Endophytic fungi, residing within plant tissues without causing disease, play critical roles in plant health and ecosystem dynamics. This study characterized the taxonomic composition, diversity, functional guilds, and spatial distribution of endophytic fungal communities in healthy spinach (Spinacia oleracea L.) leaves using ITS1 amplicon sequencing. Surface-sterilized leaf samples were processed for DNA extraction, followed by Illumina MiSeq sequencing. Sequences were clustered into 24 operational taxonomic units (OTUs) using QIIME2 and the UNITE database. Fusarium spp. dominated (77.23% of reads), followed by Thanatephorus cucumeris (19.90%) and Plectosphaerella spp. (2.73%). Alpha diversity analysis revealed moderate richness (24 OTUs) but low evenness (Shannon index: 2.097, Simpson index: 0.702). FUNGuild annotation assigned ecological roles to 21 OTUs, with saprotrophs comprising 94.82% of abundance, symbiotrophs 0.04%, and other guilds 5.14%. Multifunctional taxa, such as Fusarium (saprotroph-pathotroph-endophyte), were prevalent. Dispersion analysis revealed a highly aggregated community (VMR: 53,647.54, Morisita’s Index: 6.76), characterized by the dominance of specific genera. Rank-abundance curves and guild distribution visualizations, generated using R (ggplot2), highlighted Fusarium dominance and saprotrophic prevalence. Most taxa exhibited filamentous morphology, aligning with saprophytic strategies. These findings suggest a low-diversity, highly aggregated fungal community with saprotrophic dominance, which may potentially influence spinach health. This study provides a foundation for understanding endophytic fungal ecology in agricultural systems, with implications for crop management.
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