Novel insights into hotspots of insect vectors of GLRaV-3: Dynamics and global distribution.

Grapevine leafroll-associated virus 3 (GLRaV-3) is the most prevalent and economically damaging virus in grapevines and is found on nearly all continents, except Antarctica. Ten mealybugs act as vector insects transmitting the GLRaV-3. Understanding the potential distribution range of vector insects under climate change is crucial for preventing and managing vector insects and controlling and delaying the spread of GLRaV-3. This study investigated the potential geographical range of insect vectors of GLRaV-3 worldwide using MaxEnt (maximum entropy) based on occurrence data under environmental variables. The potential distributions of these insects were projected for the 2030s, 2050s, 2070s, and 2090s under the three climate change scenarios. The results showed that the potential distribution range of most vector insects is concentrated in Southeastern North America, Europe, Asia, and Southeast Australia. Most vector insects contract their potential distribution ranges under climate-change conditions. The stacked model suggested that potential distribution hotspots of vector insects were present in Southeastern North America, Europe, Southeast Asia, and Southeast Australia. The potential distribution range of hotspots would shrink with climate change. These results provide important information for governmental decision-makers and farmers in developing control and management strategies against vector insects of GLRaV-3. They can also serve as references for studies on other insect vectors.

The New Zealand perspective of an ecosystem biology response to grapevine leafroll disease.

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.

Application of High-Throughput Sequencing for Comprehensive Virome Profiling in Grapevines Shows Yellows in Iran.

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.

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.

Complete genome sequence of a novel closterovirus isolated from Dregea volubilis.

The complete genome sequence of a putative novel closterovirus, tentatively named "Dregea volubilis closterovirus 1" (DvCV1, GenBank accession no. MZ779122), infecting Dregea volubilis in China was determined using high-throughput sequencing (HTS). The complete genome sequence of DvCV1 consists of 16,165 nucleotides (nt) and contains nine ORFs. The genome structure of DvCV1 is typical of members of the genus Closterovirus. Complete genome sequence analysis showed that DvCV1 shares 41.4-48.4% nucleotide sequence identity with other known closteroviruses. The putative RNA-dependent RNA polymerase (RdRp), heat shock protein 70-like protein (HSP70h), and coat protein (CP) of DvCV1 share 46.80-62.65%, 31.06-51.80%, and 28.34-37.37% amino acid sequence identity, respectively, with the RdRp, HSP70h and CP of other closteroviruses. Phylogenetic analysis based on HSP70h aa sequences placed DvCV1 alongside other members of the genus Closterovirus in the family Closteroviridae. These results suggest that DvCV1 is a new member of the genus Closterovirus. This is the first report of a closterovirus infecting D. volubilis.

Scalable Early Detection of Grapevine Viral Infection with Airborne Imaging Spectroscopy.

The U.S. wine and grape industry loses $3B annually due to viral diseases including grapevine leafroll-associated virus complex 3 (GLRaV-3). Current detection methods are labor-intensive and expensive. GLRaV-3 has a latent period in which the vines are infected but do not display visible symptoms, making it an ideal model to evaluate the scalability of imaging spectroscopy-based disease detection. The NASA Airborne Visible and Infrared Imaging Spectrometer Next Generation was deployed 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 image acquisition. In September of both 2020 and 2021, industry collaborators scouted 317 hectares on a vine-by-vine basis for visible viral symptoms and collected a subset for molecular confirmation testing. Symptomatic grapevines identified in 2021 were assumed to have been latently infected at the time of image acquisition. Random forest models were trained on a spectroscopic signal of noninfected and GLRaV-3 infected grapevines balanced with synthetic minority oversampling of noninfected and GLRaV-3 infected grapevines. The models were able to differentiate between noninfected and GLRaV-3 infected vines both pre- and postsymptomatically at 1 to 5 m resolution. The best-performing models had 87% accuracy distinguishing between noninfected and asymptomatic vines, and 85% accuracy distinguishing between noninfected and asymptomatic + symptomatic vines. The importance of nonvisible wavelengths suggests that this capacity is driven by disease-induced changes to plant physiology. The results lay a foundation for using the forthcoming hyperspectral satellite Surface Biology and Geology for regional disease monitoring in grapevine and other crop species. [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.

Detecting Grapevine Virus Infections in Red and White Winegrape Canopies Using Proximal Hyperspectral Sensing.

Grapevine virus-associated disease such as grapevine leafroll disease (GLD) affects grapevine health worldwide. Current diagnostic methods are either highly costly (laboratory-based diagnostics) or can be unreliable (visual assessments). Hyperspectral sensing technology is capable of measuring leaf reflectance spectra that can be used for the non-destructive and rapid detection of plant diseases. The present study used proximal hyperspectral sensing to detect virus infection in Pinot Noir (red-berried winegrape cultivar) and Chardonnay (white-berried winegrape cultivar) grapevines. Spectral data were collected throughout the grape growing season at six timepoints per cultivar. Partial least squares-discriminant analysis (PLS-DA) was used to build a predictive model of the presence or absence of GLD. The temporal change of canopy spectral reflectance showed that the harvest timepoint had the best prediction result. Prediction accuracies of 96% and 76% were achieved for Pinot Noir and Chardonnay, respectively. Our results provide valuable information on the optimal time for GLD detection. This hyperspectral method can also be deployed on mobile platforms including ground-based vehicles and unmanned aerial vehicles (UAV) for large-scale disease surveillance in vineyards.

Discovery of a Closterovirus Infecting Jujube Plants Grown at Aksu Area in Xinjiang of China.

Chinese jujube (Ziziphus jujuba Mill.) is a widely grown fruit crop at Aksu in Xinjiang Uygur Autonomous Region of China. Viral disease-like symptoms are common on jujube plants. Here, for the first time, we report a virus tentatively named persimmon ampelovirus jujube isolate (PAmpV-Ju) infecting jujube plants. The virus was identified using high-throughput sequencing from a jujube plant (ID: AKS15) and molecularly related to viruses in the family Closteroviridae. The genomic sequences of two PAmpV-Ju variants named AKS15-20 and AKS15-17 were determined by RT-PCR amplifications. The genome structure of PAmpV-Ju was identical to that of a recently reported persimmon ampelovirus (PAmpV) and consisted of seven open reading frames. The genomes of AKS15-20 and AKS15-17 shared 83.7% nt identity with each other, and the highest nt sequence identity of 79% with two variants of PAmpV. The incidence of PAmpV-Ju on Aksu jujube plants was evaluated by RT-PCR assays. The phylogenetic analysis of amplified partial sequences coding for polymerase, HSP70h, and CP revealed two phylogenetic clades represented by AKS15-20 and AKS15-17. Our study provides important evidence for understanding viruses infecting jujube plants and establishing efficient measures to prevent virus spread.

Identification of Interactions between Proteins Encoded by Grapevine Leafroll-Associated Virus 3.

The roles of proteins encoded by members of the genus Ampelovirus, family Closteroviridae are largely inferred by sequence homology or analogy to similarly located ORFs in related viruses. This study employed yeast two-hybrid and bimolecular fluorescence complementation assays to investigate interactions between proteins of grapevine leafroll-associated virus 3 (GLRaV-3). The p5 movement protein, HSP70 homolog, coat protein, and p20B of GLRaV-3 were all found to self-interact, however, the mechanism by which p5 interacts remains unknown due to the absence of a cysteine residue crucial for the dimerisation of the closterovirus homolog of this protein. Although HSP70h forms part of the virion head of closteroviruses, in GLRaV-3, it interacts with the coat protein that makes up the body of the virion. Silencing suppressor p20B has been shown to interact with HSP70h, as well as the major coat protein and the minor coat protein. The results of this study suggest that the virion assembly of a member of the genus Ampelovirus occurs in a similar but not identical manner to those of other genera in the family Closteroviridae. Identification of interactions of p20B with virus structural proteins provides an avenue for future research to explore the mechanisms behind the suppression of host silencing and suggests possible involvement in other aspects of the viral replication cycle.

Gathered from the Vine: A Survey of Seven Grapevine Viruses Within New England Vineyards.

Vineyards in the Southeastern New England American Viticultural Area were surveyed for the incidence of seven major viruses: grapevine leafroll-associated viruses (GLRaV-1, GLRaV-2, GLRaV-3, and GLRaV-4), grapevine fanleaf virus (GFLV), tomato ringspot virus (ToRSV), and tobacco ringspot virus (TRSV). Viruses were detected by DAS-ELISA and confirmed by RT-PCR and Sanger sequencing. Multiple viruses were present in 19 out of the 25 vineyards surveyed between 2018 and 2020. GLRaV-3 (27.59%) was the most prevalent virus followed by GLRaV-4 (14.90%), GLRaV-1 (13.52%), GLRaV-2 (11.03%), ToRSV (6.34%), GFLV (5.24%), and TRSV (2.62%). Furthermore, phylogenetic analyses of the viral partial genome sequences acquired in this study revealed that the grapevine viruses present in this area are diverse, indicating that they may have been introduced from different sources. Our findings stress the need for improving the sanitary status of planting materials to avoid the introduction and dissemination of viruses to vineyards in this important wine-producing region of New England.

Diagnostic and Historical Surveys of Sweet Cherry (Prunus avium) Virus and Virus-Like Diseases in Oregon.

There are over 35 known virus and virus-like diseases of sweet cherry (Prunus avium), some with potential to cause severe economic impact by reducing vegetative growth, vigor, or fruit quality. Oregon is the second-ranked state for sweet cherry production in the United States. Statewide surveys were conducted in Oregon sweet cherry orchards for virus and virus-like diversity and distribution. Orchards in key production regions with suspected virus disease symptoms were sampled. Virus-specific enzyme-linked immunosorbent assay, isothermal amplification, or quantitative real-time PCR were used to test for the presence of common or economically important sweet cherry pathogens, including cherry leaf roll virus (CLRV), little cherry virus 2 (LChV2), prune dwarf virus (PDV), prunus necrotic ringspot virus (PNRSV), tomato ringspot virus (ToRSV), and 'Candidatus Phytoplasma pruni'. CLRV, a new virus of sweet cherry in Oregon, was found associated with enation and dieback symptoms in The Dalles. Some viruses were found in new regions, which included Hood River (PDV, PNRSV, and ToRSV) and the Umpqua Valley (PDV and PNRSV). A subsequent survey was conducted in the Mid-Columbia production region for the presence of little cherry symptoms associated with little cherry and X-Diseases. All symptomatic samples from The Dalles and Mosier, OR, or Dallesport, WA, tested positive for 'Ca. P. pruni' but not LChV2. These findings provide a foundation for the current understanding and management of virus and virus-like diseases of sweet cherry in Oregon and context for further studies into these pathogens and their vectors.

Transmission of Grapevine Leafroll-Associated Viruses and Grapevine Virus A by Vineyard-Sampled Soft Scales (Parthenolecanium corni, Hemiptera: Coccidae).

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.

Epidemiology of Yam Viruses in Guadeloupe: Role of Cropping Practices and Seed-Tuber Supply.

The epidemiology of yam viruses remains largely unexplored. We present a large-scale epidemiological study of yam viruses in Guadeloupe based on the analysis of 1124 leaf samples collected from yams and weeds. We addressed the prevalence of cucumber mosaic virus (CMV), Cordyline virus 1 (CoV1), Dioscorea mosaic associated virus (DMaV), yam asymptomatic virus 1 (YaV1), yam mosaic virus (YMV), yam mild mosaic virus (YMMV), badnaviruses, macluraviruses and potexviruses, and the key epidemiological drivers of these viruses. We provide evidence that several weeds are reservoirs of YMMV and that YMMV isolates infecting weeds cluster together with those infecting yams, pointing to the role of weeds in the epidemiology of YMMV. We report the occurrence of yam chlorotic necrosis virus (YCNV) in Guadeloupe, the introduction of YMMV isolates through the importation of yam tubers, and the absence of vertical transmission of YaV1. We identified specific effects on some cropping practices, such as weed management and the use of chemical pesticides, on the occurrence of a few viruses, but no crop-related factor had a strong or general effect on the overall epidemiology of the targeted viruses. Overall, our work provides insights into the epidemiology of yam viruses that will help design more efficient control strategies.

Transcriptomic Analyses of Grapevine Leafroll-Associated Virus 3 Infection in Leaves and Berries of 'Cabernet Franc'.

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.

A novel RNA virus, Thesium chinense closterovirus 1, identified by high-throughput RNA-sequencing of the parasitic plant Thesium chinense.

The genome sequence of a closterovirus (genus Closterovirus, family Closteroviridae), tentatively named Thesium chinense closterovirus 1 (TcCV1), was identified by performing high-throughput RNA-sequencing of the haustoria and root tissues of Thesium chinense, a parasitic plant. The TcCV1 genome was predicted to encode nine proteins, eight of which have orthologs in previously identified closteroviruses. The TcCV1 RNA-dependent RNA polymerase (RdRp) and heat shock protein 70 homolog (Hsp70h) showed 27.8-68.2% and 23.8-55.1% amino acid identity, respectively, to orthologous proteins of known closteroviruses. The putative +1 ribosomal frameshifting site required for producing RdRp was identified as GUUUAGC with UAG stop codon and the skipped nucleotide U. Phylogenetic trees based on RdRp and Hsp70h show that TcCV1 is a novel member of the genus Closterovirus, forming a subclade with a group of known closteroviruses, including mint virus 1 and carnation necrotic fleck virus. The genome sequence of TcCV1 may be useful for studying the genome evolution of closteroviruses. Keywords: Thesium chinense closterovirus 1; Closterovirus; Closteroviridae; Thesium chinense.

Molecular characterization of Cordyline virus 1 isolates infecting yam (Dioscorea spp).

Cordyline virus 1 (CoV1) is a velarivirus that has so far only been reported in ornamental Ti plants (Cordyline fruticosa). Using high-throughput sequencing, we identified CoV1 infection in yam accessions from Vanuatu. Using a specific RT-PCR assay, we found that CoV1 is also present and highly prevalent in Dioscorea alata, D. cayenensis, and D. trifida in Guadeloupe. Phylogenetic analysis showed that CoV1 isolates infecting yam in Guadeloupe display a low level of molecular diversity. These data provide insights into the transmission of CoV1 in yam in Guadeloupe.

Development of a one-step RT-qPCR assay for the detection of Grapevine leafroll-associated virus 7.

Grapevine leafroll disease (GLD) is one of the most economically important viral diseases of grapevines. GLD is caused by a complex of several ssRNA (+) viruses referred to as Grapevine leafroll-associated viruses (GLRaVs). To date, five different GLRaV species have been identified. One of those species, GLRaV-7, was first reported from a symptomless white-fruited wine grape cultivar from Albania. Since its discovery, GLRaV-7 has been reported from 14 countries. Although serological assays have been developed to detect GLRaV-7, commercially available antibodies produce high background signals making them unsuitable for regulatory testing. Furthermore, while molecular detection assays have been shown to be more sensitive when compared to the serological assays, published molecular assays, except the one Reverse Transcription-quantitaive Polymerase Chain Reaction (RT-qPCR) assay based on heat shock protein 70 homologue (HSP70h) gene, have been reported to be inadequate in detecting all reported isolates of GLRaV-7. Availability of multiple assays provides flexibility to diagnostic laboratories in cases where the chosen assay fails to detect a strain or an isolate of a pathogen due to variation in its targeted region or where additional confirmation of the results is required. In this study, we developed a sensitive and specific RT-qPCR assay, based on a region of p61 gene of GLRaV-7, which detected all available isolates.

Grapevine Leafroll-Associated Virus 3 Genotype Influences Foliar Symptom Development in New Zealand Vineyards.

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

Transmission of Grapevine Ampelo- and Vitiviruses by the Bohemian Mealybug Heliococcus bohemicus Sulc (Hemiptera: Pseudococcidae).

Grapevine-infecting ampelo- and vitiviruses are transmitted by several scale insect species, including the Bohemian mealybug, Heliococcus bohemicus Sulc. 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.

Nuances of Responses to Two Sources of Grapevine Leafroll Disease on Pinot Noir Grown in the Field for 17 Years.

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.