Compound microbial inoculant for decomposing soil insoluble phosphorus and application thereof

Abstract

The application provides a composite microbial agent for decomposing soil insoluble phosphorus and an application thereof, and relates to the technical field of microorganisms. Active ingredients of the composite microbial agent include Xanthomonas campestris, Burkholderia cenocepacia and Burkholderia parafungorum. The three strains have the ability to dissolve Ca3(PO4)2, FePO4, AlPO4, lecithin and calcium phytate, and can synergistically dissolve the above-mentioned five kinds of insoluble phosphorus sources. The phosphorus solubilizing activity of the composite microbial agent after oscillation culture can reach 53.32 mg·L ‑1 , and the phosphorus solubilizing function is stable. Meanwhile, the composite microbial agent has the characteristics of degrading lignin, producing siderophores and indole acetic acid. When applied to the rhizosphere soil of plants, the composite microbial agent has a significant phosphorus solubilizing and growth promoting effect, and solves the technical problems of single type of phosphorus source dissolved by the phosphorus solubilizing bacteria in the prior art or poor stability of the phosphorus solubilizing function.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a compound microbial agent for decomposing insoluble phosphorus in soil and its application. Background Technology

[0002] Phosphorus is an essential nutrient for plant growth and development. Although the total phosphorus content in soil is not low, most of it is adsorbed and fixed by oxides such as calcium, iron, and aluminum, or exists in the form of insoluble organic phosphorus. Globally, 43% of terrestrial ecosystems are subject to significant phosphorus limitation, making fertilizer application a crucial phosphorus-enhancing measure in modern agricultural and forestry management. However, the utilization rate of phosphate fertilizers in the current season is typically only 10%–25%, and excessive application can lead to economic losses, soil degradation, and environmental pollution. Therefore, fully activating the vast phosphorus pool in the soil is a low-cost and sustainable strategy to reduce phosphate fertilizer application and improve soil phosphorus bioavailability.

[0003] Currently reported effective methods for increasing phosphorus levels include engineering, chemical, and biotechnological approaches, with biotechnology considered both economical and effective. Microorganisms play a crucial role in regulating soil phosphorus cycling and enhancing phosphorus bioavailability. Phosphorus-solubilizing microorganisms can gradually degrade and mineralize organic phosphorus by releasing various phosphatases, or increase inorganic phosphorus dissolution by lowering soil pH through the secretion of organic acids and protons. Exploring soil phosphorus-solubilizing microbial resources and utilizing them to activate soil phosphorus pools is beneficial for the healthy cycling of phosphorus nutrients in the ecosystem. Currently reported phosphorus-solubilizing bacteria mainly focus on Bacillus, Pseudomonas, Burkholderia, and Enterobacter. However, most phosphorus-solubilizing bacteria that are effective in the laboratory face numerous challenges in production practice, particularly the issues of limited phosphorus source availability or poor stability of phosphorus-solubilizing function. Summary of the Invention

[0004] One of the objectives of this invention is to provide a composite microbial agent for decomposing insoluble phosphorus in soil, in order to solve the technical problems of existing phosphorus-solubilizing bacteria having a single type of phosphorus source or poor stability of phosphorus-solubilizing function.

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

[0006] The third objective of this invention is to provide a product.

[0007] The fourth objective of this invention is to provide the application of the above-mentioned compound microbial agent, the compound microbial agent prepared by the above-mentioned preparation method, the above-mentioned xylose-oxidizing colorless bacillus, the above-mentioned Burkholderia cepacia, or the above-mentioned parafungi Burkholderia in the decomposition of insoluble phosphorus, the preparation of products for the decomposition of insoluble phosphorus, the production of indoleacetic acid, the production of siderophores, the degradation of lignin, or the promotion of plant growth.

[0008] The fifth objective of this invention is to provide a method for promoting the release of insoluble phosphorus from soil.

[0009] The sixth objective of this invention is to provide a method for promoting plant growth.

[0010] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0011] In a first aspect, the present invention provides a compound microbial agent for decomposing insoluble phosphorus in soil, the active ingredients of which include xylose-oxidizing achromobacterium, Burkholderia cepacia, and parafungi Burkholderia.

[0012] The xylose-oxidizing achromobacter is Achromobacter xylosoxidans zafu-3, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO.M20241954.

[0013] The parafungi Burkholderia is zafu-N9, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO.M20241955.

[0014] The Burkholderia cepacia mentioned is Burkholderia cepacia zafu-111, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO.M20241956.

[0015] Furthermore, the ratio of viable bacteria of *Achromobacterium xyloseoxidans* zafu-3, *Burkholderia parafungi* zafu-N9, and *Burkholderia cepacia* zafu-111 is (344–550):(238–252):(2.82–3.52).

[0016] Preferably, the concentration of the xylose-oxidizing achromobacterium zafu-3 is 3.44–5.05 × 10⁻⁶. 12 CFU·mL -1 ;

[0017] The concentration of the parafungus Burkholderia zafu-N9 was 2.38–2.52 × 10⁻⁶. 12 CFU·mL -1 ;

[0018] The concentration of Burkholderia cepacia zafu-111 was 2.82–3.52 × 10⁻⁶.10 CFU·mL -1 ;

[0019] Preferably, the ratio of viable bacteria of *Achromobacterium xyloseoxidans* zafu-3, *Burkholderia cepacia* zafu-N9, and *Burkholderia cepacia* zafu-111 is 447:245:3.17.

[0020] Secondly, the present invention provides a method for preparing the above-mentioned composite microbial agent, comprising the following steps: mixing the xylose-oxidizing achromobacterium zafu-3 bacterial solution, the parafungi Burkholderia zafu-N9 bacterial solution, and the cephalosporin Burkholderia zafu-111 bacterial solution according to the formula amount to obtain the composite microbial agent.

[0021] Furthermore, the method for preparing the bacterial suspension includes taking the bacterial suspension in the logarithmic growth phase, centrifuging it, discarding the supernatant, and resuspending it in sterile water to obtain the OD. 600 =0.1 bacterial solution;

[0022] Preferably, the step of mixing the *Achromobacterium xyloseoxidans* zafu-3 bacterial suspension, the *Burkholderia cepacia* zafu-111 bacterial suspension, and the *Burkholderia parafungi* zafu-N9 bacterial suspension according to the formula includes mixing the *Achromobacterium xyloseoxidans* zafu-3 bacterial suspension, the *Burkholderia cepacia* zafu-111 bacterial suspension, and the *Burkholderia parafungi* zafu-N9 bacterial suspension according to the formula, shaking and culturing to the logarithmic phase, and then inoculating into fresh liquid culture medium and culturing to OD. 600 =0.32±0.02, cell count was 6.95×10 12 ±1.10×10 12 CFU·mL -1 Centrifuge, discard the supernatant, and resuspend in an equal volume of sterile water to obtain the compound microbial agent;

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

[0024] Preferably, the centrifugation conditions include centrifugation at 2500–4200 rpm for 8–12 minutes at 23–27°C, and more preferably centrifugation at 4000 rpm for 10 minutes at 25°C.

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

[0026] Preferably, the conditions for the shaking culture include shaking culture at 28–32°C and 150–200 rpm, and more preferably at 30°C and 180 rpm.

[0027] Preferably, the xylose-oxidizing achromobacterium zafu-3 bacterial solution, the Burkholderia cepacia zafu-111 bacterial solution, and the Burkholderia parafungi zafu-N9 bacterial solution are mixed in a volume ratio of 1:1:1.

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

[0029] Thirdly, the present invention provides a product whose active ingredients include the above-mentioned compound microbial agent, the compound microbial agent prepared by the above-mentioned preparation method, the above-mentioned xylose-oxidizing achromobacterium, the above-mentioned Burkholderia cepacia, or the above-mentioned parafungi Burkholderia.

[0030] Fourthly, the present invention provides the application of the above-described composite microbial agent, the composite microbial agent prepared by the above-described preparation method, or the above-described product in any of the following:

[0031] (1) Decomposes insoluble phosphorus;

[0032] (2) It produces indoleacetic acid;

[0033] (3) Iron-producing carrier;

[0034] (4) Degradation of lignin;

[0035] (5) Promote plant growth.

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

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

[0038] Preferably, the insoluble organic phosphorus includes lecithin and / or calcium phytate.

[0039] Furthermore, the promotion of plant growth is manifested in at least one of the following:

[0040] (1) Promotes an increase in plant biomass;

[0041] (2) Promotes the increase of aboveground biomass in plants;

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

[0043] Fifthly, the present invention provides a method for promoting the release of insoluble phosphorus from soil, comprising applying the above-mentioned compound microbial agent, the compound 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 includes applying the above-mentioned compound microbial agent, the compound microbial agent prepared by the above-mentioned preparation method, or the above-mentioned product to plants;

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

[0046] This invention provides a composite microbial agent for decomposing insoluble phosphorus in soil. The three strains of bacteria exhibit dissolving abilities for Ca3(PO4)2, FePO4, AlPO4, lecithin, and calcium phytate, and can synergistically dissolve these five insoluble phosphorus sources. After shaking culture, the composite microbial agent achieves a phosphorus-solubilizing activity of up to 53.32 mg·L⁻¹. -1 Furthermore, it exhibits stable phosphorus-solubilizing function and possesses the characteristics of degrading lignin, producing siderophores and indoleacetic acid. When applied near the plant rhizosphere soil, it demonstrates a significant phosphorus-solubilizing and growth-promoting effect. This solves the technical problems of existing technologies where phosphorus-solubilizing bacteria rely on a single phosphorus source or have poor stability in their phosphorus-solubilizing function. Attached Figure Description

[0047] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[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 tree of strains zafu-3, zafu-N9, and zafu-111 constructed based on the 16S rRNA sequence provided in Example 1 of this invention;

[0050] Figure 3 The phosphorus-solubilizing zones of strains zafu-3, zafu-N9, and zafu-111 provided in Example 2 of this 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 compound phosphate-solubilizing bacteria provided in Example 4 of the present invention;

[0053] Figure 6The phosphorus-solubilizing activity of the single-strain inoculant, the two-strain compound inoculant, and the three-strain compound inoculant provided in Example 5 of the present invention, and the solubility of the three-strain compound inoculant for five kinds of poorly soluble phosphorus;

[0054] Figure 7 The developmental tree 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 This is a comparison chart of the phosphorus solubilizing ability of the three-strain compound bacterial agent provided in Example 5 of the present invention and the compound bacterial agent after replacing the control strain of the same genus.

[0056] Figure 9 The bacterial concentration, acid phosphatase activity, and pH in the culture media of the single-strain bacterial agent, the two-strain compound bacterial agent, and the three-strain compound bacterial agent provided in Example 5 of the present invention;

[0057] Figure 10 A comparative graph showing the effects of temperature, resource concentration, and culture generation on the phosphorus-solubilizing ability of the three strains of compound inoculum provided in Example 6 of the present invention;

[0058] Figure 11 This is a comparative graph showing the effects of temperature, resource concentration, and evolutionary generation on the phosphorus-solubilizing ability of the compound bacterial agent after replacing the control strain of the same genus, as provided in Example 6 of the present invention.

[0059] Figure 12 A comparative diagram showing the ability of strains zafu-3, zafu-N9, and zafu-111 provided in Example 8 of the present invention to produce IAA, siderophores, and degrade lignin.

[0060] Figure 13 This is a statistical chart showing the changes in total phosphorus and available phosphorus content in soil under different phosphorus-solubilizing inoculants in Example 10 of the present invention.

[0061] Figure 14 This is a statistical chart showing the changes in aboveground and underground biomass of moso bamboo under different phosphate-solubilizing inoculants in Example 10 of the present invention. Detailed Implementation

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

[0063] Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well known in the art and as described in various general and more specific references, which are cited and discussed throughout this specification.

[0064] This invention provides a compound microbial agent for decomposing insoluble phosphorus in soil, the active ingredients of which include xylose-oxidizing achromobacterium, Burkholderia cepacia, and parafungi Burkholderia.

[0065] The *Achromobacter xylosoxidans* species is *Achromobacter xylosoxidans* zafu-3, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO. M20241954; the *Parabukholderia fungorum* species is *Parabukholderia fungorum* zafu-N9, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO. M 20241955; and the *Burkholderia cepacia* species is *Burkholderia cepacia* zafu-111, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO. M 20241956.

[0066] Preservation instructions:

[0067] Strain name: Xylose-oxidizing achromobacterium zafu-3;

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

[0069] Preservation institution: China Center for Type Culture Collection;

[0070] Abbreviation for depository institution: CCTCC;

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

[0072] Deposit date: September 11, 2024;

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

[0074] Strain name: Burkholderia parafungi zafu-N9;

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

[0076] Preservation institution: China Center for Type Culture Collection;

[0077] Abbreviation for depository institution: CCTCC;

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

[0079] Deposit date: September 11, 2024;

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

[0081] Bacterial strain name: Burkholderia cepacia zafu-111;

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

[0083] Preservation institution: China Center for Type Culture Collection;

[0084] Abbreviation for depository institution: CCTCC;

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

[0086] Deposit date: September 11, 2024;

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

[0088] Xylo-oxidizing achromobacterium zafu-3, *Burkholderia cepacia* zafu-N9, and *Burkholderia cepacia* zafu-111 can independently decompose five insoluble phosphorus sources: Ca3(PO4)2, FePO4, AlPO4, lecithin, and calcium phytate, respectively; the three strains can also synergistically dissolve these five insoluble phosphorus sources. After shaking culture, the compound microbial agent exhibited a phosphorus-solubilizing activity of up to 53.32 mg·L⁻¹. -1 Furthermore, it exhibits stable phosphorus-solubilizing function and possesses the characteristics of degrading lignin, producing siderophores and indoleacetic acid. When applied near the rhizosphere of plants growing in both barren and fertile soils, it demonstrates significant phosphorus-solubilizing and growth-promoting effects. This solves the technical problems of existing technologies where phosphorus-solubilizing bacteria rely on a single phosphorus source or have poor stability in their phosphorus-solubilizing function.

[0089] In some specific embodiments, the ratio of viable bacteria of *Achromobacterium xyloseoxidans* zafu-3, *Burkholderia cepacia* zafu-N9, and *Burkholderia cepacia* zafu-111 is (344–550):(238–252):(2.82–3.52), preferably 447:245:3.17.

[0090] In some specific embodiments, the concentration of the xylose-oxidizing achromobacterium zafu-3 is 3.44–5.05 × 10⁻⁶. 12 CFU·mL -1 The concentration of the parafungus Burkholderia zafu-N9 was 2.38–2.52 × 10⁻⁶. 12 CFU·mL -1 The concentration of Burkholderia cepacia zafu-111 was 2.82–3.52 × 10⁻⁶. 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: mixing the xylose-oxidizing achromobacterium zafu-3 bacterial solution, the parafungi Burkholderia zafu-N9 bacterial solution, and the cephalosporin Burkholderia zafu-111 bacterial solution according to the formula amount to obtain the composite microbial agent.

[0092] The bacterial strains were prepared separately and then mixed in a certain proportion to accurately control the concentration of each strain. The resulting compound microbial agent has the effect of phosphorus solubilization and growth promotion.

[0093] In some specific embodiments, the method for preparing the bacterial suspension includes taking the bacterial suspension in the logarithmic growth phase, centrifuging it, discarding the supernatant, and resuspending it in sterile water to obtain the 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 cell metabolism is vigorous. Selecting cells from this phase to prepare microbial agents can improve the effectiveness of the active ingredients in the agents and enhance their phosphorus-solubilizing and growth-promoting effects.

[0095] In some specific embodiments, the step of mixing the *Achromobacterium xyloseoxidans* zafu-3 bacterial suspension, the *Burkholderia cepacia* zafu-111 bacterial suspension, and the *Burkholderia parafungi* zafu-N9 bacterial suspension according to the formula includes mixing the *Achromobacterium xyloseoxidans* zafu-3 bacterial suspension, the *Burkholderia cepacia* zafu-111 bacterial suspension, and the *Burkholderia parafungi* zafu-N9 bacterial suspension according to the formula, shaking and culturing to the logarithmic phase, and then inoculating into fresh liquid culture medium and culturing to OD. 600 =0.32±0.02, cell count was 6.95×10 12 ±1.10×10 12 CFU·mL -1 Centrifuge, discard the supernatant, and resuspend in an equal volume of sterile water to obtain the compound microbial agent.

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

[0097] The centrifugation conditions include centrifugation at 2500–4200 rpm for 8–12 minutes at 23–27°C, preferably at 4000 rpm for 10 minutes at 25°C. The method for preparing the logarithmic phase bacterial suspension includes inoculating the bacterial strain into a liquid culture medium and culturing it with shaking until the logarithmic phase.

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

[0099] In some specific embodiments, the xylose-oxidizing achromobacterium zafu-3 bacterial solution, the Burkholderia cepacia zafu-111 bacterial solution, and the Burkholderia parafungi zafu-N9 bacterial solution 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, the active ingredients of which include the above-mentioned compound microbial agent, the compound microbial agent prepared by the above-mentioned preparation method, the above-mentioned xylose-oxidizing achromobacterium, the above-mentioned Burkholderia cepacia, or the above-mentioned parafungi Burkholderia.

[0102] Specifically, the products can be microbial agents, bio-organic fertilizers, or soil conditioners, etc., 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, the application of the above-described composite microbial agent, the composite microbial agent prepared by the above-described preparation method, or the above-described product in any of the following:

[0104] (1) Decomposes insoluble phosphorus;

[0105] (2) It produces indoleacetic acid;

[0106] (3) Iron-producing carrier;

[0107] (4) Degradation of lignin;

[0108] (5) Promote plant growth.

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

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

[0111] (1) Promotes an increase in plant biomass;

[0112] (2) Promotes the increase of aboveground biomass in plants;

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

[0114] According to another aspect of the present invention, a method for promoting the release of insoluble phosphorus from soil is also provided, comprising applying the above-described composite microbial agent, the composite microbial agent prepared by the above-described preparation method, or the above-described product to the soil. This application to the soil increases 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, comprising applying the above-described compound microbial inoculant, the compound microbial inoculant prepared by the above-described preparation method, or the above-described product to plants. When used to promote plant growth, it is applied to the soil in contact with the plant roots. By applying it to the soil near the plant rhizosphere, the available phosphorus and total phosphorus content of the soil within the root contact area can be locally increased, thereby promoting plant growth.

[0116] Specifically, the root irrigation method can be used.

[0117] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

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

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

[0120] Root samples with soil attached were placed in centrifuge tubes containing 20 mL of sterile water and shaken at 30°C and 180 rpm for 15 min to obtain a rhizosphere soil microbial suspension. The soil suspension was then diluted using a serial dilution method to obtain dilutions at different dilution factors, each dilution factor being 10 times the volume of the soil suspension. -2 10 -3 10 -4 and 10 -5 times.

[0121] Three types of Pikovskaya (PVK) inorganic phosphorus medium (calcium phosphate, iron phosphate, aluminum phosphate) and two types of Mongina (MG) organic phosphorus medium (lecithin, calcium phytate) were used as screening media (Table 1). Phosphate-solubilizing bacteria in the rhizosphere of *Phyllostachys edulis* were screened using a high-throughput microplate separation method. Specifically, 15 μL of dilution was transferred to 135 μL of 10% selection medium in each 96-well cell culture plate. Using 1% 10 mM bromocresol purple as an indicator, three cell culture plates were transferred for each dilution gradient. An equal volume of sterile water in the selection medium was added as a negative control. After two weeks of microplate incubation, wells showing significant turbidity were selected, and their culture was spread onto the corresponding selection medium. After incubation at 30°C for 7 days, colonies exhibiting distinct morphological patterns and producing phosphate-solubilizing zones on the separation medium were isolated and purified by streak plating on solid medium. The experiment ultimately yielded three phosphate-solubilizing bacteria, which were named zafu-3, zafu-N9, and zafu-111, respectively.

[0122] Table 1. Formulations of PVK, MG, and mixed phosphorus source culture media

[0123]

[0124] 2. Morphological identification

[0125] Strains zafu-3, zafu-N9, and zafu111 were inoculated onto trypticase-Soy Agar (TSA) medium and cultured in the dark at 30°C for 3 days. Their colony characteristics were then observed. Strains were also cultured in trypticase-Soy Broth (TSB) medium at 30°C with shaking at 180 rpm for 3 days. Their cell morphology was then observed under an optical microscope.

[0126] The results are as follows Figure 1 As shown (scale bar is 20 μm), strains zafu-3, zafu-N9, and zafu-111 are all Gram-negative bacteria with rod-shaped cells measuring 1.25 μm × 1.75 μm, 1.45 μm × 1.45 μm, and 1.52 μm × 2.99 μm, respectively. The colonies of all three strains are pale yellow, opaque, round, with a raised center, smooth surface, and regular, neat edges.

[0127] 3. Molecular identification

[0128] The genomes of the isolated phosphate-solubilizing bacteria were extracted using a bacterial genome kit (SPARKeasy Bacteria DNA Kit, Shandong Cisco Technology Co., Ltd., China). After the concentration was detected by a 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 analyzed by BLAST alignment in the NCBI database. Subsequently, a phylogenetic tree was constructed in MEGA software using the nearest neighbor method to determine the species of the strain.

[0129] Phylogenetic tree such as Figure 2 As shown, the results indicate that strains zafu-3, zafu-N9, and zafu-111 formed independent and stable branches with *Achromobacter xylosoxidans*, *Parabukholderia fungorum*, and *Burkholderia cepacia*, respectively, with gene sequence homology exceeding 99%. This indicates that zafu-3 is *A. xylosoxidans*, zafu-N9 is *P. fungorum*, and zafu-111 is *B. cepacia*.

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

[0131] The phosphorus-solubilizing ability of the strains was qualitatively studied using the solubilization zone method. The phosphorus-solubilizing ability of each strain was preliminarily determined based on the ratio of the solubilization zone diameter (D) to the colony diameter (d) (D / d). The phosphorus-solubilizing ability of the strains was quantitatively studied using the molybdenum-antimony colorimetric method. Figure 3 The table shows the phosphate-solubilizing zones formed by strains zafu-3, zafu-N9, and zafu-111 on calcium phytate, lecithin, and Ca3(PO4)2 media, respectively. As shown in Table 2, the three strains all exhibited certain phosphate-solubilizing abilities on Ca3(PO4)2, FePO4, AlPO4, lecithin, and calcium phytate.

[0132] Table 2. The ability of strains zafu-3, zafu-N9, and zafu-111 to dissolve different poorly soluble phosphorus sources.

[0133]

[0134] Growth curve of strain 3 in Example 3

[0135] Growth curves for strains zafu-3, zafu-N9, and zafu-111 were plotted according to the following steps: The strains were inoculated into TSB medium and cultured at 30°C with shaking at 180 rpm for 3 hours. After centrifugation at 4000 rpm for 10 minutes, the supernatant was discarded, and the bacterial cells were resuspended in sterile water. The bacterial concentration was adjusted to OD0.05. 600 =0.1 for quantification. OD was inoculated at a rate of 5% (V:V). 600 The bacterial culture with a concentration of 0.1 was re-inoculated into fresh TSB medium and cultured at 30°C with shaking at 180 rpm. The OD value of the bacterial culture was measured every 3-9 hours. 600 The values ​​were calculated, and the number of cells (CFU) in the bacterial culture was determined on TSA medium using the dilution-spread method, and a growth curve of the strain was plotted. Each sampling was performed in triplicate, and the UV spectrophotometer was calibrated using uninoculated medium. Results are as follows: Figure 4 As shown, strain zafu-3 grew rapidly from 6 to 24 hours. After 24 hours of culture, the strain's OD... 600 =3.21, cell count is 3.15×10 15 CFU·mL -1 After 24 hours, it enters a stable phase. Strain zafu-N9 grows rapidly from 9 to 48 hours; after 48 hours of culture, the strain's OD... 600 =3.13, cell count is 3.98×10 14 CFU·mL -1 After 48 hours, it entered a stable phase. Strain zafu-111 grew rapidly from 6 to 27 hours, and after 27 hours of culture, the strain's OD... 600 =3.02, cell count is 4.90×10 15 CFU·mL -1 It enters a stable period after 27 hours.

[0136] Example 4: Preparation of bacterial agent

[0137] 1. Preparation of single-strain inoculum: Three phosphate-solubilizing strains were inoculated into TSB medium and cultured with shaking at 30℃ and 180 rpm until the logarithmic growth phase. After centrifugation at 25℃ and 4000 rpm for 10 min, the supernatant was discarded, and the culture was resuspended in an equal volume of sterile water to prepare the inoculum. Alternatively, the bacterial concentration was adjusted to OD using sterile water. 600 =0.1, used for the preparation of compound microbial agents.

[0138] 2. Preparation of dual-strain compound bacterial agent: 100 μL of single-strain bacterial suspension was inoculated into fresh TSB medium at a ratio of 1:1 / V:V, and cultured with shaking at 30℃ and 180 rpm until the logarithmic phase. After centrifugation at 4000 rpm for 10 min, the supernatant was discarded and the bacterial cells were resuspended in an equal volume of sterile water to prepare the bacterial agent for later use.

[0139] 3. Preparation of the three-strain compound bacterial agent: 100 μL of single-strain bacterial suspension was inoculated into fresh TSB medium in an equal ratio (1:1:1 / V:V:V), and cultured at 30℃ and 180 rpm for 24 h with shaking. The mixture was then cultured until the logarithmic growth phase (OD50) of the compound phosphate-solubilizing bacterial suspension was reached. 600 =2.5, cell number approximately 2.29 × 10 11 CFU·mL -1 )(like Figure 5 (a. Red time point). Inoculate the compound bacterial solution into a 250mL Erlenmeyer flask containing TSB medium at a 2% inoculation rate. Incubate at 30℃ and 180rpm for 48 hours with shaking until the compound bacterial agent reaches its optimal phosphate-solubilizing state, at which point the phosphate-solubilizing activity is 53.32±4.74 mg·L⁻¹. -1 OD 600 =0.32, cell number is 6.95×10 12 ±1.10×10 12 CFU·mL -1 (like Figure 5 (b. Red time point). The compound bacterial agent was transferred to a sterile centrifuge tube and centrifuged at 4000 rpm for 10 min. After discarding the supernatant, the bacterial cells were resuspended in an equal volume of sterile water to prepare the bacterial agent. The cell count of strain zafu-3 was 4.47 × 10⁻⁶ cells. 12 ±1.03×10 12 CFU·mL -1 The cell number of strain zafu-N9 was 2.45 × 10⁻⁶. 12 ±7.00×10 10 CFU·mL -1 The cell number of strain zafu-111 was 3.17 × 10⁻⁶. 10 ±3.51×10 9 CFU·mL -1 .

[0140] Example 5: Phosphate-solubilizing ability and pathway of compound microbial agent

[0141] 100 μL of the single-strain inoculant, the two-strain (1:1 / V:V) compound inoculant, or the three-strain (1:1:1 / V:V:V) compound inoculant prepared in Example 4 were inoculated into 2 mL of mixed Monkina culture medium (mixed phosphorus source medium) containing two kinds of organic phosphorus and three kinds of inorganic phosphorus. Six replicates were performed for each treatment, with sterile water added as a blank control. The phosphorus-solubilizing activity of the single-strain inoculant, the two-strain compound inoculant, and the three-strain compound inoculant, as well as the solubility of the three-strain compound inoculant for five poorly soluble phosphorus sources, were analyzed. Simultaneously, five other phosphorus-solubilizing bacteria obtained from the rhizosphere soil of moso bamboo according to the method in Example 1 were selected as control strains. The species information is shown in Table 3, and a phylogenetic tree was constructed using the nearest neighbor method. These five control strains, belonging to the same genus, were used to replace one, two, or three strains in the three-strain compound inoculant as comparative examples to investigate the changes in the phosphorus-solubilizing ability of the compound inoculant. After incubation at 30℃ and 180 rpm for 3 days with shaking, the phosphorus solubilizing activity of each inoculation treatment was determined using the molybdenum-antimony colorimetric method. The bacterial concentration (OD) in the solutions after incubation of single-strain, two-strain, and three-strain compound inoculants was also measured. 600 ), changes in phosphatase activity and pH value, with specific results as follows: Figures 6-9 As shown.

[0142] Table 3. Other phosphorus-solubilizing strains obtained through screening and their phosphorus-solubilizing abilities.

[0143]

[0144]

[0145] As Figure 6 (Values ​​are expressed as mean ± standard deviation (n = 3), and bars with different letters indicate significant differences (p < 0.05)). It can be seen that the three-strain compound inoculant composed of zafu-3, zafu-N9, and zafu-111 exhibits the strongest phosphate-solubilizing ability, at 21.05 ± 0.37 mg·L⁻¹. -1 ·d -1 The phosphorus-solubilizing capacity was higher than that of the three strains when inoculated alone or randomly inoculated in pairs. Furthermore, [the following text appears to be incomplete and requires further context: "by..."] 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 strains of the three strains in the above compound microbial agent with other strains of the same genus, the phosphorus-solubilizing effect of the compound microbial agent composed of strains zafu-3, zafu-N9, and zafu-111 was still significantly higher than that of other groups. These results indicate that there may be a synergistic promoting effect among the member strains zafu-3, zafu-N9, and zafu-111 of this compound microbial agent.) Figure 9 (Values ​​are expressed as mean ± standard deviation (n=3), and bars with different letters indicate significant differences (p<0.05)). This indicates that when the three strains are cultured together, the bacterial concentration (OD)...600 The activity of the three strains did not decrease significantly and even promoted their growth. However, the phosphatase activity of the three strains after mixed culture was not the highest among all combinations, indicating that the compound bacterial agent may not rely mainly on secreting phosphatase to enhance its ability to activate insoluble phosphorus. The pH of the compound bacterial agent culture system was as low as 2.6, which suggests that the phosphorus-solubilizing bacterial agent may increase the solubility of insoluble phosphorus by secreting organic acids and lowering the pH in the environment.

[0146] Example 6: Stability of Phosphate-Solubilizing Function of Three-Strain Compound Inoculant

[0147] Following the steps and methods for preparing the three-strain compound bacterial agent in Example 4, the effects of culture temperature (25℃, 30℃, and 35℃), resource concentration (100% and 10% mixed phosphorus source medium), and generation number (1st, 3rd, 7th, and 10th generations) on the phosphorus solubilization ability of the three-strain compound bacterial agent were investigated. Meanwhile, *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. 100 μL of the first-generation culture was transferred to a 24-well plate containing 2 mL of fresh mixed culture medium, and the culture was continued in this manner for a total of 10 generations. The phosphate-solubilizing activity of the bacterial agent was determined using the molybdenum-antimony colorimetric method.

[0149] The results are as follows Figure 10 The values ​​are shown in the figure (values ​​are mean ± standard deviation (n=3), and bars with different letters indicate significant differences (p<0.05)). (a) represents temperature, (b) represents resource concentration, and (c) represents the generation number. Temperature, resource concentration, and evolutionary process all significantly affect the phosphorus-solubilizing ability of the inoculum. The phosphorus-solubilizing activity of the inoculum at 30℃ was 72% and 66% higher than that at 25℃ and 35℃, respectively. The phosphorus-solubilizing ability of the inoculum grown in 10% medium was significantly higher than that in 100% medium, at 22.24 ± 1.50 mg·L⁻¹. -1 ·d -1 As the number of generations increases, the interaction time lengthens, and the phosphorus-solubilizing ability decreases. After 10 generations, its phosphorus-solubilizing ability is 6.40 ± 0.19 mg·L⁻¹. -1 ·d -1 .like Figure 11(Values ​​are expressed as mean ± standard deviation (n=3), and bars with different letters indicate significant differences (p<0.05)). As shown, although the phosphorus-solubilizing activity of the three-strain compound inoculant decreased at 25℃ and 35℃, in 100% culture medium, and after 10 generations, it was still much higher than that of the control group after replacing it with a strain of the same genus. Therefore, the phosphorus-solubilizing inoculant maintained a high phosphorus-solubilizing effect under different environmental temperatures, resource concentrations, and evolutionary processes, which also indicates that its phosphorus-solubilizing ability has a certain degree of stability.

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

[0151] The pH range (3–11, in 1-unit increments) and NaCl concentration range (1%–7%, W / V, 1% intervals) for bacterial growth were determined by incubation in tryptone soybean broth (TSB) medium at 30°C and 180 rpm for 3 days with shaking. Uninoculated culture served as a blank control, with six replicates per treatment. Carbon source utilization by the strain was investigated using three carbon-free PVK and two carbon-free MG media to explore the strain's utilization of 15 common carbon sources and the formation of lysosomes. These included 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 determined at 5 g / L. -1 Add to the basal culture medium. Each treatment in the above experiments was performed in triplicate, with no inoculation as a blank control. After incubation in the dark at 30°C for 3 days, the samples were analyzed.

[0152] Table 4 shows the growth of strains zafu-3(3), zafu-N9(N9), and zafu-111(111) in five phosphorus source media after the addition of 15 carbon sources. When glucose, proline, isoleucine, oxalic acid, or succinic acid is used as the sole carbon source, the strain can grow on all five phosphorus source media, but it has difficulty utilizing serine and lactic acid. After adding eight carbon sources such as fructose, sucrose, or lysine, the strain only grows in some phosphorus source media. When glucose and succinic acid are used as the sole carbon sources, strain zafu-N9 can grow on all five phosphorus source media, but it has difficulty utilizing cellulose, glutamic acid, serine, and four organic acids other than succinic acid. After adding fructose, sucrose, xylan, or isoleucine, the strain only grows in some phosphorus source media. When glucose, sucrose, isoleucine, or succinic acid is used as the sole carbon source, the strain zafu-111 can grow on all five phosphorus-based media, but it has difficulty utilizing glutamic acid, serine, oxalic acid, malic acid, and lactic acid. With the addition of six carbon sources, including fructose, xylan, or proline, the strain only grows on some phosphorus-based media. The results show that strain zafu-3 can grow under conditions of pH 4–10 and NaCl concentrations below 6%, while strains zafu-N9 and zafu-111 can grow under conditions of pH 4–9 and NaCl concentrations below 4%. The ability of all three strains to grow under multiple carbon sources and a wide range of pH and NaCl concentrations ensures their functional stability.

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

[0154]

[0155]

[0156] The symbols are - negative, +, ++, +++, and positive. * indicates the presence of a lysing zone on the culture medium.

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

[0158] The Salkowski colorimetric method was used to determine the ability of the strain to produce indoleacetic acid (IAA). After adding the Salkowski colorimetric solution to the strain culture medium, the solution turned a distinct red color, indicating that the strain possessed the ability to produce IAA. The strain was inoculated on Chrome Azurol S CAS solid medium, and after 3 days of cultivation, the diameter of the orange iron-producing zone (D1) and the strain diameter (d1) were observed. The D1 / d1 value represented the strain's ability to produce siderophores. The strain was inoculated on aniline blue chromogenic medium, and after 8 days of cultivation, the diameter of the dissolution zone (D2) and the strain diameter (d2) were observed. The D2 / d2 value represented the strain's ability to degrade lignin.

[0159] The results are as follows Figure 12 As shown, strains zafu-3, zafu-N9, and zafu-111 can all produce IAA. Strains zafu-N9 and zafu-111 can also produce siderophores, with D1 / d1 ratios of 2.80±0.32 and 1.93±0.44, respectively. All three strains produced transparent lysozyme zones on aniline blue medium, indicating their ability to degrade lignin, with D2 / d2 ratios 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 compound microbial agent

[0161] The study on the root colonization ability of strains in the three-strain compound inoculant first involved the cultivation of sterile bamboo seedlings. Specifically, surface-sterilized seeds were placed on MS medium lined with filter paper and germinated in a 30°C incubator in the dark. After germination, the seeds were transferred to tissue culture flasks containing 50 mL of MS medium and aseptically cultured at 25°C for two weeks. Then, 0.5 mL of the compound phosphate-solubilizing inoculant was inoculated into the roots of the sterile bamboo seedlings. After further cultivation at 25°C for seven days, the bacterial strains were screened on the bamboo roots using the plate spread method, and the colony count of each strain was recorded.

[0162] Members of the inoculated compound microbial agent were isolated from both the rhizosphere and root zone of moso bamboo seedlings, demonstrating that all three strains of the agent successfully colonized the roots of moso bamboo. As shown in Table 5, strain zafu-N9 exhibited the highest colonization density in the rhizosphere, at 2.4 × 10⁻⁶. 3 CFU·g -1 The concentrations were 1.8 times and 1.2 times higher than those of zafu-3 and zafu-111, respectively. Strain zafu-111 exhibited the highest root colonization density, at 3.3 × 10⁻⁶. 3 CFU·g -1 These figures are 2.1 times and 1.1 times that of zafu-3 and zafu-N9, respectively.

[0163] Table 5 shows the colonization rates of strains zafu-3, zafu-N9, and zafu-111 in the root system of moso 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 Pot Experiment

[0166] The pot experiment was conducted in a greenhouse. The soil samples were collected from bamboo forest plots isolated from phosphate-solubilizing bacteria. The soil was sieved through a 10-mesh sieve, mixed thoroughly, and then used.

[0167] The experiment used two soil substrates with different nutrient levels: forest soil alone (nutrient-rich) and forest soil and sand in a 1:1 ratio (v / v, nutrient-poor).

[0168] The test plants were one-year-old seedlings of uniform growth, propagated from seeds of the same lineage of moso bamboo. Treatments included single-strain inoculants (three strains each), a compound phosphate-solubilizing inoculant, and a control group with added sterile water. The inoculant was applied to the rhizosphere of the moso bamboo seedlings using a root drenching method, with 20 mL of the inoculant applied three times, three days apart. The final concentration of the inoculant after each inoculation was 10. 7 CUF·kg -1 After 90 days of cultivation in a greenhouse, bamboo plants and soil were collected. The biomass of the aboveground plants and underground roots was determined by weighing. The soil was digested in H₂SO₄ and HClO₄ solutions, and the total phosphorus content (TP) was determined using the molybdenum-antimony spectrophotometric method. The available phosphorus content (AP) in the soil was determined using the Tiessen modified Hedley method.

[0169] The results of the pot experiment are as follows Figure 13 and Figure 14 As shown, under infertile soil conditions, inoculation with the compound microbial agent resulted in a decrease in total soil phosphorus and an increase in available phosphorus content compared to the control group, but neither difference was statistically significant. Under nutrient-rich soil conditions, inoculation with the compound microbial agent also increased the available phosphorus content, indicating that the compound microbial agent can activate the soil phosphorus pool and release available phosphorus. Under infertile soil conditions, the aboveground and belowground biomass of the compound microbial agent treatment significantly increased by 119.54% and 289.40% compared to the uninoculated treatment, and significantly increased by 26.14%–81.22% and 80.65%–133.80% compared to the single-strain treatment. Under fertile soil conditions, the belowground and aboveground biomass of bamboo treated with the compound microbial agent significantly increased by 117.04% and 389.77% compared to the uninoculated treatment, and significantly increased by 50.27%–74.74% and 120.65%–169.38% compared to the single-strain treatment. This indicates that inoculation with compound 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, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions 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 compound microbial inoculant for decomposing insoluble phosphorus in soil, characterized in that, Its active ingredients consist of xylose-oxidizing achromobacterium, Burkholderia cepacia, and the parafungi Burkholderia. The xylose-oxidizing achromobacter is xylose-oxidizing achromobacter ( Achromobacter xylosoxidans )zafu-3, which is deposited in the China Center for Type Culture Collection, accession number CCTCC NO. M 20241954; The parafungus Burkholderia is a parafungus Burkholderia ( Parabukholderia fungorum )zafu-N9, which is deposited in the China Center for Type Culture Collection, with accession number CCTCC NO. M 20241955; The Burkholderia cepacia mentioned is Burkholderia cepacia ( Burkholderia cepacia )zafu-111, which is deposited in the China Center for Type Culture Collection, with accession number CCTCC NO. M 20241956; Insoluble phosphorus includes insoluble inorganic phosphorus and insoluble organic phosphorus; insoluble inorganic phosphorus includes Ca3(PO4)2, FePO4 and AlPO4; insoluble organic phosphorus includes lecithin and calcium phytate.

2. The method for preparing the composite microbial agent according to claim 1, characterized in that, The process includes the following steps: mixing the xylose-oxidizing achromobacterium zafu-3 bacterial solution, the parafungi Burkholderia zafu-N9 bacterial solution, and the cephalosporin Burkholderia zafu-111 bacterial solution in equal volume ratio to obtain a composite microbial agent.

3. The preparation method according to claim 2, characterized in that, The method for preparing the bacterial suspension includes taking the bacterial suspension in the logarithmic growth phase, centrifuging it, discarding the supernatant, and resuspending it in sterile water to obtain the OD. 600 =0.1 bacterial solution.

4. The preparation method according to claim 2, characterized in that, The step of mixing the *Achromobacterium xyloseoxidans* zafu-3 bacterial suspension, the *Burkholderia cepacia* zafu-111 bacterial suspension, and the *Burkholderia parafungi* zafu-N9 bacterial suspension in equal volume ratio includes mixing the *Achromobacterium xyloseoxidans* zafu-3 bacterial suspension, the *Burkholderia cepacia* zafu-111 bacterial suspension, and the *Burkholderia parafungi* zafu-N9 bacterial suspension in equal volume ratio, shaking and culturing to the logarithmic phase, and then inoculating into fresh liquid culture medium and culturing to OD. 600 =0.32±0.02, cell number 6.95×10 12 ±1.10×10 12 CFU·mL -1 Centrifuge, discard the supernatant, and resuspend in an equal volume of sterile water to obtain the compound microbial agent.

5. The preparation method according to claim 4, characterized in that, The phosphorus-solubilizing activity of the compound microbial agent was 53.32 ± 4.74 mg·L. -1 .

6. The preparation method according to claim 4, characterized in that, The centrifugation conditions include 23~27℃, 2500~4200rpm for 8~12min.

7. The preparation method according to claim 4, characterized in that, The liquid culture medium is tryptone soybean broth liquid culture medium.

8. The preparation method according to claim 4, characterized in that, The conditions for the shaking culture include shaking culture at 28~32℃ and 150~200rpm.

9. The preparation method according to claim 4, characterized in that, The inoculation dose is 1-3%.

10. A product characterized in that, Its active ingredients include the compound microbial agent as described in claim 1 or the compound microbial agent prepared by the preparation method described in any one of claims 2 to 9.

11. The use of the compound microbial agent according to claim 1, the compound microbial agent prepared by the preparation method according to any one of claims 2 to 9, or the product according to claim 10 in any one of the following: (1) Decomposes insoluble phosphorus; (2) Produces indoleacetic acid; (3) Iron-producing carrier; (4) Degradation of lignin; (5) Promote plant growth.

12. The application according to claim 11, characterized in that, The insoluble phosphorus includes insoluble inorganic phosphorus and insoluble organic phosphorus.

13. The application according to claim 12, characterized in that, The insoluble inorganic phosphorus includes at least one of Ca3(PO4)2, FePO4, and AlPO4.

14. The application according to claim 12, characterized in that, The poorly soluble organic phosphorus includes lecithin and / or calcium phytate.

15. The application according to claim 11, characterized in that, The promotion of plant growth is manifested in at least one of the following: (1) Promotes an increase in plant biomass; (2) Promotes the increase of aboveground biomass in plants; (3) Promotes the increase of underground biomass of plants.

16. A method for promoting the release of insoluble phosphorus from soil, characterized in that, This includes applying the composite microbial agent of claim 1, the composite microbial agent prepared by the preparation method of any one of claims 2 to 9, or the product of claim 10 to the soil.

17. A method for promoting plant growth, characterized in that, This includes applying the compound microbial agent of claim 1, the compound microbial agent prepared by the preparation method of any one of claims 2 to 9, or the product of claim 10 to plants.

18. The method according to claim 17, characterized in that, The application method is root irrigation.

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