Abstract
Grapevine fanleaf virus (GFLV) is the main causal agent of fanleaf degeneration, the most damaging viral disease of grapevine. GFLV is included in most grapevine certification programs that rely on robust diagnostic tools such as biological indexing, serological methods, and molecular techniques, for the identification of clean stocks. The emergence of high throughput sequencing (HTS) offers new opportunities for detecting GFLV and other viruses in grapevine accessions of interest. Here, two HTS-based methods, i.e., RNAseq and smallRNAseq (focusing on the 21 to 27 nt) were explored for their potential to characterize the virome of grapevine samples from two 30-year-old GFLV-infected vineyards in the Champagne region of France. smallrnaseq was optimal for the detection of a wide range of viral species within a sample and RNAseq was the method of choice for full-length viral genome assembly. The implementation of a protocol to discriminate between low GFLV titer and in silico contamination (intra-lane contamination due to index misassignment) during data processing was critical for data analyses. Furthermore, we compared the performance of semi-quantitative DAS-ELISA (double antibody enzyme-linked immunosorbent assay), RT-qPCR (Reverse transcription-quantitative polymerase chain reaction), Immuno capture (IC)-RT-PCR, northern blot for viral small interfering RNA (vsiRNA) detection and RNAseq for the detection and quantification of GFLV. While detection limits were variable among methods, as expected, GFLV diagnosis was consistently achieved with all of these diagnostic methods. Together, this work highlights the robustness of DAS-ELISA, the current method routinely used in the French grapevine certification program, for the detection of GFLV and offers perspectives on the potential of HTS as an approach of high interest for certification.
Highlights
More than 80 viruses and five viroids have been identified in Vitis spp., making grapevine the most virus-infected agricultural commodity known to date (Martelli, 2017)
Samples from four grapevines from the Institut National de la Recherche Agronomique (INRA) grapevine collection in Colmar, fanleaf degeneration in the Champagne region (France) and from two grapevines showing typical fanleaf symptoms in a Champagne vineyard were selected for this study
Our results showed that grapevine fanleaf virus (GFLV) is readily detected by the five diagnostic methodologies (Figure 1)
Summary
More than 80 viruses and five viroids have been identified in Vitis spp., making grapevine the most virus-infected agricultural commodity known to date (Martelli, 2017). While some viruses might not be directly linked to a particular disease, others are of great economic concern to the grapevine industry. The grapevine fanleaf virus (GFLV) from the genus Nepovirus in the family Secoviridae (Sanfaçon, 2015) causes fanleaf degeneration disease (Schmitt-Keichinger et al, 2017). This disease is of major economic concern (Andret-Link et al, 2004). The genome of GFLV is composed of two single-stranded positive-sense RNA molecules (RNA1 and RNA2). Both genomic RNAs are necessary for infection and can be associated with a satellite RNA (RNA3) (Schmitt-Keichinger et al, 2017). GFLV exhibits strong genetic diversity with genome sequences being divergent up to 20%, with many recombination events being identified on both genomic RNAs from field isolates (Vigne et al, 2004, 2008; Zhou et al, 2017; Hily et al, 2018c)
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