Strain C5A-1 for producing lipopeptide and having plant growth promoting function and application of strain C5A-1

Abstract

The invention discloses a bacterial strain C5A-1 capable of producing lipopeptide and having a plant growth promoting function and application of the bacterial strain C5A-1, solves the technical problem of promoting plant growth, especially root length, plant height, stem diameter, leaf number, leaf area, underground dry weight and overground dry weight, and particularly discloses bacillus velezensis C5A-1 or a variant or a progeny thereof. The bacillus velezensis C5A-1 is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.32880. When the bacteria or the fermented supernate thereof or the combination thereof is applied to plants, the growth of the plants can be promoted, and the bacteria can be used for industrial production.

Description

Technical Field

[0001] The present invention specifically relates to a strain of lipid peptide-producing and plant growth-promoting functional strain C5A-1 and its applications. Background Art

[0002] Under the triple pressures of global population growth, shrinking arable land, and climate change, the coordinated development of agricultural productivity improvement and ecological sustainability has become the core issue in the 21st century. According to the prediction of the Food and Agriculture Organization of the United Nations (FAO), the global food demand will increase by 70% by 2050. However, the intensive planting mode that traditionally relies on chemical fertilizers and synthetic hormones has approached the ecological threshold. Excessive nitrogen and phosphorus loss leads to water eutrophication, soil microbial community imbalance, and increased greenhouse gas emissions, exacerbating the climate vicious cycle. At the same time, in the forestry field, the demand for fast-growing tree species has increased to cope with the shortage of wood resources, bioenergy development, and carbon neutrality goals. However, the long-term impact of chemical growth promoters on forest ecosystems remains controversial. Against this background, developing new natural plant growth-promoting active substances to activate the intrinsic growth potential of crops has become an important research direction to break through the bottleneck of the "Green Revolution".

[0003] In recent years, more and more microorganisms have been isolated from various parts of plants. It has been found that plant endophytes play important physiological roles and can be used as important resources for biological control, showing great application potential in the agricultural field. Among them, Firmicutes in the plant endophytic bacterial community accounts for about 10% and is one of the most common culturable endophytic bacterial phyla in various plants. Bacillus is one of its most representative genera. Bacillus is a very diverse microbial group with a wide distribution. It can inhibit a wide range of plant pathogens, including various diseases such as those in roots, leaves, branches, flowers, and fruits after harvest, and is an ideal biocontrol microorganism. In recent years, due to its plant growth-promoting ability, Bacillus has shown great application potential in the agricultural field and has gradually become a research hotspot at home and abroad. For example, Zhang Xiaoyun et al. watered the fermentation broth of Bacillus amyloliquefaciens PHODG36 on potatoes, which had an obvious growth-promoting effect on potato seedlings. Shi Yimeng et al. inoculated Bacillus subtilis YB-2 into alfalfa, and the plant height of alfalfa increased extremely significantly by 17.82%. The relative control effect on alfalfa root rot reached 56.83%, showing the potential to be developed into a microbial agent for controlling alfalfa root rot.

[0004] Members of the Bacillus family are also capable of producing a variety of antibacterial and antifungal antibiotics, such as bacitracin, chlorosuccinic acid, mycomycin, rhizobitoxine, bacteriocin, difficile toxin and lipopeptide. Among them, lipopeptide is an amphiphilic molecule composed of a hydrophilic peptide chain and a lipophilic fatty acid hydrocarbon chain, with characteristics such as broad-spectrum antibacterial activity, high efficiency, low toxicity and easy biodegradation by microorganisms. The important basis for distinguishing lipopeptide types is the difference in their amino acid types and the connection order. Among them, the more common cyclic lipopeptides include surfactin, fengycin, and iturin. Lipopeptides have a variety of biological activities including antibacterial activity, anti-mycoplasma, anti-inflammatory activity, antiviral activity, antitumor activity, etc. due to their high surface activity, excellent stability under extreme conditions, non-toxic and biodegradable characteristics. Therefore, they have broad application prospects in the fields of medicine, food, agriculture, environmental governance, petroleum industry, etc. In the research of HUSSAIN T et al., it was found that surfactin present in the culture filtrate of Bacillus subtilis HussainT-AMU can promote the increase in tuber yield and plant growth.

[0005] As a new research hotspot, lipopeptides have a variety of biological activities, which make them have broad application prospects in various fields. The traditional chemical synthesis method of lipopeptide analogs severely limits the overall efficiency of lipopeptide synthesis, significantly reduces the antibacterial activity, and the variability of the lipopeptide structure is not suitable for chemical synthesis. At the same time, chemical synthesis has problems such as low yield, high pollution and high cost. For a long time, high cost and low yield have been difficult problems restricting the microbial synthesis of lipopeptides. Therefore, it is necessary to screen natural high-yield strains to further promote the production and application process of lipopeptides. Summary of the Invention

[0006] The technical problem solved by the present invention is to promote plant growth, especially in the seedling stage. Using the strain C5A-1 or the lipopeptide secreted by it for plants can promote the growth of plants, especially root length, plant height, stem diameter, number of leaves, leaf area, underground dry weight, and aboveground dry weight.

[0007] In a first aspect, the present invention provides a Bacillus velezensis C5A-1 or its variant or its offspring, and the Bacillus velezensis C5A-1 is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC No. 32880.

[0008] In a specific embodiment, the variant or offspring has similar or the same function as the Bacillus velezensis C5A-1;

[0009] In a specific embodiment, the variant or progeny of Bacillus velezensis C5A-1 has similar or identical physiological and biochemical characteristics to Bacillus velezensis C5A-1;

[0010] In a specific embodiment, Bacillus velezensis C5A-1 can promote the growth of plants during the seedling stage (e.g., the seedling period) (e.g., root length, plant height, stem diameter, number of leaves, leaf area, underground dry weight, aboveground dry weight);

[0011] In a specific embodiment, the seedling stage is the seedling period;

[0012] In a specific embodiment, the seedling period is 7 days to 35 days

[0013] In a specific embodiment, the growth includes root length, plant height, stem diameter, number of leaves, leaf area, underground dry weight, and / or aboveground dry weight.

[0014] On the other hand, the present invention provides a composition comprising the above-mentioned Bacillus velezensis C5A-1 or its variant or its progeny;

[0015] In a specific embodiment, the composition further comprises one or more excipients, one or more additives, or any combination thereof;

[0016] In a specific embodiment, the excipients include agriculturally or horticulturally acceptable diluents, fillers, solvents, spontaneous promoters, carriers, emulsifiers, dispersants, thickeners, binders, or any combination thereof;

[0017] In a specific embodiment, the additives include agriculturally or horticulturally acceptable preservatives, adjuvants, synergists, microbial additives, antifreeze agents, or any combination thereof;

[0018] In a specific embodiment, the composition is a liquid formulation;

[0019] In a specific embodiment, the liquid formulation includes emulsifiable concentrates, soluble solutions, oils, dispersible liquids, electrostatic spraying liquids, suspensions, microcapsule suspensions, oil suspensions, suspension emulsions, water-in-oil emulsions, microemulsions, nanoemulsions, or any combination thereof;

[0020] In a specific embodiment, in the liquid formulation, Bacillus velezensis C5A-1 or its variant or its progeny is in an agriculturally or fertilizer-science-acceptable carrier;

[0021] In a specific embodiment, in the liquid formulation, the content of Bacillus velezensis C5A-1 or its variant or its progeny is 100 mg / L to 1000 mg / L;

[0022] In a specific embodiment, the content of Bacillus velezensis C5A-1 or its variant or its progeny is 500 mg / L;

[0023] In a specific embodiment, the liquid preparation is used for root dipping, foliar spraying, spraying, composting, seed soaking, coating, field flooding irrigation, drip irrigation of plants or plant organs, smearing of plants or plant organs, dripping of plants or plant organs, or any combination thereof;

[0024] The composition is a solid preparation;

[0025] In a specific embodiment, in the solid preparation, the Bacillus velezensis C5A-1 or its variant or its progeny is in an agriculturally or fertilizer-chemically acceptable carrier;

[0026] In a specific embodiment, the solid preparation is used for composting, smearing plants or plant organs, or any combination thereof;

[0027] In a specific embodiment, the solid preparation is used for formulating the above-mentioned liquid preparation.

[0028] On the other hand, the present invention provides a biological culture, which comprises the above-mentioned Bacillus velezensis C5A-1 or its variant or its progeny, or the above-mentioned composition;

[0029] In a specific embodiment, the biological culture further comprises a solid or liquid medium, or components of the medium;

[0030] In a specific embodiment, the biological culture further comprises live cells of Bacillus velezensis C5A-1;

[0031] In a specific embodiment, the live cells exist in the form of spores;

[0032] In a specific embodiment, the spores are present in the liquid medium of Bacillus velezensis C5A-1; preferably, the biological culture comprises the cells of Bacillus velezensis C5A-1 and the supernatant of the culture.

[0033] On the other hand, the present invention provides a fermentation supernatant, which comprises the supernatant of the above-mentioned Bacillus velezensis C5A-1 or its variant or its progeny;

[0034] In a specific embodiment, the supernatant is prepared from the Bacillus velezensis C5A-1 or its variant or its progeny described in claim 1 or 2;

[0035] In a specific embodiment, the preparation includes fermentation;

[0036] In a specific embodiment, the temperature of the fermentation is 32-40 °C (for example, 37 °C);

[0037] In a specific embodiment, the fermentation time is 1 - 6 days (e.g., 4 days);

[0038] In a specific embodiment, the rotation speed of the fermentation is 140 - 180 r (e.g., 160 r);

[0039] In a specific embodiment, the inoculation amount of the fermentation is 1 - 10% (e.g., 5%);

[0040] In a specific embodiment, the preparation includes centrifugation or natural precipitation after fermentation;

[0041] In a specific embodiment, the rotation speed of centrifugation after fermentation is 6000 rpm;

[0042] In a specific embodiment, the centrifugation time after fermentation is 20 min.

[0043] On the other hand, the present invention provides a freeze-dried powder, which includes the above-mentioned Bacillus velezensis C5A-1 or its variants or its progeny, the above-mentioned composition, the above-mentioned culture, the above-mentioned supernatant, or any combination thereof;

[0044] In a specific embodiment, the freeze-dried powder further includes a filler, a lyoprotectant, a buffer, an osmotic pressure regulator, a wetting agent, a stabilizer, an antioxidant, or any combination thereof;

[0045] In a specific embodiment, the filler includes mannitol, lactose, sucrose, trehalose, glucose, glycine, alanine, arginine, sodium chloride, disodium hydrogen phosphate, polyvinylpyrrolidone, hydroxypropyl cellulose, or any combination thereof;

[0046] In a specific embodiment, the lyoprotectant includes sucrose, trehalose, maltose, raffinose, sorbitol, mannitol, glycerin, xylitol, polyethylene glycol (PEG), dextran, hydroxypropyl-β-cyclodextrin (HP-β-CD), human serum albumin (HSA), gelatin, or any combination thereof;

[0047] In a specific embodiment, the buffer includes NaH2PO4 / Na2HPO4, K2HPO4 / KH2PO4, Tris-HCl, citrate, histidine, sodium bicarbonate, or any combination thereof;

[0048] In a specific embodiment, the stabilizer includes polysorbate 20 / 80 (Tween 20 / 80), poloxamer 188, methionine, lysine, glutamic acid, ascorbic acid, sodium thiosulfate, α-tocopherol, disodium EDTA, citric acid, or any combination thereof;

[0049] In a specific embodiment, the osmotic pressure regulator includes sodium chloride, glycerol, glucose, or any combination thereof;

[0050] In a specific embodiment, the wetting agent includes benzyl alcohol, polysorbate, or any combination thereof.

[0051] On the other hand, the present invention provides a plant growth promoter, which includes the above-mentioned Bacillus velezensis C5A-1 or its variant or its offspring, the above-mentioned composition, the above-mentioned culture, the above-mentioned supernatant, the above-mentioned freeze-dried powder, or any combination thereof.

[0052] On the other hand, the present invention provides a method for cultivating plants, which includes the steps of applying the above-mentioned Bacillus velezensis C5A-1 or its variant or its offspring, the above-mentioned composition, the above-mentioned culture, the above-mentioned supernatant, the above-mentioned freeze-dried powder, or any combination thereof to plants to obtain plants with roots, stems, leaves, flowers, fruits, and seeds;

[0053] In a specific embodiment, the application includes root dipping, foliar spraying, spraying, composting, seed soaking, coating, field flooding irrigation, drip irrigation of plants or plant organs, smearing of plants or plant organs, and dropping of plants or plant organs;

[0054] In a specific embodiment, the use concentration of Bacillus velezensis C5A-1 or its variant or its offspring in root dipping is 100 mg / L - 1000 mg / L;

[0055] In a specific embodiment, the use concentration of Bacillus velezensis C5A-1 or its variant or its offspring is 500 mg / L;

[0056] In a specific embodiment, the use concentration of the fermentation supernatant in root dipping is 10 mg / L - 100 mg / L;

[0057] In a specific embodiment, the use concentration of the fermentation supernatant is 20 mg / L, 50 mg / L, or 100 mg / L;

[0058] In a specific embodiment, the usage concentration of Bacillus velezensis C5A-1 or its variant or its progeny in the root irrigation is 100 mg / L - 1000 mg / L, and the usage concentration of the fermentation supernatant in the root irrigation is 10 mg / L - 100 mg / L;

[0059] In a specific embodiment, the usage concentration of Bacillus velezensis C5A-1 or its variant or its progeny is 50 mg / L, and the usage concentration of the fermentation supernatant in the root irrigation is 50 mg / L;

[0060] In a specific embodiment, the amount of the root irrigation is 200 ml;

[0061] In a specific embodiment, the time of the root irrigation is the plant seedling stage;

[0062] In a specific embodiment, the plant seedling stage is 7 - 35 days of plant growth.

[0063] Use of the above-mentioned Bacillus velezensis C5A-1 (Bacillus velezensis) or its variant or its progeny, the above-mentioned composition, the above-mentioned culture, the above-mentioned supernatant, the above-mentioned freeze-dried powder, the above-mentioned growth promoter in promoting plant growth;

[0064] In a specific embodiment, the uses include promoting root length, plant height, stem diameter, number of leaves, leaf area, underground dry weight, and aboveground dry weight;

[0065] In a specific embodiment, the plant is a seedling-stage plant;

[0066] In a specific embodiment, the seedling-stage plant is 7 - 35 days old.

[0067] The above-mentioned Bacillus velezensis C5A-1 or its variant or its progeny, the above-mentioned growth promoter, the above-mentioned cultivation method, the above-mentioned uses, and the plant is selected from Leguminosae, Salicaceae, Gramineae;

[0068] In a specific embodiment, the plant has one or more of the following characteristics:

[0069] (1) The Gramineae plants are selected from corn, wheat, rice, sorghum, or any combination thereof;

[0070] (2) The Leguminosae plants are selected from soybean, broad bean, pea, peanut, mung bean, chickpea, wisteria, locust tree, lupine, Amorpha fruticosa, licorice, cassia tora, Genista tinctoria, alfalfa, sophora japonica, ormosia hosiei, Hedysarum scoparium, or any combination thereof;

[0071] (3) The Salicaceae plants are selected from Populus euramericana, Populus alba, Populus euphratica, or any combination thereof;

[0072] In a specific embodiment, the soybean is Jidou 17;

[0073] In a specific embodiment, the poplar is Poplar 84;

[0074] In a specific embodiment, the corn is Jiyuan 128.

[0075] Beneficial effects

[0076] The present invention discloses a lipopeptide-producing and plant growth-promoting functional strain C5A-1 and its application, and the technical problem to be solved is to promote plant growth, especially root length, plant height, stem diameter, number of leaves, leaf area, underground dry weight, and aboveground dry weight. Specifically, it discloses a Bacillus velezensis C5A-1 or its variant or its offspring, and the Bacillus velezensis C5A-1 is deposited in the China General Microbiological Culture Collection Center, with the deposit number CGMCC No. 32880. And its fermentation supernatant. Applying the strain or its fermentation supernatant or its combination to plants can promote plant growth and can be used for industrial production.

[0077] Deposit description

[0078] Name of the strain: Bacillus velezensis

[0079] Latin name: Bacillus velezensis

[0080] Strain number: C5A-1

[0081] Deposit institution: China General Microbiological Culture Collection Center

[0082] Abbreviation of the deposit institution: CGMCC

[0083] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0084] Deposit date: December 2, 2024

[0085] Registration number of the deposit center: CGMCC No. 32880 Description of the drawings

[0086] Figure 1 It is a diagram of the screening results of the blood agar plate method for the soil sample flora.

[0087] Figure 2 It is a diagram of screening antibacterial lipopeptide-producing strains by the plate confrontation method; (a) A5A-3 (b) A5C-14 (c) A6D-11 (d) C5A-1 (e) Bv1.

[0088] Figure 3 It is a diagram of the antibacterial zone width of different strains against Rhizoctonia solani.

[0089] Figure 4 (a) Oil displacement red circles of surfactin standard solutions with different concentrations; (b) Linear fitting graph of oil displacement circle diameter vs. surfactin concentration.

[0090] Figure 5 Graphs of (a) oil displacement circle diameter and OD600, and (b) lipopeptide concentration of fermentation broths of different strains.

[0091] Figure 6 Graph of the detection results of strain C5A-1; (a) Detection results of API 20E reagent strip, (b) Phylogenetic tree.

[0092] Figure 7 Graph of MALDI-TOF MS detection results; (a) A5A-3, (b) A5C-14, (c) A6D-11, (d) C5A-1, (e) Bv1 strain.

[0093] Figure 8 Graph of the production of surfactin by different strains quantified by HPLC.

[0094] Figure 9 Graph of the growth of corn on the 20th day after treatment; (a) Seedlings treated with strain C5A-1 and lipopeptide metabolites, (b) Roots treated with strain C5A-1 and lipopeptide metabolites.

[0095] Figure 10 Bar graphs of (a) Chlorophyll SPAD value, (b) Plant height, (c) Stem diameter of corn seedlings on the 10th day after treatment; (d) Chlorophyll SPAD value, (e) Plant height, (f) Stem diameter, (g) Root length, (h) Fresh weight of the plant, (i) Fresh weight of the root, (j) Dry weight of the stem and leaf, (k) Dry weight of the root on the 20th day.

[0096] Figure 11 Graph of the growth of soybean after applying different concentrations of lipopeptide metabolites of strain C5A-1; (a) Plant, (b) Root nodule.

[0097] Figure 12 Bar graphs of various indexes of soybean after applying different concentrations of lipopeptide metabolites of strain C5A-1; (a) Number of root nodules, (b) Weight of root nodules, (c) Plant height, (d) Stem diameter, (e) Dry weight of underground part, (f) Dry weight of aboveground part.

[0098] Figure 13 Graph of the growth of poplar after different treatments; (a) Soil, (b) Liquid culture.

[0099] Figure 14 Bar graphs of the physiological indexes of poplar after applying different concentrations of strain C5A-1 or its lipopeptide metabolites; (a) Soil, (b) Physiological indexes in liquid culture. Detailed implementation methods

[0100] The present invention will be further described in detail in connection with specific embodiments below. The embodiments given are only for clarifying the present invention, rather than limiting the scope of the present invention. The following embodiments provided 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.

[0101] The experimental methods in the following embodiments 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 specifications. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0102] The following embodiments use SPSS 11.5 statistical software to process the data. The experimental results are expressed as mean ± standard deviation, and are tested by One-way ANOVA. P < 0.05 (*) indicates significant difference, P < 0.01 (**) indicates extremely significant difference, and P < 0.001 (***) indicates extremely significant difference.

[0103] Bv1 in the present application was disclosed in the patent with the application number 202110072888.X. The preservation number of the strain of Bv1 is CGMCC NO. 20317, and Bv1 is Bacillus velezensis.

[0104] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the agronomy, horticulture, pharmacy, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory operation steps used herein are all widely used terms and conventional steps in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

[0105] The term "comprising (comprising, comprises and comprised of)" used herein is synonymous with "including (including and includes)" or "containing (containing and contains)", and is inclusive or open-ended, and does not exclude additional, unstated members, elements or method steps. The term "comprising (comprising, comprises and comprised of)" also includes the term "consisting of".

[0106] As used in this disclosure, "first" and "second" are for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise specified, "a plurality" means two or more.

[0107] As used in this disclosure, "optionally", "optional" or "option" generally means that the subsequent event or condition may but does not necessarily occur, and the description includes the case where the event or condition occurs and the case where the event or condition does not occur.

[0108] As used in this disclosure, the "seedling stage" of cotton refers to the growth stage from seed germination to the initial stage of the seedling.

[0109] As used in this disclosure, "its salts" and "pharmaceutically acceptable salts" refer to derivatives of the disclosed compounds, wherein the parent compound is modified by preparing its acid addition salts or base addition salts. Examples of its salts and pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic groups such as amines; and basic salts or organic salts of acidic groups such as carboxylic acids. Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of the parent compound, which are prepared, for example, from non-toxic inorganic acids or non-toxic organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid; and salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid and hydroxyethanesulfonic acid.

[0110] As used in this disclosure, a "liquid preparation" refers to a homogeneous or heterogeneous system in which one or more active ingredients (such as drugs, nutrients, pesticides, bioactive substances, etc.) are dissolved, emulsified or suspended in a liquid dispersion medium, and auxiliary components such as stabilizers, cosolvents, preservatives, etc. may be added. Liquid preparations include, but are not limited to, true solution type (the solute is uniformly dispersed in the form of molecules or ions (particle size <1 nm), clear and stable), colloidal solution type (the solute is dispersed in the form of colloidal particles (1 - 100 nm), having a certain stability and may produce the Tyndall effect), emulsion type (two immiscible liquids (such as oil and water) form a stable emulsion (particle size 0.1 - 100 μm) through an emulsifier), and suspension type (insoluble solid particles (0.5 - 10 μm) are dispersed in a liquid, and a suspending agent is required to prevent sedimentation).

[0111] The "solid preparation" as described in the present disclosure refers to a preparation form existing in a solid state. Solid preparations include, but are not limited to, tablets, capsules, granules, powders, pills, and films.

[0112] Rhizoctonia solani Kühn is disclosed in the following literature: "Rhizoctonia solani Kühn Pathophysiology: Status and Prospects of Sheath Blight Disease Management in Rice". Its name in this literature is "Rhizoctonia solani Kühn".

[0113] Isolation and Screening of Example 1 C5A-1 and Other Strains

[0114] 1. Test Strains: Rhizoctonia solani Kühn, preserved in this laboratory.

[0115] 2. Experimental Materials

[0116] Enrichment Medium (1L): Glucose 2g, Yeast Extract 0.2g, NH4NO3 0.2g, KH2PO4 0.3g, Na2HPO4·12H2O 0.5g, MgSO4·7H2O 0.05g;

[0117] Blood Agar Plate: Manufactured by Haibo Biotechnology Co., Ltd., Product Number: 3400071;

[0118] LB Liquid Medium (1L): Yeast Extract 5g, NaCl 10g, Tryptone 10g. After preparing the medium, it needs to be autoclaved at 121°C for 20 min;

[0119] LB Solid Medium (1L): Add 15 - 20g of agar to each liter of the above LB liquid medium. After preparing the medium, it needs to be autoclaved at 121°C for 20 min.

[0120] PDA Medium (1L): PDA powder (manufactured by Beijing Aoboxing Biotechnology Co., Ltd., Product Number: 02 - 023, composition (g / L): Potato Extract Powder 3g, Glucose 20g, Agar 14g) 37g. After preparing the medium, it needs to be autoclaved at 121°C for 20 min.

[0121] 3. Experimental Methods

[0122] 3.1 Collection of Soil Samples

[0123] Soil samples from which different plants were grown were collected from 7 sampling sites across the country (Table 1), and the samples were placed in clean sampling bags, marked, and stored in a refrigerator at 4°C as rhizosphere soil samples.

[0124] Table 1 Collection locations of soil samples

[0125]

[0126]

[0127] 3.2 Isolation and primary screening of strains

[0128] 3.2.1 Enrichment-selective isolation

[0129] (1) Enrichment culture of soil and water sample flora

[0130] Weigh 1 g of soil samples from different sources and add them to a 250 mL Erlenmeyer flask containing 99 mL of water and glass beads. Incubate at 30°C and 200 rpm for 30 min. Transfer 1 mL of the activated soil suspension to 20 mL of enrichment medium and incubate on a shaker at 37°C and 200 rpm for 2 d.

[0131] (2) Screening on blood agar plates

[0132] Take the enrichment culture solution after 2 d of culture, serially dilute it to 10 -6 ~10 -8 , take 100 μL and spread it on a blood agar plate. Invert and incubate in a constant temperature incubator at 37°C for 24 h. Select strains with larger hemolytic zones and clear edges, spot them onto the blood agar plate for hemolysis verification, and streak inoculate them onto a solid LB agar plate by the end dilution method for standby. Pick a single colony and inoculate it into liquid LB medium. Incubate on a shaker at 30°C and 180 rpm for 48 h. Take 0.5 mL of the bacterial solution and mix it with 0.5 mL of 30% glycerol in an equal volume in a sterile bacterial strain preservation tube, and store it at -80°C.

[0133] 3.2.2 Selective isolation

[0134] Weigh 1 g of soil sample and put it into a 250 mL Erlenmeyer flask containing 99 mL of sterile water and a dozen glass beads (10 -2 ), shake for 37 min, then take 1 mL of the supernatant and add it to 9 mL of sterile water (10 -3 ). After mixing, take 1 mL of the supernatant and add it to a new 9 mL of sterile water (10 -4 ), and make 10 -4The soil dilution was placed in an 80 °C water bath for 10 min to kill vegetative cells. Take 100 μl and spread it on a blood agar plate, and culture it at 37 °C for 2 - 3 d. Select the strains with larger hemolytic zones and clear edges, spot them onto the blood agar plate for hemolysis verification, and at the same time, use the end dilution method to streak and inoculate a solid LB agar plate for standby. Pick a single colony and inoculate it into a liquid LB medium, culture it at 30 °C with shaking at 180 rpm for 48 h. Take 0.5 ml of the bacterial solution and mix it with 0.5 ml of 30% glycerol in an equal volume in a sterile bacterial strain preservation tube, and store it at -80 °C.

[0135] 3.3 Rescreening of lipopeptide-producing bacteria

[0136] Inoculate a Rhizoctonia solani Kühn fungal cake in the middle of a PDA medium plate. Inoculate the strains with hemolytic zones 2 cm away from the center point of Rhizoctonia solani in the upper, lower, left, and right four directions, and co-culture them upright at 25 °C for 5 - 7 d. Observe whether there is antibacterial activity and measure the bandwidth of the antibacterial zone (the width of the antibacterial zone is the distance between the edge of the Rhizoctonia solani colony and the edge of the bacterial lawn, in mm).

[0137] 4. Experimental results

[0138] 4.1 Isolation and primary screening of lipopeptide-producing strains

[0139] Because lipopeptide substances have surface activity, lipopeptide-producing strains will form hemolytic zones on blood agar plates. Therefore, the blood agar plate method was selected for preliminary screening. The results showed ( Figure 1 ), a total of seven colonies showed larger and clear hemolytic zones around them, including strains A5A - 3, A5C - 14, A6D - 11, C5A - 1, and Bv1. Since this method has false positives, it was initially judged that the above five strains could produce lipopeptide substances.

[0140] 4.2 Rescreening of the antagonistic experiment of lipopeptide-producing strains

[0141] Since lipopeptides have strong antibacterial activity, Rhizoctonia solani was used as an indicator bacterium to further screen lipopeptide-producing strains.

[0142] The experimental results showed that the above five bacteria all had antagonistic activity against Rhizoctonia solani ( Figure 2 ). All strains had relatively wide antibacterial zones against Rhizoctonia solani ( Figure 3 ). It was further proved that the above strains might produce lipopeptide substances and had strong biocontrol potential.

[0143] Example 2 Functional analysis of lipopeptide production by strains A5A - 3, A5C - 14, A6D - 11, C5A - 1, and Bv1

[0144] 1. Test strains: Strains A5A-3, A5C-14, A6D-11, C5A-1, and Bv1 isolated in Example 1.

[0145] 2. Experimental materials

[0146] (1) Culture medium

[0147] Fermentation medium: 15 g of glucose, 1 g of soluble starch, 30 g of soybean cake powder, 0.2 g of yeast powder, 1.5 g of KH2PO4, 3 g of K2HPO4, 0.5 g of MgSO4·7H2O, 0.1 g of CaCO3, 0.1 g of FeSO4, 1000 mL of distilled water;

[0148] (2) Other chemicals

[0149] Surfactin standard (≥98.0%), purchased from MCE Company, CAS No. 24730-31-2.

[0150] Oil red solution: 0.5 g of oil red was added to 100 mL of n-dodecane, fully mixed and dissolved, and then filtered through a 0.45 μm PES filter membrane. The filtrate was the oil red solution.

[0151] 3. Experimental instruments

[0152] Microplate reader: Model Infinite M200 Pro (Tecan, Switzerland).

[0153] 4. Experimental methods

[0154] 4.1 Fermentation of strain C5A-1 and other strains

[0155] (1) Single colonies were picked and inoculated into a test tube containing 6 mL of fermentation medium, and cultured at 34 °C with shaking at 200 r / min for 2 d to obtain seed liquid;

[0156] (2) The seed liquid was inoculated into a 250 mL Erlenmeyer flask containing 30 mL of fermentation medium (natural pH) at an inoculation amount of 5%, and cultured on a shaker at 34 °C with shaking at 200 r / min for 72 h, in triplicate;

[0157] (3) After fermentation, 200 μL of the fermentation broth was taken into a 96-well plate, and the OD value of each strain was measured at a wavelength of 600 nm using a microplate reader.

[0158] 4.2 Determination of lipopeptide content by the oil displacement circle method

[0159] (1) Establishment of the oil displacement circle standard curve

[0160] The diameter of the oil displacement circle was measured using a surfactin standard solution with a concentration of 0.4 - 1.4 g / L. A glass petri dish with a diameter of 9 cm was placed on graph paper, and 40 mL of deionized water was added. After the water surface stabilized, 200 μL of oil red solution was added dropwise. After the oil red spread evenly, 2.5 μL of the surfactin standard solution was added dropwise to the center of the oil red surface. After the size of the oil displacement circle stabilized, a photo was taken to record its diameter. Then, the concentration of the surfactin solution was used as the abscissa, and the diameter of the oil displacement circle was used as the ordinate to create a standard curve, and the equation of the standard curve was obtained.

[0161] (2) Determination of the surfactin production ability of the strain

[0162] For each bottle, 1 mL of the fermentation broth (the fermentation broth obtained from the above 4.1 fermentation) was centrifuged at 12,000 rpm for 10 min, and the supernatant was taken. The diameter of the oil displacement circle was measured using the oil red displacement method, and the content of lipopeptide in the strain was calculated according to the surfactin standard curve.

[0163] 4.3 Experimental results

[0164] 4.3.1 Establishment of the oil circle standard curve

[0165] As a surfactant, lipopeptide is a class of molecules with an amphiphilic structure that can spontaneously aggregate and assemble at the water - oil interface, thus effectively reducing the interfacial tension between the two phases. Therefore, measuring the diameter of the oil displacement circle using the oil red displacement method can quickly and roughly quantify the content of lipopeptide in the fermentation broth sample.

[0166] First, using surfactin as the lipopeptide standard, the oil displacement circle was measured to obtain the standard curve. The lipopeptide standard solution with a concentration of 0.4 - 1.4 g / L was used to measure the diameter of its oil displacement circle ( Figure 4 in (a) from left to right are 0.4 g / L, 0.6 g / L, 0.8 g / L, 1.0 g / L, 1.2 g / L, 1.4 g / L). Regression analysis showed that in the range of 0.4 - 1.4 g / L, the diameter of the oil displacement circle Dr (mm) and the lipopeptide concentration C srf (g / L) showed a good linear relationship: Dr = 22×C srf + 0.2, R 2 = 0.97919 ( Figure 4 in (b)).

[0167] 4.3.2 Measurement of lipopeptide concentration by the oil red displacement method

[0168] When verifying the strains screened by the fermentation - validated blood agar plate and the plate confrontation method, the oil red displacement method was used to quickly measure the lipopeptide concentration in the fermentation broth.

[0169] The experimental results showed that all of the above five strains could produce an oil displacement circle ( Figure 5(a), where A5A-3, A5C-14, A6D-11, C5A-1, and Bv1 are strains A5A-3, A5C-14, A6D-11, C5A-1, and Bv1 respectively. Among them, the diameter of the oil-displacing circle of strain C5A-1 is the largest, reaching 58 cm, indicating strong oil-displacing ability. The diameters of the oil-displacing circles of the other strains can all reach more than 40 cm, and they also have strong oil-displacing ability. In addition, the OD of strain C5A-1 after fermentation 600 is the highest ( Figure 5 (a)), reaching 2.4322, indicating that its growth condition is slightly better than that of other strains.

[0170] Substituting the diameter of the oil-displacing circle into the standard curve can roughly quantify the total amount of lipopeptides in the fermentation broth ( Figure 5 (b), where A5A-3, A5C-14, A6D-11, C5A-1, and Bv1 are strains A5A-3, A5C-14, A6D-11, C5A-1, and Bv1 respectively. Among them, the lipopeptide content of strain C5A-1 reaches the highest of 2.63 g / L. The yield of strain A5A-3 is relatively low, reaching 1.52 g / L. Based on the above experimental results, strain C5A-1 has the potential to become a chassis cell for lipopeptide production.

[0171] Example 3 Identification of Strain C5A-1

[0172] 1. Test strain: Strain C5A-1 isolated in Example 1.

[0173] 2. Experimental materials: Normal saline, API 20E reagent strip (bioMérieux, France).

[0174] 3. Experimental methods

[0175] 3.1 Physiological and biochemical experiments

[0176] Under sterile conditions, use an inoculation loop to pick a single colony into 4 mL of normal saline, mix well to prepare a bacterial suspension with an OD 600 of 0.1 - 0.2, and use the API 20E reagent strip to detect some physiological and biochemical characteristics of the target strain.

[0177] 3.2 16S rRNA gene sequencing and construction of phylogenetic tree

[0178] The strain samples were sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for amplification and sequencing of the V1-V9 region of the 16S rRNA gene using primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGCTACCTTGTTACGACTT-3′). The 16S rRNA sequences of A5A-3, A5C-14, A6D-11, C5A-1, and Bv1 are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively.

[0179] SEQ ID NO: 1 is as follows:

[0180]

[0181] SEQ ID NO: 2 is as follows:

[0182]

[0183] SEQ ID NO: 3 is as follows:

[0184]

[0185] SEQ ID NO: 4 is as follows:

[0186]

[0187] SEQ ID NO: 5 is as follows:

[0188]

[0189] The 16S rRNA sequence was submitted to EzbioCloud (https: / / www.ezbiocloud.net / ) to obtain the homology between the 16S rRNA gene of the target strain and the type strains of known species, and a phylogenetic tree was constructed using MEGA 11.0 software.

[0190] 4. Experimental results

[0191] 4.1 Physiological and biochemical characteristics of strain C5A-1

[0192] Under sterile conditions, bacteria were picked with an inoculation loop into 4 mL of normal saline, and after mixing, a bacterial suspension with an OD 600 of 0.1-0.2 was prepared, and some physiological and biochemical characteristics of the target strain were detected using an API 20E reagent strip ([[]] Figure 6 as shown in (a) below). Among them, ONPG: β-galactosidase; ADH: arginine dihydrolase; LDC: lysine decarboxylase; ODC: ornithine decarboxylase; CIT: citrate utilization; H2S: H2S production; URE: urease; TDA: tryptophan deaminase; IND: indole production; VP: production of acetoin from 3-hydroxybutanone; GEL: gelatinase; GLU: glucose (fermentation / oxidation); MAN: mannitol (fermentation / oxidation); INO: inositol (fermentation / oxidation); SOR: sorbitol (fermentation / oxidation); RHA: rhamnose (fermentation / oxidation); SAC: sucrose (fermentation / oxidation); MEL: melibiose (fermentation / oxidation); AMY: amygdalin (fermentation / oxidation); ARA: arabinose (fermentation / oxidation)). The results showed that strain C5A-1 had arginine dihydrolase activity, gelatinase activity, was able to utilize citrate, and was able to ferment or oxidize glucose and sucrose, but did not have β-galactosidase activity, lysine decarboxylase activity, ornithine decarboxylase activity, urease activity, tryptophan deaminase activity, could not produce H2S and indole, could not produce acetoin from 3-hydroxybutanone, and could not ferment or oxidize mannitol, inositol, sorbitol, rhamnose, melibiose, amygdalin, and arabinose.

[0193] 4.2 Phylogenetic analysis based on the 16S rRNA gene

[0194] Using the universal primers 27F and 1492R, PCR amplification was performed with the genomic DNA of strain C5A-1 as a template. After verification by agarose gel electrophoresis, the amplified product was sequenced. The sequence was submitted to the EzBioCloud website to obtain the homology between the 16S rRNA gene of strain C5A-1 and the type strains of known species, and a phylogenetic tree was constructed using MEGA 11.0 software ([[]] Figure 6In (b)). The results showed that strain C5A-1 belongs to the genus Bacillus and has the closest genetic relationship with Bacillus velezensis (B.velezensis CR-502 T ).

[0195] Example 4 Identification of the types of lipopeptides produced by C5A-1 and other strains

[0196] 1. Test strains: Strains A5A-3, A5C-14, A6D-11, C5A-1, and Bv1 isolated in Example 1.

[0197] 2. Experimental materials

[0198] Culture medium: 15 g of glucose, 1 g of soluble starch, 30 g of soybean cake powder, 0.2 g of yeast powder, 1.5 g of KH2PO4, 3 g of K2HPO4, 0.5 g of MgSO4·7H2O, 0.1 g of CaCO3, 0.1 g of FeSO4, 1000 mL of distilled water.

[0199] 3. Experimental instruments

[0200] Matrix-assisted laser desorption-time of flight mass spectrometer (AXIMA Performance, Shimadzu Corporation, Japan).

[0201] 4. Experimental methods

[0202] (1) Strain fermentation

[0203] The seed liquor was inoculated into the fermentation medium at an inoculation amount of 5% respectively, and cultured on a shaker at 34°C and 200 r / min for 72 h, with three replicates;

[0204] (2) Identification of the types of lipopeptides produced by the strain by MALDI-TOF MS

[0205] After fermentation, the fermentation broth was centrifuged at 12000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm PES filter membrane as the test sample. Switch to a matrix-assisted laser desorption-time of flight mass spectrometer, use α-cyano-4-hydroxycinnamic acid (CCA) as the matrix, and use a 337 nm nitrogen laser beam for excitation. The excitation voltage is 2-20 kV, and the nuclear mass ratio range of 500-2000 m / z is scanned and detected to verify the target lipopeptide ion peak and accumulate and harvest the target lipopeptide ion.

[0206] 5. Experimental results

[0207] Perform MALDI-TOF MS analysis on the five strains respectively ( Figure 7, where (a), (b), (c), (d), and (e) are the detection results of strains A5A-3, A5C-14, A6D-11, C5A-1, and Bv1, respectively). The results show (Table 2) that all strains can produce surfactin; there are also two strains that can produce fengycin, namely A6D-11 and Bv1.

[0208] Table 2 Main ion peaks of lipopeptides produced by strains determined by MALDI-TOF MS

[0209]

[0210] Example 5 Determination of the content of surfactin produced by C5A-1 and other strains

[0211] 1. Test strains: Strains A5A-3, A5C-14, A6D-11, C5A-1, and Bv1 isolated in Example 1.

[0212] 2. Experimental materials

[0213] Fermentation broth after fermentation in Example 4.

[0214] Surfactin standard (analytical pure, ≥98%)

[0215] 3. Experimental instruments

[0216] Liquid chromatography system: LC-20A HPLC system (Shimadzu, Kyoto, Japan), and the chromatographic column is a C18 column (5μm, 250×4.6mm);

[0217] 4. Experimental methods

[0218] (1) Filter the fermentation supernatant of the fermentation broth through a 0.22μm PES filter membrane and transfer it into a liquid phase bottle for detection. The mobile phase is methanol / 0.1% trifluoroacetic acid aqueous solution = (90:10, v / v), and the detection wavelength is 215nm;

[0219] (2) Dilute the 1000mg / L surfactin standard with 1% NaHCO3 aqueous solution to prepare a surfactin solution with a concentration of 75 - 500mg / L, and establish a standard curve of chromatographic absorption peak area - surfactin concentration;

[0220] (3) Substitute the chromatographic absorption peak area of the fermentation broth into the standard curve to calculate the concentration of surfactin in the solution.

[0221] 5. Experimental results

[0222] The content of surfactin in 15 samples was determined by high performance liquid chromatography, and the chromatographic peak areas of each sample were calculated. According to the obtained standard curve, the concentration of surfactin in each sample was calculated to obtain the concentration of surfactin produced by the fermentation of each strain.

[0223] The experimental results showed ( Figure 8 , where A5A-3, A5C-14, A6D-11, C5A-1, and Bv1 are the strains A5A-3, A5C-14, A6D-11, C5A-1, and Bv1 respectively). The highest yield of surfactin by strain Bv1 was 1044.88 mg / L, and the shake flask yield could reach more than 1 g / L. Followed by strains A6D-11 and C5A-1, with yields of 906.91 mg / L and 808.93 mg / L respectively, and the shake flask yields could both reach more than 800 mg / L. The yields of strains A5A-3 and A5C-14 were slightly lower than those of other strains, but also reached more than 600 mg / L. The results indicate that the above strains can all produce a large amount of surfactin and have the potential to become biological chassis cells with high yield of lipopeptides, playing an important role in the agricultural field.

[0224] Example 6 Growth promotion effect of C5A-1 and other strains on corn

[0225] 1. Test strains: Strains A5A-3, A5C-14, A6D-11, C5A-1, and Bv1 isolated in Example 1.

[0226] 2. Experimental materials

[0227] 2.1 Culture media

[0228] Fermentation medium: 7 g of corn flour, 1 g of soluble starch, 45 g of peptone, 0.2 g of yeast powder, 1.5 g of KH2PO4, 3 g of K2HPO4, 0.5 g of MgSO4·7H2O, 0.1 g of CaCO3, 0.1 g of FeSO4, 2 g of glutamic acid, 1000 mL of distilled water.

[0229] LB liquid medium: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, made up to 1000 mL with distilled water.

[0230] 2.2 Other materials

[0231] Test corn: Jiyuan 128;

[0232] Test soil: Dark brown forest soil in the field of the Three-Hectare Garden of Beijing Forestry University in Haidian District, Beijing (N39°58′40.09″, E116°20′17.37″), belonging to the temperate continental climate.

[0233] 3. Experimental instruments

[0234] Chlorophyll meter (YT-YD, Yuntang, China), forced air drying oven (DHG-9246A, Jinghong, Shanghai), digital caliper (Shanghai Shenhan), straight ruler.

[0235] 4. Experimental methods

[0236] 4.1 Fermentation of strain C5A-1 and other strains

[0237] The seed solutions (A6D-11 seed solution, C5A-1 seed solution, Bv1 seed solution) cultured in LB liquid were inoculated into 1 L of fermentation medium at an inoculation amount of 5% and fermented at 37 °C and 160 r for 4 d to obtain A6D-11 fermentation broth, C5A-1 fermentation broth, and Bv1 fermentation broth.

[0238] 4.2 Preparation of lipopeptide products and bacterial powders of strain C5A-1 and other strains

[0239] After the fermentation was completed, the fermentation broths were centrifuged at 4 °C and 6000 rpm for 20 min, and the cell precipitates were reserved to obtain A6D-11 cell precipitate, C5A-1 cell precipitate, and Bv1 cell precipitate.

[0240] The pH of the supernatant was adjusted to 2.0 with concentrated hydrochloric acid, sealed with a sealing film, and left to stand overnight at 4 °C. The acid precipitation solution was centrifuged at 12,000 rpm and 4 °C for 15 min, the supernatant was discarded, and the precipitate was collected to obtain A6D-11 supernatant acid precipitate, C5A-1 supernatant acid precipitate, and Bv1 supernatant acid precipitate. The above-mentioned cell precipitates and acid precipitates were frozen at -80 °C for 2 h and freeze-dried with a freeze dryer for 24 h to obtain A6D-11 freeze-dried bacterial powder and A6D-11 lipopeptide crude extract, C5A-1 freeze-dried bacterial powder and C5A-1 lipopeptide crude extract, and Bv1 freeze-dried bacterial powder and Bv1 lipopeptide crude extract.

[0241] 4.3 Application of lipopeptide products and freeze-dried bacterial powders of strain C5A-1 and other strains to maize seedlings

[0242] Select healthy and intact corn seeds (Era 128) and sow them in the soil for pot experiments, with four replicates set. After dissolving the lipopeptide extracts of different strains (crude lipopeptide extract of A6D-11, crude lipopeptide extract of C5A-1, crude lipopeptide extract of Bv1) in water respectively, prepare solutions with a final concentration of 50 mg / L to obtain the crude lipopeptide extract solution of A6D-11, the crude lipopeptide extract solution of C5A-1, and the crude lipopeptide extract solution of Bv1. After dissolving the freeze-dried bacterial powders of different strains (freeze-dried bacterial powder of A6D-11, freeze-dried bacterial powder of C5A-1, freeze-dried bacterial powder of Bv1) in water respectively, prepare solutions with a concentration of 500 mg / L to obtain the bacterial powder solution of A6D-11, the bacterial powder solution of C5A-1, and the bacterial powder solution of Bv1. After 7 days of growth of the above-mentioned corn, perform root irrigation treatment uniformly. There are 7 treatments set in the pot experiment, namely, the control group (CK) treated with 200 mL of clear water, two different strains treated with 200 mL of lipopeptide extract solution, two different strains treated with 200 mL of bacterial powder solution, and the C5A-1 strain treated with 200 mL of crude lipopeptide extract solution and the C5A-1 strain treated with 200 mL of bacterial powder solution.

[0243] 4.4 Determination of physiological indexes of corn seedlings

[0244] On the tenth day after the root irrigation treatment, measure the SPAD value of corn chlorophyll, plant height, and stem diameter data. On the twentieth day after the root irrigation treatment, measure the SPAD value of corn chlorophyll, plant height, and stem diameter data; gently pull out the corn seedlings from the soil, wash the roots clean with running water, and then measure their fresh weight, dry weight, and root length.

[0245] SPAD value: Uniformly measure the third leaf from the bottom to the top.

[0246] Plant height: Uniformly measure the exposed part of the plant above the ground, up to the highest leaf in the natural state of the plant.

[0247] Stem diameter data: Uniformly measure the middle part of the first stem segment above the ground.

[0248] Fresh weight: Quickly cut the plant material, put it into a self-sealing bag, bring it indoors, and weigh the fresh weight with an analytical balance.

[0249] Dry weight: Put the sample into a high-temperature oven and dry it for a period of time until the sample quality no longer changes, and then weigh its dry weight.

[0250] Root length: Dig out the root system from the soil completely, wash it clean with clear water, and then measure the length of the root with a ruler.

[0251] 5. Experimental results

[0252] Select three strains with relatively high surfactin production, and treat corn seedlings with their metabolites and bacterial powders respectively to observe the growth-promoting effects of different strains on corn.

[0253] The results showed that the above three strains and lipopeptide metabolites could all promote the growth of corn plants and roots to varying degrees (the growth conditions of corn on the 20th day after treatment were as follows Figure 9 shown, where (a) shows the situation of seedlings treated with strain C5A-1 and lipopeptide metabolites, and (b) shows the situation of roots treated with strain C5A-1 and lipopeptide metabolites (where CK is the control group, strain C5A-1 is treated with mL bacterial powder solution, and C5A-1 lipopeptide is treated with lipopeptide extract solution). On the 10th day after root irrigation treatment, the three strains and their metabolites could all positively promote the increase of chlorophyll, plant height, and stem diameter of corn ( Figure 10 in (a-c) below, where A6D-11, C5A-1, and Bv1 are the indicators after treatment with A6D-11, C5A-1, and Bv1 respectively, and CK is the control group). Among them, strain C5A-1 had a particularly obvious promoting effect on plant height and stem diameter. Its metabolites could increase the plant height and stem diameter by 1.18 times and 1.48 times respectively, and the strain could increase the plant height and stem diameter by 1.17 times and 1.76 times respectively.

[0254] On the 20th day after root irrigation treatment, the three strains and lipopeptide metabolites could still promote the growth of corn plants and roots to varying degrees ( Figure 10 in (d)-(k) below). In addition to significantly increasing chlorophyll, plant height, and stem diameter, the metabolites of strain C5A-1 could also increase the corn roots to 40.9 cm, which was 9 cm higher than the control group, enabling the plants to better absorb water and nutrients. In addition, the metabolites of strain C5A-1 could increase the dry weight of roots by 2.04 times, and the strain could increase the dry weight of stems and leaves by 1.42 times, which could enable corn to store more nutrients and improve stress resistance.

[0255] Example 7 Growth promotion effect of the metabolites of strain C5A-1 on soybeans

[0256] 1. Test strains: Strain C5A-1 isolated in Example 1.

[0257] 2. Experimental materials

[0258] Test soybeans: Jidou 17;

[0259] Test soil: Dark brown forest soil in the three-acre garden of Beijing Forestry University in Haidian District, Beijing (N39°58′40.09″, E116°20′17.37″), belonging to the temperate continental climate.

[0260] 3. Experimental instruments

[0261] Flower pots with a 15-cm diameter, tape measures, digital calipers (Shanghai Shenhan), and a forced air drying oven (DHG-9246A, Shanghai Jinghong).

[0262] 4. Experimental methods

[0263] 4.1 Application of the metabolites of strain C5A-1 to soybean seedlings

[0264] Solution of metabolites of C5A-1: The same as the steps in 4.2 above.

[0265] Sow the Jidou 17 soybean seeds evenly in flower pots with a 15-cm diameter filled with field soil, a total of 24 pots are planted. After one week, when the soybean seedlings grow out, thin the seedlings to 3 plants per pot, and then perform root irrigation on the soybeans. Apply 200 mL of C5A-1 metabolite solutions with different concentrations (20 mg / L, 50 mg / L, 100 mg / L) to the roots of each pot of soybeans, with clear water as the control. Set 6 replicates for each treatment, and water according to the growth of soybean seedlings during the cultivation period.

[0266] 4.2 Determination of physiological indexes of soybean seedlings

[0267] After four weeks, measure the plant height of soybeans with a tape measure and the stem diameter with a vernier caliper. After the measurement, carefully take out the soybeans from the flower pots, collect the root nodules on the roots, wash the soil above the root nodules with clear water and then weigh them. Divide the soybean plants into the underground part (roots) and the aboveground part (stems and leaves), put them into envelopes, blanch them at 115 °C for 30 minutes in an oven, and then dry them at 80 °C for 24 hours until constant weight, and measure the dry weights of their different tissues.

[0268] Stem diameter (stem diameter at the base of the main stem): The stem diameter at 3 cm above the ground.

[0269] 5. Experimental results

[0270] In the soybean pot experiment, applying C5A-1 metabolite solutions with different concentrations significantly promoted the growth and nodulation of soybeans ( Figure 11 , where CK is the control, and 20 mg / L, 50 mg / L, and 1000 mg / L are different concentrations of the C5A-1 metabolite solution respectively). Compared with the control group, the treatment group showed significant improvements in plant height, stem diameter, number of root nodules, weight of root nodules, and dry weights of the aboveground and underground parts ( Figure 12 , where CK is the control, and 20, 50, and 100 are the 20 mg / L C5A-1 metabolite solution, 50 mg / L C5A-1 metabolite solution, and 100 mg / L C5A-1 metabolite solution respectively). Further analysis showed that the lipopeptide at a concentration of 20 mg / L had a particularly significant promoting effect on soybeans: the plant height increased by 19.59%, the stem diameter increased by 29.54%, the number of root nodules increased by 20.45%, the weight of root nodules increased by 151.45%, the dry weight of the aboveground part increased by 23.4%, and the dry weight of the underground part increased by 56.19%.

[0271] Example 8 Growth-promoting experiment of strain C5A-1 and its metabolites on poplar

[0272] 1. Test strains: Strain C5A-1 isolated in Example 1.

[0273] 2. Experimental materials

[0274] Test poplars: Tissue culture seedlings of Populus alba×Populus glandulosa clone 84K preserved in the laboratory;

[0275] Rooting medium: 2.215 g / L MS powder, 30 g / L sucrose, 6 g / L agar, 0.02 mg / L NAA, 0.05 mg / L IBA;

[0276] Hoagland's nutrient solution: Calcium nitrate 945 mg / L, Potassium nitrate 607 mg / L, Ammonium phosphate 115 mg / L, Magnesium sulfate 493 mg / L, Iron salt solution 2.5 ml / L, Trace elements 5 ml / L, pH = 6.0.

[0277] 3. Experimental instruments

[0278] Forced air drying oven (DHG-9246A, Shanghai Jinghong), tape measure, digital caliper (Shanghai Shenhan), culture flask.

[0279] 4. Experimental methods

[0280] 4.1 Tissue culture of Populus alba×Populus glandulosa clone 84K

[0281] The tissue culture seedlings of Populus alba×Populus glandulosa clone 84K preserved in this laboratory were used for the experiment. The aseptic seedlings of Populus alba×Populus glandulosa clone 84K were cut into poplar stem segments of 2 - 3 cm and vertically inserted into a culture flask containing about 60 ml of rooting medium. The growth conditions were set as follows: light intensity 6000 - 8000 Lux, light / dark cycle 16 h / 8 h, average temperature 25 °C, relative humidity 60 - 70%. After growing for one month, the tissue culture seedlings with strong growth and consistent growth trend were selected for further experiments. The tissue culture seedlings were divided into two groups: hydroponic group: one group was used for hydroponic experiments; soil culture group: the other group was used for acclimatization and subsequent soil culture experiments.

[0282] 4.2 Application of Strain C5A-1 and its lipopeptide products to poplars

[0283] (1) Soil culture of Populus alba×Populus glandulosa clone 84K

[0284] Poplar 84K seedlings were cultivated in soil in a plant cultivation room. One week later, four treatments were set up: CK (control group), RB (50 mg / L C5A-1 bacterial powder solution), RL (50 mg / L C5A-1 lipopeptide crude extract solution), and RBL (mixed solution containing C5A-1 bacterial powder at a final concentration of 50 mg / L and lipopeptide crude extract at a final concentration of 50 mg / L). Each treatment had 8 replicates. For the treatment groups, 200 mL of the above lipopeptide solution, the above bacterial powder solution, or the above lipopeptide and bacterial powder solution was added to each pot, and 200 mL of distilled water was added to the control group. In May, they were placed at 40.00764°N, 116.33804°E and cultured under natural conditions. After four weeks, their growth was observed and experimental data were statistically analyzed.

[0285] (2) Liquid culture of Poplar 84K

[0286] Each aseptic Poplar 84K seedling was placed in a culture container containing 60 mL of nutrient solution (Haibo Biotechnology Co., Ltd., Qingdao High-Tech Industrial Park, product number: HB8870-1. Weigh 1.26 g of this product, and another 0.945 g of calcium nitrate. Heat and dissolve in 1000 mL of distilled water, dispense, and sterilize at 115°C under high pressure for 20 minutes to obtain the nutrient solution for standby) for liquid culture. After one week of culture, C5A-1 lipopeptide crude extract was added to each culture container to make the final concentrations: L (low concentration 20 μg / L, also known as L treatment), M (medium concentration 4 mg / L, also known as M treatment), and H (high concentration 20 mg / L, also known as H treatment), with no treatment as the control (CK treatment). Each treatment group had 8 replicates, and the culture solution was changed every 3 - 4 days. After four weeks, their growth was observed and experimental data were statistically analyzed.

[0287] 4.3 Sample collection and data statistics

[0288] The 84K Poplar seedlings were carefully removed from the culture containers and flower pots, and data on plant height, stem diameter, root length, plant dry weight, number of leaves, and leaf surface area were measured.

[0289] Plant height: The measurement started from the junction of the stem and root (root collar), and was measured vertically along the main stem direction to the highest growth point (apical meristem).

[0290] Stem diameter: At 2 cm from the root collar.

[0291] Root length: Gently wash the roots, remove the attached substrate, and measure the distance from the root tip to the root collar with a ruler.

[0292] Plant dry weight: The plants were treated in an oven at 105°C for 30 minutes to terminate enzyme activity, then dried at 75°C for 48 hours until constant weight (the difference in weight between two consecutive weighings < 0.5%), weighed immediately after cooling to room temperature to avoid moisture absorption.

[0293] Number of leaves: Record the total number of leaves according to the leaf position numbering, excluding withered and yellow leaves (area loss > 50%).

[0294] Leaf surface area data: Formula method: Leaf area = leaf length × leaf width × empirical coefficient (0.65).

[0295] 5. Experimental results

[0296] 5.1 Strain C5A-1 and its lipopeptides promote the growth of potted poplars

[0297] In the poplar potted experiment, compared with the control group, the three treatments of RL, RB, and RBL all significantly promoted the growth of poplars ( Figure 13 (a) in which CK, RB, RL, and RBL are CK treatment, RB treatment, RL treatment, and RBL treatment respectively). Comprehensive analysis shows ( Figure 14 (a) in which CTR, RB, RL, and RBL are CK treatment, RB treatment, RL treatment, and RBL treatment respectively)), when lipopeptides (RL) are added alone, all indicators except the number of leaves are significantly improved; among them, the leaf area is increased by 188.23%; the plant height is increased by 50.21%; the stem diameter is increased by 31.09%; the root length is increased by 17.14%; the above-ground dry weight is increased by 21.10%; the underground dry weight is increased by 25.16%, so this treatment has the best effect.

[0298] 5.2 Strain C5A-1 and its lipopeptides promote the growth of hydroponic poplars

[0299] In the poplar hydroponic experiment, lipopeptide treatments at low concentration (L), medium concentration (M), and high concentration (H) all significantly improved the phenotypic indicators of Populus alba × Populus glandulosa 84K, including the number of leaves, leaf area, plant height, stem diameter, and the dry weights of the above-ground and underground parts ( Figure 13 (b) in which CK, 20 μg / L, 4 mg / L, and 20 mg / L are CK treatment, L treatment, M treatment, and H treatment respectively). Further analysis found ( Figure 14 (b) in which CT, L, M, and H are CK treatment, L treatment, M treatment, and H treatment respectively)), the low-concentration lipopeptide treatment has the most obvious effect on promoting the growth of poplars, among which the number of leaves is increased by 22.2%; the leaf area is increased by 20.14%; the plant height is increased by 22.81%; the stem diameter is increased by 13.16%; the root length is increased by 22.34%; the above-ground dry weight is increased by 25.13%; the underground dry weight is increased by 28.08%.

[0300] The above has described the present invention in detail. For those skilled in the art, without departing from the gist and scope of the present invention and without the need for 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, in accordance with the principle of the present invention, this application intends to cover any modifications, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application and are made using conventional techniques known in the art. The application of some basic features can be made within the scope of the appended claims below.

Claims

1. A Bacillus velezensis C5A-1 or its variant or its offspring, characterized in that, The Bacillus velezensis C5A-1 is deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC No. 32880.

2. A bacterial agent, characterized in that, It contains the Bacillus velezensis C5A-1 or its variants or its progeny as described in claim 1; and one or more excipients or additives.

3. A biological culture, characterized in that, The culture is obtained by culturing the Bacillus velezensis C5A-1 or its variants or its progeny as described in claim 1 or 2 in a solid or liquid medium.

4. A fermentation broth, characterized in that, The fermentation broth contains the Bacillus velezensis C5A-1 or its variants or its progeny as described in claim 1 or 2.

5. A freeze-dried bacterial powder, characterized in that, It includes the Bacillus velezensis C5A-1 or its variants or its progeny as described in claim 1.

6. A plant growth promoter, characterized in that, The growth promoter includes the Bacillus velezensis C5A-1 (B.siamensis) or its variants or its progeny as described in claim 1, the composition as described in claim 2, the culture as described in claim 3, the fermentation broth as described in claim 4, or the freeze-dried powder as described in claim 5.

7. A cultivation method of a plant, characterized in that, The method includes the step of applying the Bacillus velezensis C5A-1 (B.siamensis) or its variants or its progeny as described in claim 1, the composition as described in claim 2, the culture as described in claim 3, the supernatant as described in claim 4, or the freeze-dried powder as described in claim 5 to a plant or a part thereof.

8. The cultivation method according to claim 7, characterized in that, The application is carried out by root irrigation during the seedling stage of the plant.

9. Use of the Bacillus velezensis C5A-1 (Bacillus velezensis) or its variants or its progeny as described in claim 1, the composition as described in claim 2, the culture as described in claim 3, the fermentation broth as described in claim 4, the freeze-dried powder as described in claim 5, or the growth promoter as described in claim 6 in promoting plant growth.

10. The Bacillus velezensis C5A-1 or its variant or its progeny according to claim 1, the growth promoter according to claim 7, the cultivation method according to claim 8, or the use according to claim 9, characterized in that, The plant is selected from any one of Leguminosae, Salicaceae, and Gramineae.

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