Long-Term Pepper ( Capsicum annuum L.) Monoculture Reshapes Rhizosphere Soil Chemistry, Microbiota, and Metabolite Profiles

Continuous monoculture alters rhizosphere soil conditions and microbial community structure, but the integrated soil biochemical, microbial, and metabolomic responses of pepper ( Capsicum annuum L.) rhizosphere soils remain insufficiently characterized. Here, we compared uncropped/non-continuously cropped pepper soil (Y0) with soil under 10 years of pepper monoculture (Y10) using soil physicochemical assays, enzyme measurements, 16S rRNA and ITS amplicon sequencing, untargeted UHPLC-Q Exactive HFX metabolomics, and predictive functional profiling. Long-term monoculture markedly separated Y10 from Y0 in multivariate analyses. Y10 soils showed higher organic matter, available nitrogen, available phosphorus, available potassium, and electrical conductivity in soil-water extracts, whereas microbial biomass carbon and pH were lower. Soil enzyme profiles also differed between treatments. Microbial alpha diversity declined under Y10, and bacterial and fungal community structures were clearly separated between treatments. At the taxonomic level, Acidobacteriota and several oligotrophic bacterial taxa were relatively enriched in Y0, whereas Proteobacteria, Bacteroidota, Chloroflexi, Bacillota, Pseudomonas , Bacillus , and several fungal genus-level taxa increased in Y10. Untargeted metabolomics revealed extensive remodeling of rhizosphere metabolites, with 413 up-regulated and 21 down-regulated differential metabolites in Y10. Differential metabolites were mainly associated with carboxylic acids and derivatives, benzene and substituted derivatives, fatty acyls, aromatic-compound transformation, sulfur metabolism, alkaloid biosynthesis, and microbial metabolism. Correlation analyses further linked key microbial taxa with soil pH, microbial biomass carbon, available nutrients, electrical conductivity, and enzyme activities. These results indicate that long-term pepper monoculture is associated with coordinated shifts in soil chemical status, microbial community composition, and metabolite profiles. The study provides an integrated basis for understanding rhizosphere changes under pepper continuous-cropping systems while recognizing that functional predictions and metabolite annotations require experimental validation.