Satellite DNA as a target for TaqMan real-time PCR detection of the pinewood nematode, Bursaphelenchus xylophilus

The pinewood nematode (PWN), Bursaphelenchus xylophilus , is a major pathogen of conifers, which impacts on forest health, natural ecosystem stability and international trade. As a consequence, it has been listed as a quarantine organism in Europe. A real-time PCR approach based on TaqMan chemistry was developed to detect this organism. Specific probe and primers were designed based on the sequence of the Msp I satellite DNA family previously characterized in the genome of the nematode. The method proved to be specific in tests with target DNA from PWN isolates from worldwide origin. From a practical point of view, detection limit was 1 pg of target DNA or one individual nematode. In addition, PWN genomic DNA or single individuals were positively detected in mixed samples in which B. xylophilius was associated with the closely related non-pathogenic species B. mucronatus , up to the limit of 0.01% or 1% of the mixture, respectively. The real-time PCR assay was also used in conjunction with a simple DNA extraction method to detect PWN directly in artificially infested wood samples. These results demonstrate the potential of this assay to provide rapid, accurate and sensitive molecular identification of the PWN in relation to pest risk assessment in the field and quarantine regulation

Sequence variability of the MspI satellite DNA family of the pinewood nematode Bursaphelenchus xylophilus at different geographic scales

Tandemly repeated sequences known as satellite DNA (satDNA) generally exhibit complex evolutionary patterns of concerted evolution in which mutations are homogenized and fixed in a stochastic process of molecular drive. Here, the nucleotidic variability of the MspI satDNA family of the pinewood nematode Bursaphelenchus xylophilus is analyzed in order to understand the evolutionary dynamics of satDNA at the intraspecific level. A total of 425 MspI monomer units, either PCR-amplified from isolates of local (Peninsula of Setúbal, Portugal) or worldwide origin, or retrieved from the B. xylophilus genome sequence, were characterized and compared. Whatever their origin, sliding window analysis of sequence variability patterns among monomers revealed low, moderate and highly variant domains, indicating that variable levels of evolutionary constraint may act upon the entire monomers. The phylogenetic inference based on the different sets of MspI satDNA family for this species shows a broad polymorphism of the individual monomers, which were distributed into four main clusters. However, such clustering appeared indepen- dent from the geographic origin of the nematodes, and could not discriminate isolates or groups of geo- graphically close isolates. Rather, the formation of different phylogenetic groups within this satDNA family suggests an a priori embodying of a set of diverging repeats from a common ancestor satDNA library, which have been differently amplified along the evolutionary pathway of this species. The present work improves knowledge on the evolutionary dynamics of satDNA at the intraspecific level, and pro- vides new information on satDNA sequence variability among natural populations sampled at a local geo- graphic scale

Satellite DNA-based species-specific identification of single individuals of the pinewood nematode Bursaphelenchus xylophilus (Nematoda : Aphelenchoididae)

International audienceThe pinewood nematode Bursaphelenchus xylophilus is a severe pest of coniferous trees, and has been designated as a quarantine organism in the European Union. From the sequence of a satellite DNA family characterized in the genome of this nematode, we developed a PCR procedure that allowed the specific discrimination of this species from closely related Bursaphelenchus species found on coniferous trees. Moreover, because of the repetitive nature of satellite DNA, positive amplification was achieved from B. xylophilus single individuals, which should contribute to an easy diagnostic procedure for assisting in the management of this major pest of conifer forests

Inference of the worldwide invasion routes of the pinewood nematode, Bursaphelenchus xylophilus, using ABC analysis on microsatellite data

Population genetics have been greatly beneficial to improve knowledge about biological invasions. Quantitative genetic methods, such as Approximate Bayesian Computation (ABC), have brought this improvement up a notch and are now essential tools to decipher the invasion routes of any invasive species. In this paper, we performed ABC analyses to shed light on the pinewood nematode (PWN) worldwide invasion routes and identify the source of the European populations. Originating from North America, this microscopic worm has been invading Asia since 1905 and Europe since 1999, causing tremendous damage on pine forests. Using microsatellite data, we demonstrated the existence of multiple independent events of introduction in Japan (one from the USA and one with an unknown origin) and China (one from the USA and one from Japan). We also found an American origin of the Portuguese samples. Although, we observed some discrepancies between traditional genetic methods and the ABC method, which are worth investigating and are discussed here, the ABC approach definitely helped clarify the worldwide history of the PWN invasion

Mariner element in the entomopathogenic Heterorhabditis bacteriophora

National audienc

First insights into the genetic diversity of the pinewood nematode in its native area using new polymorphic microsatellite loci.

The pinewood nematode, Bursaphelenchus xylophilus, native to North America, is the causative agent of pine wilt disease and among the most important invasive forest pests in the East-Asian countries, such as Japan and China. Since 1999, it has been found in Europe in the Iberian Peninsula, where it also causes significant damage. In a previous study, 94 pairs of microsatellite primers have been identified in silico in the pinewood nematode genome. In the present study, specific PCR amplifications and polymorphism tests to validate these loci were performed and 17 microsatellite loci that were suitable for routine analysis of B. xylophilus genetic diversity were selected. The polymorphism of these markers was evaluated on nematodes from four field origins and one laboratory collection strain, all originate from the native area. The number of alleles and the expected heterozygosity varied between 2 and 11 and between 0.039 and 0.777, respectively. First insights into the population genetic structure of B. xylophilus were obtained using clustering and multivariate methods on the genotypes obtained from the field samples. The results showed that the pinewood nematode genetic diversity is spatially structured at the scale of the pine tree and probably at larger scales. The role of dispersal by the insect vector versus human activities in shaping this structure is discussed

Characterization of a highly conserved satellite DNA from the parasitoid wasp Trichogramma brassicae

International audienc