Bacillus velezensis 20-1 and application thereof

Abstract

The present application relates to the field of microorganisms, and provides a bacillus velezensis 20-1 and application thereof, wherein the bacillus velezensis 20-1 is preserved in the China Center for Type Culture Collection on July 26, 2024, and the preservation number is CCTCC NO: M 20241694.The bacillus velezensis 20-1 can effectively inhibit the growth of common aquatic pathogenic fungi, has strong thermal stability and protease resistance, and can be used for preventing, relieving and / or treating diseases caused by aquatic pathogenic fungal infection.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and more specifically, to a strain of Bacillus belye 20-1 and its applications. Background Technology

[0002] Aquaculture is a vital industry in my country, but its massive production volume faces serious threats from diseases. One highly pathogenic disease in aquaculture is saprolegniasis, caused by *Saprolegnia*, a fungus belonging to the order *Saprolegniales* and family *Sprolegniaceae*, primarily including the genera *Saprolegnia* and *Achlya*. Saprolegniasis, also known as skin fungus or white hair disease, is a common pathogen in freshwater farmed animals. It is widespread both temporally and geographically: saprolegnia can occur across a wide temperature range, year-round, and in most aquaculture areas nationwide. Its spores are abundant in the natural environment and exhibit strong environmental tolerance. Furthermore, saprolegnia does not strictly select its hosts; injured hosts and eggs during incubation can both become infected. As is common with fungal diseases, once infected by *Saprolegnia*, it is difficult to cure. Therefore, saprolegniasis remains a major challenge in aquaculture, causing significant economic losses to the industry annually. Some species of the genus Achlya are aquatic pathogens of farmed fish and shrimp, the most common being Achlya americana and Achlya bisexualis.

[0003] Megaptera are holometabolous insects. The Yanhuang Star Toothfly (Protohermes xanthodes), belonging to the family Corydalidae of Megaptera, is a rare edible and medicinal insect whose commercial and scientific value has attracted the attention of entrepreneurs and researchers. In recent years, the wild population of Megaptera has declined sharply. The Yanhuang Star Toothfly is widely distributed in China and is also a dominant species in artificial breeding. However, during its artificial breeding, especially in the final instar stage, it is severely susceptible to aquatic pathogenic fungal infections, significantly impacting the industry's development. Currently, most aquatic pathogenic fungal diseases in aquaculture are treated with antibiotics. Observations show that the Yanhuang Star Toothfly almost stops feeding after infection, making antibiotic feeding ineffective. Furthermore, the use of antibiotics faces limited success and potential risks. Therefore, developing antibiotic alternatives in aquaculture is of great significance for effectively addressing antibiotic resistance issues. Utilizing intestinal probiotics to assist the host in disease prevention and control is a promising approach for the future. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent.

[0005] Therefore, in a first aspect of the present invention, a Bacillus belye is provided. Bacillus velezensis 20-1. According to an embodiment of the present invention, the Bacillus belyes 20-1 was deposited at the China Center for Type Culture Collection on July 26, 2024, with accession number CCTCC NO: M 20241694.

[0006] The Bacillus belye 20-1 strain obtained by screening in this invention can inhibit common aquatic pathogens and can be used to prevent, alleviate and / or treat diseases caused by aquatic pathogen infections.

[0007] In a second aspect, the present invention provides a fermentation broth. According to an embodiment of the invention, the fermentation broth is derived from the *Bacillus belye* 20-1 described in the first aspect. The fermentation broth of the present invention can be used to prevent, alleviate, and / or treat diseases caused by aquatic pathogenic fungal infections.

[0008] In a third aspect, the present invention provides a bacterial suspension. According to an embodiment of the invention, the bacterial suspension comprises *Bacillus belye* 20-1 as described in the first aspect. The bacterial suspension of the present invention can be used for the prevention, alleviation, and / or treatment of diseases caused by aquatic pathogenic fungal infections.

[0009] In a fourth aspect, the present invention provides the use of the *Bacillus bellis* 20-1 described in the first aspect, the fermentation broth described in the second aspect, or the bacterial suspension described in the third aspect in the preparation of feed that inhibits the activity of aquatic pathogenic fungi. The feed of the present invention can be used to prevent or inhibit diseases caused by aquatic pathogenic fungal infections.

[0010] In a fifth aspect, the present invention provides the use of *Bacillus belyssus* 20-1 as described in the first aspect, the fermentation broth as described in the second aspect, or the bacterial suspension as described in the third aspect in the preparation of a medicament for inhibiting the activity of aquatic pathogenic fungi. The medicament of the present invention can be used for the prevention, relief, and / or treatment of diseases caused by aquatic pathogenic fungal infections.

[0011] In a sixth aspect, the present invention provides a composition. According to embodiments of the invention, the composition comprises at least one of the following: *Bacillus belye* 20-1 as described in the first aspect, the fermentation broth as described in the second aspect, and the bacterial suspension as described in the third aspect. The composition of the present invention can be used for the prevention, alleviation, and / or treatment of diseases caused by aquatic pathogenic fungal infections.

[0012] In a seventh aspect, the present invention provides a method for inhibiting the growth of aquatic pathogenic fungi in vitro. According to an embodiment of the present invention, the method comprises: co-culturing at least one of the following: *Bacillus belyesii* 20-1 described in the first aspect, the fermentation broth described in the second aspect, and the bacterial suspension described in the third aspect, with aquatic pathogenic fungi. As previously stated, *Bacillus belyesii* 20-1 of the present invention can effectively inhibit the growth of aquatic pathogenic fungi.

[0013] In an eighth aspect, the present invention provides a cultivation or production apparatus for Bacillus bellis 20-1 as described in the first aspect, the fermentation broth as described in the second aspect, the bacterial suspension as described in the third aspect, or the composition as described in the sixth aspect. The cultivation or production apparatus according to embodiments of the present invention is capable of mass-producing Bacillus bellis 20-1, the fermentation broth, the bacterial suspension, or the composition described in the present invention.

[0014] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0015] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 These are morphological diagrams of healthy and diseased *S. huanghuali* larvae according to embodiments of the present invention, wherein A shows the morphology of a healthy *S. huanghuali* larvae, B shows a diseased *S. huanghuali* larvae, C shows an enlarged view of the head of a diseased *S. huanghuali* larvae, D shows an enlarged view of the middle part of a diseased *S. huanghuali* larvae, and E shows an enlarged view of the tail of a diseased *S. huanghuali* larvae.

[0017] Figure 2 According to the embodiments of the present invention Bacillus velezensis 20-1 Colony morphology after 12 hours of incubation on LB medium;

[0018] Figure 3 According to the embodiments of the present invention Bacillus velezensis 20-1 The results of the antagonistic experiment against Amphotericoptera are shown in Figure A. Figure A shows the results of normally growing Amphotericoptera, and Figure B shows the results of the experiment against Amphotericoptera. Bacillus velezensis 20-1 The results of the detection of inhibition of the growth of Amygdalina amphotericisae;

[0019] Figure 4 According to the embodiments of the present inventionBacillus velezensis 20-1 Phylogenetic tree;

[0020] Figure 5 According to the embodiments of the present invention Bacillus velezensis 20-1 The graph shows the results of the detection of the effect of fermentation broth on the germination of Amygdaloides spores. In the graph, A shows the effect of fermentation broth on the germination of Amygdaloides spores. Bacillus velezensis 20-1 The results of treating amphoteric Aureomyces spores with fermentation broth (1) and sterile water (2) for 12 hours are shown in Figure B. Figure B shows the amphoteric Aureomyces spores under a 200x microscope after treatment. Bacillus velezensis 20-1 Morphological images of fermentation broth after 12 hours of treatment; C shows the morphological image of amphoteric amygdalin spores after 12 hours of treatment with sterile water under a 200x microscope.

[0021] Figure 6 According to the embodiments of the present invention Bacillus velezensis 20-1 The results of the detection of the effect of fermentation broth on the morphology of Amygdalinia mycelia are shown in the figure. Among them, AC shows magnified images of different parts of normally growing Amygdalinia mycelia under a 100x microscope; DF shows magnified images under a 100x microscope. Bacillus velezensis 20-1 A diagram illustrating the method for treating Amphoteric Amygdalinia mycelia with fermentation broth.

[0022] Figure 7 According to the embodiments of the present invention Bacillus velezensis 20-1 The results of the antibacterial effect of the fermentation broth on *Aureomyces cerevisiae* cake are shown in the figure. In figure A, the morphology of normally growing *Aureomyces cerevisiae* cake is shown, and in figure B, the morphology of the cake is shown. Bacillus velezensis 20-1 Morphological diagram of amphoteric Amygdalin cake after fermentation broth treatment;

[0023] Figure 8 Temperature according to embodiments of the present invention Bacillus velezensis 20-1 The graph shows the results of the detection of the effect of fermentation broth on the stability of Agromycetes amphotericisae. Data are expressed as the mean ± standard deviation of the inhibition rate, with different letters indicating the results obtained through Duncan's new multiple range test. P <0.05 level significant difference;

[0024] Figure 9 For protease pairs according to embodiments of the present invention Bacillus velezensis 20-1 The results of the fermentation broth antagonizing the stability of Agromycetes are shown in the figure. Data are expressed as the mean ± standard deviation of the inhibition zone diameter. Different letters indicate the results obtained through Duncan's new multiple range test. P <0.05 level significant difference;

[0025] Figure 10 According to the embodiments of the present invention Bacillus velezensis 20-1The antagonistic effect of fermentation broth on common aquatic pathogens is shown in the figure. Data are expressed as the mean ± standard deviation of the inhibition zone diameter. Multiple t-tests were used to analyze differences between groups. "***" indicates a highly significant difference between the control group and the fermentation broth treatment group. P <0.001, df=4). Detailed Implementation

[0026] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0027] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "multiple" or "a plurality of" means at least two, two types, such as two, two, three, three, etc., unless otherwise explicitly specified.

[0028] In this document, the terms “comprising,” “having,” or “including” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0029] In this document, the term “optionally” generally means that an event or condition described below may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0030] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0031] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains.

[0032] In this document, the terms “treatment” and “relief” refer to the attainment of a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of disease or its symptoms, and / or therapeutic in terms of partial or complete cure of disease and / or adverse effects caused by disease. As used herein, “treatment” encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of disease in susceptible individuals who have not yet been diagnosed with the disease; (b) suppression of disease, such as inhibiting disease progression; or (c) relief of disease, such as reducing disease-related symptoms. As used herein, “treatment” encompasses any medication that administers a strain, fermentation broth, bacterial suspension, or composition to an individual to treat, cure, relieve, improve, reduce, or suppress the individual’s disease, including but not limited to administering to an individual in need a strain, fermentation broth, bacterial suspension, or composition described herein.

[0033] In this document, the term "carrier" includes any solvent, pharmaceutical stabilizer, or combination thereof, which are known to those skilled in the art. Except in cases where any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is covered.

[0034] Saprolegniasis is a common and highly damaging fungal infection in aquatic animals. As a fungal parasitic disease, it has a wide temporal and geographical prevalence, occurring year-round in most aquaculture areas across China. Saprolegnia does not exhibit strict host selectivity. In recent years, research on aquatic fungi has primarily focused on aquatic animals susceptible to Saprolegniasis, such as red-spotted salmon, common carp, and large silverfish. Saprolegniasis involves over 40 genera, including *Saprolegnia*, *Amygdalina*, *Cryptocoryne*, and *Cryptocoryne*. Among the more than ten genera identified in my country, *Saprolegnia* and *Amygdalina* are the two most likely to cause disease. Fish and amphibians are its primary hosts. Under artificial breeding and propagation conditions, especially during egg hatching and overwintering, *Saprolegnia* pathogens can cause mass fish mortality, resulting in significant losses to the aquaculture industry. Saprolegnia is ubiquitous in aquaculture water, with a wide temperature tolerance range (5-26℃) and an optimal reproductive temperature of 13-18℃. It exhibits strong adaptability and resilience to various aquatic environments, capable of sexual reproduction by forming spores, antheridium, and oothecae to resist adverse conditions. Therefore, it is difficult to eradicate with conventional drugs, posing a significant challenge to the prevention and control of saprolegniasis. Disinfectants and antifungal agents, while possessing broad-spectrum antibacterial properties, are widely used in fish disease treatment. However, their residues lead to water quality deterioration, microbial resistance, and an increasing use of aquaculture drugs, seriously threatening aquatic product quality and safety as well as public health safety.

[0035] strain

[0036] Therefore, in some embodiments, the present invention proposes a Bacillus belesiensis.Bacillus velezensis 20-1 The Bacillus belyssus 20-1 was deposited at the China Center for Type Culture Collection on July 26, 2024, with accession number CCTCC NO: M 20241694.

[0037] According to some specific embodiments of the present invention, the above-mentioned Bacillus belye 20-1 may further include at least one of the following additional technical features:

[0038] According to some specific embodiments of the present invention, the Bacillus belyssus 20-1 has a 16S rRNA sequence as shown in SEQ ID NO:1.

[0039]

[0040] The novel strain of Bacillus vesiculus 20-1 of the present invention was obtained by screening from diseased Saprolegnia flamingo. Bacillus vesiculus 20-1 can inhibit common aquatic pathogens and can be used to prevent, alleviate and / or treat diseases caused by aquatic pathogens. In particular, it can effectively treat Saprolegnia in aquatic animals and has high application value.

[0041] In this article, the term "Bacillus belesiensis" is used. Bacillus velezensis 20-1 “Bacillus belyssus 20-1”, “20-1 strain” and “20-1” are synonymous.

[0042] Fermentation liquid

[0043] In some embodiments, the present invention provides a fermentation broth derived from the aforementioned *Bacillus belye* 20-1. The fermentation broth of the present invention can be used for the prevention, mitigation, and / or treatment of diseases caused by aquatic pathogenic fungal infections.

[0044] It should be noted that the "fermentation broth" of the present invention refers to the solution obtained after culturing Bacillus belyss 20-1 for a period of time, which mainly contains Bacillus belyss 20-1 and its metabolites; or the supernatant after further treatment by centrifugation, filtration and other means, which mainly contains the metabolites of Bacillus belyss 20-1.

[0045] bacterial suspension

[0046] In some embodiments, the present invention provides a bacterial suspension comprising the aforementioned Bacillus belyssus 20-1. The bacterial suspension of the present invention can be used for the prevention, alleviation, and / or treatment of diseases caused by aquatic pathogenic fungal infections.

[0047] It should be noted that the bacterial suspension can be obtained by processing the above-mentioned fermentation broth through centrifugation, resuspension and other means.

[0048] use

[0049] In some embodiments, the present invention proposes the use of the aforementioned Bacillus bellis 20-1, fermentation broth, or bacterial suspension in the preparation of feed that inhibits the activity of aquatic pathogenic fungi. The feed of the present invention can be used to inhibit or prevent diseases caused by aquatic pathogenic fungi and to assist in the regulation of metabolic diseases caused by aquatic pathogenic fungi.

[0050] In some embodiments, the present invention proposes the use of the aforementioned Bacillus bellis 20-1, fermentation broth, or bacterial suspension in the preparation of medicaments for inhibiting the activity of aquatic pathogenic fungi. The medicaments of the present invention can be used for the prevention, relief, and / or treatment of diseases caused by aquatic pathogenic fungal infections.

[0051] According to embodiments of the present invention, the above-mentioned uses may further include at least one of the following additional technical features:

[0052] According to an embodiment of the present invention, the aquatic pathogenic fungi include at least one of the following: *Amygdaloides*, *Amygdaloides var. anamensis*, *Mucor*, *Mucor*, *Pythium*, *Pseudomonas aeruginosa*, *Aeromonas punctata*, *Aeromonas var. punctata*, *Pseudomonas aquaticus*, *Flexobacterium columnare*, *Edwards*, *Nocardia asteroides*, and *Streptococcus agalactiae*.

[0053] According to an embodiment of the present invention, the aquatic pathogenic fungus includes at least one of Amygdaloides, Amygdaloides 'American', Mucor 'Cryptocoryne', Mucor 'Roller', Pythium spp., and Pseudomonas spp.

[0054] Medicines and feed

[0055] This invention provides a medicine or feed comprising at least one of the aforementioned Bacillus bellis 20-1, fermentation broth, or bacterial suspension. The feed of this invention can be used to inhibit or prevent diseases caused by aquatic pathogenic fungi, and to assist in regulating metabolic disorders caused by aquatic pathogenic fungi. The medicine of this invention can be used to prevent, alleviate, and / or treat diseases caused by aquatic pathogenic fungal infections.

[0056] According to embodiments of the present invention, the above-mentioned medicine or feed may further include at least one of the following additional technical features:

[0057] According to embodiments of the present invention, it further includes pharmaceutically acceptable excipients or carriers or excipients or carriers acceptable in animal feed.

[0058] In this document, "pharmaceutical acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components of the formulation and / or the mammals to which it is treated. Preferably, "pharmaceutical acceptable" as used herein means approved by a federal regulatory agency or national government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals or humans.

[0059] In this article, “acceptable adjuvants or carriers in animal feed” refers to substances or compositions that can be consumed by animals, and these can be adjusted according to the animal feed requirements of different countries.

[0060] In this document, the term "pharmaceutically acceptable carrier" includes any solvent, drug stabilizer, or combination thereof, which are known to those skilled in the art. Except in cases where any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is covered.

[0061] In this document, the term "pharmaceuticalally acceptable excipient" may include any solvent suitable for the specific target dosage form. The use of any conventional excipients that are incompatible with the Bacillus belyssus 20-1 disclosed herein, such as any adverse biological effects produced or interactions with any other component of the pharmaceutically acceptable composition in a harmful manner, is also within the scope of this disclosure.

[0062] According to embodiments of the present invention, at least one of the aforementioned Bacillus berberis 20-1, fermentation broth, and bacterial suspension is added to or inoculated into feed or into a drug, thereby further obtaining feed that inhibits the activity of aquatic pathogenic fungi, or a drug that can prevent, alleviate, and / or treat aquatic pathogenic fungal infections or diseases caused by aquatic pathogenic fungal infections.

[0063] For example, the aforementioned drugs include, but are not limited to, human drugs and veterinary drugs. The aforementioned veterinary drugs can be for pets, livestock, wild animals, and aquatic animals.

[0064] It should be noted that the characteristics and advantages described above for Bacillus belyssus 20-1 also apply to this drug or feed, and will not be repeated here.

[0065] Composition

[0066] In some embodiments, the present invention provides a composition comprising at least one of the aforementioned Bacillus belye 20-1, fermentation broth, and bacterial suspension. The compositions of the present invention can be used for the prevention, mitigation, and / or treatment of diseases caused by aquatic pathogenic fungal infections.

[0067] According to embodiments of the present invention, the above composition may further include at least one of the following additional technical features:

[0068] According to embodiments of the present invention, the composition further includes an ingestible excipient and / or a carrier.

[0069] According to an embodiment of the present invention, the excipient includes at least one selected from adhesives, disintegrants, lubricants, flow aids, stabilizers, fillers, diluents, and sustained-release agents.

[0070] According to an embodiment of the present invention, the carrier comprises at least one selected from sugars, cellulose and its derivatives, calcium phosphates, alkaline earth metal stearates, vegetable oils, nonionic surfactants, cationic surfactants, anionic surfactants, fatty alcohols, and hydrolyzed cereal solids.

[0071] According to embodiments of the present invention, the dosage form of the composition includes at least one selected from oral liquids, powders, granules, capsules, tablets, pellets, emulsions, microemulsions, suspensions, and sprays.

[0072] method

[0073] In some embodiments, the present invention provides a method for inhibiting the growth of aquatic pathogenic fungi in vitro, the method comprising: co-culturing at least one of the aforementioned Bacillus bellis 20-1, fermentation broth, and bacterial suspension with aquatic pathogenic fungi. As previously stated, the Bacillus bellis 20-1 of the present invention can effectively inhibit the growth of aquatic pathogenic fungi.

[0074] According to an embodiment of the present invention, the above method may further include at least one of the following additional technical features:

[0075] According to an embodiment of the present invention, the aquatic pathogenic fungi include at least one of the following: *Amygdaloides*, *Amygdaloides var. anamensis*, *Mucor*, *Mucor*, *Pythium*, *Pseudomonas aeruginosa*, *Aeromonas punctata*, *Aeromonas var. punctata*, *Pseudomonas aquaticus*, *Flexobacterium columnare*, *Edwards*, *Nocardia asteroides*, and *Streptococcus agalactiae*.

[0076] According to an embodiment of the present invention, the aquatic pathogenic fungus includes at least one of Amygdaloides, Amygdaloides 'American', Mucor 'Cryptocoryne', Mucor 'Roller', Pythium spp., and Pseudomonas spp.

[0077] Device

[0078] In some embodiments, the present invention provides a culture or production apparatus for the aforementioned Bacillus bellis 20-1, fermentation broth, bacterial suspension, or composition. The culture or production apparatus according to embodiments of the present invention enables large-scale production of the Bacillus bellis 20-1, fermentation broth, bacterial suspension, or composition described herein.

[0079] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0080] Example 1: Isolation and screening of bacteria antagonistic to amphoteric Amygdalina from the intestines of *Syngonium yanhuangense*

[0081] 1.1 Isolation and screening of strains

[0082] 1.1.1 Isolation of Amphoidea: Aquatic pathogenic fungi were isolated and cultured from diseased insect bodies, and species identification was performed using ITS (Intracytoplasmic Strain Test). Diseased insect bodies were rinsed three times with sterile purified water and placed in sterile petri dishes (d=9cm). They were then incubated at 25℃ under constant humidity. Once conidia of Amphoidea showed clear growth on the insect body surface, a small amount of spores or hyphae were gently picked from the surface of the diseased insect body using an inoculation loop in a sterilized laminar flow hood and inoculated onto PDA medium. After 2 days of incubation, a small amount of spores or hyphae were transferred to fresh medium for purification until the colonies on the plates exhibited consistent morphological characteristics. The purified strain was then placed in 25% glycerol and stored at -80℃. (See the image for diseased insect body morphology.) Figure 1 .

[0083] 1.1.2 Isolation and screening of insect-derived bacteria: Fifteen healthy, late-instar larvae of the *S. yanhuangensis* were randomly selected and starved for 24 hours. They were randomly divided into three groups. After being sterilized for 30 minutes under ultraviolet light, the larvae were washed three times with sterile water, disinfected with 75% alcohol for 200 seconds, and rinsed with distilled water. Under aseptic conditions, the intestines of the late-instar larvae were dissected. The entire digestive tract was removed by cutting along the dorsal midline from the abdominal end with sterile scissors. The midgut tissue was excised and placed into a 2 mL centrifuge tube containing 1 mL of sterile water. One or two sterile metal balls with a diameter of 5 mm were added. The tube was shaken at 20 Hz for 3 minutes to break up the intestinal tissue and obtain an intestinal homogenate.

[0084] The extracted intestinal contents were diluted in five gradients (10⁻¹, 10⁻², 10⁻³, 10⁻⁴, and 10⁻⁵). The diluted intestinal homogenates were then plated onto four different culture media: MRS (Lactobacillus medium, M8330, purchased from Beijing Solarbio Science & Technology Co., Ltd.); EC (Enterococcus medium, HB0133-3, purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd.); NA (General Bacterial Medium, HB0109, purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd.); TSA (Coctidae spp. bacteria, LA0170, purchased from Beijing Solarbio Science & Technology Co., Ltd.); and LB (General Bacterial Medium): 10 g peptone, 5 g yeast extract, 10 g sodium chloride, and 15 g agar dissolved in 1 L of sterile water, adjusted to pH 7.0. The plates were then incubated at 37°C. The plates were observed every 12 hours to obtain the original bacterial strain. The isolates were classified according to differences in colony size, color, and morphology. Colonies of different morphologies were purified on LB plates for more than five generations to obtain monoclonal strains. The purified strains were stored in 50% glycerol solution at -80 ℃ for later use.

[0085] The obtained single-clone strain was grown in 500 μL liquid LB medium at 37°C for 2–3 h. The 16S rRNA sequence was amplified using the bacterial culture as a template, employing universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTAC GACTT-3') to clone the bacterial 16S rRNA. The PCR reaction system was 25 μL: 8.5 μL ddH2O, 12.5 μL Mix, 2 μL DNA template, and 1 μL each of 27F (10 mmol / L) and 1492R (10 mmol / L). PCR conditions: 95°C for 5 min, (94°C for 30 s, 56°C for 30 s, 72°C for 1 min) × 30 cycles, followed by 72°C for 10 min. The amplification products were detected by 1.0% agarose gel electrophoresis, and the amplification products (approximately 1.5 kb) were sent to Shanghai Bioengineering Co., Ltd. for sequencing.

[0086] The antagonistic effect of isolated strains against *Agromycetes* was initially screened using the plate confrontation method. After activation, the isolated monoclonal strains were inoculated onto PDA plates using a parallel streak method, with a distance of 5 cm between the two streaks. The plates were incubated at 30°C for 1 day. After colony growth, an agar block containing the pathogenic fungus (8 mm in diameter) was inoculated in the middle of the streaks. The plates were incubated at 25°C for 3 days. The shortest distance between the pathogenic fungal colony and the two streaks was measured. The average of the three replicates was taken as the final inhibition distance. Plates showing antagonistic activity were incubated for another 15 days to observe the stability and persistence of the antagonistic effect, with three replicates per group. In the control group, an agar block containing the pathogenic fungus (8 mm in diameter) was inoculated in the middle of the PDA plate and incubated at 25°C for 3 days until the pathogenic fungus had completely covered the plate. The strain numbers and final inhibition distances of the antagonistic strains were recorded.

[0087] Twenty-six bacterial strains were isolated from the midgut of the final instar larvae of *Syngonium yanhuangense*. After fermentation, five strains were found to have antagonistic effects against *Agromycetes*, with strain 20-1 showing the strongest antagonistic effect (antagonistic distance 4.25 ± 0.20 mm). The colony morphology of bacteria 20-1 after 12 hours of incubation on LB medium is shown in [Figure missing]. Figure 2 The results of the initial screening experiment on the antagonistic effect of bacteria 20-1 against amphoteric Aureomyces are shown in [the table below]. Figure 3 .

[0088] 1.2 Homology comparison and phylogenetic tree construction of isolated strains

[0089] The 16S rRNA sequences obtained from sequencing were compared with the NCBI GenBank database using BLAST. Closely related sequences from each strain were selected and sequence alignment was performed using ClustalX 1.83. A phylogenetic tree was constructed using the Neighbor-Joining algorithm with MEGA 6.0 software, and the reliability of the phylogenetic tree was verified using bootstrap. Strain 20-1 was identified as Bacillus velezensis, with a full-length sequence of 1452 bp.

[0090] The 16S sequencing results of the isolated strain 20-1 are as follows:

[0091]

[0092] See the phylogenetic tree of strain 20-1. Figure 4 .

[0093] Example 2: Determination of physiological and biochemical parameters of Bacillus velezensis 20-1 bacteria

[0094] The carbohydrate metabolism capacity and sensitivity to other compounds of *Bacillus velezensis* 20-1 isolated in Example 1 were identified. Pure cultures of *Bacillus velezensis* 20-1 were inoculated onto BiologBUG + B medium and cultured at 37°C. Single purified colonies were picked and added to inoculating fluid. The bacterial concentration was adjusted to 90%-98% using a turbidimeter. The bacterial suspension was then added to BIOLOG GENIII microplates, 100 μL per well. The microplates were incubated statically at 37°C for 48 h. The metabolic activity of *Bacillus velezensis* 20-1 to different carbon sources and its sensitivity to different antibiotics, pH values, and NaCl concentrations were assessed based on OD595 values. Changes in negative and positive wells after incubation were used as controls for verification. The experimental results are recorded in Table 1.

[0095] Table 1: Determination of physiological and biochemical parameters of Bacillus velezensis 20-1 bacteria

[0096]

[0097] Note: "+" indicates a positive result, "-" indicates a negative result, and "w" indicates a weak positive result.

[0098] Example 3: Bacillus velezensis 20-1 Antibacterial effect study

[0099] 1. Bacillus velezensis 20-1 In vitro inhibitory effect on Amphotericin Bacillus

[0100] 1.1 Effects of bacterial fermentation broth on the growth of amphoteric amygdalin spores

[0101] Inoculate bacteria at a rate of 5% in LB liquid medium, incubate at 37°C and 200 r / min for 48 h, centrifuge at 15000 rpm for 15 min, and then filter through a 0.22 nm bacterial filter membrane.

[0102] Amphoteric *Agromyces* stored at -80℃ in glycerol was activated in PDA medium at 25℃ for 3 days before use. The surface of the medium was rinsed multiple times with sterile water, filtered repeatedly through sterile multi-layered gauze, and the spore concentration was adjusted to 2 x 10⁻⁶ using a hemocytometer. 4 per ml.

[0103] In six sterilized 1.5 mL centrifuge tubes, add 500 μL of PDB (potato broth medium) and 200 μL of amphoteric Aureomyces spores (spore concentration of 2 × 10⁻⁶). 4 The bacteria (spores / mL) were randomly divided into two groups, with three replicates per group. The treatment group received 500 μL of bacterial fermentation broth, while the control group received 500 μL of sterile water. The cultures were incubated at 25°C, and the germination of amphoteric *Azolla* spores was observed under a microscope.

[0104] 1.2 Effects of bacterial fermentation broth on the growth of Amygdalinia mycelia

[0105] Amphoteric hygrophytes stored at -80℃ in glycerol were activated in PDA medium at 25℃ for 3 days before use.

[0106] The fermentation broth of the strain was mixed with PDA medium at a volume ratio of 1:10 to prepare a culture medium containing the fermentation broth of Bacillus velezensis 20-1. Sterile water was used as a blank control. An appropriate amount of the medium was taken and placed on a clean glass slide, ensuring it was evenly distributed. A small amount of hyphae was picked up with sterile tweezers and placed on the slide. The slide was then placed in a 90 mm medium, sealed, and incubated overnight at 25°C. The hyphal growth was observed under a microscope.

[0107] The fermentation broth of the strain was mixed with PDA medium at a volume ratio of 1:10 to prepare a medium containing the fermentation broth of *Bacillus velezensis* 20-1, with sterile water as a blank control. An 8 mm amphoteric *Aureomyces* var. *velezensis* was inoculated into the center of a 90 mm PDA plate, with three replicates per treatment, and incubated at 25 ℃ for 24 h. When the control group colonies had completely covered the petri dishes, the growth diameter of the colonies in both the control and treatment groups was measured using the cross-sectional method, and the inhibition rate of the 20-1 strain fermentation broth was calculated. Inhibition rate = (Correction diameter of control group colonies) / (Correction diameter of control group colonies) (Treatment group colony diameter) / (Control group colony diameter) (Diameter of mushroom cake) × 100%.

[0108] The results are shown in Table 2 below. Figures 5-7 As shown, the bacterial fermentation broth can effectively inhibit the germination of amphoteric amygdalin spores and mycelial growth, with an inhibition rate of 80.17±0.85%.

[0109] Table 2: Inhibition rate of 20-1 bacterial fermentation broth on the growth of Amygdalina amphotericis.

[0110]

[0111] Example 4: Bacillus velezensis 20-1 Antagonizing the stability of amphoteric hairy fungi

[0112] 1.1 Determination of thermal stability of fermentation broth

[0113] The fermentation broth of the strain was treated at 40℃, 50℃, 60℃, 70℃, 80℃, and 90℃ for 30 min each. After cooling, the fermentation broth without temperature treatment was used as a control, and sterile water without temperature treatment was used as a blank control. The broths were incubated at 25℃ for 24 h. Each treatment was repeated three times. The relative inhibition rate of the fermentation broth of strain 20-1 under different temperature treatments was determined. Relative inhibition rate = (colon diameter of blank control) / (colon growth diameter of blank control) (Treatment colony growth diameter) / (Blank control colony growth diameter) (Diameter of untreated colonies) × 100%. Results are as follows: Figure 8 As shown, the bacterial fermentation broth has good temperature stability, with a relative inhibition rate of over 80% at 40-90℃.

[0114] 1.2 Stability of protease in fermentation broth

[0115] The fermentation broth of the strain was treated with proteinase K (PK) and trypsin at a final concentration of 30 U / mg at 37℃ for 1 h, followed by inactivation in a metal bath at 80℃. After cooling, the fermentation broth without proteinase treatment was used as a control, and sterile water without temperature treatment was used as a blank control. The mixtures were incubated at 25℃ for 24 h. Each treatment was repeated three times. The diameter of the inhibition zone (mm) was used as an indicator of the inhibitory effect on aquatic pathogenic fungi. The experimental results are shown below. Figure 9 As shown, the bacterial fermentation broth exhibits good protease stability, and the inhibition zone diameter of the fermentation broth treated with enzyme protein is greater than 20 mm.

[0116] Example 5: Broad-spectrum antagonism of Bacillus velezensis 20-1 bacteria

[0117] Following the preparation method of bacterial fermentation broth for Bacillus velezensis 20-1 and the in vitro inhibition experiment steps against amphoteric fungi as described in Example 3, bacterial fermentation broth was prepared, and the in vitro antibacterial activity of Bacillus velezensis 20-1 against four aquatic pathogenic fungi was evaluated. The four aquatic pathogenic fungi were Mucor hiemalis, Mucorcircinelloides, Saprolegnia australis, and Phanerochaete concrescens. The pathogenic fungi were inoculated into PDA medium and activated for 3 days before use.

[0118] The diameter of the inhibition zone (mm) was used as an indicator of the effectiveness in inhibiting aquatic pathogenic fungi. The diameters of the inhibition zones in the control group and the fermentation broth treatment group were compared and the differences were statistically analyzed. See the results below. Figure 10 The bacterial fermentation broth showed a relative inhibitory effect of over 80% on *Mucor* and *Pythium*, and also had a good antagonistic effect on *Gynostemma pentaphyllum*, with an inhibitory effect of over 70%, but a poor antagonistic effect on *Mucor*.

[0119] In the description of this specification, references to terms such as "one embodiment," "some embodiments," "embodiment," or "specific embodiment," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment. Furthermore, the specific features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments and features described in this specification.

[0120] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A type of Bacillus belyssus 20-1 ( Bacillus velezensis 20-1 ), characterized in that, The Bacillus belyssus 20-1 was deposited at the China Center for Type Culture Collection on July 26, 2024, with accession number CCTCC NO:M 20241694.

2. A fermentation broth, characterized in that, The fermentation broth is obtained by fermentation of Bacillus belye 20-1 as described in claim 1.

3. A bacterial suspension, characterized in that, The bacterial suspension comprises Bacillus belyssus 20-1 as described in claim 1.

4. The use of Bacillus berleis 20-1 as described in claim 1, the fermentation broth as described in claim 2, or the bacterial suspension as described in claim 3 in the preparation of feed that inhibits the activity of aquatic pathogenic fungi, wherein the aquatic pathogenic fungi are at least one of Amphotericoptera, Mucor, Mucor, Pythium spp., and Gynostemma pentaphyllum.

5. The use of Bacillus bellis 20-1 as described in claim 1, the fermentation broth as described in claim 2, or the bacterial suspension as described in claim 3 in the preparation of a medicament for the prevention and / or treatment of diseases caused by aquatic pathogenic fungi, wherein the aquatic pathogenic fungi are at least one of Amphotericoptera, Mucor, Mucor, Pythium spp., and Gynostemma pentaphyllum.

6. A composition characterized in that, The composition comprises at least one of Bacillus belyssus 20-1 as described in claim 1, the fermentation broth as described in claim 2, and the bacterial suspension as described in claim 3.

7. The composition of claim 6, wherein, The dosage form of the composition includes at least one selected from oral liquids, powders, granules, capsules, tablets, pellets, and sprays.

8. The composition of claim 6, wherein, The dosage form of the composition includes at least one selected from water emulsions, microemulsions, and suspensions.

9. The composition of claim 6, wherein, The composition further includes excipients and / or carriers.

10. The composition according to claim 9, characterized in that, The excipients include at least one selected from adhesives, disintegrants, lubricants, flow aids, stabilizers, and diluents.

11. The composition of claim 9, wherein, The carrier includes at least one selected from sugars, calcium phosphates, stearic acid alkaline earth metal salts, vegetable oils, nonionic surfactants, cationic surfactants, anionic surfactants, fatty alcohols, and hydrolyzed cereal solids.

12. The composition of claim 9, wherein, The carrier includes those selected from cellulose.

13. A method of inhibiting the growth of an aquatic pathogenic fungus in vitro, comprising contacting the fungus with a compound of claim 1. include: At least one of the Bacillus berleis 20-1 of claim 1, the fermentation broth of claim 2, and the bacterial suspension of claim 3 is co-cultured with aquatic pathogenic fungi, wherein the aquatic pathogenic fungi are at least one of Amphoteric Amygdalina, Mucor terrestris, Mucor truncatella, Pythium spp., and Gynostemma pentaphyllum.

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