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).