【作者】 高海燕；

【导师】 宋瑞清；

【作者基本信息】 东北林业大学 ， 森林保护学， 2009， 硕士

【摘要】 本文运用植物病理学、分子生物学,生物化学、有机化学等方法,对炭团菌菌丝体发酵液中抑菌活性物质对樟子松枯梢病菌的影响、抑菌活性成分分离纯化和结构鉴定、抑菌活性物质生态安全性检验、抑菌活性物质稳定性及炭团菌与樟子松枯梢病菌分类的分子生物学验证进行研究。（1）应用通用引物ITS1与ITS4从试验菌株中分别扩增出目的片段617bp和544bp,GenBank登陆号分别为EU744544和EU796887。通过GenBank中BLAST,确认试验菌株分别为Hypoxylon perforatum和Sphaeropsis sapinea。（2）炭团菌菌丝体发酵液中活性物质的提取采用水提和酯提2种方式。水提方式采用直接提取法和超声波提取法2种方法,酯提方式选择正丁醇、乙酸乙酯、乙醇3种萃取剂。各提取物对病原菌的抑菌活性研究选用菌丝生长抑制率和孢子萌发抑制率2个指标,对病原菌菌丝生长的抑制测定采用生长速率法,对病原菌孢子萌发的抑制试验采用悬滴法。结果表明炭团菌菌丝体发酵液5种提取物对樟子松枯梢病菌均有一定的抑菌活性,其中乙酸乙酯提取物对病原菌菌丝生长的抑制率最高,达到73.20%;乙酸乙酯提取物、超声波提取物、正丁醇提取物对病原菌孢子萌发抑制率均超过90%。（3）采用生物活性跟踪技术,运用柱层析、TLC等方法对发酵液提取物中的抑菌活性成分进行了分离、纯化。结果表明该提取物中有3个组分,组分Ⅰ的抑菌率最高（72.94%）。综合气质联用（GC-MS）的检测结果,组分Ⅰ含3种化合物,其中得率最高的为对甲氧基肉桂酸甲酯（57.952%）,其他两个化合物分别为邻苯二甲酸二异丁酯（得率13.592%）和邻苯二甲酸二丁酯（得率28.455%）。（4）炭团菌液体培养菌液乙酸乙酯提取物稳定性的研究选用紫外线、温度、pH值、氧化剂与还原剂以及贮存时间5个因子。乙酸乙酯提取物对自然环境下的紫外线、温度具有很高的稳定性,并且具有一定的抗氧化还原能力和良好的耐热性,长期贮存不影响其抑菌活性。（5）小鼠毒性试验中未发生小鼠死亡情况。炭团菌发酵液乙酸乙酯提取物对小鼠的半致死剂量(LD₅₀)＞13200mg/kg,按照我国化学物质的急性毒性分级标准,该提取物为微毒。更多还原

【Abstract】 The effects of inhibiting active substance extracted from culture liquid of Hypoxylon perforatum on Sphaeropsis sapinea;isolation,purification and structure identification of inhibiting active substance; ecological safety test and stability of inhibiting active substance;the rDNA ITS sequences of Hypoxylon perforatum and Sphaeropsis sapinea were studied by methods of Phytopathology, Molecular Biology,Biochemical and Organic Chemical.The results showed as follows:（1） rDNA ITS sequences were amplified from Hypoxylon perforatum and Sphaeropsis sapinea strain with the universal primers ITS1 and ITS4.rDNA ITS sequence lenth of Hypoxylon perforatum is 617bp and GenBank Accession is EU744544;rDNA ITS sequence lenth of Sphaeropsis sapinea is 544 bp and GenBank Accession is EU796887.Through the BLAST In GenBank,the strains were confirmed Hypoxylon perforatum and Sphaeropsis sapinea.（2） Methods of water-extract and ester-extract were used for extracting active substances from culture liquid of Hypoxylon perforatum.Method of water-extract included two ways,one is extracting directly from cultural liquid,and the others is extracting by ultrasonic.N-butyl alcohol, ethyl acetate and alcohol were used as extractant in ester-extract test.Growth-inhibiting rate and germination-inhibiting rate were used as the index of antifungal activity.Growth rate method was used for determining growth-inhibiting rate and pendent drop method for determining germination-inhibiting rate.The results is that five extracts from culture liquid of Hypoxylon perforatum all have definite antifungal activities to Sphaeropsis sapinea growth and germination.Ethyl acetate-extract is the best one on inhibiting Sphaeropsis sapinea growth,the growth-inhibiting rate is 73.20%;the germination-inhibiting rates of ethyl acetate-extract,ultrasonic-extract and n-butyl alcohol-extract are all over 90%.（3） The result of isolation,purification of inhibiting active substance shows that there are three components in ethyl acetate-extract.Component I has the highest inhibiting effect on Sphaeropsis sapinea growth,the growth-inhibiting rate is 72.94%.Component I has three compounds,one is Methoxy-cinnamic acid methyl ester whose yield is highest（57.952%）,the other two are iso-butyl phthalate（yield is 13.592%） and dibutyl phthalate（yield is 28.455%）.（4） Ultraviolet rays,temperature,pH,oxidants and reductant,storage time were selected as actors of the stability of ethyl acetate-extract.In the natural environment,ethyl acetate-extract has a high stability to ultraviolet rays and temperature,definite anti-oxidation and anti-reduction capacity, and good heat resistance.Long-term storage does not affect its antifungal activity.（5） In the oral acute toxic examination,the mice’death did not happen.The LD₅₀ of the extraction is less than13200mg/kg,which was micro-toxic.更多还原