KR100797036B1 - Trichoderma asperellum dyf0328 and a biocontrol agent of plant diseases with this strain - Google Patents

Abstract

A Trichoderma asperellum DYF0328 strain is provided to extend the period of circulation by using high concentration cultivation and appropriate manufacturing and control plant diseases without tolerance against pathogenic bacteria and environmental pollution. A Trichoderma asperellum DYF0328(KACC 93038P) strain controls plant pathogenic bacteria including Rhizoctonia solani, Magnaporthe grisea, Burholderia glumae, Botry cinerea, Phytophthora capsici, Pythium sp. and Rhizoctonia solani MAFF511103. A composition for controlling plant diseases comprises 10-80 wt.% of a solid containing Trichoderma asperellum DYF0328(KACC 93038P), 15-85 wt.% of a thickener selected from talc, kaolin, bentonite, diatomite and zeolite, and 1-20 wt.% of silica powder.

Description

TRICHODERMA ASPERELLUM DYF0328 AND A BIOCONTROL AGENT OF PLANT DISEASES WITH THIS STRAIN}

1 is a Trichoderma Ars buffer relrum (Trichoderma asperellum) on rice sheath blight DYF0328 is a photograph showing the effect of hydration treatment.

Figure 2 is a photograph showing the treatment effect of Trichoderma asperellum DYF0328 hydrous treatment for tomato ash fungus.

Figure 3 is a photograph showing the effect of Trichoderma asperellum DYF0328 granules on tomato ash fungus against pepper blight.

The present invention relates to an antagonist microorganism having an antagonistic activity against specific plant pathogens, and more specifically, has a specific effect on the control of rice paddy disease (leaf blight), rice bacterial blight and cucumber, tomato gray mold, pepper blight Trichoderma asperellum DYF0328.

As it promotes eco-friendly agriculture, even if pesticides should not be used, when the disease occurs in the crop, it is necessary to spray pesticides. Recently, as interest in well-being increases, preference for eco-friendly agricultural products is increasing, and interest in eco-friendly agricultural materials that can replace pesticides is increasing, and research and development are being made.

In 1960, microorganisms began to be used in the form of pesticides since 1960.In Japan, many products were developed in the 1960s, starting with probiotic disinfectants that control tobacco waist blight using Trichoderma genus. Bacillus thuringiensis has been registered to Platanas moths, Chinese cabbages and moths, and Rhizoctonia sp , Pythium sp. , Botrytis using Trichoderma genus . Already used in the control of phytopathogens of Botrytis cineria , Fusarium sp. , Etc., there are several existing patents similar to the present invention at home and abroad, and most of them are Trichoderma viride and Trichoderma. Trichoderma koningii , Trichoderma hamatum , Trichoderma hamningum ( Trichoderma hazianum ) Trichoderma hazianum strain among the strains of Trichoderma hazianum T-35, Trichoderma hazianum T-315, Trichoderma hazianum GBF-0208, but there is no plant disease control agent using the trichoderma asperellum of the present invention, rice paddy disease No microbial species have yet been found to be effective against leaf blight, cucumber and tomato gray mold, and pepper blight. Rice paddy disease or Bacterial Bacterial Disease is widespread in the whole country and the damage is great.There are 28 registered pesticides in Rice Bite Disease, 20 items of Bacterial Bacterial Disease in Bacteria, 22 items of Cucumber Gray Mold Disease, tomato gray mold It is a lot of 20 bottles.

Pesticides are resistant to pathogens when used for a long time, but microorganisms are not resistant and do not harm the human body or the environment. However, until now, the microbial agent has a short shelf life due to the short life of the microorganisms, and has a disadvantage of low control value due to low drug efficacy.

The present inventors have isolated and studied a number of indigenous antagonistic microorganisms in order to achieve the above-mentioned object, and have succeeded in obtaining a trichoderma aperelum strain that is widely applied and excellent in effect, and named it DYF0328. The microorganism strain of the present invention can be extended to the high concentration culture technology and the appropriate manufacturing prescription to develop an eco-friendly agricultural material.

The present inventors collected the soil samples of Gyeongsangbuk-do, Gyeongsan, Qingdao, Yeongcheon and non-cultivated soil in order to find microorganisms with antagonism against phytopathogens (5-10 cm deep soil removal after topsoil removal). Potato agar medium (Potato dextrose agar (PDA)) of the following composition was sterilized at 121 ° C. for 15 minutes and dispensed into petri dishes by 20 ml to solidify.

 PDA (Potato Agar Badge) Raw material name Mixing ratio (%) Remarks (Manufacturer) Potato starch 0.4  Difco Dextrose 2 Agar 1.5 water Remaining amount

10 g of soil samples are suspended in 90 ml of sterile physiological saline, diluted 10-fold, and 0.1 ml of sterilized potato agar medium is inoculated evenly with a smear of a smear. Diluted soil samples inoculated medium was isolated separately from the Trichoderma genus strains appearing incubated for 5 days at 28 ℃ in the incubator. To the the Trichoderma (Trichoderma) in strains also use dual malbeop (Dual plating) was selected, removing the inhibitory dominant and an excellent ability in Trichoderma (Trichoderma) sp for controlling plant pathogens. The test number of the isolated strain was named DYF0328.

As a result of ITS-5.8S rDNA sequencing method, it was confirmed that it was Trichoderma asperellum by requesting the Korea Microorganism Conservation Center (KCCM) to analyze the sample name DYF0328, and it was identified as the Trichoderma asperellum . It was deposited with the Korea Agricultural Microbial Resources Center and received accession number KACC 93038P. The selected strains were formulated with a hydrating agent and assayed for their activity through laboratory and field tests.

As a result of the test, the strain of the present invention is grown in the largest area of the crops and is a pathogenic bacterium that is damaging to our stock rice, Rhizoctonia solani , Magnaporthe grisea and rice bacterial eggs. Excellent for Burholderia glumae , tomato and cucumber Botry cinerea , Phytophthora capsici , Pythium sp., Rhizoctonia solani MAFF511103) and the like also proved effective.

Antagonist microorganism Trichoderma asperellum ) A disinfectant solution, a hydrating agent , and a granule based on DYF0328 were prepared.

Rhizoctonia , a pathogen of rice paddy disease (leaf blight), was tested in packaging using the formulation. solani ) and Burholderia glumae , the pathogens of bacterial bacterial blight, and Botry cinerea , cucumber and tomato asymptomatic fungi, were superior to the pesticides, the control drug. . Details are described in the Examples section below.

Trichoderma asperellum , the main component of this microbial preparation DYF0328 was also tested on a variety of plants, and no disease caused by DYF0328. The strain, which is the main component of the microbial agent of the present invention, is used in an amount of 1 × ^{10 2} cfu / g to 1 × ^{10 12} cfu / g, and preferably in an amount of 1 × 10 ⁴ cfu / g to 1 × ^{10 9} cfu / g. Below ¹ × 10 ² cfu / g, the desired effect cannot be sufficiently obtained, and when 1 × ^{10 12} cfu / g is exceeded, it is difficult to incubate during preparation of the original agent.

In the present invention, for the formulation, first, a solid raw material at a concentration of 1 × ^{10 6} cfu / g to 1 × 10 ¹⁶ cfu / g or a liquid raw material at a concentration of 1 × ^{10 7} cfu / g to 1 × 10 ¹⁷ cfu / g is prepared, and a solution, a hydrating agent, a granule, etc. Made.

The granules are 1 to 50% by weight of the solid raw material containing the Trichoderma asperellum DYF0328 strain at a concentration of 1 × ^{10 6} cfu / g to 1 × 10 ¹⁶ cfu / g; 30 to 85% by weight of one or more extenders selected from talc, kaolin, bentonite, diatomaceous earth, and zeolites; 5 to 20 wt% surfactant; It contains 3 to 8% by weight of the pressure-sensitive adhesive. If the content of the extender is less than 30%, it is difficult to granulate, and if it is more than 85%, the dispersibility may be worse. In addition, cationic, anionic and neutral surfactants are used as surfactants to aid dispersibility of the granules, and the amount of the surfactant is preferably 5% to 20% by weight, preferably 5% to 10% by weight. If it is less than 5%, the dispersibility is bad, and if it is more than 20%, it can be wasteful. Examples of the pressure-sensitive adhesive of the granules include lactose, and the content thereof is suitably used in an amount of about 3 to 8%, preferably about 5%.

In addition, the liquid formulation is 1 to 50% by weight of a liquid raw material of Trichoderma asperellum DYF0328 strain at a concentration of 1x10 ⁷ cfu / g to 1x10 ¹⁷ cfu / g, 5 to 20% by weight of surfactant and 30 to 85% by weight of water as an extender. Contains% Formulation may be difficult to formulate if the extender content is less than 30% by weight, and dispersibility may worsen above 85%. Cationic, anionic, and neutral surfactants may be used as the surfactant, and the amount thereof is 5 to 20% by weight, preferably 5 to 10% by weight. If it is less than 5%, the dispersibility is bad, and if it is more than 20%, it can be wasteful.

The hydrating agent comprises 10 to 80% by weight of a solid raw material containing the Trichoderma asperellum DYF0328 strain at a concentration of 1 × ^{10 6} cfu / g to 1 × 10 ¹⁶ cfu / g; 15 to 85% by weight of one or more extenders selected from talc, kaolin, bentonite, diatomaceous earth, and zeolites; 3 to 20 wt% surfactant; It contains 1 to 20% by weight of silica powder. If the extender content is less than 15%, it is difficult to commercialize, and if it is more than 85%, the dispersibility may be worse. Cationic, anionic and neutral surfactants are used as the surfactant added to aid dispersibility of the hydrating agent, and the amount of the surfactant added is preferably 3% to 20%, preferably 5% to 10%. If it is less than 5%, the dispersibility is bad, and if it is more than 20%, it can be wasteful. In addition, the silica powder is used to adjust the water content of the hydrating agent, the amount is about 1% to 20%, preferably 5% to 10%.

The liquid formulation and the hydrating agent of the present invention are diluted to 1,000 times as needed, and granulation is not necessary.

The present invention will be described in more detail with reference to the following examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited to these examples.

Preparation Example 1

Trichoderma asperellum 10 ml fermentation ( Trichoderma asperellum ) containing 500 ml (1x10 ⁵ cfu / g) strain of DYF0328 containing the liquid raw material culture medium of the following Table 1 1 kg of DYF0328 liquid raw material (3 × 10 ⁸ cfu / g) was obtained.

Composition of Trichoderma asperrelum DYF0328 liquid raw material culture medium Raw material name Mixing ratio (%) Addition amount per 1kg (g) Remarks (Manufacturer) Amino Acid (Liquid) 1.2 12 Ilshin Chemical peptone 0.006 0.06 DIFCO Mult 0.03 0.3 DIFCO NaOH (saturated solution) 0.012 0.12 Dongyang Chemical glucose 0.6 6 Samyang Genex Spawn culture (1x10 ⁵ cfu / g) 50 500 water 48.152 481.52 system 100 1000

Preparation Example 2

Preparation Example 1, the liquid base agent with the following Table 2, the hot air drying of the solid base agent in the culture medium in 45 ℃ was mixed sex 4 ~ 5 hours, and pulverized to 325 mesh in Trichoderma Ars buffer relrum (Trichoderma asperellum) 1 kg of DYF0328 solid raw material (1 × 10 ⁹ cfu / g) was obtained.

Composition of Trichoderma asperellum DYF0328 solid source culture medium Raw material name Mixing ratio (%) Addition amount per 1kg (g) Remarks (Manufacturer) Diatomaceous earth 34.4 344 Wang Pyo Chemical Talc 9.2 92 Ilshin Material Starch 8.2 82 Samyang Genex glucose 1.9 19 Samyang Genex Amino Acid (Liquid) 0.4 4 Ilshin Chemical Calcium chloride 0.2 2 Dongyang Chemical Magnesium sulfate 0.1 One Gundo EDTA-Fe 0.1 One Dongyang Chemical Liquid Base (3x10 ⁸ cfu / mL) 45.5 455 system 100 1000

Preparation Example 3

Hydration Manufacturer

Using the solid raw material of Preparation Example 2, the raw materials were added and mixed homogeneously as in the unit of the following Table 3, Trichoderma asperellum 1 kg of DYF0328 (5 × 10 ⁸ cfu / g) hydrate was obtained.

Trichoderma asperellum DYF0328 Hydrating Formulation Formulation Raw material name Mixing ratio (%) Addition amount per 1kg (g) Remarks (Manufacturer) Solid ingredients 50 500 Talc 33 330 Ilshin Material Co., Ltd. Silica powder 10 100 21st Century Silica CR-CLS ^* 7 70 Yousung Hwayeon Tech system 100 1000

  CR-CLS: Calcium lignosulfonate

Preparation Example 4

Granulation

After adding kaolin to the solid raw material of Preparation Example 2 and increasing the amount to 100 times, the raw materials were added and mixed homogeneously as in the following raw unit of Table 4, added with water, kneaded, assembled into 24 mesh, dried by hot air at 45 ° C, and then tricoderma Asperelum ( Trichoderma asperellum ) 1 kg of DYF0328 (5 × 10 ⁵ cfu / g) granules was obtained.

Trichoderma asperellum DYF0328 granules manufacturing prescription Raw material name Mixing ratio (%) Addition amount per 1kg (g) Remarks (Manufacturer) Solid ingredient (increase) 5 50 Talc 76 760 Ilshin Material Lactose 10 100 Dongyang Chemical CR-CLS ^* 9 90 Yousung Hwayeon Tech system 100 1000

 * CR-CLS: Calcium Lignosulfonate

Preparation Example 5

Liquid manufacturing

Stopping the growth of bacteria by adjusting the pH to 4.5 using 5M-H ₂ SO ₄ in Preparation Example 1, preventing the generation of contaminating bacteria, and then adding a surfactant and then filtration to filter Trichoderma asperellum ) 1 kg of DYF0328 (3 × 10 ⁷ cfu / ml) liquid was obtained.

Trichoderma asperellum DYF0328 Liquid Formulation Prescription Ingredient Name Mixing ratio (%) Addition amount per 1kg (g) Remarks (Manufacturer) Liquid ingredient 10 100 water 85 850 Tween 20 3 3 Hwawang Kao corporation Span 2 2 Hwawang Kao corporation system 100 1000

Test Example 1

Rice leaf pattern blight control test

The control effect of the hydration agent ( Trichoderma asperellum DYF0328 5 × 10 ⁸ cfu / g) of Preparation Example 3 on rice leaf blight blight disease (blottle disease) was tested. Between July 1, 2005 and August 16, 2005, three sprays (7.1, 7.15, 7.28) of diluent of the above-mentioned hydrating agent were sprayed three times (7.1, 7.15, 7.28) on 135 m2 rice (a la carte rice) in Heunghae-eup, Pohang-si, Gyeongbuk. Is shown in the following Table 6 and FIG.

In the case of Pensiccuron, a registered pesticide registered by the Rural Development Administration, as a control drug, the pencicuron had a control value of 89.8%, but the microbial agent of the present invention had a control value of 92.7% when sprayed at a 250-fold dilution.

Test Example 2

Rice Bacterial Blight Control Test

The control effect of the hydration agent ( Trichoderma asperellum DYF0328 5 × 10 ⁸ cfu / g) of Preparation Example 3 against rice bacterial blight was tested. From August 23, 2005 to September 28, 2005, 20 liters of each of the diluents of the manufacture example 3 were added to 100 ㎡ of rice in Heunghae-eup, Pohang-si, Gyeongbuk, Korea on August 21, August 30, and September 6 ) And the results of the investigation on September 28 are shown in Table 7 below.

In the case of using the guard hydrating agent, a pesticide registered as a RDA, as the control drug, the control hydration was 62%, but the hydrating agent of the present invention exhibited a control value of 71% when sprayed at 250 times dilution, which was superior to the control drug.

Test Example 3

Rice Leaf Blast Control Test

The control effect of the preparation of Example 5 ( Trichoderma asperellum DYF0328 3 × 10 ⁷ cfu / ml) against rice leaf blast was tested. Between June 17, 2005 and July 8, 2005, two sprays (6.17, 6.24) of dilutions of the above solution were sprayed twice (6.17, 6.24) on 135 square meters of abundant rice in Heunghae-eup, Pohang-si, Gyeongbuk, and the results of the survey on July 14 are shown in Table 8 below. Indicated.

The control value of trizol hydrating agent was only 51% when using Trizol hydrating agent, a registered drug of the Rural Development Administration, as compared to the control agent. appear.

Test Example 4

Tomato gray mold control test

Hydration of Preparation Example 3 for tomato gray mold disease ( Trichoderma) asperellum DYF0328 5 × 10 ⁸ cfu / g) was tested for the control effect. From March 18, 2006 to April 3, 2006, Gyeongju, Gyeongbuk, Korea sprayed 10 liters of each diluent of the formulation three times (3.13, 3.20, 3.27) in 60 m2 of tomato house tomato and investigated the effects on April 3 Are shown in the following Table 9 and FIG.

When the dichroic hydration, which is a registered pesticide registered with the Rural Development Administration, is used as a control drug, the dichroic hydration has a control value of 72.8%, but when it is sprayed 250 times, an excellent control effect is recognized as 85.2%.

Test Example 5

Cucumber ash mold control test

Hydration of Preparation Example 3 for cucumber gray mold disease ( Trichoderma) asperellum DYF0328 5 × 10 ⁸ cfu / g) was tested for the control effect. Between March 10, 2006 and March 31, 2006, 10 liters of each diluent of the above formulation was sprayed three times (3.10, 3.17, 3.24) in a plastic house cucumber 60㎡ in Gyeongbuk-gun Uigun-gun, and the effects of March 31 were investigated. Is shown in Table 10 below.

When the dichrohydrate was registered as a control drug, the pesticide value was 68.1%. The formulation of the present invention had a control value of 56% at 250-fold dilution, although it was lower than the control drug. There is control.

Test Example 6

Pepper Disease Control Test

The control effect of the preparation of Preparation Example 4 ( Trichoderma asperellum DYF0328 5 × 10 ⁵ cfu / g) against pepper blight was tested. Between June 8, 2005 and July 23, 2005, 80 pepper pots (2 kg) were treated in the glass greenhouse of the Daegu University affiliated farm with the preparations of the present invention (June 8) for each pretreatment. The results of investigating the effect on the month 23 are shown in the following Table 11 and FIG. 3.

Trichoderma Asperelum DYF0328 of Red Pepper Blight Treatment formulation Byeong stock rate (%) Control price (%) Remarks Tricot Derma Asperelum DYF0328 100 g / 20 ports 28 61.1 5g per pot 50 g / 20 ports 38 47.2 2.5g per port Dimethophore, basic chlorinated hydrochloride (control agent) 1,000 times 23 68 No treatment - 72 -

Survey date: July 23 (approx. 50 days after formal treatment)

Survey Method: Examine the morbidity rate for each iteration

Bite Rate: Lee Byung-Woo / Total Research Week x 100

Control Value (Efficacy): {1- (Treatment of Blessing Rate / Treatment of Blessing Rate)} x 100

When the dimethosomorph basic copper chloride hydrochloride, a pesticide registered by the Rural Development Administration, was used as a control drug, the dimethomorph basic chloride hydrochloride was 68% for the control value, but the present invention had a control value of 61.1% for 5 g per pot. It was rather low. However, in accordance with the Rural Development Administration's "Biopesticide Review Standard", if the control is more than 50% compared to the untreated areas, this is a considerably high control price considering that it meets the registration requirements as a biopesticide.

The present invention is formulated with indigenous microbial plant fungicides can control the environmentally friendly diseases of crops. The present invention provides a non-toxic preparation that can replace pesticides with acute toxicity and crop residual toxicity.

The present invention provides an eco-friendly formulation that is excellent against the important pests of rice stock, excellent effect than conventional pesticides, such as cucumber gray mold disease, cherry tomato gray mold disease, etc., which is like a disease of the disease when growing greenhouse.

Claims (7)

Trichoderma asperellum DYF0328 strain of Accession No. KACC 93038P having control activity against causative bacteria of rice bacterial blight, rice leaf blast, cucumber, tomato gray mold and pepper blight. Phytopathogen against the causative agent of rice bacterial blight, rice leaf blast, cucumber / tomato ash fungus and pepper blight, characterized by containing the Trichoderma asperellum DYF0328 strain according to claim 1 as an active ingredient. Control agent. delete The method of claim 2, wherein the control agent comprises: 10 to 80% by weight of a solid raw material containing the Trichoderma asperellum DYF0328 strain at a concentration of 1 × ^{10 6} cfu / g to 1 × 10 ¹⁶ cfu / g; 15 to 85% by weight of one or more extenders selected from talc, kaolin, bentonite, diatomaceous earth, and zeolites; 3 to 20 wt% surfactant; A control agent of a wetting agent formulation containing 1 to 20% by weight of silica powder. The method of claim 2, wherein the control agent comprises: 1 to 50% by weight of a solid raw material containing a Trichoderma asperellum DYF0328 strain at a concentration of 1 × ^{10 6} cfu / g to 1 × 10 ¹⁶ cfu / g; 30 to 85% by weight of one or more extenders selected from talc, kaolin, bentonite, diatomaceous earth, and zeolites; 5 to 20 wt% surfactant; Control agent of the granule formulation containing 3 to 8% by weight of the adhesive. The method of claim 2, wherein the control agent comprises: 1 to 50% by weight of a liquid raw material containing Trichoderma asperellum DYF0328 strain at a concentration of 1 × ^{10 7} cfu / ml to 1 × 10 ¹⁷ cfu / ml; 30 to 85 wt.% Water as extender; And 5 to 20% by weight of the surfactant. The agent according to claim 5, wherein the pressure-sensitive adhesive is lactose.

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