【作者】 张萍；

【导师】 吕国忠；

【作者基本信息】 辽宁师范大学 ， 微生物学， 2011， 硕士

【摘要】 三裂叶豚草是重要的外来入侵生物,如何防治该杂草已成为杂草学家和社会普遍关注的问题。利用专性寄生病菌是防治三裂叶豚草的有效途径。苍耳柄锈菌三裂叶豚草专化型(Puccinia xanthii Schwein. f.sp. ambrosiae-trifidae S.W.T. Batra)对三裂叶豚草表现出较好的生防潜能。为此,本论文对该锈菌的侵染机制及在辽宁省的种群变异进行了系统研究,为利用该菌防治三裂叶豚草奠定理论基础。本论文主要取得了如下三方面的研究结果:(1)利用人工接种结合考马斯亮蓝染色技术研究苍耳柄锈菌三裂叶豚草专化型冬孢子萌发,担孢子形成以及侵染过程。侵染结果表明,该锈菌的侵染过程可以分为冬孢子的萌发、担孢子的形成、侵染前、发育、侵染和侵染后在寄主内扩展、冬孢子堆形成等阶段。(2)比较研究了该锈菌冬孢子DNA提取的9种方法,其中CTAB-钢珠法、改良的微型电钻法以及EZ-Kit改良法获得的基因组DNA经检测质量较好。在此基础上,利用ITS-PCR和ISSR引物UBC#835将待用DNA进行PCR扩增检测。结果表明,上述3种方法提取的DNA适合于后续分子生物学要求。(3)以ISSR和SRAP遗传标记方法对辽宁省16个锈菌群体进行了遗传多样性研究,11个ISSR引物和13对SRAP引物扩增出多态位点百分数分别为72.62%和71.05%。结果表明,在群体水平上ISSR法与SRAP法获得的基因多样性指数为0.0028—0.3933和0.2991—0.3869,Shannon′s信息指数为0. 0047—0. 5703和0.4445—0.4750,Nei′s分析结果显示ISSR和SRAP法的获得的种群间的遗传分化系数(Gst)为0.2537和0.2330,分子方差分析(AMOVA)显示种群间的遗传变异占总变异的5.70%和4.60%,种群内变异占总变异的94.30%和95.40%,表明锈菌在不同地理种群间存在较低水平的遗传变异,而在种群内部存在广泛的基因多样性。通过UPGMA聚类图显示,SRAP标记分析结果与两种标记复合聚类分析的结果相近,说明SRAP在锈菌的遗传多样性和亲缘关系聚类分析中可能具有更高的可信度。更多还原

【Abstract】 Ragweeds (Ambrosia L.) are alien invasive species for China. How to control the grasses has been closely noticed by weed scientists and public. It appears that it is an effective way to control the grasses by absolute parasitic pathogens. Puccinia xanthii f. sp. ambrosia-trifidae S. W. T. Batra was a microcyclic rust fungus for biological control of giant ragweed, Ambrosia trifida. Thereafter the paper is made to investigate its infection process and genetic variation in order to provide a theoretical base for biocontrol of the grasses by the fungus. The paper conducted the following research aspects:(1) The study illustrated the infection process of P. xanthii f.sp. ambrosiae-trifidae by means of light microscopy associated with Coomassie Brilliant Blue R-250 staining method. The infecting process was divided into the following phases: teliospore germination, basidiospore formation, prepenetration development, penetration and postpenetration development. The typical infection structures included germ tube, appressorium, penetration peg, intraepidermal vesicle, primary hypha, intracellular hyphae, M-haustorium (Chapter one).(2) The genomic DNA of P. xanthii f. sp. ambrosiae-trifidae was extracted by nine methods from the teliospores of the rust fungus. The results show that the methods of CTAB-steel balls, modified mini-drill and modified EZ-Kit were better than the other six methods due to their higher quality of DNA extraction with less impurity. The template DNA samples generated from these methods were detected by methods of ITS-PCR and ISSR-PCR with the primer UBC#835. They all produced expected band patterns. The research confirms that the DNA samples generated from these three methods could be used for further molecular study of the rust fungus (Chapter two).(3) The genetic diversity within and among 16 populations of P. xanthii f.sp. ambrosiae-trifidae from Liaoning province was investigated using ISSR and SRAP molecular markers. The percentages of polymorphic bands were 72.62% for 11 ISSR primers and 71.05% for 13 SRAP primers. The results showed that, the Nei′s genetic diversity and Shannon′s information indexes were 0. 0028—0. 3933 and 0. 0047—0. 5703 in ISSR assay at the population level, and the Nei′s genetic diversity and Shannon′s information indexes were 0.2991—0.3869 and 0.4445—0.4750 in SRAP assay. Based on Nei′s genetic diversity analysis, the genetic diversity coefficient (Gst) of ISSR and SRAP data was 0.2537 and 0.2330, respectively. Analysis of the molecular variance (AMOVA) showes that the genetic variance accounted for 5.70% with ISSR primers and 4.60% with SRAP primers of the total variation among the populations, while it was 94.30% with ISSR primers and 95.40% with SRAP primers within the populations. By employing both ISSR and SRAP markers, this study suggests that the level of genetic diversity among populations of rust in Liaoning province is lower than that within population. Overall, UPGMA obtained from SRAP analysis was nearly consistent with that obtained from the combination analysis of ISSR and SRAP data. In conclusion, the SRAP Marker system is a simple and efficient marker system that can be adapted for a variety of purposes in rust, including map construction, gene tagging and genomic fingerprinting (Chapter three).更多还原