Metabolomic Analysis of Xanthomonas oryzae

The sample preparation procedure was previously described [22 (link)]. In brief, overnight cultures of Z173-S and Z173-R_ZS were seeded into 50 mL of PSA liquid medium at 30 °C, 200 rpm to OD₆₀₀1.0. The Xoo thallus were collected and centrifuged at 4 °C and 10,000 rpm for 5 min, and the supernatant was discarded and washed three times with 0.9% saline. After that, 10 mL of saline and 20 mL of frozen methanol were added immediately into the sample and set for at least 1 h to terminate the metabolic process of the cells. Bacterial cells collected were resuspended with 500 μL of frozen methanol with 10 μL of ribitol at 0.2 mg/mL as an internal reference. Cells were then sonicated at power 120 W for 5 min and centrifuged at 4 °C at 12,000× g for 10 min, and 500 μL of supernatant was dried with a nitrogen blower. Samples were then silanized and derivatized with 80 μL (20 mg/mL) of methoxyamine hydrochloride and 80 μL of N-methyl-N-trimethylmethilane trifluoroacetamide (MSTFA, Sigma-Aldrich, USA) and incubated at 37 °C, 200 rpm for 30 min, respectively. The samples were centrifuged at 4 °C at 12,000× g for 5 min and then analyzed with a GC-MS system. Each sample had five biological repeats.