Efficient composite phosphate-solubilizing bacterial agent, preparation method thereof, biological bacterial fertilizer and application

Abstract

The invention relates to the field of environment and agricultural biotechnology, in particular to a high-efficiency compound phosphate-solubilizing bacterial agent, a preparation method thereof, a biological bacterial fertilizer and application, and the high-efficiency compound phosphate-solubilizing bacterial agent comprises active ingredients of enterobacter hormaechei HS6 and pantoea agglomerans CT3. The efficient composite phosphate-solubilizing bacterial agent formed by combined application of the enterobacter hormaechei HS6 and the pantoea agglomerans CT3 has efficient phosphate-solubilizing capacity and excellent stability, no antagonism exists between the two strains, the utilization efficiency of insoluble phosphorus can be greatly improved, the efficient composite phosphate-solubilizing bacterial agent has the advantages of being excellent in phosphate-solubilizing effect and high in available phosphorus content, the usage amount of chemical phosphate fertilizer can be reduced, and the production cost is reduced. The method is suitable for being used in agricultural production. The efficient composite phosphate-solubilizing bacterial agent has good prospects in application to preparation of biological bacterial fertilizer, application to agricultural soil improvement, application to phosphorus resource recovery and application to promotion of plant growth.

Description

Technical Field

[0001] The invention relates to the fields of environment and agricultural biotechnology, and in particular to a high-efficiency composite phosphate-dissolving bacterial agent and a preparation method thereof, a biological bacterial fertilizer and applications thereof. Background Art

[0002] Phosphorus is an indispensable key nutrient element for plant growth and development. It plays an important role in plant photosynthesis, cell division and metabolism. However, most soils are currently seriously deficient in phosphorus. Phosphorus in the soil exists in the form of insoluble inorganic phosphorus, which is difficult for plants to directly absorb and utilize. In order to solve the problem of insufficient phosphorus, people apply a large amount of phosphorus fertilizer, but the utilization rate of phosphorus fertilizer by plants is generally low, only 5%-25%, which leads to resource waste and environmental pollution problems such as eutrophication of water bodies and soil acidification.

[0003] There are some phosphate-dissolving microorganisms in the soil. These microorganisms convert the insoluble phosphorus in the soil into effective phosphorus that can be absorbed by plants by secreting organic acids or enzymes. However, the phosphorus-dissolving ability of a single bacterial strain is limited. Multiple bacterial strains can be used to prepare composite phosphate-dissolving agents and further optimize them. Using composite phosphate-dissolving agents to replace some chemical phosphate fertilizers can not only greatly improve the utilization rate of phosphorus, but also reduce environmental pollution, which meets the requirements of sustainable agricultural development. However, the development of composite phosphate-dissolving agents needs to be further expanded and researched to enrich the resources of phosphate-dissolving agents and promote soil improvement and agricultural development. Summary of the invention

[0004] In order to overcome the deficiencies of the prior art, the first object of the present invention is to provide a highly efficient composite phosphate-dissolving bacteria agent. The highly efficient composite phosphate-dissolving bacteria agent expands the resources of phosphate-dissolving bacteria agents, has an excellent phosphate-dissolving effect, can effectively improve the utilization rate of phosphorus in the soil, reduce the use of chemical phosphate fertilizers, and promote the green and sustainable development of agricultural production.

[0005] In order to overcome the shortcomings of the prior art, the second purpose of the present invention is to provide a method for preparing a high-efficiency composite phosphate-dissolving bacteria agent. The preparation method is simple and easy to operate. The prepared high-efficiency composite phosphate-dissolving bacteria agent expands the resources of phosphate-dissolving bacteria agents, has excellent phosphate-dissolving effect, can effectively improve the utilization rate of phosphorus in the soil, and reduce the use of phosphate fertilizers.

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

[0007] The fourth object of the present invention is to provide an application of a highly efficient composite phosphate-dissolving bacteria agent.

[0008] In order to achieve the first object of the above invention, the technical solution adopted by the present invention is as follows:

[0009] The present invention provides a highly efficient composite phosphate-dissolving bacteria agent, including Enterobacter holmesii HS6 and Pantoea agglomerans CT3;

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

[0011] The classification name of the Pantoea agglomerans CT3 is Pantoea agglomerans, which was deposited in the General Microbiological Center of the China Culture Collection Administration on January 8, 2025, with the deposit number CGMCCNO.: 33335.

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

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

[0014] Furthermore, it also includes a protective agent and a culture medium; and / or

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

[0016] Among them, the addition of protective agents can increase the number of effective live bacteria in the bacterial suspension and promote the growth and metabolism of Enterobacter huxleyi HS6 and Pantoea agglomerans CT3.

[0017] Among them, the high-efficiency composite phosphate-dissolving bacteria agent is formed by combining Enterobacter hallii HS6 and Pantoea agglomerans CT3. Not only is there no antagonism between the two strains, but they also have high-efficiency phosphate-dissolving ability and stability. It can greatly improve the utilization efficiency of insoluble phosphorus and is suitable for use in agricultural production.

[0018] In order to achieve the second purpose of the above invention, the technical solution adopted by the present invention is as follows:

[0019] The present invention provides a method for preparing a high-efficiency composite phosphate-dissolving bacteria agent, comprising the following steps:

[0020] S1. Activation culture: Refrigerated Enterobacter HS6 and Pantoea agglomerans CT3 were streaked onto LB medium plates, and incubated at 28°C for 48 hours to obtain two activated culture colonies;

[0021] S2. Prepare bacterial suspension: Use an inoculation loop to pick a single colony from the two activated bacterial colonies, and then inoculate them into LB liquid culture medium respectively. Then place them in a constant temperature shaker at 28°C and shake at 180 r / min until OD600 is 0.6-0.8, and HS6 bacterial suspension and CT3 bacterial suspension are obtained respectively;

[0022] S3. Preparation of bacterial agent: Mix the prepared HS6 bacterial suspension and CT3 bacterial suspension to form a composite bacterial liquid, then inoculate the composite bacterial liquid into NBRIP liquid culture medium, add a protective agent, and then place it in a constant temperature shaker at 28° C. and culture at 180 r / min to obtain the highly efficient composite phosphate-dissolving bacterial agent.

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

[0024] Furthermore, 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 suspension in the NBRIP liquid culture medium is 1% of the volume percentage of the culture medium; and / or

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

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

[0027] In order to achieve the third purpose of the above invention, the technical solution adopted by the present invention is as follows:

[0028] The present invention provides a biological fertilizer, which is prepared by utilizing the above-mentioned high-efficiency composite phosphate-dissolving bacterial agent.

[0029] Among them, the high-efficiency composite phosphate-dissolving bacteria agent combines the Hallii Enterobacter HS6 and the agglomerate Pantoea CT3, so that the two strains can jointly exert the phosphate-dissolving effect, and there is no antagonism between the two strains, thereby greatly improving the utilization efficiency of insoluble phosphorus. Therefore, it has a good prospect for use in the preparation of biological fertilizers in agricultural production.

[0030] In order to achieve the fourth object of the above invention, the technical solution adopted by the present invention is as follows:

[0031] The present invention provides application of the above-mentioned high-efficiency composite phosphate-dissolving bacteria agent in agricultural soil improvement.

[0032] Specifically, the high-efficiency composite phosphate-dissolving bacteria agent is applied to the soil, and the synergistic effect of Enterobacter hullii HS6 and Pantoea agglomerans CT3 is utilized to promote the dissolution and release of phosphorus in the soil, thereby improving the effective phosphorus content in the soil and promoting plant growth.

[0033] The present invention provides application of the above-mentioned high-efficiency composite phosphate-dissolving bacteria agent in phosphorus resource recovery.

[0034] Specifically, applying the high-efficiency composite phosphate-dissolving bacteria agent to the biological dissolution of phosphorus resources can improve the recovery rate of phosphorus resources.

[0035] The present invention provides the use of the above-mentioned high-efficiency composite phosphate-dissolving bacteria agent in promoting plant growth.

[0036] Among them, the high-efficiency composite phosphate-dissolving bacteria agent combines Enterobacter hullii HS6 and Pantoea agglomerans CT3, allowing the two strains to jointly exert the phosphate-dissolving effect. It has efficient phosphate-dissolving ability and stability, and can greatly improve the utilization efficiency of insoluble phosphorus. It not only helps to improve the recovery rate of phosphorus resources and reduce environmental pollution of traditional chemical treatment methods, but also can improve agricultural soil. It can also promote plant growth when used as a biological fertilizer. Therefore, it has good application prospects in the above applications.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] (1) A highly efficient composite phosphate-dissolving bacteria agent of the present invention comprises Enterobacter horneri HS6 and Pantoea agglomerans CT3. The highly efficient composite phosphate-dissolving bacteria agent is formed by the combined application of Enterobacter horneri HS6 and Pantoea agglomerans CT3. Not only is there no antagonism between the two strains, but the two strains also have highly efficient phosphate-dissolving ability and stability, which can greatly improve the utilization efficiency of poorly soluble phosphorus and is suitable for use in agricultural production. The highly efficient composite phosphate-dissolving bacteria agent not only expands the resources of phosphate-dissolving bacteria agents, but also has a high effective phosphorus content of the bacteria agent, which can reach 886.13 mg / L. Therefore, the highly efficient composite phosphate-dissolving bacteria agent has the advantages of excellent phosphate-dissolving effect, can effectively improve the utilization rate of phosphorus in the soil, and reduce the use of chemical phosphate fertilizers, thereby promoting the green and sustainable development of agricultural production.

[0039] (2) The preparation method of the high-efficiency composite phosphate-dissolving bacteria agent of the present invention has the characteristics of being simple and easy to operate. The prepared high-efficiency composite phosphate-dissolving bacteria agent expands the resources of phosphate-dissolving bacteria agents, has excellent phosphate-dissolving effect, can effectively improve the utilization rate of phosphorus in the soil, and reduce the use of phosphate fertilizers.

[0040] (3) A biological fertilizer of the present invention is prepared by using the above-mentioned efficient composite phosphate-dissolving bacteria agent, so it can promote the dissolution of insoluble phosphorus and has a good application effect. Among them, the efficient composite phosphate-dissolving bacteria agent combines Enterobacter holmesii HS6 with Pantoea agglomerans CT3, giving full play to the synergistic effect of the two strains, having efficient phosphate-dissolving ability and stability, and can effectively improve the utilization rate of phosphorus in the soil, so it has a good prospect for application in the preparation of biological fertilizers.

[0041] (4) The application of a high-efficiency composite phosphate-dissolving bacteria agent of the present invention. Since the high-efficiency composite phosphate-dissolving bacteria agent is jointly applied by Enterobacter hullii HS6 and Pantoea agglomerans CT3, there is not only no antagonism between the two strains, but also the formed high-efficiency composite phosphate-dissolving bacteria agent has high phosphate-dissolving ability and stability, which can greatly improve the utilization efficiency of insoluble phosphorus, and can also improve agricultural soil. It can also promote plant growth when used as a biological fertilizer. Therefore, it has good application prospects in agricultural soil improvement, phosphorus resource recovery and plant growth promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

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

[0044] Figure 2 It is a scanning electron microscope image of Pantoea agglomerans CT3 of the present invention.

[0045] Figure 3 This is a diagram showing the coexistence effect of Enterobacter hallii HS6 and Pantoea agglomerans CT3 of the present invention.

[0046] Figure 4 It is a growth curve diagram of Enterobacter hallii HS6 and Pantoea agglomerans CT3 of the present invention.

[0047] Figure 5 It is a graph showing the results of the phosphorus solubilization ability test of the composite bacterial solution with different inoculation amounts at different culture periods in the present invention. DETAILED DESCRIPTION

[0048] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0049] The terms used in the embodiments of the present invention are only for the purpose of describing specific embodiments, and are not intended to limit the present invention. In the present invention, the singular forms "a", "said" and "the" used in the embodiments and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0050] Unless otherwise specified, the 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, Ca 3 (PO 4 ) 2 5g, MgSO 4 0.1g, (NH 4 ) 2 SO 4 0.5g, glucose 15g, chloramphenicol 50mg, streptomycin 50mg, pH 6.8~7.0.

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

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

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

[0056] NBRIP medium (g / L): glucose 10 g, (NH 4 ) 2 SO 4 0.5g, NaCl 0.3g, MgSO 4 7H 2 O0.3g, FeSO 4 7H 2 O 0.03g, MnSO 4 ·2H 2 O 0.03g, Ca 3 (PO 4 ) 2 5g, KCl 0.3g, lecithin 0.2g, pH 7.2~7.4.

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

[0058] Example 1

[0059] A highly efficient composite phosphate-dissolving bacteria agent, comprising active ingredients Enterobacter hormaechei HS6 and Pantoea agglomerans CT3; wherein, the classification name of Enterobacter hormaechei HS6 is Enterobacter hormaechei, which has been deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration on October 19, 2023, with a deposit number of CGMCC NO.: 28680, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. wherein, the classification name of Pantoea agglomerans CT3 is Pantoea agglomerans, which has been deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration on January 8, 2025, with a deposit number of CGMCC NO.: 33335, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0060] Among them, the scanning electron micrograph of Enterobacter huoyi HS6 is shown in Figure 1 As shown in the scanning electron micrograph of Pantoea agglomerata CT3, Figure 2 shown.

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

[0062] The 16S rDNA gene sequence of Pantoea agglomerans CT3 is shown in SEQ ID No. 2. The length of the gene sequence is 1411.

[0063] In this embodiment, the high-efficiency composite phosphate-dissolving bacteria agent further comprises a protective agent and a culture medium. The culture medium is NBRIP culture medium. In this embodiment, the protective agent is sodium glutamate.

[0064] The preparation method of the high-efficiency composite phosphate-dissolving bacteria agent comprises the following steps:

[0065] S1. Activation culture: Refrigerated Enterobacter HS6 and Pantoea agglomerans CT3 were streaked onto LB medium plates, and incubated inverted at 28°C for 48 hours to obtain two activated culture colonies;

[0066] S2. Prepare bacterial suspension: Use an inoculation loop to pick a single colony from the two activated bacterial colonies, and then inoculate them into LB liquid culture medium respectively. Then place them in a constant temperature shaker at 28°C and shake at 180 r / min until OD 600 The effective viable counts of HS6 and CT3 bacterial suspensions were both 2×10 8 CFU / mL~3.4×10 9 CFU / mL;

[0067] S3, preparing bacterial agent: the prepared HS6 bacterial suspension and CT3 bacterial suspension were mixed at a volume ratio of 1:1 to form a composite bacterial solution, and then the composite bacterial solution was inoculated into NBRIP liquid culture medium, and a protective agent was added, and then placed in a constant temperature shaker at 28°C and shaken at 180r / min for 48h to obtain the highly efficient composite phosphate-dissolving bacterial agent. The inoculation amount of the composite bacterial solution in the NBRIP liquid culture medium is 1% of the volume percentage of the culture medium.

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

[0069] Example 2

[0070] A highly efficient composite phosphate-dissolving bacteria agent, the difference between this embodiment and embodiment 1 is that in this embodiment, the protective agent is trehalose. The remaining conditions and methods of this embodiment are the same as those of embodiment 1.

[0071] Example 3

[0072] A highly efficient composite phosphate-dissolving bacteria agent, the difference between this embodiment and embodiment 1 is that in this embodiment, the protective agent is potassium chloride. The remaining conditions and methods of this embodiment are the same as those of embodiment 1.

[0073] Example 4

[0074] A highly efficient composite phosphate-dissolving bacteria agent, the difference between this embodiment and embodiment 1 is that in this embodiment, the concentration of the protective agent sodium glutamate in the NBRIP liquid culture medium is 1 g / L. The remaining conditions and methods of this embodiment are the same as those of embodiment 1.

[0075] Example 5

[0076] A highly efficient composite phosphate-dissolving bacteria agent, the difference between this embodiment and embodiment 1 is that in this embodiment, the concentration of the protective agent sodium glutamate in the NBRIP liquid culture medium is 2 g / L. The remaining conditions and methods of this embodiment are the same as those of embodiment 1.

[0077] Example 6

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

[0079] Example 7

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

[0081] Example 6

[0082] A biological fertilizer is prepared by using a high-efficiency composite phosphate-dissolving bacterial agent obtained by any one of Examples 1 to 7.

[0083] Experimental testing:

[0084] (I) Detection of whether Enterobacter holmesii HS6 and Pantoea agglomerans CT3 can coexist

[0085] Prepare PVK solid culture medium, inoculate the activated cultured Enterobacter HS6 and Pantoea agglomerans CT3 strains on PVK culture medium plates in pairs, and culture at 30°C. Observe the bacterial growth at the intersection every day. If the growth of the two strains at the intersection is weak or not growing, it means that there is antagonism between the two strains; if both strains grow well at the intersection, it means that there is no inhibitory effect between the two strains.

[0086] After two days of observation, it was found that Figure 3 As shown, the intersection of Enterobacter hallii HS6 and Pantoea agglomerans CT3 grew well, indicating that there was no antagonism between the two bacteria, and they could coexist harmoniously without inhibitory effects on each other, which met the requirements for mixed culture to prepare composite bacterial agents.

[0087] (II) Determination of the growth curves of Enterobacter holmesii HS6 and Pantoea agglomerans CT3

[0088] The HS6 bacterial suspension and CT3 bacterial suspension prepared in Example 1 were inoculated into LB liquid culture medium, respectively, and placed in a constant temperature shaker at 28°C and cultured at 180r / min. The growth curve was determined, and the optical density values ​​of the HS6 bacterial suspension and the CT3 bacterial suspension were determined using an ELISA instrument, respectively, by optical density measurement. During the measurement, 200 μL of the two bacterial suspensions were respectively transferred to the ELISA strip with a pipette, and the OD value of each group of bacterial suspensions was determined at a wavelength of 600nm after transfer. During 0-8h and 14-26h, the bacterial suspension was measured once every 2h, and during 8-14h, the bacterial suspension was measured once every 1h. Each time point was measured 3 times in a row, and the average value was finally taken until the bacterial growth trend stabilized.

[0089] Among them, the growth curves of Enterobacter holmesii HS6 and Pantoea agglomerans CT3 are as follows: Figure 4 shown. Figure 4 HS6 represents the growth curve of Enterobacter hallii HS6, and CT3 represents the growth curve of Pantoea agglomerans CT3.

[0090] Depend on Figure 4It can be seen that after 12 hours of culture, Enterobacter HS6 is in the logarithmic growth phase, and after 14 hours of culture, Pantoea agglomerans CT3 is in the logarithmic growth phase. Therefore, both strains are suitable for inoculation.

[0091] (III) Determination of the phosphorus solubilizing capacity of composite bacterial solutions under different fermentation conditions

[0092] According to the preparation method of Example 1, the inoculation amount of the composite bacterial solution in the NBRIP liquid culture medium was set to 1%, 3%, 5%, 7% and 9%, respectively, and a blank control sample was set. Among them, the blank control sample is a sample without the composite bacterial solution. Three parallel samples were set for each sample. Then it was placed in a constant temperature shaker at 28°C and cultured at 180r / min for 120h. Samples were taken at 12h, 24h, 36h, 48h, 60h, 72h, 84h, 96h, 108h and 120h of culture, and the molybdenum antimony colorimetric method was used to measure the effective phosphorus content in the sampled bacterial solution. The test results are as follows Figure 5 shown.

[0093] Depend on Figure 5 It can be seen that the phosphate solubilization amount of the composite bacterial solution with an inoculation amount of 1% in NBRIP liquid medium after 48 hours of cultivation reaches 886.13 mg / L, and the phosphate solubilization amount reaches 911.90 mg / L after 120 hours of cultivation. Considering the culture efficiency and the fact that there is no significant difference in the phosphate solubilization amount between 48 hours and 120 hours, 48 ​​hours is selected as the optimal culture time. This shows that the composite phosphate solubilizing bacteria agent prepared by the present invention has efficient phosphate solubilization ability and excellent stability.

[0094] (IV) Optimization of culture conditions of composite bacterial solution

[0095] Table 1 Optimal culture conditions and effective viable bacterial counts of the obtained inoculants

[0096]

[0097]

[0098] As shown in Table 1, when the HS6 bacterial suspension and the CT3 bacterial suspension were mixed at a volume ratio of 1:1, the inoculation amount and the culture time were screened. The optimal inoculation amount was 1% and the culture time was 48h. The effective live bacteria performance of 1% inoculation amount and 48h cultured for 48h reached 6.73×10 8 CFU / mL. According to the requirements of the Agricultural Microbial Agent (GB20287-2006), the effective viable count of liquid microbial agents should be above 200 million CFU / mL. This shows that the high-efficiency composite phosphate-dissolving agent prepared by the present invention meets the requirements of the national standard and has a high-efficiency phosphate-dissolving ability.

[0099] In order to detect the optimal culture conditions of the composite bacterial liquid of Enterobacter HS6 and Pantoea agglomerans CT3 after adding protective agents, an orthogonal experimental factor level table was proposed, as shown in Table 2.

[0100] Table 2 Factor levels of three-factor orthogonal test of culture conditions

[0101]

[0102] A three-factor orthogonal test was conducted on the type of protective agent, the concentration of protective agent added and the culture time, and the results of the orthogonal test range analysis were obtained, as shown in Table 3.

[0103] Table 3 Three-factor orthogonal test range analysis results

[0104]

[0105]

[0106] As can be seen from Table 3, according to the results of the R value and by comparing the R value, the order of the effects of the various influencing factors on increasing the number of effective live bacteria is: A>C>B. Based on this, the type of protective agent has the greatest influence among the three factors, followed by the culture time, and the concentration of the protective agent added has the least influence. Comprehensively analyzing the k1, k2 and k3 values ​​of the three factors, the best combination screened out is A2B3C2, that is, the best condition combination is: the protective agent is sodium glutamate, the concentration of the protective agent is 3g / L, and the culture time is 96h. Under this optimal condition combination, the number of effective live bacteria in the high-efficiency composite phosphate-dissolving bacteria agent prepared by the present invention reaches 3.4×10 9 CFU / mL, the effective viable bacteria count of the phosphate-dissolving agent without adding protective agent increased by 4.1 times. At the same time, it was also determined that the use of sodium glutamate as a protective agent to prepare the high-efficiency composite phosphate-dissolving agent has the optimal effective viable bacteria count.

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

[0108]

[0109]

Claims

1. A highly efficient composite phosphate-dissolving bacteria agent, characterized in that: These include Enterobacter hallii HS6 and Pantoea agglomerans CT3; The classification name of the Enterobacter hormaechei HS6 is Enterobacter hormaechei, which was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on October 19, 2023, with a deposit number of CGMCC NO.: 28680; The classification name of the Pantoea agglomerans CT3 is Pantoea agglomerans, which was deposited in the General Microbiological Center of the China Culture Collection Administration on January 8, 2025, with the deposit number CGMCCNO.: 33335.

2. A highly efficient composite phosphate-dissolving bacteria agent as claimed in claim 1, characterized in that: The 16SrDNA gene sequence of the Enterobacter holmesii HS6 is shown in SEQ ID No. 1; The 16S rDNA gene sequence of the Pantoea agglomerans CT3 is shown in SEQ ID No.

2.

3. A highly efficient composite phosphate-dissolving bacteria agent as claimed in claim 1, characterized in that: Also includes a protective agent and a culture medium; and / or The protective agent is one of sodium glutamate, trehalose or potassium chloride; and the culture medium is NBRIP culture medium.

4. The method for preparing a highly efficient composite phosphate-dissolving bacteria agent according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1. Activation culture: Refrigerated Enterobacter HS6 and Pantoea agglomerans CT3 were streaked onto LB medium plates, and incubated inverted at 28°C for 48 hours to obtain two activated culture colonies; S2. Prepare bacterial suspension: Use an inoculation loop to pick a single colony from the two activated bacterial colonies, and then inoculate them into LB liquid culture medium respectively. Then place them in a constant temperature shaker at 28°C and shake at 180 r / min until OD 600 is 0.6-0.8, and HS6 bacterial suspension and CT3 bacterial suspension are obtained respectively; S3. Preparation of bacterial agent: Mix the prepared HS6 bacterial suspension and CT3 bacterial suspension to form a composite bacterial liquid, then inoculate the composite bacterial liquid into NBRIP liquid culture medium, add a protective agent, and then place it in a constant temperature shaker at 28° C. and culture at 180 r / min to obtain the highly efficient composite phosphate-dissolving bacterial agent.

5. The method for preparing a highly efficient composite phosphate-dissolving bacteria agent according to claim 4, characterized in that: The effective viable counts of the HS6 bacterial suspension and the CT3 bacterial suspension prepared in step S2 were both 2×10 8 CFU / mL~3.4×10 9 CFU / mL.

6. The method for preparing a highly efficient composite phosphate-dissolving bacteria agent according to claim 4, characterized in that: 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 suspension in the NBRIP liquid culture medium is 1% of the volume percentage of the culture medium; and / or The protective agent is one of sodium glutamate, trehalose or potassium chloride; the concentration of the protective agent in the NBRIP liquid culture medium is 1g / L to 3g / L; and / or In step S3, the culture is placed in a constant temperature shaker at 28°C and shaken at 180 r / min for 48h-144h.

7. A biological fertilizer, characterized in that: The invention is prepared by using a high-efficiency composite phosphate-dissolving bacteria agent as described in any one of claims 1 to 3.

8. Use of a high-efficiency composite phosphate-dissolving bacteria agent as described in any one of claims 1 to 3 in agricultural soil improvement.

9. Use of a high-efficiency composite phosphate-dissolving bacteria agent as described in any one of claims 1 to 3 in phosphorus resource recovery.

10. Use of a high-efficiency composite phosphate-dissolving bacteria agent as described in any one of claims 1 to 3 in promoting plant growth.

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