Experimental Design
The long-term fertilization experiment was established on a flat field in the Walaka experimental site in March 2000 using a randomized block design. Twenty-five 6×10 m2 plots of five fertilization levels with five replicates were arranged in a five-by-five matrix. A 1 m buffer strip separated the plots. The fertilization treatment was generated with different amounts of (NH4)2HPO4 fertilizer applied annually at the beginning of the growing season (usually in mid-May) starting in 2000. Fertilizer applications of 0, 30, 60, 90, and 120 g m−2 yr−1 are F0, F30, F60, F90, and F120, respectively. In 2015, N addition ceased to observe recovery dynamics. The corresponding recovery observations are R0, R30, R60, R90, and R120.
In September 2010, the ammonia oxidizers’ (AOA and AOB) response and abundances to long-term fertilization were examined. Plant biomass, plant N concentration, soil total C and N, soil extractable inorganic N pools, pH, and ammonia oxidizers (AOA and AOB) abundances were measured. In September 2021, we examined ammonia oxidizers (AOA, AOB, CladeA, and CladeB) and their communities in Tibetan alpine meadow soils that stopped receiving fertilizer for 7 years.