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.

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.

A single resistance factor to solve vineyard degeneration due to grapevine fanleaf virus.

Grapevine fanleaf disease, caused by grapevine fanleaf virus (GFLV), transmitted by the soil-borne nematode Xiphinema index, provokes severe symptoms and economic losses, threatening vineyards worldwide. As no effective solution exists so far to control grapevine fanleaf disease in an environmentally friendly way, we investigated the presence of resistance to GFLV in grapevine genetic resources. We discovered that the Riesling variety displays resistance to GFLV, although it is susceptible to X. index. This resistance is determined by a single recessive factor located on grapevine chromosome 1, which we have named rgflv1. The discovery of rgflv1 paves the way for the first effective and environmentally friendly solution to control grapevine fanleaf disease through the development of new GFLV-resistant grapevine rootstocks, which was hitherto an unthinkable prospect. Moreover, rgflv1 is putatively distinct from the virus susceptibility factors already described in plants.

High-Throughput Sequencing and the Viromic Study of Grapevine Leaves: From the Detection of Grapevine-Infecting Viruses to the Description of a New Environmental Tymovirales Member.

In the past decade, high-throughput sequencing (HTS) has had a major impact on virus diversity studies as well as on diagnosis, providing an unbiased and more comprehensive view of the virome of a wide range of organisms. Rather than the serological and molecular-based methods, with their more "reductionist" view focusing on one or a few known agents, HTS-based approaches are able to give a "holistic snapshot" of the complex phytobiome of a sample of interest. In grapevine for example, HTS is powerful enough to allow for the assembly of complete genomes of the various viral species or variants infecting a sample of known or novel virus species. In the present study, a total RNAseq-based approach was used to determine the full genome sequences of various grapevine fanleaf virus (GFLV) isolates and to analyze the eventual presence of other viral agents. From four RNAseq datasets, a few complete grapevine-infecting virus and viroid genomes were de-novo assembled: (a) three GFLV genomes, 11 grapevine rupestris stem-pitting associated virus (GRSPaV) and six viroids. In addition, a novel viral genome was detected in all four datasets, consisting of a single-stranded, positive-sense RNA molecule of 6033 nucleotides. This genome displays an organization similar to Tymoviridae family members in the Tymovirales order. Nonetheless, the new virus shows enough differences to be considered as a new species defining a new genus. Detection of this new agent in the original grapevines proved very erratic and was only consistent at the end of the growing season. This virus was never detected in the spring period, raising the possibility that it might not be a grapevine-infecting virus, but rather a virus infecting a grapevine-associated organism that may be transiently present on grapevine samples at some periods of the year. Indeed, the Tymoviridae family comprises isometric viruses infecting a wide range of hosts in different kingdoms (Plantae, Fungi, and Animalia). The present work highlights the fact that even though HTS technologies produce invaluable data for the description of the sanitary status of a plant, in-depth biological studies are necessary before assigning a new virus to a particular host in such metagenomic approaches.

Virus preparations from the mixed-infected P70 Pinot Noir accession exhibit GLRaV-1/GVA 'end-to-end' particles.

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.

A complex virome unveiled by deep sequencing analysis of RNAs from a French Pinot Noir grapevine exhibiting strong leafroll symptoms.

We have characterized the virome of a grapevine Pinot Noir accession (P70) that displayed, over the year, very stable and strong leafroll symptoms. For this, we have used two extraction methods (dsRNA and total RNA) coupled with the high throughput sequencing (HTS) Illumina technique. While a great disparity in viral sequences were observed, both approaches gave similar results, revealing a very complex infection status. Five virus and viroid isolates [Grapevine leafroll-associated viruse-1 (GLRaV-1), Grapevine virus A (GVA), Grapevine rupestris stem pitting-associated virus (GRSPaV), Hop stunt viroid (HSVd) and Grapevine yellow speckle viroid 1 (GYSVd1)] were detected in P70 with a grand total of eleven variants being identified and de novo assembled. A comparison between both extraction methods regarding their power to detect viruses and the ease of genome assembly is also provided.

A genome-wide diversity study of grapevine rupestris stem pitting-associated virus.

Over the last decade, many scientific disciplines have been impacted by the dawn of new sequencing techniques (HTS: high throughput sequencing). Plant pathology and more specifically virology have been greatly transformed by this 'metagenomics' paradigm shift. Such tools significantly facilitate disease diagnostics with tremendous sensitivity, providing invaluable information such as an exhaustive list of viruses being present in a sample as well as their relative concentration. In addition, many new plant viruses have been discovered. Using RNAseq technology, in silico reconstruction of complete viral genome sequences is easily attainable. This step is of importance for taxonomy, population structure analyses, phylogeography and viral evolution studies. Here, after assembling 81 new near-complete genome sequences of grapevine rupestris stem pitting-associated virus (GRSPaV), we performed a genome-wide diversity study of this ubiquitous virus of grapevine worldwide.

Metagenomic-based impact study of transgenic grapevine rootstock on its associated virome and soil bacteriome.

For some crops, the only possible approach to gain a specific trait requires genome modification. The development of virus-resistant transgenic plants based on the pathogen-derived resistance strategy has been a success story for over three decades. However, potential risks associated with the technology, such as horizontal gene transfer (HGT) of any part of the transgene to an existing gene pool, have been raised. Here, we report no evidence of any undesirable impacts of genetically modified (GM) grapevine rootstock on its biotic environment. Using state of the art metagenomics, we analysed two compartments in depth, the targeted Grapevine fanleaf virus (GFLV) populations and nontargeted root-associated microbiota. Our results reveal no statistically significant differences in the genetic diversity of bacteria that can be linked to the GM trait. In addition, no novel virus or bacteria recombinants of biosafety concern can be associated with transgenic grapevine rootstocks cultivated in commercial vineyard soil under greenhouse conditions for over 6 years.

Phylogenetic relationships and the occurrence of interspecific recombination between beet chlorosis virus (BChV) and Beet mild yellowing virus (BMYV).

Samples containing two viruses belonging to the genus Polerovirus, beet chlorosis virus (BChV) and beet mild yellowing virus (BMYV), were collected from French and Polish sugar beet fields. The molecular properties of 24 isolates of BChV and BMYV were investigated, and their genetic diversity was examined in the coat protein (CP)- and P0-encoding genes. For the first time, we have demonstrated that beet polerovirus populations include recombinants between BChV and BMYV containing breakpoints within the CP gene. Moreover, a partial correlation between geographic origin and phylogenetic clustering was observed for BMYV isolates.

P0 proteins of European beet-infecting poleroviruses display variable RNA silencing suppression activity.

Post-transcriptional gene silencing (PTGS), or RNA silencing, is one of the key mechanisms of antiviral defence used by plants. To counter this defence response, viruses produce suppressor proteins that are able to inhibit the PTGS pathway or to interfere with some of its function. The aim of this study was to evaluate the RNA silencing suppressor (RSS) activity of P0 proteins from selected European isolates of the beet-infecting poleroviruses beet chlorosis virus (BChV) and beet mild yellowing virus (BMYV) using two different experimental systems: (i) agro-infiltration of Nicotiana benthamiana green fluorescent protein-positive plants and (ii) mechanical inoculation of Chenopodium quinoa using a beet necrotic yellow vein virus (BNYVV, genus Benyvirus) RNA3-based replicon. The results demonstrated that P0 of most BMYV isolates exhibited RSS activity, although at various efficiencies among isolates. Conversely, P0 of BChV isolates displayed no RSS activity in either of the two systems under the experimental conditions used. These results are the first reported evidence that P0 proteins of two closely related beet poleroviruses show strain-specific differences in their effects on RNA silencing.

Retention of the virus-derived sequences in the nuclear genome of grapevine as a potential pathway to virus resistance.

BACKGROUND: Previous studies have revealed a wide-spread occurence of the partial and complete genomes of the reverse-transcribing pararetroviruses in the nuclear genomes of herbaceous plants. Although the absence of the virus-encoded integrases attests to the random and incidental incorporation of the viral sequences, their presence could have functional implications for the virus-host interactions. HYPOTHESIS: Analyses of two nuclear genomes of grapevine revealed multiple events of horizontal gene transfer from pararetroviruses. The approximately 200-800 bp inserts that corresponded to partial ORFs encoding reverse transcriptase apparently derived from unknown or extinct caulimoviruses and tungroviruses, were found in 11 grapevine chromosomes. In contrast to the previous reports, no reliable cases of the inserts derived from the positive-strand RNA viruses were found. Because grapevine is known to be infected by the diverse positive-strand RNA viruses, but not pararetroviruses, we hypothesize that pararetroviral inserts have conferred host resistance to these viruses. Furthermore, we propose that such resistance involves RNA interference-related mechanisms acting via small RNA-mediated methylation of pararetroviral DNAs and/or via degradation of the viral mRNAs. CONCLUSION: The pararetroviral sequences in plant genomes may be maintained due to the benefits of virus resistance to this class of viruses conferred by their presence. Such resistance could be particularly significant for the woody plants that must withstand years- to centuries-long virus assault. Experimental research into the RNA interference pathways involving the integrated pararetroviral inserts is required to test this hypothesis. REVIEWERS: This article was reviewed by Arcady R. Mushegian, I. King Jordan, and Eugene V. Koonin.

Biological and Molecular Characterization of an American Sugar Beet-Infecting Beet western yellows virus Isolate.

Three aphid-transmitted viruses belonging to the Polerovirus genus, Beet mild yellowing virus (BMYV), Beet chlorosis virus (BChV), and Beet western yellows virus (BWYV), have been described as pathogens of sugar beet. We present the complete biological, serological, and molecular characterization of an American isolate of Beet western yellows virus (BWYV-USA), collected from yellow beet leaves. The biological data suggested that BWYV-USA displayed a host range similar to that of BMYV, but distinct from those of BChV and the lettuce and rape isolates of Turnip yellows virus. The complete genomic RNA sequence of BWYV-USA showed a genetic organization and expression typical of other Polerovirus members. Comparisons of deduced amino acid sequences showed that P0 and the putative replicase complex (P1-P2) of BWYV-USA are more closely related to Cucurbit aphid-borne yellows virus (CABYV) than to BMYV, whereas alignments of P3, P4, and P5 showed the highest homology with BMYV. Intraspecific and interspecific phylogenetic analyses have suggested that the BWYV-USA genome may be the result of recombination events between a CABYV-like ancestor contributing open reading frame (ORF) 0, ORF 1, and ORF 2, and a beet Polerovirus progenitor providing the 3' ORFs, with a similar mechanism of speciation occurring for BMYV in Europe. Results demonstrate that BWYV-USA is a distinct species in the Polerovirus genus, clarifying the nomenclature of this important group of viruses.

A sensitive one-step real-time RT-PCR method for detecting Grapevine leafroll-associated virus 2 variants in grapevine.

Grapevine leafroll syndrome is caused by a complex of up to nine different Grapevine leafroll-associated viruses (GLRaV-1-9) with GLRaV-2 being reported as one of the most variable species of this group. Many methods, including indexing, serological and molecular procedures, have been developed for the detection of GLRaV-2. However, due to the low concentration of the virus in plants and the high variability of GLRaV-2, a method with improved sensitivity and with the capacity to detect of all known variants is required. Such improvement is essential for grapevine rootstocks, as these are suspected to harbour frequent GLRaV-2 infections difficult to detect, thus contributing to the spread of the leafroll disease. The development of new universal primers is described using a target sequence located in the 3' end of the virus genome. These primers were combined with a one-step SYBR Green real-time RT-PCR assay to achieve quantitative detection. All 43 GLRaV-2 isolates tested in this study were identified readily and reproducibly, regardless of their geographical origin or variety of grapevine. Using the procedure developed in this study, the sensitivity was increased 125 times compared to a conventional single-tube RT-PCR. This real-time method opens new perspectives for the sanitary selection of grapevine and in leafroll 2 disease monitoring.

Phylogenetic analysis of isolates of Beet necrotic yellow vein virus collected worldwide.

A study of molecular diversity was carried out on 136 sugar beets infected with Beet necrotic yellow vein virus (BNYVV, Benyvirus) collected worldwide. The nucleotide sequences of the RNA-2-encoded CP, RNA-3-encoded p25 and RNA-5-encoded p26 proteins were analysed. The resulting phylogenetic trees allowed BNYVV to be classified into groups that show correlations between the virus clusters and geographic origins. The selective constraints on these three sequences were measured by estimating the ratio between synonymous and non-synonymous substitution rates (omega) with maximum-likelihood models. The results suggest that selective constraints are exerted differently on the proteins. CP was the most conserved, with mean omega values ranging from 0.12 to 0.15, while p26 was less constrained, with mean omega values ranging from 0.20 to 0.33. Selection was detected in three amino acid positions of p26, with omega values of about 5.0. The p25 sequences presented the highest mean omega values (0.36-1.10), with strong positive selection (omega=4.7-54.7) acting on 14 amino acids, and particularly on amino acid 68, where the omega value was the highest so far encountered in plant viruses.