Comparative Metagenomic Analysis of Phyllospheric Microbial Diversity in Azolla imbricata and Azolla pinnata

Background: The aquatic fern Azolla grows rapidly, produces nutrient-rich biomass, and adapts well ecologically due to symbiotic interactions with phyllospheric microorganisms. Azolla imbricata (Aim) and Azolla pinnata (Api) are valuable for biofertilization and phytoremediation, with their ecological differences potentially influenced by species-specific microbial partnerships. This study examines their phyllospheric microbiomes under controlled conditions to explore adaptive strategies. Method and Results: High-throughput metagenomic sequencing identified 10,857 bacteria, 986 eukaryotes. Proteobacteria dominated, particularly Burkholderiales, with higher abundance in Aim. Api exhibited greater Cyanobacteria abundance, especially Nostocaceae (genus Trichormus ). Streptophyta constituted over 90% of fungal communities in both species. LEfSe analysis highlighted unique taxa in each species, including enriched Magnoliopsida and Bacillariophyta in Aim, while Api had more Marchantia and Streptophyta. KEGG pathway analysis identified ribosome, oxidative phosphorylation, photosynthesis, quorum sensing, and two-component systems among the most enriched pathways. Resistance gene analysis showed Aim had higher levels of cmlA9, qacEdelta1, tetD, and ANT3-Ii-AAC6-IId_fusion_protein, while Api had more AAC6-IIa. Conclusions: Aim’s enrichment in Proteobacteria may enhance resilience, while Api’s nitrogen-fixing Cyanobacteria suggest distinct nitrogen management. Functional and resistance gene differences highlight ecological adaptability, providing insights for optimizing Azolla’s agricultural potential.