【作者】 胡克兴；

【导师】 郭顺星；

【作者基本信息】 中国协和医科大学 ， 生药学， 2008， 博士

【摘要】 石斛属植物为附生性兰科植物,多种具有药用价值。自然条件下所有石斛属植物均与内生真菌共生,内生真菌在石斛属植物整个生活史中扮演着重要角色。针对石斛属植物内生真菌的研究得到了大量学者的重视,但是当前国内外针对石斛属植物内生真菌多样性的研究少有报道。本研究以石斛属植物为研究对象,通过形态学和分子生物学鉴定对24种石斛属药用植物中分离到的542株内生真菌进行了多样性研究。鉴于大量内生真菌不产孢无法通过形态学鉴定的现实,我们首先对内生真菌产孢诱导的方法进行了筛选。本研究选用美登木、金线莲两种药用植物来源的74株常规条件下不产孢的内生真菌进行了产孢诱导实验。结果表明低温诱导对内生真菌产孢诱导效果较为理想,因而在我们对石斛属药用植物内生真菌不产孢菌株采用低温诱导法诱导产孢。在该实验过程中,我们从金线莲内生真菌中发现一个Hansfordia的新种,定名为Hansfordia pallens。542株石斛属药用植物内生真菌中249株可产孢,占总菌株数的46%。通过形态学观察249株石斛内生真菌鉴定到21个属,其中148株鉴定到种,分别属于51个种,其中发现3个中国新纪录种(Acremonium sordidulum,Periconiella cestri,Hyalodendron lignicola ).Alternaria,Cladosporium,Aureobasidium,Chaetophoma,Periconiella,Nigrospora,Hyalodendron,Thielavia,Pleurophragmium,Melanconium,Glomerularia,Sirodesmium,Ckaetomella,Streptomyces 14个属系首次从石斛属植物中发现。虽然我们对石斛属药用植物不产孢的内生真菌进行了产孢诱导,但仍有293株不产孢,无法通过传统形态学方法进行鉴定。对于不产孢的菌株,我们采用分子生物学手段来对其进行鉴定。根据菌落培养特征和显微观察特征,将不产孢菌株划分为30个形态型,从30个形态型中选取148个代表菌株进行分子生物学鉴定。对148株内生真菌的ITS片段进行PCR扩增测序,将获得的ITS序列与参考菌种一起进行系统发育分析,结合序列碱基相似性比对来确定石斛属药用植物内生真菌的分类地位。通过分子生物学鉴定,148株代表菌株中8株鉴定到种,分别属于8个不同种;128株鉴定到属,分别属于28个属;10株鉴定到科,分别属于5个科;2株放到目。形态学鉴定的28个属中,Arthrinium,Chaetomium,Colletotrichum,Cercophora,Phomopsis,Nectria,Cosmospora,Bionectria,Nemania,Pleospora,Leptosphaerulina,Lewia,Paraconiothyrium,Ampelomyces,Davidiella,Lasiodiplodia,Fusicoccum,Botryosphaeria,Pezicula,Hypocrea20个属系首次从石斛属植物中发现。形态学鉴定结果结合分子生物学鉴定结果表明,石斛属药用植物内生真菌种类非常丰富,不同种石斛属药用植物的内生真菌在数量上和种类上差别较大,多样性丰富程度不同。更多还原

【Abstract】 Dendrobium is an adnascent plant of the orchid family, which includes many species with medicinal value. Endophytic fungi can be found in all kinds of Dendrobium under natural conditions and it plays an important role in the life history of Dendrobium. More attentions have been paid to the endophytic fungi of Dendrobium. However, very few studies have investigated the diversity of endophytic fungi of Dendrobium. The diversity of 542 isolates of endophytic fungi from 24 species medicinal Dendrobium plants was systematically studied in this thesis.Some methods in promoting sporulation were investigated using 77 isolates of endophytic fungi in Maytenus and Anoectochilus for the research material. Low temperature treatment was found to be the best method for promoting sporulation. So this method was used to promote sporulation of isolates in Dendrobium. A new species, Hansfordia pallens, isolated from living leaves of Anoectochilus roxburghii was found in this work.A total of 249 isolates sporulated of medicinal Dendrobium were identified into 21 genus based on morphological characteristics. 148 isolates were identified to species, and they were subordinated to 51 species including 3 new records Acremonium sordidulum, Periconiella cestri, Hyalodendron lignicola. Among them, 14 genus were found in Dendrobium for the first time, Alternaria, Cladosporium, Aureobasidium, Chaetophoma, Periconiella, Nigrospora, Hyalodendron, Thielavial, Pleurophragmium, Melanconium, Glomerularia, Sirodesmium, Chaetomella, Streptomyces in Dendrobium. Fusarium and Acremonium were found to be predominance populations of Dendrobium endophtic fungi.Approach 54% isolates could not produce any spore, although low temperature treatment was done for promoting sporulation. These isolates were divided to 30 morphotypes based on similar cultural characters. Our method was to identify 148 strains of these morphotypes based on phylogenetic analysis of ITS and 5.8S gene sequences, as well as sequence similarity comparison. The results showed that 8 strains were identified to species level, 128 strains to 28 generic level, 10 strains to family level, and 2 strains to order level. Among them, 21 genus were found in Dendrobium for the first time, Arthrinium, Chaetomium, Colletotrichum, Cercophora, Phomopsis, Nectria, Cosmospora, Bionectria, Nemania, Pleospora, Leptosphaerulina, Lewia, Paraconiothyrium, Ampelomyces, Davidiella, Lasiodiplodia, Fusicoccum, Botryosphaeria, Pezicula, Hypocrea. The identification result based on both morphological and molecular characters shows that the kind and amount of endophytic fungi are different according to different species of plants in medicinal Dendrobium.更多还原