Coprinellus sp. DJL-31 Promotes Both Seed Germination and Pseudobulb Growth in Cremastra appendiculata

Cremastra appendiculata is an endangered medicinal orchid with limited propagation, as its seeds depend on specific symbiotic fungi for germination, and its pseudobulbs have poor propagation efficiency. Pseudobulb-associated endophytes render ex situ cultivation of C. appendiculata more feasible than seed-associated mycorrhizal fungi, as the former are easier to culture under controlled conditions and thus better support the orchid's ex situ propagation. In this study, 35 fungal strains representing 15 genera from 12 families were isolated from C. appendiculata pseudobulbs. Six of these strains were evaluated for growth-promoting capacity, and assays on wheat and sorghum indicated five isolates demonstrated growth-promoting effects on model plants. When their specific symbiotic roles in C. appendiculata were examined, only Coprinellus sp. DJL-31 enhanced seed germination and pseudobulb development. With DJL-31, seed germination was ~85%, and protocorms formed within 45 days. DJL-31 advanced pseudobulb sprouting by 7-10 days and increased sprout number. Microscopy verified that DJL-31 colonized epidermal and cortical cells in protocorm, forming hyphal aggregates. The other four strains that promoted the growth of model crops had no significant effects on C. appendiculata. This is the first to reveal the growth-promoting potential of Coprinellus sp. DJL-31 during the "seed germination-pseudobulb growth" phase of C. appendiculata. These results provide a novel pseudobulb-based screening framework to support the ex situ propagation and conservation of C. appendiculata.