Shen_2026_J.Fungi.(Basel)_12_

Wild Polygala crotalarioides is an ethnomedicinal plant traditionally used for neurological health. Endophytic fungi associated with medicinal plants are increasingly recognized as sources of bioactive natural products; however, their diversity and biological activities in wild P. crotalarioides remain poorly understood. We used high-throughput sequencing and culture-dependent methods to explored the diversity, community composition, and bioactivities of endophytic fungi associated with wild P. crotalarioides. Culturable isolates were identified via ITS sequencing and further screened for AChE-inhibitory and antioxidant activities. Our results revealed that a total of 818 fungal OTUs were detected, revealing pronounced tissue-specific community differentiation. Stem tissues harbored the highest fungal diversity, richness, and number of unique OTUs. Ascomycota and Basidiomycota dominated fungal communities across all tissues, while distinct biomarker taxa were enriched in different organs. Functional annotation indicated marked tissue-dependent variation in trophic composition. Among 108 culturable isolates, Penicillium sp. PRP024 and Trichoderma sp. PRP008 exhibited the strongest AChE inhibitory activities (>80%), whereas Fusarium sp. PSP053, Diaporthe sp. PSM027 and Exserohilum sp. PSP042 showed notable antioxidant activities. Wild P. crotalarioides hosts highly diverse and tissue-specific endophytic fungal communities. Several isolates exhibited promising acetylcholinesterase inhibitory and antioxidant activities, highlighting medicinal plant-associated endophytic fungi as potential sources of antioxidative natural products.