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Antifungal activity and application of Bacillus tequilensis A13 in biocontrol of Rehmannia glutinosa root-rot disease



The bacterial genus Bacillus, an important group of bacteria which can suppress phytopathogens, has been widely used in agriculture. However, different species of Bacillus often displayed significant differences in probiotic efficiency and mechanism, suggesting that it is very necessary to investigate the biocontrol potential of new Bacillus isolates, especially from under-evaluated Bacillus spp. Rehmannia glutinosa (R. glutinosa), an important traditional Chinese medicinal herb, is affected by a serious root-rot disease caused by the fungus Fusarium solani (F. solani). Biocontrol agents against this root-rot disease in R. glutinosa have yet to be developed. This study explored, for the first time, the activity and biocontrol mechanism of a new Bacillus isolate, Bacillus tequilensis A13, in antagonizing F. solani.


B. tequilensis A13 displayed a strong inhibitory activity (73.49% ± 1.33%) against F. solani growth in vitro and was able to survive and multiply in the sterilized soil. The results from liquid chromatography electrospray ionization tandem mass spectrometry (LC–MS/MS) and Gene Ontology (GO) analyses indicated that B. tequilensis A13 cell-free supernatant contained six antifungal compounds, eight antifungal compound synthases, and several functional proteins involved in the processes of plant stress resistance, etc. Finally, the joint application of complex fertilizer together with B. tequilensis A13 significantly reduced the incidence of R. glutinosa root rot in the field.


B. tequilensis A13 strongly inhibited F. solani growth by producing antifungal compounds and proteins associated with plant stress resistance/tolerance, and proved to be a promising candidate biocontrol agent against R. glutinosa root-rot disease.

Graphical Abstract


As a promising alternative to crop protection chemicals, biocontrol of plant pathogens has ignited worldwide interest for its safety towards human health and its environment-friendly nature [1]. Different microbial genera (such as the bacteria Bacillus and Pseudomonas, and the fungus Trichoderma) have been reported as potential biocontrol agents against phytopathogens [2]. Among these, members of the genus Bacillus, consisting of 380 species [3], have proved to be an important source of biocontrol agents due to their distinctive abilities to form extreme environment-resistant endospore and produce a range of antimicrobial compounds [4].

At present, only a small proportion, of about 20 Bacillus species, mainly B. subtilis, B. thuringiensis, B. amyloliquefaciens, B. pumilus and B. cereus, have been investigated for biocontrol activity against phytopathogens [4, 5]. Moreover, owing to their genetic and metabolic diversity, different Bacillus species, even different strains belonging to the same species, can have markedly different inhibitory efficiencies and/or mechanisms against phytopathogens. For example, Bacillus subtilis M4 induced defense response in soybean against Macrophomina phaseolina, mediated by activities of antioxidant defense enzymes, such as superoxide dismutase, phenol peroxidase, peroxidase, and catalase [6], whereas Bacillus subtilis KLBC BS6 could also induce resistance defense response in blueberry against Botrytis cinerea, but mediated by other enzymes, such as chitinase, phenylalanine ammonia-lyase, and polyphenol oxidase [7]. B. pumilus W-7 from potato antagonized Phytophthora infestans by secreting surfactin and fengycin B [8], whereas B. pumilus MSUA3 from Fagopyrum esculentum strongly antagonized Rhizoctonia solani and F. oxysporum by producing chitinolytic enzymes and surfactin [9]. Bacillus amyloliquefaciens L3 from the watermelon produced two volatile organic compounds which inhibited F. oxysporum f.sp. niveum [10], whereas B. amyloliquefaciens 35 from cacao produced 10 different volatile organic compounds inhibiting mycelium growth and spore germination of Moniliophtora roreri [11]. All these researches suggests that it is important to investigate the biocontrol potential of some new Bacillus isolates, especially from under-evaluated Bacillus spp. Notably, these Bacillus strains in researches above were often isolated from different plants, suggesting that an effective strategy to broaden the scope of biocontrol agents is to isolate new bacterial strains within the genus Bacillus from different host plants.

Medicinal plants have been attracting increasing attentions due to their various pharmacological properties. In proper culture conditions, each medicinal plant would produce unique and structurally divergent bioactive secondary metabolites [12], some of which are frequently released into soil, shaping distinct and highly specific rhizosphere microbiome by attracting and maintaining a preferential soil microbial reservoir [13, 14]. In turn, rhizosphere microbiome can achieve direct or indirect protection for the host medicinal plant by improving the nutrient absorption, enhancing disease resistance, etc. [15, 16]. Despite the above advances, the bacteria from medicinal plant rhizosphere have not been widely explored for the use as new biocontrol agents.

R. glutinosa is one of the most important traditional Chinese medicinal herbs. It prefers to grow in the environment with sufficient light, sandy loam and good drainage, and release various root exudates (e.g., phenolic acid, phenylethanoid glycosides, and iridoid glycoside) into soil [17], which probably induce the colonization of some special functional microbes in R. glutinousa rhizosphere. In addition, during standard cultivation of R. glutinosa, root-rot disease frequently leads to yield losses of 10–80% each year. Therefore, isolating antagonistic microbes (e.g., new Bacillus strain) from the rhizosphere of healthy R. glutinousa will greatly facilitate the development of new biological control agents active against root-rot disease of R. glutinousa or other medicinal plants.

To achieve the goal, in this study, 106 bacteria strains were isolated from the R. glutinosa rhizosphere. Among these strains, one new B. tequilensis strain A13 displayed the strongest biocontrol potential against root-rot pathogen, F. solani. Furthermore, protein fractionation, LC–MS/MS and GO bioinformatics analyses were performed to identify the extracellular protein components from A13 and the potential pathways by which these proteins inhibited the growth of F. solani.

Materials and methods

Isolation of bacteria from the rhizosphere soils of R. glutinosa

To isolate the bacteria, 10 g of R. glutinosa rhizosphere soil was placed in 100-mL sterile water and shaken at 180 rpm and 37 °C. After 30 min, the 100 µL suspension was spread on Luria–Bertani (LB) agar plate. After 48 h at 37 °C, bacterial colonies of different sizes, colors and morphologies appeared on the plates, were purified by streaking and stored at − 80 °C in LB broth supplemented with 50% glycerol.

Screening for the antagonistic bacteria against F. solani

All the isolates were screened for antagonism against the pathogenic fungus F. solani from R. glutinosa. Briefly, F. solani was cultured on potato dextrose agar (PDA) medium at 28 °C. After 4 days, a mycelial plug (5.0 mm in diameter) of F. solani was cut from the leading edge of the colony and transferred onto a new PDA plate. Then, one isolate was streaked in a straight line along one edge of the PDA plate; another plate was inoculated with only F. solani to use as a control. After 4 days at 28 °C, the inhibitory activity of each isolate was calculated as described by Kumar et al. [18]. Ultimately, strain A13, identified as having the greatest inhibitory ability, was selected for further study.

Strain characteristics and identification

The physiological characteristics of strain A13, including spore formation, gram staining, and gelatin liquefaction, were determined in accordance with Bergey’s Manual of Determinative Bacteriology. The strain A13 was further identified by 16S rDNA sequencing using primers 27F (5ʹ-AGAGTTTGATCCTGGCTCAG-3ʹ)/1492R (5ʹ-GGTTCCTTGTTACGACTT-3ʹ). The total PCR reaction volume was 25 μL, containing 2 μL template DNA of the isolate bacteria, 12.5 μL of 2 × Taq PCR MasterMix, 0.8 μL of 10 μmol/L upstream and downstream primers and distilled water to a constant volume.

In accordance with the melting temperature values of the primers, the PCR amplification was started with predenaturation at 95 °C for 5 min, followed 35 cycles of denaturing at 94 °C for 30 s, annealing at 55 °C for 1 min and extension at 72 °C for 60 s, followed by holding the reaction mixture at 72 °C for 60 s to allow the complete extension of PCR products. PCR amplification products were stored at 4 °C and detected by electrophoresis on 1% agarose gels. They were then sequenced by the Sangon Biotech Company Limited (Shanghai, China).

Antifungal activity analysis of strain A13’s cell-free supernatant (CFS)

The isolated bacteria A13 was grown in liquid LB medium with continuous shaking at 180 rpm and 28 °C for 48 h. The CFS was collected at 12-h intervals by centrifugation at 10,000 rpm for 10 min at 4 °C and then passed through a 0.22-μm pore size Millipore filter to remove any cellular debris. To determine the effects of different CFS contents on antifungal activity, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mL CFS was independently mixed with PDA medium to obtain 100 mL constant volume and spread independently on 90-mm plates. Mycelial disks (5 mm in diameter) from 5-day-old fungal cultures were placed in the center of each plate. After 3–5 days at 28 °C, the diameters of the colonies were measured, and the antifungal activity of the CFS at different times was calculated. The PDA plate without CFS, but inoculated with a mycelial disk was used as the control. The experiments were repeated three times.

Effects of temperature, pH and proteases on the antifungal activity of A13 CFS

To confirm its thermal stability, the CFS was maintained independently at 30, 40, 50, 60, 70, 80, 90, 100, and 120 °C for 120 min and then cooled to room temperature. To determine pH resistance, the pH of the CFS was adjusted from 3 to 11 using 1 M HCl or 1 M NaOH [19]. To evaluate protease resistance, the CFS was treated with 1, 5 and 10 mg/mL protease (protease K/trypsin) at 37 °C for 120 min. After these treatments, the effects of temperature, pH and proteases on the antifungal activity of the CFS were determined. The experiments were repeated three times.

Fractional extraction of the CFS proteins by ammonium sulfate precipitation and the fractions’ antifungal activities

Proteins of the CFS were fractioned with ammonium sulfate at saturations of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% as described by Zhang et al. [20]. The different protein fractions were separately re-dissolved in phosphate buffer solution (pH 7.0) and desalinated by dialysis. Afterwards, 5-mm mycelial plugs from the 5 days old fungal colony were cut and transferred to PDA plates. Then, sterile Oxford Cups (6 mm in internal diameter and 10 mm high) were placed around the mycelial plugs. The different protein fractions were added independently into different cups. The fraction with the strongest antifungal activity was selected to perform LC–MS/MS analysis.

LC–MS/MS analysis of antifungal proteins

The fraction with the strongest antifungal activity was further fractioned in an ultrafiltration tube using 30-, 50-, and 100-kDa molecular weight cutoff membranes. The inhibitory activities of the protein fractions with molecular weights of < 30 kDa, 30–50 kDa, 50–100 kDa, and > 100 kDa were analyzed using the diffusion experiment with the Oxford Cup method. The compositions of the fractions exhibiting antifungal activity were stored on dry ice and sent to Shanghai Zhong Ke New Life Company for LC–MS/MS (LC: Easy-nLC 1000, Thermo Fisher, USA; MS/MS: Q Exactive, Thermo Fisher, USA) detection. Specifically, 30 µg protein was added into 30 µl STD buffer (4% sodium dodecyl sulfate, 100 mM dithiothreitol, 150 mM Tris–HCl and pH 8.0). After 5 min incubation in a boiling water bath, the dilution was mixed with 200 μL UA buffer (8 M urea, 150 mM Tris–HCl and pH 8.0) in an 30 kDa ultrafiltration tube for centrifugation (14,000×g). The protein pellet was resuspended in 100 μL 50 mM iodoacetamide and incubated in darkness. After 30 min, the protein was successively washed with 100 ml UA buffer and 25 mM NH4HCO3. Then, the protein was resuspended in 40 μL 25 mM NH4HCO3 containing 2 μg trypsin for 24 h at 37 °C, followed by centrifugation. Finally, 40 μL of 25 mM of NH4HCO3 was added, and the mixture was through centrifugation and acidification.

Protein samples were separated by Easy-nLC system at a flow rate of 300 nL/min. Mobile phases A and B were 0.1% formic acid in high-performance liquid chromatography grade water and 0.1% formic acid in 84% acetonitrile, respectively. The linear gradient was 0–60% buffer B for 50 min, 60–90% buffer B for 4 min, held in 90% buffer B for 6 min. The eluted proteins were detected by MS and MS/MS. Under positive ion mode, the 20 most abundant precursor ions were selected from the survey scan (300–1800 m/z) for high-energy collisional dissociation fragmentation. Survey scans were acquired at a resolution of 70,000 at m/z 100 and the resolution for high-energy collisional dissociation spectra was set to 17,500 at m/z 100. Normalized collision energy was 27 eV and the underfill ratio was defined as 0.1%. A raw data file was submitted to UniProtKB database [21] to obtain the qualitative identification information of the target polypeptide molecules.

Field experiments

In field experiments, two treatments (five parallel fields/each treatment) were arranged a week before R. glutinosa planting: (1) Treatment group: complex fertilizer (5 kg, Nuoweier Fertilizer Company Limited, China) combined with A13 (3.2 × 1013 cells) was spread on the field (22 m2); (2) Control group: 5 kg complex fertilizer alone was spread on the field (22 m2). The total nitrogen, phosphorus and potassium contents in the complex fertilizer were each > 45%. At harvesting stage, a total of > 110 roots for control or treatment field were randomly selected to count the root-rot incidence of R. glutinosa.

Statistical analysis

All statistical analyses were performed using origin 6.0 or excel 2010 software. Student’s T test was used for statistical comparison and P < 0.05 was considered to be a significant difference. A GO enrichment analysis of antifungal proteins was performed using the Gene Ontology database [22].


Inhibitory effects of isolate A13 on F. solani growth

We obtained 106 isolates of bacteria from the rhizosphere soil of R. glutinosa. Among these isolates, isolate A13 displayed the strongest in vitro inhibitory activity against F. solani growth, achieving 73.49% ± 1.33% inhibition (Fig. 1A, B). After being inoculated into sterilized soil, the CFU (colony forming units) number for A13 in the soil increased from (3.43 ± 0.59) × 108 CFU/g at day 0 to (1.04 ± 0.20) × 109 CFU/g at day 90, suggesting that A13 survived in the soil environment, which is important for a microbial agent that is to be used for the control of a soil-borne pathogen [23].

figure 1

Antagonistic effect of isolate A13 on F. solani. A, B The growth inhibition of A13 to F. solani. *P < 0.05

The isolate A13 characteristics and identification

After incubation on the agar culture medium for 48 h at 37 °C, the A13 colony exhibited milk white, wrinkled protuberances at the center and was opaque with an irregular edge. When observed using light microscopy, isolate A13 cells appeared rod-like (Additional file 1). The physiological characteristics of A13 were analyzed (Table 1). A13 produced positive results to most tests, such as spore formation, gram staining, and gelatin liquefaction, indicating that A13 belonged to the genus Bacillus. Furthermore, the 16S rDNA sequence indicated that isolate A13 showed 99.93% homology with strain B. tequilensis strain 10b (Accession number: NR_104919.1).

Table 1 Physiological characteristics of strain A13

Antifungal activity of B. tequilensis A13 CFS against F. solani

To examine the inhibitory effect of A13 CFS, F. solani was incubated on PDA plates containing 10% CFS for 3–5 days at 28 °C as described in “Materials and methods” section. As shown in Fig. 2A and B, CFS collected after 12 h, 24 h, 36 h, or 48 h of A13 growth (12 h-, 24 h-, 36 h-, or 48 h-A13 CFS) showed strong inhibitory effect on F. solani growth. Futhermore, 36 h-A13 CFS had the highest inhibitory ratio (up to 47.42%) against F. solani, although no significant difference was found between 36 h-A13 CFS and 24 h-/48 h- A13 CFS. As shown in Fig. 2C and D, the 36 h- A13 CFS inhibited the growth of F. solani in a concentration-dependent manner, with the average inhibitory activity increased from 12.29 to 47.42% along with the concentration range from 0.1% to 10%, respectively, suggesting that the extracellular substances from A13 contained antifungal compounds for which the concentration was positively correlated with inhibitory activity against F. solani growth.

Fig. 2
figure 2

Antifungal activity against F. solani of A13 CFS. A, B antifungal activity of CFS collected at different times of A13 growth; C, D antifungal activity of different ratios of CFS collected after 36 h of A13 growth. *P < 0.05

Furthermore, we investigated the effects of different physicochemical conditions on the inhibitory activity of 36 h-A13 CFS on F. solani growth. As shown in Fig. 3A and B, after a 120 min heat treatment at different temperatures (30–100 °C), the average inhibitory activity of 36 h-A13 CFS on F. solani growth ranged from 41.74% to 52.91%. The inhibitory activity of 36 h-A13 CFS in initial pH values ranging from 5.0 to 11.0 changed from 28.78% to 45.85% (Fig. 3C, D). Thus, the inhibitory activity of 36 h-A13 CFS was maintained at a high level over wide pH (4.0–11.0) and temperature (30–100 °C) ranges, although the antifungal activity varied with changes in pH or temperature. This stability at different pH levels and temperatures is helpful for the preservation and application of 36 h-A13 CFS when compared with traditional biological pesticides [24]. On the other hand, the inhibitory activity of 36 h-A13 CFS was severely negatively affected by proteolytic enzymes, such as proteinase K and trypsin. As shown in Fig. 3E and F, the addition of proteases decreased the inhibitory activity of 36 h-A13 CFS in a concentration-dependent manner. Both trypsin and proteinase K-treated CFS displayed significant lower inhibitory ability at a 10 mg/mL concentration than that at 1 and 5 mg/mL concentrations. Further, at a 10 mg/mL concentration, trypsin and proteinase K reduced the average inhibitory activity of 36 h-A13 CFS to 19.07% and 28.35%, respectively. Extracellular proteins play several very important roles in promoting plant growth and killing plant pathogens [25]. Thus, we speculated that some protein molecules were important for the inhibitory activity of 36 h-A13 CFS.

Fig. 3
figure 3

The effect of physicochemical properties and proteinases on inhibitory activity of A13 CFS against F. solani. A, B the effect of temperature; C, D the effect of pH; E, F the effect of different proteinases. Con, control; Tr 1–10 and Pk1-10 represent trypsin 1–10 mg/mL and proteinase K 1–10 mg/mL, respectively. *P < 0.05

Bioinformatics analysis of A13 CFS protein fractions having inhibitory activities against F. solani

Protein fractions of 36 h-A13 CFS were obtained by salting-out with ammonium sulfate at saturations of 10%–100%. As shown in Additional file 2A, the inhibitory activities of proteins against F. solani varied with different ammonium sulfate saturation levels, and the protein precipitation obtained by 70% saturated ammonium sulfate displayed the strongest inhibitory activity (Additional file 2A). This was further fractioned into four subfractions using microporous membranes: fraction A (< 30 kDa), fraction B (30–50 kDa), fraction C (50–100 kDa) and fraction D (> 100 kDa). Of these, only fractions B and D obviously inhibited F. solani growth (Additional file 2B).

To screen the potential protein components capable of inhibiting F. solani growth, we performed LC–MS/MS and bioinformatics analysis using GO bioinformatics resources. LC–MS/MS profiles of fractions B (30 − 50 kDa) and D (> 100 kDa) from B. tequilensis A13 are shown in Additional file 3. Furthermore, a total of 1,353 extracellular proteins of B. tequilensis A13 were identified in fractions B and D. Among these proteins, 500 proteins were annotated with 278, 299 and 102 terms corresponding to 8, 16 and 49 categories of molecular function, protein class and PANTHER pathway, respectively. In detail, the top categories for molecular function were catalytic activity (68%), binding (23.7%) and transporter activity (4.0%). Consistent with the molecular function, the top categories for protein class were enzymes (78%) and nucleic acid-binding proteins (11.4%). Among the enzymes, hydrolases possessed 22.4% of the total protein classes and were the most common proteins (Fig. 4). Further analysis indicated that six antimicrobial proteins, namely subtilisin, beta-1,3–1,4 glucanase, lysozyme, iturin, antimicrobial peptide LCI, and putative antimicrobiological protein were present among the A13 extracellular proteins. Notably, among the identified ligases, some synthetases of cyclic lipopeptide antibiotics and bacitracin with strong inhibition activities against pathogenic fungi, such as plipastatin, surfactin, mycosubtilin, fengycin, and linear gramicidin, were also detected in this study, suggesting that A13 has the ability to produce these antimicrobial compounds. The proteins in A13 CFS associated with plant pathogen inhibition are listed in Table 2.

Fig. 4
figure 4

GO analysis of antifungal protein fractions from A13 CFS

Table 2 The proteins related with plant pathogen inhibition in B. tequilensis A13 CFS

A metabolic pathway analysis indicated that amino acid-, vitamin-, and plant stress resistance-related metabolic categories possessed 30.60%, 5%, and 9.80% of the total 49 metabolic categories, respectively (Fig. 4). Furthermore, extracellular proteins of A13 were involved in the biosynthesis of 15 amino acids, namely phenylalanine, arginine, valine, methionine, tyrosine, lysine, threonine, isoleucine, histidine, serine, glycine, s-adenosylmethionine, cysteine, proline, and tryptophan, as well as five vitamins, thiamin, vitamin B6, flavin, biotin, and tetrahydrofolate. In addition, it has been found that some extracellular proteins of A13 were involved in plant stress resistance-related metabolisms, such as aminobutyrate and 5-hydroxytryptamine metabolic pathways.

A field application of B. tequilensis A13 for the biocontrol of R. glutinosa root-rot disease

The inoculation experiment revealed that the root-rot disease incidence in the tests simultaneously inoculated with both F. solani and B. tequilensis A13 was reduced by 42.85% when compared with the value from the test inoculated with only F. solani. Furthermore, when A13 at a density of 6.4 × 109 CFU/g was combined together with complex fertilizer and applied in the fields, the average root-rot disease incidence of R. glutinosa decreased by 13.13%, when compared with the control group (Fig. 5).

Fig. 5
figure 5

The effect of complex fertilizer combined with A13 on R. glutinosa root rot in the fields. A The field experiment of complex fertilizer (Control); B the influence of complex fertilizer combined with A13 (Treatment) on incidence of R. glutinosa root rot. *P < 0.05


As a promising alternative to chemical fungicides, the biocontrol of plant pathogens has been attracting attention. However, obtaining a strain with high biocontrol ability against a pathogenic fungus is becoming a key constraint for the biological control of plant diseases. Currently, an effective strategy to broaden the scope of biocontrol agents is to isolate novel bacterial strains from different plants in which traditional Chinese medicinal herbs, such as R. glutinosa, should be targeted due to their growth characteristics and unique root exudates.

In this study, 18 of 106 bacteria isolated from R. glutinosa rhizosphere displayed obvious antagonistic activities against the root-rot pathogen F. solani (average inhibitory ratio: 65.78%–73.49%). Among these 18 bacteria, B. tequilensis A13 displayed the strongest inhibitory activity (73.49% ± 1.33%) against F. solani in confrontation experiments, maintained an effective survival rate in sterilized soil and reduced the incidence of R. glutinosa root rot by 42.85% in the inoculation experiments. When B. tequilensis A13 was used together with a complex fertilizer in field experiments, the average incidence of root rot was significantly reduced in R. glutinosa (Fig. 5). Of course, further field experiments should be conducted to determine the optimum frequency, dose and timing of B. tequilensis A13 application. This is the first report of isolation of B. tequilensis A13 from Chinese medicinal plant R. glutinosa and demonstration of its antagonistic activities against the root-rot pathogen F. solani.

B. tequilensis is widely found in soil and water and degrades various organic contaminations [26,27,28] by producing extracellular enzymes. Recently, different strains of B. tequilensis were isolated from different plant and confirmed to possess the ability to promote plant growth or inhibit the growth of microorganisms. For example, Bhattacharya et al. indicated that endophytic B. tequilensis PBE1 isolated from tomato can effectively inhibit mycelial growth of F. oxysporum inducing tomato wilt disease and promoted tomato growth by producing indole acetic acid and hydroxymate type siderophore [29]; Nagrale et al. reported that B. tequilensis CICR-H3 isolated from cotton displayed 62.84 ± 0.50% inhibition ratio against Macrophomina phaseolina inducing cotton root rot by producing volatile organic compounds, such as Benzene, 1, 3-diethyl- and Benzene, 1, 4-diethyl [30]; Kumar et al. isolated the B. tequilensis KAS3 from Pennisetum glaucum L. which significantly protected seedlings from Fusarium sp. infection by producing lipopeptide [31]. Although the recent significant advances, only few studies are available on activity and mechanism of B. tequilensis acting as a biological control agent against phytopathogenic fungi inducing root-rot disease of medicinal plants.

Saoussen pointed out that producing antipathogenic metabolites was the best known and most important mechanism that allowed antagonistic microorganisms to inhibit plant pathogens [32]. Thus, in this study, we performed the LC–MS/MS and bioinformatics analyses of extracellular proteins from B. tequilensis A13. The results indicated that B. tequilensis A13 was capable of producing six compounds capable of antagonizing phytopathogenic fungi: subtilisin, beta-1,3–1,4 glucanase, lysozyme, iturin, antimicrobial peptide LCI, and putative antimicrobiological protein. For example, a subtilisin-like serine proteinase rTghSS42 exhibited broad-spectrum antifungal activity against F. oxysporum, Alternaria alternate, etc. by inhibiting mycelial growth [33]. Beta-1,3–1,4 glucanase caused deformity, bending, and breakage of the mycelia in both Botrytis cinerea and Alternaria panax by the hydrolyzing β-1, 3–1, 4-glucan in cell wall [34]. Antimicrobial peptide LCI inhibited the growth of both F. moniliforme and F. solani, which might be achieved by interacting with cell membranes; thus, increasing membrane permeability [35, 36]. Furthermore, we also found that A13 produced various synthetases of antimicrobial compounds, such as plipastatin, surfactin, mycosubtilin, fusaricidins, and fengycin, which were highly efficient against phytopathogenic fungi from genus Fusarium and other genera. For example, surfactin from Brevibacillus brevis inhibited the hyphal growth of the phytopathogenic fungi F. moniliforme by damaging its DNA and proteins and reducing its glutathione content [37]. Surfactin C from B. velezensis strain 1B-23 and Bacillus sp. strain 1D-12 significantly reduced the disease incidence of bacterial canker caused by Clavibacter michiganensis subsp. Michiganensis in tomato [38]. Fusaricidins, potent antifungal biocontrol agents against a broad array of phytopathogenic fungi, have frequently been used to treat plant diseases, such as fusarium wilt of cucumber and gibberella ear rot of maize [39]. Therefore, various antimicrobial compounds that were produced by B. tequilensis A13 may have cooperatively inhibited the growth of F. solani. Moreover, these compounds may also antagonize other pathogens, which was supported by B. tequilensis A13 obviously inhibiting the growth of phytopathogenic fungi Penicillium janthinellum and Aspergillus tubingensis (Additional file 4). In addition, our results indicated that some extracellular proteins of B. tequilensis A13 were involved in the synthetic pathways of 15 amino acids and five vitamins, as well as two plant stress-response pathways. Thus, it is possible that B. tequilensis A13 has multiple mechanisms by which to inhibit phytopathogenic fungi inducing root-rot and to maintain plant health.

Based on all the data, the novel strain B. tequilensis A13 showed highly effective and broad biocontrol activities against phytopathogenic fungi, especially F. solani. LC–MS/MS and GO analysis of extracellular proteins indicated that the strain could produce several antifungal compounds, which may target different species of pathogens or different pathogenesis mechanisms to play antifungal roles. This study enhanced the understanding of the antifungal mechanism in genus Bacillus and provided an excellent candidate biocontrol agent. In addition, the B. tequilensis A13 was first isolated from R. glutinosa rhizosphere, which highlighted medicinal plants as new resource pools for microorganisms with potential for developing into biocontrol agents.


In conclusion, among members of the genus Bacillus, the biocontrol efficiency of B. tequilensis against plant fungal diseases, such as root rot, has been rarely explored. In this study, we isolated a novel strain, B. tequilensis A13, from R. glutinosa rhizosphere and investigated its biocontrol potential against R. glutinosa root-rot pathogen F. solani. Our study found the following: (1) B. tequilensis A13 significantly inhibited the growth of F. solani inducing R. glutinosa root-rot disease; (2) B. tequilensis A13 significantly reduced the incidence of R. glutinosa root-rot disease in inoculation and field experiments; and (3) B. tequilensis A13 produced several antifungal compounds, such as beta-1,3–1,4 glucanase, subtilisin, and antimicrobial peptide LCI, which contribute to the antifungal activity of B. tequilensis A13. Therefore, B. tequilensis A13 is a promising candidate for development into a biocontrol agent against R. glutinosa root-rot disease.

Availability of data and materials

The data used and/or analyzed during the current study are available from the corresponding author on rational request.



Liquid chromatography electrospray ionization tandem mass spectrometry


Gene Ontology




Potato dextrose agar


Colony forming units


Cell-free supernatant

Rehmannia glutinosa :

R. glutinosa

Fusarium solani :

F. solani

Bacillus tequilensis :

B. tequilensis


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We thank International Science Editing ( for editing this manuscript.


This work was supported by China Agriculture Research System (CARS-21); The Zhongyuan high level talents special support plan-Science and Technology Innovation Leading Talents (224200510011); The Key Science and Technology Project of Henan Province, China (202102110219); Educational Commission of Henan Province of China (22B180006, 20A180015).

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Authors and Affiliations



RW performed project administration and writing-original draft, arranged funding sources. HL performed the lab experiments. ZQ performed the field experiments. YW performed data duration. QY supervised the study and writing of the manuscript, arranged funding sources. HZ arranged funding sources, contributed in review and editing of the manuscript. ML contributed in review and editing of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Qingxiang Yang.

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Supplementary Information

Additional file 1.

Colony and microscopic morphology of the strain A13.

Additional file 2.

The inhibitory activity of protein fractions from A13 CFS. A the inhibitory activity of protein fractions obtained from CFS by different ammonium sulfate concentrations; B the inhibitory activity of protein fraction obtained from CFS by 70% ammonium sulfate concentration. The protein fraction was further divided into four fractions: fraction A (< 30 kDa), fraction B (30–50 kDa), fraction C (50–100 kDa) and fraction D (> 100 kDa).

Additional file 3.

LC–MS/MS profiles of protein components of fraction B (30–50 KD) and fraction D (> 100 KD). A fraction B; B fraction D.

Additional file 4. B. tequilensis

A13 inhibited the growth of Penicillium janthinellum and Aspergillus tubingensis.

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Wang, R., Li, H., Qin, Z. et al. Antifungal activity and application of Bacillus tequilensis A13 in biocontrol of Rehmannia glutinosa root-rot disease. Chem. Biol. Technol. Agric. 10, 20 (2023).

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  • Biocontrol
  • Root-rot disease
  • Bacillus tequilensis
  • Fusarium solani
  • Rehmannia glutinosa