Brevibacillus megatherium, fungicide containing brevibacillus megatherium, application of brevibacillus megatherium and fungicide and method for preventing and treating plant diseases

Abstract

The invention relates to the technical field of microorganisms, and discloses brevibacillus megatherium, a fungicide containing the brevibacillus megatherium, application of the brevibacillus megatherium and the fungicide and a method for preventing and treating plant diseases. The preservation number of the brevibacillus formmosus is CGMCC (China General Microbiological Culture Collection Center) No.32491, and the brevibacillus formmosus can be used for preparing the bacillus formmosus. The invention also provides a method for preventing and treating plant diseases. The method comprises the step of irrigating plants by using the brevibacillus beautiful and the fungicide. The brevibacillus megaterium has the effects of promoting growth and preventing diseases, has a relatively high bacteriostasis rate on phytophthora nicotianae, can efficiently antagonize black shank caused by phytophthora nicotianae, and has relatively strong capability of secreting auxin and exopolysaccharide.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, in particular to a Brevibacillus beautifulus, a bacterial agent containing the bacterium, and their application and a method for preventing and controlling plant diseases. Background Art

[0002] Yield loss of crops due to pathogens and pests is a serious problem and poses a major threat to food security and economic development. It is estimated that the loss ranges from 17% to 30%. Plant pathogens include viruses, bacteria, oomycetes and fungi, which infect plants in different ways and affect crop growth and yield. Phytophthora is a type of oomycetes and is an important plant pathogen in the class Oomycetes. Phytophthora infestans is widely distributed. It can infect a variety of Solanaceae crops and can cause serious diseases in a variety of crops including tomatoes, strawberries and tobacco, causing significant economic losses. Tobacco Phytophthora is one of the important soil-borne pathogens of tobacco. It can infect tobacco at any growth stage, causing symptoms of root rot, leaf wilting and even plant death, which can lead to 30%-50% yield loss.

[0003] At present, chemical pesticides still play an important role in preventing and controlling various plant diseases and increasing crop yields. Although chemical pesticides show high control efficiency in the short term, they also bring about environmental pollution, pesticide residues, and increased resistance of pathogens to pesticides. In contrast, biological control, as an eco-friendly alternative, can not only effectively control diseases, but also meet the needs of sustainable agricultural development, showing great development and application potential. Bacillus is one of the most studied and applied biocontrol bacteria, with a good broad-spectrum antibacterial activity, and can effectively prevent and control diseases in various parts of plants, such as roots, stems, leaves, and fruits. Studies have found that Bacillus subtilis B3 has a significant inhibitory effect on wheat stripe blight (Rhizoctonia cerealis); Bacillus velezensis XY40-1 has a control effect of 66.13% on pepper blight (Phytophthora capsici).

[0004] Bacillus is one of the most common gram-positive bacteria and can be isolated from a variety of habitats. Some of its strains have been studied as biocontrol agents for several plant pathogens due to their antibacterial effects, such as Bacillus velez, Bacillus subtilis and Bacillus siamese. Brevibacillus was once considered a branch or subgenus of the genus Bacillus. With the progress of molecular biology, it has been gradually reclassified as an independent genus. At present, most of the research on Brevibacillus focuses on the enzyme production characteristics of the strain, and less attention has been paid to its application in the prevention and control of plant diseases, especially in the prevention and control of oomycete diseases. Summary of the invention

[0005] The purpose of the present invention is to overcome the problems existing in the prior art and provide a strain of Brevibacillus beautifulus, a bacterial agent containing the strain, and their applications and methods for preventing and controlling plant diseases. The Brevibacillus beautifulus has the effects of promoting growth and preventing diseases, has a high inhibition rate against tobacco phytophthora, can effectively antagonize the black shank disease caused by tobacco phytophthora, and has a strong ability to secrete auxin and extracellular polysaccharides.

[0006] In order to achieve the above object, the present invention provides a Brevibacillus formosus in a first aspect, wherein the deposit number of the Brevibacillus formosus is CGMCC No.32491.

[0007] The second aspect of the present invention provides a bacterial agent, which contains the above-mentioned Brevibacillus beautifulus.

[0008] Preferably, the bacterial agent contains the bacterial cells and / or metabolites of the Bacillus Brevibacillus.

[0009] Preferably, the metabolites are exopolysaccharides and / or auxins.

[0010] Preferably, the bacterial agent is a liquid bacterial agent and / or a solid bacterial agent, preferably a liquid bacterial agent.

[0011] Preferably, the liquid bacterial agent is a culture solution of Bacillus brevis, and more preferably, the OD of the culture solution is 600 It is 0.5-0.9.

[0012] The third aspect of the present invention provides a method for preparing a bacterial agent, the preparation method comprising: culturing the above-mentioned Brevibacillus beautifulus.

[0013] Preferably, the culture conditions include: inoculation amount of 0.5-2% by volume, temperature of 20-35° C., rotation speed of 150-200 rpm, and time of 20-75 h.

[0014] Preferably, the culture is liquid culture and the obtained culture fluid OD600 It is 0.5-0.9.

[0015] The fourth aspect of the present invention provides the use of the above-mentioned Brevibacillus beautifulus, the above-mentioned bacterial agent or the bacterial agent prepared by the above-mentioned preparation method in at least one of preventing and controlling plant diseases, secreting auxins and synthesizing extracellular polysaccharides.

[0016] Preferably, the controlling of plant diseases is inhibiting plant pathogens and / or inhibiting plant diseases.

[0017] Preferably, the plant pathogen is Phytophthora nicotianae; the plant disease is a plant disease mediated by Phytophthora nicotianae, more preferably black shank disease.

[0018] Preferably, the plant is a crop of the Solanaceae family, more preferably tobacco.

[0019] A fifth aspect of the present invention provides a method for preventing and controlling plant diseases, the method comprising: using the above-mentioned Bacillus brevis, the above-mentioned bacterial agent or the bacterial agent prepared by the above-mentioned preparation method to water plant plants.

[0020] Preferably, the plant is a crop of the Solanaceae family, more preferably tobacco.

[0021] Through the above technical solution, the beneficial effects of the present invention are:

[0022] The beautiful Brevibacillus provided by the present invention not only has a high inhibition rate against plant pathogens (especially tobacco phytophthora), can effectively antagonize plant diseases (especially black shank disease) caused by plant pathogens, but also has a strong ability to produce longin and extracellular polysaccharides; the beautiful Brevibacillus has the effects of promoting growth and preventing diseases. The present invention proposes for the first time that beautiful Brevibacillus can be used in the prevention and treatment of tobacco black shank disease, and has good disease resistance.

[0023] The beautiful Brevibacillus provided by the present invention is highly efficient and safe, does not cause environmental pollution problems, has simple culture conditions, and is suitable for industrial production and sustainable development needs.

[0024] Other features and advantages of the present invention will be described in detail in the following detailed description.

[0025] Biological Deposit

[0026] The strain SCTL-8 provided by the present invention is Brevibacillus formosus, and was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101; the abbreviation of the depository is CGMCC) on November 4, 2024, with a deposit number of CGMCC No. 32491. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a characteristic diagram of the colony morphology of the strain SCTL-8 in Example 1 on NA medium;

[0028] Figure 2 is the 16S rDNA phylogenetic tree of strain SCTL-8 in Example 1;

[0029] Figure 3 This is a diagram showing the effect of the strain SCTL-8 in Example 4 on inhibiting tobacco phytophthora;

[0030] Figure 4 This is the effect of SCTL-8 on preventing and treating tobacco black shank under potted conditions in Example 5. DETAILED DESCRIPTION

[0031] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0032] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0033] In a first aspect, the present invention provides a strain of Brevibacillus formosus, wherein the deposit number of the Brevibacillus formosus is CGMCC No.32491.

[0034] The beautiful Brevibacillus provided by the present invention is isolated from tobacco leaves at the cluster stage in Shunchang City, Fujian Province. The beautiful Brevibacillus can be isolated by a conventional method for isolating new strains in the art, such as a gradient dilution separation method.

[0035] The gradient dilution separation method may specifically include: collecting tobacco plant leaves, disinfecting the surface, washing in 70-80% by volume alcohol for 3-5s, and washing with sterile water for 2-4 times; placing the washed tobacco plant leaves in a sterilized mortar, adding an appropriate amount of sterile water to grind, using a pipette to draw 1-3mL as the stock solution, and then diluting with sterile water, the dilution multiples are 10 -1 and 10 -2 , take 80-120μL of the original solution and the diluted sample and evenly spread them on the enrichment medium, repeat 2-4 times with different gradients, and culture them at 20-35℃ for 2-3 days until single colonies grow; pick colonies with different shapes, sizes, colors, etc., number them and purify them for culture.

[0036] According to the present invention, the enrichment medium can be any medium that can enrich the strain. Exemplarily, the enrichment medium contains beef extract powder, peptone, NaCl and agar. Preferably, the enrichment medium contains 8-12 g / L of peptone, 2-4 g / L of beef extract powder, 3-7 g / L of NaCl and 15-20 g / L of agar.

[0037] The inventors of the present invention conducted morphological observation and molecular biological identification on the enriched and purified strain numbered SCTL-8. The results showed that the strain was Brevibacillus formosus and was deposited in the General Microbiology Center of China National Microbiological Culture Collection on November 4, 2024, with the deposit number CGMCC No. 32491.

[0038] The beautiful Brevibacillus provided by the present invention can produce a large amount of cells and / or metabolites of beautiful Brevibacillus after fermentation culture. The present invention has no particular limitation on the fermentation culture method, as long as the beautiful Brevibacillus can be proliferated in large quantities by the fermentation culture method. For example, beautiful Brevibacillus can be inoculated into a liquid culture medium, and a fermentation broth is obtained by shaking culture at a temperature of 20-35°C and a rotation speed of 150-200rpm. Among them, the liquid culture medium can be a culture medium conventionally used in the art, for example, it can contain beef extract, peptone and NaCl; preferably, the liquid culture medium contains beef extract 3-5g / L, peptone 8-12g / L and NaCl 3-5g / L.

[0039] In a second aspect, the present invention provides a bacterial agent, which contains the above-mentioned Brevibacillus beautifulus.

[0040] According to the present invention, the bacterial agent preferably contains the cells and / or metabolites of the beautiful Brevibacillus. In the present invention, the term "metabolite" refers to the metabolites (including intracellular metabolites and / or extracellular metabolites) produced by the beautiful Brevibacillus during fermentation or cultivation. Preferably, the metabolites are extracellular polysaccharides and / or auxins.

[0041] The beautiful Brevibacillus provided by the present invention has a strong ability to produce auxin and extracellular polysaccharides. The auxin has the functions of regulating the growth rate of the stem, inhibiting lateral buds, promoting rooting, etc., and the extracellular polysaccharides can promote the growth and prevent diseases of plants.

[0042] According to the present invention, there is no particular limitation on the dosage form of the bacterial agent. It can be prepared into different dosage forms according to different intended uses, and corresponding excipients (excipients) and other ingredients can be added. For example, the bacterial agent can be a liquid bacterial agent (for example, a dilution of bacterial cells and / or metabolites) and / or a solid bacterial agent (for example, the bacterial cells of Brevibacillus beautifulis), preferably a liquid bacterial agent. Among them, what kind of excipients to add to what dosage form of the bacterial agent is well known to those skilled in the art, and will not be described in detail here.

[0043] In the present invention, there is no particular restriction on the concentration of Brevibacillus in the bacterial agent, and a specific selection can be made according to specific circumstances. Preferably, the liquid bacterial agent is a culture solution of Brevibacillus, that is, Brevibacillus is directly used as a bacterial agent after being cultured in a liquid culture medium; more preferably, the OD of the culture solution is 600 It is 0.5-0.9, specifically can be 0.5, 0.6, 0.7, 0.8, 0.9, or any value between the above two values.

[0044] The bacterial agent provided in the present invention can be prepared by any conventional method in the art.

[0045] The third aspect of the present invention provides a method for preparing a bacterial agent, the preparation method comprising: culturing the above-mentioned Brevibacillus beautifulus.

[0046] The Brevibacillus beautifule provided by the present invention can produce a large number of live Brevibacillus beautifule bacteria and their metabolites after cultivation. The present invention has no particular limitation on the cultivation method, as long as the Brevibacillus beautifule can be proliferated in large quantities by the cultivation method.

[0047] According to the present invention, the Brevibacillus beautifuli can be directly cultured, or the Brevibacillus beautifuli can be activated before the culture is carried out. Preferably, the Brevibacillus beautifuli is first activated, and then the activated bacteria are inoculated into the culture medium for the culture.

[0048] The activation may be performed in any manner. Preferably, the activation process comprises: streaking Bacillus brevis into an activation medium for activation culture, then inoculating into a seed medium for seed culture to obtain a seed solution, and inoculating the seed solution into the culture medium for the culture.

[0049] In the present invention, the activation culture medium and the seed culture medium can be culture media conventionally used in the art. Further preferably, the components of the activation culture medium contain beef extract, peptone, NaCl and agar, and specifically can contain 3-5 g / L beef extract, 8-12 g / L peptone, 3-5 g / L NaCl and 15-20 g / L agar; the components of the seed culture medium contain beef extract, peptone and NaCl, and specifically can contain 3-5 g / L beef extract, 8-12 g / L peptone and 3-5 g / L NaCl.

[0050] In the present invention, the temperature, time and other conditions of the activation culture and seed culture can be selected according to the growth requirements of Bacillus brevis. Preferably, the conditions of the activation culture include: a temperature of 20-35°C and a time of 10-20h; the conditions of the seed culture include: a temperature of 20-35°C, a rotation speed of 150-200rpm, and a time of 10-20h.

[0051] In the present invention, the culture medium can be a fermentation medium conventionally used in the art, for example, it can contain beef extract, peptone and NaCl, more preferably contains 3-5 g / L beef extract, 8-12 g / L peptone and 3-5 g / L NaCl.

[0052] In the present invention, the inoculation amount, temperature, rotation speed, time and other conditions of the culture can be selected according to the growth requirements of beautiful Brevibacillus. Preferably, the conditions of the culture include: the inoculation amount is 0.5-2 volume % (volume ratio of seed liquid to culture medium), which can be 0.5 volume %, 1 volume %, 1.5 volume %, 2 volume %, or any value between the above two values; the temperature is 20-35°C, which can be 20°C, 23°C, 26°C, 29°C, 32°C, 35°C, or any value between the above two values; the rotation speed is 150-200rpm, which can be 150rpm, 160rpm, 170rpm, 180rpm, 190rpm, 200rpm, or any value between the above two values; the time is 20-75h, which can be 20h, 30h, 40h, 50h, 60h, 70h, 75h, or any value between the above two values. Under this preferred condition, it is conducive to promoting the growth and proliferation of beautiful Brevibacillus.

[0053] According to the present invention, preferably, by controlling the temperature, rotation speed, time and other conditions of the culture, OD 600 The culture solution is 0.5-0.9 and can be used directly as a bacterial agent.

[0054] In addition, the cells and metabolites of Brevibacillus beautifulus in the culture solution obtained by the above-mentioned culture can also be further separated. There is no particular limitation on the separation method, as long as the cells and metabolites can be enriched from the culture solution. Preferably, the preparation method also includes: solid-liquid separation of the culture solution obtained by the above-mentioned culture to obtain cells, and mixing the cells with water to obtain a suspension as a bacterial agent. Further preferably, the OD of the suspension is 600 It is 0.2-0.4.

[0055] In the present invention, the water in the bacterial suspension is sterile water; solid-liquid separation can be performed by any separation method, specifically centrifugation or filtration, preferably centrifugation. Exemplarily, the centrifugation conditions include: a rotation speed of 5000-7000 rpm and a time of 2-6 min.

[0056] The beautiful Brevibacillus provided by the present invention not only has a high antibacterial rate against plant pathogens (especially tobacco phytophthora), can effectively antagonize plant diseases (especially black leg disease) caused by plant pathogens, but also has a strong ability to produce auxin and extracellular polysaccharides. Among them, the auxin is specifically 3-indoleacetic acid; in order to verify the ability of beautiful Brevibacillus to secrete auxin, 0.1-0.2g / L tryptophan can be added to the culture medium of beautiful Brevibacillus, and it is found that the beautiful Brevibacillus can efficiently synthesize auxin using tryptophan as a substrate.

[0057] Based on this, in a fourth aspect, the present invention provides the use of the above-mentioned Brevibacillus beautifulus, the above-mentioned bacterial agent or the bacterial agent prepared by the above-mentioned preparation method in at least one of preventing and controlling plant diseases, secreting auxins and synthesizing extracellular polysaccharides.

[0058] According to the present invention, preferably, the plant disease control is to inhibit plant pathogens and / or inhibit plant diseases. Further preferably, the plant pathogen is Phytophthora nicotianae; the plant disease is a plant disease mediated by Phytophthora nicotianae, more preferably black shank, such as tobacco black shank.

[0059] According to the present invention, the plant can be any crop, preferably, the plant is a Solanaceae crop, more preferably tobacco (such as Nicotiana tabacum).

[0060] In a fifth aspect, the present invention provides a method for preventing and controlling plant diseases, the method comprising: using the above-mentioned Bacillus brevis, the above-mentioned bacterial agent or the bacterial agent prepared by the above-mentioned preparation method to water plant plants.

[0061] According to the present invention, the plant can be any crop, preferably, the plant is a Solanaceae crop, more preferably tobacco (such as Nicotiana tabacum).

[0062] According to the present invention, the irrigation treatment process comprises: after transplanting, 15-25 mL of a suspension formed by the cells of the beautiful Brevibacillus and water is irrigated to the root of each plant. More preferably, the OD of the suspension is 600 In this preferred embodiment, it is beneficial to improve the effect of Brevibacillus beautifulis in preventing and controlling plant diseases and better promote plant growth.

[0063] The present invention will be described in detail below through examples.

[0064] In the following examples, the components of NB medium are: 3 g / L beef extract, 10 g / L peptone and 5 g / L NaCl;

[0065] The components of NA medium are: beef extract 3g / L, peptone 10g / L, NaCl 5g / L and agar 20g / L;

[0066] The components of TSA plate medium are: tryptone 10g / L, beef extract powder 3g / L, sodium chloride 5g / L, agar powder 20g / L;

[0067] The components of oatmeal culture medium are: 30g / L oatmeal and 15g / L agar.

[0068] Tobacco Xiaohuangjin 1025 seeds were provided by the Tobacco Research Institute of the Chinese Academy of Agricultural Sciences, and tobacco phytophthora was provided by the Marine Agriculture Research Center of the Tobacco Research Institute of the Chinese Academy of Agricultural Sciences. Unless otherwise specified, other raw materials and reagents were commercially available.

[0069] Example 1 Isolation and identification of strains

[0070] (1) Strain isolation

[0071] Tobacco leaves at the cluster stage were collected from Shunchang City, Fujian Province, and then surface disinfected. They were washed in 75% alcohol for 4 seconds and then washed with sterile water for 3 times. The washed tobacco plant leaves were placed in a sterilized mortar, ground with an appropriate amount of sterile water, and about 2 mL was taken as the stock solution with a pipette, and then diluted with sterile water by 10 times. -1 and 10 -2, take 100 μL of the original solution and the diluted sample and spread them evenly on the TSA plate, repeat three times with different gradients, and culture them at 28°C for 2-3 days until single colonies grow; pick colonies with different shapes, sizes, colors, etc., number them, and purify and culture them to obtain the pure culture strain SCTL-8.

[0072] (2) Morphological observation

[0073] The strain numbered SCTL-8 was inoculated into NA solid medium and cultured at 28°C for 2 days. Figure 1 As shown, the colony morphology of strain SCTL-8 on NA medium is irregular, light yellow, opaque, flat and smooth.

[0074] (3) Molecular identification

[0075] The 16S rDNA sequence of strain SCTL-8 was analyzed. A single colony of SCTL-8 was picked, and the strain DNA was extracted using a bacterial genome extraction kit (produced by Nanjing Novozyme Biotechnology Co., Ltd.); the strain DNA was used as a template, and the primer pair (nucleotide sequence is shown in SEQ ID NO.1 and SEQ ID NO.2) was used to amplify its 16S rDNA sequence; the PCR system was 3μL 10×buffer; 3μL dNTP; 0.3μL primer; 0.15μL rTaq; 1μL DNA template; 22.25μL ddH2O; the PCR reaction conditions were pre-denaturation at 94℃ for 5min; 94℃ for 1min, 55℃ for 1min, 72℃ for 1.5min, 30 cycles; extension at 72℃ for 10min.

[0076] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 1),

[0077] 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO. 2);

[0078] The length of the partial fragment of 16S rDNA sequence of strain SCTL-8 obtained by sequencing was 671 bp, as specifically shown in SEQ ID NO.3.

[0079] 5’-CCCTAGCGGCGGACGGGTGAGTAACACGTAGGCAACCTGCCTCTCAGACTGGGATAACATAGGGAAACTTATGCTAATACCGGATAGGTTTTTGGATCGCATGATCCGAAAAGAAAAGATGGCTTCGGCTATCACTGGGAGATGGGCCTGCGGCGCATTAGCTAGTTGGTGGGGTAACGGCCTACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATTTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAACGATGAAGGTCTTCGGATTGTAAAGTTCTGTTGTTAGGGACGAATAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTGACGAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGCGCGCAGGCGGCTATGTAAGTCTGGTGTTAAAGCCCGGGGCTCAACCCCGGTTCGCATCGGAAACTGTGTAGCTTGAGTGCAGAAGAGGAAAGCGGTATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTTTCTGGTCTGTAACTGACGCT-3’(SEQ ID NO.3).

[0080] Sixteen standard strain sequences were obtained from the NCBI (GenBank) database, and the 16S rDNA sequences of the isolated strains and reference strains were analyzed using MEGA 11 to construct a Neighbor-joining phylogenetic tree, as Figure 2As shown, it can be seen that the strain SCTL-8 provided by the present invention is most closely related to Brevibacillus formosus. Combined with the colony characteristics of the strain on the plate, the isolated strain SCTL-8 of the present invention was identified as Brevibacillus formosus, named Brevibacillus formosus SCTL-8, and was deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration on November 4, 2024, with a deposit number of CGMCC No.32491.

[0081] Example 2

[0082] In this example, the ability of strain SCTL-8 to secrete auxin was determined.

[0083] (1) Strain activation: The strain SCTL-8 obtained in Example 1 was streaked onto NA solid culture medium and activated at 28°C to obtain SCTL-8 colonies; then a single colony was picked and inoculated into NB liquid culture medium, and cultured overnight at 28°C and 180 rpm to obtain SCTL-8 seed solution.

[0084] (2) Detection of growth hormone secretion ability

[0085] The content of auxin was detected by the Salkowski colorimetric method. The colorimetric solution was prepared by adding 50 mL of 35 wt% HCl solution to 1 mL of 0.5 M FeCl3 solution;

[0086] Preparation of standard curve solution: prepare auxin (3-indoleacetic acid) solutions with concentrations of 0, 20, 40, 60, 80, and 100 mg / L.

[0087] 50 mL of NB medium (0.1 g / L tryptophan added exogenously) was placed in a 150 mL Erlenmeyer flask and sterilized at 121° C. for 20 min. 500 μL of the SCTL-8 seed solution obtained in step (1) was inoculated into the Erlenmeyer flask (inoculation amount was 1 volume %), and three replicates were set up. The NB medium (0.1 g / L tryptophan added exogenously) without the strain was used as a control. The culture was placed in a 28° C. incubator and shaken at 180 rpm for 3 days to obtain a culture solution. The culture solution was centrifuged at 8000 rpm and 4° C. for 10 min, and the supernatant was taken for later use.

[0088] Take 1 mL of the supernatant and the standard curve solution respectively, add 1 mL of the colorimetric solution and let it stand in the dark for 30 minutes, then quickly measure the absorbance at a wavelength of 530 nm using a spectrophotometer; take the absorbance of the standard curve solution as y and the concentration of the standard curve solution as x, and draw a standard curve of y=4.3995x+0.2244, R 2is 0.9803;

[0089] The absorbance of the supernatant at a wavelength of 530 nm was put into the standard curve to calculate the content of auxin in the supernatant. It was found that the concentration of auxin produced in the supernatant of strain SCTL-8 was 67.17±0.003 mg / L, and there was no auxin in the supernatant of the control.

[0090] Example 3 Determination of the ability of strain SCTL-8 to secrete extracellular polysaccharides

[0091] Take 500 μL of the SCTL-8 seed solution obtained in step (1) of Example 2 and inoculate it into 50 mL of NB liquid culture medium (inoculation amount is 1 volume %), set up three repeated parallel experiments, place it in a 28°C incubator and shake culture at 180 rpm for 3 days to obtain culture solution, centrifuge the culture solution at 10000 rpm for 10 min, and take the supernatant for later use.

[0092] Preparation of standard curve solution: Dissolve 0.1g glucose in distilled water and dilute to 100mL. Accurately pipette 10ml and dilute to 100ml again to obtain a mother solution with a final concentration of 0.1g / L. Continue to dilute to prepare glucose solutions with concentrations of 0, 0.02, 0.04, 0.06, and 0.08g / L.

[0093] Take 1 mL of the supernatant of strain SCTL-8 and the standard curve solution respectively, accurately add 1 mL of 5% by weight phenol solution and 5 mL of concentrated sulfuric acid, shake well immediately, and measure the absorbance at 485 nm with a spectrophotometer; take the absorbance of the standard curve solution as y and the glucose concentration of the standard curve solution as x, and draw a standard curve of y=3.5135x+0.1794, R 2 It is 0.9918; among them, the absorbance of SCTL-8 supernatant could not be detected without being treated with phenol and concentrated sulfuric acid.

[0094] The absorbance of the supernatant at a wavelength of 485 nm was put into the standard curve to calculate the content of extracellular polysaccharides in the supernatant. It was found that the concentration of extracellular polysaccharides produced in the supernatant of strain SCTL-8 was 0.64±0.029 g / L.

[0095] Example 4 Inhibitory ability of strain SCTL-8 against tobacco phytophthora

[0096] The tobacco phytophthora was inoculated into an oat culture medium and cultured for 3 days; the SCTL-8 seed solution obtained in step (1) of Example 2 was inoculated into a NB liquid culture medium (the inoculation amount was 1 volume %), and the SCTL-8 bacterial solution was obtained by shaking the medium at a temperature of 28° C. and a rotation speed of 180 rpm for 24 hours;

[0097] A 5 mm bacterial cake was taken from the edge of the tobacco phytophthora colony and inoculated into the center of the oat solid culture medium. 3 μL of SCTL-8 bacterial solution was added 2.5 cm from the edge of the plate. Sterile water was used instead of the bacterial solution as the control group. After culturing in a 28°C incubator for 3 days, the antagonistic activity was observed and the inhibition rate was calculated by the following formula. The inhibition effect was recorded.

[0098] Inhibition rate = (pathogenic colony radius of the control group - pathogenic colony radius of the treatment group) / pathogenic colony radius of the control group × 100%;

[0099] The experimental results are as follows Figure 3 As shown, the radius of the pathogen colony in the control group was 4.2 cm, and the radius of the pathogen colony in the treatment group was 1.2 cm. Calculation showed that the inhibition rate of strain SCTL-8 against tobacco phytophthora reached 71.43%.

[0100] Example 5 Disease prevention effect of strain SCTL-8

[0101] (1) Preparation of tobacco phytophthora

[0102] Boil the hulled yellow millet with distilled water until 2 / 3 of the grains crack and turn white. Take out and drain the water, then divide into 500mL conical flasks, fill each bottle with about 200mL, and sterilize at 121℃ for 15min to obtain millet culture medium; transfer tobacco phytophthora to oat culture medium and culture at 28℃ for 3d; use a 5mm puncher to punch out bacterial cakes at the edge of the colony and insert them into the sterilized millet culture medium, 5-6 bacterial cakes per bottle, and evenly spread them on the surface of the millet in the bottle; culture at 25℃ for 14d until the mycelium fills the culture medium to obtain tobacco phytophthora millet.

[0103] (2) Biocontrol pot experiment of Brevibacillus SCTL-8

[0104] The SCTL-8 seed solution obtained in step (1) of Example 2 was inoculated into NB liquid culture medium (inoculation amount was 1 volume %), and cultured at 28°C and 180 rpm for 24 h to obtain SCTL-8 bacterial solution. The SCTL-8 bacterial solution was centrifuged at 4°C and 6000 rpm for 5 min to obtain SCTL-8 bacterial cells, which were then resuspended in sterile water to obtain an OD 600 = 0.3 of SCTL-8 bacterial suspension;

[0105] Sow tobacco seeds (small gold) into seedling trays, select tobacco seedlings with uniform growth after emergence, and culture for about 30 days; weigh 100g of substrate soil, add 2g of tobacco phytophthora fungus obtained in step (1) to each pot, stir evenly and place in a 9×9×8cm plastic flowerpot; take out the tobacco seedlings, carefully shake off the soil at the roots, retain the roots of about 5cm long, clean the root substrate, and transplant them into the flowerpot; water along the roots of the transplanted tobacco seedlings with 20mL OD 600=0.3 of SCTL-8 bacterial suspension, set up 3 replicates, each replicate 10 pots; the control group (CK) was irrigated with sterile water.

[0106] The transplanted tobacco seedlings were placed at a temperature of 28°C and a humidity of 70%. From the 4th day, the disease grade and incidence rate were counted, and the disease index and disease prevention effect were calculated. The results are shown in Tables 1 and Figure 4 .

[0107] The classification standards for tobacco black shank disease are:

[0108] Level 0, the whole plant is disease-free;

[0109] Level 1: The stem lesions do not exceed 1 / 3 of the stem circumference, or less than 1 / 3 of the leaves wilt;

[0110] Level 3: The lesions on the stems surround 1 / 2 to 2 / 3 of the stem circumference, or 1 / 3 to 1 / 2 of the leaves are slightly wilted;

[0111] Level 5: The lesions on the stem exceed 1 / 2 of the stem circumference, but do not completely surround the stem circumference, or 1 / 2 to 2 / 3 of the leaves wilt;

[0112] Level 7: The stem lesions completely surround the stem, or more than 2 / 3 of the leaves wilt;

[0113] Level 9: The diseased plants are basically dead.

[0114] Incidence rate = (number of diseased plants / total number of inoculated plants) × 100%;

[0115] Disease index = [Σ(number of diseased plants × corresponding level) / (total number of plants × highest level)] × 100%;

[0116] Control effect = [(control disease index - treatment disease index) / control disease index] × 100%.

[0117] Table 1

[0118] Group Incidence % Disease index% SCTL-8 treated group 31.11(±0.03) 26.17(±1.26) Control group 100(±0) 89.63(±7.55)

[0119] The results showed that the use of SCTL-8 bacterial solution could significantly reduce the infection of tobacco with Phytophthora nicotianae to form black shank disease (P<0.01), and the control effect reached 70.80%.

[0120] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A Brevibacillus beautifulis, characterized in that The deposit number of the Brevibacillus formosus is CGMCC No.32491.

2. A bacterial agent, characterized in that The bacterial agent contains the beautiful Brevibacillus described in claim 1.

3. The bacterial agent according to claim 2, characterized in that The bacterial agent contains the bacterial cells and / or metabolites of the beautiful Brevibacillus; Preferably, the metabolites are exopolysaccharides and / or auxins.

4. The bacterial agent according to claim 2 or 3, characterized in that: The bacterial agent is a liquid bacterial agent and / or a solid bacterial agent, preferably a liquid bacterial agent; Preferably, the liquid bacterial agent is a culture solution of Bacillus brevis, and more preferably, the OD of the culture solution is 600 It is 0.5-0.

9.

5. A method for preparing a bacterial agent, characterized in that: The preparation method comprises: culturing the beautiful Brevibacillus described in claim 1.

6. The preparation method according to claim 5, characterized in that: The culture conditions include: inoculation volume of 0.5-2% by volume, temperature of 20-35° C., rotation speed of 150-200 rpm, and time of 20-75 h; Preferably, the culture is liquid culture and the obtained culture fluid OD 600 It is 0.5-0.

9.

7. Use of the beautiful Brevibacillus according to claim 1, the bacterial agent according to any one of claims 2 to 4, or the bacterial agent prepared by the preparation method according to claim 5 or 6 in at least one of controlling plant diseases, secreting auxins, and synthesizing extracellular polysaccharides.

8. The use according to claim 7, characterized in that: The plant disease prevention and control is to inhibit plant pathogens and / or inhibit plant diseases; Preferably, the plant pathogen is Phytophthora nicotianae; the plant disease is a plant disease caused by Phytophthora nicotianae, more preferably black leg disease; Preferably, the plant is a crop of the Solanaceae family, more preferably tobacco.

9. A method for preventing and controlling plant diseases, characterized in that: The method comprises: watering plants with the beautiful Brevibacillus described in claim 1, the bacterial agent described in any one of claims 2 to 4, or the bacterial agent prepared by the preparation method described in claim 5 or 6.

10. The method according to claim 9, characterized in that The plant is a crop of the Solanaceae family, more preferably tobacco.

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