CN114317328A

CN114317328A - Streptomyces aureofaciens and application thereof

Info

Publication number: CN114317328A
Application number: CN202111487735.8A
Authority: CN
Inventors: 杨敏; 罗丽芬; 朱书生; 李玥; 叶辰; 刘屹湘; 黄惠川; 梅馨月; 何霞红; 朱有勇
Original assignee: Yunnan Agricultural University
Current assignee: Yunnan Agricultural University
Priority date: 2021-12-07
Filing date: 2021-12-07
Publication date: 2022-04-12
Anticipated expiration: 2041-12-07
Also published as: CN114317328B

Abstract

The streptomyces aureofaciens provided by the invention has stronger inhibitory activity on rust rot, which is the main root rot of panax notoginseng, can effectively reduce the occurrence of rust rot of panax notoginseng when being applied to the root of panax notoginseng as a microbial inoculum, improves the emergence rate and the fresh weight of a single plant of panax notoginseng planted in continuous cropping soil, and has extremely high economic value.

Description

Streptomyces aureofaciens and application thereof

Technical Field

The invention belongs to the field of microorganisms, and particularly relates to streptomyces aureofaciens with pseudo-ginseng root rot pathogen inhibition activity and application thereof.

Background

Notoginseng radix [ Panax notogeng (Burk.) ] is a plant of Panax of Araliaceae, and has effects of promoting blood circulation for removing blood stasis, relieving swelling and relieving pain by using root and rhizome as raw materials. Recent pharmacological and clinical researches show that the notoginseng has good curative effect on cardiovascular and cerebrovascular diseases. At present, the application of pseudo-ginseng is very wide, statistics shows that 1300 enterprises in China only take pseudo-ginseng as a raw material to obtain 3207 production documents and produce more than 400 products, wherein the varieties of Chinese patent medicines exceed 300, and almost all Chinese medicine pharmaceutical enterprises are covered.

Continuous cropping obstacle (continuous cropping obstacle) refers to a phenomenon in which, when the same kind of crop or the same family of crop is continuously cultivated in the same land, growth vigor becomes weak, yield decreases, quality decreases, and diseases and pests become more serious even under normal cultivation and management conditions (Zhang Yuan et al, 2009). At present, China mostly focuses on grain crops such as wheat, corn, sorghum and soybean, and vegetable crops such as eggplant and cucumber, and relatively few researches on continuous cropping obstacles of medicinal plants are conducted. In the existing commonly cultivated medicinal plants, the root (rhizome) medicinal materials account for about 70% (Zhang Aihua et al, 2011). However, most root (rhizome) medicinal plants have continuous cropping obstacles during cultivation, such as rhizoma atractylodis, rehmannia glutinosa, ginseng, pseudo-ginseng, radix pseudostellariae, coptis chinensis, angelica sinensis and the like (xiapinhua and the like, 2010). With the promotion of the standardized cultivation of medicinal plants and the continuous enlargement of the planting area, the continuous cropping obstacle has become a technical bottleneck in the cultivation process of medicinal plants (Guolanry et al, 2006).

Like other medicinal plants of panax, panax notoginseng has strong soil contraindication and has serious continuous cropping obstacle. Generally, it is considered that the land where the notoginseng is planted needs to be planted with crops such as corn, upland rice and alfalfa for more than 10 years before the notoginseng can be planted again (simplified friend, 2009). According to the preliminary investigation of the subject group, pseudo-ginseng can be planted again in some plots even after 30 years of rotation interval. Driven by market demands and the temporary absence of effective overcoming methods of continuous cropping obstacles, the suitable land of the traditional pseudo-ginseng production area is gradually tense, the planting area is continuously extended, and the genuine nature of medicinal material production is difficult to guarantee.

The cultivation period of the pseudo-ginseng is generally 3 years, and the seedling period is 1 year; after the seedlings are transplanted, the seedlings need to grow for 2 years, namely two years of continuous cropping, wherein the seedlings which grow for 1 year after transplantation are called two-year seven, and the seedlings which grow for 2 years are called three-year seven. In production, the two-year seven-and three-year seven-plant plants planted after continuous cropping have serious plant diseases and insect pests and low plant survival rate, and are one of the performance characteristics of pseudo-ginseng continuous cropping obstacles. On the other hand, if the land after three-year seven-harvest is transplanted (continuous cropping) with pseudo-ginseng seedlings, the incidence rate of pseudo-ginseng reaches more than 90%, and almost no healthy plants exist after one year, namely, the land is another expression characteristic of pseudo-ginseng continuous cropping obstacle. Thus, pseudo-ginseng exhibits the typical characteristic of continuous cropping obstacle-severe disease. The main diseases in the continuous cropping process of panax notoginseng include root rot, black spot, powdery mildew, damping off, epidemic disease and the like (Wangchao and the like, 2000). The incidence rate of root rot can reach 50%, and the disease is a nearly destructive disease which damages the growth of pseudo-ginseng root systems (wandering in, 2009). Therefore, the development of a technical scheme capable of inhibiting diseases and continuous cropping obstacles of panax notoginseng is urgently needed, and the economic value of planting panax notoginseng in unit soil is improved.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides streptomyces and application thereof, which are used for solving the problems of serious diseases in pseudo-ginseng planting and reduced yield and quality caused by continuous cropping obstacles in the prior art. In order to solve the technical problems, the invention specifically provides the following technical scheme:

in a first aspect of the present invention, a Streptomyces aureofaciens 90372 is provided, the preservation information of which is as follows: the preservation number is CGMCC No. 22755; the classification is named as: streptomycin aureofaciens (Streptomyces aureus); the preservation unit is China general microbiological culture Collection center; the preservation address is No. 3 of Xilu No. 1 of Beijing, Chaoyang, the area of the morning, the zip code: 100101.

in a second aspect of the present invention, a microbial inoculum for inhibiting panax notoginseng main root rot bacteria is provided, which comprises Streptomyces aureofaciens 90372 described above. In a preferred embodiment, the viable count of Streptomyces aureofaciens 90372 in the microbial inoculum is 10⁶～10⁸cfu/mL, most preferably, the viable count of streptomycin aureofaciens 90372 in the microbial inoculum is 10⁸cfu/mL。

In a third aspect of the invention, an application of the Streptomyces aureofaciens 90372 or the microbial inoculum prepared from the Streptomyces aureofaciens in resisting panax notoginseng rust rot is provided.

In one embodiment, the above use is for reducing the occurrence of root rot of panax notoginseng.

In a preferred embodiment, the application comprises applying Streptomyces aureofaciens 90372 to the soil of the root of planted panax notoginseng, wherein the viable count of Streptomyces aureofaciens 90372 is 10⁶～10⁸cfu/mL, most preferably, the viable count of Streptomyces aureofaciens 90372 at the time of administration is 10⁸cfu/mL。

In a fourth aspect of the present invention, an application of the Streptomyces aureofaciens 90372 or the microbial inoculum prepared therefrom in promoting the growth of panax notoginseng is provided.

In one embodiment, the above application is to inhibit the occurrence of pseudo-ginseng continuous cropping obstacle.

In another embodiment, the above application is to increase the emergence rate and fresh weight of panax notoginseng plant.

Compared with the prior art, the invention has the following beneficial effects:

the Streptomyces aureofaciens provided by the invention can utilize dextrin, D-galactose, N-acetyl-D-glucosamine, alpha-D-glucose, pectin and D-gluconic acid as carbon sources for growth; can grow in an environment with pH 5, pH 6, or containing 1% NaCl; the microbial inoculum is insensitive to sodium lactate, rifamycin SV, minocycline, lincomycin, guanidine hydrochloride, tetrazole violet, tetrazole blue, nalidixic acid, potassium tellurite, aztreonam and sodium butyrate, has strong inhibition activity to rust rot pathogen which is main root rot pathogen of panax notoginseng, can effectively reduce rust rot of panax notoginseng when applied to the root of panax notoginseng as the microbial inoculum, and improves the emergence rate and the fresh weight of a single plant of panax notoginseng planted in continuous cropping soil, thereby having extremely high economic value.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1 is a colony diagram of Streptomyces aureofaciens 90372 provided in example 1 of the present invention.

FIG. 2 is a gram stain of Streptomyces aureofaciens 90372 provided in example 1 of the present invention.

FIG. 3 shows the effect of Streptomyces aureofaciens 90372 on fresh weight of Panax notoginseng.

Detailed Description

The invention is further illustrated with reference to specific examples. It should be understood that the specific embodiments described herein are illustrative only and are not limiting upon the scope of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products which are not known to manufacturers and are available from normal sources.

The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples are all commercially available products unless otherwise specified.

Example 1: isolation of strains and identification of physiological and biochemical functions

A streptomyces (streptomyces sp.) is separated from healthy panax notoginseng rhizosphere soil by adopting a plate separation method, the number of the streptomyces sp is 90372, and morphological, physiological and biochemical characteristics of the streptomyces sp are identified, and specific results are as follows: the bacteria are gram-negative bacteria, chain-shaped, yellow colony, circular colony and raised edge. The method utilizes alpha-D-glucose, D-galactose, D-mannitol, acearum, rifamycin SV, L-aspartic acid, L-glutamic acid, L-arginine, L-histamine, lincomycin, guanidine hydrochloride, sodium tetradecanesulfate, D-gluconic acid, mucic acid, quinic acid, vancomycin, tetrazole blue, tetrazole violet, citric acid, L-malic acid, gamma-aminobutyric acid and sodium bromate, and meets the morphological, physiological and biochemical characteristics of streptomycete. The strain is preserved in China general microbiological culture Collection center (CGMCC) on 23.6.2021 with the preservation number of CGMCC No. 22755; the classification is named as: streptomycin aureofaciens (Streptomyces aureus); the preservation address is No. 3 of Xilu No. 1 of Beijing, Chaoyang, the area of the morning, the zip code: 100101.

the 90372 strain was examined for its ability to utilize 71 carbon source substrates on Biolog Gen III microwell plates and for its sensitivity to 23 chemicals, and the results are shown in table 1:

TABLE 1

+: indicating a positive reaction; -: a negative reaction is indicated.

As can be seen from Table 1, 90372 strain was able to grow using dextrin, D-galactose, N-acetyl-D-glucosamine, α -D-glucose, pectin and D-gluconic acid as carbon sources; can grow in an environment with pH 5, pH 6, or containing 1% NaCl; sodium lactate, rifamycin SV, minocycline, lincomycin, guanidine hydrochloride, tetrazole violet, tetrazole blue, nalidixic acid, potassium tellurite, aztreonam, and sodium butyrate are insensitive.

Example 2 Streptomyces aureomycin 90372 bacteriostatic Capacity test

In this example, 4 species of rust rot germs (Cylindrocarpon destructans) to be tested were provided by the plant pathology laboratory of the plant protection institute of Yunnan agricultural university.

The detection method comprises the following steps:

the plate confronting culture method is adopted to determine the inhibitory activity of the streptomyces aureofaciens 90372 on the growth of the hypha of the rust rot disease bacteria to be tested. Preparing the rust rot pathogen to be tested growing on a PDA culture medium on a clean bench into a bacterial cake by using a puncher with the diameter of 5mm, transferring the bacterial cake to the center of a PDA culture medium flat plate, inoculating separated and purified streptomycin streptomyces to a position 2.5cm away from the pathogenic bacteria by using a sterile toothpick, setting 10 times of repetition, taking the flat plate only containing the pathogenic bacteria cake as a control, culturing at the constant temperature of 26 ℃, measuring the diameter of a bacterial colony when the control bacterial colony grows to 2/3 of the culture medium, and calculating the bacteriostasis rate according to the following test. The bacteriostatic ratio (%) [ (radius of control colony-radius of culture colony on opposing side)/radius of control colony ] × 100%.

Wherein, the PDA culture medium formula is as follows: 200g of potato, 20g of glucose, 18g of agar powder and 1000mL of water.

The detection results are shown in Table 2, the inhibition abilities of streptomyces aureofaciens 90372 to 4 types of rust rot germs to be tested are different, and the inhibition rate is up to more than 70%. The bacterial strain has strong inhibitory activity on the main root rot of panax notoginseng, namely rust rot.

TABLE 2

Example 3 promoting action of Streptomyces aureofaciens 90372 on emergence of seeds of Panax notoginseng

1. Preparation of bacterial liquid

Activating streptomyces aureofaciens 90372 on KB solid culture medium, picking out single colony, performing shake culture in KB liquid fermentation culture medium at 160rpm and 28 ℃ for 48h, and diluting the fermentation liquid to OD₆₀₀0.3 is used for subsequent root irrigation in the field.

The KB solid medium comprises the following components in each 1L of medium: 20g peptone, 10mL glycerol, 1.5gK₂HPO₄·3H ₂0，1.5g MgSO₄·7H₂O, 17g agar, 1000mL distilled water, pH 7.0.

2. Influence of Streptomyces 90372 on emergence of seeds of Panax notoginseng

The test arrangement is carried out in a Hongdan river bridge farm pseudo-ginseng planting shed. One part of the continuous cropping soil of the panax notoginseng is subjected to damp-heat sterilization at 121 ℃ for 20min, and the other part of the continuous cropping soil of the panax notoginseng is not treated. All the continuous cropping soil is put into flowerpots respectively, 12 panax notoginseng seeds are planted in each flowerpot, and each treatment is repeated for 5 times. Sampling and investigating after three months, and measuring and recording the emergence rate of the plants.

As a result, as shown in Table 3, the emergence rate of Panax notoginseng was increased after root irrigation of Streptomyces aureofaciens 90372 under the conditions of soil sterilization/non-sterilization.

TABLE 3 influence of Streptomyces 90372 on seed emergence of Panax notoginseng

3. Influence of Streptomyces 90372 on fresh weight of Panax notoginseng

Planting pseudo-ginseng seeds into a flowerpot filled with sterilized soil, 12 seeds per bottle, then inoculating streptomyces aureofaciens 90372 suspension into the flowerpot, wherein the content of viable streptomyces aureofaciens 90372 in the suspension is 10⁶～10⁸cfu/mL perBottle 25ml, with the same volume of liquid KB medium added as a control, was measured and recorded the fresh weight of an individual plant of the plant after 3 months of culture in the natural conditions in 5 pots per treatment.

As a result, as shown in FIG. 3, the fresh weight of each individual plant of Panax notoginseng was increased in the sterilized soil after root irrigation with Streptomyces aureofaciens 90372.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims

1. A streptomycin aureofaciens 90372 is characterized in that the preservation number of the strain is CGMCC No.22755, the preservation unit is the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of West Lu No. 1 of the Beijing Korean district, and the postal code is 100101.

2. A bacterial agent for inhibiting panax notoginseng major root rot, which comprises Streptomyces aureus (Streptomyces aureus) 90372 according to claim 1.

3. The microbial preparation according to claim 2, wherein the viable count of Streptomyces aureofaciens 90372 in the microbial preparation is 10⁶～10⁸cfu/mL。

4. The microbial preparation according to claim 3, wherein the viable count of Streptomyces aureofaciens 90372 in the microbial preparation is 10⁸cfu/mL。

5. Use of Streptomyces aureofaciens (Streptomyces aureus) 90372 according to claim 1 or the microbial agent according to any one of claims 2 to 4 for combating rust rot of panax notoginseng.

6. The use of claim 5, wherein said use is for reducing the occurrence of root rot in Panax notoginseng.

7. Use of Streptomyces aureofaciens (Streptomyces aureus) 90372 according to claim 1 or the microbial inoculum according to any one of claims 2 to 4 for promoting the growth of panax notoginseng.

8. The use of claim 7, wherein said use is for inhibiting the occurrence of panax notoginseng continuous cropping disorders.

9. The use of claim 7, wherein said use is to increase the rate of emergence and the fresh weight of individual plants of Panax notoginseng.

10. The use according to any one of claims 5 to 9, wherein the use comprises applying Streptomyces aureofaciens (Streptomyces aureofaciens)90372 to the soil of the roots of planted panax notoginseng, the Streptomyces aureofaciens (Streptomyces aureofaciens)90372 viable count being 10 at the time of application⁶～10⁸cfu/mL。