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A Diverse Virome of Leafroll-Infected Grapevine Unveiled by dsRNA Sequencing.

ABSTRACT: Quebec is the third-largest wine grape producing province in Canada, and the industry is constantly expanding. Traditionally, 90% of the grapevine cultivars grown in Quebec were winter hardy and largely dominated by interspecific hybrid Vitis sp. cultivars. Over the years, the winter protection techniques adopted by growers and climate changes have offered an opportunity to establish V. vinifera L. cultivars (e.g., Pinot noir). We characterized the virome of leafroll-infected interspecific hybrid cultivar and compared it to the virome of V. vinifera cultivar to support and facilitate the transition of the industry. A dsRNA sequencing method was used to sequence symptomatic and asymptomatic grapevine leaves of different cultivars. The results suggested a complex virome in terms of composition, abundance, richness, and phylogenetic diversity. Three viruses, grapevine Rupestris stem pitting-associated virus, grapevine leafroll-associated virus (GLRaV) 3 and 2 and hop stunt viroid (HSVd) largely dominated the virome. However, their presence and abundance varied among grapevine cultivars. The symptomless grapevine cultivar Vidal was frequently infected by multiple virus and viroid species and different strains of the same virus, including GLRaV-3 and 2. Our data show that viruses and viroids associated with the highest number of grapevines expressing symptoms included HSVd, GLRaV-3 and GLRaV-2, in gradient order. However, co-occurrence analysis revealed that the presence of GLRaV species was randomly associated with the development of virus-like symptoms. These findings and their implications for grapevine leafroll disease management are discussed.

SUBMITTER: Fall ML

PROVIDER: S-EPMC7599845 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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A Diverse Virome of Leafroll-Infected Grapevine Unveiled by dsRNA Sequencing.

Fall Mamadou L ML Xu Dong D Lemoyne Pierre P Moussa Issam E Ben IEB Beaulieu Carole C Carisse Odile O

Viruses 20201008 10

Quebec is the third-largest wine grape producing province in Canada, and the industry is constantly expanding. Traditionally, 90% of the grapevine cultivars grown in Quebec were winter hardy and largely dominated by interspecific hybrid <i>Vitis</i> sp. cultivars. Over the years, the winter protection techniques adopted by growers and climate changes have offered an opportunity to establish <i>V. vinifera L</i>. cultivars (e.g., Pinot noir). We characterized the virome of leafroll-infected inter ...[more]

PMID: 33050079

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Project description:Grapevine leafroll disease (GLD) is one of the most important grapevine viral diseases affecting grapevines worldwide. The impact on vine health, crop yield, and quality is difficult to assess due to a high number of variables, but significant economic losses are consistently reported over the lifespan of a vineyard if intervention strategies are not implemented. Several viruses from the family Closteroviridae are associated with GLD. However, Grapevine leafroll-associated virus 3 (GLRaV-3), the type species for the genus Ampelovirus, is regarded as the most important causative agent. Here we provide a general overview on various aspects of GLRaV-3, with an emphasis on the latest advances in the characterization of the genome. The full genome of several isolates have recently been sequenced and annotated, revealing the existence of several genetic variants. The classification of these variants, based on their genome sequence, will be discussed and a guideline is presented to facilitate future comparative studies. The characterization of sgRNAs produced during the infection cycle of GLRaV-3 has given some insight into the replication strategy and the putative functionality of the ORFs. The latest nucleotide sequence based molecular diagnostic techniques were shown to be more sensitive than conventional serological assays and although ELISA is not as sensitive it remains valuable for high-throughput screening and complementary to molecular diagnostics. The application of next-generation sequencing is proving to be a valuable tool to study the complexity of viral infection as well as plant pathogen interaction. Next-generation sequencing data can provide information regarding disease complexes, variants of viral species, and abundance of particular viruses. This information can be used to develop more accurate diagnostic assays. Reliable virus screening in support of robust grapevine certification programs remains the cornerstone of GLD management.

Project description:Grapevine leafroll-associated virus 3 (GLRaV-3) is one of the main pathogens of grapes, causing a significant loss in yield and decrease in quality for this agricultural plant. For efficient widespread control of this infection, rapid and simple analytical techniques of on-site testing are requested as a complementary addition for the currently applied hybridization (PCR) and immunoenzyme (ELISA) approaches. The given paper presents development and approbation of the immunochromatographic assay (ICA) for rapid detection of GLRaV-3. The ICA realizes a sandwich immunoassay format with the obtaining complexes ((antibody immobilized on immunochromatographic membrane)⁻(virus in the sample)⁻(antibody immobilized on gold nanoparticles (GNP)) during sample flow along the membrane compounds of the test strip. Three preparations of GNPs were compared for detection of GLRaV-3 at different dilutions of virus-containing sample. The GNPs with maximal average diameters of 51.0 ± 7.9 nm provide GLRaV-3 detection for its maximal dilutions, being 4 times more than when using GNPs with a diameter of 28.3 ± 3.3 nm, and 8 times more than when using GNPs with a diameter of 18.5 ± 3.3 nm. Test strips have been manufactured using the largest GNPs conjugated with anti-GLRaV-3 antibodies at a ratio of 1070:1. When testing samples containing other grape wine viruses, the test strips have not demonstrated staining in the test zone, which confirms the ICA specificity. The approbation of the manufactured test strips indicated that when using ELISA as a reference method, the developed ICA is characterized by a sensitivity of 100% and a specificity of 92%. If PCR is considered as a reference method, then the sensitivity of ICA is 93% and the specificity is 92%. The proposed ICA can be implemented in one stage without the use of any additional reactants or devices. The testing results can be obtained in 10 min and detected visually. It provides significant improvement in GLRaV-3 detection, and the presented approach can be transferred for the development of test systems for other grape wine pathogens.

Project description:BackgroundGrapevine leafroll disease is one of the most economically important viral diseases affecting grape production worldwide. Grapevine leafroll-associated virus 4 (GLRaV-4, genus Ampelovirus, family Closteroviridae) is one of the six GLRaV species documented in grapevines (Vitis spp.). GLRaV-4 is made up of several distinct strains that were previously considered as putative species. Currently known strains of GLRaV-4 stand apart from other GLRaV species in lacking the minor coat protein.MethodsIn this study, the complete genome sequence of three strains of GLRaV-4 from Washington State vineyards was determined using a combination of high-throughput sequencing, Sanger sequencing and RACE. The genome sequence of these three strains was compared with corresponding sequences of GLRaV-4 strains reported from other grapevine-growing regions. Phylogenetic analysis and SimPlot and Recombination Detection Program (RDP) were used to identify putative recombination events among GLRaV-4 strains.ResultsThe genome size of GLRaV-4 strain 4 (isolate WAMR-4), strain 5 (isolate WASB-5) and strain 9 (isolate WALA-9) from Washington State vineyards was determined to be 13,824 nucleotides (nt), 13,820 nt, and 13,850 nt, respectively. Multiple sequence alignments showed that a 11-nt sequence (5'-GTAATCTTTTG-3') towards 5' terminus of the 5' non-translated region (NTR) and a 10-nt sequence (5'-ATCCAGGACC-3') towards 3' end of the 3' NTR are conserved among the currently known GLRaV-4 strains. LR-106 isolate of strain 4 and Estellat isolate of strain 6 were identified as recombinants due to putative recombination events involving divergent sequences in the ORF1a from strain 5 and strain Pr.ConclusionGenome-wide analyses showed for the first time that recombinantion can occur between distinct strains of GLRaV-4 resulting in the emergence of genetically stable and biologically successful chimeric viruses. Although the origin of recombinant strains of GLRaV-4 remains elusive, intra-species recombination could be playing an important role in shaping genetic diversity and evolution of the virus and modulating the biology and epidemiology of GLRaV-4 strains.

Dataset Information

A Diverse Virome of Leafroll-Infected Grapevine Unveiled by dsRNA Sequencing.

Publications

A Diverse Virome of Leafroll-Infected Grapevine Unveiled by dsRNA Sequencing.

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