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International audience; The cyst nematode Heterodera carotae, which parasitizes carrot roots, has been recorded in many countries in Europe (Italy, The Netherlands, Switzerland, France, Denmark, …), in South Africa and in North America (Canada, USA). To date, there is a lack of knowledge about the genetic structure of the populations of this economically important nematode. The aim of this work was to study the structuration of the genetic diversity of the carrot cyst nematode at the European scale. We have developed a set of thirteen polymorphic microsatellite markers and used it to genotype seventeen European populations of H. carotae coming from France, Switzerland, Italy, Denmark and one non-European population from Canada. As previously showed for other cyst nematode species, the H. carotae populations were characterised by a strong heterozygote deficit. A Bayesian clustering analysis revealed two distinct genetic clusters, with one group located in the north of Europe and a second one located in the south of Europe. Moreover, our results highlighted rather limited gene flow at small spatial scale and some events of long distance migration. This first investigation of the genetic diversity of H. carotae populations would be useful to develop sustainable control strategies.

Cyst Nematodes of the genus Globodera are among the most studied plant parasitic nematodes because of their economic impact on crop production. Originated from high plains of the Andes, this genus would have been imported in Europe during the late XIXth century. The existence of a cryptic species complex within G. pallida – one of the most damaging species in the genus – is questioned. The taxonomic revision of this species could have major consequences on both risk assessment and management. During this work, an integrative taxonomic approach was implemented and has involved three disciplinary fields: genetics, morphometry and biology. Genetic diversity investigation revealed two groups differing from G. pallida. This study also raises the question of the processes implied in this divergence, apparently distinct for these two groupsMorphological differentiation was also demonstrated with an automatic image analysis tool. This tool was specifically developed for this work and is an innovative approach in studying the morphometry of this genus. This differentiation is based on the combined integration of three metrics adapted to automatic morphometry. Differential study of each entity ‘s host-range showed differences between studied groups. This differentiation is observed thanks to the investigation of the quantitative aspect of the nematode multiplication. Overall, these results bring the necessary evidence to claim the existence of a new species. This discovery questions the need for adapting surveillance and management methods against nematodes of the genus Globodera., Les nématodes à kyste du genre Globodera sont parmi les phytoparasites les plus étudiés de par l’impact économique qu’ils peuvent engendrer sur les productions agricoles. Originaire des hauts plateaux de la cordillère des Andes ce genre aurait été importé en Europe à la fin du XIX siècle. L’existence d’un complexe d’espèce cryptique chez G. pallida, nématode phytoparasites d’importance au sein du genre, est questionnée. La révision taxonomique de cette espèce peut avoir des conséquences fortes en termes d’épidémiologie, d’évaluation et de gestion des risques. Durant ce travail une approche de taxonomie intégrative impliquant trois champs disciplinaires, génétique, morphométrique et biologique, a été développée sur un panel de populations sud-américaines pour délimiter de nouvelles frontières au sein de cette espèce. L’exploration de la diversité génétique a permis de révéler l’exitence de deux groupes fortement distants de G. pallidaCette étude questionne aussi les processus impliqués dans cette divergence qui semblent distincts pour ces deux groupes. L’existence d’une différenciation morphologique a été observée grâce à un outil d’analyse d’image automatisé créé spécifiquement pour ce travail. Le développement de l’outil constitue une approche novatrice pour l’étude de la morphométrie dans ce genre et plus généralement chez les nématodes. Cette différenciation s’appuie sur l’intégration combinée de trois métriques adaptées à la morphométrie automatisée. Enfin, l’étude de la capacité de ces entités à se développer aux dépens de certaines plantes a elle aussi rapporté des

Le nématode à kyste du soja (NKS), Heterodera glycines, est un nématode phytoparasite causant des pertes de rendement considérables dans la culture de soja. Il a été détecté pour la première fois au Québec en 2013. Nous présentons ici les résultats d’une enquête visant à déterminer les régions affectées par ce ravageur et la densité de populations présentes au Québec. Au cours des trois dernières années (2013-2015), le NKS a été détecté dans 54 échantillons (32 % des champs testés) répartis dans les 10 régions productrices de soja du Québec. Les densités de populations trouvées étaient par contre très faibles, à la limite de détection. Parmi les régions testées, l’Estrie présentait la plus forte proportion de champs positifs (63 %). Étonnamment, cette proportion n’était que de 7 % en Montérégie-Ouest, là où la première détection a pourtant eu lieu et où se concentre la production de soja. Dans l’ensemble, la qualité des kystes récupérés était pauvre et le nombre d’oeufs très faible. Même si les populations sont encore négligeables, le NKS est bel et bien présent au Québec et il ne pourra pas être éradiqué. Diverses mesures devraient donc être mises en oeuvre afin de limiter la dispersion et l’accroissement des populations de ce ravageur important., The soybean cyst nematode (SCN), Heterodera glycines, is a plant parasite that causes considerable yield loss in soybean crop. It was detected for the first time in Quebec in 2013. Here, we present the results of a survey aimed at identifying the regions affected by this pest and the density of its populations in Quebec. Over the last 3 years (2013-2015), SCN was detected in 54 samples (32% of fields tested) from the regions that produce soybean in Quebec. However, population density was very low, barely reaching the point of detection. Among the tested regions, the Eastern Townships had the highest proportion of fields that tested positive (63%). Surprisingly, this proportion was only 7% in Montérégie-Ouest, the centre of soybean production in Quebec, where SCN was first detected. Overall, the quality of the cysts collected was poor and the number of eggs was very low. Even if its populations are still low, SCN is present in Quebec and will not be eradicated. Therefore, different measures should be enforced to limit the spread and population growth of this important pest.

Background In hyperspecialized parasites, the ability to grow on a particular host relies on specific virulence factors called effectors. These excreted proteins are involved in the molecular mechanisms of parasitism and distinguish virulent pathogens from non-virulent related species. The potato cyst nematodes (PCN) Globodera rostochiensis and G. pallida are major plant-parasitic nematodes developing on numerous solanaceous species including potato. Their close relatives, G. tabacum and G. mexicana are stimulated by potato root diffusate but unable to establish a feeding site on this plant host. Results RNA sequencing was used to characterize transcriptomic differences among these four Globodera species and to identify genes associated with host specificity. We identified seven transcripts that were unique to PCN species, including a protein involved in ubiquitination. We also found 545 genes that were differentially expressed between PCN and non-PCN species, including 78 genes coding for effector proteins, which represent more than a 6-fold enrichment compared to the whole transcriptome. Gene polymorphism analysis identified 359 homozygous non-synonymous variants showing a strong evidence for selection in PCN species. Conclusions Overall, we demonstrated that the determinant of host specificity resides in the regulation of essential effector gene expression that could be under the control of a single or of very few regulatory genes. Such genes are therefore promising targets for the development of novel and more sustainable resistances against potato cyst nematodes. Electronic supplementary material The online version of this article (10.1186/s12864-019-5853-4) contains supplementary material, which is available to authorized users.

International audience; It is widely accepted that climate has an essential influence on the distribution of species and that temperature is the major abiotic factor that affects their life-history traits. Species with very restricted active dispersal abilities and a wide geographical distribution are thus expected to encompass distinct populations adapted to contrasted local conditions. The beet cyst nematode Heterodera schachtii is a good biological model to study temperature adaptation in populations collected from different environments. Here, we tested the effect of temperature on H. schachtii life-history traits using seven field populations from Morocco, Spain, France, Germany, Austria, Poland and Ukraine. We tested hatching and multiplication rates of each population at different temperatures, as well as hatching rates of each population after storage at different temperatures – simulating survival conditions during the inter-cropping period. Results showed a strong temperature effect on the life-history traits explored. Temperature impact on hatching (at different temperatures and after storage at different temperatures) depended on the origin of populations, separating southern from northern ones. Surprisingly, low temperatures influenced hatching less in southern populations. However, for these populations, a storage period at low temperatures strongly reduce subsequent hatching. Conversely, nematode multiplication was not differentially affected by temperatures, as favorable conditions for the host are also favorable for the parasite. Finally, a significant correlation between the genetic diversity and the level of specialization showed that the less diverse populations were more specialized than the more diverse ones..

International audience; Trade-offs between virulence (defined as the ability to infect a resistant host) and life-history traits are of particular interest in plant pathogens for durable management of plant resistances. Adaptation to plant resistances (i.e., virulence acquisition) is indeed expected to be associated with a fitness cost on susceptible hosts. Here, we investigated whether life-history traits involved in the fitness of the potato cyst nematode Globodera pallida are affected in a virulent lineage compared to an avirulent one. Both lineages were obtained from the same natural population through experimental evolution on resistant and susceptible hosts, respectively. Unexpectedly, we found that virulent lineages were more fit than avirulent lineages on susceptible hosts: they produced bigger cysts, containing more larvae and hatching faster. We thus discuss possible reasons explaining why virulence did not spread into natural G. pallida populations.

International audience; The sustainability of modern agriculture relies on strategies that can control the ability of pathogens to overcome chemicals or genetic resistances through natural selection. This evolutionary potential, which depends partly on effective population size (Ne), is greatly influenced by human activities. In this context, wild pathogen populations can provide valuable information for assessing the long-term risk associated with crop pests. In this study, we estimated the effective population size of the beet cyst nematode, Heterodera schachtii, by sampling 34 populations infecting the sea beet Beta vulgaris spp. maritima twice within a one-year period. Only 20 populations produced enough generations to analyze the variation in allele frequencies, with the remaining populations showing a high mortality rate of the host plant after only one year. The 20 analyzed populations showed surprisingly low effective population sizes, with most having Ne close to 85 individuals. We attribute these low values to the variation in population size through time, systematic inbreeding, and unbalanced sex- ratios. Our results suggest that H. schachtii has low evolutionary potential in natural environments. Pest control strategies in which populations on crops mimic wild populations may help prevent parasite adaptation to host resistance.

International audience; Studying wild pathogen populations in natural ecosystems offers the opportunity to better understand the evolutionary dynamics of biotic diseases in crops and to enhance pest control strategies. We used simulations and genetic markers to investigate the spatial and temporal population genetic structure of wild populations of the beet cyst nematode Heterodera schachtii on a wild host plant species, the sea beet (Beta vulgaris spp. maritima), the wild ancestor of cultivated beets. Our analysis of the variation of eight microsatellite loci across four study sites showed that (i) wild H. schachtii populations displayed fine-scaled genetic structure with no evidence of substantial levels of gene flow beyond the scale of the host plant, and comparisons with simulations indicated that (ii) genetic drift substantially affected the residual signals of isolation-by-distance processes, leading to departures from migration–drift equilibrium. In contrast to what can be suspected for (crop) field populations, this showed that wild cyst nematodes have very low dispersal capabilities and are strongly disconnected from each other. Our results provide some key elements for designing pest control strategies , such as decreasing passive dispersal events to limit the spread of virulence among field nematode populations.

Transfer cells are ubiquitous plant cells that play an important role in plant development as well as in responses to biotic and abiotic stresses. They are highly specialized and differentiated cells playing a central role in the acquisition, distribution and exchange of nutrients. Their unique structural traits are characterized by augmented ingrowths of invaginated secondary wall material, unsheathed by an amplified area of plasma membrane enriched in a suite of solute transporters. Similar morphological features can be perceived in vascular root feeding cells induced by sedentary plant-parasitic nematodes, such as root-knot and cyst nematodes, in a wide range of plant hosts. Despite their close phylogenetic relationship, these obligatory biotrophic plant pathogens engage different approaches when reprogramming root cells into giant cells or syncytia, respectively. Both nematode feeding-cells types will serve as the main source of nutrients until the end of the nematode life cycle. In both cases, these nematodes are able to remarkably maneuver and reprogram plant host cells. In this review we will discuss the structural, functional and morphogenetic characteristics function and formation of these specialized multinucleate cells that act as nutrient transfer cells to accumulate and synthesize components needed for survival and successful offspring of plant-parasitic nematodes. Plant cells with transfer-like functions are also a renowned subject of interest involving still poorly understood molecular and cellular transport processes.

International audience; Potato cyst nematodes induce changes in plant host gene expression following root invasion. For an accurate comparison of gene expression by reverse transcription quantitative real-time PCR (RT-qPCR), internal reference genes are necessary for transcript normalization. Very few experimental data on suitable reference genes are available for interactions between plant and root pathogens. In this study, we tested eight potential candidate reference genes for normalizing levels of potato gene transcripts by RT-qPCR after inoculation by nematodes. The ranking of candidate reference genes was performed using RefFinder WEB-based software. Four reference genes, RPN7 (26S proteasome regulatory subunit), UBP22 (ubiquitin-specific protease 22), OXA1 (OXA1 protein), and MST2 (mercaptopyruvate sulfurtransferase), were stably expressed in roots of susceptible or resistant potato plants, infected or uninfected by Globodera pallida. A normalization factor based on data from these four genes, highly homologous between potato and tomato, was used to normalize the expression of a chitinase gene, which was induced by nematodes in roots of potatoes carrying the resistance allele at a low-effect QTL, GpaXIspl. .

Les nématodes à kystes sont l’un des bioagresseurs causant le plus de dégâts sur les cultures de pommes de terre. La résistance trouvée chez l'accession spl88S.329.18, issue de l'espèce Solanum sparsipilum est caractérisée par un déterminisme oligogénique avec un QTL à effet fort localisé sur le chromosome V (GpaVspl) et un QTL mineur localisé sur le chromosome XI (GpaXIspl). Pour obtenir une résistance de haut niveau, l'effet du QTL GpaVspl, doit être complémenté par celui du QTL à effet faible GpaXIspl. Par génomique comparative, le locus GpaV a été localisé dans un intervalle compris entre 16 et 60 kb sur les génomes de la tomate et des espèces apparentées à la pomme de terre, Solanum demissum et Solanum phureja. Deux gènes ont été annotés dans cet intervalle sur les génomes de la tomate et de S. demissum: le premier appartient à la famille des TIR-NBS-LRR (TNL), famille de gènes de résistance classiques, et le second appartient à la famille des « mitochondrial, transcription termination factor » (mTERF), dont l’implication dans des mécanismes de résistance n’a jamais été démontrée. Les objectifs de ma thèse étaient d'identifier le(s) gène(s) responsable(s) de la résistance à G. pallida, conférée par le locus GpaVspl, et d'étudier sa régulation. Suite à la publication de la séquence du génome de S. phureja, en 2011, nous avons mis en évidence que le locus GpaV était dupliqué chez S. phureja et que cette duplication était également présente chez S. sparsipilum. Les quatre gènes annotés au locus GpaVspl ont été nommés Spl_mTERF18430, Spl_TNL18429, Spl_mTERF18453 et Spl_TNL18428. L'effet des deux gènes Spl_mTERF18430 et Spl_TNL18428 sur la résistance à G. pallida a été analysé via des expériences de transformation génétique suivies par des tests de résistance sur les plantes transformées. Un effet partiel du gène Spl_TNL18428 sur la résistance à G. pallida a été mis en évidence par complémentation de plantes sensibles. Aucun effet significatif n'a été détecté pour le gène Spl_mTERF18430. Des expériences d'extinction génique suggèrent que le deuxième gène TIR-NBS-LRR, Spl_TNL18429, qui est également exprimé dans les racines et qui présente un fort pourcentage d'identité de séquence avec le gène Spl_TNL18428, pourrait également être impliqué dans la résistance à G. pallida. L'expression du gène rapporteur GFP, placé sous le contrôle du promoteur du gène Spl_TNL18428, est fortement induite dans les cellules situées autour du syncytium. Ceci renforce l'hypothèse d'une implication du gène Spl_TNL18428 dans la résistance à G. pallida, car la localisation de l'expression de la GFP est similaire à celle de la nécrose, qui est caractéristique de la réaction développée par les plantes résistantes autour du syncytium induit par les nématodes. En tenant compte des données bibliographiques récentes, montrant que plusieurs gènes NBS-LRR peuvent être indispensables à l'expression d'une résistance, nos résultats suggèrent que les deux gènes Spl_TNL18428 et Spl_TNL18429 sont nécessaires à l'expression de la résistance à G. pallida.

National audience

International audience; In order to identify genes involved in pathogenicity, we compared the closely related species Globodera pallida (GP) and Globodera "mexicana" (GM) that have different host ranges and are able to produce viable and fertile hybrids. Three pioneer genes were previously identified as differentially expressed between GP and GM: GPLIA7 and GPLIB3 were found to be more highly expressed in GP, whereas GMLIVG9 was found more highly expressed in GM. In this study, we showed that Ia7 and IVg9 genes probably encode products secreted by the subventral oesophageal glands and the dorsal oesophageal gland, respectively. No Blast homolog was found in the databases, but a metridin-like ShK (Stichodactyla helianthus) toxin domain was identified in the Ia7 sequence. Analysis of the full-length sequences of these 2 genes between GP and GM revealed a high level of interspecies variability (8% for the Ia7 transcript and 17% for the IVg9 transcript) and a high proportion (90%) of nonsynonymous mutations among the substitutions observed. This suggested that these 2 pioneer genes are under strong diversifying selection pressures and therefore may be involved in pathogenicity. Further investigations of the sequence polymorphism of Ia7 and IVg9 genes were conducted in GP x GM hybrid lines that were selected in laboratory conditions for their different ability to develop on potato and black nightshade. As similar sequences were obtained for all the hybrid lines tested independently of their pathogenicity status, no correlation could be established between IA7 and IVG9 amino acid changes and the host range differences observed between GP and GM