KR100691512B1 - Insecticide and acaricide comprising benzylideneacetoneBZA and proline-tyrosinePT - Google Patents

Abstract

The invention gen drawer Douce four Mato pillar K1 ANU-Xn101 one insecticide, salbi substance is dibenzylidene acetone Isolation and Identification from the culture broth of (Xenorhabdus nematophila K1 ANU-Xn101, KACC 91136) (benzylideneacetone: BZA) and repetition (proline-tyrosine: PT). In the present invention, the insecticidal, acaricide and immunosuppressive action of the two substances BZA and PT derived from Genorabdus nematophila ANU-Xn101 are found through experiments, and in the present invention, benzylidene acetone (BZA) or phyti Insecticides and acaricides are provided as (PT) or a mixture thereof as an active ingredient.

Benzylidene Acetone, BZA, proline-tyrosine, Xenorabdus nematophila, Xn, Insect Hospital Nematodes, Insecticides, Acaricides, Immune Suppression

Description

Insecticide and acaricide comprising benzylideneacetone (BZA) and proline-tyrosine (PT)} as benzylidene acetone and phytic

FIG. 1 is a test result of BAMA and PT derived from ANU-Xn101 mixed with Beati and treated with Pabam moths, respectively.

2A and 2B show the results of analysis of cellular immunosuppression of BZA and PT through vesicle formation (nodulation) of Pabam moth blood cells.

3A and 3B show experimental results showing that BZA and PT inhibit PO activation induction for antigen (laminarin), respectively.

4A and 4B show the results of analyzing PLA2 activity inhibition through PO activity for BZA and PT, respectively.

5 is a result of comparative analysis of the inhibitory effect of PLA2 activity by combining BZA and PT material alone or mixed, respectively.

The invention gen drawer Douce four Mato pillar K1 ANU-Xn101 one insecticide, salbi substance is dibenzylidene acetone Isolation and Identification from the culture broth of (Xenorhabdus nematophila K1 ANU-Xn101, KACC 91136) (benzylideneacetone: BZA) and repetition (proline-tyrosine: PT).

Xenorhabdus nematophila (Xn), a type of insect pathogenic bacterium, coexists in the gut of insect pathogenic nematodes (Akhurst, 1980, 1986). When the host nematode invades the bloodstream of a target insect, the bacteria are released from the intestine's intestine and released into the insect bloodstream.

Hemolymph, which contains a mixture of vertebrate blood and lymph fluid, is located in the insect's blood cavity. Hemolymph is composed of plasma (hema) and blood cells (hemocyte), and has almost similar functions in addition to the oxygen transport ability of the blood of vertebrates. In particular, hematopoietic cells have cellular immune reactions against foreign invaders such as nematodes or bacteria, and humoral immune reactions with various antibiotics in plasma.

These insect immune responses are staged by adjuvant recognition, immune mediation and immune response (Gillespie et al., 1997). It is not clear how the insect's immune system recognizes foreign invaders. However, there is no specific foreign invader recognition response due to the surface protein of the major histocompatibility complex (MHC) of vertebrates and the T cell in the specific epitope, Recognition proteins capable of recognizing characteristic structures (lectins, integrins, etc.) are responsible for this task. The peculiar structure of the pathogen is peptidoglycan specific to the bacterial wall, lipopolysaccharide (LPS) present in the outer membrane of Gram-negative bacteria, β-1,3-glucans and β-1 specific to fungi. Recognition of this pattern by, 3-mannans, etc. is considered to be nonmagnetic recognition of insects (Janeway, 1994). Eicosanoids with 20 carbon atoms are considered to be the major immune mediators specific for bacterial invasion (Stanley, 2000). The enzymatic action of PLA2 (phospholipase A2) from the phospholipid, a cell membrane component, produces arachidonic acid (AA), a C20 unsaturated fatty acid, and AA is further oxidized with the help of COX (cyclooxigenase) or LOX (lipoxygenase). And conversion to eicosanoids. In addition, PSP (plasmatocyte spreading peptide), a cytokine of insects, has been known to mediate adhesion of foreign factors through structural changes of atypical blood cells by immuno-mediated immunity to atypical blood cells after recognition from granulocytes (Clark et al. , 1997). In addition to the humoral immune response by antibiotics such as lysozyme and cecropin, the immune response results in nodulation, phagocytosis and encapsulation in which blood cells directly participate.

An immediate problem with invading nematodes and symbiotic bacteria released from them is the immune defense mechanisms of insect blood lymphocytes. As a way to cope, pathogens generally exert mechanisms of immune recognition, suppression and tolerance (Dowds and Peters, 2001). However, the exact distinction between them is very difficult, and in fact, these mechanisms can be seen as immunodepression. Xenorhabdus nematophila (Xn) induces immunosuppression of PLA2 (Park and Kim, 2000). Inhibition of PLA2 by Xn does not lead to an immune response because eicosanoids are not produced by AA release inhibition even if blood cells recognize the invasion of foreign pathogens.

After suppressing immunity, Xn induces direct cell death. In particular, lethality of blood cells eventually results in irreversible inhibition of immune responses due to blood cells. On the other hand, immunosuppression of insects due to Xn can allow invasion of other pathogens in nature. Since the development of symbiotic nematodes requires monoxenic conditions of symbiotic bacteria alone, it is necessary to secrete antibiotics that can inhibit similar bacterial species or even taxonomically very heterogeneous types of bacteria and fungi. Ultimately, the infected insects in this process will be fatal and nematodes will develop in the optimum conditions for nematode development and the next generation will emerge.

Previous studies have shown that Xn inhibits PLA2 from Spodoptera exigua using specific inhibitors of the eicosanoids production circuit. In addition, the inventors identified and identified Xn from the Steinernema carpocapsae nematodes collected in Korea in another invention and named it Genorabdus nematophila ANU-Xn101 (KACC 91136). It has been shown to have a power. The present invention relates to benzylideneacetone and proline-tyrosine (PT), which are PLA2 inhibitors, isolated from the strain culture medium of Genorabdus nematophila ANU-Xn101 (KACC 91136).

In the present invention, two substances, benzylideneacetone and proline-tyrosine (PT), which inhibit PLA2, are isolated and identified from the strain culture medium of Xenorabdu nematophila ANU-Xn101 (KACC 91136).

In the present invention, the pesticidal action, acaricide action, and immunosuppression action of two substances derived from Genorabdus nematophila ANU-Xn101, BZA and PT, are found through experiments.

The present invention provides the use of BZA and PT based on the above facts.

Other objects and advantages of the invention will be described below, and will be better understood by practice of the invention.

The present invention, benzylidene acetone ( Xenorhabdus nematophila K1 ANU-Xn101, strain depositing institution: Institute of Agricultural Biotechnology, deposit date: 2004.8.17, accession number: KACC 91136) benzylideneacetone) and phytic-tyrosine (PT).

In the present invention, an insecticide comprising benzylidene acetone (BZA) or phyti (PT) or a mixture thereof as an active ingredient is provided.

Moreover, in this invention, the acaricide which uses benzylidene acetone (BZA) or phyti (PT) or a mixture thereof as an active ingredient is provided.

Xenorabdu nematophila K1 ANU-Xn101 herein may also be simply referred to as "Xenorabdu nematophila K1" or "ANU-Xn101".

Hereinafter, the present invention will be described in detail.

Benolidene nematophila ANU-Xn101 (KACC 91136) was incubated in TSB medium for 48 hours, followed by centrifugation with culture medium, and supernatant with benzylideneacetone (BZA). Piline (proline-tyrosine (PT)) was isolated and identified. The following experiments confirmed the efficacy of the two separated substances.

I. Insecticidal action

Insecticidal activity of two species, Benzylidene Acetone (BZA) and Piti (PT), was isolated from ANU-Xn101 culture medium. Artificial feed having a size of 1 cm 3 was immersed in the prepared treatment solution and then dried, and then administered to the two insects of Pabam moth. The test results are shown in Tables 1 and 2 below.

Test results show that both substances have high insecticidal properties against pabam moths. Insecticidal effect appeared after 1 day of treatment and stable insecticidal effect after 2 days. Comparing the two substances on this basis, it can be seen that there is no difference statistically (95% confidence interval overlap). These results indicate that each of these substances can be used alone as an insecticide, and it can be used as a biochemical pesticide applied to the culture medium in order to explain the causative agent of the culture solution pesticidal effect.

Based on these results, the packaging control of the two materials was analyzed. The field trials were applied with BZA and PT on the side of peanut balm. As a control drug, a biopesticide, Bti (Batis Izai granulative, Bacillus thuringiensis aizawai , Centari ™, Advance Industry) was used. The survey was conducted on 72 hours based on indoor results. The results are shown in Table 3 below.

The bitty pesticide used as a control drug showed 100% control against pabam moth at 500 ppm. Two substances from ANU-Xn101, BZA and PT, also showed 100% insecticidal at the same concentration, indicating that they could be used as novel insecticides with comparable effects to Beatty.

Based on the fact that the present inventors showed the synergistic effect of Genorabdus nematophila ANU-Xn101 with bitty pesticides, in the present invention, in order to prove the effect of this ANU-Xn101 culture solution as a specific substance, ANU-Xn101 Two derivatives, BZA and PT, were mixed with Beati and treated with Pabam moths, respectively. Test results are shown in FIG.

Test results have shown that both BZA and PT have a high cooperative effect on Beatty, and this synergistic effect can be explained by two mechanisms. The first is the insecticidal effect of the two substances, and the second is the substance extracted from the culture medium, which may be secreted into the insect cavity when the bacteria invade the insect and directly affect the insecticidal effect of the insect. In other words, Beatty assisted the transfer of the material into the body cavity of the insect, and the transferred material may have influenced the insect's pesticidal effect to accelerate.

From the above results, BZA and PT can be regarded as a causative agent of ANU-Xn101 culture medium with an excellent synergistic effect against Bt.

II. Fertilization

The effects of fertilization on the adult Tetranychus urticae of two substances, BZA and PT, derived from ANU-Xn101 culture medium were analyzed. The results are shown in Tables 4 and 5 below.

The results show that both materials BZA and PT have a high killing capacity against spotted mites. Of these two substances, BZA was found to have higher activity than PT. However, the half-dose doses of BZA and PT are higher than the results of treatment with ANU-Xn101 media, in addition to these two substances, there is a third substance in the culture or between Suggests a synergistic effect.

III. Immunosuppressive action

The cellular immunosuppressive responses of ANU-Xn101-derived BZA and PT were analyzed through nodulation reactions of Pabam moth blood cells. The results are shown in Figures 2a and 2b. Both substances have been shown to inhibit the follicle formation response of Pabam moth blood cells even at low concentrations, thereby suppressing cellular immune responses.

These two substances have also been shown to inhibit PO activation induction for antigens such as laminarin (Figures 3a and 3b). From these results, it can be seen that both BZA and PT inhibit both cellular and humoral immune responses.

Whether these two substances directly inhibit PLA2 activity was analyzed using various endpoints. First, two substances were added to the mixture of the fat cell membrane extract and the PLA2 extract of the insect, and the release of AA was analyzed by gas chromatography (GC).

1 ㎍ of arachidonic acid (AA) dissolved in 50% ethanol into gas chromatography (Hewlett-Packard SD 6890 gas chromatography, San Fernando, CA, USA) (retention time) was investigated. For GC analysis, tissues of bloody moth larvae 5 larvae (blood cells, fat bodies, intestines) that were surface sterilized in 75% ethanol were collected in microtubes. Each tissue collected was washed three times in homogenizing buffer [50 mM PBS (pH 7.0), 0.45 M NaCl]. The tissues extracted from the glass-glass homogenizer were sufficiently crushed until they became micelles. And the supernatant was collected by centrifugation for 10 minutes at 1,000 g of 4 ℃. The collected supernatant was centrifuged again at 30,000 g at 4 ° C for 30 minutes, and the precipitate and supernatant were used as membrane and PLA2 rich fraction for GC analysis, respectively. The amount of membrane and PLA2 rich fraction separated was examined by Bradford (1972) protein assay.

The amount of AA released by symbiotic bacteria was investigated by using membrane (500 mg) and PLA2 rich fraction (4 mg) separated from fat body. The symbiotic bacteria were prepared by suspension using PBS for each concentration (101, 102, 104, 106 cfu / μl), and treated with 10 μl of suspension of each bacterial concentration in a microtube treated with membrane and PLA2 rich fraction. After 30 minutes of reaction time, the amount of free AA was investigated. The symbiotic bacteria (106 cfu / μl) were heat treated at 90 ° C. for 30 minutes to treat 10 μl of the bacterial suspension in a microtube treated with membrane (500 mg) and PLA2 rich fraction (4 mg). ) And the amount of AA liberated by the dead bacteria by heat treatment. In addition, 10 μl of the organic fraction separated from the culture solution using the culture solution (tryptic soy broth, Difco, USA) and ethyl ether, which were incubated at 28 ° C. for 2 days, were treated with 10 μl of PLA 2 rich fraction in a microtube. After a minute reaction time, the amount of free AA was investigated. The results are shown in Table 6 below.

The results show that both materials (# 11-1, # 17-3 / # 18-1) significantly inhibit the release of AA (20: 4).

Next, we analyzed the inhibitory effects of PLA2 on the concentrations of BZA and PT. Previous studies have shown a direct relationship between PLA2 inhibition and PO (phenoloxidase) activity inhibition in Pabam moths. Based on this, the inhibition of PLA2 activity was analyzed through easy PO analysis. The results are shown in Figures 4A and 4B.

Even at low concentrations of BZA and PT, PABAM PLA2 was inhibited. At this point, we can hypothesize the cooperative action of the two substances. In other words, if PLA2 involved in insect immunity is heterogeneous or more, it is possible to hypothesize that these two substances have selectivity for each. This assumption can lead to the fact that both BZA and PT can inhibit different PLA2. In order to prove this, the same amount of a single or mixed inhibitor was prepared to compare the inhibitory effect, and the results are shown in FIG. 5. However, this result shows that the mixed solution of the two substances did not exert a higher inhibitory effect than the inhibition of PLA2 alone, the hypothesis that PLA2 involved in the immunity of insects may be heterogeneous or higher.

In the present invention, BZA and PT having insecticidal, acaricidal and immunosuppressive effects are isolated and identified from the strain culture medium of Genorabdus nematophila ANU-Xn101 (KACC 91136), and the efficacy of bio-derived active substances is confirmed. Enable effective pesticide development

Claims (3)

Agricultural pesticides comprising benzylidene acetone (BZA) or proty-tyrosine (PT) or a mixture thereof as an active ingredient. Acaricides containing benzylidene acetone (BZA) or proty-tyrosine (PT) or a mixture thereof as an active ingredient. A method for agricultural pesticides characterized in that the use of benzylidene acetone (BZA) or proty-tyrosine (PT) or a mixture thereof together with Bacillus thuringiensis .

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