Finding flies in the mushroom soup: host specificity of fungus-associated communities revisited with a novel molecular method

Fruiting bodies of fungi constitute an important resource for thousands of other taxa. The structure of these diverse assemblages has traditionally been studied with labour-intensive methods involving cultivation and morphology-based species identification, to which molecular information might offer convenient complements. To overcome challenges in DNA extraction and PCR associated with the complex chemical properties fruiting bodies, we developed a pipeline applicable for extracting amplifiable total DNA from soft fungal samples of any size. Our protocol purifies DNA in two sequential steps: (1) initial salt-isopropanol extraction of all nucleic acids in the sample is followed by (2) an extra clean-up step using solid-phase reversible immobilization (SPRI) magnetic beads. The protocol proved highly efficient, with practically all of our samples — regardless of biomass or other properties — being successfully PCR amplified using metabarcoding primers and subsequently sequenced. As a pro...