Potential for establishment of Bursaphelenchus xylophilus and the pine wilt disease under Nordic conditions1

Serious concern about the pinewood nematode, Bursaphelenchus xylophilus, and the pine wilt disease arose in 1984 when the nematode was discovered in wood chips imported for the Finnish pulp industry. It has been questioned whether this nematode could establish itself and actually damage pine trees under Nordic conditions. It seems likely that B. xylophilus could reproduce in Nordic forest lands. It is also likely that potential vector insects are present and could be functional in the establishment and epidemic development of the disease. Although temperatures in Nordic areas hardly seem conducive to pine wilt disease, other factors may contribute to disease expression. Research is needed on interactions of stress factors, particularly temperature and soil moisture, in the expression of pine wilt disease. A better understanding of these interactions would aid in evaluating the risk connected with an accidental introduction of the nematode into Nordic countries and provide a sound basis for trade regulations. At present, there are reasons to be most restrictive concerning any possible means of introduction of B. xylophilus.     

Survival of Bursaphelenchus xylophilus in wood chips1

Preliminary results are presented on elimination of Bursaphelenchus xylophilus from pine wood chips by dry heat, hot water treatment and immersion in dithiocarbamate solution.     

Pine Wilt Disease Caused by the Pine Wood Nematode: The Induced Resistance of Pine Trees by the Avirulent Isolates of Nematode

Since the beginning of the 20th century, pine trees in Japan have been seriously damaged by the pine wilt disease. This disease is caused by the pine wood nematode, Bursaphelenchus xylophilus, which is transmitted by the Japanese pine sawyer, Monochamus alternatus. The control of disease depends to a large extent on chemicals, but the public is now demanding environmentally friendly control methods. The virulence of B. xylophilus varies very widely. Pre-inoculation of young pine trees in a nursery with avirulent B. xylophilus has induced systemic resistance of trees against a subsequent inoculation with virulent B. xylophilus. This induced resistance was considered a hopeful means for developing a biological control for the disease. The induced resistance by the avirulent nematodes was also expressed in mature pine trees in a forest where the disease was naturally epidemic. However, the effects of induced resistance were not satisfactory for practical biological control. Since the inoculation with higher concentrations of the avirulent B. xylophilus induced the resistance more effectively, the pre-inoculation method will need to be improved to develop the biological control. The induced resistance of pine trees by avirulent B. xylophilus should be one of the candidate biological control methods against pine wilt disease. This induced resistance also provides an experimental system to clarify physiological interactions between the nematodes and pine trees.     

Differences in constitutive and inducible defences in pine species determining susceptibility to pinewood nematode

The pinewood nematode, Bursaphelenchus xylophilus, originating from North America (NA), is a major invasive pine pest in Eurasia. It was first detected in Portugal in 1999 associated with maritime pine, Pinus pinaster, and has been differently affecting the main local pine species, P. pinaster and P. pinea. Field studies and direct inoculation experiments in Pinus spp. seedlings, under controlled conditions, were performed to assess whether the differences in constitutive and inducible defences are determining the different susceptibility of pine host species to B. xylophilus. Host co‐evolution with the pathogen was also assessed, including the NA P. radiata, widely used in forestry in the northeast of the Iberian peninsula. Pine mortality in the field was positively related with the abundance of B. xylophilus, and concentration of phenolics and condensed tannins in pines. In the greenhouse assay, seedling tissues were analysed for constitutive investment in defences, as well as the potential inducibility of those defences as driven by B. xylophilus inoculation. Slower growing P. pinea presented higher levels of constitutive defences than faster growing P. pinaster, with only P. pinaster being affected by B. xylophilus. Furthermore, co‐evolution with the pathogen is important, with the fast‐growing NA P. radiata presenting an inducible and effective response to B. xylophilus. Results point to the importance of integrating data on pine life history traits, including growth rate, and production of constitutive and inducible defences, into predictive models for this invasive forest pest.     

Survey of the genus Bursaphelenchus Fuchs, 1937 (Nematoda: Aphelenchoididae) in Romania

An overview of the occurrence of species of the genus Bursaphelenchus in Romania is presented. The data is based on recent surveys conducted for the first time throughout the country, to monitor and evaluate the potential entry of the pine wood nematode, Bursaphelenchus xylophilus. Wood samples were collected from declining trees, wood‐processing companies and imported wood packaging material. Of the 895 wood samples examined, 11 contained Bursaphelenchus specimens. Morphological and molecular analyses were carried out to characterize the species detected. With respect to the possible presence of B. xylophilus, all samples were negative, confirming the absence of this quarantine pest in Romania. Nevertheless, five Bursaphelenchus species were found: B. hofmanni, B. poligraphi, B. vallesianus, B. willibaldi, and one putative new species belonging to the sexdentati group, classified here as Bursaphelenchus sp. NR512. These results constitute the first report of the genus Bursaphelenchus in Romania.     

Threats created by import of timber and chips into Norway1

In recent years, Norwegian forest industries have depended to some extent on imported coniferous timber and chips, which has to be debarked to fulfil phytosanitary requirements. However, the standard of debarking of logs and chips from North America is often low. Sunken areas on stems often develop from cankers or rot around knots, and often escape debarking. Besides, even debarked, timber and chips may carry dangerous pests (Inonotus weirii, Bursaphelenchus xylophilus). Precautions should be taken after landing to hinder further spread of such dangerous organisms.     

Direct molecular detection of the pinewood nematode, <Emphasis Type="Italic">Bursaphelenchus xylophilus</Emphasis>, from pine wood,bark and insect vector

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease. The international economic impact of the introduction of the PWN into new areas has highlighted the need for the development of accurate and reliable detection methods of B. xylophilus, which are essential to define aspects of its control and management. In the present study, a methodology was developed for the direct detection of PWN by conventional PCR assay, with a species specific set of primers based on PWN satellite DNA, using total DNA extracted directly from maritime pine, Pinus pinaster, wood and bark samples, and from the insect vector, Monochamus galloprovincialis. This methodology involves homogenisation of wood, bark and insects using liquid nitrogen, DNA extraction and one or two PCR amplification steps, which permit the rapid and direct detection of one single nematode present in 100 mg of wood and bark and in one entire insect without the preliminary steps of nematode extraction.     

Report on the survey for Bursaphelenchus xylophilus and the occurrence of other Bursaphelenchus species in Hungarian coniferous forests

An ongoing official survey to detect the pine wood nematode Bursaphelenchus xylophilus, a quarantine pest, started in 2003 in coniferous forests in Hungary. Based on the results of the study from 2003–11, B. xylophilus has not yet been detected in Hungary. Two other Bursaphelenchus species (B. mucronatus and B. vallesianus) were identified in samples in 2009. Details of the survey and the measurements of B. mucronatus and B. vallesianus are provided.     

Pinewood nematode population growth in relation to pine phloem chemical composition

The pinewood nematode, Bursaphelenchus xylophilus, native to North America (NA), is now a major invasive pine pest in Eurasia. Pine species differ greatly in their susceptibility to B. xylophilus, and differences in susceptibility might be related to differences in phytochemistry. This study addressed two major questions: (i) which plant chemical profiles (including compounds that act as plant defences and those important for nutrition) favour pinewood nematode development; and (ii) are NA and Portuguese pinewood nematodes different in their capacity to subsist on plant tissues? For this, B. xylophilus isolates from NA and Portugal were tested on pine phloem and xylem of different pine species. It was found that unaltered pine phloem can sustain the population growth of B. xylophilus, but only in a few cases, and this was clearly related to distinct chemical profiles in some pine species. No evidence was found of evolutionary divergence between B. xylophilus populations in Portugal and NA. The pine species with phloem on which pinewood nematodes could grow tended to have low levels of total phenols, condensed tannins, flavonoids and lignin. Evidence was also found that nutrients such as N may be important.     

A nested PCR assay targeting the DNA topoisomerase I gene to detect the pine wood nematode, <Emphasis Type="Italic">Bursaphelenchus xylophilus</Emphasis>

The pine wood nematode (PWN), Bursaphelenchus xylophilus, is a disastrous pathogen of the pine forests in East Asia and Europe. Plant quarantine is one of the most important ways to prevent its infection in current situation. A nested polymerase chain reaction (PCR) assay targeting the topoisomerse I gene has been developed to detect PWN in this study. To assess the specificity of the assay, 44 morphologically characterized nematode isolates including B. xylophilus, B. mucronatus, B. hofmanni, Seinura wuae, S. lii and Aphelenchoides macronucleatus were tested. Positive reactions characterized by amplification product of 509 bp were shown from all isolates of PWN. The nested PCR assay can detect 50 femtogram (fg) of template DNA or one individual nematode, as small as an egg. The validity was evaluated by analyzing the nematode samples extracted from the nematode-infested wood in the field. These results show that the assay is a specific, sensitive method for detection of PWN with the potential in relation to the pest risk assessment and quarantine regulations.     

Survival of Bursaphelenchus xylophilus and Monochamus galloprovincialis in pine branches and wood packaging material

Survival and development of Bursaphelenchus xylophilus (pine wood nematode) was studied for up to 40 weeks in Pinus pinaster sawn wood and branches: 30 boards (1200 × 100 × 25 mm), 30 long‐blocks (1200 × 95 × 95 mm), 10 pine branches with bark and nine branches without bark (1200 mm long). The nematode was found in all materials and through the entire sampling period, with higher abundance in the sawn wood (boards and long‐blocks). In the initial period B. xylophilus reproduced abundantly and a population peak was detected at 8–12 weeks. Subsequently, the populations declined and became dominated by third‐stage resistant larvae (J_III), and in the final sample nematode abundance was very low. Nematode decline in the wood was correlated with a decrease in the moisture content (MC) to below fibre saturation. Survival of the insect vector Monochamus galloprovincialis was also assessed in sawn boards (1200 × 100 × 25 mm, n = 31) and blocks (160 × 95 × 95 mm, n = 40). The majority of the larvae were killed when sawing the wood, although some adults successfully emerged from the boards (10% survival) and blocks (37%). These results represent a contribution to the quantification of the risks of dispersing pine wilt disease through wood packaging materials, confirming that untreated wood can support healthy and abundant B. xylophilus populations for sufficient time for vectors surviving the sawing process to complete their development, to emerge and disperse the nematode.     

Assessing risks of pine wood nematode Bursaphelenchus xylophilus transfer between wood packaging by simulating assembled pallets in service

The risks of Bursaphelenchus xylophilus (pine wood nematode) transfer in relation to wood material were assessed. Combinations of infested and non‐infested adjacent boards, long‐blocks and blocks of Pinus pinaster, simulating assembled pallets, were assessed. For the recipient wood, pieces with natural moisture content (MC), heat‐treatment (56°C for 30 min in the core) and kiln‐drying to <20% MC were tested, along with in‐service boards from pallets. Donor and recipient wood materials were kept in direct contact at 25°C or 10°C, with nine replicates per treatment. Bursaphelenchus xylophilus was found to transfer rapidly at 25°C when the wood had an MC above fibre‐saturation point (>30%). Nematode reproduction was rapid and sustained, gradually declining to zero at 40 weeks. Bursaphelenchus xylophilus did not transfer to kiln‐dried or to in‐service wood with an MC below fibre‐saturation point, or to wood at 10°C. The key factors determining nematode transfer were the ambient temperature, the nematode load of the donor wood and the MC of the recipient wood, with a ‘barrier’ of 20% MC below which it becomes unsuitable for nematode transfer. This finding indicates that there is a limited risk of spread of B. xylophilus in treated and untreated solid wood packaging materials.     

高效新型杀松材线虫剂的筛选及其林间防效

为筛选出新型的高效松材线虫Bursaphelenchus xylophilus防治药剂,采用浸渍法室内测定了氯氟氰虫酰胺、阿维菌素、甲维盐、灭多威、特丁硫磷、甲基异柳磷和吡虫啉7种药剂对松材线虫的毒杀作用和对其繁殖能力的影响,采用高效液相色谱仪测定氯氟氰虫酰胺在马尾松体内的输导分布,并在林间开展树干注射氯氟氰虫酰胺抗松材线虫病试验。结果表明,7种药剂中氯氟氰虫酰胺对松材线虫的毒力最强,LC_(50)为0.019 mg/L,阿维菌素和甲维盐次之,LC_(50)分别为0.0213 mg/L和0.0214 mg/L。氯氟氰虫酰胺树干注射3个月内能迅速从马尾松基部传导至树梢顶部,树干边材东、南面的药剂含量显著高于西、北面,且树干不同高度的氯氟氰虫酰胺含量均高于其LC50;氯氟氰虫酰胺制剂在林间注干后,对人工接种病原松材线虫松树的防治效果为73.33%,自然感染松材线虫病松树的死亡率降至2.00%,而对照试验中人工接种松材线虫松树和自然感染松材线虫病松树的死亡率分别为93.33%和19.67%。表明氯氟氰虫酰胺制剂能有效预防松树个体的松材线虫病发生与为害,可以推广使用。     

Improved morphological key to the species of the xylophilus group of the genus Bursaphelenchus Fuchs, 1937

Correct identification of the pine wood nematode Bursaphelenchus xylophilus becomes more difficult, as more related or similar species are described. Spread of the nematode is being monitored worldwide, and in view of the fact that several new Bursaphelenchus species have been described, identification keys should be adjusted accordingly. Based on the identification key of the xylophilus group published by Braasch (2008) and the revised intrageneric grouping of Bursaphelenchus by Braasch et al. (2009), an improved dichotome morphological key including newly described species (B. paraluxuriosae, B. populi, B. macromucronatus, B. firmae, B. koreanus and B. gillanii) is presented. The recognition of the quarantine pest B. xylophilus using morphological characteristics in laboratories of the National Plant Protection Services is facilitated by a simplified key.     

Occurrence and ITS diversity of wood‐associated Bursaphelenchus nematodes in Scots pine forests in Switzerland

The quarantine pathogen Bursaphelenchus xylophilus (pine wood nematode, PWN) represents a serious threat for Pinus species in Europe. To exclude its presence in Switzerland, in 2010 and 2011 a countrywide survey was conducted in 102 Pinus sylvestris stands, chosen according to whether they contained dying or dead trees or were located in areas at risk of PWN introduction. In total, 285 trees (1–5 per site) were sampled. Nematodes were extracted from wood chips using a standard procedure, and identified to species by internal transcribed spacer (ITS) sequencing. Bursaphelenchus species were present in 34% of the trees, but no B. xylophilus was identified, i.e. PWN is still not present in Switzerland. The nematodes found belonged to seven different species, with B. vallesianus the most frequent species, followed by B. sexdentati, B. mucronatus kolymensis and B. eggersi. Three other species (B. borealis, B. pinophilus, B. poligraphi) were each only present in one or two trees. Three groups of sequences could not be assigned to a species because of the lack of matching reference sequences. The species composition found in Switzerland suggests co‐existence of southern and central European Bursaphelenchus species. Intraspecific ITS variability differed considerably among the four most common species. Bursaphelenchus eggersi, B. mucronatus kolymensis and B. sexdentati had several variable sites in the ITS region, resulting in multiple ITS genotypes in each species. In contrast, all 99 B. vallesianus isolates had an identical ITS region. This could indicate a founder effect, and possibly that B. vallesianus is not native to the Alpine region.     

Functional analysis of the venom allergen-like protein gene from pine wood nematode Bursaphelenchus xylophilus using a baculovirus expression system

The pine wood nematode, Bursaphelenchus xylophilus, infects pine trees, leading to fatal pine wilt disease. Here, recombinant venom allergen-like protein (VAP) was obtained by expressing Bx-vap-1 in insect cells. Three-year-old Pinus massoniana were inoculated with recombinant VAP, simulating B. xylophilus esophageal gland secretions. Recombinant VAP up-regulated α-pinene synthase gene expression, the trees showed disease symptoms 15 d after inoculation and the xylem pith revealed brown tissue discoloration, indicating that recombinant VAP could damage P. massoniana cells. Recombinant VAP did not, however, lead to cavitation, indicating that the VAP secreted from B. xylophilus acts as a defense response elicitor.     

Detection of the pine wood nematode using a real-time PCR assay to target the DNA topoisomerase I gene

A species-specific real-time PCR assay targeting the DNA topoisomerase I gene has been developed to detect Bursaphelenchus xylophilus. The specificity of the assay was confirmed by the lack of amplification of genomic DNA from other Bursaphelenchus or Seinura species. The sensitivity test showed that the limit of the reaction was 0.01 ng of genomic DNA or one individual nematode, as small as an egg. The validity of the real-time PCR assay was evaluated by analyzing mixed nematode samples extracted from diseased pine trees in which B. xylophilus was associated with several closely related species, B. mucronatus, B. hofmanni, Aphelenchoides macronucleatus, S. lii and S. wuae. These results demonstrate the potential of the assay to provide rapid, specific and sensitive molecular identification of B. xylophilus for use in pest risk assessment and quarantine regulations.     

Suppression of pine wilt disease by an antibacterial agent,oxolinic acid

BACKGROUND: Pine wilt disease (PWD) is very complex and has been reported to be caused by pine wood nematode, Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle, and its accompanying bacteria. However, there is no report on the control of PWD by antibacterial agent. The present study was performed to investigate disease control efficacy of antibacterial agents against PWD. RESULTS: Among six antibacterial antibiotics tested, oxolinic acid (OA) showed the strongest antibacterial activity against five bacteria isolated from three strains of pine wood nematode. In in vivo assay, it effectively suppressed the development of PWD in three‐year‐old seedlings of Pinus densiflora Sieb. & Zucc.; it showed 71% control when injected at 3 mg per seedling. A mixture of OA and the nematicidal agent abamectin (Ab) showed higher disease control efficacy against PWD than either OA or Ab alone. In addition, OA alone and a mixture of OA and Ab also controlled PWD in approximately 20‐year‐old pine trees under field conditions. CONCLUSION: This is the first report on the suppression of PWD by OA. The result strongly indicates that PWD could be controlled by antibacterial antibiotic alone and a combination of antibacterial and nematicidal agents. Copyright © 2010 Society of Chemical Industry     

Random dispersal of Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) in Pinus densiflora branches

Bursaphelenchus xylophilus is the causative agent of pine wilt disease. These nematodes disperse in the cortical and wood resin canals of host tree branches in the early stage of infection. To determine whether the dispersal can be regarded as diffusion, we inoculated B. xylophilus into living or boiled Pinus densiflora branch sections and investigated the spatial distribution 24 hr later. Assuming that some nematodes disperse randomly in the axial direction of branch sections and the rest remain at the inoculation site, the coefficient of diffusion (D) by dispersers was estimated to be 23.7–40.7 and 109.4 cm²/day for the living and boiled branches, respectively. The proportion of nondispersers was estimated to be 0.46–0.79 and 0.52 for the living and boiled branches, respectively. When the nematodes were inoculated at the centre of upright living branch sections, they dispersed up and down at similar D values of 7.4 and 7.9 cm²/day, respectively.