Abstract: In order to explore the culturable core microbial communities in tobacco and obtain new strains with high antagonistic activity, so as to provide more microbial resources for the prevention and control of tobacco diseases, healthy tobacco plants from four ecological regions (Nanling hilly, Wuyi hilly, Qiangui Mountain and Yimeng hilly ecological region) were taken as research objects. After collecting samples from tobacco roots, stems and leaves, the tobacco culturable bacteria were isolated and cultured by traditional culture method, and species identification was carried out through 16S rRNA gene sequencing to analyze the core microbial communities. Meanwhile, the antagonistic ability of the strain against the tobacco black shank disease pathogen Phytophthora nicotianae was determined and a strain with high antibacterial activity was selected to verify its control effect by pot trials. The results showed that 856 strains of bacteria were obtained from four ecological regions, belonging to 5 phyla, 8 classes and 56 genera. Firmicutes and Proteobacteria were dominant at the phylum level, and Bacillus and Pseudomonas were the main genera at the genus level. The tobacco culturable bacterial communities were different in different ecological regions, but the main core bacteria communities were Bacillus, Pseudomonas and Priestia. Besides, there were 190 strains with antagonistic ability of black shank disease selected from 856 culturable strains of bacteria. They belong to 4 phyla, 6 classes and 14 genera, mainly dominated by Bacillus and Pseudomonas. Among them, we discovered a novel strain of Pseudomonas citri GT03. The crude extracts (1 mg/mL) of its fermentation broth could completely inhibit the growth of the pathogen of black shank disease, Phytophthora nicotianae. Pot experiments showed that the control efficiency of strain GT03 on black shank disease reached 79.87%, demonstrating great potential for development and utilization.