Lysinibacillus bacilli B1 and application thereof

Abstract

The invention discloses a strain of Lysinibacillus bacilli B1 and application thereof, and belongs to the technical field of microorganisms. The preservation number of the bacstein lysine bacillus B1 is GDMCC No: 66653, and the preservation number of the bacstein lysine bacillus B1 is GDMCC No: 66653. The bacstein lysine bacillus has an inhibition effect on penicillium expansum, aspergillus ochraceus, botrytis cinerea, escherichia coli, listeria monocytogenes, sclerotium rolfsii, root rot bacteria, ralstonia solanacearum and fusarium oxysporum. The strain can be used for preventing and treating southern blight and root rot of siraitia grosvenorii, banana fusarium wilt, mycosis of navel oranges and the like; the bacterial fertilizer can be applied to the fields of fruit preservation and bacterial fertilizer biocontrol of the plants; meanwhile, the strain can also be applied to the field of food preservation, is a bacterial strain with multiple functions and wide application fields, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a strain of Pakistani lysine spores B1 and its applications. Background Technology

[0002] Microorganisms have long been used in biopesticides, biofertilizers, and biopreservation. In practice, those skilled in the art often screen microorganisms that inhibit molds and pathogens as biocontrol agents. Because microorganisms are green and environmentally friendly, and do not easily cause chemical residues or environmental pollution, they are currently widely used. However, the biggest bottleneck in the application of biocontrol agents lies in enriching the microbial germplasm resource bank and screening more strains with good disease resistance and antibacterial properties, which is crucial for the development of this field. Currently, there are many existing technologies regarding the application of Bacillus in inhibiting the growth of other bacteria. However, in practical work, the applicant's research on the development of various plant biocontrol agents has revealed that different strains of the same genus and species have significant physiological differences. Some strains can inhibit the growth of a certain pathogen, but other strains of the same genus may not necessarily have the same antibacterial effect. Therefore, the screening and functional development of microorganisms has become a highly challenging and never-ending research topic. The discovery of new strains and their applications will provide a solid foundation for subsequent plant protection, food preservation and processing, biofertilizers, and other related bioprotection fields. Summary of the Invention

[0003] The purpose of this invention is to provide a strain of Pakistani lysine spores B1 and its applications to solve the problems existing in the prior art. This strain can inhibit a variety of fungi and bacteria and can be used in different fields such as food processing, fruit and vegetable preservation, biological control, and biofertilizer.

[0004] To achieve the above objectives, the present invention provides the following solution:

[0005] This invention provides a strain of Lysinibacillus pakistanensis B1, which was deposited on July 8, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No:66653.

[0006] The present invention also provides a preservative comprising the aforementioned Pakistan Lysine Bacillus B1 and / or its secondary metabolites.

[0007] The present invention also provides a biopesticide comprising the aforementioned Pakistan Lysine Bacillus B1 and / or its secondary metabolites.

[0008] The present invention also provides a bio-fertilizer comprising the aforementioned Bacillus lysine-containing B1 and / or its secondary metabolites.

[0009] The present invention also provides the application of the aforementioned Pakistan Lysine Bacillus B1 or its secondary metabolites in bioprotection, food processing and / or food preservation.

[0010] The present invention also provides the application of the aforementioned Pakistan Lysine Bacillus B1 or its secondary metabolites, or the aforementioned preservative, or the aforementioned biopesticide, or the aforementioned biofertilizer in the preparation of products that inhibit pathogens.

[0011] Preferably, the pathogens include fungi and bacteria; the fungi include molds, white mold fungi, root rot fungi, bacterial wilt fungi, and / or Fusarium oxysporum; the bacteria include Escherichia coli and / or Listeria monocytogenes.

[0012] The present invention also provides the application of the aforementioned Pakistan Lysine Bacillus B1 or its secondary metabolites, or the aforementioned preservative, or the aforementioned biopesticide, or the aforementioned biofertilizer in the prevention and control of plant diseases.

[0013] Preferably, the plant diseases include white mold, root rot, wilt, bacterial wilt, fungal disease, ear rot, and bakanae disease.

[0014] Preferably, the fungal diseases include plant diseases caused by gray mold, Penicillium pumilum, and Aspergillus ochraceus.

[0015] The present invention discloses the following technical effects:

[0016] The *Lysinibacillus pakistanensis* B1 strain and its metabolites of this invention exhibit broad-spectrum antibacterial and disease-resistant effects, inhibiting fungi, *Escherichia coli*, *Listeria*, *Sclerotium affine*, root rot fungi, *Ralstonia solanacearum*, and / or *Fusarium oxysporum*. In agricultural applications, this strain can be used to control *Sclerotium affine* and root rot in *Mongolia grosvenorii*, and to control *Fusarium wilt* in bananas and *Mycotoxinus* in navel oranges. It can also be applied in the fields of fruit preservation, microbial fertilizers, biopesticides, and microbial agents for the aforementioned plants, demonstrating a wide range of applications. In the food industry, due to its inhibition of *Escherichia coli* and *Listeria*, it can also be used in food preservation. This strain is multifunctional and has a wide range of applications, showing promising prospects in later stages of food processing, fruit and vegetable preservation, biological control, and biofertilizers. This multifunctional strain integrates plant protection and food safety applications, possessing significant development value and industrialization potential in multiple fields such as sustainable agricultural development and green food production. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments 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.

[0018] Figure 1 This is a morphological diagram of the Pakistan Lysine Bacillus B1 on a petri dish according to the present invention.

[0019] Figure 2 This is a phylogenetic tree diagram of Bacillus lysine B1 from Pakistan, as described in this invention.

[0020] Figure 3 This is a plate antagonistic experiment diagram of the Pakistan Lysine Bacillus B1 against Botrytis cinerea in this invention;

[0021] Figure 4 This is a photomicrograph of the confrontation between Bacillus lysine-containing Pakistani B1 and Botrytis cinerea in this invention.

[0022] Figure 5 This is a plate antagonistic experiment diagram of the Pakistan Lysine Bacillus B1 against Aspergillus ochraceus in this invention;

[0023] Figure 6 This is a photomicrograph of the confrontation results between *Bacillus lysine* B1 from Pakistan and *Aspergillus ochraceus*, as presented in this invention.

[0024] Figure 7 This is a plate antagonistic experiment diagram of the Pakistan Lysine Bacillus B1 against Penicillium patulum in this invention;

[0025] Figure 8 This is a photomicrograph of the confrontation results between *Bacillus lysine-bacterium paeonii* B1 and *Penicillium pumil* according to the present invention.

[0026] Figure 9 This is a well plate experiment result showing the inhibition of Botrytis cinerea B1 secondary metabolites on Botrytis cinerea in this invention; 0 μL (Control group), 20 μL, 40 μL, 60 μL, 80 μL, and 100 μL represent the amount of Botrytis cinerea B1 secondary metabolites added.

[0027] Figure 10 This is a graph showing the results of a plate experiment on the inhibition of Aspergillus ochraceus by secondary metabolites of Bacillus lysine in Pakistan according to the present invention; 0 μL (Control group), 20 μL, 40 μL, 60 μL, 80 μL, and 100 μL represent the amount of secondary metabolites of Bacillus lysine in Pakistan added.

[0028] Figure 11This is a well plate experiment result showing the inhibition of Penicillium patulum by secondary metabolites of Bacillus lysine in Pakistan according to the present invention; 0 μL (Control group), 20 μL, 40 μL, 60 μL, 80 μL, and 100 μL represent the amount of secondary metabolites of Bacillus lysine in Pakistan added.

[0029] Figure 12 This is a diagram showing the antibacterial test results of the Pakistan Lysine Bacillus B1 against Escherichia coli according to the present invention.

[0030] Figure 13 The figure shows the antibacterial test results of Listeria monocytogenes B1 of Pakistan in this invention.

[0031] Figure 14 The figure shows the results of the antibacterial experiment of Bacillus lysine B1 from Pakistan against Ralstonia solanacearum, a pathogen of the present invention.

[0032] Figure 15 This is a graph showing the inhibition results of the Pakistan Lysine Bacillus B1 on the gray mold of navel orange according to the present invention;

[0033] Figure 16 This is a graph showing the inhibitory effect of the Pakistan Lysine Bacillus B1 on Aspergillus ochraceus, the pathogen of navel orange, according to the present invention.

[0034] Figure 17 This is a graph showing the inhibition results of the Pakistan Lysine Bacillus B1 on Penicillium paeoniliforme, the pathogen of navel orange, according to the present invention.

[0035] Figure 18 This is a plate antagonistic experiment diagram of the Pakistan Lysine Bacillus B1 against White Sclerotium wilt in this invention;

[0036] Figure 19 This is a plate antagonistic experiment diagram of the Pakistan Lysine Bacillus B1 against root rot pathogens according to the present invention.

[0037] Figure 20 This is a plate antagonistic experiment diagram of the Pakistan Lysine Bacillus B1 against Fusarium oxysporum Foc1 of the present invention.

[0038] Figure 21 This is a plate antagonistic experiment diagram of the Pakistan Lysine Bacillus B1 against Fusarium oxysporum Foc-TR4 according to the present invention;

[0039] Figure 22 This is a plate antagonistic experiment diagram of the Pakistan Lysine Bacillus B1 against rice bakanae disease pathogens according to the present invention.

[0040] Figure 23 This is a plate antagonistic experiment diagram of the Pakistan Lysine Bacillus B1 against Fusarium effusum of the present invention. Detailed Implementation

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0043] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0044] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0045] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0046] The pathogens used in the following examples were collected by the Northwest Plateau Institute of Biology, Chinese Academy of Sciences.

[0047] Example 1: Screening of Lysinibacillus pakistanensis B1

[0048] 1. Sample collection

[0049] A new bacterial strain, B1, was collected from the feces of Tibetan wild asses kept in a zoo in 2024 and isolated using bacterial isolation and purification methods. The steps included:

[0050] Weigh 1g of Tibetan wild ass feces and place it in 100mL of sterile water. Shake at 170rpm and 28℃ for 30min. Then perform serial dilutions, selecting 3 gradients for plating, with 3 replicates for each gradient. Incubate at 28℃ for 2 days, then streak strains with different colony morphologies on LB agar. Observe colony growth regularly. Purify the strains using the streak plate method, and number and store them separately. Aseptically pick colonies with different morphological characteristics, transfer them to new PDA agar for purification, incubate at 28℃ for 2 days, and repeat the transfer 3-5 times. Pick single colonies for preservation.

[0051] PDA medium was prepared using the plate confrontation method. Five-mm diameter mycelial discs were punched from the edge of the *Botrytis cinerea* agar plate and transplanted into the center. Simultaneously, single colonies were inoculated around the perimeter of the plate. The plates were incubated at 28°C, and the inhibitory effect of the strains on the pathogen was observed daily to screen for strains with highly effective antagonistic activity against *Botrytis cinerea*. After further screening, a strain exhibiting good antagonistic activity against *Botrytis cinerea* was selected and named strain B1.

[0052] 2. Morphological classification and molecular biological identification of strain B1

[0053] (1) Morphological classification of strains

[0054] Strain B1 was inoculated onto LB medium, and colony morphology was observed, such as... Figure 1 The colony shown on the petri dish has a lace-like shape, with an uneven edge and no wrinkles in the middle. A single colony has a lace-like shape.

[0055] (2) Molecular biological identification

[0056] The above-mentioned strains were sequenced and validated: The 16S rRNA gene sequence of the strains was amplified using universal primers: 5'-CCTACGGGAGGCAGCAG-3' (SEQ ID NO. 1) and 5'-ATTACCGCGGCGCTGCTGG-3' (SEQ ID NO. 2). Then, a phylogenetic tree of the strains was constructed by comparing the results with the EzBioCloud database and the BLASTn database in GenBank. (See below) Figure 2 .

[0057] The results are as follows Figure 2 As shown, strain B1 has 97.73% similarity to Lysinibacillus pakistanensis. After morphological and sequencing identification, it is considered to be close to the species Lysinibacillus pakistanensis. Therefore, it is named Lysinibacillus pakistanensis B1.

[0058] (3) Preservation of microbial strains

[0059] The isolated Lysinibacillus pakistanensis B1 was deposited on July 8, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No:66653.

[0060] Example 2: Identification of the antagonistic effect of strain B1 against different microorganisms

[0061] I. Plate confrontation experiment against mold

[0062] 1. Counteracting effect on gray mold

[0063] ①Preparation of pathogen suspension: 10% concentration... 6 Add 1 mL of seed culture containing pathogens (at a concentration of 1 μL) to 1 L of PDA liquid medium and incubate at 30 °C and 220 rpm for 3 days. After incubation, filter the suspension through three layers of gauze and adjust the final concentration to 10 × 10⁻⁶ with distilled water. 7 cfu / mL, for later use.

[0064] ② The suspension of the above pathogens was inoculated onto PDA medium. The control group was inoculated with only pathogens, while the experimental group was inoculated with pathogens on one side of the PDA medium and the corresponding screening strain B1 was inoculated on the opposite side for antagonistic experiments.

[0065] The outcome of the standoff was as follows Figure 3 As shown, the right side is the experimental group plate and the left side is the control group plate. It can be seen that the size of the gray mold colonies in the experimental group plate is significantly smaller than that in the control group plate.

[0066] The results of the standoff were captured using microscopic photography, and the results are as follows: Figure 4 As shown in the figure, the right side is the experimental group and the left side is the control group. It can be seen that the mycelial growth of the control group is strong, while the mycelial growth of the B1 experimental group is inhibited, and the mycelium of the control group shows inhibited flattening growth. This indicates that strain B1 has an inhibitory effect on gray mold.

[0067] 2. Plate confrontation experiment against Aspergillus ochraceus

[0068] The experimental procedure is the same as the experimental method for "antagonistic effect on gray mold" mentioned above.

[0069] The outcome of the standoff was as follows Figure 5 As shown, the right side is the experimental group plate and the left side is the control group plate. It can be seen that the size of Aspergillus ochraceus colonies in the experimental group plate is significantly smaller than that in the control group plate.

[0070] The results of the standoff were captured using microscopic photography, and the results are as follows: Figure 6 As shown in the figure, the right side is the experimental group and the left side is the control group. It can be seen that the mycelium of the control group grows strongly, while the mycelium of the B1 experimental group shows inhibited growth. The mycelium of the control group shows liquefaction and bubbling. This indicates that strain B1 has an inhibitory effect on Aspergillus ochraceus.

[0071] 3. Plate confrontation experiment of Penicillium expansum

[0072] The experimental procedure is the same as the experimental method for "antagonistic effect on gray mold" mentioned above.

[0073] The outcome of the standoff was as follows Figure 7 As shown, the right side is the experimental group plate and the left side is the control group plate. It can be seen that the size of the Penicillium patulum colonies in the experimental group plate is significantly smaller than that in the control group plate.

[0074] The results of the standoff were captured using microscopic photography, and the results are as follows: Figure 8 As shown in the figure, the right side is the experimental group and the left side is the control group. It can be seen that the mycelial growth of the control group is strong, while the mycelial growth of the B1 experimental group is inhibited. The mycelia of the control group show liquefaction and bubbling and flattening growth. This indicates that strain B1 has an inhibitory effect on Penicillium patulum.

[0075] The above results indicate that strain B1 has a good inhibitory effect on molds.

[0076] II. Inhibitory effect of secondary metabolites on molds

[0077] 1. Inhibitory effect of secondary metabolites of strain B1 on Penicillium patulum

[0078] ① Preparation of secondary metabolites of strain B1: Strain B1 was inoculated into PDA liquid medium at an inoculation amount of 1% and cultured for 18 hours. After the culture was completed, the culture medium was centrifuged, and the liquid was collected and filtered through a 0.22 μm sterile filter to obtain the secondary metabolites.

[0079] ②Preparation of pathogen suspension: 10% concentration... 6 1 mL of seed culture of *Penicillium pustulans* containing the pathogen was added to 1 L of PDA liquid medium and cultured at 28 °C and 170 rpm for 2 days. After culture, the suspension was filtered through three layers of gauze and then adjusted to a final concentration of 1 × 10⁹ / L with sterile distilled water. 7 cfu / mL, for later use.

[0080] ③ The secondary metabolites prepared in step ① were added to 400 μL of PDA medium at concentrations of 0 μL (Control group), 20 μL, 40 μL, 60 μL, 80 μL, and 100 μL. Then, the PDA medium containing the secondary metabolites from step ① was dispensed into 24-well plates, with 4 wells for each concentration gradient. After that, 1 μL of the pathogen from step ② was inoculated into the 24-well plates containing the above-mentioned cell extracts at different concentrations and incubated for 3 days. After 3 days, the growth of mycelia on different well plates was observed.

[0081] The results are as follows Figure 9 As shown, when the amount of B1 metabolites added reached 60 μL, it began to inhibit the growth of Botrytis cinerea; when the amount added reached 80 μL, the B1 metabolites completely inhibited the growth of mycelium.

[0082] 2. Inhibitory effect of secondary metabolites of strain B1 on ochratoxin

[0083] The pathogen was *Aspergillus ochraceus*, and the other experimental procedures were the same as those described above for the "inhibitory effect of secondary metabolites of strain B1 on *Penicillium patulum*". The results are as follows: Figure 10 As shown. By Figure 10 It can be seen that when the amount of B1 metabolites added reaches 40 μL, it begins to have an inhibitory effect on Aspergillus ochraceus; when the amount of B1 metabolites added is 60 μL, the B1 metabolites begin to inhibit the growth of mycelium.

[0084] 3. Inhibitory effect of secondary metabolites of strain B1 on gray mold.

[0085] The pathogen was *Botrytis cinerea*. Other experimental procedures were the same as those described above for the "inhibitory effect of secondary metabolites of strain B1 on *Penicillium patulum*". The results were as follows: Figure 11 As shown. By Figure 11 It is evident that when the amount of B1 metabolites added reaches 20 μL, it begins to inhibit the growth of gray mold; when the amount of B1 metabolites added reaches 40 μL, the B1 metabolites completely inhibit the growth of mycelium.

[0086] The above results indicate that the secondary metabolites of strain B1 in this application also have a good inhibitory effect on molds.

[0087] III. Inhibition zone test of strain B1

[0088] 1. Inhibitory effect on Escherichia coli

[0089] ① Inoculate Escherichia coli into LB liquid medium and culture at 37℃ and 180 rpm for 16 h with shaking, then set aside for later use;

[0090] ② Preparation of supernatant from strain B1 culture

[0091] The B1 strain was inoculated into LB liquid medium and cultured with shaking at 37°C and 180 rpm for 24 h. The culture was then centrifuged at 3500 rpm for 10 min and stored at 4°C for later use.

[0092] ③ Preparation of pathogen indicator plates

[0093] Place two Oxford cups symmetrically on an LB solid medium plate. Mix E. coli with LB agar medium that has been melted and cooled to 50°C at a volume ratio of 1:100 (v:v). Take 20 ml of the mixture and pour it onto the surface of the LB solid medium. After spreading it evenly and cooling it to solidify, use sterile forceps to remove the Oxford cups to obtain the medium with holes.

[0094] ④ Sample loading

[0095] Experimental group: Transfer 50 μL of the supernatant of strain B1 prepared in step ② into the Oxford cup well of the indicator plate in step ③, avoiding liquid overflow.

[0096] Control group: Take 50 μL of LB culture and place it in the Oxford cup well of the plate indicated in step ③, avoiding liquid spillage.

[0097] ⑤ Cultivate

[0098] After loading the sample, the E. coli indicator plate was incubated at 37°C for 16 h.

[0099] ⑥ Processing of experimental results

[0100] After the culture is completed, take photos of the plate for observation. The plate indication results are as follows: Figure 12 As shown, the experimental group had a clear transparent inhibition zone, while the control group did not have an inhibition zone; the average diameter of the inhibition zone in the experimental group was 18 mm.

[0101] 2. Inhibitory effect against Listeria monocytogenes

[0102] The experimental procedure was basically the same as the above-mentioned "inhibitory effect on Escherichia coli". The difference was that the pathogen prepared in steps ① and ③ was Listeria. The culture conditions of Listeria in step ① were as follows: Listeria monocytogenes was inoculated into LB liquid medium and cultured with shaking at 37°C and 180 rpm for 16 h for later use. In step ③, Listeria was mixed with LB agar medium that had been melted and cooled to 50°C at a volume ratio of 1:100 (v:v) and 20 ml was poured onto the surface of the above LB solid medium.

[0103] After the culture is completed, take photos of the plate for observation. The plate indication results are as follows: Figure 13As shown, the experimental group had a clear transparent inhibition zone, while the control group did not; the size of the inhibition zone in the experimental group was 21.5 mm.

[0104] The above results indicate that strain B1 has good antibacterial activity against Escherichia coli and Listeria monocytogenes, and the next step is to use this strain in processed food preservatives.

[0105] 3. Inhibitory effect on bacterial wilt pathogen.

[0106] The experimental procedure was basically the same as the above-mentioned "inhibitory effect on Escherichia coli" experiment. The difference was that the pathogen prepared in steps ① and ③ was Ralstonia solanacearum. In step ①, the culture conditions for Ralstonia solanacearum were as follows: Ralstonia solanacearum was inoculated into LB liquid medium and cultured with shaking at 37°C and 180 rpm for 16 h for later use. In step ③, Listeria monocytogenes was mixed with LB agar medium that had been melted and cooled to 50°C at a volume ratio of 1:100 (v:v), and 20 ml was poured onto the surface of the above-mentioned LB solid medium.

[0107] After the culture is completed, take photos of the plate for observation. The plate indication results are as follows: Figure 14 As shown, after the culture was completed, the plates were photographed for observation. The plate indication results are as follows. Figure 13 As shown, the experimental group had a clear transparent inhibition zone, while the control group did not have an inhibition zone; the size of the inhibition zone in the experimental group was 19.9 mm.

[0108] The above results indicate that strain B1 has an inhibitory effect on Escherichia coli, Listeria monocytogenes, and the pathogen of bacterial wilt.

[0109] Example 3: Identification of the inhibitory effect of strain B1 on citrus plant molds

[0110] 1. Inhibition experiment on gray mold in navel orange

[0111] Mold mycelia were picked up with an inoculation needle and inoculated onto the peel of navel oranges. A total of 50 navel oranges were inoculated, of which 25 were the experimental group and 25 were the control group. The control group and the experimental group were treated as follows:

[0112] Experimental group: A bacterial suspension containing strain B1 was sprayed onto the peel of navel oranges, and the application was repeated every 72 hours. The application volume was 10 μL, and the viable count of the bacterial suspension was 1 × 10⁻⁶. 7 cfu / mL.

[0113] Control group: Distilled water was dripped onto the peel of navel oranges, and the dripping was repeated every 72 hours, with a dripping volume of 10 μL.

[0114] The above-mentioned navel oranges were cultured at room temperature, and the growth of mold spots on the orange peel was observed after 15 days.

[0115] Experimental results are as follows Figure 15 As shown, the left side represents the control group of navel oranges, and the right side represents the experimental group of navel oranges. It is clearly visible that the size of the fungal patches in the control group is significantly larger than that in the experimental group, which indicates that the strain B1 of this invention has an inhibitory effect on gray mold in navel oranges.

[0116] 2. Inhibition experiment on Aspergillus ochraceus in navel orange

[0117] Aspergillus ochraceus mycelia were picked up with an inoculation needle and inoculated onto the peel of navel oranges. A total of 50 navel oranges were inoculated, of which 25 were the experimental group and 25 were the control group. The control group and the experimental group were treated as follows:

[0118] Experimental group: A bacterial suspension containing strain B1 was sprayed onto the peel of navel oranges, and the application was repeated every 72 hours. The application volume was 10 μL, and the viable count of the bacterial suspension was 1 × 10⁻⁶. 7 cfu / μL.

[0119] Control group: Distilled water was dripped onto the peel of navel oranges, and the dripping was repeated every 72 hours, with a dripping volume of 10 μL.

[0120] The above-mentioned navel oranges were cultured at room temperature, and the growth of mold spots on the orange peel was observed after 15 days.

[0121] Experimental results are as follows Figure 16 As shown, the left side represents the control group of navel oranges, and the right side represents the experimental group of navel oranges. It is clearly visible that the size of the fungal patches in the control group is significantly larger than that in the experimental group, which indicates that the strain B1 of this application has an inhibitory effect on ochratoxin infection in navel oranges.

[0122] 3. Inhibition experiment on Penicillium patulum in navel orange

[0123] Penicillium patulum mycelium was picked up with an inoculation needle and inoculated onto the peel of navel oranges. A total of 8 navel oranges were inoculated, including 4 as the experimental group and 4 as the control group. The control and experimental groups were treated as follows:

[0124] Experimental group: A bacterial suspension containing strain B1 was sprayed onto the peel of navel oranges, and the application was repeated every 72 hours. The application volume was 10 μL, and the viable count of the bacterial suspension was 1 × 10⁻⁶. 7 cfu / μL.

[0125] Control group: Distilled water was dripped onto the peel of navel oranges, and the dripping was repeated every 72 hours, with a dripping volume of 10 μL.

[0126] The above-mentioned navel oranges were cultured at room temperature, and the growth of mold spots on the orange peel was observed after 15 days.

[0127] Experimental results are as follows Figure 17As shown, the left side represents the control group of navel oranges, and the right side represents the experimental group of navel oranges. It is clearly visible that the size of the bacterial plaque in the control group is significantly larger than that in the experimental group, which indicates that the strain B1 of this application has an inhibitory effect on Penicillium pustulosis in navel oranges.

[0128] Similarly, the applicant prepared the metabolites of the above strains into a preservative, that is: strain B1 was inoculated into PDA liquid culture medium at an inoculation amount of 1% and cultured for 12 hours. After the culture was completed, the culture medium was centrifuged and the liquid was taken and filtered through a 0.22 μm sterile filter to obtain the secondary metabolites.

[0129] The secondary metabolites were then prepared into a liquid preservative (water can be selected as the solvent). The preservative was then sprayed evenly onto the peel of the navel orange to form a protective film. After that, the orange was wrapped in a layer of plastic wrap, which greatly improved the preservation effect of the navel orange. Compared with the navel orange wrapped in plastic wrap alone, the preservation time can be extended by 20-30 days, which shows a good preservation effect on the navel orange.

[0130] Example 4: Identification of the inhibitory effect of strain B1 on the pathogens of white mold disease and root rot isolated from monk fruit plants.

[0131] In this embodiment, the pathogens of white rot and root rot were both isolated from *Siraitia grosvenorii* plants and purchased from the Guangdong Provincial Microbial Culture Collection Center. After obtaining the relevant pathogens, a plate confrontation experiment was used to study their antibacterial effects, as detailed below:

[0132] ①Preparation of pathogen suspension: 10% concentration... 6 Add 1 mL of seed culture containing pathogens (at a concentration of 1 μL) to 1 L of PDA liquid medium and incubate at 30 °C and 220 rpm for 3 days. After incubation, filter the suspension through three layers of gauze and adjust the final concentration to 1 × 10⁹ / L with distilled water. 7 cfu / μL, for later use.

[0133] ② The suspension of the above pathogens was inoculated onto PDA medium. The control group was inoculated with only pathogens, while the experimental group was inoculated with pathogens on one side of the PDA medium and the corresponding B1 strain on the opposite side for antagonistic experiments.

[0134] Among them, the inhibitory effect on the pathogen of white rot is as follows: Figure 18 As shown, the right side is the experimental group plate and the left side is the control group plate. It can be seen that the mycelium of white mold in the experimental group only occupies half of the entire plate, while the mycelium of white mold in the control group covers the entire plate.

[0135] The antibacterial effect against root rot pathogens is as follows: Figure 19As shown, the right side is the experimental group plate and the left side is the control group plate. It can be seen that the mycelium of white mold in the experimental group only occupies half of the entire plate, while the mycelium of white mold in the control group covers the entire plate. This indicates that strain B1 has an inhibitory effect on both the pathogen of white mold and the pathogen of root rot in Luo Han Guo.

[0136] Example 5: Identification of the inhibitory effect of strain B1 on Fusarium oxysporum f. sp. Foc1 and Foc-TR4, which cause banana wilt disease.

[0137] ①Preparation of pathogen suspension: 10% concentration... 6 Add 1 mL of seed culture containing the pathogen (at a concentration of 1 μL) to 1 L of PDB liquid medium and incubate at 28 °C and 170 rpm for 3 days. After incubation, filter the suspension through three layers of gauze and adjust the final concentration to 10 × 10⁻⁶ with distilled water. 7 cfu / μL, for later use.

[0138] ② The suspension of the above pathogens was inoculated onto PDA medium. The control group was inoculated with only pathogens, while the experimental group was inoculated with pathogens on one side of the PDA medium and the corresponding B1 strain on the opposite side for antagonistic experiments.

[0139] Figure 20 This is a diagram of the plate antagonism experiment between strain B1 and Fusarium oxysporum (Foc1); Figure 21 This is a diagram of the plate antagonism experiment between strain B1 and Fusarium oxysporum (Foc-TR4); Figure 20 and Figure 21 The left side of the images shows the control group without strain B1, and the right side shows the experimental group inoculated with strain B1. It is clearly visible in the images that the mycelial growth of the pathogen in the experimental group is inhibited, occupying only a part of the entire plate, while the mycelial growth in the control group is not inhibited. This indicates that strain B1 has an inhibitory effect on both Fusarium oxysporum Foc1 and Foc-TR4, which cause banana wilt disease.

[0140] Example 6: Identification of the inhibitory effect of strain B1 on Fysarium fujikuroi, which causes rice bakanae disease.

[0141] ①Preparation of pathogen suspension: 10% concentration... 6 Add 1 mL of seed culture containing the pathogen (at a concentration of 1 μL) to 1 L of PDB liquid medium and incubate at 28 °C and 170 rpm for 3 days. After incubation, filter the suspension through three layers of gauze and adjust the final concentration to 10 × 10⁻⁶ with distilled water. 7 cfu / μL, for later use.

[0142] ② The suspension of the above pathogens was inoculated onto PDA medium. The control group was inoculated with only pathogens, while the experimental group was inoculated with pathogens on one side of the PDA medium and the corresponding B1 strain on the opposite side for antagonistic experiments.

[0143] Figure 22 This is a plate antagonistic experiment between strain B1 and Fysarium fujikuroi, the causal agent of rice bakanae disease. The left side of the figure shows the control group without strain B1, and the right side shows the experimental group with strain B1. It is clearly visible in the figure that the mycelium of the pathogen in the experimental group only occupies half of the entire plate, while the mycelium in the control group fills the entire plate. This indicates that strain B1 has an inhibitory effect on Fysarium fujikuroi, which causes rice bakanae disease.

[0144] Example 7: Identification of the inhibitory effect of strain B1 on Fusarium proliferatum, the causative agent of maize ear rot.

[0145] ①Preparation of pathogen suspension: 10% concentration... 6 Add 1 mL of seed culture containing the pathogen (at a concentration of 1 μL) to 1 L of PDB liquid medium and incubate at 28 °C and 170 rpm for 3 days. After incubation, filter the suspension through three layers of gauze and adjust the final concentration to 10 × 10⁻⁶ with distilled water. 7 cfu / μL, for later use.

[0146] ② The suspension of the above pathogens was inoculated onto PDA medium. The control group was inoculated with only pathogens, while the experimental group was inoculated with pathogens on one side of the PDA medium and the corresponding B1 strain on the opposite side for antagonistic experiments.

[0147] Figure 23 This is a plate antagonism experiment between strain B1 and *Fusarium proliferatum*. The left side of the image shows the control group without strain B1, and the right side shows the experimental group inoculated with strain B1. Figure 23 It is evident that the mycelium of the pathogen in the experimental group only occupies half of the entire petri dish, while the mycelium in the control group fills the entire petri dish. This indicates that strain B1 has an inhibitory effect on Fusarium proliferatum, which causes corn ear rot.

[0148] In summary, the *Bacillus lysine* B1 strain and its metabolites of this invention exhibit inhibitory effects on a variety of pathogens, including *Penicillium patulum*, *Botrytis cinerea*, *Aspergillus ochraceus*, *Escherichia coli*, and *Listeria*. Simultaneously, they provide protection against various plant diseases, such as white mold and root rot in *Monokula japonica*, gray mold, *Penicillium patulum*, and *Aspergillus ochraceus* in navel oranges, and wilt in bananas. Therefore, the strain of this invention can be widely applied in various fields such as plant preservation, plant protection, and food processing preservation, making it an excellent strain suitable for both plant protection and food preservation.

[0149] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of *Lysinibacillus pakistanensis* B1, characterized in that, The Pakistan Lysine Bacillus B1 was deposited on July 8, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No:66653.

2. A preservative, characterized in that, It contains the Pakistan Lysine Bacillus B1 as described in claim 1 and / or its secondary metabolites.

3. A biological pesticide, characterized in that, It contains the Pakistan Lysine Bacillus B1 as described in claim 1 and / or its secondary metabolites.

4. A bio-fertilizer, characterized in that, It contains the Pakistan Lysine Bacillus B1 as described in claim 1 and / or its secondary metabolites.

5. The application of Bacillus lysine spp. B1 or its secondary metabolites as described in claim 1 in bioprotection, food processing and / or food preservation.

6. The use of the *Bacillus lysine* B1 of claim 1 or its secondary metabolites, or the preservative of claim 2, or the biopesticide of claim 3, or the biofertilizer of claim 4, in the preparation of a product for inhibiting pathogens.

7. The application as described in claim 6, characterized in that, The pathogens include fungi and bacteria; the fungi include molds, white mold fungi, root rot fungi, bacterial wilt fungi and / or Fusarium oxysporum; the bacteria include Escherichia coli and / or Listeria.

8. The application of the Pakistan Lysine Bacillus B1 or its secondary metabolites as described in claim 1, or the preservative as described in claim 2, or the biopesticide as described in claim 3, or the biofertilizer as described in claim 4, in the prevention and control of plant diseases.

9. The application as described in claim 8, characterized in that, The plant diseases mentioned include white mold, root rot, wilt, bacterial wilt, fungal diseases, ear rot, and seedling blight.

10. The application as described in claim 9, characterized in that, The fungal diseases mentioned include plant diseases caused by gray mold, Penicillium patulum, and Aspergillus ochraceus.

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