Absolute microbiome profiling highlights the links among microbial stability, soil health, and crop productivity under long‑term sod‑based rotation

High-throughput sequencing has become a critical tool for studying microbiomes by measuring relative microbiome profiling, although this typically overlooks the absolute abundance of microbiomes. Consequently, pathological, physiological, and ecological roles of microbial communities may be represented inaccurately. To address this, we estimated absolute abundances of soil microbiomes by combining amplicon sequencing with quantitative PCR. We collected soil samples (0–30 cm) at three sampling times (pre-planting, flowering, and maturity) from peanut plots subject to a long-term conventional rotation (peanut-cotton-cotton, CR) or sod-based rotation (bahiagrass-bahiagrass-peanut-cotton, SBR). Rotation and sampling time were important in shaping microbial communities. Relative to CR, SBR had greater microbial diversity, greater community stability, complexity and stability of bacterial-fungal networks, and greater richness and abundance of keystone taxa, which may make soil microbiomes more resilient to environmental changes among sampling times. SBR also showed significantly greater concentrations of total C and N, NO3−-N, resin-extractable P, Mg, Zn, Fe, and Cu, and greater potential N mineralization rates and C:N ratios, indicating that SBR’s higher rotational diversity affected soil health in the topsoil. There were more significant relationships between soil nutrients and microbial community composition as well as keystone taxa under SBR, indicating that higher rotational diversity intensified ecological connections among soil, microbes, and crops. Our results suggest that a more complex and stable microbial network with greater richness and abundance of keystone taxa (primarily bacterial communities) had critical impacts on nutrient cycling and plant health and fitness under SBR, which are the main factors contributing to crop productivity.