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2002年从贵阳市南明区采集到濒临死亡的马尾松标样,用改进的浅盘分离法对其进行了病原物的分离、纯化、形态学及分子生物学鉴定。结果表明,萎蔫死亡松树分离出的线虫形态特征与Mamiya报道的拟松材线虫形态特征相符。用伞滑刃线虫ITS区(internal transcribed spacer)通用引物VRF1(5'CGTAACAAGGTAGCTGTAG 3')和VRF2(5'TC-CTCCGCTAAATGATATG 3')对其基因组DNA进行PCR扩增,并对其PCR产物进行回收、克隆和测序。测序结果显示,此ITS序列与南京松材线虫的同源性为77.6%,与基因库已报道拟松材线虫序列U93554的同源性为93.5%,而与云南拟松材线虫江川种群、峨山种群和西畴种群的同源性为97.9%。通过以上形态学和分子生物学的研究,将贵阳市南明区采集到的马尾松萎蔫病树线虫分离物鉴定为拟松材线虫(Bursaphelenchus mucronatus)。 相似文献
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对伞滑刃线虫属部分种群包括松材线虫、拟松材线虫、豆伞滑刃线虫、莱奴尔夫伞滑刃线虫和泰国伞滑刃线虫等种的28S RNA基因中D2-D3区进行了系统的研究。经PCR扩增发现, 不同的种均产生1个约750 bp的片段。利用限制性内切酶对其进行PCR-RFLP分析, 除松材线虫与拟松材线虫的酶切图谱比较接近外, 其它伞滑刃线虫种群获得不同的酶切图谱。图谱分析发现, 限制性内切酶Bsh1236I能区分2个近似种——松材线虫和拟松材线虫。进一步分析表明:Bsh1236I对28S RNA基因D2-D3区的酶切可用于鉴定本研究中伞滑刃线虫属的不同种群。同时运用clustalx1.8和Mega 2软件对28S RNA基因D2-D3区构建部分伞滑刃线虫属种群的系统树, 显示出与形态学基本一致的聚类组群。 相似文献
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分离自对湖南郴州、耒阳、张家界地区的病死马尾松样品,采用形态鉴别和分子生物学鉴别相结合的方法对其进行鉴定.形态鉴定结果显示分离检出线虫更符合拟松材线虫特征.同时采用ITS-PCR-RELP方法对病死马尾松中分离线虫进行分子鉴定,ITS区经EcolI和HindⅢ酶切后得到110、160、242、404bp 4条特征带,与拟松材线虫相同,而与松材线虫有明显区别.而且ITS及CoI基因序列信息显示,湖南病死马尾松中分离线虫与松材线虫的同源性分别为92.9%、89.3%,而与拟松材线虫的同源性高达97.2%、95.2%.研究结果表明湖南病死马尾松中分离的线虫为拟松材线虫. 相似文献
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松材线虫组DNA条形码筛选 总被引:1,自引:0,他引:1
本文基于Gen Bank中登录的14种松材线虫组线虫的28S、18S和ITS部分基因DNA序列,利用遗传距离法及分子生物学方法评价其各自作为松材线虫组DNA条形码序列的适用性。结果显示,28S(拟松材线虫不分亚种)、28S(拟松材线虫分亚种)、18S和ITS的基因序列种内成对遗传距离平均值分别为0.0071、0.0030、0.0007、0.0043,种间成对遗传距离平均值分别为0.0476、0.0454、0.0052、0.1556,其中28S、18S基因序列种内及种间距离存在一定程度的重叠,ITS具有一定程度的Barcoding gap。3个基因序列构建的NJ进化树显示,28S、ITS构建的系统进化树具有较高的节点支持率,可以有效将14个松材线虫组线虫分离成独立分支,而且28S可以有效鉴别出拟松材线虫的2个亚种;基于18S基因构建的进化树不能对吉拉尼伞滑刃线虫、日本冷杉伞滑刃线虫、拟松材线虫进行有效区分。因此,28S、ITS区具有一定的遗传距离间隔以及相对较高的物种识别率,可以作为松材线虫组线虫候选条形码基因。 相似文献
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rDNA-ITS-PCR技术在植物寄生线虫分子诊断中的应用 总被引:13,自引:0,他引:13
20世纪80年代以来,分子生物学技术在各个研究领域得到广泛应用,分子诊断也成为植物线虫学研究的新途径,内转录间隔区ITS是位于线虫的遗传物质特别是核糖体DNA(rDNA)上18S和28S基因之间的区域片段,一般而言它在生物种和亚种间变化较大,用PCR技术扩增ITS区,可通过酶切、克隆、测序等来揭示线虫种间或群体间的差异,从而进行植物病原线虫种的快速分子鉴定.目前,国外rDNA-ITS-PCR技术已在许多重要的植物线虫如剑线虫、胞囊线虫、根结线虫、松材线虫、拟松材线虫、马铃薯金线虫、马铃薯白线虫、茎线虫等的分子诊断中广泛应用. 相似文献
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Suleyman Akbulut Paulo Vieira Alexander Ryss Besir Yuksel Akif Keten Manuel Mota Vera Valadas 《EPPO Bulletin》2006,36(3):538-542
A survey was conducted in the northern conifer forests of Turkey in 2003 and 2004 for the pinewood nematode, Bursaphelenchus xylophilus . Wood samples were collected from declining pine trees, located in the following target areas: Düzce, Ankara, Bolu, and Artvin. Nematodes were extracted from over 310 samples and were observed and identified. B. xylophilus was not detected in any samples. Bursaphelenchus species were only found in 6% of the samples. From the B. xylophilus -group, only the species B. mucronatus was reported. Species identification was performed from morphological characters, particularly male spicules, and by molecular analysis with ITS-RFLP. 相似文献
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云南松树伞滑刃线虫种类鉴定 总被引:8,自引:1,他引:8
2000~2001年从云南省34个县/市调查采集到病松树样本161份,鉴定出伞滑刃属线虫3个种即拟松材线虫(Bursaphelenchus mucronatus)、霍夫曼伞滑刃线虫(B. hofmanni)和赫列尼库斯伞滑刃线虫(B. hellenicus)。后2种为我国新记录种,其寄主分别是华山松(Pinus armandii)和云南松(P. yunnanensis),为该2种线虫的世界新寄主记录。目前云南省没有发现松材线虫,但在16个县/市发现拟松材线虫。 相似文献
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应用TaqMan-MGB探针进行松材线虫的实时荧光定量检测技术研究 总被引:7,自引:0,他引:7
松材线虫是国际公认的最重要的检疫性有害生物之一,也是我国2类检疫危险性有害生物,我国口岸多次从货物的木质包装中截获该线虫。由于松材线虫与拟松材线虫在形态上极其相似,难以区分,幼虫更无法用于鉴定。传统的形态学鉴定、生化以及其他分子技术等方法存在费时、准确度不高、灵敏度低等缺点,不易形成标准。我们设计筛选一对引物以及一条MGB探针,对松材线虫进行实时荧光PCR检测。建立了一条从1 pg到104pg标准曲线,相关系数r=0.965。该检测方法省时、准确、快速、无污染。 相似文献
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松材线虫是进境木质包装中的重要检疫对象之一。本研究以进境木质包装中截获的12种伞滑刃属线虫,包括4种不同来源的松材线虫、3种不同来源的拟松材线虫、豆伞滑刃线虫、大尖尾伞滑刃线虫、阿苏里伞滑刃线虫、伯氏伞滑刃线虫及拟小刺伞滑刃线虫为材料,进行实时荧光PCR检测。结果显示,仅松材线虫可观察到明显的荧光强度变化,而其他线虫的荧光强度没有变化。通过研磨样品的方法,提取单条松材线虫的DNA,进行实时荧光PCR试验,检测成功率为100%,且不论是雄虫、雌虫或幼虫均能检测出来。该研究对于我国口岸进境木质包装中松材线虫的检测具有一定的实际意义。 相似文献
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Bursaphelenchus species in conifers in Europe: distribution and morphological relationships 总被引:12,自引:0,他引:12
H. Braasch 《EPPO Bulletin》2001,31(2):127-142
The morphological relationship between European Bursaphelenchus species living in conifers was studied in order to provide key characters for their taxonomic identification. Several species have been newly described or recorded in Europe during the past few years and were morphologically investigated. Among the 28 conifer-inhabiting European species, four groups can be distinguished from each other by the number of lateral incisures, number and position of anal papillae of males and presence and size of a vulval flap of females. Two groups, each containing just one species show two and six lateral lines, respectively, whereas most Bursaphelenchus species belong to two groups, possessing either three or four incisures. Each of the last two groups can be divided into three subgroups, which can be differentiated by spicule shape, number and position of caudal papillae, presence and size of a vulval flap and some other features. A fifth group includes species with unclear group affiliation due to insufficient knowledge of their morphology. The B. xylophilus group ( B. xylophilus, B. mucronatus, B. fraudulentus ) with four lateral lines, unique spicule shape and characteristic position of the caudal papillae can clearly be differentiated from all other groups by morphology, also using only light microscopy. Within this group, B. xylophilus can clearly be differentiated morphologically from the other species, provided that adult specimens of both sexes are found and also provided that the round-tailed form of the species remains the only form present, as in Europe. Figures showing the important identification features of spicule shape and female tail shape are given for 28 species. A table shows the complete range of the most important morphometric measurements of these species. Data on the occurrence, host range and, as far as known, the vectors of these species were collected throughout Europe and references are given for each record. 相似文献