Revitalization of the Endophytic Fungus <i>Acremonium</i> sp. MEP2000 and Its Impact on the Growth and Accumulation of Bioactive Compounds in <i>Inonotus obliquus</i>

<i>Inonotus obliquus</i> is a large, medicinal and edible fungus known for its extremely slow growth. It has garnered significant attention due to its high socioeconomic and pharmaceutical value. Our research group previously isolated an endophytic fungus, <i>Acremonium</i> sp. MEP2000, from <i>I. obliquus</i>, and a fermentation broth of MEP2000 enhanced both the growth and bioactive compound accumulation in <i>I. obliquus</i>. However, the activity of MEP2000 gradually declined after successive subcultures on PDA medium. We therefore revitalized MEP2000 by supplementing the PDA medium with birch bark powder (H medium), <i>I. obliquus</i> fruiting body powder (Z medium), and a combination of both (H&Z medium). MEP2000 was cultured on the PDA medium as the unrejuvenated control (PDA medium). The rejuvenated MEP2000 broth exerted significant effects on the secondary metabolite production, colony morphology, and growth rate of <i>I. obliquus</i>, with the H&Z medium inducing the most uniform mycelial morphology and fastest growth. Similarly, liquid shake-flask fermentation experiments showed that the H&Z inducer had the strongest growth-promoting effect. <i>I. obliquus</i> intracellular polysaccharide content was also highest in the H&Z treatment, with polysaccharide synthesis-related gene expression following a similar trend. GC-MS analysis confirmed that the H&Z medium had the highest concentration of triterpenoid bioactive compounds, such as inotodiol. These findings suggest that the MEP2000 broth, revitalized using the H&Z medium, effectively promotes <i>I. obliquus</i> mycelial growth and bioactive compound production, providing a valuable reference for large-scale production of active endophytic fungi.