KR100657014B1 - New Bacillus thuringiensis subsp. aizawai 00-FZ52-18KCTC10715BP having simultaneously insecticidal and antifungal activity and the method of controlling insect and fungi by using it - Google Patents

Abstract

The present invention relates to a novel Bacillus thuringiensis having both insecticidal and antifungal properties, and more particularly, Bacillus churros having insecticidal and growth inhibitory effects on phytopathogenic fungi at the same time, as well as lepidoptera. Bacillus thuringiensis subsp . Aizawai 00-FZ52-18 (KCTC10715BP). Bacillus thuringiensis subsp . Aizawai has been isolated from the manure of animals and therefore has higher stability to the human body.

Bacillus thuringiensis, insecticidal, bactericidal, microbial agent

Description

      Novel Bacillus thuringiensis subspecies Aizawa 00-FZ52-18 (KCTC10715BP) having both insecticidal and antifungal properties and methods for insecticide and fungicide using the same {New Bacillus thuringiensis subsp. aizawai 00-FZ52-18 (KCTC10715BP) having simultaneously insecticidal and antifungal activity and the method of controlling insect and fungi by using it}             

1 is the colony shape of Bacillus thuringiensis subsp. Aizawai on nutrient agar plates.

FIG. 2 is a phase contrast micrograph of Bacillus thuringiensis subsp. Aizawai 00-FZ52-18 (KCTC10715BP) after 5 days of incubation after separation from the powder.

3 shows the insecticidal test results of Bacillus thuringiensis subspecies Aizawa 00-FZ52-18 (KCTC10715BP).

4 is an experimental result showing that Bacillus thuringiensis subspecies Aizawa 00-FZ52-18 (KCTC10715BP) inhibits the growth of Botrytis cinerea . Where Bt is Bacillus thuringiensis 00-FZ52-18, BC is Botrytis cinerea, and CT is a control.

5 is an experimental result showing the inhibition of the growth of Bacillus thuringiensis subspecies Aizawa 00-FZ52-18 (KCTC10715BP) Diaporthe actinideae . Where Bt is Bacillus thuringiensis subspecies Aizawa 00-FZ52-18 (KCTC10715BP) and DA is Diaporthe actinideae .

6 shows three Bacillus thuringiensis strains resistant to Penicillium digitatum (A) and Penicillium italicum (B). A is the control group, B is 00-FZ46-12, C is Bacillus thuringiensis subspecies Aizawa 00-FZ52-18 (KCTC10715BP), and D is 00-FZ19-22.

7 shows the effect of Bacillus thuringiensis crude culture to prevent strawberries from spoiling by Botrytis cinerea . A is 00-FZ46-12, B is Bacillus thuringiensis subspecies Aizawa 00-FZ52-18 (KCTC10715BP), C is 00-FZ19-22, and D is a control.

To date, chemical synthetic pesticides, which have contributed greatly to human food production, have caused serious problems such as environmental pollution and destruction of ecosystems due to excessive use and abuse.In addition, about 1,400 people die of poisoning from pesticides every year. The damage is increasing day by day. In addition, the emergence of pathogens and insects that are resistant to chemical synthetic pesticides will inevitably lead to the use of larger amounts of pesticides, which will increase the damage. In order to overcome this problem, the development of pollution-free or low pollution pesticides are urgently required.

Therefore, microbial pesticides have been developed and used as microorganisms, and microbial pesticides based on Bacillus thuringiensis are collectively referred to as BT agents. Bacillus thuringiensis is a species of insect pathogens of the genus Bacillus sp., Which has high pathogenicity and easy industrial cultivation against larvae of some pests belonging to the order of Lepidoptera, fly, and beetle.

The BT strain, which is used for the production of BT, is a Kurstaki HD-1 strain which has excellent insecticidal insect pests and high productivity of insecticidal protein toxin. BT subspecies ( Bacillus thuringiensis subsp.Kurstaki ) is a gram-positive bacterium that forms follicles and produces endotoxins that have lethal effects on lepidopteran pests.

This toxin protein is ingested with food and is activated in the alkaline environment in the intestine and binds to specific receptors and is toxic. Therefore, this toxin protein is harmless to people who do not meet these conditions, animals, plants and fishes, and is a safe future pesticide for natural ecosystems Known.

BT bacteria that have antifungal properties in addition to insecticides so far include BT IPM-215 strain disclosed in the USSR patent SU1144377 A 350830 and BP865 strain disclosed in the Republic of Korea Patent No. 054108. IPM-215 strain is described as having antifungal activity against Verticillium dahiiae , Helminthosporium sativum and Fusarium culmorum , and BP865 strain. Has been described as having antifungal activity against Rhizoctonia solani ( A ) and Fusarium solani ( B ). Although the BT agent provided in Korean Patent 154108 has been reported to have insecticidal properties against Lepidoptera, there is no report on the insecticidality to the fly tree, which limits the possible range of use as an insecticide.

 The share of BT in the pesticide market is increasing every year, but still quite low. As of 1990, the world pesticide market is about 20 trillion won, 38% of herbicides, 33% of pesticides, 21% of fungicides, 8% of growth regulators and others. Occupies the bay. Therefore, in order to increase the demand for BT agents, it is necessary to develop strains having a broader insecticidal and antifungal range and higher insecticidal and antifungal effects. There was a need for the development of new BT strains with pesticidal and antifungal and higher pesticidal and antifungal properties.

With its broad insecticidal and high antifungal properties, BT makes it possible to treat insects and plant diseases in an environmentally friendly manner with little effort and cost. It is also necessary to develop strains which are not harmful to the human body during use.

It is an object of the present invention to provide a novel Bacillus thuringiensis strain.

Another aspect of the present invention is to provide a novel culture of Bacillus thuringiensis.

Another aspect of the present invention is to provide a microbial agent containing a novel Bacillus thuringiensis or a pure culture thereof as an active ingredient.

Another aspect of the present invention is to provide a method for controlling mold, characterized by spraying an effective amount of a microbial agent containing a novel Bacillus thuringiensis or a pure culture thereof to the plant for controlling the fungus.

Another aspect of the present invention is to provide a method for controlling insects, characterized in that the effective amount of the microbial agent containing the novel Bacillus thuringiensis or its pure culture is sprayed on the plants for controlling the insects. do.

Another aspect of the present invention is the simultaneous control of fungi and insects, characterized in that the effective amount of the microbial agent containing the novel Bacillus thuringiensis or its pure culture is sprayed on the plants simultaneously for the control of fungi and insects. It is an object to provide a method.

The present invention provides a novel Bacillus thuringiensis (BT) 00-FZ52-18 (KCTC10715BP) strain having both insecticidal and antifungal properties.

More specifically, not only Lepidoptera but also fly insects, the insecticidal effect is high, the bactericidal effect is high, and the antifungal effect against a wide phytopathogenic fungus, the antifungal effect is high, the Bacillus thuringiensis subspecies Aizawai (high antifungal effect) Bacillus thuringiensis subsp. Aizawai ) to provide KCTC10715BP.

Another aspect of the present invention aims to provide a pure culture of the novel Bacillus thuringiensis subsp. Aizawai KCTC10715BP.

Another aspect of the present invention is to provide a microbial agent containing a novel Bacillus thuringiensis subsp. Aizawai KCTC10715BP or a pure culture thereof as an active ingredient.

Another aspect of the present invention is characterized by spraying an effective amount of a microbial agent containing Bacillus thuringiensis subsp. Aizawai KCTC10715BP or its pure culture to a plant for control of fungi. It is to provide a method of controlling the fungus.

Another aspect of the invention is characterized by spraying an effective amount of a microbial agent containing Bacillus thuringiensis subsp. Aizawai KCTC10715BP or its pure culture to a plant for the control of insects. It is an object to provide a method for controlling insects.

Another aspect of the present invention is to simultaneously spray an effective amount of a microbial agent containing Bacillus thuringiensis subsp. Aizawai KCTC10715BP or a pure culture thereof to plants for the control of fungi and insects. It is an object of the present invention to provide a method for simultaneously controlling fungi and insects.

 Hereinafter, the present invention will be described in detail. Definitions of terms used in the description of the present invention are as follows.

The term “fungus” includes a wide range of eukaryotic spore-forming organisms without chlorophyll. Examples of fungi include yeast, filamentous fungi, fungi, rust fungi and mushrooms.

"Pesticide" means the ability of a substance to increase mortality or inhibit growth of plant pests.

"Antifungal" means the ability of a substance to kill or inhibit fungi.

An “effective amount” is an amount sufficient to produce a beneficial or desired result. An effective amount can be administered one or more times.

“Composition” is intended to mean a combination of an active agent and another compound, such as a carrier or composition, which is inert (eg, detectable drug or label or liquid carrier) or active (eg, adjuvant).

Insects include Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Tysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera and Akari It is selected from the group consisting of (Acari). In a particularly preferred embodiment, the insect is choloptera or lepidoptera. In another particularly preferred embodiment, the insect is a black cutworm ( Agrotis ipsilon ), a fall armyworm, Spodoptera frugiperda ], a beet armyworm ( Sakaka ) S. exigua ], yellow striped armyworm [ S. ornithogalti ], Southwestern corn borer ( Diatraea ) Diatraea grandiosella ], Sugarcane Borer [D. D. saccharalis ], corn earworm, Helicoverpa zea ], mediterranean corn borer, Sesamia nonagroides , cabbage Seminar rooms [cabbage looper, Trichoplusia ni], velvetbean caterpillar, Anticarsia gemmatalis , diamondback moth, Plutella xylostella ) And tobacco moths (tobacco budworm, Heliothis virescens ).

Pathogenic fungi of crops that can be controlled are selected from the group comprising:

Oomycetes group:

-Peronosporase, specifically Plasmopara viticola (vine neutrophil), Plasmopara halsteda (sunflower neutrophil), shadowed peronospora varieties (specifically gourds and neutrophils (sedoferonospora cubensis) and hops Fungus disease (shoeferronospora mushli)), bream lactose (lettuce fungus), feronospora tabacine (tobacco disease), feronospora destructor (onion disease), feronospora parasitica (Fungal disease of cabbage), feronospora parinosa (chicken fungal disease and beetroot disease);

- Look pitop Torah (Phytophthora) in, for example pitop Torah Pace up (Phytophthora phaseoli), pitop Torah seat Rob Torah (Phytophthora citrophthora), pitop Torah kaepsi Mr. (Phytophthora capsici), pitop Torah kaek torum (Phytophthora cactorum), pitop Torah palmitate ( Phytophthora palmivora ), Phytophthora cinnamoni , Phytophthora megasperma , Pytoptora parasitka , Phytophthora fragariae , Phytophthora cryptogara , Phytophthora cryptogea popito Phytophthora porri , Phytophthora nicotianae , Phytophthora infestans (a blight of eggplant, specifically late blight of potatoes or tomatoes);

Adelomycetes (Ascomycetes) group:

-Genus Alternaria, for example Alternaria solani (eg, branched diseases of eggplant, specifically tomatoes or potatoes),

-Genus Guignardia, specifically Guignardia bidwelli (vine vine blight),

In the genus Venturia, for example Venturia inaequalis , Venturia pirina (scab in apples or pears),

-Oidium genus, eg grape powdery mildew ( Uncinula necator ); Odium of legume crops, for example Erysiphe polygoni (powdery mildew); Leveillula taurica , Erysiphe cichoracearum , Sphaerotheca fuligena ( feminine , powdery mildew of chrysanthemum and tomato); Erysiphe communis (powdery disease of cabbage and beetroot); Erysiphe pisi (powdery mildew on pea and alfalfa); Erysiphe polyphaga (powdery mildew of kidney beans and cucumbers); Erysiphe umbelliferarum (mountains and plants, specifically powdery mildew of carrots);

Sphaerotheca humuli (fungal disease of hops); Barley and wheat germ disease ( Erysiphe graminis forma specie tritici and Erysiphe graminis forma specie hordei ),

-The genus of Taprina , for example Taphrina deformans (peach leaf curling disease),

Septoria , for example Septoria nodorum or Septoria tritici ( Septaria disease of cereals),

-Genus Sclerotinia , for example Sclerotinia sclerotinium ,

Shadowed genus, such as Pseudocercosporella herpotrichoides (eyespot disease of cereals),

-Genus Botrytis cinerea (vines, vegetables and market garden crops, peas and the like),

Phomopsis viticola (vine grapevine),

-In the genus Pyrenospora,

In the genus Helmintosporium, for example Helminthosporium tritici repentis (yellow spotted leaf disease of wheat) or Helminthosporium teres (yellow spotted leaf disease of barley),

-In the genus Drechslera or Pyrenophora,

Of the basidiomycetes group:

- pussi in California, for example pussi rekon dita Nia (Puccinia recondita), or registry FORT Miss (striiformis) (wheat rust), pussi California Tea tree seeds or (Puccinia triticina), pussi California Hor Day (Puccinia hordei),

-All of Lizatonia family, for example Rhizoctonia solani .

More specifically, Botrytis cinerea , Diaporthe actinideae , Botryosphaeria dothidea, Penicillium digitatum and Penicillium itaricum Penicillium italicum ).

Isolation and Identification of Mycobacteria

BT was separated in 1986 by the method of Ohba and Aizawa in Japan.

1 g of manure was suspended in 9 ml of sterile phosphate buffered saline. In order to kill heat-sensitive bacteria, the suspension was heated at 65 ° C. for 30 minutes and placed on a nutrient broth plate. Nutritious broth is pH7.8, consisting of 25g agar and 1000ml nutrition broth (10g gravy, 10g polypeptone, 2.5g sodium chloride, 1000ml distilled water). Plates were incubated at 28 ° C. for 3-4 days and BT colonies were observed under a microscope. BT, which forms the endotoxin protein crystals among the B.cereus / B.thringiensis complex, is called.

Thus isolated strains were classified and identified through insecticidal, hemolytic, lectin activity, immunodiffusion, immunoblotting.

Such BT strain was isolated as described above and named Bacillus thuringiensis (BT) 00-FZ52-18 (KCTC10715BP), and deposited in the Genetic Resources Center of Biotechnology Research Institute, an international depository institution, Korea Institute of Science and Technology. 10715BP.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to Examples.

Example 1 Insecticidal Assay

Silkworm moth (Bombyx mori) in the Lepidoptera as the target strains of live-release testing, cabbage was used as a moth (Plutella xylostella), tobacco giant seminar room (Spodoptera litura), beet armyworm (Spodoptera exigua), mosquitoes in Diptera (Aedes aegypti ) Was used.

Bacteria were grown in nutrient agar medium consisting of 25 g agar and 1000 ml nutrient broth (10 g broth, 10 g polypeptone, 2.5 g sodium chloride, 1000 ml distilled water) at pH 7.8 for 5 days at 27 ° C. And spores were formed (see FIG. 1), which were observed under a phase contrast microscope (see FIG. 2), and serotypes were identified and endotoxins were isolated.

The activity test was conducted for silkworm moth, Chinese cabbage moth, tobacco germinal moth, parchment moth, and mosquito.The test method was Ishii, T and Ohba M. (1993) Diversity of Bacillus thuringiensis environmental isolates showing larvicidal activity specific for mosquitoes.J. Gen. Microbiol. 139, 2849-2854), and Higuchi (Higuchi, K., Saitoh, H., Mizuki, E., Ichimatsu, T. and Ohba, M. (2000) Larval susceptibility of the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), to Bacillus thuringiensis H serovars isolated in Japan, Microbial. Res. 155, 23-29).

Briefly, artificial diets infected with 0.2 ml of spores / inclusion suspension (10 ^{7-10 8} spores / ml) in one 3-year-old larva were fed at 25 ° C. for 3 to 5 days. After 3 days, the mortality was examined and the anti-lethal concentration was calculated based on this. The insecticidal assay was repeated three times, and the results are shown in the following Table 1 and FIG. 3.

Table 1 Insecticidal Effects of Bacillus thuringiensis Isolated from Manure

[Table 2] Antiprism concentration

[Embodiment Example 2] Antifungal test

(1) Antifungal activity test against Botrytis cinerea

Bacillus thuringiensis (BT) 00-FZ52-18 (KCTC10715BP) was pH7.8 for 5 days at 27 ° C., 25 g of agar and 1000 ml nutrient broth (10 g juice, 10 g polypeptone, 2.5 g sodium chloride, 1000 ml distilled water). After growing in a nutrient medium consisting of), remove the round agar block of 1cm in diameter, transfer to the plate inoculated Botrytis cinerea and continued incubation after 5 days of incubation results in Figure 4 Indicated.

(2) Antifungal activity test against Diaporthe actinideae

Bacillus thuringiensis (BT) 00-FZ52-18 (KCTC10715BP) was pH7.8 for 5 days at 27 ° C., 25 g of agar and 1000 ml nutrient broth (10 g juice, 10 g polypeptone, 2.5 g sodium chloride, 1000 ml distilled water). After growing in a nutrient medium consisting of), remove the circular agar blocks of 1 cm in diameter, transfer them to the plate inoculated with Diaporthe actinideae and continue incubation. It was.

(3) Antifungal activity test for Penicillium digitatum and Penicillium italicum

Bacillus thuringiensis (BT) 00-FZ52-18 (KCTC10715BP), 00-FZ19-22 (KCTC10716BP) and 00-FZ46-12 (KCTC10717BP) were pH7.8 for 5 days at 27 ° C., 25 g of agar and 1000 After growing in a nutrient broth consisting of nutrient broth (10 g broth, 10 g polypeptone, 2.5 g sodium chloride, 1000 ml distilled water), a round 1-cm-diameter round agar block was removed, and penicillium digitatium ( Penicillium digitatum ) and Penicillium italicum ( Penicillium italicum ) was transferred to each plate inoculated, and after 5 days of continued incubation, the culture results are shown in Figs. 6 (A) and (B), respectively. A is the control group, B is Bacillus thuringiensis 00-FZ46-12 (KCTC10717BP), C is Bacillus thuringiensis subspecies 00-FZ52-18 (KCTC10715BP), D is Bacillus thuringiensis 00-FZ19-22 (KCTC10716BP) to be.

(3) Bacillus thuringiensis (BT) 00-FZ52-18 (KCTC10715BP) The control effect of Botrytis cinerea of strawberry

During the growth of strawberries, sprinkled with the culture culture cells of Bacillus thuringiensis (BT) 00-FZ52-18 (KCTC10715BP), 00-FZ19-22 (KCTC10716BP), 00-FZ46-12 (KCTC10717BP) Each was collected and examined for infection with gray mold is shown in FIG. A is Bacillus thuringiensis 00-FZ46-12 (KCTC10717BP), B is Bacillus thuringiensis subspecies 00-FZ52-18 (KCTC10715BP), C is Bacillus thuringiensis 00-FZ19-22 (KCTC10716BP), and D is the control group to be.

(4) The antifungal action of Bacillus thuringiensis (BT) 00-FZ52-18 (KCTC10715BP) was tested in the same manner as above, and the results are shown in Table 3.

       Activity of Bacillus thuringiensis (BT) against fungi Activity level B.cinerea D.actinideae B.dothidea P.digitatum P.italicum 00-FZ52-18 (KCTC10715BP) +++ + ++ +++ + 00-FZ19-22 (KCTC10716BP) +++ +++ ++ +++ + 00-FZ46-12 (KCTC10171BP) +++ +++ + +++ ++

+++, very strong; ++, strong; +, With some

Based on the above results, the Bacillus thuringiensis strain 00-FZ52-18 of the present invention forms spores with endotoxin proteins for insects, which are harmless to the human body, but have excellent insecticidal properties and at the same time have antifungal action against plant pathogens. . Bacillus thuringiensis 00-FZ52-18 according to the present invention is isolated from the stool of the animal is harmless to the human body is excellent in stability. In particular, the BT agent known in the art has been known to have an insecticidal effect on Lepidoptera, but the BT agent according to the present invention shows an insecticidal effect to the fly tree, and thus a wide range of insecticidal effects. Therefore, the strain of the present invention has a wide range of insecticidal and antifungal activity, has a superior effect than the conventional BT agent. Therefore, the microbial agent containing the strain exhibits a wide range of insecticidal and antifungal effects at the same time, and is a non-toxic and environmentally friendly microbial agent to the human body.

Claims (9)

New strain Bacillus thuringiensis 00-FZ52-18 (KCTC10715BP). Pure culture of claim 1 strain. A microbial agent containing the strain of claim 1 or the pure culture of claim 2 as an active ingredient. A method for controlling a fungus, comprising spraying an effective amount of the microbial agent of claim 3 on a plant for controlling fungi. A method for controlling insects, characterized in that the effective amount of the microbial agent of claim 3 is sprayed on the plants for controlling the insects. A method for simultaneously controlling fungi and insects, wherein the effective amount of the microbial agent of claim 3 is sprayed on the plants at the same time for controlling fungi and insects. The method of claim 4, wherein The fungus is Botrytis cinerea , Diaporthe actinideae , Botryosphaeria dothidea, Penicillium digitatum and Penicillium itaricum ( Penicillium italicum ) is a method for controlling the fungus, characterized in that selected from the group consisting of. The method of claim 5, The insects are coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallopaga, Homoptera, Hemiptera Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, and Insect control method characterized in that it is selected from the group consisting of Akari (Acari) The method of claim 6, The fungus is Botrytis cinerea , Diaporthe actinideae , Botryosphaeria dothidea, Penicillium digitatum and Penicillium itaricum ( Penicillium italicum ), the insects are coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallopaga, Homomorph Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siponaftera (Siphonaptera), Trichoptera (Trichoptera) and Akari (Acari) selected from the group consisting of a method for the simultaneous control of fungi and insects.

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