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The first complete genome sequence of a little cherry virus-2 (LChV-2-TA) isolate from China was determined using small RNA deep sequencing combined with overlapping reverse transcriptase PCR (RT-PCR). Phylogenetic analysis revealed that LChV-2-TA grouped in a well-supported cluster with members of the genus Ampelovirus with close relationships to previously reported LChV-2 isolates.

The spatial distributions of grape plants with symptoms of grapevine leafroll in nine vineyards in the Galician wine-growing region Rías Baixas (northwestern Spain) were analyzed. In five, significant aggregations of diseased plants and/or disease gradients toward one of the borders of the plots constituted indirect evidence of vector transmission by a nonflying vector. From 1991 to 1995, vines from two vineyards were tested for grapevine leafroll associated virus 3 (GLRaV-3) by enzyme-linked immunosorbent assay. In one of these vineyards, which had been virus-free when planted, GLRaV-3 was detected 3 years after planting. In the second, the incidence of GLRaV-3 increased from 33 to 83% between 1991 and 1995. The mealybug Planococcus citri was associated with the infected plants in this vineyard. GLRaV-3 was present in P. citri, and under controlled conditions this mealybug transmitted GLRaV-3 to five of five previously healthy grape plants.

Grapevine leafroll is one of the most widespread and economically relevant viral diseases of grapevines. At least nine distinct Grapevine leafroll-associated viruses (GLRaVs), all members of the Closteroviridae family, have been associated with this disease in grapevine. Grapevine leafroll-associated virus 4 (GLRaV-4), currently classified as a Closteroviridae member under the Ampelovirus genus, was initially described in California. To determine if GLRaV-4 was present in Chilean grapevines, in addition to the previously reported GLRaV-1, -2, -3, -7, and -9 (1,2), 35 dormant cane samples from 12 different cultivars were collected from different regions of Chile and screened by reverse transcription-PCR. Two of the 35 samples (both cv. Thompson Seedless) collected from the III and VI regions of Chile were found to be infected with GLRaV-4 using two different pairs of GLRaV-4 specific primers. The first pair of primers, HSPV-F: 5′- ACA TTC TCC ACC TTG TGC TTT T -3′ and HSPC-R: 5′- CAT ACA AGC GAG TGC AAT TAC -3′ (3), was used to amplify a 321-bp fragment corresponding to a partial region of the HSP70h gene. The sequence (GenBank Accession Nos. EU746618 and EU746619) from both positive samples shared 98.4% nucleotide identity and approximately 99% identity with the corresponding fragment of a Californian GLRaV-4 isolate (GenBank Accession No. AF039553). Since there are no commercial antibodies available for GLRaV-4 detection, a second pair of primers, LR4CPINT-F: 5′- GAG AGT GAC AAG CAC CAG GTG C -3′ and LR4CPFIN-R: 5′- TCA CCT CCT GTT GCC CA -3′ (4), that amplified a 492-bp fragment of the coat protein gene was also used. The sequences of the 492-bp fragment from both Chilean samples (GenBank Accession Nos. EU746620 and EU746621) shared 99.6% nucleotide identity with one another and had 96.5% identity with an Israeli GLRaV-4 isolate (GenBank Accession No. AM176759). To our knowledge, this is the first report of GLRaV-4 in Chile. Further studies will help to establish the effects and incidence of this virus in Chilean grapevines. References: (1) E. Engel et al. Plant Dis. 92:1252, 2008 (2) N. Fiore et al. J. Plant Pathol. 90:125, 2008. (3) F. Osman et al. J. Virol. Methods 141:22, 2007. (4) P. Saldarelli et al. J. Plant Pathol. 88:203, 2006.

Grapevine leafroll associated virus-3 (GLRaV-3) and Grapevine leafroll associated virus-5 (GLRaV-5), two members of the genus Ampelovirus associated with grapevine leafroll disease, were transmitted by the mealybug Planococcus ficus and the soft scale insect Ceroplastes rusci from infected to healthy vines under experimental conditions. The efficiencies of transmission of GLRaV-3 and GLRaV-5 by P. ficus were 23.3 and 8.3%, respectively, and by C. rusci were 3.3 and 1.7%, respectively. Juvenile instars of P. ficus were more efficient in transmission of the viruses than adult females. This is the first report of the ability of C. rusci to transmit these viruses to grapevines.

Roger's Red, an interspecific hybrid between wild grape (Vitis californica, native to northern California) and the V. vinifera cv. Alicante Bouschet (1), and Claret Vine (V. vinifera cv. Purpurea Nana) are grown for their ornamental value in home gardens and other settings. We collected potted grapevines of Roger's Red and Claret Vine showing dull green-to-scarlet red leaves from two different retail nurseries in the Richland-Kennewick area and Prosser, WA, respectively. Since these symptoms ‘mimic’ grapevine leafroll disease, we tested petiole samples from four grapevines per cultivar for a panel of grapevine-infecting viruses by single-tube one-step reverse transcription (RT)-PCR (4). All samples tested positive only for Grapevine leafroll-associated virus 1 (GLRaV-1; genus Ampelovirus, family Closteroviridae). To further confirm these results, total RNA was subjected to RT-PCR to amplify a portion of the heat shock protein 70 homolog (HSP70h), coat protein duplicate 2 (CPd2), and ORF 9 (p24) of GLRaV-1. RT was performed at 52°C for 60 min, followed by 35 cycles of PCR (30 s denaturation at 94°C, 45 s annealing at 55°C, and 30 s extension at 72°C) and a 5 min final extension step at 72°C. Primers specific to HSP70h (HSP70h/416F: 5′-CAGGCGTCGTTTGTACTGTG and HSP70h/955R: 5′-TCGGACAGCGTTTAAGTTCC), CPd2 (CPd2/F: 5′-GTTACGGCCCTTTGTTTATTATGG and CPd2/R: 5′-CGACCCCTTTATTGTTTGAGTATG) and ORF 9 (p24/F: 5′-CGATGGCGTCACTTATACCTAAG and p24/R (5′-CACACCAAATTGCTAGCGATAGC) were designed based on GLRaV-1 sequence (GenBank Accession No. AF195822) to amplify 540, 398, and 633 base pair (bp) DNA fragments, respectively. To verify that the amplified products were specific to the genome of GLRaV-1, the amplicons were cloned into pCR2.1 vector (Invitrogen Corp, Carlsbad, CA) and three independent clones for each amplicon were sequenced in both directions. Pairwise comparison of HSP70h (Accession Nos. HQ833472 and HQ833473), CPd2 (Accession Nos. HQ833474 and HQ833475), and p24 (Accession Nos. HQ833476 and HQ833477) sequences from Roger's Red and Claret Vine showed 100, 96, and 99% identities, respectively, between them, and 86 to 100, 80 to 97, and 86 to 90% nucleotide sequence identities, respectively, with corresponding sequences of GLRaV-1 isolates deposited in GenBank. We further confirmed the presence of GLRaV-1 in these two ornamental grape cultivars by double antibody sandwich-ELISA using commercially available antibodies (Bioreba AG, Reinach, Germany). Previous studies have reported the presence of GLRaV-2 and -3 (1,3) and Grapevine virus A and B (2,3) in Roger's Red. To our knowledge, this study represents the first report of the occurrence of GLRaV-1 in two Vitis species distributed as ornamental grapes. It is important to prevent virus spread via the supply of virus-tested ornamental grapevines by commercial nurseries. References: (1) G. S. Dangl et al. Am. J. Enol. Vitic. 61:266, 2010. (2) D. A. Golino et al. Phytopathology (Abstr.) 99(suppl.):S44, 2009. (3) V. Klaassen et al. Online publication. doi:10.1094/PDIS-09-10-0621. Plant Dis., 2011. (4) T. A. Mekuria et al. Phytopathology (Abstr.) 99(suppl.):S83, 2009.

Grapevine leafroll ampeloviruses have been recently grouped into two major clades, one for Grapevine leafroll associated virus (GLRaV) 1 and 3 and another one grouping GLRaV-4 and its variants. In order to understand biological factors mediating differential ampelovirus incidences in vineyards, quantitative real-time polymerase chain reactions were performed to assess virus populations in three grapevine varieties in which different infection status were detected: GLRaV-3 + GLRaV-4, GLRaV-3 + GLRaV-4 strain 5, and GLRaV-4 alone. Specific primers based on the RNA-dependent RNA polymerase (RdRp) domains of GLRaV-3, GLRaV-4, and GLRaV-4 strain 5 were used. Absolute and relative quantitations of the three viruses were achieved by normalization of data to the concentration of the endogenous gene actin. In spring, the populations of GLRaV-4 and GLRaV-4 strain 5 were 1.7 × 104 to 5.0 × 105 genomic RNA copies/mg of petiole tissue whereas, for GLRaV-3, values were significantly higher, ranging from 5.6 × 105 and 1.0 × 107 copies mg–1. In autumn, GLRaV-4 and GLRaV-4 strain 5 populations increased significantly, displaying values for genome copies between 4.1 × 105 and 6.3 × 106 copies mg–1, whereas GLRaV-3 populations displayed a less pronounced boost but were still significantly higher, ranging from 4.1 × 106 to 1.6 × 107 copies mg–1. To investigate whether additional viruses may interfere in the quantifications the small RNA populations, vines were analyzed by Ion Torrent high-throughput sequencing. It allowed the identification of additional viruses and viroids, including Grapevine virus A, Hop stunt viroid, Grapevine yellow speckle viroid 1, and Australian grapevine viroid. The significance of these findings is discussed.

Pistachio (Pistacia vera L.) trees from the National Clonal Germplasm Repository (NCGR) and orchards in California were surveyed for viruses and virus-like agents by high-throughput sequencing (HTS). Analyses of sequence information from 60 trees identified a novel virus, provisionally named “Pistachio ampelovirus A” (PAVA), in the NCGR that showed low amino acid sequence identity (approximately 42%) compared with members of the genus Ampelovirus (family Closteroviridae). A putative viroid, provisionally named “Citrus bark cracking viroid-pistachio” (CBCVd-pis), was also found in the NCGR and showed approximately 87% similarity to Citrus bark cracking viroid (CBCVd, genus Cocadviroid, family Pospiviroidae). Both PAVA and CBCVd-pis were graft transmissible to healthy UCB-1 hybrid rootstock seedlings (P. atlantica × P. integerrima). A field survey of 123 trees from commercial orchards found no incidence of PAVA but five (4%) samples were infected with CBCVd-pis. Of 675 NCGR trees, 16 (2.3%) were positive for PAVA and 172 (25.4%) were positive for CBCVd-pis by reverse-transcription polymerase chain reaction. Additionally, several contigs across multiple samples exhibited significant sequence similarity to a number of other plant virus species in different families. These findings require further study and confirmation. This study establishes the occurrence of viral and viroid populations infecting pistachio trees.

Grapevine leafroll-associated virus-3 (GLRaV-3) is a major constraint on profitable grapevine cultivation. The virus is transmitted efficiently by mealybugs and soft scale insects, or through vegetative propagation by cuttings, and is present worldwide, wherever grapevines are grown. GLRaV-3 exists as a complex of genetic variants currently classified in several phylogenetic groups that can differ from each other by as much as 30% in nucleotide sequence of the whole genome. In the course of the GLRaV-3 testing of wine grapes in southern Idaho, plants of two grapevine cultivars were found to harbor a novel genetic variant of GLRaV-3, named ID45, which exhibited ≤80% nucleotide sequence identity level to the known GLRaV-3 isolates in its most conserved HSP70h gene. The ID45 variant caused no foliar symptoms in ‘Cabernet Sauvignon’ in the fall, and was demonstrated to have poor reactivity to commercial virus-specific antibodies. The entire 18,478-nt genome sequence of the GLRaV-3-ID45 was determined using a combination of high-throughput and conventional Sanger sequencing, and demonstrated to have typical organization for the genus Ampelovirus (family Closteroviridae), with only 70 to 77% identity level to the GLRaV-3 genomes from other established phylogroups. We concluded that ID45 represented a new phylogenetic group IX of GLRaV-3. Database search using ID45 nucleotide sequence as a query suggested that this novel ID45 variant is present in at least one other grape-growing state in the U.S., California, and in Brazil. An RT-PCR based test was developed to distinguish ID45 from the predominant GLRaV-3 phylogroup I found in Idaho in single and mixed infections.

The history is outlined of the steps that, starting from the establishment of the "taxonomic group Closterovirus," have brought to the erection of the family Closteroviridae, a taxon comprising plant viruses that possess very long helically constructed filamentous particles and a positive-sense single-stranded, monopartite or bipartite RNA genome and are transmitted either by aphids (genus Closterovirus), pseudococcid mealybugs/soft scale insects (genus Vitivirus), or whiteflies (genus Crinivirus) or have no known vector (genus Velarivirus).

Mealybug wilt of pineapple (MWP) is a disease of pineapple that has a long history in Hawaii, but is present throughout the world where pineapples are grown in tropical regions. The disease has an interesting etiology that is poorly understood but involves an association with virus particles, mealybug vectors, and ants which spread the mealybug vectors. Several distinct pineapple mealybug wilt-associated virus (PMWaV) species have been identified thus far with potential further member species yet to be characterized. Pineapple mealybug wilt-associated viruses are member species of the Ampelovirus genus of the Closteroviridae family. Ampeloviruses are split into two subgroups, subgroup I and subgroup II. PMWaV-2 is a subgroup II member, and these have a longer and more complex genome with additional genes on the 3’ terminus of the RNA genome compared to subgroup I ampeloviruses. PMWaV-2, along with the presence of mealybug vectors, have been shown to be necessary factors in symptom development in Hawaii. Some of these extra genes in the 3’ of PMWaV-2 have recently been shown to function as silencing suppressors, and may play a role in the virulence of PMWaV-2 and symptom development. In other regions of the world, reports of symptomatic plants without PMWaV-2 infection, but with PMWaV-1, -3 or some combination, contradict the requirement of PMWaV-2 for symptom development in MWP. It is possible that further, uncharacterized PMWaVs may be present in symptomatic pineapple plants that test negative for PMWaV-2, explaining the inconsistency in symptom development. More research is necessary to explore the confusing etiology of the MWP disease, and to perhaps shed light upon the symptom development.

International audience; P70 is a Pinot Noir grapevine accession that displays strong leafroll disease symptoms. A high-throughput sequencing (HTS)-based analysis established that P70 was mixed-infected by two variants of grapevine leafroll-associated virus 1 (GLRaV-1, genus Ampelovirus) and one of grapevine virus A (GVA, genus Vitivirus) as well as by two viroids (hop stunt viroid [HSVd] and grapevine yellow speckle viroid 1 [GYSVd1]) and four variants of grapevine rupestris stem pitting-associated virus (GRSPaV). Immunogold labelling using gold particles of two different diameters revealed the existence of 'hybrid' particles labelled at one end as GLRaV-1, with the rest labelled as GVA. In this work, we suggest that immunogold labelling can provide information about the biology of the viruses, going deeper than just genomic information provided by HTS, from which no recombinant or 'chimeric' GLRaV-1/GVA sequences had been identified in the dataset. Our observations suggest an unknown interaction between members of two different viral species that are often encountered together in a single grapevine, highlighting potential consequences in the vector biology and epidemiology of leafroll and rugose-wood diseases.

The number of virus species infecting grapevine has increased continuously and more than 60 species are described to infect grapevine. The global expansion of the grape and wine industry has seen a parallel increase in the incidence and economic impact of grapevine leafroll disease (GLD). GLD can cause reduced plant vigor and longevity, and significant losses in both fruit yield and quality. Our knowledge of the nature of the disease is still quite limited due to a variety of challenges related to the complexity of this virus disease, the association of several genetically and serologically distinct GLRaVs, and contrasting symptoms in red- and white-berried cultivars. Within grapevine leafroll complex (Grapevine leafroll-associated viruses, GLRaVs) five different species are classified in the family Closteroviridae: one is classified in the genus Closterovirus (GLRaV-2), three in the genus Ampelovirus (GLRaV-1, GLRaV -3, GLRaV -4, and his strains: GLRaV-4 strain 5, GLRaV-4 strain 6, GLRaV-4 strain 9, GLRaV-4 strain Pr, GLRaV-4 strain De and GLRaV-4 strain Car) and one in the genus Velarivirus (GLRaV-7). GLRaVs are transmitted by grafting and propagation material (rootstock and scion) and some species by mealybugs. Grapevine leafroll-associated viruses usually occur in a mixed infection, and are often present in a mixed infection with other grape viruses. Early research carried out in vineyards in Serbia determined the presence of GLRV-1, -2 and -3, with GLRaV-3 being the most prevalent. If spread of the viruses transsmited by vectors within plantings exists, roguing or removal of infected vines, mealybug control, and sanitation measures can reduce new infections. Brojnost virusa infektivnih za vinovu lozu stalno se povećava, tako da je do sada opisano više od 60 vrsta koji mogu da zaraze vinovu lozu. Paralelno sa globalnom ekspanzijom vinogradarske i vinarske industrije zapaženo je povećanje učestalosti i ekonomskog značaja oboljenja uvijenosti lišća vinove loze (Grapevine Leafroll Disease, GLD). GLD, osim što smanjuje vitalnost i dugovečnost vinove loze, značajno smanjuje prinos i kvalitet grožđa. Epidemiologija oboljenja je nedovoljno istražena zbog kompleksnosti ove bolesti, genetički i serološki različitih vrsta unutar kompleksa i različitosti simptoma kod crvenih i belih sorti vinove loze. Кompleks virusa prouzrokovača uvijenosti lišća vinove loze (Grapevine leafrollassociated viruses, GLRaVs) čini pet različitih virusa koji pripadaju familiji Closteroviridae: jedna vrsta koja pripada rodu Closterovirus (GLRaV-2), tri rodu Ampelovirus (GLRaV-1, GLRaV -3, GLRaV -4, i njegovi sojevi: GLRaV-4 soj 5, GLRaV-4 soj 6, GLRaV-4 soj 9, GLRaV-4 soj Pr, GLRaV-4 soj De i GLRaV-4 soj Car) i jedna vrsta iz roda Velarivirus (GLRaV-7). GLRaVs se prenose kalemljenjem i propagativnim materijalom (podloga i vijoka), a neke vrste i štitastim vašima. Virusi koji izazivaju uvijenost lišća se obično javljaju u mešanoj infekciji, a često su prisutni i u mešanoj infekciji sa drugim virusima vinove loze. Ranija istraživanja sprovedena u vinogorjima u Srbiji utvrdila su prisustvo GLRV-1, -2 i -3, pri čemu je GLRaV-3 bio najzastupljeniji. Ukoliko je širenje virusa unutar zasada putem vektora izraženo, krčenje ili uklanjanje zaraženih čokota, kontrola štitastih vaši i adekvatne sanitarne mere mogu redukovati nove infekcije.

Grapevine leafroll is the most complex and intriguing viral disease of grapevine (Vitis spp.). Several monopartite closteroviruses (family Closteroviridae) from grapevines have been molecularly characterized, yet their role in disease etiology is not completely resolved. Hence, these viruses are currently designated under the umbrella term of Grapevine leafroll–associated viruses (GLRaVs). This review examines our current understanding of the genetically divergent GLRaVs and highlights the emerging picture of several unique aspects of the leafroll disease pathosystem. A systems biology approach using contemporary technologies in molecular biology, -omics, and cell biology aids in exploring the comparative molecular biology of GLRaVs and deciphering the complex network of host-virus-vector interactions to bridge the gap between genomics and phenomics of leafroll disease. In addition, grapevine-infecting closteroviruses have a great potential as designer viruses to pursue functional genomics and for the rational design of novel disease intervention strategies in this agriculturally important perennial fruit crop.

Grapevine leafroll-associated virus 3 (GLRaV-3; Ampelovirus, Closteroviridae) isolates from Galicia in northwestern Spain were selected to characterize their genetic diversity according to different factors (age, origin, location, variety, etc.). The vines belonged either to local white and red varieties autochthonous to Galicia or to varieties from other Spanish regions but widely used in Galicia. These GLRaV-3 isolates came from different vineyards in Galicia located in coastal or inner areas. Multiplex RT-PCR allowed the detection of isolates belonging to groups I, II, III-V and VI. Two genomic regions were studied in the isolates, the HSP70h and the capsid protein, using specific primers that allow the detection of variants from groups I to V. Some possible recombinants could be detected; however, multiple infections with different variants indicated that they were not genuine recombinants. No differences were found in the population structure considering variety or geographical factors. Isolates belonging to four groups were found in the distinct areas surveyed: groups I and II were the most common, followed by groups VI and III, as is the case in the rest of the world. In the same surveys, the presence of insect vectors for GLRaV-3 was investigated and found lacking in inland areas but present in those with milder climate. Genetic analysis did not support isolation of the GLRaV-3 isolates in Galicia, suggesting that the uncontrolled exchange of infected vines and/or rootstocks has been a major agent of virus spread.

A robust method for removing all microorganisms from infected tissue is important for cultivar imports, germplasm maintenance and to produce healthy grafted material for the grapevine industry. In the droplet vitrification method of cryopreservation, plant tissue pre-treated with a vitrification solution is placed on aluminium foil in a droplet of vitrification solution and directly immersed in liquid nitrogen. Only highly cytoplasmic, non-vacuolar meristematic cells survive freezing. Therefore, cryopreservation can be considered a precise method of meristem culture and is developing into a new method for virus eradication in horticultural species called cryotherapy. To test the suitability of cryotherapy for virus eradication, we used ‘Chardonnay’ and ‘Lakemont Seedless’ infected with Grapevine leafroll associated virus-3 (GLRaV-3, an Ampelovirus), ‘Pinot gris’ and ‘Sauvignon blanc 316’ infected with Grapevine leafroll associated virus-2 (a Closterovirus), and another clone of ‘Sauvignon blanc’ infected with both Grapevine leafroll associated virus-1 (an Ampelovirus) and GLRaV-3. Plants regenerated after cryo-treatment tested negative (DAS-ELISA) for all three viruses, whereas untreated control plants tested positive. Droplet vitrification has the potential to be a novel and precise tool for virus eradication and establishment of high-health grapevine germplasm collections. In addition to removing virus infections, the method provides a cost-effective way of maintaining clonal plant material.

Despite being the first closterovirus documented in grapevines (Vitis sp.), the molecular biology of Grapevine leafroll-associated virus 1 (GLRaV-1, genus Ampelovirus, family Closteroviridae) is still in its infancy. In this study, the complete genome sequence of two GLRaV-1 isolates was determined to be 18,731 (isolate WA-CH) and 18,946 (isolate WA-PN) nucleotides (nt). The genome of WA-CH and WA-PN isolates encodes nine putative open reading frames (ORFs) and the arrangement of these ORFs in both isolates was similar to that of Australian and Canadian isolates. In addition to two divergent copies of the coat protein (CP), the genome of GLRaV-1 isolates contain CP-homologous domain in four genes, making the virus unique among Closteroviridae members. The 5′ and 3′ nontranslated regions (NTRs) of WA-CH and WA-PN isolates showed differences in size and sequence composition, with 5′ NTR having variable number of ∼65-nt-long repeats. Using the 5′ NTR sequences, a reverse transcription-polymerase chain reaction and restriction fragment length polymorphism method was developed to distinguish GLRaV-1 variants in vineyards. Northern analysis of total RNA from GLRaV-1-infected grapevine samples revealed three subgenomic RNAs (sgRNAs), corresponding tentatively to CP, p21, and p24 ORFs, present at higher levels, with p24 sgRNA observed at relatively higher abundance than the other two sgRNAs. The 5′ terminus of sgRNAs corresponding to CP, CPd1, CPd2, p21, and p24 were mapped to the virus genome and the leader sequence for these five sgRNAs determined to be 68, 27, 15, 49, and 18 nt, respectively. Taken together, this study provided a foundation for further elucidation of the comparative molecular biology of closteroviruses infecting grapevines.

UMR BFP - Equipe Virologie; [i]Sugarcane mild mosaic virus[/i]: towards the full characterization of this sugarcane Ampelovirus re-discovered using metagenomics-based approaches. 16. Rencontres de Virologie Végétale (RVV 2017)

Ampelovirus and Vitivirus relationships in grapevine. 16e Rencontres de Virologie Végétale

BFP - Equipe Virologie; First Report of Plum bark necrosis stem pitting-associated virus Infecting Flowering Cherry in Japan

Ampeloviruses (family Closteroviridae) are filamentous monopartite, single-stranded, positive-sense RNA genome. They are transmitted by mealybugs in semi-persistent manner and vegetative propagating material remains the major route of spread. Ampeloviruses are recent addition to the plant viruses in India. Grapevine leafroll-associated virus 3 (GLRaV-3) was first ampelovirus to be recorded from India in the year 2012. Of the nine distinct species of the genus Ampelovirus, only three, Grapevine leafroll-associated virus 1 (GLRaV-1), GLRaV-3, GLRaV-4 infecting grapevine have been reported from India. The isolates of GLRaV-3 and GLRaV-4 are diverse, a few being the recombinant ones. This chapter describes the grapevine leafroll disease caused by different ampeloviruses, their geographical distribution, characterization, diversity, management strategies and also discusses about the future course of works to be taken.

Grapevine is one of the plant species most susceptible to virus infections, which cause several complex diseases. The effects of viruses on grapevine performances are generally accepted as potentially severe, although an exact estimation is difficult because of the complexity of the factors influencing vine response (mixed infections, viral strain, environment, grape cultivar and rootstock, vineyard management, etc.). However, diseases like infectious degenerations caused by nepoviruses are clearly detrimental and dramatically affect plant vigor and yield. More controversial is the effect of members of the genera Ampelovirus, Closterovirus and Vitivirus, the causative agents of leafroll and rugose wood. In these cases, infected vines usually bear an adequate crop, so that growers are unaware of the real damage, especially to qualitative parameters. Sanitized vines usually present a more luxuriant growth and increased yield; therefore, cultivation practices (i.e., green pruning, bunch thinning, wider spacing, etc.) should be adjusted to cope with these enhanced performances. Grapevines are also affected by “minor” virus diseases (i.e., fleck, vein mosaic, rupestris stem pitting, etc.), whose impact is still unclear. Nevertheless, their presence should not be overlooked, because synergistic negative effects of their agents with other major viruses cannot be excluded. Viticulture currently faces new emerging virus diseases, such as red blotch (grapevine red blotch-associated virus) and leaf mottling and deformation (Grapevine Pinot gris virus), which threaten the profitability of the grape and wine industry. Viruses are dangerous and elusive pathogens whose presence in vines must be prevented by using clean propagation material. The new -omics technologies are expected to provide more information on plant-virus interactions.

Grapevine leafroll-associated virus 4 is a recognized species in the genus Ampelovirus, family Closteroviridae, which comprises several genetically diverse variants denominated GLRaV-4 strains 4, 5, 6, 9, De, Car, Pr, and Ob. They are collectively referred to as “grapevine leafroll-associated virus 4-like viruses” (or GLRaV-4LVs). These viruses have been found associated with leafroll disease of grapevine, their sole host known to date. They are characterized by flexuous virions up to 1800 nm in length and a genome made up of a non-polyadenylated, single-stranded, positive-sense RNA molecule of 13.6–13.8 kb in size, depending on the particular strain/molecular variant. In spite of considerable differences in nucleotide content between molecular variants, the genome of all GLRaV-4LVs consists of seven open reading frames (ORFs), flanked by relatively short 5′ and 3′ untranslated regions (UTRs), respectively, that encodes six proteins (in the 5–3′ direction): the replication-associated polyprotein (expressed via +1 ribosomal frameshift of two partially overlapping ORFs), a small hydrophobic protein (p5), the HSP70 homologue (HSP70h), the 60K protein (p60), the viral coat protein (CP), and a protein of unknown function with a molecular mass of 23K (p23). GLRaV-4LVs are phloem-limited, nonmechanically transmissible, and distributed worldwide. Several mealybug and soft scale insect species mediate their short distance spread, while long-distance dissemination occurs primarily through infected propagating material.

Plum bark necrosis stem pitting-associated virus (PBNSPaV), the causal agent of plum bark necrosis stem pitting disease, belongs to the genus Ampelovirus in the family Closteroviridae. The complete genome sequence of PBNSPaV isolates from four Prunus sources was determined by pyrosequencing. All isolates showed the same genomic organization as the PBNSPaV reference isolate. The least divergent isolate, found in a peach tree from China, showed an overall 91.8% of nucleotide identity with the type isolate. Two closely related isolates, defining a second cluster of diversity, were found in two Japanese plum lines from France and showed only 82.8% identity with the type isolate. On the other hand, they were highly homologous with two recently described PBNSPaV divergent isolates from China. The fourth and most divergent isolate, from a Chinese peach, showed only 71.2% identity to other PBNSPaV isolates and was not detected by currently available PBNSPaV reverse-transcription polymerase chain reaction detection assays. Complete sequencing of the divergent isolates allowed the development of a more broad-spectrum detection test targeting a conserved region in the P61 gene. Taken together, these results indicate a much broader diversity of PBNSPaV than previously thought and provide for a more robust detection of this still poorly characterized pathogen.

Mealybug wilt of pineapple (MWP) is a destructive viral-associated disease affecting pineapple (Ananas comosus) production in many growing regions worldwide, including Ghana (Sarpong et al., 2017; Dey et al., 2018). In Hawaii, MWP…

Little cherry disease, caused by little cherry virus 1 (LChV-1) and little cherry virus 2 (LChV-2), which are both members of the family Closteroviridae, severely affects sweet (Prunus avium L.) and sour cherry (P. cerasus L.) orchards lifelong production worldwide. An intensive survey was conducted across different geographic regions of Belgium to study the disease presence on these perennial woody plants and related species. Symptomatic as well as non-symptomatic Prunus spp. trees tested positive via RT-PCR for LChV-1 and -2 in single or mixed infections, with a slightly higher incidence for LChV-1. Both viruses were widespread and highly prevalent in nearly all Prunus production areas as well as in private gardens and urban lane trees. The genetic diversity of Belgian LChV-1 and -2 isolates was assessed by Sanger sequencing of partial genomic regions. A total RNA High-Throughput Sequencing (HTS) approach confirmed the presence of both viruses, and revealed the occurrence of other Prunus-associated viruses, namely cherry virus A (CVA), prune dwarf virus (PDV) and prunus virus F (PrVF). The phylogenetic inference from full-length genomes revealed well-defined evolutionary phylogroups with high genetic variability and diversity for LChV-1 and LChV-2 Belgian isolates, yet with little or no correlation with planting area or cultivated varieties. The global diversity and the prevalence in horticultural areas of LChV-1 and -2 variants, in association with other recently described fruit tree viruses, are of particular concern. Future epidemiological implications as well as new investigation avenues are exhaustively discussed.

During a survey conducted in vineyards in Slovenia, variety of grapevine leafroll disease symptoms were observed. Mixed infection with grapevine leafroll-associated viruses 3 and 4 (GLRaV-3, -4) in two grapevines from a vineyard in south-western part of Slovenia was confirmed by DAS-ELISA in 2010. The 3'final 1769 nucleotides of the Slovenian GLRaV-4 isolate were assembled from amplicons obtained by IC RT-PCR. The complete coat protein (CP) and p23 gene sequences were compared with other GLRaV-4 sequences from GenBank. Results showed that CP and p23 amino acid sequences of Slovenian variant (055-SI) are 88% and 85%, respectively, identical to corresponding genes of reference sequence GLRaV-4 LR106 (GenBank Acc. No. FJ467503). Phylogenetic analyses show that Slovenian variant clusters together with other corresponding strains of GLRaV-4. The sequencing results show great variability of the N-terminal part of the CP sequence indicating that this part of the genome is not suitable for molecular detection of the virus. To our knowledge this is also the first report of GLRaV-4 in Slovenian vineyards.

Grapevine fleck, rugose wood and leafroll are three grapevine viral diseases whose causal agents (or associated viruses) respectively are Grapevine fleck virus (GFkV), Grapevine virus D (GVD) and Grapevine leafroll-associated virus 5 and 6 (GLRaV-5 and -6). The objective of this work was to perform a partial molecular characterization of local isolates of these four viral species that infect grapevines. The nucleotide and deduced amino acid sequences of complete genes of the coat protein (CP) (of GFkV), the CP and the RNA binding protein (of GVD), the CP and the partial hHSP70 gene (of GLRaV-5) and the partial hHSP70 gene (of GLRaV-6) were aligned and compared in silico with other isolates. These data extend the available information about Brazilian isolates of GFkV, GLRaV-5 and -6, and reports for the first time the GVD occurrence in Brazil. Mancha das nervuras, lenho rugoso e enrolamento das folhas são três doenças virais da videira, cujos agentes causais (ou vírus associados) são o Grapevine fleck virus (GFkV), o Grapevine virus D (GVD) e os Grapevine leafroll-associated virus 5 e 6 (GLRaV-5 e -6), respectivamente. O objetivo deste trabalho foi realizar a caracterização molecular parcial de isolados locais dessas quatro espécies virais que infectam videira. As sequências de nucleotídeos e de aminoácidos deduzidos dos genes completos da proteína capsidial (CP) (do GFkV), da CP e da RNA binding protein (do GVD), da CP do GLRaV-5 e parte do gene codificador da hHSP70 do GLRaV-5 e -6 foram alinhadas e comparadas in silico com sequências de outros isolados. Os dados obtidos expandem a informação existente sobre isolados brasileiros de GFkV, GLRaV-5 e - 6 e relatam pela primeira vez a ocorrência do GVD no Brasil.

International audience; Spread patterns of a Grapevine leafrollassociated virus 1 (GLRaV-1) epidemic and a mealybug infestation survey over 10 year were recorded in two Burgundy French vineyards to investigate the relation between them. The temporal evolution of leafroll spread at both study sites was compared on disease incidence data with logisticregression models. We first tested if the spatial distribution of the disease and the mealybug were aggregated using permutation methods, then we tested the independence between the two spatial patterns by randomly shifting one pattern. In Bonzon, an increase from 5 % to 86 % of leafroll prevalence was observed over an 8-year time span, whereas leafroll prevalence remained stable around 5 % in Marsannay-la-Côte during the same period. In Bonzon, the disease spread rapidly from older neighbouring vineyards in four main patches while no spread of the disease was recorded from infected vines in Marsannay-la-Côte. The mealybug Phenacoccus aceris was recorded on 74 % of vines in Bonzon throughout the study and only 6 % of vines in Marsannay-la-Côte. In the latter location, the disease was not associated with the presence of the mealybug, so that it may have arisen from infected plant material escaping the sanitary inspection. In onzon, the significant statistical correlation between the mealybug distribution and diseased plants suggests that P. aceris was responsible for the rapid spread of GLRaV-1 in the vineyard. This is the first report of GLRaV-1 natural spread in Europe

Grapevine leafroll disease (GLRD) is one of the most economically important virus diseases of grapevine (Vitis spp.) worldwide. In this study, we used high-throughput sequencing of cDNA libraries made from small RNAs (sRNAs) to compare profiles of sRNA populations recovered from own-rooted Merlot grapevines with and without GLRD symptoms. The data revealed the presence of sRNAs specific to Grapevine leafroll-associated virus 3, Hop stunt viroid (HpSVd), Grapevine yellow speckle viroid 1 (GYSVd-1) and Grapevine yellow speckle viroid 2 (GYSVd-2) in symptomatic grapevines and sRNAs specific only to HpSVd, GYSVd-1 and GYSVd-2 in nonsymptomatic grapevines. In addition to 135 previously identified conserved microRNAs in grapevine (Vvi-miRs), we identified 10 novel and several candidate Vvi-miRs in both symptomatic and nonsymptomatic grapevine leaves based on the cloning of miRNA star sequences. Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) of selected conserved Vvi-miRs indicated that individual members of an miRNA family are differentially expressed in symptomatic and nonsymptomatic leaves. The high-resolution mapping of sRNAs specific to an ampelovirus and three viroids in mixed infections, the identification of novel Vvi-miRs and the modulation of certain conserved Vvi-miRs offers resources for the further elucidation of compatible host-pathogen interactions and for the provision of ecologically relevant information to better understand host-pathogen-environment interactions in a perennial fruit crop.

GLRaV-3, a member of the Closteroviridae family and type member of the genus Ampelovirus, is involved in the grapevine leafroll disease. Until now no RNA silencing suppressor has been found among viruses of this genus, contrary to what happens with a large number of other viral genera. In the sister genus Closterovirus, RNA silencing suppressors are present in the 3' end of the genome and have molecular weights close to 20 KDa. To test for RNA suppressing activity screening of p21, p19.6 and p19.7 proteins, coded for in an analogous genomic location of the GLRaV-3 was undertaken. Only p19.7 revealed suppressor activity demonstrated in diverse silencing inducing systems. This suppressor is able to overcome strong silencing inducers and shares several properties with the BYV p21-like family of suppressors of the closteroviruses. This is the first report of an RNA silencing suppressor in the genus Ampelovirus.

The grapevine leafroll-associated viruses (GLRaVs) (Closteroviridae) represent an emerging threat to world grape production. One group of GLRaVs within the genus Ampelovirus, the GLRaV-4-like viruses (GLRaV-4LVs), contains a fragmented collection of seven viruses only two of which (GLRaV-Pr and GLRaCV) are fully sequenced. Here in reporting the sequence of GLRaV-5, a member of GLRaV-4LVs, we identify genomic elements common to the GLRaV-4LV group. Exclusive properties include a highly conserved p5 gene product and phylogenies for complete genes that, except for the p23 gene, are reliably monophyletic. In comparison with other members of the genus Ampelovirus, GLRaV-4LVs form a tight cluster for all genes analyzed. In addition, they all possess a conserved AlkB domain which is most similar to the more distantly related GLRaV-3, suggesting recombination. In silico RNA structural analyses revealed a conserved five stem-loop structure at the 3' untranslated region that extends to all GLRaV-4LVs, and the ampeloviruses Pineapple mealybug wilt-associated virus 1 and Pineapple mealybug wilt-associated virus 3. A conserved G-U rich stem loop was also found upstream of the ORF1a stop and 1b start codons. Taken together, this work allows for a more thorough contextualization of GLRaV-5 and the GLRaV-4LVs as a group within the genus Ampelovirus.

Grapevine leafroll-associated virus 3 (GLRaV-3) is associated with grapevine leafroll disease, one of the most economically important viral diseases of grapevines. This disease impacts on both vine health and grape quality; reduction in yield, brix and wine colour are among its detrimental effects. Many methods, including serological and molecular procedures, have been developed for the detection of GLRaV-3; however, there is no PCR-based assay available to quantify virus populations within plant tissues. A real-time RT-PCR assay with TaqMan probe was developed for specific and reliable quantitative detection of GLRaV-3 in infected tissues. The designed primers and probes target the conserved sequence in the RNA-dependent RNA polymerase (RdRp) domain of the viral genome to prevent amplification of most subgenomic and defective RNAs. This protocol was used to examine the seasonal dynamics and translocation of GLRaV-3 in field-grown grapevines. The results showed that the virus spread quickly from trunks to new growing shoots and leaves early in the growing season, and most samples still harboured detectable virus during late summer and autumn. The seasonal progress of one GLRaV-3 isolate was compared in four grapevine cultivars (Chardonnay, Cabernet Sauvignon, Italia and Thompson Seedless). Within cultivars there was little variability in the distribution and translocation of GLRaV-3, except for in Thompson Seedless. This quantitative detection assay will be a valuable tool for GLRaV-3 diagnosis, disease monitoring and population ecology studies.

Pineapple mealybug wilt associated virus-2 (PMWaV-2) is a mealybug transmitted ampelovirus associated with mealybug wilt disease of pineapple (MWP) in Hawaii. The characteristics of PMWaV-2 acquisition and persistence of grey pineapple mealybugs (GPM), Dysmicoccus neobrevipes Beardsley, were measured. Some GPM were able to acquire the virus within 36 hours. Transmission efficiency of GPM in groups of 25 was 100% following a 72-hour acquisition access period (AAP). GPM remained viruliferous for up to three days after a 3-day AAP when serially transferred to virus-free pineapple plants at 24-hour intervals over a 7-day period. Access to a kabocha squash, a non-host of the virus, reduced the transmission efficiency and persistence of the virus. These data show that PMWaV-2 is transmitted by the grey pineapple mealybug in a semi-persistent manner. A potential management strategy based on these findings is discussed.

Plum bark necrosis stem pitting associated virus (PBNSPaV) has occurred globallyover the last two decades and is becoming one of the major agricultural issues ofstone fruits. The virus was detected for the first time in plums and has beenreported in the other stone fruit species. The agent, whose dissemination mode isstill unknown, has been first reported in cherries in Turkey grown in Malatyaprovince. In this study, the K1 isolate of PBNSPaV, identified in a sweet cherryplant with severe stem pitting and gumming symptoms on its trunk, was partiallycharacterised. A fragment of Hsp70h gene located on ORF3 of viral genome hasbeen cloned, sequenced and analysed phylogenetically (Accession number.FJ231498). The PBNSPaV-K1 isolate showed 93–96% nucleotide sequenceidentity to sequences of Italian and American isolates in databases. An RNAprobe has been raised for fast and reliable detection of the agent by molecularhybridisation. The studies on development of genome specific primers for thedetection of the isolate by one-tube RT-PCR have failed.

Wang, J., Sharma, A. M., Duffy, S., and Almeida, R. P. P. 2011. Genetic diversity in the 3′ terminal 4.7-kb region of Grapevine leafroll-associated virus 3. Phytopathology 101:445-450. Grapevine leafroll-associated virus 3 (GLRaV-3; Ampelovirus, Closteroviridae), associated with grapevine leafroll disease, is an important pathogen found across all major grape-growing regions of the world. The genetic diversity of GLRaV-3 in Napa Valley, CA, was studied by sequencing 4.7 kb in the 3′ terminal region of 50 isolates obtained from Vitis vinifera ‘Merlot’. GLRaV-3 isolates were subdivided into four distinct phylogenetic clades. No evidence of positive selection was observed in the data set, although neutral selection (ratio of nonsynonymous to synonymous substitution rates = 1.1) was observed in one open reading frame (ORF 11, p4). Additionally, the four clades had variable degrees of overall nucleotide diversity. Moreover, no geographical structure among isolates was observed, and isolates belonging to different phylogenetic clades were found in distinct vineyards, with one exception. Considered with the evidence of purifying selection (i.e., against deleterious mutations), these data indicate that the population of GLRaV-3 in Napa Valley is not expanding and its effective population size is not increasing. Furthermore, research on the biological characterization of GLRaV-3 strains might provide valuable insights on the biology of this species that may have epidemiological relevance.

Mealybugs (Hemiptera: Pseudococcidae) are sap-sucking insects which infest a broad range of crops worldwide. They represent an important threat to viticulture as they are vectors of viruses associated with leafroll and rugose wood complex diseases. In this study, we surveyed the presence of mealybugs and their associated viruses in vineyards of the Piemonte and Liguria regions, northwestern Italy. In order to determine the collected specimens correctly, we added a species-specific marker for Heliococcus bohemicus to an existing molecular identification key. The only species collected in Piemonte was H. bohemicus, whereas in Liguria, H. bohemicus, Planococcus ficus and Pseudococcus longispinus were found; Ps. longispinus has never before been reported in Italian vineyards. Several specimens of all three species were infected by the ampeloviruses GLRaV-1 and GLRaV-3 and the vitivirus GVA. Both nymphs and adult females tested positive for the viruses and mixed infections were commonly found within the same insect. Population levels and virus incidence were higher in Liguria than in Piemonte, suggesting a greater risk of disease spread. We conclude that the mild, Mediterranean climate of Liguria favors the development of a diverse mealybug fauna while only H. bohemicus, known to be tolerant to the severe continental winter temperatures, colonize grapevines in colder viticultural areas.

Epiphyllum spp. (orchid cactus) is a group of epiphytic plants in the cactus family grown as flowering ornamentals. Symptoms of virus infections are frequently observed in them (Chessin and Hammond 2002). In 2015, an Epiphyllum hybrid with chlorotic spots was found in California. Twelve more Epiphyllum hybrids (six with chlorotic ring spots, mottle, or mosaic and six asymptomatic) were obtained from a commercial nursery in New York. These plants were tested by RT-PCR/PCR using specific primers for Cactus mild mottle virus, Cactus virus X (CVX), Impatiens necrotic spot virus, Saguaro cactus virus, Zygocactus virus X (ZVX), or group primers for badnaviruses and potyviruses. Two potexviruses, CVX and ZVX, were detected from three and one symptomatic hybrids, respectively. The California hybrid and seven New York hybrids (two symptomatic and five asymptomatic) tested positive for a badnavirus. However, no target viruses were detected in two of the seven symptomatic hybrids. To further investigate these observations, total RNAs were extracted from stem tissue of four symptomatic hybrids (one CVX-positive, one ZVX-positive, and two PCR-negative plants) and one asymptomatic hybrid (cv. Professor Ebert) using RNeasy Plant Mini Kit (Qiagen). A pooled extract from the five samples was subjected to high-throughput RNA sequencing (SeqMatic). Analysis of the RNA reads revealed the presence of Alternanthera mosaic virus, Beet western yellows virus (BWYV), and five new viruses (one ampelovirus, one badnavirus, two distinct carlaviruses, and one caulimovirus) in addition to CVX and ZVX. Virus infections in all 13 cacti were further tested for the seven viruses (two known and five new viruses identified in this study) by RT-PCR/PCR using virus-specific primers. Results showed that the symptomless hybrid Professor Ebert was infected with five viruses (BWYV, an ampelovirus, two carlaviruses, and a caulimovirus), the most among the all hybrids. BWYV was detected by RT-PCR using primers BWYVf4972 (5′-TCTGAAGGTCGGTGAAGGAGG-3′) and BWYVr5665 (5′-ACAATGGGTAACACTCGTTGC-3′) only in Professor Ebert. To obtain the genomic sequence of this BWYV isolate, RT-PCR was conducted using seven overlapping primer pairs designed according to the alignment of the BWYV type species (GenBank no. NC_004756) and the contig obtained in this study. The RT-PCR amplicons were cloned into pGEM-T easy vector (Promega) and sequenced (MCLAB). A nearly full genomic sequence of 5,458 nt (MF543022) was assembled from seven overlapped amplicon sequences. BLAST search showed that this isolate had the highest nt sequence identity of 93% with a BWYV isolate infecting Nepenthes mirabilis (KU521325) (Miguel et al. 2016). Stem tips from the Professor Ebert plant were grafted onto four plants of hybrid King’s Ransom, which tested negative for the nine viruses included in this study. Three plants showed mild chlorotic speckling 6 months after grafting. BWYV was detected in new growth of the two grafted plants, indicating the virus was graft transmissible. Twenty additional Epiphyllum hybrids were tested by RT-PCR, and BWYV was detected from hybrids Tele Ann with mottle symptom and Vista Sun without symptoms. Thus, BWYV was detected in three different hybrids of Epiphyllum spp., an observation validated by RT-PCR, sequencing, and graft transmission. BWYV is an aphid-transmitted species of the genus Polerovirus in the family Luteoviridae. It has an extremely wide host range including legumes, crucifers, lettuce, and many weed species and can cause diseases in several crops worldwide (Duffus 1972). To our knowledge, this is the first report of BWYV in orchid cactus. It remains unclear whether BWYV causes symptoms in either single or mixed infections in other species and hybrids of Epiphyllum spp. However, these hosts can serve as a reservoir for the virus. We are currently characterizing the novel viruses detected in the hybrid Professor Ebert.