CN114480171A

CN114480171A - Rhizobium japonicum and application thereof

Info

Publication number: CN114480171A
Application number: CN202111604132.1A
Authority: CN
Inventors: 郑泽慧; 郭凯; 王庆玲; 田志喜; 任宗明; 霍毅欣; 刘书林; 黄艳华; 李哲; 郝永任
Original assignee: Shandong Jielier Fertilizer Industry Co ltd; Shandong Zhongke Food Co ltd; Venus Rizhao Agricultural Science And Technology Development Co ltd; Yantai Dadi Animal Husbandry Co ltd; Institute of Genetics and Developmental Biology of CAS; Beijing Institute of Technology BIT; Shandong Normal University; Biology Institute of Shandong Academy of Sciences
Current assignee: Shandong Jielier Fertilizer Industry Co ltd; Shandong Zhongke Food Co ltd; Venus Rizhao Agricultural Science And Technology Development Co ltd; Yantai Dadi Animal Husbandry Co ltd; Institute of Genetics and Developmental Biology of CAS; Beijing Institute of Technology BIT; Qilu University of Technology; Shandong Normal University
Priority date: 2021-12-24
Filing date: 2021-12-24
Publication date: 2022-05-13
Anticipated expiration: 2041-12-24
Also published as: CN114480171B

Abstract

The invention provides a rhizobium japonicum strain and application thereof, belonging to the technical field of microorganisms. The rhizobium japonicum is Sinorhizobium fredii DY23-9, and the strain is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation date: the preservation number is CGMCC No.23670 at 27/10/2021. The strain DY23-9 provided by the invention has the effect of promoting the growth of soybean plants under the conditions of saline-alkali soil greenhouse potting/field, the promotion effect is realized by increasing the nodulation number, plant height and stem thickness of the soybeans, and the strain DY23-9 has an important application value in the aspect of promoting the cultivation and production of the soybeans in saline-alkali soil.

Description

Rhizobium japonicum and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a rhizobium japonicum strain and application thereof.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Soybeans are important edible and feeding crops, provide high-quality grease and protein resources for human beings, and the requirements of livestock and poultry industries on soybean feeds are increased year by year along with the changes of living standards and dietary structures of China. However, cultivated land resources in China are limited, the yield per unit of soybeans is lower than that of main grain crops, and in order to ensure that the total grain yield is not reduced, the enlargement of the soybean planting area must be finished under the condition of not competing with grains for land. The saline-alkali soil is an important and indispensable reserve land resource in China, wherein the saline-alkali soil has the agricultural utilization potential of 2 hundred million acres, which accounts for 10 percent of the total cultivated land area in China, and the increase of the soybean planting area and the yield of the saline-alkali soil is a powerful way for reviving the soybean industry in China. The adverse conditions of high salt and high alkali in saline-alkali soil stress the growth of soybeans and limit the yield of the soybeans, and the difficulty that the resistance of the soybeans is improved or the saline-alkali soil is improved by technical means becomes necessary to overcome at present.

The rhizobium inoculation can enhance the tolerance of the soybean to salt stress, thereby improving the yield of the soybean in saline-alkali soil. Rhizobia is a kind of gram-negative bacteria widely distributed in soil, and can form root nodules or stem nodules with roots and stems of leguminous plants, fix nitrogen in the air into ammonia which can be absorbed and utilized by the plants, thereby promoting the growth of crops and playing an important role in improving and maintaining the soil fertility. The research on the biological geography of the rhizobia discovers that the rhizobia and host plants co-evolve under specific environmental conditions, and excellent strains mostly come from the same area where the host plants are planted. Therefore, the in-situ screening of the indigenous soybean rhizobium in the saline-alkali soil and the application thereof are effective measures for promoting the yield increase of the soybeans in the saline-alkali soil.

Disclosure of Invention

Based on the defects of the prior art, the invention provides a soybean rhizobium strain and application thereof, wherein the strain is obtained by technologies such as separation, screening, purification, identification and the like from saline-alkali-resistant soybean rhizobium and belongs to China rhizobium fredii (Sinorhizobium fredii). Experiments prove that the compound fertilizer has excellent stress resistance, short generation time and rapid growth and propagation, can effectively promote the nodulation and growth of soybeans in saline-alkali soil, achieves the effect of promoting the growth and increasing the yield of the soybeans, and has good practical application value.

In order to achieve the technical purpose, the invention relates to the following technical scheme:

the first aspect of the invention provides a Sinorhizobium fredii DY23-9 strain, which is preserved in China general microbiological culture Collection center (CGMCC for short) and has the address: the preservation date of the microbial research institute of the Chinese academy of sciences, No.3 of Xilu No.1 of Beijing, Chaoyang district, Beijing: the preservation number is CGMCC No.23670 at 27/10/2021.

The metabolite of the Sinorhizobium freundii DY23-9 also belongs to the protection scope of the invention.

In a second aspect of the invention, the culture method of the sinorhizobium freundii DY23-9 is provided, and comprises the step of inoculating the sinorhizobium freundii DY23-9 to a fermentation culture medium for fermentation culture.

Wherein the fermentation medium may be a yeast mannitol medium.

In a third aspect of the present invention, there is provided a microbial preparation comprising sinorhizobium freudenreichii DY23-9 or a fermentation product or a metabolite thereof according to the first aspect.

The metabolite of the invention comprises a thallus intracellular metabolite and/or an extracellular metabolite.

In a fourth aspect of the invention, a bacterial fertilizer is provided, which comprises the sinorhizobium freudenreichii DY23-9 or a fermentation product thereof or a metabolite thereof or a microbial inoculum of the third aspect.

In a fifth aspect of the invention, the application of the sinorhizobium freundii DY23-9, the microbial inoculum and/or the bacterial fertilizer in all or part of the following 1) -2) is also within the protection scope of the invention:

1) promoting the nodulation of the soybeans;

2) promoting the growth of soybean.

Wherein, in the applications 1) and 2), the soybean growth environment is a saline-alkali environment.

In the application 1), specific expression of promoting the nodulation of the soybeans includes but is not limited to increasing the nodulation number of the soybeans, the fresh weight of the root nodules and the diameter of the root nodules.

In the application 2), the specific expression of promoting the soybean growth includes but is not limited to increasing the plant height and the stem thickness of the plant.

In a sixth aspect of the present invention, there is provided a method for producing soybeans by cultivation in a saline-alkali soil environment, the method comprising: and when the soybeans are sown, the Sinorhizobium freundii DY23-9, a microbial inoculum and/or a bacterial fertilizer are applied.

The beneficial technical effects of one or more technical schemes are as follows:

1) according to the technical scheme, the rhizobium indigenous to the saline-alkali soil provides a new microbial germplasm resource for the soybean planting of the saline-alkali soil, and provides a foundation for developing a suitable green input product for the soybean planting of the saline-alkali soil and promoting the development of the soybean industry.

2) Adverse conditions of high salinity and high alkalinity in saline-alkali soil stress soybean plants to grow and limit the yield of soybeans. The rhizobium japonicum strain DY23-9 provided by the technical scheme can effectively promote nodulation and growth of soybeans in saline-alkali soil, and achieves the effects of promoting growth and increasing yield.

3) According to the technical scheme, core field test points are arranged in the high and new technology industry demonstration area of yellow river delta agriculture, the research on the field application effect of the strain DY23-9 in saline and alkaline land is developed, the effect is good, and the development and application prospects are good.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a colony morphology of the strain DY23-9 in YMA plate medium and a morphology under a microscope after gram staining in example 1 of the present invention;

FIG. 2 is a phylogenetic dendrogram of the 16SrDNA sequence of strain DY23-9 in example 1 of the present invention;

FIG. 3 is a growth curve and generation time of the strain DY23-9 in example 2 of the present invention;

FIG. 4 is a salinity, temperature and pH tolerance evaluation of the strain DY23-9 in example 2 of the present invention;

FIG. 5 shows the effect of inoculating strain DY23-9 to saline-alkali soil pot culture on soybean nodulation in example 3 of the present invention;

FIG. 6 is a plant phenotype diagram of a saline-alkaline earth potted inoculum strain DY23-9 in example 3 of the present invention;

FIG. 7 shows the effect of inoculation of strain DY23-9 in saline-alkali land in field for soybean growth in example 4 of the present invention;

FIG. 8 is a plant phenotype diagram of a saline-alkali soil field inoculation strain DY23-9 in example 4 of the invention.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

As previously mentioned, inoculation with rhizobia can enhance tolerance of soybeans to salt stress, thereby increasing yield of soybeans in saline and alkaline land. The special habitat of the saline-alkali soil acclimatizes rhizobia adaptive to conditions of the special habitat, the rhizobia capable of tolerating saline-alkali environment and symbiotic with saline-alkali tolerant soybeans are screened in a targeted mode, and the method is the core for effectively improving the yield of soybeans in the saline-alkali soil.

In view of the above, in an exemplary embodiment of the present invention, there is provided a strain of Sinorhizobium fredii (Sinorhizobium fredii) DY23-9, which has been deposited in the china general microbiological culture collection center (CGMCC), address: west road No.1, north west of the morning area, beijing, institute of microbiology, china academy of sciences, date of deposit: the preservation number is CGMCC No.23670 at 27/10/2021.

The characteristics of the Sinorhizobium freundii DY23-9 are as follows:

the characteristics of the thallus are as follows: under the optical microscope, the product is in the shape of short rod with length of 2-4 μm, and is red after gram staining, and is a gram-negative bacterium, and the red diaphragm is inside the thallus.

Colony characteristics: round or nearly round, viscous, smooth, slightly convex, milky colonies were formed on Yeast Mannitol Agar (YMA) plate medium. Colonies were pink on YMA medium containing Congo red dye.

The 16S rDNA sequence is shown in SEQ ID NO. 1.

In still another embodiment of the present invention, there is provided the method for culturing Sinorhizobium freundii DY23-9 as described above, wherein the method comprises inoculating the Sinorhizobium freundii DY23-9 to a fermentation medium for fermentation culture.

Wherein, the fermentation medium can be a yeast mannitol medium, and the yeast mannitol medium comprises the following components: 1g/L of yeast extract powder, 10g/L of mannitol, 0.2g/L of magnesium sulfate, 0.1g/L of sodium chloride, 0.5g/L of monopotassium phosphate and 6.8-7.2 of pH value.

In still another embodiment of the present invention, there is provided a microbial preparation comprising said Sinorhizobium freudenreichii DY23-9 or a fermented product thereof or a metabolite thereof.

In the present invention, the term "fermentate" is used to refer to a fermentation product. The corresponding fermented product can be liquid obtained from the process of fermenting and culturing Sinorhizobium freudenreichii DY23-9 bacteria, and thus, can also be called as fermentation liquid; the liquid may contain bacteria (bacteria cells), but does not necessarily need to contain bacteria. The liquid preferably contains a metabolite produced by Sinorhizobium freundii DY23-9 of the present invention.

And, in the embodiment of the present invention, the bacterial cells grown in the fermentation broth or culture broth are separated from the liquid by centrifugation, filtration, sedimentation or other means known in the art, and the liquid remaining when the bacterial cells are removed is a "supernatant", and in the present invention, the extracellular metabolite of sinorhizobium freundii DY23-9 is contained in the supernatant. In the embodiment of the present invention, the microbial agent may also contain the supernatant.

And, in the embodiments of the present invention, the fermentation liquid or culture liquid containing the bacterial cells is centrifuged, filtered, settled or separated from the liquid to obtain the bacterial cells, the bacterial cells can be disrupted by sonication (such as ultrasonic cell disruption in ice bath) or other means known in the art to obtain disrupted bacterial cells, or the disrupted bacterial cells are centrifuged to collect a supernatant, which is referred to as a cell-free bacterial extract, and the disrupted bacterial cells or cell-free extract contains intracellular metabolites of rhizobium freundii DY 23-9. In the embodiment of the present invention, the microbial inoculum may contain a disrupted microbial cell or a cell-free extract thereof.

And, in the embodiment of the present invention, the microbial inoculum may also be a solid, and more preferably a freeze-dried powder, for the convenience of storage, transportation, improvement of the survival rate of the strain, and the like. The rhizobium freundii DY23-9 or the fermentation product or the metabolite thereof is further subjected to freeze drying, and the freeze drying technology (including vacuum freeze drying technology) can be carried out by adopting a conventional method.

In another embodiment of the present invention, the microbial agent further contains a carrier in addition to the active ingredient. The carrier may be one that is commonly used in the art of microbial preparation and is biologically inert.

The carrier can be a solid carrier or a liquid carrier;

the solid carrier can be mineral materials, plant materials and/or high molecular compounds; the mineral material may be at least one of clay, talc, medical stone, kaolin, montmorillonite, white carbon, zeolite, silica and diatomaceous earth; the plant material can be at least one of corn flour, bean flour, rice hull flour and starch; the high molecular compound can be polyvinyl alcohol or/and polyglycol;

the liquid carrier can be an organic solvent, vegetable oil, mineral oil, or water; the organic solvent may be decane or/and dodecane.

The preparation formulation of the microbial inoculum can be various preparation formulations, such as liquid, emulsion, suspending agent, powder, granules, wettable powder or water dispersible granules; preferably a powder.

According to the requirement, the microbial inoculum can also be added with a surfactant (such as Tween 20, Tween 80 and the like), a binder, a stabilizer (such as an antioxidant), a pH regulator and the like.

In another embodiment of the invention, the bacterial fertilizer comprises the sinorhizobium freundii DY23-9 or the fermentation product or the metabolite thereof or the microbial inoculum of the third aspect.

In another embodiment of the invention, the application of the sinorhizobium freundii DY23-9, the microbial inoculum and/or the bacterial manure in all or part of the following 1) -2) is also within the protection scope of the invention:

1) promoting the nodulation of the soybeans;

2) promoting the growth of soybean.

Accordingly, in yet another embodiment of the present invention, there is provided a method for producing soybeans by cultivation in a saline-alkali soil environment, the method comprising: and when the soybeans are sown, the Sinorhizobium freundii DY23-9, a microbial inoculum and/or a bacterial fertilizer are applied.

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1: capture, separation, purification and identification of strain DY23-9

1.1 Capture of the Strain

The test fields are planted in the core area of the high and new technology industry demonstration area of yellow river delta agriculture in east China and east China, and 27 saline-alkali soil samples are obtained by taking the ground surface to 10cm of underground saline-alkali soil layer at random intervals of 20-30 meters.

Taking soybean seeds with proper size and complete structure, treating the soybean seeds with 95% ethanol for 1min, performing sterile water washing for 2-3 times, treating the soybean seeds with 1% sodium hypochlorite solution for 5min and performing sterile water washing for 5-7 times, placing the soybean seeds in a 0.7% agar culture medium at 28 ℃ for accelerating germination for 48h, planting the postembryonic roots downwards into a hole tray filled with Dongying soil, applying 4mL of low-nitrogen nutrient solution around the seeds, then pouring sterile water irregularly to keep the soil moist, and setting 3 times for each soil sample to repeat.

1.2 isolation and purification of the Strain

After the soybean plant enters the three-leaf period, washing out all root systems with sterile water, reducing the root systems without root nodules, treating the remaining root systems with root nodules with 95% ethanol for 5min, washing with sterile water for 2-3 times, treating with 20% sodium hypochlorite solution for 15min, and washing with sterile water for 5-7 times. Cutting off the root nodule with a sterile blade on a clean bench, picking up pink tissue inside the root nodule with a sterile inoculating needle, streaking on a starch mannitol agar (YMA) culture medium, and culturing at a constant temperature of 28 ℃ until a clear colony appears.

And selecting a single colony, streaking and purifying the single colony in a new YMA culture medium twice, selecting the single colony, transferring the single colony to a YMA inclined plane, and naming the obtained strain as DY 23-9.

The composition of YMA solid medium was (1L): 0.2g of magnesium sulfate, 1.0g of yeast extract, 10.0g of mannitol, 0.1g of sodium chloride, 0.5g of monopotassium phosphate, 15.0g of agar and 0.025g of congo red. The pH was adjusted to 7.0.

1.2 identification of the strains

(1) Morphological characterization of strains

Strain DY23-9 is inoculated on YMA solid culture medium for culture, and after culture at 28 ℃ for 72h, the colony is round or approximately round, neat in edge, translucent, viscous, and 2.0-5.0mm in diameter. The thallus under the optical microscope is in a short rod shape, two ends are round and blunt, the diameter of the thallus is 0.4-0.9 multiplied by 1.6-3.0 mu m, no spore exists, gram stain is negative, and diaphragm is common in cells.

(2) 16SrRNA gene sequence identification of strain

The 16SrRNA gene was amplified using the bacterial universal primer pair 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.2) and 1492R (5'-TACGGCTACCTTGTTACGACTT-3', SEQ ID NO. 3). PCR reaction (50. mu.L): 2 uL of each of the upstream and downstream primers, 25 uL of 2 XTAQUIMixDNA polymerase, 1 uL of Triton (x100), sterilized ddH₂O19 μ L. Reaction conditions are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 94 ℃ for 15s, annealing at 56 ℃ for 15s, extension at 72 ℃ for 45s, and amplification for 35 cycles; and (3) compensating and extending for 5min at 72 ℃. After the amplified product is detected by agarose gel electrophoresis, the amplified product is subjected to bidirectional sequencing by Shanghai Biotechnology engineering Limited company, a sequence obtained by the bidirectional sequencing is spliced by CExpress 3.0 software, about 20bp bases with unclear peak diagrams at two ends of the sequence are cut off, and the obtained sequence is the 16S rRNA gene sequence SEQ ID NO.1 of DY 23-9.

The 16S rRNA gene sequence of DY23-9 was subjected to BLAST alignment at the American bioinformatics center (NCBI), a model strain having high similarity was selected as a reference strain, and the construction of a phylogenetic tree of the 16S rRNA gene was carried out by using the Neighbor-joining method (Neighbor-joining) in MEGA 11 software, as shown in FIG. 2. Strain DY23-9 belongs to a genetic branch with the same genus as Sinorhizobium fredii sp, and has the closest genetic relationship; the sequence homology with the Sinorhizobium fredii model strain USDA205 with the accession number of AY260149 reaches 99.70 percent; the highest homology with Sinorhizobium sp. sequence with the accession number EU145985.1 is 99.78%. Thus, it was confirmed that the strain DY23-9 was a novel strain of Sinorhizobium fredii.

The strain has been deposited in the Budapest treaty International Collection of microorganisms: china general microbiological culture Collection center (CGMCC for short). And (4) storage address: xilu No.1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences. The preservation date is as follows: 2021, 10 and 27 months. The preservation number is CGMCC No. 23670.

Example 2: evaluation of physiological function of Strain DY23-9

2.1 growth curves and passage times of the strains

Inoculating strain DY23-9 into YM liquid culture medium, culturing at 28 deg.C under 150r/min, sampling every 8h, measuring OD value of bacterial liquid with enzyme-labeling instrument at 600nm wavelength, and drawing growth curve, wherein the result is shown in FIG. 3, the logarithmic growth phase is within 32h, and OD600 value tends to be stable after 32h, indicating that the growth enters stationary phase. The generation time of the strain DY23-9 is calculated to be 3.0h by the doubling time of the OD600 value in the logarithmic growth phase, and the growth speed is higher.

2.2 evaluation of stress resistance of Strain

Based on 28 ℃ culture of YMA medium with pH of 7, single-factor variable treatments such as different salt concentrations (0%, 1%, 2%, 3%, 4%, 5% NaCl), different temperatures (14 ℃, 21 ℃, 28 ℃, 35 ℃, 42 ℃), different pH values (5, 7, 9, 11), etc. were set. The strain DY23-9 and the soybean rhizobium model strain USDA110 are streaked by using a sterile toothpick in each treatment, the strain is kept for 3d, whether colonies grow or not is observed, and the growth morphology of the colonies is recorded by scanning.

As a result, as shown in FIG. 4, strain DY23-9 was more salt-tolerant than model strain USDA110 and was able to maintain normal growth on YMA medium containing 2% NaCl, and growth of strain DY23-9 was inhibited by NaCl treatment at a concentration of 3% or more. Compared with the model strain USDA110, the strain DY23-9 has wider temperature adaptability, can normally grow at 14 ℃, 21 ℃ and 35 ℃, and has higher biomass when cultured at 35 ℃ than at 28 ℃. The strain DY23-9 has stronger alkali resistance than the model strain USDA110, and can keep normal growth on an alkaline YMA medium with the pH of 9.0.

Example 3: influence of strain DY23-9 on nodulation of potted soybeans in saline-alkali soil

This example was carried out in the laboratory of the Industrial microbiology, institute of biological research, academy of sciences, Shandong province. Selecting soybean seeds with proper size and complete structure, treating the soybean seeds with 95% ethanol for 1min, performing sterile water washing for 2-3 times, treating the soybean seeds with 1% sodium hypochlorite solution for 5min, performing sterile water washing for 5-7 times, placing the soybean seeds in a 0.7% agar culture medium at 28 ℃ for accelerating germination for 48h, then transplanting radicles downwards into a tissue culture bottle containing Dongying saline-alkali soil, applying 10mL of low-nitrogen nutrient solution around the seeds, performing inoculation treatment, adding 1mL of yeast mannitol solution to cultivate DY23-9 bacterial solution for 3d around the seeds, performing blank contrast treatment, adding 1mL of sterile water around the seeds, then pouring sterile water irregularly to keep the soil moist, setting 12 times for each treatment, and culturing in an illumination incubator at 25 ℃ for 16h and dark at 20 ℃ for 8h every day. After the plant grows to the 3 rd group of three leaves and is unfolded (about 30 d), taking out a complete root system, and measuring indexes such as the number of root nodules, the fresh weight of the root nodules, the maximum diameter of the root nodules and the like.

Test microorganisms: strain DY 23-9; the test plants: and (4) soybeans TZX-805.

As a result, as shown in FIGS. 5 and 6, strain DY23-9 significantly increased (P <0.05) the number of nodules of soybean, the fresh weight of nodules, and the diameter of nodules. Compared with the control group, the number of the root nodules, the fresh weight of the root nodules and the maximum diameter of the root nodules of the soybeans in the inoculation treated group are respectively increased by 78.3 percent, 62.3 percent and 23.2 percent. The results show that the strain DY23-9 can remarkably promote the nodulation of soybeans in saline-alkali soil environment.

Example 4: influence of strain DY23-9 on growth condition of soybeans in saline-alkali soil field

The embodiment is carried out in a high and new technology industry demonstration area of yellow river delta agriculture in east China of Shandong province. And (3) taking conventional planting as a control group (CK), applying 400 mL/mu of strain DY23-9 around seeds during sowing to culture a bacterial liquid for 7d in a yeast mannitol liquid culture medium, and keeping the rest of the strain being the same as the control group. Bacterial liquid requirement OD₆₀₀The value is greater than 1.0. Each processing facility 6And (4) repeating. The planting period is 100 days in total, field investigation is carried out at 80 days, and growth phenotype data such as plant height, stem thickness and the like are measured.

The results are shown in fig. 7 and fig. 8, and strain DY23-9 significantly improves (P <0.05) the plant height and stem thickness of soybean plants. Under the condition of saline-alkali soil and field, the plant height ratio CK of the soybeans treated by the inoculated strain DY23-9 is increased by 25.1%, and the stem thickness is increased by 13.5%. The results show that the inoculated strain DY23-9 can obviously promote the growth of soybean plants in saline-alkali soil and field conditions.

It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Although the present invention has been described with reference to the specific embodiments, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.

SEQUENCE LISTING

<110> Shandong province academy of sciences biology research institute, China academy of sciences genetics and developmental biology research institute, Shandong university of sciences, Beijing university of sciences, gold Star (sunshine) agricultural science and technology development Co., Ltd, Shandong Jielier Fertilizer Co., Ltd, Yantai land animal husbandry Co., Ltd, Shandong Zhongke food Co., Ltd

<120> rhizobium japonicum strain and application thereof

<130>

<160> 3

<170> PatentIn version 3.3

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<213> Artificial sequence

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agagtttgat cctggctcag 20

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Claims

1. The strain is Chinese rhizobium fredii (Sinorhizobium fredii) DY23-9, which is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation date: the preservation number is CGMCC No.23670 at 27/10/2021.

2. The method for culturing Sinorhizobium freundii DY23-9 according to claim 1, wherein the method comprises inoculating Sinorhizobium freundii DY23-9 to a fermentation medium for fermentation culture.

3. The culture method of claim 2, wherein the fermentation medium comprises a yeast mannitol medium.

4. A microbial preparation comprising Sinorhizobium freudenreichii DY23-9 or a fermented product or a metabolite thereof according to claim 1;

preferably, the microbial inoculum also contains a carrier; it is further preferred that the carrier is a biologically inert carrier commonly used in the art of microbial preparation.

5. A bacterial fertilizer, which is characterized by comprising the Sinorhizobium freudenrz DY23-9 of claim 1 or a fermentation product or a metabolite thereof or the microbial inoculum of claim 4.

6. Use of sinorhizobium freundii DY23-9 according to claim 1, the microbial inoculum according to claim 4 and/or the bacterial manure according to claim 5 in all or part of the following 1) -2):

1) promoting the nodulation of the soybeans;

2) promoting the growth of soybean.

7. The use of claim 6, wherein in the applications 1) and 2), the soybean growth environment is a saline-alkali environment.

8. The use of claim 6, wherein in the use 1), the promotion of soybean nodulation comprises an increase in the number of soybean nodulation, the fresh weight of nodules, and the diameter of nodules.

9. The use of claim 6, wherein in the use of 2), the specific expression of soybean growth promotion comprises plant height and stem thickness increase.

10. A method for cultivating and producing soybeans in a saline-alkali soil environment, which is characterized by comprising the following steps: the Sinorhizobium freundii DY23-9 of claim 1, the microbial agent of claim 4, and/or the bacterial fertilizer of claim 5 are applied at the time of soybean sowing.