPaV-[Pk:Lah:06]-AM494976 2.482x10-01 NS NS 8.199x10-11 9.147x10-06 2.991x10-23 NS NS NS

ii) 1853- ToLCV-[IN:Luc:Phy:12]- 1974 Interspecific JX524172 PaLCuV-Luc[IN:Luc]-Y15934 1.347x10-12 8.021x10-09 4.838x10-12 2.596x10-05 6.770x10-05 6.887x10-03 NS NS 4.736x10-02 RS186 iii) 2004- ToLCPalV-[Pk:Lah:06]- 2110 Interspecific AM494976 PaLCuV-Luc[IN:Luc]-Y15934 NS NS NS 3.216x10-06 2.012x10-03 NS NS NS NS

iv) 2260- 2646 Interspecific PaLCuV-Luc[IN:Luc]-Y15934 ToLCPaV-[Pk:Lah:06]-AM494976 2.580x10-19 NS 1.438x10-17 8.077x10-15 5.316x10-04 NS NS NS 1.249x10-15

(2) i) 1683- ToLCPalV-[Pk:Lah:06]- OKELCuV- 2127 Interspecific AM494976 [IN:Odi:Okr:14]-KT390343 6.821x10-03 NS 3.426x10-03 8.979x10-04 1.688x10-2 NS NS NS NS RS101 ii) 2162- OKELCuV- 2487 Interspecific AM494976 [IN:Odi:Okr:14]- KT390343 1.503x10-12 2.096x10-17 6.957x10-10 6.130x10-09 1.176x10-07 3.484x10-07 NS NS 5.617x10-03

(2) i) 71- RaLCuV-To[IN:Bih:To:09]- ToLCKV- [IN:Sas:Tom:09]- 304 Interspecific GU732204 KP178730 2.754x10-07 2.940x10-08 3.982x10-06 7.887x10-04 4.873x10-4 4.339x10-06 NS NS 2.801x10-04 RS92 RaLCuV-To[IN:Bih:To:09]- 5.434x10-1 ii) 680-1765 Interspecific ToLCV-[IN:Luc:Zel:12]-JX987088 GU732204 2.979x10-06 1.394x10-12 3 3.557x10-13 4.215x10-08 1.053x10-22 NS NS NS

Table continue page turn over 121

(2) i) 2162-2493 Interspecific TbCSV-[IN:RAN:07]-GQ994095 KT390343 3.362x10-04 1.071x10-02 2.884x10-03 1.358x10-03 1.124x10-02 1.896x10-04 NS NS NS RS176 ii) 2619- 2696 Interspecific KT390343 AM494976 2.893x10-02 NS NS 2.244x10-02 1.115x10-03 1.155x10-04 NS NS NS

(2) i) 373- PaLCuV-IN[IN: Har2:Aca:07] - 417 Interspecific FN625926 ToLCNDV-[PK:Tom:15]-LT556076 NS 7.44ox10-11 NS 1.846x10-02 3.793x10-02 NS NS NS NS RS60 ii) 2572- PaLCuV-IN[IN: Har2:Aca:07]- 2761 Intraspecific FN625901 FN625926 1.478x10-2 9.715x10-04 3.283x10-04 NS NS 1.079x10-04 NS NS 4.742x10-02

(2) i) ToLCV-Ban[IN:IKH12:09]- 1976-2117 Intraspecific ToLCV-[PK:Tom:15]-RS187 HM803118 3.399x10-06 4.642x10-05 NS 2.232x10-5 1.959x10-05 NS NS NS 6.221x10-08 RS80 ii) 1961- ToLCV-Ban[IN:IKH12:09]- 2667 Intraspecific HM803118 ToLCV-[PK:Tom:15]-RS187 3.635x10-03 8.099x10-05 3.655x10-08 8.323x10-09 5.587x10-06 3.904x10-09 NS NS 3.015x10-08

(1) 2117- ToLCV-Ban[IN:IKH12:09]- RS179 2667 Intraspecific HM803118 ToLCV-[PK:Tom:15]-RS187 5.632x10-07 3.781x10-04 4.982x10-07 1.158x10-09 1.577x10-09 9.029x10-10 NS NS 4.464x10-08

(3) i) 388- PaLCuV-IN[IN:Har2:Aca:07] 423 Interspecific FN645926 ToLCV-Jan[IN:Jan:05]-AY754812 NS 1.500x10-06 1.394x10-07 3.701x10-02 3.466x10-02 NS NS NS NS

ii) 1063- RS33 1298 Interspecific ToLCV-[IN: Mir:99]-AF449999 ToLCV-Ban[IN:ND:09]-HM851186 6.765x10-07 2.281x10-06 2.233x10-06 4.126x10-06 3.592x10-03 9.876x10-18 NS NS 2.152x10-03

iii) 2174- 2762 interspecific ToLCV-[IN: Mir:99]-AF449999 ToLCV-Jan[IN:Jan:05]-AY754812 1.937x10-40 1.123x10-42 6.940x10-43 1.129x10-19 2.533x10-22 2.709x10-45 NS NS 4.389x10-22

(3) i) ToLCV-[IN:Luc:Phy:12]- 2689-22 Interspecific JX524172 ChiLCINV-[IN:08]-FM877858 5.727x10-22 NS 3.739x10-22 9.719x10-08 1.888x10-07 1.853x10-08 NS NS 4.335x10-06

RS82 ii) 1525- 2095 Interspecific ToLCV-Ban[IN:ban:93]-U38239 TbCSV-[BD:Tom:06]-KM383757 1.234x10-07 2.343x10-08 2.386x10-07 1.573x10-10 9.616x10-10 2.295x10-7 NS NS 2.495x10-11

iii) 2261- 2688 interspecific ChiLCINV-[IN:08]-FM877858 ToLCPalV-[Pk:Lah:06]-AM494976 1.032x10-15 3.720x10-05 2.981x10-13 4.277x10-10 2.834x10-08 1.219x10-28 NS NS 1.833x10-13

(2) i) 284- ToLCPalV-[PK:Lah:06]- 465 interspecific AM494976 ToLCV-[PK:Tom:15]-LN878121 3.669x10-7 1.340x10-04 2.763x10-06 4.699x10-05 1.208x10-05 9.852x10-19 NS NS NS RS163 ii) 1347- TbCSV-[BD:Chi:tom:06]- 1560 interspecific KM383753 ToLCPalV-[PK:Lah:06]-AM494976 NS 4.240x10-03 NS 1.732x10-02 1.415x10-03 NS NS NS NS

$ Represents number of recombination events took place in whole genome of particular recombinants and breakpoints of each recombination events # Recombination major or minor parents @ P-values for RDP, GENECONV, BootScan, MaxChi, Chimaera, SiScan, PhylPro, LARD, 3Seq algorithms of RDP for each recombination event 122

4.8 Molecular characterization of DNA-B of begomoviruses

Molecular characterization of DNA-B components of begomoviruses was done as follows.

4.8.1 Amplification and cloning of DNA-B components of begomoviruses

Genomic DNA of 75 infected samples were subjected to PCR by using RCA amplification products with newly synthesized primers, their sequences shown (Table 3.1) and 57 samples showed ~2.7 kb amplification (Figure 4.5). Amplification of DNA-B components was found positive in 21samples by B1/B2 primers pair and in 36 samples by B3/B4 primers pair.

PCR amplified product of begomoviruses after ligation into cloning vector (pTZ57/RT) were transformed into E. coli (Top-10 strain). After obtaining positive clones confirmed through restriction by using EcoRI and PstI restriction enzymes (Figure 4.6) and unique restriction enzymes i.e. HinDIII for B1/B2, KpnI for B3/B4 were also used when required. Clones after confirmation by RFLP were sent for sequencing.

4.8.2 Molecular characterization and ORFs analysis of DNA-B components of begomoviruses

All clones get sequenced, their complete nucleotides sequences were determined. A total of 41 DNA-B complete sequences were submitted in database. Complete nucleotide genomes of DNA-B consisted of 2686-2704 nucleotides, their accession numbers were (Table 4.5). For initial comparison nucleotides BLAST analysis was performed for DNA-B sequences. Isolates showed maximum homology with ToLCNDV DNA-B. Duly complete sequences were employed to determine ORFs, by using ORF finder of NCBI (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). All DNA-B components of bipartite begomoviruses were having 02 ORFs characteristics of every DNA-B, BC1 in complementary sense and BV1 in virion sense orientation, both ORFs separated by CR having IR. All sequences were found with putative stem loop structure containing nonanucleotide (TAATATTAC) sequence in their intergenic region, a characteristic of begomoviruses. Although majority of the sequences were having ORFs consists of particular number of nucleotides, few exception were there having few more or less nucleotides. Nucleotides number constituting, BC1 varied from 440 to1248 and for 123

BV11302 to 2152 among all DNA-B, coordinates for each ORF in all sequences (Table 4.5). In bipartite begomoviruses DNA-B shared same replication initiation sequences (eterons) with cognate DNA-A however, some exception were also found.

BLAST analyses performed indicated various levels of similarity of herein isolates with database, ranging from 91 to 95 % with already present sequences at NCBI database. A complete detail of BLAST results with homologies showing accession numbers and percentage values (Table 4.6).

4.9 Phylogenetic Analysis of DNA-B components of begomoviruses

Phylogenetic tree based on multiple alignments of DNA-B of herein and maximum homology sharing database sequences was constructed by Neighbor Joining method with 1000 bootstrap value. Isolates were clustered with homology sharing sequences retrieved from NCBI site; these all sequences in phylogenetic tree were arranged in particular pattern to form distinct clades.

All sequences were grouped to form 02 main clades and another clade of outgroup. First clade represented as "A" contained more isolates, was further divided into 04 subclades (a, b, c and d), isolates those form first subclade "a" were obtained from (Lahore and KP regions) samples and these were ToLCNDV DNA-B, found clustered with isolates submitted from Pakistan. Similar was also observed for isolates in subclades "b" and "c". In opposite to previous subclades "d" represented isolates grouped with isolates from Taiwan, Thailand and India. While isolates of second clade "B" were isolated from KP samples and found associated with ToLCPalV, showed grouping with maximum homology sharing isolates submitted from India. Outgroup upon which phylogenetic tree was rooted was expressed as "e" (Text Figure 4.21).

By performing multiple alignments for complete sequences of DNA-B by Clustral W method using MegAlign program of Lasergene, percentage identity and divergence were determined. Phylogenetic tree formation was further supported by percentage identities and divergence data to make sure all DNA-B isolates constituting particular clades however were distinct from other isolates forming different clades. Percentage identities vary among all present and NCBI DNA-B isolates constituting phylogenetic tree varies from 81.7 to 100 %. However all present isolates shared 82.4 to 99.9 % 124 identity. Percentage identities among all isolates of first main clade "A" ranges from 81.8 % to 99.9 %, for subclade a vary from 88.5 % to 99.7 % for present and NCBI isolates, but only present isolates shared 89.6 to 99.7 % identity. Similarly complete sequences of all isolates in b subclade shared percentage identity vary from 97.3 to 99.9 %. But present isolates of subclade a and b showed 88 % to 99.7 % percentage identities. All isolates of c subclade represent 87.4 % to 100 % identity and present isolates of this subclade showed 99.9 % identity and share 94 % to 94.1 % identity with ToLCNDV-[PK:Dar:T5/6:01]-AY150305 but with other NCBI sequences shared 87.4 % to 94.1 % identities. In d subclade, present isolates exhibited 89.7 to 97.7 % percentage identities, while NCBI and present isolates shares 84.7 % to 99.7 %. All isolates of clade B exhibited 94.9 % to 99.4 % percentage identity, while all present isolates shared 96.7 % to 99.3 % identity.

4.9.1 Comparison of isolates based on full length sequences and BV1 encoded NSP

BV1 encoded NSP protein and full length sequences based phylogenetic dandogram on comparison, indicated the presence of one main clade in NSP based tree, which further showed division in subclades originating from main clade. Isolates in a, b, c and d subclade in complete nucleotide sequences tree showed similar clades in protein based tree (Text Figure 4.22). Only RS156 that seemed distinct and grouped separately from its group d with isolates of subclade a. Grouping of RS156 with isolates of subclade a indicated similarity of BV1 of RS156 with isolates of subclade a than isolates of subclade d. Isolates of clade "B" were also showing similar grouping in NSP tree. Result derived from this analysis indicated NSP of all isolates of different species were also distinct like complete nucleotide sequences.

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Figure 4.5: Gel picture exhibiting PCR amplified fragment of ~2.7 kb sized begomoviruses. PCR amplified products were loaded with 1kb DNA marker for size specification of amplified products. Wells containing samples were designated as 01, 02, 03, 04, 05, 06, 07, 08 and 09 while wells 10 and 11 represented positive and negative controls respectively. Well marked by "M" contained 1 kb DNA marker. Previously confirmed infected plant samples, gave amplification of ~2.7 kb was used as positive control, while in well 11 PCR product of genomic DNA of healthy plant was run as negative control. Samples in wells 01 to 09 showed amplification of 2.7 kb.

Figure 4.6: Restriction of DNA-B components of bipartite begomoviruses. Gel picture indicated restriction pattern for full length DNA-B components of bipartite begomoviruses clones. Restriction product was run in 01 to 12 wells, restriction product in 03, 08, 09, 10, 11 and 12 wells give full length (~2.7 kb) fragment accumulatively, exhibiting two types of restriction patterns. Restriction product in well 03, 08 and 09 accumulatively construct 2.8 kb fragments while product in wells 10, 11 and 12 showed single fragment of ~2.7 kb, different restriction pattern indicated restriction fragment length polymorphism. "M" here was used to represent DNA. 126

Table 4.5: Coordinates of BCI and BVI Open Reading Frames (ORFs) of DNA-B component of bipartite begomoviruses, their accession numbers and origin (Longitude & Latitude) coordinates

Accession No. Clone Genome Identity in Location Longitude Latitude BV1 BC1 name length Database

LN878124 RS81 2693 ToLCNDVB Rakh Burj 31.38703 74.515358 442-1248 1305-2150 LN878126 RS84 2696 ToLCNDVB Rakh Burj 31.38703 74.515358 440-1246 1305-2150 LN886526 RS85 2696 ToLCNDVB Rakh Burj 31.38703 74.515358 440-1246 1305-2150 LT168849 RS109 2698 ToLCNDVB Kamaala 34.64119 71.79818 447-1253 1302-2147 LT168850 RS110 2692 ToLCNDVB Kamaala 34.64119 71.79818 441-1247 1303-2148 LT168851 RS111 2692 ToLCNDVB Kamaala 34.64119 71.79818 441-1247 1303-2148 LT168852 RS113 2692 ToLCNDVB Kamaala 34.64119 71.79818 441-1247 1303-2148 LT168854 RS117 2692 ToLCNDVB Khadag Zai 34.63468 71.88264 441-1247 1304-2149 LT168855 RS119 2698 ToLCNDVB Gogdarra 34.74286 72.28895 442-1248 1302-2147 LT168856 RS120 2698 ToLCNDVB Gogdarra 34.74286 72.28895 442-1248 1302-2147 LT168857 RS121 2698 ToLCNDVB Gogdarra 34.74286 72.28895 442-1248 1302-2147 LT168849 RS122 2698 ToLCNDVB Gogdarra 34.74286 72.28895 442-1248 1302-2147 LT168859 RS123 2698 ToLCNDVB Gogdarra 34.74286 72.28895 442-1248 1302-2147 LT168860 RS125 2691 ToLCNDVB Korigram 34.76807 72.02393 442-1248 1304-2149 LT168861 RS126 2693 ToLCNDVB Khadag Zai 34.63468 71.88264 442-1248 1304-2149 LT168862 RS129 2695 ToLCNDVB Korigram 34.76807 72.02393 441-1247 1305-2150 LT168863 RS130 2694 ToLCNDVB Korigram 34.76807 72.02393 440-1246 1304-2149 LT168864 RS131 2694 ToLCNDVB Korigram 34.76807 72.02393 440-1246 1304-2149 LT168865 RS133 2695 ToLCNDVB Korigram 34.76807 72.02393 440-1246 1304-2149 LT168866 RS135 2692 ToLCNDVB Shamo Zai 34.6857 72.14757 441-1247 1303-2148 LT168867 RS136 2692 ToLCNDVB Shamo Zai 34.6857 72.14757 441-1247 1303-2148

Table continue page turn over

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LT168868 RS149 2692 ToLCNDVB Jaban 34.55836 71.93477 441-1247 1303-2148 LT168869 RS152 2691 ToLCNDVB Rakh Burj 31.38703 74.515358 442-1248 1307-2152 LT168870 RS153 2692 ToLCNDVB Rakh Burj 31.38703 74.515358 442-1248 1307-2152 LT168871 RS154 2696 ToLCNDVB Rakh Burj 31.38703 74.515358 440-1246 1305-2150 LT168872 RS155 2696 ToLCNDVB Rakh Burj 31.38703 74.515358 440-1246 1305-2150 LT168873 RS156 2691 ToLCNDVB Rakh Burj 31.38703 74.515358 442-1248 1307-2152 LT168874 RS161 2694 ToLCNDVB Rakh Burj 31.38703 74.515358 442-1248 1305-2150 LT168875 RS162 2693 ToLCNDVB Rakh Burj 31.38703 74.515358 442-1248 1305-2150 LT168876 RS167 2695 ToLCNDVB Rakh Burj 31.38703 74.515358 441-1247 1306-2151 LT168877 RS168 2696 ToLCNDVB Rakh Burj 31.38703 74.515358 440-1246 1305-2150 LT168878 RS169 2696 ToLCNDVB Rakh Burj 31.38703 74.515358 440-1246 1305-2150 LT168879 RS192 2699 ToLCNDVB Tormang 34.91475 72.01043 442-1248 1305-2150 LT168880 RS194 2699 ToLCNDVB Tormang 34.91475 72.01043 442-1248 1305-2150 LT168881 RS195 2698 ToLCNDVB Tormang 34.91475 72.01043 441-1247 1304-2149 LT168882 RS196 2699 ToLCNDVB Tormang 34.91475 72.01043 442-1248 1305-2150 LT168883 RS221 2686 ToLCNDVB Kamaala 34.64119 71.79818 442-1248 1305-2150 LT168884 RS228 2692 ToLCNDVB Piraan 34.58015 71.93396 441-1247 1303-2148 LT168885 RS229 2690 ToLCNDVB Korigram 34.76807 72.02393 441-1247 1304-2149 LT168886 RS230 2690 ToLCNDVB Korigram 34.76807 72.02393 442-1248 1304-2149 LT168887 RS232 2691 ToLCNDVB Korigram 34.76807 72.02393 440-1246 1302-2147

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Table 4.6: BLAST results of current DNA-B components of begomoviruses and their percentage homology with database sequences.

Clone Accession Percentage BLAST Results Name No. homology RS81 LN878124 ToLCNDV-[IN:ND:Pum2:05]-AM286435 94 % RS84 LN878126 ToLCNDV-[PK:Kha:Chi:04]-DQ116882 94 % RS85 LN886526 ToLCNDV-[PK:Kha:Chi:04]-DQ116882 94 % RS109 LT168849 ToLCNDV-[IN:ND:AVT1]-AY438563 94 % RS110 LT168850 ToLCNDV-[IN:ND:AVT1]-AY438563 95 % RS111 LT168851 ToLCNDV-[IN:ND:AVT1]-AY438563 95 % RS113 LT168852 ToLCNDV-[IN:ND:AVT1]-AY438563 95 % RS116 LT168853 ToLCNDV-[IN:ND:AVT1]-AY438563 95 % RS117 LT168854 ToLCNDV-[IN:ND:AVT1]-AY438563 95 % RS119 LT168855 ToLCNDV-[PK:Kha:Chi:04]-DQ116882 91 % RS120 LT168856 ToLCNDV-[PK:Kha:Chi:04]-DQ116882 91 % RS121 LT168857 ToLCNDV-[PK:Kha:Chi:04]-DQ116882 91 % RS122 LT168858 ToLCNDV-[PK:Kha:Chi:04]-DQ116882 91 % RS123 LT168859 ToLCNDV-[PK:Kha:Chi:04]-DQ116882 91 % RS125 LT168860 ToLCNDV-[IN:ND:AVT1]-AY438563 95 % RS126 LT168861 ToLCNDV-[IN:ND:AVT1]-AY438563 95 % RS129 LT168862 ToLCNDV-[PK:Sam:Son:09]-FN432357 92 % RS130 LT168863 ToLCNDV-[PK:Tom:08]-AM947507 93 % RS131 LT168864 ToLCNDV-[PK:Sam:Son:09]-FN432357 92 % RS133 LT168865 ToLCNDV-[PK:Sam:Son:09]-FN432357 92 % RS135 LT168866 ToLCNDV-[IN:ND:AVT1]-AY438563 95 % RS136 LT168867 ToLCNDV-[IN:ND:AVT1]-AY438563 95 % RS149 LT168868 ToLCNDV-[IN:ND:AVT1]-AY438563 95 % RS152 LT168869 ToLCNDV-[IN:ND:AVT1]-AY438563 94 % RS153 LT168870 ToLCNDV-[IN:ND:Pum2:05]-AM286435 95 % RS154 LT168871 ToLCNDV-[PK:Kha:Chi:04]-DQ116882 94 % RS155 LT168872 ToLCNDV-[PK:Kha:Chi:04]-DQ116882 94 % RS156 LT168873 ToLCNDV-[IN:ND:05]-DQ169057 92 % RS161 LT168874 ToLCNDV-[IN:ND:Pum2:05]-AM286435 94 % RS162 LT168875 ToLCNDV-[IN:ND:Pum2:05]-AM286435 94 % RS167 LT168876 ToLCNDV-[PK:Kha:Chi:04]-DQ116882 94 % RS168 LT168877 ToLCNDV-[PK:Kha:Chi:04]-DQ116882 94 % RS169 LT168878 ToLCNDV-[PK:Kha:Chi:04]-DQ116882 94 % RS192 LT168879 ToLCNDV-[PK:Kha:Chi:04]-DQ116882 91 % RS194 LT168880 ToLCNDV-[PK:Kha:Chi:04]-DQ116882 91 % RS195 LT168881 ToLCNDV-[PK:Kha:Chi:04]-DQ116882 91 % RS196 LT168882 ToLCNDV-[PK:Kha:Chi:04]-DQ116882 91 % RS221 LT168883 ToLCNDV-[IN:ND:AVT1]-AY438563 95 % RS228 LT168884 ToLCNDV-[IN:ND:AVT1]-AY438563 95 % RS229 LT168885 ToLCNDV-[PK:Dar:T5/6:01]-AY150305 94 % RS230 LT168886 ToLCNDV-[PK:Dar:T5/6:01]-AY150305 94 % RS232 LT168887 ToLCNDV-[IN:ND:AVT1]-AY438563 95 %

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Text Figure 4.21: Phylogenetic analysis of DNA-B components. DNA-B phylogenetic dandogram was constructed by Neighbor Joining method, numbers at nodes indicated percentage bootstraps confidence values (1000 replicates). Horizontal lines were proportional to the distances among species and vertical lines were arbitrary. Isolates during present study were clustered with database sequences to form two main clades "A" and "B". "A" contained subclades (a, b, c and d). DNA-B isolates present in subclade a clustered with database sequences reported from Pakistan, similar was found for subclade b and c however subclade d represents, herein isolates grouped with Thailand, Taiwan and Indian origin while second clade "B" indicated isolates were clustered with ToLCPalV DNA-B from India. The dendrogram was rooted on the DNA-A of a distantly related NW, SiGMV indicated as e.

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Text Figure 4.22: Comparison of isolates, based on full length sequences and BV1 proteins. Phylogenetic tree was constructed by Neighbor Joining method with bootstrap (1000 replicates) value. Horizontal lines were proportional to the distances among species and vertical lines were arbitrary, full length nucleotide based dendogram ''A'' was rooted on a distantly related SIGMV-[PR:04]- AY965901full length sequence. BV1 based dendogram ''B'' was routed on BV1 protein sequence of SIGMV-[PR:04]-AY965901. Main clades in protein and full length tree were labeled as "A" (subclades, a, b, c and d) and "B" subclades.

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4.9.2 Comparison of phylogenetic tree for full length genome and BC1 encoded MP

A comparison was performed based on phylogenetic dendogram, constructed by full length DNA-B sequences of present study after multiple alignment by MUSCLE method of MEGA6 and BC1 encoded protein after multiple alignmnet by Clustral W. This comparision was to understand the different types of DNA-B as a whole and on the basis of protein and how much variation was found in BC1 sequences of present siolates. Tree based on complete sequences, indicated formation of 02 main clades represented as "A" and "B", clade "A" was further divided into subclades. Similar to this, protein based phylogenetic tree was also showing 02 main clades "A" and "B" (Text Figure 4.23). Isolates in full length tree in first subclade a, on comparing with BC1 tree indicated protein sequences of subclade a show more similarity with protein sequences of subclade d, similar was observed with BC1 sequences of isolates in subclade d. Isolates of subclade b and c have similarly grouped in BC1 tree as in full length tree. BC1 sequences in clade "B" were also grouped in similar pattern as in full length tree. These results indicated BC1 protein was conserve but minor difffernce of aminoacid sequence might be responsible for a little bit different clade pattern. However percentage identity among all BC1 proteins of present isolates varied from 90.8 to 99.6 % for all isolates, this indicated more conresve nature of BC1.

This was very obvious by this analysis that protein sequences of majority of present isolates showed conserve nature, as percentage identity among proteins of isolates was more than full length sequences, but with few exceptions. However isolates were also having minor differences in amino acids sequences, shown in multiple alignments for the proteins (results not shown here).

4.10 Recombination analysis of DNA-B

Multiple alignments of all present and maximum homology sharing isolates from NCBI database were analyzed for recombination by RDP4.2 software. RDP analysis exhibited, among 41 present isolates, 09 showed recombination and significant P- values based on majority of parameters (three or more than three algorithms). Isolates having recombination, major and minor parents, P-value for all algorithms, event numbers in every recombinant and cut off values for each recombination event were (Table 4.7). 133

Likewise RS156-[PK:Tom:15]-LT168873 was found recombinant of RS167- [PK:Tom:15]-LT168876 and major parent for this was ToLCNDV-[IN:ND:05]- DQ169057 while RS167-[PK:Tom:15]-LT168876 was minor parent. It was mapped in BC1and BV1, indicated large fragment of about 1500 base pairs and significant P- values shown (Text Figure 4.24).

Similarly RS122-[PK:Tom:13]-LT168858 ToLCNDV DNA-B component showed recombination with RS117-[PK:Tom:13]-LT168854. In this event RS117- [PK:Tom:13]-LT168854 minor while RS161-[PK:Tom:15]-LT168874 was major parent, P-value shown (Table 4.7) this recombination event was mapped in BV1 region (Text Figure 4.25).

RDP analysis of ToLCNDV DNA-B indicated RS161-[PK:Tom:15]-LT168874 showed 03 recombination events with RS117-[PK:Tom:13]-LT168854, in all 03 events major recombination parent RS122-[PK:Tom:13]-LT168858 and minor parent was RS117-[PK:Tom:13]-LT168854. First and second recombination event in BV1 region and recombination event took place in IR, P-value were shown (Table 4.7)

RS119-[PK:Tom:13]-LT168855 contained 04 recombination events with RS117- [PK:Tom:13]-LT168854 and in all recombination events it contain ToLCNDV- [PK:Sam:Son:09]-FN432357 as major parent while minor parent was RS117- [PK:Tom:13]-LT168854. All 04 events were mapped in BV1, BV1, IR and IR respectively (Text Figure 4.25).

RS129-[PK:Tom:13]-LT168862 recombine with RS230-[PK:Tom:13]-LT168886, in this recombination event ToLCNDV-[PK:Sam:Son:09]-FN432357 was as major and RS230-[PK:Tom:13]-LT168886 as minor parent, and this event was mapped in IR.

Similarly RS135-[PK:Tom:13]-LT168866 was recombinent of RS161-[PK:Tom:15]- LT168874, in this event major parent was RS136-[PK:Tom:]-LT168867 and minor parent was RS161-[PK:Tom:15]-LT168874. This recombination was located in BC1 and IR regions (Text Figure 4.25).

RS152-[PK:Tom:15]-LT168869 also recombined with 167-[PK:Tom:15]-LT168876, this recombination event contained RS153-[PK:Tom:15]-LT168870 as major and RS167 as minor parent and it was mapped in BC1 region (Text Figure 4.25). 134

RS153-[PK:Tom:15]-LT168870 recombined with RS167-[PK:Tom:15]-LT168876, major parent was ToLCNDV-[PK:FSB:Son:09]-FN432357, while RS167 as minor parent and this event was located in BC1(Text Figure 4.25).

RS192-[PK:Tom:13]-LT168879 exhibited 02 recombination events with ToLCNDV- [PK:Sam:Son:09]-FN432357, in this recombination event RS230-[PK:Tom:13]- LT168886 was major, FN432357 as minor parent. In second event RS230- [PK:Tom:13]-LT168886 was major, FN432357 as minor parent and both events were mapped in IR (Text Figure 4.25).

For all recombinant isolates, 04 recombinations took place in BC1 region, while 07 in IR region and 06 events took place in BV1 regions, so among BV1 and BC1, BV1 seemed more vulnerable for recombination or might be considered hot spot.

These all recombinants along with their recombination components were shown accumulatively (Text Figure 4.25).

135

Text Figure 4.23: Comparison of isolates based on full length nucleotide sequences and BC1 proteins. Phylogenetic tree was constructed by Neighbor Joining method with bootstrap (1000 replicates) value. Horizontal lines were proportional to the distances among species and vertical lines were arbitrary, full length nucleotide based dendogram ''A'' was rooted on a distantly related SIGMV- [PR:04]-AY965901full length sequence. BC1 based dendogram ''B'' was routed on BC1 protein sequence of SIGMV-[PR:04]-AY965901. Main clades in protein and full length tree were labeled as "A" (subclades, a, b, c and d) and "B" subclades.

136

Text Figure 4.24: RDP analysis performed for RS156 ToLCNDV DNA-B isolate. Isolate RS156- [PK:Tom:15]-LT168873 recombines with RS167-[PK:Tom:15]-LT168876. This recombination event took place in BV1 and BC1 regions. This figure exhibited recombination event in GENECONV algorithm of RDP, P-value shown on Y-axis but for above mentioned event P-value were shown over the recombination region.

Text Figure 4.25: Recombination analysis performed by RDP4.2 for DNA-B components of begomoviruses. All recombinants and recombination events detected in DNA B components through RDR have been shown accumulatively. 137

4.7: Recombination analyses of complete nucleotide sequences of DNA-B components of begomoviruses by RDP4.2 program.

Recombinants $ points No./ Break Events Recombination parents # P-values @

Major Minor RDP V GENECON Botscan MaxChi Chimaera SiScan PhylPro LARD 3Seq

3) i) 266-505 ToLCNDVB-[PK:Tom:13]- ToLCNDVB-[Tom:PK:13]- 1.074x10-03 6.568x10-04 1.969x10-03 5.488x10-04 3.605x10-04 NS NS NS NS LT168858 LT168854

RS161 ii) 954-1073 ToLCNDVB-[PK:Tom:13]- ToLCNDVB-[Tom:PK:13]- NS 4.657x10-02 4.168x10-03 1.127x10-03 1.423x10-02 NS NS NS NS LT168858 LT168854

iii) 2274- ToLCNDVB-[PK:Tom:13]- ToLCNDVB-[Tom:PK:13]- 1.796x10-02 NS 2.456x10-02 NS NS NS NS NS NS 2479 LT168858 LT168854

RS122 1) 1465-1805 ToLCNDVB-[PK:Tom:15]- ToLCNDVB-[Tom:PK:13]- 9.296x10-07 3.658x10-05 2.201x10-07 9.2960x10-05 2.599x10-04 NS NS NS 2.835x10-03 LT168874 LT168854

RS152 1) 1535-2097 ToLCNDVB-[PK:Tom:15]- ToLCNDVB-[PK:Tom:15]- 1.649x10-34 1.373x10-32 1.629x10-34 2.344x10-16 2.703x10-15 9.035x10-14 NS NS 1.391x10-34 LT168870 LT168876

4) i)1133- ToLCNDV-[PK:FSB:Son:09]- ToLCNDVB-[Tom:PK:13]- 5.324x10-04 NS 6.671x10-12 NS NS NS NS NS NS 1537 FN432357 LT168854

ii) 1554-1808 ToLCNDV-[PK:FSB:Son:09]- ToLCNDVB-[Tom:PK:13]- 1.491x10-7 3.790x10-08 1.343x10-7 1.252x10-05 9.080x10-04 NS NS NS 2.237x10-04 FN432357 LT168854 RS119 iii) 2171- ToLCNDV-[PK:FSB:Son:09]- ToLCNDVB-[Tom:PK:13]- 2.175x10-03 NS 1.264x10-02 2.633x10-02 NS NS NS NS NS 2528 FN432357 LT168854

iv) 2526-2629 ToLCNDV-[PK:FSB:Son:09]- ToLCNDVB-[Tom:PK:13]- 2.446x10-04 2.559x10-03 NS 1.832x10-04 2.361x10-03 NS NS NS NS FN432357 LT168854

RS129 1) 2535- ToLCNDVB-[PK:FSB:Son:09]- RS230-[PK:Tom:13]- 8.223x10-06 NS NS 3.578x10-06 4.201x10-03 NS NS NS NS 2658 FN432357 LT168886

RS135 1) 1269-2692 ToLCNDVB-[PK:Tom:]- ToLCNDVB-[PK:Tom:15]- 3.679 x10-02 NS NS NS NS NS NS NS 7.575x10-03 LT168867 LT168874

RS156 1) 631-2090 ToLCNDVB-[IN:ND:05]- ToLCNDVB-[PK:Tom:15]- 4.069x10-37 1.366x10-37 2.786x10-39 8.597x10-22 1.583x10-21 2.187x10-27 NS NS 3.114x10-40 DQ169057 LT168876

i) 2212-2538 RS230-[PK:Tom:13]-LT168886 ToLCNDVB- 5.719x10-11 1.028x10-05 3.053x10-09 1.559x10-11 2.915x10-12 NS NS NS 2.148x10-08 [PK:FSB:Son:09]-FN432357 RS192 ii) 2673-355 ToLCNDV B-[PK:Tom:13]- ToLCNDVB- 4.490x10-05 NS 2.879x10-05 1.767x10-8 1.423x10-8 NS NS NS 2.619x10-06 LT168886 [PK:FSB:Son:09]-FN432357

RS153 1) 1005-2217 ToLCNDVB-[PK:FSB:Son:09]- ToLCNDVB-[PK:Tom:15]- 3.571x10-2 NS NS 6.824x10-04 2.041x10-04 7.072x10-19 NS NS NS FN432357 LT168876

$ Represents number of recombination events in whole genome of particular recombinants and breakpoints of each recombination events, # Recombination major or minor parents, @ P-values for RDP, GENECONV, BootScan, MaxChi, Chimaera, SiScan, PhylPro, LARD, 3Seq algorithms of RDP for each recombination event and NS for (not significance) 138

4.11 Characterization of Betasatellites

Characterization of all present betasatellites was done as follows.

4.11.1 Amplification of betasatellites

All isolated genomic DNA were subjected to PCR to amplify betasatellites (1.4 kb fragments) associated with monopartite begomoviruses usually, causing ToLCD. Among present samples, 23 showed amplification of betasatellites (Figure 4.7A). Dilutions of amplified RCA product were also used for amplification of betasatellites by PCR. RCA amplified products after restriction by BglII were also used. Restricted products were ligated in cloning vector (pTZ57R/T) and transformed in E. coli (Top- 10 strain) for cloning. Positive clones after confirmation by RFLP, which in totality yield fragment of 1.4 kb (Figure 4.7B) were selected and sequenced.

Confirmed clones after sequencing were assembled by using Seqman software (DNA star package). Among 23, 19 sequences were completed in entirety and initially all sequences compared for species identification by BLAST, with already submitted database sequences. All 19 entirely complete sequences were submitted in database and accession numbers were acquired and shown (Table 4.9).

4.11.2 Molecular characterization of betasatellites

BLAST results exhibited 86 % to 98 % similarities of fifteen present isolates with PaLCuB from database and which were greater than threshold level, while remaining four isolates showed similarity with CLCuMuB with NCBI isolates, so far available sequences, vary from 92 % to 99 %. Cut off limit for betasatellites species demarcation was 78 %. Complete genomes of all betasatellite isolates consist of 1322 to 1370 nucleotides. Betasatellite sequences were investigated for ORFs, by means of ORF finder of NCBI (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). All betasatellites were having βC1 ORF, coordinates for βC1 of all PaLCuB and CLCuMuB isolates, their accession numbers and origin were given (Table 4.8). Beta C1 was enclosed by CR containing IR, important feature of CR of betasatellite to share homology with CR of cognitive DNA-A. A highly conserved sequence called nonanucleotide sequence TAATATTAC was also found in intergenic regions of all betasatellites. Beta C1 was 357 nucleotides and 118 amino acids long. Isolates from NCBI database which showed maximum similarity with present isolates, their accession numbers and percentage maximum similarity, were shown (Table 4.9). 139

Figure 4.7: Amplification and restriction of betasatellites. In figure, "A" represented PCR amplification of 1.4 kb sized betasatellites, amplification products were loaded in agarose gel with 1 kb DNA marker for fragment size specification and denoted by M. Wells marked as 01, 02, 03 and 04 contained PCR product of infected samples while - and +, represented negative and positive control respectively. Negative control was obtained by genomic DNA of healthy plant; already confirmed infected DNA was used for positive control. Samples loaded in wells marked 01 to 04 were found positive for infection as yield 1.4 kb fragment. Samples in well 1 and 3 showed multiple bands, required bands were eluted and purified. "B" indicating restriction pattern of betasatellites clones. Restriction product were run in 01, 02, 03, 05, 06, 08 and 10 wells gave full length 1.4 kb fragment accumulatively.

Table 4.8: Coordinates of βC1 protein of betasatellites, genome length, samples origin and GeneBank accession numbers.

Clone Accession Genome Identity in Location Longitude Latitude βC1 name No. size database RS10 LN878102 1351 CLCuMuB NIAB 31.396249 73.031816 195-551 RS19 LN878105 1350 CLCuMuB NIAB 31.396249 73.031816 195-551 RS25 LN878108 1350 CLCuMuB NIAB 31.396249 73.031816 195-551 RS31 LN878109 1350 CLCuMuB NIAB 31.396249 73.031816 195-551 RS68 LN878112 1359 PaLCuB Rakh Burj 31.38703 74.515358 195-551 RS77 LN878110 1366 PaLCuB Rakh Burj 31.38703 74.515358 200-556 RS78 LN878111 1366 PaLCuB Rakh Burj 31.38703 74.515358 200-556 RSJ88-7 LN878099 1362 PaLCuB Timergarh 34.82247 71.83608 195-551 RS88-8 LN887901 1362 PaLCuB Timergarh 34.82247 71.83608 195-551 RS104 LN901454 1360 PaLCuB Korigram 34.76807 72.02393 193-549 RS105 LN901455 1358 PaLCuB Korigram 34.76807 72.02393 193-549 RS106 LN901456 1360 PaLCuB Korigram 34.76807 72.02393 193-549 RS107 LN901457 1360 PaLCuB Korigram 34.76807 72.02393 193-549 RS108 LN901458 1322 PaLCuB Korigram 34.76807 72.02393 193-549 RS163-25 LN824093 1366 PaLCuB Badwaan 34.65394 71.7651 195-551 RS201-13 LN878100 1366 PaLCuB Onch 34.72688 72.02775 220-556 RS203 LN901459 1366 PaLCuB Onch 34.72688 72.02775 200-556 RS212 LN901460 1366 PaLCuB Badwaan 34.65394 71.7651 200-556 RS214 LN901461 1370 PaLCuB Korigram 34.76807 72.02393 201-557

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Table 4.9: Blast results and maximum homology values, shared by present isolates with database sequences.

Clone Accession Homology BLAST results name Numbers Percentage RS10 LN878102 CLCuMuB-[PK:Dho:Gos:11]-KR816003 99 % RS19 LN878105 CLCuMuB-[PK:cot:10]-HF564598 98 % RS25 LN878108 CLCuMuB-[PK:Dho:Gos:11]-KR816003 98 % RS31 LN878109 CLCuMuB-[PK:cot:10]-HF564598 98 % RS68 LN878112 PaLCB-[IN: Chi:05]-DQ118862 95 % RS77 LN878110 PaLCB-[IN: Chi:05]-DQ118862 96 % RS78 LN878111 PaLCB-[IN: Chi:05]-DQ118862 97 % RS88-7 LN878099 PaLCB-[IN: Chi:05]-DQ118862 96 % RS88-8 LN887901 PaLCB-[IN: Chi:05]-DQ118862 96 % RS104 LN901454 PaLCB-[IN: Chi:05]-DQ118862 96 % RS105 LN901455 PaLCB-[IN: Chi:05]-DQ118862 96 % RS106 LN901456 PaLCB-[IN: Chi:05]-DQ118862 96 % RS107 LN901457 PaLCB-[IN: Chi:05]-DQ118862 97 % RS108 LN901458 PaLCB-[IN: Chi:05]-DQ118862 95 % RS163-25 LN824093 PaLCB-[IN: Chi:05]-DQ118862 95 % RS201-13 LN878100 PaLCB-[IN: Chi:05]-DQ118862 96 % RS203 LN901459 PaLCB-[IN: Chi:05]-DQ118862 96 % RS212 LN901460 PaLCB-[IN: Chi:05]-DQ118862 96 % RS214 LN901461 PaLCuB-[IN:IARI:Pum:10]-JX040472 96 %

141

4.11.3 Papaya leaf curl betasatellites (PaLCuB)

A total of 15 papaya leaf curl betasatellites complete nucleotide sequences were compared with all of its isolates to determine percentage identities among them that vary from 93.3 % to 100 %. While βC1 proteins sequences showed 91.1 % to 95.9 % percentage identities, among present isolates. In multiple sequence alignments performed by using (MegAlign program of Lasergene) for present and database sequences, minor variations of amino acids were observed, and maximum homologies shared by fifteen PaLCuB isolates with NCBI database isolates were 95 to 97 % while minimum homologies 81 % to 82 %.

4.11.4 Cotton leaf curl Multan betasatellites (CLCuMuB)

Four isolates of CLCuMuB were compared for percentage identity that varies from 97.4 % to 100 %, while βC1 protein sequences showed 92.7 to 95.9 % identities among present isolates. CLCuMuB isolates (RS10 and RS25) on comparing with all isolates from database, showed maximum 99 % and minimum 92 % similarity. However RS19 and RS31 exhibited maximum 100 % and minimum 92 % homology with all isolates of NCBI database.

4.12 Analysis of conserve regions of betasatellites

Three conserve regions i.e. βC1 protein, A-Rich region and Satellite Conserve Region (SCR) were analyzed in betasatellites as follows.

4.12.1 βC1 protein

The only ORF i.e. βC1 present in betasatellites, was compared to observe amino acids sequences of βC1in all isolates. βC1 regions of CLCuMuB, PaLCuB and respective NCBI isolates indicated conserved nature of βC1 protein among all isolates, were represented as blue color, miss matches amino acids were shown as white color. Although some conserved amino acid sequences were similar in all isolates of CLCuMuB and PaLCuB represented by blue color. Species specific amino acid regions were also obvious, as in present and NCBI isolates of CLCuMuB patches of amino acids consist of one to 08 and 15 amino acids units scattered across the βC1, exhibited as white color, were particular to this species and do not match with other species. βC1of PaLCuB isolates along with NCBI isolates having conserved regions that were distinct from CLCuMuB species and blue in color, mismatched amino acids (particular to this species) shown white in this species (Text Figure 4.26). 142

4.12.2 A-Rich region

A-Rich regions of CLCuMuB, PaLCuB and respective NCBI isolates was composed of ~200 nucleotide sequences starting approximately from ~750 to 1000 base pairs. This region contained ''A'' content higher than whole genome, A-rich region of PaLCuB isolates represented 57.07 % to 57.36 % ''A'' content in A-rich region and total ''A'' content in whole genome of PaLCuB isolates varies from 35.72 % to 36.53 %. While isolates of CLCuMuB represented A-residue vary from 56.88 % to 57.08 % in A-rich region and in whole genomes of CLCuMuB isolates it vary from 28.89 % to 28.94 %. Percentage of ''A'' content in A-rich region of CLCuMuB found similar to PaLCuB, however overall distribution pattern was various in both the species and specific for each species (Text Figure 4.27). This region indicated the presence of A- rich fragments across A-rich region of present isolates, consists of various number of nucleotides.

4.12.3 Satellite conserve region (SCR) of betasatellites

Another important characteristic of betasatellites genome was the conserve nature of its particular part which remained highly conserve among species. Isolates of PaLCuB and CLCuMuB along with database sequences were compared for SCR this region consisted of ~200 nucleotides starting from ~1125-45 base pairs. Nonanucleotide sequence containing stem loop structure was also located in this region which was indicated by an arrow. SCR analysis indicated that the isolates of PaLCuB and CLCuMuB represented similar organization in this region (Text Figure 4.28).

4.13 Phylogenetic dandogram

Phylogenetic tree was constructed by multiple alignments by Clustral W method of Mega 6 software by using full nucleotide sequences of the present isolates of PaLCuB, CLCuMuB and other isolates of Tomato leaf curl betasatellites (ToLCuB), Tobacco leaf curl betasatellite (TbLCB) and Chilli leaf curl betasatellites (ChLCuB) along with sequences retrieved from database. This phylogenetic tree was constructed by using ToLCPKA-[PK:Gh:11]-HE966423 as an outgroup. Phylogenetic tree consists of 03 main clades, first clade subdivided into 03 subclades represented isolates of ToLCuB, ChLCuB and TbLCB respectively. Isolates under current study were clustered into 02 well segregated clades represented 02 distinct species of the betasatellites. Second well segregated clade represented CLCuMuB isolates of present 143 study and those obtained from the database. Third clade consists of PaLCuB isolates, all isolates in a single clade showed high level of similarity, present sequences were highlighted as bold (Text Figure 4.29).

4.13.1 Phylogenetic analysis of full length nucleotides sequence and βC1 protein

Phylogenetic tree based on full length sequences was compared with the phylogenetic tree of βC1 protein of PaLCuB and CLCuMuB isolates to study βC1 variation or consistency, among isolates of these species with respect to whole genome and to observe grouping among isolates based on βC1 protein. Phylogenetic tree based on βC1 clearly represented that all isolates were grouped into two clades, one clade contained isolates of CLCuMuB, PaLCuB, ToLCuB and TbLCB while second clade contained isolates of ChLCuB seems more distinct than other. Inspite of this, isolates of PaLCuB of present study and from database were grouped together to form clade similar to complete nucleotide sequences based tree, similarly isolates of CLCuMuB showed similar clade pattern to full length tree, but it was quite distinct from isolates of other species, similar to full length dendogram representing this species may be evolutionary more distinct. However isolates of ChLCuB, ToLCuB and TbLCB showed almost same clade pattern as was in dandogram of whole genome (Text Figure 4.30).

4.14 Recombination Recombination analysis performed by RDP4.2 indicated among all isolates only RS107 and RS163-25 isolates showed interspecific recombination.

RS 107 a PaLCuB showed interspecific recombination with CLCuMuB-[PK:Cot:1]- KR816005 where major parents for this recombination event was RS108 PaLCuB- [PK:Tom:13]-LN901458 and CLCuMuB-[PK:Cot:1]-KR816005 as minor parent, this event was mapped in SCR shown (Text Figure 4.31).

Similarly RS163-25 PaLCuB-[PK:Tom:13]-LN824093 showed single intraspecific recombination event with RS203 PaLCuB-[PK:Tom:13]-LN901459, where major parent was ToLCuB-[IN:Pum:08]-HM101175, while RS203 was minor parent and this event mapped in BC1 shown (Text Figure 4.32). Both betasatellites recombinants P-values for RDP, GENECONV, BootScan, MaxChi, Chimaera, SiScan, PhylPro, LARD, 3Seq algorithms of RDP, major and minor parents of each recombination event were given (Table 4.10). 144

Text Figure: 4.26 Multiple alignment of βC1 of CLCuMuB and PaLCuB isolates. This alignment was based on the βC1 proteins of present and NCBI isolates, CLCuMuB were shown on top, the amino acids sequences represented as white color were specific to this species, while blue colored amino acids present among white patches were conserved among both species. PaLCuB isolates were shown at the bottom of figure and demarcation among both the species was exhibited at left site of figure. βC1 of the PaLCuB showed conserved region indicated by blue colour, below the white patches of CLCuMuB enclosed by the aminoacids sequences those were conserved in both species.

145

Text Figure 4.27: A-rich region in PaLCuB and CLCuMuB. Multiple alignment of present and NCBI isolates of PaLCuB (shown at top) and CLCuMuB (at bottom). A-rich region in both of species were represented as green colored patches vary in nucleotides numbers. Figure clearly exhibited A-rich regions particular to each species and were conserved among isolates of particular species.

Text Figure 4.28: SCR region of PaLCuB and CLCuMuB. SCR of CLCuMuB shown at top and of PaLCuB at bottom, SCR was consistent in all isolates. Nonanucleotide sequences present in SCR were shown by arrow.

146

Text Figure 4.29: Phylogenetic tree of betasatellites. Phylogenetic tree was developed using multiple alignment betasatellite sequences under study along with sequences retrieved from NCBI by Neighbor Joining methods. Numbers at the nodes indicated percentage bootstraps confidence values (1000 replicates). Horizontal lines were proportional to the distances among species and vertical lines were arbitrary, dendrogram was rooted on a distantly related ToLCPKA-[PK:Gn:11]-HE966423 (green color). Isolate descriptor and betasatellites acronyms were as proposed by (Briddon et al., 2008). Isolates constructing first clade represented ToLCuB, ChLCuB and TbLCB. Present isolates were grouped into 02 distinct clades CLCuMuB and PaLCuB and represented as bold.

147

Text Figure 4.30: Comparison of βC1 and whole genome phylogeny of all isolates. Phylogenetic tree constructed by Neighbor Joining method, with bootstrap (1000 replicates) value. Horizontal lines were proportional to the distances among species and vertical lines were arbitrary, full length nucleotide based dendogram ''A'' was rooted on a distantly related ToLCPKA-[PK:Gn:11]-HE966423. βC1 based dendogram ''B'' was routed on βC1 sequence of ToLCPKA-[PK:Gn:11]-HE966423. Isolates of PaLCuB and their βC1 were shown in pink, CLCuMuB-sky blue, ToLCuB-purple, TbLCuB-orange and ChLCuB in yellow color.

148

Text Figure 4.31: RDP analysis of RS 107 PaLCuB isolate. This isolate recombined with CLCuMuB-[PK:Cot:1]-KR816005. This recombination event took place in SCR regions and P-value of this event were shown at the site where exchange took place in both genomes. This figure exhibited recombination event in GENECONV algorithm of RDP, P-value have been shown on Y-axis.

Text Figure 4.32: RDP analysis of RS163-25PaLCuB isolate. RS163-25 recombined with PaLCuB- [PK:Tom:13]-LN901459, this recombination event took place in βC1 regions, recombination site and P-value of this event shown at the site where exchange took place. This figure exhibited recombination event in GENECONV algorithm of RDP and P-values shown on Y-axis.

149

Table 4.10: Recombination analysis done for complete nucleotide sequence of betasatellites by RDP4.2 program.

Recombinants Events No./ Break points Interspecific/Intraspecific Recombination parents # P-values @

Major Parent MinorParent RDP GENECONV Botscan MaxChi Chimaera SiScan PhylPro LARD 3Seq

$

Interspecific PaLCuB-[PK:Tom:13]- CLCuMuB-[PK:Cot:1]- RS107 1247-1359 LN901458 KR816005 4.964x10-21 6.855x10-22 4.371x10-21 1.043x10-05 1.025x10-05 NS NS NS NS

Interaspecific ToLCuB-[IN:Pum:08]- PaLCuB-[PK:Tom:13]- RS163-25 322-453 HM101175 LN901459 1.198x10-02 5.929x10-03 9.724x10-03 NS 2.315x10-02 NS NS NS 7.639x10-03

$ Recombination events and their break points, NS for not significant # recombination major or minor parents and similarity % values @ P-values for RDP, GENECONV, BootScan, MaxChi, Chimaera, SiScan, PhylPro, LARD, 3Seq algorithms of RDP for all isolates. 150

4.15 Characterization of Alphasatellites

Characterization of alphasatellites was done as follows.

4.15.1 Amplification and cloning of alphasatellites

Genomic DNA of all collected samples were used as template for alphasatelliteʼs amplification, only 07 samples among those showed amplification i.e. 1.4 kb fragments for alphasatellites (Figure 4.8A). Amplified products after ligation into vector (pTZ57R/T) were transformed to the Top-10 strain of E. coli. and were cloned. Positive clones were confirmed by EcoR1and Pst1 restriction (Figure 4.8B) and sent for sequencing.

4.15.2 Molecular characterization of alphasatellites

Initial comparison for species identification by BLAST exhibited similarities of 02 present isolates with Okra leaf curl alphasatellite (OLCuA) that varied from 86 % to 98 % and was greater than threshold level for alphasatellites species demarcation. While 03 isolates showed similarity with Tomato leaf curl Pakistan alphasatellite (ToLCPKA) that varied from 91 % to 93 %. So all present isolates represented presence of 02 alphasatellite species, as according to the rules formulated for satellites classification, cut off limit for alphasatellites species demarcation was 83 %. Complete genome of all alphasatelliteʼs isolates consisted of 1362 to 1370 nucleotides, while presence of ORF was compared by means of ORF finder of NCBI (http://www.ncbi.nlm.nih.gov/gorf/gorf.html), for all alphasatellites sequences. Alphasatellites contained typical satellite specific single ORF which encoded Rep protein, coordinates for Rep in all isolates of OLCuA and ToLCPKA, accession numbers and origin were mentioned (Table 4.11). All isolates contained Rep of about 36.6 kDa, an origin of replication, an A-rich region of about 200 nucleotides and nonanucleotide sequence containing inverted repeats forming stem loop structure. A highly conserved sequence i.e. nonanucleotide TAGTATTAC similar to nanoviruses and slightly different from other begomoviruses were also found in intergenic regions of all alphasatellites. Rep was consists of (76 to 1023) nucleotides in ToLCPKA, while for isolates of OLCuA (88 to 1035) nucleotide and contained 315 amino acids.

Maximum similarity showing accession numbers with herein isolates and maximum similarity values were given (Table 4.12).

151

Figure 4.8: Alphasatellites amplification and restriction. "A" represented PCR amplification of ~1.4 kb sized alphasatellites, amplification products were loaded in agarose gel with 1 kb DNA marker for fragment size specification and denoted by "M". Wells marked as 1 and 2 contained PCR product of infected samples while 03 and 04 represented positive and negative controls. Negative control was obtained by genomic DNA of healthy plant and already confirmed infected DNA sample was used as positive control. Samples loaded in well 01 and 02 were found positive for infection, as amplified 1.4 kb fragment. "B" was indicating restriction pattern of alphasatellites clones, restriction products were run in 01, 03, 04, 06 and 07 wells gave 1.4 kb fragments.

Table 4.11: Coordinates of Rep protein, genome size, samples origin and GeneBank accession numbers of alphasatellites.

Clone Accession Identity in Genome Location Longitude Latitude Rep name numbers length database RS5 LN878101 1363 ToLCPKA NIAB 31.396249 73.031816 76-1023 RS17 LN878113 1368 ToLCPKA NIAB 31.396249 73.031816 76-1023 RS52 LN878116 1370 ToLCPKA NIAB 31.396249 73.031816 76-1023 RS69-3 LN831729 1356 OLCuA Sherpalam 34.89428 72.41253 82-1029 RS205 LN901462 1367 OLCuA Sherpalam 34.89428 72.41253 82-1029

Table 4.12: BLAST results and maximum homology percentages, shared by present isolates with database sequences.

Clone Accession BLAST Results Percentage name Numbers RS5 LN878101 ToLCPKA-[PK:Gos:11]-HE966419 92 % RS17 LN878113 ToLCPKA-[PK:Gos:11]-HE966419 91 % RS52 LN878116 ToLCPKA-[PK:Gos:11]-HE966419 91 % RS69-3 LN831729 OLCuA-[PK:Pan:Okr:12]-HG518790 98 % RS205 LN901462 OLCuA-[PK:Pan:Okr:12]-HG518790 95 % 152

4.15.3 Okra leaf curl alphasatellite (OLCuA)

Complete nucleotide sequences of two isolates of OLCuA were compared with all NCBI isolates of OLCuA to determine homologies among them that varied from 85.6 % to 99.1 %, similarly multiple alignments of Rep proteins of all isolates performed by Clustral W method to determine percentage identity that vary from 91.7 % to 100 % among present isolates. Results clearly indicated conserve nature of Rep among all isolates. However both present isolates showed 96.4 % identity with each other on comparing full length sequences, while Rep protein exhibited 98.4 % similarity.

4.15.4 Tomato leaf curl Pakistan alphasatellite (ToLCPKA)

Three isolates of ToLCPKA were compared for homologies with database squences available at NCBI, all isolates were used to perform multiple alignments by Clustral W method, their percentage identities vary from 90 % to 99.7 % among isolates, however present three isolates showed 99.6 % to 99.7 % identities with each other. Rep sequences of present and all NCBI isolates were also aligned by Clustral W method in order to confirm variations among Rep in comparision to full length sequence, percentage identities vary from 97.2 % to 99.7 % among proteins of all isolates, while in present isolates Rep percentage identities vary from 99.4 % to 99.7 %.

4.16 Conserve regions of alphasatellites

A-Rich region and Rep protein, the conserve regions of all alphasatellites were analyzed as follows.

4.16.1 Rep of all isolates

In order to indicate slight differences observed in percent identities of Rep proteins of all isolates, multiple alignments were performed to indicate species specific differences of aminoacids seuences among isolates of present species (Text Figure 4.33). It was apparent from Rep analysis that this region was extremely conserve among all isolates of a particular species.

4.16.2 A-rich region

Another characteristics of alphasatellites was the presence of ~200 nucleotides long A-rich region, for the purpose multiple alignments were performed for A-rich region by selecting sequence regions from 950 to 1225 of all present and NCBI isolates 153 together (Results not shown). However alignment of present isolates merely indicated A-rich region consisted of ~1000 to 1220 base pair. This region contained ''A'' content higher than whole genome, A-rich region of ToLCPKA isolates represented 39.86 % to 40.58 % ''A'' content, but total ''A'' content in whole genome was 30.25 % to 30.5 %. Similarly OLCuA isolates represented 43.12 % to 46.38 % ''A'' content in A-rich region and total ''A'' content in whole genome was 30.31 % to 31.24 %. Results indicated that ''A''content in A-rich region of OLCuA was higher than the ToLCPKA. A-rich fragments were different in numbers i.e. 2 to 13 base pairs and were scattered in A-rich regions of both species (Text Figure 4.34). However overall distribution pattern of A-rich fragments was various, distinct and species specific.

4.17 Phylogenetic analysis

Phylogenetic tree was constructed, using multiple alignments of full nucleotide sequences of present isolates of ToLCPKA, OLCuA and other isolates of different species, along with sequences retrieved from NCBI database. This tree was constructed by using CLCuMuB-[PK:Bur:Cot:11]-HF567946 as an outgroup. Phylogenetic tree exhibited the formation of three main clades, first clade consisted of ToLCPKA herein and NCBI isolates and Cotton leaf curl-1alphasatellite (CLCu-1). This clade was more close to Cotton leaf curl Multan alphasatellite (CLCuMuA), than other species of alphasatellites, second clade showed different subclades originated from this clade, all subclades represented different species as CLCuMuA, Bendhi yellow vein mosaic alphasatellite (BYVMA), Hibiscus leaf curl alphasatellite (HLCuA), Hollyhook yellow vein symptoms less alphasatellite (HoYVSLA), Vernonia yellow vein Fujian alphasatellite (VYVFA), Ageratum yellow vein alphasatellite (AYVA), Tomato yellow leaf curl China alphasatellites (TYLCCNA), Duranta leaf curl alphasatellite (DuLCA) and Guar leaf curl alphasatellite (GuLCuA) respectively. Third clade represented present OLCuA along with NCBI isolates were grouped separately from other species and form distinct clade, present sequences in dendogram were represented as bold (Text Figure 4.35).

154

Text Figure 4.33: multiple alignments of Rep of OLCuA and ToLCPKA. This alignment was based on Rep protein sequence of present isolates. Rep proteins of isolates of ToLCPKA (RS5, RS17 and RS52) were shown on top followed by OLCuA Rep. Blue colored amino acids presented here conserved amino acids among both species. Figure clearly depicted amino acids differences, particular for each species. Amino acid sequences, those were specific for OLCuA species were marked with black blocks and exactly above on these marked region were amino acids, those were specific to ToLCPKA.

Text Figure 4.34: A-rich region in ToLCPKA and OLCuA. Multiple alignments of ToLCPKA (shown at top, marked "A") and OLCuA (at bottom, marked ''B"). A rich region in both of species was represented as green colored patches vary in nucleotides numbers. Figure clearly exhibited A-rich regions were particular to each species and were conserved among species.

155

Text Figure 4.35: Phylogenetic analyses of Alphasatellites. Phylogenetic dendrogram was constructed by Neighbor Joining methods. Numbers at the nodes indicated percentage bootstraps confidence values (1000 replicates). Horizontal lines were proportional to the distances among species and vertical lines were arbitrary, dendrogram was rooted on a distantly related CLCuMuB- [PK:Bur:Cot:11]-HF567946. Present isolates grouped into two distinct clades representing ToLCPKA and OLCuA, sequences under study were bold.

156

4.17.1 Phylogenetic analysis based on full length nucleotide sequences and Rep proteins

Phylogenetic analysis based on full length sequences was compared with phylogenetic tree of Rep protein of OLCuA and ToLCPKA isolates to study phylogenetic relationship or grouping of isolates into clades and Rep variation or consistency among isolates of different species with respect to whole genome and to observe grouping among isolates based on Rep protein. Phylogenetic dandogram based on Rep clearly represented that all isolates were grouped into 02 clades, first clade contained ToLCPKA, CLCuA-1, CLCuMuA, BYVMA, HLCuA, AYVA, TYLCCNA, DuLCA, GuLCuA and OLCuA while second had isolates of HoYVSLA and VYVFA (Text Figure 4.36).

4.18 Recombination among alphasatellites

All present isolates were allowed to run RDP analysis via RDP4.2 program, among these 02 isolates RS5 ToLCPKA showed interspecific recombination with isolate of Cotton leaf curl Burewala alphasatellite (CLCuBuA) and RS69-3 OLCuA have intraspecific recombination.

RS5 ToLCPKA-[PK:Tom:15]-LN878101 contained 03 recombination events with CLCuBuA -[PK:Gh:11]-HE965689, having OLCuA-[PK:Ae:12]-HG518789 as major parent and CLCuBuA -[PK:Gh:11]-HE965689 as minor parent in all 03 events. All 03 events took place in Rep region shown (Text Figure 4.37), percentage similarity of recombination parents, break points and P-values (Table 4.13).

Similarly RS69-3 contained intraspecific recombination, one recombination event with OLCuA-[PK:Gh:11]-HE966420, in this event major parent was RS205- [PK:Tom:13]-LN901462 and minor parent was OLCuA-[PK:Gh:11]-HE966420 and this recombination was mapped in IR shown in (Text Figure 4.38; Table 4.13).

Recombination events number in each recombinant, events cut off values, recombination parents and P-values for RDP, GENECONV, BootScan, MaxChi, Chimaera, SiScan, PhylPro, LARD and 3Seq algorithms of RDP (Table 4.13).

157

Text Figure 4.36: Comparison of Rep proteins and full length sequences of Alphasatellites. Phylogenetic trees were constructed by Neighbor Joining method with bootstrap (1000 replicates) value. Horizontal lines were proportional to the distances among species and vertical lines were arbitrary, full length nucleotide based dendogram ''A'' was rooted on a distantly related CLCuMuB- [PK:Bur:Cot:11]-HF567946. Rep based dendogram ''B'' was rooted on Rep sequence of CLCuMuB- [PK:Bur:Cot:11]-HF567946.

158

Text Figure 4.37: RDP analysis of RS 5-ToLCPKA isolate. This isolate recombined with CLCuBuA -[PK:Gh:11]-HE965689. Recombination events took place in Rep region and recombination site has been highlighted in GENECONV algorithm of RDP, P-value were shown on Y-axis but for this event P-values were shown at the site where exchange took place in both isolates.

Text Figure 4.38: RDP analysis of RS69-3 (OLCuA) isolate. This isolate recombined with OLCuA- [PK:Gh:11]-HE966420. Recombination events took place in IR region and recombination site has been highlighted in GENECONV algorithm of RDP, P-value were shown on Y-axis but P-values of this event was shown at the site where exchange took place in both isolates.

159

Table 4.13 Recombination analysis done for complete nucleotide sequences of alphasatellites by RDP4.2 program.

Recombinants points$ Break / No. Events ecific Interspecific/Intrasp Recombination parents # P-values @

RDP GENECONV Botscan MaxChi Chimaera SiScan PhylPro LARD 3Seq

Major Minor

(3) i) 109-222 Interspecfic OLCuA-[PK:Ae:12]-HG518789 CLCuBuA -[PK:Gh:11]-HE965689 5.842x10-4 9.929x10-05 NS 1.189x10-05 5.265x10-04 NS NS NS 2.600x10-02

ii) 174- 212 Interspecfic OLCuA-[PK:Ae:12]-HG518789 CLCuBuA -[PK:Gh:11]-HE965689 NS 4.103x10-02 NS 4.688x10-03 NS NS NS NS NS RS5 iii) 912- -16 -09 -28 1134 Interspecfic OLCuA-[PK:Ae:12]-HG518789 CLCuBuA -[PK:Gh:11]-HE965689 1.615x10-16 3.286x10-18 9.184x10-15 5.022x10 5.310x10 NS NS NS 3.711x10

(1) 1116- RS69 44 Intraspecfic OLCuA-[PK:Tom:13]-LN901462 OLCuA-[PK:Gh:11]-HE966420 3.272x10-07 5.052x10-07 1.117x10-06 3.578x10-10 6.149x10-10 NS NS NS 4.731x10-10

$ recombination events number and breakpoints of recombination event, NS for not significant # recombination major or minor parents @ P-values for RDP, GENECONV, BootScan, MaxChi, Chimaera, SiScan, PhylPro, LARD, 3Seq algorithms of RDP of all recombination events

160

SECTION II

Efficiency and reliability of Ty genes carrying resistant cultivars was evaluated against bipartite begomoviruses ToLCNDV. For this purpose, Ty genes harbouring cultivars (Table 4.14) and native susceptible tomato variety (Nagina) were compared for resistance after ToLCNDV inoculation by microinfiltration.

4.19 Pathogenicity developed in Ty resistant cultivars on ToLCNDV inoculation

At 07dpi of ToLCNDV different cultivars responded in various ways. Cultivars showed variation in infection intensity and number of infected plants. Symptoms observed in plants were mild leaves rolling, growth stunting and less crumpled systemic leaves, along with this the number of symptomatic plants also increased gradually with the passage of time. In order to observe enhancement of symptoms severity and number of symptomatic plants, data was collected three times after an interval of a week. Data clearly represented increase in symptoms severity with the time as shown by bar graph (Text Figure 4.39) and in symptomatic plants number gradually. But symptoms variation was found only for those cultivars which showed symptoms after 07dpi except for RS9, all plants of R5, R6, R10, R11 and R13 cultivars remained symptomless during whole experiment, as shown (Table 4.15). However phenotypically symptoms severity scale was described as, non symptomatic plants (0), mild symptomatic (01), moderate (02), severe symptomatic (03) and very severe symptomatic (04) plants.

At 21 dpi symptoms turned severe described as severe leaf rolling, crumpling of leaves, severe curling of newly emerged leaves, marginal yellowing and growth stunting. Among all, 06 (R7, R8, R9, R12, R15 & Nagina) cultivars showed 100 % symptomatic plants, 03 cultivars (R2, R3 & R14) showed 80 % symptomatic plants and 100 % plants of 05 (R5, R6, R10, R11 and R13) cultivars were non symptomatic. Data of increase in number of infected plants against 07dpi, 14dpi and 21dpi was represented with bar graph for R2, R3, R5, R6, R7, R8, R9, R10, R11, R12, R14, R15 and native cultivar (Text Figure 4. 40). Non inoculated healthy plants and pGreen inoculated plants showed that all plants of Ty-resistant and native cultivars remained symptoms less during whole experiment (Figure 4.9 Panel A and B).

161

Table 4.14: Ty resistant cultivars with distribution codes and various Ty genes combination.

Ty genes combination Cultivars Distribution code Ty1/Ty-3 Ty-2 Ty-3 Ty-5 RS2 AVTO1010 S R R RS3 AVTO1143 R R R S RS5 AVTO1260 S R 3a R RS6 AVTO1005 - R R S RS7 AVTO1132 S R R S RS8 AVTO0922 R R S S RS9 AVTO1130 S R 3a S RS10 AVTO1122 S R S R RS11 AVTO1229 S S S R RS12 AVTO1226 - S R S RS13 AVTO1219 - R R S RS14 AVTO0301 S R S S RS15 AVTO1080 S S S S

Native Nagina

Ty-2, Ty-3 and Ty-5 genes conditioning resistance to ToYLCD R Homozygous for resistance S Homozygous for susceptibility Ty1/Ty-3 both are allelic - indicate uncertainty about Ty1 allele 3a indicate presence of the Ty-3a allele at the Ty-3 locus

162

4.5 4 3.5 3 2.5 7 dpi 2 14 dpi 1.5 21 dpi 1 0.5 0

Text Figure 4.39: Symptoms severity shown by Ty cultivars after ToLCNDV inoculation. The bar graph indicated response of Ty resistant cultivars and Nagina variety at 07dpi, 14dpi and 21dpi. X-axis indicated all studied cultivars while Y-axis increase in symptoms severity for each cultivar according to 07dpi, 14dpi and 21dpi.

100

90 80 70 60 7 dpi 50 14 dpi 40 21 dpi 30

Percentageinfectedof plants 20 10 0

Text Figure 4.40: Gradual increase in symptomatic plants number after ToLCNDV inoculation. The bar graph indicated response of Ty resistant cultivars and Nagina variety at 07dpi, 14dpi and 21dpi. X-axis indicated all cultivars while Y-axis indicates increase in number of symptomatic plants for each cultivar according to to 07dpi, 14dpi and 21dpi.

163

Figure 4.9: Pathogenicity of ToLCNDV in Ty resistant cultivars and Nagina variety. Tomato plants shown in figure were either non-inoculated (A, healthy control) mock inoculated (B) and with ToLCNDV, cultivar R2(C), R3(D), R5(E), R6(F), R7(G), R8(H), R9(I), R10(J), R11(K), R12(L), R13(M), R14(N), R15(O) and Nagina (P), photographs were taken at ~21dpi. 164

4.20 Detection of ToLCNDV in inoculated cultivars

Samples of ToLCNDV inoculated tomato plants were harvested to confirm the presence of ToLCNDV and to detect its titer as follows.

4.20.1 Confirmation of ToLCNDV in systemic leaves of inoculated plants

A total of genomic DNA was extracted by CTAB method (Chapter 3, section 3.2) from systemic leaves of each cultivar. Genomic DNA of each sample was allowed to perform PCR by using specific primer sets (ND-A qPCR F/ND-A qPCR R) sequence given (Table 3.1), for the confirmation of presence of genome of ToLCNDV DNA-A. Primers sequences used for amplification of DNA-B were (BMP qPCRF/ BMP qPCR R) sequence shown (Table 3.1).

For DNA-A, PCR gave amplification of 200 base pairs fragment and all cultivars which were symptomatic or non symptomatic, found positive except R6 cultivar which was symptomless upon inoculation (Figure 4.10). PCR for DNA samples of R6 was repeated, but it showed similar results.

Similarly presence of ToLCNDV-B component was also confirmed by PCR mediated amplification which amplified fragments of 200 base pairs. All cultivars along with native (Nagina) variety were found positive, except for R6. PCR products for all samples were run according to variation in symptoms severity i.e. non symptomatic, mild, moderate, severe, and very severe symptomatic samples (Figure 4.11). Plants inoculated by pGreen vector and healthy control were also allowed to perform PCR with same primer used for DNA-A and B showed no amplification (Results not shown).

165

Figure 4.10: Detection of ToLCNDV DNA-A in systemic leaves by PCR in inoculated tomato plants. PCR products were loaded on ethidium bromide-stained agarose gel and photographed under Jel doc. Samples were run according to ascending order of symptoms severity (non-symptomatic, mild, moderate, severe and very severe symptomatic). Wells 1-5 represent non symptomatic cultivars as R6, R5, R10, R11 and R13, wells 6-8 represented mild symptomatic cultivars i.e. R2, R3 and R9, and DNA marker (1 kb) was electrophoresed in well marked as M. Well marked 9 indicated R12 exhibiting moderate symptoms, while well marked 10 and 11 contained R7 and R8 cultivars, represented severe symptoms showing samples. Well 12-14 represented very symptomatic (R14, R15 and Nagina) variety, while - and + indicate negative, positive control respectively and PCR amplified products yield 200 bp long fragment.

Figure 4.11: Detection of ToLCNDV DNA-B in systemic leaves by PCR in inoculated tomato plants. PCR products were loaded on ethidium bromide-stained agarose gel and photographed under Jel doc. Samples were run according to ascending order of symptoms severity (non-symptomatic, mild, moderate, severe and very severe symptomatic). Well 1-5 represented non symptomatic cultivars as R6, R5, R10, R11 and R13, well 6-8 represents mild R2, R3 and R9, and DNA marker (1kb) was electrophoresed in well marked as "M". Well 9 indicated R12 exhibiting moderate symptoms, while well 10 and 11 represented samples as R7, R8 which showed severe symptoms. Very severe symptoms showing cultivars as R14, R15 and Nagina were run in wells 12-14, - as well as + indicated negative and positive controls respectively and PCR amplified products yield 200bp long fragment.

166

Table.4.15: Percentages of infectivity and types of symptoms caused by ToLCNDV in Ty resistant and Nagina plants.

Percentage of infected plants 7Dpi* 14Dpi@ 21Dpi# Symptoms PCR Persistence/ Cultivars Symptoms at 21dpi $ % % % Severity Results Symptoms index Persistant/ RS2 16.66 % 66.66 % 83.33 % Mild +ve LD, MY, GS,LR Mild Persistant/ RS3 66.66 % 83.33 % 83.33 % Mild +ve LD, LT, MY,GS Mild

RS5 0 % 0 % 0 % No +ve NS ---

RS6 0 % 0 % 0 % No -ve NS --- Persistant/ RS7 33.33 % 83.33 % 100 % Severe +ve LT, LC, MY, GS, LR Severe Persistant/ RS8 83.3 % 100 % 100 % Severe +ve LT, LC,MY,GS,LR,VT Severe Persistant/ RS9 0 % 50 % 100 % Mild +ve LC, GS, LF, LD, GS Mild

RS10 0 % 0 % 0 % No +ve NS ---

RS11 0 % 0 % 0 % No +ve NS --- Persistant/ RS12 83.33 % 100 % 100 % Moderate +ve LT, LC, GS, LR Moderate RS13 0 % 0 % 0 % No +ve NS --- Very Persistant/ RS14 83.33 % 83.33 % 83.33 % +ve LT, LC, MY,GS, LR, LD Severe Very Severe Very Persistant/ RS15 83.33 % 83.33 % 100 % +ve LT, LC, MY,GS, LR, VT Severe Very Severe Very LY, LC, MY, GS, LR, Persistant/ Nagina 100 % 100 % 100 % +ve Severe VT Very Severe pGreen ∆ 0 % 0 % 0 % Healthy -ve NS ---

Healthy ¤ 0 % 0 % 0 % Healthy -ve NS ---

*Inoculated plants were observed at 7 dpi, @ Plants symptoms observation at 14 dpi, # Symptoms observation at 21 dpi, $ Symptoms observed in inoculated plants were denoted as leaf deformation (LD), leaf curling (LC), mild yellowing (MY), growth stunting (GS), leaf rolling (LR), non symptomatic (NS), leaf yellowing (LY), vein thickening (VT), vein purpling (VP) and leaf thickening (LT), ∆ same plant numbers of each cultivar were mock inoculated, ¤ Same number of plants of each cultivar were kept uninoculated as healthy control

167

4.20.2 Southern Blot Hybridization

Southern blot hybridization was performed to determine ToLCNDV DNA-A (Figure 4.12) and DNA-B (Figure 4.13) titer, southern blot was probed with DIG labeled DNA fragment synthesized by using (ND-A F/ND-A R and ND-B F/ND-B R) primers sets respectively, sequences given (Table 3.1).

Characteristic replicative forms of begomoviruses were detected. These results indicated that among phenotypically non symptomatic Ty cultivars (R6, R10, R11, R5 and R13) R6, R10, R11 showed no virus titer, R10 and R11 however were found PCR positive in contrast to R6 which was non symptomatic, showed no PCR amplification and no virus detection was found in southern hybridization. While R5 and R13 showed very less titer of ToLCNDV DNA-A and DNA-B in southern hybridization. Cultivars R2, R3 and R9 showed mild symptoms also exhibited mild virus ToLCNDV DNA-A and DNA-B titer. Plants of R12 showed moderate symptoms severity exhibited comparable level of virus titer in Southern hybridization. R7 and R8 cultivars showed severe symptoms and similar was found for ToLCNDV DNA-A as well as DNA-B titer. For R14, R15 and Nagina variety phenotypic very severe symptoms also contain comparable level of ToLCNDV titer.

Titer of ToLCNDV-B component in Ty inoculated cultivars was also compared with symptoms via Southern analysis, results shown (Figure 4.13). Our results indicated that susceptible variety Nagina showed no resistance and highest susceptibility in this variety was characterized by severe leaf curling, growth stunting and leaf yellowing. However, R15 which contained no Ty homozygous resistance gene as compared to all other Ty resistant cultivars (which were homozygous resistant for one or more Ty genes), also showed high susceptibility characterized by very severe symptoms. Similar was found for R14, R7 and R8 which showed severe symptoms, R12 showed moderate symptoms and mild resistance. Other cultivars as R5, R6, R13, R10 and R11 were found non symptomatic and best resistant among all cultivars.

168

Figure 4.12: Southern hybridization of DNA-A component of ToLCNDV in Ty inoculated plants. Genomic DNA from systemic leaves of each cultivar was loaded on gel and was probed with DIG- labeled DNA fragment. Southern hybridization exhibitedToLCNDV replicative forms in Nagina, R15, R14, R8, R7, R12, R9, R2 and R3 symptoms showing cultivars and R5 and R13 non symptomatic plants. Healthy uninoculated (H) and ToLCNDV inoculated N. benthamiana samples (P) were used as negative and positive control respectively. Position of DNA replicative forms were designated as open circular (OC), linear (Lin), supercoiled (SC) and single stranded (SS). Genomic DNA of particular cultivars loaded in respective wells indicated at bottom of figure.

169

Figure 4.13: Southern blot detection of DNA-B component of ToLCNDV in inoculated Ty cultivars. Genomic DNAs from systemic leaves of each cultivar were loaded on gel and probed with DIG-labeled DNA fragment. Southern hybridization exhibited ToLCNDV replicative forms in Nagina, R15, R14, R8, R7, R12, R9, R2 and R3 symptoms showing cultivars and R5 and R13 non symptomatic plants in contrast to R6, R11and R10 i.e. non symptomatic plants. Healthy (H) uninoculated and ToLCNDV inoculated (P) N. benthamiana samples were used as negative and positive controls respectively. Position of DNA replicative forms were designated as open circular (OC), linear (Lin), supercoiled (SC) and single stranded (SS). Genomic DNA of particular cultivars loaded in respective wells indicated at bottom of figure.

170

4.22 Metabolites (volatile compounds) profiling of ToLCNDV inoculated resistant and susceptible Ty tomato cultivars by GCMS

Metabolite profiling of healthy and ToLCNDV inoculated Ty cultivars & Nagina variety are as follows.

4.22.1 Healthy S. lycopersicum plant

Extract of healthy plant led to the identification of 35 compounds in totality among those major compounds were Dodecane (2.74 %), Dodecane, 2,6,11-trimethyl (4.19 %), Tetradecane (9.35 %), Eicosane (7.42 %), Phenol, 3,5-bis(1,1-dimethylethyl) (5.32 %), Hexadecane, 2,6,11,15-tetramethyl (2.58 %), Hexadecane (7.74 %), Tritetracontane (2.58 %), Tetratriacontane (1.61 %), Octacosane (2.26 %), Heptadecane, 2,6,10,15-tetramethyl (4.19 %), Tetratetracontane (2.58 %), Tetrapentacontane (8.70 %), Pentatriacontane (7.09 %), Hexatriacontane (6.45 %), Tetrapentacontane, 1,54-dibromo (3.22 %) and 1, 2-Benzenedicarboxylic acid, diisooctyl ester (12.25 %), while other compounds were Benzeneethanol, .alpha.,.beta.-dimethyl and decane, 1,1′ oxybis (Table 4.16).

4.23 Tolerant or resistant cultivars

R6, R10, R11, R5 and R13 were found resistant against ToLCNDV among all Ty cultivars as follows.

4.23.1 Cultivar R6 of S. lycopersicum

GCMS analysis of R6 indicated identification of 50 volatile compounds from extract, among those abundant were, limonene (1.30 %), Dodecane (1.54 %), 2-Bromo dodecane (3.14 %), Dodecane, 2,6,11-trimethyl (1.22 %), Heptadecane, 2,6,10,15- tetramethyl (1.56 %), Tetradecane (1.79 %), Hexadecane (1.78 %), 10- Methylnonadecane (1.87 %), Eicosane (6.24 %), Tetratetracontane (5.61 %), Phenol, 3,5-bis(1,1-dimethylethyl)- (7.69 %), Hexadecane, 2,6,11,15-tetramethyl (3.01 %), Tritetracontane (1.79 %), Pentadecane, 8-hexyl (6.34), Pentatriacontane (5.45 %), Tetrapentacontane (6.83 %), Octadecane, 1-chloro (5.76 %), 1-(+)-Ascorbic acid 2,6- dihexadecanoate (4.88 %), Tetrapentacontane, 1,54-dibromo (6.83 %), 1- Hentetracontanol (4.47 %), Hexatriacontane (5.59 %) and Tetracontane (4.88 %). Other compound which was found only in this sample was 1-Hentetracontanol (Table 4.17). 171

4.23.2 Cultivar R10 of S. lycopersicum

A total of 45 compounds were identified in leaf and stem extract of R10 tomato cultivar include, Eicosane (9.07 %), Phenol, 3,5-bis (1,1-dimethylethyl)- (8.33 %), Hexadecane (7.40 %), Heneicosane (3.99 %), Pentadecane, 8-hexyl-(4.81 %), Oxirane, Tetradecyl (5.83 %), Tetratetracontane (3.70 %), Silane, trichlorooctadecyl (6.85 %), Tetrapentacontane (8.82 %), Phytol (7.31 %) and 1, 2-Benzenedicarboxylic acid, diisooctyl ester (3.89 %). Other compounds found only in this sample were 2,9- dimthylundecane, Decane, 1-iodo-, Tridecane, 4-methyl, 2,3,6,7-tetramethyl-octane, Heptane, 2,5,5-trimethyl-, Anthracene, 9-dodecyltetradecahydro-, Oxirane, tetradecyl- and Silane, trichlorooctadecyl (Table 4.18).

4.23.3 Cultivar R11 of S. lycopersicum

From this sample 32 volatile compounds identified and abundant compounds were Dodecane (2.86 %), Nonadecane (1.51 %), Tetradecane, (9.04 %), Eicosane (7.53 %), Dodecane, 2,6,11-trimethyl (3.73 %), Phenol, 3,5-bis (1,1-dimethylethyl) (5.78 %), Hexadecane, 2,6,11,15-tetramethyl (2.39 %), Tritetracontane (1.89 %), Octadecane (5.27 %), Octacosane (1.51 %), Hexadecane (8.91 %), Tetratetracontane (6.02 %), Pentatriacontane (9.04 %), Tetrapentacontane (9.79 %), Tetrapentacontane, 1,54- dibromo (7.66 %) and 1, 2-Benzenedicarboxylic acid, diisooctyl ester (11.97 %). Other compounds found only in this sample were 2-Decene, 5-methyl-, 5- Ethylundecane and 1-Decanol, 2,2-dimethyl (Table 4.19).

4.23.4 Cultivar R5 of S. lycopersicum

Leaf and stem extract of cultivar R5 after GCMS analysis led to the identification of 35 bioactive compounds and abundant among those were Dodecane (2.24 %), 2- Bromo dodecane (3.36 %), Nonadecane (2.17 %), Tetradecane (7.80 %), Eicosane (7.11 %), Phenol, 3,5-bis(1,1-dimethylethyl) (3.75 %), Hexadecane (8.14 %), Tetratriacontane (1.97 %), Tetratetracontane (2.80 %), 9-Methylnonadecane (7.51 %), Pentadecane, 8-hexyl (3.00 %), Hexadecane, 2,6,11,15-tetramethyl- (2.57 %), Octadecane (4.46 %), Hexatriacontane (6.32 %), 1-(+)-Ascorbic acid 2,6- dihexadecanoate (2.86 %), Octacosane (2.96 %), Isopropyl Palmitate (3.81 %), Tetrapentacontane (6.91 %), Pentatriacontane (4.15 %) and 1,2-benzenedicarboxylic acid, diisooctyl ester (9.39 %; Table 4.20).

172

4.23.5 Cultivar R13 of S. lycopersicum

GCMS analysis of leaf and stem extract of R13 led to the identification of 51 compounds, major compounds were 2,6,10 Trimethyl-dodecane (5.43 %), Hexadecane (8.06 %), Nonadecane (5.78 %), Tridecanol, 2-ethyl-2-methyl (2.84 %), 9-Eicosene (7.76 %), Eicosane (6.31 %), Phenol, 3,5-bis(1,1-dimethylethyl) (4.03 %), Dodecane, 2,6,11-trimethyl- (1.75 %), Heneicosane (1.70 %), Pentadecane, 8-hexyl- (2.19 %), Octadecane (4.38 %), Tetrapentacontane (5.43 %), Pentatriacontane (5.26 %), Tetracontane (6.62 %), 1-(+)-Ascorbic acid 2,6-Dihexadecanoate (1.57 %), Tetrapentacontane, 1,54-dibromo (2.45 %), Tetratriacontane (2.81 %), Nonahexacontanoic acid 2.98 % and 1, 2-Benzenedicarboxylic acid, diisooctyl ester (8.41 %). While, other compounds found only in this sample were Dodecane, 1- chloro, 2,4-Dimethyl undecane, Undecane, 3,9-dimethyl, Benzene, (1,3,3-trimethyl nonyl), trans, cis-2,6-nonadien-1-ol, 1-Octanol, 2-butyl and 9-Eicosene (Table 4.21).

4.24 Mild symptomatic cultivars

Mild symptomatic cultivars i.e. R2, R3 and R9 are as follows.

4.24.1 Cultivar R2 of S. lycopersicum

Plants of R2 cultivar showed very less symptoms, in totality 45 compounds determined from R2 extract, among those abundant compounds were 2,6,10 Trimethyl-dodecane (4.03 %), Tridecane (1.44 %), Pentadecane (9.78 %), Dodecane, 4,6-dimethyl (2.05 %), Nonadecane (4.01 %), Octacosane (1.85 %), Heneicosane (8.12 %), Tritetracontane (1.64 %), Eicosane (8.22 %), Phenol, 3,5-bis(1, 1- dimethylethyl)- (3.78 %), Tetratetracontane (9.04 %), Hentriacontane (2.05 %), Pentadecane, 8-hexyl (6.57 %), Tetrapentacontane (5.34 %), Nonacosane (3.69 %), Isopropyl palmitic acid (2.59 %), Tetracontane (7.49 %) and 1, 2-benzenedicarboxylic acid, diisooctyl ester (9.78 %). Other compounds found only in this sample were Cyclohexane, 1-methyl-2-propyl-, Cyclohexane, butyl, Decane, 3-methyl-, Dodecane, 3-cyclohexyl- and Nonacosane (Table 4.22).

4.24.2 Cultivar R3 of S. lycopersicum

This cultivar exhibited mild symptoms, 36 compounds determined from R3 extract, among those abundant were 2,6,11 Trimethyl-dodecane (3.42 %), Dodecane (1.32 %), Octacosane (4.87 %), Eicosane (7.63 %), Phenol, 3,5-bis(1,1-dimethylethyl)- (9.07 %), Tetratetracontane (2.24 %), Tetradecane (2.10 %), Pentatriacontane (1.97 %), 1- 173

Chloroheptacosane (6.58 %), 1-(+)-Ascorbic acid 2,6-dihexadecanoate (10.79 %), Phytol (8.42 %), Tetrapentacontane, 1,54-dibromo (9.34 %), nonahexacontanoic acid (6.58 %), Hexatriacontane (11.13 %) and 1,2-benzenedicarboxylic acid, diisooctyl ester (7.64 %). Other compounds which were found only in this sample were Heptane, 2,2-dimethy-, Cycloheptane, bromo and 1-Decanol, 2,2, dimethyl (Table 4.23).

4.24.3 Cultivar R9 of S. lycopersicum

GCMS analysis resultant to the identification of 49 compounds from R9 cultivar, major were p-Xylene (1.32 %), Eicosane (6.82 %), Phenol, 3,5-bis(1,1- dimethylethyl)- (10.45 %), Hexadecane (4.18 %), Heneicosane (7.26 %), Pentadecane, 8-hexyl (4.18 %), Tetrapentacontane (5.50 %), Tridecanol, 2-ethyl-2- methyl (5.94 %), Tetratetracontane (12.10 %), Tetracontane (2.64 %), Octacosane (2.17 %), Tetratriacontane (2.86 %), Pentatriacontane (4.84 %), Tritetracontane (5.28 %) and Tetrapentacontane, 1,54-dibromo-(3.96 %). Compounds which were found only in this sample were 4-Nonene, 5-methyl, 1,2,3-trimethylbenzene, Octane, 3,3- dimethyl-, 3-Methylene-1,7-octadiene and 2-Propyl-1-pentanol (Table 4.24).

4.25 Moderate symptomatic cultivar

Moderate symptomatic cultivar i.e. R12 is as follows.

4.25.1 Cultivar R12 of S. lycopersicum

This cultivar showed moderate symptoms on ToLCNDV inoculation, its GCMS analysis led to the identification of 45 compounds, major compounds were p-xylene (1.35 %), Decane, 3,7-dimethyl (1.41 %), Dodecane (1.59 %), Nonadecane (2.60 %), Hexadecane (4.24 %), Heptadecane, 2,6,10,15-tetramethyl (7.71 %), Octacosane (3.86 %), Heneicosane, 11-(1-ethylpropyl) (1.35 %), Octadecane, 2-methyl (1.83 %), Eicosane (9.05 %), Tetratriacontane (1.73 %), Phenol, 3,5-bis (1,1-dimethylethyl) (1.49 %), Hexadecane, 2,6,11,15-tetramethyl (4.11 %), Pentadecane, 8-hexyl (6.93 %), 9-Methylnonadecane (3.08 %), tetratetracontane (5.59 %), 1-Chloroheptacosane (5.20 %), Tetracontane (6.16 %), Hexatriacontane (5.97 %), 11-Decyltetracosane (5.12 %) and 1,2-benzenedicarboxylic acid, diisooctyl ester (4.96 %), while other compounds such as 3-hexanone, 2,4-dimethyl, 2-Bromo-nonane and Octadecane, 2- methyl, were found merely in this sample (Table 4.25).

174

4.26 Severe symptomatic cultivars

Severe symptomatic cultivars i.e. R7and R8 are as follows.

4.26.1 Cultivar R7 of S. lycopersicum

Among 32 compounds identified from R7 extract, major compounds were 2,6- Dimethyldecane (5.21 %), p- Xylene (5.33 %), Dodecane (5.27 %), 2-Bromo dodecane (2.55 %), Hexadecane (8.93 %), Dodecane, 2,6,10-trimethyl (1.97 %), Tetradecane (6.95 %), Heptadecane (2.08 %), Pentadecane (6.37 %), Eicosane (12.17 %), Phenol, 3,5-bis(1,1-dimethylethyl)- (4.63 %), 2-Isopropyl-5-methyl-1-hexanol (2.31 %), Heneicosane (1.85 %), Tetratetracontane (9.04 %), Tridecanol, 2-ethyl-2-methyl (3.71 %), Disulfide, di-tert-dodecyl (3.71 %) and 1, 2-benzenedicarboxylic acid, diisooctyl ester (1.24 %). While, other compounds found were Heptane, 3-methyl, 4,4- Dimethylcyclooctene, Nonane, 3,7-dimethyl- and Hexadecanal (Table 4.26).

4.26.2 Cultivar R8 of S. lycopersicum

A total of 44 compounds were identified major compounds were p-Xylene (1.59 %), 1-Chloroheptacosane (6.20 %), Dodecane, 2,6,11-trimethyl (4.07 %), Tetradecane (1.63 %), Hexadecane (1.77 %), Tetratriacontane (2.48 %), Eicosane (8.41 %), 10- Methylnonadecane (2.08 %), Phenol, 3,5-bis(1,1-dimethylethyl)- (7.97 %), Hexadecane, 2,6,11,15-tetramethyl (4.31 %), octacosane (7.97 %), Heneicosane (4.60 %), Tetrapentacontane (6.20 %), Hexadecane, 2,6,10,14-tetramethyl (4.43 %), Pentatriacontane (5.93 %),11-Decyltetracosane (5.58 %) and 1, 2- benzenedicarboxylic acid, diisooctyl ester (4.96 %). While, other compounds which were found only in this sample were pentane, 2,2,3,3-tetramethyl-, 6-Methyl-1- octanol, Dodecane, 2-cyclohexyl, 5,6-Dipropyldecane, 1-nonanol, 4,8-dimethyl-, 7- Tetradecene, p-Cymene and Decane, 2,3,4-trimethyl- (Table 4.27).

4.27 Very severe symptomatic cultivars

Very severe symptomatic cultivars i.e. R14, R15 and Nagina are as follows.

4.27.1 R14 Cultivar of S. lycopersicum

Major compounds among 49 compounds identified from GCMS analysis were Nonane, 3-methyl (1.32 %), Decane, 3,7-dimethyl (1.15 %), Undecane (1.56 %), Dodecane (4.06 %), Pentadecane (7.41 %), Hexadecane (3.59 %), Tridecane (1.56 %), Heptadecane (1.56 %), Eicosane (6.05 %), Nonadecane (2.13 %), Phenol, 3,5- 175 bis(1,1-dimethylethyl)- (2.84 %), Octacosane (5.46 %), Heneicosane (6.09 %), Octadecane (3.04 %), Tetrapentacontane (6.55 % ), Pentatriacontane (3.82 %), Isopropyl Palmitate (4.68 %), Tetratetracontane (2.49 %), Tetracontane (5.46 %), Hexatriacontane (6.55 %), Squalene (4.21 %) and 1, 2-benzenedicarboxylic acid, diisooctyl ester (6.44 %). While, other compounds which were found only in this sample were 5-Ethyl-2-methylheptane, tridecane, 6-methyl, 6-Benzoylhexanoic acid and Squalene (Table 4.28).

4.27.2 Cultivar R15 of S. lycopersicum

In R15 cultivar GCMS analysis indicated 53 compounds, major compounds among them were Tetradecane (2.43 %), Dodecane, 6-methyl (1.62 %), Hexadecane (5.63 %), 2,6,11-Trimethyl dodecane (3.39 %), Heptadecane (1.78 %), Eicosane (6.31 %), Phenol, 3,5-bis(1,1-dimethylethyl)- (3.24 %), Heneicosane (2.20 %), Octadecane (3.89 %), Tritetracontane (5.82 %), Hentriacontane (1.62 %), Pentadecane, 8-hexyl (5.82 %), Tetracosane, 11-decyl (2.43 %), Tetratetracontane (6.63 %), Tetrapentacontane (6.23 %), Tetratriacontane (2.91 %), 1-(+)-Ascorbic acid 2,6- dihexadecanoate (3.83 %), Pentatriacontane (3.56 %), Tetracontane (6.95 %) and 1, 2- benzenedicarboxylic acid, diisooctyl ester (7.65 %), while other different compounds, found only in this sample were Octadecane,6-methyl, Dodecane, 6-methyl, 2,7,10- Trimethyl dodecane and Eicosane, 10-methyl (Table 4.29).

4.27.3 Nagina variety of S. lycopersicum

GCMS analysis of susceptible native tomato variety (Nagina) extract after ToLCNDV inoculation led to the identification of 49 compounds, among those major compounds were Dodecane (3.49 %), Tridecane (1.53 %), Dodecane, 2,6,11-trimethyl (3.92 %), Dodecane, 2-methyl- (1.48 %), Tetradecane (10.43 %), Nonadecane (2.03 %), Tridecanol, 2-ethyl-2-methyl (1.97 %), Eicosane (6.81 %), Phenol, 3,5-bis(1,1- dimethylethyl)- (3.19 %), Hexadecane (6.84 %), Octadecane, 5,14-dibutyl (1.53 %), Tetratetracontane (6.45 %), Octacosane (1.87 %), Heneicosane (4.36 %), Phytol (2.62 %), Tritetracontane (3.44 %), Hexatriacontane (5.88 %), Tetrapentacontane (3.27 %), Pentatriacontane (6.67 %), Docosanoic acid, docosyl ester (9.59 %). Other compounds associated only with this cultivar were Benzene, 1, 4-dimethyl, Dodecane, 2-methyl- and Docosanoic acid, docosyl ester (Table 4.30).

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Table 4.16: Volatile compounds analyzed by GCMS from the n-hexane extract of leaves and stem of healthy S. lycopersicum plant.

Relative Sr. Retention Molecular Molecular Abundance Compound Name No. Time Formula Weight %age

1 5.55 p-Xylene C8H10 106 0.16 % 2 6.30 Benzeneethanol, .alpha.,.beta.-imethyl- C10H140 150 0.08 %

3 6.56 Hexane, 2,4-dimethyl- C8H18 114 0.06 %

4 10.28 Octane, 3,5-dimethyl C10H22 142 0.32 % 5 11.55 1-Hexanol, 2-ethyl- C8H18O 130 0.29 % 6 12.1 Butane, 2,2-dimethyl- C6H14 86 0.96 % 7 12.22 Nonane, 4,5-dimethyl- C11H24 156 0.16 % 8 12.32 Ether, heptyl hexyl C13H28O 200 0.32 % 9 13.86 Undecane C11H24 156 0.80 % 10 14.02 Hexane, 3,3-dimethyl- C8H18 114 0.04 % 11 15.76 Silane, cyclohexyldimethoxymethyl- C9H20O2Si 188 0.39 % 12 17.29 Dodecane C12H26 170 2.74 % 13 18.88 3,7-Dimethylundecane, C13H28 184 1.20 %

14 19.15 Dodecane, 2,6,11-trimethyl- C15H32 212 4.19 % 15 19.75 Tridecane C13H28 184 1.29 % 16 19.89 Decane, 1,1′ oxybis C20H42O 298 0.40 % 17 20.99 10-Methylnonadecane C20H42 282 0.48 % 18 21.11 2-Bromo dodecane C12H25Br 248 0.45 %

19 21.63 Tetradecane C14H30 198 9.35 %

20 22.94 Eicosane C20H42 282 7.42 %

21 23.13 Nonadecane C19H40 268 1.29 %

22 23.33 Phenol, 3,5-bis(1,1-dimethylethyl)- C14H22O 206 5.32 %

23 23.66 Hexadecane, 2,6,11,15-tetramethyl- C20H42 282 2.58 %

24 24.11 Dodecane, 2,6,10-trimethyl- C15H32 212 0.97 %

25 24.61 Hexadecane C16H34 226 7.74 %

26 24.93 Tetratriacontane C34H70 478 1.61 %

27 26.38 Octacosane C28H58 394 2.26 %

28 27.07 Heptadecane, 2,6,10,15-tetramethyl- C21H44 296 4.19 %

29 29.25 Tritetracontane C43H88 604 2.58 %

30 29.69 Tetratetracontane C44H90 618 2.58 %

31 30.43 Tetrapentacontane C54H110 758 8.70 %

32 32.58 Pentatriacontane C35H72 492 7.09 %

33 32.65 Hexatriacontane C21H44 506 6.45 %

34 35.10 Tetrapentacontane, 1,54-dibromo C54H108Br2 914 3.22 % 1,2-Benzenedicarboxylic acid, 35 35.6 C H O 390 12.25 % diisooctyl ester 24 38 4

177

Table 4.17: Volatile compounds isolated from the n-hexane extract of leaves and stem of S. lycopersicum (R6 cultivar which showed complete resistance after inoculation with ToLCNDV) by GCMS.

Relative Sr. Retention Molecular Molecular Compound Name abundance No. Time Formula Weight %age 1 4.04 Hexane, 2,4-dimethyl- C8H18 114 0.163 % 2 5.24 Ethylbenzene C8H10 106 0.19 % 3 5.52 p-Xylene C8H10 106 0.54 % 4 6.28 Benzene, 1,3-dimethyl C8H10 106 0.33 % 5 6.55 Decane, 2,5,6-trimethyl C13H28 184 0.24 % 6 7.77 Octane, 2,6-dimethyl C10H22 142 0.09 % 7 8.94 2,4,6-Trimethyloctane C11H24 156 0.13 % 8 9.18 Nonane, 3-methyl C10H22 142 0.16 % 9 9.65 1-Isopropyl-2,3-dimethylcyclopentane C10H20 140 0.08 % 10 10.01 Benzene, 1,2,3-trimethyl C9H12 120 0.19 % 11 10.28 Octane, 3,5-dimethyl C10H22 142 0.65 % 12 10.73 2,2,3,3-Tetramethylhexane C10H22 142 0.06 % 13 11.07 Decane, 4-methyl- C11H24 156 0.33 % 14 11.3 Limonene C10H16 136 1.30 % 15 12.23 Nonane, 4,5-dimethyl- C11H24 156 0.88 % 16 12.31 Ether, heptyl hexyl C13H28O 200 0.24 % 17 13.86 Undecane C11H24 156 0.81 % 18 15.76 Silane, cyclohexyldimethoxymethyl- C9H20O2Si 188 0.49 % 19 15.88 5-butylnonane C13H28 184 0.57 % 20 16.08 Undecane, 3,7-Dimethyl C13H28 184 0.16 % 21 16.30 3-Methyl undecane C12H26 170 0.12 % 22 17.09 1-Hexene, 3,5,5-trimethyl- C9H18 126 0.11 % 23 17.28 Dodecane C12H26 170 1.54 % 24 17.62 Undecane, 2,6-dimethyl C13H28 184 0.49 % 25 17.86 4-methyldodecane C13H28 184 0.29 % 26 18.35 Tridecane C13H28 184 0.33 % 27 19.01 2-Bromo dodecane C12H25Br 248 3.14 % 28 19.63 Nonadecane C19H40 268 1.22 % 29 19.75 Dodecane, 2,6,11-trimethyl- C15H32 212 1.22 % 30 20.15 Heptadecane, 2,6,10,15-tetramethyl- C21H44 296 1.56 % 31 20.99 Decane, 2,3,5,8-tetramethyl C14H30 198 0.49 % 32 21.64 Tetradecane C14H30 198 1.79 % 33 21.98 1-Chloroheptacosane C27H55Cl 414 0.59 % 34 22.95 10-Methylnonadecane C20H42 282 1.87 % 35 23.00 Eicosane C20H42 282 6.24 % 36 23.34 Phenol, 3,5-bis (1,1-dimethylethyl)- C14H22O 206 7.69 % 37 23.67 Hexadecane, 2,6,11,15-tetramethyl- C20H42 282 3.01 % 38 24.37 Octacosane C28H58 394 0.49 % 39 24.61 Hexadecane C16H34 226 1.78 % 40 24.68 Tritetracontane C43H88 604 1.79 % 41 25.85 Pentadecane, 8-hexyl- C21H44 296 6.34 % 42 28.32 Octadecane, 1-chloro- C18H37Cl 288 5.76 % 1-(+)-Ascorbic acid 2,6- 43 28.85 C H O 652 4.88 % dihexadecanoate 38 68 8 Table continue page turn over 178

44 30.36 Tetrapentacontane, 1,54-dibromo- C54H108Br2 914 6.83 % 45 30.43 Tetrapentacontane C54H110 758 6.83 % 46 30.65 1-Hentetracontanol C41H84O 592 4.47 % 47 30.84 Pentatriacontane C35H72 492 5.45 % 48 32.58 Tetratetracontane C44H90 618 5.61 % 49 32.66 Hexatriacontane C36H74 506 5.59 % 50 35.59 Tetracontane C40H82 562 4.88 %

179

Table 4.18: Volatile compounds isolated from the n-hexane extract of leaves and stem of S. lycopersicum (R10 cultivar which showed complete resistance after inoculation with ToLCNDV) by GCMS.

Relative Sr. Retention Molecular Molecular Compound Name Abundance No. Time Formula Weight %age

1 3.55 Hexane, 2,4-dimethyl C8H18 114 0.37 % 2 5.22 Octane, 1-chloro- C8H17Cl 148 0.51 % 3 5.5 p-Xylene C8H10 106 1.48 % 4 6.26 Ethylbenzene C8H10 106 0.93 %

5 6.53 2,2,3,3-Tetramethylhexane C10H22 142 0.37 % 6 8.79 1-Hexene, 3,5,5-trimethyl- C9H18 126 0.19 % 7 8.92 2,9-Dimthylundecane C13H28 184 0.16 % 8 10.01 Benzene, 1,2,3-trimethyl- C9H12 120 0.21 % 9 10.28 Octane, 3,5-dimethyl C10H22 142 0.74 % 10 11.05 Heptane, 5-ethyl-2-methyl C10H22 142 0.37 % 11 11.50 1-Hexanol, 2-ethyl C8H18O 130 0.56 % 12 12.20 2,6-Dimethyldecane C12H26 170 1.29 % 13 12.29 4,5- Dipropyloctane C14H30 198 0.37 % 14 12.40 Nonane, 4,5-dimethyl C11H24 156 0.46 % 15 12.58 1-Pentanol, 4-methyl-2-propyl C9H20O 144 0.28 % 16 12.79 Decane, 1-iodo- C10H21I 268 0.23 % 17 13.84 Undecane C11H24 156 0.83 % 18 15.76 Silane, cyclohexyldimethoxymethyl- C9H20O2Si 188 0.68 % 19 17.25 Dodecane C12H26 170 1.31 % 20 17.61 Undecane, 2,6-Dimethyl C13H28 184 0.56 %

21 17.85 Tridecane, 4-methyl C14H30 198 0.16 % 22 18.48 5-Butylnonane C13H28 184 0.39 % 23 18.64 Dodecane, 4,6-dimethyl- C14H30 198 0.53 % 24 18.86 Tridecanol, 2-ethyl-2-methyl- C16H34O 242 0.93 % 25 18.99 2-Bromo dodecane C12H25Br 248 0.50 % 26 19.14 Dodecane, 2,6,11-trimethyl- C15H32 212 4.26 % 27 19.45 Nonane, 1-iodo- C9H19I 254 0.56 % 28 20.5 Heptane, 2,5,5-trimethyl- C10H22 142 0.72 % 29 20.13 2,3,6,7-Tetramethyl-octane C12H26 170 2.04 % 30 21.62 Tetradecane C14H30 198 1.20 % 31 22.59 Heptadecane, 2,6,10,15-tetramethyl- C21H44 296 1.48 % 32 22.92 Dodecane, 2,6,10-trimethyl- C15H32 212 2.03 % 33 22.98 Hexadecane C16H34 226 7.41 % 34 23.4 Anthracene, 9-dodecyltetradecahydro- C20H40O3S2 392 1.67 % 35 23.32 Phenol, 3,5-bis(1,1-dimethylethyl)- C14H22O 206 8.33 % 36 23.65 Heneicosane C21H44 296 3.99 % 37 24.91 Octacosane C28H58 394 1.62 % 38 25.83 Eicosane C20H42 282 9.07 % 39 26.36 Pentadecane, 8-hexyl- C21H44 296 4.81 % 40 27.46 Oxirane, tetradecyl- C16H32O 240 5.83 % 41 28.25 Tetratetracontane C44H90 618 3.70 % 42 28.31 Silane, trichlorooctadecyl C18H37Cl3Si 386 6.85 % 43 30.35 Phytol C20H40O 296 7.31 % 44 30.42 Tetrapentacontane C54H110 758 8.82 % 45 35.55 1, 2-benzenedicarboxylic acid, C24H38O4 390 3.89 % diisooctyl ester

180

Table 4.19: Volatile compounds isolated from the n-hexane extract of leaves and stem of S. lycopersicum (R11 cultivar which showed complete resistance after inoculation with ToLCNDV) by GCMS.

Relative Sr. Retention Molecular Molecular Compound Name Abundance No Time Formula Weight %age

1 4.04 Hexane, 2,4-dimethyl- C8H18 114 0.21 % 3 5.25 Pentane, 2,2,3,4-tetramethyl- C9H20 128 0.09 % 4 5.55 p-Xylene C8H10 106 0.23 % 5 6.31 Ethylbenzene C8H10 106 0.13 % 6 6.57 Pentane, 2,2-dimethyl C7H16 100 0.10 % 7 8.82 2-Decene, 5-methyl- C11H22 154 0.75 % 8 8.95 2,2,6,6-Tetramethylheptane C11H24 156 0.09 % 9 10.3 Decane, 2,5,6-trimethyl- C13H28 184 0.06 % 10 11.09 Nonane, 2,6-dimethyl- C11H24 156 0.38 % 11 12.24 Undecane, 3,7-Dimethyl C13H28 184 0.88 % 12 13.87 5-Ethylundecane C13H28 184 0.79 % 13 15.78 Silane, cyclohexyldimethoxymethyl- C9H20O2Si 188 0.41 % 14 17.29 Dodecane C12H28 170 2.86 %

16 18.36 Tridecane C13H28 184 0.50 % 17 19.16 Dodecane, 2,6,11-trimethyl- C15H32 212 3.73 % 18 19.64 Nonadecane C19H40 268 1.51 % 19 20.99 1-Decanol, 2,2-dimethyl- C12H26O 186 0.50 % 20 21.64 Tetradecane C14H30 198 9.04 % 21 23.00 Eicosane C20H42 282 7.53 % 22 23.34 Phenol, 3,5-bis(1,1-dimethylethyl) C14H22O 206 5.78 % 23 23.67 Hexadecane, 2,6,11,15-tetramethyl- C20H42 282 2.39 % 24 24.61 Hexadecane C16H34 226 8.91 % 25 24.7 Tritetracontane C43H88 604 1.89 % 26 27.07 Octadecane C18H38 254 5.27 % 27 27.45 Octacosane C28H58 394 1.51 % 28 30.32 Pentatriacontane C35H72 492 9.04 % 29 31.20 Tetratetracontane C44H90 618 6.02 % 30 32.59 Tetrapentacontane C54H110 758 9.79 % 31 35.1 Tetrapentacontane, 1,54-dibromo- C54H108Br2 914 7.66 % 1, 2-benzenedicarboxylic acid, 32 35.6 C H O 390 11.97 % diisooctyl ester 24 38 4

181

Table 4.20: Volatile compounds isolated from the n-hexane extract of leaves and stem of S. lycopersicum (R5 cultivar which showed complete resistance after ToLCNDV inoculation) by GCMS.

Relative Sr. Retention Molecular Molecular Compound Name abundance No. Time Formula Weight %age

1 5.52 p-Xylene C8H10 106 0.25 % 2 6.65 Hexane, 2,4-dimethyl C8H18 114 0.09 % 3 11.07 Nonane, 2,6-dimethyl- C11H24 156 0.19 % 4 11.3 Limonene C10H16 136 0.29 % 5 11.44 1-Hexanol, 2-ethyl- C8H18O 130 0.19 % 6 12.08 Heptane, 5-ethyl-2-methyl C10H22 142 0.09 % 7 12.21 2,6-Dimethyldecane C12H26 170 0.59 % 8 13.28 1-Isopropyl-2,3-dimethylcyclopentane C10H20 140 0.08 % 9 13.85 Undecane C11H24 156 0.61 % 10 15.76 Silane, cyclohexyldimethoxymethyl- C9H20O2Si 188 0.28 % 11 17.26 Dodecane C12H26 170 2.24 % 12 17.86 Decane, 2,3,7-trimethyl- C13H28 184 0.59 % 13 18.65 Decane, 2,3,5,8-tetramethyl- C14H30 198 0.56 % 14 19 2-Bromo dodecane C12H25Br 248 3.36 % 15 19.75 Tridecane C13H28 184 1.07 % 16 19.87 Tridecanol, 2-ethyl-2-methyl- C16H34O 242 1.08 % 17 21.63 Tetradecane C14H30 198 7.80 % 18 21.96 Heneicosane, 11-(1-ethylpropyl)- C26H54 366 0.69 % 19 22.94 Nonadecane C19H40 268 2.17 % 20 23.0 Eicosane C20H42 282 7.11 % 21 23.32 Phenol, 3,5-bis(1,1-dimethylethyl)- C14H22O 206 3.75 % 22 24.6 Hexadecane C16H34 226 8.14 % 23 24.93 Tetratriacontane C34H70 478 1.97 % 24 25.8 9-Methylnonadecane C20H42 282 7.51 % 25 25.85 Pentadecane, 8-hexyl- C21H44 296 3.00 % 26 26.38 Hexadecane, 2,6,11,15-tetramethyl- C20H42 282 2.57 % 27 27.06 Octadecane C18H38 254 4.46 % 28 28.26 Hexatriacontane C36H74 506 6.32 % 29 28.84 1-(+)-Ascorbic acid 2,6-dihexadecanoate C38H68O8 652 2.86 % 30 29.23 Octacosane C28H58 394 2.96 % 31 29.46 Isopropyl palmitate C19H38O2 298 3.81 % 32 29.67 Tetratetracontane C44H90 618 2.80 % 33 30.42 Tetrapentacontane C54H110 758 6.91 % 34 31.2 Pentatriacontane C35H72 492 4.15 % 1, 2-benzenedicarboxylic acid, diisooctyl 35 35.59 C H O 390 9.39 % ester 24 38 4

182

Table 4.21: Volatile compounds isolated from the n-hexane extract of S. lycopersicum (R13 cultivar after ToLCNDV inoculation) by GCMS.

Sr. Retention Molecular Molecular Abundance Compound Name No. Time Formula Weight %age

1 4.05 2,4-Dimethyl undecane C13H28 184 0.08 % 2 5.55 Dodecane, 1-chloro C12H25Cl 204 0.10 % 3 6.30 p-Xylene C8H10 106 0.07 % 4 6.57 3-Methyldodecane C13H22 184 0.07 % 5 7.79 1-Pentanol, 4-methyl-2-propyl- C9H20O 144 0.05 % 6 8.81 Octane, 3,5-dimethyl C10H22 142 0.04 % 7 8.95 Undecane, 3,9-dimethyl C13H28 184 0.04 %

8 10.03 Benzene,(1,3,3-trimethyl nonyl) C18H30 246 0.26 % 9 10.29 Dodecane C12H26 170 1.05 % 10 11.08 Trans, cis-2,6-nonadien-1-ol C9H16O 140 0.22 % 11 12.31 Ether, heptyl hexyl C13H28O 200 0.52 % 12 12.43 Nonane, 4,5-dimethyl- C11H24 156 0.26 % 13 15.78 Silane, cyclohexyldimethoxymethyl- C9H20O2Si 188 0.35 % 14 15.89 5-Butylnonane C13H28 184 0.13 % 15 16.31 3-Methyl undecane C12H26 170 0.14 % 16 17.1 1-Chloroheptacosane C27H55Cl 414 0.10 % 17 17.29 Pentadecane C15H32 212 1.92 % 18 17.63 Undecane, 2,6-Dimethyl C13H28 184 0.39 % 19 18.65 Decane, 2,3,5,8-tetramethyl- C14H30 198 0.35 % 20 18.88 Heneicosane, 11-(1-ethylpropyl)- C26H54 366 0.81 % 21 19.01 Tridecanol, 2-ethyl-2-methyl- C16H34O 242 2.84 % 22 19.75 Tridecane C13H28 184 1.05 % 23 19.88 10-Methylnonadecane C20H42 282 1.05 % 24 19.97 Octane, 1,1'-oxybis C16H34O 242 0.35 % 25 20.16 1-Octanol, 2-butyl C12H26O 186 0.87 % 26 20.64 Decane, 3-cyclohexyl C16H32 224 0.11 % 27 20.71 Disulfide, di-tert-dodecyl C24H50S2 402 0.17 % 28 20.79 2-Propylheptanol C10H22O 158 0.17 % 29 21.11 Eicosane, 10-methyl- C21H44 296 0.43 % 30 21.51 Tetratetracontane C44H90 618 0.44 % 31 21.55 9-Eicosene C20H40 280 7.76 % 32 22.52 Tritetracontane C43H88 604 1.07 % 33 22.95 2,6,10 Trimethyl-dodecane C15H32 212 5.43 % 34 23.01 Eicosane C20H42 282 6.31 % 35 23.33 Phenol, 3,5-bis (1,1-dimethylethyl)- C14H22O 206 4.03 % 36 23.68 Dodecane, 2,6,11-trimethyl- C15H32 212 1.75 % 37 24.21 11-Decyltetracosane C34H70 478 0.90 % 38 24.62 Hexadecane C16H34 226 8.06 % 39 24.93 Heneicosane C21H44 296 1.70 % 40 25.81 Nonadecane C19H40 268 5.78 % 41 26.38 Pentadecane, 8-hexyl- C21H44 296 2.19 % 42 27.08 Octadecane C18H38 254 4.38 % 43 28.19 Pentatriacontane C35H72 492 5.26 % 44 28.33 Tetratriacontane C34H70 478 2.81 % 45 28.85 1-(+)-Ascorbic acid 2,6-Dihexadecanoate C38H68O8 652 1.58 % 46 29.24 Octacosane C28H58 394 0.48 %

47 30.37 Tetrapentacontane, 1,54-dibromo- C54H108Br2 914 2.46 % 48 30.43 Tetrapentacontane C54H110 758 5.44 % 49 30.73 Nonahexacontanoic acid C69H138O2 998 2.99 % 50 32.59 Tetracontane C40H82 562 6.62 % 51 35.6 1, 2-Benzenedicarboxylic acid, diisooctyl ester C24H38O4 390 8.42 % 183

Table 4.22: Volatile compounds isolated from the n-hexane extract of leaves and stem of S. lycopersicum (R2 cultivar which showed mild symptoms after ToLCNDV inoculation) by GCMS.

Relative Sr. Retention Molecular Molecular Compound Name Abundance No. Time Formula Weight %age

1 6.25 2,4,6-Trimethyloctane C11H24 156 0.07 % 2 6.65 Octane, 2,6-dimethyl C10H22 142 0.09 % 3 7.32 Cyclohexane, 1-methyl-2-propyl- C10H20 140 0.05 % 4 7.87 Benzene, 1,2,3-trimethyl C9H12 120 0.10 % 5 8.38 Dodecane C12H26 170 0.41 % 6 9.04 2,2,3,3-tetramethylhexane C10H22 142 0.07 % 7 9.30 Ether, heptyl hexyl C13H28O 200 0.11 % 8 9.51 Decane, 4-methyl C11H24 156 0.24 % 9 9.74 Limonene C10H16 136 0.11 % 10 9.9 Cyclohexane, butyl C10H20 140 0.08 % 11 10.01 1-Hexanol, 2-ethyl- C8H18O 130 0.51 % 12 10.3 Ether, heptyl hexyl C13H28O 200 0.08 % 13 11.18 Undecane C11H24 156 0.72 % 14 11.55 Pentadecane, 7-methyl- C16H34 226 0.08 % 15 11.81 Decane, 3-methyl- C11H24 156 0.15 % 16 12.33 1-Isopropyl-2,3-dimethylcyclopentane C10H20 140 0.08 % 17 14.38 Dodecane, 3-cyclohexyl- C18H36 252 0.10 % 18 15.22 Silane, cyclohexyldimethoxymethyl- C9H20O2Si 188 0.37 % 19 15.58 Hexadecane C16H34 226 0.28 % 20 15.82 3-Methyl undecane C12H26 170 0.17 % 21 17.35 2, 6-Dimethylundecane C13H28 184 0.62 % 22 17.61 4-Methyldodecane C13H28 184 0.44 % 23 16.75 1-Chloroheptacosane C27H55Cl 414 0.16 % 24 18.15 Tetradecane C14H30 198 0.54 % 25 18.47 Dodecane, 4,6-dimethyl- C14H30 198 2.05 % 26 19.01 2,6,10 Trimethyl-dodecane C15H32 212 4.03 % 27 19.63 Tridecane C13H28 184 1.44 % 28 20.05 5-Butylnonane C13H28 184 1.23 % 29 20.92 Eicosane, 10-methyl- C21H44 296 0.68 % 30 21.58 Pentadecane C15H32 212 9.78 % 31 21.75 Octacosane C28H58 394 1.85 % 32 21.92 Heneicosane, 11-(1-ethylpropyl)- C26H54 366 0.92 % 33 22.4 Tritetracontane C43H88 604 1.64 % 34 22.92 Eicosane C20H42 282 8.22 % 35 23.3 Phenol, 3,5-bis(1, 1-dimethylethyl)- C14H22O 206 3.78 % 36 24.67 Tetratetracontane C44H90 618 9.04 % 37 25.19 Hentriacontane C31H64 436 2.05 % 38 25.84 Heneicosane C21H44 296 8.12 % 39 27.05 Nonadecane C19H40 268 4.01 % 40 28.25 Pentadecane, 8-hexyl C21H44 296 6.57 %

41 29.22 Nonacosane C29H60 408 3.69 % 42 29.45 Isopropyl palmitic acid C17H34O2 256 2.59 % 43 30.40 Tetracontane C40H82 562 7.49 % 44 32.57 Tetrapentacontane C54H110 758 5.34 % 45 35.58 1,2-benzenedicarboxylic acid, diisooctyl C24H38O4 390 9.78 % ester

184

Table 4.23: Volatile compounds isolated from the n-hexane extract of leaves and stem of S. lycopersicum (R3 cultivar which showed mild symptoms after ToLCNDV inoculation) by GCMS.

Relative Sr. Retention Molecular Molecular Compound Name Abundance No. Time Formula Weight %age 1 5.25 Pentane, 2,2,3,4-tetramethyl- C9H20 128 0.11 % 2 5.54 p-Xylene C8H10 106 0.26 % 3 7.77 Heptane, 2,2-dimethy- C11H24 128 0.05 % 4 8.95 2,2,6,6-Tetramethylheptane C11H24 156 0.03 % 5 9.18 2,2,5,5-Tetramethylhexane C10H22 142 0.03 % 6 10.74 Pentane, 2,2-dimethyl C7H16 100 0.34 % 7 11.07 Nonane, 2,6-dimethyl- C11H24 156 0.19 % 8 11.48 1-Hexanol, 2-ethyl- C8H18O 130 0.28 % 9 12.10 1-Pentanol, 4-methyl-2-propyl- C9H20O 144 0.02 % 10 12.61 Butane, 2,2-dimethyl- C6H14 86 0.13 % 11 12.99 Isooctanol C8H18O 130 0.04 % 12 13.29 Cycloheptane, bromo C7H13Br 176 0.04 % 13 13.86 Undecane C11H24 156 0.53 % 14 15.78 Silane, cyclohexyldimethoxymethyl- C9H20O2Si 188 0.49 % 15 17.28 Dodecane C12H26 170 1.32 % 16 17.86 Decane, 2,5,6-trimethyl- C13H28 184 0.26 % 17 18.65 Decane, 2,3,5,8-tetramethyl- C14H30 198 0.53 % 18 19.16 Dodecane, 2,6,11-trimethyl- C15H32 212 3.42 % 19 19.47 Nonadecane C19H40 268 0.53 % 20 19.75 Tridecane C13H28 184 1.05 % 21 20.99 1-Decanol, 2,2, dimethyl C12H26O 186 0.49 % 22 21.64 Tetradecane C14H30 198 2.10 % 23 21.98 Heneicosane, 11-(1-ethylpropyl)- C26H54 366 0.74 % 24 23.34 Phenol, 3,5-bis(1,1-dimethylethyl)- C14H22O 206 9.07 % 25 24.70 Tetratetracontane C44H90 618 2.24 % 26 24.93 Tetratriacontane C34H70 478 1.05 % 27 25.85 Eicosane C20H42 282 7.63 % 28 26.39 Octacosane C28H58 394 4.87 % 29 26.65 Pentatriacontane C35H72 492 1.97 % 30 28.32 1-Chloroheptacosane C27H55Cl 414 6.58 % 31 28.84 1-(+)-Ascorbic acid 2,6-dihexadecanoate C38H68O8 652 10.79 % 32 30.36 Phytol C20H40O 296 8.42 % 33 30.65 Tetrapentacontane, 1,54-dibromo- C54H108Br2 914 9.34 % 34 32.65 Hexatriacontane C21H44 506 11.13 % 35 35.1 Nonahexacontanoic acid C69H138O2 998 6.58 % 1, 2-benzenedicarboxylic acid, 36 35.6 C H O 390 7.64 % diisooctyl ester 24 38 4

185

Table 4.24: Volatile compounds isolated from the n-hexane extract of leaves and stem of S. lycopersicum (R9 cultivar which showed mild symptoms after ToLCNDV inoculation) by GCMS.

Relative Sr. Retention Molecular Molecular Compound Name Abundance No. Time Formula Weight %age

1 5.23 Ethylbenzene C8H10 106 0.33 % 2 5.51 p-Xylene C8H10 106 1.32 % 3 6.55 2,4,6-Trimethyloctane C11H24 156 0.28 % 4 8.80 1-Hexene, 3,5,5-trimethyl- C9H18 126 0.12 % 5 8.93 1-Iodoundecane C11H23I 282 0.09 % 6 9.62 4-Nonene, 5-methyl C10H20 140 0.08 % 7 10.02 1,2,3-Trimethylbenzene C9H12 120 0.23 % 8 10.27 Octane, 3,5-dimethyl C10H22 142 0.66 % 9 11.05 Octane, 3,3-dimethyl- C10H22 142 0.28 % 10 11.29 3-Methylene-1,7-octadiene C9H14 122 0.19 % 11 11.44 1-Hexanol, 2-ethyl- C8H18O 130 0.47 % 12 11.92 1-Pentanol, 4-methyl-2-propyl- C9H20O 144 0.17 % 13 12.3 2-Propyl-1-pentanol C8H18O 130 0.22 % 14 12.21 Decane, 3,7-dimethyl- C12H26 170 1.10 % 15 12.41 Heptane, 3-ethyl-3-methyl- C10H22 142 0.41 % 16 13.85 Undecane C11H24 156 0.69 % 17 15.75 Silane, cyclohexyldimethoxymethyl- C9H20O2Si 188 0.88 % 18 17.26 Dodecane C12H26 170 1.10 % 19 17.62 Undecane, 2,6-Dimethyl C13H28 184 0.44 % 20 18.49 2-Bromo dodecane C12H25Br 248 0.41 % 21 18.64 Decane, 2,3,5,8-tetramethyl C14H30 198 0.63 % 22 18.86 Undecane, 2,4-dimethyl C13H28 184 0.99 % 23 19.14 Dodecane, 2,6,11-trimethyl C15H32 212 0.44 % 24 19.54 Decane, 4-methyl- C11H24 156 0.72 % 25 19.74 Tridecane C13H28 184 0.66 % 26 19.86 Decane, 2,3,7-trimethyl C13H28 184 0.55 % 27 20.14 5-Butylnonane C13H28 184 2.42 % 28 20.33 Nonane, 1-iodo- C9H19I 254 0.63 % 29 20.50 4-Methyldodecane C13H28 184 0.56 % 30 20.98 10-Methylnonadecane C20H42 282 0.33 % 31 21.63 Tetradecane C14H30 198 0.88 % 32 21.85 Pentadecane C15H32 212 1.10 % 33 21.96 Tetradecane, 4-methyl C15H32 212 0.47 % 34 22.6 Heptadecane, 2,6,10,15-tetramethyl- C21H44 296 1.10 % 35 22.42 Nonadecane C19H40 268 0.88 % 36 22.92 Eicosane C20H42 282 6.82 % 37 23.32 Phenol, 3,5-bis(1,1-dimethylethyl)- C14H22O 206 10.45 % 38 23.65 Hexadecane C16H34 226 4.18 % 39 25.83 Heneicosane C21H44 296 7.26 % 40 26.36 Pentadecane, 8-hexyl- C21H44 296 4.18 % 41 28.25 Tetrapentacontane C54H110 758 5.50 % 42 28.30 Tridecanol, 2-ethyl-2-methyl- C16H34O 242 5.94 % 43 28.70 Tetratetracontane C44H90 618 12.10 % 44 28.77 Tetracontane C40H82 562 2.64 % 45 29.22 Octacosane C28H58 394 2.17 % 46 29.66 Tetratriacontane C34H70 478 2.86 % 47 30.82 Pentatriacontane C35H72 492 4.84 % 48 32.64 Tritetracontane C43H88 604 5.28 % 49 35.04 Tetrapentacontane, 1,54-dibromo- C54H108Br2 914 3.96 % 186

Table 4.25: Volatile compounds isolated from the n-hexane extract of leaves and stem of S. lycopersicum (R12 cultivar which showed moderate symptoms after ToLCNDV inoculation) by GCMS.

Relative Sr. Retention Molecular Molecular Compound Name Abundance No. Time Formula Weight %age

1 4.01 Hexane, 2,4-dimethyl C8H18 114 0.19 % 2 5.50 p-Xylene C8H10 106 1.35 % 3 6.25 Ethylbenzene C8H10 106 0.93 % 4 6.52 Pentane, 2,2,3,4-tetramethyl- C9H20 128 0.36 % 5 9.15 2,2,5,5-Tetramethylhexane C10H22 142 0.19 % 6 10.28 Octane, 3,5-dimethyl C10H22 142 0.87 % 18 7 11.54 1-Hexanol, 2-ethyl- C8H O 130 0.58 % 8 11.92 1-Pentanol, 4-methyl-2-propyl- C9H20O 144 0.19 % 9 12.08 1-Hexene, 3,5,5-trimethyl C9H18 126 0.19 % 10 12.2 Decane, 3,7-dimethyl- C12H26 170 1.41 % 11 12.29 3-Hexanone, 2,4-dimethyl C8H16O 128 0.38 % 12 12.58 2,3,3-Trimethyloctane C11H24 156 0.26 % 13 12.79 Butane, 2,2-dimethyl- C6H14 86 0.35 % 14 13.98 Undecane, 3,7-Dimethyl C13H28 184 1.16 % 15 15.76 Silane, cyclohexyldimethoxymethyl- C9H20O2Si 188 0.77 % 16 17.25 Dodecane C12H26 170 1.59 % 17 17.60 Undecane, 2,6-Dimethyl C13H28 184 0.63 % 18 17.85 2-Bromo-nonane C9H19Br 206 0.24 % 19 18.47 2-Bromo dodecane C12H25Br 248 0.48 % 20 18.63 Octane, 2,6-dimethyl C10H22 142 0.59 % 21 19.14 Hexadecane C16H34 226 4.24 % 22 19.45 Hexane, 3,3-dimethyl- C8H18 114 0.77 % 23 19.61 Decane, 2,3,5,8-tetramethyl C14H30 198 0.71 % 24 19.74 Dodecane, 2,6,10-trimethyl- C15H32 212 0.96 % 25 19.86 Disulfide, di-tert-dodecyl C24H50S2 402 0.49 % 26 20.13 Nonadecane C19H40 268 2.60 % 27 21.53 2-Propylheptanol C10H22O 158 1.13 % 28 21.61 Tridecane C13H28 184 1.09 % 29 21.84 Pentadecane C15H32 212 1.16 % 30 21.96 Heneicosane, 11-(1-ethylpropyl)- C26H54 366 1.35 % 31 22.50 Octadecane, 2-methyl C19H40 268 1.83 % 32 22.92 Heptadecane, 2,6,10,15-tetramethyl- C21H44 296 7.71 % 33 22.97 Eicosane C20H42 282 9.05 % 34 23.11 Tetratriacontane C34H70 478 1.73 % 35 23.32 Phenol, 3,5-bis(1,1-dimethylethyl)- C14H22O 206 1.49 % 36 23.65 Hexadecane, 2,6,11,15-tetramethyl C20H42 282 4.11 % 37 25.83 Pentadecane, 8-hexyl- C21H44 296 6.93 % 38 25.8 9-Methylnonadecane C20H42 282 3.08 % 39 28.24 Tetratetracontane C44H90 618 5.59 % 40 28.32 1-Chloroheptacosane C27H55Cl 414 5.20 % 41 28.69 Octacosane C28H58 394 3.86 % 42 30.4 Tetracontane C40H82 562 6.16 % 43 32.62 Hexatriacontane C36H74 506 5.97 % 44 33.51 11-Decyltetracosane C34H70 478 5.12 % 1, 2-benzenedicarboxylic acid, 45 35.53 C H O 390 4.96 % diisooctyl ester 24 38 4

187

Table 4.26: Volatile compounds isolated from the n-hexane extract of leaves and stem of S. lycopersicum (R7 cultivar which showed severe symptoms after ToLCNDV inoculation) by GCMS.

Relative Sr. Retention Molecular Molecular Compound Name Abundance No. Time Formula Weight %age 1 3.47 Heptane, 3-methyl C8H18 114 0.48 % 2 4.35 Ethylbenzene C8H10 106 1.02 % 3 4.52 p-Xylene C8H10 106 5.51 % 4 7.62 Decane, 2,5,6-trimethyl- C13H28 184 0.96 % 5 9.15 2,6-Dimethyldecane C12H26 170 5.39 % 6 9.31 Heptane, 3-ethyl-3-methyl- C10H22 142 1.32 % 7 12.30 Silane, cyclohexyldimethoxymethyl- C9H20O2Si 188 1.44 % 8 12.54 Nonane, 3,7-dimethyl- C11H24 156 0.35 % 9 13.57 Dodecane C12H26 170 5.45 % 10 13.9 Undecane, 2,6-Dimethyl C13H28 184 1.19 % 11 14.55 3,7-Dimethylundecane, C13H28 184 1.19 % 12 14.69 4,4-Dimethylcyclooctene C10H18 138 1.19 % 13 15.18 2-Bromo dodecane C12H25Br 248 2.63 % 14 15.31 Hexadecane C16H34 226 9.23 % 15 15.62 Hexane, 3,3-dimethyl- C8H18 114 0.77 % 16 16.27 Dodecane, 2,6,10-trimethyl- C13H28 184 2.03 % 17 17.08 2,3,3-Trimethyloctane C11H24 156 0.48 % 18 17.71 Tetradecane C14H30 198 7.18 % 19 17.94 Heptadecane C17H36 240 2.15 % 20 18.04 Tetradecane, 4-methyl C15H32 212 0.72 % 21 19.05 Eicosane C20H42 282 12.57 % 22 19.39 Phenol, 3,5-bis(1,1-dimethylethyl)- C14H22O 206 4.79 % 23 19.46 2-Isopropyl-5-methyl-1-hexanol C10H22O 158 2.39 % 24 19.55 Nonane, 1-iodo C9H19I 254 0.48 % 25 20.74 Pentadecane C15H32 212 6.59 % 26 21.47 Heneicosane C21H44 296 1.92 % 27 21.85 Tetratetracontane C44H90 618 9.34 % 28 24.91 Tridecanol, 2-ethyl-2-methyl- C16H34O 242 3.83 % 29 25.04 Ttrapentacontane C54H110 758 1.44 % 30 25.92 Disulfide, di-tert-dodecyl C24H50S2 402 3.84 % 31 27.51 Hexadecanal C16H32O 240 0.86 % 1, 2-benzenedicarboxylic acid, 32 35.65 diisooctyl ester C24H38O4 390 1.29 %

188

Table 4.27: Volatile compounds isolated from the n-hexane extract of leaves and stem of S. lycopersicum (R8 cultivar which showed severe symptoms after ToLCNDV inoculation) by GCMS.

Relative Sr. Retention Molecular Molecular Compound Name Abundance No. Time Formula Weight %age

1 3.56 Hexane, 2,4-dimethyl C8H18 114 0.18 % 2 5.23 Ethylbenzene C8H10 106 0.39 % 3 5.51 p-Xylene C8H10 106 1.59 % 4 6.27 Benzene, 1,3-dimethyl C8H10 106 0.89 % 5 6.54 2,4,6-Trimethyloctane C11H24 156 0.35 % 6 8.8 Pentane, 2,2,3,3-tetramethyl- C9H20 128 0.22 % 7 8.93 3-Methyldodecane C13H28 184 0.18 % 8 9.16 Octane, 2,6-dimethyl C10H22 142 0.14 % 9 9.62 6-Methyl-1-octanol C9H20O 144 0.14 % 10 10.02 Benzene, 1,2,3-trimethyl C9H12 120 0.22 % 11 11.05 Decane, 4-methyl- C11H24 156 0.42 % 12 11.28 D-limonene C10H16 136 0.18 % 13 11.40 Dodecane, 2-cyclohexyl- C18H36 252 0.87 % 14 11.47 1-Hexanol, 2-ethyl C8H18O 130 0.79 % 15 11.92 1-Pentanol, 4-methyl-2-propyl- C9H20O 144 0.18 % 16 12.3 Octadecane, 1-chloro- C18H37Cl 288 0.32 % 17 12.59 5,6-Dipropyldecane C16H34 226 0.22 % 18 12.8 5- Butylnonane C13H28 184 0.18 % 19 12.95 1-Nonanol, 4,8-dimethyl- C11H24O 172 0.15 % 20 13.27 7-Tetradecene C14H28 196 0.18 % 21 13.83 Undecane C11H24 156 0.93 % 22 14.25 Octane, 1,1'-oxybis C16H34O 242 0.18 % 23 14.46 p-Cymene C10H14 134 0.17 % Silane, 24 15.76 C H O Si 188 0.58 % cyclohexyldimethoxymethyl- 9 20 2 25 15.85 2-Isopropyl-5-methyl-1-hexanol C10H22O 158 0.57 % 26 17.25 Dodecane C10H22 170 1.42 % 27 17.61 Undecane, 2,6-Dimethyl C13H28 184 0.53 % 28 17.85 Decane, 2,3,4-trimethyl- C13H28 184 0.29 % 29 18.33 Pentadecane C15H32 212 0.38 % 30 18.63 Dodecane, 4,6-dimethyl- C14H30 198 0.49 % 31 18.99 2-Bromo dodecane C12H25Br 248 0.41 % 32 19.14 Dodecane, 2,6,11-trimethyl- C15H32 212 4.08 % 33 19.75 Tridecane C13H28 184 1.08 % 34 19.85 2-Propylheptanol C10H22O 158 0.44 % 35 20.05 Disulfide, di-tert-dodecyl C24H50S2 402 0.46 % 36 21.63 Tetradecane C14H30 198 1.64 % 37 21.95 Heneicosane, 11-(1-ethylpropyl)- C26H54 366 1.33 % 38 22.58 Nonadecane C19H40 268 1.24 % 39 22.92 Tetratriacontane C34H70 478 2.48 % 40 22.98 Eicosane C20H42 282 8.41 % 41 23.13 10-Methylnonadecane C20H42 282 2.08 % 42 23.20 Tetratetracontane C44H90 618 2.04 % 43 23.32 Phenol, 3,5-bis(1,1-dimethylethyl)- C14H22O 206 7.98 % 44 23.65 Hexadecane, 2,6,11,15-tetramethyl- C20H42 282 4.32 % Table continue page turn over 189

45 23.93 Tetrapentacontane, 1,54-dibromo- C54H108Br2 914 1.21 % 46 24.6 Hexadecane C16H34 226 1.78 % 47 25.8 Octacosane C28H58 394 7.79 % 48 26.36 Heneicosane C21H44 296 4.61 % 49 28.25 Tetrapentacontane C54H110 758 6.20 % 50 28.30 1-Chloroheptacosane C27H55Cl 414 6.20 % 51 28.70 Hexadecane, 2,6,10,14-tetramethyl- C20H42 282 4.43 % 52 30.4 Pentatriacontane C35H72 492 5.94 % 53 32.64 11-Decyltetracosane C34H70 478 5.58 % 1, 2-benzenedicarboxylic acid, 54 35.55 C H O 390 4.96 % diisooctyl ester 24 38 4

190

Table 4.28: Volatile compounds isolated from the n-hexane extract of leaves and stem of S. lycopersicum (R14 cultivar which showed very severe symptoms after ToLCNDV inoculation) by GCMS.

Relative Sr. Retention Molecular Molecular Compound Name Abundance No. Time Formula Weight %age

1 5.22 Ethylbenzene C8H10 106 0.17 % 2 5.50 p-Xylene C8H10 106 0.62 % 3 6.26 Benzene, 1,4-dimethyl C8H10 106 0.47 % 4 6.53 Decane, 2,5,6-trimethyl C13H28 184 0.19 % 5 7.75 Nonane, 3-methyl C10H22 142 1.33 % 6 8.5 4,5- Dipropyloctane C14H30 198 0.06 % 7 8.79 5-Ethyl-2-methylheptane C10H22 142 0.18 % 8 8.92 Tridecane, 6-methyl C14H30 198 0.16 % 9 9.16 Octane, 2,6-dimethyl C10H22 142 0.16 % 10 9.4 6-Benzoylhexanoic acid C13H16O3 220 0.08 % 11 9.62 1-Isopropyl-2,3-dimethylcyclopentane C10H20 140 0.09 % 12 9.78 1-Pentanol, 2-ethyl-4-methyl C8H18O 130 0.07 % 13 9.99 Benzene, 1,2,3-trimethyl C9H12 120 0.23 % 14 10.26 2,4,6-Trimethyloctane C11H24 156 0.94 % 15 11.05 Nonane, 2,6-dimethyl- C11H24 156 0.47 % 16 11.29 Limonene C10H16 136 0.16 % 17 11.4 1-Hexanol, 2-ethyl- C8H18O 130 1.05 % 18 12.20 Decane, 3,7-dimethyl- C12H26 170 1.16 % 19 20.15 Dodecane, 2,6,11-trimethyl C15H32 212 1.09 % 20 12.58 Pentadecane, 7-methyl- C16H34 226 0.24 % 21 13.84 Undecane C11H24 156 1.56 % 22 15.75 Silane, cyclohexyldimethoxymethyl- C9H20O2Si 188 0.47 % 23 15.85 5-Butylnonane C13H28 184 0.25 % 24 16.06 Undecane, 2-methyl C12H26 170 0.39 % 25 17.26 Dodecane C12H26 170 4.06 % 26 17.60 Undecane, 2,6-Dimethyl C13H28 184 0.86 % 27 17.85 4-Methyldodecane C13H28 184 0.47 % 28 18.34 Tetradecane C14H30 198 0.62 % 29 21.62 Pentadecane C15H32 212 7.42 % 30 18.63 Dodecane, 4,6-dimethyl- C14H30 198 0.70 % 31 18.86 Tetradecane, 5-methyl C15H32 212 1.25 % 32 19.15 Hexadecane C16H34 226 3.59 % 33 19.74 Tridecane C13H28 184 1.57 % 34 19.86 10-Methylnonadecane C20H42 282 0.42 % 35 21.85 Heptadecane C17H36 240 1.57 % 36 23 Eicosane C20H42 282 6.06 % 37 22.93 Nonadecane C19H40 282 2.14 % 38 23.31 Phenol, 3,5-bis(1,1-dimethylethyl)- C14H22O 206 2.84 % 39 24.68 Octacosane C28H58 394 5.47 % 40 25.6 Heneicosane C21H44 296 6.09 % 41 27.05 Octadecane C18H38 254 3.05 % 42 30.40 Tetrapentacontane C54H110 758 6.56 % 43 29.23 Pentatriacontane C35H72 492 3.83 % 44 29.45 Isopropyl Palmitate C19H38O2 298 4.69 % 45 29.66 Tetratetracontane C44H90 618 2.49 % 46 28.25 Tetracontane C40H82 562 5.46 % 47 32.56 Hexatriacontane C36H74 506 6.56 % 48 35.05 Squalene C30H50 410 4.22 % 49 35.55 1, 2-benzenedicarboxylic acid, diisooctyl ester C24H38O4 390 6.45 % 191

Table 4.29: Volatile compounds isolated from the n-hexane extract of leaves and stem of S. lycopersicum (R15 cultivar which showed very severe symptoms after ToLCNDV inoculation) by GCMS.

Relative Sr. Retention Molecular Molecular Compound Name Abundance No. Time Formula Weight %age

1 5.22 Ethylbenzene C8H10 106 0.08 % 2 5.52 p-Xylene C8H10 106 0.24 % 3 6.26 Benzene, 1,3-dimethyl C8H10 106 0.23 % 4 6.54 Decane, 2,3,7-trimethyl- C13H28 184 1.05 % 5 7.76 Nonane, 3-methyl C10H22 142 0.07 % 6 9.16 Octane, 2,6-dimethyl C10H22 142 0.11 % 7 9.77 1-Pentanol, 2-ethyl-4-methyl C8H18O 130 0.06 % 8 10 Benzene, 1,2,3-trimethyl- C9H12 120 0.11 % 9 10.26 Octane, 3,5-dimethyl C10H22 142 0.40 % 10 10.71 2,2,3,3-Tetramethylhexane C10H22 142 0.089 % 11 11.05 Nonane, 2,6-dimethyl- C11H24 156 0.23 % 12 11.28 Limonene C10H16 136 0.11 % 13 11.38 1-Hexanol, 2-ethyl C8H18O 130 0.65 % 14 12.07 Octadecane, 1-chloro- C18H37Cl 288 0.12 % 15 12.21 Undecane, 3,7-Dimethyl C13H28 184 0.73 % 16 12.3 Octadecane,6-methyl C19H40 268 0.16 % 17 12.42 Dodecane, 4,6-dimethyl C14H30 198 0.29 % 18 13.84 Undecane C11H24 156 0.81 % 19 15.75 Silane, cyclohexyldimethoxymethyl- C9H20O2Si 188 0.29 % 20 15.86 5-Butylnonane C13H28 184 0.15 % 21 16.06 Undecane, 2-methyl C12H26 170 0.29 % 22 16.4 Undecane, 2,6-dimethyl C13H28 184 0.17 % 23 17.26 Tetradecane C14H30 198 2.43 % 24 17.61 Dodecane, 6-methyl C13H28 184 1.62 % 25 18.64 2,7,10-Trimethyl dodecane C15H32 212 0.65 % 26 18.8 Nonadecane C19H40 268 0.40 % 27 18.86 Tetradecane, 5-methyl C15H32 212 1.13 % 28 18.99 2-Bromo dodecane C12H25Br 248 0.35 % 29 19.15 2,6,11-Trimethyl dodecane C15H32 212 3.39 % 30 19.62 Heptadecane, 2,6,10,15-tetramethyl- C21H44 296 0.40 % 31 19.74 Tridecane C13H28 184 1.29 % 32 20.15 Pentadecane C15H32 212 1.29 % 33 20.98 Eicosane, 10-methyl- C21H44 296 0.65 % 34 21.63 Hexadecane C16H34 226 5.63 % 35 21.85 Heptadecane C17H36 240 1.78 % 36 21.96 Heneicosane, 11-(1-ethylpropyl)- C26H54 366 0.81 % 37 22.42 Tetratetracontane C44H90 618 1.29 % 38 23 Eicosane C20H42 282 6.31 % 39 23.11 Octacosane C28H58 394 1.38 % 40 23.31 Phenol, 3,5-bis(1,1-dimethylethyl)- C14H22O 206 3.24 % 41 23.65 Heneicosane C21H44 296 2.20 % 42 24.68 Tritetracontane C43H88 604 5.82 % 43 25.2 Hentriacontane C31H64 436 1.62 % 44 25.85 Pentadecane, 8-hexyl- C21H44 296 5.82 % 45 25.95 11-Decyl tetracosane, C34H70 478 2.43 %

Table continue page turn over 192

46 27.06 Octadecane C18H38 254 3.89 % 47 28.26 Tetrapentacontane C54H110 758 6.23 % 48 29.23 Tetratriacontane C34H70 478 2.91 % 1-(+)-Ascorbic acid 2,6 49 29.46 dihexadecanoate C38H68O8 652 3.83 % 50 29.67 Pentatriacontane C35H72 492 3.56 % 51 30.31 Tetracontane C40H82 562 6.96 % 52 32.59 Tetratetracontane C44H90 618 6.63 % 1, 2-benzenedicarboxylic acid, 53 35.64 diisooctyl ester C24H38O4 390 7.65 %

193

Table 4.30: Volatile compounds isolated from the n-hexane extract of leaves and stem of S. lycopersicum (Nagina variety which showed very severe symptoms after ToLCNDV inoculation) by GCMS.

Relative Sr. Retention Molecular Molecular Compound Name Abundance No. Time Formula Weight %age

1 5.242 Octane, 1-chloro- C8H17Cl 148 0.19 % 2 5.525 p-Xylene C8H10 106 0.62 % 3 6.267 Benzene, 1,4-dimethyl C8H10 106 0.44 % 4 6.55 Decane, 2,5,6-trimethyl C13H28 184 0.11 % 5 8.792 4,5- Dipropyloctane C14H30 198 0.14 % 6 8.933 1-Iodoundecane C11H23I 282 0.12 % 7 10 Benzene, 1,2,3-trimethyl C9H12 120 0.14 % 8 10.258 Octane, 3,5-dimethyl C10H22 142 0.71 % 9 10.717 Pentane, 2,2-dimethyl C7H16 100 0.08 % 10 11.033 Decane, 4-methyl- C11H24 156 0.36 % 11 11.273 Limonene C10H16 136 0.23 % 12 11.492 1-Hexanol, 2-ethyl- C8H18O 130 0.44 % 13 11.925 1-Pentanol, 4-methyl-2-propyl- C9H20O 144 0.14 % 14 12.192 Nonane, 4,5-dimethyl- C11H24 156 1.19 % 15 12.292 2,2,3,3-Tetramethylhexane C10H22 142 0.44 % 16 12.408 Decane, 3,7-dimethyl- C12H26 170 0.23 % 17 12.575 2,3,3-Trimethyloctane C11H24 156 0.19 % 18 13.833 Undecane C11H24 156 1.09 % 19 15.742 Silane,cyclohexyldimethoxymethyl C9H20O2Si 188 0.36 % 20 15.858 5-Butylnonane C13H28 184 0.11 % 21 16.058 Undecane, 3,7-dimethyl C13H28 184 0.31 % 22 16.283 3-Methylundecane C12H26 170 0.19 % 23 17.258 Dodecane C12H26 170 3.49 % 24 17.617 Undecane, 2,6-dimethyl C13H28 184 0.66 % 25 17.85 4-Methyldodecane C13H28 184 0.44 % 26 18.333 Pentadecane C15H32 212 0.65 % 27 19.733 Tridecane C13H28 184 1.53 % 28 19.125 Dodecane, 2,6,11-trimethyl- C15H32 212 3.92 % 29 18.867 Undecane, 2,4-dimethyl C13H28 184 1.28 % 30 19.85 Dodecane, 2-methyl- C13H28 184 1.48 % 31 20.975 Decane, 2,3,5,8-tetramethyl- C14H30 198 0.51 % 32 21.092 2-Bromo dodecane C12H25Br 248 0.53 % 33 21.625 Tetradecane C14H30 198 10.43 % 34 23.65 Nonadecane C19H40 268 2.03 % 35 21.958 Tetradecane, 4-methyl C15H32 212 0.79 % 36 22.917 Tridecanol, 2-ethyl-2-methyl- C16H34O 242 1.97 % 37 22.983 Eicosane C20H42 282 6.81 % Phenol, 3,5-bis(1,1- 38 23.3 C H O 206 3.19 % dimethylethyl)- 14 22 39 24.583 Hexadecane C16H34 226 6.84 % 40 24.675 Octadecane, 5,14-dibutyl C26H54 366 1.53 % 41 25.833 Tetratetracontane C44H90 618 6.45 % 42 25.408 Octacosane C28H58 394 1.87 % 43 27.05 Heneicosane C21H44 296 4.36 % 44 27.467 Phytol C20H40O 296 2.62 %

Table continue page turn over 194

45 31.183 Tritetracontane C43H88 604 3.44 % 46 28.183 Hexatriacontane C36H74 506 5.88 % 47 28.242 Tetrapentacontane C54H110 758 3.27 % 48 30.3 Pentatriacontane C35H72 492 6.67 % 49 35.542 Docosanoic acid, docosyl ester C44H88O2 648 9.59 %

CHAPTER NO. 5 DISCUSSION 195

DISCUSSION

Present study indicated the presence of bipartite begomoviruses as Tomato leaf curl New Delhi virus (ToLCNDV) and Tomato leaf curl palampur virus (ToLCPalV) which have also been reported previously from tomato in Pakistan. Along with these 03 new monopartite begomoviruses Tomato leaf curl Kerala virus (ToLCKeV), Tomato leaf curl virus (ToLCV) and Papaya leaf curl virus (PaLCuV) species, those had not been previously reported from Pakistan were also isolated. Recent disease severity and incidence seems to be linked with evolved disease complex as a result of newly identified begomoviruses. Moreover, we also found that Tomato leaf curl disease (ToLCD) complex now consists of both monopartite and bipartite begomoviruses, which makes the situation more alarming, as previously it consisted of bipartite begomoviruses only. Due to increased ToLCD incidence, this study was focused to search begomoviruses diversity infecting tomato in geographically distinct areas of Pakistan (Lahore, Faisalabad and Northern areas of Pakistan).

ToLCD is a big constraint for tomato crop in the Indian subcontinent and its incidence has increased gradually. Almost two decades ago ToLCD started affecting Punjab and Sindh provinces of Pakistan heavily, leading to 30 % to 40 % yield losses (Mansoor et al., 1997) and its incidence has increased in recent years than previous years. Previously known ToLCPalV and ToLCNDV were accountable for the disease in Pakistan (Mansoor et al., 1997; Ali et al., 2010). ToLCNDV (bipartite begomovirus) was identified in 1995 and 1997 in India (Padidam et al., 1995) and in Pakistan respectively. ToLCNDV is widespread in other regions as well, like in Bangladesh (Varma et al., 2011) and Thailand (Mandal, 2010). ToLCPalV is also a bipartite begomovirus reported from India, Iran (Heydarnejad et al., 2009) and Pakistan (Ali et al., 2010). ToLCD in India is mainly caused by several monopartite begomoviruses, such as ToLCV, ToLCKeV, Tomato leaf curl Patna virus (ToLCPatV), Tomato leaf curl Rajasthan virus (ToLCRaV), Ageratum enation virus (AEV) and Tobacco curly shoot virus (TbCSV), as well as bipartite begomoviruses like ToLCNDV and ToLCPalV (Briddon and Mansoor, 2008; Kumar et al., 2008).

Severe infection was seen in tomato fields of Rakh Burj (Lahore) a city at the border of Pakistan and India and trade route among both countries. Begomoviruses diversity from this area indicated the prevalence of viruses previously reported from India. Among 40 isolates of DNA-A, 41 of DNA-B, 15 isolates of Papaya leaf curl 196 betasatellites (PaLCuB), 04 isolates of Cotton leaf curl Multan betasatellites (CLCuMuB), 02 isolates of Okra leaf curl alphasatellite (OLCuA) and 03 isolates of Tomato leaf curl alphasatellite (ToLCPKA) were identified.

All isolates of ToLCV particularly showed maximum nucleotide identity with isolates from India. Similarly, ToLCKeV, PaLCuV and ToLCPalV isolates shared maximum nucleotide identity with respective virus isolates reported from India. But this is contrary for ToLCNDV where all 13 isolates of ToLCNDV shared maximum sequence identity with ToLCNDV isolates reported from Pakistan. All the tomato samples preceded in present study indicated a proportion of various monopartite and bipartite viruses and their distribution pattern. ToLCPalV and ToLCNDV, both bipartite, were found prevalent in Northern areas of Pakistan, a region isolated from the plains and less infestation of whiteflies; whereas monopartite begomoviruses ToLCV, PaLCuV and ToLCKeV were prevalent in Lahore and Faisalabad.

This diversity suggests a serious thought for remedy of ToLCD in both India and Pakistan. Begomoviruses can affect many hosts and over last three decades begomoviruses emerged as major constraint for solanaceous crops particularly pepper and tomato (Kenyon et al., 2014). There are at least 21 various types of ToLCV in India (Shelat et al., 2014).

Recombination analysis of DNA-A isolates revealed the presence of interspecific, intraspecific or both recombination in several isolates, with other begomoviruses. Similar results were observed for DNA-B, betasatellites and alphasatellites, during the present study.

Due to the pseudorecombination, component capture and mutation, begomoviruses keep on evolving swiftly (Roberts and Stanley, 1994; Hou and Gilbertso, 1996; Padidam et al., 1999; Saunders et al., 2001b; Singh et al., 2012), which led to increased diversification (Prasanna and Rai, 2007). As geminiviruses infect a variety of ornamental, vegetables and cereal crops, coinfections of different begomoviruses species cause recombination (Lima et al., 2013) turning existing species more virulent (Pasumarthy et al., 2010). However, quality and number of recombination events are still mysterious in various tomato-infecting begomoviruses species (Prasanna and Rai, 2007).

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ToLCKeV isolates, found for the first time in Pakistan showed recombination with Radish leaf curl virus (RaLCuV) isolates. In case of RS157 (ToLCKeV), as both recombination parents were prevalent in different regions of India, most likely recombination events occurred in India and this virus had spread from India to Pakistan in its intact form. Similar recombination was also observed in other isolate of ToLCKeV which recombine with RaLCuV-[IN:LabIso:11]. This recombination event also involved parent begomoviruses of Indian origin. ToLCV (RS186) isolate, showed interspecific recombination with PaLCuV-Luc [IN:Luc]-Y15934 and ToLCPalV- [Pk:Lah:06]-AM494976, seems the chimeric molecule of PaLCuV, ToLCV and ToLCPalV. Another ToLCV (RS110) isolate also showed interspecific recombination with OELCuV-[IN:Odi:Okr:14]-KT390343 and ToLCPalV-[Pk:Lah:06]-AM494976, one recombination event took place in India and other may occurred in India or in Pakistan as indicated by the recombination analysis during the present piece of work.

ToLCKeV was abundant in tomato crop cultivated in Kerala states in India (Pasumarthy et al., 2010). As work done by Singh et al. (2012) indicated the presence of ToLCKeV from India which is responsible for leaf curl disease in radish in India, where recombination may led to the new species emergence. Recombination has led to the emergence of several new species of begomoviruses for example Tomato leaf curl Gandhinagar virus (ToLCGNV) is the recombinant of ToLCKeV and ToLCV (Rathore et al., 2014). These kinds of recombinations usually occurred as a result of coinfection of different begomoviruses and led to emergence of new species (Davino et al., 2012).

Two intraspecific recombination events were studied in RS80 ToLCV isolate which had both recombinants belonging to the different strain of the same species and these both strains were reported from India, indicated the prevalence of ToLCV in India. RS60 PaLCuV isolate contained interspecific and intraspecific recombinations with ToLCNDV and PaLCuV respectively. A new strain of PaLCuV (RS92) indicated the presence of interspecific recombination with ToLCV and RaLCuV isolates, infecting tomato in India.

Previously PaLCuV had also recombined with other begomoviruses as it was evident from studies that PaLCuV recombined with RaLCuV and such recombination events led to the new strains or species evolution (Singh et al., 2012). Recombination due to

198 interspecific homologous events is considered a core contributing factor for diversification (Harrison and Robinson, 1999).

Present studies indicated that all viruses were not equally vulnerable for recombination, isolates of same species showed different levels of recombination. Most of the recombination events found in this study were interspecific. Recombination breakpoints were located mainly in AC1 region of begomoviruses in addition to occasional recombination in AC2, AC3, AC4, AV1, AV2 and IR. Among all DNA-B isolates, 04 recombinations took place in BC1 region, while 07 in IR region and 06 events took place in BV1 regions, so among BV1 and BC1, BV1 seemed more vulnerable for recombination or were considered as hot spot. In betasatellite recombination took place in SCR and βC1 while in alphasatellite recombination took place in IR and Rep regions.

As previous studies conducted by Hou and Gilbertso in 1996; Sanz et al. in 1999; Prasanna and Rai in 2007; Lefeuvre et al. in 2007; Silva et al. in 2014 had indicated that AC1 along with other CP, CR and REn represented recombination hot spots, present in begomoviruses.

In current study phylogenetic analysis of full length and Rep (AC1) protein indicated different clade pattern of Rep of RS82 (ToLCV) than other ToLCV isolates, which might be due to recombination. Among these RS82 contained interspecific recombination with ChLCINV in IR region, with TbCSV in Rep and with ToLCPalV in Rep and AC4 regions. These recombination events in Rep might have contributed for different clade pattern of RS82.

Previously it had been exposed as potential recombination junction in the IR and Rep protein respectively (Melgarejo et al., 2013).

Moreover clade pattern indicated that Rep sequences of ToLCPalV may have more similarity with some Rep sequences of ToLCNDV than other isolates. In AC2 phylogenetic tree as compared to full length, RS92 (PaLCuV) grouped with RS96 and RS97 (ToLCV) isolates, which was probably due to differences in proteins attributed by change of amino acids in AC2 of RS 92. In phylogenetic tree of AC3, RS96 and RS97 which formed separate subgroup, possibly due to differences in amino acids sequences of AC3 from other isolates of same species. Similarly in AV1 tree possible

199 reason of RS92 grouping with RS2 and RS157 might be the recombination of RS92, RS2 and RS157 with RaLCuV, and these recombination events were mapped in AV1 region along with other sites. As these 03 isolates were recombinant of RaLCuV, a significant feature shared by all isolates, these recombination events might have contributed towards such clade formation. AV2 clade formation revealed that newly found sequences showed maximum homology with Indian isolates which were grouped together indicating their identities among themselves. But in contrast, isolates of ToLCNDV and ToLCPalV clustered together indicating their similarity.

As previous studies conducted by Ali et al. (2010) and Mansoor et al. (1997) indicted the prevelance of ToLCNDV and ToLCPalV infecting various crops in different regions of Pakistan.

Similarly NSP of all isolates of DNA-B of different types or species were also distinct as complete nucleotide sequence, indicated highly conserved nature of this protein. All NSP on comparing for percentage identity showed 88.1% to 99.6 % sequence identity among them this supports BV1 is very conserve among all isolates. However some differences depicted by BC1 and BV1 proteins in phylogenetic tree were predicted might be due to recombination, in BC1 and BV1 regions, as many isolates were found to have recombination in these regions and seemed hot spot regions for recombination.

However Lefeuvre et al. (2009) suggested selection disfavors recombinants with breakpoints in the coding regions, but recent experimental evidences exhibited coding regions are also vulnerable to the recombination (Vuillaume et al., 2011).

In present piece of work, analysis of βC1 protein of betasatellites indicated that βC1 region was extremely conserve among isolates of a single species, but highly divergent among different species. Betasatellite protein of CLCuMuB isolates shared 24.4 % similarity with PaLCuB isolates. Similarly in contrast to whole genome, where all isolates of CLCuMuB including NCBI isolates had different overall "A" content as compare to PaLCuB, indicated conserved nature of A-rich region across species. The A-rich region indicated the presence of A-rich fragments across the genome of present isolates consisting of various numbers of nucleotides upto 07 nucleotides. Similarly in alphasatellites it was apparent from Rep analysis that, this region was extremely conserved among all isolates of a particular species. Rep of

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ToLCPKA isolates shared 99.7% to 94.3 % similarity with Rep of OLCuA isolates. However alphasatellites Rep showed more conserve nature among these species even, as compared to betasatellites, shown by protein and whole genome analysis. Phylogenetic analysis of full length and Rep sequences of alphasatellite indicated that complete genome of alphasatellite formed more distinct clades showing more divergence among isolates or species while Rep shared more identities even across species indicating conserve nature of Rep of alphasatellite as compared to whole genome.

Numerous previous studies conducted by Akhtar and coworkers in 2014 indicated that βC1 region was highly conserved among isolates of a particular species but it was extremely divergent among different species. Similarly a work conducted by Bull and his coworkers in 2004 showed the conserve nature of A-rich region across the betasatellites species. Alphasatellites Rep showed more conserve nature as compared to βC1 protein and whole genome analysis of betasatellites as indicated by the work of Bull et al. in 2003. Rep shared more identities even across species in contrast to betasatellites, indicated conserve nature of Rep of alphasatellites then whole genome (Briddon et al., 2004).

Phylogeographic analysis indicated three basic epicenters of diversity. Region1 southern India (ToLCKeV and ToLCV), Region 2 eastern subcontinent (ToLCV) and Region 3 northwestern region (PaLCuV, ToLCPalV and ToLCNDV). We have observed the spread of ToLCKeV and ToLCV from southern India to northwestern subcontinent (Pakistan). This work also confirmed the absence of ToLCKeV in northwestern region of subcontinent and also openly described that ToLCV and ToLCKeV were pre-dominant in the major tomato production region of southern- India.

Rajagopalan et al in 2012 reported the same unique direction of spread of cotton leaf curl disease complex (another begomovirus spread by whitefly) which was from the northwestern to southeastern subcontinent. However, movement of ToLCKeV and ToLCV from eastern region to western region was concurrent with the observation of the spread of Okra enation leaf curl virus (OELCuV) from southern India (Tamil- Nadu) to Pakistan (Sindh; Serfraz et al., 2015). Results were also concurrent with

201 previous comprehensive reports of leaf curl incidence of tomato in India (Chowda- Reddy et al., 2005).

Present study described that the recent begomoviruses complex may have spread from India to Pakistan. At this time, it is not clear how all of these different begomoviruses are spreading across the subcontinent. Few evidences showed the presence of geminiviruses in fruits and seeds suggested that tomato can act as a reservoir of the viruses from Indian origins and thus can spread to Pakistan. The presence of ToLCV, ToLCKeV and PaLCuV which were not reported as a part of tomato leaf curl disease complex in Pakistan previously, might be due to more active recent trade from India. However, detailed study should be done with a special focus on occurrence of isolated viruses in tomato fruit and seeds.

Begomoviruses spread due to climatic changes and enhanced international trading has increased the risk of new virus's introduction (Hanssen et al., 2010). A contributing factor to begomovirus diversity might be the movement of viruliferous whitefly from India to Pakistan or vice versa depending on the weather patterns (Rajagopalan et al., 2012). Naturally the movement of infected plant material could also be a contributing factor. However, this hypothesis must be validated by the scientific evidences.

The spread of begomovirus disease complexes is often linked with geographical proximity and trade activities. For example Cotton leaf curl Gezira virus (CLCuGV) was found in the Sindh province of Pakistan, which was previously reported from Middle East. We hypothesized that the introduction of CLCuGV was linked with the trade activity via Middle East (Tahir et al., 2011). Later investigations indeed identified that the CLCuGV virus was also found in Oman (Khan et al., 2012). It seems that Middle East is a sink of diverse begomoviruses from Asia and Africa (Khan et al., 2014). The close proximity of agricultural lands between Pakistan and India where viruliferous whiteflies can move across the border is contributing to spread of begomoviruses. Demand for fresh tomatoes in Pakistan is increasing with growing population, in order to meet the increased marketing requirement particularly in rainy season and Ramadan, tomatoes are imported from neighboring countries especially India. India has been the major source for imported tomatoes in Pakistan for the last many years, with increasing vegetables imports of worth more than US $

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120 million (Amir and Hyder, 2015). Regarding import shares, India is the major tomato exporter for Pakistan (Khan and Hussain, 2014).

Tomatoes in Pakistan come through Attari Wagha border in Lahore, from Amaritsar, India. A large proportion of exported tomatoes come from major tomato cultivating areas of India such as New Delhi and Nashik (Agricorner 2011). In tomato growing areas (New Delhi, Mirzapur, Lucknow and Bangalore etc.) of Indian subcontinent ToLCD is a major problem as numerous reports of new strains have been published (Chakraborty et al., 2003). Spread of begomovirus in Pakistan might be due to recent active trade. Pakistan is importing 40 to 45 trucks daily (Pakissan 2015) each carrying 16 tons tomato from India. A study by Kothandaraman et al. (2015) had indicated that begomovirus Mung bean yellow vein mosaic virus (MYMV) is seed born. Begomoviruses can be transmitted by seeds, as Kil et al. (2015) and Kim et al. (2015) have reported the seed transmission of TYLCV and Sweet potato leaf curly virus (SPLCV) respectively. Seed transmissible virus might be an initial inoculum source for the vector mediated transmission, which led to virus dissemination over a long range and sustained in seeds.

Begomoviruses being most devastating and emerging plant viruses are infecting a wide range of economically important vegetables, cash crops, ornamental plants and cereal crops (Sanz et al., 2000; Pasumarthy et al., 2010; Khatri et al., 2014; Zaidi et al., 2016). Their diversity is due to new species emergence through multiple infections, recombinations, mutations and pseudorecombination which is continuously increasing (Inoue-Nagata et al., 2006; Amrao et al., 2010; Singh et al., 2012).

In present study, resistance conferred by various Ty-genes pyramided in different combinations was evaluated which indicated the importance of Ty resistant cultivars against ever increasing and evolving begomoviruses species. This study showed that different combinations exhibited different resistance and susceptibility responses. Present 13 Ty resistant cultivars were developed by pyramiding Ty-1, Ty-2, Ty-3 and Ty-5, by AVRDC Taiwan, as other cultivars produced by combining Ty-1, Ty-2, Ty- 3/Ty-3a, ty-5, 'ty6' genes, were evaluated for effectiveness of pyramidal strategy.

However various recognized Ty genes might not be equally efficient for resistance against all strains or species of begomoviruses. So in order to develop tomato lines

203 suitable for wide range of environments and to attain long lasting, broad spectrum and strong resistance against numerous begomoviruses species/stains, a strategy was adapted i.e. to pyramid various Ty genes in a same tomato variety. Pyramiding previously had also been used for different plant-virus interaction (Werner et al., 2005). Similarly in tomato, pyramiding strategy had been used for multiple disease resistance (Hanson et al., 2016). So single or combinations of resistance genes were required to be assessed for different begomoviruses species/strains, in different areas to determine affectivity of single or pyramidal genes in each location (De Resende et al., 2009).

Present piece of work was the continuation of resistance evaluation against begomoviruses which were found in Pakistan. Ty cultivars were evaluated against ToLCNDV and pyramidal cultivars evidenced for showing highest resistance against bipartite begomoviruses, although Ty resistant cultivars were developed by introgression of Ty resistant genes acquired for resistance against TYLCV.

As ToLCNDV is prevalent in Pakistan infecting not only tomato, pepper (Hussain et al., 2004), bitter gourd (Tahir and Haider, 2005) and recently found associated with cotton (Zaidi et al., 2016). Although in field screening for tomato yellow leaf curl disease under high pressure, Ty resistant lines exhibited enhanced resistance (Hanson et al., 2016).

Current 13 resistant pyramidal cultivars and 01 native susceptible variety (Nagina), showed varied resistance response i.e. from non resistant to very less, to mild, to medium upto complete ones. Among 13 cultivars, 05 cultivars i.e. R6, R10, R11, R5 & R13 were found highly resistant and were absolutely normal phenotypically, healthy and vigorous like uninoculated or pGreen inoculated plants. By Southern hybridization very less or none virus titer was detected in these cultivars. All these cultivars were homozygous resistant for Ty-2 and Ty-3 or Ty-2 and Ty-5, except R11.

Prasanna et al. in 2015a reported the importance of Ty-2 and Ty-3 pyramidal lines against 03 begomoviruses i.e. 01 monopartite ToLCBV and 02 bipartite ToLCNDV and ToLCPalV species. Pyramidal lines or Ty-3 gene carrying resistant lines showed highest resistance against 01 monopartite and 02 bipartite begomoviruses, but Ty-2 resistant lines had limitations for broad spectrum resistance and pyramiding was considered significant due to the overwhelming effects of frequent recombinations.

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Among 13 cultivars, 03 i.e. R9, R2 and R3 cultivars showed moderate resistance while only 01 i.e. R12 showed mild resistance and virus titer in cultivar was found comparable with symptoms severity. Among these, R2 and R3 cultivars were homozygous resistant for Ty-2 and Ty-3 while R9 and R12 were homozygous resistant for only one Ty-2/Ty-3 gene. Cultivar R7, R8, R14, R15 & susceptible Nagina variety were found susceptible to highly susceptible, phenotypically. It was also observed that R7 was homozygous resistant for Ty-2 and Ty-3, R8 & R14 were resistant for only Ty- 2 and R15 was homozygous susceptible for all 04 genes.

Ji et al. in 2009 indicated the variation in symptoms severity, as Ty-3 had a major effect which was responsible for the 60 % of the variance of symptoms severity. Ty-5 gene was responsible for greater than 40 % of the variance of symptoms severity; this indicated that Ty-5 had less effect on resistance (Anbinder et al., 2009).

In current study, we found that among 13 Ty cultivars, R3 and R8 were homozygous resistant for Ty-1 but its contribution for resistance appeared less than Ty-2, Ty-3 and Ty-5.

Barbieri et al. in 2010 reported that among Ty-1, Ty-2 genes carrying cultivars, Ty-2 showed high level of resistance as compared to Ty-1, when were evaluated against TYLCV and TYLCSV. Similarly another study indicated effectiveness of Ty-2 gene carrying resistant cultivar were found highly resistant against 03 monopartite Honey suckel yellow vein mosaic virus (HYVMV), Tobacco leaf curl Japan virus (TbLCJV) and TYLCV (Shahid et al., 2013). Likewise AVRDC recognized Ty-2 affective against TYLCTWV but showed less resistance against TYLCTHV. Verlaan et al in 2013 reported that pyramidal lines response to natural infection indicated that ToLCD severity was higher in Ty-1 and Ty-2 carrying lines. Ty-1 lines exhibited more disease severity and incidence in fields as mixed infections were found common to natural conditions.

Results indicated that different Ty cultivars were specified by particular combinations of pyramided Ty genes which responded differently. Most resistant cultivars showed pyramidal combinations containing Ty-2 (homozygous resistance gene) along with any Ty-3 or Ty-5 homozygous resistance genes which showed complete resistance in 04 cultivars (R6, R5, R10 & R13) or showed moderate resistance (exhibited mild symptoms) in 03 cultivars i.e. R2, R3 and R9. However exceptions were still there as

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R11 was symptomatically highly resistant but here Ty genes combination was different than other highly resistant and moderate resistant cultivars. Conclusively these results interpreted that pyramidal lines homozygous for Ty-2, Ty-3 and Ty-5 might confer better resistance in high disease pressure. Cultivars those were found homozygous resistant, for only one out of Ty-1/Ty-3, Ty-2 & Ty-3 genes, were found mild resistant or nonresistant.

Study conducted on Ty lines developed by pyramiding Ty-2 and Ty-3 genes and observing pyramidal affect was found affective for ToLCNDV along with ToLCPalV bipartite begomoviruses (Prasanna et al., 2015a; Shahid et al., 2013).

In current work, among high resistant and moderate resistance exhibiting cultivars, presence of Ty-5 gene aggravated the effect of resistance. Among present cultivars, which were homozygous resistant for two or more genes showed better resistance than those cultivars which were resistant for only one gene or none.

In study herein, southern hybridization results indicated less or very less virus, even beyond detectable level in resistant lines. For susceptible cultivars a positive correlation between virus accumulation level and disease symptoms severity was found.

Barbieri et al. (2010) described that Ty-1 showed resistance to TYLCV and TYLCSV with less amounts of virus, similarly Ty-2 was found good resistant to TYLCV, less tolerant to TYLCSV and virus amount.

During present piece of work, Ty cultivars evaluated for resistance were also compared for the presence of metabolites in resistant and susceptible cultivars by GCMS.

Ibrahim et al. (2011a) performed GCMS analysis of tomato to discriminate healthy tomato from inoculated by pathogenic bacteria. Another similar study was conducted by Hussaini et al. in 2011 to compare the composition of healthy and fungi inoculated tomato fruit. Various other studies were conducted to determine the pericarp composition of transgenic tomato plants (Roessner-Tunali et al., 2003), to find metabolic diversity of different tomato species (Schauer et al., 2005). Carrari and Fernie in 2006 and Oms-Oliu et al. in 2011 determined the metabolite changes

206 occurred during fruit development and ripening. Vogg et al. in 2004 compared the composition of cuticular wax of mutant (lecer6) and wild-type tomato by GCMS.

A large number of volatile compounds, mainly aliphatic hydrocarbons, alcohols, fatty acids, esters, aldehydes, ketones, diterpenes, triterpenes and phenol were found in n- hexane extracts of present samples, by GCMS during the present study. This is however, the first report as to best of our knowledge, of metabolite profiling of Ty- resistant cultivars after ToLCNDV inoculation.

Metabolite composition of resistant cultivars i.e. R6, R5, R10, R11 & R13 indicated the presence of various compounds, however major constituents were hexadecane, 2,6,11,15-tetramethyl, tritetracontane, tetratriacontane, octacosane, dodecane, 2- bromo dodecane, 9-methylnonadecane, pentadecane, 8-hexyl, isopropyl palmitate, silane, trichlorooctadecyl, oxirane, tetradecyl, phytol, tridecanol, 2-ethyl-2-methyl, 9- eicosene, nonahexacontanoic acid and tetracontane, 1-hentetracontanol, octadecane, 1-chloro, 10-methylnonadecane, Tridecane, tetradecane, hexadecane, heptadecane, 2,6,10,15-tetramethyl, octadecane, 1-(+)-ascorbic acid 2,6-dihexadecanoate, nonadecane, eicosane, heneicosane, 2,6,10 trimethyl-dodecane, 1,2- benzenedicarboxylic acid and diisooctyl ester.

Work of Kokate Ck (2008) showed that these compounds had previously been isolated from plants which contained antimicrobial activities and had contributed in plant defense mechanism. Such compounds showed resistance in plants against microbes or pests, as root exudates of resistance tomato cultivars were known to produce increased amount of these compounds against Meloidogyne incognita (Yang et al., 2016). Similarly 2, 6, 10-trimethyldodecane (farnesane) terpenoid pathway- related parent compound, produced in infected leaves (Choi et al., 2008) was also found associated in present samples. 1, 2-benzenedicarboxylic acid, diisooctyl ester is an aromatic dicarboxylic acid contained antimicrobial activity (Igwe et al., 2016, Rajeswari et al., 2013), found in stem extract of Nothapodytes nimmoniana (Kavitha et al., 2015). Torbati et al (2016) found that 4, 6-Dimethyl-dodecane contain radical scavenging activity. Hentetracontanol was found previously associated with resistance, found in resistant cultivars (Yuanpeng et al., 2009).

Phenol, 3,5-bis (1,1-dimethylethyl) was found in all ToLCNDV, inoculated Ty cultivars. It may be associated with plant defense as reported by many workers.

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Boudet in (2007) stated that phenolics, being one among most ubiquitous secondary metaboliteʼs group found in the whole plant kingdom. Among these, phenols are the simplest phenolic compounds. This group has an antimicrobial effect, served against plants pathogens and function as plant defense mechanism (Das et al., 2010; Gurjar et al., 2012). Various external and internal factors such as wounding, pathogens attack and drought etc. influenced accumulation and synthesis of phenolics (Zaprometov, 1989; Kefeli et al., 2003). Biotic and abiotic stresses resultant to produce reactive oxygen species (ROS) to control pathogen defense, programmed cell death as well as abiotic stress response but oxidative stress occurred due to an imbalance in ROS production and antioxidant defense. This condition is overcome by antioxidants including ascorbic acid, phenolic compounds and flavonoids based impressive defense mechanism (Yadav et al., 2014). Phenols played a significant role in the reduction of ROS, hydroxide radicals, superoxide anions and singlet oxygen, ultimately stimulated defensive enzyme activation (Maffei et al., 2007; Adeosun et al., 2013).

During present study various other compounds were also detected, i.e. dodecane, pentatriacontane, hexatriacontane, tetrapentacontane, 1,54-dibromo, tetratetracontane and unique compounds were 2,9-dimethylundecane, decane, 1-iodo-, Tridecane, 4- methyl, 2,3,6,7-tetramethyl-octane, heptane, 2,5,5-trimethyl- anthracene, 9- dodecyltetradecahydro-, oxirane, tetradecyl-, silane, trichlorooctadecyl, dodecane, 1- chloro, 2,4-dimethyl undecane, undecane, 3,9-dimethyl, benzene, (1,3,3-trimethyl nonyl), trans, cis-2,6-nonadien-1-ol, 1-octanol, 2-butyl, 9-eicosene, 2-decene, 5- methyl-, 5-ethylundecane, 1-decanol, 2,2-dimethyl- and 1-hentetracontanol. These are reported for the enhancement of resistance.

However, in another study metabolites changes produced in leaves and phloem sap due to mulberry yellow dwarf disease by Phytoplasma, were investigated by Gai et al. (2014), showed different metabolite profile which had changed in quantity of various compounds.

Compounds such as octane; 2,6-dimethyl, 1-isopropyl-2, 3-dimethylcyclopentane and phytol were also found associated with resistant cultivars.

Octane; 2,6-dimethyl is a linear monoterpenoid, mainly found in plants (Connolly and Hill, 1991). It was also isolated from Olea europaea for its radical scavenging activity

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(Haloui et al., 2010; Dvořáková et al., 2011). Huang et al. (2009) had studied that octane, 2, 6-dimethyl as volatiles produced in maize induced by Ostrinia furnacalis (Asian corn borer). Similarly, phytol a linear diterpenoid alcohol was found to show antimicrobial activity (Zito et al., 2010; Maziar Bahreini, 2015). Likewise, methanolic extract of Petiveria alliaceae containg high insecticidal effect were found rich in phytol (Cruz-Estrada et al., 2013).

As mentioned earlier R2, R3 and R9 were found moderate resistant, R12 mild resistant; R7, R8, R14, R15 and Nagina were non-resistant. Their GCMS analysis indicated the identification of following major constituents, tetratetracontane, tetrapentacontane, tetrapentacontane, 1,54-dibromo, 2,6,10 trimethyl-dodecane, tridecane, dodecane, pentadecane, dodecane, 4,6-dimethyl, octacosane, nonadecane, phenol, 3,5-bis(1,1-dimethylethyl), 2,6,11 trimethyl-dodecane, isopropyl palmitic acid, tetracontane, pentatriacontane, 1-chloroheptacosane, phytol, nonahexacontanoic acid, hexatriacontane, 1,2-benzenedicarboxylic acid, diisooctyl ester, hexadecane, tridecanol, 2-ethyl-2-methyl, tetratriacontane, pentadecane, 8-hexyl, decane, 3,7- dimethyl, 9-methylnonadecane, 11-decyltetracosane, p- xylene, 2,6-dimethyldecane, 2-bromo dodecane, heptadecane, 2-isopropyl-5-methyl-1-hexanol, disulfide, di-tert- dodecyl, octadecane, 2-methyl, hexadecane, 2,6,11,15-tetramethyl, octadecane, isopropyl palmitate, dodecane, 6-methyl, tetracosane, 11-decyl, dodecane, 2-methyl and octadecane, 5, 14-dibutyl. This data showed the role of the mentioned compounds in enhancement of resistance in plants.

Studies conducted by Mohamed et al. (2014) showed that tetratetracontane and hentriacontane compounds were associated with antimicrobial and antioxidant activities. Isopropyl palmitate a fatty acid ester was isolated as prevailing component in Citrus sinensis (Esquivel-Ferriño et al., 2014) and Randia spinosa known for various biological activities (Mahabaleshwara et al., 2016). Tetradecane emitted from soybean which was infected by viruses containing aphids influenced its resistance (Zhu and Park, 2005). Many alkanes including hentriacontane, hexatriacontane nonacosane, octacosane along with other compounds were found present in leaf extract of transgenic tobacco (Choi et al., 2012), to protect plants from pests (Wagner et al., 2004). Pentatriacontane and hentriacontane also contained antioxidative activity (Sivakumar and Gayathri, 2015; Elangovan et al., 2015). While, tetrapentacontane,

209 found in leaf extract of Allamanda cathartica and A. oenotheraefolia indicated its antifungal activities (Haron et al., 2013). Similarly work of Reisenman et al. (2013) and Lee et al. (2012) indicated the presence of octadecane, hexadecane, pentadecane, tridecane, decane, undecane and dodecane with other VOCs for resistance [induced systemic resistance (ISR) and systemic resistance (SR)].

We also found 10-Methylnonadecane, 1, 3 dimethylbenzene, 1, 4 dimethyl benzene, undecane, monoterpenes (cymene, limonene), tetradecane, pentadecane, hexadecane acyclic diterpenoid (phytol), squalene, heneicosane, heneicosane, 11-(1-ethylpropyl), tritetracontane, eicosane, hexadecane, 2,6,10,14-tetramethyl, heptadecane, 2,6,10,15- tetramethyl and 1-(+)-ascorbic acid 2,6-dihexadecanoate, nonacosane, nonane, 3- methyl, along with other bioactive compounds.

As previous studies have indicated that essential oil of Trifolium pratenes contained such compounds which showed antioxidant activity (Vlaisavljevic et al., 2014). Squalene which is a triterpene was found having anti microbial activity (Zito et al., 2010) and also possesses antioxidant and antibacterial activity (Rajeswari et al., 2013). Similarly, Achillea gypsicola and A. biebersteinii extract contained main compounds as camphor, 1,8-cineole, heneicosane, eicosane, tricosane and linoleic acid, borneol, piperitone and terpineol were found to have antifungal activity (Kordali et al., 2009; Castillo et al., 2012). Extract of Azadirachta indica was rich in nonadecane, 2,6,10,14-tetramethylheptadecane, nonacosane, ascorbic acid along with other compounds showed antioxidant activity (Hossain et al., 2013), and Nonane, 3- methyl contained larvicidal activity (Maziar Bahreini, 2015).

Analysis of R2, R3, R9, R12 R7, R8, R14, R15 & Nagina cultivarsʼs extracts also showed the presence of many important compounds such as cyclohexane, 1-methyl-2- propyl, cyclohexane, butyl, dodecane, 3-cyclohexyl-, nonacosane, heptane, 2,2- dimethy-, cycloheptane, bromo, 1-decanol, 2,2, dimethyl, 4-Nonene,5-methyl, 1,2,3- trimethylbenzene, octane, 3,3-dimethyl-, 3-methylene-1,7-octadiene, 2-propyl-1- pentanol, 3-hexanone, 2,4-dimethyl, 2-bromo-nonane, octadecane, 2-methyl, heptane, 3-methyl, 4,4-dimethylcyclooctene, nonane, 3,7-dimethyl-, hexadecanal, pentane, 2,2,3,3-tetramethyl-, 6-methyl-1-octanol, dodecane, 2-cyclohexy l,5,6- dipropyldecane, 1-nonanol, 4,8-dimethyl-, 7-tetradecene, decane, 2,3,4-trimethyl, 5- ethyl-2-methylheptane, tridecane, 6-methyl, 6-benzoylhexanoic acid, squalene,

210 octadecane,6-methyl, dodecane, 6-methyl, 2,7,10-trimethyl dodecane, eicosane, 10- methyl, benzene, 1,4-dimethyl, dodecane, 2-methyl- and docosanoic acid, docosyl ester.

Previous studies indicated the association of benzene, 1,2,3-trimethyl as unique compound in spoilt African horned cucumber, while lemonene, hexadecane and heptadecane produced in tomato fruit by 03 bacterial and 03 fungi strains inoculation (Ibrahim et al., 2011b). Six-methyl-1-octanol also contained larvicidal activity (Das et al., 2015). Decane was found associated with Aspergillus flavus spoilt tomatoes (Hussaini et al., 2011). Five-ethyl-2-methylheptane was studied by Okhale et al. (2016) in leaf essential oils of Garcinia kola leaf which showed antimicrobial activity. Nonane 3, 7-dimethyl also contained antimicrobial activity (Syed and Geary 2013). Three-hexanone, 2, 4-dimethyl was also used as biological control of plant diseases (Bukvicki et al., 2012).

Chamarthi et al in 2011 reported that 4, 4-dimethyl cyclooctene caused resistance to shoot fly. Hexadecanal was studied in maize seedlings after Spodoptera littoralis exposure was found to influence resistance (Erb et al., 2011). Octane, 3, 3-dimethyl and 2-bromo dodecane contained nematocidal ability (Yang et al., 2015). Methyldecane -3, 1-Decanol, 2, 2, dimethyl, cycloheptane, bromo, also showed antimicrobial activity (De Lacy Costello et al., 1999; Singh et al., 2015; Jenecius et al., 2012). Essential oil of Alpinia galanga possessing natural insecticidal/repellent ability contained heptane, 2, 2-dimethy (Yan et al., 2014). Two-propyl-1-pentanol was also noted to be present in leaf extract of Salix viminalis with antibacterial activity (Zarger and Khatoon, 2013).

Now ToLCD, (a big constraint for the tomato crop in the Indian subcontinent), has been observed in tomato fields of different areas in Pakistan. Present enhanced ToLCD severity and its incidence led to ~50 to 80 % yield losses particularly in Lahore and Faisalabad, which were ~30 % to 40 % about two decades ago. These incidences indicated the begomovirus complex associated with the disease. Phylogenetic analyses showed maximum similarity of new sequences with Indian viruses indicating spread of these viruses from India to Pakistan. Thirteen Ty resistant cultivars and one native (Nagina) variety were evaluated against ToLCNDV which is

211 prevalent in Pakistan. Resistant and susceptible cultivars were also compared for metabolites profiling.

Conclusions

Recent begomoviruses complex which was responsible for ToLCD severity in Pakistan was consisted of 05 begomoviruses (ToLCV, ToLCKeV, PaLCuV, ToLCNDV and ToLCPalV) 02 betasatellites (PaLCuB and CLCuMuB) and 02 alphasatellites (OLCuA and ToLCPKA) species. Begomoviruses i.e. ToLCV, ToLCKeV and PaLCuV were found for the first time in Pakistan showing maximum similarity with Indian isolates. Our data suggests that the begomovirus diversity might be the result of movement of viruliferous whitefly, movement of infected plant material and trading of tomatoes from India to Pakistan or vice versa. The close proximity of agricultural lands between Pakistan and India where viruliferous whiteflies can move across the border, may have contributed in begomoviruses spread. Evaluation of 13 Ty resistant cultivars, against ToLCNDV exhibited that R5, R6, R10, R11 & R13 showed complete resistance, R12 showed mild, R2, R3 & R9 showed moderate, R7 & R8 showed very less and R14, R15 & Nagina showed no resistance at all. It was concluded from results that pyramiding of two or more Ty genes was found successful. GCMS analysis of Ty cultivars exhibited the presence of various compounds, mainly aliphatic hydrocarbons, alcohols, diterpenes, triterpenes, esters, aldehydes, fatty acids and ketones in resistant, less resistant and non resistant varieties. Majority of metabolites found in resistant varities were absent or least present in susceptible cultivars. Their repeated presence indicated that they were associated with the immunity and resistance of plants.

Future Prospects

 This study may will help scientists and researchers in better understanding of viral diseases and improved breeding of virus resistant transgenic crops.  Resistant Ty cultivars may be evaluated against other begomoviruses which are associated with recent ToLCD complex, in order to selecet cultivars with enhanced immunity.  Compounds which are present in resistant cultivars may further be filtered or narrowed down by their advanced metabolite profiling to find out the

212

exact and precise defensive ingredients, which may be used to increase the immunity of susceptible cultivars.  Legislation may be put forward by the concerned Legal Departments to stop the import of infected plants and their products in the countary.

REFERENCES

213

REFERENCES

Abdel-Farid, I., Jahangir, M., Van Den Hondel, C., Kim, H., Choi, Y. and Verpoorte, R. 2009. Fungal infection-induced metabolites in Brassica rapa. Plant Science, 176 (5): 608 - 615.

Adams, M. J., Lefkowitz, E. J., King, A. M. and Carstens, E. B. 2014. Recently agreed changes to the statutes of the International Committee on Taxonomy of Viruses. Archives of Virology, 159 (1): 175.

Adeosun, C. B., Olaseinde, S., Opeifa, A. and Atolani, O. 2013. Essential oil from the stem bark of Cordia sebestena scavenges free radicals. Journal of Acute Medicine, 3 (4): 138 - 141.

Agricorner. 2011. Indian tomatoes flooding pakistan. Retrieved from http://www.agricorner.com/indian-tomatoes-flooding-pakistan/

Agricorner. 2015. Pakistan resumes tomato import from india after one month suspension. Retrieved from http://www.agricorner.com/pakistan-resumes-tomato- import-from-india-after-one-month-suspension/

Agrios, G. N. 1997. Plant pathology, 4th edition, San Diego,California. pp 635.

Akhtar, S., Tahir, M. N., Baloch, G. R., Javaid, S., Khan, A. Q., Amin, I., Briddon, R. W. and Mansoor, S. 2014. Regional changes in the sequence of cotton leaf curl Multan betasatellite. Viruses, 6 (5): 2186 - 2203.

Akhter, A., Qazi, J., Saeed, M. and Mansoor, S. 2009. A severe leaf curl disease on chillies in Pakistan is associated with multiple begomovirus components. Plant disease, 93 (9): 962 - 962.

Alberter, B., Rezaian, M. A. and Jeske, H. 2005. Replicative intermediates of Tomato leaf curl virus and its satellite DNAs. Virology, 331 (2): 441 - 448.

Ali, I., Malik, A. and Mansoor, S. 2010. First report of Tomato leaf curl Palampur virus on bitter gourd in Pakistan. Plant disease, 94 (2): 276 - 276.

Allen, C., Prior, P. and Hayward, A. 2005. Bacterial wilt disease and the Ralstonia solanacearum species complex, ed. American Phytopathological Society (APS Press), St. Paul, MN, USA. pp 528.

214

Amarowicz, R., Pegg, R., Rahimi-Moghaddam, P., Barl, B. and Weil, J. 2004. Free- radical scavenging capacity and antioxidant activity of selected plant species from the Canadian prairies. Food Chemistry, 84 (4): 551 - 562.

Amin, I., Patil, B. L., Briddon, R. W., Mansoor, S. and Fauquet, C. M. 2011a. Comparison of phenotypes produced in response to transient expression of genes encoded by four distinct begomoviruses in Nicotiana benthamiana and their correlation with the levels of developmental mirnas. Virology Journal, 8 (1): 1

Amin, I., Hussain, K., Akbergenov, R., Yadav, J. S., Qazi, J., Mansoor, S., Hohn, T., Fauquet, C. M. and Briddon, R. W. 2011b. Suppressors of RNA silencing encoded by the components of the cotton leaf curl begomovirus-betasatellite complex. Molecular Plant-Microbe Interactions, 24 (8): 973 - 983.

Amin, I., Mansoor, S., Iram, S., Khan, M. A., Hussain, M., Y., Z., Bull, S. E., Briddon, R. W. and Markham, P. G. 2002. Association of a monopartite begomovirus producing subgenomic DNA and a distinct DNA on Croton bonplandianus showing yellow vein symptoms in Pakistan. Plant disease, 86 (4): 444 - 444.

Amir, S. S. and Hyder, S. D. 2015. Pakistan-india trade normalization: A word of caution. Retrieved from http://pbc.org.pk/wp-content/uploads/2015/07/Pakistan-India- Trade-Normalization-A-Word-of-Caution

Amrao, L., Akhter, S., Tahir, M. N., Amin, I., Briddon, R. W. and Mansoor, S. 2010. Cotton leaf curl disease in Sindh province of Pakistan is associated with recombinant begomovirus components. Virus Research, 153 (1): 161 - 165.

Anbinder, I., Reuveni, M., Azari, R., Paran, I., Nahon, S., Shlomo, H., Chen, L., Lapidot, M. and Levin, I. 2009. Molecular dissection of Tomato leaf curl virus resistance in tomato line ty172 derived from Solanum peruvianum. Theoretical and Applied Genetics, 119 (3): 519-530.

Anderson, P. K., Cunningham, A. A., Patel, N. G., Morales, F. J., Epstein, P. R. and Daszak, P. 2004. Emerging infectious diseases of plants: Pathogen pollution, climate change and agrotechnology drivers. Trends in Ecology & Evolution, 19 (10): 535 - 544.

215

Andleeb, S., Amin, I., Bashir, A., Briddon, R. W. and Mansoor, S. 2010. Transient expression of βC1 protein differentially regulates host genes related to stress response, chloroplast and mitochondrial functions. Virology Journal, 7 (1):1.

Andret-Link, P. and Fuchs, M. 2005. Transmission specificity of plant viruses by vectors. Journal of Plant Pathology, 153-165.

Antonious, G. F., Kochhar, T. S. and Simmons, A. M. 2005. Natural products: Seasonal variation in trichome counts and contents in Lycopersicum hirsutum. Journal of Environmental Science and Health Part B, 40 (4): 619 - 631.

Anwar, S. A., Zia, A., Hussain, M. and Kamran, M. 2007. Host suitability of selected plants to Meloidogyne incognita in the Punjab, Pakistan. International Journal of Nematology, 17 (2): 144.

Arguello-Astorga, G., Lopez-Ochoa, L., Kong, L. J., Orozco, B. M., Settlage, S. B. and Hanley-Bowdoin, L. 2004. A novel motif in geminivirus replication proteins interacts with the plant retinoblastoma-related protein. Journal of Virology, 78 (9): 4817 - 4826.

Argüello-Astorga, G. R., Guevara-González, L. R., Herrera-Estrella, L. R. and Rivera-Bustamante, R. F. 1994. Geminivirus replication origins have a group-specific organization of iterative elements: A model for replication. Virology, 203 (1): 90 - 100.

Asero, R. 2013. Tomato allergy: Clinical features and usefulness of current routinely available diagnostic methods. Journal of Investigational Allergology and Clinical Immunology, 23 (1): 37 - 42.

Asero, R., Antonicelli, L., Arena, A., Bommarito, L., Caruso, B., Crivellaro, M., De Carli, M., Della Torre, E., Della Torre, F. and Heffler, E. 2009. Epidemaaito: Features of food allergy in Italian adults attending allergy clinics: A multi‐centre study. Clinical & Experimental Allergy, 39 (4): 547 - 555.

Aune, D., Chan, D. S., Vieira, A. R., Rosenblatt, D. a. N., Vieira, R., Greenwood, D. C. and Norat, T. 2012. Dietary compared with blood concentrations of carotenoids and breast cancer risk: A systematic review and meta-analysis of prospective studies. The American journal of clinical nutrition, ajcn. 034165.

216

Aust, O., Stahl, W., Sies, H., Tronnier, H. and Heinrich, U. 2005. Supplementation with tomato-based products increases lycopene, phytofluene, and phytoene levels in human serum and protects against uv-light-induced erythema. International journal for vitamin and nutrition research, 75 (1): 54 - 60.

Azzam, O., Frazer, J., De La Rosa, D., Beaver, J. S., Ahlquist, P. and Maxwell, D. P. 1994. Whitefly transmission and efficient ssDNA accumulation of bean golden mosaic geminivirus requires functional coat protein. Virology, 204 (1): 289 - 296.

Bagewadi, B., Chen, S., Lal, S. K., Choudhury, N. R. and Mukherjee, S. K. 2004. PCNA interacts with Indian mung bean yellow mosaic virus rep and downregulates rep activity. Journal of Virology, 78 (21): 11890 - 11903.

Bakht, T. and Khan, I. A. 2014. Weed control in tomato (Lycopersicon esculentum mill.) through mulching and herbicides. Pakistan Journal of Botany, 46 (1): 289 - 292.

Balasundram, N., Sundram, K. and Samman, S. 2006. Phenolic compounds in plants and agri-industrial by-products: Antioxidant activity, occurrence, and potential uses. Food Chemistry, 99 (1): 191 - 203.

Baldin, E. L., Vendramim, J. D. and Lourenção, A. L. 2005. Resistance of tomato genotypes to the whitefly Bemisia tabaci (gennadius) biotype b (hemiptera: Aleyrodidae). Neotropical Entomology, 34 (3): 435 - 441.

Baliji, S., Black, M. C., French, R., Stenger, D. C. and Sunter, G. 2004. Spinach curly top virus: A newly described curtovirus species from southwest texas with incongruent gene phylogenies. Phytopathology, 94 (7): 772 - 779.

Barbieri, M., Acciarri, N., Sabatini, E., Sardo, L., Accotto, G. and Pecchioni, N. 2010. Introgression of resistance to two mediterranean virus species causing tomato yellow leaf curl into a valuable traditional tomato variety. Journal of Plant Pathology, 92: 485 - 493.

Basak, J. 2016. Tomato yellow leaf curl virus: A serious threat to tomato plants world wide. Journal of Plant Pathology & Microbiology, 7: 346.

Baulcombe, D. 2002. Viral suppression of systemic silencing. Trends in Microbiology, 10 (7): 306 - 308.

Baulcombe, D. 2004. RNA silencing in plants. Nature, 431 (706): 356 - 363.

217

Becker, N., Rimbaud, L., Chiroleu, F., Reynaud, B., Thébaud, G. and Lett, J.-M. 2015. Rapid accumulation and low degradation: Key parameters of Tomato yellow leaf curl virus persistence in its insect vector bemisia tabaci. Scientific Reports, 5: 17696.

Bedford, I. D., Briddon, R. W., Brown, J. K., Rosell, R. C. and Markham, P. G. 1994. Geminivirus transmission and biological characterisation of Bemisia tabaci (gennadius) biotypes from different geographic regions. Annals of Applied Biology, 125 (2): 311 - 325.

Bednarek, P., Schneider, B., Svatoš, A., Oldham, N. J. and Hahlbrock, K. 2005. Structural complexity, differential response to infection, and tissue specificity of indolic and phenylpropanoid secondary metabolism in arabidopsis roots. Plant Physiology, 138 (2): 1058 - 1070.

Behjatnia, S. a. A., Afsharifar, A. R., Tahan, V., Amid Motlagh, M. H., Eini, O. G., Niazi, A. and Izadpanah, K. 2011. Widespread occurrence and molecular characterization of Wheat dwarf virus in Iran. Australasian Plant Pathology, 40 (1): 12 - 19.

Behjatnia, S. a. A., Dry, I. B. and Rezaian, M. A. 1998. Identification of the replication-associated protein binding domain within the intergenic region of tomato leaf curl geminivirus. Nucleic Acids Research, 26 (4): 925 - 931.

Bellés, J. M., López-Gresa, M. P., Fayos, J., Pallás, V., Rodrigo, I. and Conejero, V. 2008. Induction of cinnamate 4-hydroxylase and phenylpropanoids in virus-infected cucumber and melon plants. Plant Science, 174 (5): 524 - 533.

Bentley, R. 1999. Secondary metabolite biosynthesis: The first century. Critical Reviews in Biotechnology, 19 (1): 1 - 40.

Bernhoft, A. 2010. A brief review on bioactive compounds in plants. Bioactive compounds in plants-benefits and risks for man and animals, 11 - 17.

Bharti, S., Rani, N., Krishnamurthy, B. and Arya, D. S. 2014. Preclinical evidence for the pharmacological actions of naringin: A review. Planta Medica, 80 (6): 437 - 451.

Bhowmik, D., Kumar, K. S., Paswan, S. and Srivastava, S. 2012. Tomato-a natural medicine and its health benefits. Journal of Pharmacognosy and Phytochemistry, 1 (1): 33 - 43.

218

Bird, J., Maramorosch, K., Lauffer, M., Bang, F. and Smith, K. 1978. Viruses and virus diseases associated with whiteflies. Advances in Virus Research, 22: 55 - 110.

Birnboim, H. C. and Doly, J. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Research, 7 (6): 1513 - 1523.

Bisaro, D. M. 2006. Silencing suppression by geminivirus proteins. Virology, 344 (1): 158 - 168.

Bisaro, D. M., Hamilton, W. D. O., Coutts, R. H. A. and Buck, K. W. 1982. Molecular cloning and characterisation of the two components of Tomato golden mosaic virus. Nucleic Acids Research, 10 (16): 4913 - 4922.

Blanca, J., Cañizares, J., Cordero, L., Pascual, L., Diez, M. J. and Nuez, F. 2012. Variation revealed by SNP genotyping and morphology provides insight into the origin of the tomato. PloS one, 7 (10): e48198.

Bleeker, P. M., Diergaarde, P. J., Ament, K., Guerra, J., Weidner, M., Schütz, S., De Both, M. T., Haring, M. A. and Schuurink, R. C. 2009. The role of specific tomato volatiles in tomato-whitefly interaction. Plant Physiology, 151 (2): 925 - 935.

Bleeker, P. M., Diergaarde, P. J., Ament, K., Schütz, S., Johne, B., Dijkink, J., Hiemstra, H., De Gelder, R., De Both, M. T. and Sabelis, M. W. 2011. Tomato- produced 7-epizingiberene and r-curcumene act as repellents to whiteflies. Phytochemistry, 72 (1): 68 - 73.

Boccafogli, A., Vicentini, L., Camerani, A., Cogliati, P., D'ambrosi, A. and Scolozzi, R. 1994. Adverse food reactions in patients with grass pollen allergic respiratory disease. Annals of allergy, 73 (4): 301 - 308.

Bohs, L. 2005. Major clades in Solanum based on ndhf sequence data. Monographs in Systematic Botany, 104: 27.

Bolton‐Smith, C., Mcmurdo, M. E., Paterson, C. R., Mole, P. A., Harvey, J. M., Fenton, S. T., Prynne, C. J., Mishra, G. D. and Shearer, M. J. 2007. Two‐year randomized controlled trial of vitamin k1 (phylloquinone) and vitamin d3 plus calcium on the bone health of older women. Journal of Bone and Mineral Research, 22 (4): 509 - 519.

Bolton, M. D. 2009. Primary metabolism and plant defense-fuel for the fire. Molecular Plant-Microbe Interactions, 22 (5): 487 - 497.

219

Bosco, D., Mason, G. and Accotto, G. P. 2004. Tylcsv DNA, but not infectivity, can be transovarially inherited by the progeny of the whitefly vector bemisia tabaci (gennadius). Virology, 323 (2): 276 - 283.

Bottcher, B., Unseld, S., Ceulemans, H., Russell, R. B. and Jeske, H. 2004. Geminate structures of African cassava mosaic virus. Journal of Virology, 78 (13): 6758-6765.

Boudet, A.-M. 2007. Evolution and current status of research in phenolic compounds. Phytochemistry, 68 (22): 2722 - 2735.

Boulton, M. I. 2002. Functions and interactions of mastrevirus gene products. Physiological and Molecular Plant Pathology, 60 (5): 243 - 255.

Boulton, M. I., Buchholz, W. G., Marks, M. S., Markham, P. G. and Davies, J. W. 1989. Specificity of agrobacterium-mediated delivery of Maize streak virus DNA to members of the Gramineae. Plant Molecular Biology, 12 (1): 31 - 40.

Boulton, M. I., Pallaghy, C. K., Chatan, M., Macfarlane, S. and Davies, J. W. 1993. Replication of Maize streak virus mutants in maize protoplasts: Evidence for a movement protein. Virology, 192 (1): 85 - 93.

Boulton, M. I., Steinkellner, H., Donson, J., Markham, P. G., King, D. I. and Davies, J. W. 1989. Mutational analysis of the virion-sense genes of Maize streak virus. Journal of General Virology, 70 (9): 2309 - 2323.

Bourgaud, F., Gravot, A., Milesi, S. and Gontier, E. 2001. Production of plant secondary metabolites: A historical perspective. Plant Science, 161 (5): 839 - 851.

Boykin, L. M. and De Barro, P. J. 2014. A practical guide to identifying members of the Bemisia tabaci species complex: And other morphologically identical species. Frontiers in Ecology and Evolution, 2: 45.

Briddon, R. W., Bull, S. E., Amin, I., Mansoor, S., Bedford, I. D., Dhawan, P., Rishi, N., Siwatch, S. S., Abdel-Salam, A. M. and Markham, P. G. 2003. Diversity of DNA β; a satellite molecule associated with some monopartite begomoviruses. Virology, 312 (1): 106 - 121.

Briddon, R. W., Bull, S. E., Amin, I., Mansoor, S., Bedford, I. D., Rishi, N., Siwatch, S. S., Zafar, Y., Abdel-Salam, A. M. and Markham, P. G. 2004. Diversity of DNA 1: A satellite-like molecule associated with monopartite begomovirus-DNA beta complexes. Virology, 324 (2): 462 - 474.

220

Briddon, R. and Mansoor, S. 2008. Beta ssDNA satellites. In Encyclopedia of Virology; Mahy, B.W.J., Van Regenmortel, M.H.V., Eds. Academic Press: Oxford, UK. pp 31-321

Briddon, R. and Markham, P. 2001. Complementation of bipartite begomovirus movement functions by Topocuviruses and Curtoviruses. Archives of Virology, 146 (9): 1811 - 1819.

Briddon, R. and Stanley, J. 2006. Subviral agents associated with plant single- stranded DNA viruses. Virology, 344 (1): 198 - 210.

Briddon, R. W. and Stanley, J. 2008. Geminiviridae. In "Encyclopedia of Life Sciences (ELS)", 1–DOI: 10.1002/9780470015902.a0000750.pub2. John Wiley & Sons, Ltd: Chichester.

Briddon, R. W., Bedford, I. D., Tsai, J. H. and Markham, P. G. 1996. Analysis of the nucleotide sequence of the treehopper-transmitted geminivirus, tomato pseudo-curly top virus, suggests a recombinant origin. Virology, 219 (2): 387 - 394.

Briddon, R. W., Brown, J. K., Moriones, E., Stanley, J., Zerbini, M., Zhou, X. and Fauquet, C. M. 2008. Recommendations for the classification and nomenclature of the DNA-β satellites of begomoviruses. Archives of Virology, 153 (4): 763 - 781.

Briddon, R. W., Heydarnejad, J., Khosrowfar, F., Massumi, H., Martin, D. P. and Varsani, A. 2010. Turnip curly top virus, a highly divergent geminivirus infecting turnip in iran. Virus Research, 152 (1): 169-175.

Briddon, R. W., Liu, S., Pinner, M. S. and Markham, P. G. 1998. Infectivity of African cassava mosaic virus clones to cassava by biolistic inoculation. Archives of Virology, 143 (12): 2487 - 2492.

Briddon, R. W., Mansoor, S., Bedford, I. D., Pinner, M. S., Saunders, K., Stanley, J., Zafar, Y., Malik, K. A. and Markham, P. G. 2001. Identification of DNA components required for induction of cotton leaf curl disease. Virology, 285 (2): 234 - 243.

Briddon, R. W. and Markham, P. G. 2000. Cotton leaf curl virus disease. Virus Research, 71 (1): 151 - 159.

Briddon, R. W., Pinner, M. S., Stanley, J. and Markham, P. G. 1990. Geminivirus coat protein replacement alters insect specificity. Virology, 177 (1): 85 - 94.

221

Briddon, R. W., Watts, J., Markham, P. G. and Stanley, J. 1989. The coat protein of Beet curly top virus is essential for infectivity. Virology, 172 (2): 628 - 633.

Brown, J., Fauquet, C., Briddon, R., Zerbini, M., Moriones, E., Navas-Castillo, J., King, A., Adams, M., Carstens, E. and Lefkowitz, E. 2012. Virus taxonomy: Ninth report of the international committee on taxonomy of viruses. Associated Press, Elsevier Inc, London. pp 351 - 373.

Brown, J., Frohlich, D. and Rosell, R. 1995. The sweetpotato or silverleaf whiteflies: Biotypes of Bemisia tabaci or a species complex? Annual Review of Entomology, 40 (1): 511 - 534.

Brown, J. and Idris, A. 2006. Introduction of the exotic monopartite Tomato yellow leaf curl virus into west Coast Mexico. Plant disease, 90 (10): 1360 - 1360.

Brown, J., Idris, A., Torres-Jerez, I., Banks, G. and Wyatt, S. 2001. The core region of the coat protein gene is highly useful for establishing the provisional identification and classification of begomoviruses. Archieve of Virology, 146 (8): 1581 - 1598.

Brown, J. K. 2007. The Bemisia tabaci complex: Genetic and phenol-typic variation and relevance to tylcv-vector interactions. Tomato yellow leaf curl virus disease.Management, molecular biology, breeding for resistance. Henryk Czosnek (ed), Springer, dordrecht. pp 25-56

Brown, J. K. and Bird, J. 1992. Whitefly-transmitted geminiviruses and associated disorders in the Americas and the Caribbean Basin. Plant disease, 76 (3): 220-225.

Brown, J. K. and Czosnek, H. 2002. Whitefly transmission of plant virues. In R. T.Plumb and J. A. Callow (eds), advances in botanical research. New york:Academic press, pp. 65–100.

Brown, J. K., Fauquet, C. M., Briddon, R. W., Zerbini, F. M., Moriones, E. and Navas-Castillo, J. 2012. Geminivirdae. In: King, a.M.Q., adams, m. J., carstens, e. B., lefkowitz, e. J (ed), virus taxonomy-ninth report of the internation committee on taxonomy of viruses. Associated press, elsevier london, waltham, san diego, pp. 351- 373.

Brown, J. K., Navas-Castillo, J., Moriones, E., Ramos-Sobrinho, R., Silva, J. C. F., Fiallo-Olive, E., Briddon, R. W., Herna´Ndez-Zepeda, C., Idris, A., Malathi, V. G., Martin, D. P., Rivera-Bustamante, R., Ueda, S. and Varsani, A. 2015. Revision of

222 begomovirus taxonomy based on pairwise sequence comparisons. Archives of Virology, 160 (6): 1593 - 1619.

Buchmann, R., Asad, S., Wolf, J., Mohannath, G. and Bisaro, D. 2009. Geminivirus AL2 and L2 proteins suppress transcriptional gene silencing and cause genome-wide reductions in cytosine methylation. Journal of Virology, 83 (10): 5005 - 5013.

Bügel, S. 2003. Vitamin k and bone health. Proceedings of the Nutrition Society, 62 (4): 839 - 843.

Bukvicki, D., Gottardi, D., Veljic, M., Marin, P. D., Vannini, L. and Guerzoni, M. E. 2012. Identification of volatile components of liverwort (Porella cordaeana) extracts using gc/ms-spme and their antimicrobial activity. Molecules, 17 (6): 6982 - 6995.

Bull, S., Briddon, R. and Markham, P. 2003. Universal primers for the PCR-mediated amplification of DNA1: A satellite-like molecule associated with begomovirus-DNA beta complexes. Molecular Biotechnology, 23 (1): 83 - 86.

Bull, S. E., Briddon, R. W., Sserubombwe, W. S., Ngugi, K., Markham, P. G. and Stanley, J. 2007. Infectivity, pseudorecombination and mutagenesis of kenyan cassava mosaic begomoviruses. Journal of General Virology, 88 (5): 1624 - 1633.

Bull, S. E., Tsai, W.-S., Briddon, R. W., Markham, P. G., Stanley, J. and Green, S. K. satellites of non-malvaceous plants in east and south east asia. Archives of Virology, 149 (6): 1193 - 1200.

Butterbach, P., Verlaan, M. G., Dullemans, A., Lohuis, D., Visser, R. G., Bai, Y. and Kormelink, R. 2014. Tomato yellow leaf curl virus resistance by ty-1 involves increased cytosine methylation of viral genomes and is compromised by Cucumber mosaic virus infection. Proceedings of the National Academy of Sciences, 111 (35): 12942 - 12947.

Byrne, D. N. 1999. Migration and dispersal by the sweet potato whitefly, Bemisia tabaci. Agricultural and Forest Meteorology, 97 (4): 309 - 316.

Byrne, D. N., Rathman, R. J., Orum, T. V. and Palumbo, J. C. 1996. Localized migration and dispersal by the sweet potato whitefly, Bemisia tabaci. Oecologia (Historical Archive), 105 (3): 320 - 328.

Campos-Olivas, R., Louis, J. M., Clerot, D., Gronenborn, B. and Gronenborn, A. M. 2002a. The structure of a replication initiator unites diverse aspects of nucleic acid

223 metabolism. Procedings of National Academy of Sciences USA, 99 (16): 10310 - 10315.

Campos-Olivas, R., Louis, J. M., Clérot, D., Gronenborn, B. and Gronenborn, A. M. 2002b. 1h, 13c, and 15n assignment of the n-terminal, catalytic domain of the replication initiation protein from the geminivirus TYLCV. Journal of Biomolecular NMR, 24 (1): 73 - 74.

Caro, M., Verlaan, M. G., Julián, O., Finkers, R., Wolters, A.-M. A., Hutton, S. F., Scott, J. W., Kormelink, R., Visser, R. G. and Díez, M. J. 2015. Assessing the genetic variation of Ty-1 and Ty-3 alleles conferring resistance to Tomato yellow leaf curl virus in a broad tomato germplasm. Molecular Breeding, 35 (6): 1 - 13.

Carrari, F., Baxter, C., Usadel, B., Urbanczyk-Wochniak, E., Zanor, M.-I., Nunes- Nesi, A., Nikiforova, V., Centero, D., Ratzka, A. and Pauly, M. 2006. Integrated analysis of metabolite and transcript levels reveals the metabolic shifts that underlie tomato fruit development and highlight regulatory aspects of metabolic network behavior. Plant Physiology, 142 (4): 1380 - 1396.

Carrari, F. and Fernie, A. R. 2006. Metabolic regulation underlying tomato fruit development. Journal of Experimental Botany, 57 (9): 1883 - 1897.

Carvalho, C. M., Machado, J. P. B., Zerbini, F. M. and Fontes, E. P. 2008. NSP- interacting GTPase: A cytosolic protein as cofactor for nuclear shuttle proteins. Plant signaling & behavior, 3 (9): 752 - 754.

Castillo, A. G., Collinet, D., Deret, S., Kashoggi, A. and Bejarano, E. R. 2003. Dual interaction of plant PCNA with geminivirus replication accessory protein (Ren) and viral replication protein (Rep). Virology, 312 (2): 381 - 394.

Castillo, A. G., Kong, L. J., Hanley-Bowdoin, L. and Bejarano, E. R. 2004. Interaction between a geminivirus replication protein and the plant sumoylation system. Journal of Virology, 78 (6): 2758 - 2769.

Castillo, F., Aguilar, C., Hernández, D., Gallegos, G. and Rodríguez, R. 2012. Antifungal properties of bioactive compounds from plants, ed. INTECH Open Access Publisher. pp. 81 - 106.

224

Chakraborty, S., Pandey, P. K. and Banerjee, M. K. 2003. A new begomovirus species causing tomato leaf curl disease in Varanasi , India. Plant disease, 87 (3): 313 - 313.

Chakraborty, S., Pandey, P. K., Banerjee, M. K., Kalloo, G. and Fauquet, C. M. 2003. Tomato leaf curl Gujarat virus, a new begomovirus species causing a severe leaf curl disease of tomato in Varanasi, India. Phytopathology, 93 (12): 1485 - 1495.

Chamarthi, S. K., Sharma, H. C., Vijay, P. M. and Narasu, M. L. 2011. Leaf surface chemistry of sorghum seedlings influencing expression of resistance to sorghum shoot fly, Atherigona soccata. Journal of Plant Biochemistry and Biotechnology, 20 (2): 211 - 216.

Chandran, S. A., Jeyabharathy, C. and Usha, R. 2013. The C2 protein of Bhendi yellow vein mosaic virus plays an important role in symptom determination and virus replication. Virus Genes, 48 (1): 203 - 207.

Chase, M. W. and Reveal, J. L. 2009. A phylogenetic classification of the land plants to accompany APG III. Botanical Journal of the Linnean Society, 161 (2): 122 - 127.

Chase, M. W., Soltis, D. E., Olmstead, R. G., Morgan, D., Les, D. H., Mishler, B. D., Duvall, M. R., Price, R. A., Hills, H. G. and Qiu, Y. L. 1993. Phylogenetics of seed plants: An analysis of nucleotide sequences from the plastid gene rbcl. Annals of the Missouri Botanical Garden, 80: 528 - 580.

Chellappan, P., Vanitharani, R. and Fauquet, C. M. 2005. MicroRNA-binding viral protein interferes with Arabidopsis development. Proceedings of the National Academy of Sciences of the United States of America, 102 (29): 10381 - 10386.

Chen, J., Song, Y. and Zhang, L. 2013. Lycopene/tomato consumption and the risk of prostate cancer: A systematic review and meta-analysis of prospective studies. Journal of Nutritional Science and Vitaminology, 59 (3): 213 - 223.

Cheng, X., Wang, X., Wu, J., Briddon, R. W. and Zhou, X. 2011. ΒC1 encoded by tomato yellow leaf curl China betasatellite forms multimeric complexes In vitro and In vivo. Virology, 409 (2): 156 - 162.

Chiang, K. P., Niessen, S., Saghatelian, A. and Cravatt, B. F. 2006. An enzyme that regulates ether lipid signaling pathways in cancer annotated by multidimensional profiling. Chemistry & Biology, 13 (10): 1041 - 1050.

225

Choi, H. W., Lee, B. G., Kim, N. H., Park, Y., Lim, C. W., Song, H. K. and Hwang, B. K. 2008. A role for a menthone reductase in resistance against microbial pathogens in plants. Plant Physiology, 148 (1): 383 - 401.

Choi, Y. E., Lim, S., Kim, H. J., Han, J. Y., Lee, M. H., Yang, Y., Kim, J. A. and Kim, Y. S. 2012. Tobacco NtLTP1, a glandular‐specific lipid transfer protein, is required for lipid secretion from glandular trichomes. The Plant Journal, 70 (3): 480 - 491.

Choudhary, D. K., Johri, B. N. and Prakash, A. 2008. Volatiles as priming agents that initiate plant growth and defence responses. CURRENT SCIENCE-BANGALORE-, 94 (5): 595.

Choudhury, N. R., Malik, P. S., Singh, D. K., Islam, M. N., Kaliappan, K. and Mukherjee, S. K. 2006. The oligomeric rep protein of Mungbean yellow mosaic india virus (MYMIV) is a likely replicative helicase. Nucleic Acids Research, 34 (21): 6362 - 6377.

Chowda-Reddy, R. V., Achenjang, F., Felton, C., Etarock, M. T., Anangfac, M. T., Nugent, P. and Fondong, V. N. 2008. Role of a geminivirus AV2 protein putative protein kinase c motif on subcellular localization and pathogenicity. Virus Research, 135 (1): 115 - 124.

Chowda Reddy, R. V., Colvin, J., Muniyappa, V. and Seal, S. 2005. Diversity and distribution of begomoviruses infecting tomato in India. Archives of Virology, 150 (5): 845 - 867.

Claye, S. S., Idouraine, A. and Weber, C. W. 1996. Extraction and fractionation of insoluble fiber from five fiber sources. Food Chemistry, 57 (2): 305 - 310.

Clerot, D. and Bernardi, F. 2006. DNA helicase activity is associated with the replication initiator protein Rep of tomato yellow leaf curl geminivirus. Journal of Virology, 80 (22): 11322 - 11330.

Clinton, S. K. 1998. Lycopene: Chemistry, biology, and implications for human health and disease. Nutrition Reviews, 56 (2): 35 - 51.

Cohen, S. and Lapidot, M. 2007. Appearance and expansion of TYLCV: A historical point of view. Tomato yellow leaf curl virus disease, Springer Netherland. pp. 3 - 12.

226

Cohen, S. N., Chang, A. C. Y. and Hsu, C. L. 1972. Nonchromosomal antibiotic resistance in bacteria: Genetic transformation of Escherichia coli by r-factor DNA. Proceedings of the National Academy of Sciences, 69 (8): 2110 - 2114.

Cohen, S. and Antignus, Y. 1994. Tomato yellow leaf curl virus, a whitefly-borne geminivirus of tomatoes. In Advances in disease vector research, Springer New York. pp. 259 - 288.

Collin, S., Fernández-Lobato, M., Gooding, P. S., Mullineaux, P. M. and Fenoll, C. 1996. The two nonstructural proteins from Wheat dwarf virus involved in viral gene expression and replication are retinoblastoma-binding proteins. Virology, 219 (1): 324 - 329.

Collmer, C. W. and Howell, S. H. 1992. Role of satellite RNA in the expression of symptoms caused by plant viruses. Annual Review of Phytopathology, 30 (1): 419- 442.

Connolly, J. D. and Hill, R. A. 1991. Dictionary of terpenoids, ed. CRC Press, London, New York, Tokoyo, Melbourne Madras. pp. 21 - 56.

Crozier, A., Jaganath, I. B. and Clifford, M. N. 2006. Phenols, polyphenols and tannins: An overview. Plant secondary metabolites: Occurrence, structure and role in the human diet. John Wiley and Sons. pp. 1 - 21.

Cruz-Estrada, A., Gamboa-Angulo, M., Bórges-Argáez, R. and Ruiz-Sanchez, E. 2013. Insecticidal effects of plant extracts on immature whitefly Bemisia tabaci genn.(hemiptera: Aleyroideae). Electronic Journal of Biotechnology, 16 (1): 6 - 6.

Cui, X., Li, G., Wang, D., Hu, D. and Zhou, X. 2005. A begomovirus DNA β- encoded protein binds DNA, functions as a suppressor of RNA silencing, and targets the cell nucleus. Journal of Virology, 79 (16): 10764 - 10775.

Cui, X., Tao, X., Xie, Y., Fauquet, C. M. and Zhou, X. 2004a. A DNA β associated with Tomato yellow leaf curl china virus is required for symptom induction. Journal of Virology, 78 (24): 13966 - 13974.

Cui, X. F., Xie, Y. and Zhou, X. P. 2004b. Molecular characterization of DNA molecules associated with Tobacco leaf curl Yunnan virus. Journal of Phytopathology, 152 (11-12): 647 - 650.

227

Czosnek, H. 2007. Tomato yellow leaf curl virus disease: Management, molecular biology, breeding for resistance. Ed. Springer Science & Business Media. pp. 241 - 249.

Czosnek, H., Ghanim, M. and Ghanim, M. 2002. The circulative pathway of begomoviruses in the whitefly vector Bemisia tabaci — insights from studies with Tomato yellow leaf curl virus. Annals of Applied Biology, 140 (3): 215-231.

Czosnek, H. and Laterrot, H. 1997. A worldwide survey of tomato yellow leaf curl viruses. Archieve of Virology, 142 1391 - 1406.

D'elia, L., Barba, G., Cappuccio, F. P. and Strazzullo, P. 2011. Potassium intake, stroke, and cardiovascular disease: A meta-analysis of prospective studies. Journal of the American College of Cardiology, 57 (10): 1210 - 1219.

Das, A. K., J, S. K. and P.S, S. 2015. Larvicidal activity and leaf essential oil composition of three species of genus Atalantia from South India. International Journal of Mosquito Research, 2 (3): 25 - 29.

Das, K., Tiwari, R. and Shrivastava, D. 2010. Techniques for evaluation of medicinal plant products as antimicrobial agent: Current methods and future trends. Journal of medicinal plants research, 4 (2): 104 - 111.

Dasgupta, A., Sinha, S. K. and Praveen, S. 2004. Structure of replication initiator protein unites diverse viruses causing tomato leaf curl disease (ToLCD). Plant Science, 166 (4): 1063 - 1067.

Davies, J. W. 1987. Geminivirus genomes. Microbial Sciences, 4 (1): 18 - 23.

Davino, S., Miozzi, L., Panno, S., Rubio, L., Davino, M. and Accotto, G. P. 2012. Recombination profiles between Tomato yellow leaf curl virus and Tomato yellow leaf curl Sardinia virus in laboratory and field conditions: Evolutionary and taxonomic implications. Journal of General Virology, 93 (12): 2712 - 2717.

De Barro, P. J., Driver, F., Trueman, J. W. H. and Curran, J. 2000. Phylogenetic relationships of world populations of bemisia tabaci (gennadius) using ribosomal ITS1. Molecular Phylogenetics and Evolution, 16 (1): 29 - 36.

De Barro, P. J., Hidayat, S. H., Frohlich, D., Subandiyah, S. and Ueda, S. 2008. A virus and its vector, Pepper yellow leaf curl virus and Bemisia tabaci, two new invaders of Indonesia. Biological Invasions, 10 (4): 411 - 433.

228

De Barro, P. J., Liu, S.-S., Boykin, L. M. and Dinsdale, A. B. 2011. Bemisia tabaci: A statement of species status. Annual Review of Entomology, 56: 1 - 19.

De La Peña, R. and Hughes, J. 2007. Improving vegetable productivity in a variable and changing climate. Journal of SAT Agricultural Research, 4 (1): 1 - 22.

De Lacy Costello, B., Evans, P., Ewen, R., Gunson, H., Ratcliffe, N. M. and Spencer- Phillips, P. T. 1999. Identification of volatiles generated by potato tubers (Solanum tuberosum cv: Maris piper) infected by Erwinia carotovora, Bacillus polymyxa and Arthrobacter sp. Plant Pathology, 48 (3): 345 - 351.

De Luca, V. and St Pierre, B. 2000. The cell and developmental biology of alkaloid biosynthesis. Trends in Plant Science, 5 (4): 168 - 173.

De Resende, J. T., Maluf, W. R., Cardoso, M. D. G., Gonçalves, L. D., Faria, M. V. and Do Nascimento, I. R. 2009. Resistance of tomato genotypes to the silverleaf whitefly mediated by acylsugars. Horticultura Brasileira, 27 (3): 345 - 348.

Deng, C., Qian, J., Zhu, W., Yang, X. and Zhang, X. 2005. Rapid determination of methyl salicylate, a plant‐signaling compound, in tomato leaves by direct sample introduction and thermal desorption followed by gc‐ms. Journal of separation science, 28 (11): 1137 - 1142.

Desbiez, C., David, C., Mettouchi, A., Laufs, J. and Gronenborn, B. 1995. Rep protein of tomato yellow leaf curl geminivirus has an atpase activity required for viral replication. Proceedings of the National Academy of Sciences USA, 92 (12): 5640 - 5644.

Desbrosses, G. G., Kopka, J. and Udvardi, M. K. 2005. Lotus japonicus metabolic profiling. Development of gas chromatography-mass spectrometry resources for the study of plant-microbe interactions. Plant Physiology, 137 (4): 1302 - 1318.

Ding, C., Qing, L., Li, Z., Liu, Y., Qian, Y. and Zhou, X. 2009. Genetic determinants of symptoms on viral DNA satellites. Applied and Environmental Microbiology, 75 (16): 5380 - 5389.

Ding, S.-W., Li, H., Lu, R., Li, F. and Li, W.-X. 2004. RNA silencing: A conserved antiviral immunity of plants and animals. Virus Research, 102 (1): 109 - 115.

Dixon, R., Harrison, M. and Lamb, C. 1994. Early events in the activation of plant defense responses. Annual Review of Phytopathology, 32 (1): 479 - 501.

229

Dixon, R. A. 2001. Natural products and plant disease resistance. Nature, 411 (6839): 843 - 847.

Dixon, R. A., Achnine, L., Kota, P., Liu, C. J., Reddy, M. and Wang, L. 2002. The phenylpropanoid pathway and plant defence—a genomics perspective. Molecular Plant Pathology, 3 (5): 371 - 390.

Dogra, S. C., Eini, O., Rezaian, M. A. and Randles, J. W. 2009. A novel shaggy-like kinase interacts with the Tomato leaf curl virus pathogenicity determinant C4 protein. Plant Molecular Biology, 71 (1-2): 25 - 38.

Dominguez, M., Ramos, P. L., Echemendia, A. L., Peral, R., Crespo, J., Andino, V., Pujol, M. and Borroto, C. 2002. Molecular charcaterization of Tobacco leaf rugose virus, a new begomovirus infecting tobacco in cuba. Plant disease, 86 (9): 1050 - 1050.

Doshi, G. M., Nalawade, V. V., Mukadam, A. S., Chaskar, P. K., Zine, S. P., Somani, R. R. and Une, H. D. 2015. Structural elucidation of chemical constituents from Benincasa hispida seeds and Carissa congesta roots by gas chromatography: Mass spectroscopy. Pharmacognosy Research, 7 (3): 282.

Doyle, J. and Doyle, J. 1990. Isolation of plant DNA from fresh tissue. Focus, 12: 13 - 15.

Dry, I. B., Krake, L. R., Rigden, J. E. and Rezaian, M. A. 1997. A novel subviral agent associated with a geminivirus: The first report of a DNA satellite. Procedings of National Academy of Sciences USA, 94 (13): 7088 - 7093.

Duan, Y.-P., Powell, C. A., Purcifull, D. E., Broglio, P. and Hiebert, E. 1997. Phenotypic variation in transgenic tobacco expressing mutated geminivirus movement/pathogenicity (BC1) proteins. Molecular Plant-Microbe Interactions, 10 (9): 1065 - 1074.

Duffus, J. E. 1987. Whitefly transmission of plant viruses. In Current topics in vector research, Springer. pp. 73 - 91.

Dvořáková, M., Valterová, I. and Vaněk, T. 2011. Monoterpenes in plants. Chemicke Listy, 105: 839 - 845.

230

Eagle, P., Orozco, B. and Hanley-Bowdoin, L. 1994. A DNA-sequence required for geminivirus replication also mediates transcriptional regulation. Plant Cell, 6 (8): 1157 - 1170.

Egelkrout, E. M., Mariconti, L., Settlage, S. B., Cella, R., Robertson, D. and Hanley- Bowdoin, L. 2002. Two e2f elements regulate the proliferating cell nuclear antigen promoter differently during leaf development. Plant Cell, 14 (12): 3225 - 3236.

El-Shabrawi, M. H., Kamal, N. M., Elhusseini, M. A., Hussein, L., Abdallah, E. A. A., Ali, Y. Z. A., Azab, A. A., Salama, M. A., Kassab, M. and Krawinkel, M. 2015. Folic acid intake and neural tube defects: Two egyptian centers experience. Medicine, 94 (37): e1395

Elangovan, N. M., Dhanarajan, M. and Elangovan, I. 2015. Evaluation of antibacterial and antifungal activity of Phyllanthus emblica leaf extract. World Journal of Pharmaceutical Research, 4 (3): 1284 - 1298.

Elmer, J. S., Brand, L., Sunter, G., Gardiner, W. E., Bisaro, D. M. and Rogers, S. G. 1988. Genetic analysis of the Tomato golden mosaic virus. Ii. The product of the al1 coding sequence is required for replication. Nucleic Acids Research, 16 (14): 7043 - 7060.

Erb, M., Balmer, D., De Lange, E. S., Von Merey, G., Planchamp, C., Robert, C. A., Roeder, G., Sobhy, I., Zwahlen, C. and Mauch‐Mani, B. 2011. Synergies and trade‐offs between insect and pathogen resistance in maize leaves and roots. Plant, Cell & Environment, 34 (7): 1088 - 1103.

Erdmann, J. B., Shepherd, D. N., Martin, D. P., Varsani, A., Rybicki, E. P. and Jeske, H. 2010. Replicative intermediates of Maize streak virus found during leaf development. Journal of General Virology, 91 (4): 1077 - 1081.

Esau, K. 1977. Virus-like particles in nuclei of phloem cells in spinach leaves infected with the Curly top virus. Journal of Ultrastructure Research, 61 (1): 78 - 88.

Esquivel-Ferriño, P. C., Clemente-Soto, A. F., Ramírez-Cabriales, M. Y., Garza- González, E., Álvarez, L. and Camacho-Corona, M. D. R. 2014. Volatile constituents identified in hexane extract of Citrus sinensis peel and anti-mycobacterial tuberculosis activity of some of its constituents. Journal of the Mexican Chemical Society, 58 (4): 431 - 434.

231

FAOSTAT. 2013. Retrieved from http://www.factfish.com/statistic- country/pakistan/tomatoes,%20yield

Fauquet, C., Briddon, R., Brown, J., Moriones, E., Stanley, J., Zerbini, M. and Zhou, X. 2008. Geminivirus strain demarcation and nomenclature. Archives of Virology, 153 (4): 783 - 821.

Fauquet, C. M., Mayo, M. A., Maniloff, J., Desselberger, U. and Ball, L. A. 2005. Virus taxonomy: Classification and nomenclature ofviruses. Eighth report of the international committee on taxonomyof viruses. Elsevier academic press, san diego, USA. pp. 392 - 396.

Fayos, J., Bellés, J. M., Lopez-Gresa, M. P., Primo, J. and Conejero, V. 2006. Induction of gentisic acid 5-o-β-d-xylopyranoside in tomato and cucumber plants infected by different pathogens. Phytochemistry, 67 (2): 142 - 148.

Fekete, K., Berti, C., Trovato, M., Lohner, S., Dullemeijer, C., Souverein, O. W., Cetin, I. and Decsi, T. 2012. Effect of folate intake on health outcomes in pregnancy: A systematic review and meta-analysis on birth weight, placental weight and length of gestation. Nutrition Journal, 11 (1): 1.

Fernie, A. R., Trethewey, R. N., Krotzky, A. J. and Willmitzer, L. 2004. Metabolite profiling: From diagnostics to systems biology. Nature Reviews Molecular Cell Biology, 5 (9): 763 - 769.

Fiallo-Olivé, E., Martínez-Zubiaur, Y., Moriones, E. and Navas-Castillo, J. 2012. A novel class of DNA satellites associated with new world begomoviruses. Virology, 426 (1): 1 - 6.

Fiehn, O. 2002. Metabolomics–the link between genotypes and phenotypes. Plant Molecular Biology, 48 (1-2): 155 - 171.

Fiehn, O., Kopka, J., Dörmann, P., Altmann, T., Trethewey, R. N. and Willmitzer, L. 2000. Metabolite profiling for plant functional genomics. Nature Biotechnology, 18 (11): 1157 - 1161.

Firdaus, S., Vosman, B., Hidayati, N., Supena, J., Darmo, E., Gf Visser, R. and Van Heusden, A. W. 2013a. The Bemisia tabaci species complex: Additions from different parts of the world. Insect Science, 20 (6): 723 - 733.

232

Firdaus, S., Van Heusden, A. W., Hidayati, N., Supena, E. D. J., Mumm, R., De Vos, R. C., Visser, R. G. and Vosman, B. 2013b. Identification and QTL mapping of whitefly resistance components in Solanum galapagense. Theoretical and Applied Genetics, 126 (6): 1487 - 1501.

Firdaus, S., Van Heusden, A. W., Hidayati, N., Supena, E. D. J., Visser, R. G. and Vosman, B. 2012. Resistance to bemisia tabaci in tomato wild relatives. Euphytica, 187 (1): 31 - 45.

Florentino, L. H., Santos, A. A., Fontenelle, M. R., Pinheiro, G. L., Zerbini, F. M., Baracat-Pereira, M. C. and Fontes, E. P. 2006. A perk-like receptor kinase interacts with the geminivirus nuclear shuttle protein and potentiates viral infection. Journal of Virology, 80 (13): 6648 - 6656.

Fontes, E. P. B., Eagle, P. A., Sipe, P. S., Luckow, V. A. and Hanley-Bowdoin, L. 1994. Interaction between a geminivirus replication protein and origin DNA is essential for viral repliation. Journal of Biological Chemistry, 269 (11): 8459 - 8465.

Foolad, M. R. 2007. Genome mapping and molecular breeding of tomato. International journal of plant genomics, 2007: 64358.

Fourie, H. and Mc Donald, A. 2000. Nematodes. ARCLNR leaflet. Crop ptotection services, 18: 4.

Fradin, E. F. and Thomma, B. P. 2006. Physiology and molecular aspects of verticillium wilt diseases caused by v. Dahliae and v. Albo‐atrum. Molecular Plant Pathology, 7 (2): 71 - 86.

Freitas, J. A., Maluf, W. R., Das Graças Cardoso, M., Gomes, L. A. and Bearzotti, E. 2002. Inheritance of foliar zingiberene contents and their relationship to trichome densities and whitefly resistance in tomatoes. Euphytica, 127 (2): 275 - 287.

Frischmuth, S., Wege, C., Hülser, D. and Jeske, H. 2007. The movement protein BC1 promotes redirection of the nuclear shuttle protein BV1 of abutilon mosaic geminivirus to the plasma membrane in fission yeast. Protoplasma, 230 (1-2): 117 - 123.

Frischmuth, T. and Stanley, J. 1992. Characterization of Beet curly top virus subgenomic DNA localizes sequences required for replication. Virology, 18 (2): 808 - 811.

233

Frischmuth, T. and Stanley, J. 1998. Recombination between viral DNA and the transgenic coat protein gene of African cassava mosaic geminivirus. Journal of General Virology, 79 (5): 1265 - 1271.

Frodin, D. G. 2004. History and concepts of big plant genera. Taxon, 53 (3): 753 - 776.

Frohlich, D., Torres‐Jerez, I., Bedford, I., Markham, P. and Brown, J. 1999. A phylogeographical analysis of the Bemisia tabaci species complex based on mitochondrial DNA markers. Molecular Ecology, 8 (10): 1683 - 1691.

Gafni, Y. and Epel, B. L. 2002. The role of host and viral proteins in intra- and inter- cellular trafficking of geminiviruses. Physiological and Molecular Plant Pathology, 60 (5): 231 - 241.

Gai, Y. P., Han, X. J., Li, Y. Q., Yuan, C. Z., Mo, Y. Y., Guo, F. Y., Liu, Q. X. and Ji, X. L. 2014. Metabolomic analysis reveals the potential metabolites and pathogenesis involved in mulberry yellow dwarf disease. Plant, Cell & Environment, 37 (6): 1474 - 1490.

Gao, H., Gordon-Kamm, W. J. and Lyznik, L. A. 2004. ASF/SF2-like maize pre- mRNA splicing factors affect splice site utilization and their transcripts are alternatively spliced. Gene, 339: 25 - 37.

Gardiner, W. E., Sunter, G., Brand, L., Elmer, J. S., Rogers, S. G. and Bisaro, D. M. 1988. Genetic analysis of Tomato golden mosaic virus: The coat protein is not required for systemic spread or symptom development. EMBO Journal, 7 (4): 899 - 904.

George, J. W. and Kreuzer, K. N. 1996. Repair of double-strand breaks in bacteriophage T4 by a mechanism that involves extensive DNA replication. Genetics, 143 (4): 1507 - 1520.

Gerszberg, A., Hnatuszko-Konka, K., Kowalczyk, T. and Kononowicz, A. K. 2015. Tomato (Solanum lycopersicum L.) in the service of biotechnology. Plant Cell, Tissue and Organ Culture (PCTOC), 120 (3): 881 - 902.

Ghanim, M., Brumin, M. and Popovski, S. 2009. A simple, rapid and inexpensive method for localization of Tomato yellow leaf curl virus and Potato leafroll virus in plant and insect vectors. Journal of Virological Methods, 159 (2): 311 - 314.

234

Ghanim, M., Morin, S. and Czosnek, H. 2001. Rate of Tomato yellow leaf curl virus translocation in the circulative transmission pathway of its vector, the whitefly bemisia tabaci. Phytopathology, 91 (2): 188 - 196.

Ghanim, M., Morin, S., Zeidan, M. and Czosnek, H. 1998. Evidence for transovarial transmission of Tomato yellow leaf curl virus by its vector, the whitefly bemisia tabaci. Virology, 240 (2): 295 - 303.

Gibon, Y., Usadel, B., Blaesing, O. E., Kamlage, B., Hoehne, M., Trethewey, R. and Stitt, M. 2006. Integration of metabolite with transcript and enzyme activity profiling during diurnal cycles in arabidopsis rosettes. Genome Biology, 7 (8): 1.

Gilbertson, R. L., Sudarshana, M., Jiang, H., Rojas, M. R. and Lucas, W. J. 2003. Limitations on geminivirus genome size imposed by plasmodesmata and virus- encoded movement protein: Insights into DNA trafficking. Plant Cell, 15 (11): 2578 - 2591.

Giovannucci, E. 1999. Tomatoes, tomato-based products, lycopene, and cancer: Review of the epidemiologic literature. Journal of the National Cancer Institute, 91 (4): 317 - 331.

Giovannucci, E. 2002. A review of epidemiologic studies of tomatoes, lycopene, and prostate cancer. Experimental Biology and Medicine, 227 (10): 852 - 859.

Glauser, G., Boccard, J., Rudaz, S. and Wolfender, J. L. 2010. Mass spectrometry‐based metabolomics oriented by correlation analysis for wound‐induced molecule discovery: Identification of a novel jasmonate glucoside. Phytochemical Analysis, 21 (1): 95 - 101.

Glick, E., Zrachya, A., Levy, Y., Mett, A., Gidoni, D., Belausov, E., Citovsky, V. and Gafni, Y. 2008. Interaction with host sgs3 is required for suppression of RNA silencing by tomato Yellow leaf curl virus V2 protein. Proceedings of the National Academy of Sciences, 105 (1): 157 - 161.

Goodman, R. M. 1977a. Single-stranded DNA genome in a whitefly-transmitted plant virus. Virology, 83 (1): 171 - 179.

Goodman, R. M. 1977b. Infectious DNA from a whitefly-transmitted virus of phaseolus vulgaris. Virology, 266: 54 - 55.

235

Gopal, P., Pravin Kumar, P., Sinilal, B., Jose, J., Kasin Yadunandam, A. and Usha, R. 2007. Differential roles of C4 and βC1 in mediating suppression of post- transcriptional gene silencing: Evidence for transactivation by the C2 of Bhendi yellow vein mosaic virus, a monopartite begomovirus. Virus Research, 123 (1): 9 - 18.

Gray, S. M. and Banerjee, N. 1999. Mechanisms of arthropod transmission of plant and animal viruses. Microbiology and Molecular Biology Reviews, 63 (1): 128 - 148.

Greathead, A. H. 1986. Host plants In bemisia tabaci-a literature survey. M. J. W. Cock (Ed.), CAB International Institute of Biological Control, Silwood Park, Ascot, Berks.,UK. pp. 17 - 25.

Griffin, J. L. and Shockcor, J. P. 2004. Metabolic profiles of cancer cells. Nature reviews cancer, 4 (7): 551 - 561.

Grimsley, N., Hohn, T., Davies, J. W. and Hohn, B. 1987. Agrobacterium-mediated delivery of infectious Maize streak virus into maize plants. Nature, 325: 177 - 179.

Gröning, B. R., Abouzid, A. and Jeske, H. 1987. Single-stranded DNA from Abutilon mosaic virus is present in the plastids of infected Abutilon sellovianum. Proceedings of the National Academy of Sciences, 84 (24): 8996 - 9000.

Gueguen, G., Vavre, F., Gnankine, O., Peterschmitt, M., Charif, D. and Chiel, E. 2010. Endosymbiont metacommunities, mtDNA diversity and the evolution of the Bemisia tabaci (hemiptera: Aleyrodidae) species complex. Molecular Ecoogyl. 19 (19): 4365 - 4378.

Guerra-Peraza, O., Kirk, D., Seltzer, V., Veluthambi, K., Schmit, A. C., Hohn, T. and Herzog, E. 2005. Coat proteins of Rice tungro bacilliform virus and Mungbean yellow mosaic virus contain multiple nuclear-localization signals and interact with importin Journal of General Virology, 86 (6): 1815 - 1826.

Guevara-González, R. G., Ramos, P. L. and Rivera-Bustamante, R. F. 1999. Complementation of coat protein mutants of pepper huasteco geminivirus in transgenic tobacco plants. Phytopathology, 89 (7): 540 - 545.

Gurjar, M. S., Ali, S., Akhtar, M. and Singh, K. S. 2012. Efficacy of plant extracts in plant disease management. Agricultural Sciences, 3 (3): 425.

Gutierrez, C. 1999. Geminivirus DNA replication. Cellular and Molecular Life Sciences, 56 (3-4): 313 - 329.

236

Gutierrez, C., Ramirez-Parra, E., Castellano, M. M., Sanz-Burgos, A. P., Luque, A. and Missich, R. 2004. Geminivirus DNA replication and cell cycle interactions. Veterinary Microbiology, 98 (2): 111 - 119.

Ha, C., Coombs, S., Revill, P., Harding, R., Vu, M. and Dale, J. 2006. Corchorus yellow vein virus, a new world geminivirus from the old world. Journal of General Virology, 87 (4): 997 - 1003.

Ha, C., Coombs, S., Revill, P., Harding, R., Vu, M. and Dale, J. 2008. Molecular characterization of begomoviruses and DNA satellites from vietnam: Additional evidence that the new world geminiviruses were present in the old world prior to continental separation. Journal of General Virology, 89 (1): 312 - 326.

Haider, M. S., Bedford, I. D., Evans, A. A. F. and Markham, P. G. 2003. Geminivirus transmission by different biotypes of the whitefly Bemisia tabaci (gennadius). Pakistan Journal of Zoology, 35 (4): 343 - 351.

Hak, H., Levy, Y., Chandran, S. A., Belausov, E., Loyter, A., Lapidot, M. and Gafni, Y. 2015. TYLCV-Is movement in planta does not require V2 protein. Virology, 477: 56 - 60.

Halket, J. M., Przyborowska, A., Stein, S. E., Mallard, W. G., Down, S. and Chalmers, R. A. 1999. Deconvolution gas chromatography/mass spectrometry of urinary organic acids–potential for pattern recognition and automated identification of metabolic disorders. Rapid communications in mass spectrometry, 13 (4): 279 - 284.

Haloui , E., Marzouk , Z., Marzouk, B., Bouftira, I., Bouraoui, A. and Fenina, N. 2010. Pharmacological activities and chemical composition of the Olea europaea l. Leaf essential oils from Tunisia. Journal of Food, Agriculture & Environment, 8 (2): 204 - 208.

Hamilton, W. D. O., Bisaro, D. M. and Buck, K. W. 1982. Identification of novel DNA forms in Tomato golden mosaic virus infected tissue. Evidence for a two component genome. Nucleic Acids Research, 10 (16): 4901 - 4912.

Hanley-Bowdoin, L., Settlage, S., Orozco, B., Nagar, S. and Robertson, D. 1999. Geminiviruses: Models of plant DNA replication, transcription, and cell cycle regulation. Crit Rev Plant Sci., 18 (1): 71 - 106.

237

Hanley-Bowdoin, L., Settlage, S. B. and Robertson, D. 2004. Reprogramming plant gene expression: A prerequisite to geminivirus DNA replication. Molecular Plant Pathology, 5 (2): 149 - 156.

Hanson, P., Green, S. and Kuo, G. 2006. Ty-2, a gene on chromosome 11 conditioning geminivirus resistance in tomato. Tomato Genet Coop Rep, 56: 17 - 18.

Hanson, P., Lu, S.-F., Wang, J.-F., Chen, W., Kenyon, L., Tan, C.-W., Tee, K. L., Wang, Y.-Y., Hsu, Y.-C. and Schafleitner, R. 2016. Conventional and molecular marker-assisted selection and pyramiding of genes for multiple disease resistance in tomato. Scientia Horticulturae, 201: 346 - 354.

Hanson P., W. J.-F., Ho F.-I., Tan C.-W. 2011. Progress towards development of bacterial wilt and begomovirus-resistant tomato lines at avrdc-the world vegetable center. In: International conference on solanaceae resistance breeding technologies genetics and genomics. Chiang mai, thailand.

Hanssen, I. M., Lapidot, M. and Thomma, B. P. 2010. Emerging viral diseases of tomato crops. Molecular Plant-Microbe Interactions, 23 (5): 539 - 548.

Hao, L., Wang, H., Sunter, G. and Bisaro, D. M. 2003. Geminivirus AL2 and L2 proteins interact with and inactivate SNF1 kinase. Plant Cell, 15 (4): 1034 - 1048.

Harrison, B. and Robinson, D. 1999. Natural genomic and antigenic variation in whitefly-transmitted geminiviruses (begomoviruses). Annu Rev Phytopathol., 37 (1): 369 - 398.

Harrison, B. and Robinson, D. 2002. Green shoots of geminivirology. Physiological and Molecular Plant Pathology, 60: 215 - 218.

Harrison, B. D., Barker, H., Bock, K. R., Guthrie, E. J., Meredith, G. and Atkinson, M. 1977. Plant viruses with circular single-stranded DNA. Nature, 270: 760 - 762.

Harrison, B. D., Swanson, M. M. and Fargette, C. 2002. Begomovirus coat protein: Serology, variation and functions. Physiological and Molecular Plant Pathology, 60 (5): 257 - 271.

Hartitz, M. D., Sunter, G. and Bisaro, D. M. 1999. The Tomato golden mosaic virus transactivator (TrAP) is a single-stranded DNA and zinc-binding phosphoprotein with an acidic activation domain. Virology, 263 (1): 1 - 14.

238

Hatta, T. and Francki, R. I. B. 1979. The fine structure of Chloris striate mosaic virus. Virology, 92 (2): 428 - 435.

Hecht, S. S., Stepanov, I. and Carmella, S. G. 2015. Exposure and metabolic activation biomarkers of carcinogenic tobacco-specific nitrosamines. Accounts of chemical research, 49 (1): 106 - 114.

Hehnle, S., Wege, C. and Jeske, H. 2004. Interaction of DNA with the movement proteins of geminiviruses revisited. Journal of Virology, 78 (14): 7698 - 7706.

Hernández-Zepeda, C., Varsani, A. and Brown, J. K. 2013. Intergeneric recombination between a new, spinach-infecting curtovirus and a new geminivirus belonging to the genus becurtovirus: First new world exemplar. Archives of Virology, 158 (11): 2245 - 2254.

Heydarnejad, J., Hosseini Abhari, E., Bolok Yazdi, H. and Massumi, H. 2007. Curly top of cultivated plants and weeds and report of a unique curtovirus from Iran. Journal of Phytopathology, 155 (6): 321 - 325.

Heydarnejad, J., Mozaffari, A., Massumi, H., Fazeli, R., Gray, A. J., Meredith, S., Lakay, F., Shepherd, D. N., Martin, D. P. and Varsani, A. 2009. Complete sequences of Tomato leaf curl Palampur virus isolates infecting cucurbits in Iran. Archives of Virology, 154 (6): 1015 - 1018.

Heyraud-Nitschke, F., Schumacher, S., Laufs, J., Schaefer, S., Schell, J. and Gronenborn, B. 1995. Determination of the origin cleavage and joining domain of geminivirus Rep proteins. Nucleic Acids Research, 23 (6): 910 - 916.

Hijazi, A., Bandar, H., Rammal, H., Hachem, A., Saad, Z. and Badran, B. 2013. Techniques for the extraction of bioactive compounds from Lebanese urtica dioica. American Journal of Phytomedicine and Clinical Therapeutics, 1 (6): 507 - 513.

Ho, E. S., Kuchie, J. and Duffy, S. 2014. Bioinformatic analysis reveals genome size reduction and the emergence of tyrosine phosphorylation site in the movement protein of new world bipartite begomoviruses. PLoS One, 9 (11): e111957.

Hofer, P., Bedford, I. D., Markham, P. G., Jeske, H. and Frischmuth, T. 1997. Coat protein gene replacement results in whitefly transmission of an insect nontransmissible geminivirus isolate. Virology, 236 (2): 288 - 295.

239

Hogenhout, S. A., Ammar, E.-D., Whitfield, A. E. and Redinbaugh, M. G. 2008. Insect vector interactions with persistently transmitted viruses*. Annual Review of Phytopathology, 46: 327 - 359.

Höhnle, M., Höfer, P., Bedford, I. D., Briddon, R. W., Markham, P. G. and Frischmuth, T. 2001. Exchange of three amino acids in the coat protein results in efficient whitefly transmission of a nontransmissible Abutilon mosaic virus isolate. Virology, 290 (1): 164 - 171.

Holzapfel, N. P., Holzapfel, B. M., Champ, S., Feldthusen, J., Clements, J. and Hutmacher, D. W. 2013. The potential role of lycopene for the prevention and therapy of prostate cancer: From molecular mechanisms to clinical evidence. International Journal of Molecular Sciences, 14 (7): 14620 - 14646.

Hong, H., Stanley, J. and Van Wezel, R. 2003. Novel system for the simultaneous analysis of geminivirus DNA replication and plant interactions in Nicotiana benthamiana. Journal of Virology, 77 (24): 13315 - 13322.

Hong, Y., Saunders, K. and Stanley, J. 1997. Transactivation of dianthin transgene expression by African cassava mosaic virus AC2. Virology, 228 (2): 383 - 387.

Hormuzdi, S. G. and Bisaro, D. M. 1995. Genetic analysis of Beet curly top virus: Examination of the roles of L2 and L3 genes in viral pathogenesis. Virology, 206 (2): 1044 - 1054.

Horning, E. and Horning, M. 1971. Human metabolic profiles obtained by gc and gc/ms. Journal of Chromatographic Science, 9 (3): 129 - 140.

Horowitz, A., Weintraub, P. G. and Ishaaya, I. 1998. Status of pesticide resistance in arthropod pests in Israel. Phytoparasitica, 26 (3): 231 - 240.

Hossain, M. A., Al-Toubi, W. A., Weli, A. M., Al-Riyami, Q. A. and Al-Sabahi, J. N. 2013. Identification and characterization of chemical compounds in different crude extracts from leaves of Omani neem. Journal of Taibah University for Science, 7 (4): 181 - 188.

Hou, Y.-M. and Gilbertso, R. L. 1996. Increased pathogenicity in a pseudorecombinant bipartite geminivirus correlates with intermolecular recombination. Journal of Virology, 70 (8): 5430 - 5436.

240

Howell, S. H. 1984. Physical structure and genetic organisation of the genome of Maize streak virus (kenyan isolate). Nucleic Acids Research, 12 (19): 7359 - 7375.

Hu, C.-C., Hsu, Y.-H. and Lin, N.-S. 2009. Satellite rnas and satellite viruses of plants. Viruses, 1 (3): 1325 - 1350.

Huang, C. H., Yan, F. M., Byers, J. A., Wang, R. J. and Xu, C. R. 2009. Volatiles induced by the larvae of the asian corn borer (Ostrinia furnacalis) in maize plants affect behavior of conspecific larvae and female adults. Insect Science, 16 (4): 311 - 320.

Huang, J., Cardoza, Y. J., Schmelz, E. A., Raina, R., Engelberth, J. and Tumlinson, J. H. 2003. Differential volatile emissions and salicylic acid levels from tobacco plants in response to different strains of Pseudomonas syringae. Planta, 217 (5): 767 - 775.

Hull, R. 2002. Matthew's plant virology, 4th ed. Academic Press, San Diego. pp. 3 - 29.

Hull, R. 2009. Comparative plant virology, 2nd ed. Academic press. pp. 13 - 29.

Hull, R. 2013. Plant virology, 4th ed. Academic press, San Diego USA. pp. 3 - 19. Hur, J., Kenneth, J. B., Lee, S. and Keith, R. D. 2007. Transcriptional activator elements for curtovirus C1 expression reside in the 3′ coding region of orf C1. Molecules and Cells, 23 (1): 80 - 87.

Hussain, M., Mansoor, S., Iram, S., Fatima, A. N. and Zafar, Y. 2005. The nuclear shuttle protein of Tomato leaf curl New Delhi virus is a pathogenicity determinant. Journal of Virology, 79 (7): 4434 - 4439.

Hussain, M., Mansoor, S., Iram, S., Zafar, Y. and Briddon, R. W. 2004. First report of Tomato leaf curl New Delhi virus affecting chilli pepper in Pakistan. Plant pathology, 53 (6): 794 - 794.

Hussain, M., Mansoor, S., Iram, S., Zafar, Y. and Briddon, R. W. 2007. The hypersensitive response to Tomato leaf curl New Delhi virus nuclear shuttle protein is inhibited by transcriptional activator protein. Molecular Plant-Microbe Interactions, 20 (12): 1581 - 1588.

Hussaini, H., Sani, A. and Aliero, A. 2011. Volatile metabolites profiling to discriminate diseases of tomato fruits inoculated with three toxigenic fungal pathogens. Research in Biotechnology, 2 (3): 14 - 22.

241

Hutton, S. F. and Scott, J. W. 2013. Fine-mapping and cloning of Ty-1 and Ty-3; and mapping of a new TYLCV resistance locus,“Ty-6”. Tomato Breeders Round Table. Retrieved from http://tgc.ifas.ufl.edu/2013/abstracts/SamOrchardAbstract%20TBRT%202013.pdf.

Hutton, S. F., Scott, J. W. and Schuster, D. J. 2012. Recessive resistance to Tomato yellow leaf curl virus from the tomato cultivar tyking is located in the same region as Ty-5 on chromosome 4. HortScience, 47 (3): 324 - 327.

Ibrahim, A., Abubakar, A., Aliero, A., Sani, A. and Yakubu, S. 2011a. Volatile metabolites profiling for discriminating tomato fruits inoculated with some bacterial pathogens. J. Pharm. Biomed. Sci, 1 (5): 79 - 84.

Ibrahim, A., Dogondaji, A., Aliero, A., Yakubu, S., Yusuf, S. and Karaye, I. 2011b. Volatile organic compounds production during spoilage of African horned cucumber fruits. International Journal of Applied Biology and Pharmaceutical Technology, 2 (3): 296 - 302.

Idris, A. M. and Brown, J. K. 2004. Cotton leaf crumple virus is a distinct western hemisphere begomovirus species with complex evolutionary relationships indicative of recombination and reassortment. Phytopathology, 94 (10): 1068 - 1074.

Idris, A. M., Shahid, M. S., Briddon, R. W., Khan, A. J., Zhu, J.-K. and Brown, J. K. 2011. An unusual alphasatellite associated with monopartite begomoviruses attenuates symptoms and reduces betasatellite accumulation. Journal of General Virology, 92 (3): 707 - 717.

Igwe, K., Okafor, P. and Ijeh, I. 2016. GC-MS analysis of phytocomponents in Vernonia amygdalina. Del leaves and its contractile potential in mammary tissue in female albino wistar rats. Global Journal of Biology, agriculture & health sciences, 5 (2): 1 - 6.

Imran, M., Khan, M. A., Fiaz, M., Azeem, M. and Mustafa, M. 2013. Influence of environmental conditions on Tomato mosaic virus disease development under natural condition. Pakistan Journal of Phytopathology, 25 (2): 117 - 122.

Ingham, D. J., Pascal, E. and Lazarowitz, S. G. 1995. Both bipartite geminivirus movement proteins define viral host range, but only BL1 determines viral pathogenicity. Virology, 207 (1): 191 - 204.

242

Inoue-Nagata, A. K., Martin, D. P., Boiteux, L. S., Giordano, L. D. B., Bezerra, I. C. and Ávila, A. C. D. 2006. New species emergence via recombination among isolates of the Brazilian tomato infecting begomovirus complex. Pesquisa Agropecuária Brasileira, 41 (8): 1329 - 1332.

Inouye, T. and Osaki, T. 1980. The first record in the literature of the possible plant virus disease that appeared in manyoshu, a japanese classic anthology, as far back as the time of the 8th century. Annals of the Phytopathological Society of Japan, 46 (1): 49- 50.

Iqbal, Z., Sattar, M. N., Kvarnheden, A., Mansoor, S. and Briddon, R. W. 2012. Effects of the mutation of selected genes of Cotton leaf curl Kokhran virus on infectivity, symptoms and the maintenance of Cotton leaf curl Multan betasatellite. Virus Research, 169 (1): 107 - 116.

Ish-Horowicz, D. and Burke, J. 1981. Rapid and efficient cosmid cloning. Nucleic Acids Research, 9 (13): 2989 - 2898.

Jackel, J. N., Buchmann, R. C., Singhal, U. and Bisaro, D. M. 2015. Analysis of geminivirus al2 and l2 proteins reveals a novel al2 silencing suppressor activity. Journal of Virology, 89 (6): 3176 - 3187.

Jenecius, A. A., Uthayakumari, F. and Mohan, V. 2012. GC-MS determination of bioactive components of Sauropus bacciformis blume (euphorbiaceae). Journal of Current Chemical and Pharmaceutical Sciences, 2 (4): 347 - 358.

Jeske, H. 2009. Geminiviruses. Current Topics in Microbiology and Immunology, 331: 185 - 226.

Jeske, H., Lutgemeier, M. and Preiss, W. 2001. DNA forms indicate rolling circle and recombination-dependent replication of abutilon mosaic virus. EMBO Journal, 20 (21): 6158 - 6167.

Ji, Y., Schuster, D. J. and Scott, J. W. 2007a. Ty-3, a begomovirus resistance locus near the Tomato yellow leaf curl virus resistance locus Ty-1 on chromosome 6 of tomato. Molecular Breeding, 20 (3): 271 - 284.

Ji, Y., Scott, J., Hanson, P., Graham, E. and Maxwell, D. 2007b. Sources of resistance, inheritance, and location of genetic loci conferring resistance to members

243 of the tomato-infecting begomoviruses. In Tomato Yellow Leaf Curl Virus Disease, Springer Netherlands. pp. 343 - 362.

Ji, Y., Scott, J. W., Schuster, D. J. and Maxwell, D. P. 2009. Molecular mapping of Ty-4, a new Tomato yellow leaf curl virus resistance locus on chromosome 3 of tomato. Journal of the American Society for Horticultural Science, 134 (2): 281 - 288.

Jiang, T. and Zhou, X. 2005. Molecular characterization of a distinct begomovirus species and its associated satellite DNA isolated from Malvastrum coromandelianum in China. Virus Genes, 31 (1): 43.

Jiang, T. and Zhou, X. P. 2004. First report of Malvastrum yellow vein virus infecting Ageratum conyzoides. Plant pathology, 53 (6): 799 - 799.

Jones, D. R. 2003. Plant viruses transmitted by whiteflies. European Journal of Plant Pathology, 109 (3): 195 - 219.

Jonsson, P., Johansson, E. S., Wuolikainen, A., Lindberg, J., Schuppe-Koistinen, I., Kusano, M., Sjöström, M., Trygg, J., Moritz, T. and Antti, H. 2006. Predictive metabolite profiling applying hierarchical multivariate curve resolution to GC-MS data a potential tool for multi-parametric diagnosis. Journal of proteome research, 5 (6): 1407 - 1414.

Jose, J. and Usha, R. 2003. Bhendi yellow vein mosaic disease in India is caused by Virology, 305 (2): 310 - 317.

Jovel, J., Preiss, W. and Jeske, H. 2007. Characterization of DNA intermediates of an arising geminivirus. Virus Research, 130 (1): 63 - 70.

Jupin, I., De Kouchkovsky, F., Jouanneau, F. and Gronenborn, B. 1994. Movement of tomato yellow leaf curl geminivirus (TYLCV): Involvement of the protein encoded by ORF C4. Virology, 204 (1): 82 - 90.

Jupin, I., Hericourt, F., Benz, B. and Gronenborn, B. 1995. DNA replication specificity of TYLCV geminivirus is mediated by the amino-terminal 116 amino acids of the Rep protein. FEBS Letters, 362 (2): 116 - 120.

Kamran, M., Anwar, S., Javed, N., Khan, S., Haq, I. and Ullah, I. 2012. Field evaluation of tomato genotypes for resistance to Meloidogyne incognita. Pakistan Journal of Zoology, 44 (5): 1355 - 1359.

244

Kanakala, S., Verma, H. N., Vijay, P., Saxena, D. R. and Malathi, V. G. 2013. Response of chickpea genotypes to Agrobacterium-mediated delivery of Chickpea chlorotic dwarf virus (CpCDV) genome and identification of resistance source. Applied Microbiology and Biotechnology, 97 (21): 9491 - 9501.

Karppi, J., Laukkanen, J. A., Mäkikallio, T. H. and Kurl, S. 2012a. Low serum lycopene and β-carotene increase risk of acute myocardial infarction in men. The European Journal of Public Health, 22 (6): 835 - 840.

Karppi, J., Laukkanen, J. A., Sivenius, J., Ronkainen, K. and Kurl, S. 2012b. Serum lycopene decreases the risk of stroke in men a population-based follow-up study. Neurology, 79 (15): 1540 - 1547.

Kassanis, B. 1962. Properties and behaviour of a virus depending for its multiplication on another. Journal of General Microbiology, 27 (3): 477 - 488.

Kavitha, S., Lincy, M. L. P., Kala, S. M. J., Mohan, V. R. and Maruthupandian, A. 2015. GC-MS analysis of ethanol extract of stem of Nothapodytes nimmoniana (graham) mabb. World Journal of Pharmaceutical Sciences, 3 (6): 1145 - 1150

Kefeli, V. I., Kalevitch, M. V. and Borsari, B. 2003. Phenolic cycle in plants and environment. Journal of Cell Molecular Biology, 2 (1): 13 - 18.

Kell, D. B., Brown, M., Davey, H. M., Dunn, W. B., Spasic, I. and Oliver, S. G. 2005. Metabolic footprinting and systems biology: The medium is the message. Nature Reviews Microbiology, 3 (7): 557 - 565.

Kelly, J. D., Afanador, L. and Haley, S. D. 1995. Pyramiding genes for resistance to bean common mosaic virus. Euphytica, 82 (3): 207 - 212.

Kenyon, L., Tsai, W.-S., Shih, S.-L. and Lee, L.-M. 2014. Emergence and diversity of begomoviruses infecting solanaceous crops in east and southeast Asia. Virus Research, 186: 104 - 113.

Khan, A., Akhtar, S., Al-Shihi, A. A., Al-Hinai, F. M. and Briddon, R. W. 2012. Identification of cotton leaf curl gezira virus in papaya in Oman. Plant disease, 96: 1199 - 1205.

Khan, A. J., Mansoor, S. and Briddon, R. W. 2014. Oman: A case for a sink of begomoviruses of geographically diverse origins. Trends in Plant Science, 19 (2): 67 - 70.

245

Khan, M. A. U., Shahid, A. A., Rao, A. Q., Shahid, N., Latif, A., Ud Din, S. and Husnain, T. 2015. Defense strategies of cotton against whitefly transmitted CLCuV and begomoviruses. Advancements in Life Sciences, 2 (2): 58 - 66.

Khan, M. U. and Hussain, S. T. 2014. Normalizing india pakistan trade relations: India pakistan agricultural trade . LUMS ECONOMICS WORKING PAPER No. 14- 10. Retrieved from http://old.lums.edu.pk/updata/publications/pdf/87.pdf

Khan, S. A. 2000. Plasmid rolling-circle replication: Recent developments. Molecular Microbiology, 37 (3): 477 - 484.

Khatri, S., Nahid, N., Fauquet, C. M., Mubin, M. and Nawaz-Ul-Rehman, M. S. 2014. A betasatellite-dependent begomovirus infects ornamental rose: Characterization of begomovirus infecting rose in Pakistan. Virus Genes, 49 (1): 124 - 131.

Khokhar, K. 2013. Present status and prospects of tomatoes in pakistan. Agricultural corner-farmers to global market. Retrieved from http://www.agricorner.com/present- status-and-prospects-of-tomatoes-in-pakistan/

Kil, E., Kim, S., Lee, Y., Byun, H., Park, J., Seo, H., Kim, C., Shim, J., Lee, J. and Kim, J. 2015. Tomato yellow leaf curl virus (TYLCV-IL): A seed-transmissible geminivirus in tomatoes. Scientific Reports, 6: 19013.

Kim, J., Kil, E. J., Kim, S., Seo, H., Byun, H. S., Park, J., Chung, M. N., Kwak, H. R., Kim, M. K. and Kim, C. S. 2015. Seed transmission of sweet Potato leaf curl virus in sweet potato (ipomoea batatas). Plant Pathology, 64 (6): 1284 - 1291.

Kimura, S. and Sinha, N. 2008. Tomato (Solanum lycopersicum): A model fruit- bearing crop. Cold Spring Harb Protocl, 3: 1 - 9.

King, A. M., Adams, M. J. and Lefkowitz, E. J. 2011. Virus taxonomy: Classification and nomenclature of viruses: Ninth report of the international committee on taxonomy of viruses, Elsevier. pp. 193 - 210.

Kirthi, N. and Savithri, H. S. 2003. A conserved zinc finger motif in the coat protein of Tomato leaf curl bangalore virus is responsible for binding to ssDNA. Archives of Virology, 148 (12): 2369 - 2380.

Kittelmann, K., Rau, P., Gronenborn, B. and Jeske, H. 2009. Plant geminivirus Rep protein induces rereplication in fission yeast. Journal of Virology, 83 (13): 6769 - 6778.

246

Kleinow, T., Tanwir, F., Kocher, C., Krenz, B., Wege, C. and Jeske, H. 2009. Expression dynamics and ultrastructural localization of epitope-tagged Abutilon mosaic virus nuclear shuttle and movement proteins in Nicotiana benthamiana cells. Virology, 391 (2): 212 - 220.

Klute, K. A., Nadler, S. A. and Stenger, D. C. 1996. Horseradisch curly top virus is a distinct subgroup ii geminivirus species with Rep and C4 genes derived from a subgroup iii ancestor. Journal of General Virology, 77 (7): 1369 - 1378.

Knapp, S., Bohs, L., Nee, M. and Spooner, D. M. 2004. Solanaceae—a model for linking genomics with biodiversity. Comparative and Functional Genomics, 5 (3): 285 - 291.

Knapp, S. and Peralta, I. E. 2016. The Tomato Genome, Compendium of Plant Genomes, M. Causse et al. (eds.), Springer-Verlag Berlin Heidelberg. pp. 07 - 21.

Kokate Ck, Purohit, A.P., Gokhale, SB. 2008. Analytical pharmacognosy, 23rd ed, Pune, Nirali Prakashan, India. pp. 111 - 112.

Kon, T., Sharma, P. and Ikegami, M. 2007. Suppressor of RNA silencing encoded by the monopartite tomato leaf curl Java begomovirus. Archives of Virology, 152 (7): 1273 - 1282.

Kong, L. and Hanley-Bowdoin, L. 2002. A geminivirus replication protein interacts with a protein kinase and a motor protein that display different expression patterns during plant development and infection. Plant Cell, 14 (8): 1817 - 1832.

Kong, L. J., Orozco, B. M., Roe, J. L., Nagar, S., Ou, S., Feiler, H. S., Durfee, T., Miller, A. B., Gruissem, W., Robertson, D. and Hanley-Bowdoin, L. 2000. A geminivirus replication protein interacts with the retinoblastoma protein through a novel domain to determine symptoms and tissue specificity of infection in plants. EMBO Journal, 19 (13): 3485 - 3495.

Kordali, S., Cakir, A., Akcin, T. A., Mete, E., Akcin, A., Aydin, T. and Kilic, H. 2009. Antifungal and herbicidal properties of essential oils and n-hexane extracts of Achillea gypsicola hub-mor and Achillea biebersteinii afan (asteraceae). Industrial crops and products, 29 (2): 562 - 570.

247

Kothandaraman, S. V., Devadason, A. and Ganesan, M. V. 2015. Seed-borne nature of a begomovirus, Mung bean yellow mosaic virus in black gram. Applied Microbiology and Biotechnology, 100 (4): 1925 - 1933.

Kotlizky, G., Boulton, M., Pitaksutheepong, C., Davie, J. W. and Epel, B. L. 2000. Intracellular and intercellular movement of maize streak geminivirus V1 and V2 proteins transiently expressed as green fluorescent protein fusions. Virology, 274 (1): 32 - 38.

Kozuma, K., Tsuchiya, S., Kohori, J., Hase, T. and Tokimitsu, I. 2005. Antihypertensive effect of green coffee bean extract on mildly hypertensive subjects. Hypertension research, 28 (9): 711 - 718.

Krake, L. R., Rezaian, M. A. and Dry, I. B. 1998. Expression of the tomato leaf curl geminivirus C4 gene produces viruslike symptoms in transgenic plants. Molecular Plant-Microbe Interactions, 11 (5): 413 - 417.

Kreuzer, K. N. 2000. Recombination-dependent DNA replication in phage T4. Trends in Biochemical Sciences, 25 (4): 165 - 173.

Kumar, J., Gunapati, S., Singh, S. P., Gadre, R., Sharma, N. C. and Tuli, R. 2013. Molecular characterization and pathogenicity of a carrot (Daucus carota) infecting begomovirus and associated betasatellite from India. Virus Research, 178 (2): 478 - 485.

Kumar, P., Ushaa, R., Zrachya, A., Levyb, Y., Spanov, H. and Gafni, Y. 2006. Protein–protein interactions and nuclear trafficking of coat protein and βC1 protein associated with bhendi yellow vein mosaic disease. Virus Research, 122 (1): 127 - 136.

Kumar, Y., Hallan, V. and Zaidi, A. A. 2008. Molecular characterization of a distinct bipartite begomovirus species infecting tomato in India. Virus Genes, 37 (3): 425 - 431.

Kumari, P., Singh, A. K., Chattopadhyay, B. and Chakraborty, S. 2010. Molecular characterization of a new species of begomovirus and betasatellite causing leaf curl disease of tomato in India. Virus Research, 152 (1-2): 19 - 29.

248

Kunik, T., Palanichelvam, K., Czosnek, H., Citovsky, V. and Gafni, Y. 1998. Nuclear import of the capsid protein of Tomato yellow leaf curl virus (TYLCV) in plant and insect cells. Plant Journal, 13 (3): 393 - 399.

Lacatus, G. and Sunter, G. 2008. Functional analysis of bipartite begomovirus coat protein promoter sequences. Virology, 376 (1): 79 - 89.

Lapidot, M. and Friedmann, M. 2002. Breeding for resistance to whitefly-transmitted geminiviruses. Annals of Applied Biology, 140 (2): 109 - 127.

Latham, J. R., Saunders, K., Pinner, M. S. and Stanley, J. 1997. Induction of cell division by Beet curly top virus gene C4. Plant Journal, 11 (6): 1273 - 1283.

Laufs, J., Jupin, I., David, C., Schumacher, S., Heyraud-Nitschke, F. and B., G. 1995. Geminivirus replication: Genetic and biochemical characterization of Rep protein function, a review. Biochimie, 77 (10): 765-773.

Lazarowitz, S. G. and Beachy, R. N. 1999. Viral movement proteins a probes for intracellular and intercellular traficking in plants. Plant Cell, 11 (4): 535 - 548.

Lecoq, K., Belloc, I., Desgranges, C. and Daignan-Fornier, B. 2001. Role of adenosine kinase in Saccharomyces cerevisiae: Identification of the ADO1 gene and study of the mutant phenotypes. Yeast, 18 (4): 335 - 342.

Lee, B., Farag, M. A., Park, H. B., Kloepper, J. W., Lee, S. H. and Ryu, C.-M. 2012. Induced resistance by a long-chain bacterial volatile: Elicitation of plant systemic defense by a C13 volatile produced by Paenibacillus polymyxa. PLoS One, 7 (11): e48744.

Lefeuvre, P., Lett, J. M., Varsani, A. and Martin, D. P. 2009. Widely conserved recombination patterns among single-stranded DNA viruses. Journal of Virology, 83 (6): 2697 - 2707.

Lefeuvre, P., Martin, D. P., Harkins, G., Lemey, P., Gray, A. J., Meredith, S., Lakay, F., Monjane, A., Lett, J.-M. and Varsani, A. 2010. The spread of Tomato yellow leaf curl virus from the Middle East to the world. PLoS Pathogens, 6 (10): e1001164.

Lefeuvre, P., Martin, D. P., Hoareau, M., Naze, F., Delatte, H., Thierry, M., Varsani, A., Becker, N., Reynaud, B. and Lett, J. M. 2007. Begomovirus 'melting pot' in the South-West Indian ocean islands: Molecular diversity and evolution through recombination. Journal of General Virology, 88 (12): 3458 - 3468.

249

Lefeuvre, P. and Moriones, E. 2015. Recombination as a motor of host switches and virus emergence: Geminiviruses as case studies. Current opinion in Virology, 10: 14 - 19.

Legg, J. and Fauquet, C. 2004. Cassava mosaic geminiviruses in Africa. Plant Mol Biol., 56 (4): 585 - 599.

Leke, W. N., Mignouna, D. B., Brown, J. K. and Kvarnheden, A. 2015. Begomovirus disease complex: Emerging threat to vegetable production systems of West and Central Africa. Agriculture & Food Security, 4 (1): 1.

Leke, W. N., Sattar, M. N., Ngane, E. B., Ngeve, J. M., Kvarnheden, A. and Brown, J. K. 2013. Molecular characterization of begomoviruses and DNA satellites associated with okra leaf curl disease in Cameroon. Virus Research, 174 (1): 116 - 125.

Li, L., Zhu, Y., Jin, S. and Zhang, X. 2014. Pyramiding bt genes for increasing resistance of cotton to two major lepidopteran pests: Spodoptera litura and Heliothis armigera. Acta Physiologiae Plantarum, 36 (10): 2717 - 2727.

Li, Z. H., Xie, Y. and Zhou, X. P. 2005. Tobacco curly shoot virus DNAβ is not necessary for infection but intensifies symptoms in a host-dependent manner. Phytopathology, 95 (8): 902 - 908.

Liedl, B. E., Lawson, D. M., White, K. K., Shapiro, J. A., Cohen, D. E., Carson, W. G., Trumble, J. T. and Mutschler, M. A. 1995. Acylsugars of wild tomato Lycopersicon pennellii alters settling and reduces oviposition of bemisia argentifolii (homoptera: Aleyrodidae). Journal of Economic Entomology, 88 (3): 742 - 748.

Lima, A. T., Sobrinho, R. R., Gonzalez-Aguilera, J., Rocha, C. S., Silva, S. J., Xavier, C. A., Silva, F. N., Duffy, S. and Zerbini, F. M. 2013. Synonymous site variation due to recombination explains higher genetic variability in begomovirus populations infecting non-cultivated hosts. Journal of General Virology, 94 (2): 418 - 431.

Lin, P.-H., Aronson, W. and Freedland, S. J. 2015. Nutrition, dietary interventions and prostate cancer: The latest evidence. BMC medicine, 13 (1): 3.

Litvak, M. E. and Monson, R. K. 1998. Patterns of induced and constitutive monoterpene production in conifer needles in relation to insect herbivory. Oecologia, 114 (4): 531 - 540.

250

Liu, H. (2008). Viral Protein–Nucleic Acid Interaction: South (North)-Western Blot. In Plant Virology Protocols: From viral sequence to Protein Function, Springer. pp. 405 - 420.

Liu, H., Boulton, M. I. and Davies, J. W. 1997. Maize streak virus coat protein binds single- and double-stranded DNA In vitro. Journal of General Virology, 78 (6): 1265 - 1270.

Liu, H., Boulton, M. I., Oparka, K. J. and Davies, J. W. 2001a. Interaction of the movement and coat proteins of Maize streak virus: Implications for the transport of viral DNA. Journal of General Virology, 82 (1): 35 - 44.

Liu, H., Boulton, M. I., Thomas, C. L., Prior, D. a. M., Oparka, K. J. and Davies, J. W. 1999. Maize streak virus coat protein is karyophyllic and facilitates nuclear transport of viral DNA. Molecular Plant-Microbe Interactions, 12 (10): 894 - 900.

Liu, H., Lucy, A. P., Davies, J. W. and Boulton, M. I. 2001b. A single amino acid change in the coat protein of Maize streak virus abolishes systemic infection, but not interaction with viral DNA or movement protein. Molecular Plant Pathology, 2 (4): 223 - 228.

Liu, Y., Robinson, D. and Harrison, B. 1998a. Defective forms of Cotton leaf curl virus DNA-a that have different combinations of sequence deletion, duplication, inversion and rearrangement. Journal of General Virology, 79 (6): 1501 - 1508.

Liu, L., Davies, J. W. and Stanley, J. 1998b. Mutational analysis of Bean yellow dwarf virus, a geminivirus of the genus mastrevirus that is adapted to dicotyledonous plants. Journal of General Virology, 79 (9): 2265 - 2274.

Liu, S.-S., Colvin, J. and De Barro, P. J. 2012. Species concepts as applied to the whitefly Bemisia tabaci systematics: How many species are there? Journal of Integrative Agriculture, 11 (2): 176 - 186.

Lizardi, P. M., Huang, X., Zhu, Z., Bray-Ward, P., Thomas, D. C. and Ward, D. C. 1998. Mutation detection and single-molecule counting using isothermal rolling-circle amplification. Nature Genetics, 19 (3): 225 - 232.

López-Gresa, M. P., Lisón, P., Kim, H. K., Choi, Y. H., Verpoorte, R., Rodrigo, I., Conejero, V. and Bellés, J. M. 2012. Metabolic fingerprinting of Tomato mosaic virus infected Solanum lycopersicum. Journal of Plant Physiology, 169 (16): 1586 - 1596.

251

López-Gresa, M. P., Torres, C., Campos, L., Lisón, P., Rodrigo, I., Bellés, J. M. and Conejero, V. 2011. Identification of defence metabolites in tomato plants infected by the bacterial pathogen Pseudomonas syringae. Environmental and Experimental Botany, 74: 216 - 228.

Lu, Q.-Y., Wu, Z.-J., Xia, Z.-S. and Xie, L.-H. 2015. Complete genome sequence of a novel monopartite geminivirus identified in mulberry (Morus alba L.). Archives of Virology, 160 (8): 2135 - 2138.

Lucatti, A. F., Van Heusden, A. W., De Vos, R. C., Visser, R. G. and Vosman, B. 2013. Differences in insect resistance between tomato species endemic to the galapagos islands. BMC Evolutionary Biology, 13 (1): 1.

Lucatti, A. F., Van Heusden, S., Broekgaarden, C., Mumm, R., Dicke, M. and Vosman, B. 2015. Quantitative resistance against Bemisia tabaci in Solanum pennellii: Genetics and metabolomics. Journal of Integrative Plant Biology, 58 (4): 397 - 412.

Lucatti, A. F., Van Heusden, S., Broekgaarden, C., Mumm, R., Dicke, M. and Vosman, B. 2016. Quantitative resistance against bemisia tabaci in solanum pennellii: Genetics and metabolomics. Journal of Integrative Plant Biology, 58 (4): 397 - 412.

Luque, A., Sanz-Burgos, A. P., Ramirez-Parra, E., M.M., C. and Gutierrez, C. 2002. Interaction of geminivirus Rep protein with replication factor C and its potential role during geminivirus DNA replication. Virology, 302 (1): 83 - 94.

Lyttle, D. and Guy, P. 2004. First record of geminiviruses in New Zealand: Abutilon mosaic virus and Honeysuckle yellow vein virus. Australasian Plant Pathology, 33 (2): 321 - 322.

Maffei, M. E., Mithöfer, A. and Boland, W. 2007. Insects feeding on plants: Rapid signals and responses preceding the induction of phytochemical release. Phytochemistry, 68 (22): 2946 - 2959.

Mahabaleshwara, K., Chandrasekhar, N. and Govindappa, M. 2016. Phytochemical investigations of methanol leaf extracts of Randia spinosa using column chromatography, HPTLC and GC-MS. Natural Products Chemistry & Research, 4: 202.

252

Mandal B. 2010. Emerging geminiviral diseases and their management, ed. Nova Science Publishers,Inc. New York. pp.

Mandal, B. 2010. Emerging geminiviral diseases and their management. Emergence of begomovirus diseases in cucurbits in India, New York: Nova Science Publishers Inc. pp. 167 - 181.

Mansoor, S., Briddon, R. W., Zafar, Y. and Stanley, J. 2003. Geminivirus disease complexes: An emerging threat. Trends in Plant Science, 8 (3): 128 - 134.

Mansoor, S., Khan, S. H., Bashir, A., Saeed, M., Zafar, Y., Malik, K. A., Briddon, R. W., Stanley, J. and Markham, P. G. 1999. Identification of a novel circular single- stranded DNA associated with cotton leaf curl disease in pakistan. Virology, 259 (1): 190 - 199.

Mansoor, S., Khan, S. H., Saeed, M., Bashir, A., Zafar, Y., Malik, K. A. and Markham, P. G. 1997. Evidence for the association of a bipartite geminivirus with tomato leaf curl disease in Pakistan. Plant Disease, 81 (8): 958 - 958.

Mansoor, S., Zafar, Y. and Briddon, R. W. 2006. Geminivirus disease complexes: The threat is spreading. Trends in Plant Science, 11 (5): 209 - 212.

Manzoor, M. T., Ilyas, M., Shafiq, M., Haider, M. S., Shahid, A. A. and Briddon, R. W. 2014. A distinct strain of Chickpea chlorotic dwarf virus (genus Mastrevirus, family geminiviridae) identified in cotton plants affected by leaf curl disease. Archives of Virology, 159 (5): 1217 - 1221.

Mariano, A. C., Andrade, M. O., Santos, A. A., Carolino, S. M. B., Oliveira, M. L., Baracat-Pereira, M. C., Brommonshenkel, S. H. and Fontes, E. P. B. 2004. Identification of a novel receptor-like protein kinase that interacts with a geminivirus nuclear shuttle protein. Virology, 318 (1): 24 - 31.

Markham, P. G., Bedford, I. D., Liu, S. and Pinner, M. S. 1994. The transmission of geminiviruses by Bemisia tabaci. Pesticide Science, 42 (2): 123 - 128.

Martinière, A., Bak, A., Macia, J.-L., Lautredou, N., Gargani, D., Doumayrou, J., Garzo, E., Moreno, A., Fereres, A. and Blanc, S. 2013. A virus responds instantly to the presence of the vector on the host and forms transmission morphs. Elife, 2: e00183.

253

Maruthi, M., Rekha, A., Mirza, S., Alam, S. and Colvin, J. 2007. PCR-based detection and partial genome sequencing indicate high genetic diversity in bangladeshi begomoviruses and their whitefly vector, bemisia tabaci. Virus Genes, 34 (3): 373 - 385.

Matthews, R. E. F. 1979. Classification and nomenclature of viruses. Third report of the international committee on taxonomy of viruses . Intervirology, 12: 132 - 296.

Mayo, M. A., Leibowitz, M. J., Palukaitis, P., Scholthof, K.-B. G., Simon, A. E., Stanley, J., Taliansky, M., 2005. Satellites. Viiith Report of The Taxonomy of International Committee on Taxonomy of Viruses, In: Fauquet, C. M., Mayo, M.A., Maniloff, J., Desselberger, U., Ball, L.A. (Eds.), Elsevier Academic Press. pp. 1163 - 1169.

Maziar Bahreini, M. O., Fereydoon Bondarian, Majid Gholibaygian. 2015. Metabolites screening of nano elicited In vitro Iranian fennel (foeniculum vulgare). American Journal of Biology and Life Sciences, 3 (5): 194 - 198.

Mccue, G. A. 1952. The history of the use of the tomato: An annotated bibliography Annals of the Missouri Botanical Garden Missouri Botanical Garden Press. 39 (4): 289 - 348.

Mcgarry, R. C., Barron, Y. D., Carvalho, M. F., Hill, J. E., Gold, D., Cheung, E., Kraus, W. L. and Lazarowitz, S. G. 2003. A novel Arabidopsis acetyltransferase interacts with the geminivirus movement protein SNP. Plant Cell, 15 (7): 1605 - 1618.

Melgarejo, T. A., Kon, T., Rojas, M. R., Paz-Carrasco, L., Zerbini, F. M. and Gilbertson, R. L. 2013. Characterization of a new world monopartite begomovirus causing leaf curl disease of tomato in Ecuador and Peru reveals a new direction in geminivirus evolution. Journal of Virology, 87 (10): 5397 - 5413.

Miller, J. and Tanksley, S. 1990. Rflp analysis of phylogenetic relationships and genetic variation in the genus lycopersicon. Theoretical and Applied Genetics, 80 (4): 437 - 448.

MINFAL. 2013. Ministry of food, agriculture and livestock pakistan agricultural research council (parc).

254

Moffat, A. S. 1999. Geminiviruses emerge as serious crop threat. Science, 286 (5446): 1835 - 1835.

Moffatt, B. A., Stevens, Y. Y., Allen, M. S., Snider, J. D., Periera, L. A., Todorova, M. I., Summers, P. S., Weretilnyk, E. A., Martin-Caffrey, L. and Wagner, C. 2002. Adenosine kinase deficiency is associated with developmental abnormalities and reduced transmethylation. Plant Physiology, 128 (3): 812 - 821.

Mohamed, A. A., Ali, S. I., El-Baz, F. K. and Hussein, S. R. 2014. Comparative study of antioxidant activities of Nitraria retusa and quantification of its bioactive components by GC/MS. International Journal of Pharmaceutical Sciences Review and Research, 29 (1): 241 - 246.

Mohr, J. J., Clark, R., Sun, S., Androphy, E. J., Macpherson, P. and Botchan, M. R. 1990. Targeting the E1 replication protein to the papillomavirus origin of replication by complex formation with E2 transactivation. Science, 250 (4988): 1694 - 1699.

Morilla, G., Castillo, A. G., Preiss, W., Jeske, H. and Bejarano, E. R. 2006. A versatile transreplication-based system to identify cellular proteins involved in geminivirus replication. Journal of Virology, 80 (7): 3624 - 3633.

Morin, S., Ghanim, M., Sobol, I. and Czosnek, H. 2000. The GroEL protein of the whitefly Bemisia tabaci interacts with the coat protein of trasmissible and nontransmissible begomoviruses in the yeast two-hybrid system. Virology, 276 (2): 404 - 416.

Moriones, E. and Navas-Castillo, J. 2000. Tomato yellow leaf curl virus, an emerging virus complex causing epidemics worldwide. Virus Research, 71 (1): 123 - 134.

Moshe, A., Pfannstiel, J., Brotman, Y., Kolot, M., Sobol, I., Czosnek, H. and Gorovits, R. 2012. Stress responses to Tomato yellow leaf curl virus (TYLCV) infection of resistant and susceptible tomato plants are different. Metabolomics S1, 6: 2153 - 0769.

Mubin, M., Amin, I., Amrao, L., Briddon, R. W. and Mansoor, S. 2010. The hypersensitive response induced by the V2 protein of a monopartite begomovirus is countered by the C2 protein. Molecular Plant Pathology, 11 (2): 245 - 254.

Mughal, S. 1985. Viral diseases of tomato and their control. Progressive Farming Pakistan, 6 (2): 20 - 23.

255

Muigai, S., Schuster, D., Snyder, J., Scott, J., Bassett, M. and Mcauslane, H. 2002. Mechanisms of resistance in lycopersicon germplasm to the whitefly Bmisia argentifolii. Phytoparasitica, 30 (4): 347 - 360.

Muigai, S. G., Bassett, M. J., Schuster, D. J. and Scott, J. W. 2003. Greenhouse and field screening of wild lycopersicon germplasm for resistance to the whitefly bemisia argentifolii. Phytoparasitica, 31 (1): 27 - 38.

Mullineaux, P. M., Donson, J., Morris-Krsinich, B. a. M., Boulton, M. I. and Davies, J. W. 1984. The nucleotide sequence of Maize streak virus DNA. EMBO Journal, 3 (13): 3063-3068.

Muniyappa, V., Venkatesh, H. M., Ramappah.K., Kulkarni, R. S., Zeidan, M., Tarba, C.-Y., Ghanim, M. and Czosnek, H. 2000. Tomato leaf curl virus from bangalore (ToLCV-Ban4): Sequence comparison with Indian ToLCV isolates, detection in plants and insects, and vector relationships. Archives of Virology, 145 (8): 1583 - 1598.

Murphy, F. A. (1995). Virus taxonomy. Sixth report of the International Committee on Taxonomy of Viruses. Archieve of Virology, 10: 313 - 314.

Nagar, S., Pedersen, T. J., Carrick, K. M., Hanley-Bowdoin, L. and Robertson, D. 1995. A geminivirus induces expression of a host DNA synthesis protein in terminally differentiated plant cells. Plant Cell, 7 (6): 705 - 719.

Nahid, N., Amin, I., Mansoor, S., Rybicki, E. P., Van Der Walt, E. and Briddon, R. W. 2008. Two dicot-infecting mastreviruses (family geminiviridae) occur in Pakistan. Archives of Virology, 153 (8): 1441 - 1451.

Nash, T. E., Dallas, M. B., Reyes, M. I., Buhrman, G. K., Ascencio-Ibañez, J. T. and Hanley-Bowdoin, L. 2011. Functional analysis of a novel motif conserved across geminivirus Rep proteins. Journal of Virology, 85 (3): 1182 - 1192.

Navas-Castillo, J., Fiallo-Olivé, E. and Sánchez-Campos, S. 2011. Emerging virus diseases transmitted by whiteflies. Annual Review of Phytopathology, 49: 219 - 248.

Navot, N., Pichersky, E., Zeidian, M., Zamir, D. and Czosnek, H. 1991. Tomato yellow leaf curl virus: A whitefly-transmitted geminivirus with a single genomic component. Virology, 185 (1): 151 - 161.

256

Nawaz-Ul-Rehman, M. S. and Fauquet, C. M. 2009. Evolution of geminiviruses and their satellites. Febs Letters, 583 (12): 1825 - 1832.

Nawaz-Ul-Rehman, M. S., Nahid, N., Mansoor, S., Briddon, R. W. and Fauquet, C. M. 2010. Post-transcriptional gene silencing suppressor activity of two non- pathogenic alphasatellites associated with a begomovirus. Virology, 405 (2): 300 - 308.

Ng, J. C. and Falk, B. W. 2006. Virus-vector interactions mediating nonpersistent and semipersistent transmission of plant viruses. Annual Review of Phytopathology, 44: 183 - 212.

Ng, T. F. F., Duffy, S., Polston, J. E., Bixby, E., Vallad, G. E. and Breitbart, M. 2011. Exploring the diversity of plant DNA viruses and their satellites using vector-enabled metagenomics on whiteflies. PLoS One 6 (4): e19050.

Nguyen, M., Francis, D. and Schwartz, S. 2001. Thermal isomerisation susceptibility of carotenoids in different tomato varieties. Journal of the Science of Food and Agriculture, 81 (9): 910 - 917.

Nicholson, J. K. and Wilson, I. D. 2003. Understanding'global'systems biology: Metabonomics and the continuum of metabolism. Nature Reviews Drug Discovery, 2 (8): 668 - 676.

Noli, E., Conti, S., Maccaferri, M. and Sanguineti, M. 1999. Molecular characterization of tomato cultivars. Seed Science and Technology, 27 (1): 1 - 10.

Nombela, G., Beitia, F. and Muñiz, M. 2000. Variation in tomato host response to Bemisia tabaci (hemiptera: Aleyrodidae) in relation to acyl sugar content and presence of the nematode and potato aphid resistance gene mi. Bulletin of Entomological Research, 90 (2): 161 - 167.

Noonari, S., Solangi, I. N. M. S. U., Wagan, M. a. L. S. A., Kalwar, A. a. S. G. Y., Jamro, A. S. K. a. S. and Panhwar, G. M. 2015. Economic implications of tomato production in Naushahro Feroze district of Sindh Pakistan. Research on Humanities and Social Sciences, 5 (7): 158 - 170.

Noris, E., Jupin, I., Accotto, G. P. and Gronenborn, B. 1996. DNA-binding activity of the C2 protein of tomato yellow leaf curl geminivirus. Virology, 217 (2): 607-612.

257

Noris, E., Vaira, A. M., Caciagli, P., Masenga, V., Gronenborn, B. and Accotto, G. P. 1998. Amino acids in the capsid protein of Tomato yellow leaf curl virus that are crucial for systemic infection, particle formation, and insect transmission. Journal of Virology, 72 (12): 10050 - 10057.

Noueiry, A. O., Lucas, W. J. and Gilbertson, R. L. 1994. Two proteins of a plant DNA virus coordinate nuclear and plasmodesmal transport. Cell, 76 (5): 925 - 932.

Novotny, M. V., Soini, H. A. and Mechref, Y. 2008. Biochemical individuality reflected in chromatographic, electrophoretic and mass-spectrometric profiles. Journal of Chromatography B, 866 (1): 26 - 47.

O'donnell, P. J., Jones, J. B., Antoine, F. R., Ciardi, J. and Klee, H. J. 2001. Ethylene‐dependent salicylic acid regulates an expanded cell death response to a plant pathogen. The Plant Journal, 25 (3): 315 - 323.

Ohnesorge, S. and Bejarano, E. R. 2009. Begomovirus coat protein interacts with a small heat-shock protein of its transmission vector (Bemisia tabaci). Insect Molecular Biology, 18 (6): 693 - 703.

Oikawa, A., Nakamura, Y., Ogura, T., Kimura, A., Suzuki, H., Sakurai, N., Shinbo, Y., Shibata, D., Kanaya, S. and Ohta, D. 2006. Clarification of pathway-specific inhibition by fourier transform ion cyclotron resonance/mass spectrometry-based metabolic phenotyping studies. Plant Physiology, 142 (2): 398 - 413.

Okhale, S. E., Buba, C. I., Oladosu, P., Egharevba, H., Ugbabe, G., Ibrahim, J. and Kunle, O. F. 2016. Chemical constituents and antimicrobial activity of the leaf essential oil of Garcinia kola heckel (clusiaceae) from Nigeria. American Chemical Science Journal, 4 (13 ): 1 - 7.

Olmstead, R. G. and Bohs, L. 2006. A summary of molecular systematic research in solanaceae: 1982-2006. In VI International Solanaceae Conference: Genomics Meets Biodiversity 745: 255 - 268.

Oms-Oliu, G., Hertog, M., Van De Poel, B., Ampofo-Asiama, J., Geeraerd, A. and Nicolaï, B. 2011. Metabolic characterization of tomato fruit during preharvest development, ripening, and postharvest shelf-life. Postharvest Biology and Technology, 62 (1): 7 KNHN- 16.

258

Orozco, B. M. and Hanley-Bowdoin, L. 1996. A DNA structure is required for geminivirus replication origin function. Journal of Virology, 7 (1): 148 - 158.

Orozco, B. M., Miller, A. B., Settlage, S. B. and Hanley-Bowdoin, L. 1997. Functional domains of a geminivirus replication protein. Journal of Biological Chemistry, 272 (15): 9840 - 9846.

Osorio, S. Do, P. T. and Fernie, A. R. 2012. Profiling primary metabolites of tomato fruit with gas chromatography/mass spectrometry. Plant Metabolomics, methods and protocols, 3: 101 - 109.

Padidam, M., Beachy, R. N. and Fauquet, C. M. 1996. The role of AV2 ("precoat") and coat protein in viral replication and movement in tomato leaf curl geminivirus. Virology, 224 (2): 390 - 404.

Padidam, M., Beachy, R. N. and Fauquet, C. M. 1995. Tomato leaf curl geminivirus from India has a bipartite genome and coat protein is not essential for infectivity. Journal of General Virology, 76 (1): 25 - 35.

Padidam, M., Sawyer, S. and Fauquet, C. M. 1999. Possible emergence of new geminiviruses by frequent recombination. Virology, 265 (2): 218 - 225.

Page, R. D. M. 1996. Treeview: An application to display phylogenetic trees on personal computers. Computer Applied Biosciences, 12: 357 - 358.

Pakissan. 2015. Tomato exports to pakistan takes a beating with soaring prices. Retrieved from http://www.pakissan.com/english/news/newsDetail.php?newsid=30181

Palanichelvam, K., Kunik, T., Citovsky, V. and Gafni, Y. 1998. The capsid protein of Tomato yellow leaf curl virus binds cooperatively to single-stranded DNA. Journal of General Virology, 79 (11): 2829 - 2833.

Palmer, K. E. and Rybicki, E. P. 1998. The molecular biology of mastreviruses. Advances in virus research, 50: 183 - 234.

Palomo, I., Fuentes, E., Padró, T. and Badimon, L. 2012. Platelets and atherogenesis: Platelet anti-aggregation activity and endothelial protection from tomatoes (Solanum lycopersicum L.)(review). Experimental and therapeutic medicine, 3 (4): 577 - 584.

259

Pandey, P., Choudhury, N. R. and Mukherjee, S. K. 2009. A geminiviral amplicon (va) derived from tomato leaf curl virus (ToLCV) can replicate in a wide variety of plant species and also acts as a vigs vector. Virology Journal, 6: 152.

Pant, V., Gupta, D., Choudhury, N. R., Malathi, V. G., Varma, A. and Mukherjee, S. K. 2001. Molecular characterization of the Rep protein of the blackgram isolate of Indian mungbean yellow mosic virus. Journal of General Virology, 82 (10): 2559 - 2567.

Paprotka, T., Metzler, V. and Jeske, H. 2010. The first DNA 1-like alpha satellites in association with new world begomoviruses in natural infections. Virology, 404 (2): 148 - 157.

Park, Y. H., West, M. A. and St. Clair, D. A. 2004a. Evaluation of AFLPS for germplasm fingerprinting and assessment of genetic diversity in cultivars of tomato (Lycopersicon esculentum L.). Genome, 47 (3): 510 - 518.

Park, J., Hwang, H., Shim, H., Im, K., Auh, C. K., Lee, S. and Davis, K. R. 2004b. Altered cell shapes, hyperplasia, and secondary growth in Arabidopsis caused by beet curly top geminivirus infection. Molecules and Cells, 17 (1): 117 - 124.

Pascal, E., Goodlove, P. E., Wu, L. C. and Lazarowitz, S. G. 1993. Transgenic tobacco plants expressing the geminivirus BL1 protein exhibit symptoms of viral disease. Plant Cell, 5 (7): 795 - 807.

Pasumarthy, K. K., Choudhury, N. R. and Mukherjee, S. K. 2010. Tomato leaf curl kerala virus (ToLCKeV) AC3 protein forms a higher order oligomer and enhances ATPase activity of replication initiator protein (Rep/AC1). Virology Journal, 7 (1): 1.

Pasumarthy, K. K., Mukherjee, S. K. and Choudhury, N. R. 2011. The presence of Tomato leaf curl kerala virus AC3 protein enhances viral DNA replication and modulates virus induced gene-silencing mechanism in tomato plants. Virolology Journal, 8 (1): 1.

Patil, B. L. and Fauquet, C. 2010. Differential interaction between cassava mosaic geminiviruses and geminivirus satellites. Journal of General Virology, 91 (7): 1871 - 1882.

Patil, B. L., Rajasubramaniam, S., Bagchi, C. and Dasgupta, I. 2004. Both Indian cassava mosaic virus and Sri Lankan cassava mosaic virus are found in India and

260 exhibit high variability as assessed by PCR-RFLP. Archives of Virology, 150 (2): 389 - 397.

Paximadis, M., Idris, A. M., Torres-Jerez, I., Villarreal, A., Rey, M. E. C. and Bron, J. K. 1999. Characterization of tobacco geminiviruses in the Old and New World. Archives of Virology, 144 (4): 703 - 717.

Peralta, I. E., & Spooner, D. M. 2006. History, origin and early cultivation of tomato (Solanaceae). Genetic improvement of Solanaceous crops, 2:1 - 27.

Permoda-Osip, Agnieszka Dorszewska, Jolanta, Skibinska, Maria Chlopocka- Wozniak, Maria Rybakowski & K, J. 2013. Hyperhomocysteinemia in bipolar depression: clinical and biochemical correlates. Neuropsychobiology, 68, 193 - 196.

Perring, T. M., Cooper, A. D., Rodriguez, R. J., Farrar, C. A. and Bellows, T. 1993. Identification of a whitefly species by genomic and behavioral studies. Science, 259 (5091): 74 - 77.

Pilartz, M. and Jeske, H. 2003. Mapping of abutilon mosaic geminivirus minichromosomes. Journal of Virology, 77 (20): 10808 - 10818.

Piroux, N., Saunders, K., Page, A. and John., S. 2007. Geminivirus pathogenicity protein C4 interacts with Arabidopsis thaliana shaggy-related protein kinase atskη, a component of the brassinosteroid signalling pathway. Virology, 362 (2): 428 - 440.

Polston, J. and Anderson, P. 1997. The emergence of whitefly-transmitted geminiviruses in tomato in the Western Hemisphere. Plant Disease, 81 (12): 1358 - 1369.

Polston, J. E., Mcgovern, R. J. and Brown, L. G. 1999. Introduction of Tomato yellow leaf curl virus in Florida and implications for the spread of this and other geminiviruses of tomato. Plant disease, 83 (11): 984 - 988.

Pooggin, M. M. 2013. How can plant DNA viruses evade sirna-directed DNA methylation and silencing? International Journal of Molecular Sciences, 14 (8): 15233 - 15259.

Pooma, W., Gillette, W. K., Jeffrey, J. L. and Petty, I. T. D. 1996. Host and viral factors determine the disspensability of coat protein for bipartite geminivirus systemic movement. Virology, 218 (1): 264 - 268.

261

Pooma, W. and Petty, I. T. D. 1996. Tomato golden mosaic virus open reading frame AL4 is genetically distinct from its C4 analogue in monopartite geminiviruses. Journal of General Virology, 77 (8): 1947 - 1951.

Power, A. G. 2000. Insect transmission of plant viruses: A constraint on virus variability. Current Opinion in Plant Biology, 3 (4): 336 - 340.

Prasanna, H., Sinha, D., Rai, G., Krishna, R., Kashyap, S. P., Singh, N., Singh, M. and Malathi, V. 2015a. Pyramiding Ty‐2 and Ty‐3 genes for resistance to monopartite and bipartite tomato leaf curl viruses of India. Plant Pathology, 64 (2): 256 - 264.

Prasanna, H., Kashyap, S. P., Krishna, R., Sinha, D., Reddy, S. and Malathi, V. 2015b. Marker assisted selection of Ty-2 and Ty-3 carrying tomato lines and their implications in breeding tomato leaf curl disease resistant hybrids. Euphytica, 204 (2): 407 - 418.

Prasanna, H. C. and Rai, M. 2007. Detection and frequency of recombination in tomato-infecting begomoviruses of South and Southeast Asia. Virology Journal, 4 (1): 111.

Preiss, W. and Jeske, H. 2003. Multitasking in replication is common among geminiviruses. Journal of Virology, 77 (5): 2972 - 2980.

Purcell, A. H. and Almeida, R. P. 2005. Insects as vectors of disease agents. Encyclopedia of Plant and Crop Science, 5.

Qazi, J., Amin, I., Mansoor, S., Iqbal, M. J. and Briddon, R. W. 2007. Contribution of the satellite encoded gene βC1 to cotton leaf curl disease symptoms. Virus Research, 128 (1): 135 - 139.

Radhakrishnan, G. K., Splitter, G. A. and Usha, R. 2008. DNA recognition properties of the cell-to-cell movement protein (mp) of soybean isolate of mungbean yellow mosaic india virus (mymiv-sb). Virus Research, 131 (2): 152 - 159.

Raja, P., Sanville, B. C., Buchmann, R. C. and Bisaro, D. M. 2008. Viral genome methylation as an epigenetic defense against geminiviruses. Journal of Virology, 82 (18): 8997 - 9007.

Raja, P., Wolf, J. N. and Bisaro, D. M. 2010. RNA silencing directed against geminiviruses: Post-transcriptional and epigenetic components. Biochimica et Biophysica Acta, 1799 (3-4): 337 - 351.

262

Rajagopalan, P. A., Naik, A., Katturi, P., Kurulekar, M., Kankanallu, R. S. and Anandalakshmi, R. 2012. Dominance of resistance-breaking Cotton leaf curl Burewala virus (CLCuBuV) in NorthWestern India. Archives of Virology, 157 (5): 855 - 868.

Rajeswaran, R., Sunitha, S., Shivaprasad, P. V., Pooggin, M. M., Hohn, T. and Veluthambi, K. 2007. The mungbean yellow mosaic begomovirus transcriptional activator protein transactivates the viral promoter-driven transgene and causes toxicity in transgenic tobacco plants. Molecular Plant-Microbe Interactions, 20 (12): 1545 - 1554.

Rajeswari, G., Murugan, M. and Mohan, V. 2013. GC-MS analysis of bioactive components of Hugonia mystax L.(linaceae). Research Journal of Pharmaceutical, Biological and Chemical Sciences, 3 (4): 301 - 308.

Rana, B., Singh, U. and Taneja, V. 1997. Antifungal activity and kinetics of inhibition by essential oil isolated from leaves of aegle marmelos. Journal of Ethnopharmacology, 57 (1): 29 - 34.

Rana, V. S., Singh, S. T., Priya, N. G., Kumar, J. and Rajagopal, R. 2012. Arsenophonus GroEL interacts with CLCuV and is localized in midgut and salivary gland of whitefly b. Tabaci. PLoS One, 7 (8): e42168.

Rao, A., Ray, M. and Rao, L. 2006. Lycopene. Advances in food and nutrition research, 51: 99 - 164.

Rao, A., Waseem, Z. and Agarwal, S. 1998. Lycopene content of tomatoes and tomato products and their contribution to dietary lycopene. Food Research International, 31 (10): 737 - 741.

Rathore, S., Bhatt, B., Yadav, B., Kale, R. and Singh, A. 2014. A new begomovirus species in association with betasatellite causing tomato leaf curl disease in Gandhinagar, Iindia. Plant disease, 98 (3): 428 - 428.

Razavinejad, S., Heydarnejad, J., Kamali, M., Massumi, H., Kraberger, S. and Varsani, A. 2013. Genetic diversity and host range studies of Turnip curly top virus. Virus Genes, 46 (2): 345 - 353.

263

Reddy, A., Patti, M., Reddy, K. and Venkataravanappa, V. 2011. Detection and diagnosis of Tomato leaf curl virus infecting tomato in Northern Karnataka. African Journal of Agricultural Research, 6 (5): 1051 - 1057.

Reisenman, C. E., Riffell, J. A., Duffy, K., Pesque, A., Mikles, D. and Goodwin, B. 2013. Species-specific effects of herbivory on the oviposition behavior of the moth Manduca sexta. Journal of Chemical Ecology, 39 (1): 76 - 89.

Rhee, Y., Gurel, F., Gafni, Y., Dingwall, C. and Citovsky, V. 2000. A genetic system for detection of protein nuclear import and export. Nature Biotechnology, 18 (4): 433 - 437.

Richter, K. S., Götz, M., Winter, S. and Jeske, H. 2016. The contribution of translesion synthesis polymerases on geminiviral replication. Virology, 488: 137 - 148.

Rick, C. M. 1978. The tomato. Scientific American Journal, 239 (8): 67 - 76.

Rigden, J. E., Dry, I. B., Mullineaux, P. M. and Rezaian, M. A. 1993. Mutagensis of the virion-sense open reading frames of tomato leaf curl virus. Virology, 193 (2): 1001 - 1005.

Rigden, J. E., Krake, L. R., Rezaian, M. A. and Dry, I. B. 1994. ORF C4 of tomato leaf curl geminivirus is a determinant of symptom severity. Virology, 204 (2): 847 - 850.

Roberts, S. and Stanley, J. 1994. Lethal mutations within the conserved stem-loop of African cassava mosaic virus DNA are rapidly corrected by genomic recombination. Journal of General Virology, 75 (11): 3203 - 3209.

Robertson, D. G. 2005. Metabonomics in toxicology: A review. Toxicological Sciences, 85 (2): 809 - 822.

Rodriguez-Negrete, E., Lozano-Duran, R., Piedra-Aguilera, A., Cruzado, L., Bejarano, E. R. and Castillo, A. G. 2013. Geminivirus Rep protein interferes with the plant DNA methylation machinery and suppresses transcriptional gene silencing. New Phytologist, 199 (2): 464 - 475.

Roessner-Tunali, U., Hegemann, B., Lytovchenko, A., Carrari, F., Bruedigam, C., Granot, D. and Fernie, A. R. 2003. Metabolic profiling of transgenic tomato plants

264 overexpressing hexokinase reveals that the influence of hexose phosphorylation diminishes during fruit development. Plant Physiology, 133 (1): 84 - 99.

Roessner, U., Luedemann, A., Brust, D., Fiehn, O., Linke, T., Willmitzer, L. and Fernie, A. R. 2001. Metabolic profiling allows comprehensive phenotyping of genetically or environmentally modified plant systems. The Plant Cell, 13 (1): 11 - 29.

Rojas, M., Hagen, C., Lucas, W. and Gilbertson, R. 2005. Exploiting chinks in the plant's armor: Evolution and emergence of geminiviruses. Annual Review of Phytopathology, 43: 361 - 394.

Rojas, M. R. and Gilbertson, R. L. 2000. Emerging plant viruses: a diversity of mechanisms and opportunities. Plant virus evolution, Berlin Heidelberg: Springer. pp. 27 - 51.

Rojas, M., Kon, T., Natwick, E., Polston, J., Akad, F. and Gilbertson, R. 2007. First report of Tomato yellow leaf curl virus associated with tomato yellow leaf curl disease in California. Plant disease, 91 (8): 1056 - 1056.

Rojas, M. R., Jiang, H., Salati, R., Xoconostle-Cazares, B., Sudarshana, M. R., Lucas, W. J. and Gilbertson, R. L. 2001. Functional analysis of proteins involved in movement of the monopartite begomovirus, Tomato yellow leaf curl virus. Virology, 291 (1): 110 - 125.

Rojas, M. R. and Gilbertson, R. L. (2008). Emerging plant viruses: a diversity of mechanisms and opportunities. In Plant virus evolution, Berlin Heidelberg, Springer. pp. 27 - 51.

Rojas, M. R., Noueiry, A. M., Lucas, W. J. and Gilbertson, R. L. 1998. Bean dwarf mosaic geminivirus movement proteins recognize DNA in a form- and size-specific manner. Cell, 95 (1): 105 - 113.

Romay, G., Chirinos, D., Geraud-Pouey, F. and Desbiez, C. 2010. Association of an atypical alphasatellite with a bipartite new world begomovirus. Archives of Virology, 155 (11): 1843 - 1847.

Rosen, R., Kanakala, S., Kliot, A., Pakkianathan, B. C., Farich, B. A., Santana-Magal, N., Elimelech, M., Kontsedalov, S., Lebedev, G. and Cilia, M. 2015. Persistent,

265 circulative transmission of begomoviruses by whitefly vectors. Current opinion in Virology, 15: 1 - 8.

Roth, B. M., Pruss , G. J. and Vance, V. B. 2004. Plant viral suppressors of RNA silencing. Virus Research, 102 (1): 97 - 108.

Rothenstein, D., Krenz, B., Selchow, O. and Jeske, H. 2007. Tissue and cell tropism of Indian cassava mosaic virus (ICMV) and its AV2 (precoat) gene product. Virology, 359 (1): 137 - 145.

Rouhibakhsh, A. and Malathi, V. G. 2005. Severe leaf curl disease of cowpea – a new disease of cowpea in Northern India caused by mungbean yellow mosaic India virus and a satellite DNA. Plant Pathology 54 (2): 259 - 259.

Ryals, J. A., Neuenschwander, U. H., Willits, M. G., Molina, A., Steiner, H.-Y. and Hunt, M. D. 1996. Systemic acquired resistance. The Plant Cell, 8 (10): 1809.

Rybicki, E. 1994. A phylogenetic and evolutionary justification for three genera of geminiviridae. Archives of Virology, 139 (1-2): 49 - 77.

Rybicki, E. P. and Pietersen, G. 1999. Plant virus disease problems in the developing world. Advances in virus research, 53: 127 - 175.

Saeed, M., Behjatania, S. a. A., Mansoor, S., Zafar, Y., Hasnain, S. and Rezaian, M. A. 2005. A single complementrary-sense transcript of a geminiviral DNA β satellite is determinant of pathogenicity. Molecular Plant-Microbe Interactions, 18 (1): 7 - 14.

Saeed, M., Mansoor, S., Rezaian, M. A., Briddon, R. W. and Randles, J. W. 2008. Satellite DNA beta overrides the pathogenicity phenotype of the C4 gene of tomato leaf curl virus but does not compensate for loss of function of the coat protein and V2 genes. Archives of Virology, 153 (7): 1367 - 1372.

Saeed, M., Zafar, Y., Randles, J. W. and Rezaian, M. A. 2007. A monopartite begomovirus-associated DNA beta satellite substitutes for the DNA b of a bipartite begomovirus to permit systemic infection. Journal of General Virology, 88 (10): 2881 - 2889.

Saghatelian, A., Trauger, S. A., Want, E. J., Hawkins, E. G., Siuzdak, G. and Cravatt, B. F. 2004. Assignment of endogenous substrates to enzymes by global metabolite profiling. Biochemistry, 43 (45): 14332 - 14339.

266

Sahu, P. P., Sharma, N., Puranik, S., Muthamilarasan, M. and Prasad, M. 2014. Involvement of host regulatory pathways during geminivirus infection: A novel platform for generating durable resistance. Functional & integrative genomics, 14 (1): 47 - 58.

Salati, R., Nahkla, M. K., Rojas, M. R., Guzman, P., Jaquez, J., Mazxwell, D. and Gilbertson, R. L. 2002. Tomato yellow leaf curl virus in the dominican republic: Charcaterization of an infectious clone, virus monitoring in whiteflies and identification of reservoir hosts. Phytopathoplogy, 92 (5): 487 - 496.

Salvaudon, L., De Moraes, C. M. and Mescher, M. C. 2013. Outcomes of co-infection by two potyviruses: Implications for the evolution of manipulative strategies. Proceedings of the Royal Society of London B: Biological Sciences, 280 (1756): 20122959.

Sambrook, J., Frisch, E. F. and Maniatis, T. 1989. Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, New York.

Samuelsson, L. M. and Larsson, D. J. 2008. Contributions from metabolomics to fish research. Molecular BioSystems, 4 (10): 974 - 979.

Sánchez-Campos, S., Martínez-Ayala, A., Márquez-Martín, B., Aragón-Caballero, L., Navas-Castillo, J. and Moriones, E. 2013. Fulfilling koch's postulates confirms the monopartite nature of Tomato leaf deformation virus: A begomovirus native to the New World. Virus Research, 173 (2): 286 - 293.

Sanderfoot, A. A. and Lazarowitz, S. G. 1996. Getting it together in plant virus movement: Cooperative interactions between bipartite geminivirus movement proteins. Trends in Cell Biology, 6 (9): 353 - 358.

Santos, A. A., Lopes, K. V., Apfata, J. A. and Fontes, E. P. 2010. NSP-interacting kinase, NIK: A transducer of plant defence signalling. Journal of Experimental Botany, erq219.

Sanz, A. I., Fraile, A., Gallego, J. M., Malpica, J. M. and Garía-Arenal, F. 1999. Genetic variability of natural populations of cotton leaf curl geminivirus, single- stranded DNA virus. Journal of Molecular Evolution, 49 (5): 672 - 681.

San , A. I., raile, A., Garc a-Arenal, F., Zhou, X., Robinson, D. J., Khalid, S., Butt, T. and Harrison, B. D. 2000. Multiple infection, recombination and genome

267 relationships among begomovirus isolates found in cotton and other plants in Pakistan. Journal of General Virology, 81 (7): 1839 - 1849.

Särkinen, T., Bohs, L., Olmstead, R. G. and Knapp, S. 2013. A phylogenetic framework for evolutionary study of the nightshades (solanaceae): A dated 1000-tip tree. BMC Evolutionary Biology, 13 (1): 1.

Sato, R., Helzlsouer, K. J., Alberg, A. J., Hoffman, S. C., Norkus, E. P. and Comstock, G. W. 2002. Prospective study of carotenoids, tocopherols, and retinoid concentrations and the risk of breast cancer. Cancer Epidemiology Biomarkers & Prevention, 11 (5): 451 - 457.

Sattar, M. N., Kvarnheden, A., Saeed, M. and Briddon, R. W. 2013. Cotton leaf curl disease - an emerging threat to cotton production worldwide. Journal of General Virology, 94 (4): 695 - 710.

Saunders, K., Bedford, I. D., Briddon, R. W., Markham, P. G., Wong, S. M. and Stanley, J. 2000. A unique virus complex causes Ageratum yellow vein disease. Proceedings of the National Academy of Sciences, 97 (12): 6890 - 6895.

Saunders, K., Bedford, I. D. and Stanley, J. 2001a. Pathogenicity of a natural recombinant associated with ageratum yellow vein disease: Implications for geminivirus evolution and disease aetiology. Virology, 282 (1): 38 - 47.

Saunders, K., Bedford, I. D., Yahara, T. and Stanley, J. 2003. Aetiology the earliest recorded plant virus disease. Nature, 422 (6934): 831-831.

Saunders, K., Lucy, A. and Stanley, J. 1991. DNA forms of the geminivirus African cassava mosaic virus consistent with a rolling circle mechanism of replication. Nucleic Acids Research, 19 (9): 2325 - 2330.

Saunders, K., Norman, A., Gucciardo, S. and Stanley, J. 2004. The DNA β satellite component associated with ageratum yellow vein disease encodes an essential pathogenicity protein (BetaC1). Virology, 324 (1): 37 - 47.

Saunders, K. and Stanley, J. 1995. Complementation of African cassava mosaic virus AC2 gene function in a mixed bipartite geminivirus infection. Journal of General Virology, 76 (9): 2287 - 2292.

268

Saunders, K. and Stanley, J. 1999. A nanovirus-like component associated with yellow vein disease of Ageratum conyzoides: Evidence for inter-family recombination between plant DNA viruses. Virology, 264 (1): 142 - 152.

Saunders, K., Wege, C., Veluthambi, K., Jeske, H. and Stanley, J. 2001b. The distinct disease phenotypes of the common and yellow vein strains of Tomato golden mosaic virus are determined by nucleotide differences in the 3′-terminal region of the gene encoding the movement protein. Journal of General Virology, 82 (1): 45 - 51.

Sauter, H., Lauer, M. and Fritsch, H. 1991. Metabolic profiling of plants: A new diagnostic technique. ACS Symposium series-American Chemical Society (USA).

Sawangjit, S., Chatchawankanphanich, O., Chiemsombat, P., Attathom, T., Dale, J. and Attathom, S. 2005. Molecular characterization of tomato-infecting begomoviruses in Thailand. Virus Research, 109 (1): 1 - 8.

Schauer, N., Zamir, D. and Fernie, A. R. 2005. Metabolic profiling of leaves and fruit of wild species tomato: A survey of the Solanum lycopersicum complex. Journal of Experimental Botany, 56 (410): 297 - 307.

Schelz, Z., Molnar, J. and Hohmann, J. 2006. Antimicrobial and antiplasmid activities of essential oils. Fitoterapia, 77 (4): 279 - 285.

Schneider, L. 1969. Satellite-like particle of Tobacco ringspot virus that resembles Tobacco ringspot virus. Science, 166 (3913): 1627 - 1629.

Schoor, S. and Moffatt, B. 2004. Applying high throughput techniques in the study of adenosine kinase in plant metabolism and development. Frontiers in bioscience: a journal and virtual library, 9: 1771 - 1781.

Schuhmacher, R., Krska, R., Weckwerth, W. and Goodacre, R. 2013. Metabolomics and metabolite profiling. Analytical and Bioanalytical Chemistry, 405 (15): 5003.

Scott, J. W., Stevens, M. R., Barten, J. H. M., Thome, C. R., Polston, J. E., Schuster, D. J. and Serra, C. A. 1996. Introgression of resistance to whitefly-transmitted geminiviruses from Lycopersicon chilense to tomato. Bemisia: 1995, taxonomy, biology, damage, control and management.

Seal, S. E., Van Den Bosch, F. and Jeger, M. J. 2006a. Factors influencing begomovirus evolution and their increasing global significance: Implications for sustainable control. Critical Reviews in Plant Science, 25 (1): 23 - 46.

269

Seal, S. E., Jeger, M. J. and Van Den Bosch, F. 2006b. Begomovirus evolution and disease management. Advances in virus research, 67: 297 - 316.

Selth, L. A., Randles, J. W. and Rezaian, M. A. 2004. Host responses to transient expression of individual genes encoded by Tomato leaf curl virus. Molecular Plant- Microbe Interactions, 17 (1): 27 - 33.

Serfraz, S., Amin, I., Akhtar, K. P. and Mansoor, S. 2015. Recombination among begomoviruses on malvaceous plants leads to the evolution of Okra enation leaf curl virus in Pakistan. Journal of Phytopathology, 163 (9): 764 - 776.

Settlage, S. B., Miller, A., Gruissem, W. and Hanley-Bowdoin, L. 2001. Dual interaction of a geminivirus replication accessory factor with a viral replication protein and a plant cell cycle regulator. Virology, 279 (2): 570 - 576.

Settlage, S. B., See, R. G. and Hanley-Bowdoin, L. 2005. Geminivirus C3 protein: Replication enhancement and protein interactions. Journal of Virology, 79 (15): 9885 - 9895.

Shahid, M., Ali, L. and Wajid, S. 2009. Cotton leaf curl Rajasthan virus infecting tomato in Pakistan. Pakistan Journal of Scientific and Industrial Research, 52 (6): 319 - 321.

Shahid, M., Rehman, A. U., Khan, A. U., Mahmood, A., Firoza, K., Shahina, F. and Soomro, M. H. 2007. Geographical distribution and infestation of plant parasitic nematodes on vegetables and fruits in the Punjab province of Pakistan. Pakistan Journal of Nematology , 25 (1): 59 - 67. Pakistan Society of Nematologists.

Shahid, M. S., Ito, T., Kimbara, J., Onozato, A., Natsuaki, K. T. and Ikegami, M. 2013. Evaluation of tomato hybrids carrying Ty‐1 and Ty‐2 loci to japanese monopartite begomovirus species. Journal of Phytopathology, 161 (3): 205 - 209.

Sharma, P. and Ikegami, M. 2009. Characterization of signals that dictate nuclear/nucleolar and cytoplasmic shuttling of the capsid protein of Tomato leaf curl Java virus associated with DNAβ satellite. Virus Research, 144 (1): 145 - 153.

Shelat, M., Murari, S., Sharma, M., Subramanian, R., Jummanah, J. and Jarullah, B. 2014. Prevalence and distribution of Tomato leaf curl virus in major agroclimatic zones of Gujarat. Advances in Bioscience and Biotechnology, 5 (1): 1.

270

Shepherd, D. N., Martin, D. P., Van Der Walt, E., Dent, K., Varsani, A. and Rybicki, E. P. 2010. Maize streak virus: An old and complex ‘emerging’pathogen. Molecular Plant Pathology, 11 (1): 1 - 12.

Shi, A., Chen, P., Li, D., Zheng, C., Zhang, B. and Hou, A. 2009. Pyramiding multiple genes for resistance to Soybean mosaic virus in soybean using molecular markers. Molecular Breeding, 23 (1): 113 - 124.

Shivaprasad, P. V., Akbergenov, R., Trinks, D., Rajeswaran, R., Veluthambi, K., Hohn, T. and Pooggin, M. M. 2005. Promoters, transcripts, and regulatory proteins of Mungbean yellow mosaic geminivirus. Journal of Virology, 79 (13): 8149 - 8163.

Shusei., Satoshi, T. H. H., Erika, A., Kenta, S., Sachiko, I., Takakazu, K.,Yasukazu, N. and Daisuke, S. M. E. 2012. The tomato genome sequence provides insights into fleshy fruit evolution. Nature, 485 (7400): 635 - 641.

Silva, F. N., Lima, A. T., Rocha, C. S., Castillo-Urquiza, G. P., Alves-Junior, M. and Zerbini, F. M. 2014. Recombination and pseudorecombination driving the evolution of the begomoviruses Tomato severe rugose virus (ToSRV) and Tomato rugose mosaic virus (ToRMV): Two recombinant DNA-A components sharing the same DNA-B. Virology Journal, 11 (1): 1.

Singh-Pant, P., Pant, P., Mukherjee, S. K. and Mazumdar-Leighton, S. 2012. Spatial and temporal diversity of begomoviral complexes in papayas with leaf curl disease. Archives of Virology, 157 (7): 1217 - 1232.

Singh, A. K., Chattopadhyay, B. and Chakraborty, S. 2012. Biology and interactions of two distinct monopartite begomoviruses and betasatellites associated with radish leaf curl disease in India. Virology Journal, 9 (1): 1.

Singh, D. K., Malik, P. S., Choudhury, N. R. and Mukherjee, S. K. 2008. MYMIV replication initiator protein (Rep): Roles at the initiation and elongation steps of MYMIV DNA replication. Virology, 380 (1): 75 - 83.

Singh, R., Kakkar, A. and Mishra, V. K. 2015. Phytochemimcal analysis of Wrightia tinctoria bark extract in water using GC-MS. International Journal of Pharmacy and Pharmaceutical Sciences, 7 (3): 470 - 472.

271

Sivakumar, V. and Gayathri, G. 2015. GC-MS analysis of bioactive components from ethanol extract of Andrographis paniculata. World Journal of Pharmacy and Pharmaceutical Sciences, 4 (11): 2031 - 2039.

Skaljac, M. and Ghanim, M. 2010. Tomato yellow leaf curl disease and plant-virus vector interactions. Israel Journal of Plant Sciences, 58 (2): 103 - 111.

Smith, A. F. 1994. The tomato in america: Early history, culture, and cookery, ed. University of Illinois Press.

Soleimani, R., Matic, S., Taheri, H., Behjatnia, S. a. A., Vecchiati, M., Izadpanah, K. and Accotto, G. P. 2013. The unconventional geminivirus Beet curly top Iran virus satisfying koch’s postulates and determining vector and host range. Annals of Applied Biology, 62 (2): 174 - 181.

Soto, M. J., Chen, L. F., Seo, Y. S. and Gilbertson, R. L. 2005. Identification of regions of the Beet mild curly top virus (family geminiviridae) capsid protein involved in systemic infection, virion formation and leafhopper transmission. Virology, 341 (2): 257 - 270.

Soto, M. J. and Gilbertson, R. L. 2003. Distribution and rate of movement of the curtovirus Beet mild curly top virus (family geminiviridae) in the beet leafhopper. Phytopathology, 93 (4): 478 - 484.

Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology, 98 (3): 503 - 508.

Stahl, W., Heinrich, U., Aust, O., Tronnier, H. and Sies, H. 2006. Lycopene-rich products and dietary photoprotection. Photochemical & Photobiological Sciences, 5 (2): 238 - 242.

Stanley, J. 1983. Infectivity of the cloned geminivirus genome requires sequences from both DNAs. Nature, 305: 643 - 645.

Stanley, J., Bisaro, D. M., Briddon, R. W., Brown, J. K., Fauquet, C. M., Harrison, B. D., Rybicki, E. P. and strenger, D. C. 2005. Geminiviriae. In Virus Taxonomy, VIIIth Report of the International Committee on Taxonomy of Viruses, Edited by C. M. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger & L. A. Ball. London: Elsevier/Academic Press. pp. 1163 - 1169.

272

Stanley, J. and Latham, J. R. 1992. A symptom variant of beet curly top geminivirus produced by mutation of open reading frame C4. Virology, 190 (1): 506 - 509.

Stanley, J., Latham, J. R., Pinner, M. S., Bedford, I. and Markham, P. G. 1992. Mutational analysis of the monopartite geminivirus Beet curly top virus. Virology, 191 (1): 396 - 404.

Stanley, J., Markham, P. G., Callis, R. J. and Pinner, M. S. 1986. The nucleotide sequence of an infectious clone of the geminivirus Beet curly top virus. EMBO Journal, 5 (8): 1761 - 1767.

Stanley, J. and Townsend, R. 1986. Infectious mutants of Cassava latent virus generated In vivo from intact recombinant DNA clones containing single copies of the genome. Nucleic Acids Research, 14 (15): 5981 - 5999.

Stenger, D. C. 1994. Complete nucleotide sequence of the hypervirulent CFH strain of Beet curly top virus. Molecular Plant-Microbe Interactions, 7 (1): 154 - 157.

Stenger, D. C. and Mcmahon, C. L. 1997. Genotypic diversity of Beet curly top virus populations in the Western United States. Phytopathology, 87 (7): 737 - 744.

Storey, H. 1936. Virus diseases of east african plants. VI-A progress report on studies of the disease of cassava. East African Agricultural Journal, 2: 34 - 39.

Storey, H. and Nichols, R. 1938. Studies of the mosaic diseases of cassava. Annals of Applied Biology, 25 (4): 790 - 806.

Story, E. N., Kopec, R. E., Schwartz, S. J. and Harris, G. K. 2010. An update on the health effects of tomato lycopene. Annual review of food science and technology, 1.

Sudarshana, M. R., Wang, H. L., Lucas, W. J. and Gilbertson, R. L. 1998. Dynamics of bean dwarf mosaic geminivirus cell-to-cell and long-distance movement in Phaseolus vulgaris revealed, using the green fluorescent protein. Molecular Plant- Microbe Interactions, 11 (4): 277 - 291.

Suh, J.-P., Jeung, J.-U., Noh, T.-H., Cho, Y.-C., Park, S.-H., Park, H.-S., Shin, M.-S., Kim, C.-K. and Jena, K. K. 2013. Development of breeding lines with three pyramided resistance genes that confer broad-spectrum bacterial blight resistance and their molecular analysis in rice. Rice, 6 (1): 1.

273

Sumner, L. W., Mendes, P. and Dixon, R. A. 2003. Plant metabolomics: Large-scale phytochemistry in the functional genomics era. Phytochemistry, 62 (6): 817 - 836.

Sun, H.-D., Qiu, S.-X., Lin, L.-Z., Wang, Z.-Y., Lin, Z.-W., Pengsuparp, T., Pezzuto, J. M., Fong, H. H., Cordell, G. A. and Farnsworth, N. R. 1996. Nigranoic acid, a triterpenoid from Schisandra sphaerandra that inhibits HIV-1 reverse transcriptase. Journal of Natural Products, 59 (5): 525 - 527.

Sung, Y. K. and Coutts, R. H. A. 1995a. Pseudorecombination and complementation between potato yellow mosaic geminivirus and tomato golden mosaic geminivirus. Journal of General Virology, 76 (11): 2809 - 2815.

Sung, Y. K. and Coutts, R. H. A. 1995b. Mutational analysis of potato yellow mosaic geminivirus. Journal of General Virology, 76 (7): 1773 - 1780.

Sung, Y. K. and Coutts, R. H. A. 1996. Potato yellow mosaic geminivirus AC2 protein is a sequence non-specific DNA binding protein. FEBS Letters, 383 (1): 51 - 54.

Sunter, G. and Bisaro, D. M. 1991. Transactivation in a geminivirus: AL2 gene product is needed for coat protein expression. Virology, 180 (1): 416 - 419.

Sunter, G. and Bisaro, D. M. 1992. Transactivation of geminivirus AR1 and BR1 gene expression by the viral AL2 gene product occurs at the level of transcription. Plant Cell, 4 (10): 1321 - 1331.

Sunter, G. and Bisaro, D. M. 1997. Regulation of a geminivirus coat protein promoter by AL2 protein (trap): Evidence for activation and derepression mechanisms. Virology, 232 (2): 269 - 280.

Sunter, G. and Bisaro, D. M. 2003. Identification of a minimal sequence required for activation of the Tomato golden mosaic virus coat protein promoter in protoplasts. Virology, 305 (2): 452 - 462.

Sunter, G., Hartitz, M. D. and Bisaro, D. M. 1993. Tomato golden mosaic virus leftward gene expression: Autoregulation of geminivirus replication protein. Virology, 195 (1): 275 - 280.

Sunter, G., Hartitz, M. D., Hormuzdi, S. G., Brough, C. L. and Bisaro, D. M. 1990. Genetic analysis of tomato golden mosaic virus: ORF AL2 is required for coat protein

274 accumulation while ORF AL3 is necessary for efficient DNA replication. Virology, 179 (1): 69 - 77.

Sunter, G., Stenger, D. C. and Bisaro, D. M. 1994. Heterologous complementation by geminivirus AL2 and AL3 genes. Virology, 203 (2): 203 - 210.

Sunter, G., Sunter, J. L. and Bisaro, D. M. 2001. Plants expressing Tomato golden mosaic virus AL2 or Beet curly top virus L2 transgenes show enhanced susceptibility to infection by DNA and RNA viruses. Virology, 285 (1): 59 - 70.

Swarnalatha, P., Mamatha, M., Manasa, M., Singh, R. and Krishnareddy, M. 2013. Molecular identification of Ageratum enation virus (AEV) associated with leaf curl disease of tomato (Solanum lycopersicum) in India. Australasian Plant Disease Notes, 8 (1): 67 - 71.

Syed, R. U. H. and Geary, B. 2013. An endophytic nodulisporium sp. From central america producing volatile organic compounds with both biological and fuel potential. Journal of microbiology and biotechnology, 23 (1): 29 - 35.

Syller, J. 2012. Facilitative and antagonistic interactions between plant viruses in mixed infections. Molecular Plant Pathology, 13 (2): 204 - 216.

Taheri, H., Izadpanah, K. and Behjatnia, S. 2012. Circulifer haematoceps, the vector of the Beet curly top Iran virus. Iranian Journal of Plant Pathology, 48 (1): 151 - 151.

Tahir, M. and Haider, M. S. 2005. First report of Tomato leaf curl New Delhi virus infecting bitter gourd in Pakistan. Plant pathology, 54: 807.

Tahir, M. N., Amin, I., Briddon, R. W. and Mansoor, S. 2011. The merging of two dynasties – identification of an African cotton leaf curl disease-associated begomovirus with cotton in Pakistan. PLoS One, 6 (5): e20366.

Tam, S. M., Mhiri, C., Vogelaar, A., Kerkveld, M., Pearce, S. R. and Grandbastien, M.-A. 2005. Comparative analyses of genetic diversities within tomato and pepper collections detected by retrotransposon-based SSAP, AFLP and SSR. Theoretical and Applied Genetics, 110 (5): 819 - 831.

Tan, P. H. N., Wong, S. M., Wu, M., Bedford, I. D., Saunders, K. and Stanley, J. 1995. Genome organization of Ageratum yellow vein virus, a monopartite whitefly- transmitted geminivirus isolated from a common weed. Journal of General Virology, 76 (12): 2915 - 2922.

275

Teng, K., Chen, H., Lai, J., Zhang, Z., Fang, Y., Xia, R., Zhou, X., Guo, H. and Xie, Q. 2010. Involvement of C4 protein of Beet severe curly top virus (family geminiviridae) in virus movement. PLoS One, 5 (6): e11280.

Thresh, J. M., Otim-Nape, G. W. and Fargette, D. 1998. The control of African cassava mosaic virus diseaes: phytosanitation and/or resistance. Plant Virus Disease Control. APS Press, St.Paul, Minnesota. pp. 670 - 677.

Tiwari, S. P., Nema, S. and Khare, M. N. 2013a. Whitefly-a strong transmitter of plant viruses. International Journal of Phytopathology, 2 (2): 102-120.

Tiwari, N., Singh, V. B., Sharma, P. K. and Malathi, V. G. 2013b. Tomato leaf curl Joydebpur virus: A monopartite begomovirus causing severe leaf curl in tomato in West Bengal. Archives of Virology, 158 (1): 1 - 10.

Torbati, M., Asnaashari, S. and Afshar, F. H. 2016. Essential oil from flowers and leaves of Elaeagnus angustifolia (elaeagnaceae): Composition, radical scavenging and general toxicity activities. Advanced Pharmaceutical Bulletin, 6 (2): 163.

Trinks, D., Rajeswaran, R., Shivaprasad, P. V., Akbergenov, R., Oakeley, E. J., Veluthambi, K., Hohn, T. and Pooggin, M. M. 2005. Suppression of RNA silencing by a geminivirus nuclear protein, AC2, correlates with transactivation of host genes. Journal of Virology, 79 (4): 2517 - 2527.

Trombetta, D., Castelli, F., Sarpietro, M. G., Venuti, V., Cristani, M., Daniele, C., Saija, A., Mazzanti, G. and Bisignano, G. 2005. Mechanisms of antibacterial action of three monoterpenes. Antimicrobial agents and chemotherapy, 49 (6): 2474 - 2478.

Tsai, W., Shih, S., Green, S., Akkermans, D. and Jan, F.-J. 2006. Molecular characterization of a distinct tomato-infecting begomovirus associated with yellow leaf curl diseased tomato in Lembang, Java Island of Indonesia. Plant disease, 90 (6): 831 - 831.

Tsai, W., Shih, S., Kenyon, L., Green, S. and Jan, F. J. 2011a. Temporal distribution and pathogenicity of the predominant tomato‐infecting begomoviruses in Taiwan. Plant Pathology, 60 (4): 787 - 799.

Tsai, W. S., Shih, S. L., Venkatesan, S. G., Aquino, M. U., Green, S. K., Kenyon, L. and Jan, F. J. 2011b. Distribution and genetic diversity of begomoviruses infecting

276 tomato and pepper plants in the Philippines. Annals of Applied Biology, 158 (3): 275 - 287.

Unseld, S., Frischmuth, T. and Jeske, H. 2004. Short deletions in nuclear targeting sequences of African cassava mosaic virus coat protein prevent geminivirus twinned particle formation. Virology, 318 (1): 90 - 101.

Unseld, S., Honle, M., Ringel, M. and Frischmuth, T. 2001. Subcellular targeting of coat protein of african cassava mosaic geminivirus. Virology, 286 (2): 373 - 383.

Vadivukarasi, T., Girish, K. R. and Usha, R. 2007. Sequence and recombination analyses of the geminivirus replication initiator protein. Journal of Biosciences, 32 (1): 17 - 29.

Van Der Greef, J. and Smilde, A. K. 2005. Symbiosis of chemometrics and metabolomics: Past, present, and future. Journal of Chemometrics, 19 (5): 376 - 386.

Van Loon, L. C., Rep, M. and Pieterse, C. 2006. Significance of inducible defense- related proteins in infected plants. Annual Review of Phytopathology, 44: 135 - 162.

Van Regenmortel, M. H. V., Fauquet, C. M., Bishop, D. H. L., Carstens, E. B., Estes, M. K., Lemon, S. M., Maniloff, J., Mayo, M. A., Mcgeoch, D. J., Pringle, C. R. and Wickner, R. B. 2000. 7th report of the International Committee on Taxonomy of Viruses. San diego, academic press.

Vanderschuren, H., Stupak, M., Fütterer, J., Gruissem, W. and Zhang, P. 2007. Engineering resistance to geminiviruses – review and perspectives. Plant Biotechnology Journal, 5 (2): 207 - 220.

Vanitharani, R., Chellappan, P., Pita, J. S. and Fauquet, C. M. 2004. Differential roles of AC2 and AC4 of cassava geminiviruses in mediating synergism and suppression of posttranscriptional gene silencing. Journal of Virology, 78 (17): 9487 - 9498.

Varma, A. and Malathi, V. G. 2003. Emerging geminivirus problems: A serious threat to crop production. Annals of Applied Biology, 142 (2): 145 - 164.

Varma, A. Mandal, B. and Singh, M. K. 2011. Global emergence and spread of whitefly (Bemisia tabaci) transmitted geminiviruses. In The Whitefly, Bemisia tabaci (Homoptera: Aleyrodidae) Interaction with Geminivirus-Infected Host Plants, Springer Netherlands. pp. 205 - 292.

277

Varsani, A., Navas-Castillo, J., Moriones, E., Hernandez-Zepeda, C., Idris, A. and Brown, J. 2014a. Establishment of three new genera in the family geminiviridae: Becurtovirus, Eragrovirus and Turncurtovirus. Archieves of Virology, 159 (8): 2193 - 2203.

Varsani, A., Martin, D. P., Navas-Castillo, J., Moriones, E., Hernandez-Zepeda, C., Idris, A., Murilo Zerbini, F. and Brown, J. K. 2014b. Revisiting the classification of curtoviruses based on genome-wide pairwise identity. Archives of Virology, 159 (7): 1873 - 1882.

Varsani, A., Monjane, A. L., Donaldson, L., Oluwafemi, S., Zinga, I., Komba, E. K., Plakoutene, D., Mandakombo, N., Mboukoulida, J., Semballa, S., Briddon, R. W., Markham, P. G., Lett, J. M., Lefeuvre, P., Rybicki, E. P. and Martin, D. P. 2009. Comparative analysis of Panicum streak virus and Maize streak virus diversity, recombination patterns and phylogeography. Virology Journal, 6 (1): 1.

Verlaan, M. G., Hutton, S. F., Ibrahem, R. M., Kormelink, R., Visser, R., Scott, J. W., Edwards, J. D. and Bai, Y. 2013. The tomato yellow leaf curl virus resistance genes Ty-1 and Ty-3 are allelic and code for DFDGD-class RNA-dependent RNA polymerases. PLoS Genetics, 9 (3): e1003399.

Verlaan, M. G., Szinay, D., Hutton, S. F., De Jong, H., Kormelink, R., Visser, R. G., Scott, J. W. and Bai, Y. 2011. Chromosomal rearrangements between tomato and Solanum chilense hamper mapping and breeding of the TYLCV resistance gene Ty-1. Plant Journal, 68 (6): 1093 - 1103.

Vidavsky, F., Leviatov, S., Milo, J., Rabinowitch, H., Kedar, N. and Czosnek, H. 1998. Response of tolerant breeding lines of tomato, Lycopersicon esculentum, originating from three different sources (L. Peruvianum, L. Pimpinellifolium and L. Chilense) to early controlled inoculation by Tomato yellow leaf curl virus (TYLCV). Plant Breeding, 117 (2): 165 - 169.

Viuda-Martos, M., Sanchez-Zapata, E., Sayas-Barberá, E., Sendra, E., Pérez-Álvarez, J. and Fernández-López, J. 2014. Tomato and tomato byproducts. Human health benefits of lycopene and its application to meat products: A review. Critical Reviews in Food Science and Nutrition, 54 (8): 1032 - 1049.

278

Vlaisavljevic, S., Kaurinovic, B., Popovic, M., Djurendic-Brenesel, M., Vasiljevic, B., Cvetkovic, D. and Vasiljevic, S. 2014. Trifolium pratense L. As a potential natural antioxidant. Molecules, 19 (1): 713 - 725.

Vlot, A. C., Dempsey, D. M. A. and Klessig, D. F. 2009. Salicylic acid, a multifaceted hormone to combat disease. Annual Review of Phytopathology, 47: 177 - 206.

Vogg, G., Fischer, S., Leide, J., Emmanuel, E., Jetter, R., Levy, A. A. and Riederer, M. 2004. Tomato fruit cuticular waxes and their effects on transpiration barrier properties: Functional characterization of a mutant deficient in a very‐long‐chain fatty acid β‐ketoacyl‐coa synthase. Journal of Experimental Botany, 55 (401): 1401 - 1410.

Voinnet, O. 2005. Induction and suppression of RNA silencing: Insights from viral infections. Nature Genetics, 6 (3): 206 - 221.

Voinnet, O., Pinto, Y. M. and Baulcombe, D. C. 1999. Suppression of gene silencing: A general strategy used by diverse DNA and RNA viruses of plants. Proceedings of the National Academy of Sciences, 96 (24): 14147 - 14152.

Von Roepenack-Lahaye, E., Newman, M.-A., Schornack, S., Hammond-Kosack, K. E., Lahaye, T., Jones, J. D., Daniels, M. J. and Dow, J. M. 2003. P- coumaroylnoradrenaline, a novel plant metabolite implicated in tomato defense against pathogens. Journal of Biological Chemistry, 278 (44): 43373 - 43383.

Vuillaume, F., Thebaud, G., Urbino, C., Forfert, N., Granier, M., Froissart, R., Blanc, S. and Peterschmitt, M. 2011. Distribution of the phenotypic effects of random homologous recombination between two virus species. PLoS Pathogens, 7 (5): e1002028.

Wagner, G., Wang, E. and Shepherd, R. 2004. New approaches for studying and exploiting an old protuberance, the plant trichome. Annals of Botany, 93 (1): 3 - 11.

Wang, A., Xia , Q. and Xie, W. 2003 The classical ubisch bodies carry a sporophytically produced structural protein (RAFTIN) that is essential for pollen development. Proceedings of the National Academy of Sciences, 100 (24): 1487 - 14492.

Wang, H., Buckley, K. J., Yang, X., Buchmann, R. C. and Bisaro, D. M. 2005. Adenosine kinase inhibition and suppression of RNA silencing by geminivirus AL2 and L2 proteins. Journal of Virology, 79 (12): 7410 - 7418.

279

Wang, H., Hao, L., Shung, C. Y., Sunter, G. and Bisaro, D. M. 2003. Adenosine kinase is inactivated by geminivirus AL2 and L2 proteins. Plant Cell, 15 (12): 3020 - 3032.

Wang, H. L., Sudarshana, M. R., Gilbertson, R. L. and Lucas, W. J. 1999. Analysis of cell-to-cell and long distance movement of a bean dwarf mosaic geminivirus-green fluorescent protein reporter in host and nonhost species: Identification of sites of resistance. Molecular Plant-Microbe Interactions, 12 (4): 345 - 355.

Wartig, L., Kheyr-Pour, A., Noris, E., De Kouchkovsky, F., Jouanneau, F., Gronenborn, B. and Jupin, I. 1997. Genetic analysis of the monopartite tomato yellow leaf curl geminivirus: Roles of V1, V2, and C2 ORFs in viral pathogenesis. Virology, 228 (2): 132 - 140.

Watanabe, T., Arai, Y., Mitsui, Y., Kusaura, T., Okawa, W., Kajihara, Y. and Saito, I. 2006. The blood pressure-lowering effect and safety of chlorogenic acid from green coffee bean extract in essential hypertension. Clinical and experimental hypertension, 28 (5): 439 - 449.

Webber, C. M. and England, R. W. 2010. Oral allergy syndrome: A clinical, diagnostic, and therapeutic challenge. Annals of Allergy, Asthma & Immunology, 104 (2): 101 - 108.

Wege, C., Gotthardt, R.-D., Frischmuth, T. and Jeske, H. 2000. ulfilling koch’s postulates for Abutilon mosaic virus. Archives of Virology, 145 (10): 2217 - 2225.

Weingart, G., Kluger, B., Forneck, A., Krska, R. and Schuhmacher, R. 2012. Establishment and application of a metabolomics workflow for identification and profiling of volatiles from leaves of Vitis vinifera by HS‐SPME‐GC‐MS. Phytochemical Analysis, 23 (4): 345 - 358.

Weingart, G. J., Lawo, N. C., Forneck, A., Krska, R. and Schuhmacher, R. 2013. Study of the volatile metabolome in plant–insect interactions. The Handbook of Plant Metabolomics, 125-153.

Were, H. K., Takeshita, M., Furuya, N. and Takanami, Y. 2005. Molecular characterization of a new begomovirus infecting honeysuckle in Sapporo, Japan. Journal of the faculty of Agriculture Kyushu University, 50 (1): 73 - 81.

280

Weretilnyk, E. A., Alexander, K. J., Drebenstedt, M., Snider, J. D., Summers, P. S. and Moffatt, B. A. 2001. Maintaining methylation activities during salt stress. The involvement of adenosine kinase. Plant Physiology, 125 (2): 856 - 865.

Werner, K., Friedt, W. and Ordon, F. 2005. Strategies for pyramiding resistance genes against the barley yellow mosaic virus complex (BaMMV, BaYMV, BaYMV-2). Molecular Breeding, 16 (1): 45 - 55.

Williams, C. E. and Clair, D. A. S. 1993. Phenetic relationships and levels of variability detected by restriction fragment length polymorphism and random amplified polymorphic DNA analysis of cultivated and wild accessions of Lycopersicon esculentum. Genome, 36 (3): 619 - 630.

Williamson, V. M. and Hussey, R. S. 1996. Nematode pathogenesis and resistance in plants. The Plant Cell, 8 (10): 1735-1745.

Willment, J. A., Martin, D. P., Palmer, K. E., Schnippenkoetter, W. H., Shepherd, D. N. and Rybicki, E. P. 2007. Identification of long intergenic region sequences involved in Maize streak virus replication. Journal of General Virology, 88 (6): 1831 - 1841.

Wink, M. 2009. Introduction: Biochemistry, physiology and ecological function of secondary metabolites. Annual Plant Reviews Vol. 40: Biochemistry of Plant Secondary Metabolism, Vol. (40), John Wiley & Sons.

Wright, E. A., Heckel, T., Groenendijk, J., Davies, J. W. and Boulton, M. I. 1997. Splicing features in Maize streak virus virion- and complementary-sense gene expression. Plant Journal, 12 (6): 1285 - 1297.

Wu, F. and Tanksley, S. D. 2010. Chromosomal evolution in the plant family Solanaceae. BMC Genomics, 11 (1): 1.

Wu, P.-J. and Zhou, X.-P. 2005. Interaction between a nanovirus-like component and the Tobacco curly shoot virus/satellite complex. Acta Biochimica et Biophysica Sinica, 37 (1): 25 - 31.

Xie, Y., Zhou, X. and Li, G. 2003. Molecular characterization of Tomato yellow leaf curl China virus and its satellite DNA isolated from tobacco. Chinese Science Bulletin, 48 (8): 766 - 770.

281

Xiong, Q., Guo, X. J., Che, H. Y. and Zhou, X. P. 2005. Molecular characterization of a distinct begomovirus species and its associated satellite DNA molecule infecting Sida acuta. Journal of Phytopathology, 153 (5): 264 - 268.

Yaakov, N., Levy, Y., Belausov, E., Gaba, V., Lapidot, M. and Gafni, Y. 2011. Effect of a single amino acid substitution in the NLS 1 domain of tomato yellow leaf curl virus-israel (TYLCV-IL) capsid protein (CP) on its activity and on the virus life cycle. Virus Research, 158 (1): 8 - 11.

Yadav, P., Kumar, S., Reddy, K. P., Yadav, T. and Murthy, I. 2014. Oxidative stress and antioxidant defense system in plants. Plant Biotechnology, Studium Press LLC, USA, 2: 261 - 281.

Yadava, P., Suyal, G. and Mukherjee, S. K. 2010. Begomovirus DNA replication and pathogenicity. Current Science, 98 (3): 360 - 368.

Yan, W., Ying, W., Zhihua, L., Chengfang, W., Jianyu, W., Xiaolan, L., Pingjuan, W., Zhaofeng, Z., Shushan, D. and Dongye, H. 2014. Composition of the essential oil from Alpinia galanga rhizomes and its bioactivity on Lasioderma serricorne. Bulletin of Insectology, 67 (2): 247 - 254.

Yang, G., Zhou, B., Zhang, X., Zhang, Z., Wu, Y., Zhang, Y., Lü, S., Zou, Q., Gao, Y. and Teng, L. 2016. Effects of tomato root exudates on Meloidogyne incognita. PLoS One, 11 (4): e0154675.

Yang, J.-Y., Iwasaki, M., Machinda, C., Machinda, Y., Zhou, X. and Chua, N.-H. 2008. Betac1, the pathogenicity factor of TYLCCNV, interacts with AS1 to alter leaf development and suppress selective jasmonic acid responses. Genes & Development, 22 (18): 2564 - 2577.

Yang, X., Baliji, S., Buchmann, R. C., Wang, H., Lindbo, J. A., Sunter, G. and Bisaro, D. M. 2007. Functional modulation of the geminivirus AL2 transcription factor and silencing suppressor by self-interaction. Journal of Virology, 81 (21): 11972-11981.

Yang, X., Caro, M., Hutton, S. F., Scott, J. W., Guo, Y., Wang, X., Rashid, M. H., Szinay, D., De Jong, H. and Visser, R. G. 2014. Fine mapping of the Tomato yellow leaf curl virus resistance gene Ty-2 on chromosome 11 of tomato. Molecular Breeding, 34 (2): 749 - 760.

282

Yang, X., Wang, X., Wang, K., Su, L., Li, H., Li, R. and Shen, Q. 2015. The nematicidal effect of camellia seed cake on root-knot nematode Meloidogyne javanica of banana. PLoS One, 10 (4): e0119700.

Yang, X., Xie, Y., Raja, P., Li, S., Wolf, J. N., Shen, Q., Bisaro, D. M. and Zhou, X. 2011. Suppression of methylation-mediated transcriptional gene silencing by BetaC1- SAHH protein interaction during geminivirus-betasatellite infection. PLoS Pathogens, 7 (10): e1002329.

Yazdi, H. R., Heydarnejad, J. and Massumi, H. 2008. Genome characterization and genetic diversity of Beet curly top Iran virus: A geminivirus with a novel nonanucleotide. Virus Genes, 36 (3): 539 - 545.

Yuanpeng, D., Qiuling, Z., Heng, Z., Enshun, J. and Zhongyue, W. 2009. Selectivity of Phylloxera viticola fitch (homoptera: Phylloxeridae) to grape with different resistance and the identification of grape root volatiles. Acta Entomologica Sinica, 52 (5): 537 - 543.

Zacarés, L., López-Gresa, M. P., Fayos, J., Primo, J., Bellés, J. M. and Conejero, V. 2007. Induction of p-coumaroyldopamine and feruloyldopamine, two novel metabolites, in tomato by the bacterial pathogen Pseudomonas syringae. Molecular Plant-Microbe Interactions, 20 (11): 1439 - 1448.

Zaidi, S. S.-E.-A., Shafiq, M., Amin, I., Scheffler, B. E., Scheffler, J. A., Briddon, R. W. and Mansoor, S. 2016. Frequent occurrence of Tomato leaf curl New Delhi virus in cotton leaf curl disease affected cotton in Pakistan. PLoS One, 11 (5): e0155520.

Zamir, D., Ekstein-Michelson, I., Zakay, Y., Navot, N., Zeidan, M., Sarfatti, M., Eshed, Y., Harel, E., Pleban, T., Van-Oss, H., Kedar, N., Rabinowitch, H. D. and Czosnek, H. 1994. Mapping and introgression of a Tomato yellow leaf curl virus tolerance gene, Ty-1. Theoretical and Applied Genetics, 88 (2): 141 - 146.

Zaprometov, M. 1989. The formation of phenolic compounds in plant cell and tissue cultures and the possibility of its regulation. Advances in cell culture (USA).

Zarger, M. S. S. and Khatoon, F. 2013 Phytochemical, GC-MS analysis and antibacterial activity of bioactive compounds of petroleum ether leaf extracts of Salix viminalis. International Journal of Science and Research, 4 (1): 494 - 498.

283

Zhang, J., Dang, M., Huang, Q. and Qian, Y. 2015. Determinants of disease phenotype differences caused by closely-related isolates of begomovirus betasatellites inoculated with the same species of helper virus. Viruses, 7 (9): 4945 - 4959.

Zhang, S. C., Ghosh, R. and Jeske, H. 2002. Subcellular targeting domains of abutilon mosaic geminivirus movement protein BC1. Archives of Virology, 147 (12): 2349 - 2363.

Zhang, S. C. and Ling, K.-S. 2011. Genetic diversity of sweet potato begomoviruses in the United States and identification of a natural recombinant between Sweet potato leaf curl virus and Sweet potato leaf curl georgia virus. Archives of Virology, 156 (6): 955 - 968.

Zhang, W., Olson, N. H., Baker, T. S., Faulkner, L., Agbandje-Mckenna, M., Boulton, M. I., Davies, J. W. and Mckenna, R. 2001. Structure of the Maize streak virus geminate particle. Virology, 279 (2): 471 - 477.

Zhang, Z., Chen, H., Huang, X., Xia, R., Zhao, Q., Lai, J., Teng, K., Li, Y., Liang, L., Du, Q., Zhou, X., Guo, H. and Xiea, Q. 2011. BSCTV C2 attenuates the degradation of SAMDC1 to suppress DNA methylation-mediated gene silencing in Arabidopsis. Plant Cell, 23 (1): 273 - 288.

Zhou, J., Zhang, L., Li, X., Chang, Y., Gu, Q., Lu, X., Zhu, Z. and Xu, G. 2012. Metabolic profiling of transgenic rice progeny using gas chromatography–mass spectrometry: The effects of gene insertion, tissue culture and breeding. Metabolomics, 8 (4): 529 - 539.

Zhou, X., Liu, Y., Calvert, L., Munoz, C., Otim-Nape, G. W., Robinson, D. J. and Harrison, B. D. 1997. Evidence that DNA-A of a geminivirus associated with sever cassava mosaic disease in Uganda has arisen by interspecific recombination. Journal of General Virology, 78 (8): 2101 - 2111.

Zhou, Y., Rojas, M. R., Park, M.-R., Seo, Y.-S., Lucas, W. J. and Gilbertson, R. L. 2011. Histone H3 interacts and colocalizes with the nuclear shuttle protein and the movement protein of a geminivirus. Journal of Virology, 85 (22): 11821 - 11832.

Zhu, J. and Park, K.-C. 2005. Methyl salicylate, a soybean aphid-induced plant volatile attractive to the predator Coccinella septempunctata. Journal of Chemical Ecology, 31 (8): 1733 - 1746.

284

Zito, P., Sajeva, M., Bruno, M., Maggio, A., Rosselli, S., Formisano, C. and Senatore, F. 2010. Essential oil composition of stems and fruits of Caralluma europaea NE Br. (apocynaceae). Molecules, 15 (2): 627 - 638.

Zrachya, A., Glick, E., Levy, Y., Arazi, T., Citovsky, V. and Gafni, Y. 2007. Suppressor of rna silencing encoded by Tomato yellow leaf curl virus-Israel. Virology, 358 (1): 159 - 165.