Relative qPCR to quantify colonization of plant roots by arbuscular mycorrhizal fungi

Abstract

Abstract 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.

Plant phenotyping relevance

植物根のAMF菌根 colonization を定量する相対qPCR法を開発・検証し、顕微鏡法との比較で性能を評価しているため、植物状態の取得手法が中心である。

Code and data availability

The paper provides two paper-specific public assets: the authors' analysis code repository on GitHub (R/DADA2/qPCR analysis workflow) and raw amplicon sequencing data deposited in the European Nucleotide Archive under study accession PRJEB20127 (sample SAMEA103939171), which contains the qPCR amplicon sequences used to