Penicillium clavatum agent composition for improving saline-alkali soil and application of penicillium clavatum agent composition

Abstract

The invention provides a penicillium clavatum agent composition for improving saline-alkali soil and application of the penicillium clavatum agent composition, and belongs to the technical field of soil improvement. The composition is prepared from penicillium kirschwskii, pseudomonas alcaligenes and an artemisia sacrorum extract. Wherein the name of the penicillium kenii strain is penicillium kenii 23301-20, the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.41049, and the preservation unit is China General Microbiological Culture Collection Center. When the microbial agent composition is applied to saline-alkali soil, the ion content and the soil hardening condition can be effectively improved.

Description

Technical Field

[0001] This invention provides a Penicillium clopidogrel agent composition for improving saline-alkali soil and its application, relating to the field of soil improvement technology. Background Technology

[0002] Penicillium, a common member of the microbial world, occupies a pivotal position in scientific research and agricultural production due to its powerful biological activity and diverse ecological functions. Among them, *Penicillium kirchowii*, as a member of the Penicillium family, has attracted much attention due to its unique properties and potential application value. However, although *Penicillium kirchowii* has shown broad applicability in agricultural production, especially in preliminary explorations of saline-alkali soil improvement, unfortunately, current technology is not yet mature, and the improvement effect is not entirely satisfactory. Penicillium, with its wide distribution and diverse functions, has become an important assistant in agricultural production. It can decompose organic matter, promote nutrient release, and provide crops with abundant nutritional sources. At the same time, Penicillium can also produce a variety of secondary metabolites, which have antibacterial and antiviral biological activities, helping to reduce crop diseases and improve crop resistance. *Penicillium kirchowii*, as a leader in the Penicillium family, has become a star strain in agricultural production due to its powerful biological activity and environmental adaptability. In agricultural production, the application of *Penicillium kirchowii* is mainly reflected in soil improvement and plant protection. Inoculating with *Penicillium clarkii* can improve the soil's microecological environment, promote the reproduction of beneficial microorganisms, and enhance soil fertility and aeration. Simultaneously, *Penicillium clarkii* can produce various antibiotics and antimicrobial peptides, which significantly inhibit pathogens in the soil, thus helping to reduce crop diseases and improve crop yield and quality. However, when scientists attempted to apply *Penicillium clarkii* to saline-alkali soil improvement, they encountered a series of challenges. Saline-alkali soil is a special type of soil with excessively high salt content and pH imbalance, which severely restricts the growth and reproduction of microorganisms. Although *Penicillium clarkii* possesses strong biological activity, it is difficult to exert its intended improvement effect in saline-alkali soil. The main reasons for this are as follows: First, the high salinity of saline-alkali soil puts immense pressure on the growth and metabolism of *Penicillium clarkii*. High salinity leads to osmotic pressure imbalance in cells, damaging cell structure and thus affecting the biological activity and improvement effect of *Penicillium clarkii*. Second, the pH imbalance of saline-alkali soil is also a significant factor restricting the application of *Penicillium clarkii*. *Penicillium clarkii* thrives in slightly acidic or neutral environments, while saline-alkali soils are typically alkaline or strongly alkaline. This pH mismatch leads to a lower survival rate for *Penicillium clarkii* in saline-alkali soils, resulting in less than ideal soil improvement effects. Despite the numerous challenges *Penicillium clarkii* faces in saline-alkali soil improvement, scientists have not been discouraged. They are continuously exploring new methods and technologies to improve its adaptability and soil improvement effects in saline-alkali soils. For example, genetic engineering techniques are being used to modify *Penicillium clarkii*, enhancing its salt tolerance and pH adaptability; or synergistic effects with other microorganisms are being explored to construct more complex microbial community structures, thereby strengthening *Penicillium clarkii*'s survival and soil improvement capabilities in saline-alkali soils. In conclusion, *Penicillium clarkii*, as a leading member of the Penicillium family, has broad application prospects in agricultural production.However, current technologies for improving saline-alkali soil are not yet mature, and the improvement results are not ideal. Summary of the Invention

[0003] To solve the above problems, the present invention provides the following specific solution:

[0004] A composition of Penicillium clavatum agent for improving saline-alkali soil includes Penicillium clavatum, Pseudomonas alkali-producing bacteria, and Artemisia annua extract.

[0005] The strain of Penicillium kloeckeri is named Penicillium kloeckeri 23301-20, classified as Penicillium kloeckeri, with accession number CGMCC No. 41049, deposited on January 22, 2024, deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] The colony characteristics of Penicillium clarithii are that the colony is initially white, then turns dark green, and the conidiophores branch multiple times to form a typical broom-like structure, continuously producing conidia at the top, with the conidia being nearly spherical.

[0007] Preferably, *Alcaligenes alkali-producing* has been disclosed in Chinese patent CN202011578994.7 and is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.21151.

[0008] Preferably, the live bacteria ratio of Penicillium clarithropii and Pseudomonas alcaligenes is (3-5):(1-3);

[0009] Preferably, the total viable count of *Penicillium kurine* and *Alcaligenes alkaligenes* is 1.5 × 10⁻⁶. 8 -5.5×10 9 cfu / mL.

[0010] The present invention also provides the application of the above-mentioned Penicillium clopidogrel agent composition for improving saline-alkali soil in improving saline-alkali soil.

[0011] Preferably, the preparation method of the Artemisia annua extract includes:

[0012] S1. Take fresh and clean Artemisia argyi roots, crush them and sieve them to obtain crushed roots;

[0013] S2. The obtained pulverized roots were soaked in an ethanol-water solution, and then heated and refluxed for extraction.

[0014] S3. After cooling the mixture to room temperature, filter it and concentrate the filtrate to obtain Artemisia annua extract.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] Preferably, the content of the Artemisia annua extract is 0.03-0.05 g / mL.

[0019] The beneficial effects of this invention are as follows:

[0020] The microbial agent composition provided by this invention, when applied to saline-alkali soil, can sustainably improve the planting conditions of saline-alkali soil. Attached Figure Description

[0021] 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;

[0022] Figure 2 This is a mass spectrum of the planting layer soil sample after treatment in Example 1 of the present invention;

[0023] Figure 3 This is a microscopic particle distribution diagram of the planting layer soil sample after treatment in Example 1 of the present invention;

[0024] Figure 4 This is a scanning electron microscope image of the planting layer soil sample after treatment in Comparative Example 3 of this invention;

[0025] Figure 5 This is a mass spectrum of the planting layer soil sample after treatment in Comparative Example 3 of this invention;

[0026] Figure 6 This is a microscopic particle distribution diagram of the planting layer soil sample after treatment in Comparative Example 3 of this invention.

[0027] Figure 7 This is a scanning electron microscope image of the planting layer soil sample after treatment in Comparative Example 6 of this invention;

[0028] Figure 8 This is a mass spectrum of the planting layer soil sample after treatment in Comparative Example 6 of this invention;

[0029] 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

[0030] 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.

[0031] 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.

[0032] 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.

[0033] Preparation Example 1: Preparation of Artemisia annua extract:

[0034] S1. Take fresh and clean Artemisia argyi roots, crush them and sieve them to obtain crushed roots;

[0035] S2. The obtained pulverized roots were soaked in an ethanol-water solution, and then heated and refluxed for extraction.

[0036] S3. After cooling the mixture to room temperature, filter it and concentrate the filtrate to obtain Artemisia annua extract.

[0037] 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.

[0038] 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.

[0039] The concentration step S3 involves rotary evaporating the extract until no more solvent evaporates, thus obtaining the extract.

[0040] Preparation Example 2: Preparation of Artemisia annua extract:

[0041] S1. Take fresh and clean Artemisia argyi roots, crush them and sieve them to obtain crushed roots;

[0042] S2. The obtained pulverized roots were soaked in an ethanol-water solution, and then heated and refluxed for extraction.

[0043] S3. After cooling the mixture to room temperature, filter it and concentrate the filtrate to obtain Artemisia annua extract.

[0044] 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.

[0045] 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.

[0046] The concentration step S3 involves rotary evaporating the extract until no more solvent evaporates, thus obtaining the extract.

[0047] Preparation of Penicillium clavatum agent composition for improving saline-alkali soil:

[0048] (1) Alcaligenes and Penicillium kuriformis were inoculated into CM0002 liquid medium and PDA solid medium, respectively, and diluted with purified water to a viable count of 1.8-3.0 × 10⁻⁶. 9 cfu / mL;

[0049] (2) The obtained bacterial suspension was mixed with Penicillium clarithromyces and Pseudomonas alkaligenes at a ratio of (3-5):(1-3), and the total number of viable bacteria was adjusted to 1.5 × 10⁻⁶. 8 -5.5×10 9 Add cfu / mL of Artemisia annua extract to the resulting mixture to obtain the fungal agent composition.

[0050] The amount of Artemisia annua extract added in step (2) is 0.03-0.05 g / mL.

[0051] Example 1

[0052] The microbial agent composition was prepared according to the above method, wherein the live bacteria ratio of *Penicillium kuriformis* and *Pseudomonas alkaligenes* in the microbial agent was 3:3; 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.

[0053] Example 2

[0054] The microbial agent composition was prepared according to the above method, wherein the live bacteria ratio of *Penicillium kuriformis* and *Pseudomonas alkaligenes* 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.

[0055] Example 3

[0056] The microbial agent composition was prepared according to the above method, wherein the live bacteria ratio of *Penicillium kurine* and *Pseudomonas alkaligenes* 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 1) was 0.03 g / mL.

[0057] Example 4

[0058] The microbial agent composition was prepared according to the above method, wherein the live bacteria ratio of *Penicillium kurine* and *Pseudomonas alkaligenes* 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 0.03 g / mL.

[0059] Example 5

[0060] The microbial agent composition was prepared according to the above method, wherein the live bacteria ratio of *Penicillium kurine* and *Pseudomonas alkaligenes* 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.

[0061] The difference between Comparative Example 1 and Example 1 is that the bacterial agent composition does not include Alcaligenes, and the number of viable Penicillium kuriformis is the same as the total number of viable bacteria in Example 1.

[0062] 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 Pseudomonas alkali-producing bacteria is the same as the total number of viable bacteria in Example 1.

[0063] The difference between Comparative Example 3 and Example 1 is that the fungal agent composition does not include Artemisia annua extract.

[0064] Comparative Example 4: Alkaloid Pseudomonas alkali-producing strains were prepared by eluting and diluting with purified water to a viable count equal to the total viable count in Example 1.

[0065] 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.

[0066] 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.

[0067] For the blank example, prepare purified water for later use.

[0068] Experimental example:

[0069] 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.)

[0070] Table 1. Results of soil total salt (soluble salt g / kg) and pH test.

[0071]

[0072]

[0073] 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.

[0074] 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.

[0075] 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 composition of Penicillium clavatum agent for improving saline-alkali soil, characterized in that: Penicillium chrysogenum, Pseudomonas alcaligenes, ironweed extract; The Penicillium chrysogenum strain is named Penicillium chrysogenum 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, Xibaheyi, Beijing, China.

2. The Paenibacillus chonosii agent composition for improving saline soil according to claim 1, characterized by: The colony of the Penicillium chrysogenum is initially white, then becomes dark green, the conidial phialae are branched multiple times to form a typical broom-like structure, and the conidia are continuously produced at the top.

3. The Paenibacillus chonosii agent composition for improving saline soil according to claim 1, characterized by: The Pseudomonas alcaligenes is preserved in China General Microbiological Culture Collection Center, and the preservation number is CGMCC NO.21151.

4. The Paenibacillus chonosii agent composition for improving saline soil according to claim 1, characterized by: The live bacteria ratio of the Penicillium chrysogenum and the Pseudomonas alcaligenes is (3-5):(1-3).

5. The Paenibacillus chonosii agent composition for improving saline soil according to claim 1, characterized by: The total viable cell count of the P. chrysogenum, P. alcaligenes is 1.5 x 10 8 -5.5 x 10 9 cfu / mL.

6. Application of the Penicillium chrysogenum agent composition for improving saline-alkali soil according to any one of claims 1-5 to improvement of saline-alkali soil.

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