Construction of a Four-Strain Synthetic Bacillus Consortium and Evaluation of Its Biocontrol Efficacy Against Tomato Fusarium Wilt Caused by Fusarium oxysporum f. sp. lycopersici

Tomato Fusarium wilt, caused by Fusarium oxysporum f. sp. lycopersici (FORL), is a major soil-borne constraint on protected tomato production worldwide. Because single biocontrol strains often show inconsistent field performance, this study aimed to construct and optimize a synthetic bacterial consortium combining endophytic and soil-derived biocontrol bacteria and to evaluate its efficacy against tomato Fusarium wilt. Using dilution plating, 582 bacterial strains were obtained from 42 rhizosphere soil samples and 195 endophytic strains from six healthy tomato plants. Initial screening by plate confrontation yielded 78 soil-derived and 46 endophytic biocontrol candidates; after a second round of screening, 45 strains with inhibition rates exceeding 35% were retained. Of these, two soil strains (SF-89, KD-54) and two endophytic strains (F-86, F-29) showed the strongest in vitro inhibition of FORL (45.9-48.7%) and were identified by morphological, physiological/biochemical and 16S rRNA gene sequence analysis as Bacillus siamensis, B. subtilis, B. velezensis and B. methylotrophicus, respectively. The four strains were compatible in co-culture. A quantitative mixture-design analysis identified F-29 (B. methylotrophicus) as the principal (dominant) strain of the consortium, and Box-Behnken response-surface optimization determined an optimal strain ratio of SF-89:F-86:KD-54:F-29 = 2:2:2:5.5, giving a predicted sterile-filtrate inhibition rate of 48.02% (validated at 48.8-49.4%; n = 3); inhibitory activity remained stable after 10^7-fold dilution of the fermentation broth. An optimal fermentation medium (10.721 g/L yeast extract powder, 12.382 g/L beef extract, 7.696 g/L MgCl2) and culture conditions were subsequently established. Application of the consortium fermentation broth promoted tomato seed germination and seedling growth, increased leaf chlorophyll content and nitrate reductase activity, and induced earlier and stronger activity of the defense enzymes superoxide dismutase, peroxidase, catalase and phenylalanine ammonia-lyase following pathogen challenge. In a pot trial, the consortium gave a relative protective efficacy of 52.87% and a relative curative efficacy of 60.0% against tomato Fusarium wilt. These results indicate that the four-strain Bacillus consortium constructed here, combining endophytic and rhizosphere-derived isolates, is a promising candidate biocontrol resource for tomato Fusarium wilt, warranting further mechanistic and field-scale evaluation.