Microbial inoculant based on strain NY3-1 and application thereof

Abstract

The application belongs to the technical field of microorganisms, and discloses a microbial agent based on a strain NY3-1 and application thereof. The application provides a microbial agent based on the strain NY3-1, wherein the microbial agent comprises a bacterial liquid of the strain NY3-1, and the bacterial liquid is obtained by culturing the strain NY3-1 in a culture medium containing sodium selenite; and the preservation number of the strain NY3-1 is CCTCC No: M20242321. The microbial agent provided by the application can effectively improve the activity of C cycle related enzymes and N cycle related enzymes in the rhizosphere soil of plants and the activity of plant self resistance related enzymes, thereby improving the growth performance and stress resistance performance of crops.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and discloses a microbial agent based on strain NY3-1 and its application. Background Technology

[0002] Patent application number 202411887824.5 disclosed strain NY3-1 on April 29, 2025. Specifically, it is Glutamicibacter endophyticus NY3-1, which was screened and isolated from the rhizosphere soil of wheat in saline-alkali land in Fuping County, Weinan City, Shaanxi Province. Its accession number is CCTCC No: M20242321. Patent 202411887824.5 describes the isolation and selection of a salt-tolerant endophytic subspecies of *Glutamicibacter endophyticus* NY3-1 from the rhizosphere soil of wheat in saline-alkali land in Fuping County, Weinan City, Shaanxi Province. This *Glutamicibacter endophyticus* NY3-1 can grow and metabolize normally in environments with pH values ​​of 6-11, and can survive in environments with 0wt%-30wt% sulfate and 0wt%-25wt% chloride. In both salt-free and salt-containing environments, the *Glutamicibacter endophyticus* NY3-1 strain can increase the chlorophyll content of plant leaves and promote plant growth. After irrigating saline-alkali soil with a solution of *Glutamicibacter endophyticus* NY3-1, the electrical conductivity of the saline-alkali soil and the free anions and cations in the soil decreased significantly, suggesting that the *Glutamicibacter endophyticus* NY3-1 strain is a symbiotic organism. Endophyticus NY3-1 can reduce soil salinity and improve the quality of saline-alkali land. Patent 202411887824.5 does not disclose that strain NY3-1 can convert inorganic selenium compounds into elemental selenium, nor does it disclose the use of strain NY3-1 in controlling common plant diseases or in promoting plant growth in selenium-enriched culture media. Summary of the Invention

[0003] The purpose of this invention is to provide more applications for strain NY3-1.

[0004] In the technical solution provided by this invention, "strain NY3-1", "NY3-1" or "endophytic glutamate bacillus NY3-1" all refer to the salt-tolerant endophytic subspecies strain Glutamicibacter endophyticus NY3-1 provided by the patent application number 202411887824.5, whose accession number is CCTCC No: M20242321, and whose depository institution is the China Center for Type Culture Collection.

[0005] On one hand, the present invention relates to a microbial agent based on strain NY3-1, wherein the microbial agent comprises a bacterial suspension of strain NY3-1, the bacterial suspension being obtained by culturing strain NY3-1 in a medium containing sodium selenite;

[0006] The preservation number of strain NY3-1 is CCTCC No: M20242321.

[0007] Furthermore, in the microbial agent based on strain NY3-1 provided by the present invention, the number of strain NY3-1 in the bacterial solution is not less than 10. 8 / mL.

[0008] Furthermore, in the microbial agent based on strain NY3-1 provided by the present invention, the concentration of sodium selenite in the sodium selenite-containing culture medium is not less than 50 mmol / L.

[0009] Furthermore, in the microbial agent based on strain NY3-1 provided by the present invention, strain NY3-1 converts inorganic selenium compounds into elemental selenium;

[0010] The preservation number of strain NY3-1 is CCTCC No: M20242321.

[0011] Furthermore, in the microbial agent based on strain NY3-1 provided by the present invention, the inorganic selenium compound is sodium selenite.

[0012] On the other hand, the present invention relates to the application of strain NY3-1 in plant cultivation, wherein strain NY3-1 has the preservation number CCTCC No: M20242321.

[0013] Furthermore, in the application of the strain NY3-1 provided by the present invention in plant cultivation, the strain NY3-1 is used for the prevention and control of plant diseases;

[0014] The pathogens of the plant diseases are selected from at least one of the following: Botrytis cinerea (tomato gray mold), Diaporthe momicola (metazoanth), and Botryosphaeria wangensis (botryosphaeria wangensis).

[0015] Furthermore, in the application of the strain NY3-1 provided by the present invention in plant cultivation, the strain NY3-1 is used to enrich selenium in plants.

[0016] Furthermore, in the application of the strain NY3-1 provided by the present invention in plant cultivation, the strain NY3-1 enhances the activity of C-cycle-related enzymes and N-cycle-related enzymes in the plant rhizosphere soil, and / or enhances the activity of plant resistance-related enzymes.

[0017] For example, C-cycle-related enzymes and N-cycle-related enzymes in rhizosphere soil include: β-1,4-glucosidase, β-D-cellulase, β-1,4-N-acetylglucosidase, and L-leucine-α-aminopeptidase.

[0018] For example, plant resistance-related enzymes include catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD).

[0019] Furthermore, the application of strain NY3-1 provided by the present invention in plant cultivation includes: applying the microbial agent to the plant.

[0020] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages.

[0021] This invention provides further applications for strain NY3-1. Strain NY3-1 can efficiently convert inorganic selenium compounds (such as sodium selenite) into elemental selenium, expanding its application in the preparation of elemental selenium. The microbial agent obtained by culturing this strain in a sodium selenite-containing medium can significantly enhance the activity of C and N cycle-related enzymes (such as β-1,4-glucosidase, β-D-cellulase, β-1,4-N-acetylglucosidase, L-leucine-α-aminopeptidase) in the plant rhizosphere soil, as well as resistance-related enzymes in the plant itself (such as catalase CAT, superoxide dismutase SOD, and peroxidase POD), thereby promoting crop growth performance and enhancing stress resistance. Simultaneously, this agent can effectively control plant diseases (such as tomato gray mold, *Gnaphalium affine*, and *Botrytis cinerea*), and enrich selenium in plants, significantly increasing the selenium content of fruits, providing an efficient and safe solution for selenium-enriched agriculture. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a diagram of a culture medium for selenium transformation and enrichment in the rhizosphere soil of wheat.

[0024] Figure 2 This is a plate image of endophytic glutamate bacillus NY3-1 transforming selenium nanoparticles.

[0025] Figure 3 This is a diagram showing the transformation of different concentrations of inorganic selenium compounds by endophytic glutamate bacillus NY3-1.

[0026] Figure 4 This is a diagram showing the inhibitory effect of endophytic glutamate bacillus NY3-1 on plant pathogens.

[0027] Figure 5 The study investigated the effects of endophytic glutamate bacillus NY3-1 selenium-enriched inoculant on rhizosphere soil indicators of greenhouse tomatoes.

[0028] Figure 6 The effect of selenium-enriched endophytic glutamate bacterium NY3-1 on enzyme activity in tomato leaves in greenhouses.

[0029] Figure 7 The effect of endophytic glutamate bacillus NY3-1 selenium-enriched inoculant on the selenium content of tomatoes grown in greenhouses. Detailed Implementation

[0030] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods and detection methods described in each embodiment are conventional methods; unless otherwise specified, the reagents and materials can be purchased commercially. Unless otherwise specified, % and ‰ in the following embodiments refer to mass percentage and mass thousandths, respectively.

[0031] Glutamicibacter endophyticus NY3-1, which was deposited at the China Center for Type Culture Collection on October 31, 2024, with accession number CCTCC No: M20242321.

[0032] Example 1

[0033] This embodiment provides an experiment on the conversion of inorganic selenium compounds into elemental selenium by endophytic glutamate bacillus NY3-1.

[0034] (1) Selenium conversion-related culture medium.

[0035] Selenium conversion enrichment medium: 0.3% beef extract, 1% peptone, 0.5% sodium chloride, 300 mmol / L sodium selenite, pH 7.4–7.6, quantification 1 L. Sterilize the medium at 121℃ for 30 min.

[0036] The selenium conversion plate medium was supplemented with 2% agar, and the remaining components were the same as those of the selenium conversion enrichment medium.

[0037] (2) Separation and screening.

[0038] In a clean bench, wheat rhizosphere soil containing endophytic glutamate bacilli NY3-1 was brushed onto sterile weighing paper using a sterile brush. 1g of the sample was then weighed and added to a pre-prepared selenium conversion enrichment medium. The medium was incubated at 30℃ and 150 rpm for 24 hours using a shaker. The enriched bacterial solution was then inoculated under sterile conditions using an inoculation needle. Figure 1 Streak the strain onto a selenium-converted agar plate and incubate at 30°C for 24 hours. Repeat this process, streaking the faster-growing strain onto fresh agar plate, until a pure culture of selenium-converted strain is obtained. Figure 2 ).

[0039] Example 2

[0040] This embodiment provides an experiment to detect the tolerance of endophytic glutamate bacillus NY3-1 to high concentrations of selenium.

[0041] Prepare six bottles of standard liquid beef extract peptone medium, adding sodium selenite to achieve selenium concentrations of 0, 50, 100, 300, 500, and 1000 mmol / L. Add these concentrations to 48-well plates, arranging the wells from lowest to highest (wells A to F), with 400 μL added to each well, and perform eight replicates. Add 50 μL of pre-cultured NY3-1 bacterial suspension (≥10⁶ cells / well) to columns 1, 2, 3, 4, 7, and 8 of the 48-well plate. 8 ( / mL), columns 5 and 6 were added with the same volume of sterile water as a control. The 48-well plate was placed in an incubator at 30℃ and incubated statically for 24 hours. Figure 3 The image shows the observation of the formation of nano-selenium.

[0042] from Figure 3 It can be seen that after adding the endophytic glutamate bacterium solution, red nano-selenium appeared in the selenium culture medium. Moreover, as the selenium concentration increased, the amount of nano-selenium produced was greater and the color was darker. The production of red nano-selenium is due to the endophytic glutamate bacterium converting and reducing inorganic selenium compounds into nano-selenium through its own reduction system. Therefore, based on this experiment, the strain described in this invention can tolerate inorganic selenium compound concentrations of at least 1000 mmol / L and can utilize inorganic selenium compound concentrations of at least 50 mmol / L, indicating that this strain has great potential for nano-selenium conversion and application.

[0043] Example 3

[0044] This embodiment provides an experiment to detect the inhibitory effect of endophytic glutamate bacillus NY3-1 on common plant pathogens.

[0045] The inhibitory effects of strain NY3-1 on common plant pathogens *Botrytis cinerea*, *Diaporthe momicola*, and *Botryosphaeria wangensis* were determined using the plate confrontation method. The three pathogens were inoculated into PDA solid medium and cultured at 25°C for 3 days. The fungi were then broken into mycelial cakes using a 9mm diameter cake beater. These mycelial cakes were inoculated in the center of the confrontation medium, with four NY3-1 colonies inoculated around the center at a 2cm perimeter. The medium was then incubated statically at 30°C for 3 days, after which the inhibitory effect of strain NY3-1 on the pathogens was observed. Figure 4 As shown in the figure, the NY3-1 strain has a significant inhibitory effect on *Metacarbamophyton floccosum*, *Botrytis cinerea*, and *Gyromyces lutea*, the pathogen of tomato gray mold.

[0046] Example 4

[0047] This embodiment provides an experiment on the preparation of selenium-enriched endophytic glutamate bacterium NY3-1 and its application effects.

[0048] Endophytic glutamate bacillus NY3-1 was activated on a slant of ordinary beef extract peptone medium, then inoculated into liquid beef extract medium and cultured in shake flasks for 24 h. It was then inoculated into liquid fermentation medium (2% glucose, 1% yeast extract, 0.05% potassium dihydrogen phosphate, 0.03% dipotassium hydrogen phosphate, 0.5% sodium chloride, and 0.01% compound vitamins) and stirred at 150 r / min with an aeration rate of 6 vvm. After fermentation for 24 h, sodium selenite was added at 500 mmol / L, and the conversion was carried out for 48 h to obtain a liquid endophytic glutamate bacillus NY3-1 selenium-enriched inoculum. The nano-selenium content was measured to be ≥35 g / L.

[0049] The prepared selenium-enriched endophytic glutamate bacterium inoculant was diluted 50-100 times and applied to the greenhouse soil before transplanting tomato seedlings, ensuring a uniform selenium application rate of 40g / mu. Relevant tests were conducted on the rhizosphere soil, leaves, and fruits (if any) at the seedling, young fruit, and fruiting stages. Soil without endophytic glutamate bacterium NY3-1 application was used as a control. Soil test indicators included β-1,4-glucosidase, β-D-cellulase, β-1,4-N-acetylglucosidase, L-leucine-α-aminopeptidase, alkaline phosphatase, and soil selenium content; leaf test indicators included catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD); and fruit test indicator included selenium content in tomatoes.

[0050] Soil enzyme activity test results as follows Figure 5NY3-1 selenium-enriched microbial agent can significantly increase the activity of β-1,4-glucosidase in rhizosphere soil during the fruiting stage, β-D-cellulase in the young fruit stage, and β-1,4-N-acetyl-glucosidase in the young fruit stage. This indicates that NY3-1 selenium-enriched microbial agent can increase the activity of enzymes related to C and N cycles in rhizosphere soil during the fruit enlargement stage, thereby improving crop growth performance.

[0051] The results of enzyme activity tests on tomato leaves are as follows: Figure 6 NY3-1 selenium-enriched microbial agent can significantly increase the activity of catalase in tomato seedlings, catalase and peroxidase in young fruit, and superoxide dismutase and peroxidase in fruiting stage, thereby enhancing the stress resistance of tomatoes by increasing the activity of stress-related enzymes in the seedling, young fruit, and fruiting stages.

[0052] Results of selenium content in tomato fruits as follows Figure 7 The NY3-1 selenium-enriched microbial agent can significantly increase the selenium content in tomatoes during the young fruit stage and the fruiting stage, indicating that the nano-selenium produced by the NY3-1 strain has good application prospects in the cultivation of selenium-enriched crops.

[0053] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. Use of a salt-tolerant strain of Gluconacetobacter diazotrophicus endophytica subsp. (Gluconacetobacter diazotrophicus endophytica subsp. NY3-1) in the preparation of selenium elemental, characterized in that, Glutamicibacter endophyticus ) NY3-1 in the preparation of selenium elemental, characterized in that, Salt-tolerant Gluconacetobacter diazotrophicus endophytic subspecies strain (Gluconacetobacter diazotrophicus endophytic subspecies) Glutamicibacter endophyticus ) NY3-1 converts inorganic selenium compounds into elemental selenium; The salt-tolerant Gluconobacter endophyticus subspecies strain (Gluconobacter endophyticus subspecies endophyticus) strain NY3-1 has a preservation number of CCTCC No: M20242321. Glutamicibacter endophyticus The salt-tolerant Gluconobacter endophyticus subspecies strain (Gluconobacter endophyticus subspecies endophyticus) 2. The salt-tolerant Glutamicibacter endophyticus subsp. strain of claim 1, Glutamicibacter endophyticus ) NY3-1 in the preparation of selenium elemental, characterized in that, The inorganic selenium compound is sodium selenite.

3. The use of salt-tolerant strain of Enterobacter glacieci endophytic subspecies (NY3-1) in the prevention and treatment of plant diseases, characterized in that, Glutamicibacter endophyticus ) NY3-1 in the prevention and treatment of plant diseases, characterized in that, The endophytic subspecies of salt-tolerant glutamate bacillus ( Glutamicibacter endophyticus The preservation number of NY3-1 is CCTCC No: M20242321, and the pathogen of the plant disease described is selected from *Botrytis cinerea*, the causal agent of tomato gray mold. Botrytis cinerea, Metasomatic spp. Diaporthe momicola, Staphylococcus aureus Botryosphaeria At least one of wangensis.

4. Endophytic subspecies of glutamate-tolerant bacteria ( Glutamicibacter endophyticus The application of NY3-1 in plant cultivation is characterized by, The salt-tolerant Gluconobacter endophyticus subspecies strain (Gluconobacter endophyticus subspecies endophyticus) strain NY3-1 Glutamicibacter endophyticus The accession number of the salt-tolerant Gluconobacter endophyticus subspecies strain (Gluconobacter endophyticus subspecies endophyticus) strain NY3-1 is CCTCC No: M20242321 The endophytic subspecies of salt-tolerant glutamate bacillus ( Glutamicibacter endophyticus NY3-1 enhances the activity of C-cycle-related enzymes and N-cycle-related enzymes in the rhizosphere soil of plants, and / or enhances the activity of plant resistance-related enzymes; wherein the C-cycle-related enzymes and N-cycle-related enzymes are at least one of β-1,4-glucosidase, β-D-cellulase, and β-1,4-N-acetylglucosidase; wherein the plant resistance-related enzymes are at least one of catalase, superoxide dismutase, and peroxidase. or the salt-tolerant Gluconacetobacter diazotrophicus endophytic subspecies strain (Gluconacetobacter diazotrophicus endophytic subspecies strain) NY3-1 Glutamicibacter endophyticus NY3-1 for use in enriching selenium in plants.

5. The salt-tolerant Glutamicibacter endophyticus subsp. strain of claim 4, Glutamicibacter endophyticus NY3-1 for use in plant cultivation, characterized in that, comprising: applying a microbial inoculant to a plant; The microbial agent comprises a strain of endophytic subspecies of Glutamicibacter halodurans (G. Glutamicibacter endophyticus ) NY3-1, and the bacterial liquid is obtained by culturing the strain of endophytic subspecies of Glutamicibacter halodurans (G. Glutamicibacter endophyticus ) NY3-1 in a sodium selenite-containing medium.

6. The salt-tolerant Glutamicibacter endophyticus subsp. strain of claim 5, Glutamicibacter endophyticus ) NY3-1 for use in plant cultivation, characterized in that, The number of strains of halotolerant Glutamicibacter endophytica subspecies in the bacterial solution is not less than 10 Glutamicibacter endophyticus / mL. 8 / mL.

7. The salt-tolerant Glutamicibacter endophyticus subsp. strain of claim 5, wherein the strain is Glutamicibacter endophyticus NY3-1 for use in plant cultivation, characterized in that The concentration of sodium selenite in the sodium selenite-containing medium is not less than 50 mmol / L.

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