Quantitative PCR determination of Trichodermaconcentration in rhizosphere soil
At the end of the trial, rhizosphere soil was collected by shaking the
roots collected from ten plants per plot. To determine theTrichoderma concentration in the rhizosphere, DNA was extracted
from the soil samples with a DNeasy®Powersoil® kit (Cat. No. 12888-50; Qiagen, Hilden,
Germany) according to Qiacube (Qiagen, Hilden, Germany) automation
procedures. To amplify the transcripts, the genus-specific primer pair
RM3/RM4 was used to detect Trichoderma spp. Every 4-µL DNA sample
was amplified in a 20-μL reaction system in the presence of 10 μL
QuaniNovaTM SYBR® Green Supermix
(2x) and 0.14 µL of 25 µM primers on a Rotor-Gene Q (Qiagen, Hilden,
Germany). The qPCR cycling conditions were as follows: initial
incubation at 95 °C for 2 min, 45 cycles of 95 °C for 5 s each, and 60
°C for 12 s. Two technical replicates were performed per sample. After
qPCR, the absolute values for the transcript quantities were calculated
by interpolation of standard curves plotted using serial DNA dilutions,
namely, undiluted and 1:1,000, 1:10,000, and 1:100,000 dilutions of
standardised liquid culture at a total Trichoderma spp.
concentration of 109 (CFU) mL-1.