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。     

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。     

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.     

Emamectin benzoate 9.7% SL as a new formulation for a trunkinjections against pine wood nematode,Bursaphelenchus xylophilus

In this study,we investigated the preventive effects of emamectin benzoate 9.7% SL,which was newly developed to reduce the injection volume and number of injection holes required to protect against pine wood nematode.None of the Pinus thunbergii trees injected with emamectin benzoate 9.7% SL at 0.3 mL/cm diameter at breast height(DBH) died within 2 years of inoculation with pine wood nematodes.Emamectin benzoate 9.7% SL injected at 0.6 mL/cm DBH resulted in no tree mortality for 3 years.Mean residue of emamectin benzoate 9.7% SL in pine twigs injected with 0.3 mL/cm DBH was 0.490 μg/g at 1 year after injection and 0.303 μg/g after 2 years.These residues values are greater than 0.031 μg/g,previously determined IC95 value for emamectin benzoate against the pine wood nematode.Our field experiment and residue analysis showed that emamectin benzoate 9.7% SL could be a substitute agent for emamectin benzoate 2.15% EC,which is widely used to prevent pine wood nematode in the field and that injection volume and number of injection holes can be greatly educed using this new formulation,which will reduce injury to the cambium,interruption of water movement,and infection of inoculation wounds by wood-decay or blue stain fungi.     

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).     

松材线虫对湖南林业和生态环境影响的风险性分析

遵循国际植物保护公约（IPPC）制定的有害生物分析准则，结合森林植物检疫对象的特点，从地理和管理标准、传入、定殖、定殖后扩散的可能性、潜在的经济和环境重要性在对松材线虫传入湖南进行了风险分析。证明松材线虫完全符合检疫性有害生物地理和管理标准，随松类植物及产品的调运从国内外疫区传入的可能性极大，在湖南完全能够定殖，定殖后能迅速扩散并暴发成灾，不仅对全省林业生产影响极大，而且严重威胁世界自然遗产武陵源风景区的安全,提出了3种降低风险的管理措施。     

Responses of water-stressedPinus thunbergii to inoculation with avirulent pine wood nematode (Bursaphelenchus xylophilus): Water relations and xylem histology

The effect of water-stress conditioning on water relations and histological features ofPinus thunbergii Parl. inoculated with avirulent isolate ofBursaphelenchus xylophilus (Steiner and Buhrer) Nickle, pine wood nematode, were investigated. Pines were kept under 8 days cycle of severe water stress. One-half of the water-stressed pines died as a result of infection by avirulent pine wood nematode and water stress tended to induce increased susceptibility and/or decreased resistance of pines to avirulent pine wood nematode. In dead pines, the water conducting function of xylem was lost, and all of the parenchyma cells died. In surviving pines, the xylem hydraulic conductivity and the xylem water content were significantly reduced (12 to 23% and 77 to 83%, respectively) compared to controls. Safranin dye perfusion of excised axis stem segments indicated that the water conductance was limited to the very narrow peripheral area of xylem. Embolism caused by cavitation in the tracheids occurred in the central part of xylem and in that dysfunctional region of the xylem the axial parenchyma cells surrounding the epithelial cells, and ray parenchyma cells partly degenerated but the epithelial cells survived. The disruption of tracheid shape observed in surviving pines indicates that avirulent pine wood nematode temporarily disturbed cell division of the cambium. Considering the differences in responses between dead pines and surviving pines after inoculation with avirulent pine wood nematode, the death of water-stressed pines apparently resulted from death of cells, in particular the vascular cambium and the loss of xylem hydraulic function by cavitation.     

Mutual influences in growth and reproduction between pine wood nematode Bursaphelenchus xylophilus and bacteria it carries

The interactions between pine wood nematode and three bacterium strains isolated from the nematode, Bursaphelenchus xylophilus, which are two strong pathogenic bacterium strains, Pseudomonas fluorescens GcM5-1A and Pseudomonas putida ZpB1-2A and a weak-pathogenic bacterium strain, Pantoea sp. ZM2C, were studied. The result showed that the strong-pathogenic GcM5-1A strain and ZpB1-2A strain significantly increased fecundity, reproduction rate, and the body volume of the adult nematode. Meanwhile, pine wood nematodes significantly promoted reproduction of the two strong-pathogenic bacterium strains. However, the weak-pathogenic bacterium strain, ZM2C, completely inhibited reproduction of pine wood nematodes. Aseptic pine wood nematodes significantly inhibited reproduction of the strain ZM2C. The results indicated that mutualistic symbiosis exists between pine wood nematodes and the two pathogenic bacteria it carries. The phenomenon showed that the pathogenic bacteria carried by the nematode were not accidentally contaminated, but rather had existed as symbionts of the nematode with which it had coevoluted over a long period. The role of mutualistic symbiosis in the process of pine wilt disease was also discussed. __________ Translated from Journal of Nanjing Forestry University, 2005, 29(3): 1–4 [译自: 南京林业大学学报, 2005, 29(3): 1–4]     

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.     

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.     

A key effector,BxSapB2, plays a role in the pathogenicity of the pine wood nematode Bursaphelenchus xylophilus

The pine wood nematode (PWN), Bursaphelenchus xylophilus, causes huge economic losses in pine forests. The plant‐parasitic nematodes have a complex life cycle that includes the secretion of effector proteins through a stylet into the host cell to promote parasitism. In this study, SignalP 4.1 and TMHMM 2.0 were used in preliminary screens for candidate effectors and were expressed in Nicotiana benthamiana through the PVX virus expression vector. The yeast signal sequence trap system was used to further study the function of the signal peptide of an effector, BxSapB2. In situ hybridization was conducted to investigate the localization of BxSapB2, followed by RNA interference technology (RNAi) to assess the functions of BxSapB2. The results demonstrate that BxSapB2 is a secreted protein that induces cell death in N. benthamiana and is highly expressed in esophageal gland cells and amphids of B. xylophilus. BxSapB2 was determined to be related to the pathogenicity of B. xylophilus. The results of this work indicate that BxSapB2 plays an important role in the interactions between B. xylophilus and the hosts.     

The pine wood nematode Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle (=B. lignicolus Mamiya and Kiyohara): an assessment of the current position

Pine wood nematode, Bursaphelenchus xylophilus (Steiner andBuhrer) Nickle, is the casual organism of pine wilt disease,a major tree killer in the Far East. This paper describes thebiology of B. xylophilus, particularly its inter-relationshipswith vector insects in the genus Monochamus (Coleoptera: Cerambycidae),tree species and environmental conditions. The principal methodof transmission, and hence of international plant health significance,is introduction of nematodes to susceptible dying or dead treesduring female oviposition. This occurs in all countries wherethe nematode occurs but is the only significant method of transmissionand survival of B. xylophilus in North America. Extensive treemortality in Japan and China is associated with presence ofhighly susceptible tree species, suitable vector species andhigh summer temperatures. Pest risk assessments have been carriedout to determine the risks to Europe; it is concluded that thenematode would undoubtedly survive in Europe but that tree mortalityis likely only in the warmer southern countries. Methods toprevent transfer of nematodes to Europe are discussed in relationto European Union legislation.     

A rapid staining‐assisted wood sampling method for PCR‐based detection of pine wood nematode Bursaphelenchus xylophilus in Pinus massoniana wood tissue

For reasons of unequal distribution of more than one nematode species in wood, and limited availability of wood samples required for the PCR‐based method for detecting pinewood nematodes in wood tissue of Pinus massoniana, a rapid staining‐assisted wood sampling method aiding PCR‐based detection of the pine wood nematode Bursaphelenchus xylophilus (Bx) in small wood samples of P. massoniana was developed in this study. This comprised a series of new techniques: sampling, mass estimations of nematodes using staining techniques, and lowest limit Bx nematode mass determination for PCR detection. The procedure was undertaken on three adjoining 5‐mg wood cross‐sections, of 0.5 × 0.5 × 0.015 cm dimension, that were cut from a wood sample of 0.5 × 0.5 × 0.5 cm initially, then the larger wood sample was stained by acid fuchsin, from which two 5‐mg wood cross‐sections (that adjoined the three 5‐mg wood cross‐sections, mentioned above) were cut. Nematode‐staining‐spots (NSSs) in each of the two stained sections were counted under a microscope at 100× magnification. If there were eight or more NSSs present, the adjoining three sections were used for PCR assays. The B. xylophilus– specific amplicon of 403 bp ( DQ855275 ) was generated by PCR assay from 100.00% of 5‐mg wood cross‐sections that contained more than eight Bx NSSs by the PCR assay. The entire sampling procedure took only 10 min indicating that it is suitable for the fast estimation of nematode numbers in the wood of P. massonina as the prelimary sample selections for other more expensive Bx‐detection methods such as PCR assay.     

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.     

Comparison of histological responses and tissue damage expansion between resistant and susceptible Pinus thunbergii infected with pine wood nematode Bursaphelenchus xylophilus

Pine wilt disease caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, has been epidemic and has had disastrous impacts on pine forests and forest ecosystems in eastern Asia. Many pine species in this area are susceptible to this disease. Pinus thunbergii is particularly susceptible. In Japan, tree breeders have selected surviving trees from severely damaged forests as resistant candidates, and have finally established several resistant varieties of P. thunbergii. However, this breeding procedure requires much time and effort due to the lack of physiological and phenotypical information about resistance. To investigate the resistance mechanisms of selected P. thunbergii, we compared histochemical responses, tissue damage expansion, and PWN distribution in resistant and susceptible clones of P. thunbergii after PWN inoculation. The results suggested that the mechanisms of resistance are as follows: damage expansion in the cortex, cambium, and xylem axial resin canals are retarded in resistant trees soon after inoculation, probably due to the induction of wall protein-based defenses. Suppression of PWN reproduction was particularly caused by inhibition of damage expansion in the cambium. The slow expansion of damage in each tissue provides time for the host to complete the biosynthesis of lignin in the walls of cells that surround the damaged regions. This lignification of cell walls is assumed to effectively inhibit the migration and reproduction of the PWNs. The mechanism of initial damage retardation is presumed to be a key for resistance.     

微波处理松材线虫病疫木技术研究

该文对利用微波处理松材线虫病疫木技术进行了研究，结果表明：不论木材厚薄大小、含水量多少、在微波处理炉腔内怎样堆放，以及环境温度、微波功率等因素的如何变化，只要SWB-Ⅱ型隧道式微波除害处理设备显示的木材表面温度大于68℃，持续30min，就能有效地杀死松木中的松褐天牛和松材线虫。     

Monitoring of xylem embolism and dysfunction by the acoustic emission technique in Pinus thunbergii inoculated with the pine wood nematode Bursaphelenchus xylophilus

Xylem dysfunction progresses rapidly in Pinus thunbergii infected with pine wilt disease. The present report deals with the timing and process of the extensive dehydration of tracheids by embolism and the subsequent desiccation of the xylem with disease development. An ultrasonic acoustic emission (AE) technique was used to detect embolisms in the xylem of pine trunks. In most of the P. thunbergii saplings inoculated with the pathogen Bursaphelenchus xylophilus, the AE frequency suddenly increased in the second week after inoculation. The high-frequency AE continued for about 3 days and into the nights. Harvesting of specimens at this time revealed that white air-filled patches, representing the dehydrated and dysfunctional areas, had just emerged in the sapwood. The AE events in the night must be due to something other than embolisms in healthy trees. Frequent embolism of tracheids, which was suggested by the elevation of the AE frequency, might occur due to the decrease in the tensile strength of xylem sap. This hypothesis is supported by previously reported data. Host cells that had reacted to infection with B. xylophilus produce and release chemicals which can lower the surface tension of xylem sap. During the second increase of AEs, most of which occurred in the third week, xylem desiccation and needle yellowing progressed. Needle fading then became distinct, and the tree was close to death when the AE frequency dropped during the fourth week. By monitoring the AE, the first physiological abnormality that took place very early after infection was detected.     

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.     

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.