DAYEH UNIVERSITY ENVIRONMENTAL ENGINEERING DEPARTMENT COLLEGE OF ENGINEERING DOCTORAL DISSERTATION Assessment of the Methods for the Detection of Hop Stunt Virus and Yellow Speckle Virus Related Grapevine Student: Nguyen Phuc Thien Advisor: Chen Yi Ching TAIWAN 106 - 6 - 30 ABSTRACT In this study a multiplexreverse-transcription polymerase chain reaction (mRT- PCR) technique is used simultaneously to detect viroids in grapevine. Fifty grapevine leaf samples with yellowing or mosaic symptoms were collected from difference vineyards at Changhua County, Taiwan during May to June, 2015-2016. Specific primer pairs for the detection of Hop stunt viroid (HSVd), Australian grapevine viroid (AGVd), Grapevine yellow speckle viroid-1 (GYSVd-1), Grapevine yellow speckle viroid-2 (GYSVd-2) and Citrus exocortis viroid (CEVd) were selected from previous reports or primers were newly designed according to the sequences obtained from NCBI Genbank. Comparison of the sequence identity of HSVd-DY with other HSVd from GenBank ranged from 37.
While the sequence identity between GYSVd-1-DY with others ranged from 82. A phylogenetic tree derived from the HSVd sequence indicated that HSVd-DY was closer to an Iran isolate (KF916041), while GYSVd-1-DY was close related to a China isolate (JF746188). Keywords: Multiplex RT-PCR, Grapevine viroids, phylogenetic tree, GYSVd-1, HSVd -iii- 中文摘要 在本研究中，多重逆轉錄聚合酶鏈反應（mRT-PCR）技術同時 被使用在檢測葡萄中的病毒。 在 2015 年及 2016 年的 5 月至 6 月期 間，從台灣彰化縣不同的葡萄園收集了五十個具有黃化或馬賽克症 狀的葡萄葉樣品。根據從 NCBI 基因庫獲得的序列，新設計的特殊 引子對被用於檢測啤酒花矮化類病毒（HSVd）､澳大利亞葡萄樹病 毒（AGVd）､葡萄黃斑類病毒-1（GYSVd-1）､葡萄黃斑類病毒-2 （GYSVd-2）及柑橘裂皮病類病毒。在比較了序列識別方面，樣品 啤酒花矮化類病毒（HSVd-DY）與基因庫的啤酒花矮化類病毒 （HSVd）之類同的範圍為 37.7％。而樣品葡萄黃斑類病毒- 1（GYSVd-1-DY）則類同範圍可達 82.7％。從啤酒花矮化 類病毒（HSVd）序列之演化樹推衍出樣品葡萄黃斑類病毒-1 （GYSVd-1-DY）更接近伊朗分離物（KF916041），樣品葡萄黃斑 類病毒-1（GYSVd-1-DY）則與中國分離物（JF746188）更密切相 關。 關鍵詞：多重逆轉錄聚合酶鏈式反應 ､葡萄類病毒､演化樹､葡萄 黃斑類病毒-1､啤酒花矮化類病毒 -iv- ACKNOWLEDGEMENTS I would like gratefully acknowledge to Prof. Chen Yi-Ching (陳宜清) in the Department of Environmental Engineering, Da-Yeh University, for his unlimited support, instruction, and encouragement.
Chen had directed me to be thorough and offering his knowledge and experience during my research. This thesis can not be completed without a great deal of help and encouragement from Prof. Several professors have contributed their time graciously on my behalf, and I would like to express my gratitude. It is a pleasure to thank the oral defense committee, Prof.
Shih Ing-Lung , Prof. Yu Shih-Chung , Prof. Yeh Philip, Prof. Lai Chi-Yung for their time and recommendations.
I would like to thank the staffs of the Department of Environmental Engineering, Da-Yeh University especially Ms Huang (馨嬅) for their help and support. I also would like to thank the Dr. Doan Quang Tri (段光智) in National Center for Hydro- Meteorological Forecasting (NCHMF). I would like to express my gratitude towards them.
Their advice and suggestions with insight throughout my work have evidently supported the consistency of my dissertation. And special thanks to all my Vietnamese and Taiwanese friends in Da-yeh for me to live a happy life during the study. Finally, I would like to desire great thanks to my family. Especially, I am indebted to my parents and my wife, who have been an inspiration throughout my entire life.
Without their constant support and understanding, I would not have had the persistence to finish this work. Nguyen Phuc Thien June 2017, Da-Yeh University -v- CONTENTS ABSTRACT. iii 中文摘要. vi LIST OF FIGURES.
iv LIST OF TABLES. x LIST OF ABBREVIATION. Background of Research. Purposes of Research.
Goals to Reach .4 Framework of Research .1 Structure and Classification of Viroid .2 Generation of Populations from Individual Viroid Variants .3 Origin and Evolution of Viroids.4 The Viroid Species Infect Grapevine .1 Hop Stunt Viroid .2 Grapevine Yellow Speckle 1, 2.3 Australian Grapevine Viroid .4 Citrus Excortis Viroid .5 The Detection Techniques in Viroid Disease .6 Elimination of Viroids from Plants .1 Source of Plant Materials .2 Extraction of RNA .3 Designation of Viroid-Specific Primer .4 The Single RT-PCR Reaction .5 Multiplex RT-PCR (mRT-PCR) .6 DNA Elution and Cloning .7 Phylogenetic Tree Construction. RESULTS AND DISCUSSION. Yield and quality of RNA extract. Primers Design for Detection Viroids.
Detection of Grapevine viroids by single RT-PCR reaction…….4 Development of multiplex RT-PCR reaction……………………….5 T&A cloning and HindIII enzyme…………………………………….6 Phylogenetic tree analysis……………………………………………….7 Nucleotide sequence identity.2 Brief Discussion of Results. CONCLUSIONS AND SUGGESTIONS. 52 APPENDIX A: The rod like secondary structure of Potato Spindle tuber viroid showing the five domains chacrateristic of members of the family Pospiviroidae: the terminal left (TL), pathogenicity (P), central (C), variable (V), and terminal right. 60 APPENDIX B: Map and Sequence reference points of T&A Cloning Vector.
61 -vii- LIST OF FIGURES Figure 1: Research framework. 4 Figure 2: Possible evolutionary relationships between viroids and ribozymes. 10 Figure 3-1: Grapevine leaves collected from Changhua County, Taiwan in June 2015. 21 Figure 3-1 (cont): Grapevine leaves collected from Changhua County, Taiwan in June 2015.
22 Figure 3-2: UV Spectrophotometer. 23 Figure 3-3: Multiple sequence alignment of HSVd by CLUSTAL-W program for primer designing. Complete sequence Hop stunt viroid was obtained from GenBank. The asterisk indicated conserved nucleotide.
Highline showed the position of the primers. 25 Figure 3-4: Multiple sequence alignment of GYSVd-1 by CLUSTAL-W program for primer designing. Complete sequence GYSVd-1 were obtained from GenBank. The asterisk indicated conserved nucleotide.
Highline shown the position of the primers. 27 Figure 3-5: RT-PCR Machine. 29 Figure 3-6: Cloning Plants. 31 Figure 4-1: Agarose gel electrophoresis analyse of single and multiplex RT-PCR reaction.
Lane 1, with specific primers for GYSVd-1; Lane 2, with specific primers for HSVd; Lane 3, with mixed primers for the detection of both GYSVd- 1 and HSVd. Lane M, molecular weight markers (GENMARK, GM100). 35 -viii- Figure 4-2: Agarose gel electrophoresis analyze T&A cloning vector kit insert DNA, Lane 1,2,4,5 with specific primers for GYSVd-1, Lane 3,6 with specific primers for HSVd. Lane M, molecular weight markers (GENMARK, GM100).
36 Figure 4-3: Restriction enzyme sites of yT&A® cloning vector…………………….38 Figure 4-4: Agarose gel electrophoresis analyzes T&A cloning vector kit insert DNA and HindIII enzyme. Lane 1,2,4,5 with specific primers for GYSVd-1, Lane 3,6 with specific primers for HSVd. Lane M, molecular weight markers (GENMARK, GM100). 39 Figure 4-5: Phylogenetic analysis of the complete sequence of HSVd-DY with other 17 HSVd retrieved from GenBank.
In the phylogenetic tree constructed using the PHYLIP software package (Felsenstein, 2005), the values adjacent to the nodes indicate the bootstrap confidence values for 1000 replicates using neighbor- joining (NJ) analyses. Values below 75% are not given. The units of the scale bar represent the nucleotide substitutions per site. 40 Figure 4-6: Phylogenetic analysis of the complete sequence of GYSVd-1-DY with other 17 HSVd retrieved from GenBank.
In the phylogenetic tree constructed using the PHYLIP software package (Felsenstein, 2005), the values adjacent to the nodes indicate the bootstrap confidence values for 1000 replicates using neighbor-joining (NJ) analyses. Values below 75% are not given. The units of the scale bar represent the nucleotide substitutions per site. 41 -ix- LIST OF TABLES Table 3-1: Primers in this work.
25 Table 4-1: Results of HSVd & GYSVd-1 analysis from Changhua County, Taiwan by mRT-PCR (MP) and single RT-PCR (SP) from 2015-2016. 34 Table 4-2: The comparison of sequence identity between HSVd-DY and other HSVd from GenBank. The accession number, infected host and isolated country are indicated. 43 Table 4-3: The comparison of sequence identity between GYSVd-1-DY and other GYSVd-1 from GenBank.
The accession number, infected host and isolated country are indicated. 44 -x- LIST OF ABBREVIATION ACRONYMS AGVd Australian grapevine viroid BMYV Beet mild yellowing virus CCR Central conserved region CEVd Citrus exocortis viroid CTV Citrus trizteza virus CPsV Citrus psorosis virus CVV Citrus infectious variegation virus CYCVD Corky vein disease of citrus PAGE Poly-acrylamide gel electrophoresis PLMVd Peach latent mosaic viroid PSTVd Potato spindle tuber viroid GYSVd-1 Grapevine yellow speckle viroid-1 GYSVd-2 Grapevine yellow speckle viroid-2 RT-PCR Reverse transcription polymerase chain reaction HSVd Hop stunt viroid MAbs Monoclonal antibodies RYMV Rice yellow mottle virus -xi- SCIENTIFIC TERMS m3/s Cubic meters per second l/s Liters per second mg/l Milligrams per liter kg/d Kilograms per day m Meters mm Millimeters -xii- Chapter 1.1 Background of Research Viroids are the smallest known agents of infectious disease – small, highly structured, single-stranded, circular RNA molecules that lack detectable messenger RNA activity. Whereas viruses supply some or most of the genetic information required for their replication, viroids are regarded as “obligate parasites of the cell‟s transcriptional machinery” and infect only plants. Four of the nearly 30 species of viroids described to date contain hammerhead ribozymes, and phylogenetic analysis suggests that viroids may share a common origin with hepatitis delta virus and several other viroid-like satellite RNAs.
Replication proceeds via a rolling-circle mechanism, and strand exchange can result in a variety of insertion/deletion events. The terminal domains of potato spindle tuber and related viroids, in particular, appear to have undergone repeated sequence exchange and/or rearrangement. Viroid populations often contain a complex mixture of sequence variants, and environmental stress (including transfer to different hosts) has been shown to result in a significant increase in sequence heterogeneity. The new field of synthetic biology offers exciting opportunities to determine the minimal size of a fully functional viroid genome.
Much of the preliminary structural and functional information necessary is already available, but formidable obstacles still remain. Sequences of viroids and viroid-like satellite RNAs were aligned separately using CLUSTAL-X and then manually edited to preserve local similarities; these partial alignments were then manually aligned before CLUSTAL-X was used to realign dissimilar regions and maximize overall similarity. -1- Virus and viroid diseases have become increasingly important constrain to sustainable crop production in the tropical countries. The climatic changes that are occurring throughout the world have an impact on plants, vectors, and viruses causing increasing instability within virus-host ecosystems.
Viroid diseases are inconspicuous compared with disease caused by fungi, bacteria and nematodes, and loss identified in comparative trials on their effects do not necessarily translate to global estimates of loss. Their effects also extend beyond direct and indirect damage associated with plant virus infection largely also applies to viroids. Some of the threatening and economically important virus diseases in tropical zone which affect the food production are tungro, yellow mottle, and hoja blanca in rice; mosaic in sugarcane, mosaic in cassava; tristeza in citrus; swollen shoot in cacao; sterility mosaic in pigeonpea; rosette, clump, and bud necrosis in peanut; necrosis in sunflower and legumes, vegetables, and ornamental crops; yellow mosaic in legumes; leaf curl in cotton and tomato; and ring spot in papaya. Key factors for emergence of new plant virus and virus-like disease include the intensification of agricultural trade (globalization), changes in cropping systems (crop diversification), and climate change.2 Purposes of Research In Taiwan, grape cultivation is a remunerative agri-business for the highly popular grape berries among local consumers in addition to the added values with the processed products.
The annual production of grapes was 102831 metric tons in 2010, and 99.5% was produced in Taichung City, Miaoli, Changhua and Nantou Counties in central Taiwan(Chiou-Chu 2013). RT-PCR has become crucial tools for detecting viroids. Simultaneous detection and identification of viruses and viroids using -2- conserved primers are possible through RT-PCR, which has a higher sensitivity than that of molecular hybridization. Our aim of this research is to isolate and identify the causal agents of the suspected disease in grapevines in Taiwan and to determine their phylogenetic relatedness to the other HSVd and GYSVd-1 strains from different geographical regions 1.3 Goals to Reach The objectives of this study were as follows: RT-PCR technique detect hop stunt viroid in grapevine (HSVd) RT-PCR technique detect grapevine yellow speckle viroid 1 in grapevine (GYSVd-1) Multiplex RT-PCR technique simultaneously detect viroids in grapevine 1.4 Framework of Research The framework of this research is shown as Figure 1-1.
HSVd and GYSVd-1 viroid are analyzed by Clustal-W solfware. Also a conserved region is found to design primers, then transferring to tube by using RT-PCR machine. Finally gel electrophoresis will be run.1 Framework of research -4- Chapter 2.1 Structure and Classification of Viroid 2.1 Structure Like many RNA viruses that infect plants or animals, individual viroids exist as complex populations of often closely related sequence variants in vivo.