The environmental conditions in Madeira Island are favorable for the presence and dissemination of the pinewood nematode (PWN), Bursaphelenchus xylophilus. Five hundred Pinus pinaster wood samples were collected in several forest areas and PWN was detected in 22.8 % of the samples. Bursaphelenchus xylophilus isolates from Madeira Island displayed the species-specific diagnostic characters. A morphological variation in the female tail terminus was detected. In most females, the tail presented a broadly rounded terminus and, occasionally, a digitate terminus with a terminal nipple-like extension resembling a mucro. PCR ITS-RFLP analysis revealed that Madeira Island isolates exhibited patterns specific to the species B. xylophilus and similar to virulent isolates. Amplified ITS regions were further sequenced and no genetic diversity was found for this genomic region among 17 Portuguese isolates (Madeira Island and Continental Portugal). Phylogenetic analysis revealed that Portuguese isolates grouped with isolates from China, Korea and one isolate from Japan.

In 2013, during a field survey conducted in Portugal on potato, Solanum tuberosum , an unusual esterase ( EST ) phenotype was detected in a root‐knot nematode ( RKN ) from potato roots collected in Coimbra. This Portuguese isolate was purified and maintained on tomato, S. lycopersicum , and morphological, biochemical and molecular characteristics were studied. Perineal pattern morphology was highly variable, similar to Meloidogyne ethiopica and not useful for identification. The EST phenotype, from young egg‐laying females, displayed three bands similar to the Brazilian M. luci (L3) and distinct from M. ethiopica (E3). Phylogenetic analyses of mitochondrial cytochrome oxidase subunit I and the mitochondrial DNA region between COII and 16S rRNA genes revealed that the Portuguese isolate grouped with M. luci isolates close to M. ethiopica isolates. However, considering the ITS 1‐5.8S‐ ITS 2 region, the Portuguese isolate grouped with isolates of M. luci , M. ethiopica and M. hispanica , which limits the confidence of this region for M. luci diagnosis, and its differentiation from other species with morphological similarities. The M. luci pathogenicity to potato was also assessed in 16 commercial cultivars and compared with M. chitwoodi , considered to be a quarantine RKN species by EPPO . All potato cultivars were susceptible to both Meloidogyne species with gall indices of 5 and higher reproduction factor values ranging from 12.5 to 122.3, which suggests that M. luci may constitute a potential threat to potato production. In the present study, M. luci is reported for the first time attacking potato in Portugal.

The pinewood nematode, Bursaphelenchus xylophilus, recognized as a worldwide major forest pest, is a migratory endoparasitic nematode with capacity to feed on pine tissues and also on fungi colonizing the trees. Bursaphelenchus mucronatus, the closest related species, differs from B. xylophilus on its pathogenicity, making this nematode a good candidate for comparative analyses. Secretome profiles of B. xylophilus and B. mucronatus were obtained and proteomic differences were evaluated by quantitative SWATH-MS. From the 681 proteins initially identified, 422 were quantified and compared between B. xylophilus and B. mucronatus secretomes and from these, 243 proteins were found differentially regulated: 158 and 85 proteins were increased in B. xylophilus and B. mucronatus secretomes, respectively. While increased proteins in B. xylophilus secretome revealed a strong enrichment in proteins with peptidase activity, the increased proteins in B. mucronatus secretome were mainly related to oxidative stress responses. The changes in peptidases were evaluated at the transcription level by RT-qPCR, revealing a correlation between the mRNA levels of four cysteine peptidases with secretion levels. The analysis presented expands our knowledge about molecular basis of B. xylophilus and B. mucronatus hosts interaction and supports the hypothesis of a key role of secreted peptidases in B. xylophilus pathogenicity.

Pine wilt disease (PWD) is a devastating forest disease caused by the pinewood nematode (PWN), Bursaphelenchus xylophilus, a migratory endoparasite that infects several coniferous species. During the last 20 years, advances have been made for understanding the molecular bases of PWN-host trees interactions. Major advances emerged from transcriptomic and genomic studies, which revealed some unique features related to PWN pathogenicity and constituted fundamental data that allowed the development of postgenomic studies. Here we review the proteomic approaches that were applied to study PWD and integrated the current knowledge on the molecular basis of the PWN pathogenicity. Proteomics has been useful for understanding cellular activities and protein functions involved in PWN-host trees interactions, shedding light into the mechanisms associated with PWN pathogenicity and being promising tools to better clarify host trees PWN resistance/susceptibility.

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease. The international economic impact of the introduction of the PWN into new areas has highlighted the need for the development of accurate and reliable detection methods of B. xylophilus, which are essential to define aspects of its control and management. In the present study, a methodology was developed for the direct detection of PWN by conventional PCR assay, with a species specific set of primers based on PWN satellite DNA, using total DNA extracted directly from maritime pine, Pinus pinaster, wood and bark samples, and from the insect vector, Monochamus galloprovincialis. This methodology involves homogenisation of wood, bark and insects using liquid nitrogen, DNA extraction and one or two PCR amplification steps, which permit the rapid and direct detection of one single nematode present in 100 mg of wood and bark and in one entire insect without the preliminary steps of nematode extraction.