Vacuum fractional distillation enhances the antibacterial activity of Mentha haplocalyx essential oil against Xanthomonas citri subsp. citri by disrupting cell membrane and inhibiting biofilm.

Citrus canker, a significant bacterial disease caused by Xanthomonas citri subsp. citri (Xcc), is currently managed primarily with copper-based treatments. However, the long-term use of these agents can result in pesticide residue and increased bacterial resistance. This study employed the vacuum fractional distillation (VFD) technique to fractionate the essential oil of Mentha haplocalyx (MHEO) to produce MHF3, which exhibited significant antibacterial activity against Xcc. The minimum inhibitory concentration (MIC) of MHF3 was 0.20 μL/mL, superior to that of the positive control, copper sulfate (156 μg/mL). Meanwhile, MHF3 also inhibited the growth of a diverse array of other pathogens with MIC values ranging from 0.78 to 100 μL/mL. Gas chromatography-mass spectrometry (GC-MS) analysis identified the primary antimicrobial components in MHF3 as isopulegol, menthol, pulegone, carvone, and piperitone, with respective MICs of 0.20, 0.78, 0.78, 0.20, and 0.39 μL/mL. The results of the antibacterial mechanism study revealed that MHF3 disrupts the bacterial cell membrane structure of Xcc, causing the leakage of intracellular proteins, nucleic acids, and AKP enzymes. MHF3 also induces ROS accumulation, inhibits biofilm formation, and significantly disrupts nucleotide, lysine, and glutathione metabolism, as well as the pentose phosphate pathway in Xcc. In vivo leaf assay results confirmed that MHF3 effectively controls citrus canker. This study reveals the potential application of the VFD technique for enriching the bioactive components of essential oils. It also provides scientific data for developing MHF3 as a green, efficient, and natural agent for managing citrus canker.