摘要为了解三叶青块根内生真菌多样性，对采自浙江省丽水市庆元县山区野生种三叶青块根进行了初步的内生真菌分离培养和鉴定试验。首先通过石蜡切片技术制作了块根横切永久装片，观察了药用植物三叶青块根的显微结构，并采用PDA平板分离技术从中分离出内生真菌，再进一步结合菌落形态、质地、生长速度及ITS序列对分离得到的内生菌进行鉴定和分类。结果显示：三叶青块根中存在多种内生真菌，实验共分离到19株内生真菌，经过Blast核苷酸分析，归属于6个属，分别是镰刀菌属(Fusarium Link)、根盘菌属(Rhizopycnis sp。)、炭疽菌属(Colletotrichum)、柱孢属(Cylindrocarpon)、翅孢壳属(Emericellopsis)、青霉属(Penicillium)，其中镰刀菌属菌为优势菌。本研究为进一步研究三叶青内生真菌的生态和功能奠定了基础。76541

Abstract In order to understand the endophytic mycobiota in Tetrastigma hemsleyanum, Endophytic fungi were isolated from the wild medicinal plant Tetrastigma hemsleyanum, which were collected from Qinyuan of Lishui, and then identified with conventional method。 First, permanent slide of Tetrastigma hemsleyanum’s root tuber was made through the technology of paraffin section, then observed the microstructure, at the same time, the endophytic fungi were isolated by the PDA plate separation technology, and gave these endophytic fungis classification and identification by further combine the colony morphology，texture, growth rate and ITS sequences。 The results showed that it indicated that the entophytes in Tetrastigma hemsleyanum were persity, during the experiment, 19 strains endophytic fungi were isolated, after Blast nucleotide analysis, which belonged to 6 genera, Fusarium Link, Rhizopycnis sp。, Colletotrichum, Cylindrocarpon, Emericellopsis, Penicillium, and Fusarium Link was the dominant species in this plant。 This study established foundation for further research on ecology and function of the endophtic fungi from Tetrastigma hemsleyanum。

毕业论文关键词：三叶青；药用植物；内生真菌；分离鉴定

Key words:  Tetrastigma hemsleyanum;  medicinal plant;  endophytic fungi;  isolation and identification

目录

目录 3

引言 4

1 材料与方法 5

1。1  材料 5

1。2  三叶青块根石蜡切片的制作 7

1。3  内生真菌的分离和纯化 7

1。3。1 培养基及配方 7

1）马铃薯葡萄糖琼脂培养基（PDA） 7

2）马铃薯葡萄糖液体培养基（PD） 7

1。3。2 内生真菌的分离和纯化 7

1。3。3 保菌 7

1。4  内生真菌的鉴定 8

1。4。1 形态鉴定 8

1。4。2 分子鉴定 8

1）真菌总DNA的提取 8

2）ITS序列鉴定 9

2 结果与分析 9

2。1三叶青根菌结构 9

2。2 内生真菌的分离鉴定结果。 10

2。2。1 形态学鉴定 10

2。2。2 分子学鉴定 11

3  讨论 13

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