Enhanced Biocontrol of Root-Knot Nematodes Through Co-Cultivation of Clonostachys rosea and Bacillus velezensis: Proline-Driven Bacterial Fitness and Synergistic Metabolite Production

The ascomycete fungus <i>Clonostachys rosea</i> is a promising biocontrol agent against root-knot nematodes. To develop a more effective and stable biocontrol strategy, we rationally constructed a co-culture system by partnering <i>C. rosea</i> with the plant growth-promoting bacterium <i>Bacillus velezensis</i>. Through systematic optimization of the medium and inoculation protocol, the co-culture demonstrated significantly enhanced performance, achieving 95.3% mortality of <i>Meloidogyne incognita</i> juveniles, a 78.0% increase in tomato shoot dry weight, and 69.2% disease control efficacy in pot trials. Metabolomic profiling indicated that the co-culture triggered a distinct metabolic profile compared to the respective monocultures. The enhanced efficacy was associated with the accumulation of two functional metabolite groups. First, the co-culture synergistically accumulated direct-effect compounds with reported nematicidal (e.g., daidzin, L-tryptophan) and plant-growth-promoting (e.g., isopentenyladenine, melatonin, and indole-3-propionic acid) activities. In parallel, L-proline emerged as a critical microbial interaction modulator. Targeted quantification showed a clear proline abundance gradient: highest in the <i>C. rosea</i> monoculture, intermediate in co-culture, and lowest in the <i>B. velezensis</i> monoculture. This gradient suggests that proline produced by <i>C. rosea</i> is likely utilized by <i>B. velezensis</i>, a finding further supported by the observation that proline enhanced bacterial biofilm formation and upregulated the matrix genes <i>epsC</i> and <i>tasA</i>. Accordingly, the co-culture itself formed significantly more robust biofilms. Thus, the enhanced biocontrol can be attributed to synergistic metabolite accumulation together with proline-mediated fitness gains in the bacterial partner, establishing a metabolic basis for rationally engineering microbial consortia.