Bacillus velezensis SCSX39 and application thereof

Abstract

The present application relates to the field of microbial technology, and particularly relates to a bacillus velezensis SCSX39 and application thereof.The bacillus velezensis SCSX39 is classified as Bacillus velezensis, and has been preserved in the China General Microbiological Culture Collection Center on September 15, 2025, with a preservation number of CGMCC No.35918.The strain is a safe strain, can show strong inhibitory activity on various mango anthracnose pathogens of anthracnose genus and gloeocercospora, and has inhibitory effect on the growth of various foodborne pathogenic bacteria from food sources such as steamed buns and sauce, indicating the potential multifunctional application prospect.The present application provides a special and efficient core strain resource for the green prevention and control of mango postharvest anthracnose and the like, provides important strain material for the development of a new type of microbial source pesticide, and has important significance for the development of mango industry.

Description

Technical Field

[0001] This invention belongs to the field of microbial control technology, specifically involving a strain of Bacillus belye SCSX39 derived from deep-sea mud in the South China Sea and its application in the control of mango anthracnose. Background Technology

[0002] Postharvest rot in mangoes is a key bottleneck restricting the improvement of industry efficiency. Anthracnose, caused by pathogens of the genus *Colletotrichum gloeosporioides*, is the most serious postharvest disease, accounting for more than 70% of postharvest losses. Currently, the mango industry mainly relies on chemical fungicides for disease control, but long-term use has led to the dual pressures of increased pathogen resistance and pesticide residue problems, restricting mango quality and safety and trade.

[0003] Employing biocontrol microorganisms for green control is a viable approach. Current research largely focuses on general-purpose biocontrol strains (such as certain Bacillus subtilis strains). These strains often exhibit limited inhibitory activity against mango anthracnose pathogens or poor adaptability to fruit surfaces, leading to unstable control efficacy. Therefore, current biocontrol strains suffer from the drawback of being "broad-spectrum but not specialized," necessitating the screening of dedicated biocontrol bacterial resources with high specificity and strong inhibitory activity against mango anthracnose pathogens.

[0004] Bacillus spp. are widely studied as biocontrol agents due to their strong resistance, diverse antimicrobial production, and safety to plants and animals. However, existing Bacillus strains used for biocontrol are mostly derived from terrestrial or agricultural environments, and their antimicrobial mechanisms are relatively common, lacking specificity against mango anthracnose. Deep-sea environments, with their extreme conditions of high pressure, low nutrition, and lack of light, often lead to the evolution of unique secondary metabolic pathways and antagonistic mechanisms in microorganisms. Discovering new Bacillus vesicle resources from this unique habitat holds promise for obtaining biocontrol strains with stronger antimicrobial activity and better environmental adaptability, providing a new approach for the green control of postharvest anthracnose in mangoes. Summary of the Invention

[0005] The technical problem to be solved by this invention is the lack of effective control of anthracnose pathogens and foodborne pathogens in the current mango industry, as well as the poor post-harvest preservation of mangoes. This invention provides a strain of Bacillus belyceae SCSX39.

[0006] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned Bacillus belyssus SCSX39.

[0007] The final technical problem to be solved by this invention is to provide a biological control agent.

[0008] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0009] In a first aspect, the present invention provides a strain of Bacillus velezensis SCSX39, classified as Bacillus velezensis, strain number SCSX39, which was deposited on September 15, 2025 at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 35918, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0010] The Bacillus belyss SCSX39 has the following morphological characteristics: milky white, slightly transparent, round colonies with a smooth surface, no wrinkles, and irregular edges; its exponential growth period is 2-15 h; it can grow at 15-45℃, tolerate salinity of 0-4%, and grow at pH 5-9; it can adapt to a variety of habitats and grow well under different conditions, thus playing a biocontrol role.

[0011] The 16S rRNA sequence of the Bacillus belyssus SCSX39 is shown in SEQ ID NO.1.

[0012] Furthermore, the in vitro safety of Bacillus belyssus SCSX39 was evaluated. Bacillus belyssus SCSX39 exhibits γ-hemolysis but no hemolytic activity; it is extremely sensitive to ceftriaxone, gentamicin, erythromycin, and chloramphenicol, and moderately sensitive to penicillin, ampicillin, and lincomycin, and does not show resistance to these antibiotics.

[0013] Secondly, this invention provides the application of Bacillus bellis SCSX39 in inhibiting the pathogenic fungus causing mango anthracnose.

[0014] The anthrax pathogenic fungi include any one or a combination of several of the following: Colletotrichum gloeosporioides, Colletotrichum coffeanum, Nigrospora sphaerica, Colletotrichum siamense, and Colletotrichum asianum.

[0015] The Bacillus bellis SCSX39 is injected into mangoes in the form of bacterial and / or sterile fermentation broth to inhibit the pathogenic fungus causing mango anthracnose.

[0016] Specifically, when the *Bacillus belyssioides* SCSX39 is injected into mangoes in the form of a fermentation broth with bacteria or a fermentation broth with and without bacteria, the bacterial concentration of the fermentation broth is 1×10⁻⁶. 7 ~1×10 9 CFU / mL.

[0017] In some embodiments of the present invention, when the *Bacillus belyssioides* SCSX39 is injected into a mango in the form of a fermentation broth with bacteria or a fermentation broth with and without bacteria, the bacterial concentration of the fermentation broth with bacteria is 1 × 10⁻⁶. 8 CFU / mL.

[0018] In some embodiments of the present invention, the fermentation broth of Bacillus belys SCSX39 with and / or without cells can effectively reduce the incidence of mango anthracnose and reduce mango rot, and its effect is better than that of the existing Bacillus belys 12Y (accession number CGMCC No. 28636, the detailed information of which has been disclosed in Chinese patent CN117660266A).

[0019] The mangoes mentioned include, but are not limited to, Kate mangoes and / or Taiwan mangoes. In the prior art, all mango varieties can be inhibited by the Bacillus bellis SCSX39 described in this invention to suppress anthrax pathogens.

[0020] Thirdly, this invention provides the application of Bacillus belye SCSX39 in inhibiting foodborne pathogenic fungi.

[0021] The foodborne pathogenic fungi include any one or a combination of several of the following from sources such as Cladosporium sp., Penicillium citrinum, Geotrichum candidum, and Schizophyllum commune, which originate from steamed buns, sauces, etc.

[0022] Fourthly, the present invention provides the application of the aforementioned Bacillus belye SCSX39 in postharvest preservation of mangoes.

[0023] Specifically, before post-harvest storage, mango fruits are soaked in Bacillus belye SCSX39 fermentation broth containing and / or without bacteria for 1-2 minutes.

[0024] In some embodiments of the present invention, in experiments on improving the storability of Taiwanese mangoes with Bacillus SCSX39 in vivo fermentation broth, it was found that treatment with the in vivo fermentation broth of strain SCSX39 can maintain the storage quality of the fruit and reduce the incidence of mango rot; can significantly extend the storage life of strawberries; can maintain the fruit's high disease resistance, maintain the integrity of the cell wall, delay the softening of the fruit, thereby extending the fruit's storage period; and can improve the microbial community structure on the fruit surface, reducing the adverse effects of harmful bacteria on the fruit during storage.

[0025] Fifthly, the present invention provides a biological control agent containing the aforementioned Bacillus berleis SCSX39.

[0026] Beneficial effects:

[0027] This invention screened and isolated a strain of *Bacillus velezensis* SCSX39 from deep-sea mud in the South China Sea. Through testing and systemic safety evaluation, the biocontrol agent prepared based on this strain effectively overcomes the shortcomings of current mango industry practices, such as poor control of anthracnose pathogens and foodborne pathogens, and poor post-harvest preservation of mangoes. This strain exhibits strong inhibitory activity against multiple *Anthracnose* species and *Ulva spp.*, showing significant inhibitory effects. Simultaneously, the strain is safe, sensitive to multiple antibiotics, and not resistant, making it suitable for food preservation. Its fermentation broth, with and / or without bacterial cells, can be directly applied to post-harvest mango preservation, with significantly improved antibacterial efficiency. Furthermore, this strain also inhibits various foodborne pathogens, indicating a rich content of active substances. This invention provides a dedicated and highly efficient core strain resource for the green control of post-harvest anthracnose in mangoes, and provides important microbial material for the development of novel microbial pesticides, which is of great significance to the development of the mango industry. Attached Figure Description

[0028] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0029] Figure 1 The inhibitory effect of strain SCSX39 on two pathogens (CK is the control group, and T is the treatment group).

[0030] Figure 2 The colony morphology of strain SCSX39 on LB medium.

[0031] Figure 3 This is a growth curve of strain SCSX39.

[0032] Figure 4The graph shows the growth of strain SCSX39 under different temperatures, pH values, and salinities. A represents different temperatures; B represents different salinities, specifically, the black, red, dark blue, green, purple, yellow, light blue, brown, and dark green curves in the graph represent the growth of strain SCSX39 after 24 h of culture at salinities of 0.0%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, and 4.0%, respectively; C represents different pH values, specifically, the black, red, blue, green, and purple curves in the graph represent the growth of strain SCSX39 after 24 h of culture at pH 3, pH 5, pH 7, pH 9, and pH 11, respectively.

[0033] Figure 5 The results show the inhibitory effect of strain SCSX39 on anthrax pathogens.

[0034] Figure 6 The results of blood plate culture of strain SCSX39 are shown.

[0035] Figure 7 A schematic diagram showing the marking of the wound pores of *Bacillus velezensis* SCSX39 on *Colletotrichum siamense* infected with Kettering mangoes and an image illustrating the preservation effect.

[0036] Figure 8 The graph shows the biofilm formation capabilities of strains SCSX39 and 12Y.

[0037] Figure 9 The graph shows the motility results of strains SCSX39 and 12Y.

[0038] Figure 10 The image shows the changes in the appearance of mangoes in each treatment group during storage.

[0039] Figure 11 The storage life of mangoes in each treatment group during storage.

[0040] Figure 12 This shows the changes in the weight loss rate of mangoes in each treatment group during storage.

[0041] Figure 13 The changes in soluble solids (TSS) content of mangoes in each treatment group during storage.

[0042] Figure 14 This shows the changes in mango cell wall metabolism-related indicators in each treatment group during storage.

[0043] Figure 15 This shows the changes in polyphenol oxidase (PPO) activity in mangoes of different treatment groups during storage.

[0044] Figure 16 The inhibitory effect of strain SCSX39 on foodborne pathogens.

[0045] Preservation Information: A strain of Bacillus velezensis, SCSX39, was deposited on September 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 35918. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0046] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0047] In the following examples, the LB broth medium was formulated as follows: 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride were added to every 1 L of water, and the mixture was sterilized at 121°C for 20 min. 2% agar powder was added to the LB agar medium, and the mixture was sterilized at 121°C for 20 min.

[0048] Example 1: Isolation, purification and identification of biocontrol bacteria strains from sea mud in the South China Sea

[0049] 1. Strains isolation and purification

[0050] Marine sediment samples were collected from the South China Sea, Sanya City, Hainan Province, China (3619 m, 120°05′E, 20°06′N), on April 8, 2022. Biocontrol bacteria were isolated using the gradient dilution plate method. Sample solutions were prepared using sterile, aged seawater and diluted. Different gradient dilutions (10⁻⁶ m, 120°05′E, 20°06′N) were then collected. -2 10 -3 10 -4 10 -5 100 μL of each culture was spread using both PDA medium and LB agar medium; the culture conditions were set at 28℃ and 37℃, respectively, and incubated at constant temperature for 48 h. Based on the colony morphology, single colonies with specific morphology were selected for streak purification in four zones, and the purified strain was obtained after two generations of culture.

[0051] Using *Colletotrichum asianum* and *Colletotrichum sp.* as indicator bacteria, the biocontrol efficacy was evaluated using a confrontation culture method. Pathogen fungal discs were inoculated into the center of PDA medium and incubated at 28°C for 7 days to activate the pathogens. Several 7 mm diameter discs were prepared on pathogen plates using a punch. Fresh PDA medium was used, and a center hole was punched to remove an agar block, which was then inoculated with a disc. Antagonistic bacteria fermentation broth was inoculated at a 2 cm equidistant point from the pathogen discs, and the plates were incubated at 28°C for 7 days. Plates without biocontrol bacteria served as a blank control. At the end of the incubation, the average distance between the pathogen discs and the test strains was measured, and the inhibition rate was used to evaluate the antibacterial effect. The strain that showed good inhibitory activity against both pathogens was selected and named SCSX39. The inhibitory effect of SCSX39 against both pathogens is shown in [the table below]. Figure 1 .

[0052] 2. Identification of strain SCSX39

[0053] (1) Colony morphology characteristics: After strain SCSX39 was cultured on LB medium at 37 ℃ for 12 h, as follows Figure 2 As shown, the single colony of the strain is round and milky white, with a smooth surface without wrinkles and irregular edges. Based on the morphological characteristics, strain SCSX39 is preliminarily identified as a bacterium.

[0054] (2) Physicochemical property analysis: The growth characteristics of strain SCSX39 were determined by shaking culture in LB broth medium at 37℃ and 180 rpm. The OD of the strain at different time points was measured. 600 The changes in OD 600 Growth curves of strain SCSX39 were plotted as evaluation indicators. Figure 3 It can be seen that the exponential growth period of strain SCSX39 is 2-15 h.

[0055] To understand the adaptability of strain SCSX39 to different growth conditions, strain SCSX39 was inoculated into LB broth medium, and its adaptability to different temperatures, pH values, and salinities was determined, using OD values ​​as the metric. 600 As an evaluation indicator. (By) Figure 4 It is known that strain SCSX39 can grow at 15~45℃, at pH 5~9, and tolerate salinity of 0~4%.

[0056] (3) Molecular biology and phylogenetic analysis

[0057] Plates containing single colonies of strain SCSX39 were sent to Qingke Biotechnology Co., Ltd. Genomic DNA was extracted using a genomic DNA extraction kit. Using this DNA as a template, the 16S rRNA gene fragment of strain SCSX39 was amplified by PCR using universal primer pairs 27F (5´-AGAGTTTGATCCTGGCTCAG-3´) and 1492R (5´-GGTTACCTTGTTACGACTT-3´). The sequence result is shown in SEQ ID NO.1. The PCR product was further sequenced, and the sequencing results were compared with the NCBI GenBank database using BLAST. The comparison results showed that strain SCSX39 had high homology with *Bacillus velezensis*. Combined with the morphological characteristics and physicochemical properties analysis of strain SCSX39, strain SCSX39 was therefore identified as *Bacillus velezensis*.

[0058] Strain SCSX39 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 15, 2025, with accession number CGMCC No. 35918. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0059] Example 2: Determination of the antibacterial activity of Bacillus velezensis SCSX39 against anthrax pathogens

[0060] Using various anthrax pathogens preserved in the laboratory (Colletotrichum gloeosporioides, Coletotrichum coffeanum, Coletotrichum sp., Nigrospora sphaerica, and Coletotrichum siamense) as indicator bacteria, the inhibitory effect of Bacillus velezensis SCSX39 on anthrax pathogens was tested using the plate confrontation method.

[0061] The specific operating steps are as follows: Select strain SCSX39 and culture it in LB broth medium at 37℃ and 180-200 rpm for 8-12 h to obtain seed culture. Inoculate the seed culture into fresh LB broth medium at a ratio of 1-2% v / v and culture at 37℃ and 180-200 rpm for 72 h to obtain SCSX39 fermentation broth. Activate the pathogen using PDA medium. Use a punch to create several 7 mm mycelial discs on the pathogen plate. Inoculate one mycelial disc into the center of each PDA plate, and then inoculate SCSX39 fermentation broth at different positions equidistant from the pathogen. Culture at 28℃ and RH 90-95% for 5-7 days to obtain the treatment group. Use plates inoculated only with the pathogen as the control group. At the end of the culture, measure the mycelial diameter of the pathogen in both the treatment and control groups. Calculate the antibacterial efficiency using the formula: Antibacterial efficiency = (Hycelial diameter of pathogen in control group - Hycelial diameter of pathogen in treatment group) / Hycelial diameter of pathogen in control group × 100%, to evaluate the antibacterial effect.

[0062] The antibacterial efficiency of strain SCSX39 against anthrax pathogens is as follows: Figure 5 As shown, strain SCSX39 exhibits excellent antibacterial efficiency against various anthrax pathogens, with an antibacterial efficiency of over 75% against Nigrospora sphaerica.

[0063] Example 3: In vitro safety evaluation of Bacillus velezensis SCSX39

[0064] To assess the safety of strain SCSX39, hemolysis and drug susceptibility tests were performed. The specific methods are as follows: Strain SCSX39 was inoculated onto blood agar plates (purchased from Guangdong Huankai Biotechnology Co., Ltd.) using the spot inoculation method and incubated at 37℃ for 48 h. Hemolysis was observed, with Staphylococcus aureus as a positive control and Lactobacillus fermentation as a negative control. The susceptibility of Bacillus to seven common antibiotics was determined using the Kb disk diffusion method to assess its resistance. 10 μL of fermentation broth from strain SCSX39 (1×10⁻⁶) was used. 8 (CFU / mL) samples were spread onto LB agar medium. Using tweezers, test strips for penicillin, ampicillin, ceftriaxone, gentamicin, erythromycin, lincomycin, and chloramphenicol were placed flat on the agar plate. The plate was incubated upside down at 37°C for 16–20 h. The diameter of the inhibition zone was measured after its appearance. The sensitivity of strain SCSX39 to each antibiotic was determined based on the diameter of the inhibition zone.

[0065] Microorganisms growing on blood agar plates exhibit three different hemolytic phenomena: α-hemolysis, where a 1-2 mm translucent, grass-green hemolytic ring appears around the colony, indicating incomplete hemolysis as red blood cells are not completely dissolved; β-hemolysis, also known as complete hemolysis, where a 2-4 mm clearly defined, completely transparent hemolytic ring appears around the colony; and γ-hemolysis, or no hemolysis, where no hemolysis is observed around the colony. Figure 6 The hemolytic results showed that the positive control *Staphylococcus aureus* exhibited β-hemolysis on blood agar plates, while the negative controls *Lactobacillus fermentum* and *Bacillus belyssae* SCSX39 showed γ-hemolysis and no hemolysis. Meanwhile, the drug susceptibility test results in Table 1 show that strain SCSX39 is extremely sensitive to ceftriaxone, gentamicin, erythromycin, and chloramphenicol, and moderately sensitive to penicillin, ampicillin, and lincomycin, and does not exhibit resistance to these antibiotics.

[0066] Based on the combined results of hemolytic activity and drug susceptibility testing of strain SCSX39, it is preliminarily determined that strain SCSX39 is safe and can be used for food preservation.

[0067] Table 1. Resistance of strain SCSX39 to multiple antibiotics

[0068]

[0069] Example 4: Preservative effect of Bacillus velezensis SCSX39 on Kettering mangoes infected with Colletotrichum siamense (Anthrax bacterium of Siam).

[0070] The mangoes used in the experiment were treated with a suspension of Bacillus velezensis SCSX39 and cell-free supernatant. The tested mango variety was Keitt mango, and mangoes of similar size and maturity with smooth surfaces and no mechanical damage were selected for treatment. The specific procedures are as follows:

[0071] 1. Preparation of pathogenic fungal spore suspension

[0072] Using a sterile pipette tip, collect 2-3 spore discs from the edge of an agar plate covered with *Anthrax sicca* pathogens. Incubate in 100 mL of PDB medium at 28 °C and 180 rpm for 3 days. Filter and resuspend to obtain a spore suspension. Determine the concentration of the spore suspension using a hemocytometer and adjust to 5 × 10⁻⁶ with sterile distilled water. 5 spores / mL.

[0073] 2. Preparation of Bacillus fermentation broth and cell-free fermentation supernatant

[0074] Single colonies of strains SCSX39 and 12Y were picked using an inoculation loop and inoculated into 100 mL of LB broth. The culture was then incubated on a shaker at 37°C and 180 rpm for 24 h to activate the seed culture. The OD of the seed culture was then measured with sterile water. 600 Adjust the pH to approximately 0.8, then inoculate the seed culture into LB medium at an inoculation rate of 2% v / v. Ferment on a shaker at 37 ℃ and 180 rpm for 72 h to obtain the fermentation broth containing bacterial cells. Centrifuge the fermentation broth at 4 ℃ and 8000 rpm, discard the precipitated bacterial cells, and collect the supernatant. Filter the supernatant through a 0.22 μm filter membrane to obtain the cell-free fermentation supernatant of Bacillus, i.e., the sterile fermentation broth.

[0075] 3. Live inoculation experiment

[0076] Wash all mangoes with sterile water, then soak them in 75% alcohol for 2 minutes for surface disinfection. Wash the mangoes three times with sterile water to remove residual alcohol, and air dry them in a well-ventilated environment at room temperature. Use a sterile toothpick to make four wounds about 5 mm deep on the surface of the mangoes (label each fruit's wounds as ①, ②, ③, and ④ from left to right and top to bottom). Inoculate 10 μL of Bacillus fermentation broth (bacterial concentration 1.0 × 10⁻⁶) into holes ① and ④ respectively. 8 Wells ② and ③ were inoculated with 10 μL of sterile Bacillus fermentation broth (CFU / mL). The inoculated fermentation broths were designated as Y (12Y) and T (SCSX39), respectively. Fruits with all four wells inoculated with 10 μL of sterile distilled water served as the control (CK). After 2 h, 10 μL of pathogen spore suspension was injected and the fruit was air-dried at room temperature. The mango fruits were then grouped and stored in a constant temperature incubator at 25±1 ℃ and 85% relative humidity. Mango disease incidence was observed and recorded daily, and the biocontrol efficacy was determined based on the disease incidence at the inoculation site.

[0077] Experimental results are as follows Figure 7 The preservation effect of *Bacillus vesicularis* on mangoes infected with *Anthracnose sicca* was investigated using the disease incidence at the inoculation site as an indicator. The figures show that both *Bacillus vesicularis* fermentation broth with and without bacterial cells showed preventative effects against mango anthracnose. Among these, *Bacillus vesicularis* SCSX39 showed better preventative effects than 12Y. Compared to the control group, *Bacillus vesicularis* SCSX39 reduced the disease incidence by approximately 70%; compared to *Bacillus vesicularis* 12Y, SCSX39 reduced the disease incidence by approximately 60%. In conclusion, strain SCSX39 can effectively inhibit the incidence of mango anthracnose caused by *Anthracnose sicca*.

[0078] Example 5: Comparative analysis of phenotypic characteristics of Bacillus velezensis SCSX39 and 12Y

[0079] Example 4 revealed that strain SCSX39 exhibited significantly better control of mango anthracnose compared to strain 12Y. Further comparisons were made of the strains' phenotypic characteristics and their colonization abilities. The specific procedures are as follows:

[0080] Preparation of Bacillus seed culture and OD 600 The adjustment method is the same as in Example 4.

[0081] 1. Biofilm formation ability

[0082] Add 196 μL of sterile LB medium to a sterile 96-well plate, and then inoculate with seed culture at a 2% v / v inoculation rate (the control group was inoculated with an equal volume of sterile LB medium). Seal the edges with sealing film and incubate at 37 ℃ for 48 h. Each treatment group should have at least 6 parallel wells, and the experiment should be repeated twice. After incubation, discard the supernatant and carefully wash with sterile water until the eluent is colorless. Then, add 200 μL of anhydrous ethanol to each well for fixation at room temperature for 15 min. After fixation, discard the fixative and wash away the fixative with sterile water. Add 200 μL of 0.05% (m / v) crystal violet staining solution and stain at room temperature for 5 min. Discard the staining solution and wash 3-4 times until the eluent is colorless. Add 200 μL of 33% (v / v) acetic acid solution to each well for dissolution for 15 min, and measure the OD using a microplate reader. 570 The biofilm formation index (BFI) is calculated using the formula.

[0083] BFI = OD(T) - OD(CK)

[0084] Wherein, OD(T) is the absorbance of the well into which the bacterial culture is introduced, and OD(CK) is the absorbance of the control group.

[0085] 2. Swimming and herd behavior

[0086] Mobility: 3 μL of bacterial seed culture of strain SCSX39 or 12Y was inoculated into the center of a 0.3% LB agar plate and incubated at 37°C for 48 h. Bacterial motility was assessed using the colony edge diameter assay and photographed for record-keeping.

[0087] Cluster movement: Adjust fermentation broth to OD 600 =0.3. Take 3 μL and inoculate it in the center of a 1.0% LB agar plate, incubate at 37℃ for 48 h. Assess the bacterial aggregation and migration ability using the colony edge diameter method, and record the data by photography.

[0088] The results of the strain phenotypic indicators are shown in Table 2. Figures 8-9 The biofilm formation ability of strain SCSX39 was significantly higher than that of strain 12Y. In the motility experiment, the diffusion diameter of strain SCSX39 was significantly larger than that of strain 12Y, indicating its stronger motility. In the swarming experiment, although there was no significant difference in the diffusion circle diameter between the two strains, after 48 hours of cultivation, the colony edge of strain 12Y was a regular circle, while the colony edge of strain SCSX39 was an irregular circle. This shows that *Bacillus belye* SCSX39 exhibits stronger motility, superior biofilm formation ability, and a unique irregular colony morphology associated with swarming. These colonization-related phenotypes not only clearly distinguish it from strain 12Y but also indicate its superior competitive potential and environmental adaptability in plant colonization, providing an intrinsic basis for its subsequent biocontrol effects.

[0089] Table 2. Phenotypic results of Bacillus belyssus SCSX39 and 12Y.

[0090]

[0091] Example 6: Postharvest preservation effect of Bacillus velezensis SCSX39 on Taiwanese mangoes

[0092] The experiment used Bacillus velezensis SCSX39 fermentation broth as the experimental medium. The tested mango variety was the Taiwanese mango. Taiwanese mangoes that were 70-80% ripe, uniform in size, and undamaged were selected for post-harvest soaking treatment. The specific procedures are as follows:

[0093] Colonies of SCSX39 were picked from LB agar plates containing the strain using an inoculation loop and inoculated into 100 mL of LB broth. The culture was then incubated at 37°C and 180 rpm for 8–12 h until the viable cell count in the fermentation broth reached 1.0 × 10⁻⁶. 8 When the concentration of CFU / mL was 2%, it was inoculated into 1000 mL of LB broth and fermented for 72 h to obtain SCSX39 fermentation broth, i.e., SCSX39 fermentation broth with bacterial cells.

[0094] The three groups of fruits were: no treatment (CK), treatment with fresh LB medium (LB), and treatment with SCSX39 fermentation broth (T). After treatment, the fruits were air-dried at room temperature under ventilation, and then placed in plastic baskets (with paper towels at the bottom) and stored at 25℃ and 90%RH. During storage, the fruits were observed and recorded every other day for the following indicators: storage life, disease incidence, disease index, weight loss, color, soluble solids content (TSS), titratable acid (TA), and firmness.

[0095] The changes in the appearance of mangoes in each group are shown in the figure. Figure 10 Storage life is shown in Figure 11 Changes in weight loss rate during storage are shown in the figure. Figure 12 Changes in soluble solids content (TSS) are shown in [reference needed]. Figure 13 The results showed that treatment with strain SCSX39 maintained good fruit appearance during storage, slowed down fruit firmness and weight loss, and maintained a high TSS level. This indicates that strain SCSX39 treatment effectively maintained mango quality, reduced mango rot, and extended fruit shelf life. Compared with the other two groups, the weight loss rate of group T fruit was significantly lower during storage, indicating that although LB medium treatment exacerbated fruit water loss, the addition of strain SCSX39 compensated for this deficiency and reduced fruit water loss, resulting in group T fruit exhibiting the lowest weight loss rate among the three groups. The soluble solids content (TSS) fluctuated little during storage, indicating that this strain has a positive effect on sugar accumulation and maintenance in mangoes. These indicators collectively demonstrate the effectiveness of Bacillus belyssus SCSX39 in extending the shelf life of Taiwanese mangoes and maintaining fruit quality.

[0096] The changes in mango cell wall metabolism-related indicators in each group during storage are shown in the figure. Figure 14 As shown in the figure, compared with the control group (CK) and the fruit treated with fresh LB medium (LB), the fruit treated with strain SCSX39 (T) experienced a slower decline in firmness during storage. Specifically, the polygalacturonase (PG) activity in group T remained at the lowest level during storage, and correspondingly, the protopectin content in group T was consistently higher than the other two groups, indicating that the degradation process of cell wall substances was significantly inhibited. This stability of cell wall structure is crucial for maintaining fruit firmness and reducing water loss. Therefore, these results explain the mechanism by which SCSX39 treatment can extend the shelf life and maintain the quality of Taiwanese mangoes from the perspective of cell wall metabolism.

[0097] During storage, the changes in polyphenol oxidase (PPO) activity in each group of fruits are shown in the figure. Figure 15 Compared to the other two groups of fruit, the T group of fruit treated with strain SCSX39 showed relatively lower polyphenol oxidase (PPO) activity. Total phenols, as important antioxidants in fruit, contribute to delaying fruit senescence and quality deterioration when their content remains stable, while reduced PPO activity effectively minimizes fruit browning. These changes indicate that Bacillus belyssus SCSX39 treatment can regulate phenolic metabolism in fruit, inhibit PPO activity, thereby reducing browning and maintaining fruit color and quality. The combined effects of these changes further confirm that Bacillus belyssus SCSX39 has a significant effect on extending the shelf life of Taiwanese mangoes and maintaining fruit quality.

[0098] Example 7: Inhibitory effect of Bacillus velezensis SCSX39 on foodborne pathogenic fungi

[0099] The foodborne pathogenic fungi used in this embodiment include Cladosporium sp., Penicillium citrinum, Geotrichum candidum, and Schizophlum commune. The specific operating steps are as follows: Strains SCSX39 were picked and cultured in LB broth at 37°C and 180-200 rpm for 8-12 hours to obtain a seed culture. The seed culture was then inoculated into fresh LB broth at a ratio of 1-2% v / v and cultured at 37°C and 180-200 rpm for 72 hours to obtain SCSX39 fermentation broth. The foodborne pathogenic fungi were activated using PDA medium. Several 7 mm mycelial cakes were punched on a plate containing the foodborne pathogenic fungi. A mycelial cake was inoculated into a hole in the center of each PDA plate. Then, SCSX39 fermentation broth was inoculated at different locations equidistant from the foodborne pathogenic fungi. The plates were cultured at 28°C and RH 90-95% for 5-7 days to obtain the treatment group. A plate inoculated only with foodborne pathogenic fungi was used as a control group. At the end of the culture, the hyphal diameters of the foodborne pathogenic fungi in the treatment group and the control group were measured. The antibacterial effect was evaluated by calculating the antibacterial efficiency using the formula: Antibacterial efficiency = (Hyphae diameter of foodborne pathogenic fungi in the control group - Hyphae diameter of foodborne pathogenic fungi in the treatment group) / Hyphae diameter of foodborne pathogenic fungi in the control group × 100%.

[0100] See results Figure 16 The diameter of the hyphae of foodborne pathogenic fungi in the treatment and control groups was measured using a ruler, and the antibacterial efficiency was calculated according to the above formula. It can be seen that strain SCSX39 has a good inhibitory effect on the above-mentioned foodborne pathogenic fungi, and on Cladosporium (…). Cladosporium sp. ), Penicillium citrinum ( Penicillium citrinum ), white mold ( Geotrichum candidum ), Schizophyllum commune ( Schizophyllum commune The antibacterial efficiencies of SCSX39 were 63.27±3.65%, 40.55±2.31%, 61.48±0.90%, and 77.02±1.49%, respectively. In addition, the mycelial growth of pathogens at the edge of the inhibition zone was sparse and deformed, indicating that SCSX39 could not only inhibit their growth but also significantly damage their mycelial morphology.

[0101] This invention provides a strain of Bacillus belyceae SCSX39 and its application, along with related ideas and methods. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A strain of Bacillus belye ( Bacillus velezensis )SCSX39, categorized and named Bacillus velezensis The strain number is SCSX39, which was deposited on September 15, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35918. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. in, The *Bacillus belyssus* SCSX39 is a smooth, wrinkle-free *Bacillus belyssus* with irregular edges.

2. The application of Bacillus belyssus SCSX39 as described in claim 1 in inhibiting the pathogenic fungus causing mango anthracnose; in, The anthrax pathogenic fungus includes: *Colletotrichum gloeosporioides*. Colletotrichum gloeosporioides Coffee anthrax Colletotrichum coffeanum spherical black spores Nigrospora sphaerica Siamese anthrax Colletotrichum siamense、 Asian anthrax Colletotrichum asianum Any one or more of the following combinations.

3. According to claim 2, the Bacillus bellis SCSX39 is injected into mangoes in the form of bacterial and / or sterile fermentation broth to inhibit the pathogenic fungus causing mango anthracnose.

4. In the application according to claim 3, when the *Bacillus belyssioides* SCSX39 is injected into a mango in the form of a fermentation broth with bacteria or a fermentation broth with and without bacteria, the bacterial concentration of the fermentation broth with bacteria is 1 × 10⁻⁶. 7 ~1×10 9 CFU / mL.

5. The application of Bacillus belyssus SCSX39 as described in claim 1 in inhibiting foodborne pathogenic fungi; in, The foodborne pathogenic fungi include Cladosporium. Cladosporium sp. Penicillium citrinum Penicillium citrinum White mold Geotrichum candidum Schizophyllum Schizophyllum commune Any one or more of the following combinations.

6. The application of Bacillus berleis SCSX39 as described in claim 1 in postharvest preservation of mangoes.

7. The application according to claim 6, characterized in that, Before postharvest storage, soak the mango fruit in Bacillus vesicles SCSX39 with and / or without bacterial cells for 1-2 minutes.

8. A biological control agent, characterized in that, The biological control agent contains Bacillus berberis SCSX39 as described in claim 1.

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