5. Liquid cultures of Tgam
Mycelium of Tgam was obtained following a two-step liquid culture procedure. Spores of Tgam were collected from 1-week-old PDA plates and inoculated in 50 mL flasks containing 25 mL of minimal medium (MM) 0.9% sucrose (9 g L-1 sucrose, 1 g/L NH4NO3, 5 g L-1C4H12N2O6, 1 g L-1 K2HPO4, 0.5 g L-1 MgSO4, 0.13 g L-1 CaCl2, 0.1 g L-1NaCl, 0.0183 g L-1 FeSO4, 0.0035 g L-1 ZnSO4, 0.002 g L-1 MnCl2) at a final concentration of 106 spores ml-1. Flasks were incubated at 28℃ on a rotatory shaker at 180 rpm for 60 h. Mycelium was collected by centrifugation at 10000 rpm for 10 min and supernatants were discarded. Mycelial pellet was resuspended in sterile distilled water and centrifugated at 10000 rpm for 10 min to wash it. Mycelium was then inoculated in 50 mL flasks containing 25 mL of MM, MM 0.9% sucrose, and MM modified by adding different stressors, such as 0.5 mM H2O2, only 0.01% of NH4NO3 and C4H12N2O6(N starvation), and 200 mM NaCl. Flasks were incubated at 28℃ on a rotatory shaker at 180 rpm for 4 days. Mycelium was collected by filtration using Miracloth (475855-1R, Millipore), frozen in liquid N2 and stored at -80℃ until RNA extraction. Three independent biological replicates were included for each condition.