Heat Stress and Soil Microbial Disturbance Influence Soybean Root Metabolite, Microbiome Profiles, and Nodulation

Heat stress is a major limiting factor for soybean productivity worldwide. Recent studies have highlighted the critical role of the plant microbiome in enhancing plant resilience to heat stress. However, our understanding of the molecular and physiological mechanisms underlying root-microbiome interactions under heat stress remains limited. To elucidate the role of native soil microbes in the heat tolerance of soybean genotypes, we analyzed rhizosphere bacterial and fungal communities via 16S rRNA and ITS sequencing, and characterized root metabolites and anatomical traits in response to microbiome composition and heat stress. Soybean plants were grown under controlled conditions in either natural soil containing native microbiota or in microbiome-disturbed soil (via 3-hour autoclaving), under both optimal and elevated temperature regimes. Alpha and beta diversity analyses revealed significant microbial shifts between treatments. Distinct clustering of bacterial, fungal, and metabolite profiles was observed under high temperature and microbial disturbance. Nodule-forming bacteria such as Rhizobium and Janthinobacterium were markedly suppressed, and belowground traits exhibited sensitivity, with significantly reduced nodule numbers and nodulation efficiency under high temperature and soil microbial perturbation. Non-targeted root metabolomics identified 372 differentially accumulated metabolites. Integrative multi-omics analysis revealed associations between metagenomic profiles, metabolite levels, and nitrogen-fixation traits, implying a coordinated modulation of root physiological processes. These findings contribute to a growing understanding of how heat stress interacts with rhizosphere microbial communities and may support future efforts in breeding climate-resilient soybean cultivars.