Pathogen Growth Assays

Total RNA was extracted from each sample using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The abundance of the fungus (R. solani) was estimated by the ITS gene copy number through qPCR using the primer pair ITS1/ITS4 (White et al., 1990). The bacterial (R. solanacearum) abundance was estimated by the 16S rRNA gene copy number through qPCR using the primer pair Eub338 and Eub518 (Rasche et al., 2011). Amplification reactions were carried out with SYBR Premix Ex Taq (TaKaRa, Japan) in a total volume of 20 μL. Standard curves were obtained using serial dilutions of a known copy number of plasmids containing an ITS or 16S rRNA gene fragment, and these curves were linear from 9.77 × 10³ to 9.77 × 10⁸ gene copies/μL (R² = 0.998; ITS gene) and gene copies/μL (R² = 0.998; 16S rRNA gene). All samples were analyzed in triplicate.