Bacillus velezensis B1-4 as well as preparation method and application thereof

Abstract

The invention discloses bacillus velezensis B1-4 as well as a preparation method and application thereof, and belongs to the technical field of microorganisms. The bacillus velezensis B1-4 is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.33418. The bacillus velezensis B1-4 is obtained through separation and screening, the strain does not have hemolytic activity, can secrete cellulase and protease and has nitrogen fixation and siderophore production capacity, and the 100x liquid of the fermentation liquor can remarkably improve the germination rate and radicle length of cucumber and small rape seeds. The B1-4 fermentation stock solution has a protection effect of 70.31% and a treatment effect of 69.53% on the gray mold of the picked grapes, and can be used for preventing and controlling the gray mold of the grapes. The problem that traditional bacillus riissii cannot effectively antagonize botrytis cinerea is solved.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a strain of Bacillus velezensis B1-4 and a preparation method and application thereof. Background Art

[0002] Gray mold is a major disease affecting grape production. The pathogen, Botrytis cinerea, infects grape leaves, inflorescences, clusters, and rachises, disrupting the normal growth of plant tissues and severely affecting grape quality and yield. Gray mold can cause yield losses, with some vineyards experiencing over 40% diseased clusters. In severe cases, some varieties can even experience crop failure. Currently, the primary method for controlling gray mold in grapes is chemical pesticides. Due to the frequent variation in the physiological subspecies of Botrytis cinerea, farmers increase the amount and frequency of pesticide applications. With this increased use of pesticides, the pathogen has developed varying degrees of resistance. Pesticide residues not only pose a health risk to humans but also disrupt the balance of the soil microbiome.

[0003] Biological control is safe, pollution-free, and drug-resistant, making it an important measure for the green prevention and control of plant diseases. Bacillus can produce antibacterial active substances and promote plant growth, playing an increasingly important role in the agricultural field. Therefore, Bacillus is considered to be a very promising biological control bacteria. Bacillus velezensis is an aerobic bacterium and a member of the genus Bacillus. The currently discovered Bacillus velezensis can secrete a variety of active substances that antagonize plant pathogens, such as antimicrobial lipopeptides, extracellular enzymes, and growth-promoting substances to exert a biocontrol effect. However, the currently known Bacillus velezensis cannot effectively antagonize grape gray mold.

[0004] Based on this, the present invention is proposed. Summary of the Invention

[0005] In view of this, the main purpose of the present invention is to provide a strain of Bacillus velezensis B1-4 and its preparation method and application, so as to solve the problem that traditional Bacillus velezensis cannot effectively antagonize grape gray mold.

[0006] The technical solution of the present invention is achieved as follows:

[0007] The present invention provides a first solution: a strain of Bacillus velez B1-4, which is deposited in the General Microbiology Center of the China Culture Collection Administration Committee of Microorganisms, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC No. 33418.

[0008] In a preferred embodiment, the 16SrDNA sequence of the Bacillus Velezii B1-4 is shown in the sequence listing as SEQ ID NO.1.

[0009] In a preferred embodiment, the physiological and biochemical characteristics of the Bacillus Velezii B1-4 are:

[0010] The colony is milky white in color, and after growing on the culture medium for a period of time, the center of the colony becomes concave;

[0011] The Gram stain of the strain is purple and the bacteria are rod-shaped.

[0012] The present invention provides a second solution: a method for preparing Bacillus Velezii B1-4, comprising the steps of:

[0013] Screening of Bacillus velezinsis B1-4 strains;

[0014] The Bacillus Velezii B1-4 strain was identified.

[0015] In a preferred embodiment, the process of screening the Bacillus Velezii B1-4 strain includes a primary screening process of the antagonistic bacteria B1-4 and a secondary screening process of the antagonistic bacteria B1-4.

[0016] In a preferred embodiment, the process of identifying Bacillus velezensis B1-4 includes morphological identification of the antagonistic bacteria B1-4, physiological and biochemical characteristics research process and molecular identification process of the antagonistic bacteria B1-4.

[0017] In a preferred embodiment, the molecular identification process of antagonistic bacteria B1-4 includes:

[0018] Strain B1-4 genomic DNA was amplified by PCR using primers 27F and 1492R;

[0019] A 25 μL reaction system was used, and the PCR amplification products were detected on 1.5% agarose gel. The amplification program was as follows: 95°C for 5 min; 94°C for 30 s, 57°C for 30 s, and 72°C for 90 s, 30 cycles; 72°C for 10 min;

[0020] The target fragment was recovered using a DNA gel recovery kit and ligated with TaKarapMD*18-T Verctor, and the positive clones obtained were screened for sequencing.

[0021] In a preferred embodiment, the sequences of the primers 27F and 1492R are:

[0022] 27F: 5′-AGTTTGATCMTGGCTCAG-3;

[0023] 1492R: 5′-GGTTACCTTGTTACGACTT-3′.

[0024] The present invention provides a third solution: application of Bacillus Velez B1-4 in preventing and controlling grape gray mold.

[0025] In a preferred embodiment, the Bacillus Velez B1-4 is used in the preparation of a fermentation broth antagonistic to grape gray mold.

[0026] Compared with the prior art, the present invention provides a strain of Bacillus velezensis B1-4 and its preparation method and application, which have the following beneficial effects:

[0027] The present invention isolates and screens Bacillus velezinis B1-4, a strain that is non-hemolytic, capable of secreting cellulase and protease, and possesses nitrogen fixation and siderophore production capabilities. A 100x fermentation broth of the strain can significantly increase the germination rate and radicle length of cucumber and rapeseed seeds. The B1-4 fermentation stock solution exhibits a protective efficacy of 70.31% and a therapeutic efficacy of 69.53% against post-harvest grape gray mold, which is not significantly different from the efficacy of a 5000x solution of 50% fludioxonil (71.88%). The strain is effective for preventing and controlling grape gray mold, resolving the problem that traditional Bacillus velezinis strains are unable to effectively antagonize grape gray mold. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained like these drawings without paying any creative work.

[0029] Figure 1 This is a diagram showing screening for antagonistic bacteria according to Example 1 of the present invention;

[0030] Figure 2 The colony morphology and Gram staining of strain B1-4 in Example 1 of the present invention are shown;

[0031] Figure 3 Phylogenetic tree diagram constructed for strain B1-4 and related strains in Example 1 of the present invention;

[0032] Figure 4 This is a growth curve diagram of strain B1-4 in Example 2 of the present invention;

[0033] Figure 5 This is a graph showing the results of the confrontation culture of strain B1-4 against six plant pathogens in Example 2 of the present invention;

[0034] Figure 6This is a graph showing the biocontrol characteristics and growth-promoting properties of strain B1-4 in Example 2 of the present invention.

[0035] Among them, Figure 2 In the figure, Figure A is the colony morphology, and Figure B is the bacterial morphology;

[0036] exist Figure 5 Figure A is a picture of the pathogen of corn leaf spot, Figure B is a picture of the pathogen of corn leaf spot, Figure C is a picture of the pathogen of corn ear rot, Figure D is a picture of the pathogen of quinoa leaf spot, Figure E is a picture of the pathogen of grape gray mold, and Figure F is a picture of the pathogen of grape anthracnose. In Figures A-F, the left side is the blank control, and the right side is the confrontation culture of the pathogen and B1-4;

[0037] exist Figure 6 Figure A is a diagram of CMC culture medium, Figure B is a diagram of casein culture medium, Figure C is a diagram of nitrogen-free culture medium, and Figure D is a diagram of CAS culture medium. DETAILED DESCRIPTION

[0038] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0039] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0040] Example 1: Screening and identification of Bacillus velezinis B1-4

[0041] 1: Test materials

[0042] Test soil samples: collected from a 5-year grape plantation at the research base of Chengde Academy of Agriculture and Forestry Sciences;

[0043] The test pathogen: B. cinerea, the pathogen of grape gray mold, strain number: HM22-5, isolated, identified and preserved by the Institute of Agricultural Ecology, Chengde Academy of Agriculture and Forestry Sciences;

[0044] Test pesticide: 50% fludioxonil wettable powder, purchased from Syngenta (Suzhou) Crop Protection Co., Ltd.;

[0045] Test culture medium:

[0046] PDA medium: 200 g potato, 20 g glucose, 20 g agar powder, 1 L distilled water; NB medium: 10 g peptone, 3 g beef extract powder, 5 g NaCl, 17 g agar powder, 1 L distilled water, pH 7.0-7.4;

[0047] NB liquid medium: peptone 10g, beef extract powder 3g, NaCl 5g, distilled water 1L, pH 7.0-7.4;

[0048] Sodium carboxymethylcellulose medium: 10 g tryptone, 5 g yeast extract powder, 10 g NaCl, 10 g sodium carboxymethylcellulose, 15 g agar, 1 L distilled water, pH 6.0;

[0049] Casein medium: casein 10g, beef extract powder 3g, NaCl 5g, NaH2PO4 2g, agar powder 15g, bromothymol blue 0.05g, pH 7.3-7.5;

[0050] Nitrogen-free culture medium: mannitol 10 g, potassium hydrogen phosphate 0.2 g, calcium sulfate dihydrate 0.2 g, calcium carbonate 5 g, sodium chloride 0.2 g, magnesium sulfate heptahydrate 0.2 g, agar powder 20 g, distilled water 1 L;

[0051] Siderophore-producing medium: CAS 60.5 mg, HDTMA 72.9 mg, FeCl3.6H2O 2.645 mg, NaH2PO42H2O295.25 mg, Na2HPO4.12H2O 1213.5 mg, ammonium chloride 125 mg, KH2PO437.5 mg, NaCl 62.5 mg.

[0052] 2: Screening of antagonistic bacteria B1-4

[0053] 2.1: Initial screening of antagonistic bacteria: Weigh 10 g of soil sample and place it in a 250 mL Erlenmeyer flask containing 90 mL of sterile water and 10 sterilized glass beads with a diameter of 6 mm. Shake well and place on a shaker at 25°C and 120 r.min. -1 After the soil suspension was allowed to stand, 1 mL of the supernatant was drawn and added to a 50 mL sterile centrifuge tube containing 9 mL of sterile water. After mixing, 10 -1 Dilution, dilute the soil suspension to 10 -5 Set aside. -3 , 10 -4 , 10 -5 200 μL of the soil suspension sample was spread onto NB plates, with three plates spread for each concentration. The plates were then incubated at 25°C. A single colony was selected and cultured against strain HM22-5. The colony with antibacterial activity was selected and stored.

[0054] 2.2: Rescreening of antagonistic bacteria: The antagonistic bacteria obtained in the initial screening were prepared into a concentration of 10 6 cfu.mL- 1 For bacterial suspension, use an 8mm diameter punch to punch out a bacterial cake from the cultured strain HM22-5 and inoculate it in the center of a PDA plate. Place 6mm diameter sterile filter paper discs at the four corners 2.5cm from the center of the plate. Add 20μL of the antagonistic bacterial suspension to each disc. Set up three replicates for each test strain. Incubate at 25°C for 4 days. Measure the size of the inhibition zone and calculate the inhibition rate.

[0055] Bacterial inhibition rate (%) = (control colony diameter - treated colony diameter) / (control colony diameter - bacterial cake diameter) × 100%.

[0056] The antibacterial effect of antagonistic bacteria B1-4 on Botrytis cinerea is shown in Table 1:

[0057] Table 1: Effects of 5 antagonistic bacteria on the antibacterial effect of Botrytis cinerea

[0058]

[0059] Note: Different lowercase letters in the same column indicate significant differences among strains at the P < 0.05 level.

[0060] A total of 142 bacterial strains were isolated from the grape rhizosphere soil, and 5 strains with antagonistic effects were obtained in the initial screening. The results of rescreening of the 5 antagonistic bacteria showed that all 5 strains had obvious inhibition zones ( Figure 1 As shown in Table 1, the inhibition rates of all five antagonistic bacterial strains were greater than 80%, with strain B1-4 having the highest inhibition rate (89.13%). This rate was not significantly different from that of strain 0-1, but significantly different from that of the other strains. The inhibition zones of the five antagonistic bacterial strains ranged from 2.42 mm to 11.08 mm, with B1-4 showing the greatest inhibition, with an inhibition zone of 11.08 mm, significantly larger than that of the other strains.

[0061] 3: Identification of antagonistic bacteria B1-4

[0062] 3.1: Morphological identification and physiological and biochemical characteristics

[0063] Morphological identification: strain B1-4 was inoculated on NB medium plates and cultured in a constant temperature incubator at 25°C for 24 hours, and the morphological characteristics of single colonies were observed; the strain was Gram-stained, and preliminary identification was performed by observing the color and morphology of the bacteria and spores.

[0064] Research on physiological and biochemical characteristics: carried out in accordance with the "Handbook of Identification of Common Bacterial Systems".

[0065] The colony morphology and Gram staining results of B1-4 were obtained as follows Figure 2 As shown by Figure 2 Strain B1-4 forms milky white colonies on NB plates. After growth on the medium for a period of time, the colony centers develop a concave shape (A). Gram staining reveals a purple color, indicating a Gram-positive bacterium with rod-shaped cells (B). Strain B1-4 tested positive in the citrate test, VP test, propionate test, nitrate reduction reaction, and gelatin liquefaction. It did not turn blue in the starch hydrolysis reaction and produced bubbles in the catalase reaction. It can utilize the carbon source D-mannitol but not D-xylose and L-arabinose. It can grow in a 7% NaCl environment and at a pH of 5.7. Based on the colony characteristics, Gram staining, and physiological and biochemical properties, strain B1-4 was tentatively identified as a Bacillus sp.

[0066] 3.2: Molecular identification

[0067] Strain B1-4 genomic DNA was extracted using a bacterial genomic DNA extraction kit from Shanghai Bioengineering. PCR amplification was performed using universal primers 27F (5′-AGTTTGATCMTGGCTCAG-3) / 1492R (5′-GGTTACCTTGTTACGACTT-3′). A 25 μL reaction system was used, and PCR products were detected on a 1.5% agarose gel. The amplification procedure was as follows: 95°C for 5 min, 94°C for 30 s, 57°C for 30 s, and 72°C for 90 s for 30 cycles, and 72°C for 10 min. The target fragment was recovered using a DNA gel extraction kit (Bioengineering) and ligated with TaKarapMD*18-T Verctor. Positive clones were screened and sent to Bioengineering for sequencing. The resulting nucleic acid sequences were aligned using the GenBank database and Blast. 16S rDNA sequences of related species were searched and downloaded to construct a phylogenetic tree.

[0068] The phylogenetic tree is obtained as Figure 3 As shown in Figure 2, after sequencing of strain B1-4, the full length of the 16S rDNA sequence was 1486 bp. This sequence was compared with sequences of the same genus with high homology in the NCBI database, and strain B1-4 was identified as Bacillus venezensis. The phylogenetic tree ( Figure 3) It can be seen that B1-4 belongs to the same branch as Bacillus velezensis (CP12855.1), with a sequence identity of 99%; combined with the colony morphology characteristics and Gram staining results, strain B1-4 was finally identified as Bacillus velezensis, and the strain of Bacillus velezensis B1-4 was deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is February 8, 2025, and the deposit number is CGMCC No. 33418.

[0069] Example 2: Gene Sequencing of Bacillus velezensis B1-4 Strain 1. Materials, Reagents, and Instruments

[0070] 1.1. Main materials and reagents

[0071] Table 2: Main materials and reagents

[0072]

[0073] 1.2. Main instruments

[0074] Table 3: Main instruments

[0075]

[0076] 2. Experimental Procedure

[0077] Primer sequences

[0078] 27F:AGAGTTTGATCMTGGCTCAG

[0079] 1492R:GGTTACCTTGTTACGACTT

[0080] ITS1:TCCGTAGGTGAACCTGCGG

[0081] ITS4-R:TCCTCCGCTTATTGATATGC

[0082] 2.2. Sample Information

[0083] Table 4: Sample information

[0084]

[0085]

[0086] DNA extraction

[0087] For detailed operation steps, please refer to the operating instructions of the Ezup column-based fungal genomic DNA extraction kit (B518259);

[0088] For detailed operation steps, please refer to the operating instructions of the Ezup column-based bacterial genomic DNA extraction kit (B518255);

[0089] DNA electrophoresis

[0090] DNA electrophoresis results (1.5% agarose, 1X TAE electrophoresis buffer, observation);

[0091] PCR amplification

[0092] PCR reaction system

[0093] Table 5: PCR reaction system

[0094]

[0095] PCR reaction conditions

[0096] Table 6: PCR reaction conditions

[0097]

[0098] 2.5.3 PCR electrophoresis

[0099] 1.5% agarose gel, 1x TAE, 150 V, 100 mA, 20 min electrophoresis observation;

[0100] Sequencing

[0101] The 16S rDNA sequence of Bacillus velezensis B1-4 was obtained as shown in the sequence listing SEQ ID NO.1:

[0102] GGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGA

[0103] CAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACG

[0104] TGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATA

[0105] CCGGATGGTTGTTTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCT

[0106] ACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGC

[0107] TCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACT

[0108] GGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCT

[0109] TCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGG

[0110] TTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAAT

[0111] AGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTG

[0112] CCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGG

[0113] CGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCT

[0114] CAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGA

[0115] GAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACA

[0116] CCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAA

[0117] GCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACG

[0118] ATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGC

[0119] ATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGA

[0120] ATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCA

[0121] ACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAG

[0122] GACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGC

[0123] TCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATC

[0124] TTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAAC

[0125] CGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGC

[0126] TACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTT

[0127] AAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGA

[0128] CTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAA

[0129] TACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAA

[0130] CACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTCGT。

[0131] Example : Activity assay of Bacillus cereus B1-4

[0132] The screened Bacillus velezensis B1-4 strain was cultured in the corresponding culture medium by shaking and then cultured into a bacterial liquid. The bacterial liquid was tested for antibacterial activity, extracellular enzyme activity, growth promotion and in vitro control efficacy against grape gray mold.

[0133] 1: Determination of the growth curve of antagonistic bacteria B1-4

[0134] Antagonistic bacteria B1-4 were streaked on NB plate medium and cultured at 25℃ for 24h. Single colonies were inoculated into NB liquid medium and cultured on a shaker for 24h (25℃, 120r.min -1 ) to obtain the strain B1-4 seed solution. Pipette 2 mL of strain B1-4 seed solution and inoculate into 500 mL Erlenmeyer flasks, each containing 200 mL of NB liquid medium, and place in a constant temperature shaker for culture (25 ° C, 120 r.min -1 ), repeated 3 times; sampling once every 4 h, and measuring the OD using a spectrophotometer 600 The value is measured with the time as the horizontal axis and the OD as the 600 The growth curve is drawn with the value as the vertical axis.

[0135] The growth curve of strain B1-4 is shown in Figure 2. Figure 4 As shown by Figure 4 It can be seen that the OD value of Bacillus Velezii B1-4 increased slowly from 0 to 12 hours, which was its delay period; the OD value of strain B1-4 increased rapidly from 12 to 44 hours, which was its logarithmic period; the OD value of the strain remained basically unchanged from 44 to 72 hours, which was its stable period; and the OD value began to decrease after 72 hours, which was its decline period.

[0136] 2: Preparation of fermentation broth of antagonistic bacteria B1-4

[0137] Pick the cultured antagonistic strains and transfer them to 250mL triangular flasks, with 100mL NB liquid culture medium in each flask, culture temperature 25℃, speed 120r.min -1 , shake culture for 24h to obtain seed solution. Pipette 2mL seed solution into triangular flask, fill each flask with 200mL NB liquid medium, shake culture (25℃, 120r.min -1 )) 48h to obtain the fermentation liquid. The fermentation liquid was centrifuged for 2min (4℃, 10000r.min -1 )), the supernatant was transferred to a sterile centrifuge tube, filtered through a 0.22 μm microporous filter membrane (water system) to obtain a sterile fermentation broth, and placed in a refrigerator (4° C.) for short-term storage.

[0138] 3: Antagonistic bacteria B1-4 antibacterial assay

[0139] Six pathogenic bacteria were cultured and 8 mm diameter cakes were taken along the edge of the colony. The cakes were picked with an inoculating needle and inoculated with the mycelial surface facing north in the center of a PDA culture medium plate. A colony of strain B1-4 was spot-inoculated 2.5 cm from the center of the cake. A PDA plate containing no fermentation broth of the pathogenic bacteria was used as a control. Three replicates were performed for each pathogen. The plates were incubated at 25°C for 5-7 days. The growth of the colonies was observed and the colony diameters were measured using the cross-hatch method. The inhibition rate (%) = (control colony diameter - treated colony diameter) / (control colony diameter - cake diameter) × 100%.

[0140] The results of the antibacterial activity of antagonistic bacteria B1-4 against 6 pathogens are as follows: Figure 5 As shown by Figure 5 It can be seen that the six pathogens grew well and formed obvious inhibition zones after being cultured against strain B1-4. This shows that Bacillus Velezii B1-4 has varying degrees of inhibitory effects on the six pathogens.

[0141] 4: Determination of extracellular enzyme activity of antagonistic bacteria B1-4

[0142] 4.1: Carboxymethyl cellulase activity test: Inoculate strain B1-4 on sodium carboxymethyl cellulose test medium and culture in an incubator at 25°C for 3 days. Pour 1g / L Congo red solution into the culture dish and soak for 30 minutes. Then wash with 1mol / L NaCl solution. If a transparent circle appears around the colony, it indicates that the strain has cellulase activity.

[0143] 4.2: Protease activity detection: Pick a single colony of the antagonistic strain B1-4, inoculate it on casein culture medium, and culture it in an incubator at 25℃ for 3 days. If a transparent circle appears around the colony, it indicates that the strain has protease activity.

[0144] The results of the extracellular enzyme activity assay of antagonistic bacteria B1-4 were obtained as follows Figure 6 As shown by Figure 6 It can be seen that strain B1-4 produces yellow and white transparent circles on sodium carboxymethyl cellulose medium (A) and casein medium (B), indicating that the strain has cellulase activity and protease activity.

[0145] 5: Determination of the growth-promoting properties of antagonistic bacteria B1-4

[0146] 5.1: Nitrogen fixation ability test: Inoculate a single colony on nitrogen-free culture medium and culture it in an incubator at 25℃ for 3 days. If the strain can grow in the nitrogen-free culture medium, it indicates that the strain has the ability to fix nitrogen.

[0147] 5.2: Siderophore production capacity test: Use an inoculation needle to pick up the cultured strain and inoculate it on a CAS culture medium plate. Incubate it in an incubator at 25°C for 2 days and observe the color change around the colony. If a transparent circle is produced, it indicates that the strain has the ability to produce siderophores.

[0148] From the results of 5.1 and 5.2 above, it can be seen that the colonies grew well on the nitrogen-free medium (C), indicating that strain B1-4 has the ability to fix nitrogen; when strain B1-4 was inoculated on CAS medium (D), a white transparent circle appeared around the colonies, indicating that the strain can produce siderophores.

[0149] 5.3 Effects of B1-4 on Seed Germination: Rapeseed and cucumber seeds were placed in sterile 15 cm diameter Petri dishes and surface disinfected by soaking in 75% ethanol for 1 min and then 0.1% mercuric chloride for 3 min. After rinsing five times with sterile water, the seeds were dried with sterile filter paper and transferred to a 15 cm Petri dish lined with two layers of sterile filter paper. The fermentation broth prepared in step 2 was diluted 50-fold, 100-fold, and 200-fold, respectively, and used to soak rapeseed and cucumber seeds. Seeds soaked in an equal amount of sterile water served as a control. Fifty seeds were placed per Petri dish, with three replicates per treatment. The seeds were incubated in a 25°C incubator with a 14-h light cycle and a 10-h dark cycle. After 3 days, the seed germination rate was assessed, and the radicle length of five germinated seeds was measured.

[0150] Germination rate = (total number of germinated seeds / total number of test seeds) × 100%;

[0151] The effects of different concentrations of B1-4 fermentation liquid on cucumber and rapeseed seed germination are shown in Table 7;

[0152] Table 7: Effects of different concentrations of B1-4 fermentation liquid on cucumber and rapeseed seed germination

[0153]

[0154] Note: Different lowercase letters in the same column indicate significant differences among treatments at the P < 0.05 level.

[0155] Table 7 shows that the fermentation broth of strain B1-4 had a certain effect on seed germination and radicle length of cucumber and rapeseed. When the fermentation broth was used to soak the seeds, it completely inhibited germination of cucumber and rapeseed seeds. When the seeds were soaked in 50× concentration of the fermentation broth, the germination rate of cucumber and rapeseed seeds was significantly lower than that of the sterile water control, and the radicle length of both seeds was significantly shorter than that of the control. This indicates that the 50× concentration of the fermentation broth significantly inhibited seed germination and radicle length of cucumber and rapeseed seeds. When the seeds were soaked in 100× concentration of the fermentation broth, the germination rate and radicle length of cucumber and rapeseed seeds were significantly higher than those of the sterile water control, indicating that the 100× concentration of the fermentation broth significantly promoted the germination rate and radicle length of cucumber and rapeseed seeds. The 200× concentration of the fermentation broth and NB liquid medium had no significant effect on the germination rate or radicle length of cucumber and rapeseed seeds.

[0156] 6. In vitro efficacy test of antagonistic bacteria B1-4 against grape gray mold

[0157] Select red grapes of uniform size and maturity, wash them with tap water, disinfect them with 1% sodium hypochlorite solution for 30 seconds, and then wash them three times with sterile water. Two treatments were set up in the experiment: (1) Protective effect: Spray the grape berries with B1-4 fermentation solution, 50× solution, 100× solution, and 200× solution. After 24 hours, the grape skin was pierced and inoculated with a gray mold cake with a diameter of 8mm; (2) Curative effect: After piercing the grape skin, inoculate the gray mold cake with a diameter of 8mm. After 24 hours, spray the grape berries with B1-4 fermentation solution, 50× solution, 100× solution, and 200× solution. The fermentation solution concentration was 1×10 8 cfu / mL; spraying with fludioxonil WP 5000× solution served as the control, and spraying with sterile water served as the blank control. Ten berries were sampled for each treatment, with three replicates. The fruit surfaces were incubated at 25°C for 5 days, and the disease status was observed and counted. The grape gray mold disease index and control efficacy were calculated.

[0158] Disease index = [∑(number of diseased fruits at each level × relative level value) / total number of fruits surveyed × 9] × 100%

[0159] Preventive effect (%) = [(control disease index - treatment disease index) / control disease index] × 100

[0160] Table 8: Grape gray mold grading standards

[0161]

[0162] The in vitro protective effects of antagonistic bacteria B1-4 are shown in Table 9;

[0163] Table 9: Control effect of Bacillus Velez B1-4 on detached grape berries

[0164]

[0165]

[0166] Note: Different lowercase letters in the same column indicate significant differences among treatments at the P < 0.05 level.

[0167] As shown in Table 9, the disease index for each treatment was significantly lower than that of the control, indicating that both Bacillus velezensis and 50% fludioxonil WP 5000× solution had effective control effects against grape gray mold. The fermentation solution had the highest control efficacy in the protective effect, with no significant difference between the control efficacy and that of the 50× fermentation solution, but significantly higher than that of the other dilutions. In the curative effect, the control efficacy of the fermentation solution was significantly higher than that of the other dilutions, and significant differences were observed between the dilutions. The control efficacy of the B1-4 fermentation solution in the two treatments was 70.31% and 69.53%, respectively, which was not significantly different from the control efficacy of 50% fludioxonil WP 5000× solution (71.88%).

[0168] The above experiments investigated the growth-promoting properties of Bacillus velezinii B1-4 and found that soaking seeds in 100× the fermentation broth significantly increased the germination rate and radicle length of cucumber and rapeseed seeds. Furthermore, the strain possesses nitrogen fixation and siderophore production capabilities. Furthermore, it effectively antagonizes grape gray mold, demonstrating the promising biocontrol potential of strain B1-4.

[0169] The technical features of the above embodiments can be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0170] The above-described embodiments merely illustrate several embodiments of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, and these modifications and improvements fall within the scope of the present invention. Therefore, the scope of the present invention shall be determined by the appended claims.

Claims

1. A strain of Bacillus Velezii B1-4, characterized in that: The Bacillus Velezii B1-4 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.33418.

2. The Bacillus Velezii B1-4 according to claim 1, characterized in that: The 16SrDNA sequence of the Bacillus Velezii B1-4 is shown in the sequence table SEQ ID NO.

1.

3. The Bacillus Velezii B1-4 according to claim 1, characterized in that: The physiological and biochemical characteristics of the Bacillus Velezii B1-4 are: The colony is milky white in color, and after growing on the culture medium, the center of the colony appears concave; The Gram stain of the strain is purple and the bacteria are rod-shaped.

4. The method for preparing Bacillus Velezii B1-4 as claimed in claim 1, characterized in that: Includes steps: Screening of Bacillus Velezii B1-4 strains; The strain Bacillus Velezii B1-4 was identified.

5. The method for preparing Bacillus Velezii B1-4 according to claim 4, characterized in that: The process of screening the Velez Bacillus B1-4 strain includes a primary screening process of the antagonistic bacteria B1-4 and a secondary screening process of the antagonistic bacteria B1-4.

6. The method for preparing Bacillus Velezii B1-4 according to claim 4, characterized in that: The process of identifying Bacillus Velez B1-4 includes the morphological identification of the antagonistic bacteria B1-4, the physiological and biochemical characteristics research process and the molecular identification process of the antagonistic bacteria B1-4.

7. The method for preparing Bacillus Velezii B1-4 according to claim 6, characterized in that: The molecular identification process of antagonistic bacteria B1-4 included: Strain B1-4 genomic DNA was amplified by PCR using primers 27F and 1492R; A 25 μL reaction system was used, and the PCR amplification product was detected by 1.5% agarose gel. The amplification program was as follows: 95°C for 5 min; 94°C for 30 s, 57°C for 30 s, and 72°C for 90 s, for 30 cycles; 72°C for 10 min; The target fragment was recovered using a DNA gel recovery kit and connected to TaKarapMD*18-T Verctor, and the positive clones obtained were screened for sequencing.

8. The method for preparing Bacillus Velezii B1-4 according to claim 7, characterized in that: The sequences of the primers 27F and 1492R are respectively: 27F: 5′-AGTTTGATCMTGGCTCAG-3; 1492R: 5′-GGTTACCTTGTTACGACTT-3′.

9. Use of Bacillus Velez B1-4 according to any one of claims 1 to 3 in preventing and controlling grape gray mold.

10. The use of Bacillus Velezii B1-4 as claimed in claim 9, characterized in that: The Velez Bacillus B1-4 is used in preparing fermentation liquid antagonizing grape gray mold.

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