The pine wood nematode (PWN), Bursaphelenchus xylophilus, the causal agent of pine wilt disease (PWD), is a major world-wide pathogen and pest of pine, with impacts on forest health, natural ecosystem stability and international trade. In Portugal, PWN was first diagnosed in 1999, the first occurrence also for Europe. The disease was recently detected on the island of Madeira and in northern Spain. In an attempt to search for more reliable and robust molecular markers that enable the study of intraspecific variability of B. xylophilus from different geographic locations, the intergenic spacer (IGS) region of the 5S rRNA gene and inter-simple sequence repeats (ISSR) analysis were used to determine the genetic relationships among 43 B. xylophilus isolates from Portugal, China, Japan, South Korea and USA. IGS sequence analysis showed that this region can only be used to establish interspecific relationships, since no differences were detected among Portuguese isolates from different geographic locations. Fingerprints obtained with ISSR show high genetic variability among Portuguese isolates, except for the ones obtained prior to 2008. The ISSR dendrogram suggests the spread of the disease inside continental Portugal and to Madeira. Until 2008, B. xylophilus populations found in continental Portugal showed low genetic diversity, pointing to a single introduction, probably from Asia, whereas recent populations from continental Portugal (2009-2010) and Madeira show high genetic diversity, suggesting multiple introductions from different origins.

A survey was conducted in the northern conifer forests of Turkey in 2003 and 2004 for the pinewood nematode, Bursaphelenchus xylophilus. Wood samples were collected from declining pine trees, located in the following target areas: Düzce, Ankara, Bolu, and Artvin. Nematodes were extracted from over 310 samples and were observed and identified. B. xylophilus was not detected in any samples. Bursaphelenchus species were only found in 6% of the samples. From the B. xylophilus‐group, only the species B. mucronatus was reported. Species identification was performed from morphological characters, particularly male spicules, and by molecular analysis with ITS‐RFLP.

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Entomopathogenic nematodes (EPN) are lethal parasites of insects, used as biocontrol agents. The objectives of this work were to survey the presence of EPN in continental Portugal and to characterize the different species. Of the 791 soil samples collected throughout continental Portugal, 53 were positive for EPN. Steinernema feltiae and Heterorhabditis bacteriophora were the two most abundant species. Analysis of EPN geographical distribution revealed an association between nematode species and vegetation type. Heterorhabditis bacteriophora was mostly found in the Alentejo region while S. feltiae was present in land occupied by agriculture with natural vegetation, broadleaved forest, mixed forest and transitional woodland-shrub, agro-forestry areas, complex cultivated patterns and non-irrigated arable land. Although no clear association was found between species and soil type, S. feltiae was typically recovered from cambisols and H. bacteriophora was more abundant in lithosols. Sequencing of the internal transcribed spacer (ITS) region indicated that S. feltiae was the most abundant species, followed by H. bacteriophora. Steinernema intermedium and S. kraussei were each isolated from one site and Steinernema sp. from two sites. Phylogenetic analyses of ITS, D2D3 expansion region of the 28S rRNA gene, as well as mitochondrial cytochrome c oxidase subunit I (COXI) and cytochrome b (cytb) genes, was performed to evaluate the genetic diversity of S. feltiae and H. bacteriophora. No significant genetic diversity was found among H. bacteriophora isolates. However, COXI seems to be the best marker to study genetic diversity of S. feltiae. This survey contributes to the understanding of EPN distribution in Europe.

Bursaphelenchus xylophilus is the causal agent of pine wilt disease and a worldwide pest with high economic impact. Since its first diagnosis in Portugal in 1999, it has been subjected to quarantine measures with impact on forest health and ecosystem stability, significantly affecting international trade of wood products. The disease was detected in the north and centre of continental Portugal and, since 2008, the whole country has been considered an affected area. Recently, it was detected in Madeira Island. In order to avoid new outbreaks, it has become of major importance to understand the patterns of spread, introduction points and to characterize the new populations from continental Portugal and Madeira Island. Mitochondrial cytochrome b (cytb) and parasitic cellulase gene sequences were used to evaluate the genetic relationships among isolates that could indicate possible origins of the new outbreaks. Portuguese isolates were compared with isolates from USA, China, Japan and South Korea, in order to investigate possible infection pathways and disease spread patterns in Portugal. Phylogenetic trees based on both genes show that Portuguese isolates group with Asian isolates. Isolates from USA are in a separate position in both gene trees. However, the phylogenetic tree based on the cellulase gene sequences shows higher differentiation among Portuguese isolates than that of cytb. These results agree with those previously obtained using inter-simple sequence repeats (ISSR). This was the first study to use cytb and cellulase genes to characterize pinewood nematode (PWN) populations. This study suggests that cellulase is a better marker than cytb to study genetic diversity in B. xylophilus.

Molecular plant breeding usually overlooks the genetic variability that arises from the association of plants with endophytic microorganisms, when looking at agronomic interesting target traits. This source of variability can have crucial effects on the functionality of the organism considered as a whole (the holobiont), and therefore can be selectable in breeding programs. However, seeing the holobiont as a unit for selection and improvement in breeding programs requires novel approaches for genotyping and phenotyping. These should not focus just at the plant level, but also include the associated endophytes and their functional effects on the plant, to make effective desirable trait screenings. The present review intends to draw attention to a new research field on functional hologenomics that if associated with adequate phenotyping tools could greatly increase the efficiency of breeding programs.

A survey for Bursaphelenchus nematodes, associated with different conifer trees, was conducted in several forest areas in the northern regions of Turkey. Only pine trees (Pinus nigra, P. pinaster and P. sylvestris) yielded Bursaphelenchus specimens. Nematodes were identified using several morphological diagnostic characters of the genus (male spicule structure, number of lateral incisures, number and distribution of the male papillae, presence of female vulval flap), and confirmed by using RFLP analysis of the internal transcriber spacer (ITS) regions of ribosomal DNA. Three different species were identified from several sampled areas, namely B. mucronatus, B. pinophilus and B. sexdentati, representing a first report of the last two species for Turkey. The association of B. pinophilus with black pine (P. nigra) is herein reported for the first time.

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