CN114891646A - A kind of tripterygium wilfordii special growth-promoting bacterial agent and its preparation method and application - Google Patents

Abstract

The invention belongs to the technical field of microbial inoculants, in particular to a special growth-promoting inoculant for Tripterygium wilfordii and a preparation method and application thereof. Described microbial inoculum is fermented by Penicillium steckii NS-12, the classification of described Penicillium steckii NS-12 is named Penicillium steckii , and this bacterial strain has been preserved on June 10, 2011 in General Microbiology Center of China Microorganism Culture Collection Management Committee, the deposit number is CGMCC No.4928. The inoculant has obvious gain effect on Tripterygium wilfordii, can significantly increase the biomass and A content of Tripterygium wilfordii, and has a good application prospect.

Description

A kind of tripterygium wilfordii special growth-promoting bacterial agent and its preparation method and application

technical field

The invention belongs to the technical field of microbial inoculants, in particular to a special growth-promoting inoculant for Tripterygium wilfordii and a preparation method and application thereof.

Background technique

Tripterygium wilfordii Hook.f. is a woody vine, distributed in Fujian, Jiangxi, Zhejiang, Hubei, Hunan, Guangdong, Taiwan and other provinces in southern China, and is a precious medicinal plant. Modern research has found that Tripterygium wilfordii contains more than 100 kinds of ingredients, mainly divided into alkaloids, diterpenes, triterpenes, sesquiterpenes and sugars, and triptolide is one of its most important active ingredients, with Anti-inflammatory, anti-cancer, immune adjustment, anti-fertility and other effects are widely used in the treatment of rheumatoid arthritis, lung disease, kidney disease, skin disease, leukemia and other diseases. main ingredient. For a long time, Tripterygium wilfordii medicinal materials have been mainly obtained from wild and artificial cultivation. Due to the extremely low content of active ingredients and long growth cycle, the reserves have dropped sharply, coupled with environmental pollution in the production area and spraying of pesticides for pest control and other reasons, the supply of raw materials has been limited. It cannot meet the needs of clinical application in both quantity and quality.

In recent years, with the deepening of research, some endophytic fungi growing in medicinal plants have attracted extensive attention of researchers. So far, domestic and foreign scholars have isolated endophytic fungi that can produce the same or similar medicinal active ingredients as the host from medicinal plants such as Taxus chinensis, Ginkgo biloba, Artemisia annua, etc., so as to obtain non-plant sources of medicinal active substances. provided the possibility. However, when these special endophytes were fermented separately, it was found that the content of medicinal active ingredients produced by most strains was extremely low, and the substrate inhibition effect was obvious, which could not meet the conditions of large-scale industrial production. Other studies have found that the interaction between this type of endophyte and the host will affect the production of their respective active components. It has good application prospects depending on the content of the host plant itself. However, because these endophytes are all screened from plants and are relatively sensitive to external environmental conditions, how to preserve and maintain a certain bacterial activity for a long time is an important factor affecting their popularization and application. In this study, an endophytic fungus of Tripterygium wilfordii with the ability to produce triptolide that was screened in the previous stage was used as the object, and it was prepared into a microbial inoculum. The results are of great significance for promoting the active ingredient content of Tripterygium wilfordii and enhancing its application value. Studies related to this are rarely reported.

SUMMARY OF THE INVENTION

In order to make up for the deficiencies of the prior art, the present invention provides a special growth-promoting bacterial agent for Tripterygium wilfordii and a preparation method and application thereof. Applying the fungicide to Tripterygium wilfordii can obviously promote the growth of Tripterygium wilfordii plants and increase the content of triptolide in the body of Tripterygium wilfordii, which has a good application prospect.

The present invention is achieved through the following technical solutions:

A special growth-promoting bacterial agent for Tripterygium wilfordii, the bacterial agent is fermented by Penicillium steckii NS-12. On June 10, 2011, it was deposited in the General Microbiology Center of China Microorganism Culture Collection Management Committee, and the deposit number is CGMCC No.4928.

Further, the above-mentioned special growth-promoting bacterial agent for Tripterygium wilfordii is a solid bacterial agent.

Further, the preparation method of the above-mentioned Tripterygium wilfordii special-purpose growth-promoting solid microbial inoculum comprises the following steps:

1) Activation of strains: Inoculate the cryopreserved Penicillium difficile NS-12 on PDA solid medium and cultivate at 28°C. After the colonies grow, pick a single colony and transfer it to a new PDA solid medium. Continue to cultivate for 5 days at 28°C;

2) Preparation of spore suspension: Inoculate the activated Penicillium difficile NS-12 on a PDA plate and expand the culture at 28°C, and elute the spores with sterile water to prepare a spore suspension with a concentration of 1.0×10 ⁸ /mL liquid;

3) Solid fermentation: take 100 mL of spore suspension and inoculate it into 1000 g of solid fermentation medium and cultivate at 28°C, stir once every 2 to 3 days under sterile conditions, and obtain solid fermentation inoculum after culturing for 9 days;

4) Drying of inoculum: put the obtained solid inoculum in a drying box at 35°C, blow drying for 40 hours, and dry the solid inoculum to a water content below 10-15%, that is, to obtain a special growth promotion for Tripterygium wilfordii solid bacteria.

Further, above-mentioned solid fermentation medium is made up of the raw material of following percentage by weight: bran 26%, bean cake flour 10%, starch 10%, coconut shell flour 10%, trehalose 3%, calcium carbonate 1%, water 40% .

Further, the viable bacteria count of the special growth-promoting solid bacteria agent for Tripterygium wilfordii prepared above is 7×10 ¹⁰ cfu/g.

Further, the application mode of the above-mentioned special growth-promoting solid inoculant for Tripterygium wilfordii is as follows: applying to the rhizosphere of crops through root irrigation.

The application of the above-mentioned special growth-promoting bacterial agent for Tripterygium wilfordii in increasing the biomass of Tripterygium wilfordii.

The application of the above-mentioned special growth-promoting bacterial agent for triptolide in increasing the content of triptolide.

The significant advantages of the present invention are:

The special growth-promoting bacterial agent for Tripterygium wilfordii involved in the invention is simple in preparation, convenient in application, and can effectively maintain the activity of bacterial species, and the number of effective viable bacteria still reaches 4.7×10 ¹⁰ cfu/g after being stored for 12 months. The application effect shows that the inoculant has obvious gain effect on Tripterygium wilfordii, can significantly increase the biomass and A content of Tripterygium wilfordii, and has a good application prospect.

Description of drawings

Figure 1 shows the special growth-promoting solid bacteria agent for Tripterygium wilfordii.

Figure 2 shows the effect of the special growth-promoting solid inoculant for Tripterygium wilfordii on the Tripterygium wilfordii plants.

Detailed ways

In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be described in further detail below with reference to the examples. The equipment and reagents used in each example and test example can be obtained from commercial channels unless otherwise specified. . The specific embodiments described herein are only used to explain the present invention, and are not intended to limit the present invention.

From the information contained in this application, various changes to the precise description of the present invention can be readily made by those skilled in the art without departing from the spirit and scope of the appended claims. It is to be understood that the scope of the invention is not limited to the processes, properties or components defined, as these embodiments and other descriptions are intended to be illustrative only of particular aspects of the invention. Indeed, various modifications of the embodiments of the invention that are obvious to those skilled in the art or related fields are intended to be within the scope of the appended claims.

The strain material Penicillium steckii NS-12 used in the present invention is classified and named Penicillium steckii , which has been registered and preserved in the General Microbiology Center of the China Microorganism Culture Collection Management Committee on June 10, 2011. The serial number is: CGMCC No.4928, and the address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The Penicillium sternum NS-12 is isolated and purified from Tripterygium wilfordii plant tissue. It is gray-green on the front and yellow-brown on the back, and single round on a flat medium; the mycelium is septate and meristem Sporophore and spores are colorless, terminally peduncles, peduncles erect, unicellular, oval, smooth surface, conidiophores occur singly from the hyphae, forming a broom-like characteristic structure. According to the above characteristics, according to the "Fungi Identification Manual", the strain was identified as Penicillium steckii .

Example 1

The preparation method of the special growth-promoting solid inoculum for Tripterygium wilfordii is as follows:

(1) Preparation of culture medium:

1) PDA solid medium: fresh potatoes 200 g/L, glucose 25 g/L, agar 15 g/L, others are sterile water, pH 7.0-7.2.

2) Solid fermentation medium (by weight percentage): bran 26%, bean cake flour 10%, starch 10%, coconut shell flour 10%, trehalose 3%, calcium carbonate 1%, water 40%.

(2) Activation of strains:

The fungal spores of Penicillium difficulum NS-12 stored at low temperature were inoculated on PDA solid medium and cultured at 28°C. After the colonies had grown, a single colony was picked and inoculated on a new PDA solid medium for 5 days.

(3) Preparation of spore suspension:

The activated Penicillium difficile NS-12 was inoculated on PDA solid medium and expanded at 28°C, and the spores on the medium were eluted with sterile water to prepare a spore suspension with a concentration of 1.0×10 ⁸ /mL.

(4) Solid fermentation:

100 mL of spore suspension was inoculated into 1000 g of solid fermentation medium and cultured at 28°C, stirring was performed under sterile conditions every 2 to 3 days, and a solid fermentation inoculum was obtained after culturing for 9 days.

(5) Drying of bacterial agent:

The obtained solid inoculum was placed in a drying box at 35°C, dried by blasting for 40 hours, and the solid inoculum was dried to a moisture content of less than 10-15% to obtain a special growth-promoting solid inoculum for Tripterygium wilfordii (Figure 1). ). The number of viable bacteria in the bacterial agent can reach 7×10 ¹⁰ cfu/g.

Example 2

The special growth-promoting solid bacteria agent for Tripterygium wilfordii obtained in Example 2 was stored in a dry place at 20-25°C. After 1, 2, 3, 6, and 12 months of storage, samples were taken to detect the number of viable bacteria. The results are shown in Table 2. After 12 months of storage, the number of spores of the inoculum was still as high as 4.7×10 ¹⁰ cfu/g, which showed good storage characteristics.

Table 1 Changes in the number of viable bacteria in the inoculum over time

Example 3

Select Tripterygium wilfordii seedlings with consistent growth and apply the special growth-promoting solid inoculant for Tripterygium wilfordii after 12 months of storage. Take 20 g of Tripterygium wilfordii special growth-promoting solid inoculant and mix it with 1000 mL of sterile water and stir evenly, irrigate the roots and apply it to the rhizosphere of Tripterygium wilfordii seedlings, and the application amount for each plant is 50 mL. Sterile water was used as a control. After 3 months of application, samples were taken to determine the whole plant biomass and A content of roots of Tripterygium wilfordii seedlings. The results are shown in Figure 2 and Table 2. The biomass of Tripterygium wilfordii seedlings in the control group was 35.86 g, and the biomass of Tripterygium wilfordii seedlings in the inoculum treatment group was 46.76 g. The results showed that the biomass in the inoculum treatment group increased by 30% compared with the control group. The content of A in the roots of the Tripterygium wilfordii seedlings in the control group was 1.06 µg/g, and the content of A in the roots of the Tripterygium wilfordii seedlings in the inoculum treatment group was 1.53 µg/g. The results showed that the biomass of the inoculum treatment group increased by 44% compared with the control group.

Table 2 The application effect of the special growth-promoting solid inoculant for Tripterygium wilfordii

Claims (8)

1. a special growth-promoting bacterial agent for Tripterygium wilfordii, is characterized in that: described bacterial agent is formed by fermenting Penicillium sternum NS-12, and the classification of described Penicillium sterilum NS-12 is named as Penicillium sterilum ( Penicillium steckii ), this strain has been deposited in the General Microbiology Center of China Microorganism Culture Collection Management Committee on June 10, 2011, and the deposit number is CGMCC No.4928. 2. The special growth-promoting microbial inoculum for Tripterygium wilfordii according to claim 1 is characterized in that: the microbial inoculum is a solid microbial inoculum. 3. Tripterygium wilfordii special-purpose growth-promoting microbial inoculum according to claim 2, is characterized in that: the preparation method of described Tripterygium wilfordii special-purpose growth-promoting solid microbial inoculum comprises the steps: 1) Activation of strains: Inoculate the cryopreserved Penicillium difficile NS-12 on PDA solid medium and cultivate at 28°C. After the colonies grow, pick a single colony and transfer it to a new PDA solid medium. Continue to culture at 28°C for 5 days; 2) Preparation of spore suspension: Inoculate the activated Penicillium difficile NS-12 on a PDA plate and expand the culture at 28°C, and elute the spores with sterile water to prepare a spore suspension with a concentration of 1.0×10 ⁸ /mL liquid; 3) Solid fermentation: Inoculate 100 mL of spore suspension into 1000 g of solid fermentation medium and culture at 28°C, stir every 2 to 3 days under sterile conditions, and obtain solid fermentation inoculum after 9 days of culture ; 4) Drying of inoculum: put the obtained solid inoculum in a drying box at 35°C, blow drying for 40 hours, and dry the solid inoculum to a water content below 10-15%, that is, to obtain a special growth promotion for Tripterygium wilfordii solid bacteria. 4. Tripterygium wilfordii special-purpose growth-promoting bacteria agent according to claim 3, is characterized in that: described solid fermentation medium is made up of the raw material of following percentage by weight: bran 26%, bean cake flour 10%, starch 10%, coconut palm Shell powder 10%, trehalose 3%, calcium carbonate 1%, water 40%. 5 . The special-purpose growth-promoting microbial inoculum for Tripterygium wilfordii according to claim 3 , wherein the number of viable bacteria in the obtained special-purpose growth-promoting microbial inoculum for Tripterygium wilfordii is 7×10 ¹⁰ cfu/g. 6 . 6. The special growth-promoting microbial inoculum for Tripterygium wilfordii according to claim 3 is characterized in that: the application mode of the special-purpose growth-promoting solid microbial inoculant for Tripterygium wilfordii is: applying to the rhizosphere of crops through root irrigation. 7. the application of the special-purpose growth-promoting microbial inoculum of Tripterygium wilfordii according to claim 1 in improving the Tripterygium wilfordii biomass. 8. the application of triptolide special-purpose growth-promoting bacterial agent according to claim 1 in improving the content of triptolide.

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