Bacillus sp. and use thereof

Abstract

The application provides a bacillus pumilus and application thereof, the bacillus pumilus is preserved in Guangdong Microbial Culture Collection Center on July 24, 2025, and the preservation number is GDMCC No: 66749;The 16S rDNA of the bacillus pumilus is as shown in SEQ ID NO.1;The application of the bacillus pumilus is to prepare microbial preparation comprising the bacillus pumilus;The bacillus pumilus has good phosphorus dissolving capacity and IAA production capacity, can enhance the resistance of plants and promote the growth of plants.

Description

TECHNICAL FIELD

[0001] The present application relates to a Bacillus vesiculus and its application, and belongs to the technical field of microorganisms. BACKGROUND

[0002] Bacillus is a kind of short rod-shaped gram-positive bacteria, commonly found in soil and water, characterized by the ability to form spores. Spores are endospores, which are bacterial dormant bodies produced to resist harsh environments. At the same time, Bacillus has various species and functions, has a wide range of ecological functions, promotes plant growth, inhibits the reproduction of harmful bacteria, decomposes organic matter and improves the environment, and is applied in the fields of agriculture, breeding, environmental protection and food industry. SUMMARY

[0003] In order to overcome the shortcomings of the prior art, the first object of the present application is to provide a Bacillus vesiculus, which has good phosphorus dissolving ability and IAA producing ability, and can enhance the resistance of plants and promote the growth of plants.

[0004] The second object of the present application is to provide the application of the above-mentioned Bacillus vesiculus, which is used for preparing a microbial preparation to realize the function of the strain.

[0005] The first object of the present application can be achieved by adopting the following technical scheme: a Bacillus vesiculus, classified and named as Cytobacillus sp . , which was preserved in Guangdong Microbial Culture Collection Center on July 24, 2025, the address of the preservation unit is: No. 59 Building, 5th Floor, Guangzhou Xianlie Middle Road 100 Courtyard, the preservation number is GDMCC No: 66749.

[0006] Further, the 16S rDNA of the Bacillus vesiculus is shown in SEQ ID NO. 1.

[0007] The second object of the present application can be achieved by adopting the following technical scheme: an application of a Bacillus vesiculus, which is used for preparing a microbial preparation comprising the Bacillus vesiculus.

[0008] Further, the preparation is prepared by the following method: the Bacillus vesiculus and Bacillus subtilis are inoculated into a fermentation medium for fermentation, and the obtained fermentation broth is dried.

[0009] Further, the mass ratio of the Bacillus vesiculus and Bacillus subtilis added is 3:(1-2).

[0010] Further, the concentration of the fermentation broth is 7x10 6 -9x10 6 cfu / mL.

[0011] Further, the microbial preparation is a plant growth promoting and / or disease resistant preparation.

[0012] Still further, the plant is at least one of kidney bean, rice, cucumber and tomato.

[0013] Still further, the pathogenic bacteria of the plant is at least one of Chaetomium globosum, Curvularia lunata and M. phaseolina.

[0014] Further, the microbial preparation is a preparation acting on at least one of seeds, roots, leaves and fruits of the plant.

[0015] Compared with the prior art, the present application has the beneficial effects that:

[0016] 1. The Bacillus vesiculus of the present application can be used as a plant treatment agent, and through the life activities of the microorganism, the supply amount of plant nutrient elements is increased, the plant nutrition is improved, and the growth efficiency and yield are improved.

[0017] 2. The Bacillus vesiculus of the present application has obvious inhibitory effect on crop pathogenic bacteria, can enhance the resistance of plants, and promote the growth of plants.

[0018] 3. The microbial preparation of the Bacillus vesiculus of the present application has flexible use mode, high strain activity, no pollution, and meets the sustainable development of the environment. BRIEF DESCRIPTION OF DRAWINGS

[0019] The Bacillus vesiculus involved in the present application is classified and named as Bacillus vesiculus sp. Cytobacillus The Bacillus vesiculus was preserved in the Guangdong Microbial Culture Collection Center on July 24, 2025, and the preservation number is GDMCC No: 66749.

[0020] Figure 1 is the colony morphology of the 2SZ(SF)-Ba5 strain;

[0021] Figure 2 is the microscopic morphology of the 2SZ(SF)-Ba5 strain;

[0022] Figure 3 is the growth curve of 2SZ(SF)-Ba5;

[0023] Figure 4 is the antibacterial test photo of Chaetomium globosum CK;

[0024] Figure 5 is the antibacterial test photo of Chaetomium globosum 6d;

[0025] Figure 6 is the antibacterial test photo of Curvularia lunata CK;

[0026] Figure 7The photo of the antibacterial test of Curvularia geniculata 6d;

[0027] Figure 8 The photo of the antibacterial test of Aspergillus flavus CK;

[0028] Figure 9 The photo of the antibacterial test of Aspergillus flavus 6d;

[0029] Figure 10 The morphology of 2SZ(SF)-Ba5 strain on the medium with 0 μg / mL sodium selenite;

[0030] Figure 11 The morphology of 2SZ(SF)-Ba5 strain on the medium with 20 μg / mL sodium selenite;

[0031] Figure 12 The morphology of 2SZ(SF)-Ba5 strain on the medium with 40 μg / mL sodium selenite;

[0032] Figure 13 The morphology of 2SZ(SF)-Ba5 strain on the medium with 80 μg / mL sodium selenite;

[0033] Figure 14 The morphology of 2SZ(SF)-Ba5 strain on the medium with 160 μg / mL sodium selenite;

[0034] Figure 15 The morphology of 2SZ(SF)-Ba5 strain on the medium with 320 μg / mL sodium selenite. DETAILED DESCRIPTION

[0035] Hereinafter, the present application will be further described in conjunction with the accompanying drawings and specific embodiments:

[0036] Example 1:

[0037] A Bacillus sp. was obtained from the following method:

[0038] 1) Isolation and purification of the strain: after sampling, the fermentation broth was prepared, 100 uL of the sample was taken in 900 uL of sterile water, and then the sample was diluted to 10 -4 , 10 -5 concentration, and placed in a 70°C water bath for 20 min. 100 uL of the liquid after water bath was spread on NA plates, and placed in a 28°C incubator for 72 h. The growth of bacteria on the plates was observed, and after the colonies grew, a typical single colony was picked and streaked on NA solid medium for isolation and purification of the strain. The single colony was inoculated into 5 mL of sterilized NB broth medium, and after 28°C static culture for 72 h, it was stored in -80°C refrigerator with 50 v / v% glycerol for standby.

[0039] The isolated and purified single colonies were identified using the following primers:

[0040] 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO. 2)

[0041] 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO. 3);

[0042] The 16S rDNA sequence of Bacillus vesicola 2SZ(SF)-Ba5 was obtained, as shown in SEQ ID NO.1, and the strain was named Bacillus vesicola 2SZ(SF)-Ba5.

[0043] 2) The sequence was compared and analyzed using the EZBioCloud database. The results showed that strain 2SZ(SF)-Ba5 was closely related to known type strains in the database. Cytobacillus massiliigabonensis Marseille -P2639 T The homology is 98.58%, which is suspected to be... Cytobacillus A new species of Bacillus vesicola.

[0044] 3) Upload the genome of the target strain to the Korean EzBioCloud website. Download the sequence of a type strain with high similarity and use the EzBioCloud online tool ANICalculator to calculate the ANI value. Use 95-96% as the cutoff value to determine whether the strains are different species. Upload the genomes of the target strain and related type strains to the online tool Genome-to-Genome Distance Calculator 3.0 on the German Culture Collection website. Use 70% as the cutoff value and calculate the dDDH value to determine whether the strains are different species.

[0045] Table 12 SZ(SF)-Ba5 genome ANI and dDDH calculation

[0046]

[0047] The genome of 2SZ(SF)-Ba5 and its closely related type strains Cytobacillus massiliigabonensis Marseille P2639 T In comparison, the ANI value was 72.08%, which is less than the threshold of 95%, and the dDDH value was 22.5%, which is less than the threshold of 70%. This indicates a new species of *Bacillus vesicatoria*, and it has been named *Bacillus vesicatoria*. Cytobacillus sp.)2SZ(SF)-Ba5. Figure 1 This refers to the colony morphology of the bacterial strain. Figure 2 It is in microscopic form.

[0048] 4) Growth curve determination of 2SZ(SF)-Ba5: the bacteria in the cryotube were inoculated into NB medium at an inoculation amount of 2% v / v, cultured in a 30°C incubator for 72 h, subcultured for 2 times, then washed twice with PBS buffer at 5000 r / min for 5 min, then adjusted the OD600nm of the strain to 1.0 with PBS buffer, inoculated the strain into NB medium at an inoculation amount of 2% v / v, placed in a growth curve determination instrument, cultured at 30°C, measured every 10 minutes, and obtained the growth curve of 2SZ(SF)-Ba5 (3 replicates).

[0049] As shown in Figure 3 , the time for 2SZ(SF)-Ba5 to grow to OD600nm of 1.0 was 22 h, and the time to reach the stationary phase was 75 h.

[0050] Detection:

[0051] 1) Inhibition effect detection of 2SZ(SF)-Ba5 strain:

[0052] The bacteria in the cryotube were inoculated into NB medium at an inoculation amount of 2% v / v, cultured in a 30°C incubator for 72 h, subcultured for 2 times, then washed twice with PBS buffer at 5000 r / min for 5 min, then adjusted the OD600nm of the strain to 1.0 with PBS buffer, inoculated the strain into NB medium at an inoculation amount of 2% v / v, cultured at 30°C for 75 h, and the obtained fermentation broth was reserved.

[0053] 100 μL of the fermentation broth of 2SZ(SF)-Ba5 strain was added to 4 holes respectively. Finally, a uniform size of bacteria block was placed in the center of the solidified plate, and cultured at 30°C for 6-10 d, and whether the strain grew was observed.

[0054] The bacteria blocks of pathogenic Sphaeria globosa, Curvularia and Sphaeropsis fuliginea were prepared respectively and repeated according to the above method.

[0055] Table 2 Inhibition experiment of 2SZ(SF)-Ba5 Inhibition rate (%)

[0056]

[0057] Figures 4-9 The photos of CK control of each pathogenic bacteria and the inhibition test of Sphaeria globosa 6d, Curvularia 6d and Sphaeropsis fuliginea 6d respectively. The 2SZ(SF)-Ba5 strain has obvious inhibition effect.

[0058] 2) Determination of effective phosphorus capacity of 2SZ(SF)-Ba5 strain:

[0059] Strains (adjusted OD = 1.0 or so) according to 2% v / v inoculation amount, inoculated to 30 mL of organic phosphorus and inorganic phosphorus in liquid medium in 50 mL centrifuge tube, with no inoculation of organic phosphorus and inorganic phosphorus in liquid medium as blank control (CK), 28°C, 200 rpm shaking culture for 5d, according to time point: 24h, 48h, 72h, 96h, 120h sampling, 5mL each time, 10000r / min, 4°C, centrifugal 5min, leave supernatant to 4°C preservation (3 repeats). The soluble phosphorus content in the supernatant at 24h, 48h, 72h, 96h, 120h was determined by molybdenum-antimony anti-colorimetric method.

[0060] Table 3 Soluble phosphorus concentration (μg / mL) of effective phosphorus capacity determination

[0061]

[0062] 3) Determination of IAA production capacity of 2SZ(SF)-Ba5 strain:

[0063] According to 2% v / v inoculation amount, OD600=1 bacterial suspension was inoculated in 5mL NB liquid medium (containing 100mg / L L-tryptophan) (using 10mL tube), placed in 28°C, 200rpm shaking incubator for 5d (3 repeats). Take 500uL of bacterial liquid at different time points: 24h, 48h, 72h, 96h, 120h in 1.5mL EP tube, 12000rpm centrifugal 5min, leave supernatant for standby. Take 50μL supernatant in 96-well plate, add 50μL Salkowski colorimetric liquid (A liquid plus B liquid mixed) at the same time. 96-well plate was placed at room temperature for 30min, then the OD530nm absorbance value was measured by enzyme marker.

[0064] Table 4 IAA concentration (mg / L)

[0065]

[0066] 4) 2SZ(SF)-Ba5 strain siderophore production capacity:

[0067] According to 2% v / v inoculation amount, OD600nm=1 bacterial suspension was inoculated in 5mL NB liquid medium (using 10mL tube), placed in 28°C, 200rpm shaking incubator for 5d (3 repeats). Take 500uL of bacterial liquid at different time points: 24h, 48h, 72h, 96h, 120h in 1.5mL EP tube, 12000rpm centrifugal 5min, leave supernatant for standby.

[0068] Take 100 μL supernatant in a 96-well plate, add an equal volume of CAS detection liquid and mix well, stand for 1 h, then measure the absorbance at 630 nm wavelength As with a microplate reader. Take an equal volume of sterile NB culture medium without inoculation and mix with an equal volume of CAS detection liquid, and measure the absorbance as the reference value Ar. Finally, calculate the amount of siderophore produced according to the formula (siderophore production = (Ar-As) / Ar x 100% siderophore production unit). If the calculation result is less than 10%, it is considered negative for siderophore secretion. Therefore, the 2SZ(SF)-Ba5 strain is positive for siderophore secretion.

[0069] Table 5 Siderophore production (%)

[0070]

[0071] 5) New species of selenium-rich (sodium selenite is converted to elemental selenium, qualitative):

[0072] Prepare NB solid medium and autoclave at 121°C for 30 min, cool to 60-80°C, add sodium selenite solution (concentration of mother liquor is 50 mg / mL) to make the concentration of sodium selenite in the medium: 0 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, 160 μg / mL, 320 μg / mL, pour the plate and cool it, then inoculate the 2SZ(SF)-Ba5 strain on the medium, incubate at 37°C for 24 h. If the strain can grow on the sodium selenite plate, absorb and convert sodium selenite, then form coffee-red elemental selenium on the surface of the plate and store it in the bacterial cells.

[0073] Figures 10-15 The morphology of 2SZ(SF)-Ba5 strain on sodium selenite plates at concentrations of 0 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, 160 μg / mL, 320 μg / mL, respectively.

[0074] Example 2:

[0075] Application of 2SZ(SF)-Ba5 strain microbial preparation:

[0076] Inoculate the 2SZ(SF)-Ba5 strain and Bacillus subtilis bacterial liquid into the fermentation medium according to a mass ratio of 3:1, and ferment at 28-32°C for 60 84h to obtain a fermentation broth with a concentration of 7x10 6 -9x10 6 cfu / mL. Centrifuge and wash, then dry to obtain the microbial preparation, which is stored in the dark at less than 30°C.

[0077] 1) Bacteriostatic effect detection of microbial preparation:

[0078] The mycelial blocks of Chaetomium globosum, Conidiobolus sp. and M. phaseolina were prepared respectively and repeated tests were carried out according to the method for detecting the bacteriostatic effect of 2SZ(SF)-Ba5 strain in Example 1.

[0079] Table 6 Bacteriostatic experiment of microbial preparation Bacteriostatic rate (%)

[0080]

[0081] The microbial preparation has a quick, stable and sustained bacteriostatic effect. It can be applied on seeds, roots, leaves and fruits by spraying, dipping, coating, encapsulation and / or dusting.

[0082] 2) Activity detection of microbial preparation in soil:

[0083] The sterilized alkaline soil (pH 7.8) was divided and mixed with the microbial preparation, and the mass ratio of the microbial preparation and the soil was 1:50. It was cultured at 28-30℃, and the soil without adding the microbial preparation was set as a control. 0 10cm soil layer sample 10g was collected at 0h, 24h and 48h, diluted with solvent and counted for the number of effective viable bacteria, and the results are shown in Table 7.

[0084] Table 7 Number of effective viable bacteria (cfu / g, 10 10 )

[0085]

[0086] From Table 7, it can be seen that the microbial preparation can effectively increase the number of effective viable bacteria in the soil, and the strains in it play a synergistic role to improve the soil environment.

[0087] 3) Detection of the influence of microbial preparation on tomato growth:

[0088] The experimental crop was selected as the 3rd tomato plant of Jinn tomato cultivated for 30d, which was transplanted into soil for planting, and divided into a control group and a preparation group, 30 plants were set in each group, and tap water was regularly irrigated. The preparation group was irrigated with the microbial preparation diluted 30 times every 7d.

[0089] After 1 month of cultivation, the plant height, stem diameter and leaf number were measured. After cultivation to the fruiting stage, the diameter of the fruit was measured. The average value was analyzed by sampling, and the results are shown in Table 8.

[0090] Table 8 Plant height, stem diameter, leaf number and fruit diameter

[0091]

[0092] 2SZ(SF)-Ba5 strain has good phosphorus conversion capacity and IAA production capacity, and after being processed into a microbial inoculant and applied to soil or plants, the growth of crops can be effectively promoted.

[0093] For those skilled in the art, other various corresponding changes and modifications can be made according to the technical solutions and concepts described above, and all these changes and modifications shall belong to the protection scope of the claims of the present application.

Claims

1. A type of vesicular Bacillus (Latin scientific name: Bacillus) Cytobacillus sp.), characterized in that, The *Bacillus vesicularis* was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 24, 2025, with accession number GDMCC No: 66749.

2. The *Bacillus vesicularis* as described in claim 1, characterized in that, The 16S rDNA of the Bacillus vesicularis is shown in SEQ ID NO.

1.

3. An application of *Bacillus vesicularis* as described in claim 1, characterized in that, The application is for preparing microbial preparations containing Bacillus vesicularis.

4. The application as described in claim 3, characterized in that, The preparation is obtained by the following method: Bacillus vesicatus and Bacillus subtilis are inoculated into a fermentation substrate for fermentation, and the obtained fermentation broth is dried.

5. The application as described in claim 4, characterized in that, The mass ratio of Bacillus vesicatus to Bacillus subtilis added is 3:(1-2).

6. The application as described in claim 4, characterized in that, The concentration of the fermentation broth is 7×10⁻⁶. 6 -9×10 6 cfu / mL.

7. The application as described in claim 3, characterized in that, The microbial preparation is a plant growth promoter and / or disease resistant agent; the pathogen causing the plant disease is Chaetomium globosum (Latin scientific name: Chaetomium globosum). Chaetomium globosum Curvularia (Latin scientific name: Curvularia ) and Coccidioides imidae (Latin scientific name: Macrophomina phaseoli At least one of the following.

8. The application as described in claim 7, characterized in that, The plant is at least one of green beans, rice, cucumber, and tomato.

9. The application as described in claim 3, characterized in that, The microbial preparation is a preparation that acts on at least one of plant seeds, roots, leaves, and fruits.

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