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The alignment of the complete genomes of genetic variants of Grapevine leafroll-associated virus 3 (GLRaV-3) representing phylogenetic groups I, II, III and VI revealed numerous regions with exceptionally high divergence between group I to III and group VI variants. Oligonucleotide primers universal for all the above groups of the virus were designed in conserved short stretches of sequences flanking the divergent regions in the helicase (Hel) and RNA-dependent RNA polymerase (RdRP) domains of the replicase gene and the divergent copy of the capsid protein ( dCP) gene. Cloning and sequencing of the 549-bp RT-PCR amplicon of the helicase domain from grapevine cv. Shiraz lead to the detection of a variant of GLRaV-3, which shared only 69.6-74.1% nt similarity with other variants, including the recently reported, new, highly divergent variant, isolate 139. This was confirmed by the results of the analysis of 517-bp amplicon of the HSP70 gene of GLRaV-3 generated in RT-nested PCR based on degenerate primers for the simultaneous amplification of members of the Closteroviridae family designed by Dovas and Katis (J Virol Methods, 109, 2003, 217). In this genomic region, the variant shares 72.3-78.7% nt similarity with other variants of GLRaV-3. This previously unreported, new, highly divergent variant was provisionally named GTG10. From the alignment of the HSP70 sequences primers for the specific RT-nested PCR amplification of the variant GTG10 and members of group VI, and specific simultaneous amplification of variants of groups I, II and III, were designed. The results obtained from brief testing of various grapevines using all these primers suggest a relatively limited presence of GTG10 variant in vineyards. [ABSTRACT FROM AUTHOR]

Diplodia seriata, Phaeomoniella chlamydospora and Phaeoacremonium aleophilum are the three main species associated with grapevine decline in Spain. AFLP markers were developed to discriminate Spanish populations of these species. The markers were used to genotype isolates of D. seriata, P. chlamydospora and P. aleophilum. AFLP markers were valuable in performing population genetic studies as genetic variability (Kx) ranged from 0.07 in the P. chlamydospora population to 0.28 in the D. seriata population. Species-specific markers obtained using only two AFLP combinations clearly discriminate D. seriata, P. chlamydospora and P. aleophilum and are a useful tool in simultaneous identification tests. [ABSTRACT FROM AUTHOR]

Vineyards in the Atacama region in Chile were surveyed from 2007 to 2009 for the presence of viruses. This region is an important area of table grape production, supplying international markets with its fruits in the off season of the Northern Hemisphere. Reverse transcription-polymerase chain reaction (RT-PCR) assay was used to detect the most economically important grapevine viruses in 1000 samples, including symptomatic and asymptomatic plants. The rate of positive samples was 8.8% for Grapevine leafroll-associated virus 1 (GLRaV-1), 46.8% for Grapevine leafroll-associated virus 2 (GLRaV-2), 9.1% for Grapevine leafroll-associated virus 3 (GLRaV-3), 12.3% for Grapevine virus A (GVA), 30.7% for Grapevine fleck virus (GFkV) and 9.6% for Grapevine fanleaf virus (GFLV). Overall virus infection was 68.7%. DNA sequencing confirmed the identification of viruses in selected samples, and comparative analysis indicated that Chilean isolates have moderate-to-high molecular identities with corresponding virus reference strains selected from GenBank. The high level of viral infection observed indicates that viruses are involved in decreasing table grape production in the region. This is the first extensive virus survey performed in the Atacama region, is also the first study of genetic comparison of grapevine viruses developed in South America with a wide spectrum of viruses and isolates and provides an assessment of grapevine viruses on table grape. [ABSTRACT FROM AUTHOR]

Surveys for virus diseases and nematode vectors were conducted in 95 commercial vineyards of four different Lebanese districts (Bekaa valley, Mount Lebanon, North and South Lebanon). Out of 915 randomly collected grapevine samples tested by ELISA, 511 (55.8%) were infected by one or more viruses. Grapevine virus A (30.9%) and Grapevine leafroll-associated virus 3 (23.7%) were the prevailing viruses, followed by Grapevine fleck virus (15.1%), Grapevine leafroll-associated virus 1 (10.6%) and Grapevine leafroll-associated virus 2 (8.7%). Arabis mosaic virus was not found whereas Grapevine fanleaf virus (GFLV) and Grapevine virus B were little represented. The most important Lebanese grapevine varieties, i.e. Maghdouchi, Tfeifihi and Beitamouni, had average infection rates between 70% and 87%, whereas varieties of foreign origin had a better sanitary status with the exception of cvs Cinsaut and Thompson ( c. 83% infection). Grapevine rupestris stem pitting-associated virus was detected in 79 of 90 (87.8%) samples tested by RT-PCR and closteroviruses were recorded in seven of 70 (10%) vines tested. One of these viruses was identified as Grapevine leafroll-associated virus 5 by ELISA and partial genome sequencing. No nepoviruses other than GFLV were detected in any of 90 samples tested using three different sets of degenerate primers. Xiphinema index was found in 23 of 89 soil samples collected from vineyards, and in three of 15 samples collected primarily under fig trees in fields where no grapevines were grown. [ABSTRACT FROM AUTHOR]

Grapevine leafroll associated virus 2 (GLRaV 2) is one of the important components in the leafroll disease complex. The coat protein gene of GLRaV 2 was cloned into a protein expression vector pMAL-c2x and the recombinant protein, consisting of the maltose binding protein (MBP) and GLRaV 2 coat protein (CP), was expressed in Escherichia coli. The recombinant MBP-CP was used to raise a high quality antiserum. When used in Western blot analysis, the anti-MBP-CP antiserum produced specific reaction to the recombinant protein as well as to the viral coat protein of GLRaV 2. In Immunosorbent electron microscopy study, the anti-MBP-CP antibodies strongly decorated the GLRaV 2 virions. Using the newly developed antiserum, an indirect plate-trapped antigen enzyme-linked immunosorbent assay method was developed and successfully implemented for virus detection. A field survey was conducted to evaluate the virus infection status by GLRaV 2 and GLRaV 3 using antibodies developed against their respective recombinant coat proteins. [ABSTRACT FROM AUTHOR]

The detectability of grapevine-leafroll-associated virus 3 (GLRaV-3) was investigated by enzyme-linked immunosorbent assay (ELISA) and reverse-transcriptase polymerase chain reaction (RT–PCR) in the domestic grapevine cultivar Sakasly, grown over two successive years in the Sahara, at Rjim-Mâatoug in Tunisia. Self-rooted cuttings, infected with GLRaV-3 were cultivated and the presence of the virus was checked over 2 years and compared with controls. During the first year, 80% of the originally infected vines were negative for GLRaV-3 using ELISA. After the second year, 93% and 95% of these plants were negative for GLRaV-3 using ELISA and RT–PCR, respectively. Furthermore, rooted cuttings derived from GLRaV-3-negative plants and grown under controlled conditions in a greenhouse (at 16–20°C) were ELISA-negative in most cases (84%). In addition, biological indexing on Vitis vinifera cv. Gamay Rouge de la Loire showed no leafroll symptoms on this indicator in 92% of the grafted vines. These results suggest a naturally occurring heat therapy in the Tunisian Sahara, which could be of practical importance for the production of GLRaV-3-free grapevine cuttings, especially as scale insect and mealybug vectors have not been observed in this area. [ABSTRACT FROM AUTHOR]

Grapevine leafroll-associated virus 3 (GLRaV-3) is an important pathogen of grapevine in the Czech Republic. A Czech isolate was studied at the molecular level in order to extend data on molecular variability of the virus from the central European region. Three conserved regions (genes encoding the RNA-dependent RNA polymerase, the heat shock protein 70 and the 55 kDa protein) were amplified using reverse transcriptase-polymerase chain reaction, sequenced and compared with related available sequences. On the sequenced regions, the Czech isolate revealed more than 99% nucleotide and amino acid identity with the published full-length sequence of a North American GLRaV-3 isolate and a similarly high identity with available partial GLRaV-3 sequences of isolates from different parts of the world. The phylogenetic position of the new and already characterized GLRaV-3 isolates within the Ampelovirus genus is discussed. [ABSTRACT FROM AUTHOR]

Grapevine leafroll-associated virus 2 (GLRaV-2) was detected by serological and molecular analyses in several grapevine accessions of different varieties from Italian, Greek, French and Brazilian vineyards in a 2001–2002 survey. In order to study the genetic variability among GLRaV-2 isolates in the open reading frame (ORF) coding the coat protein (CP), heteroduplex mobility assays were performed on 17 isolates and six strains used as reference. Eight diverse GLRaV-2 variants were identified among the infected grapevines tested. The most common variant was found in the majority of the samples characterized; it was indistinguishable from the reference strains from the Semillon and Pinot noir 95 accessions. GLRaV-2 variants found in Italian cvs Negro amaro and Vermentino were identical to the reference strain from cv. Muscat de Samos (Greece). Three other GLRaV-2 variants from Southern and Central Italy were different from all the reference strains. A grapevine accession from Tuscany was found to contain two diverse GLRaV-2 variants. None of the variants tested sample identical to the American strain H4 or the reference strains from cvs Chasselas 8386 (Switzerland) or Alphonse Lavallée 224 (France); the latter three accessions were different from one another. The estimated nucleotide homology in CP gene among 23 GLRaV-2 isolates was in some cases <88%. [ABSTRACT FROM AUTHOR]

We have studied the effect of grapevine leafroll infection on some features of the thylakoids from field grown grapevine ( Vitis vinifera L.) leaves. Changes in photosynthetic pigments, soluble proteins, ribulose-1,5-bisphosphate carboxylase (RuBP), nitrate reductase, photosynthetic activities and thylakoid membrane proteins were investigated. The level of total chlorophyll (Chl) and carotenoids were reduced in virus-infected leaves. Similar results were also observed for soluble proteins and RuBP case activity. The in vivo nitrate reductase activity was significantly reduced in infected leaves. Virus infection considerably decreased leaf net photosynthetic rate ( Pn), stomatal conductance ( gs) and transpiration rate ( E) in grapevine leaves. When various photosynthetic activities were followed in isolated thylakoids, virus infection caused marked inhibition of whole chain and photosystem (PS) II activity while the inhibition of PSI activity was only marginal. The artificial exogenous electron donors, diphenyl carbazide and hydroxylamine (NH2OH) significantly restored the loss of PSII activity in infected leaves. The same results were obtained when Fv/ Fm was evaluated by Chl fluorescence measurements. The marked loss of PSII activity in infected leaves could be due to the loss of 47, 43, 33, 28–25, 23 and 17 kDa polypeptides. It is concluded that virus infection inactivates the donor side of PSII. This conclusion was confirmed by immunological studies showing that the content of the 33 kDa protein of the water-splitting complex was diminished significantly in infected leaves. [ABSTRACT FROM AUTHOR]

Reliable and highly sensitive detection of grapevine virus A (GVA) in grapevine leaf tissue by reverse transcription-initiated polymerase chain reaction was achieved after enrichment and partial purification of the virus from grapevine crude sap by an immunocapture (IC) procedure. In comparison with ELISA the gain of sensitivity was estimated up to 1000-fold in grapevine mature leaf tissue. Using IC-reverse transcription polymerase chain reaction (IC-RT-PCR), GVA could be detected in very young grapevine tissues where the virus titre was largely below the detection limit of ELISA. The comparison of IC-RT-PCR and biological indexing results for about 60 grapevine accessions revealed a good correlation between the presence of GVA and the development of Kober stem grooving (KSG) symptoms in the specific indicator rootstock Kober 5BB. Moreover, the elimination of KSG by heat treatment in two Vitis vinifera cultivars, Savagnin rose and Servant, was accompanied by the loss of detection of GVA by PCR, thus providing further argument for the involvement of GVA in the aetiology of KSG. [ABSTRACT FROM AUTHOR]

Different fixation and embedding procedures have been tested in order to facilitate closterovirus identification on thin sections of leafroll-affected grapevine plants. Standard fixation with glutaral-dehyde followed by osmium tetroxide proved to be the most reliable for cytopathological studies while simultaneous fixation with glutaraldehyde, picric acid and osmium tetroxide facilitated the discrimination between aggregates of P-proteins and virus particles in routine e. m. work. However the identification of every single virion among P-protein filaments was only possible by means of post-embedding immunogold labelling carried out on non-osmicated tissues embedded in London Resin White. This technique has also been applied to both section sides thus allowing the identification of different closteroviruses contemporaneously present in the same phloem tissue. Numerous ultrastructural observations of different grapevine cultivars infected with either GVA, or GLRaV-I or GLRaV-III evidentiated the presence of some recurrent and peculiar cytopathic effects that can be used as diagnostic parameters for at least two of the above viruses. [ABSTRACT FROM AUTHOR]

Three hybridoma lines secreting monoclonal antibodies to a closterovirus-like particle (GLRV- 3) associated with grapevine leafroll disease were produced. One of the antibody (MAbl) reacted with one extremity of trie filamentous virus particle whereas the other two (MAb2 and 3) reacted with the entire surface of the virus particle. Using MAb2 in ELISA it is possible to detect 46 of the 50 GLRV-3 isolates. In order to detect all the 50 isolates, it is necessary to use MAbl in ELISA in conjunction with polyclonal antibodies. In immunoblotung, the three MAbs recognized a 43 Kd viral protein. [ABSTRACT FROM AUTHOR]

Four serologically unrelated closterovirus-like particles (GLRV-1, GLRV-2, GLRV-3 and GLRV-4) were isolated in our laboratory from leafroll diseased grapevines. Polyclonal antibodies raised against these particles were useful for their characterization and their detection in infected plants. The coat proteins of these four serotypes were characterized by a SDS-PAGE after denaturation, followed by a transfer on nitrocellulose sheet and immunoprinting using the specific polyclonal antibodies. The capsid of GLRV-1, GLRV-2, GLRV-3 and GLRV-4 contains a single protein species with molecular weight of about 39 Kd, 26 Kd, 43 Kd and 36 Kd respectively. No serological relation was found between these four filamentous particles either by ELISA, immuno electron microscopy or immunoblotting experiments. Serological analysis of many grapevines originating from the Middle East and Europe showed a very close association between the presence of GLRV-1, GLRV-2, GLRV-3 and GLRV-4 antigens, and leafroll symptoms on Vitis vinifera Pinot Noir. This association was confirmed by testing symptomless and diseased grapevines collected in the field, and by serological analysis of heat treated plants originally infected by GLRV-1 and GLRV-3, which are the most widespread antigens detected in leafroll infected grapevines. [ABSTRACT FROM AUTHOR]

Grapevine phloem-limited isometric virus (GPLIV) is the name proposed for a non mechanically-transmissible virus found in Italian and Tunisian grapevines. In density gradient centrifugation purified virus preparations sedimented as two components: T, made up of empty protein shells, and B, composed of intact nucleoprotein particles. B particles had a buoyant density of 1.45 g/cm³ at equilibrium in CsCl and contained 35% RNA consisting of a single molecule with an apparent size of 7.4 kb. The coat protein consisted of a single species with a mol.wt of 28,000 daltons. Purified virus preparations did not infect herbaceous hosts by manual inoculation. A specific antiserum with a titre of 1:64 raised in rabbits, was used for identification of GPLIV in field-grown Tunisian grapevines and in leafroll-affected Italian vines before and after heat treatment. Although heat treatment eliminated the virus from the majority of the plants, leaf roll symptoms persisted in several GPLIV- free vines, indicating that there is no clear-cut relationship between GPLIV and this disease. [ABSTRACT FROM AUTHOR]

A purification procedure (Zee et al. 1987) for closterovirus-like particles associated with grapevine leafroll disease was improved after various sources of plant tissues, extraction buffers, pH values, and centrifugation techniques had been examined. An antiserum against the CA-4 isolate was produced and used in ELISA. The virus-like particles of the NY-1 isolate measured in crude plant preparations were about 1,800 to 1,900 nm long. THe molecular weight of coat protein of the NY-1 and NY-2 isolates were ca. 43 x 10³ daltons in SDS-PAGE analysis; and bands having this molecular weight reacted in Western blotting tests with specific polyclonal and monoclonal antibodies. A large dsRNA molecule (ca. 10 x 106 Mr) along with lower molecular weight species were isolated from leafroll diseased grapevines, but not from healthy grapevines. Seven leafroll isolates were tested for their serological relatedness in a protein A-gold labelling immunosorbent electron microscopy assay. Results indicated that serologically distinct serotypes existed, and mixed infection of grapevines with different serotypes was common. A grouping of isolates into serotype I, II, II, IV is proposed.. [ABSTRACT FROM AUTHOR]

Surveys were carried out in Tunisian table grape vineyards for assessing the occurrence and distribution of grapevine leafroll-associated viruses (GLRaVs). Leafroll symptoms were commonly observed in most of the surveyed vineyards. Samples were randomly collected from 712 individual vines for laboratory testing. Enzyme-linked immunosorbent assay (ELISA) tests showed that 81.5% of the vines were infected by one (35.7%) or more (45.8%) viruses. GLRaV-3 was the most widespread virus (76.3%), followed by GLRaV-5 (38.5 %), GLRaV-6 (13.2%), GLRaV-1 (9.1%), GLRaV-2 (6.3%), and GLRaV-7 (0.9%). GLRaV-3 and GLRaV–5, two mealybug-transmissible Ampeloviruses, were present in mixture in 35.9% of samples. The highest infection rate was found in Cape Bon region (81.7 %), where cv. Italia had an infection rate of 79.5%. Superior seedless, the main cultivar in Sidi Bouzid, had 75% infection. GLRaV-6 and -7 were detected for the first time in Tunisia. [ABSTRACT FROM AUTHOR]

Grapevines in central Anatolia region of Turkey were surveyed for the prevalence of grapevine leafroll viruses. The field study and collection of samples were conducted in nine major grapevine-growing areas. Samples collected from 622 vines were tested for Grapevine leafroll-associated virus 1, 2, 3 and 7 (GLRaV-1, -2, -3 and -7). According to diagnostic tests and surveys, 27 of 41 cultivars and 95 of 622 samples (15.27%) were found to be infected at least one virus. GLRaV-1 (8.36%) was found to be the most frequently encountered virus associated with leafroll disease of grapes, followed by GLRaV-3 (5.78%), GLRAV-7 (3.86%) and GLRAV-2 (2.41%). [ABSTRACT FROM AUTHOR]

Isolates of grapevine virus B (GVB) and grapevine virus A (GVA) were obtained by mechanical transmission from grapevines to Nicotiana benthamiana. Western blot studies of these isolates using various antisera to GVB and GVA showed that they are serologically related. [ABSTRACT FROM AUTHOR]

Surveys were made in the main grape growing region (Southeast Anatolia) of Turkey for the occurrence of Grapevine leafroll-associated virus-5 (GLRaV-5). Plant samples with typical leafroll symptoms and mealybugs, Planococcus ficus (Signoret) were used for assessing the occurrence of GLRaV-5 by RT-PCR. A 272 bp band representing GLRaV-5 infection was successfully detected in plants and mealybugs in some vineyards of the Southeast Anatolia region and the virus is the first time reported in Turkish vineyards. [ABSTRACT FROM AUTHOR]