Antibacterial effects of volatiles produced by Bacillus strain D13 against Xanthomonas oryzae pv. oryzae

Abstract

Recent investigations have demonstrated that bacteria employ the volatile compounds they produce during interactions with other organisms, such as plants, fungi, nematodes and bacteria. However, studies focused on the antibacterial activity of plant growth-promoting rhizobacteria (PGPR) volatiles against bacterial phytopathogens are still rare. In this study, Bacillus strain D13, which is antagonistic to Xanthomonas oryzae pv. oryzae (Xoo), was isolated and screened. Volatile compounds emitted from strain D13 reduced the colony diameter and cell motility of Xoo cultured in divided Petri plates. Transmission electron micrograph analysis showed concentration in cytoplasm and altered surface morphology in the majority of Xanthomonas cells after co-cultivation with strain D13. Transcriptional expression of virulence-associated genes in Xoo was repressed. Based on gas chromatography/mass spectrometry (GC/MS) analysis, 12 volatile compounds specifically produced by strain D13 were identified. Among them, decyl alcohol and 3,5,5-trimethylhexanol inhibited the growth of Xoo at minimum inhibitory amounts of 0.48 and 2.4 mg, respectively. Furthermore, transcriptional expression of virulence-associated genes was also repressed by decyl alcohol and 3,5,5-trimethylhexanol. These results are useful for a better understanding of the biocontrol mechanisms of Bacillus.

Keywords: Bacillus; antibacterial activity; volatile compounds, Xanthomonas oryzae pv. oryzae.

Figure 1

Antibacterial activity of Bacillus spp. volatiles against Xanthomonas oryzae pv. oryzae (Xoo). Each isolated Bacillus spp. strain was cultured in one compartment of the divided plates containing Luria broth (LB) medium. Xoo was cultured in a separate compartment containing nutrient agar (NA) medium. Control plates contained Xoo on one half and no Bacillus‐inoculated LB medium on the other half. After incubation for 6 days, the rates of Xoo growth inhibition by volatiles emitted from Bacillus were measured. Inhibition rate (%) = (average colony diameter of control – average colony diameter of treatment)/average colony diameter of control × 100%. The data were statistically evaluated using analysis of variance, followed by Fisher's least‐significant difference test (P < 0.05), employing SPSS software (SPSS, Chicago, IL, USA). All error bars represent standard deviations. Asterisks above the bars indicate a significant difference (P < 0.05).

Figure 2

Phylogenetic analysis of Bacillus strain D13 based on gyrB gene sequences. The gyrB gene of Bacillus strain D13 was amplified and sequenced, and phylogenetic analysis was performed with the neighbour‐joining method using mega 4.0 software. Bars indicate the scales of genetic distances (0.02). Genetic distance measures are indicators of relatedness among species and are useful for reconstructing the historic and phylogenetic relationships among such groups.

Figure 3

Effects of volatiles produced by Bacillus strain D13 on cell motility in Xanthomonas oryzae pv. oryzae (Xoo). Xoo cultures were spotted onto the compartments of divided plates containing 0.7% (w/v) or 0.3% (w/v) nutrient agar (NA) medium; strain D13 was cultured in a separate compartment containing Luria broth (LB) medium. Control plates contained Xoo on one half and no Bacillus‐inoculated LB medium on the other half. (A) Swarming and swimming haloes of Xoo exposed to strain D13 volatiles for 3 days. (B) Real‐time polymerase chain reaction (PCR) analysis of motA, motC and rpfC expression in Xoo cells after co‐cultivation with strain D13 for 36 h. Values were normalized to the levels of 16S rRNA, an internal reference gene. Three replicates were used for each treatment, and the experiment was repeated three times. Vertical bars represent standard errors. Asterisks above the bars indicate a significant difference (P < 0.05).

Figure 4

Effects of Bacillus sp. D13 volatiles on the virulence of Xanthomonas oryzae pv. oryzae (Xoo). (A) Representative lesion lengths in rice seedling leaves (cultivar IR24, 2 months old) following inoculation with control Xoo cells and Xoo cells after co‐cultivation with strain D13. (B) Analysis of lesion lengths. The y‐axis values represent the mean lesion length ± the standard deviation (n = 3). Asterisk indicates significant difference compared with the control (P < 0.05).

Figure 5

Transmission electron micrographs of Xanthomonas oryzae pv. oryzae (Xoo) co‐cultured with strain D13. The specimens were prepared from the samples taken directly from divided Petri plates after co‐cultivation for 6 days. (A) Untreated control Xoo cells. (B) Xoo cells during co‐cultivation. Scale bars indicate 500‐nm lengths.

Figure 6

Antibacterial effects of selected volatile organic compounds (VOCs) on Xanthomonas oryzae pv. oryzae (Xoo) growth. (A) Xoo was incubated with eight volatile compounds, and the inhibition rates were measured after exposure for 6 days. (B) Xoo was incubated with DA at different amounts ranging from 0.24 to 0.8 mg, and the inhibition rates were measured after exposure for 6 days. (C) Xoo was incubated with TMH at different amounts ranging from 0.8 to 4 mg, and the inhibition rates were measured after exposure for 6 days. DA, decyl alcohol; LA, lauric acid; MA, myristic acid; MCP, 3‐methyl‐1,2‐cyclopentanediol; PA, palmitic acid; PCO, 2‐pentadecanone; TA, tridecanoic acid; TMH, 3,5,5‐trimethylhexanol. The y‐axis values represent the mean inhibition rate ± standard deviation (n = 3). *Significant difference compared with the control (P < 0.05).

Figure 7

Antibacterial activities of decyl alcohol (DA) and 3,5,5‐trimethylhexanol (TMH) against Xanthomonas oryzae pv. oryzicola (Xoc), Pseudomonas syringae pv. tomato DC3000 (Pst) and Ralstonia solanacearum (Rs); 0.48 mg DA [dissolved in 100 µL dimethylsulfoxide (DMSO)], 2.4 mg TMH (dissolved in 100 µL DMSO) and DMSO were added to one compartment of the divided plates containing Luria broth (LB) medium; Xoc, Pst and Rs were cultured in a separate compartment containing nutrient agar (NA) or King's B (KB) medium. The diameters of Xoc, Pst and Rs colonies were measured after exposure for 14, 4 and 4 days, respectively. Three independent replicates of each experiment were performed.

Figure 8

Real‐time polymerase chain reaction (PCR) analysis of motA, motC, gumD and rpfC expression in Xanthomonas oryzae pv. oryzae cells in response to 0.48 mg synthetic decyl alcohol (DA) or 2.4 mg 3,5,5‐trimethylhexanol (TMH) for 24 h. Values were normalized to the levels of 16S rRNA, an internal reference gene. The y‐axis values represent the mean expression ± standard deviation (n = 3) relative to the control.