This study describes the application of high-throughput sequencing of small RNA analysis of the efficacy of using Ribavirin to eliminate Grapevine leafroll-associated virus 1, Grapevine fleck virus and Grapevine rupestris stem pitting-associated virus from Vitis vinifera cv. Riesling. The original plant used for sanitation by Ribavirin treatment was one naturally infected with all the viruses mentioned above as confirmed by RT-PCR. A tissue cultures of the plant were established and plantlets obtained were sanitized using Ribavirin. Three years after sanitation, a small RNA sequencing method for virus detection, targeting 21, 22 and 24 nt-long viral small RNAs (vsRNAs), was used to analyze both the mother plant and the sanitized plants. The results showed that the mother plant was infected by the three mentioned viruses and additionally by two viroids - Hop stunt viroid and Grapevine yellow speckle viroid 1. After Ribavirin treatment, the plants contained only the two viroids, with the complete elimination of all the viruses previously present.
- MeSH
- nemoci rostlin prevence a kontrola virologie MeSH
- ribavirin farmakologie MeSH
- RNA virová genetika MeSH
- rostlinné viry účinky léků genetika MeSH
- sekvenční analýza DNA MeSH
- Vitis virologie MeSH
- vysoce účinné nukleotidové sekvenování * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Comprehensive next generation sequencing virus detection was used to detect the whole spectrum of viruses and viroids in selected grapevines from the Czech Republic. The novel NGS approach was based on sequencing libraries of small RNA isolated from grapevine vascular tissues. Eight previously partially-characterized grapevines of diverse varieties were selected and subjected to analysis: Chardonnay, Laurot, Guzal Kara, and rootstock Kober 125AA from the Moravia wine-producing region; plus Müller-Thurgau and Pinot Noir from the Bohemia wine-producing region, both in the Czech Republic. Using next generation sequencing of small RNA, the presence of 8 viruses and 2 viroids were detected in a set of eight grapevines; therefore, confirming the high effectiveness of the technique in plant virology and producing results supporting previous data on multiple infected grapevines in Czech vineyards. Among the pathogens detected, the Grapevine rupestris vein feathering virus and Grapevine yellow speckle viroid 1 were recorded in the Czech Republic for the first time.
- MeSH
- DNA virů chemie MeSH
- farmy MeSH
- rostlinné viry izolace a purifikace MeSH
- sekvenční analýza DNA metody MeSH
- viroidy izolace a purifikace MeSH
- Vitis virologie MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
Grapevine Syrah virus-1 (GSyV-1) was identified by small-RNA deep sequencing in Slovak grapevine co-infected by several other viruses. The RT-PCR assays developed in this work substantially improved the virus detection and allowed the identification of GSyV-1 in tested grapevine samples from Slovakia and the Czech Republic at an unexpectedly high rate (ca. 30 %). Subsequently, complete genome sequences of 3 GSyV-1 isolates (2 Slovak and 1 Czech) were determined by Sanger sequencing, showing a typical marafivirus genome organization. Analyses of complete genome sequences showed a higher intra-group diversity among these 3 central European GSyV-1 isolates (differences reaching 7.1 % at the nucleotide level) in comparison to 3 previously characterized North American isolates (only 1.2 % intra-group divergence). A substantially higher divergence among central European isolates and their clustering into two major phylogenetic groups was further confirmed by the partial genome analysis of additional 26 isolates. The CP-centered study did not support the geography-based clustering among central European and American isolates. Nevertheless, the sequence data of the highly variable 5'-proximal portion of the genome obtained for few additional isolates from Slovakia and Czech Republic showed the presence of both, "European-" and "north American-like", GSyV-1 isolates in the analyzed grapevine samples.
- MeSH
- fylogeneze MeSH
- genetická variace MeSH
- genom virový * MeSH
- molekulární sekvence - údaje MeSH
- pořadí genů MeSH
- RNA virová genetika MeSH
- sekvenční analýza DNA * MeSH
- sekvenční homologie nukleových kyselin MeSH
- shluková analýza MeSH
- Tymoviridae klasifikace genetika izolace a purifikace MeSH
- Vitis virologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
- Slovenská republika MeSH
Analysis of complete genome sequences of three Slovak isolates of grapevine Pinot gris virus (GPGV) showed their low heterogeneity (reaching 1.7 %) and a close relationship to the Italian NC_015782 isolate (4.2-4.5 % divergence). Comparison of Slovak and Italian isolates revealed an unusual accumulation of 21 indel mutations in ORF1, resulting in a localized high divergence in the encoded amino acid sequences. An elevated divergence in the 5' extremity of the GPGV genomes is suggestive of a recombination between Slovak isolates and grapevine berry inner necrosis virus. RT-PCR allowed the frequent detection of closely related GPGV isolates in grapevines from Slovakia and the Czech Republic.
- MeSH
- Flexiviridae klasifikace genetika izolace a purifikace MeSH
- fylogeneze MeSH
- genetická variace * MeSH
- genom virový MeSH
- molekulární sekvence - údaje MeSH
- nemoci rostlin virologie MeSH
- otevřené čtecí rámce MeSH
- Vitis virologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
- Slovenská republika MeSH
Genetic diversity of eight Grapevine leafroll-associated virus 1 (GLRaV-1) isolates recovered from grapevines in three distinct locations in the Czech Republic and Slovakia was characterised by restriction fragment length polymorphism (RFLP) analysis and by sequencing of cloned 540 bp fragment of the heat shock protein 70 (HSP70) gene. Comparison and phylogenetic analysis of obtained and previously available sequence data revealed the existence of two groups of GLRaV-l isolates, tentatively named A and E (genetic divergence between A and E group reached 13.9%). An RT-PCR detection method followed by simple restriction analysis was developed, showing the potential to differentiate GLRaV-1 isolates of these groups. Interestingly, a mixed infection of two GLRaV-1 groups in the same plant was frequently detected together with a high intra-group variability in some isolates.
- MeSH
- Closteroviridae genetika izolace a purifikace patogenita MeSH
- DNA virů genetika MeSH
- financování organizované MeSH
- fylogeneze MeSH
- genetická variace MeSH
- klonování DNA MeSH
- molekulární sekvence - údaje MeSH
- nemoci rostlin virologie MeSH
- polymorfismus délky restrikčních fragmentů MeSH
- proteiny tepelného šoku HSP70 genetika MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- sekvenční homologie aminokyselin MeSH
- virové geny MeSH
- virové proteiny genetika MeSH
- Vitis virologie MeSH
- Geografické názvy
- Česká republika MeSH
- Slovenská republika MeSH
