Rhizobium for promoting plant cell division, salt and alkali tolerance and alkali reduction and application thereof

Abstract

The application belongs to the field of agricultural microorganism technology, and particularly relates to rhizobium promoting plant cell division, salt and alkali tolerance and alkali reduction and application thereof. Neorhizobium alkalisoli The rhizobium has a strain number YRD01 and a preservation number CCTCC NO: M 20251990, and is preserved in the China Typical Culture Collection Center in Luo Jia Mountain, Wuhan, Hubei on September 9, 2025. The strain obtained in the application is derived from sesbania root nodule and obtained after screening and culture. Neorhizobium alkalisoli The rhizobium YRD01 obtained in the application is a new type of rhizobium with high salt and alkali tolerance, active alkali reduction function and high-efficiency growth promotion characteristics.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, specifically relating to a rhizobium that promotes plant cell division, salt and alkali tolerance, and alkali reduction, and its application. Background Technology

[0002] Soil salinization has become a core bottleneck restricting agricultural development and ecological protection, with its harms exhibiting multi-dimensional characteristics: At the agricultural production level, the saline-alkali environment damages soil aggregate structure, reduces nutrient availability, and interferes with plant metabolism through osmotic stress and ion toxicity, leading to reduced crop yields or even crop failure, thus hindering sustainable agricultural development; at the ecosystem level, salt stress alters soil microbial communities, reduces species richness, inhibits the growth of native plants, disrupts ecological balance, and exacerbates land degradation. Therefore, saline-alkali land improvement is not only an urgent need to enhance soil productivity and ensure food security, but also a strategic measure to restore the ecosystem and promote the green transformation of agriculture.

[0003] Currently, there are two main approaches to saline-alkali land management: one is the breeding of salt-tolerant plants, which has achieved some success but suffers from limitations such as long breeding cycles, insufficient adaptability and stability, and limited applicability; the other is microbial remediation technology, which has become a research hotspot due to its advantages of high efficiency, diversity, environmental friendliness, and controllable costs. By screening salt-tolerant strains or developing compound microbial agents to construct a "plant-microbe synergistic remediation system," the interaction between the two can alleviate salt stress, improve the rhizosphere microecology, and achieve comprehensive management of saline-alkali land.

[0004] Rhizobium ( Rhizobia As a typical plant rhizosphere growth-promoting bacterium (PGPR), it has great application potential in the improvement of saline-alkali land. Its mechanism of action is clear and synergistically efficient: First, it forms a symbiotic nitrogen-fixing system with leguminous plants, converting atmospheric nitrogen into ammonia nitrogen, compensating for the nitrogen deficiency in saline-alkali land and providing key nutrients; Second, it secretes hormones such as auxin and cytokinin, promoting root development and enhancing the plant's absorption efficiency of water and nutrients; Third, it regulates the synthesis of plant osmotic substances, removes reactive oxygen species, reduces salt stress damage, and activates insoluble phosphorus in the soil, improving plant salt tolerance and nutrient utilization. Currently, the resources of rhizobium that can be practically applied in production are still relatively scarce, especially strains that combine high-salt environment adaptability with highly efficient salt-promoting growth function in saline-alkali land plants, and related applications are still rare. Summary of the Invention

[0005] Based on the above research background, this invention provides a rhizobium with salt tolerance and growth-promoting ability screened from the rhizosphere soil of sesbania plants. Neorhizobium alkalisoli The strain YRD01, identified as a novel strain belonging to the genus *Rhizobium*, exhibits strong salt and alkali tolerance. This invention demonstrates that strain YRD01 can promote plant cell division, increase salt and alkali tolerance, and reduce alkalinity, thereby promoting plant growth or enhancing plant growth in saline-alkali soil environments.

[0006] In a first aspect, the present invention provides a rhizobium YRD01 that promotes plant cell division, salt and alkali tolerance, and alkali reduction. The strain has been deposited at the China Center for Type Culture Collection (CCTCC) on September 9, 2025, at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO: M 20251990.

[0007] The rhizobium ( Neorhizobium alkalisoli The nucleotide sequence of YRD01 is shown in SEQ ID NO.1.

[0008] The rhizobium ( Neorhizobium alkalisoli YRD01 has salt and alkali tolerance and alkali reduction functions, which can promote plant growth or promote plant growth in saline-alkali environments.

[0009] A second aspect of the present invention provides a salt-tolerant growth-promoting bacterial agent or plant growth-promoting bacterial agent, said salt-tolerant growth-promoting bacterial agent or plant growth-promoting bacterial agent comprising rhizobia ( Neorhizobium alkalisoli YRD01 or its fermentation broth and the metabolic supernatant of the strain.

[0010] A third aspect of the present invention provides a rhizobium ( Neorhizobium alkalisoli Application of YRD01 or its fermentation broth and the metabolic supernatant of this strain in promoting plant growth in saline-alkali land.

[0011] Furthermore, the plant includes alfalfa.

[0012] The present invention also includes rhizobia ( Neorhizobium alkalisoli Application of YRD01 or its bacterial solution in the preparation of salt-tolerant growth-promoting bacteria or plant growth-promoting bacteria.

[0013] The present invention also includes rhizobia ( Neorhizobium alkalisoli Application of YRD01 or its bacterial culture in improving the salt and alkali tolerance of alfalfa.

[0014] A fourth aspect of the present invention provides a method for promoting salt-tolerant plant growth, using rhizobia ( Neorhizobium alkalisoli The YRD01 strain was used to treat plants.

[0015] Preferably, the rhizobium ((Neorhizobium alkalisoli) The concentration of strain YRD01 should not be less than 4 × 10⁻⁶. 8 cfu / mL, inoculum size 6%.

[0016] This invention provides a rhizobium with the ability to promote plant cell division and salt tolerance, which provides a theoretical basis for promoting the salt-tolerant growth of plants in saline-alkali land and for further research on saline-alkali land improvement.

[0017] Rhizobium ( Neorhizobium alkalisoli The biological characteristics of strain YRD01 are as follows: the optimal growth temperature is 28 ℃, it is an aerobic culture, and it forms round or nearly round, viscous, smooth, raised, grayish-white or milky-white colonies on YMA plate medium.

[0018] This invention demonstrates that strain YRD01 exhibits good salt and alkali tolerance, promoting plant growth or enhancing plant growth under saline-alkali conditions. Compared to a control, soaking alfalfa seeds in the supernatant and fermentation broth of strain YRD01 showed improvements in all growth indicators by the tenth day of germination. Specifically, under different salt and alkali stress treatments, the hypocotyl diameter of alfalfa seedlings significantly increased. Treatment with the supernatant at salt contents of 6‰, 8‰, and 10‰, and alkali contents of 1‰, respectively, resulted in hypocotyl diameter increases of 34‰, 69‰, 73‰, and 36‰. Treatment with the fermentation broth resulted in increases of 39‰, 67‰, 107‰, and 62‰. Therefore, the supernatant and fermentation broth of strain YRD01 can promote crop growth and have significant economic and practical value.

[0019] Beneficial effects of the embodiments of the present invention:

[0020] 1) This invention provides a rhizobium with the ability to promote plant cell division and salt tolerance. Neorhizobium alkalisoli YRD01 analyzed the biological characteristics of this strain, such as salt tolerance. The strain has strong salt tolerance and can grow even at a salt concentration of 160‰, which has important practical significance and value for improving the ecological environment.

[0021] 2) This invention provides a rhizobium with the ability to promote plant cell division and salt tolerance. Neorhizobium alkalisoli YRD01 analyzed the biological characteristics of this strain, such as its alkali tolerance. The strain has strong alkali tolerance and can grow well in an alkaline environment of pH 11. It also has the function of reducing alkali, with a maximum alkali reduction rate of up to 34%. This has important practical significance and value for improving the ecological environment.

[0022] 3) Rhizobium ( Neorhizobium alkalisoli YRD01 can be prepared into a salt-tolerant growth promoter or a plant growth promoter;

[0023] 4) Rhizobium ( Neorhizobium alkalisoli YRD01 is pollution-free and harmless during use. It can significantly improve the salt tolerance of alfalfa, alleviate salt damage, increase biomass, and promote alfalfa growth.

[0024] 5) The salt tolerance threshold breaks through the upper limit of existing strains and adapts to extreme saline-alkali habitats. The salt tolerance concentration of salt-tolerant rhizobia reported to date is mostly concentrated in 100‰~150‰ NaCl. Most alkali-tolerant strains maintain activity in environments with pH 10 and below. However, the YRD01 strain can maintain a high cell concentration at a high salt concentration of 160‰ NaCl and can grow well in a strongly alkaline environment of pH 11 and still survive in an environment of pH 12. The salt tolerance threshold is significantly higher than that of conventional strains, and it can adapt to the harsh habitats of moderate to severe saline-alkali land, filling the gap in rhizobium resources under extreme saline-alkali environments.

[0025] 6) It has the dual functions of alkali tolerance and active alkali reduction, realizing the improvement of soil microenvironment. Most salt-alkali tolerant rhizobia only have their own stress resistance and do not have the ability to actively improve the rhizosphere alkaline environment. However, the YRD01 strain can not only tolerate the high alkaline environment, but also actively reduce alkali through its own metabolism. In the culture medium with an initial pH of 11.128, the highest alkali reduction rate can reach 34.42%, which can effectively reduce the alkalinity of the rhizosphere soil and improve the microenvironment of the plant root zone, forming a dual action mode of "strain stress resistance + environment improvement", which is different from the traditional single mechanism that only relies on plant-microbe symbiotic stress resistance.

[0026] 7) The growth-promoting pathways are diverse and synergistically efficient, enhancing plant resistance to salt and alkali stress. Most existing rhizobia only have the ability to promote growth through nitrogen fixation or secretion of a single hormone, while the YRD01 strain has three core growth-promoting functions: First, it has the ability to solubilize phosphorus (it can form a transparent zone with a radius of about 7 mm in inorganic phosphorus culture medium), which can activate insoluble phosphorus in the soil; second, it can secrete auxin (IAA), promoting plant root development; and third, it can significantly increase the cytokinin content of the host plant (the content in the treatment group under 1‰ Na2CO3 alkali stress reached 15 μg / L, an increase of more than 50% compared with the control group). Through the multiple pathways of "nutrient activation + hormone regulation", it synergistically enhances the plant's resistance to salt and alkali stress, and the growth-promoting effect is more comprehensive.

[0027] 8) Both fermentation broth and metabolic supernatant have growth-promoting properties, and the application of microbial agents is flexible. Conventional rhizobium agents are mostly applied only in the form of live bacteria fermentation broth, while the metabolic supernatant and live bacteria fermentation broth of strain YRD01 can significantly improve the salt and alkali tolerance of alfalfa: under 10‰ NaCl salt stress, the metabolic supernatant can increase the diameter of alfalfa hypocotyl by up to 73%, and the live bacteria fermentation broth can increase it by up to 107%; and both can improve the germination rate of alfalfa (the germination rate increased from 56% to 88% under 1‰ Na2CO3 alkali stress). It can be applied in the form of live bacteria agents, or the metabolites can be extracted to prepare non-live bacteria growth-promoting agents, which can meet the needs of different application scenarios and expand the industrial application path of microbial agents. Attached Figure Description

[0028] Figure 1 A morphological feature diagram of strain YRD01 provided in an embodiment of the present invention;

[0029] Figure 2 The phylogenetic tree of strain YRD01 provided in the embodiments of the present invention;

[0030] Figure 3 The growth curve of strain YRD01 provided in the embodiments of the present invention;

[0031] Figure 4 Salt tolerance curve of strain YRD01 provided in the embodiments of the present invention;

[0032] Figure 5 This is an alkali resistance curve of strain YRD01 provided in an embodiment of the present invention;

[0033] Figure 6 The graph shows the results of the phosphate-solubilizing ability test of strain YRD01 provided in the embodiments of the present invention;

[0034] Figure 7 This is a diagram showing the IAA production of strain YRD01 provided in an embodiment of the present invention.

[0035] Figure 8 This is a phenotypic effect diagram of alfalfa under different treatments provided in an embodiment of the present invention;

[0036] Figure 9 This is a graph showing the root length growth index of alfalfa under different gradient salt stress treatments provided in an embodiment of the present invention.

[0037] Figure 10 This is a graph showing the shoot growth index of alfalfa under different gradient salt stress treatments provided in an embodiment of the present invention.

[0038] Figure 11 This is a graph showing the root weight growth index of alfalfa under different gradient salt stress treatments provided in an embodiment of the present invention.

[0039] Figure 12 This is a graph showing the shoot regeneration index of alfalfa under different gradient salt stress treatments provided in an embodiment of the present invention.

[0040] Figure 13 This is a graph showing the growth index of hypocotyl diameter of alfalfa under different gradient salt stress treatments provided in an embodiment of the present invention.

[0041] Figure 14 The graph shows the root length growth index of alfalfa under 1‰ Na2CO3 alkaline stress.

[0042] Figure 15 The graph shows the growth indicators of alfalfa shoots under 1‰ Na2CO3 alkaline stress.

[0043] Figure 16 The graph shows the root weight growth index of alfalfa under 1‰ Na2CO3 alkaline stress.

[0044] Figure 17 The graph shows the bud regeneration index of alfalfa under 1‰ Na2CO3 alkaline stress.

[0045] Figure 18 The graph shows the growth index of hypocotyl diameter in alfalfa under 1‰ Na2CO3 alkaline stress.

[0046] Figure 19 The cytokinin content of alfalfa under different treatments provided in the embodiments of the present invention. Detailed Implementation

[0047] Embodiments of this embodiment will now be described in more detail with reference to the accompanying drawings. While some embodiments of this embodiment are shown in the drawings, it should be understood that this embodiment can be implemented in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of this embodiment. It should be understood that the accompanying drawings and embodiments are for illustrative purposes only and are not intended to limit the scope of protection of this embodiment.

[0048] The salt-tolerant growth-promoting bacteria provided in this embodiment of the invention are rhizobia ( Neorhizobium alkalisoli The strain, with strain number YRD01 and accession number CCTCC NO: M 20251990, was deposited on September 9, 2025, at the China Center for Type Culture Collection, Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0049] Example 1: Isolation and purification of strains

[0050] Pink, plump root nodules were collected from the main and lateral roots of *Senecio scandens* plants in the Baicao Garden of Qingdao Agricultural University. These nodules were placed in sample bags and transported to the laboratory in a 4°C ice pack insulated box. The surface of the nodules was rinsed clean with water to remove impurities, and the moisture was absorbed with sterile filter paper. In a biosafety cabinet, the nodule samples were immersed sequentially in 75% ethanol for 3 minutes, followed by 5% sodium hypochlorite solution for 4-6 minutes, and rinsed with sterile water at least 6 times. The nodules were then cut in half with a sterilized knife to expose the internal structure of the rhizobia. The nodules were then held in place with sterilized forceps and streaked onto YMA agar. The streaked agar was incubated at 28°C for 2-5 days. Typical colonies that were round, raised, sticky, with neat edges, and slightly transparent or semi-transparent were selected from the agar plates and streaked in four zones on YMA agar for purification. The purified strain was inoculated onto YMA agar slant and stored at 4°C for a short period. One of the obtained strains was named YRD01.

[0051] The composition of YMA solid medium (g / L) is as follows: 0.5g dipotassium hydrogen phosphate, 0.2g magnesium sulfate heptahydrate, 0.1g sodium chloride, 10g D-mannitol, 1.0g yeast extract, 18g agar, 1000ml distilled water, pH 6.8~7.2.

[0052] Figure 1 Morphological characteristics of strain YRD01 provided in this embodiment of the invention.

[0053] Figure 2 To utilize the strain sequences, a phylogenetic tree was constructed using MEGA after BLAST alignment via NCBI. For example... Figure 2 As shown, strain YRD01 is closely related to the new alkaline rhizobium.

[0054] Figure 3 The optimal growth curve of strain YRD01 provided in the embodiments of the present invention; Figure 3 The bacterial strain YRD01 was cultured at 28℃ with shaking at 180 r / min for 48 h. A suitable amount of bacterial solution was taken every 4 h, and the absorbance of the sample was measured at a wavelength of 600 nm. Each sample was measured in triplicate. Figure 3 As shown, the logarithmic growth phase of strain YRD01 is from 4 to 40 hours, during which the growth rate increases and then stabilizes after 40 hours.

[0055] Example 2: Salt tolerance and alkali tolerance / alkali reduction ability test of Rhizobium YRD01

[0056] Salt tolerance: Activated rhizobium YRD01 was inoculated into a 500 mL Erlenmeyer flask containing 300 mL of YMA medium and cultured on a temperature-controlled shaker for 48 h at 28℃ and 180 r / min to prepare a seed culture. The seed culture was then transferred at a 6% inoculum to 100 mL of liquid nitrogen-fixing medium containing NaCl at concentrations of 0‰, 10‰, 20‰, 30‰, 40‰, 50‰, 60‰, 70‰, 80‰, 90‰, 100‰, 110‰, 120‰, 130‰, 140‰, 150‰, 160‰, 170‰, 180‰, 190‰, and 200‰. A blank liquid medium without inoculation served as the control (CK). The culture was incubated on a shaker at 28℃ and 180 rpm for 72 h, and the OD was measured. 600 To create a curve graph, such as Figure 4 As shown, the rhizobium YRD01 maintains a high cell concentration even at a salt content of 160‰. Through... Figure 4 The results showed that the rhizobium YRD01 has a wide range of tolerance to NaCl, and can still maintain a high cell concentration even when the salt concentration reaches 160‰.

[0057] Alkali tolerance and alkalinity reduction: The seed culture of the strain from Example 2 was transferred at a 6% inoculum to 100 ml of liquid nitrogen-fixing medium with pH values ​​of 7, 8, 9, 10, 11, and 12, respectively. The uninoculated blank liquid medium served as the control (CK). The cultures were incubated in a shaker at 28°C and 180 rpm for 48 h, and the OD was measured. 600 Plot a curve with pH value, see attached. Figure 5 And Table 1, by Figure 5 It can be seen that rhizobium YRD01 maintains a high cell concentration at pH 11, and can still grow at pH 12 although the pH decreases. Table 1 shows that rhizobium YRD01 has an alkalinity-lowering ability of up to 34%.

[0058] The formula for calculating the alkalinity reduction of the microbial strain is: η = (initial pH - subsequent pH) / initial pH × 100%

[0059] The alkalinity-reducing characteristics of strain YRD01 are shown in Table 1 below.

[0060] Table 1: Alkali-reducing characteristics of strain YRD01

[0061]

[0062] Example 3: The ability of rhizobium YRD01 to produce IAA through phosphate solubilization

[0063] Phosphate solubility: Activated rhizobium YRD01 was streaked onto inorganic phosphorus medium in quadruplicate and incubated for 72 h at 28℃ in a biochemical incubator. The presence of a clear zone around the colony was observed. Results are as follows: Figure 6 As shown, the rhizobium YRD01 grew well in this medium, exhibiting a distinct clear zone with a radius of approximately 7 mm, demonstrating its phosphate-solubilizing ability.

[0064] In this embodiment, the formulation and dosage of the above-mentioned inorganic phosphorus culture medium are as follows: MgCl2·6H2O 1.0 g, (NH4)2·SO4 0.02 g, MgSO4·7H2O 0.05 g, KCl 0.04 g, Ca3(PO4)2 1.0 g, glucose 2.0 g, agar 3.6-4.0 g, pH=7.0~7.5, distilled water 200 mL

[0065] IAA Production Capacity: Rhizobium YRD01 was inoculated into IAA detection medium and cultured. The IAA production capacity of the strain was determined by reacting with a chromogenic solution in the dark. The results are as follows: Figure 7 As shown, the red color indicates that the strain has the ability to produce IAA. It can be seen that the rhizobium YRD01 turns red after treatment, indicating that it has the ability to produce IAA.

[0066] Example 4: Application of Rhizobium YRD01 in the growth of alfalfa

[0067] In this embodiment, salt-tolerant growth-promoting bacteria are used as an example of salt-tolerant growth-promoting agents for alfalfa.

[0068] 1. Preparation of Rhizobium YRD01 inoculant

[0069] Activated YRD01 was inoculated into 250 mL Erlenmeyer flasks containing 100 mL YMA medium and cultured on a temperature-controlled shaker for 48 h at 28℃ and 180 r / min to prepare seed culture. The seed culture was then inoculated at 6% into 500 mL Erlenmeyer flasks containing 300 mL YMA medium and cultured on a temperature-controlled shaker for 48 h at 28℃ and 180 r / min. The fermentation broth was then centrifuged to collect the supernatant and precipitated cells. The centrifugation temperature was 4℃, the speed was 13400 r / min, and the time was 5 min. The concentration of the fermentation broth was adjusted (OD600 = 2 ± 0.01) to obtain an effective viable count of 4 × 10⁻⁶ for *Rhizobium rhizobium* YRD01. 8 Fermentation broth with cfu / mL.

[0070] 2. Growth-promoting effect of rhizobium YRD01 on alfalfa under salt-alkali stress

[0071] To verify the effect of rhizobium YRD01 on salt tolerance and growth promotion of alfalfa under different salt concentrations, this experiment set up three salt gradients and one alkali gradient, namely 6‰, 8‰, 10‰ NaCl and 1‰ Na2CO3. The experiment included three treatments: the metabolic supernatant of YRD01 strain (YRD01-BMS) with an effective viable bacterial count of 4 × 10⁻⁶. 8 The control group (CK) consisted of two inoculants (YRD01, CFU / mL) of YRD01 fermentation broth and sterile water, with three replicates for each treatment. Plump, uniformly sized alfalfa seeds, unaffected by pests or diseases, were selected and subjected to a 10-day germination test in an incubator. The incubation conditions were set as follows: 16 h photoperiod, 28℃; 8 h dark treatment, 22℃; and 50‰–80‰ humidity.

[0072] In this embodiment, the growth status of alfalfa seedlings in the YRD01 inoculum group and the control group on day 10 is as follows: Figure 8 As shown, Figure 9 This is a graph showing the root length growth index of alfalfa under different gradient salt stress treatments provided in an embodiment of the present invention. Figure 10 This is a graph showing the shoot growth index of alfalfa under different gradient salt stress treatments provided in an embodiment of the present invention. Figure 11This is a graph showing the root weight growth index of alfalfa under different gradient salt stress treatments provided in an embodiment of the present invention. Figure 12 This is a graph showing the shoot regeneration index of alfalfa under different gradient salt stress treatments provided in an embodiment of the present invention. Figure 13 This is a graph showing the growth index of hypocotyl diameter of alfalfa under different gradient salt stress treatments provided in an embodiment of the present invention. Figure 14 The graph shows the root length growth index of alfalfa under 1‰ Na2CO3 alkaline stress. Figure 15 The graph shows the growth indicators of alfalfa shoots under 1‰ Na2CO3 alkaline stress. Figure 16 The graph shows the root weight growth index of alfalfa under 1‰ Na2CO3 alkaline stress. Figure 17 The graph shows the bud regeneration index of alfalfa under 1‰ Na2CO3 alkaline stress. Figure 18 The graph shows the growth index of hypocotyl diameter in alfalfa under 1‰ Na2CO3 alkaline stress.

[0073] The results showed that the germination rate of alfalfa was improved after applying both inoculants, as shown in Table 2.

[0074] Table 2: Germination rate of alfalfa after application of two inoculants

[0075]

[0076] Under 10‰ NaCl salt stress, the germination rate of the YRD01-BMS group was 25% higher than that of the control group. Under alkaline stress, the germination rate of both inoculant treatment groups increased from 56‰ to 88‰, showing a particularly significant improvement. Root length, shoot length, root weight, and shoot weight all showed positive growth, and the hypocotyl diameter increased significantly under different salt and alkali gradients: in 6‰, 8‰, and 10‰ NaCl salt stress environments and 1‰ Na2CO3 alkaline stress environments, the hypocotyl diameter of alfalfa seedlings treated with the strain's metabolic supernatant increased by 34%, 69%, 73%, and 36%, respectively; the hypocotyl diameter of seedlings treated with the strain's fermentation broth increased even more, by 39%, 67%, 107%, and 62%, respectively. The YRD01 inoculant effectively enhances alfalfa's salt and alkali tolerance and alleviates salt and alkali stress damage by increasing hypocotyl diameter and biomass, providing key technical support for the efficient cultivation of alfalfa in saline-alkali areas.

[0077] To further verify the salt and alkali tolerance and growth-promoting ability of the bacterial agent, the cytokinin content of its hypocotyl was measured, such as... Figure 19 As shown, after applying YRD01 inoculant, the cytokinin content of alfalfa showed a positive increase, and it was significantly improved under different salinity gradients. Figure 19 As shown in (a), under 6‰, 8‰, and 10‰ NaCl salt stress and Figure 19As shown in Figure (b), under 1‰ Na₂CO₃ alkaline stress, the cytokinin content of alfalfa seedlings treated with the supernatant of the strain (YRD01-BMS) was significantly higher than that of the control group (CK). Under 8‰ salt stress, the cytokinin content of this group reached approximately 10.2 μg / L (an increase of over 60% compared to CK). The cytokinin enhancement effect was even more pronounced in seedlings treated with the fermentation broth of the strain (YRD01), with the content reaching approximately 15 μg / L under 1‰ Na₂CO₃ alkaline stress (an increase of over 50% compared to CK). The YRD01 inoculant can effectively enhance the salt and alkali tolerance of alfalfa and alleviate salt and alkali stress damage by increasing cytokinin content and enhancing physiological activity, providing key physiological mechanism support for the efficient cultivation of alfalfa in saline-alkali areas.

[0078] YRD01 inoculant works through a pathway of action that "increases cytokinin content → enhances physiological activity → promotes growth indicators such as hypocotyl diameter → increases biomass," effectively alleviating the growth inhibition of alfalfa under salt-alkali stress, significantly enhancing its salt-alkali tolerance, and ultimately enabling robust growth of alfalfa in saline-alkali environments. This provides key technical and physiological support for the efficient cultivation of alfalfa in saline-alkali areas.

Claims

1. A rhizobium that promotes plant cell division, salt and alkali tolerance, and alkalinity reduction ( Neorhizobium alkalisoli ), characterized in that: The strain number is YRD01, and the rhizobium ( Neorhizobium alkalisoli The YRD01 strain has been deposited at the China Center for Type Culture Collection (CCTCC) on September 9, 2025. The deposit address is Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the accession number is CCTCC NO: M 20251990.

2. The rhizobium as described in claim 1 ( Neorhizobium alkalisoli The application of (or its bacterial culture) in the preparation of salt-tolerant growth-promoting bacterial agents.

3. The rhizobium as described in claim 1 ( Neorhizobium alkalisoli The application of its bacterial solution in the preparation of alfalfa growth-promoting bacterial agents.

4. The rhizobium as described in claim 1 ( Neorhizobium alkalisoli Application of its bacterial culture or its bacterial solution in improving the salt and alkali tolerance of alfalfa.

5. A salt-tolerant growth-promoting bacterial agent, characterized in that: The salt-tolerant growth-promoting bacteria agent comprises the rhizobium as described in claim 1. Neorhizobium alkalisoli (or its fermentation broth or the metabolic supernatant of the strain) 6. The application of the salt-tolerant growth-promoting bacterial agent as described in claim 5 in promoting the growth of alfalfa in saline-alkali land.

7. A method for promoting salt-tolerant plant growth, characterized in that: Using the rhizobium (as described in claim 1) Neorhizobium alkalisoli The strain was used to treat alfalfa.

8. The method as described in claim 7, characterized in that: The rhizobium ( Neorhizobium alkalisoli The concentration of the strain is not less than 4×10 8 cfu / mL, inoculum size 6%.

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