丽江老君山地衣内生菌的分离和多样性研究
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摘要
地衣是由真菌和藻类或蓝细菌共生而成的宽生态幅生物体,能够适应多种极端的环境。地衣体中含有地衣酸、地衣多糖等丰富的次生代谢产物,并且大多为地衣所特有,具有多种重要的生物活性。地衣内生菌以地衣体为生境,生长于地衣体内部,表现出丰富的生物多样性,研究发现地衣内生菌同样能够产生具有重要活性的次生代谢产物。
本文以云南丽江老君山地衣为材料分离培养地衣内生菌,调查了该地区地衣内生菌的多样性,并对地衣内生菌的次生代谢产物进行了初步的研究。首次采用刮皮层法对老君山21个属的地衣进行分离,共获得263株内生菌菌株,地衣内生菌的分离率为9.80%。根据菌落的形态特征,筛选出了68个表型特征差异较大的菌株,对其进行分子鉴定。通过GenBank的相似性搜索和系统发育分析,确定了其中68株内生真菌全部属于子囊菌门,分别属于座囊菌纲(Dothideomycetes)的多腔菌目(Myriangiales)、格孢菌目(Pleosporales)、煤炱目(Capnodiales),散囊菌纲(Eurotiomycetes)的刺盾炱目(Chaetothyriales),锤舌菌纲(Leotiomycetes)的柔膜菌目(Helotiales),茶渍纲(Lecanoromycetes)的Agyriales、微孢衣目(Acarosporales),粪壳菌纲(Sordariomycetes)的Coniochaetales、肉座菌目(Hypocreales)、炭角菌目(Xylariales)、黑痣菌目(Phyllachorales)、假毛球壳目(Trichosphaeriales)等10个目,以及少数地位未知类群。这些内生菌与15个有性型属和6个无性型属及子囊菌门有丝分裂型的3个无性型属强烈相关,表明老君山地衣内生菌群落是一个分类地位跨度广泛的生物类群。Shannon多样性指数也显示了丽江老君山地衣中内生菌群落丰富的生物多样性。
首次从地衣中分离到5株大团囊虫草属的内生菌。目前,国内外还没有关于大团囊虫草属真菌具有大团囊菌和昆虫以外寄主的报道,显示了地衣内生菌的独特性。
对8株地衣内生菌菌株进行抗菌、抗肿瘤活性的筛选。不同地衣内生菌对白色念珠菌、革兰氏阳性菌、革兰氏阴性菌的抑制活性以及KB细胞株毒性不同,其中,5株菌株抗白色念珠菌的活性明显,3株菌株抗大肠杆菌的活性明显,3株菌株抗金黄色葡萄球菌的活性明显,4株菌株抗枯草芽孢杆菌的活性较强,8株菌株对绿脓杆菌均没有活性。在抗肿瘤活性的筛选中,5株菌株具有超过50%的抑制KB肿瘤细胞生长能力。
Lichen was recognized as a symbiosis between a fungus,the mycobiont, and a photosymthetic partner, the photobiont. Lichen is a kind of worldwide distribution orgnanism which able to live in many extreme environment conditions. A large amount of secondary metabolites with variety bio-activity were found in lichen and most of them are unique to lichen. Endolichenic fungi is a sort of fungi which inhabit in lichen thallus, reflecting the relatively high biodiversity. Recent years, a large amount of secondary metabolites with variety bio- activity were found in endolichenic fungi too.
In this paper we attempted to isolate and culticate endolichenic fungi from lichen thalli and investigated endolichenic fungi diversity in Mt. Laojunshan, Lijiang, Yunnan. Some preliminary studies in the secondary metabolites of endolichenic fungi were carried out.
The innovative method- cortex scrapping was firstly usd and 263 isolates were recovered from lichen thalli of 21 genera, the isolation rate was 9.80%. Based on the the anatomical and morphological characteristics of colony, 68 stains were screened out, by the phylogenetic analysis based on ITS nrDNA sequences, these fungal isolates were identified belong to Dothideomycetes, Eurotiomycetes, Leotiomycetes, Lecanoromycetes, Sordariomycetes and some incertae sedis groups respectively. These fungal isolates strongly associated with 15 teleomorphic genera 6 anamorphic genera and 3 anamorphic genera of mitosporic Ascomycota, it indicated that endolichenic fungi is a kind of worldwide distribution orgnanism and shannon-viener index showed considerable diversity in endolichenic fungi.
Until now, there are no reports on the new host of Elaphocordyceps. However, in our study, five isolates from lichen collected from Mt. Laojunshan belong to Elaphocordyceps, the five species of Elaphocordyceps were firstly detected within lichen thalli. This result suggests that lichen is possibly the new host of Elaphocordyceps and may provide unique ecological niches for them.
The antimicrobial activity and the cytotoxic activity of 8 isolated endolichenic fungi were tested preliminarly. The result showed that 5 strains had activities against Monilia albican, 3 strains had activities against Escherichia coli, 3 strains had activities against Staphylococcus aureus, 3 strains had activities against Bacillus subtilis and no strain had activities against Pseudomonas aeruginosa. In the cytotoxic activity test, 5 strains tested had the ability to inhibit the tumor cells for 50%.
本文以云南丽江老君山地衣为材料分离培养地衣内生菌,调查了该地区地衣内生菌的多样性,并对地衣内生菌的次生代谢产物进行了初步的研究。首次采用刮皮层法对老君山21个属的地衣进行分离,共获得263株内生菌菌株,地衣内生菌的分离率为9.80%。根据菌落的形态特征,筛选出了68个表型特征差异较大的菌株,对其进行分子鉴定。通过GenBank的相似性搜索和系统发育分析,确定了其中68株内生真菌全部属于子囊菌门,分别属于座囊菌纲(Dothideomycetes)的多腔菌目(Myriangiales)、格孢菌目(Pleosporales)、煤炱目(Capnodiales),散囊菌纲(Eurotiomycetes)的刺盾炱目(Chaetothyriales),锤舌菌纲(Leotiomycetes)的柔膜菌目(Helotiales),茶渍纲(Lecanoromycetes)的Agyriales、微孢衣目(Acarosporales),粪壳菌纲(Sordariomycetes)的Coniochaetales、肉座菌目(Hypocreales)、炭角菌目(Xylariales)、黑痣菌目(Phyllachorales)、假毛球壳目(Trichosphaeriales)等10个目,以及少数地位未知类群。这些内生菌与15个有性型属和6个无性型属及子囊菌门有丝分裂型的3个无性型属强烈相关,表明老君山地衣内生菌群落是一个分类地位跨度广泛的生物类群。Shannon多样性指数也显示了丽江老君山地衣中内生菌群落丰富的生物多样性。
首次从地衣中分离到5株大团囊虫草属的内生菌。目前,国内外还没有关于大团囊虫草属真菌具有大团囊菌和昆虫以外寄主的报道,显示了地衣内生菌的独特性。
对8株地衣内生菌菌株进行抗菌、抗肿瘤活性的筛选。不同地衣内生菌对白色念珠菌、革兰氏阳性菌、革兰氏阴性菌的抑制活性以及KB细胞株毒性不同,其中,5株菌株抗白色念珠菌的活性明显,3株菌株抗大肠杆菌的活性明显,3株菌株抗金黄色葡萄球菌的活性明显,4株菌株抗枯草芽孢杆菌的活性较强,8株菌株对绿脓杆菌均没有活性。在抗肿瘤活性的筛选中,5株菌株具有超过50%的抑制KB肿瘤细胞生长能力。
Lichen was recognized as a symbiosis between a fungus,the mycobiont, and a photosymthetic partner, the photobiont. Lichen is a kind of worldwide distribution orgnanism which able to live in many extreme environment conditions. A large amount of secondary metabolites with variety bio-activity were found in lichen and most of them are unique to lichen. Endolichenic fungi is a sort of fungi which inhabit in lichen thallus, reflecting the relatively high biodiversity. Recent years, a large amount of secondary metabolites with variety bio- activity were found in endolichenic fungi too.
In this paper we attempted to isolate and culticate endolichenic fungi from lichen thalli and investigated endolichenic fungi diversity in Mt. Laojunshan, Lijiang, Yunnan. Some preliminary studies in the secondary metabolites of endolichenic fungi were carried out.
The innovative method- cortex scrapping was firstly usd and 263 isolates were recovered from lichen thalli of 21 genera, the isolation rate was 9.80%. Based on the the anatomical and morphological characteristics of colony, 68 stains were screened out, by the phylogenetic analysis based on ITS nrDNA sequences, these fungal isolates were identified belong to Dothideomycetes, Eurotiomycetes, Leotiomycetes, Lecanoromycetes, Sordariomycetes and some incertae sedis groups respectively. These fungal isolates strongly associated with 15 teleomorphic genera 6 anamorphic genera and 3 anamorphic genera of mitosporic Ascomycota, it indicated that endolichenic fungi is a kind of worldwide distribution orgnanism and shannon-viener index showed considerable diversity in endolichenic fungi.
Until now, there are no reports on the new host of Elaphocordyceps. However, in our study, five isolates from lichen collected from Mt. Laojunshan belong to Elaphocordyceps, the five species of Elaphocordyceps were firstly detected within lichen thalli. This result suggests that lichen is possibly the new host of Elaphocordyceps and may provide unique ecological niches for them.
The antimicrobial activity and the cytotoxic activity of 8 isolated endolichenic fungi were tested preliminarly. The result showed that 5 strains had activities against Monilia albican, 3 strains had activities against Escherichia coli, 3 strains had activities against Staphylococcus aureus, 3 strains had activities against Bacillus subtilis and no strain had activities against Pseudomonas aeruginosa. In the cytotoxic activity test, 5 strains tested had the ability to inhibit the tumor cells for 50%.
引文
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