Microbacterium capable of producing siderophore, dissolving potassium, promoting growth and relieving saline-alkali stress of plants and application of microbacterium

Abstract

The invention discloses a microbacterium capable of producing siderophores, dissolving potassium, promoting growth and relieving saline-alkali stress of plants and application of the microbacterium. The invention relates to the field of microorganisms, and provides a dendrobium nobile microbacterium, the dendrobium nobile microbacterium is a dendrobium nobile microbacterium, the strain number of the dendrobium nobile microbacterium is 2T1, and the registration number of the dendrobium nobile microbacterium in the China General Microbiological Culture Collection Center is CGMCC No. 32856. The strain is a new strain of microbacterium and has the capabilities of secreting siderophores and dissolving potassium. Pot experiments show that compared with uninoculated control, the root length and dry weight of wheat can be improved after the strain is inoculated under saline-alkali stress. The microbacterium dendrobii 2T1 can be used as a microbial organic fertilizer, and is used for improving the soil fertility, relieving the saline-alkali stress of crops and improving the saline-alkali adaptability of the crops.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and particularly relates to a Microbacterium strain with the abilities of siderophore production, potassium solubilization, plant growth promotion and alleviation of plant salt-alkali stress, and its application. Background Art

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

[0003] Numerous PGPR groups have been discovered at home and abroad, with functions such as secreting plant hormones, dissolving phosphorus, dissolving iron, and solubilizing potassium. Among them, members of the genus Microbacterium ( Microbacterium ) are widely present in nature and have been isolated from different habitats such as soil, plants, water, dairy products, insects, and humans. Strains of the genus Microbacterium have various functions, such as producing indole-3-acetic acid (IAA), degrading xylan, and dissolving phosphate. In addition, they also have the ability to decompose starch, good cold tolerance, heat tolerance, salt tolerance, and alkali tolerance, and at the same time have the ability to resist ultraviolet radiation, can protect plants from nematode pathogens, and promote 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 with the abilities of siderophore production, potassium solubilization, plant growth promotion and alleviation of plant salt-alkali stress, and its application.

[0005] In the first aspect, the present invention claims to protect a Microbacterium dendrobii.

[0006] The Microbacterium dendrobii claimed to be protected by the present invention is Microbacterium dendrobii ( Microbacterium dendrobii ), with the strain number 2T1 and the registration number in the China General Microbiological Culture Collection Center being CGMCC No. 32856.

[0007] Microbacterium dendrobii ( Microbacterium dendrobii ) 2T1 is a Gram-positive bacterium. After growing on TSA solid medium for 3 days, the colonies are light yellow, round, convex, with smooth edges, viscous, shiny, and the colony diameter is 1-2 mm. This strain has excellent plant growth promotion ability and can significantly alleviate the stress of salt-alkali on plants.

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

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

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

[0011] In the above-mentioned culture, the substance includes the Microbacterium dendrobii (the cells themselves) and / or its metabolites described in the first aspect above.

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

[0013] The term "culture" refers to the general term for liquid or solid culture media with microbial populations grown after artificial inoculation and culture. That is, the product obtained by growing and / or amplifying microorganisms, which can be a biological pure culture of microorganisms (i.e., the Microbacterium dendrobii), or can contain a certain amount of culture medium, metabolites, or other components produced during the culture process. The term "culture" also includes the subculture obtained by subculturing microorganisms, which can be a culture of a certain generation or a mixture of several generations.

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

[0015] In the above-mentioned bacterial agent, microecological preparation or biological fertilizer, the active ingredient of the bacterial agent, microecological preparation or biological fertilizer can be Microbacterium dendrobii described in the first aspect above, the metabolite of Microbacterium dendrobii and / or the culture of Microbacterium dendrobii. The active ingredient of the bacterial agent, microecological preparation or biological fertilizer can also contain other biological components and / or non-biological components. Those skilled in the art can determine other active ingredients of the bacterial agent, microecological preparation or biological fertilizer according to the desired effect.

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

[0017] In the above-mentioned bacterial agent, microecological preparation or biological fertilizer, the dosage form of the bacterial agent, microecological preparation or biological fertilizer can be various dosage forms, such as liquid agent, emulsion, suspension, powder, granule, wettable powder or water dispersible granule.

[0018] According to needs, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH regulators, etc. can also be added to the bacterial agent, microecological preparation or biological fertilizer.

[0019] In the bacterial agent, microecological preparation or biological fertilizer, Microbacterium dendrobii or / and the metabolite of Microbacterium dendrobii can exist in the form of cultured live cells, the fermentation broth of live cells, the filtrate of cell culture or the mixture of cells and filtrate.

[0020] In this text, the metabolite of Microbacterium dendrobii can be obtained from the fermentation broth of Microbacterium dendrobii. The metabolite of Microbacterium dendrobii can be the sterile metabolite or the bacteria-containing metabolite of Microbacterium dendrobii. The sterile metabolite (sterile fermentation filtrate) of Microbacterium dendrobii can be specifically prepared by the following method: culturing Microbacterium dendrobii in a liquid medium, and filtering to remove Microbacterium dendrobii in the liquid culture (fermentation broth), thereby obtaining the sterile metabolite of Microbacterium dendrobii. The bacteria-containing metabolite of Microbacterium dendrobii can be specifically prepared by the following method: culturing Microbacterium dendrobii in a liquid fermentation medium, and collecting the fermentation broth - containing Microbacterium dendrobii and substances secreted into the liquid medium, and this fermentation broth is the bacteria-containing metabolite of Microbacterium dendrobii.

[0021] Further, the composition may have at least one of the following properties: A1) Producing siderophores; A2) Potassium solubilization; A3) Alleviating plant saline-alkali stress; A4) Increasing the CAT activity of plants; A5) Increasing the SOD activity of plants; A6) Increasing the GSH content of plants; A7) Promoting plant growth; A8) Promoting the increase of plant underground dry weight; A9) Promoting the increase of plant aboveground dry weight; A10) Promoting the increase of plant root length.

[0022] In the third aspect, the present invention claims the application of the Microbacterium dendrobii described in the first aspect or the composition described in the second aspect, and the application can be any of the following: B1) Application in producing siderophores or preparing products for producing siderophores; B2) Application in potassium solubilization or preparing products for potassium solubilization; B3) Application in alleviating plant saline-alkali stress or preparing products for alleviating plant saline-alkali stress; B4) Application in increasing the CAT activity of plants or preparing products for increasing the CAT activity of plants; B5) Application in increasing the SOD activity of plants or preparing products for increasing the SOD activity of plants; B6) Application in increasing the GSH content of plants or preparing products for increasing the GSH content of plants; B7) Application in promoting plant growth or preparing products for promoting plant growth; B8) Application in promoting the increase of plant underground dry weight or preparing products for promoting the increase of plant underground dry weight; B9) Use in promoting the increase of the above-ground dry weight of plants or in preparing a product for promoting the increase of the above-ground dry weight of plants; B10) Use in promoting the increase of the root length of plants or in preparing a product for promoting the increase of the root length of plants.

[0023] Furthermore, the improvement of the CAT activity of plants can be the improvement of the CAT activity of plants under saline-alkali stress conditions.

[0024] Furthermore, the improvement of the SOD activity of plants can be the improvement of the SOD activity of plants under saline-alkali stress conditions.

[0025] Furthermore, the increase of the GSH content of plants can be the increase of the GSH content of plants under saline-alkali stress conditions.

[0026] Furthermore, the promotion of plant growth can be the promotion of plant growth under saline-alkali stress conditions.

[0027] Furthermore, the promotion of the increase of the underground dry weight of plants can be the promotion of the increase of the underground dry weight of plants under saline-alkali stress conditions.

[0028] Furthermore, the promotion of the increase of the above-ground dry weight of plants can be the promotion of the increase of the above-ground dry weight of plants under saline-alkali stress conditions.

[0029] Furthermore, the promotion of the increase of the root length of plants can be the promotion of the increase of the root length of plants under saline-alkali stress conditions.

[0030] Fourthly, the present invention claims to protect a method for alleviating saline-alkali stress of plants.

[0031] The method for alleviating saline-alkali stress of plants claimed by the present invention may include the following steps: treating the plant to be treated or its growth substrate with the Microbacterium dendrobii described in the first aspect above or the composition described in the second aspect above, so as to alleviate the saline-alkali stress of the plant.

[0032] In an embodiment of the present invention, the treatment is to perform root irrigation on the plant with a bacterial suspension containing the Microbacterium dendrobii.

[0033] Fifthly, the present invention claims to protect a method for promoting plant growth.

[0034] The method for promoting plant growth claimed by the present invention may include the following steps: treating the plant to be treated or its growth substrate with the Microbacterium dendrobii described in the first aspect above or the composition described in the second aspect above, so as to achieve the promotion of the growth of the plant.

[0035] Furthermore, the promotion of plant growth can be the promotion of plant growth under saline-alkali stress conditions.

[0036] In one embodiment of the present invention, the treatment is to irrigate the roots of the plant with a bacterial suspension containing the Microbacterium dendrobii.

[0037] In a sixth aspect, the present invention claims a method for culturing Microbacterium dendrobii.

[0038] The method for culturing Microbacterium dendrobii claimed by the present invention includes the step of culturing the Microbacterium dendrobii in a culture medium for culturing microorganisms; the Microbacterium dendrobii is the Microbacterium dendrobii described in the first aspect above.

[0039] In a seventh aspect, the present invention claims a method for preparing a composition.

[0040] The method for preparing a composition claimed by the present invention includes the step of using the Microbacterium dendrobii described in the first aspect above as a component of the composition; the composition is the composition described in the second aspect above.

[0041] In the present invention, the saline-alkali stress may be salt stress and / or alkali stress.

[0042] In each of the above-related aspects, in one embodiment of the present invention, the saline-alkali stress is simulated as follows: a mixed solution of NaCl, Na2SO4, and NaHCO3 with a molar ratio of 1:1:1 and a final concentration of 168 mmol / L of Na + .

[0043] In each of the above-related aspects, the plant may be any one of the following: C1) Angiosperms; C2) Monocotyledons; C3) Plants of the order Poales; C4) Gramineous plants; C5) Plants of the genus Triticum; C6) Wheat.

[0044] Experimental results show that the Microbacterium dendrobii ( Microbacterium dendrobii ) 2T1 provided by the present invention is a new strain of the genus Microbacterium. Microbacterium dendrobii ( Microbacterium dendrobii ) 2T1 has the ability to secrete siderophores and dissolve potassium. Pot experiment shows that compared with the un-inoculated negative control group, inoculation with Microbacterium dendrobii ( Microbacterium dendrobii ) 2T1 can increase the root length and dry weight of wheat. Microbacterium dendrobii ( Microbacterium dendrobii ) 2T1 can be used as a microbial organic fertilizer to improve soil fertility, alleviate the saline-alkali stress of crops, and enhance the saline-alkali adaptability of crops.

[0045] Deposit description Taxonomic name: Microbacterium dendrobii (Microbacterium dendrobii ); Biological material for reference: 2T1; Depository institution: General Microbiology Center of China Committee for Culture Collection of Microorganisms; Abbreviation of depository institution: CGMCC; Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Date of deposit: November 29, 2024; Registration number in the depository center: CGMCC No. 32856. Description of the drawings

[0046] Figure 1 Colony morphology of Microbacterium dendrobii ([[]] Microbacterium dendrobii ) 2T1 after culturing on TSA plate for 3 days.

[0047] Figure 2 Phylogenetic tree of Microbacterium dendrobii ([[]] Microbacterium dendrobii ) 2T1 and related type strains constructed by the neighbor-joining method based on 16S rRNA gene sequences. Note: The GenBank accession number of the 16S rRNA gene sequence of the strain is shown in parentheses; all reference strains in the figure are type strains of their respective species. Streptomyces showdoensis NBRC13417 T (AB184389) was used as the outgroup.

[0048] Figure 3 Genomic phylogenetic tree of Microbacterium dendrobii ([[]] Microbacterium dendrobii ) 2T1 and related Microbacterium closely related species within the genus constructed by the maximum likelihood method with 92 concatenated bacterial core genes.

[0049] Figure 4 Detection results of siderophore secretion and potassium solubilizing ability of Microbacterium dendrobii ([[]] Microbacterium dendrobii ) 2T1. The left side shows the morphology of strain 2T1 after growing on the modified potassium solubilizing medium for 2 days; the right side shows the morphology of strain 2T1 after growing on the CAS qualitative medium for 4 days.

[0050] Figure 5 Growth status of wheat seedlings inoculated with strain 2T1 and non-inoculated strain (CK) under saline-alkali stress at 27 days. The left side is CK, and the right side is the inoculated strain 2T1.

[0051] Figure 6 Root length and dry weight of wheat seedlings under 27-day saline-alkali stress. CK is the control without inoculating the bacterial solution. ** in the figure indicates P <0.01, *** indicates P <0.001.

[0052] Figure 7 Determination of the response of wheat seedlings to environmental stress and antioxidant capacity indexes under 27d saline-alkali stress. In the figure, ** indicates P <0.01. Specific implementation manners

[0053] The present invention will be further described in detail below in conjunction with specific implementation manners. The examples given are only for clarifying the present invention, rather than limiting the scope of the present invention. The following examples can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0054] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0055] The formulations of various culture media and solutions involved in the following examples are as follows: (1) TSB liquid medium: 15.0 g of tryptone, 5.0 g of soy peptone, 5.0 g of sodium chloride, 1000 mL of distilled water, adjust the pH to 7.3 ± 0.2, sterilize at 121 °C for 15 min.

[0056] (2) TSA solid medium (TSA plate): Add 20.0 g of agar to the TSB liquid medium and sterilize at 121 °C for 15 min.

[0057] (3) Starch medium: NA medium, 0.2% (w / v) soluble starch, pH 7.4, sterilize at 121 °C for 20 min.

[0058] (4) Casein medium: Solution a: 5 g of skim milk powder, 50 mL of distilled water; Solution b: 50 mL of NB, 1.5 g of agar. Sterilize solution a and solution b at 121 °C for 15 min respectively. After cooling to about 60 °C, mix them evenly and dispense into plates.

[0059] (5) CAS qualitative medium: Solution ①: Dissolve 0.012 g of CAS in 10 mL of deionized water and mix it with 2 mL of 5 mM ferric chloride.

[0060] Solution ②: Dissolve 0.015 g of cetyltrimethylammonium bromide in 8 mL of deionized water.

[0061] Dye solution ③: Slowly pour solution ① into solution ② to obtain dye solution ③, and sterilize it at 115 °C for 20 min.

[0062] Culture medium ④: Add 6.04 g of piperazine diethanol sulfonic acid and 10 mL of 0.1 M phosphate solution into an Erlenmeyer flask containing 150 mL of distilled water and mix well. Adjust the pH to 6.8 with 50% NaOH, add 4.0 g of agar powder, and sterilize at 115 °C for 20 min.

[0063] Phosphate solution: 2.427 g of disodium hydrogen phosphate, 0.5905 g of sodium dihydrogen phosphate, 0.075 g of potassium dihydrogen phosphate, 0.125 g of sodium chloride, 0.25 g of ammonium chloride, 100 mL of deionized water. Mix well and dilute 10 times before use.

[0064] Nutrient solution: 0.2 mL of 1 mM calcium chloride solution, 4 mL of 1 mM magnesium sulfate tetrahydrate solution, 6 mL of 10% (w / V) casein amino acid solution. Sterilize each at 115 °C for 20 min respectively (10 mL can be prepared when in use, and store in a 4 °C refrigerator in the dark for future use after use).

[0065] CAS qualitative medium plate: When the dye solution ③, culture medium ④, and nutrient solution are cooled to about 65 °C, add the nutrient solution to the culture medium ④, then slowly add the dye solution ③, mix well and pour into plates.

[0066] (6) Modified potassium-solubilizing medium: Aleksandrov medium: 5.0 g of sucrose, 2.0 g of disodium hydrogen phosphate, 0.5 g of magnesium sulfate heptahydrate, 5.0 mg of ferric chloride, 0.1 g of calcium carbonate, 1.0 g of potassium feldspar powder, 1000 mL of distilled water, 20.0 g of agar, pH 7.2. Sterilize at 121 °C for 15 minutes.

[0067] Modified potassium-solubilizing medium: Add 100 mg / L of bromothymol blue to the Aleksandrov medium.

[0068] Example 1. Isolation and identification of Microbacterium dendrobii ( Microbacterium dendrobii ) 2T1 I. Isolation of Microbacterium dendrobii ( Microbacterium dendrobii ) 2T1 Collect the rhizosphere soil samples of Dendrobium in Yuli County, Xinjiang Uygur Autonomous Region (41°39′7″N, 86°30′67″E). Use a 4°C ice box to bring them back to the laboratory and store them in a 4°C refrigerator. Shake off the soil attached to the plant roots, and only retain the rhizosphere soil tightly adhered to the root surface. Immerse the Dendrobium roots with rhizosphere soil in a conical flask containing 100 mL of sterile water, and oscillate at 150 rpm at room temperature in a shaker for 30 min. Centrifuge the collected suspension at 3000 rpm for 10 min at 4°C. After discarding the supernatant, the remaining is the rhizosphere soil. Weigh 1 g of rhizosphere soil and resuspend it in 10 mL of sterile water for serial dilution. Take 100 μL of the serial dilution and spread it on a TSA plate, and incubate it upside down at 30°C for 1 week. According to the physiological and morphological characteristics, use a bamboo stick to pick single colonies and purify them on the plate. After determining them as pure bacteria, transfer them to a slant for short-term storage at 4°C and transfer them to a 20% glycerol tube for long-term storage at -80°C. One of the isolated and purified strains was named 2T1.

[0069] II. Identification of Microbacterium dendrobii Microbacterium dendrobii 2T1 1. Strain morphological identification Describe the single colony state of strain 2T1, which was isolated and purified in the above step one and is in the logarithmic growth phase with a stable colony size. This mainly includes the size, color, transparency, surface state of the colony, and edge state of the colony. Use the Gram staining kit of Solarbio Science & Technology Co., Ltd. (Solarbio) in Beijing to perform smear Gram staining on strain 2T1 according to the manufacturer's instructions, and observe the morphology of the bacterial cells using an optical microscope.

[0070] The colonies of strain 2T1 on the TSA plate are light yellow, round, convex, with smooth edges, viscous, shiny, and the colony diameter is 1 - 2 mm ( Figure 1 ). The cells are Gram-positive and rod-shaped, without spore formation.

[0071] 2. Molecular identification Operate according to the instructions. Use the TIANamp Bacterial Genomic DNA Extraction Kit of TIANGEN Biochemical Technology (Beijing) Co., Ltd. to extract genomic DNA. The genomic DNA is sent to Annoroad Gene Technology (Beijing) Co., Ltd., and the Illumina NovaSeq 6000 sequencing system is used to sequence the genomic draft of strain 2T1. Use the SPAdes software for assembly to obtain 33 contigs, N 50The length is 3070005 bp. The genome size is 2.99 Mb, and the G+C content is 70.47%. The sequence of the 16S rRNA gene based on the genome was uploaded to Ezbiocloud (www.ezbiocloud.net / eztaxon) for sequence alignment. The average nucleotide identity (ANI) analysis of the whole genomes of strain 2T1 and Microbacterium strains closely related to the genus was performed using the ANIm method in the pyANI software. The digital DNA-DNA hybridization (dDDH) values between strain 2T1 and the reference strains were calculated and compared using the Genome-to-Genome Distance Calculator (GGDC) 3.0 server (https: / / ggdc.dsmz.de / ggdc.php#).

[0072] The sequencing length of the 16S rRNA gene of strain 2T1 is 1525 bp (SEQ ID No.1). The alignment results in the EzBioCloud database showed that strain 2T1 had high similarity with 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 (97.63%) sequences, and all were lower than the classification threshold (98.8%) of the described species. The 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 Neighbour-Joining method in the MEGA X software. The evolutionary distance of the NJ method was calculated using the Kimura two-parameter model, and the Bootstrap value was 1000. The phylogenetic tree constructed by the neighbour-joining method is as Figure 2 shown, in which strain 2T1 clustered with strain Microbacterium excoecariae CBS5P-1 T and formed a separate branch, indicating that strain 2T1 is Microbacterium a potential new species of the genus.

[0073] Strain 2T1 and other MicrobacteriumCompared with the type strain of the genus species, the ANI value is 80 - 81%, lower than the critical value of 95 - 96% previously proposed for species delimitation; the dDDH value between strain 2T1 and its type strain is between 22.7 - 25.0%, far lower than the species delimitation threshold of 70%. See Table 1 for details. The results of both ANI and dDDH indicate that strain 2T1 is Microbacterium a new species of the genus.

[0074]

[0075] To further clarify the taxonomic status of strain 2T1, the present invention conducted a genomic phylogenetic analysis. Using the UBCG software, strain 2T1 and Microbacterium 20 type strains of the genus were analyzed. Ninety-two bacterial core genes were concatenated, and a phylogenetic tree of gene sequences was constructed using the maximum likelihood method ( Figure 3 ), with a Bootstrap value of 1000. The results showed that strain 2T1 clustered together with strain Microbacterium gubbeenense DSM 15944 T (GCA_000422745.1) and several strains of the genus Microbacterium and formed a separate branch, indicating that strain 2T1 is Microbacterium a potential new species of the genus.

[0076] 3. Physiological and chemotaxonomic identification Drop several drops of 5% H202 on a glass petri dish, pick strain 2T1 and react with it. If bubbles are generated, it proves that the strain can produce catalase. Spot inoculate strain 2T1 on a filter paper soaked with 1% p-aminodimethylaniline hydrochloride, using Pseudomonas aeruginosa and Escherichia coli as positive and negative controls respectively. If a magenta ring is produced around the colony, it indicates that it can produce oxidase. Spot inoculate strain 2T1 at 5 points on a starch medium, set 3 parallels, and culture at 25°C for 2 - 5 days. After taking out the plate, drop iodine solution around the colony and observe the color change around the colony. If there is a colorless transparent circle around the colony, it means that the bacterium produces amylase and diffuses into the matrix, and has hydrolyzed the starch in the medium into substances that do not show color when reacting with iodine; if the area around the colony is blue, it means that the bacterium does not produce amylase. Spot inoculate strain 2T1 at 5 points on a casein medium, set 3 parallels, and culture at 25°C for 7 days. After taking out the plate, observe whether the casein around and under the colony is decomposed into a transparent circle. If it is transparent, it indicates that the strain has the ability to hydrolyze casein.

[0077] The results showed that bubbles were produced after strain 2T1 was contacted with 5% H2O2, indicating a positive catalase result; after strain 2T1 was contacted with a filter paper soaked with 1% p-dimethylaminobenzene hydrochloride, no color change occurred in the bacterial cells, and it was judged that the oxidase result was negative; no clear zones were produced on the casein medium and the starch medium added with iodine solution, indicating that strain 2T1 did not have the ability to hydrolyze casein and starch.

[0078] The API 20NE, ZYM and 50 CH test strips (bioMérieux) of bioMérieux, France were used to determine the enzymatic activity reactions and carbohydrate utilization of strain 2T1 and related reference strains.

[0079] The results of the 20NE test showed that the aesculin hydrolysis reaction, gelatin hydrolysis reaction, p-nitro- β -d-galactosidase hydrolysis reaction of strain 2T1 were positive, the nitrate reduction reaction was weakly positive, and the indole reaction, arginine hydrolysis reaction, urease hydrolysis reaction were negative. It could assimilate glucose, mannitol, N -N-acetyl-glucosamine, maltose and gluconate, and could not assimilate capric acid, adipic acid, malic acid, citric acid and phenylacetic acid.

[0080] In the ZYM enzymatic activity identification test, leucine arylamidase, β -galactosidase, α -glucosidase results were positive; alkaline phosphatase, esterase (C4), lipase (C8), lipase (C14), cystine arylamidase, trypsin, chymotrypsin, acid phosphatase, β -glucuronidase, N -N-acetyl-glucosaminidase, α -fucosidase results were negative; valine arylamidase, naphthol-AS-BI-phosphohydrolase, α -galactosidase, β -glucosidase, α -mannosidase results were weakly positive.

[0081] The results of 50 CH showed that strain 2T1 could hydrolyze l-arabinose, d-xylose, glucose, fructose, mannose, mannitol, methyl- α -d-glucopyranoside, N -N-acetylglucosamine, arbutin, aesculin, salicin, cellobiose, maltose, sucrose, melezitose, d-arabitol; it could not 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; weakly utilize mannitol, erythritol, ribose, galactose, L-rhamnose, inositol, amygdalin, lactose, trehalose, xylitol, D-gentiobiose, L-arabitol, gluconate, 2-keto-gluconate and 5-keto-gluconate.

[0082] The physiological and biochemical characteristics differences between strain 2T1 and related type strains are shown in Table 2.

[0083]

[0084] Note: "+" indicates that the test result is positive, "-" indicates that the test result is negative, and "w" indicates that the test result is weakly positive.

[0085] After the above identification, it can be confirmed that the obtained strain 2T1 of the present invention is a new strain of the genus Microbacterium ( Microbacterium ), named Microbacterium dendrobii ( Microbacterium dendrobii ), and it was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on November 29, 2024, with the registration number of CGMCC No. 32856.

[0086] Example 2. Detection of the ability of Microbacterium dendrobii ( Microbacterium dendrobii ) 2T1 to secrete siderophores and potassium-solubilizing ability Spot inoculate Microbacterium dendrobii ( Microbacterium dendrobii ) 2T1 on the CAS qualitative medium and culture it at 30 °C for 4 d, then observe whether there is an orange-yellow circle. If so, it preliminarily indicates that the strain has the ability to secrete siderophores.

[0087] Spot inoculate Microbacterium dendrobii ( Microbacterium dendrobii ) 2T1 on the modified potassium-solubilizing medium and culture it at 30 °C for 2 d, then observe whether there is a specific halo. If so, it preliminarily indicates that the strain has the potassium-solubilizing ability.

[0088] The results show that: after growing on the CAS qualitative medium for 4 d, Microbacterium dendrobii ( Microbacterium dendrobii ) 2T1 can produce an orange-yellow transparent circle; after growing on the modified potassium-solubilizing medium for 2 d, Microbacterium dendrobii ( Microbacterium dendrobii ) 2T1 can produce a specific halo, indicating that Microbacterium dendrobii ( Microbacterium dendrobii ) 2T1 has the ability to produce siderophores and potassium-solubilizing ability ( Figure 4 ).

[0089] Example 3. Microbacterium dendrobii ( Microbacterium dendrobii ) 2T1 relieves plant salt-alkali stress Pick a single colony of Microbacterium dendrobii ( Microbacterium dendrobii ), inoculate it into a 500 mL conical flask containing 200 mL of TSB liquid medium, and culture it at 30 °C for 48 h. Centrifuge at 8000 rpm for 10 min, collect the bacterial cells, and resuspend them in TSB liquid medium to obtain a bacterial suspension with an OD 600 = 1.0 (using the TSB liquid medium without inoculation as the control group for seed soaking). Select wheat (Jimai 22) seeds of uniform size, soak them in warm water at 42 °C for 6 h, take them out and drain, then soak them in the bacterial suspension for 4 h, and sow them in flower pots containing 50 g of a mixed substrate of vermiculite and nutrient soil (vermiculite: nutrient soil = 2:1), with 10 seeds per pot. Set up 9 pots for each of the experimental group and the control group (CK). When most of the wheat seeds germinate, thin out the seedlings, and keep 5 wheat seedlings with similar growth in each flower pot. Conduct saline-alkali stress treatment on the 0th day, 9th day, and 16th day after wheat sowing, and pour 50 mL of saline-alkali solution (a mixed solution of NaCl, Na2SO4, and NaHCO3 with a molar ratio of 1:1:1, and the final concentration of Na + is 168 mmol / L) into each pot; conduct root irrigation with the bacterial suspension on the 10th day and 17th day, pour 5 mL of the bacterial suspension into the roots of the wheat seedlings in the treatment group, and pour pure TSB liquid medium without inoculation into the control group. Water once every 3 days during the plant growth period. The experiment is carried out in a tissue culture room. The day and night temperatures are 25 °C / 16 °C, and the sunshine time is 14 h.

[0090] Figure 5 shows the growth status of wheat seedlings at 27 days. Under saline-alkali stress, the leaves of wheat seedlings showed symptoms such as withering, curling, and shedding, the plant height decreased significantly, and the overall showed a withering trend. Different treatments showed different degrees of salt damage. The results of the agronomic traits of wheat seedlings Figure 6 are shown. The above-ground dry weight and underground dry weight of the wheat seedlings in the experimental group (2T1) were 0.0476 ± 0.0105 g and 0.0180 ± 0.0059 g respectively, and the above-ground dry weight and underground dry weight of the control group (CK) were 0.0338 ± 0.0103 g and 0.0098 ± 0.0045 g respectively. The above-ground dry weight and underground dry weight of the experimental group were significantly higher than those of the control group (CK) ( P < 0.01). The root length of the experimental group (2T1) was 14.7100 ± 3.0410 cm, which was significantly higher than that of the control (CK) ( P < 0.001), indicating that the treatment of inoculating Microbacterium dendrobii ( Microbacterium dendrobii ) 2T1 could promote the growth of wheat seedlings under saline-alkali conditions.

[0091] To further investigate the ability of strain 2T1 to alleviate saline-alkali stress in wheat, the activities of catalase (CAT), peroxidase (POD), the content of malondialdehyde (MDA), reduced glutathione (GSH), and proline (PRO) in leaves were determined. An appropriate amount of wheat seedling leaves was cut into 2.0 mL centrifuge tubes, quickly placed in liquid nitrogen, and ground into powder. According to the methods described in the instruction manuals of the catalase (CAT) activity detection kit (Solarbio, China), peroxidase (POD) activity detection kit (Solarbio, China), malondialdehyde (MDA) content detection kit (Solarbio, China), reduced glutathione (GSH) content detection kit (Solarbio, China), and proline (Pro) content detection kit (Solarbio, China), the CAT, POD, MDA, GSH, and PRO in the wheat seedling leaves of the experimental group and the control group were extracted and determined.

[0092] The results are as Figure 7 shown. The CAT activity and SOD activity in the experimental group (2T1) were significantly higher than those in 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 important roles in the reactive oxygen species scavenging system. Better CAT and SOD activities indicate that plants have stronger antioxidant capacity and resistance to adversity. The above results show that Microbacterium dendrobii ( Microbacterium dendrobii ) 2T1 can stimulate plants to produce highly active CAT and SOD, enabling the plants to better cope with saline-alkali stress conditions and facilitating the growth of the plants. At the same time, the GSH content in the experimental group (2T1) was significantly higher than that in the control group (CK) ( P < 0.01). Reactive oxygen species (ROS) are metabolic products in plants. Stress will exacerbate the production of ROS, and its excessive accumulation leads to a decrease in cell viability and even death. The glutathione (AsA-GSH) cycle is an important pathway for scavenging ROS in plants. Glutathione reductase (GR) is a key enzyme in this pathway, catalyzing the reduction of oxidized glutathione (GSSG) to generate reduced glutathione (GSH), maintaining the GSH content and the redox state of the GSH pool in plants, and scavenging ROS. In summary, it is speculated that Microbacterium dendrobii ( 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.

[0093] The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses, or improvements to the present invention, including changes made using conventional techniques known in the art that depart from the scope disclosed in this application.

Claims

1. Microbacterium dendrobii, characterized in that: The Microbacterium dendrobii is Microbacterium dendrobii ( Microbacterium dendrobii ), its strain number is 2T1, and its registration number in the China General Microbiological Culture Collection Center is CGMCC No. 32856.

2. A composition containing the Microbacterium dendrobii described in 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 dendrobii in a microbial culture medium.

4. The composition according to claim 2, wherein: The composition is a microbial agent or a microecological agent or a bio-fertilizer.

5. The composition according to any one of claims 2-4, characterized in that: The composition has at least one of the following properties: A1) Producing siderophores; A2) Releasing potassium; A3) Alleviating saline-alkali stress of plants; A4) Increasing the CAT activity of plants; A5) Increasing the SOD activity of plants; A6) Increasing the GSH content of plants; A7) Promoting plant growth; A8) Promoting the increase of underground dry weight of plants; A9) Promoting the increase of aboveground dry weight of plants; A10) Promoting the increase of root length of plants.

6. Use of the Microbacterium dendrobii described in claim 1 or the composition described in any one of claims 2 - 5, and the use is any one of the following: B1) Use in producing siderophores or preparing products for producing siderophores; B2) Use in releasing potassium or preparing products for releasing potassium; B3) Use in alleviating saline-alkali stress of plants or preparing products for alleviating saline-alkali stress of plants; B4) Use in increasing the CAT activity of plants or preparing products for increasing the CAT activity of plants; B5) Use in increasing the SOD activity of plants or preparing products for increasing the SOD activity of plants; B6) Use in increasing the GSH content of plants or preparing products for increasing the GSH content of plants; B7) Use in promoting plant growth or preparing products for promoting plant growth; B8) Use in promoting the increase of underground dry weight of plants or preparing products for promoting the increase of underground dry weight of plants; B9) Use in promoting the increase of aboveground dry weight of plants or preparing products for promoting the increase of aboveground dry weight of plants; B10) Use in promoting the increase of root length of plants or preparing products for promoting the increase of root length of plants.

7. The application according to claim 6, wherein: The increase in the CAT activity of plants is to increase the CAT activity of plants under saline-alkali stress conditions; and / or, the increase in the SOD activity of plants is to increase the SOD activity of plants under saline-alkali stress conditions; and / or, the increase in the GSH content of plants is to increase the GSH content of plants under saline-alkali stress conditions; and / or The promotion of plant growth is to promote plant growth under saline-alkali stress conditions; and / or, the promotion of the increase of underground dry weight of plants is to promote the increase of underground dry weight of plants under saline-alkali stress conditions; and / or, the promotion of the increase of aboveground dry weight of plants is to promote the increase of aboveground dry weight of plants under saline-alkali stress conditions; and / or, the promotion of the increase of root length of plants is to promote the increase of root length of plants under saline-alkali stress conditions.

8. The application according to claim 6 or 7, characterized in that: The plant is any one of the following: C1) Angiosperm; C2) Monocotyledon; C3) Plants of Poales; C4) Gramineous plants; C5) Plants of the genus Triticum; C6) Wheat.

9. A method for alleviating saline-alkali stress of plants, comprising the following steps: treating the plant to be treated or its growth substrate with the Microbacterium dendrobii described in claim 1 or the composition described in any one of claims 2 - 5, so as to achieve alleviating the saline-alkali stress of the plant.

10. A method for promoting plant growth, comprising the following steps: treating a plant to be treated or its growth substrate with the Microbacterium dendrobii described in claim 1 or the composition described in any one of claims 2-5, so as to promote the growth of the plant.

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