Description, Identification, and Growth of Ectomycorrhizae in Tuber sinense -Mycorrhized Castanea mollissima Seedlings

The synthesis and symbiotic mechanisms of truffle ectomycorrhizae have attracted considerable scientific interest in recent decades. Although previous research has successfully identified the symbiotic partners of truffles ( Tuber spp.) and characterized their mature morphological features, the dynamic processes involved in truffle ectomycorrhizal formation remain insufficiently understood. In this study, we established an ectomycorrhizal synthesis system using Castanea mollissima seedlings inoculated with Tuber sinense spore suspensions under controlled greenhouse conditions, followed by an eight-month observation period. To systematically characterize and model the morphological changes during ectomycorrhizal development, we employed an innovative approach integrating resin sectioning with confocal microscopy. Ectomycorrhizal formation was initially observed two months post inoculation, with a colonization rate reaching 24.4 ± 5.3% by the third month. The ectomycorrhizae displayed a distinct color progression from light brown through ochre and finally dark brown, typically manifesting either monopodial or branched structures. Early developmental stages (2–3 months) were characterized by a thin mycelial membrane enveloping the root surface, accompanied by limited hyphal penetration into the root system. By the eighth month, the colonization rate stabilized at 45.2 ± 8.6%, with enhanced organization and density of the fungal mantle and extended Hartig nets reaching the periphery of outer cortical cells. The continuous growth and differentiation of mycorrhizal root tips generated repetitive root architectures, significantly enhancing symbiotic efficiency. These findings provide critical insights into the morphological development and symbiotic effectiveness of truffle ectomycorrhizae while establishing a methodological framework for investigating ectomycorrhizal associations in other economically significant plant–fungal systems.