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Cular Characterization of Viruses Infecting Potato and Vegetables in Iraq

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MOLECULAR CHARACTERIZATION OF VIRUSES INFECTING POTATO AND VEGETABLES IN IRAQ NAWRES A. SADEQ AL-KUWAITI A thesis submitted in partial fulfilment of requirements of the University of Greenwich for the degree of Doctor of Philosophy Natural Resources Institute The University of Greenwich UK June 2013 Dedication:- To My Wife, Parents & Children For the love and support Nawres DECLARATION I certify that this work has not been accepted in substance for any degree, and is not concurrently submitted for any degree other than that of Doctor of Philosophy (PhD) being studied at the University of Greenwich. I also declare that this work is the result of my own investigations except where otherwise identified by references and that I have not plagiarised the work of others. Student: …………………………………… Date…………………. Mr Nawres A. Sadeq Al-Kuwaiti Supervisor: ……………………………….. Date………………….. Dr. Susan E. Seal Supervisor ……………………………… Date………………….. Dr. Maruthi M. N. Gowda ii ACKNOWLEDGEMENTS First, I would like to express my gratitude to my supervisors, Dr. Susan E. Seal and Dr. Maruthi M. N. Gowda for providing advice, guide and support during my study, suggesting the topic of my PhD research regarding plant viruses from my country Iraq, dedicating their precious time and invaluable assistance to improve my research work and writing up my thesis. I wish to express my sincere thanks to the Iraqi Ministry of Higher Education and Scientific Research and the University of Baghdad for funding my PhD study. I do thank and appreciate the Director of Research and Enterprise Dr John Orchard and the Head of Agriculture, Health and Environment Dr Jeremy Haggar and The Director of the NRI Prof. Andrew Westby for all efforts have paid off and time been dedicated to enable such a healthy and convenient research environment for postgraduate students at the NRI. I acknowledge Dr. Carl Collins and Dr. Bettina Otto for the precious advice and support, Mr. Dudley Farman, Mr. Charles Whitfield, Mr. Simon Springate and Mrs. Natalie Morley for the technical assistance at molecular biology laboratories. Thanks in advance to the PhD examination committee professor Matthew John Dickinson and Dr. Richard Colgan for dedicating time and effort to review my PhD draft and special thanks to Dr. Rory Hillocks for the invaluable advice to improve the PhD thesis style. I thank Mr. Mark Parnell, Mrs Heather McAvoy-Marshall, Mrs Caroline Troy and J. Collins and other administrative staff to facilitate administrative issues during my PhD study. I would like to express my gratitude to my colleagues at molecular Biology laboratories, Dr. Mohammed I.U., Dr. Abarshi M. M., Mr. Aliyu Turaki and Dr. Peter Wasswa, and other NRI postgraduate students as well, for the good time spent together during my postgraduate study. iii ABSTRACT Due to the lack of published molecular information concerning plant viruses from Iraq, this study was initiated to investigate the diversity of viruses infecting potato and vegetables in Iraq on a molecular basis. Based on the economic importance and incidence worldwide, eight virus genera were investigated in 175 potato and vegetable samples collected from fields in Baghdad, Anbar and Najaf provinces in Iraq. Using genus/family specific primers, published in the literature, polymerase chain reaction (PCR) and reverse transcription-polymerase chain reaction (RT-PCR) were performed to screen samples for potyviruses, begomoviruses, carlaviruses, tombusviruses, potexviruses, cucumoviruses, tobamoviruses and alfamoviruses. Circular DNA viruses were screened by rolling circle amplification (RCA). Products resulting from PCR/RT-PCR and RCA were cloned and sequenced and data obtained were used for sequence analyses. The above approach led to the first molecular characterisation of three potyviruses; Potato virus Y (PVY), Bean yellow mosaic virus (BYMV) and Zucchini yellow mosaic virus (ZYMV), one begomovirus; Tomato yellow leaf curl virus, two carlaviruses, Potato virus S (PVS) and Cowpea mild mottle virus (CPMMV) and one tombusvirus; Grapevine Algerian latent virus (GALV) in Iraqi potato and vegetable samples. Based on nucleotide (nt) sequence analyses, BYMV from broad bean and ZYMV from zucchini were 97% and 99% identical to equivalent sequences from the GenBank sequences, respectively. Two PVY strains were distinguished when sequences from potato and tomato showed 99% maximum nt identity to equivalent PVYO: N and PVYNTN GenBank sequences, respectively. Full-length sequence from tomato amplified by RCA showed 99% maximum nt identity to equivalent TYLCV sequences from the GenBank. Sequence comparison of carlavirus sequences isolated from potato and cowpea were 99% and 96% identical to equivalents PVS and CPMMV sequences from the GenBank, respectively. All tombusvirus sequences amplified from tomato and eggplant showed 93% maximum nt identity to equivalent GALV GenBank sequences. The high similarities (93-99%) of virus sequences isolated suggest, viruses isolated may have been introduced into Iraq from other countries, through international trading of plant materials used for cultivation as Iraq import most of plant materials for agriculture. iv TABLE OF CONTENTS ABSTRACT ............................................................................................................................. iv TABLE OF CONTENTS ......................................................................................................... v FIGURES .................................................................................................................................. x TABLES .................................................................................................................................. xii APPENDICES ........................................................................................................................ xiii ABBREVIATIONS ................................................................................................................ xiv CHAPTER 1: INTRODUCTION ............................................................................................... 1 1. General introduction ........................................................................................................ 1 CHAPTER 2: LITERATURE REVIEW ............................................................................................ 6 2. Literature review .............................................................................................................. 6 2.1. The current status of potato and vegetables in Iraq .................................................... 6 2.2. Potato viruses .............................................................................................................. 9 2.3. Vegetable viruses ...................................................................................................... 13 2.4. The family Potyviridae ............................................................................................. 16 2.5. The family Geminiviridae ........................................................................................ 20 2.6. The genus Carlavirus within the family Betaflexiviridae ........................................ 21 2.7. The genus Tombusvirus (the family Tombusviridae) ............................................... 23 2.8. Conventional techniques used for virus detection .................................................... 26 2.9. Molecular techniques used in plant virus detection.................................................. 27 2.10. Nucleic acid extraction ............................................................................................. 28 2.11. PCR/RT-PCR ........................................................................................................... 30 2.12. Group specific primers ............................................................................................. 31 2.13. Rolling circle amplification (RCA) .......................................................................... 36 2.14. Gel electrophoresis and restriction fragment length polymorphism (RFLP) ........... 38 2.15. Cloning ..................................................................................................................... 39 v 2.16. Sequencing ............................................................................................................... 41 2.17. Phylogeny and sequence analyses ............................................................................ 43 2.18. Infectious clones and agro-inoculation ..................................................................... 46 CHAPTER 3: MATERIALS AND METHODS ................................................................................. 48 3. Materials and methods ................................................................................................... 48 3.1. Plant sampling and handling .................................................................................... 48 3.2. Nucleic acid extraction ............................................................................................. 48 3.3. Group specific primer sets ........................................................................................ 51 3.4. RT-PCR for RNA viruses ......................................................................................... 51 3.4.1. cDNA synthesis .................................................................................................... 51 3.4.2. RT-PCR for potyvirus sequence detection
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