Arbuscular mycorrhiza fungi (AMF) are beneficial soil fungi that can promote the growth of their host plants. Accurate quantification of AMF in plant roots is important because the level of colonization is often indicative of the activity of these fungi. Root colonization is traditionally measured with microscopy methods which visualize fungal structures inside roots. Microscopy methods are labor-intensive, and results depend on the observer. In this study, we present a relative qPCR method to quantify AMF in which we normalized the AMF qPCR signal relative to a plant gene. First, we validated the primer pair AMG1F and AM1 in silico, and we show that these primers cover most AMF species present in plant roots without amplifying host DNA. Next, we compared the relative qPCR method with traditional microscopy based on a greenhouse experiment with Petunia plants that ranged from very high to very low levels of AMF root colonization. Finally, by sequencing the qPCR amplicons with MiSeq, we experimentally confirmed that the primer pair excludes plant DNA while amplifying mostly AMF. Most importantly, our relative qPCR approach was capable of discriminating quantitative differences in AMF root colonization and it strongly correlated (Spearman Rho = 0.875) with quantifications by traditional microscopy. Finally, we provide a balanced discussion about the strengths and weaknesses of microscopy and qPCR methods. In conclusion, the tested approach of relative qPCR presents a reliable alternative method to quantify AMF root colonization that is less operator-dependent than traditional microscopy and offers scalability to high-throughput analyses.

丛枝菌根真菌（AMF）是有益的土壤真菌，可以促进宿主植物的生长。植物根部 AMF 的准确定量非常重要，因为定植水平通常可以表明这些真菌的活性。根部定植传统上是通过显微镜方法来测量的，该方法可以观察根部内部的真菌结构。显微镜方法是劳动密集型的，结果取决于观察者。在本研究中，我们提出了一种定量 AMF 的相对 qPCR 方法，其中我们将 AMF qPCR 信号相对于植物基因进行标准化。首先，我们在计算机上验证了引物对 AMG1F 和 AM1，结果表明这些引物覆盖了植物根部中存在的大多数 AMF 物种，而无需扩增宿主 DNA。接下来，我们基于矮牵牛植物的温室实验，将相对 qPCR 方法与传统显微镜法进行比较，其中 AMF 根定植水平从非常高到非常低。最后，通过使用 MiSeq 对 qPCR 扩增子进行测序，我们通过实验证实引物对排除了植物 DNA，同时扩增了大部分 AMF。最重要的是，我们的相对 qPCR 方法能够区分 AMF 根部定植的定量差异，并且与传统显微镜的定量密切相关 (Spearman Rho = 0.875)。最后，我们对显微镜和 qPCR 方法的优缺点进行了平衡的讨论。总之，经过测试的相对 qPCR 方法提供了一种可靠的替代方法来量化 AMF 根部定植，该方法比传统显微镜对操作员的依赖性较小，并且为高通量分析提供了可扩展性。