Plant probiotics with water retention, growth promotion and disease resistance effects and application thereof

Abstract

The invention discloses plant probiotics with water retention, growth promotion and disease resistance effects and application thereof. The invention provides a complex microbial inoculant, which is prepared from the following active ingredients: 1) bacillus tequilensis Bac365 (GDMCC No: 65241), 2) microbacterium azadirachta Mic250 (GDMCC No: 65244), and 3) Enterococcus querebrasis Ent274 (GDMCC No: 65242) or Pseudomonas plecoglossa Pse350 (GDMCC No: 65243), and the active ingredients of the complex microbial inoculant are as follows: 1) bacillus tequilensis Bac365 (GDMCC No: 65243), 2) microbacterium azadirachta Mic250 (GDMCC No: 65244), and 3) the active ingredients of the complex microbial inoculant. The strain provided by the invention has good nitrogen fixation, phosphorus dissolution, IAA production, HA production and disease resistance, can effectively increase the utilization efficiency of water in drought soil, significantly promote plant growth and increase crop yield, and has good application prospects in the aspects of water retention and sand stabilization, agricultural production and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a plant probiotic with water retention, growth promotion and disease resistance effects and application thereof. BACKGROUND

[0002] At present, soil water loss, land desertification and other problems are becoming more and more serious. In order to solve these problems, some researchers have made some achievements.

[0003] For example, Chinese patent application CN111172084A discloses a bacillus tectum and its application, specifically discloses that bacillus tectum 2_2a can quickly and stably colonize in soybean plants, has fast reproduction speed and strong proliferation ability, has high efficiency and broad-spectrum resistance to plant pathogenic fungi, and has a combined effect on above-ground diseases such as anthracnose, leaf spot disease, black spot disease, black spot disease, gray spot disease, and underground soil-borne diseases such as root rot, damping-off, stem rot and wilt, and has high prevention effect.

[0004] For example, Chinese patent application CN110257295A discloses bacillus tectum and its application and method in desertification prevention, specifically discloses that the strain has high exopolysaccharide production capacity, can make sand particle surface crust, fix sand particles, has excellent sand fixation capacity, and can be used for sand fixation in desertification areas.

[0005] For example, Chinese patent application CN117904012A discloses a high-efficiency compost microbial agent and its application, specifically discloses that the microbial agent is compounded by microbial agent A, microbial agent B and microbial agent C, and the microbial agent can be used for composting of agricultural production waste, improves the quality and yield of apples, and has excellent environmental and economic benefits.

[0006] For example, Chinese patent application CN115873746A discloses application of bacillus tectum SY89 in promoting plant growth, specifically discloses that the strain secretes 25.22mg / L of indole acetic acid IAA, and can secrete iron carrier. 8 The indicators of angelica root treated by bacillus tectum SY89 are improved by more than 23.35% compared with water treatment, which shows that bacillus tectum SY89 has effect of promoting growth of angelica.

[0007] However, these achievements are single-sided, such as disease resistance, growth promotion, sand fixation, etc., and there is no related technology to cover all these problems. SUMMARY

[0008] The purpose of the present application is to provide a plant probiotic with water retention, growth promotion and disease resistance effects and application thereof.

[0009] In a first aspect, the present application claims a composite microbial agent.

[0010] The composite microbial agent claimed in the present application is composite microbial agent 1 or composite microbial agent 2;

[0011] The active ingredients of the composite microbial agent 1 are Bacillus tequilensis, Microbacterium azadirachtae and Enterococcus quebecensis;

[0012] The active ingredients of the composite microbial agent 2 are Bacillus tequilensis, Microbacterium azadirachtae and Pseudomonas plecoglossicida;

[0013] The Bacillus tequilensis has a strain number of Bac365, and its registration number in Guangdong Microbial Culture Collection Center is GDMCC No: 65241;

[0014] The Microbacterium azadirachtae has a strain number of Mic250, and its registration number in Guangdong Microbial Culture Collection Center is GDMCC No: 65244;

[0015] The Enterococcus quebecensis has a strain number of Ent274, and its registration number in Guangdong Microbial Culture Collection Center is GDMCC No: 65242;

[0016] The Pseudomonas plecoglossicida has a strain number of Pse350, and its registration number in Guangdong Microbial Culture Collection Center is GDMCC No: 65243.

[0017] Further, in the composite microbial agent 1, the CFU ratio of Bacillus tequilensis, Microbacterium azadirachtae and Enterococcus quebecensis can be 1:1:1.

[0018] Further, in the composite microbial agent 2, the CFU ratio of Bacillus tequilensis, Microbacterium azadirachtae and Pseudomonas plecoglossicida can be 1:1:1.

[0019] Further, in the composite microbial agent, the effective viable bacterial content is not less than 2x10 8 CFU / mL.

[0020] Further, the composite microbial agent 1 is a microbial agent for water retention and / or sand fixation.

[0021] Further, the composite microbial agent 2 is a microbial agent for promoting plant growth.

[0022] In a second aspect, the present application claims protection of a strain or a single bacterial agent with the strain as an active ingredient.

[0023] The strain claimed in the present application is any one of the following:

[0024] (A1) Bacillus tequilensis, with strain number Bac365, and registered number GDMCC No: 65241 in Guangdong Microbial Culture Collection Center;

[0025] (A2) Microbacterium azadirachtae, with strain number Mic250, and registered number GDMCC No: 65244 in Guangdong Microbial Culture Collection Center;

[0026] (A3) Enterococcus quebecensis, with strain number Ent274, and registered number GDMCC No: 65242 in Guangdong Microbial Culture Collection Center;

[0027] (A4) Pseudomonas plecoglossicida, with strain number Pse350, and registered number GDMCC No: 65243 in Guangdong Microbial Culture Collection Center.

[0028] In the complex bacterial agent described in the first aspect above or the single bacterial agent described in the second aspect above, in addition to the active ingredient, a carrier can also be contained. The carrier can be a carrier commonly used in the agricultural 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 high molecular compound; the mineral material can be at least one of clay, talc, kaolin, montmorillonite, zeolite and diatomite; the plant material can be at least one of corn meal, bean meal and starch; the high molecular compound can be polyvinyl alcohol and / or polyglycol; the liquid carrier can be an organic solvent, a vegetable oil, a mineral oil or water; the organic solvent can be decane and / or dodecane.

[0029] The dosage form of the complex bacterial agent and the single bacterial agent can be various dosage forms, such as liquid, powder, granules, wettable powder.

[0030] According to the needs, a surfactant (such as Tween 20, Tween 80, etc.), a suspending agent, a stabilizer (such as an antioxidant), a pH regulator, etc. can also be added in the complex bacterial agent and the single bacterial agent.

[0031] In a third aspect, the present application claims the use of the composite microbial agent as described in the first aspect above, which can be any of the following:

[0032] (B1) the use of the composite microbial agent 1 as described in the first aspect above in water retention and / or sand fixation;

[0033] (B2) the use of the composite microbial agent 1 as described in the first aspect above in the preparation of a product for water retention and / or sand fixation;

[0034] (B3) the use of the composite microbial agent 1 as described in the first aspect above in the production of hyaluronic acid;

[0035] (B4) the use of the composite microbial agent 1 as described in the first aspect above in the preparation of a product for the production of hyaluronic acid;

[0036] (B5) the use of the composite microbial agent 2 as described in the first aspect above in promoting plant growth;

[0037] (B6) the use of the composite microbial agent 2 as described in the first aspect above in the preparation of a product for promoting plant growth;

[0038] (B7) the use of the composite microbial agent 2 as described in the first aspect above in the production of indole acetic acid;

[0039] (B8) the use of the composite microbial agent 2 as described in the first aspect above in the preparation of a product for the production of indole acetic acid.

[0040] Wherein the product can be a microbial agent or a microbial fertilizer.

[0041] In a fourth aspect, the present application claims the use of the strain as described in the second aspect above, which can be any of the following:

[0042] (C1) the use of Bacillus tequilensis as described in the second aspect above in the production of cellulase;

[0043] (C2) the use of Bacillus tequilensis as described in the second aspect above in the preparation of a product for the production of cellulase;

[0044] (C3) the use of Bacillus tequilensis as described in the second aspect above in nitrogen fixation;

[0045] (C4) the use of Bacillus tequilensis as described in the second aspect above in the preparation of a product for nitrogen fixation;

[0046] (C5) the use of Bacillus tequilensis as described in the second aspect above in the inhibition of pathogenic bacteria;

[0047] (C6) the use of Bacillus tequilensis as described in the second aspect above in the preparation of a product for the inhibition of pathogenic bacteria;

[0048] (C7) The use of Bacillus tequilensis as described in the second aspect above in the production of indole-3-acetic acid;

[0049] (C8) The use of Bacillus tequilensis as described in the second aspect above in the manufacture of a product for the production of indole-3-acetic acid;

[0050] (C9) The use of Bacillus tequilensis as described in the second aspect above in the production of hyaluronic acid;

[0051] (C10) The use of Bacillus tequilensis as described in the second aspect above in the manufacture of a product for the production of hyaluronic acid;

[0052] (C11) The use of Bacillus tequilensis as described in the second aspect above in promoting plant growth;

[0053] (C12) The use of Bacillus tequilensis as described in the second aspect above in the manufacture of a product for promoting plant growth;

[0054] Further, in the (C11) and the (C12), in an embodiment of the present application, the plant is soybean. The promoting plant growth specifically embodies in promoting plant height increase and / or promoting plant stem diameter increase.

[0055] (C13) The use of Microbacterium callitanthi as described in the second aspect above in nitrogen fixation;

[0056] (C14) The use of Microbacterium callitanthi as described in the second aspect above in the manufacture of a product for nitrogen fixation;

[0057] (C15) The use of Microbacterium callitanthi as described in the second aspect above in the production of indole-3-acetic acid;

[0058] (C16) The use of Microbacterium callitanthi as described in the second aspect above in the manufacture of a product for the production of indole-3-acetic acid;

[0059] (C17) The use of Microbacterium callitanthi as described in the second aspect above in the production of hyaluronic acid;

[0060] (C18) The use of Microbacterium callitanthi as described in the second aspect above in the manufacture of a product for the production of hyaluronic acid;

[0061] (C19) The use of Microbacterium callitanthi as described in the second aspect above in promoting plant growth;

[0062] (C20) The use of Microbacterium callitanthi as described in the second aspect above in the manufacture of a product for promoting plant growth;

[0063] Further, in the (C19) and the (C20), in an embodiment of the present application, the plant is soybean. The promoting plant growth specifically embodies in: promoting plant height increase and / or promoting plant root length elongation and / or promoting plant stem diameter increase and / or promoting plant underground fresh weight increase.

[0064] (C21) The use of Enterococcus quae in dissolving inorganic phosphorus according to the second aspect in the preceding;

[0065] (C22) The use of Enterococcus quae in preparing a product for dissolving inorganic phosphorus according to the second aspect in the preceding;

[0066] (C23) The use of Enterococcus quae in nitrogen fixation according to the second aspect in the preceding;

[0067] (C24) The use of Enterococcus quae in preparing a product for nitrogen fixation according to the second aspect in the preceding;

[0068] (C25) The use of Pseudomonas plecoglossicida in producing hyaluronic acid according to the second aspect in the preceding;

[0069] (C26) The use of Pseudomonas plecoglossicida in preparing a product for producing hyaluronic acid according to the second aspect in the preceding;

[0070] (C27) The use of Pseudomonas plecoglossicida in dissolving inorganic phosphorus and / or organic phosphorus according to the second aspect in the preceding;

[0071] (C28) The use of Pseudomonas plecoglossicida in preparing a product for dissolving inorganic phosphorus and / or organic phosphorus according to the second aspect in the preceding;

[0072] (C29) The use of Pseudomonas plecoglossicida in producing cellulase according to the second aspect in the preceding;

[0073] (C30) The use of Pseudomonas plecoglossicida in preparing a product for producing cellulase according to the second aspect in the preceding;

[0074] (C31) The use of Pseudomonas plecoglossicida in nitrogen fixation according to the second aspect in the preceding;

[0075] (C32) The use of Pseudomonas plecoglossicida in preparing a product for nitrogen fixation according to the second aspect in the preceding;

[0076] (C33) The use of Pseudomonas plecoglossicida in producing indole acetic acid according to the second aspect in the preceding;

[0077] (C34) The use of Pseudomonas plecoglossicida in preparing a product for producing indole acetic acid according to the second aspect in the preceding;

[0078] (C35) The use of the Pseudomonas plecoglossicida as described in the second aspect above in promoting plant growth;

[0079] (C36) The use of the Pseudomonas plecoglossicida as described in the second aspect above in the preparation of a product for promoting plant growth.

[0080] Further, in the (C35) and the (C36), in an embodiment of the present application, the plant is soybean. The promotion of plant growth specifically embodies in: promoting plant height increase and / or promoting plant root length elongation and / or promoting plant aboveground fresh weight increase.

[0081] Further, in the (C5) and the (C6), in an embodiment of the present application, the pathogenic fungus is any one of the following: Fusarium oxysporum, Fusarium solani, Aspergillus flavus, Aspergillus niger, Epicoccum latusicollum, Sarocladium oryzae, and Pestalotiopsis mangiferae.

[0082] The product can be a microbial inoculant or a microbial fertilizer.

[0083] In a fifth aspect, the present application claims a method for water retention and / or sand fixation.

[0084] The method for water retention and / or sand fixation claimed by the present application can comprise the following steps: planting a plant in an area to be watered and / or sand-fixed (such as a sandy soil area) and applying the composite microbial inoculant 1 as described in the first aspect above, so as to achieve water retention and / or sand fixation.

[0085] In an embodiment of the present application, the plant is alfalfa.

[0086] In a sixth aspect, the present application claims a method for promoting plant growth.

[0087] The method for promoting plant growth claimed by the present application can comprise the following steps: applying the composite microbial inoculant 2 as described in any one of the first aspect above to the rhizosphere soil of a plant, so as to achieve the promotion of the growth of the plant.

[0088] In an embodiment of the present application, the plant is alfalfa.

[0089] Advantages of the present application:

[0090] The plant probiotic bacteria in the application have good abilities of nitrogen fixation, phosphorus dissolution, disease resistance, indole acetic acid production, hyaluronic acid production and cellulase production, which enrich the available microbial resources in agricultural production, environmental protection and disease control.

[0091] The application of the hyaluronic acid-producing bacterial strain combination, the plant growth-promoting bacterial strain combination and the growth-promoting bacterial strain in the application can effectively increase the water utilization efficiency in dry soil, significantly promote plant growth, inhibit plant disease occurrence and improve crop yield, and has good application prospects in water and sand conservation, agricultural production and the like.

[0092] Deposit Description 1

[0093] Proposed taxonomic name: Bacillus tequilensis;

[0094] Biological material name and noted identification characteristics: Bacillus tequilensis Bac365;

[0095] Preservation agency: Guangdong Microbial Culture Collection Center;

[0096] Preservation agency abbreviation: GDMCC;

[0097] Address: 5th Floor, Building 59, 100, Jiefang Middle Road, Guangzhou, Guangdong Province;

[0098] Preservation date: October 12, 2024;

[0099] Preservation center registration number: GDMCC No: 65241.

[0100] Deposit Description 2

[0101] Proposed taxonomic name: Enterococcus quebecensis;

[0102] Biological material name and noted identification characteristics: Enterococcus quebecensis Ent274;

[0103] Preservation agency: Guangdong Microbial Culture Collection Center;

[0104] Preservation agency abbreviation: GDMCC;

[0105] Address: 5th Floor, Building 59, 100, Jiefang Middle Road, Guangzhou, Guangdong Province;

[0106] Preservation date: October 12, 2024;

[0107] Deposit accession number: GDMCC No: 65242.

[0108] Deposit Description 3

[0109] Proposed taxonomic name: Pseudomonas plecoglossicida;

[0110] Name of the biological material and the stated identifying characteristics: Pseudomonas plecoglossicida Pse350;

[0111] Collection: Guangdong Microbial Culture Collection Center;

[0112] Abbreviation of the collection: GDMCC;

[0113] Address: 5th Floor, Building 59, 100, Martyrs' Road, Guangzhou, Guangdong Province;

[0114] Date of deposit: October 12, 2024;

[0115] Deposit accession number: GDMCC No: 65243.

[0116] Deposit Description 4

[0117] Proposed taxonomic name: Microbacterium azadirachtae;

[0118] Name of the biological material and the stated identifying characteristics: Microbacterium azadirachtae Mic250;

[0119] Collection: Guangdong Microbial Culture Collection Center;

[0120] Abbreviation of the collection: GDMCC;

[0121] Address: 5th Floor, Building 59, 100, Martyrs' Road, Guangzhou, Guangdong Province;

[0122] Date of deposit: October 12, 2024;

[0123] Deposit accession number: GDMCC No: 65244. BRIEF DESCRIPTION OF DRAWINGS

[0124] Figure 1 Plate streaking diagram of four strains of probiotics. Among them, a is Bacillus tequilensis Bac365; b is Pseudomonas mutabilis Pse350; c is Microbacterium azadirachtae Mic250; d is Enterococcus quiquestris Ent274.

[0125] Figure 2 Gram staining of four strains of probiotics. Among them, a is Bacillus tequilensis Bac365; b is Pseudomonas mutabilis Pse350; c is Microbacterium arabinogalactum-reducing Mic250; d is Enterococcus quadiquadi Ent274.

[0126] Figure 3 Phylogenetic tree of Bacillus tequilensis Bac365.

[0127] Figure 4 Phylogenetic tree of Pseudomonas mutabilis Pse350.

[0128] Figure 5 Phylogenetic tree of Microbacterium arabinogalactum-reducing Mic250.

[0129] Figure 6 Phylogenetic tree of Enterococcus quadiquadi Ent274.

[0130] Figure 7 Pictures of functional verification plates of probiotics for dissolving phosphorus and producing cellulase. Among them, a is a plate picture of Pseudomonas mutabilis Pse350 dissolving inorganic phosphorus; b is a plate picture of Pseudomonas mutabilis Pse350 dissolving organic phosphorus; c is a plate picture of Enterococcus quadiquadi Ent274 dissolving inorganic phosphorus; d is a plate picture of Bacillus tequilensis Bac365 producing cellulase screening; e is a plate picture of Pseudomonas mutabilis Pse350 producing cellulase screening.

[0131] Figure 8 Nitrogenase activity determination of probiotics. Among them, a is the color development of probiotics and control hole; b is the nitrogenase concentration chart of probiotics.

[0132] Figure 9Plate confrontation pictures of probiotics and plant pathogens. Among them, a is the plate confrontation picture of Bacillus tequilensis Bac365 and Fusarium solani; b is the plate confrontation picture of Bacillus tequilensis Bac365 and Fusarium oxysporum; c is the plate confrontation picture of Bacillus tequilensis Bac365 and Aspergillus flavus; d is the plate confrontation picture of Bacillus tequilensis Bac365 and Aspergillus niger; e is the plate confrontation picture of Bacillus tequilensis Bac365 and Epicoccum latusicollum; f is the plate confrontation picture of Bacillus tequilensis Bac365 and Pestalotiopsis mangiferae; g is the plate confrontation picture of Bacillus tequilensis Bac365 and Sarocladium oryzae; and h is the plate confrontation picture of Pseudomonas plecoglossicida Pse350 and Aspergillus niger.

[0133] Figure 10 Figure for the ability of probiotics to produce plant hormone IAA. ** represents P < 0.01, and *** represents P < 0.001.

[0134] Figure 11 Figure for the content of hyaluronic acid HA produced by probiotics.

[0135] Figure 12 Soil permeability coefficient and water content of the potting soil of the combination of strains Bac365, Mic250 and Ent274. The notation “365-250-274” represents the combination of strains Bac365, Mic250 and Ent274. * represents P < 0.05.

[0136] Figure 13 Figures for the growth situation and physiological indexes of alfalfa potting of the combination of strains Bac365, Mic250 and Pse350 on sandy soil. Among them, a is the comparison figure of the alfalfa potting of the strain combination and the control; b is the comparison figure of the alfalfa plants of the strain combination and the control at the time of seedling collection; and c is the physiological index figure of the strain combination and the control. The notation “365-350-250” represents the combination of strains Bac365, Mic250 and Pse350. * represents P < 0.05.

[0137] Figure 14Figures of growth and physiological indexes of soybean plants treated by strains Bac365, Mic250 and Pse350 in field experiments, wherein, a is a figure of physiological indexes of soybean plants treated by Bac365 and control; b is a figure of physiological indexes of soybean plants treated by Pse350 and control; c is a figure of physiological indexes of soybean plants treated by Mic250 and control; d is an average number of pods of treatment groups and control groups; e is a figure of soybean plants treated by Bac365 and control; f is a figure of soybean plants treated by Pse350 and control; g is a figure of soybean plants treated by Mic250 and control; * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001. DETAILED DESCRIPTION

[0138] The application provides four strains derived from desert isolated strains. It is found through genome characteristics prediction and function verification that the strains have good abilities of nitrogen fixation, phosphorus dissolution, cellulase production, indole acetic acid production and hyaluronic acid production, and the strain Bac365 has broad-spectrum antibacterial property and can effectively inhibit the growth of pathogenic bacteria of crops such as cereals, legumes, solanaceae and melons, so as to provide new microbial strain resources for agricultural production.

[0139] The application provides application of hyaluronic acid-producing strain combination in water conservation and sand fixation. Specifically, the hyaluronic acid-producing strain combination is subjected to a potting test, and the soil used for potting is sandy soil. Soil permeability and soil water content tests are performed when seedlings are collected. It is found that the combination can reduce soil permeability and increase soil water content. It is indicated that the hyaluronic acid secreted by the strain combination can better conserve water and moisture in dry soil, increase water use efficiency, and create better conditions for plant growth.

[0140] The application provides a growth-promoting strain combination and application thereof in promoting crop growth. Specifically, a potting test is performed, and the soil used for potting is sandy soil. The strain combination is used to irrigate roots of the plants every 7 days after germination of alfalfa seedlings, and the seedlings are collected after 40 days. It is found that the strain combination can effectively increase the plant height, aboveground fresh weight and underground fresh weight of alfalfa, and it is indicated that the strain combination can effectively promote the growth of alfalfa plants in sandy soil, and has good ecological value for water and sand conservation and returning sand to forest.

[0141] The application provides application of a growth-promoting strain in promoting crop growth. Specifically, a field test is performed, and soybean seeds are used as materials. The growth-promoting strain is used for irrigation when the seeds are sown, and the strain is used to irrigate roots of the plants 7 days after germination, and the seedlings are collected after 60 days. It is found that the soybean plants treated by the growth-promoting strain grow well, and the plant height, root length and fresh weight are improved compared with the control. It is indicated that the growth-promoting agent is helpful for root strengthening, seedling promotion and yield increase of soybean, and has good application prospect in agricultural production.

[0142] The application will be described in further detail below with specific reference to the embodiments. The examples given are only intended to illustrate the present application and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the present application.

[0143] The experimental methods in the following examples are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0144] Example 1, isolation, identification and genomic characteristics of strains Bac365, Pse350, Mic250 and Ent274

[0145] 1. Isolation and purification of strains

[0146] 1g of the sand sample was weighed into 10ml of sterile water and mixed to obtain a 10 -1 dilution solution. 1ml of the dilution solution was gradient diluted into 10 -2 , 10 -3 , 10 -4 dilution solution, 0.1ml of each was spread on YMA solid medium and cultured at 28°C for 2d. Single colonies were picked and streaked to obtain purified strains, which were inoculated into YMA liquid medium for culture.

[0147] YMA liquid medium formula: mannitol 10g / L, glucose 5g / L, yeast powder 0.8g / L, NaCl 0.1g / L, MgSO4 7H2O 0.2g / L, K2HPO4 0.25g / L, KH2PO4 0.2g / L, CaCO3 2H2O 0.3g / L, Congo red 0.5% (volume percentage), pH 6.8-7.2. YMA solid medium was based on the YMA liquid medium formula with the addition of 18g / L agar.

[0148] Four strains were obtained in this step, which were designated as strain Bac365, strain Pse350, strain Mic250 and strain Ent274, respectively.

[0149] 2. Identification of strains

[0150] (1) Morphological observation:

[0151] The morphology of the strains, such as color, shape and size, was observed by plate streaking. The structural differences of bacterial cell walls were determined by Gram staining.

[0152] The morphology and Gram staining of the strains are shown in Figure 1 , Figure 2 .

[0153] ① Strain Bac365 morphological characteristics: using inoculation ring on LB solid medium, the colony is ivory white, near round, irregular edge, surface wrinkled, gram staining is positive, the bacterial body is rod-shaped.

[0154] ② Strain Pse350 morphological characteristics: the colony is ivory white round, regular edge, surface moist and smooth, gram staining is negative.

[0155] ③ Strain Mic250 morphological characteristics: the colony is light yellow, complete edge round, surface moist and smooth, gram staining is positive.

[0156] ④ Strain Ent274 morphological characteristics: the colony is light yellow, complete edge round, surface moist and smooth, gram staining is positive.

[0157] (2) Molecular identification

[0158] The 16S rDNA gene of the monoclonal strain is sequenced. The 16S rDNA gene sequence of strain Bac365 is shown as SEQ ID No. 1, the 16S rDNA gene sequence of strain Pse350 is shown as SEQ ID No. 2, the 16S rDNA gene sequence of strain Mic250 is shown as SEQ ID No. 3, and the 16S rDNA gene sequence of strain Ent274 is shown as SEQ ID No. 4.

[0159] The 16S rDNA gene sequences obtained by sequencing were aligned in the 16S rDNA gene database of EzBioCloud. The alignment results showed that the strain with the highest homology of 16S rDNA gene with strain Bac365 in the database was Bacillus tequilensis KCTC 13622 (sequence number: AYTO01000043), with a similarity of 99.86%; the strain with the highest homology of 16S rDNA gene with strain Pse350 was Pseudomonas plecoglossicida NBRC 103162 (sequence number: BBIV01000080), with a similarity of 99.85%; the strain with the highest homology of 16S rDNA gene with strain Mic250 was Microbacterium azadirachtae DSM 23848 (sequence number: JYIT01000023), with a similarity of 99.83%; the strain with the highest homology of 16S rDNA gene with strain Ent274 was Enterococcus quebecensis CCRI-16985 (sequence number: GU457262), with a similarity of 99.53%. The strain sequences were used to construct a phylogenetic tree using MAGA with a neighbor-joining method, the phylogenetic tree of strain Bac365 is shown in Figure 3 , the phylogenetic tree of strain Pse350 is shown in Figure 4 , the phylogenetic tree of strain Mic250 is shown in Figure 5 , and the phylogenetic tree of strain Ent274 is shown in Figure 6 .

[0160] (3) Genomic characteristics

[0161] The functional key genes of the four strains were obtained from the genomic information by sequencing the strains Bac365, Pse350, Mic250 and Ent274, which were predicted to have the function of promoting plant growth. Strain Bac365 was predicted to have 12272 coding genes, 7341 coding genes were annotated by KEGG, there were 3 phenazine precursor genes, and the strain was predicted to have good disease resistance, as well as nitrogen fixation, alkaline phosphatase, cellulose precursor and 2 key genes for cellulose and IAA production, iron carrier iucD related genes. Strain Pse350 sequencing obtained 6848 coding genes, 4953 coding genes were annotated by KEGG, with nitrogen fixation gene nifH450, iron carrier gene iucB, 2 key genes for IAA production, as well as alkaline phosphatase, phenazine, cellulose precursor, cellulose production and potassium solution key genes. Strain Mic250 obtained 5945 coding genes, 3746 coding genes were annotated by KEGG, with hyaluronic acid gene, 4 key genes for iron carrier, 3 key genes for IAA production, 3 cellulose precursor genes, acid phosphatase gene, and high enrichment of potassium solution gene. Strain Ent274 sequencing obtained 4408 coding genes, 2808 coding genes were annotated by KEGG, and the hyaluronic acid gene, 2 key genes for IAA production, iron carrier key gene iucA, alkaline phosphatase gene, and high enrichment of potassium solution gene were predicted.

[0162] Through the above identification, it is proved that:

[0163] Strain Bac365 is Bacillus tequilensis, which has been preserved in Guangdong Microbial Culture Collection Center on October 12, 2024, and its registration number is GDMCC No:65241.

[0164] Strain Mic250 is Microbacterium azadirachtae, which has been preserved in Guangdong Microbial Culture Collection Center on October 12, 2024, and its registration number is GDMCC No:65244.

[0165] Strain Ent274 is Enterococcus quebecensis, which has been preserved in Guangdong Microbial Culture Collection Center on October 12, 2024, and its registration number is GDMCC No:65242.

[0166] Strain Pse350 is Pseudomonas plecoglossicida, which has been deposited with the Guangdong Microbial Culture Collection Center on October 12, 2024, and the accession number is GDMCC No: 65243.

[0167] Example 2, functional determination of the strain

[0168] 1. Determination of the ability of the strain to solubilize phosphorus and produce cellulase

[0169] The strain was inoculated into LB liquid medium and cultured at 28°C with 150 rpm shaking for 48 h. 5 μL of the bacterial liquid was inoculated into each of the organic phosphorus, inorganic phosphorus, and cellulase production medium, and placed in a 28°C constant temperature incubator for culture for 5-7 d.

[0170] The appearance of a phosphorus-solubilizing ring in the phosphorus-solubilizing medium indicates that the strain has the ability to solubilize phosphorus.

[0171] The cellulase-producing strain screening was performed by adding 1 mg / mL Congo red solution (covering the colonies) to the culture dish after 5-7 d of culture, standing for 15 min, and then pouring off the Congo red dye. Then 1 mol / L sodium chloride solution was added, and after standing for 15 min, the sodium chloride solution was poured off. If a transparent ring appears around the colonies, the strain is considered to have the ability to produce cellulase.

[0172] Medium formula:

[0173] Organic phosphorus medium: glucose 10 g / L, ammonium sulfate 0.5 g / L, sodium chloride 0.3 g / L, magnesium sulfate 0.3 g / L, manganese sulfate 0.03 g / L, potassium sulfate 0.3 g / L, ferrous sulfate 0.03 g / L, calcium phosphate 5 g / L, lecithin 0.2 g / L, agar 15 g / L, pH 7.0-7.5.

[0174] Inorganic phosphorus medium: glucose 10 g / L, ammonium sulfate 0.5 g / L, sodium chloride 0.3 g / L, magnesium sulfate 0.3 g / L, manganese sulfate 0.03 g / L, potassium sulfate 0.3 g / L, ferrous sulfate 0.03 g / L, calcium phosphate 5 g / L, agar 15 g / L, pH 7.0-7.5.

[0175] Cellulase medium: peptone 10 g / L, yeast powder 10 g / L, sodium carboxymethyl cellulose 10 g / L, sodium chloride 5 g / L, potassium dihydrogen phosphate 1 g / L, agar powder 15 g / L.

[0176] The results are shown in Figure 7Strain Pse350 appeared phosphorus solubilizing halo in both organic phosphorus and inorganic phosphorus medium, and had the ability to dissolve organic phosphorus and inorganic phosphorus. Strain Ent274 appeared phosphorus solubilizing halo in inorganic phosphorus medium, and had the ability to dissolve inorganic phosphorus. In cellulase screening test, strain Bac365 and Pse350 produced transparent halo around the colonies, and thus were considered to have the ability to produce cellulase. Phosphorus solubilizing bacteria can convert the phosphorus that is difficult for plants to absorb and utilize into a form that can be absorbed and utilized by plants; cellulase-producing strains help to improve soil texture and permeability, and create a good environment for plant growth.

[0177] 2. Determination of nitrogen fixation enzyme activity of strains

[0178] Treatment of sample to be tested: 1 ml of bacterial liquid was centrifuged, and the supernatant was discarded. 0.08 ml of buffer was used for suspension, 0.08 mg of lysozyme was added, and ice bath was performed for 30 min, followed by ultrasonic treatment. The ultrasonic time was 5 seconds, the interval was 5 seconds, and a total of 5 times of ultrasonic treatment was performed. After ultrasonic treatment, centrifugation was performed at 4000 rpm for 15 min to obtain the supernatant.

[0179] Double antibody sandwich method was used for determination of nitrogen fixation enzyme activity, and the test steps were referred to the instruction manual of nitrogen fixation enzyme enzyme-linked immunoassay kit of Lianmai Biology. Within 15 min after the addition of the termination solution, the absorbance value at 450 nm was determined, and the blank well was used as the control.

[0180] The absorbance value was determined by the above method using standard solutions with concentrations of 10, 20, 40, 80, and 160 IU / L, and a standard curve was drawn.

[0181] Standard curve: y = 88.945x + 4.9326, R 2 = 0.9945.

[0182] The results are shown in Figure 8 , wells 1-5 in a are gradient-diluted standard solutions, wells 6 and 7 are CK (blank wells without adding bacterial liquid), wells 13-16 are added with samples (strains Pse350, Mic250, Ent274, and Bac365), and other well positions are other strains (non-patent strains, not embodied in this patent application). Strains Bac365, Mic250, Ent274, and Pse350 all have the ability to produce nitrogen fixation enzyme, and the concentrations are 10.98, 10.18, 9.65, and 6.27 IU / L, respectively. This indicates that the four strains have nitrogen fixation ability, and can convert molecular nitrogen in the air into ammonia for plant utilization.

[0183] 3. Determination of disease resistance of strains

[0184] A plate confrontation experiment was conducted with probiotics against four pathogens: *Fusarium oxysporum* (root rot pathogen), *Fusarium solani* (root rot pathogen), *Aspergillus flavus*, *Aspergillus niger* (root rot pathogen), *Epicoccum latusicollum* (leaf spot pathogen), *Sarocladium oryzae* (rice leaf sheath rot pathogen), and *Pestalotiops ismangiferae* (leaf spot and leaf blight pathogen). Pathogen mycelial discs (0.5 cm in diameter) were inoculated into the center of prepared LB / PDA (1:1 volume ratio) plates and incubated at 28°C for 1 day. Then, 5 μL of the activated bacterial solution from each of the four strains was inoculated around the perimeter of the plate (2 cm away from the pathogens). The control group was inoculated only with the pathogens. The plates were incubated at 28°C for 5 days, and the confrontation was observed.

[0185] The results showed that strain Bac365 could inhibit the growth of the above seven pathogenic bacteria, demonstrating good disease resistance. Figure 9 The pathogens *Fusarium oxysporum* can infect economic crops such as cereals, melons, solanaceous plants, and legumes, causing wilt and root rot; *Fusarium solani* can cause root rot in legumes and vegetables; *Aspergillus niger* can cause black mold in grapes, peanuts, and rice; *Aspergillus flavus* can infect peanuts, corn, and cereals, causing mold; *Epicoccumlatusicollum* can infect tobacco and rice, causing leaf spot; *Sarocladium oryzae* can cause leaf sheath rot in rice; and *Pestalotiopsis mangiferae* can cause leaf blight and leaf spot in economic crops such as mangoes and camellia oleifera. Strain Bac365 exhibits broad-spectrum antibacterial activity, showing varying degrees of antagonism against the aforementioned Fusarium, Aspergillus niger, Aspergillus flavus, Aspergillus parasiticus, Polychaete spp., and Sarocladium oryzae strain 7. It provides a strain resource for the biological control of leaf spot, root rot, and mildew diseases in legumes, cereals, cucurbits, solanaceous crops, fruits, and vegetables.

[0186] Similarly, strain Pse350 can inhibit the growth of Aspergillus niger, providing a strain resource for the prevention and control of black mold disease in crops such as grapes, peanuts, and rice.

[0187] 4. Determination of the strain's ability to produce indoleacetic acid (IAA)

[0188] The strains were inoculated into LB liquid medium (containing L-tryptophan 0.5 mg / mL) and cultured at 30°C with 150 rpm shaking for 3 days. The bacterial solution was centrifuged at 12000 rpm for 15 min, and 800 μL of the supernatant was mixed with an equal amount of Salkowski's reaction solution (formula: 1 mL of 0.5 mol / L ferric chloride, 49 mL of 35% perchloric acid), and inverted to mix well, and reacted in the dark for 30 min. The absorbance was measured at 530 nm using a microplate reader, and the culture medium without inoculation was treated in the same way as the control (CK). The absorbance value of the treatment group was significantly higher than that of the control (CK), indicating that the strain had the ability to produce indole acetic acid.

[0189] The absorbance values of strains Pse350, Bac365 and Mic250 at OD 530 Figure 10 The absorbance values of strains Pse350, Bac365 and Mic250 at OD

[0190] 5. Determination of the ability of strains to produce hyaluronic acid (HA)

[0191] 1 mL of bacterial solution was taken in a test tube, 2 mL of CTAB test solution (25 mg / mL) was added, and it was shaken and mixed well, and then left to stand for 10 min. The reaction solution was added to a cuvette, and the absorbance at a wavelength of 400 nm was measured using a spectrophotometer. The culture medium without inoculation was treated in the same way as the blank control. The absorbance values of HA standard solutions with concentrations of 10, 20, 30, 40 and 50 mg / L were measured using the above method, and a standard curve was drawn.

[0192] Standard curve: y = 265.25x - 5.9682, R 2 = 0.9973.

[0193] The results are shown in Figure 11 , strains Ent274, Mic250 and Bac365 all have the ability to produce HA, and the production of hyaluronic acid is 0.032, 0.049 and 0.478 mg / ml, respectively. Strain Bac365 has strong ability to produce hyaluronic acid. Hyaluronic acid has the function of chelating water molecules and locking water, and its interaction with plants can retain water and moisture in the soil, regulate soil moisture, and ensure normal plant growth.

[0194] Example 3, determination of soil permeability and water content of strain Bac365, Mic250 and Ent274 combination

[0195] ​Pot experiment was carried out with three strains (Bac365, Mic250 and Ent274) having the function of producing hyaluronic acid. The test material was alfalfa seed Algonin, and the potting soil was sand. The specific steps are as follows:

[0196] The sand was autoclaved at 121°C for 30 min, and then was divided into flowerpots (12 cm in diameter and 16.5 cm in height) with 1.8 kg of sand in each pot. The alfalfa seed Algonin was surface sterilized by soaking in 1% NaClO for 5 min and then was washed with sterile water for 5 times. 100 μL of the last washing liquid was taken and spread on LB plate to observe the growth of sterile colonies to verify the sterilization. The alfalfa seed was sowed in the flowerpot with sand, and 30 seeds were in each pot.

[0197] Preparation of bacterial suspension: the strains Bac365, Mic250 and Ent274 were activated and then were inoculated into 50 mL centrifuge tubes at 4% (volume percentage) inoculation amount, and were cultured at 150 rpm and 28°C for 3 days. After the culture, the supernatant was discarded after centrifugation at 5000 rpm for 10 min, and the bacterial suspension was prepared by resuspending the bacteria in sterile water and adjusting the absorbance at 600 nm to 0.8.

[0198] After the germination of alfalfa, the roots were irrigated. The treatment group was inoculated with the bacterial suspension (2×10 8 CFU / mL) obtained by combining the strains Bac365, Mic250 and Ent274 (volume ratio 1:1:1, and the CFU ratio of the three strains was about 1:1:1). The control (CK) was inoculated with sterile water, and 2 mL of sterile water was inoculated into the soil of each seedling. The inoculation was repeated every 7 days, and the total inoculation times were 4. The seedlings were thinned 7 days after the germination. The seedlings were harvested after 40 days. The soil permeability and soil water content test were carried out at the time of harvesting.

[0199] (1) Soil permeability determination:

[0200] The soil permeability was determined by using TST-55 type variable water head soil permeameter. The potting soil was cut by a cutting ring and was put into a sleeve. The water stone and water stop washer were installed, and the nut was tightened. The water inlet pipe and variable water head pipe of the permeation container were connected, and the air exhaust pipe and water outlet pipe were installed. The water inlet pipe valve was opened, and water flowed into the permeation container. The water overflowed from the air exhaust pipe until there was no bubble in the water flow. The air exhaust pipe was closed, and the sample was kept under the water head for a period of time. When water overflowed from the water outlet pipe, it was considered that the sample was basically saturated. Pure water was injected into the variable water head pipe until the predetermined height was reached. Then, the water source was cut off, the water inlet pipe valve was opened, and the initial water head height and initial time in the variable water head pipe were recorded when water overflowed from the water outlet pipe. The water head height was measured again after 60 s. The above steps were repeated 4 times. The soil permeability coefficient was calculated according to the formula.

[0201] Soil permeability coefficient formula: K T= (aL) x ln(hl-h2) ÷ A(t2-tl)

[0202] a: variable head pipe area;

[0203] A: sample area;

[0204] L: permeation path (sample height);

[0205] tl: initial time of reading water head;

[0206] t2: final time of reading water head;

[0207] hl: initial water head height;

[0208] h2: final water head height.

[0209] (2) Soil moisture content determination:

[0210] At the time of seedling, 100 g of potting soil sample (sand) was weighed as fresh weight before drying, and 4 soil samples were weighed as replicates for each treatment group. Put into a constant temperature oven at 65°C and dry to constant weight. Weigh the dried soil sample as dry weight. The control group is treated in the same way.

[0211] According to the formula: moisture = (m1-m2) ÷ (m1), the content of water in the soil can be obtained.

[0212] m1: weight of soil sample before drying

[0213] m2: weight of soil sample after drying

[0214] Results are shown in Figure 12 The soil permeability coefficient of the combination of strains Bac365, Mic250 and Ent274 was significantly lower than that of the control group (p<0.05, t test), because the hyaluronic acid produced by the strain combination had certain viscosity, which reduced the water flow per unit time, thus reducing the soil permeability, so that the soil had better water retention effect. Similarly, the soil moisture content of the treatment group (i.e. the combination of strains Bac365, Mic250 and Ent274) was higher than that of the control group, indicating that the hyaluronic acid secreted by the strain combination could better retain water and moisture in drought soil, and its application in drought soil could increase the utilization efficiency of water, creating better conditions for plant growth.

[0215] Example 4, Effect of strains Bac365, Mic250, Pse350 on alfalfa growth

[0216] The material used in the pot experiment was alfalfa seed Algonkin, and the soil used in the pot experiment was sand. The specific steps are as follows:

[0217] The sand was autoclaved at 121℃ for 30 minutes and then dispensed into flowerpots (12cm in diameter and 16.5cm in height), with each pot weighing 1.8kg. Algonquin surface disinfection of alfalfa seeds was performed by soaking in 1% NaClO for 5 minutes, followed by rinsing five times with sterile water. 100μL of the final rinse solution was spread onto LB agar plates, and the absence of colony growth was observed to verify thorough disinfection. 30 alfalfa seeds were then sown into each flowerpot filled with sand.

[0218] Preparation of bacterial suspension: After activation, strains Bac365, Mic250, and Pse350 were inoculated at a rate of 4% (volume percentage) and cultured in 50 mL centrifuge tubes at 150 rpm and 28 °C for 3 days. After centrifugation at 5000 rpm for 10 min, the supernatant was discarded, and the suspension was resuspended in sterile water. The absorbance at 600 nm was adjusted to 0.8 to prepare the bacterial suspension.

[0219] After alfalfa sprouted, root irrigation was performed. The treatment group used a bacterial suspension obtained from a combination of strains Bac365, Mic250, and Pse350 (volume ratio 1:1:1, CFU ratio of the three strains approximately 1:1:1) (effective viable count 2×10⁻⁶). 8 (CFU / mL) was used for the control (CK), and 2 mL of sterile water was inoculated into the soil around the roots of each seedling. Inoculation was repeated 7 days later, for a total of 4 inoculations. Thinning was performed 7 days after germination. Seedlings were harvested after 40 days. Plant height, root length, fresh weight, and dry weight were measured at harvest.

[0220] The results are as follows Figure 13 As shown, the alfalfa potted plants composed of strains Bac365, Mic250, and Pse350 grew well in sandy soil, and their plant height (17.94±5.21 cm), above-ground fresh weight (0.41±0.24 g), and underground dry weight (0.086±0.006 g) were all significantly higher than those of the control group (p<0.05, t-test). This indicates that this strain combination can effectively promote the growth of alfalfa plants in sandy soil and has good ecological value for water conservation, sand fixation, and reforestation.

[0221] Example 5: Effects of strains Bac365, Mic250, and Pse350 on soybean growth

[0222] This embodiment uses soybean seed variety Huang 13 as material for a field trial. The specific steps are as follows:

[0223] Preparation of bacterial culture: Bac365, Mic250, and Pse350 strains were inoculated into LB medium and cultured with shaking at 120 rpm and 28°C for 4-5 days. The OD of the bacterial culture was measured. 600 The value reaches 1.0. Dilute the fermented bacterial solution 20 times and set aside.

[0224] Soybean seeds were sown in the field, three seeds per hole. The treatment groups were treated with diluted bacterial solutions of Bac365, Mic250, and Pse350, respectively, 30 mL per hole. During the rooting period (10-15 days after sowing) and the growth period (30 days after sowing), the corresponding bacterial solutions were applied again, followed by root drenching with 30 mL per hole, using the same treatment method. The control group (CK) received no bacterial solution and used tap water as described above. Routine field management was followed. Seedlings were harvested after 60 days for soybean phenotypic analysis.

[0225] The results are as follows Figure 14 As shown, soybean plants treated with strains Bac365, Mic250, and Pse350 showed good growth, with improvements in plant height, root length, and fresh weight compared to the control. Figure 14 In the figure, d represents the comparison between the mean number of pods obtained from the field experiment of the three single strains and the control group. It can be seen that the mean number of pods in the treatment group is higher than that in the control group. The plant height (66.84±5.03cm) and stem diameter (0.38±0.09cm) of the Bac365 treatment group were significantly higher than those of the control (p<0.05, t-test); the plant height (67.63±4.07cm), root length (33.30±8.17cm), and above-ground fresh weight (186.41±36.14g) of the Pse350 treatment group were significantly higher than those of the control (p<0.05, t-test); the plant height (63.19±4.79cm), root length (34.79±5.92cm), stem diameter (0.38±0.08cm), and underground fresh weight (11.42±2.63g) of the Mic250 treatment group were significantly different from those of the control (p<0.05, t-test). This indicates that strains Bac365, Mic250, and Pse350 have the effects of strengthening roots, promoting seedling growth, and increasing yield, and have good application prospects in agricultural production.

[0226] The results of the above embodiments show that the strains isolated in the application have good abilities of nitrogen fixation, phosphorus dissolution, IAA production and HA production, and can provide nitrogen source, phosphorus and plant hormones for plants. The strain Bac365 has broad-spectrum antibacterial activity, and has different degrees of antagonistic effect on Fusarium, Aspergillus niger, Aspergillus flavus, Micrococcus, Pestalotiopsis, and Sarocladium oryzae 7 pathogenic strains, thereby providing bacterial resources for biological control of diseases such as leaf spot, root rot, and mildew of crops such as legumes, cereals, melons, solanaceae, and fruits and vegetables. The strain combination Bac365, Mic250, and Ent274 with HA production has the function of chelating water molecules and locking water, and can effectively conserve water and moisture in soil, thereby improving the water utilization efficiency in drought soil. The strain combination Bac365, Mic250, and Pse350 significantly promotes the growth of alfalfa in sandy soil, and the application of Bac365, Mic250, and Pse350 alone significantly promotes the growth of soybean, and has the effects of strengthening roots, promoting seedlings, and increasing yield. The above four plant probiotics provide bacterial resources for agricultural application, environmental protection, and disease control, and have good application prospects.

[0227] The application has been described in detail above. For those skilled in the art, the application can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the purpose and scope of the application, and without unnecessary experiments. Although the application gives special examples, it should be understood that the application can be further improved. In summary, according to the principle of the application, the present application is intended to include any change, use or improvement of the application, including changes made by conventional techniques known in the art, which deviates from the scope disclosed in the present application.

Claims

1. A compound microbial agent, characterized in that: The compound microbial agent is compound microbial agent 1 or compound microbial agent 2; The active ingredients of the compound microbial agent 1 are Bacillus tektii, Microbacterium neemophilus, and Enterococcus querceae. The active ingredients of the compound microbial agent 2 are Bacillus tekirae, Microbacterium neemophilus, and Pseudomonas proteus. The Bacillus tequilensis mentioned is strain number Bac365, and its registration number at the Guangdong Provincial Center for Microbial Culture Collection is GDMCC No:65241. The neem tree microbacterium is Microbacterium azadirachtae, strain number Mic250, and its registration number at the Guangdong Provincial Microbial Culture Collection Center is GDMCC No:65244. The Quebec Enterococcus quebecensis strain is numbered Ent274 and its registration number at the Guangdong Provincial Center for Microbial Culture Collection is GDMCC No:65242. The *Pseudomonas plecoglossicida* strain is designated Pse350 and its registration number at the Guangdong Provincial Center for Microbial Culture Collection is GDMCC No:65243.

2. The compound microbial agent according to claim 1, characterized in that: In the compound microbial agent 1, the CFU ratio of Bacillus tekirae, Microbacterium neemophilus, and Enterococcus querceae is 1:1:1; and / or In the compound microbial agent 2, the CFU ratio of Bacillus tekirae, Microbacterium neemophilus, and Pseudomonas proteus is 1:1:

1.

3. The compound microbial agent according to claim 1 or 2, characterized in that: The compound microbial agent 1 is a microbial agent used for water retention and / or sand fixation; and / or The compound microbial agent 2 is a microbial agent used to promote plant growth.

4. A bacterial strain or a single bacterial agent containing the said bacterial strain as an active ingredient; The strain is any one of the following: (A1) Bacillus tequilensis, strain number Bac365, is registered in Guangdong Provincial Microbial Culture Collection Center with registration number GDMCC No:65241; (A2) Microbacterium azadirachtae, strain number Mic250, is registered in Guangdong Provincial Microbial Culture Collection Center with GDMCC No:65244. (A3) Enterococcus quebecensis, strain number Ent274, is registered in Guangdong Provincial Microbial Culture Collection Center with GDMCC No:65242; (A4) Pseudomonas plecoglossicida, strain number Pse350, is registered in the Guangdong Provincial Microbial Culture Collection Center with the entry number GDMCC No:65243.

5. The application of the compound microbial agent according to any one of claims 1-3 is as follows: (B1) The application of the compound microbial agent 1 according to any one of claims 1-3 in water retention and / or sand fixation; (B2) The use of the compound microbial agent 1 according to any one of claims 1-3 in the preparation of products for water retention and / or sand fixation; (B3) The use of the compound microbial agent 1 according to any one of claims 1-3 in the production of hyaluronic acid; (B4) The use of the compound microbial agent 1 according to any one of claims 1-3 in the preparation of products for producing hyaluronic acid; (B5) The application of the compound microbial agent 2 according to any one of claims 1-3 in promoting plant growth; (B6) The use of the compound microbial agent 2 according to any one of claims 1-3 in the preparation of products for promoting plant growth; (B7) The use of the compound microbial agent 2 according to any one of claims 1-3 in the production of indoleacetic acid; (B8) The use of the compound microbial agent 2 according to any one of claims 1-3 in the preparation of products for the production of indoleacetic acid.

6. Any of the following applications: (C1) The use of Bacillus tekirae as described in claim 4 in cellulase production; (C2) The use of Bacillus tekirae as described in claim 4 in the preparation of products for producing cellulase; (C3) The use of Bacillus tekirae as described in claim 4 in nitrogen fixation; (C4) The use of Bacillus tekirae as described in claim 4 in the preparation of products for nitrogen fixation; (C5) The use of Bacillus tekirii as described in claim 4 in inhibiting pathogens; (C6) The use of Bacillus tekirae as described in claim 4 in the preparation of products for inhibiting pathogens; (C7) The use of Bacillus tekirae as described in claim 4 in the production of indoleacetic acid; (C8) The use of Bacillus tekirae as described in claim 4 in the preparation of products for the production of indoleacetic acid; (C9) The use of Bacillus tekirae as described in claim 4 in the production of hyaluronic acid; (C10) The use of Bacillus tekirae as described in claim 4 in the preparation of products for the production of hyaluronic acid; (C11) The use of Bacillus tekirae as described in claim 4 in promoting plant growth; (C12) The use of Bacillus tekirae as described in claim 4 in the preparation of products for promoting plant growth; (C13) The application of the *Melia azedarach* microbacterium as described in claim 4 in nitrogen fixation; (C14) The use of the *Melia azedarach* microbacterium as described in claim 4 in the preparation of products for nitrogen fixation; (C15) The use of the *Melia azedarach* microbacterium as described in claim 4 in the production of indoleacetic acid; (C16) The use of the *Melia azedarach* microbacterium as described in claim 4 in the preparation of products for the production of indoleacetic acid; (C17) The use of the *Melia azedarach* microbacterium as described in claim 4 in the production of hyaluronic acid; (C18) The use of the *Melia azedarach* microbacterium as described in claim 4 in the preparation of products for the production of hyaluronic acid; (C19) The application of the neem microbacterium as described in claim 4 in promoting plant growth; (C20) The use of the *Melia azedarach* microbacterium as described in claim 4 in the preparation of products for promoting plant growth; (C21) The use of Enterococcus querceae as described in claim 4 in the dissolution of inorganic phosphorus; (C22) The use of Enterococcus querceae as described in claim 4 in the preparation of products for dissolving inorganic phosphorus; (C23) The use of Enterococcus querceae as described in claim 4 in nitrogen fixation; (C24) The use of Enterococcus querceae as described in claim 4 in the preparation of products for nitrogen fixation; (C25) The use of Enterococcus querceae as described in claim 4 in the production of hyaluronic acid; (C26) The use of Enterococcus querceae as described in claim 4 in the preparation of products for the production of hyaluronic acid; (C27) The use of the *Pseudomonas mutans* as described in claim 4 in the dissolution of inorganic and / or organic phosphorus; (C28) The use of the *Pseudomonas mutans* as described in claim 4 in the preparation of products for dissolving inorganic and / or organic phosphorus; (C29) The use of Pseudomonas proteus as described in claim 4 in cellulase production; (C30) The use of the *Pseudomonas mutans* as described in claim 4 in the preparation of products for producing cellulase; (C31) The use of Pseudomonas proteus as described in claim 4 in nitrogen fixation; (C32) The use of the *Pseudomonas mutans* as described in claim 4 in the preparation of products for nitrogen fixation; (C33) The use of Pseudomonas mutans as described in claim 4 in the production of indoleacetic acid; (C34) The use of the *Pseudomonas mutans* as described in claim 4 in the preparation of products for the production of indoleacetic acid; (C35) The use of Pseudomonas proteus as described in claim 4 in promoting plant growth; (C36) The use of the *Pseudomonas proteus* as described in claim 4 in the preparation of products for promoting plant growth.

7. The application according to claim 6, characterized in that: In (C5) and (C6), the pathogen is any one of the following: Fusarium oxysporum, Fusarium solani, Aspergillus flavus, Aspergillus niger, Epicoccumlatusicollum, Sarocladium oryzae, and Pestalotiopsis mangiferae.

8. A method for water conservation and / or sand fixation, comprising the following steps: planting plants in the area to be water-conserved and / or sand-fixed and applying the compound microbial agent 1 as described in any one of claims 1-3, thereby achieving water conservation and / or sand fixation.

9. A method for promoting plant growth, comprising the following steps: applying the compound microbial agent 2 as described in any one of claims 1-3 to the rhizosphere soil of the plant, thereby promoting the growth of the plant.

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