High-throughput sequencing detection and molecular characterization of viral diseases of grapevine (Vitis vinifera L.) and their elimination by thermotherapy and meristem tissue culture

Miljanić, Vanja (author), Štajner, Nataša (mentor)

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.

• Language: English
• Keywords: grapevine, Vitis vinifera, HTS, virome, diagnosis, genetic diversity, thermotherapy, micrografting
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: BF - Biotechnical Faculty
• Year: 2022
• PID: 20.500.12556/RUL-142063
• COBISS.SI-ID: 126427651
• Publication date in RUL: 19.10.2022

Secondary language

• Language: Slovenian
• Title: Detekcija in molekularna karakterizacija virusnih bolezni vinske trte (Vitis vinifera L.) z visoko zmogljivim sekvenciranjem ter njihova eliminacija s pomočjo termoterapije in kulture meristemov

Abstract

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.

• Keywords: vinska trta, Vitis vinifera, HTS, virom, diagnostika, genetska raznolikost, termoterapija, mikrocepljenje