A strain of *Gluconobacter endophyticus* subsp. halotolerans and its application

Abstract

The present invention relates to a strain of Glutamicibacter endophyticus subsp. halotolerans. The strain Glutamicibacter endophyticus NY3-1 of Glutamicibacter endophyticus subsp. halotolerans is deposited in the China Center for Type Culture Collection with the deposit number CCTCC No: M20242321. The 16S rDNA nucleotide sequence of the strain Glutamicibacter endophyticus NY3-1 is shown as SEQ ID NO: 1. This strain can metabolize normally in both salt-free and salt-containing environments, and can tolerate environments with a pH value of 6-11. It has the effects of improving the germination rate of plant seeds and promoting plant growth in both salt-containing and salt-free environments. Moreover, this strain can improve the soil environment of saline-alkali land, has broad application prospects, and provides a new biological resource for the biological improvement of saline-alkali land.

Description

Technical Field

[0001] The present invention belongs to the technical field of agricultural microorganisms, and relates to a strain of Haloglutamicibacter endophyticus subsp. and its application. Background Art

[0002] Soil salinization inhibits crop growth and damages plant photosynthesis. In severe cases, it even causes crop failure, 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 research hotspot. Its core is to promote plant growth and the salt tolerance of plants through the interaction between salt-tolerant microorganisms and plants. At the same time, due to the metabolic effects of salt-tolerant microorganisms, the problems of saline-alkali soil are alleviated, and the sustainable utilization of land is improved.

[0004] CN118308235A discloses a strain of Bacillus megaterium and its application in agricultural production. The Bacillus megaterium was deposited at the China Center for Type Culture Collection of Wuhan University, China on April 26, 2023, with the deposit number CCTCC NO: M2023634. The Bacillus megaterium strain has strong tolerance to saline-alkali environments, can grow effectively under conditions of pH 12 and 18% salt concentration, and has significant alkali reduction ability. It can colonize the crop roots, relieve crop salt damage, promote crop growth, and increase crop yield. However, the strain provided by this invention does not have the ability to tolerate high salt, and its application range is limited. Currently, due to the lack of high salt-tolerant microbial resources, microorganisms cannot carry out normal physiological metabolism in high salt environments, resulting in an unclear effect of biological improvement of saline-alkali land. Therefore, further exploring high salt-tolerant microbial resources, especially salt-tolerant microbial resources with plant 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] To solve the above technical problems, the present invention provides a strain of Haloglutamicibacter endophyticus subsp. and its application. The present invention screened and isolated a strain of Haloglutamicibacter endophyticus subsp. from the root soil of wheat in saline-alkali land in Fuping County, Weinan City, Shaanxi Province. Through in-depth research on the salt tolerance, alkali tolerance, soil improvement performance, and plant growth-promoting function of the Haloglutamicibacter endophyticus subsp. strain, a strain of Haloglutamicibacter endophyticus subsp. is provided and applied to the improvement of saline-alkali soil, and to improve the germination rate of plant seeds and promote plant growth in salt-free and salt-containing environments.

[0006] On the one hand, the present invention provides a strain of Glutamicibacter endophyticus subsp. NY3-1.

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

[0008] Strain name: NY3-1;

[0009] Taxonomic name: Glutamicibacter endophyticus subsp.;

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

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

[0012] Preservation unit: China Center for Type Culture Collection;

[0013] Preservation number: CCTCC No: M20242321.

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

[0015] Furthermore, the nucleotide sequence of the 16S rDNA of the strain of Glutamicibacter endophyticus subsp. NY3-1 is shown in SEQ ID NO: 1.

[0016] Furthermore, the strain of Glutamicibacter endophyticus subsp. NY3-1 can survive in both salt-free and saline environments.

[0017] Furthermore, the strain of Glutamicibacter endophyticus subsp. NY3-1 can survive in sulfates with a concentration of 0 wt% - 30 wt%.

[0018] Furthermore, the strain of Glutamicibacter endophyticus subsp. NY3-1 can survive in chlorides with a concentration of 0 wt% - 25 wt%.

[0019] Furthermore, the strain Glutamicibacter endophyticus NY3-1 of Glutamicibacter endophyticus subsp. can survive in an environment with a pH value of 6 to 11.

[0020] On the other hand, the present invention provides a saline-alkali tolerant microbial agent of the strain Glutamicibacter endophyticus NY3-1 of Glutamicibacter endophyticus subsp.

[0021] It is easy for those of ordinary skill in the art to know that in order to give full play to the efficacy of the strain Glutamicibacter endophyticus NY3-1 of Glutamicibacter endophyticus subsp., it can be prepared into a preparation dosage form convenient for application according to the usage needs. Based on this, further, the present invention provides a saline-alkali tolerant microbial agent containing the strain Glutamicibacter endophyticus NY3-1 of Glutamicibacter endophyticus subsp. The saline-alkali tolerant microbial agent is processed into a preparation dosage form acceptable in agriculture, and in the preparation, there are auxiliaries for assisting the strain Glutamicibacter endophyticus NY3-1 of Glutamicibacter endophyticus subsp. to improve the germination rate of plant seeds in a salt-free environment or a saline environment, auxiliaries for promoting the growth of plants in a saline environment or a salt-free environment, and auxiliaries for improving saline-alkali soil. The saline-alkali tolerant microbial agent can be formulated into various dosage forms such as soluble liquid agents, water dispersible granules, wettable powders, dispersible oil suspensions, suspensions, microemulsions, granules, and microcapsules with a certain sustained release effect. When formulating the above different dosage forms, for those of ordinary skill in the art, in addition to using the strain Glutamicibacter endophyticus NY3-1 of Glutamicibacter endophyticus subsp. to be selected, a variety of auxiliaries also need to be selected, and different preparation auxiliary components (auxiliaries) can be selected according to needs. The auxiliary components can be one or several of a dispersion medium, a dispersant, an emulsifier, a wetting agent, a thickener, an antifoaming agent, an antifreezing agent, a disintegrant, a binder, a filler, a carrier, etc.

[0022] In addition, the present invention also provides the application of the strain Glutamicibacter endophyticus NY3-1 of Glutamicibacter endophyticus subsp. or the saline-alkali tolerant microbial agent in the improvement of saline-alkali soil, in promoting seed germination in a salt-free or saline environment, and in promoting plant growth in a salt-free or saline 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] An endophytic subspecies strain Glutamicibacter endophyticus NY3-1 of halotolerant Glutamicibacter was screened and isolated from the rhizosphere soil of wheat in saline-alkali land in Fuping County, Weinan City, Shaanxi Province. The 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 0wt%-30wt% sulfate and 0wt%-25wt% chloride. Whether in a salt-free environment or a salt-containing environment, the endophytic subspecies strain Glutamicibacter endophyticus NY3-1 of halotolerant Glutamicibacter can increase the chlorophyll content of plant leaves and promote plant growth. After the bacterial solution of Glutamicibacter endophyticus NY3-1 was irrigated into saline-alkali soil, the electrical conductivity of the saline-alkali soil decreased significantly, and the free anions and cations in the soil decreased, indicating that Glutamicibacter endophyticus NY3-1 can reduce soil salinity and improve the quality of saline-alkali land. Description of the Drawings

[0025] Figure 1 It is the colony morphology and Gram staining map of the endophytic subspecies strain Glutamicibacter endophyticus NY3-1 (×1000).

[0026] Figure 2 It is the phylogenetic tree map of the 16S rDNA sequence of the endophytic subspecies strain Glutamicibacter endophyticus NY3-1.

[0027] Figure 3 It is the tolerance map of the endophytic subspecies strain Glutamicibacter endophyticus NY3-1 to different salt types and concentrations.

[0028] Figure 4 It is the tolerance map of the endophytic subspecies strain Glutamicibacter endophyticus NY3-1 to different pH values.

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

[0030] Figure 6Effect of Glutamicibacter endophyticus NY3-1, a strain of Glutamicibacter endophyticus subsp. endophyticus, on the germination rate, root and stem length, and chlorophyll of mung bean seeds.

[0031] Figure 7 Promoting growth effect diagram of Glutamicibacter endophyticus NY3-1, a strain of Glutamicibacter endophyticus subsp. endophyticus, on salt tolerance of mung bean.

[0032] Figure 8 Effect of Glutamicibacter endophyticus NY3-1, a strain of Glutamicibacter endophyticus subsp. endophyticus, on the electrical conductivity and free salt ions of saline-alkali soil. Specific implementation mode

[0033] Next, the technical solutions of the present invention will be described in conjunction with the embodiments. However, the present invention is not limited to the following embodiments. The experimental methods and detection methods described in each embodiment are all conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified, and the % is the mass percentage unless otherwise specified.

[0034] Example 1

[0035] This example is for the screening and isolation of Glutamicibacter endophyticus NY3-1, a strain of Glutamicibacter endophyticus subsp. endophyticus.

[0036] Prepare a salt-containing screening liquid medium: Weigh beef extract 0.3%, peptone 1%, and sodium chloride at 0.5%, 1%, 2%, 5%, 10%, 15%, and 20% by mass percentage, and adjust the pH to 7.4 - 7.6. Sterilize at 121 °C for 30 min to obtain salt-containing screening liquid media with different salt concentrations. The solid medium is added with 2% agar powder on the basis of the above formula.

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

[0038] Gradual domestication of the strain: Using a sterile brush in a laminar flow hood, brush the rhizosphere soil of wheat onto a sterile weighing paper. Weigh 1 g of the rhizosphere soil sample and add it to 100 ml of a liquid medium with a salt content of 0.5% by mass percentage. Incubate in a shaking flask at 35 °C and 150 r / min for 24 h. Then, pipette 2 ml of the liquid and add it to 100 ml of fresh liquid medium with a salt content of 1% by mass percentage. Incubate in a shaking flask at 37 °C and 150 r / min for 24 h. Continue to culture the obtained culture solution under the above conditions, and increase the salt concentration of each stage of the culture solution by 0.5%, 1%, 2%, 5%, 10%, 15%, and 20%. Strain isolation and purification: Spread the diluted solution of the above gradually domesticated strain on a solid medium plate with a salt content of 20% by mass percentage. Incubate statically at 35 °C. After colonies grow, pick the colonies with rapid growth and streak them on a fresh solid medium plate with a salt content of 20% by mass percentage. Repeat the operation until a purified strain is obtained, and label it as NY3-1. As Figure 1 shown, the NY3-1 colony is milky white, with a smooth and moist surface, a viscous texture, opaque, regular edges, and the strain is elliptical, with a length of 0.5 - 0.7 μm × 0.2 - 0.4 μm, without flagella and spores. Gram stain NY3-1 and observe and record it under a 1000-fold microscope. The results are as Figure 1 shown, NY3-1 is a Gram-positive strain.

[0039] Example 2

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

[0041] (1) Physiological and biochemical tests

[0042] Prepare a bacterial suspension of the strain Glutamicibacter endophyticus NY3-1 of the subspecies Glutamicibacter endophyticus and conduct relevant physiological and biochemical tests. The results are shown in Table 1. The strain can utilize a variety of sugars and organic acids as carbon sources for growth, which is beneficial for the strain to survive in adversity. At the same time, it can produce indole acetic acid, indicating that the strain can promote the growth of plants in a salt environment.

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

[0044] Physiological and biochemical items Results Physiological and biochemical items Results Catalase + Oxidase - Glucose + Mannose - Trehalose + Maltose + Lactose - Raffinose + Mannitol - Citrate + Glycerol + Propionate + Lactate - Casein hydrolysis + Starch hydrolysis + Indole acetic acid production + <![CDATA[H2S generation]]> - M.R. test - V.P. test - Growth in 5% NaCl + Growth at 50 °C - Anaerobic growth -

[0045] (2) Molecular identification

[0046] Centrifuge the suspension of the strain Glutamicibacter endophyticus NY3-1 to obtain the bacterial cells. Extract the DNA of the strain Glutamicibacter endophyticus NY3-1 using a bacterial DNA extraction kit. Then, amplify using the 16S rDNA universal primers. Send the amplification product to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The 16S rRNA nucleotide sequence of the strain Glutamicibacter endophyticus NY3-1 measured is as shown in SEQ ID No: 1. The specific 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 on the NCBI website for searching homologous sequences. Sequences were selected for Clustal X alignment, and MEGA software was used to construct a phylogenetic tree of the strain Glutamicibacter endophyticus NY3-1. The results are as Figure 2 shown. The results show that this strain has the closest evolutionary distance to the strain Glutamicibacter endophyticus, with a sequence similarity of 99%, and it was named Glutamicibacter endophyticus NY3-1.

[0049] Example 3

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

[0051] Salty liquid media with different types and concentrations were prepared respectively. The types of salts were MgSO4, NaCl, KCl, and CaCl2, and the concentrations were 5%, 10%, 15%, 20%, 25%, and 30% (g / 100 mL). The medium and the suspension of the strain Glutamicibacter endophyticus NY3-1 were added to a 96-well plate. 100 μL of the medium and 50 μL of the bacterial suspension of the strain Glutamicibacter endophyticus NY3-1 (after centrifugation at 4000 r / min, suspended with the salt medium of the same concentration as the experimental group) were added to each well. There were three replicate wells for each salt concentration. The culture was carried out at 35°C. Every 4 h, the growth of the strain was measured with a microplate reader OD 600nm The results are as Figure 3 shown. The results show that the strain Glutamicibacter endophyticus NY3-1 can tolerate magnesium sulfate at a concentration of 30%, sodium chloride at a concentration of 25%, potassium chloride at a concentration of 25%, and calcium chloride at a concentration of 25%. It indicates that the strain Glutamicibacter endophyticus NY3-1 has strong tolerance to sulfate-type and chloride-type salts.

[0052] Example 4

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

[0054] Prepare liquid media containing 20% NaCl with initial pH values of 6, 7, 8, 9, 10, and 11 respectively. Add the media and the suspension of Glutamicibacter endophyticus NY3-1 strain into a 96-well plate. Add 100 μL of media and 50 μL of the Glutamicibacter endophyticus NY3-1 bacterial solution (after centrifuging the bacteria at 4000 r / min, resuspend with the media having the same pH as the experimental group) to each well. For each pH value, there are three replicates. Incubate at 35°C. Every 1 h, use an enzyme-linked immunosorbent assay (ELISA) reader to measure the growth of the strain (OD 600nm ). The results are as Figure 4 shown (each column represents three replicates). The results indicate that within the pH range of 6 - 11, the Glutamicibacter endophyticus NY3-1 strain can grow. In a high-pH environment, the lag phase of the strain is extended, but after the lag phase, it can still maintain growth and reproduction.

[0055] Example 5

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

[0057] Inoculate the Glutamicibacter endophyticus NY3-1 strain (abbreviated as NY3-1) into 150 mL of beef extract peptone liquid medium and culture it on a shaker at 35°C for 18 - 24 h. After the OD value reaches 1.5, take it out and store it in a 4°C refrigerator. When used, adjust the concentration to 10 8 / mL with sterile water. The control group (CK) is a blank liquid beef extract peptone medium, which is diluted by the same multiple as the treatment group.

[0058] Germinate wheat seeds in a nutrient bowl. After the wheat seedlings grow to 3 - 5 cm, transfer them into flower pots and add fruit and vegetable nutrient soil. The control group is watered with 100 mL of 3% NaCl blank medium every 5 days, and the treatment group is watered with 100 mL of Glutamicibacter endophyticus NY3-1 bacterial solution containing 3% NaCl (bacterial concentration is 10 8 / mL) every 5 days. There are three replicates for each treatment. Grow at room temperature for 21 days, and measure the root and stem length, chlorophyll, and fresh weight of the whole plant. The measurement results are shown in Table 2, and different treated plants are as 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 a 3% NaCl environment, application of the Glutamicibacter endophyticus NY3-1 bacterial solution improved wheat's tolerance to salt stress and reduced the inhibitory effects of salt on wheat growth. Application of Glutamicibacter endophyticus NY3-1 increased wheat root and stem length by 34.61%, leaf chlorophyll by 9.5%, and individual plant fresh weight by 21.1%, demonstrating that the Glutamicibacter endophyticus NY3-1 strain significantly promoted wheat growth under salt stress.

[0062] Example 6

[0063] This example tests the effect of Glutamicibacter endophyticus NY3-1 on the germination rate and growth of mung bean seeds.

[0064] The glutamicum subspecies endophyticus strain Glutamicibacter endophyticus 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. The concentration was adjusted to 10 with sterile water before use. 8 The control group (CK) consisted of blank liquid beef extract peptone medium, 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 seed surface. After disinfection, rinse the seeds repeatedly with sterile distilled water 3-5 times to remove the sodium hypochlorite residue.

[0066] The sterilized seeds were placed on a double layer of sterile filter paper in a culture dish, with 10 seeds placed in each culture dish. Germination culture was carried out at 25°C. Different groups of liquids were sprayed every day, with each culture dish sprayed with 5 mL. After 4 days, the germination rate was calculated, and the seeds were moved to a soilless culture pot and sprayed for another week. The average rhizome length and chlorophyll content were tested.

[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% (bacterial solution of Glutamicibacter endophyticus NY3-1, a strain of Glutamicibacter endophyticus, with 1% NaCl), and T-3% (bacterial solution of Glutamicibacter endophyticus NY3-1, a strain of Glutamicibacter endophyticus, with 3% NaCl).

[0068] The experimental results are as Figure 6 , Figure 7 shown. The results show that the strain Glutamicibacter endophyticus NY3-1 of Glutamicibacter endophyticus can 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 on the improvement of saline soil by the strain Glutamicibacter endophyticus NY3-1 of Glutamicibacter endophyticus.

[0071] The soil samples taken from the Weinan area of Shaanxi Province were mixed evenly, put into boxes, and divided into two groups, with 3 boxes in each group and 2 kg of soil sample in each box. They were left standing at 25°C. In the treatment group, 100 mL of the bacterial solution of Glutamicibacter endophyticus NY3-1 (bacterial concentration: 10 8 / mL) was poured into each box every 3 days, and in the control group, 100 mL of blank beef extract medium was poured into each box every 3 days. After repeating the operation for 30 days, the soil conductivity and free salt ions were detected.

[0072] The detection results are as Figure 8 shown. The results show that the soil conductivity of the soil irrigated with the bacterial solution of Glutamicibacter endophyticus NY3-1 decreased significantly, and the free anions and cations in the soil decreased. The results indicate that the fermentation broth of the strain Glutamicibacter endophyticus NY3-1 of Glutamicibacter endophyticus can effectively reduce the salinity and conductivity of the soil.

Claims

1. A strain of *Gluconobacter endophyticus* subsp. *halotolerans*, characterized in that, The strain Glutamicibacter endophyticus NY3-1 of the subspecies Glutamicibacter endophyticus is deposited in the China Center for Type Culture Collection with the deposit number CCTCC No: M20242321; the strain Glutamicibacter endophyticus NY3-1 of the subspecies Glutamicibacter endophyticus can survive in sulfates of 0 wt% to 30 wt%, can survive in chlorides of 0 wt% to 25 wt%, and can survive in an environment with a pH value of 6 to 11.

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

1.

3. A saline-alkali tolerant microbial agent containing the strain Glutamicibacter endophyticus NY3-1 of the subspecies Glutamicibacter endophyticus described in claim 1.

4. Use of the strain Glutamicibacter endophyticus NY3-1 of the subspecies Glutamicibacter endophyticus described in claim 1 or the saline-alkali tolerant microbial agent described in claim 3 in the improvement of saline-alkali soil.

5. Use of the strain Glutamicibacter endophyticus NY3-1 of the subspecies Glutamicibacter endophyticus described in claim 1 or the saline-alkali tolerant microbial agent described in claim 3 in promoting seed germination in a salt-free or salt-containing environment.

6. Use of the strain Glutamicibacter endophyticus NY3-1 of the subspecies Glutamicibacter endophyticus described in claim 1 or the saline-alkali tolerant microbial agent described in claim 3 in promoting plant growth in a salt-free or salt-containing environment.

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