Ammonia oxidizer abundances
AOA and AOB amoA copy numbers ranged from 9.3×108~2.69×109 and 8.08×106~3.97×108copies per g soil during the N addition period (Fig. 1a, b). Nutrient addition did not significantly affect AOA amoA copy numbers but increased those of AOB. AOA and AOB displayed a similar pattern during the N addition cessation period (Fig. 1c, d). AOA amoA copy numbers remained constant and ranged from 4.05×108~3.95×109copies g-1soil, while AOB amoA copy numbers increased significantly in previously fertilized plots. AOB amoA gene copy numbers were ten times higher in R90 than in the unfertilized plots (R0). Since their discovery as ammonia oxidizers in 2015, comammox strains have only been identified after terminated nutrient addition. Comammox Nitrospira clade A and B ranged from 4.32×104 to 5.30×105 copies g-1 soil, lower than AOA and AOB. Comammox Nitrospira clade B decreased in R60 but remained unchanged in other treatments (Fig. 1e, f).
AOB amoA copy numbers positively correlate with NH4+-N (r= 0.74, p < 0.001) and NO3-N (r =0.67, p < 0.001) in the N addition plots, whereas AOB amoA copy numbers were significantly negative correlated with soil pH (r = -0.47, p < 0.05) (Table S3). In contrast, AOB amoA copy numbers positively correlated with microbial biomass N (r= 0.49, p < 0.05) and negative correlated with total N (r= -0.46, p < 0.001), total C (r= -0.52, p < 0.001), and NH4+-N (r=- 0.40, p < 0.05) after nutrient addition ceased (Table S3).