Keming Zhu

Flammulina filiformis is one of the most important commercially cultivated species, and the assessment of strain quality is of paramount importance. The analysis measured physiological-biochemical indicators, morphological indicators, combined non-targeted metabolomics, and microbiomics in F. filiformis strains of different ages. The results indicate that, as the culture age increased, ergosterol content and catalase (CAT) activity declined continuously. The morphology of the mycelia gradually deteriorated, and yield continued to decrease. The relative content of lipids and lipid-like molecules decreased overall, while the proportion of organic acids and their derivatives increased; the oxidative phosphorylation pathway continued to be significantly enriched. The richness and diversity of the mycelium-associated bacterial community in the mycelia decreased significantly, and the microbial community structure changed. The community was dominated by the genus Acinetobacter from 15 to 60 days, whereas the genus Serratia was predominant in 90 days group. Correlation analysis revealed that the core differential signature metabolites MG 14:1_0:0_0:0, aspartic acid-threonine-aspartamide, and nicotinamide adenine dinucleotide (NAD⁺) were significantly correlated with multiple physiological-biochemical indicators. N-arachidonyl glycine showed significant correlations with 21 core bacterial genera; the dominant genera Acinetobacter and Serratia exhibited strong correlations with membrane homeostasis, core indicators of oxidative damage, and signature metabolites. These results indicate that the mycelial vitality of F. filiformis remains good within the first 15 days of cultivation, with significant ageing occurring after 60 days. These findings provide a theoretical basis for optimizing fungal strain preservation techniques and regulating mycelial ageing.