Table of Contents WORK PACKAGE 2: BEHAVIOUR AND DYNAMICS OF PWN IN INFESTED TREES 16 WORK PACKAGE 3: ASSESSING PHENOLOGY AND DISPERSAL CAPACITIES OF PWN VECTORS ........................................ 55 WORK PACKAGE 4: DEVELOPMENT OF NEW METHODS FOR MONITORING AND CONTROL OF MONOCHAMUS SPP AND PWN BASED ON VECTOR TRAPPING ..................................................... 83 WORK PACKAGE 5: DETERMINE RISK OF NON-VECTOR SPREAD OF PWN THROUGH VARIOUS PATHWAYS TO HEALTHY FORESTS 101 WORK PACKAGE 6: HOST TREE RESISTANCE TO PWN AND ITS VECTORS FOR FUTURE PLANTING ............................................... 151 WORK PACKAGE 7: PREDICTION OF PINE WILT EXPRESSION ACROSS ECO-CLIMATIC ZONES, TAKING ACCOUNT OF LATENCY 174 WORK PACKAGE 8: EU AND INTERNATIONAL COOPERATION AND COLLABORATION ........................................................................... 206 WORK PACKAGE 9: SYNTHESIS AND DEVELOPMENT OF PWN TOOL KIT FOR MONITORING AND MANAGEMENT OF PWN ............... 210 WORK PACKAGE 10: STAKEHOLDER ENGAGEMENT & DISSEMINATION 216 Project management during the period ........................................ 225 This was designated as WP1 in the original proposal. ............... 225 Table 2.1 Overview of features of tested P. sylvestris trees - Geographic distribution, Dt. Herkunftsgebietsverordnung (Forstvermehrungsgut) – Silvicultural regions of provenance (Forest reproduction item) (HkG), age, height, stem diameter, diameter of breast height (DBH) .................................................................................................... 19 Table 2.2 Overview of samplings with time after inoculation ....................................... 21 Table 2.3 Rating scheme of wilt classes for assessment of pine wilt (Daub 2008) ...... 22 Table 2.4 Overview of wilt class development of tested PWN inoculated pines according to sampling dates, days after inoculation and sampling intervals; Wilt classes of the sampled trees are framed for each date; Last tree tested short before wilt class 5 ................................................................................................................................... 23 Table 2.5 Wilt class development of tree B. x. 6 dependent on the week after inoculation (additional expressed as day of the year for comparison with the water potential graphs) ......................................................................................................... 41 Table 2.6 Nematode density [nematodes/gdry matter] of the analysed stem parts of the PWN inoculated trees (1: 1.5-2 cm disc of the sixth year stem segment (under the next whorl); 2: 1.5-2 cm disc of the fifth year stem segment (under the next whorl); 3: complete fourth year stem part; 4: complete third year stem part), n=6 ....................... 43 Table 2.7 Summarized moisture content MC [%] of all four sampled stem segments of the B. xylophilus (B. x.), water inoculated control (K W), drought stressed (DS W) and non-inoculated control trees (K N), n=6 (except the variant K N with n=2) .................. 44 Table 2.8 Summary of the categories and speculative function of the highly expressed genes identified in RNAseq analysis. .......................................................................... 54 Table 3.1 Total trap catches of Monochamus spp. from May to October 2013 ............ 57 Table 3.2 Correlation between weekly mean air temperature and number of trapped beetles (n = 18) ........................................................................................................... 57 Table 3.3 Distance, time and speed of flight (mean ± SE) of those M. sutor males and females that exhibited extended flight > 5 min on the flight mill. .................................. 63 Table 3.4 Details of loci characteristics and basic genetic parameters for one population (Orleans, France, n=30), including the multiplexing of primers “Multiplex” and relative amount of each primer “Amount”. (*) = potential presence of null alleles at this locus for this population. ....................................................................................... 70 Table 3.5 Transferability of the 12 loci developed for M. galloprovincialis. Values indicate the rate of positive amplification ; “_”= no amplification. ................................. 71 Table 3.6 Site characteristics for the transects across the Pyrenees. .......................... 72 Table 3.7 Geographic sources of populations of M. galloprovincialis in Europe. ......... 78 Table 4.1 Number of specimens caught in multi-funnel traps attached with Galloprotect Pack lure in six German Federal States in 2012 and 2013. ......................................... 89 Table 4.2 Number of Monochamus galloprovincialis caught in the traps in Exp 9/13 .. 95 Table 5.1 Moisture content MC (%) of stem discs at the inoculation date of the logs for each test month in 2012, n=5 .................................................................................... 104 Table 5.2 Nematode density [nematodes/gdry matter] (median, minimum, maximum) dependent on storage temperature, condition (open/ sealed) and time, n=5 ............. 109 Table 5.3 Overview of features of tested P. sylvestris trees - Geographic distribution, Dt. Herkunftsgebietsverordnung (Forstvermehrungsgut) – Silvicultural regions of provenance (Forest reproduction item) (HkG), age, height, stem diameter ............... 112 Table 5.4 Rating scheme of wilt classes for assessment of pine wilt (Daub 2008) .... 115 Table 5.5 Number of PWN infested and uninfested saplings per independent variable (a: Temperature; b: Wood chip position; c: Tree condition) without control trees ....... 128 Table 5.6 Numbers of PWN per 100 g of stem wood in plants exposed to PWN-infested wood chips ................................................................................................................ 130 Table 5.7 PWN densities over time in PWN-infested wood chips. ............................. 130 Table 5.8 Overview of features of tested P. sylvestris trees - Geographic distribution, Dt. Herkunftsgebietsverordnung (Forstvermehrungsgut) – Silvicultural regions of provenance (Forest reproduction item) (HkG), age, height, stem diameter ............... 132 Table 5.9 Wood moisture content MC [%] per pine variant with B. x. – B. xylophilus inoculated, K – Controls, N – Neighbouring trees, n=20 ............................................ 137 Table 5.10 Preliminary results of greenhouse inoculation experiments with Pinus pinaster. .................................................................................................................... 138 Table 5.11 Pairwise genetic distances (p-distances) among different Bursaphelenchus xylophilus isolates collected from the Peninsula of Setubal. ...................................... 144 Table 6.1 Rating scheme of wilt classes for assessment of pine wilt (Daub 2008) .... 154 Table 6.2 Nematode density [nematodes/gdry matter] of the PWN inoculated pine variant per provenance, n=20 ............................................................................................... 164 Table 6.3 Wood moisture content MC [%] at test end per pine variant (provenance and inoculation type) with B. x. – B. xylophilus inoculated, K – Controls (with water inoculation) and last numbers of silvicultural regions of provenance, n=20 ............... 164 Table 6.4 Number of reads and average length. ....................................................... 166 Table 6.5 Percentage of reads aligned in each sample. ............................................ 166 Table 6.6 Number of transcripts differentially expressed between samples. ............. 166 Table 6.7 germination data from the seeds from controlled crosses P. pinaster x P. halepensis ................................................................................................................ 170 Table 7.1 Parameter values for simulations in Europe. ............................................. 184 Table 7.2 Results of Sensitivity Analysis using model parameters. ........................... 187 Table 7.3 Results of Sensitivity Analysis: percentage variance in output caused by climate data. ............................................................................................................. 187 Table 7.4 Results of Sensitivity Analysis for model parameters. ............................... 190 Table 7.5 Results of Sensitivity Analysis using climate data. ..................................... 191 Table 7.6 Results of the first model. The last column represents the predicted percentage of infested trees. ..................................................................................... 198 Table 7.7 Results of the second model. The last column represents the predicted percentage of infested trees. ..................................................................................... 199 Table 7.8 Estimate of the dispersal distance according to scenarios W, M and D. Numbers in brackets indicate the standard deviation based on the dispersal simulation of 100 beetles. .......................................................................................................... 201 Table 10.1: Agenda for joint REPHRAME and Nematode Group meeting to develop the Stakeholder Observer Group and other interaction. .................................................. 221 Figure 2.1 Tree inoculation technique for Pinus sylvestris trees (here shown at 3-4 years old); a: Cutting of an I-shaped slit in the stem bark of the shoot of the previous year using a scalpel,