Biocontrol of citrus bacterial canker caused by Xanthomonas citri subsp. citri by Bacillus velezensis

Abstract

Microorganisms with biocontrol capabilities against plant pathogens are considered as one of the most promising approaches for healthy crop management. In this study, ethyl acetate extracts of 25 Bacillus strains were investigated for their antagonistic effect on Xanthomonas citri subsp. citri (Xcc), which causes the citrus bacterial canker (CBC) disease. Among them, 21 strains exerted antibacterial activity against wild-type Xcc strains. Based on the strength of the antibacterial activity, nine Bacillus strains were selected for 16S rRNA analysis. 16S rRNA sequence homology revealed that several strains were closely related to B. velezensis, where strains with no antibacterial activity grouped as the soil-associated community of B. amyloliquefaciens. B. velezensis Bv-21 exhibited the highest antibacterial activity against wild type and streptomycin resistant Xcc with inhibition zones of 22.91 ± 0.45 and 20.28 ± 0.53, respectively. Furthermore, B. velezensis Bv-21 strain was tested for biocontrol activity against a streptomycin-resistant XccM4 in detached susceptible citrus leaves. The strain reduced the incidence of CBC by 26.30% and pathogen density of XccM4 by 81.68% over control. The results of the study strongly suggest that B. velezensis can be used as an effective and eco-friendly biocontrol agent either by itself or as an active compound, against both, the wild-type and streptomycin-resistant Xcc.

Keywords: B. amyloliquefaciens; Bacillus velezensis; Biocontrol; Citrus bacterial canker; Xanthomonas citri subsp. citri.

Fig. 1

Phylogenetic analysis of 16S rRNA gene sequences that highlight the position of selected Bacillus species closely related to the representatives of the Bacillus genus, such as, B. amyloliquefaciens, B. subtilis, B. licheniformis, B. tequilensis, B. cereus, and B. pumilus. Phylogenetic tree was constructed using Maximum Likelihood method. Parentheses indicate the accession numbers obtained from the National Center for Biotechnology Information (NCBI) database. The bootstrap values (%) are mentioned at the nodes and are obtained by repeating the analysis 1000 times. The scale bar represents a nucleotide substitution rate of 0.005 per nucleotide position.

Fig. 2

In vitro antagonistic effect of the ethyl acetate extract of Bacillus spp. (1–25) and streptomycin on the virulence of X. citri subsp. citri (Xcc). (a) Bactericidal activity was conducted against the wild-type XccW1. 1–25: Ethyl acetate extract of 25 Bacillus strains (1.0 mg mL⁻¹); C: Control (methanol); S1: Streptomycin (1.0 mg mL⁻¹); S2: Streptomycin (0.1 mg mL⁻¹). (b) Measurement of inhibition zones (mm) of ethyl acetate extracts of Bacillus spp. against XccW1. The error bars in a two-tailed t-test represented the standard deviation, and the result was found to be statistically significant at p < 0.05.

Fig. 3

In vitro antagonistic effect of the ethyl acetate extract of selected Bacillus spp. and streptomycin on wild-type and streptomycin-resistant X. citri subsp. citri (Xcc) strains. (a) Bactericidal activity was conducted using the pathogenic wild-type XccW1 and XccW2. (b) Measurement of inhibition zones (mm) of ethyl acetate extracts of Bacillus spp. against XccW1 and XccW2. (c) Bactericidal activity was conducted using the pathogenic strains XccM4 and XccM6 (streptomycin-resistant). (d) Measurement of inhibition zones (mm) of ethyl acetate extracts of Bacillus spp. against the streptomycin-resistant XccW4 and XccW6. C: control (methanol); 1, 2, 6, 11, 17, 19, 21, 22, and 25: Ethyl acetate extract (1.0 mg mL⁻¹) of selected Bacillus spp.; S1: streptomycin (1.0 mg mL⁻¹); S2: streptomycin (0.1 mg mL⁻¹). The error bars in a two-tailed t-test represented the standard deviation, and the result was found to be statistically significant at p < 0.05.

Fig. 4

Development of disease symptoms in the leaves of Hwanggeum hyang citrus at 7 dpi through the inoculation of Xanthomonas citri subsp. citri (XccM4) and suspension of XccM4:Bv-21 (a) The leaves were infiltrated with 0.1 mL aliquots of OD_600nm 0.4 XccM4 mixed with SDW (XccM4); OD_600nm 0.4 XccM4 mixed with OD_600nm0.4 Bv-21 (XccM4:Bv-21) and Bv-21. As a control, leaves were inoculated with SDW (b) Diameter of disease lesions measured at 7 dpi. (c) Quantification of XccM4 from the leaves treated with XccM4 and suspension of XccM4:Bv-21. Three independent replicates were tested for each treatment. One way ANOVA (analysis of variance) test was performed and result was found significant (p < 0.0001) among the groups.