Localized co-inoculation of Bacillus subtilis and Trichoderma afroharzianum acts synergistically to reshape the root microbiome and improve plant performance in sorghum

Abstract

Despite their agricultural potential, how bacterial-fungal consortia reshape root microbiomes and improve crop performance in sorghum remains poorly understood. Here, we investigated how individual and combined inoculation with Bacillus subtilis and Trichoderma afroharzianum influenced sorghum performance and root microbiome assembly. The in vitro co-culture assay demonstrated the compatibility of B. subtilis and T. afroharzianum as a microbial consortium. The B. subtilis-T. afroharzianum consortium demonstrated the highest CPPI (composite plant performance index) and shoot fresh weight in sorghum, while all inoculation treatments improved multiple growth and physiological traits. Split-root analysis demonstrated that bilateral root co-inoculation was necessary to maximize whole-plant growth benefits. Also, B. subtilis-T. afroharzianum co-inoculation increased carbon levels in both roots and leaves, accompanied by enhanced rhizosphere siderophore production consistent with improved nutrient status. In microbial community analysis, neither bacterial nor fungal alpha or beta diversity differed significantly among treatments; instead, inoculation selectively restructured root microbial communities. The B. subtilis-T. afroharzianum consortium selectively enriched plant growth-promoting Actinoplanes, siderophore-producing Enterobacter, and the plant-beneficial fungal genus Podospora. Co-occurrence network analysis identified Rhodoplanes, Serendipita, and Zopfiella among hub taxa associated with B. subtilis-T. afroharzianum co-inoculation, suggesting potential roles in microbial community connectivity and organization. Furthermore, the persistence of Streptomyces and Serendipita, particularly the latter, suggests the presence of a beneficial microbial core that may contribute to sustained rhizosphere functioning. In addition, Bacillus and Serendipita were among the indicator taxa associated with inoculated treatment combinations, suggesting that the inoculants selectively assembled a distinct plant-beneficial microbiome. Devosia was associated with chlorophyll content, siderophore production, and shoot height, whereas Serendipita correlated with shoot biomass under the B. subtilis-T. afroharzianum co-inoculation. Taken together, B. subtilis-T. afroharzianum consortium promotes sorghum growth by selectively reshaping the root microbiome, highlighting its potential as a next-generation microbial biofertilizer.