Biocontrol bacteria, fermentation liquor, biocontrol agent for preventing and treating guava anthracnose and application thereof

Abstract

The present application belongs to the technical field of plant disease prevention and treatment, and particularly relates to a biocontrol bacterium for preventing and treating Guaiacum officinale anthracnose, a fermentation liquor, a biocontrol bacterium agent and application. Bacillus velezensis A biocontrol bacterium is isolated and screened from a microbial resource on a healthy Guaiacum officinale leaf, and is classified and named as Bacillus velezensis , which is preserved in the China General Microbiological Culture Collection Center on March 28, 2025, and has a preservation number of CGMCC No.34038; the biocontrol bacterium provided in the present application has good antagonistic effects on Colletotrichum gloeosporioides causing Guaiacum officinale anthracnose, on Stemphylium botryosum causing Guaiacum officinale leaf spot disease, on Botryosphaeria dothidea, on Alternaria alternata and on Pestalotiopsis versicolor capable of causing plant diseases. The biocontrol bacterium, the fermentation liquor and the biocontrol bacterium agent provided in the present application have potential application prospects in preventing and treating Guaiacum officinale anthracnose.

Description

Technical Field

[0001] This invention belongs to the field of plant disease control technology, specifically relating to biocontrol bacteria, fermentation broth, biocontrol agents and their applications for controlling anthracnose in *Vernicia fordii*. Background Technology

[0002] Shantongzi (scientific name: Idesia polycarpa *Vernicia fordii* (also known as water melon, oil grape, etc.) is a deciduous tree belonging to the family Flaccidae. This species is highly adaptable, able to grow in harsh environments such as high temperatures, drought, and poor soil, demonstrating remarkable resilience. Its berries are orange-yellow or purplish-red when ripe. The seeds and fruits can be pressed for oil, which is rich in unsaturated fatty acids such as linoleic acid and linolenic acid, as well as trace elements, making it a high-quality woody oilseed, often referred to as an "aerial oil depot." *Vernicia fordii* oil is a semi-drying oil, used in the industrial production of insulating varnishes, soaps, and lubricants. It is also rich in β-sitosterol, tocopherol, and polyphenols, exhibiting high antioxidant activity and possessing edible value. Due to its high linoleic acid content, *Vernicia fordii* oil shows great promise in the biodiesel field. By integrating its ecological adaptability and multifunctional oil characteristics, the *Vernicia fordii* industry can form a sustainable green economic model in the energy, food, and chemical sectors, achieving both ecological and economic benefits.

[0003] Anthracnose is a major disease of *Vernicia fordii*. Damage to the leaves leads to insufficient photosynthetic products, affecting fruit development and yield, resulting in significant economic losses. Currently, the main method for controlling *Vernicia fordii* anthracnose is the use of chemical agents. However, the overuse of chemical agents has not only led to strong resistance in the anthracnose pathogen, gradually reducing the effectiveness of control, but also may affect non-target organisms, such as beneficial insects, thereby disrupting the ecological balance.

[0004] Endophytic bacteria exist within plants as symbiotic microorganisms and have great potential in promoting plant health, enhancing crop growth, and improving the adaptability of diseased plants. Finding a safe and highly effective endophytic biocontrol agent for controlling anthracnose in *Vernicia fordii* is of significant practical importance for improving the disease resistance and quality of *Vernicia fordii*. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides biocontrol bacteria, fermentation broth, biocontrol agents, and their applications for controlling anthracnose in *Vernicia fordii*. This invention isolates and screens a strain of *Bacillus belye* from microbial resources on healthy *Vernicia fordii* leaves. Bacillus velezensisThe YBGJ strain, as demonstrated in experiments, exhibits antagonistic activity against anthracnose fungi that cause anthracnose in *Vernicia fordii*, with an inhibition rate of 59.33% ± 0.33%. The fermentation broth of the YBGJ strain achieved a maximum inhibition rate of 86.18% against anthracnose fungi, making it a safe and effective biocontrol agent for the prevention and control of anthracnose in *Vernicia fordii*. Furthermore, the YBGJ strain also shows good antagonistic activity against pathogens such as *Botrytis cinerea*, *Fusarium solani*, *Ulva niger*, *Alternaria alternata*, and *Polytrichum platysporum*.

[0006] The first aspect of this invention provides a biocontrol bacterium for controlling anthracnose in *Vernicia fordii*, wherein the biocontrol bacterium is *Bacillus belyssae* (…). Bacillus velezensis It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 34038.

[0007] A second aspect of the present invention provides a fermentation broth, which is prepared by the following steps:

[0008] The biocontrol bacteria described above were inoculated into LB liquid medium and cultured for 10-16 hours to obtain seed culture.

[0009] The seed culture was inoculated into the fermentation medium at an inoculation rate of 1 v / v% to 5 v / v%, and fermented at 20℃ to 40℃ for 1 to 4 days to obtain the fermentation broth.

[0010] Furthermore, the inoculum size is 5 v / v%, and the fermentation temperature is 35°C.

[0011] Furthermore, the number of viable biocontrol bacteria in the fermentation broth is ≥1.1×10⁻⁶. 9 CFU / mL.

[0012] Furthermore, each liter of the fermentation medium contains 4g-6g of carbon source, 8g-12g of nitrogen source, and 8g-12g of inorganic salt, with water added to make up the difference; the carbon source is yeast extract, lactose, soluble starch, sucrose, fructose, or glucose; the nitrogen source is tryptone, peptone, beef extract, ammonium sulfate, urea, or sodium nitrate; the inorganic salt is sodium chloride, ferric chloride, potassium nitrate, manganese sulfate, zinc sulfate, or magnesium sulfate.

[0013] Furthermore, each 1 liter of the fermentation medium contains 5g of carbon source, 10g of nitrogen source, 10g of inorganic salt, and water to make up the difference; the carbon source is yeast extract; the nitrogen source is tryptone; and the inorganic salt is potassium nitrate.

[0014] Furthermore, the pH value of the fermentation medium is 7-11.

[0015] A third aspect of the present invention provides a biocontrol agent containing the biocontrol bacteria or the fermentation broth described above, as well as agriculturally acceptable adjuvants or auxiliaries.

[0016] Furthermore, the biocontrol agent is a liquid preparation or a powder.

[0017] The fourth aspect of the present invention provides the application of the above-described biocontrol bacteria, fermentation broth or biocontrol agent in the prevention and control of plant pathogen diseases.

[0018] Furthermore, the disease mentioned is anthracnose of the tung tree.

[0019] Furthermore, the anthracnose of *Vernicia fordii* is caused by *Anthracnose bacterium* (…). Colletotrichum sp. Caused by ).

[0020] Furthermore, the pathogen is *Ichthyophthirius multifiliis* (…). Nigrospora musae ), Botrytis cinerea ( Botrytis cinerea ), Fusarium latifolium ( Fusarium proliferatum Alternaria ( Alternaria alstroemeriae ) and Polychaete spp. ( Pestalotiopsis telopeae Any one or more of the following.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] (1) The present invention provides a biocontrol bacterium for controlling anthracnose of *Vernicia fordii*. The biocontrol bacterium has a significant antagonistic effect on the anthracnose fungus that causes anthracnose of *Vernicia fordii*, with an inhibition rate of 59.33% ± 0.33%. The highest inhibition rate of the fermentation broth prepared based on the biocontrol bacterium against anthracnose fungus can reach 86.18%.

[0023] (2) The biocontrol bacteria provided by the present invention have good antibacterial activity. The antibacterial rates against Botrytis cinerea, Fusarium latrinum, Ichthyophthirius multifiliis, Alternaria alternata, and Polychaete spp. reached 66.67%, 48.69%, 44.33%, 51.72%, and 48.23%, respectively. It also has potential application prospects in the prevention and control of plant diseases caused by Botrytis cinerea, Fusarium latrinum, Anthracnose, Alternaria alternata, or Polychaete spp.

[0024] Instructions for the Preservation of Biological Materials

[0025] In this invention, the biocontrol bacterium is referred to as strain YBGJ and classified as Bacillus belesiensis. Bacillus velezensisIt was deposited on March 28, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34038. The address of the depository is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, 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.

[0027] Figure 1 The results of the plate confrontation experiment between YBGJ strain and anthracnose fungi; Figure 1 Figure A shows the growth of anthracnose fungi; Figure 1 Figure B shows the results of the plate confrontation experiment between strain YBGJ and anthrax fungi. The upper colonies in Figure B are strain YBGJ, and the lower colonies are anthrax fungi.

[0028] Figure 2 Phylogenetic tree of strain YBGJ.

[0029] Figure 3 Morphological characteristics of strain YBGJ.

[0030] Figure 4 The results show the antagonistic effects of the YBGJ strain against the pathogens. In the top row of plates, from left to right, the strains are *Alternaria*, *Ichthyophthirius multifiliis*, *Pseudomonas aeruginosa*, *Fusarium latrinum*, and *Botrytis cinerea*. In the bottom row of plates, from left to right, the results show the antagonistic growth of the YBGJ strain against these pathogens. In each bottom row plate, the left side of each plate represents the YBGJ strain, and the right side represents the pathogen (*Alternaria*, *Ichthyophthirius multifiliis*, *Pseudomonas aeruginosa*, *Fusarium latrinum*, and *Botrytis cinerea*).

[0031] Figure 5 The experiment shows the inhibitory effect of YBGJ strain fermentation broth on anthracnose fungi. Figures a1, a2, and a3 are plate images of anthracnose fungi grown for 8 days (control group); figures b1, b2, and b3 are plate images of anthracnose fungi grown for 8 days after treatment with 10% YBGJ strain fermentation broth; figures c1, c2, and c3 are plate images of anthracnose fungi grown for 8 days after treatment with 20% YBGJ strain fermentation broth; and figures d1, d2, and d3 are plate images of anthracnose fungi grown for 8 days after treatment with 30% YBGJ strain fermentation broth. Figure 5The three plates in each row represent three parallel experiments.

[0032] Figure 6 The results show the control effect of YBGJ strain fermentation broth on detached leaves; CK: control group only inoculated with 5mm anthracnose pathogenic fungal cake; prevention group: first inoculated with YBGJ antagonistic bacteria fermentation broth, then air-dried and inoculated with 5mm anthracnose pathogenic fungal cake; treatment group: first inoculated with 5mm anthracnose pathogenic fungal cake, then inoculated with YBGJ antagonistic bacteria fermentation broth 2 days later. Figure 6 Each row of three leaves represents three parallel experiments. Detailed Implementation

[0033] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0034] *Vernicia fordii*, a highly adaptable deciduous tree belonging to the Flammullaceae family, is a high-quality woody oilseed, often referred to as an "aerial oil depot." The *Vernicia fordii* industry can form a sustainable green economic model in the energy, food, and chemical sectors, offering both ecological and economic benefits. Anthracnose is a major disease of *Vernicia fordii*, causing leaf damage, insufficient photosynthetic product supply, and affecting fruit development and yield, resulting in economic losses. Current control methods mainly rely on chemical agents, but overuse leads to increased drug resistance in pathogens, reduced control effectiveness, and may also affect non-target organisms, disrupting the ecological balance. Endophytic bacteria are symbiotic microorganisms that exist within plants, promoting plant health, enhancing growth vigor, and improving stress resistance. Finding a safe and efficient endophytic biocontrol agent is of great significance for controlling anthracnose in *Vernicia fordii* and improving the disease resistance and quality of the fruit.

[0035] This invention isolated and screened a strain of Bacillus belye from microbial resources on healthy leaves of *Vernicia fordii*. Bacillus velezensisThe YBGJ strain, as demonstrated in experiments, exhibits antagonistic activity against anthracnose fungi that cause anthracnose in *Vernicia fordii*, with an inhibition rate of 59.33% ± 0.33%. The fermentation broth of the YBGJ strain achieved a maximum inhibition rate of 86.18% against anthracnose fungi, making it a safe and effective biocontrol agent for controlling anthracnose in *Vernicia fordii*. Furthermore, the YBGJ strain showed inhibition rates of 66.67%, 48.69%, 44.33%, 51.72%, and 48.23% against *Botrytis cinerea*, *Fusarium solani*, *Ulva niger*, *Alternaria alternata*, and *Polytrichum platyceps*, respectively. It also shows potential application prospects in controlling plant diseases caused by *Botrytis cinerea*, *Fusarium solani*, *Anthracnose*, *Alternaria alternata*, or *Polytrichum platyceps*.

[0036] Example 1: Screening and Identification of YBGJ Strains

[0037] 1. Isolation and purification of endophytic bacteria from *Vernicia fordii*

[0038] Healthy leaves of *Vernicia fordii* were collected, first rinsed with sterile water, then soaked in 75% ethanol for 15 seconds, followed by soaking in 1% sodium hypochlorite solution for 2 minutes, and then rinsed three times with sterile water. The leaves were then air-dried on sterile filter paper. The sterilized leaves were placed in a mortar, sterile water was added, and the mixture was ground into a homogenate. The homogenate was allowed to stand for 20 minutes. The supernatant obtained after standing was then subjected to 10... 1 10 2 10 3 The original solution was serially diluted, and 100 μL of both the stock solution homogenate and the serially diluted solution were spread onto the surface of LB solid medium. The sterile water used for the final rinsing during the disinfection process was also spread onto the LB solid medium as a control group to measure the surface disinfection effect. The media were incubated in the dark at 37°C. After colonies grew on the medium, single colonies of different morphologies, sizes, and colors were picked and streaked onto LB solid medium. After purification, the colonies were transferred to LB slant tubes and stored at 4°C for later use.

[0039] 2. Screening of anthrax antagonistic strains

[0040] Endophytic bacteria exhibiting antagonistic activity against the anthracnose pathogen of *Vernicia fordii* were screened using the plate confrontation method. Using *A. anthracnose* as the indicator bacterium, 5 mm bacterial discs were inoculated onto one side of a PDA medium using a punch in a clean bench. Simultaneously, purified endophytic bacteria were inoculated onto the symmetrical side of the PDA medium as the experimental group. A PDA plate inoculated only with *A. anthracnose* fungi served as the control group. The plates were incubated at 26°C for 9 days to observe the inhibitory effect of the endophytic bacteria on *Vernicia fordii*. Each group was repeated three times, and the inhibition rate was calculated. The inhibition rate formula is as follows, where the diameter unit in the inhibition rate formula is cm:

[0041] Inhibition rate (%) = [(Coronavirus diameter in control group - Coronavirus diameter in experimental group pointing towards antagonistic bacteria) / (Coronavirus diameter in control group - 0.5)] × 100%;

[0042] Among the obtained endophytic bacteria, the YBGJ strain with the strongest inhibitory effect against anthrax was screened, and its antagonistic effect against *Anthrax chinensis* was as follows: Figure 1 As shown, the antibacterial rate was 59.33% ± 0.33%.

[0043] 3. Identification of YBGJ strains

[0044] (1) Plate observation

[0045] The selected YBGJ strain was streaked on LB solid medium for 24 hours. The colony morphology, size, edge, surface, raised shape, transparency and color were observed, and the colony morphology was photographed and recorded.

[0046] like Figure 2 As shown, after culturing the strain YBGJ on LB solid medium for 24 hours, the colonies are milky white, round, raised, with a dry and wrinkled surface, irregular edges, and opaque.

[0047] (2) Molecular biological identification

[0048] Genomic DNA was extracted from strain YBGJ and analyzed using bacterial 16S rDNA and rpoB The PCR primer set of the gene was used to amplify the genomic DNA of the YBGJ strain by PCR and then sequenced.

[0049] The PCR primer set for 16S rDNA includes 27F and 1492R, and the nucleotide sequences of 27F and 1492R are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively.

[0050] SEQ ID No.1: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0051] SEQ ID No.2: 5'-GGTTACCTTGTTACGACTT-3';

[0052] rpoB The PCR primer set for the gene includes 2292F and 3354R, and the nucleotide sequences of 2292F and 3354R are shown in SEQ ID No. 3 and SEQ ID No. 4, respectively.

[0053] SEQ ID No.3: 5'-AGGTCAACTAGTTCAGTATGGAC-3';

[0054] SEQ ID No.4: 5'-AAGAACCGTAACCGGCAACTT-3';

[0055] The 16S rDNA sequence of strain YBGJ is shown in SEQ ID No. 5; strain YBGJ rpoB The sequence is shown in SEQ ID No. 6;

[0056] SEQ ID No. 5:

[0057]

[0058] SEQ ID No. 6:

[0059] .

[0060] The sequencing results were submitted to the NCBI database (https: / / www.ncbi.nlm.nih.gov.cn). Sequence homology was compared and analyzed using BLAST. MEGA X software was used to construct 16S rDNA and... rpoB Linear spliced ​​sequence evolutionary tree ( Figure 3 Based on the combined morphological and molecular biological identification results, this strain can be identified as *Bacillus belyesense*. Bacillus velezensis The strain was named YBGJ.

[0061] Example 2: Determination of the antagonistic ability of YBGJ strain

[0062] Using the isolated and purified YBGJ strain as the antagonist, and Botrytis cinerea (… Botrytis cinerea ), Fusarium latifolium ( Fusarium proliferatum ), Black spore mold ( Nigrospora musaeAlternaria ( Alternaria alstroemeriae ), Polychaete spp. ( Pestalotiopsis telopeae Using the pathogen indicator bacteria, the antagonistic effect of strain YBGJ against the pathogen was determined by the plate confrontation method. On a clean bench, a 5 mm pathogen cake was inoculated onto one side of a PDA medium using a punch, while endophytic bacteria were inoculated onto the symmetrical side of the PDA medium as the experimental group. A PDA plate inoculated only with the pathogen was used as the control group. The plates were incubated at 26℃ for 8 days, and the inhibitory effect was observed and statistically analyzed.

[0063] Botrytis cinerea ( Botrytis cinerea For the biological information of ), see “CHEN YZ, WANG SR, Li T, ZHANG GC, YANG J. Antifungal Activity of 6-Methylcoumarin against Valsa maliand Its Possible Mechanism of Action.[J].Journal of Fungi. 2023; 9(1):5.”.

[0064] Figure 4 To investigate the antibacterial activity of strain YBGJ against Alternaria alternata, Ichthyophthirius multifiliis, Polychaete spp., Fusarium moniliforme, and Botrytis cinerea, from... Figure 4 It is evident that, compared to the control group, the YBGJ strain in the treatment group exhibits significant inhibitory effects against Alternaria alternata, Ulva prolifera, Polychaete globosum, Fusarium moniliforme, and Botrytis cinerea. Calculations show that the inhibition rates of the YBGJ strain against Botrytis cinerea, Fusarium moniliforme, Ulva prolifera, Alternaria alternata, and Polychaete globosum reached 66.67%, 48.69%, 44.33%, 51.72%, and 48.23%, respectively.

[0065] Example 3: Preparation method of fermentation broth of YBGJ strain

[0066] The preparation method of YBGJ strain fermentation broth is as follows:

[0067] 1. Select colonies of the activated YBGJ strain and inoculate them into LB liquid medium. Shake and culture for 12 hours to obtain seed culture.

[0068] 2. Using LB liquid medium as the basal medium, at 30℃ and 180 r·min -1 The basic culture method was dark shaking for 24 hours. The seed culture was inoculated at 1% of the culture medium volume. The YBGJ strain was subjected to the following seven treatments. Except for the time-based treatment, the OD values ​​of the fermentation broth under different culture conditions were measured after 24 hours for each treatment. 600 value.

[0069] (1) The carbon source of LB liquid medium, yeast extract, was replaced by lactose, soluble starch, sucrose, fructose and glucose respectively. The carbon source content in LB liquid medium was 5 g / L.

[0070] (2) The nitrogen source of LB liquid medium, tryptone, was replaced by peptone, beef extract, ammonium sulfate, urea and sodium nitrate respectively. The nitrogen source content in LB liquid medium was 10 g / L.

[0071] (3) Replace the inorganic salts - sodium chloride in LB liquid medium with ZnSO4, MnSO4, KNO3, FeCl3 and MgSO4·7H2O respectively. The inorganic salt content in LB liquid medium is 10g / L.

[0072] (4) Set the fermentation broth culture temperature to five gradients in sequence: 20℃, 25℃, 30℃, 35℃, and 40℃;

[0073] (5) Adjust the pH of LB liquid culture medium to 3, 5, 7, 9, 11 and 13 respectively;

[0074] (6) Adjust the seed liquid volume (transfer inoculation volume) to 1% v / v, 2% v / v, 3% v / v, 4% v / v, or 5% v / v of the liquid culture medium volume;

[0075] (7) Set the fermentation broth culture time in six gradients: 3h, 6h, 12h, 24h, 48h and 96h.

[0076] Table 1. Effects of different carbon sources, nitrogen sources, and inorganic salts on the OD of fermentation broth 600 The influence of value

[0077]

[0078] Table 1 shows that when yeast extract is used as the carbon source in the fermentation medium of YBGJ strain, the OD of the fermentation broth... 600 The value is highest when tryptone is used as the nitrogen source in the fermentation medium of Bacillus belyssus YBGJ; the fermentation broth OD is highest when tryptone is used as the nitrogen source. 600 The value is highest when potassium nitrate is used as the inorganic salt in the fermentation medium of Bacillus belyssus YBGJ; the OD value of the fermentation broth is highest when potassium nitrate is used as the inorganic salt in the fermentation medium. 600 The value is the largest.

[0079] Therefore, the optimal fermentation medium for fermenting strain YBGJ is: 5g yeast extract, 10g tryptone, 10g potassium nitrate, water to make up to 1L, mix well and sterilize to obtain the fermentation medium.

[0080] Table 2. Effects of different fermentation times and initial pH of fermentation broth on OD of fermentation broth 600 The influence of value

[0081]

[0082] Table 3. Effects of different inoculum sizes and fermentation temperatures on the OD of fermentation broth. 600 The influence of value

[0083]

[0084] As shown in Tables 2 and 3, the optimal fermentation conditions are: initial fermentation broth pH of 7, temperature of 35℃, inoculum size of 5%, and fermentation time of 24h.

[0085] Example 4: Detection of antibacterial activity of YBGJ strain fermentation broth

[0086] YBGJ strain was fermented under the optimal fermentation conditions in Example 3 to obtain YBGJ strain fermentation broth, and the antibacterial ability of YBGJ strain fermentation broth was determined, taking anthrax fungi as an example.

[0087] 1. Preparation of fermentation broth for YBGJ strain

[0088] After culturing the YBGJ strain at 37℃ and 180 rpm for 24 h, the viable cell count was 1.1 × 10⁻⁶. 9 The fermentation broth of YBGJ strain CFU / mL was prepared by centrifuging the fermentation broth at 4℃ and 12000r / min for 20min. After centrifugation, the supernatant was filtered through a 0.22μm syringe filter to obtain sterile YBGJ strain fermentation broth.

[0089] 2. Detection of antibacterial activity of YBGJ strain fermentation broth

[0090] Take a 5.0 mm mycelial cake from the activated anthracnose fungus using a punch and place it in the center of the culture medium.

[0091] The obtained YBGJ strain fermentation broth was mixed with sterilized PDA medium at volume ratios of 1:9, 2:8, and 3:7 to obtain PDA medium supplemented with 10 v / v% YBGJ fermentation broth, 20 v / v% YBGJ fermentation broth, and 30 v / v% YBGJ fermentation broth. After the medium cooled, fresh anthracnose fungal hyphae blocks were cut using a 5.0 mm diameter sterile punch and placed in the center of an inhibition plate. The plates were incubated at 26°C for 8 days. Anthrax bacteria cultured on PDA medium without YBGJ strain fermentation broth served as a control group. The colony diameter of the pathogens was observed and recorded, and the inhibition rate was calculated using the formula below, where the diameter unit is cm. The above inhibition experiments were performed in triplicate.

[0092] Inhibition rate = (target colony diameter in control group - target colony diameter in experimental group) / (colony diameter in control group - 0.5) × 100%.

[0093] The results are as follows Figure 5 As shown, the average colony diameter of anthrax bacteria in the control group was 5.33 cm. According to calculations, the inhibition rates of anthrax bacteria by adding YBGJ fermentation broth at concentrations of 10v / v%, 20v / v%, and 30v / v% in the experimental group were 43.18%±1.48%, 68.05%±0.62%, and 86.18%±0.46%, respectively.

[0094] Example 5: Application of YBGJ strain fermentation broth on detached leaves of *Vernicia fordii*

[0095] The control effect of strain YBGJ against anthracnose of *Vernicia fordii* was tested using the detached leaf method. The experiment consisted of three treatment groups, with three leaves per group. Healthy *Vernicia fordii* leaves were selected, surface-sterilized, rinsed with distilled water, disinfected with 1% sodium hypochlorite for 3 minutes, rinsed thoroughly with sterile water, and air-dried at room temperature.

[0096] Prevention experiment: Treated leaves were punctured with an inoculation needle, and the punctured areas were coated with fermentation broth from strain YBGJ and allowed to dry. Then, anthracnose fungal cakes with a diameter of 5 mm were taken using a sterile punch and inoculated. Leaves inoculated only with anthracnose pathogen served as a blank control. After 5 days, the diameter of the lesions was measured, and the preventive effect of the fermentation broth from strain YBGJ on detached leaves of anthracnose pathogen was calculated.

[0097] Treatment Experiment: Treated leaves were punctured with an inoculation needle, and then 5 mm diameter anthracnose fungal cakes were collected using a sterile punch for inoculation. After 2 days, the cakes were removed with a toothpick. The inoculated areas were then coated with fermentation broth from strain YBGJ and allowed to dry. Leaves without the fermentation broth served as a blank control. After 4 days, the diameter of the leaf lesions was measured, and the therapeutic effect of the fermentation broth on detached leaves infected with anthracnose from *Vernicia fordii* was calculated.

[0098] Control efficacy % = (average lesion diameter of blank control - average lesion diameter of treatment) / average lesion diameter of blank control × 100%.

[0099] The results are as follows Figure 6 As shown in Table 4, treatment of detached leaves of *Vernicia fordii* with fermentation broth from strain YBGJ significantly reduced the diameter of anthracnose lesions. The lesion diameters in the treatment and prevention groups were 1.16 cm ± 0.13 cm and 1.12 cm ± 0.15 cm, respectively, with both the prevention and treatment effects exceeding 60%.

[0100] Table 4. The protective effect of antagonistic bacterial fermentation broth on detached leaves.

[0101]

[0102] Note: Different lowercase letters a and b in the figure indicate significant differences between groups; — indicates that the prevention and control effect was not statistically analyzed.

[0103] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

[0104] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A biocontrol bacterium, characterized in that, The biocontrol bacteria is Bacillus belesiensis (B. belesiensis) Bacillus velezensis (), deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No.34038.

2. A fermentation broth, characterized in that, The fermentation broth is prepared by the following steps: The biocontrol bacteria described in claim 1 were inoculated into LB liquid medium and cultured for 10-16 hours to obtain seed culture. The seed culture was inoculated into the fermentation medium at an inoculation rate of 1% v / v to 5% v / v and cultured at 20℃ to 40℃ for 1 to 4 days to obtain the fermentation broth. The number of viable biocontrol bacteria in the fermentation broth is ≥1.1×10⁻⁶. 9 CFU / mL.

3. The fermentation broth according to claim 2, characterized in that, Each liter of the fermentation medium contains 4g-6g of carbon source, 8g-12g of nitrogen source, and 8g-12g of inorganic salts, with water added to make up the difference. The carbon source is lactose, soluble starch, sucrose, fructose, or glucose. The nitrogen source is peptone, beef extract, ammonium sulfate, or sodium nitrate. The inorganic salts are sodium chloride, ferric chloride, potassium nitrate, zinc sulfate, or magnesium sulfate. The pH of the fermentation medium is 7-11.

4. A biocontrol agent, characterized in that, The biocontrol agent contains the biocontrol bacteria of claim 1 or the fermentation broth of claim 2, as well as agriculturally acceptable adjuvants or auxiliaries.

5. The biocontrol agent according to claim 4, characterized in that, The biocontrol agent is a liquid preparation or a powder.

6. The application of the biocontrol bacteria of claim 1, the fermentation broth of claim 2, or the biocontrol agent of claim 4 in the control of plant pathogenic diseases, characterized in that, The plant pathogen is Nigrospora musae , Botrytis cinerea , Fusarium proliferatum , Alternaria alstroemeriae and Pestalotiopsis telopeae Any one or more of them.

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