Restructuring tilth layers suppresses cotton Verticillium wilt through the niacinamide-mediated enrichment of beneficial Pseudomonas.

Restructuring tilth layers (RTL) is an innovative tillage practice that involves the vertical exchange of topsoil and subsoil while the deeper layer is loosened, and this practice has been verified to significantly reduce the incidence of cotton Verticillium wilt. However, the ecological mechanisms underlying disease suppression remain unclear. In this study, we integrated field experiments, metagenomic sequencing, untargeted metabolomics, and functional validation to elucidate the effects of RTL on the rhizosphere ecosystem from the perspectives of microbe and metabolite interactions. RTL significantly altered the diversity and composition of the rhizosphere microbial communities and increased their network complexity and stability. Linear discriminant analysis effect size (LEfSe) revealed that RTL promoted the enrichment of beneficial taxa such as Pseudomonas, Lysobacter, and Mesorhizobium. Metabolomic profiling revealed that the abundance of niacinamide was 19.11-fold higher (P < 0.05) in the RTL rhizosphere than in the control rhizosphere. Exogenous supplementation and antagonistic assays demonstrated that niacinamide stimulated Pseudomonas enrichment and activation in the rhizosphere. Although niacinamide did not have direct antifungal activity, its coapplication with Pseudomonas reduced the disease index of Verticillium wilt by 81.89%. Overall, RTL suppresses Verticillium wilt through two pathways, by establishing a more stable and complex microbial network and regulating rhizosphere metabolite composition, particularly niacinamide accumulation, which drives the colonization and activation of defense mediated by beneficial microbes, forming an ecological defense mechanism that links metabolite signaling, microbial response, and pathogen suppression.