Fungal Infection Assay in Rice

In this study, an Indian strain of M. oryzae, i.e., B157 (international race IC9) isolated from Hyderabad, and its cytoplasmic GFP-expressing transformant (M. oryzaeGFP) were grown on yeast extract glucose (YEG) media (Saha et al., 2020 (link)). The blast-susceptible indica rice cultivar- C0-43, was grown at 27°C under a 16:8 light/dark photoperiod. The binary vector pCAMBIA1302 was streaked onto Luria Bertani (Himedia, Mumbai, India) agar plates supplemented with 50 mg/L Kanamycin (Himedia, Mumbai, India) and incubated overnight at 37°C. Fungal growth assays were performed on a complete medium (CM) supplemented with or without dsRNA, at 28°C for 7 days. For conidiation, the fungi were grown in dark on YEG plates for 8–10 days. The fungal biomass was then scraped on 10 days after inoculation and homogenized into uniform slurry by vortexing. Spores were separated from the mycelial debris by filtering the slurry through sterile MiraCloth (Calbiochem, Darmstadt, Germany), and then centrifuged at 7,000 rpm for 7 min. The conidia were visualized and counted under a microscope using a hemocytometer (Neubauer, Marienfeld, Germany). The length and breadth of conidia were measured keeping a 20-μm scale reference using an built-in microscope camera. Microscopic examination of at least 50 conidia per replicate was done in at least three independent experiments.