A salt-tolerant strain of Bacillus belye and its application

Abstract

This invention discloses a salt-stress-tolerant Bacillus bellis and its applications. It relates to the field of agricultural microbiology. The salt-stress-tolerant Bacillus bellis ZY-2, with accession number CGMCC NO.34345, is described, along with its related applications. The salt-stress-tolerant Bacillus bellis provided by this invention has broad application prospects in resisting saline-alkali environments. Specifically, this strain can be used as the original material to breed strains with better tolerance to saline-alkali environments and more significant effects on promoting wheat growth and development through natural selection, artificial mutation breeding, and genetic engineering breeding, thereby reducing the use of chemical amendments. Furthermore, it exhibits strong tolerance to saline-alkali environments and demonstrates good promotion of wheat growth and development under salt stress.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural microorganism technology, more particularly to a salt-tolerant Bacillus velezensis and application thereof. BACKGROUND

[0002] Saline-alkali soil is a kind of obstacle land of soil degradation. The salt in the soil mainly comes from the weathering, dissolution, wind erosion of rocks and the salt in the salt-containing rock layer, which is transferred to the soil under the action of surface water and wind to form saline-alkali soil. In saline-alkali soil, due to the long-term toxic effect of salt ions on the soil, the soil is degraded, and the nutrients are lost. Moreover, due to the presence of salt ions, ion competition with nutrients occurs, limiting the absorption of nutrients such as nitrogen, phosphorus and potassium by wheat, leading to nutrient imbalance, affecting the growth and health of wheat, and even causing death. Therefore, it is very important to improve saline-alkali soil, promote crop growth, alleviate salt stress on plants and improve soil ecology for the treatment of saline-alkali soil.

[0003] At present, the improvement of saline-alkali soil is mainly completed by physical, chemical and biological remediation methods. The physical improvement of saline-alkali soil mainly includes land leveling, soil loosening, water-flood rotation, deep ploughing, sunning, watering, salt leaching, and guest soil. Chemical improvement refers to the transformation, adsorption or fixation of salt and alkali components in the soil by applying chemical modifiers and organic matter to reduce the content of salt and alkali components in the soil. Biological improvement refers to the use of microorganisms in the soil and the planting of salt-tolerant plants to improve saline-alkali soil. Physical improvement requires a large amount of manpower, material resources and financial resources, and has high investment and maintenance costs, which is difficult to maintain soil quality in the long term. Although chemical improvement has a relatively fast effect, it has high cost and is easy to cause secondary pollution to the soil, so it is not a long-term solution. Compared with other traditional methods of improving saline-alkali soil, biological improvement has low cost, does not cause secondary pollution and has sustainable development effect, although it has a slower effect. Biological remediation can significantly reduce the salt and alkali components of the soil without damaging the soil and the environment, and improve soil fertility and promote plant growth.

[0004] Microorganisms are one of the important components in the soil environment and play an important role in the composition and development of soil. Microbial remediation has the advantages of economic efficiency, wide application range and no secondary pollution, and has great application potential in the improvement of saline-alkali soil.

[0005] Therefore, it is an urgent problem for those skilled in the art to provide a salt-tolerant Bacillus velezensis and application thereof to overcome the above technical deficiencies. SUMMARY

[0006] Therefore, the present application provides a salt-tolerant Bacillus velezensis and application thereof. It has good tolerance to saline-alkali environment and good promoting effect on the growth and development of wheat under salt stress.

[0007] Bacillus velezensis ZY-2 is preserved in the China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Haidian District, Beijing, and has a preservation number of CGMCC: NO. 34345 and a preservation date of April 25, 2025.

[0008] To achieve the above-mentioned purpose, the application adopts the following technical solutions:

[0009] The application provides a salt-tolerant Bacillus velezensis strain, which is named Bacillus velezensis ZY-2 and has a preservation number of CGMCC: NO. 34345.

[0010] Further, through molecular biology analysis, the 16S rDNA sequence of the salt-tolerant strain is compared with the sequence of the gene in the database of NCBI using Blast, and a phylogenetic tree is constructed using MEGA 11 software. The 16S rDNA is identified as Bacillus velezensis.

[0011] The effective effect is that the strain is a growth-promoting bacterium isolated by the applicant from the saline-alkali soil in Huanghua City, Hebei Province, which enriches the salt-tolerant growth-promoting bacterial resource and lays a foundation for the research and development of functional growth-promoting bacterial agents.

[0012] The application also provides a soil conditioner, a fertilizer or a biocontrol agent containing the salt-tolerant Bacillus velezensis.

[0013] Preferably, the preparation method of the biocontrol agent is as follows: a single colony of Bacillus velezensis ZY-2 is picked from an NA solid culture medium plate into an NB liquid culture medium, and the medium is cultured in a shaking table at a temperature of 37 DEG C and a rotation speed of 220 r / min. When the culture reaches the logarithmic growth phase, a microbial inoculum is obtained.

[0014] The beneficial effect is that through the strain growth experiment, it is found that the strain reaches the logarithmic growth phase after being cultured in the NB culture medium for 24 hours, and the activity of the strain is the largest.

[0015] The application also provides the application of the salt-tolerant Bacillus velezensis ZY-2 in the preparation of a genetically engineered bacterium.

[0016] The application also provides the application of the salt-tolerant Bacillus velezensis ZY-2 in resisting saline-alkali stress.

[0017] Preferably, the salt-tolerant Bacillus velezensis ZY-2 is applied to promoting the resistance of crops to saline-alkali stress and the growth and development of crops in a saline-alkali environment.

[0018] The beneficial effect is that the Bacillus velezensis ZY-2 has excellent salt tolerance, and has a wide range of adaptation to salt environment.

[0019] Preferably, the crop is wheat.

[0020] The beneficial effect is that the Bacillus velezensis ZY-2 can improve the root length, aboveground fresh weight and underground fresh weight of wheat under salt stress, indicating that the strain ZY-2 can alleviate the oxidative stress caused by salt stress on plants, and provides a basis for the salt-tolerant strain of the application in promoting salt tolerance.

[0021] Through the above technical solution, compared with the prior art, the application provides a salt-tolerant Bacillus velezensis and its application, and achieves the following technical effects:

[0022] The multifunctional strain Bacillus velezensis ZY-2 is isolated and screened from saline-alkali soil, has excellent salt tolerance, and the salt tolerance of the Bacillus velezensis ZY-2 is 0-11%, and the optimal salt tolerance is 5-9%. The biocontrol preparation (bacterial liquid) prepared by using the Bacillus velezensis ZY-2 can significantly improve the salt tolerance of wheat under salt stress, alleviate the salt damage of plants, increase the biomass, promote the growth of wheat, reduce the use of chemical fertilizers, and has a wide application prospect based on its excellent biological characteristics. In addition, the strain can be used as a raw material to breed strains with better salt-alkali environment tolerance and more significant promotion effect on wheat growth and development through natural breeding, artificial mutagenesis breeding, genetic engineering breeding and other methods.

[0023] The preparation method of the biocontrol preparation provided by the application is simple, the culture conditions are low, the fermentation cost is low, and the period is short. It is suitable for the needs of industrialized mass production, and is convenient for wide range of use. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description. Obviously, the drawings in the following description are only embodiments of the application, and for those skilled in the art, other drawings can be obtained without creative labor based on the provided drawings.

[0025] Figure 1 The figure is a ZY-2 salt-tolerant strain obtained by screening and identification provided by the application.

[0026] Figure 2 The figure is a salt tolerance of the ZY-2 strain provided by the application.

[0027] Figure 3 The figure is a growth chart of the ZY-2 strain provided by the application in an NB medium.

[0028] Figure 4 The figure is a phylogenetic tree of the ZY-2 strain provided by the application.

[0029] Figure 5 The figure is a chart of the effect of the ZY-2 strain on the growth of wheat under salt stress provided by the application.

[0030] Figure 6 The figure is a growth chart of a pot experiment provided by the application, in which, from left to right, the groups are CK, NaCl, and NaCl+ZY-2. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the application will be described clearly and completely below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only some of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the application.

[0032] The embodiments of the application disclose a salt-stress-resistant Bacillus velezensis and application thereof.

[0033] Embodiment 1

[0034] 1. Isolation and screening of ZY-2 and identification

[0035] The strain isolation sample is collected from the soil in the crop growth area of Zhongjie farm in Huanghua City, Hebei Province.

[0036] 2. Culture medium

[0037] NB medium: 10.0 g of proteose peptone, 5.0 g of NaCl, 5.0 g of beef extract, 1 L of distilled water, pH 7.2-7.4, 121℃, 20 min. Add 1.5-2.0 g of agar powder to obtain NA medium.

[0038] 3. Isolation of salt-stress-resistant strains

[0039] 10 g of the soil sample is added to a conical flask containing 90 ml of sterile water, and then placed on a shaker at 200 r / min for 30 min to fully mix the sample to obtain a soil suspension. The soil suspension is gradient-diluted, and the concentration gradient is selected as 10 -2 , 10 -3 , 10 -4 , 10 -5Soil suspensions were spread onto screening medium plates to isolate salt-tolerant strains. The plates were then inverted and incubated at 37°C for 24 hours to observe for colony growth.

[0040] The results showed that one strain exhibited significant tolerance to saline environments, named ZY-2. Figure 1 ).

[0041] 4. Salt tolerance identification of strains

[0042] With the pH of the culture medium remaining constant at 7.2, a salt concentration gradient culture medium was set up, namely NA medium with NaCl contents of 5%, 7%, 9%, 11%, and 13%. After spreading the ZY-2 strain onto the above salt concentration gradient culture medium, it was placed in an incubator at 37℃ and inverted for 24–72 h, and the growth was observed.

[0043] The results showed that ZY-2 exhibited significant tolerance to saline environments; however, with increasing salt concentration, the tolerance of the ZY-2 strain gradually decreased. Figure 2 It can be seen from the data that the maximum salt tolerance concentration of strain ZY-2 is 11% ( Figure 2 ).

[0044] 5. Detection of growth characteristics of salt-tolerant bacteria

[0045] The selected salt-tolerant strains were cultured in NB medium at 37℃ and 220 rpm in a shaker. The absorbance of the strains at 600 nm was measured at regular intervals. The culture time was 72 h. Figure 3 ).

[0046] like Figure 3 As shown, strain ZY-2 reached its maximum value after 24 hours of incubation.

[0047] 6. Establishment of a phylogenetic tree of bacterial strains

[0048] The 16S rDNA sequence of strain ZY-2 was compared with the NCBI database using BLAST, and a phylogenetic tree was constructed using MEGA 11 software. Figure 4 The bacteria were identified as Bacillus velezensis by 16S rDNA analysis.

[0049] Example 2: Effects of salt-tolerant strains on wheat growth and development under salt stress

[0050] The wheat variety used in the experiment was Zhoumai 18.

[0051] Straw ZY-2 was streaked on NA medium and incubated overnight at 37°C. Single colonies were picked and cultured on NB medium at 37°C and 220 rpm until OD600 = 1.0 was reached for use.

[0052] Select the same size, full wheat seeds, treated with sterile water and placed in a culture dish water immersion 12 h after wrapping with gauze seedling 12 h for use.

[0053] The soil, vermiculite, nutrient soil was sterilized at high temperature and mixed at a volume ratio of 1:1:1, and then planted in pots (diameter 18 cm x 20 cm) for use. Three treatments were set up for the pot experiment, namely, the blank control group CK, the NaCl treatment group, and the NaCl and salt-tolerant strain ZY-2 treatment group, with three replicates for each treatment group.

[0054] The blank control group was irrigated with water, the NaCl treatment group was irrigated with 150 mM saline, and the NaCl and salt-tolerant strain ZY-2 treatment group was irrigated with salt solution (150 mM) every five days and bacterial agent (8.5 x 10 7 CFU / mL) every seven days. The plant growth condition detection was carried out on a per plant basis, and the sampling amount of each group was 25 plants. After 30 days, the agronomic traits such as plant height, root length, aboveground fresh weight, underground fresh weight, and aboveground fresh weight, underground fresh weight of wheat under different treatments were measured, and then the average values were calculated.

[0055] The results are shown in Figure 5 and Figure 6 The application of salt and bacterial agent treatment group (NaCl+ZY-2) in plant height and underground dry weight was not significant compared with the blank control group and the NaCl treatment group (P<0.05).

[0056] The root length, aboveground fresh weight, and underground fresh weight of the bacterial agent treatment group (NaCl+ZY-2) were significantly different compared with the blank control group and the NaCl treatment group (P<0.05). The bacterial agent treatment group (NaCl+ZY-2) increased by 11.95%, 19.95%,

[0057] 14.89%, 34.35% in aboveground fresh weight, and 26.03%, 33.72% in underground fresh weight compared with the blank control group and the NaCl treatment group,

[0058] The aboveground dry weight of the bacterial agent treatment group (NaCl+ZY-2) increased by 21.57%, 25.49% compared with the blank control group and the NaCl treatment group. The above results show that when wheat resists salt stress, the ZY-2 strain can effectively reduce the salt stress on wheat and promote the growth of wheat under salt stress.

[0059] Example 3

[0060] Preparation method of the biocontrol preparation (containing bacillus velezensis ZY-2):

[0061] The single colony of bacillus velezensis ZY-2 is picked from the NA solid culture medium plate into NB liquid medium (a triangular flask), and is cultured at a temperature of 37 DEG C and a rotation speed of 220 r / min (on a shaker), so that the microbial inoculum is obtained when it grows to the logarithmic growth phase.

[0062] The various embodiments in the specification are described in a progressive manner, and each embodiment focuses on the difference from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0063] The above description of the disclosed embodiments enables a person skilled in the art to implement or use the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A salt-stress tolerant B acillus velezensis strain, characterized in that, The Bacillus velezensis ZY-2 is named as Bacillus velezensis ZY-2, and the preservation number is CGMCC NO.34345.

2. A soil amendment, a fertilizer or a biocontrol preparation containing the salt-stress-tolerant Bacillus velezensis according to claim 1.

3. The biocontrol formulation of claim 2, wherein the biocontrol formulation is a biocontrol composition. The preparation method of the biocontrol preparation is as follows: single colonies of the Bacillus velezensis ZY-2 are picked from NA solid culture medium plates into NB liquid medium, and cultured in a shaking table at a temperature of 37 DEG C and a rotating speed of 220 r / min; when the logarithmic growth phase is reached, a microbial inoculum is obtained.

4. The use of the salt-stress-tolerant Bacillus velezensis ZY-2 according to claim 1 in the preparation of genetically engineered bacteria.

5. The use of the salt-stress-tolerant Bacillus velezensis ZY-2 according to claim 1 in promoting crops to resist salt stress and grow in a saline-alkali environment.

6. Use according to claim 5, wherein The crops are wheat.

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