Microsatellite and Mitochondrial Data Provide Evidence for a Single Major Introduction for the Neartic Leafhopper Scaphoideus titanus in Europe

Scaphoideus titanus, a leafhopper native to North America and invasive in Europe, is the vector of the Flavescence dorée phytoplasma, the causal agent of the most important form of grapevine yellows in European vineyards. We studied 10 polymorphic microsatellite loci and a 623 bp fragment of the mitochondrial cytochrome oxidase II gene in native S. titanus from north-eastern America and introduced European populations, to elucidate the colonization scenario. Consistent with their recent history, invasive European populations were less genetically diverse than American populations for both types of markers, suggesting a recent bottleneck. Significant isolation by distance was detected between American populations but not between European populations. None of the European mitochondrial haplotypes was found in the American vineyards, from which they are assumed to have originated. The precise source of the invasive S. titanus populations therefore remains unclear. Nevertheless, the high heterozygosity of North-East American populations (which contained 92% of the observed alleles) suggests that this region is part of the native range of S. titanus. Clustering population genetics analyses with microsatellite and mitochondrial data suggested that European populations originated from a single introduction event. Most of the introduced populations clustered with populations from Long Island, the Atlantic Coast winegrowing region in which Vitis aestivalis occurs

Twelve polymorphic expressed sequence tags-derived markers for Plasmopara halstedii, the causal agent of sunflower downy mildew

Twelve expressed sequence tags-derived markers were isolated from Plasmopara halstedii (Oomycetes), the causal agent of sunflower downy mildew. A total of 25 single nucleotide polymorphisms and five indels were detected by single-strand conformation polymorphism analysis and developed for high-throughput genotyping of 32 isolates. There was a high level of genetic diversity (HE = 0.484). Observed heterozygosity ranged from 0 to 0.143 indicating that P. halstedii is probably a selfing species. These markers were also useful in detecting significant genetic variations among French populations (FST = 0.193) and between French and Russian populations (FST = 0.23). Cross-amplification tests on three closely related species indicated that no loci amplified in other Oomycete species

Spatial distribution of lineages of oak powdery mildew fungi in France, using quick molecular detection methods

*L’oïdium est l’une des maladies fongiques les plus importantes sur chênes en Europe. Des études récentes basées sur l’internal transcribed spacer (ITS) ont suggéré l’implication de quatre lignées (espèces putatives) différentes. L’objectif de l’étude était de caractériser la distribution spatiale de ces lignées/espèces et en particulier de tester l’hypothèse d’une différenciation spatiale à plusieurs échelles : régionale (France), altitudinale (transect pyrénéen) et locale (au sein d’une parcelle forestière).*Des méthodes de détection des quatre types d’ITS ont été développées : (1) analyse SCCP ; (2) amplification par PCR de l’ITS par des amorces spécifiques. La SSCP a permis de détecter facilement les ITS correspondant à E. alphitoides et E. quercicola. Par contre, les ITS plus rares correspondant à E. hypophylla et Phyllactinia guttata (sensu lato) n’ont été détectés que par amplification spécifique.*L’étude a confirmé la forte prédominance (avec une fréquence supérieure à 80 %) de la séquence d’ITS associée à E. alphitoides. Les isolats présentant la même séquence d’ITS qu’E quercicola, espèce récemment décrite et encore non mentionnée en Europe, ont également été retrouvés dans toutes les régions françaises à une fréquence importante (15 % en moyenne pour l’échantillonnage national).*Aucune différenciation spatiale entre les espèces putatives n’a pu être mise en évidence : E. alphitoides a souvent été détectée en association avec des isolats de différents types d’ITS dans la même région, le même arbre, voire la même lésion.*Powdery mildew is a major fungal disease of oaks in Europe. Recent studies using internal transcribed spacer (ITS) sequences suggested the presence of four different lineages (putative species). The objective of the study was to investigate the spatial distribution of these lineages/species and, in particular, to test the hypothesis of a spatial differentiation, at various scales: regional (France), altitudinal (a Pyrenean transect) and local (within a forest plot).*Detection methods for the four ITS types were developed: (1) single strand conformation polymorphism analysis (SSCP); (2) PCR amplifications, for which specific primers were designed. SSCP proved to be efficient for the detection of Erysiphe alphitoides and E. quercicola types. In contrast, the rarer ITS types corresponding to E. hypophylla and Phyllactinia guttata (sensu lato) were only detected by specific amplification.*The study confirmed the strong predominance of the ITS sequence associated with E. alphitoides at all spatial scales (with a frequency higher than 80%). Isolates presumably belonging to E. quercicola (i.e. with same ITS type), a recently described species not yet recorded in Europe, were also found in all French regions at a significant frequency (15% at national level).*No pattern of spatial differentiation between the putative species could be demonstrated: E. alphitoides was often found in association with different ITS types in the same region, the same tree, and even in the same lesion

Development of new oomycete taxon-specific mitochondrial cytochrome b region primers for use in phylogenetic and phylogeographic studies

International audienceHere, we describe the development of an oomycete-specific primer pair for amplification of the cytochrome b region in plant pathogenic species that span the order Peronosporales (Phytophthora spp., downy mildews). Because of the high number of variable sites at both inter- and intra-specific levels this marker provides a powerful tool for population genetics and phylogenetic studies in this taxa. We also demonstrate its potential compared with other oomycete-specific mitochondrial markers currently available

Genetic structure of Scaphoideus titanus populations and genetic diversity of the epidemic strains of "Flavescence dorée" phytoplasma : the situation in France

International audienceThe genetic diversity and structure of the Scaphoideus titanus leafhopper populations and of the "flavescence doree" phytoplasma (FDp) they carry, was studied in parallel in southern French vineyards. Seven polymorphic microsatellite loci were used to genotype S. titanus individuals sampled in 24 healthy or FDp-infected vineyards. For each insect, FDp detection and typing was performed by amplifying and sequencing the FDp map gene. A low level of population genetic differentiation (F-ST <= 0.114) was found in S. titanus French populations. It suggests that long-distance gene flows occur due to migration between populations. In addition, it can be hypothesized that passive transport of grapevine planting material carrying eggs could play a role in homogenizing S. titanus population genetic structure. No significant correlation was found between the genetic structure of the French S. titanus populations and the distribution of the different strain types they carry

Genetic structure of Scaphoideus titanus populations and genetic diversity of the epidemic strains of "Flavescence dorée" phytoplasma : the situation in France

International audienceThe genetic diversity and structure of the Scaphoideus titanus leafhopper populations and of the "flavescence doree" phytoplasma (FDp) they carry, was studied in parallel in southern French vineyards. Seven polymorphic microsatellite loci were used to genotype S. titanus individuals sampled in 24 healthy or FDp-infected vineyards. For each insect, FDp detection and typing was performed by amplifying and sequencing the FDp map gene. A low level of population genetic differentiation (F-ST <= 0.114) was found in S. titanus French populations. It suggests that long-distance gene flows occur due to migration between populations. In addition, it can be hypothesized that passive transport of grapevine planting material carrying eggs could play a role in homogenizing S. titanus population genetic structure. No significant correlation was found between the genetic structure of the French S. titanus populations and the distribution of the different strain types they carry

Comparing the spatial genetic structures of the Flavescence dorée phytoplasma and its leafhopper vector Scaphoideus titanus

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EST-derived markers highlight genetic relationships among Plasmopara halstedii French races

Douze marqueurs dérivés d'EST ont été isolés chez Plasmopara halstedii (Oomycetes), l'agent du mildiou du tournesol. L'analyse par SSCP du polymorphisme de ces marqueurs a permis de révéler la présence de 25 single nucleotide polymorphisms (SNPs) et cinq insertion-délétions. Ces 12 marqueurs ont été utilisés pour génotyper 24 isolats de P. halstedii représentant les 14 races de mildiou du tournesol connues en France à ce jour. L'analyse génétique a mis en évidence 13 génotypes multilocus différents et l'existence d'un fort déficit en héterozygotes qui suggère que cette espèce est fortement autogame. Les isolats se structurent en trois groupes génétiquement bien différentiés organisés autour des 3 premiers pathotypes observés en France. Ce dernier résultat suggère que les populations de mildiou du tournesol pourraient résulter de trois introductions successives de P. halstedii en France

From molecular evolution to population genetics of strobilurin resistance in grapevine downy mildew populations

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Single nucleotide polymorphisms reveal multiple introductions into France of Plasmopara halstedii, the plant pathogen causing sunflower downy mildew

Plasmopara halstedii, the causal agent of sunflower downy mildew, displays a gene-for-gene interaction with its host plant, Helianthus annuus and other species of the genus. Monitoring of the evolution of virulent races in France over a 19-year period led to the identification of 14 different races (or pathotypes). Twelve expressed sequence tag (EST)-derived markers displaying SNPs and insertion-deletion variations have recently been identified in P. halstedii. We used these markers to study the genetic structure and the evolution of sunflower downy mildew races. Bayesian assignment analysis identified three genetically differentiated groups of isolates organized around the first three races described in France. Strong genetic substructuring according to geographic origin of races was observed, confirming that these three groups corresponded to three separate introductions into France of isolates with different genetic and phenotypic backgrounds. Our results suggest that multiple introductions of P. halstedii isolates may have provided the raw material for more complex processes in the evolution of races, such as recombination between races or clonal evolution through mitotic instability