Histochemical detection of H2O2 in rice leaves

The cultured bacteria re-suspended in sterilized ddH₂O at an OD₆₀₀ of 1.0 were prepared as described above, and cells were infiltrated into rice cultivar (Oryza sativa L. subsp. japonica) leaves grown for two weeks using a needleless syringe. The H₂O₂ was detected by DAB staining as previously reported [46]. Briefly, leaf sections (3–5 mm) at 12 and 24 h post-inoculation were cut and placed in water with 0.01 % Triton-X-100 and DAB at 1 mg mL⁻¹, then this solution was infiltrated with low vacuum pressure for 10 min and the leaves were incubated for 8 h at room temperature. Finally, leaves were boiled with 95 % ethanol for 10 min and then rinsed with water, and presence of H₂O₂ was visualized as reddish brown colored dark spots by a light microscope (Leica, Heidelberg, Germany).