Saline-alkali tolerant bacillus velezensis TYGC262 and application thereof in siderophore production
By screening and applying salt-alkali Bacillus Bacillus resistant Bacillus Bacillus Beles TYGC262, ferrite-philic microbial preparations were produced, which solved the problems of plant seed germination and growth in saline-alkali soil, and achieved efficient ferrite-based production and plant growth promotion effects.
Patent Information
- Application Number
- CN202510472132.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-15
- Publication Date
- 2025-07-18
AI Technical Summary
The prior art is difficult to efficiently produce ferrite, especially in saline-alkali soil, and the effect of promoting plant seed germination and growth is not significant.
A salt-alkali-resistant Bacillus Bacillus Bacillus TYGC262 was screened, and a microbial preparation containing a mixed ferrite of citric acid and oxygenamic acid was prepared through a fermentation broth, and it was applied to plant seed treatment.
This strain can efficiently produce ferrite, significantly promote the germination and growth of plant seeds, improve the solubility and mobility of iron in the soil, and improve the plant growth environment in saline-alkali soil.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of microbiology, and in particular to a strain of Bacillus velezensis TYGC262 and its application in the production of siderophores. Background Art
[0002] Iron is an essential element for the growth and development of all living organisms. Iron is an important cofactor for various enzymes in the process of cellular redox reactions, and plays an important role in many aspects such as cellular oxygen metabolism, electron transfer, and RNA synthesis. Although iron is abundant on Earth, it mostly exists in insoluble forms with extremely low bioavailability. Under such a stress, microorganisms have evolved multiple pathways to obtain the necessary iron. Siderophore-mediated iron uptake is the most common way for microorganisms to acquire iron, and many microorganisms even synthesize and secrete siderophores exceeding their own cell dry weight to meet the nutritional requirements of iron.
[0003] Siderophores are small molecular weight chelating factors secreted by organisms in the long-term evolutionary process to acquire iron under low-iron environmental conditions, and have a very strong binding ability with Fe 3+ (the formation constant can reach 1023 - 1052), and is specific. In recent years, with the in-depth study of the iron nutrition of microorganisms themselves, some physiological functions of microorganisms have attracted more and more attention from researchers in plant iron nutrition. Siderophores produced by microorganisms can activate insoluble iron in the soil, improve the solubility and mobility of iron, and thus improve the availability of iron in the soil. So far, more than 500 different siderophores have been identified. Although siderophores vary greatly in overall structure, they can be divided into three major categories according to the chemical characteristics of the chelating groups with Fe 3+ : hydroxamate-type, catecholate-type, and hydroxycarboxylate-type siderophores.
[0004] Siderophores secreted by microorganisms can inhibit the growth of pathogenic bacteria by competing with pathogenic bacteria for limited iron. Some studies have found that many siderophore-producing bacteria can promote the germination of plant seeds. As a substance with extremely strong iron-binding ability, siderophores have good application prospects in iron absorption intervention, and have important theoretical and application significance for controlling and improving iron deficiency in plants. Summary of the Invention
[0005] The purpose of the present invention is to provide a strain of salt-tolerant and alkali-tolerant Bacillus velezensis TYGC262 and its application in the production of siderophores.
[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:
[0007] The present invention provides a salt-tolerant Bacillus velezensis strain TYGC262, which is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is January 6, 2025, and the deposit number is CGMCC No. 33301.
[0008] The present invention provides a fermentation broth of the Bacillus velezensis TYGC262.
[0009] The present invention provides a method for preparing the fermentation broth, which comprises inoculating Bacillus velezensis TYGC262 into a culture medium for cultivation, performing solid-liquid separation, and taking the liquid part to obtain the fermentation broth.
[0010] Preferably, the culture medium is LB liquid medium.
[0011] Preferably, the temperature during cultivation is 28 - 32 °C, the rate is 150 - 200 rpm, and the time is 2 - 4 d.
[0012] Preferably, the solid-liquid separation method is centrifugation, and the centrifugation rate is 5000 - 8000 rpm, and the time is 3 - 7 min.
[0013] The present invention provides a microbial preparation, which comprises the Bacillus velezensis TYGC262 or the fermentation broth or the fermentation broth prepared according to the method.
[0014] The present invention provides an application of the microbial preparation in the production of siderophores.
[0015] Preferably, the siderophores are a mixture of citric acid type and hydroxamic acid type.
[0016] The present invention provides an application of the microbial preparation in promoting plant germination and growth.
[0017] Compared with the prior art, the present invention has the following beneficial effects:
[0018] In the present invention, a strain capable of producing siderophores was screened from saline-alkali soil samples. After identification, the strain belongs to Bacillus velezensis and is named Bacillus velezensis TYGC262. The Bacillus velezensis TYGC262 can efficiently produce siderophores, with a siderophore production ability of ++++, and the produced siderophores are a mixture of citric acid type and hydroxamic acid type. The present invention also confirms that the Bacillus velezensis TYGC262 has a good effect on promoting the germination and growth of plant seeds. Brief Description of the Drawings
[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.
[0020] Figure 1 The state diagram of the strain screened in Example 1 after 2 days of cultivation in LB solid medium;
[0021] Figure 2 The phylogenetic tree constructed in Example 1;
[0022] Figure 3 The growth status diagram of the Sesbania seeds cultivated in Example 3.
[0023] Deposition description
[0024] Bacillus velezensis TYGC262, this strain is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, the address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposition date is: January 6, 2025, and the deposition number is: CGMCC No. 33301. Specific implementation manners
[0025] The following will detail the technical solutions provided by the present invention in combination with the embodiments, but they cannot be construed as limiting the protection scope of the present invention.
[0026] Example 1
[0027] 1. Preparation of culture media
[0028] LB liquid medium: Add 5 g of yeast extract powder, 10 g of tryptone, and 10 g of NaCl to 1000 mL of distilled water, mix well, and autoclave at 121 °C for 20 min.
[0029] LB solid medium: Add 5 g of yeast extract powder, 10 g of tryptone, 10 g of NaCl, and 15 g of agar to 1000 mL of distilled water, mix well, and autoclave at 121 °C for 20 min.
[0030] MKB medium: Add 5 g of casein amino acids, 2.5 g of dipotassium hydrogen phosphate, and 2.5 g of magnesium sulfate to 1000 mL of distilled water, add 15 mL of glycerol, stir and dissolve at a slightly warm temperature, and autoclave at 115 °C for 30 min.
[0031] CAS detection medium: Weigh 60.5 mg of chrome azurol S (CAS), 72.9 mg of hexadecyltrimethylammonium bromide (HDTMA), 2.645 mg of ferric chloride, 295.25 mg of sodium dihydrogen phosphate, 1213.5 mg of disodium hydrogen phosphate, 125 mg of ammonium chloride, 37.5 mg of potassium dihydrogen phosphate, 62.5 mg of sodium chloride, and 9000 mg of agar, add them to 1000 mL of distilled water, mix well, and sterilize at 116 °C under high pressure for 30 min.
[0032] 2. Obtaining of siderophore-producing strains
[0033] Collect saline-alkali soil samples from the environment, coat them on the CAS detection medium by gradient dilution method with different salinity and pH concentrations. After colonies grow (tolerance salinity is 5% and pH is 9.5), pick single colonies with larger halo zones, perform plate streaking purification, pick single colonies and inoculate them into LB solid medium to obtain a strain capable of producing siderophores. The state diagram of this strain cultured at 38 °C for 2 days in LB solid medium is as Figure 1 shown.
[0034] 3. Molecular identification of the strain
[0035] Entrust Shanghai Tianhao Biotechnology Company to determine the 16S rDNA sequence of the strain. The specific method is as follows: Extract the genomic DNA of the strain, use the universal primers 27F (5’-AGAGTTTGATCCTGGCTCAG-3’, SEQ ID NO.1) and 1492R (5’-TACCTTGTTACGACT-3’, SEQ ID NO.2) of bacterial 16S rDNA for PCR amplification. The PCR reaction system is: reaction mixture (20 μL), including 10 μL of 2×GC buffer I (TAKARA), 2 μL of MgCl2 (25 mmol / L), 1.6 μL of dNTP (2.5 mmol / L), 0.4 μL + 0.4 μL of each primer (10 μmol / L), 0.2 μL of 5 U / μL HotStarTaq polymerase (TAKARA), and 1 μL of template DNA, add water to make up to 20 μL; The cycling program is: pre-denaturation at 95 °C for 2 minutes; 11 cycles (94 °C for 20 seconds, 64 °C - 0.5 °C / cycle for 40 seconds, 72 °C for 90 seconds); 24 cycles (94 °C for 20 seconds, 58 °C for 30 seconds, 72 °C for 90 seconds); extension at 72 °C for 2 minutes; hold at 4 °C constantly. Sequence the PCR amplification products. The obtained 16S rDNA sequence of the strain is:
[0036]
[0037] The 16S rDNA sequence of the strain to be tested obtained by PCR was submitted to the NCBI database for BLAST homology analysis, and then sequence alignment and phylogenetic tree construction were performed using MEGA 11.0 software.
[0038] BLAST alignment analysis showed that the strain to be tested had a high homology with Bacillus velezensis, reaching 100%. The constructed phylogenetic tree is as Figure 2 shown. It can be seen that the strain to be tested and Bacillus velezensis are in the same branch. The strain to be tested was classified into the genus Bacillus velezensis and named Bacillus velezensis TYGC262. The Bacillus velezensis TYGC262 was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date was January 6, 2025, and the deposit number was CGMCC No. 33301.
[0039] Example 2
[0040] 1. Determination of the ability of strain TYGC262 to produce siderophores
[0041] Single colonies of the TYGC262 strain screened in Example 1 were inoculated into LB liquid medium. After culturing for 24 h, they were transferred to 100 mL of MKB liquid medium at an inoculation amount of 1% and cultured with shaking at 30 °C and 180 rpm for 24 h. The cultured fermentation broth was centrifuged at 5000 rpm for 4 min by a low-speed refrigerated centrifuge and then filtered. The filtrate was mixed with an equal volume of CAS detection solution, and after standing at room temperature for 1 h, the OD 630 .
[0042] The activity unit of siderophores was calculated by the formula: SU = (Ar - As) / Ar; the ability of the strain to produce siderophores was estimated by As / Ar. Among them, Ar refers to the OD 630 measured after mixing an equal volume of MKB liquid medium and CAS detection solution and standing at room temperature for 1 h; As refers to the OD 630 measured after mixing an equal volume of the strain fermentation filtrate and CAS detection solution and standing at room temperature for 1 h.
[0043] As / Ar ranges from 1.0 to 0, with a decrease of 0.2 at intervals. For each decrease of 0.2, a "+" is added. Generally, bacteria with a relatively high siderophore-producing ability (+++) have an As / Ar lower than 0.5. The measurement results show that the siderophore activity unit SU of strain TYGC262 is 75.24%, As / Ar = 0.24, and the ability to produce siderophores is ++++. It can be seen that strain TYGC262 is a strain capable of efficiently producing siderophores.
[0044] 2. Identification of the type of siderophore produced by strain TYGC262
[0045] Inoculate strain TYGC262 into LB liquid medium and shake culture at 30 °C and 180 rpm for 3 d. Centrifuge the fermentation broth at 10,000 rpm for 5 min, take the liquid part to obtain the fermentation supernatant of strain TYGC262. Identify the type of siderophore produced by strain TYGC262 by the following methods:
[0046] (1) Detection of catechol-type siderophores by the Arnow method
[0047] Take 1 mL of the supernatant and mix it with 1 mL of 0.5 mol / L HCl, 1 mL of molybdate-sodium nitrite, and 1 mL of NaOH, then add 1 mL of water to make the volume 5 mL. If catechol-type siderophores are present, the color of the mixture will turn red, and measure the OD of the mixture within 1 h. 680 . The darker the color, the 680 greater the OD value indicates the greater the content of catechol-type siderophores.
[0048] (2) Detection method for carboxylate-type siderophores
[0049] Take 1 mL of the supernatant and mix it with 1 mL of 250 μmol / L CuSO4 and 2 mL of 0.2 mol / L pH 4.0 acetate buffer, then add 1 mL of water to make the volume 5 mL, and measure the light absorption at 190 nm - 400 nm. If carboxylate-type siderophores are present, the mixture has a maximum light absorption at 190 nm - 280 nm.
[0050] (3) Detection of hydroxamate-type siderophores by the FeCl3 method
[0051] Take 1 mL of the supernatant and mix it with 200 μL of 100 mmol / L FeCl3. If trihydroxy hydroxamate-type siderophores are present, the mixture will turn orange; if dihydroxy hydroxamate is present, the mixture will turn pink.
[0052] The results show that the siderophores produced by strain TYGC262 are a mixture of citric acid type and hydroxamate type (trihydroxy hydroxamate type).
[0053] Example 3
[0054] Promotion of Siderophore on Plant Germination Rate and Growth
[0055] Inoculate strain TYGC262 into LB liquid medium, shake culture at 30 °C and 180 rpm for 3 d, centrifuge the fermentation broth at 4000 rpm for 5 min, take the liquid part and adjust OD 600 to 0.5 - 0.6, and dilute 1 mL of the adjusted supernatant with sterile water to 100 mL.
[0056] After sterilizing Sesbania seeds (20 pieces) with alcohol, wash them clean with distilled water and place them on a culture dish with filter paper on the bottom layer. Add sterile water to the control group and add the same amount of the above-prepared supernatant dilution to the experimental group. Incubate in the dark in an incubator at 28 °C (humidity 70%), and observe the seed germination rate and growth status after 6 d. The results are shown in Table 1 and Figure 3 as follows.
[0057] Table 1 Seed Germination Rate and Growth Status
[0058] Treatment group Germination rate / % Bud length / cm Fresh weight / g Control group 52.5 5.77 0.1961 Experimental group 65.0 8.85 0.2901
[0059] As can be seen from Table 1 and Figure 3 it can be seen that the germination rate, budding length and fresh weight of Sesbania seeds in the experimental group are significantly increased compared with those in the control group, indicating that strain TYGC262 has a good effect on promoting the germination and growth of plant seeds.
[0060] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.
Claims
1. A strain of Bacillus velezensis TYGC262, which is deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposition date is January 6, 2025, and the deposition number is CGMCC No. 33301.
2. A fermentation broth of the Bacillus velezensis TYGC262 described in claim 1.
3. The preparation method of the fermentation broth according to claim 2, characterized in that, The Bacillus velezensis TYGC262 is inoculated in a medium for cultivation, and the liquid part is taken to obtain the fermentation broth.
4. The method according to claim 3, characterized in that The medium is an LB liquid medium.
5. The method according to claim 3, characterized in that, The temperature during the cultivation is 28 - 32 °C, the rate is 150 - 200 rpm, and the time is 2 - 4 days.
6. The method according to claim 3, wherein The method of solid - liquid separation is centrifugation, the rate of centrifugation is 5000 - 8000 rpm, and the time is 3 - 7 minutes.
7. A microbial preparation, characterized in that, The microbial preparation includes the Bacillus velezensis TYGC262 described in claim 1, or the fermentation broth described in claim 2, or the fermentation broth prepared by the method according to any one of claims 3 - 6.
8. An application of the microbial preparation described in claim 7 in the production of siderophore.
9. The application according to claim 8, wherein The siderophore is a mixed type of citrate - type and hydroxamate - type.
10. An application of the microbial preparation described in claim 7 in promoting plant germination and growth.