Compound microbial agent for decomposing insoluble phosphorus in soil and application of compound microbial agent

Abstract

The invention provides a composite microbial agent for decomposing insoluble phosphorus in soil and application thereof, and relates to the technical field of microorganisms, the active ingredients of the composite microbial agent comprise Achromobacter xylosoxidans, Burkholderia cepacia and Burkholderia parafungi, the Achromobacter xylosoxidans, the Burkholderia cepacia and the Burkholderia parafungi all have dissolving capacity for Ca3 (PO4) 2, FePO4, AlPO4, lecithin and calcium phytate, and the composite microbial agent can be used for decomposing insoluble phosphorus in soil. And the five insoluble phosphorus sources can be synergistically dissolved. The compound microbial agent is subjected to shake culture, the phosphate-solubilizing activity can reach 53.32 mg.L <-1 >, the phosphate-solubilizing function is stable, meanwhile, the compound microbial agent has the characteristics of degrading lignin and generating siderophore and indoleacetic acid, and the compound microbial agent has remarkable phosphate-solubilizing and growth-promoting effects when being applied to the vicinity of plant rhizosphere soil; the technical problem that in the prior art, the variety of phosphorus dissolving sources of phosphate solubilizing bacteria is single or the stability of the phosphate solubilizing function is poor is solved.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a composite microbial agent for decomposing soil insoluble phosphorus and application thereof. Background Art

[0002] Phosphorus is an essential nutrient for plant growth and development. Although the total phosphorus content in the soil is not low, most of the phosphorus is adsorbed and fixed by oxides such as calcium, iron, and aluminum, or exists in the form of insoluble organic phosphorus. 43% of the world's terrestrial ecosystems are subject to strong phosphorus restrictions, and the application of chemical fertilizers is an important phosphorus-increasing measure in modern agricultural and forestry management. However, the utilization rate of phosphate fertilizers in the season is usually only 10% to 25%, and excessive application of chemical fertilizers will cause economic losses, soil degradation, and environmental pollution. Therefore, fully activating the huge phosphorus pool in the soil is a low-cost and sustainable strategy to reduce the application of phosphate fertilizers and improve the biological effectiveness of soil phosphorus.

[0003] The effective methods reported to increase phosphorus include engineering technology, chemical technology and biotechnology, among which biotechnology is considered to be both economical and effective. Microorganisms play an important role in regulating soil phosphorus cycle and improving phosphorus bioavailability. Phosphate-solubilizing microorganisms can gradually degrade and mineralize organic phosphorus by releasing various phosphatases, or reduce soil pH by secreting organic acids and protons, thereby increasing the dissolution of inorganic phosphorus. By tapping into the resources of soil phosphate-solubilizing bacteria and using them to activate the soil phosphorus pool, it is beneficial to the benign cycle of phosphorus nutrients in the ecosystem. The phosphate-solubilizing bacteria reported today are mainly concentrated in the genera Bacillus, Pseudomonas, Burkholderia and Enterobacter. However, most of the phosphate-solubilizing bacteria that are effective in the laboratory currently face many challenges in production practice, especially the single type of dissolved phosphorus source or the poor stability of the phosphate-solubilizing function. Summary of the invention

[0004] One of the purposes of the present invention is to provide a composite microbial agent for decomposing insoluble phosphorus in soil, so as to solve the technical problems in the prior art that the types of phosphorus sources solubilized by phosphate-solubilizing bacteria are single or the stability of the phosphorus-solubilizing function is poor.

[0005] The second object of the present invention is to provide a method for preparing the above-mentioned composite microbial agent.

[0006] The third object of the present invention is to provide a product.

[0007] A fourth object of the present invention is to provide the use of the above-mentioned composite microbial agent, the composite microbial agent prepared by the above-mentioned preparation method, the above-mentioned Achromobacter xylosoxidans, the above-mentioned Burkholderia cepacia or the above-mentioned parafungus Burkholderia in decomposing insoluble phosphorus, preparing products for decomposing insoluble phosphorus, producing indoleacetic acid, producing siderophores, degrading lignin or promoting plant growth.

[0008] A fifth object of the present invention is to provide a method for promoting the release of sparingly soluble phosphorus in soil.

[0009] A sixth object of the present invention is to provide a method for promoting plant growth.

[0010] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0011] In a first aspect, the present invention provides a composite microbial agent for decomposing soil insoluble phosphorus, wherein the active ingredients include Achromobacter xylosoxidans, Burkholderia cepacia and Burkholderia paramycoides;

[0012] The Achromobacter xylosoxidans is Achromobacter xylosoxidans zafu-3, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO.M20241954;

[0013] The Burkholderia fungorum is Parabukholderia fungorum zafu-N9, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO.M20241955;

[0014] The Burkholderia cepacia is Burkholderia cepacia zafu-111, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO.M20241956.

[0015] Furthermore, the ratio of the number of live bacteria of the Achromobacter xylosoxidans zafu-3, the Burkholderia paramycoides zafu-N9 and the Burkholderia cepacia zafu-111 is (344-550):(238-252):(2.82-3.52);

[0016] Preferably, the concentration of Achromobacter xylose oxidans zafu-3 is 3.44-5.05×10 12 CFU·mL -1 ;

[0017] The concentration of the parafungus Burkholderia zafu-N9 is 2.38-2.52×10 12 CFU·mL -1 ;

[0018] The concentration of the Burkholderia cepacia zafu-111 is 2.82-3.52×1010 CFU·mL -1 ;

[0019] Preferably, the ratio of the number of live bacteria of the Achromobacter xylosoxidans zafu-3, the Burkholderia paramycoides zafu-N9 and the Burkholderia cepacia zafu-111 is 447:245:3.17.

[0020] In a second aspect, the present invention provides a method for preparing the above-mentioned composite microbial agent, comprising the following steps: mixing the bacterial liquid of Achromobacter xylosoxidans zafu-3, the bacterial liquid of Burkholderia paramycota zafu-N9 and the bacterial liquid of Burkholderia cepacia zafu-111 according to a formula amount to obtain a composite microbial agent.

[0021] Furthermore, the preparation method of the bacterial solution includes taking the bacterial solution in the logarithmic phase, discarding the supernatant after centrifugation, and resuspending it with sterile water to obtain an OD 600 = 0.1 bacterial solution;

[0022] Preferably, the mixing of the bacterial solution of Achromobacter xylose oxidans zafu-3, the bacterial solution of Burkholderia cepacia zafu-111 and the bacterial solution of Burkholderia parafungi zafu-N9 according to a formula comprises mixing the bacterial solution of Achromobacter xylose oxidans zafu-3, the bacterial solution of Burkholderia cepacia zafu-111 and the bacterial solution of Burkholderia parafungi zafu-N9 according to a formula, culturing with shaking to a logarithmic phase, and then inoculating into a fresh liquid culture medium and culturing to an OD of 0. 600 =0.32±0.02, the number of cells was 6.95×10 12 ±1.10×10 12 CFU·mL -1 , centrifuge and discard the supernatant, and resuspend with an equal amount of sterile water to obtain a composite microbial agent;

[0023] Preferably, the phosphate solubilizing activity of the composite microbial agent is 53.32±4.74 mg·L -1 ;

[0024] Preferably, the centrifugation conditions include 23-27°C, 2500-4200 rpm for 8-12 min, preferably 25°C, 4000 rpm for 10 min;

[0025] Preferably, the liquid culture medium is a tryptone soy broth liquid culture medium;

[0026] Preferably, the shaking culture conditions include 28-32°C, 150-200 rpm shaking culture, preferably 30°C, 180 rpm shaking culture;

[0027] Preferably, the bacterial solution of Achromobacter xylosoxidans zafu-3, the bacterial solution of Burkholderia cepacia zafu-111 and the bacterial solution of Burkholderia paramycoides zafu-N9 are mixed in a volume ratio of 1:1:1;

[0028] Preferably, the inoculation amount is 1-3%, preferably 2%.

[0029] In a third aspect, the present invention provides a product, the active ingredients of which include the above-mentioned composite microbial agent, the composite microbial agent prepared by the above-mentioned preparation method, the above-mentioned Achromobacter xylosoxidans, the above-mentioned Burkholderia cepacia or the above-mentioned Burkholderia paramycota.

[0030] In a fourth aspect, the present invention provides the use of the composite microbial agent, the composite microbial agent prepared by the above preparation method, or the above product in any of the following:

[0031] (1) Decompose insoluble phosphorus;

[0032] (2) Producing indoleacetic acid;

[0033] (3) siderophore production;

[0034] (4) Degradation of lignin;

[0035] (5) Promote plant growth.

[0036] Furthermore, the poorly soluble phosphorus includes poorly soluble inorganic phosphorus and poorly soluble organic phosphorus;

[0037] Preferably, the sparingly soluble inorganic phosphorus comprises at least one of Ca3(PO4)2, FePO4 and AlPO4;

[0038] Preferably, the poorly soluble organic phosphorus comprises lecithin and / or calcium phytate.

[0039] Further, the promoting plant growth is embodied in at least one of the following:

[0040] (1) Promote the increase of plant biomass;

[0041] (2) Promote the increase of plant aboveground biomass;

[0042] (3) Promote the increase of underground biomass of plants.

[0043] In a fifth aspect, the present invention provides a method for promoting the release of insoluble phosphorus in soil, comprising applying the above-mentioned composite microbial agent, the composite microbial agent prepared by the above-mentioned preparation method, or the above-mentioned product to the soil.

[0044] In a sixth aspect, the present invention provides a method for promoting plant growth, characterized in that it comprises applying the composite microbial agent, the composite microbial agent prepared by the above preparation method, or the above product to plants;

[0045] Preferably, the application method is root irrigation.

[0046] The present invention provides a composite microbial agent for decomposing soil insoluble phosphorus. The three strains of bacteria have the ability to dissolve Ca3(PO4)2, FePO4, AlPO4, lecithin and calcium phytate, and can synergistically dissolve the above five insoluble phosphorus sources. After oscillation culture, the composite microbial agent has a phosphorus-dissolving activity of 53.32 mg·L -1 , and the phosphorus solubilizing function is stable, and it has the characteristics of degrading lignin, producing siderophores and indoleacetic acid, and has a significant phosphorus solubilizing and growth-promoting effect when applied near the rhizosphere soil of plants. The technical problems of the existing technology that the types of phosphorus solubilizing bacteria that dissolve phosphorus sources are single or the stability of phosphorus solubilizing function is poor are solved. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0048] Figure 1 The colony morphology and cell morphology under an optical microscope of the strain provided in Example 1 of the present invention;

[0049] Figure 2 The developmental trees of strains zafu-3, zafu-N9 and zafu-111 constructed based on 16S rRNA sequences provided in Example 1 of the present invention;

[0050] Figure 3 Phosphate solubilization zones of the strains zafu-3, zafu-N9 and zafu-111 provided in Example 2 of the present invention on culture media with calcium phytate, lecithin and calcium phosphate as phosphorus sources, respectively;

[0051] Figure 4 The growth curves of strains zafu-3, zafu-N9 and zafu-111 provided in Example 3 of the present invention;

[0052] Figure 5 The growth curve and phosphate-solubilizing activity of the composite phosphate-solubilizing bacteria provided in Example 4 of the present invention;

[0053] Figure 6The phosphate solubilizing activity of the single-strain bacterial agent, the double-strain composite bacterial agent and the three-strain composite bacterial agent provided in Example 5 of the present invention, and the solubilizing capacity of the three-strain composite bacterial agent on five kinds of insoluble phosphorus;

[0054] Figure 7 The developmental trees of strains Sho-32, Sho-29, Sho-13, Sho-17 and Sho-25 constructed based on 16S rRNA sequences provided in Example 5 of the present invention;

[0055] Figure 8 A comparison chart of the phosphate solubilization ability of the three-strain composite bacterial agent provided in Example 5 of the present invention and the composite bacterial agent after replacing the same genus comparison strain;

[0056] Fig. 9 The bacterial concentration, acid phosphatase activity and pH in the culture solution of the single-strain bacterial agent, the double-strain composite bacterial agent and the three-strain composite bacterial agent provided in Example 5 of the present invention;

[0057] Fig.10 A comparison chart showing the effects of temperature, resource concentration and culture generation on the phosphorus solubilization capacity of a three-strain composite bacterial agent provided in Example 6 of the present invention;

[0058] Fig.11 A comparison chart showing the effects of temperature, resource concentration and evolutionary generation on the phosphorus solubilization ability of the composite bacterial agent after replacing the same genus comparison strain provided in Example 6 of the present invention;

[0059] Fig.12 A comparison chart of the ability of strains zafu-3, zafu-N9 and zafu-111 to produce IAA, produce siderophores and degrade lignin provided in Example 8 of the present invention;

[0060] Fig.13 A statistical graph showing changes in total phosphorus and available phosphorus content in soil treated with different phosphate-solubilizing bacteria inoculations provided in Example 10 of the present invention;

[0061] Fig.14 This is a statistical graph of the changes in aboveground biomass and underground biomass of bamboo treated with different phosphate-solubilizing bacteria inoculations provided in Example 10 of the present invention. DETAILED DESCRIPTION

[0062] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0063] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0064] On one hand, the present invention provides a composite microbial agent for decomposing soil insoluble phosphorus, wherein the active ingredients include Achromobacter xylosoxidans, Burkholderia cepacia and Burkholderia paramycoides;

[0065] The Achromobacter xylosoxidans is Achromobacter xylosoxidans zafu-3, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO.M20241954; the Burkholderia fungorum is Burkholderia fungorum zafu-N9, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO.M 20241955; and the Burkholderia cepacia is Burkholderia cepacia zafu-111, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO.M 20241956.

[0066] Storage Instructions:

[0067] Bacterial species name: Achromobacter xylosoxidans zafu-3;

[0068] Latin name: Achromobacter xylosoxidans zafu-3;

[0069] Depository: China Center for Type Culture Collection;

[0070] Abbreviation of depository institution: CCTCC;

[0071] Address: Wuhan University, Wuhan, Hubei Province;

[0072] Deposit date: September 11, 2024;

[0073] Collection Center Collection Number: CCTCC NO:M 20241954.

[0074] Species name: Burkholderia paramycoides zafu-N9;

[0075] Latin name: Parabukholderia fungorum zafu-N9;

[0076] Depository: China Center for Type Culture Collection;

[0077] Abbreviation of depository institution: CCTCC;

[0078] Address: Wuhan University, Wuhan, Hubei Province;

[0079] Deposit date: September 11, 2024;

[0080] Collection Center Collection Number: CCTCC NO:M 20241955.

[0081] Bacteria species name: Burkholderia cepacia zafu-111;

[0082] Latin name: Burkholderia cepacia zafu-111;

[0083] Depository: China Center for Type Culture Collection;

[0084] Abbreviation of depository institution: CCTCC;

[0085] Address: Wuhan University, Wuhan, Hubei Province;

[0086] Deposit date: September 11, 2024;

[0087] Collection Center Collection Number: CCTCC NO:M 20241956.

[0088] Achromobacter xylosoxidans zafu-3, Burkholderia paramycoides zafu-N9 and Burkholderia cepacia zafu-111 were able to independently decompose five insoluble phosphorus sources, namely Ca3(PO4)2, FePO4, AlPO4, lecithin and calcium phytate; the three strains could synergistically dissolve the above five insoluble phosphorus sources. After oscillating culture, the phosphorus-solubilizing activity of the composite microbial agent could reach 53.32 mg·L -1 , and the phosphorus solubilizing function is stable. It also has the characteristics of degrading lignin, producing siderophores and indoleacetic acid. When applied to the rhizosphere of plants growing in barren and fertile soils, it has a significant phosphorus solubilizing and growth-promoting effect. This solves the technical problems in the prior art that the types of phosphorus solubilizing bacteria that solubilize phosphorus sources are single or the stability of phosphorus solubilizing function is poor.

[0089] In some specific embodiments, the ratio of the number of live bacteria of the Achromobacter xylosoxidans zafu-3, the Burkholderia zafu-N9 and the Burkholderia cepacia zafu-111 is (344-550):(238-252):(2.82-3.52), and may preferably be 447:245:3.17.

[0090] In some specific embodiments, the concentration of Achromobacter xylosoxidans zafu-3 is 3.44-5.05×10 12 CFU·mL -1 The concentration of the parafungus Burkholderia zafu-N9 is 2.38-2.52×10 12 CFU·mL -1 The concentration of the Burkholderia cepacia zafu-111 is 2.82-3.52×10 10 CFU·mL -1 .

[0091] According to another aspect of the present invention, a method for preparing the above-mentioned composite microbial agent is also provided, comprising the following steps of mixing the bacterial solution of Achromobacter xylosoxidans zafu-3, the bacterial solution of Burkholderia parafungi zafu-N9 and the bacterial solution of Burkholderia cepacia zafu-111 according to a formula amount to obtain a composite microbial agent.

[0092] The strains are prepared into bacterial liquids respectively and mixed in a certain proportion, so that the concentration of each strain can be accurately controlled, and the prepared composite microbial agent has the effect of solubilizing phosphate and promoting growth.

[0093] In some specific embodiments, the method for preparing the bacterial solution comprises taking a bacterial solution in the logarithmic phase, discarding the supernatant after centrifugation, and resuspending the solution with sterile water to obtain an OD 600 =0.1 bacterial solution.

[0094] The logarithmic phase is the period when the number of microbial cells increases geometrically. At this time, the growth rate constant is the largest and the cell metabolism is vigorous. Selecting cells from this period to prepare microbial agents can improve the effectiveness of the active ingredients of the microbial agents and enhance their phosphate-solubilizing and growth-promoting effects.

[0095] In some specific embodiments, the mixing of the bacterial solution of Achromobacter xylose oxidans zafu-3, the bacterial solution of Burkholderia cepacia zafu-111 and the bacterial solution of Burkholderia parafungi zafu-N9 according to the formula comprises mixing the bacterial solution of Achromobacter xylose oxidans zafu-3, the bacterial solution of Burkholderia cepacia zafu-111 and the bacterial solution of Burkholderia parafungi zafu-N9 according to the formula, oscillating and culturing to the logarithmic phase, and then inoculating into a fresh liquid culture medium and culturing to OD 600 =0.32±0.02, the number of cells was 6.95×10 12 ±1.10×10 12 CFU·mL -1 , centrifuge and discard the supernatant, and resuspend with an equal amount of sterile water to obtain the composite microbial agent.

[0096] In some specific embodiments, the phosphate solubilizing activity of the composite microbial agent is 53.32±4.74 mg·L -1 .

[0097] The centrifugation conditions include 23-27°C, 2500-4200 rpm for 8-12 min, preferably 25°C, 4000 rpm for 10 min. The preparation method of the bacterial solution in the logarithmic phase includes inoculating the strain into a liquid culture medium and oscillating and culturing it to the logarithmic phase.

[0098] In some specific embodiments, the liquid culture medium is tryptone soy broth (TSB) medium; the shaking culture conditions include 28-32° C., 150-200 rpm shaking culture, preferably 30° C., 180 rpm shaking culture.

[0099] In some specific embodiments, the bacterial solution of Achromobacter xylosoxidans zafu-3, the bacterial solution of Burkholderia cepacia zafu-111 and the bacterial solution of Burkholderia paramycoides zafu-N9 are mixed in a volume ratio of 1:1:1.

[0100] In some specific embodiments, the inoculation amount is 1-3%, preferably 2%.

[0101] According to another aspect of the present invention, a product is also provided, wherein the active ingredients thereof include the above-mentioned composite microbial agent, the composite microbial agent prepared by the above-mentioned preparation method, the above-mentioned Achromobacter xylosoxidans, the above-mentioned Burkholderia cepacia or the above-mentioned Burkholderia paramycota.

[0102] Specifically, the product can be a microbial agent, a bio-organic fertilizer or a soil conditioner, etc., which is used to decompose insoluble phosphorus, produce indoleacetic acid (IAA), produce siderophores, degrade lignin or promote plant growth.

[0103] According to another aspect of the present invention, there is also provided the use of the composite microbial agent, the composite microbial agent prepared by the above preparation method or the above product in any of the following items:

[0104] (1) Decompose insoluble phosphorus;

[0105] (2) Producing indoleacetic acid;

[0106] (3) siderophore production;

[0107] (4) Degradation of lignin;

[0108] (5) Promote plant growth.

[0109] Specifically, the insoluble phosphorus includes insoluble inorganic phosphorus and insoluble organic phosphorus; the insoluble inorganic phosphorus includes at least one of Ca3(PO4)2, FePO4 and AlPO4; the insoluble organic phosphorus includes lecithin and / or calcium phytate;

[0110] In some specific embodiments, the promoting plant growth is embodied in at least one of the following:

[0111] (1) Promote the increase of plant biomass;

[0112] (2) Promote the increase of plant aboveground biomass;

[0113] (3) Promote the increase of underground biomass of plants.

[0114] According to another aspect of the present invention, a method for promoting the release of sparingly soluble phosphorus in soil is also provided, wherein the composite microbial agent, the composite microbial agent prepared by the preparation method, or the product is applied to the soil to increase the content of available phosphorus and total phosphorus in the soil.

[0115] According to another aspect of the present invention, a method for promoting plant growth is also provided, wherein the composite microbial agent, the composite microbial agent prepared by the preparation method, or the product is applied to plants. When used to promote plant growth, the agent is applied to the soil in contact with the plant root system. By applying the agent to the soil near the rhizosphere of the plant, the available phosphorus and total phosphorus content of the soil within the root contact range can be locally increased, thereby promoting plant growth.

[0116] Specifically, root irrigation method can be adopted.

[0117] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0118] Example 1 High-throughput separation, screening and identification of phosphate-solubilizing bacteria

[0119] 1. High-throughput separation and screening of phosphate-solubilizing bacteria

[0120] The root sample with soil was placed in a centrifuge tube filled with 20 mL of sterile water and shaken at 30 °C and 180 rpm for 15 min. The resulting solution was the rhizosphere soil microbial suspension. The soil suspension was diluted by continuous gradient dilution to obtain dilutions of different dilution multiples, with the dilution multiple being 10 times that of the soil suspension. -2 , 10 -3 , 10 -4 and 10 -5 times.

[0121] Three kinds of Pikovskaya inorganic phosphorus media (calcium phosphate, iron phosphate, aluminum phosphate) and two kinds of Mongina organic phosphorus media (Mongina medium, MG) (lecithin, calcium phytate) were used as screening media (Table 1), and the phosphate-solubilizing bacteria in the rhizosphere of bamboo were screened by microplate high-throughput separation and screening method. Specifically, 15 μL of dilution was respectively loaded into 96-well cell culture plates containing 135 μL of 10% screening medium, 1% 10mM bromocresol purple was used as an indicator, and each gradient of dilution was transferred to 3 cell culture plates, and the screening medium with an equal amount of sterile water was added as a negative control. After incubation of the microplate for 2 weeks, the wells that were obviously turbid after culture were selected, and their culture fluid was coated on the corresponding screening medium. After culturing at 30°C for 7 days, the colonies with phosphate-solubilizing circles and obvious morphological differences on the separation medium were separated, and the single colonies were inoculated into solid culture medium for streaking separation to purify them. The experiment eventually produced three strains of phosphate-solubilizing bacteria, which were named zafu-3, zafu-N9 and zafu-111.

[0122] Table 1 PVK, MG and mixed phosphorus source culture medium formula

[0123]

[0124] 2. Morphological identification

[0125] The strains zafu-3, zafu-N9 and zafu111 were inoculated on TSA medium at 30°C and cultured in the dark for 3 days, and then their colony characteristics were observed. The strains were cultured in TSB medium at 30°C and 180 rpm with shaking for 3 days, and then their cell morphology was observed under an optical microscope.

[0126] The results are as follows Figure 1 As shown in the figure (scale bar is 20 μm), strains zafu-3, zafu-N9 and zafu-111 are all Gram-negative bacteria, with coccoid cells and sizes of 1.25 μm × 1.75 μm, 1.45 μm × 1.45 μm and 1.52 μm × 2.99 μm, respectively. The colonies of the three strains are all light yellow, opaque, round, convex in the middle, smooth on the surface, and with regular edges.

[0127] 3. Molecular identification

[0128] The genome of the isolated phosphate-solubilizing bacteria was extracted using a bacterial genome kit (SPARKeasy Bacteria DNA Kit, Shandong Sparkjet Co., Ltd., China), and its concentration was detected by Nanodrop spectrophotometer. The extracted DNA was sent to Zhejiang Shangya Biotechnology Co., Ltd. for sequencing. The primer sequences were 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-CTACGGCTACCTTGTTACGA-3′). The sequencing results were subjected to BLAST analysis in the NCBI database, and then the phylogenetic tree was constructed using the neighbor method in MEGA software to determine the species of the strain.

[0129] Phylogenetic tree Figure 2 As shown, the results showed that strains zafu-3, zafu-N9 and zafu-111 formed independent and stable branches with Achromobacter xylosoxidans, Parabukholderia fungorum and Burkholderia cepacia, respectively, and the homology of gene sequences was more than 99%. It can be seen that zafu-3 is Achromobacter xylosoxidans, zafu-N9 is P. fungorum, and zafu-111 is B. cepacia.

[0130] Example 2 Phosphate-solubilizing ability of phosphate-solubilizing bacteria

[0131] The phosphate solubilization ability of the strains was qualitatively studied using the phosphate solubilization circle method, and the phosphate solubilization ability of each strain was preliminarily determined based on the ratio (D / d) of the phosphate solubilization circle diameter (D) and the colony diameter (d). The molybdenum antimony colorimetric method was used to quantitatively study the phosphate solubilization ability of the strains. Figure 3 The figure shows the phosphate-dissolving circles formed by strains zafu-3, zafu-N9 and zafu-111 on calcium phytate, lecithin and Ca3(PO4)2 medium, respectively. As shown in Table 2, the three strains have a certain solubility for Ca3(PO4)2, FePO4, AlPO4, lecithin and calcium phytate.

[0132] Table 2 Ability of strains zafu-3, zafu-N9 and zafu-111 to dissolve different insoluble phosphorus sources

[0133]

[0134] Example 3 Growth curve of strains

[0135] The growth curves of strains zafu-3, zafu-N9 and zafu-111 were drawn according to the following steps: the strains were inoculated into TSB medium, cultured at 30°C and 180 rpm for 3 h, centrifuged at 4000 rpm for 10 min, the supernatant was removed, the bacteria were resuspended in sterile water, and the bacterial solution concentration was adjusted to OD 600 =0.1 for quantitative analysis. 600 = 0.1 of the bacterial solution was re-inoculated into fresh TSB medium, cultured at 30°C and 180 rpm, and the OD of the bacterial solution was measured every 3h-9h. 600 The value of the strain was calculated by the dilution coating method on the TSA medium to calculate the number of cells (Colony Forming Units, CFU) in the bacterial solution and draw the growth curve of the strain. Each sampling was repeated 3 times, and the UV spectrophotometer was calibrated with the medium without bacteria. The results are shown in Figure 4 As shown, strain zafu-3 grew rapidly in 6 to 24 hours. After 24 hours of culture, the OD 600 =3.21, the number of cells is 3.15×10 15 CFU·mL -1 , and entered the stable phase after 24 hours. The strain zafu-N9 grew rapidly from 9 to 48 hours. After 48 hours of cultivation, the strain OD 600 =3.13, the number of cells is 3.98×10 14 CFU·mL -1 After 48 hours, the strain zafu-111 began to grow rapidly from 6 to 27 hours. After 27 hours of cultivation, the strain OD 600 =3.02, the number of cells is 4.90×10 15 CFU·mL -1 , and entered the stable period after 27 hours.

[0136] Example 4 Preparation of bacterial agent

[0137] 1. Preparation of single-strain inoculants: Three phosphate-solubilizing strains were inoculated into TSB medium, cultured at 30°C and 180 rpm to the logarithmic phase, centrifuged at 25°C and 4000 rpm for 10 min, discarded the supernatant and resuspended with an equal amount of sterile water to prepare the inoculant, or the concentration of the bacterial solution was adjusted to OD with sterile water. 600 =0.1, used for the preparation of composite bacterial agents.

[0138] 2. Preparation of dual-strain composite bacterial agent: inoculate 100 μL of single-strain bacterial solution in equal proportion (1:1 / V:V) into fresh TSB medium, culture at 30°C and 180 rpm with shaking until the logarithmic phase, centrifuge at 4000 rpm for 10 min, discard the supernatant and resuspend the bacteria in an equal amount of sterile water to prepare the bacterial agent for use.

[0139] 3. Preparation of three-strain composite bacterial agent: 100 μL of single strain bacterial solution was inoculated into fresh TSB medium in equal proportion (1:1:1 / V:V:V), and cultured at 30°C and 180 rpm for 24 h until the logarithmic phase (OD 600 =2.5, the number of cells is about 2.29×10 11 CFU·mL -1 )(like Figure 5 a red time point). The composite bacterial solution was inoculated into a 250 mL conical flask containing TSB medium at a 2% inoculation rate and cultured at 30°C and 180 rpm for 48 h until the composite bacterial agent reached the optimal phosphate solubilization state. At this time, the phosphate solubilization activity was 53.32±4.74 mg·L -1 , O.D. 600 =0.32, the number of cells is 6.95×10 12 ±1.10×10 12 CFU·mL -1 (like Figure 5 b red time point). The composite bacterial agent was transferred to a sterile centrifuge tube and centrifuged at 4000 rpm for 10 min. After discarding the supernatant, the bacteria were resuspended in an equal amount of sterile water to prepare a bacterial agent, in which the cell number of strain zafu-3 was 4.47×10 12 ±1.03×10 12 CFU·mL -1 The cell number of strain zafu-N9 was 2.45×10 12 ±7.00×10 10 CFU·mL -1 The cell number of strain zafu-111 was 3.17×10 10 ±3.51×10 9 CFU·mL -1 .

[0140] Example 5 Phosphate solubilization capacity and pathway of composite bacterial agent

[0141] Take 100 μL of the single-strain bacterial agent, double-strain (1: 1 / V: V) composite bacterial agent or three-strain (1: 1: 1 / V: V: V) composite bacterial agent prepared in Example 4 and inoculate them into a mixed Montana medium (mixed phosphorus source medium) containing 2 mL of the above two kinds of organic phosphorus and three kinds of inorganic phosphorus, and each treatment is repeated 6 times, and sterile water is added as a blank control to analyze the phosphorus solubilization activity of the single-strain bacterial agent, the double-strain composite bacterial agent and the three-strain composite bacterial agent, and the solubility of the three-strain composite bacterial agent for 5 insoluble phosphorus sources. At the same time, another 5 phosphate-solubilizing bacteria obtained by screening and identifying the bamboo rhizosphere soil according to the method of Example 1 were selected as comparison strains, and the strain information is shown in Table 3, and the phylogenetic tree was constructed using the neighbor method. These 5 comparison strains were used to replace one, two or three strains of the three-strain composite bacterial agent as a comparative proportion, and the changes in the phosphorus solubilization ability of the composite bacterial agent were explored. After 3 days of shaking culture at 30°C and 180 rpm, the phosphate solubilization activity of each inoculation treatment was determined by molybdenum antimony colorimetric method, and the bacterial concentration (OD) in the solution after culture of single-strain inoculum, double-strain composite inoculum, and triple-strain composite inoculum was determined. 600 ), phosphatase activity and pH changes, the specific results are as follows Figure 6 to Figure 9 shown.

[0142] Table 3 Other phosphate-solubilizing strains obtained by screening and their phosphate-solubilizing abilities

[0143]

[0144]

[0145] By Figure 6 (The values ​​are mean ± standard deviation (n = 3), and the bars marked with different letters are significantly different (p < 0.05)) It can be seen that the three-strain composite bacterial agent composed of zafu-3, zafu-N9 and zafu-111 has the strongest phosphate solubilization ability, which is 21.05 ± 0.37 mg·L -1 ·d -1 , which is higher than the phosphate solubilization capacity of the three strains inoculated alone and randomly inoculated. Figure 7 and Figure 8 (The values ​​are mean ± standard deviation (n = 3) It can be seen that after replacing any one, two or three of the three strains in the above-mentioned composite bacterial agent with other strains of the same genus, the phosphate solubilization effect of the composite bacterial agent composed of three strains, zafu-3, zafu-N9 and zafu-111, is still significantly higher than that of other groups. The above results indicate that there may be a synergistic effect between the member strains zafu-3, zafu-N9 and zafu-111 of the composite bacterial agent. Fig. 9 (The values ​​are mean ± standard deviation (n = 3), and the bars marked with different letters are significantly different (p < 0.05)) It can be seen that when the three strains were mixed and cultured, the bacterial solution concentration (OD600 ) did not significantly reduce, and even promoted each other's growth; however, the phosphatase activity of the three strains after mixed culture was not the highest among all combinations, indicating that the composite bacterial agent may not mainly rely on the secretion of phosphatase to improve its ability to activate insoluble phosphorus; and the pH of the composite bacterial agent culture system was as low as 2.6, which showed that the phosphate-solubilizing bacteria may increase the dissolution of insoluble phosphorus by secreting organic acids and lowering the pH in the environment.

[0146] Example 6 Stability of the phosphate-solubilizing function of the three-strain composite bacterial agent

[0147] According to the steps and methods of preparing the three-strain composite bacterial agent in Example 4, the effects of culture temperature (25°C, 30°C and 35°C), resource concentration (100% and 10% mixed phosphorus source medium) and evolutionary generations (1 generation, 3 generations, 7 generations and 10 generations) on the phosphorus solubilization ability of the three-strain composite bacterial agent were explored. At the same time, Achromobacter aegrifaciens Sho-32 was used instead of zafu-3, Parabukholderia tropica Sho-13 was used instead of zafu-N9, and Burkholderia sp. Sho-25 was used instead of zafu-111 as controls.

[0148] The strain was cultured for 24 hours as one generation, and 100 μL of the first generation culture solution was pipetted into a 24-well plate containing 2 mL of fresh mixed culture medium, and the culture was continued, and so on, for a total of 10 generations. The molybdenum antimony colorimetric method was used to determine the phosphate solubilization activity of the bacterial agent.

[0149] The results are as follows Fig.10 As shown (values ​​are mean ± standard deviation (n = 3), bars marked with different letters are significantly different (p < 0.05)), where (a) is temperature, (b) is resource concentration, and (c) is evolutionary generation. Temperature, resource concentration and evolutionary process all have significant effects on the phosphate solubilization ability of the bacterial agent. The phosphate solubilization activity of the bacterial agent when cultured at 30°C is 72% and 66% higher than that at 25°C and 35°C, respectively. The phosphate solubilization ability of the bacterial agent grown in 10% culture medium is significantly higher than that in 100% culture medium, which is 22.24 ± 1.50 mg·L -1 ·d -1 As the number of evolutionary generations increased, the interaction time prolonged, and the phosphate solubilization capacity decreased. After 10 generations, its phosphate solubilization capacity was 6.40±0.19 mg·L -1 ·d -1 .like Fig.11(Values ​​are mean ± standard deviation (n = 3), and bars marked with different letters are significantly different (p < 0.05)). Although the phosphate solubilization activity of the three-strain composite agent decreased at 25°C and 35°C, when grown in 100% medium, and after 10 generations of culture, it was still much higher than the control ratio after replacing the same genus control strain. Therefore, the phosphate solubilization agent maintained a high phosphate solubilization effect at different environmental temperatures, resource concentrations, and evolutionary processes, which also shows that its phosphate solubilization ability has a certain stability.

[0150] Example 7 pH, NaCl range and carbon source utilization of three phosphate-solubilizing bacteria

[0151] The pH range (3-11, in increments of 1 unit) and NaCl concentration range (1%-7%, W / V, interval 1%) for strain growth were determined by shaking culture in tryptone soy broth (TSB) medium at 30°C and 180rpm for 3 days. No inoculation was used as a blank control, and each treatment was repeated 6 times. The carbon source utilization of the strain was based on three PVK and two MG culture media without carbon sources to explore the strain's utilization of 15 common carbon sources and the production of phosphate-soluble circles, including five sugars: glucose, fructose, sucrose, cellulose, and xylan, five amino acids: glutamic acid, serine, proline, isoleucine, and lysine, and five organic acids: oxalic acid, malic acid, citric acid, succinic acid, and lactic acid. The carbon source was 5 g·L -1 Each treatment in the above experiment was repeated 3 times, with no inoculation as the blank control, and the analysis was performed after culturing in the dark at 30℃ for 3 days.

[0152] The growth of strains zafu-3(3), zafu-N9(N9) and zafu-111(111) in five phosphorus source media after adding 15 carbon sources are shown in Table 4. When strain zafu-3 used glucose, proline, isoleucine, oxalic acid or succinic acid as the sole carbon source, the strain could grow on all five phosphorus source media, but it was difficult for it to utilize serine and lactic acid. After adding eight carbon sources such as fructose, sucrose or lysine, the strain only grew in some phosphorus source media. When strain zafu-N9 used glucose and succinic acid as the sole carbon source, it could grow on all five phosphorus source media, but it was difficult for it to utilize cellulose, glutamic acid, serine and four organic acids except succinic acid. After adding fructose, sucrose, xylan or isoleucine, the strain only grew in some phosphorus source media. When the strain zafu-111 used glucose, sucrose, isoleucine or succinate as the sole carbon source, the strain could grow on five phosphorus source media, but it was difficult for it to utilize glutamate, serine, oxalic acid, malic acid and lactic acid; after adding six carbon sources such as fructose, xylan or proline, the strain only grew in some phosphorus source media. The results showed that the strain zafu-3 could grow under pH = 4-10 and NaCl concentration below 6%, and the strains zafu-N9 and zafu-111 could grow under pH = 4-9 and NaCl concentration below 4%. The three strains can grow under multiple carbon sources and a wide range of pH and NaCl concentrations, which ensures their functional stability.

[0153] Table 4 Growth of strains on different phosphorus source media after adding different carbon sources

[0154]

[0155]

[0156] Among them, - is negative, +, ++, +++ is positive. * indicates the presence of phosphate-dissolving zone on the culture medium.

[0157] Example 8 Growth-promoting properties of three phosphate-solubilizing bacteria

[0158] The ability of the strain to produce indole acetic acid (IAA) was determined by the Salkowski colorimetric method. After the culture solution of the strain was added with the Salkowski colorimetric solution, the solution turned red, indicating that the strain had the ability to produce IAA. The strain was inoculated on a solid culture medium (Chrome Azurol S CAS). After 3 days of culture, the diameter of the orange iron production ring (D1) and the strain diameter (d1) were observed. The D1 / d1 value was used to indicate the ability of the strain to produce iron carriers. The strain was inoculated on a aniline blue color development medium. After 8 days of culture, the diameter of the dissolution zone (D2) and the strain diameter (d2) were observed. The D2 / d2 value was used to indicate the strain's ability to degrade lignin.

[0159] The results are as follows Fig.12 As shown, strains zafu-3, zafu-N9 and zafu-111 can produce IAA. Strains zafu-N9 and zafu-111 can also produce siderophores, with D1 / d1 of 2.80±0.32 and 1.93±0.44, respectively. The three strains all produced transparent phosphate-dissolving circles on aniline blue medium, indicating that the three strains have the ability to degrade lignin, with D2 / d2 of 1.37±0.04, 1.37±0.11 and 1.70±0.38, respectively.

[0160] Example 9 Root colonization ability of strain members in the composite bacterial agent

[0161] The study on the ability of the strain members in the three-strain composite microbial agent to colonize the root system firstly carried out the culture of sterile bamboo seedlings. Specifically, the surface-sterilized seeds were placed on MS culture medium covered with filter paper, germinated in a 30℃ incubator in the dark, and then transferred to a tissue culture bottle containing 50mL MS culture medium. After being placed in an artificial climate of 25℃ for aseptic culture for 2 weeks, 0.5mL of the composite phosphate-dissolving bacteria agent was inoculated into the roots of the sterile bamboo seedlings. After continuing to culture at 25℃ for 7 days, the roots of the bamboo were screened for strains by the plate coating method, and the number of colonies of each strain was recorded.

[0162] The inoculated composite bacterial agent members were isolated from the rhizosphere and roots of the bamboo seedlings, proving that the three strains of the bacterial agent can successfully colonize the roots of the bamboo. As shown in Table 5, the colonization density of strain zafu-N9 in the rhizosphere was the highest, at 2.4×10 3 CFU·g -1 , which were 1.8 and 1.2 times those of zafu-3 and zafu-111, respectively. The strain zafu-111 had the highest colonization density in the roots, which was 3.3×10 3 CFU·g -1 , which are 2.1 times and 1.1 times that of zafu-3 and zafu-N9 respectively.

[0163] Table 5 Colonization of strains zafu-3, zafu-N9 and zafu-111 in the roots of bamboo seedlings.

[0164] Colonization site zafu-3 zafu-N9 zafu-111 <![CDATA[Rhizosphere (CFU·g -1 )]]> <![CDATA[2.4×10 3 ±1.2×10 2 ]]> <![CDATA[4.5×10 3 ±0.4×10 2 ]]> <![CDATA[3.7×10 3 ±1.0×10 2 ]]> <![CDATA[CFU·g in the root -1 )]]> <![CDATA[1.6×10 3 ±0.8×10 2 ]]> <![CDATA[2.9×10 3 ±0.6×10 2 ]]> <![CDATA[3.3×10 3 ±0.7×10 2 ]]>

[0165] Example 11 Potted Plant Test

[0166] The potted experiment was carried out in a greenhouse. The test soil was collected from a bamboo forest sample plot where phosphate-solubilizing bacteria were isolated. The soil was sieved through a 10-mesh sieve and mixed well for later use.

[0167] The experiment was conducted with two nutrient levels in the soil matrix: forest soil only (nutrient-rich) and forest soil and sand 1:1 (v / v, nutrient-poor).

[0168] The test plants were one-year-old seedlings with the same growth pattern obtained from the seeds of the same strain of bamboo. A single-strain inoculant of three strains, a composite phosphate-dissolving inoculant, and a control group with sterile water were set up. 20 mL of the inoculant was inoculated into the rhizosphere of the bamboo seedlings by root irrigation method. The inoculation was carried out 3 times, with an interval of 3 days each time. The final concentration of the inoculated strains was 10 7 CUF kg -1 After 90 days of cultivation in the greenhouse, bamboo plants and soil were collected, and the aboveground plant and underground root biomass of bamboo were determined by weighing method. After the soil was digested in H2SO4 and HClO4 solution, the total phosphorus content (TP) was determined by molybdenum antimony spectrophotometry. The available phosphorus content (AP) in the soil was determined by Tiessen modified Hedley method.

[0169] The results of the pot test are as follows Fig.13 and Fig.14 As shown in the results, after inoculation with the composite microbial agent under poor soil conditions, the total phosphorus content in the soil decreased and the available phosphorus content increased compared with the control group, but there was no significant difference; under nutrient-rich soil conditions, the available phosphorus content in the soil also increased after inoculation with the composite microbial agent, which showed that the composite microbial agent could activate the soil phosphorus pool and release available phosphorus. Under poor soil conditions, the aboveground biomass and underground biomass of the composite microbial agent treatment increased significantly by 119.54% and 289.40% compared with the uninoculated treatment; and increased significantly by 26.14%-81.22% and 80.65%-133.80% compared with the single strain treatment. Under rich soil conditions, the underground biomass and aboveground biomass of bamboo treated with the composite microbial agent increased significantly by 117.04% and 389.77% compared with the uninoculated treatment; and increased significantly by 50.27%-74.74% and 120.65%-169.38% compared with the single strain treatment. This shows that inoculation with composite phosphate-solubilizing bacteria can promote plant growth regardless of whether the soil is nutrient-poor or nutrient-rich.

[0170] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composite microbial agent for decomposing insoluble phosphorus in soil, characterized in that: Its active ingredients include Achromobacter xylosoxidans, Burkholderia cepacia, and Burkholderia paramycoides; The Achromobacter xylosoxidans is Achromobacter xylosoxidanszafu-3, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO.M 20241954; The Burkholderia fungorum is Parabukholderia fungorum zafu-N9, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO.M20241955; The Burkholderia cepacia is Burkholderia cepacia zafu-111, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO.M 20241956.

2. The composite microbial agent according to claim 1, characterized in that: The ratio of the number of live bacteria of the Achromobacter xylosoxidans zafu-3, the Burkholderia paramycoides zafu-N9 and the Burkholderia cepacia zafu-111 is (344-550):(238-252):(2.82-3.52); Preferably, the concentration of Achromobacter xylose oxidans zafu-3 is 3.44-5.05×10 12 CFU·mL -1 ; The concentration of the parafungus Burkholderia zafu-N9 is 2.38-2.52×10 12 CFU·mL -1 ; The concentration of the Burkholderia cepacia zafu-111 is 2.82-3.52×10 10 CFU·mL -1 ; Preferably, the ratio of the number of live bacteria of the Achromobacter xylosoxidans zafu-3, the Burkholderia paramycoides zafu-N9 and the Burkholderia cepacia zafu-111 is 447:245:3.

17.

3. The method for preparing the composite microbial agent according to claim 1 or 2, characterized in that: The method comprises the following steps: mixing the bacterial liquid of Achromobacter xylose oxidans zafu-3, the bacterial liquid of Burkholderia paramycoides zafu-N9 and the bacterial liquid of Burkholderia cepacia zafu-111 according to a formula amount to obtain a composite microbial agent.

4. The preparation method according to claim 3, characterized in that: The preparation method of the bacterial solution comprises taking a bacterial solution in the logarithmic phase, discarding the supernatant after centrifugation, and resuspending the solution with sterile water to obtain an OD 600 = 0.1 bacterial solution; Preferably, the mixing of the bacterial solution of Achromobacter xylose oxidans zafu-3, the bacterial solution of Burkholderia cepacia zafu-111 and the bacterial solution of Burkholderia parafungi zafu-N9 according to a formula comprises mixing the bacterial solution of Achromobacter xylose oxidans zafu-3, the bacterial solution of Burkholderia cepacia zafu-111 and the bacterial solution of Burkholderia parafungi zafu-N9 according to a formula, culturing with shaking to a logarithmic phase, and then inoculating into a fresh liquid culture medium and culturing to an OD of 0. 600 =0.32±0.02, the number of cells was 6.95×10 12 ±1.10×10 12 CFU·mL -1 , centrifuge and discard the supernatant, and resuspend with an equal amount of sterile water to obtain a composite microbial agent; Preferably, the phosphate solubilizing activity of the composite microbial agent is 53.32±4.74 mg·L -1 ; Preferably, the centrifugation conditions include 23-27°C, 2500-4200 rpm for 8-12 min, preferably 25°C, 4000 rpm for 10 min; Preferably, the liquid culture medium is a tryptone soy broth liquid culture medium; Preferably, the shaking culture conditions include 28-32°C, 150-200 rpm shaking culture, preferably 30°C, 180 rpm shaking culture; Preferably, the bacterial solution of Achromobacter xylosoxidans zafu-3, the bacterial solution of Burkholderia cepacia zafu-111 and the bacterial solution of Burkholderia paramycoides zafu-N9 are mixed in a volume ratio of 1:1:1; Preferably, the inoculation amount is 1-3%, preferably 2%.

5. A product, characterized in that The active ingredients include the composite microbial agent described in claim 1 or 2 or the composite microbial agent prepared by the preparation method described in claim 3 or 4.

6. Use of the composite microbial agent according to claim 1 or 2, the composite microbial agent prepared by the preparation method according to claim 3 or 4, or the product according to claim 5 in any of the following: (1) Decomposition of insoluble phosphorus; (2) Producing indoleacetic acid; (3) siderophore production; (4) Degradation of lignin; (5) Promote plant growth.

7. The use according to claim 6, characterized in that: The insoluble phosphorus includes insoluble inorganic phosphorus and insoluble organic phosphorus; Preferably, the sparingly soluble inorganic phosphorus comprises at least one of Ca3(PO4)2, FePO4 and AlPO4; Preferably, the poorly soluble organic phosphorus comprises lecithin and / or calcium phytate.

8. The use according to claim 6, characterized in that: The plant growth promotion is embodied in at least one of the following: (1) Promote the increase of plant biomass; (2) Promote the increase of plant aboveground biomass; (3) Promote the increase of underground biomass of plants.

9. A method for promoting the release of sparingly soluble phosphorus in soil, characterized in that: The method comprises applying the composite microbial agent described in claim 1 or 2, the composite microbial agent prepared by the preparation method described in claim 3 or 4, or the product described in claim 5 to the soil.

10. A method for promoting plant growth, characterized in that: The method comprises applying the composite microbial agent according to claim 1 or 2, the composite microbial agent prepared by the preparation method according to claim 3 or 4, or the product according to claim 5 to plants; Preferably, the application method is root irrigation.

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