Exploring the antibacterial efficacy of Satureja hortensis and Bacillus subtilis extracts in combating Xanthomonas citri

Biofilm plays a critical role in protecting the associated bacterial colonies. Citrus canker caused by Xanthomonas citri, resulted in remarkable yield reduction in citrus orchards. This study aims to evaluate the anti-biofilm properties of S. hortensis and B. subtilis extracts against X. citri. The antibacterial effects of both extracts were evaluated using the disk diffusion method and 96-well microdilution plates. Crystal violet and XTT procedures were employed to assess the inhibition of adhesion and antibiofilm effects of both extracts. The checkboard titration method was applied to determine the synergistic effects of the plant-bacterial extracts. The antibiofilm effects were confirmed by the light microscopy method. The results showed that the antibacterial and antibiofilm properties of both extracts varied. The MIC values of S. hortensis and B. subtilis extracts were 6.25 and 50 mg/ml, respectively. In these concentrations the inhibition of adhesion effects of S. hortensis and B. subtilis were 70% and 80%, whereas their antibiofilm effects were 60% and 72%. S. hortensis and B. subtilis extracts showed 63% and 76% antibiofilm activity, respectively, using the XTT test. The extracts showed synergistic effects, resulting in higher inhibition rates in combinatorial applications. Molecular docking results also confirmed that the secondary metabolites of S. hortensis could interact with different X. citri proteins differentially. Further large-scale studies on combinatorial antibiofilm effects of plant and bacterial extracts against citrus pathogens will provide new opportunities to develop safe and applicable reagents to control these destructive diseases.