Lysinibacillus sphaericus WSF-65, fumigation preparation and application thereof

Abstract

The invention provides lysinibacillus sphaericus WSF-65, a fumigation preparation and application of the lysinibacillus sphaericus WSF-65 and belongs to the technical field of biological prevention and control, the Latin name of the bacillus sphaericus is Lysinibacillus sphaericus, the strain number of the bacillus sphaericus is WSF-65, and the preservation number of the bacillus sphaericus is CCTCC (China Center for Type Culture Collection) NO.M20251104. The Lysinibacillus sphaericus WSF-65 disclosed by the invention can inhibit the occurrence of the citrus anthracnose through generated volatile substances, so that the Lysinibacillus sphaericus WSF-65 can be developed into a fumigation preparation for controlling the occurrence of the citrus anthracnose; the lysinibacillus sphaericus WSF-65 has a certain broad-spectrum inhibition effect on the pathogenic fungi after the citrus is picked, and fermentation liquor of the lysinibacillus sphaericus WSF-65 can be adopted to soak the picked citrus fruits, so that the rotting of the citrus fruits caused by the pathogenic fungi during the storage and transportation period after the citrus is picked is reduced; the inhibition effect of the biocontrol bacterium WSF-65 on pathogenic fungi is similar to that of a chemical bactericide commonly used in the market.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological control, and more particularly relates to a spherical lysinibacillus WSF-65, a fumigation preparation and application thereof. BACKGROUND

[0003] As a worldwide disease existing on citrus, anthracnose is one of the main diseases of citrus and a serious disease in postharvest of citrus, which often leads to rot of citrus during storage and transportation. The pathogenic bacteria of anthracnose on citrus is Colletotrichum gleosporioides in Deuteromycotina. Anthracnose belongs to a common disease on coniferous and broad-leaved trees, which frequently occurs in spring and summer and mainly endangers leaves and fruits of the aboveground part of the plant, thereby leading to early defoliation and fruit drop of the plant.

[0004] At present, the preharvest and postharvest prevention and treatment of citrus anthracnose in China is mainly spraying chemical agents, and common ones are Class A thiophanate wettable powder, carbendazim wettable powder, chlorothalonil water agent, benzene ether azole, and prochloraz. However, long-term application of the agents makes the pathogenic bacteria more resistant, and in 1996-2000, researchers selected 117 wild strains as materials to test the change of resistance of anthracnose bacteria on mango to carbendazim, which showed that part of the anthracnose bacteria on mango had resistance to carbendazim, and the resistance frequency was 48.59%. Most of the chemical agents have partial toxicity, such as carbendazim, which has microtoxicity to humans, animals, fish, bees, and the like, and causes irritation when touching the skin and eyes, and often causes nausea, dizziness, vomiting, and the like after being eaten by mistake.

[0005] With the deepening of research, prevention and treatment measures without harm to humans and the environment have gradually been paid attention by people, such as the research on antagonistic bacteria. In the 1980s, researchers used antagonistic bacteria to prevent and treat postharvest diseases of citrus due to the characteristics of no toxin production and no harm to the environment. Biological fungicides not only have no harm to the health of humans and the environment, but also can effectively inhibit harmful microorganisms on fruits and vegetables from being infected, which is a green and pollution-free efficient prevention and treatment measure. Therefore, strengthening the research on antagonistic bacteria has important significance for prevention and treatment of citrus diseases. SUMMARY

[0006] The present application aims to provide a spherical lysinibacillus WSF-65, a fumigation preparation and application thereof, so as to prevent and treat citrus anthracnose by using antagonistic bacteria and solve the problems of chemical fungicides in preventing and treating citrus anthracnose.

[0007] This invention screened 68 potentially antagonistic bacteria isolated using the in vitro plate confrontation method, obtaining an antagonistic strain, *Bacillus spheroides* WSF-65, which showed good inhibitory effects against *Colletotrichum gloeosporioides*. Simultaneously, preliminary classification and in vitro inhibition experiments were conducted, analyzing the fermentation broth, cell suspension, and cell-free supernatant of strain WSF-65. The analysis revealed that the potentially effective antibacterial substance is a volatile substance produced by strain WSF-65, which was further confirmed by a double-plate fumigation experiment, demonstrating a significant inhibitory effect. Soaking citrus fruits in the culture and fermentation broth of strain WSF-65 significantly reduced rot during storage caused by various postharvest pathogenic fungi in citrus, providing a certain guarantee for the economic benefits of citrus.

[0008] To achieve the above objectives, a first aspect of the present invention provides a biocontrol bacterium that is easy to cultivate, convenient to apply, and can effectively inhibit the occurrence of anthracnose in citrus, namely, *Lysinibacillus sphaericus* WSF-65. The Latin name of this bacterium is *Lysinibacillus sphaericus*, the strain number is WSF-65, and the accession number is CCTCC NO. M20251104. This strain was deposited on May 19, 2025, at the China Center for Type Culture Collection (CCTCC) at Wuhan University, Wuhan, Hubei Province, China.

[0009] In a second aspect, the present invention provides the use of Bacillus spheroidae WSF-65 in the production of cells and / or metabolites.

[0010] In a third aspect, the present invention provides a fumigation preparation comprising at least one of the above-described spherical lysine bacillus WSF-65 cells and metabolites.

[0011] In a fourth aspect, the present invention provides the application of the aforementioned *Bacillus spheroidans* WSF-65 in the prevention and control of plant anthracnose.

[0012] Furthermore, the plant anthracnose is citrus anthracnose.

[0013] Furthermore, the fermentation broth of Bacillus spheroides lysine-65 is used to soak citrus fruits or spray the fermentation broth of Bacillus spheroides lysine-65 onto the surface of citrus fruits or plants. During the post-harvest storage of citrus fruits, this invention uses the fermentation broth of Bacillus spheroides lysine-65 to soak freshly harvested citrus fruits, which can effectively reduce rot caused by pathogenic fungi during post-harvest storage.

[0014] In a fifth aspect, the present invention provides the application of the fumigation preparation described above in the prevention and control of anthracnose in plants.

[0015] Furthermore, the plant anthracnose is citrus anthracnose.

[0016] Furthermore, surviving *Bacillus spheroides* WSF-65 was placed within the storage space of citrus fruit, ensuring that the strain did not directly contact the fruit. This allowed the volatile gases produced by *Bacillus spheroides* WSF-65 to inhibit the growth of *Anthracnose* in citrus. This invention, through a double-plate fumigation method, demonstrated that *Bacillus spheroides* WSF-65 maintained a good inhibitory effect even without contact with the anthracnose pathogen, thus confirming that *Bacillus spheroides* WSF-65 can inhibit the growth of citrus anthracnose by producing volatile substances.

[0017] In a sixth aspect, the present invention provides the broad-spectrum application of the aforementioned *Bacillus spheroidans* WSF-65 in inhibiting pathogens, characterized in that the pathogens include *Penicillium fingernail*, *Penicillium italicum*, *Penicillium expansum*, *Penicillium chrysogenum*, *Geotrichum citrinum*, *Fusarium oxysporum*, *Aspergillus niger*, and *Colletotrichum gloeosporioides*.

[0018] Compared with the prior art, the present invention has the following technical effects:

[0019] The *Bacillus spheroides* WSF-65 of this invention can inhibit the occurrence of citrus anthracnose through the volatile substances it produces. Therefore, *Bacillus spheroides* WSF-65 can be developed into a fumigation preparation for controlling the occurrence of citrus anthracnose. At the same time, *Bacillus spheroides* WSF-65 has a certain broad-spectrum inhibitory effect on postharvest pathogenic fungi of citrus. The fermentation liquid of *Bacillus spheroides* WSF-65 can be used to soak postharvest citrus fruits to reduce the rot caused by pathogenic fungi during postharvest storage and transportation. Moreover, the inhibitory effect of the biocontrol bacterium WSF-65 of this invention on pathogenic fungi is similar to that of commonly used chemical fungicides on the market. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a diagram illustrating the screening of potential antagonistic bacteria in the root soil of plants, provided in an embodiment of the present invention.

[0022] Figure 2The colony morphology of strain WSF-65 provided in this embodiment of the invention on LB medium;

[0023] Figure 3 A phylogenetic tree of the 16S rDNA sequence of strain WSF-65 provided in this embodiment of the invention;

[0024] Figure 4 The growth curve of strain WSF-65 provided in the embodiments of the present invention in LB medium;

[0025] Figure 5 The inhibition zone formed by the confrontation culture of strain WSF-65 and Colletotrichum gloeosporioides provided in this embodiment of the invention; wherein 5A is sterile water treatment; 5B is fermentation broth treatment; 5C is cell suspension treatment; 5D is cell-free supernatant treatment;

[0026] Figure 6 After fumigation treatment with strain WSF-65 provided in this embodiment of the invention, the diameter of lesions of *Colletotrichum gloeosporioides* was statistically analyzed at 2, 4, and 6 days; where CK is the control group inoculated with sterile water.

[0027] Figure 7 The image shown is a photograph taken on day 6 after fumigation treatment of strain WSF-65 provided in this embodiment of the invention; where CK is a photograph taken after inoculation with sterile water.

[0028] Figure 8 The following diagrams illustrate the growth of the WSF-65 fermentation broth on different identification media and the identification of enzyme types provided in this embodiment of the invention; wherein 7A is the amylase identification medium; 7B is the protease identification medium; 7C is the pectinase identification medium; and 7D is the dextranase identification medium.

[0029] Figure 9 Statistical graphs of lesion diameter on days 5 and 10 after fermentation of citrus strain WSF-65 provided in this embodiment of the invention;

[0030] Figure 10 The images show citrus fruits photographed on days 5 and 10 after fermentation of the citrus strain WSF-65 provided in this embodiment of the invention.

[0031] Figure 11 The images provided in this embodiment of the invention are comparative images of the quality of citrus fruits treated with chemical agents and fermentation broth of strain WSF-65, respectively, after 90 days of storage; where 11A is a comparison image of rot rate, and 11B-D are comparison images of total soluble solids content, vitamin C content, and total titratable acid content, respectively.

[0032] Figure 12The image shows the inhibitory effect of the strain WSF-65 provided in this embodiment of the invention on common pathogenic fungi of citrus after fumigation treatment. The fungi are Aspergillus nige, Geotrichum citri-aurantii, Penicillium chrysogenum, Fusarium oxysporum, Penicillium digitatum, Penicillium expansum, and Penicillium italicum. Detailed Implementation

[0033] To make the technical problem to be solved, the technical solution, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0034] In this invention, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0035] The terminology used in the embodiments of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. The singular forms “a,” “the,” and “the” as used in the embodiments of this invention and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0036] Example 1: Isolation, purification and identification of strains

[0037] (1) Isolation and purification of strains: Rhizosphere soil collected from the rhizosphere of citrus plants in high-disease-producing areas and healthy producing areas in the past 5 years was treated as follows: 200 μL of sterilized magnesium chloride solution (10 mM) was added to a 1.5 mL centrifuge tube and vortexed thoroughly. The mixed solution was transferred to a 50 mL centrifuge tube containing 25 mL of sterile water in a laminar flow hood and allowed to stand at room temperature for 15 min after thorough shaking. After determining the optimal dilution concentration, the sample was diluted to 1000x, 2000x, 4000x, 8000x, 16000x and control group (CK). The diluted solution was shaken well in a laminar flow hood and poured into a 90 mm disposable culture dish. 160 μL of the solution was added to a 96-well culture plate. 45 culture plates were added for each treatment and 3 culture plates were added for the control group as a negative control. The 96-well culture plates were sealed with Parafilm sealing film. They were then incubated at room temperature in the dark for two weeks. After two weeks, approximately 48 cells with visibly visible turbidity between 30% and 50% were selected for subsequent plating and purification. 50 μL of the solution was taken from the 96-well cell culture plate (…). Figure 1 The diluted bacterial solution was evenly spread on LB plates and incubated at 28°C for 24 hours to obtain single colonies of the unknown bacteria. The obtained single colonies were further purified and screened to obtain a strain named WSF-65, which was then preserved.

[0038] (2) Strain identification: Strain WSF-65 was inoculated into LB medium and cultured at 28℃. The morphology was as follows: Figure 2As shown: Colonies are nearly yellowish-white, nearly round, semi-transparent with raised bumps, and have a mucous-like appearance; wrinkles often appear when they aggregate at high concentrations. Using the genome of strain WSF65 as a template, the 16S rDNA sequence was amplified by PCR. The primer sequences are as follows: 27F (SEQ ID NO: 1): AGAGTTTGATCMTGGCTCAG, 1492R (SEQ ID NO: 2): TACGGYTACCTTGTTACGACTT, yielding approximately 1000 bp DNA fragments for sequencing.The sequence is (SEQ ID NO: 3): CTTCTTCCACTTCGGCGGCTGGCTCAAAGGGTACCTCCCGACTTCGGGTGGTCAAACTCTCGTGGTGGGACGGGCGGGGGGTACCAGGGCCGGGAACGTATTCCCCGCGGCATGGTGATCCGCGATTACTAGCGATTCCGGCTTCCTGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAACGACTTTTTCGGATTAGCTCCCTCTCGCGAGTTGGCCACCGTTTGTATCGTCCATTGTAGCACGTGTGTAGCCCAGGGCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAAAGTGGCCAACTTAATGATGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACCACCATGCACCACCTGGCACCGTTGCCCCCGAAGGGGAACCATATCTCTCGGTGGTCAACGGGATGCAAGACCTGGAGGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGGGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGGGACCGTACTCCCCAGGCGGAGGGCTTATGCGTTAGCTGCAGCACTAAGGGGGGGAAACCCCCTAAACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAACCTGTTGCTCCCCACGCTTTCCGCCTCAGGTCAGTTACAACCAATAGTCCCTTCCCCTGGGTTCCCCAATCTTACGCTTTCCCGCTACCTTGGAATCCCTACCTTTTGCCTCAGTCCCCAGTTCCATGACCTCCCGGTGACCGGGGCTTCAATCAACTAAAACCCCTGCCGCCTTAGCCATATTCGGAAACGCTGCCCTAGTTACGGGTGTGCCGTGTACCGGGTTCTATAGGACGCAGGCCGCTTTCTCTGACTGTCTCCTACACAATTTCAACGA.,

[0039] The obtained 16S gene sequences were aligned using BLAST on NCBI, and sequences with high similarity were selected. A phylogenetic tree was then constructed using MEGA12 software. Figure 3 Based on the morphological characteristics and growth status of the strain, it was identified as *Lysinibacillus sphaericus*.

[0040] The strain was deposited with the China Center for Type Culture Collection on May 19, 2025. The Latin name of the strain is Lysinibacillus sphaericus, the strain number is WSF-65, the accession number is CCTCCNO.M20251104, and the deposit address is Wuhan University, Wuhan, China.

[0041] Example 2: Cultivation method of strain WSF-65

[0042] (1) Take out the original strain stored in the ultra-low temperature freezer at -80℃, spread it on LB solid medium plate, and incubate at 28℃ for 1 day. Use a sterile inoculation loop to pick 1-2 loops of strain WSF-65 and add it to 10mL LB liquid medium. Incubate at 28℃ and 180rpm for 1 day. This is used as the primary strain.

[0043] (2) The primary strain of WSF-65 was inoculated at a rate of 1% into fresh LB liquid medium and cultured at 28℃ and 180rpm for 2 days. The biomass of strain WSF-65 in the culture medium was statistically analyzed using OD600 at appropriate time points, and the growth curve of strain WSF-65 was further plotted. Figure 4 It was found that strain WSF-65 reached its biomass peak at around 24 hours in LB medium, and the bacterial culture 1 day in LB liquid medium was used as a secondary strain for subsequent treatment.

[0044] Example 3: Inhibitory effect of strain WSF-65 on Colloidal anthracnose

[0045] (1) In vitro confrontation: Using a 6mm sterile punch, make a hole in the center of a PDA plate containing the pathogen *Colletotrichum gloeosporioides*. Add 50μL of fermentation broth of strain WSF-65, cell-free supernatant, or cell suspension (with sterile water treatment as the control CK group) to the hole. Let it stand for 10-15 minutes to allow the bacterial culture to dry. Seal the petri dish and incubate at 25℃ for 2-3 days. Figure 5 ),from Figure 5 Observations revealed that the effects of WSF-65 fermentation broth, cell-free supernatant, and cell suspension were not significant. Therefore, it was suspected that other antibacterial methods might be involved, and a double-plate fumigation experiment was conducted.

[0046] The method for preparing PDA plates for *Colletotrichum gloeosporioides* is as follows: Add 200 μL of a 1×10⁻⁶ concentration of *Colletotrichum gloeosporioides* to 200 mL of melted and cooled PDA medium to 50°C. 6 Prepare a suspension of pathogenic spores per mL by shaking to mix thoroughly, then quickly pour the mixture into plates.

[0047] (2) Double-plate fumigation: Spread 100 μL of 1% concentration of primary WSF-65 strain evenly on a 90 mm LB plate, and invert the spread LB plate onto a suspension of collodion spores (1×10⁻⁶) inoculated in the center. 6 The lesions of *Colletotrichum gloeosporioides* were plated on PDA plates (2.5 μL / mL). The diameter of the lesions was counted at 2, 4, and 6 days. Figure 6 As shown, and take a photo on the 6th day, as shown. Figure 7 As shown. From Figure 6 The study observed that the mycelial growth of fungi treated with WSF-65 was significantly inhibited compared to the control group. Figure 7 It can be observed that after 6 days of growth, the outer edge of the mycelium is very sparse, and the overall growth is more irregular compared with the control group.

[0048] Example 4: Observation of identification culture medium

[0049] 2.5 μL of 1% concentration of primary WSF-65 strain was spotted in the center of each of the prepared protease identification medium, pectinase identification medium, amylase identification medium, and dextranase identification medium plates. Figure 8 Using sterile water as a blank control, observations before and after staining and elution on different identification media revealed that this strain could secrete four types of enzymes during in vitro growth: protease, pectinase, amylase, and glucanase.

[0050] The specific composition of each identification culture medium is as follows:

[0051] (1) Protease identification medium: Mix 10g casein, 3g beef extract, 2g Na2HPO4, 5g NaCl, 15g agar and 0.05g thymol blue thoroughly and bring the volume to 1000mL. Sterilize at high temperature and set aside for later use.

[0052] (2) Pectinase identification medium: 1g K2HPO4, 0.5g MgSO4-7H2O, 3g NaNO3, 0.01g FeSO4-7H2O, 2g pectin, and 15g agar are thoroughly mixed and brought to a final volume of 1000mL. After high-temperature sterilization, it is ready for use.

[0053] (3) Amylase identification medium: 10g peptone, 3g beef extract, 10g soluble starch and 20g agar are thoroughly mixed and the volume is adjusted to 1000mL. After high temperature sterilization, it is ready for use.

[0054] (4) Dextran identification medium: 0.05g glucose, 0.5g mother extract, 18g peptone, 0.5g sodium oxide, 0.01g Congo red, 18g agar powder, add double distilled water to make up to 1000mL, and adjust the pH to 7.0.

[0055] Example 5: Inhibitory effect of strain WSF-65 on anthracnose of citrus fruit

[0056] Select harvested citrus fruits that are uniform in color and size and have no obvious mechanical damage. Wash them with clean water and then soak them in a 2% sodium hypochlorite solution for 2 minutes to remove surface bacteria. Rinse thoroughly with the sodium hypochlorite solution afterward. Allow the fruits to air dry.

[0057] After the fruit is air-dried, a sterile inoculation needle with a diameter of 6 mm is used to artificially create a wound on the equatorial surface of the citrus fruit. The wound is then treated as follows: 1. Inoculate with 10 μl of a 1×10⁻⁶ solution. 6 1. A suspension of *Colletotrichum gloeosporioides* spores per mL; 2. Inoculation with 10 μL of *WSF-65* fermentation broth (with sterile water as the control group CK). Fruits treated with the same method were placed individually in a preservation box at 90% relative humidity and cultured at room temperature for 10 days. The diameter of lesions in each treatment was counted on days 5 and 10. Each treatment included 15 fruits in a single experiment, and the experiment was repeated three times. The statistical results of lesion diameter are shown below. Figure 9 As shown, photos of citrus fruits taken at 5 and 10 days were also taken. Figure 10 As shown. Figure 9 , Figure 10 This indicates that the fermentation broth of strain WSF-65 in this embodiment of the invention can effectively control the occurrence and development of citrus anthracnose.

[0058] Example 6: Effects of strain WSF-65 on the postharvest storage quality of citrus fruits

[0059] The primary strain was added to LB medium at an inoculation rate of 1%, and incubated at 180 rpm and 28℃ for 1 day. The fermented broth was then diluted 10 times with water. Citrus fruits of uniform size and without obvious mechanical damage were selected from orchards managed using scientific and effective methods and subjected to the following treatments in a ventilated storage facility in the production area:

[0060] Treatment 1: Soak in chemical reagents (50g of baicalein, 22.5g of imazalil, 7.5g of 2,4-D, and 25g of thiamethoxam in 100kg of water) for 1 minute;

[0061] Treatment 2: Soak strain WSF-65 in fermentation broth for 1 minute;

[0062] Process 3: Soak in clean water for 1 minute.

[0063] In a single storage experiment, 100 citrus fruits were soaked in each treatment, with a total of three replicates. After 90 days, the fruit rot rate was recorded. The experimental results are as follows: Figure 11 As shown, after 90 days of long-term storage, the average rot rates of fruits treated with chemical agents and those treated with biocontrol strain WSF-65 were 1.5% and 2.5%, respectively. Figure 11 A), Figure 11 The results from BD showed that, compared with citrus fruits treated with chemical agents, the total soluble solids content of citrus fruits treated with the fermentation broth of strain WSF-65 was more stable during storage and did not show significant fluctuations. Vitamin C in untreated stored citrus fruits showed a continuous downward trend, while treatment with chemical agents and WSF-65 inhibited the decline of vitamin C and showed a certain positive increase. In the statistical analysis of total titratable acid content, it was found that, with the passage of storage time, both the water-treated control group and the chemical agent-treated group showed a significant decrease, but the WSF-65-treated group showed the opposite trend.

[0064] Statistical analysis of the data using SPSS revealed no significant difference in the control effect of the two methods on the postharvest decay rate of citrus, indicating that the control effect of the biocontrol strain WSF-65 on pathogens during long-term storage of citrus fruits is similar to that of chemical agents.

[0065] Example 7: Inhibitory effect of strain WSF-65 on common postharvest pathogens of citrus

[0066] To investigate the inhibitory effect of strain WSF-65 on common postharvest pathogens of citrus other than anthracnose, the experimental method was the same as in Example 3. A double-plate fumigation experiment was conducted on different postharvest pathogens of citrus, and observations were made after treatment at 28℃ for 5 days. Figure 12 WSF-65 showed varying degrees of inhibitory effects on postharvest pathogenic fungi of citrus, such as Penicillium fingerlingum, Penicillium italicum, Penicillium expansum, Penicillium chrysogenum, Geotrichum citrinum, Fusarium oxysporum, and Aspergillus niger.

[0067] When processing large quantities of fruit, you can prepare the fermentation broth of strain WSF-65 in advance, concentrate the fermentation broth, and retain the bacterial cells and a small amount of liquid culture medium. When the fruit needs to be processed, dilute the fermentation broth according to the concentration ratio.

[0068] In this embodiment of the invention, the in vitro plate confrontation method was used to study Bacillus cereus WSF-65 isolated from citrus root soil. It was found that the WSF-65 strain can inhibit the growth of pathogenic fungus Colloidal anthracnose by producing volatile substances. Furthermore, the culture stock solution of WSF-65 can effectively reduce the rotting of citrus fruits during storage, and its effect is similar to that of commonly used chemical agents for citrus fruits.

[0069] The above embodiments merely illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this patent should be determined by the appended claims.

Claims

1. A type of spherical lysine-containing Bacillus cereus WSF-65, characterized in that, The Latin name of the spherical lysine spore bacillus is Lysinibacillus sphaericus, the strain number is WSF-65, and the preservation number is CCTCC NO.M20251104.

2. The use of the spherical lysine-containing Bacillus WSF-65 as described in claim 1 in the production of bacterial cells and / or metabolites.

3. A fumigation preparation, characterized in that, It includes at least one of the cells and metabolites of Bacillus spheroidae WSF-65 as described in claim 1.

4. The application of Bacillus spheroidans WSF-65 as described in claim 1 in the prevention and control of plant anthracnose.

5. The application of Bacillus spheroidans WSF-65 as described in claim 4 in the control of plant anthracnose, characterized in that, The plant anthracnose mentioned is citrus anthracnose.

6. The application of Bacillus spheroidans WSF-65 as described in claim 5 in the control of plant anthracnose, characterized in that, Citrus fruits can be soaked in the fermentation broth of Bacillus spheroidans WSF-65 or sprayed onto the surface of citrus fruits or plants.

7. The application of the fumigation preparation as described in claim 3 in the prevention and control of anthracnose in plants.

8. The application of the fumigation preparation as described in claim 7 in the control of plant anthracnose, characterized in that, The plant anthracnose mentioned is citrus anthracnose.

9. The application of the fumigation preparation as described in claim 8 in the control of plant anthracnose, characterized in that, Surviving Bacillus spheroidans WSF-65 was placed in the storage space of citrus fruit, without direct contact between the Bacillus spheroidans WSF-65 strain and the citrus fruit, so that the volatile gases produced by Bacillus spheroidans WSF-65 would have an inhibitory effect on citrus anthracnose.

10. The broad-spectrum application of *Bacillus spheroidans* WSF-65 in inhibiting pathogens as described in claim 1, characterized in that... The pathogens mentioned include Penicillium fingerlingum, Penicillium italicum, Penicillium expansum, Penicillium chrysogenum, Geotrichum candida, Fusarium oxysporum, Aspergillus niger, and Colletotrichum gloeosporioides.

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