CN109112071B - A kind of Trichoderma and its application - Google Patents

Abstract

The invention discloses trichoderma and application thereof. The Trichoderma of the invention is Trichoderma asperellum (Trichoderma asperellum) and has the strain number: HTTA-Z0002 with the preservation number of CGMCC No. 15677. The trichoderma can be applied to plant disease control, bacteriostasis and pesticides. The strain has stable biological characteristics and pesticide activity, is easy to culture, has short culture period and simple and feasible operation method, has greatly improved antibacterial activity and conidium production in liquid culture compared with wild strains, overcomes the disadvantage of low spore production in liquid culture of trichoderma strains, has no pollution to agricultural products and environment, and has wide and strong inhibiting effect on various pathogenic bacteria of plants.

Description

A kind of Trichoderma and its application

technical field

The invention belongs to the technical field of microorganisms, and in particular relates to a Trichoderma and its application in pesticides.

Background technique

The problem of pesticide pollution in cultivated plants is a worldwide concern. With the increasing popularity of the technology of facility cultivation of vegetables and fruits, this problem has become more prominent. The high temperature and high humidity environment of the greenhouse meets people's needs for vegetables and fruits in four seasons, and also causes serious occurrence of diseases regardless of season. Excessive pesticides and hormones cause serious pollution to vegetables and fruits, and their toxicity and potential carcinogenic effects pose a great threat to human health. Biopesticides meet the requirements of modern society for agricultural production and pesticides due to their outstanding advantages of being relatively safe and environmentally compatible with non-target organisms.

Among all biological resources, microorganisms have become the preferred resources for the development of pesticides because of their fast reproduction, low cost, and easy transformation of wild strains to rapidly increase yield. At present, the sales of microbial pesticides have reached hundreds of millions of dollars, and the annual output value has increased by 10% to 20% every year. In order to solve the current situation that terrestrial microorganisms acquire new pesticide traits, new resources of extreme marine microorganisms have begun to receive attention. The unique geographical location of the polar regions has formed a natural environment such as high salt, severe cold, and strong ultraviolet radiation, which has created a variety of adaptive survival of organisms, has unique molecular biological mechanisms and physiological and biochemical characteristics, and produces new metabolites that can be used by humans utilized.

Soil salinization in my country is relatively serious, and salinized soil accounts for about 1/3 of cultivated land. In view of the characteristics of more people and less land, these salinized soils are still used by people to grow more salt-tolerant crops. At present, microorganisms developed at home and abroad are developed Because the biocontrol bacteria all originate from the land, it is difficult to survive and colonize in the soil with high salt content, and the control effect of soil-borne diseases in the saline soil is poor. effect. The original habitat of marine microorganisms with high salinity is more likely to play a role in saline soil, showing its habitat advantages in the field of new pesticide development.

The live bacterial preparation of biological pesticides is a kind of products with relatively short development cycle, relatively small capital requirements and complete pesticide functions. The terrestrial microbial live bacteria preparations that have been registered account for a large proportion of biological pesticides and play an important role in the prevention and control of plant diseases. In order to better utilize and industrialize pesticide strains for human beings, the strains must meet the conditions of pesticide activity and production and fermentation traits. After the microbial strains are obtained through various screening models, they are usually subjected to mutation breeding, such as the success of the operation. , its economic value can be increased hundreds or even thousands of times on the basis of wild strains. Microbial liquid fermentation is the basis for low cost and easy industrial production, but Trichoderma produces less spores in liquid culture, and solid-state culture restricts large-scale production. The number of conidia is proportional to the efficacy of the drug.

The present invention is mainly aimed at the most important bacteriostatic activity and liquid culture spore yield characteristics of Trichoderma pesticide strains, and the strain HTTA-Z0002 obtained by mutation breeding not only has excellent and broad-spectrum bacteriostatic characteristics against pathogenic bacteria that are more harmful to plants, It also has the characteristics of good artificial fermentation culture production.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a kind of Trichoderma and its application.

In order to achieve the above objects, the present invention provides the following technical solutions.

A Trichoderma, the Trichoderma asperellum bacteria, strain number: HTTA-Z0002, deposit number CGMCC No.15677.

The Trichoderma asperellum of the present invention is preserved in the General Microorganism Center (CGMCC) of the China Microorganism Culture Collection Administration Committee, and the preservation address is: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing; preservation date: May 2018 17th.

The application of the above-mentioned Trichoderma in preventing and treating plant diseases.

Further, the plant diseases include Alternaia solani, Botrytis cinerea, Phytophthora infestans, Fusarium graminearum, soybean rust (hakopsora pachyrhizi), wheat leaf One of the spot disease (Zymoseptoria tritci), muskmelon blight (Muskmelon blight), cabbage powdery mildew (Cabbage powdery mildew). The Trichoderma strain of the present invention has good broad-spectrum control effect on the above diseases, especially the control effect on wheat scab is very good up to 100%, and the control effect on tomato gray mold reaches 80.1%-87.9%. The therapeutic effect is also 52.5%, the prevention of melon fusarium wilt and cabbage powdery mildew can reach 68.87%-72.26% and 59.49%-60.76%, the results are better than traditional chemical agents.

The application of the above-mentioned Trichoderma in antibacterial.

Further, the Trichoderma is resistant to Pyricularia oryzae, Alternariasalani, Fusarium oxysporrum, Botrytis cinerea, Verticillium dahliae One or more of , Cladosporium fulvum, Phytophthoracapsici, Rhizoctonia solani, Fusarium graminearum, and Sclerotina sclerotiorum have inhibitory effects. The gene detection of the Trichoderma aculeatus HTTA-Z0002 strain of the present invention proves that the strain has key genes for chitinase and antimicrobial peptide synthesis, and can inhibit pathogenic fungi by producing antimicrobial peptides and chitinase, which is common and seriously harmful to the above-mentioned agriculture. The 10 species of plant pathogenic fungi have strong and broad-spectrum antibacterial effects, the antibacterial circle is more than 30mm, and the antibacterial rate is 79.0-95.4%.

A pesticide, comprising the above-mentioned Trichoderma.

The culture method of the above-mentioned Trichoderma, the bacterial strain is cultured on a solid MPDA medium at a temperature of 20°C-23°C for 3-5 days, and the mycelium is transferred to a liquid MPDA medium for shaking and cultured for 5-7 days. Shake culture for 3-7 days.

Further, the solid MPDA medium is composed of the following raw materials: 200-250ml of potato extract, 1-2g of peptone, 25-30g of glucose, 5-8g of NaCl, 0.1-0.3g of MgCl ₂ ·6H ₂ O, 0.1g of KCl , agar 18g, water 1000ml. The strain of the invention is easy to be cultivated artificially, grows well on the MPDA medium, the medium material is cheap and easy to obtain; the strain grows vigorously on the MPDA artificial medium, the spore-producing layer and the conidiophores are dense like felt, and the green-green , occasionally white, conidiophores with septa, producing lateral branches, branches vertically opposite, wider at the base, thinner at the end, bottle-shaped, conidia monospore, spherical to ovate, colorless, in The tip forms a more obvious conidia.

The present invention has the following characteristics:

1) The artificial culture method of Trichoderma asperellum obtained by the present invention is simple, and the culture material is cheap and easy to obtain.

2) the Trichoderma asperellum (Trichoderma asperellum) that the present invention obtains, in the characteristic such as bacteriostatic activity, liquid culture to produce conidia, has greater improvement than wild strain, overcomes the fact that Trichoderma strain liquid culture sporulation is few. Disadvantage, the maximum spore production reached 4.25×10 ⁸ cfu/cm ² .

3) Trichoderma asperellum has a strong and broad-spectrum bacteriostatic effect on common and seriously harmful phytopathogenic fungi in agriculture, and has a good control effect on related plant diseases, and the control effect is obviously better than traditional chemical agents.

Description of drawings

Figure 1. Affinities branching tree.

Detailed ways

The following specific examples are further descriptions of the methods and technical solutions provided by the present invention, but should not be construed as limiting the present invention.

1. Classification and identification of Trichoderma strains

Trichoderma aculeatus HTTA-Z0002 strain, in solid MPDA medium (potato infusion 200-250ml, peptone 1-2g, glucose 25-30g, NaCl 5-8g, MgCl ₂ 6H ₂ O 0.1-0.3g, KCl 0.1g , agar 18g water 1000ml), 20 ℃-23 ℃, cultured for 3-5 days, continuous observation and recording of changes in colony morphology, color, etc., pick hyphae and observe the sporulation structure under an optical microscope. The strain grows vigorously in MPDA artificial medium, the spore layer and conidiophore are dense like felt, blue-green, or white, the conidiophore has a septum, produces lateral branches, the branches are vertically opposite, and the base is wider , the end is thin, bottle-shaped, conidial single spore, spherical to oval, colorless, forming a more obvious conidia at the tip.

The strains were cultured by the above method, the universal primers of the fungal ITS gene were used, the PCR amplification products were sequenced by conventional methods, and compared by the BLAST program of NCBI, the sequence homology was analyzed and the phylogenetic tree was used to compare the closely related species (see Fig. 1). Combined with the microscopic structure of biological colony morphology, color and sporulation structure, and the molecular biology information of the strain, referring to the relevant Trichoderma classification and identification monographs at home and abroad, the strain was identified as Trichoderma asperellum.

2. Mutagenesis breeding of Trichoderma strains

The Trichoderma strains screened by multiple pesticide activity tests were subjected to conventional UV-microwave compound mutagenesis to select vigorously growing monoclonal colonies, and Alternaria salani was used as the pathogenic target bacteria, and the antibacterial activity was tested by the confrontation culture method. , calculate the bacteriostatic rate: inhibition rate (%) = (target colony diameter of control group - target colony diameter of treatment group)/target colony diameter of control group × 100%, and compared with the inhibition rate of wild strains, select the bacteriostatic rate to increase More than 25% of the strains were first cultured in solid MPDA medium for 5 days, and the abnormal growth strains were eliminated, and then inserted into liquid MPDA, cultured for 7 days at 20°C, 23°C, and 25°C, respectively, microscopically counted and compared the number of spores produced. , select the strain with 50% increase in the number of spores, the selected strains were continuously cultured for 5 generations, and the same activity and sporulation quantity test method as above were used to observe the stability of bacteriostasis and sporulation. The final selected HTTA-Z0002 Trichoderma strain, the bacteriostatic rate was 67.2% higher than that of the wild strain, and the maximum spore production was 4.25×10 ⁸ cfu/cm ² , which was 150.7% higher than that of the wild strain.

3. Laboratory plant in vitro antibacterial activity test and gene verification test

Strain HTTA-Z0002, on MPDA medium for 7 days, adopts the same confrontation culture method above to treat 10 common and serious agricultural pathogenic fungi: Pyricularia oryzae, Alternariasalani, Fusarium oxysporrum, Botrytis cinerea, Verticillium dahliae, Cladosporium fulvum, Phytophthoracapsici, Rhizoctonia solani, Fusarium graminearum, and Sclerotinasclerotiorum were tested for antibacterial activity and calculated antibacterial activity Rate. The sequences of Trichoderma β-1,4-acetylglucosaminidase gene Nag1 and Peptaibols synthase gene Tex1 were retrieved from NCBI (http://www.ncbi.nlm.nih.gov/), and the corresponding sequences were designed using Premier 5.0 The primers JDZ-F/JDZ-R, KJT-F/KJT-R, JDZ-F (5′-CAGAACCGAATCCAGAGC-3′)/JDZ-R (5′-GAAGGAGTGTAGACGAGC-3′) were amplified by conventional PCR method, The characteristics of the strain producing antibacterial activity were verified, and the results were as follows:

Table 1 Antibacterial activity of HTTA-Z0002 against plant pathogens

Note: T1: Confrontation culture test, the size of the inhibition zone indicates the activity of the strain, and the inhibition zone above 30mm indicates strong inhibition activity; T2: The inhibition rate against pathogenic bacteria = (control colony radius - treated colony radius)/control colony radius × 100 % Phytopathogenic bacteria: Po: Pyricularia oryzae; As: Alternaria salani; Fo: Fusarium oxysporum; Bc: Botrytis cinerea; Vd: Verticillium dahliae; Cf: Cladosporium fulvum; Pc: Phytophthora capsici; : Sclerotina sclerotiorum.

From the data in the table above, it can be seen that the Trichoderma HTTA-Z0002 strain has a good bacteriostatic effect on 10 common and seriously harmful pathogenic fungi in agriculture. The circle is more than 30mm, and the antibacterial rate is 79.0-95.4%, showing strong antibacterial activity, and has the potential to develop new fungicides. The Nag1 gene and Tex1 gene were amplified by PCR from the HTTA-Z0002 strain, and the characteristic bands were about 800bp and 250bp, respectively, indicating that the strain has the key genes for chitinase and antimicrobial peptide synthesis. Enzymes inhibit pathogenic fungi.

4. Disease control test of living plants (plant pot test and field plot test)

The disease control test of living plants is carried out by conventional methods, potted plant seedlings are cultivated to 3-4 true leaves, no less than 60 seedlings are used for each potted test, the area of the field plot test plot is more than ^25m2 , and the field salty soil plot control test , The saline soil selects natural saline cultivated land near the sea in Jimo, the chloride salt is about 0.2-0.3%, the potted saline soil adopts artificially cultivated simulated saline soil, each treatment is not less than 3 replicates, inoculation disease Or natural disease, use Trichoderma viable spore dilution for prevention and control, use traditional pesticides commonly used in agricultural production to prevent and control the disease as a positive control, and treat with the same concentration of sterile fermentation broth as a blank control, investigate the disease after 6-7 days, Calculate the control effect.

Table 1 Control effect of HTTA-Z0002 strain on botrytis cinerea of potted tomato

Table 2 Control effect of HTTA-Z0002 strain on tomato gray mold in field plots

Note: Dilute the solution 200 times.

Table 3 The efficacy of salinized soil HTTA-Z0002 on melon fusarium wilt in field plots

Note: Dilute the solution 300 times.

Table 4 Field plots of salinized soil HTTA-Z0002 against cabbage powdery mildew

Note: Dilute the solution 300 times.

The data in Table 1-4 proves that the Trichoderma HTTA-Z0002 strain in ordinary soil potted plants or field plots controls tomato gray mold, and the control effect reaches 80.1%-87.9% respectively. The control of melon fusarium wilt and cabbage powdery mildew, which are difficult to control by chemical agents, can achieve 68.87%-72.26% and 59.49%-60.76% of the therapeutic effects, and the results are better than traditional chemical agents.

5. Third-party verification results

In order to confirm the pesticide potential of the strains by a third party, during the cooperation with BASF in Germany, under the premise that both parties signed a non-disclosure agreement on the strains, BASF conducted multiple plant disease control tests and verifications on the above two strains in Germany and provided the control results. Diseases tested by BASF include: early blight Alternaia solani; tomato botrytis cinerea; potato late blight Phytophthora infestans, wheat scab Fusarium graminearum; soybean rust Phakopsora pachyrhizi; wheat leaf spot Zymoseptoria tritci.

Table 5 Bioassay results of BASF in Germany

The above results prove that HTTA-Z0002 Trichoderma asperellum has a good broad-spectrum control effect on the above diseases, especially the control effect on wheat scab Fusarium graminearum is very good up to 100%. In view of the popularity of German BASF company in the field of pesticide research and development in the world, their results are the best verification of the pesticide potential of the HTTA-Z0002 Trichoderma strain.

As a result, the good pesticide potential of the strain has been confirmed in many aspects such as plant in vitro test, in vivo test, pot test, field plot test, and third-party verification.

The descriptions of the above embodiments are only used to help understand the method and the core idea of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can also be made to the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims (6)

1. A Trichoderma fungus, wherein the Trichoderma fungus is Trichoderma asperellum (Trichoderma asperellum: (A)Trichoderma asperellum) Bacteria, strain number: HTTA-Z0002 with the preservation number of CGMCC No. 15677.

2. The method for culturing trichoderma according to claim 1, wherein the trichoderma strain is cultured on a solid MPDA medium at a temperature of 20 ℃ to 23 ℃ for 3 to 5 days, and the mycelium is shake-cultured on a liquid MPDA medium for 5 to 7 days.

3. The method for culturing trichoderma as defined in claim 2, wherein said solid MPDA medium consists of the following raw materials: 250ml of potato extract, 1-2g of peptone, 25-30g of glucose, 5-8g of NaCl and MgCl₂·6H₂0.1-0.3g of O, 0.1g of KCl, 18g of agar and 1000ml of water.

4. Use of a trichoderma as defined in claim 1 for controlling plant diseases, including phytophthora infestans (a) and (b) in the control of plant diseasesPhytophthora infestans) Resulting late blight of potato、Gibberella sp（Fusarium graminearum）Resulting wheat scab、Wheat leaf blight bacterium(Zymoseptoria tritici)Caused leaf spot of wheat and fusarium wilt of muskmelon: (Muskmelon blight) The resulting blight of sweet melon, powdery mildew of cabbage: (Cabbage powdery mildew) One of the resulting powdery mildew of cabbage.

5. Use of a trichoderma as defined in claim 1, for combating fungi, characterized in that said trichoderma is a pyricularia oryzae（Pyricularia oryzae）Alternaria solani, Alternaria solani（Alternaria salani）Fusarium sp（Fusarium oxysporrum）Botrytis cinerea (Botrytis cinerea)（Botrytis cinerea）Verticillium dahliae（Verticillium dahliae）Fungus of bud（Cladosporium fulvum）Phytophthora capsici（Phytophthora capsici）Rhizoctonia solani（Rhizoctonia solani）Gibberellin, gibberellin（Fusarium graminearum）Sclerotium bacteria(Sclerotina sclerotiorum)Has an inhibitory effect.

6. A pesticide comprising the trichoderma as defined in claim 1.

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