Bacillus velezensis GJ5B6 and application thereof

Abstract

The invention discloses bacillus velezensis GJ5B6 and application thereof, and belongs to the technical field of biocontrol microorganisms, the preservation number of the bacillus velezensis GJ5B6 is CCTCC NO: M 20252105, and the bacillus velezensis GJ5B6 is preserved in China Center for Type Culture Collection on September 24, 2025. The bacillus velezensis GJ5B6 has remarkable inhibitory activity on pathogenic bacteria of the waxberry wilt disease and has a wide application prospect in the aspect of prevention and treatment of the waxberry wilt disease, and a theoretical basis and a strain resource are provided for development of environment-friendly biocontrol preparations; the biological agent prepared by taking the bacillus velezensis GJ5B6 as an active ingredient does not cause the problems of resistance of pathogenic bacteria, environmental pollution, pesticide residues and the like after being used for a long time, and is more environment-friendly and safer than a chemical control method.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biocontrol microorganisms, and particularly relates to a bacillus velezensis GJ5B6 and application thereof. BACKGROUND

[0002] Myrica rubra is a Myricaceae ( Myricaceae ) Myrica rubra Benth. Myrica rubra Lour. ) evergreen fruit tree, which is originally from China and has been cultivated for more than two thousand years. Its fruit is sweet and sour, with attractive color and rich nutritional value, and is favored in the consumer market. Myrica rubra is rich in nutrients, with lush branches and leaves, and has the functions of soil fixation, water conservation, landscaping, wind prevention and air purification, and has rich ornamental and ecological value.

[0003] Myrica rubra wilt is one of the most serious and destructive diseases in the current Myrica rubra industry. It has a fast onset, a long disease course and strong infectivity, and seriously threatens the healthy development of the Myrica rubra industry. Myrica rubra wilt can occur throughout the year, with an outbreak period from September to the following March. It first manifests as leaf discoloration and shedding, then forms dark brown lesions, and the lesions are covered with white mycelium around the leaf scars. The lesions can then spread to the entire branch and tree, causing discoloration of the wood. In most cases, infected trees die within 3-5 years. Researchers in the relevant field have conducted a large amount of research on the pathogen of Myrica rubra wilt, and have determined that it is a fungal disease caused by Pestalotiopsis versicolor ( Pestalotiopsis versicolor ), Pestalotiopsis microspora ( Pestalotiopsis microspora ).

[0004] Currently, the prevention and control of Myrica rubra wilt mainly relies on chemical control. For example, Chinese patent document CN116369086A discloses a method for preventing and controlling Myrica rubra wilt. In the spring, summer and autumn growth periods, fungicides and fertilizers are sprayed on the leaves to prevent and control Myrica rubra wilt. Chinese patent document CN103843807A discloses a method for preventing Myrica rubra wilt using prochloraz. The method uses 1% prochloraz reagent to prevent and control Myrica rubra wilt, and has good prevention and control effect on Myrica rubra wilt. Current research has found that the five pesticides, i.e. difenconazole, prochloraz, pyraclostrobin, propiconazole and iprodione, have good inhibitory effect on the growth of Myrica rubra wilt mycelium, and field tests have also proved that the spraying of the above-mentioned five pesticides has good effect on the prevention and control of Myrica rubra wilt.

[0005] Although the spraying of chemical reagents can have certain prevention and control effect on Myrica rubra wilt, long-term use can cause problems such as pathogen resistance, environmental pollution and pesticide residues. Therefore, exploring green and efficient biological control methods has become an important research direction for the prevention and control of Myrica rubra diseases. SUMMARY

[0006] In view of the shortcomings of the existing technology, the present invention provides a biocontrol strain that has a significant antagonistic effect on the wilt pathogen of bayberry, in order to provide new ideas and methods for the green prevention and control of bayberry diseases and promote the sustainable development of the bayberry industry.

[0007] The specific technical solution adopted is as follows: This invention provides a Bacillus belesiensis ( Bacillus velezensis The strain GJ5B6, with accession number CCTCC NO: M 20252105, is deposited at the China Center for Type Culture Collection on September 24, 2025. The address of the depository is China Center for Type Culture Collection, Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0008] The present invention also provides the application of the aforementioned Bacillus belyssus GJ5B6 in the production of antibacterial active substances.

[0009] The present invention also provides the application of the aforementioned Bacillus belye GJ5B6 in inhibiting plant pathogens, wherein the plant pathogen is *Pseudomonas heterophylla*.

[0010] This invention also provides the application of the aforementioned Bacillus belye GJ5B6 in the prevention and control of plant diseases caused by plant pathogens. Specifically, the plant pathogen is Polychaete heterochaete, and the plant disease is wilt of Myrica rubra.

[0011] Furthermore, in application, the culture, fermentation broth, or supernatant of Bacillus belye GJ5B6 obtained by culturing it in a culture medium is used to inhibit plant pathogens or prevent plant diseases caused by plant pathogens.

[0012] The present invention also provides a biological agent, wherein the biological agent is a *Pseudomonas heterophylla* inhibitor, and the active ingredient comprises *Bacillus belyssae* GJ5B6.

[0013] Furthermore, when the aforementioned biological agent is applied to control wilt disease of bayberry, the viable bacterial concentration of Bacillus belyceae GJ5B6 is ≥1×10⁻⁶. 8 CFU / mL.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Myrica wilt is an important disease of Myrica industry, and developing efficient biocontrol agents is the key to green prevention and control. The present application takes the rhizosphere soil of Myrica in Zhejiang Province as a sample, and obtains candidate bacteria by gradient dilution method. The strain Bacillus velezensis GJ5B6 with significant inhibitory activity against Myrica wilt pathogen is screened by plate confrontation test, which provides a theoretical basis and strain resources for the development of environmentally friendly biocontrol agents. The bio-agent prepared by using the active ingredient will not cause resistance of pathogenic bacteria, environmental pollution and pesticide residues, and is more green, environmentally friendly and safe than chemical control method.

[0015] (2) The inhibition rate of Bacillus velezensis GJ5B6 against B. andrographis is 91.21%, and the colony is milky white and gram-positive. The biochemical characteristics are consistent with Bacillus. In the in vitro leaf test, the number of diseased spots treated by 10 8 CFU / mL bacterial suspension is significantly reduced compared with the control group. In the pot experiment, the disease index of the GJ5B6 treatment group is as low as 25.99%, and the control effect is 59.50%. It has a wide application prospect in the prevention and control of Myrica wilt. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 It is a representative schematic diagram of the inhibition ability of Bacillus velezensis GJ5B6 against Myrica wilt pathogen.

[0017] Figure 2 It is a morphological characteristic diagram of Bacillus velezensis GJ5B6. The left diagram is the colony morphology diagram of GJ5B6, and the right diagram is the gram staining diagram of GJ5B6.

[0018] Figure 3 It is a phylogenetic tree of strain GJ5B6 based on 16S rRNA.

[0019] Figure 4 It is a statistical diagram of the inhibition rate of sterile fermentation filtrate of Bacillus velezensis GJ5B6 on myrica wilt pathogen mycelial growth.

[0020] Figure 5 It is a disease occurrence diagram of different treatment groups in the in vitro leaf test.

[0021] Figure 6 It is a potting tender leaf disease occurrence diagram of different treatment groups in the pot experiment.

[0022] Figure 7 It is a leaf disease occurrence diagram of different treatment groups in the pot experiment. DETAILED DESCRIPTION

[0023] To make the objectives, features, and advantages of this invention more apparent and understandable, a detailed description is provided below through specific embodiments. Many specific details are set forth in the following description to provide a thorough understanding of the invention. However, the invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below. Technical features in various embodiments of the invention can be combined appropriately without mutual conflict.

[0024] Unless otherwise specified, the operating methods in the following examples are generally performed under conventional conditions or as recommended by the manufacturer. Contents not described in detail in this specification are prior art known to those skilled in the art. Unless otherwise specified, the experimental materials used in the examples below can be purchased from conventional biochemical reagent companies.

[0025] Example 1 1. Experimental Materials 1.1 Test strains The pathogen of wilt disease in Chinese bayberry (Heteromorpha xJ27) was isolated and purified from diseased Chinese bayberry plants. The biocontrol bacteria were isolated from rhizosphere soil samples of Chinese bayberry at the research base of Zhejiang Academy of Agricultural Sciences. Both the pathogen and the biocontrol bacteria were preserved in the laboratory of the Institute of Horticulture, Zhejiang Academy of Agricultural Sciences.

[0026] 1.2 Test Culture Medium LB liquid medium: 5 g yeast extract, 10 g tryptone, 10 g NaCl, 1000 mL distilled water.

[0027] LB solid medium: 5 g yeast extract, 10 g tryptone, 10 g NaCl, 20 g agar, 1000 mL distilled water.

[0028] PDA medium: 20 g glucose, 200 g peeled potato, 20 g agar, 1000 mL distilled water.

[0029] NA medium: 3 g beef extract, 10 g peptone, 5 g NaCl, 20 g agar, 1000 mL distilled water.

[0030] MR-VP test medium: 5.0 g peptone, 5.0 g glucose, 5.0 g NaCl, 1000 mL distilled water; dispense 4-5 mL into each tube, pH 7.0-7.2.

[0031] Starch hydrolysis test medium: peptone 10 g, NaCl 5 g, beef extract 3 g, soluble starch 2 g, agar 15 g, distilled water 1000 mL, pH 7.0-7.2.

[0032] Citrate utilization medium: NaCl 1 g, MgSO4.7H2O 0.2 g, NH4H2PO4 0.5 g, sodium citrate 2 g, distilled water 1000 mL, 0.04% phenol red solution 20 mL; pH natural.

[0033] Nitrate reduction medium: peptone 10 g, glucose 10 g, beef extract 3 g, potassium nitrate 1 g, distilled water 1000 mL, pH 7.0-7.6.

[0034] Sugar fermentation medium: peptone 2 g, NaCl 5 g, K2HPO4 0.2 g, bromothymol blue (1% aqueous solution) 3 mL, glucose / sucrose / mannitol 10 g, distilled water 100 mL, pH 7.4.

[0035] Gelatin liquefaction medium: peptone 5 g, gelatin 100-150 g, distilled water 1000 mL, pH 7.2-7.4.

[0036] 2 Test method 2.1 Purification of biocontrol bacteria Using branches of ancient Yangmei trees with an age of more than 200 years in Zhejiang Province as samples, candidate bacteria were obtained by tissue isolation method. Biocontrol strains with good antibacterial effect that had been preliminarily screened were taken out from the bacterial preservation room of the Horticultural Institute of Zhejiang Academy of Agricultural Sciences. Single colonies with different shapes, colors, and sizes were picked and streaked on LB plates for purification, and the purified strains were stored. The purified single strains were inoculated in LB medium and cultured at 180 rpm and 28 °C for 24 h to collect bacterial cells, which were washed with phosphate buffer solution (PBS, pH 7.3) for 3 times, and the concentration of the bacterial solution was adjusted to 1×10 8 CFU / mL.

[0037] 2.2 Screening of biocontrol bacteria Activated Yangmei wilt fungus cakes (6 mm) were inoculated in the center of PDA medium. 5 mm sterilized filter paper pieces were attached at a distance of about 3.5 cm from the center of the pathogenic fungus, and 5 μL of bacterial solution was added on the filter paper pieces, with an equal volume of sterile water as a control. The culture dishes were placed in a 28 °C incubator for 7 d, and the antagonistic effect was determined. Each treatment was repeated 3 times, and the experiment was repeated 3 times. The diameters of the pathogenic fungus colonies in the treatment and control were measured by cross method, and the inhibition rate was calculated.

[0038] Bacteriostatic rate (%) = [(control colony diameter - treatment colony diameter)] / [(control colony diameter - bacterial cake diameter)] x 100%.

[0039] 2.3 Morphological identification and Gram staining of biocontrol bacteria The antagonistic strains screened were inoculated in LB liquid medium, and after 24 hours of culture at 28 °C, their colony morphology, pigment production around the colonies, and odor were observed. At the same time, the bacteria were picked for Gram staining, and the bacterial morphology was observed under a microscope to determine whether they were Gram-positive or Gram-negative bacteria.

[0040] 2.4 Physiological and biochemical identification of biocontrol bacteria The physiological and biochemical characteristics test was performed according to the Common Bacteria System Identification Manual.

[0041] (1) Contact enzyme experiment: the single colony of the antagonistic strain was picked with an inoculation needle and evenly smeared on a clean glass slide with 3% hydrogen peroxide on the surface. If bubbles appeared, it indicated that the contact enzyme reaction was positive, otherwise it was negative.

[0042] (2) Starch hydrolysis test: the antagonistic bacteria were spotted on starch culture medium, and after 2 days of culture at 28 °C, iodine solution was added and evenly covered on the culture medium plate. The appearance of a hydrolysis transparent circle around the colony indicated the production of amylase, which was a positive reaction. Otherwise, it was negative. The size of the transparent circle observed could be used to judge the amylase production capacity.

[0043] (3) Voges-Proskauer (V-P determination) experiment: the antagonistic bacteria were inoculated in MR-VP liquid medium, and after 2-4 days of static culture in a 28 °C incubator, equal amounts of strain fermentation broth and 40% sodium hydroxide solution were removed and contacted, a small amount of creatine was added and shaken evenly, and then left for a few minutes. If the solution turned red, the reaction was positive, otherwise it was negative.

[0044] (4) Methyl red experiment: the antagonistic bacteria were inoculated in MR-VP liquid medium, and after 2-4 days of static culture in a 28 °C incubator, one drop of methyl red reagent was added. If the solution turned red, the reaction was positive; if the solution turned yellow, the reaction was negative.

[0045] (5) Citrate utilization experiment: the antagonistic bacteria were inoculated in citrate liquid medium, and after 3-5 days of culture at 28 °C, the culture solution turned blue or pink, indicating that the antagonistic bacteria could utilize citrate, which was a positive reaction.

[0046] (6) Nitrate reduction experiment: the antagonistic bacteria were inoculated in nitrate liquid medium and cultured at 28 °C for 1, 3 and 5 days. One drop of Griess reagent A and B was added to the culture solution respectively. The pink, rose red and orange color of the culture solution indicated the presence of nitrite, which was a positive reaction. If the reaction was negative at this time, but the culture solution did not show blue reaction after adding one drop of diphenylamine reagent, it was also considered as a positive reaction of nitrate reduction.

[0047] (7) Gelatin liquefaction experiment: the selected strain was inoculated in gelatin medium and cultured at 28 °C for 48 h. The medium was placed in a refrigerator at 4 °C for 30 min. The presence of liquefaction in the medium was observed. The positive reaction was observed if the liquefaction occurred, otherwise the negative reaction was observed.

[0048] (8) Sugar fermentation test: the selected strain was inoculated in three kinds of sugar fermentation medium and cultured at 28 °C. The color change was observed at 24 h, 48 h and 72 h respectively. The positive reaction was observed if the culture solution showed yellow color, otherwise the negative reaction was observed.

[0049] 2.5 Molecular biological identification of the biocontrol bacteria (1) Extraction of genomic DNA of the biocontrol bacteria: The biocontrol bacteria liquid was taken in a 2 mL centrifuge tube. After centrifugation and removal of the supernatant, the genomic DNA was extracted according to the bacterial genomic extraction kit. The purity and concentration of the extracted DNA were measured by a spectrophotometer.

[0050] (2) Amplification of 16S rRNA of the biocontrol bacteria: The extracted DNA of the biocontrol bacteria was amplified by 1492R and 27F primers. The primer sequences were as follows: upstream primer 27F: 5'-AGAGTTTGATCMTGGCTCAG-3'; downstream primer 1492R: 5'-GGYTACCTTGTTACGACTT-3'. This combination could effectively amplify the 16S rRNA of the bacteria. The primers were synthesized by Genesee Biotech Co., Ltd. The PCR reaction system is shown in Table 1.

[0051] Table 1 PCR amplification reaction system

[0052] The PCR amplification program was as follows: pre-denaturation at 95 °C for 5 min, denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min, 30 cycles; extension at 72 °C for 7 min; The PCR products after amplification were detected by 1% agarose gel electrophoresis, and then the PCR products were sent to Genesee Biologics Co., Ltd. for DNA sequencing. The obtained sequence was compared with the NCBI database, the relevant gene sequence was downloaded in Genbank, the data was analyzed by MEGA 12 software, and the phylogenetic tree was constructed by the neighbor-joining method.

[0053] 3. Results and analysis 3.1 Screening of biocontrol bacteria The results of the plate confrontation method experiment showed that the GJ5B6 strain screened had strong inhibition effect on the mycelial growth of the Myrica moriella wilt pathogen, with an inhibition rate of 91.21% (Table 2). Figure 1 And Table 2), the next test will further identify and test the effect of GJ5B6 strain.

[0054] Table 2 Inhibition rate of antagonistic bacteria on Myrica moriella wilt pathogen

[0055] Note: The data in the table are mean ± standard deviation.

[0056] 3.2 Morphological observation and Gram reaction of biocontrol bacteria The GJ5B6 strain was semi-transparent in LB liquid medium, oval in shape, slightly viscous in texture, small and shiny in single colony, and purple in Gram staining result, with positive reaction. Figure 2 ).

[0057] 3.3 Physiological and biochemical identification of biocontrol bacteria The physiological and biochemical determination results of strain GJ5B6 are shown in Table 3. The physiological and biochemical determination results showed that the strain GJ5B6 could produce bubbles on the glass slide with the addition of H2O2, and the contact enzyme reaction was positive; the V-P determination, methyl red test and citrate reduction test results were all negative; the nitrate reduction test of strain GJ5B6 was negative reaction. In addition, the strain GJ5B6 showed positive reaction in sugar alcohol fermentation test and gelatin liquefaction test, and could hydrolyze starch.

[0058] Table 3 Physiological and biochemical test results

[0059] Note: "+" positive reaction; "-" negative reaction.

[0060] 3.4 Phylogenetic analysis of biocontrol bacteria The 16S rRNA gene of the test strain was amplified and sequenced, two groups of gene sequences were spliced using Snapgene software, the sequence was submitted to the GenBank database of NCBI, and the BLAST online sequence alignment was performed to obtain the DNA sequence with high consistency or high homology with the detection sequence. The phylogenetic tree was constructed by neighbor-joining method in MEGA12.0 software.

[0061] The analysis results show that the strain GJ5B6 and the Bacillus velezensis Bacillus velezensis are the same branch Figure 3 Therefore, combined with the morphological, physiological and biochemical characteristics of the strain, the homology and phylogeny of 16S rRNA sequence, the strain GJ5B6 is identified as Bacillus velezensis Bacillus velezensis .

[0062] The strain GJ5B6 was preserved in China Center for Type Culture Collection on September 24, 2025, with the preservation number CCTCC NO: M 20252105, and the address of the preservation unit is Luojia Mountain, China Center for Type Culture Collection, Wuhan, Hubei Province, China.

[0063] Example 2 1 Test method 1.1 Determination of mycelial growth inhibition effect of antagonistic strain The screened antagonistic GJ5B6 strain was streaked and purified on LB solid medium, a small amount of purified colony was inoculated into 100 mL of LB liquid medium, and the culture was incubated at 28 ℃ and 180 r / min for 24 h to obtain the fermentation liquid of the antagonistic strain. The fermentation liquid was transferred to a sterile 50 mL centrifuge tube and centrifuged at 4 ℃ and 11 000 r / min for 10 min. The supernatant was filtered with a 0.22 μm pore size filter membrane to obtain the sterile fermentation filtrate stock solution of the antagonistic strain. The sterile fermentation filtrate stock solution of the antagonistic strain was poured into a 250 mL conical flask containing 100 mL PDA medium at a temperature of 55 ℃ at a volume fraction of 2%, 4%, 6%, 8% and 10%, mixed thoroughly, and then poured into a plate. A 6 mm diameter Myrica nigrum wilt fungus (Pestalotiopsis versicolor) cake was inoculated in the center of each plate. The PDA plate with only Myrica nigrum wilt fungus and without addition of antagonistic strain sterile fermentation filtrate was used as a control. The plates were incubated at 25 ℃ for 7 d, the colony diameter was measured, and the inhibition rate was calculated. Each treatment was repeated 3 times.

[0064] Inhibition rate = (colony diameter of control - colony diameter of treatment) / (colony diameter of control - diameter of fungus cake) x 100%.

[0065] 1.2 In vitro leaf experiment The pathogen of wilt disease in waxberry (*Plasmodium heterophyllum*) was inoculated onto PDA solid medium plates and incubated at 28 °C inverted for 5-7 days for later use. A layer of sterile, water-moistened circular filter paper was placed in a glass petri dish. Fresh waxberry leaves of the same age (6-leaf stage) and size were taken, surface-sterilized with 75% ethanol for 30 seconds, rinsed 2-3 times with sterile water, and air-dried. Using a sterile needle, 2-3 punctures were made at the center of each leaf. Then, the leaves were sprayed with different concentrations of antagonistic bacterial suspension (1×10⁻⁶). 7 CFU / mL, 1×10 8 After drying the leaf surface, the leaves were placed in pre-prepared glass petri dishes. They were incubated for 24 hours to allow the biocontrol bacteria to spread on the leaves. Once the biocontrol solution was completely dry, the pathogen mycelial solution was sprayed onto the puncture site, and the base of the leaf stem was kept moist with a damp cotton ball. A control group was sprayed with sterile water. Each treatment consisted of 3 leaves, and the experiment was repeated 3 times. The leaves were incubated in a light incubator for 7 days (light / dark cycle 16 h / 8 h, 85% relative humidity). The number of lesions in each treatment was recorded to calculate the inhibition rate.

[0066] Inhibition rate (%) = [(Number of control lesions - Number of treated lesions) / Number of control lesions] × 100% 1.3 Pot Experiment The preserved pathogen (*Pseudomonas heterophylla*) was inoculated into PDA medium and cultured upside down at 28 °C until it covered the entire plate. The hyphae were then gently scraped off, diluted with sterile water, and filtered. The resulting filtrate was the hyphae suspension, which was stored at 4 °C for later use. The antagonistic bacterial strain was inoculated into Erlenmeyer flasks (50 mL) containing LB medium and cultured overnight at 28 °C and 180 r / min. The bacterial suspension concentration was adjusted to 10% using PBS buffer. 8 The antagonistic bacterial suspension is obtained by measuring CFU / mL.

[0067] Three-year-old bayberry plants were selected for pot experiments. Using the needle-pricking method, 5-6 wounds were made on the bayberry leaves. 10 mL of mycelial suspension was sprayed onto the pricked leaves of each plant. Four treatments were set up: 10 mL of mycelial suspension, 10 mL of antagonistic strain, and 10 mL of antagonistic strain. 8 CFU / mL 10 mL and CK (water control) 10 mL. Five pots were used for each treatment, with one bayberry plant in each pot. The leaf area and lesion area of ​​the bayberry were investigated, and the disease index and disease control effect were calculated.

[0068] Look for relevant information to design the grading standard of red bayberry wilt: 0 level: leaf intact without disease spot; 1 level: a small amount of disease spot (1%-5% of leaf area); 3 level: medium amount of disease spot (6%-10% of leaf area); 5 level: more amount of disease spot (11%-20% of leaf area); 7 level: a large amount of disease spot (21%-50% of leaf area); 9 level: a large amount of disease spot (51%-100% of leaf area).

[0069] Disease index = ∑[(number of plants at each level × value of the level) / (number of the highest level × total number of plants surveyed)] × 100%; Control effect = [(disease index of the control - disease index of the treatment) / disease index of the control] × 100%.

[0070] After 15 days of treatment, 15 leaves were investigated per pot, and a total of 5 pots of red bayberry were investigated, and the disease index of red bayberry was counted and the control effect was calculated.

[0071] 2. Results and analysis 2.1 Effect of antagonistic strains on mycelial growth The sterile fermentation filtrate of antagonistic strain GJ5B6 had an inhibitory effect on the mycelial growth of red bayberry wilt fungus, and the inhibitory effect increased with the increase of the volume fraction of the antagonistic bacteria sterile fermentation filtrate. When the volume fraction was 2%, the inhibitory effect was the lowest, with an inhibition rate of 36.77%, and when the volume fraction was 10%, the inhibitory effect was the best, with an inhibition rate of 92.33% ( Figure 4 ).

[0072] 2.2 In vitro leaf experiment To evaluate the protective effect of strain GJ5B6 on plant tissue, the effectiveness of biological control of plant tissue with different concentrations of bacterial liquid was evaluated. Red bayberry leaves showed obvious lesions 3 days after inoculation of the pathogen. After 7 days of inoculation, the red bayberry leaves of the control group of Pestalosphaeria xylarioides XJ27 showed a large number of lesions, and the leaves showed yellowing and other serious disease phenotypes ( Figure 5 ). In contrast, when the concentration of strain GJ5B6 was 1×10 7 CFU / mL, the inhibition rate was 69.23%, and when the concentration of the bacterial liquid was 1×10 8 CFU / mL, the inhibition rate was 76.92%. The results showed that strain GJ5B6 not only inhibited the growth and penetration of mycelium, but also limited the development of the disease, and the higher the concentration of the bacterial liquid, the better the control effect (Table 4). Therefore, in the subsequent pot experiment, the concentration of 1×10 8 CFU / mL of bacterial liquid was used for the experiment.

[0073] Table 4 Inhibitory effect of antagonistic strains at different concentrations on red bayberry wilt fungus

[0074] 2.3 Container seedling test To further determine the inhibitory effect of antagonistic bacteria on Myrica mori, strain GJ5B6 was subjected to container seedling control efficiency test. In the control group, the disease index was 64.18%, while the disease index of the biocontrol treatment group was 25.99%, and the control effect on Myrica mori reached 59.5%, which was better (Table 5 and Figure 6-7 ).

[0075] Table 5 Antagonistic bacteria on Myrica container seedling wilt control effect

[0076] Note: The data in the table is the average ± standard deviation.

[0077] Data analysis From the above embodiment content, it can be known that the present application selects the high-efficiency antagonistic strain GJ5B6 from the branches of ancient Myrica with an age of more than 200 years in Zhejiang Province through plate confrontation test, and the inhibition rate of the strain GJ5B6 on Myrica mori (Pseudomonas syringae) reaches 91.21%, which is significantly higher than that of the conventional biocontrol strain (usually <80%), indicating that the strain GJ5B6 has strong antagonistic potential. Pestalotiopsis

[0078] The strain GJ5B6 is a gram-positive bacterium with translucent colonies, and the physiological and biochemical characteristics include contact enzyme +, gelatin liquefaction + and sugar fermentation +.

[0079] Plant endophyte is an important biological control resource, the strain GJ5B6 belongs to Bacillus, has the biocontrol characteristics of secreting antibacterial substances and competing for nutrient sites, and can effectively inhibit most pathogenic bacteria, thereby laying a foundation for developing stable preparations. In addition, Bacillus has the advantages of easy production and convenient storage and use, and therefore occupies a large proportion in biological preparations.

[0080] When the concentration of the bacterial suspension is 10 8 CFU / mL, the inhibition rate of the strain GJ5B6 on the lesion expansion all reaches 76.92%, which is significantly better than that of 10 7 CFU / mL treatment (GJ5B6: 69.23%), and the high-concentration bacterial solution inhibits the infection of the pathogenic bacteria by rapidly colonizing the wound, thereby proving that the biocontrol agent dose is positively correlated with the control effect.

[0081] After 15 days of inoculation of the pathogenic bacteria, the disease index of the biocontrol treatment group was significantly lower than that of the control group (P<0.05). The results show that the spraying of 10 8 ​The bacterial suspension of CFU / mL: the disease index of GJ5B6 treatment group was reduced to 25.99, which was significantly lower than that of the control group (disease index 64.18). Among them, GJ5B6 showed better orchard applicability, and the yellowing of diseased leaves and the expansion rate of disease spots were significantly inhibited.

[0082] The above embodiments are used to illustrate the technical solutions of the present application in detail. It should be understood that the above description is only a specific embodiment of the present application and is not used to limit the present application. Any modification, supplement or similar replacement within the principle range of the present application should be included in the protection scope of the present application.

Claims

1. A type of Bacillus belye GJ5B6, characterized in that, The accession number is CCTCC NO: M 20252105, and it is deposited at the China Center for Type Culture Collection on September 24, 2025.

2. The application of Bacillus belyssus GJ5B6 according to claim 1 in the production of antibacterial active substances.

3. The application of Bacillus belye GJ5B6 according to claim 1 in inhibiting plant pathogens, characterized in that, The plant pathogen mentioned is *Heterophyllum heterophyllum*.

4. The application of Bacillus belye GJ5B6 according to claim 1 in the prevention and control of plant diseases caused by plant pathogens, characterized in that, The plant pathogen mentioned is *Heterophyllum heterophyllum*.

5. The application according to claim 4, characterized in that, The plant disease mentioned is bayberry wilt disease.

6. The application according to claim 3 or 4, characterized in that, Cultures, fermentation broths, or supernatants of Bacillus belye GJ5B6 obtained by culturing Bacillus belye GJ5B6 in culture media are used to inhibit plant pathogens or prevent plant diseases caused by plant pathogens.

7. A biological agent, characterized in that, The biological agent is a *Pseudomonas heterophylla* inhibitor, and the active ingredient includes *Bacillus belyssus* GJ5B6 as described in claim 1.

8. The biological agent according to claim 7, characterized in that, When the aforementioned biological agent is used to control wilt disease of bayberry, the viable bacterial concentration of Bacillus vesicularis GJ5B6 is ≥1×10⁻⁶. 8 CFU / mL.

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