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.     

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。     

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.     

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.     

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.     

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]     

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。     

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.     

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.     

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.     

Molecular characteristics and functional analysis of the β‐1,4‐endoglucanase Bm‐eng‐1 gene of Bursaphelenchus mucronatus (Nematoda: Aphelenchoididae)

The pine nematode Bursaphelenchus mucronatus has been associated with pine wilt in China. This work was intended to investigate the role of the pathogenicity‐related β‐1,4‐endoglucanase gene of B. mucronatus in the infection of pines. In this study, the full‐length cDNA of the Bm‐eng‐1 gene was cloned and characterized from a B. mucronatus isolate. Phylogenetic analysis suggested that the Bm‐eng‐1 gene of B. mucronatus may be acquired from fungi through horizontal gene transfer. The function of the gene was demonstrated using RNA interference. RNA interference indicated that Bm‐eng‐1 was involved in the dispersal, reproductive ability and pathogenicity of B. mucronatus. In situ hybridization showed that Bm‐eng‐1 was specifically expressed in the oesophageal gland of B. mucronatus. Furthermore, to investigate the relation between β‐1,4‐endoglucanase activities and virulence of different isolates, the β‐1,4‐endoglucanase activities and the expression levels of Bm‐eng‐1 were detected in six B. mucronatus isolates with different virulence. β‐1,4‐Endoglucanase activity was generally higher in pathogenic isolates than in non‐pathogenic isolates, and the Bm‐eng‐1 expression levels in different isolates were positively correlated with the β‐1,4‐endoglucanase activity. These findings suggested that β‐1,4‐endoglucanase plays important roles in the pathogenic process of B. mucronatus, and the differential expression of the gene may underlie the different activity levels of β‐1,4‐endoglucanase, subsequently influencing variations in virulence of B. mucronatus isolates.     

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.     

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.     

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.