PHRAME Final Report Page 1 QLK5-CT-2002-00672: Development of improved pest risk analysis techniques for quarantine pests, using pinewood nematode, Bursaphelenchus xylophilus, in Portugal as a model system PHRAME – Plant Health Risk And Monitoring Evaluation FINAL REPORT, JULY 2007 Chapter 1 Membership of the PHRAME Consortium and authorship of Chapters and Sections......................7 Chapter 2 Introduction............................................................................................................................................... 9 2.1 Background and context .................................................................................................................. 9 2.2 The pest problem ............................................................................................................................. 9 2.3 Approach adopted.......................................................................................................................... 10 Chapter 3 Identification and distribution of nematodes in the genus Bursaphelenchus in Europe................... 12 3.1 Introduction..................................................................................................................................... 12 3.2 Materials and Methods................................................................................................................... 12 3.2.1 Nematode sampling and isolation.............................................................................................. 12 3.2.2 Morphological characterisation .................................................................................................. 14 3.2.3 Biometric characterisation.......................................................................................................... 14 3.2.4 Molecular characterisation......................................................................................................... 14 3.3 Results ........................................................................................................................................... 16 3.3.1 Presence of Bursaphelenchus species in Europe ..................................................................... 16 3.3.1.1 Portugal ............................................................................................................................. 16 3.3.1.2 Spain ................................................................................................................................. 17 3.3.2 Morphological characterisation .................................................................................................. 18 3.3.3 Biometric characterisation.......................................................................................................... 19 3.3.4 Molecular characterisation......................................................................................................... 20 3.4 Discussion ...................................................................................................................................... 20 Chapter 4 Identification and biology of vectors of Bursaphelenchus xylophilus in Europe, particularly the Iberian Peninsula............................................................................................................................................................ 22 4.1 Introduction..................................................................................................................................... 22 4.2 Insects associated with Bursaphelenchus species in Portugal...................................................... 22 4.2.1 Materials and methods............................................................................................................... 22 4.3 Insects associated with Bursaphelenchus species in Spain.......................................................... 22 4.3.1 Materials and methods:.............................................................................................................. 22 4.3.2 Insects associated with Bursaphelenchus species in Austria.................................................... 24 4.4 Results ........................................................................................................................................... 25 4.4.1 Insects associated with Bursaphelenchus species in Portugal ................................................. 25 4.4.2 Insects associated with Bursaphelenchus species in Spain...................................................... 27 4.4.3 Insects associated with Bursaphelenchus species in Austria.................................................... 30 4.5 Discussion ...................................................................................................................................... 32 Chapter 5 Biology and pathogenesis of Bursaphelenchus xylophilus.................................................................... 35 5.1 Introduction..................................................................................................................................... 35 5.2 Materials and Methods general...................................................................................................... 36 5.2.1 Nematodes................................................................................................................................. 36 5.3 Portugal UEVORA: Nematode isolates, pine species and fungal isolation ................................... 37 5.3.1 Developmental biology and cytogenetics .................................................................................. 37 5.3.2 Hybridisation experiments.......................................................................................................... 37 5.3.3 Pathogenicity experiments......................................................................................................... 38 PHRAME Final Report Page 2 5.4 Germany: Isolates of Bursaphelenchus xylophilus and Bursaphelenchus mucronatus used for inoculations trials......................................................................................................................................... 38 5.4.1 Baermann-extraction of nematodes........................................................................................... 38 5.4.2 Rearing and multiplication of nematodes on artificial medium .................................................. 39 5.4.3 Preparation of nematodes for inoculation.................................................................................. 39 5.4.4 Fixation and counting................................................................................................................. 40 5.5 Host trees ....................................................................................................................................... 40 5.5.1 Tree species and stage of development.................................................................................... 40 5.5.2 Segmentation of trees................................................................................................................ 41 5.5.3 Bark-inoculation technique......................................................................................................... 43 5.5.4 Assessment of wilt symptoms.................................................................................................... 43 5.5.5 Sampling and handling of samples............................................................................................ 44 5.5.6 Determination of fresh, dry weight and relative water contents................................................. 44 5.6 Influence of nematode-density in the inoculum on the development of wilt and population after inoculation ................................................................................................................................................... 45 5.6.1 Materials and Methods............................................................................................................... 45 5.7 INIA Portugal: Plant material and nematode inoculation ............................................................... 45 5.7.1 Materials and methods............................................................................................................... 45 5.8 INIA Portugal: Symptom development and nematode detection ................................................... 46 5.8.1 Materials and methods............................................................................................................... 46 5.9 UEVORA, Portugal Results............................................................................................................ 47 5.9.1 Developmental biology and cytogenetics .................................................................................. 47 5.9.2 Hybridisation experiments.......................................................................................................... 47 5.9.3 Nematode reproduction on different fungi ................................................................................. 49 5.9.4 Pathogenicity experiments......................................................................................................... 49 5.10 Germany Results............................................................................................................................ 52 5.11 Susceptibility of European conifers against the Portuguese isolate of B. xylophilus in comparison