Ectoparasitic Nematode Xiphinema Index Research Articles

Selected Genotypes with the Genetic Background ofVitis aestivalisandVitis labruscaAre Resistant toXiphinema index

The ectoparasitic nematode Xiphinema index transmits grapevine fanleaf virus (GFLV) during feeding on grapevine roots, causing fanleaf degeneration in the plant. Hence, resistance breeding is a key to develop novel rootstocks to overcome such threats. In past years, various grapevine species were screened, and a few candidates with partial resistance were identified. However, they were hardly sufficient for viticulture because of their many agronomical defects. To develop reliably resistant rootstocks applicable in viticulture, multiple Vitis spp. genotypes were analyzed using root inoculation with nematodes in glass vials as an early and easy evaluation test. Resistance levels were evaluated 35 days after inoculation based on nematode reproduction factors, focusing on juveniles and eggs. Infection of grapevines with GFLV was analyzed after inoculation with viruliferous X. index. With this fast screening system, putative candidates with resistances against X. index have been identified for future breeding programs. Particularly, genotypes with the genetic background of Vitis aestivalis and Vitis labrusca were found to be nematode-resistant.

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.

THE CONTROL OF Grapevine fanleaf virus VICTOR Xiphinema index Thorne and Allen. 1950

The ectoparasitic nematode Xiphinema index Thorne and Allen is economically important in grapes Vitis vinifera L., both as a root parasite and as a vector for grapevine fan leaf virus (GFLV). The control of GFLV vector is one of the main strategies of the disease management. The using of nematicides and soil amendments is one of traditional methods for nematode controlling. The objective of this research was to evaluate the nematicidal effect of the nematicide Vyox, poultry manure and salicylic acid for controlling Xiphinema index on grapevines. The nematicidal was evaluated determining the nematode population densities in 200 ml of soil. The results revealed that both of nematicide Vyox and poultry manure have a significant activity against X. index 15.6 and 162.4 respectively compare to the positive control 557.4 nematode/200ml soil. The best results were obtained from the combination between nematicide Vyox, poultry manure and salicylic acid which significantly reduced X. index populations (P<0.05) compare to the single treatments and positive control.

Development of an in vitro Dual Culture System for Grapevine and Xiphinema index as a Tool for Virus Transmission

Grapevine fanleaf virus (GFLV) is a nepovirus that is transmitted to grapevines by the ectoparasitic nematode Xiphinema index. GFLV causes severe losses in yield and quality in viticulture worldwide. Presently, laborious and time-consuming field trials or greenhouse tests are necessary for screening putative GFLV resistance in new grape genotypes developed in breeding programmes. We developed an in vitro dual culture system for grapevines and nematode vectors that requires less time and space than inoculation experiments done in the greenhouse. Virus infection of in vitro grapevines was investigated using immunocapture-reverse transcriptase-polymerase chain reaction (IC-RT-PCR) analysis. The development of root galls induced by feeding nematodes on in vitro grapevines was also analysed. Virus infection in grapevines in the dual culture with viruliferous nematodes was detected six weeks post-inoculation. Root galls were always absent from parasitised in vitro grapevines with detectable virus infection, whereas they developed on some parasitised, but virus-negative tested grapevines. Therefore, root galls cannot be used as a reliable indicator for parasitism and virus transmission.

Local and systemic mycorrhiza-induced protection against the ectoparasitic nematode Xiphinema index involves priming of defence gene responses in grapevine

The ectoparasitic dagger nematode (Xiphinema index), vector of Grapevine fanleaf virus (GFLV), provokes gall formation and can cause severe damage to the root system of grapevines. Mycorrhiza formation by Glomus (syn. Rhizophagus) intraradices BEG141 reduced both gall formation on roots of the grapevine rootstock SO4 (Vitis berlandieri×V. riparia) and nematode number in the surrounding soil. Suppressive effects increased with time and were greater when the nematode was post-inoculated rather than co-inoculated with the arbuscular mycorrhizal (AM) fungus. Using a split-root system, decreased X. index development was shown in mycorrhizal and non-mycorrhizal parts of mycorrhizal root systems, indicating that both local and systemic induced bioprotection mechanisms were active against the ectoparasitic nematode. Expression analyses of ESTs (expressed sequence tags) generated in an SSH (subtractive suppressive hybridization) library, representing plant genes up-regulated during mycorrhiza-induced control of X. index, and of described grapevine defence genes showed activation of chitinase 1b, pathogenesis-related 10, glutathione S-transferase, stilbene synthase 1, 5-enolpyruvyl shikimate-3-phosphate synthase, and a heat shock proein 70-interacting protein in association with the observed local and/or systemic induced bioprotection against the nematode. Overall, the data suggest priming of grapevine defence responses by the AM fungus and transmission of a plant-mediated signal to non-mycorrhizal tissues. Grapevine gene responses during AM-induced local and systemic bioprotection against X. index point to biological processes that are related either to direct effects on the nematode or to protection against nematode-imposed stress to maintain root tissue integrity.

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.

Impact of Quillaja saponaria saponins on grapevine ecosystem organisms

The control of grapevine pathogens is a rising concern in Vitis vinifera culture. The current international trend is toward banning chemicals that are highly toxic to the environment and human workers, and adopting tighter regulations. We evaluated the impact of saponins on three kinds of organisms found in grapevine culture. The ectoparasitic nematode Xiphinema index, the parasitic fungus Botrytis cinerea and various yeast strains representative of the must fermentation population were incubated on synthetic media supplemented with variable concentrations of Quillaja saponaria saponins. Saponins induced reduction in the growth of B. cinerea and showed nematicide effects on X. index. The control of X. index and Botrytis cinerea is discussed in the context of the potential use of these chemicals as environmentally-friendly grapevine treatments. With Saccharomyces cerevisiae and other yeasts, saponins showed higher toxicity against S. cerevisiae strains isolated from wine or palm wine whereas laboratory strains or strains isolated from oak exhibited better resistance. This indicates that Q. saponaria saponins effects against yeast microflora should be assessed in the field before they can be considered an environmentally-safe new molecule against B. cinerea and X. index.

Transmission Competency of Single-Female Xiphinema index Lines for Grapevine fanleaf virus

Grapevine fanleaf virus (GFLV) is vectored specifically from grapevine to grapevine by the ectoparasitic nematode Xiphinema index. Limited information is available on the vector competency of X. index populations from diverse geographical origins. We determined the transmissibility of two GFLV strains showing 4.6% amino acid divergence within their coat protein (e.g., strains F13 and GHu) by seven clonal lines of X. index developed from seven distinct populations from the Mediterranean basin (Cyprus, southern France, Israel, Italy, and Spain), northern France, and California. X. index lines derived from single adult females were produced on fig (Ficus carica) plants to obtain genetically homogenous aviruliferous clones. A comparative reproductive rate analysis on Vitis rupestris du Lot and V. vinifera cv. Cabernet Sauvignon showed significant differences among clones, with the single-female Cyprus line showing the highest rate (30-fold the initial population) and the Spain and California lines showing the lowest rate (10-fold increase), regardless of the grapevine genotype. However, there was no differential vector competency among the seven X. index lines for GFLV strains F13 and GHu. The implications of our findings for the dynamic of GFLV transmission in vineyards and screening of Vitis spp. for resistance to GFLV are discussed.

Analysis of expressed sequence tags from the ectoparasitic nematode Xiphinema index

AbstractWe report the results of a small-scale expressed sequence tag project performed on the ectoparasitic nematode Xiphinema index. Approximately 1400 genes were sequenced, 70% from a cDNA library generated from dissected basal bulbs (containing the pharyngeal gland cells) and 30% from cDNA libraries generated from whole mixed stage nematodes. A large portion of the bulb library (48%) was composed of proteins with no matches in the database. Further analysis of these genes revealed that a total of 51 contigs were present, half of which encoded novel secreted proteins. By contrast, the whole nematode library contained more housekeeping and nematode-specific genes. Only one of the novel genes from the whole nematode library had a predicted signal peptide at the N-terminus. Genes encoding transthyretin-like proteins were abundant in the bulb library and in situ hybridisation confirmed that one of these is expressed in the basal bulb. Genes encoding a variety of proteases, which were shown using in situ hybridisation to be expressed in the gut, were also identified.

GRAPEVINE FANLEAF VIRUS: STILL A MAJOR THREAT TO THE GRAPEVINE INDUSTRY

SUMMARY Grapevine fanleaf virus (GFLV) is responsible for fanleaf degeneration, which is one of the most severe virus diseases of grapevines worldwide. GFLV causes substantial crop losses, reduces fruit quality and shortens the longevity of grapevines in the vineyard. GFLV is transmitted specifically from grapevine to grapevine by the ectoparasitic nematode Xiphinema index, and belongs to the genus Nepovirus in the family Comoviridae. Since the discovery of the nematode vector in the late 1950’s and the identification of GFLV as the agent responsible for fanleaf degeneration in the early 1960’s, a wealth of information has been accumulated on its transmission, biological properties and serological characteristics, as well as on the structure and expression of the GFLV genome. Although dissemination of the virus through propagation material has been drastically reduced over the past two decades by implementing rigorous certification schemes and establishing quarantine facilities, effective strategies are still needed to control GFLV in naturally infected vineyards. Recently, significant progress has been made on the elucidation of the function(s) of most GFLV proteins, in particular those involved in critical steps of the virus multiplication cycle, including RNA replication, cell-to-cell movement, and transmission by X. index. New insights have also been gained into the population structure and genomic variability among isolates from naturally infected vineyards, which have opened new avenues for designing alternative strategies to control this destructive virus. This review article offers a comprehensive overview of the most significant advances made over the past 15 years on GFLV and discusses novel control strategies for one of the major threats to the grapevine industry worldwide.

Sensitive and reliable detection of grapevine fanleaf virus in a single Xiphinema index nematode vector

Grapevine fanleaf virus (GFLV) is specifically transmitted from plant to plant by the ectoparasitic nematode Xiphinema index. A sensitive and reliable procedure was developed to readily detect GFLV in a single viruliferous X. index, regardless of the nematode origin, i.e. greenhouse rearings or vineyard soils. The assay is based on bead milling to disrupt nematodes extracted from soil samples, solid-phase extraction of total nematode RNAs, and amplification of a 555 bp fragment of the coat protein (CP) gene by reverse transcription-polymerase chain reaction with two primers designed from conserved sequences. This procedure is sensitive since the CP gene fragment is amplified from an artificial sample consisting of one viruliferous nematode mixed with 3000 aviruliferous individuals. In addition, StyI RFLP analysis of the CP amplicon enables the GFLV isolate carried by a single viruliferous X. index to be characterized. This GFLV detection assay opens new avenues for epidemiological studies and for molecular investigations on the mechanism of X. index-mediated GFLV transmission.