KR101924881B1 - Bacillus thuringiensis subsp. kyushuensis strain CAB464 having insecticidal activity and uses thereof - Google Patents

Abstract

The present invention relates to a Bacillus thuringiensis subspecies Kyushuensis CAB464 strain having insecticidal activity and a use thereof. When the Bacillus thuringiensis subsp. Kyushuensis CAB464 strain of the present invention is treated, Bradysia agrestis ), mushroom flies ( Lycoriella ingenua , Aedes albopictus , Togo forest mosquitoes ( Aedes togoi , and Culex pipiens pallens . Therefore, the composition of the present invention containing the same is an environmentally friendly preparation that minimizes the effect on crops and is effective in controlling pests occurring in crops, It is possible to obtain an excellent insecticidal effect on the Flycatcher pest by showing rapid effect, and thus it can be usefully used for the development of a pollution control material for non-pollution.

Description

Bacillus thuringiensis subsp. Bacillus thuringiensis subsp. ≪ RTI ID = 0.0 > CAB464 < / RTI > kyushuensis strain CAB464 having insecticidal activity and uses thereof}

The present invention relates to Bacillus thuringiensis subspecies Kyushuensis CAB464 strain having insecticidal activity and use thereof.

Insecticidal proteins, which are the main functional groups of Bacillus thuringiensis most commonly used as microbial insecticides, are safe as non-selective organisms and thus are used as major crop protection crops in eco-friendly agriculture and account for 80 to 90% of the world's biological control . Bacillus thuringiensis is a aerobic gram-positive bacterium that forms endogenous spores and produces insoluble crystal proteins composed of endotoxin proteins. Endotoxin proteins are mainly composed of cry and cytotoxins. Cry proteins are particularly toxic to insects such as lepidoptera, diclofenia and beetles (Bravo et al ., 2005 Elsevier BV, Amsterdam 175-806), as well as other organisms such as logging, timothy, locust, mite, (Schnepf et al. 1998 Microbiol. Mol. Biol. Rev. 62: 775-806).

The crystalline protein of Bacillus thuringiensis reacts with the insecticide medium to decompose it into a toxic protein and form a small hole in the cell membrane to produce sepsis and have a mechanism of insecticidal activity. In addition, since the toxicity of Bacillus thuringiensis has to be active, the insect must feed and kill only the larva, and it can not affect the eggs, pupa, adult of pests.

Korean Patent No. 1649139 discloses' Bacillus thuringiensis subspecies Aisawa CAB566 strain having insecticidal activity and its use ', and in Korean Patent No. 0420988,' Bacillus thuringiensis (bacillus thuringiensis) ). However, as in the case of the present invention, there is no disclosure of the 'Bacillus thuringiensis subspecies Kyushuensis CAB464 strain having insecticidal activity and its use'.

The present invention has been made in view of the above-mentioned needs. In the present invention, Bacillus thuringiensis was isolated from the soil for the purpose of controlling the insect pests of the bacterium, and the strains exhibiting excellent insecticidal effect were selected through bioassay. As a result of investigating the insecticidal activity and morphological characteristics of Bacillus thuringiensis subspecies Kyushuensis CAB464 strain selected in the present invention, it was found that Bacillus thuringiensis, Bradysia agrestis , Mushroom flies Lycoriella ingenua , Aedes albopictus , Togo forest mosquitoes ( Aedes togoi ) and red house mosquitoes ( Culex pipiens pallens ), the present inventors have completed the present invention.

In order to solve the above problems, the present invention provides a Bacillus thuringiensis subsp. Kyushuensis CAB464 strain having insecticidal activity against parasitic insects.

The present invention also provides a parasite insect pest composition comprising the above strain or a culture thereof as an active ingredient.

The present invention also provides a method for controlling parasitic insect pests by spraying an effective amount of the Bacillus thuringiensis subsp. Kyushuensis CAB464 strain on a plant, soil, or parasitic insect habitat.

When the Bacillus thuringiensis subsp. Kyushuensis CAB464 strain of the present invention is treated, the bacterium Bradysia agrestis , Lycoriella mushroom ingenua , Aedes albopictus , Togo forest mosquitoes ( Aedes togoi ) and red house mosquitoes ( Culex pipiens pallens ). Therefore, the composition of the present invention containing the same is an environmentally friendly preparation that minimizes the effect on crops and is effective in controlling pests occurring in crops, and exhibits rapid effect, It can be effectively used for the development of non-polluting pest control agents.

1 is a photograph of a phase contrast microscope (× 1000) of a crystal of Bacillus thuringiensis strain isolated in the present invention. A, Lindsey-to-N-Sys Bacillus spp difference N-Sys-study (B. thuringiensis subsp darmstadiensis.); B, Lindsay-to-N-Sys Bacillus spp Zaporozhye cis norbornene (B. thuringiensis subsp japonensis.); C, Bacillus thuringiensis subsp. Kyushuensis CAB464.
2 is an SDS-PAGE for comparing endotoxin protein characteristics of Bacillus thuringiensis strain isolated in the present invention. M; Lane 1: Bacillus thuringiensis subsp. Varieties Staphylococcus species, Bacillus thuringiensis subsp. Variant staphylococci cut with trypsin, Lane 3: Bacillus thuringiensis CAB464 Prothoxin, Lane 2 And 4: Bacillus thuringiensis subsp. ≪ RTI ID = 0.0 > Kyushuensis < / RTI >
3 is a PCR result for identifying the cry gene of Bacillus thuringiensis subspecies Kyushuensis CAB464 strain isolated in the present invention. M: 100bp leather marker, lane 1: cry4Aa, lane 2: cry4Ba, lane 3: cry10Aa, lane 4: cry11Aa, lane 5: cyt1Aa, lane 6: cyt2Aa, lane 7: cyt2Ba.
4 is electrophoresis for identifying plasmid DNA of Bacillus thuringiensis strain isolated in the present invention. M: Lamda Hind marker, lane 1: Bacillus thuringiensis subsp. Variant stdisensis, lane 4: Bacillus thuringiensis subspecies Kyushuensis CAB464, lanes 2 and 5: Bacillus thuringiensis cut with EcoR, lane 3, 6: Hind Bacillus thuringiensis.

In order to accomplish the object of the present invention, the present invention provides Bacillus thuringiensis subsp. Kyushuensis CAB464 strain having insecticidal activity against Diclofenac pests.

The Bacillus thuringiensis subspecies Kyushuensis CAB464 strain isolated from the present invention has a insecticidal activity against the parasitic insect pests and has been deposited with the Korea Research Institute of Bioscience and Human Technology with the deposit number KCTC13363BP on Oct. 17,

The present invention also provides a composition for insecticidal control of Diclofenac pests which contains, as an active ingredient, a pure culture of a novel strain of Bacillus thuringiensis subsp. Kyushuensis CAB464. In this composition, the Dicotyledonous insect pests are selected from the group consisting of Bradysia agrestis ), mosquito fly ( Lycoriella ingenua ), white mosquito ( Aedes albopictus ), Togo forest mosquitoes ( Aedes togoi ) and red house mosquitoes ( Culex pipiens pallens , preferably, but not exclusively, a small root fly ( Bradysia agrestis ) and a mushroom fly ( Lycoriella ingenua ).

The present invention also features an effective amount of a composition containing Bacillus thuringiensis subsp. Kyushuensis CAB464 or a pure culture thereof to spray on plant, soil or paralichthouse insect habitats for control of Diclofenac pests To provide a method for controlling parachute pests.

In the above control method, the present invention also provides a composition for insecticidal control of Diclofenac pests which contains, as an active ingredient, a pure culture of a novel strain of Bacillus thuringiensis subsp. Kyushuensis CAB464 . In this composition, the Flycatcher can be selected from the group consisting of Bradysia agrestis , Lycoriella ingenua , Aedes albopictus ), Togo forest mosquito ( Aedes togoi ) and red house mosquito ( Culex pipiens pallens ), preferably a small root fly ( Bradysia < RTI ID = 0.0 > agrestis ) and mushroom flies ( Lycoriella ingenua ).

Hereinafter, definitions of terms used in the description of the present invention are as follows.

"Insolvency" refers to the ability of a substance to increase the mortality or inhibit the growth rate of hygiene pests or crop pests.

An " effective amount " is an amount sufficient to produce beneficial or desired results. An effective amount can be administered more than once.

Is intended to mean a combination of an active agent and another compound, such as a carrier or composition, which is an inert (e. G., Detectable drug or label or liquid carrier) or an active (e. G., Adjuvant).

Plants to which the present invention is applied are not particularly limited and include plants such as cabbage, cabbage, cucumber, radish, perilla, pepper, lettuce, lettuce, strawberry, tomato, In addition to crops, they can be treated on plant surfaces such as flowers or special crops, or on the soil in which these plants are growing, or on the surface of vegetables being transported or stored for cultivation.

The composition for preventing parasitic insects of the present invention containing the strain Bacillus thuringiensis subsp. Kyushuensis CAB464 of the present invention, more preferably a composition for controlling pest insects such as Bradysia agrestis , Lycoriella ingenua , ( Aedes albopictus ), Togo forest mosquitoes ( Aedes togoi ) and red house mosquitoes ( Culex pipiens The present invention relates to a composition for insect pest control, which comprises a mixture of 5 to 90% by weight of a culture solution or a dry powder of Bacillus thuringiensis subsp. Kyusyuensis CAB464 strain, a surfactant, an inorganic salt, an adjuvant, a binder and an extender, can do.

The surfactant is an amphipathic substance having both a hydrophilic molecular phase and an oleophilic molecular phase in a molecule and is characterized by being excellent in washing power, dispersing power, emulsifying power, solubilizing power, wetting power, sterilizing power, foaming power and penetration power , It can be understood that BT (bitterness) in the insect pest insecticidal composition according to the present invention acts to hydrate, suspend, and disperse to effectively express the drug efficacy.

Examples of the surfactant include sodium salts of sulfonates such as alkyl benzene sulfonates, alkyl naphthalene sulfonates, dialkyl sulfosuccinates, lignin sulfonates, alkyl naphthalene sulfonate formalin condensates, polyoxyalkylene alkyl phenyl sulfonates, Sodium salt or calcium salt of sulfate such as calcium salt, alkylsulfate, polyoxyalkylene alkylsulfate, polyoxyalkylene alkylphenylsulfate, sodium salt of succinate such as naphthalene sulfosuccinate, polyoxyalkylene succinate or the like Anionic surfactants such as calcium salts, ethoxylated alkyl ethers, polyoxyalkylene alkylphenyl polymers, and polyalcohols may be used alone or in combination of two or more. These are all exemplified It is well within the skill of the art that surfactants other than these can be used Now with the knowledge of who could be easily understood.

The extender may be used in combination with the surfactant to adsorb and disperse the surfactant and to coagulate the surface of the particulate composition of the vitifier composition with the surfactant, the drug, the culture medium or the dry powder of Bacillus thuringiensis subsp. Kyushuensis CAB464 And may be used alone or as a mixture of two or more kinds thereof. The starch, soybean meal, bran, granular fiber, yuan, diatomaceous earth, zeolite, bentonite, talc, kaolin, pyrophyllite and white carbon may be used singly or in combination.

The binder binds the active agent, such as a water-soluble starch, dextrin, carboxymethyl cellulose, sodium polyacrylate, polyacrylic acid, polyvinylpyrrolidone, polyvinylpyrrolidone, Vinyl alcohol, gum arabic or xanthan gum may be used alone or in combination of two or more.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Materials and methods

Isolation and Culture of Strain

A selection test was conducted with 49 strains isolated from domestic soil in Chungnam National University Biological Pest Control Laboratory. The strains forming the endotoxin protein were selected by observation with a phase contrast microscope 1,000 times. The selected strains were diluted in sterilized water and evenly plated on nutrient agar plates. The plates were incubated in a 27 ° C incubator for 3 to 4 days and autolysis was confirmed and centrifuged at 15,000 rpm for 15 minutes using a centrifuge (Avanti J-E, Beckman).

Morphological observation of strain

(Olympus BX51) and a scanning electron microscope (Quanta FEG MK2) in order to examine the morphology of spore and endotoxin crystallin protein of Bacillus thuringiensis strains that proliferated on nutritional agar plates and spore-forming period. To observe with a phase contrast microscope, a small amount of strain was dropped on a slide glass and observed a spherical type endotoxin crystalline protein form 1,000 times (Kim et al ., 1995 Korean J. Seric. Sci ., 37 (1): 57-61).

Insect insects

Bradysia agrestis , used in this experiment, was used as food for potatoes and mushroom flies ( Lycoriella ingenua ) were used as food for mushrooms. Aedes albopictus , Aedes togoi , and Culex pipiens pallens were fed from the Department of Disease Control and Prevention, and fed with yeast extract. In the biological pest control laboratory of Chungnam National University, the temperature was 25 ± 2 ℃, the light condition was 16L: 8D, the relative humidity was 50 ~ 60% (Kim et al., 2008 Korean J. Appl. Entomol 47 (1): 87-93 ).

Biological activity test and half-life mortality

When the selected Bacillus thuringiensis strain was inoculated into nutrient agar medium and cultured at 27 ° C for 5 days, endotoxin protein protein formation was observed while observing formation of endotoxin crystal protein with a phase contrast microscope. The bacteria were collected and centrifuged. The supernatant was discarded and 5 ml of sterilized water was added to the remaining pellet. The diluted culture was used for bioassay. In order to compare the insecticidal activity, eight strains of spherical type were tested in a laboratory known to have insecticidal activity against the larvae.

1) Small root flies ( Bradysia agrestis Toxicity test

The bioactivity test for tiny root flies was carried out on Bacillus thuringiensis The strain was diluted with sterilized water and 200 μl of the culture was added to 1 g of potato. The larvae of the larvae hatched for 3-4 days were placed in a Petri dish for 3 repetitions, and the mortality was investigated for 168 hours.

2) Mushroom flies ( Lycoriella ingenua Toxicity test

The biological activity of mushroom flies was determined by adding 200 μl of Bacillus thuringiensis strain to sterilized water and adding 1 g of mushroom mushroom. The larvae of the larvae hatched for 3-4 days were placed in a Petri dish for 3 repetitions, and the mortality was investigated for 168 hours.

3) White mosquito mosquitoes ( Aedes albopictus ), Togo forest mosquitoes ( Aedes togoi ), Red house mosquitoes ( C. pipiens pallens Toxicity test

The biological activity test for mosquitoes was conducted by modifying the experimental method of LARVICIDES (2005). Ten larvae that had been hatched for 3-4 days were placed in a 90 ml plastic cup containing 30 ml of water, and 300 μl of the diluted bacterial solution was inoculated. The mortality rate was investigated for 72 hours, and three repeated experiments were performed.

4) Half Lethal concentration (LC ₅₀ )

All experiments were repeated three times and the larval mortality rate, which ranged from 5 to 7, was determined based on the probit calculation method of Finney (Finney, DJ 1971 Cambridge University Press. London . Pp 19-47) A half-life lethal concentration (LC ₅₀ ) was calculated using a PC program.

SDS-PAGE

Each of the strains used in the experiment was inoculated into a nutrient broth and incubated at 27 ° C for 5 days. After confirming that autolysis was caused by a phase contrast microscope, a centrifuge tube was placed in a centrifuge tube at 15,000 rpm at 4 ° C Followed by centrifugation for 10 minutes. After centrifugation, the supernatant was discarded and washed three times with Wash Buffer I (500 mM NaCl, 2% Triton X-100) and twice with wash buffer (500 mM NaCl). The washed parasporal inclusions were stored at -20 ° C after addition of sterile water. SDS-PAGE was performed using 12% separating gel and 5% stacking gel. The electrophoresis gel was stained with 0.5% Coomassie Brilliant Blue. The incubated subpopulations of each strain were reacted with 50 mM NaOH aqueous solution for 5 minutes at room temperature. After centrifuging, the supernatant was discarded and 1 mg / ml trypsin was treated with 10: 1 (w / w) and incubated at 37 ° C for 30 minutes. Proteins dissolved by trypsin were confirmed by SDS-PAGE.

PCR analysis

A gene-specific primer set (Table 1) was used to identify the Cry-type gene of the endotoxin gene of the selected Bacillus thuringiensis strain. PCR amplification was performed using Thermal Cycle C1000 ^™ (BIO-RAD). The reaction solution was prepared by mixing 1.0 μl of template DNA, 1.0 μl of each primer set, and 17 μl of distilled water into premix (Bioneer) containing buffer component and dNTP, and adjusting the final volume to 20 μl. PCR conditions were 95 ° C for 3 minutes, 30 cycles of 95 ° C for 1 minute, annealing at 57 ° C for 1 minute, 72 ° C for 1 minute, and 72 ° C for 5 minutes to produce PCR product DNA. And electrophoresed.

Plasmid DNA extraction

The protocol of the Qiagen midi kit was partially modified to extract plasmid DNA from Bacillus thuringiensis strain. The strain was inoculated into 5 ml of LB medium and cultured at 27 DEG C and 180 rpm for 8 hours. The culture was added to 50 ml of LB medium and incubated for 16 hours under the same conditions. The cultured bacteria were centrifuged at 6,000 g for 15 minutes at 4 ° C. The supernatant was discarded and 4 ml of P1 buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 50 μg / ml of RNase A) was added and vortexed to release the pellet. 4 ml of P2 buffer (0.2 M NaOH, 1% SDS) was added and the tube was inverted 4 ~ 6 times and mixed and incubated at 15 ~ 25 ℃ for 5 minutes. 4 ml of the cooled P3 buffer (4 M guanidine hypochloride, 0.5 M potassium acetate, pH 4.2) was added, and the mixture was inverted 4 to 6 times and mixed on ice for 15 minutes. After centrifugation for 30 minutes at 15,000 rpm and 4 ° C, the supernatant was transferred to a new tube. After centrifugation at 15,000 rpm and 4 ° C for 15 minutes, 4 ml of QBT buffer (50 mM NaCl, 50 mM MOPS, pH 7.0, 15% isopropanol, 0.15% Triton X-100) Was added to the supernatant. When the supernatant completely passed through the column, the Qiagen-tip 100 was washed with 20 ml of QC buffer. The DNA was isolated by dissolving in 5 ml of QF buffer. 3.5 ml of isopropanol at room temperature was added to precipitate the DNA, followed by centrifugation at 15,000 rpm and 4 ° C for 30 minutes. The supernatant was discarded and 2 ml of 70% ethanol was added to wash the DNA pellet, followed by centrifugation at 15,000 rpm and 4 ° C for 10 minutes. The supernatant was discarded, the air was dried for 5 to 10 minutes, and 200 μl of tertiary distilled water was added to dissolve the plasmid DNA. Plasmid DNA was mixed with tertiary distilled water and placed in a sterile Eppendorf tube and stored at -20 ° C until used in the experiment. The electrophoresis was performed by mixing agarose gel in 1 × TAE buffer at a ratio of 1%, dissolving it by heating, and then pouring it into a gel tray and agarose gel hardened for 20 minutes. The same 1 × TAE buffer as the gel was poured into the electrophoresis device 3 to 5 mm above the gel, and a mixture of a loading dye and a loading star in a ratio of 5: 1 and a plasmid DNA sample were mixed at a ratio of 1: 5 , And the mixture was placed in each well. After electrophoresis at 50 V for 60 minutes, the band pattern was confirmed by irradiating with UV.

Example 1 Isolation and Selection of Bacillus thuringiensis Strain

Forty - nine strains, which were kept in Biological Pest Control Laboratory of Chungnam National University, were cultured in NA medium at 27 ℃ and their parasporal inclusions were observed by phase contrast microscope. Of the 49 strains, 28 strains forming the bipyramidal inclusions and 9 strains producing the spherical inclusions were identified. Of these, nine of the strains that formed the inclusion bodies of multiple spherical capsules were selected (Table 2), because the inclusion bodies of multiple spheres were generally known to be effective on the flyworms. Nine strains that form the inclusions of the multiple spherical capsules were subjected to an activity test on the small root flies (Table 3). CAB464 showed more than 90% insecticidal activity, CAB117 and 469 showed more than 70% insecticidal activity, and CAB459, 462 and 490 showed more than 50% insecticidal activity. CAB464 showed more than 90% insecticidal activity, CAB117, 459 and 469 showed more than 70% insecticidal activity, and CAB490 and 492 showed more than 50% insecticidal activity. CAB463 and 467 showed insecticidal activities of less than 50% for both insects. Therefore, we selected CAB464 strain which showed high insecticidal activity over 90% against two insects of small root fly and mushroom flies.

The morphological classification of the Bacillus thuringiensis strain isolated in the present invention Morphological groups No. of B. thuringiensis  isolates Non crystal 12 Bipyramidal type 28 Spherical type 9 Total 49

The insecticidal activity test for small root fly and mushroom flies for selection of Bacillus thuringiensis strain isolated in the present invention Strain Tested insects Strain Tested insects Bradysia agrestis Lycoriella ingenua Bradysia agrestis Lycoriella ingenua Control - - CAB464 +++ +++ CAB117 ++ ++ CAB467 - - CAB459 + ++ CAB469 ++ ++ CAB462 + - CAB490 + + CAB463 - - CAB492 - +

+++: high effect, 90% insecticide rate

++: Effect, 70 ~ 89% Insecticide rate

+: Low effect, 50 ~ 69% insecticide rate

-: No effect, 0 ~ 49% Insecticide rate

Phase contrast microscopy

Crystals and spores were observed with a phase contrast microscope in order to determine the crystal form of the CAB464 strain with high insecticidal activity against the small root flies and mushroom flies. Observation of endotoxin protein morphology under phase contrast microscopy provides important information because the endotoxin protein form of Bacillus thuringiensis strains gives information that is closely related to insect host range indicating insecticidal activity. Bacillus thuringiensis Bacillus subtilis CAB464 strain was shown to form a typical polynuclear crystal protein known to exhibit insecticidal activity against Diclofen insect by observation with a phase contrast microscope (Fig. 1).

Bioactive assay

Selected Bacillus thuringiensis Eight Bacillus thuringiensis strains known to have insecticidal activity against strains and parasitic larvae Using the subspecies, the insecticidal activity was tested against five kinds of insect pests such as parasitic insects, mosquito flies, small root flies, white mosquito mosquitos, Togo forest mosquitos and red house mosquitoes. As a result, a black-to-nine strains of Bacillus Lindsay range of active N-Sys subspecies difference N-Sys-study (B. thuringiensis subsp. Darmstadiensis) and Bacillus-to Lindsay N-Sys Kyushu N-Sys subspecies (Bacillus thuringiensis subsp. Kyushuensis) CAB464 strains rootlets Paris And mosquito flies, and showed high insecticidal activity against white mosquito, Togo mosquito, and red mosquito (Table 4). Thus, Lindsay-study the insecticidal activity of Bacillus highest-to-N-Sys different subspecies of the two N-Sys pest (B. thuringiensis subsp. Darmstadiensis) and Bacillus-to Lindsay N-Sys Kyushu N-Sys subspecies (Bacillus thuringiensis subsp. Kyushuensis) by selecting a strain CAB464 bioassay by concentration . As a result, the LC ₅₀ value for the small root flies was 8.0 × 10 ⁵ (cfu / ml) in the CAB464 strain and the Bacillus thuringiensis subsp. Showed high insecticidal activity as compared to ^{1.6 × 10 6 (cfu / ml} ) of the strains. The LC ₅₀ value for the mushroom flies was 1.9 × 10 ⁵ (cfu / ml) in the CAB464 strain and the Bacillus thuringiensis subsp. Showed high insecticidal activity as compared to ^{2.8 × 10 5 (cfu / ml} ) of the strains (Table 5).

Bacillus thuringiensis CAB464 < RTI ID = 0.0 > Insecticidal Activity Assay for 5 Pests of 8 Bacillus thuringiensis strains showing insecticidal activity against strains and dicotyledonous insects Strains Tested insects Lycoriella ingenua Bradysia agrestis Aedes albopictus Aedes togoi Culex pipiens pallens Control - - - - - B. thuringiensis subsp. canadensis ++ ++ +++ +++ +++ B. thuringiensis subsp. darmstadiensis +++ +++ +++ +++ +++ B. thuringiensis subsp. fukuokaensis ++ ++ - - ++ B. thuringiensis subsp. higo ++ ++ - ++ +++ B. thuringiensis subsp. japonensis - - - - - B. thuringiensis subsp. kyushuensis CAB464 +++ +++ ++ +++ +++ B. thuringiensis subsp. morrision + + +++ +++ +++ B. thuringiensis subsp. sotto + ++ +++ ++ - B. thuringiensis subsp. tohokuensis ++ ++ +++ +++ ++

+++: high effect, 90% insecticide rate

++: Effect, 70 ~ 89% Insecticide rate

+: Low effect, 50 ~ 69% insecticide rate

-: No effect, 0 ~ 49% Insecticide rate

The Bacillus thuringiensis The half-life mortality (LC ₅₀ ) test for the bacterium Bradysia agrestis and Lycoriella ingenua Strain Tested insects Bradysia agrestis Lycoriella ingenua B. thuringiensis subsp. darmstadiensis 1.6 × 10 ⁶ 2.8 × 10 ⁵ B. thuringiensis subsp. kyushuensis CAB464 8.0 × 10 ⁵ 1.9 × 10 ⁵

SDS-PAGE

The molecular weight of the Cry protein, which constitutes a spherical type endotoxin protein with insecticidal activity against Diclofenac pests, is about 65 kDa and the molecular weight of the cyt protein is reported to be about 22-30 kDa (Ibarra and Federici , 1986. J. Bacteriol ., 165 (2): 527-533). The B. thuringiensis subsp. Israelensis strain, which shows insecticidal activity against the paralytic insect pests, has a molecular weight of 27, 65, 128, and 135 kDa when the parasporal body is hydrolyzed in the middle larva. The major proteins of the dog are produced. The 135, 128 and 65 kDa proteins are Cry4 proteins and the 27 kDa protein is the cytolytic CytA. Toxicity is caused by the synergy between the 25 kDa protein, a 27 kDa protein degradation product, and one or more proteins with higher molecular weights.

SDS-PAGE was performed in order to compare the endotoxin protein characteristics of Bacillus thuringiensis subsp. Multistandisis and Bacillus thuringiensis subsp. Subtilis strains exhibiting a high insecticidal activity in the biologically active test against Diclofenac pests. Bacillus thuringiensis subsp. The strains of about 150, 125, 70, 50, and 25 kDa were found in the strains, and the CAB464 strain of Bacillus thuringiensis subsp. Kyusyuuensis showed major protein band patterns of about 150, 70, 66, 50 and 27 kDa in size (FIG. Trypsin, a digestive enzyme, was treated to determine the active toxin pattern in the subpopulations of the two strains. Bacillus thuringiensis subsp. In the strain, a protein of about 150 kDa was degraded by trypsin to form a new protein band of about 65 and 60 kDa, and a protein of about 150 kDa was cleaved by trypsin in Bacillus thuringiensis subsp. Kyushuensis CAB464 strain to about 65, 60 kDa and 50 kDa Of the protein band.

Identification of Cry-type gene

Bacillus Turinjiensis The strain produced two types of insecticidal proteins including Cry (Crystal protein) and Cyt (cytolytic toxin) proteins in the spore formation stage. The Bacillus thuringiensis subsp. Israel Rensis strain, which is known to have high insecticidal activity against Diclofenac pests, has cry4Aa, cry4Ba, cry10, cry11Aa, cyt1Aa and cyt2Ba genes. Bacillus thuringiensis subspecies Moriensis strains are cry4A, cry4B, cry10A, cry11A And the cyt1A gene (Ibarra et al ., 1986. FEMS Microbiol Letters, 34 (1): 79-84). Many Bacillus thuringiensis strains that have insecticidal activity against mosquito larvae have genes and proteins similar to cyt1A. Cytotoxin is a protein group produced by some strains of Bacillus thuringiensis, and is classified into three types of cyt1, cyt2, and cyt3 based on amino acid homology. It has been reported that this protein has a lethal activity against larval insect larvae and has cytolytic activity on a wide range of cells including erythrocytes in vitro (Thomas et al ., 1983. FEBS Letters, 154 (2): 362-368) . A variety of cytotoxins are found in Bacillus thuringiensis The gene encoding this toxin has been identified and sequenced (Crickmore et al ., 1998. Microbiol Mol Biol Rev, 62 (3): 807-813).

In the present invention, the selected Bacillus thuringiensis subsp. The cry genes of CAB464 were identified as cyt2Aa, and the cyt2Aa gene was reported to be the only protein causing insecticidal activity in mosquito larvae. The two strains have a high insecticidal activity against small root flies and mushroom flies due to the cyt2Aa gene (Fig. 3).

Plasmid DNA analysis

Bacillus thuringiensis is involved in the formation of crystal proteins that exhibit insecticidal activity against insect pestscry The gene was mainly present on the plasmid DNA. Plasmid DNA present in Bacillus thuringiensis consists of a maximum of 17 plasmids, ranging in length from 2 to 250 kbp. The number and length of the plasmid DNA possessed by each strain vary. Likewisecry Genes were not always present in the plasmid DNA of the same size in all the strains but were present in the plasmid DNA of each strain at different positions. Bacillus thuringiensis subspecies Kyushu Nation As a control strain of CAB464 strain, a plasmid DNA pattern was confirmed using a strain of Bacillus thuringiensis subsp. Varius stadenseis (Fig. 4). Bacillus thuringiensis subspecies Kyushu Nation CAB464 strain was able to identify nine plasmid DNA bands including two plasmid DNA bands larger than 23.1 kb. In addition, Bacillus thuringiensis subsp. Strain Bacillus thuringiensis subspecies Kyushu Nation CAB464 strain exhibited three identical band patterns (Fig. 4).

In the present invention, it was difficult to distinguish between the Bacillus thuringiensis subspecies Kyushuensis CAB464 strain and the Bacillus thuringiensis subsp. Multistadense strain having a high insecticidal effect on the small root flies and mushroom flies only by the external characteristics and the characteristics of the proteins. However, Molecular biology experiments have shown that there are differences. The Bacillus thuringiensis subspecies Kyushuensis CAB464 strain exhibits a high insecticidal effect against small root flies and mushroom flies, and thus can be usefully used as a novel microbial insecticide capable of controlling both pests.

Korea Biotechnology Research Institute KCTC13363BP 20171017

<110> The Industry & Academic Cooperation in Chungnam National University (IAC) <120> Bacillus thuringiensis subsp. having kyushuensis strain CAB464 having          insecticidal activity and uses thereof <130> PN17421 <160> 36 <170> Kopatentin 2.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 cagataccct tgctggtgta a 21 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ataggcccgt gctccaccag g 21 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ccgaacaatc gaagtgaa 18 <210> 4 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 atagatggtc ctactt 16 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ccgaacaatc gaagtgaa 18 <210> 6 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 gaatcctgtg cacctaa 17 <210> 7 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 cctgaaaatt acaggcc 17 <210> 8 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 aattgatcaa tagaatc 17 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 ccgaacaatc gaagtgaa 18 <210> 10 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 attgttgccg tcaacaa 17 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 gaactgggta tggcactcaa c 21 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 ctcacaacga ttagaccctt c 21 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 gcgaggtttc ccatgtctac 20 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 gttgtagggt ggaattgtta tc 22 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 atggaagata gttctttaga t 21 <210> 16 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 ggtagatttt aattctac 18 <210> 17 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 atcatgccat ggatcaaaat aaacacggaa ttattgg 37 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 ccgcttccaa taacatcttt t 21 <210> 19 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 tcattggtat aagagttggt ctatagac 28 <210> 20 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 cctcctagac acagggatga tttcaattc 29 <210> 21 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 ctgctccctt tcaatcc 17 <210> 22 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 cctcctagac acagggatga tttcaattc 29 <210> 23 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 ccgagctcta tgaatcgaaa taatcaaaat gaat 34 <210> 24 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 cctcctagac acagggatga tttcaattc 29 <210> 25 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 25 acgcaaataa gccaaatcaa cc 22 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 26 ccaaacattt gcagggtcag 20 <210> 27 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 27 tgctgataac aatggcaatg aaa 23 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 28 tggtggtatt gttccggttc 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 29 gaatcagcgc ctgaacaatg 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 30 taatgcgagt tgctgcgata 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 31 gatgggtgct gtagtgagtt 20 <210> 32 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 32 tagcagtttc cttgcccca 19 <210> 33 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 33 acggttccat ccagtgattt a 21 <210> 34 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 34 tcaacatcca cagaaacctc a 21 <210> 35 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 35 ggcatattgc attaacagtt cca 23 <210> 36 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 36 agctgataaa ttacgccaaa caa 23

Claims (4)

The deposit number is KCTC13363BP and has insecticidal activity against Bradysia agrestis , Lycoriella ingenua , Aedes albopictus , Aedes togoi and Culex pipiens pallens . Bacillus thuringiensis subsp. Kyushuensis CAB464 strain, Bacillus thuringiensis . A method for screening a strain of the above-mentioned strain or a culture thereof containing an effective ingredient, such as Bradysia agrestis , Lycoriella ingenua , Aedes albopictus , Aedes togoi or Culex pipiens composition for insect control . delete An effective amount of a strain of Bacillus thuringiensis subsp. Israelensis CAB464 of claim 1 is selected from the group consisting of plants, soil or small root flies ( Bradysia agrestis ), Lycoriella ingenua , Aedes albopictus , Bradysia agrestis , Lycoriella ingenua , Aedes albopictus , Aedes togoi , and Togol forest mosquito by spraying them on habitat of Togo forest mosquito ( Aedes togoi ) or Culex pipiens pallens Or red house mosquito ( Culex pipiens pallens ).

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