Suchergebnisse

1. The invasive pine wood nematode (PWN) Bursaphelenchus xylophilus is one of the most serious threats to pine forests across the world. Detected in Europe in 1999, it has largely spread despite containment measures. 2. Following the European Union regulations, the requested eradication measure is to fell, remove and dispose of all susceptible plants within a clear-cut zone (CCZ) of a radius of 500 m around any infected tree. This measure is controversial since its effectiveness is questioned. 3. An individual-based model, describing the dispersal of the nematode vector and the nematode transmission, was used to estimate the relationship between the radius and the effectiveness of the CCZ at eradicating the PWN. 4. Clear-cutting of a 500-m radius is poorly effective in non-fragmented pine forests since it reduces the number of PWN transmissions by only 0.6%–11.5%. To significantly reduce the number of transmissions, the radius should be between 14 and 38 km, which is not technically nor ethically feasible. 5. Policy implications. Our results, based on model simulations at a fine spatial scale, prove that clear-cutting susceptible trees 500 m around any infested tree—as requested by EU regulation to eradicate the PWN—is not effective in large and continuous pine forests. Instead, strengthened surveillance and sanitation felling could be explored.

In Europe, species of the nematode genus Bursaphelenchus have been known and studied for a long time (Fuchs 1937; Ruhm 1956). Earlier, except from a purely biological or ecological point of view, no particular interest was paid to this group of mycophagous nematodes. In 1979, however, a study conducted in southwestern France showed that the nematode Bursaphelenchus lignicolous was associated with declining pines (Baujard et al. 1979). This report caused alarm in Europe, since B. lignicolous is a synonym of B. xylophilus ; the nematode in question was later identifi ed as B. mucronatus (De Guiran et al. 1986), which had been described as a new species that year. In 1984, a shipment of wood from North America to Finland was found to carry the pine wood nematode (PWN), B. xylophilus (Rautapaa 1986). This important interception prompted European authorities to develop more rigorous inspections at sea ports, and in particular of wood products coming from North America. However, no equivalent emphasis was placed on such products coming from East Asia. Between 1996 and 1999, an EU-funded project (RISKBURS) resulted in an updated survey of the Bursaphelenchus species in Europe (European Communities 2003). For an updated situation on the species distribution in the EU, see Braasch (2001). In 1999, the PWN, the causal agent of pine wilt disease, was fi rst detected in the European Union (EU), in Portugal (Mota 2004; Mota et al. 1999), and this immediately prompted several national and EU governments to assess the extent of the nematode's distribution, and to restrict B. xylophilus and its insect vector (Monochamus galloprovincialis ) to an area with a 30-km radius in the Setúbal Peninsula, 20 km south of Lisbon (Rodrigues 2007). The origin of the population of PWN found in Portugal remains unknown, although recent research indicates that it originated from Eastern Asia (Vieira et al. 2007). Several hypotheses have been suggested on how it entered the country, namely from North America or from Japan or China. World trade of wood products such as timber, wooden crates, and palettes play an important role in the potential dissemination of the PWN (Evans et al. 1996). In fact, human activities involving the movement of wood products may be the single most important factor in PWN spread. Despite the dedicated and concerted actions of government agencies, both the PWN and pine wilt disease continue to spread. In 2006 in Portugal, forestry and plant quarantine authorities (DGRF and DGPC) announced a new strategy for managing the problem. The plan is to establish a phytosanitary strip, 3-km wide, devoid of Pinus pinaster , surrounding the affected area, for the control and ultimately the eradication of the nematode, under the coordination of the national program for the control of the PWN (DGRF 2006). Research on the bioecology of the nematode and its insect vector, new detection methods, for example, involving real-time PCR, tree ecology and pathology, and control methods, has been underway since 1999. As well there are two major ongoing projects for the European Union (EU): PHRAME (http://www.forestresearch.gov.uk/website/forestresearch. nsf/ByUnique/INFD-63KGEF) and PortCheck (http://www.portcheck.eu.com/ index.cfm). This research has been instrumental in helping to understand the scientifi c aspects of pine wilt disease. The objective of the present paper is to highlight the progress made in Portugal and the EU. International agreements (GATT, WTO) and sharing of scientifi c information is of paramount importance for achieving effective control of the nematode and its vector, and in turn protection of our forest ecosystems and forest economies.

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 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.

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.

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.

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

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).

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), 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 root lesion nematode Pratylenchus penetrans is a migratory species that attacks a broad range of crops. Like other plant pathogens, P. penetrans deploys a battery of secreted protein effectors to manipulate plant hosts and induce disease. Although several candidate effectors of P. penetrans have been identified, detailed mechanisms of their functions and particularly their host targets remain largely unexplored. In this study, a repertoire of candidate genes encoding pioneer effectors of P. penetrans was amplified from mixed life stages of the nematode, and candidate effectors were cloned and subjected to transient expression in a heterologous host, Nicotiana benthamiana, using potato virus X-based gene vector. Among seven analyzed genes, the candidate effector designated as Ppen10370 triggered pleiotropic phenotypes substantially different from those produced by wild type infection. Transcriptome analysis of plants expressing Ppen10370 demonstrated that observed phenotypic changes were likely related to disruption of core biological processes in the plant due to effector-originated activities. Cross-species comparative analysis of Ppen10370 identified homolog gene sequences in five other Pratylenchus species, and their transcripts were found to be localized specifically in the nematode esophageal glands by in situ hybridization. RNA silencing of the Ppen10370 resulted in a significant reduction of nematode reproduction and development, demonstrating an important role of the esophageal gland effector for parasitism. ; United States Department of Agriculture Agricultural Research Service CRIS project [804221000-300-00D]; Fundacao para a Ciencia e a Tecnologia (FCT, Foundation for Science and Technology) [UIDB/05183/2020, PTDC/AGR-PRO/2589/2014]; FCT postdoctoral fellowship [SFRH/BPD/116030/2016] ; Published version ; This project was supported by the United States Department of Agriculture Agricultural Research Service CRIS project number 804221000-300-00D, by the National Funds through Fundacao para a Ciencia e a Tecnologia (FCT, Foundation for Science and Technology) under the projects UIDB/05183/2020 and PTDC/AGR-PRO/2589/2014 (to M. Mota and J. Branco) and by a FCT postdoctoral fellowship SFRH/BPD/116030/2016 (to C. S. L. Vicente). ; Public domain authored by a U.S. government employee

The invasive pine wood nematode (PWN), Bursaphelenchus xylophilus, is one of the most serious threats to pine forests across the world. Detected in Europe in 1999, it has largely spread despite containment measures. Following the European Union regulations, the requested eradication measure is to fell, remove and dispose of all susceptible plants within a clear-cut zone (CCZ) of a radius of 500 m around any infected tree. This measure is controversial since its effectiveness is questioned. An individual-based model, describing the dispersal of the nematode vector and the nematode transmission,was used to estimate the relationship between the radius and the effectiveness of the CCZ at eradicatingthe PWN. Clear-cutting of a 500 m-radius is poorly effective in non-fragmented pine forests since it reduces the number of PWN transmissions by only 0.6 % 11.5 %. To significantly reduce the number of transmissions, the radius should be between 14 and 38 km, which is obviously not technically nor ethically feasible.Policy implications. Our results, based on model simulations at a fine spatial scale,prove that clear-cutting susceptible trees 500 m around any infested tree –as requested by EU regulation to eradicate the pine wood nematode –is not effective in large and continuous pine forests. Instead, strengthened surveillance and sanitation felling could be explored. ; Le nématode du pin (PWN), Bursaphelenchus xylophilus, est l'une des plus importantes 59espèces invasives menaçant les forêts de pins dans le monde. Après avoir été détecté en Europe en 1999, il s'est rapidement propagé au Portugal puis en Espagne, malgré les mesures mises en place pour le contenir .Selon la réglementation de l'Union Européenne, la mesure relative à l'éradication consiste à abattre, retirer et éliminer tous les végétaux sensibles dans une zone de coupe à blanc (CCZ) d'un rayon de 500 m autour de tout arbre infecté par le PWN. Cette mesure est controversée car son efficacité est incertaine. Un modèle individu-centré,décrivant la dispersion de l'insecte vecteur ainsi que la transmissiondu PWN, a été utilisé pour estimer la relation entre le rayon de la coupe à blanc et son efficacité à éradiquer le PWN. Une coupe à blanc d'un rayon de 500 m est très peu efficace dans une forêt de pins non 70fragmentée car elle ne peut réduire le nombre de transmissions du PWN que de 0.6% à 11.5 71%. Pour réduire de manière significative ce nombre de transmissions, le rayon devrait être de 14 à 38 km, ce qui n'est de façon évidente pas envisageable d'un point de vue technique ou éthique. Implications politiques. Nos résultats, issus de simulations conduites à une échelle spatiale fine, prouvent que la coupe des végétaux sensibles dans un rayon de 500 m autour de tout arbre infecté –comme demandée par la réglementation de l'Union Européenne pour éradiquer le nématode du pin–n'est pas efficace dans les grandes forêts de pins non fragmentées. À la place, le renforcement de la surveillance et des coupes sanitaires ciblées devraient être envisagé