Identification and antagonistic activities of FJAT-46737
Strain FJAT-46737 is a gram-positive and endospore-forming bacterium. Its colonies on the NA plate were flat, slightly rough, nearly circular, and light yellow (Figure S2). The phylogenetic analysis of the 16S rRNA and gyrB gene results indicated that strain FJAT-46737 belongs to the genus Bacillus and is closely related to strains B. velezensis and B. amyloliquefaciens (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, respectively. The gyr B gene of FJAT-46737 displayed greater than 99.0% identity with the type species of B. amyloliquefaciens and B. velezensis. Because 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 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 the 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%, which exceeded the recommended cut-off of 96% for species delineation. This result suggested that strain FJAT-46737 was a member of B. velezensis species.
The gene clusters of bioactive secondary metabolites in strain FJAT-46737 were analyzed using the antiSMASH method. The results shown that strain FJAT-46737 possesses 7 gene clusters that are responsible for the synthesis of surfactin, fengycin, macrolactin, bacillaene, difficidin, bacilysin and bacillibactin (Figure S5). These findings indicate that strain FJAT-46737 presents strong antagonistic activities.
The agar disk diffusion method was used to evaluate the antagonistic activities of FJAT-46737 against several animal or plant pathogens. The results showed that this strain exhibited significant activities against gram-negative bacteria, including R. solanacearum and E. coli, and filamentous fungi, including 3 biotypes of . 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 strain FJAT-46737 had in vitro antibacterial activity against R. solanacearum. Thus, we attempted to investigate its biocontrol efficacy against tomato bacterial wilt by performing pot experiments under greenhouse conditions. First, we found that the DI of the tomato plants in the treatment group with whole cultures of FJAT-46737 (31.7%) was much lower than that of the control group (93.8%) (Figure 2, Table 1). The biocontrol efficacy of whole cultures of FJAT-46737 against tomato bacterial wilt could reach 66.2% in the greenhouse experiments (Table 1).
To clarify the biocontrol mechanisms of FJAT-46737, we subsequently evaluated the suppressive effects of its cell-free supernatants on tomato bacterial wilt. A preliminary experiment indicated that undiluted cell-free supernatant caused seedling injury while the 2-fold diluted supernatant had no injury effect. Thus, the 2-fold diluted cell-free supernatants was 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 entirely) attributed to its production of extracellular bioactive substances.
Previous studies indicated that the lipopeptides secreted by Bacillus strains play important roles in the 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 a high concentration (≥2.5 mg/mL) of lipopeptides led to seedling injury while 1 mg/ml lipopeptides had no injury effect. Thus, a concentration of 1 mg/mL lipopeptides was selected to perform the pot experiments. The treatment in which plantlets were soaked with the lipopeptides could significantly reduce the mortality of the tomato plants and achieve a biocontrol efficiency of 96.2% (Figure 2, Table 1). These results suggested that one of the antagonistic mechanisms of strain FJAT-46737 is the 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, namely, iturin (retention time,12.8–22.3 min), fengycin (29.0–36.0 min), and surfactin (48.3–52.0 min), were detected, and the retention times, MS and MS2 spectral data and identification results for 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 the 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 under treatment with 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). The lipopeptide concentration of 0.5 mg/mL displayed antibacterial activity against R. solanacearum FJAT-91, while the concentration 0.25 mg/mL did not (Figure S6). These results implied that the crude lipopeptides of FJAT-46737 had the strongest antibacterial activity on R. solanacearum. Moreover, under 30 mg/mL of the crude lipopeptides, the inhibition zone diameters for 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 approximately 20 mm at 48 h.
Effects of medium components and culture conditions on the antibacterial activities and contents of lipopeptides
To further improve the antagonistic activities of FJAT-46737, we analyzed the 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. The results of the antagonistic activity of the supernatant, crude lipopeptides and lipopeptide contents under different conditions are 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 for the culture supernatant from the medium A (Table 5). Moreover, the antibacterial activities of both the supernatants and crude lipopeptides from culture media C, D and E were stronger than those from culture media A, B and F (Table 5). These results suggested that the medium components could significantly affect the antibacterial activities of FJAT-46737.
Subsequently, the effects of temperature on the 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; and ii) both the cell-free supernatants and crude lipopeptides had the strongest antibacterial activities under incubated at 25 oC (Table 6).
The iturin, fengycin and surfactin contents in different culture conditions were further determined by LC-QTOF-MS and are summarized in Table 5 and Table 6. The fengycins were the most abundant lipopeptide family produced by FJAT-46737. The iturin, fengycin and surfactin contents 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 lipopeptides in culture media C and D were significantly higher than those in the other four media, indicating that media containing a rich organic nitrogen source might be benificial for producing lipopeptides. Moreover, adding yeast extract in culture medium D significantly increased the yield of lipopeptides compared with the lack of yeast extract (in culture medium B). In culture medium C, the production of lipopeptides (61.04±9.86 mg/L) was the highest compared to the other media. Moreover, the contents of the iturins, fengycins and surfactins were strongly dependent on temperature. The contents of fengycins decreased by 96.6% when the culture temperature increased from 20 oC to 40 oC while contents of surfactins increased by 59.9%. The temperature ranges of 25–30 oC, 20–25 oC and 35–40 oC were suitable for the production of iturin, fengycin and surfactin, respectively.
Subsequently, a correlation analysis was performed between the cell-free supernatant antibacterial activities and lipopeptide contents in different culture conditions (Table 7). The contents of fengycins and total lipopeptides were significantly positive correlated with the antibacterial activities of the cell-free supernatants of all samples (p < 0.05), whereas the contents of surfactins and iturins did not show this correlation. These results suggested that the antibacterial activities of the cell-free supernatants were mainly attributed to the secretion of the fengycins by FJAT-46737.
Effect of purified lipopeptides on the 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 SPE70) exhibited antibacterial activity. Therefore, the 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 fengycin plays an important role in the growth inhibition of R. solanacearum. This result was consistent with the aboved correlation analysis.