Grapevine Rupestris Stem Pitting-associated Virus Research Articles

Simultaneous detection of Brazilian isolates of grapevine viruses by TaqMan real-time RT-PCR

The aim of this work was to evaluate the efficiency of real-time RT-PCR for detection of different isolates of ten important virus species that infect grapevines in Brazil: Grapevine leafroll-associated virus (GLRaV-1, -2, -3 and -5), Grapevine virus A (GVA), Grapevine virus B (GVB), Grapevine virus D (GVD), Grapevine rupestris stem pitting-associated virus (GRSPaV), Grapevine fleck virus (GFkV) and Grapevine fanleaf virus (GFLV). The reactions consisted of individual (simplex) and simultaneous (duplex) virus detections. Thirty six grapevine accessions, regenerated after thermotherapy and tissue culture treatments, have been analysed. All the above-mentioned viruses were sensitively detected in simplex reactions in samples infected with different virus isolates. Specifically to GLRaV-1 it was necessary to mix reagents refered by different sources to achieve the amplification. GVA, GRSPaV, GLRaV-2 and GLRaV-3 combined with GVB, GFLV, GFkV, GVD and GLRaV-5 were accurately detected in duplex trials. It was shown, that real-time RT-PCR (TaqMan) is able to efficiently detect different local virus species and isolates.

The triple gene block movement proteins of a grape virus in the genus Foveavirus confer limited cell-to-cell spread of a mutant Potato virus X

Grapevine rupestris stem pitting-associated virus (GRSPaV) is a member of the genus Foveavirus in the family Betaflexiviridae. The genome of GRSPaV encodes five proteins, among which are three movement proteins designated the triple gene block (TGB) proteins. The TGB proteins of GRSPaV are highly similar to their counterparts in Potato virus X (PVX), as reflected in size, modular structure, conservation of critical amino acid sequence motifs, as well as similar cellular localization. Based on these similarities, we predicted that the TGB proteins of these two viruses would be interchangeable. To test this hypothesis, we replaced the entire or partial sequence of PVX TGB with the corresponding regions from GRSPaV, creating chimeric viruses that contain the PVX backbone and different sequences from GRSPaV TGB. These chimeric constructs were delivered into plants of Nicotiana benthamiana through agro-infiltration to test whether they were capable of cell-to-cell and systemic movement. To our surprise, viruses derived from pPVX.GFP(CH3) bearing GRSPaV TGB in place of PVX TGB lost the ability to move either cell-to-cell or systemically. Interestingly, another chimeric virus resulting from pPVX.GFP(HY2) containing four TGB genes (TGB1 from PVX and TGB1-3 from GRSPaV), exhibited limited cell-to-cell, but not systemic, movement. Our data question the notion that analogous movement proteins encoded by even distantly related viruses are functionally interchangeable and can be replaced by each other. These data suggest that other factors, besides the TGB proteins, may be required for successful intercellular and/or systemic movement of progeny viruses. This is the first experimental demonstration that the GRSPaV TGB function as movement proteins in the context of a chimeric virus and that four TGB genes were required to support the intercellular movement of the chimeric virus.

Construction and biological activities of the first infectious cDNA clones of the genus Foveavirus

Grapevine rupestris stem pitting-associated virus (GRSPaV, genus Foveavirus, family Betaflexiviridae) is one of the most prevalent viruses in grapevines and is associated with three distinct diseases: rupestris stem pitting, vein necrosis and Syrah decline. Little is known about the biology and pathological properties of GRSPaV. In this work, we engineered a full-length infectious cDNA clone for GRSPaV and a GFP-tagged variant, both under the transcriptional control of Cauliflower mosaic virus 35S promoter. We demonstrated that these cDNA clones were infectious in grapevines and Nicotiana benthamiana through fluorescence microscopy, RT-PCR, Western blotting and immuno electron microscopy. Interestingly, GRSPaV does not cause systemic infection in four of the most commonly used herbaceous plants, even in the presence of the movement proteins of two other viruses which are known to complement numerous movement-defective viruses. These infectious clones are the first of members of Foveavirus which would allow further investigations into mechanisms governing different aspects of replication for GRSPaV and perhaps related viruses.

Viral sanitary status of declining grapevine Syrah clones and genetic diversity of Grapevine Rupestris stem pitting-associated virus

In order to determine the etiology of Syrah decline, virus detection was performed on 22 Syrah clones, chosen for their various levels of sensitivity to Syrah decline. All clones, including the sensitive ones, were free of 20 main grapevine viruses. In contrast, Grapevine Syrah virus-1 and Grapevine Rupestris stem pitting-associated virus (GRSPaV), were detected in 56 % and 100 % of the analysed Syrah clones respectively. This is the first report of GSyV-1 in a French vineyard. The genetic diversity of a 380 nt region within the GRSPaV coat protein gene was studied extensively in vines differing in their sensitivity to Syrah decline. Most GRSPaV variants were scattered between the four phylogenetic groups previously identified; 65 % of the sequences analysed were found to belong to the GRSPaV—group 1, 22 % to—group 2b, 10 % to—group 2a and 2 % to—group 3. Seventy percent of the 31 plants analysed harboured mixtures of genomic variants. Statistical analyses revealed no significant correlation between sensitivity and GRSPaV sequence variation. This suggests that GRSPaV is not the direct etiological agent of the Syrah decline.

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

Grapevine rupestris stem pitting-associated virus (GRSPaV) is a widespread virus infecting Vitis spp. Although it has established a compatible viral interaction in Vitis vinifera without the development of phenotypic alterations, it can occur as distinct variants that show different symptoms in diverse Vitis species. The changes induced by GRSPaV in V. vinifera cv 'Bosco', an Italian white grape variety, were investigated by combining agronomic, physiological, and molecular approaches, in order to provide comprehensive information about the global effects of GRSPaV. In two years, this virus caused a moderate decrease in physiological efficiency, yield performance, and sugar content in berries associated with several transcriptomic alterations. Transcript profiles were analysed by a microarray technique in petiole, leaf, and berry samples collected at véraison and by real-time RT-PCR in a time course carried out at five grapevine developmental stages. Global gene expression analyses showed that transcriptomic changes were highly variable among the different organs and the different phenological phases. GRSPaV triggers some unique responses in the grapevine at véraison, never reported before for other plant-virus interactions. These responses include an increase in transcripts involved in photosynthesis and CO(2) fixation, a moderate reduction in the photosynthesis rate and some defence mechanisms, and an overlap with responses to water and salinity stresses. It is hypothesized that the long co-existence of grapevine and GRSPaV has resulted in the evolution of a form of mutual adaptation between the virus and its host. This study contributes to elucidating alternative mechanisms used by infected plants to contend with viruses.

Elimination of Grapevine rupestris stem pitting-associated virus (GRSPaV) from two Vitis vinifera cultivars by in vitro chemotherapy

The grapevine (Vitis vinifera L.) cultivars ‘Agiorgitiko’ and ‘Malagouzia’, naturally infected with Grapevine rupestris stem pitting-associated virus (GRSPaV), were subjected to in vitro chemotherapy using the antiviral inosine 5′-monophosphate dehydrogenase inhibitors tiazofurin (TR), ribavirin (RBV) and mycophenolic acid (MPA). The chemotherapy lasted 80 days and was carried out as two consecutive treatments. Severe phytotoxicity, estimated after 40 days of culture, was observed in drug-treated explants, especially when high doses of TR were used. Phytotoxicity exhibited a cultivar- and chemical compound-dependent profile. The virus eradication status of the survived plantlets was determined by nested RT-PCR using total RNA templates, after 80 days of drug treatment and one year later, after the passage of one dormancy period, in potted plants grown in a greenhouse. Data indicated that the highest GRSPaV elimination in ‘Agiorgitiko’ was obtained with 10 μg ml−1 TR, 30 μg ml−1 RBV and 20 μg ml−1 MPA. The eradication rates were lower in the case of ‘Malagouzia’, where the highest ones were achieved after treatments with 15 μg ml−1 TR and 80 μg ml−1 MPA. This is the first report on GRSPaV elimination in grapevine following treatment with antiviral compounds, which could provide an alternative to the traditional methods of virus eradication through meristem culture and thermotherapy.

Genome‐wide identification of viral and host transcripts targeted by viral siRNAs in Vitis vinifera

In plants, RNA silencing is a surveillance mechanism against invading viruses. It involves the production of virus-derived small interfering RNAs (vsiRNAs), which guide the RNA-induced silencing complex (RISC) to inactivate viruses. vsiRNAs may also promote the silencing of host mRNAs in a sequence-specific manner. In this work, vsiRNAs derived from two grapevine-infecting viruses (Grapevine fleck virus and Grapevine rupestris stem pitting-associated virus) were selected from cDNA libraries of short RNAs and were cross-referenced with the remnants of both cleaved host transcripts and viral RNAs from a degradome dataset. We identified dozens of host transcripts targeted by vsiRNAs. Among them, several encode putative proteins involved in ribosome biogenesis and in biotic and abiotic stresses. Moreover, we identified vsiRNAs which explain the cleavage sites in viral genomes. A consistent fraction of vsiRNAs did not apparently account for cleavage, suggesting that only a low percentage of vsiRNAs are involved in the antiviral response.

Viruses of grapevine in Kosovo

Surveys for virus and virus-like diseases were carried out in commercial vineyards in the provinces of Rahovec and Suhareka of Kosovo. Samples were collected at random from 306 individual vines for laboratory testing. Leafroll and rugose wood symptoms were commonly observed in the field, whereas fanleaf symptoms were very rare. A total of 68% of ELISA-tested vines (208 out of 306) was infected by one (40.9%) or multiple (27.1%) viruses. The highest infection rate was found in Suhareka province (75%). Grapevine fleck virus (GFkV) was the most widespread (52.0%), followed by Grapevine leafroll-associated virus 3 (GLRaV-3, 18.3%), Grapevine leafroll-associated virus 1 (GLRaV-1, 15.7%), and Grapevine virus A (GVA, 11.1%). Other economically relevant viruses occurred rarely, i.e. Grapevine fanleaf virus (GFLV, 1.6%), Grapevine virus B (GVB, 1.0%) and Grapevine leafroll-associated virus 2 (GLRaV-2, 0.3%). Arabis mosaic virus (ArMV) was not found. Some of the most important grapevine varieties cultivated in Kosovo, i.e. Smederevka, Vranac, Prokupac, Italian Muskat, Muskat of Hamburg, and Italian Riesling, had average infection rates that ranged from 63% to 85%. In RT-PCR, Grapevine rupestris stem pitting associated virus (GRSPaV) was detected in 80.4% of the vines. Vein mosaic and vein necrosis symptoms were detected in graft-inoculated V. riparia and 110R indicators. At least one virus-tested candidate clone of 19 different grapevine varieties were identified, that could represent a potential primary source for a grapevine certification program in Kosovo.

Magyarországon előforduló szőlővírusok és a hazai szőlő vírusbetegségek áttekintése

The past fifty years important advances have been made in the field of grapevine virus research, including characterization of pathogens and control measurements. Still the occurrence of Grapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV), Tomato black ring virus (TBRV), Grapevine chrome mosaic virus (GCMV), Alfalfa mosaic virus (AMV), Grapevine Bulgarian latent virus (GBLV), Grapevine fleck virus (GFkV), Grapevine leafroll- associated viruses (GLRaV1-4), Grapevine virus A (GVA), Grapevine virus B (GVB) and Grapevine rupestris stem pitting- associated virus (GRSPaV) have been reported in Hungary and characterized by conventional methods as woody indexing, herbaceous indexing and serological methods. Among grapevine viruses the Grapevine line pattern virus (GLPV) seems to be uncial; because it was reported only in Hungary. Causal agents of several grapevine diseases, like enation, vein necrosis and vein mosaic remained undiscovered. These virus-like diseases occurred only sporadically, without economic importance.

A new grapevine virus discovered by deep sequencing of virus- and viroid-derived small RNAs in Cv Pinot gris

Field symptoms of chlorotic mottling and leaf deformations were observed on the cv Pinot gris (PG) in the Trentino region (Italy). Extensive assays excluded the presence of widely distributed nepo-, ampelo- and vitiviruses. An analysis of small RNA populations from two PG grapevines showing or not symptoms was carried out by Illumina high throughput sequencing. The study disclosed the virus and viroids contents of the two vines that was composed by Grapevine rupestris stem pitting-associated virus (GRSPaV), two viroids Hop stunt viroid (HSVd) and Grapevine yellow speckle viroid 1 (GYSVd1), the marafiviruses Grapevine rupestris vein feathering virus (GRVFV) and Grapevine Syrah virus 1 (GSyV-1), and a hitherto unrecorded virus. This virus had a genome organization identical to that of Grapevine berry inner necrosis virus (GINV), a trichovirus reported only from Japan, with which it grouped in phylogenetic trees constructed with sequences of the RdRp domain and the coat protein gene. However, molecular differences with GINV are wide enough to warrant classification of the virus in question as a new species, for which the provisional name of Grapevine Pinot gris virus (GPGV) is proposed. A limited field survey for the presence of GPGV in diseased and symptomless plants from three different cultivars did not allow to clearly associating the virus to the observed symptoms.

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

Grapevine rupestris stem pitting-associated virus (GRSPaV) is a member of the genus Foveavirus within the new family Betaflexiviridae. GRSPaV is distributed among grapevines worldwide and is implicated in the disease rupestris stem pitting (RSP) of the rugose wood complex and two other disorders. GRSPaV is composed of a wide range of sequence variants, and so far, the complete genomes of five sequence variants have been sequenced. Quick and reliable detection of different GRSPaV variants is a critical step in the elimination and control of GRSPaV. Previously, primers designed from various genomic regions have been used in RT-PCR for the detection of GRSPaV variants. The efficiency of RT-PCR varied widely depending on the spectrum of the primers that were used. In this study, we designed a pair of degenerate primers based on the consensus sequence of the genomic region encoding the highly conserved RNA-dependent RNA polymerase domain from five reference isolates of GRSPaV for which the genome sequence are available. We demonstrate that this set of primers is comparable, if not superior, to the broad-spectrum primers RSP13&14 in detecting multiple GRSPaV variants. Using these degenerate primers, we identified two new and distinct sequence variants. The 3′ terminal genomic region of one of the new variants, GRSPaV-ML, spanning the 3′ part of ORF1, through the entire open reading frames 2–4, and the 5′ region of ORF5 were sequenced. Sequence comparison demonstrates that GRSPaV-ML is distinct from each of the five reference isolates.

Symptomatology and Effects of Viruses Associated with Rugose Wood Complex on the Growth of Four Different Rootstocks

The rootstocks Vitis rupestris cv. St. George, Kober 5BB, LN33, and Freedom were graft-inoculated with viruses associated with the rugose wood complex (RW) to study their growth response and symptom expression. The virus sources used in this study were isolates LR127, CB111, CB105, and PA94-142 for Grapevine virus A (GVA), CB120 for Grapevine virus B (GVB), LV92-07 and a field selection of Chardonnay for Grapevine virus D (GVD), and a field selection of Cabernet Sauvignon for Grapevine rupestris stem pitting-associated virus (GRSPaV). Each rootstock plant was graft-inoculated with one, two, or three virus sources using bud chips from virus-infected grapevines. Plants were planted following a partially randomized block design. After two years, plant diameter was measured, barks were removed, and the woody cylinders were observed for the presence of RW symptoms and rated based on symptom severity. Selected plants were also tested by RT-PCR. The study showed that the greatest reduction in trunk diameter was in plants inoculated with the virus source combination of GVB/GVD/GRSPaV regardless of the rootstock type. Furthermore, treatments GVB/GVD, GVA/GVB/GVD and GVB/GVD/GRSPaV had the highest effect on LN33 growth, and GVA/GVB/GRSPaV, GVB/GVD/GRSPaV, and GVA/GVB had the highest growth effects on Kober 5BB. In general, GVB source had more adverse effect on growth in multiple infections, particularly in triple combination. In treatments with single virus source, GVD showed severe growth reduction in Freedom with no symptoms on the woody cylinder of the plants. Some sources of GVA impaired the growth of both Kober 5BB and Freedom rootstocks.

Complete nucleotide sequence of a new variant of grapevine rupestris stem pitting-associated virus from southern Italy

Grapevine rupestris stem pitting-associated virus (GRSPaV), genus Foveavirus, family Betaflexiviridae, is one of the most common viruses of grapevine and is widely distributed in many, if not all, grape-growing areas of the world. This virus is thought to be involved in the ‘rugose wood’ complex (RW) as the putative agent of the disorder known as Rupestris stem pitting (RSP) [1], and some of its strains have been shown to have a very close association with vein necrosis disease (VN) [2, 3]. This virus is also associated with Syrah decline [4], and its possible involvement in other disorders has also been suggested [5, 6]. Since the characterization of GRSPaV at the end of the 1990s [1, 7], a number of studies have revealed the remarkable genetic variability of this virus, which may reflect the puzzling pattern of its biological effects. Molecular investigations have led to complete sequencing of six viral isolates, named GRSPaV-1 (accession no. AF057136), GRSPV (AF026278), GRSPaV-SG1 (AY881626), GRSPaVBS (AY881627), GRSPaV-SY (AY368590) and GRSPaV-PN (AY368172). We now report the complete nucleotide (nt) sequence of a new viral isolate (GRSPaV-MG), deposited in GenBank under the accession number FR691076. Virus source and sequencing strategy

Deep sequencing analysis of viral short RNAs from an infected Pinot Noir grapevine

Virus-derived short interfering RNAs (vsiRNAs) isolated from grapevine V. vinifera Pinot Noir clone ENTAV 115 were analyzed by high-throughput sequencing using the Illumina Solexa platform. We identified and characterized vsiRNAs derived from grapevine field plants naturally infected with different viruses belonging to the genera Foveavirus, Maculavirus, Marafivirus and Nepovirus. These vsiRNAs were mainly of 21 and 22 nucleotides (nt) in size and were discontinuously distributed throughout Grapevine rupestris stem-pitting associated virus (GRSPaV) and Grapevine fleck virus (GFkV) genomic RNAs. Among the studied viruses, GRSPaV and GFkV vsiRNAs had a 5′ terminal nucleotide bias, which differed from that described for experimental viral infections in Arabidopsis thaliana. VsiRNAs were found to originate from both genomic and antigenomic GRSPaV RNA strands, whereas with the grapevine tymoviruses GFkV and Grapevine Red Globe associated virus (GRGV), the large majority derived from the antigenomic viral strand, a feature never observed in other plant–virus interactions.

The capsid protein of Grapevine rupestris stem pitting-associated virus contains a typical nuclear localization signal and targets to the nucleus

Grapevine rupestris stem pitting-associated virus (GRSPaV) is a positive strand, ssRNA virus of the genus Foveavirus (family Betaflexiviridae; order Tymovirales). GRSPaV is distributed in table and wine grapes worldwide and comprises a large family of sequence variants. As a newly discovered virus, mechanisms of virus replication and movement of GRSPaV have not been elucidated. We recently revealed the subcellular localization of the proteins encoded by the triple gene block of GRSPaV ( Rebelo et al., 2008). However, the subcellular localization and interaction of its capsid protein (CP) have not been explored. We report here that GRSPaV CP contains a nuclear localization signal “KRKR” near its N-terminus, which is conserved among all five strains whose genomes are completely sequenced. Similar sequences were also detected in the CP of two other viruses of the same family: African oil palm ringspot virus and Cherry green ring mottle virus. Using fluorescent protein tagging, we demonstrate that the CP targets to the nucleus in tobacco protoplasts. Mutation to this nuclear localization signal abolished the nuclear localization. Using bi-molecular fluorescence complementation, we show that the capsid protein of GRSPaV engages in homologous interaction. To our knowledge, this is the first report on the nuclear localization of a CP encoded by a RNA plant virus.

First Report on the Occurrence of Grapevine leafroll-associated virus 5 in Chilean Grapevines

Grapevine leafroll is one of the most widespread and economically damaging viral diseases of grapevines. At least eight distinct Grapevine leafroll-associated viruses (GLRaVs), all members of the Closteroviridae family, have been associated with this disease (4). GLRaV-5 was recently reported in vineyards from Argentina (2). To determine if GLRaV-5 was present in Chilean grapevines, in addition to the previously reported GLRaV-1, -2, -3, -4, -7, and -9 (1), 45 dormant cane samples from 12 different cultivars were collected from different geographic regions of Chile and screened by reverse transcription-PCR. Two of the forty-five samples (cvs. Sauvignon Blanc and Superior) collected from the III (700 km north of Santiago) and VI (150 km south of Santiago) regions of Chile, respectively, were found to be infected with GLRaV-5 using two different pairs of virus-specific primers. The first pair of primers, LR5-1F: 5'-CCCGTGATACAAGGTAGGACA-3' and LR5-1R: 5'-CAGACTTCACCTCCTGTTAC-3' (3), was used to amplify a 690-bp fragment corresponding to a partial region of the coat protein gene. The sequences obtained from the two positive samples (GenBank Accession Nos. HM214148 and HM214149) shared 97 and 94% of nucleotide identities, respectively, with the corresponding fragment of a reference GLRaV-5 isolate (GenBank Accession No. EU815935). Both samples shared 99% of amino acid identity with the same reference isolate. A second pair of primers, LR5upF: 5'-CTCTGCTTTTCTGCTGGCA-3' and LR5doR: 5'-TATCTTTTATCTCCCGATAAACGAG-3' (4) that amplified a 160-bp fragment of the HSP70h gene was also used. The positive Chilean samples (GenBank Accession Nos. HM214150 and HM214151) shared in both cases 98% nucleotide and 98% amino acid identities with the corresponding fragment of a reference GLRaV-5 isolate (Accession No. AF039552). The two GLRaV-5-positive plants were additionally infected with other viruses previously reported in Chile (1). The cv. Sauvignon Blanc sample was also infected with GLRaV-2, Grapevine fleck virus, and Grapevine rupestris stem pitting-associated virus. The cv. Superior sample was also infected with GLRaV-3, GLRaV-4, and Grapevine virus A.

Rugose wood-associated viruses do not appear to be involved in Shiraz (Syrah) decline in South Africa

The presence of rugose-wood-associated viruses of the genera Foveavirus and Vitivirus in the family Betaflexiviridae was investigated in various clones of own-rooted and grafted Vitis vinifera cv. Shiraz that were affected, or not, by Shiraz decline, and in rootstocks. RT nested-PCR amplification of double-stranded RNA using degenerate primers for the simultaneous detection of foveaviruses and vitiviruses (Dovas CI, Katis NI in J Virol Meth 170:99-106, 2003), cloning of DNA amplicons, SSCP analysis of clones, sequencing and computer-assisted analysis of sequences was used to characterize viral genetic variability. A total of 1,137 clones were analysed by SSCP, and, of those, 371 clones were sequenced. The results revealed that variants of five molecular groups belonging to the species Grapevine rupestris stem pitting-associated virus (GRSPaV), including highly divergent variants related to strain SY (Lima MF et al. in Arch Virol 151:1889-1894, 2006) were present in plants of various clones of Shiraz regardless of their Shiraz decline status, and in rootstocks. Grapevine virus A (GVA) and grapevine virus B (GVB) were detected in a relatively small number of plants. This study suggested no involvement of GRSPaV, GVA or GVB in Shiraz decline.

First Report of Grapevine Syrah virus-1 in Chilean Grapevines.

At least 58 viruses have been reported to infect grapevines, causing economic damage globally. Our lab has reported previously the presence of more than 10 viral species in Chilean grapevines (2,3). Grapevine Syrah virus-1 (GSyV-1) is a novel marafivirus recently described in California vineyards (1). Grapevine virus Q (GVQ) was described shortly after GSyV-1 and both genomes share more than 99% nucleotide identity (4). Since GSyV-1 and GVQ correspond to the same viral species, the name GSyV-1 will be used in the current note to avoid confusion. Forty dormant cane samples from 12 different cultivars were collected from different regions of Chile and screened by reverse transcription-PCR. One of the 40 samples (cv. Syrah) collected from the VI region of Chile was found to be infected with GSyV-1 using two different pairs of GSyV-1-specific primers. The first pair of primers GSyV-1Det-F: 5'-CAAGCCATCCGTGCATCTGG -3' and GSyV-1Det-R: 5'-GCCGATTTGGAACCCGATGG -3' (1), was used to amplify a 297-bp fragment corresponding to a partial region of the putative methyltransferase gene. The sequence (GenBank Accession No. GU566025) shared 87% nucleotide and 100% amino acid identities with the corresponding fragment of a Californian GSyV-1 isolate (GenBank Accession No. FJ436028). Since there are no commercial antibodies available for GSyV-1 detection, a second pair of primers, GVQCP-F: 5'-TCCCAGCTTCAGGGTGAATT -3' and GVQCP-R: 5'-GCATTGCTGCGCATTGGAGG -3' (4), that amplified a 720-bp fragment of the putative coat protein gene was also used. The sequence of 720 bp from the Chilean sample (GenBank Accession No. GU566024) shared 92% nucleotide and 98% amino acid identities with the corresponding fragment of a Californian GSyV-1 isolate (GenBank Accession No. FJ436028). The GSyV-1-positive sample was also infected with Grapevine fleck virus and Grapevine rupestris stem pitting-associated virus that have been reported previously in Chile. To our knowledge, this is the first report of GSyV-1 in Chile. Further studies will help to establish the incidence and effects of this virus in Chilean grapevines.

Deep sequencing analysis of viruses infecting grapevines: Virome of a vineyard

Double stranded RNA, isolated from 44 pooled randomly selected vines from a diseased South African vineyard, has been used in a deep sequencing analysis to build a census of the viral population. The dsRNA was sequenced in an unbiased manner using the sequencing-by-synthesis technology offered by the Illumina Genome Analyzer II and yielded 837 megabases of metagenomic sequence data. Four known viral pathogens were identified. It was found that Grapevine leafroll-associated virus 3 (GLRaV-3) is the most prevalent species, constituting 59% of the total reads, followed by Grapevine rupestris stem pitting-associated virus and Grapevine virus A. Grapevine virus E, a virus not previously reported in South African vineyards, was identified in the census. Viruses not previously identified in grapevine were also detected. The second most prevalent virus detected was a member of the Chrysoviridae family similar to Penicillium chrysogenum virus. Sequences aligning to two other mycoviruses were also detected.

Detection and molecular characterization of Italian Grapevine rupestris stem pitting‐associated virus isolates

The efficiencies of two published primer pairs (RSP13‐RSP14 and RSP2‐RSP21) were compared in reverse transcription‐polymerase chain reaction (RT‐PCR) to improve the diagnosis of Grapevine rupestris stem pitting‐associated virus (GRSPaV), which has consistently been found associated with rupestris stem pitting (RSP), one of the most common infectious diseases of grapevine. Fifty‐six grapevine accessions from different locations in Northern Italy were tested. Of these, 48 were positive with primers RSP13‐RSP14 and 37 with primers RSP2‐RSP21, suggesting that primer pair RSP13‐RSP14 has a broader detection spectrum than primers RSP2‐RSP21. The coat protein (CP) gene was also amplified by RT‐PCR and the amplicons analysed by single‐strand conformation polymorphism (SSCP) and sequencing. The results support the existence of wide variability within this virus, independent of cultivar or geographic origin. Sequence analysis of the 17 Italian GRSPaV isolates examined showed a molecular identity ranging from 78·8 to 99·7%, within which, four groups of sequence variants were recognized. Flexuous rod‐shaped particles, approximately 800 nm in length, were detected in plant extracts from infected grapevines and were positively decorated with the specific antiserum As7‐276 in immunosorbent electron microscopy (ISEM). This is the first report of GRSPaV particles in grapevines observed by transmission electron microscopy (TEM) in Italy.