Salt-tolerant glutamic acid bacillus endophytic subspecies strain and application thereof

Abstract

The invention relates to an endophytic subspecies strain of salt-tolerant glutamic acid bacillus, the endophytic subspecies strain of the salt-tolerant glutamic acid bacillus is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC No: M20242321, and the 16S rDNA (ribosomal deoxyribonucleic acid) nucleotide sequence of the endophytic subspecies strain of the salt-tolerant glutamic acid bacillus is as shown in SEQ ID NO: 1. The strain can normally metabolize in a salt-free environment and a salt-containing environment, can tolerate the environment with the pH value of 6-11, has the effects of improving the germination rate of plant seeds and promoting plant growth in the salt-free environment and the salt-free environment, can improve the soil environment of the saline-alkali soil, is wide in application prospect, and provides a new biological resource for biological improvement of the saline-alkali soil.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural microorganisms and relates to a salt-tolerant glutamicum endophytic subspecies strain and application thereof. Background Art

[0002] Soil salinization inhibits crop growth, destroys plant photosynthesis, and even causes crop failure in severe cases, seriously affecting agricultural production and the ecological environment. Therefore, the improvement of saline-alkali land is particularly important and urgent.

[0003] Biological improvement of saline-alkali land is a sustainable and environmentally friendly improvement method and has always been a hot topic of research. Its core is to promote plant growth and salt resistance through the interaction between salt-tolerant microorganisms and plants. At the same time, due to the metabolic effect of salt-tolerant microorganisms, it can alleviate the problems of saline-alkali soil and improve the sustainable utilization of land.

[0004] CN118308235A discloses a strain of Bacillus megaterium and its application in agricultural production. The Bacillus megaterium has been deposited in the China Type Culture Collection of Wuhan University, China on April 26, 2023, with a deposit number of CCTCC NO: M2023634. The Bacillus megaterium strain has strong tolerance to saline-alkali environments, can effectively grow under pH12 and 18% salt concentration conditions, and has significant alkali reduction ability, can colonize crop roots, alleviate crop salt damage, promote crop growth, and increase crop yields. However, the strain provided by the invention does not have the ability to tolerate high salt, and its scope of application is limited. At present, due to the lack of high-salt-tolerant microbial resources, microorganisms cannot carry out normal physiological metabolism in a high-salt environment, resulting in an unobvious effect of biological improvement of saline-alkali land. Therefore, further exploring high-salt-tolerant microbial resources, especially salt-tolerant microbial resources with growth-promoting effects, and enriching the diversity of salt-tolerant microbial resources are of great significance for the biological improvement of saline-alkali land. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a salt-tolerant glutamicum endophytic subspecies strain and its application. The present invention screens and isolates a salt-tolerant glutamicum endophytic subspecies strain from wheat root soil in saline-alkali land of Fuping County, Weinan City, Shaanxi Province, and provides a salt-tolerant glutamicum endophytic subspecies strain through in-depth research on the salt-tolerance and alkali-tolerance properties of the salt-tolerant glutamicum endophytic subspecies strain, soil improvement properties and plant growth-promoting functions, and applies the strain to saline-alkali land soil improvement, and to improve plant seed germination rates and promote plant growth in salt-free and salty environments.

[0006] In one aspect, the present invention provides a salt-tolerant glutamicum endophytic subspecies strain Glutamicibacterendophyticus NY3-1.

[0007] Specifically, the salt-tolerant glutamicum endophytic subspecies strain Glutamicibacter endophyticus NY3-1 was screened and isolated from the rhizosphere soil of wheat in saline-alkali land of Fuping County, Weinan City, Shaanxi Province. The deposit information of the salt-tolerant glutamicum endophytic subspecies strain Glutamicibacter endophyticus NY3-1 is as follows:

[0008] Strain name: NY3-1;

[0009] Classification name: Glutamicibacter endophyticus;

[0010] Date of receipt by the depository: October 24, 2024;

[0011] Time of issuance of preservation certificate: October 31, 2024;

[0012] Depository: China Center for Type Culture Collection;

[0013] Deposit number: CCTCC No: M20242321.

[0014] Address: Wuhan University, Wuhan, China.

[0015] Furthermore, the nucleotide sequence of 16S rDNA of the salt-tolerant glutamicum endophytic subspecies strain Glutamicibacter endophyticusNY3-1 is shown in SEQ ID NO:1.

[0016] Furthermore, the salt-tolerant glutamicum endophytic subspecies strain Glutamicibacter endophyticusNY3-1 can survive in a salt-free environment and a salt-containing environment.

[0017] Furthermore, the salt-tolerant glutamicum endophytic subspecies strain Glutamicibacter endophyticusNY3-1 can survive in 0wt%-30wt% sulfate.

[0018] Furthermore, the salt-tolerant glutamicum endophytic subspecies strain Glutamicibacter endophyticusNY3-1 can survive in 0wt%-25wt% chloride salt.

[0019] Furthermore, the salt-tolerant glutamicum endophytic subspecies strain Glutamicibacter endophyticusNY3-1 can survive in an environment with a pH value of 6 to 11.

[0020] On the other hand, the present invention provides a salt-alkali resistant bacterial agent containing the salt-tolerant glutamicum endophytic subspecies strain Glutamicibacter endophyticus NY3-1 according to claim 1.

[0021] It is easy for a person skilled in the art to know that in order to give full play to the efficacy of the salt-tolerant glutamicum endophytic subspecies strain Glutamicibacter endophyticus NY3-1, it can be prepared into a preparation dosage form that is easy to apply according to the needs of use. Based on this, further, the present invention provides a salt-alkali-tolerant bacterial agent containing the salt-tolerant glutamicum endophytic subspecies strain Glutamicibacter endophyticus NY3-1. The salt-alkali-tolerant bacterial agent is processed into a preparation dosage form accepted in agriculture, and the preparation contains an auxiliary agent that assists the salt-tolerant glutamicum endophytic subspecies strain Glutamicibacterendophyticus NY3-1 to improve the germination rate of plant seeds in a salt-free environment or a salt environment, an auxiliary agent that promotes the growth of plants in a salt environment or a salt-free environment, and an auxiliary agent that improves saline-alkali soil. The salt-alkali-tolerant bacterial agent can be formulated into a variety of dosage forms such as soluble liquid, water-dispersible granules, wettable powder, dispersible oil suspension, suspension, microemulsion, granules, and microcapsules with a certain sustained release effect. When preparing the above-mentioned different dosage forms, for those skilled in the art, in addition to using the optional salt-tolerant glutamicum endophytic subspecies strain Glutamicibacterendophyticus NY3-1, it is also necessary to select a variety of auxiliary agents, and different preparation auxiliary components (auxiliaries) can be selected and used as needed. The auxiliary component can be one or more of a dispersion medium, a dispersant, an emulsifier, a wetting agent, a thickener, a defoamer, an antifreeze agent, a disintegrant, a binder, a filler, a carrier, etc.

[0022] In addition, the present invention also provides the use of the salt-tolerant glutamicum endophytic subspecies strain Glutamicibacterendophyticus NY3-1 or the salt-alkali tolerant bacterial agent in improving saline-alkali soil, promoting seed germination in a salt-free or salt-containing environment, and promoting plant growth in a salt-free or salt-containing environment.

[0023] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0024] The invention obtains a salt-tolerant glutamic acid bacterium endophytic subspecies strain Glutamicibacter endophyticus NY3-1 by screening and isolating from wheat rhizosphere soil in saline-alkali land of Fuping County, Weinan City, Shaanxi Province. The salt-tolerant glutamic acid bacterium endophytic subspecies Glutamicibacter endophyticus NY3-1 can grow and metabolize normally in an environment with a pH value of 6-11, and can survive in an environment with a sulfate content of 0wt%-30wt% and a chloride content of 0wt%-25wt%. The salt-tolerant glutamic acid bacterium endophytic subspecies strain Glutamicibacter endophyticus NY3-1 can increase the chlorophyll content of plant leaves in a salt-free environment or a salt-containing environment, and promote the growth of plants. After the glutamic acid bacterium endophyticus NY3-1 bacterial liquid is used to irrigate the saline-alkali soil, the conductivity of the saline-alkali soil is significantly reduced, and the free anions and cations in the soil are reduced, indicating that the glutamic acid bacterium endophyticus NY3-1 NY3-1 can reduce soil salinity and improve the soil quality of saline-alkali land. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the colony morphology and Gram staining image of Glutamicibacter endophyticus NY3-1 (×1000).

[0026] Figure 2 This is the evolutionary tree diagram of the NY3-116srDNA sequence of the endophytic subspecies of Glutamicibacter endophyticus.

[0027] Figure 3 This is the tolerance graph of Glutamicibacter endophyticus NY3-1 to different salt types and concentrations.

[0028] Figure 4 This is the tolerance graph of Glutamicibacter endophyticus NY3-1 to different pH values.

[0029] Figure 5 It is the promoting effect of Glutamicibacter endophyticus NY3-1 on wheat resistance to salt stress.

[0030] Figure 6The effect of Glutamicibacter endophyticus NY3-1 on the germination rate, rhizome length and chlorophyll of mung bean seeds.

[0031] Figure 7 This is a graph showing the growth-promoting effect of Glutamicibacter endophyticus NY3-1 on mung bean salt tolerance.

[0032] Figure 8 It is the effect of Glutamicibacter endophyticus NY3-1 on the electrical conductivity and free salt ions of saline-alkali soil. DETAILED DESCRIPTION

[0033] The technical scheme of the present invention is described below in conjunction with the embodiments, but the present invention is not limited to the following embodiments. The experimental methods and detection methods described in each embodiment are conventional methods unless otherwise specified; the reagents and materials are available on the market unless otherwise specified, and the % are mass percentages unless otherwise specified.

[0034] Example 1

[0035] This example is about the screening and separation of Glutamicibacter endophyticus NY3-1.

[0036] Preparation of salt-containing screening liquid culture medium: weigh 0.3% beef extract, 1% peptone, and 0.5%, 1%, 2%, 5%, 10%, 15%, and 20% of sodium chloride in liquid culture medium by mass percentage, adjust the pH to 7.4-7.6, and sterilize at 121°C for 30 minutes to prepare salt-containing screening liquid culture medium with different salt concentrations. Add 2% agar powder to the solid culture medium based on the above formula.

[0037] Sample source: Wheat was collected from the saline-alkali land in Fuping County, Weinan City, Shaanxi Province (34°47′26″N, 109°23′40″E, 344.1 meters above sea level) on April 17, 2024 and stored in a portable refrigerated box.

[0038] Strains were domesticated step by step: On a clean bench, the wheat rhizosphere soil was brushed onto sterile weighing paper using a sterile brush, 1 g of rhizosphere soil sample was weighed and added to 100 ml of liquid medium containing 0.5% salt by mass, and cultured in a flask at 35°C and 150 r / min for 24 h, and then 2 ml of liquid was taken and added to 100 ml of fresh liquid medium containing 1% salt by mass, and cultured in a flask at 37°C and 150 r / min for 24 h. The obtained culture solution was cultured under the above conditions, and the salt concentration of each culture solution was increased by 0.5%, 1%, 2%, 5%, 10%, 15%, and 20%. Strain Isolation and Purification: Spread the above graded diluted solutions on a solid culture medium plate containing 20% ​​salt by mass percentage, incubate at 35°C, and after colonies grow, pick the fast-growing colonies and streak them on a fresh culture medium plate containing 20% ​​salt by mass percentage. Repeat the operation until a purified strain is obtained, which is labeled as NY3-1. Figure 1 As shown in the figure, the NY3-1 colony is milky white, with a smooth and moist surface, a viscous texture, opaque, and regular edges. The strain is oval, 0.5-0.7μm × 0.2-0.4μm in length, and has no flagella or spores. NY3-1 was gram-stained and observed and recorded under a 1000x microscope. The results are as follows Figure 1 As shown, NY3-1 is a Gram-positive strain.

[0039] Example 2

[0040] This example is to identify the endophytic subspecies of Glutamicibacter endophyticus NY3-1.

[0041] (1) Physiological and biochemical tests

[0042] A bacterial suspension of Glutamicibacter endophyticus NY3-1 was prepared, and relevant physiological and biochemical tests were performed. The results are shown in Table 1. The strain can use a variety of sugars and organic acids as carbon sources for growth, which is beneficial to the survival of the strain in adverse conditions. At the same time, it can produce indoleacetic acid, indicating that the strain can promote the growth of plants in a salt environment.

[0043] Table 1 Physiological and biochemical results of NY3-1 strain

[0044] Physiology and Biochemistry Project result Physiology and Biochemistry Project result Catalase + Oxidase - glucose + Mannose - Trehalose + maltose + lactose - Raffinose + Mannitol - Citrate + glycerin + Propionate + Lactate - Casein hydrolysis + Starch hydrolysis + IAA production + <![CDATA[H2S generation]]> - MRI testing - VP test - 5% NaCl growth + Growth at 50℃ - Anaerobic growth -

[0045] (2) Molecular identification

[0046] The suspension of glutamicum endophytic subspecies strain Glutamicibacter endophyticus NY3-1 was centrifuged to obtain bacterial cells, and DNA of glutamicum endophytic subspecies strain Glutamicibacterendophyticus NY3-1 was extracted using a bacterial DNA extraction kit, and then amplified using 16S rDNA universal primers. The amplified product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The measured 16S rRNA nucleotide sequence of glutamicum endophytic subspecies strain Glutamicibacterendophyticus NY3-1 is shown in SEQ ID No: 1. The nucleotide sequence of SEQ ID No: 1 is as follows:

[0047]

[0048] The nucleotide sequence shown in SEQ ID No: 1 was input into the Blast tool in the NCBI website to search for homologous sequences, and the sequences were selected for Clustal X alignment. The phylogenetic tree of Glutamicibacter endophyticus NY3-1 was constructed using Mega software. The results are as follows: Figure 2 As shown, the results showed that the strain was closest to the Glutamicibacter endophyticus strain in evolution, with a sequence similarity of 99%, and was named Glutamicibacter endophyticus NY3-1.

[0049] Example 3

[0050] This example is a salinity tolerance test of Glutamicibacter endophyticus NY3-1.

[0051] Prepare different types and concentrations of salt-containing liquid culture media, the salt types are MgSO4, NaCl, KCl, CaCl2, and the concentrations are 5%, 10%, 15%, 20%, 25%, and 30% (g / 100mL), respectively. Add the culture medium and the suspension of glutamicum endophytic subspecies strain Glutamicibacter endophyticus NY3-1 to a 96-well plate, add 100 μL of culture medium and 50 μL of glutamicum endophytic subspecies strain Glutamicibacter endophyticus NY3-1 bacterial solution to each well (after centrifugation at 4000 r / min, the bacteria are suspended in a salt culture medium with the same concentration as the experimental group), and three replicate wells are added for each salt concentration. The plates are cultured at 35°C and the OD values ​​are measured every 4 hours using a microplate reader. 600nm The growth of the strain was measured. Figure 3 As shown, the results show that the endophytic subspecies of Glutamicibacter NY3-1 can tolerate 30% magnesium sulfate, 25% sodium chloride, 25% potassium chloride and 25% calcium chloride, indicating that the endophytic subspecies of Glutamicibacter NY3-1 has a strong tolerance to sulfate and chloride salts.

[0052] Example 4

[0053] This example is a pH tolerance test of Glutamicibacter endophyticus NY3-1.

[0054] Liquid culture media containing 20% ​​NaCl with initial pH values ​​of 6, 7, 8, 9, 10, and 11 were prepared respectively, and the culture media and the suspension of glutamic acid endophytic strain Glutamicibacter endophyticus NY3-1 were added to a 96-well plate, 100 μL of culture media and 50 μL of glutamic acid endophytic strain Glutamicibacter endophyticus NY3-1 bacterial solution (the bacteria were centrifuged at 4000 r / min and suspended in a culture medium with the same pH as the experimental group) were added to each well, three wells were repeated for each pH, ​​and the plates were cultured at 35° C. Every 1 h, the growth of the strain (OD 600nm ). The result is Figure 4 As shown (each column has three replicates), the results show that the endophytic subspecies of Glutamicibacter NY3-1 can grow in the pH range of 6 to 11. In a high pH environment, the strain adjustment period is prolonged, but after the adjustment period, it can still maintain growth and reproduction.

[0055] Example 5

[0056] This example is a test experiment on wheat growth promotion under salt stress by Glutamicibacter endophyticus NY3-1.

[0057] The glutamic acid bacillus subspecies strain Glutamicibacter endophyticus NY3-1 (abbreviated as NY3-1) was inoculated into 150 mL beef extract peptone liquid culture medium and cultured in a shaking incubator at 35°C for 18 to 24 h. After the OD value reached 1.5, it was taken out and stored in a refrigerator at 4°C. Sterile water was used to adjust the concentration to 10 8 / mL, the control group (CK) was blank liquid beef extract peptone medium, diluted at the same multiple as the treatment group.

[0058] Wheat seeds were germinated in nutrient pots, and after wheat seedlings (3-5 cm) grew out, they were moved into flower pots and added with fruit and vegetable nutrient soil. The control group was irrigated with 100 mL of 3% NaCl blank medium every 5 days, and the treatment group was irrigated with 100 mL of 3% NaCl Glutamicibacter endophyticus NY3-1 bacterial solution (bacterial concentration was 10 8 Each treatment was repeated 3 times, and the plants were grown at room temperature for 21 days. The root length, chlorophyll, and fresh weight of the whole plant were measured. The measurement results are shown in Table 2. Figure 5 shown.

[0059] Table 2 Effect of NY3-1 on wheat growth in 3% salt environment

[0060]

[0061] Table 2 and Figure 5 The results showed that under 3% NaCl environment, the application of Glutamicibacter endophyticus NY3-1 bacterial solution improved wheat's tolerance to salt stress and reduced the inhibitory effect of salt on wheat growth. After the application of Glutamicibacterendophyticus NY3-1, the root length of wheat increased by 34.61%, the chlorophyll in leaves increased by 9.5%, and the fresh weight of single plant increased by 21.1%, indicating that the strain Glutamicibacter endophyticus NY3-1 has a significant growth-promoting effect on wheat under salt stress.

[0062] Example 6

[0063] This example is a test experiment on the effect of glutamicum endophytic subspecies strain Glutamicibacter endophyticus NY3-1 on the germination rate and growth of mung bean seeds.

[0064] The glutamic acid bacillus endophyticus NY3-1 was inoculated into 150 mL of beef extract peptone liquid culture medium and cultured in a shaking incubator at 35 °C for 18 to 24 h. After the OD value reached 1.5, it was taken out and stored in a refrigerator at 4 °C. The concentration was adjusted to 10 with sterile water before use. 8 The control group (CK) was a blank liquid beef extract peptone medium, which was diluted at the same multiple as the treatment group.

[0065] Select plump mung bean seeds and soak them in 2% sodium hypochlorite solution for 5 minutes to disinfect the surface of the seeds. After disinfection, rinse the seeds repeatedly 3-5 times with sterile distilled water to remove the sodium hypochlorite residue.

[0066] The disinfected seeds were placed on a double layer of sterile filter paper in a culture dish, with 10 seeds placed in each culture dish. They were cultured for germination at 25°C and sprayed with different groups of liquids every day, with each culture dish sprayed with 5 mL. After 4 days, the germination rate was counted and the seeds were moved into a soilless culture pot and sprayed for another week to detect the average rhizome length and chlorophyll content.

[0067] The groups were set as CK-0 (blank medium without NaCl), CK-1% (blank medium with 1% NaCl), CK-3% (blank medium with 3% NaCl), T-0 (NY3-1 bacterial solution without NaCl), T-1% (Glutamicibacter endophyticus NY3-1 bacterial solution with 1% NaCl), T-3% (Glutamicibacter endophyticus NY3-1 bacterial solution with 3% NaCl)

[0068] The experimental results are as follows Figure 6 , Figure 7 As shown, the results showed that the endophytic subspecies of Glutamicibacter NY3-1 could promote the germination of mung bean seeds and the growth of mung bean plants under both salt-free and salt-containing conditions.

[0069] Example 7

[0070] This example is a test experiment on the improvement of saline soil by using Glutamicibacter endophyticus NY3-1, an endophytic subspecies of Glutamicibacter.

[0071] Soil samples collected from Weinan, Shaanxi Province were mixed and packed into two groups, each with 3 boxes and 2 kg soil sample per box. The samples were placed at 25 °C. Every 3 days, each box in the treatment group was watered with 100 mL of bacterial solution of Glutamicibacterendophyticus NY3-1 (bacterial concentration was 10 8 / mL), and the control group was irrigated with 100mL of blank beef extract medium per box every 3 days. The soil conductivity and free salt ions were tested after repeating the operation for 30 days.

[0072] Test results such as Figure 8 As shown, the results showed that the soil conductivity was significantly reduced after watering with the bacterial solution of Glutamicibacterendophyticus NY3-1, an endophytic subspecies of Glutamicibacter, and the free anions and cations in the soil were reduced. The results showed that the fermentation solution of Glutamicibacterendophyticus NY3-1, an endophytic subspecies of Glutamicibacter, can effectively reduce the salinity and conductivity of the soil.

Claims

1. A salt-tolerant glutamicum endophytic subspecies strain, characterized in that: The salt-tolerant glutamicum endophytic subspecies strain Glutamicibacter endophyticus NY3-1 is preserved in China Center for Type Culture Collection with a preservation number of CCTCC No: M20242321.

2. The salt-tolerant glutamicum endophytic subspecies strain Glutamicibacterendophyticus NY3-1 according to claim 1, characterized in that The nucleotide sequence of 16S rDNA of the salt-tolerant glutamicum subspecies endophyticus strain Glutamicibacterendophyticus NY3-1 is shown in SEQ ID NO:

1.

3. The salt-tolerant glutamicum endophytic subspecies strain Glutamicibacterendophyticus NY3-1 according to claim 1, characterized in that: The salt-tolerant glutamicum endophytic subspecies strain Glutamicibacterendophyticus NY3-1 can survive in a salt-free environment and a salt-containing environment.

4. The salt-tolerant glutamicum endophytic subspecies strain Glutamicibacterendophyticus NY3-1 according to claim 3, characterized in that: The salt-tolerant glutamicum endophytic subspecies strain Glutamicibacter endophyticus NY3-1 can survive in 0wt% to 30wt% of sulfate.

5. The salt-tolerant glutamicum endophytic subspecies strain Glutamicibacterendophyticus NY3-1 according to claim 3, characterized in that: The salt-tolerant glutamicum endophytic subspecies strain Glutamicibacterendophyticus NY3-1 can survive in 0wt% to 25wt% of chloride salt.

6. The salt-tolerant glutamicum endophytic subspecies strain Glutamicibacterendophyticus NY3-1 according to claim 1, characterized in that: The salt-tolerant glutamicum endophytic subspecies strain Glutamicibacterendophyticus NY3-1 can survive in an environment with a pH value of 6-11.

7. A salt- and alkali-resistant bacterial agent containing the salt-tolerant glutamicum endophytic subspecies strain Glutamicibacterendophyticus NY3-1 according to claim 1.

8. Use of the salt-tolerant glutamicum endophytic subspecies strain Glutamicibacter endophyticusNY3-1 according to claim 1 or the salt-alkali-tolerant bacterial agent according to claim 7 in improving saline-alkali soil.

9. Use of the salt-tolerant glutamicum endophytic subspecies strain Glutamicibacter endophyticus NY3-1 according to claim 1 or the salt-tolerant alkali bacterial agent according to claim 7 in promoting seed germination in a salt-free or salt-containing environment.

10. Use of the salt-tolerant glutamicum endophytic subspecies strain Glutamicibacter endophyticus NY3-1 according to claim 1 or the salt-tolerant alkali bacterial agent according to claim 7 in promoting plant growth in a salt-free or salt-containing environment.

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