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High-throughput sequencing, culture-dependent workflows, and microbiome transfer experiments reveal whether potassium phosphite (KP), an environmentally acceptable agricultural chemical, could specifically enrich the antagonistic bacterial community that inhibited the growth of the pathogen Ralstonia solanacearum. The application of KP enriched the potential antagonistic bacteria Paenibacillus and Streptomyces in soil, but depleted most dominant genera belonging to gram negative bacteria, such as Pseudomonas, Massilia, and Flavobacterium on day 7. Moreover, the KP-modulated soil microbiome suppressed R. solanacearum growth in soil. The predicted functions related to the synthesis of antagonistic substances, such as streptomycin, and the predicted functions related to tellurite resistance and nickel transport system were significantly enriched, but the synthesis of lipopolysaccharide (distinct component lipopolysaccharide in gram negative bacteria) were significantly depleted in the KP-treated soils. In addition, the copy numbers of specific sequences for Streptomyces coelicoflavus and Paenibacillus favisporus were significantly increased in the soil amended with KP, inhibited the growth of R. solanacearum, and had a higher tolerance of KP than R. solanacearum. Our study linked the application of fertilizers to the enrichment of antagonistic bacteria, which could support future work that aims to precisely regulate the soil microbiome to protect the host from infection by soil-borne pathogens. [ABSTRACT FROM AUTHOR]