Bacillus velezensis SCSX23 and fruit and vegetable postharvest biocontrol composite microbial agent and application thereof

Abstract

The present application relates to the technical field of microbial prevention and treatment, and particularly relates to a bacillus velezensis SCSX23, a fruit and vegetable postharvest biocontrol composite microbial agent and application thereof. The present application screens a bacillus velezensis SCSX23 from South China Sea mud, and is preserved in the China General Microbiological Culture Collection Center on September 15, 2025, with a preservation number of CGMCC No. 35917. The microbial agent prepared by using the strain can effectively inhibit various pathogenic bacteria, not only expands the inhibition spectrum, but also prolongs the shelf life of fruits and vegetables through the synergistic stability of spores, and has important practical significance and application value for the prevention and treatment of postharvest fungal diseases of fruits and vegetables such as strawberries and mangoes and the preservation of postharvest fruits and vegetables.

Description

Technical Field

[0001] This invention relates to the field of microbial control technology, specifically to a strain of Bacillus berleis SCSX23, a postharvest biocontrol compound microbial agent for fruits and vegetables, and its application. Background Technology

[0002] Strawberries and mangoes, as popular and high-value fruits, are highly susceptible to spoilage and deterioration during post-harvest storage and transportation, resulting in significant economic losses. Strawberries have fragile skins and soft, juicy flesh, making them highly vulnerable to mechanical damage, wilting due to dehydration, and microbial infection after harvest. Mangoes, on the other hand, are susceptible to various post-harvest diseases, with anthracnose-induced rot accounting for 20-30% of total losses, severely impacting their quality during storage.

[0003] Postharvest diseases are one of the main causes of postharvest losses in strawberries and mangoes. Among them, anthracnose, caused by fungi of the genus *Colletotrichum* spp., is one of the most destructive postharvest diseases. In addition, *Botrytis cinerea*, *Alternaria*, and other pathogenic fungi are also major causes of postharvest diseases in fruits.

[0004] Currently, postharvest disease control for fruits such as strawberries and mangoes still mainly relies on chemical fungicides, such as benzimidazole and triazole fungicides. However, long-term use of chemical substances can easily lead to increased resistance in pathogens and may cause problems such as pesticide residues, environmental pollution, and food safety risks. With the increasing awareness of food safety and environmental protection, developing efficient, safe, and environmentally friendly methods for the prevention and control of postharvest diseases in fruits and vegetables has become an urgent need for the industry.

[0005] Biological control technology, due to its environmental compatibility, lack of residue, and low likelihood of inducing resistance, is considered an important direction for replacing or supplementing chemical control. Among these, Bacillus bacteria, with their ability to produce various antimicrobial substances and form highly resistant spores, are becoming a hot topic in biocontrol research. However, single-strain biocontrol agents often suffer from unstable control effects, narrow antimicrobial spectrum, and poor persistence. In contrast, compound microbial agents, through the synergistic effect between strains, promise to achieve functional complementarity, broaden the antimicrobial spectrum, enhance control efficacy and stability, and may also possess the additional function of promoting plant growth.

[0006] Current methods for controlling fungal diseases such as anthracnose using postharvest compound biocontrol agents employ a combination of Bacillus and yeast, but these methods generally suffer from limitations such as limited inhibitory spectrum and short shelf life of fruits and vegetables. Therefore, developing a highly effective compound biocontrol agent is of significant practical importance and application value for the control of postharvest anthracnose in strawberries and mangoes. Summary of the Invention

[0007] The technical problem to be solved by the present invention is that chemical fungicides in the prior art can easily lead to increased resistance of pathogens and may bring about problems such as pesticide residues, environmental pollution and food safety risks; the fungicide control effect is unstable, the antibacterial spectrum is narrow and the persistence is poor. The present invention provides a strain of Bacillus belyssus SCSX23.

[0008] Another technical problem to be solved by the present invention is to provide the aforementioned biocontrol agent.

[0009] Another technical problem to be solved by the present invention is to provide the application of the Bacillus berleis SCSX23 or biocontrol agent in inhibiting pathogenic fungi.

[0010] Another technical problem to be solved by the present invention is to provide a postharvest biocontrol compound microbial agent for fruits and vegetables.

[0011] The final technical problem to be solved by this invention is to provide the application of the postharvest biocontrol compound microbial agent for fruits and vegetables in inhibiting pathogenic fungi and promoting the preservation of harvested fruits and vegetables.

[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 strain of Bacillus velezensis SCSX23, classified and named Bacillus velezensis, strain number SCSX23, which was deposited on September 15, 2025 at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 35917, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0014] The Bacillus belyssus SCSX23 was obtained from marine mud samples collected in the South China Sea (depth 645 m, 115°49'E, 20°20'N).

[0015] Among them, the colonies of *Bacillus belye* SCSX23 are small, opaque, pale yellow, round colonies with a relatively smooth surface and neat edges. Under a microscope (400×), the bacterial cells are cylindrical, mostly straight, with a few slightly curved, and most ends are blunt and rounded.

[0016] The 16S rRNA sequence of the Bacillus belyssus SCSX23 is shown in SEQ ID NO.1.

[0017] Secondly, the present invention provides a biocontrol agent containing the aforementioned Bacillus berleis SCSX23.

[0018] In some embodiments of the present invention, the biocontrol agent is the fermentation broth of Bacillus belye SCSX23, with a viable count of approximately 1 × 10⁻⁶. 8 CFU / mL.

[0019] Thirdly, the present invention provides the application of the Bacillus berleis SCSX23 or the biocontrol agent in inhibiting pathogenic fungi.

[0020] The pathogenic fungi include any one or a combination of several of the genera *Alternaria* sp., *Colletotrichum* sp., *Botrytis* sp., *Botryosphaeria* sp., and *Nigrosporasp.*.

[0021] Specifically, the genus *Alternaria* sp. refers to *Alternaria alternata*.

[0022] Specifically, the genus *Colletotrichum* sp. includes any one or a combination of several of the following: *Colletotrichum gloeosporioides*, *Colletotrichum coffeanum*, *Colletotrichum siamense*, and *Colletotrichum asianum*.

[0023] Specifically, the Botrytis sp. genus includes any one or a combination of several of the following: Botrytis californica, Botrytis pelargonii, Botrytis cinerea, Botrytis polyblastis, Botrytis ficariarum, and Botrytis ranunculi.

[0024] Specifically, the genus Botryosphaeria sp. includes Botryosphaeria dothidea.

[0025] Specifically, the genus *Nigrospora* sp. includes *Nigrospora sphaerica*.

[0026] In some embodiments of the present invention, the Alternaria sp. is Alternariaspecies FC.001 or Alternaria alternata CM04.

[0027] In some embodiments of the present invention, the anthrax genus Colletotrichum sp. includes any one or a combination of several of the following: Colletotrichum gloeosporioides FC.002, C1, C2; Colletotrichum coffeanum FC.008; Colletotrichum siamense HT12, MIL7; Colletotrichum asianum MIL1; and Colletotrichum sp. MIL6.

[0028] In some embodiments of the present invention, the *Botrytis* sp. includes any one or a combination of several of the following: *Botrytis californica* TB.03; *Botrytis pelargonii* TB.02 and TB.01; *Botrytis cinerea* T8; *Botrytis polyblastis* TJ.01; *Botrytis ficariarum* TJ.02; and *Botrytis ranunculi* CH.02 and CH.01.

[0029] In some embodiments of the present invention, the Botryosphaeria sp. includes Botryosphaeria dothidea HT-01.

[0030] In some embodiments of the present invention, the *Nigrospora* sp. includes *Nigrospora sphaerica* MIL2.

[0031] The inhibition of pathogenic fungi refers to inhibiting the growth of pathogenic fungi and / or destroying the hyphal morphology of pathogenic fungi.

[0032] Fourthly, the present invention provides a postharvest biocontrol compound microbial agent for fruits and vegetables, containing the aforementioned Bacillus berleis SCSX23 and Bacillus berleis SQR9.

[0033] The Bacillus belyssus SQR9 strain was donated by the team of Academician Shen Qirong of Nanjing Agricultural University. Its accession number is CGMCC NO.5808. Detailed information about this strain has been published in Chinese patent CN103539535B.

[0034] Among them, Bacillus belyssus SCSX23 and Bacillus belyssus SQR9 underwent affinity testing, and the results are as follows: Figure 4 As shown, the two have good affinity and no mutual inhibition.

[0035] The total concentration of the postharvest biocontrol compound microbial agent for fruits and vegetables is 1×10⁻⁶. 7~9 CFU / mL.

[0036] The postharvest biocontrol compound microbial agent for fruits and vegetables is prepared by fermentation broth of Bacillus vesiliflorus SCSX23 and fermentation broth of Bacillus vesiliflorus SQR9 in a volume ratio of 1:1.

[0037] In some embodiments of the present invention, the specific preparation method of the postharvest biocontrol compound microbial agent for fruits and vegetables is as follows:

[0038] (1) Activation of strains: Bacillus belyssus SCSX23 and SQR9 were inoculated into LB solid medium and cultured at 37°C for 24 h to activate the strains.

[0039] (2) Seed culture preparation: single colonies were picked and inoculated into LB liquid medium and cultured at 37℃ and 180 rpm for 12 h to obtain seed culture;

[0040] (3) Fermentation culture: Adjust the OD of the seed culture with sterile water. 600 After reaching a concentration of 1.0, the culture medium was inoculated at a rate of 1% v / v into LB liquid medium and fermented for 72 h. At this point, the viable cell count in the fermentation broth was approximately 1 × 10⁻⁶. 8 CFU / mL;

[0041] (4) Preparation of biocontrol compound microbial agent: Mix the fermentation broth of the two strains at a volume ratio of 1:1 and stir thoroughly to obtain the postharvest biocontrol compound microbial agent SCSX23+SQR9 ​​for fruits and vegetables.

[0042] Fifthly, the present invention provides the application of the postharvest biocontrol compound microbial agent for fruits and vegetables in inhibiting pathogenic fungi and promoting the preservation of postharvest fruits.

[0043] The pathogenic fungi include any one or a combination of several of the genera *Alternaria* sp., *Colletotrichum* sp., *Botrytis* sp., *Botryosphaeria* sp., and *Nigrosporasp.*.

[0044] Specifically, the genus *Alternaria* sp. refers to *Alternaria alternata*.

[0045] Specifically, the genus *Colletotrichum* sp. includes any one or a combination of several of the following: *Colletotrichum gloeosporioides*, *Colletotrichum coffeanum*, *Colletotrichum siamense*, and *Colletotrichum asianum*.

[0046] Specifically, the Botrytis sp. genus includes any one or a combination of several of the following: Botrytis californica, Botrytis pelargonii, Botrytis cinerea, Botrytis polyblastis, Botrytis ficariarum, and Botrytis ranunculi.

[0047] Specifically, the genus Botryosphaeria sp. includes Botryosphaeria dothidea.

[0048] Specifically, the genus *Nigrospora* sp. includes *Nigrospora sphaerica*.

[0049] In some embodiments of the present invention, the Alternaria sp. is Alternariaspecies FC.001 or Alternaria alternata CM04.

[0050] In some embodiments of the present invention, the anthrax genus Colletotrichum sp. includes any one or a combination of several of the following: Colletotrichum gloeosporioides FC.002, C1, C2; Colletotrichum coffeanum FC.008; Colletotrichum siamense HT12, MIL7; Colletotrichum asianum MIL1; and Colletotrichum sp. MIL6.

[0051] In some embodiments of the present invention, the *Botrytis* sp. includes any one or a combination of several of the following: *Botrytis californica* TB.03; *Botrytis pelargonii* TB.02 and TB.01; *Botrytis cinerea* T8; *Botrytis polyblastis* TJ.01; *Botrytis ficariarum* TJ.02; and *Botrytis ranunculi* CH.02 and CH.01.

[0052] In some embodiments of the present invention, the Botryosphaeria sp. includes Botryosphaeria dothidea HT-01.

[0053] In some embodiments of the present invention, the *Nigrospora* sp. includes *Nigrospora sphaerica* MIL2.

[0054] The fruits and / or vegetables mentioned are those susceptible to pathogenic fungi such as anthracnose and gray mold.

[0055] In some embodiments of the present invention, the fruits and vegetables are strawberries and / or mangoes.

[0056] In some embodiments of the present invention, the postharvest biocontrol compound microbial agent for fruits and vegetables is sprayed onto the surface of fruits and vegetables, and then stored after being ventilated and dried at 20-25°C for 1-2 hours.

[0057] Beneficial effects:

[0058] (1) The present invention isolated a strain of Bacillus belye SCSX23 from the sea mud of the South China Sea. This strain has an inhibitory effect on the growth of different pathogens and has broad-spectrum antibacterial activity. At the same time, it can inhibit the normal growth of pathogens by destroying the normal morphology of pathogen hyphae, causing the hyphal tips to swell or lyse, and local areas to show soluble damage and breakage.

[0059] (2) The present invention successfully developed a highly efficient, safe and stable biocontrol compound microbial agent SCSX23+SQR9. Through a large number of experiments, screening and verification, it was found that Bacillus belye SCSX23 and SQR9, when combined in a specific ratio, showed a significant synergistic inhibitory effect on various pathogens, expanded the antibacterial spectrum, and its control effect was significantly better than that of single microbial agent treatment, and it had good application stability.

[0060] (3) The biocontrol compound microbial agent prepared by the present invention not only significantly inhibits the growth of various pathogens such as anthracnose, effectively controls postharvest fungal diseases of strawberries and mangoes, but also effectively maintains the appearance quality and marketability of strawberries and mangoes, prolongs the shelf life of fruits and vegetables, reduces the fruit weight loss rate of strawberries and mangoes, prolongs storage life and freshness, reduces postharvest losses, and promotes the growth of strawberries and mangoes. It provides new control agents and postharvest fruit and vegetable preservation agents for the biological control of postharvest fungal diseases of fruits and vegetables.

[0061] (4) The biocontrol compound microbial agent prepared by this invention meets the requirements of green agriculture and sustainable development, and provides an effective technical means to solve the problems of post-harvest preservation of fruits and vegetables and prevention and control of fungal diseases. It has important application value and market prospects. Attached Figure Description

[0062] 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.

[0063] Figure 1 This is a colony morphology diagram of strain SCSX23 on LB agar plates.

[0064] Figure 2 The figure shows the broad-spectrum antibacterial activity of strain SCSX23 against different pathogens. In the figure, S23 refers to SCSX23.

[0065] Figure 3 The effect of strain SCSX23 on the mycelial growth of different pathogens.

[0066] Figure 4 The results show the affinity test results for strains SCSX23 and SQR9.

[0067] Figure 5The antibacterial effect of the postharvest biocontrol compound microbial agent SCSX23+SQR9 ​​on different pathogens in fruits and vegetables.

[0068] Figure 6 The effect of the postharvest biocontrol compound microbial agent SCSX23+SQR9 ​​on the preservation of strawberries.

[0069] Figure 7 The effects of the postharvest biocontrol compound microbial agent SCSX23+SQR9 ​​on the disease index, disease incidence, marketability, and weight loss of strawberries after harvest.

[0070] Figure 8 The effect of the postharvest biocontrol compound microbial agent SCSX23+SQR9 ​​on the preservation of mangoes.

[0071] Figure 9 The effects of the postharvest biocontrol compound microbial agent SCSX23+SQR9 ​​on the disease index, disease incidence, marketability, and weight loss of mangoes after harvest.

[0072] Preservation information:

[0073] A strain of Bacillus velezensis, SCSX23, was deposited on September 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 35917. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0074] A strain of Bacillus velezensis, SQR9, was deposited on February 27, 2012, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 5808, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. This strain was donated by the team of Academician Shen Qirong of Nanjing Agricultural University, and detailed information is published in Chinese Patent CN103539535B. Detailed Implementation

[0075] The present invention will be further described in detail below with reference to specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0076] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0077] Example 1: Isolation, purification and identification of Bacillus belyssus SCSX23

[0078] 1. Isolation of strains

[0079] Marine mud samples collected from the South China Sea (depth 645 m, 115°49'E, 20°20'N), courtesy of the Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences, were stored in a plastic bag as moist mud at 4 ℃. 10 g of the South China Sea mud sample was added to a sterilized Erlenmeyer flask containing 90 mL of aged seawater and several glass beads. The flask was shaken at 37 ℃ and 160 rpm for 30 min on a shaker, and the supernatant was collected; this was the 10 g sample. -1 Diluent. Using a pipette, pipette 1 mL of this diluent and add it to a sterilized 50 mL centrifuge tube containing 9 mL of aged seawater. Mix well by pipetting and aspirating. (10 mL) -2 Diluent, and so on, to prepare 10 -3 10 -4 and 10 -5 Diluent. Using a pipette, pipette 100 μL of 1×10⁻⁶ solution. -3 1×10 -4 1×10 -5 Add the diluent to LB medium, spread evenly with a sterile spreader until the surface is dry, seal with sealing film, and incubate upside down in a 28°C incubator. Observe colony growth after 1-2 days, and continue to observe for 3-4 days to prevent the appearance of slow-growing colonies. Pick single colonies with different culture characteristics using an inoculation loop, and purify them three times by streak plating on PDA and LB plates to obtain pure strains.

[0080] 2. Screening of strain SCSX23

[0081] Using *Alternaria sp.* FC.001, *Colletotrichum gloeosporioides* FC.002, *Colletotrichum coffeanum* FC.008, *Botryosphaeria dothidea* HT-01, and *Botrytis cinerea* T8 as indicator bacteria, the biocontrol potential of isolated pure strains was evaluated using a confrontation culture method. Specifically, mycelial discs of each pathogen were inoculated into the center of PDA medium and incubated at 28°C for 7 days to activate the pathogens. Several mycelial discs with a diameter of 6 mm were prepared on pathogen plates using a punch. Fresh PDA medium was used, and a hole was punched in the center of the plate to remove an agar block and inoculate it with a mycelial disc. The strains to be screened were streaked at equidistant points 2 cm away from the mycelial discs and incubated at 28°C for 5–7 days. Using culture plates inoculated with pathogens alone as control samples, when the control group colonies completely covered the surface of the culture medium, the average distance between the pathogen colony and the edge of the test strain was measured using vernier calipers. The inhibition rate was calculated to evaluate the inhibition effect, and a strain that showed good inhibitory effects against five pathogens was screened and named SCSX23.

[0082] 3. Identification of strain SCSX23

[0083] (1) Morphological observation: Strawberry strain SCSX23 was picked up with an inoculation loop and streaked onto LB plates. After incubation at 37°C for 48 h, the results were observed as follows. Figure 1 As shown, the colonies are small, opaque, pale yellow, and round, with a relatively smooth surface and neat edges. Under a microscope (400×), the bacterial cells are cylindrical, mostly straight, with a few slightly curved, and mostly blunt and rounded ends, consistent with the typical morphological characteristics of the genus Bacillus. Based on preliminary morphological identification, this strain belongs to the genus Bacillus.

[0084] (2) Molecular biological identification

[0085] The activated strain SCSX23 was sent to Qingke Biotechnology Co., Ltd., where its genome was extracted. Using SCSX23 as a template, the 16S rRNA gene fragment of strain SCSX39 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 SCSX23 was sequenced using the 16S rRNA gene fragment as the target gene. Blast alignment of the sequencing results showed that strain SCSX23 had the highest homology with *Bacillus velezensis* strain X-AB-002 / 0. Combined with morphological identification, strain SCSX23 was confirmed as *Bacillus velezensis*.

[0086] Strain SCSX23 was deposited on September 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC), classified as Bacillus velezensis, with strain number SCSX23, accession number CGMCC No. 35917, and deposited at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0087] Example 2: Determination of broad-spectrum antibacterial activity of Bacillus belyssus SCSX23

[0088] Using *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 *Colletotrichum siamense* HT12 as indicator fungi, the antibacterial activity of the isolated strain SCSX23 against the pathogen was investigated using the plate confrontation method. Using a sterile punch, 6 mm diameter bacterial discs were inoculated from the edge of a pathogen-containing agar plate and transferred to the center of a new PDA plate. The biocontrol bacteria to be tested were then inoculated along all four sides (2 cm from the center) using a cross-cutting method. One side was left uninoculated as a control. The plates were incubated at 28 °C for 6 days. The colony diameter of each group was measured, and the inhibition rate was calculated to evaluate the inhibitory effect. Furthermore, mycelia from the interface between the biocontrol bacteria and the pathogen were collected and prepared into slides. The growth status of the pathogen was observed under a microscope to assess the morphological impact of the biocontrol bacteria on the pathogen.

[0089] The results are as follows Figure 2 As shown, strain SCSX23 inhibited the growth of various pathogens, indicating its broad-spectrum antibacterial activity. The best inhibitory effects were observed against HT12, TB.03, and TJ.02, with inhibition rates of 61.4%, 57.1%, and 56.6%, respectively. When SCSX23 colonies were cultured simultaneously with pathogen colonies, the pathogen colonies ceased growth and spread due to the presence of SCSX23 colonies. Figure 3 As shown, the hyphae of all nine pathogens were damaged to some extent, with swelling or rupture at the tips of the hyphae, and local areas showing soluble damage and breakage. This indicates that strain SCSX23 can destroy the normal morphology of pathogen hyphae and inhibit their normal growth.

[0090] Example 3: Preparation of a postharvest biocontrol compound microbial agent for fruits and vegetables

[0091] Single colonies of *Bacillus belyceae* SCSX23 and other *Bacillus belyceae* species, including SQR9 (given by Academician Shen Qirong's team at Nanjing Agricultural University, accession number CGMCC No. 5808), SCSX2 (screwed in this invention), SCSX6 (screwed in this invention), SCSX9 (screwed in this invention, accession number CGMCC No. 35916), NHE16 (screwed in previous laboratory screening), 12Y (screwed in CGMCC No. 28636), and XRD006 (screwed in previous laboratory screening), were picked from LB agar plates and inoculated into 50 mL LB liquid medium. The cultures were 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 After reaching a concentration of 1.0, various cell cultures were inoculated into 750 mL of LB liquid medium at an inoculation rate of 1% v / v. Fermentation was carried out at 37°C and 180 rpm for 72 h to obtain 8 single-strain fermentation broths (i.e., 8 single-strain agents). At this point, the viable cell count in the fermentation broth was approximately 1 × 10⁻⁶. 8 CFU / mL.

[0092] The fermentation broth of strain SCSX23 was mixed with the fermentation broth of several other strains at a volume ratio of 1:1 to obtain seven kinds of postharvest biocontrol compound microbial agents for fruits and vegetables (SCSX23+SQR9, SCSX23+SCSX2, SCSX23+SCSX6, SCSX23+SCSX9, SCSX23+NHE16, SCSX23+12Y, SCSX23+XRD006).

[0093] Example 4: Determination of the antibacterial effect of postharvest biocontrol compound microbial agent for fruits and vegetables using the plate confrontation method.

[0094] 1. Test pathogens

[0095] Nigrospora sphaerica MIL2, Colletotrichum asianum MIL1, Colletotrichum sp. MIL6, Alternaria alternata CM04, Colletotrichum siamense MIL7, Colletotrichum siamense HT12, Colletotrichum gloeosporioides C1, Colletotrichum gloeosporioides C2, Botrytis cinerea T8.

[0096] 2. Antibacterial test

[0097] A 6 mm diameter pathogenic bacterial cake was inoculated into the center of a PDA culture medium. Two μL of a postharvest biocontrol compound for fruits and vegetables and two corresponding single-strain agents were added dropwise at 2.5 cm around the cake. Sterile water was used as a control (CK). After incubation at 28℃ for 6 days, the diameter of the pathogenic bacterial colonies was measured, and the inhibition rate was calculated. Inhibition rate (%) = (Coronary diameter of control group - Colony diameter of treatment group) / Colony diameter of control group × 100%.

[0098] Plate diffusion experiments (Table 1) showed that the inhibition rates of the postharvest biocontrol compound microbial agent SCSX23+SQR9 ​​against several pathogens were significantly higher than those of single-agent treatments (p<0.05). Compared with single-agent treatments, the inhibition rate of SCSX23+SQR9 ​​increased by 7.51–27.26%, demonstrating broad-spectrum and stable antibacterial activity. Furthermore, SCSX23+SQR9 ​​exhibited the best broad-spectrum antibacterial activity among all combinations, achieving an average inhibition rate of 74.42% against the nine tested pathogens, with a synergistic antibacterial effect far exceeding that of other combinations. Among them, the SCSX23+SQR9 ​​postharvest biocontrol compound for fruits and vegetables showed an inhibition rate of 77.78±1.37% against Colletotrichum siamense HT12, 79.83±0.30% against Colletotrichum asianum MIL1, 68.40±2.67% against Botrytis cinerea T8, 69.72±6.25% against Alternaria alternata CM04, and an even higher inhibition rate of 87.36±0.81% against Nigrospora sphaerica MIL2. Figure 5 The study demonstrated the antibacterial effects of the postharvest biocontrol compound microbial agent SCSX23+SQR9 ​​on different pathogens in fruits and vegetables.

[0099] Table 1. Antibacterial effects of different postharvest biocontrol compound microbial agents for fruits and vegetables

[0100]

[0101] Example 5: Application of the postharvest biocontrol compound microbial agent SCSX23+SQR9 ​​on the postharvest preservation effect of strawberries

[0102] On April 19, 2025, “Hongyan” strawberries (Fragaria × ananassa Duch.) that were 7-8 ripe, uniform in size, and free from mechanical damage and pests were purchased from Shenmin Fruit Store at the Binjiang Campus of Nanjing Agricultural University and transported to the Agricultural Product Storage and Processing Laboratory of the College of Food Science and Technology of Nanjing Agricultural University within 2 hours.

[0103] Strawberries were randomly divided into 5 groups (3 replicates per group, 8 strawberry fruits per replicate): CK1 group: sprayed with LB broth medium; CK2 group: no treatment; T1 group: sprayed with SCSX23 inoculant; T2 group: sprayed with SQR9 inoculant; T3 group: sprayed with a postharvest biocontrol compound inoculant SCSX23+SQR9. The inoculants (approximately 1×10⁻⁶ viable bacteria) were sprayed onto each group using a spray bottle. 8 (CFU / mL) was sprayed onto the surface of the strawberries in each group 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.

[0104] (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.

[0105] Disease index = ∑ (disease level × number of fruits of the corresponding level) / (total number of fruits × highest disease level) × 100%.

[0106] (2) Disease incidence and marketability: Fruit rot means loss of marketability. Disease incidence and marketability of fruit were statistically analyzed at the sampling time.

[0107] Incidence rate (%) = (Number of rotten fruits / Total number of fruits) × 100%;

[0108] Product rate (%) = 1 - incidence rate.

[0109] (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.

[0110] Weight loss rate (%) = (original weight of fruit - weight of fruit during storage) / (original weight of fruit) × 100%.

[0111] The results are as follows Figure 6 As shown, after 4 days of storage, strawberries in groups CK1 and CK2 showed obvious lesions (3-5 mm in diameter) and the flesh was soft and rotten; the number of lesions in group T3 decreased, and only 31.25% of the fruit lost its marketability, while the appearance remained good. Figure 7The results showed that after 5 days of storage, the disease index in group T3 was 28.12% lower than that in group T1 and 65.62% lower than that in group CK1; the incidence rate was 31.25% lower than that in group T1 and 62.5% lower than that in group CK1; the marketability rate was 31.25% higher than that in group T1 and 62.5% higher than that in group CK1; and the weight loss rate was 0.29% lower than that in group T1 and 4.28% lower than that in group CK1. When the fruit loses its marketability, it means the storage period has ended. Therefore, strawberry fruit treated with postharvest biocontrol compound microbial agents has the longest shelf life.

[0112] Example 6: Application of the postharvest biocontrol compound microbial agent SCSX23+SQR9 ​​on the postharvest preservation effect of mangoes

[0113] On April 19, 2025, “Tainong” mangoes (Mangifern indica L.) that were 7-8 ripe, uniform in size, and free from mechanical damage and pests were purchased from Shenmin Fruit Store at the Binjiang Campus of Nanjing Agricultural University and transported to the Tropical Fruit and Vegetable Storage and Processing Laboratory of the College of Food Science and Technology of Nanjing Agricultural University within 2 hours.

[0114] Mangoes were randomly divided into 5 groups (3 replicates per group, 8 fruits per replicate), treated in the same manner as in Example 5, and stored at 25°C and 85% RH. Relevant indicators were measured every 2 days.

[0115] The results are as follows Figure 8 As shown, after 10 days of storage, large areas of dark brown lesions began to appear on the skin of mangoes in groups CK1 and CK2, and the flesh rotted; the lesions in group T1 expanded more slowly, but the marketable rate was only 75%; the mangoes in group T3 had fewer lesions on their skin and the flesh was firm. Figure 9 The results showed that after 10 days of storage, the disease index in group T3 was 12.5% ​​lower than that in group T1 and 21.88% lower than that in group CK1; the incidence rate was 25% lower than that in group T1 and 50% lower than that in group CK1; the marketability rate was 25% higher than that in group T1 and 50% higher than that in group CK1; and the weight loss rate was 0.03% lower than that in group T1 and 0.55% lower than that in group CK1. When the fruit loses its marketability, it means that the storage period has ended. It can be seen that mango fruit treated with the postharvest biocontrol compound microbial agent SCSX23+SQR9 ​​has the longest shelf life.

[0116] This invention provides a strain of Bacillus belyceta SCSX23, a postharvest biocontrol compound microbial agent for fruits and vegetables, 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 postharvest biocontrol compound microbial agent for fruits and vegetables, characterized in that, The postharvest biocontrol compound microbial agent for fruits and vegetables was prepared from the fermentation broth of Bacillus bellis SCSX23 and Bacillus bellis SQR9 at a volume ratio of 1:1; the total concentration of the postharvest biocontrol compound microbial agent for fruits and vegetables was 1×10⁻⁶. 7 ~1×10 9 CFU / mL; Among them, the *Bacillus belyes* SCSX23 is classified and named *Bacillus belyes* (… Bacillus velezensis The strain, SCSX23, was deposited on September 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35917. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The preservation number of the Bacillus belyssus SQR9 is CGMCC No. 5808.

2. The application of the postharvest biocontrol compound microbial agent for fruits and vegetables as described in claim 1 in inhibiting pathogenic fungi and promoting the preservation of harvested fruits and vegetables; wherein, The fruits and vegetables mentioned are strawberries and / or mangoes; The pathogenic fungus is Alternaria. Alternaria sp. Anthrax genus Colletotrichum sp. Botrytis cinerea Botrytis sp. Staphylococcus spp. Botryosphaeria sp. genus *Ulva* Nigrospora sp. Any combination of one or more of them; Among them, the Alternaria species Alternaria sp. Alternaria Alternaria alternata ; Anthrax bacteria Colletotrichum sp. Colloidal anthrax bacteria Colletotrichum gloeosporioides Coffee anthrax Colletotrichum coffeanum Siamese anthrax Colletotrichum siamense Asian anthrax bacteria Colletotrichum asianum Any combination of one or more of them; The genus *Botrytis* Botrytis sp. California Botrytis cinerea Botrytis californica Geranium Botrytis cinerea Botrytis pelargonii Botrytis cinerea Botrytis cinerea Botrytis cinerea Botrytis polyblastis Botrytis cinerea fungi Botrytis ficariarum Botrytis cinerea Botrytisranunculi Any combination of one or more; The Staphylococcus species Botryosphaeria sp. Staphylococcus aureus Botryosphaeria dothidea ; The genus *Ustilago* Nigrospora sp. It is *Ulva globosum* Nigrospora sphaerica .

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