Biocontrol efficacy of Bacillus velezensis and its major bioactive metabolites against Meloidogyne incognita

The southern root-knot nematode ( Meloidogyne incognita ) is one of the predominant pathogenic nematode species that severely threatens economically important crops. The application of biocontrol microorganisms or their metabolites represents an effective and environmentally friendly strategy for the management of root-knot nematodes. This study focused on the nematicidal activity of two Bacillus velezensis strains, RKN1111 and RKN2222, against M. incognita second-stage juveniles (J2s), and the identification of bioactive metabolites associated with their differential nematicidal effects. In vitro assays showed that, under comparable viable biomass conditions, the corrected mortality of J2s caused by RKN1111 (95.6%) was significantly higher than that caused by RKN2222 (46.2%). The LC 50 values of RKN1111 and RKN2222 were 0.58 × 10 8 and 5.86 × 10 8 CFU/mL, respectively. Metabolomic analysis revealed that, compared with RKN2222, RKN1111 exhibited 150 significantly upregulated differential metabolites. Among them, glycylvaline and N-cyclohexylformamide exhibited strong nematicidal activity against J2s, with corrected mortality rates of 88.7% and 81.7%, respectively, after 24 h treatment at 100-fold dilution of their saturated solutions. Moreover, the concentrations of glycylvaline (154 μg/mL) and N-cyclohexylformamide (4.5 μg/mL) in the original fermentation supernatant of RKN1111 were significantly higher than those in RKN2222 (43 μg/mL and 2.8 μg/mL, respectively). Further concentration-gradient bioassays combined with quantitative analysis showed that the concentration of N-cyclohexylformamide in the RKN1111 supernatant was far below the concentration threshold required for effective nematicidal activity, indicating that its direct contribution to the nematicidal activity of the supernatant was limited. In contrast, glycylvaline still exhibited marked nematicidal activity at concentrations close to those measured in the RKN1111 supernatant by HPLC. However, glycylvaline alone could not fully explain the high nematicidal activity of the RKN1111 supernatant, suggesting that it may be one of the major candidate contributors to the nematicidal activity of RKN1111 and that synergistic effects among multiple metabolites may also be involved. Overall, glycylvaline was identified as the strongest candidate bioactive metabolite screened in this study. These results provide a theoretical basis for the further development of RKN1111 as a potential biocontrol strain, as well as for further investigation of glycylvaline and the high-concentration nematicidal activity of N-cyclohexylformamide.