Complex microbial inoculant for improving alkaline soil based on penicillium kkerchii as well as preparation method and application of complex microbial inoculant

Abstract

The invention provides a complex microbial inoculant for improving alkaline soil based on penicillium kkerchii and a preparation method and application thereof, and relates to the technical field of soil improvement, and the complex microbial inoculant comprises penicillium kkerchii, bacillus mucilaginosus and a tea flower extract. The ratio of the viable count of penicillium kkerchii to the viable count of bacillus mucilaginosus in the complex microbial inoculant is 1: (0.5-1), and the total viable count of penicillium kkerchii and bacillus mucilaginosus is 1.5 * 10 < 8 >-5.5 * 10 < 9 > cfu / mL. The microbial agent composition prepared by the method provided by the invention can improve alkaline soil planting conditions for a long time, the addition of baicalein regulates metabolism of penicillium kluywii, and the use of the composite microbial agent can effectively ensure the flora advantages of penicillium kluywii in soil, promote the growth of soil colonies and maintain a good plant growth microbial environment.

Description

Technical Field

[0001] The invention provides a composite bacterial agent for improving alkaline soil based on Penicillium koreana and a preparation method and application thereof, and relates to the technical field of soil improvement. Background Art

[0002] In the vast field of microbial community, Penicillium has attracted much attention due to its wide distribution range and diverse biological functions. Among them, Penicillium koreana, as an outstanding representative of the Penicillium family, is not only highly respected for its unique biological activity, but also shows extraordinary potential in agricultural production practice, especially in soil improvement and crop protection. However, although Penicillium koreana has shown its edge in the field of soil improvement, especially in the attempt to improve alkaline soil, there are still many shortcomings and challenges. Penicillium, a fungus group widely distributed in soil, air and plant surfaces, actively participates in the decomposition of soil organic matter and nutrient cycle by secreting a variety of enzymes and organic acids, and plays an indispensable role in maintaining the ecological balance of soil. Penicillium koreana, with its strong biological activity and excellent environmental adaptability, has emerged in agricultural production and become a shining star. It can produce rich secondary metabolites, which not only have antibacterial and antiviral activities, but also can effectively promote crop growth and improve crop resistance. In terms of soil improvement, the application of Penicillium koreana mainly focuses on improving soil structure and enhancing soil fertility. By inoculating Penicillium clarkii, the soil microecological environment can be significantly improved, the reproduction of beneficial microorganisms can be promoted, and the air permeability and water retention capacity of the soil can be improved. However, when scientists turned their research focus to the improvement of alkaline soil, the application of Penicillium clarkii encountered unprecedented challenges. Alkaline soil, a special type of soil, poses a great threat to the growth and reproduction of microorganisms due to its high pH value. Although Penicillium clarkii performs well in general soil, its growth and metabolic activities are severely inhibited in the high pH environment of alkaline soil, and the improvement effect is greatly reduced. Specifically, the high pH environment of alkaline soil can damage the cell structure of Penicillium clarkii, resulting in its metabolic function being blocked and its biological activity being reduced. In addition, the microbial community structure in alkaline soil is complex, and it is difficult for Penicillium clarkii to form a dominant bacterial community in it, further limiting its application effect in alkaline soil improvement. The reason is that, on the one hand, the high pH value of alkaline soil destroys the growth environment of Penicillium koreana, making it difficult for it to adapt and play its due improvement role; on the other hand, the microbial community structure in alkaline soil is complex and changeable, and it is difficult for Penicillium koreana to dominate it, thus affecting its improvement effect. Although Penicillium koreana faces many challenges in improving alkaline soil, scientific researchers have not stopped there. They are actively exploring more advanced technical means and methods and strategies to improve the adaptability and improvement effect of Penicillium koreana in alkaline soil. For example, by screening and cultivating alkali-resistant Penicillium koreana strains, or by synergizing with other microorganisms, a more stable microbial community structure can be constructed, thereby enhancing the survival ability and improvement effect of Penicillium koreana in alkaline soil. However, it is undeniable that in the field of alkaline soil improvement, the application technology of Penicillium koreana still needs to be mature and perfected, and the road to improving the improvement effect is still full of challenges and opportunities. Summary of the invention

[0003] To solve the above problems, the specific solutions provided by the present invention are as follows:

[0004] A composite bacterial agent for improving alkaline soil based on Penicillium koreana, comprising Penicillium koreana, Bacillus gelatinosa and tea tree flower extract.

[0005] Preferably, in the composite bacterial agent, the ratio of the number of live bacteria of Penicillium krusei to that of Bacillus gelatinosa is 1:(0.5-1).

[0006] Preferably, the total number of viable bacteria of Penicillium krusei and Bacillus jelly-like in the composite bacterial agent is 1.5×10 8 -5.5×10 9 cfu / mL.

[0007] Preferably, the content of tea tree flower extract in the composite bacterial agent is 0.03-0.05 g / mL.

[0008] The present invention also provides a method for preparing the composite bacterial agent, comprising the following steps:

[0009] (1) Inoculate Bacillus jelly-like and Penicillium krusei into ABM solid medium and PDA solid medium respectively, and dilute with purified water to form a bacterial suspension;

[0010] (2) mixing the obtained bacterial suspensions, and adding tea tree flower extract to the obtained mixture to obtain a bacterial agent composition;

[0011] Among them, the name of the Penicillium koreana strain is Penicillium koreana 23301-20, the preservation number is CGMCC No.41049, the preservation time is January 22, 2024, the preservation unit is the General Microbiology Center of the China Culture Collection Committee, and the preservation address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0012] Preferably, 3-5 mg / L of baicalin is added to the PDA solid culture medium used to culture Penicillium krusei in step (1).

[0013] Preferably, the viable bacterial count of the bacterial suspension in step (1) is 1.8-3.0×10 9 cfu / mL.

[0014] Preferably, the mixing in step (2) is carried out according to the ratio of live bacteria, and the total number of live bacteria is adjusted to 1.5×10 8 -5.5×10 9 cfu / mL.

[0015] The amount of tea tree flower extract added in step (2) is 0.03-0.05 g / mL.

[0016] The preparation method of the tea tree flower extract in step (2) comprises:

[0017] Take dried tea tree flowers, add ethanol aqueous solution, heat and reflux for extraction, filter and obtain the filtrate, concentrate and freeze-dry to obtain tea tree flower extract powder.

[0018] Preferably, the anhydrous ethanol content in the ethanol aqueous solution is 75-90 vol%, the material-liquid mass ratio is 1:(10-20), the extraction temperature is 80-85° C., and the extraction time is 60-90 min.

[0019] The characteristics of the Penicillium krusei colony are that the colony is white at first, then turns dark green, the conidiophores branch multiple times to form a typical broom-like structure, and conidia are continuously produced at the top, and the conidia are nearly spherical.

[0020] Preferably, the deposit number of the jelly-like Bacillus is CCTCC NO: M 20231473.

[0021] The present invention also provides application of the composite bacterial agent in improving alkaline soil.

[0022] The beneficial effects of the present invention are as follows:

[0023] The bacterial agent composition prepared by the method provided by the present invention can improve the planting conditions of alkaline soil in a long-term manner. The addition of baicalein regulates the metabolism of Penicillium koreana. The use of the composite bacterial agent can effectively ensure the flora dominance of Penicillium koreana in the soil, promote the growth of soil colonies, and maintain a good microbial environment for plant growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a scanning electron microscope image of the soil sample of the planting layer after being treated in Example 1 of the present invention;

[0025] Figure 2 This is the mass spectrum of the soil sample of the planting layer after being treated in Example 1 of the present invention;

[0026] Figure 3 This is a microscopic particle distribution diagram of the soil sample in the planting layer after being treated in Example 1 of the present invention;

[0027] Figure 4 This is a scanning electron microscope image of the soil sample of the planting layer after being treated in Comparative Example 3 of the present invention;

[0028] Figure 5 This is the mass spectrum of the soil sample of the planting layer after being treated in Comparative Example 3 of the present invention;

[0029] Figure 6 This is the microscopic particle distribution diagram of the soil sample of the planting layer after being treated in Comparative Example 3 of the present invention;

[0030] Figure 7This is a scanning electron microscope image of the soil sample of the planting layer after being treated in Comparative Example 6 of the present invention;

[0031] Figure 8 This is the mass spectrum of the soil sample of the planting layer after being treated in Comparative Example 6 of the present invention;

[0032] Fig. 9 This is the microscopic particle distribution diagram of the planting layer soil sample after being treated in Comparative Example 6 of the present invention. DETAILED DESCRIPTION

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0034] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only and are not intended to limit the content of this application.

[0035] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are purchased from commercial channels unless otherwise specified. The deposit number of the jelly-like Bacillus in the examples is CCTCC NO: M 20231473.

[0036] Preparation Example 1 Preparation of tea tree flower extract:

[0037] Take dried tea tree flowers, add ethanol aqueous solution, heat and reflux for extraction, filter and obtain the filtrate, concentrate and freeze-dry to obtain tea tree flower extract powder.

[0038] The anhydrous ethanol content in the ethanol aqueous solution is 75 vol%, the material-liquid mass ratio is 1:15, the extraction temperature is 80° C., and the extraction time is 90 min.

[0039] Preparation Example 2 Preparation of tea tree flower extract:

[0040] Take dried tea tree flowers, add ethanol aqueous solution, heat and reflux for extraction, filter and obtain the filtrate, concentrate and freeze-dry to obtain tea tree flower extract powder.

[0041] The anhydrous ethanol content in the ethanol aqueous solution is 90 vol%, the material-liquid mass ratio is 1:20, the extraction temperature is 80° C., and the extraction time is 90 min.

[0042] Preparation of the bacterial agent composition of the embodiment:

[0043] (1) Inoculate Bacillus jelly-like and Penicillium krusei into ABM solid medium and PDA solid medium respectively, and dilute with purified water to form a bacterial suspension;

[0044] (2) mixing the obtained bacterial suspensions, and adding tea tree flower extract to the obtained mixture to obtain a bacterial agent composition;

[0045] The viable bacterial count of the bacterial suspension in step (1) is 1.8-3.0×10 9 cfu / mL.

[0046] In step (2), the mixture is mixed according to the ratio of live bacteria of Penicillium koreana and Bacillus jelly-like to 1:(0.5-1), and the total live bacteria count is adjusted to 1.5×10 8 -5.5×10 9 cfu / mL.

[0047] The amount of tea tree flower extract added in step (2) is 0.03-0.05 g / mL.

[0048] Example 1

[0049] The bacterial agent composition was prepared according to the above method, wherein the ratio of live bacteria of Penicillium kosnerii and Bacillus jelly-like in the bacterial agent was 1:0.5; the total number of live bacteria in the bacterial agent composition was 1.5×10 8 The content of the tea tree flower extract (prepared in Preparation Example 1) is 0.03 g / mL.

[0050] Example 2

[0051] The bacterial agent composition was prepared according to the above method, wherein the ratio of live bacteria of Penicillium kosnerii and Bacillus jelly-like in the bacterial agent was 1:1; the total number of live bacteria in the bacterial agent composition was 1.5×10 8 The content of the tea tree flower extract (prepared in Preparation Example 1) is 0.03 g / mL.

[0052] Example 3

[0053] The bacterial agent composition was prepared according to the above method, wherein the ratio of live bacteria of Penicillium krusei and Bacillus jelly-like in the bacterial agent was 1:1; the total number of live bacteria in the bacterial agent composition was 5.5×10 9 The content of the tea tree flower extract (prepared in Preparation Example 1) is 0.03 g / mL.

[0054] Example 4

[0055] The bacterial agent composition was prepared according to the above method, wherein the ratio of live bacteria of Penicillium krusei and Bacillus jelly-like in the bacterial agent was 1:1; the total number of live bacteria in the bacterial agent composition was 5.5×10 9The content of the tea tree flower extract (prepared in Preparation Example 2) is 0.03 g / mL.

[0056] Example 5

[0057] The bacterial agent composition was prepared according to the above method, wherein the ratio of live bacteria of Penicillium krusei and Bacillus jelly-like in the bacterial agent was 1:1; the total number of live bacteria in the bacterial agent composition was 5.5×10 9 The content of the tea tree flower extract (prepared in Preparation Example 2) is 0.05 g / mL.

[0058] Example 6

[0059] The difference from Example 1 is that 3 mg / L baicalein is added to the PDA solid culture medium.

[0060] The difference between Comparative Example 1 and Example 1 is that the bacterial agent composition does not include Bacillus jelly-like, and the number of live bacteria of Penicillium krusei is the total number of live bacteria in Example 1.

[0061] The difference between Comparative Example 2 and Example 1 is that the bacterial agent composition does not include Penicillium koreana, and the number of live bacteria of Bacillus jelly-like is the total number of live bacteria in Example 1.

[0062] The difference between Comparative Example 3 and Example 1 is that the bacterial agent composition does not include tea tree flower extract.

[0063] Comparative Example 4: A jelly-like Bacillus suspension was prepared using ABM solid culture medium and diluted with purified water until the number of viable bacteria was the total number of viable bacteria in Example 1.

[0064] Comparative Example 5: A bacterial suspension of Penicillium krusei was prepared using PDA solid culture medium and diluted by eluting with pure water until the number of viable bacteria was the total number of viable bacteria in Example 1.

[0065] Comparative Example 6: The tea tree flower extract prepared in Preparation Example 1 was added to pure water in an amount of 0.03 g / mL.

[0066] For the blank case, take pure water for later use.

[0067] Experimental example:

[0068] The following experiment was conducted at the planting base of our unit in Heilongjiang Province: soil samples were tested before the experiment, with a pH of 8.77-8.92 and a total salt content of 7.66-7.91 g / kg in the soil. The products obtained in the above-mentioned embodiments, comparative examples and blank examples were mixed in the soil at 100 kg / mu for 15 days and corn was planted. Other planting measures and parameters were exactly the same. Soil samples were taken on the 15th, 30th, 60th and 90th days of planting to test soil salt content, pH and total number of microorganisms. The soil morphology was tested regularly, the soil electron microscope structure was scanned, the particle content and distribution in the soil were tested, and the results were collated and analyzed. (Soil data was tested by block detection, and the average value of 3 points was taken within the limited range. The test samples were soil samples of the planting layer - samples were taken 15 cm from the ground surface.)

[0069] Table 1 Soil pH, total salt (soluble salt g / kg) and microbial test results

[0070]

[0071]

[0072] The above test results are shown in Table 1 and the attached drawings of the specification. From the obtained results, it can be seen that the bacterial agent provided by the present invention and the preparation method thereof, the components added to the culture medium of Penicillium koreana effectively improve the growth of Penicillium koreana, and in subsequent use, the bacterial agent has a better proliferation effect in the soil, a larger number of colonies, and a more significant improvement effect on the soil. At the same time, the addition of the tea tree flower extract not only plays a certain role in regulating the pH of the soil by itself, but more importantly, it synergizes with Bacillus jelly to jointly regulate the colonization of Penicillium koreana, further enhancing the improvement effect of the bacterial agent on alkaline soil.

[0073] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

[0074] The present invention and its implementation methods are described above, which is not restrictive. The drawings are only one of the implementation methods of the present invention, and the actual application is not limited thereto. In short, if ordinary technicians in the field are inspired by it and design methods and embodiments similar to the technical solution without creativity without departing from the purpose of the invention, they should all fall within the protection scope of the present invention.

Claims

1. A composite bacterial agent for improving alkaline soil based on Penicillium koreana, characterized in that: Including Penicillium kosnerii, Bacillus gelatinosa, and tea tree flower extract.

2. The composite bacterial agent for improving alkaline soil based on Penicillium koreana according to claim 1, characterized in that: In the composite bacterial agent, the ratio of the number of live bacteria of Penicillium krusei to that of Bacillus gelatinosa is 1:(0.5-1).

3. The composite bacterial agent for improving alkaline soil based on Penicillium koreana according to claim 1, characterized in that: The total number of viable bacteria of Penicillium koreana and Bacillus jelly-like in the composite bacterial agent is 1.5×10 8 -5.5×10 9 cfu / mL.

4. The composite bacterial agent for improving alkaline soil based on Penicillium koreana according to claim 1, characterized in that: In the composite bacterial agent, the content of tea tree flower extract is 0.03-0.05 g / mL.

5. A method for preparing the composite bacterial agent according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) Inoculate Bacillus jelly-like and Penicillium krusei into ABM solid medium and PDA solid medium respectively, and dilute with purified water to form a bacterial suspension; (2) mixing the obtained bacterial suspensions, and adding tea tree flower extract to the obtained mixture to obtain a bacterial agent composition; Among them, the name of the Penicillium koreana strain is Penicillium koreana 23301-20, the preservation number is CGMCC No.41049, the preservation time is January 22, 2024, the preservation unit is the General Microbiology Center of the China Culture Collection Committee, and the preservation address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

6. The method for preparing the composite bacterial agent according to claim 5, characterized in that: In step (1), 3-5 mg / L of baicalin is added to the PDA solid culture medium used to culture Penicillium krusei.

7. The method for preparing the composite bacterial agent according to claim 5, characterized in that: The viable bacterial count of the bacterial suspension in step (1) is 1.8-3.0×10 9 cfu / mL.

8. The method for preparing the composite bacterial agent according to claim 5, characterized in that: The mixing in step (2) is carried out according to the ratio of live bacteria, and the total number of live bacteria is adjusted to 1.5×10 8 -5.5×10 9 cfu / mL; the amount of tea tree flower extract added in step (2) is 0.03-0.05g / mL.

9. The method for preparing the composite bacterial agent according to claim 5, characterized in that: The preparation method of the tea tree flower extract in step (2) comprises: Take dried tea flowers, add ethanol aqueous solution, heat and reflux extraction, filter the filtrate, concentrate and freeze-dry to obtain tea flower extract powder; wherein the anhydrous ethanol content in the ethanol aqueous solution is 75-90 vol%, the material-liquid mass ratio is 1:(10-20), the extraction temperature is 80-85°C, and the extraction time is 60-90 min.

10. Use of the composite bacterial agent according to any one of claims 1 to 4 in improving alkaline soil.

Images