Salt stress resistant bacillus velezensis and application thereof

Abstract

The invention discloses a salt stress resistant bacillus velezensis strain and application thereof. Relates to the technical field of agricultural microorganisms. The invention relates to salt stress resistant bacillus velezensis ZY-2 with the preservation number of CGMCC (China General Microbiological Culture Collection Center) NO.34345, and provides related application. The salt stress resistant bacillus velezensis provided by the invention has a wide application prospect in a salt and alkali resistant environment. Specifically, the strain can be used as an original material to breed the strain which is better in salt and alkali environment tolerance and more remarkable in wheat growth and development promotion effect through natural breeding, artificial mutation breeding, genetic engineering breeding and other methods, so that the use of chemical modifiers and the like can be reduced. In addition, the strain has a strong tolerance effect on a salt-alkali environment, and has a good promotion effect on growth and development of wheat under salt stress.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural microorganisms, and more particularly to a salt-stress-resistant Velez subtilis strain and application thereof. Background Art

[0002] Saline-alkali land is a type of land that is prone to soil degradation and is a barrier to soil degradation. Salt in the soil primarily comes from rock weathering, dissolution, wind erosion, and the transfer of salt from saline rock and soil layers into the soil under the action of surface water and wind, forming saline-alkali land. In saline-alkali soil, long-term toxicity from salt ions can lead to soil degradation and nutrient loss. Furthermore, the presence of salt ions competes with nutrients, limiting wheat's absorption of nutrients like nitrogen, phosphorus, and potassium, leading to nutritional imbalances. This impacts wheat growth and health, resulting in yield reductions and even crop death. Therefore, improving saline-alkali land, promoting crop growth, alleviating salt stress on plants, and improving soil ecology are crucial for its management.

[0003] Currently, saline-alkali land improvement is primarily accomplished through physical, chemical, and biological remediation methods. Physical improvement of saline-alkali land primarily includes land leveling, soil loosening, water-dry rotation, deep plowing, ridge drying, watering and salting, and soil addition. Chemical improvement involves applying chemical amendments and organic matter to convert, adsorb, or fix saline and alkali components in the soil, thereby reducing their content. Biological improvement utilizes soil microorganisms and the cultivation of salt- and alkali-tolerant plants to improve saline-alkali soil. Physical improvement requires significant manpower, material, and financial resources, and has high investment and maintenance costs, making it difficult to maintain soil quality over the long term. Chemical improvement, while relatively effective, is costly and prone to secondary soil contamination, making it unsuitable for long-term use. Compared to other traditional saline-alkali land improvement methods, biological improvement, while slower in effectiveness, is less costly, does not cause secondary pollution, and offers sustainable development. Bioremediation can significantly reduce soil salinity and alkali content, improve soil fertility, and promote plant growth without damaging the soil or the environment.

[0004] Microorganisms are a vital component of the soil environment and play a crucial role in its composition and development. Microbial remediation has great potential for improving saline-alkali land due to its cost-effectiveness, wide application range, and lack of secondary pollution.

[0005] Therefore, whether it is possible to provide a salt-stress-tolerant Bacillus Velez subtilis strain and its application to overcome the above technical deficiencies is an urgent problem that those skilled in the art need to solve. Summary of the Invention

[0006] In view of this, the present invention provides a salt stress-tolerant Bacillus Velezii strain and its application. The strain has good tolerance to saline-alkali environments and has a good promoting effect on the growth and development of wheat under salt stress.

[0007] Bacillus velezensis ZY-2 is deposited in the General Microbiology Center of China Culture Collection Administration, the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, the deposit number is CGMCC: NO.34345, and the deposit date is April 25, 2025.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] The invention provides a salt stress resistant Bacillus Velez subtilis strain, which is named as Bacillus Velez subtilis ZY-2 and has a preservation number of CGMCC: NO.34345.

[0010] Furthermore, molecular biological analysis was performed to compare the 16S rDNA sequence of the salt-tolerant strain in the NCBI database using Blast, and a phylogenetic tree was constructed using MEGA 11 software. The strain was identified as Bacillus velezensis by 16S rDNA.

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

[0012] The present invention also provides a soil conditioner, a fertilizer or a biocontrol agent containing the salt stress tolerant Bacillus Velez subtilis.

[0013] Preferably, the preparation method of the biocontrol agent is as follows: a single colony of Bacillus velezensis ZY-2 is picked from a NA solid culture medium plate and placed in a NB liquid culture medium, and cultured on a shaker at a temperature of 37°C and a rotation speed of 220 r / min, and a microbial agent is obtained when the colony grows to the logarithmic growth phase.

[0014] The beneficial effect is that: through the strain growth experiment, it was found that the strain reached the logarithmic growth phase after growing in the NB culture medium for 24 hours, and the activity of the strain was the highest.

[0015] The present invention also provides the use of the salt stress resistant Bacillus Velez ZY-2 in preparing genetically engineered bacteria.

[0016] The present invention also provides the use of the salt-stress-tolerant Bacillus Velez ZY-2 in resisting saline-alkali stress.

[0017] Preferred: Use of salt-stress-tolerant Bacillus Velez ZY-2 in promoting the resistance of crops to salt-alkali stress and growth and development in saline-alkali environments.

[0018] The beneficial effect is that the present invention provides Bacillus velezensis ZY-2 with excellent salt tolerance and a wide adaptability to salt environments. Through salt tolerance experiments, it was found that the strain can grow and reproduce on NA culture medium plates with salt concentrations of 5%, 7%, 9% and 11%.

[0019] Preferred: Crops: Wheat.

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

[0021] It can be seen from the above technical solutions that, compared with the prior art, the present invention discloses a salt-stress-tolerant Bacillus Velezii strain and its application, and the technical effects achieved are:

[0022] The present invention discloses a multifunctional strain of Bacillus Velez ZY-2 isolated and screened from saline-alkali soil. The strain has excellent salt tolerance, a salinity tolerance of 0-11%, and an optimum salinity tolerance of 5-9%. The biocontrol agent (bacterial solution) prepared using Bacillus Velez ZY-2 can significantly improve the salt tolerance of wheat under salt stress, slow down the salt damage of the plant, increase biomass, promote wheat growth, and reduce the use of chemical fertilizers. Based on its excellent biological properties, the salt-tolerant strain has a wide range of application prospects. In addition, the strain can be used as the starting material to breed strains with better tolerance to saline-alkali environments and more significant effects on promoting wheat growth and development through methods such as natural selection, artificial mutagenesis, and genetic engineering breeding.

[0023] The biocontrol agent provided by the present invention has a simple preparation method, low culture conditions, low fermentation cost, and a short cycle, is suitable for industrial mass production, and is easy to promote and use on a large scale. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0025] Figure 1 The accompanying drawing is a diagram of the ZY-2 salt-tolerant strain obtained by screening and identification provided by the present invention.

[0026] Figure 2 The accompanying drawing is a diagram showing the salt tolerance of the ZY-2 strain provided by the present invention.

[0027] Figure 3 The accompanying drawing is a diagram showing the growth of the ZY-2 strain provided by the present invention in NB culture medium.

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

[0029] Figure 5 The accompanying drawing shows the effect of the ZY-2 strain provided by the present invention on the growth of wheat under salt stress.

[0030] Figure 6 The accompanying drawings are diagrams showing the growth of potted plants provided by the present invention, wherein from left to right are the CK group, the NaCl group, and the NaCl+ZY-2 group. DETAILED DESCRIPTION

[0031] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0032] The embodiment of the invention discloses a salt stress-resistant Bacillus Velez subtilis and application thereof.

[0033] Example 1

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

[0035] The strain isolation samples were collected from the soil in the crop growing area of ​​Zhongjie Farm in Huanghua City, Hebei Province.

[0036] 2. Culture Medium

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

[0038] 3. Isolation of Salt Stress-tolerant Strains

[0039] Take 10g soil sample and add it into 90ml sterile water in a conical flask, place it on a shaker at 200r / min for 30min to mix the sample thoroughly and obtain soil suspension. -2 , 10 -3 , 10 -4 , 10 -5The soil suspension was spread on a screening medium plate to isolate salt-tolerant strains. The plate was inverted and placed in a 37°C incubator for 24 hours to observe whether colonies formed.

[0040] The results showed that one strain, named ZY-2 ( Figure 1 ).

[0041] 4. Identification of salt tolerance of strains

[0042] While maintaining a constant pH of 7.2, a salt gradient medium was established, i.e., NA medium containing 5%, 7%, 9%, 11%, and 13% NaCl. ZY-2 strain was plated onto this salt gradient medium and incubated upside down in a 37°C incubator for 24-72 hours, after which growth was observed.

[0043] The results showed that ZY-2 had a significant tolerance to salt environments, but with the continuous increase in salt concentration, the tolerance of ZY-2 strain to salt environments gradually decreased. Figure 2 It can be seen that the maximum salt concentration tolerated by ZY-2 strain 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°C and 220 rpm in a shaking platform. The absorbance of the strains at 600 nm was detected regularly. The culture time was 72 h ( Figure 3 ).

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

[0047] 6. Establishment of strain phylogenetic tree

[0048] The 16S rDNA sequence of ZY-2 strain was compared in the NCBI database using Blast, and the phylogenetic tree was constructed using MEGA 11 software ( Figure 4 The strain was identified as Bacillus velezensis by 16S rDNA.

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

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

[0051] The ZY-2 strain was streaked on NA medium and cultured at 37°C overnight. A single colony was picked and placed in NB medium. The culture was shaken at 37°C and 220 rpm until OD600 = 1.0.

[0052] Select wheat seeds of uniform size and fullness, treat them with sterile water, place them in a culture dish, add water and soak for 12 hours, then wrap them with gauze to promote seedling growth for 12 hours for use.

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

[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 groups were irrigated with salt solution (150 mM) every five days and the bacterial agent (8.5 × 10 7 CFU / mL) were irrigated to the roots. Plant growth was monitored on a per-plant basis, with 25 plants sampled per group. After 30 days, horticultural traits such as plant height, root length, aboveground fresh weight, belowground fresh weight, and aboveground fresh weight, belowground fresh weight, were measured for the different treatments, and the average values ​​were calculated.

[0055] The results are as follows Figure 5 and Figure 6 As shown, the plant height and underground dry weight of the salt and bacterial agent treatment group (NaCl+ZY-2) were not significantly different from those of the blank control group and the NaCl treatment group (P<0.05).

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

[0057] The aboveground fresh weight of the microbial agent treatment group (NaCl+ZY-2) increased by 14.89% and 34.35% compared with the blank control group and the NaCl treatment group, respectively. The underground fresh weight of the microbial agent treatment group (NaCl+ZY-2) increased by 26.03% and 33.72% compared with the blank control group and the NaCl treatment group, respectively.

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

[0059] Example 3

[0060] Preparation method of biocontrol agent (containing Bacillus Velez ZY-2):

[0061] A single colony of Bacillus velezensis ZY-2 was picked from the NA solid culture medium plate and placed in the NB liquid culture medium (triangular flask), and cultured at a temperature of 37°C and a rotation speed of 220 r / min (on a shaker) until it grew to the logarithmic growth phase to obtain the microbial agent.

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

[0063] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A salt stress tolerant Bacillus Velezii strain, characterized in that: It was named Bacillus velezensis ZY-2, and its deposit number was CGMCC NO.34345.

2. A soil conditioner, fertilizer or biocontrol agent containing the salt stress tolerant Bacillus Velez subtilis according to claim 1.

3. The soil conditioner, fertilizer or biocontrol agent according to claim 2, wherein The preparation method of the biocontrol agent comprises the following steps: picking a single colony of Bacillus velezensis ZY-2 from an NA solid culture medium plate and placing it in an NB liquid culture medium; culturing the colony on a shaker at a temperature of 37° C. and a rotation speed of 220 r / min; and obtaining the microbial agent when the colony grows to a logarithmic growth phase.

4. Use of the salt-stress-tolerant Bacillus Velez ZY-2 according to claim 1 in preparing genetically engineered bacteria.

5. Use of the salt-stress-tolerant Bacillus Velez ZY-2 according to claim 1 in resisting saline-alkali stress.

6. The use according to claim 5, characterized in that The application of salt-stress-tolerant Bacillus Velez ZY-2 in promoting the resistance of crops to salt-alkali stress and growth and development in saline-alkali environment.

7. The use according to claim 6, characterized in that The crop mentioned: wheat.

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