Molecular and Phenotypic Investigation on Antibacterial Activities of Limonene Isomers and Its Oxidation Derivative against Xanthomonas oryzae pv. oryzae

Xanthomonas oryzae pv. oryzae (Xoo) causes a devastating bacterial leaf blight in rice. Here, the antimicrobial effects of D-limonene, L-limonene, and its oxidative derivative carveol against Xoo were investigated. We revealed that carveol treatment at ≥ 0.1 mM in liquid culture resulted in significant decrease in Xoo growth rate (> 40%) in a concentration-dependent manner, and over 1 mM, no growth was observed. The treatment with D-limonene and L-limonene also inhibited the Xoo growth but to a lesser extent compared to carveol. These results were further elaborated with the assays of motility, biofilm formation and xanthomonadin production. The carveol treatment over 1 mM caused no motilities, basal level of biofilm formation (< 10%), and significantly reduced xanthomonadin production. The biofilm formation after the treatment with two limonene isomers was decreased in a concentration-dependent manner, but the degree of the effect was not comparable to carveol. In addition, there was negligible effect on the xanthomonadin production mediated by the treatment of two limonene isomers. Field emission-scanning electron microscope (FE-SEM) unveiled that all three compounds used in this study cause severe ultrastructural morphological changes in Xoo cells, showing shrinking, shriveling, and holes on their surface. Moreover, quantitative real-time PCR revealed that carveol and D-limonene treatment significantly down-regulated the expression levels of genes involved in virulence and biofilm formation of Xoo, but not with L-limonene. Together, we suggest that limonenes and carveol will be the candidates of interest in the development of biological pesticides.