Bacillus tequila and its application, microbial preparation and biological fertilizer

Abstract

The present application relates to a Bacillus tekila with a deposit number of CGMCC No. 31237 and its application in preventing and controlling wheat root rot, soil phosphorus solubilization, biological nitrogen fixation, iron carrier production, and salinized soil improvement. A microbial preparation and a biological fertilizer are also provided, both of which include live bacteria of the Bacillus tekila. The Bacillus tekila contains a rich secondary metabolite gene cluster, can antagonize wheat root rot Helicoverpa rapae, prevent and control wheat root rot, reduce the incidence rate, and promote wheat growth.

Description

Technical Field

[0001] The present application relates to the field of microbial technology, and in particular to a tequila bacillus and its application, a microbial preparation and a biological fertilizer. Background Art

[0002] Wheat is one of the most widely planted staple crops. Wheat planting and disease control are of great significance to national food security. Wheat root rot is a widespread soil-borne fungal disease that causes slow growth, root rot, seedling withering, stem base withering, leaf withering and ear withering of wheat, seriously affecting the emergence and growth of wheat seedlings. According to investigations, the main pathogen of wheat root rot is Helicoverpa spp.

[0003] How to inhibit the growth of wheat root rot fungus and protect the good development and growth of wheat has become an urgent problem to be solved by technical personnel in this field. Summary of the invention

[0004] In order to inhibit the growth of wheat root rot Helminthosporium and protect the good development and growth of wheat, the present application provides a tekila bacillus and its application, a microbial preparation and a biofertilizer.

[0005] To achieve the purpose of the present invention, a tequila bacillus is provided, wherein the tequila bacillus ( Bacilus tequilensis ) was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 9, 2024, with the deposit number CGMCC No. 31237.

[0006] According to another aspect of the present application, a use of Bacillus tergillosis in preventing and controlling wheat root rot is provided.

[0007] According to another aspect of the present application, there is provided a use of Bacillus tergillosis in soil phosphate solubilization.

[0008] According to another aspect of the present application, a use of Bacillus tequila in biological nitrogen fixation is provided.

[0009] According to another aspect of the present application, a use of Bacillus terequila in producing siderophore is provided.

[0010] According to another aspect of the present application, a use of Bacillus terequina in promoting plant growth is provided.

[0011] According to another aspect of the present application, a use of Bacillus terequila in improving salinized soil is provided.

[0012] According to another aspect of the present application, a microbial preparation is provided, which is the live bacterial agent of the aforementioned Bacillus terequila.

[0013] According to another aspect of the present application, a biofertilizer is provided, the biofertilizer comprising the live bacteria of the aforementioned Bacillus tequila.

[0014] The Bacillus tekila provided in the present application contains 8 secondary metabolite gene clusters, has the potential to produce rich antibiotics, and can also antagonize the pathogenic bacteria of wheat root rot - Helicobacter pylori, inhibit the growth of Helicobacter pylori, thereby preventing and controlling wheat root rot, reducing the incidence of wheat root rot, and thus protecting wheat for good growth and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 A single colony growth diagram of Bacillus tequila on LB medium provided by the present application is shown;

[0016] Figure 2 The phylogenetic tree of Bacillus tequila based on 16S rDNA sequence is shown;

[0017] Figure 3 A graph showing the growth of strains of Bacillus tequila and Helminthosporium spp. in a plate confrontation experiment;

[0018] Figure 4 A graph showing the test results of Bacillus tequila inhibiting the spore germination of Helicoverpa spp.;

[0019] Figure 5 The wheat growth diagram of Example 5 is shown;

[0020] Figure 6 The wheat root development diagram of Example 5 is shown as follows: Figure 5 A partial enlarged view of

[0021] Figure 7 From left to right, the growth of Bacillus tequila on Montgena inorganic phosphate medium (phosphate solubilization), Ashby medium (nitrogen fixation), and CAS medium (siderophore production) are shown;

[0022] Figure 8 A graph showing the test results of Bacillus tequila for tolerance to sodium chloride;

[0023] Fig. 9 A graph showing the test results of Bacillus tequila for tolerance to sodium sulfate;

[0024] Fig.10 The wheat growth diagram of Example 9 is shown;

[0025] Fig.11 A statistical diagram of the aboveground plant height of wheat in Example 9 is shown;

[0026] Fig.12A graph showing the test results of Bacillus tequila's tolerance to different concentrations of urea;

[0027] Fig.13 A graph showing the test results of Bacillus tequila's tolerance to different concentrations of phosphorus fertilizer;

[0028] Fig.14 A graph showing the test results of Bacillus tequila's tolerance to different concentrations of potassium fertilizer;

[0029] Fig.15 A graph showing the test results of the tolerance and survival rate of Bacillus tequila to the basic fertilizer of the examples of the present application. DETAILED DESCRIPTION

[0030] Various exemplary embodiments, features and aspects of the present application will be described in detail below with reference to the accompanying drawings. In order to better illustrate the present application, numerous specific details are provided in the following specific embodiments. It should be understood by those skilled in the art that the present application can also be implemented without certain specific details. In some instances, methods and means well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present application.

[0031] Based on the above problems, the present application provides a tequila bacillus. The tequila bacillus was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on July 9, 2024, with a deposit number of CGMCC No. 31237 and a classification name of: tequila bacillus Bacillus tequilensis .

[0032] In 2023, the applicant collected rhizosphere soil samples from field crops in Yongning County, Yinchuan City, Ningxia Hui Autonomous Region. Take 10 grams of soil sample and add it to 90 ml of sterile water. Oscillate on a shaker at 150 revolutions per minute for 30 minutes to disperse the microorganisms in the soil in sterile water. Use sterile water to dilute the obtained soil suspension by 10 times to obtain a gradient series of dilutions. Take 100µL of the dilution and spread it on the LB plate. After culturing in a 28°C constant temperature incubator for 48 hours, select a single colony of Microbacterium parvum and purify it on a new LB plate until the pure strain is obtained, which is the Tekila Bacillus strain provided in this application. Preserve with sterile glycerol at a final concentration of 20% and store it in a -80°C refrigerator for standby use.

[0033] The LB medium used in the present application includes: 5 g / L yeast powder, 10 g / L peptone and 10 g / L NaCl, the same below.

[0034] The single colony of Bacillus tequila Figure 1 As shown, on the LB plate, it is white / off-white, the colonies are irregular in shape, the edges are burr-like, and the surface of the colonies is dry and granular.

[0035] The applicant conducted molecular identification on the purified single colony and confirmed that it was Bacillus tequila, and named it according to the molecular identification results. Bacilus tequilensis FD15131, hereinafter referred to as FD15131 strain, or Bacillus tequila FD15131 strain. Specifically:

[0036] The genomic DNA of the purified single colony was extracted and collected, and the 16S rDNA was amplified using the universal bacterial identification primers 27 F and 1492 R, and the sequence was sent to a third-party company for sequencing.

[0037] The 16S rDNA sequence of the strain was obtained after forward and reverse sequencing and splicing as shown in SEQ ID NO. 1. The sequence was compared with the NCBI database by Blast, and a phylogenetic tree was further drawn as shown in Figure 2 As shown, this strain Bacilus tequilensis KCTC13622 is on the same branch and has the closest evolutionary distance, so the strain was identified as Bacillus tequila and named Bacilus tequilensis FD15131, translated into Chinese as Bacillus tequila FD15131.

[0038] The applicant sequenced the genome of the FD15131 strain and used the gene clusters in the whole genome to predict the functions of the FD15131 strain. It was found that the FD15131 strain can synthesize secondary metabolites such as spore-forming killing factors, antimicrobial peptide surfactant, antimicrobial active peptides, fengin, antimicrobial peptides, siderophores, pulcherriminic acid, bacitracin A and lysobactin, which can improve the survival rate of the FD15131 strain and thus better prevent and control wheat root rot.

[0039] Using whole genome sequencing and gene clusters, secondary metabolite analysis of the FD15131 strain was performed as shown in Example 1 and Table 1.

[0040] Embodiment 1:

[0041] Generally, the secondary metabolites produced by microorganisms have multiple biological activities such as anti-infection, anti-tumor, and immunomodulation, and are an important source for the development of new drugs. Secondary metabolites are generally controlled by multiple genes, and their encoding genes usually exist in clusters in the genome. This gene is called a secondary metabolite synthesis gene cluster. Usually, by analyzing the secondary metabolite data of the whole genome of the microorganism, the secondary metabolite gene cluster genes contained in the strain can be obtained to predict whether the strain has the ability to synthesize a certain secondary metabolite. Therefore, the applicant further sequenced the whole genome of FD15131 and used the secondary metabolite database antiSMASH 7.1.0 online tool to predict and analyze its secondary metabolite gene cluster.

[0042] The FD15131 genome was extracted and assembled using third-generation sequencing. FD15131 contains one chromosome and no plasmid. Its genome size is 4279769 bases, and the guanine (G) + cytosine (C) content is 43.57%.

[0043] Through online analysis, the gene cluster analysis results of the FD15131 genome are shown in Table 1. FD15131 contains 14 gene clusters, of which 8 gene clusters have a similarity of 100% with known gene clusters, and one gene cluster has a similarity of 82% with known gene clusters.

[0044] Table 1 Prediction results of secondary metabolite gene clusters of strain FD15131

[0045]

[0046] Sporulation killing factor synthesized by gene cluster 1 and subtilosin A synthesized by gene cluster 11 are natural products of the secptide class. Secptides are a class of natural ribosomal peptides with a variety of biological activities, such as antibacterial function. Subtilosin A is an antibacterial peptide with a very broad antibacterial spectrum, which has inhibitory effects on Listeria, Klebsiella pneumoniae, Staphylococcus aureus and Escherichia coli.

[0047] The antimicrobial peptides surfactin synthesized by gene cluster 2, bacillaene synthesized by gene cluster 4, fengycin synthesized by gene cluster 5, and bacillibactin synthesized by gene cluster 9 are non-ribosomal peptides. These small-molecule peptides have antibacterial and anti-tumor activities. Their synthesis pathway does not go through the ribosome pathway, but is catalyzed by non-ribosomal peptide synthetases. Their structures often contain a variety of "non-protein amino acids" and a variety of cyclic or branched structures, and are an important source of antibiotic drugs. Bacillaene can inhibit protein synthesis and has a wide spectrum of antibacterial effects. Fengycin has excellent antifungal ability, especially against filamentous fungi. Bacillibactin can inhibit the growth of pathogens.

[0048] The gene cluster 7 synthesized sublancin168 belongs to the glycoprotein class and is a new type of antimicrobial glycopeptide that can improve the survival rate of Bacillus in the environment.

[0049] Pulcherriminic acid, synthesized by gene cluster 10, is an iron-binding red pigment produced by bacteria and yeast. It can protect Bacillus from oxidative stress during biofilm formation, play a role in biocontrol microbial interactions, iron metabolism and stress response, and has potential application prospects.

[0050] Bacilysin synthesized by gene cluster 12 belongs to the other category and is one of the simplest peptide antibacterial substances synthesized by Bacillus. Its target 6-phosphoglucosamine synthetase is a key enzyme in the synthesis of microbial cell walls. It has a broad-spectrum antibacterial activity against both fungi and bacteria. Therefore, bacilysin has broad application prospects in agriculture and medicine.

[0051] It can be seen that the FD15131 strain contains a very rich synthetic gene cluster that produces antibiotic substances. Under certain conditions, the bacteria can produce a variety of antibacterial substances, which can protect and promote the growth and proliferation of Bacillus, enabling it to antagonize crop pathogens and prevent crop diseases. It has broad application prospects in agriculture.

[0052] In addition, among the multiple secondary metabolite gene clusters contained in FD15131, the gene sequences of 8 gene clusters have 100% similarity with known antibiotics. This indicates that the strain has strong activity in synthesizing antibiotics. This means that the whole genome of the FD15131 strain contains abundant synthetic gene clusters that produce antibiotic substances, which can produce antibiotics or precursors for preparing antibiotics. Antibiotics or precursors for preparing antibiotics can be obtained through simple extraction and purification, and have broad application prospects in the field of preparing antibiotics.

[0053] The applicant analyzed the functional properties of the strain and found that Bacillus tekila FD15131 can antagonize the pathogenic bacteria of wheat root rot, Helicobacterium tekila, and inhibit the growth of Helicobacterium tekila. The antagonistic and inhibitory properties of Bacillus tekila FD15131 are shown in Examples 3 and Figure 3 ; Example 4 and Figure 4 Embodiment 5, Figure 5 and Figure 6 .

[0054] Embodiment 2:

[0055] The purified Bacillus tequila FD15131 was inoculated into LB medium, shaken at 30°C and 180 rpm for 24 hours, centrifuged at 7000 rpm for 15 minutes at room temperature, the supernatant was discarded, the centrifugal precipitate was collected as the strain cells, and then the centrifugal precipitate was resuspended with sterile water to obtain a live bacterial suspension of the FD15131 strain, also called FD15131 spore suspension. Then the bacterial concentration (OD 600 ), adjust the amount of sterile water to change the bacterial concentration OD 600 The value of OD 600 The concentration of FD15131 spore suspension was 1.0.

[0056] Embodiment 3:

[0057] In the plate confrontation experiment, the wheat root rot flat umbilical helminth spore was inoculated in the center of the PDA medium solid plate. 5 μL of the FD15131 spore suspension of Example 2 was inoculated 1.5 cm away from the center of the plate and cultured at 30°C for 5 days. The culture results are shown in FIG. Figure 3 As shown, the FD15131 strain can significantly inhibit the growth of Helicoverpa spp., forming an obvious inhibition ring. This indicates that the FD15131 strain has the effect of antagonizing Helicoverpa spp., preventing and controlling wheat root rot, and reducing the incidence of wheat root rot.

[0058] The PDA medium used in this application includes: 200 g / L peeled potatoes (cut into small pieces and boiled for 20 minutes, then filtered through 6 layers of gauze to obtain the filtrate), 20 g / L glucose and 15 g / L agar.

[0059] Embodiment 4:

[0060] Verification of the inhibitory effect of FD15131 on Helminthosporium spp.

[0061] In this example, the applicant used a microscope to observe the two microorganisms to further observe the inhibitory effect of the FD15131 strain on Helicoverpa spp.

[0062] The fungal spore suspension was prepared by washing the spores of Helicoverpa graminis cultured for 7 days with sterile water.

[0063] In this embodiment, two treatment groups are set up, namely: a blank control group and a FD15131 strain treatment group. Among them:

[0064] Blank control group: Use concave glass slides to culture the fungus, and take 10 2 The fungal spores were placed in a concave glass slide and cultured at 30°C for 24 hours.

[0065] FD15131 strain treatment group: Use concave glass slides to culture the fungus, and take 10 2 fungal spores were placed in a concave glass slide, and 10 2 -10 3 The bacterial spores were placed in a concave slide and cultured at 30°C for 24 hours.

[0066] The germination of fungal spores in the two treatment groups was observed under an optical microscope. Figure 4 As shown in the figure, the spores of Helicoverpa spp. in the blank control group germinated and grew hyphae. The spores of Helicoverpa spp. in the FD15131 strain treatment group did not germinate, and the hyphae growth was significantly inhibited.

[0067] This shows that the FD15131 strain can inhibit the germination and growth of wheat root rot fungus, and can antagonize and inhibit wheat root rot fungus. It has great potential and broad prospects in reducing its infection of wheat and preventing and controlling wheat root rot diseases.

[0068] Embodiment 5:

[0069] Prepare diseased soil. Cultivate Helicospora spp., which can cause wheat root rot, on PDA medium for one week. Scrape off the mycelium and spores with a sterile scalpel, dissolve them in sterile water, and then evenly mix them into the soil sample to prepare diseased soil containing wheat root rot pathogens. Use the diseased soil to conduct a wheat pot experiment.

[0070] Three treatment groups were designed in the potted experiment: CK group (blank control group), Helicoverpa spp. treatment group, and Helicoverpa spp. + FD15131 strain treatment group.

[0071] The planting soil used in the blank control group was the original soil sample that did not contain the pathogenic bacteria of wheat root rot. The planting soil used in the other two groups was the diseased soil prepared previously. All wheat seeds (of the same batch and of the same quality) were surface disinfected and then subjected to the same germination treatment. The next day, the white wheat seeds were sown in the soil of the flower pot. Five wheat seeds were sown in each group, that is, five replicates were set up for each group.

[0072] The blank control group was irrigated with 10 mL of sterile water per pot each time on the day of sowing, 7 days after sowing, and 14 days after sowing, for a total of three times. On the day of sowing, 7 days after sowing, and 14 days after sowing, while the blank control group was irrigated with sterile water, the wheat root soil was irrigated with 10 mL of sterile water per pot each time in the wheat root soil treated with the wheat root soil treated with the wheat root soil treated with the FD15131 strain, and 10 mL of OD in Example 2 was irrigated per pot each time in the wheat root soil treated with the wheat root soil treated with the FD15131 strain. 600 The application time, frequency and dosage of FD15131 spore suspension were consistent with those of the blank control group. Except for the different soil and irrigation solution, the planting environment, cultivation conditions and light conditions of the three treatment groups were consistent.

[0073] The wheat growth of the three treatment groups is as follows Figure 5 As shown. The growth of wheat in the blank control group was better than that in the group treated with Helminthosporium spp. + FD15131 strain, and the growth of wheat in the group treated with Helminthosporium spp. + FD15131 strain was better than that in the group treated with Helminthosporium spp. The wheat leaves in the group treated with Helminthosporium spp. turned yellow.

[0074] Pull out the wheat Figure 5 and Figure 6 It was found that the wheat roots in the blank control group were well developed, and there was no blackening and rot at the base of the stem (the enlarged picture of the base of the stem is shown in Figure 6 In addition, in the wheat root rot group, the wheat roots wilted, the root system was shallow, the stem base turned black, and some of them rotted (the enlarged picture of the stem base is shown in Figure 6 The wheat root rot group (the enlarged picture of the stem base is shown in the middle picture) was diagnosed with root rot. Figure 6 As shown in the bottom layer of pictures), the root system development is not as good as the blank control group, with shallow roots, but it is significantly better than the group treated with Helicoverpa spp., with thicker roots and deeper roots. In addition, the stem base of the group treated with Helicoverpa spp. + FD15131 strain only showed slight yellowing, without blackening or rot.

[0075] This shows that the FD15131 strain can antagonize the wheat root rot fungus, inhibit the infection of wheat root rot fungus to wheat, prevent and control the wheat root rot caused by its infection of wheat, and reduce the incidence of wheat root rot.

[0076] In addition, the functional analysis of the FD15131 strain by the applicant also found that the FD15131 strain has a phosphate solubilizing effect (see Examples 6 and Figure 7 ), nitrogen fixation effect (see Example 7 and Figure 7 ), and the siderophore production effect (see Examples 8 and Figure 7 ). Specifically:

[0077] Embodiment 6:

[0078] After activating and culturing the FD15131 strain in LB liquid medium for 24 hours using a test tube, the bacterial solution was aspirated and 2 μL of the bacterial solution was inoculated on the Montana inorganic phosphate medium.

[0079] After the bacterial solution is absorbed by the plate, place the plate upside down in a 28℃ constant temperature incubator away from light for incubation. Observe the results after 72 hours. Figure 7 shown.

[0080] The plate of the Montkina inorganic phosphate medium is a screening plate for phosphate-solubilizing bacteria. If the FD15131 strain can grow on the Montkina inorganic phosphate medium, it means that the strain has the characteristics of phosphate solubilization. If a transparent circle appears around the strain, it means that the strain has a strong ability to solubilize phosphate. Figure 7 As shown in the left picture in the middle (with inorganic phosphorus on top), the strain grew well and produced an obvious transparent zone of phosphorus dissolution on the inorganic phosphorus medium of Montkina. This shows that the FD15131 strain has phosphorus dissolving characteristics and strong phosphorus dissolving ability, and has broad application prospects in phosphorus dissolution.

[0081] Therefore, the present application provides an application of Bacillus tekila in soil phosphate solubilization, mainly its application in the preparation of a phosphate solubilizing agent, which is a live bacterial agent of Bacillus tekila FD15131. The preparation method and application method are shown in the following microbial preparation.

[0082] Embodiment 7:

[0083] After activating and culturing the FD15131 strain in LB liquid medium for 24 hours using a test tube, the bacterial solution was aspirated and 2 μL of the bacterial solution was inoculated on Ashby medium.

[0084] Ashby (nitrogen-fixing) medium includes: mannitol 10 g / L, KH2PO4 0.2 g / L, MgSO4·7H2O 0.2 g / L, NaCl 40 g / L, CaSO4·2H2O 0.2 g / L, CaCO3 5 g / L, and agar 15 g / L.

[0085] After the bacterial solution is absorbed by the plate, place the plate upside down in a 28℃ constant temperature incubator away from light for incubation. Observe the results after 72 hours. Figure 7 shown.

[0086] Ashby medium is a screening plate for nitrogen-fixing bacteria. If the FD15131 strain can grow on the Ashby medium, it means that it has the ability to fix nitrogen spontaneously. Figure 7 As shown in the middle figure (with nitrogen fixation on top), the strain grew well, which shows that the FD15131 strain has the characteristic of self-generated nitrogen fixation and has broad application prospects in biological nitrogen fixation.

[0087] Therefore, the present application provides an application of Bacillus tekila in biological nitrogen fixation. It is mainly used in the preparation of nitrogen-fixing bacteria. The nitrogen-fixing bacteria is a live bacterial agent of Bacillus tekila FD15131. The preparation method and application method are shown in the following microbial preparation.

[0088] Embodiment 8:

[0089] After activating and culturing the FD15131 strain in LB liquid medium for 24 hours using a test tube, the bacterial solution was aspirated and 2 μL of the bacterial solution was inoculated on the CAS detection medium.

[0090] CAS detection medium (1L): prepare 800mL of component ① solution, including 100g of glucose, 20g of peptone, 0.5g of MgSO4·7H2O, 0.5g of CaCl2, 15g of agar, and adjust the pH to 6.6~7.0; sterilize 800mL of component ① solution and cool it to 50~60℃, add 100mL of sterile component ② and 100mL of component ③ to obtain 1L of CAS detection medium. Among them:

[0091] Component ② is 10× PBS buffer;

[0092] 100 mL of component ③ includes: chrome azurol S (CAS) 0.06 g, FeCl3·5H2O 0.0027 g, and HDTMA 0.073 g.

[0093] After the bacterial solution is absorbed by the plate, place the plate upside down in a 28℃ constant temperature incubator away from light for incubation. Observe the results after 72 hours. Figure 7 shown.

[0094] CAS medium is a common medium for iron carrier detection. The growth of FD15131 strain on CAS medium is shown in Figure 7 As shown in the figure on the right (with iron carrier production on it), the strain grew well and produced an iron carrier secretion circle on the CAS medium. This shows that the growth of FD15131 strain produces iron carriers, which are a class of low molecular weight metal ion chelates that can chelate Fe 3+ Promote plant growth. Therefore, the FD15131 strain is a siderophore-producing bacterium and has broad application prospects in producing siderophores and preparing siderophore-producing bacterial agents.

[0095] Therefore, the present application provides an application of Bacillus tergila in producing siderophores. It is mainly used for preparing a siderophore-producing bacterial agent. The siderophore-producing bacterial agent is a live bacterial agent of Bacillus tergila FD15131. The preparation method and application method are shown in the following microbial preparation.

[0096] In addition, based on the verified characteristics of the FD15131 strain itself, the applicant has also conducted further research on the application of some characteristics of the FD15131 strain itself. Among them, the phosphorus solubilizing effect of the FD15131 strain shows that the FD15131 strain may have application potential in improving salinized soil and promoting plant growth in salinized soil. In order to verify the ability of the FD15131 strain to promote wheat growth in salinized soil, the present application designed Example 9 to verify the tolerance of the FD15131 strain to a high-salt growth environment, and found that the FD15131 strain can grow in a high-salt environment with a high tolerance. Subsequently, Example 10 was designed to verify the growth-promoting effect of the FD15131 strain on wheat plants in salinized soil. Specifically:

[0097] Embodiment 9:

[0098] The FD15131 strain provided in this application mainly converts phosphides in the soil that are difficult for plants to absorb and utilize into phosphate fertilizers that can be directly absorbed and utilized by plants through its phosphorus solubilizing properties, providing fertilizer for plants and also desalinating salinized soils, mainly saline soils. Therefore, this application mainly verifies the tolerance of the FD15131 strain to common sodium salts in saline soils, mainly sodium chloride and sodium sulfate.

[0099] Prepare sodium chloride culture medium of different concentrations: add sodium chloride to the basic LB medium (the composition is shown in the above-mentioned LB medium) to prepare LB culture medium with sodium chloride concentration (mass volume ratio) of 1%, 5% and 10% as sodium chloride culture medium.

[0100] Prepare sodium sulfate culture medium of different concentrations: add sodium sulfate to the basic LB medium (the ingredients are shown in the above LB medium) to prepare LB culture medium with sodium sulfate concentrations (mass-to-volume ratio) of 1%, 5% and 10% as sodium sulfate culture medium.

[0101] The salt concentration tolerance experiment of the present invention is carried out in a 96-well plate. First, an experimental group and a control group are set up, wherein the experimental group: the live strain of FD15131 is inoculated into the culture solution, and the FD15131 bacterial solution is shaken in advance to obtain the live bacterial solution of FD15131. And the control group: the live strain of FD15131 is inoculated into the culture solution to obtain an inoculum, and the inoculum is treated at high temperature for inactivation to obtain an FD15131 inactivated bacterial solution. It should be noted that the present application carried out streak culture on the inactivated bacterial solution of FD15131 to verify the inactivation effect. After culture, no bacteria grew, and no single colony growth was found. It can be considered that the FD15131 strain in the inactivated bacterial solution of FD15131 is completely inactivated.

[0102] Then, the experimental group inoculated 198 μL / well of the medium with different salt (sodium chloride or sodium sulfate) concentrations into the 96-well plate, and then inoculated 2 μL / well of the FD15131 live bacterial solution obtained by shaking in advance. (Specifically, 198 μL / well of the medium with different sodium chloride concentrations was inoculated into the 96-well plate, and then 2 μL / well of the aforementioned FD15131 live bacterial solution was inoculated; at the same time, 198 μL / well of the medium with different sodium sulfate concentrations was inoculated into the 96-well plate, and then 2 μL / well of the FD15131 live bacterial solution was inoculated.) At the same time, the control group inoculated the basal LB medium with the FD15131 inactivated bacterial solution in equal proportion (2 μL inactivated bacterial solution / 198 μL basal LB medium).

[0103] Then, the inoculated 96-well plate was placed in an incubator and cultured at 30°C for 48 hours. The OD was then counted. 600 value.

[0104] The growth of FD15131 strain in sodium chloride medium is as follows Figure 8 As shown in the figure, the strain grew best when the sodium chloride concentration was 1%. As the sodium chloride concentration increased, the strain's growth became worse, and it could tolerate a maximum sodium chloride concentration of 10%. This shows that the strain can maintain activity in a high-salt environment and has certain application prospects in the improvement of salinized soil.

[0105] The growth of FD15131 strain using sodium sulfate medium is as follows Fig. 9 As shown in the figure, the strain grew best when the sodium sulfate concentration was 1%. As the sodium sulfate concentration increased, the strain's growth became worse, and it could tolerate a maximum sodium sulfate concentration of 15%. This shows that the strain can maintain activity in a high-salt environment and has certain application prospects in the improvement of salinized soil.

[0106] Embodiment 10:

[0107] Soil from a greenhouse in Shandong was collected for potted experiments. The pH of the soil sample was measured to be 7.5, which is neutral / alkaline soil; the conductivity was 1500μS / cm, which is severely salinized soil.

[0108] The collected soil samples were placed in flower pots for potted experiments. The plants used in the potted experiments were wheat. All wheat seeds (from the same batch, with consistent seed quality) were disinfected on the same surface and all wheat seeds were subjected to the same germination treatment. The next day, white wheat seeds were selected and transplanted into the soil. The temperature for wheat growth is 20-25 degrees, with 12 hours of alternating light during the day and night.

[0109] Two treatment groups were designed, each with two pots planted to repeat the experiment, and five wheat seeds were planted in each pot. The two treatment groups were CK (Control Check) blank control group and FD15131 treatment group.

[0110] The CK blank control group was watered with 10 ml of sterile water per pot each time on the day of wheat seed sowing (first time), 7 days after sowing (second time), and 14 days after sowing (third time). While the CK blank control group was applying sterile water, the FD15131 treatment group was watered with the FD15131 spore suspension in Example 2 to the soil in the flowerpot, and the application time, number of times and dosage were consistent with the blank control group. That is to say, on the day of wheat seed sowing (first time), 7 days after sowing (second time), and 14 days after sowing (third time), each pot of the FD15131 treatment group was watered with 10 ml of the FD15131 spore suspension with an OD of 1.0 in Example 2, and a total of three treatments were performed.

[0111] The two treatment groups were cultured for 20 days after sowing. The results of wheat growth were as follows: Fig.10 The statistical results of wheat aboveground plant height are shown in Fig.11 As shown. The wheat in the blank control CK group grew slowly, and the branches and leaves drooped. The growth of wheat treated with FD15131 was significantly better than that of the CK blank control group. The wheat plant height was significantly higher than that of the CK blank control group, and the branches and leaves were straight without drooping. The results show that the FD15131 strain can use its phosphorus removal characteristics to improve the soil ecology of saline-alkali soil, improve the growth environment of saline-alkali soil, provide crops with a nitrogen source that can be directly absorbed and utilized, and can also perform biological nitrogen fixation and produce iron carriers to provide energy for plants, thereby promoting crop growth in saline-alkali soil.

[0112] Therefore, the present application provides an application of Bacillus tequila FD15131 in improving salinized soil, especially in preparing a salinized soil conditioner. The spore suspension of Bacillus tequila FD15131 is poured into the salinized soil. Or the remaining bacterial agents and biofertilizers containing live bacteria of Bacillus tequila FD15131 are applied to the salinized soil. Further, the application of Bacillus tequila FD15131 in promoting plant growth in salinized soil is provided, and the salinized soil conditioner is applied to the soil at the root system of the plant, for example, the spore suspension of Bacillus tequila FD15131 is poured into the salinized soil at the root system of the crop. Or the remaining bacterial agents and biofertilizers containing live bacteria of Bacillus tequila FD15131 are applied to the salinized soil at the root system of the crop.

[0113] In addition, the present application also provides an application of Bacillus tequila FD15131 in promoting plant growth, especially an application of Bacillus tequila FD15131 in preparing a plant growth promoter. The spore suspension of Bacillus tequila FD15131 is poured into the soil at the root of the plant.

[0114] According to another aspect of the present application, a microbial preparation is provided, which is suitable for use as a wheat root rot prevention and control drug, a phosphate-dissolving bacteria agent, a nitrogen-fixing bacteria agent, an iron carrier-producing bacteria agent, a growth promoter or a salinized soil conditioner. The microbial preparation is a live bacterial preparation of Bacillus tekila FD15131. The present application does not specifically limit other active components, adjuvants and dosage forms of the live bacterial preparation, as long as the Bacillus tekila FD15131 in the preparation remains active and can be inoculated and applied in the form of live bacteria. Bacillus tekila FD15131 can be combined with other probiotics to prepare a composite bacterial preparation, and beneficial metabolites of other probiotics can be added to the bacterial preparation, as long as the microbacterium in the microbial preparation remains active. The live bacterial preparation can be solid, such as live bacterial powder, or liquid, such as a live bacterial spore suspension with different OD values ​​in Example 2.

[0115] In a possible implementation, the microbial preparation is a live bacterial suspension of Bacillus tequila FD15131, and the application method is root irrigation treatment or soil watering. The present application does not specifically limit the solvent of the live bacterial suspension, as long as it does not kill or destroy the live bacteria and can ensure the survival of the live bacteria. The solvent of the live bacterial suspension can be a culture medium or sterile water. Furthermore, the solvent of the live bacterial suspension is sterile water, that is, the spores of Bacillus tequila FD15131 are suspended in sterile water to obtain a live bacterial suspension. The concentration of the Bacillus tequila FD15131 in the live bacterial suspension is OD 600 =0.8~1.5, preferably 1.

[0116] The present application also provides a method for preparing a live bacterial suspension of Bacillus tergrillii FD15131, comprising the following steps:

[0117] The aforementioned Bacillus tergillosis FD15131 was inoculated into the culture medium, and cultured at 28° C. and 180 rpm for 36 hours to obtain a first fermentation liquid;

[0118] The first fermentation liquid is centrifuged, and the centrifuged precipitated bacteria are resuspended in a solvent to obtain a live bacteria suspension, which is used as a microbial preparation. The present application does not specifically limit the centrifugation conditions. In principle, the culture medium is removed as much as possible to obtain a live bacteria precipitate, with the survival of live bacteria being ensured. The centrifugation conditions are preferably 7000 rpm and centrifuged at room temperature for 15 minutes, which will not cause the death of live bacteria.

[0119] According to the above embodiments, the applicant believes that Bacillus tekila FD15131 can utilize its growth, phosphorus dissolving characteristics, nitrogen fixation characteristics and iron carrier production characteristics to provide nutrients for plants and promote plant growth. To some extent, the live bacteria of FD15131 strain is the "fertilizer" of crops. Therefore, FD15131 strain has broad application prospects in the biological fertilizer of crops. According to another aspect of the present application, a biological fertilizer is provided, which includes live bacteria of Bacillus tekila FD15131.

[0120] In one possible implementation, the biological fertilizer includes a basic fertilizer and live bacteria of Bacillus subtilis FD15131. It should be noted that the present application does not specifically limit the components and proportions of the basic fertilizer, nor does it specifically limit the mixing method and application method of the basic fertilizer and Bacillus subtilis FD15131. The basic fertilizer can be any one of potash fertilizer, sodium fertilizer, phosphate fertilizer and nitrogen fertilizer, or a compound fertilizer of at least two, and can be any basic fertilizer for wheat. The basic fertilizer can be pre-mixed with the live bacteria of the FD15131 strain for transportation and storage, or it can be packaged separately and prepared (mixed) for use. The basic fertilizer and Microbacterium exigua can be liquid or solid, as long as it can ensure that Bacillus subtilis FD15131 is a live bacterium when fertilizing.

[0121] At this time, whether the FD15131 strain can still maintain high vitality and good growth after the fertilizer is applied has become an urgent problem to be solved. Therefore, the present application designs Examples 22 and 23 to verify the tolerance of the FD15131 strain to fertilizer.

[0122] Embodiment 11:

[0123] Urea, phosphate fertilizer (consisting of monoammonium phosphate) and potash fertilizer (consisting of potassium phosphate) sold on the market were selected as solutes, and LB (liquid) culture medium was used as solvent. Fertilizer solutions of 1%, 5% and 10% were prepared according to the mass-to-volume ratio, respectively.

[0124] A 96-well plate was used to carry out the tolerance experiment. First, 198 μL of fertilizer solution of different concentrations was pipetted into the well, and then 2 μL of FD15131 spore suspension with an OD of 1 in Example 2 was added. After mixing, the mixture was placed in a 30°C incubator and the OD was read after 24 hours. 600 The absorbance value.

[0125] The results are as follows Figure 12-14 As shown, in general, FD15131 has different degrees of tolerance to urea, phosphate fertilizer and potash fertilizer. FD15131 grows well at a urea concentration of 1% and can tolerate a urea concentration of 5%, but when the urea concentration increases to 10%, the growth of the strain is inhibited and it is difficult to grow. FD15131 grows well at a phosphate fertilizer concentration of 1% and can tolerate a phosphate fertilizer concentration of 5%, but when the phosphate fertilizer concentration increases to 10%, the growth of the strain is inhibited and it is difficult to grow. FD15131 grows well at potassium fertilizer concentrations of 1% and 5%. When the potassium fertilizer concentration increases to 10%, the growth of the strain decreases, but it can still grow. The above results show that FD15131 can tolerate a variety of concentrations of chemical fertilizers and can be used after mixing with chemical fertilizers within a certain range. In particular, potassium fertilizer can be used as a basic fertilizer in combination with biological fertilizers.

[0126] In a possible implementation, the basic fertilizer includes the following components and their mass percentages: 15%~20% amino acid solution, 13%~18% urea, 10%~12% nitrogen-potassium fertilizer, 20%~25% potassium chloride, 7%~10% diammonium phosphate, 0.1%~0.5% EDTA-Zn, 0.3%~0.8% EDTA-Cu, 0.3%~0.8% boric acid, and the rest is clean water.

[0127] Embodiment 12:

[0128] The present embodiment provides a basic fertilizer, comprising the following components and their mass percentages: 18% amino acid solution, 15% urea, 11% nitrogen-potassium fertilizer, 22% potassium chloride, 9.3% diammonium phosphate, 0.3% EDTA-Zn, 0.5% EDTA-Cu, 0.5% boric acid, and 23.4% clean water.

[0129] After culturing the FD15131 strain with the culture medium, centrifugation was performed, the supernatant was removed, and the precipitate was evenly added to the basic fertilizer provided in this embodiment to obtain a biological fertilizer. After the addition, the initial concentration of the FD15131 strain in the biological fertilizer was 2 x 10 6 The survival period of FD15131 strain in basic fertilizer was detected by using biological fertilizer.

[0130] The aforementioned biofertilizer was stored at 30°C. The biofertilizer was sampled immediately after mixing and adding, and after adding and standing for 1, 2, 3, 5, 7, 14, 21, and 28 days. 1 mL of the biofertilizer was taken as a sample each time, and the ten-fold gradient dilution method was used for spreading and counting. The dilutions of different dilution multiples were spread on LB medium, and the number of single colonies was counted to obtain the number of cells of FD15131 surviving in the basic fertilizer.

[0131] The results are as follows Fig.15 As shown in Figure 2, after adding FD15131 strain to the basic fertilizer, the initial concentration of FD15131 strain in the biofertilizer was 2×10 6 After 5 days, the number of bacteria decreased to 1.48×10 6 / mL, tended to be stable in the later period and remained until the 14th day. On the 21st day, the number of bacteria was 1.09×10 6 / mL, and on the 28th day, the number of bacteria was 0.85×10 6 Compared with the initial value, the number of bacteria decreased by 57.5% after one month, that is, it was reduced by half.

[0132] The embodiments of the present application have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or improvements to the technology in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

[0133] Sequence Listing

[0134] SEQ ID No: 1 is as follows:

[0135] agtcgagcgg acagatggga gcttgctccc tgatgttagc ggcggacggg tgagtaacac60

[0136] gtgggtaacc tgcctgtaag actgggataa ctccgggaaa ccggggctaa taccggatgg120

[0137] ttgtttgaac cgcatggttc aaacataaaa ggtggcttcg gctaccactt acagatggac180

[0138] ccgcggcgca ttagctagtt ggtgaggtaa cggctcacca aggcaacgat gcgtagccga240

[0139] cctgagaggg tgatcggcca cactgggact gagacacggc ccagactcct acgggaggca 300

[0140] gcagtaggga atcttccgca atggacgaaa gtctgacgga gcaacgccgc gtgagtgatg360

[0141] aaggttttcg gatcgtaaag ctctgttgtt agggaagaac aagtaccgtt cgaatagggc420

[0142] ggtaccttga cggtacctaa ccagaaagcc acggctaact acgtgccagc agccgcggta480

[0143] atacgtaggt ggcaagcgtt gtccggaatt attgggcgta aagggctcgc aggcggtttc540

[0144] ttaagtctga tgtgaaagcc cccggctcaa ccggggaggg tcattggaaa ctggggaact600

[0145] tgagtgcaga agaggagagt ggaattccac gtgtagcggt gaaatgcgta gagatgtgga660

[0146] ggaacaccag tggcgaaggc gactctctgg tctgtaactg acgctgagga gcgaaagcgt720

[0147] ggggagcgaa caggattaga taccctggta gtccacgccg taaacgatga gtgctaagtg780

[0148] ttagggggtt tccgcccctt agtgctgcag ctaacgcatt aagcactccg cctggggagt840

[0149] acggtcgcaa gactgaaact caaaggaatt gacgggggcc cgcacaagcg gtggagcatg 900

[0150] tggtttaatt cgaagcaacg cgaagaacct taccaggtct tgacatcctc tgacaatcct960

[0151] agagatagga cgtccccttc gggggcagag tgacaggtgg tgcatggttg tcgtcagctc1020

[0152] gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccttgatct tagttgccag1080

[0153] cattcagttg ggcactctaa ggtgactgcc ggtgacaaac cggaggaagg tggggatgac1140

[0154] gtcaaatcat catgcccctt atgacctggg ctacacacgt gctacaatgg acagaacaaa1200

[0155] gggcagcgaa accgcgaggt taagccaatc ccacaaatct gttctcagtt cggatcgcag1260

[0156] tctgcaactc gactgcgtga agctggaatc gctagtaatc gcggatcagc atgccgcggt1320

[0157] gaatacgttc ccgggccttg tacacaccgc ccgtcacacc acgagagttt gtaacacccg1380

[0158] aagtcggtga ggtaacc1397

Claims

1. A bacillus tequila, characterized in that The Bacillus tequila ( Bacillus tequilensis ) is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number CGMCC No.31237.

2. An application of Bacillus tequila, characterized in that: Use of the Bacillus tequila described in claim 1 in preventing and treating wheat root rot.

3. An application of Bacillus tequila, characterized in that: Use of the Bacillus tergillosis described in claim 1 in soil phosphate solubilization.

4. An application of Bacillus tequila, characterized in that: Use of the Bacillus tequila described in claim 1 in biological nitrogen fixation.

5. An application of Bacillus tequila, characterized in that: Use of the Bacillus tergiornicotus described in claim 1 in producing siderophores.

6. An application of Bacillus tequila, characterized in that: Use of the Bacillus tequila described in claim 1 in promoting plant growth.

7. An application of Bacillus tequila, characterized in that: Use of the Bacillus tequila described in claim 1 in improving salinized soil.

8. A microbial preparation, characterized in that: The microbial preparation is the live bacterial preparation of Bacillus tergillosis according to claim 1.

9. The microbial preparation according to claim 8, characterized in that The microbial preparation is a live bacterial suspension of the Bacillus tequila.

10. A biological fertilizer, characterized in that: The biological fertilizer comprises live bacteria of Bacillus tequila according to claim 1.

Images