Chloroform

Three replicate entire second leaf blades of each treatment were dipped in 10 mL of chloroform (Tianjin Guangfu Fine Chemical Research Institute, Tianjin, China) for 45 s to extract the total wax. As an internal standard, 20 μL n-tetracosane (1 μg μL⁻¹, Aladdin, Shanghai, China) was added. The solution was concentrated to 1 mL under nitrogen. Then, 20 μL of N, O-Bis(trimethylsilyl)-trifluoroacetamide (GC Derivatization Reagent, ≥98.0%, Aladdin, Shanghai, China), and 20 μL pyridine (Standard for GC, >99.9%, Aladdin, Shanghai, China) were added, and the samples were maintained at 70 °C in a water bath for 1 h for derivatization. The solution was transferred to GC sample vials (1.5 mL) through organic filtration (0.45 μm) and blown dry under nitrogen. Finally, the samples were redissolved with 0.5 mL of chloroform for GC−MS analysis.

Tetrabutyl titanate [Ti(OBu)₄], silver nitrate (99.8%, AgNO₃), absolute ethanol (99%), glacial acetic acid (98%), zinc nitrate hexahydrate, 2-methylimidazole, N,N-dimethylformamide (DMF), dichloromethane (DCM) and chloroform were purchased from Tianjin Guangfu Fine Chemical Research Institute Co., Ltd. China. Escherichia coli (E. coli) and B. subtilis (B. subtilis) were obtained from the private collection of the Biology Department, School of Life Science and Technology, Changchun University of Science and Technology. All biological reagents were purchased from Ob-xing Biological Co., Ltd. China. Deionized water was used throughout the experiments except for antibacterial activity evaluation experiments when sterile water was used. All chemicals were used directly without further purification.