KR20220127995A - Bacillus thuringiensis IMBL-B9 strain having insecticidal activities and uses thereof - Google Patents

Abstract

The present invention relates to a Bacillus thuringiensis IMBL-B9 strain having insecticidal activity and uses thereof. The Bacillus thuringiensis IMBL-B9 strain according to the present invention exhibits a strong insecticidal effect against lepidoptera pests. The composition containing the strain of the present invention is an eco-friendly agent and is effective in minimizing the impact on crops and controlling pests occurring in the crops. In addition, it is possible to obtain an excellent insecticidal effect on lepidoptera pests by showing fast-acting properties, so that it can be usefully used for the development of eco-friendly pest control agents.

Description

Bacillus thuringiensis IMBL-B9 strain having insecticidal activity and uses thereof {Bacillus thuringiensis IMBL-B9 strain having insecticidal activities and uses thereof}

The present application relates to a novel Bacillus thuringiensis IMBL-B9 strain having insecticidal activity and uses thereof.

Insects not only damage crops, causing economic damage, but also have a serious impact on mankind by mediating various pathogens that cause disease in humans. Chemical pesticides most often used to control these pests are increasing day by day due to problems such as environmental pollution through residue and the appearance of toxic and resistant pests to livestock.

In recent years, biological pesticides have fewer side effects to the human body and are highly safe, and due to their environmentally friendly properties, research on new environmentally friendly pesticides using plants and microorganisms is being actively conducted. Insecticidal protein, which is the main mechanism of action of Bacillus thuringiensis, which is most used as a microbial insecticide, is safe against non-selective organisms, so it is used as a major crop protection agricultural material in eco-friendly agriculture and accounts for 80-90% of global biological control. .

Bacillus thuringiensis is an aerobic gram-positive bacillus that forms endospores and produces insecticidal crystal proteins composed of endotoxin proteins. Endotoxin proteins are mainly composed of cry and cyt toxins. Cry protein is particularly toxic to insects such as Lepidoptera, Diptera and Coleoptera (Bravo et al., 2005 Elsevier BV, Amsterdam 175-806), and in addition to various pests such as Hymenoptera, Hemiptera, Grasshopper, mites, nematodes, and protozoa. It has also been reported for activity against (Schnepf et al. 1998 Microbiol. Mol. Biol. Rev. 62: 775-806). The crystal protein of Bacillus thuringiensis reacts with the insect's heavy intestinal juice and is decomposed into a toxic protein to form a small hole in the cell membrane, causing sepsis, and has a mechanism of action showing insecticidal activity.

Korean Patent No. 10-1555124 discloses a gene encoding a novel Bacillus thuringiensis endotoxin protein with improved insecticidal activity against Lepidopteran pests and a manufacturing method thereof, and Korean Patent No. 10-0458765 discloses a control effect on Diptera pests Bacillus thuringiensis strain and a method for producing a microbial pesticide using the same are disclosed, but the Bacillus thuringiensis IMBL-B9 strain showing high toxicity to lepidopteran pests, particularly tropical spider moth, as in the present application, and its use there is no bar

Due to the emergence of resistant individuals, it is necessary to discover new strains with stronger control activity against specific pests.

Republic of Korea Patent Registration 10-1555124 Republic of Korea Patent Registration 10-0458765

The present application intends to provide a novel Bacillus thuringiensis strain having strong insecticidal activity against Lepidoptera pests and their use as marine control agents.

In one aspect, the present application provides a Bacillus thuringiensis ( BT) IMBL-B9 strain deposited as KCTC18882P at the Korea Institute of Biotechnology and Biotechnology Life Resource Center.

In particular, the IMBL-B9 strain according to the present application contains both Cry1Ac and Cry1Ea as Cry toxins, which is a novel combination that cannot be found in the existing BT strains, and has excellent control effects compared to existing strains, as well as controllable pests. It is considered that the scope is widened.

In another aspect, the present application provides a composition for controlling pests comprising the Bacillus thuringiensis IMBL-B9 strain according to the present application.

The spores of the Bacillus thuringiensis IMBL-B9 strain contained in the composition according to the present application, the vegetative cells of the strain, or a cell free extract that does not contain the cell residue of the strain may include

In one embodiment, the Bacillus thuringiensis IMBL-B9 strain comprising the spores of the Bacillus thuringiensis IMBL-B9 strain in the composition according to the present application, the feeder cells of the strain or the cell-free extract of the strain is about 10 to 40 wt% may include

Pests that can be controlled by the composition according to the present application are pests belonging to the order Lepidoptera, and in particular include pests belonging to the family Lepidoptera, the family Moth, or the family Moth.

In one embodiment, the lepidopteran pests are pests belonging to the lepidoptera moth (Helicoverpa armigera), white fire moth (Hyphantria cunea) or corn light moth (Ostrinia furnacalis), chestnut moth (Spodoptera exigua), tropical spider moth (Spodoptera) frugiperda), or tobacco spider moth (Spodoptera litura), Plutella xylostella as a pest belonging to the family Moth, and Plodia interpunctella as a pest belonging to the family Moth, Chilo suppressalis, Including Cnaphalocrocis medinalis, or Galleria mellonella.

In another aspect, the present application provides a composition according to the present disclosure to a pest, a larva of a pest or an egg of a pest; all or part of a plant, or the seed of a plant; Or it provides a method for controlling pests, comprising the step of contacting the pest or the habitat of the plant.

Pests in which the composition according to the present application can be used for control effect may refer to the above.

Bacillus thuringiensis IMBL-B9 strain having insecticidal activity according to the present application shows a strong insecticidal effect against Lepidoptera pests. The composition comprising the strain of the present application is an eco-friendly formulation, and is effective in minimizing the effect on crops and controlling pests occurring in crops.

In addition, the strain of the present application shows an excellent insecticidal effect against chestnut moths and pests such as sphagnum moth and tropical spider moth, which are known to show high resistance to existing Bacillus thuringiensis preparations or organic synthetic pesticides, thereby controlling heating agent lepidoptera pests can be used effectively for

In addition, the composition for controlling pests comprising the strain of the present application can obtain an excellent insecticidal effect against lepidopteran pests or lepidopteran pests by showing fast-acting properties, and can be usefully used in the development of eco-friendly pest control agents.

1 is a phase contrast micrograph before (a) and after (b) natural degradation of the Bacillus thuringiensis IMBL-B9 strain according to an embodiment of the present application, where S is a spore and C is an endotoxin protein crystal.
Figure 2 is a scanning electron micrograph of Bacillus thuringiensis IMBL-B9 strain according to an embodiment of the present application, where S is a spore and bC is a bi-pyramidal form (bi-pyramidal), cC is a cube form ( cuboidal) endotoxin protein crystals.
3 is a transmission electron micrograph of a Bacillus thuringiensis IMBL-B9 strain according to an embodiment of the present application, where S is a spore and bC is a bi-pyramidal form (bi-pyramidal), cC is a cube form ( cuboidal) endotoxin protein crystals.
Figure 4 is the SDS-PAGE result of the endotoxin protein produced by the Bacillus thuringiensis IMBL-B9 strain according to an embodiment of the present application, wherein column M is a protein standard molecular weight label, and column H is Kurstaki HD It is the endotoxin protein of strain -1, and column B is the endotoxin protein of the Bacillus thuringiensis IMBL-B9 strain.
5 is a Bacillus thuringiensis IMBL-B9 strain centered on the Bacillus thuringiensis IMBL-B9 strain according to an embodiment of the present application. The result of 3-genome analysis of chromosomal DNA is shown.
6 shows the homology analysis results of the entire genome including the chromosomes and plasmid DNA of the MBL-B9 strain, HD-1 and AM65-52 according to an embodiment of the present application.

The present application is a new highly toxic Bacillus thuringiensis IMBL-B9 strain and strain having different characteristics from the previously reported Bacillus thuringiensis strain, and a microorganism preparation containing the endotoxin protein produced therefrom as an active ingredient It is based on the finding that can be used as an insecticide.

Accordingly, in one aspect, the present application relates to the Bacillus thuringiensis IMBL-B9 strain deposited as KCTC18882P in the gene bank affiliated with the Korea Research Institute of Bioscience and Biotechnology on January 18, 2021.

The Bacillus thuringiensis IMBL-B9 strain according to the present application was isolated from soil as a microorganism native to Korea, and as a result of genomic analysis of chromosomal DNA herein, Bacillus thuringiensis kurstaki genus HD-1 and 92%, Bacillus thuringiensis kurstaki It was found to be a novel strain showing 84% similarity to thuringiensis israelensis genus AM65-52. Accordingly, the strain isolated from the soil was named Bacillus thuringiensis IMBL-B9 strain, and on January 18, 2021, it was deposited with the Korea Research Institute of Bioscience and Biotechnology and was given KCTC18882P.

Bacillus thuringiensis is an aerobic Gram-positive bacillus, which forms endospores and expresses endotoxin proteins in the absence of sufficient nutrients for growth. When the nutrients necessary for growth are sufficient, vegetative cells are formed. The endotoxin protein is encoded by a plasmid, not the bacterial genome. It is usually composed of cry and cyt toxins.

The strain according to the present application expresses Cry1, Cry2 form and Vip (vegetative insecticidal proteins), and is specifically as described in Table 4. It has a gene that encodes this toxin protein, and the toxin protein is produced from this gene. The Bacillus thuringiensis IMBL-B9 strain according to the present application has cry1Aa, cry1Ac, cry1Be, cry1Ea, cry1Ha and cry2Aa, cry2Ab, cry2Ah, and vip3Aa type toxin protein genes, and has double pyramidal and cubic crystalline endotoxin proteins. can be produced, and it can be effectively used for controlling lepidopteran pests and Diptera pests.

In another aspect, the present application relates to a composition or insecticidal composition for controlling lepidopteran pests comprising the Bacillus thuringiensis IMBL-B9 strain.

Pests for which the strain according to the present application or the composition for controlling pests comprising the same are used refers to invertebrates or microorganisms that harm plants or animals through colonizing, damage induction, or infection. Pests are also organisms that damage or spread disease and compete with the host for nutrients.

In another embodiment according to the present application, the pest is particularly a pest associated with a plant, and includes a lepidopteran pest, in particular a pest belonging to the Lepidoptera family Lepidoptera, Chestnut or Lepidoptera.

In one embodiment, as the lepidopteran pest, the pest belonging to the chestnut family is Spodoptera exigua, tropical spider moth (Spodoptera frugiperda), or tobacco spider moth (Spodoptera litura), and a pest belonging to the moth family. Silver is a Chinese cabbage moth (Plutella xylostella), and the pests belonging to the Myung moth family are Plodia interpunctella, Chilo suppressalis, Cnaphalocrocis medinalis, or Galleria mellonella, Others include insects including Helicoverpa armigera, Hyphantria cunea, or Ostrinia furnacalis, which belong to Lepidoptera and belong to Lepidoptera.

Other embodiments include, inter alia, chestnut moths and tropical spider moths.

The tropical spider moth occurred in 113 countries around the world, including 43 countries in Africa in 2016, and has rapidly spread to 8 countries in Southeast Asia in 2018 and Japan and China in 2019. In particular, the tropical spider moth is classified as a pest that can migrate over long distances and cause great loss to crops when it occurs in large quantities. In Korea, tropical spider moth larvae have been found in corn farms across the country following the first outbreak in a corn cultivation field in Gujwa-eup, Jeju in 2018, but there is no effective eco-friendly control method.

As a pest indigenous to Korea, the green leaf moth has a wide host range such as vegetables, flowers, and fruit trees, and it occurs extensively in facility cultivation areas such as green onions, Chinese cabbage, and watermelons, causing severe damage nationwide. In particular, the leaf moth has difficulties in treating conventional synthetic pesticides due to the ecological characteristics that caterpillars inflict from the inside of the crop, and it is known as a representative heating agent pest because of its high resistance expression.

In particular, the strain according to the present application is 19-fold and 22-fold, respectively, for the tropical spider moth belonging to the family Chestnut family and 22-fold compared to the previously widely used Bacillus thuringiensis Kurstaki HD-1, as described in Example 5 of the present application. By showing a low LC ₅₀ value, it was found that the insecticidal effect against these pests was significantly higher than that of the existing strains.

Bacillus thuringiensis IMBL-B9 strain according to the present application used as a composition for controlling pests, the culture (or culture or bacteria) of the strain, the spore of the strain or the vegetative cell of the strain (vegetative cell), Or an extract of the strain, or a cell-free extract of the strain from which cells are removed.

In one embodiment, the culture or the culture itself may be used. Although not limited thereto, in this case, the cell membrane and the like are disrupted in the environment in which the cultured body is treated, and the substance in the cell is discharged out of the cell to exhibit an effect. In another embodiment, resistant spores are used. In other embodiments, one or more combinations of cultures, resistant spores and cell-free extracts may be used. In another embodiment, an extract of the culture body, for example a cell-free extract, may be used. The culture extract contains the endotoxin protein of the Bacillus thuringiensis IMBL-B9 strain of the present application.

In one embodiment, the composition according to the present disclosure comprises the Bacillus thuringiensis IMBL-B9 strain in an appropriate medium, a medium suitable for spore production, for example, a GYS medium (0.1% glucose, 0.2% yeast extract, 0.05% potassium phosphate, 0.2% Ammonium Sulfate, 0.002% Magnesium Sulfate, 0.005% Manganese Sulfate, 0.008% Calcium Chloride) After culturing for 3 to 5 days at 160 to 220 rpm under 30 to 70%, preferably 60% of oxygen partial pressure at 28 to 30° C., preferably 30° C., the culture solution is centrifuged to harvest the cells, A microbial pesticide can be prepared by mixing 10 to 40% by weight of the cells, preferably 20% by weight, with an additive commonly used in microbial pesticides.

The composition for controlling pests or pesticides of the present invention includes as an active ingredient extract of the strain and/or the culture of the strain, but may further include excipients and additives included in other conventional compositions for pesticides. Examples include, but are not limited to, plasticizers, colorants, brighteners, surfactants, dispersants, emulsifiers, glidants, and the like.

The composition for pesticides of the present invention may be formulated as a conventional pesticide or pesticide formulation. For example, the composition for pesticides of the present invention may be formulated as an emulsion, liquid, wettable powder, water-soluble agent, powder, granule, fumigant, coating agent, and the like. Specifically, the compounds of Formulas 1 to 8 are included in an appropriate manner to formulate an agrochemical preparation. These composition formulations are described in "Catalogue of pesticide formulation types and international coding system", Technical Monograph No. 2, 6th Ed. May 2008, CropLife International]. Compositions are described in Mollet and Grubemann, Formulation technology, Wiley VCH, Weinheim, 2001; or Knowles, New developments in crop protection product formulation, Agrow Reports DS243, T&F Informa, London, 2005.

Specifically, a surfactant, an extender, a disintegrant, a nutrient, etc. are mixed with the extract of the strain and/or the culture of the strain and pulverized to be used as a wetting agent or dissolved to form a liquid formulation. Surfactants include polycarboxylate, sodium lignosulfonate, calcium lignosulfonate sodium dialkyl sulfosuccinate, sodium alkyl sulfonate, polyoxyethylene alkyl phenyl Ethers, sodium tripolyphosphate, sodium alkyl aryl sulfonate, polyoxyethylene alkyl phenyl ether, polyoxyethylene aryl phosphoric ester, Polyoxyethylene alkyl aryl ether, polyoxyethylene alkyl aryl polymer, polyoxyethylene alkyl aryl polymer special, polyoxyalkylone alkyl phenyl ether, polyoxy One or two or more selected from the group consisting of ethylene nonyl phenyl ether, sodium sulfonate naphthalene formaldehyde, Triton 100 and Tween 80 may be used. Examples of surfactants are listed in McCutcheon's, Vol. 1: Emulsifiers & Detergents, McCutcheon's Directories, Glen Rock, USA, 2008 (International Ed. or North American Ed.). As a disintegrant, bentonite, talc, kaolin, calcium carbonate, etc. may be used, and as an extender, one or two or more of ocher, diatomaceous earth, dextrin, glucose, and starch may be used.

The composition for controlling pests or pesticides of the present invention may contain an insecticidal effective amount of the compound, for example, the amount used per pesticide formulation may be 0.01 to 100 (mg/kg). The term “effective amount” means an amount of a composition or compound that is sufficient to control larvae on cultivated plants or is sufficient for the protection of substances and does not cause substantial damage to the treated plant. Such amounts can vary within wide limits and depend on various factors, such as the pest species to be controlled, the cultivated plant or material treated, the climatic conditions and the particular compound used. In the case of formulation, it can be used at any time when pests occur.

The control agent or insecticide comprising the Bacillus thuringiensis IMBL-B9 strain according to the present application is the pest itself in need of control, or the area in need of control, its larvae or eggs, or all or part of the area, habitat or plant in need of control. can be applied.

Plants to which the pesticide according to the invention can be applied can be applied to the whole plant or parts thereof, for example roots, stems, leaves, and seeds, as well as cells or tissues of the plant body.

Crops to which the pesticide according to the present application can be applied include vegetables (cabbage, radish, green onion, spinach, lettuce, asparagus, cabbage, onion, tomato, potato, paprika, etc.), grain (corn, wheat, barley, rye, oat, rice, sugarcane, etc.), cucumber plants (cucumber, melon, melon, etc.), fruits (apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries, blackberries), legumes (beans, lentils, peas, soybeans), beets (sugar beets and fodder beets), grasses (orchard grasses, laver hairs, etc.), oil plants (rapeseed, mustard, poppy, olives, sunflowers, coconuts, castor oil plants, cocoa beans, peanuts, etc.), fiber Plants (cotton, flax, hemp, jute), citrus fruits (tangerines, oranges, lemons, grapefruit, etc.) , oak, alder, poplar, birch, fir, larch, pine), or plants such as tobacco, nuts, coffee, sugarcane, tea, vines, hops, bananas, natural rubber, or ornamental plants.

Plants or crops to which the composition according to the present application is applied also include both planted or growing crops and harvested crops.

As used herein, extermination, control, control or control of pests by pesticides includes eradication of targeted pests or inhibiting, interfering with, physically or their growth or behavior of pests.

In another aspect, the present application provides a composition for pesticides comprising the Bacillus thuringiensis IMBL-B9 strain and/or a culture extract thereof as a pest or a terrestrial habitat of the pest, or a plant or soil or water requiring control of the pest. It relates to a method of combating or controlling pests comprising contacting the plant's habitat.

Pests and crops to which the present method can be applied are as described above.

The extermination or control of pests of the present application refers to removing the pests or preventing or controlling crops from pests to drive out the pests.

The method herein can be used to determine the habitat of the pest or larva; Or all or part of a plant or its seeds, plants or pests or their larvae inhabit habitat, such as soil or water, by any application method known in the art can be contacted with the composition for pesticides of the present invention. . In this case, "contact" means direct contact (applying the strain or composition directly to the pest or plant—typically directly to the leaves, stem or root of the plant) and indirect contact (applying the strain or composition to the locus of the pest or plant) ) includes both.

The methods herein may also be used to protect a plant/crop from attack or infestation by pests by contacting the growing plant or crop with a pesticidally effective amount of the composition. As used herein, the term plant or crop is intended to include both growing and harvested crops.

The methods herein are utilized by treating a plant, plant propagation material, such as seed, soil, surface, material or space, to be protected from attack by pests with a pesticidally effective amount of the composition herein. The methods herein can be performed both before and after infection of plants, plant propagation materials, such as seeds, soil, surfaces, materials or spaces by pests.

The method of the present invention can be applied prophylactically to places where the occurrence of pests is expected.

Hereinafter, examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the present invention is not limited thereto.

Example

Example 1. Isolation of Bacillus thuringiensis IMBL-B9 strain according to the present application from soil

Bacillus thuringiensis IMBL-B9 strain was isolated from soil samples collected in Suwon, Gyeonggi-do, Korea. 1 g of the soil sample was placed in 10 ml of sterile distilled water, stirred vigorously for 2 minutes, bathed at 65° C. for 30 minutes, and diluted to a concentration of 10 ³ CFU/ml with sterile distilled water. After spreading 300 μl of the dilution on a nutrient plate medium (Nutrient agar), it was cultured at 30° C. for 3 days. Among the generated colonies, the formation of endotoxin protein and spores was observed under a phase contrast microscope for colonies whose appearance was similar to that of Bacillus thuringiensis. Colonies forming endotoxin proteins were selected as Bacillus thuringiensis strains, cultured in pure water, and stored at 4°C.

Example 2. Identification of the isolated strain

In order to primarily identify the Bacillus thuringiensis IMBL-B9 strain isolated according to Example 1, flagellar antigenicity against 33 known Bacillus thuringiensis flagellated antibodies was performed using the method of Ohba [Ohba & Aizawa, J. Invertebr. . Pathol., 1978, 32, 303-309]. Bacillus thuringiensis IMBL-B9 strain was inoculated in LB medium (1% tryptone, 0.5% yeast extract and 1% sodium chloride) and cultured for 12 hours at 30 ℃, 100rpm, Bacillus thuringia diluted 100 times in the culture medium After adding the same amount of the dilution of Ncis flagellate antibody, it was reacted at 37° C. for 1 hour, and the agglutination reaction was investigated. The results are shown in Table 1. Bacillus thuringiensis IMBL-B9 strain of the present application was shown to have the same flagellar antigenicity as the Bacillus thuringiensis Kurstaki strain.

[Table 1]

Example 3. Endotoxin protein morphology analysis of Bacillus thuringiensis IMBL-B9 strain

The size and shape of the endotoxin protein produced by the Bacillus thuringiensis IMBL-B9 strain isolated according to Example 1 was observed with a phase contrast microscope, a scanning electron microscope, and a transmission electron microscope.

To this end, in order to obtain an endotoxin protein sample, a single colony of the Bacillus thuringiensis IMBL-B9 strain obtained in Example 1 was treated with 100 ml of GYS medium (0.1% glucose, 0.2% yeast extract, 0.05% potassium phosphate, 0.2% sulfuric acid). After inoculation with ammonium, 0.002% magnesium sulfate, 0.005% manganese sulfate, 0.008% calcium chloride; Nickerson et al., Appl. Environ. Microbiol., 28, 124-128 (1974)), spores and endotoxin proteins are formed and Shaking culture was performed at 30 °C and 150 rpm for 5 days until autolysis. The culture solution was suspended in sterile distilled water, dropped on a slide glass, covered with a cover glass, and observed under a phase contrast microscope on the emulsion oil (1,000 times magnification). In addition, the crystals of the strain were observed with a scanning electron microscope and a transmission electron microscope. The results are shown in Figures 1-3.

Spores (S) and endotoxin proteins (C) of the Bacillus thuringiensis IMBL-B9 strain before (a) and after (b) natural degradation were observed in the phase contrast micrograph of FIG. 1 . In the scanning electron micrograph of FIG. 2 , it was confirmed that the endotoxin protein was present as a double pyramidal (bC) or cube-shaped (cC) crystal. In the transmission electron micrograph of FIG. 3 , it was confirmed that the endotoxin protein was present as a double pyramidal (bC) or cube-shaped (cC) crystal. These observation results indicate that the endotoxin protein derived from the Bacillus thuringiensis IMBL-B9 strain is a well-defined double pyramidal crystal, consistent with the typical morphology of the endotoxin protein toxic to Lepidoptera.

Example 4. Endotoxin protein electrophoretic analysis of Bacillus thuringiensis IMBL-B9 strain

The endotoxin protein crystals according to Example 3 were electrophoresed (SDS-PAGE) by Laemmli's method (Laemmli, Nauter, 227, 680-685 (1970)) as follows. To a constant amount of endotoxin protein crystals, 5 volumes of sample buffer (1M Tris-HCl (pH6.8) 0.6 ml, 50% glycerol 5 ml, 10% SDS 2 ml, 2-mercaptoethanol 0.5 ml, 1% bromophenol Blue 1ml, distilled water 0.9ml) was added, heated at 100° C. for 5 minutes, left at room temperature for a while, centrifuged to recover the supernatant, and then 10% SDS-electrophoresis was performed.

At this time, for comparison, an endotoxin protein sample was obtained from the Kurstaki HD-1 strain in the same manner as in Example 3 and electrophoresed together with the endotoxin protein sample of the Bacillus thuringiensis IMBL-B9 strain. The results are shown in FIG. 4 . Column M is the protein standard molecular weight label, Column H is the endotoxin protein of the Kurstaki HD-1 strain, and Column B is the endotoxin protein of the Bacillus thuringiensis IMBL-B9 strain. Kurstaki HD-1 strain (H) has about 130 kDa family corresponding to cry1 type and cry2 type endotoxin protein (Jaoua S., et al., FEMS Microbiol. Lett. 145: 349-354 (1996)), respectively. It showed a band of 65 kDa, and the endotoxin protein of the Bacillus thuringiensis IMBL-B9 strain (B) according to the present application also showed a similar pattern.

Example 5. Analysis of insecticidal effect of Bacillus thuringiensis IMBL-B9 strain according to the present application

In order to test the insecticidal power of the Bacillus thuringiensis IMBL-B9 strain according to Example 1, the strain was inoculated in GYS medium to form spores and endotoxin proteins and cultured for 5 days at 28°C until autolysis did. Then, the culture medium was collected through centrifugation, the supernatant was discarded, and the obtained pure pellet was released using distilled water, and then, various spore concentrations (10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ and 10 ⁸ spores (CFU)/ml) ), and each diluted solution was added to cabbage leaves (2 cm × 2 cm). Lepidoptera insects, the Chinese cabbage moth ( Plutella xylostella ) and spore moth ( Spodoptera exiuga ), and tropical spider moth ( Spodoptera frugiperda ) The Chinese cabbage treated with the culture dilution solution was added to each treatment group containing 20 larvae and bred at 25° C., The lethality of the larvae was investigated for 72 hours, respectively.

A Bacillus thuringiensis Kurstaki HD-1 strain, which is a representative strain having an insecticidal effect on Lepidoptera, was used as a comparison group, and the culture solution and untreated group of the Bacillus thuringiensis Kurstaki HD-1 strain at the same concentration were tested together. As a result, the half-lethal concentration of the Bacillus thuringiensis IMBL-B9 strain for Lepidoptera pests is shown in Table 2.

[Table 2]

The LC ₅₀ value of the Bacillus thuringiensis IMBL-B9 strain against the diamondback moth was found to be lower than that of the Bacillus thuringiensis Kurstaki HD-1 strain. Furthermore, the LC ₅₀ value for the nightshade moth was also about 22 times lower in the strain according to the present application than the Bacillus thuringiensis Kurstaki HD-1 strain, and about 19 times lower for the tropical spider moth.

These results indicate that the Bacillus thuringiensis IMBL-B9 strain of the present application has a very excellent insecticidal effect against all of the Chinese dragonfly moth, scabbard moth and tropical spider moth. In particular, the insecticidal effect of the Bacillus thuringiensis IMBL-B9 strain on the Chinese cabbage moth is lower than that of the Bacillus thuringiensis Kurstaki HD-1 strain, which is known to have the best effect. The insecticidal effect was also found to be very good.

Example 6. Endotoxin protein gene composition of Bacillus thuringiensis IMBL-B9 strain

To determine the genotype of the endotoxin protein possessed by the Bacillus thuringiensis IMBL-B9 strain, chromosomal DNA and plasmid DNA of the Bacillus thuringiensis IMBL-B9 strain were extracted, followed by Next Generation sequencing (Hiseq2000, ilumina). ) was used for genome analysis. The total size in the chromosomal DNA analysis was 5,620,474 bp, the total number of subread bases in the plasmid analysis was 1,051,776,107 bp, and the contig obtained as a result of the de novo assembly was 15 in total. The total number of contig bases is 900,886 bp, and the average contig size is 60,059 bp. Reads analyzed through Hiseq were assembled through RS HGAP Assembly (v3.0), one of the analysis tools, and error correction was performed using a tool called Pilon. The annotation of the genome was analyzed using the Prokka (v1.13) analysis tool, and the results of the genome sequencing of the Bacillus thuringiensis IMBL-B9 strain, which summarized the whole, are shown in Table 3.

[Table 3]

The insecticidal protein genes possessed by the Bacillus thuringiensis IMBL-B9 strain revealed through next-generation sequencing are shown in Table 4. Bacillus thuringiensis IMBL-B9 strain has a total of 9 insecticidal protein genes, 5 Cry1-forms (cry1Aa, cry1Ac, cry1Be, cry1Ea, cry1Ha), 3 Cry2-forms (cry2Aa, cry2Ab, cry2Ah) and It was shown to have one Vip toxin protein gene. Among them, the amino acid sequences of the Cry1Ac, Cry1Be and Cry1Ha proteins were confirmed to have a similarity of 99.6 to 99.9% with the previously reported homologous proteins.

[Table 4]

The genome analysis results were analyzed using the program SyMAP (SyMAP v5.0.6) to perform a sequence analysis (Syntheny analysis) with other previously reported Bacillus thuringiensis strains. In order to compare with the Bacillus thuringiensis IMBL-B9 strain, Bacillus thuringiensis kurstaki genus HD-1, and Bacillus thuringiensis israelensis genus AM65-52 type strain was compared as a reference.

The results are shown in FIG. 5 . As a result of 3-genome analysis of chromosomal DNA, the IMBL-B9 strain was found to have 92% and 84% similarity to HD-1 and AM65-52, respectively. Also, as shown in FIG. 6, homology of the entire genome including chromosomes and plasmid DNA was 93% for IMBL-B9 and HD-1 strains, and 86% for IMBL-B9 and AM65-52 strains.

Although not limited to this theory, the IMBL-B9 strain according to the present application is a strain belonging to Bacillus thuringiensis kurstaki, and it contains Cry1Ea in addition to Cry1Aa, 1Ac, etc. present in most kurstaki strains, and thus, it is particularly suitable for chestnut moths. It is thought that the insecticidal effect was shown to be high.

In particular, the IMBL-B9 strain according to the present application contains both Cry1Ac and Cry1Ea, which is a novel combination of Cry toxins, which showed excellent insecticidal power against diamondback moths, balm moths and tropical spider moths, and was controlled by one strain by this combination It is judged that the effect of broadening the range of pests that can be used is shown.

Korea Institute of Bioscience and Biotechnology KCTC18882P 20210118

Claims (12)

Bacillus thuringiensis IMBL-B9 strain deposited as KCTC18882P at the Korea Research Institute of Bioscience and Biotechnology Life Resource Center.
A composition for controlling pests comprising the Bacillus thuringiensis IMBL-B9 strain according to claim 1.
3. The method of claim 2,
The Bacillus thuringiensis IMBL-B9 strain is a spore of the strain, the vegetative cells of the strain or a cell-free extract of the strain, a composition for controlling pests.
3. The method of claim 2,
The Bacillus thuringiensis IMBL-B9 strain comprises 10 to 40% by weight, pest control composition.
5. The method according to any one of claims 2 to 4,
The pest is a pest belonging to the order Lepidoptera, pest control composition.
6. The method of claim 5,
The pests are moths belonging to the order Lepidoptera, chestnut moths, or Myungmoths pests, composition for controlling pests.
6. The method of claim 5,
The lepidopteran pest is an insect including a lepidoptera moth (Helicoverpa armigera), a white fire moth (Hyphantria cunea) or a corn light moth (Ostrinia furnacalis), a composition for controlling pests.
7. The method of claim 6,
Pests belonging to the chestnut family are Spodoptera exigua, tropical spider moth (Spodoptera frugiperda), or tobacco spider moth (Spodoptera litura),
The pest belonging to the moth family is a Chinese cabbage moth (Plutella xylostella),
Pests belonging to the Myungmoth family are Hwaranggok moth (Plodia interpunctella), Chilo suppressalis, Cnaphalocrocis medinalis, or Honeybee fantail moth (Galleria mellonella), a composition for controlling pests.
The Bacillus thuringiensis IMBL-B9 strain according to claim 1 or the composition according to claim 2 is used as a pesticide, pest larva or pest egg; all or part of a plant, or the seed of a plant; or contacting the pest or plant habitat.
10. The method of claim 9,
The Bacillus thuringiensis IMBL-B9 strain is a spore (spore) of the strain, the vegetative cells (vegetative cells) of the strain or a method for controlling pests comprising a cell-free extract of the strain.
11. The method according to claim 9 or 10,
The pest is a chestnut moth and pests of the order Lepidoptera, a pest control method.
12. The method of claim 11,
The pests are insects, including Spodoptera exigua, tropical spider moth (Spodoptera frugiperda), and tobacco spider moth (Spodoptera litura).

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