Leafroll Research Articles

Identification of a receptor for the sex pheromone of the vine mealybug, Planococcus ficus

The vine mealybug, Planococcus ficus, is a significant pest of vineyards in all major grape growing regions of the world. This pest causes significant aesthetic damage to berry clusters through its feeding behavior and secretion of "honeydew", which leads to significant decreases in crop marketability. More importantly, the vine mealybug is a vector of several grapevine viruses which are the causal agent of grapevine leafroll disease, one of the most destructive and economically devastating diseases of the grape industry worldwide. As there is no cure for grapevine leafroll disease, the only control measures available to reduce its spread are to remove infected vines whilst simultaneously controlling mealybug populations. Using transcriptomic libraries prepared from male and female mealybugs and a draft genome, we identified and evaluated expression levels of members of the odorant receptor gene family. Interestingly, of the 50 odorant receptors identified from these P. ficus genetic resources, only 23 were found to be expressed in females, suggesting this flightless life stage has a decreased reliance on the olfactory system. In contrast, 46 odorant receptors were found to be expressed in the alate male life stage. Heterologous expression of eight of these receptors, along with the obligate co-receptor, Orco, in HEK293 cells allowed for the identification of two receptors that respond to lavandulyl senecioate, the sole constituent of the sex pheromone used by this species. Interestingly, one of these receptors, PficOR8, also responded to the sex pheromone used by the Japanese mealybug, Planococcus kraunhiae. The data presented here represent the first report of odorant receptor gene family expression levels, as well as the identification of the first sex pheromone receptor, in soft-scale insects. The identification of a receptor for the vine mealybug sex pheromone will allow for the development of novel, species-specific pest control tools and monitoring devices.

Effects of meteorological variables on potato leaf roll virus and potato virus y infections on different potato varieties under natural and mulched conditions

After rice and wheat, potatoes are the third most important staple crop in Pakistan, but due to various virus diseases attacks, yield remains very low. Among viral pathogens, potato leaf roll virus (PLRV) and potato virus Y (PVY) are the most destructive viruses present in all potato-growing areas. A field experiment was conducted at the Research Farm of Plant Virology Section, Ayub Agricultural Research Institute, Faisalabad, to evaluate the effects of meteorological variables on the development of virus infections (PLRV and PVY) on different potato varieties under natural and mulched conditions. Results indicated that the development of infections of PVY and PLRV was delayed in different potato varieties under mulched conditions. The potato variety Simply Red had the highest PLRV and PVY disease incidences under unmulched and mulched circumstances, while Sahiwal Red and Ruby had the lowest infection rates. The least PVY disease incidence was noted under mulched conditions on FD-81-1. Mulched conditions were better in the reduction of diseases than unmulched conditions. The average high temperature (HT) ranged from 21.9 ◦C to 27 ◦C was most favorable for the development of PLRV and PVY on all six tested potato varieties in both conditions. The average low temperature (LT) ranged from 8 ◦C to 15 ◦C was favorable for the progression of PLRV and PVY diseases in both conditions. The relative humidity (RH) range of 50-60% was conducive for the development of PLRV and PVY diseases. It is concluded that mulching application is an essential tool to enhance the yield of potato crops by reducing their specific diseases.

Clonal Preselection in Grape (Vitis vinifera L.) Varieties of Ekşi Kara and Gök Üzüm

Ekşi Kara (functional female flowers) and Gök Üzüm (hermaphroditic flowers) are the two most important autochthonous varieties of middle Anatolia. This clone selection study started with mass-selection in producer vineyards consisting of approximately 5000 vines by The International Organization of Vine and Wine (OIV) clonal selection procedure. Two-years genetic and sanitation were examined visually in population and 220 clone candidates were ampelography and fertilization biology and bud fertility determined for Ekşi Kara variety. The clone candidates were ranked at the level of sums, with weighted grading of three-year yield, growth, and quality records. Sanitation analyses of the superior clones were made. 17 clones in the Ekşi Kara grape variety were selected according to their superior scores in genetic selection and sanitation analyses. Eleven clones were selected by mass selection from Gök Üzüm carried out in a single location, and 2 clones were selected with genetic selection scores and health tests. Grapevine fleck virus (GfKV) was the most common ( 29%) in the samples tested, Grapevine leafroll-associated virus 1+3 (GLRaV-1 + 3,  26%), Grapevine virus A (GVA, 12%), Grapevine leafroll associated virus-2 (GLRaV-2,  3%), Arabis mosaic virus (ArMV) / Grapevine fanleaf virus (GFLV) ( 1%) are fallowed with indicated percentage. Although virus and bacterial infections are common in the vineyards, enough healthy clones were selected. 17 Ekşi Kara and 2 Gök Üzüm clones selected as pollinators were grafted onto the 110R rootstock for clone comparison in homogeneous conditions, and a "Clone Comparison Vineyard" was established in Selçuk University.

Genetic Diversity of Grapevine Virus A in Three Australian Vineyards Using Amplicon High Throughput Sequencing (Amplicon-HTS).

Shiraz disease (SD) is one of the most destructive viral diseases of grapevines in Australia and is known to cause significant economic loss to local growers. Grapevine virus A (GVA) was reported to be the key pathogen associated with this disease. This study aimed to better understand the diversity of GVA variants both within and between individual SD and grapevine leafroll disease (LRD) affected grapevines located at vineyards in South Australia. Amplicon high throughput sequencing (Amplicon-HTS) combined with median-joining networks (MJNs) was used to analyze the variability in specific gene regions of GVA variants. Several GVAII variant groups contain samples from both vineyards studied, suggesting that these GVAII variants were from a common origin. Variant groups analyzed by MJNs using the overall data set denote that there may be a possible relationship between variant groups of GVA and the geographical location of the grapevines.

Grapevine Virome of the Don Ampelographic Collection in Russia Has Concealed Five Novel Viruses.

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).

Investigating Protein-Protein Interactions Between Grapevine Leafroll-Associated Virus 3 and Vitis vinifera.

Grapevine leafroll disease (GLD) is a globally important disease that affects the metabolic composition and biomass of grapes, leading to a reduction in grape yield and quality of wine produced. Grapevine leafroll-associated virus 3 (GLRaV-3) is the main causal agent for GLD. This study aimed to identify protein-protein interactions between GLRaV-3 and its host. A yeast two-hybrid (Y2H) library was constructed from Vitis vinifera mRNA and screened against GLRaV-3 open reading frames encoding structural proteins and those potentially involved in systemic spread and silencing of host defense mechanisms. Five interacting protein pairs were identified, three of which were demonstrated in planta. The minor coat protein of GLRaV-3 was shown to interact with 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase 02, a protein involved in primary carbohydrate metabolism and the biosynthesis of aromatic amino acids. Interactions were also identified between GLRaV-3 p20A and an 18.1-kDa class I small heat shock protein, as well as MAP3K epsilon protein kinase 1. Both proteins are involved in the response of plants to various stressors, including pathogen infections. Two additional proteins, chlorophyll a-b binding protein CP26 and a SMAX1-LIKE 6 protein, were identified as interacting with p20A in yeast but these interactions could not be demonstrated in planta. The findings of this study advance our understanding of the functions of GLRaV-3-encoded proteins and how the interaction between these proteins and those of V. vinifera could lead to GLD.

Optimization of a Protocol for Launching Grapevine Infection with the Biologically Active cDNA Clones of a Virus.

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.

Detection and elimination of grapevine leafroll-associated viruses (GLRaVs) from Vitis vinifera cultivars of Pakistan by meristem tip culture

Detection and elimination of grapevine leafroll-associated viruses (GLRaVs) from Vitis vinifera cultivars of Pakistan by meristem tip culture

A NitroPure Nitrocellulose Membrane-Based Grapevine Virus Sampling Kit: Development and Deployment to Survey Japanese Vineyards and Nurseries.

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.

Molecular and Metagenomic Analyses Reveal High Prevalence and Complexity of Viral Infections in French-American Hybrids and North American Grapes.

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.

Mycoinsecticides applied in late afternoon to avoid solar UV damage are more efficacious against rice leaf-rolling insect pests than those applied in the morning

Beauveria bassiana ZJU435 (Bb-ZJU435) and Metarhizium anisopliae CQ421 (Ma-CQ421) registered as mycoinsecticides in China were tested for control efficacies against the rice leaf-rolling pests Cnaphalocrocis medialis and Parnara guttata, which migrated to the tillering stage of rice crop in three rice-shrimp rotation paddy fields, where chemical control is prohibited. Either mycoinsecticide was sprayed twice at a recommended rate after 5:00 pm to avoid solar UV avoidance (UVavo) or before 10:00 am to increase solar UV exposure (UVexp). The first spray, although followed by two-week harsh weather, reduced significantly leaf roll densities (damages) in the fungal treatments compared to blank control. The UVavo and UVexp treatments resulted in control efficacies of 63 % and 35 % for Ma-CQ421 and 28 % and 16 % for Bb-ZJU435 on day 14. The second spray was followed by two weeks of weather favorable for fungal action. Consequently, Bb-ZJU435 and Ma-CQ421 showed significantly higher efficacies in their UVavo treatments (60 % and 55 % on day 21; 66 % and 64 % on day 28) than in corresponding UVexp treatments (47 % and 43 % on day 21; 53 % and 50 % on day 28). The UVavo versus UVexp efficacies after the second spray were corrected to ∼95 % versus ∼85 % for Bb-ZJU435, and 75–84 % versus 54–68 % for Ma-CQ421 by verifying whether the leaf rolls were empty (damage-transferred) or occupied by a living or mycosis-killed larva in all treatments. The corrected efficacies of Bb-ZJU435 were significantly higher than those of Ma-CQ421. Conclusively, either mycoinsecticide was more efficacious against the rice leaf rollers when applied in late afternoon to avoid solar UV damage.

The First Virome of a Russian Vineyard.

Among other pathogens, more than 80 viruses infect grapevine. The aim of this work was to study the virome diversity of grapevine viruses and mycoviruses of a vineyard using high-throughput sequencing technologies. The grapevine virome was studied in symptomatic vines of the Rkatsiteli cultivar (V. vinifera) collected at the vineyards of the Krasnodar Krai in Russia. Ribosomal-depleted total RNA and isolated small RNAs were used for library preparation and high-throughput sequencing. Six grapevine-infecting viruses and two viroids were validated by RT-PCR and analyzed phylogenetically. We identified the presence of grapevine leafroll-associated virus 3, grapevine Pinot gris virus, grapevine virus T, grapevine rupestris stem-pitting-associated virus, grapevine fleck virus, and grapevine rupestris vein feathering virus, as well as two viroids, grapevine yellow speckle viroid 1 and hop stunt viroid. We also studied the mycovirome of the vineyard and identified nine viruses with single-stranded positive-sense RNA genomes: alternaria arborescens mitovirus 1, botrytis cinerea mitovirus 1, botrytis cinerea mitovirus 2, botrytis cinerea mitovirus 3, botrytis cinerea mitovirus 4, sclerotinia sclerotiorum mitovirus 3, botrytis cinerea hypovirus 1, grapevine-associated narnavirus 1, and botrytis virus F. In addition, we identified botrytis cinerea hypovirus 1 satellite-like RNA and two single-stranded negative-sense RNA viruses. This is the first study of grapevine mycoviruses in Russia. The obtained result will contribute to the development of biocontrol strategies in the future.

Diverse spectra of virus infection identified through high throughput sequencing in nursery plants of two Indian grapevine cultivars

Grapevine (Vitis vinifera L.) is a valuable fruit crop known to be infected by more than 80 viruses and viroidal pathogens globally. In view of absence of indexing system for viruses and lack of information on viruses and viroids in grafted grapevine nursery plants in India, high throughput sequencing (HTS) based virome analysis of four libraries from sample pools and single plants from grafted nursery plants of two commonly grown grapevine cultivars namely Super Sonaka and Anushka was undertaken. RNA sequencing and analysis revealed the infection of 18 viruses and 5 viroids. Grapevine virus A and grapevine virus L (vitiviruses), grapevine leafroll-associated virus-3 (GLRaV-3) and GLRaV-4 (ampeloviruses) were the most predominant in all the grafted nursery plants based on the number of contigs, depth of coverage and copy number. Presence of grapevine virus D, grapevine virus H, and GLRaV-7 have been recorded for the first time in the Indian grapevine. Infection of these viruses obtained in HTS data was confirmed through RT-PCR and sequencing. Using de novo assembly and reference-based mapping, 43 complete or near complete genomes of the identified viruses and viroids were reconstructed from the sequenced grapevine samples. The phylogenetic analysis indicated the possible introduction of identified viruses through the import of infected planting materials which probably escaped quarantine procedure. The study showed the importance of HTS in detection of known and unknown viruses in plant propagules of clonally propagated perennial crops.

Trouble-free detection of grapevine leafroll-associated virus-3 employing reverse transcription-recombinase polymerase amplification assay

Trouble-free detection of grapevine leafroll-associated virus-3 employing reverse transcription-recombinase polymerase amplification assay

An Assessment of the Virome of Nigerian Vineyards

The virome of Nigerian vineyards was assessed via diagnostic surveys for viruses and viroids in 40 sites across six states in 2016. Leaf tissues from 360 grapevines were preserved dried under CaCl2. High-throughput sequencing of total RNA extracts from two composite dry leaf samples resulted in the detection of grapevine leafroll-associated virus 1 (GLRaV-1), hop stunt viroid (HSVd), grapevine yellow speckle viroid 1 (GYSVd-1), and GYSVd-2. The incidences and distribution of these agents were assessed via RT-PCR with specific primers and two additional primer pairs targeting grapevine rupestris stem pitting-associated virus (GRSPaV) and grapevine red blotch virus (GRBV). Approximately 47% (169/360) of the samples had mixed infections of two to five agents, ∼22% (78/360) had single infections, and ∼31% (113/360) tested negative. HSVd was the most common, with its occurrence in ∼53% (192/360; 39 sites) of samples, followed by GYSVd-2 (∼43%; 155/360; 34 sites), GYSVd-1 (∼33%; 120/360; 26 sites), GLRaV-1 (∼24%; 85/360; 26 sites), and GRSPaV (∼3%; 10/360; 7 sites). All 360 samples were negative for GRBV. In pairwise comparisons, GLRaV-1 isolates from Nigeria shared 97 to 100% nucleotide (nt) and amino acid identities among themselves based on partial HSP70h and complete p24 sequences. The nearly complete genome (18,689 nt) of one GLRaV-1 isolate was determined and it shared 76.5 to 94.5% identity with other fully sequenced GLRaV-1 isolates. The results revealed a simpler virome of Nigerian vineyards and underscore the need to strengthen phytosanitary measures and promote propagation of clean plant materials for vineyard establishment in Nigeria to forestall the introduction of additional viruses and viroids. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Scalable early detection of grapevine virus infection with airborne imaging spectroscopy.

The US wine and grape industry suffers $3B in damages and losses annually due to viral diseases such as Grapevine Leafroll-associated Virus Complex 3 (GLRaV-3). Current detection methods are labor intensive and expensive. GLRaV-3 undergoes a latent period in which the vines are infected but do not yet display visible symptoms, making it an ideal model to evaluate the scalability of imaging spectroscopy-based disease detection. We deployed the NASA Airborne Visible and Infrared Imaging Spectrometer Next Generation (AVIRIS-NG) to detect GLRaV-3 in Cabernet Sauvignon grapevines in Lodi, CA in September 2020. Foliage was removed from the vines as part of mechanical harvest soon after imagery acquisition. In both Sept. 2020 and 2021, industry collaborators scouted 317ac on a vine-by-vine basis for visible viral symptoms and collected a subset for molecular confirmation testing. Grapevines identified as visibly diseased in 2021, but not 2020, were assumed to have been latently infected at time of acquisition. We trained spectral models with random forest and synthetic minority oversampling technique to distinguish non-infected and GLRaV-3-infected grapevines. Non-infected and GLRaV-3 infected vines could be differentiated both pre- and post-symptomatically at 1m through 5m resolution. The best-performing models had 87% accuracy distinguishing between non-infected and asymptomatic vines, and 85% accuracy distinguishing between non-infected and asymptomatic + symptomatic vines. The importance of non-visible wavelengths suggests this capacity is driven by disease-induced changes to overall plant physiology. Our work sets a foundation for using the forthcoming hyperspectral satellite Surface Biology and Geology for regional disease monitoring.

Occurrence and distribution of Grapevine pinot gris virus and other grapevine viruses in German viticultural regions

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.

ПРОБЛЕМАТИКА И РАСПРОСТРАНЕННОСТЬ ВИРУСА СКРУЧИВАНИЯ ЛИСТЬЕВ ВИНОГРАДА

The grapevine leafroll-associated virus is currently one of the most dangerous phytopathological diseases of grapevine culture, it is listed to the list of quarantine diseases of the EU and Russia. The article deals with the problems of the spread of the grapevine leafroll-associated virus in Russia and in the world, its biodiversity in different countries and ways of control are considered. Statistics on the damage caused by this phytopathological disease to medium and large wineries are given. Special attention is paid to the importance of early identification of the grapevine leafroll-associated virus in order to prevent its further spread and to the most important link in RT-PCR, the isolation of the virus RNA from plant material.

High-Throughput Sequencing of Grapevine in Mexico Reveals a High Incidence of Viruses including a New Member of the Genus Enamovirus.

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.

Serological relationships among strains of grapevine leafroll-associated virus 4 reflect the evolutive behavior of its coat protein gene

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. Highlights: The occurrence of GLRaV-4 strains -5, -6 and -9 was recorded in Argentina. The coat protein sequence showed a highly variable N-terminal region among the strains. An evolutionary analysis inferred the occurrence of sites under positive diversifying selection pressure. The most variable region of the coat protein presented the highest probability of occurrence of linear epitopes.