UNIVERSITY OF LJUBLJANA BIOTECHNICAL FACULTY Vanja MILJANIĆ HIGH-THROUGHPUT SEQUENCING DETECTION AND MOLECULAR CHARACTERIZATION OF VIRAL DISEASES OF GRAPEVINE ( Vitis vinifera L.) AND THEIR ELIMINATION BY THERMOTHERAPY AND MERISTEM TISSUE CULTURE DOCTORAL DISSERTATION Ljubljana, 2022 UNIVERSITY OF LJUBLJANA BIOTECHNICAL FACULTY Vanja MILJANIĆ HIGH-THROUGHPUT SEQUENCING DETECTION AND MOLECULAR CHARACTERIZATION OF VIRAL DISEASES OF GRAPEVINE ( Vitis vinifera L.) AND THEIR ELIMINATION BY THERMOTHERAPY AND MERISTEM TISSUE CULTURE DOCTORAL DISSERTATION DETEKCIJA IN MOLEKULARNA KARAKTERIZACIJA VIRUSNIH BOLEZNI VINSKE TRTE ( Vitis vinifera L.) Z VISOKO ZMOGLJIVIM SEKVENCIRANJEM TER NJIHOVA ELIMINACIJA S POMOČJO TERMOTERAPIJE IN KULTURE MERISTEMOV DOKTORSKA DISERTACIJA Ljubljana, 2022 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Based on the Statute of the University of Ljubljana and the decision of the Biotechnical Faculty senate, as well as the decision of the Commission for Doctoral Studies of the University of Ljubljana adopted on 27th session, June 30th, 2020, it has been confirmed that the candidate meets the requirements for pursuing a PhD in the interdisciplinary doctoral programme in Biosciences, Scientific Field Biotechnology. Prof. dr. Nataša Štajner is appointed as supervisor. Doctoral dissertation was conducted at the Chair of Genetics, Biotechnology, Statistics, and Plant Breeding at the Agronomy Department, Biotechnical Faculty, University of Ljubljana. Commission for assessment and defense: President: Prof. dr. Jernej JAKŠE University of Ljubljana, Biotechnical Faculty Member: Prof. dr. Mario LEŠNIK University of Maribor, Faculty of Agriculture and Life Science Member: Assist. prof. dr. Maja RAVNIKAR National Institute of Biology Date of defense: II Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 KEY WORDS DOCUMENTATION DN Dd DC UDC 634.8:632.38:601.4:577.2(043.3) CX grapevine, Vitis vinifera L., HTS, virome, diagnosis, genetic diversity, thermotherapy, micrografting AU MILJANIĆ, Vanja AA ŠTAJNER, Nataša (supervisor) PP SI-1000 Ljubljana, Jamnikarjeva 101 PB University of Ljubljana, Biotechnical Faculty, Interdisciplinary Doctoral Programme in Biosciences, Scientific field Biotechnology PY 2022 TI HIGH-THROUGHPUT SEQUENCING DETECTION AND MOLECULAR CHARACTERIZATION OF VIRAL DISEASES OF GRAPEVINE ( Vitis vinifera L.) AND THEIR ELIMINATION BY THERMOTHERAPY AND MERISTEM TISSUE CULTURE DT Doctoral dissertation NO XII, 103, [3] p., 8 fig., 1 ann., 242 ref. LA en AL en/sl AB We studied the virome of preclonal candidates obtained after mass selection of grapevines using HTS technology. Nine viruses (GFLV, GLRaV-3, GRSPaV, GFkV, GSyV-1, GRVFV, GRGV, GPGV, and RBDV) and two viroids (HSVd and GYSVd-1) were identified. GRGV, GRVFV, and GSyV-1 were detected for the first time in Slovenia. All in silico predicted viruses and viroids were validated with RT-PCR and Sanger sequencing. We obtained a comprehensive insight into genetic diversity, phylogeny and co-infections. In the second part of the dissertation, we investigated the viruses and viroids elimination efficacy by in vivo thermotherapy and in vitro meristem tip micrografting. Heat therapy was performed at 36-38 °C for at least six weeks. Meristem tips (0.1-0.2 mm) were aseptically isolated and micrografted onto the sectioned, etiolated hypocotyls of Vialla ( Vitis labrusca × Vitis riparia). The overall regeneration rate was low (8.53%). The higher regeneration rate was observed in the white varieties. The regenerated plants were micropropagated several times. The sanitation status was checked with RT-PCR. All viruses were completely eliminated, while the elimination of viroids was less successful (39.2% for HSVd and 42.6% for GYSVd-1). It is important to emphasize that plant growth regulators (hormones) were not used. In the third part of the thesis, we studied the virome of samples that were not part of the clonal selection process. We detected: GLRaV-1, GLRaV-2, GLRaV-3, GFkV, GRVFV, GRSPaV, GFLV (in association with its satellite RNA), GPGV, GV-Sat, HSVd, and GYSVd-1. GV-Sat was also detected for the first time in Slovenia. We developed a protocol for high-throughput validation of in silico predicted infections, including various combinations of viruses, viroids, and satellites. III Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 KLJUČNA DOKUMENTACIJSKA INFORMACIJA ŠD Dd DK UDC 634.8:632.38:601.4:577.2(043.3) KG vinska trta, Vitis vinifera L., HTS, virom, diagnostika, genetska raznolikost, termoterapija, micrografting (mikrocepljenje) AV MILJANIĆ, Vanja SA ŠTAJNER, Nataša (mentorica) KZ SI-1000 Ljubljana, Jamnikarjeva 101 ZA Univerza v Ljubljani, Biotehniška fakulteta, Interdisciplinarni doktorski študijski program Bioznanosti, znanstveno področje Biotehnologija LI 2022 IN DETEKCIJA IN MOLEKULARNA KARAKTERIZACIJA VIRUSNIH BOLEZNI VINSKE TRTE ( Vitis vinifera L.) Z VISOKO ZMOGLJIVIM SEKVENCIRANJEM TER NJIHOVA ELIMINACIJA S POMOČJO TERMOTERAPIJE IN KULTURE MERISTEMOV TD Doktorska disertacija OP XII, 103, [3] str., 8 sl., 1 pril., 242 vir. IJ en JI en/sl AI Na osnovi visokozmogljivega sekvenciranja smo preučevali virom predklonskih kandidatov (82 vzorca, 6 sorti), pridobljenih po množični selekciji vinske trte. Identificirali smo devet virusov (GFLV, GLRaV-3, GRSPaV, GFkV, GSyV-1, GRVFV, GRGV, GPGV in RBDV) in dva viroida (HSVd in GYSVd-1). Virusi GRGV, GRVFV in GSyV-1 so bili prvič odkriti v Sloveniji. Vsi in silico napovedani virusi in viroidi so bili potrjeni z RT-PCR in Sangerjevim sekvenciranjem. Na osnovi sekvenc posameznih delov virusov in viroidov smo dobili podatke o genetski raznolikosti, filogeniji in sočasnih okužbah. V drugem delu doktorske naloge smo raziskali učinkovitost eliminacije virusov in viroidov po in vivo termoterapijo vinske trte ter po mikrograftingu izoliranih meristemov v in vitro pogojih. Toplotno terapijo smo izvajali pri 36-38 °C vsaj šest tednov. Meristemsko tkivo velikosti 0,1-0,2 mm smo aseptično izolirali in cepili na etolirane hipokotile sorte Vialla ( Vitis labrusca × Vitis riparia). Celotna stopnja regeneracije vzorcev iz meristemov je bila 8,53 %. Višjo stopnjo regeneracije smo opazili pri belih sortah. Regenerirane rastline smo večkrat subkultivirali in mikropropagirali. Stanje okuženosti po eliminaciji smo preverili z RT-PCR. Vsi virusi so bili odstranjeni iz vseh analiziranih vzorcev, medtem ko je bila eliminacija viroidov manj uspešna (39,2 % z HSVd in 42,6 % z GYSVd-1). Pomembno je poudariti, da pri regeneraciji in mikropropagaciji regulatorji rasti niso bili uporabljeni. V tretjem delu doktorskega dela smo proučevali virom vzorcev, ki niso bili del klonskega selekcijskega procesa. Potrdili smo prisotnost virusov GLRaV-1, GLRaV-2, GLRaV-3, GFkV, GRVFV, GRSPaV, GFLV (v povezavi s svojo satelitsko RNA), GPGV, GV-Sat, ter viroidov HSVd in GYSVd-1. GV-Sat je bil tokrat prvič potrjen v Sloveniji. Razvili smo protokol za visoko zmogljivo validacijo in silico predvidenih okužb, na osnovi hkratnega pomoževanja z RT-PCR (multiplex RT-PCR) za različne kombinacije virusov, viroidov in satelitov. IV Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 TABLE OF CONTENTS KEY WORDS DOCUMENTATION ...................................................................... III KLJUČNA DOKUMENTACIJSKA INFORMACIJA ............................................ IV TABLE OF CONTENTS .......................................................................................... V TABLE OF CONTENTS OF SCIENTIFIC WORKS ............................................ VII LIST OF FIGURES ............................................................................................... VIII LIST OF ANNEXES ................................................................................................ IX ABBREVIATIONS AND SYMBOLS ..................................................................... X 1 INTRODUCTION .................................................................................................... 1 1.1 GRAPEVINE AND GRAPEVINE VIRAL PATHOGENS ...................................... 1 1.1.1 Grapevine fanleaf virus ............................................................................................ 2 1.1.2 Grapevine leafroll-associated virus 3 ...................................................................... 3 1.1.3 Grapevine rupestris stem pitting-associated virus ................................................ 5 1.1.4 Grapevine fleck and fleck-similar viruses .............................................................. 6 1.1.5 Grapevine Pinot gris virus ....................................................................................... 6 1.1.6 Raspberry bushy dwarf virus .................................................................................. 7 1.1.7 Grapevine viroids ..................................................................................................... 8 1.2 METHODS FOR DETECTION OF GRAPEVINE VIRAL PATHOGENS ............. 9 1.3 METHODS FOR ELIMINATION OF GRAPEVINE VIRAL PATHOGENS ....... 12 1.4 AIMS AND HYPOTHESES .................................................................................... 14 2 SCIENTIFIC WORKS........................................................................................... 15 2.1 PUBLISHED SCIENTIFIC WORKS ...................................................................... 15 2.1.1 Virome Status of Preclonal Candidates of Grapevine Varieties ( Vitis vinifera L.) From the Slovenian Wine-Growing Region Primorska as Determined by High-Throughput Sequencing ............................................................................... 15 2.1.2 First Report of Grapevine Red Globe Virus, Grapevine Rupestris Vein Feathering Virus, and Grapevine Syrah Virus-1 Infecting Grapevine in Slovenia .................................................................................................................... 27 2.1.3 Elimination of Eight Viruses and Two Viroids from Preclonal Candidates of Six Grapevine Varieties ( Vitis vinifera L.) through In Vivo Thermotherapy and In Vitro Meristem Tip Micrografting ............................... 29 2.1.4 Small RNA Sequencing and Multiplex RT-PCR for Diagnostics of Grapevine Viruses and Virus-like Organisms ..................................................... 44 V Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 2.1.5 First report of grapevine satellite virus in Slovenia ............................................ 56 3 DISCUSSION AND CONCLUSIONS ................................................................. 59 3.1 DISCUSSION .......................................................................................................... 59 3.2 CONCLUSIONS ...................................................................................................... 71 4 SUMMARY (POVZETEK) ................................................................................... 73 4.1 SUMMARY ............................................................................................................. 73 4.2 POVZETEK ............................................................................................................. 78 5 REFERENCES ....................................................................................................... 86 ACKNOWLEDGEMENTS ANNEX VI Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 TABLE OF CONTENTS OF SCIENTIFIC WORKS Miljanić V., Jakše J., Kunej U., Rusjan D., Škvarč A., Štajner N. 2022. Virome Status of Preclonal Candidates of Grapevine Varieties ( Vitis vinifera L.) From the Slovenian Wine-Growing Region Primorska as Determined by High-Throughput Sequencing. Frontiers in Microbiology, 13, doi: 10.3389/fmicb.2022.830866: 11 p. Miljanić V., Jakše J., Kunej U., Rusjan D., Škvarč A., Štajner N. 2022. First Report of Grapevine Red Globe Virus, Grapevine Rupestris Vein Feathering Virus, and Grapevine Syrah Virus-1 Infecting Grapevine in Slovenia. Plant Disease, 106, 9: 2538 Miljanić V., Rusjan D., Škvarč A., Chatelet P., Štajner N. 2022. Elimination of Eight Viruses and Two Viroids from Preclonal Candidates of Six Grapevine Varieties ( Vitis vinifera L.) through In Vivo Thermotherapy and In Vitro Meristem Tip Micrografting. Plants, 11, 8: 1064, doi:10.3390/plants11081064: 14 p. Miljanić V., Jakše J., Rusjan D., Škvarč A., Štajner N. 2022. Small RNA Sequencing and Multiplex RT-PCR for Diagnostics of Grapevine Viruses and Virus-like Organisms. Viruses, 14, 5: 921, doi: 10.3390/v14050921: 11 p. Miljanić V., Jakše J., Beber A., Rusjan D., Škvarč A., Štajner N. 2021. First report of grapevine satellite virus in Slovenia. Journal of Plant Pathology, 103: 1329–1330 VII Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 LIST OF FIGURES Figure 1: Symptoms present on grapevine infected with GFLV: (a) chromatic alteration ('Refošk' variety); (b) shoots malformation ('Volovnik' variety) .......................... 3 Figure 2: Symptoms present on grapevine infected with GLRaV-3: (a) reddening of the interveinal areas while veins remain green ('Cabernet Sauvignon' variety); (b) leaves yellowing ('Zeleni Sauvignon' variety); (c) downward rolling of leaf margins ('Merlot' variety) ..................................................................................... 4 Figure 3: Diseases associated with GRSPaV: (a) Vein necrosis on Richter 110R (Reproduced from Bouyahia et al., 2005); (b) Declining of 'Syrah' variety (necrosis of stem wood) (Reproduced from Al Rwahnih et al., 2009). ............................................. 5 Figure 4: Symptoms present on grapevine infected with GPGV: (a) leaves mottling, deformation and shoot stunting ('Zeleni Sauvignon' variety); (b) uneven fruit set ('Cabernet Sauvignon' variety) ............................................................................... 7 Figure 5: Curved line patterns on leaves infected with RBDV ('Zeleni Sauvignon' variety). ............................................................................................................................... 8 Figure 6: (a) Yellow speckle symptoms present on grapevine infected with GYSVd-1 and GYSVd-2; (b) vein-banding symptoms present on grapevine infected with GYSVd-1, GYSVd-2 in co-infection with GFLV (Reproduced from Hajizadeh et al., 2015) ................................................................................................................ 9 Figure 7: Biogenesis and function of: (a) miRNAs; (b) siRNAs (Reproduced from Khraiwesh et al., 2012) ....................................................................................... 12 Figure 8: Developed shoot from the meristem (no callus formation) ................................. 13 VIII Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 LIST OF ANNEXES ANNEX A: Statement on publisher permissions for the inclusion of own published articles in the printed and electronic versions of the doctoral thesis. IX Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 ABBREVIATIONS AND SYMBOLS aa amino acid AGO argonaute protein AGVd australian grapevine viroid AlkB alkylation B domain CCR central conserved region cDNA complementary deoxyribonucleic acid CEVd citrus exocortis viroid CP coat protein CTV citrus tristeza virus DCL Dicer enzyme DNA deoxyribonucleic acid dsRNA double-stranded ribonucleic acid ELISA enzyme-linked immunosorbent assay EM electron microscopy GAMaV grapevine asteroid mosaic-associated virus GaMV grapevine‐associated marafivirus GC guanine-cytosine GFDD grapevine fanleaf degeneration disease GFLV grapevine fanleaf virus GFkV grapevine fleck virus GHVd hammerhead viroid-like RNA GINV grapevine berry inner necrosis virus GLD grapevine leafroll disease GLRaV-3 grapevine leafroll-associated virus 3 GLRaV-1-9 grapevine leafroll-associated viruses 1-9 GLMD grapevine leaf mottling and deformation GLVd grapevine latent viroid GPGV grapevine Pinot gris virus GPoV-1 grapevine polero virus 1 GRBV grapevine red blotch virus GRGV grapevine red globe virus GRSPaV grapevine rupestris stem pitting-associated virus X Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 GRVFV grapevine rupestris vein feathering virus GSyV-1 grapevine Syrah virus-1 GVN grapevine virus N GVO grapevine virus O GV-Sat grapevine satellite virus GYSVd-1 grapevine yellow speckle viroid 1 GYSVd-2 grapevine yellow speckle viroid 2 Hel helicase HP homing protein HSP70h heat shock 70 protein homologue HSVd hop stunt viroid HTS high-throughput sequencing IC-RT-PCR immunocapture-reverse transcription-polymerase chain reaction ISEM immunosorbent electron microscopy LFIA lateral flow immunoassay L-Pro papain-like leader protease mCP minor coat protein miRNA micro ribonucleic acid MP movement protein mRT-PCR multiplex reverse transcription-polymerase chain reaction MTR methyltransferase nt nucleotide ORF open reading frame OTU ovarian tumor cysteine protease PCR polymerase chain reaction P-Pro papain-like cysteine protease Pro protease PTGS post-transcriptional gene silencing QGB quintuple gene block RdRp RNA-dependent RNA polymerase RGB replication gene block RISC RNA-induced silencing complex RITS RNA-induced initiation of transcriptional gene silencing XI Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 RNA ribonucleic acid RSP rupestris stem pitting RT-LAMP reverse transcription-loop-mediated isothermal amplification RT-nPCR reverse transcription-nested polymerase chain reaction RT-PCR reverse transcription-polymerase chain reaction RT-qPCR reverse transcription-quantitative polymerase chain reaction satGFLV GFLV satellite RNA siRNA short interfering RNA sRNA small RNA sRNA-seq small RNA sequencing ssRNA single-stranded RNA TCH terminal conserved hairpin TCR terminal conserved region TGB triple-gene block TGS transcriptional gene silencing Tm temperature melting TMV tobacco mosaic virus UTR untranslated region VPg viral genome-linked protein YS yellow speckle XII Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 1 INTRODUCTION 1.1 GRAPEVINE AND GRAPEVINE VIRAL PATHOGENS The grape family ( Vitaceae) consists of 16 genera and about 950 species (Wen et al., 2018). The genus Vitis encompasses roughly 60 species, and Vitis vinifera L. is the most important, with more than 10,000 varieties, and the number of new varieties is constantly growing due to grapevine breeding programs (Reynolds, 2017). It is mainly used for wine production, but also for fresh fruit, raisins, juice, spirits, seed oils, vinegar, and other products. According to the OIV (International Organization of Vine and Wine Intergovernmental Organization), 7.3 million hectares of land worldwide were planted with vines in 2020, with five countries (Spain, France, China, Italy, and Turkey) accounting for 50% of the total vineyard area. In the same year, the largest wine producers were Italy, France, Spain, the United States and Argentina. In Slovenia, the area under vines (15,075 ha) is divided into three wine-growing regions (Primorska, Podravje and Posavje) with a total of nine smaller districts (Štajerska, Prekmurje, Bizeljsko-Sremč, Dolenjska, Bela krajina, Goriška brda, Vipavska dolina, Kras, and Slovenska Istra). Among them, Primorska is the largest wine-growing region (6,428 ha), followed by Podravje (6,160 ha) and Posavje (2,487 ha). In 2021, 3,700.12 ha in Primorska were planted with white varieties and 2,727.54 ha with red varieties (Database of Ministry of Agriculture, Forestry and Food, 2021). One of the most important limiting factors for sustainable viticulture worldwide are diseases caused by viral pathogens. In 2020, it was published that 86 viruses are infectious to grapevines (Fuchs, 2020). Since then a few new viruses have been found, such as grapevine polerovirus 1 (GPoV-1) (Chiaki and Ito, 2020), grapevine‐associated marafivirus (GaMV) (Fan et al., 2021), grapevine virus O (GVO), grapevine virus N (GVN) (Read et al., 2022). Most grapevine viruses have a positive or a negative single-stranded RNA genome (ssRNA), a few have a double-stranded RNA genome (dsRNA) or DNA genome (Martelli, 2017). Four major disease complexes are infectious degeneration and decline, leafroll, rugose wood, and fleck disease complex. In Slovenia, according to the Official Gazette of the RS N°93/05 and 101/20, all vine propagation material must be compulsorily tested on: grapevine fanleaf virus (GFLV), arabis mosaic virus (ArMV), tomato black ring virus (TBRV), raspberry ringspot virus (RpRSV), grapevine leafroll-associated virus 1 and 3 (GLRaV-1, -3), grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine virus A (GVA), grapevine virus B (GVB), and grapevine fleck virus (GFkV) (only for rootstocks). In preclonal candidates, we detected and characterized the following viruses: GFLV, GLRaV-3, GRSPaV, GFkV, grapevine red globe virus (GRGV), grapevine rupestris vein feathering virus (GRVFV), grapevine Syrah virus-1 (GSyV-1), grapevine Pinot gris virus (GPGV), and raspberry bushy dwarf virus (RBDV), as well as two viroids: hop stunt viroid (HSVd) and grapevine yellow speckle 1 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 viroid 1 (GYSVd-1). This chapter describes their taxonomic status, genome organization, symptomatology, hosts, and transmission. 1.1.1 Grapevine fanleaf virus Grapevine fanleaf virus (GFLV) is the oldest and most important causal agent of grapevine fanleaf degeneration disease (GFDD) and one of the most detrimental grapevine viruses with worldwide distribution (Martelli, 1993, 2014; Andret-Link et al., 2004a). GFLV belongs to the genus Nepovirus and the family Secoviridae. Its particles are polyhedral and have a diameter of about 30 nm. The genome is bipartite and consists of two positive-sense (+) RNAs: RNA1 (7,326–7,342 nt) and RNA2 (3,730–3,817 nt). Both RNAs have a viral genome-linked protein (VPg) at the 5' end and a polyA tail at the 3' end. The RNA1 encodes a polyprotein (P1) containing the RNA-dependent RNA polymerase (RdRp) at the N-terminus, the cysteine protease, the genome-linked protein (VPg), the nucleotide triphosphate-binding domain (helicase; Hel), the protease cofactor, and a protein of 46 kDa at the C-terminus (Martelli, 2014; Digiaro et al., 2017). The RNA2 encodes a polyprotein (P2) that includes the homing protein (HP), the movement protein (MP), and the coat protein (CP) (Martelli, 2014; Digiaro et al., 2017). An additional GFLV satellite RNA (satGFLV) associated with some GFLV isolates, has been identified (Pinck et al., 1988; Fuchs et al., 1989; Gottula et al., 2013; Lamprecht et al., 2013; Čepin et al., 2016). Symptoms caused by GFLV vary widely depending on the virus strain, variety and environmental factors. The virus causes leaves distortion (fan shape, asymmetry, puckering with toothed margins), chromatic alterations (yellow spots, yellow mosaic, chlorotic mottling, partial or total chrome leaves yellowing, vein banding) (Figure 1a), shoots malformation (fasciation, zigzag growth, bifurcation, double nodes, shorten internodes) (Figure 1b), clusters are smaller, berries ripen irregularly, sugar content and acid concentration are altered, root is less developed, grafting success is lower (Martelli, 1993, 2014; Andret-Link et al., 2004a; Digiaro et al., 2017; Panno et al., 2021). The presence of endocellular cordons (trabeculae) in tracheids is a specific internal symptom of GFLV infection (Martelli, 1993, 2014; Andret-Link et al., 2004a; Digiaro et al., 2017). Yield losses can exceed 80% (Andret-Link et al., 2004a). GFLV was detected in herbaceous weeds ( Aristolochia clematitis, Lagenaria siceraria, Cynodon dactylon, Sorghum halepense, Melilotus sp., Plantago lanceolata, Polygonum sp., Rubus sp . ) that may serve as reservoirs for infection (Horvath et al., 1994; Izadpanah et al., 2003; Zaki-Aghl et al., 2015). The virus is transmitted non-circulatively and semipersistently by the ectoparasitic dagger nematode Xiphinema index (Nematoda: Longidoridae) (Hewitt et al., 1958; Andret-Link et al., 2004b; Demangeat et al., 2010). GFLV can be mechanically transmitted from infected vines to various herbaceous hosts (Cadman et al., 1960; Martelli, 1993, 2014; Digiaro et al., 2017). GFLV was detected in pollen and seeds, but with conflicting reports (Martelli, 1993, 2014; Digiaro et al., 2017). Over long distances, it is transmitted through infected propagating material (Martelli, 1993, 2014; Andret-Link et al., 2004a; Digiaro et al., 2017). 2 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 (a) (b) Figure 1. Symptoms present on grapevine infected with GFLV: (a) chromatic alteration ('Refošk' variety); (b) shoots malformation ('Volovnik' variety). 1.1.2 Grapevine leafroll-associated virus 3 Grapevine leafroll-associated virus 3 is the main etiological agent of grapevine leafroll disease (GLD) (Maree et al., 2013). GLRaV-3 belongs to the genus Ampelovirus and the family Closteroviridae (Martelli et al., 2012). GLRaV-3 particles are flexuous, filamentous, 1,800 nm in length and 12 nm in diameter. The genome is a (+) ssRNA. It is capped at the 5′ end and it is not polyadenylated at the 3′ end (Maree et al., 2013). GLRaV-3 has the largest genome (18,433–18,671 nts) among plant viruses after citrus tristeza virus (CTV) (Burger et al., 2017). All known GLRaV-3 genomes have unusually long 5′ UTRs with considerable length variation and very high uracil content (Maree et al., 2008; Fei et al., 2013; Maree et al., 2013; Burger et al., 2017). Different groups of GLRaV-3 genetic variants exist. Isolates of groups I–III have 12 ORFs (ORF1a, ORF1b, ORF2-12), isolates of groups IV and V have not yet been fully sequenced, isolates of groups VI and VII have not ORF2. ORF1a and ORF1b form the replication gene block (RGB) which contains domains for MTR, Hel, and RdRp. ORF1a also contains a papain-like leader protease (L-Pro) (associated with RNA accumulation, systemic virus spread and invasiveness), and an alkylation B domain (AlkB) (repair RNA from methylation damage). The function of ORF2 is unknown but probably not important, as it is absent in some isolates. ORF3-7 are conserved among members of the Closteroviridae family, and they form the quintuple gene block (QGB). ORF3 encodes a small transmembrane protein. ORF4 encodes the heat shock 70 protein homolog (HSP70h), while ORF5 encodes a ~60 kDa protein that likely has a similar function to HSP70h. CP is encoded by ORF6, while minor CP (mCP) is encoded by ORF7 and is the main component 3 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 of the virion head. ORF8-12 are unique to the genus Ampelovirus. ORF8-10 encode proteins that potentially may be involved in suppression of the host RNA silencing defense mechanism and long-distance virus transport. Small ORF11 and ORF12 are unique and highly diverse among GLRaV-3 variants (Maree et al., 2013; Burger et al., 2017). Symptoms vary depending on season, variety, scion-rootstock combinations and environmental conditions. GLRaV-3 in red varieties causes reddening of the interveinal areas, while veins remain green (Figure 2a). White varieties show yellowing or chlorotic mottling (Figure 2b). At the end of the season (in late autumn) leaves roll downward (Figure 2c). The virus may also be latent (Maree et al., 2013; Naidu et al., 2014; Martelli, 2014; Burger et al., 2017). GLRaV-3 reduces cluster size, ripening, alters sugar content, acidity, pigments and aromatic components (Lee and Martin, 2009; Vega et al., 2011; Maree et al., 2013; Alabi et al., 2016; Burger et al., 2017), resulting in significant economic losses (Cheon et al., 2020). There is no evidence of mechanical transmission of GLRaV-3, it is mainly transmitted by propagation of infected material, and by grafting. Known vectors are soft scale insects (Homoptera: Coccidae) and mealybugs (Homoptera: Pseudococcidae) (Mahfoudhi et al., 2009; Tsai et al., 2010; Maree et al., 2013; Naidu et al., 2014; Martelli, 2014; Burger et al., 2017). (a) (b) (c) Figure 2. Symptoms present on grapevine infected with GLRaV-3: (a) reddening of the interveinal areas while veins remain green ('Cabernet Sauvignon' variety); (b) leaves yellowing ('Zeleni Sauvignon' variety); (c) downward rolling of leaf margins ('Merlot' variety). In addition to GLRaV-3, grapevine leafroll-associated viruses 1-9 (GLRaV-1-9), GLRaV-Car, GLRaV-De, and GLRaV-Pr have also been associated with GLD. Further analysis revealed that GLRaV-7 is novel virus, GLRaV-8 is not viral origin, and GLRaV-4-6, -9, - Car, -De, and -Pr are different strains of GLRaV-4 (Martelli et al., 2012). 4 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 1.1.3 Grapevine rupestris stem pitting-associated virus Grapevine rupestris stem pitting-associated virus (GRSPaV) was discovered in 1998 in association with graft-transmissible Rupestris stem pitting (RSP), one of the four causes of rugose wood disease complex (Zhang et al., 1998). GRSPaV is a member of the genus Foveavirus and the Betaflexiviridae family (Martelli and Jelkmann, 1998; Martelli, 2014; Meng and Rowhani, 2017). GRSPaV particles are flexuous, filamentous, 723 nm in length and 12 nm in diameter (Petrovic et al., 2003). The genome is a (+) ssRNA of ~8,725 nt. The 5' end is capped and the 3' end is polyadenylated. GRSPaV has five ORFs. ORF1 encodes the replicase protein: MTR, Hel, RdRp, and additionally three unique domains: papain-like cysteine protease (P-Pro), ovarian tumor cysteine protease (OTU), and AlkB domain. ORF2, ORF3 and ORF4 form a triple-gene block (TGB) encoding MPs. ORF5 encodes CP. In addition, the putative ORF, which overlaps the CP gene at the 3' end, encodes a 14 kDa protein with unknown function (Zhang et al., 1998; Meng et al., 1998; Martelli, 2014; Meng and Rowhani, 2017). The virus is restricted only to Vitis and its hybrids (Meng and Rowhani, 2017). GRSPaV has a wide range of genetic variants (Meng et al., 1999, 2006; Nolasco et al., 2006; Alabi et al., 2010; Terlizzi et al., 2010, 2011; Glasa et al., 2017; Hily et al., 2018; Rai et al., 2021). There are reports that GRSPaV has no major effects on grape yield and growth (Reynolds et al., 1997), or may even be beneficial to grapevine (Gambino et al., 2012). But it was also found in association with RSP (Zhang et al., 1998; Meng et al., 1998), vein necrosis disease (Bouyahia et al., 2005) (Figure 3a), and with the severe decline of 'Pinot noir' (Lima et al., 2009) and 'Syrah' varieties (Figure 3b) (Al Rwahnih et al., 2009; Beuve et al., 2013). Therefore, its actual effect on grapevines is poorly known. It is graft-transmissible, and no vectors have been identified (Martelli, 2014; Meng and Rowhani, 2017). It was detected in pollen and seeds, but the highest transmissibility to seedlings reported to date is 0.4% (Lima et al., 2006). (a) (b) Figure 3. Diseases associated with GRSPaV: (a) Vein necrosis on Richter 110R (Reproduced from Bouyahia et al., 2005); (b) Declining of 'Syrah' variety (necrosis of stem wood) (Reproduced from Al Rwahnih et al., 2009). 5 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 1.1.4 Grapevine fleck and fleck-similar viruses Grapevine fleck and similar viruses are classified into two genera: grapevine fleck virus (GFkV) and grapevine red globe virus (GRGV) belong to the genus Maculavirus, while grapevine rupestris vein feathering virus (GRVFV), grapevine asteroid mosaic-associated virus (GAMaV), and grapevine Syrah virus-1 (GSyV‐1) belong to the genus Marafivirus (Sabanadzovic et al., 2017) . Grapevine fleck and fleck-like viruses are evolutionarily related and share similar characteristics. These viruses are phloem-limited, there is no evidence of mechanical transmission, and they spread through infected propagating material. Regarding vectors and natural spread, GSyV-1 was detected in leafhopper ( Erythroneura variabilis), but transmissibility to grapevine has not yet been reported (Al Rwahnih et al., 2009), and there are few reports of natural field spread of GFkV, which have never been confirmed experimentally (Martelli, 2014; Sabanadzovic et al., 2017). GFkV is not seed-borne, and transmission by dodder has no epidemiological significance (Martelli, 2014; Sabanadzovic et al., 2017). Complete genomes are known for all of them and they differ slightly in the number and organization of cistrons, but they all share common features: (i) isometric particles about 30 nm in diameter, (ii) (+) ssRNA, (iii) capped 5′ end, (iv) polyadenylated 3′ end, (v) large polyprotein essential for their replication, (vi) synthesis of 3' coterminal subgenomic RNA (sgRNA) as a template for CP translation, (vii) unusually high cytidine content (Martelli et al., 2002; Sabanadzovic et al., 2017). These viruses are latent or semi-latent in most Vitis species and rootstock hybrids (Martelli, 2014; Sabanadzovic et al., 2017). GFkV and related viruses infect only Vitis species, with the exception of GSyV-1 which was also detected in wild blackberry ( Rubus sp.) (Sabanadzovic et al., 2009). Moreover, GSyV-1 is unique among plant viruses due to the specific permutation of motifs in the RdRp gene (Sabanadzovic et al., 2009). 1.1.5 Grapevine Pinot gris virus Another dangerous and emerging grapevine virus is the grapevine Pinot gris virus (GPGV), which belongs to the genus Trichovirus and the family Betaflexiviridae (Giampetruzzi et al., 2012). The virus is associated with grapevine leaf mottling and deformation (GLMD). It was discovered in Italy in 2012 using Illumina small RNA sequencing (Giampetruzzi et al., 2012). It is similar to grapevine berry inner necrosis virus (GINV). GPGV has a (+) ssRNA genome. The complete sequence encompasses 7,258 nt (excluding 3′ polyA tail) (Giampetruzzi et al., 2012). The genome is organized into 3 ORFs. ORF1 encodes a replication protein (MTR, Hel, RdRp) and also contains an AlkB domain; ORF2 encodes a MP; and ORF3 encodes the CP (Giampetruzzi et al., 2012). An additional putative small ORF, encoding an 11.5 kDa protein, has been identified within ORF1 which bears no similarity to known proteins (Glasa et al., 2014). Recent studies showed that GPGV particles are located in deep parenchyma cells (Tarquini et al., 2018), and it is likely that the virus originated in China (Hily et al., 2020). Many symptoms are associated with GPGV (Figure 4), such as chlorotic mottling, leaf deformation, puckering, low vigor, shortened internodes, 6 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 stunting, inner necrosis of berries, low quality, and low yield (Giampetruzzi et al., 2012; Cho et al., 2013; Mavrič Pleško et al., 2014; Saldarelli et al., 2015; Gazel et al., 2016). The presence of the virus has been observed both in asymptomatic plants and in plants with characteristic symptoms (Giampetruzzi et al., 2012; Bianchi et al., 2015; Saldarelli et al., 2015; Shvets and Vinogradova, 2022). GPGV is a graft-transmissible virus (Saldarelli et al., 2015), and the known vector is the eriophyid mite Colomerus vitis (Acari: Eriophyidae) (Malagnini et al., 2016). GPGV is not only infecting grapevine as Gualandri et al. (2017) reported that this virus also infects two herbaceous plants ( Chenopodium album and Silene latifolia), making the epidemiology of this virus much more complex (Saldarelli et al., 2017). (a) (b) Figure 4. Symptoms present on grapevine infected with GPGV: (a) leaves mottling, deformation and shoot stunting ('Zeleni Sauvignon' variety); (b) uneven fruit set ('Cabernet Sauvignon' variety). 1.1.6 Raspberry bushy dwarf virus The first report that grapevine is a natural host of raspberry bushy dwarf virus (RBDV) was found in Slovenia on two white varieties, 'Laški rizling' and 'Štajerska belina' (Mavrič et al., 2003). Later, its occurrence was reported on grapevines from Serbia (Jevremovic and Paunovic, 2011), Hungary (Mavrič Pleško et al., 2012; Czotter et al., 2018) and Russia (Navrotskaya et al., 2021). RBDV is a pollen- and seed-borne virus and belongs to the genus Idaeovirus. It has quasi-spherical particles about 33 nm in diameter and a bipartite genome. RNA1 encodes a polyprotein with MTR, Hel, and RdRp domains (Ziegler et al., 1992). In addition, a small overlapping ORF was observed near the 3′ end of RNA1 that encodes 12K protein (Wood et al., 2001). RNA2 encodes MP at the 5′ end and CP at the 3′ end (Natsuaki et al., 1991). The CP is expressed from the subgenomic RNA3 (Mayo et al., 1991). The virus causes yellowing of leaves and curved line patterns (Mavrič et al., 2003; Jevremovic and 7 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Paunovic, 2011) (Figure 5). In raspberry, it infects seedlings progeny via pollen (up to 77%), but in grapevine ('Laški rizling' variety) it is not transmissible via seeds (Mavrič Pleško et al., 2009). It was detected in the nematode Longidorus juvenilis (Nematoda: Longidoridae), but its transmissibility to grapevine has not yet been experimentally confirmed (Mavrič Pleško et al., 2009). Figure 5. Curved line patterns present on grapevine infected with RBDV ('Zeleni Sauvignon' variety). 1.1.7 Grapevine viroids Viroids are small, single-stranded, circular, non-encapsidated, and nonprotein-coding RNAs. Six viroids and one viroid-like RNA have been identified in grapevines: grapevine yellow speckle viroid 1 (GYSVd-1), grapevine yellow speckle viroid 2 (GYSVd-2), grapevine latent viroid (GLVd), australian grapevine viroid (AGVd), hop stunt viroid (HSVd), citrus exocortis viroid (CEVd), and grapevine hammerhead viroid-like RNA (GHVd) (Di Serio et al., 2017). They are members of the genus: Apscaviroid (GYSVd-1, GYSVd-2, GLVd, and AGVd); Hostuviroid (HSVd), and Pospiviroid (CEVd), and all belong to the family Pospiviroidae (Di Serio et al., 2017). GHVd shares characteristics with members of the family Avsunviroidae (Wu et al., 2015). Members of the family Pospiviroidae replicate and accumulate in the nucleus, whereas members of the family Avsunviroidae replicate and accumulate in the chloroplast. HSVd and GYSVd-1 are distributed worldwide, and they are the only two viroids found in grapevines in Slovenia. HSVd was first detected in hop plants (Sasaki and Shikata, 1977), and since then it has been reported to infect various plants from different botanical families (Sano et al., 1985, 1989; Astruc et al., 1996; Yakoubi et al., 2007; Zhang et al., 2009; Elbeaino et al., 2012; Elleuch et al., 2013; Pirovano et al., 2014; Marquez-Molins et al., 2021). In 2012 it was published that this viroid infects hops in Europe (Radisek et al., 2012). Although HSVd is asymptomatic in grapevines it can be transmitted to hops and cause epidemics (Sano et al., 2001; Kawaguchi-Ito et al., 2009). The genome is 296-302 nt in size and assume rod-like conformation containing the typical central conserved region (CCR) and terminal conserved hairpin (TCH) (Di Serio et al., 2017). HSVd in grapevine exhibits the typical features of a 8 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 quasispecies (Sano et al., 2001). GYSVd-1 and GYSVd-2 are associated with yellow speckle (YS), a disease that causes yellow spots on leaves induced by high temperatures (Koltunow et al., 1989; Hajizadeh et al., 2015) (Figure 6a). GYSVd-1 and/or GYSVd-2 in co-infection with GFLV may cause vein banding (Figure 6b) (Hajizadeh et al., 2015). GYSVd-1 has a rod-like conformation that contains the CCR and terminal conserved region (TCR) (Di Serio et al., 2017). GYSVd-1 in grapevine exhibits characteristics of a quasispecies (Polivka et al., 1996). Viroids have no vectors and are spread by propagating material and grafting. They can also be spread through pruning tools in the vineyard (Di Serio et al., 2017). Figure 6. (a) Yellow speckle symptoms present on grapevine infected with GYSVd-1 and GYSVd-2; (b) vein-banding symptoms present on grapevine infected with GYSVd-1, GYSVd-2 in co-infection with GFLV (Reproduced from Hajizadeh et al., 2015). In the part of the dissertation where we worked with randomly collected samples from Ampelographic collection Kromberk, that were not included in the clonal selection process, we found: GFLV (and its satellite RNA), GLRaV-1, GLRaV-2, GLRaV-3, GRSPaV, GFkV, GRVFV, GPGV, GV-Sat, HSVd, and GYSVd-1. 1.2 METHODS FOR DETECTION OF GRAPEVINE VIRAL PATHOGENS Visual examination of symptoms is certainly the first approach to diagnosis of viral pathogens. This approach is unreliable because different abiotic and biotic factors can elicit similar symptoms. For example, leaves reddening can be caused by leafroll viruses (in red varieties), grapevine red blotch virus (GRBV) (Sudarshana et al., 2015), phytoplasmas (Grapevine flavescence dorée) (Chuche and Thiéry, 2014), mechanical injuries, pesticides, mineral nutrition disorders, etc. The expression of symptoms also depends on the virus strain, weather conditions, and viral titer. In addition, infected plants may be asymptomatic. Reliable diagnosis of viral pathogens is crucial to take measures to control their spread. Diagnostic methods can be divided into biological indexing, electron microscopy, immunodiagnosis and diagnostic methods based on nucleic acids amplification. 9 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Biological testing is one of the oldest diagnostic methods, and takes a lot of time and space. In biological testing indicators plants are artificially inoculated with grapevine sap (mechanical inoculation) or by grafting. The inoculation technique depends on both the virus and the plant indicator. After inoculation, the indicator plants are incubated for a period ranging from a few days to several weeks or even longer (in the case of woody indicators), followed by evaluation of manifested symptoms. For example, V. rupestris St George is often used as an indicator plant for fanleaf, fleck, asteroid mosaic, and RSP diseases; red varieties for leafroll disease; Kober 5BB for Kober stem grooving; LN33 for LN33 stem grooving, Corky bark, and enations; V. riparia Glorie de Montpellier for vein mosaic; 110R for vein necrosis; Baco 22A for the stunting component of leafroll disease; V. vinifera Mataro or Mission for leafroll, yellow speckle; etc (Martelli et al., 1993). Herbaceous indicators are frequently from the families Chenopodiaceae, Amaranthaceae, Solanaceae, Cucurbitaceae, and Leguminosae. Biological tests are being replaced by faster, more accurate and less labor-intensive diagnostic methods. However, this method is still important for certification programs and testing of new viruses or new strains. Electron microscopy (EM) is one of the most significant achievements that has contributed to the development of virology, as it allows the visibility of virus particles. The first EM was developed in 1931 by Ernst Ruska. This achievement was awarded the Nobel Prize in 1986. EM has found wide application in the study of virus morphology, size and structure. The first virus visualized using EM was tobacco mosaic virus (TMV) (Goldsmith and Miller, 2009). Immunoassays (serological methods) rely on the binding of an antigen to specific antibodies produced against that antigen. Various immunoassays are used for the detection of grapevine viruses such as: immunosorbent electron microscopy (ISEM) (Scagliusi et al., 2002; Petrovic et al., 2003); enzyme-linked immunosorbent assay (ELISA) (Fiore et al., 2008; Cogotzi et al., 2009; Komínek, 2009; Vončina et al., 2011; Zindović et al., 2014); lateral flow immunoassays (LFIAs) (Liebenberg et al., 2009); western blot (Saldarelli et al., 2000; Maliogka et al., 2009a; Alkowni et al., 2011). The ELISA test is the most commonly used. It is performed on microtiter plates, and reagents are added in the following order: specific antibody, antigen (homogenized sample in extraction buffer), specific enzyme-labeled antibody and substrate (usually para-nitrophenylphosphate) with appropriate incubation time and washing of the plate. When the sample is infected, the color of the reaction changes. The color intensity is precisely proportional to the concentration of antigens in the plant. The results are analyzed visually and by spectrophotometric measurements at 405 nm (Clark and Adams, 1977). The most sensitive and reliable diagnostic methods are based on the detection of nucleic acids. One such technique is the polymerase chain reaction (PCR), which was developed in 1983 and awarded the Nobel Prize in 1993. PCR is based on the amplification of complete 10 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 or part of the viral genome by the heat-stable DNA polymerase enzyme. PCR is used to detect viruses that possess a DNA genome (Maliogka et al., 2015; Čarija et al., 2022). Most grapevine viruses, as well as virus-like organisms, have an RNA genome; therefore, reverse transcription is required for the synthesis of complementary DNA (cDNA) prior to PCR amplification, and this method is called reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR is used for confirmation of serological results (Fiore et al., 2008; Mavrič Pleško et al., 2012), for screening (Fattouh et al., 2014; Glasa et al., 2015; Porotikova et al., 2021; Čarija et al., 2022), and for validation of HTS results (Diaz-Lara et al., 2018; Czotter et al., 2018; Eichmeier et al., 2019; Demian et al., 2020; Turcsan et al., 2020; Navrotskaya et al., 2021). For higher accuracy immunocapture-RT-PCR (IC-RT-PCR) which is a combination of serological and molecular methods (Wetzel et al., 2002; Mavrič et al., 2003; Koolivand et al., 2014; Kumar et al., 2015), and nested RT-PCR (RT-nPCR) in which the amplified product from the first reaction is used as a template in the second reaction (Farooq et al., 2013; Fan et al., 2015), can be used. Quantitative RT-PCR (RT-qPCR) allows quantification in addition to detection (Osman et al., 2007; Čepin et al., 2010; Bianchi et al., 2015). Multiplex RT-PCR (Nassuth et al., 2000; Gambino and Gribaudo, 2006; Digiaro et al., 2007; Hajizadeh et al., 2012; Gambino, 2015; Ahmadi et al., 2017; Komínková and Komínek, 2020) and multiplex RT-qPCR (Osman et al., 2013; López-Fabuel et al., 2013; Aloisio et al., 2018) are used to amplify multiple viral organisms in a single reaction. Other methods such as RT loop-mediated isothermal amplification (RT-LAMP) (Walsh and Pietersen, 2013) and microarrays (Engel et al., 2010) can also be used for diagnostics. All of the above diagnostic methods require prior knowledge of the potential organisms, with the exception of high-throughput sequencing (HTS). HTS allows detection of known and novel viral pathogens in symptomatic or asymptomatic plants (Al Rwahnih et al., 2009; Fajardo et al., 2017; Massart et al., 2017; Hily et al., 2018). HTS of small virus- and viroid-derived RNAs (small RNA sequencing; sRNA-seq) relies on RNA silencing-an antiviral defense mechanism of plants. Upon viral infection, Dicer enzymes (DCL) cleave long dsRNAs and microRNA (miRNA) precursors into short interfering (si)RNA and miRNA duplexes (miRNA/miRNA∗), respectively (Bernstein et al., 2001; Baulcombe, 2004; Bartel, 2004). Small RNAs (sRNAs) are loaded into Argonaute proteins (AGOs), which are core catalytic component of the RNA-induced silencing complex (RISC) or RNA-induced initiation of transcriptional gene silencing (RITS) (Parker and Barford, 2006). RISC (post-transcriptional gene silencing-PTGS) targets mRNA through complementary sequence-specific mechanisms and leads to its degradation or inhibits its translation (Hammond et al., 2001). RITS (transcriptional gene silencing-TGS) is involved in the formation of heterochromatin (Verdel et al., 2004) (Figure 7). Upon viral infection sRNAs accumulate in plants and can be detected by sRNA-seq. sRNA-seq has been shown to be highly efficient in grapevine virology (Navarro et al., 2009; Giampetruzzi et al., 2012; Czotter et al., 2018; Turcsan et al., 2020; Demian et al., 2020; Li et al., 2021; Navrotskaya et al., 2021). 11 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Figure 7. Biogenesis and function of: (a) miRNAs; (b) siRNAs (Reproduced from Khraiwesh et al., 2012). 1.3 METHODS FOR ELIMINATION OF GRAPEVINE VIRAL PATHOGENS Viruses and viroids are obligate pathogens and cannot be controlled by phytopharmaceutical measures like fungi and bacteria, so planting of healthy material is essential. Selection of healthy material requires effective therapeutic methods and permanent controlling of sanitary status of vines for propagation. Various biotechnological methods for producing healthy vine material have been used experimentally and routinely. Among them, thermotherapy/heat therapy and meristem or shoot tip tissue culture are the most commonly used. Heat therapy is a treatment in which plants are kept at an elevated temperature for a period of time, which allows the plants to survive and slows down virus replication or even degrades the virus (Panattoni et al., 2013). High temperatures trigger an RNA silencing mechanism (Szittya et al., 2003; Qu et al., 2005; Chellappan et al., 2005; Wang et al., 2008; Velázquez et al., 2010; Liu et al., 2015, 2016; Kim et al., 2021). Meristem cells are smaller and proliferate rapidly, therefore they have the ability to exclude pathogenic organisms from donor plants. In addition, shoot development from the meristem avoids the formation of callus tissue (Figure 8), which reduces the risk of off-types (Grout, 1999). To 12 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 accelerate the regeneration of plants from meristems, the technique of micrografting (placing an excised meristem/shoot tip on a seedling growing in vitro) can be used (Jonard et al., 1983; Hussain, et al., 2014). Figure 8. Developed shoot from the meristem (no callus formation). Other sanitation strategies are: cryotherapy, chemotherapy, somatic embryogenesis, and electrotherapy. Cryotherapy is a promising tool based on freezing shoot tips in liquid nitrogen (-196 °C) for a short period of time, usually 1 hour (Bettoni et al., 2016). Because meristem cells have less water, denser cytoplasm, and smaller vacuoles compared to other cells, they can survive in liquid nitrogen, whereas other cells die upon ice crystallization (Bettoni et al., 2016). After freezing, shoot tips are thawed and placed in an appropriate regeneration medium (Bettoni et al., 2016). Chemotherapy is a method for virus elimination with mixed success. It consists of placing plant explants in a medium containing antiviral compounds that interfere viral replication. The most common antiviral chemicals are: ribavirin, tiazofurin, oseltamivir, 6-tioguanine, and mycophenolic acid (Panattoni et al., 2011; Skiada et al., 2013; Komínek et al., 2016). Somatic embryogenesis is a strategy for virus elimination with excellent results but with a high risk of somatic variations/mutations. In this technique, plants are regenerated from somatic embryos produced directly or indirectly from floral explants (Gambino et al., 2006, 2009, 2011; Bouamama-Gzara et al., 2017; Turcsan et al., 2020). Electrotherapy is a method with limited success. It consists of exposing herbaceous cuttings or rooted plants to an electric current for a short period of time, where the electric field inactivates the viruses by heating the tissue, followed by in vitro regeneration (Guţa et al., 2010, 2019). In addition, different elimination methods were combined to evaluate the efficacy of virus elimination, including thermotherapy with meristem tip culture (Maliogka et al., 2009b; Salami et al., 2009), thermotherapy with shoot tip culture (Maliogka et al., 2009b; Bota et 13 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 al., 2014), thermotherapy with shoot apices micrografting (Spilmont et al., 2012), thermotherapy with chemotherapy (Hu et al., 2020, 2021), and thermotherapy with somatic embryogenesis (Goussard and Wiid, 1992). Sanitation success depends on various factors such as variety, virus/viroid species, sanitation method used, treatment conditions, etc. 1.4 AIMS AND HYPOTHESES Given the importance of rapid and accurate detection and molecular characterization of viruses and virus-like organisms and the development of effective methods for virus elimination, the objectives of this dissertation were (i) to perform virome screening of different grapevine varieties using HTS of small RNAs; (ii) to validate in silico results; (iii) to study the genetic diversity of viruses/viroids, phylogeny and co-infections; (iv) to develop a protocol for the production of healthy plants. We set up four research hypotheses: 1) Vines are infected with different viruses and viroids, which can be adequately determined using HTS of small RNAs. 2) Based on the sequences information of viruses and viroids obtained by the HTS approach specific primers could be designed for amplification and validation of the predicted viral pathogens by RT-PCR and Sanger sequencing. 3) Predicted infections will be confirmed with Sanger sequencing and additional information about strain-specific polymorphisms related to different host grapevines could be obtained. 4) Using thermotherapy and meristem/shoot tip culture virus-free material could be established, but the percentage of elimination will vary depending on variety and viral pathogen. 14 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 2 SCIENTIFIC WORKS 2.1 PUBLISHED SCIENTIFIC WORKS 2.1.1 Virome Status of Preclonal Candidates of Grapevine Varieties ( Vitis vinifera L.) From the Slovenian Wine-Growing Region Primorska as Determined by High-Throughput Sequencing Miljanić V., Jakše J., Kunej U., Rusjan D., Škvarč A., Štajner N. 2022. Virome Status of Preclonal Candidates of Grapevine Varieties ( Vitis vinifera L.) From the Slovenian Wine-Growing Region Primorska as Determined by High-Throughput Sequencing. Frontiers in Microbiology, 13, doi: 10.3389/fmicb.2022.830866: 11 p. Diseases caused by viruses and virus-like organisms are one of the major problems in viticulture and grapevine marketing worldwide. Therefore, rapid and accurate diagnosis and identification is crucial. In this study, we used HTS of virus- and viroid-derived small RNAs to determine the virome status of Slovenian preclonal candidates of autochthonous and local grapevine varieties ( Vitis vinifera L.). The method applied to the studied vines revealed the presence of nine viruses and two viroids. All viral entities were validated and more than 160 Sanger sequences were generated and deposited in NCBI. In addition, a complete description into the co-infections in each plant studied was obtained. No vine was found to be virus- and viroid-free, and no vine was found to be infected with only one virus or viroid, while the highest number of viral entities in a plant was eight. 15 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 16 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 17 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 18 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 19 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 20 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 21 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 22 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 23 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 24 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 25 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 26 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 2.1.2 First Report of Grapevine Red Globe Virus, Grapevine Rupestris Vein Feathering Virus, and Grapevine Syrah Virus-1 Infecting Grapevine in Slovenia Miljanić V., Jakše J., Kunej U., Rusjan D., Škvarč A., Štajner N. 2022. First Report of Grapevine Red Globe Virus, Grapevine Rupestris Vein Feathering Virus, and Grapevine Syrah Virus-1 Infecting Grapevine in Slovenia. Plant Disease, 106, 9: 2538 The study of the virome of preclonal candidates of six grapevine varieties revealed three viruses: grapevine rupestris vein feathering virus (GRVFV), grapevine red globe virus (GRGV), and grapevine Syrah virus-1 (GSyV-1), which were not previously confirmed in Slovenia. GRVFV was the most widespread, being detected in 11 out of 12 libraries. GRGV was detected in two libraries of the variety 'Refošk', and GSyV-1 was also detected in two libraries of the varieties 'Laški rizling' and 'Malvazija'. In silico results were validated with RT-PCR and Sanger sequencing. Forty-four samples were infected with GRVFV and 3 with GRGV and GSyV-1. Twenty-eight sequences were generated and deposited in NCBI (Acc. numbers MW446914-MW446941). 27 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 28 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 2.1.3 Elimination of Eight Viruses and Two Viroids from Preclonal Candidates of Six Grapevine Varieties ( Vitis vinifera L.) through In Vivo Thermotherapy and In Vitro Meristem Tip Micrografting Miljanić V., Rusjan D., Škvarč A., Chatelet P., Štajner N. 2022. Elimination of Eight Viruses and Two Viroids from Preclonal Candidates of Six Grapevine Varieties ( Vitis vinifera L.) through In Vivo Thermotherapy and In Vitro Meristem Tip Micrografting. Plants, 11, 8: 1064, doi: 10.3390/plants11081064: 14 p. Viruses and virus-like organisms are a major problem in viticulture worldwide. They cannot be controlled by standard plant protection measures, and once infected, plants remain infected throughout their life; therefore, the propagation of healthy vegetative material is crucial. In vivo thermotherapy at 36–38 °C for at least six weeks, followed by meristem tip micrografting (0.1–0.2 mm) onto in vitro-growing seedling rootstocks of Vialla ( Vitis labrusca × Vitis riparia), was successfully used to eliminate eight viruses (grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine Pinot gris virus (GPGV), grapevine fanleaf virus (GFLV), grapevine leafroll-associated virus 3 (GLRaV-3), grapevine fleck virus (GFkV), grapevine rupestris vein feathering virus (GRVFV), grapevine Syrah virus-1 (GSyV-1), and raspberry bushy dwarf virus (RBDV)), as well as two viroids (hop stunt viroid (HSVd) and grapevine yellow speckle viroid 1 (GYSVd-1)) from preclonal candidates of six grapevine varieties (Vitis vinifera L.). A half-strength MS medium including vitamins supplemented with 30 g/L of sucrose and solidified with 8 g/L of agar, without plant growth regulators, was used for the growth and root development of micrografts and the subsequently micropropagated plants; no callus formation, hyperhydricity, or necrosis of shoot tips was observed. Although the overall regeneration was low (higher in white than in red varieties), a 100% elimination was achieved for all eight viruses, whereas the elimination level for viroids was lower, reaching only 39.2% of HSVd-free and 42.6% GYSVd-1-free vines. To the best of our knowledge, this is the first report of GPGV, GRVFV, GSyV-1, HSVd, and GYSVd-1 elimination through combining in vivo thermotherapy and in vitro meristem tip micrografting, and the first report of RBDV elimination from grapevines. The virus-free vines were successfully acclimatized in rockwool plugs and then transferred to soil. 29 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 30 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 31 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 32 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 33 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 34 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 35 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 36 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 37 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 38 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 39 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 40 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 41 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 42 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 43 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 2.1.4 Small RNA Sequencing and Multiplex RT-PCR for Diagnostics of Grapevine Viruses and Virus-like Organisms Miljanić V., Jakše J., Rusjan D., Škvarč A., Štajner N. 2022. Small RNA Sequencing and Multiplex RT-PCR for Diagnostics of Grapevine Viruses and Virus-like Organisms. Viruses, 14, 5: 921, doi: 10.3390/v14050921: 11 p. Metagenomic approaches used for virus diagnostics allow for rapid and accurate detection of all viral pathogens in the plants. In order to investigate the occurrence of viruses and virus-like organisms infecting grapevine from the Ampelographic collection Kromberk in Slovenia, we used Ion Torrent small RNA sequencing (sRNA-seq) and the VirusDetect pipeline to analyze the sRNA-seq data. The used method revealed the presence of: Grapevine leafroll-associated virus 1 (GLRaV-1), Grapevine leafroll-associated virus 2 (GLRaV-2), Grapevine leafroll-associated virus 3 (GLRaV-3 ), Grapevine rupestris stem pitting-associated virus (GRSPaV), Grapevine fanleaf virus (GFLV) and its satellite RNA (satGFLV), Grapevine fleck virus (GFkV), Grapevine rupestris vein feathering virus (GRVFV), Grapevine Pinot gris virus (GPGV), Grapevine satellite virus (GV-Sat), Hop stunt viroid (HSVd), and Grapevine yellow speckle viroid 1 (GYSVd-1). Multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed for validation of sRNA-seq predicted infections, including various combinations of viruses or viroids and satellite RNA. mRT-PCR could further be used for rapid and cost-effective routine molecular diagnosis, including widespread, emerging, and seemingly rare viruses, as well as viroids which testing is usually overlooked. 44 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 45 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 46 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 47 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 48 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 49 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 50 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 51 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 52 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 53 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 54 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 55 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 2.1.5 First report of grapevine satellite virus in Slovenia Miljanić V., Jakše J., Beber A., Rusjan D., Škvarč A., Štajner N. 2021. First report of grapevine satellite virus in Slovenia. Journal of Plant Pathology, 103: 1329–1330 The study of the virome of 13 samples of six grapevine varieties not included in the clonal selection process, revealed the presence of grapevine satellite virus, which was not previously confirmed in Slovenia. In silico results were validated with RT-PCR and Sanger sequencing. The infection was confirmed in three samples of variety 'Cipro'. The sequences were deposited in NCBI under the numbers MW446942-MW446944. 56 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 57 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 58 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 3 DISCUSSION AND CONCLUSIONS 3.1 DISCUSSION Preclonal candidates (the 'best' vines with certain characteristics), obtained after a mass selection of grapevines that did not show visible symptoms associated with the diseases, were analyzed in the first part of the dissertation using HTS technology. HTS is a powerful metagenomic approach that allows the detection of all viral entities even in asymptomatic plants, and even if they are present in low titer. sRNA-seq of 12 libraries yielded 112,647,551 reads. In the VirusDetect pipeline, 107,016,121 reads were processed. Of the total reads, 2.24% mapped to viral genomes. Nine viruses: GFLV, GLRaV-3, GRSPaV, GFkV, GSyV-1, GRVFV, GRGV, GPGV, RBDV, and two viroids: HSVd and GYSVd-1 were detected by the method used. Considering that a considerable number of viral entities were detected, including those that require mandatory testing in Slovenia, in the second part of the dissertation we developed a protocol for the generation of healthy vines by in vivo thermotherapy and in vitro meristem tip micrografting. Meristem tip culture is effective for viruses restricted to the phloem, whereas thermotherapy is desirable for eliminating viruses that can infect various tissues. In preclonal candidates we detected both (phloem and non-phloem-limited viruses), therefore to increase the efficiency of elimination, we combined both methods. Here we present HTS results on preclonal candidates, genetic diversity studies, potential introduction of the detected viruses and viroids into Slovenia, co-infections, and elimination rates achieved with the chosen biotechnological approach. GFLV, one of the most detrimental viruses, was detected in library 013 ('Zeleni Sauvignon' variety) and library 016 ('Pokalca' variety). In library 013, VirusDetect (BLASTN search) identified 11 complete RNA1 segment sequences, which were 78.7-92% covered with 23-31 contigs and sequencing depth of 149.6X-155.7X; and 4 complete RNA2 segment sequences, which were 77.4-90.7% covered with 11 or 12 contigs and sequencing depth of 211.9-218.2X. In library 016, the VirusDetect identified 7 complete and 1 partial RNA1 sequences which were 58.8-71.7% covered with 13-21 contigs and a sequencing depth of 719.8X-785.8X; and 16 complete RNA2 segments that were 73-92.1% covered with 13-20 contigs and a sequencing depth of 752.5X-890.3X. Predicted infections were validated using the primer pair C2647/H2042 targeting a 606 bp long fragment of RNA2 containing a partial CP gene (Fattouch et al., 2001). One sample from library 013 (Zeleni Sauvignon 16/3P) and three samples from library 016 (Pokalca 3/4P, 3/5P, 3/6P) were infected. Each sample infected with GFLV was sequenced, and nt differences between red and white varieties were more than 12%. Analyzing the same genome fragment as we did, Fattouch et al. (2005) reported that the differences between sequences were more than 11%. Naraghi-Arani et al. (2001) found that genetic diversity in the 1557 bp genome region of RNA2 in 14 isolates were from 11 to 13%. While Elbeaino et al. (2014) found that sequence variability in the HP was as high as 41% among isolates. In Slovenia, high genetic variability was found within the RNA2 segment of nine samples of the variety 'Volovnik' (Pompe-Novak et al., 2007). 59 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 The high genetic variability indicates the quasispecies nature of the genome (Naraghi-Arani et al., 2001). In addition, we found that the sequenced fragments were longer than the expected 606 bp, which is consistent with the results of Fattouch et al. (2005). The authors reported that this may indicate that RT-PCR products represented a population structure with more restrictotypes. Considering that the generated sequences showed large differences among varieties and also with the sequences from GenBank database, a novel primer pair was designed in this study, also in the RNA2 segment, and additional two samples from library 013 (Zeleni Sauvignon 26/1P and 26/2P) and four samples from library 016 (Pokalca 9/2P, 9/3P, 9/26G, 9/27G) were confirmed as infected. Phylogenetic analysis showed that the GFLV isolates were grouped into two clusters, consistent with the results of Panno et al. (2021). The Slovenian isolates generated in this study grouped into major clade in 2 subclades (one subclade 'Zeleni Sauvignon'; the other 'Pokalca'). Moreover, the phylogenetic tree showed certain degree of variability between our isolates and those from GenBank, and our isolates were closer to those from Italy and France than to those from Slovenia that had been previously characterized (Pompe-Novak et al., 2007). Testing on GFLV is obligatory in all certification programs. Therefore, various methods have been used for its elimination from grapevines: thermotherapy (Křižan et al., 2009; Salami et al., 2009; Panattoni and Triolo, 2010), meristem tissue culture (Youssef et al., 2009; Salami et al., 2009), combination of thermotherapy and meristem tissue culture (Salami et al., 2009), combination of thermotherapy with shoot apices micrografting (Spilmont et al., 2012), chemotherapy (Weiland et al., 2004; Guţa et al., 2017), somatic embryogenesis (Gambino et al., 2009), and combination of thermotherapy and somatic embryogenesis (Goussard and Wiid, 1992). In our study, 3 preclonal candidates of the 'Pokalca' variety were included in the elimination process (Pokalca 3/4P, 3/6B, and 9/2G). Pokalca 9/2G did not regenerate. Pokalca 3/4P and 3/6P, yielded one meristem per candidate, and both regenerated plants were GFLV-free. In addition, the 'Pokalca' variety had the lowest regeneration rate (3.3%) compared to the other varieties. Among the preclonal candidates, GLRaV-3, a representative virus of the genus Ampelovirus, was detected only in library 010 ('Refošk' variety). VirusDetect (BLASTN search) identified only one complete genome sequence (GQ352631) from South Africa. The reference was 98.6% covered with 23 contigs and a sequencing depth of 39.6X. ORF2 is known to be absent in some isolates (Burger et al., 2017). In the 010 library, a unique viral contig was observed at position 9243-9811 that corresponded to ORF2. The presence of the virus was validated with RT-PCR using two primer pairs LR3-8504V/LR3-9445C (Fajardo et al., 2007) amplifying the CP gene and LC1/LC2 (Turturo et al., 2005) amplifying the HSP70h gene, and only Refošk 11/4P was infected. It was found in co-infection with GRSPaV, GPGV, GFkV, GRVFV, HSVd, and GYSVd-1. RT-PCR product obtained with LR3-8504V/LR3-9445C was Sanger sequenced. The 822 nt sequence (partial CP) was compared with isolates available in the database and it showed 99.76% nt identity with isolates from Europe (Portugal and Greece), North America (United States and Canada), Asia (Pakistan), 60 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 and three isolates of unknown origin. The phylogenetic tree was constructed based on the CP sequences and included our isolate and 32 isolates from GenBank. The phylogenetic tree showed that the isolate Refošk 11/4P belonged to the major phylogroup and was the closest to the isolate from Portugal. CP gene is commonly used in phylogenetic studies (Turturo et al., 2005; Jooste et al., 2010; Sharma et al., 2011; Wang et al., 2011; Gouveia et al., 2011; Kumar et al., 2012; Bester et al., 2012; Liu et al., 2013; Lehad et al., 2015; Crnogorac et al., 2021), as well as HSP70h (Turturo et al., 2005; Fuchs et al., 2009; Jooste et al., 2010; Kumar et al., 2012; Lehad et al., 2015; Čarija et al., 2022). Considering that GLRaV-3 is one of the most economically important viruses, numerous studies have been published on its elimination, including: thermotherapy (Panattoni and Triolo, 2010; Panattoni et al., 2011; Hu et al., 2020), combination of thermotherapy and shoot apices micrografting (Spilmont et al., 2012), cryotherapy (Bi et al., 2018), somatic embryogenesis (Gambino et al., 2006; Bouamama-Gzara et al., 2017), chemotherapy (Panattoni et al., 2011; Hu et al., 2020), and combination of thermotherapy and chemotherapy (Hu et al., 2020). In our study, 28 meristems were isolated from the infected mother plant (Refošk 11/4P) and micrografted; 2 plants were regenerated and were both free of GLRaV-3. In preclonal candidates, GRSPaV was detected in 10 libraries based on sRNA-seq data. In 8 libraries, VirusDetect identified 4 (008 library; 'Rebula' variety) to 40 (005 library; 'Laški rizling' variety) complete and partial (mainly CP gene) reference sequences per library. The highest coverage of complete reference sequences was 48.4% (013 library; 'Zeleni Sauvignon' variety) - 99.1% (006 library; 'Refošk' variety). They were covered with a high number of contigs (41-86), and a low sequencing depth (5.3X-16.6X). Only partial and/or complete CP gene sequences were identified in libraries 012 ('Zeleni Sauvignon' variety), and 015 ('Malvazija' variety). The virus was not detected in libraries 007 ('Rebula' variety) and 014 ('Malvazija' variety). A primer pair targeting the highly conserved CP gene (RSP 52/RSP 53) was used to validate GRSPaV (Nolasco et al., 2000). GRSPaV was confirmed in 10 libraries where it was predicted and additionally in two libraries where it was not predicted. Problems in detecting this virus with sRNA-seq technology have also been reported in several studies (Czotter et al., 2018; Turcsan et al., 2020; Demian et al., 2020). The possibility that its concentration was low was ruled out because each sample in library 007 was infected. The reason why GRSPaV was not detected with sequencing of sRNAs may have a deeper biological background, because it is known that GRSPaV can be beneficial to grapevine and that mutual adaptation exists (Gambino et al., 2012). Overall, 88.61% of the preclonal candidates were infected with GRSPaV. Direct sequencing of the RT-PCR products was not possible due to the presence of different genetic variants, so the RT-PCR products were ligated into the vector and transformed into bacterial competent cells. GRSPaV exhibits high heterogeneity and has a wide range of sequence variants (Meng et al., 1999, 2006; Nolasco et al., 2006; Glasa et al., 2017). In our study, the results showed that at least 3 genetic variants were present in the same sample. The same result was obtained by Glasa et al. (2017). Laški rizling 3/45B had the highest genetic variability (17.14%) and 61 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Malvazija 20/48P (6.32%) had the lowest. Interestingly, VirusDetect identified the highest number (40) of sequences from the database in the 'Laški rizling' library, while in one 'Malvazija' library (014) GRSPaV was not detected with sRNA-seq and in the other 'Malvazija' library (015) only one CP gene sequence was identified. Although the virus was mainly studied in the highly conserved Hel domain or CP, several groups of sequence variants were found. Additionally, new variants were found in the RdRp domain (Meng and Rowhani, 2017). Our phylogenetic analysis showed differential clustering of genetic variants even when they originated from the same sample, and no clustering by geographic origin was observed, which is consistent with other studies (Nolasco et al., 2006; Alabi et al., 2010). In plant virology, GRSPaV is thought to be benign and may also be beneficial to grapevine. However, because GRSPaV has high genetic variability, and was found in association with RSP (Zhang et al., 1998; Meng et al., 1998), vein necrosis disease (Bouyahia et al., 2005), and the severe decline of 'Pinot noir' (Lima et al., 2009) and 'Syrah' varieties (Al Rwahnih et al., 2009; Beuve et al., 2013), its real impact on grapevine is not yet known, and testing is therefore mandatory in all certification programs. Previous studies indicated that GRSPaV is difficult to eliminate, because it is presumed that this virus is able to infects meristems (Gribaudo et al., 2006; Meng and Rowhani, 2017; Hu et al., 2021). However, it should be noted that its elimination also strongly depends on the variety. For example, the combination of thermotherapy and shoot tip culture resulted in 39.62% and 92.85% GRSPaV-free plants in two Greek varieties, 'Mantilaria' and 'Prevezaniko', respectively (Maliogka et al. 2009b). Other methods used for its elimination so far are: somatic embryogenesis (Gambino et al., 2006; Gribaudo et al., 2006; Bouamama-Gzara et al., 2017; Turcsan et al., 2020), chemotherapy (Skiada et al., 2013; Komínek et al., 2016; Hu et al., 2021), combination of thermotherapy and chemotherapy (Hu et al., 2021), and combination of chemotherapy and shoot tip culture (Hu et al., 2018). In our study, 26 of 28 samples included in the elimination process were infected with GRSPaV, and by in vivo thermotherapy (36-38 °C) and in vitro meristem tip micrografting (0.1-0.2 mm), all regenerated plants (49) were GRSPaV-free. GFkV was predicted in eight libraries. In seven libraries, VirusDetect identified 4-10 reference sequences from the database per library. In library 011 ('Refošk' variety) only one partial replicase gene was identified, with a coverage of 33.3%. For the complete reference genome sequences, the lowest coverage was in the 'Malvazija' variety (libraries 014 and 015), 30.4% and 32.2%, respectively, while coverage in the other libraries was 76.9-82.1%. Many contigs (36-60) and their short length were observed in all libraries. The HTS results were validated with RT-PCR using the primer pair GFkV-U279/GFkV-L630 targeting the replicase gene (Shi et al., 2003). Thirty-four products were obtained and Sanger sequenced but 7 products belonged to fleck-similar virus (GRVFV), which is consistent with the results reported by Czotter et al. (2018). Phylogenetic analysis, performed on the partial replicase gene sequences of 25 Slovenian isolates and 18 reference isolates from GenBank, showed clustering into two groups. The Slovenian isolates were within the both groups. Interestingly, the isolates clustered according to variety, with the exception of 'Laški rizling'. GFkV is 62 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 graft-transmissible; the vectors are unknown (Sabanadzovic et al., 2017). Isolates analyzed in this study had the highest nt identity and clustered with isolates from neighboring countries and the United States, from which untested rootstocks were imported (Hrček, 1977) and predecessors of our isolates were grafted onto these rootstocks, suggesting that infection occurred via propagation material and grafting. In addition, propagation material produced in Slovenia was exported to geographically nearby countries. In Slovenia, testing on GFkV is obligatory only for rootstocks. GFkV is a phloem-limited virus, and with shoot/meristem tissue culture different eliminations efficacy have been reported depending mainly on the size of the shoots/meristems. Shoots (1-3 mm) resulted in 20% or 25% of GFkV-free plants (in combination with thermotherapy in the growth chamber and thermotherapy during summer in the field, respectively) (Bota et al., 2014); meristems (0.8 mm) resulted in 50% GFkV-free plants, while meristems (0.3 mm) resulted in 100% GFkV elimination (Kim et al., 2017). Complete eradication was also achieved by combining thermotherapy and shoot apices micrografting (Spilmont et al., 2012). Thermotherapy alone (Panattoni and Triolo, 2010; Hu et al., 2021), somatic embryogenesis (Turcsan et al., 2020), chemotherapy (ribavirin, repeated ribavirin treatment, combination of ribavirin and oseltamivir) (Komínek et al., 2016; Guţa et al., 2017; Hu et al., 2021), and ribavirin in combination with thermotherapy (Hu et al., 2021) have also been used for its elimination. GRGV was discovered in 'Red Globe' variety from Southern Italy and in two samples of an unknown variety from Albania, and the virus was studied in the domains RdRp and MTR (Sabanadzovic et al., 2000). Recently, the whole genome of GRGV was obtained (Cretazzo and Velasco, 2017). Comparing the whole GRGV genome sequences with the available RdRp and MTR domains, Cretazzo and Velasco (2017) found that the RdRp gene region corresponded to GRGV, while the MTR gene corresponded better to GRVFV, suggesting that the same samples were simultaneously infected with both viruses. This virus is less studied because it is asymptomatic in V. vinifera and V. rupestris (Cretazzo and Velasco, 2017). When our analysis was performed, only four complete or nearly complete GRGV sequences were available in NCBI, three from Spain (KX109927, KX171167, and NC_030693) and one from Brazil (KX828704). In this study, GRGV was detected in two libraries of 'Refošk' variety (006 and 010). In both libraries, unique viral contigs were compared with these four sequences; the highest coverage in library 006 was 63.6% (KX171166) and in library 010 was 45.8% (KX109927). In addition, the MTR domain (AJ249360) was identified in library 010, which is indeed the domain of GRVFV (Cretazzo and Velasco, 2017). HTS results were validated with primers RG6061F/RG6801R (Cretazzo and Velasco, 2017) and 2 samples from library 006 (Refošk 10/2B and Refošk 10/3B) and 1 sample from library 010 (Refošk 9/5P) were infected. The virus was amplified at 40 cycles. Partial CP gene was Sanger sequenced and phylogenetic analysis showed that our isolates clustered separately, probably due to the few sequences deposited in NCBI. No studies regarding its elimination have been published. 63 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 El Beaino et al. (2001) reported that an unknown pathogen in Greek vines, that caused unusual symptoms reminiscent of asteroid mosaic upon grafting onto V. rupestris, was related to grapevine fleck virus-like viruses, but it was different enough which required further studies. The Greek virus was later characterized at the 3′ end and its name - GRVFV was proposed (Abou Ghanem-Sabanadzovic et al., 2003). In our study, GRVFV was detected in eleven libraries, being absent only in library 009 ('Malvazija' variety). The VirusDetect (BLASTN search) identified between 5 and 25 complete and partial reference sequences per library, with the exception of library 013, where only one partial reference was identified with a coverage of 30.2%. Complete reference sequences were covered with 30-94 contigs and sequencing depth of 5.4X-93.3X. The low genome coverage (61%) and low sequencing depth (13X) for this virus was also obtained in the study by Saldarelli et al. (2015) using sRNA-seq. The authors reported that the data indicated limited GRVFV replication in the analyzed tissue. The presence of the virus was validated in all predicted libraries. GRVFV was confirmed in the 44 samples by RT-PCR, and all of them were Sanger sequenced. Twenty-two sequences were of lower quality, likely due to the presence of different genetic variants in the same samples (a similar situation was also observed for GRSPaV), and were excluded from further analysis. The other 22 high-quality sequences were deposited in NCBI. The overall average divergence between the 22 Slovenian GRVFV polyprotein partial sequences was 10.9% ± 0.9%. High average divergence was also reported for Slovak GRVFV isolates (11.9% ± 0.9%) (Glasa et al., 2019). Phylogenetic analysis of 22 partial sequences from GenBank and 22 Slovenian isolates suggested the existence of two molecular groups, consistent with the results reported by Glasa et al. (2019), and some of our isolates clustered with isolates from Slovakia and France, but the majority clustered separately. Although GRVFV has been known for two decades, only one report of its elimination by somatic embryogenesis in 'Muscat Ottonel' variety has been published recently (Turcsan et al., 2020). In our study, 19 candidates infected with GRVFV were included in therapy process, 33 regenerated plants were obtained and all were GRVFV-free. GSyV-1 was discovered in California in 2009 during the study of severe decline of 'Syrah' variety using HTS (Al Rwahnih et al., 2009). In a contemporary study conducted in Southeastern United States, the same virus named as grapevine virus Q (GVQ) was discovered in asymptomatic muscadine grape ( V. rotundifolia Michx.) (Sabanadzovic et al., 2009). The same study reported that this virus is also infective for summer grape ( V. aestivalis) and wild blackberry ( Rubus sp.). Two complete genome sequences derived from the above studies, FJ436028 (GSyV-1) and FJ977041 (GVQ), deposited in NCBI, have 99.09% nt identity (99% query coverage), confirming that these two viruses are the same species. In addition, GSyV-1 exhibits permuted and non-canonical organization of RdRp motifs (C → A → B), a feature not previously reported in plant virology, as this feature is associated with animal viruses (Sabanadzovic et al., 2009). GSyV-1 was found in North America and has been detected in only nine European countries (Italy, France, Hungary, Slovakia, Czech Republic, Spain, Turkey, Croatia, and Russia) (Giampetruzzi et al., 2012; 64 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Beuve et al., 2013; Czotter et al., 2015; Glasa et al., 2015; Ruiz-García et al., 2017; Caglayan et al., 2017; Vončina et al., 2017; Navrotskaya et al., 2021). However, knowledge about the distribution and genome organization of GSyV-1 on the European continent is still very limited. GSyV-1 was found in two of our libraries: library 005 ('Laški rizling' variety) and library 009 ('Malvazija' variety). In library 005, VirusDetect (BLASTN search) identified only one partial polyprotein reference sequence (KP221269) with a length of 334 nt, which was 44.9% covered by 3 contigs (69 nt, 35 nt and 47 nt) and a sequencing depth of 20.9X. Four reference sequences (3 complete and 1 partial) were identified in library 009. The complete genome sequence from Slovakia (KP221256) had the highest coverage of 48.8%. Validation of the predicted GSyV-1 was performed with primers SY5922F/SY6295R (Glasa et al., 2015) targeting a fragment of the CP gene. One sample from the 005 library (Laški rizling 3/45B) and two samples from the 009 library (Malvazija 32/2B and Malvazija 32/3B) were infected. The virus was amplified at 40 cycles, the same as GRGV, which may indicate that the concentration of these two viruses was very low, which could explain the results of HTS (scarce genome coverage and low sequencing depth). In Laški rizling 3/45B, GSyV-1 was found in co-infection with RBDV, GRSPaV, GPGV, GRVFV, GFkV, HSVd, and GYSVd-1; while in Malvazija 32/2B and 32/3B it was found in co-infection with GRSPaV, GPGV, HSVd, and GYSVd-1. Phylogenetic analysis of the partial CP gene showed that GSyV-1 isolates clustered into two clades, and the major clade consisting of two major lineages, consistent with the results of Glasa et al. (2015). Our isolates clustered together with isolates from Hungary and Slovakia. All three samples were included in the elimination process, Laški rizling 3/45B did not regenerate. From Malvazija 32/2B and 32/3B, only two meristems regenerated (one meristem per candidate), and both regenerated plants were free of GSyV-1. To date, only one study considering GSyV-1 elimination by meristem tissue culture and somatic embryogenesis has been published with an elimination success of 100% (Turcsan et al., 2020). GPGV was detected in all libraries. The lowest reference genome coverage (95.3%), the lowest sequencing depth (10.7X), and the highest number of contigs (38) were observed in library 014 ('Malvazija' variety); while in the other 11 libraries the reference genomes were 97-100% covered with 5-16 contigs and sequencing depth ranging from 33.2X-487.4X. The sRNA-seq results were validated with primers targeting partial MP and partial CP genes. Among the preclonal candidates, GPGV was the most abundant, and 91.14% of the tested plants were infected. High incidence of GPGV was also found in neighboring countries: Italy (Saldarelli et al., 2015; Bianchi et al., 2015), Hungary (Czotter et al., 2018), Croatia (Hančević et al., 2021). In addition, it was detected in all countries of the former Yugoslavia (Montenegro, Serbia, Bosnia and Herzegovina, and North Macedonia) (Bertazzon et al., 2016). Forty RT-PCR products were sequenced. MP sequences obtained from 40 Slovenian preclonal GPGV isolates showed a specific C/T polymorphism at position 6,685, that introduced the premature stop codon. The C/T polymorphism was also found in GPGV survey in different countries. Studies from Italy (Saldarelli et al., 2015; Marra et al., 2020) 65 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 and Switzerland (Reynard, 2015) showed that GPGV isolates from asymptomatic vines had 18 extra nt (6 aa), while a study from Hungary (Czotter et al., 2018) showed that asymptomatic isolates had 18 nt (6 aa) shorter MP due to this polymorphism. In our study, all preclonal candidates were asymptomatic, and the 18 nt (6 aa) shorter MP was found in 13 vines, while the MP with these 18 nt (6 aa) residues was observed in 27 isolates. The results of several studies indicate that the C/T polymorphism in the stop codon is most likely not responsible for symptom expression. Interestingly, it seems to be a silent mutation in the CP gene, since the same polymorphism is also part of the overlapping CP gene. In addition, a mutation responsible for 5 aa shortening of MP has been found in Spanish and Russian isolates (Morán et al., 2018; Shvets and Vinogradova, 2022). Using sRNA-seq, Saldarelli et al. (2015), examined viromes from symptomatic and asymptomatic grapevines. The authors constructed two sRNA libraries (one symptomatic and one asymptomatic vine) and found that GPGV was co-infected with two viruses (GRSPaV and GRVFV), and two viroids (HSVd and GYSVd-1). GYSVd-1 was present only in asymptomatic sample while GRVFV was present only in symptomatic sample. To investigate the possible involvement of GRVFV in symptomatology, asymptomatic and symptomatic samples were tested with RT-PCR. GRVFV was confirmed in symptomatic and asymptomatic samples, which ruled out an association of this virus with symptoms. Bianchi et al. (2015) reported that three viruses (GRSPaV, GRVFV, and GSyV-1), and two viroids (HSVd and GYSVd‐1) were present in plants with or without symptoms, although GRVFV and GSyV-1 were present in a lower percentage. Bertazzon et al. (2017) reported that most samples with or without symptoms were infected with the same viruses. In our study we found GPGV only in co-infection with HSVd (Rebula 24/2B, Rebula 26/3B), but also in Laški rizling 3/45B with five viruses (RBDV, GRSPaV, GFkV, GSyV-1, GRVFV) and two viroids (HSVd and GYSVd-1). In most cases (18.99%) it was found in co-infection with GRSPaV, HSVd, and GYSVd-1. Therefore, we also did not find any correlation between the expression of symptoms and co-infection of GPGV with other viral pathogens. In addition, Saldarelli et al. (2015) performed biological assay for better understanding the role of GPGV in the etiology of GLMD, and suggested that different GPGV strains exist with diverse biological traits. Bianchi et al. (2015) and Bertazzon et al. (2017) also disclosed that symptomatic plants had significantly higher GPGV concentration than asymptomatic plants, whereas Morán et al. (2018) and Shvets and Vinogradova (2022) found no association between symptoms and virus titer. The above facts suggest that GPGV has a complex epidemiology. Therefore, additional analysis of symptom expression and general epidemiology of the disease are needed. The MP/CP region is commonly used in published GPGV research to infer phylogenetic relationships, although some authors have also included sequences of RdRp. For example, Bertazzon et al. (2017) and Al Rwahnih et al. (2021) performed a phylogenetic analysis with partial region spanning MP and CP genes which was also analyzed in our study. Eichmeier et al. (2017, 2018) and Saldarelli et al. (2015) included besides MP/CP sequences also RdRp region. Phylogenetic analysis showed that our isolates clustered into two clades, although all were asymptomatic. Isolates with shorter MP clustered in the same clade, whereas isolates with 66 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 longer MP clustered in both clades. Our isolates grouped with those from countries that are relatively close geographically, which may suggest that infection spreads geographically through the dissemination of infected propagation material, but the high prevalence also suggests that the vector plays an important role. Testing the grapevine propagation materials on GPGV is not mandatory. However, based on the complex etiology and epidemiology of the disease and its impact on grapevine production, testing on GPGV should be mandatory. To date, three studies have been carried out considering its elimination through meristem tip culture with and without thermotherapy, chemotherapy, and somatic embryogenesis (Gualandri et al., 2015; Komínek et al., 2016; Turcsan et al., 2020). In our study, 26 out of 28 samples included into sanitation process were GPGV-infected. Forty-nine regenerated plants were obtained and all were GPGV-free. In 2001 and 2002, unusual symptoms (curved line patterns and leaf yellowing) were observed on the variety 'Laški rizling' (Mavrič et al., 2003). Samples were negative on nepoviruses, but were positive on RBDV. Later, RBDV was detected in 'Štajerska belina' variety (Mavrič et al., 2003). Since then, it was detected in all Slovenian viticulture regions in the period between 2003 and 2005, but only in white varieties (Mavrič Pleško et al., 2009). In 2006, it was detected in the red variety 'Pinot Noir' in the Podravje region (Mavrič Pleško et al., 2009). A more recent study showed that the lowest incidence of RBDV was found in the Primorska viticultural region (5.1%) compared to Podravje and Posavje (Mavrič Pleško et al., 2020). Infection of grapevine with RBDV has also been reported in Serbia (Jevremovic and Paunovic, 2011), Hungary (Mavrič Pleško et al., 2012; Czotter et al., 2018), and recently in Russia (Navrotskaya et al., 2021). In our study, RBDV was predicted only in library 005 ('Laški rizling' variety). Although the complete genome of RBDV isolates from grapevine has not yet been obtained, the assembled contigs were compared with the reference isolate J1 from Rubus idaeus. RNA1 was 100% covered with two contigs (one of which was sufficient to cover the complete RNA1 segment) and a sequencing depth of 1556.2X; the RNA2 segment was 98.7% covered with four contigs and a sequencing depth of 513.8X. The bulk samples showed nt identity of 93.40% and 96.10% with the reference RNA1 and RNA2, respectively. A 5' end (941 bp) of a Serbian isolate from grapevine showed 93.62% identity at the nt level with isolate R15 from raspberry (Jevremovic and Paunovic, 2011). The presence of the virus was validated using RT-PCR and all 4 samples (Laški rizling 3/34B, 3/45B, 3/54B, and 3/64B) were infected. Partial RNA2 (partial MP and partial CP) was Sanger sequenced and a phylogenetic tree was constructed based on the partial CP nucleotide sequences (438 bp) of our 4 and 29 RBDV isolates from grapevine and raspberry from the GenBank database. The RNA2 region is commonly used in studies of the phylogenetic relationship of RBDV, although some authors have also included sequences from RNA1. Mavrič Pleško et al. (2009) performed a phylogenetic analysis based on MP and CP amino acid sequences, and later on nucleotide sequences of the almost complete RNA2 segment and also only for the CP gene (Mavrič Pleško et al., 2020). These two Slovenian studies (Mavrič Pleško et al., 2009, 2020), as well as studies from Hungary 67 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 (Czotter et al., 2018), Belarus and Sweden (Valasevich et al., 2011) showed that grapevine isolates were clearly separated from Rubus sp. isolates, which is in agreement with our results. In addition, phylogenetic studies in Hungary showed that partial RNA1 from grapevine was also clustered separately from RNA1 sequences from Rubus sp. (Czotter et al., 2018). With serological methods, using monoclonal antibodies it is also possible to distinguish isolates from grapevine and raspberry (Mavrič Pleško et al., 2009). In various studies, using different elimination methods such as thermotherapy, meristem tissue culture, chemotherapy, cryotherapy, and their different combinations, it was reported that elimination of RBDV from raspberry is very difficult (Murant et al., 1974; Theiler‐Hedtrich and Baumann, 1989; Wang et al., 2008; Mathew et al., 2021). It is presumed that this pollen-transmitted virus infects meristematic tissues except for the least differentiated cells of the apical dome (Wang et al., 2008). To our knowledge, this is the first report of elimination of this virus from grapevine. 103 meristems were isolated, 11 regenerated (2 from 3/34B, 5 from 3/56B, and 4 from 3/64B), all of which were free of RBDV. Six viroids and one viroid-like RNA have been reported from grapevine (Di Serio et al., 2017). HSVd and GYSVd-1 are globally distributed and are the only two viroids known to occur in grapevine in Slovenia. In our study using sRNA-seq, HSVd was detected in all libraries. In all libraries, VirusDetect (BLASTN search) identified only one reference isolate per library (KJ810551 or KY508372). HSVd was validated in all libraries using HSV-78P/HSV-83M primers that amplified the entire genome (Sano et al., 2001), and all (79) samples were infected. Considering that HSVd is latent in grapevines, and that testing is usually overlooked, this contributed to its high prevalence in many countries. Forty RT-PCR products were selected and Sanger sequenced. Interestingly, 38 sequences were 100% identical. Two other sequences (Pokalca 3/4P and Pokalca 3/6P) were identical and had 98% identity with the other 38 isolates. These two isolates clustered separately from the other 38 sequences from this study and 29 sequences from different hosts from the GenBank database ( Vitis sp., Humulus lupulus, Ficus carica, Morus alba, Citrus sp., Prunus sp.). HSVd is latent in grapevines but can be transmitted to hop plants and cause epidemics (Sano et al., 2001; Kawaguchi-Ito et al., 2009). This is particularly important because Slovenia is one of the largest hop producers. GYSVd-1 was also detected in all libraries, and only one reference sequence was identified per library (AB028466, KP010010, or KJ466324). GYSVd-1 predicted by sRNA-seq was validated in all libraries using primers that amplified the whole genome (Ward et al., 2011), and 71 samples were positive (89.87%). It should be noted that in order to validate GYSVd-1, the number of amplification cycles for all libraries was 40. The increased number of amplification cycles (45) in order to validate sRNA-seq predicted GYSVd-1, was reported from Russia (Navrotskaya et al., 2021). Thirty-five RT-PCR products were selected and Sanger sequenced. GYSVd-1 had higher genetic diversity in comparison with the HSVd. InDel mutations were also observed. GYSVd-1 and/or GYSVd-2 in co-infection with GFLV may elicit vein banding symptoms (Hajizadeh et al., 2015). In our study GYSVd-1 in co-infection with GFLV was found in two samples of 'Zeleni 68 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Sauvignon' variety (26/1P and 26/2P), and five samples of 'Pokalca' variety (3/4P, 3/5P, 3/6P, 9/2G, and 9/3G), but symptoms were not observed. Simultaneous infection with GYSVd-1 and GFLV also did not induce visible symptoms of vein banding or yellow speckles in two varieties of V. vinifera and one variety of V. labrusca in Brazil (Fajardo et al., 2016). Phylogenetic analysis of 35 Slovenian GYSVd-1 isolates generated in this study and 28 sequences from database showed that our isolates clustered in different phylogroups, independently of variety or geographic distribution. Testing of material for clonal selection and vegetative propagation on viroids is not obligatory, therefore they are often overlooked. Three methods for viroids eradication from grapevines have been used so far: thermotherapy (Gambino et al., 2011), meristem tissue culture (Duran-Vila et al., 1988; Turcsan et al., 2020), and somatic embryogenesis (Gambino et al., 2011; Turcsan et al., 2020). Thermotherapy alone (Gambino et al., 2011) and treatment with ribavirin (Eichmeier et al., 2019) were unsuccessful. Several studies reported different elimination success with meristem tissue culture (Duran-Vila et al., 1988; Turcsan et al., 2020). The best results were obtained with somatic embryogenesis (Gambino et al., 2011; Turcsan et al., 2020). In our study, 28 samples infected with HSVd and 27 infected with GYSVd-1 were included in the elimination process. Fifty-one and 47 regenerated plants were tested on HSVd and GYSVd-1, respectively. Elimination of viroids was lower because heat therapy induced their replication; 39.2% of HSVd-free, and 42.6% of GYSVd-1-free plants were obtained. Considering that nine viruses and two viroids were detected and validated in the asymptomatic preclonal samples, in the third part of the dissertation we wanted to investigate the virome of samples not included in clonal selection programs. Thirteen samples from six grapevine varieties ('Malvazija', 'Rebula', 'Pokalca', 'Cipro', 'Volovnik', and 'Poljšakica') were analyzed. Four libraries were constructed and sequenced. A total of 70,902,637 reads were generated, of which 7.44% mapped to viral reference sequences. The method used revealed the presence of: GLRaV-1, GLRaV-2, GLRaV-3, GFLV, satGFLV, GRSPaV, GPGV, GFkV, GRVFV, GV-Sat, HSVd, and GYSVd-1. Among the preclonal candidates, GLRaV-3 was detected in only one library and only one sample was infected, while other viruses belonging to the leafroll disease complex were not detected. In these samples, GLRaV-3 was more prevalent as it was detected in three libraries (L2, L3, and L4). In addition, two viruses from the leafroll complex, GLRaV-1 and GLRaV-2, were detected in the variety 'Cipro' (L1). GFLV was found in two libraries (L2 and L4) and its satellite RNA was also detected in both. GPGV, the emerging and most abundant virus in preclonal candidates, was also found here in all libraries. Two viruses (GRGV and GSyV-1), found for the first time in Slovenia in preclonal candidates, were not detected in these samples, while the third (GRVFV) was found in all libraries. In preclonal candidates, the viroids were detected in all libraries, while RT-PCR showed that GYSVd-1 was less abundant. In these sample set, HSVd was detected in all libraries, while GYSVd-1 was absent in one library (L2). GRSPaV was also detected in all libraries, whereas it was not detected in preclonal candidates with sRNA-seq in 2 out of 12 libraries, although it was confirmed in all libraries 69 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 with RT-PCR. In addition, GV-Sat was detected, which is the first report in Slovenia. GV-Sat was detected in a very low percentage (3%; 346 samples were analyzed) in California with HTS of dsRNAs (Al Rwahnih et al., 2013). Four years later, it was reported on Iranian variety 'Askeri' held in the INRA collection in France (Candresse et al. 2017). In European vineyards has been found only in Hungary (Czotter et al., 2018). To date, only eleven sequences have been deposited in NCBI, including three generated in this study. The stop codon of ORF1 and the start codon of ORF2 overlap, and multimeric forms exist (Candresse et al., 2017). GV-Sat require the help of another virus(es) to replicate in plants. The helper virus is still unknown, but in published studies (Al Rwahnih et al., 2013; Czotter et al., 2018) it was found in co-infections with vitiviruses and leafroll-associated viruses. In the 'Cipro' library, we detected two viruses belonging to the leafroll complex (GLRaV-1, GLRaV-2), but we did not detect vitiviruses. This result may suggest that GLRaVs play a role in amplification, which could also explain why GV-Sat was not detected in the preclonal candidates. In addition, GRSPaV, GPGV, GRVFV, HSVd, and GYSVd-1 were detected in the same library. As we expected, more viruses with mandatory and recommended tests were detected in samples that were not included in certification programs. Therefore, our goal was to develop a multiplex RT-PCR for validation of sRNA-seq data that could be used for rapid and cheaper routine diagnostics. The KAPA2G Fast Multiplex PCR Kit was used. According to the protocol, the primers used should have a similar melting temperature (Tm) and a guanine-cytosine content (GC) of 40-60%. We chose primer pares with different amplicon sizes that allowed differentiation on the agarose gel. Although the selected primers had different Tm and some primers did not have optimal GC content, successful amplification was achieved in all cases. mRT-PCR has been used in several studies to detect grapevine viruses and viroids (Nassuth et al., 2000; Gambino and Gribaudo, 2006; Digiaro et al., 2007; Hajizadeh et al., 2012; Gambino, 2015; Ahmadi et al., 2017; Komínková and Komínek, 2020). 70 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 3.2 CONCLUSIONS Viral pathogens are one of the major obstacles in grapevine production. Rapid and accurate detection, molecular characterization, and implementation of viral elimination methods are crucial for viticulture worldwide. In this doctoral dissertation, we aimed to study the virome of different grapevine varieties (included or not in the clonal selection process) using HTS of virus- and viroid-derived small RNAs, and to validate all predicted pathogens with RT-PCR and Sanger sequencing. We also aimed to study their genetic diversity, phylogeny, and co-infections, as well as to investigate the efficiency of their elimination by thermotherapy and meristem tip micrografting. We set up four research hypotheses: 1) Vines are infected with different viruses and viroids, which can be adequately determined using HTS of small RNAs. The virome status of 82 preclonal candidates and 13 samples not included in the clonal selection was examined by high-throughput sequencing of small RNAs. For the preclonal candidates, 12 libraries were constructed, and for the samples not included in the clonal selection, 4 libraries were constructed. Sequencing was performed using the IonTorrent System. In total, 12 viruses were detected: GRSPaV, GFLV (in association with its satellite RNA), GLRaV-1, GLRaV-2, GLRaV-3, GPGV, GFkV, GSyV-1, GRVFV, GRGV, RBDV and GV-Sat, as well as two viroids: HSVd and GYSVd-1. GRGV, GRVFV, GSyV-1 and GV-Sat have been found for the first time in Slovenia. 2) Based on the sequences information of viruses and viroids obtained by the HTS approach specific primers could be designed for amplification and validation of the predicted viral pathogens by RT-PCR and Sanger sequencing. Based on the sequences obtained by the HTS approach, primers were selected for validation of the predicted viral pathogens. The primers corresponded to those found in the literature or were newly designed in this study. The predicted viral pathogens in the preclonal candidates were validated using RT-PCR and Sanger sequencing. The predicted viral pathogens in samples not included in the clonal selection were validated using the multiplex RT-PCR developed in this study. 3) Predicted infections will be confirmed with Sanger sequencing and additional information about strain-specific polymorphisms related to different host grapevines could be obtained. 71 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 In preclonal candidates, 191 Sanger sequences were generated. Genetic diversity was studied in different viral genomic regions. Detailed insights into genetic diversity, phylogeny, and co-infections were obtained. 4) Using thermotherapy and meristem/shoot tip culture virus-free material could be established, but the percentage of elimination will vary depending on variety and viral pathogen. Twenty-eight preclonal candidates infected with eight viruses and two viroids were selected for elimination experiment by in vivo thermotherapy and in vitro meristem tip micrografting. Efficient protocols were established for hypocotyl production (used as rootstocks), micrografts and micropropagated plants separated from rootstocks. The overall regeneration rate was 8.53%, the elimination rate for viruses was 100%, while it was lower for viroids (39.2% for HSVd and 42.6% for GYSVd-1). The regenerated plants were successfully acclimatized. 72 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 4 SUMMARY (POVZETEK) 4.1 SUMMARY In the PhD thesis, we investigated the virome of grapevine ( Vitis vinifera L.) using high-throughput sequencing technology, studied the genetic diversity of viruses and viroids, their phylogeny, and co-infections, developed a protocol for healthy vines production by in vivo thermotherapy and in vitro meristem tip micrografting, and developed a multiplex RT-PCR for validation of sRNA-seq data, that could be used for rapid, reliable, sensitive and cheaper diagnostics. Grapes are considered one of the most widely grown fruit crops in the world (7.3 million hectares). Viruses are one of the major obstacles to sustainable viticulture. Viral pathogens can be latent, but also can cause severe symptoms and high economic losses. Therefore, rapid and accurate detection, characterization and application of biotechnological approaches for viral elimination are of great importance. The aim of the first part of our study was to investigate the virome of preclonal candidates in the Primorska viticultural region, where clonal selection has been carried out for decades. Eighty-two samples of six grape varieties (2 red and 4 white) were analyzed using small RNA sequencing technology. Micro RNAs were isolated, twelve libraries were constructed, and then sequenced using the Ion Proton™ System. VirusDetect was used for data analysis. Nine viruses: GFLV, GLRaV-3, GRSPaV, GFkV, GSyV-1, GRVFV, GRGV, GPGV, and RBDV, and two viroids: HSVd and GYSVd-1 were identified. Three viruses (GRGV, GRVFV, GSyV-1) have never been reported in Slovenia before. In silico results were validated by RT-PCR and Sanger sequencing. Total RNA from all samples (82) was extracted and reverse transcribed, followed by amplification with specific primers selected based on sequences obtained by sRNA-seq. The amplification products were analyzed by gel electrophoresis, and the remaining reactions were sequenced in both directions after purification (Exo-Sap treatment). The sequences were trimmed and assembled. A total of 191 sequences were generated and analyzed. All generated Sanger sequences were deposited in NCBI under the accession numbers: GSyV-1 (MW446939-MW446941), GRVFV (MW446917-MW446938), GRGV (MW446914-MW446916), GLRaV-3 (OK138920), GRSPaV (OK138921-OK138934), HSVd (OK138935-OK138974), GYSVd-1 (OK138975-OK139009), GFkV (OK139010-OK139034), GFLV (OK139035-OK139038), RBDV (OK139039-OK139042), GPGV (OK139043-OK139082). GLRaV-3 was predicted only in library 010 ('Refošk' variety). The reference sequence was an isolate from South Africa (GQ352631). A unique viral contig corresponding to ORF2 (known to be absent in some isolates) was observed. The predicted virus was validated with two primer pairs that amplified two different genomic regions (CP and HSP70h), and only one sample was infected. Prior to our analysis, all preclonal vines were tested by ELISA on 73 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 viruses from the leafroll complex, and all infected vines were excluded from further analysis, which explains why GLRaV-3 was detected in only one sample and why other leafroll-associated viruses were not detected. CP gene of Refošk 11/4P was sequenced and was 99.76% identical with 18 sequences originating from different continents, but phylogenetic study showed that our isolate was clustered with a Portuguese isolate. RBDV was also detected only in one library ('Laški rizling'). Reference RNA1 was 100% covered, while RNA2 was 98.7% covered. All four samples were infected. Part of the MP and part of the CP gene were sequenced. The phylogenetic tree was constructed based on 438 bp of the partial CP gene, and the sequences of grapevine were clearly separated from those of raspberry. Four viruses from the grapevine fleck complex were detected, three of which (GSyV-1, GRVFV, GRGV) were detected for the first time in Slovenian vineyards. GFkV was predicted in eight libraries. In seven libraries, VirusDetect identified 4-10 reference sequences from the GenBank database per library, while in one library (011; 'Refošk' variety) only one partial replicase gene was identified, with 33.3% coverage. Many contigs and their short length were observed in all libraries. GFkV was validated in all predicted libraries. Genetic diversity was examined in the replicase gene. Interestingly, phylogenetic studies showed that isolates clustered according to variety, the only exception was 'Laški rizling'. Based on nt identity and phylogenetic studies, we concluded that GFkV infection occurred through the exchange of infected propagation material. Among the viruses detected for the first time in Slovenia, GRVFV was more prevalent than the other two. GRVFV was detected in 11 libraries. In all libraries, mainly scarce reference genomes coverage with many short contigs were obtained. The virus was validated in all libraries where it was predicted. Forty-four RT-PCR products were sequenced (partial polyprotein), and 22 sequences were of lower quality, probably due to the presence of different genetic variants in the same sample, but these sequences were not cloned; only the high quality ones were further analyzed. We found high genetic variability between sequences (10.9% ± 0.9%) and their partitioning into two molecular groups, in different subclades. GRGV was detected in two libraries of 'Refošk' variety. In both libraries, references from Spain had the highest genome coverage. GRGV was validated in both libraries, and a total of 3 samples were infected. Due to the limited number of sequences deposited in NCBI, we could not conclude the possible introduction of this virus in Slovenia. GSyV-1 was also detected in two libraries, library 005 ('Laški rizling' variety) and library 009 ('Malvazija' variety) with very scarce reference genomes coverage. GSyV-1 was validated in both libraries. Phylogenetic analysis (partial CP gene of our sequences and those retrieved from NCBI) showed that isolates clustered into two clades, and our three isolates belonged to the major clade and clustered together with isolates from Hungary and Slovakia. It should be noted that for validation of GRGV and GSyV-1, the number of amplification cycles for all libraries was 40, which could explain the HTS results (very low coverage of the reference genomes and very low sequencing depth). 74 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 From the infectious degeneration and decline disease complex, GFLV was detected. GFLV was predicted in library 013 ('Zeleni Sauvignon' variety) and library 016 ('Pokalca' variety). In both libraries, VirusDetect (BLASTN search) identified more sequences from the database for both the RNA1 and RNA2 segments. The virus was validated in both predicted libraries, and four samples were positive with primers amplifying the 606 bp RNA2 region, including the partial CP gene. Sequence analysis revealed high genetic diversity (more than 12%) between red and white varieties. Based on the high genetic diversity between our isolates and isolates from GenBank, a new primer pair was designed, and an additional six samples were found infected. The phylogenetic tree showed that the isolates included in the analysis clustered into two clades; our isolates belonged to the major clade and were closer to the isolates from Italy and France than to the previously characterized isolates from Slovenia (variety 'Volovnik'). From the rugose wood disease complex, only GRSPaV was detected. GRSPaV was detected in ten libraries. In eight libraries 4 to 40 complete and partial (mainly CP gene) reference sequences were identified. The highest coverage of complete reference sequences ranged from 48.4% to 99.1%. They were covered by a high number of contigs (41-86) and a low sequencing depth (5.3X-16.6X). Only partial sequences were identified in two libraries (012 and 015). The virus was not detected in libraries 007 and 014. The virus was validated in all libraries where it was predicted and additionally in both libraries where it was not predicted. This may have a profound biological background and requires further investigation, as the hypothesis that this occurred due to low concentration and bulk sequencing strategy was ruled out. Overall, 88.61% of the samples were infected. Sequencing of the whole RT-PCR product was not possible, so cloning was performed. At least three genetic variants were found to be present in the same sample, and the mean distance between the 14 sequences was 14.06%. The variety 'Laški rizling' showed the highest diversity (17.14%), interestingly, VirusDetect identified the highest number of reference sequences from the database in the 'Laški rizling' variety (40). Phylogenetic analysis showed that the genetic variants clustered differently even if they were from the same sample, and that there was no clustering by geographic origin. GPGV is an emerging virus that may be latent or cause severe damage. It is of concern that this virus was predicted in all libraries and that 72 samples (91.14%) were infected. Genetic diversity was examined in 40 partial MP and partial CP sequences. A specific C/T polymorphism at position 6,685 was observed in 13 samples, which caused premature stop codon, and shorter MP by 18 nt (6 aa). The data obtained here indicate that the C/T polymorphism is not responsible for the expression of symptoms. Synergism with different viruses/viroids is also not responsible for symptomatology, as we found GPGV only in co-infection with HSVd, but also with up to five viruses (GRSPaV, GFkV, GSyV-1, GRVFV, and RBDV) and two viroids (HSVd and GYSVd-1). Phylogenetic analysis of the partial sequences of the MP/CP genes showed that our isolates clustered into two clades, although all were asymptomatic. Our isolates clustered mainly with those from countries relatively 75 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 close geographically, which may suggest that infections spread through the dissemination of infected propagation material, but the high prevalence also suggests that vectors play an important role. Testing on GPGV is only recommended but should be mandatory due to the complex etiology and epidemiology of the disease and its impact on vine production. Considering viroids, HSVd and GYSVd-1 are distributed worldwide, and are the only two viroids known to occur on grapevines in Slovenia. HSVd was detected and validated in all libraries. With RT-PCR, HSVd was detected in all samples. Forty RT-PCR products were selected and Sanger sequenced. Interestingly, 38 sequences were identical, while two sequences showed 98% identity with other isolates but clustered separately in the phylogenetic tree from other isolates generated in this study and from other isolates retrieved from NCBI. Considering that HSVd is latent in grapevines and was found in all samples, and that isolates from grapevines have been shown to cause epidemics in hops, this viroid requires close attention. GYSVd-1 was also detected in all libraries, and only one reference sequence per library was also identified. Seventy-one samples were positive (89.87%). Thirty-five RT-PCR products were selected and Sanger sequenced. Sequences were 95.35-100% identical, and InDel mutations were also observed. Overall, it was more genetically diverse than HSVd. Phylogenetic analysis showed that our isolates clustered in different phylogroups regardless of variety or geographic distribution. Overall, we obtained a complete insight into the virome of the preclonal candidates. GLRaV-3 was the rarest (1.27%), followed by GRGV and GSyV-1 (3.80%), RBDV (5.06%), GFLV (12.66%), GFkV (34.18%), GRVFV (55.70%), GRSPaV (88.61%), GYSVd-1 (89.87%), GPGV (91.14%), HSVd (100%). Most samples were simultaneously infected with five viral pathogens, while eight viral pathogens were detected in one sample. Infections with GRSPaV, GPGV, HSVd and GYSVd-1 were the most frequent (18.99%). Considering that the preclonal candidates were infected with nine viruses and two viroids, the objective of the second part of this dissertation was to develop a protocol for the production of healthy vines. Twenty-eight preclonal candidates from six varieties infected with the viral pathogens predicted and validated above (except GRGV) were selected for virus/viroid elimination. Heat therapy was performed from six weeks to three months at 36-38 °C. After in vivo thermotherapy and surface disinfection of apical and axillary segments, meristem tips (0.1-0.2 mm) were isolated and immediately micrografted onto etiolated and sectioned hypocotyls of Vialla ( Vitis labrusca × Vitis riparia). The overall regeneration rate was very low (8.53%). Four preclonal candidates did not regenerate. A higher regeneration rate was observed in white varieties. 'Rebula' had the highest regeneration rate (16.7%), and 'Pokalca' had the lowest (3.3%). The regenerated plants were micropropagated several times to increase their number and to have enough material for testing, acclimatization and in vitro storage. The in vitro plants were tested after seven months using RT-PCR. A 100% 76 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 elimination rate was achieved for eight viruses. Elimination rates for viroids were lower (39.2% for HSVd and 42.6% for GYSVd-1), probably because the high temperature promoted viroid replication and accumulation. Although this method is difficult, labor-intensive, and time-consuming, it has many advantages over other biotechnological approaches for viral elimination, such as chemotherapy, which can cause severe phytotoxicity, or somatic embryogenesis, which carries a high risk of mutations. In our study, we reduced the risk of genetic instability by in vivo thermotherapy (shortened duration of in vitro cultivation), meristem isolation (no callus formation), micrografting (accelerated regeneration), and the use of a medium without plant growth regulators/hormones. The aim of the third part of this dissertation was to study the virome of symptomatic samples not included in clonal selection programs. Thirteen samples were analyzed. Four libraries were constructed and sequenced. GLRaV-3 was more prevalent (compared to preclonal candidates) and was detected in three libraries. In addition to GLRaV-3, two viruses from the leafroll disease complex (GLRaV-1 and GLRaV-2) were identified. Other viruses with obligatory testing (GRSPaV, GFLV, GFkV) as well as GPGV, GRVFV and GV-Sat were also detected. GV-Sat is the first report in Slovenia, the second in Europe and the fourth in the world. We also developed a multiplex RT-PCR for validation of sRNA-seq data (viruses, viroids and satellites). In the first part of the dissertation, we studied the virome of 82 preclonal candidates of six grapevine varieties. 191 sequences were generated, genetic diversity and phylogenetic studies were performed. In the second part of the dissertation, the efficacy of virus and viroid elimination rates of six grapevine varieties was studied using in vivo thermotherapy and in vitro micrografting of meristem tips. In the third part of the dissertation, the virome of grapevine samples not involved in clonal selection was studied and a multiplex RT-PCR was developed for efficient validation of HTS predicted organisms. 77 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 4.2 POVZETEK V doktorski disertaciji smo raziskali virom vinske trte ( Vitis vinifera L.), na osnovi tehnologije visokozmogljivega sekvenciranja, proučevali genetsko raznolikost virusov in viroidov, njihovo filogenijo in sočasne okužbe. Poleg tega smo tudi razvili protokol za eliminacijo virusov in viroidov vinske trte in vivo s termoterapijo in in vitro z mikrograftingom meristemov ter razvili hkratni RT-PCR (multipleksni) za validacijo podatkov sRNA-seq (sekvenciranje malih RNA), za namen hitre, zanesljive in cenejše diagnostike. Vinska trta velja za eno najbolj razširjenih kulturnih rastlin na svetu (goji se na površinah, ki obsegajo 7,3 milijona hektarjev). Ena izmed glavnih ovir za trajnostno vinogradništvo so okužbe z virusi in z njimi povezana obolenja. Virusni patogeni so lahko latentni, a vendar lahko povzročijo tudi hude simptome in veliko gospodarsko izgubo. Zato je velikega pomena hitro in natančno odkrivanje virusov, karakterizacija ter uporaba metod za zatiranje virusov. Cilj prvega dela naše raziskave je bil raziskati virom predklonskih kandidatov v vinogradniški regiji Primorska, kjer se klonska selekcija izvaja že desetletja. Analizirali smo 82 vzorcev šestih sort vinske trte (2 rdeči in 4 bele sorte) z uporabo tehnologije sekvenciranja malih RNA. Izolirali smo male RNA iz 12 združenih vzorcev, v katerih so bili zbrani predklonski kandidati posamezne sorte, pripravili knjižnice in jim določili nukleotidno zaporedje s sistemom Ion Proton; v primeru, da smo imeli znotraj sorte veliko število predklonskih kandidatov, smo za isto sorto naredili dva ali tri združena vzorca. Za analizo podatkov smo uporabili sklop orodij programskega cevovoda VirusDetect, ki so prosto dostopna na spletu. Odkrili smo devet virusov: GFLV, GLRaV-3, GRSPaV, GFkV, GSyV-1, GRVFV, GRGV, GPGV, RBDV in dva viroida: HSVd in GYSVd-1 (Poglavje 2.1.1; Tabela 1). Trije virusi (GSyV-1, GRVFV, GRGV) v Sloveniji pred tem še niso bili odkriti. In silico rezultati so bili potrjeni z RT-PCR in sekvenciranjem po Sangerju. V nadaljevanju smo izolirali celokupno RNA tudi iz vseh posameznih vzorcev. Najprej smo celotno RNA reverzno prepisali in jo nato v reakciji PCR pomnožili s specifičnimi začetnimi oligonukleotidi, ki smo jih izbrali glede na sekvence pridobljene z sRNA-seq. Pomnožke RT-PCR smo analizirali z elektroforezo in jih po čiščenju (tretiranje z Exo-Sap) sekvencirali v obe smeri (sekvenciranje po Sangerju). Pridobljena zaporedja smo obrezali in sestavili, tako da smo dobili 191 sekvenc, ki smo jih analizirali. Vse generirane sekvence smo deponirali v NCBI pod sledečimi pristopnimi številkami: GSyV-1 (MW446939-MW446941), GRVFV (MW446917-MW446938), GRGV (MW446914-MW446916), GLRaV-3 (OK138920), GRSPaV (OK138921-OK138934), HSVd (OK138935-OK138974), GYSVd-1 (OK138975-OK139009), GFkV (OK139010-OK139034), GFLV (OK139035-OK139038), RBDV (OK139039-OK139042), GPGV (OK139043- OK139082). 78 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Virus GLRaV-3, pripadnik rodu Ampelovirus, smo odkrili le v knjižnici 010 (sorta 'Refošk') (Poglavje 2.1.1; Tabela 1). Preko aplikacij VirusDetect (BLASTN) smo zanj identificirali samo eno celotno sekvenco genoma (GQ352631), ki izhaja iz Južne Afrike. Naš genom se je z referenčnim genomom ujemal v 98,6 %, s 23 soseskami in 39,6 kratno (X) globino sekvenciranja. Opazili smo eno virusno sosesko, ki ustreza ORF2 (za katerega je znano, da ga v nekaterih izolatih ni). Predvideni virus smo potrdili z dvema paroma začetnih oligonukleotidov, ki sta pomnožila dve različni genomski regiji (CP in HSP70h). Ugotovili smo, da je bil z virusom GLRaV-3 okužen le en vzorec. Pred tem, so bili vsi vzorci testirani s testom ELISA na prisotnost virusov ki povzročajo zvijanje listov vinske trte, in vse okužene trte so bile izključene iz nadaljnjih analiz. To pojasnjuje, zakaj je bil GLRaV-3 odkrit samo v enem vzorcu in zakaj nismo zaznali drugih virusov ki so povezani z zvijanjem listov. Sekvencirali smo genomsko regijo CP vzorca Refošk 11/4P, pri čemer smo ugotovili 99,76 % nukleotidno podobnost z 18 sekvencami, ki izhajajo iz različnih celin. Vendar je filogenetska študija pokazala, da je naš izolat najbolj podoben izolatu s Portugalske (Poglavje 2.1.1; Slika 5 v prilogi). Virus RBDV smo odkrili v knjižnici 005 ('Laški rizling') (Poglavje 2.1.1; Tabela 1), kar je zanimivo, saj je bil v Sloveniji prvič odkrit pred dvajsetimi leti pri isti sorti. Pri sekvenciranju smo potrdili visoko pokritost segmentov RNA1 in RNA2. Čeprav celoten genom izolatov RBDV iz vinske trte še ni na razpolago, so sestavljeni soseski primerjani z izolatom J1 iz maline ( Rubus idaeus). RNA1 se je z referenco 100 % ujemal v 2 soseskah in 1556,2 X globino sekvenciranja; segment RNA2 se je ujemal v 98,7 % s 4 soseskami in 513,8 X globino sekvenciranja. Podobnost sekvenc združenih vzorcev glede na referenco RNA1 je bila 93,40 % in glede na RNA2 96,10 %. Vsi štirje vzorci iz knjižnice 005 so bili okuženi. Sekvencirali smo del gena MP in del gena CP. Sekvence gena MP naših izolatov so si 100 % podobne, medtem ko so imele sekvence regije CP 98,18–99,55 % podobnost (97,95–100 % aminoksilinska podobnost). Filogenetsko drevo smo narisali na podlagi dela gena CP dolgega 438 bp, pri čemer so bile sekvence izolatov iz vinske trte jasno ločene od sekvenc izolatov iz malin (Poglavje 2.1.1; Slika 1 v prilogi). Iz kompleksa virusov, ki povzročajo bolezen (kompleks bolezni) imenovano 'fleck' smo potrdili štiri viruse (GFkV, GSyV-1, GRVFV in GRGV) (Poglavje 2.1.1; Tabela 1), od tega so bili trije (GSyV-1, GRVFV in GRGV) v Sloveniji odkriti prvič. Virus marmoriranosti vinske trte (GFkV) smo odkrili v osmih knjižnicah (Poglavje 2.1.1; Tabela 1). V sedmih knjižnicah smo z orodjem VirusDetect na posamezno knjižnico identificirali 4-10 referenčnih nukleotidnih zaporedij. V knjižnici 011 (sorta 'Refošk') je bila s 33,3 % pokritostjo določena le ena delna sekvenca gena ki kodira replikazo. Pokritost celotnih referenčnih sekvenc je bila 76,9-82,1 %. Izjema sta bili dve knjižnici sorte 'Malvazija', katerih je bila pokritost sekvenc le 30,4 % oziroma 32,2 %. V vseh knjižnicah smo opazili veliko sosesk (36-60) s kratko dolžino. Z RT-PCR smo pomnožili del sekvence za replikazo virusa GFkV. Z reakcijo PCR smo namnožili 34 produktov, ki smo jih analizirali s sekvenciranjem po Sangerju, pri čemer je 7 produktov pripadalo virusu GRVFV. Dve 79 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 sekvenci sta bili slabše kakovosti in sta bili izključeni iz nadaljnje analize. Potrjenih pa je bilo petindvajset sekvenc GFkV, katerih nukleotidna podobnost je bila 91,6–100 % (93,7– 100 % aminokislinska podobnost). Filogenetska analiza, ki smo jo opravili na delu nukleotidnega zaporedja gena za replikazo, je 25 slovenskih izolatov in 18 referenčnih izolatov virusa iz GenBank, razvrstila v dve skupini. Slovenski izolati so bili prisotni v obeh skupinah. Zanimivo je, da so se, z izjemo sorte 'Laški rizling', izolati združili glede na sorto (Poglavje 2.1.1; Slika 6 v prilogi). Naši izolati so se združili z izolati iz sosednjih držav in iz ZDA, iz katerih so bile uvožene podlage in na katere so bili cepljene gostiteljske trte. Predvidevamo, da je do širjenja virusnih okužb prišlo preko razmnoževanega materiala in cepljenja na okužene podlage. Poleg tega je bilo veliko cepljenk nato iz Slovenije izvoženih v sosednje države. Med virusi, ki so bili prvič odkriti v Sloveniji, je bil virus GRVFV bolj razširjen kot druga dva. Virus GRVFV smo odkrili v 11 knjižnicah (Poglavje 2.1.1; Tabela 1). V posamezni knjižnici smo z orodjem VirusDetect identificirali od 5 do 25 popolnih in delnih referenčnih sekvenc, z izjemo knjižnice 013, kjer je bila identificirana samo ena delna referenca. Največja pokritost celotnega referenčnega genoma je bila v knjižnici 008 (KY513702; 85 %), medtem ko je bila pokritost delne referenčne sekvence v knjižnici 013 (MH544692) 30,2 %. Virus smo potrdili v vseh predvidenih knjižnicah. Vsem produktom RT-PCR (44) smo določili nukleotidno zaporedje (delni poliprotein), pri čemer je bilo 22 zaporedij nižje kakovosti, verjetno zaradi prisotnosti različnih genetskih variant v istem vzorcu. Za nadaljnje analize smo uporabili le tiste z visoko kakovostjo. Ugotovili smo visoko genetsko variabilnost med nukleotidnimi zaporedji (10,9 % ± 0,9 %), pri čemer so se razdelili v dve molekularni skupini z različnimi podskupinami. Virus GRGV je bil potrjen v dveh knjižnicah sorte 'Refošk' (006 in 010) in največja pokritost in identičnost je bila z izolati iz Španije; 63,6 % v knjižnici 006 (KX171166) in 45,8 % v knjižnici 010 (KX109927) (Poglavje 2.1.1; Tabela 1). GRGV smo potrdili v obeh knjižnicah, pri čemer so bili okuženi 3 vzorci. Zaradi relativno majhnega števila sekvenc, deponiranih v NCBI, nismo mogli sklepati o morebitni vnosu tega virusa v Slovenijo. V dveh knjižnicah smo odkrili tudi virus GSyV-1, in sicer v knjižnici 005 (sorta 'Laški rizling') in knjižnici 009 (sorta 'Malvazija'), vendar je bila pokritost genoma zelo nizka (Poglavje 2.1.1; Tabela 1). V knjižnici 005 je bila identificirana samo ena delna poliproteinska referenčna sekvenca (KP221269) z dolžino 334 nt, ki je imela pokritost 44,9 % s 3 kontigi in globino sekvenciranja 20,9 X, medtem ko so bile v knjižnici 009 določene štiri referenčne sekvence (3 popolne in 1 delna). Pri tem je največjo pokritost (48,8 %) imelo zaporedje iz Slovaške (KP221256). Virus GSyV-1 smo potrdili v obeh knjižnicah. Filogenetska analiza na osnovi dela gena CP je izolate združila v dve skupini, kjer so trije slovenski izolati pripadali glavni skupini, združenimi z izolati iz Madžarske in Slovaške. Naj še omenimo, da smo za validacijo virusov GRGV in GSyV-1 za vse knjižnice uporabili 40 ciklov pomnoževanja (za ostale viruse pa le 35 ciklov), kar sovpada z zelo nizko pokritostjo referenčnega genoma in zelo nizko globino sekvenciranja. Virus pahljačavosti listov vinske trte (GFLV) je bil potrjen v knjižnici 013 (sorta 'Zeleni Sauvignon') in 016 (sorta 'Pokalca') (Poglavje 2.1.1; Tabela 1). V knjižnici 013 smo 80 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 identificiranih 11 zaporedij segmenta RNA1. Poleg njih smo določili 4 popolne sekvence segmenta RNA2. V knjižnici 016 smo identificirali 7 popolnih in 1 delno sekvenco segmenta RNA1, v tej knjižnici smo določili tudi 16 popolnih sekvenc segmenta RNA2. Virus smo potrdili v obeh predvidenih knjižnicah. Pri pomnoževanju 606 bp dolge regije RNA2 z vključenim delom gena CP, smo dobili pozitiven rezultat pri 4 vzorci. Trije izolati sorte 'Pokalca' so imeli 99,67-99,84 % nukleotidno podobnost (99,5 ali 100 % aminokislinsko podobnost), medtem ko se je izolat Zeleni Sauvignon 16/3P močno razlikoval od izolatov 'Pokalca', s 87,27-87,44 % podobnostjo (96,49 ali 96,99 % aminokislinska podobnost). Poleg razlik med našimi izolati, smo potrdili tudi razlike z izolati, ki so dostopni v bazi podatkov NCBI. Zaradi velikih sekvenčnih razlik med našimi izolati in izolati iz NCBI baze podatkov smo oblikovali nov par začetnih oligonukleotidov, specifičen za zaporedja naših izolatov in z RT-PCR analizo potrdili okužbo pri dodatnih šestih vzorcih. Na osnovi filogenetske analize so se naši izolati in izolati iz baze podatkov NCBI razvrstila v dve skupini. Naši izolati so bili razvrščeni v glavno skupino in so bili bližje izolatom iz Italije in Francije, kakor že predhodno analiziranim izolatom iz Slovenije (sorta 'Volovnik') (Poglavje 2.1.1; Slika 4 v prilogi). Virus razbrazdanja lesa vinske trte (GRSPaV) je bil odkrit v desetih knjižnicah (Poglavje 2.1.1; Tabela 1). V osmih knjižnicah smo z orodjem VirusDetect identificirali od 4 do 40 popolnih in delnih referenčnih sekvenc (večinoma gen CP). Pokritost referenčnih sekvenc je bila 48,4-99,1 %, pri čemer smo dobili veliko število sosesk (41-86) in nizko globino pokritosti (5,3 X-16,6 X). V dveh knjižnicah, 012 (sorta 'Zeleni Sauvignon') in 015 (sorta 'Malvazija') smo identificirali samo delne in/ali popolne sekvence gena CP. V knjižnicah 007 (sorta 'Rebula') in 014 (sorta 'Malvazija') virusa nismo zaznali. Za potrditev rezultatov sRNA-seq smo izbrali par začetnih oligonukleotidov, ki pomnožujejo visoko ohranjeno regijo CP gena. Virus smo potrdili v vseh knjižnicah, kjer je bil predviden, in tudi v dveh knjižnicah, kjer ni bil predviden. Predvidevamo, da do tega ni prišlo zaradi nizke koncentracije in strategije sekvenciranja združenih vzorcev, ampak, da je razlog za to kompleksno ozadje biologije razmnoževanja virusa in okuževanja, kar zahteva nadaljnje študije. Skupno je bilo okuženih 88,61 % vzorcev (Poglavje 2.1.1; Slika 1). Direktno sekvenciranje produkta RT-PCR ni bilo možno izvesti, zato smo produkte reakcije RT-PCR ligirali v vektor in transformirali v kompetentne bakterijske celice. Skupno smo sekvencirali 14 produktov. Najvišja skupna povprečna genetska razdalja je bila odkrita med tremi različicami Laškega rizlinga 3/45B (17,14 %), najnižja pa za Malvazijo 20/48P (6,32 %). Povprečna skupna genetska razdalja med vsemi 14 sekvenciranimi različicami je bila 14,06 %. Pri sorti 'Laški rizling', kjer smo potrdili največjo pestrost je bilo preko VirusDetect aplikacije poravnanih z našimi zaporedij tudi največje število referenčnih sekvenc iz baze (40). Na osnovi filogenetske analize so bile genetske različice razvrščene v različne skupine, kljub temu, da so izvirale iz istega vzorca, prav tako se niso združili glede na geografski izvor (Poglavje 2.1.1; Slika 3 v prilogi). 81 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Virus GPGV je nedavno odkrit virus, ki je lahko latenten ali zelo škodljiv. Zaskrbljujoče je, da smo ta virus odkrili v vseh knjižnicah (95,3–100 % pokritost celotnih referenčnih sekvenc) (Poglavje 2.1.1; Tabela 1) in da je bilo z njim okuženih 72 vzorcev (91,14 %) (Poglavje 2.1.1; Slika 1). Genetsko raznolikost smo preučevali v 40 vzorcih zaporedja delov gena MP in CP. Podobnost nukleotidnega zaporedja gena MP je bila pri 40 slovenskih izolatih 93,94-100 % (87,79-100 % aminokislinska podobnost), podobnost gena CP pa 94,53-100 % (97,86-100 % aminokislinska podobnost). V 13 vzorcih smo ugotovili, da je na mestu 6,685 specifičen polimorfizem C/T, in polimorfno zaporedje kodira prezgodnji stop kodon, zato je v teh primerih gen MP 18 nt (6 ak) krajši. Pridobljeni podatki kažejo, da polimorfizem C/T ni odgovoren za spremembo izražanja simptomov oziroma, da na izražanje simptomov poleg tega polimorfizma vplivajo še nekateri drugi dejavniki. Sinergizem z različnimi virusi/viroidi prav tako ni odgovoren za nastanek simptomov, saj smo virus GPGV potrdili pri sočasni okužbi s samo enim viroidom (HSVd), kot tudi s kar petimi virusi (RBDV, GRSPaV, GFkV, GRVFV, GSyV-1) in dvema viroidoma (HSVd in GYSVd-1) in so bili vsi vzorci asimptomatični (Poglavje 2.1.1; Slika 2). Na osnovi filogenetske analize nukleotidnih zaporedij dela genov MP/CP so se naši izolati razdelili v dve skupini, čeprav so bili vsi asimptomatični. Izolati s krajšim genom MP so bili združeni v isto skupino, medtem ko so bili izolati z daljšim genom MP uvrščeni v obe skupini. Naši izolati so bili združeni predvsem s tistimi iz držav, ki so geografsko relativno blizu (Poglavje 2.1.1; Slika 2 v prilogi), kar lahko nakazuje, da se okužba širi z širjenjem okuženega sadilnega materiala, vendar visoka razširjenost kaže tudi na pomembno vlogo vektorjev. Testiranje na prisotnost virusa GPGV je samo priporočljivo, vendar ugotavljamo, da bi zaradi kompleksne epidemiologije in etiologije bolezni ter njenega vpliva na pridelavo vina ter velike razširjenosti in velikega števila latentnih oblik virusa, testiranje na virus GPGV moralo biti obvezno. Glede viroidov sta HSVd in GYSVd-1 razširjena po vsem svetu in sta edina znana viroida, ki se pojavljata na vinski trti v Sloveniji. Viroid HSVd smo odkrili v vseh knjižnicah, in sicer smo v posamezni knjižnici identificirali le eno referenčno sekvenco (Poglavje 2.1.1; Tabela 1). Napovedan viroid HSVd smo potrdili v vseh knjižnicah, pri čemer so bile z RT-PCR potrjene okužbe pri vseh vzorcih. Izbrali smo 40 produktov RT-PCR in jim določili zaporedje s sekvenciranjem po Sangerju. Zanimivo je, da je bilo 38 nukleotidnih zaporedij identičnih, medtem ko sta bili dve zaporedji (Pokalca 3/4P in Pokalca 3/6P) enaki in sta pokazali 98-odstotno podobnost z drugimi izolati (Poglavje 2.1.1; Slika 9 v prilogi). Glede na to, da smo viroid HSVd odkrili v vseh vzorcih in je latenten v vinski trti ter da je dokazano, da lahko izolati iz vinske trte okužujejo hmelj, je potrebno temu viroidu posvetiti veliko pozornost. Viroid GYSVd-1 smo z sRNA-seq metodo prav tako odkrili v vseh knjižnicah in tudi identificirali samo eno referenčno sekvenco na posamezno knjižnico (Poglavje 2.1.1; Tabela 1). Z RT-PCR smo potrdili prisotnost viroida GYSVd-1 v vseh knjižnicah, pri čemer je bilo pozitivnih 71 vzorcev (89,87 %) (Poglavje 2.1.1; Slika 1). 35 produktom RT-PCR smo 82 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 določili nukleotidna zaporedja s sekvenciranjem po Sangerju. Zaporedja so si bila 95,35-100 % podobna. Opazili smo tudi mutacije tipa indel na 4 mestih v genomu (63, 92, 163 in 287) (Poglavje 2.1.1; Slika 10 v prilogi). Na splošno je ta viroid genetsko bolj raznolik kot HSVd. Filogenetska analiza je naše izolate razvrstila v različne skupine, ne glede na sorto ali geografsko razširjenost (Poglavje 2.1.1; Slika 8 v prilogi). Na splošno smo dobili popoln vpogled v virom predklonskih kandidatov. Najmanj razširjen virus je bil GLRaV-3 (1,27 %), sledila sta mu GRGV in GSyV-1 (3,80 %), RBDV (5,06 %), GFLV (12,66 %), GFkV (34,18 %), GRVFV (55,70 %), GRSPaV (88,61 %), GYSVd-1 (89,87 %), GPGV (91,14 %) in HSVd (100 %) (Poglavje 2.1.1; Slika 1). Večina vzorcev je bila hkrati okužena s petimi, en vzorec (Laški rizling 3/45B) pa z osmimi virusnimi patogeni (Poglavje 2.1.1; Slika 3). Najpogostejše so bile sookužbe z GPGV, GRSPaV, HSVd in GYSVd-1 (18,99 %) (Poglavje 2.1.1; Slika 2). Cilj drugega dela naše študije je bil razviti protokol in preučiti učinkovitost eliminacije virusov in viroidov in vivo s termoterapijo ter in vitro z izolacijo mersitemov in mikrograftingom. V postopek eliminacije smo vključili 28 predklonskih kandidatov šestih različnih sort, ki so bili okuženi z napovedanimi in potrjenimi virusnimi patogeni, katere smo navedli v zgornjem odstavku (razen GRGV) (Poglavje 2.1.3; Tabela S1 v prilogi). Toplotno terapijo smo izvajali od 6 tednov do 3 mesece pri temperaturi 36-38 °C (Poglavje 2.1.3; Slika 6a). Po termoterapiji in vivo smo vzorčili apikalne in aksilarne segmente rastlin (Poglavje 2.1.3; Slika 6b), jih površinsko razkužili (z 1,66 % raztopino natrijevega dikloroizocianurata), meristeme (0,1-0,2 mm) aseptično izolirali pod stereomikroskopom (Poglavje 2.1.3; Slika 6c) ter jih takoj nacepili na etiolirane hipokotile vinske trte Vialla ( Vitis labrusca × Vitis riparia) (Poglavje 2.1.3; Slika 5c). Skupno smo izolirali in nacepili 598 meristemov, od skupno se je regeneriralo 51 rastlin (8,53 %) (Poglavje 2.1.3; Tabela 1). Da bi povečali njihovo število, smo regenerante večkrat mikropropagirali, pri čemer nismo nikoli opazili tvorjenja kalusa, vitrifikacije ali nekroze. Pri belih sortah je bila stopnja regeneracije večja kot pa pri rdečih (Poglavje 2.1.3; Tabela 1, Slika 2). Samo en vzorec bele sorte 'Laški rizling' (3/45B), ki je bil okužen z osmimi virusnimi patogeni in z vsaj tremi genetskimi različicami virusa GRSPaV, se ni regeneriral. Med rdečimi sortami se ni regeneriral vzorec 'Pokalca' (9/2G) in dva vzorca 'Refoška' (12/3P in 12/6P). Najvišjo stopnjo regeneracije je imela 'Rebula' (16,7 %), sledita 'Laški rizling' in 'Zeleni Sauvignon' (10,7 %) (Poglavje 2.1.3; Slika 2). Čeprav je imela 'Rebula' najvišjo stopnjo regeneracije, se je 'Zeleni Sauvignon' med mikropropagacijo veliko hitreje obnavljal in rasel. Najnižjo stopnjo regeneracije je imela 'Pokalca' (3,3 %) (Poglavje 2.1.3; Slika 2). Učinkovitost eliminacije virusov in viroidov iz regeneriranih rastlin, ki smo jih gojili 7 mesecev v in vitro pogojih, smo določili z RT-PCR. Za vse viruse je bila dosežena 100 % eliminacija. Kar pa ne drži za viroide, saj je bila eliminacija viroidov HSVd in GYSVd-1 bistveno nižja, in sicer 39,2 % za HSVd in 42,6 % za GYSVd-1 (Poglavje 2.1.3; Tabela 2), verjetno zato, ker visoka temperatur (termoterapija) spodbuja replikacijo in kopičenje viroidov. Rastline brez virusov, vzgojene v in vitro pogojih, so bile uspešno aklimatizirane v kockah iz kamene volne, ki so 83 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 se izkazale kot odličen substrat za rast in razvoj korenin (Poglavje 2.1.3; Slika 3). Rastline smo gojili v mini rastlinjaku v rastni komori, nato pa jih presadili v lončke (Poglavje 2.1.3; Slika 4). Vsi predklonski kandidati brez virusov bodo ponovno testirani po približno treh letih, preden bodo tudi uradno uveljavljeni kot certificirani kloni. Čeprav je ta metoda zahtevna, delovno intenzivna in dolgotrajna, ima veliko prednosti pred drugimi biotehnološkimi pristopi za eliminacijo virusov, kot je na primer kemoterapija, ki lahko povzroči hudo fitotoksičnost, ali tudi somatska embriogeneza, pri kateri obstaja veliko tveganje na pojav mutacij. V naši raziskavi smo zmanjšali tveganje za genetsko nestabilnost z in vivo termoterapijo (skrajšali čas gojenja in vitro), izolacijo meristema (brez tvorbe kalusa), mikrograftingom (pospešili regeneracijo) in uporabili gojišče, ki ne vsebuje regulatorjev rasti za rastline. Glede na to, da smo pri predklonskih kandidatih, ki niso imeli vidnih simptomov, odkrili devet virusov in dva viroida, je bil cilj tretjega dela naše študije, raziskati virom vzorcev, ki niso vključeni v programe klonske selekcije. Analizirali smo 13 vzorcev in pripravili ter sekvencirali štiri knjižnice (Poglavje 2.1.4; Tabela 2 in 3). Virus GLRaV-3 je bil bolj razširjen, saj smo ga odkrili v treh knjižnicah. Poleg GLRaV-3 sta bila identificirana še dva virusa iz kompleksa bolezni zvijanja listov vinske trte (GLRaV-1 in GLRaV-2). Odkrili smo tudi druge viruse, ki so na seznamu za obvezno testiranje (GRSPaV, GFLV, GFkV), pa tudi GPGV, GRVFV in GV-Sat. To je tudi prvo poročilo o virusu GV-Sat v Sloveniji, drugo v Evropi in četrto v svetu. Zaznali smo ga samo v kultivarju 'Cipro'. Najvišja pokritost (91,4 %) in podobnost na nukleotidnem nivoju (95,74 %) je bila z zaporedjem genoma ameriškega izolata AUD46129 (KC149510). V knjižnici 'Cipro' smo zaznali tudi viruse GLRaV-1, GLRaV-2, GRSPaV, GPGV, GRVFV in viroida HSVd ter GYSVd-1. Razvili smo tudi hkratni RT-PCR (multipleks) za validacijo podatkov sRNA-seq, ki vključuje različne kombinacije virusov, viroidov in satelitov, ki bi jih lahko uporabili za stroškovno učinkovito, visoko zmogljivo in hitro diagnostiko, kot je to potrebno pri analiziranju velikega števila vzorcev z mešanimi okužbami. Za mRT-PCR smo izbrali kombinacije začetnih oligonukleotidov, ki pomnožujejo fragmente različnih dolžin, kar omogoča določitev razlik na agaroznem gelu. Da bi lahko hkrati določili več različnih virusov in viroidov, smo za vzpostavitev najboljših pogojev PCR reakcije, optimizirali več parametrov, kot je koncentracija začetnih oligonukleotidov (0,04–0,2 µM), temperatura prileganja (55–60 °C), število ciklov (30–35) in količina vhodne cDNA (1 µL in 2 µL). Za najboljše so se izkazali naslednji pogoji: 0,08 µM koncentracija začetnih oligonukleotidov (0,04 µM samo za virus GRVFV), temperatura prileganja 58 °C, 35 ciklov in 1 µL cDNA. Različne kombinacije virusov smo hkrati pomnožili v vseh štirih knjižnicah: L1 (GLRaV-1, GLRaV-2, GRSPaV, GPGV, GRVFV in GV-Sat); L2 (GLRaV-3, GFLV, GRSPaV, GPGV in GRVFV); L3 (GLRaV-3, GRSPaV, GPGV, GFkV in GRVFV); L4 (GLRaV-3, GFLV, GRSPaV, GPGV, GFkV in GRVFV) (Poglavje 2.1.4; Slika 3). Poleg tega smo istočasno pomnožili tudi različne kombinacije viroidov/satGFLV: L1 in L3 (HSVd, GYSVd-1), L2 (satGFLV, HSVd), L4 (satGFLV, HSVd in GYSVd-1) (Poglavje 2.1.4; Slika 3). 84 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 V prvem delu disertacije smo dobili popoln vpogled v virom 82 predklonskih kandidatov šestih sort vinske trte. Pridobili smo veliko število sekvenc, opravili filogenetske analize in analize genetske raznolikosti. V drugem delu disertacije smo raziskali učinkovitost eliminacije virusov in viroidov pri šestih sortah vinske trte, kjer smo uporabili in vivo termoterapijo ter in vitro mikrografting meristemov. V tretjem delu disertacije smo preučili virom sort vinske trte, ki niso vključene v program klonske selekcije in razvili multipleks RT-PCR za učinkovito validacijo virusov, viroidov in satelitov, ki so bili napovedani na osnovi visoko zmogljivega sekvenciranja malih RNA. 85 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 5 REFERENCES Abou Ghanem-Sabanadzovic N., Sabanadzovic S., Martelli G. P. 2003. Sequence Analysis of the 3′ end of Three Grapevine fleck virus-like viruses from Grapevine. Virus Genes, 27: 11–16 Ahmadi G., Hajizadeh M., Roumi V. 2017. A multiplex RT-PCR for simultaneous detection of the agents of yellow speckle and vein banding diseases in grapevine. Journal of Plant Pathology, 99, 1: 261-266 Alabi O. J., Casassa L. F., Gutha L. R., Larsen R. C., Henick-Kling T., Harbertson J. F., Naidu R. A. 2016. Impacts of Grapevine Leafroll Disease on Fruit Yield and Grape and Wine Chemistry in a Wine Grape ( Vitis vinifera L.) cultivar. PLoS ONE, 11, 2: e0149666, doi: 10.1371/journal.pone.0149666: 18 p. Alabi O. J., Martin R. R., Naidu R. A. 2010. Sequence diversity, population genetics and potential recombination events in grapevine rupestris stem pitting-associated virus in Pacific North-West vineyards. Journal of General Virology, 91: 265–276 Alkowni R., Zhang Y. P., Rowhani A., Uyemoto J. K., Minafra A. 2011. Biological, molecular, and serological studies of a novel strain of grapevine leafroll-associated virus 2. Virus Genes, 43: 102–110 Aloisio M., Morelli M., Elicio V., Saldarelli P., Ura B., Bortot B., Severini G. M., Minafra A. 2018. Detection of four regulated grapevine viruses in a qualitative, single tube real-time PCR with melting curve analysis. Journal of Virological Methods, 257: 42-47 Al Rwahnih M., Daubert S., Golino D., Rowhani A. 2009. Deep sequencing analysis of RNAs from a grapevine showing Syrah decline symptoms reveals a multiple virus infection that includes a novel virus. Virology, 387, 2: 395–401 Al Rwahnih M., Diaz-Lara A., Arnold K., Cooper M. L., Smith R. J., Zhuang G., Battany M. C., Bettiga L. J., Rowhani A., Golino D. 2021. Incidence and Genetic Diversity of Grapevine Pinot gris Virus in California. American Journal of Enology and Viticulture, 72, 2: 164-169 Al Rwahnih M., Daubert S., Sudarshana M. R., Rowhani A. 2013. Gene from a novel plant virus satellite from grapevine identifies a viral satellite lineage. Virus Genes, 47: 114– 118 Andret-Link P., Laporte C., Valat L., Ritzenthaler C., Demangeat G., Vigne E., Laval V., Pfeiffer P., Stussi-Garaud C., Fuchs M. 2004a. Grapevine fanleaf virus: Still a major threat to the grapevine industry. Journal of Plant Pathology, 86, 3: 183–195 Andret-Link P., Schmitt-Keichinger C., Demangeat G., Komar V., Fuchs M. 2004b. The specific transmission of Grapevine fanleaf virus by its nematode vector Xiphinema index is solely determined by the viral coat protein. Virology, 320, 1: 12–22 Astruc N., Marcos J. F., Macquaire G., Candresse T., Pallás V. 1996. Studies on the diagnosis of hop stunt viroid in fruit trees: Identification of new hosts and application of a nucleic acid extraction procedure based on non-organic solvents. European Journal of Plant Pathology, 102: 837–846 86 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Bartel D. P. 2004. MicroRNAs: Genomics, Biogenesis, Mechanism, and Function. Cell, 116, 2: 281–297 Baulcombe D. 2004. RNA silencing in plants. Nature, 431: 356–363 Bernstein E., Caudy A. A., Hammond S. M., Hannon G. J. 2001. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature, 409: 363–366 Bertazzon N., Filippin L., Forte V., Angelini E. 2016. Grapevine Pinot gris virus seems to have recently been introduced to vineyards in Veneto, Italy. Archives of Virology, 161: 711–714 Bertazzon N., Forte V., Filippin L., Causin R., Maixner M., Angelini E. 2017. Association between genetic variability and titre of Grapevine Pinot gris virus with disease symptoms. Plant Pathology, 66, 6: 949–959 Bester R., Maree H. J., Burger J. T. 2012. Complete nucleotide sequence of a new strain of grapevine leafroll-associated virus 3 in South Africa. Archives of Virology, 157: 1815– 1819 Bettoni J. C., Costa M. D., Gardin J. P. P., Kretzschmar A. A., Pathirana R. 2016. Cryotherapy: a new technique to obtain grapevine plants free of viruses. Revista Brasileira de Fruticultura, 38, 2: e-833, doi: 10.1590/0100-29452016833: 13 p. Beuve M., Moury B., Spilmont A. S., Sempé-Ignatovic L., Hemmer C., Lemaire O. 2013. Viral sanitary status of declining grapevine Syrah clones and genetic diversity of Grapevine Rupestris stem pitting-associated virus. European Journal of Plant Pathology, 135: 439–452 Bi W. L., Hao X. Y., Cui Z. H., Pathirana R., Volk G. M., Wang Q. C., 2018. Shoot tip cryotherapy for efficient eradication of grapevine leafroll-associated virus-3 from diseased grapevine in vitro plants. Annals of Applied Biology, 173, 3: 261–270 Bianchi G. L., De Amicis F., De Sabbata L., Di Bernardo N., Governatori G., Nonino F., Prete G., Marrazzo T., Versolatto S., Frausin C. 2015. Occurrence of Grapevine Pinot gris virus in Friuli Venezia Giulia (Italy): field monitoring and virus quantification by real-time RT-PCR. Bulletin OEPP/EPPO Bulletin, 45, 1: 22–32 Bota J., Cretazzo E., Montero R., Rosselló J., Cifre J. 2014. Grapevine fleck virus (GFkV) elimination in a selected clone of Vitis vinifera L. cv. Manto Negro and its effects on photosynthesis. Journal International des Sciences de la Vigne et du Vin, 48, 1: 11–19 Bouamama-Gzara B., Selmi I., Chebil S., Melki I., Mliki A., Ghorbel A., Carra A., Carimi F., Mahfoudhi N. 2017. Elimination of Grapevine leafroll associated virus-3, Grapevine rupestris stem pitting associated virus and Grapevine virus A from a Tunisian Cultivar by Somatic Embryogenesis and Characterization of the Somaclones Using Ampelographic Descriptors. Plant Pathology Journal, 33, 6: 561–571 Bouyahia H., Boscia D., Savino V., La Notte P., Pirolo C., Castellano M. A., Minafra A., Martelli G. P. 2005. Grapevine rupestris stem pitting-associated virus is linked with grapevine vein necrosis. Vitis, 44, 3: 133–137 Burger J. T., Maree H. J., Gouveia P., Naidu R. A. 2017. Grapevine leafroll-associated virus 3. In: Grapevine Viruses: Molecular Biology, Diagnostics and Management. Meng B., Martelli G. P., Golino D. A., Fuchs M. (eds.). Springer: 167–195 87 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Cadman C. H., Dias H. F., Harrison B. D. 1960. Sap-Transmissible viruses associated with diseases of grape vines in Europe and North America. Nature, 187, 4737: 577–579 Caglayan K., Gazel M., Kocabag H. D. 2017. First report of Grapevine Syrah virus 1 in grapevine in Turkey. Journal of Plant Pathology, 99, 1: 303 Candresse T., Marais A., Theil S., Faure C., Lacombe T., Boursiquot J. M. 2017. Complete Nucleotide Sequence of an Isolate of Grapevine Satellite Virus and Evidence for the Presence of Multimeric Forms in an Infected Grapevine. Genome Announcements, 5, 16: e01703-16, doi: 10.1128/genomeA.01703-16: 2 p. Chellappan P., Vanitharani R., Ogbe F., Fauquet C. M. 2005. Effect of Temperature on Geminivirus-Induced RNA Silencing in Plants. Plant Physiology, 138, 4: 1828–1841 Cheon J. Y., Fenton M., Gjerdseth E., Wang Q., Gao S., Krovetz H., Lu L., Shim L., Williams N., Lybbert T. J. 2020. Heterogeneous benefits of virus screening for grapevines in California. American Journal of Enology and Viticulture, 71, 3: 231-241 Chiaki Y., Ito T. 2020. Complete genome sequence of a novel putative polerovirus detected in grapevine. Archives of Virology, 165: 1007–1010 Cho I. S., Jung S. M., Cho J. D., Choi G. S., Lim H. S. 2013. First report of Grapevine Pinot gris virus infecting grapevine in Korea. New Disease Reports, 27: 10 Chuche J., Thiéry D. 2014. Biology and ecology of the Flavescence dorée vector Scaphoideus titanus: a review. Agronomy for Sustainable Development, 34: 381–403 Clark M. F., Adams A. N. 1977. Characteristics of the Microplate Method of Enzyme-Linked Immunosorbent Assay for the Detection of Plant Viruses. Journal of General Virology, 34, 3: 475-483 Cogotzi L., Giampetruzzi A., Nölke G., Orecchia M., Elicio V., Castellano M. A., Martelli G. P., Fischer R., Schillberg S., Saldarelli P. 2009. An assay for the detection of grapevine leafroll-associated virus 3 using a single-chain fragment variable antibody. Archives of Virology, 154: 19–26 Cretazzo E., Velasco L. 2017. High-throughput sequencing allowed the completion of the genome of grapevine Red Globe virus and revealed recurring co-infection with other tymoviruses in grapevine. Plant Pathology, 66, 7: 1202–1213 Crnogorac A., Panno S., Mandic A., Gašpar M., Caruso A. G., Noris E., Davino S., Matić S. 2021. Survey of five major grapevine viruses infecting Blatina and Žilavka cultivars in Bosnia and Herzegovina. PLoS ONE, 16, 1: e0245959. doi: 10.1371/journal.pone.0245959: 20 p. Czotter N., Molnar J., Szabó E., Demian E., Kontra L., Baksa I., Szittya G., Kocsis L., Deak T., Bisztray G., Tusnady G. E., Burgyan J., Varallyay E. 2018. NGS of Virus-Derived Small RNAs as a Diagnostic Method Used to Determine Viromes of Hungarian Vineyards. Frontiers in Microbiology, 9, 122, doi: 10.3389/fmicb.2018.00122: 13 p. Czotter N., Szabó E., Molnar J., Kocsis L., Deák T., Bisztray G., Tusnády G. E., Burgyán J., Várallyay E. 2015. First description of Grapevine Syrah virus 1 in vineyards of Hungary. Journal of Plant Pathology, 97: 74 88 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Čarija M., Radić T., Černi S., Mucalo A., Zdunić G., Vončina D., Jagunić M., Hančević K. 2022. Prevalence of Virus Infections and GLRaV-3 Genetic Diversity in Selected Clones of Croatian Indigenous Grapevine Cultivar Plavac Mali. Pathogens, 11, 2: 176, doi: 10.3390/pathogens11020176: 13 p. Čepin U., Gutiérrez-Aguirre I., Balažic L., Pompe-Novak M., Gruden K., Ravnikar M. 2010. A one-step reverse transcription real-time PCR assay for the detection and quantitation of Grapevine fanleaf virus. Journal of Virological Methods, 170, 1-2: 47–56 Čepin U., Gutiérrez-Aguirre I., Ravnikar M., Pompe-Novak M. 2016. Frequency of occurrence and genetic variability of Grapevine fanleaf virus satellite RNA. Plant Pathology, 65, 3: 510-520 Demangeat G., Komar V., Van-Ghelder C., Voisin R., Lemaire O., Esmenjaud D., Fuchs M. 2010. Transmission Competency of Single-Female Xiphinema index Lines for Grapevine fanleaf virus. Phytopathology, 100, 4: 384–389 Demian E., Jaksa-Czotter N., Molnar J., Tusnady G. E., Kocsis L., Varallyay E. 2020. Grapevine rootstocks can be a source of infection with non-regulated viruses. European Journal of Plant Pathology, 156: 897–912 Diaz-Lara A., Golino D., Al Rwahnih M. 2018. Genomic characterization of grapevine virus J, a novel virus identified in grapevine. Archives of Virology, 163: 1965–1967 Digiaro M., Elbeaino T., Martelli G. P. 2007. Development of degenerate and species-specific primers for the differential and simultaneous RT-PCR detection of grapevine-infecting nepoviruses of subgroups A, B and C. Journal of Virological Methods, 141, 1: 34-40 Digiaro M., Elbeaino T., Martelli G. P. 2017. Grapevine fanleaf virus and Other Old World Nepoviruses. In: Grapevine Viruses: Molecular Biology, Diagnostics and Management. Meng B., Martelli G. P., Golino D. A., Fuchs M. (eds.). Springer: 47–82 Di Serio F., Izadpanah K., Hajizadeh M., Navarro B. 2017. Viroids Infecting the Grapevine. In: Grapevine Viruses: Molecular Biology, Diagnostics and Management. Meng B., Martelli G. P., Golino D. A., Fuchs M. (eds.). Springer: 373–392 Duran-Vila N., Juárez J., Arregui J. M. 1988. Production of Viroid-Free Grapevines by Shoot Tip Culture. American Journal of Enology and Viticulture, 39: 217–220 Eichmeier A., Kominkova M., Pecenka J., Kominek P. 2019. High-throughput small RNA sequencing for evaluation of grapevine sanitation efficacy. Journal of Virological Methods, 267: 66–70 Eichmeier A., Peňázová E., Muljukina N. 2018. Survey of Grapevine Pinot gris virus in certified grapevine stocks in Ukraine. European Journal of Plant Pathology, 152: 555– 560 Eichmeier A., Pieczonka K., Peňázová E., Pečenka J., Gajewski Z. 2017. Occurrence of Grapevine Pinot gris virus in Poland and description of asymptomatic exhibitions in grapevines. Journal of Plant Diseases and Protection, 124: 407–411 Elbeaino T., Kiyi H., Boutarfa R., Minafra A., Martelli G. P., Digiaro M. 2014. Phylogenetic and recombination analysis of the homing protein domain of grapevine fanleaf virus 89 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 (GFLV) isolates associated with ‘yellow mosaic’ and ‘infectious malformation’ syndromes in grapevine. Archives of Virology, 159: 2757–2764 Elbeaino T., Kubaa R. A., Choueiri E., Digiaro M., Navarro B. 2012. Occurrence of Hop Stunt Viroid in Mulberry ( Morus alba) in Lebanon and Italy. Journal of Phytopathology, 160, 1: 48–51 El Beaino T., Sabanadzovic S., Digiaro M., Abou-Ghanem-Sabanadzovic N., Rowhani A., Kyriakopoulou P. E., Martelli G. P. 2001. Molecular detection of Grapevine fleck virus-like viruses. Vitis, 40, 2: 65–68 Elleuch A., Hamdi I., Ellouze O., Ghrab M., Fkahfakh H., Drira N. 2013. Pistachio ( Pistacia vera L.) is a new natural host of Hop stunt viroid. Virus Genes, 47: 330–337 Engel E. A., Escobar P. F., Rojas L. A., Rivera P. A., Fiore N., Valenzuela, P. D. T. 2010. A diagnostic oligonucleotide microarray for simultaneous detection of grapevine viruses. Journal of Virological Methods, 163, 2: 445–451 Fajardo T. V. M., Dianese É. C., Eiras M., Cerqueira D. M., Lopes D. B., Ferreira M. A. S. V., Martins, C. R. F. 2007. Variability of the coat protein gene of Grapevine leafroll-associated virus 3 in Brazil. Fitopatologia Brasileira, 32, 4: 335–340 Fajardo T., V. M., Eiras M., Nickel O. 2016. Detection and molecular characterization of Grapevine yellow speckle viroid 1 isolates infecting grapevines in Brazil. Tropical Plant Pathology, 41: 246–253 Fajardo T. V. M., Silva F. N., Eiras M., Nickel O. 2017. High-throughput sequencing applied for the identification of viruses infecting grapevines in Brazil and genetic variability analysis. Tropical Plant Pathology, 42: 250–260 Fan X., Hong N., Dong Y., Ma Y., Zhang Z. P., Ren F., Hu G., Zhou J., Wang G. 2015. Genetic diversity and recombination analysis of grapevine leafroll-associated virus 1 from China. Archives of Virology, 160: 1669–1678 Fan X., Zhang Z., Li C., Ren F., Hu G., Zhang B., Dong Y. 2021. High-Throughput Sequencing Indicates a Novel Marafivirus in Grapevine Showing Vein-Clearing Symptoms. Plants, 10, 7: 1487, doi: 10.3390/plants10071487: 10 p. Farooq A. B. U., Ma Y. X., Wang Z., Zhuo N., Wenxing X., Wang G. P., Hong N. 2013. Genetic diversity analyses reveal novel recombination events in Grapevine leafroll-associated virus 3 in China. Virus Research, 171, 1: 15–21 Fattouch S., Acheche H., M’Hirsi S., Mellouli L., Bejar S., Marrakchi M., Marzouki N. 2005. RT-PCR-RFLP for genetic diversity analysis of Tunisian Grapevine fanleaf virus isolates in their natural host plants. Journal of Virological Methods, 127, 2: 126–132 Fattouch S., M’Hirsi S., Acheche H., Marrakchi M., Marzouki N. 2001. RNA Oligoprobe capture RT-PCR, a sensitive method for the detection of Grapevine fanleaf virus in Tunisian grapevines. Plant Molecular Biology Reporter, 19: 235–244 Fattouh F., Ratti C., El Ahwany A. M. D., Abdel Aleem E., Babini A. R., Rubies Autonell C. 2014. Detection and molecular characterization of Egyptian isolates of grapevine viruses. Acta Virologica, 58, 2: 137–145 90 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Fei F., Lyu M. D., Li J., Fan Z. F., Cheng Y. Q. 2013. Complete nucleotide sequence of a Chinese isolate of Grapevine leafroll-associated virus 3 reveals a 5′ UTR of 802 nucleotides. Virus Genes, 46: 182–185 Fiore N., Prodan S., Montealegre J., Aballay E., Pino A. M., Zamorano A. 2008. Survey of grapevine viruses in Chile. Journal of Plant Pathology, 90, 1: 125-130 Fuchs M. 2020. Grapevine viruses: a multitude of diverse species with simple but overall poorly adopted management solutions in the vineyard. Journal of Plant Pathology, 102: 643–653 Fuchs M., Martinson T. E., Loeb G. M., Hoch H. C. 2009. Survey for the Three Major Leafroll Disease-Associated Viruses in Finger Lakes Vineyards in New York. Plant Disease, 93, 4: 395–401 Fuchs M., Pinck M., Serghini M. A., Ravelonandro M., Walter B., Pinck L. 1989. The Nucleotide Sequence of Satellite RNA in Grapevine Fanleaf Virus, strain F13. Journal of General Virology, 70, 4: 955-962 Gambino G. 2015. Multiplex RT-PCR Method for the Simultaneous Detection of Nine Grapevine Viruses. In: Plant Virology Protocols. Methods in Molecular Biology. Uyeda I., Masuta C. (eds.). Humana Press, New York, NY: 39-47 Gambino G., Bondaz J., Gribaudo I. 2006. Detection and Elimination of Viruses in Callus, Somatic Embryos and Regenerated Plantlets of Grapevine. European Journal of Plant Pathology, 114: 397–404 Gambino G., Cuozzo D., Fasoli M., Pagliarani C., Vitali M., Boccacci P., Pezzotti M., Mannini F. 2012. Co-evolution between Grapevine rupestris stem pitting-associated virus and Vitis vinifera L. leads to decreased defence responses and increased transcription of genes related to photosynthesis. Journal of Experimental Botany, 63, 16: 5919–5933 Gambino G., Gribaudo I. 2006. Simultaneous Detection of Nine Grapevine Viruses by Multiplex Reverse Transcription-Polymerase Chain Reaction with Coamplification of a Plant RNA as Internal Control. Phytopathology, 96, 11: 1223-1229 Gambino G., Di Matteo D., Gribaudo I. 2009. Elimination of Grapevine fanleaf virus from three Vitis vinifera cultivars by somatic embryogenesis. European Journal of Plant Pathology, 123: 57–60 Gambino G., Navarro B., Vallania R., Gribaudo I., Di Serio F. 2011. Somatic embryogenesis efficiently eliminates viroid infections from grapevines. European Journal of Plant Pathology, 130: 511–519 Gazel M., Caglayan K., Elçi E., Öztürk L. 2016. First report of Grapevine Pinot Gris virus in Grapevine in Turkey. Plant Disease, 100, 3: 657 Giampetruzzi A., Roumi V., Roberto R., Malossini U., Yoshikawa N., La Notte P., Terlizzi F., Credi R., Saldarelli P. 2012. A new grapevine virus discovered by deep sequencing of virus- and viroid-derived small RNAs in Cv Pinot gris. Virus Research, 163, 1: 262–268 Glasa M., Predajňa L., Komínek P., Nagyová A., Candresse T., Olmos A. 2014. Molecular characterization of divergent grapevine Pinot gris virus isolates and their detection in Slovak and Czech grapevines. Archives of Virology, 159: 2103–2107 91 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Glasa M., Predajňa L., Šoltys K., Sihelská N., Nagyová A., Wetzel T., Sabanadzovic S. 2017. Analysis of Grapevine rupestris stem pitting-associated virus in Slovakia Reveals Differences in Intra-Host Population Diversity and Naturally Occurring Recombination Events. Plant Pathology Journal, 33, 1: 34–42 Glasa M., Predajňa L., Šoltys K., Sabanadzovic S., Olmos A. 2015. Detection and molecular characterisation of Grapevine Syrah virus-1 isolates from Central Europe. Virus Genes, 51: 112–121 Glasa M., Predajňa L., Wetzel T., Rheinpfalz D. L. R., Šoltys K., Sabanadzovic S. 2019. First Report of Grapevine Rupestris Vein Feathering Virus in Grapevine in Slovakia. Plant Disease, 103, 1: 170 Goldsmith C. S., Miller S. E. 2009. Modern Uses of Electron Microscopy for Detection of Viruses. Clinical Microbiology Reviews, 22, 4: 552-563 Gottula J., Lapato D., Cantilina K., Saito S., Bartlett B., Fuchs M. 2013. Genetic Variability, Evolution, and Biological Effects of Grapevine fanleaf virus Satellite RNAs. Phytopathology, 103, 11: 1180–1187 Goussard P. G., Wiid J. 1992. The Elimination of Fanleaf Virus from Grapevines Using in vitro Somatic Embryogenesis Combined with Heat Therapy. South African Journal of Enology and Viticulture, 13, 2: 81-83 Gouveia P., Santos M. T., Eiras-Dias J. E., Nolasco G. 2011. Five phylogenetic groups identified in the coat protein gene of grapevine leafroll-associated virus 3 obtained from Portuguese grapevine varieties. Archives of Virology, 156: 413-420 Gribaudo I., Gambino G., Cuozzo D., Mannini F. 2006. Attempts to eliminate grapevine rupestris stem pitting-associated virus from grapevine clones. Journal of Plant Pathology, 88, 3: 293–298 Grout B. W. W. 1999. Meristem-Tip Culture for Propagation and Virus Elimination. In: Plant Cell Culture Protocols. Methods in Molecular Biology. Hall R. D. (ed.). Humana Press: 115–125 Gualandri V., Asquini E., Bianchedi P., Covelli L., Brilli M., Malossini U., Bragagna P., Saldarelli P., Si-Ammour A. 2017. Identification of herbaceous hosts of the Grapevine Pinot gris virus (GPGV). European Journal of Plant Pathology, 147: 21–25 Gualandri V., Bianchedi P., Morelli M., Giampetruzzi A., Valenzano P., Bottalico G., Campanale A., Saldarelli P. 2015. Production of Grapevine Pinot gris virus-free germplasm: techniques and tools. In: Proceedings of the 18th Congress of the International Council for the Study of Virus and Virus-like Diseases of the Grapevine (ICVG), Ankara, Turkey: 246-247 Guţa I. C., Buciumeanu E. C., Gheorghe R. N., Teodorescu A. 2010. Solutions to eliminate grapevine leafroll associated virus serotype 1+3 from V. vinifera L. cv. Ranâi Magaraci. Romanian Biotechnological Letters, 15, 1: 72–78 Guţa I. C., Buciumeanu E. C., Tataru L. D., Oprescu B., Topala C. M. 2019. New approach of electrotherapy for grapevine virus elimination. Acta Horticulturae, 1242: 697–702 Guţa I. C., Buciumeanu E. C., Tataru L. D., Topala C. M. 2017. Regeneration of grapevine virus-free plants by in vitro chemotherapy. Acta Horticulturae, 1188: 319–322 92 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Hajizadeh M., Navarro B., Bashir N. S., Torchetti E. M., Di Serio F. 2012. Development and validation of a multiplex RT-PCR method for the simultaneous detection of five grapevine viroids. Journal of Virological Methods, 179, 1: 62-69 Hajizadeh M., Torchetti E. M., Sokhandan-Bashir N., Navarro B., Doulati-Baneh H., Martelli G. P., Di Serio F. 2015. Grapevine viroids and grapevine fanleaf virus in North-West Iran. Journal of Plant Pathology, 97, 2: 363–368 Hammond S. M., Boettcher S., Caudy A. A., Kobayashi R., Hannon G. J. 2001. Argonaute2, a Link Between Genetic and Biochemical Analyses of RNAi. Science, 293, 5532: 1146-1150 Hančević K., Saldarelli P., Čarija M., Černi S., Zdunić G., Mucalo A., Radić T. 2021. Predominance and Diversity of GLRaV-3 in Native Vines of Mediterranean Croatia. Plants, 10, 1: 17, doi: 10.3390/plants10010017: 14 p. Hewitt W. B., Raski J. D., Goheen A. C. 1958. Nematode vector of soil-borne fanleaf virus of grapevines. Phytopathology, 48, 11: 586–595 Hily J. M., Candresse T., Garcia S., Vigne E., Tannière M., Komar V., Barnabé G., Alliaume A., Gilg S., Hommay G., Beuve M., Marais A., Lemaire O. 2018. High-Throughput Sequencing and the Viromic Study of Grapevine Leaves: From the Detection of Grapevine-Infecting Viruses to the Description of a New Environmental Tymovirales member. Frontiers in Microbiology, 9, 1782, doi: 10.3389/fmicb.2018.01782: 16 p. Hily J. M., Poulicard N., Candresse T., Vigne E., Beuve M., Renault L., Velt A., Spilmont A. S., Lemaire O. 2020. Datamining, Genetic Diversity Analyses, and Phylogeographic Reconstructions Redefine the Worldwide Evolutionary History of Grapevine Pinot gris virus and Grapevine berry inner necrosis virus. Phytobiomes Journal, 4, 2: 165–177 Horvath J., Tobias I., Hunyadi K. 1994. New natural herbaceous hosts of grapevine fanleaf nepovirus. Kertészeti Tudomány, 26, 1: 31–32 Hrček L. (1977). Vinogradništvo. Ampelografija. II. del. Ljubljana: VTOZD Agronomski oddelek, 130 p. Hu G., Dong Y., Zhang Z., Fan X., Ren F., Li Z., Zhang S. 2018. Elimination of Grapevine rupestris stem pitting-associated virus from Vitis vinifera ‘Kyoho’ by an antiviral agent combined with shoot tip culture. Scientia Horticulturae, 229: 99–106 Hu G., Dong Y., Zhang Z., Fan X., Ren F. 2020. Efficiency of chemotherapy combined with thermotherapy for eliminating grapevine leafroll-associated virus 3 (GLRaV-3). Scientia Horticulturae, 271: 109462, doi: 10.1016/j.scienta.2020.109462: 5 p. Hu G., Dong Y., Zhang Z., Fan X., Ren F. 2021. Elimination of grapevine fleck virus and grapevine rupestris stem pitting-associated virus from Vitis vinifera 87-1 by ribavirin combined with thermotherapy. Journal of Integrative Agriculture, 20, 9: 2463–2470 Hussain G., Wani M. S., Mir M. A., Rather Z. A., Bhat K. M. 2014. Micrografting for fruit crop improvement. African Journal of Biotechnology, 13, 25: 2474-2483 Izadpanah K., Zaki-Aghl M., Zhang Y. P., Daubert S. D., Rowhani A. 2003. Bermuda Grass as a Potential Reservoir Host for Grapevine fanleaf virus. Plant Disease, 87, 10: 1179– 1182 93 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Jevremovic D., Paunovic S. 2011. Raspberry bushy dwarf virus-A grapevine pathogen in Serbia. Pesticides and Phytomedicine, 26, 1: 55–60 Jonard R., Hugard J., Macheix J. J., Martinez J., Mosella-Chancel L., Poessel J. L., Villemur P. 1983. In vitro micrografting and its applications to fruit science. Scientia Horticulturae, 20, 2: 147–159 Jooste A. E. C., Maree H. J., Bellstedt D. U., Goszczynski D. E., Pietersen G., Burger J. T. 2010. Three genetic grapevine leafroll-associated virus 3 variants identified from South African vineyards show high variability in their 5′UTR. Archives of Virology, 155: 1997-2006 Kawaguchi-Ito Y., Li S. F., Tagawa M., Araki H., Goshono M., Yamamoto S., Tanaka M., Narita M., Tanaka K., Liu S. Y., Shikata E., Sano T. 2009. Cultivated Grapevines Represent a Symptomless Reservoir for the Transmission of Hop Stunt Viroid to Hop Crops: 15 Years of Evolutionary Analysis. PLoS ONE, 4, 12: e8386, doi: 10.1371/journal.pone.0008386: 13 p. Khraiwesh B., Zhu J. K., Zhu J. 2012. Role of miRNAs and siRNAs in biotic and abiotic stress responses of plants. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 1819, 2: 137-148 Kim M. Y., Cho K. H., Chun J. A., Park S. J., Kim S. H., Lee H. C. 2017. Elimination of Grapevine fleck virus from infected grapevines 'Kyoho' through meristem-tip culture of dormant buds. Journal of Plant Biotechnology, 44, 4: 401–408 Kim Y., Kim Y. J., Paek K. H. 2021. Temperature-specific vsiRNA confers RNAi-mediated viral resistance at elevated temperature in Capsicum annuum. Journal of Experimental Botany, 72, 4: 1432–1448 Koltunow A. M., Krake L. R., Johnson S. D., Rezaian M. A. 1989. Two Related Viroids Cause Grapevine Yellow Speckle Disease Independently. Journal of General Virology, 70, 12: 3411–3419 Komínek P. 2009. Distribution of grapevine viruses in vineyards of the Czech Republic. Journal of Plant Pathology, 90, 2: 357–358 Komínek P., Komínková M., Jandová B. 2016. Effect of repeated Ribavirin treatment on grapevine viruses. Acta Virologica, 60, 4: 400–403 Komínková M., Komínek P. 2020. Development and validation of RT-PCR multiplex detection of grapevine viruses and viroids in the Czech Republic. Journal of Plant Pathology, 102: 511-515 Koolivand D., Sokhandan-Bashir N., Behjatnia S. A. A., Jafari Joozani R. A. 2014. Detection of Grapevine fanleaf virus by immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) with recombinant antibody. Archives of Phytopathology and Plant Protection, 47, 17: 2070–2077 Křižan B., Ondrušiková E., Holleinová V., Moravcová K., Bláhová L. 2009. Elimination of Grapevine fanleaf virus in Grapevine by in vivo and in vitro thermotherapy. Horticultural Science, 36, 3: 105–108 94 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Kumar S., Baranwal V. K., Singh P., Jain R. K., Sawant S. D., Singh S. K. 2012. Letter to the Editor: Characterization of a Grapevine leafroll-associated virus 3 from India showing incongruence in its phylogeny. Virus Genes, 45: 195-200 Kumar S., Rai R., Baranwal V. K. 2015. Development of an immunocapture–reverse transcription–polymerase chain reaction (IC-RT-PCR) using modified viral RNA release protocol for the detection of Grapevine leafroll-associated virus 3 (GLRaV-3). Phytoparasitica, 43: 311–316 Lamprecht R. L., Spaltman M., Stephan D., Wetzel T., Burger J. T. 2013. Complete Nucleotide Sequence of a South African Isolate of Grapevine Fanleaf Virus and Its Associated Satellite RNA. Viruses, 5, 7: 1815–1823 Lee J., Martin R. R. 2009. Influence of grapevine leafroll associated viruses (GLRaV-2 and -3) on the fruit composition of Oregon Vitis vinifera L. cv. Pinot noir: Phenolics. Food Chemistry, 112, 4: 889–896 Lehad A., Selmi I., Louanchi M., Aitouada M., Mahfoudhi N. 2015. Genetic diversity of grapevine leafroll-associated virus 3 in Algeria. Journal of Plant Pathology, 97, 1: 203-207 Li H., Wei L., Qin C., Cheng J., Zhang X., Chen W., Ali T., Yu Z., Zhang P., Wu J., Shi N. 2021. Characterization of viruses and viroids in Vitis vinifera ‘Kyoho’ in Hangzhou China by small RNA deep sequencing and molecular detection. The Journal of Horticultural Science and Biotechnology, 96, 3: 400–406 Liebenberg A., Freeborough M. J., Visser C. J., Bellstedt D. U., Burger J. T. 2009. Genetic variability within the coat protein gene of Grapevine fanleaf virus isolates from South Africa and the evaluation of RT-PCR, DAS-ELISA and ImmunoStrips as virus diagnostic assays. Virus Research, 142, 1-2: 28–35 Lima M., Alkowni R., Uyemoto J. K., Rowhani A. 2009. Genomic study and detection of a new variant of grapevine rupestris stem pitting associated virus in declining California Pinot noir grapevines. Journal of Plant Pathology, 91, 1: 155-162 Lima M. F., Rosa C., Golino D. A., Rowhani A. 2006. Detection of Rupestris stem pitting associated virus in seedlings of virus-infected maternal grapevine plants. In: 15th Meeting of the International Council for the Study of Virus and Virus-like Diseases of the Grapevine (ICGV), Stellenbosch, South Africa: 244–245 Liu M. H., Li M. J., Qi H. H., Guo R., Liu X. M., Wang Q., Cheng Y. Q. 2013. Occurrence of Grapevine Leafroll-Associated Viruses in China. Plant Disease, 97, 10: 1339–1345 Liu J., Zhang X. J., Yang Y. K., Hong N., Wang G. P., Wang A., Wang L. P. 2016. Characterization of virus-derived small interfering RNAs in Apple stem grooving virus- infected in vitro-cultured Pyrus pyrifolia shoot tips in response to high temperature treatment. Virology Journal, 13, 166, doi: 10.1186/s12985-016-0625-0: 11 p. Liu J., Zhang X. J., Zhang F. P., Hong N., Wang G. P., Wang A., Wang L. P. 2015. Identification and characterization of microRNAs from in vitro-grown pear shoots infected with Apple stem grooving virus in response to high temperature using small RNA sequencing. BMC Genomics, 16, 945, doi: 10.1186/s12864-015-2126-8: 16 p. 95 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 López-Fabuel I., Wetzel T., Bertolini E., Bassler A., Vidal E., Torres L. B., Yuste A., Olmos A. 2013. Real-time multiplex RT-PCR for the simultaneous detection of the five main grapevine viruses. Journal of Virological Methods, 188, 1-2: 21-24 Mahfoudhi N., Digiaro M., Dhouibi M. H. 2009. Transmission of Grapevine Leafroll Viruses by Planococcus ficus (Hemiptera: Pseudococcidae) and Ceroplastes rusci (Hemiptera: Coccidae). Plant Disease, 93, 10: 999–1002 Malagnini V., de Lillo E., Saldarelli P., Beber R., Duso C., Raiola A., Zanotelli L., Valenzano D., Giampetruzzi A., Morelli M., Ratti C., Causin R., Gualandri V. 2016. Transmission of grapevine Pinot gris virus by Colomerus vitis (Acari: Eriophyidae) to grapevine. Archives of Virology, 161: 2595–2599 Maliogka V. I., Dovas C. I., Lotos L., Efthimiou K., Katis N. I. 2009a. Complete genome analysis and immunodetection of a member of a novel virus species belonging to the genus Ampelovirus. Archives of Virology, 154: 209–218 Maliogka V. I., Skiada F. G., Eleftheriou E. P., Katis N. I. 2009b. Elimination of a new ampelovirus (GLRaV-Pr) and Grapevine rupestris stem pitting associated virus (GRSPaV) from two Vitis vinifera cultivars combining in vitro thermotherapy with shoot tip culture. Scientia Horticulturae, 123, 2: 280–282 Maliogka V. I., Olmos A., Pappi P. G., Lotos L., Efthimiou K., Grammatikaki G., Candresse T., Katis N. I., Avgelis A. D. 2015. A novel grapevine badnavirus is associated with the Roditis leaf discoloration disease. Virus Research, 203: 47-55 Maree H. J., Almeida R. P. P., Bester R., Chooi K. M., Cohen D., Dolja V. V., Fuchs M. F., Golino D. A., Jooste A. E. C., Martelli G. P., Naidu R. A., Rowhani A., Saldarelli P., Burger J. T. 2013. Grapevine leafroll-associated virus 3. Frontiers in Microbiology, 4, 82, doi: 10.3389/fmicb.2013.00082: 21 p. Maree H. J., Freeborough M. J., Burger J. T. 2008. Complete nucleotide sequence of a South African isolate of grapevine leafroll-associated virus 3 reveals a 5′UTR of 737 nucleotides. Archives of Virology, 153: 755–757 Marquez-Molins J., Gomez G., Pallas V. 2021. Hop stunt viroid: A polyphagous pathogenic RNA that has shed light on viroid–host interactions. Molecular Plant Pathology, 22, 2: 153–162 Marra M., Giampetruzzi A., Abou Kubaa R., de Lillo E., Saldarelli P. 2020. Grapevine Pinot gris virus variants in vines with chlorotic mottling and leaf deformation. Journal of Plant Pathology, 102: 531 Martelli G. P. 1993. Grapevine degeneration - fanleaf. In: Graft-transmissible diseases of grapevines. Handbook for detection and diagnosis. Martelli G. P. (ed.). International Council for the Study of Viruses and Virus Diseases of the Grapevine. Food and Agriculture Organization of the United Nations: 9-18 Martelli G. P. 2014. Directory of Virus and Virus-like Diseases of the Grapevine and their Agents. Journal of Plant Pathology, 96: 1-136 Martelli G. P. 2017. An Overview on Grapevine Viruses, Viroids, and the Diseases They Cause. In: Grapevine Viruses: Molecular Biology, Diagnostics and Management. Meng B., Martelli G. P., Golino D. A., Fuchs M. (eds.). Springer: 31–46 96 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Martelli G. P., Abou Ghanem-Sabanadzovic N., Agranovsky A. A., Al Rwahnih M., Dolja V. V., Dovas C. I., Fuchs M., Gugerli P., Hu J. S., Jelkmann W., Katis N. I., Maliogka V. I., Melzer M. J., Menzel W., Minafra A., Rott M. E., Rowhani A., Sabanadzovic S., Saldarelli P. 2012. Taxonomic revision of the family Closteroviridae with special reference to the grapevine leafroll-associated members of the genus Ampelovirus and the putative species unassigned to the family. Journal of Plant Pathology, 94, 1: 7–19 Martelli G. P., Jelkmann W. 1998. Foveavirus, a new plant virus genus. Archives of Virology, 143: 1245–1249 Martelli G. P., Sabanadzovic S., Abou-Ghanem Sabanadzovic N., Edwards M. C., Dreher T. 2002. The family Tymoviridae. Archives of Virology, 147: 1837–1846 Martelli G. P., Savino V., Walter B. 1993. Indexing on Vitis indicators. In: Graft-transmissible diseases of grapevines: Handbook for detection and diagnosis. Martelli G. P. (ed.). International Council for the Study of Viruses and Virus Diseases of the Grapevine. Food and Agriculture Organization of the United Nations: 137–156 Massart S., Candresse T., Gil J., Lacomme C., Predajna L., Ravnikar M., Reynard J. S., Rumbou A., Saldarelli P., Škorić D., Vainio E. J., Valkonen J. P. T., Vanderschuren H., Varveri C., Wetzel T. 2017. A Framework for the Evaluation of Biosecurity, Commercial, Regulatory, and Scientific Impacts of Plant Viruses and Viroids Identified by NGS Technologies. Frontiers in Microbiology, 8, 45, doi: 10.3389/fmicb.2017.00045: 7 p. Mathew L., Tiffin H., Erridge Z., McLachlan A., Hunter D., Pathirana R. 2021. Efficiency of eradication of Raspberry bushy dwarf virus from infected raspberry ( Rubus idaeus) by in vitro chemotherapy, thermotherapy and cryotherapy and their combinations. Plant Cell, Tissue and Organ Culture, 144: 133–141 Mavrič I., Viršček Marn M., Koron D., Žežlina I. 2003. First Report of Raspberry bushy dwarf virus on Red Raspberry and Grapevine in Slovenia. Plant Disease, 87, 9: 1148 Mavrič Pleško I., Lamovšek J., Lešnik A., Viršček Marn M. 2020. Raspberry bushy dwarf virus in Slovenia - geographic distribution, genetic diversity and population structure. European Journal of Plant Pathology, 158: 1033–1042 Mavrič Pleško I., Viršček Marn M., Seljak G., Žežlina I. 2014. First Report of Grapevine Pinot gris virus Infecting Grapevine in Slovenia. Plant Disease, 98, 7: 1014 Mavrič Pleško I., Viršček Marn M., Širca S., Urek G. 2009. Biological, serological and molecular characterisation of Raspberry bushy dwarf virus from grapevine and its detection in the nematode Longidorus juvenilis. European Journal of Plant Pathology, 123: 261–268 Mavrič Pleško I., Višček Marn M., Nyerges K., Lázár J. 2012. First Report of Raspberry bushy dwarf virus Infecting Grapevine in Hungary. Plant Disease, 96, 10: 1582 Mayo M. A., Jolly C. A., Murant A. F., Raschke J. H. 1991. Nucleotide sequence of raspberry bushy dwarf virus RNA-3. Journal of General Virology, 72, 2: 469–472 Meng B., Pang S. Z., Forsline P. L., McFerson J. R., Gonsalves D. 1998. Nucleotide sequence and genome structure of grapevine rupestris stem pitting associated virus-1 reveal similarities to apple stem pitting virus. Journal of General Virology, 79, 8: 2059– 2069 97 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Meng B., Rebelo A. R., Fisher H. 2006. Genetic diversity analyses of grapevine Rupestris stem pitting-associated virus reveal distinct population structures in scion versus rootstock varieties. Journal of General Virology, 87, 6: 1725–1733 Meng B., Rowhani A. 2017. Grapevine rupestris stem pitting-associated virus. In: Grapevine Viruses: Molecular Biology, Diagnostics and Management. Meng B., Martelli G. P., Golino D. A., Fuchs M. (eds.). Springer: 257–287 Meng B., Zhu H. Y., Gonsalves D. 1999. Rupestris stem pitting associated virus-1 consists of a family of sequence variants. Archives of Virology, 144: 2071–2085 Morán F., Olmos A., Lotos L., Predajňa L., Katis N., Glasa M., Maliogka V., Ruiz-García A. B. 2018. A novel specific duplex real-time RT-PCR method for absolute quantitation of Grapevine Pinot gris virus in plant material and single mites. PLoS ONE, 13, 5: e0197237, doi: 10.1371/journal.pone.0197237: 14 p. Murant A. F., Chambers J., Jones A. T. 1974. Spread of raspberry bushy dwarf virus by pollination, its association with crumbly fruit, and problems of control. Annals of Applied Biology, 77, 3: 271–281 Naidu R., Rowhani A., Fuchs M., Golino D., Martelli G. P. 2014. Grapevine Leafroll: A Complex Viral Disease Affecting a High-Value Fruit Crop. Plant Disease, 98, 9: 1172– 1185 Naraghi-Arani P., Daubert S., Rowhani A. 2001. Quasispecies nature of the genome of Grapevine fanleaf virus. Journal of General Virology, 82, 7: 1791–1795 Nassuth A., Pollari E., Helmeczy K., Stewart S., Kofalvi S. A. 2000. Improved RNA extraction and one-tube RT-PCR assay for simultaneous detection of control plant RNA plus several viruses in plant extracts. Journal of Virological Methods, 90, 1: 37-49 Natsuaki T., Mayo M. A., Jolly C. A., Murant A. F. 1991. Nucleotide sequence of raspberry bushy dwarf virus RNA-2: A bicistronic component of a bipartite genome. Journal of General Virology, 72, 9: 2183–2189 Navarro B., Pantaleo V., Gisel A., Moxon S., Dalmay T., Bisztray G., Di Serio F., Burgyán J. 2009. Deep Sequencing of Viroid-Derived Small RNAs from Grapevine Provides New Insights on the Role of RNA Silencing in Plant-Viroid Interaction. PLoS ONE, 4, 11: e7686, doi: 10.1371/journal.pone.0007686: 12 p. Navrotskaya E., Porotikova E., Yurchenko E., Galbacs Z. N., Varallyay E., Vinogradova S. 2021. High-Throughput Sequencing of Small RNAs for Diagnostics of Grapevine Viruses and Viroids in Russia. Viruses, 13, 12: 2432, doi: 10.3390/v13122432: 19 p. Nolasco G., Mansinho A., Teixeira Santos M., Soares C., Sequeira Z., Sequeira C., Correia P.K., Sequeira O.A. 2000. Large Scale Evaluation of Primers for Diagnosis of Rupestris Stem Pitting Associated Virus-1. European Journal of Plant Pathology, 106: 311–318 Nolasco G., Santos C., Petrovic N., Teixeira Santos M., Cortez I., Fonseca F., Boben J., Nazaré Pereira A. M., Sequeira O. 2006. Rupestris stem pitting associated virus isolates are composed by mixtures of genomic variants which share a highly conserved coat protein. Archives of Virology, 151: 83-96 98 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Osman F., Hodzic E., Omanska-Klusek A., Olineka T., Rowhani A. 2013. Development and validation of a multiplex quantitative PCR assay for the rapid detection of Grapevine virus A, B and D. Journal of Virological Methods, 194, 1-2: 138-145 Osman F., Leutenegger C., Golino D., Rowhani A. 2007. Real-time RT-PCR (TaqMan®) assays for the detection of Grapevine Leafroll associated viruses 1-5 and 9. Journal of Virological Methods, 141, 1: 22–29 Panattoni A., Luvisi A., Triolo E. 2011. Selective chemotherapy on Grapevine leafroll-associated virus-1 and -3. Phytoparasitica, 39: 503–508 Panattoni A., Luvisi A., Triolo E. 2013. Review. Elimination of viruses in plants: Twenty years of progress. Spanish Journal of Agricultural Research, 11, 1: 173–188 Panattoni A., Triolo E. 2010. Susceptibility of grapevine viruses to thermotherapy on in vitro collection of Kober 5BB. Scientia Horticulturae, 125, 1: 63–67 Panno S., Caruso A. G., Bertacca S., Pisciotta A., Lorenzo R. D., Marchione S., Matić S., Davino S. 2021. Genetic Structure and Molecular Variability of Grapevine Fanleaf Virus in Sicily. Agriculture, 11, 6: 496, doi: 10.3390/agriculture11060496: 16 p. Parker J. S., Barford D. 2006. Argonaute: a scaffold for the function of short regulatory RNAs. Trends in Biochemical Sciences, 31, 11: 622–630 Petrovic N., Meng B., Ravnikar M., Mavric I., Gonsalves D. 2003. First Detection of Rupestris stem pitting associated virus Particles by Antibody to a Recombinant Coat Protein. Plant Disease, 87, 5: 510–514 Pinck L., Fuchs M., Pinck M., Ravelonandro M., Walter B. 1988. A Satellite RNA in Grapevine Fanleaf Virus Strain F13. Journal of General Virology, 69, 1: 233-239 Pirovano W., Miozzi L., Boetzer M., Pantaleo V. 2014. Bioinformatics approaches for viral metagenomics in plants using short RNAs: Model case of study and application to a Cicer arietinum population. Frontiers in Microbiology, 5, 790, doi: 10.3389/fmicb.2014.00790: 13 p. Polivka H., Staub U., Gross H. J. 1996. Variation of viroid profiles in individual grapevine plants: Novel grapevine yellow speckle viroid 1 mutants show alterations of hairpin I. Journal of General Virology, 77, 1: 155–161 Pompe-Novak M., Gutiérrez-Aguirre I., Vojvoda J., Blas M., Tomažič I., Vigne E., Fuchs M., Ravnikar M., Petrovič N. 2007. Genetic variability within RNA2 of Grapevine fanleaf virus. European Journal of Plant Pathology, 117: 307–312 Porotikova E., Terehova U., Volodin V., Yurchenko E., Vinogradova S. 2021. Distribution and Genetic Diversity of Grapevine Viruses in Russia. Plants, 10, 6: 1080, doi: 10.3390/plants10061080: 14 p. Qu F., Ye X., Hou G., Sato S., Clemente T. E., Morris T. J. 2005. RDR6 Has a Broad-Spectrum but Temperature-Dependent Antiviral Defense Role in Nicotiana benthamiana. Journal of Virology, 79, 24: 15209–15217 Radisek S., Majer A., Jakse J., Javornik B., Matoušek J. 2012. First Report of Hop stunt viroid Infecting Hop in Slovenia. Plant Disease, 96, 4: 592 99 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Rai R., Sharma S. K., Kumar P. V., Baranwal V. K. 2021. Evidence of novel genetic variants of Grapevine rupestris stem pitting-associated virus and intra-host diversity in Indian grapevine cultivars. Tropical Plant Pathology, 46: 576-580 Read D. A., Thompson G. D., Le Cordeur N., Swanevelder D., Pietersen G. 2022. Genomic characterization of grapevine viruses N and O: novel vitiviruses from South Africa. Archives of Virology, 167: 611-614 Reynard J. S. 2015. Survey of emerging viruses in Switzerland. In: Proceedings of the 18th Congress of the International Council for the Study of Virus and Virus-like Diseases of the Grapevine (ICVG), Ankara, Turkey: 223–224 Reynolds A. G. 2017. The Grapevine, Viticulture, and Winemaking: A brief Introduction. In: Grapevine Viruses: Molecular Biology, Diagnostics and Management. Meng B., Martelli G. P., Golino D. A., Fuchs M. (eds.). Springer: 3–29 Reynolds A. G., Lanterman W. S., Wardle D. A. 1997. Yield and Berry Composition of Five Vitis Cultivars as Affected by Rupestris Stem Pitting Virus. American Journal of Enology and Viticulture, 48, 4: 449–458 Ruiz-García A. B., Sabaté J., Lloria O., Laviña A., Batlle A., Olmos A. 2017. First Report of Grapevine Syrah virus-1 in Grapevine in Spain. Plant Disease, 101, 10: 1830 Sabanadzovic S., Abou-Ghanem N., Castellano M. A., Digiaro M., Martelli G. P. 2000. Grapevine fleck virus-like viruses in Vitis. Archives of Virology, 145: 553–565 Sabanadzovic S., Aboughanem-Sabanadzovic N., Martelli G. P. 2017. Grapevine fleck and similar viruses. In: Grapevine Viruses: Molecular Biology, Diagnostics and Management. Meng B., Martelli G. P., Golino D. A., Fuchs M. (eds.). Springer: 331–349 Sabanadzovic S., Abou Ghanem-Sabanadzovic N., Gorbalenya A. E. 2009. Permutation of the active site of putative RNA-dependent RNA polymerase in a newly identified species of plant alpha-like virus. Virology, 394, 1: 1–7 Salami S. A., Ebadi A., Zamani Z., Habibi M. K. 2009. Incidence of Grapevine Fanleaf Virus in Iran: A survey Study and Production of Virus-Free Material Using Meristem Culture and Thermotherapy. European Journal of Horticultural Science, 74, 1: 42–46 Saldarelli P., Giampetruzzi A., Morelli M., Malossini U., Pirolo C., Bianchedi P., Gualandri V. 2015. Genetic Variability of Grapevine Pinot gris virus and Its Association with Grapevine Leaf Mottling and Deformation. Phytopathology, 105, 4: 555–563 Saldarelli P., Gualandri V., Malossini U., Glasa M. 2017. Grapevine Pinot gris virus. In: Grapevine Viruses: Molecular Biology, Diagnostics and Management. Meng B., Martelli G. P., Golino D. A., Fuchs M. (eds.). Springer: 351–363 Saldarelli P., Minafra A., Castellano M. A., Martelli G. P. 2000. Immunodetection and subcellular localization of the proteins encoded by ORF 3 of grapevine viruses A and B. Archives of Virology, 145: 1535–1542 Sano T., Hataya T., Terai Y., Shikata E. 1989. Hop Stunt Viroid Strains from Dapple Fruit Disease of Plum and Peach in Japan. Journal of General Virology, 70, 6: 1311–1319 100 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Sano T., Mimura R., Ohshima K. 2001. Phylogenetic Analysis of Hop and Grapevine Isolates of Hop Stunt Viroid Supports a Grapevine Origin for Hop Stunt Disease. Virus Genes, 22: 53–59 Sano T., Uyeda I., Shikata E., Meshi T., Ohno T., Okada Y. 1985. A viroid-like RNA Isolated from Grapevine has High Sequence Homology with Hop Stunt Viroid. Journal of General Virology, 66, 2: 333–338 Sasaki M., Shikata E. 1977. On Some Properties of Hop Stunt Disease Agent, a Viroid. Proceedings of the Japan Academy Series B, 53: 109–112 Scagliusi S. M. M., Vega J., Kuniyuki H. 2002. Cytopathology of callus cells infected with grapevine leafroll-associated virus 3. Fitopatologia Brasileira, 27, 4: 384–388 Sharma A. M., Wang J., Duffy S., Zhang S., Wong M. K., Rashed A., Cooper M. L., Daane K. M., Almeida R. P. P. 2011. Occurrence of Grapevine Leafroll-Associated Virus Complex in Napa Valley. PLoS ONE, 6, 10: e26227, doi: 10.1371/journal.pone.0026227: 7 p. Shi B. J., Habili N., Symons R. H. 2003. Nucleotide sequence variation in a small region of the Grapevine fleck virus replicase provides evidence for two sequence variants of the virus. Annals of Applied Biology, 142, 3: 349–355 Shvets D., Vinogradova S. 2022. Occurrence and Genetic Characterization of Grapevine Pinot Gris Virus in Russia. Plants, 11, 8: 1061, doi: 10.3390/plants11081061: 15 p. Skiada F. G., Maliogka V. I., Katis N. I., Eleftheriou E. P. 2013. Elimination of Grapevine rupestris stem pitting-associated virus (GRSPaV) from two Vitis vinifera cultivars by in vitro chemotherapy. European Journal of Plant Pathology, 135: 407–414 Spilmont A. S., Ruiz A., Grenan S. 2012. Efficiency of Micrografting of Shoot Apices as a Sanitation Method Against Seven Grapevine Viruses (ArMV, GFLV, GLRaV-1, -2, -3, GFkV, GVA). In: Proceedings of the 17th Congress of the International Council for the Study of Virus and Virus-like Diseases of the Grapevine (ICVG), Davis, California, USA: 270–271 Sudarshana M. R., Perry K. L., Fuchs M. F. 2015. Grapevine Red Blotch-Associated Virus, an Emerging Threat to the Grapevine Industry. Phytopathology, 105, 7: 1026–1032 Szittya G., Silhavy D., Molnár A., Havelda Z., Lovas A., Lakatos L., Bánfalvi Z., Burgyán J. 2003. Low temperature inhibits RNA silencing-mediated defence by the control of siRNA generation. The EMBO Journal, 22, 3: 633–640 Tarquini G., Ermacora P., Bianchi G. L., De Amicis F., Pagliari L., Martini M., Loschi A., Saldarelli P., Loi N., Musetti R. 2018. Localization and subcellular association of Grapevine Pinot Gris Virus in grapevine leaf tissues. Protoplasma, 255: 923–935 Terlizzi F., Li C., Ratti C., Qiu W., Credi R., Meng B. 2011. Detection of multiple sequence variants of Grapevine rupestris stem pitting-associated virus using primers targeting the polymerase domain and partial genome sequencing of a novel variant. Annals of Applied Biology, 159, 3: 478-490 Terlizzi F., Ratti C., Filippini G., Pisi A., Credi R. 2010. Detection and molecular characterization of Italian Grapevine rupestris stem pitting-associated virus isolates. Plant Pathology, 59, 1: 48-58 101 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Theiler‐Hedtrich R., Baumann G. 1989. Elimination of Apple Mosaic Virus and Raspberry Bushy Dwarf Virus from Infected Red Raspberry ( Rubus idaeus L.) by Tissue Culture. Journal of Phytopathology, 127, 3: 193–199 Tsai C. W., Rowhani A., Golino D. A., Daane K. M., Almeida R. P. P. 2010. Mealybug Transmission of Grapevine Leafroll Viruses: An Analysis of Virus-Vector Specificity. Phytopathology, 100, 8: 830-834 Turcsan M., Demian E., Varga T., Jaksa-Czotter N., Szegedi E., Olah R., Varallyay E. 2020. HTS-Based Monitoring of the Efficiency of Somatic Embryogenesis and Meristem Cultures Used for Virus Elimination in Grapevine. Plants 9, 12: 1782, doi: 10.3390/plants91217821–10: 10 p. Turturo C., Saldarelli P., Yafeng D., Digiaro M., Minafra A., Savino V., Martelli G. P. 2005. Genetic variability and population structure of Grapevine leafroll-associated virus 3 isolates. Journal of General Virology, 86, 1: 217–224 Valasevich N., Kukharchyk N., Kvarnheden A. 2011. Molecular characterisation of Raspberry bushy dwarf virus isolates from Sweden and belarus. Archives of Virology, 156: 369–374 Vega A., Gutiérrez R. A., Peña-Neira A., Cramer G. R., Arce-Johnson P. 2011. Compatible GLRaV-3 viral infections affect berry ripening decreasing sugar accumulation and anthocyanin biosynthesis in Vitis vinifera. Plant Molecular Biology, 77: 261–274 Velázquez K., Renovell A., Comellas M., Serra P., García M. L., Pina J. A., Navarro L., Moreno P., Guerri J. 2010. Effect of temperature on RNA silencing of a negative-stranded RNA plant virus: Citrus psorosis virus. Plant Pathology, 59, 5: 982–990 Verdel A., Jia S., Gerber S., Sugiyama T., Gygi S., Grewal S. I. S., Moazed D. 2004. RNAi-Mediated Targeting of Heterochromatin by the RITS Complex. Science, 303, 5658: 672-676 Vončina D., Badurina D., Preiner D., Vjetkovic B., Maletic E., Kontic J. K. 2011. Incidence of virus infections in grapevines from Croatian collection plantations. Phytopathologia Mediterranea, 50, 2: 316-326 Vončina D., Al Rwahnih M., Rowhani A., Gouran M., Almeida R. P. P. 2017. Viral Diversity in Autochthonous Croatian Grapevine Cultivars. Plant Disease, 101, 7: 1230– 1235 Walsh H. A., Pietersen G. 2013. Rapid detection of Grapevine leafroll-associated virus type 3 using a reverse transcription loop-mediated amplification method. Journal of Virological Methods, 194, 1-2: 308–316 Wang Q., Cuellar W. J., Rajamäki M. L., Hirata Y., Valkonen J. P. T. 2008. Combined thermotherapy and cryotherapy for efficient virus eradication: Relation of virus distribution, subcellular changes, cell survival and viral RNA degradation in shoot tips. Molecular Plant Pathology, 9, 2: 237–250 Wang J., Sharma A. M., Duffy S., Almeida R. P. P. 2011. Genetic Diversity in the 3′ Terminal 4.7-kb Region of Grapevine leafroll-associated virus 3. Phytopathology, 101, 4: 445-450 102 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Ward L. I., Burnip G. M., Liefting L. W., Harper S. J., Clover G. R. G. 2011. First Report of Grapevine yellow speckle viroid 1 and Hop stunt viroid in Grapevine ( Vitis vinifera) in New Zealand. Plant Disease, 95, 5: 617 Weiland C. M., Superior E. P., Cantos M., Troncoso A., Perez-Camacho F. 2004. Regeneration of virus-free plants by in vitro chemotherapy of GFLV ( Grapevine fanleaf virus) infected explants of Vitis vinifera L. cv 'Zalema'. Acta Horticulturae, 652: 463–466 Wen J., Lu L. M., Nie Z. L., Liu X. Q., Zhang N., Ickert-Bond S., Gerrath J., Manchester S. R., Boggan J., Chen Z. D. 2018. A new phylogenetic tribal classification of the grape family (Vitaceae). Journal of Systematics and Evolution, 56, 4: 262-272 Wetzel T., Jardak R., Meunier L., Ghorbel A., Reustle G. M., Krczal G. 2002. Simultaneous RT/PCR detection and differentiation of arabis mosaic and grapevine fanleaf nepoviruses in grapevines with a single pair of primers. Journal of Virological Methods, 101, 1-2: 63– 69 Wood N. T., McGavin W. J., Mayo M. A., Jones A. T. 2001. Studies on a putative second gene in RNA-1 of Raspberry bushy dwarf virus. Acta Horticulturae, 551: 19–22 Wu Q., Ding S. W., Zhang Y., Zhu S. 2015. Identification of Viruses and Viroids by Next-Generation Sequencing and Homology-Dependent and Homology-Independent Algorithms. Annual Review of Phytopathology, 53: 425-444 Yakoubi S., Elleuch A., Besaies N., Marrakchi M., 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 Youssef S. A., Al-Dhaher M. M. A., Shalaby A. A. 2009. Elimination of Grapevine fanleaf virus (GFLV) and Grapevine leaf roll-associated virus-1 (GLRaV-1) from infected grapevine plants using meristem tip culture. International Journal of Virology, 5, 2: 89– 99 Zaki-Aghl M., Izadpanah K., Gholampour Z., Kargar M., Mehrvar M. 2015. Molecular characterization of grapevine fan leaf virus from non Vitis hosts. In: Proceedings of the 18th Congress of the International Council for the Study of Virus and Virus-like Diseases of the Grapevine (ICVG), Ankara, Turkey: 149–150 Zhang B., Liu G. Y., Liu C., Wu Z., Jiang D., Li S. 2009. Characterisation of Hop stunt viroid (HSVd) isolates from jujube trees ( Ziziphus jujuba). European Journal of Plant Pathology, 125: 665–669 Zhang Y. P., Uyemoto J. K., Golino D. A., Rowhani A. 1998. Nucleotide Sequence and RT-PCR Detection of a Virus Associated with Grapevine Rupestris Stem-Pitting Disease. Phytopathology, 88, 11: 1231–1237 Ziegler A., Natsuaki T., Mayo M. A., Jolly C. A., Murant A. F. 1992. The nucleotide sequence of RNA-1 of raspberry bushy dwarf virus. Journal of General Virology, 73, 12: 3213–3218 Zindović J., Viršček Marn M., Mavrič Pleško I. 2014. Phytosanitary status of grapevine in Montenegro. Bulletin OEPP/EPPO Bulletin, 44, 1: 60-64 103 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 ACKNOWLEDGEMENTS First and foremost, I would like to thank my supervisor Prof. dr. Nataša Štajner. I am very grateful for the patient support, help, knowledge, comments and suggestions and for all the opportunities I was given during my research. I am also very grateful to Prof. dr. Jernej Jakše for his knowledge and help in acquiring and refining a range of laboratory and bioinformatics skills. I would like to express gratitude to my great colleagues from the Department of Genetics, Biotechnology, Statistics and Plant Breeding, especially for making me feel like I was at home. I am also very grateful for the wonderful collaboration and working atmosphere, the great support and the indispensable morning coffee. In the end, I am very happy to say that they have become my second family, my Slovenian family. My special thanks go to Prof. dr. Jelena Zindović (Biotechnical Faculty, University of Montenegro), who introduced me to plant virology and was my biggest support in enrolling for my PhD studies abroad. I would like to thank all my friends, especially dr. Ana Vučurović, who accompanied me through all stages of my student life in Ljubljana. Finally, I would like to thank my family who believes in me and tirelessly supports me in all aspects of my life. This dissertation is dedicated to them. Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 ANNEX A Statement on publisher permissions for the inclusion of own published articles in the printed and electronic versions of the doctoral thesis: - Miljanić V., Jakše J., Kunej U., Rusjan D., Škvarč A., Štajner N. 2022. Virome Status of Preclonal Candidates of Grapevine Varieties ( Vitis vinifera L.) From the Slovenian Wine-Growing Region Primorska as Determined by High-Throughput Sequencing. Frontiers in microbiology, 13, 830866, doi: 10.3389/fmicb.2022.830866: 11 p. - Miljanić V., Jakše J., Kunej U., Rusjan D., Škvarč A., Štajner N. 2022. First Report of Grapevine Red Globe Virus, Grapevine Rupestris Vein Feathering Virus, and Grapevine Syrah Virus-1 Infecting Grapevine in Slovenia. Plant Disease, 106, 9: 2538 Miljanić V. High-throughput sequencing detection and molecular…thermotherapy and meristem tissue culture. Doct. dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 - Miljanić V., Rusjan D., Škvarč A., Chatelet P., Štajner N. 2022. Elimination of Eight Viruses and Two Viroids from Preclonal Candidates of Six Grapevine Varieties (Vitis vinifera L.) through In Vivo Thermotherapy and In Vitro Meristem Tip Micrografting. Plants, 11, 8: 1064, doi: 10.3390/plants11081064: 14 p. - Miljanić V., Jakše J., Rusjan D., Škvarč A., Štajner N. 2022. Small RNA Sequencing and Multiplex RT-PCR for Diagnostics of Grapevine Viruses and Virus-like Organisms. Viruses, 14, 5: 921, doi: 10.3390/v14050921: 11 p. - Miljanić V., Jakše J., Beber A., Rusjan D., Škvarč A., Štajner N. 2021. First report of grapevine satellite virus in Slovenia. Journal of Plant Pathology, 103: 1329–1330