Strawberry endophytic bacillus velezensis NR10 and application thereof

Abstract

The invention provides bacillus velezensis NR10, and the preservation number of the bacillus velezensis NR10 is CGMCC (China General Microbiological Culture Collection Center) NO.31771. The bacillus velezensis NR10 is a strawberry endophyte, has good bacteriostatic activity, has good antagonistic action on botrytis cinerea, fusarium oxysporum, colletotrichum gloeosporioides, colletotrichum oxysporum, trichoderma harzianum, trichoderma longibrachiatum and the like, is a potential biological preparation for preventing and treating strawberry diseases and the like, and has broad application prospects. And the bacteriostatic action of the bacillus velezensis NR10 has thermal stability. In addition, the bacillus velezensis also has good capacities of producing siderophores, fixing nitrogen and dissolving potassium, producing protease and cellulase and the like. The bacillus velezensis NR10 provided by the invention has wider-spectrum disease resistance, wide development space and good development and application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a strawberry endophytic Bacillus Velez subtilis NR10 and an application thereof. Background Art

[0002] Strawberry is a perennial herbaceous plant of the genus Fragaria in the Rosaceae family and is deeply loved by people. However, the yield and quality of strawberry cultivation are affected by a variety of diseases. At present, the strawberry planting industry mainly relies on chemical control, but due to the increasingly prominent problems of pesticide residues caused by chemical pesticides, environmental pollution, enhanced resistance of pathogenic bacteria, and gradual decline in control effects, people have gradually shifted their attention to more environmentally friendly biological control methods. Using biological control to control diseases is safe and efficient. In order to adapt to green agriculture and maintain sustainable agricultural development, the pursuit of green, safe and efficient biological pesticides has become a top priority. Biological control is considered to be one of the most important control methods with the most development potential.

[0003] Chinese patent document CN117106615A discloses a microbial agent for preventing and treating gray mold of strawberry and its application, wherein the microbial agent comprises one or more of Bacillus vallismortis, Bacillus amyloliquefaciens, Bacillus megaterium and Streptomyces luteoverticillatus, and the agent is mainly used for preventing and treating gray mold of strawberry by spraying, but the effect of preventing and treating other strawberry diseases is not mentioned. Chinese patent document CN117165492A discloses a strain of Bacillus velez and its application, wherein the agent can be used for preventing and treating anthracnose, wilt and root rot of strawberry, but its inhibition rate is about 50%.

[0004] Strawberry diseases have a serious impact on strawberry yields, so isolating and screening out biocontrol bacteria that have preventive and therapeutic effects on strawberries is an important way to prevent and control strawberry diseases. Strawberry endophytes, as a special beneficial microorganism existing in strawberry tissues, can prevent crop diseases by colonizing in the body. They produce a wide variety of secondary metabolites that can promote growth and have antibacterial effects on strawberries, which are beneficial to plant growth. In addition, strawberry endophytes have long coexisted with strawberry hosts and evolved with them, forming a long-term mutually beneficial symbiotic relationship. Compared with other sources, microbial agents used in strawberry cultivation are safer, avoiding food safety issues, and can replace pesticides and fertilizers to achieve environmentally friendly and increased production effects, maintaining the stability and diversity of the ecosystem. Summary of the invention

[0005] The object of the present invention is to overcome the shortcomings of the prior art and provide a strawberry endophytic Bacillus Velez subtilis NR10 capable of simultaneously inhibiting multiple pathogens.

[0006] In order to achieve the above object, the present invention provides a Bacillus velezensis NR10, with a deposit number of CGMCCNO.31771. Bacillus velezensis NR10 (Bacillus velezensis NR10), classification name: Bacillus yelezensis, the Bacillus velezensis NR10 was received and registered by the General Microbiology Center of the China Microbiological Culture Collection Administration on August 28, 2024, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the telephone number is 010-64807355.

[0007] The present invention also provides a method for preparing the Bacillus Velez NR10, wherein the Bacillus Velez NR10 is separated from the inside of strawberry tissue, comprising the following steps:

[0008] Step 1: Select healthy strawberry plants with no surface damage, wash with clean water, clean the surface with 75% ethanol, soak in 5% NaClO solution for 5 minutes, rinse with sterile water three times, and dry under sterile conditions;

[0009] Step 2: Perform the following operations under sterile conditions: cut roots, stems, leaves, and fruits, grind them into homogenate, and then dilute them in a gradient to obtain 10-1, 10-2, and 10-3 dilutions;

[0010] Step 3: The stock solution homogenate and the dilution obtained in step 2 are evenly spread on NA, PDA, and GSM culture media, and the sterile water-coated plate washed for the last time in step 1 is used as the control group;

[0011] Step 4: Place the culture medium upside down in a 28°C incubator for several days and observe and record. Describe the characteristics of the colonies based on their color, morphology, gloss, odor, and moisture content, and number them. After purification, draw a slant on a test tube and store at 4°C.

[0012] in:

[0013] NA solid culture medium (g / L): 3 g beef extract, 5 g peptone, 5 g sodium chloride, 15 g agar powder.

[0014] PDA solid culture medium (g / L): potato powder 10g, glucose 20g, agar 15g.

[0015] GSM medium (Gao's synthetic medium No. 1) was purchased from Beijing Luqiao Technology Co., Ltd.

[0016] The present invention also provides a fermentation and culture method of the Bacillus Velezii NR10, comprising the following steps:

[0017] Step a: taking the Velez subtilis NR10 described above and inoculating it into LB liquid culture medium, culturing it at 25-37° C. for 12-14 hours, and obtaining Velez subtilis NR10 fermentation seed liquid;

[0018] Step b: taking the fermentation seed liquid prepared in step a, inoculating it into a fermentation medium at a volume percentage of 2-5%, and culturing it at a shaking table at 25-37° C. and a stirring speed of 100-220 rpm for 40-96 hours to obtain a fermentation liquid of Bacillus Velez NR10;

[0019] Step c: culturing for 40 to 96 hours under the conditions of 25 to 37° C., a stirring speed of 100 to 1200 rpm, and a ventilation volume of 0.5 to 3 vvm in a fermenter to obtain a fermentation liquid of Bacillus Velez NR10.

[0020] Preferably, the fermentation medium in step b comprises: 20.0-150.0 g / L of glucose, 1-10.0 g / L of sodium glutamate, 2-20 g / L of peptone, 0.1-2.0 g / L of magnesium sulfate heptahydrate, 0.1-2.0 g / L of potassium chloride, 0.5-3.0 g / L of potassium dihydrogen phosphate, 0.1-10.0 mg / L of ferrous sulfate, 0.1-10.0 mg / L of manganese sulfate, and 0.05-10.0 mg / L of copper sulfate.

[0021] The present invention also provides the fermentation liquid of the Bacillus Velezii NR10.

[0022] The invention also provides the aseptic fermentation filtrate of the Bacillus Velez NR10.

[0023] The invention also provides application of the Bacillus Velezii NR10 in preventing and controlling plant diseases.

[0024] Preferably, the Bacillus Velez NR10 antagonizes at least one of Botrytis cinerea, Fusarium oxysporum, Colletotrichum gloeosporioides, Colletotrichum acutatum, Trichoderma harzianum or Trichoderma longibrachiatum.

[0025] Preferably, the Bacillus Velezii NR10 simultaneously antagonizes multiple pathogens including Botrytis cinerea, Fusarium oxysporum, Colletotrichum gloeosporioides, Colletotrichum acutatum, Trichoderma harzianum or Trichoderma longibrachiatum, and simultaneously prevents and controls diseases caused by the above multiple pathogens.

[0026] The present invention also provides the use of the Bacillus Velezii NR10 in preparing a preparation for preventing and controlling plant diseases.

[0027] Preferably, the plant diseases include at least one disease caused by Botrytis cinerea, Fusarium oxysporum, Colletotrichum gloeosporioides, Colletotrichum acutatum, Trichoderma harzianum or Trichoderma longibrachiatum.

[0028] The present invention also provides the use of the Bacillus Velezii NR10 in at least one of the following aspects, including producing iron carriers, nitrogen fixation, potassium release, preparing protease or preparing cellulase.

[0029] The present invention also provides the use of the Bacillus Velez NR10 in preparing at least one preparation for producing iron carriers, fixing nitrogen and dissolving potassium.

[0030] The present invention also provides the use of the Bacillus Velezii NR10 in the preparation of protease and / or cellulase preparations.

[0031] The present invention also provides a preparation, comprising the Bacillus subtilis NR10, the fermentation liquid of the Bacillus subtilis NR10, and the sterile fermentation filtrate of the Bacillus subtilis NR10.

[0032] The present invention also provides use of the Bacillus Velez NR10, the fermentation liquid of the Bacillus Velez NR10, and the sterile fermentation filtrate of the Bacillus Velez NR10 in antagonizing at least one of the following pathogens, wherein the pathogen is at least one of Botrytis cinerea, Fusarium oxysporum, Colletotrichum gloeosporioides, Colletotrichum acutatum, Trichoderma harzianum, or Trichoderma longibrachiatum.

[0033] The present invention also provides use of the Bacillus Velez NR10, the fermentation liquid of the Bacillus Velez NR10, and the sterile fermentation filtrate of the Bacillus Velez NR10 in preparing a method for preventing and treating diseases caused by at least one of the following pathogens, wherein the pathogens include at least one of Botrytis cinerea, Fusarium oxysporum, Colletotrichum gloeosporioides, Colletotrichum acutatum, Trichoderma harzianum, and Trichoderma longibrachiatum.

[0034] The present invention also provides the 16S rRNA gene sequence of the Bacillus Velezii NR10, as shown in SEQ ID NO:1.

[0035] The strawberry endophytic Bacillus Velez NR10 of the present invention has good antibacterial activity, has good antagonistic effect on Botrytis cinerea, Fusarium oxysporum, Colletotrichum gloeosporioides, Colletotrichum acutatum, Trichoderma harzianum, Trichoderma longibrachiatum and the like, is a potential biological agent for preventing and treating strawberry diseases, and the Bacillus Velez NR10 of the present invention also has good ability of producing siderophore, fixing nitrogen and potassium, producing protease, cellulase and the like. Compared with the prior art, the Bacillus Velez NR10 of the present invention can produce antagonistic effect on multiple pathogenic bacteria at the same time, has a broader spectrum of disease resistance, has a broad development space, and has a good development and application prospect. The strawberry endophytic Bacillus Velez subtilis NR10 of the present invention has thermal stability. Preferably, the inhibition rate of the Bacillus Velez subtilis NR10 on the above-mentioned pathogens of the present invention can maintain the same antibacterial effect under high temperature conditions of 40° C. to 121° C. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 These are the morphological characteristics of strain NR10 in preferred embodiment 1 of the present invention.

[0037] Figure 2 This is the result of the determination of iron carrier production by strain NR10 in preferred embodiment 2 of the present invention.

[0038] Figure 3 The preferred embodiment 2 of the present invention shows the results of nitrogen fixation and potassium solubilization of strain NR10.

[0039] Figure 4 The preferred embodiment 2 of the present invention shows the enzyme production assay result of strain NR10.

[0040] Figure 5 This is the phylogenetic tree of the strain NR10 described in the preferred embodiment 3 of the present invention.

[0041] Figure 6 This is the antagonistic result of strain NR10 in preferred embodiment 4 of the present invention.

[0042] Figure 7 The antibacterial results of the aseptic fermentation filtrate of strain NR10 in the preferred embodiment 5 of the present invention are as follows:

[0043] Figure 8 This is the thermal stability result of the sterile fermentation filtrate of strain NR10 in preferred embodiment 6 of the present invention. DETAILED DESCRIPTION

[0044] The present invention will be further described with reference to the accompanying drawings in conjunction with specific embodiments to better understand the present invention. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in the art or in accordance with the product instructions are used. Where the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0045] The bacterial culture medium used in the following examples is as follows:

[0046] NA solid culture medium (g / L): 3 g beef extract, 5 g peptone, 5 g sodium chloride, 15 g agar powder.

[0047] PDA solid culture medium (g / L): potato powder 10g, glucose 20g, agar 15g.

[0048] King medium formula (g / L): peptone 20.0 g, K2HPO4 1.725 g, glycerol 15.0 mL, MgSO4·7H2O 1.5 g, tryptophan 0.1 g, pH 7.2.

[0049] Gao's synthetic medium (GSM) was purchased from Beijing Luqiao Technology Co., Ltd.; CAS detection medium was purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.; Ashby nitrogen-free medium was purchased from Ruichu Biotechnology (Jiangsu) Co., Ltd.

[0050] Potassium-dissolving culture medium (g / L): sucrose 1.5g, MgSO4·7H2O 0.02g, CaSO4·7H2O 0.01g, NaCl 0.02g, potassium feldspar powder 0.5g, agar 15g, pH 7.0~7.5.

[0051] Potassium feldspar powder treatment method: Add potassium feldspar powder to 10% HCl and soak for 8 to 10 hours (stir once every 1 to 2 hours), and soak overnight. After discarding the supernatant, rinse with deionized water, stir thoroughly, discard the supernatant after standing, and then add deionized water to rinse; repeat this several times until the pH value test shows no change. Dry the treated potassium feldspar powder, grind it, pass it through a 100-mesh sieve, and put it into a bottle for later use.

[0052] Sodium carboxymethyl cellulose (CMC) culture medium (g / L): CMC-Na 5g, MgSO4·7H2O 0.1g, (NH4)2SO40.5g, K2HPO4 0.25g, agar powder 15g. Prepare 0.3mg / mL Congo red reagent and 1mol NaCl solution in advance.

[0053] Skim milk powder culture medium (g / L): 8 g of skim milk powder was dissolved in 300 mL of water and sterilized at 115°C for 10 min.

[0054] Add water to 9 g of agar to make up to 300 mL, sterilize at 121°C for 20 min, and sterilize A and B separately before mixing.

[0055] Unless otherwise specified, the reagents described in the present invention are conventional reagents in the prior art and can be purchased through commercial channels.

[0056] In a preferred embodiment of the present invention, the fermentation culture method of the Bacillus Velezii NR10 is:

[0057] Step a: inoculating Bacillus Velez NR10 into LB liquid culture medium, culturing for 12 to 14 hours at 25 to 37° C. and a shaking speed of 100 to 220 rpm to obtain fermentation seed liquid of Bacillus Velez NR10;

[0058] Step b: taking the fermentation seed liquid prepared in step a, inoculating it into a fermentation medium at a volume percentage of 2-5%, and culturing it at a shaking table at 25-37° C. and a stirring speed of 100-220 rpm for 40-96 hours to obtain a fermentation liquid of Bacillus Velez NR10;

[0059] Step c: culturing for 40 to 96 hours under the conditions of 25 to 37° C., a stirring speed of 100 to 1200 rpm, and a ventilation volume of 0.5 to 3 vvm in a fermenter to obtain a fermentation liquid of Bacillus Velez NR10.

[0060] The fermentation medium of Bacillus Velez NR10 is: glucose 20.0-150.0 g / L, sodium glutamate 1-10.0 g / L, peptone 2-20 g / L, magnesium sulfate heptahydrate 0.1-2.0 g / L, potassium chloride 0.1-2.0 g / L, potassium dihydrogen phosphate 0.5-3.0 g / L, ferrous sulfate 0.1-10.0 mg / L, manganese sulfate 0.1-10.0 mg / L, and copper sulfate 0.05-10.0 mg / L.

[0061] Example 1

[0062] Obtaining endophytic Bacillus Velez NR10 from strawberry.

[0063] 1. Sample source

[0064] In December 2023, strawberry plants were provided by Tianjin Academy of Agricultural Sciences.

[0065] 2. Isolation and screening of strains

[0066] Select healthy strawberry plants with no surface damage, wash with clean water, clean the surface with 75% ethanol, soak in 5% NaClO solution for 5 min, rinse with sterile water 3 times, and dry with sterile filter paper. Use a sterile knife to cut the roots, stems, leaves, and fruits, place them in a sterile mortar, add sterile water and grind them into a homogenate, and dilute them in a gradient to obtain 10 -1 , 10 -2 , 10 -3 Dilution. Pipette 100μL of the stock homogenate and dilution onto NA, PDA, and GSM, and evenly spread on the surface of the culture medium. Take another 100μL of sterile water from the last wash to coat the plate as a control group. If no bacteria grow, it means that the sample surface is sterile. Repeat the experiment 3 times. Invert the culture medium and incubate it at 28℃ for several days to observe and record. Describe the characteristics of the colonies according to their color, morphology, gloss, odor, and moisture content, and number them. After purification, draw a slant on the test tube and store it at 4℃.

[0067] Figure 1 Shown are the morphological characteristics of the obtained strain NR10.

[0068] Example 2

[0069] Determination of biological characteristics of strain NR10

[0070] The plate method was used to determine the ability of strain NR10 to produce iron carriers, fix nitrogen, solubilize potassium, and produce cellulase and protease.

[0071] (1) Determination of iron carrier production, nitrogen fixation, and potassium dissolution

[0072] The isolated endophytes were inoculated in a square pattern on CAS test medium, Ashby nitrogen-free medium, and potassium-dissolving medium by the plate method. The growth status of the strain and the presence of transparent circles were observed, and the diameter was recorded. The ratio of transparent circle diameter D (mm) to colony diameter d (mm) was calculated, and the iron carrier production reached 1.67. Growing in Ashby nitrogen-free medium and potassium-dissolving medium indicates that it has nitrogen-fixing and potassium-dissolving functions.

[0073] (2) Enzyme production assay

[0074] The isolated endophytes were inoculated on the cellulase detection medium by the plate method. After 5 days of culture, the colony diameter was measured and recorded. Congo red solution was added to the medium for staining for 15 minutes. After discarding the stain, 1M NaCl solution was used for decolorization for 15 minutes. The transparent circle was observed and the diameter was recorded. The endophytes were inoculated on the skim milk powder medium in a triangle shape, and the transparent circle was observed and recorded. The ratio of the transparent circle diameter D (mm) to the colony diameter d (mm) was calculated. The cellulase production reached 4.67 and the protease production reached 3.00.

[0075] The results are as follows Figure 2-4 shown.

[0076] Example 3

[0077] 16S rRNA gene sequencing of strain NR10.

[0078] The extracted genomic DNA was used as a template, and the bacterial 16S rDNA gene primers: 27F primer sequence as shown in SEQ ID NO: 2 and 1492R primer sequence as shown in SEQ ID NO: 3 were used for PCR amplification and sequencing. The sequencing results were shown in SEQ ID NO: 1. The sequencing results were compared by BLAST on the NCBI website to determine the species of closely related bacteria. The phylogenetic tree of strain NR10 constructed based on the 16S rRNA gene sequence is shown in Figure 5 As shown, it was identified as Bacillus velezensis.

[0079] Based on the above test results, combined with morphological, cultural and biological characteristics, strain NR10 was identified as a new species of Bacillus velezensis and named Bacillus velezensis NR10. It has been deposited in the General Microbiology Center of China Microbiological Culture Collection Administration with the deposit number CGMCC No.31771.

[0080] Example 4

[0081] Antagonism assay of strain NR10

[0082] The isolated and purified strain NR10 was used as the antagonistic bacteria, and Botrytis cinerea, Fusarium oxysporum, Colletotrichum gloeosporioides, Colletotrichum acutatum, Trichoderma harzianum and Trichoderma longibrachiatum were used as pathogen indicator bacteria. The antagonistic effect of strain NR10 on pathogens was determined by the plate confrontation method. The bacterial cake was punched with a 5mm diameter puncher, the mycelium surface was in contact with the culture medium, and it was placed in the center of the PDA culture dish. The pathogens were dotted around the periphery. The bacterial cake containing only the tested pathogens was used as the control (CK). Each group was repeated 3 times and cultured at 28°C for 5-7 days. The inhibition zones were observed and recorded. The inhibition rates were 88.46%, 72.82%, 87.69%, 77.91%, 65.70%, and 69.23%, respectively. Figure 6 shown.

[0083] Corrected inhibition rate = (colony diameter of control group - colony diameter of experimental group) / (colony diameter of control group - 5 mm)*100%.

[0084] Example 5

[0085] Antibacterial activity of the aseptic fermentation filtrate of strain NR10, taking Botrytis cinerea as an example.

[0086] After the strain NR10 was cultured at 28°C and 180rpm for 48h, the fermentation liquid was centrifuged at 4°C and 12000r / min for 10min. The supernatant after centrifugation was filtered with a 0.22μm syringe filter to obtain a sterile fermentation filtrate. A 5mm bacterial cake of the activated pathogen (taking Colletotrichum gloeosporioides as an example) was taken with a puncher and placed in the center of the culture medium. 9mm holes were punched around the periphery to remove the agar blocks and then 50μL, 100μL, 150μL, and 200μL of the fermentation filtrate were added respectively. Each group was repeated 3 times. After culturing at 28°C for 5-7d, the growth diameter of the pathogen hyphae was measured and the inhibition rate was calculated. The results were 47.69%, 73.08%, 73.34%, and 74.36%, respectively. Figure 7 shown.

[0087] Corrected inhibition rate = (colony diameter of control group - colony diameter of experimental group) / (colony diameter of control group - 5 mm)*100%.

[0088] Example 6

[0089] Thermal stability of sterile fermentation filtrates of strain NR10, using Botrytis cinerea as an example.

[0090] Preparation of fermentation broth of strain NR10: Inoculate single colonies of the strain into LB medium, culture overnight at 28°C and 180 rpm in a shaking incubator. After obtaining the seed solution, inoculate it into PDB liquid medium and culture it at 28°C and 180 rpm for 48 h for use.

[0091] The effect of temperature on the antibacterial active substances of strain NR10 was detected. The sterile filtrate of strain NR10 was treated at 6 different temperatures: (a) left at room temperature without treatment; (b) treated in a water bath at 40℃ for 20 minutes; (c) treated in a water bath at 60℃ for 20 minutes; (d) treated in a water bath at 80℃ for 20 minutes; (e) treated in a water bath at 100℃ for 20 minutes; (f) treated in an autoclave at 121℃ for 20 minutes; after the treatment, a plate punching antibacterial test was carried out. A 5mm diameter pathogen cake was placed in the center of the culture medium, 9mm holes were punched around it to remove the agar block, and 200μL fermentation filtrate was added. Each group was repeated 3 times, and the culture was placed at 28℃ for 5-7d. The colony diameter was measured and the antibacterial rate was calculated. The results were 80.56%, 79.37%, 79.37%, 79.37%, 82.14%, and 77.38% respectively.

[0092] As shown in the figure, the temperature has little effect on the filtrate of strain NR10. As the treatment temperature gradually increases, the inhibitory effect of the aseptic fermentation filtrate of strain NR10 on pathogens (taking Colletotrichum gloeosporioides as an example) does not change much. The inhibitory effect of the fermentation filtrate of strain NR10 on pathogens is 80.56% when the filtrate is not treated at room temperature; after 100°C water bath treatment and 121°C high temperature treatment, the inhibition rates on Colletotrichum gloeosporioides are 82.14% and 76.59%, respectively. The experiment shows that for other pathogens described in the present invention, the antibacterial effect of Bacillus Velezii NR10 also has the same thermal stability.

[0093] The specific embodiments of the present invention are 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 modification and substitution of the practical is also within the scope of the present invention. Therefore, the equalization transformation and modification made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

[0094] The above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A Bacillus Velez NR10, deposited with CGMCC NO.31771.

2. The method for preparing Bacillus Velez NR10 according to claim 1, characterized in that: The Velez Bacillus NR10 is isolated from the inside of strawberry tissue, comprising the following steps: Step 1: Select healthy strawberry plants with no surface damage, wash with clean water, clean the surface with 75% ethanol, soak in 5% NaClO solution for 5 minutes, rinse with sterile water three times, and dry under sterile conditions; Step 2: Perform the following operations under sterile conditions: cut the roots, stems, leaves, and fruits, grind them into homogenate, and gradient dilute to obtain 10 -1 , 10 -2 , 10 -3 dilution; Step 3: The stock solution homogenate and the dilution obtained in step 2 are evenly spread on NA, PDA, and GSM culture media, and the sterile water-coated plate washed for the last time in step 1 is used as the control group; Step 4: Place the culture medium upside down in a 28°C incubator for several days and observe and record. Describe the characteristics of the colonies based on their color, morphology, gloss, odor, and moisture content, and number them. After purification, draw a slant on a test tube and store at 4°C.

3. The fermentation culture method of Bacillus Velez NR10 according to claim 1, comprising the following steps: Step a: taking the Velez subtilis NR10 described above and inoculating it into LB liquid culture medium, culturing it at 25-37° C. for 12-14 hours, and obtaining Velez subtilis NR10 fermentation seed liquid; Step b: taking the fermentation seed liquid prepared in step a, inoculating it into a fermentation medium at a volume percentage of 2-5%, and culturing it at a shaking table at 25-37° C. and a stirring speed of 100-220 rpm for 40-96 hours to obtain a fermentation liquid of Bacillus Velez NR10; Step c: culturing for 40 to 96 hours under the conditions of 25 to 37° C., a stirring speed of 100 to 1200 rpm, and a ventilation volume of 0.5 to 3 vvm in a fermenter to obtain a fermentation liquid of Bacillus Velez NR10.

4. The fermentation and culturing method of Bacillus Velez NR10 according to claim 3, characterized in that: The fermentation medium in step b comprises: 20.0-150.0 g / L of glucose, 1-10.0 g / L of sodium glutamate, 2-20 g / L of peptone, 0.1-2.0 g / L of magnesium sulfate heptahydrate, 0.1-2.0 g / L of potassium chloride, 0.5-3.0 g / L of potassium dihydrogen phosphate, 0.1-10.0 mg / L of ferrous sulfate, 0.1-10.0 mg / L of manganese sulfate, and 0.05-10.0 mg / L of copper sulfate.

5. Use of the Bacillus Velez NR10 as claimed in claim 1 in preventing and controlling plant diseases.

6. The use of Bacillus Velez NR10 in preventing and controlling plant diseases as claimed in claim 5, characterized in that: The Bacillus Velez NR10 antagonizes at least one of Botrytis cinerea, Fusarium oxysporum, Colletotrichum gloeosporioides, Colletotrichum oxysporioides, Trichoderma harzianum or Trichoderma longibrachiatum.

7. Use of the Bacillus Velez NR10 as claimed in claim 1 in preparing a preparation for preventing and controlling plant diseases.

8. The use of Bacillus Velez NR10 according to claim 7 in preparing a preparation for preventing and treating plant diseases, characterized in that: The plant diseases include at least one disease caused by Botrytis cinerea, Fusarium oxysporum, Colletotrichum gloeosporioides, Colletotrichum oxysporioides, Trichoderma harzianum and Trichoderma longibrachiatum.

9. Use of the Bacillus Velez NR10 as claimed in claim 1 in at least one of the following aspects, including producing siderophores, nitrogen fixation, potassium solubilization, preparing protease or preparing cellulase.

10. The preparation for use according to any one of claims 5 to 9, characterized in that: The preparation includes at least one of the Bacillus oryzae NR10, the sterile fermentation broth of the Bacillus velezii NR10, or the sterile fermentation filtrate of the Bacillus velezii NR10.

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