Tobacco–barley rotation reshapes soil microbial communities and enriches culturable bacteria with plant growth-promoting potential

Introduction Crop rotation is an important agronomic practice for alleviating the adverse effects of continuous tobacco cropping, yet the effects of preceding gramineous crops on the microbial communities and potential functions of tobacco-growing soils have not been systematically verified. Methods Based on a three-year continuous field experiment, this study used a flue-cured tobacco–fallow system (CK) as the control and established a flue-cured tobacco–barley rotation system (DM). Soil chemical properties and enzyme activities, bacterial 16S rRNA and fungal ITS amplicon sequencing, culturable bacterial isolation, and greenhouse pot reinoculation experiments were integrated to evaluate changes in the soil environment and microbial communities following barley rotation and to verify the plant growth-promoting potential of candidate enriched isolates. Results Compared with CK, DM significantly ( P < 0.05) increased soil dissolved organic carbon, available phosphorus, available potassium, and hydrolyzable nitrogen by 19.65%, 3.23%, 5.45%, and 5.90%, respectively; sucrase, β-glucosidase, acid phosphatase, and N-acetyl-β-D-glucosaminidase activities increased by 23.80%, 66.28%, 15.23%, and 176.32%, respectively. Amplicon sequencing showed that barley rotation significantly altered soil bacterial and fungal community. Sphingomonas and Burkholderia-Caballeronia-Paraburkholderia were relatively enriched under DM and were significantly associated with multiple soil nutrient and enzyme activity variables; therefore, they were defined as candidate enriched bacterial taxa. Six culturable isolates belonging to these taxa were subsequently obtained from DM soil. In vitro , these isolates exhibited different combinations of functional phenotypes, including growth on nitrogen-free medium, phosphorus transformation, potassium solubilization, zinc solubilization, siderophore production, and organic-substrate degradation. Greenhouse pot experiments showed that mixed inoculation significantly promoted flue-cured tobacco growth and increased plant nitrogen, phosphorus, and potassium accumulation. Conclusion Barley rotation was accompanied by significant changes in soil available nutrients, enzyme activities, and microbial community composition, whereas culturable isolates obtained from rotation-enriched taxa showed plant growth-promoting potential under controlled conditions. These findings support the possibility that rotation-enriched microorganisms participate in subsequent plant–soil nutrient feedbacks, although their direct mediating role in field rotation effects requires further verification. This study provides a theoretical basis and candidate microbial resources for understanding the microbial ecological effects of barley–tobacco rotation and screening potential plant growth-promoting bacteria.