Department of Biology, McMaster University.
Department of Biology, McMaster University;
J Vis Exp. 2022 May 27(183). doi: 10.3791/63396.
Soil is host to an incredible amount of microbial life, with each gram containing up to billions of bacterial, archaeal, and fungal cells. Multicellular fungi such as molds and unicellular fungi, broadly defined as yeasts, fulfill essential roles in soil ecosystems as decomposers of organic material and as food sources for other soil dwellers. Fungal species diversity in soil is dependent on a multitude of climatic factors such as rainfall and temperature, as well as soil properties including organic matter, pH, and moisture. Lack of adequate environmental sampling, especially in regions of Asia, Africa, South America, and Central America, hinders the characterization of soil fungal communities and the discovery of novel species. We characterized soil fungal communities in nine countries across six continents using ~4,000 soil samples and a protocol developed in the laboratory for the isolation of yeasts and molds. This protocol begins with separate selective enrichment for yeasts and the medically relevant mold Aspergillus fumigatus, in liquid media while inhibiting bacterial growth. Resulting colonies are then transferred to solid media and further processed to obtain pure cultures, followed by downstream genetic characterization. Yeast species identity is established via sequencing of their internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene cluster, while global population structure of A. fumigatus is explored via microsatellite marker analysis. The protocol was successfully applied to isolate and characterize soil yeast and A. fumigatus populations in Cameroon, Canada, China, Costa Rica, Iceland, Peru, New Zealand, and Saudi Arabia. These findings revealed much-needed insights on global patterns in soil yeast diversity, as well as global population structure and antifungal resistance profiles of A. fumigatus. This paper presents the method of isolating both yeasts and A. fumigatus from international soil samples.
土壤是数量惊人的微生物生命的宿主，每克土壤中可能含有数亿个细菌、古菌和真菌细胞。多细胞真菌，如霉菌和单细胞真菌，广义上被定义为酵母，它们在土壤生态系统中作为有机物质的分解者和其他土壤生物的食物来源，发挥着至关重要的作用。土壤中真菌物种的多样性取决于多种气候因素，如降雨和温度，以及土壤特性，包括有机质、pH 值和湿度。缺乏足够的环境采样，特别是在亚洲、非洲、南美洲和中美洲地区，阻碍了对土壤真菌群落的特征描述和新物种的发现。我们使用约 4000 个土壤样本和在实验室中开发的用于分离酵母和霉菌的方案，对六大洲九个国家的土壤真菌群落进行了特征描述。该方案首先在液体培养基中分别对酵母和与医学相关的霉菌烟曲霉进行选择性富集，同时抑制细菌生长。然后将得到的菌落转移到固体培养基上，并进一步处理以获得纯培养物，然后进行下游遗传特征描述。酵母物种的身份是通过其核核糖体 RNA 基因簇的内部转录间隔区（ITS）区域的测序来确定的，而烟曲霉的全球种群结构则通过微卫星标记分析来探索。该方案成功地应用于从喀麦隆、加拿大、中国、哥斯达黎加、冰岛、秘鲁、新西兰和沙特阿拉伯的土壤中分离和鉴定土壤酵母和烟曲霉种群。这些发现为我们提供了有关土壤酵母多样性的全球模式、烟曲霉的全球种群结构和抗真菌耐药性特征的急需的见解。本文介绍了从国际土壤样本中分离酵母和烟曲霉的方法。
