*2.6. Real-Time Quantitative PCR Analysis (qPCR)*

qPCR was performed in triplicate to evaluate bacterial 16S rRNA gene copy numbers using an Applied Biosystems 7500 detection system (Applied Biosystems, Foster City, CA, USA) with 341 primer pairs, forward (5 -CCTACGGGNGGCWGCAG-3 ) and 785 primer pairs, reverse (5 -GACTACHVGGGTATCTA ATCC-3 ) [26]. The reaction mixture (20 μL) and amplification conditions were performed based on the methods of Pascazio et al. [27]. Reference strain DNA of *Azospirillum irakense* [28] was used to create the standard curve and the gene copy number in the samples was calculated using the regression equation that related the cycle threshold (Ct) value to the number of copies in the standard curve [29].
