DNA based analysis of thrips diversity and thrips-borne Iris yellow spot virus (Tospovirus: Bunyaviridae) from Pakistan Submitted in partial fulfillment of Doctor of Philosophy By Romana Iftikhar 2015 Department of Biotechnology (NIBGE) Pakistan Institute of Engineering and Applied Sciences Nilore-45650 Islamabad, Pakistan National Institute for Biotechnology and Genetic Engineering P. O. BOX 577, JHANG ROAD, FAISALABAD. (Affiliated with PIEAS, Islamabad) Declaration of Originality I hereby declare that the work accomplished in this thesis is the result of my own research carried out in Agricultural Biotechnology Division (NIBGE). This thesis has not been published previously nor does it contain any material from the published resources that can be considered as the violation of international copyright law. Furthermore, I also declare that I am aware of the terms “copyright” and “plagiarism” and if any copyright violation was found out in this work, I will be held responsible of the consequences of any such violation. Signature: _______________ Name of the Student: Romana Iftikhar Registration No. 10-7-1-017-2008 Date: ______________ Place: NIBGE, Faisalabad National Institute for Biotechnology and Genetic Engineering P. O. BOX 577, JHANG ROAD, FAISALABAD. (Affiliated with PIEAS, Islamabad) Research Completion Certificate Certified that the research work contained in this thesis entitled “DNA based analysis of thrips diversity and thrips-borne Iris yellow spot virus (Tospovirus: Bunyaviridae) from Pakistan” has been carried out and completed by “Romana Iftikhar” under my supervision during her PhD studies in the subject of Biotechnology. ---------------------------- ---------------------------- Date Dr. Muhammad Ashfaq (FFP) Research Supervisor Submitted Through ------------------------ --------------------------- Dr. Shahid Mansoor Controller of Examination Director NIBGE Certificate of Approval This is to certify that the work contained in this thesis titled “DNA based analysis of thrips diversity and thrips-borne Iris yellow spot virus (Tospovirus: Bunyaviridae) from Pakistan” was carried out by “Romana Iftikhar” in our opinion is fully adequate in scope and quality for the degree of Doctor of Philosophy in Biotechnology from Pakistan Institute of Engineering and Applied Sciences (PIEAS). Approved by: Signature: _____________________ Dr. Muhammad Ashfaq Internal Examiner/Supervisor Verified by: Signature: _____________________ Dr. Shahid Mansoor Head, Department of NIBGE (Biotechnology) Stamp: ACKNOWLEDGEMENT All praises are for Almighty Allah (the most affectionate, the most merciful), and Holy Prophet Muhammad (May Allah Bless Muhammad & Descendants of Muhammad). I bow before Almighty Allah with limitless humility and modesty that He granted me with potential and ability to this material contribution to already existing ocean of knowledge. I thank from the deep core of heart to Holy Prophet Muhammad (May Allah bless and peace be upon him) forever a torch of guidance and knowledge for humanity as a whole. Special gratitude is due to Higher Education Commission (HEC) of Pakistan for providing a scholarship under the “Indigenous 5000 fellowship programme Phase IV” with out which such a costly research project could not be completed. I crave to thank my eminent supervisor Prof Dr. Muhammad Ashfaq, HEC foreign faculty member for accepting me as his PhD student. He is a wonderful man and scientist. He taught me from pipetting to data analysis and how to present scientific data. I have learned a lot from his experiences not only in research but also in other fields of life. I am really obliged to him for holding me to a high research standard and enforcing strict validations for each research result, and thus teaching me how to do research. His supervision and suggestions helped me in overcoming many technical difficulties and for all the useful insightful discussions to improve my research. His presence was always a source of confidence for me. It was indeed an honor to work under his guidance. It is my pleasure to thank Dr. Zafar Mehmood Khalid and Dr. Sohail Hameed former Directors, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, who have been very kind to provide every possible facility to carry out this research work. I express my deepest gratitude to current Director Dr. Shahid Mansoor Deputy chief Scientist for facilitating me during research work. My sincerest appreciations are due to Dr. Hanu R. Pappu, Department of Plant Pathology, Washington State University, Pullman, USA for my training in virology. I am very thankful for his precious suggestions, dedicated efforts, kind supervision, affectionate criticism, extremely encouraging behavior, and inspiring guidance provided to me throughout the course of virus study. He helped me in each step of research during my stay in USA. I can never forget the invaluable help and guidance of Dr. S.V. Ramesh and Dr. Sudeep bag during my research work in USA and Dr. Khalid Naveed for his helping and friendly behavior to make my stay in USA more comfortable. I am very grateful to Dr. Paul Hebert, Director, Biodiversity Institute of Ontario, University of Guelph Canada and his co-workers at the Canadian Centre for DNA Barcoding for sequencing barcodes of my thrips specimens. I am extremely thankful to Dr. R. Srinivasn, University of Georgia, Tifton, USA for giving the opportunity to learn the morphological identification and thrips rearing in his laboratory and Mr. Stan Diffie, University of Georgia, Tifton, USA for giving me training in morphological identification and helping me with thrips identification. I thank to Mr. Sueo Nakahara, USDA ARS, Beltsville MD (retired) for reviewing the i thrips species identification. I am also thankful to Valerie Lynch-Holm, Ph.D. Franceschi Microscopy & Imaging Center, Washington State University, Pullman, USA for giving me training and help in electron microscopy of thrips. I am thankful to my lab colleagues Dr. Akhtar Rasool, Mr. Saleem Akhtar, Dr. Innam Ullah, Mr. Tayyib Naseem, Dr. Arif M. Khan, Mr. Qamar Abbas, Mr. Shah Sawar and all others for their cooperation and help in experimental work. I want to thank to my dear friend, Afshan Mashkoor. The time spent with you is really memorable. I cannot forget the moments spent with you. I have no words to pay sincerest thanks to classfellows and hostelmates for their help, encouragement. Their company made my stay comfortable at NIBGE. No acknowledgements would ever adequately express my obligation to my loving brothers, Arbab Iftikhar and Usman Iftikhar, and my cute, loving sister Asna Iftikhar who always wished me health and success. Last but not the least, I find it hard to express my gratitude and appreciations in words to my affectionate parents Mr. Muhammad Iftikhar (Late) and Farhat Iftikhar who always wished to see me glittering high on the skies of success. None of this would have been possible without their love, patience and prayers. I am in short of words to thank my Appo Ji (Late) and uncles, Mr. Israr Ahmad (Late) and Mr. Isar Javed and my grandparents (Late) for their love, care and support. Finally, it has been a great pleasure for me to conduct my PhD work but I still have a long way to go…. Romana Iftikhar August, 2014 ii Table of Contents Contents Page # Acknowledgment I Table of contents iii List of Figures viii List of Tables ix List abbriviations x Abstract xiii Chapter 1 .................................................................................................................................... 1 INTRODUCTION AND REVIEW OF LITERATURE ........................................................... 1 1.1 Thrips ................................................................................................................... 1 1.1.1 Morphology ......................................................................................................... 1 1.1.2 Feeding behavior ................................................................................................. 1 1.1.3 Dispersal .............................................................................................................. 3 1.1.4 Reproduction....................................................................................................... 4 1.1.5 Life cycle of thrips ............................................................................................... 4 1.2 Taxonomic classification .................................................................................... .5 1.3 Thrips diversity……………..……………………..………………………...……………………………. 6 1.4 Importance of thrips ........................................................................................... 8 1.5 Identification of thrips ........................................................................................ 9 1.5.1 Morphological identification ............................................................................. 10 1.5.2 Molecular identification .................................................................................... 10 1.5.2.1 Use of COI and emergence of DNA Barcoding .................................................. 11 1.6 Thrips and Tospovirus transmission .................................................................. 12 1.7 Tospoviruses ..................................................................................................... 13 1.7.1 Genome Organization ....................................................................................... 14 1.8 Importance