Opening the black box: in situ imaging of arbuscular mycorrhizal fungal structures in soil using synchrotron-based micro-CT.

Arbuscular mycorrhizal fungi (AMF) contribute to plant nutrient and water uptake via their extraradical hyphal networks. However, in situ methodologies to quantify architectural and morphological traits of these networks in soil are largely lacking, limiting our understanding of AMF-mediated resource transport. Using synchrotron-based X-ray computed microtomography (micro-CT), we established a workflow to cultivate, noninvasively image, and quantitatively analyze AMF hyphosphere and rhizosphere structures in the interaggregate space across two soil textures and biological contexts. We developed a pipeline for quantitative three-dimensional (3D) assessment of key architectural and morphological traits including structure counts, hyphal length, branching frequency, volume, and surface area. Our method further permits (1) measurement of AMF-soil and AMF-root interface areas and (2) microscale quantification of pore space occupancy by AMF. Micro-CT offers a tool for noninvasively visualizing AMF in air-filled soil pore space. We outline how such quantitative 3D information can be incorporated into image-based and functional-structural soil-plant models, thereby supporting a better mechanistic understanding of AMF-mediated processes in soils and plants.