Strain of Steterbacter sp. And application thereof

Abstract

The invention relates to the technical field of microorganisms, and discloses a strain of bacillus stergeri and application thereof, the classification name of the bacillus stergeri is bacillus stergeri, the bacillus stergeri is preserved in the China General Microbiological Culture Collection Center on December 1, 2025, the preservation number of the bacillus stergeri is CGMCC No.36867, and the gene sequence of 16S rDNA of the bacillus stergeri is as shown in SEQ ID No.1. According to the invention, a microbiological method is utilized to separate and identify a strain of Steterobacteria with special functions from a tobacco endophytic environment, and a series of experiments prove that the strain has remarkable effects in the aspects of promoting tobacco growth, improving tobacco yield and quality and the like, so that a new biological growth promoting means is provided for the field of tobacco planting; the green and sustainable development of the tobacco planting industry can be promoted.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a strain of Serratia and its application. BACKGROUND

[0002] At present, tobacco is one of the important economic crops, which plays a vital role in agricultural production and national economic development. Its planting range is wide, not only is the main source of income for many local tobacco farmers, but also provides strong support for local economic growth, tax contribution and employment. However, with the expansion of planting scale and the increase of planting years, tobacco planting is facing many serious problems, especially the continuous cropping obstacle is becoming increasingly serious, which leads to frequent occurrence of soil-borne diseases. For example, tobacco bacterial wilt and other diseases can cause whole plant death during the growth period, causing huge economic losses to tobacco farmers.

[0003] In order to solve the above problems, the existing technology mainly adopts the measures of applying chemical fertilizer and chemical pesticide. Although the use of chemical fertilizer can supplement soil nutrients to a certain extent, meet the demand of tobacco growth for nitrogen, phosphorus, potassium and other elements, and improve yield, but long-term and large-scale use of chemical fertilizer can destroy soil structure, cause soil compaction, and easily cause water eutrophication and other environmental pollution, and destroy ecological balance. At the same time, although chemical pesticides can effectively control the occurrence and spread of diseases and pests in the short term, long-term dependence on pesticides not only can lead to drug resistance of diseases and pests, reduce the control effect, but also can lead to pesticide residues exceeding the standard in tobacco, endanger the health of consumers, and cause continuous damage to the ecological system.

[0004] In view of the many disadvantages brought by the use of chemical fertilizer and pesticide, the development of environmentally friendly and sustainable biological control and microbial growth promotion technology has become a new trend to solve the problem of tobacco planting. Rhizosphere growth promoting bacteria (PGPR) as a kind of beneficial microorganism which can colonize in the rhizosphere of plants and promote plant growth has received extensive attention in recent years. PGPR has multiple mechanisms of action: first, they can convert atmospheric nitrogen into plant available nitrogen through nitrogen fixation, and convert soil insoluble nutrients into soluble nutrients through phosphorus and potassium solubilization, etc., to improve soil fertility; second, PGPR can produce plant hormones such as auxin and cytokinin, regulate plant growth and development, promote root growth, and enhance the plant's ability to absorb nutrients; in addition, PGPR can inhibit the growth and reproduction of harmful pathogenic bacteria in soil through competition or secretion of antibiotics, reduce the incidence of diseases, and enhance the plant's disease resistance.

[0005] As a potential rhizosphere growth-promoting bacteria, Bacillus stercoris has unique biological characteristics and functions. In-depth research and development of Bacillus stercoris are expected to provide an efficient, green and sustainable growth-promoting and disease-preventing means for tobacco planting, and have important theoretical value and practical significance for solving the problems of continuous cropping obstacles and abuse of chemical fertilizers and pesticides in current tobacco planting. SUMMARY

[0006] Therefore, the purpose of the present application is to provide a Bacillus stercoris and its application, which can effectively improve the yield of tobacco, improve the quality of tobacco, and has a certain prevention and treatment effect on bacterial wilt, and enhances the resistance of tobacco to bacterial wilt.

[0007] The present application solves the above technical problems through the following technical means:

[0008] In a first aspect, the present application provides a Bacillus stercoris, which is named Zst-12 and classified as Bacillus stercoris. The Bacillus stercoris was deposited with China General Microbiological Culture Collection Center on December 01, 2025, and the strain deposit number is CGMCC No. 36867. The 16S rDNA gene sequence of the Bacillus stercoris is shown as SEQ ID No. 1.

[0009] In a second aspect, the present application provides a microbial preparation, characterized in that the microbial preparation comprises the Bacillus stercoris of claim 1 or a bacterial liquid thereof.

[0010] Preferably, the concentration of Bacillus stercoris Zst-12 in the microbial preparation is 10 9 -10 10 CFU / mL.

[0011] In a third aspect, the present application provides the application of the Bacillus stercoris or the microbial preparation in any one or more of the following (a) to (e):

[0012] (a) inhibiting plant diseases;

[0013] (b) promoting plant growth;

[0014] (c) improving soil;

[0015] (d) improving the tolerance of plants to drought, salinization and high temperature;

[0016] The plant disease is tobacco bacterial wilt; and the plant is a tobacco plant.

[0017] In a fourth aspect, the present application provides a product of the Bacillus stercoris or the microbial preparation.

[0018] Preferably, the product includes biofertilizers, biocontrol agents, and biopesticides.

[0019] Fifthly, the present invention provides the use of the Sterculia salina or the preparation containing the microorganism in the preparation of biofertilizers.

[0020] Preferably, the bio-fertilizer has at least one of the following functions: (1) nitrogen fixation; (2) dissolving inorganic phosphorus; (3) potassium solubilization; (4) iron carrier production; (5) indoleacetic acid production; and (6) inhibition of bacterial wilt.

[0021] In a sixth aspect, the present invention provides a method for preventing and controlling tobacco bacterial wilt, the method comprising spraying or drenching tobacco plants with Sterculia lysate or the microbial preparations described above.

[0022] In a seventh aspect, the present invention provides a method for promoting the growth of tobacco plants, the method comprising spraying or drenching the tobacco plants with the aforementioned Stellobacterium or the aforementioned microbial preparation.

[0023] The beneficial effects of this invention are:

[0024] (1) The strain of the present invention has a significant growth-promoting effect. By inoculating the roots of tobacco with Sterculia esculenta Zst-12, the growth and development of tobacco seedlings can be effectively promoted, specifically by significantly increasing the plant height, stem diameter, number of leaves and leaf area. At the same time, this strain can improve the photosynthetic efficiency of tobacco, enhance root vigor, and thus enhance the plant's ability to absorb nutrients, thereby achieving a significant increase in tobacco yield.

[0025] (2) The strain of this invention exhibits excellent performance in biological control, particularly showing a significant inhibitory effect on tobacco bacterial wilt. Experimental verification has shown that co-culturing this strain with Ralstonia solanacearum can significantly reduce the abundance of Ralstonia solanacearum in the culture medium, indicating that it can effectively inhibit the growth of pathogens and enhance tobacco's resistance to common diseases and pests. In addition, this strain also has the potential to improve tobacco's tolerance to adverse stresses such as drought, salinity, and high temperature, which helps to enhance the crop's stress resistance. Attached Figure Description

[0026] Figure 1 A plate image of Stellobacter Zst-12 on TSB medium;

[0027] Figure 2 Micrograph of Stellaria Zst-12;

[0028] Figure 3 Phylogenetic tree of 16S rDNA from Sterculia Zst-12;

[0029] Figure 4Figure showing the effect of Stellaria media Zst-12 plate growth promotion experiment on tobacco plant growth.

[0030] Figure 5 Figure showing the effect of Stellobacter Zst-12 on tobacco plant growth in a pot experiment.

[0031] Figure 6 This is a diagram of a plate confrontation experiment between Sterculia esculenta Zst-12 and Ralstonia solanacearum. Detailed Implementation

[0032] The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0033] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods in the art.

[0034] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0035] TSB medium: tryptone 17.0 g / L, sodium chloride 5.0 g / L, soybean papain hydrolysate 3.0 g / L, dipotassium hydrogen phosphate 2.5 g / L, glucose (monohydrate / anhydrous) 2.5 g / L, pH 7.3±0.2, autoclaved at 121℃, cooled to 30℃, ready for use.

[0036] LB medium: yeast extract 5g / L, tryptone 15g / L, sodium chloride 10g / L, pH 7.0-7.2, autoclaved at 121℃, cooled to 30℃, ready for use.

[0037] Culture medium B: 10g peptone, 1g yeast powder, 1g acid-hydrolyzed casein, 1000ml water. Sterilize by autoclaving at 121℃ and cool to 30℃ for later use.

[0038] BG plate medium: Based on B medium, add 15 g / L agar and 20 ml / L sterile glucose solution with a mass fraction of 25%.

[0039] Assumption medium: sucrose 5 g / L, glucose 5 g / L, ammonium sulfate 0.5 g / L, yeast extract 0.5 g / L, magnesium sulfate 0.3 g / L, potassium dihydrogen phosphate 2 g / L, ferrous sulfate 0.03 g / L, manganese sulfate 0.03 g / L, potassium feldspar 2 g / L, agar 15 g / L, pH 7.2 ± 0.2. The medium is autoclaved at 121°C and then poured into plates for later use.

[0040] Phosphate-soluble medium: glucose 10g / L, ammonium sulfate 0.5g / L, sodium chloride 0.3g / L, magnesium sulfate 0.3g / L, manganese sulfate 0.03g / L, potassium sulfate 0.3g / L, ferrous sulfate 0.03g / L, calcium phosphate 5g / L, agar 15g / L, pH 7.0-7.5. Sterilize by autoclaving at 121℃ and pour into plates for later use.

[0041] Potassium-solubilizing medium: 0.2 g / L potassium dihydrogen phosphate, 0.2 g / L magnesium sulfate, 0.2 g / L sodium chloride, 5 g / L calcium carbonate, 10 g / L mannitol, 0.1 g / L calcium sulfate, 15 g / L agar. Sterilize by autoclaving at 121°C and pour into plates for later use.

[0042] CAS medium: Chromium azurite S 60.5 mg / L, cetyltrimethylammonium bromide 72.9 mg / L, ferric chloride hexahydrate 2.645 mg / L, sodium dihydrogen phosphate dihydrate 295.25 mg / L, disodium hydrogen phosphate dodecahydrate 1213.5 mg / L, ammonium chloride 125 mg / L, potassium dihydrogen phosphate 37.5 mg / L, sodium chloride 62.5 mg / L, agar 9000 mg / L, pH 6.8±0.1. The medium was autoclaved at 121℃ and then plated for later use.

[0043] Example 1: Isolation of Stellaria media Zst-12

[0044] The tobacco endophytic environment was screened using a high-throughput sorting system for endophytic bacteria.

[0045] First, tobacco stalks were chopped and ground, then soaked and washed with PBS buffer. The ground tissue was then diluted, and the optimal dilution gradient was selected (five gradients were used: 1 / 6000, 1 / 18000, 1 / 54000, 1 / 162000, and 1 / 486000). The diluted bacterial solutions were inoculated into 10% TSB medium and cultured for two weeks. After culture, the optimal dilution gradient was determined (1 / 162000). High-throughput sequencing was performed on the microorganisms in 96-well plates corresponding to this gradient (1 / 162000) and the next gradient (1 / 486000). Simultaneously, the microorganisms in the 96-well plates corresponding to gradient 1 / 54000 were preserved in glycerol for subsequent single-cell purification. A strain with growth-promoting effects was screened by measuring growth-promoting physiological and biochemical indicators and named Zst-21. This strain is *Bacillus subtilis*, also known as *Bacillus compostii*. On TSB agar plates, it presents continuous white, wrinkled colonies with raised surfaces and small, shallow spreading rings around the perimeter. It grows well within a temperature range of 28-35°C and requires 2-3 days for streak incubation. It is a Gram-negative bacterium; under a microscope, the bacteria appear as thin rods with flagella and spores (see...). Figure 1 and Figure 2 ).

[0046] Example 2: Detection of the growth-promoting ability of the strain

[0047] Nitrogen fixation index detection: 10 μL of overnight cultured Stellobacter Zst-12 was added to Assumption medium plates. The formation of a clear zone indicates that the strain has nitrogen fixation ability.

[0048] Phosphate solubility index detection: 10 μL of overnight cultured Stellobacterium Zst-12 was added to a phosphate solubility medium plate. The appearance of a clear zone indicates that the strain has phosphate solubility.

[0049] Potassium solubilization index detection: 10 μL of overnight cultured Stellobacterium Zst-12 was added to a potassium solubilization medium plate. The appearance of a clear zone indicates that the strain has potassium solubilization ability.

[0050] Siderophore index detection: 10 μL of overnight cultured Stellobacterium Zst-12 was added to a CAS medium plate. The appearance of an orange-yellow halo indicates that the strain has the ability to produce siderophores.

[0051] Indoleacetic acid (IAA) detection: The Salkowski colorimetric method was used. The strain was inoculated into liquid LB medium containing 0.5 g / L tryptophan and cultured at 30 ℃ with shaking at 180 r / min for 2-3 days. 1 mL of bacterial suspension was centrifuged at 12000 r / min for 2 min, and 100 μL of the supernatant was collected. 100 μL of Salkowski colorimetric solution was added, and the mixture was reacted in the dark at room temperature for 30 min. The color change was observed; a red or pink color indicated a positive result, meaning the strain possessed the ability to produce IAA. The results are shown in Table 1.

[0052] Table 1 Results of growth-promoting indicators of Sterculia esculenta Zst-12

[0053]

[0054] Note: "+" indicates a positive result, and "-" indicates a negative result.

[0055] Example 3: Identification method of Sterculia esculenta Zst-12

[0056] The 16S rDNA gene of the strain was amplified by PCR and sent to Chongqing Qingke Biotechnology Co., Ltd. for sequencing analysis. The primers used for amplifying the 16S rDNA gene are as follows:

[0057] The upstream primer sequence is: 5'-AGAGTTTGATCCTGGCTCAG-3' (as shown in SEQ ID No. 2);

[0058] The downstream primer sequence is: 5'-GGTTACCTTGTTACGACTT-3' (as shown in SEQ ID No. 3).

[0059] The PCR reaction conditions were: 95℃ pre-denaturation for 4 min, 95℃ denaturation for 40 s, 55℃ annealing for 50 s, 72℃ extension for 1 min, 32 cycles, followed by a 72℃ extension for 10 min. Sequence alignment in the GeneBank database using NCBI's BLAST function revealed that the strain screened in this invention showed the highest homology (99.76%) with *Bacillus stercoris* subsp. *stercoris* G124. Therefore, this strain was identified as *Bacillus stercoris* and named *Bacillus stercoris* Zst-12 (see...). Figure 3 The nucleotide sequence is shown in SEQ ID NO.1. This strain was deposited on December 1, 2025 at the China Microbiological Culture Collection Center, with accession number CGMCC No. 36867.

[0060] The 16S rDNA sequence of Stenotrophomonas Zst-12 (SEQ ID NO.1) is as follows:

[0061]

[0062] Example 4: Preparation of liquid bacterial agent of Stellaria media Zst-12

[0063] The preserved Stellariae Zst-12 strain was activated on TSA medium, and the activated Stellariae Zst-12 was inoculated into Erlenmeyer flasks containing seed medium (TSB medium). Sterile air was introduced at an aeration ratio of 1:0.6 (V / V·m), the mixture was stirred at 180 rpm, and the culture was carried out at 30-32℃ for 36 hours. The culture was then shaken until the logarithmic growth phase was reached to obtain the bacterial strain.

[0064] The bacterial strain was inoculated into a seed tank containing seed culture medium at an inoculation rate of 8% (v / v) and cultured until the logarithmic growth phase to obtain Stellaria dichotoma Zst-12 seed culture.

[0065] The obtained Sterculia esculenta Zst-12 seed culture was then inoculated into a production tank containing fermentation medium (LB medium) at an inoculation rate of 8% (v / v). The culture conditions were the same as those used to prepare the Sterculia esculenta Zst-12 seed culture. The concentration of Sterculia esculenta Zst-12 in the fermentation broth was determined to be 5 × 10⁻⁶. 8 When the concentration of CFU / ml is reached, Stellaria media Zst-12 liquid bacterial agent with growth-promoting effect is formed immediately upon discharge from the container.

[0066] Experimental Example 5: Growth-promoting effect of Sterculia salina Zst-12 on tobacco plants

[0067] Using 1 / 2 MS medium as the plant culture medium, *Stemonas stercoralis* Zst-12 fermentation agent was inoculated into the root system of tobacco plants. The experimental group consisted of tobacco seedlings inoculated with *Stemonas stercoralis* Zst-12 fermentation agent, while the control group consisted of tobacco seedlings inoculated only with an equal volume of sterile water. The inoculation volume for both groups was 10 μL. After 7 days, the tobacco seedlings in the experimental group showed significantly better growth than those in the control group (see...). Figure 4 ).

[0068] Experimental Example 6: Effects of Sterculia pyrenes Zst-12 on tobacco plants in a pot experiment.

[0069] Tobacco seedlings were transplanted into small flowerpots, each 25cm in diameter at the top and 20cm in diameter at the bottom, filled with 250g of soil. A fermentation agent of *Stemonas stercoralis* Zst-12 was inoculated into the soil around the tobacco plants. The experimental group consisted of tobacco seedlings inoculated with *Stemonas stercoralis* Zst-12 fermentation agent, while the control group consisted of tobacco seedlings inoculated only with an equal volume of sterile water. The inoculum was prepared according to Example 4, with an inoculation volume of 5 ml per pot. A second inoculation was performed 7 days later. After 30 days, the experimental group of tobacco seedlings showed significantly better growth than the control group (see [link to example]. Figure 5 ).

[0070] Experimental Example 7: Antagonistic effect of Sterculia salina Zst-12 against Ralstonia solanacearum

[0071] This embodiment demonstrates the antagonistic effect of the Stellobacterium Zst-12 liquid inoculum prepared in Example 3 against Ralstonia solanacearum through an inhibition zone experiment.

[0072] Inhibition zone experiment: Ralstonia solanacearum OE-1 cultured overnight in B medium was spread onto a BG plate. Then, Stellaria media Zst-12 cultured overnight in TSB was dropped onto filter paper and placed on the spread BG plate. The plates were incubated at 32°C for 3 days. A clear transparent zone appeared around the filter paper, indicating that under the plate culture conditions, Stellaria media Zst-12 effectively inhibited the growth of Ralstonia solanacearum OE-1 (see...). Figure 6 ).

[0073] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.

Claims

1. A strain of Sterculia, characterized in that, The strain of *Bacillus stercoris* is classified as *Bacillus stercoris*. It was deposited at the China Microbial Culture Collection Center on December 1, 2025, with the accession number CGMCC No. 36867. The 16S rDNA gene sequence of *Bacillus stercoris* is shown in SEQ ID No.

1.

2. A microbial preparation, characterized in that, The microbial preparation includes the Stellobacterium or its bacterial culture as described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, The concentration of Sterculia lysate in the microbial preparation is 10. 9 -10 10 CFU / mL.

4. The use of the Sterculia lysate of claim 1 or the microbial preparation of claims 2-3 in any one or more of the following (a)-(e): (a) Suppressing plant diseases; (b) Promotes plant growth; (c) Soil improvement; (d) Improve the plant's tolerance to drought, salinity, and high temperature; The plant disease is tobacco bacterial wilt; the plant is a tobacco plant.

5. A product containing the Sterculia salina according to claim 1 or containing any of the microbial preparations according to claims 2-3.

6. The product as described in claim 4, characterized in that, The products include biofertilizers, biocontrol agents, and biopesticides.

7. The use of the Sterculia salina according to claim 1 or the microbial preparation containing any one of claims 2-3 in the preparation of biofertilizer.

8. The application as described in claim 7, characterized in that, The bio-fertilizer has at least one of the following functions: (1) nitrogen fixation; (2) dissolving inorganic phosphorus; (3) potassium solubilization; (4) iron carrier production; (5) indoleacetic acid production; and (6) inhibition of bacterial wilt.

9. A method for preventing and controlling tobacco bacterial wilt, characterized in that, The method includes spraying or drenching tobacco plants with Stellaria media as described in claim 1 or a microbial preparation as described in any one of claims 2-3.

10. A method for promoting the growth of tobacco plants, characterized in that, The method includes spraying or drenching tobacco plants with Stellaria media as described in claim 1 or a microbial preparation as described in any one of claims 2-3.

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