Pseudomonas entomophila 23S Produces a Novel Antagonistic Compound against Clavibacter michiganensis subsp. michiganensis, a Pathogen of Tomato Bacterial Canker

Biopesticides, which consist of antagonistic microorganisms, or the bioactive compounds they produce offer attractive alternatives to synthetic agrochemicals as a means of reducing plant diseases in crop production systems. In our previous study, a strain of Pseudomonas entomophila 23S was reported to have a strong antagonistic activity toward Clavibacter michiganensis subsp. michiganensis (Cmm), a pathogen of tomato bacterial canker disease, which causes serious damage to the tomato production industry. This study focused on purification and characterization of the anti-Cmm compound(s) produced by P. entomophila 23S. The cell-free supernatant was first subjected to butanol extraction, fractioned by solid-phase extraction (SPE), and purified by high-performance liquid-chromatography (HPLC). The semi-preparative purified compounds retained anti-Cmm activity. These compounds were then characterized by liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-HRMS/MS) and nuclear magnetic resonance (NMR). Two anti-Cmm compounds, C15H16N2O and C16H18N2O, were isolated by this procedure. From the LC/MS spectra, the compounds were predicted to have an isoquinoline ring, the presence of which was confirmed by NMR. The compounds differed with respect to the presence of a methyl group in a side chain. In addition, P. entomophila 23S grown in different culture media produced the greatest amounts of these anti-Cmm compounds when in Nutrient Broth (NB), where bacterial growth was the slowest. NB composition is more favorable in stimulating P. entomophila 23S to produce higher amounts of these anti-Cmm compounds.