【作者】 傅科鹤；

【导师】 林福呈； 章初龙；

【作者基本信息】 浙江大学 ， 微生物学， 2005， 硕士

【摘要】 植物内生真菌(Endophytic fungi)是指在某一时期生活在植物体内,但对寄主植物组织并不引起明显病害症状的真菌。由于长期生长于植物内部,往往能产生许多具有与共生植物相似的活性代谢产物。植物内生真菌作为一种新的微生物资源引起了人们广泛的关注,从内生真菌中寻找和发现新的活性化合物,如抗肿瘤、抗真菌等活性化合物是植物内生菌研究的一个热点。 本文主要以红豆杉科植物(红豆杉、榧树)为研究对象,研究其内生真菌多样性,同时寻找具有抗植物病原菌的活性菌株。 主要研究结果如下: 1、从来源于江西与浙江两地的南方红豆杉、榧树的针叶、茎、树皮中分离得到507株内生真菌,其中有289株菌株分别鉴定为下列13个属:拟盘多毛孢(Pestalotiopsis sp.)55株(占总数10.8%)、拟茎点霉(Phomopsis sp.)43株(占总数8.5%)、镰孢霉(Fusarium sp.)43株(占总数8.5%)、链格孢(Alternaria sp.)34株(占总数6.7%)、炭疽菌(Colletotrichum sp.)30株(占总数5.9%)、木霉(Trichoderma sp.)20株(占总数3.9%)、茎点霉(phoma sp.)19株(占总数3.7%)、拟青霉(Paecilomyces sp.)15株(占总数3.0%)、炭角菌(Xylaria sp.)10株(占总数2.0%)、青霉(Penicillium sp.)9株(占总数1.8%)、曲霉(Aspergillus sp.)4株(占总数0.8%)、盘单毛孢(Monochaetia sp.)4株(占总数0.8%)、根霉(Rhizopus sp.)3株(占总数0.6%)。另有子囊菌(Ascomycete sp.)29株(占总数5.7%)。同时有189株(占总数37.3%)在培养中未见产孢,暂定为无孢菌群(Mycelia sterilia)。结果表明不同的植物来源、同一植物的不同部位其内生真菌的数量、种群分布等都有明显差异:①南方红豆杉中共分离到407株菌株(占总数的80.3%),优势菌群为拟盘多毛孢52株,其次为拟茎点霉36株,链格孢34株;而榧树中共分离到100株菌株(占总数的19.7%),优势菌群为镰孢霉22株,其次为拟茎点霉7株,拟盘多毛孢3株。②从两种植物的针叶中分离到144株菌株(占总数的28.4%),优势菌群为拟盘多毛孢15株(占总数10.4%),其次为炭疽菌14株(占总数9.7%),茎点霉10株(占6.9%);茎中共分离到310株(占总数的61.1%),优势菌群为拟盘多毛孢40株(占总数的12.9%),其次为拟茎点霉39株(占总数12.6%),镰孢霉34更多还原

【Abstract】 An endophytic fungus colonizes symptomlessly the living, internal tissues of the host plants. Many of endophytic fungi produce compounds with novel structure and specific bioactivity for their long period of living in host tissues. Furthermore, endophytic fungi could be a reliable source of materials of agriculture and pharmacy as exemplified by anti-tumor and anti-fungal agents.In this paper, the occurrence of endophytic fungi from Taxus mairei and Torreya grandis, members of the family Taxaceae, was analyzed to find out endophytes diversity in the host T. mairei and To. Grandis. Bioassays were carried out on the inhibitory activity of the endophytic fungi against phytopathogenic fungi as well in order to search for active strains.The results obtained were as following:Five hundred and seven strains of endophytic fungi were isolated from needle, stem and bark of T. mairei and To. grandis. Out of them, two hundred and eighty-nine strains were identified as thirteen generas: Pestalotiopsis sp.(about 10.8% of the total 507 isolates), Phomopsis sp.(about 8.5% of the total), Fusarium sp.(about 8.5% of the total), Alternaria sp.(about 6.7% of the total), Colletotrichum sp.(about 5.9% of the total), Trichoderma sp.(about 3.9% of the total), Phoma sp.(about 3.7% of the total), Paecilomyces sp.(about 3.0% of the total), Xylaria sp.(about 2.0% of the total), Penicillium sp.(about 1.8% of the total), Aspergillus sp.(about 0.8% of the total), Monochaetia sp.(about 0.8% of the total), Rhizopus sp.(about 0.6% of the total);while 29 strains were identified as Ascomycete sp.(about 5.7% of the total) and another 189 strains (37.3% out of the total) were tentatively classified as Mycelia sterilia, with no spores observed during culture. It has showed that the diversity of the endophytic fungi varied in different plants as well as different tissues of the same plant. First, 407 endophytic fungi isolated from T. mairei were mostly composed of Pestalotiopsis sp., Phomopsis sp. and Alternaria sp.. In contrast, 100 endophytic fungi strains isolatedfrom T. mairei were mostly composed of Fusarium sp., Phomopsis sp. and Pestalotiopsis sp.. Second, 144 strains (composed mostly of Pestalotiopsis sp. and Colletotrichum sp.), 310 strains (composed mostly of Pestalotiopsis sp., Phomopsis sp. and Fusarium sp.) and 53 strains (composed mostly of Fusarium sp., Trichoderma sp. and Paecilomyces sp.) were isolated from needle, stem and bark, respectively.Of the 507 strains, the bioactivity targeted against Pythium ultimum, Rhizoctonia solani and Botrytis cinerea was tested. The results revealed that crude fermentation broths of 36 strains (about 7.1%) of the total presented significant activity with ID50 beyond 10. Out of those strains, 34 strains came from T. mairei, composed mostly of Mycelia sterilia, Paecilomyces sp., Penicillium sp. and Trichoderma sp., while 2 strains from To. grandis.The strains F0028, which has high inhibitory rate against P. ultimum (ID50 of 278 ± 15.0) and R. solani (ID50 of 108 + 18.6) was identified as Aspergillus clavatonanicus. Its crude fermentation broths showed strong inhibitory activity at pH value range from 1.0 to 7.0 and retained certain inhibitory activity of 65% when autoclaved. It also has cytotoxic activity to the SMMC-7721, SPC-A1 and DU-145 by the MTT assay. Five compounds were isolated from the crude fermentation broths by HPLC. Bioassays were carried out on the inhibitory activity of the compounds against two pathogenic fungi. The results showed that all the five compounds have certain inhibitory activity with IC50 between 7.0 and 45 ug/ml. Then the cultural characteristics of F0028 was preliminarily studied. The results showed that the optimum condition for fermentation was 1 OOmL MID media with soytone in 250ml flasks (pH6.5) at 25 °C in still condition.更多还原