LMGE, Laboratoire 'Microorganismes: Génome & Environnement', UMR CNRS 6023, Université Blaise Pascal (Clermont-Ferrand II), BP 80026, 63171 Aubière Cedex, France.
J Microbiol Methods. 2010 Apr;81(1):69-76. doi: 10.1016/j.mimet.2010.02.002. Epub 2010 Feb 11.
Recently, molecular environmental surveys of the eukaryotic microbial community in lakes have revealed a high diversity of sequences belonging to uncultured zoosporic fungi. Although they are known as saprobes and algal parasites in freshwater systems, zoosporic fungi have been neglected in microbial food web studies. Recently, it has been suggested that zoosporic fungi, via the consumption of their zoospores by zooplankters, could transfer energy from large inedible algae and particulate organic material to higher trophic levels. However, because of their small size and their lack of distinctive morphological features, traditional microscopy does not allow the detection of fungal zoospores in the field. Hence, quantitative data on fungal zoospores in natural environments is missing. We have developed a quantitative PCR (qPCR) assay for the quantification of fungal zoospores in lakes. Specific primers were designed and qPCR conditions were optimized using a range of target and non-target plasmids obtained from previous freshwater environmental 18S rDNA surveys. When optimal DNA extraction protocol and qPCR conditions were applied, the qPCR assay developed in this study demonstrated high specificity and sensitivity, with as low as 100 18S rDNA copies per reaction detected. Although the present work focuses on the design and optimization of a new qPCR assay, its application to natural samples indicated that qPCR offers a promising tool for quantitative assessment of fungal zoospores in natural environments. We conclude that this will contribute to a better understanding of the ecological significance of zoosporic fungi in microbial food webs of pelagic ecosystems.
最近，对湖泊真核微生物群落的分子环境调查揭示了大量属于未培养的游动孢子真菌的序列。尽管游动孢子真菌在淡水系统中被认为是腐生生物和藻类寄生虫，但它们在微生物食物网研究中被忽视了。最近，有人提出，游动孢子真菌通过被浮游动物消耗其游动孢子，可以将能量从大型不可食用的藻类和颗粒有机物质转移到更高的营养水平。然而，由于它们的体积小，而且缺乏独特的形态特征，传统的显微镜无法在现场检测到真菌的游动孢子。因此，自然环境中真菌游动孢子的定量数据是缺失的。我们开发了一种用于定量检测湖泊中真菌游动孢子的定量 PCR（qPCR）检测方法。使用从以前的淡水环境 18S rDNA 调查中获得的一系列靶标和非靶标质粒，设计了特异性引物并优化了 qPCR 条件。当应用最佳 DNA 提取方案和 qPCR 条件时，本研究中开发的 qPCR 检测方法表现出高特异性和灵敏度，检测到的最低反应 18S rDNA 拷贝数低至 100 个。尽管目前的工作重点是新 qPCR 检测方法的设计和优化，但它在自然样本中的应用表明 qPCR 是一种很有前途的工具，可用于定量评估自然环境中的真菌游动孢子。我们得出结论，这将有助于更好地理解游动孢子真菌在浮游生态系统微生物食物网中的生态意义。
