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Shiraz disease (SD) is an economically important virus-associated disease that can significantly reduce yield in sensitive grapevine varieties and has so far only been reported in South Africa and Australia. In this study, RT-PCR and metagenomic high-throughput sequencing was used to study the virome of symptomatic and asymptomatic grapevines within vineyards affected by SD and located in South Australia. Results showed that grapevine virus A (GVA) phylogroup II variants were strongly associated with SD symptoms in Shiraz grapevines that also had mixed infections of viruses including combinations of grapevine leafroll-associated virus 3 (GLRaV-3) and grapevine leafroll-associated virus 4 strains 5, 6 and 9 (GLRaV-4/5, GLRaV-4/6, GLRaV-4/9). GVA phylogroup III variants, on the other hand, were present in both symptomatic and asymptomatic grapevines, suggesting no or decreased virulence of these strains. Similarly, only GVA phylogroup I variants were found in heritage Shiraz grapevines affected by mild leafroll disease, along with GLRaV-1, suggesting this phylogroup may not be associated with SD. [ABSTRACT FROM AUTHOR]

Grapevine leafroll disease (GLD) is the most common and economically destructive grapevine viral disease in South African vineyards and throughout the world. There are many GLD-associated virus variants with Grapevine leafroll-associated virus 3 (GLRaV-3) being the main causative agent of GLD. The vine mealybug, Planococcus ficus, is the most widespread and problematic vector of GLRaV-3. Roguing, pesticides and sanitary measures are common control strategies used in South African vineyards. In this paper, we propose an age-structured mathematical model for the transmission of (GLRaV-3) by Planococcus ficus in the South African context. The model is investigated in terms of the transmission thresholds and control strategies. A simplified model is used to shed light into the qualitative dynamics of transmission and assess the effectiveness of roguing as the main control strategy for GLRaV-3 spread. [ABSTRACT FROM AUTHOR]

With the aim to characterize changes caused by grapevine leafroll-associated virus 3 (GLRaV-3) singly or in coinfection with other viruses and to potentially determine genotype-specific or common markers of viral infection, thirty-six parameters, including nutrient status, oxidative stress parameters, and primary metabolism as well as symptoms incidence were investigated in 'Cabernet Franc,' 'Merlot,' 'Pinot Noir,' and 'Tribidrag' grapevine varieties. Host responses were characterized by changes in cellular redox state rather than disturbances in nutrient status and primary metabolic processes. Superoxide dismutase, hydrogen peroxide, and proteins were drastically affected regardless of the type of isolate, the host, and the duration of the infection, so they present cellular markers of viral infection. No clear biological pattern could be ascertained for each of the GLRaV-3 genotypes. There is a need to provide a greater understanding of virus epidemiology in viticulture due to the increasing natural disasters and climate change to provide for global food production security. Finding grape varieties that will be able to cope with those changes can aid in this task. Among the studied grapevine varieties, autochthonous 'Tribidrag' seems to be more tolerant to symptoms development despite numerous physiological changes caused by viruses. [ABSTRACT FROM AUTHOR]

Grapevine leafroll-associated virus 3 (GLRaV-3) is one of the most important viruses affecting global grape and wine production. GLRaV-3 is the chief agent associated with grapevine leafroll disease (GLRD), the most prevalent and economically destructive grapevine viral disease complex. Response of grapevine to GLRaV-3 infection at the gene expression level is poorly characterized, limiting the understanding of GLRaV-3 pathogenesis and viral-associated symptom development. In this research, we used RNA-Seq to profile the changes in global gene expression of Cabernet franc, a premium red wine grape, analyzing leaf and berry tissues at three key different developmental stages. We have identified 1457 differentially expressed genes (DEGs) in leaves and 1181 DEGs in berries. The expression profiles of a subset of DEGs were validated through RT-qPCR, including those involved in photosynthesis (VvPSBP1), carbohydrate partitioning (VvSUT2, VvHT5, VvGBSS1, and VvSUS), flavonoid biosynthesis (VvUFGT, VvLAR1, and VvFLS), defense response (VvPR-10.3, and VvPR-10.7), and mitochondrial activities (ETFB, TIM13, and NDUFA1). GLRaV-3 infection altered source–sink relationship between leaves and berries. Photosynthesis and photosynthate assimilation were inhibited in mature leaves while increased in young berries. The expression of genes involved in anthocyanin biosynthesis increased in GLRaV-3-infected leaves, correlating with interveinal tissue reddening, a hallmark of GLRD symptoms. Notably, we identified changes in gene expression that suggest a compromised sugar export and increased sugar retrieval in GLRaV-3-infected leaves. Genes associated with mitochondria were down-regulated in both leaves and berries of Cabernet franc infected with GLRaV-3. Results of the present study suggest that GLRaV-3 infection may disrupt mitochondrial function in grapevine leaves, leading to repressed sugar export and accumulation of sugar in mature leaf tissues. The excessive sugar accumulation in GLRaV-3-infected leaves may trigger downstream GLRD symptom development and negatively impact berry quality. We propose a working model to account for the molecular events underlying the pathogenesis of GLRaV-3 and symptom development. [ABSTRACT FROM AUTHOR]

Grapevine-infecting ampelo- and vitiviruses are transmitted by several scale insect species, including the Bohemian mealybug, Heliococcus bohemicus Šulc. Virus infectivity experiments were performed with this species to study the transmission ability of natural populations living in infected vineyards in Alsace, France. Mealybugs were sampled on vines infected by grapevine leafroll-associated viruses (GLRaV-1, -2, and -3) and by grapevine virus A (GVA), either alone or in combinations. Out of six natural populations tested, only one, located at Bennwihr, was able to transmit GLRaV-1 and -3 to healthy vines, though with low transmission rates (1.6 and 11.8%, respectively). Mealybugs from Bennwihr were also able to transmit GLRaV-3 from grapevines of another location where H. bohemicus was not a vector. Conversely, mealybugs from two other locations did not transmit any virus acquired from infected grapevines at Bennwihr. These results suggest differences in vector ability between H. bohemicus populations. Moreover, laboratory experiments were developed to estimate the minimal acquisition and inoculation access periods (AAP and IAP, respectively) for virus transmission of GLRaV-1 and -3, and GVA. First instar nymphs transmitted GLRaV-1 after 6 h AAP, GLRaV-3 and GVA together after 1 h AAP, and the three viruses after only 1 h IAP, supporting a semi-persistent mode of transmission. Second instar nymphs fed on multi-infected grapevine for 72 h then starved or fed on potatoes tested positive by RT-PCR for GLRaV-1 and -3 after up to 35 and 40 days, respectively, contrasting with the short retention times generally observed for mealybugs. These findings provide new knowledge of the vector ability of H. bohemicus. [ABSTRACT FROM AUTHOR]

Grapevine leafroll disease (GLD) constrains wine production worldwide. In New Zealand, the main causal agent of GLD is grapevine leafroll-associated virus 3 (GLRaV-3). To control GLD, an integrated management program is used and includes removing (roguing) GLRaV-3-infected vines from the vineyard. The classical foliar symptoms from virus-infected red-berry cultivars are leaves with dark red intervein, green veins, and downward rolling of margins. Growers use these phenotypic cues to undertake visual symptom identification (VSI) for GLD. However, the influence of the known large genetic variation among GLRaV-3 isolates on the foliar symptoms from different grapevine cultivars remains undescribed, especially in cool-climate growing environments, such as New Zealand. Over three vintages (2015, 2016, and 2017), VSI for GLD was undertaken at three field sites in New Zealand (Auckland, Hawke's Bay, and Marlborough), each including four cultivars (Merlot, Pinot noir, Sauvignon blanc, and Pinot gris) infected with three GLRaV-3 genotypes (Groups I, VI, and X) or GLRaV-3-uninfected control plants. Throughout this study, no visual symptoms were observed on white-berry cultivars infected with GLRaV-3. For red-berry cultivars, the greatest variability in observed foliar symptoms among regional study sites, cultivars, and GLRaV-3 genotypes was observed early in the growing season. In particular, Group X had significantly delayed symptom expression across all three sites compared with Groups I and VI. As the newly infected, young vines matured in years 2 and 3, the GLRaV-3 genotype, cultivar, region, and environmental conditions had minimal influence on the accuracy of VSI, with consistently high (>95%) within-vintage identification by the end of each vintage. The results from this study strongly support the use of VSI for the GLD management of red-berry cultivar grapevines, Merlot and Pinot noir, as a reliable and cost-effective tool against GLD. [ABSTRACT FROM AUTHOR]

Recuperation and genetic diversity preservation of local cultivars have acquired a huge interest in viticulture areas worldwide. In the Balearic Islands, most of the old cultivars are only preserved in grapevine germplasm banks, and so far, the sanitary status of these local cultivars has remained unexplored. The aim of this study was to survey and detect the virus incidence of all conserved cultivars in the government Grapevine Germplasm Bank of the Balearic Islands and to promote the sanitary recovery of two important minor cultivars, Argamussa and Gorgollassa. Enzyme-linked immunosorbent assay (ELISA) screenings were performed on 315 vines of 33 local cultivars. It was shown that the local cultivars were highly infected with simple (39.7%) and mixed infections (52.1%) and only 8.25% of them were free from the viruses tested. Grapevine leafroll-associated virus 3 (GLRaV-3) infection was the most common (82%). Moreover, Grapevine fanleaf virus (GFLV) and Grapevine fleck virus (GFkV) were also present with considerable incidence (25.4% and 43.5%, respectively). In addition, two sanitation protocols were used: shoot tip culture (ST) and thermotherapy in combination with shoot tip culture (CT). Virus elimination using only ST was effective to obtain "healthy" vines of cvs. Argamussa and Gorgollassa. It is important to emphasize that the methods described in the current study were rapid and effective in eliminating both GLRaV-3 and GFLV, also in combination. [ABSTRACT FROM AUTHOR]

This study aimed to survey the occurrence of eight grapevine viruses in commercial vineyards located in the Calchaquíes Valleys in the northwest region of Argentina. A total of 103 samples of mature canes of vines showing either none or some viral-like symptoms were randomly collected. The samples were tested by RT-PCR/PCR-based assays for the screening of the following viruses: Grapevine fanleaf virus (GFLV), Grapevine leafroll-associated viruses (GLRaV-1, -2, -3, -4), Grapevine virus A (GVA), Grapevine rupestris stem pitting-associated viruses (GRSPaV), and Grapevine red blotch virus (GRBV). Sixty percent of the analyzed samples showed infection with some of the analyzed viruses, except GRBV. GLRaV-3 and GFLV were the most frequent viruses, present in 34% and 21% of the positive samples, respectively. This study represents the first survey report of the presence of grapevine viruses in the region of the Calchaquíes Valleys and contributes to the knowledge to maintain the sanitary status of commercial vineyards in Argentina. [ABSTRACT FROM AUTHOR]

Objective: Grapevine (Vitis spp.) viral infections, including those by Grapevine leafroll-associated virus 3 (GLRaV-3) and Grapevine red blotch virus (GRBV), greatly reduce fruit yields and quality. Evidence exists that host chemistry shifts result in reductions in fruit quality. However, changes over the season in foliar chemistry has not been well examined. Therefore, phenolic and amino acid levels were examined in leaves collected in grapevines with different rootstocks that were healthy or were infected with GLRaV-3 or GRBV. This was part of an effort to assess changes that different pathogens cause in grapevine tissues. Results: Month and year appeared to account for the greatest variability in grapevine foliar phenolic or amino acid levels, followed by differences in rootstock, and then differences in infection status. GLRaV-3 infection significantly lowered levels of total and individual hydroxycinnamic acid derivatives, and GRBV lowered total phenolic levels, total and individual hydroxycinnamic acids. Amino acid levels were increased over controls in vines infected by GLRaV-3, but not with GRBV. Overall, changes within grapevine leaves due to viral infection were likely too small to overcome variability due to sampling time or rootstock cultivar, and therefore such factors should be considered in determining infection effects on plant foliar chemistry. [ABSTRACT FROM AUTHOR]

Grapevine collections play an important role, especially in the study of viruses and virus-like pathogens. In 2009, after an initial ELISA screening for eight viruses (arabis mosaic virus, grapevine fanleaf virus, grapevine fleck virus, grapevine leafroll-associated viruses 1, 2, and 3, and grapevine viruses A and B), a collection of 368 grapevine accessions representing 14 different Croatian autochthonous cultivars and containing single or mixed infection of viruses was established to further characterize the viral pathogens. Subsequently, Western blot, RT-PCR, cloning, and sequencing revealed that grapevine rupestris stem pitting-associated virus was frequently found in accessions of the collection, with isolates showing substantial genetic diversity in the helicase and coat protein regions. High-throughput sequencing of 22 grapevine accessions provides additional insight into the viruses and viroids present in the collection and confirms the fact that Croatian autochthonous grapevine cultivars have high infection rates and high virome diversity. The recent spread of "flavescence dorée" phytoplasma in Europe has not spared the collection. After the first symptoms observed in 2020 and 2021, the presence of phytoplasma was confirmed by LAMP in six grapevine accessions and some of them were lost. Single or multiple viruses and viroids, as well as own rooted grapevines in the collection, make the plants susceptible to various abiotic factors, which, together with the recent occurrence of "flavescence dorée", makes the maintenance of the collection a challenge. Future efforts will be directed towards renewing the collection, as 56% of the original collection has been lost in the last 13 years. [ABSTRACT FROM AUTHOR]

Background: the most widely distributed and virulent Grapevine leafroll-associated viruses (GLRaV) that affect grapevine are GLRaV-1 and GLRaV-3, transmitted semi-persistently by different mealybugs and soft scales, mainly causing downward rolling of the leaf margins and interveinal reddening. Methods: the main objectives of this study were to investigate the genetic structure and molecular diversity of GLRaV-1 and GLRaV-3 in 617 samples from 11 autochthonous Sicilian grapevine cultivars, ascertaining their presence and spread. The detection was implemented by serological and molecular analyses and subsequently phylogenetic analyses on selected Sicilian isolates were conducted. Results: in total, 33 and 138 samples resulted positive to GLRaV-1 and GLRaV-3, with an incidence of 5.34% and 22.36%, respectively; 9 out of the 11 cultivars resulted positive, while the presence of both viruses was not found in 'Grillo' and 'Moscato' cultivars. Conclusions: phylogenetic analyses of the coat protein (CP) gene of 12 GLRaV-1 selected sequences showed a close relationship with European isolates; the discrete nucleotide differentiation and positive selection could demonstrate a current increase in population fitness. The phylogenetic analyses of the CP gene of 31 GLRaV-3 Sicilian CP sequences demonstrates a close relationship between Sicilian and different countries isolates; a certain stability of GLRaV-3 in the different cultivars analyzed is suggested by the discrete differentiation nucleotide and negative selection of the Sicilian isolates. [ABSTRACT FROM AUTHOR]

The Ampelovirus Grapevine leafroll-associated virus 3 (GLRaV-3) and the Nepovirus Grapevine fanleaf virus (GFLV) are pathogens reported in many grapevine-growing areas all over the world, main causal agents of grapevine leafroll disease and grapevine fanleaf disease, respectively. Prevention of virus spread thanks to rapid diagnosis of infected plants is a key factor for control of both diseases. Although serological (e.g., enzyme-linked immunosorbent assay-ELISA test) and molecular methods are available to reveal the presence of the viruses, they turn out to be quite expensive, time-consuming and laborious, especially for large-scale health screening. Here we report the optimization of a lab-on-a-chip (LOC) for GLRaV-3 and GFLV detection, based on an electrochemical transduction and a microfluidic multichamber design for measurements in quadruplicate and simultaneous detection of both targets. The LOC detect GLRaV-3 and GFLV at dilution factors more than 15 times higher than ELISA, providing a higher sensitivity in the detection of both viruses. Furthermore, the platform offers several advantages as easy-to-use, rapid-test, portability and low costs, favoring its potential application for large-scale monitoring programs. Compared to other grapevine virus biosensors, our sensing platform is the first one to provide a dose-dependent calibration curve combined with a microfluidic module for sample analysis and a portable electronics providing an operator-independent read-out scheme. [ABSTRACT FROM AUTHOR]

The incidence of grapevine virus infections in Korean vineyards was investigated from July to October, 2020. A total of 177 petiole samples were collected from two or three different cultivars in each of four different regions; these were examined by reverse transcription-polymerase chain reaction assay for the presence of 14 major viruses. The overall occurrence of grapevine viruses was 91.0%, and the level of incidence was high irrespective of region or cultivar. The predominant viruses were grapevine leafroll-associated virus 3 (80.2%), grapevine fleck virus (70.6%), and grapevine rupestris stem pitting-associated virus (49.2%). Most grapevines were infected with multiple viruses, suggesting that Korean vineyards are likely to suffer economic losses resulting from viral diseases. This is the first extensive survey performed in Korea to observe the outbreak status of diverse grapevine viruses; surveys of this type can provide important information for the management of grapevine viruses in Korea. [ABSTRACT FROM AUTHOR]

Grapevine leafroll‐associated virus (GLRaV) infections are accompanied by symptoms influenced by host genotype, rootstock, environment, and which individual or combination of GLRaVs is present. Using a dedicated experimental vineyard, we studied the responses to GLRaVs in ripening berries from Cabernet Franc grapevines grafted to different rootstocks and with zero, one, or pairs of leafroll infection(s). RNA sequencing data were mapped to a high‐quality Cabernet Franc genome reference assembled to carry out this study and integrated with hormone and metabolite abundance data. This study characterized conserved and condition‐dependent responses to GLRaV infection(s). Common responses to GLRaVs were reproduced in two consecutive years and occurred in plants grafted to different rootstocks in more than one infection condition. Though different infections were inconsistently distinguishable from one another, the effects of infections in plants grafted to different rootstocks were distinct at each developmental stage. Conserved responses included the modulation of genes related to pathogen detection, abscisic acid (ABA) signalling, phenylpropanoid biosynthesis, and cytoskeleton remodelling. ABA, ABA glucose ester, ABA and hormone signalling‐related gene expression, and the expression of genes in several transcription factor families differentiated the effects of GLRaVs in berries from Cabernet Franc grapevines grafted to different rootstocks. These results support that ABA participates in the shared responses to GLRaV infection and differentiates the responses observed in grapevines grafted to different rootstocks. [ABSTRACT FROM AUTHOR]

Grapevine leafroll-associated virus 3 (GLRaV-3), an economically significant pathogen of grapevines, is transmitted by Pseudococcus calceolariae , a mealybug commonly found in New Zealand vineyards. To help inform alternative GLRaV-3 control strategies, this study evaluated the three-way interaction between the mealybug, its plant host and the virus. The retention and transmission of GLRaV-3 by P. calceolariae after access to non- Vitis host plants (and a non-GLRaV-3 host) White clover (Trifolium repens L. cv. "Grasslands Huia white clover"), Crimson clover (T. incarnatum), and Nicotiana benthamiana (an alternative GLRaV-3 host) was investigated. For all experiments, P. calceolariae first instars with a 4 or 6 days acquisition access period on GLRaV-3-positive grapevine leaves were used. GLRaV-3 was detected in mealybugs up to 16 days on non- Vitis plant hosts but not after 20 days. GLRaV-3 was retained by second instars (n = 8/45) and exuviae (molted skin, n = 6/6) following a 4 days acquisition period on infected grapevines leaves and an 11 days feeding on non- Vitis plant hosts. Furthermore, GLRaV-3 was transmitted to grapevine (40−60%) by P. calceolariae second instars after access to white clover for up to 11 days; 90% transmission to grapevine was achieved when no alternative host feeding was provided. The 16 days retention period is the longest observed in mealybug vectoring of GLRaV-3. The results suggest that an alternative strategy of using ground-cover plants as a disrupter of virus transmission may be effective if mealybugs settle and continue to feed on them for 20 or more days. [ABSTRACT FROM AUTHOR]

The sanitary status of grapevines has not yet been considered sufficiently in vineyards throughout Bosnia and Herzegovina (BiH). An extensive survey of five major grapevine viruses in the country was carried out in 2019. A total of 630 samples from the two dominant autochthonous cultivars, named Žilavka and Blatina, were tested by DAS-ELISA for the presence of grapevine leafroll-associated viruses (GLRaV-1 and 3), grapevine fleck virus (GFkV), grapevine fanleaf virus (GFLV) and Arabis mosaic virus (ArMV). Eighty-eight % of the samples were positive for at least one virus, and all five viruses were detected, thought with different incidence, i.e. GLRaV-3 (84%), GFLV (43%), GLRaV-1 (14%), GFkV (10%) and ArMV (0.2%). The majority of infected plants (about 75%) were asymptomatic. Specific virus symptoms were observed in the remaining infected plants, together with the reported GLRaV vectors, Planococcus ficus and Parthenolecanium corni, while nematodes of the Xiphinema genus were not found in the GFLV- or ArMV-infected vineyards. The GLRaV-3 CP phylogenetic analyses showed 75–100% nucleotide identity between the BiH and reference isolates, and the BiH isolates clustered into the major group. The dNS/dS ratio indicated a negative selection of the virus population, and the lack of geographical structuring within the population was observed. In addition, putative GLRaV-3 recombinants with breakpoints in the 5' of the CP gene were detected, while no recombinant strains were identified for the other four viruses. The obtained results indicate a deteriorated sanitary status of the cultivated grapevines, the prevalence and intraspecies genetic diversity of GLRaV-3 throughout the country. The establishment of certified grapevine material and adequate virus vector control is therefore of primary importance to prevent further spread of these viruses. This study presents the results of the first molecular characterisation of grapevine viruses in Bosnia and Herzegovina. [ABSTRACT FROM AUTHOR]

Several virus diseases cause damage to Vitis vinifera L., but information on their incidence and impact on hybrid cultivars is scarce, particularly under cool-climate conditions. In Nova Scotia (NS), the wine industry is based predominantly on interspecific hybrid cultivars. To understand the occurrence of major grapevine viruses in NS, surveys were conducted in 2016, 2017 and 2018. A total of 965 composite five-vine samples, collected from 35 hybrids and 18 V. vinifera vineyard blocks, were tested for grapevine leafroll-associated virus 1 (GLRaV-1), GLRaV-2, GLRaV-3 and GLRaV-4, grapevine fanleaf virus (GFLV), grapevine red blotch virus (GRBV) and grapevine Pinot gris virus (GPGV) by PCR/RT-PCR. Overall, 3.4% of the samples were positive for GLRaV-1, 22.8% for GLRaV-3, 0.9% for GFLV, 4.6% for GRBV and 3.2% for GPGV. None of the 575 samples collected in 2016 and 2017 tested positive for GLRaV-2 or GLRaV-4. Mixed infections by more than one virus occurred in 3% of the composite samples. Of 671 hybrid and 294 V. vinifera samples tested, 38.3% and 27.6% were positive for at least one of the viruses (GLRaV-1, −3, GFLV, GRBV and GPGV), respectively. Phylogenetic analysis of GLRaV-1, −3, GFLV and GPGV revealed the presence of global variants. Complete genome characterization and phylogenetic analysis of nine GRBV isolates grouped three into clade I and six into clade II, indicating the presence of two variants. These findings, along with preliminary reports of insect vectors, establish the first epidemiological framework of the major viral diseases in NS, highlighting the need for long-term management strategies. [ABSTRACT FROM AUTHOR]

Grapevine leafroll-associated virus 3 (GLRaV-3) is an economically significant virus of grapevines, with secondary spread mediated by several species of mealybug and soft scale insects. To better understand virus-vector interactions, sensitive virus detection in these insects is a key tool. In this research, two new hydrolysis-probe-based real-time assays for GLRaV-3 detection were developed and compared to three existing assays. Of the five assays compared, the one-step RT-qPCR probe-based assay was the most sensitive and reliable, with as few as 10 virus RNA copies detected. This is the first description of a real-time molecular assay for virus detection in mealybugs with such sensitivity. [ABSTRACT FROM AUTHOR]

Grapevines from 13 vineyards in Pakistan were surveyed for the prevalence of several pathogens. Using RT-qPCR, 257 samples were tested for 19 viruses, phytoplasmas and Xylella fastidiosa. Prevalent viruses were: grapevine virus A (GVA, 47.8%), grapevine leafroll-associated virus 2 (GLRaV-2, 37.3%), grapevine rupestris stem pitting-associated virus (GRSPaV, 36.1%), and grapevine fleck virus (GFkV, 35%). Other viruses detected were: grapevine leafroll-associated virus 1 (GLRaV-1, 2.3%), grapevine leafroll-associated virus 2RG (GLRaV-2RG, 5%), grapevine leafroll-associated virus 3 (GLRaV-3, 7%), grapevine leafroll-associated virus 4 (GLRaV-4) and its strains (5, 6, and Pr, 16.6%), grapevine leafroll-associated virus 7 (GLRaV-7, 4.2%), grapevine fanleaf virus (GFLV, 11.6%), grapevine virus B (GVB, 4.2%), grapevine virus D (GVD, 0.7%), grapevine virus E (GVE, 1.1%), and grapevine Pinot gris virus (GPGV, 1.9%). Mixed infections were detected in 75.9% of samples. Pathogens tested for, but not detected include GLRaV-4 strains 9 and Car, grapevine red blotch virus (GRBV), tomato ringspot virus (ToRSV), tobacco ringspot virus (TRSV), Arabis mosaic virus (ArMV), grapevine virus F (GVF), phytoplasmas and X. fastidiosa. Additionally, 16 samples were analyzed by high-throughput sequencing (HTS) to confirm RT-qPCR results. In this paper we present an extensive survey for grapevine pathogens and thus the first report of GVA, GVB, GVD, GVE, GRSPaV, GFkV, GLRaV-1, GLRaV-2, GLRaV-2RG, GLRaV-3, GLRaV-4, GLRaV-4 strains 5, 6, and Pr, and GLRaV-7 in Pakistan. [ABSTRACT FROM AUTHOR]

Grapevine leafroll disease (GLD) is present in all grape-growing regions of the world and is considered the most significant grapevine viral disease. Grapevine leafroll-associated virus 3 (GLRaV-3) is considered the primary cause of GLD and in South African vineyards five genetic variant groups (I, II, III, VI and VII) have been confirmed. Biological distinctions between GLRaV-3 variants have not been fully validated. By characterising virus concentration and stress-responsive microRNA expression in GLRaV-3 infected plants, this study aimed to glean a better understanding of the possible biological distinctions between GLRaV-3 variants. Quantitative reverse transcription PCR was utilised for virus concentration ratio (VCR) determination and miRNA quantitation in GLRaV-3 positive and negative grapevines grown under greenhouse and field conditions. This study found statistically significant differences in VCRs in plants singly infected with different GLRaV-3 variants. Interestingly, no difference in mean VCRs were observed between data sets, despite notable differences in plant age, duration of GLRaV-3 infection, scion/rootstock combination and growing conditions. Several miRNAs showed statistically significant expression modulation between infected and healthy samples. miRNA expression between data sets varied substantially and a greater overall miRNA response was observed in plants with more established GLRaV-3 infections. The lack of significant differences in mean VCRs between data sets, coupled with the consistent modulation of certain miRNAs in plants that have likely been infected for longer is a promising result. This finding could indicate that successful inhibition of further virus replication by plant defence mechanisms occurred, and that these miRNAs are implicated in this response. [ABSTRACT FROM AUTHOR]

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We describe a droplet‐vitrification cryotherapy method for the eradication of grapevine leafroll‐associated virus‐3 (GLRaV‐3) from diseased in vitro shoots of Vitis plants. The procedure involved pre‐culture of 1.0‐mm shoot tips containing five to six leaf primordia (LPs) for 3 days with a pre‐culture medium containing 0.3 M sucrose, 0.16 mM glutathione and 0.14 mM ascorbic acid, treatment of the pre‐cultured shoot tips for 20 min at room temperature with a loading solution composed of 2 M glycerol and 0.4 M sucrose and exposure to plant vitrification solution 2 (PVS2), prior to freezing in liquid nitrogen of dehydrated shoot tips contained in 2.5‐μL PVS2 droplets on aluminium foil strips. Virus localisation showed GLRaV‐3 was not present in apical dome (AD) and LPs 1–4, but it was detected in the basal shoot tip region, approximately 0.5 mm from the AD, as well as in LP 5 and more mature tissues. Histological observations identify that only freezing in liquid nitrogen results in the death of all cells in areas of shoot tips harbouring virus, whereas PVS2 treatment does not. Thus, freezing in liquid nitrogen is a necessary step that eradicates GLRaV‐3. This cryotherapy procedure produced shoot regrowth levels that ranged from 43% to 59%, and all plants recovered after cryotherapy were free of GLRaV‐3 in two wine, one table and one rootstock cultivars. Thus, this procedure can be considered to be efficient and wildly applicable for eradication of GLRaV‐3 from Vitis spp. A shoot tip cryotherapy was described for efficient eradication of grapevine leafroll‐associated virus‐3. Mechanism for eradication of GLRaV‐3 by shoot tip cryotherapy was provided. [ABSTRACT FROM AUTHOR]

P70 is a Pinot Noir grapevine accession that displays strong leafroll disease symptoms. A high-throughput sequencing (HTS)-based analysis established that P70 was mixed-infected by two variants of grapevine leafroll-associated virus 1 (GLRaV-1, genus Ampelovirus) and one of grapevine virus A (GVA, genus Vitivirus) as well as by two viroids (hop stunt viroid [HSVd] and grapevine yellow speckle viroid 1 [GYSVd1]) and four variants of grapevine rupestris stem pitting-associated virus (GRSPaV). Immunogold labelling using gold particles of two different diameters revealed the existence of ‘hybrid’ particles labelled at one end as GLRaV-1, with the rest labelled as GVA. In this work, we suggest that immunogold labelling can provide information about the biology of the viruses, going deeper than just genomic information provided by HTS, from which no recombinant or ‘chimeric’ GLRaV-1/GVA sequences had been identified in the dataset. Our observations suggest an unknown interaction between members of two different viral species that are often encountered together in a single grapevine, highlighting potential consequences in the vector biology and epidemiology of leafroll and rugose-wood diseases. [ABSTRACT FROM AUTHOR]

The distribution and diversity of grapevine red blotch virus (GRBV) and wild Vitis virus 1 (WVV1) (genus Grablovirus; family Geminiviridae) were determined in free-living Vitis spp. in northern California and New York from 2013 to 2017. Grabloviruses were detected by polymerase chain reaction in 28% (57 of 203) of samples from California but in none of the 163 samples from New York. The incidence of GRBV in free-living vines was significantly higher in samples from California counties with high compared with low grape production (χ² = 83.09; P < 0.001), and in samples near (<5 km) to compared with far (>5 km) from vineyards (χ² = 57.58; P < 0.001). These results suggested a directional spread of GRBV inoculum predominantly from vineyards to freeliving Vitis spp. WVV1 incidence was also significantly higher in areas with higher grape production acreage (y2 = 16.02; P < 0.001). However, in contrast to GRBV, no differential distribution of WVV1 incidence was observed with regard to distance from vineyards (χ² = 0.88; P = 0.3513). Two distinct phylogenetic clades were identified for both GRBV and WVV I isolates from free-living Vitis spp., although the nucleotide sequence variability of the genomic diversity fragment was higher for WWV1 (94.3 to 99.8% sequence identity within clade 1 isolates and 90.1 to 100% within clade 2 isolates) than GRBV (98.3% between clade 1 isolates and 96.9 to 100% within clade 2 isolates). Additionally, evidence for intraspecific recombination events was found in WVV 1 isolates and confirmed in GRBV isolates. The prevalence of grabloviruses in California free-living vines highlights the need for vigilance regarding potential grablovirus inoculum sources in order to protect new vineyard plantings and foundation stock vineyards in California. [ABSTRACT FROM AUTHOR]

Vineyard surveys were conducted for three consecutive seasons in eastem Washington State, the major grapevine-growing region in the state, to document the occurrence of Grapevine leafroll-associated virus 3 (GLRaV-3) and Grapevine red blotch virus (GRBV). The majority of samples were collected from red-berried wine grape (Vitis vinifera) cultivars exhibiting symptoms of or suspected for grapevine leafroll (GLD) and red blotch (GRBD) diseases. A limited number of samples from white-berried cultivars were collected randomly due to the lack of visual symptoms. Samples were collected from a total of 2.063 grapevines from 18 red-berried cultivars and seven white-berried cultivars planted in eight American Viticultural Areas and tested for GLRaV-3 and GRBV using RT-PCR and PCR, respectively. The results showed 67.77% and 6.01% of total samples positive for GLRaV-3 and GRBV, respectively, and 9.06% of samples positive for both viruses. About 17% of samples tested negative for the two viruses, but some of these samples were positive for GLRaV-2 and GLRaV-4. Overall results indicated that GLRaV-3 was more common than GRBV, independent of cultivars and the geographic origin of samples. Due to variability in symptoms in redberried cultivars, virus-specific diagnostic assays were deemed necessary for reliable identification of GLRaV-3 and GRBV and to differentiate GLD and GRBD symptoms from those induced by biotic and abiotic stresses in vineyards. A multiplex PCR protocol was developed for simultaneous detection of GLRaV-3 and GRBV in grapevine samples. A global phylogenetic analysis of GRBV genome sequences revealed segregation of virus isolates from Washington State vineyards into two distinct clades, with the majority of isolates belonging to clade II. [ABSTRACT FROM AUTHOR]

Main conclusion: Foliar sugar accumulation in grapevines with leafroll disease was correlated with lower photosynthesis, likely due to feedback inhibition. However, cold acclimation of dormant tissues remained unaffected by the virus status.Grapevine leafroll-associated viruses (GLRaV) contribute to losses in fruit yield and quality worldwide. Visually, leafroll disease symptoms appear similar to those associated with an imbalance in source/sink relations and a concomitant feedback inhibition of photosynthesis, which is often caused by an impasse in sugar translocation. In order to test this potential relationship and related physiological responses, leaf water status, gas exchange, non-structural carbohydrates, and dormant tissue cold hardiness were examined over 2 years in healthy and GLRaV-3-infected, field-grown Merlot grapevines. Diurnal and seasonal changes in leaf water status and gas exchange were dominated by variations in water availability, temperature, and leaf age, while GLRaV-3 infection contributed less to the overall variation. By contrast, foliar carbohydrates increased markedly in infected plants, with starch accumulating early in the growing season, followed by soluble sugar accumulation, leaf reddening, and declining gas exchange. Photosynthesis correlated negatively with leaf sugar content. However, dormant-season cold hardiness of buds and cane vascular tissues was similar in healthy and infected vines. These findings support the idea that visible symptoms of grapevine leafroll disease are a consequence of carbohydrate accumulation which, in turn, may lead to feedback inhibition of photosynthesis. In addition, this study provided evidence that GLRaV-3 infection is unlikely to alter the susceptibility to moderate water deficit and winter damage in mature Merlot grapevines. [ABSTRACT FROM AUTHOR]

Grapevine red blotch virus (GRBV) is a recently described virus that infects grapevine. Little information is available on the possible occurrence and distribution outside North America. Therefore, we surveyed commercial vineyards from the three major grape-growing regions in Switzerland to determine the presence or absence of GRBV. In total, 3,062 vines were analyzed by polymerase chain reaction. None of the vines tested positive for GRBV, suggesting the absence of GRBV from Swiss vineyards. We also investigated whether GRBV was present in 653 grapevine accessions in the Agroscope grapevine virus collection at Nyon, including dominantly Swiss (457) but also international accessions. Only six referential accessions were infected by GRBV, all originating from the United States, whereas all others from 10 European and 8 non-European origins tested negative. High-throughput sequencing analysis of Zinfandel A2V13, in the collection since 1985, confirmed close similarity of GRBV isolate Z_A2V 13 to American isolates according to genomes deposited in GenBank. Because the Zinfandel A2V13 reference was also maintained grafted on the leafroll virus indicator Vitis vinifera 'Gamay', we evaluated the effect of GRBV on viticultural performance over a 3-year period. Our results showed clear detrimental effects of GRBV on grapevine physiology (vine vigor, leaf chlorophyll content, and gas exchange) and fruit quality. These findings underscore the importance of implementation of GRBV testing worldwide in certification and quarantine programs to prevent the dissemination of this virus. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Grapevine leafroll disease (GLD) is a virus disease that quickly propagates through vineyards under appropriate weather conditions and can reduce grape production worldwide by nearly 60 %. Therefore, the accurate diagnosis and reliable evaluation of GLD distribution, particularly at the early stage of GLD infection, is important to prevent the spread of this disease. This study applied the ant colony clustering algorithm (ACCA) to detect GLD spectral anomalies on 4 GLD-infected vineyards according to multi-spectral imagery for precision disease management. GLD was classified into three stages: GLD1, GLD2 and GLD3 according to its infection severity. An 11-index feature vector and its stacked image were generated to enhance the spectral differences and spectral discrimination between diseased and healthy grapevines. ACCA was then designed to solve the fuzziness of the multi-spectral image for GLD-infected grapevines and successfully identify GLD from healthy grapevines. Finally, a field survey with 49 samples and pixel purity index technology were applied to validate the effectiveness, efficiency and accuracy of ACCA. Field results indicated that an early stage of the GLD infection (GLD1) could be successfully discriminated from GLD2-, GLD3- and non-infected grapevines. The classification accuracies of non-, GLD1-, GLD2- and GLD3-infected grapevines were 94.4, 75, 84.6 and 83.3 %, respectively. Hence, the method based on an 11-index image and ACCA may significantly detect GLD at an early stage from healthy grapevines for precision disease management at the field level. [ABSTRACT FROM AUTHOR]

Several mealybug species are vectors of grapevine leafroll-associated viruses (GLRaV), which cause the economically important grapevine leafroll disease in grape-producing regions worldwide. The mealybug Ferrisia gilli Gullan is a new pest of grapevines in El Dorado County, located in the Sierra Foothill wine-growing region of California. GLRaV species 1, 2, 3, and 4LV have been detected in vineyards with symptomatic vines in the Sierra Foothills. We conducted controlled virus acquisition and transmission experiments using source vine accessions infected with different combinations of GLRaV. We determined that F. gilli acquired GLRaV 1, 2, 3, and 4LV, and transmitted GLRaV-3 and GLRaV-4LV to uninfected recipient vines. Like numerous other mealybug species, in addition to causing direct damage to vines, F. gilli poses a threat to the grape industry as a vector of economically damaging viruses. [ABSTRACT FROM AUTHOR]

Grapevine leafroll disease (GLD) is an economically important virus disease affecting wine grapes (Vitis vinifera L.), but little is known about its effect on wine chemistry and sensory composition of wines. In this study, impacts of GLD on fruit yield, berry quality and wine chemistry and sensory features were investigated in a red wine grape cultivar planted in a commercial vineyard. Own-rooted Merlot vines showing GLD symptoms and tested positive for Grapevine leafroll-associated virus 3 and adjacent non-symptomatic vines that tested negative for the virus were compared during three consecutive seasons. Number and total weight of clusters per vine were significantly less in symptomatic relative to non-symptomatic vines. In contrast to previous studies, a time-course analysis of juice from grapes harvested at different stages of berry development from symptomatic and non-symptomatic vines indicated more prominent negative impacts of GLD on total soluble solids (TSS) and berry skin anthocyanins than in juice pH and titratable acidity. Differences in TSS between grapes of symptomatic and non-symptomatic vines were more pronounced after the onset of véraison, with significantly lower concentrations of TSS in grapes from symptomatic vines throughout berry ripening until harvest. Wines made from grapes of GLD-affected vines had significantly lower alcohol, polymeric pigments, and anthocyanins compared to corresponding wines from grapes of non-symptomatic vines. Sensory descriptive analysis of 2010 wines indicated significant differences in color, aroma and astringency between wines made from grapes harvested from GLD-affected and unaffected vines. The impacts of GLD on yield and fruit and wine quality traits were variable between the seasons, with greater impacts observed during a cooler season, suggesting the influence of host plant × environment interactions on overall impacts of the disease. [ABSTRACT FROM AUTHOR]

Some diseases manifest as one characteristic set of symptoms to the host, but can be caused by multiple pathogens. Control treatments based on plant symptoms can make it difficult to effectively manage such diseases, as the biology of the underlying pathogens can vary. Grapevine leafroll disease affects grapes worldwide, and is associated with several viral species in the family Closteroviridae. Whereas some of the viruses associated with this disease are transmitted by insect vectors, others are only graft-transmissible. In three regions of California, we surveyed vineyards containing diseased vines and screened symptomatic plants for all known viral species associated with grapevine leafroll disease. Relative incidence of each virus species differed among the three regions regions, particularly in relation to species with known vectors compared with those only known to be graft-transmitted. In one region, the pathogen population was dominated by species not known to have an insect vector. In contrast, populations in the other surveyed regions were dominated by virus species that are vector-transmissible. Our survey did not detect viruses associated with grapevine leafroll disease at some sites with characteristic disease symptoms. This could be explained either by undescribed genetic diversity among these viruses that prevented detection with available molecular tools at the time the survey was performed, or a misidentification of visual symptoms that may have had other underlying causes. Based on the differences in relative prevalence of each virus species among regions and among vineyards within regions, we expect that region and site-specific management strategies are needed for effective disease control. [ABSTRACT FROM AUTHOR]

In our study, in addition to GV-Sat, grapevine leafroll-associated virus 1 (GLRaV-1), grapevine leafroll-associated virus 2 (GLRaV-2), grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine Pinot gris virus (GPGV), hop stunt viroid (HSVd), and grapevine yellow speckle viroid-1 (GYSVd-1) were also identified. The helper virus of GV-Sat was not identified, but previous studies reported co-infection of this virus with vitiviruses and GLRaVs, which potentially indicates that these viruses have helper virus function for GV-Sat. "Cipro" Helper virus 1329 1330 2 11/29/21 20211101 NES 211101 I Grapevine satellite virus i (GV-Sat) is a member of the genus I Virtovirus. i It was first identified in California with the application of high-throughput sequencing. [Extracted from the article]

The vine mealybug, Planococcus ficus (Signoret), and the longtailed mealybug, Pseudococcus longispinus (Targioni Tozzetti), are vectors of grapevine leafroll-associated virus 3 (GRLaV-3), one of the most abundant viruses associated with grapevine leafroll disease. To elucidate the transmission biology in South Africa, acquisition access periods (AAPs), inoculation access periods (IAPs) and the retention of the virus in starving and feeding first- to second instar nymphs were determined. The rootstock hybrid LN33 served as virus source and grapevines (Vitis vinifera L., cv. Cabernet franc) served as recipient plants. An AAP of 15 min or an IAP of 15 min was sufficient for Pl. ficus to acquire or transmit GLRaV-3, respectively. Nymphs of PL ficus retained the virus for at least eight days when feeding on a non-virus host and grapevine, and for at least two days when starving, and were then capable of transmitting it successfully to healthy grapevine plants. Nymphs of Ps. longispinus transmitted the virus after an AAP of 30 min and an IAP of 1 h. They retained the virus for at least three days when feeding on virus-free vines or starving. The GLRaV-3 infection rates of plants with Pl. ficus as vector varied with AAPs. These were lower (20 to 60%) for AAPs of 12 h or less than for AAPs of 24 h or more (80 to 100%). The findings are of importance for understanding the transmission biology of mealybug vectors and devising management strategies for grapevine leafroll. [ABSTRACT FROM AUTHOR]

To characterize the prevalence of viruses associated with grapevine leafroll disease in China, 249 grapevine (Vitis spp.) samples (86 popular cultivars and a rootstock) from 19 provinces and regions were collected and tested for Grapevine leafroll-associated virus 1 (GLRaV-1), GLRaV-2, GLRaV-3, GLRaV-4, and GLRaV-4 strain 5 by SYBR Green real-time reverse-transcription polymerase chain reaction (RTPCR), and RT-PCR and sequencing. GLRaV-3 was found in 100% of the samples while GLRaV-1, GLRaV-2, and GLRaV-4 were detected in 24.9% (62/249), 15.3% (38/249), and 0.80% (2/249) of the samples, respectively. Single infections with GLRaV-3 were found in 66.3% (165/249) of the samples, and the remaining samples were mixed infections of GLRaV-3 with one or two other GLRaVs, those with GLRaV-1 being the most common (18.5%, 46/249). The genetic variability of Chinese GLRaV-3 isolates was characterized based on the coat protein (CP) gene. In total, 153 full-length CP gene sequences (94 sequences newly generated) of Chinese GLRaV-3 isolates from different grapevine-growing regions showed 89.3 to 100.0% and 92.7 to 100.0% identity at the nucleotide and amino acid levels, respectively. The average nucleotide diversity for the population of Chinese GLRaV-3 isolates was estimated at 0.037 (standard error = 0.0032). GLRaV-3 isolates from China segregated into five distinct phylogenetic groups and two novel recombination events were found in the viral population. This is the first and most extensive report of the prevalent species of GLRaV in China, which also provides an assessment of genetic variability of GLRaV-3 Chinese isolates. [ABSTRACT FROM AUTHOR]

Vitis californica and Vitis californica x Vitis vinifera hybrids are hosts for grapevine leafroll-associated virus -2 and -3 and grapevine virus a and B. Plant Dis. 10.3389/fmicb.2013.00082 4 Osman F, Leutenegger C, Golino D, Rowhani A. Real-time RT-PCR (TaqMan) assays for the detection of grapevine Leafroll association viruses 1-5 and 9. Keywords: GLRaV-3; Grapevine leafroll disease; GLD; Southeastern US EN GLRaV-3 Grapevine leafroll disease GLD Southeastern US 385 386 2 02/25/21 20210201 NES 210201 Grapevine leafroll-associated virus 3 (GLRaV-3) belongs to the genus I Ampelovirus i and is considered the economically most impactful grapevine virus worldwide (Maree et al. [3]). [Extracted from the article]

Prevalence and distribution of Grapevine leafroll-associated virus 7 in China detected by an improved reverse transcription polymerase chain reaction assay. Negative ELISA samples had been randomly tested by RT-PCR, but no amplicon was obtained in these samples using the same primer pair. A total of 436 samples was collected and tested for the detection of five GLRaVs using five commercial ELISA kits (GLRaV-1 DAS, GLRaV-2 DAS, GLRaV-3 DAS, GLRaV-4 DAS and GLRaV-7 DAS, Invitrogen™, U.S.A.). [Extracted from the article]

Grapevine leafroll disease ( GLRD) is one of the most economically important virus diseases of grapevine ( Vitis spp.) worldwide. In this study, we used high-throughput sequencing of c DNA libraries made from small RNAs (s RNAs) to compare profiles of s RNA populations recovered from own-rooted Merlot grapevines with and without GLRD symptoms. The data revealed the presence of s RNAs specific to Grapevine leafroll-associated virus 3, Hop stunt viroid ( Hp SVd), Grapevine yellow speckle viroid 1 ( GYSVd-1) and Grapevine yellow speckle viroid 2 ( GYSVd-2) in symptomatic grapevines and s RNAs specific only to Hp SVd, GYSVd-1 and GYSVd-2 in nonsymptomatic grapevines. In addition to 135 previously identified conserved micro RNAs in grapevine ( Vvi-mi Rs), we identified 10 novel and several candidate Vvi-mi Rs in both symptomatic and nonsymptomatic grapevine leaves based on the cloning of mi RNA star sequences. Quantitative real-time reverse transcriptase-polymerase chain reaction ( RT-PCR) of selected conserved Vvi-mi Rs indicated that individual members of an mi RNA family are differentially expressed in symptomatic and nonsymptomatic leaves. The high-resolution mapping of s RNAs specific to an ampelovirus and three viroids in mixed infections, the identification of novel Vvi-mi Rs and the modulation of certain conserved Vvi-mi Rs offers resources for the further elucidation of compatible host-pathogen interactions and for the provision of ecologically relevant information to better understand host-pathogen-environment interactions in a perennial fruit crop. [ABSTRACT FROM AUTHOR]

Grapevine leafroll-associated virus 3 (GLRaV-3) is one of the agents responsible for leafroll disease of cultivated grapevines. To secure information on the molecular variability of GLRaV-3 isolates from central Europe, a 3,477 nucleotide long genome fragment coding for the end of a 55kDa protein, the complete capsid protein (CP), the minor CP (CPm), a 21kDa protein, and the N-terminal part of the p20A protein was sequenced from an atypical Slovak viral isolate, inducing no leafroll symptoms on the original Vitis vinifera host. In the sequenced ca. 3.5 kb region, the nucleotide identities with previously characterized isolates from different parts of the world ranged from 89.4 (Group 3 isolates) to 99.5% (Group 1 isolates). The Slovak SK04 isolate clustered within the largest Group 1, although in a slightly separated branch. Vegetative propagation by infected cuttings did not lead to changes of the dominant GLRaV-3 sequence, determined from the mother plant, when compared with the sequences obtained from the newly established 5-year-old self-rooted plants, indicating the high genetic stability of the SK04 viral population. [ABSTRACT FROM AUTHOR]

Grapevine leafroll-associated virus 3 (GLRaV-3) is associated with grapevine leafroll disease, one of the most economically important viral diseases of grapevines. This disease impacts on both vine health and grape quality; reduction in yield, brix and wine colour are among its detrimental effects. Many methods, including serological and molecular procedures, have been developed for the detection of GLRaV-3; however, there is no PCR-based assay available to quantify virus populations within plant tissues. A real-time RT-PCR assay with TaqMan probe was developed for specific and reliable quantitative detection of GLRaV-3 in infected tissues. The designed primers and probes target the conserved sequence in the RNA-dependent RNA polymerase (RdRp) domain of the viral genome to prevent amplification of most subgenomic and defective RNAs. This protocol was used to examine the seasonal dynamics and translocation of GLRaV-3 in field-grown grapevines. The results showed that the virus spread quickly from trunks to new growing shoots and leaves early in the growing season, and most samples still harboured detectable virus during late summer and autumn. The seasonal progress of one GLRaV-3 isolate was compared in four grapevine cultivars (Chardonnay, Cabernet Sauvignon, Italia and Thompson Seedless). Within cultivars there was little variability in the distribution and translocation of GLRaV-3, except for in Thompson Seedless. This quantitative detection assay will be a valuable tool for GLRaV-3 diagnosis, disease monitoring and population ecology studies. [ABSTRACT FROM AUTHOR]

Background and Aims: Australian certification programs that provide high-health planting material depend on accurate virus detection methods. The reliability of enzyme-linked immunosorbent assays (ELISAs) and reverse transcription-polymerase chain reaction (RT-PCR) tests for virus detection was compared in Australian conditions. Methods and Results: Replicate trials were established in a hot climate and a cool climate with grapevines that were uninoculated or inoculated with Grapevine virus A, Grapevine fleck virus, Grapevine leafroll-associated virus (GLRaV)-2 and GLRaV-3. Grapevines were tested monthly for virus during 3 years. RT-PCR detected viruses more frequently than ELISA, and the reliability of both tests increased after 12 months and up to 3 years post-inoculation in both climates. Conclusions: Viruses may not be consistently detected until 12 months after an infection event. RT-PCR is more reliable than ELISA for virus detection during spring and summer. However, detection of viruses was rarely 100% efficient, and retesting of grapevines is recommended to improve the rate of detection. Significance of the Study: Validated diagnostic procedures were developed to improve the reliability of grapevine virus detection in Australia. [ABSTRACT FROM AUTHOR]

New insights into the genetic structure and variability of grapevine leafroll-associated viruses (GLRaVs) gained through worldwide efforts in the last decade or so, and the production and use of new sets of serological reagents, have provided the solid foundation on which the present revision of the taxonomic structure of the family Closteroviridae, and the genus Ampelovirus in particular, is based. A comparative examination of the amino acid sequence divergence of three taxonomically relevant genes [RNA-dependent RNA polymerase (polymerase), heat shock protein 70 homologue (HSP70h) and coat protein (CP)] disclosed a difference among Grapevine leafroll-associated virus 4 (GLRaV-4), -5, -6 and -9 and a group of more recently described viruses (GLRaV-Pr, GLRaV-De and GLRaV-Car) below the 25% limit recently set by the International Committee on Taxonomy of Viruses (ICTV) as a discriminating criterion for the identification of species in the family Closteroviridae. This, plus the recognition that GLRaV- 4, -5, -6 and -9 are serologically related, have similar biological and epidemiological traits, and that these viruses and GLRaV-Pr, GLRaV-De, GLRaV-Car have a genome with the same structure and size, supports the notion that they are all genetically divergent variants of a single species, GLRaV-4. The genus Ampelovirus is split into two subgroups designated I and II in recognition of the wide difference in the size and structure of the genome of the present members. Finally, the establishment of a fourth genus within the family Closteroviridae, comprising the unassigned putative species Grapevine leafrollassociated virus 7 (GLRaV-7), Little cherry virus 1 (LChV-1) and Cordyline virus 1 (CoV-1), is justified based on their molecular and biological characteristics that differ from those of members of the other three genera of the family. [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]

putative new ampelovirus was detected in Vitis vinifera cv. Carnelian showing mild leafroll symptoms and molecularly characterized. The complete genome consisted of 13,625 nt and had a structure similar to that of members of subgroup I in the genus Ampelovirus (fam. Closteroviridae). In-depth analyses showed that the virus from cv. Carnelian is the most distinct member of the 'GLRaV-4 lineage' of ampeloviruses, which comprises GLRaV-4, -5, -6, -9, and the recently characterized GLRaV-Pr, and GLRaV-De. This virus appears to be a new member of the family Closteroviridae, for which the provisional name grapevine leafroll-associated Carnelian virus is proposed. [ABSTRACT FROM AUTHOR]

The spread of grapevine leafroll-associated virus 3 (GLRaV-3) through a newly planted block of Merlot and Chardonnay grapevines in a commercial vineyard was measured between 1998 and 2003. The population changes of the mealybug vector (Pseudococcus longispinus) were recorded over the same period in 12–17-year-old GLRaV-3-infected grapevines immediately adjacent to two sides of the new block. Relationships between the spread of GLRaV-3 and mealybug numbers were examined. Mealybugs developed through three generations a year and populations were low for 4 of the 6 years. Numbers were usually higher in the 12–17-year-old Sauvignon Blanc vines and adjacent young 1–6-year-old Merlot vines than in the 12–17-year-old Breidecker and adjacent young 1–6-year-old Chardonnay vines on the other side of the block. However, GLRaV-3 spread more rapidly in young Chardonnay vines than in young Merlot vines. New infections in both Merlot and Chardonnay initially occurred at random, but newly infected vines were more often clustered from 2002 to 2003, and especially in Merlot. Most newly infected Chardonnay were recorded in the years following the highest numbers of mealybugs, in 1997–98 and 2000–01, but the disease spread more gradually in the Merlot. The data indicated that GLRaV-3 did not spread into the new block when third generation mealybug populations in adjacent infected vines were lower than ~5 mealybugs/leaf. Three methods for virus transmission by mealybug were proposed: one method was from natural, crawling dispersal of infected mealybugs, leading predominantly to within-row, vine-to-vine transmission; another method was from human-assisted movement of mealybug crawlers (such as on vineyard machinery) leading to random but localised infection on a within- or between-block scale; and a third method was through aerial dispersal of infectious mealybugs, resulting in random infections of vines and particularly operating on a between-block or between-vineyard scale. Infection in any vineyard probably occurs by all three methods, mediated by the poorly understood, and possibly site- and grape variety-specific, transmission ecology that underpins the mealybug-virus-vine relationships. [ABSTRACT FROM AUTHOR]