A spindle-shaped lysinibacillus strain N-16 and its derivatives and their application in preventing and controlling scab of crops

Abstract

The present invention provides a spindle-shaped Lysinibacillus fusiformis strain N-16, a biocontrol agent and its application in preventing and controlling fusarium head blight of crops, belonging to the field of functional microbial technology. The present invention isolates and purifies a strain N-16 from wheat flowers, and the deposit number is CGMCC No.30199, which has a biocontrol effect on fusarium head blight. Experiments show that the culture solution, bacterial suspension and supernatant of strain N-16 can effectively reduce the disease index of wheat head blight, with good prevention and control effects. At the same time, strain N-16 has a significant inhibitory effect on the germination of fusarium head blight spores. In addition, the strain N-16 has a growth-promoting effect on crops. The strain N-16 provided by the present invention enriches the biocontrol strain resource library of various plant pathogens, and provides a new means for green and efficient biological control microbial resources.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional microorganisms, and in particular relates to a Lysinibacillus fusiformis strain N-16 and its derivatives, and applications in preventing and controlling fusiform blight of crops. Background Art

[0002] Wheat Fusarium head blight is a major and common wheat disease worldwide, resulting in reduced or even complete wheat harvests and causing severe economic losses. Furthermore, when the fusarium graminearum pathogen infects wheat ears, it produces mycotoxins, trichothecenes, and zearalenone, posing a serious threat to human and animal health.

[0003] Currently, no wheat varieties resistant to ergot have been discovered, and chemical control remains the primary means of prevention. However, due to drawbacks such as pesticide residues, resistance development, and environmental pollution, biological control is gaining increasing attention. A review of literature and other databases reveals that over ten biocontrol strains have been published as effective against wheat ergot: Bacillus velezensis, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus psychrophilus, Paenibacillus polymyxa, Streptomyces, Trichoderma, Pseudomonas fluorescens, Pseudomonas aeruginosa, Bacillus licheniformis, and Bacillus brevis. However, only two bacterial agents have been registered for wheat ergot prevention: Bacillus subtilis and Paenibacillus polymyxa. Compared to the literature, the number of commercially available strains is relatively small, failing to meet the demand for safe and effective disease control. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a spindle-shaped Lysinibacillus strain N-16, which not only has the effect of effectively preventing and controlling wheat fusarium wilt, but also can promote crop growth, and can be widely used as an industrial strain.

[0005] The invention provides a lysinophilic Bacillus strain N-16, which has a deposit number of CGMCC No. 30199.

[0006] The present invention provides a biocontrol agent, wherein the active ingredients include the bacterial body of the lysinophilic Bacillus strain N-16 and / or the metabolites of the lysinophilic Bacillus strain N-16.

[0007] Preferably, the bacterial cells of the fusiform Lysinibacillus strain N-16 include a bacterial suspension of the fusiform Lysinibacillus strain N-16;

[0008] The metabolites of the Lysinibacillus fusiformis strain N-16 include the culture supernatant of the Lysinibacillus fusiformis strain N-16.

[0009] Preferably, the active ingredient comprises the culture broth and / or fermentation broth of the Lysinibacillus fusiformis strain N-16.

[0010] Preferably, the active ingredient further comprises at least one of the following bacterial species and / or metabolites thereof: Bacillus velezensis, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus psychrophilus, Bacillus polymyxa, Streptomyces, Trichoderma, Pseudomonas fluorescens, Pseudomonas aeruginosa, Bacillus licheniformis and Bacillus brevis.

[0011] Preferably, the dosage form of the biocontrol agent includes at least one of the following: powder, aqueous solution, granule and microcapsule.

[0012] The present invention provides the use of the lysinibacillus fusiformis strain N-16 or the biocontrol agent in preventing and controlling fusarium head blight in crops.

[0013] Preferably, the prevention and control of crop ergot disease includes inhibiting the germination of ergot fungus spores.

[0014] The present invention provides the use of the lysinibacillus fusiformis strain N-16 or the biocontrol agent in promoting the growth of crops.

[0015] 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.

[0016] The present invention provides a fusiform Lysinibacillus strain N-16, with a deposit number of CGMCC No. 30199. The present invention isolates and purifies a strain N-16 from wheat flowers. After primary screening and rescreening, the strain N-16 has a biocontrol effect on fusiform scab. Molecular identification shows that the strain N-16 is fusiform Lysinibacillus fusiformis. The present invention uses the culture solution, bacterial suspension and supernatant of the strain N-16 to detect the biocontrol effect on wheat fusiform scab. The results show that compared with the control, the culture solution, bacterial suspension and supernatant can significantly reduce the disease index of wheat fusiform scab, 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 N-16 has a significant inhibitory effect on the germination of fusiform spores. In addition, the strain N-16 has a growth-promoting effect on crops grown in a natural environment. It can be seen that the strain N-16 provided by the present invention enriches the biocontrol strain resource library of various plant pathogens and provides a new means for green and efficient biocontrol microbial resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1The results of the isolation and purification of wheat fusarium head blight biocontrol strain N-16 are shown. The left picture shows the plate morphology of each strain screened, and the right picture shows the plate morphology of strain N-16.

[0018] Figure 2 The biocontrol effect of the biocontrol strain N-16 on wheat fusarium head blight on coleoptiles;

[0019] Figure 3 The biocontrol effect of the biocontrol strain N-16 on wheat head rust was measured.

[0020] Figure 4 Phylogenetic tree of biocontrol strain N-16 (A) and strain XYG1 (B)

[0021] Figure 5 This is the result of the biocontrol strain N-16 inhibiting the growth of fusarium spores;

[0022] Figure 6 The control results (A) and statistical results (B) of the culture solution, bacterial suspension and supernatant of the biocontrol strain N-16 on wheat scab;

[0023] Figure 7 This is the growth promotion effect of biocontrol strain N-16 on wheat.

[0024] Biological deposit information

[0025] The strain Lysinibacillus fusiformis was deposited at the General Microbiology Center of China 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. 30199, and the strain number is N-16. DETAILED DESCRIPTION

[0026] The invention provides a lysinophilic Bacillus strain N-16, which has a deposit number of CGMCC No. 30199.

[0027] In the present invention, strain N-16 was isolated and purified from wheat flowers, and its effectiveness in preventing and controlling wheat fusarium head blight was verified using coleoptiles and spikes. Using molecular identification methods, the amplified PCR product was compared with databases and subjected to phylogenetic cluster analysis, confirming that strain N-16 belongs to Lysinibacillus fusiformis.

[0028] The present invention provides a biocontrol agent, wherein the active ingredients include the bacterial body of the lysinophilic Bacillus strain N-16 and / or the metabolites of the lysinophilic Bacillus strain N-16.

[0029] In the present invention, the cells of the fusiform Lysinibacillus strain N-16 preferably include a bacterial suspension of the fusiform Lysinibacillus strain N-16. The metabolites of the fusiform Lysinibacillus strain N-16 include a culture supernatant of the fusiform Lysinibacillus strain N-16. When the active ingredient contains both the cells of the strain N-16 and the metabolites, the active ingredient preferably includes a culture solution and / or fermentation solution of the fusiform Lysinibacillus strain N-16.

[0030] In the present invention, the method for preparing the culture broth and / or fermentation broth of the spindle-shaped Lysinibacillus strain N-16 is preferably performed by inoculating the activated strain N-16 into LB medium and shaking culture. The shaking culture time is 24 to 720 hours, more preferably 26 to 56 hours, and more preferably 48 hours. The shaking culture speed is preferably 120 to 220 rpm, more preferably 180 to 210 rpm, and more preferably 200 rpm. The shaking culture temperature is preferably 28 to 50°C, and more preferably 37°C.

[0031] In the present invention, the method for preparing the culture supernatant of the spindle-shaped lysine Bacillus strain N-16 is preferably to subject the culture solution and / or fermentation solution prepared above to solid-liquid separation, and collect the liquid phase. 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 is 10% to 90%, more preferably 20% to 80%, further preferably 40% to 70%, and further preferably 50%. The bacterial suspension of the spindle-shaped lysine Bacillus strain N-16 is preferably to subject the culture solution and / or fermentation solution prepared above to solid-liquid separation, collect the solid phase, and resuspend the solid phase in sterile water. The concentration of the active ingredient in the biocontrol agent is preferably (1 to 100) × 10 9 CFU / ml, more preferably (5 to 80)×10 9 CFU / ml, more preferably (5-50)×10 9 CFU / ml.

[0032] The prevention and control effect test experiment of wheat coleoptile fusarium ergot showed that compared with the control, the culture medium, bacterial suspension and supernatant can significantly reduce the disease index of wheat fusarium ergot, have good prevention and control effects, and there is no significant difference in the prevention and control effects of the three.

[0033] In the present invention, the active ingredient preferably also includes at least one of the following bacterial species and / or metabolites thereof: Bacillus velezensis, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus psychrophilus, Paenibacillus polymyxa, Streptomyces, Trichoderma, Pseudomonas fluorescens, Pseudomonas aeruginosa, Bacillus licheniformis, and Bacillus brevis. Given that prior art reports indicate that the above-mentioned bacterial species are common biocontrol agents for the prevention and control of fusarium scurf. Therefore, the Lysinibacillus spindle-shaped strain N-16 developed in the present invention is compounded with at least one of the above-mentioned biocontrol agents to prepare a novel biocontrol agent. When compounding the active ingredients, the number of viable bacteria of each bacterial species is preferably mixed in equal proportions, or the culture or fermentation broth of each bacterial species is preferably mixed in equal volumes.

[0034] 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.

[0035] The powder is preferably prepared by mixing the active ingredient's bacteria and / or metabolites with a freeze-dried protective agent and freeze-drying. The present invention does not impose any particular restrictions on the type of freeze-dried protective agent, and freeze-dried protective agents well known in the art can be used, such as substances that regulate osmotic pressure, such as glucose and mannose. 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 prepared by culturing the active ingredient, and the resulting culture solution is subjected to bacterial cell separation or is not separated and then mixed with an antifreeze agent. 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 particular restrictions on the preparation method of the granules or microcapsules, and they can be prepared using biocontrol agent granules or microcapsules well known in the art.

[0036] The present invention provides the use of the lysinibacillus fusiformis strain N-16 or the biocontrol agent in preventing and controlling fusarium head blight in crops.

[0037] In the present invention, the control of fusobacterium scab in crops preferably includes at least one of the following: inhibiting spore germination of fusobacterium scab. In an embodiment of the present invention, the effects of biocontrol strains on fusobacterium scab conidia germination were investigated. The results showed that the fusobacterium Lysinibacillus strain N-16 significantly inhibited conidia germination compared to a control group and strains screened from the same batch.

[0038] The present invention provides the use of the lysinibacillus fusiformis strain N-16 or the biocontrol agent in promoting the growth of crops.

[0039] In the present invention, the crops preferably include crops grown under natural planting conditions. The crops preferably include at least one of the following: wheat, oats, barley, corn, millet, rice, sorghum, soybean, cotton, sugarcane, sugar beet, eggplant, apple, grape, potato and sweet potato.

[0040] In an embodiment of the present invention, a growth promotion experiment of wheat planted in a natural environment was carried out using the spindle-shaped Lysinibacillus strain N-16. Compared with the control group, after treatment with the strain N-16, the root length, plant height, aboveground fresh weight and underground fresh weight 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 wheat planted in salt land using the spindle-shaped Lysinibacillus strain N-16. Compared with the control group, after treatment with the strain N-16, the root length, plant height, aboveground fresh weight and underground fresh weight of the wheat were significantly different, and compared with the strains screened in the same batch, it had significant advantages.

[0041] The following examples provide a detailed description of the lysinophilic Bacillus strain N-16 and its derivatives and their use in preventing and controlling scab of crops, but they should not be construed as limiting the scope of protection of the present invention.

[0042] Example 1

[0043] Isolation, purification and preservation of a biocontrol strain

[0044] 1.1 Isolation of bacteria from wheat flowers

[0045] Use sterile tweezers to remove the wheat flowers and place them in a sterile mortar. When there are 20 wheat flowers, add 1 mL of sterile water and start grinding. Grind until it is homogenous. Pipette 100 μL of grinding liquid 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℃ for 3-4 days.

[0046] 1.2 Purification and preservation of bacteria from wheat flowers

[0047] Observe the growth of the bacteria on the culture medium. Once the colony count stabilizes, select colonies of different shapes and colors and streak them onto new LB solid medium until a single colony grows. After shaking the purified and stable strain in liquid LB, mix it with 50% glycerol at a 1:1 ratio and store in a -80°C freezer.

[0048] 78 strains of bacteria were isolated from wheat flowers collected from the wheat experimental field. The morphology of the isolated strain N-16 is shown in Figure 1 The surface of the colony of strain N-16 on LB solid medium was smooth, and the single colony was white and nearly round.

[0049] 2. Preliminary screening of wheat scab biocontrol bacteria using the coleoptile method

[0050] (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;

[0051] (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 on the filter paper with the ventral groove facing down. Place the seeds in a 52 × 36 × 14.5 cm white sealed box and place them in a 12 h photoperiod, 25 °C light incubator. Keep them moist for 2 days until they germinate.

[0052] (3) Prepare a culture dish containing soaked filter paper according to (2) and place the germinated seeds into the culture dish one by one with the ventral groove facing downwards, with 10 seeds placed in one culture dish;

[0053] (4) Place four layers of gauze on 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 it, 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 about 3-4 cm long, it can be inoculated.

[0054] (5) Cut the wheat leaf about 3 mm below the junction of the coleoptile and the leaf with sterilized scissors, and apply 2 μL of biocontrol bacteria culture solution (strain N-16) to the wound. After the culture solution is dried, cover the lid and place it in a light incubator with a 12-h photoperiod and 25°C for 24 h to keep it moist and then inoculate the pathogen spore suspension.

[0055] (6) After treating the wheat leaves according to the method in (5), 2 μL of 1.0×10 5 A 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.

[0056] (7) After 7 days of incubation, the disease was graded based on the ratio of the lesion length to the entire coleoptile length (a). The disease index was calculated according to Formula I, and the control efficacy was calculated according to Formula II. The disease was recorded and photographed. The grading method is as follows:

[0057] Level 0: a = 0;

[0058] Level 1: 0 <a<1 / 4;

[0059] Level 2: 1 / 4≤a≤1 / 2;

[0060] Level 3: 1 / 2 <a<2 / 3;

[0061] Level 4: 2 / 3≤a≤1;

[0062]

[0063] The results for strain N-16 are shown in Figure 2 The disease index of strain N-16 was 12.33, and the control efficacy was 81.22%.

[0064] 3. Wheat ear bioassay of wheat scab biocontrol bacteria

[0065] (1) 10 wheat plants in the flowering stage with uniform growth were selected and bundled into bundles;

[0066] (2) Dilute the cultured bacterial solution (strain N-16 and strain XYG1) twice and spray it to ensure that the bacterial solution completely soaks the bundled wheat ears;

[0067] (3) After 24 hours, the concentration of 1.0×10 5 Spray the suspension containing 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;

[0068] (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;

[0069] (5) After 10 to 15 days, the crops are graded based on the percentage of the diseased area of ​​the ear to the whole ear area. The grading method is as follows (according to the industry standard "GB / T 15796-2011"):

[0070] Level 0: no disease;

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

[0072] Level 2: The number of diseased ears accounts for 1 / 4-1 / 2 of the total number of ears;

[0073] Level 3: The number of diseased ears accounts for less than 1 / 2-3 / 4 of the total number of ears;

[0074] Level 4: The number of diseased ears accounts for more than 3 / 4 of the total number of ears.

[0075] Calculate the disease index and prevention efficacy according to the above method and take photos for record.

[0076] See the results Figure 3The disease index of strain N-16 was 17.36, with a control efficacy of 78.47%. The disease index of strain XYG1 was 20.37, with a control efficacy of 69.95%.

[0077] 4 Molecular identification of wheat scab biocontrol bacteria

[0078] 4.1 Extraction of bacterial genomic DNA

[0079] (1) Use a pipette to draw 1.5 mL of bacterial suspension of strain N-16 or strain XYG1 into a 2 mL centrifuge tube, centrifuge at 12000 rpm for 1 min, discard the supernatant, and collect the bacterial cells;

[0080] (2) Add 500 μL ddH2O and vortex for 30-40 seconds until no bacterial clumps are visible;

[0081] (3) Add 60-100 μL of lysozyme (20 mg / mL) according to the amount of bacteria, vortex and mix thoroughly, then place in a 37°C water bath for 30 min to 1 h, turning it upside down several times every 20 min.

[0082] (4) Add 650 μL of nucleic acid lysis buffer and mix thoroughly by pipetting;

[0083] (5) Add 250 μL of protein precipitation solution and vortex for 1 min;

[0084] (6) Centrifuge at 12000 rpm for 5 min and transfer 900 μL of supernatant to a new 2 mL centrifuge tube;

[0085] (7) Add 600 μL of pre-chilled isopropanol to precipitate the DNA, mix thoroughly by inverting the tube several times, and place in a -20°C refrigerator for 3–5 h.

[0086] (8) Centrifuge at 12000 rpm for 5 min and discard the supernatant;

[0087] (9) Add 500 μL of pre-chilled 70% ethanol, mix by inverting several times, centrifuge at 14,000 rpm for 2 min, and discard the supernatant;

[0088] (10) Repeat step (9);

[0089] (11) Use a pipette to absorb the remaining liquid and place the centrifuge tube in a clean bench to blow for 30 minutes;

[0090] (12) Add 100 μL of ddH 2 O to dissolve the DNA and store it in a -20 °C refrigerator for PCR amplification and verification.

[0091] 4.2 PCR amplification and verification

[0092] PCR amplification: DNA of bacterial N-16 was used as a template to amplify the corresponding 16S rDNA and gyrA gene fragments. The PCR reaction system is shown in Table 1, and the PCR amplification program is shown in Table 2:

[0093] Table 1 PCR reaction system

[0094]

[0095] Among them, the primers for 16S rDNA are as follows:

[0096] 63F: CAGGCCTAACACATGCAAGTC (SEQ ID NO: 1);

[0097] 1397R: GGGCGGWGTGTACAAGGC (SEQ ID NO: 2);

[0098] The primers for the gyrA gene are as follows:

[0099] gyrA-F: CAGTCAGGAAATGCGTACGTCCTT (SEQ ID NO: 3);

[0100] gyrA-R: CAAGGTAATGCTCCAGGCATTGCT (SEQ ID NO: 4).

[0101] Table 2 PCR amplification program

[0102]

[0103] 4.3 Construction of phylogenetic tree

[0104] PCR products were sent to Nosai Sequencing for sequencing. Once the sequencing results were obtained, the sequences were aligned for homology on the NCBI website, and a phylogenetic tree was constructed using Mega5 software. The phylogenetic tree for strain N-16 and its associated strains was constructed using the 16S rDNA gene sequence. The phylogenetic tree for strain XYG1 and its associated strains was constructed using the spliced ​​16S rDNA and gyrA gene sequences.

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

[0106] ACGTTAGCGGCGGACGGGTGAGTAACACGTGGGCAACCTACCTTA

[0107] TAGTTTGGGATAACTCCGGGAAACCGGGGCTAATACCGAATAATCTGTT

[0108] TCACCTCATGGTGAAACACTGAAAGACGGTTTCGGCTGTCGCTATAGG

[0109] ATGGGCCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAG

[0110] GCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGAC

[0111] TGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTC

[0112] CACAATGGGCGAAAGCCTGATGGAGCAACGCCGCGTGAGTGAAGAAG

[0113] GATTTCGGTTCGTAAAACTCTGTTGTAAGGGAAGAACAAGTACAGTAG

[0114] TAACTGGCTGTACCTTGACGGTACCTTATTAGAAAGCCACGGCTAACTA

[0115] CGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAAT

[0116] TATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAA

[0117] GCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAG

[0118] TGCAGAAGAGGATAGTGGAATTCCAAGTGTAGCGGTGAAATGCGTAGA

[0119] GATTTGGAGGAACACCAGTGGCGAAGGCGACTATCTGGTCTGTAACTG

[0120] ACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTG

[0121] GTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGC

[0122] CCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACG

[0123] GTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCG

[0124] GTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGT

[0125] CTTGACATCCCGTTGACCACTGTAGAGATATGGTTTCCCCTTCGGGGGC

[0126] AACGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATG

[0127] TTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCATCA

[0128] TTTAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAG

[0129] GTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACAC

[0130] GTGCTACAATGGACGATACAAACGGTTGCCAACTCGCGAGAGGGAGCT

[0131] AATCCGATAAAGTCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTA

[0132] CATGAAGCCGGAATCGCTAGTAATCGCGATCAGCA(SEQ ID NO:5).

[0133] The nucleotide sequence of the 16S rDNA gene amplified from strain XYG1 is as follows:

[0134] GCTGGATCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCA

[0135] GACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCG

[0136] CGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAG

[0137] GTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTT

[0138] GTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAG

[0139] ATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACAC

[0140] GAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGG

[0141] ACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTC

[0142] TTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCC

[0143] CCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGA

[0144] GTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACA

[0145] CTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGT

[0146] TCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGT

[0147] CGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTAC

[0148] ACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAA

[0149] TGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAAC

[0150] CGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCT

[0151] ACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTA

[0152] GGTACCGTCAAGGTGCCGCCCTATTCGAACGGTACTTGTTCTTCCCTAA

[0153] CAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGC

[0154] TCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCG

[0155] TAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTC

[0156] AGGTCGGCTACGCATCGTTGCCTTGGTGAGCCATTACCTCACCAACTAG

[0157] CTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACCTTTTA

[0158] TGTTTGAACCATGCGGTTCAAACAACCATCCGGTATTAGCCCCGGTTTC

[0159] CCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCC

[0160] GTCCGCCGCTAACATCAGGAGCAG (SEQ ID NO:6).

[0161] The nucleotide sequence of the gyrA gene amplified from strain XYG1 is as follows:

[0162] CATGAGCGTTATCGTGTCCCGTGCTCTTCCGGATGTTCGAGACGGTTTA

[0163] AAACCGGTTCATAGACGGATTTTGTATGCAATGAATGATTTAGGCATGA

[0164] CAAGTGACAAGCCTTATAAAAAATCCGCGCGTATCGTTGGAGAAGTTAT

[0165] CGGGAAATACCACCCGCACGGTGATTCAGCGGTATATGAATCCATGGTC

[0166] AGAATGGCTCAGGATTTCAACTACCGTTATATGCTCGTTGACGGTCACG

[0167] GAAACTTCGGTTCTGTTGACGGAGACTCAGCGGCGGCCATGCGTTATA

[0168] CAGAAGCAAGAATGTCTAAAATCTCAATGGAGATTCTTCGCGACATCA

[0169] CAAAAGACACAATCGATTACCAGGATAACTATGACGGGTCAGAAAGAG

[0170] AACCTGTCGTTATGCCTTCAAGGTTCCCGAATCTGCTCGTGAACGGTGC

[0171] TGCCGGCATTGCGGTAGGTATGGCAACAAACATTCCTCCGCACCAGCT

[0172] GGGAGAAATCATTGACGGTGTACTTGCTGTCAGTGAGAATCCGGACAT

[0173] TACAATTCCAGAGCTTATGGAAGTCATTCCAGGGCCTGATTTCCCGACT

[0174] GCGGGTCAAATCTTGGGACGCAGCGGTATCCGGAAAGCATACGAATCA

[0175] GGCCGAGGCTCTATCACGATCCGGGCAAAAGCTGAGATCGAACAAACA

[0176] TCTTCGGGTAAAGAAAGAATTATCGTTACAGAGTTACCTTACCAAGTAA

[0177] ATAAGGGCGAAATTAATTGAGAAAATTGCTGATCTCGTAAGGGACAAAA

[0178] AGATAGAGGGTATCACAGATCTGCGTGATGAGTCAGATCGTACAGGTAT

[0179] GAGAATTGTCATTGAAATCAGACGCGATGCCAATGCGAATGTCATCTTA

[0180] AACAATCTGTACAAACAAACTGCTCTACAAACATCTTTTGGCATCAACC

[0181] TGCTTGCACTAGTTGATGACAGCCGA (SEQ ID NO:7).

[0182] See the results Figure 4 .Depend on Figure 4 From A in the figure, we can see that N-16 is Lysinibacillus fusiformis through the phylogenetic tree clustering and comparison results. Figure 4 As shown in Figure B, strain XYG1 and Bacillus subtilis are clustered together with a confidence level of 100%, indicating that strain XYG1 belongs to Bacillus subtilis.

[0183] Study on the mechanism of biocontrol bacteria in controlling wheat scab

[0184] 5.1 Detection of the inhibitory effect of biocontrol bacteria on spore germination of Fusarium spores

[0185] (1) The biocontrol bacteria (strain N-16 and strain XYG1) were inoculated into a glass test tube containing 5 mL of LB liquid medium, incubated at 37°C for 8 h, and centrifuged at 5000 rpm for 10 min to obtain the supernatant of the strain;

[0186] (2) Equal volumes of the biocontrol strain supernatant and the spore suspension of Fusarium fusogenum were mixed and placed in a dark incubator at 25°C for 8 h, with three biological replicates;

[0187] (3) The mixed solution was observed under an optical microscope, and the germination status of 100 sporangia was counted each time, and the counts were recorded and photographed.

[0188] See the results Figure 5 The results showed that the spores of Gibberella fusca were abnormally swollen and inhibited from germination after treatment with biocontrol strains N-16 and XYG1, while the number of Gibberella fusca spores was significantly reduced after treatment with biocontrol strain N-16.

[0189] 5.2 Effects of biocontrol bacteria metabolites on the biocontrol effect of wheat scab

[0190] 5.2.1 Preparation of biocontrol bacteria metabolites

[0191] Biocontrol bacteria (strain N-16, strain XYG1) were inoculated into 300 mL Erlenmeyer flasks containing 100 mL LB liquid medium and cultured at 37°C, 200 rpm, and then treated as follows:

[0192] (1) Culture medium: The above shake culture medium was uniform in OD 600 ;

[0193] (2) Supernatant: Pipette 1 mL of the above bacterial solution into a 2 mL centrifuge tube and centrifuge at 5000 rpm for 10 min. The supernatant was collected and filtered through a 0.22 μm bacterial filter to obtain a sterile supernatant.

[0194] (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 with 1 mL of sterile water three times, add appropriate amount of sterile water and vortex, and calculate the OD value. 600 Consistent with the culture medium.

[0195] 5.2.2 Coleoptile bioassay of biocontrol bacteria metabolites

[0196] The three metabolites of the above-mentioned biocontrol strain N-16 were used to detect the biocontrol effect of wheat fusarium scaber by the coleoptile method, as shown in step 2.

[0197] See the results Figure 6 and Table 3.

[0198] Table 3 Biocontrol effects of three metabolites of biocontrol strain N-16

[0199]

[0200] To investigate whether the secondary metabolites produced by biocontrol bacteria are the primary factors in their disease prevention, this example used culture fluid, bacterial suspension, and supernatant of biocontrol bacteria strain N-16 to conduct biocontrol efficacy testing. The results showed that there was no significant difference in the biocontrol efficacy of the metabolic fluid, bacterial suspension, and culture fluid of strain N-16, but the efficacy was significantly better than that of the control group. The bacterial suspension of biocontrol bacteria strain N-16 was significantly better than that of strain XYG1.

[0201] 5.3 Growth-promoting effects of biocontrol bacteria on wheat

[0202] (1) The soil for greenhouse wheat cultivation was prepared by mixing loess, vermiculite, and nutrient soil in a ratio of 3:3:3. The mixed soil was added to small pots with a diameter of 5 cm and a height of 10 cm. Each 5 small pots were 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. The boxes were then watered and left for use.

[0203] (2) The two biocontrol bacteria strains (strain N-16 and strain XYG1) were inoculated into 5 mL of LB liquid medium and cultured with shaking at 37°C overnight;

[0204] (3) Inoculate the biocontrol bacteria solution into a 250 mL Erlenmeyer flask containing 50 mL LB liquid medium at a ratio of 1:100, shake and incubate at 37°C for 48 h, and adjust the OD 600 consistent;

[0205] (4) Place equal amounts of healthy wheat seeds in a 50 mL centrifuge tube and add 40 mL of sterile water to soak the seeds for 24 h until the seeds turn white. A total of ten 50 mL centrifuge tubes were processed.

[0206] (5) After 24 h, the sterile water was poured out, and 40 mL of biocontrol solution was added to nine 50 mL centrifuge tubes and the seeds were soaked for 1 h. Another 50 mL centrifuge tube was added with 40 mL of liquid LB and the seeds were soaked for 1 h as a blank control.

[0207] (6) After 1 hour, pour out the bacterial solution and pour the wheat seeds in each centrifuge tube onto absorbent paper and allow the wheat seeds to dry.

[0208] (7) Plant the dried wheat seeds in the soil in (1), planting 4 wheat seeds in each small pot, with five replicates for each treatment. After planting, cover each small pot with a layer of dry soil, and pour 700 mL of water into the plastic box containing the small pots. Water once every two weeks.

[0209] (8) One month later, the plant height, root length, aboveground fresh weight and underground fresh weight of wheat were investigated and recorded by taking photos.

[0210] The results are shown in Table 4 and Figure 7The results showed that compared with the wheat in the control group treated with LB medium only, the root length, plant height, aboveground fresh weight and underground fresh weight of wheat treated with strain N-16 were significantly different, while strain XYG1 had no effect on promoting crop growth compared with the control group.

[0211] Table 4 Growth-promoting effect of strain N-16 on wheat

[0212]

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

[0214] 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 principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A spindle-shaped lysinophilic Bacillus ( Lysinibacillus fusiformis ) strain N-16, characterized in that The deposit number is CGMCC No.30199.

2. A biocontrol agent, characterized in that The active ingredient comprises the bacterial body of the Lysinibacillus fusiformis strain N-16 according to claim 1 and / or the metabolites of the Lysinibacillus fusiformis strain N-16; The metabolite of the lysinophilic Bacillus strain N-16 is the culture supernatant of the lysinophilic Bacillus strain N-16.

3. A biocontrol agent, characterized in that The active ingredient comprises the bacterial suspension of the lysinophilic Bacillus strain N-16 according to claim 1.

4. A biocontrol agent, characterized in that The active ingredient comprises the fermentation broth of the lysinophilic Bacillus strain N-16 according to claim 1.

5. The biocontrol agent according to any one of claims 2 to 4, characterized in that The active ingredient also includes at least one of the following bacterial species and / or metabolites of the bacterial species: Bacillus velez, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus psychrophilus, Bacillus polymyxa, Streptomyces, Trichoderma, Pseudomonas fluorescens, Pseudomonas aeruginosa, Bacillus licheniformis and Bacillus brevis.

6. 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.

7. Use of the Lysinibacillus fusiformis strain N-16 according to claim 1 or the biocontrol agent according to any one of claims 2 to 6 in preventing and controlling wheat fusarium head blight.

8. The application according to claim 7, characterized in that: The prevention and control of wheat fusarium head blight includes inhibiting the germination of fusarium head blight spores.

9. Use of the Lysinibacillus fusiformis strain N-16 according to claim 1 or the biocontrol agent according to any one of claims 2 to 6 in promoting wheat growth.

Images