The Effect of Citrus Essential Oils and Their Constituents on Growth of Xanthomonas citri subsp. citri

Abstract

Citrus bacterial canker (CBC) caused by Xanthomonas citri subsp. citri (Xcc), is the most devastating of the citrus diseases worldwide. During our study, we found that Essential oils (EOs) of some citrus cultivars are effective on Xcc. Therefore, it prompted us to determine the plant metabolites responsible for the antibacterial properties. We obtained EOs from some locally cultivated citrus by using a Clevenger apparatus and their major constituents were identified by gas chromatography/mass spectrometry (GC-MS). The effect of Citrus aurantium, C. aurantifolia, Fortunella sp. EOs and their major constituents were evaluated against Xcc-KVXCC1 using a disk diffusion assay. Minimal inhibitory and bactericidal concentration of the EOs and their constituents were determined using the broth microdilution method. C. aurantium, C. aurantifolia Eos, and their major constituents including citral, linalool, citronellal, geraniol, α-terpineol, and linalyl acetate indicated antibacterial effects against Xcc. The C. aurantifolia EO and citral showed the highest antibacterial activity among the tested EOs and constituents with inhibition zones of 15 ± 0.33 mm and 16.67 ± 0.88 mm, respectively. Synergistic effects of the constituents were observed between α-terpineol-citral, citral-citronellal, citral-geraniol, and citronellal-geraniol by using a microdilution checkerboard assay. Transmission electron microscopy revealed that exposure of Xcc cells to citral caused cell wall damage and altered cytoplasmic density. We introduced C. aurantifolia and C. aurantium EOs, and their constituents citral, α-terpineol, citronellal, geraniol, and linalool as possible control agents for CBC.

Keywords: C. aurantium; Citrus aurantifolia; citral; synergistic effect; transmission electron microscopy.

Figure 1

Antibacterial effect of essential oils and their constituents against Xcc-KVXCC1 isolates of Xanthomonas citri subsp. citri. Different letters indicate significant differences according to Duncan analysis using SPSS software (p = 0.05). All data represent means ± standard error of the mean (SEM) from 3 independent experiments. All data represent means ± standard error of the mean (SEM) from 3 independent experiments.

Figure 2

Transmission electron micrographs of Xcc-KVXCC1 isolate of Xanthomonas citri subsp. citri exposed to MIC value of citral for 4 h. (A,B), control with complete cell wall; (C) bacterial cells exposed to streptomycin sulfate (25 mg·mL⁻¹); (D–F), bacterial cells exposed to citral with apparent the complete destruction of the cell (arrows), damage of cell wall (arrows with lozenge), alteration in cytoplasm density (arrows with square), swelling and colored nuclear area (arrows with circle).

Figure 3

Time-kill kinetics of EO constituents against Xcc-KVXCC1 Xanthomonas citri subsp. citri at (A). MIC value and (B). 2.5× MIC value. All data represent means ± standard error of the mean (SEM) from three independent experiments.

Figure 4

Schematic overview of the chemical structure of major essential oils. The straight black arrow shows the toxicity of the constituents subsequently. The curved red and blue arrows indicates different positions for double bands which makes two different isomers for citral. The circle dashed lines demonstrate the hydroxyl groups which probably attach to the bacteria. The cross red lines shows no possibility for citronellal to make any isomer.