福建农林大学 硕士学位论文 柑橘半穿刺线虫不同地理种群形态学、遗传多样性研究及分子 检测 姓名：陈娟 申请学位级别：硕士 专业：植物病理学 指导教师：刘国坤;张绍升 20100401 福建农林大学硕士学位论文 1 摘摘 要要 柑橘半穿刺线虫(Tylenchulus semipenetrans)引起柑橘慢衰病(citrus slow decline disease)是重要的柑橘病害线虫，广泛分布于世界柑橘产区。柑橘半穿刺 线虫在我国广泛分布，危害严重。本研究对不同地理群体的柑橘半穿刺线虫从形 态学、交叉接种寄生性到分子生物学几个方面进行了比较和分析，并建立了柑橘 半穿刺线虫特异性检测技术。 对我国 4 个省，寄主分别为杉木、柑橘 5 个地理群体柑橘半穿刺线虫的形态 学进行了显微观测。各种群测量值与前人测量数据基本一致，表明柑橘半穿刺线 虫种群间虫体形态保守；所有群体中均有发现近末端钝圆，呈棍状，表面呈缢缩 状类型。 通过室内、野外交叉接种发现，杉木林地的柑橘半穿刺线虫种群可侵染杉木 苗根系，但未能侵染柑橘苗根系；柑橘地的柑橘半穿刺线虫可侵染柑橘根系，但 未侵染杉木根系，这与前人的结果不一致。 首次对采自我国 4 个省份柑橘或杉木上 27 个柑橘半穿刺线虫种群的 rDNA-ITS 进行测序，提交 GenBank，获得序列号，丰富了柑橘半穿刺线虫的核 酸数据库；经过对我国 4 个省的 27 个柑橘半穿刺线虫种群的 rDNA-ITS 区进行 测序和比对分析，结果表明：我国不同地区的柑橘半穿刺线虫的 rDNA-ITS 序列 保守， 差异极小， 线虫种群间的序列相似性在 98.4%以上， 序列变异率为 01.6%， 序列多态性分析表明序列特征上存在差异，具有丰富的多样性；根据序列相似度 与系统进化树显示我国柑橘半穿刺线虫的 ITS 序列与地理位置及寄主无相关性。 寄生于杉木或柑橘的柑橘半穿刺线虫种群之间并不能通过rDNA-ITS序列区分是 否存在生理小种。 本研究根据柑橘半穿刺线虫的rDNA-ITS的序列特征，设计出了一对柑橘半穿 刺线虫特异性引物，Ts-SF(正向: 5- TACCAGGTTGAGCAGAGTTCTT -3 )和 Ts-SR (反向: 5- TCCTACCCTTCCACAGCG3)。特异性引物灵敏度高，可扩增出 单条柑橘半穿刺线虫二龄线虫的DNA；特异性强及可靠性好，对采自全国4个省 份16个不同地区寄生柑橘或杉木的柑橘半穿刺线虫种群均能扩增出特异条带， 对 非靶标线虫未能扩增出条带； 可从改良贝尔曼漏斗法分离的线虫混合群体中检测 福建农林大学硕士学位论文 2 出单条靶标线虫。本研究建立了柑橘半穿刺线虫特异性检测体系，对柑橘半穿刺 线虫的防治具有重要意义和应用价值。 关键词：关键词：柑橘半穿刺线虫；形态学；交叉接种寄生性；rDNA-ITS；特异性检测 福建农林大学硕士学位论文 3 Abstract The citrus nematode, Tylenchulus semipenetrans (TS) Cobb, 1913, causing agent of slow decline of citrus is considered one of major nematode pests of citrus. TS widely distributes in China, causes significant economic losses in all citrus-growing regions of China. In this study, TS from different geographies in China were compared and analyzed based on morphology, cross-inoculation parasitism and molecular biology, to develop a diagnostic protocol with a single step PCR using the primers to detect low number of TS in soil nematode community extracted by the Modified Baermann funnel method. 5 populations of TS were collected from root and soil samples in different citrus orchards in 4 provinces and Chinese-fir(Cunninghamia lanceolata) plantations in Fujian province in China. And then, the samples were observed and measured with the microscope. The results showed that the measurement of TS was consistent with previous study. The morphologic of TS is conservative. In all populations, there is another type of tail. The near terminal of tail is obtuse circle, like stick, constriction reduced. The results enriched morphometrics of TS. Several cross-inoculations through indoor, the field or the forest land showed that TS can infect Chinese fir, but failed to infect citrus seedling roots when Chinese fir and citrus were all planted in Chinese-fir plantations which were infected with TS. However, TS can infect citrus, but failed to infect Chinese fir seedling roots when Chinese fir and citrus were all planted in the citrus orchards. The result is inconsistent with the predecessors results. The 27 rDNA-ITS sequences of TS from 4 provinces were sequenced, submitted to the GenBank, and obtained the accession numbers, so the results enriched nucleic acid database of TS. rDNA-ITS sequences of TS were compared and analyzed. The results indicated that the rDNA-ITS sequences of TS are conservative with minimum differences. The sequence similarity between the TS populations was above 98.4% and the sequence variation rate is between 0 and 1.6%. Phylogenetic analysis of sequences indicated that there was differene in sequence characteristics and rich multiplicity. According to the sequence similarity and the system cladogram, it was demonstrated that TSs ITS sequence was unrelated with geographical position and the 福建农林大学硕士学位论文 4 host. It proved that it could not distinguish the different physiologic race of TS. Species-specific primers were designed based on the sequences of rDNA ITS regions of 14 populationa of TS collected in different citrus groves or Chinese fir plantations in China. The primers were sufficiently sensitive to yield an expected size band for individual second-stage juvenile (J2) or different life stages of TS. Specificity and reliability of species-specific primers were validated by further 16 additional populations of TS collected from 4 provinces in China. Non-target nematode specificity of species-specific primers was demonstrated by testing 14 non-target nematode species respectively which were commonly found in citrus groves. The sensitivity and reliability of primers were also confirmed to detect low numbers of J2 of TS in nematode soil communities containing large numbers of non-target nematode extracted by the Baermann funnel method. It appears that the diagnostic primers will be useful for accurate identification and also as a decision management tool for growers in China. Keywords: Tylenchulus semipenetrans; morphologic; cross-inoculation; rDNA-ITS; sensitive detection 独创性声明独创性声明 本人声明，所呈交的学位（毕业）论文，是本人在指导教师的指导下独 立完成的研究成果，并且是自己撰写的。尽我所知，除了文中作了标注和致 谢中已作了答谢的地方外，论文中不包