Soybean seed solid fungicide pre-inoculated coating and preparation method thereof

Abstract

The invention discloses a soybean seed solid fungicide pre-inoculated coating and a preparation method thereof. The coating is prepared from an adhesive, a protective agent and compound microorganisms, the adhesive is vegetable oil; the protective agent is skim milk powder; the compound microorganism is any one or more than two of bacillus velezensis, soybean fast-growing type rhizobium and soybean slow-growing type rhizobium; the total bacterial count of the compound microorganisms is greater than or equal to 10 < 9 > cfu / mL. The invention solves the problem of poor application effect caused by low survival rate and short survival time of the existing microorganisms on the surface of the seed. According to the method, the rhizobium and various bacilli are compounded and pre-inoculated on the surfaces of the soybean seeds, so that the soybean sowing efficiency can be effectively improved, the nitrogen fixation capability of crops can be improved, and the stress resistance of the crops can also be improved. In addition, the compound microorganisms on the surfaces of the seeds can be stably stored for a long time, and the defects of low survival rate and short survival time of microorganisms on the surfaces of the seeds in the prior art are overcome.

Description

Technical Field

[0001] The invention relates to a solid bacterial agent coating, in particular to a soybean seed solid bacterial agent pre-inoculation coating and a preparation method thereof. Background Art

[0002] Seed coating is a high-tech technology that promotes agricultural production and harvest. After sowing, seeds coated with seed coating agents can quickly absorb water and swell. With the gradual development of the embryo in the seed and the continuous growth of the seedling, the seed coating agent will slowly release the various active ingredients it contains and be gradually absorbed into the body by the seed seedlings, thereby achieving the purpose of preventing and controlling seedling diseases and pests, promoting growth and development, and increasing crop yields.

[0003] Rhizobium agent refers to a microbial preparation product made with rhizobia as the production strain. It can fix nitrogen (N2) in the air and provide a large amount of nitrogen fertilizer to the host plant, thereby achieving the purpose of increasing production. When legumes have not been planted for many years or when legumes are planted in newly reclaimed land, inoculating rhizobia can ensure the good growth of legumes. Rhizobium agent, as the most important variety of microbial fertilizer, is the earliest studied and most stable microbial preparation in agricultural production research.

[0004] At present, the inoculation rate of rhizobia in leguminous crops in my country is very low, and the seed coating technology for leguminous crops is not mature enough. Existing document 1 (patent application number is 202211465818.1) reports a soybean seed coating agent and its application, which belongs to the field of soybean disease control technology. The invention directly prepares seed coating by forming a composite bacterial liquid with biocontrol bacteria, which overcomes the problem of chemical pollution in seed coating for preventing and controlling soybean Phytophthora root rot. However, the coating agent is a liquid bacterial agent, and the coating technology lacks a protective agent. It needs to be prepared and used immediately when used, and the seeds after coating cannot be stored for a long time. Document 2 (Tu Liang. Preparation and performance study of microcapsule suspension seed coating agent based on Baillus subtilis SL-13. [Master's thesis]. Xinjiang: Shihezi University, 2016) prepares microencapsulated suspended microbial seed coating agent and encapsulates Bacillus subtilis in microcapsules, but the technology is also based on liquid bacterial agent, and the process is complex and the cost is high. Due to the poor stress resistance of rhizobia, the survival rate on the seed surface is often low and the survival time is short. At present, there is a lack of pre-inoculation technology for coating rhizobia (or composite coating of rhizobia and Bacillus) on soybean seeds, making it difficult to efficiently achieve the seed coating effect of improving soybean nodulation and nitrogen fixation and reducing the use of chemical fertilizers. One of the reasons for the low survival rate of microorganisms on the seed surface is the lack of suitable protective agents and reasonable coating processes and methods, which leads to low survival rate of microorganisms on the seed surface; the second is the traditional method of using rhizobia liquid formulations for seed dressing, which requires seed dressing and use on the same day, with low work efficiency and easy delay of sowing time, thus limiting its large-scale application and rapid promotion; the third is that the application effect of single-component rhizobia agents is often not ideal. Summary of the invention

[0005] The purpose of the present invention is to provide a solid bacterial agent pre-inoculation coating for soybean seeds and a preparation method thereof, which solves the problem that the existing microorganisms have low survival rate and short survival time on the seed surface resulting in poor application effect. The composite of rhizobia and multiple bacilli not only has a high survival rate and a long survival time, but also can improve the efficiency of soybean sowing.

[0006] In order to achieve the above-mentioned object, the present invention provides a soybean seed solid bacterial agent pre-inoculation coating, which is composed of an adhesive, a protective agent and a composite microorganism; the adhesive is vegetable oil; the protective agent is skimmed milk powder; the composite microorganism is any one or more of Bacillus velez, soybean fast-growing rhizobium and soybean slow-growing rhizobium; the total bacterial count of the composite microorganism is ≥10 9 cfu / mL.

[0007] Preferably, the mass ratio of the adhesive to the protective agent is 1: (10-15); the mass ratio of the adhesive to the composite microorganism is 3: 1. Too much adhesive will reduce the seed germination rate, while too little will cause the bacterial powder to fail to adsorb to the seed surface.

[0008] The present invention provides a method for preparing the solid bacterial agent pre-inoculation coating for soybean seeds as described above, the method comprising:

[0009] (1) resuspending the composite microorganism with a protective agent, prefreezing it into a solid at -20°C to -80°C, freeze-drying it, and grinding it with a sterile mortar to obtain freeze-dried bacterial powder;

[0010] (2) The soybean seeds and the binder are mixed and stirred thoroughly, and the soybean seeds coated with the binder are mixed with the freeze-dried bacterial powder and shaken repeatedly to obtain pre-inoculated coated seeds containing the solid bacterial agent.

[0011] Preferably, the concentration of the protective agent is 10%; the volume ratio of the protective agent to the composite microorganism is 2:25.

[0012] Preferably, the freeze-drying is carried out at a cold trap temperature of -52°C and a vacuum degree of 10Pa to 20Pa.

[0013] Preferably, the mass ratio of the soybean seeds to the adhesive is 200:(1-3).

[0014] Preferably, the mass ratio of the soybean seeds coated with the adhesive to the freeze-dried bacterial powder is 20:1.

[0015] Preferably, the composite microorganism is prepared by:

[0016] (1) First, Bacillus velezensis, fast-growing soybean rhizobium, and bradyrhizobium are activated and cultured to the logarithmic growth phase, and then inoculated into liquid fermentation medium for culture until the total bacterial count reaches 10 9 cfu / mL or more, and a microbial fermentation culture fluid is obtained;

[0017] (2) The microbial fermentation culture fluid is centrifuged to remove the supernatant, and the bacterial cells are collected to obtain the composite microorganism.

[0018] Preferably, the liquid fermentation medium is BPY medium and BSE medium.

[0019] Preferably, during the inoculation, the volume of the Bacillus Velez subtilis, fast-growing soybean rhizobium or bradyrhizobium soybean is 2% of the volume of the liquid fermentation medium.

[0020] The solid bacterial agent pre-inoculation coating for soybean seeds and the preparation method thereof of the present invention solve the problem that the existing microorganisms have low survival rate and short survival time on the seed surface, resulting in poor application effect, and have the following advantages:

[0021] 1. The existing rhizobium liquid formulation seed dressing method is adopted, and the survival time of rhizobium does not exceed one week. The present invention freeze-dries rhizobium and multiple bacillus to make solid bacterial powder and then coats it on the surface of soybean seeds, which can not only effectively improve the efficiency of soybean sowing and the survival time of rhizobium (more than 28 days), but also increase the nitrogen fixation ability of crops, improve the stress resistance of crops, thereby increasing the yield of soybeans and improving the economic benefits of soybean crop planting.

[0022] 2. The composite microorganism-coated soybean seeds of the present invention can greatly reduce the use of chemical fertilizers and pesticides, and significantly improve soil fertility without harming the environment. In addition, the composite microorganisms on the surface of the seeds can be stably stored for a long time, improving the defects of low survival rate and short survival time of microorganisms on the seed surface in the past. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram for comparing the freeze-dried bacterial powder, the inner coating of soybean seeds and the outer coating of soybean seeds in Example 1 of the present invention.

[0024] Figure 2 This is a potted growth diagram of soybean seeds coated with solid bacterial agent in Experimental Example 2 of the present invention.

[0025] Figure 3 This is a statistical diagram of the potted phenotype of soybean seeds coated with solid bacterial agent in Experimental Example 2 of the present invention.

[0026] Figure 4 This is a potted growth diagram of soybean solid bacterial agent coated seeds in Experimental Example 3 of the present invention.

[0027] Figure 5 This is a statistical diagram of the potted phenotype of soybean seeds coated with solid bacterial agent in Experimental Example 3 of the present invention. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] The composition and sources of the reagents involved in the following examples are as follows:

[0030] Fahraeus nitrogen-free nutrient solution: 0.10 g of CaCl2·2H2O, 0.12 g of MgSO4·7H2O, 0.10 g of KH2PO4, 0.15 g of Na2HPO4·12H2O, 5.0 mg of ferric citrate and 1.0 mL of Gibson trace element solution, add ddH2O to 1000 mL.

[0031] Gibson trace element solution: 2.86 g H3BO 3、 2.03 g of MnSO4·4H2O, 0.22 g of ZnSO4·7H2O, 0.13 g of Na2MoO4·2H2O, and 0.08 g of CuSO4·5H2O, add ddH2O to 1000 mL.

[0032] Vegetable oil was purchased from Hubei Fubon Technology Co., Ltd.

[0033] Skim milk powder was purchased from Beijing Solebow Technology Co., Ltd.

[0034] Bacillus Velez, fast-growing soybean rhizobia and slow-growing soybean rhizobia were all purchased from Huachuang Jianong Biotechnology (Wuhan) Co., Ltd., with the trade names of Bacillus agent, fast-growing soybean rhizobia agent, and slow-growing soybean rhizobia agent, respectively.

[0035] BSE culture medium formula: 10.0 g of mannitol / sucrose, 0.5 g of K2HPO4, 0.2 g of MgSO4·7H2O, 0.1 g of NaCl, 0.1 g / 0.064 g of CaCl2·6H2O / CaCl2·2H2O, 4.0 mL of Rh trace element solution and bean sprout juice.

[0036] Bean sprout juice was prepared by boiling 500 g soybean sprouts and 1500 mL dH2O for 30 min and filtering twice with gauze.

[0037] Rh trace element solution is obtained by adding 5g Na2MoO4, 5g H3BO3 and ddH2O to 1000mL.

[0038] BPY medium formula: 5.0 g beef extract, 10.0 g peptone, 5.0 g yeast powder, 5.0 g glucose, 5.0 g NaCl, add ddH2O to 1000 mL.

[0039] Example 1

[0040] A method for preparing a solid bacterial agent pre-inoculation coating for soybean seeds, the method comprising:

[0041] (1) First, Bacillus velez, fast-growing soybean rhizobium, and bradyrhizobium were activated and cultured to the logarithmic growth phase, and then inoculated into a liquid fermentation medium at a volume percentage of 2% until the total bacterial count reached 10 9 cfu / mL or more, and a microbial fermentation culture medium is prepared. The liquid fermentation culture medium is BPY culture medium and BSE culture medium.

[0042] (2) The microbial fermentation culture liquid is centrifuged to remove the supernatant, centrifuged at 6000-8000 r / min for 5-10 minutes, the bacterial cells are collected, and the bacterial cells are resuspended in skim milk with a concentration of 10%, and the resuspended bacterial cells are placed in a glass plate, pre-frozen at -80°C to -20°C for 12 hours to 24 hours to freeze into a solid, and quickly transferred to a freeze dryer with a cold trap temperature of -52°C and a vacuum degree of 10Pa to 20Pa, and freeze-dried for 24 hours, and then ground with a sterile mortar to obtain freeze-dried bacterial powder. The volume ratio of skim milk to microbial fermentation liquid is 2:25.

[0043] (3) Mix soybean seeds and vegetable oil at a mass ratio of 200:3, stir thoroughly and repeatedly until each seed surface is evenly coated with a layer of vegetable oil film, then mix the soybean seeds coated with vegetable oil film with freeze-dried bacterial powder at a mass ratio of 20:1, shake repeatedly to allow the freeze-dried bacterial powder to be fully adsorbed onto the seeds, and obtain pre-inoculated coated seeds containing solid bacterial agent. Among them, seeds coated with Bacillus Velezii are denoted as HNA3 coating, seeds coated with fast-growing soybean rhizobium are denoted as HN01 coating, and seeds coated with slow-growing soybean rhizobium are denoted as NW5-3 coating.

[0044] like Figure 1 As shown, a schematic diagram comparing the freeze-dried bacterial powder, the inner coating of soybean seeds and the outer coating of soybean seeds in Example 1 of the present invention, wherein A is the freeze-dried bacterial powder; B is the inner coating of soybean seeds (vegetable oil film); and C is the outer coating of soybean seeds. Figure 1 It can be seen that using vegetable oil as an adhesive can adsorb the freeze-dried bacterial powder on the surface of soybean seeds to form a stable and non-detachable bacterial agent coating film.

[0045] Example 2

[0046] The preparation method of a solid bacterial agent pre-inoculation coating for soybean seeds is basically the same as that in Example 1, except that:

[0047] In step (1), Bacillus velezensis and fast-growing soybean rhizobium cultured to the logarithmic growth phase are inoculated into the same liquid fermentation medium at a volume percentage of 2% respectively and cultured until the total bacterial count reaches 10 9 cfu / mL or more, and a microbial fermentation culture medium is prepared. Alternatively, Bacillus velezensis and Bradyrhizobium sojae cultured to the logarithmic growth phase are inoculated into the same liquid fermentation medium at a volume percentage of 2% respectively, and cultured until the total bacterial count reaches 10 9 cfu / mL or more, and a microbial fermentation culture medium is prepared. The liquid fermentation culture medium is BPY culture medium and BSE culture medium.

[0048] The same operation as in Example 1 was performed to obtain coated seeds, wherein the coated seeds containing Bacillus Velez subtilis and fast-growing soybean rhizobium were recorded as HNA3+HN01 coating, and the coated seeds containing Bacillus Velez subtilis and slow-growing soybean rhizobium were recorded as NW5-3+HNA3 coating.

[0049] Experimental Example 1 Study on the ratio of soybean to adhesive

[0050] The coated seeds of Example 1, the blank control group (CK) and the comparative experimental group were sown in vermiculite, and each treatment was repeated 3 times, and 20 soybean seeds were used for each repetition, and grown in a light culture room (22°C, 16h light / 8h dark period). After 5 days of cultivation, the plant germination rate and the root length and bud length of the germinated plants were measured. The seeds of the blank control group (CK) were the same as those of Example 1 and were not coated. The comparative experimental group was a soybean (the same as the seeds of Example 1) mixed with an adhesive in other proportions, taken out and mixed with freeze-dried bacterial powder at a mass ratio of 20:1, and repeatedly shaken to fully adsorb the freeze-dried bacterial powder to obtain freeze-dried powder coated seeds with different amounts of adhesive.

[0051] Table 1 Germination rate, root length and tooth length of different treatment groups

[0052]

[0053]

[0054] From the seed germination data in Table 1, it can be seen that the ratio of soybean to adhesive is 200:3, which is a key point. When the adhesive is lower than this ratio, it has no significant effect on the germination rate, root length, and sprout length of soybeans. When the adhesive is higher than this ratio, it will significantly reduce the root length and sprout length of the seeds, affecting the normal germination of the seeds. Therefore, in order to ensure the normal germination of seeds and the absorption of more bacterial powder, the optimal mass ratio of soybean to adhesive is determined to be 200:3.

[0055] Experimental Example 2: Detection of the number of viable bacteria on the pre-inoculated coating of solid bacterial agent for soybean seeds

[0056] The coated seeds of Example 1 were stored at room temperature, and the number of viable bacteria on the surface of the seeds was measured. The sampling time was 28 days, and the results are shown in Table 2 below.

[0057] Table 2 Results of viable bacteria count test on soybean seed solid inoculant pre-inoculated coating

[0058]

[0059] From the results in Table 2, it can be seen that the number of viable bacteria in the seeds coated with Bacillus Velezii (HNA3 coating) in Example 1 reached 1×10 after 28 days. 8 The above results show that the number of live bacteria in soybean fast-growing rhizobium-coated seeds (HN01 coating) and soybean slow-growing rhizobium-coated seeds (NW5-3 coating) can reach 1×10 after 28 days. 5 The above shows that the coating formula and method provided by the present invention can ensure that rhizobia and bacillus can survive on the surface of soybean seeds for a long time.

[0060] The comparative experiment adopts the existing soybean rhizobium liquid formulation seed dressing method, which is as follows:

[0061] First, the fast-growing soybean rhizobia were activated and cultured to the logarithmic growth phase, and then inoculated into the liquid fermentation medium at a volume percentage of 2% until the total bacterial count reached 10 9 cfu / mL or more, and a microbial fermentation culture medium is prepared, wherein the liquid fermentation medium is a BSE medium. The HN01 bacterial solution of soybean fast-growing rhizobium cultured to the stable period and soybean seeds are mixed evenly at a ratio of 1:20, and stirred repeatedly until the coating is visually uniform, and blow-dried in a clean bench for 30 minutes. Store at room temperature, and take samples to determine the number of viable bacteria on the 0th and 7th days after the coating is completed.

[0062] Table 3 Results of live bacteria count test of rhizobium liquid formulation seed dressing (CFU / particle)

[0063]

[0064] From the test results in Table 3, it can be seen that after the existing liquid bacterial agent seed dressing method was stored at room temperature for 7 days, there was no live bacteria on the surface of the soybean seeds. Therefore, the existing liquid bacterial agent seed dressing method cannot keep the rhizobia alive on the seed surface for a long time.

[0065] Experimental Example 3 Tumor weight, number of tumors and nitrogenase activity of seeds treated with coating sown on vermiculite

[0066] Vermiculite was placed in a flower pot, and the soybean seeds containing solid bacterial agent pre-inoculation coating in Example 1 and Example 2 were sown, 4 per pot, 6 pots per treatment, and the seedlings were removed after 1 week, leaving one seedling with similar growth in each pot. Grow in a light culture room (22°C, 16h light / 8h dark period), and water with nitrogen-free nutrient solution once every two days. After 28d of cultivation in the light room, the nodulation was observed, and the fresh weight of the aboveground part of the plant, plant height, number of nodules, fresh weight of nodules, nitrogenase activity and other indicators were measured. Uncoated soybean seeds were used as blank controls, and soybean seeds inoculated with 1mL USDA110 liquid rhizobium agent after 7 days of sowing were used as positive controls.

[0067] like Figure 2 As shown, the potted growth diagram of soybean solid bacterial agent coated seeds in Experimental Example 2 of the present invention, wherein A is uncoated soybean seeds, blank control; B is USDA110 liquid inoculation, positive control; C is HNA3 coating of Example 1; D is HN01 coating of Example 1; E is HNA3+HN01 coating of Example 2; F is NW5-3 coating of Example 1; G is NW5-3+HNA3 coating of Example 2. Figure 2 It can be seen that compared with the blank control, the soybeans in Example 1 and Example 2 are more robust and have greener leaves, which indicates that the plants grown after coating treatment are better.

[0068] like Figure 3 As shown, the potted phenotype statistical diagram of soybean solid bacterial agent coated seeds in Experimental Example 2 of the present invention, wherein each bar graph is blank control, positive control (USDA110 liquid), HNA3 coating of Example 1, HN01 coating of Example 1, HNA3+HN01 coating of Example 2, NW5-3 coating of Example 1 and NW5-3+HNA3 coating of Example 2 from left to right. Figure 3 It can be seen that compared with the blank control (CK), the tumor weight, tumor number and nitrogenase activity of the coating treatments of Example 1 and Example 2 were all higher.

[0069] Experimental Example 4 Tumor weight, number of tumors and nitrogenase activity of coated seeds sown on soil

[0070] The soil was sieved through a 2 mm sieve and placed in a flower pot. The soybean seeds containing solid bacterial agent pre-inoculation coating of Example 1 and Example 2 were sown, 4 per pot, 6 replicates per soil sample, and seedlings with similar growth were retained after pruning after 1 week. Grow in a light culture room (22 ° C, 16 h light / 8 h dark period), irrigate with tap water once every two days to maintain soil water holding capacity. After 28 days of cultivation in the light room, the nodulation situation was observed, and the fresh weight of the aboveground part of the plant, the number of nodules, the fresh weight of nodules, the activity of nitrogenase and other indicators were measured. Uncoated soybean seeds were used as blank controls, and soybean seeds inoculated with 1 mL of USDA110 liquid rhizobium agent after 7 days of sowing were used as positive controls.

[0071] like Figure 4 As shown, the potted growth diagram of soybean solid bacterial agent coated seeds in Experimental Example 3 of the present invention, wherein A is uncoated soybean seeds, blank control; B is USDA110 liquid inoculation, positive control; C is HNA3 coating of Example 1; D is HN01 coating of Example 1; E is HNA3+HN01 coating of Example 2; F is NW5-3 coating of Example 1; G is NW5-3+HNA3 coating of Example 2. Figure 4 It can be seen that compared with the blank control, the soybean plants of Example 1 and Example 2 are more robust and have greener leaves, which indicates that the plants of Example 1 and Example 2 grow better after being coated.

[0072] like Figure 5 As shown, the potted phenotype statistical diagram of soybean solid bacterial agent coated seeds in Experimental Example 3 of the present invention, wherein each bar graph is blank control (CK), positive control (USDA110 liquid), HNA3 coating of Example 1, HN01 coating of Example 1, HNA3+HN01 coating of Example 2, NW5-3 coating of Example 1 and NW5-3+HNA3 coating of Example 2 from left to right. Figure 5 It can be seen that compared with the blank control (CK), the tumor weight, tumor number and nitrogenase activity of the coating treatments of Example 1 and Example 2 (HN01 coating, NW5-3 coating, HN01+HNA3 coating, NW5-3+HNA3 coating) were all higher.

[0073] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. A solid bacterial agent pre-inoculation coating for soybean seeds, characterized in that: The coating is composed of a binder, a protective agent and composite microorganisms; The adhesive is vegetable oil; The protective agent is skimmed milk powder; The composite microorganism is any one or more of Bacillus velez, fast-growing soybean rhizobium and bradyrhizobium; The total bacterial count of the composite microorganisms is ≥ 10 9 cfu / mL.

2. The solid bacterial agent pre-inoculation coating for soybean seeds according to claim 1, characterized in that: The mass ratio of the adhesive to the protective agent is 1:(10-15); the mass ratio of the adhesive to the composite microorganism is 3:

1.

3. A method for preparing a solid bacterial agent pre-inoculation coating for soybean seeds as claimed in any one of claims 1 to 2, characterized in that: The method includes: (1) resuspending the composite microorganism with a protective agent, prefreezing it into a solid at -20°C to -80°C, freeze-drying it, and grinding it with a sterile mortar to obtain freeze-dried bacterial powder; (2) The soybean seeds and the binder are mixed and stirred thoroughly, and the soybean seeds coated with the binder are mixed with the freeze-dried bacterial powder and shaken repeatedly to obtain pre-inoculated coated seeds containing solid bacterial agent.

4. The preparation method according to claim 3, characterized in that: The concentration of the protective agent is 10%; the volume ratio of the protective agent to the composite microorganism is 2:

25.

5. The preparation method according to claim 3, characterized in that: The freeze drying is carried out at a cold trap temperature of -52°C and a vacuum degree of 10Pa to 20Pa.

6. The preparation method according to claim 3, characterized in that: The mass ratio of the soybean seeds to the adhesive is 200:(1-3).

7. The preparation method according to claim 3, characterized in that: The mass ratio of the soybean seeds coated with the adhesive to the freeze-dried bacterial powder is 20:

1.

8. The preparation method according to claim 3, characterized in that: The composite microorganism is prepared by the following method: (1) First, Bacillus velezensis, fast-growing soybean rhizobium, and bradyrhizobium are activated and cultured to the logarithmic growth phase, and then inoculated into liquid fermentation medium for culture until the total bacterial count reaches 10 9 cfu / mL or more, and a microbial fermentation culture fluid is obtained; (2) The microbial fermentation culture fluid is centrifuged to remove the supernatant, and the bacterial cells are collected to obtain the composite microorganism.

9. The preparation method according to claim 8, characterized in that: The liquid fermentation medium is BPY medium and BSE medium.

10. The preparation method according to claim 8, characterized in that: During the inoculation, the volume of the Velez subspecies, fast-growing soybean rhizobium or bradyrhizobium is 2% of the volume of the liquid fermentation medium.

Images