松花江底泥真菌的分离培养及分子鉴定

Isolation and Molecular Identification of Fungi from the Sediment of Songhua River,China

筛选出合适的培养基对底泥真菌进行计数,分析不同培养基的真菌数量与理化环境的差异性,并对典型真菌进行分离鉴定,探讨其解决环境问题的可能性。选用马铃薯葡萄糖琼脂(PDA)培养基、察氏琼脂(CA)培养基和沙保氏琼脂(SDA)培养基3种真菌培养基对底泥真菌进行分离培养,通过方差分析和主成分分析比较了各培养基之间可培养真菌数量的差别及其与环境因子的关系,并对代表性真菌分离纯化,进行真菌核糖体基因转录间隔区(ITS)序列的测序和进化树分析。在同一采样点之间,3种不同培养基的可培养真菌在数量上没有显著差别,而在环境因子的关联上,PDA培养基的可培养真菌数量与沉积物有机质浓度、硝氮、总氮显著相关,CA培养基的可培养真菌数量与硝氮显著相关,SDA培养基的可培养真菌数量与总氮相关,PDA培养基的可培养真菌数量与环境因子的相关性优于其他两种培养基;分离纯化得到20株代表性真菌,系统进化树表明所有的菌株能得到鉴定,实验结果显示ITS序列能很好地应用于真菌的鉴定及生态分析。

The current study aims to select proper media to determine the concentration of sediment fungi in the Songhua River, analyze the differences in environmental factors, identify the representative fungi, and explore their potential environmental applications. Different media [i.e. Potato Dextrose agar(PDA) , Czapek aga(r CA) and Sabouraud′s aga(r SDA) ] were used to determine the concentration of sediment fungi, and the results were compared using Analysis of Variance and Principal Component Analysis. The internal transcribed spacer (ITS) of isolated fungi was sequenced and a phylogenetic analysis was carried out. There were no significant differences among the fungal concentrations at the same sampling point when cultivated using three kinds of different media. However, there were clear differences in their correlation to environmental factors. The number of fungi cultivated on PDA medium exhibited a significant correlation with organic matter, nitrate nitrogen and total nitrogen, while the fungi cultivated on CA medium showed correlation only with nitrate nitrogen. In contrast, fungi cultivated on SDA medium were significantly associated with the total nitrogen content. The number of fungi cultivated on PDA medium was more closely related with environmental variables than that cultivated on the other two media. Hence, PDA was a more suitable medium in the term of environmental monitoring. 20 representative fungi were isolated and subsequent phylogenetic analysis showed that all of the strains could be identified. The results also demonstrated that the ITS region is a reliable element for fungal identification and ecological analysis.