【作者】 秦楠；

【导师】 曹挥；

【作者基本信息】 山西农业大学 ， 植物病理学， 2013， 硕士

【摘要】 本研究以危害番茄的灰霉病菌为研究对象,通过提取、萃取、柱层析等分离方法,对广泛分布于我国长江以南各地区的植物-决明子进行了活性成分分离,最终获得了活性流份L3,并对其抑菌机理进行了系统的研究,实验结果如下：1.对决明子颗粒用三种不同极性的溶剂分别进行提取,提取率由高到低依次为乙醇>氯仿>石油醚。同时测定了各提取物对番茄灰霉病菌的生物活性,结果表明：决明子提取物在72h和48h对供试菌的菌丝生长和孢子萌发都有较强的抑制作用,浓度为2mg·mL-1在对菌丝的抑制率和1mg·mL-1时对孢子萌发抑制率中,决明子氯仿提取物分别达到80.55%和100%。决明子提取物在96h和48h对菌丝生长和孢子萌发的ECso分别为0.60mg·mL-1和0.24mg·mL-1。因此,选用决明子氯仿提取物进行进一步分离纯化。2.对决明子氯仿提取物三种不同溶剂萃取物的研究结果表明：氯仿萃取率最高,达到47.73%；甲醇次之,为24.47%；水和石油醚最低,分别为12.33%和8.47%。对萃取物进行生物活性测定,结果表明,氯仿萃取物对番茄灰霉菌菌丝生长和孢子萌发均有较高的抑制作用,抑制率分别为83.33%和100%。石油醚和甲醇萃取物也有较高的生物活性,水溶液萃取物的生物活性最低。其中氯仿萃取物对菌丝生长和孢子萌发的ECso分别为0.40mg·mL-1和0.19mg·mL-1。3.决明子氯仿萃取物经柱层析分离和TLC检测,合并相同流分后得到7个流分,对所得7个流分分别进行生物活性测定,其结果表明,L3对病原菌菌丝有很高的生物活性,在72h时抑制率为100%。在96h时,L3对番茄灰霉病病菌菌丝生长的ECso为0.24mg·mL-1。4.L3处理番茄灰霉病菌菌丝后,经显微观察发现,培养基上生长着少量菌丝,菌丝匍匐在药基上,菌丝稀少杂乱；在电镜下观察,菌丝粗细不一、杂乱,菌丝干瘪,细胞壁皱缩、局部有肿瘤状突起。经分析,L3可能影响了细胞膜和细胞壁的形成。5.通过L3处理番茄灰霉病菌菌丝后,在测定细胞膜渗透性中发现,L3增大了菌丝细胞膜渗透性,导致电解质渗漏。同时在对细胞壁中的几丁质酶、蛋白酶测定中,发现L3在1mg·mL-1时,3h后,对几丁质酶活性最高,同时N-乙酰葡萄糖胺含量达到最大,随后开始逐渐下降,表明N-乙酰葡萄糖胺有可能在参与细胞壁形成过程中受阻。而蛋白酶活性的测定中,在3、6、12h时,酶活性与对照无明显差异。对细胞膜上麦角甾醇含量测定中,L3抑制了麦角甾醇的合成,说明L3在番茄灰霉病病菌的细胞膜上存在作用位点。.实验研究结果表明,决明子提取物对番茄灰霉病菌有很强的抑菌活性,在新型植物源杀菌剂开发方面有着广阔的前景。更多还原

【Abstract】 A plant, Cassia obtusifolia L.. widely distributed in south of the Yangtze river of China, was investigated for its disinfectanted activity against Botrytis cinerea Pers, crude extract extracted by respective immersion, was separated by extraction and purified through column chromatograghy, and its active constitute,L3,was separated. Its activities to active methods and inhibitory mechanism were studied. The results indicated:1. The Semen Cassiae extracted by alcohol, chloroform, petroleum ether,alcohol had highest extraction rate, and lowest was petroleum ether. The result of bioassay revealed that there were the effect of inhibitory effect in varying degrees, the inhibitory effect of chloroform extract was80.55%,when the pharmacy concentration was2mg.ml-1.The inhibition rate of chloroform extract was100%, when the pharmacy concentration was1mg.ml-1in the tests of inhabiting effect of the different menstruum of Semen Cassiae siebotdii extract against conidiao production of Botrytis cinerea.The MIC was0.60mg.ml and0.24mg.ml-1,the result was more obvious than other extractions.2. The antifungal activities of the extract from Semen Cassiae against Botrytis cinerea was tested.The result indicated that the chloroform ether extract of Semen Cassiae against mycelium growth and spores germination were83.33%and100%respectively. The MIC was0.40mg.ml-1and0.19mg.ml-1.The water extract of Semen Cassiae against spores germination and mycelium growth were the lowest. So extraction of chloroform ether was studiey further more.3. The silicagel column was used to separate and purify active constitutes of the extraction of various fractions form the cacetic ether. A total of7fractions were seen by TLC analysis. The bioassay showed that the3th fraction have strong antifungal activities, after72h of which to Botrytis cinerea was100%, the MIC was0.24mg.ml-1after96h.4. L3deal with B. cinerea and found that the growth of its mycelium sparse, prostrate on the medium; in the electron microscope, tumor-like hyphae on the health and abnormal enlargement of an analysis, L3may affect the cell membrane and cell wall formation.5. Through L3after treatment B. cinerea. is found in the determination of membrane permeability, L3mycelium increased membrane permeability, leading to electrolyte leakage. At the same time in the cell wall of chitinase, protein determination, we found L3in the1mg·mL-1,3h later, on the highest chitinase activity,3h when the N-acetyl glucosamine content reached the maximum, and then began to decline, indicating that N-acetyl-glucosamine may be involved in cell wall formation is blocked. The protease activity in the treatment of3,6,12h, the activity was no difference with the control.lt significantly inhibited the synthesis of ergosterol,so indicate the existence of role of cell membrane sites.Based on the results, we discussed perspectives of using Semen Cassiae for the Botrytis cinerea Pers control.更多还原