Preparation method of penicillium clavatum agent composition for improving saline-alkali soil
Patent Information
- Application Number
- CN202411993155.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-31
- Publication Date
- 2026-03-17
Smart Images
Figure CN121674071A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application provides a preparation method of a paecilomyces variotii microbial agent composition for improving saline-alkali soil, and relates to the technical field of soil improvement. BACKGROUND
[0002] In the vast world of microorganisms, Penicillium is known for its wide distribution and diverse functions. Among them, Penicillium chrysogenum, as an important member of the Penicillium family, not only attracts attention due to its unique biological activity, but also shows broad application prospects in agricultural production. However, although P. chrysogenum shows potential in soil improvement and crop protection, especially in the preliminary exploration of saline-alkali soil improvement, current technological development is not perfect, and its improvement effect still needs to be improved. Penicillium, a widely existing fungal group in soil, air and plant surface, actively participates in the decomposition of organic matter and nutrient cycling in soil by secreting various enzymes and organic acids. P. chrysogenum, as one of the Penicillium, has become a new star in agricultural production due to its strong biological activity and environmental adaptability. It can produce a variety of secondary metabolites, which not only have antibacterial and antiviral activity, but also can promote crop growth and improve crop stress resistance. In agricultural production practice, the application of P. chrysogenum mainly focuses on two major fields of soil improvement and disease control. By inoculating P. chrysogenum, the microecological environment of soil can be effectively improved, the reproduction of beneficial microorganisms can be promoted, and the fertility and air permeability of soil can be improved. At the same time, the secondary metabolites produced by P. chrysogenum can effectively inhibit the pathogenic bacteria in the soil, reduce the occurrence of crop diseases, and thus ensure the healthy growth and high yield of crops. However, when scientists turn their attention to saline-alkali soil improvement, the application of P. chrysogenum faces unprecedented challenges. Saline-alkali soil, a special type of soil, has a high salt content and an unbalanced pH, which poses a great threat to the growth and reproduction of microorganisms. Although P. chrysogenum has strong biological activity, its growth and metabolic activity are severely inhibited in the high-salt environment and unbalanced pH of saline-alkali soil, and the improvement effect is naturally not satisfactory. The reason is that, on the one hand, the high salt content of saline-alkali soil can damage the cell structure of P. chrysogenum, leading to blocked metabolic activity and reduced biological activity. On the other hand, the unbalanced pH of saline-alkali soil also affects the growth and reproduction of P. chrysogenum, making it difficult to colonize and function in saline-alkali soil. In addition, the microbial community structure in saline-alkali soil is complex, and P. chrysogenum is difficult to form a dominant flora in it, which further limits its application effect in saline-alkali soil improvement. Although P. chrysogenum faces many challenges in saline-alkali soil improvement, scientists have not given up. They are constantly exploring new technologies and methods to improve the adaptability and improvement effect of P. chrysogenum in saline-alkali soil. For example, by screening and cultivating P. chrysogenum strains resistant to saline-alkali, or by synergistic action with other microorganisms, a more stable microbial community structure is constructed to enhance the survival and improvement ability of P. chrysogenum in saline-alkali soil. In summary, P. chrysogenum, as a star in the Penicillium family, has broad application prospects in agricultural production. However, in the aspect of saline-alkali soil improvement, the current technology is not mature, and the improvement effect still needs to be improved. SUMMARY
[0003] To solve the above problems, the specific scheme provided by the present application is as follows:
[0004] A preparation method of a penicillium kloeckeri microbial agent composition for improving saline-alkali soil, comprising the following steps:
[0005] (1) Bacillus polymyxa and Penicillium kloeckeri are inoculated into NA solid culture medium and PDA solid culture medium respectively, and pure water is diluted into a bacterial suspension;
[0006] (2) The obtained bacterial suspension is mixed, and an artemisia ferruginea extract is added to the obtained mixture to obtain a microbial agent composition;
[0007] The strain name of the Penicillium kloeckeri is Penicillium kloeckeri 23301-20, the preservation number is CGMCC No. 41049, the preservation time is January 22, 2024, the preservation unit is China General Microbiological Culture Collection Center, and the preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing.
[0008] Preferably, the viable bacterial count of the bacterial suspension in step (1) is 1.8-3.0×10 9 cfu / mL.
[0009] Preferably, the mixing in step (2) is carried out at a viable bacterial ratio of Penicillium kloeckeri to Bacillus polymyxa of (3-5):(1-3), and the total viable bacterial count is adjusted to 1.5×10 8 -5.5×10 9 cfu / mL.
[0010] The artemisia ferruginea extract is added in step (2) at an addition amount of 0.03-0.05 g / mL.
[0011] The colony characteristics of the Penicillium kloeckeri are that the colony is initially white and then becomes dark green, the conidial phialide is branched multiple times to form a typical broom-like structure, and the conidia are continuously produced at the top, and the conidia are nearly spherical.
[0012] Preferably, the Bacillus polymyxa is preserved in China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 18386.
[0013] Preferably, the viable bacterial ratio of the Penicillium kloeckeri to the Bacillus polymyxa is (3-5):(2-3);
[0014] Preferably, the total viable bacterial count of the microbial agent composition is 1.5×10 8 -5.5×10 9 cfu / mL.
[0015] Preferably, the preparation method of the Artemisia annua extract includes:
[0016] S1. Take fresh and clean Artemisia argyi roots, crush them and sieve them to obtain crushed roots;
[0017] S2. The obtained pulverized roots were soaked in an ethanol-water solution, and then heated and refluxed for extraction.
[0018] S3. After cooling the mixture to room temperature, filter it and collect the filtrate, then concentrate it to obtain Artemisia annua extract.
[0019] Preferably, the sieving in step S1 is through a 30-60 mesh sieve; the ethanol content in the ethanol aqueous solution in step S2 is 70-75 vol%; and the mass / volume ratio of the pulverized material to the ethanol aqueous solution in step S2 is 1 g:(10-20) mL.
[0020] Preferably, the soaking temperature in step S2 is 20-35℃, and the soaking time is 18-24h; the heating and reflux is carried out at a reflux temperature of 78.5℃ and maintained in reflux state for 2-3h.
[0021] Preferably, the concentration in step S3 involves rotary evaporating the extract until no more solvent evaporates, obtaining the extract, which is then stored at 4°C for later use.
[0022] Preferably, the content of the Artemisia annua extract is 0.03-0.05 g / mL.
[0023] The beneficial effects of this invention are as follows:
[0024] The microbial agent composition prepared by the method provided by this invention can effectively improve planting conditions in saline-alkali land over a long period of time. Attached Figure Description
[0025] Figure 1 This is a scanning electron microscope image of a soil sample from the planting layer after treatment in Example 1 of the present invention;
[0026] Figure 2 This is a mass spectrum of the planting layer soil sample after treatment in Example 1 of the present invention;
[0027] Figure 3 This is a microscopic particle distribution diagram of the planting layer soil sample after treatment in Example 1 of the present invention;
[0028] Figure 4 This is a scanning electron microscope image of the planting layer soil sample after treatment in Comparative Example 3 of this invention;
[0029] Figure 5 This is a mass spectrum of the planting layer soil sample after treatment in Comparative Example 3 of this invention;
[0030] Figure 6 This is a microscopic particle distribution diagram of the planting layer soil sample after treatment in Comparative Example 3 of this invention.
[0031] Figure 7 This is a scanning electron microscope image of the planting layer soil sample after treatment in Comparative Example 6 of this invention;
[0032] Figure 8 This is a mass spectrum of the planting layer soil sample after treatment in Comparative Example 6 of this invention;
[0033] Figure 9 This is a microscopic particle distribution diagram of the planting layer soil sample after treatment in Comparative Example 6 of this invention. Detailed Implementation
[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.
[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.
[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the experimental materials used in the following examples are all purchased from commercial channels.
[0037] Preparation Example 1: Preparation of Artemisia annua extract:
[0038] S1. Take fresh and clean Artemisia argyi roots, crush them and sieve them to obtain crushed roots;
[0039] S2. The obtained pulverized roots were soaked in an ethanol-water solution, and then heated and refluxed for extraction.
[0040] S3. After cooling the mixture to room temperature, filter it and collect the filtrate, then concentrate it to obtain Artemisia annua extract.
[0041] The sieving in step S1 is through a 60-mesh sieve; the ethanol content in the ethanol-water solution in step S2 is 70 vol%; the mass / volume ratio of the pulverized material to the ethanol-water solution in step S2 is 1 g: 10 mL.
[0042] The soaking temperature in step S2 is 25°C and the soaking time is 24 hours; the heating and reflux is carried out by heating until the solvent boils and maintaining the reflux state for 2 hours.
[0043] The concentration step S3 involves rotary evaporating the extract until no more solvent evaporates, thus obtaining the extract.
[0044] Preparation Example 2: Preparation of Artemisia annua extract:
[0045] S1. Take fresh and clean Artemisia argyi roots, crush them and sieve them to obtain crushed roots;
[0046] S2. The obtained pulverized roots were soaked in an ethanol-water solution, and then heated and refluxed for extraction.
[0047] S3. After cooling the mixture to room temperature, filter it and collect the filtrate, then concentrate it to obtain Artemisia annua extract.
[0048] The sieving in step S1 is through a 60-mesh sieve; the ethanol content in the ethanol-water solution in step S2 is 75 vol%; the mass / volume ratio of the pulverized material to the ethanol-water solution in step S2 is 1 g: 20 mL.
[0049] The soaking temperature in step S2 is 35°C and the soaking time is 18 hours; the heating and reflux process involves heating the solvent until it boils and maintaining the reflux state for 3 hours.
[0050] The concentration step S3 involves rotary evaporating the extract until no more solvent evaporates, thus obtaining the extract.
[0051] Example: Preparation of the microbial agent composition:
[0052] (1) Inoculate Bacillus polymyxa and Penicillium kuribesti into NA solid medium and PDA solid medium respectively, and dilute with purified water to form bacterial suspensions;
[0053] (2) Mix the obtained bacterial suspension, add Artemisia annua extract to the mixture to obtain a bacterial agent composition;
[0054] The viable bacterial count in the bacterial suspension in step (1) is 1.8-3.0 × 10⁻⁶. 9 cfu / mL.
[0055] The mixing in step (2) is carried out at a live bacteria ratio of (3-5):(1-3) for Penicillium clarithromyces and Bacillus polymyxa, and the total live bacteria count is adjusted to 1.5 × 10⁻⁶. 8 -5.5×10 9 cfu / mL.
[0056] The amount of Artemisia annua extract added in step (2) is 0.03-0.05 g / mL.
[0057] Example 1
[0058] The microbial agent composition was prepared according to the above method, wherein the live bacteria ratio of Penicillium clarkii and Bacillus polymyxa in the microbial agent was 3:1; the total live bacteria count of the microbial agent composition was 1.5 × 10⁻⁶. 8 The concentration of the Artemisia annua extract (prepared in Preparation Example 1) was 0.03 g / mL.
[0059] Example 2
[0060] The microbial agent composition was prepared according to the above method, wherein the live bacteria ratio of Penicillium clarkii and Bacillus polymyxa in the microbial agent was 5:1; the total live bacteria count of the microbial agent composition was 1.5 × 10⁻⁶. 8 The concentration of the Artemisia annua extract (prepared in Preparation Example 1) was 0.03 g / mL.
[0061] Example 3
[0062] The microbial agent composition was prepared according to the above method, wherein the live bacteria ratio of Penicillium clarkii and Bacillus polymyxa in the microbial agent was 5:1; and the total live bacteria count of the microbial agent composition was 5.5 × 10⁻⁶. 9 The concentration of the Artemisia annua extract (prepared in Preparation Example 1) was 0.03 g / mL.
[0063] Example 4
[0064] The microbial agent composition was prepared according to the above method, wherein the live bacteria ratio of Penicillium clarkii and Bacillus polymyxa in the microbial agent was 5:1; and the total live bacteria count of the microbial agent composition was 5.5 × 10⁻⁶. 9 The concentration of the *Artemisia annua* extract (prepared in Preparation Example 2) was 0.03 g / mL.
[0065] Example 5: The microbial agent composition was prepared according to the above method. The live bacteria ratio of Penicillium clarkii and Bacillus polymyxa in the microbial agent was 5:1; the total live bacteria count of the microbial agent composition was 5.5 × 10⁻⁶. 9 The concentration of the Artemisia annua extract (prepared in Preparation Example 2) was 0.05 g / mL.
[0066] The difference between Comparative Example 1 and Example 1 is that the bacterial agent composition does not include Bacillus polymyxa, and the number of viable Penicillium kuribda is the same as the total number of viable bacteria in Example 1.
[0067] The difference between Comparative Example 2 and Example 1 is that the bacterial agent composition does not include Penicillium kuribda, and the number of viable Bacillus polymyxa is the same as the total number of viable bacteria in Example 1.
[0068] The difference between Comparative Example 3 and Example 1 is that the fungal agent composition does not include Artemisia annua extract.
[0069] Comparative Example 4: Polymyxin Bacillus suspension was prepared using NA solid medium, washed and diluted with purified water to a viable count equal to the total viable count of Example 1.
[0070] Comparative Example 5: Penicillium clarithromyces was prepared using PDA solid culture medium and diluted with purified water to a viable count equal to the total viable count in Example 1.
[0071] Comparative Example 6: The Artemisia annua extract prepared in Preparation Example 1 was added to purified water at a concentration of 0.03 g / mL.
[0072] For the blank example, prepare purified water for later use.
[0073] Experimental example:
[0074] The following experiment was conducted at our planting base in Heilongjiang Province: Soil samples were tested before the experiment; the pH was 8.77-8.92, and the total salt content was 7.66-7.91 g / kg. Corn was planted 100 kg / mu of the product obtained in the above-mentioned examples, comparative examples, and blank examples after mixing with the soil. Other planting measures and parameters were completely identical. Soil samples were taken on days 15, 30, 60, and 90 after planting to test soil salinity and pH. Soil morphology was also periodically examined, and the soil structure was scanned using electron microscopy to detect the particle content and distribution. The results were compiled and analyzed. (Soil data were collected by dividing the soil into blocks, and the average value of three points within a defined range was taken. The test samples were from the planting layer soil sample taken 15 cm below the soil surface.)
[0075] Table 1. Results of soil total salt (soluble salt g / kg) and pH test.
[0076]
[0077]
[0078] The above test results are shown in Table 1 and the attached figures in the specification. As can be seen from the results, the microbial agent provided by the present invention enhances the improvement effect of the core microbial species Penicillium kuribii on saline-alkali soil through the synergistic effect of additives and auxiliary microbial species.
[0079] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.
[0080] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.
Claims
1. A process for the preparation of a Penicillium chrysogenum agent composition for the improvement of saline-alkali soils, characterized by: The method comprises the following steps: (1) Bacillus polymyxa and Paecilomyces lilacinus are inoculated into NA solid medium and PDA solid medium respectively, and pure water is diluted to obtain a bacterial suspension; (2) The obtained bacterial suspension is mixed, and an extract of Artemisia sacopula is added to the obtained mixture to obtain a bacterial agent composition; The Paecilomyces lilacinus strain is named Paecilomyces lilacinus 23301-20, the preservation number is CGMCC No. 41049, and the preservation unit is China General Microbiological Culture Collection Center.
2. The process for the preparation of Paenibacillus chlorellae agent composition for the reclamation of saline soil as claimed in claim 1 wherein: The viable cell count of the bacterial suspension in step (1) is 1.8-3.0 x 10 9 cfu / mL.
3. The process for the preparation of Paenibacillus chlorellae agent composition for the reclamation of saline soil as claimed in claim 1 wherein: The mixing in step (2) is carried out in a ratio of viable Penicillium chrysogenum to Bacillus polymyxa of (3-5) : (1-3) and the total viable count is adjusted to 1.5 x 10 8 -5.5 x 10 9 cfu / mL.
4. The process for the preparation of Paenibacillus chlorellae agent composition for the reclamation of saline soil as claimed in claim 1 wherein: The extract of Artemisia sacopula is added in step (2), and the addition amount is 0.03-0.05 g / mL.
5. The process for the preparation of Paenibacillus chlorellae agent composition for the reclamation of saline soil as claimed in claim 1 wherein: The Paecilomyces lilacinus colony has the following characteristics: the colony is initially white, then becomes dark green, the conidial phialide is branched multiple times to form a typical broom-like structure, and the conidial phialide continuously produces conidia at the top, and the conidia are nearly spherical.
6. The process for the preparation of Paenibacillus chlorellae agent composition for the reclamation of saline soil as claimed in claim 1, wherein: The Bacillus polymyxa is preserved in China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 18386.
7. The process for the preparation of Paenibacillus chlorellae agent composition for the reclamation of saline soil as claimed in claim 1, wherein: Preferably, the preparation method of the extract of Artemisia sacopula comprises: S1, taking fresh and clean Artemisia sacopula roots, crushing and sieving to obtain crushed roots; S2, the obtained crushed roots are soaked with an ethanol aqueous solution, and then heated to reflux extraction; S3, the obtained mixture is cooled to room temperature, filtered to obtain a filtrate, and concentrated to obtain the extract of Artemisia sacopula.