Metagenomic Insights into Microbial Community Response to <i>Melilotus officinalis</i> Green Manuring in Degraded Steppe Soils

Single-season legume green manuring is widely promoted for soil fertility restoration in degraded agricultural lands, yet its effectiveness in alkaline semi-arid soils remains poorly understood. This study investigated the impact of first-year sweet clover (<i>Melilotus officinalis</i> (L.)) green manuring on soil microbiome structure and agrochemical properties in southern carbonate chernozem soils of Northern Kazakhstan. Using shotgun metagenomics, we analyzed microbial communities from sweet clover-amended soils, clean fallow, and virgin steppe reference sites. Contrary to expectations, sweet clover green manuring did not enhance soil nitrogen availability, with nitrate-N content (9.1 mg/kg) remaining lower than clean fallow (10.5 mg/kg), likely due to temporary immobilization during initial decomposition. While sweet clover significantly increased archaeal diversity (<i>p</i> = 0.01) and enriched nitrogen-cycling taxa, including <i>Nitrospirae</i> and <i>Thaumarchaeota,</i> overall microbial richness remained unchanged (ACE index, <i>p</i> > 0.05). Surprisingly, functional analysis revealed only five significant metabolic differences between sweet clover and fallow systems, indicating functional convergence of agricultural microbiomes regardless of management practice. Correlation analysis identified phosphorus as the master regulator of microbial metabolism (r = 1.0, <i>p</i> < 0.0001), while elevated pH (9.0), K₂O (>1000 mg/kg), and <inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mrow><mo mathvariant="normal">N</mo><msubsup><mrow><mo mathvariant="normal">O</mo></mrow><mrow><mn>3</mn></mrow><mrow><mo>−</mo></mrow></msubsup></mrow></semantics></math></inline-formula> showed strong negative correlations with essential metabolic pathways, revealing previously unrecognized nutrient toxicity thresholds. Virgin steppe maintained 69 unique metabolic pathways lost in agricultural systems, highlighting the ecological cost of cultivation. These findings demonstrate that sweet clover green manuring in alkaline steppe soils induces selective rather than comprehensive microbiome restructuring, with limited immediate benefits for soil fertility. This study provides critical insights for developing sustainable agricultural practices in the world’s extensive semi-arid regions facing similar edaphic constraints.