KR100835535B1 - Composition of new microbial pesticide using technique suppressing insect immune capacity - Google Patents

Abstract

A juvenile hormone or an analogue thereof is provided to inhibit immunity of an insect, thereby being used as a novel biotic pesticide. A method for sterilizing insects is provided to enhance the activity of a conventional biotic pesticide by using the juvenile hormone or the analogue thereof in combination with Bacillus thuringiensis. An agricultural insecticide comprises a juvenile hormone or an analogue thereof, which enhances the activity of a microbial pesticide, and Bacillus thuringiensis, wherein the analogue is pyriproxyfen. To kill insects, the juvenile hormone or the analogue thereof is used together with Bacillus thuringiensis. The juvenile hormone or the analogue thereof is used together with Bacillus thuringiensis in order to sterilize insects.

Description

Composition of New Microbial Pesticide Using Technique Suppressing Insect Immune Capacity

1 is a Chinese cabbage moth larva ( Plutella) Hemocytosis of xylostella larvae) was observed by phase contrast microscope.

Figures 2a and 2b graphically shows the effect of the hormone on the hemagglutination of the cabbage moth larvae.

3A and 3B show the results of analyzing the interaction of JH and 20E on hemagglutination.

Figure 4 is a pyriproxyfen ('PYR') and Beati ( Bacillus ) targeting four cabbage moths thuringiensis subsp. kurstaki : Result of testing the synergistic effect of '(Bt').

Beckage, NE and LM Riddiford. 1982.Effects of parasitism by Apanteles congregatus on the endocrine physiology of the tobacco hornworm, Manduca sexta . Gen. Comp. Endocrinol. 47: 308-322.

Beckage, NE, FF Tan, KW Schleifer, RD Lane and LL Cherubin. 1994. Characterization and biological effects of Cotesia congregata polydnavirus on host larvae of the tobacco hornworm, Manduca sexta . Arch. Insect Biochem. Physiol. 26: 165-195.

Clark, K.D., Y. Kim and M.R. Strand. 2005. Plasmatocyte sensitivity to plasmatocyte spreading peptide (PSP) fluctuates with the larval molting cycle. J. Insect Physiol. 51: 587-596.

Cole, T. J., N. E. Beckage, F. F. Tan, A. Srinivasan and S.B. Ramaswamy. 2002. Parasitoid-host endocrine relationships: self-reliance or co-optation Insect Biochem. Mol. Biol. 32: 1673-1679.

Fathpour, H. and DL Dahlman. 1995. Polydnavirus of Microplitis croceipes prolongs the larval period and changes hemolymph protein content of the host, Heliothis virescens . Arch. Insect Biochem. Physiol. 28: 33-48.

Figueiredo, MB, DP Castro, NFS Nogueira, ES Garcia and P. Azambuja. 2006.Cellular immune response in Rhodnius prolixus : role of ecdysone in hemocyte phagocytosis. J. Insect Physiol. 52: 711-716.

Fleming, J-A.G.W. 1992. Polydnaviruses: mutualists and pathogens. Annu. Rev. Entomol. 37: 401-425.

Franssens, V., G. Smagghe, G. Simonet, I. Claeys, B. Breugelmans, A. De Loof and JV Broeck. 2006. 20-Hydroxyecdysone and juvenile hormone regulate the laminarin-induced nodulation reaction in larvae of the flesh fly, Neobellieria bullata . Dev. Comp. Immunol. 30: 735-740.

Hoffmann, JA 1970. Endocrine regulation of production and differentiation of haemocytes in an orthopteran insect Locusta migratoria migratoroides . Gen. Comp. Endocrinol. 15: 198-219.

Ibrahim, AMA and Y. Kim. 2006. Parasitism by Cotesia plutellae alters the hemocyte population and immunological function of the diamondback moth, Plutella xylostella . J. Insect Physiol. 52: 943-950.

Jones, JC 1967. Effects of repeated haemolymph withdrawals and of ligaturing the head on differential haemocyte counts of Rhodnius prolixus . J. Insect Physiol. 13: 1351-1360.

Kaeslin, M., R. Pfister-Wilhelm and B. Lanzrein. Influence of the parasitoid Chelonus inanitus and its polydnavirus on host nutritional physiology and implications for parasitoid development. J. Insect Physiol. 51: 1330-1339.

Lavin, M.D. and M.R. Strand. 2002. Insect hemocytes and their role in immunity. Insect Biochem. Mol. Biol. 32: 1295-1309.

Lee, S. and Y. Kim. 2004.Juvenile hormone esterase of diamondback moth, Plutella xylostella , and parasitism of Cotesia plutellae . J. Asia-Pacific Entomol. 7: 283-287.

Loret, SM and MR Strand. 1998.Follow-up of protein release from Pseudoplusia includens hemocytes: a first step to identification of factors mediating encapsulation in vitro. Eur. J. Cell. Biol. 76: 146-155.

Nakahara, Y., Y. Kanamori, M. Kiuchi and M. Kamimura. 2003. In vitro studies of hematopoiesis in the silkworm: cell proliferation in and hemocyte discharge from the hematopoietic organ. J. Insect Physiol. 49: 907-916.

Pech, L.L. and M.R. Strand. 1996. Granular cells are required for encapsulation of foreign targets by insect haemocytes. J. Cell Sci. 109: 2053-2060.

Plantevin, G., G. Bosquet, B. Calvez and C. Nardon. 1987.Relationship between juvenile hormone levels and synthesis of major haemolymph protein in Bombyx mori larvae. Comp. Biochem. Physiol. 86B: 455-461.

Raymond, M. 1985. Presentation d'un program d'analyse log-probit pour micro-ordinateur. Cah. ORSTOM. Ser. Ent. Med. Parasitol. 22: 117-121.

Riddiford, L.M. 1994. Cellular and molecular actions of juvenile hormone. I. General considerations and premetamorphic actions. Adv. Insect Physiol. 24: 213-274.

Sakurai, S. 1983. Temporal organization of endocrine events underlying larval-pupal ecdysis in the silkworm, Bombyx mori . J. Insect Physiol. 29: 919-932.

SAS Institute. 1989. SAS / STAT User's Guide, Release 6.03. Ed. Cary, N.C.

Schopf, A. and H. Rembold. 1993.Changes in juvenile hormone titer of gypsy moth larvae by parasitism of Glyptapanteles liparidis . Naturwissenschaften 80: 527-528.

Sorrentino, RP, Y. Carton and S. Govind. 2002. Cellular immune response to parasite infection in the Drosophila lymph gland is developmentally regulated. Dev. Biol. 243: 65-80.

Strand, MR and BA Dover. 1991.Development disruption of Pseudoplusia includens and Heliothis virescens larvae by the calyx fluid and venom of Microplitis demolitor . Arch. Insect Biochem. Physiol. 18: 131-145.

Strand, M.R. and L.L. Pech. 1995. Immunological basis for compatibility in parasitoid-host relationships. Annu. Rev. Entomol. 40: 31-56.

Vinson, SB 1990. Physiological interactions between the host genus Heliothis and its guild of parasitoids. Arch. Insect Biochem. Physiol. 13: 63-81.

Vinson, SB, KM Edson and DB Stoltz. 1979.Effect of a virus associated with the reproductive system of the parasitoid wasp, Campoletis sonorensis , on host weight-gain. J. Invertebr. Pathol. 34: 133-137.

Wyatt, G.R. and K.G. Davey. 1996. Cellular and molecular actions of juvenile hormone. II. Roles of juvenile hormone in adult insects. Adv. Insect Physiol. 26: 1-155.

The present invention relates to a novel microbial pesticide composition using insect immunosuppression, and more particularly, to an insect immunosuppressive effect of juvenile hormone (JH) and to enhancing microbial pesticide activity using the same.

The relative concentrations of juvenile hormone (JH) and molten hormone determine insect growth and metamorphosis (Riddiford, 1994). The steroid substance molting hormone promotes peeling and transformation, while the relative JH concentration determines the character of the peeling. In other words, secretion of molten hormone under a distinct JH concentration induces another larval peel, but if JH does not appear during susceptibility, molting leads to dragon or adult transformation (Sakurai, 1983; Plantevin et al ., 1987). On the other hand, JH and molting hormones can act as gonadotropin during adulthood (Wyatt and Davey, 1996). In addition, it has been reported that molten hormones have a promoting effect on hematopoiesis in relation to immunity (Nakahara et. al ., 2003).

Some internal parasitic rods disrupt the endocrine of host insects to maintain their parasitic life. Heliothis virescens , parasitic by Campoletis sonorensis , has been shown to delay larval escape and suppress metamorphosis (Vinson et al ., 1979; Fleming, 1992). In addition, injection of CsIV, a symbiotic virus possessed by the parasitic rods, showed similar growth inhibition (Vinson, 1990). Injecting McBV , a polydnavirus from the microplitis croceipes, cocoons, resulted in weight loss, reduced larval storage protein production, and lowered solubility (Fathpour and Dahlman, 1995). As another example of cocoon bees, MdBV , a symbiotic virus of M. demolitor , was injected into Pseudoplusia includens , resulting in overgrowth as well as delayed larval development, and even coexisting larvae and dragon forms. Causes malformations (Strand and Dover, 1991). Chelonus inanitus , which parasites eggs and reduces host larval development, causes premature transformation when parasitized in Spodoptera littoralis and stops development in a dedicated stage. Nutrients are converted into parasitic rod development (Kaeslin et. al ., 2005). Manduca parasitic by Cotesia congregata sexta ) alters immunity, growth, development, color pigmentation and hemolymph proteins (Beckage et. al ., 1994).

Cotesia plutellae induces specific parasites against Plutella xylostella , and parasitic cabbage moths induce larval prolongation, metamorphosis and immunodeficiency (Lee and Kim, 2004; Ibrahim and Kim, 2006). Recently, Clark et al. al .) (2005) reported that glaze hormone lowered the hematopoietic behavioral response to cytokines of atypical blood cells, one of the insect blood cells, while molar hormones had an antagonistic effect. In other words, it is hypothesized that the control of the endocrine system can affect not only the larval development but also immunity from the fact that several larval parasites regulate the development of the host by regulating the endocrine system. Can be presented.

In the present invention, focusing on the fact that the fruit worm cocoon beetle lengthens the larvae of Chinese cabbage moth to match its life cycle, it is estimated that the cocoon bee raises the concentration of the glaze hormone of the Chinese cabbage moth and suppresses the release of skin hormones. . In this case, the cocoon bee may be thought to exhibit immunosuppressive mechanisms by glaze hormone in addition to larval period extension. In the present invention, in order to prove this hypothesis, the interaction of these two hormones was analyzed in blood cell slid phenomena of Chinese cabbage moth. In the present invention, the experiment confirms the fact that the glaze hormone suppresses the immunity of insects, and further confirms the synergistic effect of the insecticide effect by the combination of biological pesticides, such as glaze hormone and Beati ( Bacillus thuringiensis ). The present invention provides a technique for enhancing the activity of microbial pesticides using glaze hormone based on these results.

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

Juvenile hormone (JH) and molten hormone affect a variety of physiological processes, including insect growth and metamorphosis. Several internal parasitic rods regulate the concentrations of JH and molten hormones in the host, and parasitic hosts show delayed development or reduced immunity. In the present invention, the Chinese cabbage moth ( Plutella) to find out how this endocrine regulation affects immune function The effects of these two hormones were analyzed in the blood cell swelling of xylostella ). JH and JH analogs (pyriproxyfen) suppressed blood cell sticking in a concentration-dependent manner. On the other hand, molten hormone (20-hydroxyecdysone: 20E) did not have any effect when treated alone. When JH and 20E were treated together, the two hormones showed characteristic antagonism against hematopoietic activity. JH, on the other hand is of 10 ^-9 M 20E showing the inhibition in a concentration-dependent manner when present, 20E showed a concentration-dependent manner to promote blood cell survival effect on the 10 ^-9 M JH.

In the present invention, it is confirmed that the insecticide inhibitory effect of the JH and the analogs thus identified can be used for insect pests. In addition, in the present invention, JH and analogues through experiments, and the conventionally used Beati ( Bacillus Confirm interactions with microbial pesticides such as thuringiensis . The combination of Pyriproxyfen and Bitty showed a markedly enhanced insecticidal effect than the treatment with Bitty alone. Based on these results, the present invention provides a technique for enhancing the activity of existing microbial pesticides, that is, a new microbial pesticide composition, using JH and analogs.

Blood cell sticking is an important behavior for cellular immune response and can be directly linked to immune capacity (Strand and Pech, 1995). In the case of encapsulation of blood cells, granulocytes first adhere to the surface of the invading foreign factors and form a primary coating (Pech and Strand, 1996; Loret and Strand, 1998). Then, when the atypical blood cells are attracted and their multiple swellings are formed, the formation of the final single film by granulocytes ends, which leads to the end of the cyst formation (Lavine and Strand, 2002). Dividing the regulation effect of the endocrine into two parts can be divided into the effect on the production and differentiation of blood cells and the effect of regulating cellular immune response. Rhodnius as the first endocrine control study It has been reported that molten hormones play an important role in the production and differentiation of blood cells through nodular treatment that blocks the hormonal supply of the prothymus in prolixus (Jones, 1967). Hoffmann (1970) supported this in Locusta migratoria . In Drosophila, as a modulator of cellular immune response, it has been reported that the inhibition of molten hormone decreases the encapsulation ability of parasite eggs (Sorrentino et. al ., 2002). The fly, Neobellieria bullata , causes vesicle formation against laminarin treatment, and the vesicle-forming reaction revealed that JH and molpy hormone act antagonistically as shown in the present invention (Franssens et. al ., 2006). R. prolixus inhibited blood cell phagocytosis when treated with azadiractin , which acts on the thymus and inhibits the biosynthesis of molten hormones, but the addition of molten hormones restores the phagocytosis again (Figueiredo et. al ., 2006). The regulatory effects of these endocrines on blood cell lubrication have also recently become known. That is, the blood cell responsiveness to cytokines of PSP1 (plasmatocyte-spreading peptide 1) of the insects due to differ depending on the developmental timing of the insect control effect of the analyzed JH and break hormone (Clark et al ., 2005). Here as in the present invention, JH was inhibited in reactivity to PSP1, and 20E was promoted.

It is difficult to explain the molecular level mechanisms of JH and escape hormones on the slidability of blood cells from the present or existing reported mechanisms of these hormones. However, some reports on the membranes of JH (Sevala et al. al ., 1995; Kim et al ., 1999), a functional analogy can be made. In follicular cells, the JH membrane receptor activates Na ⁺ -K ⁺ ATpase using protein kinase C (PKC) signaling, which leads to a decrease in cell volume (Ilenchuk and Davey, 1987; Sevala). and Davey, 1989). Although it cannot be confirmed at present, when the JH membrane receptor of blood cells causes such volume reduction for JH, the effect of inhibiting the adhesion of blood cells may be induced. At this time, if the escape hormone inhibits the PKC signaling process again, an antagonistic relationship between the two hormones can be established.

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the scope of the present invention is not limited by the following examples, and those skilled in the art to which the present invention pertains should be within the equivalent scope of the technical concept of the present invention and the claims to be described below. Of course, various modifications and variations are possible.

Materials and methods

Analysis reagent

Both JH III and 20-hydroxyecdysone (20E) were made from Sigma (Sigma, MO, USA). The pyriproxyfen, a JH analogue, was obtained from Dong-Agro (Seoul, Korea) and used 98% of the active ingredient. JH III and pyriproxyfen were both dissolved in dimethylsulfoxide and 20E was dissolved in 50% ethanol. As an anticoagulant buffer, 4 mg of L-cysteine hydrochloride (Sigma) was added to 5 ml of TBS (Tris-buffered saline: 50 mM Tris-HCl, 100 mM dextrose, 5 mM KCl, 2.5 mM MgCl 2). , 50 mM NaCl, pH 7.5, 300 mOsm).

Chinese Cabbage Moth Breeding and Fruit Crust

Chinese cabbage larvae were bred as cabbage leaves, and adults were fed 10% sugar water. Fruitel cocoon bee was cultivated under indoor conditions (temperature about 25 ° C, photoperiod 16: 8 (L: D) h) from outdoor cabbage moth collected from Andong cabbage package. Cabbage moth 2 larvae were used for the parasitization of fruitel cocoon bees. The parasites of the cabbage moth larvae were parasitic by exposing the cabbage moth larvae for 24 hours to adult groups that were allowed to mate in advance for one day after allegory. Since the parasitic cabbage moth was bred in the general breeding method, the adult adult in the cocoon formed was fed 50% sugar water.

Blood lymph collection

To collect enough hemolymph, cabbage moth larvae 7-8 days after spawning with relatively high blood cell density were used. After the larvae were sterilized with 70% ethanol, the tweezers were excised with thin tweezers and blood was collected. Collected 100 μl of blood lymphocytes were mixed with 1 ml of cold (about 4 ° C.) anticoagulant buffer solution.

Hemagglutination Bioassay

50 μl blood cell solution was dispensed on the sterilized slidegrass, and the formation of monocytic hemocytosis was performed for 40 minutes at 25 ° C. under humid conditions. Hormones were treated in various concentrations in blood cell solutions. The adhesion of blood cells was determined according to the cytoplasmic process of blood cells (FIG. 1). These blood cell structures were observed at 40 times magnification (unspreading cells) or 100 times magnification (spreading cells) using a phase contrast microscope (Olympus BX41, Tokyo, Japan). The degree of hemagglutination was randomly selected from about 100 cells in the microscope field of view, and their degree of adhesion was determined, and each repetition was performed independently. The degree of adhesion thus obtained was converted into a percentage, and the treatment effect assay was analyzed by one-way ANOVA using SAS PROC GLM (SAS Institute, 1989) after Arcsin transformation.

Figures 1a to 1c is observed by the phase contrast microscope of the spread hemocytes (%) of the blood cells of the Chinese cabbage moth larvae ( Plutella xylostella larvae). Figure 1a is a blood cell that did not appear lubrication, 1b is a granulocyte ('GR') showing the swelling phenomenon, 1c is an atypical blood cell ('PL') showing the swelling phenomenon. Cabbage moth blood cells showed characteristic lubrication of cytoplasmic processes without coagulation in a buffer solution containing cysteine hydrochloride. All cells showed sticking, but clear sticking was seen in granulocytes ('GR') and atypical blood cells ('PL'). In the case of granulocytes ('GR'), cytoplasmic projections such as filopodia ('fp') were produced, whereas atypical blood cells ('PL') formed pseudopodia ('pp').

2a and 2b is a hormone larvae of Chinese cabbage ( Plutella) The effect of xylostella larvae on blood cell adhesion is shown graphically. Figure 2a (graph A) shows the effect of the glaze hormone ('JH Ⅲ') and glare hormone analogs (pyriproxyfen) by the concentration of blood cells, and Figure 2b (graph B) shows the effect of 20E blood cell The effect is shown by concentration. Each measurement was performed in three replicates. Blood cells of Chinese cabbage moth larvae showed high activity of about 80% under these test conditions. However, glaze hormone ('JH III') or glaze hormone analogue (pyriproxyfen) was added in the range of 1-10 ³ nM, which is considered to be the body's concentration of this hormone, which markedly inhibited blood cell adhesion (FIG. 2A). However, 20E did not inhibit blood cell sticking in the same concentration range (FIG. 2B).

JH's suppression of hemagglutination is an unfavorable phenomenon for insects in the threat of pathogen invasion, and there is another signal to suppress it, suggesting the possibility of maintaining a complementary relationship. In order to clarify this, the interaction of JH and 20E on the adhesion of blood cells was analyzed, and the results are shown in FIGS. 3A (graph A) and 3B (graph B). Interactions were tested under two conditions. In other words, the JH III concentration was constant (10 ⁻⁹ M) and 20E was increased to test whether 20E relieved the inhibitory action of JH (FIG. 3A). In addition, the concentration of 20E was constant (10 ⁻⁹ M) and JH III was increased while increasing the concentration of JH III. Each measurement was performed three times. In the presence of low JH III, 20E significantly increased hemagglutination in a concentration dependent manner. On the other hand, at low concentrations of 20E, JH III again inhibited hemagglutination.

With pyriproxyfen Beatty  Mixed Effect Bioassay

We confirmed whether the immunosuppressive effects of JH identified above affect the pathogenicity of Beatty. First, Pyriproxyfen was previously dissolved in 1 ml of acetone in an amount, and then suspended in water to prepare a suspension for each concentration. Bitty formulations are described in B. thuringiensis subsp. kurstaki (Thuricide: Misung Inc., Korea), the formulation contained 3.0 x 10 ¹⁰ cfu / g of bacterial counts and other toxins.

10% lethal dose (LC ₁₀ , ppm) of each preparation was obtained by repeating three 3 cabbage moths 4 insects. In other words, the cabbage leaf (1 x 1 cm) was immersed for 1 minute at the concentration of each drug, dried and placed in a breeding container of 9 cm in diameter and then treated with Chinese cabbage moth. Pyriproxyfen was diluted at a rate of 2 to 2,000-0 ppm and analyzed for 15 concentrations. Beati analyzed 7 concentrations in the same concentration range. The insecticidal effect was analyzed at 24 hour intervals, and the death rate at 5 days after treatment was determined as the insecticide rate. LC ₁₀ was calculated by probit analysis (Raymond, 1985). The 10% lethal doses of both drugs were calculated to be 7.81 ppm for pyriproxyfen and 250 ppm for betty.

The mixing effect was compared to the pesticides after the treatment with a mixture of 7.81 ppm of pyriproxyfen, 250 ppm of Beati alone based on the concentration of LC ₁₀ of each drug. Each measurement was performed in three replicates.

The results are shown in FIG. 10% lethal dose of Pyriproxyfen ('PYR') and Beati ( Bacillus) thuringiensis subsp. kurstaki : Treatment with '(Bt') alone showed about 10% insecticidal activity. On the other hand, the treatment resulted from the mixing of the two drugs showed about 70% of insecticide, showing a great synergistic effect.

The immunosuppressive effects of glaze hormones and analogs identified in the present invention allow the use of new biopesticide compositions in the agricultural sector. In particular, the cooperative action of microbial pesticides and glaze hormones and analogues identified in the present invention can be greatly utilized in the agricultural field as a technique for increasing the insecticidal effect of BTI and similar microbial pesticides, and despite the many advantages of the conventional pesticides It may be an opportunity to activate the use of microbial pesticides, which are less effective.

Claims (4)

delete An agricultural insecticide comprising juvenile hormone or its analogues and beta ( Bacillus thuringiensis ) together as an active ingredient to enhance the activity of microbial pesticides. The agricultural insecticide according to claim 2, wherein the glaze hormone analogue is Pyriproxyfen. A pesticidal method for agriculture, characterized by using juvenile hormone or an analog thereof with Bacillus thuringiensis .

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