2.5. Generation of Standard Curve and phlD Quantification in Samples

Quantitative PCR with SYBR green technology was used to quantify the phlD gene copy numbers from the root and rhizosphere soil samples of OSR and wheat plants. A standard curve of phlD copy number was generated using genomic DNA of the phlD P. ogarae F113 bacterial strain. Genomic DNA of this bacteria was serially diluted ten-fold in three separated series to obtain standards from 3 × 10⁶ to 30 fg DNA µL-1. One microliter of each standard dilution (i.e., from approximately 4 × 10⁵ to 4 phlD copies) was used for qPCR analysis. QPCR assays were conducted using 96-well white microplates, Roche SYBR green master mix in a final volume of 10 µL and a LightCycler 96 (Roche Applied Science, Meylan, France).

The reaction mixture contained 5 µL of Roche SYBR Green I Master Vial 1, 3 µL of Vial 2 (Roche Applied Science), 0.5 µL of primer B2BF (1 µM), 0.5 µL of primer B2BR3 (1 µM) and 1 µL of DNA. The sequences of the primers used are detailed in Table 1. The final cycling program included a 10 min incubation at 95 °C, 50 amplification cycles of 30 s at 94 °C, 7 s at 67 °C and 15 s at 72 °C. Amplification specificity was checked by melting curve analysis of the amplification product using a fusion program consisting of an initial denaturing step of 5 s at 95 °C, an annealing step of 1 min at 65 °C and a denaturing temperature ramp from 65 to 97 °C with a rate of 0.11 °C s⁻¹. The cycle threshold (Ct) of each individual sample was calculated using the second derivative maximum method in the LightCycler 96 software v 1.5 (Roche Applied Science).

Primers used for phlD qPCR optimization.

The standard curve was obtained by plotting the mean Ct value of the three replicates (per DNA concentration) against the log-transformed DNA concentration. Amplification efficiency (E), calculated as E = 10 (−1/slope) −1, and the error of the method (mean squared error of the standard curve) were determined using the LightCycler software v1.5 (Roche Applied Science). The equivalence between DNA amount and phlD copy number was estimated based on (i) a Pseudomonas genome of approximately 7.26 fg DNA and (ii) the occurrence of one phlD copy per genome. The detection limit was determined as the number of phlD copies giving 3 positive results out of 3 replicates. The amplification curve, melting curve and standard graph are displayed in Supplementary Figure S1. Melting curve calculation and Tm determination were performed using the Tm Calling Analysis module of LightCycler 96 software v1.5 (Roche Applied Science). The standard curve thus generated from genomic DNA of P. ogarae F113 was subsequently used as the external standard curve for determination of the phlD copy number in DNA samples. The equimolar concentration (10 ng µL⁻¹) of the DNA samples of root and rhizosphere soil were analysed by qPCR in triplicate (following the above protocol), and the mean Ct value was reported in the external standard curve to infer the phlD copy number in the sample, using the LightCycler 96 software and the ‘standard curve’ option for the absolute quantification. Positive control (P. ogarae F113 genomic DNA 30 ng µL⁻¹), water control and three DNA standards from the genomic DNA of P. ogarae F113 (3000 pg, 30 pg and 0.3 pg corresponding to approximately 4 × 10⁵, 4 × 10³ and 40 copies) in triplicate were included as a reference in each run to detect between-run variations.