Bacillus brevis strain SXM-17, biocontrol inoculant and application of bacillus brevis strain SXM-17 in prevention and control of crop diseases

Abstract

The invention provides a Brevibacillus formmosus strain SXM-17, a derivative product of the Brevibacillus formmosus strain SXM-17 and application of the Brevibacillus formmosus strain SXM-17 to prevention and control of crop diseases, and belongs to the technical field of functional microorganisms. The strain SXM-17 is separated and purified from wheat plants, the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.30198, and the strain SXM-17 has a good inhibition effect on gibberellic disease bacteria, physalospora piricola, botrytis cinerea, alternaria solani, stem rot bacteria, gaeumannosis and the like. Experiments show that a culture solution, a bacterial suspension and a supernatant of the strain SXM-17 have good prevention and treatment effects. In addition, the strain SXM-17 also has a growth promoting effect on crops planted in a natural planting environment and a salinization environment. The strain SXM-17 provided by the invention enriches a gibberellic disease biocontrol strain resource library and green and efficient biological control microbial resources.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional microorganisms, and specifically relates to a Brevibacillus beautifulus strain SXM-17, a biocontrol agent and an application 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 such as 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 statistics of 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 velezensis, 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 Brevibacillus beautiful strain SXM-17, which not only has the function of effectively preventing and controlling ergot disease, but also can prevent and control a variety of other crop diseases, and can be widely used as an industrial strain in the preparation of biocontrol products.

[0004] The invention provides a Brevibacillus formosus strain SXM-17, whose preservation number is CGMCC No.30198.

[0005] The invention provides a biocontrol agent, wherein the active ingredients include the bacterial body of the Brevibacillus beautifulus strain SXM-17 and / or the metabolites of the Brevibacillus beautifulus strain SXM-17.

[0006] Preferably, the cells of the Brevibacillus beautifulus strain SXM-17 are in the form of a bacterial suspension of the Brevibacillus beautifulus strain SXM-17;

[0007] The metabolites of the Brevibacillus beautifule strain SXM-17 include the culture supernatant of the Brevibacillus beautifule strain SXM-17;

[0008] Preferably, the active ingredient comprises the culture broth and / or fermentation broth of the Brevibacillus beautifulus strain SXM-17.

[0009] 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, Bacillus licheniformis and Bacillus brevis.

[0010] Preferably, the dosage form of the biocontrol agent includes at least one of the following dosage forms: powder, aqueous solution, granule and microcapsule.

[0011] The present invention provides the use of the Brevibacillus beautifulus strain SXM-17 or the biocontrol agent in preventing and controlling crop diseases.

[0012] 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;

[0013] Preferably, the prevention and control of Fusarium fusarium disease in crops includes at least one of the following: inhibiting the germination of fusarium fusarium spores, inhibiting the growth of fusarium fusarium hyphae and increasing the activity of crop defense enzymes.

[0014] The present invention provides the use of the Brevibacillus beautifulus strain SXM-17 or the biocontrol agent in promoting the growth of crops.

[0015] Preferably, the crops include crops grown under salinization conditions and crops grown under non-salinization conditions.

[0016] 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.

[0017] The present invention provides a Brevibacillus formosus strain SXM-17, with a deposit number of CGMCC No.30198. The present invention separates and purifies a strain SXM-17 from wheat plants, and after primary screening and rescreening, the strain SXM-17 has a biocontrol effect on fusarium scurf, and the strain SXM-17 is identified as Brevibacillus formosus by molecular identification. The present invention respectively uses the culture solution, bacterial suspension and supernatant of the strain SXM-17 to detect the biocontrol effect of wheat fusarium scurf, and the results show that compared with the control, the culture solution, bacterial suspension and supernatant can significantly reduce the disease index of wheat fusarium scurf, have good prevention and control effects, and there is no significant difference in the prevention and control effects of the three. At the same time, the strain SXM-17 also has obvious inhibitory effects on pathogens such as ring rot, gray mold, early blight, and wheat stem base rot.

[0018] At the same time, the strain SXM-17 provided by the present invention has the effect of promoting the growth of crops planted in natural planting environments and salinized environments. It can be seen that the strain SXM-17 provided by the present invention enriches the biological control strain resource library of fusarium head blight and the green and efficient biological control microbial resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The results of the separation and purification of wheat fusarium rust biocontrol strains are shown in the figure on the left, the figure on the middle is the strain SXM-17, and the figure on the right is the XYG1 plate morphology;

[0020] Figure 2 The biocontrol effect of the biocontrol strain SXM-17 on wheat fusarium head blight on coleoptiles;

[0021] Figure 3 The biocontrol effect of the biocontrol strain SXM-17 on wheat head blight;

[0022] Figure 4 This is the phylogenetic tree of the biocontrol strain SXM-17;

[0023] Figure 5 This is the result of the biocontrol strain SXM-17 inhibiting the growth of spores of Fusarium head blight;

[0024] Figure 6 It is the plate antagonistic activity of the biocontrol strain SXM-17;

[0025] Figure 7 The results of the broad-spectrum antimicrobial activity test of the biocontrol strain SXM-17 are shown;

[0026] Figure 8The results of the culture solution, bacterial suspension and supernatant of the biocontrol strain SXM-17 on wheat fusarium head blight are shown;

[0027] Fig. 9 The growth promotion effect of the biocontrol strain SXM-17 on naturally grown wheat;

[0028] Fig.10 The results of promoting wheat growth under salt stress by the biocontrol strain SXM-17 are shown;

[0029] Fig.11 This is the result of the effect of biocontrol strain SXM-17 on the activities of SOD, APX and CAT in wheat roots.

[0030] Biological deposit information

[0031] The Brevibacillus formosus 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.30198, and the strain number is SXM-17. DETAILED DESCRIPTION

[0032] The invention provides a Brevibacillus beautifulus strain SXM-17, whose preservation number is CGMCC No.30198.

[0033] In the present invention, the strain SXM-17 is isolated and purified from wheat plants, and its wheat fusarium rust prevention and control effect is verified by using coleoptiles and spikes. Using molecular identification methods, the amplified PCR product is compared with the database and clustered by phylogenetic tree analysis to determine that the strain SXM-17 belongs to Brevibacillus formosus.

[0034] The invention provides a biocontrol agent, wherein the active ingredients include the bacterial body of the Brevibacillus beautifulus strain SXM-17 and / or the metabolites of the Brevibacillus beautifulus strain SXM-17.

[0035] In the present invention, the cell of the beautiful Brevibacillus strain SXM-17 is preferably present in the form of a bacterial suspension of the beautiful Brevibacillus strain SXM-17. The metabolites of the beautiful Brevibacillus strain SXM-17 include the culture supernatant of the beautiful Brevibacillus strain SXM-17. When the active ingredient contains both the SXM-17 cell and the metabolites of SXM-17, the active ingredient preferably includes the culture solution and / or fermentation solution of the beautiful Brevibacillus strain SXM-17. The preparation method of the culture solution and / or fermentation solution of the beautiful Brevibacillus strain SXM-17 is preferably obtained by inoculating the activated strain SXM-17 in LB medium and oscillating culture. The time of the oscillating culture is 24 to 72 hours, more preferably 36 to 56 hours, and most preferably 48 hours. The speed of the oscillating culture is preferably 120 to 220 rpm, more preferably 200 rpm. The temperature of the oscillating culture is preferably 28 to 50°C, more preferably 37°C. The method for preparing the culture supernatant of the beautiful Brevibacillus strain SXM-17 is preferably to subject the culture solution prepared above to solid-liquid separation, and collect the liquid phase to obtain it. 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 volume concentration of the culture supernatant in the biocontrol agent is 10% to 90%, more preferably 20% to 80%, further preferably 40% to 70%, and further preferably 50%. The bacterial suspension of the beautiful Brevibacillus strain SXM-17 is preferably obtained by subjecting the culture solution prepared above to solid-liquid separation, collecting the solid phase, and resuspending the solid phase in sterile water or PBS buffer. The concentration of the active ingredient in the biocontrol agent is preferably (1 to 100)×10 9 CFU / ml, more preferably (10-90)×10 9 CFU / ml, the most preferred is 50×10 9 The prevention effect test experiment of wheat coleoptile fusarium head blight showed that compared with the control, the culture solution, bacterial suspension and supernatant could significantly reduce the disease index of wheat head blight, and had a good prevention and control effect, and there was no significant difference in the prevention and control effects of the three.

[0036] In the present invention, the active ingredient preferably also includes at least one of the following strains or metabolites of the strains: Bacillus velezensis, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus psychrophilus, Bacillus polymyxa, Streptomyces, Trichoderma, Pseudomonas fluorescens, Pseudomonas aeruginosa, Bacillus licheniformis and Bacillus brevis. In view of the fact that the prior art reports that the above strains are all common biocontrol bacteria for the prevention and control of fusarium scurf, the beautiful brevicaulis Bacillus strain SXM-17 developed by the present invention is compounded with at least one of the above biocontrol bacteria to prepare a new type of biocontrol agent. When the active ingredient is compounded, the number of live bacteria of each strain is preferably mixed in equal proportions or the culture solution or fermentation solution of each strain is mixed in equal volumes.

[0037] In the present invention, the dosage form of the biocontrol agent preferably includes at least one of the following: powder, aqueous solution, granule and microcapsule. The powder is preferably obtained by mixing the bacteria and / or metabolites of the active ingredient with a freeze-dried protective agent and freeze-drying. The present invention does not impose any special restrictions on 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 aqueous solution 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 impose any special restrictions on the preparation method of the granule or microcapsule, and the granule or microcapsule of the biocontrol agent known in the art is used to prepare it.

[0038] The present invention provides the use of the Brevibacillus beautifulus strain SXM-17 or the biocontrol agent in preventing and controlling crop diseases.

[0039] In the present invention, the crop diseases preferably include at least one of the following: fusarium head blight (caused by Botrytis cinerea), gray mold (caused by Botrytis cinerea), early blight (caused by Alternaria solani), stem base rot (caused by Fusarium graminearum) and total erosion. 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. Prevention and control of fusarium head blight of crops includes at least one of the following: inhibiting the germination of fusarium head blight spores, inhibiting the growth of fusarium head blight hyphae and increasing the activity of crop defense enzymes. In an embodiment of the present invention, the plate confrontation method was used to detect the effect of the biocontrol strain on the growth of fusarium head blight hyphae. The results showed that compared with the control group, the beautiful Brevibacillus strain SXM-17 can significantly inhibit the growth of hyphae. The effects of biocontrol strains on the germination of conidia of Fusarium fusogenum were investigated, and the results showed that the Brevibacillus beautifulus strain SXM-17 could significantly inhibit the germination of conidia compared with the control group and strains screened in the same batch.

[0040] The present invention provides the use of the Brevibacillus beautifulus strain SXM-17 or the biocontrol agent in promoting the growth of crops.

[0041] In the present invention, the crops preferably include crops grown under salinization conditions and crops grown under non-salinization conditions. The crops preferably include at least one of the following: wheat, oats, barley, corn, millet, rice, sorghum, soybean, cotton, beet, eggplant, apple, grape, potato and sweet potato.

[0042] In an embodiment of the present invention, the beautiful Brevibacillus strain SXM-17 was used to promote the growth of wheat planted in a natural environment. Compared with the control group, after treatment with the strain SXM-17, the root length, plant height, fresh weight of the aboveground part and fresh weight of the underground part of the wheat were significantly different, and compared with the strains screened in the same batch, it had significant advantages. At the same time, the present invention also carried out a growth promotion experiment of the beautiful Brevibacillus strain SXM-17 on wheat planted in salt land. Compared with the control group, after treatment with the strain SXM-17, the root length, plant height, fresh weight of the aboveground part and fresh weight of the underground part of the wheat were significantly different, and compared with the strains screened in the same batch, it had significant advantages.

[0043] The following is a detailed description of a Brevibacillus beautifulus strain SXM-17, 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.

[0044] Example 1

[0045] Isolation, purification and preservation of a biocontrol strain

[0046] 1.1 Isolation of bacteria from wheat plants

[0047] 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.

[0048] 1.2 Purification and preservation of bacteria on wheat plants

[0049] 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.

[0050] The morphology of strain SXM-17 isolated from the collected wheat plants is shown in Figure 1When cultured on LB solid medium, the colony surface of strain SXM-17 was smooth, the single colony was white, and the shape was irregular. Similarly, strain XYG1 isolated from wheat plants had a smooth colony surface on LB solid medium, the single colony was white, and the shape was irregular.

[0051] 2. Preliminary screening of wheat fusarium rust biocontrol bacteria using coleoptile method

[0052] (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;

[0053] (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.

[0054] (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;

[0055] (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.

[0056] (5) Cut off the wheat leaf with sterilized scissors at about 3 mm below the seam between the coleoptile and the leaf, apply 2 μL of the biocontrol bacteria culture solution to the wound, cover the lid after the culture solution is dried, place in a 12-h photoperiod, 25°C light incubator for 24 h to keep the moisture incubated, and then inoculate the pathogen spore suspension;

[0057] (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.

[0058] (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:

[0059] Level 0: a=0;

[0060] Level 1: 0 <a<1 / 4;

[0061] Level 2: 1 / 4≤a≤1 / 2;

[0062] Level 3: 1 / 2 <a<2 / 3;

[0063] Level 4: 2 / 3≤a≤1;

[0064]

[0065] The results for strain SXM-17 are shown in Figure 2 The disease index of strain SXM-17 was 12.56, and the prevention efficacy was 80.47%.

[0066] 3. Wheat ear bio-test of wheat fusarium wilt biocontrol bacteria

[0067] (1) selecting 10 wheat plants in the flowering stage and with uniform growth and tying them into bundles;

[0068] (2) diluting the cultured bacterial solution (strain SXM-17) twice and spraying it to ensure that the bacterial solution completely soaks the bundled wheat ears;

[0069] (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;

[0070] (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;

[0071] (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"):

[0072] Level 0: no disease;

[0073] Level 1: The number of diseased ears accounts for less than 1 / 4 of the total number of ears;

[0074] Level 2: The number of diseased ears accounts for 1 / 4-1 / 2 of the total number of ears;

[0075] Level 3: The number of diseased ears accounts for less than 1 / 2-3 / 4 of the total number of ears;

[0076] Level 4: The number of diseased ears accounts for more than 3 / 4 of the total number of ears.

[0077] Calculate the disease index and prevention efficacy according to the above method and take photos for record.

[0078] Results Figure 3The disease index of strain SXM-17 was 16.92, and the control effect was 75.03%.

[0079] 4 Molecular biological identification of wheat scab biocontrol bacteria

[0080] 4.1 Extraction of bacterial genomic DNA

[0081] (1) Use a pipette to draw 1.5 mL of the bacterial solution of strain SXM-17 into a 2 mL centrifuge tube, centrifuge at 12000 rpm for 1 min, discard the supernatant, and collect the bacteria;

[0082] (2) Add 500 μL ddH2O and vortex for 30-40 seconds until no bacterial clumps are evident;

[0083] (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;

[0084] (4) Add 650 μL of nucleic acid lysis buffer and mix well by pipetting;

[0085] (5) Add 250 μL protein precipitation solution and vortex for 1 min;

[0086] (6) Centrifuge at 12000 rpm for 5 min and transfer 900 μL of supernatant into a new 2 mL centrifuge tube;

[0087] (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;

[0088] (8) Centrifuge at 12000 rpm for 5 min and discard the supernatant;

[0089] (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;

[0090] (10) Repeat step (9);

[0091] (11) Use a pipette to absorb the remaining liquid and place the centrifuge tube in a clean bench to blow for 30 minutes;

[0092] (12) Add 100 μL ddH2O to dissolve the DNA and store it in a -20°C refrigerator for PCR amplification and verification.

[0093] 4.2 PCR amplification and verification

[0094] PCR amplification: Using the DNA of strain SXM-17 or strain XYG1 as a template, the corresponding 16S rDNA and gyrA gene fragments were amplified. The PCR reaction system is shown in Table 1, and the PCR amplification program is shown in Table 2:

[0095] Table 1 PCR reaction system

[0096]

[0097] The primers for 16S rDNA are as follows:

[0098] 63F: CAGGCCTAACACATGCAAGTC (SEQ ID NO: 1);

[0099] 1397R: GGGCGGWGTGTACAAGGC (SEQ ID NO: 2);

[0100] The primers for the gyrA gene are as follows:

[0101] gyrA-F: CAGTCAGGAAATGCGTACGTCCTT (SEQ ID NO: 3);

[0102] gyrA-R: CAAGGTAATGCTCCAGGCATTGCT (SEQ ID NO: 4).

[0103] Table 2 PCR amplification program

[0104]

[0105]

[0106]

[0107] The nucleotide sequence of the 16S rDNA gene amplified from strain XYG1 is as follows:

[0108] GCTGGATCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCA

[0109] GACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCG

[0110] CGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAG

[0111] GTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTT

[0112] GTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAG

[0113] ATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACAC

[0114] GAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGG

[0115] ACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTC

[0116] TTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCC

[0117] CCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGA

[0118] GTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACA

[0119] CTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGT

[0120] TCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGT

[0121] CGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTAC

[0122] ACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAA

[0123] TGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAAC

[0124] CGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCT

[0125] ACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTA

[0126] GGTACCGTCAAGGTGCCGCCCTATTCGAACGGTACTTGTTCTTCCCTAA

[0127] CAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGC

[0128] TCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCG

[0129] TAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTC

[0130] AGGTCGGCTACGCATCGTTGCCTTGGTGAGCCATTACCTCACCAACTAG

[0131] CTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACCTTTTA

[0132] TGTTTGAACCATGCGGTTCAAACAACCATCCGGTATTAGCCCCGGTTTC

[0133] CCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCC

[0134] GTCCGCCGCTAACATCAGGAGCAG(SEQ ID NO:6).

[0135] The nucleotide sequence of the gyrA gene amplified from strain XYG1 is as follows:

[0136] CATGAGCGTTATCGTGTCCCGTGCTCTTCCGGATGTTCGAGACGGTTTA

[0137] AAACCGGTTCATAGACGGATTTTGTATGCAATGAATGATTTAGGCATGA

[0138] CAAGTGACAAGCCTTATAAAAAATCCGCGCGTATCGTTGGAGAAGTTAT

[0139] CGGGAAATACCACCCGCACGGTGATTCAGCGGTATATGAATCCATGGTC

[0140] AGAATGGCTCAGGATTTCAACTACCGTTATATGCTCGTTGACGGTCACG

[0141] GAAACTTCGGTTCTGTTGACGGAGACTCAGCGGCGGCCATGCGTTATA

[0142] CAGAAGCAAGAATGTCTAAAATCTCAATGGAGATTCTTCGCGACATCA

[0143] CAAAAGACACAATCGATTACCAGGATAACTATGACGGGTCAGAAAGAG

[0144] AACCTGTCGTTATGCCTTCAAGGTTCCCGAATCTGCTCGTGAACGGTGC

[0145] TGCCGGCATTGCGGTAGGTATGGCAACAAACATTCCTCCGCACCAGCT

[0146] GGGAGAAATCATTGACGGTGTACTTGCTGTCAGTGAGAATCCGGACAT

[0147] TACAATTCCAGAGCTTATGGAAGTCATTCCAGGGCCTGATTTCCCGACT

[0148] GCGGGTCAAATCTTGGGACGCAGCGGTATCCGGAAAGCATACGAATCA

[0149] GGCCGAGGCTCTATCACGATCCGGGCAAAAGCTGAGATCGAACAAACA

[0150] TCTTCGGGTAAAGAAAGAATTATCGTTACAGAGTTACCTTACCAAGTAA

[0151] ATAAGGGCGAAATTAATTGAGAAAATTGCTGATCTCGTAAGGGACAAAA

[0152] AGATAGAGGGTATCACAGATCTGCGTGATGAGTCAGATCGTACAGGTAT

[0153] GAGAATTGTCATTGAAATCAGACGCGATGCCAATGCGAATGTCATCTTA

[0154] AACAATCTGTACAAACAAACTGCTCTACAAACATCTTTTGGCATCAACC

[0155] TGCTTGCACTAGTTGATGACAGCCGA(SEQ ID NO:7)

[0156] 4.3 Construction of phylogenetic tree

[0157] The PCR products were sent to Nosai Sequencing Company for sequencing. After the sequencing results were obtained, the sequencing sequences were compared for homology on the NCBI website, and the phylogenetic tree was constructed using Mega5 software. The phylogenetic tree of strain SXM-17 and its aligned strains was constructed using the 16S rDNA 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.

[0158] Results Figure 4 .Depend on Figure 4It can be seen from the phylogenetic tree clustering and comparison results that strain SXM-17 is Brevibacillus formosus. Strain XYG1 and Bacillus subtilis are clustered into one branch with a confidence of 100%. Therefore, strain XYG1 belongs to Bacillus subtilis.

[0159] Study on the mechanism of controlling wheat scab by 5 biocontrol bacteria

[0160] 5.1 Detection of the inhibitory effect of biocontrol bacteria on spore germination of Fusarium fusiformis

[0161] (1) The biocontrol bacteria (strain SXM-17, 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;

[0162] (2) Equal volumes of the supernatant of the biocontrol strain and the spore suspension of Fusarium fusae were mixed and placed in a dark incubator at 25°C for 48 h, with three biological replicates;

[0163] (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.

[0164] Results Figure 5 The results showed that both strain SXM-17 and strain XYG1 could inhibit the germination of spores, and strain SXM-17 could also reduce the number of spores.

[0165] 5.2 Detection of the inhibitory effect of biocontrol bacteria on the mycelial growth of Fusarium fusiformis

[0166] Antagonism was determined using the plate standoff method:

[0167] (1) The biocontrol bacteria (strain SXM-17) were activated on solid LB medium and cultured in an inverted state in a 37°C incubator overnight;

[0168] (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;

[0169] (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;

[0170] (4) Inoculate the activated biocontrol bacteria 2 cm from the left and right sides of the bacterial cake. Repeat three times for each treatment, with only the bacterial cake inoculated as the blank control.

[0171] (5) Place the plate upside down in a 25°C incubator in the dark for 4 days.

[0172] (6) Measure the width of the inhibition zone, calculate the inhibition rate and take photos for record.

[0173] Results Figure 6 The biocontrol fungus strain SXM-17 has antagonistic activity against Fusarium fusiformis hyphae.

[0174] 5.3 Determination of the antimicrobial spectrum of biocontrol bacteria

[0175] (1) The biocontrol bacteria (strain SXM-17 and strain XYG1) were activated on solid LB medium and cultured in an inverted state in a 37°C incubator overnight;

[0176] (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;

[0177] (3) Use a 5 mm punch to punch out a fungal cake and inoculate it in the center of the PDA plate;

[0178] (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.

[0179] (5) Place the plate upside down in a 25°C incubator in the dark and incubate for 5 days.

[0180] (6) Observe whether an inhibition zone is formed and take photos to record it.

[0181] Results Figure 7 .Depend on Figure 7 It can be seen that strain SXM-17 has obvious antibacterial effect on ergot, ring rot, gray mold, early blight, stem base rot and take-all pathogen, and the inhibitory effect is significantly better than that of strain XYG1.

[0182] 5.4 Effect of the metabolites of strain SXM-17 on the biocontrol of wheat fusarium head blight

[0183] 5.4.1 Preparation of biocontrol bacteria metabolites

[0184] The biocontrol bacteria (strain SXM-17) were inoculated into a 300 mL Erlenmeyer flask containing 100 mL LB liquid medium, and cultured at 37°C and 200 rpm for 48 h before the following treatments:

[0185] (1) Culture medium: unify the OD of the above shake culture medium 600 ;

[0186] (2) Supernatant: Pipette 1 mL of the above bacterial solution into a 2 mL centrifuge tube and centrifuge at 5000 rpm for 10 min. Take the supernatant and filter it through a 0.22 μm bacterial filter to obtain a sterile supernatant.

[0187] (3) Bacterial suspension: Pipette 1 mL of the above bacterial suspension into a 2 mL centrifuge tube, centrifuge at 5000 rpm for 10 min, discard the supernatant, resuspend three times with 1 mL of sterile water, add an appropriate amount of sterile water and vortex, and calculate the OD 600 Consistent with the culture medium.

[0188] 5.4.2 Coleoptile bioassay of biocontrol bacteria metabolites

[0189] The three metabolites of the above-mentioned biocontrol bacteria SXM-17 were used to detect the biocontrol effect of wheat fusarium rust by the coleoptile method, as shown in step 2.

[0190] Results Figure 8 and Table 3.

[0191] Table 3 Biocontrol effects of three metabolites of biocontrol bacteria SXM-17

[0192]

[0193] 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 SXM-17 to conduct a biocontrol effect detection experiment. The results show that there is no significant difference in the biocontrol effect of the metabolic fluid, bacterial suspension, and culture fluid of the strain SXM-17, but they are all significantly better than the control group.

[0194] 5.5 Effects of biocontrol bacteria on wheat growth promotion

[0195] (1) The soil for growing wheat in the greenhouse is made of loess, vermiculite and nutrient soil in a ratio of 3:3:3. The mixed soil is added to small pots with a diameter of 5 cm and a height of 10 cm. Every 5 small pots are placed in a white plastic box with a length of 28.5 cm, a width of 21 cm and a height of 7.5 cm for easy watering and standby;

[0196] (2) The two strains of biocontrol bacteria were inoculated into 5 mL of LB liquid medium and cultured at 37°C overnight with shaking;

[0197] (3) The biocontrol bacteria solution (strain SXM-17, strain XYG1) was inoculated into a 250 mL Erlenmeyer flask containing 50 mL LB liquid medium at a ratio of 1:100 and incubated at 37°C for 48 h to adjust the OD 600 Consistency;

[0198] (4) Place an equal amount of healthy wheat seeds in a 50 mL centrifuge tube, add 40 mL of sterile water and soak the seeds for 24 h until the seeds turn white. A total of ten 50 mL centrifuge tubes are processed;

[0199] (5) After 24 hours, the sterile water was poured out, and 40 mL of biocontrol solution was added to nine 50 mL centrifuge tubes to soak the seeds for 1 hour. Another 50 mL centrifuge tube was added with 40 mL of liquid LB to soak the seeds for 1 hour as a blank control.

[0200] (6) After 1 h, pour out the bacterial solution and pour the wheat seeds in each centrifuge tube onto absorbent paper and allow the wheat seeds to dry;

[0201] (7) Plant the dried wheat seeds in the soil in (1), with 4 wheat seeds in each small pot, and five replicates for each treatment. After planting, a layer of dry soil is attached to each small pot, and 700 mL of water is poured into the plastic box containing the small pots. Water is applied once every two weeks.

[0202] (8) One month later, the plant height, root length, aboveground fresh weight, and underground fresh weight of wheat were investigated and photographed for record.

[0203] The results are shown in Table 4 and Fig. 9 The results showed that compared with wheat treated with LB medium only, the root length, plant height, aboveground fresh weight and underground fresh weight of wheat treated with strain SXM-17 were significantly different, while the XYG1 strain did not have a corresponding effect on promoting wheat growth.

[0204] Table 4 Growth-promoting effect of strain SXM-17 on wheat

[0205]

[0206] Note: Data are expressed as "mean ± standard deviation" and the same letters indicate no significant difference.

[0207] 5.6 Effects of biocontrol bacteria on wheat growth under salt stress

[0208] Methods (1) to (6) are the same as those in (1) to (6) in 5.3;

[0209] (7) Plant the dried wheat seeds in the soil described in (1), with 4 wheat seeds in each small pot, and five replicates for each treatment. After planting, a layer of dry soil was attached to each small pot, and 700 mL of 100 mmol / L salt water was poured into the plastic box containing the small pots;

[0210] (8) Ten days later, investigate the plant height, root length, aboveground fresh weight, and underground fresh weight of the wheat and record them by taking photos.

[0211] Table 5 Growth-promoting effect of strain SXM-17 on wheat treated with salt stress

[0212]

[0213] Note: Data are expressed as "mean ± standard deviation" and the same letters indicate no significant difference.

[0214] The results are shown in Table 5 and Fig.10 In this example, a high-salt ion environment was created by pouring a 100 mmol / L NaCl aqueous solution into the soil of potted wheat, and the wheat seeds were treated with the biocontrol strain. After one week, the root length, plant height, aboveground fresh weight and underground fresh weight of the wheat were investigated. After analyzing the data, it was found that after the application of the strain SXM-17, the growth of wheat was significantly higher than that of the control, while the XYG1 strain did not have a corresponding effect of promoting wheat growth.

[0215] 5.7 Effects of biocontrol bacteria on defense enzymes in wheat subjected to salt stress

[0216] The detection of wheat defense enzyme activity was carried out according to the instructions provided by Solebow Biotechnology Co., Ltd.

[0217] 5.7.1 Determination of catalase (CAT) activity

[0218] (1) Weigh 0.1 g of wheat root and grind it in a mortar containing liquid nitrogen. Place a 2 mL centrifuge tube in liquid nitrogen for precooling and place the ground product in the centrifuge tube on an ice box.

[0219] (2) Add 1 mL of the extract to the centrifuge tube, vortex to mix, and centrifuge at 8000 g for 10 min at 4 °C. Take the supernatant and place it in a new 2 mL centrifuge tube on ice for testing.

[0220] (3) Take an appropriate amount of CAT working solution and place it in a water bath at 25°C for 10 min;

[0221] (4) Turn on the spectrophotometer, adjust the wavelength to 240 nm after preheating, and calibrate with distilled water;

[0222] (5) Mix 35 μL of sample with 1 mL of test solution in a cuvette and measure the absorbance immediately as A1. Measure again 1 minute later and the absorbance is A2.

[0223] (6) Calculate the value of CAT according to formula III.

[0224] CAT(U / g)=678×△A÷W Formula III

[0225] Where, W: sample mass; △A = A1-A2.

[0226] 5.7.2 Determination of superoxide dismutase (SOD) activity

[0227] (1) Weigh 0.1 g of wheat root and grind it in a mortar containing liquid nitrogen. Place a 2 mL centrifuge tube in liquid nitrogen for precooling and place the ground product in the centrifuge tube on an ice box.

[0228] (2) Add 1 mL of the extract to the centrifuge tube, vortex to mix, and centrifuge at 8000 g for 10 min at 4°C. Take the supernatant and place it in a new 2 mL centrifuge tube on ice for testing;

[0229] (3) Take appropriate amounts of reagents 1, 3, and 4 and place them in a water bath at 25°C for 10 min;

[0230] (4) Turn on the spectrophotometer, adjust the wavelength to 560 nm after preheating, and calibrate with distilled water;

[0231] (5) Add 90 μL of sample, 240 μL of reagent 1, 60 μL of reagent 2, 180 μL of reagent 3, 400 μL of distilled water and 30 μL of reagent 4 to the assay tube; add 90 μL of sample, 240 μL of reagent 1, 180 μL of reagent 3, 460 μL of distilled water and 30 μL of reagent 4 to the control tube; add 240 μL of reagent 1, 60 μL of reagent 2, 180 μL of reagent 3, 490 μL of distilled water and 30 μL of reagent 4 to blank tube 1; add 240 μL of reagent 1, 180 μL of reagent 3, 550 μL of distilled water and 30 μL of reagent 4 to blank tube 2

[0232] (6) After mixing the components in the above tubes, place them in a water bath at 37°C for 30 min, and use a cuvette to measure the absorbance of the components in each tube at 560 nm, which are recorded as: A assay, A control, A1 blank, A2 blank, ΔA assay = A assay - A control, Δ blank = A1 blank - A2 blank

[0233] (7) Calculate SOD value according to formula IV

[0234] SOD (U / g) = 11.11 × inhibition percentage ÷ (1-inhibition percentage) ÷ W Formula IV

[0235] Where W: sample mass;

[0236] Inhibition percentage = (ΔA blank - ΔA assay) ÷ ΔA blank × 100%.

[0237] 5.7.3 Determination of ascorbate peroxidase (APX) activity

[0238] (1) Weigh 0.1 g of wheat root and grind it in a mortar containing liquid nitrogen. Place a 2 mL centrifuge tube in liquid nitrogen for precooling and place the ground product in the centrifuge tube on an ice box.

[0239] (2) Add 1 mL of the extract to the centrifuge tube, vortex to mix, and centrifuge at 8000 g for 10 min at 4 °C. Take the supernatant and place it in a new 2 mL centrifuge tube on ice for testing.

[0240] (3) Take an appropriate amount of reagent and place it in a water bath at 25°C for 30 minutes;

[0241] (4) Turn on the spectrophotometer, adjust the wavelength to 290 nm after preheating, and calibrate with distilled water;

[0242] (5) Add 100 μL of sample, 700 μL of reagent 1, 100 μL of reagent 2, and 100 μL of reagent 3 to the assay tube; add 100 μL of distilled water, 700 μL of reagent 1, 100 μL of reagent 2, and 100 μL of reagent 3 to the blank tube;

[0243] (6) The absorbance value measured after 10 seconds of mixing the components is A1, and the absorbance value measured after 130 seconds is A2.

[0244] (7) Calculate the APX value according to formula V.

[0245] APX (U / g) = 1.79 × (△A measurement light - △A blank tube) ÷ W formula V

[0246] Where W: sample mass; △A measurement tube = A1 measurement value - A2 measurement value, △A blank tube = A1 blank tube - A2 blank tube.

[0247] Results Fig.11 . Reactive oxygen species (ROS) are signal molecules of defense responses in plants. Under salt stress, hydrogen peroxide and oxygen anions accumulate in plants. In order to explore whether strain SXM-17 can induce homeostasis of reactive oxygen species in wheat roots under salt stress, this example measured the activities of SOD, CAT, and APX in wheat roots after treatment with strain SXM-17. The results showed that compared with the control, the activities of SOD and APX enzymes in wheat roots were significantly increased after treatment with strain SXM-17, while the activity of CAT did not change significantly.

[0248] 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 Brevibacillus fornosus strain SXM-17, characterized in that: The deposit number is CGMCC No.30198.

2. A biocontrol agent, characterized in that: The active ingredients include the bacterial body of the Brevibacillus beautiful strain SXM-17 according to claim 1 and / or the metabolites of the Brevibacillus beautiful strain SXM-17.

3. The biocontrol agent according to claim 2, characterized in that: The bacterial body of the Brevibacillus beautifule strain SXM-17 includes a bacterial suspension of the Brevibacillus beautifule strain SXM-17; The metabolites of the Brevibacillus beautifule strain SXM-17 include the culture supernatant of the Brevibacillus beautifule strain SXM-17; Preferably, the active ingredient comprises the culture broth and / or fermentation broth of the Brevibacillus beautifulus strain SXM-17.

4. 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, Bacillus polymyxa, Streptomyces, Trichoderma, Pseudomonas fluorescens, Pseudomonas aeruginosa, Bacillus licheniformis and Bacillus brevis.

5. The biocontrol agent according to any one of claims 2 to 4, characterized in that: The dosage form of the biocontrol agent includes at least one of the following: powder, aqueous solution, granule and microcapsule.

6. Use of the Brevibacillus beautiful strain SXM-17 according to claim 1 or the biocontrol agent according to any one of claims 2 to 5 in preventing and controlling crop diseases.

7. The use according to claim 6, 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; Preferably, the prevention and control of Fusarium fusarium disease in crops includes at least one of the following: inhibiting the germination of fusarium fusarium spores, inhibiting the growth of fusarium fusarium hyphae and increasing the activity of crop defense enzymes.

8. Use of the Brevibacillus beautiful strain SXM-17 according to claim 1 or the biocontrol agent according to any one of claims 2 to 5 in promoting the growth of crops.

9. The use according to claim 8, characterized in that: The crops include crops grown under salinization conditions and / or crops grown under non-salinization conditions.

10. The use according to any one of claims 6 to 9, characterized in that: The crop includes at least one of the following: wheat, oats, barley, corn, millet, rice, corn, soybean, cotton, sugar beet, eggplant, apple, grape, potato and sweet potato.

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