Deciphering chemometric diversity in aerial and subterranean endophytic fungal communities of Thymus vulgaris via LC-MS/MS based untargeted metabolomics and in-silico therapeutic target prediction of marker metabolites.

Endophytic fungi associated with medicinal plants represent an important and largely untapped source of structurally diverse secondary metabolites. This study explores the tissue-specific metabolic diversity of endophytic fungi isolated from aerial and root tissues of Thymus vulgaris using LC-MS/MS-based untargeted metabolomics to enable the accelerated prioritization of biosynthetically competent strains for natural product discovery. A total of eighteen fungal endophytes were isolated and taxonomically characterized by 18S rDNA sequencing, followed by metabolomic profiling of extracellular extracts. Comprehensive data analysis of more than 11,000 detected molecular features led to the annotation of 1164 metabolites. Among these, 269 metabolites exhibited significant differential abundance between tissue-derived groups, with 230 showing marked upregulation in root-associated isolates. Multivariate statistical analyses revealed clear metabolic segregation according to tissue origin. Functional pathway enrichment analysis demonstrated that root-derived fungi possess enhanced tyrosine metabolism and terpenoid biosynthesis potential compared with aerial counterparts. Notably, Ganoderiol C emerged as a defining chemometric marker metabolite for subterranean endophytes. Subsequent in silico target prediction indicated strong binding and stable interactions of Ganoderiol C with NF-κB1 and GLRA1, suggesting potential biological relevance. Collectively, these results demonstrate pronounced metabolic compartmentalization between aerial and root endophytes in T. vulgaris and highlight the utility of metabolomics-driven strategies for niche-informed strain selection. This integrative approach provides a robust framework for prioritizing endophytic fungi with elevated biosynthetic capacity and strengthens the prospects of discovering novel bioactive compounds from plant-associated microbial communities.