Assessement of the method for the detection of hop stunt virus and yellow speckle virus related grapevine

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 - - 30 ABSTRACT In this study a multiplexreverse-transcription polymerase chain reaction (mRTPCR) 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 37.6% to 99.7% While the sequence identity between GYSVd-1-DY with others ranged from 82.9% to 99.7% 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 月至 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 37.6％〜99.7％。而樣品葡萄黃斑類病毒1（GYSVd-1-DY）則類同範圍可達 82.9％〜99.7％。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 Prof 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 Chen 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 Lee B S., and 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 HydroMeteorological 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 iv ACKNOWLEDGEMENTS v CONTENTS vi LIST OF FIGURES iv LIST OF TABLES x LIST OF ABBREVIATION xii Chapter INTRODUCTION 1.1 Background of Research 1.2 Purposes of Research 1.3 Goals to Reach 1.4 Framework of Research Chapter 2.1 LITERATURE REVIEWS Structure and Classification of Viroid 2.1.1 Structure 2.1.2 Classification 2.2 Generation of Populations from Individual Viroid Variants 2.3 Origin and Evolution of Viroids 2.4 The Viroid Species Infect Grapevine 10 2.4.1 Hop Stunt Viroid 11 2.4.2 Grapevine Yellow Speckle 1, 13 2.4.3 Australian Grapevine Viroid 13 2.4.4 Citrus Excortis Viroid 13 2.5 The Detection Techniques in Viroid Disease 13 2.6 Elimination of Viroids from Plants 20 Chapter STUDY METHODS 21 3.1 Source of Plant Materials 21 3.2 Extraction of RNA 22 3.3 Designation of Viroid-Specific Primer 23 3.4 The Single RT-PCR Reaction 28 3.5 Multiplex RT-PCR (mRT-PCR) 29 3.6 DNA Elution and Cloning 30 -vi- 3.7 Phylogenetic Tree Construction 31 Chapter 4.1 RESULTS AND DISCUSSION 33 Results 33 4.1.1 Yield and quality of RNA extract 33 4.1.2 Primers Design for Detection Viroids 33 4.1.3 Detection of Grapevine viroids by single RT-PCR reaction…… ………33 4.1.4 Development of multiplex RT-PCR reaction……………………… ……34 4.1.5 T&A cloning and HindIII enzyme…………………………………… …35 4.1.6 Phylogenetic tree analysis……………………………………………… 39 4.1.7 Nucleotide sequence identity ……………………………………………42 4.2 Brief Discussion of Results 46 Chapter CONCLUSIONS AND SUGGESTIONS 49 Conclusions 49 Suggestions 51 REFERENCES 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 Figure 2: Possible evolutionary relationships between viroids and ribozymes 10 Figure 3-1: Grapevine leaves collected from Changhua County, Taiwan in June 2015 (A) Hop stunt viroid (HSVd) and Grapevine yellow speckle (GYSVd-1) were detected in the leaf samples; (B) Only GYSVd 21 Figure 3-1 (cont): Grapevine leaves collected from Changhua County, Taiwan in June 2015 (A) Hop stunt viroid (HSVd) and Grapevine yellow speckle (GYSVd-1) were detected in the leaf samples; (B) Only GYSVd 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 GYSVd1 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 neighborjoining (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- In our collected leaf samples, all of them were found to have infected by HSVd This is reasonable since HSVd is widespread in the world and it may be prevalent in the vineyards GYSVd-1 showed 16% of infection in collected samples The frequency of its infection is much lower as compare to that of HSVd No other grapevine viroids, including GYSVd-2, AGVd, and CEVd were found in the collected samples It is interesting to find that a DNA fragment obtained from RT-PCR analysis when using GYSVd-2 primers and the resulting sequence showed that it was GYSVd1 GYSVd-1 and GYSVd-2 are very closely related and have high sequence identity to each other It is important to carefully check if the amplicon is the correct target when detected between these two viroids -48- Chapter CONCLUSIONS AND SUGGESTIONS 5.1 Conclusions In designing primers for simultaneously detection viroids in plant samples, the following specifications needed to notice The designed primers needed to be capable to specific amply a single viroid target Primers sharing a similar melting temperature so as to facilitate setting the appropriate conditions for amplification of all the expected viroid target The DNA products after amplified needed to show in different size from each other to easily identify each of them by standard agarous gel electrophorsis When designed for the primers, it needs to consider the intraspecific sequence variability because of the quasispecies exist in the natural viroid population We have designed the primers for amplified different size of DNA fragment from different viroids The design of the primers were based on conserved regions in the sequence from several plant, in the hope that the same primers could be used in control reactions for all those plants and even for plants for which currently no sequence data are available In order to calculate accurately the melting temperature, the users have to specify which buffer is used for the amplification They can choose between using pre-set polymerase buffers or to specify the ionic content of the buffer and the algorithms they want using the custom-buffer mode The library of sequences to amplify should be provided in the form of a FASTA file The software returns a result page in which all primers are shown and can be downloaded as a FASTA or a -49- CSV file The validation plot showing the performance of the melting temperature algorithm used on experimental data and the logs of the program are also provided Most likely some viroids in these extracts is so high that, with the primer concentrations and RT-PCR conditions used, the amplification of the viroid-specific fragment out-competes the amplification of the control fragment This does not pose a real problem in practice, because the purpose of the control is to determine if the extract can support the RT-PCR reaction The finding that prolonged storage of plant tissue and plant extracts under the proper conditions (-80°C) is possible means that plants can be tested for additional viruses when their sequences become available and specific primers can be designed This study showed the effectiveness of a multiplex RT-PCR protocol for the contemporary identification of two grapevine infecting viroids The method was validated by testing naturally infected vines from Da-Yeh University of Taiwan and proved to be sensitive and reliable both in the absence and presence of the primer pair designed to amplify a host-derived internal control The formation of secondary structures by the target sequence, a major determinant affecting its interaction with other molecules, affects availability for hybridization(Murashige 1972) Random amplification approaches, such as the one used in this work, inherently generate non-targeted amplicons which can affect the sensitivity and specificity of hybridization reaction The genomic sequences of tospoviruses used for comparison were obtained from the National Center for Biotechnology Information (NCBI) databases (http://www ncbi.nlm.nih.gov/) Multiple sequence alignments, comparison of the newly determined sequences to the reference sequences, and translation of the nt sequences to aa residues were performed using the Clustal-W program, the Bl2seq program, and the Six frame program, respectively, of Biology Workbench The homologies of nt and aa sequences were calculated using the Gap program of SeqWeb Phylogenetic analysis was done using Philip 3.69 Bootstrapping was -50- repeated 100 times to generate multiple data sets, and versions of the input data sets were resampled with the Seqboot program of Philip 3.69 A distance matrix for the amino acid sequences was produced using the dnadist.exe program of Phylip 3.69 using the PAM matrixes of the Dayhoff model Phylogenetic branches were set by the Neighbor program of Phylip 3.69 using the neighbor-joining method Finally, phylogenetic trees were produced using the Consense program of Philip 3.69 5.2 Suggestions We compared then HSVd and GYSVd-1 from grapevine in Dacun, Yuanlin, Taiwan samples with previously published sequences in GenBank Results show the mutations on the closest sequence; the similarity was between 99% and 95 % The entire sequences of HSVd-DY and GYSVd-1-DY have 301 nt and 367 nt, respectively However, it shares the highest nt (95%-98%) identity with those AB742224, AB742225, GQ995466, GQ995464, Y14050 for HSVd and nt (95%-99%) identity with those JF746190, JF746188, GU170805, AY639607, AB028466, Z17225 for GYSVd-1 Phylogenetic analysis of the sequences of all of the viral proteins indicated that HSVd_DY and GYSVd-1-DY are closely related to GQ995464 and JF746188, AB028466, Z17225, respectively -51- REFERENCES Ambrós S, H C., Desvignes, JC and Flores, R (1998) "Genomic structure of three phenotypically different isolates of Peach latent mosaic viroid: Implication of the existence of constraints limiting the heterogeneity of viroid quasi-species." J Virol 72: 7397–7406 Baumstark, T., Schröder, A.R.W and Riesner, D (1997) "Viroid processing: switch from cleavage to ligation is driven by a change from a tetraloop to a loop E conformation." EMBO J(16): 599–610 Blanco-Urgoiti, B., Tribodet, M., Leclere, S., Ponz, F., Pérez de san román, C., Legorburu, F J and Kerlan, C (1998) "Characterization of potato potyvirus Y (PVY) isolates from seed potato batches Situation of the NTN, Wilga and Z isolates." European Journal of Plant Pathology 104(8): 811–819 Bowers, G R and Goodman, R M (1979) "Soybean mosaic virus: Infection of soybean seed parts and seed transmission." Phytopathology 69: 569-572 Burke, J M and Hamrick, J L (2002) "Genetic variation and evidence of hybridization in the genus Rhus (Anacardiaceae)." J Hered 93: 37-41 Canto, T., Ellis, P., Bowler, G., and Lopez-Abella, D (1995) "Production of monoclonal antibodies toPotato virus Y helper component protease and their use for strain differentiation." Plant Dis 79: 234-237 Chiou-Chu, S., Chung Jan, Chang., Che-Ming, Chang., Hsien-Tzung, Shih., Kuo-Ching, Tzeng., Fuh-Jyh, Jan., Chin-Wen, Kao and Wen-Ling Deng (2013) "Pierce's Disease of Grapevines in Taiwan: Isolation, Cultivation and Pathogenicity of Xylella fastidiosa." Journal of phytopathology 161(6):389396 -52- Crandall, K A., Perez-Losada, M., Christensen, RG., McClellan, DA and Viscidi, R P (2005) "Phylogenomics andmolecular evolution of polyomaviruses." In: Ahsan, N., (ed.) Polyomavirus and Human Diseases Landes BioScience; Georgetown, TX De Ávila, A C., Huguenot, C., Resende, R O., Kitajima, E W and Goldbach, R W (1990) "Serological differentiation of 20 isolates of Tomato spotted wilt virus." J Gen Vir 71(12): 2801-2807 10 Deyong, Z., Willingmann, P., Heinze, C., Adam, G., Pfunder, M., Frey, B and Frey, J E (2005) "Differentiation of Cucumber mosaic virus isolates by hybridization to oligonucleotides in a microarray format." J.Virol Meth 123: 101-108 11 Di Serio F, M M., Alioto, D., Ragozzino, A and Flores, R (2002) "Apple dimple fruit viroid: Sequence variability and its specific detection by multiplex fluorescent RT-PCR in the presence of Apple scar skin viroid." J Plant Pathol 84: 27–34 12 Diener, T O (1989) "Circular RNAs: relics of precellular evolution?" Proc Natl Acad Sci 86: 9370–9374 13 Diener, T O (2003) "Discovering viroids – a personal perspective." Nature Reviews Microbiology 1: 75-80 14 Duret, L and Abdeddaim, S (2000) "Multiple alignment for structural, functional, or phylogenetic analyses of Homologous Sequences." In: Higgins, D and Taylor, W (Ed.), Bioinformatics, Sequence, Structure and Databanks Oxford University Press, Oxford 15 Eiras, J C., Takemoto, R M and Pavanelli, G C (2000) "Métodos de estudo e técnicas laboratoriais em parasitologia de peixes / Methods of study and laboratorial techniques in parasitology of fish." EDUEM, Maringá 85: 45-55 -53- 16 Fagoaga, C., Semancik, JS and Duran-Vila, N (1995) "A citrus exocortis viroid variant from broad bean (Vicia faba L.): infectivity and pathogenesis." Journal of general virology 76(9): 2271-2277 17 Fagoaga, C and Duran-Vila, N (1996) "Naturally occurring variants of citrus exocortis viroid in vegetable crops." Plant pathology 45(1): 45-53 18 Fawcett, H and Klotz, J (1948) "Exocortis of trifoliate orange." CALIFORNIA AGRICULTURE: 13 19 García-Arenal, F., Pallás, Vicente., and Flores, Ricardo (1987) "The sequence of a viroid from grapevine closely related to severe isolates of citrus exocortis viroid." Nucleic Acids Res 15(10): 4203-4210 20 Gazel, M., Ulubaş, C., and Çağlayan, K (2008) "Detection of hop stunt viroid in sweet and sour cherry trees in turkey by RT-PCR." Journal of Plant Pathology 90(1): 23-28 21 Goldbach, R and Kuo, G (1996) "Introduction: Proceedings of the international symposium on tospo-virus and thrips and vegetable crops." Acta Hortic 431: 21-36 22 Góra-Sochacka, A., Kierzek, A., Candresse, T and Zágorski W (1997) "The genetic stability of potato spindle tuber viroid (PSTVd) molecular variants ." RNA 3: 68-74 23 Góra, A., Candresse, T and Zagórski, W (1994) "Analysis of the population structure of three phenotypically different PSTVd isolates." Arch Virol 138: 223-245 24 Hajizadeh, M., Navarro, B., Bashir, N S., Torchetti, E M and Di Serio, F (2012) "Development and validation of a multiplex RT-PCR method for the simultaneous detection of five grapevine viroids." J Virol Methods 179(1): 6269 -54- 25 Hammond, R (1994) "Agrobacterium-mediated inoculation of PSTVd cDNAs onto tomato reveals the biological effect of apparently lethal mutations." Virology 201: 36-45 26 Hampell, K J and Burke, J.M (2001) "A conformational change in the “loop E-like” motif of the hairpin ribozyme is coincidental with domain docking and is essential for catalysis." Proc Natl Acad Sci(40): 3723–3729 27 Horst, R and Cohen, D (1980) Amantadine supplemented tissue culture medium: a method for obtaining chrysanthemums free of Chrysanthemum stunt viroid V International Symposium on Virus diseases of Ornamental Plants 110 28 Felsentein, J (2005) "PHYLIP: phylogeny inference package version 3.6 Seattle (WA):." University of Washington 29 Jan, F J., Chen, T C and Yeh, S D (2003) "Occurrence, importance, taxonomy and control of thrips-borne tospoviruses." in: Advances in Plant Disease Management Research Signpost: 391-411 30 Juhasz, A., Hegyi, H and Solymosy, F (1988) "A novel aspect of the information content of viroids ." FEBS Biochim Biophys Acta(950): 455–458 31 Keese, P and Symons, RH (1985) " Domains in viroids: evidence of intermolecular RNA rearrangements and their contribution to viroid evolution." Proc Natl Acad Sci(82): 4582–4586 32 Kobayashi, Y., Kobayashi, A., Hagiwara, K., Uga, H., Mikoshiba, Y., Naito, T., Honda, Y and Omura, T (2005) "Gentian mosaic virus: a new species in the genus Fabavirus." Phytopathology 95(2): 192-197 33 Kolunow, A M., Krake, L R., Johnson, S D and Rezaian, M A (1988) "Two related viroids cause grapevine yellow speckle disease independently." J Gen Virol 70 ( Pt 12): 3411-3419 -55- 34 Konaté, G., Traoré, O and Coulibaly, M M (1997) "Characterization of rice yellow mottle virus isolates in sudano-sahelian areas." Archives of virology 142: 1117-1124 35 Lin, Y H., Chen, T C.; Hsu, H T., Liu, F L., Chu, F H., Chen, C C., Lin, Y Z and Yeh, S D (2005) "Serological comparison and molecular characterization for verification of Calla lily chlorotic spot virus as a new Tospovirus species belonging to Watermelon silver mottle virus serogroup ." Phytopathology 95: 1482–1488 36 Mahmood, T and Rush, C M (1999) "Evidence of cross-protection between beet soilborne mosaic virus and beet necrotic yellow vein virus in sugar beet." Plant Dis 83: 521-526 37 Malfitano, M., Di Serio F., Covelli, L., Ragozzino, A., Hernandez, C and Flores, R (2003) "Peach latent mosaic viroid variants inducing peach calico (extreme chlorosis) contain a characteristic insertion that is responsible for this symptomatology." Virology 313: 492–501 38 Malinowski, T K., B and Candresse, T (1998) "Characterisation of SX/2 an apple chlorotic leaf spot virus isolates showing unusual coat protein properties." Acta Hort 472: 43-50 39 Mandic B, A R M., Myrta, A., Gomez, G and Pallás, V (2007) "Incidence and genetic diversity of Peach latent mosaic viroid and Hop stunt viroid in stone fruits in Serbia." Eur J Plant Pathol.120(2):167-176 40 Mishra, M., Hammond, Rosemarie W., Owens, RA., Smith, DR and Diener, TO (1991) "Indian bunchy top disease of tomato plants is caused by a distinct strain of citrus exocortis viroid." J Gen Virol 72: 1781-1785 41 Morris, T and Smith, EM (1977) "Potato spindle tuber disease: procedures for the detection of viroid RNA and certification of disease-free potato tubers." Phytopathology 67(2): 145-150 -56- 42 Murashige, T., Bitters, WP., Rangan, EM., Nauer, EM., Roistacher, CN and Holliday, PB (1972) "A technique of shoot apex grafting and its utilization toward recovering virus-free Citrus clones." HortScience 7: 118-119 43 Narváez, G., Skander, B S., Ayllón, M A., Rubino, L., Guerri, J and Moreno, P (2000) "A new procedure to differentiate Citrus tristeza virus isolates by hybridization with digoxigenin-labeled cDNA probes." J Virol Meth 85: 8392 44 Newbury, H J and Possingham, J V (1977) "Factors affecting the extraction of intact ribonucleic acid from plant tissues containing interfering phenolic compounds." Plant Physiology 60: 543-547 45 Niblett, C., Dickson, Elizabeth., Horst, RK and Romaine, CP (1980) "Additional hosts and an efficient purification procedure for four viroids." Phytopathology 70(7): 610-615 46 Notredame, C (2002) "Recent progresses in multiple sequence alignment: A survey." Pharmacogenomics 3: 1-14 47 Owens, R A and Diener, T O (1981) "Sensitive and rapid diagnosis of potato spindle tuber viroid disease by nucleic Acid hybridization." Science 213(4508): 670-672 48 Page (1996) "TreeView: an application to display phylogenetic trees on personal computers." Oxford University Press 49 Pelchat, M., Rocheleau, Lynda., Perreault, Jonathan and Perreault, JeanPierre (2003) "SubViral RNA: a database of the smallest known autoreplicable RNA species." Nucleic Acids Res 31(1): 444-445 50 Polivka, H., Staub, U and Gross, H.J (1996) "Variation of viroid profiles in individual grapevine plants: novel grapevine yellow speckle viroid mutants show alterations of hairpin I." J Gen Virol 77 ( Pt 1): 155-161 -57- 51 Qi Y, P T., Itaya, A., Hunt, E., Wassenegger, M and Ding, B (2004) "Direct role of a viroid RNA motif in mediating directional RNA trafficking across a specific cellular boundary." Plant Cell 16: 1741-1752 52 Rosner, A and Bar- J., M (1984) "Diversity of Citrus tristeza virus strains indicated by hybridization with cloned cDNA sequences." Virology 139: 189– 193 53 Rosner, A., Lee, R F and Bar-Joseph, M (1986) "Differential hybridization with cloned cDNA sequences for detecting a specific isolate to Citrus tristeza virus." Phytopathology 76: 820-824 54 Roy, A and Ramanchada, P (2003) " Occurrence of a Hop stunt viroid (HSVd) variant in yellow corky vein disease of citrus in India." Curr Sci 85: 1608–1612 55 Roy, A and Ramachadran, P (2006) "Characterization of a Citrus exocortis viroid variant in yellow corky vein disease in India." Curr Sci 91: 798–803 56 Sano, T., Kudo, H., Sugimoto, T and Shikata, E (1988) "Synthetic oligonucleotide hybridization probes to diagnose hop stunt viroid strains and citrus exocortis viroid." J Virol Methods 19(2): 109-119 57 Sherwood, J L., Sanborn, M R., Keyser, G C and Myers, L D (1989) "Use of monoclonal antibodies in detection of tomato spotted wilt virus." Phytopathology 79: 61-64 58 Smith, H G., Barker, I., Brewer, G., Stevens, M and Hallsworth, P B (1996) "Production and evaluation of monoclonal antibodies for the detection of beet mild yellowing luteovirus and related strains." European Journal of Plant Pathology 102(2): 163-169 59 Stevens, M., Smith, H G and Hallsworth, P B (1994) "The host range of beet yellowing viruses among common arable weed species." Plant pathology 43: 579-588 -58- 60 Szychowski, J., Credi, R., Reanwarakorn, K and Semancik, JS (1998) "Population diversity in grapevine yellow speckle viroid-1 and the relationship to disease expression." Virology 248(2): 432-444 61 Teruo, S., Tatsuji, hataya., Yasuo, Terai and Eishiro, Shokita (1989) "Hop Stunt Viroid Strains from Dapple Fruit Disease of Plum and Peach in Japan." J.gen Virol 70(6): 1311-1319 62 Thompson, J D., Gibson, T J., Plewniak,F., Jeanmougin, F and Higgins, D G (1997) "The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignement aided by quality analysis tools." Nucleic Acids Res 25: 4876–4882 63 Thompson, J D., Higgins, D G and Gibson, T J (1994) "CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positionspecific gap penalties and weight matrix choice." Nucleic Acids Res 22: 4673-4690 64 Walia Y, K Y., Rana T., Bhardwaj, P., Raja Ram, Thakur PD., Usha Sharma, Hallan V and Zaidi, AA (2009) "Molecular characterization and variability analysis of Apple scar skin viroid in India." J Gen Plant Pathol 75: 307–311 65 Wassenegger M, H S a S., HL (1994) "An infectious RNA replicon evolved from an in vitro-generated non-infectious viroid deletion mutant via a complementary deletion in vivo." EMBO J 13: 6172-6177 66 Woodham, R.C and Taylor, R H (1972) "grapevine yellow speckle a newly recognized graft-transmissible disease of vitis." J.Agric Res 23: 447-452 67 Yakoubi, S., Elleuch, A., Besaies, N., Marrakchi, M and Fakhfakh, H (2007) "First report of Hop stunt viroid and Citrus exocortis viroid on fig with symptoms of fig mosaic disease." Journal of phytopathology 155(2): 125-128 -59- 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 (TR) -60- -61- Appendix B Map and Sequence reference points of T&A Cloning Vector -62- ... 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... 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... virus (CVYV) and Cucurbit yellow stunting disorder virus (CYSDV) in individual adults of the whitefly vector Bemisia tabaci The method includes an internal control for the detection of a gene from