【作者】 李海莲；

【导师】 侯喜林；

【作者基本信息】 南京农业大学 ， 蔬菜学， 2005， 硕士

【摘要】 本文对来自我国不同地区的14个茄子黄萎病菌进行了形态、致病力、致病机制的研究及ISSR的指纹分析。 1 茄子黄萎病菌的收集及形态学鉴定:对采集到的5个茄子黄萎病植株进行分离纯化后的病原菌,经鉴定表明均为大丽轮枝菌。生长温度的测定表明,大丽轮枝菌在PDA培养基上,25℃菌落生长最好,在30℃仍可生长,但在33℃时,均不能生长。采用黄萎病菌双层盘法接种,对来自不同的寄主分离来的5个大丽轮枝菌在茄子、番茄、辣椒和棉花几种作物致病力的结果,发现在不同作物上的致病力存在明显的分化现象。对来源不同的14个黄萎病菌进行形态观察发现:根据其性状的不同可以将其分为丝核型、疏核型和菌核型三种类型;分生孢子和微菌核的大小存在差异（p<0.0001）,此外,微菌核的大小在我国的南方和北方存在差异分化（p<0.0001）。 2 茄子黄萎病菌致病力的鉴定:采用孢子悬浮液接种与毒素接种对14个菌株进行致病力的鉴定,结果表明二者的鉴定结果高度吻合。并根据致病力的不同将14个菌株分为强、中、弱三个类型,且三个类型之间达到了极显著的差异,进一步对致病力、病原菌的形态类型、地理来源、落叶与否等之间的关系进行分析,结果发现,病原菌的形态类型与气生菌丝、微菌核形态之间;气生菌丝的生长的状况与分生孢子的长;分生孢子的长和宽、微菌核的长和宽;落叶与否与菌落颜色、微菌核形态及形态类型等均达到了显著的相关;尽管3个有强致病力的菌株均属于落叶型,但数据未显示出它们之间的相关性。致病类型与形态类型、地理来源之间均未表现出相关性。 3 黄萎病菌粗毒素接种对感病和抗病茄子品种的一些酶类活性和光合特性的影响:以黄萎病菌粗毒素接种不同茄子品种的结果表明接种后,抗病品种比感病品种的过氧化物酶（POD）、超氧化物歧化酶（SOD）活性高,而多酚氧化酶（PPO）和苯丙氨酸解氨酶（PAL）活性则表现相对稳定。同时,接种茄子黄萎病菌粗毒素后,感病品种比抗病品种在茄子叶片净光合速率（P_n）、气孔导度（G_s）、蒸腾速率（T_r）和细胞间CO₂浓度（C_i）的变化幅度大,但二者的叶片净光合速率（P_n）、气孔导度（G_s）、蒸腾速率（T_r）在72h后都出现了明显的下降趋势。 4 茄子黄萎病菌基因组ISSR指纹图谱分析:对茄子大丽轮枝菌基因组的DNA提取、适合于ISSR反应体系的建立中的几个问题、ISSR多态性的扩增及其与不同的地理来源、形态类型、致病力和落叶型之间的关系进行了探讨。结果表明,改进的CTAB法提取的DNA的质量较高;建立了适应茄子大丽轮枝菌的ISSR反应体系:20μL的PCR更多还原

【Abstract】 The objective of this study is to characterize 14 eggplant isolates of Verticillium dahliae Kleb. from different cropping areas in China. Four independent experiments, covering the morphological characterization, pathogenicity test, mechanism of virulence, and PCR-based ISSR fingerprinting analysis, were carried out. The results of each part were respectively described as follows:1. To characterize the morphology of V. dahliae isolates from affected eggplants, 5 isolates of Verticillium were involved in this study. The results indicated that all of the collected fungi are belonging to the single species of V. dahliae Kleb., and the optimal temperature for single clone development was 25℃; however, restrained under 30℃, and no surviving beyond 33℃.The further inoculation on different hosts, e.g. eggplant, tomato, pepper, and cotton using double-layer-plate method, displayed that the 5 isolates differed in pathogenicity to each host. According to the morphological characteristics, 14 single spore isolates were therefore classified into three morphotypes: HS （Hypha-Sclerotia）, Sc （Sclerotia）, and SS （Sparse Sclerotia）; moreover, there were significant differences in mean size of conidia and sclerotia （p<000001） among 14 isolates. Interestingly, there are also significant differences in the size of sclerotia between southern and northern China.2. To identify the differentiation of pathogenicity among the eggplant isolates of V. dahlia, all of the 14 isolates were inoculated to eggplant by simultaneously using conidial suspension and crude toxin. Significant differences among isolates were consensus in both inoculations. Therefore, the isolates were divided into three pathotypes, e.g. strongly, moderately and weakly pathogenic, depending on the integrating performance on G24, 85m, and Sm90017. Further correlation analysis revealed that closely associations were detected among numbers of morphological characters, including between morphotype and both aerial hypha and microsclerotia, between microsclerotia length and width, between conidia length and width, between Defoliating/Non-Defoliating （D/ND） and sclerotia and morphotype, and between aerial hypha and conidial length. Unexpectedly, there was no significant correlation between pathogenicity and D/ND type, morphological characteristics, and geographical origin, even all three isolates involved in Defoliating type demonstrated strong pathogenicity.3 To insight into impacts of crude toxin to activities of enzyme and photosynthetic characters, both susceptible and resistant eggplants were inoculated with crude toxin isolated from Verticillium dahliae Kleb.. The result showed that theperoxidase（POD） and superoxide dismutase（SOD） activities of resistant eggplant were higher than that of susceptible one. However, polyphenol oxidase （PPO） and phenylalanine ammonia lyase （PAL） activities were relatively stable. Meanwhile, the Net photosynthetic rate（P_n）, Stomata conductance（G₅）, Transpiration rate（T_r） and Intercellular CO₂ concentration （C,）of resistant eggplant were less than that of susceptible. While both eggplants displayed descending trend in P_n, G_s, and T_r after inoculation for 72 hours.4. To distinguish the eggplant isolates of Verticillium dahliae at molecular level, the ISSR-based fingerprinting analysis was carried out using genomic DNA from 12 isolates collected from different areas. The results showed that the optimal ISSR reaction system comprised 20ng template DNA, 0.25 u mol/L ISSR primer, 2.5 mmol/L MgCl₂, 250 \x mol/L dNTP, 1U Taq DNA-polymerase, and 1 X PCR buffer in a total volume of 20μL reaction mixture, with the procedure of one cycle denaturing at 94℃ for 5 min;40 cycles each involved denaturing at 94℃ for 30s, annealing at 48℃ for 45s;extending at 72℃ for 2 min; a final extension at 72℃ for 6 min after the last cycle; and then remaining at 4℃. The abundant amplification products implied that ISSR （inter simple sequence repeat） was ubiquity in genome of Verticillium dahilae isolates from eggplant. The fingerprint更多还原