sharply in the first week of incubation with an average rate of 5.98 mg N kg_1 day_1 and only a small amount of ammonium (1.24 _ 0.56 mg N kg_1) was left for nitrification. The change of NH4+-N was similar for RNT, as the NH4+-N decrease slowed down through the first two weeks with a significantly lower average nitrification rate of 2.57 mg N kg_1 day_1 for RNT than CT (p = 0.002; F = 27.660). NH4+-N concen-trations did not vary significantly in the last two weeks of incubation (p = 0.065; F = 5.087)
3.3. Impacts of tillage method on N nitrification dynamics
After pre-incubation, (NH4)2SO4 (264 mg kg_1 dry soil) was added to investigate nitrification dynamics. For CT, NH4+-N concentrations decreased sharply in the first week of incubation with an average rate of 5.98 mg N kg_1 day_1 and only a small amount of ammonium (1.24 _ 0.56 mg N kg_1) was left for nitrification. The change of NH4+-N was similar for RNT, as the NH4+-N decrease slowed down through the first two weeks with a significantly lower average nitrification rate of 2.57 mg N kg_1 day_1 After the addition of 264 mg (NH4)2SO4 kg_1 dry soil, NO3_-N increased sharply in the first week of incubation with an average nitrification rate of 10.7 mg N kg_1 day_1 for CT, then increased again until the end of incubation. Similar nitrate accumulation patterns were also observed for RNT (Fig. 2b). The net nitrification rate was 13% lower for RNT than CT during the 28-day incubation. During the first week of incubation, the average nitrification rate was 6.68 mg N kg_1 day_1 for RNT, which was 42% lower than CT. Significant increases in NO3_-N concentrations lasted for the first two weeks, then increased slightly until the end of incubation for RNT. Though the nitrification rates were different for CT and RNT, the dynamic changes of nitrification were similar. Regression analysis showed that the time-dependent kinetics of net nitrification was best fitted by first-order kinetic model for both CT and RNT. Potential nitrification (Np) did show significant differences between CT and RNT (p = 0.045; F = 6.391). Potential nitrification rates (Vp) and nitrification reaction rates (k1) were also two times higher for CT than for RNT (Table 2).
3.4. Abundance of ammonia-oxidizing bacterial (AOB) and ammonia-oxidizing archaeal (AOA) amoA gene copies
After pre-incubation, AOB amoA gene copy numbers were two times higher for RNT than CT (p < 0.05). The dynamic changes of AOB abundance during incubation showed similar trends for CT and RNT (Fig. 3A). AOB abundance increased to its highest numbers after 2 weeks’ incubation, then decreased in the last two weeks of incubation for both tillage regimes. The dynamic change of AOB amoA gene copy numbers for treatments with (NH4)2SO4 application had similar trends to the treatments without N application (Fig. 3B). AOB amoA gene copy numbers were significantly higher for RNT than CT after pre- incubation (p < 0.05), and all increased to their highest numbers after 2 weeks’ incubation, then decreased in the last two weeks.
AOA amoA gene copy numbers were 27.6 and 18.2 _ 106 copies g_1 dry soil for RNT and CT after pre-incubation, respectively, and were significantly different for soils with different tillage regimes, and AOA numbers were also significantly higher than AOB (p < 0.05). AOA dynamics during incubation showed similar trends for all soils, with or without NH4+ application (Fig. 3C and D). 3.5. Soil pH changes in mineralization and nitrification dynamics
The pH changes during 28 d mineralization and nitrification are shown in Fig. 4a and b, respectively. The pH of RNT was significantly lower than CT by 0.5 units (p < 0.05) during the incubation. The dynamic changes of soil pH in mineralization and nitrification showed a similar declining pattern for RNT and CT. Generally, soil pH decreased during the first 3 weeks of incubation. The largest pH decrease in mineralization was about 0.6 units, whereas for nitrification it was 1.1 units.
4. Discussion
Present results provide evidence to support our hypothesis that soil nitrification rates decreased after 22 years of RNT treatment compared to CT. Soil samples were collected from field, and chemical and microbial analysis performed in the laboratory reflected the soil’s state after long-term imposition of tillage regimes, thus the measured differences in soil properties between tillage regimes reflect field conditions and the response of the microbial community to long-term tillage management. Soil nitrification rates decreased after 22 years of RNT treatment, which may prove that RNT is a promising management technique for improving soil fertility and preventing N from leaching. Microbial ammonia oxidation is the first and rate-limiting step of nitrification, a primary process controlling soil nitrate concen-trations. The key enzyme for chemolithotrophic ammonium oxidation is the membrane-bound AMO for which ammonia (NH3), rather than ammonium (NH4+), is the substrate (Suzuki et al., 1974). Therefore, factors affecting NH3 and nitrifying microorganisms will affect the nitrification process. In the