Biocontrol of tomato bacterial wilt by a new strain Bacillus velezensis FJAT-46737 and its lipopeptides

doi:10.21203/rs.2.17222/v2

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Biocontrol of tomato bacterial wilt by a new strain Bacillus velezensis FJAT-46737 and its lipopeptides

https://doi.org/10.21203/rs.2.17222/v2

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published 15 Jun, 2020

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Background: There is an urgent need to discover alternative antimicrobial agents to control bacterial wilt. The objectives of this study were to report a new lipopeptide-producing biocontrol strain FJAT-46737 and its lipopeptides, and to investigate their antagonistic effects.

Results: Based on the whole genome sequence analysis, the new strain FJAT-46737 was finally identified as Bacillus velezensis and seven gene clusters that responsible for synthesis of bioactive secondary metabolite in the FJAT-46737 were predicted. Antimicrobial results demonstrated that FJAT-46737 exhibited broad-spectrum antimicrobial activities in vitro against bacteria and fungi . The pot experiments showed that the tomato bacterial wilt control efficiencies of the whole cultures, the 2-fold diluted supernatants and the crude lipopeptide of FJAT-46737 were 66.2%, 82.0%, and 96.2%, respectively. The above results suggested that one of the antagonistic mechanisms of FJAT-46737 was secretion of lipopeptides that consisted of iturins, fengycins and surfactins. The crude lipopeptides had significant antagonistic activities on several pathogen in a dosage-dependent manner, and the fengycins confirmed to play a major role in antibacterial abilities in vitro of the lipopeptides against Ralstonia solanacearum . Furthermore, it was found that the rich organic nitrogen sources (especially yeast extracts) in the media were beneficial for FJAT-46737 to produce fengycin and surfactin. The secretion of these two lipopeptides were also adjusted by the culture temperature: the content of the fengycins decreased by 96.6% and that of the surfactins ascended by 59.9% from 20 o C to 40 o C. The temperature of 20 o C~25 o C is the suitable temperature for FJAT-46737 to produce lipopeptide.

Conclusions: The B. velezensis strain FJAT-46737 and its lipopeptides would used as new sources of potential biocontrol agents against plant pathogens, especially the bacterial wilt pathogen R. solanacearum .

Applied & Industrial Microbiology

Bacillus velezensis FJAT-46737

lipopeptides

antagonistic activities

bacterial wilt

fengycin

culture condition

Bacterial wilt caused by Ralstonia solanacearum is the devastating diseases of almost 250 plant species, which seriously threaten plant growth and lead to huge losses around the world [1, 2]. Traditional agricultural practices, like crop rotation, field sanitation, plant resistant varieties, and chemical bactericide have been widely applied to control the bacterial wilt but obtained limited success [3]. In factlly, the long-term or repeated using of chemical bactericide caused pathogen resistance and has its adverse effects on the environment, beneficial organisms, and human health [3]. To seek safe, effective and alternative methods for controlling plant pathogen diseases are become more and more important in improving the output and quality of agricultural products. Furthermore, biological control agents, the use of antagonistic microorganisms, including bacteria, yeasts, and filamentous fungi, fight plant pathogens, has been proved as a safe, effective and sustainable alternative method comparing to chemical bactericide [4-6]. Members of the genus Bacillus have been used as effectively biocontrol agents to reduce damage caused by bacterial wilt and their derived products occupy approximately half of the commercially available biopesticides [7]. However, the control of bacterial wilt becomes difficult and ineffective since the high genetic variability, persistence in the environment and broad host range of R. solanacearum, highlighting the urgent need for discovering alternative antimicrobial agents [3, 8].

Studies on the suppression mechanism of the Bacillus agents indicated that the biocontrol effects of species primarily attribute to their production of various bioactive molecules [9]. Lipopeptide is one of the most important bioactive substances and exhibits excellent properties, such as broad-spectrum antibiotic activity, good stability, low toxicity, high biodegradability, and less prone to drug resistance [10]. Bacillus lipopeptides include three classes, namely iturin (bacillomycin D/F/L/Lc, iturin A/C/D/E, and mycosubtilin), fengycin (fengycin A/B, and plipastatin A/B) and surfactin (halobacillin, pumilacidin and surfactin) [11, 12, 13]. They all share a common amphiphilic molecule structure composed of a fatty acid side chain linking to a cyclic peptide ring (Figure S1). Among them, the iturins exhibit strong antifungal activities against various types of yeast and filamentous fungi, but with limited antibacterial activity; the fengycins have strong antifungal activities, especially on filamentous fungi, but with no effect on the inhibition of bacteria until the new finding reported recently by Villegas-Escobar et al. [14]; the surfactins display effectively bactericidal activities. Besides, surfactin can also effectively reduce the surface tension in plant root to facilitate the swimming ability and biofilm formation of the Bacillus strains to protect plants from attack by pathogens [15].

The well-studied lipopeptides were mainly focused on control of the plant fungal pathogens, such as Rhizoctonia solani [16], Pythium ultimum [4], Botrytis cinerea [17], Podosphaera fusca [17], Fusarium graminearum [18, 19], Fusarium oxysporum [19], and Sclerotinia sclerotiorum [20]. Few studies were devoted to bacterial pathogens of R. solanacearum. Zhu et al. [21] reported that the lipopeptide mixtures (surfactin and iturin A) produced by B. amyloliquefaciens XZ-173 could effectively inhibit growth of R. solanacearum. Furthermore, the lipopiptides secreted by XZ-173 were used to manufacture lipopeptide-mineral composites to achieve an 87.76% biocontrol efficacy on controlling bacterial wilt of tomato [22]. The suppression mechanism of lipopeptides against plant diseases is either by impeding the pathogens directly or acting as elicitors for the induced systemic resistance in the host plants [17, 23, 24]. A strong correlation between defense-inducing activity and content of the surfactins formed by the Bacillus strains have been reported [25]. In addition, the lipopeptide secretion of the Bacillus strains relies on not only the differences of the strains themselves but also of the culture medium components as well as fermentation conditions [26, 27, 28].

The present study is aim to report a new lipopeptide-producing biocontrol strain FJAT-46737. The strain FJAT-46737 was identified through the whole genome sequence analysis and the bioactive secondary metabolite gene clusters in the strain FJAT-46737 were predicted. The suppression effects of FJAT-46737 and its lipopeptides in vitro and in vivo were evaluated. Furthermore, the lipopeptide components were identified, and effects of culture conditions on antibacterial activity of the strain FJAT-46737 and its lipopeptides were investigated. This work would promote application of the strain FJAT-46737 and its lipopeptides as new sources of potential biocontrol agents against plant pathogens, especially the bacterial wilt pathogen R. solanacearum.

Strains and Chemicals

The strain FJAT-46737 was isolated from a soil sample of Huanggang Mountain, Fujian Province, China, which had been preserved at the China General Microbiological Culture Collection Center (CGMCC No. 14661). The indicator strains R. solanacearum FJAT-91 (CGMCC No. 10692, tomato pathogens), R. solanacearum FJAT-77 (peanut pathogens), Escherichia coli FJAT-301, F. oxysporum f. sp. capsicum FJAT-831, F. oxysporum f. sp. niveum FJAT-30265 and F. oxysporum f. sp. melonis FJAT-9230 were preserved at the Agricultural Bioresources Research Institute, Fujian Academy of Agricultural Sciences, Fujian, China. All the culture media used for bacteria and fungi growth were purchased from AoBoXing Biological Technology Co., Ltd. (Beijing, China).The cultivation substrates used for the pot experiments were purchased from Xiamen Jiang Ping biological Technology Co., Ltd. (Xiamen, China). The reference standards of iturin and surfactin that produced from B. subtilis were purchased from Sigma (St. Louis, MO, USA).

Antimicrobial spectra of the strain FJAT-46737

Using the agar disk diffusion method, the strain FJAT-46737 was evaluated for its in vitro potential to inhibit several animal and plant pathogens, including R. solanacearum, E. coli, F. oxysporum f. sp. capsicum, F. oxysporum f. sp. niveum and F. oxysporum f. sp. melonis. For fungal pathogen strains, a 7 mm-diameter block of mycelium was placed onto the centre of a new potato dextrose agar (PDA) plate, the cultures of the strain FJAT-46737 were then streaked with sterilised toothpicks at a distance 2.5 cm away from the marginal of the mycelia colony and cultivated at 28^oC for seven days.

The antagonistic effects of the strain FJAT-46737 against bacterial pathogen srains were carried out using a two layer plating method. The semi-solid (NA)/Luria–Bertani (LB) media containing pathogenic bacterial culture suspensions were poured in Petri plates (90 mm) coated with the corresponding solid media. After solidification, a colony of the strain FJAT-46737 was streaked on the plated, and then were co-cultivated at 30^oC for two days.

Identification of the strain FJAT-46737

The strain FJAT-46737 was streaked on the NA agar plates and cultured at 30^oC for 48 h for investigating the colony and cell morphology. The genomic deoxyribonucleic acid (DNA) of FJAT-46737 was extracted with the sodium dodecyl sulfate (SDS) method and detected by the agarose gel electrophoresis and quantified by Qubit. The strain FJAT-46737 was further identified through analysis of its 16S rRNA and partial gyrase subunit B (gyrB) gene sequences and whole-genome sequence. The 16S rRNA gene was amplified and sequenced using the universal primers 27F and 1492R. The gyrB gene was amplified using the specific primers F (5'-GAAGTCATCATGACC-3') and R (5'-AGCAGGGTACGGAT-3'). The whole-genome sequencing was performed on the Illumina HiSeq PE150 platform at the Beijing Novogene Bioinformatics Technology Co., Ltd. Average nucleotide identity (ANI) was calculted using the OrthoANIu algorithm [29]. The ANI value cut-off of 96% is recommended for species delineation [30, 31].The 16S rRNA, gyrB gene and the whole-genome sequences of strain FJAT-46737 had been deposited in the GenBank with the accession number of MG924092, MH470338 and CP044133, respectively. The secondary metabolite gene clusters in the strain FJAT-46737 were predicted using the antibiotics Secondary Metabolite Analysis Shell (antiSMASH) tool.

Extraction and identification of the lipopeptides

The FJAT-46737 was grown at 30 °C in 50 mL of culture medium (Table S1) in a shaking incubator (170 rpm). After 48 h, the culture was centrifuged at 6000 g for 5 min to remove the cell. The supernatant was adjusted to pH 2.0 with 3 mol/L HCl to obtain the crude lipopeptides. The extract lipopeptides were dissolved in phosphate buffer and then were lyophilized. The obtained lipopeptide power were dissolved in methanol for for antimicrobial activity tests, qualitative and quantitative analyses, and dissolved in water for pot experiment in the further study.

The qualitative and quantitative analyses of lipopeptides using liquid chromatography quadrupole time-of-flight tandem mass spectrometry (LC-QTOF-MS/MS) technology were carried out using previous methods[32]. The structure of lipopeptide was estimated by their accurate mass and MS/MS fragmentation patterns according to literatures [33–37]. The content of each type lipopeptide was defined as: milligram of standard/litre of culture supernatant (mg/L).

Biocontrol evaluation of FJAT-46737 against tomato bacterial wilt

The strain FJAT-46737 was cultured at 30 °C for 48 h in medium E (Table S1) in a shaking incubator (170 rpm). The tomato seedlings with 3-4 emerged leaves were transplanted into 15 cm diameter pots at 30°C. After 2 days, two groups (each with thirty seedlings) were drenched with 100 mL/pot of the whole FJAT-46737 cultures (10⁸cfu/mL) and the double diluted fermentation supernatant, respectively. The lipopeptide treated group was carried out as following: thirty tomato seedlings were uprooted carefully and the roots were dipped in the crude lipopeptide solution (1mg/mL, pH 6.5) for one hour, then the seedlings were transplanted into pots. Each pot was drenched with 100 mL of the R. solanacearum cultures (10⁸ cfu/mL) after three days. Two control groups were also included: one was treated only by the R. solanacearum cultures and the other was treated only with water. Each treated group was performed three times repeatedly. Survivability of the seedlings was monitored at a regular interval. Above pot experiments were carried out at June to August in 2017 under greenhouse with the following incubated conditions: illumination of 16-h day-8-h night cycle; temperature of 28–32°C; and relative humidity of 60–80%. The disease index (di), disease incidence (DI) and biocontrol efficacy of bacterial wilt was calculated according to the method described by Xiong et al [38].

Antimicrobial spectrum determination of the lipopeptides

The crude lipopeptide extracts from FJAT-46737 were dissolved in methanol to the final concentrations of 2.5 – 30 mg/mL. The antimicrobial activities of the crude lipopeptides were evaluated against eight indicator strains using the oxford cup method [39]. The indicator strains were grown at 30^oC in the corresponding liquid media for 48 h. A two layer plate containing fungal spores or bacterial culture suspensions were prepared. Then, four oxford cups were placed onto each plate and 150 μL of the crude lipopeptides or cell-free supernatants were added to each oxford cup. Methanol was served as a negative control. Finally, the plates were cultivated at 30 ^oC for 48 h and the inhibition zone diameter was measured. All experiments were repeated by three times.

Effects of fermentation parameters on the antagonistic activities of the lipopeptides

R.solanacearum FJAT-91 was selected as an indicator strain. Culture media of A, B, C, D, E and F in Table S1 and temperatures of 20, 25, 30, 35 and 40°C were selected for cultivating the strain FJAT-46737. The antagonistic activities of the lipopeptides produced under different culture conditions were determined by the aforementioned agar disk diffusion method.

Purification of antibacterial lipopeptides

The lipopeptides were loaded onto a solid phase extraction (SPE) C₁₈ cartridge (6g/60mL) and washed with 60 mL of water and different proportions of methanol (10% to 100%), respectively. After evaporation, the fraction was dissolved in water and used for antibacterial activity test.

Statistical analysis

Analysis of variance (ANOVA) of the quantitative data were carried out using statistical software SPSS 19.0. Duncan’s multiple range tests were employed to determine the significant differences among the data (p < 0.05).

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 13613^T, Bacillus velezensis CR-502^T and B.amyloliquefaciens DSM7^T. 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 DSM7^T, B. siamensis KCTC 13613^T, and B. velezensis KCTC 13012^T 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 C₁₆fengycin 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 40^oC, on the contrary, that of the surfactins ascended by 59.9%. The temperature ranges of 25–30^oC, 20–25^oC 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.

Nowadays, it has been recognized that the traditional phenotypic methods and phylogenetic analysis of conserved gene are hardly to distinguish the species including B. subtilis, B. amyloliquefaciens, B. velezensis, B. siamensis, B. licheniformis, and B. pumilus due to their tight relatedness [40]. With the increasing lots of available complete genome sequences, it became easy to discriminate these species. The average nucleotide identity (ANI) based on the complete genome sequences was calculated and cut-off of 96% was proposed for species delineation [30, 40]. In present study, the strain FJAT-46737 displayed 98.26% similarity to the B. velezensis type strain KCTC 13012^T, which is well above of the recommended threshold of 96% for species delineation. Thus, the FJAT-46737 was finally identified as B. velezensis.

The occurrence of bacterial wilt in crop has brought great economic losses in the word. Chemicals are often used in controlling the disease but lead to environmental pollution and pathogen resistance in plants [3]. Many studies proved that the use of Bacillus strains as biological control agent is as a promising and safe strategy for the effective tomato bacterial wilt management. Kwon and Kim reported that B. subtilis JW-1 could result in >80% reduction in bacterial wilt disease, which can use as a potential biocontrol agent of bacterial wilt [41]. Xiong et al. had isolated B. amyloliquefaciens JK6 that could effectively suppress tomato bacterial wilt with the biocontrol efficacies of 58.6% and 52.9% in two greenhouse conditions [38]. B. subtilis 916 had been found to exhibit a biocontrol efficiency of 55.6% on the tomato bacterial wilt [42]. In present study, we had obtained a new biocontrol strain B. velezensis FJAT-46737 that exhibited broad-spectrum antimicrobial activities against gram-negative bacteria and filamentous fungi. The biocontrol efficacy of the strain FJAT-46737 on controlling the potato bacterial wilt was 66.2%, which was higher than those of many previously reported Bacillus spp. strains, such as the strains APF1 (60.3%), JK6 (58.6%) and 916 (55.6%) [38, 42, 43].

The biocontrol ability of the Bacillus strains against the plant pathogens has been confirmed to be achieved by induction of host systemic resistance, antibiotic production, secretion of siderophores and lytic enzymes, biofilm formation, or competition for niches within the rhizosphere [44]. Bais et al reported that B. subtilis 6051 was able to control Pseudomonas syringae root infection in Arabidopsis because of the secretion of surfactin and the formation of biofilm on the plant roots [15]. Kwon and Kim demonstrated that B. subtilis JW-1 induced a significant bacterial wilt disease suppression effect in vivo due to the production of cyclic lipopeptide [41]. Xiong et al. reported that secretion of the surfactin lipopeptides played important roles in biocontrol of tomato bacterial wilt by the strain JK6 [38]. In the present study, biocontrol efficiency of cell-free supernatant against the tomato bacterial wilt was 82.0%, while that of lipopeptide reached up to 96.2%, inferring that one of the mechanisms of disease suppression by B. velezensis FJAT-46737 was lipopeptide secretion.

The Bacillus species produces lipopeptides depending on the strains themselves: some strains can co-produce two or three classes of lipopeptides, while others can yield only one class [11-13]. Bacillus licheniformis MB01 only produce surfactin [12], B. subtilis K1 co-produce surfactins and iturins, while B. amyloliquefaciens SYBC H47 yield three types of lipopeptides including bacillomycin, fengycin and surfactin [45]. In present study, the strain B. velezensis FJAT-46737 could also co-produce three types of lipopeptides including C₁₄–C₁₆ iturin A, C₁₆ fengycin A/B, C₁₆fengycin A₂/ B₂, as well as C₁₄–C₁₅ surfactin. Furthermore, yield of the lipopeptides is significantly affected by the medium components (such as carbon and nitrogen sources, trace metals, etc.) and fermentation conditions (such as culture temperature, incubation time, rotary speed, etc.) [46]. In this study, B. velezensis FJAT-46737 could yield three types of lipopeptides in the all six common culture media (A~F). Whereas, the antibacterial activities of the crude lipopeptides (10 mg/mL) produced from culture media A, B or F were much weaker than the other three media. Carbonsource had been believed as a primary factor for lipopeptide production, for an example, Li et al found that variation of carbonsourse in the culture medium changed the type of lipopeptides produced by B. licheniformis HSN221 [47]. However, the types of lipopeptide produced by FJAT-46737 were not changed whether the culture medium containing glucose or not. It was reported that nitrogen sources played an important role in the regulation of biosurfactant synthesis [48]. Our results showed that rich organic nitrogen sources in the media were benificial for producing fengycin and surfactin. Furthermore, influence of yeast extracts on production of fengycin was more significant than that of iturin and surfactin. This might be owing to the fact that response of the enzymatic complex for fengycin biosynthesis to nitrogen sources would be less sensitive than that of the enzymatic complexs for iturin and surfactin [49].

Interestingly, effects of the culture temperatures on contents of the fengycins and surfactins generated by the strain FJAT-46737 displayed a different manner: with the temperature increasing, content of the fengycins decreased, but that of the surfactin rised. These results were not consistent with the report demonstrated by Monteiro et al., who found that the low temperature (15 ^oC) was suitable temperature for producing surfactins and the amount of fengycin was not affected by temperature changes [46]. Fengycin and surfactin are synthesized nonribosomally by fengycin synthetases and surfactin synthetases, respectively [50]. Usually, the optimum temperature for enzyme activity is 37^oC, this can explain why the content of surfactin ascend along with increase of the culture temperature. The high temperature leading to the few fengycin could attribute to the decreasing activity of fengycin syntheases. Generally, the suitable temperatures are 20 ^oC–30 ^oC for crop growing in the field. Our results indicated that 25^oC was a favorable temperature for the strain FJAT-46737 to yield lipopeptides with the strongest antibacterial activities, implying that the strain FAJT-46737 and its lipopeptides have good ecosystem adaptability and prospect in future agricultural application. Previous studies demonstrated that the surfactins display antibacterial activities, whereas iturins showed strong antifungal activities with limited antibacterial activity. The fengycin family is specific against filamentous fungi until two cases recently reported by Villegas-Escobar et al.[14] and Chen et al., [32], who found that the fengycins exhibited strong antibacterial activity against R. solanacearum in vitro. In present study, the correlation analysis between the cell-free supernatant antibacterial activities and lipopeptide contents indicated that the antibacterial activities of cell-free supernatant were significantly positive correlated with content of the fengycins. This is agreement with that the report that the significant relationship between the strong antibacterial activity and the production of fengycin and surfactin of Bacillus isolates [51]. In addition, only the purified fraction SPE70 exhibited antibacterial activity in vitro, which just consisted of fengycins, indicating that the antibacterial activity of lipopeptide mixture could be due to the fengycins. These results further confirmed antibacterial activity of the fengycins first reported by Villegas-Escobar et al. [14] and Chen et al., [32].

A new strain B. velezensis FJAT-46737 with broad-spectrum antimicrobial activities was confirmed to have strong antibacterial activity against the bacterial wilt pathogen R. solanacearum by both the in vivo and in vitro experiments. Moreover, suppressive effects of FJAT-46737 were associated with to the lipopeptide secretion, especially being attributed to content of the fengycins. Therefore, FJAT-46737 and its lipopeptides would have good application prospects for biocontrol of the bacterial wilt.

CGMCC: China General Microbiological Culture Collection Center

PDA: potato dextrose agar

LB: Luria–Bertani

NA: nutrient agar

SDS: sodium dodecyl sulfate

gyr B: gyrase subunit B

DNA: deoxyribonucleic acid

RNA: ribonucleic acid

ANI: average nucleotide identity

antiSMASH: antibiotics Secondary Metabolite Analysis Shell

di: disease index

DI: disease incidence

SPE: solid phase extraction

ANOVA: analysis of variance

LC-QTOF-MS/MS: liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry.

Ethics approval and consent to participate

No animals, human subjects, human material, or human data are used in this study.

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary information files. The 16S rRNA, gyrB and whole genome sequences of this study have been submitted to NCBI GenBank database (accessions MG924092, MH470338 and CP044133).

Competing interests

The authors declare that they have no competing interests.

Funding

This work was funded by the Natural Science Foundation of Fujian Province (No. 2017J01050); the Young Elite Scientists Sponsorship Program by CAST (No. 2017QNRC001); the Science and Technology Innovation Team Program (No. STIT2017-1-11), the Agrobiogenome Program (No. A2015-3, No. A2017-4), and the Youth Fund (No. YC2015-11) of Fujian Academy of Agricultural Sciences. These organazations provided finance support for this study, but did not involved in the study design, data analysis, decision to publish, or in writing this manuscript.

Authors’ Contributions

MC, JW, BL and YZ conceived and designed the experiments. MC, JW, YZ, RX and BL were responsible for drafting the article. MC, RX, WY, CG, and ZC were involved in the experiments preformation and data analysis. All authors approved the final version of the manuscript

Acknowledgements

Not applicable

Authors’ information

Affiliations

Agricultural Bioresources Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou, 350003, China

Meichun Chen, Jieping Wang,Bo Liu, Yujing Zhu, Rongfeng Xiao, Cibin Ge& Zheng Chen

College of Biological Science and Engineering, Fuzhou University, Fuzhou, 350001, China

Wenjing Yang

Corresponding author

Correspondence to Bo Liu

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Table 1. The biocontrol efficacies and the disease incidence in differently treatment groups of plants.

Treatment group	biocontrol efficiency	Disease incidence (DI)
Control group	0%	93.8%
Whole culture	66.2%	31.7%
2-fold diluted cell-free supernatants	82.0%	17.5%
Lipopeptide (1mg/mL)	96.2%	3.7%

Table 2. Identification of lipopeptides extracted from strain Bacillus velezensis FJAT-46737 through LC-QTOF-MS/MS

Retention time (min)	MS m/z [M+H]⁺ /[M+Na]⁺	Identified Compounds
12.833	1065.5^a	C₁₄Iturin A
16.632	1079.7^a	C₁₅Iturin A
17.536	1079.7^a	C₁₅Iturin A
21.244	1093.6^a	C₁₆Iturin A
22.284	1093.6^a	C₁₆Iturin A
29.067	1449.9^b	C₁₆FengycinA₂
30.423	1449.9^b	C₁₆FengycinA₂
30.514	1463.9^b	C₁₆FengycinA
31.056	1463.9^b	C₁₆FengycinA
31.735	1449.9^b	C₁₆FengycinA₂
32.413	1477.9^b	C₁₆FengycinB₂
33.182	1463.9^b	C₁₆FengycinA
34.132	1477.9^b	C₁₆FengycinB₂
34.991	1477.9^b	C₁₆FengycinB₂
35.262	1491.8^b	C₁₆FengycinB
35.986	1491.8^b	C₁₆FengycinB
48.376	1030.8^a	C₁₃Surfactin
50.26	1044.8^a	C₁₄Surfactin
50.757	1044.8^a	C₁₄Surfactin
51.345	1030.8^a	C₁₃Surfactin
51.978	1058.8^a	C₁₅Surfactin

Note: a is [M+Na]⁺and b is [M+H]⁺.

Table 3. Antibacterial ability of lipopeptides from strain Bacillus velezensis FJAT-46747

		Diameter of inhibition zone (mm)
indicator strains		Ralstonia solanacearum FJAT-91 (tomato pathogens)	Escherichia coli FJAT-301	Ralstonia solanacearum FJAT-77 (peanut pathogens)
concentration of crude lipopeptides (mg/mL)	10	18.52±0.73^a	14.55±0.23^a	14.57±1.85^a
	5	16.04±0.26^b	12.32±1.14^b	13.90±0.43^a
	2.5	13.87±0.99^b	10.02±0.01^c	11.19±0.03^a
	1	12.51±0.58^b	weak	-

Note: Values were expressed as mean ± standard deviation (n = 3).

The difference letter in the same column indicated that the difference between the grades is significantly through Duncan test (p <0.05).

Table 4. Antifungal ability of lipopeptides from strain Bacillus velezensis FJAT-46747

	Diameter of inhibition zone (mm)
indicator strains		Fusarium oxysporum f. sp. capsicum FJAT-831	Fusarium oxysporum f. sp. melonis FJAT-9230	Fusarium oxysporum f. sp. niveum FJAT-30265
concentration of crude lipopeptides (mg/mL)	30	17.87±0.11^a	18.77±0.25^a	20.47±3.08^a
	20	16.62±0.78^a	18.36±0.44^a	17.28±0.64^a
	10	13.84±0.58^b	16.00±0.22^b	14.51±1.35^a

Note: Values were expressed as mean ± standard deviation (n = 3).

The difference letter in the same column indicated that the difference between the grades is significantly through Duncan test (p <0.05).

Table 5. Diameter of inhibition zone of the culture supernatant and crude lipopeptides (10 mg/mL) produced by FJAT-46737 against Ralstonia solanacearum FJAT-91 and the content of lipopeptide under different culture medium.

Diameter of inhibition zone (mm)	Medium	A	B	C	D	E	F
	supernatant	-	-	15.75±0.38^ab	14.93±0.77^b	16.36±0.60^a	12.77±0.88^c
	Lipopeptides	13.88±2.54^b	-	21.41±1.60^a	19.15±0.78^a	19.75±0.93^a	15.67±1.00^b
Yield of lipopeptide in the supernatant (mg/L)	iturin	0.53±0.20^cd	0.31±0.09^d	0.98±0.02^b	0.68±0.14^c	1.99±0.18^a	0.38±0.02^d
	fengycin	22.17±11.94^b	9.03±1.69^b	58.66±9.81^a	50.37±12.46^a	24.34±2.75^b	9.17±1.21^b
	surfactin	0.65±0.42^a	0.55±0.23^a	1.39±0.06^a	1.21±0.35^a	0.61±0.06^a	0.59±0.13^a
	Total lipopeptides	23.34±12.54^b	10.07±1.80^b	61.04±9.86^a	52.27±12.70^a	27.54±3.90^b	10.22±1.29^b

Note: Values were expressed as mean ± standard deviation (n = 3).

The difference letter in the same row indicated that the difference between the grades is significantly through Duncan test (p <0.05). The effects of medium components on the antibacterial activities and contents of lipopeptides were carried out at 25^oC

Table 6. Diameter of inhibition zone of the culture supernatant and crude lipopeptides (10 mg/mL) produced by FJAT-46737 against Ralstonia solanacearum FJAT-91 and the content of lipopeptide under different culture temperature.

Diameter of inhibition zone (mm)	Temperature	20^oC	25^oC	30^oC	35^oC	40^oC
	supernatant	12.36±0.46^b	14.47±1.06^a	12.06±0.26^b	-	-
	Lipopeptides	14.34±0.90^b	17.16±0.46^a	15.53±1.83^ab	11.00±0.49^c	-
Yield of lipopeptide in the supernatant (mg/L)	iturin	0.65±0.03^b	0.78±0.07^a	0.86±0.10^a	0.61±0.07^b	0.45±0.01^c
	fengycin	55.11±4.74^a	56.47±6.59^a	23.62±6.34^b	9.55±0.27^c	1.88±0.28^c
	surfactin	0.59±0.06^b	0.83±0.19^b	0.78±0.13^b	1.22±0.21^a	1.47±0.15^a
	Total lipopeptides	56.30±4.76^a	57.94±4.76^a	25.41±6.37^b	11.39±0.53^c	3.72±0.36^c

Note: Values were expressed as mean ± standard deviation (n = 3).

The difference letter in the same row indicated that the difference between the grades is significantly through Duncan test (p <0.05). The effects of temperature on the antibacterial activities and contents of lipopeptides were carried out using the culture media C.

Table 7. Correlation coefficients between antibacterial activities of the fermentation supernatant and the lipopeptide contents.

	Pearson correlation coefficient	Kendall correlation coefficient	Spearman correlation coefficient
Iturin	0.570	0.597^*	0.740^*
Fengycin	0.695^*	0.559^*	0.726^*
Surfactin	-0.100	0.078	0.033
Total lipopeptide	0.705^*	0.559^*	0.726^*

Test of significance by two-tailed

*p < 0.05;

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Biocontrol of tomato bacterial wilt by a new strain Bacillus velezensis FJAT-46737 and its lipopeptides

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