A microbacterium capable of producing siderophores, dissolving potassium, promoting growth and alleviating plant saline-alkali stress and its application

Abstract

The present invention discloses a microbacterium capable of producing siderophores, dissolving potassium, promoting growth and alleviating plant saline-alkali stress and its application. The present invention relates to the field of microorganisms and provides a microbacterium dendrobium, wherein the microbacterium dendrobium is a microbacterium dendrobium ( Microbacterium dendrobii ), with the strain number 2T1, and the registration number at the General Microbiology Center of the China Culture Collection Administration of Microorganisms is CGMCC No. 32856. This strain is a new species of the genus Microbacterium, which has the ability to secrete siderophores and dissolve potassium. Potted plant experiments showed that inoculation with this strain under saline-alkali stress increased the root length and dry weight of wheat compared to the uninoculated control. Microbacterium dendrobii )2T1 can be used as a microbial organic fertilizer to improve soil fertility, alleviate crop saline-alkali stress, and enhance crop adaptability to saline-alkali environments.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a microbacterium capable of producing siderrophores, dissolving potassium, promoting growth and alleviating plant saline-alkali stress, and applications thereof. Background Art

[0002] The rhizosphere microbiome plays a key role in plant growth and health, improving nutrient utilization, and protecting plants from biotic and abiotic stresses. Plant growth-promoting rhizobacteria (PGPR) have been increasingly used as bioagents and are playing an increasingly important role in agricultural production. Recent studies have shown that PGPR not only promote plant growth, prevent diseases, and increase crop yields, but also enhance plant resistance to various abiotic stresses, such as drought, salt, and heavy metals, improving plant adaptability to various environmental stresses.

[0003] Many PGPR groups have been discovered at home and abroad, which have the functions of secreting plant hormones, dissolving phosphorus, dissolving iron, and dissolving potassium. Among them, Microbacterium spp. Microbacterium Members of the genus Microbacterium are widespread in nature and have been isolated from diverse habitats, including soil, plants, water, dairy products, insects, and humans. Microbacterium strains possess diverse functions, such as producing indole-3-acetic acid (IAA), degrading xylan, and solubilizing phosphates. They also possess starch-degrading abilities and exhibit good tolerance to cold, heat, salt, and alkali. They also exhibit resistance to ultraviolet radiation, protecting plants from nematode pathogens and promoting plant growth. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a new strain of Microbacterium capable of producing siderophores, dissolving potassium, promoting growth and alleviating plant saline-alkali stress, and application thereof.

[0005] In a first aspect, the present invention claims protection for a Microbacterium dendrobium.

[0006] The Microbacterium dendrobium claimed in the present invention is Microbacterium dendrobium ( Microbacterium dendrobii ), the strain number is 2T1, and the registration number of the General Microbiology Center of China Culture Collection Administration is CGMCC No.32856.

[0007] Microbacterium dendrobium ( Microbacterium dendrobii 2T1 is a Gram-positive bacterium. After three days of growth on TSA solid medium, its colonies are pale yellow, round, convex, with smooth edges, a sticky, and shiny appearance, and a diameter of 1-2 mm. This strain has excellent plant growth-promoting properties and can significantly alleviate saline-alkali stress in plants.

[0008] In a second aspect, the present invention claims a composition containing the Microbacterium dendrobium described in the first aspect above.

[0009] Furthermore, the composition may be a culture, which is a substance obtained by culturing the Microbacterium dendrobium in a microbial culture medium (all substances in the culture container, i.e., fermentation products, such as the substance containing the Microbacterium dendrobium and secreted into the liquid culture medium, i.e., fermentation liquid, or the substance containing the Microbacterium dendrobium and secreted into the solid culture medium, i.e., solid fermentation product).

[0010] The microbial culture medium may be a bacterial culture medium, and the bacterial culture medium may be a solid culture medium or a liquid culture medium.

[0011] In the above culture, the substance includes the Microbacterium dendrobium (the bacteria itself) and / or its metabolites described in the first aspect above.

[0012] The term "metabolite" refers to the primary and / or secondary metabolites produced during microbial metabolism. Primary metabolism refers to the process by which microorganisms absorb various nutrients from the outside world and, through catabolism and anabolism, generate substances and energy to sustain life activities. The products of primary metabolism are primary metabolites, such as monomers such as monosaccharides or monosaccharide derivatives, nucleotides, vitamins, amino acids, fatty acids, and various macromolecular polymers composed of them, such as proteins, nucleic acids, polysaccharides, and lipids. Secondary metabolism refers to the process by which microorganisms, during a certain growth period, use primary metabolites as precursors to synthesize substances with no clear function in the microorganism's life activities. The products of secondary metabolism are secondary metabolites, which are mostly compounds with relatively complex molecular structures. Based on their functions, they can be divided into types such as antibiotics, hormones, alkaloids, and toxins.

[0013] The term "culture" refers generally to liquid or solid media containing microbial communities following artificial inoculation and cultivation. This refers to products obtained by growing and / or amplifying microorganisms. These can be biologically pure cultures of the microorganisms (i.e., the Microbacterium dendrobium) or contain a certain amount of culture medium, metabolites, or other components produced during the culture process. The term "culture" also includes subcultures obtained by subculturing microorganisms, which can be cultures of a single generation or a mixture of several generations.

[0014] Furthermore, the composition may be a bacterial agent, a microecological preparation, or a biological fertilizer.

[0015] In the above-mentioned microbial agent or probiotic preparation or biofertilizer, the active ingredient of the microbial agent or probiotic preparation or biofertilizer can be the Microbacterium dendrobium described in the first aspect above, a metabolite of the Microbacterium dendrobium and / or a culture of the Microbacterium dendrobium, and the active ingredient of the microbial agent or probiotic preparation or biofertilizer can also contain other biological components or / and non-biological components. Other active ingredients of the microbial agent or probiotic preparation or biofertilizer can be determined by those skilled in the art according to the desired effect.

[0016] In the above-mentioned microbial agent, microecological preparation or biofertilizer, the microbial agent, microecological preparation or biofertilizer may further contain a carrier in addition to the active ingredient. The carrier may be a carrier commonly used in the field of pesticides and is biologically inert. The carrier may be a solid carrier or a liquid carrier; the solid carrier may be a mineral material, a plant material or a polymer compound; the mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica and diatomaceous earth; the plant material may be at least one of wheat flour, soy flour and starch; the polymer compound may be polyvinyl alcohol and / or polyglycol; the liquid carrier may be an organic solvent, vegetable oil, mineral oil or water; the organic solvent may be decane and / or dodecane.

[0017] The above-mentioned microbial agent, microecological preparation or biofertilizer can be in various dosage forms, such as liquid, emulsion, suspension, powder, granule, wettable powder or water-dispersible granule.

[0018] As needed, surfactants (such as Tween 20, Tween 80, etc.), adhesives, stabilizers (such as antioxidants), pH regulators, etc. may be added to the bacterial agent, microecological preparation, or biological fertilizer.

[0019] In the bacterial agent, probiotic preparation or biofertilizer, the Microbacterium dendrobium and / or its metabolites can be present in the form of cultured living cells, fermentation liquid of living cells, filtrate of cell culture or a mixture of cells and filtrate.

[0020] Herein, the metabolites of Microbacterium dendrobii can be obtained from the fermentation broth of Microbacterium dendrobii. The metabolites of Microbacterium dendrobii can be sterile metabolites of Microbacterium dendrobii or bacterial metabolites of Microbacterium dendrobii. The sterile metabolites of Microbacterium dendrobii (sterile fermentation filtrate) can be prepared by culturing Microbacterium dendrobii in a liquid culture medium and filtering out the Microbacterium dendrobii from the liquid culture (fermentation broth) to obtain the sterile metabolites of Microbacterium dendrobii. The bacterial metabolites of Microbacterium dendrobii can be prepared by culturing Microbacterium dendrobii in a liquid fermentation medium and collecting the fermentation broth, which contains the Microbacterium dendrobii and substances secreted into the liquid culture medium. This fermentation broth is the bacterial metabolites of Microbacterium dendrobii.

[0021] Furthermore, the composition may have at least one of the following properties:

[0022] A1) siderophore production;

[0023] A2) potassium removal;

[0024] A3) Alleviate plant salinity and alkali stress;

[0025] A4) Increase CAT activity in plants;

[0026] A5) Increase the SOD activity of plants;

[0027] A6) Increase the GSH content of plants;

[0028] A7) Promote plant growth;

[0029] A8) Promote the increase of plant underground dry weight;

[0030] A9) Promote the increase of plant aboveground dry weight;

[0031] A10) Promotes the growth of plant roots.

[0032] In a third aspect, the present invention claims the use of the Microbacterium dendrobium described in the first aspect or the composition described in the second aspect, and the use may be any of the following:

[0033] B1) Use in the production of siderophores or in the preparation of products for the production of siderophores;

[0034] B2) Use in potassium dissolution or in the preparation of products for potassium dissolution;

[0035] B3) Use in alleviating saline-alkali stress in plants or in preparing products for alleviating saline-alkali stress in plants;

[0036] B4) Use in increasing CAT activity in plants or in preparing products for increasing CAT activity in plants;

[0037] B5) Use in increasing the SOD activity of plants or in preparing products for increasing the SOD activity of plants;

[0038] B6) Use in increasing the GSH content of plants or in preparing a product for increasing the GSH content of plants;

[0039] B7) Use in promoting plant growth or preparing products for promoting plant growth;

[0040] B8) Use in promoting the increase of underground dry weight of plants or in preparing products for promoting the increase of underground dry weight of plants;

[0041] B9) Use in promoting the increase of aboveground dry weight of plants or in preparing products for promoting the increase of aboveground dry weight of plants;

[0042] B10) Use in promoting the increase of plant root length or in preparing a product for promoting the increase of plant root length.

[0043] Furthermore, the increasing of the CAT activity of the plant may be increasing the CAT activity of the plant under saline-alkali stress conditions.

[0044] Furthermore, the increasing the SOD activity of the plant may be increasing the SOD activity of the plant under saline-alkali stress conditions.

[0045] Furthermore, increasing the GSH content of the plant may be increasing the GSH content of the plant under saline-alkali stress conditions.

[0046] Furthermore, the promoting plant growth may be promoting plant growth under saline-alkali stress conditions.

[0047] Furthermore, the promoting the increase of underground dry weight of plants may be promoting the increase of underground dry weight of plants under saline-alkali stress conditions.

[0048] Furthermore, the promoting the increase of the aboveground dry weight of the plant may be promoting the increase of the aboveground dry weight of the plant under saline-alkali stress conditions.

[0049] Furthermore, the promoting the increase of plant root length may be promoting the increase of plant root length under saline-alkali stress conditions.

[0050] In a fourth aspect, the present invention claims a method for alleviating saline-alkali stress in plants.

[0051] The method for alleviating plant saline-alkali stress claimed in the present invention may include the following steps: treating the plant to be treated or its growth medium with the Microbacterium dendrobium described in the first aspect or the composition described in the second aspect, thereby alleviating the saline-alkali stress of the plant.

[0052] In one embodiment of the present invention, the treatment is to irrigate the roots of the plants with a bacterial suspension containing the Microbacterium dendrobium.

[0053] In a fifth aspect, the present invention claims a method for promoting plant growth.

[0054] The method for promoting plant growth claimed in the present invention may include the following steps: treating the plant to be treated or its growth substrate with the Microbacterium dendrobium described in the first aspect or the composition described in the second aspect, thereby promoting the growth of the plant.

[0055] Furthermore, the promoting plant growth may be promoting plant growth under saline-alkali stress conditions.

[0056] In one embodiment of the present invention, the treatment is to irrigate the roots of the plants with a bacterial suspension containing the Microbacterium dendrobium.

[0057] In a sixth aspect, the present invention claims a method for culturing Microbacterium dendrobium.

[0058] The method for culturing Microbacterium dendrobium claimed in the present invention comprises the step of culturing the Microbacterium dendrobium in a culture medium for culturing microorganisms; the Microbacterium dendrobium is the Microbacterium dendrobium described in the first aspect above.

[0059] In a seventh aspect, the present invention claims a method of preparing a composition.

[0060] The method for preparing the composition claimed in the present invention comprises the step of using the Microbacterium dendrobium described in the first aspect as a component of the composition; the composition is the composition described in the second aspect.

[0061] In the present invention, the saline-alkali stress may be salt stress and / or alkali stress.

[0062] In the above-mentioned related aspects, in one embodiment of the present invention, the saline-alkali stress is simulated by using the following: NaCl, Na2SO4 and NaHCO3 in a molar ratio of 1:1:1, Na + The final concentration of the mixed solution is 168mmol / L.

[0063] In the above related aspects, the plant may be any of the following:

[0064] C1) angiosperms;

[0065] C2) Monocots;

[0066] C3) Gramineae;

[0067] C4) Grasses;

[0068] C5) Triticum;

[0069] C6) Wheat.

[0070] Experimental results show that the Microbacterium dendrobium provided by the present invention ( Microbacterium dendrobii )2T1 is a new species of Microbacterium. Microbacterium dendrobii )2T1 has the ability to secrete iron carriers and dissolve potassium. The pot experiment showed that compared with the negative control group without inoculation, the cells inoculated with Microbacterium dendrobium ( Microbacterium dendrobii ) 2T1 can increase the root length and dry weight of wheat. Microbacterium dendrobii )2T1 can be used as a microbial organic fertilizer to improve soil fertility, alleviate saline-alkali stress of crops, and enhance the adaptability of crops to saline-alkali environments.

[0071] Preservation Instructions

[0072] Classification name: Microbacterium dendrobium ( Microbacterium dendrobii );

[0073] Reference biological materials: 2T1;

[0074] Depository: General Microbiology Center of China Culture Collection Administration of Microorganisms;

[0075] Abbreviation of depository institution: CGMCC;

[0076] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0077] Deposit date: November 29, 2024;

[0078] Registration number of the CGMCC Collection Center: CGMCC No. 32856. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 Microbacterium dendrobium ( Microbacterium dendrobii ) Colony morphology of 2T1 after 3 days of culture on TSA plates.

[0080] Figure 2 Microbacterium dendrobium was constructed using the neighbor-joining method based on the 16S rRNA gene sequence. Microbacterium dendrobii Phylogenetic tree of 2T1 and related model strains. Note: The GenBank sequence numbers of the strains' 16S rRNA gene sequences are in parentheses; all reference strains in the figure are model strains of their respective species. Streptomyces showdoensis NBRC13417 T (AB184389) served as the outgroup.

[0081] Figure 3A gene encoding Microbacterium dendrobii ( Microbacterium dendrobii ) 2T1 and Microbacterium Phylogenetic tree of genomes of closely related species within the genus.

[0082] Figure 4 Microbacterium dendrobium ( Microbacterium dendrobii ) 2T1 siderophore secretion and potassium-solubilizing ability assay results. The left image shows the morphology of strain 2T1 after 2 days of growth on a modified potassium-solubilizing medium; the right image shows the morphology of strain 2T1 after 4 days of growth on a CAS qualitative medium.

[0083] Figure 5 The growth status of wheat seedlings inoculated with strain 2T1 and uninoculated strain (CK) under saline-alkali stress at 27 days. The left side is CK, and the right side is inoculated with strain 2T1.

[0084] Figure 6 The root length and dry weight of wheat seedlings under 27 days of saline-alkali stress. CK is the control without bacterial solution inoculation. P <0.01, *** indicates P <0.001.

[0085] Figure 7 The figure shows the response of wheat seedlings to environmental stress and the determination of antioxidant capacity under 27 days of saline-alkali stress. P <0.01. DETAILED DESCRIPTION

[0086] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0087] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0088] The formulas of various culture media and solutions involved in the following examples are as follows:

[0089] (1) TSB liquid medium: 15.0 g tryptone, 5.0 g soytone, 5.0 g sodium chloride, 1000 mL distilled water, adjust pH to 7.3 ± 0.2, and sterilize at 121°C for 15 min.

[0090] (2) TSA solid medium (TSA plate): Add 20.0 g agar to TSB liquid medium and sterilize at 121°C for 15 min.

[0091] (3) Starch medium: NA medium, 0.2% (w / v) soluble starch, pH 7.4, 121°C, sterilization for 20 min.

[0092] (4) Casein culture medium: Solution a: 5 g skim milk powder, 50 mL distilled water; Solution b: 50 mL NB, 1.5 g agar. Sterilize solution a and solution b separately at 121°C for 15 min, cool to about 60°C, mix thoroughly, and dispense onto plates.

[0093] (5) CAS qualitative culture medium:

[0094] Solution ①: 0.012 g CAS was dissolved in 10 mL deionized water and mixed with 2 mL 5 mM ferric chloride.

[0095] Solution ②: 0.015 g of hexadecyltrimethylammonium bromide was dissolved in 8 mL of deionized water.

[0096] Dye solution ③: Slowly pour solution ① into solution ② to obtain dye solution ③, and sterilize at 115℃ for 20 min.

[0097] Culture medium (4): Add 6.04 g of piperazine diethanolsulfonic acid and 10 mL of 0.1 M phosphate solution to a conical flask containing 150 mL of distilled water, mix thoroughly, adjust the pH to 6.8 with 50% NaOH, add 4.0 g of agar powder, and sterilize at 115°C for 20 min.

[0098] Phosphate solution: 2.427g disodium hydrogen phosphate, 0.5905g sodium dihydrogen phosphate, 0.075g potassium dihydrogen phosphate, 0.125g sodium chloride, 0.25g ammonium chloride, 100mL deionized water, mix well, and dilute 10 times before use.

[0099] Nutrient solution: 0.2 mL of 1 mM calcium chloride solution, 4 mL of 1 mM magnesium sulfate tetrahydrate solution, and 6 mL of 10% (w / v) casamino acid solution, sterilized at 115°C for 20 min (10 mL can be prepared before use and stored in a 4°C refrigerator away from light after use).

[0100] CAS qualitative culture medium plate: When the dye solution ③, culture medium ④, and nutrient solution have cooled to approximately 65°C, add the nutrient solution to the culture medium ④, then slowly add the dye solution ③, mix thoroughly, and pour onto the plate.

[0101] (6) Improved potassium-dissolving medium:

[0102] Alexander Baugh medium: sucrose 5.0 g, disodium hydrogen phosphate 2.0 g, magnesium sulfate heptahydrate 0.5 g, ferric chloride 5.0 mg, calcium carbonate 0.1 g, potassium feldspar powder 1.0 g, distilled water 1000 mL, agar 20.0 g, pH 7.2, high temperature sterilization at 121°C for 15 min.

[0103] Modified potassium-lysing medium: Add 100 mg / L bromothymol blue to Alexander Bauman medium.

[0104] Example 1, Microbacterium dendrobium ( Microbacterium dendrobii ) Isolation and identification of 2T1

[0105] 1. Microbacterium dendrobium ( Microbacterium dendrobii ) Separation of 2T1

[0106] Rhizosphere soil samples of Dendrobium officinale were collected in Yuli County, Xinjiang Uygur Autonomous Region (41°39′7″N, 86°30′67″E), stored in a 4°C refrigerator, and brought back to the laboratory for storage at 4°C. Soil adhering to the plant roots was shaken off, retaining only the rhizosphere soil tightly adhering to the root surface. The Dendrobium officinale roots, containing the rhizosphere soil, were immersed in 100 mL of sterile water in a conical flask and shaken at 150 rpm for 30 minutes at room temperature. The collected suspension was centrifuged at 3000 rpm at 4°C for 10 minutes, and the remaining supernatant was discarded as the rhizosphere soil. Weigh 1 g of rhizosphere soil and resuspend it in 10 mL of sterile water for serial dilution. Spread 100 μL of the serial dilutions onto TSA plates and incubate them upside down at 30°C for 1 week. Based on their physiological and morphological characteristics, single colonies were picked with bamboo sticks and attached to the plates for purification. Once confirmed as pure, the colonies were transferred to slants for short-term storage at 4°C or to 20% glycerol tubes for long-term storage at -80°C. One of the isolated and purified strains was designated 2T1.

[0107] 2. Microbacterium dendrobium ( Microbacterium dendrobii ) Identification of 2T1

[0108] 1. Morphological identification of strains

[0109] Strain 2T1, isolated and purified in step 1 above, which is in the logarithmic growth phase and has a stable colony size, was characterized for individual colonies, including colony size, color, transparency, surface condition, and edge condition. Strain 2T1 was smeared and Gram-stained using a Gram staining kit (Solarbio) according to the manufacturer's instructions. Bacterial morphology was observed under a light microscope.

[0110] The colonies of strain 2T1 on TSA plates are light yellow, round, convex, with smooth edges, sticky, and shiny. The diameter of the colonies is 1-2 mm ( Figure 1 The cells were Gram-positive, rod-shaped, and did not form spores.

[0111] 2. Molecular identification

[0112] According to the instructions, genomic DNA was extracted using the TIANamp bacterial genomic DNA extraction kit from Beijing Tiangen Biochemical Company (TIANGEN). The genomic DNA was sent to Annoroad Gene Technology (Beijing) Co., Ltd., and the draft genome of strain 2T1 was sequenced using the Illumina NovaSeq 6000 sequencing system. SPAdes software was used to assemble the genome, resulting in 33 contigs, N 50 The length is 3070005bp. The genome size is 2.99Mb, with a G+C content of 70.47%. The 16S rRNA gene sequence of the genome was uploaded to Ezbiocloud (www.ezbiocloud.net / eztaxon) for sequence alignment. The strain 2T1 was compared with the strain 2T1 using the ANIm method in the pyANI software. Microbacterium The average nucleotide identity (ANI) of the whole genome of closely related strains was analyzed. The digital DNA-DNA hybridization (dDDH) value between strain 2T1 and the reference strain was calculated using the Genome-to-Genome Distance Calculator (GGDC) 3.0 server (https: / / ggdc.dsmz.de / ggdc.php#).

[0113] The sequencing length of the 16S rRNA gene of strain 2T1 was 1525bp (SEQ ID No. 1). The comparison results of EzBioCloud database showed that strain 2T1 was similar to Microbacterium excoecariae CBS5P-1 T (98.14%), Microbacterium karelineae TRM 80801 T (97.71%), Microbacterium suaedae YZYP 306 T (97.49%) and Microbacterium indicum AM158907 T The sequences of strain 2T1 (97.63%) were highly similar and all were below the taxonomic threshold for species description (98.8%). 16S rRNA gene sequences with high similarity to strain 2T1 were retrieved from the EzBioCloud server and aligned using MUSCLE. A phylogenetic tree was constructed using the neighbor-joining method using MEGA X software. The evolutionary distances were calculated using the Kimura two-parameter model using the NJ method, with a bootstrap value of 1000. The phylogenetic tree constructed using the neighbor-joining method is shown in Figure 2. Figure 2 As shown, strain 2T1 and strainMicrobacterium excoecariae CBS5P-1 T clustered together and formed a separate branch, indicating that strain 2T1 was Microbacterium A potential new species of the genus.

[0114] strain 2T1 and other strains with publicly available genome sequences Microbacterium Compared with the type strains of the genus species, the ANI value is 80-81%, which is lower than the previously proposed critical value of 95-96% for species delimitation; the dDDH values ​​of strain 2T1 and its type strain are between 22.7-25.0%, which is far below the species delimitation threshold of 70%. See Table 1 for details. The results of ANI and dDDH indicate that strain 2T1 is Microbacterium A new species of the genus.

[0115]

[0116] In order to further clarify the taxonomic status of strain 2T1, the present invention conducted genome phylogenetic analysis and used UBCG software to classify strains 2T1 and Microbacterium genus were analyzed, 92 bacterial core genes were concatenated, and a gene sequence phylogenetic tree was constructed using the maximum likelihood method ( Figure 3 ), Bootstrap value is 1000. The results showed that strain 2T1 and strain Microbacterium gubbeenense DSM 15944 T (GCA_000422745.1) and Microbacterium Several strains of the genus clustered together and formed a separate branch, indicating that strain 2T1 was Microbacterium A potential new species of the genus.

[0117] 3. Physiological and chemical classification and identification

[0118] Add several drops of 5% H₂O₂ to a glass culture dish and select strain 2T1 to react with it. If bubbles are generated, this strain can produce catalase. Apply a spot of strain 2T1 onto filter paper soaked in 1% p-aminoxylidine hydrochloride, using Pseudomonas aeruginosa and Escherichia coli as positive and negative controls. If rose-red circles form around the colonies, this indicates oxidase production. Inoculate strain 2T1 at five locations on starch culture medium, setting up three replicates. Incubate at 25°C for 2-5 days. After removing the plate, add iodine solution around the colonies and observe the color changes around them. If a colorless, transparent circle forms around the colonies, this indicates that the bacteria is producing amylase and has diffused into the substrate, hydrolyzing the starch in the culture medium into a substance that does not react with iodine. If the colonies are blue, this indicates that the bacteria are not producing amylase. Strain 2T1 was inoculated at 5 points on casein culture medium, with 3 parallel plates set up. Cultured at 25°C for 7 days. After removing the plate, observe whether the casein around and below the colonies is decomposed into a transparent circle. If it is transparent, it means that the strain has the ability to hydrolyze casein.

[0119] The results showed that after strain 2T1 came into contact with 5% H202, bubbles were produced, indicating that its catalase result was positive; after strain 2T1 came into contact with filter paper soaked with 1% p-aminoxylidine hydrochloride, the bacteria did not change color, and its oxidase result was judged to be negative; the strain did not produce a transparent circle on casein culture medium or starch culture medium with iodine solution added, indicating that strain 2T1 did not have the ability to hydrolyze casein and starch.

[0120] The enzyme activities and carbohydrate utilization of strain 2T1 and related model strains were determined using API 20NE, ZYM, and 50 CH test strips (bioMérieux, France).

[0121] The results of 20NE test showed that the hydrolysis reaction of esculin, gelatin, and nitro- β -d-galactoside hydrolysis reaction is positive, nitrate reduction reaction is weakly positive, indole reaction, arginine hydrolysis reaction, urease hydrolysis reaction is negative, and it can assimilate glucose, mannitol, N -Acetyl-glucosamine, maltose and gluconate, cannot assimilate capric acid, adipic acid, malic acid, citric acid and phenylacetic acid.

[0122] In the ZYM enzyme activity identification test, leucine arylaminease, β -galactosidase, α - Glucosidase results were positive; alkaline phosphatase, esterase (C4), lipid esterase (C8), lipase (C14), cystine arylaminease, trypsin, chymotrypsin, acid phosphatase, β -uronidase, N -Acetyl-glucosaminidase,α - Fucosidase results were negative; valine arylaminease, naphthol-AS-BI-phosphohydrolase, α -galactosidase, β -glucosidase, α - Mannosidase result was weakly positive.

[0123] The results of 50 CH showed that strain 2T1 could hydrolyze l-arabinose, d-xylose, glucose, fructose, mannose, mannitol, methyl- α -d-glucopyranoside, N -Acetyl glucosamine, arbutin, esculin, salicin, cellobiose, maltose, sucrose, melezitose, d-arabinol; cannot hydrolyze d-arabinose, l-xylose, β- Methyl-d-xyloside, sorbose, dulcitol, sorbitol, methyl- α -d-mannopyranoside, d-melibiose, inulin, raffinose, starch, glycogen, d-lyxose, d-tagatose, d-fucose, l-fucose; weak utilization of mannitol, erythritol, ribose, galactose, l-rhamnose, inositol, amygdalin, lactose, trehalose, xylitol, d-gentiobiose, l-arabinol, gluconate, 2-keto-gluconate and 5-keto-gluconate.

[0124] The differences in physiological and biochemical characteristics between strain 2T1 and related model strains are shown in Table 2.

[0125]

[0126] Note: “+” indicates the test result is positive, “-” indicates the test result is negative, and “w” indicates the test result is weakly positive.

[0127] After the above identification, it can be confirmed that the strain 2T1 obtained in the present invention belongs to the genus Microbacterium ( Microbacterium ) a new species, named Microbacterium dendrobii ( Microbacterium dendrobii ), and deposited it in the General Microbiology Center of China Culture Collection Administration on November 29, 2024, and its registration number is CGMCC No.32856.

[0128] Example 2, Microbacterium dendrobium ( Microbacterium dendrobii ) Detection of 2T1's ability to secrete siderophores and dissolve potassium

[0129] Microbacterium dendrobium ( Microbacterium dendrobii ) 2T1 was spotted on CAS qualitative culture medium and cultured at 30℃ for 4 days to observe whether orange-yellow circles were produced. If so, it preliminarily indicated that the strain had the ability to secrete siderophores.

[0130] Microbacterium dendrobium (Microbacterium dendrobii ) 2T1 was spot-inoculated on a modified potassium-solubilizing medium and cultured at 30°C for 2 days to observe whether specific halos were produced. If so, it preliminarily indicated that the strain had the ability to solubilize potassium.

[0131] The results showed that after 4 days of growth on CAS qualitative medium, Microbacterium dendrobium ( Microbacterium dendrobii ) 2T1 can produce an orange-yellow transparent circle; after growing on the modified potassium-dissolving medium for 2 days, Microbacterium dendrobium ( Microbacterium dendrobii )2T1 can produce specific halos, indicating that Microbacterium dendrobium ( Microbacterium dendrobii ) 2T1 has the ability to produce iron carriers and dissolve potassium ( Figure 4 ).

[0132] Example 3, Microbacterium dendrobium ( Microbacterium dendrobii ) 2T1 alleviates plant saline-alkali stress

[0133] Pick up Microbacterium dendrobium ( Microbacterium dendrobii ) A single 2T1 colony was inoculated into a 500 mL conical flask containing 200 mL TSB liquid medium and cultured at 30°C for 48 h. The cells were collected by centrifugation at 8000 rpm for 10 min and resuspended in TSB liquid medium to an OD of 600 = 1.0 bacterial suspension (TSB liquid medium without bacteria was used as the control group for soaking seeds). Wheat seeds (Jimai 22) of uniform size were selected, soaked in 42℃ warm water for 6h, fished out and drained, and then soaked in bacterial solution for 4h, and sown in pots filled with 50g of vermiculite and nutrient soil mixed matrix (vermiculite: nutrient soil = 2:1), 10 seeds per pot. Set up 9 pots each for the experimental group and the control group (CK). When most of the wheat seeds germinated, thinning was carried out, and 5 wheat seedlings with similar growth were retained in each pot. Salt-alkali stress treatment was carried out on the 0th, 9th and 16th days after wheat sowing, and saline-alkali solution (NaCl, Na2SO4 and NaHCO3, the molar ratio of which is 1:1:1 mixed solution, Na + The bacterial suspension was then applied to the roots of wheat seedlings in the treated group (5 mL per plant) at a final concentration of 168 mmol / L (50 mL). Plants in the control group were watered with uninoculated TSB liquid medium. Plants were watered every three days during growth. The experiment was conducted in a tissue culture room. Day and night temperatures were 25°C / 16°C, with a 14-hour daylight period.

[0134] Figure 5 The growth status of wheat seedlings at 27 days old. Under saline-alkali stress, the leaves of wheat seedlings showed symptoms such as drying, curling, and falling off. The height of the plants was significantly reduced, and the plants showed an overall trend of withering. Different treatments showed different degrees of salt damage. Figure 6As shown, the aboveground dry weight and underground dry weight of wheat seedlings in the experimental group (2T1) were 0.0476±0.0105g and 0.0180±0.0059g, respectively, while those in the control group (CK) were 0.0338±0.0103g and 0.0098±0.0045g, respectively. The aboveground dry weight and underground dry weight of the experimental group were significantly higher than those in the control group (CK) ( P <0.01). The root length of the experimental group (2T1) was 14.7100±3.0410cm, which was significantly higher than that of the control (CK) ( P <0.001), indicating that the inoculation of Microbacterium dendrobium ( Microbacterium dendrobii )2T1 treatment can promote the growth of wheat seedlings under saline-alkali conditions.

[0135] To further investigate the ability of strain 2T1 to alleviate saline-alkali stress in wheat, catalase (CAT) activity, peroxidase (POD) activity, malondialdehyde (MDA) content, and reduced glutathione (GSH) and proline (PRO) content in leaves were measured. Appropriate amounts of wheat seedling leaves were cut into 2.0 mL centrifuge tubes, quickly placed in liquid nitrogen, and ground into powder. CAT, POD, MDA, GSH, and PRO were extracted and measured from wheat seedling leaves in the experimental and control groups according to the instructions provided in the Catalase (CAT) activity assay kit (Solarbio, China), the Peroxidase (POD) activity assay kit (Solarbio, China), the MDA (MDA) content assay kit (Solarbio, China), the GSH (GSH) content assay kit (Solarbio, China), and the Pro (Pro) content assay kit (Solarbio, China).

[0136] The results are as follows Figure 7 As shown, the CAT activity and SOD activity of the experimental group (2T1) were significantly higher than those of the control group (CK) ( P <0.01), SOD catalyzes the dismutation of superoxide anions to generate H2O2 and O2, and CAT catalyzes H2O2 to generate H2O and O2. SOD and CAT play an important role in the active oxygen scavenging system. Higher CAT and SOD activities indicate that plants have stronger antioxidant capacity and resistance to adversity. The above results show that Microbacterium dendrobium ( Microbacterium dendrobii ) 2T1 can stimulate plants to produce high-activity CAT and SOD, enabling them to better cope with saline-alkali stress conditions and benefiting their growth; at the same time, the GSH content of the experimental group (2T1) was significantly higher than that of the control group (CK) ( P<0.01), reactive oxygen species (ROS) are metabolic products in plants. Stress will aggravate the production of ROS, and its excessive accumulation will lead to reduced cell viability and even death. The glutathione (AsA-GSH) cycle is an important pathway for removing ROS in plants. Glutathione reductase (GR) is a key enzyme in this pathway, catalyzing the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH), maintaining the GSH content and the redox state of the GSH pool in the plant, and removing ROS. In summary, it is speculated that Microbacterium dendrobium ( Microbacterium dendrobii )2T1 alleviates the growth stress of wheat seedlings under saline-alkali conditions and improves the saline-alkali adaptability of wheat by inducing plants to produce a large amount of GSH and highly active CAT and SOD.

[0137] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. Microbacterium dendrobium, characterized by: The Microbacterium dendrobium is Microbacterium dendrobium ( Microbacterium dendrobii ), the strain number is 2T1, and its registration number at the China General Microbiology Center is CGMCC No.32856.

2. A composition containing the Microbacterium dendrobium according to claim 1.

3. The composition according to claim 2, characterized in that: The composition is a culture, and the culture is a substance obtained by culturing the Microbacterium dendrobium in a microbial culture medium.

4. The composition according to claim 2, characterized in that: The composition is a bacterial agent, a microecological preparation or a biological fertilizer.

5. The composition according to any one of claims 2 to 4, characterized in that: The composition has at least one of the following properties: A1) siderophore production; A2) potassium removal; A3) Alleviate plant salinity and alkali stress; A4) Increase CAT activity in plants; A5) Increase the SOD activity of plants; A6) Increase the GSH content of plants; A7) Promote plant growth; A8) Promote the increase of plant underground dry weight; A9) Promote the increase of plant aboveground dry weight; A10) Promotes the growth of plant roots; The promoting of plant growth is to promote plant growth under saline-alkali stress conditions; the promoting of increase in underground dry weight of plants is to promote increase in underground dry weight of plants under saline-alkali stress conditions; the promoting of increase in above-ground dry weight of plants is to promote increase in above-ground dry weight of plants under saline-alkali stress conditions; the promoting of increase in plant root length is to promote increase in root length of plants under saline-alkali stress conditions.

6. Use of the Microbacterium dendrobium according to claim 1 or the composition according to any one of claims 2 to 5, wherein the use is any one of the following: B1) Use in alleviating saline-alkali stress in plants or in preparing products for alleviating saline-alkali stress in plants; B2) Use in promoting plant growth or preparing products for promoting plant growth; B3) Use in promoting the increase of underground dry weight of plants or in preparing products for promoting the increase of underground dry weight of plants; B4) Use in promoting the increase of aboveground dry weight of plants or in preparing products for promoting the increase of aboveground dry weight of plants; B5) Use in promoting the increase of plant root length or in the preparation of a product for promoting the increase of plant root length; The promoting of plant growth is to promote plant growth under saline-alkali stress conditions; the promoting of increase in underground dry weight of plants is to promote increase in underground dry weight of plants under saline-alkali stress conditions; the promoting of increase in above-ground dry weight of plants is to promote increase in above-ground dry weight of plants under saline-alkali stress conditions; the promoting of increase in plant root length is to promote increase in root length of plants under saline-alkali stress conditions.

7. The use according to claim 6, characterized in that: The plant is an angiosperm.

8. The use according to claim 6, characterized in that: The plant is a monocotyledonous plant.

9. The use according to claim 6, characterized in that: The plant is a plant of the order Poaceae.

10. The use according to claim 6, characterized in that: The plant is a grass plant.

11. The use according to claim 6, characterized in that: The plant is a plant of the genus Triticum.

12. The use according to claim 6, characterized in that: The plant is wheat.

13. A method for alleviating saline-alkali stress in plants, comprising the steps of treating a plant to be treated or its growth medium with the Microbacterium dendrobium according to claim 1 or the composition according to any one of claims 2 to 5, thereby alleviating the saline-alkali stress in the plant.

14. A method for promoting plant growth, comprising the steps of: treating a plant to be treated or a growth substrate thereof with the Microbacterium dendrobium of claim 1 or the composition of any one of claims 2 to 5, thereby promoting the growth of the plant; The promoting plant growth is promoting plant growth under saline-alkali stress conditions.

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