Identification of Bioactive Metabolites from Dust-like Seeds of Cremastra appendiculata via Metabolomics, UPLC-Q-TOF-MS/MS, and Molecular Networking

Orchid seeds are dust-like and lack endosperm, which limits their capacity to support germination using endogenous nutrient reserves. Successful germination therefore depends on the establishment of a compatible symbiotic association with germination-promoting orchid mycorrhizal fungi (OMF). In plant-root symbioses, host-derived small molecules, such as strigolactones and flavonoids, function as early chemical signals that recruit microbial partners and stimulate their growth. However, the chemical constituents that may facilitate fungal recognition, growth, or colonization during orchid seed germination remain poorly understood. In this report, we combined metabolomics, UPLC-Q-TOF-MS/MS, molecular networking, phytochemical isolation, and bioactivity assays to characterize bioactive metabolites from the seeds of the medicinal orchid Cremastra appendiculata. Metabolomic profiling revealed abundant primary metabolites, including lipids, amino acids, organic acids, saccharides, and nucleosides. And then, nineteen secondary metabolites were isolated and identified, including two new structures. Functional assays showed that selected organic acids, saccharides, and lignanamides promoted the growth of Coprinellus disseminatus, a fungus required for seed germination, whereas lignanamides inhibited the plant pathogen Fusarium oxysporum. These findings provide the first systematic chemical and functional characterization of metabolites from C. appendiculata seeds and offer new insight into the molecular basis of symbiosis between orchids and fungi.