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

Specific mRT-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 GYSV

DAYEH UNIVERSITY

ENVIRONMENTAL ENGINEERING DEPARTMENT

COLLEGE OF ENGINEERING

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

DOCTORAL DISSERTATION

ABSTRACT

In this study a multiplexreverse-transcription polymerase chain reaction 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

(mRT-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

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 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

37.6％〜99.7％。而樣品葡萄黃斑類病毒-（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

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

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

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 1 INTRODUCTION 1

1.1 Background of Research 1

1.2 Purposes of Research 2

1.3 Goals to Reach 3

1.4 Framework of Research 3

Chapter 2 LITERATURE REVIEWS 5

2.1 Structure and Classification of Viroid 5

2.1.1 Structure 5

2.1.2 Classification 7

2.2 Generation of Populations from Individual Viroid Variants 7

2.3 Origin and Evolution of Viroids 8

2.4 The Viroid Species Infect Grapevine 10

2.4.1 Hop Stunt Viroid 11

2.4.2 Grapevine Yellow Speckle 1, 2 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 3 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

3.7 Phylogenetic Tree Construction 31

Chapter 4 RESULTS AND DISCUSSION 33

4.1 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 5 CONCLUSIONS AND SUGGESTIONS 49

5 1 Conclusions 49

5 2 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

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

(A) Hop stunt viroid (HSVd) and Grapevine yellow speckle 1 (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 1 (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 GYSVd-

1 and HSVd Lane M, molecular weight markers (GENMARK, GM100) 35

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

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

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

SCIENTIFIC TERMS

m3/s Cubic meters per second

mg/l Milligrams per liter

Chapter 1 INTRODUCTION

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

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

1.2 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

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

Figure 1.1 Framework of research

Chapter 2 LITERATURE REVIEWS

2.1 Structure and Classification of Viroid

2.1.1 Structure

Like many RNA viruses that infect plants or animals, individual viroids exist as complex populations of often closely related sequence variants in vivo A number of studies have examined natural variability within viroid populations, and the Subviral RNA Database now contains the complete sequences of more than 1,100 viroid variants In many cases, multiple sequence variants have been isolated from a single infected plant

Viroids are circular RNAs, infectious agents with a small, single-strand RNA molecules, viroids do not code for proteins, the length of viroids is about 250 to 400 nucleotides Viroids are able to replicate and move through infected plants, which cause frequently severe diseases in plants to severe stunting, leaf necrosis, corky-bark, leaf-roll and fruit deformation depending on host plant and viroid species

They contain five structural regions: left terminal region (T1), pathogenic region (P), central conserved region (C), variable region (V) and right terminal region (T2)

All species in the family Pospiviroidae have a rod-like secondary structure that

contains five structural/functional domains (Keese 1985) and replicates in the nucleus

Three of the four members of the Avsunviroidae have a branched secondary structure, and all replicate/accumulate in the chloroplast All members of the Avsunviroidae

contain hammerhead ribozymes in both the infectious (+) strand and complementary (−) strand RNAs Figure 2-1 compares the secondary structures of PSTVd (rod-like,

Pospiviroidae) and Peach latent mosaic viroid (PLMVd; branched, Avsunviroidae)

With the possible exception of PLMVd, viroids do not appear to contain any modified

nucleotides or unusual phosphodiester bonds Nucleic acid extracts from infected leaf tissue contain a variety of viroid-related RNAs of both polarities Some of these molecules – especially those having a complementary or (−) strand polarity – are considerably longer than the infectious circular viroid (+) strand Northern analysis using strand-specific probes and/or primer extension has shown that these molecules represent the intermediates expected for a “rolling-circle” mechanism of replication

2.1.2 Classification

Up to now, viroid species are classified into two families Pospiviroidae and

Avsunviroidae, which are composed of five and two genera, respectively

Pospiviroidae possess a thermodynamically stable rod-like secondary structure with a CCR (central conserved region) and do not self-cleave, including the genera

Pospiviroid, Coleviroid, Hostuviroid, Cocadviroid and Apscaviroid Avsunviroidae do

not possess a CCR and self-cleave via hammerhead ribozyme, with the genera

Avsunviroid and Pelamoviroid

Nucleic acid extracts from infected leaf tissue contain a variety of viroid-related RNAs of both polarities Some of these molecules – especially those having a complementary or (−) strand polarity – are considerably longer than the infectious circular viroid (+) strand Northern analysis using strand-specific probes and/or primer extension has shown that these molecules represent the intermediates expected for a

“rolling-circle” mechanism of replication

2.2 Generation of Populations from Individual Viroid Variants

Several different approaches have been used to monitor the genetic stability of individual viroid sequence variants in vivo These include inoculation with

recombinant plasmid DNAs (Góra-Sochacka 1997), Agrobacterium-mediated introduction of nondisarmed recombinant Ti plasmids (Hammond 1994), and Agrobacterium-mediated plant transformation (Wassenegger M 1994) When working with highly debilitated variants that are only weakly infectious, constitutive expression from an integrated transgene provides an effective means to detect the rare events that can restore viroid infectivity (Góra 1994) used a reverse-transcription PCR strategy to generate, in a single step, infectious full-length cDNAs from three phenotypically dissimilar isolates of PSTVd When this method was applied to a

“mild” isolate, only a single sequence variant was recovered “Intermediate” and

“severe” isolates yielded three and four variants, respectively Not all of the variants recovered from the severe isolate produced severe symptoms when inoculated onto Rutgers tomato; thus, the presence of milder variants in a mixed inoculum may be masked by variants that are more severe Follow-up studies of (Góra-Sochacka 1997) revealed that many of these naturally occurring PSTVd sequence variants were unstable when inoculated alone – sometimes disappearing within a single 5~6 week passage in tomato This finding supports one of the basic tenets of the quasis-pecies theory, that mixtures of variants can complement each other, and hence the whole population is in essence a single entity analogous to an individual with thousands of alleles rather than just two In most cases, the new variants detected induced symptoms that were less severe than those of the parent The number of sequence changes detected in both studies was relatively limited, confined almost exclusively to the pathogenicity and variable domains with only a few changes located in the terminal right domain

One important advantage of screening assays that involve mechanical inoculation of full-length viroid cDNAs or RNA transcripts is that the results are usually available within a few weeks Many point mutations in PSTVd and other viroids, however, appear to abolish infectivity via mechanical inoculation In some

cases, these mutations have been shown to inhibit replication; in other cases, cell or long-distance transport is disrupted(Qi Y 2004)

cell-to-2.3 Origin and Evolution of Viroids

Several possible origins for viroids have been proposed Viroids could be primitive ancestors or highly degenerate derivatives of conventional viruses, but as discussed by (Diener 1989), their unusual molecular structure and biological properties together with a lack of sequence similarity Evolution argues against this possibility, of viroids from transposable elements, plasmids, or in-trons has also been proposed At present, the balance of evidence suggests that viroids could represent

“relics of precellular RNA evolution,” and several reviews exploring this area have been published (Diener 2003) In essence, the argument for viroid origin in the RNA world is straightforward: RNA is the only known biological macromolecule that can function as both genotype and phenotype, allowing evolution to occur in the absence

of DNA or protein As described by (Diener 1989), a simple hammerhead ribozyme

similar to those found in ASBVd and other members of the Avsunviroidae is

theoretically capable of performing all the polymerization, cleavage, and ligation steps required for viroid replication The circular structure of the viroid genome and the rolling-circle mechanism of replication eliminate the need for replication to initiate at

a specific site; likewise, the apparently polyploid nature of viroid genomes (Juhasz 1988) would have favored their survival under the error-prone conditions of the prebiotic world

It can be compared (shown as Figure 2-1) that the structure of the first intermediate in the PSTVd cleavage-ligation pathway (Baumstark 1997) with those of the hammerhead and hairpin ribozymes The upper portion of the pospiviroid central conserved region contains a short sequence motif (GAAA) that is also present in

hammerhead ribozymes (Diener 1989) Moving from the level of RNA primary/secondary structure to tertiary structure However, one can see that pospiviroid share an even greater degree of similarity with ribozymes The hairpin

ribozyme found in (-) strand satellite RNA of Tobacco ringspot virus contains two

domains that interact in the transition state Like the central conserved region of pospiviroids, the loop B domain of the hairpin ribozyme also contains a loop E motif Loop E motifs are found in many different contexts, often acting as “organizers” for multi helix loops in ribosomal RNAs; in the case of the hairpin ribozyme, a conformational change in the loop E motif accompanies domain docking and is essential for catalysis(Hampell 2001) In addition to sequence-specific cleavage, the hairpin ribozyme also catalyzes RNA ligation Recent experimental work with the hammerhead and hairpin ribozymes suggests that they have more similar than previously thought(Burke 2002), and the possibility that viroids are “relics of precellular evolution” continues to be very much alive

Pospiviroids and hammerhead ribozymes (lower left) both contain a short conserved GAAA (shaded) sequence, suggesting to (Diener 1989) the possible existence of a common ancestor in the prebiotic RNA world The presence of a loop E motif in the hairpin ribozyme (lower right) provides additional support for such a relationship The processing structure involved in the initial PSTVd cleavage (above) does not contain a loop E motif, however Cleavage sites in the respective RNAs are denoted by arrows

Figure 2-1-Possible evolutionary relationships between viroids and ribozymes

2.4 The Viroid Species Infect Grapevine

Five viroids, Hop stunt viroid (HSVd), Australian grapevine viroid (AGVd),

Grapevine yellow speckle viroid-1 (GYSVd-1), Grapevine yellow speckle viroid-2

(GYSVd-2) and Citrus exports viroid (CEVd) have been reported to infect grapevines

(Sano 1988) although only GYSVd-1 and 2 have been shown to induce yellow speckled symptom expression (Kolunow 1988) HSVd, AGVd, and CEVd produce no

obvious disease symptoms and infect in the grapevine unnoticed, acting as a symptomless reservoir, which represents a potential threat to other crops

2.4.1 Hop Stunt Viroid

The first viroid reported in grapevines was an isolate of Hop stunt viroid (HSVd) from Japan HSVd is a member of the Potato spindle tuber viroid group, belonging to the family Pospiviroid HSVd apparently has a wide host range and besides hopping

it can propagate in cucumber, grapevine, citrus, plum, peach, pear (Teruo 1989),apricot and almond plants (Polivka 1996) Eighty-four HSVd sequences are present in

the subviral RNA database (Pelchat 2003) Hop stunt viroid (HSVd) infects a large

number of woody plant hosts such as Prunus spp., Citrus spp., and Vitis spp HSVd along with Citrus exocortis viroid (CEVd) has been detected in both citrus and grapevines Hence, to differentiate these two viroids, total RNA from leaves of grapevine Vitis vinifera „Cabernet Sauvignon‟ and V labrusca „Niagara Rosada‟ was used as a template for RT-PCR assay Primers specific for HSVd, CEVd, Grapevine speckle viroid 1 (GYSVd-1), Grapevine speckle viroid 2 (GYSVd-2) and Australian grapevine viroid (AGVd) were employed The PCR amplicons were cloned and sequenced The grapevine samples analyzed showed the presence of both HSVd and CEVd Phylogenetic analysis showed that Brazilian grapevine HSVd variants clustered with other grapevine HSVd variants, forming a specific group separated from citrus variants On the other hand, the Brazilian CEVd variants clustered with other citrus and grapevine variants (Eiras 2000) Molecular characterization of Hop stunt viroid (HSVd) isolates from different naturally infected Prunus sources including apricot, plum and peach were performed by determining the nucleotide sequences of eleven isolates Five new sequence variants of 296-nt (3 variants) or 297-nt (2 variants) comparable to the known HSVd isolates were identified

Association of Hop stunt viroid (HSVd) with yellow corky vein disease of citrus

(CYCVD) occurring in India was investigated to establish its causal relationship with the disease In silico analysis showed that HSVd has 295 nucleotides and that the

isolates exhibited nearly 100% nucleotide identity with six citrus cachexia isolates of

HSVd This variant was tentatively designated Hop stunt viroid-ycv (Roy 2003) Later

Citrus exocortis viroid (CEVd) was also found to be associated with CYCVD

BLAST analysis revealed the alignment of the sequences with a different CEVd The isolates from CYCVD-infected plants was tentatively named as a variant of CEVd-

ycv This variant showed close relationship with CEVd Gynura variants reported from

Australia (Roy 2006)

Phylogenetic analyses indicated that one apricot isolate clustered with a recombinant group, whereas all others (one apricot, two plum and one peach isolate) clustered with the hop-group, confirming the genetic diversity of HSVd isolates The sequence variability appeared to be more related to the geographical origin of the

isolates than to their hosts (Gazel 2008)

2.4.2 Grapevine Yellow Speckle 1, 2

The first described Grapevine yellow speckle disease to be caused by viroids in grapevine to date(Kolunow 1988) Some of the infected plants developed yellow speckle symptoms indicating that both viroids can cause Grapevine yellow speckle disease GYSVd-1 is the causal agent of yellow speckle disease They are direct or indirect associated with plant virus infection in the field (Woodham.R.C 1972) The foliar symptoms of yellow speckle are often absent in confined to a few yellowish spots or flecks scattered in tissue along major or minor veins of leaves (Szychowski 1998) GYSVd-1 is the most closely related viroid to GYSVd-2 with an overall sequence Similarly, that occurs between the two species

2.4.3 Australian Grapevine Viroid

Australian grapevine viroid (AGVd) is a novel viroid with less than 50% sequence similarity with any known viroid Nevertheless, its entire sequence can be divided into regions, each with a high sequence similarity with segments from one of citrus exocortis, potato spindle tuber, apple scar skin, and Grapevine yellow speckle viroids AGVd contains the entire central conserved region of the apple scar skin viroid group and is proposed as a member of this group(Kolunow 1988)

2.4.4 Citrus Excortis Viroid

CEVd has a wide host-range, that is, in addition to citrus (Fawcett 1948) and

grapevine (García-Arenal 1987), it infects some species in the family Compositae (Niblett 1980), Solanaceae (Morris 1977) Leguminosae, Brassicaceae, and

Moraceae, such as chrysanthemum (Niblett 1980), tomato (Mishra 1991), broad bean

(Fagoaga 1995), eggplant, turnip carrot (Fagoaga 1996), and fig (Yakoubi 2007), respectively

2.5 The Detection Techniques in Viroid Disease

Viroids are commonly detected by electron microscopy and biological characterization, based on host range, bioassay, poly-acrylamide gel electrophoresis (PAGE), molecular hybridization, RT-PCR-enzyme-linked immune-sorbent assay (RT-PCR-ELISA), real-time PCR and RT-PCR

A real-time polymerase chain reaction (qPCR) is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR) It

time, and not at its end, as in conventional PCR Real-time PCR can be used quantitatively (Quantitative real-time PCR), and semi-quantitatively, i.e above/below

a certain amount of DNA molecules (Semi quantitative real-time PCR)

Two common methods for the detection of PCR products in real-time PCR are: (1) non-specific fluorescent dyes that intercalate with any double-stranded DNA, and (2) sequence-specific DNA probes consisting of oligonucleotides that are labeled with

a fluorescent reporter which permits detection only after hybridization of the probe with its complementary sequence

The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines proposes that the abbreviation qPCR be used for quantitative real-time PCR and that RT-qPCR is used for reverse transcription–qPCR The acronym "RT-PCR" commonly denotes reverse transcription polymerase chain reaction and not real-time PCR, but not all authors adhere to this convention The usefulness of ELISA tests for differentiation of strains of several viruses has been reported: Tomato spotted wilt virus (Sherwood 1989, De Ávila 1990), Beet mild yellowing virus (Stevens 1994, Smith 1996), Soybean mosaic virus (Bowers 1979),Potato virus Y (Canto 1995), Apple chlorotic leaf spot virus (Malinowski 1998) and Beet necrotic yellow vein virus (Mahmood 1999) In addition to the coat protein (CP)

of Potato virus Y (PVY), a helper component protein (HC-Pro) that helps in the aphid transmission is also synthesized in the infected plants MAbs and PAbs generated against the HC-Pro could be used to differentiate the strains of PVY (Canto 1995, Blanco-Urgoiti 1998) A MAb was employed to differentiate Beet mild yellowing virus (BMYV) with a differing host range from other strains of BMYV commonly observed under field conditions (Smith 1996)

Monoclonal antibodies (MAbs) have the greater discriminating capacity as they can react with different specific epitopes present on the viral coat protein or other virus-associated protein Plant viruses and their strains have been assigned to different serotypes based on their reactivity to different MAbs The serotypes may have similar

biological or structural characteristics in common By employing DAS-ELISA format using a combination of the universal MAb5B, and MAbs specific for serotypes, the West African Rice yellow mottle virus (RYMV) isolates (73) were grouped into three distinct serogroups These serogroups were correlated to two RYMV pathotypes that were differentiated based on their reaction on a set of differential rice cultivars (Konaté G.; Traoré 1997)

A threshold of 90% amino acid sequence identity of tospoviral nucleocapsid protein (NP) that Encapsidates viral RNAs is one of the important criteria for species designation (Goldbach 1996) Based on the serological relationships and phylogenetical analysis of NPs, the genus Tospovirus has been assigned with 16 official and tentative virus species that can be clustered into three major serogroups and four monospecies serotypes (Jan 2003) Tomato spotted wilt virus (TSWV), and Watermelon silver mottle virus (WSMoV) are the representatives of TSWV and WSMoV serogroups respectively Calla Lily chlorotic spot virus (CCSV) isolated from Taiwan was identified as a tospovirus serologically but distantly related to WSMoV based on serological relationship established by employing PAbs and MAbs

to WSMoV NP and CCSV NP in indirect ELISA format and low-intensity banding in immunoblotting The MAbs produced against CCSV NP, or WSMoV NP reacted specifically with homologous antigens, but not with heterologous antigens in both ELISA and immune blot analyses (Lin 2005)

Hybridization with cDNA or cRNA probes is an easy and powerful method for detecting differences located in any region of the genome (Rosner 1984, Rosner 1986) However, the need for RNA purification and for radioactive probes which have

a short life and safety hazards, limit the wider use of hybridization methods Availability of non-radioactive probes like digoxigenin (DIG)-labeled DNA and RNA probes in the recent years, has enlarged the scope of their wider applicability A non-isotopic hybridization procedure was developed to differentiate isolates of Citrus

RNA Hybridization of DIG-labeled probes with purified dsRNA or concentrated total RNA extracts spotted on nylon membranes allowed detection of CTV nucleic acid equivalent to as little as 0.1–1.0 mg infected tissues Comparatively, the sensitivity level was similar or slightly better than that was obtained by hybridization with a 32P-labeled probe Hybridization of tissue prints with DIG- labeled probes under stringent conditions (60°C and 50% formamide) could differentiate CTV isolates in citrus plants grown either in the greenhouse or the field (Narváez 2000)

A system of microarrays has been developed for detecting and differentiating plant viruses and their strains The amplicons from plant viral RNA are used for their differentiation by hybridization to synthetic oligonucleotide probes arranged in a two-dimensional array on a glass slide Cucumber mosaic virus (CMV) known to be highly heterologous in its coat protein (CP) was used as the model pathogen

The CP genes of 14 different isolates were amplified using cy3-labeled generic, but species-specific primers These amplicons were hybridized against a set of five different serotype and subgroup-specific 24-mer oligonucleotides (probes) bound to

an aldehyde-coated glass slide via an amino linker The probes targeted regions optimal for the differentiation between subgroups 1 and 2 or between subgroups 1a and 1b This microarray procedure allowed a clear differentiation of the 14 different CMV isolates into serogroup 1 and 2 and it was also able to assign nine out of ten different serogroups/isolates correctly into subgroup 1a and 1b Such a clear differentiation was not attainable using RFLP analysis using the restriction enzyme MspI The differentiation hybridization against five specifically selected nucleotides clearly demonstrates the high potential of oligonucleotide-based microarray technology for the virus isolate level detection and differentiation This report presents the development of a diagnostic chip for plant viruses for the first time (Deyong 2005)

However, these methods have limited practicality Bioassays are associated with constraints of time and space, while PAGE is restricted by the number of samples for

analysis Quantitative, or real-time, PCR (qPCR) is standard PCR with the advantage

of detecting the amount of DNA formed after each cycle with either fluorescent dyes

or fluorescently-tagged oligonucleotide probe qPCR results can be obtained faster and with less variability than standard PCR due to sensitive fluorescent chemistry and elimination of post-PCR detection procedures, Real-time PCR for specific detection and quantification of virus and viroid

Reverse transcription followed by real-time PCR assays based on TaqMan®

chemistry have been developed for the detection and quantification of Cucumber vein

yellowing 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 B.tabaci to compensate for variations

in extraction efficiency The assays designed were used to estimate proportions of viruliferous whiteflies collected from commercial greenhouse-grown crops in Spain

In a significant number of whiteflies, both viruses were detected and their amounts were estimated The assays could be used to assist risk assessment of CVYV and CYSDV which constitute limiting factors in cucurbit crops They are also suited to investigating the epidemiology and plant–virus–vector relationships in these diseases Polymerase chain reaction (PCR) is a biochemical process for amplifying specific DNA sequences from relatively small amounts of starting material This technology mimics DNA replication in a test tube with one key enzyme, thermophilic DNA polymerase PCR takes place in a thermal cycler machine and involves three steps: Melting: denaturing of the DNA duplex at a high temperature to yield single stranded DNA, Annealing: primers anneal to the single-stranded target DNA sequence, Elongation: DNA polymerase extends the primers by adding dNTPs to the phosphate backbone These steps complete one PCR cycle, and the cycle repeats until

a sufficient DNA concentration is reached

Apple dimple fruit viroid (ADFVd) induces a serious fruit disorder that reduces

developed with differently labeled primers in conjunction with a universal primer for

detection and differentiation of isolates of ADFVd and Apple scar skin viroid

(ASSVd) which also induces similar (dapple) symptoms on the apple fruits as ADFVd The sequence variability of two ADFVd field isolates from two commercial apple cultivars was investigated Sequencing of 18 full-length cDNA clones revealed five new sequence variants Sequence comparison showed nine polymorphic positions distributed in different regions of the ADFVd molecule As ADFVd and ASSVd cause similar symptoms, it is essential to ascertain the extent of infection by these two viroids Simultaneous detection of both viroids in a multiplex format was accomplished Reverse transcription with primer ADA-36 was followed by a single PCR reaction with this primer and two viroid specific primers AD-38 rd and AS-3ft The resulting products were analyzed by agarose gel electrophoresis and examined under UV The sequence conservation existing between certain regions of the ADFVd and ASSVd, as well as their sequence divergence in other regions were used to design viroid-specific primers, each labeled with a different fluorescent dye for rapid detection and discrimination of the two viroids by RT-PCR amplification A cDNA of the expected size and fluorescence (254-bp and red, and 330-bp and green) was amplified from tissues infected by ADFVd and ASSVd respectively This protocol could also differentiate these two viroids in double infections in which one of them was present in higher concentration than the other viroid(Di Serio F 2002)

The incidence of Apple scar skin viroid (ASSVd) was recorded for the first time

in Himachal Pradesh, India The genetic diversity of the isolates of ASSVd was assessed by performing RT-PCR assay The amplicons (~330-bp) were cloned and sequenced Ten clones so generated showed significant variability (94–100%) with each other Further, variability was found to be more common in the pathogenic domain of the viroid genome Four clones had 330-nt, while the other six clones had

an additional nucleotide Seven clones were considered as new sequence variants of ASSVd Two clones showed 100% identity to a Chinese isolate, whereas six clones

exhibited 99% nucleotide similarities to a Korean isolate The remaining two clones were more similar to the Chinese and Japanese isolates of ASSVd The RT-PCR protocol developed in this investigation was useful for detection and discrimination of

isolates of ASSVd (Walia Y 2009)

Peach latent mosaic viroid (PLMVd) belonging to the genus Pelamoviroid in the

family Avsunviroidae and Hop stunt viroid (HSVd) belonging to the genus

Hostuviroid in the family Pospiviroidae cause important diseases in Prunus spp

PLMVd isolates (348–351 nt) causing peach calico (PC) disease were reported to have

an additional sequence that contains the PC pathogenicity determinant (Malfitano 2003) Phylogenetic studies indicated that PLMVd isolates could be divided into three main groups (Ambrós S 1998) Sequencing of the PCR products showed that nine new PLMVd variants were present in the 177 peach samples examined The length of the sequenced PLMVd isolates ranged between 335- and 338-nt All the obtained sequences clustered together in group III HSVd isolates have been divided into three groups primarily based on overall homology, as plum-type, hop-type and citrus-type The length of the isolates sequenced was 297 and 298nt Two HSVd isolates from apricot cv Saturn were not clustered in one of the previously proposed groups, suggesting a novel putative group of HSVd isolates Both HSVd isolates were found

to be derived from recombination(Mandic B 2007)

RT-PCR has become important tools for viroid (Owens 1981) Simultaneous detection and identification of virus and viroid using conserved primers The sensitivity of RT-PCR is higher than Molecular hybridization

2.6 Elimination of Viroids from Plants

Grape is one of the most popular in the world In Taiwan, the grape is the most

grapevine cultivation Because these diseases and virus are difficult to eliminate from grape plants by conventional apical meristem culture methods, much effort has been made to generate grape plants completely free of diseases

A long period of low-temperature treatment is effective in generating grape plants Alternatively, grape plants can be generated from shoot tips cultured on a medium containing anti-virus agents Currently, methods of detecting various diseases have improved, and many sensitive methods, such as nested PCR or dot blot hybridization, have been established for viroid detection

Chapter 3 STUDY METHODS

3.1 Source of Plant Materials

Young leaves of 50 grapevine samples were collected during the hotter months of summer from difference vineyards in Changhua, Taiwan during summer of 2015 Plant leaves showed either yellow speckle or line patterns or asymptomatic plants Samples from four or five neighboring plants of every second row in the vineyard were analyzed Leaves were collected and stored at -80oC until use (shown as Figure 3-1)

Figure 3-1: Grapevine leaves collected from Changhua County, Taiwan in June 2015

(A) Hop stunt viroid (HSVd) and Grapevine yellow speckle 1 (GYSVd-1) were detected in the leaf samples; (B) Only GYSVd

Figure 3-1 (conti.): Grapevine leaves collected from Changhua County, Taiwan in

June 2015 (A) Hop stunt viroid (HSVd) and Grapevine yellow speckle 1 (GYSVd-1) were detected in the leaf samples; (B) Only GYSVd

3.2 Extraction of RNA

Grapevine leaves (100 mg) were homogenized in a mortar with 450 µl PRX

buffer (add ten µl β-mercaptoethanol) After centrifugation at full speed (13000 x g )

for 2 minutes, transfer flow-through sample from the Collection tube to a new tube, add 230 µl 98-100% ethanol to the clear lysate and mix by pipette, apply 680 µl of the ethanol added sample to a Plant Total RNA Mini Column sitting in a Collection Tube,

close cap, centrifuge at 10000 x g for 1 minute, and discard the filtrate Wash the

column once with 0.5 ml of WF buffer by centrifuging at full speed for 1 minute and discard the filtrate Wash the column twice with 0.7 ml of WS buffer by centrifuging

at full speed for 1 minute and discard the filtrate then centrifuge at full speed for 3 minutes to remove traces of WS buffer Finally, transfer the column to an RNase-free 1.5 ml Elution tube, add 50 µl of RNase-free dd H2O, and centrifuge at full speed for

2 minutes to elute RNA The quality of the extracted RNA was measured using UV Spectrophotometer (Figure 3-2) with ratio 1.8~2.0

Figure 3-2: UV Spectrophotometer

3.3 Designation of Viroid-Specific Primer

In fact, all of the available methods use approximations (heuristics) Moreover, observed performance differences in comparative analyses (see later) usually emerge

as average estimates; hence, approaches that work well for a certain gene or protein family may not work as well for a different one Therefore, as a standard procedure, one should use multiple alignment approaches and parameter sets and carefully inspect the results (reviewed in (Duret 2000, Notredame 2002) Here we will review

different global alignment procedures (i.e., for sequences related to their whole length)

to perform MSA Alignment is one of the most important but ironically underappreciated and neglected aspects of sequence analysis (Crandall 2005, J 2005); hence, we will endeavor to explain here the strategies underlying some of the most commonly used algorithms, as well as their strengths and caveats

Progressive alignment algorithms are by far the most widely used because of their speed, simplicity, and efficiency The basic strategy of these methods is first to estimate a tree and then to construct a pairwise alignment of the subtrees found at each internal node More sophisticated algorithms (e.g., iterative algorithms) also use this basic strategy in the initial or final steps of their routines The most frequently used progressive algorithm is the one implemented in Clustal-W (Thompson 1994) and its window interface Clustal-X (Thompson 1997)

Since the sequences of an increasing number of viroids are available in Genbank

on the world wide web, it is now possible to design primers specific for the detection

of a large number of viroids In this analysis, 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 synthesized according to a previous report(Table3-1) Four pairs of primers corresponding to HSVd-DY ( HSVd-(O) Old & HSVd-New) and GYSVd-1-DY( GYSVd-1-(O) Old & GYSVd-1-(N) New) were newly designed according to the sequences available at the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov) under the accession numbers: HSVd-New-DY (AB742225, HE575348, AB742224, JX401927, AY594202, FJ716178, JX418270, FJ716190, HQ386721, GU327606), HSVd-Old

was designed by Sano et al (2001), GYSVd-1-New-DY (JF746188, JF746176,

JF746193, JF746185, JF746182, JF746184, JF746189, JF746180, JF746183, JF746190), and GYSVd-1-O was designed by (Hajizadeh, Navarro et al 2012)

Complete sequence alignment was conducted by Clustal-W software (Figure 3 & 4) and specific primers for RT-PCR were designed(Table 3-1)

3-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