Tuber encompasses ectomycorrhizal fungi (EMF) of significant ecological and economic importance. This study reports the first controlled synthesis of ectomycorrhizae between the near-threatened species T. umbilicatum and Quercus glauca, confirmed through molecular analysis and detailed morphological characterization. Colonization dynamics, assessed over eight months, revealed substantial physiological benefits for the host. At six months post-inoculation, seedling height and above-ground biomass increased by 20.8% and 27.1%, respectively; these increments persisted to eight months, with above-ground biomass remaining 16.9% higher and below-ground biomass elevated by 25.4%. Concomitantly, the photosynthetic performance was markedly improved: a net photosynthetic rate (A) rose by 136.8% and stomatal conductance (g_s) by 36.5% at six months. Available phosphorus (AP) in the mycorrhizosphere was concurrently enhanced, exhibiting a 10.9% increment at eight months. These results underscore the agronomic and conservation utility of T. umbilicatum inoculation for Q. glauca and provide a critically experimental foundation for the ex situ preservation and sustainable truffle cultivation of this threatened fungal taxon.