Fungal inoculum preparation

The inoculum used was obtained from leaves of C. arabica naturally infected with H. vastatrix. The pustules of these leaves were scraped and placed in microtubes, were frozen in liquid nitrogen, and stored in a freezer at -80°C. To prepare the inoculum, the stored spores were submitted to 40°C thermal shock for 10 min, added in sterile distilled water and the suspension was calibrated at 1 x 10⁶ urediniospores/mL. The viability of inoculum was verified by observing the spore germination in glass cavity slides. After preparing the suspension for plant inoculation, three drops were transferred to glass cavity slides, which were incubated at 25°C for 48 hours. After the incubation, the spores were visualized under an optical microscope, so their germination could be observed (S1 Fig).