Endophytes are microorganisms that inhabit plant tissues, colonizing partially or completely within the tissues or intercellular spaces of various organs in healthy plants without causing any apparent harm (Vandenkoornhuyse et al., 2015; Zhang et al., 2019). They constitute a vital component of the plant microecosystem. Studies have shown that endophytic microorganisms are abundantly present in roots, stems, leaves, flowers, and fruits of plants, where they play essential roles in plant growth, health and response to biotic stresses (Emami et al., 2019; Matsumoto et al., 2021). Increasing evidences has demonstrated that plant endophytes are an important component of the internal environment of medicinal plants (Pandey et al., 2023; Wu et al., 2021). On one hand, they secrete phytohormones and growth-promoting factors that enhance plant development. On the other hand, they modulate the biosynthese and accumulation of bioactive compounds in medicinal plants. Endophytes can increase the host plant’s active constituents through multiple mechanisms: by synthesizing metabolites identical or structurally analogous to the host’s bioactive compounds, or by biotransforming the plant’s original metabolites into novel derivatives, thereby enriching the diversity and yield of pharmacologically active substances (Li et al., 2022; Ouyang et al., 2024; Yan et al., 2019).

Endophytic bacteria are beneficial microorganisms that establish symbiotic relationships with plants by colonizing their tissues without causing disease. They enhance plant health through multiple mechanisms, including improved nutrient uptake, phytohormone modulation, and promotion of growth, stress tolerance, and overall fitness (Afzal et al., 2019; Taulé et al., 2021). These bacteria exhibit remarkable colonization capacity, inhabiting nearly all plant organs of roots, stems, leaves, flowers, and seeds. Among them, key genera such as Enterobacter, Pseudomonas, Burkholderia, Bacillus, and Streptomyces play critical roles in plant growth promotion, stress resilience, and biosynthesis of bioactive metabolites (Ek-Ramos et al., 2019; Laranjeira et al., 2022; Taghavi et al., 2010; Vurukonda et al., 2018). Endophytic fungi also colonize the internal tissues of nearly all plant species without causing disease, creating a mutually beneficial relationship where the host plant gains enhanced growth and resilience, while the fungi obtain nutrients and shelter (Baron and Rigobelo, 2022; Zhang J. et al., 2024). Dark Septate Endophytes (DSE) is key symbiotic fungi that significantly enhance plant growth and contribute to ecosystem functionality (Bi et al., 2022). DSE is a group of endophytic fungi commonly found in plant root systems, characterized by melanized septate hyphae and microsclerotia (Santos et al., 2021). They play a crucial role in plant-fungi symbiosis, particularly under stressful conditions like salinity and drought, enhancing plant resilience and adaptation (Akhtar et al., 2022). DSE colonization modulates plant growth by influencing cell metabolism, signaling pathways, and phytohormone biosynthesis, enhancing plant development and stress adaptation (Wu et al., 2020).

The rhizosphere, a highly dynamic zone surrounding plant roots, is enriched with root-derived metabolites that recruit a diverse array of microorganisms, creating a hotspot for microbial activity and interactions. Rhizosphere microorganisms are integral to this ecosystem, playing a vital role in mediating plant–soil interactions (Qu et al., 2020; Wang et al., 2017). This microbial community, which includes bacteria, fungi, archaea, actinomycetes, and other functionally diverse microbes, thrives in the soil adjacent to plant roots (Song et al., 2023). These microorganisms drive essential processes such as soil organic matter decomposition, humus formation, and nutrient transformation, thereby playing a fundamental role in producing high-quality medicinal materials and enhancing the accumulation of secondary metabolites (Jacoby et al., 2017; Noman et al., 2021; Song et al., 2023). Investigating the composition and diversity of rhizosphere microbial communities is essential for unraveling the intricacies of plant growth and stress tolerance. This exploration not only deepens our comprehension of plant–microbe interactions but also paves the way for innovative agricultural practices and sustainable ecosystem management.

Ophioglossum vulgatum L. is a small, rare, ancient and perennial fern belonging to the genus Ophioglossum and the family Ophioglossaceae. It is known by many folk names, such as Yizhijian, Shexucao, and Maoduncao (Hao et al., 2024; Xu G. et al., 2024). The herb is predominantly distributed across the northern hemisphere, typically thriving in moist mountainous areas, riverbanks, and ditches (Hao et al., 2021). O. vulgatum is primarily used medicinally in its entirety, valued for its properties of clearing heat, reducing inflammation, detoxifying, acting as an expectorant, and providing sedative effects (Clericuzio et al., 2014; Hao et al., 2024; Zhu et al., 2025). O. vulgatum synthesizes diverse bioactive substances, including flavonoids, sterols, polysaccharides, and alkaloids (e.g., sinoacutine, acutumine, michelalbine, stepharine, sinomerine, and disinomenine), which collectively mediate its pharmacological effects. Flavonoids and alkaloids are the two most important pharmacological substances; however, there are conflicting reports regarding its composition, and its synthesis is influenced by various environmental factors, including endophytes (Xu G. et al., 2024; Yousaf et al., 2024). Studies have revealed that climatic factors, including harvesting period, temperature, hours of sunshine, and rainfall, significantly influence the variations in the content of active ingredients in O. vulgatum (Qiu et al., 2024).

Plant–microbe interactions in natural environments are complex, further complicated by microbial abundance and diversity, as well as the heterogeneity of their composition (Sarkar et al., 2022). Notably, current research on endophytic communities in O. vulgatum remains limited, with particularly scarce investigations into their functional contributions to bioactive compound biosynthesis. In this study, we addressed this gap by analyzing the microbial diversity and community structure in O. vulgatum rhizosphere soils, roots, and leaves, identifying the dominant fungi and bacteria, and establish the relationship between bioactive substances and correlating with probiotic bacteria and fungi. This study lays a foundation for elucidating the diversity and community structure of endophytic and rhizosphere microorganisms, as well as the composition of the main bioactive substances. Furthermore, it explores the correlation between microbial communities and the synthesis and accumulation of bioactive substances. These findings provide both theoretical insights and practical methodologies for cultivating O. vulgatum with enhanced yields of bioactive constituents.

Plant endophytic and rhizospheric microorganisms play pivotal roles in mediating host growth and secondary metabolite synthesis, with profound implications for both natural ecosystems and commercial applications (Li et al., 2023). Our study reveals that O. vulgatum harbors a remarkably diverse endophytic and rhizospheric microbiome, comprising 3,564 bacterial ASVs (spanning 35 phyla) and 2,120 fungal ASVs (13 phyla), exhibiting distinct compartment partitioning across rhizosphere soil, roots, and leaves (Figure 1). The observed microbial richness gradient, with rhizosphere soils as the most biodiverse compartment, followed by roots and leaves for bacteria, and leaves surpassing roots for fungi that aligns with established ecological theory predicting stronger environmental filtering in plant interior tissues. This pattern likely reflects the combined effects of (1) soil as a microbial source habitat, (2) root exudate-mediated selection, and (3) leaf-specific physicochemical constraints (Dang et al., 2023).

The predominance of Proteobacteria, Actinobacteria, and Bacteroidetes among bacterial communities mirrors findings in other medicinal plants like Lonicera japonica (Xu Q. et al., 2024) and Pinus bungeana (Yang et al., 2022), suggesting conserved phylum-level associations across phylogenetically distant hosts (Figure 2A). Notably, the functional specialization of these taxa, including nitrogen fixation (Proteobacteria), bioactive metabolite production (Actinobacteria), and organic matter decomposition (Bacteroidetes), considering them as key players in potentially enhancing O. vulgatum’s medicinal properties through direct synthesis or stimulation of plant metabolic pathways (Narsing et al., 2022; Pan et al., 2023). The fungal community’s distinct distribution pattern (rhizosphere > leaves > roots), contrasting with bacterial trends, may reflect the difference in the roles of active compound biosynthesis or compartment-specific resource availability, warranting further investigation into fungal-plant biochemical crosstalk (Figure 4B).

The predominance of Ascomycota and Basidiomycota across all examined compartments of O. vulgatum aligns with observations in other plant systems such as Stipa purpurea and Picea abies (Marčiulynas et al., 2022; Yang et al., 2020), suggesting conserved functional roles of these phyla in plant–microbe ecosystems (Figure 2B). Notably, their compartment-specific distribution patterns reveal distinct ecological strategies: Ascomycota demonstrated particularly high abundance in rhizosphere soils, consistent with their well-documented saprophytic capabilities in decomposing recalcitrant organic compounds (Beimforde et al., 2014; Gaiero et al., 2013). In contrast, Basidiomycota showed pronounced dominance in roots and leaves, likely reflecting their dual ecological roles as both decomposers and symbiotic partners. Their prevalence in plant tissues may be attributed to their unique enzymatic machinery for breaking down complex plant polymers like lignin and cellulose (Manici et al., 2024), as well as their ability to form mycorrhizal associations that enhance host nutrient acquisition (Frey et al., 2004). This compartment partitioning between Ascomycota (soil specialists) and Basidiomycota (plant tissue specialists) suggests complementary functional contributions to plant health—while Ascomycota maintain soil nutrient pools through organic matter decomposition, Basidiomycota directly facilitate nutrient transfer to the host plant. The conservation of these phylum-level associations across diverse plant species, including our findings in O. vulgatum, highlights their fundamental importance in plant–microbe systems. Network analysis also identified Basidiomycota as the keystone microbial group in O. vulgatum, exhibiting the highest relative abundance among four fungal and six bacterial phyla (Figure 6). Its central network position and numerous positive correlations highlight its critical role in maintaining this stable, cooperative microbiome, likely through organic matter decomposition, nutrient cycling functions and active compound biosynthesis (Banerjee et al., 2018).

Our genus-level analysis revealed distinct compartmentalization of microbial communities in O. vulgatum, with each compartment harboring specialized taxa exhibiting unique functional attributes (Figure 2). The rhizosphere was dominated by Candidatus Udaeobacter, a trace gas-scavenging bacterium known to maintain soil microbial equilibrium through antibiotic production (Willms et al., 2020). Root endophytes were characterized by Streptomyces, the prolific producers of bioactive metabolites that account for approximately 65% of known microbial-derived plant growth regulators (Law et al., 2018; Pang et al., 2022). This genus’s prevalence suggests its potential role in mediating O. vulgatum’s secondary metabolism and stress resistance. The leaf microbiome showed strong selection for Methylobacterium, nitrogen-fixing symbionts that enhance host disease resistance through phytohormone modulation (Sanjenbam et al., 2022). The consistent presence of plant growth-promoting bacteria (PGPB) across compartments, including nitrogen-fixing Allorhizobium groups, phosphorus-solubilizing Pseudoxanthomonas, and siderophore-producing Sphingomonas (Singh et al., 2022), demonstrates O. vulgatum’s ability to cultivate beneficial consortia throughout its tissues. Fungal communities exhibited parallel compartment specialization, with the rhizosphere-enriched Archaeorhizomyces enhancing mineral nutrient acquisition (Ma et al., 2022), while the root/leaf-associated Homophron (Basidiomycota) conferred stress resilience (Rosas-Medina et al., 2020). The widespread colonization by DSEs like Cladophialophora and Exophiala suggests their potential involvement in both nutrient mobilization and bioactive compound synthesis (Wang et al., 2022). LEfSe analysis confirmed strong compartment differentiation, identifying 112 bacterial and 69 fungal biomarkers. Acidobacteria, Basidiomycota, and Ascomycota were distinctly enriched in O. vulgatum’s rhizosphere soil, roots, and leaves, respectively (Figure 3). This compartmentalization parallels patterns seen in Cycas panzhihuaensis (Zheng and Gong, 2019), suggesting conserved evolutionary strategies for microbiome partitioning across phylogenetically distant plants. The consistent presence of PGPB in all compartments implies that O. vulgatum actively maintains beneficial microbial functions throughout its tissues, potentially contributing to its medicinal properties.

Our study reveals a sophisticated compartmentalization of microbial communities in O. vulgatum, with distinct diversity patterns and functional specialization across different plant tissues and rhizospheric soil. The alpha diversity analysis demonstrated that bacterial communities followed a clear rhizosphere soil > roots > leaves gradient, while fungal communities exhibited a modified pattern of rhizosphere soil > leaves > roots (Figure 4). These findings align with previous observations in Senecio vulgaris L. (Cheng et al., 2019) and support the emerging paradigm of plant-mediated microbiome assembly across phylogenetically diverse species (Trivedi et al., 2020). Beta diversity analysis further confirmed significant compositional differences between compartments (P < 0.01), underscoring the strong selective pressures exerted by plant tissues through their unique physicochemical microenvironments (Zhang et al., 2023). The functional predictions provided deeper insights into potential microbe–host interactions, revealing that bacterial metabolic profiles were particularly enriched in human disease-related pathways and metabolic processes (Figure 5A), potentially linked to O. vulgatum’s characteristic accumulation of flavonoids and alkaloids. Fungal communities displayed particularly interesting compartment specialization patterns, with the rhizosphere dominated by multifunctional Pathotroph-Saprotroph-Symbiotrophs, reflecting the complex nutrient dynamics of soil ecosystems (Figure 5B). In contrast, roots and leaves were enriched in Saprotrophs, likely supporting organic matter decomposition in these tissues. This guild distribution mirrors patterns observed in agricultural systems (Clocchiatti et al., 2020), where saprotrophs are known to enhance soil stability through their extensive hyphal networks (Abiven et al., 2007). The dynamic equilibrium between different fungal nutritional modes appears particularly significant for plant health, where symbiotroph dominance enhances host resistance while potentially suppressing saprotrophic activity, and pathotroph prevalence correlates with disease susceptibility.

These findings collectively suggest that O. vulgatum has evolved to maintain compartmentalized microbial functions that likely contribute to its medicinal properties through several mechanisms: the rhizosphere microbiome facilitates nutrient mobilization, root endophytes may participate in secondary metabolite production, and leaf-associated microbes could influence defense compound synthesis. Through comprehensive metabolite profiling, we identified 2,261 secondary metabolites, with flavonoids (583 compounds), amino acids and derivatives (246 compounds), and organic acids (104 compounds) representing the most abundant classes (Figure 7A and Supplementary Table 2). These results align with previous reports on the chemical composition of O. vulgatum (Yousaf et al., 2024), confirming its rich pharmacologically active constituents. Mantel test analysis revealed significant microbe-metabolite associations, with bacterial communities showing particularly strong correlations (P < 0.01) with alkaloids, steroids, and lipids, while fungal communities were more closely associated (P < 0.05) with flavonoids, terpenoids, and phenolic acids (Figure 7B). The strong flavonoid-microbiome correlation may reflect their dual roles in plant defense and microbial signaling (Zeng et al., 2022), while the alkaloid-bacteria associations suggest specialized interactions related to nitrogen metabolism (Fairbairn and Paterson, 1966). These patterns mirror observations in other medicinal plants such as Panax notoginseng (Burkill) F. H. Chen ex C. H. Chow and Paris polyphylla var. yunnanensis, supporting the emerging understanding that endophytic and rhizospheric microorganisms play vital roles in the biosynthesis and accumulation of active compounds (Dai et al., 2024; Shu et al., 2025; Zhang X. K. et al., 2024). In addition, significant correlations were identified between microbial taxa and specialized metabolites: Actinobacteria exhibited a highly significant positive association (P < 0.01) with flavonoids, lipids, and quinones, while Acidobacteria, Basidiomycota, and Ascomycota showed marked positive correlations (P < 0.05) with steroids and tannins (Figure 7C). These findings that align with previous research demonstrating the critical role of Actinomycetes in flavonoid biosynthesis in Dendrobium species (Yuan et al., 2024).

The distribution of microbial functions suggests a coordinated system where: (1) rhizosphere communities facilitate nutrient mobilization and provide precursor molecules, (2) root endophytes (particularly Streptomyces) likely participate in alkaloid biosynthesis and nitrogen metabolism, and (3) leaf-associated microbes (notably Methylobacterium) may influence flavonoid synthesis and plant defense responses. These compartmentalized interactions appear to have co-evolved to enhance O. vulgatum’s medicinal properties through both direct microbial biosynthesis and microbiome-induced plant metabolic pathways.

Taken together, these findings provide valuable insights into the ecological roles of secondary metabolites and establish a foundation for future research to: (1) isolate and characterize key microbial taxa responsible for metabolite production, (2) elucidate the molecular mechanisms of microbe-induced metabolite synthesis, and (3) develop microbiome-based cultivation strategies to enhance medicinal compound yields in O. vulgatum and related species.

This study elucidates the diversity and functional specialization of endophytic and rhizospheric microorganisms in O. vulgatum and their pivotal role in bioactive compound synthesis. High-throughput sequencing revealed distinct microbial communities across plant compartments, with Proteobacteria, Actinobacteria, and Bacteroidetes dominating bacterial assemblages, while Ascomycota and Basidiomycota prevailed among fungi. Basidiomycota emerged as a keystone taxon, maintaining network stability. Functional predictions linked endophytic and rhizospheric microorganisms to critical metabolic pathways, particularly in flavonoid and alkaloid biosynthesis, supported by strong microbe-metabolite correlations. Notably, biomarker taxa including Actinobacteria, Acidobacteria, Basidiomycota, and Ascomycota demonstrated robust microbe-metabolite interactions, suggesting their critical regulatory role in biosynthesis pathways. These findings demonstrate how compartmentalized microbial functions, from rhizosphere nutrient mobilization to root/leaf metabolite production, collectively enhance bioactive compound accumulation. This integrated microbiome-metabolome framework provides novel insights for harnessing plant–microbe interactions to optimize medicinal yields in O. vulgatum and related species.