Endophyte-Mediated Elicitation Enhances Picrosides Production in Suspension Cultures of Picrorhiza kurroa Royle ex Benth

In the present investigation, three endophytic fungi isolated from rhizome tissues of wild Picrorhiza kurroa Trichoderma sp. (EE1), Fusarium acuminatum (EE2), and Fusarium sp. (EE3) were used to modulate picroside (P-I, P-II, P-III) biosynthesis in leaf- and root-derived cell suspension cultures. ITS-based molecular identification confirmed the taxonomic affiliation of the isolates, and mycelial extracts were applied as elicitors at 0.5–1.5 ml/L for up to 192 h. All three elicitors triggered concentration- and tissue-dependent shifts in metabolite accumulation, with pronounced stimulation of picroside accumulation and precursor biosynthesis under specific treatment regimes. EE1 elicitation produced moderate increases in picrosides, with maximal accumulation of picroside I and II at 1.5 ml/L in both leaf- and root-derived cultures. In contrast, picroside III peaked at 1.0 ml/L in leaf-derived cell cultures. EE2 exhibited the strongest elicitation potential, markedly enhancing metabolite production at 1.5 ml/L, including picroside I (4.71 mg/g DW), picroside II (3.93 mg/g DW), picroside III (0.48 mg/g DW), aucubin (0.4 mg/g DW), and catalpol (4.0 mg/g DW) in leaf-derived suspension cultures. In contrast, EE3 induced comparatively lower picroside accumulation but preferentially stimulated precursor metabolites, including catalpol, vanillic acid, and tissue-specific accumulation of caffeic and cinnamic acids. Vanillic acid was detected across all treatments, while p-hydroxybenzoic acid and 4-hydroxyacetophenone further indicated endophyte-driven remodelling of secondary metabolism. Collectively, these findings demonstrate that endophytic fungal elicitors exert distinct, dose-dependent, and tissue-specific effects on specialized metabolite biosynthesis in P. kurroa , identifying F. acuminatum (EE2) elicitation as a potential approach for enhancing picroside accumulation in vitro .