A strain of Brevibacillus pulchrificus SXM-17, a biocontrol agent and its application in controlling crop diseases

Abstract

The present invention provides a Brevibacillus formosus strain SXM-17, its derivative products and applications in preventing and controlling crop diseases, belonging to the technical field of functional microorganisms. In the present invention, a strain SXM-17 is isolated and purified from wheat plants, and the preservation number is CGMCC No. 30198, which has good inhibitory effects on Gibberella zeae, Physalospora piricola, Botrytis cinerea, Alternaria solani, Rhizoctonia solani and Gaeumannomyces graminis var. tritici, etc. Experiments show that the culture solution, bacterial suspension and supernatant of the strain SXM-17 all have good control effects. In addition, the strain SXM-17 also has a growth-promoting effect on crops planted in natural planting environments and salinized environments. The strain SXM-17 provided by the present invention enriches the resource library of biocontrol strains against Gibberella zeae and the resources of green and efficient biological control microorganisms.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional microorganisms, and specifically relates to a Brevibacillus formosus strain SXM-17, a biocontrol agent, and their application in controlling crop diseases. Background Art

[0002] Wheat Fusarium Head Blight is a major and frequently-occurring wheat disease worldwide. The disease causes wheat yield reduction or even no harvest, resulting in serious economic losses. In addition, the Fusarium graminearum that infects wheat ears 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 control method. However, due to its disadvantages such as pesticide residues, drug resistance, and environmental pollution, biological control has received increasing attention. By consulting literature and other databases, it is statistically found that there are more than 10 biocontrol strains with efficacy against Wheat Fusarium Head Blight published in the current literature: Bacillus velezensis, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus psychrophilus, Paenibacillus polymyxa, Streptomyces, Trichoderma, Pseudomonas fluorescens, Pseudomonas aeruginosa, Bacillus licheniformis, Brevibacillus brevis, etc. And there are only two registered bacterial agents for controlling Wheat Fusarium Head Blight: Bacillus subtilis and Paenibacillus polymyxa. Compared with the literature reports, the number of industrialized strains is small and cannot meet the demand for safe and efficient control of diseases. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a Brevibacillus formosus strain SXM-17, which not only has high efficiency in controlling Fusarium Head Blight, but also can control other various crop diseases, and can be widely used as an industrialized strain in the preparation of biocontrol products.

[0004] The present invention provides a Brevibacillus formosus strain SXM-17 with a preservation number of CGMCC No. 30198.

[0005] The present invention provides a biocontrol agent, the active ingredient of which includes the cells of the Brevibacillus formosus strain SXM-17 and / or the metabolites of the Brevibacillus formosus strain SXM-17.

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

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

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

[0009] Preferably, the active ingredient further includes at least one of the following strains and / or metabolites of the strains: Bacillus velezensis, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus psychrophilus, Paenibacillus polymyxa, Streptomyces, Trichoderma, Pseudomonas fluorescens, Pseudomonas aeruginosa, Bacillus licheniformis, and Brevibacillus 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 application of the Brevibacillus pulchrum strain SXM-17 or the biocontrol agent in controlling crop diseases.

[0012] Preferably, the crop diseases include at least one of the following: scab, ring rot, gray mold, early blight, basal rot, and take-all;

[0013] Preferably, controlling crop scab includes at least one of the following: inhibiting the spore germination of Gibberella zeae, inhibiting the mycelial growth of Gibberella zeae, and enhancing the activity of crop defense enzymes.

[0014] The present invention provides the application of the Brevibacillus pulchrum strain SXM-17 or the biocontrol agent in promoting crop growth.

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

[0016] Preferably, the crops include at least one of the following: wheat, oats, barley, corn, millet, rice, sorghum, soybeans, cotton, sugar beets, eggplants, apples, grapes, potatoes, and sweet potatoes.

[0017] The present invention provides a strain SXM-17 of Brevibacillus formosus, with the preservation number of CGMCC No. 30198. A strain SXM-17 was isolated and purified from wheat plants in the present invention. After primary screening and re-screening, the strain SXM-17 has the biocontrol effect on Fusarium head blight. Through molecular identification, the strain SXM-17 is Brevibacillus formosus. The culture solution, bacterial suspension and supernatant of the strain SXM-17 were respectively used to detect the biocontrol effect on Fusarium head blight of wheat. The results show that, compared with the control, the culture solution, bacterial suspension and supernatant can all significantly reduce the disease index of Fusarium head blight of wheat, have good control effects, and there is no significant difference in the control effects among the three. At the same time, the strain SXM-17 also has obvious inhibitory effects on pathogens such as Physalospora piricola, Botrytis cinerea, Alternaria solani, and wheat basal rot pathogen.

[0018] Meanwhile, 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 biocontrol strain resource library of Fusarium head blight and the green and efficient biological control microbial resources. Description of the Drawings

[0019] Figure 1 It is the isolation and purification result of the biocontrol strain for Fusarium head blight of wheat. Among them, the left figure is the plate morphology diagram of each screened strain, the middle figure is the strain SXM-17, and the right figure is the plate morphology diagram of XYG1;

[0020] Figure 2 It is the biocontrol effect of the biocontrol strain SXM-17 on Fusarium head blight of wheat on coleoptiles;

[0021] Figure 3 It is the biocontrol effect of the biocontrol strain SXM-17 on Fusarium head blight of wheat on wheat ears;

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

[0023] Figure 5 It is the growth inhibition result of the biocontrol strain SXM-17 on the spores of Fusarium graminearum;

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

[0025] Figure 7 It is the detection result of the antibacterial broad spectrum of the biocontrol strain SXM-17;

[0026] Figure 8Control results of the culture solution, bacterial suspension and supernatant of biocontrol strain SXM-17 against wheat scab;

[0027] Figure 9 Growth promotion results of biocontrol strain SXM-17 on naturally growing wheat;

[0028] Figure 10 Growth promotion results of biocontrol strain SXM-17 on wheat under salt stress;

[0029] Figure 11 Results of the effects of biocontrol strain SXM-17 treatment on the activities of SOD, APX, and CAT in wheat roots.

[0030] Biological deposit information

[0031] Brevibacillus formosus strain, deposited in the General Microbiology Center of the China National Committee for Culture Collection of Microorganisms. The deposit date is 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 implementation manners

[0032] The present invention provides a Brevibacillus formosus strain SXM-17 with a deposit number of CGMCC No. 30198.

[0033] In the present invention, the strain SXM-17 is isolated and purified from wheat plants, and its control effect on wheat scab is verified using coleoptiles and ears. Using molecular identification methods, the amplified PCR products are compared with the database and subjected to cluster analysis of phylogenetic trees to determine that the strain SXM-17 belongs to Brevibacillus formosus.

[0034] The present invention provides a biocontrol agent, the active ingredients of which include the cells of the Brevibacillus formosus strain SXM-17 and / or the metabolites of the Brevibacillus formosus strain SXM-17.

[0035] In the present invention, the cells of the Brevibacillus pulchrum strain SXM-17 preferably exist in the form of a bacterial suspension of the Brevibacillus pulchrum strain SXM-17. The metabolites of the Brevibacillus pulchrum strain SXM-17 include the culture supernatant of the Brevibacillus pulchrum strain SXM-17. When the active ingredient simultaneously contains the SXM-17 cells and the metabolites of SXM-17, the active ingredient preferably includes the culture broth and / or fermentation broth of the Brevibacillus pulchrum strain SXM-17. The preparation method of the culture broth and / or fermentation broth of the Brevibacillus pulchrum strain SXM-17 preferably comprises inoculating the activated strain SXM-17 into an LB medium and culturing it with shaking. The shaking culture time is 24 to 72 h, more preferably 36 to 56 h, and most preferably 48 h. The shaking culture speed is preferably 120 to 220 rpm, more preferably 200 rpm. The shaking culture temperature is preferably 28 to 50 °C, more preferably 37 °C. The preparation method of the culture supernatant of the Brevibacillus pulchrum strain SXM-17 preferably comprises subjecting the above-prepared culture broth to solid-liquid separation and collecting the liquid phase. The solid-liquid separation method 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 still further preferably 50%. The bacterial suspension of the Brevibacillus pulchrum strain SXM-17 is preferably obtained by subjecting the above-prepared culture broth 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, further preferably (10 to 90)×10 9 CFU / ml, and most preferably 50×10 9 CFU / ml. The efficacy detection experiment on the control of Fusarium head blight of wheat coleoptiles shows that, compared with the control, the culture broth, the bacterial suspension, and the supernatant can all significantly reduce the disease index of wheat Fusarium head blight, have good control effects, and there is no significant difference in the control effects among the three.

[0036] In the present invention, the active ingredient preferably further includes at least one of the following strains or the metabolites of the strains: Bacillus velezensis, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus psychrophilus, Paenibacillus polymyxa, Streptomyces, Trichoderma, Pseudomonas fluorescens, Pseudomonas aeruginosa, Bacillus licheniformis, and Brevibacillus. In view of the fact that the above strains are all common biocontrol bacteria for the prevention and control of Fusarium head blight reported in the prior art, a novel biocontrol agent is prepared by compounding the Brevibacillus pulchrum strain SXM-17 developed in the present invention with at least one of the above biocontrol bacteria. When the active ingredients are compounded, the viable cell numbers of each strain are preferably mixed in equal proportions or the culture broths or fermentation broths of each strain are 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 prepared by mixing the cells and / or metabolites of the active ingredient with a freeze-drying protectant and then freeze-drying. The present invention does not impose special restrictions on the type of the freeze-drying protectant, and well-known freeze-drying protectants in the art can be used, such as substances for adjusting osmotic pressure like glucose and mannose. The addition concentration of the freeze-drying protectant is preferably 5% - 20%, more preferably 8% - 18%, further preferably 10% - 15%, and most preferably 12%. The aqueous solution is preferably prepared by culturing the active ingredient and then mixing the obtained culture solution with or without cell separation with an antifreeze agent. The antifreeze agent includes glycerol. The volume concentration of the antifreeze agent is 20% - 80%, and most preferably 40%. The present invention does not impose special restrictions on the preparation method of the granule or microcapsule, and they can be prepared by using well-known preparation methods of biocontrol agent granules or microcapsules in the art.

[0038] The present invention provides the application of the Brevibacillus pulchrum 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 Botryosphaeria dothidea), gray mold (caused by Botrytis cinerea), early blight (caused by Alternaria solani), basal stem rot (caused by Fusarium pseudograminearum), and take-all. 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. Preventing and controlling Fusarium head blight of crops includes at least one of the following: inhibiting the spore germination of Fusarium graminearum, inhibiting the mycelial growth of Fusarium graminearum, and enhancing the activity of crop defense enzymes. In the examples of the present invention, the plate confrontation method was used to detect the effect of the biocontrol strain on the mycelial growth of Fusarium graminearum, and the results showed that, compared with the control group, the Brevibacillus pulchrum strain SXM-17 could significantly inhibit the mycelial growth. By studying the effect of the biocontrol strain on the conidial germination of Fusarium graminearum, the results showed that, compared with the control group and the strains screened in the same batch, the Brevibacillus pulchrum strain SXM-17 could significantly inhibit the conidial germination.

[0040] The present invention provides the application of the Brevibacillus pulchrum strain SXM-17 or the biocontrol agent in promoting crop growth.

[0041] In the present invention, the crops preferably include crops growing under salinized conditions and crops growing under non-salinized 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 the embodiments of the present invention, a growth-promoting experiment of the Brevibacillus pulchrum strain SXM-17 on wheat planted in a natural environment was carried out. Compared with the control group, after treatment with the strain SXM-17, there were significant differences in the root length, plant height, above-ground fresh weight, and underground fresh weight of wheat, and it had significant advantages compared with the strains screened in the same batch. At the same time, the present invention also carried out a growth-promoting experiment of the Brevibacillus pulchrum strain SXM-17 on wheat planted in saline land. Compared with the control group, after treatment with the strain SXM-17, there were significant differences in the root length, plant height, above-ground fresh weight, and underground fresh weight of wheat, and it had significant advantages compared with the strains screened in the same batch.

[0043] The following is a detailed description of a Brevibacillus pulchrum strain SXM-17, a biocontrol agent, and its application in preventing and controlling crop diseases provided by the present invention in combination with embodiments, but they cannot be understood as limiting the protection scope 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 sterilized scissors to cut off the above-ground part of the wheat plant and place it in a sterilized mortar. Add a small amount of sterilized quartz sand and start grinding. Add 1 mL of sterile water during grinding and continue grinding until it becomes a homogeneous slurry. Pipette 100 μL of the grinding liquid into a 2 mL centrifuge tube and dilute it successively with sterile water to concentrations of 10 -1 、10 -2 、10 -3 、10 -4 . Take 100 μL each of the original solution and the diluted grinding liquid and spread them on TSA and 1 / 10 TSA media respectively. Each treatment has 3 replicates and is cultured at 30 °C for 3 - 4 d.

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

[0049] Observe the growth of bacteria on the medium. After the number of colonies stabilizes, select colonies with different morphologies and colors and streak them on a new LB solid medium until a single colony grows. Mix the purified and stable strain with 50% glycerol at a ratio of 1:1 after shaking culture in liquid LB and store it in a -80 °C ultra-low temperature refrigerator.

[0050] The morphological diagram of the strain SXM-17 isolated from the collected wheat plants is shown in Figure 1Cultured on LB solid medium, the colony surface of strain SXM-17 is smooth, the single colony is white, and its shape is irregular. Similarly, the colony surface of strain XYG1 isolated from wheat plants is smooth on LB solid medium, the single colony is white, and its shape is irregular.

[0051] 2 Screening of biocontrol bacteria against Fusarium head blight of wheat by coleoptile method

[0052] (1) Select an appropriate amount of healthy wheat seeds, disinfect them with 75% ethanol for 2 min, twice, rinse them with sterile water multiple times, and soak them in sterile water for 24 h until the seeds show white tips.

[0053] (2) Place 2 sterilized filter papers at the bottom of a 9-cm petri dish, moisten them with an appropriate amount of sterile water, place the soaked wheat seeds with the ventral groove facing down on the filter papers, put them in a white sealed box of 52×36×14.5 cm, and place them in a light incubator with a 12-h light cycle and a temperature of 25 °C for moisturized cultivation. They can germinate after 2 days.

[0054] (3) After preparing the petri dish containing the moistened filter paper as in (2), place the germinated seeds with the ventral groove facing down into the petri dish in sequence, with 10 seeds in one petri dish.

[0055] (4) Lay four layers of gauze at the bottom of the sealed box and add 250 mL of sterile water for moisturization. Place the treatment in (3) in the sealed box, cover the lid, and place it in a light incubator with a 12-h light cycle and a temperature of 25 °C for moisturized cultivation for 3 - 4 days. When the coleoptile length is about 3 - 4 cm, inoculation can be carried out.

[0056] (5) Use sterilized scissors to cut off the leaves at about 3 mm below the joint of the wheat coleoptile and the leaf. Drop 2 μL of the biocontrol bacteria culture solution on the wound. After the culture solution dries, cover the lid and place it in a light incubator with a 12-h light cycle and a temperature of 25 °C for moisturized cultivation for 24 h, then inoculate the pathogen spore suspension.

[0057] (6) After treating the wheat leaves according to the method in (5), drop 2 μL of the suspension with a concentration of 1.0×10 5 spores / mL on the wound. After the suspension dries, cover the lid and place it in a light incubator with a 12-h light cycle and a temperature of 25 °C for moisturized cultivation for 7 days.

[0058] (7) After 7 days of cultivation, grade the diseases according to the ratio (a) of the lesion length to the total coleoptile length, calculate the disease index according to formula I, calculate the control efficacy according to formula II, and take pictures for record. The grading method is as follows:

[0059] Grade 0: a = 0;

[0060] Grade 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 of strain SXM-17 are shown in Figure 2 . The disease index of strain SXM-17 was 12.56, and the control effect was 80.47%.

[0066] 3 Wheat head blight biocontrol bacteria wheat head bioassay

[0067] (1) Select 10 wheat plants at the flowering stage with consistent growth and tie them into bundles;

[0068] (2) Dilute the cultured bacterial liquid (strain SXM-17) two-fold and spray it to ensure that the bacterial liquid completely wets the tied wheat ears;

[0069] (3) After 24 h, spray a suspension with a concentration of 1.0×10 5 spores / mL in the same way as in (2) to ensure that the bacterial liquid wets the tied wheat ears, and immediately 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 lower than 25 °C, remove the plastic bags 5 days after bagging; if the temperature is higher than 25 °C, remove the plastic bags 3 days after bagging;

[0071] (5) After 10 - 15 days, classify according to the percentage of the diseased area of the wheat ear accounting for the whole ear area. The classification method is as follows (according to the industry standard "GB / T 15796-2011):

[0072] Level 0: Disease-free;

[0073] Level 1: The number of diseased ears is 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 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 control effect according to the above method and take pictures for record.

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

[0079] 4 Molecular Biological Identification of Biocontrol Bacteria against Wheat Head Blight

[0080] 4.1 Extraction of Bacterial Genomic DNA

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

[0082] (2) Add 500 μL of ddH2O and vortex for 30 s - 40 s until there are no obvious bacterial clumps;

[0083] (3) Add 60 - 100 μL of lysozyme (20 mg / mL) according to the amount of bacteria, vortex to mix evenly, then place it in a 37 °C water bath for 30 min - 1 h, and invert it several times every 20 min during this period;

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

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

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

[0087] (7) Add 600 μL of pre-cooled isopropanol to precipitate DNA, invert it several times to mix evenly, and place it in a -20 °C refrigerator for 3 - 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, invert it several times to mix evenly, centrifuge at 14000 rpm for 2 min, and discard the supernatant;

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

[0091] (11) Use a pipette to suck out the remaining liquid, and then place the centrifuge tube in a laminar flow hood and blow it for 30 min;

[0092] (12) Add 100 μL of ddH2O to dissolve the DNA, and store it in a -20 °C refrigerator for use in 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 respectively. 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] Among them, 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] ATAAGGCGAAATTAATTGAGAAAATTGCTGATCTCGTAAGGGACAAAA

[0152] AGATAGAGGGTATCACAGATCTGCGTGATGAGTCAGATCGTACAGGTAT

[0153] GAGAATTGTCATTGAAATCAGACGCGATGCCAATGCGAATGTCATCTTA

[0154] AACAATCTGTACAAACAAACTGCTCTACAAACATCTTTTGGCATCAACC

[0155] TGCTTGCACTAGTTGATGACAGCCGA(SEQ ID NO:7)

[0156] 4.3 Construction of phylogenetic tree

[0157] Send the PCR products to Novogene for sequencing. After obtaining the sequencing results, perform homology alignment of the sequencing sequences on the NCBI website, and use Mega5 software to construct a phylogenetic tree. Among them, strain SXM-17 and the strains obtained by alignment are constructed into a phylogenetic tree using the 16S rDNA gene sequence. Strain XYG1 and the strains obtained by alignment are constructed into a phylogenetic tree using the spliced sequence of the 16S rDNA gene and the gyrA gene.

[0158] The results are shown in Figure 4 . From Figure 4It can be seen from the phylogenetic tree clustering and alignment results that strain SXM-17 is Brevibacillus formosus. Strain XYG1 clusters with Bacillus subtilis with a confidence of 100%. Therefore, strain XYG1 belongs to Bacillus subtilis.

[0159] Investigation on the Mechanism of Biocontrol Bacteria against Wheat Head Blight

[0160] 5.1 Detection of the Inhibitory Effect of Biocontrol Bacteria on the Spore Germination of Fusarium graminearum

[0161] (1) Inoculate the biocontrol bacteria (strain SXM-17, strain XYG1) into a glass test tube containing 5 mL of LB liquid medium, shake culture at 37 °C for 8 h, centrifuge at 5000 rpm for 10 min to obtain the supernatant of the strain;

[0162] (2) Mix equal volumes of the supernatant of the biocontrol strain and the spore suspension of Fusarium graminearum, and place them in a 25 °C dark incubator for static culture for 48 h, with 3 biological replicates;

[0163] (3) Observe the mixture under an optical microscope, count the germination of 100 sporangia each time, and record by counting and taking pictures.

[0164] The results are shown in Figure 5 . The results show that both strain SXM-17 and strain XYG1 can inhibit the spore germination, and strain SXM-17 can also reduce the spore number.

[0165] 5.2 Detection of the Inhibitory Effect of Biocontrol Bacteria on the Mycelial Growth of Fusarium graminearum

[0166] The antagonistic assay was performed by the plate confrontation method:

[0167] (1) Activate the biocontrol bacteria (strain SXM-17) on solid LB medium and incubate it overnight in an inverted position in a 37 °C incubator;

[0168] (2) Activate the fungal strain Fg 0609 used in the experiment on solid PDA medium and incubate it in a 25 °C incubator for 7 d;

[0169] (3) Use a 5 mm punch to punch out a mycelial plug of Fusarium graminearum Fg 0609 and inoculate it in the center of a PDA plate;

[0170] (4) Inoculate the activated biocontrol bacteria 2 cm to the left and right of the mycelial plug. Each treatment has three replicates, and only inoculating the mycelial plug is used as the blank control

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

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

[0173] The results are shown in Figure 6 . The biocontrol strain SXM-17 has antagonistic activity against the hyphae of Fusarium graminearum.

[0174] 5.3 Determination of the antibacterial spectrum of the biocontrol bacterium

[0175] (1) Activate the biocontrol bacteria (strain SXM-17, strain XYG1) on solid LB medium and incubate them upside down in an incubator at 37 °C overnight;

[0176] (2) Activate the fungal strains used in the experiment on solid PDA medium and incubate them in an incubator at 25 °C for 7 d;

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

[0178] (4) Inoculate the activated biocontrol bacteria 2 cm to the left and right of the fungal disc. Each treatment has three replicates, and only inoculating the fungal disc is used as the blank control;

[0179] (5) Invert the plate and incubate it in the dark in an incubator at 25 °C for 5 d.

[0180] (6) Observe whether an antibacterial zone is produced and take pictures for record.

[0181] The results are shown in Figure 7 . It can be seen from Figure 7 that strain SXM-17 has obvious antibacterial effects on Fusarium graminearum, Physalospora piricola, Botrytis cinerea, Alternaria solani, Rhizoctonia solani and Gaeumannomyces graminis, 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 head blight

[0183] 5.4.1 Preparation of the metabolites of the biocontrol bacterium

[0184] Inoculate the biocontrol bacterium (strain SXM-17) into a 300-mL Erlenmeyer flask containing 100 mL of LB liquid medium. After shaking culture at 37 °C and 200 rpm for 48 h, perform the following treatments:

[0185] (1) Culture broth: Make the OD of the above-mentioned shaken culture broth uniform 600 ;

[0186] (2) Supernatant: Pipette 1 mL of the above-mentioned bacterial liquid into a 2-mL centrifuge tube, 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-mentioned bacterial liquid into a 2 mL centrifuge tube, centrifuge at 5000 rpm for 10 min, discard the supernatant, resuspend it 3 times with 1 mL of sterile water, and then add an appropriate amount of sterile water for vortexing until the OD 600 is consistent with that of the culture medium.

[0188] 5.4.2 Bioassay experiment of the metabolites of biocontrol bacteria on coleoptiles

[0189] Detect the biocontrol effect of the three metabolites of the above biocontrol bacterium SXM-17 on Fusarium head blight of wheat by the coleoptile method. The method is shown in step 2.

[0190] The results are shown in Figure 8 and Table 3.

[0191] Table 3 Results of the biocontrol effect of the three metabolites of biocontrol bacterium SXM-17

[0192]

[0193] In order to explore whether the secondary metabolites produced by biocontrol bacteria are the main factors for their disease prevention, in this example, the culture medium, bacterial suspension, and supernatant of biocontrol bacterium strain SXM-17 were used for the biocontrol effect detection experiment. The results showed that there was no significant difference in the biocontrol effects of the metabolite solution, bacterial suspension, and culture medium of strain SXM-17, but they were all significantly better than the control group.

[0194] 5.5 Growth promotion effect of biocontrol bacteria on wheat

[0195] (1) The soil for growing wheat in the greenhouse is a mixture of loess, vermiculite, and nutrient soil in a ratio of 3:3:3. Add the mixed soil into small pots with a diameter of 5 cm and a height of 10 cm. Place every 5 small pots 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. Wait for use.

[0196] (2) Inoculate the above two biocontrol bacteria into 5 mL of LB liquid medium and shake culture overnight at 37 °C.

[0197] (3) Inoculate the biocontrol bacteria solution (strain SXM-17, strain XYG1) into a 250 mL Erlenmeyer flask containing 50 mL of LB liquid medium at a ratio of 1:100 and shake culture at 37 °C for 48 h to adjust the OD 600 to be consistent.

[0198] (4) Put several equal amounts of healthy wheat seeds in a 50 mL centrifuge tube, add 40 mL of sterile water to soak the seeds for 24 h until the seeds show white tips. A total of ten 50 mL centrifuge tubes are processed.

[0199] (5) After 24 hours, pour out the sterile water. Add 40 mL of the biocontrol bacterial solution to each of the nine 50-mL centrifuge tubes and soak the seeds for 1 hour, and add 40 mL of liquid LB to the other 50-mL centrifuge tube and soak the seeds for 1 hour as a blank control;

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

[0201] (7) Plant the dried wheat seeds in the soil in (1). Plant 4 wheat seeds in each small pot, with five replicates for each treatment. After planting, cover a layer of dry soil on each small pot, pour 700 mL of water into the plastic box containing the small pots, and water every two weeks;

[0202] (8) After one month, investigate the plant height, root length, aboveground fresh weight, and underground fresh weight of the wheat and take pictures for record.

[0203] The results are shown in Table 4 and Figure 9 . The results show that compared with the wheat treated only with LB medium, after treatment with strain SXM-17, there are significant differences in the root length, plant height, aboveground fresh weight, and underground fresh weight of the wheat, while strain XYG1 has no corresponding effect on promoting wheat growth.

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

[0205]

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

[0207] 5.6 Growth-promoting effect of biocontrol bacteria on wheat under salt stress treatment

[0208] (1)-(6) The methods are the same as (1)-(6) in 5.3;

[0209] (7) Plant the dried wheat seeds in the soil in (1). Plant 4 wheat seeds in each small pot, with five replicates for each treatment. After planting, cover a layer of dry soil on each small pot, and pour 700 mL of brine with a concentration of 100 mmol / L into the plastic box containing the small pots;

[0210] (8) After ten days, investigate the plant height, root length, aboveground fresh weight, and underground fresh weight of the wheat and take pictures for record.

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

[0212]

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

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

[0215] 5.7 Effects of biocontrol bacteria on the defense enzyme system of wheat under salt stress

[0216] The detection of the activity of the wheat defense enzyme system was carried out according to the instructions provided by Solarbio Science & Technology Co., Ltd.

[0217] 5.7.1 Determination of catalase (CAT) activity

[0218] (1) Weigh 0.1 g of wheat roots and place them in a mortar containing liquid nitrogen for grinding. After pre-cooling a 2 mL centrifuge tube with liquid nitrogen, transfer the ground product into the centrifuge tube and place it on an ice box.

[0219] (2) Add 1 mL of extraction solution to the centrifuge tube. After vortex mixing, centrifuge at 8000 g for 10 min using a 4°C centrifuge. Take the supernatant and transfer it to a new 2 mL centrifuge tube, and place it on an ice box for further measurement.

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

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

[0222] (5) Mix 35 μL of the sample with 1 mL of the detection solution and transfer it to a cuvette. Immediately measure the absorbance value as A1, and then measure it again after 1 min. The absorbance value 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 roots and place them in a mortar containing liquid nitrogen for grinding. After pre-cooling a 2 mL centrifuge tube with liquid nitrogen, transfer the ground product into the centrifuge tube and place it on an ice box.

[0228] (2) Add 1 mL of extraction solution to the centrifuge tube. After vortex mixing, centrifuge at 8000 g for 10 min at 4 °C. Transfer the supernatant to a new 2 mL centrifuge tube and place it on an ice box for further measurement.

[0229] (3) Take appropriate amounts of Reagent One, Three, and Four and water-bath them in a water bath at 25 °C for 10 min.

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

[0231] (5) Add 90 μL of the sample, 240 μL of Reagent One, 60 μL of Reagent Two, 180 μL of Reagent Three, 400 μL of distilled water, and 30 μL of Reagent Four to the measurement tube; add 90 μL of the sample, 240 μL of Reagent One, 180 μL of Reagent Three, 460 μL of distilled water, and 30 μL of Reagent Four to the control tube; add 240 μL of Reagent One, 60 μL of Reagent Two, 180 μL of Reagent Three, 490 μL of distilled water, and 30 μL of Reagent Four to Blank Tube 1; add 240 μL of Reagent One, 180 μL of Reagent Three, 550 μL of distilled water, and 30 μL of Reagent Four to Blank Tube 2.

[0232] (6) After mixing the components in the above tubes, water-bath them in a water bath at 37 °C for 30 min. Then, use a cuvette to measure the absorbance values of the components in each tube at 560 nm, and record them as: Ameasurement, Acontrol, A1blank, A2blank. ΔAmeasurement = Ameasurement - Acontrol, Δblank = A1blank - A2blank.

[0233] (7) Calculate the 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 = (ΔAblank - ΔAmeasurement) ÷ ΔAblank × 100%.

[0237] 5.7.3 Determination of Ascorbate Peroxidase (APX) Activity

[0238] (1) Weigh 0.1 g of wheat roots and place them in a mortar containing liquid nitrogen for grinding. After pre-cooling a 2 mL centrifuge tube with liquid nitrogen, transfer the ground product into the centrifuge tube and place it on an ice box.

[0239] (2) Add 1 mL of extraction solution into a centrifuge tube. After vortexing and mixing evenly, centrifuge at 8000 g for 10 min using a 4 °C centrifuge. Take the supernatant and transfer it into a new 2 mL centrifuge tube, and place it on an ice box for further measurement;

[0240] (3) Take an appropriate amount of Reagent 1 and water bath it in a 25 °C water bath for 30 min;

[0241] (4) Turn on the spectrophotometer. After preheating is completed, adjust the wavelength to 290 nm and calibrate it with distilled water;

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

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

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

[0245] APX (U / g) = 1.79 × (ΔA measurement - ΔA blank tube) ÷ W Formula V

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

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

[0248] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A Brevibacillus pulchrum Brevibacillus formosus strain SXM-17, characterized in that The preservation number is CGMCC No. 30198.

2. A preparation, characterized in that, The active ingredient is the cells of Brevibacillus formosus strain SXM-17 described in claim 1 and / or the metabolites of Brevibacillus formosus strain SXM-17.

3. The preparation according to claim 2, wherein, The preparation is a bacterial suspension of Brevibacillus formosus strain SXM-17 and / or the culture supernatant of Brevibacillus formosus strain SXM-17.

4. The preparation according to claim 2, wherein The active ingredient is the culture broth of Brevibacillus formosus strain SXM-17.

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

6. The application of Brevibacillus formosus strain SXM-17 described in claim 1 or the preparation described in any one of claims 2 to 5 in preventing and controlling crop diseases, wherein the crop diseases are at least one of the following: scab, ring rot, gray mold, early blight, basal rot, and take-all.

7. The application according to claim 6, characterized in that, Preventing and controlling crop scab includes inhibiting the spore germination of Gibberella zeae and / or inhibiting the mycelial growth of Gibberella zeae.

8. The application of Brevibacillus formosus strain SXM-17 described in claim 1 or the preparation described in any one of claims 2 to 5 in promoting the growth of crops, wherein the crop is wheat.

9. The application according to claim 8, wherein The crops include crops grown under salinized conditions and / or crops grown under non-salinized conditions.

10. The application according to claim 9, wherein The Brevibacillus formosus strain SXM-17 has the ability to increase the activity of defense enzymes in crops grown under salinized conditions.

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