2.6. Quantitative PCR

The quantification of functional genes involved in the nitrogen cycles and 16S rRNA genes (total bacterial abundance) was performed on bulk soil samples only. The same DNA extracts as described above were used. Quantitative PCR (qPCR) was conducted on a Rotor-Gene 3000 (Corbett Research, Sydney, Australia) by using Absolute qPCR SYBR Green Mix (ThermoFischer Scientific). The different primer pairs and the PCR conditions used to amplify the genes are listed in Table S2. Standards for the different genes were prepared from pure cultures or environmental clones as described by Kampmann et al. [44], and 10-fold serial dilutions of the standards were used as template, in triplicate, to determine the calibration curves. Total gene copy numbers of the standards were calculated according to Kampmann et al. [44]. For comparison of the abundance, the target numbers of the functional nitrogen cycles genes were expressed as relative to the total 16S rRNA gene copy number. Statistical comparisons were done with ANOVA and Tukey HSD Test, using SPSS version 20. The model included the factors “treatment”, “soil habitat”, their interaction, and “block” as random factor.