Protocorm-Derived Fungus, Ceratobasidium sp., Significantly Enhances Seed Germination in Anoectochilus roxburghii (Wall.) Lindl. (Orchidaceae)

Orchid seed germination is heavily dependent on orchid mycorrhizal fungi (OMF) for nutrient acquisition in the field. Employing OMF to promote the germination and reproductive success of orchids is increasingly recognized as an effective conservation strategy. However, the success of this approach depends on identifying the most compatible fungal partners and integrating them properly into conservation programs. In this study, seeds of Anoectochilus roxburghii (Wall.) Lindl., a medicinal terrestrial orchid with Chinese national Level-II protected status, were co-cultured in vitro with 12 fungal strains from diverse sources to test seed preference to fungi and identify germination-promoting fungi. One strain (P2), isolated from host protocorms and identified as Ceratobasidium sp. based on rDNA-ITS phylogeny, showed the highest germination-promoting efficacy in in vitro symbiotic seed germination (SSG) experiments, yielding 41.09 ± 3.04% protocorm formation and 13.83 ± 3.15% seedling development at 60 days after sowing. Both values were significantly higher than those of other fungal treatments and the uninoculated control. Pilot trials of ex vitro and ex situ symbiotic seed germination demonstrated that strain P2 enhanced seedling development despite a low germination percentage caused by seed loss in artificial medium. These findings highlight the strong symbiotic preference of A. roxburghii seeds for strain P2 and demonstrate its potential as a valuable microbial resource for increasing seedling density in large-scale seedling propagation programs.