Leymus chinensis rhizosphere growth-promoting bacterium and application thereof in growth promotion and drought resistance of Leymus chinensis

Abstract

The invention relates to a Chinese wildrye rhizosphere growth-promoting bacterium and application of the Chinese wildrye rhizosphere growth-promoting bacterium in growth promotion and drought resistance of Chinese wildrye, and belongs to the technical field of microorganisms, the strain is classified and named as Paraburkholderia graminis, the strain number is B24, and the preservation number is CGMCC No.31907. The strain provided by the invention is derived from the intergrassland soil of leymus chinensis in an excessive grazing grassland, has good growth promoting characteristics of producing IAA, producing ACC deaminase, fixing nitrogen, dissolving phosphorus and the like, and also can obviously enhance the drought resistance of the leymus chinensis. When the strain is inoculated to the rhizosphere of a leymus chinensis seedling, the growth of the leymus chinensis can be obviously promoted, and the overground and underground biomass of the leymus chinensis is increased; the activity of antioxidant enzymes is reduced and the hormone content in leymus chinensis leaves is changed, so that drought stress is relieved, the photosynthesis of leymus chinensis is enhanced, and the drought resistance of leymus chinensis is remarkably improved. Therefore, the Burkholderia parakholderia can be used for development of a microbial fertilizer for grassland plants and applied to a drought stress habitat, so that the drought resistance of the grassland plant leymus chinensis is improved, and grassland management is optimized.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to a plant rhizosphere growth-promoting bacterium, in particular to a paraburkholderia B24 and an application thereof in growth promotion and drought resistance in a grassland plant Leymus chinensis. Background Art

[0002] Plant Growth-Promoting Rhizobacteria (PGPR) refers to a class of microorganisms that live in or inside the rhizosphere of plants and can directly or indirectly promote plant growth through a variety of mechanisms. These mechanisms mainly include nitrogen fixation, phosphorus solubilization, plant hormone production, and induced systemic resistance (ISR), which enhance the tolerance of plants to environmental stresses such as drought, salinity, and high temperature, thereby improving plant stress resistance. Therefore, PGPR is widely used in 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 contaminated soils. With the deepening of research on PGPR, its potential in molecular mechanisms, bacterial 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 an inoculant has the advantages of being environmentally friendly and cost-effective.

[0003] Drought stress is one of the main environmental stresses that affect plant growth and development. Its effects on plants are multifaceted, including physiological, biochemical, molecular and morphological levels. Drought stress inhibits the photosynthetic efficiency and stomatal conductance of plants, thereby reducing carbon assimilation. Under drought conditions, a large amount of reactive oxygen species (ROS) will be produced 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. In order to adapt to drought stress, plants will initiate a series of stress resistance regulatory mechanisms, such as closing stomata by activating the abscisic acid (ABA) signaling pathway to reduce water transpiration; increasing the accumulation of osmotic regulating substances (such as proline and soluble sugars) to maintain cell osmotic balance. Inoculation of growth-promoting rhizobacteria (PGPR) under drought stress has a significant positive effect on plants and is an effective strategy to alleviate the negative impact of drought stress on plants. PGPR can promote the water use efficiency of plants and improve the plant's ability to absorb limited water resources by enhancing the development of plant roots and expanding the root absorption range. PGPR can also regulate the opening and closing of plant stomata and the growth and development process by secreting plant hormones such as indoleacetic acid (IAA), cytokinin and abscisic acid (ABA), thereby reducing water transpiration loss and optimizing physiological metabolism. In addition, PGPR can enhance the osmotic regulation ability of plants, and its secreted metabolites such as proline and soluble sugars can help plants maintain water balance in cells and reduce cell dehydration and stress damage caused by drought stress. PGPR can also activate the plant antioxidant system, remove the accumulation of reactive oxygen 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 by inducing systemic resistance (ISR), making them show stronger tolerance and adaptability under drought conditions. PGPR can also improve the soil microenvironment, for example, by improving the availability of soil nutrients through nitrogen fixation, phosphorus dissolution, potassium decomposition and other functions, providing plants with more sufficient nutrient support. Although the plant growth promotion and stress resistance mechanisms mediated by PGPR are not fully understood, related research mainly focuses on crops. A large number of literatures have shown that PGPR inoculation can promote plant nutrient absorption, growth, yield, stress resistance and plant hormone homeostasis. PGPR inoculation can effectively alleviate the adverse effects of drought stress on plants, promote plant growth and development, improve plant adaptability to drought environments, and provide new solutions for the sustainable development of agricultural ecosystems.

[0004] Leymus chinensis Leymus chinensis) is a perennial herb that is drought-resistant, cold-resistant, and salt-alkali-tolerant, and is widely distributed in grasslands and semi-arid areas. At the same time, Leymus chinensis has high nutritional value and palatability, and is an important feed for livestock. Leymus chinensis also has strong stress resistance and ecological adaptability, and can grow in degraded grasslands, saline-alkali lands, and semi-arid areas. It is a high-quality grass species for grassland ecological restoration and vegetation restoration. By studying the role of PGPR in promoting the growth of Leymus chinensis, it is not only helpful to increase forage yields, but also to reduce the use of chemical fertilizers and promote sustainable agricultural development. In arid areas or degraded grasslands, drought-resistant Leymus chinensis can better adapt to harsh environments, restore vegetation coverage, and prevent further desertification of the land. By studying technologies to improve the drought resistance of Leymus chinensis (such as the addition of bacterial liquid or bacterial fertilizer, etc.), technical support can be provided for the restoration of ecosystems, especially for vegetation restoration in arid and semi-arid areas. Summary of the invention

[0005] In view of the increasingly serious situation of large-scale grassland degradation and drought problems, the present invention provides a strain of Burkholderia parasitica suitable for use in drought stress habitats. The strain effectively solves the problem of converting insoluble phosphates into soluble phosphates and producing the plant hormone IAA. It also has growth-promoting properties such as nitrogen fixation and production of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, and can significantly promote the growth of sheepgrass under drought conditions and improve the drought resistance of sheepgrass. Based on this, the present invention isolated a strain of Burkholderia parasitica B24 from the rhizosphere of grazing degraded grassland sheepgrass. After inoculation, the strain not only has a growth-promoting function for sheepgrass, but also can significantly improve the drought resistance of sheepgrass. It is a strain with great application potential, providing strain resources and theoretical basis for the subsequent analysis of plant drought resistance mechanisms, optimization of grassland management, restoration of degraded grasslands, and the development of microbial fertilizers. Specifically, the present invention adopts the following technical solution: the strain of the present invention has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration (referred to as CGMCC, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101) on September 9, 2024, and is classified and named Paraburkholderia graminis (Burkholderia parasitica), strain number B24, deposit number CGMCC No.31907.

[0006] The colony characteristics and bacterial morphology of the para-Burkholderia B24: The colonies of B24 on the LB plate are light yellow, the texture of the colonies is relatively moist, and the edges are relatively smooth. Under a microscope, the bacteria are spherical, 0.5~0.8um×0.5~0.8um, red, and Gram-negative. It can grow under both normoxic and anaerobic conditions, but it grows better under normoxic conditions, and is a facultative aerobic bacterium.

[0007] The physiological and biochemical characteristics of the paraburkholderia are as follows: the B24 strain has a positive catalase reaction, glucose oxidation is a fermentation type, the hydrogen sulfide test is negative, the methyl red test is negative, the VP assay is weakly positive, the starch hydrolysis test is negative, the nitrate reduction test is weakly positive, the citrate utilization test is positive, and the glucose gas production is positive.

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

[0009] The 16S rDNA sequence of strain B24 was compared with the sequences in the GenBank database by BLAST. 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 identified as Paraburkholderia graminis.

[0010] A transparent circle of 1.77 ± 0.28 cm was formed around the colony of the paraburkholderia B24, indicating that the strain can degrade insoluble phosphate into a form that can be absorbed and utilized by plants, and the degradation amount is 179.90 ± 2.59 μg / mL, thereby improving the plant's ability to obtain nutrients and promoting the growth and development of the plant root system.

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

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

[0013] The Burkholderia parakholderiae B24 has the ability to produce indoleacetic acid (IAA) with a yield of 25.33 ± 3.48 μg / mL.

[0014] Obviously, the fermentation product of strain B24 and the gene construct based on the gene of strain B24 also have the above-mentioned functions of degrading inorganic phosphorus, fixing nitrogen, producing indoleacetic acid, and synthesizing ACC deaminase. Further, strain B24 and the fermentation product can also be prepared into microbial fertilizer or microbial agent for large-scale application.

[0015] The application method of the rhizosphere growth-promoting bacteria B24 of the present invention for promoting the growth of sheep fescue and improving the drought resistance of sheep fescue can be applied by preparing a suspension of rhizosphere growth-promoting bacteria of sheep fescue, and the preparation thereof can include the following steps: inoculating the strain into LB liquid culture medium and culturing (constant temperature culture at 30°C and 180rpm) for 48 hours to prepare a fermentation liquid, centrifuging at 8000 rpm and room temperature for 10 minutes, suspending and washing twice with sterile water, and finally resuspending with sterile water to prepare an OD 600 =1.0 bacterial suspension for standby use, 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 rhizosphere growth-promoting bacteria or gene constructs or fermentation products or microbial fertilizers to the rhizosphere of Leymus chinensis. The application to the rhizosphere of Leymus chinensis includes any one or more application methods of spraying, irrigation, and burying.

[0017] The para-Burkholderia B24 of the present invention is separated from the rhizosphere soil of Leymus chinensis in an overgrazing plot, and is obtained by artificial separation and purification. Drought stress is effectively alleviated by improving the water use efficiency of plants, promoting root growth, promoting light action, regulating proline metabolism and plant hormone levels, and reducing the occurrence of oxidative damage. It reflects the protective effect of B24 on plants, helping plants to better adapt to drought conditions. Therefore, the para-Burkholderia B24 of the present invention has a strong growth-promoting function, has a significant growth-promoting effect on the aboveground and underground roots of Leymus chinensis, and can significantly improve the drought resistance of Leymus chinensis, and can be used as a microbial inoculant in arid habitats, providing strain resources and theoretical basis for subsequent plant drought resistance mechanism analysis, optimization of grassland management, restoration of degraded grasslands, and the development of microbial fertilizers.

[0018] Beneficial effects of the present invention: Under potted conditions, the Burkholderia parabacterium B24 of the present invention promotes the growth of Leymus chinensis under drought stress by increasing the plant height, stem diameter, leaf number, leaf width, chlorophyll content, aboveground 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 photosynthesis of Leymus chinensis.

[0019] The Burkholderia parabacterium B24 effectively helps plants alleviate drought stress by reducing stress-related hormone content, antioxidant enzyme activity and proline content under drought stress, allowing plants to reduce their dependence on proline or respond to drought stress through other mechanisms (such as improving water absorption and producing other protective substances). This shows that PGPR B24 can help plants better adapt to drought environments by promoting plant growth, improving drought resistance or regulating plant metabolic pathways.

[0020] The Burkholderia parasitica provided by the invention can provide a new strain resource for the microbial fertilizer industry, and shows potential application prospects in soil phosphorus activation, plant growth promotion, yield increase, drought resistance and grassland degradation restoration. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The colony morphology and optical microscope morphology of strain B24 of the present invention are shown (the scale bar in the lower right corner of the left picture is 10 μm).

[0022] Figure 2 This is the phylogenetic tree of B24 based on 16S rDNA.

[0023] Figure 3 The figure shows the effect of strain B24 of the present invention on the morphological indicators of Leymus chinensis (plant height, leaf width, stem thickness, and number of leaves).

[0024] Figure 4 This is the effect of strain B24 of the present invention on the aboveground and underground biomass of Leymus chinensis.

[0025] Figure 5 The figure shows the effect of strain B24 of the present invention on the chlorophyll content and ribulose bisphosphate carboxylase (Rubisco) activity of Leymus chinensis.

[0026] Figure 6 This is the effect of strain B24 on the activity of antioxidant enzymes (superoxide dismutase, peroxidase, catalase, and malondialdehyde) of the present invention.

[0027] Figure 7 This is the effect of strain B24 of the present invention on the proline content of Leymus chinensis.

[0028] Figure 8 The figure shows the effect of strain B24 of the present invention on the content of hormones (abscisic acid, indoleacetic acid, gibberellin and jasmonic acid) in leaves of Leymus chinensis. DETAILED DESCRIPTION

[0029] Example 1 (Screening, identification and growth-promoting characteristics of drought-resistant rhizosphere strains of Leymus chinensis) 1. Test materials (1) Collection of soil samples The rhizosphere soil samples of Leymus chinensis were collected from the long-term overgrazed grassland in Bayinxile, Ordos City, Inner Mongolia. About 10 Leymus chinensis plants were carefully dug 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 to be the rhizosphere soil, which was collected into a sterile self-sealing bag with a brush, stored in a refrigerator at 4°C, and the strain was isolated and purified as soon as possible.

[0030] (2) Preparation of culture medium and reagents ①LB solid medium Tryptone 10.0 g, yeast powder 5.0 g, NaCl 10 g, agar 15 g, distilled water 1000 mL, pH = 7.0-7.2, sterilize at 121℃ for 20 min.

[0031] ②Axubei solid culture medium KH2PO4 0.2g, CaCO3 5.0g, MgSO4·7H2O 0.2g, glucose 10.0g, NaCl 0.2g, CaSO4·2H2O 0.1g, agar 18.0g, distilled water 1L, pH=6.8-7.0 ③PKO solid medium 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 ④Reagents Salkowski color developer: slowly pour 150 mL H2SO4 into 250 mL distilled water, wait for the H2SO4 solution to cool, then add 7.5 mL 0.5 M FeCI3·6H2O solution to prepare the color developer. This color developer cannot be stored for a long time.

[0032] 2. Test methods (1) Initial screening of drought-resistant strains Under sterile conditions, 10 g of rhizosphere soil of Leymus chinensis was weighed and dissolved in 90 mL of sterile water. The mixture was shaken on a vibrating screen at 120 r / min for 1 h and then graded and diluted to 10 -3 , 10 -4 , 10 -5 , 10 -6Take 50 μL of each dilution and spread it evenly on LB solid medium with different concentrations of PEG (polyethylene glycol) (5%-15%), and culture it in a 30°C incubator. The grown single colonies with different morphologies are streaked for further purification until they are completely purified.

[0033] (2) Screening of phosphate-solubilizing and nitrogen-fixing strains The purified single colony was spotted on inorganic phosphorus solid medium and Axubei nitrogen-free medium and cultured in an incubator at 30°C. Among them, B24 can produce both phosphate-dissolving circles on inorganic phosphorus solid medium and nitrogen-fixing circles on Axubei medium, and was transferred into a glycerol tube with a final concentration of 30% and stored in a -20°C refrigerator.

[0034] (2) Determination of IAA production characteristics Using the Salkowski colorimetric method, the strain was inoculated into LB liquid medium containing L-tryptophan (100 mg / L) and cultured at 30°C and 200 r / min for 24 h. The bacterial suspension was centrifuged at 4°C and 5000 rpm for 10 minutes, 1 mL of the supernatant was added to 3 mL of the Salkowski colorimetric agent, and the mixed solution of the uninoculated LB liquid medium and the colorimetric agent was used as a blank control. After being placed in the dark at room temperature for 30 min, the color turned red, indicating that IAA could be secreted. The IAA standard solution was prepared into different concentrations to make a standard curve, and the OD of the reaction solution was measured after the reaction under the above conditions. 530 , calculate the concentration of IAA in the supernatant.

[0035] (4) Determination of phosphate solubilization capacity The phosphate solubilizing ability of the strain was determined by qualitative and quantitative methods.

[0036] Qualitative determination by phosphate solubilization circle method: strain B24 was inoculated on PKO inorganic phosphorus solid culture medium, with 3 replicates for each strain. The diameter of the phosphate solubilization circle (D) and colony diameter (d) formed by each strain was measured, and the phosphate solubility index PSI (D / d) value was calculated to make a preliminary judgment on the phosphate solubilization ability of the strain.

[0037] Quantitative determination of molybdenum antimony resistance by colorimetric method: The activated strain (1 mL) was inoculated into PKO medium (50 mL), and the control group was inoculated with an equal amount of sterile water. -1 The cells were cultured at constant temperature for 7 days and then at 11000 r·min -1Centrifuge for 5 min and take the supernatant. Add 1-2 drops of dinitrophenol indicator to the supernatant (30 mL), and adjust with a small amount of NaOH solution (1M, 10 M) and HCl solution (1 M, 5 M) until the solution just turns slightly yellow, then accurately add 5 mL of molybdenum antimony mixed color development solution, shake well, and make up to 50 mL with deionized water. After reacting at room temperature above 15°C for 30 min (within 8 h), measure and record the OD 700 The phosphorus content (mg·L -1 ).

[0038] (5) Determination of nitrogenase activity of strains The nitrogenase (NITS) enzyme-linked immunosorbent assay kit was used for determination. The kit uses the double antibody sandwich method to determine the nitrogenase (NITS) level in the sample. The microplate was coated with purified nitrogenase (NITS) antibodies to prepare solid-phase antibodies. Nitrogenase (NITS) was added to the microwells coated with monoclonal antibodies in turn, and then combined with HRP-labeled nitrogenase (NITS) antibodies to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB was added for color development. TMB was converted into blue under the catalysis of HRP enzyme, and converted into the final yellow under the action of acid. The depth of color was positively correlated with the nitrogenase (NITS) in the sample. The absorbance (OD value) was measured at a wavelength of 450 nm using an enzyme reader, and the nitrogenase (NITS) activity concentration in the sample was calculated using the standard curve.

[0039] (6) Determination of ACC deaminase activity of strains The ACC deaminase (ACCD) enzyme-linked immunosorbent assay kit was used for determination. The kit uses a double antibody sandwich method to determine the level of ACC deaminase (ACCD) in the sample. The microplate is coated with purified ACC deaminase (ACCD) antibody to prepare a solid-phase antibody. ACC deaminase (ACCD) is added to the microwells coated with the monoclonal antibody in sequence, and then combined with the HRP-labeled ACC deaminase (ACCD) antibody to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB is added for color development. TMB is converted into blue under the catalysis of HRP enzyme and converted into the final yellow under the action of acid. The depth of color is positively correlated with the ACC deaminase (ACCD) in the sample. The absorbance (OD value) is measured at a wavelength of 450 nm using an enzyme reader, and the ACC deaminase (ACCD) activity concentration in the sample is calculated using the standard curve.

[0040] (7) Physiological and biochemical characteristics According to the Manual of Identification of Common Bacteria Systems, a series of physiological and biochemical characteristics of B24 were tested, including Gram staining, catalase, glucose oxidation and fermentation, methyl red, VP, nitrate reduction, oxidase, starch hydrolysis, H2S, citrate utilization, glucose gas production, indole test, and gelatin liquefaction test.

[0041] (8) 16S rDNA sequence identification Total bacterial DNA was extracted, and 16S rDNA was amplified using universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5 '-TACCTTGTTACGACTT-3′). The PCR amplification product was sent to Nanjing Qingke Technology Co., Ltd. for sequencing, and the primers used were synthesized by Nanjing Qingke Technology Co., Ltd. The sequence obtained by sequencing was submitted to the GenBank gene library of the online website NCBI (https: / / www.ncbi.nlm.nih.gov / ) for homology search and comparison.

[0042] 3. Identification results of strain B24 (1) Morphological characteristics identification results like Figure 1 As shown in the figure, the colonies of Burkholderia parabacterium B24 on the LB plate are light yellow, moist in texture, and smooth at the edges. Under the microscope, the bacteria are spherical, 0.5~0.8um×0.5~0.8um, and appear red. They are Gram-negative facultative aerobic bacteria.

[0043] (2) Identification of physiological and biochemical characteristics As shown in Table 1 , the catalase reaction of strain B24 was positive, glucose oxidation was fermentative, hydrogen sulfide test was negative, methyl red test was negative, VP assay was weakly positive, starch hydrolysis test was negative, nitrate reduction test was weakly positive, citrate utilization test was positive, and glucose gas production was positive.

[0044] Table 1 Identification results of physiological and biochemical characteristics of strain B24

[0045] Note: “+” means the reaction is positive or the plant can grow and be used; “-” means the reaction is negative or the plant cannot grow and be used.

[0046] (3) 16S rDNA sequence identification results The 16SrDNA sequence of strain B24 was amplified and sequenced, and the sequencing result is shown in SEQ ID NO: 1 in the sequence list. The sequence was compared with the sequence in the GenBank database by BLAST, and the results showed that strain B24 had the highest homology with Paraburkholderia graminis strain, which was 99.92%. Figure 2 The results of phylogenetic analysis showed that B24 had a 100% similarity with Paraburkholderia graminis strain. Based on the 16S rDNA sequence, combined with phylogenetic analysis, morphology, physiological and biochemical characteristics, B24 was identified as Paraburkholderia graminis. The strain was deposited in the General Microbiology Center of China Microbiological Culture Collection (CGMCC, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101) on September 9, 2024, with the deposit number CGMCC No. 31907.

[0047] The 16S rDNA gene sequence of the paraburkholderia is shown in SEQ ID No. 1: (4) Growth-promoting properties The results of the growth-promoting properties of strain B24 are shown in Table 2 , indicating that the strain has high phosphate solubilization, nitrogen fixation, IAA production and ACC deaminase activity.

[0048] Table 2 Identification results of growth-promoting properties of strain B24

[0049] Example 2 (Preparation of B24 bacterial suspension and its drought resistance effect on Leymus chinensis) 1. Preparation of B24 strain suspension The activated strain was inoculated into sterile LB liquid medium (50 mL) and cultured (30°C, 180 rpm constant temperature culture) for 48 h to prepare a fermentation broth. Centrifuged at 8000 rpm and room temperature for 10 min, washed twice with sterile water, and finally resuspended with sterile water to prepare OD 600 =1.0 bacterial suspension for standby use, the concentration of the B24 strain in the bacterial suspension is 1×10 8 CFU / mL.

[0050] 2. Verification of the effect of B24 bacterial suspension on the growth promotion and drought resistance of Leymus chinensis 1. Experimental materials and methods (1) Potted plant test The variety of Leymus chinensis seeds used in the experiment was Xiwuzhumuqin Leymus chinensis, which was provided by the Grassland Research Institute of the Chinese Academy of Agricultural Sciences. Select the same size and full Leymus chinensis seeds for surface disinfection (soak in 75% ethanol for 2 minutes, rinse with sterile water 3 times, soak in 2% NaClO solution for 10 minutes, and rinse thoroughly with sterile water), and plant them in plastic pots (30 cm high; 15 cm in diameter). Each pot is added with 400g of equal amount of soil sample and 400g of nutrient soil. Twenty sterile seeds were planted in each pot, and each treatment contained 4 pots of replication. It was divided into four treatments: control (CK), inoculation with B24 (B), drought treatment (D), and inoculation with B24 under drought (BD). The pots were randomly placed in the greenhouse (16 / 8 h light / dark cycle, 30°C / 18°C (day / night), relative humidity 60-70%), and the position was randomly changed every 3 days. One week after the emergence of the seedlings, thinning was carried out, and seedlings with consistent growth were selected, leaving 8 plants in each pot. After 3 days of growth, 80 ml of bacterial solution was injected into the rhizosphere of each pot in groups BD and B, and 10 ml was injected into each plant of Leymus chinensis. The colony concentration was 1×10 8CFU / ml. CK and D groups were injected with 80 ml LB culture solution per pot in the same way. Drought treatment was carried out 5 days later. The drought treatment group maintained a water content of 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; measured with a ruler; leaf width and stem diameter were measured with a vernier caliper; the number of leaves was counted by direct counting method; chlorophyll content was measured using a (SPAD)-502 Plus chlorophyll meter (Konica Minolta, Japan). Leymus chinensis plants were harvested 30 days after inoculation with the bacterial solution, and the aboveground biomass and underground biomass were measured.

[0051] (2) Determination of photosynthesis of individual plants By using an open gas exchange system (Li-6800) and 6 cm 2 The leaf cup (Li-COR Inc., Lincoln, NE, USA) was measured on a clear day from 10:00 to 12:00 am, when the photosynthesis of the plant reached its maximum in the daily dynamics. Four plants were selected from each pot, and the fully expanded healthy leaves on each plant were selected for gas exchange measurement. During the measurement, the Li-6800 infrared gas analyzer was set to the specification of light availability (1500PAR). The gas exchange parameters obtained by measurement included net photosynthetic rate (A), transpiration rate (E), stomatal conductance (gsw), and intercellular carbon dioxide concentration (Ci).

[0052] (3) Determination of plant physiological indicators Liquid chromatography-tandem mass spectrometry (LC-MS / MS) (Rigol L3000, Beijing, China) was used to determine the contents of plant hormones such as abscisic acid (ABA), jasmonic acid (JA), indole-3-acetic acid (IAA) and gibberellin (GA3) in fresh leaves. The activities of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), malondialdehyde (MDA) and ribulose diphosphate carboxylase (Rubisco) in leaves of Leymus chinensis were determined using corresponding detection kits (Suzhou Keming Biotechnology Co., Ltd., China).

[0053] 2. Test results (1) Effects of inoculation with B24 on morphological indicators, biomass and chlorophyll content of Leymus chinensis like Figure 3 and Figure 4As shown, compared with the control, after the paraburkholderia B24 of the present invention was inoculated into the rhizosphere of leymus chinensis, the plant height, leaf width, aboveground biomass and underground biomass of leymus chinensis were significantly increased, increasing by 8.67%, 19.42%, 45.69% and 92.06% respectively. In addition, under drought stress, B24 inoculation significantly increased the plant height, leaf width, stem thickness, leaf number and underground biomass of leymus chinensis, increasing by 9.40%, 16.60%, 33.18%, 30.43% and 97.50% respectively. This shows that regardless of drought, B24 inoculation significantly promotes the growth of leymus chinensis above and below ground, but under drought conditions, the promotion effect of inoculation of B24 on the growth of leymus chinensis is more significant.

[0054] (2) Effect of B24 inoculation on photosynthesis of Leymus chinensis like Figure 5 As shown, compared with the control, the inoculation of the paraburkholderia B24 of the present invention significantly increased the chlorophyll content and Rubisco enzyme activity of Leymus chinensis by 15.53% and 92.06%, respectively. In addition, under drought stress, B24 inoculation significantly increased the chlorophyll content of Leymus chinensis by 15.23% and increased the Rubisco enzyme activity of Leymus chinensis by 14.37%. This shows that regardless of drought, B24 inoculation significantly promotes photosynthesis of Leymus chinensis.

[0055] (3) Effect of B24 inoculation on photosynthesis of individual Leymus chinensis As shown in Table 3, regardless of whether there is drought stress or not, inoculation with B24 can significantly increase the net photosynthetic rate, transpiration rate, stomatal conductance and intercellular CO2 concentration of Leymus chinensis. High temperature stress will increase ROS, destroy the chloroplast membrane structure, reduce photosynthetic efficiency, and affect the growth of Leymus chinensis. In addition, under drought conditions, stomata are closed, the ability to fix carbon dioxide is reduced, and leaf senescence is accelerated. Inoculation with B24 can improve the photosynthetic index of plants, improve photosynthetic efficiency, and enhance the tolerance of Leymus chinensis to drought stress.

[0056] Table 3 Effects of inoculation with B24 on photosynthesis of individual Leymus chinensis

[0057] Note: Different lowercase letters indicate significant differences among groups ( P <0 .05).

[0058] (4) Effects of B24 inoculation on Leymus chinensis and antioxidant enzyme activities like Figure 6As shown in the figure, there was no significant difference in the effect of B24 inoculation on the antioxidant enzymes in the leaves of Leymus chinensis under non-drought conditions, but B24 inoculation under drought conditions significantly reduced the SOD (superoxide dismutase) and POD (peroxidase) in the leaves of Leymus chinensis by 20.23% and 12.29%, respectively. Inoculation with B24 under drought conditions reduced the CAT (catalase) and MDA (malondialdehyde) contents in the leaves of Leymus chinensis by 9.11% and 2.36%, respectively, but there was no significant difference. This shows that with the help of PGPR, plants can more effectively maintain the redox balance in cells, reduce the accumulation of reactive oxygen, and thus reduce the demand for antioxidant enzymes.

[0059] (5) Effect of inoculation with B24 on the proline content of Leymus chinensis like Figure 7 As shown in the figure, regardless of whether there is drought stress or not, the proline content of Leymus chinensis was significantly reduced after inoculation with B24, which shows that B24 successfully helped the plant alleviate water stress, so that the plant did not need to synthesize a large amount of proline to cope with drought. In other words, B24 effectively helped the plant alleviate drought stress by enhancing the plant's drought resistance and reducing the plant's demand for proline synthesis.

[0060] (6) Effect of inoculation with B24 on hormone content in Leymus chinensis like Figure 8 As shown in the figure, regardless of whether there is drought stress or not, the IAA content of the leaves of Leymus chinensis increased significantly after inoculation with B24, which increased by 132.75% and 146% under non-drought and drought treatments, respectively. In addition, the ABA and GA3 contents were significantly reduced under drought stress, by 63.71% and 50.73%, respectively, and the JA content was reduced by 15.37%. This shows that B24 effectively alleviates drought stress and reduces the damage of plant cells under stress conditions by regulating the hormone levels of plants, especially IAA and ABA.

[0061] In summary, the Paraburkholderia graminis provided by the present invention has strong characteristics of decomposing inorganic phosphorus, fixing nitrogen, producing IAA, and producing ACC deaminase, which can significantly promote the growth of sheep grass, promote the absorption and utilization of soil nutrients by plants, thereby improving the quality and yield of sheep grass. The growth of sheep grass is further promoted under drought conditions, and drought stress is alleviated by significantly reducing the proline content, antioxidant enzyme activity and changing the hormone content in the leaves of sheep grass; in addition, the chlorophyll content, net photosynthetic rate, transpiration rate and stomatal conductance of sheep grass are significantly increased under drought stress, and the photosynthesis of sheep grass is enhanced, thereby significantly improving the drought resistance of sheep grass.

[0062] The Burkholderia parakholderiae B24 of the present invention provides an excellent plant rhizosphere growth-promoting bacteria species for multifunctional growth-promoting bacteria, and also provides an excellent strain resource for improving the drought resistance of the grassland plant Leymus chinensis. It can be used to prepare microbial fertilizers for grassland plants, and shows potential application prospects in 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. The 16SrDNA encoding gene of the rhizosphere growth-promoting bacteria of Leymus chinensis according to claim 1 is SEQ ID No. 1 in the sequence table.

3. A fermentation product obtained by fermentation culture of the rhizosphere growth-promoting bacteria of Leymus chinensis according to claim 1.

4. The fermentation and culturing method of the fermentation product according to claim 3, characterized in that: The rhizosphere growth-promoting bacteria of Leymus chinensis described in claim 1 are cultured in LB liquid culture medium at 30° C. and 180 r / min for 48 h.

5. A microbial fertilizer comprising the rhizosphere growth-promoting bacteria of Leymus chinensis according to claim 1, or the fermentation product according to claim 3, or the fermentation product prepared by the method according to claim 4.

6. Use of the rhizosphere growth-promoting bacteria of Leymus chinensis according to claim 1, the fermentation product according to claim 3, or the microbial fertilizer according to claim 5 in promoting the growth of Leymus chinensis.

7. Use of the rhizosphere growth-promoting bacteria of Leymus chinensis according to claim 1, the fermentation product according to claim 3, or the microbial fertilizer according to claim 5 in improving the drought resistance of Leymus chinensis.

8. Use of the rhizosphere growth-promoting bacteria of sheep fescue according to claim 1, the fermentation product according to claim 3, or the microbial fertilizer according to claim 5 in at least one of the following: production of indoleacetic acid, synthesis of ACC deaminase, nitrogen fixation, and degradation of inorganic phosphorus.

9. Use of the rhizosphere growth-promoting bacteria of sheep fescue according to claim 1 or the fermentation product according to claim 3 or the microbial fertilizer according to claim 5 in at least one of the following: increasing the chlorophyll content of sheep fescue, increasing the Rubisco enzyme activity of sheep fescue, reducing the antioxidant enzyme activity of sheep fescue, reducing the proline content of sheep fescue, and regulating the hormone content of sheep fescue.

10. A method for promoting the growth of Leymus chinensis and / or improving the drought resistance of Leymus chinensis, characterized in that: The rhizosphere growth-promoting bacteria according to claim 1, the fermentation product according to claim 3, or the microbial fertilizer according to claim 5 is applied to the rhizosphere of Leymus chinensis.

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