Pine wilt disease (PWD) is one of the most damaging events affecting conifer forests (in particular Pinus spp.), in the Far East (Japan, China and Korea), North America (USA and Canada) and, more recently, in the European Union (Portugal). In Japan it became catastrophic, damaging native pine species (Pinus thunbergii and P. densiflora), and becoming the main forest problem, forcing some areas to be totally replaced by other tree species. The pine wilt nematode (PWN) Bursaphelenchus xylophilus, endemic, with minor damage, to North America, was introduced in Japan in the early XX century and then spread to Asia (China and Korea) in the 1980s. In 1999 it was detected for the first time in Portugal, where, due to timely detection and immediate government action, it was initially (1999-2008) contained to a small area 30 km SE of Lisbon. In 2008, the PWN spread again to central Portugal, the entire country now being classified as "affected area". Being an A1 quarantine pest, the EU acted to avoid further PWN spreading and to eradicate it, by actions including financial support for surveyes and eradication, annual inspections and research programs. Experience from control actions in Japan included aerial spraying of insecticides to control the insect vector (the Cerambycid beetle Monochamus alternatus), injection of nematicides to the trunk of infected trees, slashing and burning of large areas out of control, beetle traps, biological control and tree breeding programs. These actions allowed some positive results, but also unsuccessful cases due to the PWN spread and virulence. Other Asian countries also followed similar strategies, but the nematode is still spreading in many regions. In Portugal, despite lower damage than Asia, PWD is still significant with high losses to the forestry industry. New ways of containing PWD include preventing movement of contaminated wood, cutting symptomatic trees and monitoring. Despite a national and EU legislative body, no successful strategy to control and eventually eradicate the nematode and the disease will prevail without sound scientific studies regarding the nematode and vector(s) bioecology and genetics, the ecology and ecophysiology of the pine tree species, P. pinaster and P. pinea , as well as the genomics and proteomics of pathogenicity (resistance/ susceptibility).
According to the European Plant Protection Organization, the pinewood nematode (PWN), Bursaphelenchus xylophilus is a quarantine organism at the top of the list of the pathogenic species. PWN may be found in North America (Canada, USA and Mexico) and in East Asia (Japan, Korea, China and Taiwan) and has a highly destructive capability towards conifers, in a relatively short time, causing serious economic damage in Japan, China and Korea. This nematode surveying is extremely important and a correct identification of nematode species associated with conifers is essential. Several conifers species are very susceptible (Pinus sylvestris, Pinus nigra and Pinus pinaster), the last one (maritime pine) is a particulary important economic specie in Portugal, and in southern Europe. In 1999, this nematode was found and identified for the first time in Portugal and in Europe. During 1999 and 2000, the Portuguese government, following an alert provided to European Community officials initiated an extensive national survey. During 2000 and 2001, research has been carried out regarding the morphobiometric as well as molecular (DNA: ITS-RFLP) characterization of the collected populations, as well as closely related species of Bursaphelenchus. The book details the Proceedings of an international workshop held at the University of Évora in 2001, covering all major aspects of the bioecology of the pinewood nematode (PWN), Bursaphelenchus xylophilus, its insect vectors and effects on the tree. Topics covered are: global issues and national surveys; morphology and molecular methods of identification of PWN; ecology and epidemiology; quarantine issues; tree physiology, resistance and histopathology; biology of PWN and relationships to its cerambycid vectors; control methods. General quarantine and political views are also presented.
Comparative studies between Portuguese (T and HF) and Japanese (S10, T4, C14-5 and OKD-1) isolates of the pinewood nematode Bursaphelenchus xylophilus have been made in order to provide information to better understand the possible origin of the Portuguese isolates, recently introduced in the European Union. The main comparative aspects investigated were pathogenicity (seedling mortality ratio), sexual compatibility, and DNA sequences of the rDNA region. Four-year-old Japanese black pine (Pinus thunbergii) seedlings were used as host plants for pathogenicity tests. The Portuguese isolates, and in particular isolate "T," propagated in higher numbers than the Japanese isolates within pine seedlings. All combinations of crossings produced viable progeny, with higher numbers obtained when crossings were made between Japanese and Portuguese isolates, a possible situation of heterosis and/or inbreeding depression. Reciprocal crossings yielded different values, which may reflect a sex effect (maternal inheritance, mtDNA). Regarding DNA sequencing, both Portuguese isolates displayed nearly identical ITS 1, ITS2, and 5.8S rDNA base sequences as the Japanese isolates. Although biologically very similar, and possibly reflecting a common origin, the Portuguese isolates may present a serious threat to Japanese black pine, due to their higher virulence.
Comparative studies between Portuguese (T and HF) and Japanese (S10, T4, C14-5 and OKD-1) isolates of the pinewood nematode Bursaphelenchus xylophilus have been made in order to provide information to better understand the possible origin of the Portuguese isolates, recently introduced in the European Union. The main comparative aspects investigated were pathogenicity (seedling mortality ratio), sexual compatibility, and DNA sequences of the rDNA region. Four-year-old Japanese black pine (Pinus thunbergii) seedlings were used as host plants for pathogenicity tests. The Portuguese isolates, and in particular isolate quot;T,quot; propagated in higher numbers than the Japanese isolates within pine seedlings. All combinations of crossings produced viable progeny, with higher numbers obtained when crossings were made between Japanese and Portuguese isolates, a possible situation of heterosis and/or inbreeding depression. Reciprocal crossings yielded different values, which may reflect a sex effect (maternal inheritance, mtDNA). Regarding DNA sequencing, both Portuguese isolates displayed nearly identical ITS 1,ITS2, and 5.8S rDNA base sequences as the Japanese isolates. Although biologically very similar, and possibly reflecting a common origin, the Portuguese isolates may present a serious threat to Japanese black pine, due to their higher virulence.
Comparative studies between Portuguese (T and HF) and Japanese (S10, T4, C14-5 and OKD-1) isolates of the pinewood nematode Bursaphelenchus xylophilus have been made in order to provide information to better understand the possible origin of the Portuguese isolates, recently introduced in the European Union. The main comparative aspects investigated were pathogenicity (seedling mortality ratio), sexual compatibility, and DNA sequences of the rDNA region. Four-year-old Japanese black pine (Pinus thunbergii) seedlings were used as host plants for pathogenicity tests. The Portuguese isolates, and in particular isolate "T," propagated in higher numbers than the Japanese isolates within pine seedlings. All combinations of crossings produced viable progeny, with higher numbers obtained when crossings were made between Japanese and Portuguese isolates, a possible situation of heterosis and/or inbreeding depression. Reciprocal crossings yielded different values, which may reflect a sex effect (maternal inheritance, mtDNA). Regarding DNA sequencing, both Portuguese isolates displayed nearly identical ITS 1, ITS2, and 5.8S rDNA base sequences as the Japanese isolates. Although biologically very similar, and possibly reflecting a common origin, the Portuguese isolates may present a serious threat to Japanese black pine, due to their higher virulence.
The pinewood nematode (PWN) is one of the worst tree‐killing exotic pests in East‐Asian countries. The first European record of establishment in Portugal in 1999 triggered extensive surveys and contingency plans for eradication in European countries, including immediate removal of large areas of conifer host trees. Using Norway as an example, we applied a simulation model to evaluate the chance of successful eradication of a hypothetical introduction by the current contingency plan in a northern area where wilting symptoms are not expected to occur. Despite a highly variable spread of nematode infestations in space and time, the probability of successful eradication in 20 years was consistently low (mean 0.035, SE 0.02). The low success did not change significantly by varying the biological parameters in sensitivity analyses (SA), probably due to the late detection of infestations by the survey (mean 14.3 years). SA revealed a strong influence of management parameters. However, a high probability of eradication required unrealistic measures: achieving an eradication probability of 0.99 in 20 years required 10,000 survey samples per year and a host tree removal radius of 8,000 m around each detection point.
Bursaphelenchus xylophilus is the main causal agent of the pine wilt disease and a worldwide pest with high economic impact. As a serious invasive and destructive species, it is listed as a quarantine pest in the legislation of more than 40 countries. B. xylophilus was reported for the first time in Europe in Portugal in 1999 and in 2008 the whole country has been considered an affected area. In 2008, presence of B. xylophilus was confirmed in Spain. This paper reports on the biology, introduction, spread and the control of B. xylophilus as potential risk element in European coniferous forests. Keywords: Bursaphelenchus; pine wilt disease; pathway spread survey
Der Kiefernholznematode, Bursaphelenchus xylophilus (Steiner und Buhrer) Nickle 1970, führt in anfälligen Koniferenarten, hauptsächlich Pinus spp., außerhalb seines natürlichen Verbreitungsgebietes (Nordamerika) und in nicht heimischen Kiefernarten zur sogenannten Kiefernwelkekrankheit. Er ist seit seiner Einschleppung nach Asien und Europa zum schädlichsten pflanzenparasitären Nematoden von Bäumen geworden. Erkrankte Bäume welken und sterben. Derzeit betroffene europäische Länder sind Portugal und Spanien. B. xylophilus ist in der Europäischen Union (EU) aufgrund der zu beobachteten Gefahr in Befallsländern als ein Quarantäneschädling gelistet. In dieser Doktorarbeit, die Teil eines EU-Forschungsvorhabens der EU-Kommission ist, wurden Untersuchungen zur Unterstützung der Schädlingsrisikoanalyse, Managementstrategien und Notfallplanung der EU-Pflanzengesundheitspolitik durchgeführt. Die Übertragung von B. xylophilus zu neuen Wirtsbäumen findet üblicherweise über Vektorkäfer der Gattung Monochamus statt. Es wurden jedoch B. xylophilus in importierten Holzhackschnitzeln mit Ursprung Nordamerika in der EU beanstandet und die Nachfrage nach Hackschnitzelimporten aus Nordamerika steigt. Daher ist das phytosanitäre Risiko der nicht-vektorassoziierten Übertragung von B. xylophilus mittels Hackschnitzeln von Interesse. Darüberhinaus stellte sich heraus, dass Pinus sylvestris, eine in Deutschland und Nordosteuropa weitverbreitete Baumart, in Gewächshausversuchen mit Sämlingen höchst anfällig gegenüber B. xylophilus ist. In Europa wurden bisher noch keine erwachsenen Bäume dieser Art getestet. Der dritte Teil dieser Doktorarbeit bestand aus einer Bewertung von potenziell toleranten oder resistenten Wirtsbaumherkünften als eine Managementoption der Kiefernwelkeerkrankung in betroffenen Ländern. Das Langzeitüberleben von B. xylophilus in Hackschnitzeln and seine nicht-vektorassoziierte Übertragung von befallenen Hackschnitzeln zu unbefallenen Bäumen wurden untersucht. Mit B. xylophilus befallene Hackschnitzel wurden hergestellt, indem eine Suspension bestehend aus Nematoden und Leitungswasser in P. sylvestris Stämme inokuliert wurde. Während des Langzeitlagerungstests wurde das Überleben von B. xylophilus in versiegelten und offen gelagerten P. sylvestris Hackschnitzeln bei 15°C und 25°C untersucht. Für die Untersuchung der nicht-vektorassoziierten Übertragung wurden mit B. xylophilus befallene Hackschnitzel unter unterschiedlichen Testbedingungen an P. sylvestris Sämlinge platziert. Es wurden Untersuchungen mit sieben- bis achtjährigen Bäumen durchgeführt, um die Aussagefähigkeit der auf Sämlingen basierenden Analysen zur Populationsdynamik und Pathgenität des Schädlings für erwachsene P. sylvestris Bäume zu überprüfen. Die Bäume wurden mit einer B. xylophilus Suspension bestehend aus Nematoden und Leitungswasser künstlich inokuliert. Zur Nematodenextraktion während der Populationsdynamikuntersuchung wurden die Kiefern in 48Segmente geteilt. Physiologische Änderungen und die Entwicklung von Welkesymptomen wurden bis zum Tod der Bäume aufgenommen. Die Pathogenität von B. xylophilus wurde gegenüber unterschiedlichen deutschen Kiefernherkünften (gemäß der Deutschen Herkunftsgebietsverordnung) untersucht. Hierfür wurden P. sylvestris Sämlinge mit einer B. xylophilus Suspension bestehend aus Nematoden und Leitungswasser künstlich inokuliert. In den versiegelten Hackschnitzeln wurde B. xylophilus bei 15°C und 25°C mehr als 1Jahr lang gefunden. Dies war signifikant länger als die zu beobachtende Dauer für die Variante offen gelagert bei 25°C. Weiterhin war die nicht-vektorassoziierte Übertragung mittels Hackschnitzeln durch Temperatur, Baumzustand und Hackschnitzelposition beeinflusst. Hauptsächlich waren Bäume bei 25°C mit Stamm- oder Wurzelverletzungen plus Direktkontakt des verwundeten Pflanzenabschnitts mit befallenen Hackschnitzeln mit B. xylophilus befallen und zeigten eindeutige Symptome der Kiefernwelkekrankheit. Darüberhinaus war für stamm- und wurzelverletzte Kiefern nicht immer ein Direktkontakt mit befallenen Hackschnitzeln für die nicht-vektorassoziierte Übertragung nötig. Bei 15°C wies eine mit B. xylophilus befallene Kiefer eindeutige Symptome der Kiefernwelkekrankheit auf. Zu Beginn der Populationsdynamikuntersuchung war B. xylophilus in der Inokulationsstelle und den benachbarten Segmenten lokalisiert. Bevor externe Welkesymptome entstanden, war B. xylophilus im gesamten Stamm, den benachbarten Astsegmenten, Wurzelhals und Wurzeln lokalisiert. Schließlich wurde B. xylophilus in allen Holz- und Wurzelsegmenten in Kombination mit einer Zunahme der Kiefernwelkeerkrankung und hohen Nematodendichten festgestellt. Kurz vor dem vollständigen Absterben der Bäume war die Baumspitze teilweise nematodenfrei und die darunter liegenden Baumsegmente waren stark mit Nematoden befallen. Während der Pathogenitätsuntersuchung starben alle mit B. xylophilus inokulierten Kiefern und zeigten einen signifikanten, aber unterschiedlich verlaufenden Abfall des Wasserpotentials in den Nadeln im Vergleich zu einer trockengestressten Variante. Alle getesteten P. sylvestris Herkünfte zeigten eine Mortalität von 100%. Es wurde jedoch eine zeitlich signifikant unterschiedlich verlaufende Krankheitsentwicklung bei wenigen P. sylvestris Herkünften gefunden. Schlussfolgernd betrachtet wurden das Langzeitüberleben in Hackschnitzeln und die nicht-vektorassoziierte Übertragung von befallenen Hackschnitzeln zu beschädigten Bäumen klar aufgezeigt, obwohl eine solche Etablierung weniger wahrscheinlich als eine Ausbreitung über Vektoren sein sollte. Diese Ergebnisse sollten in Freilandversuchen getestet werden. P. sylvestris Sämlinge sind gute Indikatorbäume für Untersuchungen der Populationsdynamik und Pathogenität von B. xylophilus, weil die Ergebnisse in sieben- bis achtjährigen P. sylvestris Bäumen vergleichbar zu denen in Sämlingen waren, auch wenn das Erreichen eines Populationsmaximums und die Entwicklung von Welkesymptomen zeitlich verzögert waren. Die Phänomene verspäteter Symptomentwicklung und verspäteten Baumtodes einiger Kiefernherkünfte sollten hinsichtlich potenzieller Abwehreigenschaften näher überprüft werden. Es wird eine fortlaufende Suche nach toleranten oder resistenten Herkünften oder Individuen für Kreuzungsvorhaben als Teil einer phytosanitären Langzeitstrategie gegen B. xylophilus vorgeschlagen. Alles in allem müssen die von befallenen Hackschnitzeln ausgehende Gefahr und das hohe Risiko für P. sylvestris Wälder bei der Schädlingsrisikoanalyse, den Managementstrategien und der Notfallplanung Berücksichtigung finden. ; The pinewood nematode, Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle 1970, is the causal agent of the so-called pine wilt disease in susceptible conifer species, mainly Pinus spp., outside its natural range (North America) and in non-native pine species. Since its introduction to Asia and Europe, it has become the most harmful plant parasitic nematode of trees. Diseased trees wilt and die. Currently affected European countries are Portugal and Spain. Because of the observed threat in infested countries, B. xylophilus is listed as a quarantine pest in the European Union. In this PhD thesis, which is part of a European Union research project of the European Union Commission, investigations were conducted to support the pest risk analysis, management strategies and contingency planning of European Union Plant Health policy. The transmission of B. xylophilus to new host trees is commonly by vector beetles of the genus Monochamus. However, B. xylophilus have been complained in wood chips with origin North America imported to the European Union, and the demand for wood chip imports from North America is increasing. Therefore, the phytosanitary risk of non-vector transmission of B. xylophilus by wood chips is of interest. Moreover, Pinus sylvestris, a widespread tree species in Germany and northeastern Europe, was found to be highly susceptible to B. xylophilus in greenhouse trials using saplings. In Europe, mature trees of this species have not yet been tested. The third part of this thesis was an evaluation of potentially tolerant or resistant host tree provenances as an option for management of pine wilt disease in affected countries. The long-term survival of B. xylophilus in wood chips and its non-vector spread from infested wood chips to non-infested trees were investigated. B. xylophilus-infested wood chips were produced by inoculating a nematode-tap water suspension into P. sylvestris logs. During the long-term storage test, the survival of B. xylophilus was studied in sealed and openly stored P. sylvestris wood chips at 15°C and 25°C. For the investigation of non-vector spread, B. xylophilus-infested wood chips were placed on P. sylvestris saplings under different conditions. Investigations using seven- to eight-year-old trees were conducted to examine the significance of sapling-based analyses of the population dynamics and pathogenicity of the pest for mature P. sylvestris trees. The trees were artificially inoculated with B. xylophilus using a nematode-tap water suspension. For nematode extraction during the population dynamics investigation, the pines were divided into 48 segments. Physiological changes and the development of wilt symptoms were recorded until tree death. The pathogenicity of B. xylophilus towards different German pine provenances (according to the German Legal Ordinance on Regions of Provenance) was studied. For this purpose, P. sylvestris saplings were artificially inoculated with B. xylophilus using a nematode-tap water suspension. In the sealed wood chips, B. xylophilus was found for more than 1year at 15°C and 25°C. This was significantly longer than the duration observed for the variant openly stored at 25°C. Furthermore, non-vector spread through wood chips was influenced by temperature, tree condition and wood chip location. Trees with stem or root injuries plus direct contact of the wounded part with infested wood chips at 25°C were primarily B. xylophilus-infested and showed clear symptoms of pine wilt disease. Moreover, for stem- and root-injured pines, direct contact with infested wood chips was not always necessary for non-vector spread. At 15°C, one B. xylophilus-infested pine exhibited clear symptoms of pine wilt disease. At the start of the population dynamics investigation, B. xylophilus was located at the inoculation site and in adjacent segments. Before any external wilt symptoms developed, B. xylophilus was located in the entire stem, adjacent branch segments, root collar and roots. Finally, B. xylophilus was detected in all wood and root segments in combination with an increase in pine wilt disease and high nematode densities. Shortly before tree death, the treetop was partly nematode-free, and the subjacent tree segments were highly nematode-infested. During the pathogenicity investigation, all B. xylophilus-inoculated pines died and exhibited a significant but variable decline in the water potential in the needles compared to a drought-stressed variant. All tested P. sylvestris provenances showed a mortality of 100%. However, significant differences in the time course of disease development were found for a few provenances. In conclusion, long-term survival in wood chips and non-vector transmission from infested wood chips to damaged trees were clearly shown, although such establishment should be less likely than spread via vectors. These findings should be tested in outdoor trials. P. sylvestris saplings are good indicator trees for investigations of B. xylophilus population dynamics and pathogenicity because the results in seven- to eight-year-old P. sylvestris trees were comparable to those in saplings, although delayed in reaching a population peak and developing wilt symptoms. The phenomena of delayed symptom development and delayed tree death of some pine provenances should be more closely examined with respect to potential defence traits. An ongoing search for tolerant or resistant provenances or individuals for cross-breeding purposes is suggested as part of a long-term phytosanitary strategy against B. xylophilus. Overall, the threat based on infested wood chips and the high risk for P. sylvestris forests must be considered in pest risk analysis, management strategies and contingency planning.
The pinewood nematode, Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle 1970, is the causal agent of the so-called pine wilt disease in susceptible conifer species, mainly Pinus spp., outside its natural range (North America) and in non-native pine species. Since its introduction to Asia and Europe, it has become the most harmful plant parasitic nematode of trees. Diseased trees wilt and die. Currently affected European countries are Portugal and Spain. Because of the observed threat in infested countries, B. xylophilus is listed as a quarantine pest in the European Union. In this PhD thesis, which is part of a European Union research project of the European Union Commission, investigations were conducted to support the pest risk analysis, management strategies and contingency planning of European Union Plant Health policy. The transmission of B. xylophilus to new host trees is commonly by vector beetles of the genus Monochamus. However, B. xylophilus have been complained in wood chips with origin North America imported to the European Union, and the demand for wood chip imports from North America is increasing. Therefore, the phytosanitary risk of non-vector transmission of B. xylophilus by wood chips is of interest. Moreover, Pinus sylvestris, a widespread tree species in Germany and northeastern Europe, was found to be highly susceptible to B. xylophilus in greenhouse trials using saplings. In Europe, mature trees of this species have not yet been tested. The third part of this thesis was an evaluation of potentially tolerant or resistant host tree provenances as an option for management of pine wilt disease in affected countries. The long-term survival of B. xylophilus in wood chips and its non-vector spread from infested wood chips to non-infested trees were investigated. B. xylophilus-infested wood chips were produced by inoculating a nematode-tap water suspension into P. sylvestris logs. During the long-term storage test, the survival of B. xylophilus was studied in sealed and openly stored P. sylvestris wood chips at 15 °C and 25 °C. For the investigation of non-vector spread, B. xylophilus-infested wood chips were placed on P. sylvestris saplings under different conditions. Investigations using seven- to eight-year-old trees were conducted to examine the significance of sapling-based analyses of the population dynamics and pathogenicity of the pest for mature P. sylvestris trees. The trees were artificially inoculated with B. xylophilus using a nematode-tap water suspension. For nematode extraction during the population dynamics investigation, the pines were divided into 48 segments. Physiological changes and the development of wilt symptoms were recorded until tree death. The pathogenicity of B. xylophilus towards different German pine provenances (according to the German Legal Ordinance on Regions of Provenance) was studied. For this purpose, P. sylvestris saplings were artificially inoculated with B. xylophilus using a nematode-tap water suspension. In the sealed wood chips, B. xylophilus was found for more than 1 year at 15 °C and 25 °C. This was significantly longer than the duration observed for the variant openly stored at 25 °C. Furthermore, non-vector spread through wood chips was influenced by temperature, tree condition and wood chip location. Trees with stem or root injuries plus direct contact of the wounded part with infested wood chips at 25 °C were primarily B. xylophilus-infested and showed clear symptoms of pine wilt disease. Moreover, for stem- and root-injured pines, direct contact with infested wood chips was not always necessary for non-vector spread. At 15 °C, one B. xylophilus-infested pine exhibited clear symptoms of pine wilt disease. At the start of the population dynamics investigation, B. xylophilus was located at the inoculation site and in adjacent segments. Before any external wilt symptoms developed, B. xylophilus was located in the entire stem, adjacent branch segments, root collar and roots. Finally, B. xylophilus was detected in all wood and root segments in combination with an increase in pine wilt disease and high nematode densities. Shortly before tree death, the treetop was partly nematode-free, and the subjacent tree segments were highly nematode-infested. During the pathogenicity investigation, all B. xylophilus-inoculated pines died and exhibited a significant but variable decline in the water potential in the needles compared to a drought-stressed variant. All tested P. sylvestris provenances showed a mortality of 100 %. However, significant differences in the time course of disease development were found for a few provenances. In conclusion, long-term survival in wood chips and non-vector transmission from infested wood chips to damaged trees were clearly shown, although such establishment should be less likely than spread via vectors. These findings should be tested in outdoor trials. P. sylvestris saplings are good indicator trees for investigations of B. xylophilus population dynamics and pathogenicity because the results in seven- to eight-year-old P. sylvestris trees were comparable to those in saplings, although delayed in reaching a population peak and developing wilt symptoms. The phenomena of delayed symptom development and delayed tree death of some pine provenances should be more closely examined with respect to potential defence traits. An ongoing search for tolerant or resistant provenances or individuals for cross-breeding purposes is suggested as part of a long-term phytosanitary strategy against B. xylophilus. Overall, the threat based on infested wood chips and the high risk for P. sylvestris forests must be considered in pest risk analysis, management strategies and contingency planning. ; Der Kiefernholznematode, Bursaphelenchus xylophilus (Steiner und Buhrer) Nickle 1970, führt in anfälligen Koniferenarten, hauptsächlich Pinus spp., außerhalb seines natürlichen Verbreitungsgebietes (Nordamerika) und in nicht heimischen Kiefernarten zur sogenannten Kiefernwelkekrankheit. Er ist seit seiner Einschleppung nach Asien und Europa zum schädlichsten pflanzenparasitären Nematoden von Bäumen geworden. Erkrankte Bäume welken und sterben. Derzeit betroffene europäische Länder sind Portugal und Spanien. B. xylophilus ist in der Europäischen Union (EU) aufgrund der zu beobachteten Gefahr in Befallsländern als ein Quarantäneschädling gelistet. In dieser Doktorarbeit, die Teil eines EU-Forschungsvorhabens der EU-Kommission ist, wurden Untersuchungen zur Unterstützung der Schädlingsrisikoanalyse, Managementstrategien und Notfallplanung der EU-Pflanzengesundheitspolitik durchgeführt. Die Übertragung von B. xylophilus zu neuen Wirtsbäumen findet üblicherweise über Vektorkäfer der Gattung Monochamus statt. Es wurden jedoch B. xylophilus in importierten Holzhackschnitzeln mit Ursprung Nordamerika in der EU beanstandet und die Nachfrage nach Hackschnitzelimporten aus Nordamerika steigt. Daher ist das phytosanitäre Risiko der nicht-vektorassoziierten Übertragung von B. xylophilus mittels Hackschnitzeln von Interesse. Darüberhinaus stellte sich heraus, dass Pinus sylvestris, eine in Deutschland und Nordosteuropa weitverbreitete Baumart, in Gewächshausversuchen mit Sämlingen höchst anfällig gegenüber B. xylophilus ist. In Europa wurden bisher noch keine erwachsenen Bäume dieser Art getestet. Der dritte Teil dieser Doktorarbeit bestand aus einer Bewertung von potenziell toleranten oder resistenten Wirtsbaumherkünften als eine Managementoption der Kiefernwelkeerkrankung in betroffenen Ländern. Das Langzeitüberleben von B. xylophilus in Hackschnitzeln and seine nicht-vektorassoziierte Übertragung von befallenen Hackschnitzeln zu unbefallenen Bäumen wurden untersucht. Mit B. xylophilus befallene Hackschnitzel wurden hergestellt, indem eine Suspension bestehend aus Nematoden und Leitungswasser in P. sylvestris Stämme inokuliert wurde. Während des Langzeitlagerungstests wurde das Überleben von B. xylophilus in versiegelten und offen gelagerten P. sylvestris Hackschnitzeln bei 15 °C und 25 °C untersucht. Für die Untersuchung der nicht-vektorassoziierten Übertragung wurden mit B. xylophilus befallene Hackschnitzel unter unterschiedlichen Testbedingungen an P. sylvestris Sämlinge platziert. Es wurden Untersuchungen mit sieben- bis achtjährigen Bäumen durchgeführt, um die Aussagefähigkeit der auf Sämlingen basierenden Analysen zur Populationsdynamik und Pathgenität des Schädlings für erwachsene P. sylvestris Bäume zu überprüfen. Die Bäume wurden mit einer B. xylophilus Suspension bestehend aus Nematoden und Leitungswasser künstlich inokuliert. Zur Nematodenextraktion während der Populationsdynamikuntersuchung wurden die Kiefern in 48 Segmente geteilt. Physiologische Änderungen und die Entwicklung von Welkesymptomen wurden bis zum Tod der Bäume aufgenommen. Die Pathogenität von B. xylophilus wurde gegenüber unterschiedlichen deutschen Kiefernherkünften (gemäß der Deutschen Herkunftsgebietsverordnung) untersucht. Hierfür wurden P. sylvestris Sämlinge mit einer B. xylophilus Suspension bestehend aus Nematoden und Leitungswasser künstlich inokuliert. In den versiegelten Hackschnitzeln wurde B. xylophilus bei 15 °C und 25 °C mehr als 1 Jahr lang gefunden. Dies war signifikant länger als die zu beobachtende Dauer für die Variante offen gelagert bei 25 °C. Weiterhin war die nicht-vektorassoziierte Übertragung mittels Hackschnitzeln durch Temperatur, Baumzustand und Hackschnitzelposition beeinflusst. Hauptsächlich waren Bäume bei 25 °C mit Stamm- oder Wurzelverletzungen plus Direktkontakt des verwundeten Pflanzenabschnitts mit befallenen Hackschnitzeln mit B. xylophilus befallen und zeigten eindeutige Symptome der Kiefernwelkekrankheit. Darüberhinaus war für stamm- und wurzelverletzte Kiefern nicht immer ein Direktkontakt mit befallenen Hackschnitzeln für die nicht-vektorassoziierte Übertragung nötig. Bei 15 °C wies eine mit B. xylophilus befallene Kiefer eindeutige Symptome der Kiefernwelkekrankheit auf. Zu Beginn der Populationsdynamikuntersuchung war B. xylophilus in der Inokulationsstelle und den benachbarten Segmenten lokalisiert. Bevor externe Welkesymptome entstanden, war B. xylophilus im gesamten Stamm, den benachbarten Astsegmenten, Wurzelhals und Wurzeln lokalisiert. Schließlich wurde B. xylophilus in allen Holz- und Wurzelsegmenten in Kombination mit einer Zunahme der Kiefernwelkeerkrankung und hohen Nematodendichten festgestellt. Kurz vor dem vollständigen Absterben der Bäume war die Baumspitze teilweise nematodenfrei und die darunter liegenden Baumsegmente waren stark mit Nematoden befallen. Während der Pathogenitätsuntersuchung starben alle mit B. xylophilus inokulierten Kiefern und zeigten einen signifikanten, aber unterschiedlich verlaufenden Abfall des Wasserpotentials in den Nadeln im Vergleich zu einer trockengestressten Variante. Alle getesteten P. sylvestris Herkünfte zeigten eine Mortalität von 100 %. Es wurde jedoch eine zeitlich signifikant unterschiedlich verlaufende Krankheitsentwicklung bei wenigen P. sylvestris Herkünften gefunden. Schlussfolgernd betrachtet wurden das Langzeitüberleben in Hackschnitzeln und die nicht-vektorassoziierte Übertragung von befallenen Hackschnitzeln zu beschädigten Bäumen klar aufgezeigt, obwohl eine solche Etablierung weniger wahrscheinlich als eine Ausbreitung über Vektoren sein sollte. Diese Ergebnisse sollten in Freilandversuchen getestet werden. P. sylvestris Sämlinge sind gute Indikatorbäume für Untersuchungen der Populationsdynamik und Pathogenität von B. xylophilus, weil die Ergebnisse in sieben- bis achtjährigen P. sylvestris Bäumen vergleichbar zu denen in Sämlingen waren, auch wenn das Erreichen eines Populationsmaximums und die Entwicklung von Welkesymptomen zeitlich verzögert waren. Die Phänomene verspäteter Symptomentwicklung und verspäteten Baumtodes einiger Kiefernherkünfte sollten hinsichtlich potenzieller Abwehreigenschaften näher überprüft werden. Es wird eine fortlaufende Suche nach toleranten oder resistenten Herkünften oder Individuen für Kreuzungsvorhaben als Teil einer phytosanitären Langzeitstrategie gegen B. xylophilus vorgeschlagen. Alles in allem müssen die von befallenen Hackschnitzeln ausgehende Gefahr und das hohe Risiko für P. sylvestris Wälder bei der Schädlingsrisikoanalyse, den Managementstrategien und der Notfallplanung Berücksichtigung finden.
Pine wilt disease (PWD) is perhaps the most serious threat to pine forests worldwide. Since it´s discovery in the early XXth century by Japanese forest researchers, and the relationship with its causative agent, the pinewood nematode (PWN) Bursaphelenchus xylophilus, in the 1970s, PWD has wreaked havoc wherever it appears. Firstly in the Far East (Japan, China and Korea) and now, more recently in 1999, in the EU (Portugal). The forest sector in Portugal plays a major role in the Portuguese economy with a 12% contribution to the industrial gross domestic product, 3.2% of the gross domestic product, 10% of foreign trade and 5% of national employment. Maritime pine (Pinus pinaster) is one of the most important pine productions, and industrial activity, such as the production of wood and resin, as well as coastal protection associated with sand dunes. Also, stone pine (Pinus pinea) plays an important role in the economy with a share derived from the exports of high-quality pineon seed. Thus, the tremendous economical and ecological impact of the introduction of a pest and pathogen such as the PWN, although as far as is known, the only species susceptible to the nematode is maritime pine. Immediately following detection, the research team involved (Univ. Évora, INIAP) informed the national plant quarantine and forest authorities, which relayed the information to Brussels and the appropriate EU authorities. A task force (GANP), followed by a national program (PROLUNP) was established. Since then, national surveys have been taking place, involving MADRP (Ministry of Agriculture), the University of Évora and several private corporations (e.g. UNAC). Forest growers in the area are particularly interested and involved since the area owned by the growers organizations totals 700 000 ha, largely affected by PWD. Detection of the disease has led to serious consequences and restrictions regarding exploration and commercialization of wood. A precautionary phytosanitary strip, 3 km-wide, has been recently (2007) established surrounding the affected area. The Portuguese government, through its national program PROLUNP, has been deeply involved since 1999, and in conjunction with the EU (Permanent Phytosanitary Committee, and FVO) and committed to controlling this nematode and the potential spread to the rest of the country and to the rest of the EU. The global impact of the presence of Bursaphelenchus xylophilus or the threat of its introduction and the resulting pine wilt disease in forested areas in different parts of the world is of increasing concern economically. The concern is exacerbated by the prevailing debate on climate change and the putative impact this could have on the vulnerability of the world's pine forests to this disease. The scientific and regulatory approach taken in different jurisdictions to the threat of pine wilt disease varies from country to country depending on the perceived vulnerability of their pine forests to the disease and/or to the economic cost due to lost trade in wood products. Much of the research surrounding pine wilt disease has been located in the northern hemisphere, especially in southern Europe and in the warmer, coastal, Asian countries. However, there is an increased focus on this problem also in those countries in the southern hemisphere where plantations of susceptible pine have been established over the years. The forestry sector in Australia and New Zealand are on "high alert" for this disease and are practicing strict quarantine procedures at all ports of entry for wood products. As well, there is heightened awareness, as there is worldwide, for the need to monitor wood packaging materials for all imported goods. In carrying out the necessary monitoring and assessment of products for B. xylophilus and its vectors substantial costs are incurred especially when decisions have to be made rapidly and regardless of whether the outcome is positive or negative. Australia's response recently to the appearance of some dying pines in a plantation illustrated the high sensitivity of some countries to this disease. Some $200,000 was spent on the assessment in order to save a potential loss of millions of dollars to the disease. This rapid, co-ordinated response to the report was for naught, because once identified it was found not to be B. xylophilus. This illustrates the particular importance of taking the responsibility at all levels of management to secure the site and the need of a rapid, reliable diagnostic method for small nematode samples for use in the field. Australia is particularly concerned about the vulnerability of its 1million hectares of planted forests, 80% of which are Pinus species, to attack from incursions of one or more species of the insect vector. Monochamus alternatus incursions in wood pallets have been reported from Brisbane, Queensland. The climate of this part of Australia is such that the Pinus plantations are particularly vulnerable to the potential outcome of such incursions, and the state of Queensland is developing a risk management strategy and a proactive breeding programme in response to this putative threat. New Zealand has 1.6 million hectares of planted forests and 89% of the commercial forest is Pinus radiata. Although the climate where these forests are located tends to be somewhat cooler than that in Australia the potential for establishment and development of the disease in that country is believed to be high. The passage alone of 200,000 m³/year of wood packaging through New Zealand ports is itself sufficient to require response. The potential incursion of insect vectors of pinewood nematode through the port system is regarded as high and is monitored carefully. The enormous expansion of global trade and the continued use of unprocessed/inadequately-processed wood for packaging purposes is a challenge for all trading nations as such wood packaging material often harbours disease or pest species. The extent of this problem is readily illustrated by the expanding economies and exports of countries in south-east Asia. China. Japan and Korea have significant areas of forestland infested with B. xylophilus. These countries too are among the largest exporting countries of manufactured goods. Despite the attempts of authorities to ensure that only properly treated wood is used in the crating and packaging of goods B. xylophilus and/or its insect vector infested materials is being recorded at ports worldwide. This reminds us, therefore, of the ease with which this nematode pest can gain access to forest lands in new geographic locations through inappropriate use, treatment or monitoring of wood products. It especially highlights the necessity to find an alternative to using low-grade lumber for packaging purposes. Lest we should believe that all wood products are always carriers of B. xylophilus and its vectors, it should be remembered that international trade of all kinds has occurred for thousands of years and that lumber-born pests and diseases do not have worldwide distribution. Other physico-biological factors have a significant role in the occurrence, establishment and sustainability of a disease. The question is often raised as to why the whole of southern Europe doesn't already have B. xylophilus and pine wilt disease. European countries have traded with countries that are infested with B. xylophilus for hundreds of years. Turkey is an example of a country that appears to be highly vulnerable to pine wilt disease due to its extensive forests in the warm, southern region where the vector, Monochamus galloprovincialis, occurs. However, there is no record of the presence of B. xylophilus occurring there despite the importation of substantial quantities of wood from several countries In many respects, Portugal illustrates both the challenge and the dilemma. In recent times B. xylophilus was discovered there in the warm coastal region. The research, administrative and quarantine authorities responded rapidly and B. xylophilus appears to have been confined to the region in which it was found. The rapid response would seem to have "saved the day" for Portugal. Nevertheless, it raises again the long-standing questions, how long had B. xylophilus been in Portugal before it was found? If Lisbon was the port of entry, which seems very likely, why had B. xylophilus not entered Lisbon many years earlier and established populations and the pine wilt disease? Will the infestation in Portugal be sustainable and will it spread or will it die out within a few years? We still do not have sufficient understanding of the biology of this pest to know the answers to these questions.
The first report of the disease ("pine wilt disease") associated with the pinewood nematode, goes back to 1905, when Yano reported an unusual decline of pines from Nagasaki. For a long time thereafter, the cause of he disease was sought, but without success. Because of the large number of insect species that were usually seen around and on infected trees, it had always been assumed that the causal agent would prove to be one of these. However, in 1971, Kiyohara and Tokushike found a nematode of the genus Bursaphelenchus in infected trees. The nematode found was multiplied on fungal culture, inoculated into healthy trees and then re-isolated from the resulting wilted trees. The subsequent published reports were impressive: this Bursaphelenchus species could kill fully-grown trees within a few months in the warmer areas of Japan, and could destroy complete forests of susceptible pine species within a few years. Pinus densiflora, P. thunbergii und P. luchuensis were particularly affected. In 1972, Mamiya and Kiyohara described the new species of nematode extracted from the wood of diseased pines; it was a named Bursaphelenchus lignicolus. Since 1975, the species has spread to the north of Japan, with the exception of the most northerly prefectures. In 1977, the loss of wood in the west of the country reached 80%. Probably as a result of unusually high summer temperatures and reduced rainfall in the years 1978 and 1979, the losses were more than 2 million m3 per year. From the beginning, B. lignicolus was always considered by Japanese scientists to be an exotic pest. But where did it come from? That this nematode could also cause damage in the USA became clear in 1979 when B. lignicolus was isolated in great numbers from wood of a 39 year-old pine tree (Pinus nigra) in Missouri which had suddenly died after the colour of its needles changed to a reddish-brown colour (Dropkin und Foudin, 2 1979). In 1981, B. lignicolus was synonymised by Nickle et al. with B. xylophilus which had been found for the first time in the USA as far back as 1929, and reported by Steiner and Buhrer in 1934. It had originally been named Aphelenchoides xylophilus, the wood-inhabiting Aphelenchoides but was recognised by Nickle, in 1970,to belong in the genus Bursaphelenchus. Its common name in the USA was the "pine wood nematode" (PWN. After its detection in Missouri, it became known that B. xylophilus was widespread throughout the USA and Canada. It occurred there on native species of conifers where, as a rule, it did not show the symptoms of pine wilt disease unless susceptible species were stressed eg., by high temperature. This fact was an illuminating piece of evidence that North America could be the homeland of PWN. Dwinell (1993) later reported the presence of B. xylophilus in Mexico. The main vector of the PWN in Japan was shown to be the long-horned beetle Monochamus alternatus, belonging to the family Cerambycidae. This beetle lays its eggs in dead or dying trees where the developing larvae then feed in the cambium layer. It was already known in Japan in the 19th century but in the 1930s, it was said to be present in most areas of Japan, but was generally uncommon. However, with the spread of the pine wilt disease, and the resulting increase of weakened trees that could act as breeding sites for beetles, the populations of Monochamus spp. increased significantly In North America, other Monochamus species transmit PWN, and the main vector is M. carolinensis. In Japan, there are also other, less efficient vectors in the genus Monochamus. Possibly, all Monochamus species that breed in conifers can transmit the PWN. The occasional transmission by less efficient species of Monochamus or by some of the many other beetle genera in the bark or wood is of little significance. In Europe, M. galloprovincialis and M. sutor transmits the closely related species B. mucronatus. Some speculate that these two insect species are "standing by" and waiting for the arrival of B. xylophilus. In 1982, the nematode was detected and China. It was first found in dead pines near the Zhongshan Monument of Nanjing (CHENG et. al. 1983); 265 trees were then killed by pine wilt disease. Despite great efforts at eradication in China, the nematode spread further and pine wilt disease has been 3 reported from parts of the provinces of Jiangsu, Anhui, Guangdong, Shandong, Zhejiang and Hubei (YANG, 2003). In 1986, the spread of the PWN to Taiwan was discovered and in 1989, the nematode was reported to be present in the Republic of Korea where it had first been detected in Pinus thunbergii and P. densiflora. It was though to have been introduced with packing material from Japan. PWN was advancing. In 1984, B. xylophilus was found in wood chips imported into Finland from the USA and Canada, and this was the impetus to establish phytosanitary measures to prevent any possible spread into Europe. Finland prohibited the import of coniferous wood chips from these sources, and the other Nordic countries soon followed suit. EPPO (the European and Mediterranean Plant Protection Organization) made a recommendation to its member countries in 1986 to refuse wood imports from infested countries. With its Directive of 1989 (77/93 EEC), the European Community (later called the European Union or EU) recognised the potential danger of B. xylophilus for European forests and imposed restrictions on imports into the Europe. PWN was placed on the quarantine list of the EU and also of other European countries. Later, in 1991, a dispensation was allowed by the Commission of the EU(92/13 EEC) for coniferous wood from North America provided that certain specified requirements were fulfilled that would prevent introduction.
The pinewood nematode (PWN), Bursaphelenchus xylophilus, is a quarantine organism in the European Union and the causal agent of pine wilt disease (PWD), a serious threat to pine forests worldwide, leading to rapid decline and death. In Europe, this invasive pest was first reported in Portugal in 1999, on Pinus pinaster. Due to its economic importance and worldwide distribution, an enormous effort has been devoted to research on B. xylophilus and PWD. However, relating the presence and abundance of this pest with actual forest decline and mortality is not straightforward. In the present work, we assess the interaction between B. xylophilus populations and pine decline at the tree and landscape level. To test this, we determined PWN population densities from different sections of healthy and declining P. pinaster – considered very susceptible to PWN – and P. pinea – considered resistant, in two consecutive years. The studied site was Herdade da Apostiça (Sesimbra, Portugal), a 4 thousand ha forest that exhibits areas of moderate to severe decline. An approximate 500 m long transect was set in four different areas of the experimental site, along which healthy and declining P. pinaster trees were randomly selected. Wood samples from lower (DBH), middle (M) and upper (T) sections of trees were retrieved whenever possible, and kept in individual plastic bags to avoid cross contamination. Twigs from the canopy were also sampled. When present, samples from P. pinea were collected as well. Nematodes were extracted from wood material using a modified Baermann tray method, and counted under a stereoscopic microscope. With only few exceptions, M and T sections consistently displayed the lowest PWN densities, although samples collected at breast height (DBH) also had relatively low numbers of B. xylophilus. On the other hand, twigs had the highest numbers of nematodes in all areas. Surprisingly, nematodes were extracted from the canopy of apparently healthy P. pinaster trees, including the PWN, although in low densities. Expectedly, areas with no visible tree decline had low population densities of B. xylophilus, and declining trees tended to present higher numbers. P. pinea samples were mostly free of the PWN. To further understand the complex dynamics shaping pine forest decline caused by the PWN, more sampling will be carried out in the upcoming months. This will allow us to build predictive models on the spread and damage of PWD.
The pinewood nematode (PWN), Bursaphelenchus xylophilus, the causal agent of pine wilt disease (PWD), is a serious pest and pathogen of forest tree species, in particular among the genus Pinus. It was first reported from Japan in the beginning of the XXth century, where it became the major ecological catastrophe of pine forests, with losses reaching over 2 million m3/ year in the 1980s. It has since then spread to other Asian countries such as China, Taiwan and Korea, causing serious losses and economic damage. In 1999, the PWN was first detected in the European Union (EU), in Portugal, and immmediately prompted several government (national and EU) actions to assess the extent of the nematode's presence, and to contain B. xylophilus and its insect vector (Monochamus galloprovincialis) to an area with a 30km radius in the Setúbal Peninsula, 20 km south of Lisbon. International wood trade, with its political as well as economic ramifications, has been seriously jeopardized. The origin of the population of PWN found in Portugal remains elusive. Several hypotheses may be considered regarding pathway analysis, basically from two general origins: North America or the Far East (Japan or China). World trade of wood products such as timber, wooden crates, palettes, etc… play an important role in the potential dissemination of the pinewood nematode. In fact, human activities involving the movement of wood products may be considered the single most important factor in spreading of the PWN. Despite the dedicated and concerted actions of government agencies, this disease continues to spread. Very recently (2006), in Portugal, forestry and phytosanitary authorities (DGRF and DGPC) have announced a new strategy for the control and ultimately the erradication of the nematode, under the coordination of the national program for the control of the pinewood nematode (PROLUNP). Research regarding the bioecology of the nematode and insect as well as new detection methods, e.g., involving real-time PCR, has progressed since 1999. International agreements (GATT, WTO) and sharing of scientific information is of paramount importance to effectively control the nematode and its vector, and thus protect our forest ecosystems and forest economy.
