Surveillance of Pine Wood Nematode (PWN) Bursaphelenchus xylophilus

Transcription

1 Bioforsk Report Vol. 2 No Surveillance of Pine Wood Nematode (PWN) Bursaphelenchus xylophilus Norwegian Surveys ) MAGNUSSON, C., 2) THUNES, K. H., 2) NYEGGEN, H., 1) OVERGAARD, H., 1) RAFOSS, T., 1) HAUKELAND, S., 1) BRURBERG, M. B, 1) RASMUSSEN, I., 1) STRANDENÆS, K-A., 3) ØKLAND, B. & 1) HAMMERAAS, B. 1) Norwegian Institute for Agricultural and Environmental Research, Høgskoleveien 7, N-1432 Ås, Norway. 2) Norwegian Forest and Landscape Institute, Fanaflaten 4, N-5244 Fana, Norway. 3) Norwegian Forest and Landscape Institute, Høgskoleveien 8, N-1431 Ås, Norway.

2 Sammendrag Påvisningen av furuvednematoden (FVN) Bursaphelenchus xylophilus i Portugal i 1999 førte til surveyer i flere Europeiske land. Prøvetaking i Norge ble gjennomført i henhold til en Nordisk manual, hvor prøveantallet (n) beregnes ut fra anslått sannsynlighet for et positivt funn (p) og ønsket statistisk sikkerhet (ε) i henhold til likningen: n = ln ε / ln (1-p). Forekomsten i norsk hogstavfall av den utbredte vednematoden Bursaphelenchus mucronatus ble anslått til 0,01 eller 1%. Ved å så anta at FVN ville forekomme i en 10 ganger lavere frekvens (p=0,001) og kreve et 95 % konfidensintervall (ε=0,05) for statistisk sikkerhet til et positivt funn ga et minimum prøveantall på prøver for et sikkert utsagn om frihet for FVN i Norge. I henhold til manualen ble prøver tatt ut fra 10 sirkulære sone områder med 50 km radius og med sentra i et sted hvor importert risikomateriale er blitt håndtert. I perioden ble totalt prøver, fra furu (Pinus sylvestris), 279 fra gran (Picea abies) og 6 fra ukjent ved samlet inn fra totalt 446 hogstflater i 84 kommuner i fylkene Aust-Agder, Buskerud, Hedmark, Møre og Romsdal, Nord-Trøndelag, Oppland, Oslo and Akershus, Sør-Trøndelag, Telemark, Troms, Vestfold, Vest-Agder and Østfold. Sørøst- og Midt-Norge er vel dekket av sone områder. Flest prøver ble tatt i årene med et årlig prøvemengde på 600 prøver. Østfold med 688 prøver er det fylke som er mest undersøkt. I gjennomsnitt ble det på landsbasis tatt ut 7 prøver per hogstflate. Sponprøver ble tatt ut ved hjelp av bærbar drill med spiralbor (Ø=17 mm). Hogstavfall, tømmer og liggende trær utgjorde mer enn 90 % av den totale prøvemengden. Ved med angrep av furubukk (Monochamus) utgjorde 73 % av prøvene. Alle prøver ble innkubert ved 25 o C i to uker før ekstraksjon med Baermanntrakt i 48 timer. Forekomst av nematoder ble konstatert i 85 % av prøvene. Orden Rhabditida var mest frekvent, følgt av ordrene Aphelenchida, Tylenchida og Dorylaimida. Nematoder i Rhabditida var like vanlige i furu som gran, mens Aphelenchida (Aphelenchus, Aphelenchoides, Cryptaphelenchus, Seinura og Bursaphelenchus), og Tylenchida (Filenchus, Lelenchus, Ditylenchus, Deladenus, Nothotylenchus) viste en tendens til å være mer frekvente i prøver fra furuved. Aphelenchoides var den vanligst førekommende slekten. Bursaphelenchus ble påtruffet i totalt 32 prøver tilsvarende 1 % av materialet. Mesteparten av artene i denne slekten hørte til undergruppene borealis og sexdentati. Den eneste representant for xylophilus-gruppen som ble påtruffet var arten B. mucronatus, en i Norden, Europa (inkl. Russland) hyppig forekommende nær slekting til FVN. B. mucronatus ble påvist i 11 prøver tilsvarende 0,3 % av materialet, og oftest i hogstavfall av furu. På landsbasis ble B. mucronatus oppdaget på 2,5 % av hogstflatene. De fleste funnene ble gjort i Aust-Agder, hvor nematoden ble påvist i prøver fra 6 hogstflater. Forekomsten av B. mucronatus i Norge er betraktelig lavere enn i Fennoskandia generelt. Furuvednematoden, B. xylophilus, kunne ikke påvises i noen av de prøvene. Den uventet lave forekomsten av B. mucronatus indikerer at antallet potensielle nisjer i Norsk skog er mindre enn forventet, hvilket også vil gjelde for FVN B. xylophilus. Hvis vi fortsatt anslår at FVN vil påtreffes i en frekvens som er 10 ganger lavere enn for B. mucronatus, dvs , og fortsatt har et krav på 95 % konfidensintervall for et positivt funn, vil det i henhold til likningen ovenfor trenges et minimum av 9984 prøver for et sikkert utsagn om frihet for FVN i Norge. Konklusjoner: (1) Fastsatt prøvevolum på prøver er nådd; (2) Furuvednematoden (FVN) B. xylophilus er ikke påtruffet; (3) Vednematoden B. mucronatus forekommer i uventet lav frekvens; (4) Kartleggingen av furuvednematoden bør videreføres inntil et prøvevolum på ca prøver; (5) Eksisterende soneområder i Midt- og Sør-Norge bør bli permanente observasjonsområder og kompletteres med nye områder sentrert i Voss-Bergen regionen; (6) Alle prøver skal tas med forsterket fokus på hogstavfall, vindfall og tømmer av furu som viser tydelige tegn på angrep av furubukk. Videreføring: I 2007 planlegges prøvetakingen for kommende år. Observasjonsområdet med sentrum i Voss opprettes slik at det dekker skråningene nord for Sognefjorden. En veiledning for identififikasjon av sportegn av furubukk utarbeides. Utstyret kompletteres med mer vanntolerante driller og digital kamera. Prøvetaking av Voss-området startes høsten 2007.

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4 Contents Abstract Introduction Methods 5 4 Results Discussion 8 6. Conclusions References Tables Figures Appendices 22 Cover plate: 4 th dispersal juveniles of Bursaphelenchus mucronatus, extracted from a female of Monochamus galloprovincialis from Hornsö in the province of Småland southern Sweden. Photo: C. Magnusson SLU, Uppsala. 2

5 1. Abstract The detection in 1999 of the pine wood nematode (PWN), Bursaphelenchus xylophilus, in Portugal triggered survey activities in many European countries. The assumption that PWN would reach frequency 10 times lower than the native B. mucronatus and the requirement of a 95 % confidence limit suggested samples to be required for a safe statement on the absence of PWN from the territory surveyed. Samples were taken from 10 circular areas with 50 km diameter erected from a point of import of risk materials. In the period wood samples, from Pinus sylvestris, 279 from Picea abies and 6 from unknown wood, were collected from 446 logging sites in 84 municipalities and 13 counties. Of the total material 85 % of the samples came from cutting wastes, timber or lying trees. Wood showing signs of insect activity (incl. Monochamus) formed 73 % of the total material. Nematodes were recorded in 85 % of the samples. The order Rhabditida was most frequent, followed by the orders Aphelenchida, Tylenchida and Dorylaimida. Rhabditid nematodes were equally frequent in pine and spruce, while Aphelenchida (Aphelenchus, Aphelenchoides, Cryptaphelenchus, Seinura and Bursaphelenchus) and Tylenchida (Filenchus, Lelenchus, Ditylenchus, Deladenus and Nothotylenchus) tended to be more common in pinewood. Aphelenchoides was the most common genus. The genus Bursaphelenchus occurred in 1 % of the samples. B. mucronatus was detected in 0.3 % of the samples and most often in cutting waste of pine. The pine wood nematode (PWN), B. xylophilus, was not detected in this survey. The unexpected low natural occurrence of B. mucronatus indicates that the number of potential niches for PWN also is lower than expected. A hypothetical PWN frequency of 0,0003 and a 95 % confidence limit suggests samples to be required for a reliable statement on the absence of PWN. Hence, it will be necessary to continue this surveillance program. The present zone sites in central and south eastern Norway will be supplemented with 1-2 zone sites in south western region. In the future these zone sites will function as permanent observation areas. Care will also be taken to collect all samples from detached wood with signs of Monochamus activity. Magnusson et al

6 2. Introduction The detection in 1999 of the pine wood nematode (PWN), Bursaphelenchus xylophilus, in Portugal (Mota et al. 1999) has changed the earlier view on Europe as an area free from this pest (Evans et al. 1996). In 2000 the Standing Committee on Plant Health of the European Union (EU) decided on obligating each member state to conduct a survey of their territories for PWN (Anon. 2000). In accordance with international agreements, a draft manual for a Nordic PWN survey was developed (Appendix 1) and included an estimation of the minimal number of samples required for a safe statement on the absence of the nematode from the territory surveyed. According to this manual the detection of PWN would rely upon: (a) the close association of the nematode with its vector insects (Monochamus spp.); (b) the transmission of PWN at oviposition of the vector insects on weakened trees or detached wood; (c) the capacity of PWN to increase its population in wood; From previous experience it was estimated that the occurrence of the native, and to PWN closely related species Bursaphelenchus mucronatus in cutting wastes attacked by Monochamus would correspond to a frequency of 0.01, i.e. it was expected to detect this species in 1 sample out of 100 samples taken from wood with signs of Monochamus activity. If PWN had been introduced into a given territory the additional assumption was made that this species would occur in 10 times lower a frequency than B. mucronatus, corresponding to Hence, it was assumed that PWN should be detected in one sample out of 1000 samples of wood showing Monochamus activity. The minimal number of samples (n) required is defined from the probability of a positive find (p) and the degree of confidence (ε) according to the equation: n = ln ε / ln (1-p) p ε In the tabulation above, the selection of ε = 0.05 and p = gives a minimal sample volume of 2 995, so samples per country was agreed upon between the Nordic countries, and with modifications this protocol was adopted also by the EU (Anon. 2000). Norway has 5.14 mill ha productive forests of conifers, which represents a significant monitary value. This report summarizes the results presented by Magnusson et al. (2001, 2002, 2004abc) and supplies complementary data from the survey activities. 4 Magnusson et al. 2007

7 3. Methods In the period of June 2000 to May 2007, wood samples were collected from 1-2 years-old logging sites within 10 circular areas (i.e. zone sites A-J) with 50 km radius centred in points of exposure to potentially infested wood materials (Fig 1). Zone site A was centred in Tofte, the location of a major pulp factory and situated in the county of Vestfold. The centre of area B was a major timber yard in Drammen in the county of Buskerud. Area C was centred in Greåker, a major timber yard close to Sarpsborg in the county of Østfold. The zone site D was centred in Hunsfoss close to Vennesla in the county of Vest-Agder. The centre of area E was the village Rykene close to Arendal in the county of Aust-Agder. Both the latter localities have a previous history of handling import wood. Area F was erected out from Skogn (the location of a major paper mill) in the county of North-Trøndelag. Area G was centred in Elverum in the county of Hedmark, and covers points of wood imports to military installations and saw mills etc. The most northern area H had its centre in Skjold garrison in Målselv in the county of Troms. Area I was erected in the county of Møre og Romsdal, and centred in Surnadalsøra the location of an important saw mill. The area J was centred in Skien the location of a previous important paper mill in the county of Telemark. As stated above, the sampling was focused on Scots pine (Pinus sylvestris), which made up 91% of the total material. The ambition was to collect samples from wood attacked by beetles in the genus Monochamus. Each sample consisted normally of 500 ml of wood shavings, obtained by portable Bosch GSR 24 VE-2 and Milwaukee LokTor S18TX reversible electric drilling machines set at low speed and fitted with a 17 mm diameter bite (Fig. 2-3). The geographical coordinates were collected with GPSequipment and mapped in ArcGIS. The wood shavings of each sample was put into a plastic bag and transported to the nematode laboratory of the Norwegian Crop Research Institute, Plant Protection Centre in Ås. In the laboratory, all wood samples were incubated at +25 o C in their plastic bags for two weeks prior to extraction in Ø 28.5 cm Baermann funnels. To avoid cross contamination between the samples funnels were covered by plastic plates (Fig. 4). After 48 hours the water was removed and the nematode suspension was allowed to settle. Samples were screened in a Leica M10 stereomicroscope, and nematodes picked for closer investigation were killed at +65 o C, mounted in water on objective slides and examined in a Leitz DMRB interference microscope fitted with the Leica Q500MC and Leica Qwin image processing and analysis system. Specimens belonging to the Bursaphelenchus xylophilus-group (Braasch 2003) were identified by morphology and in some instances by molecular techniques involving a multiplex PCR reaction with specific primers for the species B.xylophilus, B. mucronatus and B. fraudulentus (Appendix 2). Statistical testing was made with 2-sample test. Magnusson et al

8 4. Results Sampling volume and intensity In this survey samples were analysed. The material contained samples (91 %) from Scots pine (Pinus sylvestris), 279 samples (9 %) from Norway spruce (Picea abies) and 6 samples from unidentified wood (Tab. 1). The sampling intensity between years is shown in Fig. 5. In 2000, the first year of this survey, 214 samples were obtained from forest sites. The highest sampling activity occurred in the years , when 600 samples were analysed annually. In the last two years 2005 and 2006 the sampling intensity declined to 300 and 250 samples respectively. Areas and localities In this survey most of the zone sites, i.e. A, B, C, D, E, G and J, were established in southeastern Norway, two sites F and I were erected in central Norway and one site H was situated in the northern part (Fig. 1). In the southeast region the distribution of samples within the zone sites was rather even (Fig. 6), while the sampling was more aggregated in the central (Fig. 6) and very local in the northern area H (Fig. 7). Samples were collected from the counties of Aust-Agder, Buskerud, Hedmark, Møre og Romsdal, Nord-Trøndelag, Oppland, Oslo and Akershus, Sør-Trøndelag, Telemark, Troms, Vestfold, Vest- Agder and Østfold (Tab.1). Most samples were collected in Østfold (688), Telemark (423) and Buskerud (421), but also Aust-Agder (364) and Hedmark (355) had a good volume of samples. With regard to pine most samples came from the county of Østfold (633) followed by Telemark (390) and Buskerud (388). Most spruce samples were collected in Østfold (55) followed by Aust- Agder (38), Nord-Trøndelag (34) and Telemark (33). Samples were collected from 84 municipalities and 446 logging sites (Tab. 2). The number of logging sites varied greatly between counties and municipalities (Tab. 2). Most logging sites were situated in Buskerud (82), but many sites were sampled also in Østfold (60), Hedmark (59), Telemark (56) and Aust-Agder (55). The highest number of logging sites (15) was sampled in the municipalities of Aremark in Østfold, in Øvre Eiker (13) and Hurum (13) in Buskerud, Skien (12) in Telemark and Åmot (12) in Hedmark. On the average 34 samples were taken per municipality. Østfold (68.8) and Telemark (52.9) are the counties with the most samples collected on a municipality basis (Tab. 3). The number of samples, however, varied considerably, with the highest numbers collected in Marker (235), Eidsberg (122) in Østfold, and from Notodden (153) in Telemark. The average number of samples taken per logging site was 7.2 and the mean number of samples per logging site for most municipalities was well below 20. However, in the case of Marker (78.3) in Østfold and Notodden (38.3) in Telemark, there is a strong divergence from the respective county mean of 11.5 and 7.6 samples per logging site (Tab. 4). In the total material (Tab.5) samples, equal to 73.4 %, were taken from detached wood. Detached wood contains objects like logging debris, branches, tops, bolts, timber (including timber piles). The 567 lying trees (often wind thrown) at a frequency of 17.9 % formed the next largest type of objects. Standing trees (107) and stumps (167) corresponded to 3.4 % and 5.3 % of the total samples. For the total material, and for the samples of pine and the 279 samples of spruce (Tab. 5) the category detached wood was the major object sampled with a frequency of 73.2 % in pine and 74.6 % in spruce. Lying trees and standing trees formed about 18 % and 3% regardless of the species of wood sampled. Stumps were sampled mainly in the first year of the survey. For pine and spruce stumps constituted 4.8 and 10.4 % respectively. 6 Magnusson et al. 2007

9 Samples with Monochamus activity Of the total material samples (73.9 %) were taken from wood showing signs wood boring insect activity (including the PWN vector insects in the genus Monochamus). The proportion of such samples varied between years (Fig. 8). In samples, which is less than half (42.5 %) of the annual volume, showed signs of wood insect attack. In 2001 and 2002 the proportion of such samples was 56.1 % and 66.5 % respectively. For samples taken in 2003, 2004 and 2005 signs of activity from Monochamus were recorded in 98 % or more. In the final year % of the samples were from Monochamus wood. Occurrence of nematodes In the total material of the survey nematodes were recorded in 85.4 % of the analysed samples (Tab.6). The nematodes extracted from wood represented the orders Rhabditida, Tylenchida, Aphelenchida and Dorylaimida. Members of the order Rhabditida were the most frequent and occurred in 71.0 % of the samples, followed by Aphelenchida at a frequency of 43.6 % and Tylenchida at 21.0 %. The occasional occurrence of Dorylaimida corresponded to only 0.4 %. In comparing the occurrence of nematodes in pine and spruce the two nematode negative samples collected from spruce in 2005 were omitted. Nematodes were more (p=0.042) frequent in pine with a mean value of 86.7 % compared to a mean value in spruce of 73.1 %. The order Rhabditida (genera not determined) showed no difference (p=0.189) in occurrence between pine (73.1 %) and spruce (60.3 %). The next group in falling frequency was the order Aphelenchida (gen. Aphelenchus, Aphelenchoides, Seinura, Bursaphelenchus and Cryptaphelenchus), which showed a tendency (p=0.092) of a higher frequency in pine (46.0 %) compared to spruce (30.0 %). This was also true of Tylenchida (gen. Filenchus, Lelenchus, Ditylenchus, Deladenus, Nothotylenchus and others), which tended (p=0.076) to be more frequent in pine (21.6 %) than in spruce (14.1 %). The frequency of Dorylaimida showed no difference (p=0.227) between pine (0.3 %) and spruce (1.0 %). The genus Aphelenchoides was the most common genus in wood. In this material there was no difference (p=0.166) in frequency of this genus between wood of pine (38.2 %) and spruce (27.1 %). The genus Bursaphelenchus was recorded in 32 samples, which corresponded to 1.0 % of the total number of samples. This genus occurred in both pine (1.0 %) and spruce (0.4 %). In this material no difference (p=0.311) could be detected in the occurrence of this genus between species of wood. Within the genus Bursaphelenchus several sub-groups have been distinguished (Braasch 2003). In this study species in the borealis and the sexdentati groups were recorded in 21 samples collected from lying stems, timber piles, wind-thrown trees and detached wood of pine, and in one stump of spruce. Nematodes in these sub-groups were not determined to species. In the xylophilus-group the only species detected was B. mucronatus. B. mucronatus, which occurred exclusively in pinewood with signs of Monochamus activity, was detected in 11 samples (Tab. 7), corresponding to a frequency of 0.3 %. The pinewood nematode B. xylophilus was not detected in the examined samples. The geographical position of samples positive for B. mucronatus is shown in Fig. 9. In 2001 B. mucronatus was detected in a wood sample from a lying tree of P. sylvestris at Ombudstvedt in the municipality of Våler. In 2002 it was observed in cutting waste of pine at Bjørdalen, in the municipality of Eidsberg. Both logging sites are situated in the county of Østfold in the zone site C. Most finds of B. mucronatus were made during the survey of 2003 in the southern county of Aust-Agder (Tab.7). In the municipality of Evje and Hornnes, within zone site D, B. mucronatus was encountered in cutting waste of pine at a logging area in Skjerkelia and Sutestad (Fig 9). In the municipality of Birkenes the nematode was detected in a sample from a pine stem at Vågsdalen. In Froland municipality it was found in two separate pieces of pine cutting waste on a logging site at Budalsfjellet, as well as in cutting waste of pine at Mjålandsvatn. These finds were all located within the overlapping area of zone sites D and E. In 2004 B. mucronatus again was found in eastern Norway in cutting wastes of pine at Grønnvollsmyra in the municipality of Åmot situated in the county of Hedmark in zone site G. In 2005 two finds were made of B. mucronatus in the county of Møre og Romsdal zone site I. Both detections were made in cutting waste of pine at a logging site in Hjellnes, Surnadal municipality, and at Hanemsvatnet in the municipality of Tingvoll. All the sampled objects had signs of Monochamus activity. Magnusson et al

10 5. Discussion Sampling intensity The sampling intensity varied between years, reflecting the funds allocated for the survey activities. A volume of 600 samples is readily handled at the nematode laboratory with its current personnel and equipment. If necessary this capacity can easily be increased by investments in better suited microscopes, by increasing the technical staff and strengthening their competence. Areas and localities The south eastern region of Norway is well covered by the sampling activities. In fact, the surveys in Østfold and Hedmark are complementary to the Swedish samplings in this region (Anon. 2006). The focus in erecting the zone sites was the exposure of a specific location to imported risk commodities. Because of this some forest dens areas have not been included in the study due to the strong dependence of the industry on domestic and local wood. This pertains for example to the vast forests in parts of the county of Hedmark. No zone sites have been established in the more southern parts of the west coast, and a complementary zone site could be established in Voss/Bergen to cover the valleys east from the Bergen area. Bergen has an important harbour, which might have been much exposed to import risk materials. These zone sites should cover the Hardangerfjord area in the south and the southern slopes of the Sognefjord valley in the north. There is a clear difference in the density and pattern of samples taken in zone sites in the southeast region compared to other regions. This often relates to topography and to the smaller areas of pine forests. In the Trøndelag area spruce is a more common forest tree than pine, and this is especially true for the county of Nord-Trøndelag. In the case of zone site I, the topography and the small pine logging activity can explain the aggregated sampling. In the northernmost zone site H, in the county of Troms, the pine logging sites with Monochamus occurred only in a restricted area in the Målselv municipality. This survey has covered 13 counties. Counties so far not covered by the sampling program, are Finnmark, Hordaland, Nordland, Rogaland and Sogn og Fjordane. When comparing the sampling activity between counties, the county of Østfold emerges as the county most intensively studied out from the number of logging sites, number of samples on a surface basis, number of samples per municipality and per logging site. For most counties samples were rather evenly distributed between logging sites. However, in the case of Marker in Østfold and Notodden in Telemark the very high mean number of samples reflects the exceptionally high number of samples collected at a few specific logging sites. In Marker 101 samples were collected from lying trees at Ørje (Magnusson et al. 2002) and 113 samples from lying trees, timber and debris at Mærrakleiva (Magnusson et al. 2004b). In the Notodden municipality in Telemark a total number of 153 samples were collected mostly from detached wood of pine. Here 143 samples of were taken mainly from two logging sites i.e. 73 samples at Nystul and 70 samples at Amerika. Objects sampled The main focus throughout the survey period has been wood of pine. In this respect the year of 2005 was exceptional with only 2 samples out of 300 being obtained from spruce. Scots pine formed more than 90 % of the total number of samples taken in this survey. Pine trees are the main host trees for the pine wood nematode (PWN) B. xylophilus, and are the most common breeding material for the most abundant insect vector in Norway Monochamus sutor (Bakke & Kvamme 1992). Since there are indications that wood of Scots pine offers better conditions for 8 Magnusson et al. 2007

11 the development of B. xylophilus compared to wood of Norway spruce (Magnusson unpubl.), the selective sampling of lying trees and detached wood of pine is biologically well-founded and would facilitate the detection of PWN. In fact, detached wood and lying (wind-thrown) trees of pine made up 83.5 % of the total number of samples taken in this survey. Monochamus frequency M. sutor is distributed widely in Norway (Bakke & Kvamme 1992). This species has been demonstrated to transmit the domestic wood nematode B. mucronatus to both Scots pine and Norway spruce, and is likely to be a potential vector also for PWN (Schroeder & Magnusson 1992). In this survey 74 % of the total material had signs of wood boring insect activity. In the first three years of the study a substantial proportion of the sampled wood objects did not show signs of insect activity. In the last four years, however, the situation improved resulting in a clear majority of samples being taken from wood with cerambycid activity mostly from Monochamus. In Swedish surveys % of the samples collected from forest logging sites showed signs of Monochamus attack (Anon. 2004, 2006). Occurrence of nematodes It can be concluded that nematodes are common inhabitants of wood. Nematodes recovered from wood contain insect parasites and microbivorous species with a phoretic relationship to wood inhabiting insects. The microbivorous species include both the bacterial feeders in the order Rhabditida and the fungal feeders in the orders Aphelenchida, Tylenchida and Dorylaimida. The microbivorous nematodes belong to the normal decomposer food chain and have important key-functions in the natural decomposition processes. Rhabditid, aphelenchid and tylenchid/neotylenchid nematodes occurred in a falling order of dominance in the wood. All these forms live in insect galleries and feed on bacteria and fungi occurring in insect frass. Rhabditids mostly graze from the bacterial populations, but some diplogasterid species may also be predatory on other members of the micro fauna. The aphelenchids contain specialized fungal feeders like many species in the genus Aphelenchoides. The genus Cryptaphelenchus was observed occasionally, and species in this genus are known to have phoretic relationships with bark beetles and cerambycid beetles like Monochamus (Hunt 1993). Also aphelenchid predators like species in the family Seinuridae were present to a various extent. Representatives of the order Tylenchida include invasive and mycetophagous stages of insect parasitic nematodes like species in the genus Deladenus, and fungal feeding species of Filenchus, Lelenchus. Ditylenchus and Nothotylenchus. In wood samples fungal feeding forms in Tylenchus sensu lato are quantitatively inferior to aphelenchid fungivores. The occasional finding of nematodes in the order Dorylaimida was unexpected. Forest wood seems to offer a large number of niches for nematode existence. The reason for nematodes to be more common in pine wood compared to spruce wood is not quite clear, but suggests that pine trees may offer a higher density of potential microhabitats for nematode existence. Rhabditida, one important order of bacterial feeders, did not show a difference in occurrence between pine and spruce. That the orders Aphelenchida and Tylenchida tended to be more frequent in pine samples can indicate a higher selectivity among fungal feeders in the choice of habitat. Aphelenchoides is the genus most consistently encountered in wood. In our material there is no significant difference in its frequency of occurrence between wood of pine and spruce. Several studies have demonstrated species of Aphelenchoides as very common in coniferous forest soils (Sohlenius et al. 1977, Magnusson & Sohlenius 1980, Magnusson 1983). This genus contains species which tolerate dry conditions well, and some species have been recorded present in forest microhabitats with occasionally low water levels like moss cushions on stones and tree stems etc. (Overgaard Nielsen 1967). Ditylenchus is another example of a nematode genus with species occurring in both soil and wood. Species in the Bursaphelenchus sub-groups borealis and sexdentati have been recorded frequently in European forests (Braasch et al. 2000). In Norway the survey of McNamara and Støen (1988) recorded the genus Bursaphelenchus in 6 % of the samples. In Germany, Braasch (1999) reported on a frequency of 15 % in samples taken from forest trees with insect damage. These frequencies are higher than the frequencies recorded in the present study, which did not target deteriorating trees or trees with insect damage in a broad sense, but specifically breeding material of Monochamus. Magnusson et al

12 The pine wood nematode (PWN) B. xylophilus was not detected in this survey. The only representative of the xylophilus-group was B. mucronatus. This nematode is well established in the Nordic area, where it was first reported from Norway living in cut logs at Hanestad in Østerdalen (McNamara & Støen 1988). Magnusson and Schroeder (1989) reported B. mucronatus dauer juveniles both from M. sutor and M. galloprovincialis emerging from wood of P. sylvestris. In Sweden the nematode occurs from the province of Småland in the south to the province of Lappland in the north (Schroeder & Magnusson 1989). Also in Finland (Tomminen et al. 1989, Tomminen 1990) B. mucronatus occurs in the whole country. This species is also widely distributed in Europe (Braasch et al. 2000) and in Russia (Kulinich et al. 1994, Kulinich & Orlinskii 1998). Within the areas sampled in the Norwegian survey B. mucronatus occurred in a frequency of 0.3 %, which is in line with the 0.2 % frequency in the previous Norwegian data of McNamara & Støen (1988). It is lower, however, than frequencies reported in the Swedish surveys for PWN, where B. mucronatus occurred in % of the samples (Anon. 2002, 2006). The 11 finds of B. mucronatus in 446 Norwegian logging sites demonstrates that 2.5 % of the localities sampled were positive for the nematode, to be compared to 45% of the forest sites in Finland surveyed by Tomminen et al. (1989). It is important to note the association of this nematode with pinewood and in particular cutting wastes attacked by Monochamus. In Finland Tomminen et al. (1989) and Tomminen (1990) reported a similar consistent association between Monochamus and B. mucronatus. Similarly, Swedish data demonstrated that the area of Särna and Idre, in the province of Dalarna, had a high population of B. mucronatus, with 41 % of the Monochamus individuals recorded to carry dauer juveniles (Schroeder & Magnusson 1989). The most probable explanation for the low detection of B. mucronatus in Norway is the combination of a low natural occurrence and a possible failure in detecting consistently Monochamus symptomatic wood. These concerns draw the attention to the outmost importance of securing a correct diagnosis of the signs of Monochamus activity in sampling for PWN. The monitoring system in Norway In our material B. mucronatus occurred in 3 out of 1000 samples of wood, which is lower than the estimated frequency of At the outset of this survey we assumed a hypothetical frequency of PWN to be 10 times lower than that of B. mucronatus. The unexpected low occurrence of B. mucronatus means that the number of potential niches for PWN also is lower than expected. With a natural frequency of for B. mucronatus, and maintaining a 10 time lower hypothetical frequency for PWN (p = ) and a 95 % confidence level (ε = 0.05), samples would be the minimum number required for a safe statement on the absence of the nematode from Norway. In view of this it will be necessary to continue this surveillance program. 10 Magnusson et al. 2007

13 6. Conclusions The volume of samples decided on (n=3 000) was reached in The pinewood nematode B. xylophilus was not detected in the analysed samples. Bursaphelenchus mucronatus occurred in 0.3 % of the samples. A hypothetical frequency for B. xylophilus of and a confidence limit at 95 % gives a requirement of samples as a minimum for a safe statement on the absence of the nematode. It is recommended to continue the program for surveillance of the pinewood nematode (PWN) B. xylophilus in Norway. The existing zone sites in the central and southern part of Norway should be permanent areas of observation. One or two new zone sites should be erected in the region of Bergen and Voss. Cutting waste and timber of pine attacked by Monochamus should be the first priority for sampling. Magnusson et al

14 7. References ANONYMOUS (2000). E.C. Pinewood Nematode Survey Protocol European Commission, Directorate-General Health and Consumer Protection, Directorate E- Public, animal and plant health. Unit E1. Legislation relating to crop products and animal nutrition. SANCO E/1 D(00): 7 pp. ANONYMOUS (2002). Report on the Pine Wood Nematode Survey in Sweden 1 November October Swedish Board of Agriculture Report: 3 pp. ANONYMOUS (2004). Report on the Pine Wood Nematode Survey in Sweden 1 November October Swedish Board of Agriculture Report: 3 pp. ANONYMOUS (2006). Report on the Pine Wood Nematode Survey in Sweden 1 November October Swedish Board of Agriculture Report: 3 pp. BAKKE, A. & KVAMME, T. (1992). The pine sawyer (Monochamus sutor): Distribution and life history in South Norway. Medd. Skogforsk. 44:1-16 BRAASCH, H. (1999). Bursaphelenchus-Arten (Nematoda: Parasitaphelenchidae) in Nadelgehølzen In Deutschland und ihre ITS-RFLP-Munster. Nachrichtenbl. Deut. Pflanzenschutzd. 51: BRAASCH, H. (2003). Morphology of Bursaphelenchus xylophilus compared with other Bursaphelenchus species. Nematology Monographs & Perspectives 1: BRAASCH, H., BURGERMEISTER, W., HARMEY, M.A., MICHALOPOULOS-SKARMOUTSOS, H., TOMICZEK, C. & CAROPPO, S. (2000). Pest risk analysis of pinewood nematode related Bursaphelenchus species in view of South European pine wilting and wood imports from Asia. Final Report of EU research project Fair CT pp. EVANS, H.F., McNAMARA, D.G., BRAASCH, H., CHADOEUF, J. & MAGNUSSON, C. (1996). Pest Risk Analysis (PRA) for the territories of the European Union (as PRA area) on Bursaphelenchus xylophilus and its vectors in the genus Monochamus. EPPO Bulletin 26: HUNT, D.J. (1993). Aphelenchida, Longidoridae and Trichodoridae. Their Systematics and Bionomics. CAB International. Oxford, UK. 352 pp. KULINICH, O.A. & ORLINSKII, P.D. (1998). Distribution of conifer beetles (Scotylidae, Curculionidae, Cerambycidae and wood nematodes (Bursaphelenchus spp.) in European and Asian Russia. EPPO Bulletin 28: KULINICH, O.A., KRUGLIC, I.A., EROSHENKO, A.S. & KOLOSSOVA, N.V. (1994). Occurrence and distribution of the nematode Bursaphelenchus mucronatus in the Russian Far East. Russian Journal of Nematology 2: MAGNUSSON, C. (1983). Abundance, distribution and feeding relations of root/fungal feeding nematodes in a Scots pine forest. Holarct. Ecol. 6: MAGNUSSON, C. & SCHROEDER, L.M. (1989). First record of a Bursaphelenchus species (Nematoda) in Monochamus beetles in Scandinavia. Anzeiger für Schädlingskunde, Pflanzenschutz und Umweltschutz 62: MAGNUSSON, C. & SOHLENIUS, B. (1980). Root consumption in a year old scots pine stand, with special regard to phytophagous nematodes. In: Persson, T. (ed.). Structure and Function of Northern Coniferous Forests An Ecosystem Study. Ecol. Bull. (Stockholm) 32: MAGNUSSON, C., THUNES, K., HAUKELAND SALINAS, S. & ØKLAND, B. (2001). Survey of the pine wood nematode, Bursaphelenchus xylophilus, in Norway Planteforsk Rapport 07/2001: MAGNUSSON, C., THUNES, K., HAUKELAND SALINAS, S. & HAMMERAAS, B. (2002). Survey of the pine wood nematode, Bursaphelenchus xylophilus, in Norway Grønn forskning 26/2002: MAGNUSSON, C., THUNES, K., HAUKELAND SALINAS, S. & ØKLAND, B. (2004a). Survey of the pine wood nematode, Bursaphelenchus xylophilus, in Norway Nematology Monographs and Perspectives 1: MAGNUSSON, C., NYEGGEN, H., THUNES, K., HAUKELAND SALINAS, S. & HAMMERAAS, B. (2004b). Survey of the pine wood nematode, Bursaphelenchus xylophilus, in Norway Internal Report Planteforsk Plantevernet Ås, : Magnusson et al. 2007

15 MAGNUSSON, C., OVERGAARD, H., NYEGGEN, H., THUNES, K. H., HAUKELAND SALINAS, S. & HAMMERAAS, B. (2004c). Survey of the pine wood nematode, Bursaphelenchus xylophilus, in Norway Internal Report Planteforsk Plantevernet Ås, : MCNAMARA, D.G, & STØEN, M. (1988). A survey for Bursaphelenchus spp. in pine forests in Norway. EPPO Bulletin 18: MOTA, M. M., BRAASCH, H., BRAVO, M. A., PENAS, A. C., BURGERMEISTER, W., METGE, K. & SOUSA, E. (1999). First report of Bursaphelenchus xylophilus in Portugal and Europe. Nematology 1: OVERGAARD-NIELSEN, C. (1967). Nematoda. In: Burges, A. & Raw, F. (eds.) Soil Biology. Acad. Press Inc. (London) Ltd. 2 nd print: SCHROEDER, M. & MAGNUSSON, C. (1989). Tallvednematoden ett hot mot svensk skog? Skogsfakta, Biologi och skogsskötsel 64. SLU Uppsala, Sweden: 4 pp. SCHROEDER, L.M. & MAGNUSSON, C. (1992). Transmission of Bursaphelenchus mucronatus to branches and bolts of Pinus sylvestris and Picea abies by the ceranbycid beetle Monochamus sutor. Scand. J. For. Res. 7: SOHLENIUS, B., PERSSON, H. & MAGNUSSON, C. (1977). Distribution of roots and nematodes in a young scots pine stand in central Sweden. In: Lohm, U. & Persson, T. (eds.) Soil Organisms as Components of Ecosystems. Ecol. Bull. (Stockholm) 25: TOMMINEN, J. (1990). Presence of Bursaphelenchus mucronatus (Nematoda: Aphelenchoididae) fourth dispersal stages in selected conifer beetles in Finland. Silva Fennica 24: TOMMINEN, J., NUORTEVA, M., PULKKINEN, M. & VÄKEVÄ, J. (1989). Occurrence of the nematode Bursaphelenchus mucronatus Mamiya & Enda 1979 (Nematoda: Aphelenchoididae) in Finland. Silva Fennica 23: Magnusson et al

16 8. Tables Table 1. Survey of the pine wood nematode (PWN), Bursaphelenchus xylophilus, in Norway Number of samples distributed on counties and tree species. COUNTY PINE SPRUCE UNIDENTIFIED COUNTY WOOD TOTAL Aust-Agder Buskerud Hedmark Møre og Romsdal Nord-Trøndelag Oppland Oslo og Akershus Sør-Trøndelag Telemark Troms Vestfold Vest-Agder Østfold SURVEY TOTAL Table 2. Survey of the pine wood nematode (PWN), Bursaphelenchus xylophilus, in Norway Counties, municipalities and logging sites sampled. COUNTY MUNICIPALITIES (LOGGING SITES) COUNTY LOGGING SITES Aust-Agder Arendal(4), Birkenes(8), Evje og Hornnes(7), Froland(9), Gjerstad(5), 55 Grimstad(4), Iveland(5), Risør(1), Vergårshei(5), Åmli(7); Buskerud Drammen(7), Flesberg(11), Hole(4), Hurum(13), Kongsberg(9), 82 Lier(3), Modum(8), Nedre Eiker(4), Ringerike(8), Røyken(2), Øvre Eiker(13); Hedmark Elverum(11), Hamar(3), Løten(5), Stange(6), Stor Elvdal(7), Trysil(1), 59 Våler(6), Åmot(12), Åsnes(8), Møre og Romsdal Rindal(2), Surnadal(9), Tingvoll(5); 16 Nord-Trøndelag Levanger(5), Mosvik(3), Steinkjer(4), Stjørdal(1), Verdal(3); 16 Oppland Jevnaker(3), Lunner(3); 6 Oslo og Akershus Asker(2), Aurskog-Høland(9), Bærum(2), Enebakk(2), Fet(2), Frogn(1), 30 Nesodden(6), Rælingen(1), Ski(2); Sørum(3); Sør-Trøndelag Malvik(3), Selbu(6), Trondheim(3); 12 Telemark Bamble(6), Bø(8), Drangedal(6), Kragerø(5), Nome(8), Notodden(4), 56 Sauherad(7), Skien(12); Troms Målselv(4); 4 Vestfold Hof(5), Lardal(3),Sandefjord(1); 9 Vest-Agder Audnedal(9), Hægebostad(4), Kristiansand(4), Lindesnes(2), Marnadal(6), 41 Songdalen(6), Søgne(1), Vennesla(6), Åseral(3); Østfold Aremark(15), Eidsberg(6), Halden(11), Marker(3), Moss(1), Rakkestad(4), 60 Rygge(5), Rømskog(4), Trøgstad(5), Våler(6); TOTAL Magnusson et al. 2007

17 Table 3. Survey of the pine wood nematode (PWN), Bursaphelenchus xylophilus, in Norway Number of samples distributed on municipalities. COUNTY MUNICIPALITIES (SAMPLES) COUNTY MUNICIPALITY MEAN Aust-Agder Arendal(15), Birkenes(58), Evje og Hornnes(70), Froland(38), 36.4 Gjerstad(13), Grimstad(37), Iveland(36), Risør(2), Vergårshei(45), Åmli(50); Buskerud Drammen(48), Flesberg(44), Hole(60), Hurum(67), Kongsberg(50), 38.3 Lier(14), Modum(20), Nedre Eiker(12), Ringerike(69), Røyken(4), Øvre Eiker(33); Hedmark Elverum(49), Hamar(30), Løten(32), Stange(23), Stor Elvdal(51), 39.4 Trysil(2), Våler(42), Åmot(78), Åsnes(48), Møre og Rindal(6), Surnadal(69), Tingvoll(27); 34.0 Romsdal Nord-Trøndelag Levanger(38), Mosvik(43), Steinkjer(36), Stjørdal(6), Verdal(6); 25.8 Oppland Jevnaker(24), Lunner(46); 35.0 Oslo og Asker(10), Aurskog-Høland(40), Bærum(8), Enebakk(9), Fet(10), 19.7 Akershus Frogn(3), Nesodden(56), Rælingen(1), Ski(39); Sørum(21); Sør-Trøndelag Malvik(35), Selbu(52), Trondheim(31); 39.3 Telemark Bamble(14), Bø(26), Drangedal(22), Kragerø(45), Nome(46), 52.9 Notodden(153), Sauherad(47), Skien(70); Troms Målselv(10); 10.0 Vestfold Hof(37), Lardal(13),Sandefjord(2); 17.3 Vest-Agder Audnedal(42), Hægebostad(28), Kristiansand(33), Lindesnes(2), 26.2 Marnadal(27), Songdalen(11), Søgne(2), Vennesla(55), Åseral(36); Østfold Aremark(98), Eidsberg(122), Halden(63), Marker(235), Moss(13), 68.8 Rakkestad(25), Rygge(52), Rømskog(16), Trøgstad(23), Våler(41); TOTAL MUNICIPALITY MEAN 34.1 Table 4. Survey of the pine wood nematode (PWN), Bursaphelenchus xylophilus, in Norway Number of samples per logging site (in parenthesis) in surveyed municipalities. COUNTY MUNICIPALITIES (SAMPLES PER LOGGING SITE) COUNTY MEAN Aust-Agder Arendal(3,8), Birkenes(7,3), Evje og Hornnes(10,0), Froland(4,2), 6.6 Gjerstad(2,6), Grimstad(9,3), Iveland(7,2), Risør(2,0), Vergårshei(9,0), Åmli(7,1); Buskerud Drammen(6,9), Flesberg(4,0), Hole(15,0), Hurum(5,2), Kongsberg(5,6), 5.1 Lier(4,7), Modum(2,5), Nedre Eiker(3,0), Ringerike(8,6), Røyken(2,0), Øvre Eiker(2,5); Hedmark Elverum(4,5), Hamar(10,0), Løten(6,4), Stange(3,8), Stor Elvdal(7,3), 6.0 Trysil(2,0), Våler(7,0), Åmot(6,5), Åsnes(6,0), Møre og Romsdal Rindal(3,0), Surnadal(7,7), Tingvoll(5,4); 6.4 Nord-Trøndelag Levanger(7,6), Mosvik(14,3), Steinkjer(9,0), Stjørdal(6,0), Verdal(2,0); 8.1 Oppland Jevnaker(8,0), Lunner(15,3); 11.7 Oslo og Akershus Asker(5,0), Aurskog-Høland(4,4), Bærum(4,0), Enebakk(4,5), Fet(5,0), 6.6 Frogn(3,0), Nesodden(9,3), Rælingen(1,0), Ski(19,5); Sørum(7,0); Sør-Trøndelag Malvik(11,7), Selbu(8,7), Trondheim(10,3); 9.8 Telemark Bamble(2,3), Bø(3,3), Drangedal(3,7), Kragerø(9,0), Nome(5,8), 7.6 Notodden(38,3), Sauherad(6,7), Skien(5,8); Troms Målselv(2,5); 2.5 Vestfold Hof(7,4), Lardal(4,3),Sandefjord(2,0); 5.8 Vest-Agder Audnedal(4,7), Hægebostad(7,0), Kristiansand(8,3), Lindesnes(1,0), 5.8 Marnadal(4,5), Songdalen(1,8), Søgne(2,0), Vennesla(9,2), Åseral(12,0); Østfold Aremark(6,5), Eidsberg(20,3), Halden(4,2), Marker(78,3), Moss(13,0), 11.5 Rakkestad(6,3), Rygge(10,4), Rømskog(4,0), Trøgstad(4,6), Våler(6,8); TOTAL MEAN 7.2 Magnusson et al

18 Table 5. Survey of the pine wood nematode (PWN), Bursaphelenchus xylophilus, in Norway Number of samples distributed kinds of objects and year of sampling. WOOD SAMPLES TOTAL PINE SAMPLES SPRUCE SAMPLES OBJECTS TOTAL % Standing trees Lying trees Detached wood ,4 Stumps Roots Standing trees Lying trees Detached wood Stumps Roots Standing trees Lying trees Detached wood Stumps Table 6. Survey of the pine wood nematode (PWN), Bursaphelenchus xylophilus, in Norway Relative frequency of nematode groups in different sampling years. WOOD SAMPLES TOTAL PINE SAMPLES SPRUCE SAMPLES GROUP TOTAL Nematoda Rhabditida Tylenchida Aphelenchida Aphelenchoides Burasphelenchus B. mucronatus Dorylaimida Nematoda Rhabditida Tylenchida Aphelenchida Aphelenchoides Burasphelenchus B. mucronatus Dorylaimida Nematoda Rhabditida Tylenchida Aphelenchida Aphelenchoides Burasphelenchus B. mucronatus Dorylaimida Magnusson et al. 2007

19 Table 7. Survey of the pine wood nematode (PWN), Bursaphelenchus xylophilus, in Norway Locations and parameters of samples with Bursaphelenchus mucronatus. The pine wood nematode was not recorded in this survey. YEAR ZONE SITE COUNTY MUNICIPALITY LOCATION TREE SPECIES OBJECT SAMPLED MONOCHAMUS ACTIVITY 2001 C Østfold Våler Ombudstvedt Pine Lying tree Yes 2002 C Østfold Eidsberg Bjørdalen Pine Cutting waste Yes 2003 D Aust-Agder Evje og Skjerkelia Pine Cutting waste Yes Hornnes D Aust-Agder Evje og Sutestad Pine Cutting waste Yes Hornnes D/E Aust-Agder Birkenes Vågsdalen Pine Stem Yes D/E Aust-Agder Froland Budalsfjellet Pine Cutting waste Yes D/E Aust-Agder Froland Budalsfjellet Pine Cutting waste Yes D/E Aust-Agder Froland Mjålandsvatn Pine Cutting waste Yes 2004 G Hedmark Åmot Grønnvollsmyra Pine Cutting waste Yes 2005 I Møre og Romsdal Surnadal Hjellnes Pine Cutting waste Yes I Møre og Romsdal Tingvoll Hanemsvatnet Pine Cutting waste Yes Magnusson et al

20 9. Figures Figure 1. Survey of the pine wood nematode (PWN), Bursaphelenchus xylophilus, in Norway The 10 zone sites sampled are shown as grey circular areas with a radius of 50 km. A. Tofte; B. Drammen; C. Greåker; D. Hunsfoss; E. Rykene; F. Skogn; G. Elverum; H. Skjold; I. Surnadalsøra; J. Skien. Green colour shows the forest area, while yellow colour indicates agricultural land. Figure 2. Survey of the pine wood nematode (PWN), Bursaphelenchus xylophilus, in Norway Dr. Thunes sampling wood with signs of Monochamus activity using a battery powered electric drilling machine. Figure 3. Wood shavings for nematode analysis obtained from a 17 mm bit. 18 Magnusson et al. 2007

21 Figure 4. Baermann funnel technique for the extraction of pine wood nematode (PWN). Bursaphelenchus xylophilus. Wood shavings for extraction are placed on a filter paper supported by a metal net in a plastic funnel (Ø=28,5 cm). Each funnel is fitted with plastic tubing closed by a metal clip. Funnels are filled with water and left for 48 hrs at +25 o C. A transparent plastic lid to avoid cross contamination of samples covers each funnel. Figure 5. Survey of the pine wood nematode (PWN), Bursaphelenchus xylophilus, in Norway Sampling intensity shown as the number of samples taken from wood of Scots pine (Pinus sylvestris) and Norway spruce (Picea abies). Magnusson et al

22 Figure 7. Survey of the pine wood nematode (PWN), Bursaphelenchus xylophilus, in Norway Map showing the northernmost zone site H in the county of Troms. Note the very restricted sampling activity in Målselv municipality. Figure 6. Survey of the pine wood nematode (PWN), Bursaphelenchus xylophilus, in Norway Map showing the zone sites and sampling positions in the central and southern regions the country. Green colour shows the distribution of forests, while yellow colour indicates agricultural land. 20 Magnusson et al. 2007

23 Figure 8. Survey of the pine wood nematode (PWN), Bursaphelenchus xylophilus, in Norway Samples with and without Monochamus activity distributed over sampling years. Figure 9. Survey of the pine wood nematode (PWN), Bursaphelenchus xylophilus, in Norway Map showing the position of samples positive for B. mucronatus. The find at Hanestad in Østerdalen was reported previous to the present survey by McNamara and Støen (1988). Magnusson et al

24 10. Appendices Summary over appendix No Subject 1 Nordic Pine Wood Nematode Survey Draft Manual 2 Detection and differentiation of Bursaphelenchus species using multiplex PCR 22 Magnusson et al. 2007

25 Appendix 1: Nordic Pine Wood Nematode Survey - Draft Manual Christer MAGNUSSON (1), Martin SCHROEDER (2) and Jyrki TOMMINEN (3) (1)The Norwegian Crop Research Institute, Høgskoleveien 7, NO-1432 Aas, Norway (2) Swedish University of Agricultural Sciences, Department of Entomology, Box 7044, SE Uppsala, Sweden (3) KTTK-Plant Protection Service, Box 42, FIN Helsinki, Finland Background The recent detection of the pine wood nematode (PWN) Bursaphelenchus xylophilus in Portugal has changed the earlier view on Europe as an area free from this pest (Evans et al. 1996), and made the European perspective on forest health considerably wider. The infestation in Portugal is suspected date back 2-3 years (Anonymous 1999) and there is now a growing concern about the possible presence of PWN also in other countries. The Standing Committee on Plant Health of EU has reached a decision on obligating each member state to conduct a survey of their territories for PWN. The Nordic countries, including Norway, strongly support that decision. In previous surveys in the Nordic countries (McNamara & Støen 1988, Magnusson & Schroeder 1989, Tomminen et al. 1989, Tomminen 1990) PWN was not detected. However, in the Nordic area PWN is not expected to cause a large-scale pine mortality. Because of this PWN infections could easily be overlooked. Only sampling strategies designed to give a high probability of detection can form the basis for confident statements on the presence or absence of PWN. This document presents the outlines of an extended and coordinated PWN survey of the Nordic forests. Objectives The objectives of the proposed work are to survey: 1. Zone sites: 1.1. Forests adjacent to points of wood import (harbours) Forests adjacent to points of handling and storage of imported wood (saw mills and pulp mills) Forests adjacent to points of handling and storage of imported wood packaging material. 2. General sites: 2.1. Forests in general Clear cuts burnt for nature conservation 2.3. Areas of forest decline.

26 Sites of sampling. Zone sites: Forests situated within an area with a 50 km radius centred in points of handling and storage of high risk wood import materials. Before the survey activity starts each country shall identify such centres and map the potential sampling objects within each zone. The sites for sampling are forest blocks logged 1-2 year before sampling. The sampling shall be focused on cutting wastes of Pinus sylvestris oviposited by Monochamus spp, or any other conifer wood showing Monochamus activity. In winter or spring, after the first sampling of year 2000 and 2001, each forest block should be provided with four bait-logs of freshly cut P.sylvestris, preferably felled in exposed situations. Bait-logs would serve as traps for Monochamus spp. and should be sampled after one year. This strategy allows for an increased probability of detection by taking advantage of the natural association of PWN with its vector insect, and the lag phase of spread from the point of a possible introduction. It is essential that sample sites are distributed as even as possible over the circular area. General sites: Forest sites of normal health, clearings burnt for natural conservation and sites of forest decline. As a consequence of the sampling strategy, general forest sites will be selected in areas naturally infested by Monochamus spp. Instances of forest decline could be identified by Forest Service officers. Samples should be taken from trees showing various symptoms of branch die-back and wilt. If detected, trees, logs or any conifer wood showing Monochamus activity is a primary target for sampling. Local conditions Conditions may vary between countries, with regard to acerage of pine forests, density of vector insects ect. Therefore, each country need to decide how the sampling activity should be allocated to sampling objects. In a situation where P.sylvestris is a minor forest tree species the sampling activity may be directed towards stands of another conifer host plant, like Picea abies. Wood of P. abies will provide suitable conditions for reproduction of PWN. Sample size It is essential to find a way of a correct assessment of the sample size required to allow for confident statements on the hypothetical presence of PWN in each of the Nordic countries. It is also of outmost importance that the sample size is determined primarily on biological criteria. Economy is of secondary importance, as it relates to political concerns. Assumptions: There is a similar probability of finding PWN regardless of the region sampled. In wood attacked by Monochamus spp. PWN is assumed to occur in a frequency 0,001, equal to one find out of 1000 samples of wood showing Monochamus activity. The minimal number of samples (n) required is defined from the probability of a positive find (p) and the degree of confidence (ε) according to:

27 n = ln ε / ln (1-p) Table 1. Minimal number of samples determined by the value of ε and p. p \ ε 0,10 0,05 0,01 0,001 0,0001 0, , , , , According to Tab.1. the selection of ε = 0,05 and p = 0,001 gives a sample size of Hence, the suggestion is to take samples for each country, regardless of differences in the forest area between countries Allocation of sampling activities Each country should define and map the zones of interest. Within each zone at least 10 locations for sampling collection should be selected. Zone sites have the highest priority for sampling, and equal interest should be paid to the categories 1.1., 1.2. and 1.3. General sites (forests in general, clear cuts burnt for nature conservation and areas of forest decline) are of second priority. The whole survey includes 3000 samples per country. This sampling activity should be executed preferably during a period of 3 years. The duration of the project depends, however, on funding and the capacity for sampling and analysis. Sample collection, handling and extraction. Samples should be collected in May - October 2000, 2001 and Logs, branches and cutting wastes : A suitable spiral drill (diameter 25 mm) should be used to obtain wood chips in a minimal volume of 100 ml from each object sampled. Samples should be packed in plastic bags, marked and sent for analysis. Samples should be incubated for a minimum of 2 weeks at +25 o C before extraction in water by immersion. Trees: For each area of forest decline, 5 symptomatic trees are felled. From each tree trunk and branch wood is sampled. The trunk is sampled with a spiral drill (diameter 25 mm) to obtain wood chips in a minimal volume of 100 ml per drill hole. Ten such drillings evenly distributed along the trunk are combined to form one ml trunk sample. Wood from branches showing symptoms of die-back is sampled with the similar technique, and 10 subsamples of 100 ml are combined to form one ml branch sample per tree. The samples are packed in plastic bags, marked and sent for analysis. Samples should be incubated for a minimum of 2 weeks at +25 o C before extraction in water by immersion. The survey efforts should be focused on Monochamus/wilt symptomatic trees regardless of the nature of the site, decline or not decline.

28 Identification of PWN PWN can be identified on morphological criteria, or by various techniques based on DNA. For the time being, morphology is considered to support to the molecular techniques. Progression of survey Preliminary results from the year 2000 survey will be made available before October 15 th References Anonymous Draft report of a mission carried out in Portugal from September 1999 to assess the measures taken by the Portuguese authorities in response to the outbreak of pine wood nematode (Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle). European Commission, Directorate-General Health and Consumer Protection, Directorate D-Food and Veterinary Office. Doc. DG (SANCO)/1223/99-MR-Draft 23/09/1999: 18 pp. Evans, H.F., McNamara, D.G., Braasch, H., Chadoeuf, J. & Magnusson, C Pest Risk Analysis (PRA) for the territories of the European Union (as PRA area) on Bursaphelenchus xylophilus and its vectors in the genus Monochamus. EPPO Bulletin 26: McNamara, D.G. & Støen, M A survey for Bursaphelenchus spp. in pine forests in Norway. EPPO Bulletin 18: Magnusson, C. & Schroeder, LM First record of a Bursaphelenchus-species (Nematoda) in a Monochamus beetles in Scandinavia. Anzeiger für Schädlingskunde,Pflanzenschutz und Umweltschutz 62: Tomminen, J Presence of Bursaphelenchus mucronatus (Nematoda: Aphelenchoididae) fourth dispersal stages in selected conifer beetles in Finland. Silva Fennica 24: Tomminen, J., Nuorteva, M., Pulkkinen, M. & Väkevä, J Occurrence of the nematode Bursaphelenchus mucronatus Mamiya & Enda 1979 (Nematoda: Aphelenchoididae) in Finland. Silva Fennica 23:

29 Appendix 2: and differentiatio