Rhizosphere growth-promoting bacteria of Leymus chinensis and their application in promoting growth and drought resistance of Leymus chinensis

Abstract

The present invention relates to a plant growth-promoting rhizobacterium of Leymus chinensis and its application in promoting the growth and drought resistance of Leymus chinensis, belonging to the field of microbial technology. The strain is classified and named as Paraburkholderia graminis , with the strain number B24 and the preservation number CGMCC No. 31907. The strain of the present invention is derived from the rhizosphere soil of Leymus chinensis in overgrazed grasslands. This strain not only has good plant growth-promoting characteristics such as IAA production, ACC deaminase production, nitrogen fixation, and phosphorus solubilization, but also can significantly enhance the drought resistance of Leymus chinensis. Inoculating this bacterium into the rhizosphere of Leymus chinensis seedlings can significantly promote the growth of Leymus chinensis, increase the aboveground and underground biomass of Leymus chinensis; reduce the antioxidant enzyme activity and change the hormone content in the leaves of Leymus chinensis, thereby alleviating drought stress, enhancing the photosynthesis of Leymus chinensis, and thus significantly improving the drought resistance of Leymus chinensis. Therefore, the Burkholderia parakniphia of the present invention can be used for the development of microbial fertilizers for grassland plants and applied in arid stress habitats, so as to improve the drought resistance of the grassland plant Leymus chinensis and optimize grassland management.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology and relates to a plant growth-promoting rhizobacterium, specifically, Burkholderia paraunii B24 and its application in promoting the growth and drought resistance of the grassland plant Leymus chinensis. Background Art

[0002] Plant growth-promoting rhizobacteria (PGPR) refer to a class of microorganisms that live in the rhizosphere or inside plants and can directly or indirectly promote plant growth through various mechanisms. These mechanisms mainly include nitrogen fixation, phosphorus solubilization, production of plant hormones, induction of systemic resistance (ISR), etc., enhancing the tolerance of plants to environmental stresses such as drought, salinity, and high temperature, thereby improving the stress resistance of plants. Therefore, PGPR are widely used in the fields of agriculture and ecological restoration. They can reduce the use of chemical fertilizers and pesticides, promote the development of sustainable agriculture, and play an important role in the ecological restoration of degraded and polluted soils. With the in-depth study of PGPR, their potential in molecular mechanisms, flora screening, and practical applications has been continuously explored, providing new solutions for improving agricultural production efficiency and protecting the ecological environment. Compared with chemical fertilizers, PGPR as inoculants have the advantages of environmental friendliness and economic efficiency.

[0003] Drought stress is one of the main environmental stresses affecting plant growth and development, and its impact on plants is multi-faceted, including multiple levels such as physiological, biochemical, molecular, and morphological. Drought stress inhibits the photosynthesis efficiency and stomatal conductance of plants, thereby reducing carbon assimilation. Under drought conditions, a large amount of reactive oxygen species (ROS) are generated inside plants, causing membrane lipid peroxidation, protein denaturation, and DNA damage, leading to cellular oxidative stress. At the same time, drought stress inhibits root growth and weakens the plant's ability to absorb water and nutrients. To adapt to drought stress, plants will initiate a series of stress resistance regulation mechanisms. For example, by activating the abscisic acid (ABA) signaling pathway to close stomata and reduce water transpiration; increasing the accumulation of osmoregulatory substances (such as proline, soluble sugars) to maintain cell osmotic balance. Inoculation with plant growth-promoting rhizobacteria (PGPR) under drought stress has a significant positive impact on plants and is an effective strategy to alleviate the negative effects of drought stress on plants. PGPR can promote the water use efficiency of plants, improve the plant's ability to absorb limited water resources by enhancing the development of plant roots and expanding the absorption range of roots. PGPR can also regulate the opening and closing of plant stomata and the growth and development process by secreting plant hormones such as indole acetic acid (IAA), cytokinin, and abscisic acid (ABA), thereby reducing water transpiration loss and optimizing physiological metabolism. In addition, PGPR can enhance the osmoregulation ability of plants, and its secreted metabolites such as proline, soluble sugars, etc. can help plants maintain the water balance inside cells and reduce cell dehydration and stress damage caused by drought stress. PGPR can also activate the plant antioxidant system, clear the accumulation of reactive oxygen species caused by drought by increasing the activity of antioxidant enzymes, and protect cell structure and function. In addition, PGPR improves the overall stress resistance of plants through induced systemic resistance (ISR), making it show stronger tolerance and adaptability under drought conditions. PGPR can also improve the soil microenvironment, for example, by functions such as nitrogen fixation, phosphorus solubilization, and potassium decomposition to enhance the availability of soil nutrients and provide more sufficient nutrient support for plants. Although the mechanisms of plant growth promotion and stress resistance mediated by PGPR are not fully understood, related research mainly focuses on crops. A large number of literatures have proved that inoculation with PGPR can promote plant nutrient absorption, growth, yield, stress resistance, and plant hormone homeostasis. Inoculation with PGPR can effectively alleviate the adverse effects of drought stress on plants, promote plant growth and development, improve the plant's adaptability to drought environment, and provide new solutions for the sustainable development of agricultural ecosystems.

[0004] Leymus chinensis Leymus chinensis) is a perennial herbaceous plant that is drought-tolerant, cold-tolerant, and salt-alkali tolerant, widely distributed in grasslands and semi-arid regions. At the same time, Leymus chinensis has high nutritional value and palatability and is an important forage for livestock. Leymus chinensis also has strong stress resistance and ecological adaptability and can grow in degraded grasslands, saline-alkali lands, and semi-arid regions. It is an excellent grass species for grassland ecological restoration and vegetation restoration. By studying the promoting effect of PGPR on the growth of Leymus chinensis, it not only helps to increase forage yield but also reduces the use of chemical fertilizers and promotes the development of sustainable agriculture. In arid regions or degraded grasslands, Leymus chinensis with strong drought resistance can better adapt to harsh environments, restore vegetation cover, and prevent further desertification of the land. By studying techniques to improve the drought resistance of Leymus chinensis (such as adding bacterial liquid or bacterial fertilizer), it can provide technical support for the restoration of the ecosystem, especially for vegetation restoration in arid and semi-arid regions. Summary of the Invention

[0005] In view of the increasingly serious problems of large-scale grassland degradation and drought, the present invention provides a Burkholderia parakniphofiae strain suitable for application in drought-stressed habitats. This strain effectively solves the problem of converting insoluble phosphate into soluble phosphate, produces the plant hormone IAA, and at the same time has growth-promoting characteristics such as nitrogen fixation and production of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, and can significantly promote the growth of Leymus chinensis and improve the drought resistance of Leymus chinensis under drought conditions. Based on this, the present invention isolates a strain of Burkholderia parakniphofiae B24 from the rhizosphere of Leymus chinensis in a grazing-degraded grassland. After inoculation with this strain, it not only has a growth-promoting function on Leymus chinensis but also can significantly improve the drought resistance of Leymus chinensis. It is a strain with great application potential, providing strain resources and theoretical basis for subsequent analysis of plant drought resistance mechanisms, optimization of grassland management, restoration of degraded grasslands, and development of microbial fertilizers. Specifically, the present invention adopts the following technical solutions: The strain of the present invention was deposited on September 09, 2024, at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms (abbreviation: CGMCC, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101), and was classified and named as Paraburkholderia graminis (Burkholderia parakniphofiae), strain number B24, deposit number CGMCC No. 31907.

[0006] Colony characteristics and cell morphology of the Burkholderia parakniphofiae B24: The colonies of B24 on the LB plate are light yellow, the colony texture is relatively moist, and the edge is relatively smooth. Under the microscope, the cells are spherical, 0.5~0.8um×0.5~0.8um, showing red, and are Gram-negative. It can grow under both aerobic and anaerobic conditions, but grows better under aerobic conditions and belongs to facultative aerobes.

[0007] Physiological and biochemical characteristics of the Paraburkholderia sp.: For strain B24, the catalase reaction is positive, glucose oxidation is of the fermentative type, the hydrogen sulfide test is negative, the methyl red test is negative, the V-P determination is weakly positive, the starch hydrolysis test is negative, the nitrate reduction test is weakly positive, the citrate utilization test is positive, and glucose gas production is positive.

[0008] The 16S rDNA gene sequence of the strain is shown in SEQ ID No. 1. Obviously, related gene constructs containing this coding gene, including but not limited to one or more of genetically engineered bacteria and recombinant proteins, also have the related functions of strain B24.

[0009] The 16S rDNA sequence of the tested strain B24 was BLAST-aligned with the sequences in the GenBank database. The results showed that strain B24 had the highest homology with Paraburkholderia graminis strain, which was 99.92%. Based on the 16S rDNA sequence, combined with phylogenetic analysis, morphology, physiological and biochemical characteristics, B24 was determined to be Paraburkholderia graminis.

[0010] A transparent zone with a diameter of 1.77 ± 0.28 cm was formed around the colonies of the Paraburkholderia sp. B24, indicating that the strain could degrade insoluble phosphate into a form that can be absorbed and utilized by plants, and the degradation amount was 179.90 ± 2.59 μg / mL. Thus, it can improve the ability of plants to obtain nutrients and promote the growth and development of plant roots.

[0011] The Paraburkholderia sp. B24 has a strong ability to synthesize ACC deaminase, and the enzyme activity is 178.12 ± 3.48 IU / L. It can hydrolyze 1-aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene synthesis under stress conditions, reduce the level of ethylene, increase the nutrient absorption and light capture area, promote plant growth and improve plant stress resistance.

[0012] The Paraburkholderia sp. B24 grows well on Ashby medium and has a strong nitrogen fixation ability. The nitrogenase NITS activity is 199.13 ± 0.74 IU / L. By improving the efficiency of biological nitrogen fixation (BNF), it helps plants assimilate nitrogen more efficiently, thereby further enhancing the growth ability of plants.

[0013] The Paraburkholderia sp. B24 has the ability to produce indole-3-acetic acid (IAA), and the yield reaches 25.33 ± 3.48 μg / mL.

[0014] Obviously, the fermentation product of the strain B24 and the gene construct based on the B24 gene also have the functions of degrading inorganic phosphorus, nitrogen fixation, indole acetic acid production, and ACC deaminase synthesis described above. Further, the strain B24 and the fermentation product can also be prepared into microbial fertilizers or microbial agents for large-scale application.

[0015] The application method of the plant growth-promoting rhizobacterium B24 of the present invention for promoting the growth of Leymus chinensis and improving its drought resistance. Among them, it can be applied by preparing a suspension of the plant growth-promoting rhizobacterium of Leymus chinensis. The preparation may include the following steps: inoculating the strain into an LB liquid medium and culturing (constant temperature culture at 30 °C and 180 rpm) for 48 h to prepare a fermentation broth, centrifuging at 8000 rpm and room temperature for 10 min, washing twice with sterile water suspension, and finally resuspending with sterile water to prepare a bacterial suspension with an OD 600 = 1.0 for standby. The concentration of the B24 strain in the bacterial suspension is 1×10 8 CFU / mL.

[0016] The above-mentioned application method for promoting the growth of Leymus chinensis and improving its drought resistance is to apply the plant growth-promoting rhizobacterium or the gene construct or the fermentation product or the microbial fertilizer to the rhizosphere of Leymus chinensis. The application to the rhizosphere of Leymus chinensis includes any one or more application methods such as spraying, irrigation, and burying.

[0017] The Paraburkholderia B24 of the present invention is isolated from the rhizosphere soil of Leymus chinensis in overgrazed plots and obtained through artificial isolation and purification. By improving the plant's water use efficiency, promoting root growth, promoting photosynthesis, regulating proline metabolism and plant hormone levels, etc., it effectively alleviates drought stress and reduces the occurrence of oxidative damage. It reflects the protective effect of B24 on plants and helps plants better adapt to drought conditions. Therefore, the Paraburkholderia B24 of the present invention has a strong plant growth-promoting function, has a significant plant growth-promoting effect on the above-ground and underground roots of Leymus chinensis, and can significantly improve the drought resistance of Leymus chinensis. It can be used as a microbial inoculant in arid habitats, providing strain resources and theoretical basis for subsequent analysis of plant drought resistance mechanisms, optimization of grassland management, restoration of degraded grasslands, and development of microbial fertilizers.

[0018] The beneficial effects of the present invention:

[0019] Under pot culture conditions, the Paraburkholderia B24 of the present invention promotes the growth of Leymus chinensis under drought stress by increasing the plant height, stem diameter, number of leaves, leaf width, chlorophyll content, above-ground biomass and underground biomass of Leymus chinensis under drought stress. At the same time, it can increase the net photosynthetic rate (A), transpiration rate (E), stomatal conductance (gsw), intercellular carbon dioxide concentration (Ci), chlorophyll content and Rubisco enzyme activity of Leymus chinensis under drought stress, thereby improving the photosynthesis of Leymus chinensis.

[0020] Under drought stress, Burkholderia parakusensis B24 effectively helps plants alleviate drought stress by reducing the contents of stress-related hormones, antioxidant enzyme activities, and proline content, enabling plants to reduce their dependence on proline or cope with drought stress through other mechanisms (such as improving water absorption and producing other protective substances). This indicates that PGPR B24 can help plants better adapt to the drought environment by promoting plant growth, enhancing drought resistance, or regulating plant metabolic pathways.

[0021] The Burkholderia parakusensis provided by the present invention can provide a new strain resource for the microbial fertilizer industry and shows potential application prospects in aspects such as soil phosphorus activation, plant growth promotion, yield increase, drought resistance, and restoration of grassland degradation. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the colony morphology diagram and the morphology diagram under an optical microscope of strain B24 in the embodiment of the present invention (the scale in the lower right corner of the left figure is 10 μm).

[0023] Figure 2 It is the phylogenetic tree of B24 based on 16S rDNA.

[0024] Figure 3 It is the influence of strain B24 in the embodiment of the present invention on the morphological indexes (plant height, leaf width, stem diameter, number of leaves) of Leymus chinensis.

[0025] Figure 4 It is the influence of strain B24 in the embodiment of the present invention on the above-ground and underground biomass of Leymus chinensis.

[0026] Figure 5 It is the influence of strain B24 in the embodiment of the present invention on the chlorophyll content and ribulose bisphosphate carboxylase (Rubisco) activity of Leymus chinensis.

[0027] Figure 6 It is the influence of strain B24 in the embodiment of the present invention on the antioxidant enzyme (superoxide dismutase, peroxidase, catalase, malondialdehyde) activities of Leymus chinensis.

[0028] Figure 7 It is the influence of strain B24 in the embodiment of the present invention on the proline content of Leymus chinensis.

[0029] Figure 8 It is the influence of strain B24 in the embodiment of the present invention on the contents of leaf hormones (abscisic acid, indoleacetic acid, gibberellin, and jasmonic acid) of Leymus chinensis. DETAILED DESCRIPTION OF THE INVENTION

[0030] Example 1 (Screening, Identification, and Analysis of Growth-Promoting Characteristics of Drought-Resistant Strains in the Rhizosphere of Leymus chinensis)

[0031] 1. Experimental Materials

[0032] (1) Collection of soil samples

[0033] The rhizosphere soil samples of Leymus chinensis were collected from a long-term overgrazed grassland in Bayin Xile, Ordos City, Inner Mongolia. About 10 Leymus chinensis plants were carefully dug out with a shovel, and the roots were shaken to remove large pieces of soil. The remaining soil on the root surface (about 1 - 3 mm thick) was considered as rhizosphere soil, which was collected into a sterile self-sealing bag using a brush, stored in a refrigerator at 4°C, and the strain isolation and purification were carried out as soon as possible.

[0034] (2) Preparation of culture media and reagents

[0035] ① LB solid medium

[0036] Tryptone 10.0 g, yeast extract 5.0 g, NaCl 10 g, agar 15 g, distilled water 1000 mL, pH = 7.0 - 7.2, sterilized at 121°C for 20 min.

[0037] ② Ashby solid medium

[0038] KH2PO4 0.2 g, CaCO3 5.0 g, MgSO4·7H2O 0.2 g, glucose 10.0 g, NaCl 0.2 g, CaSO4·2H2O 0.1 g, agar 18.0 g, distilled water 1 L, pH = 6.8 - 7.0

[0039] ③ PKO solid medium

[0040] Glucose 10.0 g, (NH4)2SO4 0.5 g, NaCl 0.3 g, KCl 0.3 g, MgSO4·7H2O 0.3 g, FeSO4·7H2O 0.03 g, MnSO4·4H2O 0.03 g, Ca3(PO4)2 5.0 g, yeast extract powder 0.5 g, agar 18.0 g, distilled water 1 L, pH = 7.0 - 7.2

[0041] ④ Reagents

[0042] Salkowski color reagent: Slowly pour 150 mL of H2SO4 into 250 mL of distilled water. After the H2SO4 solution cools, add 7.5 mL of 0.5 M FeCI3·6H2O solution to obtain the color reagent. This color reagent cannot be stored for a long time.

[0043] 2. Test methods

[0044] (1)Primary screening of drought-resistant strains

[0045] Under sterile conditions, weigh 10 g of the rhizosphere soil of Leymus chinensis and dissolve it in 90 mL of sterile water. Shake it on a shaker at 120 r / min for 1 h, and dilute it stepwise into suspensions of 10 -3 、10 -4 、10 -5 、10 -6 times. Take 50 μL from each dilution and spread it evenly on LB solid media with different concentrations of PEG (polyethylene glycol) (5% - 15%). Incubate in an incubator at 30°C. Streak the morphologically different single colonies for further purification until completely purified.

[0046] (2)Screening of phosphate - solubilizing and nitrogen - fixing strains

[0047] Spot - inoculate the purified single colonies onto inorganic phosphate solid media and Ashby nitrogen - free media, and incubate in an incubator at 30°C. Among them, B24 can produce a phosphate - solubilizing circle on inorganic phosphate solid media and a nitrogen - fixing circle on Ashby media. Transfer it into a glycerol tube with a final concentration of 30% and store it in a - 20°C refrigerator.

[0048] (2)Determination of IAA - producing characteristics

[0049] Using the Salkowski colorimetric method, inoculate the strain into LB liquid medium containing L - tryptophan (100 mg / L), and culture it on a shaker at 30°C and 200 r / min for 24 h. Centrifuge the bacterial suspension at 4°C and 5000 rpm for 10 minutes. Take 1 mL of the supernatant and add 3 mL of Salkowski color reagent. Use the mixed solution of non - inoculated LB liquid medium and color reagent as a blank control. Observe after standing in the dark at room temperature for 30 min. A red color indicates the ability to secrete IAA. Prepare standard curves with different concentrations of IAA standard solution. After reacting under the above conditions, measure the OD 530 of the reaction solution, and calculate the concentration of IAA in the supernatant.

[0050] (4)Determination of phosphate - solubilizing ability

[0051] Determine the phosphate - solubilizing ability of the strain through qualitative and quantitative assays.

[0052] Qualitative determination by the phosphate - solubilizing circle method: Inoculate strain B24 onto PKO inorganic phosphate solid media, with 3 replicates for each strain. Measure the diameter (D) of the phosphate - solubilizing circle and the diameter (d) of the colony formed by each strain, and calculate the phosphate - solubilization index PSI (D / d) value to preliminarily judge the phosphate - solubilizing ability of the strain.

[0053] Quantitative determination by the molybdenum antimony anti - colorimetric method: Inoculate the activated strain (1 mL) into PKO medium (50 mL). The control group is inoculated with an equal volume of sterile water. Incubate at 30°C and 150 r·min-1 Incubate at a constant temperature for 7 days; then centrifuge at 11000 r·min -1 for 5 minutes and take the supernatant. Add 1 - 2 drops of dinitrophenol indicator to the supernatant (30 mL), and use NaOH solutions (1M, 10 M) and HCl solutions (1 M, 5 M) to micro - drip and adjust until the solution just turns slightly yellow. Then accurately add 5 mL of molybdenum antimony anti - mixed chromogenic solution, shake well, make up the volume to 50 mL with deionized water. After reacting at room temperature above 15℃ for 30 minutes (within 8 hours), measure and record the OD 700 value. Calculate the phosphorus content (mg·L -1 ) according to the standard curve.

[0054] (5) Determination of the nitrogenase activity of the strain

[0055] Use a nitrogenase (NITS) enzyme - linked immunosorbent assay kit for determination. This kit uses the double - antibody sandwich method to determine the level of nitrogenase (NITS) in the specimen. Coat the microplate with purified nitrogenase (NITS) antibody to make a solid - phase antibody. Sequentially add nitrogenase (NITS) to the microplate coated with the monoclonal antibody, then bind it with the HRP - labeled nitrogenase (NITS) antibody to form an antibody - antigen - enzyme - labeled antibody complex. After thorough washing, add the substrate TMB for color development. TMB is converted into blue under the catalysis of HRP enzyme and into the final yellow under the action of acid. The depth of the color is positively correlated with the nitrogenase (NITS) in the sample. Measure the absorbance (OD value) with an enzyme - labeled instrument at a wavelength of 450 nm, and calculate the activity concentration of nitrogenase (NITS) in the sample through the standard curve.

[0056] (6) Determination of the ACC deaminase activity of the strain

[0057] Use an ACC deaminase (ACCD) enzyme - linked immunosorbent assay kit for determination. This kit uses the double - antibody sandwich method to determine the level of ACC deaminase (ACCD) in the specimen. Coat the microplate with purified ACC deaminase (ACCD) antibody to make a solid - phase antibody. Sequentially add ACC deaminase (ACCD) to the microplate coated with the monoclonal antibody, then bind it with the HRP - labeled ACC deaminase (ACCD) antibody to form an antibody - antigen - enzyme - labeled antibody complex. After thorough washing, add the substrate TMB for color development. TMB is converted into blue under the catalysis of HRP enzyme and into the final yellow under the action of acid. The depth of the color is positively correlated with the ACC deaminase (ACCD) in the sample. Measure the absorbance (OD value) with an enzyme - labeled instrument at a wavelength of 450 nm, and calculate the activity concentration of ACC deaminase (ACCD) in the sample through the standard curve.

[0058] (7) Physiological and biochemical characteristics

[0059] A series of physiological and biochemical characteristics of B24 were determined according to the Manual for the Systematic Identification of Common Bacteria. The determination indexes included: Gram staining, catalase, glucose oxidation fermentation, methyl red, V-P, nitrate reduction, oxidase, starch hydrolysis, H2S, citrate utilization, glucose gas production, indole test, and gelatin liquefaction test.

[0060] (8)16S rDNA sequence sequencing identification

[0061] Total bacterial DNA was extracted, and 16S rDNA was amplified using the universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5 '-TACCTTGTTACGACTT-3′). The PCR amplification products were sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing, and the primers used were synthesized by Nanjing Qingke Biotechnology Co., Ltd. The sequenced sequences were submitted to the GenBank gene bank of the online website NCBI (https: / / www.ncbi.nlm.nih.gov / ) for homology search and comparison.

[0062] 3. Identification results of strain B24

[0063] (1)Identification results of morphological characteristics

[0064] As Figure 1 shown, the colonies of Burkholderia paraphytica B24 on the LB plate were light yellow, the colony texture was relatively moist, and the edge was relatively smooth. Under the microscope, the bacteria were spherical, 0.5~0.8um×0.5~0.8um, showing red, and were Gram-negative facultative aerobic bacteria.

[0065] (2)Physiological and biochemical characteristics identification

[0066] As shown in Table 1, the catalase reaction of strain B24 was positive, glucose oxidation was of the fermentative type, the hydrogen sulfide test was negative, the methyl red test was negative, the V-P determination was weakly positive, the starch hydrolysis experiment was negative, the nitrate reduction experiment was weakly positive, the citrate utilization experiment was positive, and glucose gas production was positive.

[0067] Table 1 Identification results of physiological and biochemical characteristics of strain B24

[0068]

[0069] Note: “+” indicates a positive reaction or growth and utilization; “—” indicates a negative reaction or non-growth and non-utilization.

[0070] (3)Identification results of 16S rDNA sequence sequencing

[0071] The 16S rDNA sequence of the tested strain B24 was amplified and sequenced, and the sequencing result is shown as SEQ ID NO:1 in the sequence listing. The sequence was subjected to BLAST alignment with the sequences in the GenBank database, and the result showed that the strain B24 had the highest homology with Paraburkholderia graminis strain, which was 99.92%. As Figure 2 , the results of phylogenetic analysis showed that the similarity between B24 and Paraburkholderia graminis strain was 100%. Based on the 16S rDNA sequence, combined with phylogenetic analysis, morphological, physiological and biochemical characteristics, B24 was determined to be Paraburkholderia graminis. This strain was deposited on September 9, 2024 at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms (abbreviated as CGMCC, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, postal code: 100101), and the deposit number was CGMCC No. 31907.

[0072] The 16S rDNA gene sequence of the said Paraburkholderia is as shown in SEQ ID No. 1:

[0073]

[0074] (4) Results of growth-promoting characteristics

[0075] The results of the growth-promoting characteristics of strain B24 are shown in Table 2, indicating that this strain has high phosphate-solubilizing, nitrogen-fixing, IAA-producing, and ACC deaminase activities.

[0076] Table 2 Identification results of the growth-promoting characteristics of strain B24

[0077]

[0078] Example 2 (Preparation of B24 bacterial suspension and its drought resistance effect on Leymus chinensis)

[0079] I. Preparation of B24 bacterial suspension

[0080] The activated strain was inoculated into a sterilized LB liquid medium (50 mL) and cultured (constant temperature culture at 30 °C and 180 rpm) for 48 h to prepare a fermentation broth. Centrifuge at 8000 rpm and room temperature for 10 min, suspend and wash twice with sterile water, and finally resuspend with sterile water to prepare a bacterial suspension with an OD 600 = 1.0 for standby. The concentration of the B24 strain in the bacterial suspension is 1×10 8 CFU / mL.

[0081] II. Verification of the growth promotion and drought resistance effects of B24 bacterial suspension on Leymus chinensis

[0082] 1. Experimental materials and methods

[0083] (1) Pot experiment

[0084] The variety of Leymus chinensis seeds used in the experiment was Xiwuzhumuqin Leymus chinensis, provided by the Institute of Grassland Research, Chinese Academy of Agricultural Sciences. Select Leymus chinensis seeds of the same size and plumpness for surface disinfection (soak in 75% ethanol for 2 minutes, rinse 3 times with sterile water, then soak in 2% NaClO solution for 10 minutes, and rinse thoroughly with sterile water), and plant them in plastic flowerpots (height 30 cm; diameter 15 cm). Add 400 g of equal soil samples and 400 g of nutrient soil to each flowerpot. Plant 20 sterile seeds in each pot, and each treatment contains 4 pot replicates. Divide into four treatments, namely: control (CK), inoculated with B24 (B), drought treatment (D), inoculated with B24 under drought (BD). The flowerpots were randomly placed in the greenhouse (16 / 8 h light / dark cycle, 30 °C / 18 °C (day / night), relative humidity 60 - 70%), and the positions were randomly changed every 3 days. One week after emergence, thin out the seedlings, select seedlings with consistent growth, and leave 8 plants in each pot. After growing for 3 days, 80 ml of bacterial liquid was injected into the rhizosphere of plants in each pot of the BD and B groups with a syringe, 10 ml was injected into each Leymus chinensis plant, and the colony concentration was 1×10 8CFU / ml. CK and group D were injected with 80 ml of LB culture solution per pot according to the same method. After 5 days, drought treatment was carried out. The water content of the drought treatment group was maintained at 10% ± 5%, and the soil of the non-drought treatment group was watered every two days, and the water content was controlled at 70% ± 5%. The phenotype was measured every 7 days, and the plant height, stem diameter, number of leaves, leaf width, leaf length, and chlorophyll content of Leymus chinensis were measured. Plant height was measured with a ruler; leaf width and stem diameter were measured with a vernier caliper; the number of leaves was counted by the direct counting method; the chlorophyll content was measured using a (SPAD)-502 Plus chlorophyll meter (Konica Minolta, Japan). After 30 days of inoculation with the bacterial solution, the Leymus chinensis plants were harvested, and the above-ground biomass and underground biomass were measured.

[0085] (2)Measurement of individual plant photosynthesis

[0086] Measurements were carried out from 10:00 to 12:00 am on sunny days using an open gas exchange system (Li-6800) and a 6 cm 2 clip-on leaf chamber (Li-COR Inc., Lincoln, NE, USA), when the photosynthesis of the plants reaches the maximum value in the daily dynamics. Four plants were selected from each pot, and healthy leaves that were completely flat on the plants were selected for gas exchange measurement for each plant. During the measurement, the Li-6800 infrared gas analyzer was set with the specification of light availability (1500 PAR). The gas exchange parameters obtained by measurement include net photosynthetic rate (A), transpiration rate (E), stomatal conductance (gsw), and intercellular carbon dioxide concentration (Ci).

[0087] (3)Measurement of plant physiological indicators

[0088] The contents of plant hormones such as abscisic acid (ABA), jasmonic acid (JA), indole-3-acetic acid (IAA), and gibberellin (GA3) in fresh leaves were measured by liquid chromatography-tandem mass spectrometry (LC-MS / MS) (Rigol L3000, Beijing, China). The activities of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and malondialdehyde (MDA) and ribulose bisphosphate carboxylase (Rubisco) activity in the leaves of Leymus chinensis were measured using the corresponding detection kits (Suzhou Keming Biotechnology Co., Ltd., China).

[0089] 2. Experimental results

[0090] (1)Effects of inoculation with B24 on the morphological indexes, biomass, and chlorophyll content of Leymus chinensis

[0091] As Figure 3 and Figure 4As shown, compared with the control, after inoculating Burkholderia parakusensis B24 of the present invention into the rhizosphere of Leymus chinensis, the plant height, leaf width, aboveground biomass and underground biomass of Leymus chinensis were significantly increased, by 8.67%, 19.42%, 45.69% and 92.06% respectively. In addition, under drought stress, inoculation with B24 significantly increased the plant height, leaf width, stem diameter, number of leaves and underground biomass of Leymus chinensis, by 9.40%, 16.60%, 33.18%, 30.43% and 97.50% respectively. This indicates that whether there is drought or not, inoculation with B24 significantly promotes the growth of Leymus chinensis above and below the ground, but under drought conditions, the promoting effect of inoculating B24 on the growth of Leymus chinensis is more significant.

[0092] (2)Effect of inoculating B24 on photosynthesis of Leymus chinensis

[0093] As Figure 5 shown, compared with the control, after inoculating Burkholderia parakusensis B24 of the present invention, the chlorophyll content and Rubisco enzyme activity of Leymus chinensis were significantly increased, by 15.53% and 92.06% respectively. In addition, under drought stress, inoculation with B24 significantly increased the chlorophyll content of Leymus chinensis by 15.23% and increased the Rubisco enzyme activity of Leymus chinensis by 14.37%. This indicates that whether there is drought or not, inoculation with B24 significantly promotes the photosynthesis of Leymus chinensis.

[0094] (3)Effect of inoculating B24 on individual photosynthesis of Leymus chinensis

[0095] As shown in Table 3, regardless of whether there is drought stress or not, after inoculating B24, the net photosynthetic rate, transpiration rate, stomatal conductance and intercellular CO2 concentration of Leymus chinensis can be significantly increased. High temperature stress will lead to an increase in ROS, damage the chloroplast membrane structure, reduce the photosynthetic efficiency, affect the growth of Leymus chinensis, and under drought conditions, the stomata close, reducing the carbon dioxide fixation ability and accelerating leaf senescence. Inoculating B24 can improve the photosynthetic indexes of plants, improve the photosynthetic efficiency and enhance the tolerance of Leymus chinensis to drought stress.

[0096] Table 3 Effect of inoculating B24 on individual photosynthesis of Leymus chinensis

[0097]

[0098] Note: Different lowercase letters indicate significant differences between groups ( P <0.05).

[0099] (4)Effect of inoculating B24 on Leymus chinensis and antioxidant enzyme activity

[0100] As Figure 6As shown in the figure, there was no significant difference in the effect of inoculating B24 on the antioxidant enzymes in the leaves of Leymus chinensis under non-drought conditions. However, under drought conditions, inoculating B24 significantly reduced the SOD (superoxide dismutase) and POD (peroxidase) in the leaves of Leymus chinensis by 20.23% and 12.29% respectively. Under drought conditions, inoculating B24 reduced the contents of CAT (catalase) and MDA (malondialdehyde) in the leaves of Leymus chinensis by 9.11% and 2.36% respectively, but there was no significant difference. This indicates that with the help of PGPR, plants can more effectively maintain the redox balance within cells, reduce the accumulation of reactive oxygen species, and thus reduce the demand for antioxidant enzymes.

[0101] (5)Effect of inoculating B24 on the proline content of Leymus chinensis

[0102] As Figure 7 shown in the figure, regardless of whether there was drought stress or not, after inoculating B24, the proline content of Leymus chinensis was significantly reduced. This indicates that B24 successfully helped the plants relieve water stress, so that the plants did not need to synthesize a large amount of proline to cope with drought. That is to say, B24 effectively helped the plants relieve drought stress by enhancing the drought resistance of the plants and reducing the synthesis demand of the plants for proline.

[0103] (6)Effect of inoculating B24 on the hormone content of Leymus chinensis

[0104] As Figure 8 shown in the figure, regardless of whether there was drought stress or not, after inoculating B24, the IAA content in the leaves of Leymus chinensis increased significantly, increasing by 132.75% and 146% under non-drought and drought treatments respectively. In addition, under drought stress, the contents of ABA and GA3 decreased significantly, by 63.71% and 50.73% respectively, and the JA content decreased by 15.37%. This indicates that B24 effectively relieved drought stress and reduced the damage of plant cells under stress conditions by regulating the hormone levels of plants, especially IAA and ABA.

[0105] In summary, the Paraburkholderia graminis provided by the present invention has strong characteristics of decomposing inorganic phosphorus, nitrogen fixation, producing IAA, and producing ACC deaminase, and can significantly promote the growth of Leymus chinensis, promote the absorption and utilization of soil nutrients by plants, and thus improve the quality and yield of Leymus chinensis. Under drought conditions, it further promoted the growth of Leymus chinensis, relieved drought stress by significantly reducing the proline content, antioxidant enzyme activity and changing the hormone content in the leaves of Leymus chinensis; in addition, under drought stress, it significantly increased the chlorophyll content, net photosynthetic rate, transpiration rate and stomatal conductance of Leymus chinensis, enhanced the photosynthesis of Leymus chinensis, and thus significantly improved the drought resistance of Leymus chinensis.

[0106] Burkholderia paramultivorans B24 of the present invention provides excellent plant growth-promoting rhizobacteria for multifunctional growth-promoting bacteria, and also provides excellent strain resources for improving the drought resistance of Leymus chinensis, a grassland plant. It can be used to prepare microbial fertilizers for grassland plants, showing potential application prospects in aspects such as soil phosphorus activation, plant growth promotion, yield increase, drought resistance, and grassland degradation restoration.

Claims

1. A rhizosphere growth-promoting bacterium of Leymus chinensis, characterized in that: The rhizosphere growth-promoting bacteria are classified as Paraburkholderia graminis , strain number B24, was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 9, 2024, with the deposit number CGMCC No.31907.

2. Microbial fertilizer, comprising the rhizosphere growth-promoting bacteria of Leymus chinensis according to claim 1.

3. Use of the rhizosphere growth-promoting bacteria of Leymus chinensis according to claim 1 or the microbial fertilizer according to claim 2 in promoting the growth of Leymus chinensis.

4. Use of the rhizosphere growth-promoting bacteria of Leymus chinensis according to claim 1 or the microbial fertilizer according to claim 2 in improving the drought resistance of Leymus chinensis.

5. Use of the rhizosphere growth-promoting bacteria of Leymus chinensis according to claim 1 or the microbial fertilizer according to claim 2 in at least one of the following: production of indoleacetic acid, synthesis of ACC deaminase, nitrogen fixation and degradation of inorganic phosphorus.

6. Use of the rhizosphere growth-promoting bacteria of sheep fescue according to claim 1 or the microbial fertilizer according to claim 2 in at least one of the following: increasing the chlorophyll content of sheep fescue, increasing the activity of Rubisco enzyme of sheep fescue, reducing the activity of antioxidant enzymes of sheep fescue, reducing the proline content of sheep fescue, and promoting the secretion of indoleacetic acid by sheep fescue.

7. A method for promoting the growth of Leymus chinensis and / or improving the drought resistance of Leymus chinensis, characterized in that: Apply the rhizosphere growth-promoting bacteria described in claim 1 or the microbial fertilizer described in claim 2 to the rhizosphere of Leymus chinensis.

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