Phylogeography of the vector nematode Xiphinema index using mitochondrial and microsatellite markers highlights its Eastern origin closely linked to grapevine domestication.

The economic impact of the dagger nematode Xiphinema index is high in Western vineyards by transmitting the damaging Grapevine fanleaf virus. Our phylogeographical study based on mitochondrial sequences and microsatellite loci used more than 80 X. index representative samples collected from the Middle- and Near East, the Eastern-, Central- and Western Mediterranean, and the Western countries (Europe and the Americas). In this mainly (meiotic) parthenogenetic species, the mitochondrial marker CytB was first considered for comparison of X. index with the related amphimictic vector species X. diversicaudatum. Xiphinema index exhibits a significantly lower intraspecific molecular variability than X. diversicaudatum, in agreement with the respective reproduction modes of both nematodes. We then showed that CytB, concatenated with additional mitochondrial genes ATP6, ND4 and COI, display a robust phylogeographical pattern consisting in three clades grouping Eastern Mediterranean, Nearand Middle Eastern samples and a single clade grouping samples from Western Mediterranean, Europe and the Americas. The highest mitochondrial polymorphism is observed in one clade of Middle- and Near-East samples that overlaps the Transcaucasia and Southern Caspian Sea region from where grapevine has been presumably domesticated and that likely overlaps the nematode native area. East-to-west nematode dissemination appears to match that of its domesticated grapevine host during the Antiquity mainly by the Greeks and then the Romans. In Western Mediterranean, Europe and the Americas, two close and almost exclusive mitochondrial haplotypes were detected. The first haplotype, found in vineyards from the Southern Iberian Peninsula, Bordeaux and Provence, exhibits a high microsatellite polymorphism. By contrast, the second haplotype contains a single predominant microsatellite genotype surprisingly widespread in most Western countries. This is almost certainly due to its recent dispersal during the massive grapevine replants following the 19th century phylloxera crisis. Our data provide an improved knowledge of X. index diversity for future pest control strategies

Origine et gestion des populations du nématode Xiphinema index, vecteur du Grapevine fanleaf virus (GFLV) de la vigne

Diplôme : Dr. d'UniversitéLa maladie du court-noué, virose la plus grave de la vigne, est répandue dans la quasi-totalité des vignobles du monde. Cette maladie est due à un complexe de 15 nepovirus, dont le principal est le Grapevine fanleaf virus (GFLV), transmis spécifiquement de plante à plante par le nématode ectoparasite Xiphinema index. Le GFLV est responsable de l'arrachage précoce des parcelles de vigne, notamment dans les crus très anciens où la maladie se propage de façon lente mais régulière au cours des générations successives de culture. Après replantation, la recontamination des parcelles est ainsi de plus en plus rapide et économiquement grave. Nos travaux portent sur le nématode vecteur X. index et s’intéressent d’une part à l’origine des nématodes par une étude de variabilité génétique et d’autre part à la gestion des populations dans le contexte régional du vignoble bordelais. Dans le premier volet nous souhaitons tout d’abord tenter de retracer l’histoire de la colonisation des principaux vignobles par le nématode, qui aurait été disséminé en même temps que la culture de la vigne à partir du Moyen-Orient. Préciser le berceau d’origine de cette espèce peut avoir un intérêt appliqué primordial dans la lutte contre le nématode. En effet, cela pourrait permettre de mettre en évidence la réduction de la variabilité génétique (multiples goulots d'étranglement ? ; effets fondateurs ?) et ainsi de guider le choix des populations les plus aptes à refléter la variabilité génétique de l’espèce, notamment pour évaluer les méthodes alternatives en cours d’étude (en particulier le matériel résistant d’intérêt dans les travaux de création de porte-greffe initiés à l’INRA). Nous souhaitons d’autre part comprendre comment se font les contaminations entre vignobles, entre parcelles de vignes, ou au sein de parcelles de vignes, afin de proposer des mesures prophylactiques pour éviter les contaminations de parcelle à parcelle. Le deuxième volet de cette thèse s’intéresse à la gestion des populations de nématode pour réduire le plus possible leur impact dans le contexte du vignoble bordelais. Il s’agit dans un premier temps de préciser la distribution spatiale du nématode et de la virose au sein de parcelles d’étude puis d’utiliser ces informations pour la mise en place de plusieurs études à objectif appliqu

Origine, variabilité et gestion des populatins de Xiphinema index, le nématode vecteur du Grapevine, fanleaf virus (GFLV)

La maladie du court-noué, virose la plus grave de la vigne, est principalement due au Grapevine fanleaf virus (GFLV). Ce virus est transmis de plante à plante, dans le sol, par le nématode ectoparasite Xiphinema index. Une approche pluridisciplinaire combinant démographie spatiale, génétique des populations, phylogéographie et agronomie a été réalisée afin de mieux comprendre le fonctionnement des populations de ce nématode à trois échelles spatiales : la parcelle de vigne, la région bordelaise et le monde. L étude de démographie spatiale, conduite dans trois parcelles de vigne, montre que les nématodes se situent majoritairement entre 40 et 100 cm de profondeur, dans les horizons où les radicelles sont les plus abondantes. Elle révèle un patron agrégé et une structuration en voisinage des effectifs, avec des foyers de 6 à 8 m de diamètre. Nos résultats suggèrent également la formation d un foyer primaire due à l introduction initiale des nématodes en bordure de parcelle, suivie du développement d un foyer secondaire, préférentiellement dans le sens des rangs de vigne, vraisemblablement par le travail du sol, et illustrent la corrélation entre les répartitions spatiales du virus et du nématode. Le génotype, avec sept loci microsatellites de nématodes prélevés sur six parcelles du Bordelais permet de préciser le régime de reproduction de X. index. Tous les indices d un mode de reproduction asexué sont retrouvés (notamment un fort déséquilibre de liaison et un excès en hétérozygotes) mais l existence de rares évènements de reproduction sexuée associés à de très forts taux de clonalité (95 % pour une parcelle ; 99 à 100 % pour les autres) est également mise en évidence. Un échantillonnage hiérarchérisé estimant la diversité et la structure génétique aux niveaux échantillon de sol, parcelle de vigne et région, et combinant analyses Bayésiennes, analyses multivariées et reconstruction phylogénétiques, ainsi qu une dimension spatiale, a permis de détecter et de localiser finement les individus issus de reproduction sexuée aux zones de contact entre groupes génétiques différents, ce qui suggère que la reproduction sexuée est favorisée entre deux foyers de nématodesThe nematode Xiphinema index has the greatest economic impact in vineyards, as it transmits the Grapevine fanleaf virus. GFLV is transmitted in the soil, grapevine, by the nematode feeding on growing root tips. We realised a multidisciplinary approach, combining spatial demography, population genetics, phylogeography and agronomy, to better understand the population functioning of this nematode at three spatial scales : the grapevine field the region of the Bordeaux vineyard and the world. In the spatial demography study, conducted in three grapevine fields, we showed that the highest numbers of individuals occurred at 40 to 110 cm depth, corresponding to the two layers where the highest densities of fine roots were observed. We revealed an aggregative spatial pattern, together with a significant neighbourhood structure of nematode densities, with patches of 6 to 8 m diameter. The nematode distribution suggests an initial introduction of nematodes in primary patches and then the onsed and development of secondary patches, preferentially in the direction of vine rows. This culd be an effect of tillage during mechanical weeding which is performed exclusively in the direction of vine rows. Finally, nematode patches correlated significantly with those of GFLV-infected grapevine plants. By genotyping, with seven microsatellites, nematodes individuals collected in six grapevine fields, we précised the reproductive mode of X. index. All cues of an asexual reproductive mode are found (high linkage disequilibrium and heterozygous excess) but the existence of some rare sexual reproduction events associated with high levels of clonality (95 % for one given plot ; 99 to 100% for the others) is also revealed. A hierarchical sampling, estimating the genetic diversity and structure at the scales of soil-sample, grapevine plot and vineyard region, and combining Bayesian and multivariate analysis and phylogenetic reconstructions, as well a spatial dimension, allowed to detct region, and combining Bayesian and multivariate analysis and phylogenetic reconstructions, as well a spatial dimension, allowed to detect and finally localise sexually produced individuals at the contact zones between different genetic clusters, which suggest that sexual reproduction is favoured between two nematode patches.RENNES-Agrocampus-CRD (352382323) / SudocSudocFranceF

Development of nine polymorphic microsatellite markers for the phytoparasitic nematode Xiphinema index, the vector of the grapevine fanleaf virus

International audienceWe report isolation, characterization and cross-species amplification of nine microsatellite loci from the phytoparasitic nematode Xiphinema index, the vector of grapevine fanleaf virus. Levels of polymorphism were evaluated in 62 individuals from two X. index populations. The number of alleles varies between two and 10 depending on locus and population. Observed heterozygosity on loci across both populations varied from 0.32 to 0.857 (mean 0.545). The primers were tested for cross-species amplification in three other species of phytoparasitic nematodes of the Xiphinema genus. These nine microsatellite loci constitute valuable markers for population genetics and phylogeographical studies of X. inde

Control of <em>Xiphinema</em> index populations by fallow plants under greenhouse and field conditions

International audienceThe dagger nematode Xiphinema index has a high economic impact in vineyards by direct pathogenicity and above all by transmitting the Grapevine fanleaf virus (GFLV). Agrochemicals have been largely employed to restrict the spread of GFLV by reducing X. index populations but are now banned. As an alternative to nematicides, the use of fallow plants between two successive vine crops was assessed. We selected plant species adapted to vineyard soils and exhibiting negative impact on nematodes and we evaluated their antagonistic effect on X. index in greenhouse using artificially infested soil, and in naturally infested vineyard conditions. The screening was conducted with plants belonging to the families Asteraceae (sunflower, marigold, zinnia, and nyjer), Poaceae (sorghum and rye), Fabaceae (white lupin, white melilot, hairy vetch, and alfalfa), Brassicaceae (rapeseed and camelina), and Boraginaceae (phacelia). In the greenhouse controlled assay, white lupin, nyjer, and marigold significantly reduced X. index populations compared with that of bare soil. The vineyard assay, designed to take into account the aggregative pattern of X. index distribution, revealed that marigold and hairy vetch are good candidates as cover crops to reduce X. index populations in vineyard. Moreover, this original experimental design could be applied to manage other soilborne pathogen

Tandem-Repeat Patterns and Mutation Rates in Microsatellites of the Nematode Model Organism Pristionchus pacificus

Modern evolutionary biology requires integrative approaches that combine life history, population structure, ecology, and development. The nematode Pristionchus pacificus has been established as a model system in which these aspects can be studied in one organism. P. pacificus has well-developed genetic, genomic, and transgenic tools and its ecologic association with scarab beetles is well described. A recent study provided first mutation rate estimates based on mitochondrial genome sequencing and mutation accumulation line experiments that help resolve rather ancient evolutionary branches. Here, we analyzed the tandem-repeat pattern and studied spontaneous mutation rates for microsatellite markers by using the previously generated mutation accumulation lines. We found that 0.59%–3.83% of the genome is composed of short tandem repeats. We developed 41 microsatellite markers, randomly chosen throughout the genome and analyzed them in 82 mutation accumulation lines after 142 generations. A total of 31 mutations were identified in these lines. There was a strong correlation between allele size and mutation rate in P. pacificus, similar to Caenorhabditis elegans. In contrast to C. elegans, however, there is no evidence for a bias toward multistep mutations. The mutation spectrum of microsatellite loci in P. pacificus shows more insertions than deletions, indicating a tendency toward lengthening, a process that might have contributed to the increase in genome size. The mutation rates obtained for individual microsatellite markers provide guidelines for divergence time estimates that can be applied in P. pacificus next-generation sequencing approaches of wild isolates

Genetic signature of amphimixis allows for the detection and fine scale localization of sexual reproduction events in a mainly parthenogenetic nematode

International audienceAsexuality is an important mode of reproduction in eukaryotic taxa and has a theoretical advantage over sexual reproduction because of the increased ability to propagate genes. Despite this advantage, hidden signs of cryptic sex have been discovered in the genomes of asexual organisms. This has provided an interesting way to address the evolutionary impact of sex in plant and animal populations. However, the identification of rare sexual reproduction events in mainly asexual species has remained a challenging task. We examined the reproductive history in populations of the plant parasitic nematode Xiphinema index by genotyping individuals collected from six grapevine fields using seven microsatellite markers. A high level of linkage disequilibrium and heterozygous excess suggested a clonality rate of 95-100%. However, we also detected rare sexual reproduction events within these highly clonal populations. By combining highly polymorphic markers with an appropriate hierarchical sampling, and using both Bayesian and multivariate analysis with phylogenetic reconstructions, we were able to identify a small number of sexually produced individuals at the overlapping zones between different genetic clusters. This suggested that sexual reproduction was favoured when and where two nematode patches came into contact. Among fields, a high degree of genetic differentiation indicated a low level of gene flow between populations. Rare genotypes that were shared by several populations suggested passive dispersal by human activities, possibly through the introduction of infected plants from nurseries. We conclude that our method can be used to detect and locate sexual events in various predominantly asexual species