脉冲磁场对松材线虫生理生化及超微结构的影响

松材线虫病又称松树枯萎病,是由病原松材线虫(Bursaphelenchus xylophilus)引起的一种毁灭性森林病害。随着国际贸易日益频繁,松材线虫随木包装以及木材的进出口,反复传播的可能性越来越大(张星耀等,2003),而传统的熏蒸、热处理和杀线虫剂处理效率低、易造成环境污染(陈劲松等,     

脉冲磁场对葡萄座腔菌生长及侵染的影响

利用脉冲磁场研究在固定脉冲前沿宽度10ms条件下,不同脉冲时间对葡萄座腔菌生长及侵染的影响。结果表明:短时间内的脉冲磁场处理即可有效抑制葡萄座腔菌的生长及侵染能力,处理时间越长,抑制作用越强。当脉冲处理45min时,菌落生长48h的抑菌率为54.11%,接种葡萄座腔菌的毛白杨愈伤组织72h后的褐化指数仅为26.89%。当脉冲处理60min时,抑菌效果最好,菌落全部死亡。扫描电镜观察结果可知:与对照相比,脉冲磁场处理的葡萄座腔菌的菌丝粗细不均,表面皱缩;菌丝体之间相互粘连,部分菌丝分枝、干瘪。透射电镜观察可知:脉冲处理后菌丝的细胞壁变薄,细胞内部组成紊乱、降解,细胞结构破坏严重,胞内空腔增多;同时毛白杨愈伤组织的细胞结构受到葡萄座腔菌不同程度的破坏,细胞壁变形、解体,中胶层分解,质膜断裂,质壁分离;细胞器分解沉积于细胞壁附近,细胞内菌丝降解,管腔空化。脉冲磁场对葡萄座腔菌的生长及侵染具有较强的抑制作用,可改变菌丝超微结构。     

脉冲磁场对马尾松愈伤组织生长和生理生化特性的影响

以马尾松愈伤组织为试验材料,通过不同脉冲前沿宽度和处理时间的磁场处理,观察了马尾松愈伤组织在磁场处理后的生长变化,探讨了磁场处理对马尾松愈伤组织外部形态、可溶性蛋白和可溶性糖含量、保护酶活性和细胞膜透性的影响。结果表明:(1)脉冲磁场处理后,马尾松愈伤组织的鲜质量生长与对照相比有明显增加,其趋势为先缓慢上升,再急速下降;(2)脉冲磁场处理后,可溶性蛋白的含量、可溶性糖的含量与对照相比都有增加的趋势,当脉冲前沿宽度为5 ms、处理时间为8 h时,马尾松愈伤组织可溶性蛋白和可溶性糖的含量增加到峰值;(3)脉冲磁场处理后,与对照相比,由POD、SOD、CAT构成的保护酶系统的活性随脉冲前沿宽度、处理时间的增加而增强,在脉冲前沿宽度为5 ms、处理时间为8 h时,三种酶活性达到一定的峰值后又出现不同程度的下降;(4)在一定的脉冲前沿宽度和处理时间内,马尾松愈伤组织细胞膜仍可保持较强的稳定性,马尾松愈伤组织细胞对脉冲磁场具有一定的抗性和耐性。     

Mutualistic symbiosis between Bursaphelenchus xylophilus and bacteria of the genus Pseudomonas

Interactions between the pine wood nematode (PWN), Bursaphelenchus xylophilus, and bacteria of the genus Pseudomonas were examined by cultivating axenic PWN and bacterial strains using callus of Pinus thunbergii. Ten (Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas cepacia and Pseudomonas spp.) of the 29 bacterial strains tested, significantly increased the reproduction of PWN. The rest of the bacteria (19 strains of 10 species) inhibited the reproduction of PWN completely. The growth of 18 of the 29 bacterial strains tested, including the 10 strains promoting PWN reproduction, was significantly increased by the presence of PWN. It indicated a mutualistic symbiotic relationship between PWN and the 10 bacterial strains in the genus Pseudomonas. The bacterial mutualistic symbionts are organisms, which may have co‐evolved with PWN rather than being accidentally associated. The finding provides further evidence for our hypothesis that pine wilt disease is complex, induced by both PWN and associated phytotoxin‐producing bacteria.     

α-三噻吩和恶喹酸混合应用对松材线虫病的防治作用

为了探明α-T与OA的混合溶液对松材线虫病的防治方法和作用机理,采用α-三噻吩(α-T)与恶喹酸(OA)混合应用的方式对松材线虫的毒杀作用以及对其繁殖的影响进行研究,并研究α-T与OA 1︰1的混合溶液对接种松材线虫的离体黑松枝存活的影响.结果表明:当混合溶液浓度为5×10-7 g/mL时,对松材线虫的致死率可高达99...     

松材线虫病致病机理的研究进展

该文对松材线虫病的致病机制的研究进行了概述。目前对松材线虫病的病原有两种看法，一种认为松材线虫是惟一病原，另一种认为病原为松材线虫和细菌两种生物。对于松材线虫的致病机理，目前存在3种观点。第1种观点认为松材线虫的酶使松树薄壁细胞的细胞壁和细胞膜遭到破坏，树脂不正常地从树脂道中渗漏并扩散到相邻的管胞中，使水分输导受阻，导致萎蔫。第2种观点认为松树感染了松材线虫后，木质部内挥发性萜烯类物质的含量增加，这些物质进入管胞在管胞中形成空洞，致使水分输导受阻。第3种观点认为松树感染松材线虫后，体内产生有毒物质，这些物质使松树萎蔫。     

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.     

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.     

Correlation of seedling size and branch number with disease resistance of Pinus thunbergii seedlings to Bursaphelenchus xylophilus

Japanese black pine (Pinus thunbergii) seedlings resistant to pine wood nematode (PWN; Bursaphelenchus xylophilus) are routinely selected in Japanese field inoculation trials. Correlations between morphological factors (such as height, stem diameter at ground level and number of branches on seedlings) and disease resistance were examined to improve the production efficiency of 1‐year‐old black pine seedlings for inoculation. Family relatedness and environmental conditions strongly affected seedling resistance; accordingly, logistic regression analysis was used to separate effects of these two variables. Height and stem diameter at ground level significantly correlated with disease resistance in seedlings inoculated with PWN. Because (a) interactions between stem diameter at ground level and environmental condition were significant and (b) height did not interact with any other factor, it was concluded that height of 1‐year‐old Japanese black pine seedlings independently correlated with PWN resistance. Thus, field inoculation tests should use tall seedlings to achieve enhanced survival rates.     

Within-tree distribution and attractant sampling of propagative pinewood nematode, Bursaphelenchus xylophilus: An early diagnosis approach

Early detection is of primary importance to enable rapid actions to prevent the spread and introduction of invasive species. The pinewood nematode (PWN), Bursaphelenchus xylophilus, a serious invasive and destructive species, is listed as a quarantine pest in the legislation of more than 40 countries. However, Baermann funnel extractions of wood from discs cut from trees at breast-height often do not detect the presence of PWN in infested trees. A serious consequence of such false negatives is the loss of the best window for implementation of eradication or quarantine measures to prevent establishment of incipient PWN infestations. Here we document the within-tree horizontal and vertical distribution of PWN in infested stands in China, using a newly developed kairomonal trapping technique. Our results provide a simple, effective, rapid and non-destructive sampling method that takes into account the changes of PWN within-tree distribution in relation to pine wilt disease (PWD) symptom development. When 60–80% of the foliage has become pale green, PWN is recovered from larger diameter branches. As disease symptoms progress, PWN moves into and down the trunk. As the needles turn yellow, PWN was recovered from the trunk at 1–2 m above the ground. The correlation between the within-tree distribution of PWN and the expression of symptoms indicated a strong association between the distribution of PWN and physiological and pathological changes that develop in attacked pines through the interaction between PWN and tree. This systematic sampling technique takes into account the within-tree distribution of the nematode and should greatly enhance early detection of PWN in field surveys, monitoring and phytosanitary inspections.     

The population structure and sex ratios of Bursaphelenchus xylophilus under a-pinene stress

Pine wood nematode(PWN), Bursaphelenchus xylophilus, is a serious pathogen of pines throughout the world. Previous work indicated that different concentrations of a-pinene could affect nematode reproduction,however the mechanism of that influence is not clear. In order to examine the reproductive strategies of PWN in response to the stress of the volatile material a-pinene, we investigated different aspects of population changes of B.xylophilus under two concentrations of a-pinene. The results show that a high concentration(214.5 mg ml-1)promoted population growth while a low concentration(56.33 mg ml-1) decreased the population. Population structure was analyzed and it was found that a high concentration of a-pinene decreased the percentage of adults but increased the percentages of larvae and eggs.Furthermore, from the results of an evaluation of sex ratios(female/male), it was determined that a high concentration could elevate sex ratios but a low concentration decreased ratios sharply. These results suggest that the PWN could regulate its population by changing sex ratios under stress of a-pinene. This study has provided a theoretical basis for the prevention and control of pine wilt disease caused by the pine wood nematode.     

松材线虫侵染对马尾松苯丙烷类代谢的影响

1.5年生马尾松植株接种松材线虫后,苯丙氨酸解氨酶(PAL)、过氧化物酶(POD)、多酚氧化酶(PPO)的活性水平均高于对照,活性高峰通常出现在症状表现前夕,并在植株发病后下降.叶内PAL和POD活性高峰的出现晚于茎,与叶部病害发展一致.茎中酚类物质的含量随着线虫侵染时间的延长逐渐增加,至发病时达到高峰.酚类物质含量的变化与PAL、POD、PPO 3种酶活性的变化趋势基本一致.松材线虫的侵染明显影响寄主植物的苯丙烷类代谢,由此可知,松材线虫病的发生发展与松材线虫诱导感病寄主苯丙烷类代谢的异常变化有关.     

松材线虫病的潜伏侵染及松墨天牛传播新途径

在南京用松材线虫接种 7种松树 ,于第 2a在无任何症状的松树的接种点上方 10cm处取样 ,分离线虫。结果表明松材线虫病的潜伏侵染现象比较普遍。在不同的松树品种上潜伏侵染现象有所差异。感病黑松和赤松很快死亡 ,只有在接种量小的情况下才有潜伏侵染现象发生。抗病性强的火炬松和湿地松感病后潜伏侵染现象比较普遍 ,并且样品中的线虫量也较高。而抗病性中等的马尾松和刚松感病后潜伏侵染现象的普遍性及样品中的线虫量均处于中等。短针松接种松材线虫后既不枯死 ,也无潜伏侵染现象。松墨天牛传播松材线虫的试验结果表明 ,未携带松材线虫的松墨天牛成虫在松材线虫病松枝上取食后 ,再到健康松枝上取食 ,有传播松材线虫的可能。这些结果表明在松材线虫病新病区清理病死木时 ,应采取早期诊断技术 ,把未表现症状的松树一起清除 ,以达到防治目的。     

Localized within‐ and between‐tree variation in nematode distribution during latent state of pine wilt disease makes the disease status cryptic

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease, an epidemic disease that has severely damaged pine forests in East Asia. The disease has spread to northern areas in Asia and parts of Europe. To prevent disease spread as the forefront of damage prevention, a better understanding of infection status is highly important. Not all infected trees show disease symptoms, and such asymptomatic PWN‐carrying trees are likely to be overlooked and can become a pathogen reservoir. To elucidate PWN infection status in asymptomatic trees, we performed PWN inspection of branches and trunks in 21 test trees in two different conditions: trees that had experienced PWN inoculation and those with suspected PWN infection that had experienced transient foliage discoloration. We detected PWNs in eight test trees (38%) and in 13 (1.5%) of a total of 843 samples. The difference in these percentages suggests that nematode inhabitation was highly localized within the trees, possibly owing to the restricted migration of PWNs. Our data demonstrated that trees that were once weakened but recovered their vigour can persist, as the asymptomatic carriers, in the forest. The implications for disease control are also discussed.     

开展松材线虫病抗性选育 保护我国松林资源

松材线虫病是我国松林毁灭性病害,已造成数千万松株死亡,发生区经济损失巨大,目前此病害正威胁着南方重点林区及重要风景名胜区松林的安全。我国松属资源丰富,科技人员队伍组织体系完备,抗性选育研究已有较好基础。因此尽快开展抗性选育有利于发生区森林恢复、非发生区林分改造和结构调整,有利于保护我国森林资源。本文对开展抗性选育提出了具体建议。     

蓝变真菌引起的欧洲云杉木质部解剖学特征及纤维素酶活性的变化

选择奥地利境内阿尔卑斯山健康欧洲云杉为对象,研究室内接种蓝变真菌(Ceratocystis polonica)引起的寄主树木韧皮部和木质部解剖学特征的变化,揭示蓝变真菌引起欧洲云杉枯萎的机制.结果表明:接种1周后的4株欧洲云杉的木质部组织内,蓝变区域显著增加,4～6周后蓝变区域不再增加;而在接种无菌琼脂的2株对照欧洲云杉的木质部组织内,没有检测到蓝变区域.采用生物化学分析和组织定位技术,确定接种真菌和无菌琼脂的欧洲云杉木质部区域纤维素酶的分布和活性变化.接种2周后剥皮取样检测,接种真菌的4株欧洲云杉的木质部组织内纤维素酶含量大幅度增加,其等电聚焦电泳显示明显的纤维素酶酶带;而在接种无菌琼脂的欧洲云杉木质部区域,纤维素酶含量分布较少,其等电聚焦电泳显示微弱的纤维素酶酶带.进一步证明蓝变真菌分泌的纤维素酶能利用寄主欧洲云杉木质部的纤维素,蓝变真菌是致死阿尔卑斯山境内欧洲云杉的重要病原菌.     

加工活性炭处理松材线虫病死木技术的研究

经３ａ的试验和实践结果表明，加工活性炭处理松材线虫病死木中松褐天牛成、幼虫等媒介昆虫是一项简单易行、灭害彻底、效益较高的处理松材线虫病死木的新技术，值得在疫区推广应用。     

A loop‐mediated isothermal amplification‐based method for detecting Bursaphelenchus xylophilus from Monochamus alternatus

The introduction of the pine wood nematode (PWN), Bursaphelenchus xylophilus, to new areas has impacted on the international economy. Therefore, accurate and reliable detection methods for PWN are essential for the control and management of this pest. A rapid and economic method for detecting PWN may be developed focusing on the PWN vector (Monochamus alternatus). This work standardized a loop‐mediated isothermal amplification (LAMP) method using newly designed primer sequences based on the syg‐2 gene, which encodes the synaptogenesis protein syg‐2. Loop‐mediated isothermal amplification was conducted at 63°C for 60 min using six sets of primers. The result was confirmed by visual observation. A positive reaction was confirmed by SYBR Green I fluorescence dye under light thermal cycling. The lower limit of DNA detection was 51.4 pg/μl in both LAMP and 51.4 ng/μl in PCR. Therefore, the LAMP was 1,000 times more sensitive in DNA detection than PCR. The LAMP is a relatively new, highly accurate and rapid molecular technique that can rapidly detect infectious agents in the field without requiring sophisticated instruments, giving a visually readable result. The method greatly improves detection, without requiring professional knowledge and expensive, sophisticated equipment. Therefore, this system is suitable for quarantine and field detection.     

A new on‐site detection method for Bursaphelenchus xylophilus in infected pine trees

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease (PWD), which is a major problem in East Asia and West Europe. Quick identification of PWN is needed to prevent the dispersal of PWD to healthy forests. Various detection methods of PWN have been developed using anatomical characters and molecular markers. These methods are not suitable for rapid diagnosis because it is difficult to distinguish B. xylophilus from the non‐pathogenic species Bursaphelenchus mucronatus based on morphological characters without expertise in nematode taxonomy and most PCR or isothermal amplification detection methods require time‐consuming processes. In this study, we developed an on‐site PWN detection method using a recombinase polymerase amplification (RPA) assay with a novel extraction buffer (DAP buffer). This new PWN detection method is able to extract genomic DNA from PWN in pinewood by simple buffer consisting of sodium hydrate, polyethylene glycol 200 and dimethyl sulfoxide in 10 min without using the experimental devices and able to distinguish between B. xylophilus and other Bursaphelenchus spp. by amplifying the species‐specific 5S rDNA fragment of B. xylophilus in 10 min. Taken together, our protocol can obtain the result for the detection of PWN in pine tree samples within 30 min. This result suggests that RPA/DAP assay is much faster, easier and cheaper than the conventional methods for detecting PWN.     

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.