Abstract
<title>Abstract</title> <p> Endophytic fungi confer diverse fitness benefits to host plants and play a critical role in sustaining plant microecological balance. In this study, we investigated the community composition and tissue distribution of endophytic fungi in the tree peony cultivar <italic>Paeonia suffruticosa</italic> ‘Luoyang Hong’ (PSLH), screened endophytic strains with antagonistic activity against common fungal pathogens of tree peony and wheat, and further explored the physiological biocontrol mechanism of elite antagonistic strains. A total of 146 endophytic fungal strains were isolated from 480 PSLH tissue segments, with an overall colonization rate of 25.20% and isolation rate of 30.42%. Fungal colonization and isolation rates varied significantly among tissues, reaching the highest levels in roots (56.25% and 80%, respectively) while no endophytic fungi were obtained from seeds. Based on morphological characteristics and ITS sequence analysis, 139 strains were taxonomically classified into 23 species across 17 genera, 17 families, nine orders, four classes, and two phyla. <italic>Fusarium</italic> , Graphiopsis, <italic>Cephalosporium</italic> , and <italic>Paraconiothyrium</italic> dominated the endophytic community of PSLH, with relative abundances of 17.81%, 11.64%, 10.96%, and 6.85%, respectively. The calculated Shannon diversity index (H′ = 2.83) and Margalef richness index (D = 3.21) revealed a relatively high endophytic fungal biodiversity in PSLH. Dual-culture assays against five phytopathogens (the tree peony pathogens <italic>Alternaria alternata</italic> and <italic>A. suffruticosa</italic> , and wheat pathogens <italic>Fusarium pseudograminearum</italic> , <italic>Gaeumannomyces graminis</italic> var. tritici (Ggt), and <italic>Rhizoctonia cerealis</italic> ) yielded ten antagonistic strains, including eight antibiotic-producing isolates and two mycoparasitic isolates. The strain <italic>Bartalinia robillardoides</italic> T2-07 exhibited the most prominent biocontrol potential, forming a 15 mm inhibition zone against the wheat take-all pathogen Ggt. Physiological assays demonstrated that the fermentation filtrate of T2-07 significantly increased the relative electrical conductivity and malondialdehyde content in Ggt, inhibited the activities of antioxidant enzymes (SOD, POD, CAT), and suppressed key energy metabolic enzymes (HK, PK, SDH). These findings indicate that strain T2-07 inhibits Ggt via a synergistic multi-mechanism, including cell membrane damage, antioxidant system disruption, and energy metabolism blockade. This study enriches the germplasm resources of tree peony endophytic fungi and provides a valuable strain resource and theoretical basis for the development of microbial biocontrol agents against crop fungal diseases. </p>