2.2 │ Pathogen culture and plant inoculation
Aggressive isolate of P. melonis , obtained from naturally
infected cucumber plants exhibiting post-emergence damping-off and root
rot symptoms and identified as P. melonis (MH924841). Inoculum
was obtained by growing of P. melonis isolate in 500 mL flasks
containing water soaked wheat seed, which had been autoclaved at 120°C
for 30 min on 2 consecutive days. The flasks were inoculated with 5 cm
disks cut, from 10-day-old culture of P. melonis isolate, and
placed in the dark for 2 weeks at 25±2°C. 45-day-old seedlings were
inoculated by 10-g of wheat seed (106 sporangia/ml) and incubated for
two days under saturated moist condition in the greenhouse (Nasr
Esfahani et al., 2018, 2020, hashemi et al., 2019). In seedling stage,
inoculated and control root samples were harvested in three biological
replicates over the time courses of 7, 14 and 21 days after inoculation
(DAI) and then immediately transferred to liquid nitrogen and kept in
-80°C. All experiments were repeated 3 times.