Objective The medium formulas and preparation methods currently adopted for the spawn production of Stropharia rugosoannulata are unreasonable, which frequently cause weak mycelial growth, slow or even stagnant mycelial growth in the middle and late stages of cultivation, and failure of mycelia to fully colonize spawn bags. This study aims to optimize the cultivation conditions for Stropharia rugosoannulata spawn.

Methods In this study, the carbon-nitrogen ratio of stock culture media was screened, the fermentation parameters of liquid spawn (rotational speed, mycelium dispersion treatment and nutrient supplementation) were optimized, and the adaptability of solid cultivation substrates was evaluated. The growth status of mycelia and mycelial pellets was measured.

Results The stock medium supplemented with trisodium phosphate (Na₃PO₄) and peptone could significantly boost the mycelial growth of specific strains such as DQG001, with a growth rate 18.7% higher than the control (CK). The suitable concentration range of jujube powder supplementation was 1–40 g/L. For liquid culture, the combination of mycelium fragmentation, 3% corn steep liquor and glass bead-assisted dispersion, followed by static culture for 48 h and shaking culture at 140 r/min, remarkably increased the biomass of mycelial pellets. The cultivation substrate mixed with corncob and licorice residue at a ratio of 1∶1.7 accelerated mycelial growth, shortened the spawn cultivation cycle to 20–30 d, and greatly reduced contamination rate.

Conclusion Precise regulation of carbon and nitrogen sources, optimization of liquid fermentation conditions and matching of physically and chemically suitable cultivation substrates can markedly improve the spawn production efficiency of Stropharia rugosoannulata. This research provides key technical support for large-scale production of Stropharia rugosoannulata.