Diversity, function and assembly of the Trifolium repens L. root-associated microbiome under lead stress

Root-associated microbial layers provide unique niches that drive specific microbe assemblies. While the rhizosphere microbiome has long received much attention, endophytic microbes remain largely elusive. Characterizing metal-tolerant plants' strategies for assembling different root-associated microbial layers is important for optimizing phytoremediation. Here, a pre-stratified rhizo-box assay was conducted with Trifolium repens L. under greenhouse conditions with artificial Pb-contaminated soil. Cultivation compensated for the pollution-driven loss of soil microbial biomass carbon, enzyme activities and abundance. The acid-soluble Pb proportion increased in the rhizosphere (from 6.5-13.7% to 7.1-18.0%) compared with bulk soil. Under stress, root-layer variants were a considerable source of variation in the microbiome, with the endosphere representing a unique and independent niche. A core set of root microbes were selected by T. repens, with Proteobacteria and Actinobacteria composed of diverse plant-growth-promoting bacteria (PGPBs) and metal-tolerant members. Cluster analysis revealed endosphere-enriched genera, with Rhizobium, Nocardioides, Novosphingobium, Phyllobacterium, and Sphingomonas being the most dominant. Finally, inferred microbial metabolic pathways suggested that these potential metal-tolerant PGPB species provide critical services to hosts, enabling them to tolerate and even flourish in contaminated soil. Our results provide novel insights for understanding how root-associated microbes help metal-tolerant plants cope with abiotic stress.