Novel salt-tolerant rhizobium and application thereof

Abstract

The present application relates to a kind of novel salt-tolerant rhizobium and its application, the rhizobium preservation number is GDMCC No:65918.The rhizobium of the application has host broad spectrum, can be symbiotic nodulation with leguminous plants including purple winged bean and peanut etc., improve the nitrogen fixation capacity of plant, and growth speed is faster.Especially, the rhizobium has high salt, acid, alkali tolerance, can grow in extreme salt, acid, alkali environment, improve soil, significantly promote the growth survival of plant in high salt, acid, alkali environment with symbiosis.The rhizobium of the application has very important application value to the development and utilization of leguminous plants, salt-alkali land (such as beach sand), ecological environment repair and improvement, and agricultural production.

Description

Technical Field

[0001] This invention belongs to the field of microbiology technology, and relates to rhizobia, specifically a novel salt-tolerant rhizobium and its applications. Background Technology

[0002] With the increasing salinization of land globally, agricultural production faces severe challenges. Saline-alkali soil conditions limit crop growth, impacting yield and quality. Traditional methods of improvement, such as fertilizer application and irrigation projects, are costly and have limited effectiveness.

[0003] Rhizobia are a class of Gram-negative bacteria that can fix nitrogen in symbiosis with legumes and play an important role in sustainable agricultural development. However, existing rhizobium species have limited adaptability to high-salt-alkali environments, which limits their application in the improvement of saline-alkali soils.

[0004] Therefore, developing a new type of salt-tolerant rhizobium is of great significance for improving the yield of leguminous crops in saline-alkali land and for ecological restoration. Summary of the Invention

[0005] Based on this, the purpose of the present invention is to provide a novel salt-tolerant rhizobium and its application, wherein the rhizobium can form nodules with leguminous plants to help the plants tolerate high salt and alkali environments and promote plant growth in saline and alkali environments.

[0006] The first aspect of the present invention provides a rhizobium (Enterobacter sp.) ZH6, the accession number of which is GDMCC No: 65918.

[0007] A second aspect of the invention provides the application of Enterobacter sp. ZH6, as described above, in plant nitrogen fixation.

[0008] A third aspect of the invention provides the application of Enterobacter sp. ZH6, as described above, in improving the salt, acid and alkali tolerance of plants.

[0009] In some embodiments, the plant is a legume, preferably soybean, peanut, broad bean, pea, red bean, mung bean, cowpea, kidney bean, or lentil.

[0010] A fourth aspect of the invention provides the use of Enterobacter sp. ZH6, as described above, in the preparation of soil conditioners.

[0011] The fifth aspect of the invention provides the use of Enterobacter sp. ZH6, as described above, in the preparation of a repair agent.

[0012] A sixth aspect of the present invention provides a biological agent for improving the nitrogen fixation capacity of plants, wherein the active ingredient of the biological agent comprises Enterobacter sp. ZH6 as described above.

[0013] A seventh aspect of the present invention provides a soil conditioner or remediation agent, wherein the active ingredient of the soil conditioner or remediation agent comprises Enterobacter sp. ZH6 as described above.

[0014] The eighth aspect of the present invention provides a method for culturing Enterobacter sp. ZH6 as described above, comprising the following steps: inoculating the Enterobacter sp. ZH6 into a culture medium and culturing it at 26°C to 30°C.

[0015] In some embodiments, the culture medium is YMA medium.

[0016] In some embodiments, the culture is carried out at 26°C to 28°C.

[0017] The rhizobium (Enterobacter sp.) ZH6 described in this invention was deposited on February 19, 2025, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province), a depositary unit designated by the State Intellectual Property Office. The deposit date was February 19, 2025, and the deposit number was GDMCC No. 65918.

[0018] This invention provides a salt-tolerant, fast-growing rhizobium (Enterobacter sp.) ZH6, which contains the nodC (symbiotic gene) and nif (nitrogen-fixing gene) genes, exhibiting broad host spectrum and the ability to form nodules in symbiotic relationships with legumes, including winged beans and peanuts. The rhizobium ZH6 grows rapidly and exhibits resistance to streptomycin, gentamicin, rifampin, ampicillin, kanamycin, and hygromycin. In particular, the rhizobium demonstrates high tolerance to salt, acid, and alkali, enabling it to grow in extreme saline, acidic, and alkaline environments, improving soil conditions, and significantly promoting the growth and survival of its symbiotic plants in high-salt and alkaline environments.

[0019] The rhizobium provided by this invention is not only significant for the development and utilization of leguminous plants, including purple winged beans and peanuts, but also has important application value in the protection of saline-alkali land (such as coastal sandy land), the restoration and improvement of the ecological environment, and agricultural production. The discovery of this rhizobium provides a new solution for coping with saline-alkali land and extreme climates in agricultural production. Attached Figure Description

[0020] Figure 1 The results of isolation and identification of Enterobacter sp. ZH6 are as follows: a: growth morphology of Enterobacter sp. ZH6 in YMA medium; b: identification of nodC (symbiotic gene) and nif (nitrogen fixation gene) in Enterobacter sp. ZH6.

[0021] Figure 2 The 16S rRNA gene sequence alignment and evolutionary analysis of Enterobacter sp. ZH6 are presented. a: 16S rRNA gene sequence alignment results; b: evolutionary analysis results.

[0022] Figure 3 The results show the growth rate of Enterobacter sp. ZH6.

[0023] Figure 4 The results are for antibiotic resistance testing of Enterobacter sp. ZH6.

[0024] Figure 5 The acid-base tolerance of Enterobacter sp. ZH6 was tested; where a: ZH6; b: SMH12; c: NGR234; d: USDA110.

[0025] Figure 6 Salt tolerance test for Enterobacter sp. ZH6; where a: ZH6; b: SMH12; c: NGR234; d: USDA110.

[0026] Figure 7The results show the host broadness detection of Enterobacter sp. ZH6; among them, a: nodulation phenotype of purple winged bean (Siratro) after 25 days of inoculation with Enterobacter sp. ZH6 and d: magnified image of nodule; b: nodulation phenotype of peanut variety Zhongkaihua 1 (ZKH1H) after 25 days of inoculation with Enterobacter sp. ZH6 and e: magnified image of nodule; c: nodulation phenotype of peanut variety Caiyi (CY) after 25 days of inoculation with Enterobacter sp. ZH6 and f: magnified image of nodule.

[0027] Figure 8 The results of experiments on how Enterobacter sp. ZH6 enhances the salt tolerance of host plants and promotes their growth are shown. -Rh represents the control group without inoculation with Enterobacter sp. ZH6, and +Rh represents the experimental group inoculated with Enterobacter sp. ZH6.

[0028] Figure 9 The results of experiments on how Enterobacter sp. ZH6 can enhance the alkali tolerance of host plants and promote their growth; -Rh represents the control group without inoculation with Enterobacter sp., and +Rh represents the experimental group inoculated with Enterobacter sp. ZH6.

[0029] Figure 10 To detect the symbiotic nitrogenase activity of Enterobacter sp. ZH6 and purple winged bean. Detailed Implementation

[0030] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0031] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in the fourth edition of *Molecular Cloning: A Laboratory Manual*, edited by Green and Sambrook, published in 2013, or according to the manufacturer's recommendations. All commonly used chemical reagents used in the examples are commercially available products.

[0032] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0033] Furthermore, as used herein, the term "or" is an inclusive "or" sign and is equivalent to the term "and / or" unless the context clearly specifies otherwise. The term "based on" is not exclusive and allows for basing on other factors not described unless the context clearly specifies otherwise. Additionally, throughout the specification, the meanings of "an," "a," and "the" include plural indicators. The meaning of "in" includes both "in" and "on."

[0034] The present invention will be further described in detail below with reference to specific embodiments.

[0035] Example 1

[0036] 1. Isolation and Identification of Strains

[0037] (1) Isolation of rhizobium (Enterobacter sp.) ZH6.

[0038] The host plant, *Pterocarya purpurea*, was cultivated in the Tropical Saline-Alkali Resource Conservation Center of the South China Botanical Garden, Chinese Academy of Sciences. The specific procedure involved harvesting healthy, large, and clearly patterned root nodules from the roots of *Pterocarya purpurea*, and rinsing them thoroughly with clean water to remove surface sand and gravel. In a clean bench, the nodules were disinfected in the following sequence: 75% ethanol for 5 minutes, sodium hypochlorite for 5 minutes, and then rinsed 10 times with sterile water. After disinfection, the nodules were placed on sterile filter paper to absorb excess moisture. Once completely dry, the outer epidermis of the nodule was cut open with a sterile blade, and the central red tissue was selected, sectioned, and inoculated onto YMA solid medium. The nodules were incubated at 28°C for one week, during which time the grown colonies were streaked onto fresh YMA solid medium to isolate single colonies. Finally, the isolated single colonies were preserved in 50% glycerol at -80°C for subsequent strain identification.

[0039] The formulation of YMA medium (1L, 1% Agar, pH = 6.8–7.0) is shown in Table 1 below.

[0040] Table 1

[0041]

[0042]

[0043] (2) Amplification of the 16S rRNA gene of Enterobacter sp. ZH6.

[0044] Add 5 μl of frozen bacterial culture to 4 ml of YMA liquid medium and incubate at 28°C and 220 rpm for 1 day. Prepare a PCR amplification system (50 μl) for amplifying bacterial 16S rRNA according to the dosages described in Table 2 below.

[0045] Table 2

[0046]

[0047] Note: The polymerase used in this study was Novizan 2×Phanta UniFi Master Mix (Dye Plus) (product number 526-01).

[0048] The PCR amplification system is shown in Table 3 below.

[0049] Table 3

[0050]

[0051] The amplified products were sent to Qingke Biotechnology Co., Ltd. for sequencing.

[0052] The 16S amplification primer sequences can be found in the following literature: Gisele Laguerre, et al. Classification of rhizobia based on nodC and nifH gene analysis reveals a close phylogenetic relationship among Phaseolus vulgaris symbionts; Microbiology (2001), 147, 981-993.

[0053] (3) Detection of the nodC symbiotic gene and the nif nitrogen-fixing gene of Enterobacter sp. ZH6.

[0054] Take the bacterial culture prepared in (2) and amplify the nodC and nif genes in the isolated bacteria according to the system and PCR procedure in (2). Primer information can be found in the following literature: Gisele Laguerre, et al. Classification of rhizobia based on nodC and nifH gene analysis reveals a close phylogenetic relationship among Phaseolus vulgaris symbionts; Microbiology (2001), 147, 981-993.

[0055] (4) Construction of the phylogenetic tree of Enterobacter sp. ZH6

[0056] The DNA sequence of the 16S rRNA gene of the control rhizobium used in this invention was downloaded from NCBI, and a phylogenetic tree of the four species was constructed using MEGA7.

[0057] The DNA sequence of the 16S rRNA gene of Enterobacter sp. ZH6 described in this invention, as determined by sequencing, is shown in SEQ ID NO.1.

[0058]

[0059]

[0060] 2. Detection of growth rate of Enterobacter sp. ZH6

[0061] Prepare YMA liquid medium containing 0.5% BTB (bromothymol blue) and sterilize at 121℃ for later use. Take 5 μl of frozen bacterial suspensions of Enterobacter sp. ZH6 and control rhizobia (control rhizobia are fast-growing rhizobia SMH12, NGR234 and slow-growing rhizobia USDA110), and add them to 4 ml of YMA liquid medium respectively. Incubate at 28℃ and 220 rpm for 1 day. Take out the cultured bacterial suspensions, centrifuge at 12000 rpm for 2 min, discard the supernatant, and resuspend in sterile water to OD. 600 =0.2. Take 200 μl of the resuspended bacterial solution and add it to 5 ml of YMA medium containing BTB. Incubate at 28℃ and 220 rpm for 2 days. Observe the color change and take pictures.

[0062] 3. Antibiotic resistance detection of Enterobacter sp. ZH6

[0063] After high-temperature sterilization of YMA solid medium, YMA solid medium containing antibiotics including streptomycin, gentamicin, rifampicin, ampicillin, kanamycin, hygromycin B, spectinomycin, and chloramphenicol were prepared according to the standard of 20 ml / plate. Take 5 μl of OD... 600 The bacterial suspension at 0.2 g / mL was streaked onto culture media with different resistance levels, incubated at 28°C for 3 days, and the growth was observed and photographed.

[0064] 4. Detection of salt and acid / alkali tolerance characteristics of Enterobacter sp. ZH6

[0065] (1) Acid-base tolerance test.

[0066] Prepare YMA culture media with pH values ​​of 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 respectively. After autoclaving, prepare bacterial culture media at a standard rate of 20 ml / plate. Take 5 μl of OD... 600 The bacterial suspension at 0.2 g / mL was streaked onto culture media with different resistance levels, incubated at 28°C for 3 days, and the growth was observed and photographed.

[0067] (2) Salt tolerance test.

[0068] Prepare YMA culture media (pH = 6.8) with NaCl concentrations of 0, 50, 100, 200, 300, 400, 500, and 600 mM / L respectively. After autoclaving, prepare bacterial culture media in 20 ml / plates. Take 5 μl of O D 600 The bacterial suspension at 0.2 g / mL was streaked onto culture media with different resistance levels, incubated at 28°C for 3 days, and the growth was observed and photographed.

[0069] 5. Broad-spectrum host activity test of Enterobacter sp. ZH6

[0070] Purple-flowered winged bean, peanut variety Zhongkai Flower No. 1, and Caiyi were selected as broad-spectrum host testing subjects. First, purple-flowered winged bean and peanut seeds were sterilized with chlorine for 4 hours (sodium hypochlorite: concentrated hydrochloric acid = 100ml: 4.2ml). After sterilization, the seeds were transferred to a clean bench to remove any remaining chlorine. Before planting, the seeds were soaked in sterile water for 1 hour, and then sown at a uniform depth (2cm) into culture pots (10cm x 10cm) containing sterilized vermiculite. Six pots were planted for each sample.

[0071] When the seedlings reach day 20, each seedling is inoculated with 10 ml of OD. 600 =0.1% bacterial suspension. After inoculation, continue culturing for 25 days, observe the nodulation phenotype, and take photographs. Use 1 / 4 Hoagland solution for culturing.

[0072] The formulation of Hogrange solution is shown in Table 4 below (1L).

[0073] Table 4

[0074]

[0075]

[0076] 6. Experiment on the promotion of purple-flowered winged bean growth by rhizobium (Enterobacter sp.) ZH6 under salt stress

[0077] Liquid culture media with NaCl concentrations of 0, 50, and 100 mM / L were prepared. After high-temperature sterilization, they were placed in sterile hydroponic bags for later use. The sterilized seeds of *Pterocarya stenoptera* were then soaked in sterile water and OD245 solution, respectively. 600 Soak in a bacterial suspension at a concentration of 0.1 g / L for 1 hour, then transfer to a hydroponic bag for cultivation.

[0078] 7. Experiment on the promotion of purple-flowered winged bean growth by rhizobium (Enterobacter sp.) ZH6 under alkaline stress

[0079] Sterilized purple winged bean seeds were sown in sterilized quartz sand and cultured in a 28℃ light incubator at a light / dark ratio of 16h / 8h. On day 20, the experimental group was inoculated with OD... 600 10 ml of a bacterial suspension with a concentration of 0.1 was used, and the control group was inoculated with the same amount of sterile water. On day 7 of inoculation, the culture medium was replaced with nutrient solutions of different pH values. The nutrient solutions of different pH values ​​were prepared by mixing NaHCO3 and Na2CO3 in a specific ratio. Photos were taken on day 8 of treatment and day 2 of recovery.

[0080] 8. Detection of nitrogenase activity in the symbiotic relationship between Enterobacter sp. ZH6 and purple winged bean

[0081] Root nodules of *Lysimachia christinae* (purple-flowered broad bean) at the early flowering stage were collected and placed in 20 ml sample vials. 2 ml of acetylene gas was injected into the vials, and the reaction was carried out at 28°C for 3 hours. After the reaction was completed, the vials were immediately placed on ice to terminate the reaction. Nitrogenase activity was detected using a gas chromatograph at the South China Botanical Garden's public platform. 1 ml of reaction gas was injected into each sample for analysis. Enzyme activity was calculated using the following formula:

[0082]

[0083] In the formula: t℃: air temperature, 28℃; P: air pressure, usually taken as 760 mmHg.

[0084] 22.4: The volume of 1 mol of gas under standard conditions is 22.4 liters; 273 is the absolute temperature.

[0085] result

[0086] 1. Identification

[0087] Figure 1 The results of isolation and identification of Enterobacter sp. ZH6 are shown. Figure 1 As shown, Figure 1 Image a shows the colony morphology of Enterobacter sp. ZH6 on YMA medium after 2 days of incubation at 28°C. The colonies of this bacterium are raised, with neat edges, milky white in color, and odorless. Figure 1 Figure b shows the amplification results of the symbiotic gene *nodC* and the nitrogen-fixing gene *nif* in the genome of *Enterobacter sp.* ZH6. The results show the presence of genes related to symbiosis and nitrogen fixation in the *Enterobacter sp.* ZH6 genome, indicating that this bacterium can establish a symbiotic relationship with plants and perform nitrogen fixation, which corroborates the nodulation experiment described later. In this result, the symbiotic gene *nodC* amplified two clear, independent bands. One band was approximately 930 bp, consistent with previously reported results; the other amplified fragment was approximately 2000 bp, indicating the presence of a larger *nodC* gene in the *Enterobacter sp.* ZH6 genome. Whether this is related to the bacterium's wide host range requires further experimental verification. As for the nitrogen-fixing gene *nif*, in addition to amplifying a band consistent with the reported 780 bp band, several fragments of similar size were amplified, which may be due to low primer specificity.

[0088] Figure 2 The results of sequence alignment and evolutionary analysis of the 16S rRNA gene of Enterobacter sp. ZH6 are presented. Figure 2 Figure a shows the DNA sequence alignment results of the 16S rRNA gene of *Enterobacter sp.* ZH6 and control rhizobia (fast-growing rhizobia SMH12, NGR234, and slow-growing rhizobia USDA110). The results show that the *Enterobacter sp.* ZH6 isolated in this invention exhibits a very high degree of conservation in its 16S DNA sequence compared to the control rhizobia (sequences marked with a black background in the figure). Figure 2The phylogenetic tree in section b shows that *Enterobacter sp.* ZH6 is closely related to USDA110, followed by NGR234, with SMH12 being the most distantly related. The *Enterobacter sp.* 16S rRNA gene sequence was downloaded from publicly available data published on the NCBI website (https: / / www.ncbi.nlm.nih.gov / ).

[0089] 2. Growth rate

[0090] Figure 3 This is the result of the growth rate detection for Enterobacter sp. ZH6. Bromothymol blue (BTB) is a commonly used acid-base indicator. It is green under neutral conditions, yellow under acidic conditions, and blue under alkaline conditions. Fast-growing rhizobia grow rapidly on YMA medium, generally forming visible colonies in 2-3 days, while simultaneously producing acid. Slow-growing rhizobia grow slowly on YMA medium, generally taking 5-7 days to form visible plaques, while producing alkali. From... Figure 3 The results show that Enterobacter sp. ZH6 can turn the medium containing BTB yellow, which is consistent with the color change of the medium of two other fast-growing rhizobia, SMH12 and NGR234, proving that this bacterium is a fast-growing rhizobium.

[0091] 3. Antibiotic resistance

[0092] Figure 4 The results show the antibiotic resistance test results for Enterobacter sp. ZH6, a rhizobium isolated from the root nodules of *Enterobacter spp.*. The results indicate that Enterobacter sp. ZH6, a fast-growing rhizobium isolated from the root nodules of *Enterobacter spp.*, can grow on YMA agar plates containing streptomycin, gentamicin, rifampin, ampicillin, kanamycin, and hygromycin, but cannot grow on media containing spectinomycin and chloramphenicol. These results demonstrate that this bacterium is resistant to six antibiotics: streptomycin, gentamicin, rifampin, ampicillin, kanamycin, and hygromycin.

[0093] 4. Acid and alkali tolerance

[0094] Figure 5 The results are for the acid-base tolerance test of Enterobacter sp. ZH6. Figure 5The results showed that, compared with the other three control rhizobia, the fast-growing rhizobium (Enterobacter sp.) ZH6 could grow normally within a pH range of 4–13. Under acid-base stress, this bacterium performed similarly to another broad-host fast-growing rhizobium, NGR234, but significantly better than the fast-growing rhizobium SMH12 and the slow-growing rhizobium USDA110. Based on its growth rate and growth status, this bacterium is suitable for growth in an environment with pH > 7.0. On YMA medium at pH 10.0, the bacterium grew rapidly and maintained good growth status. This may be due to its acid-producing characteristics. Figure 5 In the table, a: ZH6; b: SMH12; c: NGR234; d: USDA110.

[0095] 5. Salt tolerance of Enterobacter sp. ZH6

[0096] Figure 6 This presents the salt tolerance test results for Enterobacter sp. ZH6. The salt stress resistance test results for Enterobacter sp. ZH6 show that this bacterium can grow on YMA medium containing a high salt concentration (NaCl: 600 mM, approximately the concentration of seawater). The performance of Enterobacter sp. ZH6 under salt stress is consistent with that of the broad-host-type fast-growing rhizobium NGR234, and significantly superior to the fast-growing rhizobium SMH12 and the slow-growing rhizobium USDA110. Figure 6 In the table, a: ZH6; b: SMH12; c: NGR234; d: USDA110.

[0097] 6. Broad host spectrum

[0098] Figure 7 The nodulation phenotypes of purple-flowered winged bean, Zhongkai Flower No. 1, and colored-coat (peanut) 25 days after inoculation are shown in the figure. As can be seen from the figure, although the nodulation mode of peanut is mainly fissure infection, which is completely different from the root hair infection mode of purple-flowered winged bean, the rhizobium (Enterobacter sp.) ZH6 isolated in this invention can still induce nodulation in purple-flowered winged bean and peanut. Figure 7 a~ Figure 7 (c). This result not only confirms that ZH6 isolated in this invention belongs to rhizobia, but also proves that rhizobia ZH6 has a broad host spectrum, greatly expanding the application value of rhizobia in agricultural production. Figure 7In the middle: a: Nodulation phenotype of purple winged bean (Siratro) after inoculation with Enterobacter sp. ZH625d and d: magnified image of root nodules; b: Nodulation phenotype of peanut variety Zhongkaihua 1 (ZKH1H) after inoculation with Enterobacter sp. ZH625d and e: magnified image of root nodules; c: Nodulation phenotype of peanut variety Caiyi (CY) after inoculation with Enterobacter sp. ZH625d and f: magnified image of root nodules.

[0099] 7. Enterobacter sp. ZH6 enhances the salt tolerance of host plants.

[0100] Figure 8 The growth phenotypes of plants inoculated with (+Rh) / uninoculated with (-Rh) rhizobium ZH6 were compared under different salt concentrations (0, 50, 100 mM / L). The figures show that without salt stress, inoculation with (+Rh) / uninoculated with (-Rh) rhizobium had no effect on host plant growth, exhibiting consistent aboveground and belowground phenotypes. However, under salt stress, inoculation with (+Rh) rhizobium significantly promoted host plant growth. This phenomenon became more pronounced with increasing salt concentration. The results indicate that Enterobacter sp. ZH6 can significantly improve plant salt tolerance and promote plant growth and development under high-salt conditions.

[0101] 8. Enterobacter sp. ZH6 enhances the host plant's alkali tolerance.

[0102] Figure 9 The figure shows the aboveground and belowground growth phenotypes of the host plant after 8 days of alkali stress treatment and 2 days of recovery treatment. As can be seen from the figure, the inoculation treatment significantly improved the survival rate of the plant in a highly alkaline environment (pH≥8). In the uninoculated (-Rh) control group, even after resuming normal culture after 8 days of treatment, the lethal damage caused by alkali stress could not be reversed. However, under alkali stress at pH=7, the inoculated group did not show senescence or wilting in the aboveground leaves; after 2 days of recovery culture, the alkali-treated group inoculated with rhizobia (pH=7) rapidly developed new lateral roots (indicated by the red arrows in the figure).

[0103] 9. Detection of nitrogenase activity in the symbiotic relationship between *Enterobacter sp.* ZH6 and *Vigna purpurea*.

[0104] Figure 10The figure shows the average nitrogenase activity of three purple-flowered winged beans at the initial flowering stage, with a value of 0.19. The results of the nitrogenase activity determination indicate that the rhizobium (Enterobacter sp.) ZH6 isolated in this invention can not only form root nodules in symbiosis with purple-flowered winged beans, but also effectively enhance their nitrogen-fixing capacity.

[0105] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A type of rhizobium Enterobacter sp. ZH6, characterized in that, The rhizobium Enterobacter sp. ZH6 has the accession number GDMCC No: 65918.

2. The rhizobium as described in claim 1 Enterobacter sp. Application of ZH6 in nitrogen fixation in purple winged beans and / or peanuts.

3. The rhizobium as described in claim 1 Enterobacter sp. Application of ZH6 in improving the salt and alkali tolerance of purple winged beans and / or peanuts.

4. A biological agent for enhancing nitrogen fixation capacity in plants, characterized in that, The active ingredient of the biological agent comprises the rhizobium as described in claim 1. Enterobacter sp. ZH6; The plants include purple winged beans and / or peanuts.

5. The rhizobium as described in claim 1 Enterobacter sp. The method for cultivating ZH6 is characterized by, Includes the following steps: The rhizobium Enterobacter sp. ZH6 was inoculated into the culture medium and cultured at 26 ℃~30 ℃.

6. The cultivation method as described in claim 5, characterized in that, The culture medium is YMA medium.

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