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.     

Emamectin benzoate as a candidate for a trunk‐injection agent against the pine wood nematode,Bursaphelenchus xylophilus

In order to develop an effective trunk‐injection agent against pine wood nematode, Bursaphelenchus xylophilus, an in vitro assay was used to examine the antinematodal activity of 58 commercially available compounds with known modes of action. Among compounds tested, the GABA receptor agonists had better anti‐nematodal activity than compounds influencing glutamate, N‐methyl‐D ‐aspartate, β‐adrenergic, dopamine, muscarinic acetylcholine and nicotinic acetylcholine receptors, as well as those inhibiting acetylcholinesterase, monoamine oxidase, 5‐hydroxytryptamine uptake and Ca²⁺, K⁺, Na⁺ and Cl⁻ channels. Avermectins and milbemycins strongly inhibited propagation of the nematode. Emamectin benzoate proved to be the most active (IC₉₅ 0.050 µM ) being over 140 times more active than the active ingredient of conventional trunk‐injection agents. It is concluded that emamectin benzoate is a strong candidate for an anti‐nematodal trunk injection agent. © 2000 Society of Chemical Industry     

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.     

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.     

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.     

木质包装热处理国际标准对松材线虫有效性的试验研究

使用温度为65～85℃、相对湿度50%～90%的热空气,对不同规格和含水率的携带松材线虫(Bursaphelenchus xylophilus)的马尾松(Pinus massoniana)木板进行热处理验证试验,结果显示,当木材中心温度达到56℃并持续30min以上,10cm×10cm×10cm木块、5cm厚度木板以及高含水率10cm厚度木板中线虫死亡几率值达到9的要求,验证了国际植物检疫措施标准第15号所推荐的热处理技术指标对杀灭松材线虫的有效性.试验中也发现10cm厚度含水率低于30%木板的线虫死亡率达不到几率值9的要求,其原因可能是中心温度或持续时间没有达到该标准要求.     

Susceptibility evaluation of Picea abies and Cupressus lusitanica to the pine wood nematode (Bursaphelenchus xylophilus)

Pine wilt disease (PWD), recently introduced into Europe, is caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus and is a devastating illness that affects mainly pine trees. It is known that the PWN is capable of infecting other conifers; however, there is currently no information on which other plant species may be susceptible to PWD. In this study, the potential susceptibility of two common species of European forests, Picea abies and Cupressus lusitanica, to PWN was assessed through the monitoring of visual external symptoms, dimension and localization of the nematode population in stems, quantification of total chlorophyll, total soluble phenolics and lignin, at 7, 14, 21 and 28 days after inoculation. The degree of susceptibility was established through the comparison of symptoms with Pinus pinaster, a well‐known PWN host. Furthermore, the stem ultrastructure of P. abies, C. lusitanica and Pn. pinaster was analysed by scanning electron microscopy. The results suggest that P. abies and C. lusitanica are resistant to PWN, and that lignin biosynthesis in these species is affected at an early stage of the infestation. Nevertheless, P. abies seems to be a compatible host that could act as a repository for PWN.     

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.     

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.     

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     

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.     

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.     

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.     

Pest survey of softwood boxes,with special emphasis on pinewood nematode,Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae)

Wood packaging material (WPM) is an important pathway for the spread of non‐native plant pests. To reduce the likelihood of plant pest movement with WPM, the International Standard for Phytosanitary Measures No. 15: “Regulation of Wood Packaging Material in International Trade” (ISPM 15) was developed in the framework of the International Plant Protection Convention. To be compliant with this Standard, WPM shipped internationally must be either heat‐treated or fumigated, regardless of any specific characteristics of the WPM. The objective of this survey was to determine if Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae) or insects, especially Monochamus spp. (Coleoptera: Cerambycidae), were present in a specific subset of softwood boxes. While not compliant with ISPM 15, these boxes had been treated with wood preservatives, stored indoors for over five years, and, in addition to meeting various quality standards, almost all had a moisture content below 20 percent. United States Department of Agriculture staff inspected a sample of 630 softwood boxes, focusing on those showing signs of possible pest infestation. Based on a binomial distribution with an efficacy of detection equal to 0.95, a sample size of 630 provides a 95 percent confidence of detecting pests if >0.5 % of boxes are infested. No B. xylophilus or insect pests were found in the boxes, though other nematode genera (Aphelenchoides, Aphelenchus, and Filenchus), which feed on decomposing fungi in wood and do not damage trees, were found in 21 boxes. This study demonstrates that not all types of WPM present a high phytosanitary risk. It may be worthwhile to consider an amendment to ISPM 15 to differentiate between various risk categories of WPM in order to minimize costs and environmental impacts associated with treatments currently prescribed in ISPM 15.     

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.     

Experiences with Bursaphelenchus xylophilus in Finland1

In 1984 a heavy Bursaphelenchus xylophilus infestation was observed in pinewood chips imported from USA and Canada into Finland. Because there are good reasons to suppose that B. xylophilus, if spread to pine forests in North Europe, can cause significant damage, the import of coniferous wood into Finland has been prohibited since autumn 1984. In surveys B. xylophilus has not been found in roundwood or in chips imported from European countries into Finland. Neither has B. xylophilus been detected in Finnish pine trees around harbours nor in paper mills handling North American wood.     

Nematode response to methyl bromide and 1,3‐dichloropropene soil fumigation at different temperatures

Several heat‐based methods, such as soil solarization, are being developed as alternative practices for managing soil‐borne pests and pathogens. The effectiveness of these practices is often inconsistent or marginal, thus commanding the need for their integration with other methods. The main objective of this study was to determine synergistic interaction between soil fumigants and temperature. Soil infested with citrus nematode Tylenchulus semipenetrans was exposed to methyl bromide or 1,3‐dichloropropene at various temperatures. Fumigant degradation was concurrently measured and concentration‐time index (ct) was calculated and correlated to the recovered nematode population. In untreated soil, nematode survival was not affected by temperatures of 20–30 °C, but was strongly reduced at ≥ 40 °C. In fumigated soil, nematode suppression was much greater at 30 °C than at 20 °C, and the ct required for nematode elimination at 30 °C was < 50% of that needed at 20 °C for both fumigants. These results suggest that these fumigants became more active with increasing temperature in the sub‐lethal temperature range. It also implies that, when integrated with a heat‐based practice, reduced rates of fumigants may provide adequate pest control, thus minimizing the environmental input of chemical fumigants. © 2000 Society of Chemical Industry     

Short communication: Satellite DNA-based species-specific identification of single individuals of the pinewood nematode <Emphasis Type="Italic">Bursaphelenchus xylophilus</Emphasis> (Nematoda: Aphelenchoididae)

The pinewood nematode Bursaphelenchus xylophilus is a severe pest of coniferous trees, and has been designated as a quarantine organism in the European Union. From the sequence of a satellite DNA family characterized in the genome of this nematode, we developed a PCR procedure that allowed the specific discrimination of this species from closely related Bursaphelenchus species found on coniferous trees. Moreover, because of the repetitive nature of satellite DNA, positive amplification was achieved from B. xylophilus single individuals, which should contribute to an easy diagnostic procedure for assisting in the management of this major pest of conifer forests.     

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.