Priming of rhizobial nodulation signaling in the mycosphere accelerates nodulation of legume hosts

Summary The simultaneous symbiosis of leguminous plants with two root mutualists, endophytic fungi and rhizobia is common in nature, yet how two mutualists interact and co‐exist before infecting plants and the concomitant effects on nodulation are less understood. Using a combination of metabolic analysis, fungal deletion mutants and comparative transcriptomics, we demonstrated that Bradyrhizobium and a facultatively biotrophic fungus, Phomopsis liquidambaris , interacted to stimulate fungal flavonoid production, and thereby primed Bradyrhizobial nodulation signaling, enhancing Bradyrhizobial responses to root exudates and leading to early nodulation of peanut ( Arachis hypogaea ), and such effects were compromised when disturbing fungal flavonoid biosynthesis. Stress sensitivity assays and reactive oxygen species (ROS) determination revealed that flavonoid production acted as a strategy to alleviate hyphal oxidative stress during P. liquidambaris – Bradyrhizobial interactions. By investigating the interactions between P. liquidambaris and a collection of 38 rhizobacteria, from distinct bacterial genera, we additionally showed that the flavonoid‐ROS module contributed to the maintenance of fungal and bacterial co‐existence, and fungal niche colonization under soil conditions. Our results demonstrate for the first time that rhizobial nodulation signaling can be primed by fungi before symbiosis with host plants and highlight the importance of flavonoid in tripartite interactions between legumes, beneficial fungi and rhizobia.