Molecularly Supported Isolation of Russula griseocarnosa Mycelium from Fruiting-Body Tissue

Russula griseocarnosa is an economically valuable ectomycorrhizal mushroom, but obtaining and maintaining its mycelium under axenic, host-free culture conditions remains challenging. In this study, fresh fruiting-body tissue was subjected to a tissue-cleaning and isolation procedure, followed by culture under different nutritional conditions. Mycelial growth emerging from the isolated tissue was subsequently evaluated by molecular identification using Oxford Nanopore Technologies (ONT)-based ITS amplicon sequencing. The sequencing analysis generated 307 raw reads, of which 217 high-quality reads were retained after quality filtering. Taxonomic classification using Kraken2 assigned 207 of the 217 clean reads (95.39%) to the genus Russula, indicating that Russula represented the dominant fungal component of the sequenced sample. A high-accuracy ITS consensus sequence was generated from the dominant sequence population and compared against the NCBI nucleotide (NT) database. The consensus sequence showed high similarity to multiple reference sequences of Russula griseocarnosa, including 99.718% identity to accession OM760493.1 and 99.444% identity to accession MN275663.1, both with E-values of 0.0. It also showed 98.613% identity to the R. griseocarnosa type-material sequence NR_158880.1. The independent sequencing report therefore identified the dominant fungal component as R. griseocarnosa. Preliminary culture observations further indicated that conventional media did not consistently support vigorous mycelial development, whereas selected carbon and nutritional supplements were associated with improved visible hyphal development under the tested conditions. These observations suggest that the nutritional requirements of R. griseocarnosa in host-free culture may differ substantially from those of commonly cultivated saprotrophic fungi. The present study provides molecularly supported evidence for the recovery of R. griseocarnosa-associated mycelial growth from fruiting-body tissue and provides preliminary observations relevant to its host-free cultivation. Further experiments involving repeated subculture, independent biological replicates, additional molecular verification, and longer-term culture stability will be necessary to determine whether a stable axenic culture can be reproducibly established.