Standard curves for each assay were generated by serial dilutions of linearized plasmids with cloned fragments of 16S rRNA genes from Pseudomonas aeruginosa PAO1, nirS from Pseudomonas stutzeri (ATCC 14405), nirK from Sinorhizobium meliloti 1021, nosZI from Bradyrhizobium japonicum USDA 110, and nosZII from Gemmatimonas aurentica 27-T. Standard curves were linear (R2 ¼ 0.999) in the range used and amplification efficiency was 97% for the 16S rRNA gene and varied between 73 and 85% for the functional genes.