Leafroll Research Articles

Incidence and genetic diversity of grapevine leafroll-associated virus 3 (GLRaV-3) isolates in Turkey

Grapevine leafroll-associated virus 3 (GLRaV-3) is the most important virus species within the grapevine leafroll complex. It causes significant losses to growers and vineries because of its effect on grape and wine quality. A survey was conducted in the major grape-growing provinces of Turkey in 2018 to investigate distribution and the genetic diversity of GLRaV-3 in local and foreign grapevine cultivars. The genetic diversity of GLRaV-3 isolates based on partial heat-shock protein 70 homologue (Hsp70 h), partial coat protein (CP) and partial RNA-dependent RNA polymerase (RdRp) genes were determined by RT-PCR and sequencing. The infection rate of GLRaV-3 in 141 tested grapevine samples was 13.47% by DAS-ELISA and 28.36% by RT-PCR analysis. Totally, 17 amplicons from Hsp70 h, 22 isolates from RdRp and 16 isolates from CP genes were sequenced in both directions. The sequence analysis of three genes revealed that the Turkish GLRaV-3 isolates shared 91–100% nucleotide identities with the sequences of GLRaV-3 isolated deposited in the GenBank from other parts of the world without any correlation between the distribution and geographical origin.

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.

Returns to public investments in clean plant centers: A case study of leafroll virus-tested grapevines in support of cost-effective grape production systems

AbstractViruses and related graft-transmissible pathogens cause diseases that cost the grape industry billions of dollars annually if left uncontrolled. The National Clean Plant Network (NCPN), a USDA Farm Bill program, is an organization of clean plant centers that produce and maintain virus-tested foundation vine stocks and distribute propagation material derived thereof to nurseries and growers to minimize the introduction of viruses and virus-like diseases into the vineyard. Foundation Plant Services (FPS) is the major NCPN-grapes center. We examined the economic impacts of public investments in FPS from 2006 to 2019. By focusing on grapevine leafroll disease, our analyses revealed a benefit-cost ratio ranging from 22:1 to 96:1, with a 5% and a 20% disease infection rates in commercial vineyards, respectively. A welfare analysis was consistent with grape growers and nurseries capturing most (64–98%) of the benefits from adopting clean planting material compared with winemakers and other actors in the downstream wine supply chain system. This study provided new insights into the returns to public investments in a clean plant center and documented strong financial incentives for higher adoption of clean vines derived from virus-tested stocks, while justifying continued support of NCPN centers from public and private sectors.

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.

Recovering Ancient Grapevine Cultivars in the Balearic Islands: Sanitary Status Evaluation and Virus Elimination.

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.

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

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.

Survey for Major Grapevine Viruses in Commercial Vineyards of Northwestern Argentina.

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.

Genome editing (CRISPR-Cas)-mediated virus resistance in potato (Solanum tuberosum L.).

Plant viruses are the major pathogens that cause heavy yield loss in potato. The important viruses are potato virus X, potato virus Y and potato leaf roll virus around the world. Besides these three viruses, a novel tomato leaf curl New Delhi virus is serious in India. Conventional cum molecular breeding and transgenics approaches have been applied to develop virus resistant potato genotypes. But progress is slow in developing resistant varieties due to lack of host genes and long breeding process, and biosafety concern with transgenics. Hence, CRISPR-Cas mediated genome editing has emerged as a powerful technology to address these issues. CRISPR-Cas technology has been deployed in potato for several important traits. We highlight here CRISPR-Cas approaches of virus resistance through targeting viral genome (DNA or RNA), host factor gene and multiplexing of target genes simultaneously. Further, advancement in CRISPR-Cas research is presented in the area of DNA-free genome editing, virus-induced genome editing, and base editing. CRISPR-Cas delivery, transformation methods, and challenges in tetraploid potato and possible methods are also discussed.

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.

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.

Occurrence of a 16SrII subgroup D phytoplasma strain associated with leaf roll of greenhouse tomato in Iran

Occurrence of a 16SrII subgroup D phytoplasma strain associated with leaf roll of greenhouse tomato in Iran

Incidence of Grapevine Fanleaf Virus (GFLV) and Grapevine Leafroll-Associated Viruses (GLRaV 1−3) in Vojvodina Province

Summary During May and June of 2021, a total of 123 grapevine leaf samples were collected and analyzed for infection by grapevine fanleaf virus (GFLV) and three viruses from the grapevine leafroll-associated virus complex -1, -2 and -3 (GRLaV-1, GRLaV-2 and GRLaV-3, respectively). The samples were collected from commercial vineyards, small backyard vineyards and grapevine nurseries located across the entire Vojvodina Province (Bačka, Banat and Srem). OEPP/EPPO sampling protocols were followed during the sampling of leaf tissues showing possible virus infection symptoms. Among the 123 samples, 47 were collected from Bačka region (Bačko Gradište − 11, Bečej − 10, Temerin − 15, Vrbas − 1, Hajdukovo − 10), 50 from Banat region (Srpska Crnja − 10, Vojvode Stepe − 10, Čoka − 10, Uljma −20) and 26 from Srem region (Šid − 6, Banoštor − 10, Sremski Karlovci − 10) and serological ELISA tests were performed for virus detection. GFLV was detected in five samples (4.06%), GLRaV-1 was detected in six samples (4.87%), while GLRaV-2 was not detected in any of the analyzed grapevine samples. GLRaV-3 was present in five samples (4.06%). When infection rates were examined in relation to cultivars, GFLV was detected in Cardinal, Zalagyöngye, Black Muscat, Italian Riesling and Dornfelder. GRLaV-1 was detected in cultivars Cardinal, Black Muscat, Italian Riesling and Merlot, and GRLaV-3 in cultivars Othello and Italian Riesling. Based on these results, it can be concluded that GFLV and GRLaV-1 and GRLaV-3 are present in vineyards across Vojvodina Province and affect different grapevine cultivars. To effectively control virus infections and their spreading, continuous monitoring of these viruses and their vectors is required along with the planting of healthy propagation material.

Virus and Virus-like Pathogens in the Grapevine Virus Collection of Croatian Autochthonous Grapevine Cultivars.

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.

Metagenomic analysis for detection and discovery of plant viruses in wild Solanum spp. in South Africa

AbstractWild plant species play an important role in plant virus epidemiology, mainly by harbouring viruses pathogenic to cultivated plant species. The genus Solanum includes important crop species as well as some highly abundant and widely distributed wild plant species. The aim of the present study was to profile the viromes of 11 wild Solanum spp. found in five provinces of South Africa. Metatranscriptomic analysis of pooled RNA samples from each of the Solanum spp. detected viruses belonging to 13–19 families. Reverse transcription PCR confirmed the detection of potato virus Y (PVY), potato leaf roll virus (PLRV), tomato torrado virus (ToTV) and tomato chlorosis virus (ToCV) for the first time in some of the Solanum spp. studied. Phylogenetic analyses indicated that the PVY and PLRV strains from the wild Solanum spp. were divergent from those reported in cultivated crops, while there was limited divergence among ToTV and ToCV isolates. Viruses detected for the first time in South Africa included tobacco mild green mosaic virus, tomato matilda virus, a pepper enamovirus from Rwanda and a cytorhabdovirus from potato in Kenya. Potentially novel viruses were also detected in S. lichtensteinii, S. mauritianum and S. viarum. This study demonstrates the role of Solanum spp. in harbouring viruses pathogenic to solanaceous crops, and in the emergence of new virus strains. It may therefore inform strategies for virus disease management by control of wild solanums that harbour viruses pathogenic to important Solanum crops.

First Report of Grapevine Polerovirus 1 in Grapevine in China.

Xinjiang Uygur Autonomous Region is the largest grape-producing area in China, with a grape output of 3.05 million tons in 2020, accounting for nearly 20% of the total grape output in China (National Bureau of Statistics, 2021). Viral disease is a major factor threatening the grape industry and results in large economic losses by affecting the quality of grapes and wines. Actually, nearly 80 different viruses have been recorded in grapevine (Fuchs, 2020). To identify viruses that infect grapevine in Xinjiang, leaves of four vines of cultivar Cabernet Sauvignon with symptoms of chlorotic spots and crinkling were collected from a vineyard at Shihezi University in Shihezi City in May 2019 and pooled for total RNA extraction (Invitrogen™ PureLink ® Plant RNA Reagent, USA). After ribodepletion, a cDNA library was prepared using the Ribo-ZeroTM Kit (Illumina, San Diego, USA) and subjected to high-throughput sequencing (HTS) on the Illumina NovaSeq 6000 platform (Novogene, China). In total, 41,799,420 paired-end reads (150 nt × 2) were obtained after performing quality control using Trimmomatic version 0.39 (Bolger et al., 2014). These reads were de novo assembled into 154,716 contigs using the rnaSPAdes method in SPAdes software with default parameters (Bankevich et al., 2012). BLASTn analysis of these contigs led to the identification of 59 viral-related contigs from 248 -18476 bp. These contigs belonged to six positive-stranded RNA viruses, namely, grapevine polerovirus 1 (GPoV-1; 2 contigs), grapevine berry inner necrosis virus (GBINV; 4 contigs), grapevine leafroll-associated virus 3 (GLRaV-3; 2 contigs), grapevine Pinot gris virus (GPGV; 3 contigs), grapevine rupestris stem pitting-associated virus (GRSPaV; 4 contigs), and grapevine fleck virus (GFkV; 17 contigs); one DNA virus, grapevine geminivirus A (GGVA; 2 contigs); and one viroid, Australian grapevine viroid (AGVd; 1 contig). Among them, GPoV-1 is a newly discovered grape-infecting virus that has recently been reported from Japan and France (Candresse et al., 2020; Chiaki and Ito, 2020). The two contigs of GPoV-1 were assembled manually into a 5627-nt scaffold that covers 99.6% of the genome of the reference GPoV-1 isolate (MT008025). The scaffold shared 98.5% and 98.2% nucleotide (nt) sequence identities with the French GPoV-1 isolate KT (MT008025) and the Japanese GPoV-1 isolate KC (LC505098), respectively. To confirm the GPoV-1 infection of the grapevines used for HTS analysis, we designed a primer pair targeting the coding region of the P1 protein GP30F (5'-CCTCTTTCGCTGCCATAGGC-3') and GP2180R (5'-CCTGGAGCCTTAAGCTGGTG-3') and applied them in revere transcription (RT)-PCR using a PrimeScriptTM One Step RT-PCR Kit (Takara, China) to detect GPoV-1. The expected 2151-bp fragment was amplified from one of the four grapevine samples. The amplicon was cloned into the pMD19-T vector (TaKaRa, China) and Sanger sequenced. BLASTn analysis showed that the sequence of the amplicon (GenBank accession no. OK574336) shared 98% identity with the scaffold obtained from HTS and shared 98.5% and 97.4% identity with the GPoV-1 isolates KT and KC, respectively. To determine the occurrence of GPoV-1 in the vineyard, 8 and 20 leaves were randomly collected from grapevines of cvs. Black Monukka and Cabernet Sauvignon, respectively. We designed a primer pair of GPoV4264F (5'-ACTGCACAGACTCTCACACG-3') and GPoV4657R (5'- TCCTTCGCGCAGTCACTATC-3'), which target the coding region of the P3-P5 fusion protein. An expected 394-bp amplicon was detected in 2 out of the 8 Black Monukka and 7 out of the 20 Cabernet Sauvignon leaf samples. Sanger sequencing confirmed the GPoV-1 identity of the amplicons. Although all the samples used for HTS analysis displayed symptoms, 4 of 9 samples in which GPoV-1 infection was detected were asymptomatic, suggesting that GPoV-1 may be latent, as reported previously (Candresse et al., 2020). To the best of our knowledge, this is the first report of GPoV-1 infection of grapevine in China. Although most members of the genus Polerovirus (family Solemoviridae) are transmitted by aphids, how GPoV is transmitted remains unknown, representing an increased risk for its spread. Therefore, attention should be given to reducing the prevalence of GPoV-1 in grape-producing areas in China, especially in Xinjiang.

Planococcus ficus and the spread of grapevine leafroll disease in vineyards: a 30-year-long case study in north-West Spain

The mealybug Planococcus ficus is one of the main vectors of Grapevine leafroll associated virus-3 (GLRaV-3), which was commonly detected in cv “Albariño” planting material before certified stock was available. Mealybug infestations were rare in vineyards in southern Galicia (NW Spain) during the 1990s (2.2% of the vineyards surveyed) and are still rare in inland zones. However, mealybug infestations have spread since 2000, with 15% of surveyed vineyards infested in 2004 and 80% of surveyed vineyards infested in 2016. The spatial and temporal distributions of plants infected with GLRaV-3 were quantified over a 30-year period in an experimental plot established in 1989. The disease progress curve (DPC) was linear for 25 years, with a slow constant rate of spread of less than one newly infected plant per year (0.6%). Since 1992, >82% of infected plants were located on the west side of the plot as were 84% of newly infected plants. Newly infected plants were in contact with infected plants, suggesting vector-mediated transmission, but no potential vectors were found. In 2013, a small mealybug infestation was detected and identified as Pl. ficus. Between 2014 and 2016, the infection rate increased to >21% per year, and in 2019 all plants tested positive for GLRaV-3. This is a valuable case study illustrating how changes to the vector fauna can increase the rate of spread of an economically important virus of grapevine.

GLRaV-2 protein p24 suppresses host defenses by interaction with a RAV transcription factor from grapevine.

Grapevine leafroll-associated virus 2 (GLRaV-2) is a prevalent virus associated with grapevine leafroll disease, but the molecular mechanism underlying GLRaV-2 infection is largely unclear. Here, we report that 24-kDa protein (p24), an RNA-silencing suppressor (RSS) encoded by GLRaV-2, promotes GLRaV-2 accumulation via interaction with the B3 DNA-binding domain of grapevine (Vitis vinifera) RELATED TO ABSCISIC ACID INSENSITIVE3/VIVIPAROUS1 (VvRAV1), a transcription factor belonging to the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) superfamily. Salicylic acid-inducible VvRAV1 positively regulates the grapevine pathogenesis-related protein 1 (VvPR1) gene by directly binding its promoter, indicating that VvRAV1 may function in the regulation of host basal defense responses. p24 hijacks VvRAV1 to the cytoplasm and employs the protein to sequester 21-nt double-stranded siRNA together, thereby enhancing its own RSS activity. Moreover, p24 enters the nucleus via interaction with VvRAV1 and weakens the latter's binding affinity to the VvPR1 promoter, leading to decreased expression of VvPR1. Our results provide a mechanism by which a viral RSS interferes with both the antiviral RNA silencing and the AP2/ERF-mediated defense responses via the targeting of one specific host factor.

Epidemiological Survey of Grapevine Leafroll-Associated Virus 1 and 3 in Sicily (Italy): Genetic Structure and Molecular Variability

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.

Small RNA Sequencing and Multiplex RT-PCR for Diagnostics of Grapevine Viruses and Virus-like Organisms

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.

Developing a mealybug pheromone monitoring tool to enhance IPM practices in New Zealand vineyards

Mealybugs are phloem-feeding insects found on many crops worldwide. In New Zealand vineyards, they transmit the economically important Grapevine leafroll-associated virus 3 (GLRaV-3). For some mealybug species, synthetic sex pheromones have been commercialised, and are used as monitoring tools. The mealybugs Pseudococcus longispinus and Pseudococcus calceolariae are major pests in many New Zealand vineyards. We present work on the development of a combined P. longispinus and P. calceolariae pheromone lure. The optimal dose for monitoring P. longispinus was found to be 10 µg of the (S)-(+)-enantiomer, either alone or in the racemic mixture. Addition of the corresponding alcohol did not improve trap catch of P. longispinus. Both the P. longispinus and the P. calceolariae pheromone lures remained active in the field for 90 days. Combining the 2 species’ pheromones had no negative effects on male mealybug trap catch for either species. We conclude that the pheromone ester alone is the best lure for the male P. longispinus. Combining the two mealybug species’ pheromones into a single lure provides the New Zealand viticultural industry with an efficient monitoring tool. Late-vintage deployment of baited lures will provide information on mealybug abundance and local distribution that will inform the scope of future insecticide programmes, to target areas based on need rather than an area-wide application by default.