Microbial complex microbial inoculant for soil improvement and preparation method of microbial complex microbial inoculant

Abstract

The invention discloses a composite microbial inoculant for soil improvement, which is prepared from the following raw materials: Providencia retinii S17-1, Serratia sp. VL03-3, Pantoea eucallypti P2FPb1 (Pantoea eucallypti P2FPb1) and Bacillus proteolyticus 1372 (Bacillus proteolyticus 1372), and the composite microbial inoculant is prepared from the following raw materials: Pantoea eucallypti P2FPb1, Pantoea eucallypti P2FPb1, Pantoea eucallypti P2FPb1, Pantoea eucallypti P2FPb2, Pantoea eucallypti, Pantoea eucallypti, Pantoea eucallypti On the other hand, the invention discloses a preparation method of the compound microbial inoculant. The preparation method comprises the following steps: acquiring a single colony, preparing a single strain solution, mixing the single strain solution, and inoculating and culturing. The composite microbial inoculant has the composite growth promoting capacities of dissolving phosphorus, dissolving potassium, secreting plant hormones and the like, does not cause plant diseases, has good ecological adaptability, is safe to use and high in economic benefit, and does not threaten the ecological environment. The compound microbial inoculant disclosed by the invention can be used for increasing the plant yield, improving the soil enzyme activity and improving the soil quality and the rhizosphere micro-ecological environment.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural microorganisms, and in particular relates to a microbial composite agent for soil improvement and a preparation method thereof. Background Art

[0002] Microbial agents such as plant growth-promoting bacteria are different from traditional fertilizers and organic fertilizers that are directly applied to plants. They promote plant growth and increase crop yields by improving soil fertility and improving the rhizosphere microecological environment. Their main functions are: (1) secreting plant growth-promoting substances such as indoleacetic acid, cytokinin and gibberellin during metabolism; (2) improving the nutritional status of the plant rhizosphere through nitrogen fixation, phosphorus solubilization, potassium solubilization and activation of other nutrients; (3) improving the disease resistance of plants through biological control. With the improvement of people's living standards and the change of consumption concepts in recent years, green food has become more and more popular, and organic agriculture and pollution-free agriculture have also developed rapidly. Therefore, developing a richer and more efficient soil improvement agent to improve the healthiness and safety of plant-based foods is in line with the needs of modern agricultural development. Summary of the invention

[0003] In view of this, the present invention screens out multiple strains with growth-promoting functions such as producing indoleacetic acid (IAA), solubilizing phosphate and potassium, and prepares them into a microbial composite agent. By comprehensively increasing the soil nutrient content and soil enzyme activity, the soil activity is enhanced, the nutritional status of the plant rhizosphere is improved, and plant growth is promoted.

[0004] To achieve the above object, the present invention provides a microbial composite agent for soil improvement, comprising:

[0005] Providencia rettgeri S17-1, with the accession number of CCTCC No.M20242149;

[0006] Serratia sp. VL03-3, with a deposit number of CCTCC No. M20242150;

[0007] Pantoea eucalypti P2FPb_1, with the accession number of CCTCC No.M20242148;

[0008] and Bacillus proteolyticus 1372, with the accession number being CCTCCNo.M20242147.

[0009] The preservation unit of the above-mentioned microorganisms is the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China; the preservation date is: October 9, 2024.

[0010] Preferably, Providencia rettgeri S17-1, Serratia sp. VL03-3, Pantoea eucalypti P2FPb_1 and Bacillus proteolyticus 1372 are cultured using LB medium, and the pH of the LB medium is 6.5-7.5.

[0011] Preferably, the effective viable counts of Providencia rettgeri S17-1, Serratia asp. VL03-3, Pantoea eucalypti P2FPb_1 and Bacillus proteolyticus 1372 are all at least 10 9 cfu / mL.

[0012] Another aspect of the present invention provides an application of the above-mentioned microbial composite agent, wherein the microbial composite agent is applied to plant seedlings by root irrigation, and the application amount is 20 mL / plant.

[0013] Another aspect of the present invention provides an application of the above-mentioned microbial composite agent, wherein the microbial composite agent is applied to plant seeds by a seed dressing method.

[0014] Another aspect of the present invention provides a method for preparing the above-mentioned microbial composite agent, comprising the following steps:

[0015] S1: Obtaining single colonies of Providencia rettgeri S17-1, Serratia sp. VL03-3, Pantoea eucalypti P2FPb_1 and Bacillus proteolyticus 1372;

[0016] S2: inoculating the single colonies of Providencia rettgeri S17-1, Serratia sp. VL03-3, Pantoea eucalypti P2FPb_1 and Bacillus proteolyticus 1372 into corresponding culture media respectively, and preparing corresponding single strain culture solutions respectively;

[0017] S3: After mixing the single strain culture fluids in a volume ratio of 1:1:1:1, the mixed fluid is inoculated into a high-density mixed fermentation special medium to obtain the microbial composite agent; the high-density mixed fermentation special medium includes LB medium, and the pH of the high-density mixed fermentation special medium is 6.5-7.5.

[0018] Preferably, in step S2: the culture medium is LB liquid culture medium; the culture conditions for preparing the single strain culture solution are 28°C, 210r / min shaking culture for 48h, and the viable bacteria concentration after culture is greater than 10 8 cfu / mL; after culturing, the OD600 of the single strain culture solution was adjusted to 0.8 with sterile water.

[0019] Preferably, in step S3: the inoculation amount during inoculation is 5%-10%, and the culture conditions are 30° C. and 180 r / min for 48 hours.

[0020] The beneficial effects of the present invention are:

[0021] The microbial composite agent of the present invention has the ability to dissolve phosphorus, potassium, secrete plant hormones and other composite growth-promoting abilities, does not cause plant diseases, has good ecological adaptability, is safe to use, has high economic benefits, and does not threaten the ecological environment. The microbial composite agent of the present invention can increase plant yield, increase soil enzyme activity, and improve soil quality and rhizosphere microecological environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, 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.

[0023] Figure 1 Schematic diagram of the process of preparing the composite microbial agent according to an embodiment of the present invention;

[0024] Figure 2This is the seedling growth status measurement result table of Example 2;

[0025] Figure 3 This is a comparison chart of the plant growth promotion effect of Example 2 (with the non-inoculated treatment group as 100%);

[0026] Figure 4 The table is the result of the measurement of the rhizosphere microecological environment of Example 3;

[0027] Figure 5 and Figure 6 This is a comparison chart of the rhizosphere microecological environment conditions in Example 3 (with the uninoculated treatment group as 100%). DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. 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] Embodiment 1

[0030] Figure 1 The preparation process of the microbial composite agent of the present invention is shown, which includes the following steps:

[0031] S1: strain selection: the following strains were screened and purified: Providencia rettgeri S17-1, Serratia sp. VL03-3, Pantoea eucalypti P2FPb_1 and Bacillus proteolyticus 1372; by co-culture method, it was determined that there was no antagonism between the strains; among the above strains, Providencia rettgeri S17-1 and Serratia sp. VL03-3 mainly played the role of producing IAA, while Pantoea eucalypti P2FPb_1 mainly played the role of dissolving phosphorus, and Bacillus proteolyticus 1372 mainly played the role of dissolving potassium; after purification, single colonies of the above strains were obtained respectively;

[0032] S2: Prepare the single strain culture solution of each of the above strains, specifically: inoculate the single colonies of Providencia rettgeri S17-1, Serratia sp. VL03-3, Pantoeaeucalypti P2FPb_1 and Bacillus proteolyticus 1372 into the corresponding 250mL Erlenmeyer flasks containing 100mL LB liquid culture medium, and culture them at 28°C and 210r / min shaking table for 48h, so that the live bacteria concentration of the liquid inoculum is greater than 10 8 cfu / mL, and the OD600 value was adjusted to 0.8 with sterile water to obtain the corresponding single strain culture solution;

[0033] S3: preparing a microbial composite agent: mixing the single strain culture solutions of Providenciarettgeri S17-1, Serratia sp. VL03-3, Pantoea eucalypti P2FPb_1 and Bacillus proteolyticus 1372 obtained in step S2 in a volume ratio of 1:1:1:1; inoculating the mixed solution into a high-density mixed fermentation special medium with an inoculation amount of 5%-10%, culturing at 30°C and 180r / min for 48h, and obtaining the microbial composite agent, wherein the number of effective live bacteria in the microbial composite agent is ≥10 9 cfu / mL; the special medium for high-density mixed fermentation includes the following components: LB medium (1% peptone, 0.5% yeast extract, 1% NaCl), pH value is 6.5-7.5.

[0034] Embodiment 2

[0035] This example discloses the steps of applying the aforementioned microbial composite agent to corn plants by root irrigation and the growth-promoting effect on the corn plants after application.

[0036] Corn seeds with full grains and uniform size were selected, disinfected with 3% hydrogen peroxide for 15 minutes, washed three times with sterile water, and planted in plastic flower pots (specifications: bottom diameter 12.3 cm × diameter 15.5 cm × height 12.0 cm) filled with 1.2 kg of flower soil. Two corn plants were planted in each pot. After emergence, the seeds were divided into three groups and treated as follows: (a) deionized water treatment (uninoculated treatment group); (b) single watering with Serratia sp. VL03-3 culture solution with corresponding content (single bacteria group); (c) watering with the above-mentioned microbial composite bacterial agent treatment (composite bacteria group); during the treatment, the roots were irrigated with the same inoculum volume, 20 mL per pot, and each treatment was repeated twice, i.e., 20 mL / plant in total.

[0037] All plants were harvested after 30 days of growth, and the dry weight, total nitrogen, total phosphorus and total potassium of the seedlings were measured. During this period, the soil moisture content was maintained at 60%.

[0038] The results of the test are as follows Figure 2 and Figure 3 As shown. Figure 2 and Figure 3 It can be seen that after applying the microbial composite agent of the embodiment of the present invention, the dry weight, total nitrogen, total phosphorus and total potassium of corn increased by 22.90%, 32.23%, 35.94% and 30.87% compared with the non-inoculated treatment, and increased by 11.58%, 19.15%, 35.94% and 26.41% compared with the single bacteria group. It can be seen from the results that the microbial composite agent provided by the present invention can effectively promote the growth of corn and increase the nutrient content of the plant, and the effect of the composite agent is better than that of the single bacteria.

[0039] Embodiment 3

[0040] This example discloses the effect of improving the nutritional status of the rhizosphere of plants after applying the aforementioned microbial composite agent by root irrigation.

[0041] The soil culture experiment used root bags to plant plants. Two root bags made of nylon mesh (300 mesh, 5 cm in diameter, 10 cm in height) were filled with 0.1 kg of soil and placed in a soil culture pot (specifications: bottom diameter 12.3 cm × diameter 15.5 cm × height 12.0 cm). 0.9 kg of soil was filled around the two root bags in each pot. A soil solution sampler (RhizosphereRHIZON MOM, the Netherlands) was inserted inside the root bag to collect in situ rhizosphere soil solution. Healthy corn seedlings with consistent growth and good growth were selected and transplanted into root bags, with one plant in each root bag. After transplanting, each pot of seedlings was treated as follows: (a) deionized water treatment (uninoculated treatment group); (b) Providencia rettgeri S17-1 culture solution with corresponding content was treated (single bacteria group); (c) the above-mentioned microbial composite agent was treated (composite bacteria group). The roots were irrigated with the same inoculum volume, 20 mL per pot, and each treatment was repeated twice, i.e., 20 mL / plant in total.

[0042] All plants were harvested after 30 days of growth, and the soil was collected to determine the available phosphorus, effective potassium and acid phosphatase activity, and the rhizosphere soil solution was collected to determine the phosphorus and potassium contents.

[0043] The results of the test are as follows Figures 4 to 6 As shown. Figures 4 to 6It can be seen that after applying the microbial composite agent of the embodiment of the present invention, the effective nutrient content of the soil is higher than that of the single bacteria and non-inoculated treatments. Compared with the non-inoculated treatment, the effective phosphorus in the soil increased by 146.65%, and the effective potassium increased by 18.24%; the effective phosphorus in the soil solution increased to 16.57 times, and the effective potassium increased to 1.73 times; at the same time, the soil acid phosphatase activity increased to 1.38 times. From the results, it can be seen that the microbial composite agent of the embodiment of the present invention can effectively increase the nutrient content of the soil and improve the vitality of the soil.

[0044] In addition to the above-mentioned application methods, the microbial composite agent of the embodiment of the present invention can also be applied to plant seeds by seed dressing method and other methods, which will not be described in detail here.

[0045] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A microbial composite agent for soil improvement, characterized in that: include: Providencia rettgeri S17-1, with the accession number of CCTCC No.M20242149; Serratia sp. VL03-3, with a deposit number of CCTCC No. M20242150; Pantoea eucalypti P2FPb_1, with the accession number of CCTCC No.M20242148; and Bacillus proteolyticus 1372, with the accession number being CCTCC No.M20242147.

2. The microbial composite agent according to claim 1, characterized in that: Providencia rettgeri S17-1, Serratia sp. VL03-3, Pantoeaeucalypti P2FPb_1 and Bacillus proteolyticus 1372 are cultured in LB medium, and the pH of the LB medium is 6.5-7.

5.

3. The microbial composite agent according to claim 1, characterized in that: The effective viable counts of Providencia rettgeri S17-1, Serratia sp. VL03-3, Pantoea eucalypti P2FPb_1 and Bacillus proteolyticus 1372 are all at least 10 9 cfu / mL.

4. A use of the microbial composite agent according to any one of claims 1 to 3, characterized in that: The microbial composite agent was applied to plant seedlings by root irrigation, with an application amount of 20 mL / plant.

5. A use of the microbial composite agent according to any one of claims 1 to 3, characterized in that: The microbial composite agent is applied to plant seeds by a seed dressing method.

6. A method for preparing the microbial composite agent according to any one of claims 1 to 3, characterized in that: The steps include: S1: Obtaining single colonies of Providencia rettgeri S17-1, Serratia sp. VL03-3, Pantoea eucalypti P2FPb_1 and Bacillus proteolyticus 1372; S2: inoculating the single colonies of Providencia rettgeri S17-1, Serratia sp. VL03-3, Pantoea eucalypti P2FPb_1 and Bacillus proteolyticus 1372 into corresponding culture media respectively, and preparing corresponding single strain culture solutions respectively; S3: After mixing the culture solutions of the single strains in a volume ratio of 1:1:1:1, the mixed solution is inoculated into a high-density mixed fermentation special culture medium to culture the microbial composite agent; The high-density mixed fermentation special medium includes LB medium, and the pH of the high-density mixed fermentation special medium is 6.5-7.

5.

7. The preparation method according to claim 6, characterized in that: In step S2: the culture medium is LB liquid culture medium; the culture conditions for preparing the single strain culture solution are 28°C, 210r / min shaking culture for 48h, and the viable bacteria concentration after culture is greater than 10 8 cfu / mL; after culturing, the OD600 of the single strain culture solution was adjusted to 0.8 with sterile water.

8. The preparation method according to claim 6, characterized in that: In step S3: the inoculation amount during inoculation is 5%-10%, and the culture conditions are 30° C. and 180 r / min for 48 hours.

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