Antimicrobial mechanism and biocontrol effect of Bacillus cereus XZ30-2 on Aspergillus niger

Yanjie Yi1,2,3*, Zhipeng Hou1,2,3, Qian Yang1,3, Liuqing Cui1,2,3, Heng Lu1, Ruifang Li1,3, Yang Liu1,2,3, Yuyanqiao Zhang1,3, Yuan Chen1,3

1School of Biological Engineering, Henan University of Technology, Zhengzhou, China;

2Food Laboratory of Zhongyuan, Luohe, China;

3The Key Laboratory of Functional Molecules for Biomedical Research, Zhengzhou, China


Aspergillus niger is a major mold-causing spoilage in cereals, fruits and vegetables. Controlling of mold in stored grains is essential for safety of food. Currently, application of microorganisms to control A. niger is a safer and more effective method. In this study, strain XZ30-2 against A. niger was isolated and identified as Bacillus cereus according to morphological and biochemical characteristics as well as 16 Svedberg ribosomal ribonucleic acid (16S rRNA) gene sequence analysis. The investigation of action mechanism showed XZ30-2 culture filtrate caused the mycelia inflated or contract, increasing the membrane permeability, leading to the intracellular leakage and nucleic acids release, disrupting the proton pump, decreasing the ergosterol content, inducing the membrane lipid peroxidation and reactive oxygen species (ROS) accumulation in A. niger. Moreover, B. cereus XZ30-2 culture filtrate could produce hydrolases and lipopeptides, including iturin, surfactin and fengycin. This work also evaluated the control effect of XZ30-2 on A. niger in wheat grains, and indicated that 40 μL/g of culture filtrate significantly controlled the infection of A. niger. Therefore, B. cereus XZ30-2 can be developed as a biological agent for controlling A. niger in stored grains.

Key words: Aspergillus niger, Bacillus cereus XZ30-2, culture filtrate, antimicrobial mechanism, biocontrol effect

*Corresponding Author: Yanjie Yi, School of Biological Engineering, Henan University of Technology, Zhengzhou 450001, China. Email: [email protected]

Received: 28 August 2023; Accepted: 8 October 2023; Published: 27 October 2023

DOI: 10.15586/qas.v15i4.1379

© 2023 Codon Publications
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0). License (


Wheat (Triticum aestivum L.) is the most widespread global crop; however, up to half of the global harvested grain is lost to poor storage, wastage, and mycotoxin contamination (Mesterházy et al., 2020). During storage, Aspergillus niger causes mold in wheat with high water content (Gherbawy et al., 2021; Mannaa and Kim, 2017). Furthermore, A. niger produces a lot of mycotoxins and other secondary metabolites, which have been known to cause various diseases and ailments in humans, such as nephrotoxic, immunosuppressive, teratogenic and carcinogenic disorders (Frisvad et al., 2018; Gil-Serna et al., 2019). In addition to health fallouts, A. niger infection also causes substantial economic losses. Stored grains and food products infected with mold may become unsuitable for consumption, resulting in economic difficulties and losses for farmers, food producers and distributors (Wilson et al., 2002).

In the conventional agricultural production, the control of A. niger mainly depends on physical and chemical methods (Agriopoulou et al., 2020). Physical measures required to prevent the growth of A. niger include controlling temperature and water activity of stored grains (Fleurat-Lessard, 2017). Nevertheless, there are deficiencies in the practical application of physical measures. For example, incidental fungal spoilage in stored grains also occurs due to water condensation because of ‘cold wall contact’ effect and moisture transfer resulting from temperature differences in bulk grains. The chemical method mainly inhibits propagation of Aspergillus by fumigation (Thorpe, 2008). Common chemical reagents used are ozone gas, phosphine, propionic acid and plant essential oils (Rutenberg et al., 2018). However, these chemical agents have persistent toxicity when used to control fungal spoilage in stored grains. Hence, a safer and more effective method is needed to prevent mold propagation in stored grains.

At present, biological control presents a promising approach for combating A. niger infestation, which is more reliable ecologically and economically sustainable. Researchers mainly use microorganisms or their metabolites to reduce the impact of other harmful organisms to prevent and control mold in stored grains (de Andrade Santiago et al., 2018; Santra and Banerjee, 2020). Kluyveromyces thermotolerans (Ponsone et al., 2011) and some antagonistic bacteria (Paraburkholderia fungorum, Bacillus subtilis, and Lactobacillus) were found to inhibit A. niger during grain storage (Rahayu et al., 2021; Yassein and Elamary, 2021). Antagonistic yeast Cyberlindnera jadinii volatiles were reported to inhibit the growth of A. niger and had a significant biocontrol ability against the pathogen (Alkuwari et al., 2022). Meanwhile, most bioactive metabolites, such as alkaloids, lipopeptides and lactones could also control A. niger (Li et al., 2020, 2022; Wang et al., 2017).

Recently, B. cereusis has been reported to play an important role in controlling pathogens and molds. For instance, B. cereus B25 inhibited the growth of the phytopathogen Fusarium verticillioides P03 (Báez-Astorga et al., 2022). B. cereus F-BC26 promoted pepper growth and protected it against Xanthomonas euvesicatoria (Hernandez-Huerta et al., 2023). Also, B. cereus can eliminate zearalenone (a nonsteroidal estrogenic mycotoxin) in mice and regulate gut microbiota (Wang et al., 2018). B. cereus secretes various secondary metabolites against bacterial and fungal growths, including lipopeptide antibiotics synthesized by nonribosomal peptide synthetase (NRPS). Some studies on the antifungal mechanisms of biocontrol bacteria have shown their effects on fungal cells, such as cell membrane disorder, membrane potential imbalance, and effect on physiological metabolism (Ju et al., 2023; Molina-Hernández et al., 2022; Niu et al., 2022).

Bacillus spp. plays an important role in inhibiting fungal growth by changing the cell structure and disrupting energy, transportation, and cell membrane permeability in fungi (Zhang et al., 2022a). Further research in this field could contribute to the development of effective biocontrol strategies for management of A. niger. Compared to other microorganisms, little is known about the antifungal potential of B. cereus against food spoilage fungi. Therefore, in this study, antagonistic bacteria B. cereus XZ30-2 were screened and identified against A. niger. The action mechanism of strain XZ30-2 was analyzed and its biocontrol efficacy on A. niger in wheat grains was evaluated to develop a new biological agent for controlling the pathogen.

Materials and Methods


In all, 48 of soil samples from Tibet, China were provided by Dr. Yu Shi of the Henan University on 30 August 2017 for isolating biocontrol bacteria. Antagonistic bacteria B. cereus XZ30-2 and A. niger were stored at -20°C in the laboratory of the School of Biological Engineering, Henan University of Technology. The wheat seeds of Zhengmai 366 were obtained from Henan Academy of Agricultural Sciences. Potato dextrose agar (PDA) medium (20-g glucose, 200-g potato, 15-g agar, and 1-L sterile water; pH 7) was used to culture A. niger. Luria-Bertani (LB) medium (5-g yeast extract, 10-g peptone, 10-g NaCl, and 1-L sterile water; pH 7) was used to culture bacteria.

Isolation and screening of antagonistic bacteria

Bacteria were isolated and screened according to the method described by Zhai et al. (2021) with modification. In brief, 10-g soil samples were added into a series of flasks and gradually diluted. The diluted suspensions were coated onto the LB medium and incubated at 35°C for 48 h. Antagonistic ability of all isolates against A. niger were tested using the plate confrontation method. The screened bacteria were inoculated onto liquid LB medium and cultured in a shaker at 180 rpm and 35°C for 48 h. Then the culture filtrate of bacteria was centrifuged at 10,000 rpm and prepared by filtering through a 0.22-µm filter. The inhibitory activity of culture filtrate against A. niger was tested using the mycelial growth rate method (Yi et al., 2022). PDA inoculated with A. niger without culture filtrate treatment was treated as a control. Following formula was used for calculating the inhibition rate:

Inhibition rate (%) = [(CT)/C] × 100(1)

Where C represents the mycelial radial diameter of A. niger in the control group, and T is the mycelial radial diameter of A. niger in the treatment group. Antagonistic bacteria with the strongest inhibitory effect were selected for further study.

Identification of antagonistic strain

Antagonistic bacteria XZ30-2 were cultured onto LB medium at 37°C for 24 h to observe colony characteristics, including morphology, color and growth properties. Physiological and biochemical tests were carried out according to Berger’s Manual of Systematic Bacteriology (Vos et al., 2009).

Molecular identification of strain XZ30-2 was performed by 16 Svedberg ribosomal ribonucleic acid (16S rRNA) gene sequence analysis. The genome DNA of antagonistic strain was extracted by the cetyltrimethylammonium bromide (CTAB) method (Yan et al., 2020). The 16S rRNA was amplified using the universal primers F (5’-AGAGTTTGATCATGGCTCAG-3’) and R (5’-ACG-GTTACCTTGTTACGACTT-3’). The conditions for poly-merase chain reaction (PCR) were as follows: pre- denaturation at 94°C for 5 min, then 32 cycles of denaturation at 94°C for 40 s, annealing at 60°C for 1 min, extension at 72°C for 1.5 min, followed by a final extension at 72°C for 10 min. PCR amplification products were verified by 1.2% agarose gel electrophoresis and sent to Sangon Biotech Co. Ltd. (Shanghai, China) for sequencing. Sequences were searched and compared by the Basic Local Alignment Search (BLAST) tool in GenBank (Bethesda, MD, USA; Sequence alignment and phylogenetic tree construction were performed through the MEGA 7.0 program using the neighbor-joining method (Saitou and Nei, 1987).

Action analysis of XZ30-2 culture filtrate on A. niger cells

Inhibition of XZ30-2 culture filtrate on mycelial morphology of A. niger

The effects of culture filtrate on mycelial morphology were observed using an optical microscope based on the method described by Tso et al. (2021) with minor modifications. Briefly, A. niger was grown on a PDA plate containing XZ30-2 culture filtrate (200-μL/mL) at 28°C for 2 days. A. niger cultured on PDA plate not containing culture filtrate was taken as a control. Then the mycelia of A. niger were collected and observed.

Detection for cell membrane permeability of A. niger treated with culture filtrate

Cell membrane permeability reflects the degree of cell membrane injury. A fluorescent probe propidium iodide (PI) was used to detect cell membrane permeability. A. niger mycelia were treated with 200-μL/mL culture filtrate for 48 h. Then the mycelia were collected and incubated in 2-μmol/L prodidium iodide (PI) staining solution for 20 min and rinsed with phosphate buffer solution (PBS), followed by visualization with fluorescence microscope. Mycelia without any culture filtrate treatment were set as a control.

Detection for intracellular leakage of A. niger treated with culture filtrate

The effects of culture filtrate on intracellular leakage of A. niger were determined using the intracellular conductivity detection method with minor modifications (He et al., 2021). After culturing for 5 days at 28°C, A. niger spores (106 colony forming unit [CFU]/mL) were inoculated with 100-mL potato dextrose broth (PDB) and cultured in a shaker at 110 rpm and 28°C for 48 h. Then XZ30-2 culture filtrate was added to the mold culture fluid to make the final concentration of 200-μL/mL. The mold culture fluid without XZ30-2 culture filtrate was set as a control. After shaking for an additional 24 h, the mycelia of A. niger were collected on double gauze and rinsed twice with sterile water; 0.5-g dried mycelia were suspended in 20-mL sterile water for 0, 5, 10, 20, 40, 80, 100, 120, 140, 180, 200, and 220 min at 25°C. Then the electric conductivity was measured using a conductivity meter to assess intracellular leakage through cell membranes. The relative conductivity of mycelia was calculated using the following formula:

Relative conductivity (%) = (Conductivity/Final conductivity) × 100 (2)

Determination of release of nucleic acids in A. niger

The release of cellular components was performed by the method described by Ke et al. (2022). The culture of the mold was prepared described previously. After centrifugation for 25-min at 4,000-rpm to obtain mold supernatant; absorbance (OD260) of the supernatant was measured using UV spectrophotometer.

Assay for the extracellular pH of A. niger treated with culture filtrate

Proton pump of the cell membrane can transfer intracellular protons to extracellular environment. Activities of A. niger proton pump were detected by monitoring the glucose-induced acidification of external medium by determining pH according to the method described by Tao et al. (2014) with slight modifications. A. niger was cultured and mycelia were prepared by the method described previously; 1.0-g mycelia was treated with 40-mL KCl (50-mmol/L) for 18 h at 4°C; Then XZ30-2 culture filtrate was added to mycelia culture to make the final concentration of 200-μL/mL. Mycelia without treatment of culture filtrate were set as a control. The mixture was incubated at 25°C for 10 min and treated with 20-mL 10% glucose solution to make medium acidification for 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.0, 3.5, and 4.0 h. The value of extracellular pH was obtained by using pH meter.

Determination of ergosterol content in A. niger

Ergosterol is found in fungal cell membranes, and the growth of fungus is evaluated based on the content of its ergosterol. Ergosterol content in the plasma membrane of A. niger was determined by the method described by Prakash et al. (2014). In brief, A. niger mycelia were treated with 200-μL/mL culture filtrate for 48 h. Mycelia without culture filtrate treatment were set as a control. After treatment, the mycelia were harvested and washed twice with sterile water. The wet weight of the mycelia was recorded. Then, 5 mL of 25% alcoholic potassium hydroxide solution (25-g KOH and 35-mL sterile distilled water, diluted to 100-mL with anhydrous ethanol) was added to each sample. The mixture was vortexed for 2 min and incubated at 85 ± 2°C in a water bath for 5 h. Subsequently, a mixture of 2-mL sterile distilled water and 5-mL n-hexane was added to extract sterols. After 1 h, the n-hexane layer was analyzed using a scanning spectrophotometric method between 230 nm and 300 nm. The amount of ergosterol was determined using the formula provided by Tian et al. (2012).

Determination of lipid peroxidation of A. niger

Malondialdehyde (MDA) concentration was used as an indicator of lipid peroxidation level. In brief, mycelia of A. niger were treated with 200-μL/mL culture filtrate for 48 h. Mycelia without culture filtrate treatment were set as a control. After cultivation, mycelia were collected and rinsed twice with sterile water and its wet weight was determined. Then the MDA content was measured using a lipid peroxidation (MDA) assay kit (Solarbio, Beijing, China).

Detection of reactive oxygen species (ROS) accumulation in A. niger

Reactive oxygen species are primarily produced in the mito-chondria by the oxidative respiratory chain. Oxidative stress might cause damage to plasma membrane damage and enhance membrane permeability. The accumulation of ROS was monitored by following the method described by Yi et al. (2022). Mycelia of A. niger were treated with 200-μL/mL culture filtrate for 48 h. The mycelia were stained with 10-μM DCFH-DA solution (Beyotime, Shanghai, China). After incubation in the dark at 28°C for 30 min, the samples were observed and photographed using a fluorescence microscope.

Detection of antimicrobial substances in strain XZ30-2

The antimicrobial substances produced by XZ30-2, including protease and cellulase, were determined by using skimmed milk and carboxymethyl cellulose (CMC) according to the protocol described by Ben Khedher et al. (2021). Amylase was determined by the starch agar plate method (Nxumalo et al., 2020). Surfactin was determined by the oil spreading assay (Rani et al., 2020).

PCR detection of lipopeptides in strain XZ30-2

The genome DNA of strain XZ30-2 was extracted by the CTAB method (Yan et al., 2020). Ten pairs of primers of lipopeptide genes (sfp, srfAA, srfAD, fenA, fenB, fenD, ituA, ituB, ituC and ituD) were used for PCR amplification of lipopeptide synthesis-related genes (Table 1). Sequencing of PCR-amplified products was performed at Sangon Biotech Co. Ltd. (Shanghai, China), and the sequences were compared with the NCBI database (Bethesda, MD, USA; to analyze sequence homology with lipopeptide genes.

Table 1. Ten pairs of primers of lipopeptide synthesised genes.

Genes Primers Sequences (5’ →3’) Amplified size / bp

Assay of XZ30-2 culture filtrate for controlling A. niger infection

The control effect of strain XZ30-2 was evaluated by the method described by Duan et al. (2023). In this test, sterilized 120-g wheat seeds were divided into three groups, including the control group, in different tubes. XZ30-2 culture filtrate, 20 and 40-μL/g, was added into tubes. In the control group, culture filtrate was replaced by LB medium. Next, 100-μL of A. niger spores suspension (107-CFU/mL) was added into each tube containing wheat seeds and shaken well; the tubes were sealed and stored at 28°C. After 2 weeks, the number of fungal colonies in wheat seeds was calculated.

Statistical analysis

All tests are performed in triplicate. The data were expressed as the mean ± standard error (SE), and analyzed statistically by independent-sampled test of variance (ANOVA) using SPSS 20.0 (SPSS Co. Ltd., Chicago, IL, USA). Significant differences between mean values at p < 0.05 were determined by multiple comparisons using Duncan’s multiple range test (DMRT).

Results and Discussion

Isolation and identification of antagonistic strain

Of the 30 bacteria strains isolated, six strains had antifungal effect on A. niger (Figure 1A). Among them, strain XZ30-2 showed higher antagonistic effects for A. niger. When strain XZ30-2 was cultured on LB plate, Gram-positive bacteria presented big, rod-shaped, milky white, opaque irregular edges. In addition, XZ30-2 was positive for nitrate reduction, glucose reaction, citrate, indole, amylolysis, and catalase test, whereas it was negative for methyl red test. Meanwhile, the 16S rRNA gene sequence of XZ30-2 (GenBank accession No.: MZ646135) was arranged in a phylogenetic tree (Figure 1B). Results of morphological and biochemical characteristics and molecular identification discovered strain XZ30-2 as Bacillus cereus, showing 100% resemblance to B. cereus ATCC 14579.

Figure 1. Screening and identification of bacteria against A. niger. (A) Inhibition proportions of six strains against A. niger. (B) Phylogenetic tree obtained from 16S rRNA gene sequences of strain XZ30-2 and other closely related bacteria. Numbers at branch points are bootstrap values of parsimony-based analysis. Different lowercase letters in each column represent extremely significant differences at p < 0.05.

Effects of B. cereus XZ30-2 culture filtrate on A. niger

Changes of mycelial morphology of A. niger

Effects of XZ30-2 culture filtrate on mycelial morphology of A. niger, observed using optical microscope (Figure 2A), showed that mycelia were either inflated or compacted, compared to the control, particularly at mycelial tips. It showed that culture filtrate could remarkably affect the mycelial morphology of A. niger.

Figure 2. Effects of XZ30-2 culture filtrate on the mycelia of A. niger. (A) Effects on the morphology of A. niger mycelia. (B) Effects on the serosal membrane injury of A. niger. Red fluorescence represents PI-stained mycelia. Micrographs were taken using a fluorescence inverted microscope with 40× magnification.

Cell membrane permeability of A. niger

The growth of A. niger was inhibited due to cell membrane damage (Ju et al., 2020). In damaged cells, PI dye entered the nucleus through cell membrane. After PI staining, red fluorescence was obviously observed in the mycelia of A. niger treated with culture filtrate (Figure 2B). The result indicated that the membrane permeability of mycelia was increased by XZ30-2 culture filtrate. It was consistent with the inhibitory effects of cinnamaldehyde vapor on the cell membranes of A. niger HY2 (Niu et al., 2022).

Intracellular leakage of A. niger

In case fungal cell membrane is damaged, intracellular electrolyte solution flows out and increases the conductivity of extracellular solution. Hence, changes in the electric conductivity of extracellular solution are important indicators reflecting intracellular leakage (Yan et al., 2023). In this study, electric conductivity increased after exposure to culture filtrate (Figure 3A), which indicated that XZ30-2 culture filtrate resulted in the intracellular leakage of A. niger.

Figure 3. Effects of XZ30-2 culture filtrate on the (A) relative conductivity and (B) OD260 of A. niger mycelia. Each value is presented as the average of three replicates. Different lowercase letters in each column represent extremely significant differences at p < 0.05.

Release of nucleic acids of A. niger

Release of nucleic acids after culture filtrate treatment was measured by spectroscopy at 260 nm. As shown in Figure 3B, compared to OD260 (0.123) in the control group, the absorbance value of DNA treated with culture filtrate was 0.211. The results indicated that culture filtrate showed a significant effect on the intracellular leakage of A. niger. The absence of nucleic acids in cells could disrupt the normal activities of cells and even cause cell death (Niu et al., 2022).

Extracellular pH of A. niger

As shown in Figure 4A, the extracellular pH declined slowly after treatment with culture filtrate (200-μL/mL) at different periods, compared to that of the control group. Decrease in extracellular pH reflects disruption of the proton pump of A. niger (Tian et al., 2012). The results showed that XZ30-2 culture filtrate could disrupt the proton pump of A. niger.

Figure 4. Effects of XZ30-2 culture filtrate on (A) pH, (B) ergosterol content, and (C) MDA content of the mycelia of A. niger. C indicates the control group and T indicates the mycelia treated with culture filtrate. Different lowercase letters on the bars indicate significant difference at p < 0.05.

Ergosterol content of A. niger

Changes in ergosterol content are presented in Figure 4B. Compared to the control, treatment with culture filtrate at 200-μL/mL resulted in a 26.90% decrease in the ergosterol content of A. niger. Fungal ergosterol is a classical drug target because it is important for plasma membrane structure and functioning as well as localization of plasma membrane proteins (Georgopapadakou and Tkacz, 1995). Therefore, XZ30-2 culture filtrate could decrease the biosynthesis of ergosterol, thereby affecting the growth of A. niger.

Lipid peroxidation of A. niger

The MDA level reflects the extent of lipid peroxidation (Ju et al., 2020). As shown in Figure 4C, compared to the control, treatment with culture filtrate at 200-μL/mL resulted in an increase in the MDA content of A. niger by 0.339. This result showed that culture filtrate treatment induced membrane lipid peroxidation of A niger.

Reactive oxygen species accumulation of A. niger

Reactive oxygen species plays a crucial regulatory role in normal cellular metabolism and under stress conditions. However, excessive ROS levels can lead to oxidation stress and cellular damage (Ju et al., 2020). As shown in Figure 5, treatment with culture filtrate (200 μL/mL) significantly increased the level of green fluorescence, compared to the control group. Therefore, culture filtrate resulted in a significant accumulation of ROS in A. niger and could lead to cell damage.

Figure 5. Effects of XZ30-2 culture filtrate on ROS. C indicates the control group, and T indicates the treatment group. The samples were visualized/photographed using microscope at 40× magnification.

Active substances produced by B. cereus XZ30-2

Antimicrobial action through the production of hydrolase is probably an important mechanism of biocontrol bacteria to inhibit pathogens (van Schie et al., 2021). Hydrolase of B. cereus XZ30-2 were detected and exhibited transparent circles on identification media (Figures 6A–6C), which indicated that XZ30-2 could produce protease, cellulase and amylase. Meanwhile, the diameter of the oil drainage circle was 7.35 cm if 20-μL culture filtrate was added to the media, indicating the presence of surfactin in the culture filtrate (Figure 6D). Similarly, antimicrobial substances produced by B. cereus had an inhibitory effect and could be taken as antifungal agents (Durval et al., 2021; Ming et al., 2022).

Figure 6. Active substances of B. cereus XZ30-2. (A) Protease, (B) cellulase, (C) amylase and (D) surfactin.

Lipopeptides produced from B. cereus XZ30-2

Among the useful metabolites produced by Bacillus spp., the main antimicrobial substances are lipopeptides (Hu et al., 2023; Zhang et al., 2022b). After the PCR amplification of 10 known primer pairs, five fragments of ituA, ituC, ituD, srfAD and fenB genes were analyzed according to sequence comparison with the lipopeptide genes of NCBI database (Table 2). It indicated that B. cereus XZ30-2 could produce lipopeptides, the main antimicrobial substances, including iturin, surfactin, and fengycin (Ayangbenro and Babalola, 2020; Li et al., 2021).

Table 2. PCR amplification of fragments from B. cereus XZ30-2.

Genes Product size(bp) Probable genes Identity (%)
srfAD 1300 srfAD of B. subtilis 98.56%
fenB 1600 fenB of B. amyloliquefaciens 97.22%
ituA 1047 ituA of B.velezensis 99.26%
ituC 423 ituC of B.velezensis 97.11%
ituD 1203 ituD of B.amyloliquefaciens 97.82%

Control effects of XZ30-2 culture filtrate on A. niger infection in wheat grains

The control effects of XZ30-2 on wheat grains are shown in Figure 7. The A. niger infection in wheat grains was obviously visible with different moldy levels. Colony counting of A. niger in wheat samples treated with different concentrations of culture filtrate showed the following results: colony count in the control group was 1.68 CFU×108/g; colony count in the treatment group with 20-μL/g of culture filtrate was 9.4 CFU×107/g; colony count in the treatment group with 40 μL/g of culture filtrate was 6.69 CFU×105/g. The results showed that B. cereus XZ30-2 effectively controlled A. niger and reduced moldiness in stored wheat grains. Comparatively, the control effect of XZ30-2 culture filtrate was similar to or better than other candidate biocontrol agents reported previously (Oztopuz et al., 2018; Ul Hassan et al., 2019).

Figure 7. Control effects of XZ30-2 culture filtrate on A. niger in stored wheat grains. (A) Mold infection on wheat in different treatment groups. (B) A. niger colony count after 2 weeks when exposed to different concentrations of culture filtrate. Different lowercase letters on the bars indicate significant difference at P < 0.05.


According to morphological, biochemical and molecular analysis, antagnostic strain XZ30-2 of B. cereus was identified to combat A. niger. B. cereus XZ30-2 controlled A. niger by inhibiting mycelia development and increasing membrane permeability, thus leading to intracellular leakage and nucleic acid release, disrupting proton pump, decreasing the ergosterol content, inducing membrane lipid peroxidation and ROS accumulation, and producing hydrolases and lipopeptides, including iturin, surfactin, and fengycin. The culture filtrate of strain XZ30-2 effectively controlled the infection of A. niger and reduced moldiness in stored grains. In summary, the present study demonstrated the controlled potential of B. cereus XZ30-2 and provided a new microbial resource for controlling A. niger.


This work was supported by the Henan Provincial Science and Technology Major Project (221100110100 and 221100110700), Henan Provincial Key R&D and Promotion Special Project (Science and Technology Research) (232102111011), and Innovative Funds Plan of Henan University of Technology (2020ZKCJ23).

Conflicts of interest

The authors stated that they had no conflict of interest to declare.

Author Contributions

The manuscript was written and revised by Yanjie Yi and Zhipeng Hou. Experiment design and execution were performed by Zhipeng Hou, Qian Yang, Heng Lu, Yang Liu, Yuyanqiao Zhang, and Yuan Chen. Data collection and analysis were performed by Zhipeng Hou, Liuqing Cui, Yang Liu, and Ruifang Li. All authors read and approved the published version of the manuscript.

Data Availability

All original contributions presented in the study are included in the article; further inquiries can be directed to the corresponding author.


Agriopoulou, S., Stamatelopoulou, E. and Varzakas, T., 2020. Advances in occurrence, importance, and mycotoxin control strategies: prevention and detoxification in foods. Foods 9(2): 137. 10.3390/foods9020137

Alkuwari, A., Hassan, Z.U., Zeidan, R., Al-Thani, R. and Jaoua, S., (2022) Occurrence of mycotoxins and toxigenic fungi in cereals and application of Yeast volatiles for their biological control. Toxins (Basel), 14(6). 10.3390/toxins14060404

Ayangbenro, A.S. and Babalola, O.O., 2020. Genomic analysis of Bacillus cereus NWUAB01 and its heavy metal removal from polluted soil. Scientific Reports 10(1): 19660. 10.1038/s41598-020-75170-x

Báez-Astorga, P.A., Cázares-Álvarez, J.E., Cruz-Mendívil, A., Quiroz-Figueroa, F.R., Sánchez-Valle, V.I. and Maldonado-Mendoza, I.E., 2022. Molecular and biochemical characterisation of antagonistic mechanisms of the biocontrol agent Bacillus cereus B25 inhibiting the growth of the phytopathogen Fusarium verticillioides P03 during their direct interaction in vitro. Biocontrol Science and Technology 32(9): 1074–1094. 10.1080/09583157.2022.2085662

Ben Khedher, S., Mejdoub-Trabelsi, B. and Tounsi, S., 2021. Biological potential of Bacillus subtilis V26 for the control of Fusarium wilt and tuber dry rot on potato caused by Fusarium species and the promotion of plant growth. Biological Control 152: 104444. 10.1016/j.biocontrol.2020.104444

de Andrade Santiago, J., Cardoso, M.D.G., Batista, L.R., Santiago, W.D., Passamani, F.R.F., Rodrigues, L.M.A. and Nelson, D.L., 2018. Effect of the essential oils from Melaleuca alternifolia, Melaleuca quinquenervia and Backhousia citriodora on the synthesis of ochratoxin A by Aspergillus niger and Aspergillus carbonarius isolated from tropical wine grapes. Journal of Food Science and Technology-Mysore 55(1): 418–423. 10.1007/s13197-017-2857-4

Duan, W., Zhang, S., Lv, Y., Zhai, H., Wei, S., Ma, P., Cai, J. and Hu, Y., 2023. Inhibitory effect of (E)-2-heptenal on Aspergillus flavus growth revealed by metabolomics and biochemical analyses. Applied Microbiology and Biotechnology 107(1): 341–354. 10.1007/s00253-022-12320-3

Durval, I.J.B., Meira, H.M., de Veras, B.O., Rufino, R.D., Converti, A. and Sarubbo, L.A., 2021. Green synthesis of silver nanoparticles using a biosurfactant from Bacillus cereus UCP 1615 as stabilizing agent and its application as an antifungal agent. Fermentation (Basel) 7(4): 233. 10.3390/fermentation7040233

Fleurat-Lessard, F., 2017. Integrated management of the risks of stored grain spoilage by seedborne fungi and contamination by storage mould mycotoxins–an update. Journal of Stored Products Research 71: 22–40. 10.1016/j.jspr.2016.10.002

Frisvad, J.C., Moller, L.L.H., Larsen, T.O., Kumar, R. and Arnau, J., 2018. Safety of the fungal workhorses of industrial biotechnology: update on the mycotoxin and secondary metabolite potential of Aspergillus niger, Aspergillus oryzae, and Trichoderma reesei. Applied Microbiology and Biotechnology 102(22): 9481–9515. 10.1007/s00253-018-9354-1

Georgopapadakou, N.H. and Tkacz, J.S., 1995. The fungal cell wall as a drug target. Trends in Microbiology 3(3): 98–104. 10.1016/S0966-842X(00)88890-3

Gherbawy, Y.A., Maghraby, T.A., Hamza, L.H.A. and El-Dawy, E., 2021. New morphological criteria and molecular characterization of black aspergilli aggregate from corn, sorghum and wheat grains. Archives of Microbiology 203(1): 355–366. 10.1007/s00203-020-02024-5

Gil-Serna, J., Garcia-Diaz, M., Vazquez, C., Gonzalez-Jaen, M.T. and Patino, B., 2019. Significance of Aspergillus niger aggregate species as contaminants of food products in Spain regarding their occurrence and their ability to produce mycotoxins. Food Microbiology 82: 240–248. 10.1016/

He, R., Yang, Y., Hu, Z., Xue, R. and Hu, Y., 2021. Resistance mechanisms and fitness of pyraclostrobin-resistant isolates of Lasiodiplodia the obromae from mango orchards. PLoS One 16(6): e0253659. 10.1371/journal.pone.0253659

Hernandez-Huerta, J., Tamez-Guerra, P., Gomez-Flores, R., Delgado-Gardea, M.C.E., Robles-Hernandez, L., Gonzalez-Franco, A.C. and Infante-Ramirez, R., 2023. Pepper growth promotion and biocontrol against Xanthomonas euvesicatoria by Bacillus cereus and Bacillus thuringiensis formulations. PeerJ 11: e14633. 10.7717/peerj.14633

Hu, J., Dong, B., Wang, D., Meng, H., Li, X. and Zhou, H., 2023. Genomic and metabolic features of Bacillus cereus inhibiting the growth of Sclerotinia sclerotiorum by synthesizing secondary metabolites. Archives of Microbiology 205(1): 1–13. 10.1007/s00203-022-03351-5

Ju, J., Lei, Y., Guo, Y., Yu, H., Cheng, Y. and Yao, W., 2023. Eugenol and citral kills Aspergillus niger through the tricarboxylic acid cycle and its application in food preservation. Food Science and Technology (LWT) 173: 114226. 10.1016/j.lwt.2022.114226

Ju, J., Xie, Y., Yu, H., Guo, Y., Cheng, Y., Zhang, R. and Yao, W., 2020. Synergistic inhibition effect of citral and eugenol against Aspergillus niger and their application in bread preservation. Food Chemistry 310: 125974. 10.1016/j.foodchem.2019.125974

Ke, Y., Ding, B., Zhang, M., Dong, T., Fu, Y., Lv, Q., Ding, W. and Wang, X ., 2022. Study on inhibitory activity and mechanism of chitosan oligosaccharides on Aspergillus Flavus and Aspergillus Fumigatus. Carbohydrate Polymers 275: 118673. 10.1016/j.carbpol.2021.118673

Li, T., Li, L., Du, F., Sun, L., Shi, J., Long, M., and Chen, Z., 2021. Activity and mechanism of action of antifungal peptides from microorganisms: a review. Molecules 26(11): 3438. 10.3390/molecules26113438

Li, S., Xu, X., Zhao, T., Ma, J., Zhao, L., Song, Q. and Sun, W., 2022. Screening of Bacillus velezensis E2 and the inhibitory effect of its antifungal substances on Aspergillus flavus. Foods 11(2): 140. 10.3390/foods11020140

Li, F., Zeng, Y., Zong, M., Yang, J. and Lou, W., 2020. Bioprospecting of a novel endophytic Bacillus velezensis FZ06 from leaves of Camellia assamica: production of three groups of lipopeptides and the inhibition against food spoilage microorganisms. Journal of Biotechnology 323: 42–53. 10.1016/j.jbiotec.2020.07.021

Mannaa, M. and Kim, K.D., 2017. Influence of temperature and water activity on deleterious fungi and mycotoxin production during grain storage. Mycobiology 45(4): 240–254. 10.5941/MYCO.2017.45.4.240

Mesterházy, Á., Oláh, J. and Popp, J., 2020. Losses in the grain supply chain: causes and solutions. Sustainability 12(6): 2342. 10.3390/su12062342

Ming, S., Chen, X., Zhang, N., Li, S., Zhu, Z. and Cheng, S., 2022. Structure and stability analysis of antibacterial substance produced by selenium enriched Bacillus cereus BC1. Archives of Microbiology 204(3): 196. 10.1007/s00203-022-02798-w

Molina-Hernández, J.B., Scroccarello, A., Della Pelle, F., De Flaviis, R., Compagnone, D., Del Carlo, M. and Lόpez, C. C., 2022. Synergistic antifungal activity of catechin and silver nanoparticles on Aspergillus niger isolated from coffee seeds. Food Science and Technology (LWT) 169: 113990. 10.1016/j.lwt.2022.113990

Niu, A., Wu, H., Ma, F., Tan, S., Wang, G. and Qiu, W., 2022. The antifungal activity of cinnamaldehyde in vapor phase against Aspergillus niger isolated from spoiled paddy. Food Science and Technology (LWT) 159: 113181. 10.1016/j.lwt.2022.113181

Nxumalo, C.I., Ngidi, L.S., Shandu, J.S.E. and Maliehe, T.S., 2020. Isolation of endophytic bacteria from the leaves of Anredera cordifolia CIX1 for metabolites and their biological activities. BMC Complementary Medicine and Therapies 20(1): 300. 10.1186/s12906-020-03095-z

Oztopuz, O., Pekin, G., Park, R.D. and Eltem, R., 2018. Isolation and evaluation of new antagonist Bacillus strains for the control of pathogenic and mycotoxigenic fungi of fig orchards. Applied Biochemistry and Biotechnology 186(3): 692–711. 10.1007/s12010-018-2764-9

Ponsone, M.L., Chiotta, M.L., Combina, M., Dalcero, A. and Chulze, S., 2011. Biocontrol as a strategy to reduce the impact of ochratoxin A and Aspergillus section Nigri in grapes. International Journal of Food Microbiology 151(1): 70–77. 10.1016/j.ijfoodmicro.2011.08.005

Prakash, B., Singh, P., Goni, R., Raina, A.K.P. and Dubey, N.K., 2014. Efficacy of Angelica archangelica essential oil, phenyl ethyl alcohol and α-terpineol against isolated molds from walnut and their antiaflatoxigenic and antioxidant activity. Journal of Food Science and Technology 52(4): 2220–2228. 10.1007/s13197-014-1278-x

Rahayu, E.S., Triyadi, R., Khusna, R.N.B., Djaafar, T.F., Utami, T., Marwati, T. and Hatmi, R. U., 2021. Indigenous yeast, lactic acid bacteria and acetic acid bacteria from cocoa bean fermentation in Indonesia can inhibit fungal-growth-producing mycotoxins. Fermentation (Basel ) 7(3): 192. 10.3390/fermentation7030192

Rani, M., Weadge, J.T. and Jabaji, S., 2020. Isolation and characterization of biosurfactant-producing bacteria from oil well batteries with antimicrobial activities against food-borne and plant pathogens. Frontiers in Microbiology 11: 64. 10.3389/fmicb.2020.00064

Rutenberg, R., Bernstein, S., Fallik, E., Paster, N. and Poverenov, E., 2018. The improvement of propionic acid safety and use during the preservation of stored grains. Crop Protection 110: 191–197. 10.1016/j.cropro.2017.09.005

Saitou, N. and Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4(4): 406–425. 10.1093/oxfordjournals.molbev.a040454

Santra, H.K. and Banerjee, D., 2020. Natural products as fungicide and their role in crop protection. Springer, Singapore. 10.1007/978-981-15-3024-1_9

Tao, N., OuYang, Q. and Jia, L., 2014. Citral inhibits mycelial growth of Penicillium italicum by a membrane damage mechanism. Food Control 41: 116–121. 10.1016/j.foodcont.


Thorpe, G.R., 2008. The application of computational fluid dynamics codes to simulate heat and moisture transfer in stored grains. Journal of Stored Products Research 44(1): 21–31. 10.1016/j.jspr.2007.07.001

Tian, J., Ban, X., Zeng, H., He, J., Chen, Y. and Wang, Y., 2012. The mechanism of antifungal action of essential oil from dill (Anethum graveolens L.) on Aspergillus flavus. Plos One, 7(1): e30147. 10.1016/j.foodchem.2011.07.061

Tian, J., Huang, B., Luo, X., Zeng, H., Ban, X., He, J. and Wang, Y., 2012. The control of Aspergillus flavus with Cinnamomum jensenianum Hand.-Mazz essential oil and its potential use as a food preservative. Food Chemistry 130(3): 520–527. 10.1016/j.foodchem.2011.07.061

Tso, K.H., Lumsangkul, C., Ju, J., Fan, Y. and Chiang, H.I., 2021. The potential of peroxidases extracted from the spent mushroom (Flammulina velutipes) substrate significantly degrade mycotoxin deoxynivalenol. Toxins (Basel) 13(1): 72. 10.3390/toxins13010072

Ul Hassan, Z., Al Thani, R., Alnaimi, H., Migheli, Q. and Jaoua, S., 2019. Investigation and application of Bacillus licheniformis volatile compounds for the biological control of toxigenic Aspergillus and Penicillium spp. ACS Omega 4(17): 17186–17193. 10.1021/acsomega.9b01638

van Schie, L., Borgers, K., Michielsen, G., Plets, E., Vuylsteke, M., Tiels, P., Festjens, N. and Callewaert, N., 2021. Exploration of synergistic action of cell wall-degrading enzymes against mycobacterium tuberculosis. Antimicrobial Agents and Chemotherapy 65(10): e00659–00621. 10.1128/AAC.00659-21

Vos, P., Garrity, G.M., Jones D., Krieg, N.R., Ludwig, W., Rainey, F.A., Schleifer KH. and Whitman, W.B. (Eds.). 2009 Bergey’s manual of systematic bacteriology, 2nd ed. Springer, New York, NY.

Wang, Q., Lin, Q., Peng, K., Cao, J., Yang, C. and Xu, D., 2017. Surfactin variants from Bacillus subtilis natto CSUF5 and their antifungal properities against Aspergillus niger. Journal of Biobased Materials and Bioenergy 11(3): 210–215. 10.1166/jbmb.2017.1665

Wang, Y., Zhang, J., Wang, Y., Wang, K., Wei, H. and Shen, L., 2018. Isolation and characterization of the Bacillus cereus BC7 strain, which is capable of zearalenone removal and intestinal flora modulation in mice. Toxicon 155: 9–20. 10.1016/j.toxicon.2018.09.005

Wilson, D.M., Mubatanhema, W. and Jurjevic, Z., 2002. Biology and ecology of mycotoxigenic Aspergillus species as related to economic and health concerns. Advances in Experimental Medicine and Biology 504: 3–17. 10.1007/978-1-4615-0629-4_2

Yan, H., Meng, X., Lin, X., Duan, N., Wang, Z. and Wu, S., 2023. Antifungal activity and inhibitory mechanisms of ferulic acid against the growth of Fusarium graminearum. Food Bioscience 52: 102414. 10.1016/j.fbio.


Yan, P., Zhang, X., Hu, L., Wang, Y., Zhu, M.L., Wu, X. and Chen, F., 2020. Two novel strains, Bacillus albus JK-XZ3 and B. velezensis JK-XZ8, with activity against Cerasus crown gall disease in Xuzhou, China. Australasian Plant Pathology 49(2): 127–136. 10.1007/s13313-020-00682-z

Yassein, A.S. and Elamary, R.B., 2021. Efficacy of soil Paraburkholderia fungorum and Bacillus subtilis on the inhibition of Aspergillus niger growth and its ochratoxins production. Egyptian Journal of Botany 61(1): 319–334. 10.21608/ejbo.2021.68481.1656

Yi, Y., Luan, P., Liu, S., Shan, Y., Hou, Z., Zhao, S., Jia, S. and Li, R., 2022. Efficacy of Bacillus subtilis XZ18-3 as a biocontrol agent against Rhizoctonia cerealis on wheat. Agriculture 12(2): 258. 10.3390/agriculture12020258

Zhai, Y., Zhu, J., Tan, T., Xu, J., Shen, A., Yang, X., Li, J., Zeng, L., and Wei, L., 2021. Isolation and characterization of antagonistic Paenibacillus polymyxa HX-140 and its biocontrol potential against Fusarium wilt of cucumber seedlings. BMC Microbiology 21(1): 75. 10.1186/s12866-021-02131-3

Zhang, D., Qiang, R., Zhou, Z., Pan, Y., Yu, S., Yuan, W., Cheng, J., Wang, J., Zhao, D., Zhu, J. and Yang, Z., 2022a. Biocontrol and action mechanism of Bacillus subtilis lipopeptides’ fengycins against alternaria solani in potato as assessed by a transcriptome analysis. Frontiers in Microbiology 13: 861113. 10.3389/fmicb.2022.861113

Zhang, R.X., Wu, Z.W., Cui, H.Y., Chai, Y.N., Hua, C.W., Wang, P., et al., 2022b. Production of surfactant-stable keratinase from Bacillus cereus YQ15 and its application as detergent additive. BMC Biotechnology 22(1): 1–13. 10.1186/s12896-022-00757-3