Incidence and Distribution of Grapevine Leafroll-associated viruses in Tunisian Vineyards

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.     

Occurrence of grapevine leafroll-associated virus complex in Napa Valley

Grapevine leafroll disease (GLD) is caused by a complex of several virus species (grapevine leafroll-associated viruses, GLRaV) in the family Closteroviridae. Because of its increasing importance, it is critical to determine which species of GLRaV is predominant in each region where this disease is occurring. A structured sampling design, utilizing a combination of RT-PCR based testing and sequencing methods, was used to survey GLRaVs in Napa Valley (California, USA) vineyards (n = 36). Of the 216 samples tested for GLRaV-1, -2, -3, -4, -5, and -9, 62% (n = 134) were GLRaV positive. Of the positives, 81% (n = 109) were single infections with GLRaV-3, followed by GLRaV-2 (4%, n = 5), while the remaining samples (15%, n = 20) were mixed infections of GLRaV-3 with GLRaV-1, 2, 4, or 9. Additionally, 468 samples were tested for genetic variants of GLRaV-3, and of the 65% (n = 306) of samples positive for GLRaV-3, 22% were infected with multiple GLRaV-3 variants. Phylogenetic analysis utilizing sequence data from the single infection GLRaV-3 samples produced seven well-supported GLRaV-3 variants, of which three represented 71% of all GLRaV-3 positive samples in Napa Valley. Furthermore, two novel variants, which grouped with a divergent isolate from New Zealand (NZ-1), were identified, and these variants comprised 6% of all positive GLRaV-3 samples. Spatial analyses showed that GLRaV-3a, 3b, and 3c were not homogeneously distributed across Napa Valley. Overall, 86% of all blocks (n = 31) were positive for GLRaVs and 90% of positive blocks (n = 28) had two or more GLRaV-3 variants, suggesting complex disease dynamics that might include multiple insect-mediated introduction events.     

Development of immunodiagnostics for the detection of <Emphasis Type="Italic">Grapevine leafroll</Emphasis>-<Emphasis Type="Italic">associated virus</Emphasis> 3 (GLRaV-3) in grapevine using in vitro expression and purification of its coat protein gene

A previously cloned coat protein (CP) gene of Grapevine leafroll-associated virus 3 (GLRaV-3) from cultivar Cabernet Souvignon was over-expressed in Escherichia coli strain BL21 expression system as ~ 43 kDa fusion protein containing polyhistidine tag (6His) at its N terminal. The protein was purified from insoluble fraction and reacted positively in western blotting with commercial anti GLRaV-3 polyclonal antiserum (Bioreba, Switzerland) and hence, used as immunogen for the production of polyclonal antisera in New Zealand white rabbits. Polyclonal antiserum specific to GLRaV-3 detected the virus by double antibody sandwich enzyme linked immunosorbent assay using commercial alkaline phosphatase (ALP) conjugated globulin fraction (Bioreba, Switzerland) in GLRaV-3 positive grapevine samples. The immunoreactivity of the antiserum was confirmed through western blotting. The purified antiserum was conjugated with ALP. The primary antiserum along with ALP conjugate successfully detected the GLRaV-3 from the infected sample at 1:8000 and 1:10,000 dilutions, respectively. To the best of our knowledge, it is the first global study wherein the CP of GLRaV-3 was cloned in pET28a(+) expression vector having many advantages over the earlier used expression vectors. The cloned CP gene was expressed, purified and subjected to the production of immunoreagents. The developed immunoreagents will be useful for certification programmes as well as for large scale virus screening to produce GLRaV-3 free grapevines. The indigenously developed immunereagents will provide a cost-effective way of managing grapevine leafroll disease in Indian sub-continent.     

In Vivo Inhibition of Trans-Plasma Membrane Electron Transport by Antiviral Drugs in Grapevine

Electrophysiological techniques were applied to investigate the action of antiviral drugs during trans-plasma events in in vivo grapevine cells infected by GLRaV-1 and GLRaV-3. Carbon fiber microelectrodes and redox-sensitive dyes were used to measure trans-plasma membrane electron transport (t-PMET) activity in healthy and infected samples treated with ribavirin, tiazofurin and oseltamivir. Each drug caused a reduction in oxidation current (expressed as Δ[Fe²⁺]) in healthy samples, indicating t-PMET inhibition. In almost all infected samples, the effect of drugs on t-PMET activity was significantly lower, suggesting that higher content of NADH in infected plants can interfere with t-PMET inhibition caused by drugs. Moreover, virus-infected samples exhibited elevated t-PMET activity compared to healthy samples. Analogous effects were observed by dye tests. Considering the effects of drugs on trans-plasma membrane potential, tests showed the activity of a proton pump during drug treatments with no significant difference with regard to health status.     

In vitro introduction of healthy and virus-infected genotypes of native Croatian grapevine cultivars

We evaluated the response of eight economically important Croatian grapevine cultivars and studied the impact of their sanitary status on in vitro introduction, by comparing the response of healthy and virus-infected genotypes of one cultivar. Nodal explant survival on three media, M1 (half-strength MS), M2 (full-strength MS) or M3 (full-strength MS with 4.4 µM L^?1 benzylaminopurine) was measured after 2 weeks and regrowth after 8 weeks. After 8 weeks, average shoot length and node number were significantly higher on M2 compared to M1 and M3. M3 induced significantly shorter average internode length, compared to M1 and M2. Survival of one healthy and of five cultivar Plavac mali genotypes infected with GFLV, GLRaV-1, GLRaV-3, GLRaV-3+GVA and GLRaV-1+GLRaV-3 was 97.5 and 82.8–87.5%, respectively. Regrowth of the healthy genotype reached 95.5%, but dropped to 5.5–31.4% in infected ones. The healthy genotype showed significantly higher shoot length (6.3 cm) and node number (7.3) compared to infected genotypes, with shoot length between 1.2–2.6 cm and node number between 1.2–3.0. By contrast, internode length was not significantly different between the healthy and the infected genotypes. The present work represents the first successful in vitro introduction for three of the eight native Croatian cultivars studied.     

Phylogenomic Analysis Reveals Deep Divergence and Recombination in an Economically Important Grapevine Virus

The evolutionary history of the exclusively grapevine (Vitis spp.) infecting, grapevine leafroll-associated virus 3 (GLRaV-3) has not been studied extensively, partly due to limited available sequence data. In this study we trace the evolutionary history of GLRaV-3, focussing on isolate GH24, a newly discovered variant. GH24 was discovered through the use of next-generation sequencing (NGS) and the whole genome sequence determined and validated with Sanger sequencing. We assembled an alignment of all 13 available whole genomes of GLRaV-3 isolates and all other publicly available GLRaV-3 sequence data. Using multiple recombination detection methods we identified a clear signal for recombination in one whole genome sequence and further evidence for recombination in two more, including GH24. We inferred phylogenetic trees and networks and estimated the ages of common ancestors of GLRaV-3 clades by means of relaxed clock models calibrated with asynchronous sampling dates. Our results generally confirm previously identified variant groups as well as two new groups (VII and VIII). Higher order groups were defined as supergroups designated A to D. Supergroup A includes variant groups I-V and supergroup B group VI and its related unclassified isolates. Supergroups C and D are less well known, including the newly identified groups VII (including isolate GH24) and VIII respectively. The inferred node ages suggest that the origins of the major groups of GLRaV-3, including isolate GH24, may have occurred prior to worldwide cultivation of grapevines, whilst the current diversity represents closely related isolates that diverged from common ancestors within the last century.     

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

Summary 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.     

Expression of disease resistance in genetically modified grapevines correlates with the contents of viral sequences in the T-DNA and global genome methylation

Increased tolerance to pathogens is an important goal in conventional and biotechnology-assisted grapevine breeding programs worldwide. Fungal and viral pathogens cause direct losses in berry production, but also affect the quality of the final products. Precision breeding strategies allow the introduction of resistance characters in elite cultivars, although the factors determining the plant’s overall performance are not fully characterized. Grapevine plants expressing defense proteins, from fungal or plant origins, or of the coat protein gene of grapevine leafroll-associated virus 3 (GLRaV-3) were generated by Agrobacterium-mediated transformation of somatic embryos and shoot apical meristems. The responses of the transformed lines to pathogen challenges were investigated by biochemical, phytopathological and molecular methods. The expression of a Metarhizium anisopliae chitinase gene delayed pathogenesis and disease progression against the necrotrophic pathogen Botrytis cinerea. Modified lines expressing a Solanum nigrum osmotin-like protein also exhibited slower disease progression, but to a smaller extent. Grapevine lines carrying two hairpin-inducing constructs had lower GLRaV-3 titers when challenged by grafting, although disease symptoms and viral multiplication were detected. The levels of global genome methylation were determined for the genetically engineered lines, and correlation analyses demonstrated the association between higher levels of methylated DNA and larger portions of virus-derived sequences. Resistance expression was also negatively correlated with the contents of introduced viral sequences and genome methylation, indicating that the effectiveness of resistance strategies employing sequences of viral origin is subject to epigenetic regulation in grapevine.     

The symptomless leaf infection with grapevine leafroll associated virus 3 in grown in vitro plants as a simple model system for investigation of viral effects on photosynthesis

The photosynthetic changes evaluated by oxygen evolution, chlorophyll fluorescence, photoacoustics, and delayed fluorescence (DF) were studied in leaves of grown in vitro for 8 weeks grapevine plants (Vitis vinifera) infected by grapevine leafroll-associated virus 3 (GLRaV-3). The infected leaves were characterized during the viral infection without visible disease symptoms. The symptomless infection led to a decrease in plant biomass. The non-photochemical fluorescence quenching, qN, declined, whereas the photochemical quenching, qP, and the Chl a/b ratio were not significantly affected. Photoacoustic and oxygen evolution measurements showed that the energy storage and oxygen evolution rate decreased in the infected leaves. Enhanced alternative electron sinks during the symptomless viral infection were also estimated. The changes in fluorescence and DF temperature curves demonstrated an enhanced stability of the thylakoid membranes in the infected leaves. This effect was clearly expressed at high actinic light intensities. The viral infected in vitro grown grapevine plants were used in the present study as a simplified model system that allow to avoid the involvement of different environmental factors that could interfere with the GLRaV infection and the virus-grapevine interactions. Thus, the 'pure' impact of the viral infection on photosynthesis could be investigated.     

Detection of Grapevine Viruses in Poland

During a 3‐year study, grapevines from 23 vineyards in Poland were surveyed for virus diseases and tested to determine the prevalence of the most economically important viruses by RT‐PCR. The rate of positive samples was 2.2% for grapevine leafroll‐associated virus 1 (GLRaV‐1), 1.9% for grapevine leafroll‐associated virus 2 (GLRaV‐2), 1.5% grapevine leafroll‐associated virus 3 (GLRaV‐3), 1.9% for grapevine virus A (GVA), 0.2% for grapevine virus B (GVB), 0.2% for grapevine virus E (GVE), 0.65% for grapevine fanleaf virus (GFLV), 20.4% for grapevine fleck virus (GFkV) and 71.9% for grapevine rupestris stem pitting‐associated virus (GRSPaV). These viruses were found to occur as single or mixed infections of different combinations in individual grapevines. The overall viral infection rate in the surveyed grapevines was 82.6%. GRSPaV is the most widely distributed virus of all the viruses currently detected in the region. DNA sequencing confirmed the identification of the viruses in selected samples, and analysis indicated that the Polish isolates shared a close molecular identity with the corresponding isolates in GenBank. To our knowledge, this is the first detection of GLRaV‐1, ‐2, ‐3, GVA, GVB, GVE, GFLV, GFkV and GRSPaV in Poland.     

Occurrence and Distribution of Grapevine Leafroll-associated Viruses 1, 2, 3 and 7 in Turkey

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%).     

Putative new plant viruses associated with Plasmopara viticola-infected grapevine samples

In this study, we analysed a total of 16 libraries from over 150 grapevine leaf and grape samples infected with Plasmopara viticola (downy mildew of grapevine) to characterise the virome associated to this oomycete. Samples were collected in five distinct regions in Italy and in four different regions in Spain, representative of different pedoclimatic conditions and different grapevine cultivars during 2018 growing season. Due to the metagenomics nature of the samples (containing at least both downy mildew hyphae and spores, and grapevine cells residues), we were able to assemble several plant viruses and a few possible novel plant virus genomes with our in silico analysis. We detected several plant virus variants already reported in grapevine, and a putative new ilarvirus previously unreported in grapevine. Furthermore, we characterised three new phenui-like viruses (in the order Bunyavirales), one of which shares some commonalities with plant coguviruses. Finally, we report a new strict association of three viral segments (one flavi-like and two virga-like) that we propose to be a new virus taxon named jivivirus.     

Comparison of the effects of different virus infections on performance of three Majorcan grapevine cultivars in field conditions

The effects of viruses on grape production and must quality are not fully understood. In this study, the evaluation of the impact of different virus infections on performance of the main autochthonous grapevine varieties of Mallorca (Callet, Manto Negro and Moll) was pursued. Therefore, a large number of vines were observed in field conditions over 4 years and tested by enzyme-linked immunosorbent assay for viruses listed by the international certification programmes. In each variety, some specific virus infections resulted to be more effective than the others in inducing losses in production. In Callet, yield (Y) reduction was over 20% in plants infected by Grapevine fanleaf virus (GFLV). In Moll, plants subject to more than one infection showed over 40% Y decrease. In Manto Negro, the most surprising results were obtained, because plants showed almost 40% Y reduction because of Grapevine leafroll-associated virus-1 (GLRaV-1) infection. In addition, virus infections were linked to some must quality parameter increase in Manto Negro and Moll, but in the majority of cases it was an indirect effect, because the decrease in production parameters played a predominant role by producing an important concentration effect. However, in Manto Negro, anthocyanin content decrease was directly related to GFLV infection.     

Effect of host plant tissue on the vector transmission of grapevine leafroll-associated virus 3

Many biotic and abiotic factors affect the transmission efficiency of vector-borne plant pathogens. Insect vector within-plant distribution and host tissue preference are known to affect pathogen acquisition and inoculation rates. In this study, we first investigated whether feeding tissue affects the transmission of Grapevine leafroll-associated virus 3 by Planococcus ficus (Signoret) (Hemiptera: Pseudococcidae) and the effect of mealybug within-plant distribution on virus transmission under greenhouse conditions. Results showed no significant effect on transmission efficiency after insect confinement on leaf blades, petioles or stems of virus source or healthy test plants for either acquisition or inoculation trials. Transmission efficiency of a single mealybug varied from 4 to 25% in those trials. Second, we tested whether leaf position affected transmission efficiency due to potentially variable virus populations within acquisition plant tissues. No significant differences of transmission rate among acquisition leaf position were observed, probably because there were no differences in the virus population within source tissues. Finally, we examined the seasonality of the virus in field-collected samples and found that GLRaV-3 prevalence varied along a growing season, such that GLRaV-3 translocated along expanding shoots to leaves. Similarly, mealybug populations are known to increase in spring, and then mealybugs spread to cordons and leaves. This coordination of spatial and temporal dynamics of the virus and its vector may increase the risk of GLRaV-3 transmission during late spring and early summer. Further integration of information about pathogen populations in plants, vector feeding behavior and vector population seasonality could lead to more effective management practices.