Grapevine Leafroll-associated Closterovirus Research Articles

Early detection of grapevine leafroll virus in Vitis vinifera using in vitro micrografting

Micrografting of grapevine was investigated for its use as a tool in virus indexing of grapevine stock. Cabernet franc and Cabernet sauvignon scions infected with grapevine leafroll-associated closterovirus III (GLRaVIII) were grafted on to virus-free indicator rootstocks of LN 33 and Cabernet sauvignon growing in tissue culture. The two rootstocks and two scions were grafted in all four possible combinations along with two control grafts (virus-free scion on virus-free rootstock). A modified MS Murashige and Skoog (1962) tissue culture medium supplemented with 0.5 mg l−1 6-benzylaminopurine was sufficient to induce multiple shoots. Shoots and micrografts readily produced roots in the basal medium. Micrografting gave an overall success rate of 77.8%, with no significant difference between LN 33 rootstock and Cabernet sauvignon. When leafroll infected scion material was micrografted on to virus-free rootstock, the rootstock leaf turned red (23.5% in LN 33 and 63.9% in Cabernet sauvignon) or it showed leafrolling (28.5%, no significant difference between LN 33 and Cabernet sauvignon) within 2–3 weeks. After 12 weeks in culture, the extent of viral symptoms in the micrografted material was high (81.3%), with no significant difference between LN 33 and Cabernet sauvignon; however, the expression of symptoms was more severe on Cabernet sauvignon than on LN 33 rootstock. Double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) was used to validate the visual symptoms and the presence of virus was confirmed in 80% of the rootstock with visual symptoms of infection. Results indicate that micrografting is an effective method for viral indexing of grapevines. The method can be used in conjunction with wood indexing for post-entry quarantine to identify infected material and reject it much earlier than is currently possible.

Detection of Grapevine A vitivirus in Tunisian grapevines

Grapevine A vitivirus (GVA), Grapevine B vitivirus (GVB), Grapevine leaf‐roll associated closterovirus 3 (GLRaV3) and Grapevine fanleaf nepovirus (GFLV) were detected by DAS‐ELISA in samples of grapevine showing symptoms of rugose wood from different viticultural areas of Tunisia. After optimization of experimental conditions, PCR and IC‐RT–PCR were found to be more efficient and sensitive than DAS‐ELISA for the detection of GVA.

Grapevine leafroll-associated closterovirus 7 in Greece

An extensive survey was carried out to assess, by ELISA, the occurrence of grapevine closteroviruses in several grape-growing areas of Greece, with particular regard to GLRaV-7. Samples were collected in commercial vineyards and varietal collections from apparently symptomless and symptomatic leafroll vines. GLRaV-7 was widely distributed and frequently associated with GLRaV-1 and GLRaV-3. ELISA was suitable for GLRaV-7 detection but the reagents utilized in this study must be improved.

Development of Monoclonal Antibodies Reactive to a New Grapevine Leafroll-Associated Closterovirus.

Monoclonal antibodies reactive to a previously uncharacterized grapevine leafroll-associated virus (GLRaV) coat protein were developed. A novel 37-kDa protein was found associated with grapevine leafroll disease in a mixed virus infection. The polypeptide was separated from the 38-kDa polypeptide associated with GLRaV-1 using a Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis system. Concentrated p37 preparations were used to immunize mice for the production of monoclonal antibodies (MAb). Serum collected from the immunized mice showed reactivity with the 37-kDa polypeptide but no reactivity with the 38-kDa polypeptide associated with GLRaV-1 using the Western blot assay. The polyclonal antiserum reacted to both native and heat-denatured virus preparations. Two sets of anti-p37 MAbs were developed. One set binds p37 and also cross reacts with other GLRaV proteins, such as p36 of GLRaV-4 and -5, while the second set recognizes p37 specifically. We obtained the partial nucleotide sequence of the putative p37 viral protein. The predicted amino acid sequence revealed the presence of three out of the five conserved amino acids present in closterovirus capsid and diverged duplicate capsid proteins. We propose the name grapevine leafroll-associated virus-8 for the new virus with a 37-kDa capsid protein.

Effective application of DAS-ELISA for detection of grapevine leafroll associated closterovirus-3 using a polyclonal antiserum developed from recombinant coat protein

A polyclonal antiserum (As163) specific to grapevine leafroll associated closterovirus-3 (GLRaV-3) was developed using a recombinant coat protein expressed in E. coli from a cDNA clone identified after immunoscreening of a cDNA library. Specificity of the antiserum to GLRaV-3 was shown by Western blot and immunosorbent electron microscopy. With this antiserum, an effective double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for GLRaV-3 detection. To evaluate the sensitivity of the antiserum in DAS-ELISA for virus detection, different combinations of antibodies were compared. Although best results were obtained when As163 was used for coating and a monoclonal antibody (MabNY1.1) was used as an enzyme conjugate, good results were also obtained when As163 was used both for coating and as an enzyme conjugate. Using this As163–Mab system in DAS-ELISA, we confirmed the presence of GLRaV-3 in a diverse collection of leafroll infected vines.

Transformation of five grape rootstocks with plant virus genes and a virE2 gene from Agrobacterium tumefaciens

To facilitate the development of transgenic grapevines that are resistant to grapevine fanleaf virus (GFLV), grapevine leafroll-associated closterovirus (GLRaV-3) and crown gall diseases, we developed a rapid system for regenerating root-stocks: Couderc 3309, Vitis riparia ‘Gloire de Montpellier’, Teleki 5C, Millardet et De Grasset 101-14, and 110 Richter via somatic embryogenesis. Embryo culture and grape regeneration were accomplished with four media. Embryogenic calluses from anthers were induced in the initiation medium [MS basic medium containing 20 g sucrose per L, 1.1 mg 2,4-dichlorophenoxyacetic acid (2,4-D) per L, 0.2 mg N6-benzyladenine (BA) per L, and 0.8% Noble agar). The percentage of anthers that developed into embryogenic calli ranged from 2 to 16.3% depending on the rootstock. Calluses with early globular stage embryos were cocultivated with Agrobacterium tumefaciens strain C58Z707 containing the gene constructs of interest. The genes were sense-oriented translatable and antisense coat protein genes from GFLV and GLRaV-3, a truncated HSP90-related gene of GLRaV-3 (43K), and a virE2 del B gene from A. tumefaciens strain C58. Twenty independent transformation experiments were performed on five rootstocks. After 3–4 mo. under kanamycin selection, secondary embryos were recovered on differentiation medium (1/2 MS salts with 10 g sucrose per L, 4.6 g glycerol per L, and 0.8% Noble agar). Embryos that were transformed were regenerated on a medium containing MS salts with 20 g sucrose per L, 4.6 g glycerol per L, 1 g casein hydrolysate per L, and 0.8% Noble agar. Elongated embryos were then transferred to a rooting medium supplemented with 0.1 mg BA per L, 3 g activated charcoal per L, 1.5% sucrose, and 0.65% Bacto agar. A total of 928 independent putative transgenic plants were propagated in the greenhouse. All plants were tested for neomycin phosphotransferase II expression by enzyme-linked immunosorbent assay (ELISA). The presence of transgenes was assessed by polymerase chain reaction and Southern analysis. ELISA revealed various levels of expression of GFLV coat protein in transgenic plants of Couderc 3309. The transgenic rootstocks that have been generated are being screened to determine whether transgenes have conferred resistance to the virus and crown gall diseases.

Leafroll Virus Is Common in Cultivated American Grapevines in Western New York.

American grapevines (Vitis labrusca L. 'Niagara'; Vitis × labruscana L. H. Bailey 'Concord' and 'Catawba'; V. labrusca × V. riparia Michx. 'Elvira') from 24 vineyards in the New York portion of the Lake Erie production region (>13,000 ha cultivated) were tested to explore a possible relationship between virus infection and an unexplained fruit set malady in the district. One-year-old cane segments were collected 4 to 6 weeks before budbreak from 65 individual vines, which previously had been identified as malady positive or negative. Preparations from bark scrapings were tested for the presence of double-stranded (ds) RNA and for fan leaf degeneration virus, tobacco streak virus, and grapevine leafroll associated closterovirus-3 (GLRaV-3) by enzyme-linked immunosorbent assay (ELISA). Mechanical transmission of other potential viruses to Chenopodium quinoa was attempted with sap extracted from young shoots forced from intact segments of sampled canes. GLRaV-3 was detected in 17 (26%) of the sampled vines from eight (33%) of the vineyards, but there was no apparent relationship between infected vines and the fruit set malady. Vines of all four cultivars were infected. dsRNA was detected in all 17 samples positive for GLRaV-3 plus four additional samples. No other viruses were detected. Near harvest, nine vines (from two vineyards) previously testing positive for GLRaV-3 were examined and retested; all nine tested positive again, although none showed any overt symptoms of viral infection. This is believed to be the first report of GLRaV-3 from American grape vineyards in New York. The source of these infections is unknown: all vines were self rooted, the individual vineyards had been planted independently at different times, and V. vinifera and its hybrids are rare in the district. Wild grapevines (primarily V. riparia) are abundant in the region, although it has been reported that leafroll disease does not occur naturally in wild North American grapes (1). Nevertheless, our results indicate that cultivated American grapevines can be common reservoirs of GLRaV-3, and furthermore suggest the need to reassess the possibility that wild grapes also may serve as reservoirs of the virus. Trials are currently underway to determine possible effects of GLRaV-3 on cv. Concord, the most widely planted variety in the region. Reference: (1) A. C. Goheen. 1988. Leafroll. Page 52 in: Compendium of Grape Diseases. R. C. Pearson and A. C. Goheen, eds. American Phytopathological Society, St. Paul, MN.

Nucleotide sequence of the 3'-terminal two-thirds of the grapevine leafroll-associated virus-3 genome reveals a typical monopartite closterovirus.

The RNA genome of grapevine leafroll-associated closterovirus-3 (GLRaV-3) was cloned as a cDNA generated from GLRaV-3-specific dsRNA, and a partial genome sequence of 13154 nucleotides (nt) including the 3' terminus was determined. The sequenced portion contained 13 open reading frames (ORFs) potentially encoding, in the 5'-3' direction, proteins of > 77 kDa (ORF1a; helicase, HEL), 61 kDa (ORF1b; RNA-dependent RNA polymerase, RdRp), 6 kDa (ORF2), 5 kDa (ORF3, small transmembrane protein), 59 kDa (ORF4; heat shock protein 70, HSP70), 55 kDa (ORF5), 35 kDa (ORF6; coat protein, CP), 53 kDa (ORF7; diverged coat protein, CPd), 21 kDa (ORF8), 20 kDa (ORF9), 20 kDa (ORF10), 4 kDa (ORF11), 7 kDa (ORF12), and an untranslated region of 277 nt. ORF1b is probably expressed via a +1 ribosomal frameshift mechanism, most similar to that of lettuce infectious yellows virus (LIYV). Phylogenetic analysis using various gene sequences (HEL, RdRp, HSP70 and CP) clearly demonstrated that GLRaV-3, a mealybug-transmissible closterovirus, is positioned independently from aphid-transmissible monopartite closteroviruses (beet yellows, citrus tristeza and beet yellows stunt) and whitefly-transmissible bipartite closterovirus (lettuce infectious yellows, LIYV). However, another alleged mealybug-transmissible closterovirus, little cherry virus, was shown to be more closely related to the whitefly-transmissible LIYV than to GLRaV-3.

Characterization of Grapevine Rugose Wood Disease Sources from Italy.

A collection of 27 sources of grapevine rugose wood (RW) disease from a viticultural region in northern Italy was analyzed by graft-inoculating vines of three selective Vitis indicators (V. rupestris cv. St. George, V. berlandieri × V. riparia cv. Kober 5BB, and hybrid cv. LN 33). On the basis of stem reactivity, different groups were identified among the selected RW inoculum sources: nine isolates induced pitting only on cv. St. George, whereas four induced grooving only on cv. Kober 5BB. These two groups were classified as isolates of rupestris stem pitting and Kober stem grooving. Three of the remaining isolates induced wood abnormalities on cvs. LN 33 and Kober 5BB, seven induced wood abnormalities on cvs. St. George and Kober 5BB, and four induced symptoms on all three indicators. These groups may represent RW sources with various disease combinations. RW-affected grapevine clones used as inoculum sources also were tested for virus infections by enzyme-linked immunosorbent assay (ELISA). ELISA revealed the presence of grapevine fleck virus, grapevine leafroll-associated closterovirus 1 and 3, and grapevine trichovirus A. These viruses infected most of the selected RW sources. However, eight of the latter were ELISA-negative. The findings are discussed, and the biological and etiological complexity of the RW phenomena in grapevine is confirmed.

The coat protein gene of grapevine leafroll associated closterovirus-3: cloning, nucleotide sequencing and expression in transgenic plants.

A lambda ZAP II cDNA library was constructed by cloning cDNA prepared from a high molecular weight double-stranded RNA (dsRNA, ca. 18 kb) isolated from grapevine leafroll associated closterovirus-3 (GLRaV-3) infected tissues. This cDNA library was immuno-screened with GLRaV-3 coat protein specific polyclonal and monoclonal antibodies and three immuno-positive clones were identified. Analysis of nucleotide sequences from these clones revealed an open reading frame (ORF) which was truncated at the 3' end; the remainder of this ORF was obtained by sequencing a fourth clone that overlapped with one of the immunopositive clones. A total of 2028 bp was sequenced. The putative GLRaV-3 coat protein ORF, 939 bp, encodes a protein (referred to as p35) with a calculated M(r) of 34866. Multiple alignment of the p35 amino acid sequence with coat protein sequences from other closteroviruses revealed that the consensus amino acid residues (R and D) of filamentous plant viruses are preserved in the expected locations. The GLRaV-3 coat protein gene was then engineered for sense and antisense expression in transgenic plants. Transgenic Nicotiana benthamiana plants that contain the sense GLRaV-3 coat protein gene produced a 35 kDa protein that reacted with GLRaV-3 antibody in Western blot.

A spot-PCR technique for the detection of phloem-limited grapevine viruses

Specific amplification of genomic fragments of grapevine trichovirus A (GVA), grapevine trichovirus B (GVB) and grapevine leafroll-associated closterovirus 3 (GLRaV3) was obtained by reverse transcription-PCR on total nucleic acid solubilized from small pieces of charged nylon membrane, on which a drop of crude infected grapevine sap was spotted (spot-PCR). A thermal treatment (95 ° for 10') of spotted sap in a buffered solution improved the release of viral template. Consistent amplification was obtained with three viruses from fragments of the same respective blots up to 1 month after spotting, while a detection threshold limit comparable with standard PCR techniques was found for this method. Duplex PCR (i.e. amplification of different viruses from a mixed-infected grapevine source) was also found to be effective, since GVA and GLRaV3 were amplified by a mixture of specific primers in the same reaction. This rapid and easy sampling technique, using leaf petioles to express crude sap, may have a wide field application for screening grapevine viruses.

Improved RNA Extraction from Woody Plants for the Detection of Viral Pathogens by Reverse Transcription-Polymerase Chain Reaction.

An efficient procedure for the extraction of high-quality RNA from woody plants without the use of phenol, organic solvents, or alcohol precipitation is described. The method employs commercially available spin-column matrices and mitigates the inhibitory effects of plant polysaccharides and polyphenolic compounds commonly observed on subsequent polymerase chain reaction amplification when conventional extraction methods are applied to woody plant species. The method described has been successfully used in the development of highly sensitive reverse transcription-polymerase chain reaction (RT-PCR) techniques for the detection of a number of viruses in their woody hosts. The viruses detected included apple stem grooving capillovirus (ASGV), apple stem pitting virus, Prunus necrotic ringspot ilarvirus (PNRSV), grapevine fanleaf and Arabis mosaic nepoviruses, and grapevine leafroll-associated closterovirus type 3. The method described was equally effective for the extraction of viral RNA from either budwood, leaves, or flower blossoms as determined by the equivalent RT-PCR detection of ASGV and PNRSV from these tissues. Detection of viral RNA in samples of total plant RNA prepared using this method was found to be as sensitive as was previously described for the immunocapture RT-PCR technique.

Viruses and virus diseases of grapevine in Lebanon

Grapevines were surveyed for the presence of virus and virus‐like diseases in the main viticultural areas of Lebanon (Bekaa valley, Mount Lebanon, South and North Lebanon). Symptoms of rugose wood were observed in vines ofall cultivars and areas surveyed, whereas leafroll was observed only in some vineyards of the Bekaa valley and, to a lesser extent, in South Lebanon on cvs Tfaifihi, Cinsaut and Cardinal. Symptoms of fanleaf and of phytoplasma‐induced yellows were also observed with low frequency in the Bekaa valley on wine‐grape cultivars. ELISA tests showed that 53% of 1536 Vitis vinifera vines individually checked were infected by one or more viruses. Grapevine trichovirus A (GVA) was the prevailing virus (32.4%), followed by grapevine fleck virus (GFkV) (19.5%) and grapevine leafroll‐associated closterovirus 3 (GLRaV‐3) (12.4%). Grapevine leafroll‐associated closterovirus 1 (GLRaV‐l), grapevine trichovirus B (GVB) and grapevine fanleaf nepovirus (GFLV) were also detected to a lesser extent, their incidence ranging between 1.1 and 3.6%.