Bacillus velezensis and application thereof in preventing and treating pine wilt disease

Abstract

The application discloses bacillus velezensis and application thereof in prevention and treatment of pine wilt disease, the bacillus velezensis is named bacillus velezensis QZ10-4, the preservation number is CGMCC No.30401, and the application is preserved in China General Microbiological Culture Collection Center on April 22, 2024.The strain QZ10-4 disclosed in the application not only has inhibiting effect on pine wilt disease fungus, but also has high inhibiting activity on various plant pathogenic bacteria, has broad-spectrum bacteriostasis, and has the potential of preventing and treating various plant diseases.

Description

Technical Field

[0001] This invention relates to a bacterium and its application, specifically to Bacillus belye and its application in the prevention and control of pine shoot blight. Background Technology

[0002] Pine twig dieback is one of the most widespread and common pine tree diseases worldwide, and was listed as an important forest disease in China in 1999. The pathogen, *Pinus pineneatus*, is one of the most common fungal pathogens in over 65 countries across Asia, Africa, Europe, Oceania, North America, and South America. Red pine, Scots pine, Masson pine, Chinese pine, and larch are the main hosts of pine twig dieback. Once these trees are infected, their branches will exhibit a series of symptoms such as dieback, cankers, and dead needles. *Pinus pineneatus* can directly invade young pine shoots, causing them to wither. As the disease progresses, non-current-year branches will also be affected, eventually leading to the death of the entire tree. Simultaneously, *Pinus pineneatus* reproduces through conidiophores during infection, further expanding the range of pine twig dieback.

[0003] Pine shoot blight is a host-dominant disease that typically infects weak or poorly growing trees. It is characterized by its insidious and sudden onset, causing infected pine trees to wither, develop stem cankers, die at the tips, exhibit blue staining, and even die. The pathogen, *Pinus pinensis*, is a common fungal pathogen that causes shoot blight and canker in naturally occurring and planted pine and other coniferous trees worldwide. Severe cases result in shoot death, causing considerable losses. Studies show that timber loss due to felling immature diseased trees reaches 28%, with potential timber loss reaching 55%. Over the years, the host range of pine shoot blight in China has been continuously expanding, and its damage has been increasing. Mild cases affect the growth of individual seedlings, while severe cases cause large-scale disease outbreaks and even death of trees, resulting in significant ecological damage and economic losses. It has become a major forest disease, making its prevention and control increasingly important.

[0004] Currently, based on the self-control capacity of the ecosystem and the epidemic pattern of pine blight, the main approach adopted is a comprehensive control technology that combines silvicultural control with biological and chemical control.

[0005] The review of pine dieback disease indicates that silvicultural control measures mainly include: removing dead, dying, and crushed trees from the forest floor; pruning the lower branches of severely diseased trees; removing diseased leaves; and sweeping up fallen diseased leaves and cones. It also includes creating mixed coniferous and broadleaf forests and isolation belts, avoiding large-scale pure pine plantations; and spraying Bordeaux mixture during the spring growing season. Research on pine dieback disease control strategies clarifies the importance of selecting resistant varieties, planting suitable trees in suitable locations, and strengthening quarantine measures. However, the application of silvicultural measures for pine dieback disease control is currently still theoretical and requires further promotion and practical application.

[0006] Chemical control is a practical and widespread method for pest and disease control in forestry, capable of quickly and effectively preventing the spread of diseases. Zhao Meiling et al. found that fungicides such as Senbao No. 1 + Miebingwei (1:2) at a dilution of 1000 times and sodium pentachlorophenate fumigant had good control effects on larch dieback. Chang Jiayu et al. found that most quaternary ammonium compounds containing dihydronorbutyl groups achieved inhibition rates of over 90% against *Pinus pinenulata* at a concentration of 50 mg / L, significantly exceeding the inhibitory effects of commonly used fungicides such as chlorothalonil and carbendazim. While chemical control can provide emergency control in severely affected forest stands, effectively controlling the continued spread of diseases in the short term and bringing significant benefits to forestry production, it also brings numerous drawbacks such as environmental pollution and disruption of the natural balance of the ecosystem.

[0007] Biological control is an important new disease control technology that neither impacts the ecological environment nor induces drug resistance in host plants. It is also a crucial component of integrated pest management, and its role in forestry pest and disease control is increasingly recognized, indicating a promising future for research in this area. Currently, biological control of pine blight mainly involves two aspects: screening antagonistic bacteria and isolating and extracting antimicrobial substances produced by biological metabolites. However, no commercially available biocontrol agents are currently available for the control of pine blight. Summary of the Invention

[0008] Purpose of the invention: The purpose of this invention is to address the important role of chemical control in integrated pest management. However, long-term use of stable chemical pesticides not only enhances the resistance of pathogens and reduces the effectiveness of control, but also pollutes agricultural products, air, soil, and water, endangering human and animal health and safety, as well as the ecological environment.

[0009] Due to the large area and tall trees in most pine forests, manual clearing of pine shoot blight is difficult, labor-intensive, inefficient, and costly, and its effectiveness is far inferior to other control measures. As a result, it remains largely theoretical.

[0010] Technical solution: The Bacillus velezensis described in this invention is named Bacillus velezensis QZ10-4, with accession number CGMCC No.30401, and was deposited at the China General Microbiological Culture Collection Center on April 22, 2024.

[0011] The application of Bacillus berleis in the prevention and control of pine shoot blight.

[0012] The aforementioned Bacillus belye appears blue-purple under a microscope and is a Gram-positive bacterium.

[0013] The method for culturing Bacillus belyssus uses a culture medium. The carbon source is selected from tryptone, sucrose, soluble starch, mannitol, glycerol, and corn flour. The nitrogen source is selected from soybean peptone, ammonium sulfate, beef extract, ammonia, peptone, and yeast extract. The inorganic salts are selected from sodium chloride, calcium carbonate, potassium dihydrogen phosphate, zinc sulfate, calcium chloride, and ferrous sulfate.

[0014] The culture method described uses TSB medium for culturing.

[0015] The application of *Bacillus belyssus* in the preparation of plant antibacterial agents that inhibit one or more of the following bacteria: *Pinus pinecone*, *Plasmodium spp.*, *Fusarium oxysporum*, *Fusarium graminearum*, *Botrytis cinerea*, *Alternaria ovalis*, *Pinus pinecone*, and *Alternaria ovalis*.

[0016] The application described herein is that the antibacterial agent is used in agriculture or forestry.

[0017] A fermentation broth obtained by fermenting and culturing the aforementioned Bacillus belye.

[0018] A sterile fermentation filtrate is obtained by centrifuging and filtering the supernatant of the Bacillus belye after fermentation culture.

[0019] A bacterial suspension is obtained by fermenting and culturing the aforementioned Bacillus belye, followed by centrifugation, precipitation, and resuspension with water.

[0020] The fermentation broth is prepared by co-fermenting Bacillus belye with trypsin, soybean peptone, and potassium dihydrogen phosphate.

[0021] The application of the fermentation broth, the sterile fermentation filtrate, or the bacterial suspension in the prevention and control of pine shoot blight.

[0022] The key points and points to be protected in this invention are: 1. QZ10-4 has strong antibacterial activity against pine shoot blight. The antibacterial rate can reach 62.34%, 76.99%, and 83.29% respectively after inoculation with bacteria at 0h, 24h, and 48h. The antibacterial rate can reach 100% after inoculation with pine cone spores 72h after bacterial inoculation.

[0023] 2. The aseptic fermentation filtrate of QZ10-4 still has an inhibitory effect on *Pinus pinecone* when diluted 200 times, and the inhibition rate remains above 90% when diluted 80 times or less.

[0024] 3. After optimization of the components of the culture medium, the antibacterial rate of QZ10-4 aseptic fermentation filtrate can reach 86.35% when diluted 100 times.

[0025] 4. In the potted plant efficacy test, QZ10-4 can reduce the symptoms of pine blight in the early stage of pine seedlings and basically stop the development of the disease in the later stage. Its bacterial suspension, fermentation broth and fermentation filtrate all have good control effects on Pinus pinensis, with relative control efficacy of over 70%.

[0026] Beneficial effects: (1) Biological control is an important new disease control technology that will not affect the ecological environment or cause drug resistance in host plants. It is also an important part of integrated management. Plant endophytic bacteria have a high survival space in the plant body, are not easily affected by external environmental conditions, reproduce quickly and in large numbers; and most bacteria can produce a variety of secondary metabolites, which have the effect of resisting pathogens and promoting plant growth. Bacillus has the advantages of secreting a variety of secondary metabolites, good root colonization ability, and large and stable sporulation. The bacteria screened in this experiment are endophytic bacteria of healthy pine needles, which can be identified as Bacillus belesii. (2) The strain QZ10-4 used in this study is an endophytic bacterium from Pinus massoniana in Quanjiao, Anhui Province. It is non-pathogenic to plants, has simple culture conditions, strong antibacterial activity, and good control effect on pine shoot blight. After culturing in LB solid medium for 24h, 48h, and 72h, the inhibition rate of *Pinus pinensis* mycelial growth can reach 76.99%, 83.29%, and 100%, respectively. The 72h fermentation filtrate of strain QZ10-4 has an extremely high inhibition rate against *Pinus pinensis*. After optimization of the culture medium composition, the aseptic fermentation filtrate can still achieve an inhibition rate of 86.35% against *Pinus pinensis* when diluted 200 times, showing potential for development and utilization as a biological pesticide. (3) Strain QZ10-4 not only has an inhibitory effect on pine shoot blight pathogens, but also has high antibacterial activity against a variety of plant pathogens, showing broad-spectrum antibacterial activity and potential for controlling a variety of plant diseases. It can be used for the development and application of biological control of various garden plant diseases. Tobacco trials, alfalfa trials, and antibiotic sensitivity tests are all important indicators for assessing the safety of potential biocontrol bacteria, and QZ 10-4 can be considered safe. (4) In pot experiments, the bacterial suspension, fermentation broth, and fermentation filtrate of strain QZ10-4 all showed good control effects against Pinoceros pinecone, with relative control efficacy reaching over 70%, and also showed good relative therapeutic effects. Attached Figure Description

[0027] Figure 1 Screening for antagonistic bacteria against Pinoceros pinecone;

[0028] Figure 2 The results of morphological observation of strain QZ10-4;

[0029] Figure 3 A phylogenetic tree of QZ10-4 constructed based on gyrb sequences;

[0030] Figure 4 The inhibitory effect of fermentation filtrate of strain QZ10-4 on *Pinococcus pineae*;

[0031] Figure 5 The effect of six different fermentation media on the inhibition rate;

[0032] Figure 6 The effect of different carbon sources on the antibacterial rate of fermentation filtrate;

[0033] Figure 7 The effect of different nitrogen sources on the antibacterial rate of fermentation filtrate;

[0034] Figure 8 The effect of different inorganic salts on the antibacterial rate of fermentation filtrate;

[0035] Figure 9 A comparative experiment was conducted to examine the antibacterial rate of bacterial strains before and after culture medium optimization.

[0036] Figure 10 For the bacterial safety testing of strain QZ10-4;

[0037] Figure 11 The results of the antibacterial broad-spectrum experiment for strain QZ10-4;

[0038] Figure 12 The results of pot control efficacy determination of strain QZ10-4 are as follows (A: CK1: spraying only sterile water, CK2: spraying only liquid LB medium, CK3: inoculation only with *Pinus pinecone*; B: inoculation with pathogen after 3 days of application of QZ10-4 bacterial suspension, fermentation broth, and sterile fermentation filtrate; C: application of QZ10-4 bacterial suspension, fermentation broth, and sterile fermentation filtrate 3 days after inoculation with pathogen). Detailed Implementation

[0039] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0040] Example 1

[0041] Isolation and screening of endophytic bacteria in pine needles

[0042] Take healthy pine needles, rinse with clean water, disinfect with 75% alcohol for 1 minute, and rinse three times with sterile water. Use the sterile water from the last rinse as a blank control and spread it on the surface of the culture medium. Take thoroughly sterilized sample tissue blocks and grind them thoroughly in a mortar. Prepare different concentrations of dilutions with sterile water. Spread 30 μL of the sample dilutions onto NA, LB, and PDA agar plates, spread evenly, and invert them in a constant temperature incubator at 28°C in the dark for 2–3 days. Pick colonies with different morphological characteristics and streak them on the culture medium. After purification three times, store for later use.

[0043] 188 bacterial strains were isolated and purified from healthy pine needles of *Pinus massoniana* using the dilution plating method. Using *Pinus pinenulata* A1-2 as a target, initial screening was performed using the plate confrontation method, yielding 8 endophytic bacteria with antagonistic effects, exhibiting inhibition rates between 62.34% and 74.38%. Because the pathogenic bacteria grew faster than the antagonistic bacteria, subsequent rescreening involved inoculating the pathogenic bacteria 24 h, 48 h, and 72 h after inoculation with the antagonistic bacteria, and the inhibition rates were calculated.

[0044] After secondary screening, four strains (QX3-4, QX5-2, QZ10-4, and QZ10-12) were selected as having strong antagonistic effects against the pathogen. Their inhibition rates reached 93.84-100% after 72 hours of inoculation with the antagonistic bacteria. Among them, QZ10-4 showed the strongest inhibitory effect against *Pinococcus pineae*, directly causing lethality to the pathogen after 72 hours of inoculation with the antagonistic bacteria.

[0045] Inhibition rate = (Control colony diameter - Treated colony diameter) / (Control colony diameter - Mycelial cake diameter) × 100%

[0046] Five-point confrontation method: *Chaetoceros pinecone* strains cultured for 3 days were selected. The five-point confrontation method was used to screen for strains with antibacterial activity. 5mm mycelial discs were prepared using a punch and inoculated into the center of PDA medium. 2μL of purified endophytic bacterial suspension was added dropwise from each of the four directions (top, bottom, left, and right) of the mycelial disc. Each treatment was repeated three times. LB liquid medium was added as a control group. After incubation at 25℃ for 3 days, the diameter of the pathogen colonies was measured using the cross-sectional method, and the inhibition rate was calculated. Strains with an inhibition rate greater than 50% were considered antagonistic bacteria and subsequently subjected to secondary screening.

[0047] Example 2

[0048] Identification of antagonistic bacteria

[0049] (1) Morphological identification

[0050] The selected antagonistic bacteria were streaked onto the corresponding culture medium plates and incubated at 28°C for 3 days. Once single colonies grew, the size, color, smoothness of the edges, smoothness of the surface, transparency, and elevation of the colonies were observed, and Gram staining was performed simultaneously.

[0051] The results are as follows Figure 2 The colonies of strain QZ10-4 are milky white, round, dull, translucent, and easily picked up. The colony surface has irregular small protrusions, smooth edges, and a pungent odor. Gram staining results show that strain QZ10-4 appears blue-purple under a microscope, indicating it is a Gram-positive bacterium.

[0052] (2) Physiological and biochemical identification

[0053] The physiological and biochemical test results of strain QZ10-4 are shown in Table 1. Based on the morphological characteristics and physiological and biochemical reaction characteristics, according to Bergey's Manual of Bacterial Identification, QZ10-4 was preliminarily identified as a species of Bacillus.

[0054] Table 1. Results of physiological and biochemical tests on strain QZ10-4

[0055]

[0056] Note: ("+" represents positive, "-" represents negative)

[0057] (3) Molecular biological identification

[0058] DNA of antagonistic bacteria was extracted using the freeze-thaw method, and the specific procedures are as follows:

[0059] (1) The selected single colonies were inoculated into liquid LB medium and cultured overnight at 28°C with shaking.

[0060] (2) Centrifuge 1 mL of bacterial culture at 12000 r / min for 1 min, discard the supernatant, and resuspend in 100 μL of ultrapure water. Incubate at 100℃ for 5 min, freeze at -20℃ for 10 min, repeat the above operation once, centrifuge again at 12000 r / min for 10 min, and collect the supernatant, which is the bacterial DNA. Measure the concentration and then perform PCR amplification.

[0061] The gyrb fragment of strain QZ10-4 was amplified and sequenced. The sequence was then compared with the NCBI database using BLAST. The results showed that strain QZ10-4 had the highest similarity to Bacillus velezensis, with a similarity of up to 88%.

[0062] Based on the combined morphological characteristics, physiological and biochemical properties, and molecular biological identification results, strain QZ10-4 was identified as Bacillus belye.

[0063] Example 3

[0064] Inhibitory effect of antagonistic bacteria sterile fermentation filtrate on *Pinococcus pineae*

[0065] The strain was streaked onto LB solid medium and activated at 28°C for 24 h. Single colonies were picked and inoculated into LB liquid medium and cultured at 28°C with shaking at 180 rpm for 12 h to obtain a seed culture. The seed culture was then cultured in shake flasks at 28°C with shaking at 180 rpm for 72 h at an inoculation rate of 0.2% and a liquid volume of 50%, followed by centrifugation at 10000 rpm for 15 min. The supernatant was filtered through a 0.22 μm bacterial filter to obtain a sterile fermentation filtrate. The fermentation filtrate was mixed with PDA medium and diluted 5-500 times to prepare plates. After the medium solidified, pathogenic bacterial cakes were inoculated into the center of the plates, with the mixed LB liquid medium serving as a control. The inhibition rate of the strain's fermentation filtrate against *Chaetoceros pinecone* was calculated.

[0066] Depend on Figure 4 It can be seen that the aseptic fermentation filtrate of strain QZ10-4 has a high inhibition rate of 100% against *Bacillus pinecone* when diluted 5-80 times, and still has a certain inhibitory effect on *Bacillus pinecone* when diluted 200 times.

[0067] Preparation of fermentation broth: The strain was streaked onto LB solid medium and activated at 28°C for 24 h. A single colony was picked and inoculated into LB liquid medium and cultured at 28°C with shaking at 180 r / min for 12 h to obtain the seed culture. The seed culture was then cultured in shake flasks at 28°C with shaking at 180 r / min for 72 h to obtain the fermentation broth of the strain.

[0068] Preparation of bacterial suspension: The above-obtained bacterial fermentation broth is centrifuged at 5000 r / min for 10 min, the supernatant is discarded, and the resulting precipitate is resuspended in an appropriate volume of sterile water to obtain the bacterial suspension.

[0069] Example 4

[0070] Optimization of fermentation media for antagonistic bacteria

[0071] (1) Screening of basic fermentation culture medium

[0072] Based on literature review, the following six culture media were selected for screening of basic culture media, with the inhibition rate as the screening index.

[0073] Modified culture medium 1: glucose 20.0 g / L, soluble starch 20.0 g / L, ammonium chloride 10.0 g / L, peptone 10.0 g / L, magnesium sulfate 1.0 g / L;

[0074] Modified culture medium 2: sucrose 30.0 g / L, ammonium sulfate 10.0 g / L, yeast extract 3.0 g / L, potassium dihydrogen phosphate 0.3 g / L, magnesium sulfate 5.0 g / L;

[0075] TSB: Tryptone 15.0 g / L, Soy Peptone 5.0 g / L, Sodium Chloride 5.0 g / L;

[0076] NA: Beef extract 3.0 g / L, peptone 10.0 g / L, sodium chloride 5.0 g / L;

[0077] Modified culture medium 5: glucose 10.0 g / L, peptone 5.0 g / L, yeast extract 8.0 g / L, potassium chloride 5.0 g / L;

[0078] Modified medium 6: peptone 20.0 g / L, dipotassium hydrogen phosphate 1.5 g / L, magnesium sulfate heptahydrate 1.5 g / L.

[0079] Bacillus QZ10-4 was cultured in 50 mL Erlenmeyer flasks at 28°C with an inoculum of 0.2% at 50% broth volume and a rotation speed of 200 rpm for 72 hours to obtain fermentation filtrate. The filtrate was then diluted 200 times with PDA medium and plated. Each treatment was repeated 3 times. The diameter of the pathogen colony was measured, the inhibition rate was calculated, and the optimal basal fermentation medium was determined.

[0080] The screening results of the basic fermentation medium are as follows: Figure 5 As shown in the figure, among the six selected basal media, strain QZ10-4 showed the highest inhibition rate on TSB medium, therefore TSB medium was chosen as the basal medium for media optimization.

[0081] (2) Optimization of different components of the culture medium

[0082] Several carbon sources (glucose, sucrose, soluble starch, mannitol, and corn flour), nitrogen sources (protein, yeast extract, beef extract, soybean protein, ammonium sulfate, ammonia, and yeast extract), and inorganic salts (potassium dihydrogen phosphate, calcium carbonate, calcium chloride, ferrous sulfate, and zinc sulfate) were selected and replaced with equal amounts of the corresponding components in the basal culture medium. Bacillus QZ10-4 was cultured in 50 mL Erlenmeyer flasks at 50% concentration, 0.2% inoculum, 200 rpm, and 28°C for 72 hours. Following the steps in Example 3, sterile fermentation filtrate was prepared and diluted 200 times with PDA medium, then plated. Each treatment was repeated three times. The diameter of the pathogen colonies was measured, the inhibition rate was calculated, and the optimal carbon source, nitrogen source, and inorganic salts were determined.

[0083] Depend on Figure 6 It can be seen that when using TSB medium as the basic fermentation medium, the fermentation filtrate of strain QZ10-4 showed the highest inhibition rate against *Chaetoceros pinecone*, reaching 45.51%, when tryptone was used as the carbon source. Therefore, tryptone should be selected as the optimal carbon source for the medium in which strain QZ10-4 antagonizes *Chaetoceros pinecone*.

[0084] Depend on Figure 7 It can be seen that, based on the TSB medium optimized with carbon source as the fermentation medium, the fermentation filtrate of strain QZ10-4 showed the highest inhibition rate against *Chaetoceros pinecone*, reaching 75.87%, when soybean peptone was used as the nitrogen source. Therefore, soybean peptone should be selected as the optimal nitrogen source for the medium in which strain QZ10-4 antagonizes *Chaetoceros pinecone*.

[0085] Depend on Figure 8 It can be seen that, based on the TSB medium optimized with carbon and nitrogen sources as the fermentation medium, the fermentation filtrate of strain QZ10-4 showed the highest inhibition rate against *Pinococcus pineae*, reaching 84.44%, when potassium dihydrogen phosphate was used as the inorganic salt. Therefore, potassium dihydrogen phosphate should be selected as the optimal inorganic salt for the medium in which strain QZ10-4 antagonizes *Pinococcus pineae*.

[0086] Figure 9 The results show that after optimization of each component of the culture medium, the antibacterial rate of QZ10-4 aseptic fermentation filtrate increased from 23.89% to 86.35% when diluted 200 times.

[0087] Example 5

[0088] Bacterial safety testing

[0089] like Figure 10 As shown, alfalfa and tobacco experiments were conducted on QZ10-4, with sterile water added to the control group. The results all demonstrated that the QZ10-4 bacterial solution did not have any adverse effects on the growth of tobacco and alfalfa, indicating that QZ10-4 does not pose a biosafety risk.

[0090] The sensitivity of bacterial strains to antibiotics is an important indicator for evaluating their safety. QZ10-4 showed sensitivity to all 10 antibiotics within the quality control range, and was highly sensitive to 9 of them (inhibition zones exceeding 15 mm in diameter). Detailed drug sensitivity results are shown in Table 1.

[0091] Table 2. Antimicrobial susceptibility test results of strain QZ10-4

[0092]

[0093] s. Highly sensitive I. Moderately sensitive R. Drug resistant

[0094] 1. Broad-spectrum antibacterial activity

[0095] Under the optimized fermentation conditions described above, fermentation broth was obtained. Seven plant pathogens (*Polytrichum gloeosporioides*, *Fusarium oxysporum*, *Fusarium graminearum*, *Botrytis cinerea*, *Alternaria ovalis*, *Discocephala pineinae*, and *Alternaria*) were tested as inhibitory targets, and the inhibition rates were determined using the method described in Example 4. The inhibitory effects of strain QZ10-4 on the seven tested plant pathogens were statistically analyzed, and all showed good inhibitory activity (see Example 4). Figure 11 QZ10-4 has broad-spectrum antibacterial activity.

[0096] 2. QZ10-4 potted plant efficacy

[0097] Bacterial fermentation broth, sterile fermentation filtrate, and bacterial suspension of QZ10-4 were prepared separately, with the bacterial concentration of the fermentation broth and bacterial suspension adjusted to OD600 = 0.8. Among them, (1) the treatment method for the efficacy determination test: two-year-old healthy Masson pine potted seedlings with similar growth were taken, and 2 ml of each of the QZ10-4 fermentation broth, sterile fermentation filtrate, and bacterial suspension were evenly sprayed on the surface of Masson pine needles. After 3 days, 2 ml of Pinus massoniana mycelial liquid was evenly sprayed on each treatment; (2) the treatment method for the efficacy determination test: two-year-old healthy Masson pine potted seedlings with similar growth were taken, and 2 ml of Pinus massoniana mycelial liquid was evenly sprayed on each treatment. After 3 days, 2 ml of each of the QZ10-4 bacterial fermentation broth, sterile fermentation filtrate, and bacterial suspension were evenly sprayed on the surface of Masson pine needles. The control was sprayed with an equal amount of sterile water and liquid LB medium. Moisturizing treatment was carried out, and each treatment was repeated 4 times. Record the severity of illness 30 days after vaccination.

[0098] The severity of the disease is divided into four levels: Level 0 is when the whole plant is disease-free, Level 1 is when the whole plant has ≤25% yellowing, Level 2 is when the whole plant has ≥25.1% to 50% yellowing, Level 3 is when the whole plant has ≥59.1% to 75% yellowing, and Level 4 is when the whole plant has ≥75.1% yellowing.

[0099] Disease index = 100 × ∑(number of diseased plants at each level × representative value at each level) / (total number of plants surveyed × highest representative value)

[0100] Relative control effect = 100 × (Disease index of pathogen only - Disease index of combined application of biocontrol bacteria and pathogen) / Disease index of pathogen only

[0101] The pot control efficacy test results of strain QZ10-4 are shown below. Figure 12 As shown in Table 3, the incidence rate of Masson pine seedlings inoculated with the pathogen reached 100%. The bacterial suspension, fermentation broth, and sterile fermentation filtrate of QZ10-4 all showed good control effects against Pinus pinecone.

[0102] Table 3. Pot control efficacy of strain QZ10-4

[0103] Handling method Incidence rate / % Disease index Relative prevention and control effect / % Inoculate with pathogens 3 days after applying QZ10-4 bacterial suspension. 25 6.25 90.90％ Inoculate with pathogens 3 days after applying QZ10-4 fermentation broth. 50 18.75 72.72 Inoculate with pathogens 3 days after applying QZ10-4 aseptic fermentation filtrate. 25 6.25 90.9 Administer QZ10-4 bacterial suspension 3 days after inoculation with pathogens 50 31.25 54.54 Three days after inoculation with pathogens, QZ10-4 fermentation broth was applied. 75 43.75 36.36 Three days after inoculation with pathogens, apply QZ10-4 aseptic fermentation filtrate. 50 12.5 81.81 CK3 100 68.75 /

Claims

1. A strain QZ10-4, characterized by: Taxonomically named Bacillus belesii ( Bacillus velezensis The specimen, with accession number CGMCC No.30401, was deposited at the China General Microbiological Culture Collection Center on April 22, 2024.

2. The Bacillus belye as described in claim 1 appears blue-purple under a microscope and is a Gram-positive bacterium.

3. The application of Bacillus berberis as described in claim 1 in the prevention and control of pine shoot blight.

4. The method for culturing Bacillus belye according to claim 1, characterized in that: The culture was carried out using TSB medium, with trypsin as the carbon source, soybean peptone as the nitrogen source, and potassium dihydrogen phosphate as the inorganic salt.

5. The use of Bacillus berreatus as described in claim 1 in the preparation of a plant antibacterial agent that inhibits one or more of the following bacteria: Corylus pinecone, Corylus pseudodisparus, Fusarium oxysporum, Fusarium graminearum, Botrytis cinerea, Alternaria ovalis, Corylus pinecone, and Alternaria.

6. A fermentation broth, characterized in that: It is obtained by fermenting and culturing the Bacillus berleis described in claim 1.

7. The fermentation broth according to claim 6, characterized in that: The aforementioned Bacillus belye was co-fermented with trypsin, soybean peptone, and potassium dihydrogen phosphate.

8. A sterile fermentation filtrate, characterized in that: It is obtained by fermenting and culturing the Bacillus berberis described in claim 1, followed by centrifugation and filtration of the supernatant through a bacterial filter.

9. A bacterial suspension, characterized in that: It is obtained by fermenting and culturing the Bacillus berberis described in claim 1, followed by centrifugation, precipitation, and resuspension in water.

10. The application of the fermentation broth of claim 6, the aseptic fermentation filtrate of claim 8, or the bacterial suspension of claim 9 in the prevention and control of pine shoot blight.

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