Measurement of Reactive Oxygen Species

The production of reactive oxygen species was measured following a described procedure [36 (link),37 (link)]. Menadione was used as a positive control. This molecule has been described as an inducer of ROS generation, since it is capable of both redox cycling and arylating nucleophilic substrates by Michael addition [37 (link),38 (link)]. The ROS was evaluated using ROS-GLOtm H₂O₂ assay kit (Promega, Madison, WI, USA) [37 (link)]. Briefly, conidia (79 µL) were plated in a 96-well plate at a concentration of 1 × 10⁵ conidiam L⁻¹/well. Later, each well was incubated in presence of each compound at their IC₅₀ concentration, 20 µL of buffer substrate H₂O₂ and cultured for 2 h at 21 ± 1 °C. After this period of incubation, 100 µL of ROS-GLOtm reagent was added to each well and incubated for 20 min at room temperature. The ROS production was measured using a luminometer (Tecan infinite m200pro; Tecan Group Ltd., Hombrechtikon, Switzerland). Mean values with at least significant difference (p < 0.05) were considered.