WO2016111480A1

WO2016111480A1 - Entomopathogenic beauveria bassiana, and agricultural pest control liquid preparation using same

Info

Publication number: WO2016111480A1
Application number: PCT/KR2015/013883
Authority: WO
Inventors: 김재수; 이세진; 유정선; 김시현
Original assignee: 전북대학교 산학협력단
Priority date: 2015-01-06
Filing date: 2015-12-17
Publication date: 2016-07-14
Also published as: KR20160084968A; KR101666968B1

Abstract

The present invention relates to strain JFE007 of Beauveria bassiana, which is an entomopathogenic fungus, and a liquid preparation for controlling agricultural pests, especially garden and orchard pests, by using the same. The agricultural pest control preparation according to the present invention is prepared using strain JFE007 of Beauveria bassiana, which is an entomopathogenic fungus, thereby facilitating handling of the preparation, being harmless to the human body, and controlling agricultural pests in an environmentally friendly manner. Additionally, the liquid preparation of the strain is simply and economically prepared using a filter bag.

Description

Insect Pathogenic Boberia Bassiana and Liquid Preparation for Control of Agricultural Pests Using the Same

The present invention relates to an insect pathogenic fungus Boberia Bassiana JEF007 strain and a liquid formulation for controlling agricultural pests, in particular horticultural or fruit pests using the same.

It is widely distributed in Korea, Japan, Taiwan, and other plants, such as sawtooth ants, yellow bugs, yellow locust, beetle and rice weevil, horticultural crops such as beans, soybeans, peas, and watermelons. It is a major agricultural pest that causes enormous losses in the yield of crops, which directly absorbs rice and crops such as rice, blood, and crude, and directly causes physical damage. In order to control pests damaging such crops, the use of chemical pesticides has increased and the spreading area has been widened with the development of agricultural technology and the enlargement of the crop size. The emergence of organic synthetic insecticides after World War II marked a major turning point in two aspects: increased crops by agricultural pest control and improved human health by sanitary pest control. Insecticides used for pest control in Korea are increasing every year, of which organophosphorus and carbamate account for about 90% of total consumption.

However, decades of abuse and abuse of these existing organic synthetic pesticides have caused a number of side effects, such as the emergence of resistant pests, toxic toxicity, disruption of biological control by natural enemies, residual poisoning and environmental pollution. With increasing interest in the adverse effects and environmental impacts of the use of synthetic insecticides is a trend that is increasingly limited. Therefore, biological control is actively researched as part of the development of next-generation pesticides, which can replace synthetic insecticides, but biological control is a barrier to commercialization because it is not easy to handle using microorganisms, natural enemies or natural extracts. In addition, studies on biological control using insect pathogenic fungi have been actively conducted, but studies on mass production and formulation, which are an important part of commercialization, are insufficient. Currently, preparations using entomopathogenic fungi are used as a method of separating the spores produced through solid culture from the culture and then spraying them in the form of a liquid formulation and a hydrating agent. There is a necessary problem, which is an obstacle to commercialization.

Therefore, there is an urgent need for the development of a new agricultural pest control formulation that is harmless to the human body, environmentally friendly, easy to handle, and easy to manufacture and commercially available.

The inventors of the present invention, while searching for agricultural pest control agents, newly isolated the Beauveria bassiana JEF007 strain, and confirmed that the strain has insect pathogenicity and excellent spore forming ability, when used for agricultural pest control It has been confirmed that it has an excellent control effect against agricultural pests, and that the liquid formulation of the strain can be produced simply and economically by using a filter bag, and completed the present invention.

Accordingly, the present invention is to provide a B. bassiana JEF007 strain, insect spores, microorganisms and liquid formulations of insect pathogenic fungi for controlling agricultural pests.

In order to achieve the above object,

The present invention provides Boveria bassiana JEF007 strain (KCCM11592P) having insect pathogenicity.

In addition, the present invention provides a microbial agent for controlling agricultural pests, including Boveria bassiana JEF007 strain (KCCM11592P) or its spores.

In addition, the present invention comprises the steps of culturing the Boberia Bassiana JEF007 strain (KCCM11592P) in a solid culture medium; It provides a method for producing a microbial preparation for controlling agricultural pests; and harvesting the culture.

In addition, the present invention comprises the steps of culturing the Beauveria bassiana ( Beauveria bassiana ) JEF007 strain (KCCM11592P) in a solid culture medium; Encapsulating the culture in a filterable bag; And impregnating the filter bag with water to elute the Boveria bassiana JEF007 strain or its spores, and provides a method for preparing a liquid pest control agent for agricultural pest control.

In another aspect, the present invention provides a liquid formulation for controlling agricultural pests prepared by the production method.

Agricultural pest control agent according to the present invention is manufactured using the insect pathogenic fungus Beauveria bassiana JEF007 strain, it is easy to handle, harmless to humans and environmentally friendly to control agricultural pests, and also filter bag By using the liquid formulation of the strain there is an effect that can be produced simply and economically.

FIG. 1 is a diagram showing an image observed with an optical microscope (400-fold) of Boberia Bassiana JEF007 strains cultured in Sabraud dextrose agar (SDA) medium for 25 days in a dark at 25 ℃.

Figure 2 (a) -2 (i) is a diagram showing the DNA sequencing (DNA sequencing) results of the Boberia Bassiana JEF007 strain.

Figure 3 is a view showing a microbial preparation of a plastic container for solid culture and strains of Boberia Bassiana JEF007, respectively.

Figure 4 is a view showing a method of preparing a diluted spray liquid formulation using the Boberia Bassiana JEF007 strain.

Figure 5 is a diagram showing the control effect on the sawtooth ant hindlin rinse of the diluted spray type liquid preparation using the Boberia Bassiana JEF007 strain.

Hereinafter, the present invention will be described in detail.

In addition, the present invention provides a composition for controlling pests of agricultural plants, including Beauveria bassiana JEF007 strain (KCCM11592P) or its spores.

" Beauveria bassiana JEF007 strain" of the present invention, the present inventors show an excellent insecticidal effect against agricultural pests, such as sawtooth ant, yellow bugs, flower yellow whiskers, etc. among the fungi isolated from natural soil After isolation of the Boberia Bassiana strain through bioassay experiments, the isolated strain was identified morphologically, and the genomic DNA sequence was analyzed to show about 98% homology with the known Boberia Bassiana strain. The strain was identified as Boberia Bassiana strain, named JEF007, and was deposited on the Korea Microbiological Conservation Center of Korea Spawn Association on October 28, 2014 (Accession No. KCCM11592P).

The Boberia Bassiana JEF007 strain forms a white colony and has morphological characteristics of forming spores (see FIG. 1). The JEF007 strain has particularly excellent sporulation ability, and the spores of the JEF007 strain have better spore storage stability than the known strains, Boberia bassiana or GHA.

The insect pathogenicity (insect pathogenicity) means the ability to cause the disease by infecting the pathogen to the target insect (host insect), in the present invention, the strains of the Boberia Bassia or JEF007 is infected with agricultural pests to the pest It means to cause illness.

The insect pathogenicity may be pesticidal activity. Specifically, when the germinated spores of the Boberia Bassiana JEF007 strain or the above strains are attached to the outer shell of the host insect, chitinase, protease, lipase or esterase, etc. are produced. It penetrates through the cuticle layer of the insect, and proliferates in the body of the insect while producing a small molecular weight of toxic proteins such as toxic substances or glycoproteins to block the insect's immune action and have insecticidal activity to kill host insects.

The Boberia Bassiana JEF007 strain has been identified and separated to have a significantly superior pesticidal activity against agricultural pests with excellent spore forming ability, it can be usefully used for the control of agricultural pests.

The insect may be a pest, and the pest includes both hygienic pests, forest pests and agricultural pests.

The insect may preferably be an agricultural pest, and the agricultural pest is meant to include all horticultural pests, fruit pests and rice pests. The agricultural pests are preferably sawtooth ants, loin bugs, flowers yellow locust beetle, Chinese cabbage insects, greenhouse powder, tobacco powder, pear moth, peach pure moth, apple pattern leaf moth, apple mother's eye pattern leaf moth, larvae, rice plant, It may be a white light bulb, rice weevil, rice oyster flies, rice leaf oyster flies, and the like, and more preferably, may be a sawtooth ant hind henling or flower yellow whiskers.

Spores of the strain Boberia Bassiana JEF007 may be produced by culturing the inoculation bacteria of the strains Boberia Bassiana JEF007 in a solid culture medium. Boberia Bassiana JEF007 strain of the present invention can exhibit a two to three times the spore productivity compared to the conventional Boberia Bassiana GHA strain.

The microbial preparation for controlling agricultural pests may be provided as granular preparation, liquid preparation or compost. The microbial preparation for controlling agricultural pests shows an excellent insecticidal effect when sprayed on agricultural pests, in particular, compared to the case of spraying the microbial preparations of the known strains, Boberia basia or GHA strains for sawtooth ants In the short term, it showed better insecticidal effect.

The microbial agent may preferably be a granular agent, and if the agent is a granular agent, the microorganism agent may be easily handled, and the granular agent may be easily manufactured by using excellent spore forming ability of Boberia basia or JEF007 strain, and thus agricultural pests. The production efficiency of a control agent can be improved.

In addition, the present invention comprises the steps of culturing the Boberia Bassiana JEF007 strain in a solid culture medium; And it provides a method for producing a microbial preparation for controlling agricultural pests comprising the step of harvesting the culture.

The solid culture medium preferably contains any one selected from a bath, millet, white rice, bran, sorghum and combinations thereof, and more preferably includes a bath.

The method for preparing a microbial preparation for controlling agricultural pests may further include preparing a solid culture medium by mixing any one selected from organic acids, crude, millet, white rice, bran, sorghum and combinations thereof.

The organic acid may be used without particular limitation as long as it can be used as a carbon source. More specifically, it is preferable that it is any one selected from citric acid, propionic acid, butyric acid, lactic acid, succinic acid, gluconic acid, valeric acid, caproic acid, and combinations thereof, and more preferably citric acid.

Mixing of the organic acid and the solid culture medium may be performed by adding an organic acid to the solid culture medium and mixing.

The culture vessel of the strain may use a culture vessel commonly used in the art to which the present invention belongs, but is not limited thereto. For example, the oxygen supply filter may be attached to a plastic container or a polyvinyl bag for solid culture and used for culturing.

Cultivation of the Boberia Bassiana JEF007 strain is preferably carried out at 20 to 30 ℃ for 4 to 8 days, more preferably at 25 ℃ for 6 days. In the present invention, the solid culture medium mixed with the organic acid was heated and cooled at 110 to 130 ° C. for 20 to 40 minutes for sterilization, and then cultured by inoculating the medium with Boberia bassia or JEF007 strain.

The inoculating bacteria to be used in the solid culture medium can be obtained by subculture of Boberia bassiana JEF007 strain, the culture of the JEF007 strain can be carried out according to the conventional subculture method. The medium required for the passage is not limited to the type thereof.

As an example of the passage medium, a liquid medium including PDB (Potato Dextrose Broth) medium, rice bran or bran may be used, but is not limited thereto. In addition, the subculture medium may include corn flour, rice bran or corn stiff powder.

In addition, the present invention comprises the steps of culturing the Beauveria bassiana (Fauveria bassiana ) JEF007 strain in a solid culture medium; Encapsulating the culture in a filterable bag; And impregnating the filter bag with water to elute the Boveria bassiana JEF007 strain or its spores, and provides a method for preparing a liquid pest control agent for agricultural pest control.

The solid culture medium preferably contains any one selected from a bath, millet, white rice, bran, sorghum and combinations thereof, and more preferably includes a bath. The culture may be used as it is encapsulated in a bag, but preferably used by grinding. Since increasing the surface area of the culture through the grinding process is to extend the surface in contact with the water, it is easy to elute the strain and its spores from the filter bag.

The filterable bag is a bag made of a water-permeable filterable material. The filter bag is a woven or nonwoven fabric made of a single or a composite fiber of synthetic fibers of polyester, nylon, polyethylene, polypropylene and vinylon, semi-synthetic fibers such as rayon, natural fibers of mulberry and cedar, and manila hemp, It is preferably formed of at least one selected from the group consisting of wood pulp, kneaded paper made of polypropylene fibers, and paper.

When the Boberia Basia or JEF007 strain, its spores, microbial preparations or liquid formulations are sprayed on agricultural pests, compared to the case of spraying the existing Boberia Basia or GHA strains, excellent control rate against the splinters It has the effect of indicating.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.

실시예 1. 균주의 분리 및 동정Example 1. Isolation and Identification of Strains

1-1. 균주의 분리1-1. Isolation of Strains

Beauveria bassiana JEF007 strain is a fungus isolated from natural soils.Biobaria bassiana strains show excellent insecticidal effects against horticultural and fruit pests, such as sawtooth ant bugs. Separated.

1-2. 생육 및 형태학적 동정1-2. Growth and Morphological Identification

An optical microscope (400-fold) image of Boberia Bassiana JEF007 strain incubated in Sabraud Dextrose Agar (SDA) for 7 days in a dark room at 25 ° C. is shown in FIG. 1.

As shown in FIG. 1, the formation of white fungi, spores, and spores, typical of Boberian strains, was well observed.

1-3. 염기서열 분석1-3. Sequencing

After PCR (polymerase chain reaction) amplification by ITS1 forward primer and ITS4 reverse primer, DNA sequencing of the Boberia bassiana JEF007 strain is shown in FIGS. 2 (a) -2 (i).

실시예 2. 농업 해충 방제용 미생물 제제의 제조Example 2 Preparation of Microbial Formulation for Agricultural Pest Control

200 g of Italian millet was placed in a plastic container for solid culture (1 L), followed by additional 0.32 ml of 50% citric acid. Then it was heated at 121 ° C. for 30 minutes for sterilization. The microbial preparation of the JEF007 strain was prepared by cooling the sterilized bath to room temperature, inoculating 1 ml of Boberia bassiana JEF007 strain liquid cultured in PDB medium at room temperature for 3 days in a tank, shaking and incubating for 6 days at 25 ° C. .

The microorganism preparation of the plastic container for the solid culture and the strain of Boberia bassiana JEF007 are shown in Figure 3, respectively.

실시예 3. 포자 생성능 확인Example 3. Confirmation of spore generation ability

After diluting 1 g of the microbial preparation of JEF007 strain with 10 ml of 0.3% silwet, spore productivity was measured by counting spores using a hemocytometer. Spore counts of Boberia bassiana JEF007 strain are shown in Table 1.

Table 1

Strain name	Spore count
B. bassiana JEF007	2.11 x 10 ⁸ conidia / g
B. bassiana GHA	8.23 x 10 ⁷ conidia / g

As shown in Table 1, the Boberia bassiana JEF007 strain showed more than twice the spore productivity compared to the conventional commercialized Boberia Bassiana GHA strain.

실시예 4. 희석살포형 액상제제의 제조Example 4 Preparation of Dilute Spray Type Liquid Formulation

10 g of the microbial preparation of JEF007 strain was placed in a filter bag (8 × 12 cm ² ) and sealed. Thereafter, the sealed filter bag was impregnated with 20 L of water, and then shaken for several seconds to release a strain or spores thereof in the filter bag into water to prepare a dilute spray type liquid formulation.

4 shows a method of preparing a diluted spray type liquid preparation using the Boberia bassiana JEF007 strain.

실험예 1. 희석살포형 액상제제의 톱다리개미허리노린재에 대한 방제효과 확인Experimental Example 1. Confirmation of the control effect on the sawtooth antler norin material of the dilute spray type liquid preparation

Diluted spray liquid preparation prepared according to Example 4 was sprayed using a sprayer to 30 sawtooth ant hindnorrin material 3 nymphs generated from Gangnam beans. After 3, 7 and 10 days after the sparging, the live insects were examined, respectively. The above process was repeated three times, and the control effect of the sawtooth ant loin erin was shown in FIG. 5. In addition, B. bassiana GHA strain was prepared as a control treatment in the same manner as JEF007 formulation, and then treated with a spore suspension.

As shown in FIG. 5, the diluted spray formulation using the Boberia bassiana JEF007 strain showed a superior control effect on the sawtooth ant hindlining material than the diluted spray formulation using the existing microorganism Boberia bassiana GHA strain. As a result of examining the mortality rate 7 days after the suspension suspension treatment, the GHA strain treated group showed a low mortality rate of 20%, but the JEF007 treated group showed a higher mortality rate of 65% or more. In addition, the 10-day survey showed a similar trend.

(Accession number)

Depositary Name: Korea Microorganism Conservation Center

Accession number: KCCM11592P

Trust Date: 20141028

Claims

Beauveria bassiana JEF007 strain (KCCM11592P) with insect pathogenicity.
The method of claim 1,

The insects are sawtooth ants, yellow bugs, flowers yellow locust beetle, Chinese cabbage beetle, greenhouse powder, tobacco powder, pear moth, peach pure moth, apple pattern leaf moth, apple mother-of-law pattern leaf moth, larvae, rice plant, white light bulb, rice plants Beauveria bassiana JEF007 strain (KCCM11592P), which is one of the pests selected from weevil, rice bran fly, rice leaf oyster fly and combinations thereof.
Beauveria bassiana ( Beauveria bassiana ) JEF007 strain (KCCM11592P) or microorganisms for controlling pests, including spores thereof.
Culturing the Boberia bassiana JEF007 strain (KCCM11592P) in a solid culture medium; And

Harvesting the culture; manufacturing method of a microbial preparation for agricultural pest control comprising a.
The method of claim 4, wherein

The solid culture medium comprises any one selected from crude, millet, white rice, bran, sorghum and combinations thereof and organic acids, a method for producing a microbial preparation for controlling pests of agriculture.
The method of claim 4, wherein

The culturing is to be carried out for 4 to 8 days at 20 ~ 30 ℃, a method for producing a microbial preparation for agricultural pest control.
Culturing Beauveria bassiana JEF007 strain (KCCM11592P) in a solid culture medium;

Encapsulating the culture in a filterable bag; And

Impregnating the filter bag with water to elute Boveria bassiana (Fauveria bassiana ) JEF007 strain or its spores, a method for producing a liquid pest control agricultural pest control.
The method of claim 7, wherein

The filter bag is a woven or nonwoven fabric made of a single or a composite fiber of synthetic fibers of polyester, nylon, polyethylene, polypropylene and vinylon, semi-synthetic fibers such as rayon, natural fibers of mulberry and cedar, and manila hemp, A method for producing a liquid pest control agent for controlling agricultural pests, which is formed by any one selected from the group consisting of wood pulp, polypropylene fibers, and paper.
A liquid formulation for controlling agricultural pests prepared by the manufacturing method of claim 7.