A salt-tolerant liuzhenghensis strain and its application in promoting growth of phragmites australis

Abstract

This invention discloses a salt-tolerant strain of *Liu Zhiheng* and its application in promoting the growth of wetland reeds, belonging to the field of microbial technology. The salt-tolerant *Liu Zhiheng* strain has the preservation number CGMCC No. 38351. This strain possesses multiple growth-promoting functions, including IAA production, siderophore production, and phosphate solubilization. Experiments show that this strain can significantly promote reed seed germination and seedling growth, increasing plant height and aboveground dry weight under normal conditions; under salt stress, proline content increases, as do MDA and Na content. + / K + The ratio decreased; under combined salt and dust stress, it significantly improved photosynthetic efficiency and alleviated stress damage. Wetland simulation verification showed that the survival rate of this strain reached 42.5% on day 28 under flooded and hypoxic conditions, and its vitality-promoting effect was significantly better than that of the control strain. This invention also provides a bacterial agent preparation scheme and standardized application method, providing a complete technical solution for the growth of dominant wetland plants and ecological restoration.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a salt-tolerant strain of *Liu Zhiheng* and its application in promoting the growth of wetland reeds. Background Technology

[0002] Zhihengliuella halotolerans It is a salt-tolerant strain with multiple growth-promoting functions. Existing studies have shown that this strain exhibits good plant growth-promoting effects in arid / desert habitats. Research by Najafi Zilaie et al. found that... Z. halotolerans SB strain inoculated with desert halophyte Nitraria tangutorum (Syngonium nitidae) Nitraria tangutorum After [a certain event], under 300 mmol / L NaCl stress, total dry biomass increased by 31%, and chlorophyll a content increased by 25%. In halophytes... Seidlitzia rosmarinus Studies have also confirmed that this strain can increase its dry biomass by 30% and proline content by 74% under dust stress conditions. Furthermore, isolates from the rhizosphere of halophytes... Z. halotolerans When applied to wheat, it exhibits multiple growth-promoting properties, including IAA production, siderophore production, ACC deaminase activity, and phosphate solubilization. It can significantly increase chlorophyll content, proline content, and antioxidant enzyme activity in wheat leaves under salt stress. This phosphate-solubilizing bacterium was isolated from the rhizosphere of halophytes in the saline-alkali land of the Qaidam Basin. Z. halotolerans The P2 strain possesses the ability to dissolve both organic and inorganic phosphorus, and it promoted rapeseed germination under salt stress.

[0003] In wetland plants reeds ( Phragmites australis In the research on microbial growth promotion of bacteria, existing technologies mainly focus on the application of other bacterial species. For example, salt-tolerant rhizosphere bacteria. Glutamicibacter sp. Inoculation with dark-colored septate endophytic fungi (DSE) can alleviate salt stress damage in reeds by producing extracellular polysaccharides, and can increase the biomass and antioxidant enzyme activity of reed seedlings. Furthermore, US Patent 10721936B2 discloses endophytic bacteria isolated from reed seeds (…). Pseudomonas spp. , Microbacterium spp. Applications of (etc.) in grasses. However, Zhihengliuella halotolerans This strain, which has abundant growth-promoting properties, has not yet been applied to reeds.

[0004] Will Zhihengliuella halotolerans The application of reeds in dryland / desert habitats to wetland plants faces objective technical obstacles. Firstly, the rhizosphere microenvironment of wetlands differs fundamentally from that of drylands: the oxygen transfer rate in wetland systems is approximately one ten-thousandth that of drylands, exhibiting a complex characteristic of coexistence of aerobic, anoxic, and anaerobic microzones. Zhihengliuella halotoleransAs aerobic bacteria, their survival and function in wetland environments are difficult to predict directly. Secondly, reed root exudates are mainly composed of organic acids such as oxalic acid and citric acid, which are significantly different from those of dryland plants, potentially affecting the chemotactic colonization and functional expression of the strain. Furthermore, it remains uncertain whether the growth-promoting metabolic pathways of dryland-derived PGPR can maintain their activity under hypoxic conditions in the rhizosphere of wetlands.

[0005] Therefore, the existing Zhihengliuella halotolerans The growth-promoting effect on dryland crops cannot be sufficient evidence for its similar effectiveness on wetland plants like reeds. There is an urgent need to develop methods that can be effectively applied to reeds to achieve wetland ecological restoration. Zhihengliuella halotolerans Strains and related technical solutions. Summary of the Invention

[0006] The purpose of this invention is to provide a salt-tolerant strain of *Liu Zhiheng* and its application in promoting the growth of wetland reeds, in order to solve the problems existing in the prior art. This strain can significantly promote the growth of wetland reeds under combined salt and dust stress conditions, which is of great significance for promoting the growth of wetland plants in semi-arid and degraded wetlands and improving their stress resistance.

[0007] To achieve the above objectives, the present invention provides the following solution: This invention provides a salt-tolerant strain of Liu Zhiheng Bacterium ( Zhihengliuella halotolerans The salt-tolerant Liu Zhiheng bacteria has the preservation number CGMCC No.38351.

[0008] This invention also provides the use of the salt-tolerant Liu Zhiheng bacteria in any of the following: (1) Application in promoting the growth of wetland reeds; (2) Application in promoting the growth of wetland reeds under combined salt and dust stress; (3) Application in improving the survival and establishment ability of reeds in wetland flooding environments.

[0009] Preferably, the promotion of wetland reed growth refers to promoting reed seed germination and promoting reed seedling growth.

[0010] Preferably, the growth promotion is to promote the growth of wetland reed seedlings under combined salt and dust stress.

[0011] Preferably, the salt-tolerant Liu Zhiheng bacteria are applied in the form of bacterial suspension, liquid bacterial agent or solid bacterial agent.

[0012] Preferably, the concentration of the salt-tolerant Liu Zhiheng bacteria in the bacterial suspension or liquid bacterial agent is 1×10⁻⁶. 6 CFU / mL ~ 1×10 9 CFU / mL.

[0013] Preferably, the solid microbial agent is obtained by mixing peat moss and liquid microbial agent at a mass ratio of 2:1 and then drying it until the moisture content is less than 10%.

[0014] The present invention also provides a method for promoting the growth of wetland reeds under combined salt and dust stress, comprising the step of treating wetland reeds with the aforementioned salt-tolerant Liu Zhiheng bacteria; wherein the treatment method includes one or more combinations of seed soaking, root irrigation, and foliar spraying.

[0015] Preferably, the salt-tolerant Liu Zhiheng bacteria are in the form of bacterial suspension, liquid bacterial agent or solid bacterial agent.

[0016] The present invention also provides a microbial agent, comprising the aforementioned salt-tolerant Liu Zhiheng bacteria and a carrier, wherein the microbial agent is in the form of a liquid agent or a solid agent; The concentration of the liquid bacterial agent is 1×10⁻⁶. 9 CFU / mL, the solid inoculant is obtained by mixing peat moss and liquid inoculant at a mass ratio of 2:1 and then drying until the moisture content is less than 10%.

[0017] The present invention discloses the following technical effects: (1) The salt-tolerant Liu Zhiheng fungus was applied to wetland plants for the first time, filling the gap in homologous inoculation technology. Current research on the application of salt-tolerant Liu Zhiheng bacteria is highly limited to dryland crops (wheat) and desert halophytes (Nitraria tangutorum, etc.). Seidlitzia rosmarinus This invention has not yet covered wetland plants such as reeds. Furthermore, existing rhizosphere screening of growth-promoting bacteria (such as nitrogen-fixing bacteria and phosphate-solubilizing bacteria) obtained from reeds has not been further applied to the reeds themselves, resulting in a disconnect between screening and application. This invention is the first to apply this strain to reeds, utilizing the "homological matching" co-adaptation advantage between the strain and the host in saline-alkali environments, filling the technological gap in the application of this strain in wetland halophytes and in the field of homologous inoculation of reeds.

[0018] (2) The strain has growth-promoting function Existing salt-tolerant growth-promoting bacteria in the rhizosphere of reeds mostly possess only single or a few functions (nitrogen fixation, phosphorus solubilization, IAA production, etc.), and practical applications often require the combination of multiple strains, increasing complexity. The salt-tolerant Liu Zhiheng bacterium used in this invention integrates multiple functions such as IAA production (51.06 mg / L), siderophore production (77.17%), and phosphorus solubilization (3.10 mg / L), and can synergistically promote reed growth from multiple levels such as hormone regulation, nutrient activation, and stress relief, avoiding the cumbersome combination of multiple strains.

[0019] (3) Complete microbial agent preparation schemes and standardized application methods are provided to facilitate industrialization. Existing research mainly focuses on exploratory work involving direct inoculation of bacterial suspensions in the laboratory, lacking standardized inoculation methods and inoculation protocols. This invention provides specific preparation protocols for liquid inoculation agents (containing 10% glycerol protectant) and solid inoculation agents (peat carrier, water content <10%), and establishes a soaking method for reeds (1×10⁻⁶). 8 CFU / mL, soaking for 2-4 hours), root irrigation method (1×10 8 Three standardized application methods—CFU / mL (20-50 mL per plant) and spraying—provide a complete technical solution for moving from laboratory to engineering applications.

[0020] (4) Provide effective microbial technologies for the ecological restoration of arid and semi-arid regions and degraded wetlands. Arid and semi-arid regions and degraded wetlands face multiple problems such as drought, salinization, and vegetation degradation, leading to a weakening of their carbon sequestration function. This invention, using reed as a representative species, systematically verified the growth-promoting effects of a strain under normal conditions and salt stress (increasing plant height by 24.1% and aboveground dry weight by 31.0%), providing a complete technical solution encompassing strain screening, functional verification, inoculant preparation, and application methods. The strain's indigenous characteristics give it better environmental adaptability, overcoming the challenge of low survival rates when transplanted from exogenous strains.

[0021] (5) Possesses unique functions to cope with the combined stress of saline-alkali and dust. In addition to salt-alkali stress, arid and semi-arid regions often experience frequent dust activity and strong winds, which can clog stomata and reduce photosynthetic efficiency. Most existing salt-tolerant growth-promoting bacteria only address single salt-alkali stresses. The strain of this invention has been verified to significantly improve the net photosynthetic rate, stomatal conductance, transpiration rate, and relative leaf water content of reeds under combined stress, alleviating damage from combined stress and improving biomass and seedling quality. This dual function of "salt-tolerant growth promotion + dust-resistant photosynthetic enhancement" is a unique advantage not possessed by conventional salt-tolerant growth-promoting bacteria.

[0022] (6) Validation of strain adaptability under simulated wetland environment To address the limitations of existing methods for validating salt-tolerant *P. pekinensis* strains under aerobic conditions in drylands, this invention specifically designed a wetland flooding simulation experiment. Results showed that on day 28 post-inoculation, the strain survival rate remained above 42%, and IAA production (18.5 mg / L) and siderophore activity (48.5%) remained at high levels, significantly superior to the control strain DSM 17364T (survival rate 35.5%, IAA 10.8 mg / L). These data demonstrate that the strain of this invention overcomes the technical obstacles of difficult colonization and functional degradation of *PGPR* in hypoxic wetland environments, achieving a technological leap into the field of wetland ecological restoration. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 for Zhihengliuella halotolerans Image of a single colony culture of the strain on a culture medium; Figure 2 Phylogenetic tree; Figure 3 This is the IAA standard curve; Figure 4 For qualitative testing of iron production capacity; Figure 5 For qualitative testing of phosphorus solubility; Figure 6 This is a standard curve for phosphorus solubility. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0028] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0029] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0030] Example 1: Screening, Identification and Biological Function Determination of Strains 1. Strains Isolation The present invention Zhihengliuella halotolerans The strain was isolated from saline-alkali soil samples in Daqing City, Heilongjiang Province, by our research group. The strain number is NEFU-LAWJH-001. The specific isolation method was as follows: 10 g of saline-alkali soil sample was weighed, added to 90 mL of sterile physiological saline, and shaken at 28℃ and 180 r / min for 30 min. After standing, the supernatant was serially diluted (10⁻⁶ ppm). -1 ~10 -6 ); Take 100 μL of soil suspension at each dilution and spread it on LB solid agar plates with pH 8 and 2% salinity. Incubate upside down in a constant temperature incubator at 28℃ for 48 h. Pick single colonies with different morphologies and repeatedly streak them on LB solid plates for purification more than 3 times to obtain pure culture strains.

[0031] 2. Morphological characteristics and molecular identification of the strain The NEFU-LAWJH-001 strain was streaked onto LB solid plates and incubated upside down in a 28°C incubator for 24-48 h. Colony morphology was then observed.

[0032] like Figure 1 As shown, the results indicate that the colonies are opaque, round, pale yellow, approximately 1 mm in diameter, with regular edges and a smooth, moist surface. The bacterial cells are short rod-shaped and Gram-positive.

[0033] Genomic DNA was extracted from strain NEFU-LAWJH-001 and amplified by PCR using the universal 16S rDNA primers 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.1); 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO.2). The amplified products were then sequenced.

[0034] Blast analysis was performed on the sequencing results and compared with known sequences in the NCBI database. The results showed that the NEFU-LAWJH-001 strain was similar to... Zhihengliuella halotolerans The 16S rDNA sequence of the type strain YIM 70185ᵀ (DSM 17364ᵀ) showed over 99.85% homology. Based on this, a phylogenetic tree was constructed, see [link to phylogenetic tree]. Figure 2Morphological characteristics and molecular identification results confirmed that the NEFU-LAWJH-001 strain of this invention belongs to the Micrococcidales suborder of the Micrococcidae family, and is a salt-tolerant strain of *Liu Zhiheng*. Zhihengliuella halotolerans This strain was deposited on April 21, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 38351.

[0035] 3. Salt tolerance test of the strain LB liquid medium containing different concentrations of NaCl (0, 50, 100, 200, 300 mmol / L) was prepared, and the pH was adjusted to 7.0. The bacterial suspension was inoculated into the above medium at a 1% (v / v) inoculation rate, and cultured at 28℃ with shaking at 180 r / min for 24 h. The OD of each treatment group at 600 nm was measured. 600 The absorbance values ​​were used to plot the growth curves of the strain at different salt concentrations. The results are shown in Table 1.

[0036] Table 1. Salt tolerance test results of NEFU-LAWJH-001 strain Note: Different lowercase letters in the same row indicate significant differences between treatments (P<0.05). Duncan's method was used for multiple comparisons.

[0037] The strain grows well in the range of 0–200 mmol / L NaCl, with the optimal growth salinity being 100 mmol / L NaCl (OD). 600 (The value can reach above 0.65); growth activity (OD) can still be detected under 300 mmol / L NaCl conditions. 600 ≥0.25).

[0038] 4. Determination of IAA production capacity of the strain The activated NEFU-LAWJH-001 strain was inoculated into LB liquid medium containing 100 mg / L L-tryptophan and cultured at 28℃ with shaking at 180 r / min for 3 days. 1 mL of culture medium was centrifuged at 4℃ and 10000 r / min for 10 min, and the supernatant was collected. 0.5 mL of the supernatant was added to 1 mL of Salkowski colorimetric solution (35% HClO4 solution containing 0.5 mol / L FeCl3), and incubated at room temperature in the dark for 30 min. The uninoculated culture medium supernatant served as a blank control. [The text abruptly ends here, likely due to an incomplete sentence or missing information.] 530 The absorbance value was measured at the specified wavelength. The IAA content in the supernatant was calculated based on the IAA standard curve.

[0039] IAA standard curve plotting: Standard curves were plotted using 10, 20, 50, and 100 μg / mL IAA standard solutions. Results are shown below. Figure 3 .

[0040] The IAA standard curve is obtained as y = 0.032 + 0.0139x, RA 2 = 0.9927. The experiment yielded IAA production results for strain NEFU-LAWJH-001, OD... 530 The value was 0.74168±0.00514. After calculation, the IAA yield was 51.06±0.37 mg / L.

[0041] 5. Determination of the strain's ability to produce siderophores (1) Qualitative detection of siderogenetic agents: The NEFU-LAWJH-001 strain was inoculated onto CAS solid medium and cultured at 28℃ for 48-72 h. The presence of an orange-yellow transparent ring around the colony was observed. Each L of CAS solid medium contained: 60.5 mg of Chromium Azurite S, 2.7 mg of FeCl3·6H2O, 72.9 mg of hexadecyltrimethylammonium bromide (HDTMA), and 15-18 g of agar. It was added to MM9 basal salt medium at pH 7.0.

[0042] (2) Quantitative detection: The NEFU-LAWJH-001 strain was inoculated into MKB iron-limited medium and cultured at 28℃ and 180 r / min for 3 days with shaking. The culture medium was centrifuged to obtain the supernatant. 0.5 mL of the supernatant was mixed with 0.5 mL of CAS detection solution (same as the CAS solid medium formula, without agar). The mixture was incubated at room temperature in the dark for 30 min, and the OD was measured. 630 Using uninoculated MKB iron-limited medium supernatant as a control, siderophore activity units were calculated. Each 1 L of MKB iron-limited medium contained: 5 g casein amino acids, 2.5 g K2HPO4·3H2O, 2.5 g MgSO4·7H2O, 15 mL glycerol, and pH 7.2.

[0043] like Figure 4 As shown, strain NEFU-LAWJH-001 was able to grow on CAS solid plates, and the qualitative detection on CAS plates was positive. The ratio of the diameter of the clear zone to the diameter of the colony (D / d) was ≥2.4, proving that it has the ability to produce siderophores. The quantitative detection of siderophore activity units (SU) was 77.17% ± 1.7%.

[0044] 6. Determination of the phosphorus solubilization ability of the strain (1) Qualitative detection: The NEFU-LAWJH-001 strain was inoculated onto inorganic phosphorus solid medium (PKO medium) plates and cultured at 28℃ for 5-7 days. The appearance of the clear zone was observed. Each 1 L of PKO medium contained: 10 g glucose, 2.5 g Ca3(PO4)2, 0.5 g (NH4)2SO4, 0.2 g NaCl, 0.2 g KCl, 0.03 g MgSO4·7H2O, 0.03 g MnSO4, 0.003 g FeSO4·7H2O, 0.5 g yeast extract, 15-18 g agar, and pH 7.0-7.2.

[0045] (2) Quantitative detection: The NEFU-LAWJH-001 strain was inoculated into liquid inorganic phosphorus medium (same formula as PKO inorganic phosphorus solid medium, without agar) and cultured at 28℃ and 180 r / min for 5 days with shaking. The culture medium was centrifuged and the supernatant was collected. The soluble phosphorus content in the supernatant was determined by the molybdenum antimony colorimetric method. The amount of phosphorus solubilized was calculated using the uninoculated medium as a blank control.

[0046] Experimental results: such as Figure 5 As shown, a distinct transparent ring can be seen forming on the PKO inorganic phosphorus plate.

[0047] Quantitative detection experiments, such as Figure 6 As shown, the standard curve obtained is y = -0.0166 + 0.220 x, R0 2 =0.99937. The OD value of the NEFU-LAWJH-001 strain was obtained experimentally. 700 The value was 0.79357±0.00215. After calculation, the phosphorus solubility was 3.095±0.00811 mg / L, proving that the NEFU-LAWJH-001 strain has a certain phosphorus solubility.

[0048] Example 2: Fermentation culture of bacterial strains and preparation of bacterial suspension (1) Activation: Take out the cryopreservation tube of NEFU-LAWJH-001 strain stored in an ultra-low temperature freezer at -80℃ and thaw it slowly at 4℃. In a clean bench, use an inoculation loop to dip the frozen bacterial solution and streak it onto LB solid plate medium. Incubate in an inverted incubator at 28℃ for 24-48 h.

[0049] (2) Seed culture preparation: Activated single colonies were picked and inoculated into 250 mL Erlenmeyer flasks containing 50 mL of LB liquid medium. The flasks were then incubated at 28℃ and 180 r / min in a constant-temperature shaker for 18-24 h until the logarithmic growth phase (OD200) was reached. 600 The concentration is approximately 0.8-1.0%, which is the seed solution.

[0050] (3) Expanded culture: The seed culture was transferred to LB liquid medium at an inoculation rate of 2% (volume ratio) and expanded cultured in a constant temperature shaker at 28℃ and 180 r / min until the logarithmic growth phase.

[0051] (4) Preparation of bacterial suspension: The fermentation broth was centrifuged at 8000 r / min for 10 min at 4℃, and the bacterial precipitate was collected. The precipitate was washed twice with sterile physiological saline or phosphate buffer and resuspended to prepare a bacterial suspension. The concentration of the bacterial suspension was determined by measuring the OD value. 600 The concentration is calibrated using the plate count method or the microbial count method, and adjusted to the required concentration (usually about 1 × 10⁻⁶). 8 (CFU / mL), used for subsequent inoculation experiments.

[0052] Example 3: Germination Experiment of Reed Seed Inoculation 1. Treatment of reed seeds Select plump, disease-free reed seeds and rinse them under running water for 1 hour to remove surface impurities. Soak the seeds in a 5% NaClO solution for 5 minutes and rinse them 3-5 times with sterile water. Place the sterilized seeds on sterile moist filter paper and germinate them at 28℃ and 60% humidity for 2-3 days until they show signs of germination.

[0053] 2. Inoculum suspension soaking treatment Take approximately 1 × 10⁻⁶ of the NEFU-LAWJH-001 strain suspension prepared in Example 2. 8 (CFU / mL) Seeds in the experimental group were soaked in bacterial suspension for 2-4 h, while seeds in the control group were soaked in sterile water for the same amount of time. Double-layered filter paper was laid flat in a petri dish and thoroughly moistened with bacterial suspension (T group) or uninoculated / sterile water (CK group). The soaked seeds were then evenly arranged on the moistened filter paper, 20 seeds per dish, with 3 replicates per group.

[0054] 3. Germination culture and observation records The petri dishes were covered and placed in an artificial climate chamber for cultivation under the following conditions: temperature 25℃ / 20℃ (day / night), photoperiod 14 h / 10 h, and relative humidity 60%. Germination was observed and recorded daily starting from day 2, with the germination rate calculated as the radicle breaking through the seed coat by 1 mm. On day 7, the final germination rate, germination potential, and germination index were calculated. On day 14, the stem and root lengths of the seedlings were measured (accurate to mm), and the fresh and dry weights were determined (sterilized at 105℃ for 30 min, then dried at 80℃ to constant weight). The results are shown in Table 2.

[0055] 4. Experimental Results Table 2. Germination experiment of reed seeds Compared with the control group, the germination rate of reed seeds treated with the NEFU-LAWJH-001 strain of the present invention increased by 12.4%, germination potential increased by 15.9%, and germination index increased by 18.3%. Seedling stem length increased by 23.1%, root length increased by 18.4%, seedling fresh weight increased by 25.2%, and dry weight increased by 27.6% (p<0.05). The results indicate that the NEFU-LAWJH-001 strain of the present invention has a significant promoting effect on reed seed germination and early seedling growth.

[0056] Example 4: Experiment on promoting the growth of reed seedlings in pots (normal conditions) 1. Experimental Design This embodiment verifies the growth-promoting effect of the NEFU-LAWJH-001 strain on reed seedlings under salt-free stress conditions. Two treatment groups were set up: one group inoculated with the NEFU-LAWJH-001 strain (T group) and the other group uninoculated / sterile water (CK group). Each treatment was replicated in 5 replicates (plants).

[0057] 2. Experimental Procedure (1) Seedling raising: Disinfected reed seeds are sown in seedling trays containing a mixture of vermiculite and peat moss in a 1:2 ratio (by volume). The seeds are then raised in a greenhouse until they reach the 2-3 leaf stage. During this period, Hoagland nutrient solution is regularly applied to maintain normal growth. (2) Transplanting: Select reed seedlings with uniform growth and transplant them into flower pots filled with sterilized substrate; (3) Inoculation: A bacterial suspension of NEFU-LAWJH-001 strain (approximately 1×10⁻⁶) was prepared using the method described in Example 2. 8 (CFU / mL). A root drenching inoculation method was used: each plant in group T was drenched with 20 mL of bacterial suspension, and each plant in group CK was drenched with an equal volume of sterile water. A second inoculation was performed on the 7th day after transplanting to enhance the colonization effect of the strain in the rhizosphere of reeds; (4) Cultivation and management: Irrigate with Hoagland nutrient solution every 3-4 days to keep the soil moisture content at about 70% of field capacity.

[0058] 3. Growth index measurement After the final harvest, the following indicators were measured. The specific experimental methods and measurement time points are shown in Table 3, and the measurement results are shown in Table 4.

[0059] Table 3. Determination of experimental indicators and methods for promoting growth of potted reed seedlings (under normal conditions) 4. Experimental Results Table 4. Results of the experiment on promoting the growth of reed seedlings in pots (under normal conditions) Compared with the control group, the reed plants treated with the strain of this invention showed an increase in plant height of 24.1%, stem diameter of 20.0%, aboveground fresh weight of 28.6%, underground fresh weight of 30.0%, aboveground dry weight of 31.0%, underground dry weight of 40.0%, chlorophyll content of approximately 32.0%, and root length of approximately 20.5% (p<0.05). The results indicate that under normal growth conditions, inoculation with the NEFU-LAWJH-001 strain of this invention can significantly promote the growth and development of reed seedlings.

[0060] Example 5: Potted plant growth promotion experiment of reed seedlings (salt stress conditions) 1. Experiment on promoting growth of reed seedlings in pots under salt stress The effect of NEFU-LAWJH-001 strain on promoting stress resistance in reed seedlings under salt stress was verified. A two-factor, completely randomized block design was used. The inoculation treatments were NEFU-LAWJH-001 strain inoculation (T) and no-inoculation control group (CK). Salt stress conditions were: no salt stress (0 mmol / L NaCl); moderate salt stress (100 mmol / L NaCl); and severe salt stress (200 mmol / L NaCl).

[0061] Four treatments were set up after combination: T100, T200, CK100, and CK200. CK100 represents the group without +100 mmol / L NaCl treatment, T100 represents the group treated with +100 mmol / L NaCl, CK200 represents the group without +200 mmol / L NaCl treatment, and T200 represents the group treated with +200 mmol / L NaCl. Each treatment was set up in 5 replicates. Data from the 0 mmol / L NaCl treatment (no salt stress) group and the control group were directly quoted from the results of Example 4.

[0062] 2. Experiment on promoting growth of reed seedlings under combined salt and dust stress in pots 2.1 Verification of the promoting effects of NEFU-LAWJH-001 strain on photosynthesis, water balance, and growth of reed seedlings under combined salt-alkali and dust stress. 100 mmol / L NaCl was selected as the representative salt stress concentration. In addition to salt stress, a dust stress treatment group was added. Dust stress simulation method: Sterilized and sieved soil dust was evenly sprayed onto the surface of reed leaves, once every 3 days, with a dosage of 2 g / m³ each time. 2 The treatment lasted for 21 consecutive days.

[0063] There are a total of 7 processing groups, configured as follows: (1) CK: No salt or alkali, no dust, no inoculation; (2) S: 100 mmol / L NaCl salt stress, no dust, no inoculation; (3) D: Dust stress, no salt or alkali, no inoculation; (4) S+T: 100 mmol / L NaCl salt stress, dust-free, inoculated with NEFU-LAWJH-001 strain; (5) D+T: Dust stress, no salt and alkali, inoculated with NEFU-LAWJH-001 strain; (6) S+D: 100 mmol / L NaCl + dust combined stress, no inoculation; (7) S+D+T: 100 mmol / L NaCl + dust combined stress, inoculated with NEFU-LAWJH-001 strain.

[0064] 2.2 The measurement indicators include the following: Photosynthetic parameters: net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), and intercellular CO2 concentration (Ci) were measured using a portable photosynthesis system. Moisture parameters: Relative water content of leaves (RWC); Growth parameters: aboveground dry weight, seedling quality index, chlorophyll content; Stress resistance: Proline and MDA content.

[0065] 3. Experimental Procedure The transplanting and inoculation methods are the same as in Example 4. Approximately 14 days after transplanting, a second inoculation is performed, and salt stress treatment is initiated simultaneously.

[0066] The moderate salt stress group was irrigated with Hoagland nutrient solution containing 100 mmol / L NaCl, the severe salt stress group was irrigated with Hoagland nutrient solution containing 200 mmol / L NaCl, and the no-salt stress group was irrigated with conventional Hoagland nutrient solution. Irrigation was carried out every 3-4 days, and the soil moisture content was maintained at about 70% of field capacity using the weighing method.

[0067] 4. Measurement of physiological indicators Physiological parameters were measured on day 28 after salt stress treatment. The details are shown in Table 5, and the results are shown in Tables 6-9.

[0068] Table 5. Determination of indicators and methods for potted plant growth promotion experiment of reed seedlings (salt stress conditions) 5. Growth index measurement After the final harvest, the following growth indicators were measured: plant height and aboveground / underground dry weight, using the same method as in Example 4.

[0069] 6. Experimental Results Table 6. Results of Physiological Experiments on Potted Growth Promotion of Reed Seedlings (Salt Stress Conditions) Table 7. Results of the growth experiment of potted reed seedlings under salt stress. Under salt stress, inoculation with NEFU-LAWJH-001 strain significantly improved the osmotic regulation capacity, antioxidant enzyme activity, reduced membrane lipid peroxidation damage, improved ion homeostasis, and significantly alleviated the growth-inhibiting effect of salt stress on reed seedlings.

[0070] Table 8 Results of the potted plant growth promotion experiment of reed seedlings (compound stress conditions) – photosynthetic and water parameters Note: Different lowercase letters in the same row indicate significant differences (P<0.05).

[0071] Table 9 Results of the potted plant growth promotion experiment of reed seedlings (compound stress conditions) – growth and stress resistance parameters Under combined stress conditions of salinity, alkalinity, and dust, inoculation with the NEFU-LAWJH-001 strain significantly improved the photosynthetic efficiency, stomatal conductance, transpiration rate, and relative leaf water content of reed seedlings, alleviating the damage caused by the combined stress and thus increasing biomass and seedling quality. This function is a unique advantage not possessed by conventional salt-tolerant growth-promoting bacteria.

[0072] Example 6: Preparation of Microbial Inoculants 1. Preparation of liquid bacterial agent The fermentation broth of the NEFU-LAWJH-001 strain obtained from the expanded culture in Example 2 was centrifuged at 8000 r / min for 10 min at 4℃, and the bacterial cells were collected. The bacterial cells were resuspended in sterile buffer, and glycerol was added as a cryoprotectant to a final concentration of 10% (v / v). The volume of resuspending buffer was 10%~20% of the original fermentation broth volume, and the concentration was adjusted to approximately 1×10⁻⁶. 9 CFU / mL. The prepared liquid bacterial agent was dispensed into sterile containers and stored at 4°C in the dark.

[0073] 2. Preparation of solid microbial agents The prepared liquid microbial agent was mixed with a sterilized solid carrier in a certain proportion. The solid carrier was peat moss, and the mass ratio of carrier to liquid microbial agent was 2:1. After uniform mixing, the mixture was dried at low temperature until the moisture content was below 10%. The prepared solid microbial agent was vacuum-packed and stored at 4°C in the dark.

[0074] Example 7: Application method of microbial agent 1. Soaking method Reed seeds were soaked in a bacterial suspension prepared in Example 6 and diluted 10 times (approximately 1×10⁻⁶). 8 After soaking in water (CFU / mL) for 2-4 hours, remove and air dry before sowing.

[0075] 2. Root irrigation method After transplanting the reeds, the liquid inoculant prepared in Example 6 was diluted to approximately 1 × 10⁻⁶. 8 Apply CFU / mL to the roots of the plant at a dose of 20-50mL per plant, once every 7-10 days, for 2-3 consecutive applications.

[0076] 3. Spraying method The liquid microbial agent prepared in Example 6 was diluted 10 times and sprayed evenly on the surface of reed leaves. It is suitable for microbial treatment of reed seedlings in large-scale wetland restoration projects.

[0077] Example 8: Comparison of inoculation effects with different bacterial suspension concentrations To determine the optimal inoculation concentration, this example included treatments with different concentrations of bacterial suspension to investigate their effects on reed seed germination and seedling growth. The NEFU-LAWJH-001 bacterial suspension prepared in Example 2 was diluted to 1×10⁻⁶. 6 CFU / mL, 1×10 7 CFU / mL, 1×108 CFU / mL and 1×10 9 Four concentration gradients of CFU / mL were used, with sterile water as a control. Reed seed germination experiments were conducted according to the method in Example 3. It can be seen that 1×10⁻⁶ CFU / mL... 8 At a CFU / mL concentration, the NEFU-LAWJH-001 strain showed the best growth-promoting effect. Specific experimental results are shown in Table 10.

[0078] Table 10. Inoculation effects of different bacterial suspension concentrations Example 9: Comparison of the stress resistance effects of different salt concentration strains on reeds To investigate the stress-promoting effect of NEFU-LAWJH-001 strain under different salt stress intensities, this example follows the experimental method of Example 5, conducting pot experiments at four salt concentration gradients of 50 mmol / L, 100 mmol / L, 150 mmol / L, and 200 mmol / L NaCl, and measuring the changes in some salt stress-related indicators to evaluate the growth-promoting effect of the bacterial suspension on reeds under different salt concentrations. Some results under the 0 mmol / L NaCl treatment are referenced in Example 4.

[0079] The experimental results are shown in Table 11.

[0080] Table 11. Data on the stress resistance effect of different NaCl concentrations Note: ① In absolute value data, different lowercase letters in the same column indicate significant differences among all treatments (p<0.05). Duncan's method was used for multiple comparisons.

[0081] As shown in Table 11, the NEFU-LAWJH-001 strain of this invention exhibits significant stress resistance and growth-promoting effects within the range of 50–200 mmol / L NaCl. Among these, the osmotic regulation and antioxidant effects are most significant under moderate salt stress of 100–150 mmol / L; and the biomass increase rate is highest under severe salt stress of 200 mmol / L, indicating that the strain can effectively alleviate growth inhibition even under extreme salt stress.

[0082] At a NaCl concentration of 200 mmol / L, the absolute dry weight of 2.10 g / strain was still lower than that at 100 mmol / L (3.00 g / strain), but the CK200 baseline was only 1.38 g / strain, resulting in a growth rate as high as 52.2%. The proline growth rate peaked at 78.3% at 150 mmol / L and then decreased to 61.2% at 200 mmol / L, indicating that the strain's regulatory capacity tends to reach its limit under extreme stress, and non-enzymatic protective mechanisms cannot be infinitely amplified. The MDA reduction rate remained stable at 39%–43% within the 100–200 mmol / L range, indicating that the strain's membrane protection function remained effective within this salinity range and did not fail due to increased salinity.

[0083] Example 10: Adaptability Verification and Comparative Experiment of Strains under Wetland Simulation Environment 1. Select Zhihengliuella halotolerans The type strain DSM 17364T was used as the control strain. This strain was isolated from saline soil and is widely recognized as... Zhihengliuella halotolerans The standard representative strain has been publicly reported to possess growth-promoting characteristics such as IAA production, siderophore production, ACC deaminase production, and phosphate solubilization, and is available for comparative experiments. This strain is a strict aerobic bacterium, and its metabolic activities are highly dependent on a sufficient oxygen supply. This strain was provided by the China Center for Type Culture Collection (CCTCC).

[0084] The experiment was set up with three treatment groups, each with three replicates (flowerpots), one reed seedling per pot, and 20 mL (1×10⁻⁶) of bacterial suspension per pot. 8 (CFU / mL), see Table 12.

[0085] Table 12 Wetland Simulation Environmental Treatment 2. Reed seedling cultivation and transplanting Same as Example 4. Sterilized reed seeds were sown in seedling trays containing a 1:2 (volume ratio) mixture of vermiculite and peat moss. The seedlings were cultivated in a greenhouse until the 2-3 leaf stage (approximately 3-4 weeks), during which time Hoagland nutrient solution was regularly applied to maintain normal growth. Reed seedlings with uniform growth were selected and transplanted into flowerpots containing sterilized substrate.

[0086] 3. Wetland flooding treatment Pots from the Wet-EXP and Wet-CK groups were placed in water troughs, maintaining a 2-3 cm water layer above the substrate surface, and cultured for 7 days to allow the rhizosphere environment to reach wetland characteristics, namely a decrease in rhizosphere redox potential, activation of oxygen secretion from reed roots, and a significant decrease in rhizosphere dissolved oxygen content. The Dry-CK group was managed with conventional water, maintaining soil moisture content at approximately 70% of field capacity.

[0087] 4. Inoculation with strains On the 7th day of flooding, the bacterial suspension was inoculated into the rhizosphere of the reeds at a dose of 20 mL per plant using the root irrigation method. (1) Wet-EXP group: Inoculated with NEFU-LAWJH-001 strain suspension (1×10 8 (CFU / mL) (2) Wet-CK group: Inoculated with control strain DSM 17364T bacterial suspension (1×10⁻⁶) 8 (CFU / mL) (3) Dry-CK group: Inoculated with control strain DSM 17364T bacterial suspension (1×10⁻⁶) 8 (CFU / mL).

[0088] 5. Sample collection and testing Rhizosphere soil samples (0–5 cm from the root base) of reeds were collected from each treatment group on days 3, 7, 14, and 28 post-inoculation for the following tests: (1) Determination of strain survival rate Weigh 1 g of fresh rhizosphere soil, add 9 mL of sterile physiological saline, shake to mix, then serially dilute, spread on LB solid agar plates, incubate at 28°C for 48 h, count the colonies, and calculate the viable cell count (CFU / g dry soil). With the same initial inoculum, use the viable cell count on day 1 after inoculation as the initial value to calculate the survival rate at each time point.

[0089] (2) Detection of the growth-promoting functional activity of the strain On day 28 after inoculation, rhizosphere soil samples were collected from each treatment group, and the following indicators were tested. For specific experimental methods, please refer to Example 1.

[0090] IAA secretory activity was determined using the Salkowski colorimetric method; Ferrite production: determined using the CAS detection method.

[0091] 6. Experimental Results The results of the strain survival rate experiment are shown in Table 13, and the results of the growth-promoting functional activity test are shown in Table 14.

[0092] Table 13 Survival rate of strains under water flooding treatment Table 14 Growth-promoting ability of bacterial strains under water flooding treatment Note: Different lowercase letters in the same row indicate significant differences between treatments (P<0.05). Duncan's method was used for multiple comparisons.

[0093] conventional Zhihengliuella halotolerans The survival rate of the model strain DSM 17364T decreased in the anoxic environment of flooded wetlands, and its growth-promoting activity was significantly reduced. The NEFU-LAWJH-001 strain of this invention showed significantly better survival rate and growth-promoting activity than the control strain under the same simulated wetland conditions. These results demonstrate that the NEFU-LAWJH-001 strain of this invention is superior to the conventional strain. Zhihengliuella halotolerans Overcoming the technical bottlenecks in the application of strains in hypoxic wetland environments has led to improved effectiveness in wetland ecological restoration, moving beyond dryland / desert habitats.

[0094] 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 salt-tolerant strain of Liu Zhiheng fungus ( Zhihengliuella halotolerans ), characterized in that, The preservation number of the salt-tolerant Liu Zhiheng bacterium is CGMCC No. 38351.

2. The application of the salt-tolerant Liu Zhiheng bacterium as described in claim 1 in any of the following: (1) Application in promoting the growth of wetland reeds; (2) Application in promoting the growth of wetland reeds under combined salt and dust stress; (3) Application in improving the survival and establishment ability of reeds in wetland flooding environment.

3. The application as described in claim 2, characterized in that, The promotion of wetland reed growth refers to promoting the germination of reed seeds and the growth of reed seedlings.

4. The application as described in claim 2, characterized in that, The growth promotion refers to promoting the growth of wetland reed seedlings under combined salt and dust stress.

5. The application as described in claim 2, characterized in that, The salt-tolerant Liu Zhiheng bacteria are applied in the form of bacterial suspension, liquid inoculum or solid inoculum.

6. The application as described in claim 5, characterized in that, In the bacterial suspension or liquid inoculum, the concentration of the salt-tolerant Liu Zhisheng bacteria is 1×10 6 CFU / mL ~ 1×10 9 CFU / mL.

7. The application as described in claim 5, characterized in that, The solid inoculant is obtained by mixing peat moss and liquid inoculant at a mass ratio of 2:1 and then drying it until the moisture content is less than 10%.

8. A method for promoting the growth of wetland reeds under combined salt and dust stress, characterized in that, The method includes the step of treating wetland reeds with the salt-tolerant Liu Zhiheng bacteria as described in claim 1; wherein the treatment method includes one or more combinations of seed soaking, root irrigation, and foliar spraying.

9. The method as described in claim 8, characterized in that, The salt-tolerant Liu Zhiheng bacteria can be in the form of bacterial suspension, liquid inoculum, or solid inoculum.

10. A microbial inoculant, characterized in that, Includes the salt-tolerant Liu Zhiheng bacteria as described in claim 1 and the carrier, wherein the microbial agent is in the form of a liquid agent or a solid agent; The liquid bacterial agent has a bacterial concentration of 1x10 9 The solid bacterial agent is obtained by mixing the grass carbon and the liquid bacterial agent in a mass ratio of 2:1 and drying to a water content of less than 10%.

Images