High-efficiency composite phosphorus-solubilizing bacterial agent, preparation method thereof, biological bacterial fertilizer and application thereof

Abstract

The present application relates to the field of environment and the field of agricultural biotechnology, and in particular to a kind of high-efficiency composite phosphorus-solubilizing bacterial agent and its preparation method, biological bacterial fertilizer and application, the high-efficiency composite phosphorus-solubilizing bacterial agent has active ingredient enterobacter cloacae HS6 and pantoea agglomerans CT3. The high-efficiency composite phosphorus-solubilizing bacterial agent formed by the combined application of enterobacter cloacae HS6 and pantoea agglomerans CT3 has high-efficiency phosphorus-solubilizing capacity and excellent stability, there is no antagonism between the two strains, but also can greatly improve the utilization efficiency of insoluble phosphorus, has the advantages of excellent phosphorus-solubilizing effect and high available phosphorus content, can reduce the use amount of chemical phosphorus fertilizer, suitable for use in agricultural production. The high-efficiency composite phosphorus-solubilizing bacterial agent has good prospects in the application of preparing biological bacterial fertilizer, in agricultural soil improvement, in phosphorus resource recovery and in promoting plant growth.

Description

Technical Field

[0001] This invention relates to the fields of environmental and agricultural biotechnology, specifically to a highly efficient compound phosphate-solubilizing agent and its preparation method, as well as bio-fertilizers and their applications. Background Technology

[0002] Phosphorus is an essential nutrient for plant growth and development, playing a vital role in photosynthesis, cell division, and metabolism. However, most soils currently suffer from severe phosphorus deficiency. Phosphorus in soil exists in the form of insoluble inorganic phosphorus, which plants cannot directly absorb and utilize. To address this deficiency, large amounts of phosphate fertilizers are applied, but plant utilization rates are generally low, ranging from only 5% to 25%. This leads to resource waste and environmental pollution problems, such as eutrophication and soil acidification.

[0003] Soil contains phosphorus-solubilizing microorganisms that convert insoluble phosphorus into available phosphorus for plant absorption by secreting organic acids or enzymes. However, the phosphorus-solubilizing capacity of a single microbial strain is limited. Therefore, compound phosphorus-solubilizing agents can be prepared using multiple strains and further optimized. Replacing some chemical phosphate fertilizers with these compound agents can not only significantly improve phosphorus utilization but also reduce environmental pollution, meeting the requirements of sustainable agricultural development. However, the development of compound phosphorus-solubilizing agents requires further research and development to enrich phosphorus-solubilizing agent resources and promote soil improvement and agricultural development. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, the first objective of this invention is to provide a highly efficient compound phosphate-solubilizing agent. This highly efficient compound phosphate-solubilizing agent expands the resources of phosphate-solubilizing agents, has excellent phosphate-solubilizing effects, can effectively improve the utilization rate of phosphorus in the soil, reduce the amount of chemical phosphate fertilizer used, and promote the green and sustainable development of agricultural production.

[0005] To overcome the shortcomings of the prior art, the second objective of this invention is to provide a method for preparing a high-efficiency compound phosphorus-solubilizing agent. This method is simple and easy to operate, and the resulting high-efficiency compound phosphorus-solubilizing agent expands the resources of phosphorus-solubilizing agents, has excellent phosphorus-solubilizing effect, can effectively improve the utilization rate of phosphorus in the soil, and reduce the amount of phosphate fertilizer used.

[0006] The third objective of this invention is to provide a bio-fertilizer.

[0007] The fourth objective of this invention is to provide an application of a highly efficient compound phosphate-solubilizing agent.

[0008] To achieve the first objective of the invention, the technical solution adopted by the present invention is as follows:

[0009] This invention provides a highly efficient compound phosphate-solubilizing agent, comprising Enterobacter holmieae HS6 and Pantotheca cumulus CT3;

[0010] The classification name of the Enterobacter hormaechei HS6 is Enterobacter hormaechei, which was deposited at the China General Microbiological Culture Collection Center on October 19, 2023, with the accession number CGMCC NO.: 28680;

[0011] The aforementioned Pantoea agglomerans CT3 is classified as Pantoea agglomerans and was deposited on January 8, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.: 33335.

[0012] Furthermore, the 16S rDNA gene sequence of the *Enterobacter holmieae* HS6 is shown in SEQ ID No. 1;

[0013] The 16S rDNA gene sequence of the CT3 clump is shown in SEQ ID No. 2.

[0014] Furthermore, it also includes protectants and culture media; and / or

[0015] The protective agent is one of monosodium glutamate, trehalose, or potassium chloride; the culture medium is NBRIP medium.

[0016] The addition of the protectant can increase the number of viable bacteria in the bacterial suspension and promote the growth and metabolism of Enterobacter holmieae HS6 and Pantotheca clumps CT3.

[0017] This highly efficient compound phosphate-solubilizing agent is formed by combining Enterobacter holmium HS6 and Pantotheca cumulus CT3. The two strains not only have no antagonistic effect, but also have highly efficient phosphate-solubilizing ability and stability, which can greatly improve the utilization efficiency of insoluble phosphorus and is suitable for use in agricultural production.

[0018] To achieve the second objective of the invention, the technical solution adopted by the present invention is as follows:

[0019] This invention provides a method for preparing a highly efficient compound phosphate-solubilizing agent, comprising the following steps:

[0020] S1. Activation culture: Refrigerated Enterobacter holmieae HS6 and Pantotheca cumulus CT3 were streaked onto LB agar plates and incubated upside down at 28°C for 48 h to obtain the two colonies in activated culture.

[0021] S2. Preparation of bacterial suspension: Using an inoculation loop, pick single colonies from each of the two activated bacterial cultures, then inoculate them separately into LB liquid medium, and then place them in a constant temperature shaker at 28℃ and shake at 180 r / min until OD.600 The concentrations were 0.6-0.8, resulting in HS6 and CT3 bacterial suspensions, respectively.

[0022] S3. Preparation of bacterial agent: Mix the prepared HS6 bacterial suspension and CT3 bacterial suspension to form a compound bacterial solution. Then, inoculate the compound bacterial solution into NBRIP liquid medium, add a protectant, and then place it in a constant temperature shaker at 28°C and shake at 180 r / min to obtain the high-efficiency compound phosphate-solubilizing bacterial agent.

[0023] Furthermore, the effective viable count of both the HS6 bacterial suspension and the CT3 bacterial suspension obtained in step S2 is 2 × 10⁻⁶. 8 CFU / mL ~3.4×10 9 CFU / mL.

[0024] Further, in step S3, the volume ratio of the HS6 bacterial suspension to the CT3 bacterial suspension is 1:1; the inoculation amount of the composite bacterial solution in the NBRIP liquid medium is 1% of the culture medium volume; and / or

[0025] The protective agent is one of monosodium glutamate, trehalose, or potassium chloride; the concentration of the protective agent in the NBRIP liquid medium is 1 g / L to 3 g / L; and / or

[0026] In step S3, the food is placed in a constant temperature shaker at 28°C and oscillated at 180 r / min for 48 h-60 h.

[0027] To achieve the third objective of the invention, the technical solution adopted by the present invention is as follows:

[0028] This invention provides a bio-fertilizer, which is prepared using the aforementioned highly efficient compound phosphate-solubilizing agent.

[0029] This highly efficient compound phosphorus-solubilizing agent combines Enterobacter holmium HS6 and Pantotheca cumulus CT3, allowing both strains to work together to solubilize phosphorus without antagonism, thus greatly improving the utilization efficiency of insoluble phosphorus. Therefore, it has great potential for use in the preparation of bio-fertilizers in agricultural production.

[0030] To achieve the fourth objective of the invention, the technical solution adopted by the present invention is as follows:

[0031] This invention provides the application of the above-mentioned high-efficiency compound phosphate-solubilizing agent in agricultural soil improvement.

[0032] Specifically, the highly efficient compound phosphorus-solubilizing agent is applied to the soil, and the synergistic effect of Enterobacter holmieae HS6 and Pantotheca cumulus CT3 promotes the dissolution and release of phosphorus in the soil, thereby improving the available phosphorus content in the soil and promoting plant growth.

[0033] This invention provides the application of the aforementioned high-efficiency compound phosphate-solubilizing agent in phosphorus resource recovery.

[0034] Specifically, applying the aforementioned high-efficiency composite phosphorus-solubilizing agent to the biodissolution of phosphorus resources can improve the recovery rate of phosphorus resources.

[0035] This invention provides the application of the above-mentioned high-efficiency compound phosphate-solubilizing agent in promoting plant growth.

[0036] This highly efficient compound phosphorus-solubilizing agent combines Enterobacter holmium HS6 and Pantotheca cumulus CT3, allowing the two strains to work together to solubilize phosphorus. It has a high phosphorus-solubilizing capacity and stability, which can greatly improve the utilization efficiency of insoluble phosphorus. This not only helps to improve the recovery and utilization rate of phosphorus resources and reduce the environmental pollution of traditional chemical treatment methods, but also improves agricultural soil. When used as a bio-fertilizer, it can also promote plant growth. Therefore, it has a promising application prospect in the above applications.

[0037] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0038] (1) This invention provides a highly efficient compound phosphate-solubilizing agent comprising *Enterobacter holmium* HS6 and *Pantotheca acuminata* CT3. The combined application of *Enterobacter holmium* HS6 and *Pantotheca acuminata* CT3 forms a highly efficient compound phosphate-solubilizing agent. The two strains not only exhibit no antagonistic effect but also possess highly efficient phosphate-solubilizing ability and stability, significantly improving the utilization efficiency of insoluble phosphorus, making it suitable for use in agricultural production. This highly efficient compound phosphate-solubilizing agent not only expands the resources of phosphate-solubilizing agents but also has a high effective phosphorus content, reaching 886.13 mg / L. Therefore, this highly efficient compound phosphate-solubilizing agent has the advantages of excellent phosphate-solubilizing effect, effectively improving the utilization rate of phosphorus in the soil, reducing the amount of chemical phosphate fertilizer used, and thus promoting the green and sustainable development of agricultural production.

[0039] (2) The preparation method of the high-efficiency compound phosphorus-solubilizing agent of the present invention has the characteristics of simple preparation method and easy operation, and the high-efficiency compound phosphorus-solubilizing agent obtained expands the phosphorus-solubilizing agent resources, has excellent phosphorus-solubilizing effect, can effectively improve the utilization rate of phosphorus in soil and reduce the amount of phosphate fertilizer used.

[0040] (3) The bio-fertilizer of the present invention is prepared using the above-mentioned highly efficient compound phosphorus-solubilizing agent, thus promoting the dissolution of insoluble phosphorus and having a good application effect. Specifically, this highly efficient compound phosphorus-solubilizing agent combines Enterobacter holmium HS6 and Pantotheca agglomerata CT3, fully utilizing the synergistic effect of the two strains, exhibiting highly efficient phosphorus-solubilizing ability and stability, and effectively improving the utilization rate of phosphorus in the soil. Therefore, it has a promising future in the preparation of bio-fertilizers.

[0041] (4) The application of the high-efficiency compound phosphate-solubilizing agent of the present invention is as follows: Since the high-efficiency compound phosphate-solubilizing agent is applied by the combined application of Enterobacter holmium HS6 and Pantotheca cumulus CT3, there is no antagonistic effect between the two strains. Moreover, the high-efficiency compound phosphate-solubilizing agent formed has high phosphorus-solubilizing ability and stability, which can greatly improve the utilization efficiency of insoluble phosphorus, improve agricultural soil, and promote plant growth when used as a bio-fertilizer. Therefore, it has good application prospects in agricultural soil improvement, phosphorus resource recovery and plant growth promotion. Attached Figure Description

[0042] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0043] Figure 1 This is a scanning electron microscope image of Enterobacter HS6 of the present invention.

[0044] Figure 2 This is a scanning electron microscope image of the CT3 cluster of pantothecin bacteria according to the present invention.

[0045] Figure 3 This is a diagram illustrating the coexistence effect of Enterobacter holmieae HS6 and Pantotheca cumulus CT3 according to the present invention.

[0046] Figure 4 This is a growth curve diagram of Enterobacter holmieae HS6 and Pantotheca cum Caulis CT3 of the present invention.

[0047] Figure 5 This is a graph showing the results of phosphorus-solubilizing ability tests of compound bacterial solutions with different inoculation amounts at different cultivation periods in this invention. Detailed Implementation

[0048] To make the technical problem to be solved, the technical solution, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0049] The terminology used in the embodiments of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. In this invention, the singular forms “a,” “the,” and “the” as used in the embodiments and appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0050] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0051] The culture medium formulations described in the following examples are as follows:

[0052] PVK medium (g / L): NaCl 0.2g, Ca3(PO4)2 5g, MgSO4 0.1g, (NH4)2SO4 0.5g, glucose 15g, chloramphenicol 50mg, streptomycin 50mg, pH 6.8~7.0.

[0053] Solid medium for PVK: Add 18g to 20g of agar powder to every 1L of PVK medium.

[0054] LB medium (g / L): 10g peptone, 5g yeast extract, 10g NaCl, pH 7.0-7.2.

[0055] LB solid medium: Add 18g to 20g of agar powder to every 1L of LB medium.

[0056] NBRIP medium (g / L): glucose 10g, (NH4)2SO4 0.5g, NaCl 0.3g, MgSO4·7H2O 0.3g, FeSO4·7H2O 0.03g, MnSO4·2H2O 0.03g, Ca3(PO4)2 5g, KCl 0.3g, lecithin 0.2g, pH 7.2~7.4.

[0057] Solid medium for NBRIP: Add 18g to 20g of agar powder to every 1L of NBRIP medium.

[0058] Example 1

[0059] A highly efficient compound phosphate-solubilizing agent contains the active ingredients *Enterobacter hormaechei* HS6 and *Pantoea agglomerans* CT3. *Enterobacter hormaechei* HS6 was deposited on October 19, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 28680, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. *Pantoea agglomerans* CT3 was deposited on January 8, 2025, at the same center with accession number CGMCC NO. 33335, also located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0060] Among them, the scanning electron microscope image of Enterobacter holmie HS6 is shown below. Figure 1 As shown. Scanning electron micrograph of CT3 clusters of pantothenic bacteria, as shown. Figure 2 As shown.

[0061] The 16S rDNA gene sequence of Enterobacter holmieae HS6 is shown in SEQ ID No. 1. The gene sequence length is 1476.

[0062] The 16S rDNA gene sequence of *Pantotheca cumulus* CT3 is shown in SEQ ID No. 2. The gene sequence length is 1411.

[0063] In this embodiment, the highly efficient compound phosphate-solubilizing agent also includes a protectant and a culture medium. The culture medium is NBRIP medium. In this embodiment, the protectant is monosodium glutamate.

[0064] The preparation method of this highly efficient compound phosphate-solubilizing agent includes the following steps:

[0065] S1. Activation culture: Refrigerated Enterobacter holmieae HS6 and Pantotheca cumulus CT3 were streaked onto LB agar plates and incubated upside down at 28°C for 48 h to obtain the two colonies in activated culture.

[0066] S2. Preparation of bacterial suspension: Using an inoculation loop, pick single colonies from each of the two activated bacterial cultures, then inoculate them separately into LB liquid medium, and then place them in a constant temperature shaker at 28℃ and shake at 180 r / min until OD. 600 The concentrations were 0.6-0.8, resulting in HS6 and CT3 bacterial suspensions, respectively; the effective viable counts of both HS6 and CT3 bacterial suspensions were 2 × 10⁻⁶. 8 CFU / mL ~3.4×10 9 CFU / mL;

[0067] S3. Preparation of bacterial agent: The prepared HS6 bacterial suspension and CT3 bacterial suspension are mixed at a volume ratio of 1:1 to form a composite bacterial solution. This composite bacterial solution is then inoculated into NBRIP liquid medium, a protectant is added, and the medium is incubated at 28°C with shaking at 180 rpm for 48 hours to obtain the highly efficient composite phosphate-solubilizing bacterial agent. The inoculation amount of the composite bacterial solution in the NBRIP liquid medium is 1% of the medium volume.

[0068] In this experiment, the concentration of the protective agent sodium glutamate in NBRIP liquid medium was 3 g / L.

[0069] Example 2

[0070] A highly efficient compound phosphate-solubilizing agent is disclosed. The difference between this embodiment and Example 1 is that the protectant in this embodiment is trehalose. All other conditions and methods in this embodiment are the same as in Example 1.

[0071] Example 3

[0072] A highly efficient compound phosphate-solubilizing agent is disclosed. The difference between this embodiment and Example 1 is that the protectant in this embodiment is potassium chloride. All other conditions and methods in this embodiment are the same as in Example 1.

[0073] Example 4

[0074] A highly efficient compound phosphate-solubilizing agent is disclosed. The difference between this embodiment and Example 1 is that in this embodiment, the concentration of the protectant sodium glutamate in the NBRIP liquid medium is 1 g / L. All other conditions and methods in this embodiment are the same as in Example 1.

[0075] Example 5

[0076] A highly efficient compound phosphate-solubilizing agent is disclosed. The difference between this embodiment and Example 1 is that in this embodiment, the concentration of the protectant sodium glutamate in the NBRIP liquid medium is 2 g / L. All other conditions and methods in this embodiment are the same as in Example 1.

[0077] Example 6

[0078] A highly efficient compound phosphate-solubilizing agent. The difference between this embodiment and Example 1 is that in this embodiment, in step 3, the bacteria are placed in a constant temperature shaker at 28°C and shaken at 180 r / min for 96 h. The remaining conditions and methods of this embodiment are the same as those of Example 1.

[0079] Example 7

[0080] A highly efficient compound phosphate-solubilizing agent. The difference between this embodiment and Example 1 is that in this embodiment, in step 3, the bacteria are placed in a constant temperature shaker at 28°C and shaken at 180 r / min for 144 h. The remaining conditions and methods of this embodiment are the same as those of Example 1.

[0081] Example 6

[0082] A bio-fertilizer is prepared using a highly efficient compound phosphate-solubilizing agent obtained from any one of Examples 1 to 7.

[0083] Experimental testing:

[0084] (I) Detection of whether Enterobacter holmie HS6 and Pantotheca clumps CT3 can coexist

[0085] Prepare PVK solid medium. Streak the activated *Enterobacter holmieae* HS6 and *Pantotheca cumulus* CT3 strains pairwise on PVK agar plates and incubate at 30°C. Observe the bacterial growth at the crossover points daily. If the growth of both strains at the crossover points is weak or absent, it indicates an antagonistic effect between the two strains; if both strains grow well at the crossover points, it indicates no inhibitory effect between the two strains.

[0086] After two days of observation, it was found that, Figure 3 As shown, the growth at the intersection of Enterobacter holmieae HS6 and Pantotheca cumulus CT3 was good, indicating that there was no antagonism between the two bacteria, they could coexist harmoniously, and there was no inhibition between them, which meets the requirements for the preparation of compound bacterial agents by mixed culture.

[0087] (II) Determination of growth curves of Enterobacter holmie HS6 and Pantotheca cumulus CT3

[0088] The HS6 and CT3 bacterial suspensions prepared in Example 1 were inoculated into LB liquid medium and cultured in a constant temperature shaker at 28°C with shaking at 180 rpm. Growth curves were measured using an optical density reader (OD) to determine the optical density values ​​of the HS6 and CT3 bacterial suspensions. For measurement, 200 μL of each bacterial suspension was pipetted into the OD strip of the OD reader, and the OD value of each suspension was measured at 600 nm. Measurements were taken every 2 hours from 0-8 h and 14-26 h, and every hour from 8-14 h, with three consecutive measurements at each time point. The average value was then taken until the bacterial growth trend stabilized.

[0089] The growth curves of Enterobacter holmieae HS6 and Pantotheca cum Caulis CT3 are shown below. Figure 4 As shown. Figure 4 In the figure, HS6 represents the growth curve of Enterobacter holmieae HS6, and CT3 represents the growth curve of Pantotheca clumps CT3.

[0090] Depend on Figure 4 As can be seen, Enterobacter holmium HS6 was in the logarithmic growth phase after 12 hours of culture, and Pantotheca cumulus CT3 was in the logarithmic growth phase after 14 hours of culture. Therefore, both strains are suitable for inoculation.

[0091] (III) Determination of the phosphorus-solubilizing capacity of compound bacterial cultures under different fermentation conditions

[0092] Following the preparation method of Example 1, the inoculation amounts of the compound bacterial solution in NBRIP liquid medium were set at 1%, 3%, 5%, 7%, and 9%, respectively, and blank control samples were also prepared. The blank control samples were those without inoculation of the compound bacterial solution. Three replicates were prepared for each sample. The samples were then incubated in a constant temperature shaker at 28°C with shaking at 180 rpm for 120 h. Samples were taken at 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, 96 h, 108 h, and 120 h of incubation, and the available phosphorus content in the sampled bacterial solution was measured using the molybdenum-antimony colorimetric method. The test results are as follows: Figure 5 As shown.

[0093] Depend on Figure 5It is evident that the phosphate-solubilizing agent prepared by the composite bacterial culture at an inoculum of 1% reached 886.13 mg / L after 48 hours of cultivation in NBRIP liquid medium, and 911.90 mg / L after 120 hours. Considering the cultivation efficiency and the lack of significant difference in phosphate-solubilizing capacity between 48 and 120 hours, 48 ​​hours was selected as the optimal cultivation time. This demonstrates that the composite phosphate-solubilizing bacterial agent prepared in this invention possesses high phosphate-solubilizing capacity and excellent stability.

[0094] (iv) Optimization of culture conditions for compound bacterial culture

[0095] Table 1. Optimal culture conditions and effective viable bacteria count of the prepared inoculum.

[0096]

[0097]

[0098] As shown in Table 1, when HS6 and CT3 bacterial suspensions were mixed at a 1:1 volume ratio, the optimal inoculum size and incubation time were determined by screening. An inoculum size of 1% and an incubation time of 48 hours were found to be optimal, with an effective viable cell count of 6.73 × 10⁻⁶ at a 1% inoculum size and 48 hours of incubation. 8 CFU / mL. According to the requirements of the agricultural microbial inoculants (GB20287-2006), the effective viable count of liquid microbial inoculants should be above 200 million CFU / mL. This indicates that the high-efficiency compound phosphate-solubilizing inoculant prepared in this invention meets the national standard requirements and has a high phosphate-solubilizing capacity.

[0099] To determine the optimal culture conditions for the combined bacterial culture of Enterobacter holmie HS6 and Pantotheca cum Caulis CT3 after the addition of a protectant, an orthogonal experimental factor level table was proposed, as shown in Table 2.

[0100] Table 2. Factor Levels of the Three-Factor Orthogonal Experiment for Cultivation Conditions

[0101]

[0102] A three-factor orthogonal experiment was conducted to determine the type of protectant, the concentration of protectant, and the culture time. The results of the orthogonal experiment range analysis are shown in Table 3.

[0103] Table 3. Results of Range Analysis of Three-Factor Orthogonal Experiment

[0104]

[0105]

[0106] Table 3 shows that, based on the R-values ​​and comparison, the order of influence of each factor on increasing the effective viable count is: A>C>B. Therefore, among the three factors, the type of protectant has the greatest impact, followed by the culture time, while the concentration of the protectant has the least impact. Comprehensive analysis of the k1, k2, and k3 values ​​of the three factors reveals the optimal combination to be A2B3C2, i.e., the optimal condition combination is: sodium glutamate as the protectant, a protectant concentration of 3 g / L, and a culture time of 96 h. Under this optimal condition combination, the effective viable count in the high-efficiency compound phosphate-solubilizing agent prepared in this invention reaches 3.4 × 10⁻⁶. 9 The effective viable count of the compound phosphate-solubilizing agent was increased by 4.1 times compared to the agent without a protectant, at a concentration of CFU / mL. It was also determined that using monosodium glutamate as a protectant resulted in the optimal effective viable count for this highly efficient compound phosphate-solubilizing agent.

[0107] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

[0108]

[0109]

Claims

1. A highly efficient compound phosphate-solubilizing agent, characterized in that, The medium includes Enterobacter holmieae HS6, Pantotheca clumps CT3, a protectant, and a culture medium; the protectant is monosodium glutamate; the culture medium is NBRIP liquid medium. The classification name of the Enterobacter HS6 is... Enterobacter hormaechei It was deposited at the China General Microbiological Culture Collection Center on October 19, 2023, with accession number CGMCC NO.28680; The classification name of the pantothecin CT3 cluster is... Pantoea agglomerans It was deposited at the China General Microbiological Culture Collection Center on January 8, 2025, with accession number CGMCC NO.33335; The preparation method of the high-efficiency compound phosphate-solubilizing agent includes the following steps: S1. Activation culture: Refrigerated Enterobacter holmieae HS6 and Pantotheca cumulus CT3 were streaked onto LB agar plates and incubated upside down at 28°C for 48 h to obtain the two colonies in activated culture. S2. Preparation of bacterial suspension: Using an inoculation loop, pick single colonies from each of the two activated bacterial cultures, then inoculate them separately into LB liquid medium, and then place them in a constant temperature shaker at 28℃ and shake at 180 r / min until OD. 600 The concentrations were 0.6-0.8, resulting in HS6 and CT3 bacterial suspensions, respectively. S3. Preparation of bacterial agent: Mix the prepared HS6 bacterial suspension and CT3 bacterial suspension to form a compound bacterial solution, then inoculate the compound bacterial solution into NBRIP liquid medium, add a protectant, and then place it in a constant temperature shaker at 28°C and shake at 180 r / min to obtain the high-efficiency compound phosphate-solubilizing bacterial agent. The effective viable count of both the HS6 bacterial suspension and the CT3 bacterial suspension obtained in step S2 is 2 × 10⁻⁶. 8 CFU / mL ~3.4×10 9 CFU / mL; In step S3, the volume ratio of the HS6 bacterial suspension to the CT3 bacterial suspension is 1:1; the inoculation amount of the composite bacterial solution in the NBRIP liquid culture medium is 1% of the culture medium volume. The concentration of the protective agent in the NBRIP liquid medium is 1 g / L to 3 g / L; In step S3, the food is placed in a constant temperature shaker at 28°C and oscillated at 180 r / min for 48 h-144 h.

2. A bio-fertilizer, characterized in that, It is prepared using the highly efficient compound phosphate-solubilizing agent described in claim 1.

3. The application of the high-efficiency compound phosphate-solubilizing agent as described in claim 1 in agricultural soil improvement.

4. The application of the high-efficiency compound phosphate-solubilizing agent as described in claim 1 in phosphorus resource recovery.

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