2.5. Quantification of the Bacterial Community by Quantitative PCR (qPCR)

The abundances of 16S rRNA and nifH genes were quantified by real-time PCR (qPCR) using the primers 6S-27F/338R and POLF/POLR (Table S1), respectively [21, 22, 30]. The qPCR reaction was performed in a CFX96 Touch Real-Time PCR (Bio-Rad) equipment, and all measurements were performed using the SYBR Green approach. The PCR mixture was 12.5 μl of the iQSYBR Green Supermix (Bio-Rad), 1 μM of each primer, and 4 to 25 ng of DNA template, within a total volume of 25 μl. The qPCR cycle for the 16S rRNA coding gene consisted of a denaturation step for 10 min at 95°C, 40 cycles for 15 s at 95°C, 30 s at 58°C, and 30 s at 72°C. The qPCR cycle for the nifH gene consisted of a denaturation step for 5 min at 95°C and 40 cycles for 10 s at 95°C, for 10 s at 59°C, and 30 s at 72°C. Product specificity was confirmed by melting curve analysis (58–95°C, 0.5°C per read, 5 s hold) and visualization in agarose gels, which showed specific product bands at the expected size of 180 bp for the 16S rRNA gene and 360 bp for the nifH gene.

For both genes, three replicates in duplicate were used. For the standard curve, triplicates were employed for every run using a known number of each gene from the genome of Herbaspirillum seropedicae SmR1 [31], from 6.62 × 10¹ to 6.62 × 10⁵ copies of the nifH gene and from 1.99 × 10³ to 1.99 × 10⁶ copies of the 16S rRNA encoding gene.

For the standard curve, mass concentrations of standard DNA were converted into copy concentrations using the following equation [32]:

For statistical analyses, an ANOVA test was performed using the InfoStat programme and in those circumstances where significant differences were confirmed, the means were compared using the Tukey test with a P < 0.05 [29].