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Myzus Persicae) Molecular aspects of Potato Virus Y transmission by aphids (Myzus persicae) Saman Bahrami Kamangar A dissertation submitted in partial fulfillment of the requirements for the PhD degree in Bioscience Engineering Promoters: Prof. dr. ir. Guy Smagghe, Department of Plants and Crops, Faculty of Bioscience Engineering, Ghent University Dr. ir. Kris De Jonghe, - Plant Sciences Unit, Flanders Research institute for Agriculture, Fisheries and Food (ILVO) Dr. ir. Nji Tizi Clauvis Taning, Department of Plants and Crops, Faculty of Bioscience Engineering, Ghent University Dutch translation of the title: Moleculaire aspecten van aardappel virus Y transmissie door bladluizen (Myzus persicae) Please refer to this thesis as follows: Bahrami Kamangar, S. (2021). Molecular aspects of Potato Virus Y transmission by aphids (Myzus persicae). PhD thesis. Ghent University, Belgium. ISBN: 9789463574259 Ghent University: Rector: Prof. dr. ir. Rik Van de Walle Faculty of Bioscience Engineering: Dean: Prof. dr. ir. Marc Van Meirvenne II Aknowledgment During the preparation of this thesis, I received great support and assistance. First, I would like to thank the Agricultural Research Education and Extention Organization (AREEO) in Iran for funding a part of my study. I would like to extend my sincere thanks to my supervisors Prof. dr. ir. Guy Smagghe, Dr. ir. Kris De Jonghe and Dr. ir. Nji Tizi Clauvis Taning for their consistent encouragement, support and guidance over the running of this research. I also wish to thank the Jury members Prof. Dr. Daisy Vanrompay, Dr. Olivier Christiaens, Dr. Jochem Bonte, and Dr. Stephan Steyer for reviewing and improving this thesis. I am grateful to Steve Baeyen, Lab Manager at the Institute for Agricultural and Fisheries Research, for his technical support. My wife Touba and son Siamand deserve special thanks for their patience, spiritual and emotional support. Without their understanding and encouragement in the past few years, it would have been impossible for me to complete my doctoral research. I wish to thank my parents and sisters who supported and always gave me positive energy. I owe a great debt of gratitude to my brother Dr. Barzan Bahrami Kamangar for his encouragement and assistance when I was working on my PhD. I gratefully acknowledge the help of my friends Dr. Khosro Mehdi Khanlou, Dr. Asad Maroufi, Serkewt Safaei, Dr. Amir Sadeghi, Hemen Piri and Dr. Hossein Hosseini Moghaddam. Saman Bahrami Kamangar III Contents List of abbreviations ........................................................................................................... 1 Doctoral project context and research questions ............................................................. 5 Chapter 1: General introduction ......................................................................................... 8 1.1. The potato plant as staple food ................................................................................... 9 1.2. Plant viruses ............................................................................................................... 10 1.2.1. Plant virus transmission ......................................................................................... 15 Non-circulative non-persistent transmission (NCNP) .................................................. 23 Persistent circulative, non-propagative transmission (CNP) ...................................... 23 Non-circulative semi-persistent transmission (NCSP) ................................................ 24 Persistent circulative, propagative transmission (CPP) .............................................. 25 1.2.2. Plant virus symptoms ............................................................................................. 25 1.2.3. Important potato viruses ......................................................................................... 26 1.3. Potato virus Y (PVY) and its characteristics ............................................................. 37 1.3.2. Genome organization and proteins ........................................................................ 38 1.3.3. Genetic diversity and strains .................................................................................. 40 1.3.4. Host range ................................................................................................................ 46 1.3.5. Transmission ........................................................................................................... 47 Strain specific aspects and virus plant sources .......................................................... 48 Impact of the virus receiving host plants ..................................................................... 49 Aphid and transmission efficiency ............................................................................... 50 Winged versus wingless aphids .................................................................................... 54 Impact of the feeding behavior ...................................................................................... 54 Environmental conditions .............................................................................................. 56 Plant volatiles, color and nutrients ............................................................................... 58 Control measures; Insecticides, straw mulch and mineral oil .................................... 59 Vector plant hosts before virus acquisition ................................................................. 60 Starving, acquisition accession period (AAP), inoculation accession period (IAP) and virus retention ............................................................................................................ 61 Molecular aspects of PVY transmission by aphids ...................................................... 63 1.4. Techniques to study virus transmission .................................................................. 68 1.4.2. Electric penetration graph ...................................................................................... 70 1.4.3. Electron microscopy (EM) and labeling ................................................................. 71 1.4.4. Immunological methods ......................................................................................... 71 1.4.5. Molecular methods (q(RT-)PCR and (RT-)PCR) ..................................................... 72 1.4.7. RNAi (RNA interference) ......................................................................................... 75 IV 1.4.8. Statistical methods and calculation bottlenecks .................................................. 79 Chapter 2: Potato virus Y (PVY) strains in Belgian seed potatoes and first molecular detection of the N-Wi strain .............................................................................................. 82 2.1. Introduction ................................................................................................................ 82 2.2. Materials and Methods ............................................................................................... 84 2.2.1. PVY screening and collection of PVY isolates ...................................................... 84 2.2.2. Strain determination ................................................................................................ 85 2.3. Results ........................................................................................................................ 87 2.3.1. Prevalence of PVY types in Belgium ...................................................................... 87 2.3.2. Strain determination ................................................................................................ 89 2.3.3. Symptoms, strain and potato cultivar relation ...................................................... 94 2.4. Discussion .................................................................................................................. 95 Chapter 3: Quantity and transmission efficacy of an isolate of the Potato virus Y-Wilga (PVY N-Wi) by aphid species reared on different host plants ........................................... 99 3.1. Introduction ................................................................................................................ 99 3.2. Material and Methods ............................................................................................... 100 3.2.1. Aphids, plants and virus isolate ........................................................................... 100 3.2.2. RNA‑transcript synthesis ........................................................................................ 101 3.2.3. PVYN−Wi transmission bioassays ............................................................................. 102 3.2.4. PVY N-Wi quantification in the aphid stylet and intact whole body ................................. 103 3.2.5. Preparation of aphid and plant samples, and RNA extraction ........................... 104 3.3. Results ....................................................................................................................... 104 3.3.1. RNA transcript and standard ................................................................................ 104 3.3.2. Transmission of PVYN−Wi by aphids ...................................................................... 105 3.2.3. Quantification of PVYN−Wi in the aphid stylet and intact whole body .............................. 107 3.3. Discussion ...............................................................................................................
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