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Viruses can adversely affect vines' vitality, longevity, quality, and quantity in single or mixed infections. In this work, to detect single and mixed viral infections of grapevine in two central viticulture regions of Azerbaijan, a total of 166 samples showing virus-like symptoms including yellows, reddening, fan leaf, mosaic, leaf rolling, and shortening of internodes were collected during the growing seasons of 2022-2023 from different vineyards and subjected to ELISA and RT-PCR analyses. Plant samples were initially assayed using the one-step assay AgriStrip based on lateral flow immunochromatography and DAS-ELISA with specific antisera for Grapevine fanleaf virus (GFLV), Grapevine leafroll-associated virus 1 (GLRaV-1), Grapevine leafroll-associated virus 2 (GLRaV-2), Grapevine leafroll-associated virus 3 (GLRaV-3), and Grapevine leafroll-associated virus 4 (GLRaV-4). Following serological tests, 65% (108 out of 166) of the examined samples exhibited a positive result for one or more of the specified viruses. Total RNA was extracted, and synthesized cDNA was used to amplify a part of the coat protein (CP) gene in RT-PCR using specific primers. Amplicons of the expected size of 1560 bp, 401 bp, 600 bp, 942 bp, 500 bp, and 272 bp were amplified for the mentioned viruses, respectively (but not from healthy samples). The result of this study can be used in the management strategies of these viruses in grapevine-producing areas of Azerbaijan. [ABSTRACT FROM AUTHOR]

The objective of this study was to investigate the response of the grapevine variety 'Tribidrag' to virus infection over two vegetation seasons. Virus-free plants were greenhouse cultivated and green grafted with five different virus inocula composed of grapevine leafroll-associated virus 3 (GLRaV-3) singly or in coinfection with other most economically important grapevine viruses. Changes in nutrient status and photosynthesis-related parameters, along with symptom development, were measured. Using the quantitative PCR method, the relative concentration of five selected Vitis genes was determined. Cluster analysis and ANOVA revealed the reduction in phosphorus concentration (P) and photosynthesis-related parameters in infected plants in both seasons, even in the absence of symptom expression, indicating P and assimilation rate (Photo (A)) as stable markers of virus infection. Plants infected with inoculum Y composed of five different viruses provoked major significant changes in the first season while, in the second, fewer changes were measured. The sucrose synthase 3 gene was upregulated in infected plants confirming disturbed sugar metabolism related to virus-induced stress. This study showed that virus-induced changes in 'Tribidrag' plants even in the absence of symptoms are dependent on plant age, as well as on the composition of virus inocula. [ABSTRACT FROM AUTHOR]

A comprehensive study on the whole spectrum of viruses and viroids in five Iranian grapevine cultivars was carried out using sRNA libraries prepared from phloem tissue. A comparison of two approaches to virus detection from sRNAome data indicated a significant difference in the results and performance of the aligners in viral genome reconstruction. The results showed a complex virome in terms of viral composition, abundance, and richness. Thirteen viruses and viroids were identified in five Iranian grapevine cultivars, among which the grapevine red blotch virus and grapevine satellite virus were detected for the first time in Iranian vineyards. Grapevine leafroll-associated virus 1 (GLRaV1) and grapevine fanleaf virus (GFLV) were highly dominant in the virome. However, their frequency and abundance were somewhat different among grapevine cultivars. The results revealed a mixed infection of GLRaV1/grapevine yellow speckle viroid 1 (GYSVd1) and GFLV/GYSVd1 in grapevines that exhibited yellows and vein banding. We also propose a threshold of 14% of complete reconstruction as an appropriate threshold for detection of grapevine viruses that can be used as indicators for reliable grapevine virome profiling or in quarantine stations and certification programs. [ABSTRACT FROM AUTHOR]

In this study, an analysis of the virome of 51 grapevines from the Don ampelographic collection named after Ya. I. Potapenko (Russia) was performed using high-throughput sequencing of total RNA. A total of 20 previously described grapevine viruses and 4 viroids were identified. The most detected were grapevine rupestris stem pitting-associated virus (98%), hop stunt viroid (98%), grapevine Pinot gris virus (96%), grapevine yellow speckle viroid 1 (94%), and grapevine fleck virus (GFkV, 80%). Among the economically significant viruses, the most present were grapevine leafroll-associated virus 3 (37%), grapevine virus A (24%), and grapevine leafroll-associated virus 1 (16%). For the first time in Russia, a grapevine-associated tymo-like virus (78%) was detected. After a bioinformatics analysis, 123 complete or nearly complete viral genomes and 64 complete viroid genomes were assembled. An analysis of the phylogenetic relationships with reported global isolates was performed. We discovered and characterized the genomes of five novel grapevine viruses: bipartite dsRNA grapevine alphapartitivirus (genus Alphapartitivirus, family Partitiviridae), bipartite (+) ssRNA grapevine secovirus (genus Fabavirus, family Secoviridae) and three (+) ssRNA grapevine umbra-like viruses 2, -3, -4 (which phylogenetically occupy an intermediate position between representatives of the genus Umbravirus and umbravirus-like associated RNAs). [ABSTRACT FROM AUTHOR]

To determine the occurrence and distribution of prevalent viruses in commercially important vineyards, a survey was carried out in all thirteen wine-growing regions in Germany. Results reveal that the recently emerged Grapevine pinot gris virus (GPGV) was the most abundant virus with a percentage of 18% prevalence, followed by 13% Grapevine fleck virus (GFkV), 9% Grapevine leafroll-associated virus 1 (GLRaV-1), 4% Grapevine fanleaf virus (GFLV), 2% Raspberry ringspot virus (RpRSV), 2% Arabis mosaic virus (ArMV) and 2% Grapevine leafroll-associated virus 3 (GLRaV-3). Distribution of some viruses varies greatly between individual regions, thus regional hotspots or gradients were detected. GPGV for example is mostly found in southeastern Germany, while its incidence decreases to the north along the river Rhine. The findings of this survey provide an overview of the allocation of the most prevalent grapevine viruses in Germany and can support regional virus management and national risk assessment especially GPGV. [ABSTRACT FROM AUTHOR]

Grapevine leafroll disease (GLRD) is the most globally prevalent and destructive disease complex responsible for significant reductions in grape yield and quality as well as wine production. GLRD is associated with several positive-strand RNA viruses of the family Closteroviridae, designated as grapevine leafroll-associated viruses (GLRaVs). However, the specific etiological role of any of these GLRaVs in GLRD has not been demonstrated. Even though GLRaV-3 is considered the chief GLRD agent, little is known about the molecular, cellular, and pathological properties of this virus. Such a knowledge gap is due to multiple factors, including the unavailability of biologically active virus cDNA clones and the lack of reliable experimental systems for launching grapevine infection using such clones. In this work, we tested four methods for inoculating tissue-cultured grapevine plantlets with cDNA clones of GLRaV-3: (i) vacuum agro-infiltration; (ii) agro-pricking; (iii) agro-drenching; and (iv) agro-injection. We showed that vacuum agro-infiltration was the most effective of these methods. Furthermore, we examined the impacts of different experimental conditions on the survival and infectivity rate of grapevines after infiltration. To verify the infectivity rate for different treatments, we used RT-PCR, RT-qPCR, and Western blotting. We found that humidity plays a critical role in the survival of plantlets after agro-infiltration and that the use of RNA silencing suppressor and dormancy treatment both had strong effects on the infection rates. To our knowledge, the experimental protocol reported herein is the most effective system for launching the infection of grapevine using cDNA clones of grapevine viruses featuring up to a 70% infection rate. This system has strong potential to facilitate grapevine virology research including the fulfillment of Koch's postulates for GLRD and other major virus diseases as well as identifying the molecular, cellular, and pathological properties of GLRaVs and, potentially, other important grapevine viruses. [ABSTRACT FROM AUTHOR]

Virus diseases in grapevine 'Shine Muscat', an economically significant table grape cultivar in China, have become a major concern for grapevine growers. To investigate which viruses are frequent in 'Shine Muscat' and screen for healthy propagation material for this cultivar, a total of 140 samples (dormant canes) of 'Shine Muscat' plants were collected from 45 vineyards in different viticultural areas in China. RT-PCR was conducted to detect the presence of grapevine leafroll-associated virus 1 (GLRaV-1), GLRaV-2, GLRaV-3, grapevine fanleaf virus (GFLV), grapevine fleck virus (GFkV), grapevine virus A (GVA), grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine Pinot gris virus (GPGV) and grapevine berry inner necrosis virus (GINV). No virus-free cane was found and all samples were infected with a mixture of at least two viruses; double, triple and quadruple infections accounted for about 31%, 30%, and 22% of the infections, respectively. The most frequent viruses in 'Shine Muscat' plants were GPGV (67.86%), GVA (59.29%) and GRSPaV (55.0%), and GFLV and GLRaV-1 were not detected in any of the samples. In vitro-grown 'Shine Muscat' plantlets carrying GFkV/GINV/GVA/GRSPaV were subjected to thermotherapy or chemotherapy followed by shoot-tip culture. GINV, GFkV and GVA were easily eliminated from the cultivar by both sanitation methods, whereas the frequency of GRSPaV elimination was relatively low. The results presented here contribute to our understanding of virus diseases in grapevine cv. Shine Muscat and to improving the sanitary status of the cultivar's planting material. [ABSTRACT FROM AUTHOR]

We developed a NitroPure Nitrocellulose (NPN) membrane-based method for sampling and storing grapevine sap for grapevine virus detection. We devised an efficient nucleic acid extraction method for the NPN membrane, resulting in 100% amplification success for grapevine leafroll-associated virus 2 (GLRaV2) and 3 (GLRaV3), grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine virus A, grapevine virus B, and grapevine red blotch virus (GRBV). This method also allowed the storage of recoverable nucleic acid for 18 months at room temperature. We created a sampling kit to survey GLRaV2, GLRaV3, and GRBV in Japanese vineyards. We tested the kits in the field in 2018 and then conducted mail-in surveys in 2020–2021. The results showed a substantial prevalence of GLRaV3, with 48.5% of 132 sampled vines being positive. On the other hand, only 3% of samples tested positive for GLRaV2 and none for GRBV. [ABSTRACT FROM AUTHOR]

French-American hybrids and North American grape species play a significant role in Canada's grape and wine industry. Unfortunately, the occurrence of viruses and viral diseases among these locally important non-vinifera grapes remains understudied. We report here the results from a large-scale survey to assess the prevalence of 14 viruses among 533 composite samples representing 2665 vines from seven French-American hybrid wine grape cultivars, two North American juice grape cultivars (Concord and Niagara), and the table grape cultivar Sovereign coronation. Based on reverse transcription polymerase chain reaction (RT-PCR) assays, ten viruses were detected. Grapevine rupestris stem pitting-associated virus, grapevine leafroll-associated virus 3, grapevine Pinot gris virus and grapevine red blotch virus were detected with the highest frequency. As expected, mixed infections were common; 62% of the samples contained two or more viruses. Overall, hybrid wine grapes were infected with more viruses and a higher prevalence of individual viruses than juice and table grapes. To validate these findings and to refine the virome of these non-European grapes, high-throughput sequencing (HTS) analyses of five composite samples representing each category of grapevine cultivars was performed. Results from HTS agreed with those from RT-PCR. Importantly, Vidal, a widely grown white-wine grape with international recognition due to its use in the award-winning icewine, is host to 14 viruses, four of which comprise multiple and distinct genetic variants. This comprehensive survey represents the most extensive examination of viruses among French-American hybrids and North American grapes to date. [ABSTRACT FROM AUTHOR]

This is the first viral metagenomic analysis of grapevine conducted in Mexico. During the summer of 2021, 48 plants displaying virus-like symptoms were sampled in Queretaro, an important grapevine-producing area of Mexico, and analyzed for the presence of viruses via high-throughput sequencing (HTS). The results of HTS were verified by real-time RT-PCR following a standardized testing scheme (Protocol 2010). Fourteen different viruses were identified, including grapevine asteroid mosaic-associated virus (GAMaV), grapevine Cabernet Sauvignon reovirus (GCSV), grapevine fanleaf virus (GFLV), grapevine fleck virus (GFkV), grapevine Pinot gris virus (GPGV), grapevine red globe virus (GRGV), grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine rupestris vein feathering virus (GRVFV), grapevine Syrah virus 1 (GSyV-1), grapevine virus B (GVB), and grapevine leafroll-associated viruses 1, 2, 3, 4 (GLRaV1, 2, 3, 4). Additionally, divergent variants of GLRaV4 and GFkV, and a novel Enamovirus-like virus were discovered. This is the first report of GAMaV, GCSV, GLRaV4, GPGV, GRGV, GRVFV, and GSyV-1 infecting grapevines in Mexico; the impact of these pathogens on production is unknown. [ABSTRACT FROM AUTHOR]

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Background and Aims. Shiraz disease (SD) is a viral disease associated with Grapevine virus A that causes significant yield loss in economically important grape cultivars in Australia such as Shiraz and Merlot. Current diagnostic methods are time-consuming and costly. This study evaluates an alternative methodology using visible remote sensing imagery to detect SD in Shiraz grapevines. Methods and Results. High-resolution visible remote sensing images were captured of Shiraz grapevines in two South Australian viticultural regions over two seasons. The projected leaf area (PLA) of individual grapevines was estimated from the images. Virus-infected vines had significantly lower PLA than healthy vines in the early season but fewer difference after veraison. The lower PLA was only observed in grapevines coinfected with grapevine leafroll-associated viruses (GLRaVs) and Grapevine virus A (GVA). Shiraz vines infected with either GLRaVs or GVA had similar PLA to healthy vines. Conclusions. High-resolution RGB remote sensing technology has the potential to rapidly estimate SD infection in Shiraz grapevines. Our observations of shoot devigouration only in coinfected vines calls into question the etiology of SD. Further validation of the PLA technique incorporating different regions, seasons, cultivars, and combinations of viruses is needed for improving the robustness of the method. Significance of the Study. This preliminary study presents a new rapid and low-cost surveillance method to estimate SD infections in Shiraz vineyards, which could significantly lower the cost for growers who conduct on-ground SD visual assessments or lab-based tissue testing at the vineyard scale. [ABSTRACT FROM AUTHOR]

Grapevine leafroll disease affects the health status of grapevines worldwide. Most studies in Australia have focused on grapevine leafroll-associated viruses 1 and 3, while little attention has been given to other leafroll virus types, in particular, grapevine leafroll-associated virus 2 (GLRaV-2). A chronological record of the temporal occurrence of GLRaV-2 in Australia since 2001 is reported. From a total of 11,257 samples, 313 tested positive, with an overall incidence of 2.7%. This virus has been detected in 18 grapevine varieties and Vitis rootstocks in different regions of Australia. Most varieties were symptomless on their own roots, while Chardonnay showed a decline in virus-sensitive rootstocks. An isolate of GLRaV-2, on own-rooted Vitis vinifera cv. Grenache, clone SA137, was associated with severe leafroll symptoms after veraison with abnormal leaf necrosis. The metagenomic sequencing results of the virus in two plants of this variety confirmed the presence of GLRaV-2, as well as two inert viruses, grapevine rupestris stem pitting-associated virus (GRSPaV) and grapevine rupestris vein feathering virus (GRVFV). No other leafroll-associated viruses were detected. Among the viroids, hop stunt viroid and grapevine yellow speckle viroid 1 were detected. Of the six phylogenetic groups identified in GLRaV-2, we report the presence of four groups in Australia. Three of these groups were detected in two plants of cv. Grenache, without finding any recombination event. The hypersensitive reaction of certain American hybrid rootstocks to GLRaV-2 is discussed. Due to the association of GLRaV-2 with graft incompatibility and vine decline, the risk from this virus in regions where hybrid Vitis rootstocks are used cannot be overlooked. [ABSTRACT FROM AUTHOR]

The negative effects of viruses and the positive effects of arbuscular mycorrhizal fungi (AMF) on grapevine performance are well reported, in contrast to the knowledge about their interactive effects in perennial plants, e.g., in grapevine. To elucidate the physiological consequences of grapevine–AMF–virus interactions, two different AMF inoculum (Rhizophagus irregularis and 'Mix AMF') were used on grapevine infected with grapevine rupestris stem pitting virus, grapevine leafroll associated virus 3 and/or grapevine pinot gris virus. Net photosynthesis rate (AN), leaf transpiration (E), intercellular CO2 concentration (Ci) and conductance to H2O (gs) were measured at three time points during one growing season. Furthermore, quantum efficiency in light (ΦPSII) and electron transport rate (ETR) were surveyed in leaves of different maturity, old (basal), mature (middle) and young (apical) leaf. Lastly, pigment concentration and growth parameters were analysed. Virus induced changes in grapevine were minimal in this early infection stage. However, the AMF induced changes of grapevine facing biotic stress were most evident in higher net photosynthesis rate, conductance to H2O, chlorophyll a concentration, total carotenoid concentration and dry matter content. The AMF presence in the grapevine roots seem to prevail over virus infection, with Rhizophagus irregularis inducing greater photosynthesis changes in solitary form rather than mixture. This study shows that AMF can be beneficial for grapevine facing viral infection, in the context of functional physiology. [ABSTRACT FROM AUTHOR]

Introduction: Grapevine leafroll-associated viruses (GLRaVs) and grapevine red blotch virus (GRBV) cause substantial economic losses and concern to North America's grape and wine industries. Fast and accurate identification of these two groups of viruses is key to informing disease management strategies and limiting their spread by insect vectors in the vineyard. Hyperspectral imaging offers new opportunities for virus disease scouting. Methods: Here we used two machine learning methods, i.e., Random Forest (RF) and 3D-Convolutional Neural Network (CNN), to identify and distinguish leaves from red blotch-infected vines, leafroll-infected vines, and vines co-infected with both viruses using spatiospectral information in the visible domain (510-710nm). We captured hyperspectral images of about 500 leaves from 250 vines at two sampling times during the growing season (a pre-symptomatic stage at veraison and a symptomatic stage at mid-ripening). Concurrently, viral infections were determined in leaf petioles by polymerase chain reaction (PCR) based assays using virus-specific primers and by visual assessment of disease symptoms. Results: When binarily classifying infected vs. non-infected leaves, the CNN model reaches an overall maximum accuracy of 87% versus 82.8% for the RF model. Using the symptomatic dataset lowers the rate of false negatives. Based on a multiclass categorization of leaves, the CNN and RF models had a maximum accuracy of 77.7% and 76.9% (averaged across both healthy and infected leaf categories). Both CNN and RF outperformed visual assessment of symptoms by experts when using RGB segmented images. Interpretation of the RF data showed that the most important wavelengths were in the green, orange, and red subregions. Discussion: While differentiation between plants co-infected with GLRaVs and GRBV proved to be relatively challenging, both models showed promising accuracies across infection categories. [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]

Worldwide, virus infections in grapevines are of concern due to the potential for economic loss. Although the grape industry in Mexico is relatively small and focused mainly on the local market, production dates back to the time of the Spanish colonization. This manuscript discusses the findings on grapevine viruses in Mexico. Nine viruses have been identified in the last fifty years, including grapevine red blotch virus (GRBV), grapevine leafroll-associated virus 3 (GLRaV-3), grapevine fanleaf virus (GFLV), and grapevine virus A (GVA). Important information is provided about these viruses and viral pathogens that have not yet been reported in Mexico, but represent an ongoing threat to plant health and grapevine production in other viticultural regions of the world. Strategies for virus control in vineyards are described. The information discussed here should be shared with growers and stakeholders to prevent future negative impacts on the Mexican grapevine industry and to save ancient grapevine accessions. [ABSTRACT FROM AUTHOR]

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 (Vitis vinifera L.) is one of the most important crops in the world due to its economic and social impact. Like many other crops, grapevine is susceptible to different types of diseases caused by pathogenic microorganisms. Grapevine leafroll-associated virus 1 (GLRaV-1) is a virus associated with grapevine leafroll disease and it is considered at the national and European level as a pathogen that must be absent in propagative plant material. For this reason, the availability of specific, sensitive and reliable detection techniques to ascertain the sanitary status of the plants is of great importance. The objective of this research was the development of a new GLRaV-1 detection method based on a TaqMan quantitative real-time RT-PCR targeted to the coat protein genomic region and including a host internal control in a duplex reaction. To this end, three new GLRaV-1 full genomes were recovered by HTS and aligned with all sequences available in the databases. The method has been validated following EPPO standards and applied for the diagnosis of field plant material and transmission vectors. The new protocol designed has turned out to be highly sensitive as well as much more specific than the current available methods for the detection and absolute quantitation of GLRaV-1 viral titer. [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 is a widely grown fruit crop that is seriously affected by different viruses, reducing grape yield and quality, as well as threatening profitability. Vineyard disease management requires accurate identification of viral infections. This study aimed to survey the presence of ten grapevine viruses in four geographic sites in the Mendoza province of Argentina. Two hundred twenty-three composite cane samples from 1060 plants of six cultivars were collected from 26 blocks distributed across 11 vineyards. The cane samples were screened by RT-PCR for the following viruses: grapevine leafroll-associated viruses 1–4 (GLRaV 1, 2, 3, and 4), grapevine fanleaf virus (GFLV), grapevine fleck virus (GFkV), grapevine virus A (GVA) and B (GVB), grapevine rupestris stem pitting associated virus (GRSPaV), and arabis mosaic virus (ArMV). The results showed an uneven occurrence of viruses through the sampled regions, with GRSPaV being prevalent (71.1%), followed by GFLV (28.9%), GFkV (20.6%), and GLRaV-2 (14.7%). GVB was not detected. This study revealed a moderate prevalence of viruses associated with economically impactful diseases in the vineyards surveyed. [ABSTRACT FROM AUTHOR]

This research studied serological relationships and genetic diversity of Argentinean isolates of grapevine leafroll-associated virus-4 (GLRaV-4). Phylogenetic analysis of coat protein (CP) sequences from 19 local isolates revealed clustering with the previously described GLRaV-4 strain 5, strain 6, and strain 9 groups. Evolutionary sequence analysis of the obtained and database-available sequences showed evidence of recombination events. Additionally, both CP N- and C-terminal regions appeared to be under purifying selection, but the N-terminal region presented seven sites under positive selection, with a dN/dS ratio 5-fold greater than that of the C-terminal region. Serological reactivity against monoclonal antibodies supports a higher occurrence probability for linear epitopes in the N-terminal region, as inferred by the sequence analysis. The obtained results reflect an unusual evolutionary behavior of the CP that, together with protein serological reactivity, suggests biological significance of the observed variability. [ABSTRACT FROM AUTHOR]

Grapevine virus G (GVG) and grapevine virus H (GVH) (genus Vitivirus) are recently discovered viruses. Analysis of 38 samples from Sardinian grapevine cultivars for the presence of GVG and GVH was carried out using RT-PCR. All samples were also tested for grapevine Pinot gris virus (GPGV) using RT-PCR, and for grapevine leafroll virus -1, -2 and -3, grapevine virus A (GVA) and B (GVB), arabis mosaic virus (ArMV), grapevine fanleaf virus (GFLV) and grapevine fleck virus (GFkV) using multiplex RT-PCR. GVG was confirmed in four vines, and GVH was detected in only one sample. In phylogenetic analyses of the coat protein (CP) region, the Sardinian GVG isolates clustered separately from isolates from Croatia and New Zealand. The Sardinian GVH isolate clustered with most sequences from other countries, but with greater affinity to isolates from California (USA) for the CP region, whereas it clustered with isolates from Croatia in the RNA-dependent RNA polymerase (RdRp) region. In addition to GVG and GVH, many samples were coinfected with GVA, viruses from the leafroll complex, and GPGV. This is the first record of GVG and GVH occurring in Italy. [ABSTRACT FROM AUTHOR]

Grapevine-infecting ampelo- and vitiviruses are transmitted by scale insects belonging to several species, among which is the European fruit lecanium, Parthenolecanium corni (Bouché) (Hemiptera Coccidae). Our objective was to characterize the transmission biology of grapevine leafroll-associated viruses (GLRaV) and grapevine virus A (GVA) by this soft scale species in order to evaluate its ability to spread these viruses. In transmission experiments with nymphs sampled from different vineyards infected with GLRaV 1, 2, 3 and GVA, P. corni transmitted only GLRaV 1 and GVA to healthy vines. GVA was predominantly transmitted along with GLRaV 1, whereas the latter could be transmitted alone from single or co-infected vines. Vineyard-sampled second instar nymphs were more efficient than first instars at transmitting GLRaV 1, whereas both instars displayed similar transmission rates for GVA. Short virus inoculation access periods and the absence of virus in eggs of females living on infected grapevines fulfilled the criteria of non-circulative semi-persistent transmission mode. [ABSTRACT FROM AUTHOR]

In this study, we analyzed the virome of 73 grape samples from two Dagestan ampelographic collections in Russia using high-throughput sequencing of total RNAs. Fourteen viruses and four viroids were identified, with one to eleven of them detected in each plant. For the first time in Russia, we identified grapevine leafroll-associated virus 7 and grapevine Kizil Sapak virus. A total of 206 genomes of viruses and viroids were obtained, and their phylogenetic analysis was carried out. The de novo assembly and tblastx analysis allowed us to obtain contigs of a novel (+) ssRNA genome of a plant virus from the genus Umbravirus, which was tentatively named grapevine umbra-like virus (GULV), as well as contigs of a novel dsDNA pararetrovirus from the genus Caulimovirus, which was tentatively named grapevine pararetrovirus (GPRV). Complete genomes of these viruses were obtained and used for Sequence Demarcation Tool (SDT) analysis and phylogeny studies. GULV and GPRV were detected in 16 and 33 germplasm samples from the Dagestan collections, respectively. [ABSTRACT FROM AUTHOR]

Grapevine leafroll-associated virus 3 (GLRaV-3) is a graft-transmissible virus present in every viticultural region of the world and poses a large threat to grapevine production. Frequent coinfections with other viruses, the large number of grapevine varieties, the complexity of processes involved in plant response to virus infection, and the lack of studies on GLRaV-3 replication limit our knowledge of GLRaV-3 damaging effects and their background. In this study, five different inocula, one containing GLRaV-3 and others containing GLRaV-3 in combination with different grapevine viruses were green grafted to 52 different grapevine plants of four varieties to analyze the influence of the phenological stage and virus composition on GLRaV-3 replication. Relative concentration analysis by quantitative PCR conducted over a 16-month period revealed that other viruses as well as plant stage had a significant effect on GLRaV-3 replication and symptoms expression. The replication was most pronounced in the deep dormancy stage at the beginning of the infection, and the least at the exit of the dormancy stage. This study brings new insight into GLRaV-3 replication and discusses about viral interactions in one of the most economically important perennial plants, the grapevine. [ABSTRACT FROM AUTHOR]

Five grapevine viruses, including grapevine geminivirus A (GGVA), grapevine Syrah virus 1 (GSyV-1), grapevine Cabernet Sauvignon reovirus (GCSV), grapevine virus F (GVF) and grapevine Red Globe virus (GRGV) have been detected in four South Australian vineyards using metagenomic high throughput sequencing for the first time in Australia. Most of them were present in asymptomatic grapevines and some were present in grapevines with leafroll disease that were infected by several other viruses, including grapevine leafroll-associated viruses. Therefore, the association of GGVA, GSyV-1, GCSV, GVF and GRGV with disease in Australia is unknown. [ABSTRACT FROM AUTHOR]

• First comprehensive report on virome analysis of Indian grapevine rootstocks. • The Study revealed the presence of nine important grapevine viruses and five viroids. • HTS analysis indicated the possible role of rootstocks in viruses and viroids dissemination. • Grapevine virus B was the highly prevalent virus in rootstocks and scions. • Recombinase polymerase amplification assay was adapted for GVB diagnosis. Grapevine stocks are generally vegetatively propagated for the commercial cultivation. The propagation is predominantly done on rootstocks with >90 % of plant stocks propagated on Dogridge rootstock alone in India. Three additional rootstocks i.e. Paulsen, SO4 and Ramsey are also used for grapevine propagation on a limited scale. However, no information is available on the health status of these rootstocks being used by nurserymen and growers. Therefore, these rootstocks were collected from nurseries of five different locations and subjected to high-throughput sequencing (HTS) to determine the spectrum of viruses and viroids. The de novo assembly of unaligned reads revealed the presence of genome sequences of nine viruses viz. grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine leafroll-associated virus 3 (GLRaV-3), grapevine leafroll-associated virus 4 (GLRaV-4), grapevine virus A (GVA), grapevine virus B (GVB), grapevine virus D (GVD), grapevine virus F (GVF), grapevine virus L (GVL), grapevine fleck virus (GFkV) and five viroids viz. hopstunt viroid (HSVd), grapevine yellow speckle viroid 1 and 2 (GYSVd-1 and GYSVd-2), Australian grapevine viroid (AGVd) and grapevine latent viroid (GLVd). The identified viruses and viroids were further confirmed through RT-PCR and Sanger sequencing. Indexing for the presence of these graft-transmissible pathogens in 43 rootstocks and 23 scion samples collected through field surveys indicated that infected rootstocks are the probable source for dissemination of these viruses and viroids in grapevine scions. As GVB was the most prevalent virus in all the rootstocks and scions as observed in RT-PCR testing, a simplified crude sap-based recombinase polymerase amplification (RPA) assay was optimized for its rapid detection. The present study highlights that grapevine rootstocks are reservoir of viruses and viroids and source for their dissemination in grapevine vineyards. Findings of the study including the optimized simplified cost-effective isothermal nucleic acid-based detection technique for GVB will be highly useful for production of certified clean rootstocks of grapevine in India. [Display omitted] [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]

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 leafroll disease (GLD) is one of the most economically damaging virus diseases in grapevine, with grapevine leafroll-associated virus 1 (GLRaV-1) and grapevine leafroll-associated virus 3 (GLRaV-3) as the main contributors. This study complements a previously published transcriptomic analysis and compared the impact of two different forms of GLD to a symptomless control treatment: a mildly symptomatic form infected with GLRaV-1 and a severe form with exceptionally early leafroll symptoms (up to six weeks before veraison) infected with GLRaV-1 and GLRaV-3. Vine physiology and fruit composition in 17-year-old Pinot noir vines were measured and a gradient of vigor, yield, and berry quality (sugar content and berry weight) was observed between treatments. Virome composition, confirmed by individual RT-PCR, was compared with biological indexing. Three divergent viromes were recovered, containing between four to seven viruses and two viroids. They included the first detection of grapevine asteroid mosaic-associated virus in Switzerland. This virus did not cause obvious symptoms on the indicators used in biological indexing. Moreover, the presence of grapevine virus B (GVB) did not cause the expected corky bark symptoms on the indicators, thus underlining the important limitations of the biological indexing. Transmission of GLRaV-3 alone or in combination with GVB by Planococcus comstocki mealybug did not reproduce the strong symptoms observed on the donor plant infected with a severe form of GLD. This result raises questions about the contribution of each virus to the symptomatology of the plant. [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]

Metagenomic approaches used for virus diagnostics allow for rapid and accurate detection of all viral pathogens in the plants. In order to investigate the occurrence of viruses and virus-like organisms infecting grapevine from the Ampelographic collection Kromberk in Slovenia, we used Ion Torrent small RNA sequencing (sRNA-seq) and the VirusDetect pipeline to analyze the sRNA-seq data. The used method revealed the presence of: Grapevine leafroll-associated virus 1 (GLRaV-1), Grapevine leafroll-associated virus 2 (GLRaV-2), Grapevine leafroll-associated virus 3 (GLRaV-3), Grapevine rupestris stem pitting-associated virus (GRSPaV), Grapevine fanleaf virus (GFLV) and its satellite RNA (satGFLV), Grapevine fleck virus (GFkV), Grapevine rupestris vein feathering virus (GRVFV), Grapevine Pinot gris virus (GPGV), Grapevine satellite virus (GV-Sat), Hop stunt viroid (HSVd), and Grapevine yellow speckle viroid 1 (GYSVd-1). Multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed for validation of sRNA-seq predicted infections, including various combinations of viruses or viroids and satellite RNA. mRT-PCR could further be used for rapid and cost-effective routine molecular diagnosis, including widespread, emerging, and seemingly rare viruses, as well as viroids which testing is usually overlooked. [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]

Viruses and virus-like organisms are a major problem in viticulture worldwide. They cannot be controlled by standard plant protection measures, and once infected, plants remain infected throughout their life; therefore, the propagation of healthy vegetative material is crucial. In vivo thermotherapy at 36–38 °C for at least six weeks, followed by meristem tip micrografting (0.1–0.2 mm) onto in vitro-growing seedling rootstocks of Vialla (Vitis labrusca × Vitis riparia), was successfully used to eliminate eight viruses (grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine Pinot gris virus (GPGV), grapevine fanleaf virus (GFLV), grapevine leafroll-associated virus 3 (GLRaV-3), grapevine fleck virus (GFkV), grapevine rupestris vein feathering virus (GRVFV), grapevine Syrah virus-1 (GSyV-1), and raspberry bushy dwarf virus (RBDV)), as well as two viroids (hop stunt viroid (HSVd) and grapevine yellow speckle viroid 1 (GYSVd-1)) from preclonal candidates of six grapevine varieties (Vitis vinifera L.). A half-strength MS medium including vitamins supplemented with 30 g/L of sucrose and solidified with 8 g/L of agar, without plant growth regulators, was used for the growth and root development of micrografts and the subsequently micropropagated plants; no callus formation, hyperhydricity, or necrosis of shoot tips was observed. Although the overall regeneration was low (higher in white than in red varieties), a 100% elimination was achieved for all eight viruses, whereas the elimination level for viroids was lower, reaching only 39.2% of HSVd-free and 42.6% GYSVd-1-free vines. To the best of our knowledge, this is the first report of GPGV, GRVFV, GSyV-1, HSVd, and GYSVd-1 elimination through combining in vivo thermotherapy and in vitro meristem tip micrografting, and the first report of RBDV elimination from grapevines. The virus-free vines were successfully acclimatized in rockwool plugs and then transferred to soil. [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]

Nearly 80 different viruses belonging to different genera and families have been identified in grapevines, but their seed transmissibility remains largely unclear. Thus, the specific objective of this work is to monitor the presence of viruses in seedlings grown from seeds of virus-infected grapevine plants. Using reverse-transcription polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR, we showed the presence of grapevine leafroll-associated virus 2 (GLRaV-2), GLRaV- 3, grapevine 'Pinot gris' virus (GPGV), grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine virus A (GVA), and grapevine fleck virus (GFkV) in all tested parts including mature cane, leaf, petiole, young shoot, flower, berry skin, and seed of infected grapevine 'Moldova' plants. Furthermore, GLRaV-2 and GPGV were found in all tested seedlings developed from seeds of 'Moldova' plants, while the other four viruses were not detected. Our results provide the first evidence that GLRaV-2 and GPGV can be transmitted to progeny seedlings from seeds of infected grapevine plants. [ABSTRACT FROM AUTHOR]

Background: Grapevine, as an essential fruit crop with high economic values, has been the focus of molecular studies in diverse areas. Two challenges exist in the grapevine research field: (i) the lack of a rapid, user-friendly and effective RNA isolation protocol for mature dark-skinned berries and, (ii) the lack of validated reference genes that are stable for quantification of gene expression across desired experimental conditions. Successful isolation of RNA with sufficient yield and quality is essential for downstream analyses involving nucleic acids. However, ripe berries of dark-skinned grape cultivars are notoriously challenging in RNA isolation due to high contents of polyphenolics, polysaccharides, RNase and water. Results: We have optimized an RNA isolation protocol through modulating two factors at the lysis step that could impact results of RNA isolation - 2-ME concentration and berry mass. By finding the optimal combination among the two factors, our refined protocol was highly effective in isolating total RNA with high yield and quality from whole mature berries of an array of dark-skinned wine grape cultivars. Our protocol takes a much shorter time to complete, is highly effective, and eliminates the requirement for hazardous organic solvents. We have also shown that the resulting RNA preps were suitable for multiple downstream analyses, including the detection of viruses and amplification of grapevine genes using reverse transcription-polymerase chain reaction (RT-PCR), gene expression analysis via quantitative reverse transcription PCR (RT-qPCR), and RNA Sequencing (RNA-Seq). By using RNA-Seq data derived from Cabernet Franc, we have identified seven novel reference gene candidates (CYSP, NDUFS8, YLS8, EIF5A2, Gluc, GDT1, and EF-Hand) with stable expression across two tissue types, three developmental stages and status of infection with grapevine leafroll-associated virus 3 (GLRaV-3). We evaluated the stability of these candidate genes together with two conventional reference genes (actin and NAD5) using geNorm, NormFinder and BestKeeper. We found that the novel reference gene candidates outperformed both actin and NAD5. The three most stable reference genes were CYSP, NDUFS8 and YSL8, whereas actin and NAD5 were among the least stable. We further tested if there would be a difference in RT-qPCR quantification results when the most stable (CYSP) and the least stable (actin and NAD5) genes were used for normalization. We concluded that both actin and NAD5 led to erroneous RT-qPCR results in determining the statistical significance and fold-change values of gene expressional change. Conclusions: We have formulated a rapid, safe and highly effective protocol for isolating RNA from recalcitrant berry tissue of wine grapes. The resulting RNA is of high quality and suitable for RT-qPCR and RNA-Seq. We have identified and validated a set of novel reference genes based on RNA-Seq dataset. We have shown that these new reference genes are superior over actin and NAD5, two of the conventional reference genes commonly used in early studies. [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 disease (GLD) affects grapevines worldwide. The primary causal agent of GLD is grapevine leafroll-associated virus 3 (GLRaV-3), which spreads to uninfected grapevines via mealybugs and soft-scale insects. Pseudococcus calceolariae (Hemiptera: Pseudococcidae) is a mealybug vector of GLRaV-3 in New Zealand. P. calceolariae also colonizes clovers (Trifolium spp.) growing naturally as vineyard ground cover. Separating mealybug from GLRaV-3 grapevine host could be enhanced by a trap plant: an alternative host attractive to and retentive of the target pest. We evaluated the association between P. calceolariae and ‘Grasslands Huia’ white clover (GHWC). GHWC seed was sown under grapevines in a commercial vineyard (14 × 0.4 ha plots); the control was under-vine herbicide use (7 × 0.4 ha plots, where only few Trifolium spp. plants grew). After 2 years, GHWC cover peaked at 40% mealybug infestation in 2019. From 2018 to 2021, P. calceolariae detection and abundance on GHWC was significantly higher than plants from the control plots. There was no treatment effect for mealybug infestation of grapevine leaves nor of GLRaV-3 incidence, independent of vintage. A glasshouse trial found no transmission of GLRaV-3 by P. calceolariae to any of 256 plants among five clover cultivars tested (Trifolium spp.), including GHWC; mealybug transmitted GLRaV-3 to 35 of 107 Nicotiana benthamiana plants. The results showed that in the 5-year period, added GHWC did not decouple P. calceolariae from the grapevine to reduce GLRaV-3 incidence, but rapid colonization of GHWC by mealybug and the lack of GLRaV-3 transmission to GHWC are encouraging. Further evaluation is needed to assess whether plant biodiversity can benefit a GLRaV-3 ecological management objective. [ABSTRACT FROM AUTHOR]

The detrimental effects of grapevine leafroll disease (GLD) have been documented mainly in young plants and little data is available on infected grapevines when they reach maturity. This study examined the influence of the rootstock on the effect of GLRaV-3 in a 20-year-old Vitis vinifera cv Albariño vineyard in which the virus has been spread by Planococcus ficus. Plants grafted on Richter 110 or Castell 196.17 with similar development were analysed for grapevine leafroll-associated viruses. In particular, 25 GLRaV-3-infected and 25 leafroll-free and asymptomatic vines were selected and monitored during a period of three years to determine grape yield and must components. Although the virus infection affected plants on both rootstocks, it caused a greater effect on plants grafted onto 110R, with average accumulated yield losses of up to 33%, relative to losses of 16% for plants on 196.17C. The sugar content was lower in the must from infected plants on 110R (-2.1°Brix) than in the must from plants on 196.17C (-1.5°Brix). The presence of the virus was associated with decreased concentrations of primary amino nitrogen (PAN) and ammonium (NH4+), with the greatest effect for 110R (-35%). As 110R is the most used rootstock in Spain, particular care must be taken to prevent the transmission of GLRaV-3 to certified virus-free plants on 110R in new vineyards. In sensitive varieties such as Albariño, particularly in areas with an active spread of the virus by mealybugs, grafting on 196.17C seems to minimise the effect that GLD has in a medium to long term. [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]

Grapes secondary metabolites content mainly depends on variety, but also on climate and cultural conditions, including sanitary status. This study aimed to use a metabolomic fingerprinting approach for grouping 72 wine grape biotypes, Negro amaro n. (N), Malvasia nera di Brindisi/Lecce n. (M), and Uva di Troia n. (U), on the basis of their cultivar and virological conditions. The skins were extracted and analysed by flow injection mass spectrometry; a one-way ANOVA/Principal Component Analysis (PCA) allowed to efficiently cluster the samples, recognizing M from N and U biotypes. Conversely, the clusterisation of the biotypes affected by different virus complexes was really more tough and a clear distinction among infected plants was not always observed. However, very interestingly, by applying ANOVA/PCA to the biotypes of each varieties, singularly, healthy biotypes were sharply separated in all the varieties and a relationship between anthocyanin compounds and Grapevine leafroll associated virus (GLRaV3) slightly appeared. [ABSTRACT FROM AUTHOR]

Grapevine leafroll-associated virus 3 (GLRaV-3) is a worldwide pathogen of Vitis negatively affecting wine production. In red berry cultivars grown in New Zealand, the foliar changes to GLRaV-3-infected vines inform decisions on vine removal (roguing). However, roguing does not always contain GLRaV-3 spread in the presence of an insect vector like the mealybug, Pseudococcus calceolariae. Virus incidence and mealybug abundance data collected annually over 7 years were modelled under differing scenarios to ascertain support for roguing. In a simulated 1 ha vineyard planted in 2500 mature Merlot vines, simulations over 20 years evaluated the effectiveness of roguing, 'rogue 1 + 2' (concurrent roguing of symptomatic plus both within-row neighbouring vines), inefficient roguing (50% of symptomatic vines rogued), and 'no-action'. The model used variable initial GLRaV-3 incidence (0.4, 5, 10, 15 and 20%), and low, median and high vector densities (6, 26 and 75 mealybugs per 100 vine leaves, respectively). Roguing was the optimal response to GLRaV-3, independent of the initial incidence, but results were vector density dependent. At a low vector density, roguing relative to the other responses tested, sustained the lowest annual GLRaV-3 incidence, the least need to plant replacement vines and the lowest estimated average annual costs plus loss of income. At median and high vector densities, roguing remained the most favourable response but virus control was less effective and the costs incurred were higher. Thus, for vineyards affected by GLRaV-3, achieving economic sustainability relies on integrating efficient roguing with effective vector management. [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]