Nelumbo nucifera rhizosphere growth promoting bacteria Nn211 and application thereof

Abstract

The application discloses rhizosphere growth promoting bacteria Nn211 of Nelumbo nucifera and application thereof. The gibbsiella dentisursi Nn211 provided by the application is preserved in the China General Microbiological Culture Collection Center on November 4, 2022, and the preservation number is CGMCC No. 26047. The Nn211 strain has the characteristics of producing ACC deaminase, producing urease, secreting growth hormone IAA and tolerating high-concentration cadmium; the strain is inoculated into the rhizosphere of Nelumbo nucifera seedlings and adult plants, can effectively promote the growth of the Nelumbo nucifera seedlings and adult plants, and improve the stress resistance of the Nelumbo nucifera under cadmium stress; in addition, the strain can promote the absorption and enrichment of cadmium by the Nelumbo nucifera and reduce the cadmium content in the sediment environment. The application has important significance for the development of multifunctional microbial fertilizer (promoting plant growth and improving plant cadmium tolerance) and the remediation of cadmium-polluted environment.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of garden plants, microorganisms and water environment remediation, and particularly relates to a lotus rhizosphere growth-promoting bacteria Nn211 and application thereof. BACKGROUND

[0002] Lotus (Nelumbo nucifera Gaertn.), also known as lotus, is a perennial aquatic flower of Nelumbonaceae, and is one of the ten traditional famous flowers in China, with high ornamental value, economic value and cultural value. Lotus is elegant and refined, and blooms in the summer when there are few flowers, and is an important plant material indispensable for modern landscape water scene construction. Lotus likes fertilizer, and in order to promote its growth and ensure that flowers and leaves are lush, chemical fertilizer is often applied. However, long-term application of chemical fertilizer can cause soil compaction and decline in fertility, and the residual chemical fertilizer releases a large amount of harmful substances into the environment, causing serious pollution to water bodies. Therefore, it is urgent to reduce the use of chemical fertilizer and develop new fertilizer sources to replace chemical fertilizer.

[0003] Heavy metal pollution of water bodies has become an important environmental problem faced by the whole world. Among them, cadmium (Cd) is one of the main heavy metal pollutants in water bodies in China, which can easily accumulate in animals and plants, affect the normal growth of animals and plants, and ultimately endanger human health through the food chain. Therefore, the treatment of cadmium pollution of water bodies is imminent. Compared with traditional physical and chemical remediation technologies, plant remediation technology is concerned due to its green environmental protection and low cost, and especially the use of ornamental plants to remediate contaminated environment not only has ecological effect, but also beautifies the environment, and has become a research hotspot in recent years. However, aquatic ornamental plant lotus will appear toxic phenotype under high concentration cadmium stress for a long time, therefore, it is very important to enhance the cadmium tolerance of lotus for improving plant remediation technology.

[0004] Plant growth-promoting rhizobacteria (PGPRs) are a class of beneficial microorganisms that live in the rhizosphere soil or on the root surface of plants. Currently identified PGPRs include Bacillus, Pseudomonas, Arthrobacter, Enterobacter, and Klebsiella. Compared to chemical fertilizers, microbial fertilizers made from plant growth-promoting rhizobacteria have advantages such as high fertilizer efficiency and improved soil structure. They can increase plant biomass and stress resistance, and can mitigate environmental pollution caused by excessive application of chemical fertilizers. However, related research mainly focuses on crops. Furthermore, the use of plant-microbe co-remediation technology to remediate heavy metal pollution has become one of the cutting-edge areas of research in bioremediation of environmental pollution. In recent years, research on the remediation of heavy metal-contaminated soils using terrestrial plants in conjunction with microorganisms has made some progress. However, research on the effects of rhizosphere growth-promoting bacteria on the growth and stress resistance of aquatic ornamental plants under heavy metal stress, as well as their impact on the remediation of heavy metal-contaminated environments, is still quite scarce. As an important resource for biofertilizers and plant-microbe co-remediation technologies, the isolation and application of rhizosphere growth-promoting bacteria has become a current research hotspot. This has significant practical implications for accelerating ecological environment construction and promoting sustainable development. However, to date, there are no publicly available reports on rhizosphere growth-promoting bacteria specifically for lotus. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the above-mentioned problems and provide a lotus rhizosphere growth-promoting bacterium Nn211 and its application. This bacterium is a new plant rhizosphere growth-promoting bacterium that can be applied to the preparation of multifunctional microbial fertilizers (promoting plant growth and improving plant cadmium tolerance) and to improve the remediation technology of cadmium-polluted environments.

[0006] A rhizosphere growth-promoting bacterium for lotus, Nn211, classified as Gibbsiella dentisursi, was deposited on November 4, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 26047.

[0007] The lotus rhizosphere growth-promoting bacterium Nn211 described in this invention is characterized by the following: Nn211 colonies on LB agar are round, white, raised, opaque, and mucous-like, with a moist surface and neat edges; Nn211 is a Gram-negative bacterium, negative for methyl red, VP, tryptophan deaminase, and cellulose degradation tests, and positive for catalase and urease tests, and grows well at a NaCl concentration of 2-7%.

[0008] The lotus rhizosphere growth-promoting bacterium Nn211 described in this invention has ACC deaminase activity, with an enzyme activity of 0.242 U / mg; it has a strong IAA secretion capacity, reaching 23.91 μg / mL; and it can maintain good growth in a high concentration (400 mg / L) cadmium environment, exhibiting good cadmium tolerance.

[0009] The 16S rDNA nucleotide sequence of the lotus rhizosphere growth-promoting bacterium Nn211 is shown in SEQ ID NO:1 in the sequence listing.

[0010] Inoculants, bio-fertilizers, bacterial suspensions, culture media, or fermentation products containing the lotus rhizosphere growth-promoting bacterium Nn211 described in this invention.

[0011] The application of the lotus rhizosphere growth-promoting bacterium Nn211 or the bacterial agent, bio-fertilizer, bacterial suspension, culture medium or fermentation product described in this invention in any one of the following (1) to (11):

[0012] (1) Promotes plant growth;

[0013] (2) Prepare products that promote plant growth;

[0014] (3)Production of IAA;

[0015] (4) Prepare products that produce IAA;

[0016] (5) Produces ACC deaminase;

[0017] (6) Prepare products that produce ACC deaminase;

[0018] (7) Alleviate the damage of cadmium stress to plants and promote plant growth under cadmium stress;

[0019] (8) Prepare products that improve the cadmium tolerance of plants;

[0020] (9) Enhance the absorption and accumulation of cadmium by plants;

[0021] (10) Enhance the ability of plants to remediate cadmium-polluted environments;

[0022] (11) Prepare products that enhance the ability of plants to remediate cadmium-polluted environments.

[0023] In the application described in this invention, the plant is a lotus.

[0024] The application described in this invention, wherein:

[0025] Inoculation with Nn211 can promote the growth of lotus seedlings and potted mature lotus, and increase the biomass, number of leaves, chlorophyll content, and number of underground buds of lotus.

[0026] Inoculation with Nn211 under cadmium stress can alleviate the inhibition of plant height, number of leaves and leaf area of ​​potted mature lotus by cadmium, significantly increase the biomass and chlorophyll content of lotus seedlings and mature lotus, and reduce malondialdehyde content, thereby enhancing the cadmium tolerance of lotus.

[0027] Inoculation with Nn211 can promote the absorption and accumulation of cadmium in various organs of adult lotus, while limiting the transfer of cadmium from underground to aboveground parts. In addition, inoculation with Nn211 can enhance the lotus's ability to accumulate cadmium, significantly reduce the cadmium content in the sediment, and improve the sediment environment.

[0028] The method for preparing a bacterial suspension containing lotus rhizosphere growth-promoting bacteria Nn211 according to the present invention includes the following steps: Inoculating Nn211 strain into LB liquid medium, culturing at 30℃ and 200 r / min for 14-18 h, centrifuging at 8000 rpm and room temperature for 10 min, resuspending and washing twice with sterile water, and finally resuspending in sterile water to prepare OD. 600 A bacterial suspension with a concentration of 1.0 was prepared for use, wherein the concentration of the Nn211 strain in the bacterial suspension was 1 × 10⁻⁶. 8 CFU / mL.

[0029] A method for promoting the growth of lotus seedlings includes the following steps: Select plump and round lotus seeds, disinfect their surface with 3% sodium hypochlorite and 75% alcohol, and after cracking the shell, place them in distilled water to germinate for 2-3 days. Select seedlings with hypocotyls that have spread to 2cm and transfer them to 2L plastic pots with 3 seeds per pot. Soak the seedlings in 1L of the bacterial suspension for 1 hour, repeating the soaking every 3 days for a total of 3 times. Cultivate the seedlings at 30℃, under 16h light / 8h darkness and a light intensity of 18000Lx. After 12 days, lotus seedlings with varying degrees of increase in leaf number, leaf area, root length, and biomass are obtained.

[0030] A method for improving the cadmium tolerance of lotus seedlings includes the following steps: Select plump and round lotus seeds, disinfect their surface with 3% sodium hypochlorite and 75% alcohol, and after cracking the shells, place them in 2L plastic pots with 3 seeds per pot. Cultivate them at 30℃ and 18000Lx long-day conditions, changing the water every 5 days. When the seedlings have grown 4-5 leaves, select hydroponic seedlings with uniform growth and spray 15mL of the bacterial suspension described in this invention around their roots, spraying once every 2 days for a total of 3 times. After treatment, lotus seedlings with enhanced cadmium tolerance can be obtained when the cadmium concentration in the environment is 0-25μmol / L.

[0031] A method for promoting the growth of mature lotus flowers and enhancing their ability to remediate cadmium-polluted environments includes the following steps: Selecting three-year-old lotus rhizomes of uniform size with terminal buds, and planting them in white, non-porous plastic buckets with a diameter of 12.5 cm and a volume of 5 L, with 2.5 kg of soil per bucket and one rhizome per bucket. During the lotus growth period, normal water and fertilizer management is carried out; after the lotus flowers have grown two upright leaves, injecting 150 mL of the bacterial suspension described in this invention around the root zone for 3 consecutive days, and then injecting 150 mL of the bacterial suspension every 7 days for a total of 6 injections. After treatment, lotus flowers with increased biomass, improved cadmium tolerance, and enhanced ability to remediate cadmium-polluted environments can be obtained when the cadmium concentration in the environment is 0-30 mg / kg.

[0032] The present invention differs from the prior art in that:

[0033] 1. The *Gibbsiella dentisursi* Nn211 provided by this invention is the first strain isolated from lotus rhizosphere soil. Routine physiological and biochemical characterization tests and 16S rDNA sequencing of this strain confirmed it to be a novel *Gibbsiella dentisursi* strain with growth-promoting and high cadmium resistance, named *Gibbsiella dentisursi* Nn211. To date, there have been no reports of *Gibbsiella dentisursi* exhibiting growth-promoting ability and resistance to high cadmium concentrations.

[0034] 2. The *Gibberella ursoderm* Nn211 provided by this invention can efficiently secrete IAA and possess ACC deaminase activity, which can reduce ethylene synthesis in plants, thus effectively promoting plant growth as a rhizosphere growth-promoting bacterium. Simultaneously, Nn211 exhibits tolerance to high concentrations of cadmium, giving it strong survival capabilities and enabling it to continue promoting plant growth even under cadmium stress. To date, there have been no reports on *Gibberella ursoderm* promoting plant growth and enhancing plant cadmium tolerance.

[0035] 3. This invention provides a strain that can promote lotus growth and improve lotus's tolerance to cadmium. When this strain is inoculated into the rhizosphere of lotus seedlings and mature plants, it can effectively promote the growth and development of lotus seedlings and mature plants, significantly alleviate the damage of cadmium stress to lotus, and significantly increase the biomass of lotus under cadmium stress. It can be applied to lotus cultivation to improve yield and stress resistance.

[0036] 4. This invention provides a strain that can enhance the ability of lotus to remediate cadmium-polluted environments. When this strain is inoculated into the rhizosphere of mature lotus plants, it can effectively promote the absorption and accumulation of cadmium in various organs of the lotus under cadmium stress, significantly reduce the cadmium content in the sediment, and improve the sediment environment.

[0037] The lotus rhizosphere growth-promoting bacterium Nn211 of the present invention will be further described below with reference to the accompanying drawings. Attached Figure Description

[0038] Figure 1 This is a plate colony characteristic diagram of strain Nn211 from an embodiment of the present invention;

[0039] Figure 2 This is a diagram of the IAA production test of strain Nn211 in an embodiment of the present invention;

[0040] Figure 3 This invention demonstrates the growth-promoting effect of strain Nn211 on lotus seedlings in an embodiment of the present invention.

[0041] Figure 4 This invention illustrates the effect of strain Nn211 on the leaf growth of lotus seedlings under cadmium stress.

[0042] Figure 5 This invention illustrates the effect of strain Nn211 on root vigor of lotus seedlings under cadmium stress.

[0043] Figure 6 This invention illustrates the effect of strain Nn211 on the chlorophyll content of lotus seedling leaves under cadmium stress.

[0044] Figure 7 This invention illustrates the effect of strain Nn211 on malondialdehyde content in lotus seedling leaves under cadmium stress.

[0045] Figure 8 This invention illustrates the effect of strain Nn211 on the chlorophyll content of potted mature lotus leaves under cadmium stress.

[0046] Figure 9 This invention illustrates the effect of strain Nn211 on malondialdehyde content in potted mature lotus leaves under cadmium stress.

[0047] Figure 10 This invention illustrates the effect of strain Nn211 on the cadmium content of various organs and individual plants of potted mature lotus under cadmium stress.

[0048] Figure 11 The effect of strain Nn211 on cadmium content in sediment is illustrated in this embodiment of the invention. Detailed Implementation

[0049] Example 1: Screening and Identification of Rhizosphere Growth-Promoting Bacteria in Lotus

[0050] 1. Test materials

[0051] (1) Soil sample collection

[0052] Soil samples were collected from the rhizosphere soil of lotus flowers in Hehuatang, Yancheng City, Jiangsu Province. The samples were collected using a 5-point sampling method and placed in sterile self-sealing bags for immediate separation.

[0053] (2) Preparation of culture medium and reagents

[0054] ① PAF medium

[0055] 10g peptone, 10g casein hydrolysate, 1.5g anhydrous MgSO4, 1.5g K2HPO4, 10mL glycerol, 1000mL distilled water, pH 7-7.2, sterilized at 121℃ for 20min.

[0056] ②DF culture medium

[0057] Component 1: H3BO3 10mg, ZnSO4·7H2O 124.6mg, MnSO4·H2O 11.19mg, MoO3 10mg, CuSO4·5H2O 78.22mg. Dissolve the above components in 100mL of distilled water and store at 4℃ for later use.

[0058] Component 2: 100 mg FeSO4·7H2O, dissolved in 10 mL distilled water, refrigerated at 4°C for later use.

[0059] Take 0.1 mL of each of component one and component two, and add the following reagents: 4 g KH2PO4, 15.12 g Na2HPO4·12H2O, 0.2 g MgSO4·7H2O, 2 g glucose, 4 mL 50% D-glucose solution, 2 g citric acid, 2 g (NH4)2SO4, 15 g agar (solid culture medium), pH 7.0-7.2, 1000 mL distilled water, and sterilize at 121℃ for 20 min.

[0060] ③ADF medium

[0061] 0.5 mol / L ACC: Weigh 0.505 g of ACC and dissolve it in 10 mL of sterile water. Sterilize by passing it through a 0.22 nm bacterial filter and store at -20 °C.

[0062] The prepared 0.5 mol / L ACC solution was added to the sterilized and cooled (NH4)2SO4-free DF medium at a rate of 6 ml / L, replacing (NH4)2SO4 as the sole nitrogen source in the medium.

[0063] ④LB medium

[0064] 10.0g tryptone, 5.0g yeast extract, 10g NaCl, 15g agar (solid culture medium), 1000mL distilled water, pH 7.0-7.2, sterilized at 121℃ for 20min.

[0065] ⑤TSB medium

[0066] 3g soybean peptone, 17g tryptone, 5g NaCl, 2.5g glucose, 2.5g K2HPO4, 1000mL distilled water, pH 7.0-7.2, sterilized at 121℃ for 20min.

[0067] ⑥ Cadmium-resistant solid culture medium

[0068] Weigh out CdCl2·2.5H2O at cadmium concentrations of 200, 300, and 400 mg / L and add it to LB solid medium. Sterilize at 121℃ for 20 min.

[0069] ⑦ Urease activity assay medium

[0070] 3g L-tryptophan, 1g KH2PO4, 1g K2HPO4, 5g NaCl, 10mL 95% ethanol, 900mL distilled water, add phenol red until slightly orange-yellow, pH=6.8-6.9, sterilize at 121℃ for 20min.

[0071] ⑧ Reagents

[0072] Salkowski colorimetric reagent: Slowly pour 150 mL of H₂SO₄ into 250 mL of distilled water. After the H₂SO₄ solution cools, add 7.5 mL of 0.5 M FeCl₃·6H₂O solution to prepare the colorimetric reagent. This colorimetric reagent cannot be stored for a long time.

[0073] Coomassie Brilliant Blue Solution: Weigh 100 mg of Coomassie Brilliant Blue, dissolve it in 50 mL of 90% ethanol, add 100 mL of 85% (w / v) phosphoric acid, and finally add distilled water to bring the volume to 1000 mL.

[0074] 2. Test methods

[0075] (1) Isolation of rhizosphere bacteria from lotus

[0076] Weigh 1g of lotus rhizosphere soil and add it to 50mL of sterile water, then shake to prepare a soil suspension. Inoculate 1mL of the soil suspension into 50mL of PAF liquid medium and culture at 30℃ and 200r / min for 24h. Then, transfer 1mL of the PAF culture to 50mL of DF liquid medium and culture for 24h. Next, inoculate 1mL of the DF bacterial suspension into 50mL of ADF liquid medium and culture for 24h. Dilute the ADF culture and spread it on ADF agar plates, incubating at 30℃ for 3 days until single colonies appear. Purify the single colonies from the ADF plates and transfer them to TSB slant medium for storage. The strain with ACC deaminase activity, as initially screened, is named Nn211.

[0077] (2) ACC deaminase activity assay

[0078] Strain Nn211 was inoculated into TSB liquid medium and cultured overnight at 30°C and 200 rpm. After centrifugation at 4°C for 10 min, the cells were collected and washed three times with DF medium. The cells were then resuspended in ADF medium and cultured with shaking for 48 h. The cells were then collected by centrifugation at 4°C. The cells were washed three times with 0.1 mol / L Tris-HCl buffer (pH 7.6) and resuspended in 600 μL of 0.1 mol / L Tris-HCl buffer (pH 8.0). 30 μL of toluene was added and the mixture was rapidly shaken for 30 s to lyse the cells. 200 μL of the toluene-containing cell extract was added to 20 μL of 0.5 mol / L ACC and mixed thoroughly. A blank assay without ACC was also performed. The mixture was then incubated at 37°C for 15 min. Then, 1 mL of 0.56 mol / L HCl was added, and the mixture was centrifuged at 5000 r / min for 5 min. 1 mL of the supernatant was collected, and 800 μL of 0.56 mol / L HCl and 300 μL of 2,4-dinitrophenylhydrazine were added. The mixture was then incubated in a water bath at 30℃ for 30 min. Finally, 2 mL of 2 mol / L NaOH was added to terminate the reaction, and the absorbance at 540 nm was measured. α-Ketobutyric acid standards were prepared at different concentration gradients, and the absorbance at 540 nm was measured after the same reaction to plot a standard curve.

[0079] Take another 100 μL of cell extract containing toluene, add 0.15 mol / L NaCl to a final volume of 1 mL, and finally add 5 mL of Coomassie Brilliant Blue reagent and mix well. Simultaneously prepare a blank control without bovine serum albumin. Measure the OD after the reaction. 595 Bovine serum albumin standard samples were prepared into different concentration gradients, and the absorbance at 595 nm was measured after the reaction to plot a standard curve. The specific activity (U / mg) is obtained by dividing the unit enzyme activity by the total protein mass, which represents the bacterial ACC deaminase activity.

[0080] Formula for calculating the specific activity of ACC deaminase:

[0081]

[0082] (3) IAA Characteristics Measurement

[0083] The Salkowski colorimetric method was used to inoculate strain Nn211 into LB liquid medium containing L-tryptophan (100 mg / L) and culture it at 30°C and 200 rpm for 24 h. The bacterial suspension was centrifuged at 4°C and 5000 rpm for 10 min, and 1 mL of the supernatant was added to 3 mL of Salkowski colorimetric reagent. A mixture of uninoculated LB liquid medium and the colorimetric reagent served as a blank control. After incubation at room temperature in the dark for 30 min, observation was performed; a red color indicated the secretion of IAA. Standard curves were prepared by using IAA standard solutions of different concentrations. The OD of the reaction solution was measured after reacting under the above conditions. 530Calculate the concentration of IAA in the supernatant.

[0084] (4) Cadmium resistance test

[0085] Strains Nn211 were inoculated onto LB plates with cadmium concentrations of 200, 300, and 400 mg / L, respectively, and their growth was observed after incubation at 30°C for 3 days.

[0086] (5) Physiological and biochemical characteristics

[0087] According to the *Manual of Systematic Identification of Common Bacteria*, a series of physiological and biochemical characteristics of Nn211 were determined. The measured indicators included: Gram staining, methyl red, VP, tryptophan deaminase, cellulose degradation, catalase, salt requirement, and salt tolerance tests. Urease activity was determined by dissolving 20g of urea in 100mL of water, filtering and sterilizing, and then mixing it with the above sterile culture medium. Nn211 was inoculated into the culture medium and cultured for 2-3 days. The culture medium turning from yellow to red indicates the presence of urease.

[0088] (6) Identification by 16S rDNA sequence sequencing

[0089] Total bacterial DNA was extracted, and 16S rDNA was amplified using universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACCTTGTTACGACTT-3′). The PCR amplification products were sent to Nanjing Qingke Technology Co., Ltd. for sequencing. The primers used were synthesized by Nanjing Qingke Technology Co., Ltd. The obtained 16S rDNA gene sequence was compared with the NCBI and Ezibiocloud databases for homology.

[0090] 3. Identification results of strain Nn211

[0091] (1) Morphological characteristics identification results

[0092] The colony morphology of strain Nn211 on LB agar plates is as follows: Figure 1 As shown, the colonies are round, white, raised, opaque, and slimy, with a moist surface and neat edges.

[0093] (2) Identification of physiological and biochemical characteristics

[0094] Strain Nn211 is a Gram-negative bacterium. Other physiological and biochemical tests are shown in Table 1.

[0095] Table 1. Identification results of physiological and biochemical characteristics of strain Nn211

[0096]

[0097] Note: + indicates positive; - indicates negative.

[0098] (3) 16S rDNA sequence sequencing identification results

[0099] The 16S rDNA sequence of strain Nn211 was amplified and sequenced, and the sequencing results are shown in SEQ ID NO:1 in the sequence listing. The obtained 16S rDNA sequence of strain Nn211 was compared with that of *Gibbsiella dentisursi* on the NCBI website. The results showed that this strain had the highest homology with *Gibbsiella dentisursi*, reaching 99.32%; a comparison on the Ezibiocloud website confirmed that the strain was *Gibbsiella dentisursi*, with a similarity of 99.38%. Therefore, the strain obtained by screening in this invention was identified as *Gibbsiella dentisursi*, named *Gibbsiella dentisursi* Nn211, and this strain Nn211 was deposited at the China General Microbiological Culture Collection Center on November 4, 2022, with the accession number CGMCC No. 26047.

[0100] (4) Results of identification of growth-promoting characteristics

[0101] The growth-promoting characteristics of the strains are shown in Table 2; Figure 2 This is a diagram of the IAA production test of strain Nn211 in an embodiment of the present invention.

[0102] Table 2. Results of identification of growth-promoting characteristics of strain Nn211

[0103]

[0104] Note: + indicates positive; - indicates negative.

[0105] (5) Results of cadmium resistance test

[0106] The results of the cadmium resistance test of the strain are shown in Table 3, indicating that the strain has good cadmium resistance.

[0107] Table 3 Results of cadmium tolerance test for strain Nn211

[0108]

[0109] Note: + indicates that the strain is growing well; - indicates that the strain is not growing.

[0110] Example 2: Preparation of Nn211 bacterial suspension and its effects on lotus seedling growth and cadmium tolerance.

[0111] I. Preparation of Nn211 bacterial suspension

[0112] The *Gibberella ursoderm* strain Nn211 was inoculated into LB liquid medium and cultured on a shaker (30℃, 200 rpm) for 14-18 h. After centrifugation at 8000 rpm at room temperature for 10 min, the suspension was washed twice with sterile water and then resuspended in sterile water to prepare a bacterial suspension with OD600 = 1.0 for later use. The concentration of *Gibberella ursoderm* strain Nn211 in the bacterial suspension was 1 × 10⁻⁶. 8 CFU / mL.

[0113] II. Effects of Nn211 bacterial suspension on the growth of lotus seedlings

[0114] 1. Experimental Materials and Methods

[0115] Select plump, round seeds of Weishan Lake red lotus and disinfect their surface with 3% sodium hypochlorite and 75% alcohol. After cracking the shell, place them in distilled water to germinate for 2-3 days. Select seedlings with hypocotyls that have spread to 2cm and transplant them into small plastic pots with a diameter of 20cm and a volume of 2L, with 3 seeds per pot. Use 1L of 1×10⁻⁶ solution. 8 Seedlings were soaked in CFU / mL bacterial suspension for 1 hour, once every 3 days for a total of 3 times. Soaking in sterile water served as a control. The two treatments were designated as CK and Nn211, respectively. Each treatment was replicated in 3 replicates, with each pot constituting one replicate. Seedlings were cultured at 30℃ and 18000Lx long-day conditions (16h light / 8h dark). After 12 days, the number of leaves, leaf area, root length, and biomass were measured.

[0116] 2. Test Results

[0117] The results show that: Figure 3 As shown in Table 4, compared with the control, Nn211 significantly promoted the growth of lotus seedlings. The number of leaves, leaf area and root length of lotus seedlings treated with Nn211 increased by 50.2%, 12.1% and 6.7% respectively compared with the control. The fresh weight and dry weight of lotus seedlings also increased significantly compared with the control.

[0118] Table 4. Effects of strain Nn211 on the growth of lotus seedlings

[0119]

[0120] Note: * indicates significant differences between different treatments (P<0.05), the same applies below.

[0121] III. Effects of Nn211 bacterial suspension on the growth of lotus seedlings under cadmium treatment

[0122] 1. Experimental Materials and Methods

[0123] Plump and rounded seeds of Weishan Lake red lotus were selected and surface-sterilized with 3% sodium hypochlorite and 75% alcohol. After hatching, the seeds were placed in small plastic pots with a diameter of 20cm and a volume of 2L, with 3 seeds per pot. They were cultured at 30℃ and 18000Lx long day (16h light / 8h dark), with water changed every 5 days. The experiment included 4 treatments: (1) Nn211 inoculation under normal conditions (Nn211); (2) Nn211 inoculation under cadmium stress (Cd+Nn211); (3) Uninoculated cadmium treatment (Cd); (4) Uninoculated and untreated cadmium control (CK). The specific treatment methods are as follows: When the seedlings have grown 4-5 leaves, select hydroponic seedlings with uniform growth and spray them around the roots with a concentration of 1×10 8 15 mL of CFU / mL bacterial suspension was sprayed every 2 days for a total of 3 times. The untreated group was injected with sterile water instead. A cadmium concentration of 25 μmol / L was set, and CdCl₂·2.5H₂O was prepared as an aqueous solution and applied to the pots. The untreated group was treated with an equal volume of sterile water. Each treatment was repeated in triplicate, with each pot constituting one replicate. After 48 hours of treatment, the biomass of various organs of the lotus seedlings, root cell viability, and leaf chlorophyll and malondialdehyde content were measured.

[0124] 2. Test Results

[0125] The results show that: Figure 4 , Figure 5 As shown in Table 5, compared with the control, cadmium stress caused toxic phenotypes in the leaves and roots of lotus seedlings, specifically manifested as chlorosis and brown patches on leaves, massive death of root tip cells, and significant reduction in plant fresh weight and dry weight. However, inoculation with Nn211 under cadmium stress could greatly alleviate these toxic effects of cadmium stress on lotus seedlings and significantly increase the biomass of lotus seedlings under cadmium stress.

[0126] Table 5. Effects of strain Nn211 on the growth of lotus seedlings under cadmium stress.

[0127]

[0128] Note: Different lowercase letters in the same column indicate significant differences between different treatments (P<0.05), and the same applies below.

[0129] like Figure 6 and Figure 7 As shown, compared with the control, under normal conditions, inoculation with Nn211 significantly increased the chlorophyll a and chlorophyll b content of lotus seedling leaves, while under cadmium stress, the chlorophyll content of lotus seedling leaves significantly decreased and the malondialdehyde content significantly increased. Compared with the uninoculated cadmium treatment, inoculation with Nn211 under cadmium stress significantly increased the chlorophyll content of lotus seedling leaves and significantly reduced the malondialdehyde content, thus alleviating the toxic effects of cadmium on lotus seedlings.

[0130] Example 3: Effects of Nn211 bacterial suspension on the growth and cadmium tolerance of potted mature lotus flowers

[0131] 1. Experimental Materials and Methods

[0132] After inoculating the Nn211 strain into LB liquid medium and culturing for 14-18 hours, the bacterial cells were collected by centrifugation and resuspended in sterile water to prepare OD. 600 Prepare a bacterial suspension with a concentration of 1 × 10⁻¹⁰ for later use. 8 CFU / mL. Using three-year-old lotus rhizomes from Weishan Lake as material, select rhizomes of similar size with terminal buds and plant them in white, non-porous plastic buckets with a diameter of 12.5cm and a volume of 5L. Each pot contains 2.5kg of soil and one rhizome. During the lotus growth period, normal water and fertilizer management is carried out.

[0133] The pot experiment was conducted in a plastic greenhouse on the campus of Nanjing Agricultural University. The experiment included four treatments: (1) Nn211 inoculation under normal conditions (Nn211); (2) Nn211 inoculation under Cd stress (Cd+Nn211); (3) Cd-free treatment (Cd); and (4) a control group (CK) without inoculation or Cd treatment. Each treatment was replicated four times. The specific treatment methods were as follows: When each pot of lotus had two upright leaves, healthy and uniformly growing lotus plants were selected, and for three consecutive days, a concentration of 1×10⁻⁶ was injected around the root zone using a syringe. 8 A bacterial suspension of CFU / mL was injected once daily, 150 mL per pot per injection. For the untreated group, sterile water was used instead. After 3 consecutive days of inoculation, the lotus flowers were treated with cadmium at a concentration of 30 mg / kg. CdCl₂·2.5H₂O was dissolved in sterile water and applied to the pots as an aqueous solution. For the untreated group, an equal volume of sterile water was used instead. Day 0 was the day after cadmium treatment, and injections were repeated every 7 days at a concentration of 1×10⁻⁶. 8 150 mL of bacterial suspension with a concentration of CFU / mL was injected three times. Morphological indicators such as plant height, number of leaves, leaf area, underground buds, and biomass of potted lotus were measured 28 days after cadmium treatment. Physiological indicators such as leaf chlorophyll and malondialdehyde were also measured, along with cadmium content in various lotus organs and the bottom mud.

[0134] 2. Test Results

[0135] The results, as shown in Table 6, indicated that compared to the control, inoculation with Nn211 under normal conditions significantly increased the number of leaves, underground buds, rhizome fresh weight, plant fresh weight, and dry weight of mature potted lotus, demonstrating a significant growth-promoting effect. Cadmium stress significantly inhibited the growth of mature lotus plant height, number of leaves, leaf area, and the increase in plant biomass. However, compared to the uninoculated cadmium treatment, inoculation with Nn211 under cadmium stress significantly increased the plant height, number of floating leaves, and leaf area of ​​both floating and upright leaves in mature lotus. Furthermore, the fresh weight of all lotus organs, as well as the fresh and dry weight of the plant, all significantly increased. This suggests that inoculation with Nn211 can significantly alleviate the toxic effects of cadmium stress on mature lotus and promote the growth of mature lotus under cadmium stress.

[0136] Table 6. Effects of strain Nn211 on the growth of mature potted lotus under normal conditions and cadmium stress.

[0137]

[0138]

[0139] like Figure 8 and Figure 9 As shown, compared with the control, cadmium stress significantly reduced chlorophyll content and significantly increased malondialdehyde (MDA) content in the leaves of mature lotus plants. However, after inoculation with Nn211 under cadmium stress, the chlorophyll content of lotus leaves significantly increased and the MDA content significantly decreased compared with the uninoculated cadmium stress, with no significant differences between the indicators and the control. This indicates that inoculation with Nn211 can significantly alleviate the physiological damage of cadmium stress to mature lotus plants and enhance their cadmium tolerance.

[0140] like Figure 10 As shown in Table 7, the cadmium content in various organs of lotus under cadmium stress was significantly increased compared with the control, with the highest cadmium content in the roots, followed by the leaves and rhizomes. After inoculation with Nn211 under cadmium stress, the cadmium content in individual lotus plants and various organs was significantly increased compared with the uninoculated cadmium treatment, while the translocation coefficient was significantly reduced. This indicates that inoculation with Nn211 promoted the absorption and accumulation of cadmium in various organs of lotus and could resist cadmium stress by limiting the transfer of cadmium from the underground parts to the aboveground parts.

[0141] like Figure 11 As shown in Table 7, the cadmium content in the bottom mud of mature potted lotus plants under cadmium treatment was 14.02 mg / kg. After inoculation with Nn211 under cadmium stress, the cadmium content in the bottom mud significantly decreased, while the enrichment coefficient and the enrichment amount per plant both significantly increased. This indicates that inoculation with Nn211 significantly promoted the enrichment of cadmium in the bottom mud by potted lotus plants, reduced the cadmium content in the environment, and played a role in improving and remediating cadmium-polluted environments.

[0142] Table 7. Transfer coefficient, enrichment coefficient, and cadmium accumulation per plant of lotus under different treatments.

[0143]

[0144] In summary, the *Gibbsiella dentisursi* Nn211 strain provided by this invention possesses the characteristics of producing ACC deaminase, urease, secreting IAA, and tolerating heavy metal cadmium. It can significantly promote the growth of lotus seedlings and mature plants, and improve the lotus's resistance to cadmium stress. Furthermore, this strain can effectively promote the absorption and accumulation of cadmium in mature lotus plants, reducing the cadmium content in the sediment environment. This invention provides an excellent rhizosphere growth-promoting strain for multifunctional growth-promoting bacteria, which can be applied to the preparation of multifunctional microbial fertilizers for lotus (promoting plant growth and improving plant cadmium tolerance) and to improving cadmium-polluted environmental remediation technologies.

[0145] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A rhizosphere growth-promoting bacterium for lotus, Nn211, is classified and named *Gibberella ursodesiforme* (…). Gibbsiella dentisursi It was deposited on November 4, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 26047.

2. A bio-fertilizer, bacterial suspension, or culture medium containing the lotus rhizosphere growth-promoting bacterium Nn211 as described in claim 1.

3. The application of the lotus rhizosphere growth-promoting bacterium Nn211 as described in claim 1 or the bio-fertilizer, bacterial suspension or culture medium as described in claim 2 in any one of the following (1) to (11): (1) Promotes plant growth; (2) Prepare products that promote plant growth; (3) Produce IAA; (4) Prepare products that produce IAA; (5) Produces ACC deaminase; (6) Prepare products that produce ACC deaminase; (7) Alleviate the damage of cadmium stress to plants and promote plant growth under cadmium stress; (8) Prepare products that improve the cadmium tolerance of plants; (9) Enhances the absorption and accumulation of cadmium by plants; (10) Enhance the ability of plants to remediate cadmium-polluted environments; (11) Prepare products that enhance the ability of plants to remediate cadmium-polluted environments; in, The plant in question is a lotus.

4. The application according to claim 3, characterized in that: Inoculation with Nn211 can promote the growth of lotus seedlings and potted mature lotus, and increase the biomass, number of leaves, chlorophyll content and number of underground buds of lotus. Inoculation with Nn211 under cadmium stress can alleviate the inhibition of plant height, number of leaves and leaf area of ​​potted mature lotus by cadmium, significantly increase the biomass and chlorophyll content of leaves of lotus seedlings and mature lotus, and reduce malondialdehyde content, thereby enhancing the cadmium tolerance of lotus. Inoculation with Nn211 can promote the absorption and accumulation of cadmium in various organs of adult lotus, while limiting the transfer of cadmium from underground to aboveground parts. In addition, inoculation with Nn211 can enhance the lotus's ability to accumulate cadmium, significantly reduce the cadmium content in the sediment, and improve the sediment environment.

5. The method for preparing the bacterial suspension containing lotus rhizosphere growth-promoting bacteria Nn211 as described in claim 2, characterized in that: The process includes the following steps: Inoculate the Nn211 strain into LB liquid medium, incubate at 30°C and 200 rpm for 14-18 hours, centrifuge at 8000 rpm at room temperature for 10 minutes, resuspend and wash twice with sterile water, and finally resuspend again with sterile water to prepare OD. 600 A bacterial suspension with a concentration of 1.0 was prepared for use, and the concentration of the Nn211 strain in the bacterial suspension was 1×10⁻⁶. 8 CFU / mL.

6. A method for promoting the growth of lotus seedlings, characterized in that: Includes the following steps: Select plump and round lotus seeds, disinfect their surface with 3% sodium hypochlorite and 75% alcohol, and after cracking the shell, place them in distilled water to germinate for 2-3 days. Select seedlings with hypocotyls that have spread to 2cm and transfer them to 2L plastic pots with 3 seeds per pot. Soak the seedlings in 1L of the bacterial suspension described in claim 5 for 1 hour, once every 3 days, for a total of 3 times. Cultivate them at 30℃, 16h light / 8h darkness, and a light intensity of 18000Lx. After 12 days, lotus seedlings with varying degrees of increase in leaf number, leaf area, root length, and biomass were obtained.

7. A method for improving the cadmium tolerance of lotus seedlings, characterized in that: Includes the following steps: Select plump and round lotus seeds, disinfect them with 3% sodium hypochlorite and 75% alcohol, and after cracking the shells, place them in 2L plastic pots with 3 seeds per pot. Cultivate them at 30℃ and 18000Lx long day, changing the water every 5 days. When the seedlings have grown 4-5 leaves, select hydroponic seedlings with uniform growth and spray 15mL of the bacterial suspension described in claim 5 around their roots. Spray once every 2 days for a total of 3 times. After treatment, lotus seedlings with enhanced cadmium tolerance can be obtained when the cadmium concentration in the environment is 25μmol / L.

8. A method for promoting the growth of mature lotus flowers and enhancing the ability of lotus flowers to remediate cadmium-polluted environments, characterized in that: Includes the following steps: Three-year-old lotus rhizomes of uniform size with terminal buds were selected and planted in white, non-porous plastic buckets with a diameter of 12.5 cm and a volume of 5 L. Each bucket contained 2.5 kg of soil and one rhizome. Normal water and fertilizer management was carried out during the lotus growth period. After the lotus grew two upright leaves, 150 mL of the bacterial suspension described in claim 5 was injected around the root zone for 3 consecutive days. Then, 150 mL of the bacterial suspension was injected every 7 days for a total of 6 injections. After treatment, lotuses with increased biomass, improved cadmium tolerance, and enhanced ability to remediate cadmium-polluted environments were obtained when the cadmium concentration in the environment was 30 mg / kg.

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