A mycoheterotrophic orchid uses very limited soil inorganic nitrogen in its natural habitat

Mycoheterotrophic plants acquire nitrogen (N) directly from the soil and through their symbiotic fungi. The fungi-derived N has received considerable attention, but the contribution of soil-derived N has been largely overlooked. We investigated how the leafless, rootless, and almost mycoheterotrophic orchid Cymbidium macrorhizon obtains soil N by applying ¹⁵N-labeled ammonium nitrate in its natural habitat, and tracking metabolite accumulation and mycorrhizal fungal association after N application. The decline of N in the rhizome from flowering to fruiting indicated a transfer of N from the rhizome to fruits. At current dose of N application (0.6 g NH₄NO₃ each plant), only 1.5% of the plant's N was derived from fertilizer, resulting in a low nitrogen use efficiency of 0.27%. The majority of those newly absorbed N (88.89%) was found sank in the rhizome. Amino acids (or their derivatives) and alkaloids were predominant differentially accumulated nitrogenous metabolites after N application, with amino acids occurring in both fruits and the rhizome, and alkaloids primarily in the fruits. The addition of N did not alter the richness of mycorrhizal fungi, but did affect their relative abundance. Our findings suggest that Cymbidium macrorhizon uses very limited soil inorganic nitrogen in its natural habitat, and the root-like rhizome primarily stores N rather than absorbs its inorganic forms, offering new insights into how mycoheterotrophic plants utilize soil N, and the influence of nutrient availability on the orchid-fungi association.