Nesterennet rhizobium and application thereof

Abstract

The invention discloses a strain of Nesterenznets rhizobium and application of the strain of Nesterenznets rhizobium. The Nerennets rhizobium is preserved in the China General Microbiological Culture Collection Center on September 2, 2024, and the preservation number of the Nerennets rhizobium is CGMCC (China General Microbiological Culture Collection Center) No. 31818. The Nerennets rhizobium provided by the invention grows fast, and has good nitrogen fixation function and salt tolerance. The Nerennets rhizobium provided by the invention can significantly improve the germination rate of alfalfa seeds at different times, and has a significant promotion effect on the growth of lettuce seedlings.

Description

Technical Field

[0001] This invention belongs to the field of biological technology, specifically relating to a strain of Skärnevize rhizobium and its applications. Background Technology

[0002] my country has a vast area of ​​saline-alkali land. Soil salinization damages soil structure, reduces fertility, inhibits the activity of key microorganisms, and causes multiple harms to plants, including osmotic stress, ion toxicity, and metabolic disturbance, severely restricting agricultural production. Against this backdrop, utilizing functional microorganisms for ecological restoration has become an important strategy. Among them, nitrogen-fixing bacteria, especially rhizobia that live in symbiosis with legumes, have shown unique advantages and application potential. They can convert atmospheric nitrogen into ammonia, which plants can directly utilize, through their nitrogenase system, providing a valuable biological nitrogen source for saline-alkali and infertile soils. Some rhizobium strains possess a certain degree of salt and alkali tolerance, maintaining activity under adverse conditions. The growth-promoting mechanisms of rhizobia are multifaceted: firstly, they provide direct nutrient supply, effectively reducing dependence on chemical nitrogen fertilizers; secondly, they can synergistically improve plant nutrition by dissolving elements such as phosphorus and potassium; furthermore, some strains can secrete plant hormones such as auxins and cytokinins to alleviate environmental stress, directly stimulating root development and enhancing overall stress resistance. Introducing salt-tolerant rhizobia can not only enhance the overall activity and diversity of soil microorganisms and promote the accumulation of organic matter and the formation of soil aggregates, but also gradually drive the saline-alkali land ecosystem to recover towards a virtuous cycle.

[0003] Therefore, screening and utilizing efficient salt-tolerant nitrogen-fixing bacteria resources is a key approach to developing microbial agents for saline-alkali land and achieving synergistic improvement in ecology and productivity. Summary of the Invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a strain of Rhizobium skierniewicense CNBG-PGPR-28, which can improve the salt tolerance and nitrogen fixation of plants, with the accession number CGMCCNo.31818.

[0005] Another technical problem that this invention aims to solve is to provide a microbial inoculant.

[0006] Another technical problem to be solved by the present invention is to provide the application of Skørnevize rhizobium or its microbial inoculants in the degradation of inorganic phosphorus.

[0007] Another technical problem to be solved by the present invention is to provide the application of Skørnevize rhizobium or its microbial inoculants in nitrogen fixation.

[0008] Another technical problem that this invention aims to solve is to provide the application of Skørnevize rhizobium or its microbial inoculants in plant cultivation.

[0009] Technical Solution: To solve the above-mentioned technical problems, the present invention provides a strain of Rhizobium skierniewicense CNBG-PGPR-28, which was deposited on September 2, 2024 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 31818 and classified as Rhizobium skierniewicense. The deposit address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, China.

[0010] The present invention also provides a microbial inoculant containing the aforementioned Rhizobium skierniewicense CNBG-PGPR-28.

[0011] The present invention also provides the application of the aforementioned Rhizobium skierniewicense CNBG-PGPR-28 or the aforementioned microbial inoculant in improving plant salt tolerance.

[0012] Among them, the Rhizobium skierniewicense CNBG-PGPR-28 can grow at a salinity of 1% to 6%.

[0013] The present invention also provides the application of the aforementioned Rhizobium skierniewicense CNBG-PGPR-28 or the aforementioned microbial agent in the degradation of inorganic phosphorus.

[0014] The inorganic phosphorus is derived from one or more of tricalcium phosphate, calcium phosphate, magnesium phosphate, iron phosphate, or aluminum phosphate.

[0015] The present invention also provides the application of the aforementioned Rhizobium skierniewicense CNBG-PGPR-28 or the aforementioned microbial inoculant in nitrogen fixation.

[0016] The present invention also provides the application of the aforementioned Rhizobium skierniewicense CNBG-PGPR-28 or the aforementioned microbial inoculant in plant cultivation.

[0017] The application of Rhizobium skierniewicense CNBG-PGPR-28 or the microbial agent in promoting plant seed germination and seedling growth.

[0018] The plant in question is alfalfa or lettuce. Other plants, such as tomatoes, spinach, and bok choy, may also be used.

[0019] Beneficial Effects: Compared with existing technologies, the present invention has the following advantages: The Rhizobium skierniewicense CNBG-PGPR-28 of the present invention exhibits rapid growth, good stability, and can be scaled up in large-volume devices. It is highly salt-tolerant, growing at 30°C on media with salinity of 1%, 3%, and 5%, and demonstrates excellent phosphorus solubilization and nitrogen fixation capabilities. Simultaneously, the Rhizobium skierniewicense CNBG-PGPR-28 of the present invention can significantly improve the germination time of alfalfa seeds, and significantly increase the germination rate at all time points. The Rhizobium skierniewicense CNBG-PGPR-28 of the present invention has a significant promoting effect on the growth of lettuce seedlings. Attached Figure Description

[0020] Figure 1 The colony morphology of *Skelnevize rhizobium* CNBG-PGPR-28 on agar plate. Figure 2 The cell morphology of Rhizobium Scärnevizei CNBG-PGPR-28 under a scanning electron microscope; Figure 3 Image showing the results of CNBG-PGPR-28 Gram staining of Rhizobium skärnevizei; Figure 4 Figure showing the growth of Skelnevize rhizobium CNBG-PGPR-28 on 5% salt medium; Figure 5 Figure showing the growth of Skelnevize rhizobium CNBG-PGPR-28 on inorganic phosphorus plate medium; Figure 6 Figure showing the growth of Skelnevize rhizobium CNBG-PGPR-28 on nitrogen-fixing agar plates; Figure 7 The image shows the effect of Skärnevize rhizobium CNBG-PGPR-28 on the growth of lettuce seedlings. Detailed Implementation

[0021] In the following embodiments, various processes and methods not described in detail are all conventional methods known in the art. Furthermore, the terminology used in this invention, unless otherwise stated, generally has the meanings commonly understood by those skilled in the art.

[0022] Example 1: Isolation and identification of Rhizobium skelnevizei

[0023] (1) Cultivation of alfalfa and pretreatment of root samples

[0024] Mature, plump alfalfa seeds were selected and sown in plastic flowerpots (200 g of saline-alkali soil per pot) using the spot sowing method, with approximately 15 seeds sown per pot. When the seedlings reached a height of about 30 cm, healthy seedlings were selected, and their root systems were collected. The saline-alkali soil used in the experiment was taken from Guangtan, Lianyungang New City (34°46'33"N, 119°15'38"E), with a salinity of 2‰. The tested alfalfa variety was 'Zhongmu No.1' (Medicago sativa Linn.cv. Zhongmu No.1), provided by the College of Grassland Agriculture Science and Technology, Lanzhou University. Root samples of alfalfa were collected using the root-shaking method. Rinse the root surface with sterile water, then soak in 75% ethanol solution for 30 min. Rinse again with sterile water, then soak in 90% ethanol solution for 30 min. Rinse the root surface with sterile water again. Place approximately 1 g of the rinsed roots into a 50 mL centrifuge tube, add zircon and 20 mL of 1×PBS buffer, and vortex twice in a FastPrep-24™ rapid sample preparation instrument (MP Biomedicals, USA) at a speed of 6.5 m / s for 45 s each time. Collect the supernatant. Dilute the supernatant with sterile water in a 10-fold serial dilution series to obtain 10... -1 10 -2 10 -3 10 -4 10 -5 10 -6 Solution. Take 100 µL of each graded dilution and spread it evenly on the surface of TTC nutrient agar plates. Invert the plates and incubate at 30℃ for 18–24 h. The TTC nutrient agar plate culture medium consists of: 10.0 g peptone, 3.0 g beef extract, 5.0 g sodium chloride, and 15.0 g agar (pH 7.3±0.1) per liter of water, and 0.01 g 2,3,5-triphenyltetrazolium chloride (TTC).

[0025] (2) Isolation and purification of strains

[0026] From culture plates of different dilution gradients, plates with distinct colony morphological characteristics and colony counts between 30 and 300 were selected for their suitability. Single colonies exhibiting typical morphological differences were picked using a sterile inoculation needle and transferred to TTC nutrient agar plates for isolation and purification using the four-zone streak method. This purification process was repeated until single colony cultures with completely identical morphology were obtained. To ensure strain purity, the final single colonies were subjected to at least five consecutive streak subculturings, and their culture stability was verified by microscopic morphological observation.

[0027] (3) Staining and preservation

[0028] Gram staining identification of the strain was performed using a standard method: single colonies were picked and evenly spread on clean glass slides, and then treated sequentially with ammonium oxalate crystal violet solution (primary staining for 1 min), Lugol's iodine solution (mordanting for 1 min), 75% (v / v) ethanol solution (decolorizing for 30 s), and safranin solution (counter-staining for 1 min). The staining characteristics were observed and recorded under an optical microscope (100× oil immersion). The identified pure cultures were inoculated into sterile NA liquid medium and incubated at 200 r / min for 24±2 h at 30℃. The logarithmic growth phase bacterial culture was mixed with an equal volume of pre-cooled 60% (v / v) glycerol protectant (final concentration 30% glycerol), aliquoted into 2 mL sterile cryovials (1.5 mL per tube), labeled, and placed in an ultra-low temperature freezer at -80℃ to establish a bacterial culture bank. The NA medium formula is as follows (per liter): 10.0g peptone, 3.0g beef meal, 5.0g sodium chloride, 15.0g agar, pH 7.3±0.1. The NB liquid medium has the same composition as NA, but does not contain agar.

[0029] (4) Strain identification

[0030] Remove the plate containing the corresponding numbered strain from the 4°C freezer, pick a single colony as a DNA template, and perform PCR amplification, 16S rDNA sequencing, and sequence alignment. The specific procedures are as follows:

[0031] 1) Amplification system:

[0032] The 50 μL amplification system consisted of the following components: 2X Taq enzyme Master Mix (25 μL, Aikerui Biotechnology, 2X Accurate Taq premix), upstream primer 27F (1 μL), downstream primer 1492R (1 μL), DNA template (1 μL), and ddH2O (22 μL). The 27F sequence was 5'-AGAGTTTGATCMTGGCTCAG-3', and the 1492R sequence was 5'-GGYTACCTTGTTACGACTT-3'.

[0033] 2) Amplification conditions:

[0034] Pre-denaturation temperature: 94℃, first step denaturation: 94℃ for 30 s, second step annealing: 56℃ for 60 s, third step extension: 72℃ for 45 s, number of cycles: 30, fourth step final extension: 72℃ for 10 min, fifth step storage: 4℃.

[0035] 3) Agarose gel electrophoresis identification, nucleic acid sequencing and identification.

[0036] Weigh 1 g of agarose and dissolve it in 100 mL of TBE electrophoresis buffer. Microwave the solution until clear and transparent. Add 1‰ GelRed nucleic acid dye, shake well, and let stand until no bubbles remain. Slowly pour the solution into a gel plate and let it stand for about 1 hour. Remove the solidified gel block and place it in an electrophoresis tank. Add 5 μL of PCR amplification product to each well and run the gel at 120V for 20 minutes. Remove the gel block and place it in a gel imaging system. Select UV light and take a picture. By comparison, if there is a clear band around 1400 bp on the gel block, the PCR amplification is considered successful. Send the PCR amplification product to Nanjing Qingke Biotechnology Co., Ltd. for sequencing. The 16S rDNA sequence of Rhizobium skiernwicense is shown in SEQ ID NO.1. Enter the returned sequence into the online sequence search tool The Basic Local Alignment Search Tool on the National Center for Biotechnology Information (NCBI) website. A search using BLAST revealed that the bacterial strain provided in this invention is *Rhizobium skierniewicense*, belonging to the phylum Pseudomonadota, class Alphaproteobacteria, order Hyphomicrobiales, family Rhizobiaceae, and genus *Rhizobium*. This *Rhizobium skierniewicense* strain (CNBG-PGPR-28) was deposited on September 2, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 31818, classified as *Rhizobium skierniewicense*, and located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China. The colony morphology is as follows: on the surface of agar plates, colonies are round with neat edges, appearing as convex lenses, pale yellow, with a uniform texture, moist and glossy. Figure 1The cells of *Skelnevize rhizobium* CNBG-PGPR-28, observed under an oil immersion microscope, appeared as short rods. Scanning electron microscopy images showed that the bacterial cells were approximately 1.3 μm long and 0.5 μm wide, with an uneven surface. Figure 2 Gram staining results showed it to be a Gram-negative bacterium. Figure 3 ).

[0037] Example 2: Salt tolerance test of Skelnevize rhizobium CNBG-PGPR-28

[0038] Different concentrations of sodium chloride were added to NA nutrient agar medium (containing 0.5% sodium chloride) to achieve final sodium chloride concentrations of 1%, 2%, 3%, 4%, 5%, 6%, 7%, and 8%. A small amount of Skørnevize rhizobium CNBG-PGPR-28 was picked up with an inoculation loop and streaked onto the NA medium for inverted culture. Figure 4 The culture temperature was set at 30℃. The results showed that when the culture temperature was 30℃, *Skelnevize rhizobium* CNBG-PGPR-28 could grow on nutrient agar medium containing up to 5% sodium chloride, but the growth rate was slower than that on nutrient agar medium with low sodium chloride content.

[0039] A small amount of *Skelnevize rhizobium* CNBG-PGPR-28 was picked up with an inoculation loop and added to NB liquid medium, which was then incubated overnight until turbidity was observed. Different concentrations of sodium chloride were added to NB liquid medium (containing 0.5% sodium chloride) to achieve final concentrations of 2%, 4%, 6%, and 8%. *Skelnevize rhizobium* CNBG-PGPR-28 was then inoculated at a 5% inoculation rate into NB liquid medium containing 2%, 4%, 6%, and 8% sodium chloride, and incubated at 30°C for 72 h. The results showed that *Skelnevize rhizobium* CNBG-PGPR-28 could still grow normally in medium containing 6% sodium chloride. In medium containing 8% sodium chloride, *Skelnevize rhizobium* CNBG-PGPR-28 showed almost no vegetative growth.

[0040] Skernevich rhizobium CNBG-PGPR-28 can grow in NA nutrient agar medium with up to 5% sodium chloride and NB liquid medium with 6% sodium chloride, indicating that Skernevich rhizobium CNBG-PGPR-28 has good salt tolerance.

[0041] Example 3: Inorganic phosphorus degradation capacity of Rhizobium skelnevizei CNBG-PGPR-28

[0042] 1) Preparation of culture medium

[0043] Prepare inorganic phosphorus solid culture medium plates with the following components: 10 g glucose, 2 g tricalcium phosphate, 0.1 g calcium carbonate, 0.5 g anhydrous magnesium sulfate, 0.5 g ammonium sulfate, 0.005 g ferric chloride, 1.7 g potassium chloride, 20 g agar, and 1 L water (Note: The calcium carbonate solution needs to be sterilized and cooled separately before being mixed with the other components of the culture medium to prepare the plate culture medium).

[0044] 2) Qualitative experiment on inorganic phosphorus degradation ability

[0045] Select the bacterial suspension from overnight culture in Example 2, add an appropriate amount of bacterial suspension to inorganic phosphorus solid plate culture medium, air dry naturally, and then invert the plate for culture in a 30℃ incubator for 14 consecutive days.

[0046] Experiments showed that the strain *Skelnevize rhizobium* CNBG-PGPR-28 could grow rapidly on inorganic phosphorus solid plates, and the plate color began to lighten after 7 days, indicating that it had a weak phosphorus-solubilizing ability. Figure 5 ).

[0047] Example 4: API Reagent Kit Test Results

[0048] Single colonies of *Skelnevize* rhizobium CNBG-PGPR-28 were picked from the plates. The fermentable carbohydrate content in the bacterial culture was qualitatively detected using the bioMérieux API 50CH Carbohydrate Identification Kit (Biomérieux, France), and the activity of enzymes in the bacterial culture was qualitatively detected using the bioMérieux APIZYM Enzyme Activity Strips (Biomérieux, France). All procedures were performed according to the kit instructions. The results are shown in Table 1.

[0049] Table 1. API reagent kit test results

[0050]

[0051] The results in Table 1 show that *Skelnevize rhizobium* CNBG-PGPR-28 can utilize glycerol, D-arabinose, L-arabinose, D-ribose, D-xylose, L-xylose, D-calendol, methyl-β-D-xylanoside, D-galactose, D-glucose, D-fructose, D-mannose, L-rhamnose, inositol, mannitol, sorbitol, methyl-α-D-glucopyranoside, N-acetylglucosamine, amygdalin, arbutin, ferric citrate of aesculin, and salicylates. It contains glycosides, D-cellobiose, D-maltose, D-lactose, D-miciose, D-sucrose, D-trehalose, xylitol, gentiobiose, mesobiose, D-lysose, D-tagatose, D-fucose, L-fucose, D-arabinol, and L-arabinol. It can also metabolize alkaline phosphatase, esterase (C4), lipoesterase (C8), leucine aromatic aminoaminase, valine aromatic aminoaminase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-glucosidase, and β-glucosidase.

[0052] Example 5: Nitrogen fixation growth experiment of *Skelnevize* rhizobium CNBG-PGPR-28

[0053] 1) Preparation of culture medium

[0054] Prepare Ashby nitrogen-fixing plate medium without any nitrogen source components. The specific components are: 10 g glucose, 0.2 g potassium dihydrogen phosphate, 0.2 g magnesium sulfate heptahydrate, 0.2 g sodium chloride, 0.1 g calcium sulfate dihydrate, 5 g calcium carbonate, 20 g agar, and add water to 1 L.

[0055] 2) Nitrogen fixation growth experiment

[0056] Single colonies of *Skelnevize rhizobium* CNBG-PGPR-28 were picked using an aseptic inoculation loop and inoculated onto Ashby nitrogen-free solid medium plates, which were then incubated statically at 30°C for 48 hours. After incubation, well-grown colonies were selected and transferred to fresh Ashby medium using the three-zone streak method. This purification process was repeated three times for subculturing. The results showed that the strain exhibited stable growth characteristics during continuous subculturing and formed typical colonies under nitrogen-free conditions, indicating that it possesses a complete biological nitrogenase system. This validation experiment confirms that *Skelnevize rhizobium* CNBG-PGPR-28 has a significant biological nitrogen-fixing capacity and can meet its nitrogen nutrition requirements for growth through nitrogen fixation under conditions lacking combined nitrogen sources. Figure 6 ).

[0057] Example 6: Trial of the effect of Skärnevize rhizobium CNBG-PGPR-28 on promoting seed germination and growth of alfalfa

[0058] 1) Experimental setup

[0059] The alfalfa variety used for testing was Zhongmu No. 1, provided by the College of Grassland Agriculture Science and Technology, Lanzhou University. The seeds were first treated with 95% alcohol for 5 seconds, then with 75% alcohol for 5 minutes to sterilize the seed surface. After sterilization, the seeds were thoroughly rinsed five times in sterile water.

[0060] The experimental design involved three treatment conditions: a stable-phase culture of *Skelnevize rhizobium* CNBG-PGPR-28 (concentration approximately 10...). 8 Inoculate 5% (v / mL) of the culture medium with 1,000 cells per mL of NB liquid medium and incubate overnight at 30°C. Dilute with sterile water to a final volume of 10. 7 Cell suspensions were prepared using 100 cells per mL. Sterile water was used to treat surface-sterilized seeds as a control group (CK) instead of bacterial culture. Alfalfa seeds were soaked in the cell suspension and sterile water at 30°C for 8 hours. Two sheets of sterile filter paper were placed in a petri dish, and sterile water and 3 mL of 100 mM sodium chloride were added. The soaked seeds were then evenly distributed on the filter paper. Seeds were germinated for 5 days at 25–30°C. The number of germinated seeds was counted daily. Each treatment had 30 replicates, with three replicate groups.

[0061] 2) Experimental Results

[0062] Table 2 Experimental Results

[0063]

[0064] Table 2 shows that in petri dishes without sodium chloride, *Skelnevize rhizobium* CNBG-PGPR-28 still increased the germination rate of alfalfa seeds by 2.67% on the third day. In petri dishes with 100 mM sodium chloride, *Skelnevize rhizobium* CNBG-PGPR-28 increased the germination rate of alfalfa seeds by 1.33% on the third day. In conclusion, *Skelnevize rhizobium* CNBG-PGPR-28 significantly improves the germination rate of alfalfa seeds, and can still improve the germination rate under salt stress, demonstrating extremely high application value.

[0065] Example 7: The effect of Skärnevize rhizobium CNBG-PGPR-28 on the growth promotion of lettuce.

[0066] The tested lettuce (Lactuca sativa var. ramosa Hort.) is an Italian year-round bolting resistant lettuce, purchased from Hebei Lihong Seed Industry Co., Ltd.

[0067] The soil was botanical garden soil. The cultivation substrate was an organic cultivation substrate produced by Jiangsu Xingnong Substrate Technology Co., Ltd., with an organic matter content ≥50% and nitrogen, phosphorus, and potassium content ≥2.5%. Lettuce seeds were sown in the organic cultivation substrate, and the growing environment was 25℃, 14 h light / 8 h dark. After 14 days of growth, the seeds were transplanted into pots containing 500 g of soil per pot. CNBG-PGPR-28 was streaked onto NA medium and incubated at 30℃ for 1-2 days. Single clones were then picked and cultured in 5 mL NB medium for 1-2 days. The culture was then scaled up stepwise at a 10% inoculum to 50 mL and 250 mL medium for later use. The pot experiment included a control group (CK) and a treatment group (T). Treatment group T was watered with bacterial solution, while the control group was watered with an equal volume of sterile water. Bacterial solution was watered 3 days after transplanting. The bacterial solution was treated using the bacterial resuspension method. Centrifuge the bacterial culture at 6000 rpm for 5 minutes, discard the supernatant, resuspend the bacterial cells in an appropriate amount of sterile water, repeat twice, and finally adjust the bacterial concentration to 10 with sterile water. 7 The concentration of CFU / mL was 12.5 mL per pot, and inoculation was performed once every 7 days. Each treatment was replicated in triplicate. Observations were made daily, and the plants were harvested after 28 days. The aboveground plant height and fresh weight were measured using an analytical balance. The results are shown in Table 3.

[0068] Table 3 Results of CNBG-PGPR-28 on promoting lettuce growth

[0069]

[0070] Table 3 shows that while the treatment group did not show an increase in plant height, it did show a significant increase in above-ground fresh weight, increasing by approximately 70%. This indicates that *Skelnevize rhizobium* CNBG-PGPR-28 has a significant effect on improving the growth of lettuce. Figure 7 ).

Claims

1. A strain of Rhizobium skierniewicense CNBG-PGPR-28, characterized in that, The Rhizobium skierniewicense CNBG-PGPR-28 was deposited at the China General Microbiological Culture Collection Center on September 2, 2024, with accession number CGMCCNo.31818.

2. A microbial inoculant, characterized in that, It contains Rhizobium skierniewicense CNBG-PGPR-28 as described in claim 1.

3. The application of Rhizobium skierniewicense CNBG-PGPR-28 as described in claim 1 or the microbial agent as described in claim 2 in improving plant salt tolerance.

4. The application according to claim 3, characterized in that, The Rhizobium skierniewicense CNBG-PGPR-28 can grow at salinity levels of 1% to 6%.

5. The application of Rhizobium skierniewicense CNBG-PGPR-28 as described in claim 1 or the microbial agent as described in claim 2 in the degradation of inorganic phosphorus.

6. The application according to claim 5, characterized in that, The inorganic phosphorus is derived from one or more of tricalcium phosphate, calcium phosphate, magnesium phosphate, ferric phosphate, or aluminum phosphate.

7. The application of Rhizobium skierniewicense CNBG-PGPR-28 as described in claim 1 or the microbial agent as described in claim 2 in nitrogen fixation.

8. The application of Rhizobium skierniewicense CNBG-PGPR-28 as described in claim 1 or the microbial inoculant as described in claim 2 in plant cultivation.

9. The application according to claim 8, characterized in that, The application of Rhizobium skierniewicense CNBG-PGPR-28 or the microbial agent of claim 2 in promoting plant seed germination and seedling growth.

10. The application according to claim 8, characterized in that, The plant in question is alfalfa or lettuce.

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