Anaerobic lotus endophytic growth-promoting bacteria L985 and application thereof

Abstract

The present application is an anaerobic lotus endophytic growth-promoting bacteria L985, which is classified as Clostridium mangenotii (C. Metaclostridioides mangenotii ), and was preserved in China General Microbiological Culture Collection Center on June 3, 2024, with the preservation number of CGMCC No. 30853. The L985 strain of the present application is isolated from lotus for the first time, has the characteristics of producing ACC deaminase, producing IAA, nitrogen fixation, phosphorus dissolution, anaerobic, tolerance to high concentration of salt and alkali, and cadmium, etc. The present application has important significance for the development of multifunctional microbial fertilizer (promoting the growth of aquatic plants and improving the stress resistance of aquatic plants) suitable for aquatic plants.

Description

Technical Field

[0001] This invention relates to the fields of aquatic ornamental plants, microorganisms, and aquatic ecological restoration, specifically to an anaerobic endogenous growth-promoting bacterium L985 isolated from lotus, and its application in promoting the growth of lotus and water lilies and enhancing their stress resistance. Background Technology

[0002] lotus ( Nelumbo nucifera Gaertn.) and water lilies ( Nymphaea L. (Leymus chinensis) are important aquatic ornamental flowers, possessing outstanding ornamental, economic, and cultural value, and are widely planted in modern landscape water bodies. They not only beautify the aquatic environment but also have multiple ecological functions, including water purification, algae control, and maintaining biodiversity. However, in the cultivation process, to ensure abundant flowering and foliage, chemical fertilizers are often applied. Long-term application of chemical fertilizers can easily lead to sediment compaction and fertility decline, and residual fertilizer entering the water body can exacerbate environmental pollution. Therefore, developing new, environmentally friendly bio-fertilizers that can replace chemical fertilizers has become an urgent need for the sustainable development of the aquatic ornamental plant industry.

[0003] Meanwhile, with the acceleration of urbanization, water pollution problems are becoming increasingly severe, especially salinization and heavy metal pollution. Salinity stress can cause osmotic imbalance, ion toxicity, and oxidative damage in plants; heavy metals such as cadmium damage plant cell structure, interfere with enzyme activity, and accumulate through the food chain, threatening ecological security and human health. These adverse conditions severely inhibit the normal growth of aquatic ornamental plants such as lotus and water lilies, and also limit their widespread application in aquatic ecological restoration. Therefore, effectively improving the growth capacity and adaptability of aquatic plants to stressful environments is key to achieving the synergistic development of their ornamental and ecological functions, and is also an important prerequisite for promoting phytoremediation technology. Compared with traditional physical and chemical remediation methods, bioremediation technology, especially plant-microbe combined remediation systems, has advantages such as low cost, environmental friendliness, and strong sustainability, showing promising application prospects.

[0004] Plant growth-promoting endophytic bacteria (PGPEBs) are a class of beneficial bacteria that colonize plants without causing disease. However, related research has largely focused on crops and terrestrial plants. Furthermore, the use of plant-microbe co-remediation technologies to control salinity or heavy metal pollution has become a research hotspot in the environmental field, but research on the interaction between aquatic ornamental plants and PGPEBs under stress is still quite scarce, with insufficient attention paid to anaerobic PGPEBs. It is noteworthy that aquatic plants are often exposed to hypoxic or anaerobic water and sediment environments, which may limit the colonization and function of traditional aerobic strains under such conditions. Therefore, screening for anaerobic PGPEBs adapted to hypoxic environments can not only provide new strains for the development of microbial fertilizers for aquatic ornamental plants but also potentially enhance the adaptability and remediation potential of host plants under stresses such as salinity, heavy metals, and other pollutants.

[0005] Isolating and developing PGPEB resources suitable for aquatic plants such as lotus and water lily is of great significance for promoting their green cultivation, enhancing landscape sustainability, constructing an efficient plant-microbe co-remediation system, and achieving ecological treatment of polluted water bodies. Currently, there are no systematic reports on research into endophytic growth-promoting bacteria in lotus, especially anaerobic strains. This invention has significant innovation and application value in this field. Summary of the Invention

[0006] 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 endophytic growth-promoting bacterium L985 and its application. This bacterium is a new plant endophytic growth-promoting bacterium that can be used to prepare microbial fertilizers to promote the growth of aquatic plants and improve the host plant's adaptability and repair potential under stresses such as salinity, alkali and heavy metals.

[0007] An anaerobic endophytic growth-promoting bacterium for lotus, L985, is classified as *Clostridium mangenotti*. Metaclostridioides mangenotii L985 was deposited on June 3, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 30853. L985 is an anaerobic microorganism, better adapted to the hypoxic environment of water, and suitable for aquatic plants. In 2024, Metaclostridioides mangenotii Classified into a new genus Metaclostridioides Original name: Clostridioides mangenotii .

[0008] The anaerobic endophytic growth-promoting bacterium L985 for lotus described in this invention comprises: colonies that are round, translucent, moist, with neat edges and milky-white protrusions, isolated and cultured from lotus roots, stems, and leaves; anaerobic characteristics, negative for catalase and cellulase tests, and positive for methyl red, VP, urease, and amylase tests; nitrogen-fixing and phosphorus-solubilizing abilities; growth-promoting properties by producing ACC deaminase and IAA, with ACC deaminase activity and IAA yield of 0.286 U / mg and 40.28 μg / mL, respectively; and salt and alkali tolerance and cadmium tolerance, resistant to salt (3-7% NaCl), alkali (pH=8-10), mixed salt and alkali (3% NaCl and pH=8, 5% NaCl and pH=9, 7% NaCl and pH=9, 5% NaCl and pH=10, 7% NaCl and pH=10), and cadmium (200 mg·L⁻¹). -1 It can grow well in any environment.

[0009] The 16S rDNA nucleotide sequence of the lotus endophytic growth-promoting bacterium L985 is shown in SEQ ID NO:1 in the sequence listing.

[0010] Bio-fertilizers, bacterial suspensions, or culture media containing the anaerobic lotus endophytic growth-promoting bacteria L985 described in this invention.

[0011] Application of the anaerobic lotus endophytic growth-promoting bacteria L985 described in this invention or the bio-fertilizer, bacterial suspension or culture medium described therein in any one of the following (1) to (12): (1) Produce IAA; (2) Prepare products that produce IAA; (3) Production of ACC deaminase; (4) Prepare products that produce ACC deaminase; (5) Promotes the growth of aquatic plants; (6) Prepare products that promote the growth of aquatic plants; (7) Alleviate the damage of salt and alkali stress to aquatic plants and promote the growth of aquatic plants under salt and alkali stress; (8) Prepare products that improve the salt and alkali tolerance of aquatic plants; (9) Salt-alkali stress reduces the absorption and accumulation of sodium ions by aquatic plants; (10) Under salt and alkali stress, aquatic plants can enhance the absorption and accumulation of potassium ions; (11) Alleviate the damage of cadmium stress to aquatic plants and promote the growth of aquatic plants under cadmium stress; (12) Prepare products that improve the cadmium tolerance of aquatic plants.

[0012] The application described in this invention, wherein: The aquatic plant is either lotus or water lily; Inoculation with L985 can promote the growth of lotus seedlings, potted mature lotus, and potted water lilies, increase the biomass and chlorophyll content of lotus leaves, and increase the number of leaves and chlorophyll content of water lilies. Inoculation with L985 under salt-alkali stress can alleviate the inhibition of plant height, root length, number of leaves and leaf area of ​​potted mature lotus under salt-alkali stress, significantly increase the biomass, chlorophyll content, membrane stability index and soluble protein content of lotus seedlings and potted mature lotus, and reduce hydrogen peroxide content, thereby enhancing the salt-alkali tolerance of lotus. Inoculation with L985 under salt-alkali stress significantly inhibited the Na+ oxidative stress of mature potted lotus flowers. + Absorption and accumulation, enhancing K + Absorption and accumulation, maintaining a low Na + / K + The ratio promotes ion balance, thereby promoting the growth and development of lotus under salt and alkali stress; Inoculation with L985 under cadmium stress can alleviate the inhibition of leaf number and leaf area caused by cadmium stress in potted water lilies, and significantly increase the chlorophyll content of leaves, thereby enhancing the cadmium tolerance of water lilies.

[0013] The application described in this invention, wherein: The method for preparing ACC deaminase includes the following steps: inoculating strain L985 into LB liquid medium at 30 °C and 200 r·min -1 After activating the cells under the specified conditions for 12 h, the cells were collected by centrifugation at 5000 r / min for 10 min at 4 ℃. The cells were then washed twice with SM liquid medium and transferred to ADF liquid medium. After shaking culture for 48 h, the induced culture product was collected, which yielded ACC deaminase. The method for preparing IAA includes the following steps: inoculating strain L985 into LB liquid medium containing L-tryptophan, and incubating at 30 °C and 200 r·min. -1 After 24 hours of incubation, the induced culture product was collected, which yielded IAA.

[0014] The method for preparing a bacterial suspension containing the anaerobic lotus endophytic growth-promoting bacterium L985 according to the present invention includes the following steps: inoculating strain L985 into LB liquid medium and incubating at 30 °C and 180 r·min -1 After 14 h of shaking culture under the specified conditions, centrifuge at 8000 rpm for 10 min at room temperature, resuspend in deionized water, wash twice, and then resuspend again in deionized water to prepare OD200. 600 =1.0, concentration is 1×10 8 cfu·mL -1 Bacterial suspension.

[0015] A method for promoting the growth and salt tolerance of hydroponic lotus seedlings includes the following steps: Select plump lotus seeds, disinfect their surface with 3% sodium hypochlorite and 75% alcohol, and after hatching, place them in a 20cm diameter, 2L volume plastic bucket filled with deionized water for cultivation. The cultivation conditions are 30℃, 18000 Lx long-day, 16h light / 8h dark, with water changed every 3 days. When the lotus seedlings have 3-4 leaves, select hydroponic seedlings with uniform growth and spray 15 ml of the bacterial suspension described in this invention around the roots of the seedlings once a day for 3 consecutive days. After treatment, seedlings with an environmental salt and alkali concentration ≤75 mmol·L⁻¹ can be obtained. -1 At that time, the biomass and leaf chlorophyll content of lotus seedlings increased, and their salt and alkali tolerance was enhanced. The salt and alkali was NaCl:NaHCO3=2:1.

[0016] A method for promoting the growth and salt tolerance of potted mature lotus flowers includes the following steps: Selecting robust, uniformly sized, two-year-old lotus rhizomes with terminal buds, and planting them in white plastic buckets with a diameter of 12.5 cm and a volume of 5 L containing 2.5 kg of soil. One rhizome is planted per pot, and normal water and fertilizer management is carried out during the lotus growth period. After the potted lotus flowers have grown two upright leaves, select potted lotus flowers with good and uniform growth, and inject the bacterial suspension described in this invention around the root zone of the lotus flowers using a syringe. Inject 150 mL per pot once a day for 3 consecutive days. After treatment, a solution with an environmental salt and alkali concentration ≤75 mmol·L⁻¹ can be obtained. -1 At that time, potted mature lotus plants showed varying degrees of increase in plant height, root length, number and area of ​​leaves, leaf chlorophyll and biomass, and enhanced salt and alkali tolerance. The salt and alkali was NaCl:NaHCO3=2:1.

[0017] A method for promoting the growth and cadmium tolerance of potted water lilies includes the following steps: Selecting robust water lily tubers of uniform size and planting them in white plastic pots with a diameter of 23 cm and a volume of 7 L filled with bottom soil, one tuber per pot; providing normal water and fertilizer management during the water lily growth period; after one month of cultivation, selecting water lilies of similar size and uniform growth, and injecting the bacterial suspension described in this invention around the root zone of the water lilies using a syringe, once every 2 days, with 150 mL injected per pot each time, for a total of 3 inoculations; after treatment, a cadmium concentration ≤100 μmol·L⁻¹ in the environment can be obtained. -1 Potted water lilies exhibit increased leaf number and chlorophyll content, along with enhanced cadmium tolerance.

[0018] The present invention differs from the prior art in that: 1. The *Clostridium mangenotti* L985 provided by this invention is the first strain isolated from lotus roots, stems, and leaves. Routine physiological and biochemical characterization tests and 16S rDNA sequencing of this strain confirmed it to be a novel anaerobic microorganism, *Clostridium mangenotti*, and named it *Clostridium mangenotti* (…). Metaclostridioides mangenotii L985. To date, there have been no reports of Clostridium mangenotti being isolated from plants. L985 is an anaerobic bacterium, which is more adapted to the hypoxic underwater environment than common aerobic growth-promoting bacteria, and is more suitable for the cultivation of aquatic plants.

[0019] 2. The *Clostridium mangenotti* L985 provided by this invention possesses growth-promoting properties such as ACC deaminase activity, IAA secretion, nitrogen fixation, and phosphorus solubilization. Simultaneously, L985 exhibits tolerance to high concentrations of salt, alkali, and cadmium, enabling it to survive strongly in saline-alkali or cadmium-stressed environments. To date, there have been no reports of *Clostridium mangenotti* possessing growth-promoting abilities, as well as high tolerance to salt, alkali, and cadmium.

[0020] 3. The *Clostridium mangenotti* L985 provided by this invention can effectively promote the growth of aquatic plants as an endophytic growth-promoting bacterium; at the same time, L985 can enhance the growth and stress resistance of aquatic plants under saline-alkali or cadmium stress. To date, there have been no reports on *Clostridium mangenotti* promoting plant growth and improving plant tolerance to saline-alkali or cadmium.

[0021] 4. This invention provides a strain L985 that can promote lotus growth and improve lotus's salt and alkali tolerance. When this strain is inoculated into lotus seedlings and mature plants, it can effectively promote the growth of lotus seedlings and mature plants, significantly alleviate the damage of salt and alkali stress to lotus, and significantly increase the biomass of lotus under salt and alkali stress and maintain ion balance. It can be applied to lotus cultivation to improve yield and stress resistance.

[0022] 5. This invention provides a strain L985 that can promote the growth of water lilies and improve their cadmium tolerance. When this strain is inoculated into water lilies, it can effectively promote the growth of water lilies and significantly alleviate the damage caused by cadmium stress. It can be applied to water lily cultivation to promote their growth and stress resistance.

[0023] The anaerobic lotus endophytic growth-promoting bacterium L985 of the present invention will be further described below with reference to the accompanying drawings. Attached Figure Description

[0024] Figure 1 This is a diagram showing the growth status of strain L985 on Assumption medium in an embodiment of the present invention. Figure 2 This is a diagram showing the growth status of strain L985 on inorganic phosphorus medium in an embodiment of the present invention. Figure 3This is a diagram illustrating the activity identification of IAA production by strain L985 in this embodiment of the invention. Figure 4 This is a diagram showing the effect of strain L985 on the leaf growth of hydroponic lotus seedlings under normal conditions and salt-alkali stress in this embodiment of the invention. Figure 5 This is a diagram showing the effect of strain L985 on the leaf growth of potted mature lotus under normal conditions and salt-alkali stress in the embodiments of the present invention. Figure 6 This is a diagram showing the effect of strain L985 on the leaf growth of potted water lilies under normal conditions and cadmium stress in an embodiment of the present invention. Detailed Implementation

[0025] Example 1: Screening and identification of L985, an endophytic growth-promoting bacterium in lotus.

[0026] 1. Experimental materials and reagents

[0027] (1) Collection of plant samples

[0028] The plant materials were obtained from the Baima Lotus Base of Nanjing Agricultural University in Jiangsu Province. The lotus roots, stems and leaves were stored in a -80℃ refrigerator and endophytic bacteria were isolated as soon as possible.

[0029] (2) Preparation of culture medium and reagents

[0030] LB medium: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 15 g agar (solid medium), 1 L deionized water, pH 7.0-7.2, sterilized at 121 ℃ for 20 min.

[0031] PAF medium: 10 g peptone, 10 g casein hydrolysate, 1.5 g MgSO4, 1.5 g K2HPO4, 10 mL glycerol, 1 L deionized water, pH 7.0-7.2, sterilized at 121 ℃ for 20 min.

[0032] SM medium: Component 1: H3BO3 10 mg, ZnSO4·7H2O 124.6 mg, MnSO4·H2O 11.19 mg, MoO3 10 mg, CuSO4·5H2O 78.22 mg, deionized water 100 mL, refrigerated at 4 ℃ for later use. Component 2: FeSO4·7H2O 100 mg, deionized water 10 mL, refrigerated at 4 ℃ for later use. Take 0.1 mL of each of Component 1 and Component 2, and add the following reagents: KH2PO4 4 g, Na2HPO4·12H2O 15.12 g, MgSO4·7H2O 0.2 g, glucose 2 g, 50% D-gluconic acid solution 4 mL, citric acid 2 g, pH 7.0-7.2, deionized water 1 L, sterilize at 121 ℃ for 20 min.

[0033] DF medium: 1 L of SM medium, 2 g of (NH4)2SO4, 15 g of agar (solid medium), pH 7.0-7.2, sterilized at 121℃ for 20 min.

[0034] ADF medium: 1 L SM medium, 15 g agar (solid medium), pH 7.0-7.2, sterilized at 121 ℃ for 20 min, and after cooling, add 0.5 mol / L... -1 ACC (sterilized with a 0.22 nm bacterial filter) 6 mL.

[0035] Inorganic phosphorus medium: Ca3(PO4)2 5 g, MgSO4·7H2O 0.25 g, MgCl2 5 g, KCl 0.2 g, (NH4)2SO4 0.1 g, glucose 10 g, distilled water 1000 mL, agar 15 g, pH 7.0, sterilized at 121 ℃ for 20 min.

[0036] Ashube medium: glucose 10 g, CaCO3 5 g, KH2PO4 0.2 g, MgSO4·7H2O 0.2 g, NaCl 0.2 g, CaSO4·2H2O 0.1 g, distilled water 1000 mL, agar 15 g, pH 7.0, sterilized at 121 ℃ for 20 min.

[0037] NaCl solid medium: Weigh out NaCl at contents of 3%, 5%, 7%, and 9% and add it to LB solid medium. Sterilize at pH 7.0 and 121 °C for 20 min.

[0038] Alkaline solid culture medium: Adjust the alkalinity of LB solid culture medium with 2 mol / L NaOH solution to pH 8.0, 9.0, and 10.0, and sterilize at 121 ℃ for 20 min.

[0039] Salt-alkali mixed solid culture medium: Prepare LB solid culture medium containing 3% NaCl (pH=8), 5% NaCl (pH=9), 7% NaCl (pH=9), 5% NaCl (pH=10) and 7% NaCl (pH=10) by mixing NaCl solid and 2 mol / L NaOH solution, and sterilize at 121℃ for 20 min.

[0040] Cadmium solid culture medium: 200 mg·L -1 Cadmium was added to LB solid medium, pH 7.0, and sterilized at 121 °C for 20 min.

[0041] Urease activity assay medium: 1g peptone, 1g glucose, 5g NaCl, 2g KH2PO4, 6mL 0.2% phenol red solution, 20g agar, 1L distilled water, pH 6.9. Autoclave at 121℃ for 15 min. When cooled to 55℃, add one-tenth of a 20% urea solution (sterilized by filtration) to make the urea content 2% (i.e., 20g per 1000ml).

[0042] Amylase activity assay medium: 10g peptone, 3g beef extract, 5g NaCl, 2g starch, 20g agar, 1000ml distilled water, pH 7.4-7.8, sterilized at 121℃ for 20 min.

[0043] Cellulase activity assay medium: Na2HPO4 1.2g, KH2PO4 0.9g, MgSO4 0.5g, yeast extract powder 0.5g, acid-hydrolyzed casein 0.5g, Congo red 0.2g, cellulose powder 5.0g, agar 15.0g, distilled water 1000ml, pH 6.9-7.1, autoclaved at 121℃ for 15 min.

[0044] Salkowski colorimetric reagent: Slowly pour 150 mL of H2SO4 into 250 mL of deionized water. After the H2SO4 solution cools, add 7.5 mL of 0.5 M FeCl3·6H2O solution and store in a brown reagent bottle protected from light.

[0045] 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 deionized water to bring the volume to 1 L.

[0046] 2. Test methods

[0047] (1) Isolation of endophytic growth-promoting bacteria in lotus

[0048] Take 0.2 g of lotus tissue (roots, stems, and leaves), soak it in 75% alcohol for 2 min and 3% sodium hypochlorite for 10 min in a clean bench, then rinse it 5 times with distilled water. Grind the plant tissue with distilled water, then transfer it to a 2 mL centrifuge tube and centrifuge at 4 °C and 5000 rpm for 10 min. Next, take 1 mL of the supernatant and add it to 50 mL of PAF medium, then centrifuge at 30 °C and 200 rpm for 10 min. -1 After culturing under shaking conditions for 24 h, 1 mL of PAF culture medium was transferred to DF liquid medium and incubated at 30 °C and 200 r·min. -1 After culturing under shaking conditions for 24 h, 1 mL of DF culture medium was transferred to ADF liquid medium and incubated at 30°C and 200 r·min. -1 Under the specified conditions, the culture was shaken and incubated for 48 h. Finally, 1 mL of ADF culture medium was taken and diluted to 10⁻⁶. -6 Take 100 μL of the diluted solution and spread it evenly on an ADF solid culture plate. Incubate at 30 °C for 3-5 days until single colonies appear. Purify the single colonies 2-3 times on LB solid culture plates, then name and store them.

[0049] (2) Determination of nitrogen fixation and phosphorus solubility

[0050] Strain strain L985 was inoculated onto Ashby medium and incubated at 30°C for 3-5 days, during which bacterial growth was observed. Strain strain L985 was also spot-inoculated onto inorganic phosphorus solid medium and incubated at 30°C for 3-5 days; the appearance of a clear zone was then observed. (3) ACC deaminase activity assay Strain strain L985 was inoculated into LB liquid medium at 30 ℃ and 200 r·min. -1 After activating the cells under the specified conditions for 12 h, the cells were collected by centrifugation at 5000 r / min for 10 min at 4 ℃. The cells were then washed twice with SM liquid medium and transferred to ADF liquid medium. After shaking culture for 48 h, the cells were collected by centrifugation at 5000 rpm for 20 min at 4 ℃. The cells were then treated with 0.1 mol·L⁻¹... -1 Cells were washed twice with Tris-HCl (pH=7.6), centrifuged (5000 rpm, 20 min) to collect the cells, and resuspended in 600 μL of 0.1 mol·L⁻¹ solution. -1 Add 30 μL of toluene to Tris-HCl (pH=8.0) and shake rapidly for 30 s. Take two 200 μL portions of the toluene cell lysis buffer, and add 20 μL of 0.5 mol·L⁻¹ to one portion. -1One sample was vortexed with ACC, and the other was incubated without ACC. Both samples were then incubated in a water bath at 37 °C for 15 min. Then, 1 mL of 0.56 mol·L⁻¹ HCl was added, and the samples were centrifuged at 5000 r·min⁻¹ for 5 min. 1 mL of the supernatant was collected, and 800 μL of 0.56 mol·L⁻¹ HCl and 300 μL of 2,4-dinitrophenylhydrazine were added. The samples were incubated in a water bath at 30 °C for 30 min. Finally, 2 mL of 2 mol·L⁻¹ HCl was added. -1 The reaction was terminated with NaOH, and the absorbance at 540 nm was measured. Different concentration gradients of α-ketobutyric acid (α-ketobutyric acid) standards were prepared, and the absorbance at 540 nm was measured after the reaction to plot a standard curve. The ACC deaminase content was determined using the Coomassie Brilliant Blue method.

[0051] (4) IAA production determination

[0052] Strain strain L985 was inoculated into LB liquid medium containing L-tryptophan and cultured for 24 h (30 ℃, 200 r·min). -1 After centrifugation at 4°C (5000 rpm, 10 min), 1 mL of the supernatant was mixed with 3 mL of Salkowski reagent. LB liquid medium with Salkowski reagent was used as a blank control. IAA standard solutions of different concentrations were also added to the same volume of reagent. All reaction solutions were incubated at room temperature in the dark for 30 min before measuring the OD of the reaction solution. 530， IAA concentration was determined using a standard curve.

[0053] (5) Determination of salt and alkali tolerance

[0054] Strain strain L985 was inoculated onto LB agar plates subjected to three different concentrations of stress: salt stress (3% NaCl, 5% NaCl, 7% NaCl, 9% NaCl); alkali stress (pH=8, pH=9, pH=10); and salt-alkali stress (3% NaCl and pH=8, 5% NaCl and pH=9, 7% NaCl and pH=9, 5% NaCl and pH=10, 7% NaCl and pH=10). The LB agar plates were then incubated at 30°C for 3-5 days, and colony growth was observed.

[0055] (6) Determination of cadmium tolerance

[0056] Strain strain L985 was inoculated into an environment with a cadmium concentration of 200 mg·L⁻¹. -1 Place the culture medium on LB medium and incubate at 30 ℃ for 3-5 days, then observe the growth of the colonies.

[0057] (7) Morphological identification

[0058] Bacteria were inoculated onto LB solid culture plates using the four-zone streak method and incubated at 30°C for 24 h before observing the morphological characteristics of single colonies.

[0059] (8) Physiological characteristics identification

[0060] The strains were identified based on physiological characteristics such as methyl red, VP, catalase, urease, cellulase, and amylase, according to the "Manual of Systematic Identification of Common Bacteria".

[0061] (9) 16S rDNA sequencing identification

[0062] Using universal bacterial primers: PCR amplification was performed using the total DNA of the isolated strains as templates, with primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-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 for homology with the NCBI and Ezibiocloud databases.

[0063] 3. Identification results of strain L985

[0064] (1) Results of morphological and physiological characteristics identification

[0065] Colonies of strain L985 on LB agar plates are round, milky white, raised, translucent, moist, and have neat edges. The physiological and biochemical tests of strain L985 are shown in Table 1. Notably, this strain was negative in the catalase test, indicating that it is an anaerobic bacterium.

[0066]

[0067] (2) 16S rDNA sequencing identification results

[0068] The 16S rDNA sequence of strain L985 was amplified and sequenced, and the sequencing result is shown in SEQ ID NO:1 in the sequence listing. The obtained 16S rDNA sequence of strain L985 was compared with that of Clostridium mangenotti (Clostridium perfringens) on the NCBI website. The results showed that L985 was similar to Clostridium mangenotti (Clostridium mangenotti). Metaclostridioides mangenotii The highest homology was found, reaching 99.93%; comparison on the Ezibiocloud website showed that this strain was *Clostridium mangenotti* (…). Metaclostridioides mangenotii The similarity was 99.93%. Therefore, the strain obtained by screening in this invention was identified as *Clostridium mangenotti* (…). Metaclostridioides mangenotii ), named Clostridium mangenotti ( Metaclostridioides mangenotii) L985.

[0069] SEQ ID NO:1 Rank: (3) Results of identification of growth-promoting characteristics The results of the identification of the growth-promoting characteristics of the strain are shown in Table 2. Figure 1 The fact that strain L985 can grow continuously on Assumption medium indicates that it has nitrogen-fixing ability; Figure 2 The fact that strain L985 can show a clear zone on inorganic phosphorus medium indicates that it has phosphorus-solubilizing ability; Figure 3 This is a diagram of the IAA production test of strain L985.

[0070]

[0071] (4) Results of salt and alkali resistance and cadmium resistance assessment

[0072] The results of the tests on the salt, alkali, mixed salt and alkali tolerance and cadmium tolerance of strain L985 are shown in Table 3, indicating that the strain has good salt and alkali tolerance and cadmium tolerance.

[0073]

[0074] Example 2: Preparation of L985 bacterial suspension and its effects on the growth and salt tolerance of lotus seedlings.

[0075] I. Preparation of L985 bacterial suspension

[0076] Strain strain L985 was inoculated into LB liquid medium and incubated at 30 ℃ and 180 r·min. -1 After 14 h of shaking culture under the specified conditions, centrifuge at 8000 rpm for 10 min at room temperature, resuspend in deionized water, wash twice, and then resuspend again in deionized water to prepare OD200. 600 =1.0, concentration is 1×10 8 cfu·mL -1 Bacterial suspension.

[0077] II. Effects of L985 bacterial suspension on the growth and salt tolerance of lotus seedlings

[0078] 1. Experimental Materials and Methods

[0079] Select plump and round 'Weishanhu Red Lotus' lotus seeds, disinfect them with 3% sodium hypochlorite and 75% alcohol, and after hatching, place them in a 20 cm diameter, 2 L volume plastic bucket filled with deionized water for cultivation. The cultivation conditions are 30℃, 18000 Lx long day (16 h light / 8 h dark), and the water is changed every 3 days.

[0080] Once the lotus seedlings have grown 3-4 leaves, select hydroponic seedlings with uniform growth and conduct the following experimental treatments: (1) Inoculation with L985 under normal conditions (L985); (2) Inoculation with L985 under salt-alkali stress (salt-alkali + L985); (3) Salt-alkali treatment without inoculation (salt-alkali); (4) Control (CK) with no inoculation and no salt-alkali treatment. Each group has 4 replicates, and each pot is a replicate. The specific treatment method is as follows: Spray around the roots of the lotus seedlings with a concentration of 1×10 8 cfu·mL -1 15 ml of L985 bacterial suspension was sprayed once daily for 3 consecutive days. For uninoculated groups, an equal volume of deionized water was used instead. The saline-alkali treatment concentration was set at 75 mmol·L⁻¹. -1 (NaCl:NaHCO3 = 2:1), for those not treated with saline-alkali, an equal volume of deionized water was used instead. After 48 h of treatment, the biomass of lotus seedlings, leaf chlorophyll content, anthocyanin content, membrane stability index, hydrogen peroxide content, and soluble protein content were measured.

[0081] 2. Test Results

[0082] The results showed that, compared with the control, inoculation with L985 under normal conditions significantly increased the fresh weight of roots and leaves of lotus seedlings, as well as the fresh weight and dry weight of the plants (Table 4), indicating that this strain can promote the growth of lotus seedlings.

[0083] Furthermore, salt-alkali stress significantly reduced the fresh weight of various organs in lotus seedlings and the biomass of the plants, caused chlorosis and even reddening of leaves, and significantly decreased the contents of chlorophyll a, b, and total chlorophyll, while significantly increasing the contents of anthocyanins (Tables 4, 5, and 6). Figure 4 Under salt-alkali stress, inoculation with L985 significantly increased the fresh weight of lotus seedling roots, leaves, and petioles, as well as plant biomass, compared to salt-alkali stress alone. Leaf chlorosis was significantly alleviated, and the contents of chlorophyll a, b, and total chlorophyll significantly increased, while the anthocyanin content significantly decreased (Tables 4, 5, and 6). Figure 4 Therefore, inoculation with L985 greatly alleviated the toxic effects of salt-alkali stress on the growth of lotus seedlings and improved their tolerance to salt-alkali stress.

[0084]

[0085] As shown in Table 6, compared with the control, the membrane stability index of lotus seedlings under salt-alkali stress decreased significantly, while the contents of hydrogen peroxide and soluble protein increased significantly. Compared with salt-alkali treatment alone, inoculation with L985 under salt-alkali stress significantly increased the membrane stability index and soluble protein of lotus seedlings and significantly reduced the hydrogen peroxide content, effectively alleviating the oxidative damage and osmotic stress of lotus seedlings under salt-alkali stress.

[0086]

[0087] Example 3: Effects of L985 bacterial suspension on the growth and salt tolerance of potted mature lotus flowers.

[0088] 1. Experimental Materials and Methods

[0089] Using two-year-old rhizomes of the 'Weishan Lake Red Lotus' variety as material, select robust rhizomes of similar size with terminal buds and plant them in white plastic buckets with a diameter of 12.5cm and a volume of 5L containing 2.5kg of soil. Plant one rhizome in each bucket and manage water and fertilizer normally during the lotus's growth period.

[0090] After the potted lotus plants had grown two upright leaves, potted lotus plants with good and uniform growth were selected for the following experimental treatments: (1) Inoculation with L985 under normal conditions (L985); (2) Inoculation with L985 under salt-alkali stress (salt-alkali + L985); (3) Salt-alkali treatment without inoculation (salt-alkali); (4) Control (CK) with no inoculation and no salt-alkali treatment. Each treatment was repeated 4 times, with each pot as one replicate. The specific treatment method is as follows: Inject a concentration of 1×10⁻⁶ around the root zone of the lotus plant using a syringe. 8 cfu·mL -1 The L985 bacterial suspension was injected once daily, 150 mL per pot each time. Uninoculated plants were injected with an equal volume of deionized water instead. After 3 consecutive days of inoculation, the lotus flowers were treated with a concentration of 75 mmol·L⁻¹. -1 For saline-alkali treatment (NaCl:NaHCO3 = 2:1), use an equal volume of deionized water instead of saline-alkali treatment. Day 0 is the day after saline-alkali treatment, and injections at a concentration of 1×10⁻⁶ are continued every 7 days. 8 150 mL of bacterial suspension with a concentration of CFU / mL was injected three times. After 21 days of saline-alkali treatment, the following parameters were measured in potted lotus plants: plant height, root length, number and area of ​​leaves, biomass, chlorophyll content, membrane stability index, hydrogen peroxide content, proline content, soluble protein content, AsA-GSH cycle-related antioxidant content and antioxidant enzyme activity, as well as ion content.

[0091] 2. Test Results

[0092] The results showed that, compared with the control, inoculation with L985 under normal conditions significantly increased the plant height, root length, number and area of ​​upright and floating leaves of mature potted lotus plants, as well as the fresh weight of each organ (root, stem, leaf, and petiole) and the fresh and dry weight of the plant (Tables 7 and 8). Simultaneously, inoculation with L985 significantly increased the chlorophyll a, b, and total chlorophyll content of lotus leaves (Table 9). This indicates that this strain can effectively promote photosynthesis in mature potted lotus plants, thereby promoting their growth.

[0093] Furthermore, salt-alkali stress significantly reduced the height, root length, number of leaves, leaf area, fresh weight of various organs, and biomass of mature potted lotus plants. Leaves also showed chlorosis and curling, and the contents of chlorophyll a, b, and total chlorophyll were significantly decreased. These indicators, which were inhibited by salt-alkali stress, were all significantly improved after inoculation with L985 (Tables 7-9 and 10). Figure 5 It is evident that inoculation with L985 significantly alleviated the toxic effects of salt-alkali stress on photosynthetic pigments and growth in potted mature lotus plants, thereby improving their tolerance to salt-alkali stress.

[0094]

[0095] As shown in Table 10, compared with the control, after inoculation with L985 under normal conditions, there were no significant changes in the membrane stability index, hydrogen peroxide, proline, and soluble protein content of potted mature lotus. Salt-alkali treatment led to a significant decrease in the lotus membrane stability index and a significant increase in the content of hydrogen peroxide, proline, and soluble protein. At the same time, it also had a certain impact on the AsA-GSH cycle in the antioxidant network (Tables 10 and 11), indicating that salt-alkali treatment caused oxidative stress and osmotic stress.

[0096] Compared with saline-alkali treatment alone, inoculation with L985 under saline-alkali stress significantly increased the membrane stability index of mature potted lotus flowers, significantly reduced hydrogen peroxide content, and further enhanced proline and soluble protein content. Further analysis of the AsA-GSH cycle revealed that inoculation with L985 under saline-alkali stress significantly increased AsA content, AsA / DHA ratio, GSSG content, APX and GR activity, while decreasing GSH content and GSH / GSSG ratio (Tables 10 and 11). Therefore, inoculation with L985 can resist oxidative damage caused by saline-alkali treatment in mature potted lotus flowers by regulating antioxidant enzymes and antioxidants in the AsA-GSH cycle, and also effectively alleviate osmotic stress caused by saline-alkali treatment by inducing the synthesis of osmotic regulators.

[0097]

[0098] As shown in Table 12, compared with the control, inoculation with L985 under normal conditions significantly reduced the calcium content in mature potted lotus flowers. 2+ Content, but for Na + K + The content was not significantly affected; saline-alkali treatment led to an increase in Na content in lotus flowers. + Ca 2+ Content and Na + / K + The ratio increased significantly, K+ The content decreased significantly. Compared with salt-alkali treatment alone, inoculation with L985 under salt-alkali stress reduced Na content. + Ca 2+ Content and Na + / K + The ratio decreased significantly, K + The content was significantly increased; therefore, inoculation with L985 alleviated the Na+ stress caused by salt and alkali stress. + Poisoning and promoting K + Absorption and maintaining low Na + / K + The ratio and the promotion of ion balance can help mature potted lotus flowers grow normally under salt and alkali stress.

[0099]

[0100] Example 4: Effects of L985 bacterial suspension on the growth and cadmium tolerance of potted water lilies

[0101] 1. Experimental Materials and Methods

[0102] Using 'Golden Light' water lily tubers as material, select robust tubers of roughly the same size and plant them in white plastic pots with a diameter of 23cm and a volume of 7L filled with bottom soil. Plant one tuber in each pot and carry out normal water and fertilizer management during the water lily's growth period.

[0103] After one month of water lily cultivation, water lilies of similar size and growth were selected for the following experimental treatments: (1) L985 inoculation under normal conditions (L985); (2) L985 inoculation under cadmium stress (Cd+L985); (3) Uninoculated cadmium treatment (Cd); (4) Uninoculated and untreated cadmium was used as the control (CK). Each treatment was repeated 4 times, with each pot constituting one replicate. The specific treatment method is as follows: a concentration of 1×10⁻⁶ was injected around the root zone of the water lily using a syringe. 8 CFU·mL -1 L985 bacterial suspension was injected every 2 days, with 150 mL injected per pot each time. Sterile water was used instead of inoculated groups in this manner. After three consecutive inoculations, the water lilies were treated with a concentration of 100 μmol·L⁻¹. -1 Cadmium treatment: An aqueous solution of CdCl2·2.5H2O was prepared and applied to the basins. The untreated group was treated with an equal volume of sterile water. Day 0 was the day after cadmium treatment, and injections at a concentration of 1×10⁻⁶ were repeated every 7 days. 8 150 mL of bacterial suspension with a concentration of CFU / mL was injected three times. The number of leaves, stem length, and chlorophyll content (SPAD value) of potted water lilies were measured 28 days after cadmium treatment.

[0104] 2. Test Results

[0105] The results showed that, compared with the control, inoculation with L985 under normal conditions significantly increased the number of leaves in potted water lilies, effectively promoting their growth and development (Table 13).

[0106] Furthermore, compared to the control, cadmium stress significantly reduced the number and area of ​​leaves in potted water lilies, caused obvious chlorosis and even necrosis, and also significantly reduced the chlorophyll content of water lily leaves. Compared to cadmium stress alone, inoculation with L985 under cadmium stress significantly increased the number and area of ​​leaves in water lilies, significantly alleviated leaf chlorosis and necrosis, and significantly increased the chlorophyll content of water lilies (Table 13 and 14). Figure 6 Therefore, inoculation with L985 effectively reduced the toxic effects of cadmium stress on photosynthetic pigments and growth of water lilies, and improved their tolerance to cadmium stress.

[0107]

[0108] In summary, the *Clostridium mangenotti* strain provided by this invention (… Metaclostridioides mangenotii L985 possesses characteristics such as producing ACC deaminase, secreting IAA, nitrogen fixation, phosphorus solubilization, salt and alkali tolerance, cadmium tolerance, and anaerobic activity. Inoculation with strain L985 significantly promotes the growth of lotus seedlings and mature plants and enhances their resistance to salt and alkali stress; simultaneously, this strain can promote potassium absorption by mature lotus plants under salt and alkali stress. + Ions and maintain low Na + / K + The ratio promotes ion balance. Furthermore, inoculating strain L985 significantly promotes the growth of water lilies and their cadmium tolerance. This invention provides an excellent plant endophytic strain for multifunctional growth-promoting bacteria in aquatic plants, which can be applied to the preparation of multifunctional microbial fertilizers for aquatic plants such as lotus and water lilies (promoting aquatic plant growth, improving aquatic plant tolerance to salt and alkali, and enhancing cadmium tolerance).

[0109] 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. An anaerobic endophytic growth-promoting bacterium for lotus, L985, is classified as *Clostridium mangenotti* (…). Metaclostridioides mangenotii It was deposited at the China General Microbiological Culture Collection Center on June 3, 2024, with accession number CGMCC No. 30853.

2. The anaerobic lotus endophytic growth-promoting bacterium L985 according to claim 1, characterized in that: Colonies that are round, translucent, moist, with neat edges and milky white protrusions were isolated and cultured from lotus roots, stems and leaves; they are anaerobic, negative for catalase and cellulase, and positive for methyl red, VP, urease and amylase. The 16S rDNA nucleotide sequence of the anaerobic lotus endophytic growth-promoting bacterium L985 is shown in SEQ ID NO:1 in the sequence listing.

3. A bio-fertilizer, bacterial suspension, or culture medium containing the anaerobic lotus endophytic growth-promoting bacterium L985 as described in claim 1.

4. The application of the anaerobic lotus endophytic growth-promoting bacterium L985 as described in claim 1 or the bio-fertilizer, bacterial suspension or culture medium as described in claim 3 in any one of the following (1) to (12): (1) Produce IAA; (2) Prepare products that produce IAA; (3) Production of ACC deaminase; (4) Prepare products that produce ACC deaminase; (5) Promotes the growth of aquatic plants; (6) Prepare products that promote the growth of aquatic plants; (7) Alleviate the damage of salt and alkali stress to aquatic plants and promote the growth of aquatic plants under salt and alkali stress; (8) Prepare products that improve the salt and alkali tolerance of aquatic plants; (9) Salt-alkali stress reduces the absorption and accumulation of sodium ions by aquatic plants; (10) Under salt and alkali stress, aquatic plants can enhance the absorption and accumulation of potassium ions; (11) Alleviate the damage of cadmium stress to aquatic plants and promote the growth of aquatic plants under cadmium stress; (12) Prepare products that improve the cadmium tolerance of aquatic plants.

5. The application according to claim 4, characterized in that: The aquatic plant is either lotus or water lily; Inoculation with L985 can promote the growth of lotus seedlings, potted mature lotus, and potted water lilies, increase the biomass and chlorophyll content of lotus leaves, and increase the number of leaves and chlorophyll content of water lilies. Inoculation with L985 under salt-alkali stress can alleviate the inhibition of plant height, root length, number of leaves and leaf area of ​​potted mature lotus under salt-alkali stress, significantly increase the biomass, chlorophyll content, membrane stability index and soluble protein content of lotus seedlings and potted mature lotus, and reduce hydrogen peroxide content, thereby enhancing the salt-alkali tolerance of lotus. Inoculation with L985 under salt-alkali stress significantly inhibited the Na+ oxidative stress of mature potted lotus flowers. + Absorption and accumulation, enhancing K + Absorption and accumulation, maintaining a low Na + / K + The ratio promotes ion balance, thereby promoting the growth and development of lotus under salt and alkali stress; Inoculation with L985 under cadmium stress can alleviate the inhibition of leaf number and leaf area caused by cadmium stress in potted water lilies, and significantly increase the chlorophyll content of leaves, thereby enhancing the cadmium tolerance of water lilies.

6. The application according to claim 4, characterized in that: The method for preparing ACC deaminase includes the following steps: inoculating strain L985 into LB liquid medium at 30 °C and 200 r·min. -1 Activation was achieved by incubation for 12 h at 4 ℃ and 5000 r·min. -1 After centrifuging for 10 min to collect the bacterial cells, wash them twice with SM liquid medium and transfer them to ADF liquid medium. After shaking culture for 48 h, collect the induced culture product to obtain ACC deaminase. The method for preparing IAA includes the following steps: inoculating strain L985 into LB liquid medium containing L-tryptophan, and incubating at 30 °C and 200 r·min. -1 After 24 hours of incubation, the induced culture product was collected, which yielded IAA.

7. The method for preparing the bacterial suspension containing anaerobic lotus endophytic growth-promoting bacteria L985 as described in claim 3, characterized in that: The steps include: inoculating strain L985 into LB liquid medium and incubating at 30 °C and 180 r·min. -1 After 14 h of shaking culture under the specified conditions, centrifuge at 8000 rpm for 10 min at room temperature, resuspend in deionized water, wash twice, and then resuspend again in deionized water to prepare OD200. 600 =1.0, concentration is 1×10 8 cfu·mL -1 Bacterial suspension.

8. A method for promoting the growth and salt tolerance of hydroponically grown lotus seedlings, characterized in that: The steps include: selecting plump lotus seeds, surface disinfecting them with 3% sodium hypochlorite and 75% alcohol, and after hatching, placing them in a 20 cm diameter, 2 L volume plastic bucket filled with deionized water for cultivation. The cultivation conditions are 30 ℃, 18000 Lx long-day, 16 h light / 8 h dark, with water changed every 3 days. When the lotus seedlings have grown 3-4 leaves, select hydroponic seedlings with uniform growth, and spray 15 ml of the bacterial suspension described in claim 7 around the roots of the seedlings once a day for 3 consecutive days. After treatment, a solution with an environmental salt concentration ≤75 mmol·L⁻¹ can be obtained. -1 At that time, the biomass and leaf chlorophyll content of lotus seedlings increased, and their salt and alkali tolerance was enhanced. The salt and alkali was NaCl:NaHCO3=2:

1.

9. A method for promoting the growth and salt tolerance of mature potted lotus flowers, characterized in that: The process includes the following steps: Select robust, uniformly sized, two-year-old lotus rhizomes with terminal buds, and plant them in white plastic buckets with a diameter of 12.5 cm and a volume of 5 L containing 2.5 kg of soil. Plant one rhizome per pot, and provide normal water and fertilizer management during the lotus growth period. After the potted lotus plants have grown two upright leaves, select potted lotus plants with good and uniform growth, and inject the bacterial suspension described in claim 7 around the base of the lotus plants using a syringe. Inject once a day, 150 mL per pot each time, for 3 consecutive days. After treatment, a solution with an environmental salt and alkali concentration ≤75 mmol·L can be obtained. -1 At that time, potted mature lotus plants showed varying degrees of increase in plant height, root length, number and area of ​​leaves, leaf chlorophyll and biomass, and enhanced salt and alkali tolerance. The salt and alkali was NaCl:NaHCO3=2:

1.

10. A method for promoting the growth and cadmium tolerance of potted water lilies, characterized in that: The steps include: selecting healthy, uniformly sized water lily tubers and planting them in white plastic pots with a diameter of 23cm and a volume of 7L filled with bottom soil, one tuber per pot; providing normal water and fertilizer management during the water lily growth period; after one month of cultivation, selecting water lilies of similar size and uniform growth, and injecting the bacterial suspension described in claim 7 around the root zone of the water lilies using a syringe, once every 2 days, 150mL per pot each time, for a total of 3 inoculations; after treatment, a solution with an environmental cadmium concentration ≤100 μmol·L⁻¹ can be obtained. -1 Potted water lilies exhibit increased leaf number and chlorophyll content, along with enhanced cadmium tolerance.

Images