Long‐term survival and non‐vector spread of the pinewood nematode,Bursaphelenchus xylophilus,via wood chips

The pinewood nematode, Bursaphelenchus xylophilus, is the causal agent of pine wilt disease and is transmitted to new host trees by beetles of the genus Monochamus. The increasing interest in imported wood chips from North America for paper production and energy purposes and the corresponding phytosanitary risk of non‐vector transmission of B. xylophilus has been discussed since 1984, the year of the first interception of B. xylophilus in wood chips in the European Union. The long‐term survival of B. xylophilus in wood chips and its non‐vector spread from infested wood chips to non‐infested trees were studied. Pinus sylvestris logs were inoculated with a suspension of B. xylophilus to produce infested wood chips. During the long‐term storage test, B. xylophilus in P. sylvestris wood chips were examined. Four variants, including sealed and openly stored wood chips at both 15°C and 25°C, were studied. For the test of non‐vector spread, B. xylophilus ‐infested wood chips were placed on three‐ to four‐year‐old P. sylvestris saplings under different conditions. Bursaphelenchus xylophilus survived for more than 1 year at both temperatures in the sealed wood chips, which was significantly longer than for the openly stored variant at 25°C. Temperature, tree condition and wood chip location all influenced non‐vector spread through wood chips. Of the 480 trees that were in contact with infested wood chips and showed clear symptoms of pine wilt disease, B. xylophilus were extracted from 42 pines at 25°C and one pine at 15°C. The highest B. xylophilus infestation rates resulting in clear pine wilt disease symptoms (75%) were found in infested wood chips directly attached to stem‐wounded trees at 25°C. However, more variants exhibited B. xylophilus infestation at this temperature; trees with stem or root injuries plus direct contact with infested wood chips to the wounded part were primarily affected. Moreover, non‐vector spread was also detected in stem‐ and root‐injured pines without any direct contact with infested wood chips. Our results confirmed that B. xylophilus can survive for long periods in wood chips and can be transmitted from infested wood chips to damaged trees, but the likelihood of such PWN establishment should be low compared to spread through vectors. These findings must be considered in the pest risk analysis of B. xylophilus, and studies using outdoor trials should be carried out to complete this pest risk analysis.     

Identification and genetic analysis of the pinewood nematode Bursaphelenchus xylophilus from Pinus yunnanensis

In China, pinewood nematode (PWN), Bursaphelenchus xylophilus, was first discovered from Pinus thunbergii in 1982. Thus far, 14 species in the genus Pinus have been reported to be infected by PWN under natural conditions. Pinus yunnanensis, a pine species native to south‐western China, is considered a pioneer tree for barren hill afforestation in areas undergoing rocky desertification. In this study, we detected PWN in dead P. yunnanensis trees in Anlong County, Guizhou Province, China, using both morphological and molecular methods. To our knowledge, this is the first report of PWN from P. yunnanensis in China. To investigate the possible origin of this new outbreak, mitochondrial cytochrome oxidase gene subunit I and cellulase gene sequences were used to evaluate genetic relationships among worldwide PWN isolates. Phylogenetic tree and haplotype networks revealed that the Anlong isolate (BxChQAL008) sequence was identical to those of seven Chinese isolates collected from Sichuan, Chongqing, Zhejiang, Anhui and Shandong (372–1500 km from Anlong County), but different from the isolate BxChQZY030 collected from the same province (330 km from Anlong County). It is suggested, therefore, that more than one introduction of PWN into Guizhou Province has taken place. The Anlong isolate was likely introduced from neighbouring or more distant provinces rather than from outside China. Moreover, the absence of a correlation between geographic and genetic distance was observed using Mantel test analysis, providing evidence that human‐induced dispersal plays a fundamental role in the spread of the PWN in this region.     

Development of two reverse transcription‐PCR methods to detect living pinewood nematode,Bursaphelenchus xylophilus,in wood

Pinewood nematode, Bursaphelenchus xylophilus, is an inhabitant of native pine species of North America, where its presence in trees is non‐pathogenic. By contrast, the introduction of this nematode to forests overseas has devastated some pine stands and is recognized as a pest of phytosanitary concern by some countries' National Plant Protection Organizations. The ability to detect B. xylophilus in internationally traded wood products is crucial to reduce the spread of this organism. Current molecular techniques for the detection of B. xylophilus rely on the presence of genomic DNA and thus will detect both living and dead nematodes without differentiation. The detection of dead nematodes could lead to unnecessary trade disruption. Therefore, accurate techniques for the detection of and differentiation between live and dead B. xylophilus are critical. We have developed an endpoint RT‐PCR assay and a SYBR Green 1 real‐time RT‐PCR assay, both of which selectively identify living pinewood nematode by detecting the presence of Hsp70 mRNA as a viability marker. Both of these assays may help overcome or resolve disputes involving the detection of pinewood nematode at the port of entry and can also be used to evaluate the efficiency of wood treatment procedures.     

Chitosan as a biocontrol agent against the pinewood nematode (Bursaphelenchus xylophilus)

The pine wilt disease (PWD) is caused by Bursaphelenchus xylophilus and poses great environmental and economic challenges. Thus, the development of sustainable techniques for the control of this epidemic disease is of major importance. This work aimed at evaluating if the application of different molecular weight (MW) chitosans as a soil amendment could be used to control the PWD in maritime pine (Pinus pinaster, very susceptible to the disease) and stone pine (Pinus pinea, less susceptible). At the end of the experimental period (24 days after inoculation), P. pinaster and P. pinea untreated plants presented ca. 3825 ± 100 and 70 ± 47 nematodes, respectively. In P. pinaster, the high‐MW chitosan prompted the most drastic results, inducing a 21.9‐fold reduction in nematodes numbers, whereas in P. pinea, the most effective was the low MW chitosan, which reduced nematodes numbers up to 7‐fold, compared with untreated plants. P. pinea seems to be highly resistant to the disease, presenting nematode numbers up to 54.6‐fold lower than P. pinaster and less severe chlorophyll loss (ca. 2‐fold).     

Avirulent isolate of the pinewood nematode Bursaphelenchus xylophilus,survives 7 months in asymptomatic host seedlings

An avirulent isolate (C14‐5) of the pinewood nematode, Bursaphelenchus xylophilus, was inoculated onto 45 seedlings of the susceptible host Japanese black pine (Pinus thunbergii) and its viability was investigated. The nematode survived inside host seedlings for approximately 7 months even when the host seemed to overcome the infection based on lack of foliar wilting and the observation of normal oleoresin flow.     

A SCAR molecular marker to distinguish Bursaphelenchus mucronatus from the pinewood nematode,B. xylophilus

Bursaphelenchus mucronatus is closely related to the pinewood nematode Bursaphelenchus xylophilus, the causative agent of pine wilt disease. B. xylophilus became a devastating pest when it was introduced in the Far East; however, B. mucronatus is considered to have low virulence. Morphological similarities between B. xylophilus and B. mucronatus make the accurate morphological identification of both species difficult. Thus, it has become important to pay attention towards B. mucronatus impact and the need of discrimination of these two species. To distinguish among the two species, a B. mucronatus‐specific sequence‐characterized amplified region (SCAR) marker has been developed. The specific Random amplified polymorphic DNA (RAPD) fragment of B. mucronatus, OPY₀₁‐M₈₅₀ was excised from agarose gels and purified. The gel‐purified fragment was cloned into the pGEM^®‐T Vector and subjected to sequencing. Based on the sequenced RAPD fragments, a number of SCAR primers were designed. It is demonstrated that OPY₀₁‐M₈₅₀ through primers Y₀₁F/R can be transformed into a B. mucronatus‐specific SCAR‐Y₀₁‐M₆₀₉ marker. Primers set Y₀₁F/R had high specificity that could be used for the discriminative identification of B. mucronatus versus B. xylophilus.     

Lethal temperature for pinewood nematode, Bursaphelenchus xylophilus, in infested wood using radio frequency (RF) energy

We evaluated radio frequency (RF) dielectric heating for eradication of pinewood nematodes (PWN) in infested wood. Thirteen temperatures were tested (from ambient to 70 °C) on small wood samples (2.5 × 3.8 × 0.64 cm) to determine the minimum lethal temperature (100 % mortality), which was 56 °C [based on infra-red (IR) thermal images data 55.5–57.4 °C] with a 1 min hold time. We also used thermal probes inside the wood to confirm that temperatures were ≥56 °C. Thirty additional samples were tested bracketing the minimum lethal temperature using 54, 56 and 58 °C with additional replications to produce the minimum sample size equivalent of 100 % mortality of at least 93,616 nematodes to satisfy the Probit 9 efficacy requirement. This minimum lethal temperature was further verified by treating infested large wood blocks (10.2 × 10.2 × 25.4 cm). All samples that met or exceeded the 56 °C lethal temperature for the required 1 min hold time (as measured by probes inserted in the wood and on the wood surface by IR) produced 100 % mortality. The sample size required to show Probit 9 efficacy was also satisfied. This study supports the consideration of RF in addition to microwave (MW) dielectric heating as alternative treatments of wood packaging material for inclusion in ISPM No. 15, provided the treatment delivers the target lethal temperature throughout the profile of the material in industrial scale operations.     

Canadian conifers as hosts of the pinewood nematode (Bursaphelenchus xylophilus): Results of seedling inoculations

Seedlings of 22 species of conifers from across Canada were inoculated with m and r form isolates of the pinewood nematode (Bursaphelenchus xylophilus). In an experiment made under ambient (summer‐fall) temperatures in a shadehouse at Victoria, British Columbia, Canada, 8 of the 22 conifer species were killed by the nematodes, but mortality was low, i.e. 4–30%. Pines (eastern white, Jack and red) were more susceptible than other conifers. Yellow cypress, eastern white cedar, western red cedar and western hemlock were not killed by the nematodes. In a second experiment made at elevated temperatures (30°C‐16h long days, 25°C‐8 h long nights) in a greenhouse, 18 of the 22 conifer species died following nematode inoculation. Again, pines (lodgepole, eastern white, western white and red) were among the most susceptible tree species and the four conifers that were unaffected in the first experiment were not killed. Tamarack and western larch, both immune at ambient temperatures, were the two most susceptible conifers at elevated temperatures. Compared to ambient temperatures, seedlings at elevated temperatures died quicker and contained more nematodes. M and r form nematodes were equally pathogenic in both experiments.     

Impact of the pinewood nematode, Bursaphelenchus xylophilus, on gross calorific value and chemical composition of Pinus pinaster woody biomass

The pinewood nematode (PWN), Bursaphelenchus xylophilus, the causal agent of the pine wilt disease, has been detected in several regions of Portugal affecting Pinus pinaster, a coniferous species of a great economic value. The nematodes, migrating through resin canals and feeding on parenchyma cells, induce rapid metabolic changes in ray parenchyma cells, cavitation areas, and denaturation and necrosis of parenchyma and cambial cells. To understand how anatomic changes and biochemical incidences of tree defense reactions affect the technological parameters of the wood, the gross calorific value (GCV) and chemical composition of PWN-infected and -uninfected P. pinaster wood were evaluated. The GCV was determined using Parr 6300 Automatic Isoperibol Calorimeter, and chemical composition analysis was performed by determining the contents of carbon, hydrogen, nitrogen, sulfur and Ash on complete and instant oxidation of samples by “flash” combustion. The Student’s t test with Welch correction was used for statistical data analysis. The difference between the GCV and chemical composition for PWN-infected and -uninfected P. pinaster wood was statistically significant for the GCV and for hydrogen and nitrogen contents. The carbon, oxygen, sulfur and Ash contents did not differ statistically. The GCV of PWN-infected wood varied between the highest value of hardwood and the lowest value of softwood. This interdisciplinary study stresses the important technological and economic aspects, namely the impact of PWN on wood properties and the suitability of infected P. pinaster wood for use in the wood-processing and energy industries.     

Basic density,extractive content and moisture sorption properties of Pinus pinaster wood infected with the pinewood nematode,Bursaphelenchus xylophilus

The pinewood nematode(PWN), Bursaphelenchus xylophilus, has become one of the most severe threats to pine forest worldwide. Nematodes, migrating through resin canals and feeding on the living cells, induce rapid metabolic changes in ray parenchyma cells, create cavitation areas, decrease xylem water content and oleoresin exudation, and cause necrosis of parenchyma and cambial cells. This study focused on the impact of PWN infection on technological parameters of wood and evaluated the impact of anatomic and biochemical incidences of tree defense reactions on basic density, extractive content and moisture sorption properties of Pinus pinaster wood.Samples of infected and uninfected wood were studied.The presence of nematodes reduced wood basic density by2 % and decreased the total content of extractives in infected wood as compared with uninfected(5.98 and8.90 % of dry wood mass, respectively). Extractives in infected trees had inverse distribution along the trunk as compared with uninfected trees. The adsorption isotherms for infected and uninfected wood had similar positioning.We recorded differences(some statistically significant) in the equilibrium moisture content of infected and uninfected wood under varying environmental conditions. Despite the verified differences in wood basic density, extractive content and moisture sorption properties, the overall conclusion is that the PWN had a slight impact on these characteristics of wood.     

松材线虫低温冷冻保存的影响因子

比较了保护剂平衡时间、解冻温度、线虫浓度及线虫发育期对低温冷冻松材线虫存活的影响.结果表明,平衡时间对冷冻保存有较大影响,以20％甘油为保护剂,在4℃下平衡12h后转入-80℃超低温冰箱,1d后线虫存活率为(41.7±4.9)％;冻存存活率与线虫发育期有一定关系,幼虫显著高于成虫,幼虫、雌成虫和雄成虫的存活率分别为(31.5±7.4)％,(7.9％±3.5)％,(6.6±2.8)％.在所选择的解冻温度(30 ～45℃)和线虫浓度(10 500 条/mL～8 4000条/mL)范围内,解冻温度及线虫浓度对存活率影响差异不明显.     

Comparison of the potential spread of pinewood nematode (Bursaphelenchus xylophilus) in Finland and Iberia simulated with a cellular automaton model

Pinewood nematode (PWN) is one of the most threatening invasive pests in the pine forests of Europe, and it has recently spread to the Iberian Peninsula via import of timber and wooden packaging material from East Asia. A cellular automaton (CA) model was developed to simulate and compare the potential spread of PWN by transportation and its vectors, Monochamus beetles in the pine forests of Finland and Iberian Peninsula. The model assumes that all pines are equally sensitive to PWN. The CA is a spatio‐temporal grid‐based model, which can easily be applied on different geographical scales. The effects of climate warming and number of entries from ports on the spread of PWN were studied. A sensitivity analysis was conducted on the most uncertain model parameters. Twenty years after hypothetical entries, the predicted area of symptomatic PWN infection (pine wilt disease, PWD) was very low in Finland compared to Iberia. This was because of the low probability of warm July in Finland. The increase in the mean July temperature increased the area of PWD‐infected pine forest relatively more in Finland than in Iberia. An increase in the number of entries also increased the area of PWD‐infected pine forest relatively more in Finland than in Iberia. The probability of PWD infection was the highest in pine forests that were close to entry points and in areas with low elevation and high human population density.     

松材线虫发病条件的研究概况

研究和综述了寄生松科植物的伞滑刃属(Bursaphelenchus)的线虫23种,松材线虫的寄主松科植物108种、携带昆虫40种及发病的环境条件。     

Pine wilt disease: detection of the pinewood nematode (Bursaphelenchus xylophilus) as a tool for a pine breeding programme

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is a serious quarantine pest first detected in Portugal and Europe in 1999. It is the causal agent of pine wilt disease (PWD). A resistance breeding programme has been initiated to contribute to control the evolution of the disease. Five hundred and four adult maritime pine, Pinus pinaster, trees were phenotypically selected as candidate trees for this programme from an area affected by PWD. To identify tolerance to the nematode, the selected trees were monitored monthly. Over the course of 1 year, 57 candidate trees died and were tested for the presence/absence of the PWN. As accuracy of detection is of major importance, an ITS‐PCR‐based method applied directly to wood from adult maritime pine trees was tested and compared with a standard morphological identification method. The results showed that the use of PCR to detect the pathogen provided more rapid and accurate results in comparison with the standard morphological identification. Thus, this method is suitable to be used in the survey of the breeding population for resistance/tolerance to PWD.     

日本松材线虫病研究的最新动向

该文重点对近2a日本松材线虫病的研究文献进行整理分析。目前松材线虫病的研究仍是日本国森林病虫害的主要研究课题。在松树的抗（感）病机理、线虫在树体内的活动与增殖规律、线虫与天牛的关系、生物防治以及新技术在研究中的应用等方面取得较大进展。     

Direct PCR‐based method for detecting Bursaphelenchus xylophilus,the pine wood nematode in wood tissue of Pinus massoniana

Pine wilt disease (PWD), caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus, leads to serious losses to pine forestry around the world. Pinus massoniana, which is vulnerable to be attacked by the PWN, is the dominant species used in pine forestry in China. The objective of this study is to develop a direct PCR‐based method for detecting B. xylophilus in the wood of P. massoniana without a separate nematode extraction step. A simple procedure was first developed for isolating B. xylophilus DNA in 5 mg pine wood tissue samples harbouring PWN for detection by PCR amplification. A B. xylophilus‐specific amplicon of 403 bp (DQ855275) was generated by PCR from the infested wood tissue. The entire procedure can be completed within 5 h with one pair of primers. This assay can serve as a rapid, cheap and environmentally friendly method to detect B. xylophilus in samples of P. massoniana.