Streamlining the isolation of fungal hyphae: a semi-automated approach for soil substrates.

Extracting fungal hyphae with their naturally associated microbiota from soil samples presents a significant challenge due to their small size, typically in the micrometer range, and the formation of dynamic fungal networks. We combined elements of previous protocols and automated the wet-sieving steps of the methodology to efficiently extract fungal hyphae from various soil types, including natural loamy soils. This approach reduces manual handling, minimizes operator-dependent variability, and shortens processing time by up to 2.5-fold. Unlike earlier methods that require sand or glass bead supplementation, which can introduce artificial conditions and limit large-scale field applications, our sieving and sucrose centrifugation (SSC) method avoids these drawbacks. The SSC technique both enables quantification of hyphal length density (HLD) and, importantly, preserves surface-associated microbes for downstream analyses. Among the tested methods, SSC yielded the highest HLD. Using a combination of microscopy, molecular techniques, and next-generation sequencing (NGS), we demonstrate that this method allows targeted study of bacteria tightly attached to fungal hyphae. Furthermore, the SSC approach effectively enriched fungal hyphae from a highly diverse soil community, establishing a dependable tool for advancing research on fungal hyphae as microbial hotspots in soil ecosystems.