Abstract

Cordyceps militaris is a valuable medicinal fungus; however, large-scale cultivation is frequently constrained by strain degeneration during repeated subculturing. This study investigated the relationship between mating-type (MAT) locus imbalance and degeneration of C. militaris across seven consecutive subculture generations. Phenotypic characteristics, including mycelial growth, fruiting body development, and biomass, were evaluated alongside molecular analysis of MAT loci. Multiplex Polymerase Chain Reaction (PCR) combined with densitometric analysis was used to monitor changes in the semi-quantitative band-intensity pattern of MAT1-1-1, MAT1-1-2, and MAT1-2- 1, and sequence analysis was performed to detect potential mutations. Degenerative phenotypes became evident after the third generation and were accompanied by a progressive decline of MAT1-1 loci, which became undetectable from the fifth generation onward, while MAT1-2-1 was continuously detectable throughout cultivation. No sequence variations were detected in the analyzed MAT loci, suggesting that degeneration may be associated with changes in MAT locus balance rather than detectable mutations in these regions. Fruiting body formation was primarily observed in early generations with relatively comparable detection of MAT1-1 and MAT1-2 loci. Collectively, these results suggest that disruption of MAT balance may be associated with the degeneration process in C. militaris. Monitoring the MAT locus distribution may therefore provide a useful approach for the early detection of strain deterioration during prolonged cultivation.

Key words: Cordyceps militaris; degeneration; mating-type locus; MAT imbalance; serial subculturing; fruiting body development