Seed Metabolites Recruit Beneficial <i>Pseudomonas</i> During Imbibition to Promote Protocorm Development in the Terrestrial Orchid <i>Gymnadenia conopsea</i>

Orchids rely on symbiotic microorganisms for nutrient acquisition throughout their life cycle, from seed germination to plant maturity. In the terrestrial orchid Gymnadenia conopsea, beneficial Pseudomonas species have been previously identified as associated with seed germination and enriched in protocorms. Yet, the specific metabolites that mediate this microbial recruitment remain unknown. In this study, integrated transcriptomic and metabolomic analyses revealed that seed imbibition activates the phenylpropanoid biosynthesis along with starch and sucrose metabolism pathways, resulting in increased secretion of trehalose and sinapyl alcohol. These metabolites were found to attract Pseudomonas sp. and facilitate their colonisation. We further assessed the effects of these metabolites in the presence of the germination-promoting fungus Ceratobasidium sp. GS2, with or without Pseudomonas. Our results indicated that trehalose enhanced protocorm development when combined with the fungus, and this effect was significantly strengthened by the addition of Pseudomonas. In contrast, sinapyl alcohol promoted protocorm development only when both the fungus and Pseudomonas were present. These findings uncover a metabolite-mediated synergy that coordinates beneficial microbes to orchestrate early development in G. conopsea, advancing our understanding of metabolite-fungus-bacteria interactions and benefiting cultivation practices.