【作者】 孙剑秋；

【导师】 迟德富； 郭良栋；

【作者基本信息】 东北林业大学 ， 森林保护， 2007， 博士

【摘要】 药用植物中含有一些重要的化学成分，某些成分对人体疾患及动物病害具有保健和治疗作用，在农业生产上具有杀虫、杀菌和除草等功效。内生真菌是指在生活史中的某一个阶段存在于健康植物组织内部，不会引起宿主明显病症或者对宿主造成明显伤害的真菌，是生物多样性的重要组成部分。由于内生真菌的次级代谢产物中含有与药用植物相同或相似的生物活性物质，所以研究药用植物内生真菌多样性及其生态分布规律，大量收集内生真菌资源，具有重要的生态学意义和潜在的经济价值。本篇论文在北京市中国科学院植物研究所植物园、辽宁省清原满族自治县英额门镇椽子沟村、吉林省抚松县松郊乡马路村和黑龙江省五大连池风景区等采样地点，选取我国北方地区15科18种药用植物作为研究对象。研究的药用植物包括杜仲科的杜仲，木犀科的连翘、卵叶连翘、秦岭连翘，漆树科的青麸杨，小檗科的细叶小檗，芸香科的白藓，菊科的苍术，薯蓣科的穿龙薯蓣，毛莨科的黄花乌头，龙胆科的龙胆草，马兜铃科的细辛，百合科的玉竹，伞形科的柴胡，唇形科的藿香，五加科的人参、刺五加，木兰科的五味子。药用植物内生真菌具有丰富的多样性。从15科18种药用植物的3199个表面消毒的组织块中，分离获得2618株内生真菌。根据形态特征，将能够产生孢子的内生真菌鉴定为34属68个分类单元，包括28种腔孢菌、12种子囊菌、26种丝孢菌、1种接合菌、1种酵母菌；其中有5种我国新记录种，即Sporormiella intermedia(Auersw.)Ahmed & Cain、Sporormiella minima (Auersw.) Ahmed & Cain, Phoma jolyana Pirozynski & Morgan-Jones, Microsphaeropsis conielloides Sutton、Drechslera halodes (Drechslera) Subram. & Jain。对北京的杜仲、连翘、卵叶连翘、秦岭连翘、青麸杨、细叶小檗6种药用植物内生真菌比较分析后发现，细叶小檗的内生真菌定植率最高(63.1％)，其余依次是卵叶连翘(60％)、杜仲(54.8％)、连翘(54.4％)、秦岭连翘(51.3％)、青麸杨(47.9％)；内生真菌分离率最高的是连翘(0.93)，其余依次是细叶小檗(0.92)、杜仲(0.87)、青麸杨(0.83)、卵叶连翘(0.82)、秦岭连翘(0.7)。Alternaria alternata是6种药用植物内生真菌的优势种。6种药用植物内生真菌的总体多样性指数不同，即秦岭连翘内生真菌的多样性指数为1.6、青麸杨为1.46、卵叶连翘为1.42、杜仲为1.28、连翘为1.21、细叶小檗为0.98；内生真菌组成的相似性系数在0.35-0.80的范围内，其中连翘与青麸杨间内生真菌的相似性系数最低，而连翘与卵叶连翘间内生真菌的相似性系数最高，漆树科的青麸杨与其他3科5种药用植物内生真菌的相似性系数相对较低，但木樨科的连翘、秦岭连翘、卵叶连翘之间内生真菌的相似性系数很高。以内生真菌的种类组成及其定植率为参数进行聚类分析，分析结果表明内生真菌在植物中的存在受到宿主种类的影响而且不同植物组织影响内生真菌的种类组成、数量和分布，内生真菌在一定程度上表现出宿主专一性和组织专一性。对东北地区的人参、刺五加、五味子3种药用植物内生真菌比较分析后发现，刺五加内生真菌定植率(84.3％)和分离率(0.93)最高，而五味子和人参的内生真菌定植率(58.9％和61.3％)和分离率(0.66和0.65)比较相近。根据内生真菌的相对频率，Phomopsissp.是五味子、刺五加内生真菌的优势种，而人参优势内生真菌为Nodulisporium sp.。3种药用植物内生真菌组成的相似性系数在0.18到0.22的范围内，刺五加与人参内生真菌组成的相似性系数最高；五味子与刺五加和人参的相似性系数相对较低。3种药用植物的总体多样性指数不同，五味子内生真菌多样性最丰富。另外，五味子和刺五加枝条中内生真菌的多样性指数高于叶片，而人参植株中茎与叶片和根的内生真菌多样性指数相比最高。杜仲、连翘、卵叶连翘、秦岭连翘、青麸杨、细叶小檗、五味子和刺五加等药用植物叶片内生真菌的定植率明显低于枝条，但是人参植株中，叶片内生真菌的定植率和分离率比茎、根高得多，表现出与木本植物不同的分布规律。杜仲、连翘、卵叶连翘、秦岭连翘、青麸杨、细叶小檗、五味子和刺五加的1年生、2年生、3年生枝条内生真菌定植率和分离率逐渐增加，表现出植物内生真菌定植率和分离率随植物组织年龄的增长而逐渐增加的生态分布规律。总之，北方地区药用植物资源丰富，全面调查该地区药用植物组织中的内生真菌，可以为通过发酵的方法生产有益生物活性物质提供大量真菌资源，进一步探索植物内生真菌与宿主植物之间的生态关系，对于药用植物的保护、开发和利用具有重要意义。更多还原

【Abstract】 There were some important components in medicinal plants, and some had an effect ofhealth protection and treatment on human and animals diseases, at the same time had an effectto kill insects, germs and grasses in agricultural production. Endophytic fungi were organismsinhabiting plant organs that at some time in their life can colonize internal plant tissues withoutcausing apparent harm to the host and were important components of biodiversity. Becausethere were the same or similar active substances in metabolites of endophytic fungi as those inmedicinal plants, so it had an importantly ecological significance and potential economicvalure to study the diversity and ecological distribution of endophytic fungi in medicinal plantsand to collect endophytic fungi resources.In the paper, samples were collected in Beijing Botanical Garden located in BotanyInstitute of the Chinese Academy of Sciences, Chuanzigou Village of Yingemen Town ofQingyuan County in Liaoning Province, Malu Village of Songjiao town of Fusong County inJilin Province and Wudalianchi Beauty Spot in Heilongjiang Province. The 18 species in 15families medicinal plants in the North were selected as research objects, such as Eucommiaulmoides Oliv in Eucommiaceae, Forsythia suspense (Thunb.) Vahl, F. ovata Nakai and F.giraldiana Lingelsh in Oleaceae, Rhus potanini Maxim in Anacardiaceae, Berberis poiretiiSchneid in Berberidaceae, Dictamnus dasycarpu Turcz. in Rutaceae, Atractylodes chinensisKoidz. in Compositae, Dioscorea fipponica Makino in Dioscoreaceae, Aconitum coreanum(Levl.) Rapalcs in Ranunculaceae, Gentiana scabra Bunge in Gentianaceae, Asarumheterotropoides Fr. in Aristolochiaceae, Polygonatum odoratum (Mill.) Druce in Liliaceae,Bupleurum chinense DC. in Umbelliferae, Agastache rugosa (Fisch. et Mey.) O. Ktze. inLabiatae, Panax ginseng C. A. Meyer and Acanthopanax senticosus (Rupr.et Maxim.) Harms.in Araliaceae, Schisandra chinensis (Turcz.) Baill in Magnoliaceae.Endophytic fungi of medicinal plants had abundantdiversity. A total of 2618 isolates ofendophytic fungi were recovered from 3199 segments and discs of the 18 species of medicinalplants in 15 families plants, of those isolates which could produce spores were identified to 68taxa under 34 genera by morphological characteristics. Among the 68 taxa, 28 taxa belonged toCeolomycetes, 12 to Ascomycetes, 26 to Hyphomycetes, 1 to Zygomycete and 1 to yeast. The5 species were found to be new records in China, viz. Sporormiella intermedia (Auersw.)Ahmed & Cain, Sporormiella minima (Auersw.) Ahmed & Cain, Phoma jolyana Pirozynski& Morgan-Jones, Microsphaeropsis conielloides Sutton, Drechslera halodes (Drechslera)Subram. & Jain。The endophytic fungi of Eucommia ulmoides, Forsythia suspense, F. ovata, F. giraldiana, Rhus potanini and Berberis poiretii in Beijing were analyzed, and were found that thecolonization rate of endophytic fungi in B. poiretii was the highest in the six species ofmedicinal plants(63.1％). In the other plants, the colonization rate from high to low wasForsythia ovata (60％), E. ulmoides (54.8％), F. suspense (54.4％), F. giraldiana (51.3％), R.potanini (47.9％). The isolation rate of endophytic fungi in Forsythia suspense (0.93) washighest, and in the other plants was Berberis poiretii (0.92), E. ulmoides (0.87), R. Potanini(0.83), Forsythia ovata (0.82) and F. giraldiana (0.7) from high to low. Alternaria alternatawas the most common species in E. ulmoides, F. suspense, F. giraldiana, F. ovata, B. poiretiiand R. potanini. The Shannon-Weiner diversity indices of endophytic fungi in six species ofmedicinal plants was from high to low F. giraldiana(1.6)＞R. potanini(1.46)＞F. ovata (1.42)＞E. ulmoides (1.28)＞F. suspense (1.21)＞B. poiretii (0.98). The Sorenson’s coefficientspectrum of endophytic fungi community was 0.35-0.80 among six species in four families ofplants in Beijing. The lowest similarity was between F. suspense and R. potanini, and thehighest similarity was between F. suspense and F. ovata. Lower similarities of endophyticfungi composition were found between Rhus potanini in Anacardiaceae and the other fivespecies in three families of plants, and higher similarities arrived at 0.71-0.80 among Forsythiasuspense, F. giraldiana and F. ovata in Oleaceae. The dendrogram was built by endophyticfungus composition and colonization rate. The results showed that the presence of endophyticfungus were affected by kinds of host plants and endophytic fungus composition, number anddistributing were affected by characteratics of plants tissues. At a certain extent, the endophyticfungi presented host and tissue specificity.The endophytic fungi in the three medicinal plants of Panax ginseng, Acanthopanaxsenticosus and Schisandra chinensis in the Northeast were analyzed, then the colonization(84.3％) and isolation (0.93) rate of endophytic fungi in Acanthopanax senticosus were foundto be the highest and the data between Panax ginseng and Schisandra chinensis were similar.On the basis of relative frequency, Phomopsis sp. was the most common species inAcanthopanax senticosus and Schisandra chinensis, and the most common species in Panaxginseng was Nodulisporium sp. The Sorenson’s coefficient spectrum of endophytic fungicommunity was 0.18-0.22 among three species. The highest similarity was between Schisandrachinensis and Panax ginseng, and lower similarities of endophytic fungus composition werefound between Schisandra chinensis and Acanthopanax senticosus, and at the same timebetween Schisandra chinensis and Panax ginseng. The Shannon-Weiner diversity indices ofendophytic fungi in three species of medicinal plants were different, and the species ofendophytic fungi in Schisandra chinensis were the most abundant. In addition, the Shannon-Weiner diversity indices of endophytic fungi in twigs of Schisandra. chinensis andAcanthopanax senticosus was higher than in their leaves, but the Shannon-Weiner diversity indices of endophytic fungi in stems of Panax ginseng was highest than in their leaves androots.The colonization rates of endophytes in Eucommia ulmoides, Forsythia suspense, F. ovata,F. giraldiana, Rhus potanini, Berberis poiretii, Acanthopanax senticosus and Schisandrachinensis were apparently lower in leaves than in twigs. The colonization and isolation rates ofendophytes in leaves of Panax ginseng was far more higher than in stems and roots. The resultsshowed the endophytes of Panax ginseng compared with those in other wood plants possesseddifferent distributing rule. Colonization and isolation rates of endophytes in Eucommiaulmoides, Forsythia suspense, F. ovata, F. giraldiana, Rhus potanini, Berberis poiretii,Acanthopanax senticosus and Schisandra chinensis had been found to increase with the age ofthe plant tissues in hosts and the distributing trend of increasing infection with tissue aging wasmore validated.In conclusion, the results showed that abundant endophytic fungi were in medicinal plantsin the North. It was necessary to make a comprehensive investigation in order to obtain largenumbers of endophytic fungi producing active substances, at same time to make clear theecological relation between hosts and endophytic fungi. The result would have an importantsignificance on protecting, exploiting and utilizing medicinal plants in North.更多还原