Soil extracellular DNA fragments show variable degradation rates among sequences and environmental conditions

While extracellular DNA (eDNA) persistence substantially influences soil microbiome investigations, its degradation kinetics remain poorly quantified. Here, we developed a primer-labeled DNA approach coupled with microcosm incubation to determine the overall and sequence-specific degradation rates of eDNA amplicon fragments across China. We observed substantial variations in the overall degradation rates of extracellular 16S rRNA gene amplicon fragments among the study sites, with degradation rate constants ranging from 0.05 to 0.16 day −1 . The overall degradation rate constants showed significant correlations with soil moisture content, prokaryotic abundance, prokaryotic community profiles, and mean annual precipitation. The significant influences of moisture content on the overall degradation rates were further verified by a moisture gradient microcosm experiment. The sequence-specific degradation rate constant profiles were additionally correlated with pH, nitrogen content, and mean annual temperature. Furthermore, propidium monoazide-based exclusion of eDNA signals significantly altered soil prokaryotic abundance, richness, and prokaryotic community profiles, and the pool sizes of sequence-specific extracellular 16S rRNA gene amplicon fragments were significantly correlated with their respective degradation rates. This study developed a methodology for determining the overall and sequence-specific degradation rates of eDNA amplicon fragments, highlighting the profound influences of eDNA on soil microbial research and informing the optimization of environmental DNA technologies.