Background: Stress-induced hormones are essential for plants to modulate their microbiota and dynamically adjust to the environment. Despite the emphasis of the role of the phytohormone ethylene in the plant physiological response to heterospecific neighbour detection, less is known about how this activated signal mediates focal plant rhizosphere microbiota to enhance plant fitness. Here, using 3 years of peanut (Arachis hypogaea L.), a legume, and cyanide-containing cassava (Manihot esculenta Crantz) intercropping and peanut monocropping field, pot and hydroponic experiments in addition to exogenous ethylene application and soil incubation experiments, we found that ethylene, a cyanide-derived signal, is associated with the chemical identification of neighbouring cassava and the microbial re-assemblage in the peanut rhizosphere. Results: Ethylene production in peanut roots can be triggered by cyanide production of neighbouring cassava plants. This gaseous signal alters the microbial composition and re-assembles the microbial co-occurrence network of peanut by shifting the abundance of an actinobacterial species, Catenulispora sp., which becomes a keystone in the intercropped peanut rhizosphere. The re-assembled rhizosphere microbiota provide more available nutrients to peanut roots and support seed production. Conclusions: Our findings suggest that root ethylene acts as a signal with a dual role. It plays a role in perceiving biochemical cues from interspecific neighbours, and also has a regulatory function in mediating the rhizosphere microbial assembly, thereby enhancing focal plant fitness by improving seed production. This discovery provides a promising direction to develop novel intercropping strategies for targeted manipulations of the rhizosphere microbiome through phytohormone signals.

Endophytic fungi play important roles in the modification of ecosystem productivity; however, the underlying mechanisms are only partly understood. A 2-year field plot experiment verified that the endophytic fungus Phomopsis liquidambaris increased peanut (Arachis hypogaea L.) yields and significantly increased nodulation and N2 fixation regardless of whether N fertilizers were added. Root exudates collected from P. liquidambaris-colonized plants significantly improved nodulation and N2 fixation. Rhizosphere stimulation experiments further showed that colonized root exudates had significantly decreased soil nitrate (NO3−) concentrations, with decreased abundance and diversity of ammonia oxidizing archaea (AOA). In contrast, the abundance and diversity of diazotrophs significantly increased, and most diazotrophs identified were peanut nodulation-related strains (Bradyrhizobium sp.). P. liquidambaris symbiosis increased the expression of phenolic and flavonoid synthesis-related genes, and the derived phenolics and flavonoids could effectively increase the chemotaxis, biofilm formation, and nodC gene expression (nodulation-related biological processes) of the Bradyrhizobium strain. Metabolic pattern analysis showed that phenolics and flavonoids are more likely to accumulate to higher levels in the rhizosphere soil of peanuts colonized with P. liquidambaris. Finally, a synthetic root exudate experiment further confirmed the underlying mechanisms for the P. liquidambaris-induced improvement in nodulation and N2 fixation, i.e., that the specific root exudates derived from P. liquidambaris colonization decrease nitrate concentration and increase the population and biological activities of peanut nodulation-related Bradyrhizobium species, which beneficially enhance peanut-Bradyrhizobium interactions. Therefore, this study is the first to provide new insight into a positive relationship between an exotic endophytic fungus, crop nodulation, and N2 fixation increase. IMPORTANCE Endophytic fungi play an important role in balancing the ecosystem and boosting host growth; however, the underpinning mechanisms remain poorly understood. Here, we found that endophytic fungal colonization with P. liquidambaris significantly increased the productivity, nodulation, and N2 fixation of peanuts through the secretion of specific root exudates. We provide a reasonable mechanism explaining how P. liquidambaris promotes peanut nodulation and N2 fixation, whereby the specific root exudates produced by P. liquidambaris colonization decrease rhizosphere soil nitrate (NO3−) and increase the population and biological activities of peanut-nodulating-related Bradyrhizobium strains, which is beneficial to enhancing the peanut-Bradyrhizobium symbiotic interaction. Our study provides reliable empirical evidence to show the mechanism of how an exotic endophytic fungus drives an increase in nodulation and N2 fixation, which will be helpful in erecting a resource-efficient and sustainable agricultural system.

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