This study investigated the effects of soil physicochemical properties on bacterial community structure and function in tobacco rhizosphere (GJ) and bulk soil (GW), aiming to elucidate key environmental drivers and their mechanisms. Field experiments were conducted in five-year continuous tobacco cultivation fields in Lichuan City, Hubei Province. Soil samples were collected from healthy (JK) and diseased (FB) plants at 45, 75, and 105 days post-transplantation. Soil parameters, including pH, organic matter (OM), macronutrients (AN, P, K), and micronutrients (Fe, Mn) were analyzed. Bacterial communities were characterized via 16S rRNA gene sequencing, and interactive effects of microhabitat, soil status, and sampling time were assessed using linear mixed-effects models (LMMs). Results demonstrated that microhabitat exerted the strongest influence on bacterial diversity (β = 1.70 - 1.83, p < 0.0001), surpassing soil type and sampling time. Rhizosphere soils exhibited significant increases in AN, K, pH, TN, Ca, Mg, and Mn, but decreases in P, OM, Fe, and Cu. Microhabitat differentiation elevated the abundance of beneficial phyla including Chloroflexi (β = 1.9), Actinobacteria (β = 1.7), and Bacteroidetes (β = 1.3), while reducing Proteobacteria (β = ？1.6), Cyanobacteria (β = ？0.8), and Firmicutes (β = ？0.5). At the genus level, Bacillus (β = 0.35) and Streptomyces (β = 0.96) were enriched, whereas Pseudomonas (β = ？1.27) was depleted. Mantel tests revealed significant correlations between Fe, Mn, P, and microbial composition (r = 0.42 - 0.58, p < 0.01), suggesting micronutrients drive community assembly through direct metabolic regulation or indirect environmental modulation. These findings highlight that soil microhabitats critically shape root-associated microbial diversity and functional guilds via physicochemical property mediation, providing a theoretical foundation for optimizing soil health management and microbial ecological regulation in tobacco production systems.
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