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The identification of a new compound active against Agrobacterium tumefaciens.The culture conditions of a newly isolated Bacillus subtilis strain, designed 14B, were optimized, as a first step, to produce its bacteriocin (termed Bac 14B) for the biocontrol of Agrobacterium spp., the causal agents of the crown gall disease. Bac 14B was then partially purified and biochemically characterized. Bacillus subtilis 14B was observed to produce an antibacterial compound having a protinaceous nature. As estimated by sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE), the semi-purified bacteriocin substance was found to be a monomeric protein with a molecular weight of 21 kDa. While the latter's antimicrobial activity was completely stable during exposure to a temperature range of up to 100°C for 2 h, its initial activity was totally lost at 121°C for 20 min. The maximum bacteriocin production (4096 AU ml−1) was recorded after 96 h-incubation in an optimized Luria Bertani medium supplemented with 10 g l−1 glucose, 15 g l−1 K2HPO4 and 5 g l−1 MgSO4 7H2O at 30°C in a shaking flask culture. Interestingly, the B. subtilis 14B culture supernatant that contained the bacteriocin under study was proved efficient in reducing both the percentage of galled plants and the number of galls in tomato.The findings revealed that B. subtilis 14B and its bacteriocin are efficient in reducing the percentage of infections in plants caused by Ag. tumefaciens.The results could be useful for the nurserymen who are particularly interested in the biocontrol of the crown gall disease.