未培养真菌分离培养方法初探

据估计自然界中真菌有220万到380万种,目前已经描述的真菌仅约12万种,不超过总数的8%。大量基于高通量测序的研究显示,自然环境中蕴藏的真菌多样性可能远远超出我们的预估。然而基于传统的分离培养技术的研究中,大量真菌却因难以获得纯培养而未被认知。因此探索新的真菌分离技术有助于提高我们对自然界中真菌多样性的认识,并获得可供开发利用的全新生物遗传资源。本研究以淡水湖底泥为调查对象,从优化培养条件和原位培养两个方面探索未培养真菌的分离培养方法,并与传统培养方法及免培养的高通量测序结果比较,评估各方法的分离效果。结果显示,低温分离显著影响获得的真菌组成,有利于嗜冷真菌的获得;无论在4℃低温还是25℃常温条件下,在培养基中添加维生素都能显著提高分离获得的真菌多样性,在属级水平上提高比例分别高达207%和81%。相较于传统25℃稀释平板法,基于分离芯片技术的原位培养在分离纯化效率、未知真菌捕获率以及物种多样性和均匀度等方面具有显著优势,显示原位培养技术在未来真菌分离培养中可能具有极大应用前景。

Attempt to isolate hitherto uncultured fungi

Global species diversity of fungi has been estimated to be 2.2 million to 3.8 million species. Currently, only about 120 000 species were described, less than 8% of the total species number. A number of most recent studies based on high-throughput sequencing technologies have shown that the diversity of fungi in the natural environment may even largely exceed our previous estimates. However, in studies based on the traditional plating method, it is very hard to obtain the pure cultures of numerous fungal species. Therefore, exploring new fungal isolation methods is of great significance in improving our understanding on fungal diversity, as well as obtaining new biological resources for exploitation. In this study, we collected the sediment from a freshwater lake and isolated the samples through optimized culture conditions and in situ cultivation. The isolation efficiencies of these methods were evaluated by comparing the results obtained from traditional 25°C dilution plate and culture-independent metabarcoding sequencing method. Lower temperature was found to be very useful for capturing psychrophilic fungi and obviously affected composition of fungi captured. At both 4°C and 25°C, the addition of vitamins in the media significantly increased the diversities of isolated fungi, and the increment reached 207% and 81% at the generic level, respectively. In situ cultivation showed significant advantages in the efficiency of isolation, capture rate of unknown fungi, diversity and evenness of isolation, indicating that in situ cultivation method may have great potential in fungal isolation in future studies.