Biocontrol of Tomato Bacterial Wilt by Foliar Spray Application of a Novel Strain of Endophytic Bacillus sp

Abstract

The aim of the present study was to identify a strain of endophytic Bacillus species that control tomato bacterial wilt by foliar spray application. Fifty heat-tolerant endophytic bacteria were isolated from the surface-sterilized foliar tissues of symptomless tomato plants that had been pre-inoculated with the pathogen Ralstonia pseudosolanacearum. In the primary screening, we assessed the suppressive effects of a shoot-dipping treatment with bacterial strains against bacterial wilt on tomato seedlings grown on peat pellets. Bacillus sp. strains G1S3 and G4L1 significantly suppressed the incidence of tomato bacterial wilt. In subsequent pot experiments, the biocontrol efficacy of foliar spray application was examined under glasshouse conditions. G4L1 displayed consistent and significant disease suppression, and, thus, was selected as a biocontrol candidate. Moreover, the pathogen population in the stem of G4L1-treated plants was markedly smaller than that in control plants. A quantitative real-time PCR analysis revealed that the foliar spraying of tomato plants with G4L1 up-regulated the expression of PR-1a and LoxD in stem and GluB in roots upon the pathogen inoculation, implying that the induction of salicylic acid-, jasmonic acid-, and ethylene-dependent defenses was involved in the protective effects of this strain. In the re-isolation experiment, G4L1 efficiently colonized foliar tissues for at least 4‍ ‍weeks after spray application. Collectively, the present results indicate that G4L1 is a promising biocontrol agent for tomato bacterial wilt. Furthermore, to the best of our knowledge, this is the first study to report the biocontrol of bacterial wilt by the foliar spraying with an endophytic Bacillus species.

Keywords: Ralstonia pseudosolanacearum; biological control; endophytic Bacillus; foliar spray; induced systemic resistance.

Fig. 1.

Forest plot of the comparison between bacterial treatments and control for the incidence of tomato bacterial wilt in the primary screening experiment. Wilt incidence data (the number of wilted seedlings at 9 dai) obtained from four independent repeated trials were analyzed by a frequentist network meta-analysis. The grey boxes indicate the relative risk (RR) for individual treatments and the horizontal bars indicate the corresponding 95% confidence interval (95% CI).

Fig. 2.

Phylogenetic tree derived from 16S rRNA gene sequence data of two selected strains (G1S3 and G4L1) and their relatives. The tree was generated by the neighbor-joining method, and genetic distances were calculated by the Kimura 2-parameter method using MEGA ver. 7.0.27 (Tamura et al., 2013). Escherichia coli (ATCC 11775) was used as an outgroup to root the tree. Numbers at nodes are percentage bootstrap values (only bootstrap values higher than 60% are shown from 1,000 replications). The scale bar represents 0.02 nucleotide substitutions per nucleotide position.

Fig. 3.

Forest plot of a network meta-analysis comparing bacterial treatments and the control treatment for the incidence of tomato bacterial wilt in the pot experiment. Wilt incidence data (the number of wilted seedlings at 14 dai) obtained from four independent repeated trials were analyzed by a frequentist network meta-analysis. The grey boxes indicate the relative risk (RR) for individual treatments and the horizontal bars indicate the corresponding 95% confidence interval (95% CI).

Fig. 4.

Population dynamics of Ralstonia pseudosolanacearum in the stem of tomato plants spray-treated with the biocontrol Bacillus strain G4L1. Bars represent the mean±standard error. N.D., not detected. N.T., not tested; n.s., not significant (P<0.05 using the Student’s t-test).

Fig. 5.

Population of dynamics of the biocontrol Bacillus strain G4L1 on leaves (A) and stems (B) of tomato plants. Bars represent the mean±standard error.

Fig. 6.

Expression of defense-related genes in main roots and stems of G4L1-treated or mock-treated tomato plants inoculated without or with Ralstonia pseudosolanacearum 3 days after the pathogen inoculation. The housekeeping gene β-tubulin was used for normalization. The expression levels of the target genes in different samples were calculated using the formula 2^–ΔΔCT (Livak and Schmittgen, 2001), given as a value relative to mock-treated control plants (not inoculated with the pathogen). Bars represent the mean±standard error of three biological replicates per treatment with two or three technical repetitions for each sample. Different letters indicate significant differences between treatments according to Tukey’s test at P<0.05.