Bacillus licheniformis with salt-tolerant and growth-promoting functions and application of bacillus licheniformis

Abstract

The invention belongs to the technical field of agricultural microorganisms, and particularly relates to bacillus licheniformis with salt-tolerant and growth-promoting functions and application of the bacillus licheniformis. The invention discloses a bacillus licheniformis K4 with salt-tolerant and growth-promoting functions, which is preserved in the China General Microbiological Culture Collection Center on January 20, 2025, and the preservation number is CGMCC No. 33460. The invention also discloses a method for preparing the bacillus licheniformis K4 with salt-tolerant and growth-promoting functions. Experiments prove that the bacillus licheniformis K4 disclosed by the invention has various growth promoting characteristics such as phosphate solubilization and phosphate solubilization as well as high salt tolerance, can improve the utilization rate of soil, and provides an excellent strain resource for saline-alkali farmland soil remediation.

Description

Technical Field

[0001] The present invention belongs to the technical field of agricultural microorganisms, and particularly relates to a Bacillus licheniformis K4 with salt tolerance and plant growth promotion functions and its applications. Background Art

[0002] With the changes in the global ecological environment and the diversification of the ways humans utilize land resources, the problem of soil salinization has become increasingly prominent, becoming one of the key factors restricting agricultural development and disrupting the ecological balance. For most organisms, a high-salt environment is an extremely severe survival challenge. Adverse conditions such as high osmotic pressure and ionic toxicity make it difficult for general organisms to grow and reproduce in such an environment. However, salt-tolerant and plant growth-promoting bacteria have survived tenaciously in soils, water bodies with relatively high salt content and other extreme environments by virtue of their excellent salt tolerance mechanisms and unique physiological characteristics. The special environment of saline-alkali soil with high salt and poor nutrition contains many rich microbial resources adapted to this special environment, especially plant growth-promoting strains with high salt tolerance characteristics. By exploring the native salt-tolerant and plant growth-promoting functional microorganisms in saline-alkali land and utilizing their plant growth promotion functions such as phosphorus solubilization, potassium solubilization, phosphorus dissolution, and nitrogen fixation, the plant flora environment can be effectively improved, the available nutrient elements in the soil can be increased, the improvement and restoration of saline-alkali land can be accelerated, and crop growth can be promoted, thereby improving the utilization rate of saline-alkali soil. Salt-tolerant and plant growth-promoting bacteria will play a more important role in solving global ecological environment problems and promoting sustainable development. Although salt-tolerant and plant growth-promoting bacteria have broad application prospects, the current research on them is still in the stage of continuous in-depth exploration. Scientists are further exploring issues such as the salt tolerance mechanism of salt-tolerant and plant growth-promoting bacteria, the molecular mechanism of their interaction with plants, and how to more effectively exert their application potential in various fields. Summary of the Invention

[0003] Aiming at the deficiencies of the prior art, the problem to be solved by the present invention is to provide a Bacillus licheniformis with salt tolerance and plant growth promotion functions.

[0004] The technical solution of this application is as follows: In the first aspect, the present invention provides a Bacillus licheniformis with salt tolerance and plant growth promotion functions, and the Bacillus licheniformis is named Bacillus licheniformis ( Bacillus licheniformis ) K4. This strain was deposited in the "General Microbiological Center of the China Committee for Culture Collection of Microorganisms" (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing) on January 20, 2025, and the deposit number is CGMCC No. 33460.

[0005] According to the specific implementation manners of the present invention, the Bacillus licheniformis can maintain good growth under the condition of 1-14% salt concentration.

[0006] In a second aspect, the Bacillus licheniformis of the present invention has growth-promoting characteristics of phosphorus solubilization and phosphorus dissolution.

[0007] The Bacillus licheniformis with salt-tolerant growth-promoting function of the present invention has the following biological characteristics: the cell morphology is rod-shaped, single, the cell size is 0.8 µm × (1.5 µm - 3.5 µm), producing nearly central elliptical spores, and the sporangium is slightly enlarged. When cultured on solid medium at 30 °C, the colony is milky white, opaque, with a rough and wrinkled surface, easy to pick, and the edge is irregular; its physiological and biochemical characteristics are: Gram-positive staining, aerobic, the optimal growth temperature is 28 - 34 °C, the optimal growth pH value is 6.0 - 8.0, the salt tolerance range is 1 - 14%, and it has the characteristics of phosphorus solubilization and phosphorus dissolution.

[0008] The result of determining the 16S rRNA gene sequence of the Bacillus licheniformis K4 of the present invention shows that its gene length is 1474 bp, and the corresponding nucleotide sequence is shown in SEQ ID NO.1.

[0009] By using the BLASTN program of the National Center for Biotechnology Information (NCBI) in the United States for alignment and phylogenetic analysis, it is determined that the strain K4 of the present invention is a Bacillus licheniformis ( Bacillus licheniformis ).

[0010] Select the 16S rDNA sequences of 10 sequences with relatively high homology as the reference objects, and use the Neighbour-Joining method to construct the phylogenetic tree between strain K4 and the reference strains by using Mega 7 software. In the phylogenetic tree, strain K4 and the type strain of Bacillus licheniformis Bacillus licheniformis DSM13 form a separate intra-cluster evolutionary branch ( Figure 6 ).

[0011] The composition of the LB liquid medium used for the study of the biological and physiological and biochemical characteristics of the strain is: 10 g of peptone, 5 g of yeast powder, 10 g of sodium chloride in every 1000 mL of distilled water, and the pH is adjusted to 7.0.

[0012] The composition of the LB solid medium used for the study of the biological and physiological and biochemical characteristics of the strain is: 10 g of peptone, 5 g of yeast powder, 10 g of sodium chloride, 10 g of agar powder in every 1000 mL of distilled water, and the pH is adjusted to 7.0.

[0013] The basic method for breeding the Bacillus licheniformis K4 with salt-tolerant growth-promoting function of the present invention is: Collect the rhizosphere soil of Suaeda salsa from the saline-alkali land in the Yellow River Delta Nature Reserve, oscillate and mix it evenly to make a soil suspension, dilute it in gradient and coat it on an LB plate containing 8% NaCl, and statically culture it at 28-30 °C. Pick single colonies onto an LB culture plate with the same salt concentration, obtain the strain after two purifications, and preserve the bacteria by glycerol freezing.

[0014] Advantages of the present invention: The present invention discloses a Bacillus licheniformis K4 with salt tolerance and growth promotion functions. The strain simultaneously has characteristics such as salt tolerance, phosphorus solubilization and phosphorus dissolution. It has a wide salt tolerance range and can maintain good growth under the condition of 1-14% salt concentration, and has great potential for application in saline-alkali soil remediation and agricultural production. The innovation of the present invention lies in obtaining a salt-tolerant strain with multiple growth promotion functions through multiple rounds of screening. This salt-tolerant growth-promoting strain provides excellent strain resources for the remediation of saline-alkali farmland soil, and the strain and its bacterial liquid have high agricultural application prospects and economic value.

[0015] Deposition description: The Bacillus licheniformis ( Bacillus licheniformis ) K4 of the present invention was deposited in the "China General Microbiological Culture Collection Center" on January 20, 2025, and the deposition number is CGMCC No. 33460. Description of the drawings

[0016] Figure 1 Figure 1 shows the cell morphology of Bacillus licheniformis K4 under the microscope after Gram staining; Figure 2 Figure 2 shows the colony morphology of Bacillus licheniformis K4 on an LB solid medium; Figure 3 Figure 3 shows the growth curves of Bacillus licheniformis K4 under different salt concentration conditions; where a is 1% salt concentration; c is 4% salt concentration; d is 6% salt concentration; e is 8% salt concentration; f is 10% salt concentration; g is 12% salt concentration; h is 14% salt concentration; Figure 4 Figure 4 shows the effect diagram of phosphorus solubilization (organic phosphorus) of Bacillus licheniformis K4; Figure 5 Figure 5 shows the effect diagram of phosphorus dissolution (inorganic phosphorus) of Bacillus licheniformis K4; Figure 6 Figure 6 shows the phylogenetic tree between Bacillus licheniformis K4 and reference strains. Detailed implementation manners

[0017] The content of the present invention will be described in detail below in conjunction with specific drawings and embodiments. The following examples are only the preferred embodiments of the present invention. It should be noted that the following description is only for explaining the present invention and does not impose any formal restrictions on the present invention. Any simple modifications, equivalent changes and modifications made to the embodiments based on the technical essence of the present invention all fall within the scope of the technical solution of the present invention.

[0018] In the following embodiments, the materials, reagents, etc. used are obtained from commercial channels without special instructions.

[0019] Example 1 Screening of Halotolerant Bacillus licheniformis (1) Collect root soil samples of salt-tolerant plants such as Suaeda salsa and Phragmites australis from saline-alkali land in the Yellow River Delta Nature Reserve, put them into clean sampling bags, make marks, place them in an ice box and bring them back to the laboratory, and store them at -20 °C for later use. Weigh 1 g of soil sample respectively in a 100 mL triangular flask containing glass beads and 20 mL of sterile water in a sterile ultra-clean bench, and culture it in a shaking incubator at 28 °C and 180 rpm for 1 hour to fully disperse and mix the soil sample. Under sterile conditions, respectively pipette 0.5 mL of the soil suspension into 4.5 mL of sterile water and mix well, then sequentially dilute it to make sample solutions with different dilution factors of 10 -1 、10 -2 、10 -3 . Respectively pipette 0.2 mL of the sample solution and spread it on the solid medium plate for salt tolerance screening, and incubate it upside down in a constant temperature incubator at 28 °C for 2 - 4 days until obvious single colonies appear. Pick the single colonies respectively and transfer them to the same solid medium plate and number them in sequence for culture. After streaking twice, pure culture strain single colonies are obtained respectively.

[0020] (2) Use an inoculation loop to pick single colonies and transfer them to test tubes containing 5 mL of salt tolerance screening liquid medium, and culture them by shaking at 28 °C and 180 rpm for 24 h, and conduct strain preservation and physiological and biochemical analysis respectively. The strain preservation method adopts the glycerol tube freezing preservation method. Add 200 μL of glycerol and 800 μL of the bacterial solution to the freezing storage tube respectively, mix well and place it in an ultra-low temperature refrigerator at -80 °C for storage.

[0021] Among the screened strains, one strain has a rod-shaped cell morphology, with cell size of 0.8 µm × (1.5 µm - 3.5 µm), produces nearly central elliptical spores, and the sporangium is slightly swollen. When cultured on solid medium at 30°C, the colony is milky white, opaque, with a rough and wrinkled surface, easy to pick, and the edge is irregular; Gram-positive, aerobic, with an optimal growth temperature of 28 - 34°C, an optimal growth pH value of 6.0 - 8.0, a salt tolerance range of 1 - 14%, and has phosphorus-solubilizing and phosphorus-dissolving characteristics. This strain is numbered K4 and is preliminarily determined to be the selected strain of the present invention.

[0022] The composition of the above salt-tolerant screening solid medium is: 10 g of peptone, 5 g of yeast powder, 80 g of sodium chloride, and 10 g of agar powder in every 1000 mL of distilled water, and the pH is adjusted to 7.5 - 8.0.

[0023] The composition of the above salt-tolerant screening liquid medium is: 10 g of peptone, 5 g of yeast powder, 80 g of sodium chloride in every 1000 mL of distilled water, and the pH is adjusted to 7.5 - 8.0.

[0024] Example 2 Observation of the morphology and identification of the physiological and biochemical characteristics of strain K4 The morphology of strain K4 was observed using the oil immersion lens of an OLYMPUS microscope.

[0025] The culture temperature for the identification test of the physiological and biochemical characteristics of strain K4 was set at 30°C. At the same time, the optimal temperature, optimal growth pH value, and optimal salt concentration of strain K4 during growth in LB liquid medium were analyzed.

[0026] The biological characteristics of strain K4 are: rod-shaped, single, with cell size of 0.8 µm × (1.5 µm - 3.5 µm), produces nearly central elliptical spores, and the sporangium is slightly swollen ( Figure 1 ). When cultured on solid medium at 30°C, the colony is irregular in shape, milky white, opaque, with a rough and wrinkled surface, easy to pick, and the edge is irregular ( Figure 2 ).

[0027] The physiological and biochemical characteristics of strain K4 are: Gram-positive, aerobic, positive in esterase, lipase, leucine arylamidase, chymotrypsin, naphthol-AS-BI-phosphohydrolase, β-galactosidase, β-glucosidase tests, and negative in chitin hydrolysis test. The optimal growth temperature is 28 - 34°C, the optimal growth pH value is 6.0 - 8.0, and the salt tolerance range is 1 - 14% ( Figure 3 a - h).

[0028] The result of the determination of the 16S rRNA gene sequence of strain K4 of the present invention shows that its gene length is 1474 bp, and the corresponding nucleotide sequence is as shown in SEQ ID NO.1.

[0029] By using the BLASTN program of the National Center for Biotechnology Information (NCBI) in the United States for alignment and phylogenetic analysis, it was determined that the strain K4 described in the present invention is a Bacillus licheniformis ( Bacillus licheniformis ).

[0030] Select the 16S rDNA sequences of 10 sequences with relatively high homology as the reference objects, and use the Neighbour-Joining method to construct a phylogenetic tree between strain K4 and the reference strains using Mega 7 software ( Figure 6 ). In the phylogenetic tree, strain K4 and the type strain of Bacillus licheniformis Bacillus licheniformis DSM13 form a separate intra-cluster evolutionary branch ( Figure 6 ).

[0031] The composition of the LB liquid medium used for the study of the morphological, physiological and biochemical characteristics of the strain is: 10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride in every 1000 mL of distilled water, and the pH is adjusted to 7.0.

[0032] The composition of the LB solid medium used for the biological study of the strain is: 10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride, 10 g of agar powder in every 1000 mL of distilled water, and the pH is adjusted to 7.0.

[0033] Example 3 Analysis of the salt tolerance effect of strain K4 Transfer the single colony of strain K4 obtained by isolation in Example 1 to a test tube containing 5 mL of LB liquid medium, and shake culture at 30 °C and 200 rpm for 24 h. Then, take 50 µL of the bacterial liquid and transfer it to LB liquid media with different salt concentrations. Set 3 parallels for each salt concentration gradient, and measure its OD 600 value, and draw the growth curve of strain K4 at salt concentrations of 1%, 2%, 4%, 6%, 8%, 10%, 12% and 14% ( Figure 3 a-h). Set the salt concentration in the initial LB medium to 1%.

[0034] The results show that strain K4 can maintain good growth at salt concentrations of 1-14%, and has a wide salt tolerance range.

[0035] The composition of the initial LB liquid medium used is: 10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride in every 1000 mL of distilled water, and the pH is adjusted to 7.0.

[0036] Example 4 Analysis of the growth-promoting effects such as phosphorus solubilization and phosphorus release of strain K4 The single colony of strain K4 obtained by separating in Example 1 was transferred to a test tube containing 5 mL of LB liquid medium and cultured with shaking at 30 °C and 200 rpm for 24 h. 5 μL of the seed liquid was taken and spot-inoculated onto an organic phosphorus solid culture plate, and then cultured in a constant temperature incubator at 30 °C. Whether there was a degradation clear zone around the colony was observed. The results showed that after culturing strain K4 on the organic phosphorus solid medium for 8 days, a degradation clear zone appeared around the colony, indicating that strain K4 could dissolve organic phosphorus and had the characteristic of phosphorus solubilization ( Figure 4 ).

[0037] The single colony of strain K4 obtained by separation was transferred to a test tube containing 5 mL of LB liquid medium and cultured with shaking at 30 °C and 200 rpm for 24 h. 5 μL of the seed liquid was taken and spot-inoculated onto an inorganic phosphorus solid culture plate, and then cultured in a constant temperature incubator at 30 °C. After culturing for 8 days, a transparent degradation zone was produced around the colony, indicating that strain K4 could dissolve inorganic phosphorus and had the characteristic of phosphorus solubilization ( Figure 5 )

[0038] The components and concentrations of the used organic phosphorus solid medium were: 10 g L -1 glucose, 0.5 g L -1 (NH 4 ) 2 SO 4 , 0.3 g L -1 NaCl, 0.3 g L -1 MgSO 4 .7H 2 O, 0.03 g L -1 MnSO 4 .H 2 O, 0.3 g L -1 KCl, 0.03 g L - 1 FeSO 4 .7H 2 O, 2.0 g L -1 lecithin, 15 g L -1 agar.

[0039] The components and concentrations of the used inorganic phosphorus solid medium were: 10 g L -1 glucose, 0.5 g L -1 (NH 4 ) 2 SO 4 , 0.3 g L -1 NaCl, 0.3 g L -1 MgSO 4 .7H 2 O, 0.03 g L-1 MnSO 4 .H 2 O, 0.3 g L -1 K 2 SO 4 , 0.03 g L - 1 FeSO 4 .7H 2 O, 5.0 g L -1 Ca 3 (PO 4 ) 2 , 15 g L -1 agar.

[0040] 16S rRNA Gene Identification Method of Strain K4 in Example 5 Transfer the single colony of strain K4 isolated in Example 1 into a test tube containing 5 mL of LB liquid medium, and culture it with shaking at 30 °C and 200 rpm for 24 h. Collect the bacterial liquid and send it to a sequencing company for 16S rRNA sequencing.

[0041] The result of measuring the 16S rRNA gene sequence of strain K4 shows that its gene length is 1474 bp, and the corresponding nucleotide sequence is shown in SEQ ID NO.1.

[0042] Submit the 16S rRNA gene sequence obtained by sequencing to the NCBI database (http: / / www.ncbi.nlm.nih.gov) for BLASTN alignment. The result shows that the 16S rRNA of this strain has 99% similarity with Bacillus licheniformis Bacillus licheniformis DSM13. Select the 16S rDNA sequences of 10 sequences with relatively high homology as the reference objects, and use the Neighbour-Joining method to construct a phylogenetic tree between strain K4 and the reference strains using Mega 7 software ( Figure 6 ). It is determined that the strain K4 described in the present invention is a Bacillus licheniformis ( Bacillus licheniformis ), so it is named Bacillus licheniformis K4.

Claims

1. A strain of Bacillus licheniformis having salt-tolerant growth-promoting function, characterized in that: The Bacillus licheniformis is named Bacillus licheniformis ( Bacillus licheniformis )K4, which was deposited in the "General Microbiology Center of China Microbiological Culture Collection Administration" on January 20, 2025, with the deposit number CGMCC No. 33460.

2. The Bacillus licheniformis having salt-tolerant growth-promoting function according to claim 1, characterized in that: The Bacillus licheniformis can maintain good growth under the condition of 1-14% salt concentration.

3. The salt-tolerant growth-promoting Bacillus licheniformis according to claim 1 or 2 has the growth-promoting properties of solubilizing and dissolving phosphate.

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