Fanleaf Virus Research Articles

Molecular detection of Grapevine fanleaf Virus by the isolation of ssRNA and dsRNA from Xiphinema index

Xiphinema index is an important grapevine pathogen nematode which also vectors Grapevine fanleaf virus. The viral genes involved in transmission by the vector nematode are mapped to the C-terminal residues of RNA2-encoded polyprotein. To recognize viruliferous nematodes, there are some serological and molecular methods. In this study, we extract RNA and dsRNA of the virus, then Reverse transcription-polymerase Chain Reaction was done with virus specific primers to detect virus in its vector. The virus was detected by visualizing the desired 350 and 750 bp gene fragments in electrophoresis. This study reduces the virus detection time to only couple of hours with least imposed charges, and could be employed in transmission experiments as well.

The 50 distal amino acids of the 2AHP homing protein of Grapevine fanleaf virus elicit a hypersensitive reaction on Nicotiana occidentalis.

Avirulence factors are critical for the arm's race between a virus and its host in determining incompatible reactions. The response of plants to viruses from the genus Nepovirus in the family Secoviridae, including Grapevine fanleaf virus (GFLV), is well characterized, although the nature and characteristics of the viral avirulence factor remain elusive. By using infectious clones of GFLV strains F13 and GHu in a reverse genetics approach with wild-type, assortant and chimeric viruses, the determinant of necrotic lesions caused by GFLV-F13 on inoculated leaves of Nicotiana occidentalis was mapped to the RNA2-encoded protein 2AHP , particularly to its 50 C-terminal amino acids. The necrotic response showed hallmark characteristics of a genuine hypersensitive reaction, such as the accumulation of phytoalexins, reactive oxygen species, pathogenesis-related protein 1c and hypersensitivity-related (hsr) 203J transcripts. Transient expression of the GFLV-F13 protein 2AHP fused to an enhanced green fluorescent protein (EGFP) tag in N.occidentalis by agroinfiltration was sufficient to elicit a hypersensitive reaction. In addition, the GFLV-F13 avirulence factor, when introduced in GFLV-GHu, which causes a compatible reaction on N.occidentalis, elicited necrosis and partially restricted the virus. This is the first identification of a nepovirus avirulence factor that is responsible for a hypersensitive reaction in both the context of virus infection and transient expression.

Application of the Enzyme-linked Immunosorbent Assay Procedure to the Detection of Grapevine Fanleaf Virus

Enzyme-linked immunosorbent assay was used to detect grapevine fanleaf virus (GFV) directly in grapevine leaf tissue. The addition of nicotine or nicotine and sodium diethyldithiocarbamate to the extracting buffer solution greatly enhanced the sensitivity of the procedure and enabled detection of GFV concentrations as low as 12 ng/m t The conjugated GFV gamma-globulin detected the GFV strains, grapevine vein banding and grapevine yellow mosaic but failed to detect the GFV serotype arabis mosaic in grapevine leaf tissue. The technique makes possible the collection and processing of numerous samples throughout the growing season and is, therefore, particularly suitable for studies on the incidence and spread of GFV in the field. It also facilitates the resolution of disease syndromes with which GFV is associated.

The Effectiveness of in vitro Somatic Embryogenesis in Eliminating Fanleaf Virus and Leafroll Associated Viruses from Grapevines

Somatic embryos were successfully regenerated from callus tissue of anthers and ovaries extracted from inflorescences of grapevines infected with grapevine fanleaf virus (GFLV) and grapevine leafroll associated viruses (GLR) respectively. Production of pro-embryogenic masses (PEMS) was controlled by specific growth regulators and culture conditions. Somatic embryos (containing roots and cotyledons) and plantlets were subjected to immunosorbent electron microscopy (ISEM) as well as serological tests (ELISA). Results indicated that somatic embryogenesis derived from ovary tissue of infected grapevines is an effective technique to eliminate grapevine Ieafroll associated viruses from grapevines but the procedure was not successful in the elimination of GFL V from anther source material.

First Report of Grapevine fabavirus in Grapevines in China

Recently, a novel virus tentatively named grapevine fabavirus (GFabV) was reported in North America, it currently classified as a putative member of the genus Fabavirus (Al Rwahnih et al. 2016). Currently, its effects on grapevines remain unknown. In this study, leaves of three virus-eliminating ‘Beta’ grapevines (Vitis riparia×V.labrusca) showed chlorotic mottling and deformation after being grafted with ‘Ruby seedless’ grapevine (Vitis vinifera L.) scion. Before grafting, the ‘Beta’ and ‘Ruby seedless’ grapevines had been screened by RT-PCR for Grapevine leafroll-associated virus -1-4 and 7 (GLRaV-1-4 and 7), Grapevine fanleaf virus, Grapevine rupestris stem pitting-associated virus, Grapevine fleck virus, Grapevine virus A (GVA), GVB, GVE, Grapevine pinot gris virus, and Grapevine berry inner necrosis virus. No viruses were detected in the ‘Beta’ grapevines, except for GLRaV-3 in the ‘Ruby seedless’ grapevine. Additionally, small RNAs from the diseased ‘Beta’ leaves were sequenced as described previous...

The Elimination of Fanleaf Virus from Grapevines Using in vitro Somatic Embryogenesis Combined with Heat Therapy

Somatic embryos were successfully regenerated from callus tissue of anthers and ovaries excised from inflorescences of grapevines infected with grapevine fanleaf virus (GFLV). Production of pro-embryogenic masses (PEMS) was controlled by specific growth regulators and culture conditions, including heat incubation at 35°C. Somatic embryos (containing roots and cotyledons) and plantlets were subjected to immunosorbent electron microscopy (ISEM) and serological tests (ELISA). Results show that somatic embryogenesis in combination with heat therapy of the cultures is an effective procedure to eliminate GFLV from anther and ovary source material.

Viral Diversity in Autochthonous Croatian Grapevine Cultivars.

A survey was conducted on nine autochthonous grapevine cultivars grown along the Croatian coastal region. In total, 48 vines (44 from germplasm collection, 4 from vineyards) originating from 23 sites were tested for 26 viruses using molecular methods. Results revealed high infection rates with Grapevine leafroll-associated virus 3 (GLRaV-3); Grapevine virus A (GVA, both 91.7%); Grapevine fleck virus (GFkV, 87.5%); and Grapevine rupestris stem pitting-associated virus (GRSPaV, 83.3%). Other detected viruses were: Grapevine fanleaf virus (GFLV); Grapevine leafroll-associated viruses 1, 2, and strains of 4 (GLRaV-1, GLRaV-2, GLRaV-4); Grapevine viruses B, D, F (GVB, GVD, GVF); Grapevine red globe virus (GRGV); Grapevine vein feathering virus (GVFV); Grapevine Syrah virus 1 (GSyV-1); and Grapevine Pinot gris virus (GPGV). No virus-free vine was found. Mixed infections were determined in all vines, the number of viruses in a single vine ranged from three to nine. GLRaV-3 variant typing confirmed presence of group I, II, and III. Four vines with leaf deformation and mottling were positive for GPGV. Seven viruses (GLRaV-4-like group, GVD, GVE, GVF, GRGV, GSyV-1, and GVFV) were detected for the first time in Croatia. This survey confirmed the deteriorated sanitary status of autochthonous Croatian grapevine cultivars.

ICTV Virus Taxonomy Profile: Secoviridae.

Members of the family Secoviridae are non-enveloped viruses with mono- or bipartite (RNA-1 and RNA-2) linear positive-sense ssRNA genomes with the size of the RNAs combined ranging from 9 to 13.7 kb. They are related to picornaviruses and are classified in the order Picornavirales. The majority of known members infect dicotyledonous plants and many are important plant pathogens (e.g. grapevine fanleaf virus and rice tungro spherical virus). This is a summary of the current International Committee on Taxonomy of Viruses (ICTV) report on the taxonomy of the family Secoviridae available at www.ictv.global/report/secoviridae.

In vitro propagation of Vitis vinifera L. cv. ‘Monastrell’

Normal 0 false false false ES X-NONE X-NONE Background: A protocol for micropropagation of the grape ( Vitis vinifera L.) cultivar ‘Monastrell' was developed. Initial plant material was based on sanitary selection of grapevine plants performed by real-time RT-PCR, to confirm the absence of Grapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV), Grapevine leafroll-associated virus 1 (GLRaV-1), Grapevine leafroll-associated virus 3 (GLRaV-3), and Grapevine fleck virus (GFkV). Results: The effects of the salt composition (comparing Lloyd and McCown woody plant medium and Murashige and Skoog medium ½ macronutrients) and the growth regulator benzylaminopurine (BAP), at 0 and 8.9 µM, on the multiplication step were evaluated using nodes as explants. The most-efficient procedure consisted of bud induction in the medium with Lloyd and McCown woody plant salts and 8.9 µM BAP for 30 days along with elongation in cytokinin-free medium for 60 days, which gave 22 nodes/explant (174 plants/initial plant). A second cycle of multiplication in a medium without the growth regulator for another 60 days could give around 10,000 nodes; this quantity of rooted plants could be obtained after another two months of culture. All plants acclimatized after the second cycle of multiplication were successfully transferred to soil. Conclusion: We developed an optimal protocol for V. vinifera cv. Monastrell micropropagation, the first described for this cultivar. /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Calibri,sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Times New Roman; mso-bidi-theme-font:minor-bidi; mso-ansi-language:ES; mso-fareast-language:ES;}

A MULTIPLEX RT-PCR FOR SIMULTANEOUS DETECTION OF THE AGENTS OF YELLOW SPECKLE AND VEIN BANDING DISEASES IN GRAPEVINE

Six viroids have been reported to infect grapevine and among them only Grapevine yellow speckle viroid-1 (GYSVd-1) and Grapevine yellow speckle viroid-2 (GYSVd-2) are able to induce yellow speckle (YS) disease, characterized by yellowish flecks scattered on the leaf blade or massed along the veins. The mixed infection of GYSVd-1 or GYSVd-2 with Grapevine fanleaf virus (GFLV) may elicit vein banding (VB), a syndrome characterized by chrome-yellow flecks along the main veins of mature leaves and expanding through the interveinal tissues of infected leaves. In the present study, we have developed a multiplex RT-PCR (mRT-PCR) for simultaneous detection of GYSVd-1, GYSVd-2 and GFLV. Hop stunt viroid (HSVd), another viroid that infecting most grapevines worldwide is used as a positive control instead of a host- derived internal control. The test developed here proved to be reliable for simultaneous detection of three pathogens, i.e. GYSVd-1, GYSVd-2 and GFLV, in grapevine; hence it is useful for detection of YS and VB in large-scale surveys and certification programs.

Sanitary Status of the Grapevine Germplasm Collection in Republic of Srpska

In July 2015, 179 grapevine plants belonging to 16 grapevine autochthonous cultivars were assessed for sanitary status using DAS ELISA test for the presence of: Grapevine fanleaf virus (GFLV), Grapevine leafroll-associated virus 1 (GLRaV-1), Grapevine leafroll-associated virus 2 (GLRaV-2)and Grapevine leafroll-associated virus 3 (GLRaV-3). Furthermore, surveyfor the phytoplasma presence and laboratory analyses using nested-PCR/RFLP assay was conducted at the beginning of September 2015 on grapevine cultivars which were not positive in DAS ELISA test for the presence of the four viruses. Out of 179 tested plants with DAS ELISA test, 146 (81%) were positive for the presence of at least one virus. The most widespread viruses were GFLaV- 1 and GFLaV- 3 with approximately 80 % of grapevines infected. Nested–PCR/RFLP assay showed that out of 33 tested samples 2 were positive for the presence of phytoplasmas from 16SrXII group. Sanitation of infected grapevine cultivars is needed in near future.

Dynamics of the population structure and genetic variability within Iranian isolates of grapevine fanleaf virus: evidence for polyphyletic origin.

To determine the genetic diversity and population structure of grapevine fanleaf virus (GFLV), the complete nucleotide sequence of the coat protein gene of 41 isolates from different regions in Iran was determined. Phylogenetic analyses of these isolates together with those available in the GenBank revealed two evolutionary divergent lineages, designated GFLV-G and GFLV-Ir that reflect origin of the isolates. Analysis of the genetic variability in the coat protein of these isolates revealed 37 genotype groups in GFLV population. Analyses indicate that GFLV-G and GFLV-Ir clades are significantly differentiated populations of GFLV. Also, geographical subpopulations of the virus in Iran were completely distinct from each other. Examination of nonsynonymous/synonymous nucleotide diversity showed that the CP gene has been under purifying selection. The neutrality tests indicate balancing selection operating within isolates of the northwest of Iran and purifying selection within the other populations.

Candidate nematicidal proteins in a new Pseudomonas veronii isolate identified by its antagonistic properties against Xiphinema index.

The nematode Xiphinema index affects grape vines and transmits important viruses associated with fanleaf degeneration. Pseudomonas spp. are an extensive bacterial group in which important biodegradation and/or biocontrol properties can occur for several strains in the group. The aim of this study was to identify new Pseudomonas isolates with antagonist activity against X. index. Forty bacterial isolates were obtained from soil and root samples from Chilean vineyards. Thirteen new fluorescent pseudomonads were found and assessed for their antagonistic capability. The nematicide Pseudomonas protegens CHA0 was used as a control. Challenges of nematode individuals in King's B semi-solid agar Petri dishes facilitated the identification of the Pseudomonas veronii isolate R4, as determined by a 16S rRNA sequence comparison. This isolate was as effective as CHA0 as an antagonist of X. index, although it had a different lethality kinetic. Milk-induced R4 cultures exhibited protease and lipase activities in cell supernatants using both gelatin/tributyrin Petri dish assays and zymograms. Three proteins with these activities were isolated and subjected to mass spectrometry. Amino acid partial sequences enabled the identification of a 49-kDa protease similar to metalloprotease AprA and two lipases of 50 kDa and 69 kDa similar to LipA and ExoU, respectively. Electron microscopy analyses of challenged nematodes revealed degraded cuticle after R4 supernatant treatment. These results represent a new and unexplored property in this species associated with the presence of secretable lipases and protease, similar to characterized enzymes present in biocontrol pseudomonads.

Comparative detection of a large population of grapevine viruses by TaqMan® RT-qPCR and ELISA

Grapevine (Vitis spp.) can be infected by numerous viruses that are often widespread and of great economic importance. Reliable detection methods are necessary for sanitary selection which is the only way to partly control grapevine virus diseases. Biological indexing and ELISA are currently the standard methods for screening propagation material, and PCR-methods are becoming increasingly popular. Due to the diversity of virus isolates, it is essential to verify that the tests allow the detection of the largest possible virus populations. We developed three quadruplex TaqMan® RT-qPCR assays for detecting nine different viruses that cause considerable damage in many vineyards world-wide. Each assay is designed to detect three viruses and the grapevine Actin as an internal control.A large population of grapevines from diverse cultivars and geographic location was tested for the presence of nine viruses: Arabis mosaic virus (ArMV), Grapevine fleck virus (GFkV), Grapevine fanleaf virus (GFLV), Grapevine leafroll-associated viruses (GLRaV-1, -2, -3), Grapevine rupestris stem pitting-associated virus (GRSPaV), Grapevine virus A (GVA), and Grapevine virus B (GVB). In general, identical results were obtained with multiplex TaqMan® RT-qPCR and ELISA although, in some cases, viruses could be detected by only one of the two techniques.

Development of an in vitro Dual Culture System for Grapevine and Xiphinema index as a Tool for Virus Transmission

Grapevine fanleaf virus (GFLV) is a nepovirus that is transmitted to grapevines by the ectoparasitic nematode Xiphinema index. GFLV causes severe losses in yield and quality in viticulture worldwide. Presently, laborious and time-consuming field trials or greenhouse tests are necessary for screening putative GFLV resistance in new grape genotypes developed in breeding programmes. We developed an in vitro dual culture system for grapevines and nematode vectors that requires less time and space than inoculation experiments done in the greenhouse. Virus infection of in vitro grapevines was investigated using immunocapture-reverse transcriptase-polymerase chain reaction (IC-RT-PCR) analysis. The development of root galls induced by feeding nematodes on in vitro grapevines was also analysed. Virus infection in grapevines in the dual culture with viruliferous nematodes was detected six weeks post-inoculation. Root galls were always absent from parasitised in vitro grapevines with detectable virus infection, whereas they developed on some parasitised, but virus-negative tested grapevines. Therefore, root galls cannot be used as a reliable indicator for parasitism and virus transmission.

Xiphinema index and its Relationship to Grapevines: A review

The dagger nematode, Xiphinema index, is considered a major pest in grape growing countries. Xiphinema index is especially important because of its ability to transmit Grapevine fanleaf virus when feeding on grapevine roots. This paper provides a comprehensive and updated review of the classification, genetics and biology of Xiphinema index, and its relationship with grapevine fanleaf virus. Current control measures, as well as past and present efforts to breed resistant grapevine rootstocks, are presented.

Complete nucleotide sequence of a new variant of grapevine fanleaf virus from northeastern China.

The complete RNA1 and RNA2 sequences of a new grapevine fanleaf virus isolate (GFLV-SDHN) from northeastern China were determined. The two RNAs are 7,367 and 3,788 nucleotides (nt) in length, respectively, excluding the poly(A) tails. Compared to other GFLV isolates, GFLV-SDHN has a 22- to 24-nt insertion in the RNA1 5' untranslated region, and there was 19.1-20.1% and 11.7%-13.0% sequence divergence in RNA1, and 15.5%-20.5% and 8.5-13.5% in RNA2, at the nt and amino acid level, respectively. Phylogenetic analysis revealed that the origins of GFLV-SDHN are distinct from those of other GFLV isolates. One recombination event was identified in the 2AHP region of RNA2 in GFLV-SDHN.

Production of Polyclonal Antibody against Grapevine fanleaf virus Movement Protein Expressed in Escherichia coli.

The genomic region of Grapevine fanleaf virus (GFLV) encoding the movement protein (MP) was cloned into pET21a and transformed into Escherichia coli strain BL21 (DE3) to express the protein. Induction was made with a wide range of isopropyl-β-D-thiogalactopyranoside (IPTG) concentrations (1, 1.5, and 2 mM) each for duration of 4, 6, or 16 h. However, the highest expression level was achieved with 1 mM IPTG for 4 h. Identity of the expressed protein was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting. The expressed 41 kDa protein was purified under denaturing condition by affinity chromatography, reconfirmed by Western blotting and plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA) before being used as a recombinant antigen to raise polyclonal antibodies in rabbits. Purified anti-GFLV MP immunoglobulines (IgGs) and conjugated IgGs detected the expressed MP and GFLV virions in infected grapevines when used in PTA-ELISA, double antibody sandwich-ELISA, and Western blotting. This is the first report on the production of anti-GFLV MP polyclonal antibodies and application for the virus detection.

Діагностика вірусів скручування листя винограду і коротковузля в процесі виробництва сертифікованого садивного матеріалу винограду

Діагностика вірусів скручування листя винограду і коротковузля в процесі виробництва сертифікованого садивного матеріалу винограду

Display of whole proteins on inner and outer surfaces of grapevine fanleaf virus-like particles.

SummaryVirus‐like particles (VLPs) derived from nonenveloped viruses result from the self‐assembly of capsid proteins (CPs). They generally show similar structural features to viral particles but are noninfectious and their inner cavity and outer surface can potentially be adapted to serve as nanocarriers of great biotechnological interest. While a VLP outer surface is generally amenable to chemical or genetic modifications, encaging a cargo within particles can be more complex and is often limited to small molecules or peptides. Examples where both inner cavity and outer surface have been used to simultaneously encapsulate and expose entire proteins remain scarce. Here, we describe the production of spherical VLPs exposing fluorescent proteins at either their outer surface or inner cavity as a result of the self‐assembly of a single genetically modified viral structural protein, the CP of grapevine fanleaf virus (GFLV). We found that the N‐ and C‐terminal ends of the GFLV CP allow the genetic fusion of proteins as large as 27 kDa and the plant‐based production of nucleic acid‐free VLPs. Remarkably, expression of N‐ or C‐terminal CP fusions resulted in the production of VLPs with recombinant proteins exposed to either the inner cavity or the outer surface, respectively, while coexpression of both fusion proteins led to the formation hybrid VLP, although rather inefficiently. Such properties are rather unique for a single viral structural protein and open new potential avenues for the design of safe and versatile nanocarriers, particularly for the targeted delivery of bioactive molecules.