A bacterial agent containing Bacillus velezensis and its application

Abstract

The present invention provides a bacterial agent containing Bacillus velezensis, which is named Bacillus velezensis QN3NO-3 and is registered and preserved in the China Center for Type Culture Collection with the preservation number CCTCC NO: M 2021304. The Bacillus velezensis of the present invention has broad-spectrum antibacterial activity and has good antagonistic effects against Fusarium oxysporum f. sp. lycopersici, etc. This strain has the abilities of efficient nitrogen fixation and IAA production, which can promote plant growth. It also has activities such as producing cellulase, chitinase, and β-1,3-glucanase, etc., and can efficiently degrade cellulose and destroy the fungal cell wall. The bacterial agent prepared by using this bacterium in the present invention can give full play to its antibacterial, nitrogen fixation abilities and other biological characteristics, can reduce the dependence on pesticides and fertilizers, improve the yield and quality of crops, and at the same time reduce the negative impact on the environment, and has broad application potential in the fields of agriculture and the environment.

Description

Technical Field

[0001] The present invention relates to a bacterial agent, and particularly to a bacterial agent containing Bacillus velezensis and its application. Background Art

[0002] Nitrogen (N) in NPK fertilizers is one of the most crucial nutrients in agricultural production. It is an essential chemical element necessary for the life activities of plants and animals. However, the problem of nitrogen deficiency is widespread in farmland soils. Increasing the nitrogen content in organic fertilizers and chemical fertilizers to improve the productivity of the ecosystem is an effective way to achieve yield increase benefits. However, excessive application of nitrogen will cause environmental pollution. It will not only reduce the utilization efficiency of nitrogen, but also have a negative impact on the water and atmospheric environmental quality, posing a threat to the balance of the animal and plant ecosystem and human health. China is one of the countries with the largest global consumption of fertilizer nitrogen. Although the use of a large amount of nitrogen fertilizer has promoted the development of agriculture in China, it has also caused problems such as water eutrophication. With the growth of the population and the increase in the demand for agricultural products, the consumption of fertilizer nitrogen will continue to increase. Therefore, improving the utilization efficiency of nitrogen, avoiding overuse of fertilizer nitrogen, preventing adverse effects on environmental quality (such as soil compaction, etc.), and taking into account economic and environmental benefits have become the basic principles and important tasks of agricultural nitrogen management. While ensuring the crop yield, we should actively explore methods to cultivate soil nitrogen fertility to achieve the sustainable development of agriculture.

[0003] Microorganisms are the oldest organisms on Earth, with a history of at least 4.1 billion years. They exist in environments such as soil, water, air, and inside animals and plants in different forms, physiological characteristics, and metabolic capabilities. The diversity of earth microorganisms such as bacteria, fungi, and viruses enriches the microbial resource library, providing valuable resources for scientific research and applications in fields such as agriculture, medicine, and the environment. As a special type of bacteria, nitrogen-fixing bacteria play an important role among earth microorganisms. It can convert atmospheric nitrogen (N2) into nitrogen compounds that can be utilized by plants, such as ammonia (NH3) and nitrate (NO3 -, is crucial for the nitrogen cycle of the Earth's ecosystem. Nitrogen is an important element for organisms to synthesize essential substances for life such as proteins and nucleic acids, but most organisms cannot directly utilize nitrogen gas in the atmosphere. Nitrogen-fixing bacteria convert nitrogen gas into forms that can be utilized by other organisms through nitrogen fixation, thereby promoting biodiversity and the healthy development of the ecosystem. Nitrogen-fixing bacteria are widely present in environments such as soil, water bodies, and the rhizosphere of plants, forming a symbiotic relationship with plants. For example, rhizobia in leguminous plants combine with the roots of leguminous plants to jointly promote nitrogen fixation and plant growth. In addition, nitrogen-fixing bacteria can also live together with other microorganisms to form complex microbial communities, participating in key ecological processes such as the decomposition of soil organic matter and nutrient cycling. Conducting research on nitrogen-fixing bacteria in aspects such as classification, physiological characteristics, nitrogen fixation mechanisms, and interactions with plants and other microorganisms helps to reveal the nitrogen cycle mechanism of the Earth's ecosystem, improve agricultural production efficiency, and promote sustainable development. However, the research on nitrogen-fixing bacteria still faces some challenges, such as the exploration and rational utilization of nitrogen-fixing bacteria resources, the development and utilization of nitrogen-fixing bacteria, the improvement of nitrogen fixation efficiency, and the impact of environmental factors on nitrogen fixation. Therefore, further research still requires continuous efforts to give full play to the important role of nitrogen-fixing bacteria among the Earth's microorganisms.

[0004] Plant endophytes are considered to be microorganisms that live in healthy plants at least at a certain stage or throughout the entire life cycle without causing obvious diseases in the plants. Since they were first mentioned by Kleopper et al. in 1992, they have attracted people's attention. Their species diversity determines their wide applications in the fields of biological control, medicine, forestry, etc. Under the double impact of the strengthening of the concept of agricultural sustainable development and the ineffectiveness of chemical control, microorganisms have received an increasingly wide research and development space due to their advantages such as wide sources, large quantities, and low costs, including improving soil fertility, repairing soil, increasing crop yield and quality, and enhancing crop stress resistance. Microorganisms with antagonistic functions, after entering the soil, establish a competition mechanism with plant pathogens, occupy ecological sites, and compete for nutrients, oxygen, etc., or inhibit pathogens by parasitism or dissolution, inducing the crop itself to produce resistance and reducing the incidence of diseases. Previous reports have shown that the metabolites produced by antagonistic strains during metabolism can improve the disease resistance of plants, or by destroying the cell structure of pathogens, causing hyphae to undergo fission and distortion and losing their infectivity. Zeng Yibo studied the growth-promoting and disease-resistant functions of photosynthetic bacterium PSB06 and found that the extracellular proteins produced by the strain can effectively inhibit rice blast. Anming et al. isolated a biocontrol strain Y-1 that resists Pestalotiopsis from the stems of Dendrobium nobile Lindl. and determined the mechanism of action of its polypeptide metabolites in inhibiting energy production and amino acid biosynthesis in the respiratory chain of pathogens through proteomics research. Bacillus velezensis has certain advantages as a biological pesticide in controlling crop diseases. It can produce a series of antibacterial substances, such as antibiotics, enzymes, and antibacterial peptides, which have inhibitory effects on a variety of plant pathogens. Exploring and developing Bacillus velezensis biological pesticide products can effectively reduce the use of chemical pesticides, reduce environmental pollution and harm to human health, and at the same time increase crop yield and quality. However, for specific application situations, it is necessary to select appropriate Bacillus velezensis strains and application methods according to different crops and diseases. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a bacterial agent containing Bacillus velezensis and its application.

[0006] The first aspect of the present invention is to provide the application of Bacillus velezensis or its fermentation broth in nitrogen fixation and / or cellulose degradation. The Bacillus velezensis is named Bacillus velezensis QN3NO-3, which is registered and preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 2021304.

[0007] The second aspect of the present invention is to provide a bacterial agent of Bacillus velezensis. The Bacillus velezensis is named Bacillus velezensisQN3NO-3 is registered and preserved at the China Center for Type Culture Collection, with the preservation number CCTCC NO: M 2021304.

[0008] The "bacterial agent" in the present invention, also known as microbial pesticide, refers to a biogenic pesticide with living microorganisms as the active ingredient.

[0009] Those skilled in the art can prepare the required pesticide dosage forms according to actual usage requirements, such as suspension, solid particles, etc. Therefore, the bacterial agent of the present invention may also include excipients acceptable in pesticides.

[0010] The "excipient" in the present invention, also known as additive, is the general term for all additional materials in a pesticide formulation other than the active ingredient.

[0011] The third aspect of the present invention is to provide a preparation method of the bacterial agent as described in the second aspect of the present invention, including the following steps:

[0012] (1) Inoculate the Bacillus velezensis into a fermentation medium for subculture to obtain a fermentation broth;

[0013] (2) Centrifuge the fermentation broth and pour off the supernatant to obtain the product.

[0014] Those skilled in the art can further prepare the precipitate obtained by centrifugation into various dosage forms according to actual usage requirements.

[0015] The Bacillus velezensis QN3NO-3 of the present invention has broad-spectrum antibacterial properties and has good inhibitory effects on Fusarium oxysporum f. sp. lycopersici, Fusarium oxysporum f. sp. cubense race 4, Fusarium oxysporum f. sp. cubense race 1, Fusarium oxysporum f. sp. cucumerinum, Colletotrichum capsici, Colletotrichum fragariae, Colletotrichum litchii, Colletotrichum musae, Colletotrichum parachuticum, Colletotrichum gloeosporioides, Curvularia lunata, Alternaria alternata, Gibberella zeae, and / or Cryphonectria parasitica, Pestalotiopsis mangiferae, Magnaporthe oryzae, Colletotrichum gloeosporioides Penz. et Sacc. Therefore, the bacterial agent containing the Bacillus velezensis of the present invention also has inhibitory effects on the above-mentioned pathogenic bacteria.

[0016] Therefore, the fourth aspect of the present invention is to provide the application of the bacterial agent as described in the second aspect of the present invention in antagonizing Fusarium oxysporum f. sp. lycopersici, and / or Fusarium oxysporum f. sp. cubense race 4, and / or Fusarium oxysporum f. sp. cubense race 1, and / or Fusarium oxysporum f. sp. cucumerinum, and / or Colletotrichum capsici, and / or Colletotrichum fragariae, and / or Colletotrichum litchii, and / or Colletotrichum musae, and / or Colletotrichum parachuticum, and / or Colletotrichum gloeosporioides, and / or Curvularia lunata, and / or Alternaria alternata, and / or Gibberella zeae, and / or Cryphonectria parasitica, and / or Pestalotiopsis mangiferae, and / or Magnaporthe oryzae, and / or Colletotrichum gloeosporioides Penz. et Sacc.

[0017] The fifth aspect of the present invention is to provide the application of the bacterial agent as described in the second aspect of the present invention in preventing and treating diseases caused by Fusarium oxysporum f. sp. lycopersici, and / or Fusarium oxysporum f. sp. cubense race 4, and / or Fusarium oxysporum f. sp. cubense race 1, and / or Fusarium oxysporum f. sp. cucumerinum, and / or Colletotrichum capsici, and / or Colletotrichum acutatum, and / or Colletotrichum gloeosporioides, and / or Colletotrichum musae, and / or Colletotrichum parachuticum, and / or Curvularia lunata, and / or Alternaria alternata, and / or Gibberella zeae, and / or Cryphonectria parasitica, and / or Pestalotiopsis mangiferae, and / or Magnaporthe oryzae, and / or Colletotrichum gloeosporioides Penz.

[0018] The cell wall components of pathogenic fungi are mainly composed of chitin and glucan. Bacillus velezensis QN3NO-3 of the present invention can produce chitinase and β-1,3-glucanase at different culture stages, which can destroy the fungal cell wall. Therefore, the bacterial agent containing the Bacillus velezensis of the present invention can also destroy the fungal cell wall. Therefore, the sixth aspect of the present invention is to provide the application of the bacterial agent as described in the second aspect of the present invention in destroying the fungal cell wall.

[0019] Bacillus velezensis QN3NO-3 of the present invention ferments to produce cellulolytic enzymes (exoglucanase, exoglucanase, β-glucosidase, filter paper cellulase, etc.), which can efficiently degrade cellulose. Therefore, the bacterial agent containing the Bacillus velezensis of the present invention can also efficiently degrade cellulose. Therefore, the seventh aspect of the present invention is to provide the application of the bacterial agent as described in the second aspect of the present invention in degrading cellulose.

[0020] Bacillus velezensis QN3NO-3 of the present invention has nitrogen-fixing function. Therefore, the bacterial agent containing the Bacillus velezensis of the present invention can also fix nitrogen. Therefore, the eighth aspect of the present invention is to provide the application of the bacterial agent as described in the second aspect of the present invention in nitrogen fixation.

[0021] Bacillus velezensis QN3NO-3 of the present invention has nitrogen-fixing function and can also secrete IAA, and can promote plant growth through nitrogen fixation and / or secretion of IAA. Therefore, the bacterial agent containing the Bacillus velezensis of the present invention can also promote plant growth. Therefore, the ninth aspect of the present invention is to provide the application of the bacterial agent as described in the second aspect of the present invention in promoting plant growth.

[0022] Bacillus velezensis QN3NO-3 of the present invention can promote seed germination, promote the seed to enter the white bud stage in advance, promote the growth of plumule and radicle, increase the fresh weight of the above-ground and underground parts of plants, promote the growth of plant root crown and root system, and increase the number of lateral roots of plants. Therefore, the microbial agent containing the Bacillus velezensis of the present invention can also promote seed germination, promote the seed to enter the white bud stage in advance, promote the growth of plumule and radicle, increase the fresh weight of the above-ground and underground parts of plants, promote the growth of plant root crown and root system, and increase the number of lateral roots of plants. Therefore, the tenth aspect of the present invention is to provide the application of the microbial agent as described in the second aspect of the present invention in promoting seed germination, and / or promoting the seed to enter the white bud stage in advance, and / or promoting the growth of plumule, and / or promoting the growth of radicle, and / or increasing the fresh weight of the above-ground part of plants, and / or increasing the fresh weight of the underground part of plants, and / or promoting the growth of plant root crown, and / or promoting the growth of plant root system, and / or increasing the number of lateral roots of plants.

[0023] Lateral roots play an important role in plant drought tolerance. Bacillus velezensis QN3NO-3 of the present invention can increase the number of lateral roots of plants, and can improve the drought resistance of plants. Therefore, the microbial agent containing the Bacillus velezensis of the present invention can also improve the drought resistance of plants. Therefore, the eleventh aspect of the present invention is to provide the application of the microbial agent as described in the second aspect of the present invention in improving the drought resistance of plants.

[0024] Bacillus velezensis of the present invention, its fermentation broth and the volatile substances of the strains produced by sealed culture have broad-spectrum antibacterial activity, and have good inhibitory effects on Fusarium oxysporum f. sp. lycopersici, Fusarium oxysporum f. sp. cubense race 4, Fusarium oxysporum f. sp. cubense race 1, Fusarium oxysporum f. sp. cucumerinum, Colletotrichum capsici, Colletotrichum fragariae, Colletotrichum litchii, Colletotrichum musae, Colletotrichum parachinense, Colletotrichum gloeosporioides, Curvularia lunata, Alternaria alternata, Gibberella zeae, Cryphonectria parasitica, Pestalotiopsis mangiferae, Magnaporthe oryzae, Colletotrichum gloeosporioides Penz. et Sacc. var. mangiferae, etc. Moreover, this strain has the ability of efficient nitrogen fixation and IAA production, and can promote the growth of plants, such as effectively promoting seed germination, promoting the seed to enter the white bud stage in advance, promoting the growth of plumule and radicle, increasing the fresh weight of plants, promoting the growth of plant root crown and root system, and the number of lateral roots of plants, and can also improve the drought resistance of plants. This strain also has activities such as producing cellulase (endo-glucanase, exo-glucanase, β-glucosidase, filter paper cellulase, etc.), chitinase and β-1,3-glucanase, and can efficiently degrade cellulose and destroy the fungal cell wall.

[0025] The Bacillus velezensis of the present invention is prepared into a microbial agent, which can give full play to its antibacterial, nitrogen fixation ability and other biological characteristics, can reduce the dependence on pesticides and fertilizers, improve the yield and quality of crops, and at the same time reduce the negative impact on the environment, and has broad application potential in the fields of agriculture and environment. Description of the Drawings

[0026] Figure 1 They are the scanning electron microscope results and Gram staining results of QN3NO-3 bacteria.

[0027] Figure 2 They are partial physiological and biochemical experiments of strain QN3NO-3.

[0028] Figure 3 They are the determination results of the IAA production ability of strain QN3NO-3.

[0029] Figure 4 They are the standard curves of IAA content.

[0030] Figure 5 They are the determination results of the cellulolytic ability of strain QN3NO-3 (screenshots of Image J).

[0031] Figure 6 They are the determination of cellulase activity in the fermentation broth of strain QN3NO-3.

[0032] Figure 7 They are the germination situations of corn seeds after being treated with strain QN3NO-3. Left figure: germination situations from 1 to 3 days; right figure: germination situation on the third day.

[0033] Figure 8 They are the determination of the antibacterial activity of the volatile substances of the strain by the double-dish buckling method.

[0034] Figure 9 They are the antagonism determination of the supernatant of the fermentation broth of strain QN3NO-3 and ammonium sulfate crude protein against Fusarium oxysporum f. sp. lycopersici.

[0035] Figure 10 They are the determination of the activities of chitinase and β-1,3-glucanase in the supernatant of the fermentation broth of strain QN3NO-3.

[0036] Figure 11 They are the research results of the broad-spectrum antibacterial activity of strain QN3NO-3. Specific embodiments

[0037] The present invention will be further described below with reference to the accompanying drawings and in combination with specific embodiments for better understanding. For those not specifying specific techniques or conditions in the embodiments, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0038] 1 Materials

[0039] 1.1 Sample source and treatment

[0040] Collect multi-point samples of noni live plant fruits from the noni plantation in Qiaotou Town, Chengmai County, Hainan Province (N 19°58′9″, E 109°55′19″), bag and number them, and store them at 4 °C.

[0041] 1.2 Culture media

[0042] LB medium: 5 g of yeast powder, 10 g of tryptone, 10 g of sodium chloride, 20 g of agar, 1 L of distilled water, pH 7.2 - 7.5;

[0043] Ashby's medium: 10 g of glucose, 0.2 g of potassium dihydrogen phosphate, 0.2 g of sodium chloride, 0.2 g of magnesium sulfate, 0.1 g of calcium sulfate, 5 g of calcium carbonate, 20 g of agar, 1 L of distilled water, pH 7.0 - 7.2;

[0044] Cellulose Congo red medium: 1.88 g of microcrystalline cellulose, 1 g of Congo red, 2.0 g of gelatin, 20 g of agar, 1 L of distilled water, pH 7.0.

[0045] 1.3 Main reagents:

[0046] DNS reagent: Weigh 10 g of 3,5-dinitrosalicylic acid and dissolve it in 500 ml of distilled water. Add 20 g of sodium hydroxide and 200 g of potassium sodium tartrate. After heating and dissolving the solution, add 2 g of phenol and 0.5 g of anhydrous sodium sulfite. After the solute dissolves, cool it to room temperature and make up the volume to 1000 ml. The reagent is stored in a brown volumetric flask and filtered for use after standing for one week;

[0047] CMC substrate solution: Weigh 0.625 g of sodium carboxymethylcellulose and dissolve it in 100 ml of sodium acetate buffer solution, and heat and stir until dissolved;

[0048] Microcrystalline cellulose substrate solution: Weigh 0.5 g of microcrystalline cellulose and dissolve it in 100 ml of sodium acetate buffer solution;

[0049] Filter paper substrate solution: Weigh 0.5 g of filter paper and immerse it in 100 ml of sodium acetate buffer solution;

[0050] Standard glucose solution: Dissolve 27.0 g of glucose in 25 ml of distilled water to prepare a 6 mol / L glucose solution. Prepare the glucose solution standard curve according to Table 1;

[0051] 1.10 mol / L sodium hydroxide: Weigh 400 g of analytical grade sodium hydroxide and dissolve it in 500 ml of distilled water, and then make up the volume to 1 L;

[0052] 2% Boric Acid - Indicator Solution: Weigh 20 g of boric acid and dissolve it in 1000 mL of distilled water. Then add 5 mL of methyl red - bromocresol green mixed indicator, and adjust it to a slightly purplish - red color with dilute acid or dilute alkali. This reagent should be prepared and used immediately.

[0053] High - efficiency Kjeldahl nitrogen determination catalyst tablets.

[0054] 1.4 Test Crops:

[0055] The test materials are corn, with the variety being Meiyu 16, a sweet - waxy variety provided by Hainan Lvchuan Seedling Co., Ltd.

[0056] 1.5 Data Processing

[0057] Use Image J (Version 1.38) software to measure the gray - scale area, use Microsoft Excel 2007 software and one - way analysis of variance for experimental data, and use Duncan's new multiple - range test method (DMRT method) for significant difference analysis.

[0058] 2 Experimental Methods and Results

[0059] 2.1 Isolation and Screening of Endophytic Bacteria

[0060] Sample Disinfection: Wash and air - dry the noni fruits for later use. Sterilize them with 75% ethanol for 5 min, then treat them with sodium hypochlorite in the dark for 20 min. Take them out and soak them in 10% sodium bicarbonate for 10 min, rinse them 5 times with sterile water, dry them on sterile filter paper and label them.

[0061] Sample Imprint Experiment: Adhere the sample to the LB medium for 5 min using the tissue imprint method, and culture it at 28 °C for 3 d for sterility inspection.

[0062] Sample Grinding: After slicing the sample, grind it into a paste in a sterilized mortar. Take 1 mL of the juice from each and put it into a centrifuge tube, add 4 mL of LB medium, culture it at room temperature at 180 r / min for 1 h, and vortex - oscillate.

[0063] Isolation and Screening: Make a suspension by gradient dilution (10 -1 、10 -2 、10 -3 )with sterile water, fully oscillate it, then take 200 μL of the diluted solution and evenly inoculate it on Ashby's medium by the spread - plate method. Repeat 3 times, and culture it in an inverted dark box at 28 °C for 5 d. Select different colonies with better morphology, growth vigor, and faster growth rate, and purify them multiple times on a new solid Ashby's medium by the streak - plate method with an inoculation loop until pure culture.

[0064] In this study, noni fruit samples were disinfected to prepare suspensions. Through the dilution plate method, 71 strains of endophytic bacteria were initially isolated and purified. Five dominant strains were selected for re-screening of nitrogen fixation and growth promotion functions (for the re-screening of nitrogen fixation function, refer to "2.2 Identification and Activity Determination of Nitrogen Fixation Ability", and for the re-screening of growth promotion function, refer to "2.6 Biological Activity of Strains on Maize Growth"). One strain with the best effect was selected as the research object and named QN3NO-3. The colony was irregularly round, yellowish-gray, opaque, with a smooth and wrinkled surface, a wavy-curved edge, and a central elevation on the LB medium. The bacteria were elliptical or short rod-shaped, and Gram-stained purple-red, belonging to positive bacteria ( Figure 1 ). As the culture time extended, the colony tended to be dry and flat, with irregular wrinkles at the edge.

[0065] 2.2 Physiological and Biochemical Identification of Strains

[0066] The screened antagonistic strains were subjected to physiological and biochemical identification according to Bergey's Manual of Determinative Bacteriology and Manual of Systematic Bacteriology of Common Bacteria.

[0067] (1) Single carbon source utilization experiment: In the physiological and biochemical identification of bacteria, the utilization of carbon sources by strains is an important indicator. Different bacteria have different utilization of carbon sources. In the single carbon source utilization experiment, a single carbon source is added to Pugh's basal medium at a concentration of 1%, and then the strain to be identified is inoculated. It is cultured at 28 °C for one to two weeks. In addition, the strain is inoculated onto the basal medium without any carbon source as a blank control, and the growth of the strain on each medium is observed. If it can grow, it is positive, indicating that the strain has the ability to utilize this carbon source; if it cannot grow, it is negative, indicating that it does not have the ability to utilize this carbon source. The carbon sources include: α-lactose, D-fructose, D-galactose, D-mannitol, glucose, mannitol, anhydrous lactose, soluble starch, trehalose, sorbitol, inositol, melezitose, rhamnose, xylose, maltose, sucrose, melibiose, etc.

[0068] (2) Single nitrogen source utilization experiment: Similar to the utilization of carbon sources, different bacteria have different utilization of nitrogen sources. A single nitrogen source is added to the nitrogen source basal medium at a concentration of 0.5% to make a plate, and then the strain is inoculated. In addition, the strain is inoculated onto the basal medium without any nitrogen source as a blank control, and it is cultured at 28 °C for one to two weeks, and the growth of the strain is observed. If it can grow, it is positive, indicating that the strain has the ability to utilize this nitrogen source; if it cannot grow, it is negative, indicating that it does not have the ability to utilize this nitrogen source. The added nitrogen sources include: tryptone, asparagine, valine, histidine, oxamic acid, ammonium sulfate, ammonium acetate, ammonium chloride, ammonium molybdate tetrahydrate, ammonium nitrate, phenylalanine, glycine, cysteine, alanine, arginine, serine, tryptophan, methionine, and tyrosine.

[0069] (3) Starch hydrolysis experiment: Use nutrient agar medium as the basic medium and add 1% soluble starch to it. Adopt the spot inoculation method (i.e., the inoculation diameter < 0.5 cm) to inoculate the antagonistic strain onto the prepared plate. After culturing at 28 °C for one week, add iodine solution around the colonies. If there is a clear zone around the colonies, the result is positive, indicating that the strain can produce amylase, and the diameter of the clear zone represents the activity of the produced amylase; if no amylase is produced, the whole plate will show blue.

[0070] (4) Gelatin liquefaction experiment: Inoculate the strain to be identified into a test tube containing gelatin medium, and then culture it at 28 °C. Observe the liquefaction of gelatin on the 5th, 10th, 20th, and 30th days. If there is a liquefaction phenomenon, it is positive, indicating that the strain has the ability to liquefy gelatin; otherwise, it is negative.

[0071] (5) Cellulose decomposition experiment: Prepare cellulose decomposition medium according to the formula. Then immerse one end of a filter paper strip into the liquid medium, sterilize the medium, inoculate the strain into the medium, and culture it statically. After one month, observe whether the filter paper strip is decomposed. If it is decomposed, it is positive, indicating that cellulase is produced; otherwise, it is negative.

[0072] (6) Nitrate reduction: Prepare nitrate reduction medium according to the formula. Inoculate the strain into the medium and culture it statically at 28 °C for 7 d and 14 d, using the medium without inoculating the strain as a blank control. Take blank test tubes, add a little of the 7 d and 14 d culture solutions into different test tubes respectively, and at the same time add the previously prepared Solution A and Solution B drop by drop. If the solution shows pink, rose red, brown or orange, the nitrate reduction is positive; if no such colors appear, add 1 or 2 drops of diphenylamine reagent. If the solution turns blue, it is still negative; otherwise, it is regarded as positive.

[0073] (7) Urease experiment: Prepare urease medium according to the formula. Inoculate the strain to be tested onto the urease medium and culture it in an inverted position at 28 °C for 4 d, then observe the color change of the medium. If the strain to be tested has the ability to produce urease, the medium will turn pink, which is positive; if it does not change color, it is negative.

[0074] (8) Lipase (Tween 20, 40, 80) experiment: After preparing the lipase medium, sterilize the separately packaged Tween 20, 40, and 80 together, and mix the Tween with the medium to make plates. Inoculate the strain onto the plate and culture it for one to two weeks. Observe the plate. If there is a blurred halo around the colonies, it is positive; otherwise, it is negative.

[0075] (9)Salt tolerance experiment: Media with different NaCl concentrations (1%, 3%, 5%, 7%, 9%, 11%, 13%, 15%) were prepared, with the other components of the media being the same. The strain to be identified was inoculated onto these media and cultured at 28 °C. Observation was carried out once a week for four weeks, and finally, it was recorded whether the strain grew on the media to determine the upper and lower limit concentrations of the NaCl tolerance ability of the strain to be identified.

[0076] (10)pH tolerance experiment: The pH values of the liquid media were adjusted to 4, 5, 6, 7, 8, 9, and 10 respectively. The strains to be tested were inoculated into the media with different pH values, ensuring that other culture conditions were the same. They were cultured with shaking at 28 °C. Observation was carried out once a week for four weeks, and finally, the growth of the strains on each medium was determined. The upper and lower limits of the pH values at which the strains could grow and the optimal pH value were determined.

[0077] The experimental determination results showed that QN3NO-3 was positive in starch hydrolysis, gelatin liquefaction, urease test, and enzymatic test; it was only sensitive to Tween 40 in the enzyme lipid experiment; it had no reaction in methyl red, malonate, phosphate reduction, and hydrogen sulfide tests and was negative. It could grow at pH 4 - 10, and the optimal growth environment was pH 9; in the salt tolerance experiment, it could grow at NaCl concentrations of 1% - 9% and did not grow at 11% - 15%.

[0078] According to the results of the carbon and nitrogen source utilization experiment, QN3NO-3 could utilize carbon sources such as α-lactose, D-fructose, D-galactose, D-mannitol, glucose, mannitol, anhydrous lactose, soluble starch, trehalose, sorbitol, inositol, melezitose, rhamnose, xylose, maltose, sucrose, and melibiose; it could utilize fewer nitrogen sources, such as tryptone, asparagine, valine, histidine, and oxamic acid, and could not utilize ammonium sulfate, ammonium acetate, ammonium chloride, ammonium molybdate tetrahydrate, ammonium nitrate, phenylalanine, glycine, cysteine, alanine, arginine, serine, tryptophan, methionine, and tyrosine.

[0079] Based on the morphological, cultural, and physiological and biochemical characteristics, this bacterium was preliminarily identified as Bacillus velezensis QN3NO-3 (China Center for Type Culture Collection. Wuhan, China, Wuhan University, CCTCC NO: M 2021304, March 30, 2021).

[0080] Table 1 Analysis of the physiological and biochemical characteristics of QN3NO-3 strain

[0081]

[0082] 2.3 Identification and activity determination of nitrogen fixation ability

[0083] Use a sterilized toothpick to pick the strain to be tested into Ashby nitrogen-free solid medium and subculture it for 6 generations. Good growth indicates nitrogen fixation ability, while failure to grow indicates no nitrogen fixation ability.

[0084] Determine the nitrogen fixation activity of the strain by the Kjeldahl method. Use an inoculation loop to pick the dominant nitrogen-fixing bacteria and ferment them in LB culture medium for 2 days. Centrifuge the fermentation broth at 10000 rmp for 10 min at 4°C to remove the supernatant. Place the bacterial cells in a mortar and quickly grind them with liquid nitrogen. Weigh 1 g and place it at the bottom of a dry digestion tube, moisten it with a small amount of water, add 5 mL of concentrated sulfuric acid and 2 high-efficiency Kjeldahl digestion catalyst tablets. Digest the digestion tube until the solution in the digestion tube turns grayish-white with a slightly green color, and then continue to digest for some time. Remove the digestion tube. Use an automatic Kjeldahl nitrogen analyzer to measure the nitrogen content in each digested culture broth.

[0085] Use a Kjeltec 8200 Kjeldahl nitrogen analyzer to determine the nitrogen content in the bacterial suspension. Under the action of a catalyst, digest the sample with concentrated sulfuric acid to convert organic nitrogen into inorganic ammonium salts, then convert the ammonium salts into ammonia under alkaline conditions, distill it out with water vapor and absorb it with an excessive boric acid solution, and then titrate it with standard hydrochloric acid. Calculate the nitrogen content in the sample to be 152.75 mg / L, and the protein content to be 954.69 mg (protein content = nitrogen content / 16%), indicating that the strain has high-efficiency nitrogen fixation ability.

[0086] 2.4 Identification and Activity Determination of IAA Production

[0087] Prepare liquid LB containing 100 mg / L α-aminoindolepropionic acid, inoculate the strain to be tested, set 3 replicates, and culture it at 180 r / min at room temperature for 2 d. Pipette 50 μL of the cultured bacterial suspension onto a white spot plate. Set up 2 control groups: 50 μL of uninoculated LB as the negative control, and 50 μL of IAA solution with a concentration of 50 μg / mL as the positive control. Add 50 μL of Salkowski colorimetric solution to all three at the same time, and let it stand in the dark at room temperature for 15 min. If the color does not change, it is negative and does not produce IAA; if it turns pink, it is an IAA-producing bacterium, and the IAA content can be determined.

[0088] Prepare an IAA stock solution (100 μg / mL), and make up IAA solutions with concentrations of 0, 10, 20, 30, 40, 50, and 60 μg / mL in 25 mL volumetric flasks respectively. Measure the OD with a UV spectrophotometer 530nm and draw an IAA standard curve. Pipette the bacterial suspension into a centrifuge tube and place it in a centrifuge with an initial speed of 12000 r / min for 10 min. Centrifuge and discard the precipitate. Pipette 4 mL of the supernatant and mix it with an equal volume of Salkowski colorimetric solution. Use the supernatant-free solution as the blank control. The determination method is the same as above. Calculate the IAA production of the strain according to the IAA standard curve.

[0089] According to the identification results of the Salkowski colorimetric method ( Figure 3 ), the mixture of strain QN3NO-3 and the IAA positive control group showed a pink color, while the uninoculated negative control group had no color change, indicating that strain QN3NO-3 could secrete IAA. The IAA ( Figure 4 ) was determined using an ultraviolet spectrophotometer, and the amount of IAA secreted by QN3NO-3 in the L-tryptophan-containing LB medium was calculated to be 40.61 ± 0.43 μg / mL.

[0090] 2.5 Identification of cellulolytic ability and determination of cellulase activity

[0091] The bacterial sample was streaked on the cellulose Congo red medium using an inoculation loop. After incubating at 28 °C for 2 - 3 d, whether a clear zone was produced around the colonies on the medium was observed after the colonies grew, and three replicates were set. The production of a clear zone indicated that the strain could produce cellulase, and the cellulase activity was preliminarily judged according to the size of the clear zone. After photographing and collecting, the gray area (pix) of the irregular clear zone and the colony area (ar) on the plate were measured using Image J software, and the ratio of the two was calculated, and the AR / ar ratio was calculated. The size of the ratio could preliminarily reflect the activity of the cellulase produced by the strain. Excel 2007 and variance analysis software were used for experimental data conversion and analysis.

[0092] Formula for calculating the "ratio":

[0093] Ratio (AR / ar) = colony area (pix) / clear zone area (pix).

[0094] According to the observation results of the plate streaking ( Figure 5 ), a large clear zone was produced on the cellulose Congo red medium, indicating that QN3NO-3 could produce cellulase. Using Image J (Version 1.38) software, the average value of the area (AR) of the irregular clear zone on the plate was measured to be 12690.33 pix, and the average value of the colony area (ar) was 823.33 pix. The ratio AR / ar of the two was 15.41. It can be seen that the strain has strong cellulase activity and can be identified and used for subsequent experiments.

[0095] The bacterial suspension cultured for 2 d (10 8Take 2 mL (CFU / mL) and inoculate it into a 250 mL flask containing 100 mL of CMC liquid medium. Incubate it at 28 °C with shaking at 150 r / min for 2 days. The fermentation broth is centrifuged at 10000 rmp for 5 minutes at 4 °C, and the supernatant is used as the enzyme extraction solution. The activities of exoglucanase, endoglucanase, and β-glucosidase are determined by measuring the reducing sugar release of microcrystalline cellulose (MCC), carboxymethyl cellulose (CMC), and salicylic acid (SAL). The total cellulase activity is expressed as the filter paper cellulase activity (FPA), and is determined by the filter paper analysis method described by Ghose (1987), using Whatman No. 1 filter paper as the substrate. The cellulase activity is determined according to the dinitrosalicylic acid (DNS) method at 540 nm, and monosaccharide analysis is carried out. A calibration curve is made with D-glucose solutions of different concentrations. All samples are measured with a UV-visible spectrophotometer at 540 nm. The unit of enzyme activity (U) is defined as the amount of enzyme that releases 1 μmol (2 μmol for β-glucosidase) of reducing sugar (calculated as glucose) per minute per milliliter of fermentation supernatant.

[0096] The results of cellulase activity determination are as Figure 6 shown. After culturing for 2 d, the exoglucanase activity is 61.81 IU / mL, the endoglucanase activity is 47.11 IU / mL, the β-glucosidase activity is 53.31 IU / mL, and the filter paper cellulase activity is 29.47 IU / mL, indicating that the strain QN3NO-3 has good enzyme activities such as endoglucanase, exoglucanase, and β-glucosidase, suggesting that this strain has the ability to efficiently degrade cellulose.

[0097] 2.6 Effect of the strain on the biological activity of maize growth

[0098] Place maize seeds in the refrigerator at 4 °C for vernalization for 3 days, take them out and place them in 10% (v / v) sodium hypochlorite for surface disinfection for 15 min, and wash them repeatedly with sterile water 5 times. Immerse the disinfected maize seeds in the bacterial suspension (×10 -1 ) for 60 min, then arrange them in a petri dish with sterilized filter paper and culture them in the dark at 28 °C for 3 days, wait for germination, and count the germination rate. Then transfer them to a transparent incubator for 7 days, collect the whole seedlings, and slowly wash the filter paper attachments in clean water, record the lengths and fresh weights of the roots and above-ground parts, and calculate the seedling growth indexes (above-ground fresh weight g, underground fresh weight g, germ length cm, radicle length cm, number of lateral roots pcs, root-shoot ratio). The control group is the culture solution without inoculating the strain, and each treatment is repeated 3 times, with 10 maize seeds each time.

[0099] Germination rate = (number of germinated seeds / total number of seeds) × 100%; Root-shoot ratio = root fresh weight / above-ground fresh weight.

[0100] Maize seeds were treated with a bacterial suspension (10 -1 ), with the culture medium as the control. After 3 days of cultivation, the germination rates of the seeds were 93.99% and 77.78% respectively. And on the first day of cultivation, 93.99% of the seeds treated with the bacterial suspension showed white tips, while only 66.67% of the seeds in the control group did so; on the second day, the QN3NO-3 bacterial suspension promoted the growth of the radicles of maize seeds, and the plumules also swelled rapidly. 80% of the seeds treated with the culture medium showed white tips. There were significant differences between the two treatments, indicating that the strain could make maize seeds enter the white tip stage in advance, with good stability and a germination-promoting effect on maize ( Figure 7 ).

[0101] The quality of maize seedlings greatly affects their later growth and yield, and generally, robust seedlings have stronger biological activities. After maize seeds were inoculated with Bacillus velezensis QN3NO-3 and grown for 7 days, the biological activity indexes of maize seedlings were measured. The results showed that compared with the non-inoculated control group, the maize seedlings inoculated with QN3NO-3 had significant promoting effects on the lengths and fresh weights of the plumules and radicles, and also had a certain increasing effect on the number of lateral roots. The QN3NO-3 strain promoted the aboveground and underground fresh weights of maize seedlings to increase by 35.29% and 80% respectively compared with the non-inoculated seedlings, and the root-shoot ratio also increased from 0.60 to 0.80, significantly promoting the growth of the underground part of the maize seedlings and then promoting the growth of the aboveground part. The plumule of the strain treatment group was 6.04 cm long, 2.22 cm longer than that of the culture medium treatment; the average length of the radicle was 11.45 cm, 1.56 times that of the culture medium group, significantly enhancing the root function activity. Lateral roots play an important role in the drought tolerance of maize. The number of lateral roots in the culture medium group during the seedling stage was 8.07, and it was 8.3 after being treated with the strain. The above results indicate that QN3NO-3 can promote the growth and development of maize seedlings and play an important role in root growth.

[0102] 2.7 Determination of the broad-spectrum antibacterial activity of the strain by the plate confrontation culture method

[0103] Test pathogens: Fusarium oxysporum f. sp. lycopersici, Fusarium oxysporum f. sp. cubense Race 4 (ATCC 76255), Fusarium oxysporum f. sp. cubense Race 1 (ATCC MYA-3244), Fusarium oxysporum sp. cucumerinum (ACCC 30220), Colletotrichum capsici (ATCC 96157), Colletotrichum fragariae (ATCC 58718), Colletotrichum gloeosporioides (ATCC 58691), Colletotrichum musae (ATCC 96167), Colletotrichum higginsianum (KACC 40807), Colletotrichum gloeosporioides (ATCC 58222), Curvularia lunata (ATCC 60937), Alternaria tenuissima (ATCC 26276), Fusarium graminearum Schwabe (ATCC MYA-4620), Cryphonectria parasitica, Pestalotiopsis mangiferae (MTCC 3412), Magnaporthe grisea (ATCC 208987), and Colletotrichum gloeosporioides (ATCC MYA-4131).

[0104] Prepare PDA medium plates. Use a borer to cut out pathogen discs (Φ = 5 mm) that have been cultured for 5 days and are in good growth condition, and place them in the center of the PDA plates. Draw a "plus" sign with the center of the plate as the center. At a distance of 2.5 cm from the center along the four sides of the "plus" sign, inoculate the test strains, with 4 inoculation sites per plate, and set 3 replicates. Use only inoculating the pathogen as the control. After incubating in an inverted position at 28°C for 5 - 7 days, calculate the inhibition rate. Inhibition rate = (control colony diameter - treated colony diameter) / control colony diameter × 100%. The results are shown in Table 2 and Figure 11As shown, strain QN3NO-3 has broad-spectrum resistance to 17 host plants / pathogenic bacteria such as banana, tomato, mango, rubber, and rice.

[0105] Table 2 Research results on the broad-spectrum antibacterial activity of the strain

[0106] Serial number Chinese name of the pathogen Latin name Inhibitory rate 1 Fusarium oxysporum f. sp. lycopersici sp.(Sacc. ) Snyder et Hansen. 61.18% 2 Fusarium oxysporum f. sp. cubense race 4 (E.F.Sm.) Snyd.et Hans,Foc 4 64.12% 3 Fusarium oxysporum f. sp. cubense race 1 (E.F.Sm.) Snyd.et Hans,Foc 1 55.29% 4 Fusarium oxysporum f. sp. cucumerinum (Schl.).cucumerinum Owen. 61.76% 5 Colletotrichum capsici 82.35% 6 Colletotrichum acutatum Brooks 62.94% 7 Colletotrichum gloeosporioides Penz. 68.82% 8 Colletotrichum musae Berk.et Curt. 72.35% 9 Colletotrichum parachutis Sacc. 63.53% 10 Colletotrichum gloeosporioides Penz. 64.12% 11 Curvularia lunata (Wakker) Boed 70.59% 12 Alternaria alternata 65.88% 13 Gibberella zeae 65.88% 14 Cryphonectria parasitica 77.65% 15 Pestalotiopsis mangiferae (P.Henn.) Steyaert 74.71% 16 Magnaporthe oryzae (Hebert) Barr 81.76% 17 Colletotrichum gloeosporioides Penz. 72.35%

[0107] 2.8 Determination of the antibacterial activity of the volatile substances of the strain by the double-dish counterposition method

[0108] The "double-dish counterposition method" is used to determine the antibacterial activity of microbial-derived volatile antibacterial substances. The QN3NO-3 strain was streaked on an LB medium and incubated in an incubator at 28 °C for 2 d. Then, the mycelial discs (Φ = 5 mm) of Fusarium oxysporum f. sp. lycopersici and Fusarium oxysporum f. sp. cubense race 4 were respectively inoculated at the center of PDA plates, and then counterposed with the QN3NO-3 strain grown for 2 days. The two plates were sealed with a sealing film, and the plate counterposed with an empty LB culture dish was used as a control. Incubate in an incubator at 28 °C for another 7 d. Repeat 3 times. It was found that the QN3NO-3 strain could release volatile substances to inhibit the growth of Fusarium oxysporum f. sp. lycopersici and Fusarium oxysporum f. sp. cubense race 4, and the diameter of the pathogenic bacteria was measured using ImageJ software.

[0109] Antibacterial rate = (control colony diameter - treated colony diameter) / control colony diameter × 100%

[0110] The antibacterial rates were 49.22% and 42.16% respectively.

[0111] 2.9 Research on the supernatant and crude protein of the strain fermentation broth

[0112] (1) Antagonistic assay of the supernatant of QN3NO-3 fermentation broth and ammonium sulfate crude protein against Fusarium oxysporum f. sp. lycopersici

[0113] Take 2 mL of the bacterial suspension (10 8 CFU / mL) cultured for 2 d and inoculate it into a 250 mL flask containing 100 mL of CMC liquid medium, and culture it at 28 °C with shaking at 150 r / min for 2 days. The fermentation broth was centrifuged at 10000 rmp for 5 minutes at 4 °C, and the supernatant was the supernatant of the QN3NO-3 fermentation broth, and the antibacterial rate was measured.

[0114] Using ammonium sulfate fractional precipitation method, centrifuge 1 L of the original fermentation broth in batches at high speed in a centrifuge at 4 °C, 12,000 r / min for 30 min. After discarding the precipitate, slowly add the corresponding amount of ammonium sulfate to the supernatant in portions to a final concentration of 20%, 30%, 50%, 70%, and 80% solutions, stir until clear, and obtain the supernatant with different saturation concentrations. Let it stand and precipitate in a 4 °C refrigerator for 24 h. Aliquot it into 50 mL centrifuge tubes and centrifuge at 4 °C, 12,000 r / min for 30 min to collect the precipitate. Add a small amount of 10 mmol / L PBS buffer to each of the above precipitates in a beaker until dissolved, place it in a dialysis bag containing the same concentration of PBS buffer and let it stand overnight at 4 °C. The solution after dialysis and desalting is sterilized with a filter to obtain the crude protein solution of the fermentation broth, and the antibacterial rate is measured.

[0115] Antibacterial rate determination: Using sterile water as a control, the antibacterial activities of the supernatant of the QN3NO-3 fermentation broth and the crude protein solution of the fermentation broth were measured by the plate punching antibacterial test. Use a punching tool with a diameter of 7 mm to punch holes at a distance of 25 mm from the edge of the center of the PDA plate. Add 100 μL of each treatment solution to each hole, and repeat each treatment 3 times. Add the pathogenic fungus (Fusarium oxysporum f. sp. lycopersici) to the center position of the plate. After culturing at room temperature until the control plate is covered with fungi, use ImageJ software to measure the diameter of the pathogenic bacteria in the treatment solution of the QN3NO-3 fermentation broth and the crude protein solution with different concentration gradients. Antibacterial rate = (control colony diameter - treatment colony diameter) / control colony diameter × 100%.

[0116] The results are as Figure 9 shown. The supernatant of QN3NO-3 can antagonize Fusarium oxysporum f. sp. lycopersici, and the antibacterial rate is 49.11%. The crude protein of ammonium sulfate with different concentrations also has an obvious inhibitory effect on Fusarium oxysporum f. sp. lycopersici, and the antibacterial rate reaches more than 46.39%.

[0117] (2) Determination of chitinase and β-1,3-glucanase activities in the supernatant of the strain fermentation broth

[0118] Incubate the QN3NO-3 strain in a sterile LB liquid medium at 28 °C and 180 rpm for 5 d to collect the bacterial suspension, and centrifuge at 8,000 rpm for 15 min. Collect the supernatant every day to measure the chitinase activity and β-1,3-glucanase activity.

[0119] The results are as Figure 10As shown. The cell wall components of pathogenic fungi are mainly composed of chitin and glucan. Experimental data show that Bacillus velezensis QN3NO-3 can produce chitinase and β-1,3-glucanase at different culture stages. Incubated for 5 days, the activities of chitinase and β-1,3-glucanase are the highest, reaching 19.79 ± 0.52 U / mL and 15.19 ± 0.38 U / g respectively. Combined with the antibacterial experiment, it shows that chitinase can weaken or destroy the structural integrity of the fungal cell wall by degrading chitin, directly inhibiting the growth and development of various plant pathogenic fungi. In addition, β-1,3-glucanase degrades another key component of the fungal cell wall, β-1,3-glucan. These two enzymes cause osmotic imbalance, cell lysis and fungal growth inhibition. The activities of chitinase and β-1,3-glucanase provide a basis for the antibacterial mechanism of biocontrol agents. Understanding the existence and activities of these enzymes can further support the development and optimization of biocontrol strategies for plant pathogen management.

[0120] 2.10 Pesticide Hypersensitivity Experiment

[0121] The drug sensitivity experiment of strain QN3NO-3 was carried out by the agar dilution method: Prepare agar dilution plates containing various fungicides (3 concentrations, 3 replicates for each concentration) for streak inoculation of QN3NO-3, and observe the growth of the strain. The results are shown in Table 3. According to the results, strain QN3NO-3 can grow in 7 alternative broad-spectrum fungicides, namely thiophanate-methyl, zhongshengmycin, mancozeb, carbendazim, dexon, metalaxyl, and hymexazol. It is inhibited and grows poorly when the concentration of mancozeb is too high, and grows extremely well when the concentration of carbendazim reaches 500%. This shows that strain QN3NO-3, as a microbial fertilizer, has certain development potential in the current agricultural industry with serious pesticide residue problems. It can replace fungicides to inhibit bacterial and fungal pathogens and can also reproduce normally in farmland contaminated with pesticide residues to promote plant growth.

[0122]

[0123] Note: +: Slightly grow, ++: Normal growth, +++: Abundant growth, -: No growth.

[0124] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to this utility are also within the scope of the present invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. Use of Bacillus velezensis or a fermentation broth containing Bacillus velezensis in nitrogen fixation and / or cellulose degradation, wherein the Bacillus velezensis is named Bacillus velezensis QN3NO-3, registered and preserved in the China Center for Type Culture Collection, with the preservation number CCTCC NO: M 2021304.

2. A bacterial agent containing Bacillus velezensis, wherein the Bacillus velezensis is named Bacillus velezensis QN3NO-3, registered and preserved in the China Center for Type Culture Collection, with the preservation number CCTCC NO: M2021304.

3. Preparation method of the bacterial agent according to claim 2, characterized in that comprises the following steps: (1) Inoculating the Bacillus velezensis into a fermentation medium for scale-up culture to obtain a fermentation broth; (2) Centrifuging the fermentation broth and pouring off the supernatant to obtain a precipitate.

4. According to the preparation method described in claim 3, characterized in that It further comprises step (3): preparing the precipitate into a required pesticide formulation.

5. Application of the bacterial agent according to claim 2 in antagonizing Fusarium oxysporum f. sp. lycopersici, and / or Fusarium oxysporum f. sp. cubense race 4, and / or Fusarium oxysporum f. sp. cubense race 1, and / or Fusarium oxysporum f. sp. cucumerinum, and / or Colletotrichum capsici, and / or Colletotrichum fragariae, and / or Colletotrichum litchii, and / or Colletotrichum musae, and / or Colletotrichum parachinense, and / or Colletotrichum gloeosporioides, and / or Curvularia lunata, and / or Alternaria alternata, and / or Gibberella zeae, and / or Cryphonectria parasitica, and / or Pestalotiopsis mangiferae, and / or Magnaporthe oryzae, and / or Colletotrichum gloeosporioides Penz.

6. Application of the bacterial agent according to claim 2 in preventing and controlling diseases caused by Fusarium oxysporum f. sp. lycopersici, and / or Fusarium oxysporum f. sp. cubense race 4, and / or Fusarium oxysporum f. sp. cubense race 1, and / or Fusarium oxysporum f. sp. cucumerinum, and / or Colletotrichum capsici, and / or Colletotrichum fragariae, and / or Colletotrichum litchii, and / or Colletotrichum musae, and / or Colletotrichum parachinense, and / or Colletotrichum gloeosporioides, and / or Curvularia lunata, and / or Alternaria alternata, and / or Gibberella zeae, and / or Cryphonectria parasitica, and / or Pestalotiopsis mangiferae, and / or Magnaporthe oryzae, and / or Colletotrichum gloeosporioides Penz.

7. Application of the bacterial agent according to claim 2 in destroying the fungal cell wall by producing chitinase and β-1,3-glucanase; or application of the bacterial agent according to claim 2 in nitrogen fixation and / or cellulose degradation.

8. Application of the bacterial agent according to claim 2 in promoting plant growth and / or improving plant drought resistance.

9. The application according to claim 8, characterized in that Bacillus velezensis promotes plant growth by nitrogen fixation and / or secreting IAA.

10. The use of the microbial agent according to claim 2 in promoting seed germination, and / or promoting the seed to enter the white-emergence stage in advance, and / or promoting plumule growth, and / or promoting radicle growth, and / or increasing the fresh weight of the above-ground part of the plant, and / or increasing the fresh weight of the underground part of the plant, and / or promoting the growth of the plant root cap, and / or promoting the growth of the plant root system, and / or increasing the number of lateral roots of the plant.

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