First report of Bursaphelenchus xylophilus on Pinus sylvestris in Spain

Досліджено, що стовбурова нематода Bursaphelenchus mucronatus зумовлює всихання верхівок сосни звичайної на Поліссі та ялини європейської в Українських Карпатах. Найбільшу чисельність нематод виявлено у стовбурі дерева нижче ділянки всихання. У деревині всохлих верхівок, окрім стовбурових нематод, іноді у великій кількості розмножуються бактеріофаги, які разом із бактеріями є першими агентами розкладання відмерлої деревини. Збільшення тривалості теплого періоду сприяє інтенсивнішому розвитку стовбурових нематод у деревині хвойних порід. У природних деревостанах смуги ялинових лісів на верхній межі лісу зареєстровано частіше їх трапляння у відмерлій деревині ялини. Наявність на значних площах вторинних монодомінантних деревостанів осередків всихання ялини європейської та сосни звичайної сприяє покращенню функціонуванню біотичної системи стовбурових нематод, а відтак і збільшенню їхньої шкодочинності та всихання дерев.

Pine wood nematode (PWN), Bursaphelenchus xylophilus, causes pine wilt disease (PWD) in Pinus species given suitable climatic conditions. A model was run for Germany to assess the potential that Pinus sylvestris trees succumb to pine wilt once B. xylophilus has been introduced. The following climate scenarios have been modelled: current climatic conditions, an exceptional hot year (2003) and two future climatic conditions with target year 2050 for low (B1) and middle (A1B) emission scenarios based on the IPCC classification. Additional parameters included in the model were nematode inoculum, time of infestation and tree physiological conditions (healthy ors stressed). Under the current climatic conditions, P. sylvestris would not develop PWD assuming that trees are healthy. However, water-stressed trees in the river Rhine area south-west of Germany may succumb to PWD in the current climate. Climatic conditions like those experienced in 2003 would support the expression of PWD in healthy trees in the south of Germany and in almost the whole of Germany if the trees are stressed. Predicted future temperature increases up to 2050 under both emission scenarios will lead to more stress, placing extensive areas of Germany at risk of PWD, including the north-eastern part where Pinus sylvestris is the dominant tree species.

I: Pine wilt disease: global issues, surveys, trade and economic impact. Session Summary. I.1. Eradication program for the pinewood nematode in Portugal. I.2. Incursion management in the face of multiple uncertainties: a case study of an unidentified nematode associated with dying pines near Melbourne, Australia. I.3. The risk of pine wilt disease to Australia and New Zealand.I.4. Potential threat and present status of survey of pine wood nematode in Turkey. I.5. Investigations on wood-inhabiting nematodes of the genus Bursaphelenchus in pine forests in the province Brandenburg, Germany. I.6. Official survey for Bursaphelenchus xylophilus carried out on the territory of the Republic of Poland. I.7. Bursaphelenchus spp. in wood packaging intercepted in China. II: Pine wood nematode: biology and microbial inter-relationships. Session Summary. II.1. Developmental biology and cytogenetics of B. xylophilus. II.2. The relationship between PWN and fungi cohabiting in pine trees inoculated with thee PWN. II.3. Influence of fungi on multiplication and distribution of the pinewood nematode. III: PWN taxonomy and detection methods. Session Summary. III.1. Taxonomic databases for Bursaphelenchus and other aphelenchoid nematodes. III.2. The enlargement of the xylophilus group in the genus Bursaphelenchus. III.3. Interspecific variation in ITS rDNA of Bursaphelenchus species of different groups. III.4. Molecular characterization of isolates of the Bursaphelenchus sexdentati group using ITS-RFLP and ribosomal DNA sequences. III.5. Analysis of Bursaphelenchus xylophilus provenances using ISSR and RAPD fingerprints. III.6. Satellite DNA as a versatile genetic marker for Bursaphelenchus xylophilus. III.7. An effective PCR-based diagnostic method for the detection of PWN in wood samples. IV: The insect vectors: biology, ecology and interaction with PWN. Session Summary IV.1. Biological studies relevant to the vector role of Monochamus species for pinewood nematode. IV.2. Potentialinsect vectors of Bursaphelenchus spp. in Spanish pine forests. IV.3. Genetic structure of Monochamus alternatus in Japan. IV.4. Distribution of nematodes (Bursaphelenchus xylophilus) in the beetle of Monochamus alternatus and its exiting transmission way. V: Ecology and modelling. Session Summary. V.1. Modelling PWN-induced wilt expression: a mechanistic approach. V.2. Field diagnosis of the asymptomatic carrier of pinewood nematode. VI: The tree: physiology, resistance and histopathology as a result of pine wilt disease. Session Summary.VI.1. Inoculation of pine trees with avirulent pinewood nematode under experimental conditions: risk-benefit analysis.VI.2. Rapidity of disease development seems to result in high mortality - insight from an inoculation test using hybridized populations between a virulent and an avirulent isolates of Bursaphelenchus xylophilus.VI.3. Defense systems of Pinus densiflora cultivars selected as resistant to pine wilt disease.VI.4. Histological observations of Bursaphelenchus xylophilus in symptomatic tissues of pinewood. VI.5. Development of external and internal symptoms in pine seedlings (Pinus sylvestris) due to inoculation with Bursaphelenchus vallesianus.VII: PWN and insect vector control methods. Session Summary. VII.1. Screening and isolation of antinematodal metabolites against Bursaphelenchus xylophilus produced by fungi and plant. VII.2. Microbial control of Bursaphelenchus xylophilus by fungi. VII.3. Attraction trap for monitoring Monochamus alternatus adults - its usefulness and limitations. VII.4. Studies on Scleroderma guani to control the pine sawyer, Monochamus alternatus. VII.5. Effect of aerial spraying insecticide as a control measure of pine wilt disease. VII. 6. Control program of pine wilt disease for landscape conservation - the case of Amanohashidate in Kyoto, Japan.

Two isolates of the pinewood nematode, Bursaphelenchusxylophilus (Steiner and Buhrer), from Canada (an r form from Ontario and an m form from Quebec) and one from Japan (an r form) were inoculated into Scots pine, Pinussylvestris L., western larch, Larixoccidentalis Nutt., and black spruce, Piceamariana (Mill.) B.S.P. to determine the pathogenicity of this nematode and to observe changes in its numbers as well as the effect it had on seedling water and tannin content. Attractiveness of water, ethyl ether, and ethyl acetate stem extracts of these conifers to the pinewood nematode was also determined. The r form of the pinewood nematode was pathogenic on all three conifers: the Japanese isolate killed more Scots pine and the Ontario isolate, more black spruce. The Quebec isolate was as pathogenic as the other two isolates to western larch, weakly pathogenic to black spruce, and nonpathogenic to Scots pine. In general, there was a positive relationship between nematode population buildup and seedling mortality. No clear relationship was found between the water content of seedling stems and changes in nematode populations. Tannin content of stems increased following inoculation with pinewood nematode, but whereas correlation values between tannin content and nematode numbers were positive and significant, they were small. Although all three isolates were attracted to all host extracts, regardless of solvent used, ethyl acetate extracts were generally more attractive. The Japanese and Ontario isolates, which originated from pines, showed no particular affinity for Scots pine extracts; however, the Ontario isolate, which was especially attracted to black spruce extracts, was also most pathogenic to black spruce.

Pine wilt disease (PWD), caused by pinewood nematodes, is highly destructive to pine forests in Asia and Europe, including Korean white pine (Pinus koraiensis). The microbiome in the needles and trunk of Pinus spp. are recognized to play key roles in resistance against PWD. However, the role of root and soil microbiomes in the resistance remains unclear. This study compares bacterial and fungal communities in the root endosphere, rhizosphere soil, and bulk soil of diseased versus healthy P. koraiensis. Results showed that PWD increased the α-diversity of fungi in rhizosphere soil but did not affect the microbial diversity in the root endosphere or bulk soil. The composition of bacterial and fungal communities in rhizosphere and bulk soils was significantly altered by PWD. Specifically, the relative abundance of Planctomycetes decreased, and the relative abundance of Tremellomycetes increased, while Agaricomycetes decreased in both rhizosphere and bulk soils after infestation with PWD, respectively. Relative abundances of Chloroflexi and Verrucomicrobia increased, while Proteobacteria decreased in bulk soil following PWD. Relative abundances of Leotiomycetes and Eurotiomycetes increased in the rhizosphere soil and bulk soil following PWD, respectively. Furthermore, with the host plant infestation by PWD, the relative abundance of ectomycorrhizal fungi decreases, while that of saprotrophic fungi increases in both rhizosphere and bulk soils. Our results revealed that PWD significantly affects the soil microbiomes of P. koraiensis, with varying impacts across different plant-soil compartments. This study provides insights into how root and soil microbiomes respond to PWD, enhancing our understanding of the disease's ecological consequences.IMPORTANCEThe belowground microbiome is often sensitive to infection of forest diseases and is also recognized as a potential reservoir for selection of microbial agents against PWD. Our study demonstrates that the dynamics of belowground microbiome following natural infection of PWD are compartment and taxa specific, with varying degrees of responses in both diversity and composition of bacterial or fungal communities across the root endosphere, rhizosphere soil, and bulk soil. The results highlight the importance of utilizing appropriate plant-soil compartments and microbial taxa to understand the ecological consequences of the destructive PWD.

Pine wilt disease (PWD) caused by pine wood nematode (PWN), Bursaphelenchus xylophilus, is a serious threat to global forest populations of conifers, in particular Pinus spp. Recently, the presence of PWN was reported in dead Yunnan pine (Pinus yunnanensis) trees under natural conditions. To further understand the potential impact caused by PWN in Yunnan pine populations, a transcriptional profiling analysis was performed over different time points (0 hours (h), 6 h, 24 h, 48 h, and 7 days) after PWN inoculation. A total of 9961 differentially expressed genes were identified after inoculation, which suggested a dynamic response against the pathogen, with a more intense pattern at 48 h after inoculation. The results also highlighted a set of biological mechanisms triggered after inoculation that provide valuable information regarding the response of Yunnan pine to PWN infection. When compared with maritime pine (Pinus pinaster), the Yunnan pine response was less complex and involved a smaller number of differentially expressed genes, which may be associated with the increased degree of resistance to PWN displayed by Yunnan pine. These results revealed different strategies to cope with PWN infection by these two pine species, which display contrasting degrees of susceptibility, especially in the timely perception of the infection and response magnitude.

Pinewood nematode (PWN), Bursaphelenchus xylophilus, the causal agent of pine wilt disease (PWD), was detected in Spain in 2008. This gives rise to serious concern, as the disease has caused severe environmental and economic losses in Portugal and in Asian countries. We studied interspecific variation in susceptibility to pine wilt disease and differences in constitutive chemical compounds in the xylem tissue of the seven pine species -P. canariensis, P. halepensis, P. pinaster, P. pinea, P. sylvestris, P. radiata and P. taeda. Two-year-old trees were inoculated with B. xylophilus. Water potential and nematode densities were measured for each species on specific dates; whereas, wilting symptoms were recorded weekly until the end of the assay. Chemical compounds in the xylem were determined prior to inoculation. Three different resistance groups can be established in terms of the pine species susceptibility to PWN: non- to slightly-susceptible (P. canariensis, P. halepensis, P. taeda and P. pinea), susceptible (P. pinaster and P. radiata), and highly-susceptible (P. sylvestris). Nematodes migrated downward to the roots in all seven species. Constitutive xylem nitrogen, total polyphenols, and marginally phosphorus were negatively correlated with mortality caused by PWN. The most susceptible species, Pinus sylvestris, presented high levels of constitutive lipid-soluble substances and low levels of manganese, pointing to a possible relation between these components and PWN susceptibility. The results suggest P. sylvestris, P. pinaster and P. radiata forests could be severely damaged by PWN in Spain and highlight how constitutive chemical compounds such as nitrogen might play a role in resistance mechanisms against PWN.

We examined the efficacy of the insecticide/nematicide abamectin to prevent pine wilt disease caused by the pinewood nematode (Bursaphelenchus xylophilus) in Scots pine (Pinus sylvestris). Pinewood nematode movement was inhibited (>80% death or paralysis) following a 48 hr exposure to abamectin concentrations as low as 0.1 μL a.i. per L (100 ppb). A commercial formulation of abamectin (Avid™) was injected into Scots pine using a pressurized systemic trunk injection tube (STIT) technique. Fifteen to 30 mL (0.45 to 0.90 fl oz) of Avid per STIT could be injected into the trees in less than 1 hr. Trees were successfully injected throughout February, March, and April at temperatures above 4.4°C (40°F). Survival after 1 year of 10 cm diameter (4 in) at breast height (dbh) Scots pines injected with Avid and subsequently inoculated with pinewood nematode was higher (75%) than in pines injected with water (42%). Similarly, survival after 3 years of large Scots pines (30 to 60 cm [12 to 24 in] dbh)] injected with Avid and exposed to a natural epidemic of pine wilt was higher (96%) than in noninjected pines (33%) or those injected with water (71%). These results indicate that preventive injections of Scots pine with Avid are effective in protecting against pine wilt disease.

The pine wood nematode (PWN) Bursaphelenchus xylophilus is the pathogen that causes pine wilt disease (PWD), a devastating forest disease. PWN-associated bacteria may play a role in PWD. However, little is known about the endo-bacteria in PWN. We analyzed the diversity of endo-bacteria in nine isolates of PWNs from Pinus massoniana Lamb. in nine epidemic areas from three Chinese provinces by high-throughput sequencing of 16S rDNA and isolated and identified culturable endo-bacteria through construction of a 16S rDNA phylogenetic tree and Biolog microbial identification. We also examined the effects of endo-bacteria on PWN fecundity, antioxidant capacity, and virulence using sterile nematodes as a control. While the dominant endo-bacteria in PWNs from different regions exhibited no significant difference in the classification levels of class and genus, their proportions differed. Pseudomonas and Stenotrophomonas were highly abundant in all PWN isolates. A total of 15 endo-bacterial strains were successfully isolated and identified as six species: Stenotrophomonas maltophilia, Pseudomonas fluorescens, Kocuria palustris, Microbacterium testaceum, Rhizobium radiobacter, and Leifsonia aquatica. We also found that P. fluorescens significantly increased the egg production of PWN, and that both P. fluorescens and S. maltophilia enhanced the mobility of PWN under oxidative stress and reduced the content of reactive oxygen species by increasing antioxidant enzyme activity in PWN. These strains also accelerated the development of PWD, and P. fluorescens had a more beneficial effect on PWN than S. maltophilia. Diversity exists among the endo-bacteria in PWNs from different regions, and some endo-bacteria can promote PWN infestation by enhancing the fecundity and antioxidant capacity of the nematode. Our study contributes to clarifying the interaction between endo-bacteria and PWN.

Estimate global risks of a forest disease under current and future climates using species distribution model and simple thermal model – Pine Wilt disease as a model case

The invasive pine wood nematode (PWN), Bursaphelenchus xylophilus, causal agent of pine wilt disease, was first reported in Europe, near Lisbon, in 1999, and has since then spread to most of Portugal. We here modelled the spatiotemporal patterns of future PNW natural spread in the Iberian Peninsula, as dispersed by the vector beetle Monochamus galloprovincialis, using a process-based and previously validated network model. We improved the accuracy, informative content, forecasted period and spatial drivers considered in previous modelling efforts for the PWN in Southern Europe. We considered the distribution and different susceptibility to the PWN of individual pine tree species and the effect of climate change projections on environmental suitability for PWN spread, as we modelled the PWN expansion dynamics over the long term (>100 years). We found that, in the absence of effective containment measures, the PWN will spread naturally to the entire Iberian Peninsula, including the Pyrenees, where it would find a gateway for spread into France. The PWN spread will be relatively gradual, with an average rate of 0.83% of the total current Iberian pine forest area infected yearly. Climate was not found to be an important limiting factor for long-term PWN spread, because (i) there is ample availability of alternative pathways for PWN dispersal through areas that are already suitable for the PWN in the current climatic conditions; and (ii) future temperatures will make most of the Iberian Peninsula suitable for the PWN before the end of this century. Unlike climate, the susceptibility of different pine tree species to the PWN was a strong determinant of PWN expansion through Spain. This finding highlights the importance of accounting for individual tree species data and of additional research on species-specific susceptibility for more accurate modelling of PWN spread and guidance of related containment efforts.

Pine wood nematode (PWN), Bursaphelenchus xylophilus, is a major pathogen of pine wilt disease (PWD), which is a devastating disease affecting pine trees. Eco-friendly plant-derived nematicides against PWN have been considered as promising alternatives to control PWD. In this study, the ethyl acetate extracts of Cnidium monnieri fruits and Angelica dahurica roots were confirmed to have significant nematicidal activity against PWN. Through bioassay-guided fractionations, eight nematicidal coumarins against PWN were separately isolated from the ethyl acetate extracts of C. monnieri fruits and A. dahurica roots, and they were identified to be osthol (Compound 1), xanthotoxin (Compound 2), cindimine (Compound 3), isopimpinellin (Compound 4), marmesin (Compound 5), isoimperatorin (Compound 6), imperatorin (Compound 7), and bergapten (Compound 8) by mass and nuclear magnetic resonance (NMR) spectral data analysis. Coumarins 1–8 were all determined to have inhibitory effects on the egg hatching, feeding ability, and reproduction of PWN. Moreover, all eight nematicidal coumarins could inhibit the acetylcholinesterase (AChE) and Ca2+ ATPase of PWN. Cindimine 3 from C. monnieri fruits showed the strongest nematicidal activity against PWN, with an LC50 value of 64 μM at 72 h, and the highest inhibitory effect on PWN vitality. In addition, bioassays on PWN pathogenicity demonstrated that the eight nematicidal coumarins could effectively relieve the wilt symptoms of black pine seedlings infected by PWN. The research identified several potent botanical nematicidal coumarins for use against PWN, which could contribute to the development of greener nematicides for PWD control.

Pine wilt is a disease of pine (Pinus spp.) caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus. However, the pathogenic mechanism of pine wilt disease (PWD) remains unclear. Although the PWN was thought to be the only pathogenic agent associated with this disease, a potential role for bacterial symbionts in the disease process was recently proposed. Studies have indicated that aseptic PWNs do not cause PWD in aseptic pine trees, while PWNs associated with bacteria cause wilting symptoms. To investigate the pathogenicity of the PWN and its associated bacteria, 3-month-old microcuttings derived from certain clones of Pinus densiflora Siebold & Zucc. produced in vitro were inoculated under aseptic conditions with aseptic PWNs, non-aseptic PWNs and bacteria isolated from the nematodes. Six-month-old aseptic P. densiflora microcuttings and 7-month-old P. massoniana seedlings were also inoculated under aseptic conditions with aseptic PWNs and non-aseptic PWNs. The results showed that the aseptic microcuttings and seedlings inoculated with aseptic PWNs or non-aseptic PWNs wilted, while those inoculated with bacterial isolates did not wilt. Nematodes were recovered from wilted microcuttings and seedlings inoculated with aseptic PWNs and non-aseptic PWNs, and the asepsis of nematodes recovered from aseptic PWN-inoculated microcuttings and seedlings was reconfirmed by culturing them in NB liquid medium at 30°C for more than 7 days. Taken together, the results indicate that the asepsis of PWN did not cause the loss of pathogenicity.

湖北宜昌松林景观格局与松材线虫流行的关系及驱动力

Pine wood nematode (PWN), Bursaphelenchus xylophilus, is a threat for pine species (Pinus spp.) throughout the world. The nematode is native to North America, and invaded Japan, China, Korea, and Taiwan, and more recently Portugal and Spain. PWN enters new areas through trade in wood products. Once established, eradication is not practically feasible. Therefore, preventing entry of PWN into new areas is crucial. Entry risk analysis can assist in targeting management to reduce the probability of entry. Assessing the entry of PWN is challenging due to the complexity of the wood trade and the wood processing chain. In this paper, we develop a pathway model that describes the wood trade and wood processing chain to determine the structure of the entry process. We consider entry of PWN through imported coniferous wood from China, a possible origin of Portuguese populations, to Europe. We show that exposure increased over years due to an increase in imports of sawn wood. From 2000 to 2012, Europe received an estimated 84 PWN propagules from China, 88% of which arose from imported sawn wood and 12% from round wood. The region in Portugal where the PWN was first reported is among those with the highest PWN transfer per unit of imported wood due to a high host cover and vector activity. An estimated 62% of PWN is expected to enter in countries where PWN is not expected to cause the wilt of pine trees because of low summer temperatures (e.g., Belgium, Sweden, Norway). In these countries, PWN is not easily detected, and such countries can thus serve as potential reservoirs of PWN. The model identifies ports and regions with high exposure, which helps targeting monitoring and surveillance, even in areas where wilt disease is not expected to occur. In addition, we show that exposure is most efficiently reduced by additional treatments in the country of origin, and/or import wood from PWN-free zones. Pathway modelling assists plant health managers in analyzing risks along the pathway and planning measures for enhancing biosecurity.