Microbial agent as well as preparation method and application thereof

Abstract

The invention relates to the field of microbial fertilizers, in particular to a microbial agent and a preparation method and application thereof. The preparation method of the microbial agent comprises the following steps: base material sterilization: mixing corn starch, peptone, yeast powder and water, then heating to 121 DEG C for steam sterilization, and cooling to obtain a sterilized base material; bacillus subtilis, bacillus amyloliquefaciens, bacillus licheniformis, bacillus polymyxa and a protective agent are put into the sterilized base material to form a fermentation mixture, then sterile air is introduced for fermentation culture, and a microbial agent is obtained; the protective agent comprises at least one of D-sodium erythorbate, tocotrienol and tea polyphenol. The protective agent is added to scavenge free radicals and relieve the influence of oxidative stress on the strain, so that the growth rate, metabolic activity and survival rate of the strain are improved, and the yield and production efficiency of the strain are remarkably improved.

Description

Technical Field

[0001] The present application relates to the field of microbial fertilizers, and in particular to a microbial agent and a preparation method and application thereof. Background Art

[0002] Under the current situation, agricultural non-point source pollution, as a negative product of agriculture, has become increasingly prominent as a shackle to the sustainable development of agriculture, and has been highly valued by the whole society. A large number of existing studies have shown that excessive use of chemical fertilizers is one of the main causes of agricultural non-point source pollution at this stage.

[0003] Agricultural microbial agents refer to live bacterial preparations made by industrially expanding and multiplying target microorganisms (effective bacteria). They have the functions of directly or indirectly improving soil, restoring soil fertility, maintaining the balance of rhizosphere microbial flora, and degrading toxic and harmful substances. They are applied to agricultural production and, through the life activities of the microorganisms contained therein, increase the supply of plant nutrients or promote plant growth, improve the quality of agricultural products and the agricultural ecological environment. Compared with traditional fertilizers, microbial fertilizers have advantages in protecting the ecology, utilizing agricultural waste resources, maintaining soil health, and improving fertilizer utilization and agricultural product quality.

[0004] The bacteria used for biocontrol are mainly Bacillus, including Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus polymyxa. Bacillus has the ability to inhibit plant diseases, is a non-pathogenic bacterium widely found in nature, is harmless to humans and animals, and does not pollute the environment, so it has attracted much attention.

[0005] Many Bacillus only target a certain type of pathogen, have a narrow antibacterial spectrum, and are of a single variety, so they are usually prepared together with multiple Bacillus. In actual operation, various Bacillus are stored separately, which is inconvenient. Therefore, people consider co-fermenting multiple Bacillus, but co-fermenting multiple Bacillus is likely to affect the overall yield after co-fermentation due to the mutual influence of their metabolites. Summary of the invention

[0006] In order to solve the problem that the co-fermentation of multiple Bacillus subtilis affects the overall yield after fermentation, the present application provides a method for preparing a microbial agent, which improves the growth rate, metabolic activity and survival rate of the strain by adding a protective agent to scavenge free radicals and reduce the impact of oxidative stress on the strain, thereby significantly improving the yield and production efficiency of the strain.

[0007] In the first aspect, the present application provides a method for preparing a microbial agent, using the following technical solution: A method for preparing a microbial agent comprises the following steps: Sterilization of base material: corn starch, peptone, yeast powder and water are mixed, and then heated to 121°C for steam sterilization, and the sterilized base material is obtained after cooling; Feeding and fermentation: Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus polymyxa as well as a protective agent are added to a sterilized base material to form a fermentation mixture, and then sterile air is introduced for fermentation culture to obtain a microbial agent; The protective agent includes at least one of sodium D-isoascorbate, tocotrienol and tea polyphenols.

[0008] By adopting the above technical scheme, during the simultaneous fermentation of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus polymyxa, a large number of free radicals will be generated due to the metabolic activities and interactions of each strain. Free radicals are highly reactive molecules or atomic groups that can attack biological molecules such as lipids, proteins and nucleic acids in cells, causing damage to cell structure and function. For strains in the fermentation process, the generation of free radicals will induce oxidative stress, affect the growth rate, metabolic activity and survival rate of the strains, and thus reduce the yield of the strains.

[0009] 1. Protective agents such as sodium isoascorbate, tocotrienols and tea polyphenols have powerful antioxidant functions. They can neutralize free radicals by providing electrons or hydrogen atoms, thereby preventing the occurrence of free radical chain reactions. This antioxidant effect helps protect biological molecules in cells from free radical attacks and maintain the integrity of cell structure and function.

[0010] Because protectants can scavenge free radicals and reduce the effects of oxidative stress on bacteria, they can significantly increase the growth rate and metabolic activity of bacteria. During the fermentation process, bacteria can reproduce faster and accumulate metabolites, thereby increasing the yield of bacteria. In addition, the use of protectants can also reduce the number of bacteria that die due to oxidative stress, further improving the survival rate and final yield.

[0011] With the increase in strain production, the production efficiency of the entire fermentation process has also been significantly improved. The addition of protective agents makes the fermentation process more stable and controllable, reducing production delays and cost increases caused by strain death or slow growth. At the same time, since the use of protective agents can optimize fermentation conditions and promote strain growth and accumulation of metabolites, it can also improve the quality and performance of microbial agents.

[0012] The present invention adds protective agents (sodium D-isoascorbate, tocotrienols and tea polyphenols) to remove free radicals and reduce the impact of oxidative stress on the strains, thereby increasing the growth rate, metabolic activity and survival rate of the strains, thereby significantly increasing the yield and production efficiency of the strains. This makes the preparation process of the microbial agent more efficient and controllable, and helps to improve the quality and performance of the final product.

[0013] Preferably, the protective agent is sodium D-isoascorbate, and the mass fraction of sodium D-isoascorbate in the fermentation mixture is 0.8%-1.2%.

[0014] By adopting the above technical solution, the main function of sodium D-isoascorbate is to remove free radicals generated during the fermentation process and protect the strains from damage by oxidative stress. If its content is too low, it may not provide sufficient antioxidant capacity to neutralize all the free radicals generated, resulting in oxidative damage to the strains, affecting growth and accumulation of metabolites. Due to insufficient antioxidant capacity, the survival rate, growth rate and metabolic activity of the strains during the fermentation process may be negatively affected, thereby reducing the yield of the strains.

[0015] When the content of sodium D-erythorbate is too high, the yield of each strain cannot be further increased. For production cost considerations, there is no need to further increase the amount of sodium D-erythorbate added.

[0016] Preferably, the protective agent is tocotrienol, and the mass fraction of the tocotrienol in the fermentation mixture is 0.5%-1%.

[0017] By adopting the above technical scheme, tocotrienol has the structure of vitamin E and contains three carbon-carbon double bonds, which makes it have strong antioxidant capacity. Therefore, its used content is slightly lower than D-sodium isoascorbate and can effectively improve the yield and production efficiency of each strain. If its content is too low, it may not provide enough antioxidant protection to neutralize the free radicals produced in the fermentation process, thereby causing the strain to be oxidatively damaged, affecting the growth rate and the accumulation of metabolites. The accumulation of free radicals can damage the cell structure and function of the strain and reduce the survival rate. The shortage of tocotrienol may aggravate this damage and further reduce the yield of the strain. When the content of tocotrienol is too high, the yield of each strain cannot be further increased, and for production cost considerations, there is no need to further increase the addition amount of tocotrienol.

[0018] Preferably, the protective agent is tea polyphenols, and the mass fraction of the tea polyphenols in the fermentation mixture is 1.2%-1.8%.

[0019] By adopting the above technical solution, tea polyphenols is the general name of polyphenols in tea leaves, and its effective ingredients for scavenging free radicals may be lower than those of sodium D-erythorbate and tocotrienols. Therefore, the required content is higher than those of sodium D-erythorbate and tocotrienols.

[0020] If the content of tea polyphenols is too low, it may not provide sufficient antioxidant protection to neutralize the free radicals produced during the fermentation process, resulting in oxidative damage to the strains, affecting growth and the accumulation of metabolites. When the content of tea polyphenols is too high, the yield of each strain cannot be further increased. For production cost considerations, there is no need to further increase the amount of tea polyphenols added.

[0021] Preferably, the protective agent is a protective agent mixture of tocotrienol and tea polyphenols.

[0022] By adopting the above technical scheme, tocotrienols and tea polyphenols are both effective antioxidants, which can remove free radicals and protect cells from oxidative damage. When they are used in combination, they can enhance each other's antioxidant capacity and form a stronger antioxidant barrier. This synergistic effect makes the mixed protective agent show higher efficiency in removing free radicals and so on.

[0023] Tocotrienols and tea polyphenols have different antioxidant mechanisms and targets. The mixed use of these two protective agents can expand the antioxidant range, cover more biological molecules and cell structures, and thus provide more comprehensive protection. Tocotrienols can play an antioxidant role more effectively because of its unsaturated side chain. It can not only scavenge free radicals, but also protect key enzymes such as cytochrome P450 from oxidative damage. In addition to its antioxidant effect, tea polyphenols also have multiple biological activities such as antibacterial, anti-inflammatory, and weight loss. These functions enable tea polyphenols to protect bacteria from oxidative damage while also improving the fermentation environment, promoting the growth of bacteria and the accumulation of metabolites.

[0024] When tocotrienols and tea polyphenols are used in combination, their synergistic effects and their unique antioxidant properties are superimposed on each other, making the mixed protective agent show a higher effect in the preparation of microbial inoculants. This mixed protective agent can not only more effectively remove free radicals and protect the strains from oxidative damage, but also improve the fermentation environment, increase the growth rate of the strains and the accumulation of metabolites, thereby significantly improving the yield and production efficiency of microbial inoculants.

[0025] Preferably, the mass ratio of tocotrienol to tea polyphenols in the protective agent mixture is 1:0.3-0.5.

[0026] By adopting the above technical scheme, the mixed use of tocotrienols and tea polyphenols can produce a synergistic antioxidant effect and improve the overall antioxidant efficiency. If the proportion of tea polyphenols is too low, this synergistic effect may be weakened, resulting in a decrease in the antioxidant capacity of the mixed protective agent. In addition to its antioxidant effect, tea polyphenols also have a variety of biological activities such as antibacterial and anti-inflammatory. These functions help to improve the fermentation environment, promote the growth of bacteria and the accumulation of metabolites. Too low a tea polyphenol content may affect the performance of these positive effects, thereby reducing the yield and production efficiency of the bacteria.

[0027] The antioxidant property of tocotrienols is higher than that of tea polyphenols. If the proportion of tea polyphenols is too high, it will reduce the antioxidant property of the protective agent, thereby reducing the yield and production efficiency of the strain.

[0028] In a second aspect, the present application provides a microbial agent, which adopts the following technical solution: A microbial agent is prepared by the above-mentioned preparation method of the microbial agent.

[0029] In a third aspect, the present application provides an application of a microbial agent, using the following technical solution: An application of a microbial agent, comprising the application of the above-mentioned microbial agent in rice and corn production.

[0030] By adopting the above technical solution, the beneficial microorganisms in the microbial agent can decompose organic matter in the soil, release nutrients such as nitrogen, phosphorus, potassium, etc., and improve soil fertility. These beneficial microorganisms can also promote the formation of soil aggregate structure, increase soil aeration and water retention, and provide a good environment for crop root growth. The beneficial microorganisms in the microbial agent can secrete plant growth hormones, such as auxin, gibberellin, etc., to stimulate the root growth of rice and corn, make their root systems more developed, and enhance the ability to absorb soil nutrients.

[0031] The soil fertility of rice fields using the microbial agent has been significantly improved, the rice root system is well developed, the leaves are dark green, and the growth is vigorous. The microbial agent can also improve the photosynthesis efficiency of rice and increase the yield and quality of rice. The results of the field test showed that the rice yield using the microbial agent was significantly higher than that of the control group without the agent.

[0032] In corn cultivation, the microbial agent also showed significant effects. In corn fields where the agent was used, the soil structure was improved, the corn plants grew stronger, and the incidence of diseases was reduced. The microbial agent can also promote the growth and development of corn, delay plant aging, and increase corn yield and quality. The test report shows that the per mu yield of corn can be significantly increased by applying a certain amount of microbial agent per mu of land.

[0033] Preferably, the microbial agent is applied during the tillering stage, jointing stage and flowering stage of rice.

[0034] By adopting the above technical solution, the tillering stage is an important stage of rice nutritional growth. At this time, rice begins to tiller and form more tiller seedlings, laying the foundation for subsequent panicle development. Rice needs a lot of nutrients during the tillering stage to support the growth and development of tiller seedlings. The beneficial microorganisms in the microbial agent can decompose organic matter in the soil, release nutrients such as nitrogen, phosphorus, and potassium, and meet the nutrient needs of rice.

[0035] The jointing stage is a critical period for rice to transition from vegetative growth to reproductive growth. At this time, the rice stems begin to elongate and thicken, laying the foundation for subsequent heading and filling. During the jointing stage, the demand for nutrients of rice further increases, and microbial agents can continuously provide nutrient support for rice to meet its growth needs.

[0036] The flowering stage is an important stage of rice reproductive growth, when rice begins to bloom, pollinate, and form grains. During the flowering stage, rice still has a high demand for nutrients, especially phosphorus, potassium and other nutrients that are essential for the formation and development of grains. Microbial agents can continue to provide nutrient support for rice and promote the fullness and quality of its grains.

[0037] Preferably, the microbial agent is applied during the corn tasseling stage and the early heading stage.

[0038] By adopting the above technical solution, after corn enters the trumpet stage, the plant grows rapidly, the leaves are wide, and the photosynthesis ability is strong. It is a critical period for both vegetative growth and reproductive growth. At this time, the demand for nutrients of corn increases greatly, especially nutrients such as nitrogen, phosphorus, and potassium. The beneficial microorganisms in the microbial agent can decompose organic matter in the soil, release these nutrients, and meet the growth needs of corn.

[0039] The pre-heading period is a critical period for the reproductive growth of corn, when corn begins to head and form grains. The application of microbial agents can provide sufficient nutrient support for corn and promote the fullness and quality of its grains. The beneficial microorganisms in microbial agents may indirectly increase the pollination rate and fruiting rate of corn by improving the growth environment and nutritional status of corn, thereby increasing yield.

[0040] In summary, this application has the following beneficial effects: 1. Since the present invention removes free radicals and reduces the effect of oxidative stress on the strain by adding protective agents (sodium D-isoascorbate, tocotrienols and tea polyphenols), the growth rate, metabolic activity and survival rate of the strain are increased, thereby significantly improving the yield and production efficiency of the strain; 2. The present application uses a mixture of tocotrienols and tea polyphenols, which can not only more effectively remove free radicals and protect bacteria from oxidative damage, but also improve the fermentation environment, increase the growth rate of bacteria and the accumulation of metabolites, thereby significantly improving the yield and production efficiency of microbial agents. DETAILED DESCRIPTION

[0041] The raw materials in this application include the following parts: Corn starch: a commercially available product is used. This application uses a commercially available product of Suzhou Hanlin Chemical Co., Ltd. as an example; Peptone: a commercially available product is used. This application uses the commercially available product of Yiweilong (Xiamen) Biotechnology Co., Ltd. as an example; Yeast powder: a commercially available product is used. This application uses a commercially available product of Shandong Xinzhuoyuan Biotechnology Co., Ltd. as an example for illustration. Water: a commercially available product with a CAS number of 7732-18-5 is used. Bacillus subtilis: commercially available products are used. This application uses the commercially available products of Henan Yangshao Biotechnology Co., Ltd. as an example for illustration; Bacillus amyloliquefaciens: a commercially available product is used. This application uses a commercially available product of Henan Yangshao Biotechnology Co., Ltd. as an example; Bacillus licheniformis: commercially available products are used. This application uses the commercially available products of Hubei Qiming Bioengineering Co., Ltd. as an example; Bacillus polymyxa: commercially available products are used. This application uses the commercially available products of Henan Yangshao Biotechnology Co., Ltd. as an example; D-sodium isoascorbate: a commercially available product with CAS number 7378-23-6; Tocotrienol: a commercial product with CAS number 490-23-3 was used; Tea polyphenols: a commercially available product with CAS number 84650-60-2 was used; The present application is further described in detail below in conjunction with embodiments and comparative examples.

[0042] Example 1 A method for extracting a microbial agent comprises the following steps: Sterilization of base material: 40 g corn starch, 40 g peptone, 20 g yeast powder and 1380 g water were mixed, and then heated to 121°C for 30 min steam sterilization, and then cooled to 37°C to obtain a sterilized base material; Feeding and fermentation: 100 g of Bacillus subtilis (200 billion CFU / g), 50 g of Bacillus amyloliquefaciens (100 billion CFU / g), 50 g of Bacillus licheniformis (100 billion CFU / g), 50 g of Bacillus polymyxa (10 billion CFU / g) and 12 g of sodium D-isoascorbate are added to 738 g of sterilized base material to form a fermentation mixture, wherein the weight of the sterilized base material needs to be adjusted to ensure that the total weight of the fermentation mixture is 1000 g, and then sterile air is introduced for fermentation and culture for 30 minutes, maintaining the dissolved oxygen concentration at 20%, to obtain a microbial agent.

[0043] Embodiment 2-4 Example 2-3 Based on the preparation method of Example 1, the content of sodium D-isoascorbate was adjusted, and the specific adjustment is shown in Table 1.

[0044] Comparative Example 1 Comparative Example 1 Based on the preparation method of Example 1, 12 g of D-sodium isoascorbate and 738 g of sterilized base material were replaced with 750 g of sterilized base material, and the other conditions remained unchanged.

[0045] Table 1 Content and performance test table of sodium D-isoascorbate of Examples 1-5 and Comparative Example 1 Performance testing The microbial agents of Examples 1-5 and Comparative Example 1 were subjected to the following performance tests, and the test results are shown in Table 1: The total colony count in the microbial inoculant was determined according to GB 4789.2-2016.

[0046] Referring to Table 1, by comparing Examples 1-5 with Comparative Example 1, it can be seen that the use of sodium D-isoascorbate and Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus polymyxa for simultaneous fermentation can increase the total colony count of the microbial agent, because the main function of sodium D-isoascorbate is to remove free radicals generated during the fermentation process and protect the strains from damage by oxidative stress, thereby increasing the total colony count of the microbial agent.

[0047] As the content of sodium D-isoascorbate continued to increase, the total colony count of the microbial inoculant showed a trend of first increasing and then tending to a stable state. It may be that as the content of sodium D-isoascorbate continued to increase, the free radicals produced were continuously neutralized to avoid oxidative damage to the bacteria, thereby continuously increasing the total colony count of the microbial inoculant.

[0048] After comparison, Example 1 can be regarded as a preferred embodiment.

[0049] Embodiment 6-9 Example 6 Based on the preparation method of Example 1, 12 g of sodium D-isoascorbate was replaced with 10 g of tocotrienol, and the other conditions remained unchanged.

[0050] In Examples 7-9, based on the preparation method of Example 6, the amount of tocotrienol added was adjusted. The specific adjustments are shown in Table 2.

[0051] The microbial agents of Examples 6-9 were subjected to the above performance tests, and the test results are shown in Table 2.

[0052] Table 2 The addition amount and performance test table of tocotrienol in Example 1 and Examples 6-9 project Example 1 Example 6 Example 7 Example 8 Example 9 Tocotrienols / g / 10 5 8 12 Total colony count / (100 million CFU / mL) 806 807 705 769 807 Referring to Table 2, it can be seen from Comparative Example 1 and Examples 6-9 that the use of tocotrienols and Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus polymyxa for simultaneous fermentation can increase the total colony count of the microbial agent, because the main function of tocotrienols is to remove free radicals generated during the fermentation process and protect the strains from damage by oxidative stress, thereby increasing the total colony count of the microbial agent. Tocotrienols have the structure of vitamin E and contain three carbon-carbon double bonds, which makes it have a strong antioxidant capacity. Therefore, its content is slightly lower than that of sodium D-isoascorbate, which can also effectively improve the yield and production efficiency of each strain.

[0053] As the content of tocotrienols continued to increase, the total colony count of the microbial inoculant showed a trend of first increasing and then tending to a stable state. It may be that as the content of tocotrienols continued to increase, the free radicals produced were continuously neutralized to avoid oxidative damage to the bacteria, thereby continuously increasing the total colony count of the microbial inoculant.

[0054] Examples 10-13 Example 10 Based on the preparation method of Example 1, 12 g of sodium D-isoascorbate is replaced with 18 g of tea polyphenols, and the other conditions remain unchanged.

[0055] In Examples 11-13, based on the preparation method of Example 10, the content of tea polyphenols was adjusted, and the specific adjustments are shown in Table 3.

[0056] The microbial agents of Examples 10-13 were subjected to the above performance tests, and the test results are shown in Table 3.

[0057] Table 3 Content and performance test table of tea polyphenols in Example 1 and Examples 10-13 project Example 1 Example 10 Embodiment 11 Example 12 Embodiment 13 Tea polyphenols / g / 18 12 15 20 Total colony count / (100 million CFU / mL) 806 806 734 785 807 Referring to Table 3, it can be seen from the comparison between Example 1 and Examples 10-13 that the use of tea polyphenols and Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus polymyxa for simultaneous fermentation can increase the total colony count of the microbial agent, because the main function of tea polyphenols is to remove free radicals generated during the fermentation process and protect the strains from damage by oxidative stress, thereby increasing the total colony count of the microbial agent. Tea polyphenols is a general term for polyphenols in tea leaves, and its effective ingredients for removing free radicals may be lower than sodium D-isoascorbate and tocotrienols. Therefore, the required content is higher than sodium D-isoascorbate and tocotrienols.

[0058] As the content of tea polyphenols continues to increase, the total colony count of the microbial inoculant shows a trend of first increasing and then tending to a stable state. This may be because as the content of tea polyphenols continues to increase, the free radicals produced are continuously neutralized to avoid oxidative damage to the bacteria, thereby continuously increasing the total colony count of the microbial inoculant.

[0059] Examples 14-18 Example 14 Based on the preparation method of Example 1, 12g of sodium D-isoascorbate is replaced with 10g of a protective agent mixture of tocotrienols and tea polyphenols, the mass ratio of tocotrienols and tea polyphenols in the protective agent mixture is 1:0.4, and other conditions remain unchanged.

[0060] In Examples 15-18, based on the preparation method of Example 1, the mass ratio of tocotrienols and tea polyphenols was adjusted, and the specific adjustment is shown in Table 4.

[0061] The microbial agents of Examples 14-18 were subjected to the above performance tests, and the test results are shown in Table 4.

[0062] Table 4 Mass ratio and performance test table of tocotrienols and tea polyphenols of Example 1 and Examples 14-18 Referring to Table 4, it can be seen from the comparison between Example 1 and Examples 14-18 that the mixed protective agent composed of tocotrienols and tea polyphenols shows higher efficiency in scavenging free radicals, etc. This is mainly because tocotrienols and tea polyphenols have different antioxidant mechanisms and targets. The mixed use of these two protective agents can expand the antioxidant range, cover more biological molecules and cell structures, and thus provide more comprehensive protection. Tocotrienols can play an antioxidant role more effectively because of its unsaturated side chain. It can not only scavenge free radicals, but also protect key enzymes such as cytochrome P450 from oxidative damage. In addition to its antioxidant effect, tea polyphenols also have multiple biological activities such as antibacterial, anti-inflammatory, and weight loss. These functions enable tea polyphenols to improve the fermentation environment while protecting strains from oxidative damage, and promote the growth of strains and the accumulation of metabolites.

[0063] As the proportion of tea polyphenols gradually increased, the total colony count of the microbial agent showed a trend of first increasing and then decreasing. This may be because as the proportion of tea polyphenols gradually increased, the effect of tea polyphenols in improving the fermentation environment gradually increased, thereby increasing the total colony count of the microbial agent. When it exceeds a certain range, the antioxidant property of tocotrienols is higher than that of tea polyphenols. If the proportion of tea polyphenols is too high, it will reduce the antioxidant property of the protective agent, thereby reducing the total colony count of the microbial agent.

[0064] Application Examples The above-mentioned embodiments 1-18 are all capable of being put into practical application, and the following application examples are illustrated by embodiment 1.

[0065] Application of rice Test variety: Tsuen Yu 822 There are 4 treatments, each with 3 replicates. The plot area of ​​each treatment is 54 square meters, and each plot is randomly arranged with a protection row.

[0066] Treatment 1: conventional fertilization + spraying of microbial agents.

[0067] Treatment 2: Conventional fertilization + spraying of inactivated microbial agents.

[0068] Treatment 3: conventional fertilization.

[0069] Treatment 4: blank control.

[0070] Fertilization method: Conventional fertilization: base application of farmyard manure 1100kg / mu, 48% (16-16-16) compound fertilizer 50kg / mu.

[0071] Spraying of microbial agents: Use 200 mL of microbial agents per mu each time, dilute 500 times, and spray three times on July 28, 2024, during the rice tillering stage, August 11, during the jointing stage, and August 31, during the flowering stage.

[0072] Spraying of inactivated microbial agents: the time, method and dosage are the same as those for spraying microbial agents.

[0073] Field management: The rice seedlings in the experimental field were raised on May 6, 2024; the land was prepared and base fertilizer was applied on June 14, and the field was soaked; the rice seedlings were transplanted on June 15; 15 kg / mu of urea was applied on August 25; and the rice was harvested on October 28.

[0074] The mean values ​​of rice traits after each treatment were statistically analyzed and are shown in Table 5.

[0075] Table 5 Mean values ​​of rice traits after treatment 1-4 project Number of grains per ear Thousand-grain weight / g Yield per mu / kg Process 1 174.32 28.20 816.1 Process 2 172.81 26.99 759.3 Process 3 172.80 26.97 755.6 Process 4 168.70 22.24 602.5 As shown in Table 5, by comparing treatments 1-4, it can be seen that the performance of treatment 1 is higher than that of treatments 2 and 3, and the performance of treatments 2 and 3 is similar, and higher than that of treatment 4. This shows that spraying microbial agents during the tillering stage, jointing stage and flowering stage of rice can effectively improve the various traits of rice, mainly because of the four microorganisms in the microbial agents.

[0076] Application of corn Test variety: Denghaiyuyu 812 There are 4 treatments, each with 3 replicates. The plot area of ​​each treatment is 54 square meters, and each plot is randomly arranged with a protection row.

[0077] Treatment 5: conventional fertilization + spraying of microbial agents.

[0078] Treatment 6: Conventional fertilization + spraying of inactivated microbial agents.

[0079] Treatment 7: Conventional fertilization.

[0080] Treatment 8: blank control.

[0081] Fertilization method: Conventional fertilization: 700kg / mu of bottom soil fertilizer, 40kg / mu of biological fertilizer, and 50kg / mu of 48% (26-12-10) compound fertilizer.

[0082] Spraying of microbial agents: Use 200 mL of microbial agents per mu each time, dilute 500 times, and spray on the leaves twice on July 28, 2024, when the corn is in the tassel stage, and on August 10, before heading.

[0083] Spraying of inactivated microbial agents: the time, method and dosage are the same as those for spraying microbial agents.

[0084] The mean values ​​of corn traits after each treatment were statistically analyzed and are shown in Table 6.

[0085] Table 6 Mean values ​​of corn traits after treatment 5-8 project Ear weight / g 100-grain weight / g Yield per mu / kg Process 5 314.42 33.94 514.8 Process 6 293.37 31.55 480.2 Process 7 293.19 31.53 477.8 Process 8 290.27 29.69 380.2 As shown in Table 6, by comparing treatments 5-8, it can be seen that the performance of treatment 5 is higher than that of treatments 6 and 7, and the performance of treatments 6 and 7 is similar and higher than that of treatment 8. This shows that spraying microbial agents at the tasseling stage and the early heading stage of corn can effectively improve the various traits of corn, mainly because of the four microorganisms in the microbial agents.

[0086] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A method for preparing a microbial agent, characterized in that: The following steps are involved: Sterilization of base material: corn starch, peptone, yeast powder and water are mixed, and then heated to 121°C for steam sterilization, and the sterilized base material is obtained after cooling; Feeding and fermentation: Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus polymyxa as well as a protective agent are added to a sterilized base material to form a fermentation mixture, and then sterile air is introduced for fermentation culture to obtain a microbial agent; The protective agent includes at least one of sodium D-isoascorbate, tocotrienol and tea polyphenols.

2. The method for preparing the microbial agent according to claim 1, characterized in that: The protective agent is sodium D-isoascorbate, and the mass fraction of the sodium D-isoascorbate in the fermentation mixture is 0.8%-1.2%.

3. The method for preparing the microbial agent according to claim 1, characterized in that: The protective agent is tocotrienol, and the mass fraction of the tocotrienol in the fermentation mixture is 0.5%-1%.

4. The method for preparing the microbial agent according to claim 1, characterized in that: The protective agent is tea polyphenols, and the mass fraction of the tea polyphenols in the fermentation mixture is 1.2%-1.8%.

5. The method for preparing the microbial agent according to claim 1, characterized in that: The protective agent is a protective agent mixture obtained by mixing tocotrienol and tea polyphenols.

6. The method for preparing the microbial agent according to claim 5, characterized in that: The mass ratio of tocotrienol to tea polyphenols in the protective agent mixture is 1:0.3-0.

5.

7. A microbial agent, characterized in that: The microbial agent is prepared by the preparation method of the microbial agent described in any one of claims 1 to 6.

8. An application of a microbial agent, characterized in that: Use of the microbial agent described in claim 7 in rice and corn production.

9. The use of the microbial agent according to claim 8, characterized in that: The microbial agent is applied during the tillering stage, jointing stage and flowering stage of rice.

10. The use of the microbial agent according to claim 8, characterized in that: The microbial agent is applied during the corn tasseling stage and the early heading stage.

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