A novel qPCR approach reveals distinctions in abundance of arbuscular mycorrhizal fungi in roots of Zea mays L.

Julia Schneider1, Nicolas Tyborski1, Benedikt Bartel1, Bastian Fichtl2, Andreas J. Wild2, Tina Koehler3, Franziska Steiner4, Shu-Yin Tung5, Johanna Pausch2, Tillmann Lueders1
1 Ecological Microbiology, Bayreuth Center of Ecology and Environmental Research (BayCEER), University of Bayreuth, Bayreuth, Germany
2 Agroecology, Bayreuth Center of Ecology and Environmental Research (BayCEER), University of Bayreuth, Bayreuth, Germany
3 Soil Physics, Bayreuth Center of Ecology and Environmental Research (BayCEER), University of Bayreuth, Bayreuth, Germany and Physics of Soils and Terrestrial Ecosystems, Department of Environmental Systems Science, ETH Zürich, Zürich, Switzerland
4 Soil Science, Technical University of Munich, Freising, Germany
5 Institute for Agroecology and Organic Farming Management, Bavarian State Research Center for Agriculture (LfL), Freising, Germany and School of Life Sciences, Technical University of Munich, Freising, Germany

P 3.10 in Zooming in: Small scale findings

Mutualistic interaction with arbuscular mycorrhizal fungi (AMF) is key for nutrient and water supply of most plant species. Reproducible and precise quantification of root colonization by AMF is fundamental for understanding their effect on host plants and to investigate factors impacting AMF. Conventionally, AMF abundance is assessed by microscopy-based methods which are time-consuming and prone to observer bias. Here, we present a genomic approach utilizing quantitative polymerase chain reaction (qPCR) to measure abundance of a wide taxonomic range of AMF in root tissue of maize (Zea mays L.).

Roots of twelve maize varieties grown in different settings and displaying a wide range of mycorrhization rates were used for establishment and validation of the qPCR protocol. Axenically grown plants and a genotype incapable of establishing symbiosis with AMF were used to test the behavior of the reaction in absence of AMF. Amplicons were sequenced via the Illumina platform to test specificity of the assay. Taxonomic classification was resolved by long-read amplicon sequencing via the Nanopore platform. Results were compared with counts obtained by microscopy.

In all colonized samples, amplicons generated by qPCR were exclusively assigned to taxa belonging to AMF, proving high specificity of the approach. Non-target amplicons observed for control samples in late PCR cycles were easily distinguishable by melt curve analysis. Amplicon sequencing showed dominance of multiple AMF taxa (Funneliformis, Rhizophagus, Dominikia) in our samples. AMF abundance ranged from low to high mycorrhization. This suggests general applicability of the approach for different AMF taxa and for a wide range of mycorrhization rates. From this, we conclude that qPCR offers a viable alternative to traditional microscopy-based methods for quantification of AMF, with better reproducibility, avoidance of observer bias and higher throughput, especially when DNA extracts are available from other assays.

Keywords: AMF abundance, mycorrhiza, qPCR, metabarcoding, maize
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