[Show abstract][Hide abstract] ABSTRACT: Grapevine virus A (GVA; genus Vitivirus, family Betaflexiviridae) has been implicated with the Kober stem grooving disorder of the Rugose Wood disease complex. In this study, 26 isolates of GVA recovered from wine grape (Vitis vinifera L.) cultivars from California (CA) and Washington (WA) were analyzed for their genetic diversity. An analysis of a portion of the RNA-dependent RNA polymerase (RdRp) and complete coat protein (CP) sequences revealed intra- and inter-isolate sequence diversity. Our results indicated that both RdRp and CP are under strong negative selection based on the normalized values for the ratio of nonsynonymous substitutions per nonsynonymous site to synonymous substitutions per synonymous site. A global phylogenetic analysis of CP sequences revealed segregation of virus isolates into four major clades with no geographic clustering. In contrast, the RdRp-based phylogenetic tree indicated segregation of GVA isolates from CA and WA into six clades, independent of geographic origin or cultivar. Phylogenetic network coupled with recombination analyses showed putative recombination events in both RdRp and CP sequence data sets, with more of these events located in the CP sequence. The preponderance of divergent variants of GVA co-replicating within individual grapevines could increase viral genotypic complexity with implications for phylogenetic analysis and evolutionary history of the virus. The knowledge of genetic diversity of GVA generated in this study will provide a foundation for elucidating the epidemiological characteristics of virus populations at different scales and implementing appropriate management strategies for minimizing the spread of genetic variants of the virus by vectors and via planting materials supplied to nurseries and grape growers.
[Show abstract][Hide abstract] ABSTRACT: We have identified the genome of a novel viral satellite in deep sequence analysis of double-stranded RNA from grapevine. The genome was 1,060 bases in length, and encoded two open reading frames. Neither frame was related to any known plant virus gene. But translation of the longer frame showed a protein sequence similar to those of other plant virus satellites. Other than in commonalities they shared in this gene sequence, members of that group were extensively divergent. The reading frame in this gene from the novel satellite could be translationally coupled to an adjacent reading frame in the -1 register, through overlapping start/stop codons. These overlapping AUGA start/stop codons were adjacent to a sequence that could be folded into a pseudoknot structure. Field surveys with PCR probes specific for the novel satellite revealed its presence in 3 % of the grapevines (n = 346) sampled.
[Show abstract][Hide abstract] ABSTRACT: In the Napa Valley of California, vineyards of Cabernet Franc (CF) clone 214, Cabernet Sauvignon clone 337 and Zinfandel clone 1A (Z1A) with grapevines exhibiting foliar symptoms of red blotches, marginal reddening, and red veins, that were accompanied by reduced sugar accumulation in fruits at harvest, were initially suspected to be infected with leafroll-associated viruses. However, reverse transcription-polymerase chain reaction tests were negative for all known leafroll-associated viruses, with the exception of Grapevine leafroll-associated virus 2 in Z1A. Metagenomic analysis of cDNA libraries obtained from double-stranded RNA enriched nucleic acid (NA) preparations from bark scrapings of dormant canes on an Illumina platform revealed sequences having distant relationship with members of the family Geminiviridae. Sequencing of products obtained by PCR assays using overlapping primers and rolling circle amplification (RCA) confirmed the presence of a single circular genome of 3,206 nucleotides which was nearly identical to the genome of a recently reported Grapevine cabernet franc-associated virus found in declining grapevines in New York. We propose to call this virus 'Grapevine red blotch-associated virus' (GRBaV) to describe its association with grapevine red blotch disease. Primers specific to GRBaV amplified a product of expected size (557 bp) from NA preparations obtained from petioles of several diseased source vines. Chip bud inoculations successfully transmitted GRBaV to test plants of CF, as confirmed by PCR analysis. This is the first report of a DNA virus associated with red blotch disease of grapevines in California.
[Show abstract][Hide abstract] ABSTRACT: A novel virus-like sequence from grapevine was identified by Illumina sequencing. The complete genome is 7,551 nucleotides in length, with polyadenylation at the 3' end. Translation of the sequence revealed five open reading frames (ORFs). The genomic organization was most similar to those of vitiviruses. The polymerase (ORF1) and coat protein (ORF4) genes shared 31 to 49% nucleotide and 40 to 70% amino acid sequence identities, respectively, with other grapevine vitiviruses. The virus was tentatively named grapevine virus F (GVF).
Journal of Virology 09/2012; 86(17):9545. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nine isolates of Grapevine leafroll-associated virus 7 (GLRaV-7) from diverse geographical regions were sequenced to design more sensitive molecular diagnostic tools. The coat protein (CP) and heat shock protein 70 homologue (HSP70h) genes of these nine isolates were sequenced. Sequences were then used to design more sensitive molecular diagnostic tools. Sequence identity among these isolates ranged between 90 to 100% at the nucleotide and amino acid levels. One RT-PCR and two qRT-PCR assays were used to survey 86 different grapevines from the University of California, Davis Grapevine Virus Collection, the Foundation Plant Services collection and the USDA National Clonal Germplasm Repository, Davis, CA with primers designed in conserved regions of the CP and HSP70h genes. Results revealed that qRT-PCR assays designed in the HSP70h gene was more sensitive (29.07% positives) than that designed in the CP gene (22.09% positives) and both qRT-PCR assays proved to be more sensitive than RT-PCR.
Journal of virological methods 12/2011; 179(2):383-9. · 2.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Deep sequencing analysis of an asymptomatic grapevine revealed a virome containing five RNA viruses and a viroid. Of these, Grapevine leafroll-associated virus 7 (GLRaV-7), an unassigned closterovirus, was by far the most prominently represented sequence in the analysis. Graft-inoculation of the infection to another grape variety confirmed the lack of the leafroll disease symptoms, even though GLRaV-7 could be detected in the inoculated indicator plants. A 16,496 nucleotide-long genomic sequence of this virus was determined from the deep sequencing data. Its genome architecture and the sequences encoding its nine predicted proteins were compared with those of other closteroviruses. The comparison revealed that two other viruses, Little cherry virus-1 and Cordyline virus-1 formed a well supported phylogenetic cluster with GLRaV-7.
Virus Research 10/2011; 163(1):302-9. · 2.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The genetic diversity of 34 isolates of Grapevine leafroll-associated virus 1 (GLRaV-1) from different wine, table, and ornamental grape cultivars in California, New York, and Washington States in the United States was investigated. Segments of the heat-shock protein 70 homolog (HSP70h) gene, coat protein (CP) gene, coat protein duplicate 2 (CPd2) gene, and open reading frame 9 (p24) were amplified by reverse-transcription polymerase chain reaction, cloned, and sequenced. A pairwise comparison of nucleotide sequences revealed intra- and interisolate sequence diversity, with CPd2 and HSP70h being the most and the least divergent, respectively, among the four genomic regions studied. The normalized values for the ratio of nonsynonymous substitutions per nonsynonymous site to synonymous substitutions per synonymous site indicated different purifying selection pressures acting on each of the four genomic regions, with the CP and CPd2 being subjected to the strongest and weakest functional constraints, respectively. A global phylogenetic analysis of sequences from the four genomic regions revealed segregation of GLRaV-1 isolates into three major clades and a lack of clearly defined clustering by geographical origin. In contrast, only two lineages were apparent when the CP and CPd2 gene sequences were used in phylogenetic analyses. Putative recombination events were revealed among the HSP70h, CP, and p24 sequences. The genetic landscape of GLRaV-1 populations presented in this study provides a foundation for better understanding of the epidemiology of grapevine leafroll disease across grape-growing regions in the United States. In addition, this study will benefit grape clean plant programs across the country in improving the sanitary status of planting materials provided to nurseries and grape growers.
[Show abstract][Hide abstract] ABSTRACT: 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.
Archives of Virology 12/2010; 156(3):397-403. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Until very recently isolates of American plum line pattern virus (APLPV) had not been reported from outside North America. The nucleotide sequences corresponding to the movement (MP) and coat (CP) proteins of eight APLPV isolates from five Mediterranean countries were determined. Sequence analysis showed that both MP and CP genes are highly conserved irrespective of geographic origin. The study of the distribution of synonymous and nonsynonymous changes along both open reading frames revealed that these proteins are under the effect of negative purifying selection. The MP and CP of APLPV possess most of the functional motifs described for other members of the genus Ilarvirus.
Archives of Virology 02/2008; 153(2):367-73. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tissue-imprint hybridization (TIH) assay was validated for large-scale detection of Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd). All 72 collected leaves (100%) from 2 PLMVd- and 2 HSVd-infected trees were positive in TIH, regardless of the geographic
orientation of the scaffold, level of the canopy and position of the leaf in the shoot. In a large-scale survey in Serbia,
we tested by TIH 871 trees of stone fruits, representing 602 cultivars from fruit collections in Belgrade, Čačak and Novi
Sad. PLMVd was detected in 185 (50%) peach trees or 95 (54%) cultivars and HSVd in 2 apricot trees and cultivars (2%). The
occurrence of HSVd is a new report for Serbia. No viroid infection was found in European plums, sweet cherries, sour cherries
and wild Prunus spp. PLMVd-infected peach cultivars originated from the world’s main breeding centres of this crop. Western European and
Asian cultivars were the most infected (58%) followed by those originating from North America (50%). Nine PLMVd and two HSVd
isolates were sequenced and analyzed. All showed PMLVd sequences clustered together in the previously reported phylogenetic
group III. Both HSVd isolates were found to be derived from recombinant events, but that of the cv. Saturn represented a putative
new phylogenetic group of HSVd.
European Journal of Plant Pathology 01/2008; 120(2):167-176. · 1.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Archives of Virology 02/2007; 152(12):2197-206. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Stone fruit trees were surveyed from June to october (2003 and 2004) to determine the incidence of viroid, phytoplasma, and fungal diseases in the eastern anatolia region of turkey. Molecular hybridisation test (tissue printing and dot-blot) was used to investigate the presence of Peach latent mosaic viroid (plMVd) and Hop stunt viroid (HSVd). a total of 16 trees out of 491 were positive for viroids. plMVd was found in 15 peaches (Prunus persica) (3%) and a unique HSVd isolate was found only in an apricot (Prunus armeniaca) tree (0.1%). t he average incidence of viroid infection was 3.2%. HSVd was detected for the first time in eastern Anatolia, whereas no PLMVd infection was encountered in the main apricot growing provinces (Malatya, elazig). p c r analyses of the few symptomatic apricots for the presence of "Candidatus phytoplasma prunorum" were negative. isolates of Armillaria mellea, Cytospora spp., Monilinia laxa, Stigmina carpophila, Chondrostereum purpureum, Fusarium spp., Rosellinia spp., and Phytophthora spp. were identified from symptomatic samples collected from the region.
New Zealand Journal of Crop and Horticultural Science 01/2006; 34(1):1-6. · 0.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Field surveys were carried out in the main stone-fruit growing areas of Morocco to evaluate the sanitary status of commercial orchards, varietal collections and nurseries. The presence of virus and virus-like diseases was checked by ELISA, sap transmission to herbaceous hosts, testing on woody indicators and molecular hybridization (dot-blot and tissue-printing). 1211 samples (382 almond, 339 peach, 291 plum, 150 apricot and 49 cherry) were tested by ELISA for the presence of Prunus necrotic ring spot virus (PNRSV), Prune dwarf virus (PDV), Apple chlorotic leaf spot virus (ACLSV), Apple mosaic virus (ApMV) and Plum pox virus (PPV). The overall average of virus infection rate was 16.4%, whereas that of single species was: 22.6% for almond, 17.8% for plum, 15% for peach, 10.2% for cherry, and 2.7% for apricot. The following viruses were detected: PNRSV, PDV, ACLSV and ApMV. 565 samples were tested by dot-blot and tissue-printing hybridization for the presence of Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd). 48 samples were infected, 41 by PLMVd and 7 by HSVd. In addition, nested-PCR tests identified Plum bark necrosis and stem-pitting associated virus (PBNSPaV) in a few almond trees affected by stem pitting.
[Show abstract][Hide abstract] ABSTRACT: SUMMARY A filamentous virus was isolated by mechanical sap transmission from GF 305 seedlings showing asteroid ringspots to Nicotiana occidentalis. The GF 305 seedlings were graft-inoculated with buds from an apricot tree cv Mistikawi, from Palestine. The electrophoretic dsRNA pattern from symptomatic GF 305 and N. occidentalis showed a major band of about 9.5 kbp. Bundles of elon- gated virus-like particles were present in the cytoplasm of infected N. occidentalis. Dissociated coat protein from partially purified virus preparations consisted of a single subunit with an estimated Mr of ca. 50 kDa. RT-PCR us- ing primers specific for Apricot latent virus (ApLV) RNA amplified a DNA product of about 200 bp from infected GF 305 and N. occidentalis plants. Sequence analysis of this fragment showed 90-93% identity in the encoded amino acid sequence with corresponding sequences from different ApLV isolates. This is the first record of ApLV from the southern Mediterranean. An ApLV-spe- cific digoxygenin-labelled riboprobe was produced and used for molecular hybridisation tests. A survey of apri- cot orchards in Southern Italy did not show the presence of ApLV.
[Show abstract][Hide abstract] ABSTRACT: SUMMARY In a recent survey of the sanitary status of stone fruits in Palestine, a virus (isolate PL27) was transmitted to Nicotiana occidentalis by mechanical inoculation from Japanese plum. Purified virus consisted of quasi-spheri- cal particles 26-35 nm in diameter which sedimented as 3-4 components in density gradient centrifugation. Virus coat protein had an estimated molecular mass of ca. 25 kDa, while four encapsidated RNA bands were visible in electrophoregrams of purified nucleic acid. The nucleotide sequence of a 385 bp PCR-generated amplicon from RNA-3 was determined showing 99% identity at the nucleic acid level and 98.9% similarity at the aminoacid sequence level with a previously charac- terized RNA-3 region of American plum line pattern virus (APLPV). A digoxigenin-labelled probe was syn- thesized which specifically recognized virus isolates in extracts from herbaceous and woody samples but did not cross-hybridize with Prunus necrotic ringspot virus (PNRSV), Apple mosaic virus (ApMV), and Prune dwarf virus (PDV). An antiserum to PL27 was raised, which recognized homologous antigens but not PDV and PNRSV. An ELISA kit prepared with this antiserum was successfully used for the detection of PL27 in in- fected Prunus species. Based on particle morphology, biological, serological, and molecular properties, PL27 was identified as an isolate of APLPV.