A kind of rhizosphere growth-promoting bacteria of Leymus chinensis and its application

Abstract

The invention discloses a rhizosphere growth-promoting bacterium of sheep grass and its application, belonging to the field of microbial technology. The strain is classified and named as Phyllobacterium myrsinacearum, strain number B68, and deposit number CGMCC No.31906. The strain of the present invention is separated from the rhizosphere soil of sheep grass in an overgrazing sample, and is obtained by artificial separation and purification. The strain has high yield of 3-indoleacetic acid, has strong solubility for insoluble phosphates, and has good growth-promoting properties such as nitrogen fixation, ACC deaminase production, and IAA production. The prepared strain fermentation liquid is inoculated into sheep grass seedlings, and the strain can be colonized in the rhizosphere of sheep grass, regulate the bacterial community structure of rhizosphere soil of sheep grass; significantly promote the growth of sheep grass, enhance photosynthesis, and increase chlorophyll content; significantly increase the hormone and nutrient content in plant leaves and roots; significantly increase the content of available phosphorus, total carbon and organic matter in rhizosphere soil. Therefore, the myrsinacearum leaf bacillus of the present invention can be used for the development and related research of subsequent microbial fertilizers for grazing grasslands.

Description

Technical Field

[0001] The invention belongs to the field of microbial technology, and specifically relates to the field of growth-promoting rhizobacteria (PGPR), and in particular to a rhizosphere growth-promoting Bacillus argyi and its application in promoting the growth of grassland plant Leymus chinensis. Background Art

[0002] Leymus chinensis Leymus chinensis ) is a rhizome-type perennial grass plant, and is also one of the main forage and dominant plants in the grasslands of eastern Eurasia. At the same time, sheepgrass has high nutritional value and palatability. It is a high-quality grass species that is beneficial to grassland ecological restoration. It has the characteristics of salt-alkali resistance, barrenness resistance, cold resistance, drought resistance, and wind and sand resistance. Through the continuous growth and reproduction of the main stem, tillers, and underground horizontal rhizomes, the aboveground coverage and biomass of sheepgrass have increased significantly, and the underground root network is dense, thereby increasing the carbon sink of degraded grasslands, which has important ecological and economic value. With the continuous improvement of the level of economic and social development, residents' demand for high-quality protein sources such as meat, eggs, and milk has gradually increased, which in turn has led to a corresponding increase in demand for forage, and grassland degradation has also become increasingly severe. Therefore, promoting the growth and development of sheepgrass, a native grass species in domestic grasslands, is of great significance to promoting the development of the grass seed industry, effectively repairing and managing degraded grasslands, and protecting and restoring ecosystems.

[0003] Growth-promoting rhizobacteria (PGPR) are a type of beneficial bacteria that live in the rhizosphere of plants, promote plant nutrient absorption and growth, and inhibit harmful organisms. These bacteria mainly have a positive impact on plants through various direct or indirect pathways, such as synthesizing hormones (indoleacetic acid, gibberellins, etc.), producing iron carriers, dissolving phosphorus, fixing nitrogen, and secreting antibacterial substances. Through various processes, PGPR greatly promotes plant growth and development, and provides adaptive advantages for host plants by mobilizing nutrients (such as nitrates, phosphates, and iron), improving soil properties, and enhancing plant tolerance. The PGPRs that have been discovered so far include Bacillus sp., Pseudomonas sp., Arthrobacter sp., Enterobacter sp., Klebsiella sp., etc. Compared with chemical fertilizers, plant growth-promoting rhizobacteria as inoculants have the advantages of being environmentally friendly and cost-effective. Although the plant growth mechanism mediated by PGPR is not fully understood, related research is mainly focused 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. In addition, PGPR can improve the ecological environment of the rhizosphere soil for plant growth, especially promote the diversity of the community structure of the rhizosphere soil, and promote plant growth and dry matter accumulation.

[0004] Phyllobacterium sp., also known as Phyllobacterium, is a genus of aerobic or facultative anaerobic fermentation-type Gram-negative rods in the family Phyllobacterales, with a straight rod shape (in vitro). It appears in leaf tumors of higher plants (Myrsinaceae and Robiniaceae-Rubia). Most of the studied Phyllobacterium sp. are isolated from plant nodules or soil, and some strains can promote crop growth by nitrogen fixation, phosphorus solubilization and IAA production. At present, the isolation and application of plant rhizosphere growth-promoting bacteria has become a hot topic in current research, which has important practical significance for accelerating ecological environment construction and promoting sustainable development. However, there has been no public report on rhizosphere growth-promoting bacteria specifically for grassland plants such as Leymus chinensis. Summary of the invention

[0005] The purpose of the present invention is to isolate a strain of Bacillus argenteus from the rhizosphere of grazing degraded grassland Leymus chinensis, strain number B68, which can effectively solve the problem of converting insoluble phosphates into soluble phosphates, decomposing organic phosphorus, and producing plant hormone IAA. It also has growth-promoting properties such as nitrogen fixation and ACC deaminase production, and can regulate the bacterial community structure of the rhizosphere soil of Leymus chinensis, increase the richness of beneficial bacteria, and have a significant growth-promoting effect on Leymus chinensis. Specifically, the technical solution adopted by the present invention is as follows:

[0006] The strain of the present invention was isolated, purified and screened from grazing grassland in Xilinhot City, Inner Mongolia. The strain was deposited in the General Microbiological Center of China Microbiological Culture Collection (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 was classified and named Phyllobacterium myrsinacearum, strain number B68, and deposit number CGMCC No. 31906.

[0007] The colony of the rhizosphere growth-promoting bacteria B68 of the present invention on the LB plate is translucent or gray-brown and mucous. Under the microscope, the bacteria are spherical, 0.5~0.8um×0.5~0.8um, facultative aerobic, Gram-negative; catalase, glucose oxidation and fermentation, methyl red, VP positive, nitrate reduction positive, oxidase test are all positive, starch hydrolysis, H2S, citrate utilization, glucose gas production, indole test, gelatin liquefaction are all negative.

[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 B68.

[0009] The 16SrDNA sequence was compared with the sequences in the GenBank database by BLAST. The results showed that the strain B68 had the highest homology with Phyllobacterium myrsinacearum, which was 98.3%. The strain B68 was identified as Phyllobacterium myrsinacearum based on its morphological, physiological and biochemical characteristics and 16SrDNA sequence.

[0010] The strain B68 formed a transparent circle of 1.85±0.16 cm around the colony, indicating that it has the ability to degrade inorganic phosphorus, and the degradation amount is 87.83 μg / mL. This shows that the bacteria has a dissolving effect on tricalcium phosphate, grows with insoluble inorganic phosphate as a phosphorus source, and converts it into soluble phosphate.

[0011] The fermentation product can be obtained by inoculating the B68 strain into a liquid fermentation medium for fermentation culture, wherein the liquid fermentation medium is preferably an LB liquid medium. Obviously, the fermentation product also has the relevant functions of strain B68. The fermentation culture conditions can be specifically as follows: the strain stored on the slant is transferred to a test tube containing 5 mL of LB liquid medium, and cultured in a constant temperature shaker for 12 hours, temperature: 37°C, speed: 180rpm; the activated bacterial liquid is inoculated into 50 mL of LB liquid medium at a 1% inoculation amount, and cultured in a shaker for 24 hours, temperature: 37°C, speed: 180rpm, to prepare a fermentation liquid; the LB liquid medium composition can be: 0.5% yeast powder, 1% peptone, 1% sodium chloride and 1000 mL of distilled water, pH=7.0, and the prepared culture base is sterilized at 121°C for 20 minutes.

[0012] The strain B68 has a strong ability to produce indoleacetic acid (IAA), with a yield of 35.49 μg / mL.

[0013] The strain B68 can grow well in the Axubei culture medium, has a strong nitrogen fixation ability, and the nitrogenase NITS activity is 183.43 IU / L.

[0014] The strain B68 has a strong ability to synthesize ACC deaminase, with an enzyme activity of 138.29 IU / L. The strain can hydrolyze 1-aminocyclopropane-1-carboxylic acid (ACC), a precursor of ethylene synthesis, thereby reducing the level of ethylene, thereby inhibiting the triple reaction of ethylene to plants, promoting the elongation and growth of plant roots and stems, increasing nutrient absorption and light capture area, increasing dry matter accumulation, and the amplified root tissue can enhance the plant's effective utilization of growth-promoting substances and nutrients secreted or decomposed by rhizosphere microorganisms.

[0015] Obviously, the fermentation product of strain B68 and the gene construct based on the gene of strain B68 also have the above-mentioned functions of degrading inorganic phosphorus, producing indoleacetic acid, synthesizing ACC deaminase, and producing nitrogenase. Furthermore, strain B68 and the fermentation product can also be prepared into microbial fertilizer or microbial agent for large-scale application.

[0016] The application method of the rhizosphere growth-promoting bacteria B68 of the present invention in promoting the growth of sheep grass can be applied by preparing a suspension of rhizosphere growth-promoting bacteria of sheep grass, and the preparation thereof can include the following steps: inoculating the strain into LB liquid culture medium 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 B68 strain in the bacterial suspension is 1×10 8 CFU / mL.

[0017] The above-mentioned application method for promoting the growth of Leymus chinensis 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.

[0018] The strain of the present invention is isolated from rhizosphere soil of Leymus chinensis in an overgrazing sample site, and is obtained by artificial separation and purification. The strain has high yield of 3-indoleacetic acid, has strong solubility for insoluble phosphates, and has good growth-promoting properties such as nitrogen fixation, production of ACC deaminase, and production of IAA. The prepared strain fermentation liquid is inoculated into Leymus chinensis seedlings, and the strain can colonize in the rhizosphere of Leymus chinensis, regulate the bacterial community structure of the rhizosphere soil of Leymus chinensis; significantly promote the growth of Leymus chinensis, enhance photosynthesis, and increase the chlorophyll content; significantly increase the hormone and nutrient content in the leaves and roots of the plants; and significantly increase the content of available phosphorus, total carbon and organic matter in the rhizosphere soil.

[0019] This strain has relatively comprehensive growth-promoting functions and strong growth-promoting abilities. It can successfully colonize in the rhizosphere of sheep fescue and change its microbial community. It has a significant growth-promoting effect on the above-ground and underground roots of sheep fescue. It is a strain with great application potential, and provides strain resources and theoretical basis for the subsequent development of microbial fertilizers for repairing degraded grasslands.

[0020] Beneficial effects of the present invention:

[0021] Under potted conditions, after the rhizosphere growth-promoting bacteria B68 of the sheep grass described in the present invention are inoculated into the rhizosphere of sheep grass, it can significantly promote the plant height, leaf length, leaf width, stem thickness, aboveground biomass and underground biomass of sheep grass; and significantly enhance the enhanced photosynthesis of sheep grass, which is manifested by increased chlorophyll content and enhanced Rubisco enzyme activity.

[0022] After the above-mentioned sheepgrass rhizosphere growth-promoting bacteria B68 were inoculated into the rhizosphere of sheepgrass, the contents of soil organic matter, total carbon and available phosphorus were significantly increased; the content of hormones such as auxin, gibberellin, jasmonic acid, salicylic acid, etc. produced by sheepgrass was significantly increased; the absorption and utilization of plant nutrients such as total nitrogen and total phosphorus by sheepgrass was significantly promoted; and the content of soluble sugar and glucose was significantly increased.

[0023] Analysis of soil bacterial communities showed that the bacterial diversity in the rhizosphere soil of Leymus chinensis decreased after inoculation of the strain of the present invention, and the abundance of the strain B68 increased significantly, indicating that the strain can successfully colonize in the rhizosphere of Leymus chinensis, thereby occupying more ecological niches and resulting in reduced bacterial diversity.

[0024] The Myrdalubiella argentea provided by the invention can provide a new strain resource for the microbial fertilizer industry, and shows potential application prospects in soil phosphorus activation, growth promotion, yield increase, grassland degradation restoration, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The optical microscope morphology (scale 10 μm in the lower right corner) and Gram staining morphology of strain B68 of the present invention are shown;

[0026] Figure 2 This is a test diagram of the bacterial strain B68 in the embodiment of the present invention for decomposing inorganic phosphorus and producing IAA;

[0027] Figure 3 This is a graph showing the effect of strain B68 of the present invention on promoting aboveground and underground growth of Leymus chinensis;

[0028] Figure 4 The effect of strain B68 of the present invention on the content of Rubisco enzyme and chlorophyll in the leaves of Leymus chinensis;

[0029] Figure 5 The effect of strain B68 of the present invention on the content of hormones (auxin, abscisic acid, jasmonic acid, salicylic acid) in roots and leaves of Leymus chinensis;

[0030] Figure 6 The effect of strain B68 of the present invention on the soluble sugar and glucose content in roots and leaves of Leymus chinensis is shown in the figure.

[0031] Figure 7 The effect of strain B68 of the present invention on the nutrient content (total carbon, total nitrogen, total phosphorus, total potassium) in the roots and leaves of Leymus chinensis;

[0032] Figure 8 This is a diagram showing the abundance of Phyllobacterium myrsinacearum in rhizosphere soil after inoculation of strain B68 in an embodiment of the present invention;

[0033] Fig. 9The effect of inoculation of strain B68 of the present invention on the α diversity (Shannon) index of rhizosphere soil;

[0034] Fig.10 This is the effect of inoculation of strain B68 of the present invention on different bacterial genera in the rhizosphere. DETAILED DESCRIPTION

[0035] Example 1: Screening, identification and analysis of growth-promoting properties of rhizosphere bacteria of Leymus chinensis

[0036] 1. Test materials

[0037] (1) Collection of soil samples

[0038] Soil samples were collected from the Bayinxile grazing grassland in Ordos City, Inner Mongolia. The samples were taken using the 5-point sampling method and placed in sterile ziplock bags, stored in a refrigerator at 4°C and separated as soon as possible.

[0039] (2) Preparation of culture medium and reagents

[0040] ①LB medium

[0041] Tryptone 10.0 g, yeast powder 5.0 g, NaCl 10 g, agar 15 g (solid culture medium), distilled water 1000 mL, pH = 7.0-7.2, sterilize at 121℃ for 20 min.

[0042] ②Asubei culture medium

[0043] 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

[0044] ③PKO medium

[0045] 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

[0046] ④Reagents

[0047] 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 0.5M FeCI3·6H2O solution to prepare the color developer. This color developer cannot be stored for a long time.

[0048] 2. Test methods

[0049] (1) Screening of Phosphate-solubilizing and Nitrogen-fixing Strains in the Rhizosphere of Leymus chinensis

[0050] 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 -6 times suspension, take 50 μL of each dilution and evenly spread it on inorganic phosphorus solid culture medium, culture it in a 30℃ incubator, and observe whether there is a transparent phosphorus-solubilizing circle on the plate. Pick the strain with a transparent circle on the plate for further purification, and finally streak the purified strain on the Axubei nitrogen-free medium. The strain that can produce a nitrogen-fixing circle on the Axubei medium is transferred to a glycerol tube with a final concentration of 30% and stored in a -20℃ refrigerator, which is strain B68.

[0051] (2) Determination of IAA production characteristics

[0052] Indoleacetic acid can be secreted by qualitative and quantitative determination.

[0053] Qualitative determination: The strain B68 isolated and purified in (1) was inoculated into LB liquid medium containing L-tryptophan (100 mg / L) at 30°C and 180 r·min. -1 Incubate on a shaker for 1 day. Take 50 μL of bacterial suspension and drop it on a white ceramic plate, and add 50 μL of Salkowski colorimetric solution. Add 50 μL of 50 mg / L indoleacetic acid colorimetric solution as a positive control. Place the white ceramic plate at room temperature and away from light for 30 minutes, then observe. If the color turns red, it means that indoleacetic acid can be secreted.

[0054] Quantitative determination: The culture conditions are the same as above. Using the Salkowski colorimetric method, the strain was inoculated into LB liquid culture medium containing L-tryptophan (100 mg / L) and cultured in a shaking incubator at 30°C and 200 r / min for 24 hours. 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 culture medium and the colorimetric agent was used as a blank control. After 30 minutes of observation at room temperature away from light, the color turns red, indicating that IAA can be secreted. The IAA standard solution is prepared into different concentrations to make a standard curve, and the OD of the reaction solution is measured after the reaction according to the above conditions. 530 , calculate the concentration of IAA in the supernatant.

[0055] (4) Determination of the phosphate solubilization ability of strains:

[0056] The phosphate solubilizing ability of the strain was determined by qualitative and quantitative methods.

[0057] Phosphate solubility circle method qualitative determination: strain B68 was inoculated on PKO inorganic phosphorus solid medium, with 3 replicates for each strain, and the diameter of the phosphate solubility circle (D) and colony diameter (d) formed by each strain was measured, and the phosphate solubility ability of the strain was preliminarily judged according to the size of the D / d value. Strains with a phosphate solubility index PSI (D / d) value greater than 1.5 were used as primary screening strains, and the phosphate solubility ability of these strains was quantitatively determined.

[0058] 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 -1 Centrifuge 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 ).

[0059] (5) Determination of ACC deaminase activity of strains

[0060] 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 make 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 450nm using an enzyme reader, and the ACC deaminase (ACCD) activity concentration in the sample is calculated using the standard curve.

[0061] (6) Determination of nitrogenase activity of strains

[0062] 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 is coated with purified nitrogenase (NITS) antibodies to make solid-phase antibodies. Nitrogenase (NITS) is 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 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 nitrogenase (NITS) in the sample. The absorbance (OD value) is measured at a wavelength of 450nm using an enzyme reader, and the nitrogenase (NITS) activity concentration in the sample is calculated using the standard curve.

[0063] (7) Physiological and biochemical characteristics

[0064] According to the Manual of Identification of Common Bacteria Systems, a series of physiological and biochemical characteristics were measured for the strain B68 that was screened out for its ability to degrade inorganic phosphorus, produce high IAA, produce ACC deaminase and nitrogenase activity. The measurement indicators included: Gram staining, catalase, glucose oxidation fermentation, methyl red, VP, nitrate reduction, oxidase, starch hydrolysis, H2S, citrate utilization, glucose gas production, indole test, and gelatin liquefaction test.

[0065] (8) 16S rDNA sequence identification

[0066] 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. 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.

[0067] 3. Identification results of strain B68

[0068] (1) Morphological characteristics identification results

[0069] like Figure 1 As shown, the colonies of strain B68 are round, light yellow, translucent, mucous, moist, and have neat edges. Under the microscope, the bacteria are spherical, 0.5-0.8um×2.5-10, and are Gram-negative facultative anaerobic bacteria.

[0070] (2) Identification of physiological and biochemical characteristics

[0071] Table 1 Identification results of physiological and biochemical characteristics of strain Nn211

[0072]

[0073] 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.

[0074] (3) 16S rDNA sequence identification results

[0075] The 16S rDNA sequence of strain B68 was amplified and sequenced, and the sequencing result is shown in SEQ ID NO: 1 in the sequence table. The 16S rDNA sequence of strain B68 obtained was compared on the NCBI website, and the results showed that the strain had the highest homology with Phyllobacterium myrsinacearum, which was 98.3%; the strain B68 was identified as Phyllobacteriummyrsinacearum based on morphological, physiological and biochemical characteristics and 16SrDNA sequence. It was 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, with the deposit number CGMCC No. 31906.

[0076] The 16S rDNA gene sequence of the Bacillus argenteus B68 is shown in SEQ ID No. 1:

[0077]

[0078] (4) Results of identification of growth-promoting properties

[0079] The identification of the growth-promoting properties of the strains is shown in Table 2 ; Figure 2 This is a test chart of the inorganic phosphorus decomposition and IAA production of the strain B68 in the embodiment of the present invention.

[0080] Table 2 Identification results of growth-promoting properties of strain B68

[0081]

[0082] Example 2: Preparation of B68 bacterial suspension and its growth-promoting effect on Leymus chinensis

[0083] 1. Preparation of B68 strain suspension

[0084] The preserved strains were transferred to a test tube containing 5 mL of LB liquid culture medium and cultured in a constant temperature shaker for 12 hours at 37°C and 180 rpm. The activated bacterial solution was transferred to 50 mL of LB liquid culture medium at a 1% inoculation rate and cultured in a shaker for 24 hours at 37°C and 180 rpm to prepare a fermentation liquid. Centrifuge at 8000 rpm and room temperature for 10 minutes, rinse twice with sterile water, and finally resuspend with sterile water to prepare an OD 600 =1.0 bacterial suspension for standby use, the concentration of the B68 strain in the bacterial suspension is 1×10 8 CFU / mL.

[0085] 2. Effect of B68 bacterial suspension on the growth of Leymus chinensis

[0086] 1. Experimental materials and methods

[0087] (1) Potted plant test

[0088] The seeds of Leymus chinensis were soaked in 75% ethanol and 2% NaClO solution for 2 min and 10 min, respectively, and then thoroughly washed. The seeds of uniform size were cultured in petri dishes for 10 days. After the seedlings grew to the clover stage, they were transplanted into pots (20 cm high; 18 cm in diameter) containing about 3 kg of field soil, substrate soil and vermiculite (4:1:1 volume ratio) and placed for 30 days. There were 8 seedlings per pot, and 6 pots were replicated for inoculation and non-inoculation. The pots were randomly placed in a 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. The soil moisture was maintained at 60%. The B68 suspension was inoculated in the soil around the roots of 4-week-old Leymus chinensis plants, or not inoculated (control inoculation with water). After 4 weeks, plant height, stem diameter, leaf width, leaf length, chlorophyll content, fresh weight of aboveground parts, fresh root weight, aboveground dry weight and root dry weight were measured; the contents of hormones such as abscisic acid (ABA), jasmonic acid (JA), indole-3-acetic acid (IAA), gibberellin (GA3) and salicylic acid (SA) and the contents of nutrients such as total carbon, total nitrogen, total phosphorus, total potassium and soluble sugar were measured in fresh leaves and roots; and rhizosphere soil was collected for analysis of rhizosphere soil physical and chemical properties and microbiome.

[0089] (2) Determination of plant physiological indicators

[0090] Soluble sugar content, glucose, plant total carbon and total phosphorus content, and RuBisCO activity in B68-inoculated and non-inoculated root and leaf samples were determined using corresponding test kits (Suzhou Keming Biotechnology Co., Ltd., China). About 0.3 g of dried plants were ground into powder, and the total nitrogen content of plants was determined by graphite digestion; the total potassium content of plants was determined by flame photometry; ABA, JA, IAA, GA3, and SA in fresh plants were determined by liquid chromatography-tandem mass spectrometry (LC-MS / MS) (Rigol L3000, Beijing, China). Chlorophyll content was determined using a (SPAD)-502 Plus chlorophyll meter (Konica Minolta, Japan).

[0091] 2. Test results

[0092] (1) Effects on traits, biomass, hormones, nutrients and sugar content of Leymus chinensis

[0093] As shown in Table 3 and Figure 3As shown in Table 2, compared with the control, under potted conditions, after the rhizosphere growth-promoting bacteria B68 of the present invention were inoculated into the rhizosphere of Leymus chinensis, the height, leaf length, leaf width, and stem thickness of Leymus chinensis increased by 15.96%, 9.00%, 13.89%, and 12.21%, respectively; as shown in Table 2, compared with the control, the aboveground dry weight increased by 45.49%, the aboveground fresh weight increased by 23.45%, the underground dry weight increased by 31.57%, and the underground fresh weight increased by 35.92%. This shows that B68 significantly promotes the growth of Leymus chinensis above and below the ground.

[0094] Table 3 Effects of inoculated strain B68 on phenotypic traits of Leymus chinensis

[0095]

[0096] Note: In the same column, * indicates significant difference (p<0.05), ** indicates significant difference (p<0.01), and *** indicates extremely significant difference (p<0.001); the same below.

[0097] Table 4 Effects of inoculation with strain B68 on aboveground and underground biomass of Leymus chinensis

[0098]

[0099] Depend on Figure 4 As shown, after inoculation of the above-mentioned rhizosphere growth-promoting bacteria B68, the photosynthesis of sheep fescue was significantly enhanced, as manifested by an increase of 13.83% in chlorophyll content and a significant enhancement of Rubisco enzyme activity, as high as 185.86%.

[0100] Depend on Figure 5 , Figure 6 , Figure 7 As shown, after inoculation with the above-mentioned rhizosphere growth-promoting bacteria B68, the IAA and JA contents in leaves and roots were significantly increased by 55.70-80.18% and 39.94-42.24%, respectively; the soluble sugar content in leaves and roots was significantly increased by 14.48 ~ 29.56%, the glucose content was 41.14 ~ 42.50%, the total carbon content of plants was 13.92 ~ 19.28%, the total nitrogen content of plants was 9.16 ~ 10.22%, and the total phosphorus content of plants was 18.96 ~ 24.34%; the ABA content in leaves, the SA in roots and the total potassium content of plants were also significantly increased by 14.71%, 39.17% and 59.39%, respectively.

[0101] It can be seen that the growth-promoting bacteria B68 significantly promoted the growth of sheepgrass and enhanced photosynthesis. This may be because B68 promoted the acquisition of nitrogen and phosphorus nutrients by sheepgrass, thereby increasing the hormone content of sheepgrass, especially increasing the levels of growth hormone IAA and jasmonic acid JA to promote the growth of aboveground and underground parts of sheepgrass.

[0102] (2) Effects on the physical and chemical properties and microbial communities of rhizosphere soil of Leymus chinensis

[0103] The effects of the above-mentioned rhizosphere growth-promoting bacteria B68 on the physical and chemical properties of rhizosphere soil after inoculation are shown in Table 5. The results showed that the inoculation significantly increased the contents of soil organic matter, total carbon and available phosphorus by 4.85%, 5.09% and 13.17%, respectively.

[0104] Table 5 Effects of B68 inoculation on soil physical and chemical indicators

[0105]

[0106] Figure 8-10 BCK was used as blank control. Figure 8 As shown, after the above-mentioned rhizosphere growth-promoting bacteria B68 were inoculated with Leymus chinensis, the abundance of Phyllobacterium myrsinacearum in the rhizosphere soil increased significantly, indicating that Phyllobacterium myrsinacearum B68 can successfully colonize in the rhizosphere of Leymus chinensis.

[0107] Depend on Fig. 9 As shown, B68 inoculation significantly reduced the α diversity of the microbial community, leading to changes in the structure of the microbial community. This was mainly due to the successful colonization of B68 after inoculation, which occupied too many ecological niches, resulting in a decrease in other bacteria, thereby reducing the α diversity of the microbial community.

[0108] Depend on Fig.10 As shown in the figure, 10 bacterial genera were significantly enriched after B68 inoculation, most of which are beneficial to plants, including RamLibacter sp., Luteimonas sp., and Ohtaekwangia sp. For example, RamLibacter sp. has phosphate solubilizing activity and can promote plant growth. Luteimonas sp. can degrade complex organic compounds, thereby enhancing the absorption of N and P and promoting early plant development. This shows that B68 can not only successfully colonize in the rhizosphere of Leymus chinensis after inoculation, but also attract other beneficial bacteria, change the rhizosphere microenvironment, and reshape the rhizosphere microbial community.

[0109] In summary, the Phyllobacterium myrsinacearum provided by the present invention has strong characteristics of dissolving inorganic phosphorus, producing IAA, ACC deaminase and nitrogenase, can significantly promote the growth of sheep grass, produce plant hormones that promote growth and improve resistance, and improve the photosynthetic capacity of sheep grass and the ability to obtain nitrogen and phosphorus nutrients. The present invention provides excellent plant rhizosphere growth-promoting bacteria for multifunctional growth-promoting bacteria, which can be used to prepare microbial fertilizers applied to grassland plants to promote plant growth, improve nutrient acquisition ability, and further repair degraded grasslands. In addition, there are currently few reports on the application of Phyllobacterium in promoting the growth of grassland plants, which shows that the strains screened out by the present invention have enriched the strain resources and have very large application potential.

[0110] The embodiments described above are merely descriptions of preferred implementation modes of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A rhizosphere growth-promoting bacterium of sheep grass, classified and named Phyllobacterium myrsinacearum, strain number B68, deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on September 9, 2024, with the deposit number CGMCC No.31906.

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

3. The fermentation product according to claim 2, characterized in that The fermentation culture adopts LB liquid medium.

4. A microbial fertilizer, comprising the rhizosphere growth-promoting bacteria of Leymus chinensis according to claim 1 or the fermentation product according to claim 2.

5. The use of the rhizosphere growth-promoting bacteria of sheep fescue according to claim 1 or the fermentation product according to claim 2 or the microbial fertilizer according to claim 4 in any one or more of the following: use in promoting the growth of sheep fescue; use in degrading inorganic phosphorus; use in secreting indoleacetic acid; use in synthesizing ACC deaminase; use in producing nitrogenase.

6. A method for promoting the growth of Leymus chinensis, characterized in that: The rhizosphere growth-promoting bacteria according to claim 1, the fermentation product according to claim 2, or the microbial fertilizer according to claim 4 is applied to the rhizosphere of sheep fescue.

7. The method according to claim 6, characterized in that The application to the rhizosphere of Leymus chinensis includes any one or more application methods of spraying, irrigation, and burying.

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