Bacillus licheniformis strain Y15, biocontrol inoculant and application of biocontrol inoculant in prevention and control of crop diseases

Abstract

The invention provides a bacillus licheniformis strain Y15, a biocontrol inoculant and application of the biocontrol inoculant in prevention and control of crop diseases, and belongs to the technical field of functional microorganisms. The bacillus licheniformis strain Y15 is separated and purified from soy sauce residues, the preservation number is CGMCC No.30197, and the bacillus licheniformis strain Y15 has a gibberellic disease biocontrol effect. Experiments show that the strain Y15 has an obvious inhibiting effect on spore germination of gibberellic disease and hypha growth of gibberellic disease. Besides, the strain Y15 provided by the invention not only has a good inhibition effect on gibberellic disease bacteria, but also has a good inhibition effect on physalospora piricola, botrytis cinerea, alternaria solani, stem rot pathogen and gaeumannosis, so that a biocontrol strain resource library of various phytopathogens is enriched, and the biocontrol strain Y15 has a good application prospect. And a new means is provided for green and efficient biological control of microbial resources.

Description

Technical Field

[0001] The invention belongs to the technical field of functional microorganisms, and specifically relates to a Bacillus licheniformis strain Y15, a biocontrol agent and applications thereof in preventing and controlling crop diseases. Background Art

[0002] Wheat Fusarium Head Blight is a major common wheat disease worldwide, which leads to reduced wheat production or even total crop failure, causing serious economic losses. In addition, when the Fusarium graminearum pathogen infects the wheat ear, it produces mycotoxins, trichothecenes and zearalenone, which seriously threaten the health of humans and animals. At present, no wheat varieties resistant to Fusarium Head Blight have been found, and chemical control is still the main means of prevention and control. However, due to its disadvantages such as pesticide residues, resistance and environmental pollution, biological control has received more and more attention. According to the literature and other databases, there are more than 10 kinds of biocontrol strains that are effective against wheat fusarium rust published in the literature: Bacillus Velez, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus psychrophilus, Bacillus polymyxa, Streptomyces, Trichoderma, Pseudomonas fluorescens, Pseudomonas aeruginosa, Bacillus licheniformis, Bacillus brevis, etc., while there are only two registered bacterial agents for wheat fusarium rust prevention: Bacillus subtilis and Bacillus polymyxa. Compared with the literature reports, there are fewer industrialized strains, which cannot meet the needs of safe and efficient disease prevention and control. Summary of the invention

[0003] In view of this, the purpose of the present invention is to provide a Bacillus licheniformis strain Y15, which not only has the effect of effectively preventing and controlling wheat fusarium rust, but also can promote crop growth, and can be widely used as an industrial strain.

[0004] The invention provides a Bacillus licheniformis strain Y15, whose deposit number is CGMCC No.30197.

[0005] The invention provides a biocontrol agent, wherein the active component comprises the Bacillus licheniformis strain Y15.

[0006] Preferably, the Bacillus licheniformis strain Y15 is present in any form of a bacterial suspension, a culture solution and a fermentation solution of the Bacillus licheniformis strain Y15.

[0007] Preferably, the concentration of the active ingredient is (1-100)×10 9 CFU / ml.

[0008] Preferably, the active ingredient further comprises at least one of the following bacterial species and / or metabolites thereof: Bacillus velez, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus psychrophilus, Bacillus polymyxa, Streptomyces, Trichoderma, Pseudomonas fluorescens, Pseudomonas aeruginosa and Bacillus brevis.

[0009] Preferably, the dosage form of the biocontrol agent includes at least one of the following: powder, aqueous solution, granule and microcapsule.

[0010] The present invention provides application of the Bacillus licheniformis strain Y15 or the biocontrol agent in preventing and controlling crop diseases.

[0011] Preferably, the crop diseases include at least one of the following: head blight, ring rot, gray mold, early blight, stem base rot and take-all disease.

[0012] Preferably, the prevention and control of Fusarium fusae disease in crops includes inhibiting the germination of spores of Fusarium fusae and / or the growth of mycelium of Fusarium fusae.

[0013] Preferably, the crops include at least one of the following: wheat, oats, barley, corn, millet, rice, sorghum, soybean, cotton, sugar beet, eggplant, apple, grape, potato and sweet potato.

[0014] The present invention provides a Bacillus licheniformis strain Y15, with a deposit number of CGMCC No. 30197. The present invention separates and purifies a strain Y15 from soy sauce residue, and after primary screening and rescreening, the strain Y15 has a biocontrol effect on fusarium spores, and the strain Y15 is identified as Bacillus licheniformis by molecular identification. The results of the embodiments of the present invention show that the strain Y15 has a significant inhibitory effect on the spore germination and hyphae growth of fusarium spores. It can be seen that the strain Y15 provided by the present invention enriches the biocontrol strain resource library of various plant pathogens, and provides a new means for green and efficient biological control microbial resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The results of the separation and purification of wheat fusarium rust biocontrol strain Y15 are shown in the figure below. The left picture shows the plate morphology of each strain screened, the middle picture shows the plate morphology of strain Y15, and the right picture shows the plate morphology of strain XYG1.

[0016] Figure 2 The biocontrol effect of the biocontrol strain Y15 on wheat fusarium head blight on coleoptile;

[0017] Figure 3 The biocontrol effect of the biocontrol strain Y15 on wheat head blight;

[0018] Figure 4This is the phylogenetic tree of the biocontrol strain Y15;

[0019] Figure 5 This is the result of the biocontrol strain Y15 inhibiting the growth of fusarium spores;

[0020] Figure 6 The plate antagonism results of biocontrol strain Y15 against Fusarium fusogenum;

[0021] Figure 7 The results of the antimicrobial spectrum experiment of biocontrol strain Y15;

[0022] Figure 8 The biocontrol effects of three cultures of biocontrol strain Y15.

[0023] Biological deposit information

[0024] The Bacillus licheniformis strain is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on March 29, 2024. The address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, the biological deposit number is CGMCC No.30197, and the strain number is Y15. DETAILED DESCRIPTION

[0025] The invention provides a Bacillus licheniformis strain Y15, whose preservation number is CGMCC No.30197.

[0026] In the present invention, the strain Y15 is isolated and purified from soy sauce residue, and further screened by a coleoptile screening method to determine that it has a wheat fusarium rust prevention and control effect. Using a molecular identification method, the amplified PCR product is compared with a database and clustered by a phylogenetic tree to determine that the strain Y15 belongs to Bacillus licheniformis.

[0027] The invention provides a biocontrol agent, wherein the active component comprises the Bacillus licheniformis strain Y15.

[0028] In the present invention, the Bacillus licheniformis strain Y15 is preferably embodied in the form of a bacterial suspension, culture solution and / or fermentation solution of the Bacillus licheniformis strain Y15. The method for preparing the culture solution and / or fermentation solution of the Bacillus licheniformis strain Y15 is preferably to inoculate the activated strain Y15 in LB medium for shaking culture. The shaking culture time is 24 to 72 hours, more preferably 36 to 56 hours, and more preferably 48 hours. The shaking culture speed is preferably 120 to 220 rpm, more preferably 180 to 210 rpm, and more preferably 200 rpm. The shaking culture temperature is preferably 28 to 50°C, and more preferably 37°C. The bacterial suspension of the Bacillus licheniformis strain Y15 is preferably obtained by separating the culture solution prepared above into solid and liquid, collecting the solid phase, and resuspending the solid phase in sterile water. The method for solid-liquid separation is preferably membrane filtration or centrifugation. The pore size of the filter membrane is preferably 0.22 μm. The concentration of the active ingredient in the biocontrol agent is preferably (1 to 100)×10 9 CFU / ml, more preferably (5 to 90)×10 9 CFU / ml, more preferably (5-50)×10 9 The prevention effect detection experiment of wheat coleoptile fusarium head blight showed that, compared with the control, the licheniformis strain Y15 could significantly reduce the disease index of wheat head blight and had a good prevention and control effect.

[0029] In the present invention, the active ingredient preferably also includes at least one of the following bacteria and / or metabolites of the bacteria: Bacillus velez, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus psychrophilus, Bacillus polymyxa, Streptomyces, Trichoderma, Pseudomonas fluorescens, Pseudomonas aeruginosa and Bacillus brevis. In view of the fact that the prior art reports that the above-mentioned strains are all common biocontrol bacteria for the prevention and control of fusarium wilt, the Bacillus licheniformis strain Y15 developed by the present invention is compounded with at least one of the above-mentioned biocontrol bacteria to prepare a new type of biocontrol agent.

[0030] In the present invention, the dosage form of the biocontrol agent preferably includes at least one of the following: powder, water, granules and microcapsules. The powder is preferably obtained by mixing the bacteria or culture solution (fermentation solution) of the active ingredient with a freeze-dried protective agent and freeze-drying. The present invention does not specifically limit the type of the freeze-dried protective agent, and freeze-dried freeze-dried protective agents known in the art can be used, such as glucose, mannose and other substances that adjust osmotic pressure. The added concentration of the freeze-dried protective agent is preferably 5% to 20%, more preferably 8% to 18%, further preferably 10% to 15%, and most preferably 12%. The water is preferably obtained by culturing the active ingredient, and the obtained culture solution is separated from the bacteria or mixed with an antifreeze agent without separation. The antifreeze agent includes glycerol. The volume concentration of the antifreeze agent is 20% to 80%, and most preferably 40%. The present invention does not specifically limit the preparation method of the granules or microcapsules, and the granules or microcapsules of the biocontrol agent known in the art are used to prepare them.

[0031] The present invention provides application of the Bacillus licheniformis strain Y15 or the biocontrol agent in preventing and controlling crop diseases.

[0032] In the present invention, the crop diseases controlled preferably include at least one of the following: fusarium head blight, ring rot (caused by Botrytis cinerea), gray mold (caused by Botrytis cinerea), early blight (caused by Alternaria solani), stem base rot (caused by Fusarium graminearum) and take-all disease. The crops preferably include at least one of the following: wheat, oats, barley, corn, millet, rice, sorghum, soybean, cotton, sugar beet, eggplant, apple, grape, potato and sweet potato. The crop fusarium control preferably includes at least one of the following: inhibiting the germination of fusarium head blight spores and / or mycelial growth.

[0033] In the embodiment of the present invention, the effect of the biocontrol strain on the germination of conidia of Fusarium fusogenum was tested, and the results showed that the licheniformis strain Y15 could significantly inhibit the germination of conidia compared with the control group and the strains screened in the same batch. The present invention also uses a plate confrontation method to detect the effect of the biocontrol strain on the mycelial growth of Fusarium fusogenum, and the results showed that the licheniformis strain Y15 could significantly inhibit the mycelial growth of Fusarium fusogenum compared with the control group. At the same time, the antimicrobial spectrum experiment results showed that the strain Y15 had a better inhibitory effect on Fusarium fusogenum, ring spot pathogen, gray mold, early blight pathogen, stem base rot pathogen and wheat take-all pathogen than the control group.

[0034] The following is a detailed description of a Bacillus licheniformis strain Y15, a biocontrol agent and its application in preventing and controlling crop diseases provided by the present invention in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1

[0036] Isolation, purification and preservation of a biocontrol strain

[0037] 1. Isolation, purification and preservation of bacterial strain Y15 from soy sauce residue

[0038] Use a sterile spoon to scoop 5g of soy sauce residue into a sterile sampling bag, then crush it into a muddy state, take 5mL of sterile water for preliminary mixing, draw 100μL of the mixture into a 2mL sterile centrifuge tube, and dilute it with sterile water to a concentration of 10 -1 , 10 -2 , 10 -3 , 10 -4 100 μL of the preliminary mixture and the diluted solution were spread on PCA and MRS culture media respectively. Each treatment had 3 replicates and cultured at 37°C for 3 to 4 days.

[0039] Observe the growth of bacteria on the culture medium. When the number of colonies is stable, select colonies of different shapes and colors and streak them on new PCA and MRS solid culture media until a single colony grows. After the purified and stable strain is shaken in the broth liquid culture medium, mix it evenly with a 40% glycerol water mixture in a ratio of 1:1 and store it in a -80℃ ultra-low temperature refrigerator.

[0040] The morphology of the isolated strain Y15 is shown in Figure 1 Middle figure. The surface of the colony of strain Y15 on LB solid medium is moist and smooth, and the single colony is white and rod-shaped.

[0041] 2. Isolation, Purification and Preservation of Bacterial Strain XYG1 in Wheat

[0042] 2.1 Isolation of bacteria from wheat plants

[0043] Use sterile scissors to cut the above-ground part of the wheat plant and place it in a sterile mortar. Add a small amount of sterilized quartz sand and start grinding. Add 1 mL of sterile water and continue grinding until it becomes a homogenous slurry. Pipette 100 μL of the grinding solution into a 2 mL centrifuge tube and dilute it with sterile water to a concentration of 10 -1 , 10 -2 , 10 -3 , 10 -4 , 100 μL of the original solution and the diluted grinding solution were spread on TSA and 1 / 10 TSA culture media respectively. Each treatment had 3 replicates and cultured at 30°C for 3 to 4 days.

[0044] 2.2 Purification and preservation of bacteria on wheat plants

[0045] Observe the growth of bacteria on the culture medium. When the number of colonies is stable, select colonies of different shapes and colors and streak them on a new LB solid culture medium until a single colony grows. After shaking the purified stable strain in liquid LB, mix it with 50% glycerol at a ratio of 1:1 and store it in a -80℃ ultra-low temperature refrigerator.

[0046] The morphology of the isolated strain XYG1 is shown in Figure 1 The right picture shows that the bacterial strain XYG1 isolated from wheat plants has a smooth colony surface on LB solid medium, and the single colony is white and irregular in shape.

[0047] 3. Preliminary screening of wheat fusarium rust biocontrol bacteria using the coleoptile method

[0048] (1) Select an appropriate amount of healthy wheat seeds, sterilize them with 75% ethanol for 2 min, twice, rinse them with sterile water several times, and soak them in sterile water for 24 h until the seeds turn white;

[0049] (2) Place two sterilized filter papers on the bottom of a 9 cm culture dish and soak them with an appropriate amount of sterile water. Place the soaked wheat seeds with their ventral groove facing downward on the filter paper and place them in a 52×36×14.5 cm white sealed box. Place the seeds in a 12 h photoperiod, 25°C light incubator to keep them moist for 2 days before germination.

[0050] (3) Prepare a petri dish containing soaked filter paper according to (2) and place the germinated seeds with their ventral groove facing downward into the petri dish one by one, with 10 seeds placed in one petri dish;

[0051] (4) Place four layers of gauze at the bottom of the sealed box and add 250 mL of sterile water for moisturizing. Place the treatment in (3) in the sealed box, cover the lid, and place it in a light incubator with a 12-h photoperiod and 25° C. for moisturizing culture for 3-4 days. When the coleoptile is approximately 3-4 cm long, it can be inoculated.

[0052] (5) Cut off the leaves about 3 mm below the joint between the wheat coleoptile and the leaves with sterilized scissors, and apply 2 μL of biocontrol bacteria culture solution (strain Y15 and strain XYG1) to the wound. After the culture solution is dried, cover the lid and place it in a light incubator with a 12-h photoperiod and 25°C for 24 h to keep it moist and then inoculate the pathogen spore suspension;

[0053] (6) After treating wheat leaves according to the method in (5), 2 μL of 1.0×10 5 The suspension of spores / mL was applied to the wound, and after the suspension was dried, the lid was covered and placed in a light incubator with a 12-h photoperiod and 25°C for 7 days to maintain moisture.

[0054] (7) After 7 days of cultivation, the disease was graded according to the ratio of the length of the lesion to the length of the entire coleoptile (a), the disease index was calculated according to Formula I, and the control effect was calculated according to Formula II and photographed and recorded. The grading method is as follows:

[0055] Level 0: a=0;

[0056] Level 1: 0 <a<1 / 4;

[0057] Level 2: 1 / 4≤a≤1 / 2;

[0058] Level 3: 1 / 2 <a<2 / 3;

[0059] Level 4: 2 / 3≤a≤1;

[0060]

[0061] The results for strain Y15 are shown in Figure 2 The disease index of strain Y15 was 19.46 and the protection efficiency was 70.35%.

[0062] 4. Wheat ear bioassay of wheat fusarium rust control bacteria

[0063] (1) selecting 10 wheat plants in the flowering stage and with uniform growth and tying them into bundles;

[0064] (2) Dilute the cultured bacterial solution (strain Y15 and strain XYG1) twice and spray it to ensure that the bacterial solution completely soaks the bundled wheat ears;

[0065] (3) After 24 hours, the concentration of 1.0×10 5 Spray the suspension containing 10 spores / mL in the same manner as in (2), ensuring that the bacterial solution soaks the bundled wheat ears, and promptly cover them with white plastic bags to keep them moist;

[0066] (4) Remove the white plastic bags at an appropriate time according to the field temperature: if the average temperature is below 25°C, remove the plastic bags 5 days after bagging; if the temperature is above 25°C, remove the plastic bags 3 days after bagging;

[0067] (5) After 10 to 15 days, the crops are graded according to the percentage of the diseased area of ​​the ear to the whole ear area. The grading method is as follows (based on the industry standard "GB / T 15796-2011" issued by the Ministry of Agriculture of the People's Republic of China):

[0068] Level 0: no disease;

[0069] Level 1: The number of diseased ears accounts for less than 1 / 4 of the total number of ears;

[0070] Level 2: The number of diseased ears accounts for 1 / 4-1 / 2 of the total number of ears;

[0071] Level 3: The number of diseased ears accounts for less than 1 / 2-3 / 4 of the total number of ears;

[0072] Level 4: The number of diseased ears accounts for more than 3 / 4 of the total number of ears.

[0073] Calculate the disease index and prevention efficacy according to the above method and take photos for record.

[0074] Results Figure 3 The disease index of strain Y15 was 17.36, and the control effect was 77.75%. The disease index of strain XYG1 was 20.37, and the control effect was 69.95%.

[0075] 5. Molecular identification of wheat fusarium rust biocontrol bacteria

[0076] 5.1 Extraction of bacterial genomic DNA

[0077] (1) Use a pipette to draw 1.5 mL of strain Y15 bacterial solution into a 2 mL centrifuge tube, centrifuge at 12000 rpm for 1 min, discard the supernatant, and collect the bacterial cells;

[0078] (2) Add 500 μL ddH2O and vortex for 30-40 seconds until no bacterial clumps are evident;

[0079] (3) Add 60-100 μL of lysozyme (20 mg / mL) according to the amount of bacteria, vortex and mix, then place in a 37°C water bath for 30 min to 1 h, turning upside down several times every 20 min;

[0080] (4) Add 650 μL of nucleic acid lysis buffer and mix well by pipetting;

[0081] (5) Add 250 μL protein precipitation solution and vortex for 1 min;

[0082] (6) Centrifuge at 12000 rpm for 5 min and transfer 900 μL of supernatant into a new 2 mL centrifuge tube;

[0083] (7) Add 600 μL of pre-chilled isopropanol to precipitate the DNA, mix by inverting several times, and place in a -20°C refrigerator for 3 to 5 h;

[0084] (8) Centrifuge at 12000 rpm for 5 min and discard the supernatant;

[0085] (9) Add 500 μL of pre-cooled 70% ethanol, mix by inverting several times, centrifuge at 14,000 rpm for 2 min, and discard the supernatant;

[0086] (10) Repeat step (9);

[0087] (11) Use a pipette to absorb the remaining liquid and place the centrifuge tube in a clean bench to blow for 30 minutes;

[0088] (12) Add 100 μL ddH2O to dissolve the DNA and store it in a -20°C refrigerator for PCR amplification and verification.

[0089] 5.2 PCR amplification and verification

[0090] PCR amplification: DNA of bacteria Y15 was used as a template to amplify the corresponding 16S rDNA and gyrA gene fragments. The PCR reaction system is shown in Table 1, and the PCR amplification program is shown in Table 2:

[0091] Table 1 PCR reaction system

[0092]

[0093] The primers for 16S rDNA are as follows:

[0094] 63F: CAGGCCTAACACATGCAAGTC (SEQ ID NO: 1);

[0095] 1397R: GGGCGGWGTGTACAAGGC (SEQ ID NO: 2).

[0096] The primers for the gyrA gene are as follows:

[0097] gyrA-F: CAGTCAGGAAATGCGTACGTCCTT (SEQ ID NO: 3);

[0098] gyrA-R: CAAGGTAATGCTCCAGGCATTGCT (SEQ ID NO: 4)

[0099] Table 2 PCR amplification program

[0100]

[0101] 5.3 Construction of phylogenetic tree

[0102] The PCR product was sent to Nosai Sequencing Company for sequencing. After the sequencing results were obtained, the sequences were compared for homology on the NCBI website, and the phylogenetic tree was constructed using Mega5 software. The phylogenetic tree of strain Y15 and its aligned strains was constructed using the 16SrDNA gene sequence. The phylogenetic tree of strain XYG1 and its aligned strains was constructed using the spliced ​​sequence of the 16S rDNA gene and the gyrA gene.

[0103] Among them, the nucleotide sequence of the 16S rDNA gene amplified by strain Y15 is as follows:

[0104]

[0105] The nucleotide sequence of the 16S rDNA gene amplified from strain XYG1 is as follows:

[0106] GCTGGATCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCA

[0107] GACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCG

[0108] CGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAG

[0109] GTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTT

[0110] GTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAG

[0111] ATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACAC

[0112] GAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGG

[0113] ACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTC

[0114] TTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCC

[0115] CCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGA

[0116] GTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACA

[0117] CTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGT

[0118] TCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGT

[0119] CGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTAC

[0120] ACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAA

[0121] TGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAAC

[0122] CGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCT

[0123] ACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTA

[0124] GGTACCGTCAAGGTGCCGCCCTATTCGAACGGTACTTGTTCTTCCCTAA

[0125] CAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGC

[0126] TCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCG

[0127] TAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTC

[0128] AGGTCGGCTACGCATCGTTGCCTTGGTGAGCCATTACCTCACCAACTAG

[0129] CTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACCTTTTA

[0130] TGTTTGAACCATGCGGTTCAAACAACCATCCGGTATTAGCCCCGGTTTC

[0131] CCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCC

[0132] GTCCGCCGCTAACATCAGGAGCAG(SEQ ID NO:6)。

[0133] The nucleotide sequence of the gyrA gene amplified from strain XYG1 is as follows:

[0134] CATGAGCGTTATCGTGTCCCGTGCTCTTCCGGATGTTCGAGACGGTTTA

[0135] AAACCGGTTCATAGACGGATTTTGTATGCAATGAATGATTTAGGCATGA

[0136] CAAGTGACAAGCCTTATAAAAAATCCGCGCGTATCGTTGGAGAAGTTAT

[0137] CGGGAAATACCACCCGCACGGTGATTCAGCGGTATATGAATCCATGGTC

[0138] AGAATGGCTCAGGATTTCAACTACCGTTATATGCTCGTTGACGGTCACG

[0139] GAAACTTCGGTTCTGTTGACGGAGACTCAGCGGCGGCCATGCGTTATA

[0140] CAGAAGCAAGAATGTCTAAAATCTCAATGGAGATTCTTCGCGACATCA

[0141] CAAAAGACACAATCGATTACCAGGATAACTATGACGGGTCAGAAAGAG

[0142] AACCTGTCGTTATGCCTTCAAGGTTCCCGAATCTGCTCGTGAACGGTGC

[0143] TGCCGGCATTGCGGTAGGTATGGCAACAAACATTCCTCCGCACCAGCT

[0144] GGGAGAAATCATTGACGGTGTACTTGCTGTCAGTGAGAATCCGGACAT

[0145] TACAATTCCAGAGCTTATGGAAGTCATTCCAGGGCCTGATTTCCCGACT

[0146] GCGGGTCAAATCTTGGGACGCAGCGGTATCCGGAAAGCATACGAATCA

[0147] GGCCGAGGCTCTATCACGATCCGGGCAAAAGCTGAGATCGAACAAACA

[0148] TCTTCGGGTAAAGAAAGAATTATCGTTACAGAGTTACCTTACCAAGTAA

[0149] ATAAGGGCGAAATTAATTGAGAAAATTGCTGATCTCGTAAGGGACAAAA

[0150] AGATAGAGGGTATCACAGATCTGCGTGATGAGTCAGATCGTACAGGTAT

[0151] GAGAATTGTCATTGAAATCAGACGCGATGCCAATGCGAATGTCATCTTA

[0152] AACAATCTGTACAAACAAACTGCTCTACAAACATCTTTTGGCATCAACC

[0153] TGCTTGCACTAGTTGATGACAGCCGA (SEQ ID NO:7).

[0154] Results Figure 4 .Depend on Figure 4 From A in the figure, we can see that Y15 is Bacillus licheniformis through the phylogenetic tree clustering and comparison results. Figure 4 As shown in Figure B, strain XYG1 and Bacillus subtilis are clustered into one branch with a confidence level of 100%, so strain XYG1 belongs to Bacillus subtilis.

[0155] 6. Study on the mechanism of biocontrol bacteria in controlling wheat scab

[0156] 6.1 Detection of the inhibitory effect of biocontrol bacteria on spore germination of Fusarium fusiformis

[0157] (1) The biocontrol bacteria (strain Y15 and strain XYG1) were inoculated into a glass test tube containing 5 mL of LB liquid medium, incubated at 37°C for 8 h, and centrifuged at 5000 rpm for 10 min to obtain the supernatant of the strain;

[0158] (2) Equal volumes of the supernatant of the biocontrol strain and the spore suspension of Fusarium fusogenum were mixed and placed in a dark incubator at 25°C for 8 h, with three biological replicates;

[0159] (3) Observe the mixed solution under an optical microscope and count the germination status of 100 sporangia each time, count them and take photos to record.

[0160] Results Figure 5 The results showed that the spores of Fusarium fusogenum were abnormally swollen and inhibited from spore germination after the treatment of biocontrol bacteria strains Y15 and XYG1, while the number of spores of Fusarium fusogenum was significantly reduced after the treatment of biocontrol bacteria Y15.

[0161] 6.2 Detection of the inhibitory effect of biocontrol bacteria on the mycelial growth of Fusarium fusiformis

[0162] Antagonism was determined using the plate standoff method:

[0163] (1) The biocontrol bacteria (strain Y15) were activated on solid LB medium and cultured in an inverted state in a 37°C incubator overnight;

[0164] (2) The fungal strain Fg 0609 used in the experiment was activated on solid PDA medium and cultured in an incubator at 25°C for 7 days;

[0165] (3) Use a 5 mm puncher to punch out a cake of Fusarium fusiformis Fg 0609 and inoculate it in the center of the PDA plate;

[0166] (4) Inoculate the activated biocontrol bacteria 2 cm from the left and right sides of the bacterial cake. Each treatment was repeated three times, with only the bacterial cake inoculated as a blank control.

[0167] (5) Place the plate upside down in a 25°C incubator in the dark for 4 days.

[0168] (6) Measure the width of the inhibition zone, calculate the inhibition rate and take photos for record.

[0169] Results Figure 6 The biocontrol fungus strain Y15 has antagonistic activity against Fusarium fusiformis hyphae.

[0170] 6.3 Determination of the antimicrobial spectrum of biocontrol bacteria

[0171] (1) The biocontrol bacteria (strain Y15) were activated on solid LB medium and cultured in an inverted state in a 37°C incubator overnight;

[0172] (2) The fungal strains used in the experiment were activated on solid PDA medium and cultured in an incubator at 25°C for 7 days;

[0173] (3) Use a 5 mm punch to punch out a fungal cake and inoculate it in the center of the PDA plate;

[0174] (4) Inoculate the activated biocontrol bacteria 2 cm from the left and right sides of the bacterial cake. Each treatment was repeated three times, with only the bacterial cake inoculated as a blank control.

[0175] (5) Place the plate upside down in a 25°C incubator in the dark and incubate for 5 days.

[0176] (6) Observe whether an inhibition zone is formed and take photos to record it.

[0177] Results Figure 7 .Depend on Figure 7 It can be seen that strain Y15 has obvious antibacterial effects on wheat fusarium rust, apple ring rot, grape gray mold, potato early blight and wheat stem rot.

[0178] 6.4 Coleoptile bioassay of biocontrol bacteria metabolites

[0179] The three metabolites of the above-mentioned biocontrol bacteria strain Y15 were used to detect the biocontrol effect of wheat fusarium rust by the coleoptile method, as shown in step 2.

[0180] Results Figure 8 and Table 3.

[0181] Table 3 Biocontrol effects of metabolites of biocontrol bacteria strain Y15

[0182]

[0183] In order to explore whether the secondary metabolites produced by biocontrol bacteria are the main factors for their disease prevention, this example uses the culture fluid, bacterial suspension, and supernatant of the biocontrol bacteria strain Y15 to carry out a biocontrol effect detection experiment. The results show that the biocontrol effects of the 48h fermentation culture fluid and supernatant of the strain Y15 are significantly higher than those of the bacterial suspension, and the biocontrol effects are higher than those of the control group, and the biocontrol effect of the supernatant is better than that of the strain XYG1.

[0184] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Bacillus licheniformis strain Y15, characterized in that: The deposit number is CGMCC No.30197.

2. A biocontrol agent, characterized in that: The active ingredient comprises the Bacillus licheniformis strain Y15 according to claim 1.

3. The biocontrol agent according to claim 2, characterized in that: The Bacillus licheniformis strain Y15 exists in any form of bacterial suspension, culture solution and fermentation solution of the Bacillus licheniformis strain Y15.

4. The biocontrol agent according to claim 2, characterized in that: The concentration of the active ingredient is (1-100)×10 9 CFU / ml.

5. The biocontrol agent according to claim 2, characterized in that: The active ingredients also include at least one of the following bacterial species and / or metabolites thereof: Bacillus Velez, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus psychrophilus, Paenibacillus polymyxa, Streptomyces, Trichoderma, Pseudomonas fluorescens, Pseudomonas aeruginosa and Bacillus brevis.

6. The biocontrol agent according to any one of claims 2 to 5, characterized in that: The dosage form of the biocontrol agent includes at least one of the following: powder, aqueous solution, granule and microcapsule.

7. Use of the Bacillus licheniformis strain Y15 according to claim 1 or the biocontrol agent according to any one of claims 2 to 6 in preventing and controlling crop diseases.

8. The use according to claim 7, characterized in that: The crop diseases include at least one of the following: head blight, ring rot, gray mold, early blight, stem base rot and take-all disease.

9. The use according to claim 8, characterized in that: Preventing and controlling Fusarium fusae disease in crops includes inhibiting the germination of fusarium fusae spores and / or the growth of fusarium fusae hyphae.

10. The use according to any one of claims 7 to 9, characterized in that: The crops include at least one of the following: Wheat, oats, barley, corn, millet, rice, sorghum, soybeans, cotton, sugar beets, eggplant, apples, grapes, potatoes and sweet potatoes.

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