KR102251508B1 - Novel Beauveria bassiana KNU-101 Strain with Improved Insecticidal Effect and Spore Production and Uses thereof - Google Patents

Abstract

The present invention relates to a strain of Beauveria bassiana KNU-101, and exhibits excellent insecticidal effect in controlling thrips, a soil pest that harms crops with insect pathogenicity, and excellent spore production ability, so environmentally friendly pest control It can be usefully used as a means.

Description

[Novel Beauveria bassiana KNU-101 Strain with Improved Insecticidal Effect and Spore Production and Uses thereof]

The present invention is a novel Boberia bassiana (Beauveria bassiana) KNU-101 strain (accession number KCTC 13660BP) having excellent insecticidal effect and sporulation ability, a mass production method of the strain, an insecticidal microorganism preparation using the strain, and the microorganism It relates to an insecticidal method and the like using a formulation.

Insects currently account for the largest number of known creatures on the planet, and are known to have excellent adaptability to the environment. Because some of these insects cause a lot of damage to various agricultural crops that are commercially important, chemical pesticides have been widely used in the past to control these pests. However, chemical pesticides also eradicated non-purpose microorganisms such as beneficial insects and parasites in pests due to the non-specific spectrum of insecticidal capacity, and the frequent use of chemical pesticides promotes the acquisition of resistance of the target pests repeatedly exposed to chemical pesticides. In addition, chemical pesticide components have disadvantages that are harmful to the human body.

In order to reduce the use of chemical pesticides, a method using entopathogenic fungi as an eco-friendly pest control agent has been used. These insect pathogenic fungi have no harm to humans, including plants, but have specific insecticidal powers only for host insects, so they have the advantage of being able to effectively control target pests.

On the other hand, Beauveria bassiana is known as a representative insect pathogenic fungi, and it is known that spores dehydrate the insect by dehydrating the insect's wax layer, leading to death, and to continue the next generation by using their corpses as nutrients. For this reason, it is known that both oral and contact toxicity to insects are effective and can infect a variety of insect hosts. For the mass cultivation of spores of fungi for controlling such pests, solid cultures that mainly produce conidia are used.

However, unlike bacteria, the production of spores for fungi is not sufficient despite a long cultivation period of at least 7 days, so the commercialization of fungi for controlling pests is delayed. It is known to be at a low level to be used as. Therefore, for industrialization, it becomes a priority to select fungi that exhibit excellent insecticidal power and have both requirements capable of producing large amounts of spores.

On the other hand, Thrips are representative soil pests that lay eggs on soft tissues such as young leaves, stems, and petals, and begin to appear as the temperature rises, harming the surface of stems and leaves, and inflicting flower buds during flowering. It seriously degrades the quality of the crop. Damaged crops show lesions in which the leaves are not straightened and deformed or leaf veins contract. Thrips have a wide host range such as cucumber, lettuce, tomato, eggplant, and chicory, and the generation period is very short, ranging from a minimum of 12 days to a maximum of 26 days, depending on temperature conditions, and it is difficult to control because it has strong fertility.

Research on various substances is being conducted for the above-described pest control, but studies on biopesticides compared to chemical pesticides are still insufficient.

Korean Patent Registration Publication 10-1444214

The inventors of the present invention were searching for insect pathogenic microorganisms for controlling pests that are commercially important and harmful to various agricultural crops, while confirming the thrips killing effect and remarkable sporulation ability of the Beauveria bassiana KNU-101 strain. The present invention was completed by preparing a microorganism preparation for controlling pests including the strain.

Accordingly, one object of the present invention is to provide a Boberia bassian a (Beauveria bassian a) KNU-101 strain (accession number KCTC 13660BP) having an insecticidal effect.

Another object of the present invention is to provide a method for mass production of a strain comprising the step of culturing the Beauveria bassiana KNU-101 strain (accession number KCTC 13660BP).

Another object of the present invention is one selected from the group consisting of a Boberia bassiana KNU-101 strain (accession number KCTC 13660BP), a culture of the strain, a concentrate of the culture, and a dried product of the culture It is to provide an insecticidal microbial preparation comprising the above as an active ingredient.

Another object of the present invention is to provide an insecticidal method comprising the step of treating the microbial preparation to soil or plants.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems that are not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, one aspect of the present invention provides a Boberia bassian (Beauveria bassian a) KNU-101 strain (accession number KCTC 13660BP) having an insecticidal effect.

According to an embodiment of the present invention, the strain may include an ITS (Internal Transcribed Spacer Region) nucleotide sequence represented by SEQ ID NO: 1.

Another aspect of the present invention provides a method for mass production of a strain comprising the step of culturing the Beauveria bassiana KNU-101 strain (accession number KCTC 13660BP).

According to one embodiment of the present invention, the cultivation may be performed at 28 ℃ to 30 ℃.

Another aspect of the present invention is one selected from the group consisting of a Boberia bassiana KNU-101 strain (accession number KCTC 13660BP), a culture of the strain, a concentrate of the culture, and a dried product of the culture It provides an insecticidal microbial preparation comprising the above as an active ingredient.

According to an embodiment of the present invention, the microbial preparation may have insecticidal activity against thrips.

Another aspect of the present invention provides an insecticidal method comprising the step of treating the microbial agent to soil or plants.

The Boberia bassiana KNU-101 strain of the present invention has insect pathogenicity and exhibits excellent insecticidal effect on thrips, which is a soil pest that harms crops, and has excellent spore production, so it is useful as an eco-friendly pest control means. Can be used.

1 is a view showing a morphological feature (A) and a photograph (B) observed with a microscope after pure separation of the Boberia bassiana KNU-101 strain of the present invention.
2 is a diagram showing a schematic phylogenetic diagram using a gene encoding an Internal transcribed spacer (ITS) region for the Beauveria bassiana KNU-101 strain of the present invention.
3 is a diagram showing thrips killed by the Boberia bassiana KNU-101 strain of the present invention, and the arrows represent thrips killed by the Boberia bassiana KNU-101 strain.
Figure 4 is a graph comparing the insect pathogenicity of the Boberia bassiana KNU-101 strain of the present invention with other strains against thrips.
Figure 5 is a graph showing the amount of spore production after 7 days of culture of the Boberia bassiana KNU-101 fungi of the present invention.
6 is a graph showing the control against thrips in a field experiment using pepper of the Boberia bassiana KNU-101 fungus of the present invention.

The present inventors were searching for insect pathogenic microorganisms for the control of pests that are commercially important and harmful to various agricultural crops, by confirming the thrips killing effect and excellent spore-generating ability of the Beauveria bassiana KNU-101 strain. The present invention was completed by preparing a microorganism preparation for controlling pests including the strain.

In an embodiment of the present invention, it was confirmed that the Boberia bassiana KNU-101 strain was treated with a soil pest of thrips, showing 100% mortality and excellent insect pathogenicity to thrips (see Example 2). .

In another embodiment of the present invention, it was confirmed that Boberia bassiana KNU-101 has excellent spore production capacity from a minimum of 100 times to a maximum of 10,000 times compared to other pest pathogenic fungi (see Example 3).

In one embodiment of the present invention, it was confirmed that Boberia bassiana KNU-101 has about 30% better control than other pest pathogenic fungi (see Example 4).

Hereinafter, the present invention will be described in detail.

One aspect of the present invention provides a Boberia bassiana (Beauveria bassian a) KNU-101 strain (accession number KCTC 13660BP) having an insecticidal effect.

According to an embodiment of the present invention, the strain may include an ITS (Internal Transcribed Spacer Region) nucleotide sequence represented by SEQ ID NO: 1.

In the present invention, strains having an insecticidal effect against thrips are selected from thrips, crops and soils inhabited by thrips, and insects killed by infection with insect pathogenic fungi from the crops and soils inhabited by entomopathogenic fungi. The soil was collected to isolate a strain having an insecticidal effect on thrips, and a strain exhibiting excellent insecticidal effect from thrips was selected (see FIG. 1), and the strain was referred to as'Beauveria bassiana KNU-101'. (See Fig. 2), and as of October 16, 2018, it was deposited with the Korea Research Institute of Bioscience and Biotechnology Microbial Resource Center (KCTC) (accession number: KCTC 13660BP).

The Boberia bassiana KNU-101 strain of the present invention can have optimal growth conditions at 30°C and pH 7.0, is an aerobic bacteria, can be freeze-dried and preserved, and contains 0.02% polysorbate 80. After mixing the 40% glycerol aqueous solution and the fungal culture solution 1 to 1, it can be stored by a conventional method such as freezing at -80 °C.

Another aspect of the present invention provides a method for mass production of a strain comprising the step of culturing the Beauveria bassiana KNU-101 strain (accession number KCTC 13660BP).

The basic medium mixed with water and bran, etc. for mass-producing the Boberia bassiana KNU-101 strain of the present invention in an economical way is one or two or more selected from the group consisting of glycerin, glucose, yeast extract, phosphate, etc. May be added, but is not limited thereto. When cultivated in a solid medium, the greatest impact on the growth of fungi is ventilation, temperature, and moisture content. When the temperature is 30℃, the better the contact with oxygen, the better the number of viable cells produced and the Production is also improving. The yeast extract added to the medium is used to provide trace elements and nitrogen sources, and to maintain the insecticidal power of the activated strain, glycerin is used as an adjuvant to improve the physical properties and water content of the medium, and phosphate is used as a pH buffer. .

According to one embodiment of the present invention, the cultivation may be performed at 28 ℃ to 30 ℃.

Another aspect of the present invention is one selected from the group consisting of a Boberia bassiana KNU-101 strain (accession number KCTC 13660BP), a culture of the strain, a concentrate of the culture, and a dried product of the culture It provides an insecticidal microbial preparation comprising the above as an active ingredient.

The microbial preparation is Boberia bassiana (Beauveria bassiana) KNU-101 strain (accession number KCTC 13660BP), a culture of the strain, a concentrate of the culture, or a dried product of the culture, such as surfactants, weight agents, nutrients It can be prepared by adding an additive. At this time, as surfactants, polycarboxylate, sodium lignosulfonate, calcium lignosulfonate, sodium dialkyl sulfosuccinate, sodium alkyl aryl sulfonate, polyoxyethylene alkyl phenyl ether, sodium tripolyphosphate, polyoxyethylene The group consisting of alkyl arylphosphoric esters, polyoxyethylene alkyl aryl ethers, polyoxyethylene alkyl aryl polymers, polyoxyalkylone alkyl phenyl ethers, polyoxyethylene nonyl phenyl ethers, sodium sulfonate naphthalene formaldehyde, Triton 100 and Tween 80 At least one selected from the group consisting of soybean flour, rice, wheat, loess, diatomaceous earth, dextrin, glucose and starch is used as an extender and nutrient, and as a disintegrant, bentonite, At least one selected from the group consisting of talc, dialite, kaolin and calcium carbonate may be used.

The insecticidal microbial preparation may be prepared in the form of a granule, a hydrating agent, or an encapsulated formulation for the purpose of stable formulation of the strain, but is not limited thereto, and in the case of the granule, a surface active agent, an inert carrier, a preservative, a wetting agent, a supply accelerator, Attracting agents, encapsulating agents, binders, emulsifiers, dyes, UV protective agents, buffers and flow agents, low-dispersing powders, fine powders, can be prepared by further selecting one or more selected from the group consisting of a sleep-developer.

According to an embodiment of the present invention, the microbial preparation may have insecticidal activity against thrips.

Boberia bassiana (Beauveria bassiana) KNU-101 strain (accession number KCTC 13660BP) of the present invention exhibits about 30% or more superior thrips insect control rate compared to the cognate Boberia bassiana strain, for the control of thrips It can be usefully used.

Another aspect of the present invention provides an insecticidal method comprising the step of treating the microbial agent to soil or plants.

The insecticidal method of the present invention is one selected from the group consisting of a Boberia bassiana KNU-101 strain (accession number KCTC 13660BP), a culture of the strain, a concentrate of the culture, and a dried product of the culture. An insecticidal microbial preparation comprising the above as an active ingredient may be made by a conventional method of treating the microbial preparation on soil or plants in the art.

Hereinafter, a preferred embodiment is presented to aid in understanding of the present invention. However, the following examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1. Isolation and identification of Boberia bassiana KNU-101 strain

In the present invention, in order to isolate fungi having insecticidal power against soil pests, a method of collecting insects infected by pathogenic fungi outdoors and a method of separating them from crops and soil samples inhabited by soil pests were performed in parallel.

Specifically, the infected and dead thrips were weighed and diluted 10 times sequentially in 0.85% saline solution to take 0.1 ml of the suspension, and saburod dextrose agar medium (SDA medium) as shown in Table 1 below. ; Sabouraud Dextrose Agar media). The components of the medium indicated in the following examples are for separating the Boberia bassiana of the present invention, and there is no limitation on the addition or subtraction of the components presented below.

ingredient amount(%) Glucose 4.0 Yeast extract 1.0 Peptone 1.0 Polysorbate 80, tween 80 0.01 Ampicillin (100 mg/mL) 0.01 Kanamycin (100 mg/mL) 0.01 Agar powder 1.5 pH 6.9 ± 0.2

In addition, 1.0 g of samples collected to separate fungi from soil, crops, feces, and feed samples were sequentially diluted in 9 ml 0.85% saline solution, 0.1 ml for each dilution factor, and spread on a medium having the composition shown in Table 1. , After incubation at 30° C. for 5 days, only fungi morphologically similar to the commonly known Boberia bassiana were selectively isolated, inoculated into a new SDA medium, and cultured under the same conditions.

As a result, 13 insect pathogenic fungi having insecticidal power against thrips were selected, and the fungi were identified as Beauveria bassiana. The present inventors named the fungus that appeared to have the best insecticidal power among the insect pathogenic isolates isolated above as'Boberia bassiana KNU-101', and as of October 16, 2018, it was reported to the Microbial Resource Center of Korea Research Institute of Bioscience and Biotechnology. Deposited (accession number; KCTC 13660BP).

After pure separation of the Boberia bassiana KNU-101, the morphological characteristics of FIG. 1A and the photograph observed under a microscope are shown in FIG. 1B, and the sequence analysis result of the Internal transcribed spacer (ITS) region in FIG. 2 The schematic diagram of the phylogenetic system created from is shown. The phylogenetic schematic diagram of the strain was confirmed by the maximum likelihood method using Molecular Evolutionary Genetics Analysis 7.0 version.

Example 2. Verification of pathogenicity to soil pests of Boberia bassiana KNU-101 strain

Cucumber thrips (Parthenothrips dracaenae ) were used as test insects among thrips, which are pests that are a problem in the cultivation of cucumbers, tomatoes and lettuce, and kidney beans were used as host crops for adult insects. While doing, the experiment was conducted within 2-4 generations.

In order to verify the insecticidal power of the fungus Boberia bassiana isolated in the present invention against thrips insects, the fungus was inoculated with about 10 ⁴ spores in the saburod dextrose liquid medium and spot-cultivated about 10 ⁵ spores. The number was incubated for 5 days in a solid medium made of bran. As a result of observation with a hemocytometer after incubation, about 3.1×10 ⁷ spores were obtained per 1 g. ^{The obtained spores were treated with a number of about 10 5} spores on thrips obtained from farms, and observed for 7 days. The number of thrips in the untreated group and each treatment group was set to about 30, and Aspergillus flavus ATCC 9643 was used as a negative control, and Beauveria bassiana ATCC 74040 was used as a positive control.

As a result, as shown in Figures 3 and 4, Boberia bassiana KNU-101 strain showed 100% mortality to thrips, and it was confirmed that the insect pathogenicity to thrips was superior to other isolated fungi and positive controls. .

Example 3. Comparison and verification of spore production of Boberia bassiana KNU-101

In order to mass-produce the Boberia bassiana KNU-101 fungi of the present invention in an economical manner, a basic medium was prepared in which water and bran were mixed.

The medium may be additionally added one or two or more selected from the group consisting of glycerin, white chinensis, yeast extract, phosphate, and the like. Among the additives of the medium, yeast extract provides trace elements and nitrogen source, glycerin is used as an adjuvant to improve the physical properties and water content of the medium, and white napkin is used to maintain the insecticidal power of the activated strain, and phosphate is used as a pH buffer. Is used.

As test strains, Aspergillus falvus ATCC 9643 and Beauveria bassiana ATCC 74040 were used. Boberia bassiana (Beauveria bassiana) KNU-101 and the strains isolated in the present invention were ^{inoculated in a solid medium at a spore concentration of about 10 4} spore/ml per 1 g, and cultured for 7 days, and the number of spores was confirmed. The experimental group of each strain was set to three.

As a result, as shown in Figure 5, as a result of culturing at 28 to 30 °C for 7 days with a hemocytometer and observed, about 1.8 × 10 per 1 g of the culture medium of the present invention Boberia bassiana (Beauveria bassiana) KNU-101 ¹¹ spores/g spores were observed, and when compared with the specimen strain Boberia bassiana ATCC 74040, it was confirmed that the spore-producing ability was predominantly at least 100 times and at most 10,000 times.

Example 4. Demonstration of control effect of Boberia bassiana KNU-101

An empirical experiment was conducted to verify the control of soil pests in the actual crop of Boberia bassiana KNU-101 isolated in the present invention.

The test crop was carried out for a total of 27 days using red pepper as a test crop, and Frankliniella occidentalis as a test worms, and three repeated tests were performed by a completely random batch method in a ^{total area of 45 m 2.}

Boberia bassiana KNU-101 of the present invention was treated as shown in Table 2 below, and Boberia bassiana ATCC 74040 was used as a positive control. In the field experiment, a potted plant with red pepper seedlings was placed in the test area blocked from the outside, and 300 flower thrips reared indoors were administered and inoculated. Control was confirmed by examining the density ratio of the flower yellow thrips in the field 27 days after the start of the test.

Test drug Main ingredient content (%) Drug efficacy test Dilution multiple and usage When and how to process ATCC 74040 granular formulation 50% 300g/10a Soil drench treatment after planting KNU-101 granular formulation 50% 300g/10a Soil drench treatment after planting No treatment - - -

As a result, as shown in FIG. 6, it was confirmed that the KNU-101 formulation of the present invention showed about 80% of control, and even in actual packaging, it had a higher insecticidal ability compared to the published strain.

The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that other specific forms can be easily modified without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative and non-limiting in all respects.

Korea Research Institute of Bioscience and Biotechnology KCTC13660BP 20181016

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Claims (7)

As a Cucumber thrips or flower yellow thrips insecticidal effect, Boberia bassiana (Beauveria bassian a) KNU-101 strain (accession number KCTC 13660BP);
The strain is a strain that grows up ^{to 10 11} spore/g of spores when cultured at 28 to 30° C. for 7 days.
The method of claim 1,
The strain is a strain containing the ITS (Internal Transcribed Spacer Region) nucleotide sequence represented by SEQ ID NO: 1.
The method of mass production of a strain comprising the step of culturing the Boberia bassiana of claim 1 KNU-101 strain (accession number KCTC 13660BP).
The method of claim 3,
The cultivation is made at 28 ℃ to 30 ℃ mass production method of the strain.
Claim 1 Boberia bassiana (Beauveria bassiana) KNU-101 strain (accession number KCTC 13660BP), a culture of the strain, a concentrate of the culture, and at least one selected from the group consisting of a dried product of the culture is an active ingredient Cucumber thrips or flower yellow thrips insecticide microbial preparations containing as.
The method of claim 5,
The microbial preparation is a microbial preparation for insecticidal cucumber thrips or flower yellow thrips that has insecticidal activity against thrips.
Cucumber thrips or flower thrips insecticide method comprising the step of treating the microbial preparation of claim 5 on soil or plants.

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