Diversity and functional roles of endophytic and rhizospheric microorganisms in Ophioglossum vulgatum L.: implications for bioactive compound synthesis

Abstract

Background: Ophioglossum vulgatum L. is a widely utilized medicinal plant, with the entire plant being used for medicinal purposes. This study systematically characterized the endophytic and rhizospheric community structure, taxonomic diversity, and symbiotic networks within distinct compartments of O. vulgatum, while evaluating their potential associations with the accumulation of pharmacologically active metabolites.

Methods: Endophytic and rhizospheric community profiling was conducted via Illumina sequencing, while bioactive compounds were identified using UPLC-ESI-MS/MS.

Results: Roots and leaves harbored beneficial bacteria (e.g., Methylobacterium, Streptomyces, Sphingomonas, and Flavobacterium). Dominant fungi included Archaeorhizomyces (rhizosphere soil) and Homophron (roots/leaves). Dark septate endophytes (DSEs; e.g., Cladosporium, Cladophialophora, and Chaetomium) were abundant across rhizosphere soil, roots, and leaves. Alpha/beta diversity analyses showed higher microbial richness in rhizosphere soil than in plant tissues. Functional predictions (PICRUSt2/FUNGuild) linked endophytic and rhizospheric bacteria to metabolism, human diseases, and biological systems. Network analysis highlighted Basidiomycota as keystone taxa, with modular community structure. Functional predictions revealed that endophytic and rhizospheric microorganisms were associated with critical metabolic pathways, particularly in the biosynthesis of flavonoids and alkaloids (primary bioactive compounds). LEFSe analyses highlighted compartment-specific biomarkers: Acidobacteria, Basidiomycota, and Ascomycota were enriched in distinct zones (rhizosphere, roots, and leaves), with Actinobacteria exhibiting highly significant correlations (P < 0.01) with flavonoids, lipids, and quinones, while Acidobacteria, Basidiomycota, and Ascomycota were strongly linked to steroids and tannins (P < 0.05).

Conclusion: The diversity and abundance of microbial communities in O. vulgatum exhibited tissue-specific and rhizosphere-dependent variations, with distinct patterns strongly correlating to bioactive compound accumulation. Notably, biomarker taxa including Actinobacteria, Acidobacteria, Basidiomycota, and Ascomycota demonstrated robust microbe-metabolite interactions, suggesting their critical regulatory role in biosynthesis pathways. These findings establish endophytic-rhizospheric microbiota as key biosynthetic modulators, proposing innovative approaches for enhancing phytochemical production through targeted microbial community manipulation.

Keywords: Ophioglossum vulgatum; bioactive compounds; community diversity; plant endophytes; rhizosphere soil.

FIGURE 1

Comparative analysis of endophytic microbial communities in plant compartments and rhizosphere soil. (A) Venn diagram of shared bacterial ASVs among rhizosphere soil, root, and leaf samples. (B) Venn diagram of shared fungal ASVs. (C) Rarefaction curves for endophytic bacterial communities. (D) Rarefaction curves for endophytic fungal communities. Rhizosphere soil exhibited the highest ASV richness for both bacteria and fungi, with root-leaf overlaps comprising 30 bacterial and 77 fungal ASVs (>30% of total sequences; fungal overlap reached 50%). Rarefaction analysis confirmed sufficient sequencing depth and reinforced the rhizosphere’s dominance in microbial diversity.

FIGURE 2

Microbial community composition and structure across O. vulgatum compartments. Bacterial/fungal composition at phylum (A,B) and genus (C,D) levels. (E,F) Top 50 genera heatmaps. Proteobacteria dominated all bacterial niches (28.5%–88.1%), contrasting fungal compartmentalization: Ascomycota in rhizosphere (75.0%) vs. Basidiomycota in roots (86.0%)/leaves (90.1%). Key specialists: Candidatus Udaeobacter (rhizosphere, 4.0%), Streptomyces (roots, 17.5%), and Methylobacterium (leaves, 43.5%); Archaeorhizomyces (rhizosphere, 21.9%) and Homophron (roots/leaves, 40.6%–63.3%). Heatmaps indicate bacterial rhizosphere-leaf convergence vs. fungal root-leaf clustering, suggesting cross-niche microbial transfer.

FIGURE 3

Compartment-specific microbial enrichment patterns revealed by LEfSe analysis. (A) Bacterial and (B) fungal community differences among O. vulgatum rhizosphere soil, roots, and leaves. The cladograms (phylum to genus level) depict taxonomic units with node sizes proportional to relative abundance. Colored nodes represent significantly enriched taxa per compartment (yellow: non-significant). Letters denote key discriminative taxa. Rhizosphere soil contained the highest indicator species counts (54 bacterial and 49 fungal), followed by roots (36 bacterial and 6 fungal), and leaves (22 bacterial and 14 fungal), indicating pronounced compartment-specific selection in O. vulgatum-microbe interactions.

FIGURE 4

Microbial diversity patterns across O. vulgatum compartments. Alpha diversity of (A) bacterial and (B) fungal communities showing significantly higher richness (Chao1) and diversity (Shannon/Simpson) in rhizosphere soil vs. roots/leaves (P < 0.05, ANOVA). PCoA plots of (C) bacterial (84.9% variance explained) and (D) fungal (90.7% variance) communities based on Bray–Curtis distances, demonstrating clear compartment differentiation.

FIGURE 5

Functional profiling of endophytic communities in O. vulgatum and rhizosphere soil. (A) PICRUSt2 analysis revealed that genes associated with human diseases, metabolism, and organismal systems exhibited high relative abundance. A detailed predictive classification of ecological functions identified a total of 46 metabolic pathways across the samples. (B) FUNGuild analysis showed that Saprotroph and Pathotroph-Saprotroph-Symbiotroph functional groups dominated in rhizosphere soil, roots, and leaves. Additionally, a detailed predictive categorization of species ecological functions highlighted distinct distributions of dominant functional taxa across different compartments.

FIGURE 6

Bacterial-fungal co-occurrence network in O. vulgatum ecosystems. The network visualization displays ASVs (97% similarity threshold) as nodes colored by phylum affiliation, with node size proportional to connectivity degree. Significant correlations (P < 0.05, |r| > 0.70) are shown as edges (positive: pink; negative: green). Key topological features include: (1) dominance of Basidiomycota (largest nodes) as keystone taxa, (2) network complexity (116 nodes, 2,585 edges; average degree = 44.6), and (3) structural robustness (modularity = 0.441 > 0.40 threshold; clustering coefficient = 0.875). The 3.2:1 ratio of positive:negative edges (74.8% vs. 25.2%) highlights predominantly mutualistic microbial interactions across rhizosphere, roots, and leaves. Graph density (0.388) further confirms tight ecological connectivity within this microbiome system.

FIGURE 7

Bioactive compound profiles and microbe-metabolite correlations in O. vulgatum. (A) Relative abundance of 2,261 identified metabolites (normalized values × 108), showing flavonoids as the dominant class followed by amino acid derivatives and organic acids. (B) Mantel test results demonstrating significant positive correlations between microbial communities and key metabolites: bacterial associations with alkaloids, steroids and lipids (P < 0.01), and fungal links to flavonoids and phenolic acids (P < 0.05). (C) Actinobacteria exhibited highly significant positive correlations with flavonoids, lipids, and quinones (P < 0.01), whereas Acidobacteria along with Basidiomycota and Ascomycota fungi showed significant associations with steroids and tannins (P < 0.05). Significant interactions were observed among the bioactive compounds themselves, revealing potential co-regulation within metabolic networks.