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.