Species of Bursaphelenchus Fuchs, 1937 (Nematoda : Parasitaphelenchidae) associated with maritime pine in Portugal

Species of Bursaphelenchus associated with maritime pine, Pinus pinaster, from Portugal - within and outside the quarantine restricted demarcated zone of B. xylophilus - are described and characterised both morphologically (LM and SEM) and with the use of molecular biology (ITS-RFLP). A new staining method for spicules is proposed. Species include B. hellenicus, B. hylobianum, B. leoni, B. pinophilus, B. sexdentati, B. tusciae, B. teratospicularis, B. xylophilus and Bursaphelenchus sp. 1. Bursaphelenchus hylobianum was collected from the insect Hylobius sp. The most frequent species in the demarcated zone, besides B. xylophilus, was Bursaphelenchus sp. 1. Morphological characterisation is compared with the original descriptions and discussed. The differentiation between B. pinophilus and B. sexdentati is not clear in the literature and is discussed. Since differentiation of B. xylophilus (mucronate form) from B. mucronatus, and B. pinophilus from B. sexdentati, as well as their juvenile forms, is almost im possible on the basis of morphological features, a molecular approach based on ITS-RFLPs was used. Ribosomal DNA containing the 5.8S gene, the internal transcribed spacer region 1 and 2, and partial regions of 18S and 28S gene were amplified by PCR. Restriction profiles of the amplified products generated species-specific differences, leading to the unambiguous identification of isolates belonging to B. xylophilus, B. mucronatus, B. sexdentati, B. tusciae and B. hylobianum.