Compound microbial agent as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of microbial control, and particularly relates to a compound microbial agent as well as a preparation method and application thereof. The preparation method of the compound microbial agent comprises the following steps: (1) respectively activating bacillus megatherium, bacillus marinus N6-2 and lactobacillus plantarum A37 to obtain various strain activating solutions; mixing the activation solutions of the strains to prepare a mixed bacterial solution; (2) inoculating the mixed bacteria solution into a seed culture medium to prepare a seed solution; and (3) inoculating the seed solution into a fermentation culture medium for fermentation, and then drying and crushing to obtain the compound microbial agent. According to the prepared compound microbial agent, the total number of effective viable bacteria is increased by optimizing the composition of the culture medium. After the compound microbial agent is used, the contents of organic matters, quick-acting nitrogen, quick-acting phosphorus and quick-acting potassium in soil can be remarkably increased, and the soil fertility is improved, so that the growth of tomatoes is promoted, the yield and quality of the tomatoes are improved, and the compound microbial agent has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial control, and specifically relates to a composite microbial inoculant, a preparation method thereof, and an application thereof. Background Art

[0002] In the growth of crops and vegetables, long-term excessive application of chemical fertilizers can lead to a series of problems such as soil compaction, acidification, reduction of organic matter content, and degradation of soil communities, which in turn affect the quality of crops and vegetables and restrict the soil's ability to sustain growth. Therefore, researching new fertilizers to replace chemical fertilizers has become a research hotspot and difficulty in the fields of agricultural resources and the environment. Against this background, microbial fertilizers have emerged.

[0003] As a green and environmentally friendly agricultural input, microbial inoculants have received extensive attention in recent years. By introducing beneficial microorganisms, microbial inoculants can improve the soil microbial community structure, promote the transformation and release of nutrients such as nitrogen, phosphorus, and potassium in the soil, and increase soil fertility. At the same time, some microorganisms also have antibacterial and insecticidal functions, which can effectively control crop diseases and reduce the use of chemical pesticides. However, there are still some problems in the actual application of existing microbial inoculants, such as low activity of strains, poor adaptability, single function, etc., resulting in unstable effects and difficulty in meeting the diverse needs of modern agricultural production.

[0004] In the preparation process of microbial inoculants, the activity and quantity of strains are the key factors determining their effects. Traditional microbial fermentation technologies often have difficulty ensuring the high-efficiency proliferation and activity maintenance of strains. Especially in large-scale production, the activity and stability of strains are easily affected by factors such as the composition of the culture medium and fermentation conditions. In addition, the functions of single-strain microbial inoculants are limited, and it is difficult to achieve multiple goals such as soil improvement, disease control, and crop growth promotion simultaneously. Therefore, developing a composite microbial inoculant by optimizing the strain combination and fermentation process, increasing the number of effective viable bacteria in the inoculant, and endowing it with multiple functions has important practical significance. Summary of the Invention

[0005] In order to fill the gaps in the existing technology, one of the purposes of the present invention is to provide a preparation method of a composite microbial inoculant.

[0006] Another purpose of the present invention is to provide a composite microbial inoculant prepared by the above preparation method.

[0007] The third purpose of the present invention is to provide the application of the above composite microbial inoculant in promoting the growth of crops.

[0008] In order to achieve the above purposes, the technical solution adopted by the present invention is: A preparation method of a composite microbial inoculant, comprising the following steps: (1) Activate Bacillus megaterium, Bacillus marinus N6-2, and Lactobacillus plantarum A37 respectively to obtain the activated liquid of each strain; mix the activated liquids of each strain to prepare a mixed bacterial liquid. (2) Inoculate the mixed bacterial liquid obtained in step (1) into a seed medium to prepare a seed liquid. (3) Inoculate the seed liquid obtained in step (2) into a fermentation medium for fermentation, and then dry and crush it to obtain a compound microbial inoculant.

[0009] Further, the seed medium in step (2) comprises the following components by mass fraction: 1.5 - 2.5% lactose, 0.5 - 1.5% yeast extract, 0.5 - 1.5% magnesium sulfate, 0.2 - 1.0% trisodium 1,6-diphosphate fructose, and the balance is water.

[0010] Further, the fermentation medium in step (3) comprises the following components by mass fraction: 0.1 - 1.5% starch, 0.5 - 2% soybean cake powder, 0.5 - 2% ammonium sulfate, 0.5 - 1.0% peptone, 0.3 - 0.5% yeast powder, 0.1 - 0.3% calcium carbonate, 0.5 - 2.5% potassium dihydrogen phosphate, 0.5 - 1.5% 6-amino-3-methylpurine, and the balance is water.

[0011] Further, the ratio of the effective bacterial concentration of Bacillus megaterium, Bacillus marinus N6-2, and Lactobacillus plantarum A37 in the mixed bacterial liquid in step (1) is 1:(1 - 3):(1 - 3).

[0012] Further, the total effective bacterial concentration of the compound microbial inoculant ≥ 10 9 CFU / g.

[0013] Further, the inoculation amount of the seed liquid in step (3) is 4 - 6%.

[0014] Further, the fermentation temperature in step (3) is 25 - 35°C, and the fermentation time is 25 - 40 h.

[0015] A compound microbial inoculant is prepared by the method described above.

[0016] Use of the compound microbial inoculant described above in promoting the growth of crops.

[0017] Compared with the prior art, the beneficial effects of the present invention mainly lie in: The present invention provides a method for preparing a compound microbial inoculum. By adding trisodium 1,6-diphosphate fructose and 6-amino-3-methylpurine components to the seed culture medium and the fermentation culture medium respectively, the effective viable count of Bacillus megaterium, Bacillus marinus N6-2 and Lactobacillus plantarum A37 in the compound microbial inoculum is increased. After applying the compound microbial inoculum to the soil, it can significantly increase the contents of organic matter, available nitrogen, available phosphorus and available potassium in the soil, improve soil fertility, thereby promoting the growth of tomatoes, increasing the yield and quality of tomatoes, and having broad application prospects. Brief Description of the Drawings

[0018] Figure 1 It is a result diagram of the influence of the compound microbial inoculum prepared by the present invention on the tomato yield. Detailed Embodiments

[0019] The following further describes the technical solutions of the present invention in combination with specific embodiments. However, those skilled in the art should understand that the following examples are only used to illustrate the present invention and should not be regarded as a limitation of the present invention. The specific conditions not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are conventional products obtained through commercial channels.

[0020] The preservation number of Bacillus megaterium of the present invention is: CGMCC No. 3770, the preservation number of Bacillus marinus N6-2 is: CCTCC NO: M2014586, and the preservation number of Lactobacillus plantarum A37 is: CCTCC NO: M2019559.

[0021] Example 1 A method for preparing a compound microbial inoculum includes the following steps: (1) After activating Bacillus megaterium, Bacillus marinus N6-2 and Lactobacillus plantarum A37 respectively, the activated liquid of each strain is obtained; the activated liquids of each strain are mixed to prepare a mixed bacterial liquid; the viable count ratio of Bacillus megaterium, Bacillus marinus N6-2 and Lactobacillus plantarum A37 in the mixed bacterial liquid is 1:2:2, and the viable count of Bacillus megaterium in the mixed bacterial liquid is 1×10 8 CFU / g.

[0022] (2) Transfer the mixed bacterial liquid obtained in step (1) to a seed culture medium containing the following mass fraction components: 2.0% lactose, 1.0% yeast extract, 1.0% MgSO4 magnesium sulfate, 0.6% trisodium 1,6-diphosphate fructose, and the balance is water. Place it on a shaker at 30 °C and culture at 200 rpm until the OD 600 is about 5 to obtain a seed liquid.

[0023] (3) Inoculate the seed liquid obtained in step (2) into a fermentation medium containing the following components by mass fraction: 0.8% starch, 1.2% soybean cake powder, 1.2% ammonium sulfate, 0.7% peptone, 0.4% yeast powder, 0.2% calcium carbonate, 1.5% potassium dihydrogen phosphate, 1.0% 6-amino-3-methylpurine, and the balance is water. Place it on a shaker at 30 °C and ferment at 200 rpm for 35 h. Dry and crush the fermentation culture at 38 °C, and pass through a 200-mesh sieve to obtain a compound microbial inoculant; the total viable count in the compound microbial inoculant ≥ 10 9 CFU / g.

[0024] This example also provides a compound microbial inoculant obtained by the above preparation method.

[0025] Example 2 A preparation method of a compound microbial inoculant, comprising the following steps: (1) After activating Bacillus megaterium, Bacillus marinus N6-2, and Lactobacillus plantarum A37 respectively, obtain the activated liquid of each strain; mix the activated liquids of each strain to prepare a mixed bacterial liquid; the viable count ratio of Bacillus megaterium, Bacillus marinus N6-2, and Lactobacillus plantarum A37 in the mixed bacterial liquid is 1:1:1, and the viable count of Bacillus megaterium in the mixed bacterial liquid is 1×10 8 CFU / g.

[0026] (2) Transfer the mixed bacterial liquid obtained in step (1) to a seed medium containing the following components by mass fraction: 1.5% lactose, 0.5% yeast extract, 0.5% magnesium sulfate, 0.2% trisodium 1,6-diphosphate fructose, and the balance is water. Place it on a shaker at 25 °C and culture at 200 rpm until the OD 600 is about 4 to obtain a seed liquid.

[0027] (3) Inoculate the seed liquid obtained in step (2) into a fermentation medium containing the following components by mass fraction: 0.1% starch, 0.5% soybean cake powder, 0.5% ammonium sulfate, 0.5% peptone, 0.3% yeast powder, 0.1% calcium carbonate, 0.5% potassium dihydrogen phosphate, 0.5% 6-amino-3-methylpurine, and the balance is water. Place it on a shaker at 25 °C and ferment at 200 rpm for 25 h. Dry and crush the fermentation culture at 38 °C, and pass through a 200-mesh sieve to obtain a compound microbial inoculant; the total viable count in the compound microbial inoculant ≥ 10 9 CFU / g.

[0028] This example also provides a compound microbial inoculant obtained by the above preparation method.

[0029] Example 3 A preparation method of a compound microbial inoculum, comprising the following steps: (1) After activating Bacillus megaterium, Bacillus marinus N6-2 and Lactobacillus plantarum A37 respectively, mix the activated solutions of each strain to prepare a mixed bacterial solution; the ratio of the viable counts of Bacillus megaterium, Bacillus marinus N6-2 and Lactobacillus plantarum A37 in the mixed bacterial solution is 1:3:3, and the viable count of Bacillus megaterium in the mixed bacterial solution is 1×10 8 CFU / g.

[0030] (2) Transfer the mixed bacterial solution obtained in step (1) to a seed culture medium containing the following components by mass fraction: 2.5% lactose, 1.5% yeast extract, 1.5% magnesium sulfate, 1.0% trisodium 1,6-diphosphate fructose, and the balance is water. Place it on a shaker at 35°C and culture at 200 rpm until the OD 600 is about 6 to obtain a seed solution.

[0031] (3) Inoculate the seed solution obtained in step (2) into a fermentation medium containing the following components by mass fraction at an inoculation amount of 6%: 1.5% starch, 2% soybean cake powder, 2% ammonium sulfate, 1.0% peptone, 0.5% yeast powder, 0.3% calcium carbonate, 2.5% potassium dihydrogen phosphate, 1.5% 6-amino-3-methylpurine, and the balance is water. Place it on a shaker at 35°C and ferment at 200 rpm for 45 h. Dry and crush the fermentation product at 38°C and pass through a 200-mesh sieve to obtain a compound microbial inoculum; the total viable count in the compound microbial inoculum ≥ 10 9 CFU / g.

[0032] This example also provides a compound microbial inoculum obtained by the above preparation method.

[0033] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that trisodium 1,6-diphosphate fructose is omitted from the seed culture medium in step (2).

[0034] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that 6-amino-3-methylpurine is omitted from the fermentation medium in step (3).

[0035] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that Bacillus marinus N6-2 and Lactobacillus plantarum A37 are omitted in step (1), and the dosage of Bacillus megaterium is adjusted to the sum of the three.

[0036] Comparative Example 4 The difference between Comparative Example 4 and Example 1 is that Lactobacillus plantarum A37 is omitted in step (1), while the dosages of Bacillus marinus N6-2 and Bacillus megaterium are the same as those in Example 1.

[0037] Comparative Example 5 The difference between Comparative Example 5 and Example 1 is that in step (1), Bacillus marinus N6-2 is omitted, while the dosages of Lactobacillus plantarum A37 and Bacillus megaterium are the same as those in Example 1.

[0038] Test Example 1 Viable count of different compound microbial inoculants Weigh 10 g of each of the compound microbial inoculants prepared in Examples 1-3 and Comparative Examples 1-5, add them to 100 mL of sterile water respectively, let stand for 20 min, and fully oscillate for 30 min at 20 r / min on a rotary shaker to prepare bacterial suspensions. Then, the above-mentioned bacterial suspensions of each group are serially diluted, and the total number of viable bacteria of different compound microbial inoculants is determined by the spread plate counting method. The results are shown in Table 1.

[0039] Table 1 Determination of viable count of compound microbial inoculant As can be seen from Table 1, compared with the groups of Comparative Examples 1-2, the total number of viable bacteria in the compound microbial inoculants prepared in Examples 1-3 of the present invention is higher. It shows that the two nutritional components, fructose 1,6-diphosphate trisodium salt and 6-amino-3-methylpurine, play a very important role in microbial fermentation and can significantly increase the total number of viable bacteria in the compound microbial inoculant.

[0040] Test Example 2 Effect of compound microbial inoculant on soil properties Alkaline farmland soil samples were selected as the research object in the experiment. The collected soil samples were air-dried, ground and sieved, and divided into a control group, Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group, Comparative Example 2 group, Comparative Example 3 group, Comparative Example 4 group, and Comparative Example 5 group. According to 8 g / kg of soil, the compound microbial inoculants prepared in Examples 1-3 and Comparative Examples 1-5 were mixed with the soil. The control group did not add the compound microbial inoculant. After mixing, they were filled into culture dishes, about 300 g of soil in each dish. The inoculated soil was cultured at 28 °C and 60% relative humidity for 1 month. After the cultivation was completed, samples were randomly taken from the cultivated soil every week. The soil organic matter was determined by the dichromate volumetric method in NY / T 52 Determination of Soil Organic Matter, the available nitrogen content of the soil was determined by NY / T53 Determination of Alkaline Hydrolyzable Nitrogen in Soil, the available phosphorus content of the soil was determined by the Olsen method (sodium bicarbonate extraction-molybdenum antimony anti-colorimetric method), and the available potassium content of the soil was determined by the ammonium acetate extraction-flame photometry method. All experiments were repeated three times, and the results are shown in Table 2.

[0041] Table 2 Nitrogen fixation, phosphorus solubilization and potassium solubilization of compound microbial inoculant As can be seen from Table 2, compared with the control groups 1-5, the composite microbial inoculants prepared in Examples 1-3 of the present invention can significantly increase the contents of organic matter, available nitrogen, available phosphorus, and available potassium in the soil, improve soil nutrients, and further enhance soil fertility, which helps to promote plant growth. Among them, the composite microbial inoculant prepared in Example 1 has the most obvious effect on improving soil nutrients and enhancing soil fertility.

[0042] Test Example 3 Field experiment of composite microbial inoculant A field experiment of the composite microbial inoculant was carried out in a tomato greenhouse. The greenhouse covers an area of 8 mu and is divided into Example 1 group, Example 2 group, Example 3 group, Control group 1, Control group 2, Control group 3, Control group 4, and Control group 5, each group covering an area of 1 mu and planting 1,800 tomato seedlings. Before transplanting the tomato seedlings, the composite microbial inoculants prepared in Examples 1-3 and Controls 1-5 were diluted with water at 1 kg / mu and sprayed on the field, and then rotary tillage was carried out; unified management was implemented for each group. After the tomatoes matured, the total yield of tomatoes in each group was measured, and the results are as Figure 1 shown. When the tomatoes grew for 90-100 days, fruits were picked. 20 fruits were selected from each group to measure indexes such as soluble sugar content, soluble protein, vitamin C, titratable acid, and lycopene. The results are shown in Table 3.

[0043] Table 3 Effects of composite microbial inoculant on tomato fruit quality From Figure 1 and Table 3, it can be seen that compared with the control groups 1-5, the composite microbial inoculants prepared in Examples 1-3 of the present invention can increase the yield of tomatoes and the contents of soluble sugar, soluble protein, vitamin C, and lycopene in tomatoes. It shows that by using the composite microbial inoculant prepared in the present invention, the growth of tomatoes can be significantly promoted, and the yield and quality of tomatoes can be improved. Among them, the composite microbial inoculant prepared in Example 1 has the most obvious effect on promoting the growth of tomatoes and improving the yield and quality of tomatoes.

[0044] Further analysis shows that, compared with Example 1, the seed culture medium of Comparative Example 1 omits trisodium 1,6-diphosphate fructose; the fermentation medium of Comparative Example 2 omits 6-amino-3-methylpurine; in step (1) of Comparative Example 3, Bacillus marinus N6-2 and Lactobacillus plantarum A37 are omitted, and the dosage of Bacillus megaterium is adjusted to the sum of the three; in step (1) of Comparative Example 4, Lactobacillus plantarum A37 is omitted, while the dosages of Bacillus marinus N6-2 and Bacillus megaterium are the same as those in Example 1; in step (1) of Comparative Example 5, Bacillus marinus N6-2 is omitted, while the dosages of Lactobacillus plantarum A37 and Bacillus megaterium are the same as those in Example 1. The tomato quality of the Comparative Example 1-5 groups has decreased, which indicates that the two nutritional components, trisodium 1,6-diphosphate fructose and 6-amino-3-methylpurine, play very important roles in microbial fermentation. The two nutritional components, trisodium 1,6-diphosphate fructose and 6-amino-3-methylpurine, further increase the content of organic matter, available nitrogen, available phosphorus, and available potassium in the soil by increasing the total number of effective viable bacteria in the compound microbial inoculant, thereby promoting tomato growth and improving its yield and quality. In the compound microbial inoculant, Bacillus megaterium, Bacillus marinus N6-2, and Lactobacillus plantarum A37 can effectively promote tomato growth, improve tomato yield and quality only when they cooperate together.

[0045] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them. The basic principles and main features of the present invention have been described in the above with specific implementation schemes. On the basis of the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the scope of the present invention claimed.

Claims

1. A preparation method of a compound microbial inoculum, characterized in that It includes the following steps: (1) Activate Bacillus megaterium, Bacillus marinus N6-2, and Lactobacillus plantarum A37 respectively to obtain the activated solutions of each strain; Mix the activated solutions of each strain to prepare a mixed bacterial solution; (2) Inoculate the mixed bacterial solution obtained in step (1) into a seed medium to prepare a seed solution; (3) Inoculate the seed solution obtained in step (2) into a fermentation medium for fermentation, and then dry and pulverize it to obtain a compound microbial inoculant.

2. The preparation method of a composite microbial inoculum according to claim 1, characterized in that, The seed medium described in step (2) includes the following components by mass fraction: 1.5-2.5% lactose, 0.5-1.5% yeast extract, 0.5-1.5% magnesium sulfate, 0.2-1.0% trisodium 1,6-diphosphate fructose, and the balance is water.

3. The preparation method of a composite microbial inoculum according to claim 1, characterized in that, The fermentation medium described in step (3) includes the following components by mass fraction: 0.1-1.5% starch, 0.5-2% soybean cake powder, 0.5-2% ammonium sulfate, 0.5-1.0% peptone, 0.3-0.5% yeast powder, 0.1-0.3% calcium carbonate, 0.5-2.5% potassium dihydrogen phosphate, 0.5-1.5% 6-amino-3-methylpurine, and the balance is water.

4. The preparation method of a composite microbial inoculum according to claim 1, characterized in that, In the mixed bacterial solution described in step (1), the ratio of the effective bacteria concentrations of Bacillus megaterium, Bacillus marinus N6-2, and Lactobacillus plantarum A37 is 1:(1-3):(1-3).

5. The preparation method of a composite microbial inoculum according to claim 1, characterized in that, The total effective bacteria concentration of the composite microbial inoculum ≥ 10 9 CFU / g.

6. The preparation method of a compound microbial inoculum according to claim 1, characterized in that, The inoculation amount of the seed solution described in step (3) is 4-6%.

7. The preparation method of a composite microbial inoculum according to claim 1, characterized in that, The fermentation temperature described in step (3) is 25-35°C, and the fermentation time is 25-40 h.

8. A compound microbial inoculum, characterized in that, Prepared according to the method described in any one of claims 1 to 7.

9. Application of the compound microbial inoculant described in claim 8 in promoting the growth of crops.

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