Pleurotus ostreatus is one of the widely cultivated edible fungi across the world. Mycelial subculture is an indispensable part in the process of cultivation and production for all kinds of edible fungi. However, successive subcultures usually lead to strain degeneration. The degenerated strains usually have a decrease in stress resistance, yield, and an alteration in fruiting time, which will subsequently result in tremendous economic loss. Through proteomic analysis, we identified the differentially expressed proteins (DEPs) in the mycelium of Pleurotus ostreatus from different subcultured generations. We found that the DNA damage repair system, especially the double-strand breaks (DSBs), repairs via homologous recombination, was impaired in the subcultured mycelium, and gradual accumulation of the DSBs would lead to the strain degeneration after successive subculture. The TUNEL assay further confirmed our finding about the DNA breaks in the subcultured mycelium. Interestingly, the enzyme activity of laccase, carboxylic ester hydrolase, α-galactosidase, and catalase directly related to passage number could be used as the characteristic index for strain degeneration determination. Our results not only reveal for the first time at the molecular level that genomic instability is the cause of degeneration, but also provide an applicable approach for monitoring strain degeneration in process of edible fungi cultivation and production.

Cordycepin is the primary active compound of Cordyceps militaris. However, the definitive genetic mechanism governing cordycepin synthesis in fruiting body growth and development remains elusive, necessitating further investigation. This study consists of 64 C. militaris strains collected from northeast China. The high-yielding cordycepin strain CMS19 was selected for the analysis of cordycepin production and the genetic basis of cordycepin anabolism. First, the whole-genome sequencing of CMS19 yielded a final size of 30.96 Mb with 8 contigs and 9781 protein-coding genes. The genome component revealed the presence of four additional secondary metabolite gene clusters compared with other published genomes, suggesting the potential for the production of new natural products. The analyses of evolutionary and genetic differentiation revealed a close relationship between C. militaris and Beauveria bassiana. The population of strains distributed in northeast China exhibited the significant genetic variation. Finally, functional genes associated with cordycepin synthesis were identified using a combination of genomic and transcriptomic analyses. A large number of functional genes associated with energy and purine metabolism were significantly enriched, facilitating the reconstruction of a hypothetical cordycepin metabolic pathway. Therefore, our speculation of the cordycepin metabolism pathway involved 24 genes initiating from the glycolysis and pentose phosphate pathways, progressing through purine metabolism, and culminating in the core region of cordycepin synthesis. These findings could offer fundamental support for scientific utilizations of C. militaris germplasm resources and standardized cultivation for cordycepin production.