Bacterial Inoculation and Rice Seedling Evaluation

The bacterial strain used in this study, except those referenced in Fig. 6, Supplementary Figs. S1 and S11, was B. glumae MAFF301682 (MAFF designates strains from the culture collection of the National Agriculture and Food Research Organization (NARO) Genebank, formerly the culture collection of the Ministry of Agriculture, Forestry and Fisheries, Japan). Bacterial inocula were incubated on Luria–Bertani (LB) media with 2% agar at 28 °C for 4 days and then suspended in sterilized, deionized water at a concentration of 10⁸ CFU/ml. The rice seeds were sterilized by soaking in a chlorine bleach solution (available chlorine 2.5%) for 30 min, rinsed carefully with sterilized water, and then soaked in sterilized water for 3 days in a plant growth chamber at 28 °C. The sterilized seeds were subsequently placed in a freshly prepared bacterial suspension and held under vacuum (0.2 MPa) for 3 min. The inoculated seeds were dried for 2 h, sown in sterilized soil (Bonsol No. 2, Sumitomo Kagaku Kougyo, Osaka, Japan) and incubated in a growth chamber at 28 °C with 80% humidity under a 14-h photoperiod. The disease symptoms were measured 8 days after sowing on a scale of 1–3, where 1 = no symptoms, 2 = sheaths with reddish-brown lesions (mild infection), and 3 = necrotic seedlings or seeds that did not germinate (severe infection). The BSR severity was calculated from these scores as follows: $$\text{BSR}\text{severity}\left(\%\right)=\left(N_3-N_1-N_2/2\right)\times 100/N_3,$$ where N₁ = number of seedlings with a score of 1, N₂ = number of seedlings with a score of 2, and N₃ = number of seeds per replication. There were three or four replications per inoculation. As a control, we germinated uninoculated seeds and confirmed that the average germination rate was > 90%. The bacterial strain for evaluation of resistance to bacterial seedling blight was B. plantarii MAFF301723. The method for inoculating seeds was the same as that used for B. glumae. Five inoculated seeds and 95 uninoculated seeds were sown in sterilized soil in the same cell tray, and the disease severity of B. plantarii (Fig. 6) was calculated as described above. The bacterial strain shown in Supplementary Fig. S11 was B. glumae MAFF302744, and the panicle disease severity assay was conducted according to a previously described metohd^{53 (link)}.