Beyond Mycorrhizae: A Multi‐Kingdom Community in Epiphytic Orchid Roots Reveals Cyanobacteria Intracellular and a Velamen Microbial Refuge

Orchid biology has long been dominated by the paradigm of mycorrhizal dependency; however, the nutrient acquisition strategies of epiphytic orchids in canopy niches remain poorly understood. Using an integrated approach of multilocus metabarcoding (16S, ITS, 18S) and advanced microscopy (SEM, TEM, CLSM), we characterised the root microbial community of the threatened epiphytic orchid Cattleya purpurata. We report the discovery of intracellular Nostoc-like cyanobacteria within cortical cells, coexisting with the mycorrhizal fungus Tulasnella at a root colonisation rate of 18%. Furthermore, we identified: (1) a diverse velamen-associated microbiome hosting plant growth-promoting bacteria (e.g., the Burkholderia-Caballeronia-Paraburkholderia complex), nematodes and microalgae; and (2) a vast unknown fungal diversity, with over 39 genera and numerous ASVs unmatched in databases. We demonstrate that C. purpurata roots sustain a complex multi-kingdom community. We propose that the root velamen acts as a multi-kingdom microbial refuge, expanding the concept of symbiosis in orchids beyond fungi. Based on the intracellular presence of Nostoc-like cyanobacteria and their known diazotrophic capacity in other plant symbioses, we hypothesise a previously little-known nitrogen pathway mediated by cyanobacteria in a canopy environment, to be functionally tested in future studies.