Abstract

Background/Objectives: Wheat root and crown rot, caused by Fusarium pseudograminearum, Fusarium graminearum, and Bipolaris sorokiniana, are devastating soil-borne diseases that cause substantial yield losses worldwide. Endophytic fungi are promising sources of bioactive metabolites for agricultural applications. This study aimed to isolate and characterize an endophytic fungus with antifungal activity against major wheat pathogens, identify its active compound, and investigate the underlying transcriptional response. Methods: An endophytic strain Y2 was isolated from Hedyotis diffusa leaves and identified through morphological and phylogenetic analysis based on TEF-1α and RPB2 sequences. Pathogenicity of strain Y2 was evaluated on wheat stem bases. The bioactive compound was purified by HPLC and identified by HR-ESI-MS and NMR. Antifungal activity was assessed using dual-culture and microbroth dilution assays. Transcriptomic analysis (RNA-seq) was performed on F. pseudograminearum treated with equisetin, with qRT-PCR validation of seven representative differentially expressed genes. Results: Strain Y2 was identified as Fusarium incarnatum or a closely related member of the F. incarnatum–equiseti species complex (FIESC) and confirmed to be non-pathogenic to wheat. The purified bioactive compound was characterized as equisetin, which exhibited significant antifungal activity with MIC values of 16, 32, and 64 μg/mL against F. pseudograminearum, B. sorokiniana, and F. graminearum, respectively. Transcriptomic analysis revealed that equisetin treatment induced a polarized transcriptional response in F. pseudograminearum, characterized by strong upregulation of ribosome and translation-related genes and widespread downregulation of other metabolic pathways, particularly nitrogen metabolism. qRT-PCR validation of seven representative genes confirmed the reliability of the RNA-seq data. Conclusions: Our findings demonstrate that equisetin is the active antifungal metabolite produced by F. incarnatum Y2, with potent in vitro activity against major wheat root and crown rot pathogens. The transcriptomic data provide insights into the potential mechanism of action, while the non-pathogenic nature of strain Y2 supports its biosafety. Although these results highlight equisetin as a promising lead compound for antifungal development, further in planta efficacy and safety studies are required before it can be considered for practical biocontrol.

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