KR101194127B1 - Insecticide composition having enhanced insecticidal activity of an entomopathogenic fungus, Beauveria bassiana and insect control method using it - Google Patents

Abstract

The present invention relates to an insecticidal composition that improves the insecticidal power of Buberia abasiana and a control method using the same. In the present invention, it was confirmed that the benzylidene acetone inhibits the immune response of pabam moth to rapidly increase the penetration force of burberry abasiana and significantly increase the mortality rate. Also, this insecticidal effect is the lepidopteran moth ( Plutella) xylostella ), it was confirmed that it shows an empirical control effect. Based on these results, the present invention provides a pesticide composition comprising buberia abaciana and benzylidene acetone as active ingredients.

Description

Insecticide composition having enhanced insecticidal activity of an entomopathogenic fungus, Beauveria bassiana and insect control method using it}

The present invention relates to pesticides and methods for controlling pesticides, and more particularly, to pesticide compositions and methods for controlling pesticides using Burberry avaciana.

The insect-pathogenic fungus Beauveria bassiana is used as a biological control agent with a relatively wide range of target pests. However, compared to chemical pesticides, the expression of insecticidal effect is slow, and it is not widely used due to the inconsistent medicinal properties based on the pathology according to natural environmental conditions (Wraight et al., 2001).

[references]

Gho HK, Lee SG, Lee BP, Choi KM, Kim JH. 1991.Simple mass-rearing of beet armyworm, Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae), on an artificial diet. Kor. J. Appl. Entomol. 29: 180-183.

Ji D, Yi Y, Kang GH, Choi YH, Kim P, Baek NI, Kim Y. 2004.Identification of an antibacterial compound, benzylideneacetone, from Xenorhabdus nematophila against major plant-pathogenic bacteria. FEMS Microbiol, Lett. 239: 241-248.

Kwon B, Kim Y. 2008. Benzylideneacetone, an immunosuppressant, enhances virulence of Bacillus theuringiensis against beet armyworm (Lepidoptera: Noctuidae). J. Econ. Entomol. 101: 36-41.

Wraight SP, Jackson MA, de Kock SL. 2001. Production, stabilization and formulation of fungal biological agents. In; Butt TM, Jackson C, Magan N(eds), fungi as biocontrol agents. CABI, Wallingford, pp. 253-287.

An object of the present invention is to provide a pesticide composition and a control method capable of rapidly expressing the insecticidal effect of Beauveria bassiana . In addition, an object of the present invention is to provide an insecticidal composition and control method using buberia abasiana, which enhances the insecticidal power of buberia abasiana and can reproducibly exhibit a certain insecticidal power even in natural environmental conditions.

Benzylideneacetone (trans-4-phenyl-3-buten-2-one) is a monoterpene compound that was identified and derived from a culture of the insect pathogen Xenorhabdus nematophila (Ji et al). ., 2004). It has been shown to block the biosynthesis of icosanoids, which are important for the mediation of insect immune signals, leading to immunosuppression (Kwon and Kim, 2008).

In the present invention, when using the immunosuppressive effect of benzylidene acetone (BZA), focusing on the fact that it is possible to increase the insecticidal power and early expression of the insecticidal effect of Burberry avaciana, it is used in combination with benzylidene acetone (BZA). We investigated whether it can improve the hospitality of Bassiana. As a result, it was confirmed that benzylidene acetone suppresses the immune response of pabam moth, thereby speeding up the blood-settlement rate of burberry avaciana and significantly increasing the lethality. In addition, in the present invention, it was confirmed that this insecticidal effect exhibits an empirical control effect against Pleella xylostella , a lepidopteran pest that has occurred outdoors.

Based on these results, in the present invention,

Provided is an insecticide composition comprising buberia abasiana and benzylidene acetone as active ingredients. Benzylidene acetone, used in conjunction with buberia abasiana, increases immunity of pabam moth by increasing the penetration and viability of buberia abasiana, thereby increasing its pathogenicity. The mixing ratio of buberia abaciana and benzylidene acetone is preferably, buberia abasiana 1.0x10 ⁵ to 1.0x10 ⁶ benzylidene acetone per cfu/mL is used in 0.5 to 5 mg, more preferably buberia abashi Ana 1.0x10 ⁵ cfu/mL of benzylidene acetone is used at about 1 mg.

Also in the present invention,

A pest control method is provided, which is characterized by using buberia abasiana and benzylidene acetone together. Here, “to be used together” means that the crops are processed at the same time or sequentially at regular time intervals. Preferably, the treatment of Buberia abasiana culture (1.0x10 ⁵ ~1.0x10 ⁶ cfu/mL) and benzylidene acetone is performed at a concentration of 400 to 4,000 ppm, respectively, and more preferably at a concentration of about 1,000 to 2,000 ppm. .

In the present invention, when benzylidene acetone is used together with buberia abaciana, it can increase the pathogenicity of buberia abaciana and the synergistic effect of benzylidene acetone is the cellular and humoral immunity of pabam moth against buberia abaciana. It was found that it was caused by suppressing each reaction, thereby increasing the ability of Buberia abasiana to settle for the fava moth bloodstream. The mixed insecticide composition of the present invention using this action of benzylidene acetone rapidly expresses the insecticidal effect of buberia abaciana and enhances the insecticidal power, so it can exhibit reproducible insecticidal power even in natural environmental conditions.

Figure 1a is a test result of the insecticidal effect on the papaver moth of buberia abasiana (Bb), 1b is a result of comparing the respective insecticidal effects of buberia abasiana (Bb) and benzylidene acetone (BZA). Different characters on the standard deviation bar mean that there is a statistically significant difference at the 95% significance level.
Figure 2 is the experimental results for the effect of benzylidene acetone (BZA) on the increase in the invasion of the bloodstream of the insects of Buberia abasiana (Bb). The asterisk (*) means that the mixed treatment was statistically different at a 95% significant difference (LSD test) compared to the Bb alone treatment.
Figure 3a is a photograph showing the formation of vesicles by the cellular immune response of papam moth against the invasion of buberia abasiana (Bb). Figure 3b is a result of blood cell vesicle formation by concentration of pabam moth for buberia abasiana (Bb), Figure 3c is a result of blood cell vesicle formation by time, and Figure 3d is a mixed use with BZA (1,000 ppm) to inhibit vesicle formation The effect is compared with that of Buberia Abasiana (Bb) alone.
Figure 4 is the effect of benzylidene acetone (BZA) on the humoral immune suppression of pabam moth, Figure 4a is a result of the expression of the immune-related gene (RT-PCR) according to the treatment with buberia vasiana (Bb), Figure 4b Is a result of suppressing the expression of the immune gene according to the BZA feeding treatment.
FIG. 5 is a mixed control effect of buberia abasiana (Bb) and benzylidene acetone (BZA) on outdoor cabbage moth.

Beauveria bassiana , a white beetle that causes disease specifically for insects, is a Spodoptera exigua ) was shown (Fig. 1a). However, it had to take about a week to show effective control (Fig. 1B). When benzylideneacetone (trans-4-phenyl-3-buten-2-one), which induces immunosuppression of insects, was treated with a mixture of buberia abaciana, the fatality rate of papam moth was significantly promoted (FIG. 1B). ). This synergistic effect of benzylidene acetone was attributed to an increase in the ability of Buberia abasiana to settle into the blood cells of the pabam moth. And this increase in the ability to settle was shown by benzylidene acetone suppressing the cellular and humoral immune responses of pabam moth to buberia abaciana, respectively. Based on these results, as a result of experimenting with the control effect on the outdoor moth ( Plutella xylostella ), benzylidene acetone was found to significantly improve the insecticidal activity of Buberia abasiana. These results suggest that benzylidene acetone can be used as a co-operative to enhance the insecticidal effect of buberia vasiana. The pesticidal composition and control method of the present invention comprising buberia abasiana and benzylidene acetone as active ingredients may be applied to the target pest range of buberia abasiana, but also not limited thereto, according to an increase in the pesticidal power of buberia abasiana The pesticidal effect can be applied to all recognized pest ranges.

[ Example ]

Hereinafter, the present invention will be described in more detail through specific examples. However, these examples are only intended to illustrate the present invention in more detail, and the scope of the present invention is not limited by these examples.

Materials and methods

1. Breeding insects

The papam moth larvae were bred as artificial feed (Gho et al., 1991). All adults were supplied with 10% sugar water. During the whole breeding period, the temperature was maintained at 25±1°C, relative humidity 60±10%, and photoperiod 16:8 h (L:D). For the Chinese cabbage moth, the larvae collected in the cabbage field located in Songcheon-dong, Andong-si were used for indoor breeding without chemical treatment. The larvae were fed cabbage in a culture medium with a temperature of 25±1°C, a photoperiod of 16:8 h (L:D) and a relative humidity of 40 to 60%. Adults induced spawning with Chinese cabbage leaves by feeding 10% sugar water.

2. Proliferation and bioassay of Buberia abasiana

Buberia Abasiana was purely separated from BotanyGuard™ (Buberia Abasiana H.A.S. suspension), sold by Arista LifeScience Korea. Botanical Guard™ is a microbial fertilizer that uses B. bassiana GHA (1.0x10 ⁸ cfu/mL) as an active ingredient and is an emulsion type containing minerals. Buberia abasiana was propagated through passage culture in a medium of potato dextrose agar (PDA): potato starch 20.0 g, dextrose 15.0 g, agar 15.0 g, 1 L distilled water). 1 ml of water (1.0x10 ⁵ cfu/mL) was added to 1 ml of buberia abaciana to prepare 1,000 ppm. In the same way, benzylidene acetone (Sigma-Aldrich Korea, Seoul, Korea) was dissolved in dimethyl sulfoxide (DMSO) to prepare a high concentration solution of 100,000 ppm, mixed with distilled water, diluted to a low concentration, and used in the experiment. Did. After making Buberia abasiana and benzylidene acetone at 2,000 ppm each, immersing cabbage leaves (1 cm 2) in a solution mixed in a 1:1 ratio for 10 minutes, and then processing cabbage in a filtered container (9 cm in diameter). The leaves were dried for 1 minute. Each treated cabbage leaf was treated with 3 repeats of 10 papam moths, and the number of survivals was counted for 7 days with a cycle of 24 hours.

3. The effect of enhancing the initial settling power of Buberia abasiana on insect blood

After sprinkling 20 μL of a mixture of buberia abasiana (1,000 ppm) and benzylidene acetone (1,000 ppm) evenly on cabbage leaves (1 cm 2 ), 5 insects that were fasted for 5 hours were fed for 5 days. Each time, only the chestnut moth fed the cabbage leaves was selected to extract the blood lymph and the plasma was discarded through centrifugation (10,000 rpm, 10 minutes) to obtain a precipitate. After adding 20 μL of distilled water and dissolving the precipitate, 20 μL of the precipitate dissolved in PDA medium was added and cultured for 7 days in a 25°C incubator.

4. Analysis of vesicle formation

The viewberry abarsiana cultured in PDA was collected and mixed with distilled water to remove the suspension, the hyphae was filtered through filter paper and only spores were separated. This was filtered again through a syringe to separate only pure spores. The number of spores was calculated using a hemocytometer (Hemocytometer: 0.0025 포, Marienfeld, Germany) and concentrated for each concentration. The separated spore suspensions were inoculated with 5 μL of each of the five insects of Pam moth.

5. Antibiotic protein expression analysis

The heat-treated Buberia abasiana was inoculated with 5 μL of each of the five insects of the Pam moth. Fats were separated by time, and total RNA was extracted therefrom. CDNA was prepared based on the extracted RNA, and expression patterns were examined through PCR (Polymerase Chain Reaction) using primers specific to the gene (Table 1). PCR was conducted in 35 cycles, and each cycle consisted of 1 minute at 95°C, 1 minute at 48°C, and 1 minute at 72°C.

6. Investigation of packaging control power

Experiments were conducted using a mixture of buberia abasiana (1,000 ppm) alone and benzylidene acetone (1,000 ppm), and a control and control drug. Chinese cabbage moths generated on Chinese cabbage grown in the field were treated with 3 repetitions of the egg mass method. Survival was counted daily for a total of 7 days.

result

1. The effect of cooperation between Buberia Abasiana and Benzylidene Acetone

The results of investigating the pathogenicity of papam moth are shown in FIGS. 1A and 1B. Figure 1a is a result of experimenting the pathogenicity of the papaver moth of Burberry abasiana, the insecticidal power was investigated for 7 days after treating Burberry abasiana by concentration by leaf immersion, and as a result, the insecticidal power increased in proportion to the concentration I could confirm. Figure 1b is a comparison of the insecticidal power of the papaver moth of the view (A), a mixture of buberia abaxiana alone (○), a mixture of buberia abaxiana and benzylidene acetone (▼) and benzylidene acetone alone (●) by treatment day. In the case of Buberia abasiana alone (○), it showed a low control efficiency until 6 days after treatment, and the insecticidal effect increased rapidly on the 7th day. However, in the case of treatment with a mixture of benzylidene acetone in buberia abasiana (▼), a clear control effect was observed after 1 day of treatment, and the insecticidal effect increased with time. On the other hand, benzylidene acetone alone (●) did not show an insecticidal effect.

2. The effect of enhancing the initial settling power of Buberia abasiana on insect blood

The effect of benzylidene acetone on the increase in body invasion of buberia abasiana (Bb) was analyzed, and the results are shown in FIG. 2. It has been found that the increased pathogenicity of benzylidene acetone to buberia vasiana is due to the compound's ability to promote fungal penetration into the bloodstream of insects and early settling ability. When mixed with benzylidene acetone compared to treatment with fungi alone, there was a clear difference in initial fixation ability at 24 hours after treatment, which is the initial infection time.

3. follicular formation

As a result of the analysis of vesicle formation, pabam moth showed vesicle formation, a cellular immune response against the invasion of buberia vasiana (FIG. 3A). When more than 106 conidia spores were injected into each of the five nightworms, the maximum vesicle formation density was about 60 per individual (FIG. 3B). In addition, a certain number of spores were injected and the number of vesicles was examined hourly. As a result, the maximum vesicle density was observed between 4-11 hours after treatment, and the formed vesicles were then removed (FIG. 3C). Based on the above results, 10 ⁶ spores were injected per individual, benzylidene acetone was treated together, and vesicles 8 hours after treatment were examined. As a result, when the benzylidene acetone was treated together, it was found that the vesicle-forming ability of pabam moth was significantly lowered (FIG. 3D).

4. Antibiotic protein expression analysis

It was investigated whether benzylidene acetone inhibits the expression of an immunogene formed by pabam moth against buberia abaciana. The results for the humoral immunosuppressive effect of benzylidene acetone (BZA) are shown in FIG. 4. As a result of investigating ten immunogenes derived from papam moth, they were all expressed in the Burberry avaciana treatment group (FIG. 4A ). However, the expression of apoLpIII, attacin A and cecropin genes was suppressed when inoculation with buberia avaciana again after 24 hours after feeding benzylidene acetone (FIG. 4B).

From the above results, it was judged that benzylidene acetone increased humoral immunity by inhibiting humoral immunity of pabam moth, thereby increasing the penetration and viability of buberia vasiana.

5. Packaging control power

Based on the above experimental results, it was investigated whether the mixed use with benzylidene acetone enhances the control effect against Chinese cabbage moths generated in outdoor farmland, and the results are shown in FIG. 5. In the case of the mixed treatment, there was a clear difference in the control at the 3rd day of the treatment, which is the initial effect, compared to the treatment with Buberia Abasiana alone.

The pesticide composition of the present invention rapidly expresses the insecticidal effect of the biopesticides, Buberia Abasiana, enhances the insecticidal power, and exhibits reproducible insecticidal power even in natural environmental conditions, replacing conventional biological control agents and chemical insecticides. Can be widely used. The pesticidal composition and control method of the present invention using buberia abasiana and benzylidene acetone as active ingredients can be basically applied to the target pest range of buberia abasiana, and further, not limited thereto, all pest ranges for which insecticidal effects are recognized Can be applied to.

Claims (4)

In the pesticidal composition using buberia abaciana as an active ingredient, the insecticidal composition further comprising benzylidene acetone as an active ingredient that rapidly expresses and enhances the insecticidal power of buberia abasiana. The insecticide composition of claim 1, comprising 0.5 to 5 mg of benzylidene acetone per 1.0x10 ⁵ to 1.0x10 ⁶ cfu/mL of Buberia abasiana. In the pest control method using buberia abasiana as an active ingredient, a pest control method characterized by using benzylidene acetone as an active ingredient that rapidly expresses and enhances the insecticidal power of buberia abasiana. [4] The method of controlling pests according to claim 3, wherein the buberia abasiana culture solution (1.0x10 ⁵ to 1.0x10 ⁶ cfu/mL) and benzylidene acetone are used at concentrations of 400 to 4,000 ppm, respectively.

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