Limited Genetic Variability Among American Isolates of Grapevine virus E from Vitis spp.

A survey for the presence of Grapevine virus E (GVE, genus Vitivirus, family Betaflexiviridae) in vineyards in New York and California was conducted using macroarray hybridization or reverse-transcription polymerase chain reaction (RT-PCR) assays. In New York, GVE was detected in 10 of 46 vines of Vitis labrusca, one V. riparia, and one Vitis hybrid. All GVE-infected New York vines were coinfected with Grapevine leafroll-associated virus-3. In California, GVE was detected in 8 of 417 vines of V. vinifera. All GVE-infected California vines were also coinfected by one of the leafroll-associated viruses and other vitiviruses. In order to assess the genetic diversity among GVE isolates, a viral cDNA was amplified by RT-PCR, and a 675-nucleotide region that included the 3' terminus of the coat protein gene, a short intergenic region, and the 5' terminus of the putative nucleic acid binding protein gene was sequenced. All 20 GVE isolates sequenced in this study were very closely related, with >98% nucleotide identity to the SA94 isolate from South Africa. These findings confirm the presence of GVE in major grape-growing regions of the United States and indicate a very low level of genetic diversity.

Deep sequencing evidence from single grapevine plants reveals a virome dominated by mycoviruses.

We have characterized the virome in single grapevines by 454 high-throughput sequencing of double-stranded RNA recovered from the vine stem. The analysis revealed a substantial set of sequences similar to those of fungal viruses. Twenty-six putative fungal virus groups were identified from a single plant source. These represented half of all known mycoviral families including the Chrysoviridae, Hypoviridae, Narnaviridae, Partitiviridae, and Totiviridae. Three of the mycoviruses were associated with Botrytis cinerea, a common fungal pathogen of grapes. Most of the rest appeared to be undescribed. The presence of viral sequences identified by BLAST analysis was confirmed by sequencing PCR products generated from the starting material using primers designed from the genomic sequences of putative mycoviruses. To further characterize these sequences as fungal viruses, fungi from the grapevine tissue were cultured and screened with the same PCR probes. Five of the mycoviruses identified in the total grapevine extract were identified again in extracts of the fungal cultures.

Vitis californica and Vitis californica × Vitis vinifera Hybrids are Hosts for Grapevine leafroll-associated virus-2 and -3 and Grapevine virus A and B.

Vitis and non-Vitis spp. surrounding nine Napa Valley vineyards were surveyed for Grapevine leafroll-associated virus (GLRaV)-1 to -5 and -9, Grapevine virus A (GVA), Grapevine virus B (GVB), and Grapevine virus D (GVD). Vitis spp. from three riparian areas not adjacent to vineyards were also included. DNA fingerprinting and probability analyses indicated that the Vitis samples consisted primarily of Vitis californica followed by V. californica × V. vinifera hybrids. Single and mixed infections of GLRaV-2, -3, GVA, or GVB were detected by conventional or quantitative reverse-transcription polymerase chain reaction in 6 of the 66 V. californica and 11 of the 19 V. californica × V. vinifera hybrids. GLRaV-1, -4, -5, -9, and GVD were not detected. Phylogenetic analysis of GLRaV-2 and -3 partial coat protein gene nucleotide sequences indicated that the isolates from V. californica and V. californica × V. vinifera hybrids were closely related to isolates from V. vinifera.

A putative new ampelovirus associated with grapevine leafroll disease.

A putative new ampelovirus was detected in Vitis vinifera cv. Carnelian showing mild leafroll symptoms and molecularly characterized. The complete genome consisted of 13,625 nt and had a structure similar to that of members of subgroup I in the genus Ampelovirus (fam. Closteroviridae). In-depth analyses showed that the virus from cv. Carnelian is the most distinct member of the "GLRaV-4 lineage" of ampeloviruses, which comprises GLRaV-4, -5, -6, -9, and the recently characterized GLRaV-Pr, and GLRaV-De. This virus appears to be a new member of the family Closteroviridae, for which the provisional name grapevine leafroll-associated Carnelian virus is proposed.

Deep sequencing analysis of RNAs from a grapevine showing Syrah decline symptoms reveals a multiple virus infection that includes a novel virus.

In a search for viruses associated with decline symptoms of Syrah grapevines, we have undertaken an analysis of total plant RNA sequences using Life Sciences 454 high-throughput sequencing. 67.5 megabases of sequence data were derived from reverse-transcribed cDNA fragments, and screened for sequences of viral or viroid origin. The data revealed that a vine showing decline symptoms supported a mixed infection that included seven different RNA genomes. Fragments identified as derived from viruses or viroids spanned a approximately ten thousand fold range in relative prevalence, from 48,278 fragments derived from Rupestris stem pitting-associated virus to 4 fragments from Australian grapevine viroid. 1527 fragments were identified as derived from an unknown marafivirus. Its complete genome was sequenced and characterized, and an RT-PCR test was developed to analyze its field distribution and to demonstrate its presence in leafhoppers (vector for marafiviruses) collected from diseased vines. Initial surveys detected a limited presence of the virus in grape-growing regions of California.

First Report of Grapevine leafroll-associated virus-9 in Washington State Vineyards.

Grapevine leafroll disease (GLD) has been recognized as one of the major constraints to the production of wine grapes in Washington State. At least nine distinct Grapevine leafroll-associated viruses (GLRaV-1 to -9) have been detected in grapevines showing GLD symptoms in grape-growing areas of several countries. Previous studies documented the presence of GLRaV-1, -2, and -3 in Washington State (3). We initiated a program to test grapevine cultivars with GLD symptoms for the occurrence of the other GLRaVs. Leaf samples were collected from individual grapevines of red-berried grapevine cultivars showing typical GLD symptoms and tested by single-tube reverse transcription (RT)-PCR. Of nearly 300 samples from 13 cultivars in 19 vineyards, 14 samples from 5 cultivars (Cabernet Sauvignon, Merlot, Pinot Noir, Mourvedre, and Lagrein) in different vineyards tested positive for GLRaV-9 using primers LR9 F/F (5'-CGG CAT AAG AAA AGA TGG CAC-3') and LR9 R/R (5'-TCA TTC ACC ACT GCT TGA AC-3'), specific for the HSP-70h gene of GLRaV-9 (1). To confirm the identity of the RT-PCR products, the 393-bp amplicons obtained from each of these five cultivars were cloned individually into the pCR2.1 plasmid (Invitrogen Corp., Carlsbad, CA). Two independent clones per amplicon were sequenced from both orientations. Pairwise comparisons of these sequences (GenBank Accession Nos. EF101737, EF101738, EF101739, EF101740, and EU252530) with corresponding sequences of other GLRaVs in GenBank showed 94 to 100 and 96 to 100% identity at the nucleotide and amino acid level, respectively, with the sequence of HSP-70h gene of GLRaV-9 (GenBank Accession No. AY297819). Antiserum specific to GLRaV-9 was not accessible, therefore, an additional 540-nucleotide fragment specific to the coat protein (CP) gene of GLRaV-9 was amplified from cv. Lagrein using primers LR9-CP-F (5' TAC CGT CGA CAC TTT CGA AGC ACT 3') and LR9-CP-R (5' TGA GGC GTC GTA ACC GAA CAA TCT 3'). PCR amplified fragments were cloned and sequenced. A comparison of this sequence (GenBank Accession No. EU251512) with corresponding nucleotide sequences of other GLRaVs in GenBank showed 96% identity with CP of GLRaV-9 (GenBank Accession No. AY297819), further confirming the presence of GLRaV-9. Previously, GLRaV-9 was reported in grapevines in California (1), Tunisia (2), and Western Australia (4). To our knowledge, our results are the first evidence for the occurrence of GLRaV-9 in Washington State vineyards. Results from our study and previous reports (1,2,4) indicate the wide distribution of GLRaV-9 in several Vitis vinifera cultivars. The economic impact of GLRaV-9 on wine grape cultivars, however, remains to be determined. References: (1) R. Alkowni et al. J. Plant Pathol. 86:123, 2004. (2) N. Mahfoudhi et al. Plant Dis. 91:1359, 2007. (3) R. R. Martin et al. Plant Dis. 89:763, 2005. (4) B. K. Peake et al. Aust. Plant Pathol. 33:445, 2004.

Molecular characterization and detection of plum bark necrosis stem pitting-associated virus.

The complete RNA genome of plum bark necrosis stem pitting-associated virus (PBNSPaV) was cloned and sequenced and was determined to be 14, 214 nts long. The genome structure revealed seven major open reading frames (ORFs), and nontranslated regions at the 5' and 3' ends. PBNSPaV represents the simplest genome organization in the genus Ampelovirus, family Closteroviridae. The ORFs 1a and 1b encode, respectively, a large polyprotein with a molecular mass (Mr) of 259.6 kDa containing conserved domains characteristic of a papain-like protease, methyltransferase and helicase (ORF1a) and a 64.1-kDa protein of eight conserved motifs characteristic of viral RNA-dependent RNA polymerase (RdRp) (ORF1b). ORF1b is presumably expressed via a +1 ribosomal frameshift mechanism. ORF2 encodes a small 6.3-kDa hydrophobic protein of unknown function. ORF3 encodes a 57.4-kDa protein, a homologue of the HSP70 family of heat shock proteins. ORF4 encodes a 61.6-kDa protein with unknown function. ORF5 encodes a 35.9-kDa capsid protein (CP). Lastly, ORF6 encodes a 25.2-kDa minor capsid protein (CPm). Phylogenetic analyses performed on sequences of the HSP70h RdRp and CP support classification of the virus in the genus Ampelovirus. A real-time TaqMan RT-PCR assay and a one-step RT-PCR were developed for PBNSPaV detection and compared using three different sample preparation methods.

Improved Efficiency for Quantitative and Qualitative Indexing for Citrus tristeza virus and Citrus psorosis virus.

Reverse transcription-polymerase chain reaction (RT-PCR) assays were developed for the detection of Citrus tristeza virus (CTV; genus Closterovirus) and Citrus psorosis virus (CPsV; genus Ophiovirus) in citrus trees. Real-time TaqMan RT-PCR was also developed for the detection of CTV. Three different sample preparation methods were compared. The total RNA extraction method by Qiagen was found to be more reliable than the other two methods consisting of crude plant sap extraction and total nucleic acid trapping on a silica bed. Of 287 samples tested for CTV, 210 samples tested positive by RT-PCR and 198 samples by enzyme-linked immunosorbent assay (ELISA). Furthermore, the results from monthly tests of a selected number of field-grown CTV-infected trees showed that RT-PCR detected the virus in 100% of the infected trees in winter and summer, whereas ELISA did not. The one-tube RT-PCR detection was developed for CPsV and was more sensitive than ELISA. Notably, three of 10 CPsV isolates were not detected by ELISA. As demonstrated here, our approach allows the efficacious detection of different viruses in citrus plants using a minimal amount of tissue during all seasons. The molecular methods described could be used in citrus certification programs and to test trees in nurseries and commercial orchards.

Molecular analysis of a California strain of Rupestris stem pitting-associated virus isolated from declining Syrah grapevines.

The sequence of the genome of a Rupestris stem pitting-associated virus (RSPaV) isolated from a declining Syrah grapevine in California, designated the Syrah strain (RSPaV-SY) was determined. The genome of this strain had an overall nucleotide identity of 77% in comparison with RSPaV sequences in GenBank; the coat protein was the most conserved gene among RSPaV sequences and the replicase was the least conserved gene. Phylogenetic analysis of partial coat protein and replicase gene sequences showed RSPaV-SY clustered independently from the majority of RSPaV isolates.

Bermuda Grass as a Potential Reservoir Host for Grapevine fanleaf virus.

Grapevine fanleaf virus (GFLV) was detected in samples of Bermuda grass (BG) from Iran by reverse transcription-polymerase chain reaction (RT-PCR) using two different pairs of GFLV-specific primers, and also by enzyme-linked immunosorbent assay (ELISA) using antiserum specific for a North American isolate of the virus. RT-PCR detected GFLV in both fresh and dried BG tissues and in virus preparations purified from these plants. Cloning and sequencing of the RT-PCR products confirmed that the amplified sequences were sections of the GFLV coat protein gene. Similar results were obtained when random and oligo(dT) primers were used on viral RNA templates recovered from BG to synthesize cDNA for cloning and sequencing. The virus induced few or no symptoms in BG, but could nonetheless be transmitted from BG to Chenopodium quinoa by mechanical inoculation. Some isolates induced systemic chlorotic spots and leaf deformation; others remained symptomless in this plant. Both symptomatic and symptomless C. quinoa plants were found to be infected with GFLV, giving positive ELISA and RT-PCR tests. A North American isolate of GFLV was found to be mechanically transmissible to BG as indicated by positive RT-PCR results from root samples of inoculated plants. GFLV-infected BG was widely distributed in the Fars province of Iran.

Etiology and Host Range of a Closterovirus Associated with Plum Bark Necrosis-Stem Pitting Disease.

Diseased plum (Prunus salicina) cv. Black Beaut trees developed stem gumming, severe bark necrosis, and stem pitting symptoms on the woody cylinder of the main trunk and scaffold branches. The sucker shoots of the peach (Prunus persica) cv. Nemaguard understocks exhibited oak-leaf patterns, but lacked the wood or bark markings. Other susceptible hosts included almond (Prunus dulcis), sweet (Prunus avium) and Japanese flowering (Prunus serrulata) cherries, and several plum (Prunus salicina) and prune (Prunus domestica) varieties. A purified preparation containing high molecular size dsRNAs was obtained initially from diseased cherry (P. avium × Prunus pseudocerasus) cv. Colt tissues. Healthy preparations were devoid of similar sized dsRNAs. Reverse transcriptase-polymerase chain reaction (RT-PCR) assays with degenerate oligonucleotide Closterovirus primers, designed from the HSP70 gene, were used to amplify two DNA fragments of 0.67 and 0.56 kbp. The larger cDNA product was cloned, sequenced (AF195501), and compared with other viral sequences. Depending on the number of nucleotides used in the comparisons, identities ranged from 77% for Grapevine leafroll associated virus to 3 to 44% for Little cherry virus-1. Specific primers from the 0.67 kbp cDNA sequence were designed and used in subsequent RT-PCR assays. The associated 0.67 kbp HSP70 amplicon of Plum bark necrosis-stem pitting associated virus was detected in all graft-inoculated Prunus species and varieties except prune (P. domestica var. French Improved).

A strategy for rapid cDNA cloning from double-stranded RNA templates isolated from plants infected with RNA viruses by using Taq DNA polymerase.

A fast and efficient cDNA cloning procedure for plant RNA viruses was developed. In this procedure, double-stranded RNA (dsRNA) was used as a template source. Standard cDNA synthesis reagents and random hexamers were then used for making cDNAs. Taq DNA polymerase was used to add additional (A) at the ends of cDNAs, a TA cloning kit to ligate the cDNAs to vectors, and an electroporator to transform the DNAs to E. coli cells. dsRNAs were extracted from grapevine tissues infected with four different viruses and used for cloning. These viruses included grapevine rupestris stem pitting associated virus, grapevine leafroll associated virus 5, and two uncharacterized grapevine viruses, one each closely related to marafivirus and vitivirus groups. Selected cDNA clones were sequenced and PCR primers were developed for RT-PCR detection of these viruses in host plants.

Nucleotide sequence and rt-PCR detection of a virus associated with grapevine rupestris stem-pitting disease.

ABSTRACT Grapevine rupestris stem pitting (RSP) is a graft-transmissible disease of unknown etiology. We have characterized a virus associated with this disease. The entire genomic sequence (GenBank accession number AF026278) consisted of 8,725 nucleotides excluding a poly(A) tail. Six open reading frames (ORF) were found. ORF1 potentially encodes a polypeptide with a methyltransferase domain, a papain-like proteinase domain, a helicase domain, and a RNA-dependent RNA polymerase domain; ORF2, ORF3, and ORF4 compose a triple-gene block; ORF5 encodes a coat protein; and ORF6 is located near the 3' end with unknown function. Sequence analysis indicated that the virus is most similar to apple stem-pitting virus and may be allied with the carla- and potexviruses and grouped with other viruses that infect woody hosts. A specific reverse-transcription polymerase chain reaction (RT-PCR)-based detection method was developed. Among 62 grapevine sources known to be infected with rupestris stem-pitting disease, 60 sources tested positive by RT-PCR. Among 43 healthy vines tested, all were negative. The name grapevine rupestris stem-pitting-associated virus is proposed.

Use of Degenerate Primers for Partial Sequencing and RT-PCR-Based Assays of Grapevine Leafroll-Associated Viruses 4 and 5.

ABSTRACT Double-stranded RNA (dsRNA) was purified from grapevines infected with grapevine leafroll-associated viruses 4 (GLRaV-4) and 5 (GLRaV-5), two putative closteroviruses. Reverse-transcriptase polymerase chain reaction (RT-PCR) was performed on this dsRNA using degenerate oligonucleotides designed to amplify an approximately 550- to 650-nucleotide fragment from the heat shock protein 70 homolog (HSP70) of the known closteroviruses. RT-PCR products of the appropriate molecular weight were gel-isolated and cloned into the plasmid vector pGEM-T. Clones of RT-PCR products generated by using these primers on dsRNA isolated from a plant infected with GLRaV-4 were sequenced. This sequence was used to develop an immunocapture RT-PCR (IC-RT-PCR) detection protocol capable of detecting GLRaV-4. Similar clones were made from dsRNA isolated from a plant infected with GLRaV-5. These clones were also sequenced. The two sequences were compared, and RT-PCR primers were developed that were able to amplify cDNA from both. These experiments demonstrate that degenerate primers that amplify closterovirus HSP70 sequences can be used to successfully generate sequences useful for IC-RT-PCR detection of these viruses. These data also suggest that it is feasible to use HSP70 sequences to design PCR primers capable of more general PCR detection of multiple GLRaV serotypes. Lastly, the presence of closterovirus-like HSP70 sequences in these putative closteroviruses implies that they are indeed members of this taxonomic group.

First Report of Grapevine Kober Stem Grooving in the United States.

The last decade has brought extensive new plantings of grapevines (Vitis vinifera L.) to California vineyards. In some vineyards, severe viruslike problems have been observed (2). To ascertain possible causal agent(s), extensive testing for grapevine viruses was initiated. Testing included a 2-year woody index on indicators V. rupestris cv. Saint George, LN 33, V. vinifera cv. Cabernet Franc, and Kober 5BB. To our knowledge, this is the first field survey for grape virus diseases in the United States that includes woody indexing on Kober 5BB, a relatively new indicator for the grapevine rugose wood diseases (1). A Sonoma County field selection of Sauvignon Blanc grafted to the rootstock Freedom was included in these tests; the 2-year-old vines exhibited stunting, leaf rolling, leaf yellowing, wood necrosis, and pitting symptoms at the graft union. When indexed on the four indicators, the following disease symptoms were observed: stem pitting on V. rupestris; corky bark on LN 33; leafroll on Cabernet Franc; and stem grooving on Kober 5BB. An enzyme-linked immunosorbent assay confirmed the presence of grapevine leafroll-associated viruses 2, and 3, as well as Grapevine Virus A, which is associated with Kober stem grooving. The Sauvignon Blanc selection appears to be infected with several grapevine viruses, as is often the case when virus symptoms in vineyards are severe. Among these disease agents, only Kober stem grooving is reported to cause the severe pitting and grooving symptoms observed on the indicator Kober 5BB (1). This is the first report of Kober stem grooving in the United States. References: (1) R. Garau et al. Vitis 33:161, 1994. (2) D. A. Golino. Am. J. Enol. Vitic. 44:148, 1993.

Long, nearly identical untranslated sequences at the 3' terminal regions of the genomic RNAs of cherry leafroll virus (walnut strain).

Hybridization analyses of cDNA clones derived from the two genomic RNAs, RNA1 and RNA2, of the walnut strain of the nepovirus cherry leafroll nepovirus (wCLRV) demonstrated a long region of high homology between the two viral RNAs. Subsequent mapping and nucleotide sequencing revealed a long, noncoding, presumably untranslated, region (3' UTR) immediately 5' of the terminal polyadenylate, a region that is almost identical in the two RNAs. This 3' UTR is 1567 nucleotide residues long in RNA1. Homologies of about 80% were found with corresponding regions of genomic RNAs from other strains of CLRV, but not with the corresponding regions of other nepovirus genomic RNAs.

Purification and characterization of potato leafroll virus.

Potato leafroll virus (PLRV) was purified from infected potato (Solanum tuberosum L.) with yields of 0.4-0.6 mg/kg of foliage. The virus sedimented as a single component of 127 S. An antiserum prepared against purified virus had a maximum titer of 1:1024 in agar gel double diffusion tests. PLRV had a buoyant density of 1.39 g/ml and an estimated nucleic acid content of 28%. The nucleic acid had a molecular weight of 2.0 x 10(6) and was degraded by RNase but not by DNase, indicating that the PLRV nucleic acid is RNA. The sedimentation coefficient of the RNA molecule was 34.5 S and after treatment with formaldehyde, 20.7 S. Dissociated coat protein migrated as a single band in polyacrylamide gel electrophoresis and the average subunit molecular weight was 26,300. PLRV should be considered a member of the luteovirus group.