Growth characteristics and phylogenetic analysis of the isolate mycelium, Ophiocordyceps sinensis.

Ophiocordyceps sinensis is a highly valued medicinal fungus and has been used as a traditional Chinese medicine (TCM) to treat asthma and respiratory, kidney, and other diseases for centuries. Due to its medical benefit and scarcity of natural resource, the anamorph mycelium is considered to be a useful substitute for natural O. sinensis. Here, the isolate mycelium from the fruit body of natural O. sinensis was identified to be the anamorph of O. sinensis using single-gene (wc-1 gene) and multi-locus sequence typing (MLST) of 3 genes (ITS, nrSSU, and β-tubulin) based molecular phylogenetic analysis. Biomass assay showed that the log phase occurred in 30–50 days of culture in media. Lots of aerial mycelium was produced on the 60-day potato dextrose agar (PDA) culture and was found to be sticky into lumps with numerous conidia under a scanning electron microscope (SEM) and an optical microscopy. Conidia were polymorphic, measuring about 1.94–5.83 × 1.58–5.57 µm in size, and could be distinguished from other filamentous fungi. Besides, the number of blastospores increased in the 30–60 day potato dextrose broth (PD) liquid culture. No conidia or blastospores were observed in the early stages in PDA media. So the mycelium would initially grow in a polar growth pattern, followed by budding growth or/and conidiogenesis in media. Moreover, some interesting and significant observations revealed that conidial and hyphal fusion occurred, which would play important roles in nutrition utilization, reproduction, and hyphal growth in this isolate. Moreover, 12 sporulation-related genes were screened using our RNA-seqs of O. sinensis, and the mRNA levels were assayed between 45-day and 90-day culture using quantitative real-time PCR(qPCR). Four of them, including fbA, skn7, abaA, and serine/threonine-protein kinase MAK gene, had higher levels of expression in the 45-day culture than in the 90-day culture. Besides, nucleoside components were detected using high-performance liquid chromatography analysis (HPLC). Our findings would aid in understanding O. sinensis's asexual reproduction and hyphal growth in media, as well as the use of the anamorph mycelium as a substitute for natural O. sinensis. [ABSTRACT FROM AUTHOR]