Abstract:[Background] Plant growth-promoting agents have been widely used in agricultural production, while the knowledge to their impacts on the root microbial community of crops in substrate culture is limited. [Objective] The objective is to investigate the effects of Pseudomonas sp. JP2-3 and Bacillus subtilis Z54 on the growth and root bacterial community of tomato in substrate culture.[Methods] The two strains were inoculated by root dipping and root irrigation for the tomato plants in substrate culture. We then measured the growth indexes of tomato seedlings to explore the effect of growth promoting bacteria on tomato growth. The strain effects on the composition and structure of the root bacterial community were analyzed by 16S rRNA gene amplicon sequencing technology.[Results] The two strains promoted tomato growth. Four growth indexes (plant height, stem diameter, maximum leaf length, and maximum leaf width) in strain JP2-3 group were higher than those in CK group. Except stem diameter, the other three indexes in strain Z54 group were higher than those in CK group. Both strains JP2-3 and Z54 changed the α diversity of bacterial community and decreased the bacterial richness and diversity compared with the CK group. Particularly, the decrease was more significant in strain Z54 group (P<0.05). The β diversity of root bacterial community was significantly changed by strain Z54 while slightly affected by strain JP2-3. The dominant bacterial phyla included Proteobacteria, Actinobacteria, Firmicutes, and Bacteriodetes, and the dominant genera were Streptomyces, Pseudomonas, Ideonella, Devosia, etc. The composition of root bacterial community of tomato in substrate culture was similar to that in soil culture at the phylum level while different at the genus level. Inoculation with strain JP2-3 increased the relative abundance of Ideonella, Actinoplanes, and Aquincola, while strain Z54 increased the relative abundance of Brevundimonas and Flavobacterium. Among the genera with increased relative abundance, many bacteria are beneficial to plants. LEfSe showed that the biomarkers of strain JP2-3 group were mainly distributed in Betaproteobacteria and Actinobacteria, while those of strain Z54 group in Gammaproteobacteria. [Conclusion] Pseudomonas sp. JP2-3 and B. subtilis Z54 promoted the growth of tomato in substrate culture and had different impacts on the composition and structure of the bacterial community in tomato roots. The two strains increased the relative abundance of indigenous beneficial microorganisms and cooperated with indigenous beneficial microorganisms to promote tomato growth.