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

Extracting fungal hyphae with their natural 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. Previous methods, which involved supplementing soil substrates with sand or glass beads, have proven technically challenging for large-scale field applications and tend to create artificial conditions that disrupt the plant-microbe-soil continuum. In this study, we introduce a semi-automated approach for efficiently extracting fungal hyphae from a variety of soil types, including natural loamy soils. The Sieving and Sucrose Centrifugation (SSC) technique enables the enrichment of fungal hyphae, spores, and their surface-associated bacteria, with subsequent analysis of hyphal length density and the identification of tightly attached surface bacteria via next-generation sequencing (NGS). A comparison of different hyphal extraction techniques revealed that the SSC method yielded maximal hyphal length density. Furthermore, the SSC approach effectively enriched fungal hyphae from a highly diverse community, establishing a dependable method for advancing soil microbial research.