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.