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Phylogenetic inference of grapevine leafroll-associated virus 3 (GLRaV-3) isolates. Maximum likelihood tree generated based on complete or near-complete genomes (above 16,000 nts) available in GenBank and/or the FPS database. Bootstrap values less than 50% are not shown. Horizontal branch length is proportional to genetic distance and the scale bars represent changes per site. Red stars denote the divergent variants identified in this study. https://doi.org/10.1371/journal.pone.0208862.g004
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Grapevine leafroll-associated virus 3 (GLRaV-3) is the most widely prevalent and economically important of the complex of RNA viruses associated with grapevine leafroll disease (GLD). Phylogenetic studies have grouped GLRaV-3 isolates into nine different monophyletic groups and four supergroups, making GLRaV-3 a genetically highly diverse virus spe...
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... Following this discovery, New Zealand and other researchers worldwide collaborated with the commercial provider of the antibody to improve the ELISA antisera to ensure continued reliability of the assay for all diagnostic laboratories worldwide (Bioreba, 2022;Chooi et al., 2018). Notably, a similar activity to improve molecular diagnostics for GLRaV-3 was undertaken in 2018 by researchers around the world to validate a RT-qPCR assay for the detection of all known GLRaV-3 genotypes that was corroborated by high-throughput sequencing (Diaz-Lara et al., 2018). These activities highlight the need for continuous confirmation all genotypes are detected by the diagnostic tests. ...
Grapevine leafroll-associated virus 3 (GLRaV-3) is a major pathogen of grapevines worldwide resulting in grapevine leafroll disease (GLD), reduced fruit yield, berry quality and vineyard profitability. Being graft transmissible, GLRaV-3 is also transmitted between grapevines by multiple hemipteran insects (mealybugs and soft scale insects). Over the past 20 years, New Zealand has developed and utilized integrated pest management (IPM) solutions that have slowly transitioned to an ecosystem-based biological response to GLD. These IPM solutions and combinations are based on a wealth of research within the temperate climates of New Zealand's nation-wide grape production. To provide context, the grapevine viruses present in the national vineyard estate and how these have been identified are described; the most pathogenic and destructive of these is GLRaV-3. We provide an overview of research on GLRaV-3 genotypes and biology within grapevines and describe the progressive development of GLRaV-3/GLD diagnostics based on molecular, serological, visual, and sensor-based technologies. Research on the ecology and control of the mealybugs Pseudococcus calceolariae and P. longispinus, the main insect vectors of GLRaV-3 in New Zealand, is described together with the implications of mealybug biological control agents and prospects to enhance their abundance and/or fitness in the vineyard. Virus transmission by mealybugs is described, with emphasis on understanding the interactions between GLRaV-3, vectors, and plants (grapevines, alternative hosts, or non-hosts of the virus). Disease management through grapevine removal and the economic influence of different removal strategies is detailed. Overall, the review summarizes research by an interdisciplinary team working in close association with the national industry body, New Zealand Winegrowers. Teamwork and communication across the whole industry has enabled implementation of research for the management of GLD.
... At CDFA, total nucleic acid (TNA) was extracted from 0.2 g of petioles and midribs ground in 2 ml of lysis buffer using a MagMAX-96 viral RNA isolation kit (Ambion, Austin, TX, U.S.A.) according to the procedures described by Diaz-Lara et al. (2018). At the Plant Pathogen Confirmatory Diagnostic Laboratory (PPCDL) in Laurel, Maryland, total RNA was extracted from 0.1 g of petioles and midribs using the RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. ...
Citrus yellow vein clearing virus (CYVCV) is a previously reported citrus virus from Asia with widespread distribution in China. In 2022 the California Department of Food and Agriculture (CDFA) conducted a multi-pest citrus survey targeting multiple citrus pathogens including CYVCV. In March 2022, a lemon tree with symptoms of vein clearing, chlorosis and mottling in a private garden in the city of Tulare, California tested positive for CYVCV, which triggered an intensive survey in the surrounding areas. A total of 3,019 plant samples, including citrus and non-citrus species, were collected, and tested for CYVCV using conventional RT-PCR, RT-qPCR, and Sanger sequencing. Five hundred eighty-six citrus trees tested positive for CYVCV, including eight citrus species not previously recorded infected under field conditions. Comparative genomic studies were conducted using seventeen complete viral genomes. Sequence analysis revealed two major phylogenetic groups. Known Asian isolates and five California isolates from this study comprised the first group, whereas all other CYVCV isolates from California formed a second group, distinct from all worldwide isolates. Overall, CYVCV population shows rapid expansion and high differentiation indicating a population bottleneck typical of a recent introduction into a new geographic area.
... It is the main causal agent of grapevine leafroll disease (GLD), provoking symptoms in grapevine hosts while disturbing sugar and anthocyanin metabolism (Song et al., 2021). So far, genomic variants of GLRaV-3 are classified into eight phylogenic groups (Diaz-Lara et al., 2018), with biological properties of individual variants still unknown in terms of symptom development and changes in plant metabolism, unlike those defined in other members of Closteroviridae family. Complex GLRaV-3 interactions with other viruses, scionrootstock combinations and environmental factors pose as limiting factors in assessing and defining mechanisms related to the disease development (Naidu et al., 2014). ...
... Importantly, while one of the GLRaV-3 variants matched isolate WA-MR with 97-100% sequence identity, the other variant differed significantly in nucleotide sequence from all GLRaV-3 isolates for which genome sequences were available at the time in GenBank. This variant whose complete genome sequence was subsequently obtained and deposited in GenBank under the accession number MK032068 was designated as Vdl [14]. Similarly, Vidal samples were also infected with two distinct variants of GVE, one of which potentially represents a new and distinct variant that is only 73-83% identical to isolate WAHH2 from Cabernet Sauvignon [15]. ...
... Collectively, RNA-seq data revealed seven viruses including GLRaV-3, GRBV, and four viruses of the Betaflexiviridae family (Table 5). Strikingly, the sequence contigs related to GLRaV-3 belong to five distinct genetic variants, including one that is more closely related to isolate Vdl from Vidal [14]. ...
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 eight 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.
... Although downward leaf rolling is a diagnostic symptom of GLD and mostly appears at the end of the season, it is not exhibited in all the whiteberried cultivars leading to difficulties in visual diagnosis of GLRaV-3 in these cultivars (Maree et al. 2013;Naidu et al. 2015). So, to detect this virus in different grapevine cultivars, several serological (ELISA) and molecular detection techniques (RT-PCR and RT-qPCR) along with low-density PCR arrays (LDA) have been employed by researchers (Bester et al. 2012;Diaz-Lara et al. 2018, 2019Kumar et al. 2015;Osman et al. 2008;Sidharthan et al. 2022). Although these techniques are sensitive and specific for targeted detection of GLRaV-3, some aspects like requirement of purified nucleic acid, costly laboratory equipments and reagents limit their application in resource-poor setups. ...
Grapevine leafroll-associated virus-3 (GLRaV-3) is a viral pathogen of grapevine leafroll disease (GLD) complex, responsible for debilitating viticulture across the globe. The only available options for management of this important virus are vector control and selection of clean plant propagating materials. To detect this virus in planting material and field samples, it is necessary to develop a quick, reliable, sensitive, and cost-effective detection method. In this study, a simple crude plant extract-based reverse transcription-recombinase polymerase amplification (RT-RPA) assay was developed for efficient detection of GLRaV-3. This assay is quick and trouble-free in sample preparation and can be performed at a wide range of temperatures (20–45 °C) and time periods (20–35 min) although best results were obtained at 40 °C for 30 min of incubation. The assay does not require tedious RNA isolation as grapevine leaf sap simply prepared in NaOH: EDTA (1:1) buffer is directly used as template. The developed RT-RPA assay has been shown to be highly sensitive and specific for the detection of GLRaV-3 in grapevine samples, with a limit of detection as low as 10–5 dilutions. Field validation and application of the developed assay further confirmed the sensitivity and reliability as 76% of samples were tested positive for GLRaV-3. The developed assay requires less than 60 min for its completion, making it a rapid and efficient tool for screening large numbers of grapevine samples and planting materials. The simplicity and efficiency of the developed RT-RPA assay make it a promising tool for detecting GLRaV-3 in grapevines, allowing for timely and accurate diagnosis and control of this economically significant virus.
... accessed on 1 June 2023). These molecular tests have been described previously [24][25][26][27][28]. Some assays have been recently updated and are available from FPS by request. ...
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.
... Using all available GLRaV-3 sequences from GenBank, and Australian isolates from this study, a tree with five phylogroups was produced and new naming of these proposed based on the aa sequence similarity of the CP gene from high (phylogroup I) to low (phylogroup V) to the exemplar isolate NY1. This finding contrasts to several previous studies, including our preliminary study, which identified seven phylogroups [1], and that described by Diaz-Lara et al. (2018) that identified 10 phylogroups [44]. It appears the addition of a larger number of full-length GLRaV-3 genomes and CP gene sequences in our most recent phylogenetic analysis has more clearly defined the evolutionary relationship between isolates, resulting in the collapse of previously described phylogroups to a smaller number. ...
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.
... As a single-stranded positive RNA virus, viral RNA-dependent RNA polymerase contributes to genetic variability leading to the accumulation of genetically diverse variants, often called "quasi-species" [2]. Based on the phylogenetic analyses, GLRaV-3 variants are classified into eight basic phylogenetic groups: I, II, III, V, VI, VII, IX, and X [3], with those belonging to phylogenetic groups I and II, globally the most represented. With more samples analyzed, more variants are continuously being discovered [4], pushing the need to systematize viral genome regions. ...
... Thirteen different virus isolates for grafting were used in this study (Table 1), composed solely of GLRaV-3 (LR) or LR in combination with other wild-type viruses (WT). Five inoculums were composed of monophyletic GLRaV-3 isolates and included genomic variants from phylogenetic groups I, II, III, VI, and VII, according to Diaz-Lara et al. [3]. An additional four were prepared as mutual combinations of selected monophyletic GLRaV-3 isolates (I/II, I/III, II/III, I/II/III; Table 1). ...
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.
... Total nucleic acid extracts were prepared from symptomatic and asymptomatic pawpaw leaf tissue, as previously described [31]. Briefly, approximately 0.2 g of pawpaw leaf tissue was spiked with leaf tissue of black turtle soup dry bean plants infected by PvEV1 and PvEV2 (5%, wt:wt), and homogenized using a Homex grinder (Bioreba, Reinach, Switzerland). ...
Pawpaw (Asimina triloba) trees exhibiting stunting and foliar mosaic, chlorosis, or distortions were observed in New York. In 2021, leaf samples from two symptomatic trees and a sapling, as well as two asymptomatic trees, were tested for the presence of viruses and viroids by high-throughput sequencing (HTS) using total RNA after ribosomal RNA depletion. HTS sequence information revealed tobacco ringspot virus (TRSV) and tomato ringspot virus (ToRSV) in symptomatic but not in asymptomatic leaves. HTS reads and de novo-assembled contigs covering the genomes of both viruses were obtained, with a higher average read depth for RNA2 than RNA1. The occurrence of TRSV and ToRSV was confirmed in the original leaf samples used for HTS and 12 additional trees and saplings from New York and Maryland in 2022 by RT-PCR combined with Sanger sequencing, and DAS-ELISA. Single infections by TRSV in 11 of 14 trees and dual infections by TRSV and ToRSV in 3 of 14 trees were identified. The nucleotide sequence identity of partial gene fragments of TRSV and ToRSV was high among pawpaw isolates (94.9–100% and 91.8–100%, respectively) and between pawpaw isolates and isolates from other horticultural crops (93.6–100% and 71.3–99.3%, respectively). This study is the first to determine the virome of pawpaw.
... The use of high-throughput sequencing (HTS) has also been applied to explore the virome of vegetatively propagated crop germplasm, including berry crops [18], strawberry [19], grapevine [20,21], and pistachio [22]. These HTS studies have been useful in clarifying disease etiology, characterizing new viruses, and associating virus presence with symptom development. ...
Pineapple (Ananas comosus L. [Merr.]) accessions from the U.S. Tropical Plant Genetic Resources and Disease Research (TPGRDR) in Hilo, Hawaii were subjected to RNA-sequencing to study the occurrence of viral populations associated with this vegetatively propagated crop. Analysis of high-throughput sequencing data obtained from 24 germplasm accessions and public domain transcriptome shotgun assembly (TSA) data identified two novel sadwaviruses, putatively named “pineapple secovirus C” (PSV-C) and “pineapple secovirus D” (PSV-D). They shared low amino acid sequence identity (from 34.8 to 41.3%) compared with their homologs in the Pro-pol region of the previously reported PSV-A and PSV-B. The complete genome (7485 bp) corresponding to a previously reported partial sequence of the badnavirus, pineapple bacilliform ER virus (PBERV), was retrieved from one of the datasets. Overall, we discovered a total of 69 viral sequences representing ten members within the Ampelovirus, Sadwavirus, and Badnavirus genera. Genetic diversity and recombination events were found in members of the pineapple mealybug wilt-associated virus (PMWaV) complex as well as PSVs. PMWaV-1, -3, and -6 presented recombination events across the quintuple gene block, while no recombination events were found for PMWaV-2. High recombination frequency of the RNA1 and RNA2 molecules from PSV-A and PSV-B were congruent with the diversity found by phylogenetic analyses. Here, we also report the development and improvement of RT-PCR diagnostic protocols for the specific identification and detection of viruses infecting pineapple based on the diverse viral populations characterized in this study. Given the high occurrence of recombination events, diversity, and discovery of viruses found in Ananas germplasm, the reported and validated RT-PCR assays represent an important advance for surveillance of viral infections of pineapple.
