Two-week-old Arabidopsis plants were inoculated with V. dahliae strain JR2 as described above. After visible symptom development at 19–29 d post-inoculation, for each experiment and for each Arabidopsis genotype all above-ground tissues were harvested per plant and flash-frozen in liquid nitrogen. The samples were ground to a powder, of which an aliquot of approximately 100 mg was used for DNA isolation (Fulton et al., 1995 ). Quantitative real-time PCR was conducted using an ABI7300 PCR machine (Applied Biosystems, Foster City, USA) with the qPCR Core kit for SYBR Green I (Eurogentec Nederland BV, Maastricht, NL). To measure V. dahliae biomass, the internal transcribed spacer region of the ribosomal DNA was targeted using the fungus-specific ITS1-F primer (AAAGTTTTAATGGTTCGCTAAGA; Gardes and Bruns, 1993 (link)) in combination with the V. dahliae-specific reverse primer ST-VE1-R (CTTGGTCATTTAGAGGAAGTAA; Lievens et al., 2006 ), generating a 200 bp amplicon. For sample equilibration, the Arabidopsis large subunit of the RuBisCo gene was targeted using the primer set At-RuBisCo-F3 and -R3 (GCAAGTGTTGGGTTCAAAGCTGGTG and CCAGGTTGAGGAGTTACTCGGAATGCTG, respectively), generating a 120 bp amplicon. Real-time PCR conditions consisted of an initial 95 °C denaturation step for 4 min, followed by 30 cycles of denaturation for 15 s at 95 °C, annealing for 30 s at 60 °C, and extension for 30 s at 72 °C. The average fungal biomass was determined using at least four Verticillium-inoculated plants for each genotype.