6. Tgam interactions in FHB scenario
Wheat seeds were sown in pots in a potting mix and incubated in a growth
chamber with photoperiod 16 h light/8 h dark, at 20℃/22℃ respectively,
until plants reached the anthesis stage (5 weeks). Three biological
replicates of three plants each were inoculated per each condition, i.e.Tgam alone, Fgra alone and Tgam + Fgratheses. For Tgam inoculation, spores were collected by washing
1-week-old PDA plates with 20 mL of sterile 0.01% Tween-80 solution,
and a 107 spores mL-1 suspension was
sprayed on spikes of Tgam alone and Tgam + Fgraplants. Plants were covered with a white bag, previously moistened
inside with water to maintain humidity, and with a black bag to
facilitate penetration by the fungus (Dufresne M., personal
communication). Plants were incubated in a growth chamber for 48 h in
the same conditions described above. For inoculation of the pathogen,
conidia of Fgra were collected by washing 2-week-old PDA plates
with 20 mL of sterile 0.01% Tween-80 solution, and a
105 spores mL-1 suspension was
sprayed on spikes of Fgra alone and Tgam + Fgraplants. Plants were covered with a white bag, previously moistened
inside with water, and a black bag was placed above to facilitate
penetration by the pathogen. Plants were incubated in a growth chamber
in the same conditions described above for additional 24 h. Bags were
then removed for plant aeration and after 1h, white bags were placed
back for additional 24 h. Six days after inoculation of Fgra , ten
spikes colonized by fungi were collected from each condition, frozen in
liquid N2 and stored at -80℃ until RNA extraction.
Reduction of FHB symptoms was evaluated by calculating the percentage of
healthy and symptomatic spikelets (Disease severity – DS), inFgra alone and Fgra + Tgam plants. Differences on
DS values were determined statistically by ANOVA after angular
transformation, (P value (P) < 0.05).