Bacillus velezensis strain from marine source SCSX9, biocontrol composite microbial agent, and preparation method and application thereof

Abstract

The present application relates to the technical field of microorganism technology and postharvest disease biological control of fruits and vegetables, and particularly relates to a marine source bacillus velezensis SCSX9, a biocontrol composite microbial agent, and a preparation method and application thereof.The marine source bacillus velezensis SCSX9 is obtained by separation and screening, and the strain, fermentation product (bacterial suspension, bacterial fermentation broth) and green biocontrol microbial agent thereof can effectively inhibit various pathogenic bacteria, thereby safely and effectively controlling the occurrence and development of plant diseases.Further, the biocontrol composite microbial agent composed of the bacillus velezensis 12Y can produce various antibacterial substances, through the synergistic effect between the strains, significantly improves the broad-spectrum bacteriostasis, stability and control efficiency, prolongs the fruit preservation period and storage time, improves the quality of strawberries and mangoes, and has high economic significance in agricultural production.

Description

Technical Field

[0001] This invention relates to the fields of microbial technology and biological control technology of postharvest diseases of fruits and vegetables, specifically to a marine-derived Bacillus berleis SCSX9 strain, a biocontrol compound microbial agent, and its preparation method and application. Background Technology

[0002] Postharvest fungal diseases are a significant factor affecting the storage quality and commercial value of fruits. Common postharvest diseases include strawberry gray mold (caused by *Botrytis cinerea*), strawberry black spot (caused by *Alternaria alternata*, etc.), strawberry anthracnose, and mango anthracnose (mainly caused by fungi of the *Colletotrichum* genus). The typical symptoms of these diseases are brown or black spots on the fruit surface, which gradually expand and lead to fruit rot. Strawberry skins are soft and fragile, and mangoes are easily damaged mechanically after harvest. Both fruits have soft, juicy flesh rich in bioactive substances, making them even more susceptible to postharvest pathogen infection. Therefore, the incidence of fungal diseases in strawberries reaches 25-30%, and the postharvest loss rate in mangoes reaches 20-30%, which seriously affects the storage quality and commercial value of the fruits.

[0003] The main control methods for the above-mentioned diseases include: physical methods: such as low-temperature storage, heat treatment, and radiation, which can inhibit the growth of pathogens but easily damage fruit quality; chemical methods: such as fungicides like benzimidazole and carbendazim, which are effective but easily produce harmful residues, promote the development of drug resistance in pathogens, and also lead to environmental pollution; biological methods: using biocontrol bacteria, such as Bacillus, yeast, and Pseudomonas, to control diseases through nutrient competition and antibiotic production, which has the advantages of safety and sustainability. However, the commonly used biocontrol bacteria are mostly derived from the soil or plant endogenous environment, which has obvious shortcomings: (1) the spectrum of inhibition is narrow, making it difficult to deal with the complex infection caused by different fungi such as Botrytis cinerea, Alternaria alternata, and Colletotrichum; (2) the control effect is unstable and easily affected by different environmental factors; (3) the fruit is often cross-infected by multiple pathogens after harvest, lacking the comprehensive ability to deal with complex disease systems.

[0004] Therefore, although biological control is considered a promising green control approach, existing biocontrol agents still have significant shortcomings in terms of broad-spectrum antibacterial activity, environmental stability, and practical application effectiveness. There is an urgent need to develop new biocontrol agents with a wider antibacterial spectrum, stronger environmental adaptability, and synergistic control effects to meet the control needs of complex fungal diseases after fruit harvest. Summary of the Invention

[0005] The technical problem to be solved by the first aspect of the present invention is to provide a marine-derived Bacillus belye, which addresses the shortcomings of existing biocontrol bacteria, such as narrow antibacterial spectrum, poor stability, and poor practical application effect.

[0006] The second technical problem to be solved by the present invention is to provide a microbial agent.

[0007] The technical problem to be solved by the third aspect of the present invention is to provide the application of the marine-derived Bacillus belye in inhibiting pathogens.

[0008] The fourth technical problem to be solved by the present invention is to provide the application of the marine-derived Bacillus vesiculosus in postharvest preservation of fruit and prevention and control of fungal diseases.

[0009] The technical problem to be solved by the fifth aspect of the present invention is to provide a biocontrol compound microbial agent.

[0010] The technical problem to be solved by the sixth aspect of the present invention is to provide a method for preparing the biocontrol compound microbial agent.

[0011] The technical problem to be solved by the seventh aspect of the present invention is to provide the application of the biocontrol compound microbial agent in postharvest preservation of fruit and prevention and control of fungal diseases.

[0012] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0013] In a first aspect, the present invention provides a marine-derived Bacillus velezensis strain, classified and named Bacillus velezensis, strain number SCSX9, which was deposited on September 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 35916, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0014] Among them, the marine-derived Bacillus belye colonies are opaque and pale yellow; the colonies are round and small; the surface is relatively smooth and the edges are neat. Under a microscope (400×), the bacterial cells are cylindrical, mostly straight, with a few slightly curved, and the ends of the bacterial cells are mostly blunt and rounded.

[0015] Molecular biological identification confirmed that the 16S rRNA sequence of the marine-derived Bacillus belesii is shown in SEQ ID NO. 1.

[0016] Secondly, the present invention provides a microbial agent containing the aforementioned marine-derived Bacillus belye.

[0017] Thirdly, the present invention provides the application of the marine-derived Bacillus vesiculosus or the bacterial agent in inhibiting pathogenic bacteria.

[0018] The pathogens include any one or a combination of several of the genera *Alternaria*, *Anthracis*, *Staphylococcus*, *Fusarium*, *Botrytis*, and *Ulva*.

[0019] In some embodiments of the present invention, the *Alternaria* genus includes *Alternaria*; the *Anthrax* genus includes any one or a combination of several of *Colletotrichum gloeosporioides*, *Anthrax caeruleus*, *Anthrax asiaticus*, and *Anthrax siamensis*; the *Botrytis* genus includes *Botrytis cinerea*; the *Fusarium* genus includes *Fusarium latae*; the *Botrytis* genus includes any one or a combination of several of *Botrytis cinerea*, *Botrytis cinerea*, *Botrytis pelargonifolia*, *Botrytis cinerea*, and *Botrytis ranunculus*; and the *Ulva* genus includes *Ulva*.

[0020] In some embodiments of the present invention, the *Alternaria* genus includes *Alternaria sp.* FC.001 and *Alternaria alternata* CM04; the *Anthrax* genus includes any one or a combination of several of the following: *Colletotrichum sp.* MIL6, *Colletotrichum gloeosporioides* FC.002, *Colletotrichum gloeosporioides* C1, *Colletotrichum gloeosporioides* C2, *Colletotrichum coffeanum* FC.008, *Colletotrichum asianum* MIL1, *Colletotrichum siamense* MIL7, and *Colletotrichum siamense* HT12; the *Botryosphaeria* genus includes *Botryosphaeria*. The *Fusarium* genus includes *Fusarium proliferatum* HT-04; the *Botrytis* genus includes any one or a combination of several of the following: *Botrytis cinerea* T8, *Botrytis californica* TB.03, *Botrytis pelargonii* TB.01, *Botrytis pelargonii* TB.02, *Botrytis polyblastis* TJ.01, *Botrytis ficariarum* TJ.02, *Botrytis ranunculi* CH.01, *Botrytis ranunculi* CH.02, and *Botrytis ranunculi* CH.03; the *Nigrospora* genus includes *Nigrospora sphaerica* MIL2.

[0021] Among them, the bacterial suspension and / or bacterial fermentation broth of the marine-derived Bacillus vesiculosus are used to inhibit pathogenic bacteria.

[0022] Specifically, the concentration of the bacterial suspension is 1×10⁻⁶. 5~7 The concentration of the bacterial fermentation broth was 1×10 CFU / mL. 7~9 CFU / mL.

[0023] In some embodiments of the present invention, the concentration of the bacterial suspension is 1×10⁻⁶. 6 The concentration of the bacterial fermentation broth was 1×10 CFU / mL. 8 CFU / mL.

[0024] Fourthly, this invention provides the application of the marine-derived Bacillus vesiculosus in postharvest preservation of fruits and prevention of fungal diseases.

[0025] The fungal diseases mentioned include, but are not limited to, any one or a combination of several of gray mold, black spot, and anthracnose.

[0026] In some embodiments of the present invention, the fungal disease includes any one or a combination of several of strawberry gray mold, strawberry black spot, strawberry anthracnose, and mango anthracnose.

[0027] In some embodiments of the present invention, the fruit is a strawberry and / or a mango.

[0028] Fifthly, the present invention provides a biocontrol compound microbial agent containing the marine-derived Bacillus vesicularis and Bacillus vesicularis 12Y described in the present invention.

[0029] The *Bacillus velezensis* 12Y strain, classified as *Bacillus velezensis*, was deposited on October 16, 2023, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 28636, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Detailed information about this strain is published in Chinese Patent CN117660266A.

[0030] In a sixth aspect, the present invention provides a method for preparing the biocontrol compound microbial agent, wherein the fermentation broth of marine-derived Bacillus belye and the fermentation broth of Bacillus belye 12Y are mixed at a volume ratio of 1:1 to obtain the biocontrol compound microbial agent.

[0031] The total concentration of the biocontrol compound microbial agent is 1×10⁻⁶. 7~9 CFU / mL.

[0032] In some embodiments of the present invention, the total concentration of the biocontrol compound microbial agent is 1×10⁻⁶. 8 CFU / mL.

[0033] In a seventh aspect, the present invention provides the application of the aforementioned biocontrol compound microbial agent in postharvest preservation of fruits and prevention and control of fungal diseases.

[0034] In some embodiments of the present invention, spraying a biocontrol compound microbial agent on the surface of the fruit enables post-harvest fruit preservation and fungal disease control.

[0035] Beneficial effects:

[0036] (1) This invention obtained a marine-derived Bacillus SCSX9 strain from the South China Sea through isolation and screening. This strain and its fermentation products (bacterial suspension, bacterial fermentation broth) and green biocontrol agents can effectively inhibit various pathogens and effectively prevent and control fungal diseases of strawberries and mangoes. In particular, it can effectively prevent and control strawberry gray mold, strawberry black spot, strawberry anthracnose and mango anthracnose, thereby safely and effectively controlling the occurrence and development of plant diseases.

[0037] (2) This invention provides a biocontrol compound microbial agent composed of marine-derived Bacillus belyssus SCSX9 and Bacillus belyssus 12Y. This biocontrol compound microbial agent can produce a variety of antibacterial substances. Utilizing its unique metabolic diversity and environmental adaptability, through the synergistic effect between strains, it significantly improves the broad spectrum of antibacterial activity, stability, and control efficiency, overcoming the limitations of single strains. It exhibits excellent synergistic antibacterial effects against pathogenic fungi such as Botrytis cinerea, Alternaria alternata, and Colletotrichum, with inhibition rates exceeding 65%.

[0038] (3) The biocontrol compound microbial agent described in this invention can form a stable biofilm on the fruit surface, enhancing the fruit's resistance and durability, and significantly reducing the incidence and disease index of postharvest fungal diseases in strawberries and mangoes. Simultaneously, through the synergistic effect of the strains, it can effectively control postharvest fungal diseases such as gray mold, black spot, and anthracnose in strawberries and anthracnose in mangoes, significantly extending the fruit's shelf life and storage time, improving the quality of strawberries and mangoes, and possessing high economic significance in agricultural production. Attached Figure Description

[0039] 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.

[0040] Figure 1 The results of the initial screening of the strain showed inhibition against different pathogens.

[0041] Figure 2 Morphological identification of marine-derived Bacillus cereus strain SCSX9.

[0042] Figure 3 The results show the inhibition of mycelial growth of the pathogen by marine-derived Bacillus belye SCSX9 bacterial suspension.

[0043] Figure 4 The results validate the affinity of different Bacillus species.

[0044] Figure 5 The results of plate validation of different biocontrol compound bacterial agents against pathogenic fungi.

[0045] Figure 6 The results show the effects of different postharvest treatments on the appearance quality of strawberries. CK1 was sprayed with only LB broth medium; CK2 received no treatment; T1 was sprayed with SCSX9 inoculant; T2 was sprayed with 12Y inoculant; and T3 was sprayed with a biocontrol compound inoculant SCSX9+12Y.

[0046] Figure 7 The results show the disease index, incidence rate, marketable rate, and weight loss rate of strawberries after different post-harvest treatment groups.

[0047] Figure 8 The results show the effects of different post-harvest treatments on the appearance quality of mangoes. CK1 was sprayed with only LB broth medium; CK2 received no treatment; T1 was sprayed with SCSX9 microbial agent; T2 was sprayed with 12Y microbial agent; and T3 was sprayed with a biocontrol compound microbial agent SCSX9+12Y.

[0048] Figure 9 The results show the disease index, morbidity rate, marketable rate, and weight loss rate of mangoes after different post-harvest treatment groups.

[0049] Preservation information:

[0050] A marine-derived Bacillus velezensis strain, classified as Bacillus velezensis with strain number SCSX9, was deposited on September 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35916. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0051] A strain of Bacillus velezensis 12Y, classified as Bacillus velezensis, was deposited on October 16, 2023, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 28636, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0052] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0053] The culture medium formulations used in the following examples are as follows:

[0054] LB solid medium (LB plate): 5 g yeast extract, 10 g tryptone, 10 g NaCl, 20 g agar, bring the volume to 1000 mL with deionized water, and autoclave at 121°C for 25 min.

[0055] LB liquid medium: 5 g yeast extract, 10 g tryptone, 10 g NaCl, bring the volume to 1000 mL with deionized water, and autoclave at 121 °C for 25 min.

[0056] PDA medium (PDA plate): 200 g potato, 20 g glucose, 20 g agar, deionized water to a final volume of 1000 mL, autoclave at 121°C for 20 min.

[0057] Example 1: Isolation and identification of marine-derived Bacillus belye SCSX9

[0058] 1. Isolation of strains

[0059] Marine mud sampling: The soil samples used in this experiment were collected from the South China Sea (depth 645 m, 115°49'E, 20°20'N), kindly provided by the Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences. They were stored in a plastic bag in the form of moist marine mud and kept in a refrigerator at 4 ℃.

[0060] Isolation of the bacterial strain: 10 g of South China Sea mud sample was added to a sterilized Erlenmeyer flask containing 90 mL of seawater and several glass beads. The flask was shaken at 37 ℃ and 160 r / min for 30 min, and the supernatant was collected. The sample was then serially diluted 10-fold to a final volume. -2 10 -3 10 -4 and 10 -5 For each concentration, 100 μL was applied evenly to LB solid medium using a sterile spreader until the surface was dry. The medium was then sealed with sealing film. Each concentration was repeated three times. The medium was incubated upside down in a 28 ℃ incubator. Colony growth was observed after 1–2 days, and continued for another 3–4 days to prevent the appearance of slow-growing colonies.

[0061] Purification of strains: Select single colonies with different morphologies, and purify each single colony three times by streaking on LB plates. Select strains based on different colony morphologies and assign numbers. At the same time, store them in 50% sterile glycerol tubes at -80℃ for later use.

[0062] 2. Screening of strain SCSX9

[0063] Activation of pathogens: Take mycelial cakes of Alternaria sp. FC.001, Colletotrichum gloeosporioides FC.002, Colletotrichum coffeanum FC.008, Botryosphaeria dothidea HT-01, Botrytis cinerea T8, and Fusarium proliferatum HT-04 and inoculate them into the center of PDA medium. Incubate at 28°C for 5-7 days for later use.

[0064] Initial screening: After culturing the pathogens in PDA medium for 7 days, several 6 mm mycelial cakes were created on Alternaria sp. FC.001, Colletotrichum gloeosporioides FC.002, Colletotrichum coffeanum FC.008, Botryosphaeria dothidea HT-01, and Botrytis cinerea T8 plates using a punch. One mycelial cake was inoculated into the center of each PDA plate. Then, the isolated and purified strains were streaked parallel to the pathogens on both sides (2 cm above and below the mycelial cake) using an inoculation needle. The plates were incubated at 28 ℃ for 5–7 days. The antibacterial activity of each plate was observed, and the results are as follows: Figure 1 As shown.

[0065] Secondary screening: Using *Alternaria sp.* FC.001, *Colletotrichum gloeosporioides* FC.002, *Colletotrichum coffeanum* FC.008, *Botryosphaeria dothidea* HT-01, and *Fusarium proliferatum* HT-04 as indicator bacteria, secondary screening was conducted on biocontrol bacteria that showed significant inhibitory effects in the initial screening. Using a sterile perforator, 6 mm diameter bacterial discs were taken from the edge of the pathogen agar plate and inoculated into the center of a new PDA agar plate. Biocontrol bacteria were inoculated around the four sides (2 cm from the center disc). Sterile pure water was used as a control. Each pathogen inoculation was performed in triplicate. The colony diameter of each group was measured, and the inhibition rate (%) was calculated. Each experiment was repeated in triplicate. A marine-derived biocontrol strain, SCSX9, was selected to inhibit gray mold and anthracnose.

[0066] 3. Identification of strain SCSX9

[0067] (1) Colony morphology identification: Strain SCSX9 was picked with an inoculation loop and streaked onto LB plates. After incubation at 37°C for 48 h, the results were observed as follows. Figure 2 As shown, the colonies of strain SCSX9 are opaque and pale yellow; the colonies are round and small; the surface is relatively smooth and the edges are neat. Under a microscope (400×), the bacterial cells are cylindrical, mostly straight, with a few slightly curved, and the ends of the bacterial cells are mostly blunt and rounded, which preliminarily conforms to the morphological characteristics of Bacillus. Morphological identification confirms that the strain belongs to the genus Bacillus.

[0068] (2) Molecular biological identification

[0069] The activated strain SCSX9 was sent to Qingke Biotechnology Co., Ltd., where its genome was extracted. Using SCSX9 as a template, the 16S rRNA gene fragment of strain SCSX9 was amplified using universal primer pairs 27F (5´-AGAGTTTGATCCTGGCTCAG-3´) and 1492R (5´-GGTTACCTTGTTACGACTT-3´). The 16S rRNA sequence of strain SCSX9 is shown in SEQ ID NO.1. Strain SCSX9 was sequenced using the 16S rRNA gene fragment as the target gene. BLAST alignment of the sequencing results showed that strain SCSX9 had the highest homology with Bacillus velezensis strain C-11. Combined with morphological identification, strain SCSX9 was confirmed as Bacillus velezensis.

[0070] The strain SCSX9 was deposited on September 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC), classified as Bacillus velezensis, with the strain number SCSX9, accession number CGMCC No. 35916, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0071] Example 2: Inhibitory effect of marine-derived Bacillus belye SCSX9 bacterial suspension on pathogenic mycelial growth

[0072] 1. Preparation of bacterial suspension

[0073] The bacterial discs of strain SCSX9 were inoculated into LB solid medium and cultured at 37 °C for 5–7 days. The surface of the medium was rinsed with sterile water to suspend strain SCSX9 in sterile water. The SCSX9 suspension was collected in centrifuge tubes, and the cell count was determined using a hemocytometer. The concentration of the SCSX9 suspension was adjusted to 1 × 10⁻⁶ cells / mL with sterile water. 6CFU / mL.

[0074] 2. Flat-panel standoff

[0075] Inoculate each pathogenic bacterial cell into a well in the center of the PDA medium, and take 2 µL of 1×10⁻⁶ bacterial culture medium. 6 CFU / mL SCSX9 bacterial suspension was inoculated at four symmetrical points 2.5 cm away from the bacterial cake, with sterile water added as a control. Each group was repeated in triplicate. The culture medium was placed in a constant temperature incubator at 28 ℃ for incubation.

[0076] The inhibition rate of SCSX9 bacterial suspension on pathogenic mycelial growth was calculated using the mycelial growth rate method. Inhibition rate (%) = (control colony growth diameter - treatment colony growth diameter) / control colony growth diameter × 100%.

[0077] The results are as follows Figure 3 As shown, SCSX9 bacterial suspension has a significant inhibitory effect on the mycelial growth of different pathogens. Calculations showed that SCSX9 significantly inhibited the growth of *Botrytis californica* TB.03, *Botrytis pelargonii* TB.02, *Botrytis pelargonii* TB.01, *Botrytis cinerea* T8, *Botrytis polyblastis* TJ.01, *Botrytis ficariarum* TJ.02, *Botrytis ranunculi* CH.02, *Botrytis ranunculi* CH.01, and *Botrytis... The mycelial growth inhibition rates of *Ranunculi* CH.03 were 45.90%, 53.60%, 52.10%, 45.16%, 54.7%, 50.0%, 34.6%, 44.9%, and 51.4%, respectively.

[0078] Example 3: Affinity verification of different Bacillus species

[0079] The affinity of eight *Bacillus belyceae* strains (SCSX2, SCSX6, SCSX9, SCSX23, NHE16, 12Y, XRD006, and SQR9) was tested using the agar diffusion method. Among them, strains SCSX2, SCSX6, and SCSX23 were obtained during the screening of strain SCSX9 in this invention; strains NHE16 and XRD006 were obtained from previous laboratory screenings; strain 12Y has the accession number CGMCC No. 28636, published in Chinese patent CN117660266A; and strain SQR9 has the accession number CGMCC No. 5808, published in Chinese patent CN103539535A. Figure 4 As shown, none of the eight strains of Bacillus belyssus formed inhibition zones, indicating that there was no mutual inhibition between the strains and that they had the potential for co-cultivation.

[0080] Example 4: Preparation of single-strain agents and biocontrol compound microbial agents

[0081] Single colonies of *Bacillus belyssus* SCSX2, SCSX6, SCSX9, SCSX23, NHE16, 12Y, XRD006, and SQR9 were picked from LB plates and inoculated into 50 mL of LB liquid medium. The culture was incubated at 37°C and 180 rpm for 12 h to obtain seed culture. The OD of the seed culture was adjusted with sterile water. 600 =1.0. The adjusted seed culture was inoculated into 750 mL LB liquid medium at an inoculation rate of 1% v / v, and fermented at 37℃ and 180 rpm for 72 h to obtain fermentation broths of 8 single strains, i.e., 8 single-strain agents. At this point, the viable cell count in each fermentation broth was approximately 1 × 10⁻⁶. 8 CFU / mL.

[0082] The two fermentation broths were mixed at a volume ratio of 1:1 to obtain a total of 13 compound bacterial fermentation broths, namely 13 marine-derived biocontrol compound bacterial agents.

[0083] Example 5: Verification Experiment of Marine-Derived Biocontrol Compound Microbial Agent on Anti-Pathogenic Fungi in Plates

[0084] 1. Test pathogens

[0085] Nigrospora sphaerica MIL2, Colletotrichum asianum MIL1, Colletotrichum sp. MIL6, Colletotrichum siamense MIL7, Alternaria alternata CM04, Colletotrichum gloeosporioides C1, Colletotrichum gloeosporioides C2, Botrytis cinerea T8, Colletotrichum siamense HT12.

[0086] 2. Validation experiment on resistance to pathogenic fungi

[0087] A 6 mm diameter pathogenic fungal disc was inoculated into the center of the PDA medium, and 2 μL of 1×10⁻⁶ bacteria were dropped around the disc at 2.5 cm intervals. 8 CFU / mL marine-derived biocontrol compound bacterial agent and two corresponding single bacterial agents were incubated at 28℃ for 6 days with sterile water as the control. The diameter of pathogen colonies was measured, and the inhibition rate was calculated. Inhibition rate (%) = (colon diameter of control group - colony diameter of treatment group) / colony diameter of control group × 100%.

[0088] The results are as follows Figure 5 As shown in Table 1, the plate diffusion experiment demonstrated that the biocontrol compound microbial agent possessed broad-spectrum antibacterial activity. Among them, only the biocontrol compound microbial agent SCSX9+12Y exhibited inhibition rates exceeding 65% against all tested pathogenic fungi. For example, it showed an inhibition rate of 76.80±1.23% against *Colletotrichum siamense* HT12, 79.64±1.11% against *Colletotrichum asianum* MIL1, 65.36±1.56% against *Botrytiscinerea* T8, and 65.92±2.29% against *Alternaria alternata* CM04. Compared to its own single-agent treatment, the biocontrol compound microbial agent SCSX9+12Y also showed excellent synergistic antibacterial effect, with the antibacterial rate increased by 3.75~21.20%, significantly improving the broad spectrum of antibacterial activity, stability and control efficiency.

[0089] In conclusion, the broad-spectrum antibacterial activity of the biocontrol compound microbial agent SCSX9+12Y is the best among all combinations, and this combination will be used for subsequent experiments.

[0090] Table 1. Antibacterial effects of different marine-derived biocontrol compound microbial agents

[0091]

[0092] Example 6: Study on the effect of biocontrol compound microbial agent SCSX9+12Y on postharvest preservation of strawberries

[0093] 1. Test materials

[0094] 120 "Hongyan" strawberries (Fragaria × ananassa Duch.) that were 7-8 ripe, uniform in size, and free from mechanical damage and pests were selected as experimental materials.

[0095] 2. Post-harvest treatment of strawberries

[0096] The effect of the biocontrol compound microbial agent SCSX9+12Y on postharvest preservation of strawberries was evaluated by spraying. Specifically, 120 strawberries were randomly divided into 5 treatment groups: T1 group: sprayed with SCSX9 microbial agent; T2 group: sprayed with 12Y microbial agent; T3 group: sprayed with the biocontrol compound microbial agent SCSX9+12Y; control group 1 (CK1): sprayed with LB broth medium only; control group 2 (CK2): no treatment. Each treatment group had three replicates, with 8 strawberries per replicate. The microbial agent (with a viable count of approximately 1×10⁻⁶) was used in each group. 8 (CFU / mL) was sprayed onto the surface of each group of strawberries using a spray bottle until the fermentation liquid dripped down. The strawberries were dried in a ventilated place at 20℃ and then packed into boxes. They were stored at 20℃ and 90% RH. The strawberries were observed and photographed daily, and the disease index, morbidity, marketability, shelf life, and weight loss were measured.

[0097] (1) Disease index: The diameter of the lesions on the fruit was determined by the cross-multiplication method. The diseased area was divided into 5 grades according to the percentage of the fruit surface area: Grade 0, no disease; Grade 1, 0.1~10.0%, slight disease; Grade 2, 10.1~25.0%, moderate disease; Grade 3, 25.1~50.0%, moderate to severe disease; Grade 4, 50.1~100%, severe disease.

[0098] Disease index = ∑ (disease level × number of fruits of the corresponding level) / (total number of fruits × highest disease level) × 100%.

[0099] (2) Disease incidence and marketability: Fruit rot means loss of marketability. Disease incidence and marketability of fruit were statistically analyzed at the sampling time.

[0100] Incidence rate (%) = (Number of rotten fruits / Total number of fruits) × 100%;

[0101] Product rate (%) = 1 - incidence rate.

[0102] (3) Storage life and weight loss rate: The number of days the fruit loses its marketability is the storage life of the fruit, and the weight loss rate is calculated according to the formula.

[0103] Weight loss rate (%) = (original weight of fruit - weight of fruit during storage) / (original weight of fruit) × 100%.

[0104] like Figure 6 As shown, group T3 exhibited superior biocontrol efficacy, in stark contrast to the control group. After 5 days of storage, over 90% of the strawberry samples in the control group were covered with a thick layer of white mold, and the red color faded significantly, indicating rapid infection by pathogens (such as gray mold). In contrast, the mold coverage in group T3 was significantly reduced, with only a few strawberry samples showing white mold, and most samples maintaining their intact shape and bright red sheen. Compared to groups T1 and T2, group T3 showed more uniform and thorough antibacterial activity, demonstrating the synergistic effect of the biocontrol compound microbial agent SCSX9+12Y, which effectively inhibits mold growth and is suitable for disease control in fruits such as strawberries, showing potential application in low-temperature and high-humidity environments.

[0105] like Figure 7 The results showed that the biocontrol compound microbial agent SCSX9+12Y significantly reduced the postharvest disease incidence and disease index of strawberries, inhibited fruit spoilage, maintained marketability, and reduced weight loss. On the 5th day of storage, the biocontrol compound microbial agent SCSX9+12Y reduced the disease incidence by 18.75% and the disease index by 17.19% compared to SCSX9; and reduced the disease incidence by 18.75% and the disease index by 14.06% compared to SCSX9+12Y. This combination showed outstanding performance in inhibiting strawberry weight loss, reducing it by 4.33% compared to the CK1 group.

[0106] Example 7: Study on the postharvest preservation effect of biocontrol compound microbial agent SCSX9+12Y on mangoes

[0107] 1. Test materials

[0108] 120 "Tainong" mangoes (Mangifernindica L.) that were 7-8 ripe, uniform in size, and free from mechanical damage and pests were selected as experimental materials.

[0109] 2. Post-harvest processing of mangoes

[0110] The effect of the biocontrol compound microbial agent SCSX9+12Y on postharvest preservation of mangoes was evaluated by spraying. Specifically, 120 mangoes were randomly divided into 5 treatment groups: T1 group: sprayed with SCSX9 microbial agent; T2 group: sprayed with 12Y microbial agent; T3 group: sprayed with the biocontrol compound microbial agent SCSX9+12Y; control group 1 (CK1): sprayed with LB broth medium only; control group 2 (CK2): no treatment. Each treatment group had three replicates, with 8 mangoes per replicate. The microbial agents (with a viable count of approximately 1×10⁻⁶) were used in each group. 8 (CFU / mL) was sprayed onto the surface of each group of mangoes using a spray bottle until the fermentation liquid dripped down. The mangoes were dried in a ventilated place at 20℃ and then packed into boxes. They were stored at 20℃ and 90% RH. The mangoes were observed and photographed daily, and the disease index, morbidity, marketability, shelf life, and weight loss were measured.

[0111] like Figure 8 As shown, group T3 exhibited a significant biocontrol effect, in stark contrast to the control group. In the control group, over 90% of the fruit was covered with black lesions of varying sizes, with severe lesion spread, faded yellow peel, and deformed shape, indicating rapid infection by pathogens (such as anthracnose). Group T3 showed almost no visible lesion coverage, with only a few fruits exhibiting faint spots on the edges, maintaining a bright yellow sheen, clean surface, and intact shape. Compared to groups T1 and T2, group T3 showed more uniform and thorough inhibition of the bacteria, with no obvious infection points. This demonstrates that the biocontrol compound microbial agent SCSX9+12Y formulation has a significant synergistic effect, effectively inhibiting the germination and spread of pathogenic spores.

[0112] like Figure 9 The results showed that the biocontrol compound microbial agent SCSX9+12Y significantly reduced the postharvest disease incidence and disease index of mangoes, inhibited fruit decay, maintained marketability, and reduced weight loss. At day 10 of storage, the biocontrol compound microbial agent SCSX9+12Y reduced the disease incidence by 25% and the disease index by 10.94% compared to SCSX9; and reduced the disease incidence by 25% and the disease index by 11.5% compared to SCSX9+12Y. The application of this combination to mangoes significantly improved the marketability of mangoes, increasing it by 62.5% compared to the control group (CK1).

[0113] This invention provides a marine-derived Bacillus belye SCSX9 strain, a biocontrol compound bacterial agent, and its preparation and application 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 biocontrol compound microbial agent, characterized in that, Marine-derived Bacillus bailesi ( Bacillus velezensis The fermentation broth of Bacillus vesiculosus and Bacillus belesi ( Bacillus velezensis The biocontrol compound microbial agent is obtained by mixing the fermentation broth of 12Y cells at a volume ratio of 1:

1. Among them, the marine-derived Bacillus belyes is classified and named Bacillus belyes. Bacillus velezensis The strain number is SCSX9, which was deposited on September 15, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35916. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The Bacillus belyssus 12Y has the accession number CGMCC No. 28636.

2. The preparation method of the biocontrol compound microbial agent according to claim 1, characterized in that, The biocontrol compound microbial agent is obtained by mixing the fermentation broth of marine-derived Bacillus vesicularis and the fermentation broth of Bacillus vesicularis 12Y at a volume ratio of 1:

1.

3. The preparation method according to claim 2, characterized in that, The total concentration of the compound microbial agent is 1×10⁻⁶. 7 CFU / mL ~ 1×10 9 CFU / mL.

4. The application of the biocontrol compound microbial agent according to claim 1 in postharvest preservation of fruit or prevention and control of fungal diseases; in, The fruit is strawberry and / or mango; the fungal disease is any one or a combination of several of the following: strawberry gray mold, strawberry black spot, strawberry anthracnose, and mango anthracnose.

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