【作者】 杨国栋；

【导师】 宋晓平；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2009， 硕士

【摘要】 瑞香狼毒(Stellera Chamaejasme L)是一种在我国广泛分布的有毒植物。瑞香狼毒不仅可引起家畜中毒,而且还与牧草争肥、水和光照,从而抑制优良牧草生长,导致草地生态呈恶性循环,加速天然草原退化。目前草原瑞香狼毒的防除仍存在一定困难。内生真菌是指整个生活史或者生活史中的某一个阶段存在于植物组织内部,且不会引起宿主出现明显病症或者没有对宿主造成明显伤害的一类真菌。现有研究表明,有的内生真菌自身可分泌多种入侵、定植到宿主植物内部和降解宿主植物成分所需的多种酶类,且有的内生真菌还亲自参与宿主植物成分的降解。为了给综合防治瑞香狼毒提供新的思路和基础资料,本试验从瑞香狼毒茎叶中分离瑞香狼毒内生真菌,并进行了瑞香狼毒内生真菌降解宿主成分的研究,取得了以下结果:1.瑞香狼毒化学成分预试验及糖类成分检测:采用系统预试验方法对瑞香狼毒茎叶和根的化学成分进行检测,结果表明,瑞香狼毒根部主要含有黄酮类、甾体、萜类、有机酸、挥发油、内酯香豆素、糖及其苷类化合物;可能含有皂苷、强心苷;不含蒽醌、生物碱。瑞香狼毒茎叶主要含有黄酮类、甾体、萜类、有机酸、挥发油、蒽醌、内酯香豆素、糖及其苷类;可能含有皂苷、强心苷、蛋白质;不含生物碱。采用薄层色谱法,以V(氯仿):V(冰醋酸):V(水)=18:21:3为展开剂,苯胺-二苯胺磷酸丙酮溶液为显色剂,对瑞香狼毒茎叶中的可溶性糖进行定性检测。采用苯酚-硫酸比色法,以葡萄糖标准液制作标准曲线,对瑞香狼毒茎叶中可溶性糖和总糖含量进行测定,结果表明瑞香狼毒茎叶中的可溶性糖主要为葡萄糖和蔗糖,茎叶中可溶性糖和总糖含量分别为34.2 g/kg和77.8 g/kg。2瑞香狼毒内生真菌的分离与鉴定:采用组织块培养法对瑞香狼毒茎、叶、花、根中的内生真菌进行分离培养,共分离到内生真菌178株,其中叶部91株,茎部59株,根部17株,花11株。根据菌株的菌落形态、大小、颜色、生长速率、质地、培养基颜色变化以及菌丝体和孢子的形态特征对其中分离的68株内生真菌进行鉴定。结果表明,藻菌纲毛霉目毛霉科根霉属黑根霉(Rhizopus nigricans Ehrenb)1株。半知菌纲丛梗孢目丛梗孢科的青霉属(Penicillium link)6株,曲霉属(Aspergillus link)4株,头孢霉属(Cephalosporium Corda)1株,丛梗孢属(Monilia Pers.)2株;半知菌纲丛梗孢目暗梗孢科的交链孢属(Alternaria Nees ex Wallr.)36株,绕顶霉属(Acrospeira B. et Br.)1株;半知菌纲丛梗孢目瘤座孢科的镰孢属(Fusarium Lk..ex Fr.)2株,枝孢霉属(Ramulispora Miura)1株;半知菌纲无孢目无孢菌群的丝核菌属(Rhizoctonia DC. Ex Fr.)1株;另有未产孢内生真菌13株。3.降解瑞香狼毒化学成分的内生真菌的筛选:以瑞香狼毒茎叶乙醇提取物为碳源制作筛选培养基,以能否在筛选培养基上生长为判定标准,对分离到的内生真菌进行初步筛选,共筛选到能在筛选培养基上生长的内生真菌17株。用D101大孔吸附树脂对瑞香狼毒茎叶醇提取物进行了处理,除去了其中的可溶性糖类成分,制备得到醇洗部分(瑞香狼毒精提物,JSW)。以JSW为唯一碳源制作复选培养基,对初选的内生真菌进行进一步筛选,共筛选出5株对瑞香狼毒化学成分有降解作用的内生真菌。经鉴定分别为交链孢属2株,无孢目1株,曲霉属1株,枝孢霉属1株。4.内生真菌降解瑞香狼毒成分降解率的测定:通过对试验组和对照组瑞香狼毒内生真菌曲霉属C1-Y7-4株培养液中黄酮类和香豆素类化合物含量的测定,发现试验组液体培养基中的黄酮类和香豆素类化合物含量均比对照组的低,且试验组和对照组间差异极显著。表明瑞香狼毒内生真菌曲霉属C1-Y7-4在生长过程中对黄酮类化合物和香豆素类化合物均有降解作用,在试验条件下其降解率分别为27.54%和26.70%。本试验首次发现瑞香狼毒内生真菌对原植物成分具有降解作用。更多还原

【Abstract】 Stellera Chamaejasme L is a poisonous plant, which is widely distributed in China. It not only can cause livestock poisoning, but also compete with grass for fertilizer,water and light, thus inhibiting the growth of forage grass, resulting a vicious cycle of grassland ecosystem to accelerate the deterioration of natural grassland. At present, it is still difficult to control Stellera chamaejasme L in the grassland. Endophytic fungi are fungi which spend the whole or part of their life cycle inside the healthy plant tissues without any discernible infectious symptoms. Some existing studies have shown that endophytic fungi can secrete a variety of enzymes which is necessary for it to invade, colonization within the hose plants. Even endophytic fungi act a part in the degradation of the host plant’s components. In order to provide new ideas and basic data for the integrated control of Stellera Chamaejasme L, in this study, we isolate, identify the endophytic fungi of Stellera Chamaejasme L, and research the degradation of the host plant’s components by its endophytic fungi.The results are as follows.1. The chemical composition pre-tests of Stellera chamaejasme L and the determination of its saccharide compositions. The systematic preliminary tests of Stellera chamaejasme L’s leaf, stem and root had been carried out. The results showed that the root of Stellera chamaejasme L contained flavonoids, steroid, terpene, organic acids, volatile oil, lactone coumarin, sugar and its glycosides. In addition, the root might contain saponins, cardiac glycosides, and did not contain alkaloids, anthraquinone; The mixture of leaf and stem of Stellera chamaejasme L contains flavonoids, steroid, terpene, organic acids, volatile oil, anthraquinone, lactone coumarin, sugar and its glycosides. In addition, it might contain saponins, cardiac glycosides, protein, and non-alkaloids. The soluble sugar was detected by thin layer chromatography (TLC).The plate was developed with chloroform-glacial acetic acid-distilled water(V:V:V=18:21:3). The spots were located by exposing the plate to the solution of aniline, diphenylamine, phosphoric acid and acetone. The content of sugar was determined with phenol-sulfuric acid method and by reference to glucose. The main compositions of soluble sugar in the leaf and stem of Stellera chamaejasme L were glucose and sucrose,and the contents of soluble sugar and total sugar in the leaf and stem of Stellera chamaejasme L were 34.2 g/kg and 77.8 g/kg, respectively.2. Isolation and identification of the endophytic fungi of Stellera chamaejasme L. The 178 strains of endophytic fungi were isolated from the stem, leaf, flower and root of Stellera chamaejasme L with the surface sterilization method. There are 91 strains in the leaf, 59 strains in the stem, 17 strains in the root and 11 strains in the flower. The endophytic fungi were identified according to the characteristic of their colony’s configuration, size, color, growth speed, texture, color changing of medium, the microphological characteristics of mycelium and spore. There were 1 strain in genera of Rhizopus Ehrenberg, 6 strains in gerera of Ramulispora Miura, 4 strains in gerera of Aspergillus link, 1 strain in gerera of Cephalosporium Corda, 2 strains in gerera of Monilia Pers),36 strains in gerera of Alternaria Nees ex Wallr,1 strain in gerera of Acrospeira B. et Br, 2 strains in gerera of Fusarium Lk..ex Fr,1 strain in gerera of Ramulispora Miura, and 1 strain in gerera in Rhizoctonia DC. Ex Fr, and there were 13 strains of endophytic fungi without producing spore.3. Screening of endophytic fungi with biodegrading ability for the chemical constituents of Stellera chamaejasme L. The ethanol extract of the leaf and stem of Stellera chamaejasme L as carbon source was used to make medium to screen endophytic fungi, 17 strains of endophytic fungi which could grow on the primary selection medium were chosen. The ethanol extract of the leaf and stem of Stellera chamaejasme L was processed with D101 macroporous resin to remove the soluble carbohydrate composition. The ethanol eluting materials from D101 macroporous resin was marked as JSW. The 17 strains of the endophytic fungi were screened again by the reselection medium which was made of JSW. Finally, a total of 5 strains of the endophytic fungi which have the ability of biodegrading the chemical constituents of Stellera chamaejasme L were chosen. The 5 strains of the endophytic fungi belonged to 4 generas. There were 2 strains in gerera of Alternaria Nees ex Wallr,1 strain in gerera of Aspergillus link, 1 strain in gerera of Ramulispora Miura, and 1 strain in gerera in Rhizoctonia DC. Ex Fr.4. Determination of the degradation rate of chemical constituents in the leaf and stem of Stellera chamaejasme L by endophytic fungi. Determination of the content of flavonoids and coumarins in the liquid culture medium of test group and control group had been done with the standard curve method. The results proved that the strain C1-Y7-4 of endophytic fungus can degrade the flavonoids and coumarins component of Stellera chamaejasme L. Under this experimental condition, the degradation rates were 27.54% and 26.70%, respectively. It was the first time to find the degradation of Stellera chamaejasme L’s composition by its endophytic Fungi.更多还原