A highly salt-tolerant marine sediment Bacillus LS06 and its application

Abstract

The present invention belongs to the technical field of microbiology, and discloses a highly salt-tolerant marine sediment Bacillus LS06 and its application, which has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on August 2, 2024, with a deposit number of CGMCC No. 31524. The marine sediment Bacillus LS06 has the ability to dissolve potassium, fix nitrogen, degrade protein and tolerate salt, which can help wheat resist saline-alkali environment and promote wheat growth.

Description

Technical Field

[0001] The invention belongs to the technical field of microbiology, and specifically relates to a highly salt-tolerant marine sediment Bacillus LS06 and an application thereof. Background Art

[0002] The statements herein merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] Salt-alkali tolerant microorganisms can help plants adapt to saline-alkali environments and improve their salt-alkali tolerance. Salt-tolerant bacteria absorb extracellular potassium ions and excrete intracellular sodium ions through the sodium-potassium pump on the membrane, which reduces the concentration of intracellular sodium ions and helps plants adapt to high-salt environments. In abiotic adversities, growth-promoting bacteria can improve the absorption of mineral nutrients and water by plants and enhance stress resistance. At the same time, rhizosphere growth-promoting bacteria can change the morphological or physiological characteristics of plants by fixing nitrogen, dissolving phosphorus, dissolving potassium, producing iron carriers, secreting secondary metabolites and plant hormones, and inducing the expression of plant stress resistance genes, thereby increasing the utilization rate of nutrients by plants and improving plant yield, quality and stress resistance. Therefore, rhizosphere growth-promoting bacteria have broad application prospects in agriculture.

[0004] Currently, the research on Bacillus sp. Cytobacillus oceanisedimins ) are rarely studied. In terms of plant stress resistance, Vaishali Umrao found that marine sediment Bacillus can improve the drought resistance of guar plants, but there are no reports that marine sediment Bacillus can promote the salt-alkali tolerance of wheat. Summary of the invention

[0005] In view of the shortcomings of the prior art, the object of the present invention is to provide a highly salt-tolerant marine sediment Bacillus LS06 and applications thereof.

[0006] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0007] In a first aspect, the present invention provides a highly salt-tolerant marine sediment Bacillus ( Cytobacillus oceanisedimins )LS06, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on August 2, 2024, with the deposit number CGMCC No. 31524.

[0008] In a second aspect, the present invention provides a mixed bacterial agent, wherein the mixed bacterial agent at least includes the highly salt-tolerant marine sediment Bacillus LS06.

[0009] In a third aspect, the present invention provides a product comprising the highly salt-tolerant marine sediment Bacillus LS06 or the mixed bacterial agent.

[0010] In some embodiments, the product is in the form of a powder, a solution, a suspension, a tablet, or a granule.

[0011] Preferably, the product comprises a carrier, and the carrier is liquid or solid.

[0012] In a fourth aspect, the present invention provides the use of the highly salt-tolerant marine sediment Bacillus LS06 or the mixed bacterial agent in promoting the growth of wheat seedlings.

[0013] In some embodiments, the marine sediment Bacillus LS06 or the mixed bacterial agent of the present invention is used to promote the growth of wheat seedlings in a saline-alkali environment.

[0014] In a fifth aspect, the present invention provides the use of the highly salt-tolerant marine sediment Bacillus LS06 or the mixed bacterial agent in potassium solution.

[0015] In a sixth aspect, the present invention provides the use of the highly salt-tolerant marine sediment Bacillus LS06 or the mixed bacterial agent in nitrogen fixation.

[0016] In a seventh aspect, the present invention provides the use of the highly salt-tolerant marine sediment Bacillus LS06 or the mixed bacterial agent in degrading protein.

[0017] The beneficial effects achieved by one or more embodiments of the present invention are as follows:

[0018] The highly salt-tolerant marine sediment Bacillus LS06 of the present invention has the abilities of dissolving potassium, fixing nitrogen, degrading protein and being salt-tolerant, and can help wheat resist saline-alkali environment and promote the growth of wheat. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0020] Figure 1 This is a colony morphology diagram of strain LS06 in an embodiment of the present invention;

[0021] Figure 2 It is a comparison diagram of the colony morphology of the strain LS06 in the embodiment of the present invention in different salt concentration environments, wherein A is 7% sodium chloride concentration; B is 9% sodium chloride concentration;

[0022] Figure 3 The graphs are the potassium-solubilizing ability test graph (A), nitrogen-fixing ability test graph (B) and protein-degrading ability test graph (C) of the strain LS06 in the embodiment of the present invention;

[0023] Figure 4is the genetic identification evolutionary tree of strain LS06 in the embodiment of the present invention;

[0024] Figure 5 is a comparison chart of the effect of strain LS06 on wheat seedling height in the embodiment of the present invention;

[0025] Figure 6 This is a comparison chart of the effects of strain LS06 on aboveground and underground fresh and dry weights of wheat in the embodiment of the present invention;

[0026] Figure 7 It is a comparison diagram of the effect of strain LS06 on the growth of wheat plants in the embodiment of the present invention, wherein A is the planting state; and B is the overall diagram after the wheat plants are taken out. DETAILED DESCRIPTION

[0027] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0028] The present invention will be further described below in conjunction with the embodiments.

[0029] Materials and methods

[0030] Culture media and reagents

[0031] The conventional bacterial culture media in this experiment were solid LB medium and liquid LB medium with sodium chloride concentrations of 5%, 7%, 9%, and 11%, respectively.

[0032] Table 1 Culture medium composition

[0033]

[0034] Experimental methods

[0035] Determination of salt tolerance of strains

[0036] Solid LB culture medium with a sodium chloride concentration of 5%, 7%, 9%, and 11% was poured into the plate, and a single colony was picked onto the culture medium. The plate was placed in a 37°C constant temperature incubator for 24 h, and the growth of the colony was observed.

[0037] Verification of the growth-promoting function of strains

[0038] The operation of the growth-promoting function of the strain is shown in Table 2.

[0039] Table 2 Verification of the growth-promoting function of strains

[0040]

[0041] Molecular Biology Identification

[0042] The 16S rDNA of single colonies was extracted for Blast comparison and analyzed using MEGA 11 software to construct a phylogenetic tree.

[0043] Single bacteria pot experiment

[0044] (1) Potting soil and wheat preparation

[0045] Soil was collected from the experimental field of Shandong Agricultural University, sieved and mixed evenly with vermiculite in a volume ratio of 1:2 to make nutrient soil for later use; plump and healthy wheat seeds were picked, and the surface dust was washed with distilled water. The seeds were soaked in water for 4 h, and then wrapped with wet gauze and placed at room temperature away from light. After white buds grew, they were placed in a 4°C refrigerator and vernalized for 3 days for later use.

[0046] (2) Planting method

[0047] The control group was set up with salt and without salt. The salt-free control group was irrigated with tap water, and the salt-containing control group and the experimental group were irrigated with an equal amount of mixed salt water. The molar ratio of each component in the mixed salt water was NaCl: Na2SO4: NaHCO3: Na2CO3 = 1:9:9:1.

[0048] For bacterial liquid preparation, the activated strain LS06 was inoculated into liquid LB and cultured at 37°C for 12 h to prepare seed liquid, and then transferred to new LB at a 2% inoculation rate and cultured at 37°C for 24 h to prepare fermentation liquid for use.

[0049] After the wheat breaks through the soil, dilute the fermentation liquid 100 times before use.

[0050] The experimental group was poured with fermentation broth dilution; the salt-free control group and the salt control group were poured with an equal amount of LB dilution.

[0051] Both the experimental group and the control group were placed in an incubator with a day and night temperature of 20 ℃ / 18 ℃, humidity of 50% RH, and light of 40,000 Lx for 12 days. They were regularly watered with an equal amount of tap water, and the plant height was measured every 3 days. On the 12th day, the above-ground and underground dry and fresh weights of wheat were weighed and recorded.

[0052] result

[0053] Colony morphology of strain LS06

[0054] A salt-tolerant strain was screened and named Marine Sediment Bacillus ( Cytobacillus oceanisedimins )LS06, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on August 2, 2024, with the deposit number CGMCC No. 31524. The colony morphology is shown in Figure 1 .

[0055] Salt tolerance of strain LS06

[0056] The strain LS06 was streaked in different salt concentrations to observe the salt tolerance of the strain. The results showed that the strain LS06 could tolerate a maximum salt concentration of 9% by mass, which means it is a salt-tolerant bacterium. Figure 2 As shown in A and B.

[0057] Functional verification of strain LS06

[0058] The activated strain LS06 was inoculated into the functional identification medium, and the strain's ability to dissolve inorganic phosphorus, degrade organic phosphorus, degrade cellulose, degrade protein, fix nitrogen, produce galactin and solubilize potassium was determined, see Table 2.

[0059] The results showed that in silicate bacterial culture medium, oily substances were observed on the surface of the strain, indicating that the strain had the function of dissolving potassium; in both the axoplasmite culture medium and the casein culture medium, transparent circles were observed, indicating that the strain had the functions of nitrogen fixation and protein degradation, respectively. Therefore, strain LS06 has the function of dissolving potassium (see Figure 3 A), nitrogen fixation (see Figure 3 B) and degraded proteins (see Figure 3 C) ability.

[0060] Molecular biological identification of strain LS06

[0061] Through the identification of the growth-promoting ability of the strains, strain LS06 was selected for molecular biological identification, and the 16SrDNA gene sequence of the single colony was extracted for Blast comparison. MEGA 11 software was used for analysis and phylogenetic tree was constructed. The analysis results are shown in Figure 4 The results showed that strain LS06 was similar to marine sediment Bacillus ( Cytobacillus oceanisedimins ) with a homology of 69%, and the strain LS06 was preliminarily identified as marine sediment Bacillus ( C. oceanisedimins ).

[0062] The 16S rDNA sequence of strain LS06 is shown in SEQ ID No.1:

[0063]

[0064] Effect of strain LS06 on salt-alkali tolerance and growth promotion of wheat

[0065] Salt-free CK group: put the mixed nutrient soil into a 5.5cm×5.5cm×7.5cm flower pot, then add 50 ml of salt-free tap water, and add 100-fold diluted sterilized liquid LB after the wheat seeds emerge from the soil;

[0066] CK group with salt: put the mixed nutrient soil into a 5.5cm×5.5cm×7.5cm flower pot, then add 50 ml of 130 mmol / L mixed salt water (the molar ratio of each component in the mixed salt water is NaCl: Na2SO4: NaHCO3: Na2CO3 = 1:9:9:1), and add 100-fold diluted sterilized liquid LB after the wheat seeds emerge from the soil;

[0067] LS06 group: Pour the mixed nutrient soil into a 5.5cm×5.5cm×7.5cm flowerpot, then add 50 ml of 130mmol / L mixed salt water, and add the fermentation liquid of strain LS06 diluted 100 times after the wheat seeds emerge.

[0068] The volume of nutrient soil in the salt-free CK group, salt-containing CK group, and LS06 group was the same.

[0069] like Figure 5 As shown in Figure 2, in terms of plant height, the wheat plant with strain LS06 increased by 127.96% ( P <0.05); on the 6th day, the wheat with strain LS06 increased by 122.68% compared with the salt CK ( P <0.05); on the 9th day, the wheat with strain LS06 increased by 68.35% compared with the salt CK ( P <0.05); on the 12th day, the wheat with strain LS06 increased by 99.22% compared with the salt CK ( P <0.05).

[0070] like Figure 6 As shown, when the wheat was grown to 12 days, the aboveground dry fresh weight of wheat in the salt-free CK group, salt-containing CK group and LS06 group was measured. In terms of aboveground fresh weight, the wheat with strain LS06 increased by 80.15% compared with the salt-containing CK group ( P <0.05); in terms of underground fresh weight, the wheat with strain LS06 increased by 72.14% compared with the salt CK group ( P <0.05); in terms of aboveground dry weight, the wheat with strain LS06 increased by 82.27% compared with the salt CK group ( P<0.05); in terms of underground dry weight, the wheat with strain LS06 increased by 35.67% compared with the salt CK group ( P <0.05).

[0071] like Figure 7 As shown in A and B, the potted biomass with the addition of strain LS06 was higher than that of the two control groups without the addition of strains (no-salt CK group and salt CK group); and the plant height and root length of wheat seedlings with the addition of strain LS06 were greater than those of the salt CK, respectively.

[0072] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A highly salt-tolerant marine sediment Bacillus ( Cytobacillus oceanisediminis )LS06, characterized in that: It was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on August 2, 2024, with the deposit number CGMCC No. 31524.

2. A mixed bacterial agent comprising at least the highly salt-tolerant marine sediment Bacillus LS06 according to claim 1.

3. A product comprising the highly salt-tolerant marine sediment Bacillus LS06 of claim 1 or the mixed bacterial agent of claim 2, characterized in that: The product is in the form of powder, solution, suspension, tablet or granules.

4. The product according to claim 3, characterized in that: The product comprises a carrier, which is liquid or solid.

5. Use of the highly salt-tolerant marine sediment Bacillus LS06 according to claim 1 or the mixed bacterial agent according to claim 2 in promoting the growth of wheat seedlings.

6. Use of the highly salt-tolerant marine sediment Bacillus LS06 according to claim 1 or the mixed bacterial agent according to claim 2 in promoting the growth of wheat seedlings in a saline-alkali environment.

7. Use of the highly salt-tolerant marine sediment Bacillus LS06 according to claim 1 or the mixed bacterial agent according to claim 2 in potassium solution.

8. Use of the highly salt-tolerant marine sediment Bacillus LS06 according to claim 1 or the mixed bacterial agent according to claim 2 in nitrogen fixation.

9. Use of the highly salt-tolerant marine sediment Bacillus LS06 according to claim 1 or the mixed bacterial agent according to claim 2 in protein degradation.

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