Abstract
Tobacco Fusarium wilt (TFW), caused by Fusarium spp., is a destructive disease affecting tobacco crops and resulting in significant economic losses. The current chemical fungicide-based management strategy poses a risk of resistance development in pathogens. In order to identify key factors in the tobacco rhizosphere for sustainable TFW control, untargeted metabolomics and microbiomics were conducted to analyze the key metabolites and microbes between healthy and diseased rhizosphere soils. The results revealed a total of 65 differential metabolites (DMs), with 40 upregulated and 25 downregulated were identified in the diseased group. Among these, Aesculin, 8-Deoxy-11-hydroxy-13-chlorogrosheimin, and N-Gluconyl ethanolamine phosphate were identified as key DMs exhibiting a strong negative correlation with healthy soil status. The microbiome analysis revealed that bacterial diversity (OTUs: 3278 vs. 3039; Shannon: 6.72 vs. 6.65) and fungal diversity (OTUs: 919 vs. 701; Shannon: 3.91 vs. 3.44) were significantly higher in healthy soils. Furthermore, co-occurrence networks in healthy soils were larger and more stable, with bacteria comprising 431 nodes compared to 405 in diseased soils, and fungi comprising 112 nodes compared to 101 in diseased soils. Additionally, the analysis of the microbial community assembly process revealed that deterministic processes dominated microbial assembly in healthy soils during the first 75 d, subsequently shifting toward stochastic processes. In contrast, diseased soils exhibited a consistently deterministic assembly. Furthermore, we observed that variations in soil types influenced the distribution of key microbial groups. Chloroflexi, Bryobacter, Bacillus, Preussia, and Tausonia, were enriched in healthy soils while Lysobacter, Arthrobacter, Fusarium, and Lectera were dominated in diseased soils. The combined analysis results indicated that the three key DMs correlated positively with disease-enriched microbes but negatively with health-associated taxa. This study provides insights into the changes in and microbe-metabolite interactions within the tobacco rhizosphere under pathogen stress and supports the future development of TFW control strategies based on key metabolites and microbes. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1038/s41598-026-40653-w.