几种物质对松材线虫的作用

松材线虫病是松树的一种毁灭性病害。本文选用了几种物质，采用浸泡生测法和棉球生测法测定了其对松材线虫的作用。结果表明，阿维菌素对松材线虫的杀线活性较强，米乐尔次之，狼牙刺种子粗提物和氯化铵的杀线活性均较弱，氯化钙则无杀线活性。阿维菌素和米乐尔对松材线虫繁殖均有较强的抑制作用，狼牙刺种子粗提物的抑制作用较弱，氯化铵无抑制作用，而氯化钙对松材线虫的繁殖具有促进作用。     

松材线虫致病性最佳接种量的研究

松材线虫是引起松树死亡的一种毁灭性病害的病原。为了研究松材线虫的致病性,该文对松材线虫的接种量进行研究,对3年生黑松苗接种松材线虫后的发病程度进行了等级划分以及发病等级的描述。3年生黑松苗,接种松材线虫LN001后,每株接种量为10000条时,发病率、感病指数、死亡率3个指标均达到最大值。接种松材线虫LN002后,每株接种量为8000条的松苗,在接种第47d时,感病指数达到100。3年生黑松苗接种松材线虫8000~10000条,为适合进行松材线虫接种试验的接种量。     

双稠哌啶类生物碱分子结构与杀线活性间关系

使用培养基法和棉球法测定了苦豆碱、野靛碱、甲基野靛碱、苦参碱对松材线虫的杀线活性。两种生测方法测得的结果一致,表明苦豆碱的杀线活性最强,其ｌｏｇ（１／ＩＤ５０）（ｍｏｌ／棉球）＝８．６７,与文献中用同样方法测定的对松材线虫杀线活性的其它７种双稠哌啶类生物碱的杀线活性相比较,它也是最强。本文提出了双稠哌啶类生物碱对松材线虫的杀线活性强弱主要受其分子结构中官能团对的类型及其中的官能团的类型控制的假说。根据这一假说,推测了具高杀线活性的生物碱的分子结构。     

山西平陆枯死油松线虫分离物鉴定

对山西平陆县28年生枯死油松进行了取样、分离、镜检,发现13个样品中有4个样品分离到线虫。通过对分离物的形态测量与比对,鉴定为拟松材线虫,且样品中分离线虫的数量较少。所分离的拟松材线虫株系为东亚型拟松材线虫。     

生防细菌杀松材线虫的作用机制及应用

由松材线虫Bursaphelenchus xylophilus引起的松树枯萎病是一种世界性森林病害,严重威胁松属Pinus植物的健康。生防细菌可以通过多种作用方式杀死松材线虫,如产生毒素、分泌胞外蛋白酶消解线虫体表组织、干扰线虫行为模式以及捕食线虫与动员真菌捕食线虫等。围绕上述几种主要作用机制,介绍生防细菌杀线虫作用机理研究的最新进展以及杀线菌株在松材线虫病防治中的应用现状,指出生防细菌杀线虫的作用机理研究与应用中存在的问题,展望未来杀线虫机理研究与松材线虫病生防细菌制剂的研发前景。     

松材线虫和拟松材线虫繁殖能力差异研究

松材线虫作为外来入侵种,逐步取代拟松材线虫占据生态位。初步研究表明这可能与两种线虫的繁殖能力差异关系密切。本研究采取了两种方法,即离体条件竞争试验和体内竞争试验比较二者繁殖力。结果表明,松材线虫的繁殖水平明显高于拟松材线虫。松材线虫和拟松材线虫混合物长时间培养后,松材线虫逐步竞争淘汰拟松材线虫。除此以外,离体培养和体内竞争结果均表明,竞争条件下松材线虫的繁殖力高于非竞争条件。     

疫木中松材线虫的免疫学检测方法的研究

以松材线虫的粉碎虫体免疫家兔制备抗血清 ,通过免疫印迹考察抗血清的特异性。利用固相松材线虫抗原和游离线虫抗原同线虫抗体相竞争 ,建立竞争型ELISA法用于疫木中松材线虫的分析。该方法可以直接检测出 10mg的木屑中 0 1μg微量的线虫蛋白 ,约 7条线虫的存在。利用该方法对 2 0个不同地区、树种及类型的松材样品进行检测 ,疫木中松材线虫的检出率为 10 0 % ,但拟松材线虫也呈现了一定的交叉反应。研究结果表明 ,以松材线虫抗原蛋白为指标的免疫学检测方法简便快速、灵敏度高 ,为松材线虫的快速检疫提供一种可能的途径     

无菌和带菌松材线虫对赤松的致病性

【目的】探讨无菌和带菌松材线虫对赤松的致病性,明确松材线虫在松树萎蔫病中的地位,为致病机制研究及病害防控提供依据。【方法】以赤松胚性愈伤组织为材料,每块组织接种无菌松材线虫250条,培养12天后,利用2,3,5-三苯基氯化四氮唑(TTC)染色法对愈伤组织进行染色,分析无菌松材线虫对赤松愈伤组织细胞活性的影响。以赤松组培苗和4年生盆栽实生苗为对象,每株接种无菌松材线虫和带菌松材线虫(组培苗250条,4年生苗3 000条),分别于18天和35天后统计2种类型线虫接种所致松苗萎蔫率,并对植株体内线虫进行再分离,分析2种类型松材线虫对赤松幼苗的致病能力。【结果】无菌松材线虫接种导致赤松胚性愈伤组织严重水渍化,TTC染色为米黄色或淡粉色,证明愈伤组织已失去活性。无菌松材线虫和带菌松材线虫接种均能导致赤松组培苗萎蔫,接种18天后,萎蔫率分别为70%和60%;萎蔫植株中均可再分离到松材线虫,其每株平均分别为599±567条和365+240条,二者差异显著(P0.01)。剩余外观健康的赤松组培苗中也分离到线虫,但数量较少(仅10~20条)。2种类型松材线虫接种引起赤松盆栽苗同等程度的萎蔫,接种35天后的萎蔫率均达80%,无菌和带菌松材线虫致萎蔫植株中均可再分离到松材线虫,其每株平均分别为34 733±34 162条和25 057±21 410条,但差异不显著(P=0.508)。其余外观尚健康的松苗也进行线虫分离,2株接种无菌松材线虫的植株体内线虫分别为486条和22条;接种带菌松材线虫的2株松苗中,1株分离到646条线虫,另1株没有分离到线虫。【结论】无菌和带菌松材线虫均对赤松具有致病性,松材线虫是赤松萎蔫的直接原因,伴生细菌并非赤松萎蔫的必须因子。     

无细菌松材线虫对马尾松的致病性

【目的】探讨松材线虫及其伴生细菌在松材线虫病中的作用,为其致病机制研究及病害防治提供理论依据。【方法】以5年生温室健康马尾松盆栽苗为材料,人工接种无菌松材线虫和带菌松材线虫(8 000条·株-1),观察松苗萎蔫情况,并于接种5、10和30天后取茎段横切面观察松脂分泌情况,对接种20天后的茎段组织解剖结构进行显微观察,接种后30天对植株取样进行线虫再分离,统计线虫在树体内种群增殖情况。【结果】无菌松材线虫和带菌松材线虫均能引起5年生马尾松盆栽苗发病,且症状一致,接种35天后致萎蔫率均达到90%,对照植株保持健康状态;茎段横切面观察结果显示,无菌松材线虫接种5天后松脂停止分泌,10天后茎段开始出现少量空洞,30天时空洞化严重;带菌线虫接种5天后尚有少量松脂分泌,10天松脂分泌停止,30天后茎段产生空洞;对照松树松脂分泌正常,茎段横切面完整平滑。石蜡切片结果显示,2组线虫接种致萎蔫松树茎段的组织解剖结构均发生显著变化,皮层及木质部细胞、髓心细胞大多破碎变形,树脂道上皮细胞破碎解体。无菌松材线虫在松苗体内种群增殖速率高于带菌松材线虫,萎蔫松苗体内线虫数量分别为(8 105±5 661)条·g-1和(4 317±1 896)条·g-1,但二者差异不显著(P=0.361 9)。【结论】无菌松材线虫和带菌松材线虫接种均能使5年生马尾松苗发病,无菌化处理未使松材线虫失去对马尾松苗的致病性,松材线虫的致病性与其伴生细菌无关。     

国内部分地区松材线虫和拟松材线虫基因多态性的RAPD分析

采用SDS法提取线虫基因组DNA,分别选取了6种随机引物对部分地区的10株松材线虫和10株拟松材线虫进行随机扩增和亲缘关系比较。RAPD的结果可以明显区分松材线虫和拟松材线虫,与形态学分类相吻合。聚类分析的结果也表明,线虫种内和种间均存在差异,但种间差异明显大于种内差异,同种线虫在同一地区或临近地区的同源性明显高于其它地区,同一地区内寄主对线虫种类分布的影响较小。该方法具有所需样品量少、检测灵敏度高、结果准确等优点,为松材线虫种群的亲缘关系、传播途径及其检测防疫等方面提供一种灵敏、可靠的研究方法。     

Pathogenicity of the pine wood nematode at different developmental stages

The ability of the pine wood nematode,Bursaphelenchus xylophilus, a pathogen that causes pine wilt disease, to kill cortical cells of Japanese black pine,Pinus thunbergii, during early development of the disease was conjectured to be a function of nematode developmental stage. A tangential segment of bark was removed from a 2-cm-long current-year stem. The cortex-exposed segments with cut cortical resin canals were designated as + RC-segments and those without them as − RC-segments. When a nematode population containing many older juveniles and adults (NL) was inoculated onto the cut surface, the − RC-segments were still alive 4 d after inoculation, as were non-inoculated control segments. When cortex-exposed segments were inoculated with either a nematode population containing many younger nematodes (NS) or with nematodes isolated from inoculated pine cuttings that also contained many younger juveniles, most tissue cells in − RC-segments died 4 d after inoculation, suggesting that younger juveniles killed pine cells directly, in contrast with older juveniles and adults. When nematodes were inoculated onto + RC-segments in which they could easily enter resin canals, both NL and NS killed the segment tissues. This suggests that NL is pathogenic to pine cells while living in resin canals. Such differences in the pathogenicity of NL and NS to pine parenchymatous cells were also demonstrated in a pathogenicity assay system using bark peelings, which allowed an estimate of direct attack on the cambial cells by nematodes. Based on these results, we hypothesize that younger juveniles are pathogenic to pine parenchymatous cells, while adults and older juveniles are not pathogenic. This work was supported in part by Grants-in-Aid for Scientific Research (No.01440012 and 06454088) and for Young Scientists (to K.I.) from Ministry of Education, Science, Culture, and Sports of Japan, and by a grant from PROBRAIN.     

无菌松材线虫对10年生黑松致病性的研究

2001年9月,用无菌的松材线虫对10年生黑松进行接种.2002年8月,检查黑松发病情况,并对接种的松树进行线虫和细菌的再分离.试验发现:接种的8株黑松11个月后仍有5株表现正常,没有出现任何症状,而从其木质部却能分离到数量较多的无菌松材线虫.结果说明:无菌的松材线虫虽然能侵入松树木质部,但在不携带细菌的情况下对松树是没有致病力的.     

越南松树枯死病症状及原因初步研究

为查明越南松树枯死的原因,对越南南部的林同省,中部的承天-顺化省,北部的广宁省的思茅松、马尾松和南亚松枯死症状和原因进行了调查,分别从枯死、濒死、健康松树木质部中抽取52份样品,用于线虫分离镜检。调查发现松树在感病后,树冠部分针叶失去光泽,逐渐褪绿变黄,进而褐红干枯,最后整个树冠变为褐色或红褐色,全株枯死,针叶下垂且当年不落;随着病程的发展,松脂分泌逐渐减少,直至停止;树干上有天牛产卵刻槽,死亡的松树上有天牛的羽化孔。在抽取的松树样品中分离鉴定到了泰国伞滑刃线虫(Bursaphelenchus thailandae BrasschBrassch-Bidasak,2002),为病原的确定和防治工作提供初步的依据。     

Migration of pine wood nematodes in the tissues of <Emphasis Type="Italic">Pinus thunbergii</Emphasis>

The distribution of pine wood nematodes (Bursaphelenchus xylophilus, PWNs) in Japanese black pine (Pinus thunbergii) tissues was investigated by staining with fluorescein isothiocyanate-conjugated wheat germ agglutinin. After PWNs were inoculated to current-year stems of pine seedlings, their distribution at about 5 cm below the inoculation site was confined only to cortical resin canals 1 day after inoculation, and then spread to other tissues, including resin canals of short branches. When PWNs were inoculated onto cross or tangentially cut surfaces of stem segments, maximal PWN migration speed was estimated to be faster through cortical resin canals and xylem axial resin canals vertically (>6.7 and <2.3 mm/h, respectively) than through cortical tissues both vertically and horizontally (<1.2 and <0.2 mm/h). To examine whether PWNs in cortical resin canals could invade surrounding tissues, segments in which PWNs resided only in cortical resin canals were prepared by removing the top portion 6 h after inoculation. Additional incubation of such segments caused extended PWN distribution to xylem axial resin canals and then to other tissues. A similar experiment with top portions of girdled segments removed 12 h after inoculation also showed extended PWN distribution from xylem axial resin canals and pith to cortical resin canals and then to other tissues. These results provided direct evidence that PWNs have the ability to migrate from cortical resin canals and xylem axial resin canals to other tissues.     

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。     

徂徕山病死松树中线虫的分离及鉴定

徂徕山林场地处鲁中山区的中心位置,具有重要的生态价值。近年徂徕山林场出现了松树的枯死现象,影响了徂徕山松林生态系统的健康和景观。为探究徂徕山松树死亡的原因,分析松树死亡是否与松材线虫病有关系,本研究调查了徂徕山10个林区,采集了969个样品,从线虫形态学、分子序列测定方面进行了鉴定和分析。研究显示,在徂徕山衰弱、病死木中未发现松材线虫,但是发现了大量的拟松材线虫(Bursaphelenchusmucronatus)。     

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]     

“九五”期间重大森林病虫害发生动态及原因分析

文章重点叙述了我国“九五”期间森林病虫害发生的特点和原因，并对松毛虫，美国白蛾、杨树天牛、杨树食叶害虫，松材线虫病，森林鼠害等主要病虫害发生变化及原因进行了分析。     

Aseptic Bursaphelenchus xylophilus does not reduce the mortality of young pine tree

To assess the role of bacteria in pine wilt disease (PWD), aseptic M form (with a mucronated tail) and R form (with a round tail) of Bursaphelenchus xylophilus and B. mucronatus were obtained and compared, in terms of reproduction and pathogenicity, with non‐aseptic nematode. In addition, bacteria isolated from non‐aseptic nematodes and pine trees inoculated with non‐aseptic nematodes were identified. The results indicated that the bacteria associated with nematodes significantly lowered the reproduction of R form of B. xylophilus and B. mucronatus. Both the non‐aseptic and aseptic R forms of B. xylophilus induced death in all infected 7‐ to 8‐year‐old pine trees, while the non‐aseptic and aseptic M forms of B. xylophilus and B. mucronatus caused almost no plant mortality. High numbers of the non‐aseptic and aseptic R forms of B. xylophilus were distributed throughout the inoculated trees, while B. mucronatus and M form of B. xylophilus nematodes were lower in number and their distribution in stems limited within the inoculation site. Bacteria isolated from non‐aseptic nematodes were not recovered from the pine trees inoculated with these same kinds of nematodes. Two species of bacteria were both isolated from non‐aseptic B. mucronatus and from R form of B. xylophilus. Microbacterium trichotecenolyticum was common to both the control and inoculated pine trees. These results suggest that R form of B. xylophilus is the causal agent of PWD and that bacteria cannot increase the virulence of B. xylophilus and B. mucronatus.