A Bacillus velezensis and its application in preventing and controlling grape diseases

Abstract

The present invention discloses a Bacillus velezensis and its application in the prevention and control of plant diseases, belonging to the field of microbial strains. The present invention discloses Bacillus velezensis GSBZ09, with the preservation number of CGMCC NO. 23947; the present invention also discloses the application of the above-mentioned Bacillus velezensis or its culture solution or its bacterial suspension or its fermentation product in promoting plant growth and inhibiting bacteria. The present invention can be used for the prevention and control of grape diseases, especially grape white rot disease.

Description

Technical Field

[0001] The present invention belongs to the field of microbial strains, and particularly relates to a Bacillus velezensis and its application in the prevention and control of plant diseases. Background Art

[0002] Plant growth-promoting rhizobacteria (PGPR) are a type of bacteria that can promote plant growth. After colonizing the plant roots, they can inhibit the invasion of harmful pathogens by promoting plant nutrient absorption, inducing the production of hormones on the root surface, or secreting antibacterial substances, etc., so as to directly or indirectly affect plant growth and development. The growth-promoting mechanisms of PGPR mainly include nitrogen fixation, phosphorus solubilization, potassium solubilization, secretion of siderophores, secretion of plant hormones and related regulatory substances, and release of volatile substances, etc. The research on the enhancement of plant disease resistance by rhizosphere growth-promoting bacteria has always been a hot topic. Growth-promoting strains that can enhance plant disease resistance have been isolated from the genus Bacillus. Research shows that inoculating stress-tolerant PGPR strains under drought conditions can induce the production of plant hormones, promote the growth and development of lateral roots, and enhance the absorption and utilization rate of nutrients and water by plants; inoculating the rhizosphere growth-promoting bacterium Bacillus tequilensis U36 that can secrete IAA can promote the formation of root hairs and root growth of seedlings, thereby increasing the uptake of water and nutrients and helping plants cope with water shortage conditions. Grape white rot is also one of the main diseases of grapes, occurring in grape-growing areas across the country and showing an increasing trend. It mainly damages the fruit clusters and can also infect the branches and leaves. Grape white rot is caused by the infection of wounds by Coniella diplodiella, and is one of the main diseases causing fruit rot. It occurs relatively commonly in grape gardens across the country, with a fruit loss rate of 10-15%. In severe years, the loss can be more than 60%, and even result in no harvest. In the high-temperature and high-humidity season, the damage of this disease is quite serious. At present, the prevention and control of grape white rot mainly rely on chemical control. There is relatively little research on biocontrol bacteria.

[0003] Although domestic and foreign scholars have conducted a large number of studies on biocontrol Bacillus, in the prevention and control of grape diseases, especially grape white rot, the research on biocontrol Bacillus is relatively less. Screening Bacillus with high antagonistic activity is the key link in the research and development of biocontrol agents and the realization of biological control. Summary of the Invention

[0004] The technical problem to be solved by the present invention is: how to effectively prevent and control grape white rot.

[0005] To solve the above technical problems, in a first aspect, the present invention provides Bacillus velezensis, which is Bacillus velezensis, with the strain number GSBZ09 and the deposit number CGMCC No. 23947 at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms.

[0006] To solve the above technical problems, in a second aspect, the present invention provides a microbial agent, which contains the above-mentioned Bacillus velezensis and / or a culture of the above-mentioned Bacillus velezensis.

[0007] The microbial agent can be a microbial agent that produces indole-3-acetic acid (IAA), a phosphorus-degrading microbial agent, a plant growth promoter, a plant pathogen inhibitor, and / or a plant white rot disease control agent.

[0008] The term "culture" refers to the general term for liquid or solid products (all substances in the culture container) with a microbial population after artificial inoculation and cultivation. That is, the product obtained by growing and / or amplifying microorganisms, which can be a pure biological culture of microorganisms or can contain a certain amount of culture medium, metabolites, or other components produced during the cultivation process.

[0009] In the present invention, the culture is the substance (all substances in the culture container) obtained by culturing Bacillus velezensis GSBZ09 in a microbial culture medium.

[0010] To solve the above technical problems, in a third aspect, the present invention provides an application, which can be any one of the following A1)-A9):

[0011] A1) Application of the above-mentioned Bacillus velezensis in the preparation of a product for promoting plant growth;

[0012] A2) Application of the above-mentioned Bacillus velezensis or the above-mentioned microbial agent in promoting plant growth;

[0013] A3) Application of the above-mentioned Bacillus velezensis in the preparation of a plant pathogen inhibitor;

[0014] A4) Application of the above-mentioned Bacillus velezensis or the above-mentioned microbial agent in inhibiting plant pathogens;

[0015] A5) Application of the above-mentioned Bacillus velezensis in the preparation of a product for preventing and / or treating plant white rot disease;

[0016] A6) Application of the above-mentioned Bacillus velezensis or the above-mentioned microbial agent in preventing and / or treating plant white rot disease;

[0017] A7) Use of the above-mentioned Bacillus velezensis or the above-mentioned microbial agent in degrading phosphorus;

[0018] A8) Use of the above-mentioned Bacillus velezensis or the above-mentioned microbial agent in preparing indole-3-acetic acid (IAA);

[0019] A9) Use of the above-mentioned Bacillus velezensis or the above-mentioned microbial agent in preparing siderophore.

[0020] Furthermore, in the above-mentioned use, the plant can be any one of the following P1)-P4):

[0021] P1) Dicotyledonous plants;

[0022] P2) Vitaceae plants;

[0023] P3) Vitis plants;

[0024] P4) Grape.

[0025] Furthermore, in the above-mentioned use, the promotion of plant growth can be the promotion of vegetative growth of plants.

[0026] Specifically, it can be the promotion of the growth of the above-ground part of the plant, the promotion of the increase in fresh weight of the plant and / or the promotion of the increase in dry weight of the plant.

[0027] Furthermore, in the above-mentioned use, it is characterized in that: the pathogenic bacteria in A3) or A4) can be fungi or bacteria;

[0028] The fungi can be at least one of Coniella diplodiella (the causative agent of grape white rot), Gloeosporium fructigrum (the causative agent of grape anthracnose), Fusarium oxysporum, Pestalotia menezesiana (the causative agent of grape twig blight), Phomopsis viticola (the causative agent of grape trunk blight), Alternaria viticola (the causative agent of grape rachis blight), Botryosphaeria dothidea (the causative agent of grape canker), Botrytis cinerea (the causative agent of grape gray mold);

[0029] And / or, the bacteria can be Agrobacterium vitis.

[0030] To solve the above technical problems, in the fourth aspect, the present invention provides a method for promoting plant growth, the method comprising culturing the above-mentioned Bacillus velezensis in a microbial culture medium, collecting the culture, and treating the plant with the culture.

[0031] To solve the above technical problems, in a fifth aspect, the present invention provides a method for preventing and / or treating plant diseases, the method comprising culturing the above-mentioned Bacillus velezensis in a microbial culture medium, collecting the culture, and treating the plant with the culture.

[0032] Further, in the above method, the pathogenic bacteria can be fungi or bacteria;

[0033] The fungi can be at least one of Coniella diplodiella, Gloeosporium fructigrum, Fusarium oxysporum, Pestalotia menezesiana, Phomopsis viticola, Alternaria viticola, Botryosphaeria dothidea, Botrytis cinerea;

[0034] and / or, the bacteria can be Agrobacterium vitis.

[0035] More specifically, the plant disease can be plant white rot, and the pathogenic bacteria can be Coniella diplodiella.

[0036] Further, in the above method, the microbial culture medium can be prepared from the following raw materials: 2% starch, 1% beef extract, 0.5% yeast extract, 0.5% NaCl, 0.1% ZnSO4, the percentages being mass percentages, and the rest being water.

[0037] To solve the above technical problems, in a seventh aspect, the present invention provides a method for culturing the above-mentioned Bacillus velezensis, the culturing method may include the step of culturing the Bacillus velezensis using a microbial culture medium, and the formula of the microbial culture medium is: 2% starch, 1% beef extract, 0.5% yeast extract, 0.5% NaCl, 0.1% ZnSO4, the percentages being mass percentages, and the rest being water.

[0038] In the present invention, the microbial culture medium can be a solid culture medium or a liquid culture medium.

[0039] In the present invention, the solid medium can be prepared by adding 15 g / L of agar powder to the liquid medium.

[0040] In the present invention, the treatment of plants with the culture specifically refers to root irrigation of plants with the fermentation broth or fermentation supernatant of Bacillus velezensis, or contacting the surface of plants with the fermentation broth or fermentation supernatant of Bacillus velezensis.

[0041] In the present invention, the treatment of pathogenic bacteria with the culture specifically refers to spraying plants with the fermentation broth or fermentation supernatant of Bacillus velezensis before the pathogenic bacteria contact the plants, or contacting the surface of plants with the fermentation broth or fermentation supernatant of Bacillus velezensis before and / or after the pathogenic bacteria contact the plants.

[0042] In this study, a Bacillus strain GSBZ09 with good control efficacy against Coniella diplodiella was isolated and screened from the rhizosphere soil of a grape plantation. This strain has a broad antibacterial spectrum and good antibacterial activity against common pathogenic bacteria in grape cultivation, especially against Coniella diplodiella and Agrobacterium vitis. Through the directional screening of grape rhizosphere soil microorganisms, this study aimed to preliminarily obtain Bacillus strains with high efficiency in antagonizing Coniella diplodiella, providing basic materials for the biological control of grape white rot.

[0043] Preservation Description

[0044] Strain name: Bacillus velezensis

[0045] Latin name: Bacillus velezensis

[0046] Strain number: GSBZ09

[0047] Depository institution: China General Microbiological Culture Collection Center

[0048] Abbreviation of depository institution: CGMCC

[0049] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postcode 100101

[0050] Date of deposit: November 22, 2021

[0051] Accession number in the depository center: CGMCC No. 23947 Description of the drawings

[0052] Figure 1 Colony morphology of strain GSBZ09

[0053] Figure 2 Phylogenetic tree of strain GSBZ09 based on 16S rDNA sequence.

[0054] Figure 3For the growth rate and pH change of strain GSBZ09.

[0055] Figure 4 For the antibacterial situation of strain GSBZ09 against different pathogenic bacteria.

[0056] Figure 5 For the abilities of strain GSBZ09 to produce cellulase and protease

[0057] Figure 6 For the siderophore circle and phosphate-solubilizing circle of strain GSBZ09 on CAS and NBRIP media respectively.

[0058] Figure 7 For the reaction phenomena of strain GSBZ09 with Solution I and Solution II in DF+ and DF media.

[0059] Figure 8 For the growth promotion effect of strain GSBZ09 on grapes.

[0060] Figure 9 For the detection of the effect of strain GSBZ09 in controlling grape white rot. Specific implementation manners

[0061] The present invention will be further described in detail below in conjunction with specific implementation manners. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0062] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0063] The experimental methods used in the following embodiments are all conventional methods unless otherwise specified.

[0064] The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0065] The test media in the following embodiments are as follows:

[0066] Beef extract peptone solid medium (NA) medium: 3 g of beef extract, 10 g of peptone, 5 g of NaCl, 15 g of agar, made up to 1 L with deionized water, pH 7.0 - 7.2;

[0067] Beef extract peptone liquid (NB) medium: 3 g of beef extract, 10 g of peptone, 5 g of NaCl, 1 L of distilled water; Agrobacterium rhizogenes (YEB) medium: 1 g of yeast extract, 5 g of beef extract, 5 g of peptone, 5 g of sucrose, 0.5 g of MgSO4·7H2O, add distilled water to 1 L, with or without 15 g of agar, pH 7.8.

[0068] Tryptone liquid medium (LB): 5 g of yeast powder, 10 g of tryptone, 10 g of NaCl, 1000 mL of distilled water. Tryptone solid medium (LB): 5 g of yeast powder, 10 g of tryptone, 10 g of NaCl, 15 g of agar, made up to 1 L with deionized water.

[0069] Potato dextrose medium (PDA): 200 g of potato, 20 g of glucose, 15 g of agar, made up to 1 L with deionized water.

[0070] DF (Dworkin and Foster) medium (1 L): 5.0 g of peptone, 1.5 g of yeast extract, 1.5 g of beef extract, 5 g of NaCl, pH 7.0, made up to 1000 mL with deionized water; DF+ medium (1 L): DF medium supplemented with 500 mg / L of tryptophan.

[0071] NBRIP solid medium: 10.0 g of glucose, 5.0 g of Ca3(PO4)2, 0.5 g of (NH4)2SO4, 0.2 g of NaCl, 0.2 g of KCl, 0.1 g of MgSO4·7H2O, 0.03 g of FeSO4·7H2O, 0.03 g of MnSO4·4H2O, 0.5 g of yeast powder, made up to 1000 mL with deionized water, pH 6.8 - 7.2.

[0072] Cellulase detection medium: 10 g of CMC-Na, 1.31 g of K2HPO4, 3 g of NaNO3, 0.5 g of KCl, 0.5 g of MgSO4·7H2O, 0.01 g of FeSO4, made up to 1000 mL with deionized water, pH 6.5.

[0073] Protease detection medium: 2% skim milk powder, 2% agar powder, made up to 1 L with deionized water.

[0074] The above media were all sterilized at 121 °C for 20 min.

[0075] Example 1: Isolation, purification and identification of strain GSBZ09

[0076] 1.1 Isolation of strain GSBZ09

[0077] This strain was isolated from the rhizosphere soil of grape plants. Weigh 10 g of soil and add it to an Erlenmeyer flask containing 90 mL of sterilized normal saline. Cultivate it on a shaker at 180 r / min for 1 h, then heat it in a water bath at 80 °C for 20 min. Use the dilution plate method to isolate the strain and cultivate it at 28 °C for 48 h. After colonies grow, pick single colonies and purify them by streaking on NA solid medium to obtain strain GSBZ09. Cultivate it at 28 °C for 48 h and store it in a refrigerator at 4 °C for later use.

[0078] 1.2 Identification of Strain GSBZ09

[0079] The screened strain GSBZ09 was identified as follows:

[0080] 1) Morphological observation and physiological characteristic determination of strain GSBZ09

[0081] Inoculate the isolated strain by streaking on LB medium and cultivate it at 28 °C for 48 h to observe the colony morphology of the strain. Identify the physiological and biochemical characteristics according to "Bergey's Manual of Determinative Bacteriology" and "Manual of Systematic Identification of Common Bacteria".

[0082] 2) Identification using the Biolog microbial automatic identification system Use the BIOLOG GENIII kit (operate according to the kit instructions) to measure the utilization of the sole carbon source. All instruments and consumables are products of the Biolog company. The Biolog results are shown in Table 1 Table 1. BIOLOG identification of strain GSBZ09

[0083]

[0084] Note: In the table, "+" indicates positive and "-" indicates negative.

[0085] After strain GSBZ09 was cultured on LB medium for 4 days, the colonies were round, milky white, with irregular edges, protruding surfaces, and semi-moist. As the culture time extended, the colonies gradually turned yellowish-brown, and the outer edges had obvious wrinkles. Observed under a microscope, the bacteria were short rod-shaped and Gram-positive. And no hemolysis zone was produced in the blood agar plate, indicating that strain GSBZ09 is a non-pathogenic bacterium and is safe for humans.

[0086] The culture morphology of the strain is as Figure 1 shown, Figure 1 The first picture from the left in [Figure] is the culture morphology of strain GSBZ09 on LB medium, Figure 1 The second picture from the left in [Figure] is the culture morphology of strain GSBZ09 on the blood agar plate (purchased from Beckman Biotechnology Co., Ltd.) medium.

[0087] The physiological and biochemical characteristics of strain GBA are shown in the table: GSBZ09 can utilize xylose, arabinose, and lactose as carbon sources. The glucose oxidation fermentation type is oxidative. It can grow in a medium containing 2% - 5% NaCl, cannot utilize inorganic nitrogen sources such as (NH4)2SO4 and NH4Cl, cannot produce fluorescent pigments and 3-ketolactose, cannot utilize malonate and tartrate, cannot decompose cellulose and pectin. The result of the litmus milk test is peptonization. It can hydrolyze starch, liquefy gelatin, utilize citrate, and is positive for oxidase, catalase, methyl red test, V-P determination, nitrate reduction, and lipase.

[0088] Table 2. Physiological and Biochemical Indexes of Strain GBA

[0089]

[0090] Note: In the table, "+" indicates positive and "-" indicates negative.

[0091] 1.3. Molecular Identification

[0092] Omega genomic DNA miniprep kit was used to extract DNA. The 16S rRNA sequence of the pathogen was amplified with primers 27F and 1492R. PCR reaction conditions: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 45 s, for 34 cycles; finally, extension at 72°C for 8 min. The PCR product was detected by 1% agarose gel electrophoresis, and the molecular weight of the product was detected using DNA Marker BM5000 as a control. A 1387 bp 16S rDNA target band was obtained. After sequencing, the nucleotide sequence of 16S rDNA is SEQ ID No.1.

[0093] The nucleotide sequences of the primers are as follows:

[0094] 27F: 5’-AGAGTTTGATCCTGGCTCAG-3’;

[0095] 1492R: 5’-CTACGGCTACCTTGTTACGA-3’.

[0096] The obtained sequence was aligned with the data in the NCBI ribosomal RNA sequence database using the NCBI Blast program, and a phylogenetic tree between strain GSBZ09 and other similar strains was constructed using the maximum likelihood method in MEGA 6.0 software. It was found that the genetic relationship between strain GSBZ09 and Bacillus velezensis reached 99%. Combining the observation of morphological characteristics and the analysis of physiological and biochemical characteristics, strain GSBZ09 was identified as Bacillus velezensis of the genus Bacillus. Figure 2 Phylogenetic tree based on 16S rDNA.

[0097] Strain GSBZ09 was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on November 22, 2021, with the deposit number CGMCC No. 23947 and the taxonomic name Bacillus velezensis.

[0098] 1.4 Determination of growth rate and pH value of Bacillus velezensis

[0099] Strain GSBZ09 was inoculated into liquid LB medium and cultured with shaking at 28 °C for 16 h. The bacterial suspension cultured for 16 h was added to LB medium, with a total of 3 replicates, and placed in a shaker at 28 °C for culture. It was measured once every 4 h, and the OD value and pH value measured each time were recorded, with a total of 11 measurement points.

[0100] The growth curve and pH change curve of strain GSBZ09 are as Figure 3 shown. Strain GSBZ09 was in the exponential growth phase before 12 h, and the absorbance reached 1.523 at 12 h. After 12 h, it entered the stable growth phase. The pH value gradually increased with time and reached 8.116 at 32 h and remained stable.

[0101] 1.5 Antibiotic determination of strain GSBZ09

[0102] 1) Determination of resistance to different antibiotics

[0103] Nine antibiotics, namely Ampicillin, Vancomycin, Chloramphenicol, Gentamicin, Tetracycline, Spectinomycin, Kanamycin, Streptomycin, and Rifampicin, were added to 5 mL of liquid LB medium at a ratio of 0.1%. A single colony of strain GSBZ09 was picked and inoculated into 5 mL of liquid LB medium containing different antibiotics, and cultured with shaking at 28°C for 24 h.

[0104] Strain GSBZ09 can grow in LB medium containing Spectinomycin and cannot grow in other antibiotics.

[0105] 2) Determination of resistance to different concentrations of Spectinomycin

[0106] Spectinomycin at concentrations of 50, 100, 200, 400, and 800 μg / mL was added to 4 mL of liquid LB medium at a ratio of 0.1%. A single colony of GSBZ09 was picked and inoculated into 5 mL of liquid LB medium containing different concentrations of Spectinomycin, and cultured with shaking at 28°C for 24 h.

[0107] Strain GSBZ09 can tolerate Spectinomycin, with a maximum tolerance of 2048 μg / mL. The lethal end concentration (EC50) was calculated to be 512 μg / mL.

[0108] Example 2. Functional study of strain GSBZ09

[0109] 2.1 Application of strain GSBZ09 in antibacterial

[0110] 2.1.1 Determination of the antibacterial spectrum of strain GSBZ09 against fungi

[0111] Using the plate confrontation method, fungal discs with a diameter of 7 mm were inoculated at the center of 90 mm PDA plates and cultured at 25°C for 1 day. Strain GSBZ09 was inoculated into liquid LB medium and cultured with shaking at 28°C for 16 h to obtain a GSBZ09 bacterial suspension. 2.5 μL of the GSBZ09 bacterial suspension was inoculated at four points 10 mm from the edge of the culture dish. Inoculating liquid LB medium was used as a blank control. The culture was incubated at 25°C for 3 days. When the blank control was about to cover the entire culture dish, the growth of the target bacteria in the control (colony radius) and the treated group (growth radius after inoculating GSBZ09) were measured, and the antibacterial rate was used to represent.

[0112] Antibacterial rate (%) = (control growth - treated growth) / control growth × 100%

[0113] The sources of pathogenic fungi are as follows:

[0114] Coniella diplodiella, the pathogen of grape white rot; Gloeosporium fructigrum, the pathogen of grape anthracnose; Fusarium oxysporum; Pestalotia menezesiana, the pathogen of grape twig blight; Phomopsis viticola, the pathogen of grape trunk blight; Alternaria viticola, the pathogen of grape rachis blight have been disclosed in the literature "Yin Xiangtian, Su Ling, Wu Xinying, Yang Liying, Zhang Jiuhui. Antibacterial activity and identification of Bacillus sp. GSBMO5 against Coniella diplodiella. Chinese Agricultural Science Bulletin, 2018, 34(1): 134-141.", and the public can obtain the above-mentioned pathogenic bacteria from Shandong Grape Research Institute.

[0115] Botryosphaeria dothidea, the pathogen of grape canker; Botrytis cinerea, the pathogen of grape gray mold have been disclosed in the literature "Yin Xiangtian, Yang Liying, Wang Chaoping, Chen Yingchun, Wu Xinying. Indoor toxicity determination of different fungicides against 6 kinds of grape pathogenic bacteria. Jiangsu Agricultural Sciences, 2021, 49(03): 99-104.", and the public can obtain them from Shandong Grape Research Institute.

[0116] 2.1.2 Determination of antibacterial spectrum of bacteria

[0117] Using the double-layer plate method, inoculate strain GSBZ09 in liquid LB medium and shake-culture at 25 °C for 16 h to obtain a GSBZ09 bacterial suspension. Inoculate 2.5 μL of the GSBZ09 bacterial suspension at the center point of a 90 mm PDA plate, use the inoculated liquid LB medium as a blank control, culture at 25 °C for 3 d, invert the petri dish, add 3 mL of chloroform to the petri dish in a fume hood, and let it stand for 12 h to evaporate the chloroform and inactivate the GSBZ09 strain. Shake-culture the selected pathogenic bacteria in YEB medium at 28 °C for 24 h, add 5 mL of the bacterial suspension to 200 mL of 5% (m / v) WA medium, mix well and pour it onto the PDA plate as the upper layer. Culture in an incubator for 48 h, observe and measure the size of the inhibition zone.

[0118] The sources of pathogenic bacteria are as follows:

[0119] Agrobacterium vitis has been disclosed in the literature "Yin Xiangtian, Wei Yanfeng, Xu Liang, Yang Yang. Screening and Identification of Pathogens of Grape Crown Gall Disease. Acta Agriculturae Sinica, 2019, 9(02): 24-30.", and the public can obtain it from the Shandong Grape Research Institute.

[0120] The determination results of the antifungal spectrum of strain GSBZ09 are as Figure 4 shown in Table 3 below.

[0121] Table 3. Inhibitory effect of strain GSBZ09 on grape pathogens

[0122]

[0123]

[0124] Note: +++ indicates that the diameter of the bacterial inhibition zone is greater than or equal to 5.0 cm or the antifungal rate of fungi is greater than or equal to 60%; ++ indicates that the diameter of the bacterial inhibition is greater than or equal to 4.0 cm and less than 5.0 cm or the antifungal rate of fungi is greater than or equal to 40% and less than 60%.

[0125] 2.2. Determination of the enzyme-producing ability of strain GSBZ09

[0126] Cellulase detection: Inoculate strain GSBZ09 into a test tube containing 5 mL of liquid LB medium, shake culture at 28 °C and 170 r / min for 24 h, and then prepare a bacterial suspension of 10 7 CFU / mL. Drop 2.5 μL of the bacterial suspension onto the center of the cellulase detection medium plate, culture for 3 d, add 10 mL of 0.1% Congo red solution to the plate for staining for 1 h, and wash twice with 1 M NaCl, 30 min each time. Observe the size of the clear zone. The results are as Figure 5 , Bacillus velezensis GSBZ09 was cultured in the cellulase detection medium for 3 d, and the diameter of the clear zone was 1.9 cm, indicating that strain GSBZ09 has the ability to degrade cellulose.

[0127] Protease detection: Drop 2.5 μL of the above bacterial suspension onto the protease detection medium, and observe the diameter of the clear zone after 3 d. The results are as Figure 5 , Bacillus velezensis GSBZ09 was cultured in the protease medium for 3 d, and the diameter of the clear zone was 2.9 cm, indicating that strain GSBZ09 has the ability to produce protease.

[0128] 2.3. Detection of the growth-promoting effect of strain GSBZ09

[0129] 2.3.1. Qualitative detection of phosphorus-solubilizing plate

[0130] The strain GSBZ09 was inoculated into a test tube containing 5 mL of liquid LB medium and cultured with shaking at 28 °C and 170 r / min for 24 h. Then, a bacterial suspension with a concentration of 10 7 CFU / mL was prepared. 2.5 μL of the bacterial suspension was spot-inoculated in the center of an NBRIP solid medium plate and cultured for 7 d. The phosphate-solubilizing circle was observed and its diameter was measured. Each treatment was repeated 3 times.

[0131] The results are as Figure 6 shown. Figure 6 The second figure from the left is the result of the qualitative detection of the phosphate-solubilizing plate. The results show that: After culturing Bacillus velezensis GSBZ09 in NBRIP medium for 7 d, the diameter of the phosphate-solubilizing circle is 1.2 cm, indicating that Bacillus velezensis GSBZ09 has the ability to degrade phosphorus.

[0132] 2.3.2, Determination of IAA (Indole-3-acetic acid)

[0133] A single colony of the strain GSBZ09 was cultured with shaking in LB medium for 12 h and then inoculated into DF and DF+ media at an inoculation amount of 0.1% and cultured at 28 °C and 180 r·min -1 for 24 h. Centrifuge at 12000 r·min -1 for 5 min, take 1 mL of the supernatant and place it in a test tube. Then, 1 mL of standard solution was taken from the prepared IAA aqueous solutions of 5, 10, 15, 20, 25, and 30 μg / mL and placed in a test tube. 50 μL of Solution I (10 mmol / L phosphoric acid) and 2 mL of Solution II (1 mL of 0.5 mol / L FeCl3 dissolved in 50 mL of 35% HClO4) reaction solutions were added respectively. After mixing, the reaction was carried out at room temperature for 30 min, and the absorbance at 530 nm was measured. Deionized water was used as a blank control to draw the standard curve of IAA content and calculate its IAA production.

[0134] The results are as Figure 7 shown: A: The strain GSBZ09 in DF+ medium; B: DF+ medium; C: The strain GSBZ09 in DF medium; DF medium. The reaction phenomena of the strain GSBZ09 with Solution I and Solution II in DF+ and DF media indicate that the strain GSBZ09 can produce a large amount of IAA when cultured with shaking in DF medium containing 500 mg / L tryptophan. The concentration can reach 25.56 μg / mL after 24 h of culture. While in DF medium without tryptophan, the IAA concentration produced by the strain is 4 μg / mL. The results show that tryptophan is beneficial for the strain GSBZ09 to produce IAA.

[0135] 2.3.3, Siderophore detection

[0136] General CAS flat plate detection, and the preparation of the CAS detection solution is as follows:

[0137] Place 6 mL of 10 mmol·L-1 cetyltrimethylammonium bromide (HDTMA) in a 100 mL volumetric flask and appropriately dilute it with double-distilled water. Mix 1.5 mL of 1 mmol·L-1 FeCl3·6H2O and 7.5 mL of 2 mmol·L -1 CAS solution and slowly add it to the volumetric flask along a glass rod. Dissolve 4.3 g of anhydrous piperazine in water and add 6.25 mL of 12 mol·L-1 hydrochloric acid to obtain a buffer solution with a pH of 5.6. Then transfer this solution to the aforementioned volumetric flask and make up the volume to 100 mL with double-distilled water. After sterilization at 121 °C for 15 min, it is reserved for use.

[0138] Preparation of the CAS detection flat plate: Sterilize the prepared 1 mol·L-1 CaCl2 solution, 1 mmol·L -1 MgSO4·6H2O solution, and 10% acid-hydrolyzed casein solution separately at 121 °C for 15 min and reserve for use. Take 0.2 mL of the CaCl2 solution, 0.2 mL of the MgSO4·7H2O solution, and 6 mL of the 10% acid-hydrolyzed casein solution respectively, add the biological buffer solution Pipes, and adjust the pH value to 6.8 - 7.0. Make up the volume to 100 mL with deionized water, add 2 g of agar powder, and after sterilization at 121 °C for 15 min, when the temperature drops to 60 °C, add 5 mL of the prepared CAS blue detection solution and mix evenly. Note that no bubbles should be generated to affect the flat plate detection experiment. Then pour 25 mL into each petri dish. The positive reaction is recorded by the change of the CAS color from blue to yellow or orange. Record the formation of a halo around the colonies on the CAS agar plate.

[0139] The results are as Figure 6 shown Figure 6 In the first attached figure on the left, the result of the siderophore detection is shown. The results indicate that after culturing the strain GSBZ09 on the CAS agar plate for 7 days, a yellow halo was produced around the colonies, indicating the production of siderophores.

[0140] 2.3.4. Grape growth promotion test

[0141] After transplanting one-year-old Red Globe grape seedlings, take 50 seedlings with consistent growth for the experiment. Uniformly pour the reserved 10 6 CFU / mL fermentation broth and the supernatant of the fermentation broth into the rhizosphere of the seedlings. At the same time, set up a control group with clear water. There are 10 plants in each treatment, 50 mL of the bacterial solution for each plant, and root irrigation is carried out once every 7 days for a total of 5 times. Regularly observe the growth status of the plants. After 60 days, pull out all the plants with their roots, wash them, dry them, and respectively count the plant height, root length, fresh weight, and dry weight. All values are statistically averaged.

[0142] Among them, the preparation methods of the fermentation broth and the supernatant of the fermentation broth are as follows:

[0143] (1) The preparation method of the fermentation broth of strain GSBZ09 is as follows:

[0144] Pick a single colony of strain GSBZ09 into 50 mL of LB medium and culture it at 28 °C and 180 r·min -1 for 24 h. Transfer it to LB medium at a ratio of 0.1% and culture it at 28 °C and 180 r·min -1 for 48 h. Obtain the fermentation broth of strain GSBZ09. The content of strain GSBZ09 in the fermentation broth of this strain GSBZ09 is 1×10 8 cfu / mL.

[0145] The preparation method of the fermentation broth of strain GSBM05 is the same as above. The only difference is that the strain used is GSBM05.

[0146] Strain GSBM05 was deposited in the China General Microbiological Culture Collection Center CGMCC on January 10, 2017 for patent procedures, with the deposit number CGMCC NO.13556, and was disclosed in the Chinese patent document CN107058183B announced on March 12, 2021. The public can obtain this strain from the applicant. The obtained strain can only be used for the experimental verification of the present invention and cannot be used for other purposes. (2) The preparation method of the supernatant of the fermentation broth is as follows:

[0147] Centrifuge the fermentation broth of strain GSBZ09 prepared in step (1) at 12000 rpm for 10 min and take the supernatant. Filter the supernatant through a 0.22 μm microporous filter membrane to remove bacteria to obtain the supernatant of the GSBZ09 fermentation broth.

[0148] The preparation method of the supernatant of the fermentation broth of strain GSBM05 is the same as above. The only difference is that the fermentation broth used is the fermentation broth of GSBM05.

[0149] The results are as Figure 8 shown. The results show that both the fermentation broth and the supernatant of the fermentation broth can significantly promote the growth of grape plants after root irrigation, and the fresh weight, dry weight, and length of the above-ground part and roots have all increased. Among them, the growth-promoting effect of the fermentation broth is better than that of the supernatant of the fermentation broth. Compared with the control, the fresh weight and dry weight of the above-ground part treated with the fermentation broth increased by 88.30% and 59.40% respectively. The fresh weight and dry weight of the roots increased by 17.93% and 30.89% respectively. The root length and shoot length increased by 12.29% and 30.40% respectively. The growth-promoting effects of the fermentation broth and the supernatant of the fermentation broth of strain GSBM05 on grapes are both lower than those of strain GSBZ09.

[0150] Table 4. Growth-promoting effect of Bacillus velezensis GSBZ09 on grapes

[0151]

[0152] Note: The lowercase letters after the numbers indicate the significance of differences. The same letters indicate no significant differences between the results, and different letters indicate significant differences (P < 0.05).

[0153] 2.3.5, Effects of Strain GSBZ09 on the Activities of Defense Enzymes in Grapes

[0154] For each grape seedling at 60 days of treatment in 2.3.4, take the leaves of each plant (the second and third fully expanded leaves from the bottom), and use a kit (Suzhou Keming Biotechnology Co., Ltd.) to measure the activities of superoxide dismutase (SOD), polyphenol oxidase (PPO), phenylalanine ammonia-lyase (PAL), total protein content, proline content, and malondialdehyde content.

[0155] 1) Determination of SOD Activity

[0156] Use a superoxide dismutase (SOD) test kit (Suzhou Keming Biotechnology Co., Ltd., product number SOD-2-W) to measure SOD activity.

[0157] The determination of SOD activity adopts the nitroblue tetrazolium method. The 3 mL reaction system contains 2.2 mL of 50 mmol / L pH 7.8 phosphate buffer, 0.2 mL of 60 μmol / L riboflavin, 0.2 mL of 195 mmol / L methionine, 0.1 mL of 3 μmol / L EDTA-Na2, 0.1 mL of SOD crude extract (replaced by buffer for the control), and 0.2 mL of 1.125 mmol / L NBT. The reaction system is placed under 4000 lx sunlight for 20 min. After the reaction, cover it with a black cloth to terminate the reaction, measure the absorbance at a wavelength of 560 nm, and take the inhibition of 50% of the photochemical reduction of NBT as 1 enzyme activity unit.

[0158] Among them, the preparation method of SOD crude extract is: weigh 0.1 g of tissue, add 1 mL of extract, perform ice bath homogenization, centrifuge at 8000 g at 4 °C for 10 min, take the supernatant, and place it on ice for later measurement.

[0159] The calculation formula for SOD activity: SOD activity (U / g fresh weight) = [inhibition percentage ÷ (1 - inhibition percentage) × Vtotal reaction] ÷ (W × Vsample ÷ Vtotal sample);

[0160] In the formula, inhibition percentage = (A control tube - A determination tube) ÷ A control tube × 100%; V total reaction: total volume of the reaction system; V sample: volume of the sample added to the reaction system; V total sample: volume of the extraction solution added; C pr: protein concentration of the sample; W: sample mass.

[0161] 2) Determination of PPO enzyme activity

[0162] The PPO activity was determined using a polyphenol oxidase (PPO) test kit (Suzhou Keming Biotechnology Co., Ltd., product number PPO - 2 - Y).

[0163] Specifically, it includes: 1 g of plant tissue was ground with 1 mL of pre - cooled pH 7.8 (0.05 M) phosphate buffer, then another 1 mL of buffer was added, and it was poured into a 5 mL centrifuge tube. Centrifugation was carried out at 4°C and 10,000 rpm for 20 min, and the supernatant was collected. The reaction system was 3 mL of 0.2 M catechol (prepared with pH 7.8 phosphate buffer), 1 mL of enzyme solution. The inactivated enzyme solution was used as the blank control. It was incubated in a water bath at 30°C for 10 min, and the reaction was immediately terminated with 20% trichloroacetic acid. Centrifugation was carried out at 5,000 rpm for 10 min, and the absorbance was measured at 525 nm.

[0164] PPO enzyme activity = (U / g fresh weight) = 60 × ΔA ÷ W

[0165] In the formula, ΔA = A determination tube - A control tube OD 525nm is the absorbance measured at 525 nm for the reaction solution to be measured; W: sample mass.

[0166] 3) Determination of PAL enzyme activity

[0167] The PAL activity was determined using a phenylalanine ammonia - lyase (PAL) test kit (Suzhou Keming Biotechnology Co., Ltd., product number PAL - 2 - Y).

[0168] Specifically, weigh 0.2 g of grape leaves, add 6 mL of 0.05 mol / L borax-hydrochloric acid buffer solution (containing 5 mmol / L mercaptoethanol) with pH 8.8, 0.2 g of polyvinylpyrrolidone (PVP), grind and homogenize in an ice bath. The homogenate is centrifuged at 4°C and 12,000 r / min for 15 min. Take 0.2 mL of the supernatant, 1 mL of 0.02 mol / L phenylalanine, and 3.8 mL of 0.05 mol / L borax-hydrochloric acid buffer solution to detect PAL activity. The control group adds only 0.2 mL of the supernatant and 4.8 mL of borax-hydrochloric acid buffer solution without adding phenylalanine. After mixing, place it in a constant temperature water bath at 30°C and let it stand for 30 min, then add 0.5 mL of 6 mol / L hydrochloric acid solution to terminate the reaction. Measure the absorbance (A 290nm) at a wavelength of 290 nm. Taking a change of 0.1 in the A290nm value per hour as one enzyme activity unit (U), calculate the PAL activity according to the following formula.

[0169] PAL (U / g fresh weight) = ΔA × Vtotal reaction ÷ (W × Vsample ÷ Vtotal extract) ÷ 0.1 ÷ T = 17.3 × ΔA ÷ W

[0170] In the formula, Vtotal reaction: total volume of the reaction system, 1.04 mL; Vsample: volume of the sample added, 0.02 mL; Vtotal extract: volume of the extraction solution added, 1 mL; T: reaction time, 30 min; Cpr: sample protein concentration, mg / mL; W: sample mass,

[0171] 4) Determination of total protein

[0172] The total protein content is determined using a Coomassie Brilliant Blue method protein content test kit (Suzhou Keming Biotechnology Co., Ltd., product number KMSP-2-W).

[0173] The determination of total protein includes:

[0174] (1) Drawing of the standard curve: Take 100 μl of BSA protein standard solution (0, 25, 125, 250, 500, 750, 1000, 1500, 2000 μg / mL) into a 5 mL centrifuge tube, add 2 mL of BCA working solution, mix thoroughly, and place it at 37°C for 30 min. Measure the OD value at a wavelength of 562 nm. Plot the OD value against the BSA protein mass concentration to obtain the BSA protein standard curve.

[0175] (2) Determination of protein content in grape leaves: Weigh 100 mg of grape leaves and dissolve them in 50 mL of distilled water to prepare a protein test solution with a concentration of 1 mg / mL. Take 100 μL of the test solution into a 5 mL centrifuge tube, add 2 mL of BCA working solution, mix thoroughly, incubate at 37 °C for 30 min, measure the OD value at a wavelength of 562 nm, and substitute it into the standard curve equation to calculate the protein mass concentration.

[0176] Cpr (mg / g) = C standard × (A measurement tube - A blank tube) ÷ (A standard tube - A blank tube) ÷ sample mass = 0.5 × (A measurement tube - A blank tube) ÷ (A standard tube - A blank tube) ÷ sample mass.

[0177] 5) Proline determination

[0178] Use Proline (PRO) Content Test Kit (Suzhou Keming Biotechnology Co., Ltd., product number PRO - 2 - Y) to determine the proline content.

[0179] The proline determination includes: (1) Take 0.5 mL of the sample + 0.5 mL of glacial acetic acid + 0.5 mL of reagent two into a covered test tube, place it in a boiling water bath for 30 min (cover tightly to prevent water loss), and shake it once every 10 min. (2) After cooling, add 1 mL of reagent three to the test tube, shake for 30 s, let it stand for a while to transfer the pigment to toluene; absorb 0.8 mL - 1 mL of the upper layer solution into a 1 mL glass cuvette, and measure the absorbance at a wavelength of 520 nm, record the absorbance value A.

[0180] Pro content (μg / g fresh weight) = [(A 520nm + 0.0021) ÷ 0.0521 × V1] ÷ (W × V1 ÷ V2) = 19.2 × (A 520nm + 0.0021) ÷ W.

[0181] Among them, the sample preparation method is: Weigh about 0.1 g of tissue, add 1 mL of extraction solution, perform ice bath homogenization; then place it in a shaker at 90 °C for 10 min; centrifuge at 10000 g and 25 °C for 10 min, take the supernatant, and wait for measurement after cooling.

[0182] 6) Malondialdehyde determination

[0183] Use a malondialdehyde (MDA) test kit (Suzhou Keming Biotechnology Co., Ltd., product number MDA - 2 - Y) to determine the proline content.

[0184] The determination of malondialdehyde includes: taking 0.5 g of leaves, adding 2 ml of precooled 0.05 mol / l phosphate buffer solution with pH 7.8, adding a small amount of quartz sand, grinding into a homogenate in a mortar cooled in an ice bath, transferring to a 5 ml centrifuge tube, making up the volume to 5 ml with the buffer solution, centrifuging at 4500 r / min for 10 min, and the supernatant is the malondialdehyde extract. Absorb 2 ml of the extract into a graduated test tube, add 3 ml of 5% trichloroacetic acid solution of thiobarbituric acid, boil in a water bath for 10 min, centrifuge at 4500 r / min for 10 min, and measure the absorbance of the supernatant at wavelengths of 532 and 600 nm, using distilled water as the blank control.

[0185] MDA content (nmol / g fresh weight) = [ΔA × V total reaction ÷ (ε × d) × 10^9] ÷ (W × V sample ÷ V total sample) = 25.8 × ΔA ÷ W.

[0186] In the formula, ΔA = A 532nm -A 600nm , and W represents the sample mass.

[0187] The measurement results are as described in the table. The results show that: compared with the control, the activities / content of the three leaf protective enzymes measured after treatment with the fermentation broth and the supernatant of the fermentation broth of strain GSBZ09 have all increased. Among them, in the determination of the activity of phenylalanine ammonia-lyase (PAL), the effect of the supernatant of the fermentation broth is better than that of the fermentation broth, and for SOD and PPO, the effect of the fermentation broth is better than that of the supernatant of the fermentation broth. The contents of total protein and proline after treatment with the fermentation broth and the supernatant of the fermentation broth have also increased, and the malondialdehyde content has decreased. The contents of SOD, PPO, PAL, total protein and proline after treatment with the fermentation broth and the supernatant of the fermentation broth of strain GSBZ05 are all lower than those of strain GSBZ09.

[0188] Table 5. Effects of strain GSBZ09 on grape leaf protective enzymes

[0189]

[0190] Note: The lowercase letters after the numbers indicate the significance of differences. The same letters indicate no significant differences between the results, and different letters indicate significant differences (P < 0.05).

[0191] Example 3. Optimization of the fermentation medium components of strain GSBZ09

[0192] 3.1. Determination content and methods

[0193] 1) Method for determining cell biomass: Dilute the original fermentation broth of strain GSBZ09 5 times with 0.85% normal saline, and measure its D at a wavelength of 600 nm with a 721-type spectrophotometer 600nm .

[0194] 2) Bacteriostatic activity detection method: The strain fermentation broth was centrifuged at 4°C and 10,000 r / min for 15 min, and the supernatant was filtered through a 0.22-μm microporous membrane to obtain a sterile fermentation broth. The spores of Coniella diplodiella cultured on PDA medium for 7 d were scraped and prepared into a suspension with a concentration of 10 7 spores / mL. The spore suspension was mixed with PDA medium at a volume ratio of 1:50 and then poured into a petri dish. After solidification, a hole with a diameter of 7 mm was punched in the middle of the petri dish, and 60 μL of the fermentation broth was added. After 3 d, the diameter of the bacteriostatic zone was measured.

[0195] 3.2 Screening of carbon sources

[0196] 1) 1% lactose, mannitol, glucose, soluble starch, citric acid, maltose, sucrose, malt extract, glycerol, corn flour, and oat flour were respectively added to the basic culture medium as carbon sources in the NB medium, and other components remained unchanged. They were dispensed into 250-mL Erlenmeyer flasks at 100 mL / bottle.

[0197] 2) The seed liquid was inoculated into 250-mL Erlenmeyer flasks containing 100 mL of the liquid medium prepared in step 1) at an inoculation amount of 5%, and cultured at 28°C and 180 r / min for 48 h. The biomass and bacteriostatic activity of the bacteria were measured according to the method in 3.1, with 3 replicates for each treatment group.

[0198] Among them, the formula of the basic culture medium was: 10 g of sucrose, 10 g of peptone, 0.4 g of dipotassium hydrogen phosphate, 0.4 g of magnesium sulfate, 1 L of deionized water, and pH 7.0.

[0199] The preparation method of the seed liquid was: After the strain GSBZ09 was activated on the LB plate, a single colony was picked and cultured in LB liquid medium at 28°C and 180 r / min for 12 h to obtain the seed liquid.

[0200] Table 6. Effects of different carbon sources on Bacillus velezensis GSBZ09

[0201]

[0202] Effects of different carbon sources on the growth of the strain and the production of bacteriostatic active substances: Different carbon sources had significant effects on the metabolism of bacteriostatic active substances by the strain GSBZ09. The bacteriostatic activity and viable bacteria count of the fermentation broth with citric acid as the carbon source were the minimum values, while the bacteriostatic activity and viable bacteria count of the fermentation broth with starch as the carbon source were the highest, and the diameter of the bacteriostatic zone reached 15.5 mm. Therefore, starch was selected as the carbon source for the medium.

[0203] 3.3 Screening of nitrogen sources

[0204] Using 1% peptone, yeast extract, beef extract, yeast paste, (NH4)2SO4, NH4Cl, tryptone, soybean meal, and soybean flour to replace the nitrogen source in the basal medium, the cell biomass and antibacterial activity were measured according to the method in 3.1, with 3 replicates for each treatment group.

[0205] Table 7. Effects of different nitrogen sources on Bacillus velezensis GSBZ09

[0206] Index Peptone Yeast Extract Beef Extract Yeast Paste Ammonium Sulfate Ammonium Chloride Tryptone Soybean Meal Soybean Flour <![CDATA[OD 600nm > 0.515 0.786 0.832 0.440 0.005 0.008 0.266 1.178 1.075 Inhibition Zone Diameter (mm) 13.2 12.5 32.0 31.5 10.0 10.0 22.0 11.5 12.9

[0207] Effects of different nitrogen sources on the growth of the strain and the production of antibacterial active substances: Using starch as the fixed carbon source, the fermentation filtrates with different nitrogen sources all had antibacterial activity. When (NH4)2SO4 and NH4Cl were used as nitrogen sources, the strain hardly grew, and the antibacterial effect of the fermentation filtrate was not strong. When yeast paste was used as the nitrogen source, the antibacterial activity of the fermentation filtrate was the highest, with an inhibition zone diameter of 31.5 mm, but the viable cell count was not large. When beef extract was used as the nitrogen source, the viable cell count in the fermentation broth was relatively high, and the antibacterial activity of the fermentation filtrate was also strong, with an inhibition zone diameter of 32.0 mm. Therefore, yeast paste and beef extract, which can improve the production of antibacterial active substances, were jointly used as the nitrogen source of the medium.

[0208] 3.4. Screening of inorganic salts

[0209] Using 0.5% NaCl, CuSO4, CaCl, MgSO4, ZnSO4, FeSO4, CaNO3, K2HPO4, and CaCO3 to replace the inorganic salts in the basal medium, the cell biomass and antibacterial activity were measured according to the method in 3.1, with 3 replicates for each treatment group.

[0210] Table 8. Effects of different inorganic salts on Bacillus velezensis GSBZ09

[0211] Index NaCl <![CDATA[Copper sulfate]]> <![CDATA[CaCl2]]> <![CDATA[MgSO4]]> <![CDATA[ZnSO4]]> <![CDATA[FeSO4]]> <![CDATA[CaNO3]]> <![CDATA[K2HPO4]]> <![CDATA[CaCO3]]> <![CDATA[OD 600 > 1.333 0.435 0.957 1.233 0.160 0.863 1.342 1.351 1.136 Inhibition Zone Diameter (mm) 15.2 14.0 13.2 312.0 31.0 10.2 15.1 112.3 13.1

[0212] Effects of different inorganic salts on the growth of the strain and the production of antibacterial active substances: Different inorganic salts had different effects on the viable cell count and antibacterial activity of the strain. NaCl could significantly improve the antibacterial activity of the strain, but the viable cell count did not reach the highest. ZnSO4 also had strong antibacterial activity, but the viable cell count was very low. When CuSO4, ZnSO4, and FeSO4 were used as inorganic salts, the viable cell count and antibacterial activity in the fermentation broth were both relatively low. Therefore, NaCl and ZnSO4 in the fermentation broth were selected as the inorganic salts.

[0213] 3.5. Multi-factor orthogonal experiment

[0214] Using the optimal carbon source, nitrogen source, and inorganic salts screened by single-factor experiments as variable factors, an orthogonal experiment with the L16(45) orthogonal table was used to optimize the medium and determine the optimal ratio of each component of the medium.

[0215] Table 9. Results of L16(45) orthogonal experiment

[0216]

[0217]

[0218] Note: K1 represents the sum of the results of 4 tests at the first level of each factor, and K2 represents the sum of the results of 4 tests at the second level of each factor

[0219] Through the above research on single-factor experiments of carbon source and nitrogen source, the optimal types of carbon source, nitrogen source and inorganic salts in the liquid fermentation medium were determined. To further optimize the medium formula, an L16(45) orthogonal table was used to design a 5-factor 4-level orthogonal experiment. Taking the antibacterial activity of the fermentation filtrate as the judgment standard, the optimal ratio of each component in the medium was determined. The experimental results showed that the influence degree of different medium components on the antibacterial activity of strain GSBZ09 was A > C > B > D > E, and the optimal level combination was A3B2C1D3E1.

[0220] According to the results of orthogonal experiment optimization, the optimal medium formula for GSBZ09 to produce antibacterial substances is 2% starch, 1% beef extract, 0.5% yeast extract, 0.5% NaCl, 0.1% ZnSO4, and the rest is water.

[0221] A control experiment was conducted with the optimized medium and the initial medium. The specific steps are as follows:

[0222] 1) Preparation of seed liquid

[0223] After the strain GSBZ09 was activated on the LB plate, a single colony was picked and cultured in the LB liquid medium at 28 °C and 180 r / min for 12 h to obtain the seed liquid.

[0224] 2) Preparation of medium

[0225] The medium formula for the experimental group is: 2% starch, 1% beef extract, 0.5% yeast extract, 0.5% NaCl, 0.1% ZnSO4, and the rest is water.

[0226] The initial medium formula is: 10 g of sucrose, 10 g of peptone, 0.4 g of dipotassium hydrogen phosphate, 0.4 g of magnesium sulfate, 1 L of deionized water, pH 7.0.

[0227] The prepared medium was dispensed into 250 mL Erlenmeyer flasks at 100 mL / bottle, and 3 bottles were dispensed for each treatment group.

[0228] 3) Inoculate the seed solution into 250 mL Erlenmeyer flasks containing 100 mL of liquid medium prepared in step 2) at an inoculation amount of 5%, and culture at 28 °C and 180 r / min for 48 h. Determine the biomass and antibacterial activity of the bacteria according to the method in 3.1, with 3 replicates for each treatment group.

[0229] The results showed that: under the optimal fermentation medium, the antibacterial ability of strain GSBZ09 increased by 40.1%.

[0230] Example 4. Detection of the control effect of strain GSBZ09 against white rot

[0231] 4.1. Test agents

[0232] 4.1.1 Spore suspension of Coniella diplodiella

[0233] Preparation of spore suspension of Coniella diplodiella: Coniella diplodiella was cultured on a PDA plate at 28 °C. After sufficient sporulation, 10 mL of sterile 1% glucose solution was added, and the spores were washed off with a sterile cotton swab, and then made into a suspension with sterile 1% glucose solution, and the spore content was adjusted to 1×10 7 cfu / mL

[0234] 4.1.2 Preparation of the fermentation broth of strain GSBZ09

[0235] After activating strain GSBZ09 on an LB plate, pick a single colony and culture it in an LB liquid medium at 28 °C and 180 r / min for 12 h to obtain a seed solution. Transfer the seed solution to the optimized medium (2% starch, 1% beef extract, 0.5% yeast extract, 0.5% NaCl, 0.1% ZnSO4, and the rest is water) at a ratio of 0.1%, and culture at 28 °C and 180 r / min for 48 h. The fermentation broth of strain GSBZ09 was obtained, and the content of strain GSBZ09 in the fermentation broth of this strain was 1×10 8 cfu / mL.

[0236] 4.1.3 Preparation of the supernatant of the fermentation broth

[0237] Centrifuge the fermentation broth of strain GSBZ09 prepared in 4.1.2 at 12000 rpm for 10 min, take the supernatant, and filter the supernatant through a 0.22 μm microporous filter membrane to remove bacteria to obtain the supernatant of the fermentation broth.

[0238] 4.2. Prevention and treatment of grape white rot

[0239] The experiment had 7 treatments, namely the treatment with Coniella diplodiella, the treatment with fermentation broth, the treatment with supernatant of fermentation broth, the treatment with fermentation broth + Coniella diplodiella (prevention), the treatment with supernatant of fermentation broth + Coniella diplodiella (prevention), the treatment with fermentation broth + Coniella diplodiella (treatment), and the treatment with supernatant of fermentation broth + Coniella diplodiella (treatment). Select Red Globe grape fruits with neat appearance, no diseases, pests and injuries. First, wash them clean with tap water, then spray and disinfect them with 75% alcohol. After 2 minutes, dry them with sterile filter paper. Put the fruits into a petri dish with a diameter of 100 mm lined with sterile filter paper, add sterile water to keep them moist, and prick a wound about 1 mm on each fruit with a sterilized inoculation needle. There are 20 fruits for each treatment group, and repeat 3 times.

[0240] The specific experimental methods are as follows:

[0241] 1) Treatment with Coniella diplodiella: Inoculate 40 μL of Coniella diplodiella spore suspension at the wound.

[0242] 2) Treatment with fermentation broth: Inoculate 40 μL of the fermentation broth of strain GSBZ09 at the wound

[0243] 3) Treatment with supernatant of fermentation broth: Inoculate 40 μL of the supernatant of the fermentation broth of strain GSBZ09 at the wound

[0244] 4) Treatment with fermentation broth + Coniella diplodiella (prevention): After inoculating 20 μL of the fermentation broth of strain GSBZ09 at the wound for 24 hours, then inoculate 20 μL of Coniella diplodiella spore suspension.

[0245] 5) Treatment with supernatant of fermentation broth + Coniella diplodiella (prevention): After inoculating 20 μL of the supernatant of the fermentation broth of strain GSBZ09 at the wound for 24 hours, then inoculate 20 μL of Coniella diplodiella spore suspension.

[0246] 6) Treatment with fermentation broth + Coniella diplodiella (treatment): After inoculating 20 μL of Coniella diplodiella spore suspension at the wound for 24 hours, then inoculate 20 μL of the fermentation broth of strain GSBZ09

[0247] 7) Treatment with supernatant of fermentation broth + Coniella diplodiella (treatment): After inoculating 20 μL of Coniella diplodiella spore suspension at the wound for 24 hours, then inoculate 20 μL of the supernatant of the fermentation broth of strain GSBZ09

[0248] For the control group with the fermentation broth of GSBM05 and the supernatant of the fermentation broth of GSBM05 of strain GSBM05, the treatments of the experimental group are the same as those in 2)-7) above, and the only difference is that the fermentation broth of strain GSBZ09 is replaced with the fermentation broth of strain GSBM05, and the supernatant of the fermentation broth of strain GSBZ09 is replaced with the supernatant of the fermentation broth of strain GSBM05.

[0249] The above treatments are all cultured at 28 °C with moisturizing for 7 days, and the disease statistics are carried out and the control effect is calculated.

[0250] Classification Standard for White Rot Disease

[0251] Grade 0: No disease

[0252] Grade 1: The diseased area accounts for ≤ 5% of the fruit area

[0253] Grade 3: The diseased area accounts for 5% - 20% of the fruit area

[0254] Grade 5: The diseased area accounts for 21% - 50% of the fruit area

[0255] Grade 7: The diseased area accounts for 51% - 75% of the fruit area

[0256] Grade 9: The diseased area accounts for 76% - 100% of the fruit area

[0257]

[0258] Control efficacy = [(Disease index of sterile water control - Disease index of treatment) / Disease index of sterile water control] × 100%

[0259] The experimental data was analyzed for significant differences using DPS software and graphed using Microsoft Excel software

[0260] The results are shown in Table 10 and Figure 9 as follows. The results show that compared with inoculation with the white rot pathogen alone, the fermentation broth and supernatant of strain GSBZ09 both had good control effects on white rot disease of Red Globe grape fruits. The incidence rate and disease index were significantly reduced. And the preventive effect was greater than the therapeutic effect. The control efficacy of the fermentation broth and supernatant of control strain GSBM05 on grape fruits was lower than that of strain GSBZ09

[0261] Table 10. Control Efficacy of Bacillus velezensis GSBZ09 and GSBM05 against Grape White Rot Disease

[0262] Serial Number Treatment Incidence Rate / % Disease Index Control Effect / % A Coniella diplodiella 72.32a 28.95a / B Fermentation Broth of GSBZ09 1.67f 0.18f / C Supernatant of Fermentation Broth of GSBZ09 1.67f 0.18f / D Fermentation Broth of GSBZ09 + White Rot (Prevention) 15.79e 5.93e 89.97a E Supernatant of Fermentation Broth of GSBZ09 + White Rot (Prevention) 15.79e 3.70ef 87.41a F Fermentation Broth of GSBZ09 + White Rot (Treatment) 20.00e 5.93d 79.31b G Supernatant of Fermentation Broth of GSBZ09 + White Rot (Treatment) 60.00b 18.56c 35.32 H Fermentation Broth of GSBM05 3.33f 0.37f / I Supernatant of Fermentation Broth of GSBM05 3.33f 0.37f / J Fermentation Broth of GSBM05 + White Rot (Prevention) 20.00e 4.62d 84.04a K Supernatant of Fermentation Broth of GSBM05 + White Rot (Prevention) 30.00d 6.96d 75.96b L Fermentation Broth of GSBM05 + White Rot (Treatment) 41.67c 24.33b 15.96c M Supernatant of Fermentation Broth of GSBM05 + White Rot (Treatment) 68.33a 24.88a 16.35c

[0263] Note: The lowercase letters after the numbers in the table indicate significant differences. Treatments with at least one same letter have no significant difference at the 0.05 level, and treatments with no same letter have a significant difference at the 0.05 level

[0264] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any changes, uses or improvements to the present invention, including those that depart from the scope disclosed in this application and are made by using conventional techniques known in the art. The application of some basic features can be carried out according to the scope of the appended claims below. Sequence Listing <110> Shandong Academy of Grape <120> Bacillus velezensis and its application in controlling grape diseases <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1387 <212> DNA <213> Bacillus velezensis <400> 1 tgggagcttg ctccctgatg ttagcggcgg acgggtgagt aacacgtggg taacctgcct 60 gtaagactgg gataactccg ggaaaccggg gctaataccg gatggttgtc tgaaccgcat 120 ggttcagaca taaaaggtgg cttcggctac cacttacaga tggacccgcg gcgcattagc 180 tagttggtga ggtaacggct caccaaggcg acgatgcgta gccgacctga gagggtgatc 240 ggccacactg ggactgagac acggcccaga ctcctacggg aggcagcagt agggaatctt 300 ccgcaatgga cgaaagtctg acggagcaac gccgcgtgag tgatgaaggt tttcggatcg 360 taaagctctg ttgttaggga agaacaagtg ccgttcaaat agggcggcac cttgacggta 420 cctaaccaga aagccacggc taactacgtg ccagcagccg cggtaatacg taggtggcaa 480 gcgttgtccg gaattattgg gcgtaaaggg ctcgcaggcg gtttcttaag tctgatgtga 540 aagcccccgg ctcaaccggg gagggtcatt ggaaactggg gaacttgagt gcagaagagg 600 agagtggaat tccacgtgta gcggtgaaat gcgtagagat gtggaggaac accagtggcg 660 aaggcgactc tctggtctgt aactgacgct gaggagcgaa agcgtgggga gcgaacagga 720 ttagataccc tggtagtcca cgccgtaaac gatgagtgct aagtgttagg gggtttccgc 780 cccttagtgc tgcagctaac gcattaagca ctccgcctgg ggagtacggt cgcaagactg 840 aaactcaaag gaattgacgg gggcccgcac aagcggtgga gcatgtggtt taattcgaag 900 caacgcgaag aaccttacca ggtcttgaca tcctctgaca atcctagaga taggacgtcc 960 ccttcggggg cagagtgaca ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt 1020 gggttaagtc ccgcaacgag cgcaaccctt gatcttagtt gccagcattc agttgggcac 1080 tctaaggtga ctgccggtga caaaccggag gaaggtgggg atgacgtcaa atcatcatgc 1140 cccttatgac ctgggctaca cacgtgctac aatggacaga acaaagggca gcgaaaccgc 1200 gaggttaagc caatcccaca aatctgttct cagttcggat cgcagtctgc aactcgactg 1260 cgtgaagctg gaatcgctag taatcgcgga tcagcatgcc gcggtgaata cgttcccggg 1320 ccttgtacac accgcccgtc acaccacgag agtttgtaac acccgaagtc ggtgaggtaa 1380 cctttta 1387

Claims

1. Bacillus velezensis, characterized in that: The Bacillus velezensis is Bacillus velezensis ( Bacillus velezensis ), its strain number is GSBZ09, and its preservation number in the China General Microbiological Culture Collection Center is CGMCC No. 23947.

2. Bacterial agent, characterized in that: The microbial agent contains the Bacillus velezensis described in claim 1 and / or a culture containing the Bacillus velezensis described in claim 1.

3. Application, characterized in that: The applications are any one of the following A1)-A9): A1) Application of the Bacillus velezensis described in claim 1 in the preparation of a product for promoting grape growth; A2) Application of the Bacillus velezensis described in claim 1 or the microbial agent described in claim 2 in promoting grape growth; A3) Application of the Bacillus velezensis described in claim 1 in the preparation of an inhibitor of grape pathogenic bacteria; A4) Application of the Bacillus velezensis described in claim 1 or the microbial agent described in claim 2 in inhibiting grape pathogenic bacteria; A5) Application of the Bacillus velezensis described in claim 1 in the preparation of a product for preventing and / or treating grape white rot; A6) Application of the Bacillus velezensis described in claim 1 or the microbial agent described in claim 2 in preventing and / or treating grape white rot; A7) Application of the Bacillus velezensis described in claim 1 or the microbial agent described in claim 2 in degrading phosphorus; A8) Application of the Bacillus velezensis described in claim 1 or the microbial agent described in claim 2 in the preparation of indoleacetic acid; A9) Application of the Bacillus velezensis described in claim 1 or the microbial agent described in claim 2 in the preparation of siderophore; The pathogenic bacteria in A3) or A4) are fungi or bacteria; The fungus is at least one of Coniella diplodiella (( Coniella diplodiella ), Fusarium oxysporum (( Fusarium oxysporum ), Phomopsis viticola (( Phomopsis viticola ), Alternaria viticola (( Alternaria viticola ), Botryosphaeria dothidea (( Botryosphaeria dothidea ), Botrytis cinerea (( Botrytis cinerea )); the bacterium is Agrobacterium vitis (( Agrobacterium vitis )); The pathogen of the grape white rot disease described in A5) or A6) is Coniella diplodiella( Coniella diplodiella ).

4. A method for promoting grape growth, characterized in that: The method includes culturing the Bacillus velezensis described in claim 1 in a microbial culture medium, collecting the culture, and treating grapes with the culture.

5. The method according to claim 4, characterized in that: The microbial culture medium is prepared from the following raw materials: 2% starch, 1% beef extract, 0.5% yeast extract, 0.5% NaCl, 0.1% ZnSO4, and the percentages are mass percentages, and the rest is water.

6. A method for preventing and / or treating grape diseases, characterized in that: The method includes culturing the Bacillus velezensis described in claim 1 in a microbial culture medium, collecting the culture, and treating grapes with the culture; The grape disease is caused by infection of pathogenic bacteria, and the pathogenic bacteria are fungi or bacteria; The fungus is at least one of Coniella diplodiella ( Coniella diplodiella ), Fusarium oxysporum ( Fusarium oxysporum ), Phomopsis viticola ( Phomopsis viticola ), Alternaria viticola ( Alternaria viticola ), Botryosphaeria dothidea ( Botryosphaeria dothidea ), Botrytis cinerea ( Botrytis cinerea ); and / or, the bacterium is Agrobacterium vitis ( Agrobacterium vitis ).

7. The method according to claim 6, characterized in that: The microbial culture medium is prepared from the following raw materials: 2% starch, 1% beef extract, 0.5% yeast extract, 0.5% NaCl, 0.1% ZnSO4. The percentages are by mass, and the rest is water.

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