The application of slow-release nitrogen fertilizer not only economizes labor input, but also decreases the frequency of use of mechanical intakes, with significant implications in advancing modern intensive agricultural production. Whether slow-release nitrogen fertilizer application can influence the association between microbial diversity and soil multifunctionality remains controversial. This study analyzed the spatial variances of soil environmental factors, soil multifunctionality, and their correlations with bacterial and fungal communities under five nitrogen application rates. The key factors influencing the dominant microbial species and community structures at different spatial locations were determined by the slow-release nitrogen fertilizer application rate, and the driving factors and dominant species of soil multifunctionality were identified. In contrast to the control group, moderate slow-release nitrogen fertilizer application enhanced soil multifunctionality and ameliorated the resilience of microbial diversity loss at diverse spatial locations resulting from irrational nitrogen fertilizer application. The resilience of the fungal community to disturbances caused by fertilization was lower than that of the bacterial community. Bacterial diversity exhibited a significant correlation with soil multifunctionality, and the soil multifunctionality intensity under 240 kg ha−1 treatment increased by 159.01% compared to the CK. The main dominant bacterial communities and the dominant fungal community Ascomycota affected soil multifunctionality through slow-release nitrogen fertilizer application. Structural equation modeling and random forest analysis demonstrated that bacterial community diversity, particularly in bulk soil and the rhizosphere, community composition, and soil nitrogen form are the primary driving factors of soil multifunctionality. Results indicated that the microbial niche alterations induced by slow-release nitrogen fertilizer application positively affect soil multifunctionality.
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