Bacillus velezensis and application thereof in prevention and treatment of pine blight

Abstract

The bacillus velezensis is named as bacillus velezensis QZ10-4, the preservation number of the bacillus velezensis QZ10-4 is CGMCC (China General Microbiological Culture Collection Center) No.3040l, and the bacillus velezensis is preserved in China General Microbiological Culture Collection Center on April 22, 2024. The strain QZ10-4 disclosed by the invention not only has an inhibition effect on the oxysporum brassicae, but also has relatively high bacteriostatic 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] The invention relates to a bacterium and an application thereof, in particular to a Bacillus Velezii and an application thereof in preventing and treating pine dieback disease. Background Art

[0002] Pine dieback is one of the most common and widespread pine diseases in the world. It was listed as an important forest disease in China in 1999. The pathogen of Aspergillus niger is one of the most common fungal pathogens in more than 65 countries including Asia, Africa, Europe, Oceania, North America and South America. Red pine, Scots pine, Masson pine, Chinese pine, larch and other tree species are the main hosts of pine dieback. Once these trees are infected, their branches will show a series of symptoms such as dieback, ulcer spots and dead needles. Aspergillus niger can directly invade the tender shoots of pine trees, causing the tender shoots to wilt. As the symptoms of the disease continue to deepen, branches that are not born that year will also be affected, eventually leading to the death of the entire tree. At the same time, Aspergillus niger will reproduce through conidiophores during the infection process, further expanding the scope of pine dieback.

[0003] Pine dieback is a host-dominant disease that usually infects trees with poor growth and weak vigor. It is hidden and sudden. Infected pine branches wither, stem ulcers, dead tops, blue discoloration and even death. Its pathogen Aspergillus pinaster is a common fungal pathogen that can cause dieback and ulcers in naturally occurring and planted pine and other coniferous trees around the world. In severe cases, branches and branches die back, causing considerable losses. According to research, the loss of timber caused by felling immature diseased trees due to the disease is as high as 28%, and the potential loss of timber is as high as 55%. Over the years, the host range of pine dieback has continued to expand in China, and the damage has continued to increase. In mild cases, it affects the growth of individual seedlings, and in severe cases, it causes large-scale disease and even death of trees, causing major ecological damage and economic losses. It has become a major forest disease, so the prevention and control of the disease is becoming increasingly important.

[0004] At present, based on the self-control ability of the ecosystem and the epidemic patterns of pine dieback disease, the main prevention and control technology adopted is a comprehensive prevention and control technology that focuses on forestry prevention and control and combines biological control and chemical control.

[0005] In the review of pine dieback, it is pointed out that the main measures for afforestation and prevention of pine dieback include: clearing dead trees, dying trees and depressed trees in the forest, pruning the lower branches of seriously diseased plants, removing diseased leaves, cleaning up fallen diseased leaves and cones, etc.; creating mixed coniferous and broad-leaved forests and isolation belts to avoid planting large areas of pure pine forests; at the same time, Bordeaux liquid can be sprayed during the spring growth period. In the study of pine dieback prevention and control strategies, it is clear that the prevention and control of this disease should pay attention to the selection of planting resistant varieties, suitable trees for suitable places, and strengthening quarantine. However, the current application of afforestation measures for the prevention and control of pine dieback is still in theory, and further promotion and practice are needed.

[0006] Chemical control is a practical and common method of pest control in forestry, which can quickly and timely prevent the spread of diseases. Zhao Meiling et al. found that fungicides such as Senbao No. 1 + Miebingwei (1:2) 1000 times liquid and sodium pentachlorophenol smoke agent have good control effects on larch dieback. Chang Jiayu et al. found that most quaternary ammonium salt compounds containing dihydrogenated nopyl have an inhibition rate of more than 90% on pinocytic Aspergillus at a mass concentration of 50 mg / L, and the inhibition effect is much higher than the commonly used fungicides thiophanate-methyl and carbendazim. Chemical control can play an emergency control role in forest stands with serious disease, and control the continued spread of diseases in a short period of time. Although this has brought high benefits to forestry production, many disadvantages such as environmental pollution and destruction of the natural balance of the ecosystem have also followed.

[0007] Biological control is an important new disease control technology that neither affects the ecological environment nor produces drug resistance in host plants. It is also an important part of comprehensive management. People are gradually paying attention to its role in forestry pest control. The research on biological control has a good development prospect. At present, the biological control of pine dieback is mainly carried out from two aspects, namely the screening of antagonistic bacteria and the separation and extraction of antibacterial substances produced by biological metabolism. However, there are currently no commercial biocontrol agents used in the control of pine dieback. Summary of the invention

[0008] Purpose of the invention: The purpose of the present invention is that chemical control plays an important role in the integrated pest control. However, the long-term use of stable chemical pesticides will not only enhance the resistance of pathogens and reduce the control effect, but also pollute agricultural products, air, soil and water, endangering the health and safety of humans and animals and the ecological environment.

[0009] As most pine forests are large and the trees are tall, manual clearing is difficult, labor-intensive, inefficient and costly, and the effects are not as good as other prevention and control measures. Therefore, it is difficult to implement and remains largely at the theoretical level.

[0010] Technical solution: The Bacillus velezensis described in the present invention is named Bacillus velezensis QZ10-4, with a preservation number of CGMCC No.30401, and was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on April 22, 2024.

[0011] The application of the Bacillus Velez in preventing and treating pine dieback.

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

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

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

[0015] The Bacillus Velezii is used in the preparation of a plant antibacterial agent for inhibiting one or more bacteria selected from Conchosporon pinecones, Polytrichomonas pseudodiscus, Fusarium oxysporum, Fusarium graminearum, Botrytis cinerea, Alternaria ovata, Discospora pine needles, and Alternaria alternata.

[0016] The application, the antibacterial agent is used in agriculture or forestry.

[0017] A fermentation liquid is obtained after fermenting and culturing the Velez bacillus.

[0018] The invention discloses a sterile fermentation filtrate, which is obtained by fermenting and culturing the Velez bacillus, centrifuging the filtrate and filtering the supernatant with a bacteria filter.

[0019] A bacterial suspension is obtained by fermenting and culturing the Velez bacillus, centrifuging and precipitating the bacterium and then resuspending the bacterium in water.

[0020] The fermentation liquid, the Velez bacillus, tryptone, soy peptone and potassium dihydrogen phosphate are fermented and cultured together.

[0021] The application of the fermentation liquid, the sterile fermentation filtrate or the bacterial suspension in preventing and treating pine dieback disease.

[0022] The key points and points to be protected of the present invention are: 1. QZ10-4 has strong antibacterial activity against Pinus massoniana dieback disease, and the antibacterial rates can reach 62.34%, 76.99% and 83.29% respectively when inoculated with Conchocera pine at 0h, 24h and 48h after inoculation of bacteria, and the antibacterial rate can reach 100% when inoculated with Conchocera pine 72h after inoculation of bacteria.

[0023] 2. The sterile fermentation filtrate of QZ10-4 still had an inhibitory effect on Aspergillus niger when diluted 200 times, and the inhibition rate remained above 90% when diluted 80 times or less.

[0024] 3. After the components of the culture medium were optimized, the antibacterial rate of the QZ10-4 sterile fermentation filtrate could reach 86.35% when diluted 100 times.

[0025] 4. In the potted plant efficacy test, QZ10-4 can alleviate the symptoms of pine dieback disease in Masson pine seedlings in the early stage, and basically stop the development of the disease in the later stage. Its bacterial suspension, fermentation liquid and fermentation filtrate have good control effects on Ascochyta pinaster, and the relative control efficacy is more than 70%.

[0026] Beneficial effects: (1) Biological control is an important new disease control technology that will neither affect the ecological environment nor produce drug resistance in host plants. It is also an important part of comprehensive management. Endophytic bacteria in plants have a high living 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 fighting pathogens and promoting plant growth. Bacillus has the advantages of being able to secrete a variety of secondary metabolites, good root colonization ability, and large and stable spore production. The bacteria screened in this experiment are endophytic bacteria of healthy Pinus massoniana needles, and they can be identified as Bacillus Velezii. (2) The strain QZ10-4 used in this study is an endophytic bacterium from Pinus massoniana in Quanjiao, Anhui. It is non-pathogenic to plants, has simple culture conditions, strong antibacterial activity, and good prevention effect on pine dieback. After culturing in LB solid medium for 24h, 48h, and 72h, the inhibition rate of mycelial growth of Aspergillus niger can reach 76.99%, 83.29%, and 100%, respectively; the 72h fermentation filtrate of strain QZ10-4 has a very high inhibition rate on Aspergillus niger. After the optimization of the culture medium components, the inhibition rate of its sterile fermentation filtrate on Aspergillus niger can still reach 86.35% when diluted 200 times, which has the potential for development and utilization as a biological pesticide. (3) Strain QZ10-4 not only has an inhibitory effect on pine dieback pathogens, but also has high antibacterial activity against a variety of plant pathogens. It has a broad spectrum of antibacterial activity and has the potential to prevent and control a variety of plant diseases. It can be used for the development and application of biological control of a variety of garden plant diseases. Tobacco tests, alfalfa tests, and antibiotic sensitivity tests are all important indicators for evaluating the safety of potential biocontrol bacteria, and QZ10-4 can be considered safe. (4) In pot experiments, the bacterial suspension, fermentation liquid, and fermentation filtrate of strain QZ10-4 all had good control effects on Aspergillus niger, with relative control rates exceeding 70%, and also had good relative efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Screening of antagonistic bacteria against Aspergillus pinecone;

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

[0029] Figure 3 Phylogenetic tree constructed for QZ10-4 based on gyrb sequence;

[0030] Figure 4 The inhibitory effect of the fermentation filtrate of strain QZ10-4 on Aspergillus pinocene.

[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] Fig. 9 This is a comparative test of the antibacterial rate of the strains before and after the optimization of the culture medium;

[0036] Fig.10 It is a bacterial safety test for strain QZ10-4;

[0037] Fig.11 The results of the broad-spectrum antibacterial experiment of strain QZ10-4 are shown;

[0038] Fig.12 These are the results of potted plant protection efficacy test of strain QZ10-4 (A: CK1: spraying only sterile water, CK2: spraying only liquid LB medium, CK3: inoculating only Aspergillus niger; B: applying QZ10-4 bacterial suspension, fermentation broth, and sterile fermentation filtrate for 3 days before inoculating the pathogen; C: applying QZ10-4 bacterial suspension, fermentation broth, and sterile fermentation filtrate for 3 days after inoculating the pathogen). DETAILED DESCRIPTION

[0039] The technical solution of the present invention is further illustrated and described below through specific implementation modes in combination with the accompanying drawings.

[0040] Example 1

[0041] Isolation and Screening of Endophytic Bacteria from Pine Needles

[0042] Take healthy Masson pine needles, rinse with water, disinfect with 75% alcohol for 1 minute, rinse with sterile water 3 times, and take the sterile water from the last rinse as a blank control and apply it on the surface of the culture medium. Take the sample tissue blocks with thorough surface sterilization and put them into a mortar to grind them thoroughly, and use sterile water to prepare the grinding liquid into dilutions of different concentrations. Take 30μL of the sample dilution and apply it to NA, LB, and PDA culture medium plates respectively. After evenly applying, invert and place in a constant temperature incubator at 28℃ in the dark for 2 to 3 days. Pick colonies with different morphological characteristics and streak them on the culture medium. After purification 3 times, store them for later use.

[0043] By dilution plate method, 188 strains of bacteria were isolated and purified from healthy pine needle samples of Masson pine. Using Aspergillus niger A1-2 as the target, a total of 8 strains of endophytic bacteria with antagonistic effects were obtained by initial screening by plate confrontation method, with an inhibition rate of 62.34-74.38%. Since the growth rate of pathogens is faster than that of antagonistic bacteria, the subsequent re-screening selected the pathogens to be inoculated 24h, 48h, and 72h after the inoculation of antagonistic bacteria, and the inhibition rate was calculated.

[0044] After repeated screening, a total of 4 strains (QX3-4, QX5-2, QZ10-4, QZ10-12) with strong antagonistic effects on pathogens were screened out in this experiment. The antibacterial rates reached 93.84-100% when the antagonistic bacteria were first inoculated for 72 hours. Among them, QZ10-4 had the strongest inhibitory effect on Aspergillus niger, and could directly kill the pathogen when the antagonistic bacteria were first inoculated for 72 hours.

[0045] Inhibition rate = (control colony diameter - treated colony diameter) / (control colony diameter - bacterial cake diameter) × 100%

[0046] Five-point confrontation method: Select the pine cone fungus that has been cultured for 3 days, and use the five-point confrontation method to screen strains with antibacterial activity. Use a hole puncher to prepare a 5mm bacterial cake and inoculate it in the center of the PDA culture medium. Add 2μL of purified endophytic bacterial solution in the four directions of the upper, lower, left, and right of the bacterial cake. Repeat each treatment 3 times, and add LB liquid culture medium as a control group. After constant temperature culture at 25℃ for 3 days, the cross method is used to measure the colony diameter of the pathogen and calculate the inhibition rate. Among them, the strain with an inhibition rate greater than 50% is the antagonistic bacteria, and then re-screening is carried out.

[0047] Example 2

[0048] Identification of antagonistic bacteria

[0049] (1) Morphological identification

[0050] The selected antagonistic bacteria were streaked onto the corresponding culture medium plate and cultured at 28°C for 3 days. When single colonies grew out, the colony size, color, edge flatness, surface smoothness, transparency and ridges were observed, and Gram staining was performed at the same time.

[0051] The results are as follows Figure 2 The colonies of strain QZ10-4 are milky white, round, matte, translucent and easy to pick up. There are irregular small protrusions on the surface of the colonies, with smooth edges and a pungent smell. The results of Gram staining showed that strain QZ10-4 appeared blue-purple under a microscope and was a Gram-positive bacterium.

[0052] (2) Physiological and biochemical identification

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

[0054] Table 1 Physiological and biochemical test results of strain QZ10-4

[0055]

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

[0057] (3) Molecular biological identification

[0058] The freeze-thaw method was used to extract the antagonistic bacterial DNA. The specific operation was as follows:

[0059] (1) Inoculate the screened single colony into liquid LB medium and culture at 28°C with shaking overnight.

[0060] (2) Take 1 mL of bacterial solution and centrifuge at 12000 r / min for 1 min, discard the supernatant, and resuspend in 100 μL of ultrapure water. Place in a 100°C water bath for 5 min, freeze at -20°C for 10 min, repeat the above steps, centrifuge again at 12000 r / min for 10 min, and take 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. It was found that strain QZ10-4 had the highest similarity with Bacillus velezensis, with a similarity of 88%.

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

[0063] Example 3

[0064] Inhibitory Effects of Antagonistic Bacteria Aseptic Fermentation Filtrate on Aspergillus pinocene

[0065] The strain was streaked and inoculated on LB solid medium, cultured and activated at 28℃ for 24h, a single colony was picked and inoculated in LB liquid medium, cultured at 28℃, 180r / min for 12h to obtain seed liquid, the seed liquid was cultured in a flask at 0.2% inoculation volume and 50% liquid volume at 28℃, 180r / min shaking for 72h, and centrifuged at 10000r / min for 15min. The supernatant was filtered and sterilized using a 0.22μm bacterial filter to obtain a sterile fermentation filtrate, the fermentation filtrate was mixed with PDA medium, and prepared into plates diluted 5-500 times. After the culture medium solidified, the pathogen cake was inoculated in the center of the plate, and the mixed LB liquid medium was used as a control. The antibacterial rate of the strain fermentation filtrate against Aspergillus pine cones 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 on Conchocera pinecone when diluted 5-80 times, all reaching 100%, and still has a certain inhibitory effect on Conchocera pinecone when diluted 200 times.

[0067] Preparation of fermentation broth: streak the strain on LB solid culture medium, activate it at 28°C for 24 h, pick a single colony and inoculate it into LB liquid culture medium, culture it at 28°C, 180 r / min for 12 h to obtain seed solution, culture the seed solution in a shake flask at 0.2% inoculation volume and 50% filling volume, and culture it at 28°C, 180 r / min for 72 h to obtain the strain fermentation broth.

[0068] Preparation of bacterial suspension: centrifuge the fermentation broth of the strain obtained above at 5000r / min for 10min, discard the supernatant, and resuspend the obtained precipitate with a corresponding volume of sterile water to obtain a strain suspension.

[0069] Example 4

[0070] Optimization of Fermentation Medium for Antagonistic Bacteria

[0071] (1) Screening of basic fermentation medium

[0072] By consulting the literature, the following 6 culture media were selected for screening of basic culture media, with the inhibition rate as the screening index.

[0073] Improved 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] Improved 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] Improved 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] Improved culture 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 shaken in a 50 mL Erlenmeyer flask in the above basic fermentation medium at a liquid volume of 50%, an inoculation amount of 0.2%, a rotation speed of 200 r / min, and 28°C for 72 h to obtain the fermentation filtrate, which was diluted 200 times with PDA medium and poured onto plates. Each treatment was repeated 3 times, the colony diameter of the pathogen was determined, the inhibition rate was calculated, and the optimal basic fermentation medium was determined.

[0080] The screening results of basic fermentation medium are as follows Figure 5 Among the six selected basic culture media, the inhibition rate of strain QZ10-4 using TSB medium was the highest, so TSB medium was selected as the basic culture medium for culture medium optimization.

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

[0082] Select several carbon sources (glucose, sucrose, soluble starch, mannitol and corn flour), nitrogen sources (protein pulse, yeast powder, beef extract, soybean protein pulse, ammonium sulfate, ammonia water and yeast extract) and inorganic salts (potassium dihydrogen phosphate, calcium carbonate, calcium chloride, ferrous sulfate and zinc sulfate) for testing, and replace the corresponding components in the basic culture medium in equal amounts. Bacillus QZ10-4 was shaken in a 50 mL Erlenmeyer flask at a liquid volume of 50%, an inoculation amount of 0.2%, a rotation speed of 200 r / min, and 28°C for 72 hours. According to the steps of Example 3, a sterile fermentation filtrate was prepared, and the PDA culture medium was diluted 200 times and poured into a plate. Each treatment was repeated 3 times, and the colony diameter of the pathogen was measured, the antibacterial rate was calculated, and the optimal carbon source, nitrogen source and inorganic salt type were determined.

[0083] Depend on Figure 6 It can be seen that when TSB medium is used as the basic fermentation medium, when trypsin is used as the carbon source, the fermentation filtrate of strain QZ10-4 has the highest inhibition rate against Aspergillus niger, reaching 45.51%. Therefore, trypsin should be selected as the optimal carbon source for strain QZ10-4 to antagonize Aspergillus niger culture medium.

[0084] Depend on Figure 7 It can be seen that when TSB medium after carbon source optimization was used as the basic fermentation medium, the fermentation filtrate of strain QZ10-4 had the highest inhibition rate against Aspergillus pinecone, reaching 75.87%. Therefore, soy peptone should be selected as the optimal nitrogen source for the culture medium of strain QZ10-4 to antagonize Aspergillus pinecone.

[0085] Depend on Figure 8 It can be seen that when TSB medium after carbon and nitrogen sources are optimized is used as the basic fermentation medium, when potassium dihydrogen phosphate is used as the inorganic salt, the fermentation filtrate of strain QZ10-4 has the highest inhibition rate against Aspergillus pinecone, reaching 84.44%. Therefore, potassium dihydrogen phosphate should be selected as the optimal inorganic salt for the culture medium of strain QZ10-4 to antagonize Aspergillus pinecone.

[0086] Fig. 9 This is the result of optimization of each component of the culture medium. The antibacterial rate of QZ10-4 sterile fermentation filtrate increased from 23.89% to 86.35% when diluted 200 times.

[0087] Example 5

[0088] Bacterial safety testing

[0089] like Fig.10 As shown, QZ10-4 was subjected to alfalfa and tobacco tests respectively, and sterile water was added to the control group. The results showed that the QZ10-4 bacterial solution did not have an adverse effect on the growth of tobacco and alfalfa, indicating that QX10-4 does not pose a biosafety risk.

[0090] Studying the sensitivity of strains to antibiotics is an important indicator for evaluating the safety of strains. The sensitivity of QZ10-4 to 10 antibiotics was within the quality control range, and it was highly sensitive to 9 antibiotics (the diameter of the inhibition zone was above 15 mm). See Table 1 for the drug sensitivity results.

[0091] Table 2 Results of drug sensitivity test of strain QZ10-4

[0092]

[0093] s. Highly sensitive I. Moderately sensitive R. Resistant

[0094] 1. Broad spectrum of bacterial inhibition

[0095] Under the above optimized fermentation culture conditions, a fermentation liquid was obtained, and the seven plant pathogens (Pseudodiscus hirsutus, Fusarium oxysporum, Fusarium graminearum, Botrytis cinerea, Alternaria ovata, Discospora pine needles, and Alternaria alternata) were used as the inhibition objects, and the inhibition rate was determined and calculated using the method in Example 4. The antibacterial effects of strain QZ10-4 on the seven plant pathogens tested were statistically analyzed, and the results showed good inhibitory activity (see Fig.11 ), QZ10-4 has a broad-spectrum antibacterial activity.

[0096] 2. QZ10-4 potted plant prevention effect

[0097] The bacterial fermentation liquid, sterile fermentation filtrate and bacterial suspension of QZ10-4 were prepared respectively, wherein the concentration of the fermentation liquid and bacterial suspension was adjusted to OD600 = 0.8. Among them, (1) the treatment method of the prevention effect determination test: take two-year-old healthy Masson pine potted seedlings with similar growth, take 2 ml of QZ10-4 fermentation liquid, sterile fermentation filtrate and bacterial suspension respectively, and spray them evenly on the surface of Masson pine needles. After 3 days, spray 2 ml of pine cone conch mycelium liquid evenly for each treatment; (2) the treatment method of the efficacy determination test: take two-year-old healthy Masson pine potted seedlings with similar growth, spray 2 ml of pine cone conch mycelium liquid evenly for each treatment. After 3 days, take 2 ml of QZ10-4 bacterial fermentation liquid, sterile fermentation filtrate and bacterial suspension respectively, and spray them evenly on the surface of Masson pine needles. Spraying equal amounts of sterile water and liquid LB culture medium was used as a control, and moisturizing treatment was done well. Each treatment was repeated 4 times. The severity of disease was recorded 30 days after inoculation.

[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 withered and yellowed part of the whole plant is ≤25%, Level 2 is when the withered and yellowed part of the whole plant is ≥25.1%-50%, Level 3 is when the withered and yellowed part of the whole plant is ≥59.1%-75%, and Level 4 is when the withered and yellowed part of the whole plant is ≥75.1%.

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

[0100] Relative control effect = 100 × (disease index of pathogens only - disease index of biocontrol bacteria and pathogens) / disease index of pathogens only

[0101] The potted plant efficacy test results of strain QZ10-4 are shown in Fig.12 As shown in Table 3, the incidence of Masson pine seedlings inoculated with pathogens alone reached 100%. The bacterial suspension, fermentation liquid and sterile fermentation filtrate of QZ10-4 all had good effects on the prevention and control of Aspergillus niger.

[0102] Table 3 Potted plant control effect of strain QZ10-4

[0103] Processing Incidence / % Disease index Relative control effect / % Pathogen inoculation 3 days after application of QZ10-4 bacterial suspension 25 6.25 90.90％ Pathogen inoculation 3 days after application of QZ10-4 fermentation liquid 50 18.75 72.72 Pathogen inoculation 3 days after application of QZ10-4 sterile fermentation filtrate 25 6.25 90.9 Apply QZ10-4 bacterial suspension 3 days after inoculation of pathogens 50 31.25 54.54 Apply QZ10-4 fermentation liquid 3 days after inoculation of pathogens 75 43.75 36.36 Apply QZ10-4 sterile fermentation filtrate 3 days after inoculation of pathogens 50 12.5 81.81 CK3 100 68.75 /

Claims

1. A Bacillus Velez, characterized in that: It was named Bacillus Velezii QZ10-4, with the deposit number CGMCC No.30401, and was deposited in the General Microbiology Center of China Culture Collection Administration on April 22, 2024.

2. Use of Bacillus Velezii as claimed in claim 1 in preventing and treating pine dieback.

3. The Bacillus Velezii as claimed in claim 1 presents bluish purple under a microscope and is a Gram-positive bacterium.

4. The method for culturing Bacillus Velezii according to claim 1, wherein: The culture medium is used for culturing, wherein the carbon source is selected from tryptone, sucrose, soluble starch, mannitol, glycerol, and corn flour, the nitrogen source is selected from soy peptone, ammonium sulfate, beef extract, ammonia water, peptone, and yeast extract, and the inorganic salt is selected from sodium chloride, calcium carbonate, potassium dihydrogen phosphate, zinc sulfate, calcium chloride, and ferrous sulfate.

5. Use of the Bacillus Velezii as claimed in claim 1 in the preparation of a plant antibacterial agent for inhibiting one or more bacteria selected from the group consisting of Conchosporon pinicola, Polytrichomoniasis spp., Fusarium oxysporum, Fusarium graminearum, Botrytis cinerea, Alternaria ovata, Discospora piniformis, and Alternaria alternata.

6. A fermentation liquid, characterized in that: The product is obtained by fermenting and culturing the Bacillus Velez subtilis described in claim 1.

7. A sterile fermentation filtrate, characterized in that: The method is obtained by fermenting and culturing the Bacillus Velez subtilis according to claim 1, centrifuging the fermented product, and filtering the supernatant with a bacteria filter.

8. A bacterial suspension, characterized in that: The invention is obtained by fermenting and culturing the Bacillus Velez subtilis described in claim 1, centrifuging the precipitate and resuspending the precipitate in water.

9. The fermentation broth according to claim 6, characterized in that: The Velez bacillus is fermented and cultured together with tryptone, soy peptone and potassium dihydrogen phosphate.

10. Use of the fermentation liquid according to claim 6, the sterile fermentation filtrate according to claim 7 or the bacterial suspension according to claim 8 in preventing and treating pine dieback disease.

Images