CN114806892A - Trichoderma atroviride strain and application thereof in preventing and treating panax notoginseng root rot - Google Patents
Trichoderma atroviride strain and application thereof in preventing and treating panax notoginseng root rot Download PDFInfo
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Abstract
The invention discloses a trichoderma atroviride strain and application thereof in preventing and treating panax notoginseng root rot. The Trichoderma atroviride strain is PRWS21 with the preservation name of Trichoderma atroviride, is preserved in China general microbiological culture Collection center (CGMCC), has the preservation unit address of No. 3 Hospital No.1 North Chen West Lu of the sunward area in Beijing, and has the preservation date: 2021, 4 months and 6 days; the preservation number is: CGMCC No. 21478. The trichoderma atroviride is separated from rhizosphere soil of paris polyphylla, and can simultaneously have good inhibition effect on panax notoginseng rhizoctonia rot (fusarium oxysporum, fusarium solani and phytophthora infestans).
Description
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 panax notoginseng root rot.
Background
In recent years, with the rapid increase of the demand of traditional Chinese medicine resources, the traditional Chinese medicine wild resources are seriously damaged, and the change of wild into cultivation becomes a necessary way for meeting the market demand. Wild traditional Chinese medicinal materials rarely cause diseases, even if the wild traditional Chinese medicinal materials do not cause disasters, the growth environment of the wild traditional Chinese medicinal materials is changed into artificial cultivation, the wild traditional Chinese medicinal materials are single in type and high in density in an agricultural ecological system, management measures such as high water and high fertilizer, artificial remote seed regulation and the like create conditions for epidemic diseases, the diseases are easy to spread and spread in the population, between fields and between regions, and the diseases of cultivated medicinal materials are aggravated.
Pseudo-ginseng (Panax notoginseng) is a perennial herb of Panax of Araliaceae, and is a rare Chinese medicinal material in China. The pseudo-ginseng is perennial root plant, prefers warm and humid environment, and provides favorable conditions for the occurrence of diseases due to the single planting in large area in successive years in production. The main diseases of pseudo-ginseng are root rot, and the pathogenic bacteria of pseudo-ginseng mainly comprise: alternaria alternata (Alternaria alternata), Fusarium solani (Fusarium solani), Fusarium oxysporum (Fusarium oxysporum), Phoma stipulatum (Phoma herbare), Rhizoctonia solani (Rhizoctonia solani), Phytophthora infestans (Phytophora cactorum), Pseudomonas sp, Meloidogyne sp, and the like. Wherein Fusarium solani, Fusarium oxysporum, Alternaria alternata and phytophthora infestans are also taken as main panax notoginseng root rot pathogenic bacteria which are prevalent in Yunnan Wenshan. Root rot can cause the yield of pseudo-ginseng to be reduced by 5-20%, and can reach more than 70% in serious cases, accounting for 70-85% of various diseases of pseudo-ginseng, which is one of the important restriction factors of the pseudo-ginseng planting industry at present.
At present, the prevention and treatment of the root diseases of the pseudo-ginseng are mainly based on chemical prevention and treatment, and comprise the following steps: (1) and (5) fumigating and disinfecting the soil. The principle is that the chemical fumigant is used for fumigating soil to achieve the purpose of killing soil pathogenic bacteria. Although the incidence of root rot of panax notoginseng can be obviously reduced by the soil fumigation treatment, the surrounding environment is easily polluted due to the toxicity of the chemical fumigant, and the micro-ecological community of the soil is damaged. (2) A chemical bactericide. Mainly comprises carbendazim, thiophanate-methyl, metalaxyl, procymidone and other chemical pesticides. The long-term use of the chemical pesticide can cause environmental pollution, damage to an ecological system and generation of drug resistance of pathogenic bacteria, and even bring harm to traditional Chinese medicinal materials such as pesticide residue and heavy metal pollution, so that the use of the pesticide 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 disease of panax notoginseng is serious at present, and the chemical pesticide prevention and control is easy to cause environmental pollution, pesticide residue, drug resistance and the like, a Trichoderma atroviride (Trichoderma harzianum) strain for efficiently preventing and controlling the root rot disease of panax notoginseng and application thereof are provided.
In order to achieve the purpose, the invention adopts the following technical scheme: the Trichoderma atroviride strain is PRWS21 with the preservation name of Trichoderma atroviride, is preserved in China general microbiological culture collection center (CGMCC), has the preservation unit address of No. 3 Hospital No.1 North Chen West Lu of the sunward area in Beijing, and has the preservation date: 2021, 4 months and 6 days; the preservation number is: CGMCC No. 21478.
Further, the ITS gene sequence of the Trichoderma atroviride strain is the nucleotide sequence shown in SEQ ID No. 1.
The trichoderma atroviride microbial inoculum prepared by the trichoderma atroviride strain is provided by the invention.
Further, the active ingredient is at least one of the following (a), (b) and (c):
(a) a fermentation culture of said trichoderma atroviride;
(b) obtaining spore suspension of trichoderma atroviride;
(c) ultrasonic lysis precipitation of the resulting trichoderma atroviride cells.
The preparation method of the trichoderma atroviride fermentation liquor comprises the following steps: inoculating 5 trichoderma PRWS21 fungus cakes with the diameter of 6mm into 100mL PDA liquid culture medium, culturing on a constant temperature shaking table at 28 ℃ and 180r/min for 7d, filtering the fungus liquid by using filter paper, and filtering and sterilizing by using a sterile filter with the diameter of 0.22 mu m to obtain sterile fermentation filtrate.
Further, the PDA liquid culture medium consists of the following components: 100-200 g of potatoes and 5-20 g of glucose, diluting the mixture to 1L of distilled water with constant volume, controlling the pH value to 6-8, and sterilizing the mixture for 20min at 121 ℃.
The preparation method of the spore suspension of trichoderma atroviride comprises the following steps: inoculating Trichoderma atroviride strain PRWS21 on PDA solid culture medium plate, culturing at 28 deg.C for 7 days, preparing activated strain into spore suspension with distilled water, and counting spore concentration under microscope to make the spore suspension concentration be 1.0 × 10 5 cfu/mL。
Further, the PDA solid culture medium consists of the following components: 100-200 g of potatoes, 5-20 g of glucose and 10-20 g of agar, and sterilizing at 121 ℃ for 20min with constant volume in 1L of distilled water and pH of 6-8.
The trichoderma atroviride strain or trichoderma atroviride microbial inoculum is applied to production of products for preventing and treating panax notoginseng root rot.
Further, root irrigation treatment is carried out on the pseudo-ginseng by using trichoderma atroviride spore suspension or fermentation liquor.
Has the advantages that: the paris polyphylla rhizosphere soil is obtained by separation, and can simultaneously have good inhibition effect on panax notoginseng rhizoctonia solani (fusarium oxysporum, fusarium solani and phytophthora infestans). Therefore, the strain fermentation liquor of the strain can effectively prevent and treat the root rot of panax notoginseng.
Compared with the prior art, the invention has the following advantages:
the bacterial strain PRWS21 has broad-spectrum resistance, can antagonize various panax notoginseng root rot germs simultaneously, can be developed into a high-efficiency biocontrol microbial inoculum, and effectively reduces the use of chemical pesticides.
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The following will be further explained in conjunction with the attached drawings, in which:
FIG. 1 shows the colony morphology of Trichoderma atroviride (Trichoderma harzianum) PRWS21 according to the present invention.
FIG. 2 is a tree phylogenetically of Trichoderma atroviride (Trichoderma harzianum) PRWS21 NJ according to the invention.
FIG. 3 shows the effect of the Trichoderma atroviride (Trichoderma harzianum) PRWS21 plate confrontation according to the present invention.
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 these examples at all.
Example 1
The Trichoderma atroviride strain is PRWS21 with the preservation name of Trichoderma atroviride, which is preserved in China general microbiological culture Collection center (CGMCC), the preservation unit address is No. 3 of the No.1 Hospital of Beijing Kogyo Chen-Yang district, the preservation date is as follows: 2021, 4 months and 6 days; the preservation number is: CGMCC No. 21478.
The ITS gene sequence of the trichoderma atroviride strain is a nucleotide sequence shown in SEQ ID No. 1.
The trichoderma atroviride microbial inoculum prepared by the trichoderma atroviride strain is provided.
The active component is at least one of the following components (a), (b) and (c):
(a) a fermentation culture of said trichoderma atroviride;
(b) obtaining spore suspension of trichoderma atroviride;
(c) ultrasonic lysis precipitation of the resulting trichoderma atroviride cells.
The preparation method of the trichoderma atroviride fermentation liquor comprises the following steps: inoculating 5 trichoderma PRWS21 fungus cakes with the diameter of 6mm into 100mL PDA liquid culture medium, culturing on a constant temperature shaking table at 28 ℃ and 180r/min for 7d, filtering the fungus liquid by using filter paper, and filtering and sterilizing by using a sterile filter with the diameter of 0.22 mu m to obtain sterile fermentation filtrate.
The PDA liquid culture medium consists of the following components: 100g of potato and 20g of glucose, diluting to 1L of distilled water with constant volume, adjusting pH to 6, and sterilizing at 121 ℃ for 20 min.
The preparation method of the spore suspension of trichoderma atroviride comprises the following steps: inoculating Trichoderma atroviride strain PRWS21 on PDA solid culture medium plate, culturing at 28 deg.C for 7 days, preparing activated strain into spore suspension with distilled water, and counting spore concentration under microscope to make the spore suspension concentration be 1.0 × 10 5 cfu/mL。
The PDA solid culture medium consists of the following components: 100g of potato, 20g of glucose and 20g of agar are added to 1L of distilled water, the pH value is 6, and the mixture is sterilized at 121 ℃ for 20 min.
The trichoderma atroviride strain or trichoderma atroviride microbial inoculum is applied to production of products for preventing and treating panax notoginseng root rot.
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 and 5g of glucose, diluting to 1L of distilled water with constant volume, adjusting pH to 8, and sterilizing at 121 ℃ for 20 min. The PDA solid culture medium consists of the following components: 200g of potato, 10g of glucose and 10g of agar are added, the mixture is added into 1L of distilled water with constant volume, the pH value is 8, and the mixture is sterilized at 121 ℃ for 20 min.
Example 3
Example 3 differs from example 1 in that: the PDA liquid culture medium consists of the following components: 150g of potatoes and 15g of glucose, diluting to 1L of distilled water with constant volume, adjusting the pH value to 7, and sterilizing at 121 ℃ for 20 min. The PDA solid culture medium consists of the following components: 150g of potatoes, 5g of glucose and 12g of agar, diluting the mixture to 1L of distilled water, naturally adjusting the pH to 7, and sterilizing the mixture for 20min at 121 ℃.
Example 4
Obtaining of Strain
Trichoderma atroviride PRWS21, which is rhizosphere soil collected from Paris polyphylla planted in Songhonging of Yunnan province, 10g of rhizosphere soil is dissolved in 100mL of Paris polyphylla root secretion, the solution is cultured at the temperature of 28 ℃ by a shaker at 200rpm, and soil suspension is taken every 24 hours according to the condition that 10 percent of the soil suspension is mixed with the soil suspension -5 After dilution, the mixture is respectively coated on PDA culture medium added with Yunnan paris polyphylla root exudates (10mg/mL) for culturing fungi. After the bacteria grow out, the bacteria are purified for 3 times and then used for an antagonistic bacteria screening test.
Example 5
Identification of strains
1 morphological feature Observation
The bacterial strain PRWS21 cultured on a PDA culture medium for 2-7 days is observed to observe the colony morphology, the bacterial colony hyphae are white velvet within 2 days of culture, conidia begin to be generated near an inoculation point on the fourth day, the surfaces of the spore-forming clusters are granular or powdery, the spore-forming clusters are initially white, and are converted into green to dark green at the later stage without pigment (fig. 1a and b). Conidiophores have tree branches, and the number of secondary branches is large, and one main branch usually has 5-7 secondary branches. The top of the secondary branch produces phialides which are short, thin at the base and thick in the middle. Usually 6-8 clusters (FIG. 1 c); conidia are oval or nearly spherical in shape and about 3 μm in diameter (FIG. 1 d).
2ITS sequence analysis
After extracting the genomic DNA of the strain by a liquid nitrogen grinding method, adopting a primer: ITS 1: 5'-TCCGTAGGTGAACCTGCGG-3', respectively; ITS 4: 5'-TCCTCCGCTTATTGATATGC-3', PCR amplification was performed. After gel electrophoresis, the gel is sent to bioengineering (Shanghai) Limited company for sequencing, and the total sequence length is 558bp (shown as SEQ). The obtained sequence was submitted to GenBank database for BLAST analysis and alignment, and the strain with the highest homology to PRWS21 was Trichoderma atroviride strain OL989154 with homology of 99.65%. The ITS sequence was used to construct an NJ phylogenetic tree, PRWS21 branching from Trichoderma atroviride (FIG. 2). According to the morphological feature analysis, ITS analysis and NJ phylogenetic tree, the strain is preliminarily identified as Trichoderma atroviride.
Example 6
Bacteriostatic activity of bacterial strain PRWS21 on panax notoginseng pathogenic bacteria
By adopting a plate-confrontation culture method, Fusarium solani (Fusarium solani), Fusarium oxysporum (Fusarium oxysporum), phytophthora infestans (Phytophora cacorum) and trichoderma strain PRWS21 are respectively inoculated at two ends of a 90mm PDA plate, and only the pathogenic bacteria of panax notoginseng are inoculated to be used as CK control. Each treatment was repeated 3 times. Calculating the bacteriostasis rate by a PDA culture medium for 7 days: inhibition (%) - (control target colony diameter-treatment target colony diameter)/control target colony diameter × 100%. The results of the bacteriostatic activity of PRWS21 on the pathogenic bacteria of panax notoginseng are shown in Table 1. The growth inhibition rate of trichoderma strain PRWS21 on notoginseng pathogens 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 on PRWS21 pseudo-ginseng fusarium solani, pseudo-ginseng fusarium oxysporum and pseudo-ginseng phytophthora infestans is 81.11-68.05%. The trichoderma PRWS21 can inhibit the growth of the panax notoginseng pathogenic bacteria through space and nutrition competition, and the trichoderma has good broad-spectrum effect of resisting the panax notoginseng pathogenic bacteria.
Example 7
Preparation of bacterial strain PRWS21 zymocyte liquid
Inoculating 5 trichoderma PRWS21 fungus cakes with the diameter of 6mm into a 100m LPDA liquid culture medium, culturing on a constant-temperature shaking table at 28 ℃ and 180r/min for 7d, filtering the fungus liquid by using filter paper, and filtering and sterilizing by using a sterile filter with the diameter of 0.22 mu m to obtain sterile fermentation filtrate.
The PDA liquid culture medium consists of the following components: 160g of potatoes and 16g of glucose are added, the volume is fixed to 1L of distilled water, the pH value is 6.8, and the mixture is sterilized for 20min at 121 ℃.
Example 8
Bacteriostatic activity of bacterial strain PRWS21 zymocyte liquid on panax notoginseng pathogenic bacteria
The PRWS21 fermentation broth from example 4 was added to PDA solid medium to prepare PDA plates containing 30% fermentation broth. Respectively inoculating the pseudo-ginseng fusarium solani, fusarium oxysporum and phytophthora infestans on a PDA (personal digital assistant) plate added with 30% fermentation liquor to serve as a treatment group; the pathogenic bacteria of notoginseng are inoculated on a PDA plate without adding fermentation liquor to be used as a CK control group. Each group was repeated 3 times, the diameter of the colony was measured and the inhibition rate was calculated after 7 days of constant temperature culture at 28 ℃, and the growth inhibition rate of the fermentation broth of trichoderma strain PRWS21 on panax notoginseng pathogens was shown in table 2:
TABLE 2
As shown in Table 2, the addition of 30% PRWS21 fermentation broth can significantly inhibit the growth of pathogenic bacteria of Panax notoginseng, with an inhibition rate of 61.82-42.01%.
Example 9
Preparation of spore suspension of Strain PRWS21
Inoculating Trichoderma atroviride strain PRWS21 on PDA solid culture medium plate, culturing at 28 deg.C for 7 days, preparing activated strain into spore suspension with distilled water, and counting spore concentration under microscope to make the spore suspension concentration be 1.0 × 10 5 cfu/mL；
The PDA solid culture medium consists of the following components: 100g of potato, 20g of glucose and 18g of agar are added to 1L of distilled water, the pH value is 6.8, and the mixture is sterilized at 121 ℃ for 20 min.
Example 710
Control effect of trichoderma atroviride PRWS21 spore suspension on fusarium solani
1. Preparation of a fusarium solani spore suspension of panax notoginseng: inoculating Fusarium solani to PDA solid culture medium plate, culturing at 28 deg.C for 7 days, preparing activated strain into spore suspension with distilled water, counting spore concentration under microscope, and adjusting the spore concentrationThe concentration of the sub-suspension was 1.0X 10 5 cfu/mL。
2. A suspension of Trichoderma atroviride PRWS21 spores was prepared as in example 6.
3. And (4) selecting 1-year-old panax notoginseng seedlings to carry out root irrigation experiments. Experiment is as follows: CK group, using inoculated sterile distilled water as control 10 mL; in the SI group, only 10mL of pseudo-ginseng fusarium solani is inoculated; DI group: simultaneously inoculating 10mL of pseudo-ginseng fusarium solani and 10mL of trichoderma atroviride. Each group had 3 replicates, each replicate inoculated with 20 seedlings of notoginseng. After 14 days, the morbidity, disease index and prevention rate are counted.
4. The disease index and the prevention effect are calculated according to the following formulas
Disease index ∑ (number of diseased plant at each stage × disease-grade value)/(total number of investigated plants × highest-grade value) × 100
Severity grading criteria: level 0: no disease at root; level 1: the scab accounts for less than 20% of the surface area of the root; and 2, stage: the scab accounts for 21-40% of the surface area of the root; and 3, level: the spots account for 41% -65% of the root surface area, and the appearance is seriously affected; 4, level: lesions account for more than 66% of the surface area of the root or are completely rotten.
The control rate (%) (control disease index-treatment disease index)/control disease index is multiplied by 100
5. The control effect of trichoderma atroviride PRWS21 on panax notoginseng root rot is shown in table 2, and the biocontrol effect of trichoderma atroviride PRWS21 spore suspension on panax notoginseng fusarium solani is shown in table 3:
TABLE 3
As shown in Table 3, the pot experiment of the invention shows that the application of the strain for preventing and controlling the fusarium solani can effectively reduce the disease index, and the prevention and control rate of the fusarium solani reaches 75.29%.
The foregoing shows and describes the general principles, essential 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 embodiments described above, which are described in the foregoing description only for the purpose of illustrating the principles of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims, specification, and equivalents thereof.
Sequence listing
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Claims (10)
1. A Trichoderma atroviride strain is characterized in that: the Trichoderma atroviride strain is PRWS21, the preservation name is Trichoderma atroviride, the Trichoderma atroviride strain is preserved in China general microbiological culture collection center (CGMCC), the preservation unit address is No. 3 of Siro No.1 of Beijing, Chaoyang district, the preservation date is as follows: 2021, 4 months and 6 days; the preservation number is: CGMCC No. 21478.
2. The trichoderma atroviride strain of claim 1, wherein: the ITS gene sequence of the trichoderma atroviride strain is a nucleotide sequence shown in SEQ ID No. 1.
3. A Trichoderma atroviride bacterial agent produced by the Trichoderma atroviride 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) and (c):
(a) a fermentation culture of trichoderma atroviride of claim 1;
(b) a spore suspension of trichoderma atroviride obtained according to claim 1;
(c) an ultrasonic lysis pellet of trichoderma atroviride cells obtained according to claim 1.
5. A process for the preparation of a fermentation broth of Trichoderma atroviride as claimed in claim 4, comprising the steps of: inoculating 5 trichoderma PRWS21 fungus cakes with the diameter of 6mm into a 100m LPDA liquid culture medium, culturing on a constant-temperature shaking table at 28 ℃ and 180r/min for 7d, filtering the fungus liquid by using filter paper, and filtering and sterilizing by using a sterile filter with the diameter of 0.22 mu m to obtain sterile fermentation filtrate.
6. The method of claim 5, wherein the fermentation broth of Trichoderma atroviride is prepared by the following steps: the PDA liquid culture medium consists of the following components: 100-200 g of potatoes and 5-20 g of glucose, diluting to a constant volume of 1L of distilled water, adjusting the pH value to 6-8, and sterilizing at 121 ℃ for 20 min.
7. A spore suspension of Trichoderma atroviride as claimed in claim 4The preparation method is characterized by comprising the following steps: inoculating Trichoderma atroviride strain PRWS21 on PDA solid culture medium plate, culturing at 28 deg.C for 7 days, preparing activated strain into spore suspension with distilled water, and counting spore concentration under microscope to make the spore suspension concentration be 1.0 × 10 5 cfu/mL。
8. The method for preparing a spore suspension of trichoderma atroviride according to claim 7, wherein: the PDA solid culture medium consists of the following components: 100-200 g of potatoes, 5-20 g of glucose and 10-20 g of agar, and sterilizing at 121 ℃ for 20min with constant volume in 1L of distilled water and pH of 6-8.
9. Use of a trichoderma atroviride strain or a trichoderma atroviride inoculant according to any one of claims 1 to 8 in the manufacture of a product for controlling root rot of panax notoginseng.
10. Use according to claim 9, characterized in that: root irrigation treatment is carried out on the pseudo-ginseng by using trichoderma atroviride spore suspension or fermentation liquor.
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| CN110699301A (en) * | 2019-11-15 | 2020-01-17 | 云南大学 | Bacterial strain and application thereof in preventing and treating root rot of panax notoginseng |
| CN113913302A (en) * | 2021-11-15 | 2022-01-11 | 中国农业科学院特产研究所 | Trichoderma atroviride and application thereof in inhibiting ginseng pathogenic bacteria |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110699301A (en) * | 2019-11-15 | 2020-01-17 | 云南大学 | Bacterial strain and application thereof in preventing and treating root rot of panax notoginseng |
| CN113913302A (en) * | 2021-11-15 | 2022-01-11 | 中国农业科学院特产研究所 | Trichoderma atroviride and application thereof in inhibiting ginseng pathogenic bacteria |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116855390A (en) * | 2023-08-18 | 2023-10-10 | 西南大学 | Trichoderma asperellum SWU B077R1 for preventing and treating loquat root rot and application thereof |
| CN116855390B (en) * | 2023-08-18 | 2024-03-19 | 西南大学 | Trichoderma asperellum SWU B077R1 for preventing and treating loquat root rot and application thereof |
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