Grapevine Fanleaf Nepovirus Research Articles

Bazı Bağ Hastalıklarının Faster R-CNN Modeli ile Otomatik Tespit Edilmesi ve Sınıflandırılması

Türkiye is one of the countries with the most important vineyard areas in the world, where the most grape production is made. Vineyard diseases are one of the most important reasons that adversely affect the productivity in viticulture. In this study, some vineyard diseases were detected and classified using the Faster R-CNN deep learning model, which is an artificial intelligence approach. These diseases are powdery mildew, downy mildew, dead arm disease, grapevine leaf roll-associated virus disease (GLRaV) and grapevine fan leaf nepovirus (GFLV) diseases that are common and cause economic problems. The proposed method is trained and tested using 11000 images. At the end of the study, the overall accuracy rate was found to be 92%. The proposed approach gave better results than similar methods in the literature. Therefore, it was concluded that the method can be used reliably in the detection and classification of some vineyard diseases.

Mapping of the exchangeable and dispensable domains of the RNA 2-encoded 2AHP protein of arabis mosaic nepovirus

The N-terminal domains of the RNA 2-encoded 2AHP proteins of the arabis mosaic (ArMV) and grapevine fanleaf (GFLV) nepoviruses were shown to be highly variable and a hotspot for intra- and inter-species recombination events. Chimeric ArMV-NW clones in which the N-terminal domain of 2AHP or the entire 2AHP of GFLV isolates replaced the corresponding domains of ArMV retained their infectivity, showing that the 2AHP proteins of ArMV-NW and GFLV are exchangeable. ArMN-NW clones with deletions of the N-terminal, core, or C-terminal domains of the ArMV-NW 2AHP were infectious in Chenopodium quinoa although viral RNA (especially RNA 2) accumulated at reduced levels. In contrast, deletion of the entire 2AHP protein or of the C-terminal two thirds of the protein abolished infectivity of the ArMV-NW clones. These results suggest that multiple functional domains are distributed throughout the 2AHP protein and are essential for the accumulation of viral RNA 2.

Susceptibility of grapevine viruses to thermotherapy on in vitro collection of Kober 5BB

A number of virus elimination protocols have been developed to obtain healthy plants but their success rates have been variable due to the strong virus–host relationship, treatment performances and experimental condition. An in vitro collection of plants of Vitis berlandieri × Vitis riparia Kober 5BB single-infected by Grapevine vitivirus A (GVA), Grapevine fanleaf nepovirus (GFLV), Grapevine fleck maculavirus (GFkV), Grapevine leafroll ampelovirus 1 (GLRaV-1) and Grapevine leafroll ampelovirus 3 (GLRaV-3) was established to carry out thermotherapy treatments. The experimental system allowed to control the effects of host genotype by using the same genetic background, and the environmental condition such as relative humidity, soil and temperature conditions. Trials were conducted in a growth chamber set at 37 ± 0.5 °C for 48 days using in vitro cultures for all infections. ELISA and RT-PCR were used to check sanitary conditions. Results showed complete eradication of GFLV and no effect against GFkV. Different sanitation rates were obtained for other phloematic viruses: 70.2% for GVA, 25.1% for GFLaV-1 and 24.7% for GLRaV-3. The results on phloematic viruses showed a gradient of elimination efficiency which could not be completely explained with the location of viral particles that seems to be only one of the several factors affecting virus susceptibility to heat stress. Moreover, the advantages of this experimental system provide the opportunity to obtain a greater understanding of the thermotherapeutic effects on phloematic viruses.

Pathogen‐tested material of grapevine varieties and rootstocks

Pathogen‐tested material of grapevine varieties and rootstocks

Detection and characterization of two strains of Grapevine fanleaf nepovirus in Tunisia

The nematode‐borne Grapevine fanleaf nepovirus (GFLV) causes severe degeneration of grapevines in vineyards worldwide. In a recent survey of the sanitary status of grapevine plants in north Tunisian vineyards, we were interested to study the polymorphism of GFLV. Purified virus, from mechanically inoculated Chenopodium quinoa, was used to produce anti‐GFLV antiserum, which specifically recognized GFLV in different Tunisian grapevine samples using the DAS‐ELISA technique. Positive samples were subjected to oligoprobe‐RT‐PCR to amplify a 606 bp region of the viral coat protein sequence. PCR products used for RFLP analysis after digestion with endonuclease AluI produced 3 restriction profiles. RFLP data allowed clear distinction of two GFLV strains in Tunisia. The nucleotide sequence of the PCR‐generated amplicons from each strain was determined showing 93.4% identity at the nucleic acid level and 97.5% similarity at the aminoacid sequence level compared to the previously characterized GFLV‐F13 French isolate. This paper is the first report on molecular variability of GFLV in Tunisia.

Detection of Grapevine fanleaf Nepovirus capsid protein gene by RT-PCR and DNA Hybridization

Detection of Grapevine fanleaf Nepovirus capsid protein gene by RT-PCR and DNA Hybridization

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.

Simultaneous RT/PCR detection and differentiation of arabis mosaic and grapevine fanleaf nepoviruses in grapevines with a single pair of primers

The movement protein genes from several isolates of ArMV and GFLV of different geographical origins were amplified by RT/PCR using degenerate primers, cloned and sequenced. A single pair of degenerate primers was designed from these sequences to allow the simultaneous amplification of parts of the movement protein genes of ArMV and GFLV. Their use in an immunocapture-RT/PCR for the detection of ArMV or GFLV in infected grapevines proved to be ten times more sensitive than the corresponding ArMV or GFLV ELISA tests. A Sph1 restriction site found in the sequences corresponding to the amplified products from the GFLV isolates, but not in the amplified products from the ArMV isolates, allowed the differentiation between ArMV and GFLV in the infected grapevines by a Sph1 restriction digestion of the amplified products.

Protein 2A of Grapevine Fanleaf Nepovirus Is Implicated in RNA2 Replication and Colocalizes to the Replication Site

RNA2 of grapevine fanleaf virus is replicated in trans by the RNA1-encoded replication machinery. Full processing of the RNA2-encoded polyprotein P2 yields protein 2A of unknown function, the movement protein 2BMP, and the coat protein 2CCP. Analysis of a set of deletion mutants in the P2-coding sequence revealed that protein 2A is necessary but not sufficient for RNA2 replication. In addition to the 5′ and 3′ noncoding sequences and the 2A-coding sequence, an additional sequence coding for 2BMP and/or 2CCP or the green fluorescent protein (GFP) is necessary for RNA2 replication. When 2A fused to GFP (2AGFP) was transiently expressed in uninfected T-BY2 protoplasts, 2AGFP appeared as punctate structures evenly distributed in the cytoplasm. However, in cells cotransfected with grapevine fanleaf virus RNAs and the 2AGFP construct, 2AGFP was predominantly found in a juxtanuclear location along with 1Dpro and 1CVPg, two RNA1-encoded proteins involved in RNA replication. Viral RNA replication as traced by 5-bromouridine 5′ triphosphate (BrUTP) incorporation into newly synthesized RNA occurred at the same location. This colocalization is consistent with the hypothesis that 2A enables RNA2 replication through its association with the replication complex assembled from RNA1-encoded proteins.

The nine C-terminal residues of the grapevine fanleaf nepovirus movement protein are critical for systemic virus spread.

The grapevine fanleaf virus (GFLV) RNA2-encoded polyprotein P2 is proteolytically cleaved by the RNA1-encoded proteinase to yield protein 2A, 2B(MP) movement protein and 2C(CP) coat protein. To further investigate the role of the 2B(MP) and 2C(CP) proteins in virus movement, RNA2 was engineered by alternatively replacing the GFLV 2B(MP) and 2C(CP) genes with their counterparts from the closely related Arabis mosaic virus (ArMV). Transcripts of all chimeric RNA2s were able to replicate in Chenopodium quinoa protoplasts and form tubules in tobacco BY-2 protoplasts in the presence of the infectious transcript of GFLV RNA1. Virus particles were produced when the GFLV 2C(CP) gene was replaced with its ArMV counterpart, but systemic virus spread did not occur in C. quinoa plants. In addition, chimeric RNA2 containing the complete ArMV 2B(MP) gene was neither encapsidated nor infectious on plants, probably because polyprotein P2 was incompletely processed. However, chimeric RNA2 encoding ArMV 2B(MP), in which the nine C-terminal residues were those of GFLV 2B(MP), formed virus particles and were infectious in the presence of GFLV but not ArMV 2C(CP). These results suggest that the nine C-terminal residues of 2B(MP) must be of the same virus origin as the proteinase for efficient proteolytic processing of polyprotein P2 and from the same virus origin as the 2C(CP) for systemic virus spread.

Association of grapevine Fanleaf nepovirus poly-protein P2 to membranes by a GFP-tagged protein 2A

Association of grapevine Fanleaf nepovirus poly-protein P2 to membranes by a GFP-tagged protein 2A

Viruses and virus diseases of grapevine in Tunisia

Mature canes were collected from vines in the main grapevine‐growing areas in Tunisia (Cape Bon, Bizerte, Ben Arous), from commercial vineyards and mother‐plant plots, to assess the presence of virus and virus‐like diseases. Biological (mechanical transmission onto herbaceous hosts and grafting onto indicator woody plants) and serological detection (ELISA) methods were applied. ELISA showed that 96.4% of 669 vines tested were infected, most of them (88.1%) by at least two viruses. Grapevine leafroll‐associated 3 closterovirus (GLRaV‐3) was the most widespread virus (87.9%), followed by grapevine A vitiviras (GVA, 69.4%), grapevine fleck virus (GFkV, 51.9%), grapevine leafroll‐associated 1 closterovirus (GLRaV‐1, 36.8%), grapevine leafroll‐associated 2 closterovirus (GLRaV‐2, 19.1%), grapevine fan leaf nepovirus (GFLV, 18.2%) and grapevine B vitiviras (GVB, 14.8%). ELISA tests yielded negative results for grapevine leafroll‐associated 7 closterovirus (GLRaV‐7) and potato X potexvirus (PVX). The highest infections were found in Bizerte and Cape Bon regions (100 and 99.2%), and in vineyards aged over 20 years (98.5%) as compared with the younger ones (81.1%). Rootstocks in mother‐plant plots were practically free from all the viruses tested (1 plant infected out of 81), whereas severe infections were found in Vitis vinifera mother plants (67.4% of 341 samples), in particular table grapes (92.6%) compared with wine grapes (47.9%). In these mother‐plant plots, the prevailing viruses were GLRaV‐3 (41.3%), followed by GFkV (36.7%), GVA (27.9%), GLRaV‐1 (17%) and GLRaV‐2 (15.2%). GFLV and GVB were far more limited (1.5 and 0.6%, respectively). The presence of vein necrosis and vein mosaic was ascertained by transmission onto 110R and Vitis riparia indicators, whereas only GFLV was mechanically transmitted onto herbaceous hosts (from about 20% of the samples).

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%.

Transformation of grapevine rootstocks with the coat protein gene of grapevine fanleaf nepovirus

Control of fanleaf disease induced by the Grapevine Fanleaf Nepovirus (GFLV) today is based on sanitary selection and soil disinfection with nematicides. This way of control is not always efficient and nematicides can be dangerous pollutants. Coat protein (CP) mediated protection could be an attractive alternative. We have transferred a chimeric CP gene of GFLV-F13 via Agrobacterium tumefaciens LBA4404 into two rootstock varieties: Vitis rupestris and 110 Richter (V. rupestris X V. Berlandieri). Transformation was performed on embryogenic callus obtained from anthers and on hypocotyl fragments from mature embryos. Success of the transformation was assessed by polymerase chain reaction and Southern analyses. Transformants with a number of copies of the CP gene varying from one to five were obtained. Enzyme-linked immunosorbent assay with virus-specific antibodies revealed various levels of expression of the coat protein in the different transformants.

Grapevine fanleaf nepovirus P38 putative movement protein is not transiently expressed and is a stable final maturation product in vivo.

The putative 38 kDa movement protein (P38) gene, located on the RNA2 of grapevine fanleaf nepovirus (GFLV), was cloned in Escherichia coli and expressed as a fusion protein fused to six histidines (6HisP38). This protein was purified and used to produce a specific polyclonal antiserum from which immunoglobulins were isolated by immunoaffinity against the recombinant 6HisP38. Western immunoblot analyses on GFLV RNA2 in vitro maturation products showed that the antibodies were specific for P38 protein. This protein was detected as early as 18 h post-inoculation in GFLV-infected Chenopodium quinoa protoplasts and accumulated to very high levels. Tissue-prints and time course experiments on infected C. quinoa plants confirmed that P38 is present at a high level late in infection and is a final maturation product of the GFLV RNA2 polyprotein in vivo. P94 and P66 intermediates of maturation and polyprotein P2 were also detected in vivo but in very low concentrations. No significant difference was observed in the relative amounts of P66 and P94 detected in vivo, contrary to what occurs in vitro. Subcellular fractionation studies showed that P38, although mainly cytosolic, is also found in association with cell wall and membranes. Thought to be the GFLV movement protein, P38 would thus behave in an 'atypical' manner.

Grapevine Fanleaf Nepovirus P38 Putative Movement Protein Is Located on TubulesIn Vivo

Grapevine Fanleaf Nepovirus P38 Putative Movement Protein Is Located on Tubules<i>In Vivo</i>

Agroecological characterization of Xiphinema index in Spain1

The agroecological characteristics of Xiphinema index were determined by studying its spatial and temporal distribution in different environmental conditions in Spain. The host range, life cycle in the Mediterranean continental climate and its relationships with grapevine fanleaf nepovirus are also reported. X. index is widespread in grape-growing areas in a wide range of soil textures and pH, even at high percentage of carbonates. However, it seems to prefer sandy-loam and sandy-clay loam soils, and populations decrease when soil carbonates increase. Its spatial distribution is random but it has a tendency to be found in the deepest clay horizons, where soil moisture is retained. Availability of moisture seems to be the limiting factor for nematode development. Grapevine and fig are the main host plants but X. index is also associated with fruit trees, ornamental crops, vegetable crops, woodlands and other uncultivated soils. Its life cycle in central Spain, where temperature ranges from <0°C until March to above 40°C from May—June until September, takes 6–8 weeks to complete. X. index was found in 14% of all vineyards sampled and in 50% of the fanleaf virus-infected vineyards. Transmission tests using bait plants were successful and the detection of the virus by means of ELISA test in a mimimun of five nematodes was achieved. Fanleaf-like symptoms often occurred in the absence of the virus, as demonstrated by ELISA.

Differential Proteolytic Activities of Precursor and Mature Forms of the 24K Proteinase of Grapevine Fanleaf Nepovirus

The presence of a genome-linked protein (VPg) at the RNA 5′-end of the genome is a characteristic of different groups of animal and plant positive-sense single-stranded RNA viruses. These viruses express their structural and functional proteins from polyproteins that are sequentially processed by at least one viral proteinase. The grapevine fanleaf nepovirus 24K chymotrypsin-like cysteine proteinase, located between the VPg and the RNA polymerase in the RNA-1 encoded polyprotein P1, is active in its free form and in various precursors forms. The VPg proteinase precursor (VPg-Pro) constitutes a stable protein and its maturation in the reticulocyte lysate system occurs at a very low rate. Differences on cleavage activity were observed between the proteinase and its VPg-Pro precursor forms, depending upon the cleavage site considered. The proteinase alone has a greater cleavage efficiency than VPg-Pro at the Arg605/Gly606 and Cys257/Ala258 sites of polyprotein P2. On the other hand, the presumed Cys415/Ala416 site, present at the amino terminus of polyprotein P1, was preferentially cleaved by the VPg-Pro precursor. During their in vitro maturation, proteins containing the VPg proteinase-polymerase coding region or the proteinase-polymerase region were similar in their ability to cleave in cis between the proteinase and the RNA polymerase.

Protection against virus infection in tobacco plants expressing the coat protein of grapevine fanleaf nepovirus

Grapevine fanleaf nepovirus (GFLV) is responsible for the economically significant "court-noué" disease in vineyards. Its genome is made up of two single-stranded RNA molecules (RNA1 and RNA2) which direct the synthesis of polyproteins P1 and P2 respectively. A chimeric coat protein gene derived from the C-terminal part of P2 was constructed and subsequently introduced into a binary transformation vector. Transgenic Nicotiana benthamiana plants expressing the coat protein under the control of the CaMV 35S promoter were engineered by Agrobacterium tumefaciens-mediated transformation. Protection against infection with virions or viral RNA was tested in coat protein-expressing plants. A significant delay of systemic invasion was observed in transgenic plants inoculated with virus compared to control plants. This effect was also observed when plants were inoculated with viral RNA. No coat protein-mediated cross-protection was observed when transgenic plants were infected with arabis mosaic virus (ArMV), a closely related nepovirus also responsible for a "court-noué" disease.

Viruses of grapevine in Albania

Grapevines were surveyed for the presence of virus and virus‐like diseases in the Albanian viticultural districts of Shkoder, Lesh, Kruje, Durres, Tirana, Elbasan, Lushnje and Vlora. Symptoms of grapevine degeneration, leafroll and rugose wood were observed in all areas surveyed, whereas fleck was found in volunteer plants of Vitis rupestris at Elbasan, and enation disease in a few vines near Durres. Viruses identified were grapevine fanleaf nepovirus, grapevine fleck virus, grapevine virus A, and grapevine leafroll‐associated closteroviruses I and III. ELISA tests showed that 83.5% of 530 Vitis vinifera vines and 46% of 24 American rootstocks individually checked were infected by one or more viruses. The presence of Xiphinema index, the major vector of grapevine fanleaf nepovirus, was recorded from vineyards affected by yellow mosaic.