【作者】 尉研；

【导师】 龙章富；

【作者基本信息】 四川大学 ， 微生物学， 2007， 硕士

【摘要】 山葵墨入菌病随着20世纪90年代后期山葵的引种而传入我国，该病作为传入我国大陆的新病害，目前药剂防治效果不明显，而且造成了一定程度的环境污染。应用植物源抑菌剂对植物病害进行生物防治是比较理想的方法。因此，本文拟从中药中筛选出对山葵墨入菌具有抑菌活性的物质，并对其进行活性和成分的初步研究。对中药黄芩、射干、百部、郁金进行清水、95％乙醇提取并浓缩得到浸膏，以山葵墨入菌作为指示菌，应用生长速率法测定抑菌活性。初步研究表明，郁金的95％乙醇热搅拌提取率为4.42％，其浸膏对山葵墨入菌具有一定的抑菌活性，药基浓度为1.0 mg／ml时，抑菌率为67.08％。为了进一步研究郁金95％乙醇提取物的抑菌活性，对郁金醇提浸膏进行正己烷、乙酸乙酯、甲醇的分步萃取，并对其进行抑菌活性分析。结果表明，抑菌的活性成分主要存在于郁金正己烷萃取物中，药基浓度为1.0 mg／ml时，对山葵墨入菌的抑菌率为79.70％，而乙酸乙酯和甲醇的萃取物抑菌活性较低。郁金正己烷萃取物以山葵墨入菌为指示菌，毒力回归方程为：Y=1.5427x+5.7430，EC₅₀为0.3299 mg／ml，在药液浓度为10 mg／ml时，对孢子萌发抑制率为97.2％。郁金95％乙醇提取物和各萃取物经乙醇—浓硫酸显色后，结果表明，郁金中的主要成分姜黄素存在于乙酸乙酯萃取物中，结合抑菌活性实验确定姜黄素对山葵墨入菌的抑菌活性较低，乙酸乙酯萃取物药基浓度为1.0 mg／ml时，抑菌率仅为37.09％。采用GC-MS技术对郁金正己烷萃取物进行系统分析后，选择鉴定出其中含量较高的40种化合物，其中含量最高的为土青木香烯（23.91％），其次是1，2-去氢睾酮（23.63％），还含有一些挥发油类的化合物、长链脂肪酸、长链脂肪酸酯、生物碱等。在抑菌谱的测定中，选用具有代表性的7种病原菌：小麦赤霉菌、油菜菌核菌、轮枝孢子菌、疣孢霉、橄榄绿霉、苍白青霉、番茄灰霉病菌等作为供试菌，用菌丝生长速率法测定郁金95％乙醇浸膏正己烷萃取物对多种病原真菌的抑制作用，并测定了最低抑菌浓度（MIC）和最低杀菌浓度（MBC）。结果表明，郁金正己烷萃取物对多种病原真菌均具有抑菌活性，药基浓度为1.0mg／ml时，对番茄灰霉病菌的抑菌率达到100％，对油菜菌核菌的抑菌率为86.58％，对疣孢霉的抑菌率为71.72％，对轮枝孢子菌的抑菌率为78.19％，对小麦赤霉菌的抑菌率为76.82％，对苍白青霉的抑菌率为74.85％，对橄榄绿霉的抑菌率为54.78％。郁金正己烷萃取物对灰霉的MIC和MBC值相同均为1.56 mg／ml；油菜菌核菌和苍白青霉MIC和MBC均为3.13 mg／ml；山葵墨入菌、小麦赤霉菌、疣孢霉、轮枝孢子菌的MIC值为6.25 mg／ml，而对橄榄绿霉的MIC值为12.5 mg／ml。以山葵墨入菌、油菜菌核菌、疣孢霉作为指示菌，研究各种物理、化学条件下郁金正己烷萃取物的稳定性及对抑菌能力的影响，并在透射电镜下观察正己烷萃取物作用前后山葵墨入菌和油菜菌核菌超微结构的改变。结果表明：郁金正己烷萃取物处理温度在60℃以上，pH＞6.0时，抑菌率有所降低，紫外照射对其抑菌率影响不大。0.5 mg／ml的郁金正己烷萃取物作用后的山葵墨入菌细胞质解体、出现空腔，细胞器肿胀，甚至坏死溶解；油菜菌核菌细胞内线粒体出现严重肿胀，嵴大面积断裂，部分细胞壁缺失。更多还原

【Abstract】 Wasabi（Eutrema wasabi Maxim） had been planted in China in 1990s later periods and taken along with the pathogen Phoma wasabiae Yokogi at the same time. Wasabi steak was the new plant disease in our country. The prevention against it was through agricultural and chemical measures since 1990s, but the effect of prevention was not obvious. Application of chemical and pesticides has brought entironment pollution, so biological-control for natural antagonistic organisms is a potential measure against plant diseases. One kind of traditional Chinese herb which could inhibit P. wasabiae Yokogi was screened and further study results were reported in this paper.Four Chinese herbs, including Scutellaria baicalensis Gerorgi, Belamcanda chinensis （L.） DC, Stemona japonica （Bl） Miq, Curcuma phaeocaulis Val, were extracted by different ways. During the extraction, water and 95% ethanol were used as solvents. Then those extracts were dissolved in water for the experiment in order to systematically investigate their fungistasis by growth rate method while P.wasabiae Yokogi was used as the pathogenic fungi. The antifungal activities of extraction by different solvents were quite different, and 95% ethanol extracts from C. phaeocaulis gave the best result. Results showed that the inhibition rate of 95% ethanol extracts from C. phaeocaulis was 67.08% on P. wasabiae Yokogi at the concentration of 1.0 mg/ml. But the extraction rate of C.phaeocaulis by 95% ethanol was only 4.42%. The 95% ethanol extracts of C.phaeocaulis were extracted by skellysolve B, acetic ether and methanol step by step and the fungistasis were tested further. Acetone was used as the solvent for the extracts. The results showed that skellysolve B extracts was the active component and it had 79.9% inhibiting rate on P. wasabiae Yokogi at the concentration of 1.0mg/ml with EC₅₀ being 0.3299 mg/ml, and 97.2% inhibiting rate on spore germination of P.wasabiae Yokogi at the concentration of 10.0 mg/ml. The chromogenic result of different solvent extracts from C. phaeocaulis indicated that curcumine was in the acetic ether extracts with no fungistasis.The chemical components of skellysolve B extracts of C. phaeocaulis had been studied systemically by GC-MS analysis. One hundred and eight peaks appeared in GC. Forty compounds were identified and quantified. The main contents （%） of the skellysolve B extracts of C. phaeocaulis were based on the percent total peak area of each component peak. The major compounds were Aristolene （23.91%） and 1, 2-Dehydrotestosterone （23.63%）.In the antimicrobial spectrum test, the growth rate method was adopted to test the fungistasis on seven kinds of pathogenic fungi, including Fusarium graminearum, Sclerotinia sclerotiorum, Chaetomium olivaceum, Penicilium pallidum Smith, Mycogone peniciosa , Botrytis Cirerea, Verticillium dahliae Kled. The study showed that broad-spectrum antifungal active components were present in C. phaeocaulis. Mycelium growth declined when the concentration increased. The EC50 of extracts with skellysolve B of C. phaeocaulis to Fusarium graminearum, Sclerotinia sclerotiorum, Chaetomium olivaceum Cooket Ell, penicilium pallidum Smith, Mycogone peniciosa , Botrytis Cirerea, Verticillium dahliae Kled, were 0.2517 mg/ml, 0.2809 mg/ml, 0.8778 mg/ml, 0.2322 mg/ml, 0.7206 mg/ml, 0.1749 mg/ml, 0.4080 mg/ml, respectively. The skellysolve B extracts of C. phaeocaulis had 100% inhibition rate against Botrytis Cirerea at the concentration of 1.0 mg/ml. Its fungistasis on pathogenic fungi has potential pharmaceutical values, which provides experimental proof for the general application and research of C. phaeocaulis.The stability of skellysolve B extracts from C. phaeocaulis was studied by growth rate method against P. wasabiae Yokogi, Sclerotinia sclerotiorum, and Mycogone peniciosa . When the skellysolve B extracts was treated at 60℃for 10 minutes, or in pH 6.0～10.0, the fungistasis diminished distinctly, but the substance still had inhibitive effect on the pathogen. It is suggested that high temperature and alkalescence treatment should destroy the active components of skellysolve B extracts. Results indicated that the active compound was stable against UV but influenced by high temperature and alkalescence condition.After the treatment with skellysolve B extracts from C. phaeocaulis, the morphology and ultrastructure of P. wasabiae Yokogi and Sclerotinia sclerotiorum showed obvious changes, for example, their cell wall ruptured, intracellar components dissolved, mitochondria swollen and cristae dissolved. There was no change observed in morphology and ultrastructure of samples treated with water or acetone.更多还原