Hyphosphere microorganisms facilitate hyphal spreading and root colonization of plant symbiotic fungus in ammonium-enriched soil

Anthropogenic nitrogen inputs lead to a high ammonium (NH₄⁺)/nitrate (NO₃^-) ratio in the soil, which restricts hyphal spreading of soil fungi. Access of symbiotic fungi to roots is a prerequisite for plant-fungal interactions. Hyphosphere bacteria protect fungi from environmental stress, yet the impact of hyphosphere bacteria on adaptation of host fungi to NH₄⁺-enriched conditions remains unclear. By developing soil microcosm assays, we report that a plant-symbiotic fungus, Phomopsis liquidambaris, harbors specific hyphosphere bacteria that facilitate hyphal spreading and assist in the root colonization in NH₄⁺-enriched soil. Genetic manipulation, 16S rRNA gene analysis and coinoculation assays revealed that the genus Enterobacter was enriched in the hyphosphere of NH₄⁺-sensitive wild-type compared to NH₄⁺-preferring nitrite reductase-deficient strain. The representative Enterobacter sp. SZ2-promoted hyphal spreading is only evident in nonsterilized soil. We further identified an increased abundance and diversity of ammonia-oxidizing archaea (AOA) and a synchronously decreased NH₄⁺:NO₃^- ratio following SZ2 inoculation. Microbial supplementation and inhibitor assays showed that AOA-mediated reduction in NH₄⁺:NO₃^- ratio is responsible for SZ2-enhanced fungal adaptation to NH₄⁺-enriched conditions. The Ph. liquidambaris-Enterobacter-AOA triple interaction promoted rice growth in NH₄⁺-enriched soil. Our study reveals the essential role of hyphosphere microorganism-based hyphal spreading in plant-fungal symbiosis establishment within nitrogen-affected agroecosystems.