Six-Year Biochar Experiment Reduces Soil N₂O Emissions in Eucalyptus Plantations: Associations with Microbial N-Cycle...

Nitrous oxide (N₂O) is a major greenhouse gas, and terrestrial ecosystems are among the primary sources of its emissions. Biochar is recognized as an effective soil amendment for mitigating N₂O emissions, but its long-term residual effects and microbial mechanisms in subtropical plantations remain unclear. Therefore, this study evaluated the residual effects of Eucalyptus-derived biochar on soil N₂O emissions six years after a single application and explored associations with nitrogen cycle functional genes. A field experiment was conducted in a Eucalyptus plantation in northern Guangxi with biochar applied at six rates (0-6% w/w). Soil N₂O fluxes were measured in the fifth and sixth years (2022-2023); soil chemical parameters, soil enzyme activities, and N₂O-related microbial functional genes (amoA, nirK, nirS and nosZ) abundance were analyzed. Biochar application significantly reduced ammonium nitrogen content but enhanced nitrate nitrogen content. Urease, protease, and sucrase activities increased, while nitrate reductase, nitrite reductase, and hydroxylamine reductase activities decreased. Furthermore, quantitative analysis revealed substantial variations in functional gene abundances. The abundance of ammonia-oxidizing archaea (AOA-amoA) exhibited a unimodal response, whereas ammonia-oxidizing bacteria (AOB-amoA) showed a robust dose-dependent accumulation. Notably, annual N₂O emissions were suppressed by up to 35.2%, driven by a 3.4-fold increase in nosZ gene abundance and a significant reduction in the (nirK + nirS)/nosZ ratio. This mitigation was attributed to enhanced N₂O consumption by nosZ-harboring denitrifiers and reduced heterotrophic ammonia oxidation. Overall, these findings highlight the pivotal role of long-term organic amendments in steering nitrogen transformation pathways, providing a theoretical basis for sustainable soil management in subtropical plantations.