中国兰属植物菌根真菌的rDNA ITS分析

rDNA ITS analysis of mycorrhizal fungi of Cymbidium plants

【目的】对30株中国兰属植物菌根真菌进行分子鉴定及遗传多样性分析,进一步探讨中国兰属植物与其菌根真菌的专一性。【方法】以真菌通用引物ITS1/ITS4,扩增分离自云南6种兰属植物的30株菌根真菌的rD-NA ITS序列,再与GenBank中的菌株rDNA ITS序列进行Blast比对,并采用非加权组平均法(UPGMA)进行聚类分析。【结果】rDNA ITS序列Blast比对结果显示,分离的30株菌根真菌分别隶属于镰刀菌属、毛壳菌属、柱孢霉属和瘤菌根菌属,与形态学鉴定结果一致;菌根真菌ITS1-5.8S-ITS2序列总长429～563bp,GC含量为41.67%～58.06%,其中ITS1长92～259bp,GC含量为47.66%～58.62%;5.8S长155～158bp,GC含量为40.81%～52.05%;ITS2长146～192bp,GC含量为38.78%～55.56%;聚类分析显示,30株菌根真菌可分为4组,其中,分离自送春、蕙兰、建兰的菌根真菌皆属于镰刀菌属,聚在Ⅰ组上;分离自春剑的菌根真菌除CJ7单独聚在Ⅳ组外,其他均聚在Ⅰ组上;分离自莲瓣兰、春兰的菌根真菌分别聚在Ⅱ和Ⅲ与Ⅰ和Ⅱ2个分支上,分离自莲瓣兰的菌根真菌聚在Ⅱ、Ⅲ2个分支上。【结论】在供试的6种兰属植物中,春剑、春兰、莲瓣兰和蕙兰菌根真菌的遗传多样性较送春、建兰丰富;蕙兰、送春、建兰与菌根真菌的专一性较强,而春剑、春兰、莲瓣兰与菌根真菌的专一性相对较弱。

[Objective] The research was conducted to identify 30 mycorrhizal fungi associated with Cymbidium plants,analyze genetic diversity of them, and explore fungal specificity. [Method] rDNA ITS sequences from 30 mycorrhizal fungi of six Cymbidium plants amplified using the eukaryotic universal primer pair ITS1/ITS4 were compared with sequences in GenBank through a Blast research before being estimated by UPGMA. [Result] The Blast results of rDNA ITS sequences showed that these fungi belonged to the genus Fusarium, Chaetomium, Cylindrocarpon, and Epulorhiza, which was fully consistent with identification by morphological method, rDNA ITS sizes of 30 mycorrhizal fungi ranged from 429 to 563 with GC contents of 41.67%-58.06%. ITS1 were 92-259 bp with GC of 47.66%-58.62%,5.8S were 155-158 bp,with GC of 40.81%0-52. 05%,and ITS2 were 146-192 bp with GC of 38. 78%- 55.56%. The phylogenetical tree showed that 30 mycorrhizal fungi were divided into four groups,which of mycorrhizal fungi in C. cyperifolim, C. faberi, C. ensifolium belonged to a single clade of Fusarium. Mycorrhizal fungi associates of C. longibracteatum belonged to Clade I except CJ7,and mycorrhizal fungi isolated from C. goeringii and C. tortisepalum distributed in Clade I ,Clade Ⅰ and Clade Ⅱ ,Clade Ⅲ, respectively. [Conclusion] Mycorrhizal fungi in C. longibracteatum, C. goeringii , C. tortisepalum , and C. f aberi had greater genetic diversities than C. cyperi folim, C. ensi folium. C. faberi, C. cyperi folim, C. ensi folium had higher fungal specificities than C. longibracteatum, C. goeringii and C. tortisepalum.