Exogenous substances promote the formation of Phallus dongsun primordia by remodeling the mycosphere microenvironment.

Exogenous substance application (ESA) directly influences subterranean processes through the action of macrofungal mycelia, garnering considerable attention for its role in enhancing crop yields and resource utilization. However, the intricate mechanisms of mycosphere metabolite-microbe interactions governing macrofungal development remain unclear. This study employed Phallus dongsun for plate culture and pot experiments, integrating multi-omics analysis to investigate the regulatory mechanisms governing the growth and development of this macrofungus through functional interactions between the macrofungus, metabolites, and microorganisms under ESA. Single-factor plate cultures revealed that 60 mg/L ZnSO4·7H2O exhibited the most pronounced effect on promoting mycelial growth and biomass in all three P. dongsun strains, while 1.5 g/L MgSO4 demonstrated a slightly weaker yet still significant effect. To verify the performance of each ESA under natural conditions, single-factor pot experiments were conducted. Pot experiments showed that 1.5 g/L MgSO4 was optimal for primordia formation and earliness. Furthermore, triacontanol (TRIA, 6 mg/L), vitamin B6 (VB6, 6 mg/L) and ZnSO4·7H2O (60 mg/L) also exhibited promotional effects on primordia formation and earliness, respectively. ESA significantly altered the mycosphere metabolite composition and microbial community structure, enhancing metabolite chemodiversity and keystone taxa diversity (P < 0.05). Moreover, ESA significantly enhanced the accumulation of the keystone taxa Paraburkholderia and major metabolites-such as lipids and organic acids-within the mycosphere. Notably, ESA promoted coupling of carbon (C) and phosphorus (P) cycling, while decoupling nitrogen (N) cycling in the mycosphere. Partial least squares modeling (PLS-PM) further indicated that ESA-induced N cycling constraints significantly stimulated P. dongsun primordia formation and P uptake. Furthermore, ESA indirectly enhanced P. dongsun primordia number by increasing keystone taxa diversity. This finding provided new theoretical insights into understanding the interaction mechanisms between macrofungi and their microenvironments, as well as their microbial ecology.