CN114806892B

CN114806892B - Trichoderma atroviride strain and application thereof in preventing and treating root rot of pseudo-ginseng

Info

Publication number: CN114806892B
Application number: CN202210463044.2A
Authority: CN
Inventors: 郑宽瑜; 魏治镭; 张仲凯; 苏晓霞; 郑雪; 周凤; 陈永对; 吴阔
Original assignee: Changdu Agricultural Science Research Institute Of Tibet; Biotechnology and Germplasm Resource Institute of Yunnan Academy of Agricultural Sciences
Current assignee: Changdu Agricultural Science Research Institute Of Tibet; Biotechnology and Germplasm Resource Institute of Yunnan Academy of Agricultural Sciences
Priority date: 2022-04-28
Filing date: 2022-04-28
Publication date: 2023-05-16
Anticipated expiration: 2042-04-28
Also published as: CN114806892A

Abstract

The invention discloses a trichoderma atroviride strain and application thereof in preventing and treating root rot of pseudo-ginseng. The trichoderma atroviride strain is PRWS21, the preservation name is trichoderma atroviride Trichoderma atroviride, the trichoderma atroviride strain is preserved in China general microbiological culture collection center (CGMCC), the preservation unit address is North Chen Xie Lu No.1, 3 in the Korean region of Beijing city, and the preservation date is as follows: 2021, 4 and 6; preservation number: CGMCC No.21478. The trichoderma atroviride disclosed by the invention is separated from paris polyphylla rhizosphere soil, and can simultaneously have a good inhibition effect on pseudo-ginseng root rot fungi (fusarium oxysporum, fusarium putrescens and phytophthora capsici).

Description

Trichoderma atroviride strain and application thereof in preventing and treating root rot of pseudo-ginseng

Technical Field

The invention relates to the technical field of microorganism application, in particular to a trichoderma atroviride strain and application thereof in preventing and treating root rot of pseudo-ginseng.

Background

In recent years, with the rapid increase of the demand of Chinese medicine resources, the damage of Chinese medicine wild resources is serious, and the wild cultivation becomes a necessary way for meeting the market demand. The wild Chinese herbal medicines rarely generate diseases, if any, the diseases can not form disasters, after the wild Chinese herbal medicines are changed into artificial cultivation, the growth environment of the wild Chinese herbal medicines is changed, in an agricultural ecological system, the variety is single, the density is increased, and the conditions that the diseases are popular are created by high water and high fertilizer, artificial remote seed regulation and other management measures, so that the diseases are easy to spread and spread inside a population, among fields and among areas, and the diseases of the cultivated medicinal materials are aggravated.

Notoginseng radix (Panax notoginseng) is perennial herb of Panax of Araliaceae, and is a rare Chinese medicinal material. Pseudo-ginseng is perennial root plant, is a warm and damp environment, and provides favorable conditions for disease occurrence because of large-area single planting in successive years in production. The main diseases of pseudo-ginseng are root rot, and pathogenic bacteria of the pseudo-ginseng mainly comprise: alternaria (Alternaria alternaria), fusarium solani (Fusarium solani), fusarium oxysporum (Fusarium oxysporum), phoma (Phoma herbarum), rhizoctonia solani (Rhizoctonia solani), phytophthora (Phytophthora cactorum), pseudomonas sp, meloidogyne sp, etc. Wherein Fusarium solani, fusarium oxysporum, alternaria alternata and Phytophthora are used as main root rot pathogen of pseudo-ginseng which is popular in Yunnan mountain. The root rot can reduce the yield of the pseudo-ginseng by 5-20%, and can reach more than 70% when serious, and accounts for 70-85% of various diseases of the pseudo-ginseng, and one of important restriction factors of the pseudo-ginseng planting industry is caused at present.

At present, chemical control is mainly used for controlling root diseases of pseudo-ginseng, and the method comprises the following steps: (1) soil fumigation treatment. The principle is that the chemical fumigant is utilized to fumigate the soil so as to achieve the purpose of killing pathogenic bacteria in the soil. Although the soil fumigation treatment can obviously reduce the incidence rate of the root rot of pseudo-ginseng, the chemical fumigant is easy to pollute the surrounding environment and destroy the micro-ecological community of the soil due to the toxicity of the chemical fumigant. (2) chemical germicides. Mainly comprises chemical pesticides such as carbendazim, thiophanate-methyl, metalaxyl, procymidone and the like. The long-term use of chemical pesticides can cause environmental pollution, ecological system destruction and pathogenic bacteria drug resistance, and even bring harm such as pesticide residue and heavy metal pollution to traditional Chinese medicinal materials, so that the use of pesticides is reasonably reduced, a green and environment-friendly prevention and control mode is found, and the method has important significance for preventing and controlling diseases of the traditional Chinese medicinal materials.

Disclosure of Invention

Aiming at the problems that the root rot of the pseudo-ginseng is serious at present, and chemical pesticide prevention and treatment easily causes environmental pollution, pesticide residue, drug resistance and the like, the trichoderma atroviride (Trichoderma harzianum) strain for effectively preventing and treating the root rot of the pseudo-ginseng and the application thereof are provided.

In order to achieve the above purpose, the present invention adopts the following technical scheme: the invention discloses a trichoderma atroviride strain, which is PRWS21, the preservation name is trichoderma atroviride Trichoderma atroviride, the trichoderma atroviride strain is preserved in China general microbiological culture collection center CGMCC, the preservation unit address is North Chen Xie No.1, 3 of the Korean region of Beijing city, and the preservation date is as follows: 2021, 4 and 6; preservation number: CGMCC No.21478.

Further, the ITS gene sequence of the trichoderma atroviride strain is the nucleotide sequence shown as SEQ ID No. 1.

The invention discloses a trichoderma atroviride bacterial agent prepared from trichoderma atroviride bacterial strain.

Further, the active ingredients are at least one of the following (a), (b) and (c):

(a) The fermentation culture of the trichoderma atroviride;

(b) The spore suspension of the obtained trichoderma atroviride;

(c) Ultrasonic lysis precipitation of the obtained Trichoderma atroviride cells.

The preparation method of the fermentation liquor of trichoderma atroviride disclosed by the invention comprises the following steps of: after 5 trichoderma PRWS21 bacterial cakes with the diameter of 6mm are inoculated into 100mL PDA liquid culture medium and cultured on a constant temperature shaking table at the temperature of 28 ℃ and the speed of 180r/min for 7d, the bacterial liquid is filtered by filter paper, and then the bacterial liquid is filtered and sterilized by a sterile filter with the diameter of 0.22 mu m, so as to obtain the sterile fermentation filtrate.

Further, the PDA liquid culture medium consists of the following components: the potato is 100 g-200 g, the glucose is 5 g-20 g, the volume is fixed to 1L distilled water, the pH is 6-8, and the sterilization is carried out for 20min at 121 ℃.

The preparation method of the spore suspension of trichoderma atroviride disclosed by the invention comprises the following steps of: inoculating Trichoderma atroviride strain PRWS21 on PDA solid culture medium plate, culturing at 28deg.C for 7 days, preparing spore suspension with distilled water, and counting spore concentration under microscope to obtain spore suspension with concentration of 1.0X10 ⁵ cfu/mL。

Further, the PDA solid culture medium consists of the following components: 100 g-200 g of potato, 5 g-20 g of glucose, 10 g-20 g of agar, constant volume of the potato in 1L of distilled water, pH of the potato is 6-8, and sterilization is carried out for 20min at 121 ℃.

The trichoderma atroviride strain or trichoderma atroviride microbial inoculum disclosed by the invention is applied to the production of products for preventing and treating the root rot of pseudo-ginseng.

Further, root irrigation treatment is carried out on the pseudo-ginseng by using trichoderma atroviride spore suspension or fermentation liquor.

The beneficial effects are that: the paris polyphylla rhizosphere soil is separated, and can simultaneously have good inhibition effect on root rot fungi (fusarium oxysporum, fusarium putrescens and phytophthora capsici). Therefore, the strain fermentation liquor of the strain can effectively prevent and treat the root rot of pseudo-ginseng.

Compared with the prior art, the invention has the following advantages:

the strain PRWS21 has broad-spectrum resistance, can simultaneously antagonize various pseudo-ginseng root rot fungi, can be developed into a high-efficiency biocontrol microbial inoculum, and effectively reduces the use of chemical pesticides.

Drawings

Further description will be made with reference to the accompanying drawings, in which:

FIG. 1 shows the colony morphology of Trichoderma atroviride (Trichoderma harzianum) PRWS21 of the invention.

FIG. 2 is a P.dark green (Trichoderma harzianum) PRWS21 NJ phylogenetic tree of the invention.

FIG. 3 shows the effect of the invention on the facing of Trichoderma atroviride (Trichoderma harzianum) PRWS21 plates.

Detailed Description

The present invention is further illustrated in detail by the following examples, but it should be noted that the scope of the present invention is not limited by any of these examples.

Example 1

The invention discloses a trichoderma atroviride strain, which is PRWS21, the preservation name is trichoderma atroviride Trichoderma atroviride, the trichoderma atroviride strain is preserved in China general microbiological culture collection center CGMCC, the preservation unit address is North Chen Xie No.1, 3 of the Korean region of Beijing city, and the preservation date is as follows: 2021, 4 and 6; preservation number: CGMCC No.21478.

The ITS gene sequence of the trichoderma atroviride strain is the nucleotide sequence shown as SEQ ID No. 1.

The active ingredients are at least one of the following (a), (b) and (c):

(a) The fermentation culture of the trichoderma atroviride;

(b) The spore suspension of the obtained trichoderma atroviride;

The PDA liquid culture medium consists of the following components: the potato is 100g, the glucose is 20g, the volume is fixed to 1L of distilled water, the pH is 6, and the sterilization is carried out for 20min at 121 ℃.

Spore suspension of trichoderma atroviride of the inventionThe preparation method of (2) comprises the following steps: inoculating Trichoderma atroviride strain PRWS21 on PDA solid culture medium plate, culturing at 28deg.C for 7 days, preparing spore suspension with distilled water, and counting spore concentration under microscope to obtain spore suspension with concentration of 1.0X10 ⁵ cfu/mL。

The PDA solid culture medium consists of the following components: 100g of potato, 20g of glucose and 20g of agar, and sterilizing for 20min at 121 ℃ in 1L of distilled water with a pH of 6.

Root irrigation treatment is carried out on the pseudo-ginseng by using trichoderma atroviride spore suspension or fermentation liquor.

Example 2

Example 2 differs from example 1 in that: the PDA liquid culture medium consists of the following components: 200g of potato, 5g of glucose, constant volume to 1L of distilled water, pH 8 and sterilizing at 121 ℃ for 20min. The PDA solid culture medium consists of the following components: 200g of potato, 10g of glucose and 10g of agar, and sterilizing for 20min at 121 ℃ in 1L of distilled water with a constant volume and a pH of 8.

Example 3

Example 3 differs from example 1 in that: the PDA liquid culture medium consists of the following components: the potato is 150g, the glucose is 15g, the volume is fixed to 1L of distilled water, the pH is 7, and the sterilization is carried out for 20min at 121 ℃. The PDA solid culture medium consists of the following components: 150g of potato, 5g of glucose and 12g of agar, fixing the volume to 1L of distilled water, sterilizing for 20min at 121 ℃ and naturally pH 7.

Example 4

Obtaining of strains

Trichoderma atroviride (Trichoderma atroviride) PRWS21 is prepared from rhizosphere soil obtained from Yunnan rhizoma paridis planted in Songming province of Yunnan, 10g rhizosphere soil is dissolved in 100mL of paris polyphylla root system secretion, and cultured at 28 ℃ with shaking table at 200rpm, and once every 24 hours, the soil suspension is taken according to 10 ^-5 After dilution, the plates are respectively smeared on PDA culture medium added with paris polyphylla root system secretion (10 mg/mL)The fungus is cultivated. After 3 times of purification after bacterial colony growth, the bacterial colony is used for antagonistic bacteria screening test.

Example 5

Identification of strains

1 morphological feature observations

Bacterial strain PRWS21 cultured on PDA culture medium for 2-7 days to observe colony morphology, bacterial colony hypha is white velvet in 2 days, conidium starts to be produced near inoculation point in fourth day, surface of spore-producing cluster is granular or powdery, spore-producing cluster is white initially, and later turns green to dark green, and pigment is not produced (figures 1a and b). Conidiophores have tree-shaped branches, the secondary branches are more, and one main branch usually has 5-7 secondary branches. The top end of the secondary branch is provided with a bottle stem, the bottle stem is short, the base part is thin, and the middle is thick. Usually 6-8 clusters (FIG. 1 c); conidia were elliptical or nearly spherical with a diameter of about 3 μm (FIG. 1 d).

2ITS sequence analysis

After extracting genome DNA of the strain by a liquid nitrogen grinding method, adopting a primer: ITS1:5'-TCCGTAGGTGAACCTGCGG-3'; ITS4:5'-TCCTCCGCTTATTGATATGC-3', PCR amplification was performed. After gel electrophoresis, the sequence is sent to a biological engineering (Shanghai) limited company for sequencing, and the full length of the sequence is 558bp (SEQ). The obtained sequence was submitted to the GenBank database for BLAST analysis and alignment, and the strain having the highest homology with PRWS21 was found to be Trichoderma atroviride strain OL989154, and the homology was found to be 99.65%. The NJ phylogenetic tree was constructed with ITS sequences, PRWS21 and Trichoderma atroviride in the same branch (FIG. 2). The strain was initially identified as Trichoderma atroviride based on the morphological feature analysis, ITS analysis and NJ phylogenetic tree described above.

Example 6

Antibacterial activity of strain PRWS21 on Notoginseng radix pathogenic bacteria

Fusarium solani (Fusarium solani), fusarium oxysporum (Fusarium oxysporium), phytophthora (Phythophthora cactorum) and Trichoderma strain PRWS21 are inoculated at two ends of a 90mm PDA plate respectively by adopting a plate facing culture method, and only the pseudo-ginseng pathogenic bacteria are inoculated as CK control. Each treatment was repeated 3 times. The bacteriostatic rate was calculated 7 days on PDA medium: inhibition (%) = (control group target colony diameter-treatment group target colony diameter)/control group target colony diameter x 100%. The results of the antibacterial activity of PRWS21 against Notoginseng radix pathogenic bacteria are shown in Table 1. The growth inhibition rate of trichoderma strain PRWS21 against pseudo-ginseng pathogenic bacteria is shown in table 1:

TABLE 1

As shown in table 1, the experimental results of the present invention show that: the growth inhibition rate of the strain to PRWS21 Fusarium solani, fusarium oxysporum and Phytophthora pseudoginseng is 81.11-68.05%. The trichoderma strain PRWS21 can inhibit the growth of pseudo-ginseng pathogenic bacteria through space and nutrition competition, and has good broad-spectrum pseudo-ginseng pathogenic bacteria resisting effect.

Example 7

Preparation of PRWS21 fermentation broth

5 trichoderma PRWS21 bacterial cakes with the diameter of 6mm are inoculated into a 100mLPDA liquid culture medium, and after 7d of culture on a constant temperature shaking table at the temperature of 28 ℃ and the speed of 180r/min, bacterial liquid is filtered by filter paper, and then sterile filtration and sterilization are carried out by a sterile filter with the diameter of 0.22 mu m, so that sterile fermentation filtrate is obtained.

The PDA liquid culture medium consists of the following components: 160g potato, 16g glucose, constant volume to 1L distilled water, pH6.8, and sterilizing at 121deg.C for 20min.

Example 8

Antibacterial activity of PRWS21 fermentation broth for pseudo-ginseng pathogenic bacteria

The PRWS21 broth from example 4 was added to PDA solid medium to make PDA plates containing 30% broth. Fusarium pseudoginseng, fusarium oxysporum and phytophthora capsici are respectively inoculated on a PDA plate added with 30% fermentation liquor to be used as a treatment group; notoginseng radix pathogenic bacteria are inoculated on PDA plate without fermentation liquid as CK control group. The colony diameters were measured and the inhibition rates were calculated after 3 replicates of each group and incubation at 28℃for 7 days, and the growth inhibition rates of the fermentation broth of Trichoderma strain PRWS21 against the pathogenic bacteria of Notoginseng radix were as shown in Table 2:

TABLE 2

As shown in Table 2, the addition of 30% PRWS21 fermentation broth significantly inhibited the growth of pseudo-ginseng pathogenic bacteria, with an inhibition rate of 61.82-42.01% or more.

Example 9

Preparation of bacterial PRWS21 spore suspension

Inoculating Trichoderma atroviride strain PRWS21 on PDA solid culture medium plate, culturing at 28deg.C for 7 days, preparing spore suspension with distilled water, and counting spore concentration under microscope to obtain spore suspension with concentration of 1.0X10 ⁵ cfu/mL；

The PDA solid culture medium consists of the following components: 100g of potato, 20g of glucose and 18g of agar, and sterilizing the potato in 1L of distilled water with pH of 6.8 and at 121 ℃ for 20min.

Example 710

Prevention and treatment effect of trichoderma atroviride PRWS21 spore suspension on Fusarium solani

1. Preparing a fusarium spore suspension of pseudo-ginseng: inoculating Fusarium solani of Notoginseng radix on PDA solid culture medium plate, culturing at 28deg.C for 7 days, preparing spore suspension from activated strain with distilled water, counting spore concentration under microscope, and adjusting the spore suspension concentration to 1.0X10 ⁵ cfu/mL。

2. Preparation of Trichoderma atroviride PRWS21 spore suspension was performed as in example 6.

3. And (5) selecting 1-year-old pseudo-ginseng seedlings for root irrigation experiments. The experiment is as follows: CK group, inoculated sterile distilled water as control 10mL; SI group, inoculated with 10mL of fusarium solani only; DI group: simultaneously inoculating 10mL of Fusarium solani and 10mL of trichoderma atroviride. Each group was repeated 3 times, and each repeated inoculation was performed with 20 seedlings of notoginseng. After 14 days, the morbidity, the disease index and the prevention and treatment rate are counted.

4. The disease index and the prevention effect are calculated according to the following formulas

Disease index = Σ (number of disease plants at each stage x the number of disease stages)/(total number of investigation x highest stage) ×100

Severity grading criteria: level 0: the root is free from diseases; stage 1: the disease spots account for less than 20% of the root surface area; 2 stages: the disease spots account for 21% -40% of the root surface area; 3 stages: the spots account for 41-65% of the surface area of the root, and the appearance is seriously affected; 4 stages: lesions account for over 66% of the root surface area or are completely rotted.

Control rate (%) = (control disease index-treated disease index)/control disease index x 100

5. The control effect of trichoderma atroviride PRWS21 on root rot of pseudo-ginseng is shown in Table 2, and the biocontrol effect of trichoderma atroviride PRWS21 spore suspension on fusarium solani is shown in Table 3:

TABLE 3 Table 3

As shown in Table 3, the potted plant test of the invention shows that the application of the strain to control Fusarium solani can effectively reduce the disease index and the control rate of Fusarium solani on Fusarium solani reaches 75.29%.

The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the foregoing embodiments, which have been described in the foregoing embodiments and description merely illustrates the principles of the invention, and various changes and modifications may be made therein without departing from the spirit and scope of the invention, the scope of which is defined in the appended claims, specification and their equivalents.

Sequence listing

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Claims

1. A trichoderma atroviride strain is characterized in that: the Trichoderma atroviride strain is PRWS21 and has the preservation name of Trichoderma atrovirideTrichoderma atrovirideThe culture medium is preserved in China general microbiological culture Collection center (CGMCC), and the preservation unit address is 1 # 3 of Beichen Xiyu in the Chaoyang area of Beijing city, and the preservation date is: 2021, 4 and 6; preservation number: CGMCC No.21478.

2. The trichoderma atroviride strain of claim 1, wherein: the ITS gene sequence of the trichoderma atroviride strain is the nucleotide sequence shown as SEQ ID No. 1.

3. The trichoderma atroviride bacterial preparation prepared by trichoderma atroviride bacterial strain of claim 1.

4. The trichoderma atroviride microbial agent of claim 1, wherein the active ingredient is at least one of the following (a) (b) (c):

(a) A fermentation culture of trichoderma atroviride of claim 1;

(b) A spore suspension of trichoderma atroviride obtained according to claim 1;

(c) Ultrasonic lysis precipitation of Trichoderma atroviride cells obtained in claim 1.

5. A process for producing a fermentation culture of Trichoderma atroviride according to claim 4, comprising the steps of: after 5 pieces of trichoderma PRWS21 bacterial cake with the diameter of 6mm were inoculated into 100mL of PDA liquid medium and cultured on a constant temperature shaker at 28 ℃ and 180r/min for 7d, bacterial liquid was filtered by filter paper, and then sterilized by a sterile filter with the diameter of 0.22 μm, to obtain a sterile fermentation culture.

6. The method for producing a fermentation culture of Trichoderma atroviride according to claim 5, wherein: the PDA liquid culture medium consists of the following components: the potato is 100 g-200 g, the glucose is 5 g-20 g, the volume is fixed to 1L distilled water, the pH is 6-8, and the sterilization is carried out for 20min at 121 ℃.

7. The method for preparing the spore suspension of trichoderma atroviride according to claim 4, comprising the steps of: inoculating Trichoderma atroviride strain PRWS21 on PDA solid culture medium plate, culturing at 28deg.C for 7 days, preparing spore suspension with distilled water, and counting spore concentration under microscope to obtain spore suspension with concentration of 1.0X10 ⁵ cfu/mL。

8. The method for preparing spore suspension of trichoderma atroviride according to claim 7, wherein: the PDA solid culture medium consists of the following components: 100 g-200 g of potato, 5 g-20 g of glucose, 10 g-20 g of agar, constant volume of the potato in 1L of distilled water, pH of the potato is 6-8, and sterilization is carried out for 20min at 121 ℃.

9. Use of a trichoderma atroviride strain according to claim 1 or 2 for the production of a product for controlling root rot of pseudo-ginseng.

10. Use of the trichoderma atroviride microbial inoculum of claim 3 or 4 in the production of a product for controlling root rot of pseudo-ginseng.