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.

Characterization of genetic determinants of the resistance to phylloxera, Daktulosphaira vitifoliae, and the dagger nematode Xiphinema index from muscadine background.

BACKGROUND: Muscadine (Muscadinia rotundifolia) is known as a resistance source to many pests and diseases in grapevine. The genetics of its resistance to two major grapevine pests, the phylloxera D. vitifoliae and the dagger nematode X. index, vector of the Grapevine fanleaf virus (GFLV), was investigated in a backcross progeny between the F1 resistant hybrid material VRH8771 (Vitis-Muscadinia) derived from the muscadine R source 'NC184-4' and V. vinifera cv. 'Cabernet-Sauvignon' (CS). RESULTS: In this pseudo-testcross, parental maps were constructed using simple-sequence repeats markers and single nucleotide polymorphism markers from a GBS approach. For the VRH8771 map, 2271 SNP and 135 SSR markers were assembled, resulting in 19 linkage groups (LG) and an average distance between markers of 0.98 cM. Phylloxera resistance was assessed by monitoring root nodosity number in an in planta experiment and larval development in a root in vitro assay. Nematode resistance was studied using 10-12 month long tests for the selection of durable resistance and rating criteria based on nematode reproduction factor and gall index. A major QTL for phylloxera larval development, explaining more than 70% of the total variance and co-localizing with a QTL for nodosity number, was identified on LG 7 and designated RDV6. Additional QTLs were detected on LG 3 (RDV7) and LG 10 (RDV8), depending on the in planta or in vitro experiments, suggesting that various loci may influence or modulate nodosity formation and larval development. Using a Bulked Segregant Analysis approach and a proportion test, markers clustered in three regions on LG 9, LG 10 and LG 18 were shown to be associated to the nematode resistant phenotype. QTL analysis confirmed the results and QTLs were thus designated respectively XiR2, XiR3 and XiR4, although a LOD-score below the significant threshold value was obtained for the QTL on LG 18. CONCLUSIONS: Based on a high-resolution linkage map and a segregating grapevine backcross progeny, the first QTLs for resistance to D. vitifoliae and to X. index were identified from a muscadine source. All together these results open the way to the development of marker-assisted selection in grapevine rootstock breeding programs based on muscadine derived resistance to phylloxera and to X. index in order to delay GFLV transmission.

Structural basis of nanobody recognition of grapevine fanleaf virus and of virus resistance loss.

Grapevine fanleaf virus (GFLV) is a picorna-like plant virus transmitted by nematodes that affects vineyards worldwide. Nanobody (Nb)-mediated resistance against GFLV has been created recently, and shown to be highly effective in plants, including grapevine, but the underlying mechanism is unknown. Here we present the high-resolution cryo electron microscopy structure of the GFLV-Nb23 complex, which provides the basis for molecular recognition by the Nb. The structure reveals a composite binding site bridging over three domains of one capsid protein (CP) monomer. The structure provides a precise mapping of the Nb23 epitope on the GFLV capsid in which the antigen loop is accommodated through an induced-fit mechanism. Moreover, we uncover and characterize several resistance-breaking GFLV isolates with amino acids mapping within this epitope, including C-terminal extensions of the CP, which would sterically interfere with Nb binding. Escape variants with such extended CP fail to be transmitted by nematodes linking Nb-mediated resistance to vector transmission. Together, these data provide insights into the molecular mechanism of Nb23-mediated recognition of GFLV and of virus resistance loss.

Detection of Multiple Variants of Grapevine Fanleaf Virus in Single Xiphinema index Nematodes.

Grapevine fanleaf virus (GFLV) is responsible for a widespread disease in vineyards worldwide. Its genome is composed of two single-stranded positive-sense RNAs, which both show a high genetic diversity. The virus is transmitted from grapevine to grapevine by the ectoparasitic nematode Xiphinema index. Grapevines in diseased vineyards are often infected by multiple genetic variants of GFLV but no information is available on the molecular composition of virus variants retained in X. index following nematodes feeding on roots. In this work, aviruliferous X. index were fed on three naturally GFLV-infected grapevines for which the virome was characterized by RNAseq. Six RNA-1 and four RNA-2 molecules were assembled segregating into four and three distinct phylogenetic clades of RNA-1 and RNA-2, respectively. After 19 months of rearing, single and pools of 30 X. index tested positive for GFLV. Additionally, either pooled or single X. index carried multiple variants of the two GFLV genomic RNAs. However, the full viral genetic diversity found in the leaves of infected grapevines was not detected in viruliferous nematodes, indicating a genetic bottleneck. Our results provide new insights into the complexity of GFLV populations and the putative role of X. index as reservoirs of virus diversity.

From a Movement-Deficient Grapevine Fanleaf Virus to the Identification of a New Viral Determinant of Nematode Transmission.

Grapevine fanleaf virus (GFLV) and arabis mosaic virus (ArMV) are nepoviruses responsible for grapevine degeneration. They are specifically transmitted from grapevine to grapevine by two distinct ectoparasitic dagger nematodes of the genus Xiphinema. GFLV and ArMV move from cell to cell as virions through tubules formed into plasmodesmata by the self-assembly of the viral movement protein. Five surface-exposed regions in the coat protein called R1 to R5, which differ between the two viruses, were previously defined and exchanged to test their involvement in virus transmission, leading to the identification of region R2 as a transmission determinant. Region R4 (amino acids 258 to 264) could not be tested in transmission due to its requirement for plant systemic infection. Here, we present a fine-tuning mutagenesis of the GFLV coat protein in and around region R4 that restored the virus movement and allowed its evaluation in transmission. We show that residues T258, M260, D261, and R301 play a crucial role in virus transmission, thus representing a new viral determinant of nematode transmission.

Viruses in the phytobiome.

The phytobiome, defined as plants and all the entities that interact with them, is rich in viruses, but with the exception of plant viruses of crop plants, most of the phytobiome viruses remain very understudied. This review focuses on the neglected portions of the phytobiome, including viruses of other microbes interacting with plants, viruses in the soil, viruses of wild plants, and relationships between viruses and the vectors of plant viruses.

Phylogeography of the soil-borne vector nematode Xiphinema index highly suggests Eastern origin and dissemination with domesticated grapevine.

The soil-borne nematode Xiphinema index is closely linked to its main host, the grapevine, and presents a major threat to vineyards worldwide due to its ability to transmit Grapevine fanleaf virus (GFLV). The phylogeography of X. index has been studied using mitochondrial and microsatellite markers in samples from most regions of its worldwide distribution to reveal its genetic diversity. We first used the mitochondrial marker CytB and illustrated the low intraspecific divergence of this mainly meiotic parthenogenetic species. To generate a higher polymorphism level, we then concatenated the sequences of CytB and three mitochondrial markers, ATP6, CO1 and ND4, to obtain a 3044-bp fragment. We differentiated two clades, which each contained two well-supported subclades. Samples from the eastern Mediterranean and the Near and Middle East were grouped into three of these subclades, whereas the samples from the western Mediterranean, Europe and the Americas all belonged to the fourth subclade. The highest polymorphism level was found in the samples of one of the Middle and Near East subclades, strongly suggesting that this region contained the native area of the nematode. An east-to-west nematode dissemination hypothesis appeared to match the routes of the domesticated grapevine during Antiquity, presumably mainly dispersed by the Greeks and the Romans. Surprisingly, the samples of the western subclade comprised only two highly similar mitochondrial haplotypes. The first haplotype, from southern Iberian Peninsula, Bordeaux and Provence vineyards, exhibited a high microsatellite polymorphism level that suggests introductions dating from Antiquity. The second haplotype contained a highly predominant microsatellite genotype widespread in distant western countries that may be a consequence of the massive grapevine replanting following the 19th-century phylloxera crisis. Finally, our study enabled us to draw a first scaffold of X. index diversity at the global scale.

Vector-transmission of plant viruses and constraints imposed by virus-vector interactions.

Because plants are sessile and their cells protected by a cell wall, the contact transmission of plant viruses is very rare. Almost all plant viruses are transmitted by vectors, which can be insects, nematodes, mites or fungi. Although very efficient, this mode of transmission is not trivial and imposes numerous constraints on viruses. In this review we show that these constraints apply at all stages of the transmission process and at all scales, from the molecular to ecological interactions. We discuss several viral adaptations that likely reflect sophisticated means to alleviate these constraints and to maximize transmission, and we point at gaps and future directions in this field of research.

High Risk Blueberry Viruses by Region in North America; Implications for Certification, Nurseries, and Fruit Production.

There is limited information on the distribution of blueberry viruses in the U.S. or around the world other than where the viruses were first discovered and characterized. A survey for blueberry viruses was carried out in the U.S. in 2015⁻2017. Most blueberry viruses have been characterized to the point that sensitive diagnostic assays have been developed. These assays are based on ELISA or variations of PCR, which were employed here to determine the presence of blueberry viruses in major blueberry production and nursery areas of the U.S. The viruses included in this study were: blueberry fruit drop (BFDaV), blueberry latent (BlLV), blueberry leaf mottle (BLMoV), blueberry mosaic (BlMaV), blueberry red ringspot (BRRV), blueberry scorch (BlScV), blueberry shock (BlShV), blueberry shoestring (BlSSV), blueberry virus A (BVA), peach rosette mosaic (PRMV), tobacco ringspot (TRSV), and tomato ringspot (ToRSV). In the Pacific Northwest BlShV was the most widespread virus, with BlScV and ToRSV detected in a limited number of fields in Oregon and Washington, but BlScV was widespread in British Columbia. In the upper midwest, the nematode-borne (ToRSV, TRSV), aphid-transmitted (BlSSV and BVA) and pollen-borne (BLMoV) viruses were most widespread. In the northeast, TRSV, ToRSV, and BlScV, were detected most frequently. In the southeast, BRRV and BNRBV were the most widespread viruses. BlLV, a cryptic virus with no known symptoms or effect on plant growth or yield was present in all regions. There are other viruses present at low levels in each of the areas, but with the lower incidence they pose minimal threat to nursery systems or fruit production. These results indicate that there are hotspots for individual virus groups that normally coincide with the presence of the vectors. The information presented highlights the high risk viruses for nursery and fruit production each pose a different challenge for control.

Novel RNA viruses within plant parasitic cyst nematodes.

The study of invertebrate-and particularly nematode-viruses is emerging with the advancement of transcriptome sequencing. Five single-stranded RNA viruses have now been confirmed within the economically important soybean cyst nematode (SCN; Heterodera glycines). From previous research, we know these viruses to be widespread in greenhouse and field populations of SCN. Several of the SCN viruses were also confirmed within clover (H. trifolii) and beet (H. schachtii) cyst nematodes. In the presented study, we sequenced the transcriptomes of several inbred SCN populations and identified two previously undiscovered viral-like genomes. Both of these proposed viruses are negative-sense RNA viruses and have been named SCN nyami-like virus (NLV) and SCN bunya-like virus (BLV). Finally, we analyzed publicly available transcriptome data of two potato cyst nematode (PCN) species, Globodera pallida and G. rostochiensis. From these data, a third potential virus was discovered and called PCN picorna-like virus (PLV). PCN PLV is a positive-sense RNA virus, and to the best of our knowledge, is the first virus described within PCN. The presence of these novel viruses was confirmed via qRT-PCR, endpoint PCR, and Sanger sequencing with the exception of PCN PLV due to quarantine restrictions on the nematode host. While much work needs to be done to understand the biological and evolutionary significance of these viruses, they offer insight into nematode ecology and the possibility of novel nematode management strategies.

Assessing Potato Cultivar Sensitivity to Tuber Necrosis Caused by Tobacco rattle virus.

Tobacco rattle virus (TRV) causes the economically important corky ring spot disease in potato. Chemical control is difficult due to the soilborne nature of the TRV-transmitting nematode vector, and identifying natural host resistance against TRV is considered to be the optimal control measure. The present study investigated the sensitivity of 63 cultivars representing all market types (evaluated at North Dakota and Washington over 2 years) for the incidence of TRV-induced tuber necrosis and severity. This article also investigates the cultivar-location interaction (using a mixed-effects model) for TRV-induced necrosis. TRV-induced tuber necrosis (P < 0.0001) and severity (P < 0.0001) were significantly different among cultivars evaluated separately in North Dakota and Washington trials. Mixed-effects model results of pooled data (North Dakota and Washington) demonstrated that the interaction of cultivar and location had a significant effect (P = 0.03) on TRV-induced necrosis. Based on the virus-induced tuber necrosis data from both years and locations, cultivars were categorized into sensitive, moderately sensitive, insensitive, and moderately insensitive groups. Based on data from North Dakota, 10 cultivars, including Bintje, Centennial Russet, Ciklamen, Gala, Lelah, Oneida Gold, POR06V12-3, Rio Colorado, Russian Banana, and Superior, were rated as insensitive to TRV-induced tuber necrosis. Similar trials assessing TRV sensitivity among cultivars conducted in Washington resulted in a number of differences in sensitivity rankings compared with North Dakota trials. A substantial shift in sensitivity of some potato cultivars to TRV-induced tuber necrosis was observed between the two locations. Four cultivars (Centennial Russet, Oneida Gold, Russian Banana, and Superior) ranked as insensitive for North Dakota trials were ranked as sensitive for Washington trials. These results can assist the potato industry in making cultivar choices to reduce the economic impact of TRV-induced tuber necrosis.

Metagenomics reshapes the concepts of RNA virus evolution by revealing extensive horizontal virus transfer.

Virus metagenomics is a young research filed but it has already transformed our understanding of virus diversity and evolution, and illuminated at a new level the connections between virus evolution and the evolution and ecology of the hosts. In this review article, we examine the new picture of the evolution of RNA viruses, the dominant component of the eukaryotic virome, that is emerging from metagenomic data analysis. The major expansion of many groups of RNA viruses through metagenomics allowed the construction of substantially improved phylogenetic trees for the conserved virus genes, primarily, the RNA-dependent RNA polymerases (RdRp). In particular, a new superfamily of widespread, small positive-strand RNA viruses was delineated that unites tombus-like and noda-like viruses. Comparison of the genome architectures of RNA viruses discovered by metagenomics and by traditional methods reveals an extent of gene module shuffling among diverse virus genomes that far exceeds the previous appreciation of this evolutionary phenomenon. Most dramatically, inclusion of the metagenomic data in phylogenetic analyses of the RdRp resulted in the identification of numerous, strongly supported groups that encompass RNA viruses from diverse hosts including different groups of protists, animals and plants. Notwithstanding potential caveats, in particular, incomplete and uneven sampling of eukaryotic taxa, these highly unexpected findings reveal horizontal virus transfer (HVT) between diverse hosts as the central aspect of RNA virus evolution. The vast and diverse virome of invertebrates, particularly nematodes and arthropods, appears to be the reservoir, from which the viromes of plants and vertebrates evolved via multiple HVT events.

ICTV Virus Taxonomy Profile: Nyamiviridae.

The Nyamiviridae is a family of viruses with unsegmented, negative-sense RNA genomes of 11.3-12.2 kb that produce enveloped, spherical virions. Viruses of the genus Nyavirus are tick-borne and some also infect birds. Other nyamiviruses infecting parasitoid wasps and plant parasitic nematodes have been classified into the genera Peropuvirus and Socyvirus, respectively. This is a summary of the current International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of Nyamiviridae, which is available at www.ictv.global/report/nyamiviridae.

Disparate gain and loss of parasitic abilities among nematode lineages.

Plant parasitism has arisen time and again in multiple phyla, including bacteria, fungi, insects and nematodes. In most of these organismal groups, the overwhelming diversity hampers a robust reconstruction of the origins and diversification patterns of this trophic lifestyle. Being a moderately diversified phylum with ≈ 4,100 plant parasites (15% of total biodiversity) subdivided over four independent lineages, nematodes constitute a major organismal group for which the genesis of plant parasitism could be mapped. Since substantial crop losses worldwide have been attributed to less than 1% of these plant parasites, research efforts are severely biased towards this minority. With the first molecular characterisation of numerous basal and supposedly harmless plant parasites as well as their non-parasitic relatives, we were able to generate a comprehensive molecular framework that allows for the reconstruction of trophic diversification for a complete phylum. In each lineage plant parasites reside in a single taxonomic grouping (family or order), and by taking the coverage of the next lower taxonomic level as a measure for representation, 50, 67, 100 and 85% of the known diversity was included. We revealed distinct gain and loss patterns with regard to plant parasitism per se as well as host exploitation strategies between these lineages. Our map of parasitic nematode biodiversity also revealed an unanticipated time reversal in which the two most ancient lineages showed the lowest level of ecological diversification and vice versa.

Detection of Nepovirus Vector and Nonvector Xiphinema Species in Grapevine.

Fanleaf degeneration is considered the most damaging viral disease of grapevine. The two major nepoviruses involved are Grapevine fanleaf virus (GFLV) and Arabis mosaic virus (ArMV) which are respectively and specifically transmitted by the dagger nematodes Xiphinema index and X. diversicaudatum. The methods described below are aimed at detecting four prevalent grapevine Xiphinema species: the vector species previously mentioned and two nonvector species X. vuittenezi and X. italiae.

Status and prospects of plant virus control through interference with vector transmission.

Most plant viruses rely on vector organisms for their plant-to-plant spread. Although there are many different natural vectors, few plant virus-vector systems have been well studied. This review describes our current understanding of virus transmission by aphids, thrips, whiteflies, leafhoppers, planthoppers, treehoppers, mites, nematodes, and zoosporic endoparasites. Strategies for control of vectors by host resistance, chemicals, and integrated pest management are reviewed. Many gaps in the knowledge of the transmission mechanisms and a lack of available host resistance to vectors are evident. Advances in genome sequencing and molecular technologies will help to address these problems and will allow innovative control methods through interference with vector transmission. Improved knowledge of factors affecting pest and disease spread in different ecosystems for predictive modeling is also needed. Innovative control measures are urgently required because of the increased risks from vector-borne infections that arise from environmental change.

Nyamiviridae: proposal for a new family in the order Mononegavirales.

Nyamanini virus (NYMV) and Midway virus (MIDWV) are unclassified tick-borne agents that infect land birds and seabirds, respectively. The recent molecular characterization of both viruses confirmed their already known close serological relationship and revealed them to be nonsegmented, single- and negative-stranded RNA viruses that are clearly related to, but quite distinct from, members of the order Mononegavirales (bornaviruses, filoviruses, paramyxoviruses, and rhabdoviruses). A third agent, soybean cyst nematode virus 1 (SbCNV-1, previously named soybean cyst nematode nyavirus), was recently found to be an additional member of this new virus group. Here, we review the current knowledge about all three viruses and propose classifying them as members of a new mononegaviral family, Nyamiviridae.

RNA viruses and the host microRNA machinery.

Gene silencing by small RNAs (sRNAs) occurs in all three domains of life. In recent years, our appreciation of the diverse functions of sRNAs has increased, and we have identified roles for these RNAs in cellular differentiation, fitness and pathogen defence. Interestingly, although plants, nematodes and arthropods use sRNAs to combat viral infections, chordates have replaced this defence strategy with one based exclusively on proteins. This limits chordate use of sRNAs to the silencing of genome-encoded transcripts and has resulted in viruses that do not perturb sRNA-related cellular processes. This evolutionary phenomenon provides an opportunity to exploit the pre-existing chordate sRNA pathways in order to generate a range of virus-based biological tools. Here, I discuss the relationship between sRNAs and RNA viruses, detail how microRNA expression can be harnessed to control RNA viruses and describe how RNA viruses can be designed to deliver sRNAs.

Structural insights into viral determinants of nematode mediated Grapevine fanleaf virus transmission.

Many animal and plant viruses rely on vectors for their transmission from host to host. Grapevine fanleaf virus (GFLV), a picorna-like virus from plants, is transmitted specifically by the ectoparasitic nematode Xiphinema index. The icosahedral capsid of GFLV, which consists of 60 identical coat protein subunits (CP), carries the determinants of this specificity. Here, we provide novel insight into GFLV transmission by nematodes through a comparative structural and functional analysis of two GFLV variants. We isolated a mutant GFLV strain (GFLV-TD) poorly transmissible by nematodes, and showed that the transmission defect is due to a glycine to aspartate mutation at position 297 (Gly297Asp) in the CP. We next determined the crystal structures of the wild-type GFLV strain F13 at 3.0 Å and of GFLV-TD at 2.7 Å resolution. The Gly297Asp mutation mapped to an exposed loop at the outer surface of the capsid and did not affect the conformation of the assembled capsid, nor of individual CP molecules. The loop is part of a positively charged pocket that includes a previously identified determinant of transmission. We propose that this pocket is a ligand-binding site with essential function in GFLV transmission by X. index. Our data suggest that perturbation of the electrostatic landscape of this pocket affects the interaction of the virion with specific receptors of the nematode's feeding apparatus, and thereby severely diminishes its transmission efficiency. These data provide a first structural insight into the interactions between a plant virus and a nematode vector.

Tobraviruses--plant pathogens and tools for biotechnology.

The tobraviruses, Tobacco rattle virus (TRV), Pea early-browning virus (PEBV) and Pepper ringspot virus (PepRSV), are positive-strand RNA viruses with rod-shaped virus particles that are transmitted between plants by trichodorid nematodes. As a group, these viruses infect many plant species, with TRV having the widest host range. Recent studies have begun to dissect the interaction of TRV with potato, currently the most commercially important crop disease caused by any of the tobraviruses. As well as being successful plant pathogens, these viruses have become widely used as vectors for expression in plants of nonviral proteins or, more frequently, as initiators of virus-induced gene silencing (VIGS). Precisely why tobraviruses should be so effective as VIGS vectors is not known; however, molecular studies of the mode of action of the tobravirus silencing suppressor protein are shedding some light on this process.