Role of nitrate-dependent anaerobic methanotrophs in reducing methane emissions under delayed flooding in straw-amended paddy soil

Abstract Straw incorporation is commonly adopted to improve soil fertility in rice paddies, yet it leads to substantial increases in methane (CH 4 ) emissions. Although water management is recognized as an effective mitigation strategy, the impacts of delayed flooding on CH 4 emissions and the underlying microbial mechanisms under straw incorporation remain poorly understood. To fill this knowledge gap, a laboratory mesocosm study was conducted involving three straw treatments (no straw, maize leaf, and maize stem) and two water regimes (direct and delayed flooding). Results showed that straw addition significantly increased CH 4 emissions, with greater stimulation observed in leaf-amended soil. Relative to direct flooding, delayed flooding reduced CH 4 emissions by 83.3% and 85.6% in leaf- and stem- amended soils, respectively. Delayed flooding significantly decreased methanogenic mcrA and aerobic methanotrophic pmoA gene abundances but increased M. nitroreducens -like archaeal mcrA abundance, with no significant effect on M. oxyfera -like bacterial pmoA . CH 4 emissions were positively associated with methanogenic mcrA abundance and negatively associated with M. nitroreducens -like archaeal mcrA abundance. Random forest identified methanogenic mcrA abundance as the strongest predictor of CH 4 emissions. SHAP analysis linked higher methanogenic mcrA abundance to higher predicted emissions, whereas higher NO 3 ⁻-N content and greater M. nitroreducens -like archaeal mcrA gene abundance were generally associated with lower predicted emissions. PLS-PM analysis further revealed that the reduced CH 4 emissions caused by delayed flooding under straw addition was primarily mediated by increasing NO 3 ⁻-N content, which subsequently stimulated M. nitroreducens -like archaea. Collectively, our findings demonstrate that delayed flooding serves as an effective strategy to mitigate CH 4 emissions in straw-incorporated paddy ecosystems.