CN105368719A - Trichoderma koningii for preventing and treating alfalfa anthracnose and application thereof - Google Patents

Abstract

The invention discloses a Trichoderma strain for preventing and treating alfalfa anthracnose and application thereof. The invention provides a kind of Trichoderma, and the preservation number of the Trichoderma (Trichoderma? koningii) is CGMCC? No. 11221. The present invention also provides a composite microbial inoculum, the active ingredients of which include the above-mentioned Trichoderma and Bacillus subtilis. The present invention also provides the application of the above-mentioned Trichoderma or the above-mentioned microbial agent in the prevention and treatment of alfalfa anthracnose. Through the above technical scheme, the present invention can effectively prevent the occurrence of alfalfa anthracnose.

Description

A Trichoderma strain for preventing and controlling alfalfa anthracnose and its application

technical field

The invention relates to the field of microbes, in particular to a Trichoderma koningii, microbial inoculants containing the Trichoderma koningii and their application in the prevention and treatment of alfalfa anthracnose.

Background technique

Alfalfa Medicagosativa L. is a perennial leguminous plant known as the "King of Grass". Because of its rich nutrition, high yield, good palatability, high protein content and multivitamins needed for animal growth, it is widely used in animal husbandry. plays an important role in.

Alfalfa anthracnose is a major disease in alfalfa cultivation, which seriously affects the quality and yield of alfalfa. Alfalfa anthracnose mainly affects the above-ground parts of plants, especially near-ground vines. At the beginning, small yellow-brown spots appeared on the stems, and then gradually expanded to grow into ovals. When the lesions expanded, they merged with each other and circled the stems for a week, causing the branches to die. Alfalfa anthracnose is a widely distributed and devastating disease in many countries, and it also occurs in many areas of my country. It is known that the pathogenic bacteria causing anthracnose in alfalfa are mainly three common species of Neptonia, namely Colletotrichum trifolii, Colletotrichum destructivum and Colletotrichum truncatum. Among them, Echinococcus flathead is widely distributed all over the world, infecting a variety of leguminous plants, including various leguminous forages, including alfalfa.

In recent years, due to the increasingly serious pollution of chemical pesticides, biological control has attracted increasing attention due to its advantages of no pollution, no residue, and safety to the ecological environment and human health. Among them, the application of antagonistic microorganisms in the biological control of plant diseases much attention. Trichoderma is one of the most competitive biocontrol bacteria among many antagonistic microorganisms. It is a type of filamentous fungus that exists widely in nature. It has strong adaptability, many antagonists, wide parasiticity and low environmental impact. With many characteristics, the application potential in plant disease and biological control has been paid more and more attention, and breakthroughs have been made constantly. At present, there are many kinds of antagonistic Trichoderma studied, mainly including Trichoderma harzianum, Trichoderma hamatum, Trichoderma longibrachiatum and Trichodermaviride. It is known that the biological control mechanism of Trichoderma mainly includes competition for nutrients and space of pathogenic bacteria, reparasitization of pathogenic bacteria, production of antibiotics to inhibit or kill pathogenic bacteria, and promotion of plant-induced autoimmunity. The control targets are mainly plant diseases caused by soil-borne pathogens, such as bean root rot (Fusarium graminerarum) and anthracnose (Colletotrichum truncatum), cotton wilt (Fusarium oxysporium) and watermelon wilt (Didymelabryoniae), etc.

As a Gram-positive bacterium, Bacillus subtilis is widely distributed in nature. It can secrete a variety of antibiotics and enzymes with antibacterial activity, and has strong antibacterial and disease-preventing capabilities. Its biological control mechanism mainly includes colonizing in the rhizosphere, body surface or body of plants, competing for nutrients with pathogenic bacteria, secreting antibacterial substances and enzymes, etc., inhibiting the growth of pathogenic bacteria, and at the same time can promote plant growth and induce plant resistance, etc. . The control targets are mainly plant diseases caused by filamentous fungi, such as cotton wilt (Fusarium oxysporium) and Verticillium dahliae (Verticillium dahliae), soybean root rot (Fusarium graminerarum), rice sheath blight (Rhizoctonia solani) and tomato gray mold (Botrytiscinerea) Wait.

Today, environmental pollution is becoming more and more serious, and biological control has great potential for the control of alfalfa anthracnose. Using the antagonistic effect on the pathogenic fungus of alfalfa anthracnose to screen biocontrol microorganisms, and using microbial agents to act together on the pathogenic fungus, is the key to the control of alfalfa anthracnose. effective way of prevention.

Contents of the invention

The inventors of the present invention have screened a strain of Trichoderma and found that it can significantly inhibit the growth of the pathogenic bacteria of alfalfa anthracnose and effectively prevent the occurrence of alfalfa anthracnose, thus obtaining the present invention.

The invention provides a Trichoderma fungus, the Trichoderma fungus is Trichodermakoningii (TKmx) and its preservation number is CGMCC No.11221.

The present invention also provides a composite microbial inoculum, wherein the active ingredients of the composite microbial inoculum include the above-mentioned Trichoderma and Bacillus subtilis with the product number ACCC03221.

The present invention also provides the use of the above-mentioned Trichoderma or the above-mentioned composite microbial agent in the prevention and treatment of alfalfa anthracnose.

Through the above technical scheme, the Trichoderma or composite microbial inoculum provided by the present invention has high control effect on alfalfa anthracnose, and has the advantages of being green and environment-friendly.

Other features and advantages of the present invention will be described in detail in the following detailed description.

biological material deposit

Trichoderma koningii of the present invention is obtained by the inventor of the present invention from isolation and screening of alfalfa planting soil in Huanghua City, Hebei Province. Its preservation number is CGMCCNo.11221, and the preservation date is August 11, 2015. The preservation unit is The General Microbiology Center of the China Committee for the Collection of Microbial Cultures is located at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The classification is named Trichodermakoningii.

detailed description

Specific embodiments of the present invention will be described in detail below. It should be understood that the specific embodiments described here are only used to illustrate and explain the present invention, and are not intended to limit the present invention.

The invention provides a Trichoderma fungus, the Trichoderma fungus is Trichodermakoningii (TKmx) and its preservation number is CGMCC No.11221.

The present invention also provides a composite microbial inoculum, wherein the active ingredients of the composite microbial inoculum include the above-mentioned Trichoderma and Bacillus subtilis with a product number of ACCC03221.

Wherein, relative to 1 g of microbial agent, the content of Trichoderma can be 10 ⁵ -10 ⁹ CFU, preferably 10 ⁶ -10 ⁸ CFU.

Wherein, the CFU ratio of the Trichoderma and the Bacillus subtilis is preferably 1: (10-20).

Wherein, the microbial bacterial agent can be prepared by the preparation method of the following steps:

(1) Trichoderma with the preservation number CGMCCNo.11221 and the Bacillus subtilis (Bacillus subtilis) bacterial classification with the commodity number ACCC03221 were respectively inoculated and cultivated in the activation medium to obtain respective activated bacterial classifications;

(2) the activated strain of Trichoderma is cultivated in the Trichoderma liquid fermentation medium, and the activated bacterial strain of Bacillus subtilis is cultivated in its liquid fermentation medium simultaneously, obtains the bacterium liquid after fermentation respectively;

(3) the Trichoderma and Bacillus subtilis bacterium liquid obtained in step (2) are adsorbed, dried and pulverized by an inert carrier respectively to obtain the bacteria powder of each strain;

(4) Mix the bacterial powders of Trichoderma and Bacillus subtilis in proportion to obtain a composite microbial bacterial agent.

Wherein, in step (1), there are no special restrictions on the activation culture conditions of the strain and the state of the strain after cultivation, which can be common conditions in the cultivation process of Trichoderma and Bacillus subtilis, for example, the cultivation temperature can be 28-32°C, and the cultivation The time can be 1-5 days. During the cultivation process, the state of the activated strain can be determined by conventional methods, such as microscopic observation.

Wherein, in step (2), there is no special requirement for the concentration of live bacteria in the fermentation conditions and the fermented material, which can be the conditions and concentrations commonly used in the fermentation process of Trichoderma and Bacillus subtilis, as a preferred embodiment of the present invention , in step (2), the fermentation conditions are such that the concentration of viable bacteria in the fermented material is (5-500) × 10 ⁸ CFU/mL, under the concentration of viable bacteria in this preferred embodiment, the obtained fermented The bacterial liquid is used as a raw material for the preparation of microbial bacterial agents. For example, the fermentation time may be 20-200 hours, and the temperature may be 28-32°C.

Wherein, the activation medium can be a variety of conventional medium capable of cultivating Trichoderma and Bacillus subtilis, such as including but not limited to PDA medium, beef extract peptone medium, broth medium and LB medium at least one.

Wherein, the Trichoderma liquid fermentation medium can be various conventional liquid mediums capable of fermenting and expanding the number of Trichoderma, for example, the liquid medium can contain starch, yeast powder, at least one of corn steep liquor; and the The liquid culture medium may also contain 0.01-0.02% ZnSO ₄ and 0.01-0.03% MgSO ₄ to supplement the inorganic salts required by Trichoderma for rapid growth.

Wherein, the Bacillus subtilis liquid fermentation medium can be conventional various liquid mediums capable of fermenting and expanding the number of Bacillus subtilis, for example, the liquid medium can contain cornstarch, yeast extract powder, peanut cake powder, cottonseed cake At least one of powder; and the liquid medium can also contain 0.2-0.5% ammonium sulfate, 0.02-0.05% magnesium sulfate, 0.01-0.05% sodium chloride, to supplement the inorganic salts needed for the rapid growth of Bacillus subtilis .

Wherein, as a particularly preferred embodiment of the present invention, based on the total weight of the Trichoderma liquid fermentation medium, calculated by weight ratio, the Trichoderma liquid fermentation medium contains 2.0-3.5% starch, 1.0- 2.0% yeast powder, 4.0-8.0% corn steep liquor, 0.1-0.5% CaCO ₃ , 0.01-0.02% ZnSO ₄ and 0.01-0.03% MgSO ₄ . Wherein, the culture medium may also contain the rest of water and/or other components. In this preferred mode, on the one hand, Trichoderma can be allowed to grow rapidly during the fermentation process to produce a sufficient number of chlamydospores; application in prevention and treatment.

Among them, as a particularly preferred embodiment of the present invention, the Bacillus subtilis liquid fermentation medium contains 1-1.5% corn starch, 1-1.5% peanut cake powder, 0.2-0.5% ammonium sulfate, 0.1-0.4% carbonic acid Calcium, 0.02-0.05% magnesium sulfate and 0.01-0.05% sodium chloride. Wherein, the culture medium may also contain the rest of water and/or other components. In this preferred mode, on the one hand, the rapid growth of Bacillus subtilis in the fermentation process can produce a sufficient number of spores; application.

Wherein, the fermented bacterial liquid can be directly used as a microbial bacterial agent, and can also be further processed into a microbial bacterial agent in a dosage form that is more convenient for storage through steps including carrier adsorption, drying, crushing and/or granulation. The amount of the inert carrier can make the content of live bacteria in Trichoderma powder reach (2-5)×10 ⁸ CFU/g, and the content of live bacteria in Bacillus subtilis powder reach (2-10)×10 ⁹ CFU/g respectively. Mix Trichoderma and Bacillus subtilis bacteria powder; make the CFU ratio of the Trichoderma and the Bacillus subtilis 1: (10-20). The preparation of the two bacterial agents in the preparation of the composite microbial bacterial agent is not limited to the above ratio, and can be mixed in other ratios.

Wherein, the inert carrier may include at least one of calcium carbonate, loess, diatomite, bentonite, talcum powder, mica powder, kaolin and calcite powder.

The present invention also provides the use of the above-mentioned Trichoderma or the above-mentioned composite microbial agent in the prevention and treatment of alfalfa anthracnose.

Wherein, in a preferred embodiment of the present invention, the above-mentioned Trichoderma or the above-mentioned composite microbial inoculum is applied in the prevention and treatment of alfalfa anthracnose.

Among them, in a particularly preferred embodiment of the present invention, Trichoderma or the above-mentioned composite microbial inoculum is applied in the farmland where alfalfa is planted; wherein, before planting alfalfa, Trichoderma or the above-mentioned composite microbial inoculum and The fine soil is mixed and applied to the planting soil; or at the emergence stage of the alfalfa, the Trichoderma or the above-mentioned composite microbial agent is mixed with the fine soil and applied to the planting soil.

The present invention is further described in detail by the following examples:

Example 1

This embodiment is used to illustrate the cultivation of Trichoderma of the present invention and the preparation of microbial inoculum.

The Trichoderma (Trichodermakoningii (TKmx)) whose preservation number is CGMCCNo.11221 is inoculated into the activated medium PDA medium (get the potato 20g after peeling and boil for 20 minutes to get the juice, the juice is mixed with 2g glucose, 1.5g agar and water) into 100ml, pH6.8-7.0, put into 2 eggplant bottles, each 50ml, sterilized and made on a slant), and cultivated at 28°C for 6 days. Inoculate the activated Trichoderma obliquus bacterial classification into the sterilized liquid fermentation medium (by weight, containing 2.5% starch, 1.5% yeast powder, 5.0% corn steep liquor, 0.4% CaCO ₃ , 0.01% ZnSO ₄ , 0.03% MgSO ₄ ), cultured at 28° C. for 144 hours, until 90% of mycelia formed chlamydospores, and Trichoderma liquid was obtained.

Centrifuge 10000mL of the above-mentioned Trichoderma liquid, collect the wet cells, and fully mix with an appropriate amount of carrier diatomaceous earth. Place the uniformly mixed material in an oven at 50°C and bake for 4-7 hours to make the water content less than 5%. Utilize the plate culture counting method to detect the number of viable bacteria in the bacteria powder, then use diatomaceous earth to adjust until the number of Trichoderma viable bacteria contained in each gram of bacterial agent is 500 million, and the obtained material is the Trichoderma microbial bacterium of this embodiment agent.

Example 2

This embodiment is used to illustrate the preparation of the composite microbial bacterial agent of the present invention.

The product number is ACCC03221 Bacillus subtilis (Bacillus subtilis) was inoculated onto LB medium (containing 10g/L tryptone, 5g/L yeast extract, 10g/L NaCl and 15g/L agar) slant, at 30 Cultivate at ℃ for 18 hours to obtain activated strains. Inoculate the liquid fermentation medium (by weight, containing 1% cornstarch, 1.5% peanut powder, 0.5% ammonium sulfate, 0.4% calcium carbonate, 0.03% magnesium sulfate, 0.02% chlorine NaCl), cultured at 35°C until the concentration of viable bacteria was 5-10×10 ⁹ CFU/mL to obtain a Bacillus subtilis bacterial liquid.

Centrifuge or suction filter 10000mL of the above-mentioned Bacillus subtilis bacteria liquid, collect the wet bacteria, and then mix it with an appropriate amount of carrier diatomaceous earth. Place the well-mixed and uniform material in an oven at 60°C for 4-7 hours to keep the water content below 5%. The number of bacteria in the bacteria powder is detected by the plate culture counting method, and then adjusted with diatomaceous earth until the number of viable bacillus subtilis contained in each gram of the bacteria agent is 2 billion.

Mix the Trichoderma powder obtained in Example 1 and the Bacillus subtilis powder obtained in this embodiment at a weight ratio of 1:3, and the obtained material is the composite microbial agent of this embodiment.

Comparative example 1

According to the method for embodiment 2, compound microbial bacterial agent is prepared, and the difference is that what Trichoderma uses is literature (Xiao Ye etc., the antagonism of Trichoderma to several plant pathogenic bacteria, "Journal of Hunan Agricultural University", 2007 01 period) Trichoderma harzianum No. 26 in Trichoderma harzianum.

Comparative example 2

The composite microbial agent was prepared according to the method of Example 2, except that the Bacillus subtilis (Bacillus subtilis) with the product number ACCC03221 was replaced with the Bacillus cereus with the product number ACCC11109.

Test Example 1

In this test example, the effect of the microbial agent of the present invention on the prevention and treatment of alfalfa anthracnose was determined in a field test.

The experimental field is located in the experimental base of the Animal Husbandry Institute of the Chinese Academy of Agricultural Sciences in Langfang City, Hebei Province. The tissues of the alfalfa plants that died of anthracnose were mashed, soaked in water of equal weight for 48 hours, and filtered to obtain the soaking solution containing the pathogenic bacteria of anthracnose. Alfalfa (No. 1 in middle alfalfa, provided by the National Plantation Grass Medium-Term Bank, the product catalog number is 00068) seeds were soaked in the above-mentioned soaking solution containing anthracnose pathogenic bacteria for 24 hours, and the consumption of the soaking solution was enough to submerge the alfalfa seeds, and obtained Soaked seeds.

Sow the soaked alfalfa seeds in the farmland, set the Trichoderma inoculum prepared in Example 1, the composite microbial inoculum prepared in Example 2 and the composite microbial inoculum prepared in Comparative Examples 1 and 2 and five blank controls Each treatment was repeated 3 times, a total of 15 plots were randomly arranged. After 10 days of alfalfa emergence, the Trichoderma inoculum prepared in Example 1, the composite microbial inoculum prepared in Example 2 and Comparative Examples 1 and 2 were fully mixed with sterilized fine soil, so that the content of viable bacteria in the fine soil 2×10 ⁶ CFU/g, with the same treatment, 100g of fine soil per square meter was evenly applied to the farmland, and the same weight of diatomite was used for the same soil-doped treatment as a control. Field management routines. The incidence of alfalfa anthracnose was investigated after 40 days of growth. The condition of alfalfa anthracnose is divided into 5 levels, immunity (level 0): disease index 0; disease resistance (level 1): disease index 0.1-20.0; disease resistance (level 2): disease index 20.1-40.0; susceptible (level 3 ): disease index 40.1-60.0; high sensitivity (level 4): disease index above 60.0 or completely dead. Disease index=∑(representative value of disease level×the number of plants of this level)/(representative value of the most severe disease level×total number of plants)×100; control effect=(control disease index-treatment disease index)/control disease index×100%. The results are shown in Table 1.

Table 1

deal with Disease index Control effect control 90.31±5.74 - Embodiment 1 Trichoderma agent 19.65±3.17 78.24% Embodiment 2 composite microbial bacterial agent 15.82±2.85 82.48% Comparative Example 1 Composite microbial bacterial agent 35.28±5.03 60.93% Comparative Example 2 Composite microbial bacterial agent 29.63±4.52 67.19%

Visible by the data of table 1, Trichoderma inoculum of the present invention, composite microbial inoculum all have significant control effect to alfalfa anthracnose, and the control effect of composite microbial inoculum of the present invention is higher than Trichoderma inoculum.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention. These simple modifications All belong to the protection scope of the present invention.

In addition, it should be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable way if there is no contradiction. The combination method will not be described separately.

In addition, various combinations of different embodiments of the present invention can also be combined arbitrarily, as long as they do not violate the idea of the present invention, they should also be regarded as the disclosed content of the present invention.

Claims (10)

1. A Trichoderma, characterized in that: the Trichoderma is Trichodermakoningii (Trichodermakoningii), and its preservation number is CGMCCNo.11221. 2. A composite microbial inoculum, characterized in that: the active ingredient of the composite microbial inoculum contains the Trichoderma claimed in claim 1 and the product number is ACCC03221 Bacillus subtilis (Bacillus subtilis). 3. The composite microbial agent according to claim 2, characterized in that: relative to 1 g of the microbial agent, the content of the Trichoderma is 10 ⁵ -10 ⁹ CFU, preferably 10 ⁶ -10 ⁸ CFU. 4. The compound microbial bacterial agent according to claim 2 or 3, characterized in that: the CFU ratio of the Trichoderma and the Bacillus subtilis is 1: (10-20). 5. according to claim 2 or 3 described compound microbial inoculums, it is characterized in that: the preparation method that this microbial inoculums comprises the following steps is prepared: (1) the Trichoderma described in claim 1 and the product number are inoculated respectively in the activated medium and cultivated to obtain respective activated bacterial classifications for the Bacillus subtilis (Bacillus subtilis) bacterial classification of ACCC03221; (2) the activated strain of Trichoderma is cultivated in the Trichoderma liquid fermentation medium, and the activated bacterial strain of Bacillus subtilis is cultivated in its liquid fermentation medium simultaneously, obtains the bacterium liquid after fermentation respectively; (3) the Trichoderma and Bacillus subtilis bacterium liquid obtained in step (2) are adsorbed, dried and pulverized by an inert carrier respectively to obtain the bacteria powder of each strain; (4) Mix the bacterial powders of Trichoderma and Bacillus subtilis in proportion to obtain a composite microbial bacterial agent. 6. The compound microbial inoculant according to claim 5, characterized in that: the consumption of the inert carrier makes the content of viable bacteria in the Trichoderma bacterial powder reach (2-5) × 10 ⁸ CFU/g, in the Bacillus subtilis bacterial powder The live bacteria content reaches (2-10)×10 ⁹ CFU/g, and the bacterial powders of Trichoderma and Bacillus subtilis are mixed so that the CFU ratio of Trichoderma and Bacillus subtilis is 1:(10-20). 7. The compound microbial bacterial agent according to claim 5, characterized in that: the inert carrier comprises at least one of calcium carbonate, loess, diatomaceous earth, talcum powder, mica powder, kaolin and calcite powder. 8. composite microbial bacterial agent according to claim 5, is characterized in that: take the gross weight of described Trichoderma liquid fermentation medium as a basis, by weight, described Trichoderma liquid fermentation medium contains 2.0-3.5% Starch, 1.0-2.0% yeast powder, 4.0-8.0% corn steep liquor, 0.1-0.5% CaCO ₃ , 0.01-0.02% ZnSO ₄ and 0.01-0.03% MgSO ₄ . 9. composite microbial bacterial agent according to claim 5, is characterized in that: take the gross weight of described Bacillus subtilis fermentation medium as a basis, by weight, the used liquid fermentation medium of Bacillus subtilis contains 1-1.5 % corn starch, 1-1.5% peanut cake powder, 0.2-0.5% ammonium sulfate, 0.1-0.4% calcium carbonate, 0.02-0.05% magnesium sulfate and 0.01-0.05% sodium chloride. 10. the purposes of the trichoderma described in claim 1 or any one described microbial inoculum agent in claim 2-9 in the control of alfalfa anthracnose.