4.5. Pathogenicity Test

The sheath inoculation method was performed to test for pathogenicity as described previously [20,30]. A freshly harvested spore suspension (5.0 × 10⁵ mL⁻¹) of P. oryzae (INA168, INA168:BAS4:eGFP, and PO6-6) was inoculated onto 4–5 cm rice leaf sheaths (n = 4) from 5–6-week old rice cv. NB and HY. The inoculated rice leaf sheaths were incubated at 25 °C in the dark for 48 h in a moistened chamber. The infection phenotype was determined in isolated chlorophyll-free thin epidermal layer using fluorescence microscopy (Zeiss equipped with Axioplan 2; Campbell, CA, USA) with bright field. The infected cells were categorized into two phenotypes: cells with invasive hyphae (IH) and cells with dead hyphae/hypersensitive response (HR). The infection assay and quantification of infected cells were performed at least three times with similar results.