2.5. QPCR Quantification of F. culmorum Biomass

Quantification of F. culmorum biomass was performed in samples from solid substrate (rice) cultures and in the kernel samples from semi-field trials by using a Real-Time PCR (qPCR) approach.

For the qPCR assay, DNA was isolated both from 50 mg powder-ground test samples and 50 mg F. culmorum powder mycelia as well as from 50 mg each serial dilution of mixture of F. culmorum powder mycelia and F. culmorum-free rice/wheat kernels, using the Wizard^® Genomic DNA Purification Kit (Promega, Madison, WI, USA). Homogeneous, powdered F. culmorum mycelium was obtained by culturing the KF846 strain on a PDA medium supplemented with 100 μg/mL streptomycin, lyophilization and grinding. Mixed samples were obtained from 10 mg of F. culmorum mycelia powder and 990 mg of F. culmorum-free rice/wheat kernel powder. A series of 10-fold dilutions (from 1 mg/g to 0.01 mg/g) of this mixture were prepared by sequentially combining 100 mg of each mixture with 900 mg of F. culmorum-free rice-wheat kernel powder. DNA obtained from pure mycelium and mixed samples was used initially to confirm the specificity and sensitivity of the qPCR assay and to optimize the reaction, including primer concentration and annealing temperature by analyzing the amplification plots, dissociation curves and excluding the primer dimers formation.

Finally, a Real-Time PCR was performed in 10 μL using 7.5 μL AmpliQ Real-Time PCR Opti Probe Kit (Novazym, Poland) and 100 nM of FAM-labelled probe and 300 nM of forward and reverse primers proposed by Waalwijk et al. [44]. Reactions were run using the C1000 Thermal Cycler with CFX96 Real-Time System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The established thermal cycling parameters were 2 min at 50 °C, 95 °C for 2 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s. Nuclease-free water (Merck KGaA, Darmstadt, Germany) was used as the no-template control. The positive control was F. culmorum genomic DNA. A standard curve was generated by plotting the Ct value for each sample of a standard series of the amount of fungal biomass. All samples were tested in triplicate.