Identification and antagonistic activities of FJAT-46737
The strain FJAT-46737 was a gram-positive and endospore-forming bacterium. Its colonies on the NA plate were flat, slightly rough, nearly circular, light yellow (Figure S2). The phylogenetic analysis of 16S rRNA and gyrB gene results displayed that the strain FJAT-46737 belongs to the genus Bacillus, with a close relationship to the strain B. velezensis and B. amyloliquefaciens, respectively (Figure S3, S4). The 16 S rRNA gene of FJAT-46737 exhibits 99.72%, 99.71% and 99.44% similarity to the type strains Bacillus siamensis KCTC 13613T, Bacillus velezensis CR-502T and B.amyloliquefaciens DSM7T. The gyr B gene of FJAT-46737 displayed exceeding 99.0% identity to the type species of B.amyloliquefaciens and B. velezensis. Take into consideration of the high degree of relatedness among B. amyloliquefaciens, B. siamensis and B. velezensis, the ANI method based on the whole genome sequence was used to discriminate the strain FJAT-46737.
The whole genome sequence of strain FJAT-46737 contains 3995340 bp, and the G + C content of the chromosomal DNA was 46.5 mol%. The ANI values between the strain FJAT-46737 and those type strains B. amyloliquefaciens DSM7T, B. siamensis KCTC 13613T, and B. velezensis KCTC 13012T were calculated as 94.16%, 94.36% and 98.26%, respectively. The last one displayed ANI values of >98% exceeding the recommended cut-off of 96% for species delineation. This result suggested that the strain FJAT-46737 was a member of B. velezensis species.
The gene clusters of bioactive secondary metabolites in strain FJAT-46737 was analyzed using antiSMASH method. Results shown that the strain FJAT-46737 possess 7 gene clusters that responsible for synthesis of surfactin, fengycin, macrolactin, bacillaene, difficidin, bacilysin and bacillibactin (Figure S5), which indicate that the strain FJAT-46737 could display the strong antagonistic activities.
The agar disk diffusion method was used to evaluate antagonistic activities of FJAT-46737 against several animal or plant pathogens. The results showed that this strain exhibited significant activities against the gram-negative bacteria including R. solanacearum and E. coli, and the filamentous fungi including 3 biotypes of Fusarium oxysporum (F. oxysporum f. sp. capsicum, F. oxysporum f. sp. niveum and F. oxysporum f. sp. melonis) (Figure 1).
Biocontrol efficacy of FJAT-46737 against tomato bacterial wilt
The above results showed that the B. velezensis FJAT-46737 had in vitro antibacterial activity against R. solanacearum. Thus, we attempted to investigate its biocontrol efficacy against the tomato bacterial wilt by using the pot experiments in the greenhouse condition. Firstly, we found that DI of the tomato plants in the treatment group with the whole cultures of FJAT-46737 (31.7%) was much lower than that in the control group (93.8%) (Figure 2, Table 1). The biocontrol efficacy of whole cultures of FJAT-46737 against tomato bacterial wilt could reach up to 66.2% in the greenhouse experiments (Table 1).
In order to clarify the biocontrol mechanisms of FJAT-46737, we subsequently evaluated the suppressive effects of its cell-free supernatants on the tomato bacterial wilt. A preliminary experiment indicated the undiluted cell-free supernatant caused seedling injury, but the 2-fold diluted one had no injury effect. Thus, the 2-fold diluted cell-free supernatants were selected to perform the pot experiments. The results showed that an approximately 82.0% biocontrol efficiency could be achieved in the cell-free supernatant treatment group (Figure 2, Table 1), which indicated that the biocontrol ability of FJAT-46737 could be mainly (if not all) attributed to its production of extracellular bioactive substances.
Some previous studies indicated that the lipopeptides secreted by Bacillus strains play important roles in biocontrol of tomato bacterial wilt. Thus, we further tested the biocontrol efficiency of the crude lipopeptide extracts from FJAT-46737 against tomato root infection by R. solanacearum. A preliminary experiment indicated that high concentration (≥2.5 mg/mL) of lipopeptide led to seedling injury, but 1 mg/ml lipopeptide had no injury effect. Thus, the lipopeptide with concentration of 1 mg/mL was selected to perform the pot experiments. The directly plantlet-soaking treatment with the lipopeptides could significantly reduce mortality of the tomato plants and achieve a biocontrol efficiecy of 96.2% (Figure 2, Table 1). All, these results suggested that one of the antagonistic mechanisms of the strain FJAT-46737 was secretion of lipopeptides.
LC-QTOF-MS/MS analyses of the lipopeptides produced by FJAT-46737
In this study, we used the LC-ESI-MS/MS method to determine the lipopeptide profile of FJAT-46737. Three classes of cyclic lipopeptides iturin (retention time,12.8–22.3 min), fengycin (29.0–36.0 min), and surfactin (48.3–52.0 min) could be detected in present study. The retention times, MS and MS2 spectral data and identification results of the lipopeptides from B. velezensis FJAT-46737 are summarized in Table 2. The results show that the lipopeptides consisted of C14–C16 iturin A, C13–C15 surfactins, and C16 fengycin A/B and C16 fengycin A2/B2) .
Inhibitory spectra of the antagonistic lipopeptides
We used an agar well diffusion assay to further evaluate antimicrobial activities of the crude lipopeptides from FJAT-46737. The results showed that the crude lipopeptides had significant activities against R. solanacearum, E. coli, and F. oxysporum in a dosage-dependent manner (Table 3 and Table 4). At 48 h, the inhibition zone diameters caused by 10 mg/mL of the crude lipopeptides could reach up to 18.52±0.73 mm, 14.55±0.23 mm, and 14.57±1.85 mm against the pathogens R. solanacearum FJAT-91 and FJAT-77, and E. coli FJAT-301, respectively (Table 3). It was found that the lipopeptide with the concentration of 0.5 mg/mL displayed antibacterial activity against R. solanacearum FJAT-91, but not at 0.25 mg/mL (Figure S6). These results implied that the crude lipopeptides of FJAT-46737 had the strongest antibacterial activity on R. solanacearum. Moreover, the inhibition zone diameters caused by 30 mg/mL of the crude lipopeptides on 4 biotypes of F. oxysporum (F. oxysporum f. sp. capsicum FJAT-831, F. oxysporum f. sp. niveum FJAT-9230 and F. oxysporum f. sp. melonis FJAT-30265) were all about 20 mm at 48 h.
Effects of medium components and culture conditions on the antibacterial activities and contents of lipopeptides
To further improve antagonistic activities of FJAT-46737, we analyzed effects of the medium components and temperatures on the antibacterial activities of the fermentation supernatants and crude lipopeptides. Given that the crude lipopeptides of FJAT-46737 had strong antibacterial activity on R. solanacearum, FJAT-91 was used as the indicator bacterium. Results of the antagonistic activity of the supernatant and crude lipopeptides and lipopeptide contents under different conditions were shown in Table 5 and Table 6.
Using the selected culture media A~F (Table S1), the supernatants and crude lipopeptides of FJAT-46737 exhibited good antibacterial activities against R. solanacearum FJAT-91, except the culture supernatant from the medium A (Table 5). Moreover, the antibacterial activities of both the supernatants and crude lipopeptide from the culture media C, D and E were stronger than those from the culture media A, B and F (Table 5). These results suggested that the medium components could significantly affect antibacterial activities of FJAT-46737.
Subsequently, effects of temperature on antibacterial activities of the cell-free supernatants and crude lipopeptides were determined using the culture medium C. The results showed that the culture temperature had significant effects on the antibacterial activities of the cell-free supernatants and crude lipopeptides: i) the cell-free supernatants did not exhibit antibacterial activity when the temperature was higher than 35 oC; ii) when it was incubated at 25 oC, both the cell-free supernatants and crude lipopeptides had the strongest antibacterial activities (Table 6).
Contents of the iturins, fengycins and surfactins in different culture conditions were further determined by LC-QTOF-MS and summarized in Table 5 and Table 6. The fengycins were the most abundant lipopeptide family produced by FJAT-46737. Contents of the iturins, fengycins and surfactins in the six culture media varied within the range of 0.31–1.99 mg/L, 9.03–58.66 mg/L and 0.55–1.39 mg/L, respectively. The contents of lipopeptide in the culture medium C and D were significantly higher than those in other four media, indicating the media containing rich organic nitrogen source might be benificail for producing lipopeptide. It was found that adding of yeast extract in culture medium D significantly increased yield of lipopeptide compared with the one without yeast extract adding (in culture medium B). In the culture medium C, production of the lipopeptides (61.04±9.86 mg/L) was the highest compared to those in the other media. Moreover, contents of the iturins, fengycins and surfactins strongly depended on temperature. Content of the fengycins decreased by 96.6% when the culture temperature was increased from 20 oC to 40oC, on the contrary, that of the surfactins ascended by 59.9%. The temperature ranges of 25–30 oC, 20–25oC and 35–40 oC were suitable for the strain FJAT-46737 to produce iturin, fengycin and surfactin, respectively.
Subsequently, a correlation analysis between the cell-free supernatant antibacterial activities and lipopeptide contents in the different culture conditions were carried out (Table 7). It was observed that contents of the fengycins and total lipopeptides were significantly positive correlated with the antibacterial activities of the cell-free supernatants of all the samples (p < 0.05), but those of the surfactins and iturins not. These results suggested that the antibacterial activities of the cell-free supernatants were mainly attributed to secretion of the fengycins by FJAT-46737.
Effect of purified lipopeptides on inhibition of R. solanacearum growth
The antibacterial activities of the purified lipopeptides against R. solanacearum were tested. The results showed that only the fraction obtained by SPE with 70% MeOH (named as SPE70) exhibited antibacterial activity. Therefore, composition of the fraction SPE70 was further determined by LC-QTOF-MS/MS (Figure S7). The results showed that the fraction SPE70 contained only fengycin, indicating that the fengycin plays an important role in the growth inhibition of R. solanacearum. This result was consistent with the aboved correlation analysis.