Resumen
Laminaria japonica was extracted with ethanol, and tested for antimicrobial activity against Clavibacter michiganensis subsp. michiganensis (Cmm). This gram-positive bacterium is the agent of a serious tomato disease called bacterial canker. On in vitro antimicrobial assays, L. japonica extracts showed intensive antimicrobial activity to inhibit Cmm. Extracting parameters (time, temperature, and solid/liquid ratio) were investigated and further optimized integratedly with L27(313) orthogonal array design (OAD). Our data implied that the optimum extraction conditions were A3B3C3, that were extracting time 12 h, extracting temperature 80 °C, and solid/liquid ratio 1:30 (g:mL). Extracting temperature was the significant factor affecting extracting condition. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of L. japonica extracts against Cmm were 2.5 mg/mL and 5 mg/mL. Scanning electron microscopy (SEM) observation indicated L. japonica extracts caused considerable morphological alterations in Cmm cells, such as protoplast leakage, cell membrane disruption, cell clumped, and cell fragmented. Reactive oxygen species (ROS) level determination and ATPase activity assay revealed that L. japonica extracts did cause significant accumulation in ROS and reduce the intracellular ATPase activity rapidly. Accordingly, these results indicated that L. japonica extracts could open a new promising opportunity for this tomato disease control.