Development of Bursaphelenchus xylophilus-specific microsatellite markers to assess the genetic diversity of populations from European forests.

The pinewood nematode (PWN), Bursaphelenchus xylophilus (Steiner & Buhrer, 1934), Nickle (Nematoda: Aphelenchoididae) is the causal agent of the pine wilt disease and is currently considered as one of the most important pests and pathogens in the world. Its introduction and spread in new forest ecosystems have considerable consequences both economically and environmentally. Therefore, it is of crucial importance to identify its invasion routes, to determine the origin of new outbreaks and to understand the invasion process of this species to prevent further dissemination of the disease in Europe. In order to address these questions using population genetic approaches, we have been developing a set of PWN-specific microsatellite markers, usable in routine conditions at the individual level, thanks to multiplex PCR coupled with a fast DNA extraction method. Microsatellites were isolated from a genomic library using a procedure combining DNA enrichment and high throughput pyrosequencing as recently described by Malausa et al. (2011). Primers were designed for 71 and 23 perfect and compound microsatellites, respectively, 26 of which were experimentally validated so far. Among them, 18 markers exhibited polymorphism after several rounds of amplification tests. Preliminary results on a set of 190 nematodes from 13 populations indicate a very low level of polymorphism in PWN populations from Portugal and Madeira Island, compared to populations from the native area in North America. The genotyping of a wide collection of samples from Europe, Asia and North America is currently underway in the laboratory. Assessing the genetic diversity of populations indeed constitutes the cornerstone to determine whether the European invasive PWN populations are the result of a single or several independent events of introduction

Variability among four populations of Meloidogyne javanica from Brazil

La morphologie, les profils isoenzymatiques, la caryolgie, la gamme d'hôtes et l'analyse de l'ADN (PCR-RAPD) de quatre populations atypiques de #Meloidogyne javanica originaires du Brésil (P1-P4) ont été comparés à ceux de deux populations typiques appartenant à la même espèce (S1 et S2). Ces approches ont permis la séparation de ces populations en deux groupes : P1, S1 et S2, d'une part et P3 et P4, d'autre part. En se basant sur l'analyse de l'ADN, la population P2 présente une position intermédiaire, mais semble très proche de P1 d'après ses caractères morphologiques et le nombre de ses chromosomes. Elle en diffère cependant par son phénotype estérasique et par sa gamme d'hôtes. La technique de PCR-RAPD a permis la détection d'une variabilité intraspécifique notable. Cela représente un avantage certain par rapport à l'utilisation des isoenzymes et des gammes d'hôtes, dans la mesure où le nombre d'hôtes différentiels et de systèmes enzymatiques disponsibles est limité. Par comparaison avec d'autres études, nos résultats mettent en évidence une variabilité intraspécifique élevée chez #M. javanica, espèce considérée jusque là comme relativement homogène. (Résumé d'auteur

Analysis of the transcriptome of the infective stage of the beet cyst nematode, H. schachtii

The beet cyst nematode, Heterodera schachtii, is a major root pest that significantly impacts the yield of sugar beet, brassicas and related species. There has been limited molecular characterisation of this important plant pathogen: to identify target genes for its control the transcriptome of the pre-parasitic J2 stage of H. schachtii was sequenced using Roche GS FLX. Ninety seven percent of reads (i.e., 387,668) with an average PHRED score > 22 were assembled with CAP3 and CLC Genomics Workbench into 37,345 and 47,263 contigs, respectively. The transcripts were annotated by comparing with gene and genomic sequences of other nematodes and annotated proteins on public databases. The annotated transcripts were much more similar to sequences of Heterodera glycines than to those of Globodera pallida and root knot nematodes (Meloidogyne spp.). Analysis of these transcripts showed that a subset of 2,918 transcripts was common to free-living and plant parasitic nematodes suggesting that this subset is involved in general nematode metabolism and development. A set of 148 contigs and 183 singletons encoding putative homologues of effectors previously characterised for plant parasitic nematodes were also identified: these are known to be important for parasitism of host plants during migration through tissues or feeding from cells or are thought to be involved in evasion or modulation of host defences. In addition, the presence of sequences from a nematode virus is suggested. The sequencing and annotation of this transcriptome significantly adds to the genetic data available for H. schachtii, and identifies genes primed to undertake required roles in the critical pre-parasitic and early post-parasitic J2 stages. These data provide new information for identifying potential gene targets for future protection of susceptible crops against H. schachtii

First insights into the genetic diversity of the pinewood nematode in its native area and around the world.

The pinewood nematode (PWN), Bursaphelenchus xylophilus is the causal agent of the pine wilt disease and is currently considered as one of the most important pests and pathogens in forest ecosystems. Native to North America, it has been introduced and it has spread in pine forests in Asia and more recently in Europe where it has now considerable economic and environmental impacts (annual loss of millions of pine trees worldwide). Anticipating the possibility of expansion of the PWN in European forests is essential. It is therefore important to decipher the invasion routes and better understand the invasion process of this species. To do this, 16 microsatellite markers have been developed and the study of genetic variability of the PWN was undertaken

Evaluation expérimentale de stratégies de déploiement de gènes de résistance pour la gestion durable des nématodes à galles

Dans le cadre de projets soutenus par l'ANR Systerra et le GIS PICLeg (projets "Sysbiotel" et "Neoleg") menés en collaboration entre l'INRA PACA, l'IRD, l'APREL, la Chambre d'Agriculture du 06 et des entreprises privées de sélection de semences, plusieurs stratégies de déploiement de gènes de résistance ont été évaluées pendant 3 ans sur le terrain en conditions agronomiques pour mettre au point une gestion raisonnée des cultivars résistants permettant de gérer de manière durable les problèmes de nématodes à galles des racines. L'alternance des gènes de résistance dans la rotation et le "pyramiding" de gènes dans un même cultivar se sont révélés extrêmement efficaces pour supprimer l'émergence de populations virulentes et réduire les taux d'infestation du sol de plus de 80% en 3 ans. Un nouveau projet INRA "Gedunem", mis en place dans le cadre du Métaprogramme INRA SMaCH (Sustainable Management of Crop Health), vise maintenant à associer ces innovations variétales aux autres méthodes de lutte disponibles (gestion de l'interculture, plantes non hôtes, prophylaxie) afin de maintenir une pression parasitaire faible, tout en évaluant ces nouveaux systèmes de culture du point de vue agronomique et socio-économique