KR101671885B1 - Escin compound inhibiting secretion system of phytopathogenic Gram-negative bacteria and biocontrol agent of plant diseases with this compound - Google Patents

Abstract

The present invention relates to a plant disease control agent containing an acid component as an active ingredient which inhibits the secretory system of phytopathogenic Gram-negative bacteria.
The acid compounds of the present invention can selectively inhibit the secretory system associated with the pathogenic induction of plants without affecting the growth of phytopathogenic gram-negative bacilli. Therefore, the eczynic compounds of the present invention are environmentally friendly plants for controlling plant diseases caused by gram- Can be used as an active ingredient of a disease inhibitor.

Description

[0001] The present invention relates to a phytopathogenic Gram-negative bacteria and a biocontrol agent for plant diseases with this compound, which comprises an escin compound which inhibits the secretory system of phytopathogenic Gram-

The present invention relates to a plant disease control agent containing an acid component as an active ingredient which inhibits the secretory system of phytopathogenic Gram-negative bacteria.

 For a long time, human food production and crop protection have been improved by cultivar improvement, fertilizer application, soil improvement, use of chemically synthesized pesticides and mechanization of farming. However, due to repeated use of pesticides, the advent of drug-resistant bacteria, residual toxicity of agricultural products, and environmental pollution have increased day by day, and they have become a major social problem, and the incidence of resistant strains to existing drugs has increased, It is known that conventional chemical synthetic pesticides are not controllable, and the need for a new control agent is increasing. In accordance with the United Nations Conference on Environment and Development (UNCED) Convention in 1992, domestic use of chemical synthetic pesticides currently in use within the next 10 years is expected to be drastically reduced, and an alternative means of maintaining agricultural productivity is urgently required.

Despite the growing social demand, the speed of development of new agricultural antibiotics is delayed, and unlike methods of inhibiting or killing bacteria that have been widely used, only the factors that cause pathogenesis are selectively targeted Efforts are being made to develop new antibiotics through the approach. In particular, studies on the type 3 secretion system (TTSS), which is involved in the pathogenesis of Gram negative bacteria in plants and plants, have been recognized as new pathogen-based targets and are becoming increasingly important in advanced countries (Nomura, K. et al., Science, 313: 220-223, 2006; Kauppi, AM et al., Chem. Biol., 10: 241-249, 2003). Most of the Gram negative bacteria ( Xanthomonas campestris , Erwinia species, Pseudomonas syringae , Ralstonia solanacearum, Salmonella , Yersinia , Pseudomonas aeruginosa, Chlamydia ) showing pathogenicity to plants and plants were treated with needle / (TTSS) and its mechanism is reported to be similar to each other [Cornelis, GR (1999)). et al., Annu. Rev. Microbiol., 54: 735-774,2000).

This type 3 secretion system is a powerful pathogenetic induction mechanism widely distributed in Gram-negative bacteria that cause pathogenicity in plants and plants. It injects pathogen-inducible protein into the cytosol of the host cell, ≪ / RTI > Keyser, P., et al., J. Intern. Med., 264: 17-29, 2008]. Bacterial secretion systems have been reported to exist in various forms ranging from type 1 to type 7 secretion system [Buttner, D. et al., Trends Microbiol., 10: 186-192, 2002; Vidal, JE et al., Cell. Microbiol., 10: 1975-1986, 2008; Bonemann, G. et al., EMBO J., 28: 315-325, 2009; Bitter, W. et al., Trends Microbiol., 17: 337-338, 2009). In particular, Yersinia (Y. pestis, Y. pseudo tuberculosis and Y. enterocolitica), Salmonella species (Salmonella species), Shigella flex Neri (Shigella flexneri), Rouge Pseudomonas labor (Pseudomonas aeruginosa), E. coli bowel disease (enteropathogenic Escherichia coli) and bacterial species such as Chlamydia (Chlamydia species) that is known to cause an infection via the third type secretion system in the human body (Muschiol, S. et al, Proc Natl Acad Sci, 103.....: J. Bacteriol., 191: 563-570, 2009; Keyser, P. et al., J. Intern. Med., 264: 17-29, 2008). In addition, in plants, gram-negative bacteria such as Xanthomonas campestris , Erwinia species, Pseudomonas syringae and Ralstonia solanacearum are classified into the third type It has been reported to cause pathogenicity in host plants using the secretion system [Kauppi, AM, et al., Chem. Biol., 10: 241-249, 2003). This type 3 secretion system is a translocation channel involving more than 20 different proteins, and these proteins show considerable base equivalence among pathogenic Gram-negative bacteria [Yip CK, et al ., Trends Biochem. Sci., 31: 223-230, 2006]. In addition, these pathogenic effector proteins have been shown to be essential for bacterial pathogenicity, as they have the ability to inhibit signals associated with host defense mechanisms [Grant, JE et al., Annu. Rev. Microbiol., 60: 425-449, 2006).

Previous strategies for developing antibiotics are essential for the growth of bacteria and inhibit metabolic pathways that are common to all bacteria, inhibit targets involved in cell-cell signaling systems, and kill or inhibit pathogenic bacteria Have been focused on the development of compounds [Baron, C. et al., Infect. Disord. Drug Targets, 7: 19-27, 2007]. However, the antibiotics developed through this approach showed excellent pharmacological effects at the beginning, but the emergence of resistant strains due to repeated use and misuse led to many social problems, and in recent years, they have not been treated with conventional antibiotics The development of new antibiotics has been steadily increasing in recent years [Keyser, P. et al., J. Intern. Med., 264: 17-29, 2008; Baron, C. et al., Infect. Disord. Drug Targets, 7: 19-27, 2007].

On the other hand, the existing approach has the disadvantage that not only the pathogenic bacteria but also the beneficial bacteria around them are killed together, but the strategy for the pathogenic factors of the pathogenic bacteria which appeared from the mid 1980's had no effect on the growth of bacteria And selectively inhibits pathogenic bacteria because it inhibits only pathogenic agents [Kauppi, AM et al., Chem. Biol., 10: 241-249, 2003] and is expected to overcome tolerance problems [Clatworthy, A. E., et al., Nat. Chem. Biol., 3: 541-48, 2007], it is known that it is also applicable to pathogenic bacteria that are resistant to conventional antibiotics.

Although studies on inhibitors that inhibit this type 3 secretion system have been reported in human pathogenic gram-negative bacteria [Nordfelth, R. et al., Infect. Immun., 73: 3104-3114, 2005; Hudson, D. L. et al., Antimicrob. Agents Chemother., 51: 2631-2635, 2007; Keyser, P. et al., J. Intern. Med., 264: 17-29, 2008; Kauppi, A. M. et al., Chem. Biol., 10: 241-249, 2003], a study of inhibitors targeting the third type of germ-negative germ-negative bacteria has not yet been activated.

Thus, the development of new antibiotics for the last decades has made a great contribution to the health and welfare of mankind. However, the new mechanism of action and the effective use of existing antibiotics due to the emergence of resistant bacteria due to repeated use and abuse of antibiotics The need for new antibiotics is constantly being sought.

On the other hand, it is known that the ethene compound is a compound of triterpen saponin which is one of the main components of the widely distributed westerly tree ( Aesculus hippocastamum ) in the world, and is effective for chronic venous dysfunction, hemorrhoids, postoperative swelling and inflammation (Sirtori, C., Pharmacol. Res., 44: 183-193, 2001). It is also known to be effective against transient focal cerebral ischemia-induced apoptosis [Hu, XM et al., Acta Pharmacol. Sin., 25: < / RTI > 1267-1275, 2004].

Korean Patent No. 228,750 discloses that heavy metal ions can be separated from soil contaminated with heavy metals by using an ethyne compound as a metal ion capturing agent. In Japanese Patent Laid-Open No. 2007-238468, an ethyne compound has a lipase inhibiting activity , And U.S. Patent No. 2010-227830 discloses an effect on allergic diseases.

However, the content of the compound inhibits virulence factors of gram-negative bacteria.

Accordingly, the present inventors have sought an acid compound having an excellent inhibitory activity against the pathogen-related secretory system of phytopathogenic Gram-negative bacteria, and confirmed that the compound can be used as a plant disease control agent through in vitro and in vivo activity assays Thereby completing the present invention.

Accordingly, it is an object of the present invention to provide a plant disease control agent containing, as an active ingredient, an acid compound capable of inhibiting the pathogenic secretory system of phytopathogenic Gram-negative bacteria.

In order to achieve the above object, the present invention is characterized by a plant disease controlling agent comprising an acetic acid compound represented by the following formula (1) as an active ingredient.

The acid compounds of the present invention can selectively inhibit the secretory system associated with the pathogenic induction of plants without affecting the growth of phytopathogenic gram-negative bacilli. Therefore, the eczynic compounds of the present invention are environmentally friendly plants for controlling plant diseases caused by gram- Can be used as an active ingredient of a disease inhibitor.

FIG. 1 is a schematic diagram showing an example of the construction of a vector containing HpaG and Gus genes and the search for a secretion system inhibitor.
Fig. 2 is an ESI-MS spectrum of an acid compound isolated from a Japanese chestnut ( Aesculus hippocastanum ).
Fig. 3 is a ¹ H-NMR spectrum of an eshine compound isolated from a Japanese chestnut tree ( Aesculus hippocastanum ).
Fig. 4 is a ¹³ C-NMR spectrum of an eshine compound isolated from a Japanese chestnut ( Aesculus hippocastanum ).
FIG. 5 shows the effect of the acidic compound on the growth of the soybean flour cultured by using the Trypticase soy broth medium.
FIG. 6 shows Western blotting results of the GUS fusion protein by treatment with an acid compound [secreted GUS fusion proteins were separated by SDS-PAGE and then subjected to Western blotting].
Fig. 7 shows symptoms of leaf lesions after inoculation with rice blast fungus on a leaf of rice blast and treatment with an acetic acid compound (observed after 14 days from inoculation).
Fig. 8 is a comparison of the length of lesion lesions after inoculation of the rice blast fungus with the acacia var.

Hereinafter, the present invention will be described in detail.

The present invention is based on the finding that an escin compound represented by the following formula 1 exhibits excellent inhibitory activity against the pathway-related secretory system of gram-negative bacteria without affecting the growth of phytopathogenic gram negative bacteria, It can be used to suppress disease outbreaks for the first time.

[Chemical Formula 1]

Formula 1 of esin compounds phytopathogenic gram plant diseases that caused by the negative bacteria, in particular soybean fire blight (Xanthomonas axonopodis pv. Glycines), tomato stains disease (Pseudomonas syringae pv. Tomato DC3000), rice al blight (Burkholdera glumae BGR1 and Burkholdera glumae mutant The controlling effect of the BGS6), pepper cheonggobyeong (Ralstonia solanacearum SL341 and Ralstonia solanacearum SL2029), rice huinip blight (Xanthomonas oryzae pv. Oryzae KX085) or jeommunuibyeong pepper (Xanthomonas axonopodis pv. Vesicatoria) is excellent.

Accordingly, the present invention is characterized by a plant disease control agent containing the above-mentioned escin compound of Chemical Formula 1 as an active ingredient.

Such a plant disease controlling agent may be prepared by using a carrier which can be used as an agricultural chemical preparation together with an active ingredient. As a carrier, conventionally used pesticide formulations can be used as long as it is an acceptable carrier, but in particular, freezing preservative skim milk, yeast extract, electrodeposition agent SOF70, tween 20, surfactant TD20A, AS65D and the like are used .

The active ingredient according to the present invention is preferably used in an amount of 0.003 to 2.0% by weight, more preferably 0.003 to 0.5% by weight, based on 100% by weight of the whole plant disease controlling composition. The above-mentioned acid compounds may be used in powder form or in a liquid form, and they are diluted 10 to 1000 times when used. However, the effective amount can be adjusted depending on the type of Gram negative bacteria to be applied, the degree of infection of the pathogens and the environmental conditions. At this time, diluent which is conventionally used including purified water is used.

It is preferable that the plant disease control agent thus prepared is treated with a foliar treatment method at 30 to 40 days after the plant is planted, or at the plant just before the onset of the disease.

In the present invention, it is meaningful that a specific composition containing an acidic compound as an active ingredient is provided as a practical framework that can be used as an effective inhibitor for controlling plant diseases caused by gram-negative bacteria without affecting the growth of Gram-negative bacteria at all.

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

Example 1: Purchase of a plant extract library

The acid compounds used in the present invention were obtained from a plant extract library from the Korean plant extract bank of the National Institute of Bioscience and Human-Technology, Korea Research Institute of Bioscience and Biotechnology.

Example 2 Activity Assay and Derivation of Inhibitors that Inhibit the Secretion System of Gram-Negative Bacteria from the Extracted Plant Extract Library

To the present invention, in order to test whether inhibition of the secretion system of the plant pathogenic Gram negative bacterium of esin compound, causing the fire blight of bacterial the beans jaento Pseudomonas evil sono's capsule discharge glycine (Xanthomonas axonopodis pv. Glycines) (HapG) gene [Kim, JG et al., J. Bacteriol., 185: 3155-3166, 2003], a kind of harpin gene known to be involved in the secretory system from a strain, Sono discharge port glycine's 8ra established the (Xanthomonas axonopodis pv. glycines 8ra, or less Xag8ra) test system [1].

A recombinant strain containing HpaG-Gus fusion protein Xag8ra The strain was cultured overnight at 28 ° C. in 10 ml of LB medium (10 μg / ml of tetracycline, 50 μg / ml of rifampicin), and then the XMC2 medium (NaCl 20 mM, (NH ₄ ) ₂ SO ₄ 10 mM, MgSO _{4 4?} 7H ₂ O 5 mM, CaCl _2? 2H ₂ O 10 mM, CaCl _2? 2H ₂ O 1 mM, KH ₂ PO ₄ 0.16 mM, K ₂ HPO ₄ 0.32 mM, FeSO _4? 7H ₂ O 0.01 mM, casamino 2% inoculum was incubated with 1 ml of LB medium overnight in 1 ml of a solution containing 0.03% acid, 10 mM fructose, 10 mM sucrose, 10 μg / ml tetracycline and 50 μg / ml rifampicin. For 10 to 12 hours. Cultured Xag8ra The broth was transferred to an eppendorf tube, centrifuged at 12,000 rpm for 1 minute, the supernatant was transferred to a new Eppendorf tube and well stirred, then 100 μl was added to a 96 well plate. After the addition, 100 μl of X-GIcA (cyclohexylammonium), which is a substrate purchased from Duchefa Co., was added and GUS staining was carried out to check whether the HpaG2-Gus fusion protein was inhibited. After 24 hours of reaction in the field of view, compounds changing from blue to yellow were selected and the absorbance at 590 nm was measured. The inhibition (%) was calculated from the following equation (1). The following natural products were selected as the subject of the activity test, and the results are shown in Table 1 below.

Examples of secretion system inhibitor activity assays Target natural product Final concentration (μg / ml) Inhibition of secretion system (%) Western hardwood 100 56.5 Paddy herb 400 14.5 Dwarf tree 400 6.6 White Duck Duck 400 5.3

Example 3: Preparation of crude extract from western three-leafed leaves and separation of escin compound

In order to isolate and purify the active inhibitor compound which showed inhibitory activity against the secretion system of Xag8ra, a gram-negative bacterium recombinant in Example 2, 500 g of Aesculus hippocastanum powder was purchased from the natural plant utilization technology development group, 2.5 L of methanol was added to the powder, and the mixture was extracted at room temperature for 4 days. The mixture was concentrated under reduced pressure using a vacuum concentrator to obtain 50 g of methanol crude extract. The methanol crude extract was dissolved in 1 L of distilled water and subjected to HP-20 column chromatography. The methanol eluate was sequentially eluted with 20% methanol to 0-100% methanol, followed by methanol 50-80% 0% of eluate fractions were collected and tested for inhibition of secretion system and then concentrated under reduced pressure.

Second-order HP-20 column chromatography was performed on the 50-80% eluted fractions which showed inhibitory activity by the above inhibition activity assay, in which methanol was eluted with increasing 10% to 50-100%. After the eluted fractions were collected to confirm the inhibitory activity, the fraction subjected to the third HP-20 column chromatography was collected and subjected to Sephadex LH-20 chromatography. To further separate and purify the fractions, 30% acetonitrile and Watchers 22.5 mg of a pure white single compound having a retention time of 11.3 minutes was obtained by high performance liquid chromatography (HPLC) using a 120 ODS-BP column (5 占 퐉, 4.6 占 250 mm).

Example 4: Structural analysis of isolated compounds

As a result of structural analysis using nuclear magnetic resonance (Varian UNITY 300 MHz, 500 MHz NMR) and ESI-MS in order to determine the molecular weight and molecular formula of the separated compounds, It was confirmed that the molecular weight and the NMR data agree with the saponin-based? -Esin compound having a molecular weight of 1130 (Yoshikawa, M. et al., Chem. Pharm. Bull., 44: 1454-1464, 1996). The above ESI-MS spectrum and NMR spectrum are shown in Figs. 2 to 4. Fig.

Example  5: of the isolated acetic compound Slobber  Inhibition activity assay

As a result of assaying the secretory system inhibitory activity of the acetic acid separated pure by using the method described in Example 2, the IC ₅₀ of this compound was 5.2 μg / ml, and the results are shown in Table 2.

Secretion system inhibitory activity of the acid compound compound IC ₅₀ (μg / ml) Escin 5.2 Nalidixic acid ^* 3

* Positive control

Example 6: Antimicrobial activity test of an acid compound for phytopathogenic Gram-negative bacteria

In order to investigate whether the escin compound isolated in Example 4, which was confirmed to exhibit inhibitory activity against bacterial secretion system, would inhibit bacterial growth and affect the secretory system, Xanthomonas axonopodis pv glycines , Pseudomonas syringae pv. tomato DC3000, Burkholdera glumae BGR1 and Burkholdera glumae mutant BGS6), Office of pepper gobyeonggyun (Ralstonia solanacearum SL341 and Ralstonia solanacearum SL2029), rice huinip blight bacteria (Xanthomonas oryzae pv. Oryzae KX085) and bacterial strains jeommunuibyeong of pepper (Xanthomonas axonopodis pv. vesicatoria ) and 8 kinds of phytopathogenic bacteria. The phytopathogenic bacteria were cultured overnight at 30 ° C and the number of cells was adjusted to 1.5 × 10 ⁹ CFU (colony forming unit) / ml so that the absorbance at 660 nm was 1.0. The ethyne compound was dissolved in sterilized distilled water in each well of a 96-well plate, followed by successive two-fold dilution series with a trypticase soy broth medium, followed by addition of 3.0 x 10 ⁷ CFU / ml of final cells per well And cultured at 30 to 37 ° C for 1 to 2 days. The minimum growth inhibition concentration (MIC) was determined by observing the growth of the microorganism after cultivation. The results are shown in Table 3 below. At this time, nalidixic acid was used as a positive control.

           The minimum inhibitory concentration (μg / ml) of the ethene compound against phytopathogenic bacteria Tested organism   Naridisan Ashin Burkholderia glumae (BGR1) 8 > 512 Burkholderia glumae (BGS6) 4 > 512 Pseudomonas syringae pv. tomato DC3000 32 > 512 Ralstonia solanacearum SL341 32 > 512 Ralstonia solanacearum SL2029 16 > 512 Xanthomonas oryzae pv. oryzae KX085 16 > 512 Xanthomonas axonopodis pv. vesicatoria 16 > 512 Xanthomonas axonopodis pv . glycines 32 > 512

As shown in Table 3 above, the acidic compound did not exhibit the antimicrobial activity even at a concentration of 512 μg / ml for all of the 8 tested bacteria, while the nalidixic acid used in the control group contained 8 kinds of test bacteria , Respectively, at a concentration of 4 ~ 32 / / ml. Therefore, it was confirmed that the above-mentioned acid compounds did not affect the growth of the plant pathogenic bacteria.

Example 7: Test for inhibiting growth of phytopathogenic gram negative bacteria by an acid compound

In order to confirm whether the escin compound inhibited the growth of the Xag8ra strain used for assay of the secretory system activity, the acidic compound was added to the Tyrpticase soy broth liquid medium on the basis of the MIC results to obtain an inhibitory concentration of 256 μg / ml And the growth was inhibited at the intervals of 2 hours while culturing the cells. The results are shown in Fig. At this time, the control group was compared with no compound added.

As the time elapsed, the growth of the microorganism increased in both the control and the compound-added groups. The change in the number of viable cells after 10 hours incubation was 4.0 × 10 ⁸ CFU / ml in the control group without the compound On the other hand, when the acid compound was added, it was found to be 4.0 × 10 ⁸ CFU / ml. From the above results, it was confirmed that GUS staining was not inhibited by the inhibition of the growth of the microorganism, since it did not affect the growth of the microorganism at a concentration of 256 μg / ml, which is a concentration at which the secretory system is inhibited .

Example 8: Secretion system inhibition test of phytopathogenic Gram-negative bacteria by Western blotting

Western blotting was performed in order to investigate whether an escin compound strongly inhibiting the secretion system of gram-negative bacteria actually inhibited the secretion of pathogenic factors through the secretory system.

First, 10 ml of LB medium (tetracycline 10 μg / ml, rifampicin 50 μg / ml) containing antibiotics was added to Xag8ra The strain was inoculated and cultured at 28 ° C for 12 hours with shaking. The strain was inoculated 2% in 100 ml of XVM2 medium (containing 10 μg / ml of tetracycline and 50 μg / ml of rifampicin) supplemented with antibiotics and cultured in LB medium , And cultured with shaking at 28 DEG C for 10 hours. The cultured bacteria were centrifuged at 12,000 rpm for 20 minutes, and then 10 ml of 100% TCA (trichloroacetic acid) was added to 90 ml of the supernatant. The mixture was allowed to stand on ice for 30 minutes and centrifuged at 12,000 rpm for 20 minutes at 4 ° C Separately, the supernatant was discarded, followed by addition of 2 ml of acetone and centrifugation again at 4 ° C for 10 minutes. After centrifugation, the supernatant was discarded, acetone was added once again, centrifugation was performed, the supernatant was removed and sufficiently dried. SDS-PAGE was performed to separate the dried protein by size, and the gel-like protein was transferred to a PVDF membrane (pore: 0.45 μm, Millipore). The protein-transferred PVDF membrane was added to 5% skim milk, and the mixture was shaken at room temperature for 1 hour to block the reaction. The GUS antibody was diluted with TBST (Tris-buffered saline Tween-20) Respectively. After stirring, the GUS antibody was discarded, and the PVDF membrane was washed three times for 5 minutes using TBST, and a second antibody (anti-rabbit IgG, HRP-linked Antibody) diluted with TBST was added and stirred for 2 hours. The second antibody was discarded and the PVDF membrane was washed with TBST three times for 5 minutes using TBST. Supersignal was uniformly sprayed on the PVDF membrane using West Pico Chemiluminescent (Thermo Co.) reagent and detected using a LAS-3000 system (FujiFilm Co., Japan) (Fig. 6).

As a result, HpaG-GUS fusion protein secreted out of the XVM2 medium was secreted normally through the secretory system in the cytoplasm of the control group. In contrast, in the case of treatment with the acidic compound, HpaG- The band of GUS fusion protein was significantly reduced compared to the control group, and it was not detected at a concentration of 50 μM or more, indicating that HpaG-GUS fusion protein was reduced in a concentration-dependent manner. As a result, it was clearly confirmed that the escine compound inhibited the secretory system of bacteria.

Example 9: Using rice in vivo Active black

In order to investigate whether the escine compound, which has been shown to strongly inhibit the secretory system of bacteria, exhibits inhibitory activity in vivo , activity test was carried out using Dongjin rice grown in a pot for 5 weeks or more.

First, the ethyl benzene mixed with the twin-20 (mixed to make the final concentration of tween-20 to 200 ppm) was sprayed to Dongjin resin for 5 weeks at the concentration of 300, 600 and 900 ppm respectively, and the negative control was sprayed with distilled water Sprayed and allowed to stand for 24 hours. After 24 hours, the causative bacteria of Xanthomonas oryzae pv. oryzae strains were cultured at 28 ° C for 48 hours, sterilized scissors were soaked in the bacterial solution prepared to have a bacterial concentration of 2 × 10 ⁶ cfu / ml, and then Dongjin rice was cut and inoculated. In the positive control group, the scissors were sterilized well and the end of the rice paddy was cut to prevent the inoculation of the bacteria.

After 48 hours of inoculation, the cells were again sprayed with the same concentration, and only the distilled water was sprayed on the positive control and the negative control. The humidity of the incubator was maintained at 70%, the incubation was carried out at 28 ° C for 16 hours, in darkness at 25 ° C for 8 hours, and after 2 weeks, the length of the lesion on the Dongjin rice paddy was measured.

As a result, treated with distilled water, In the positive control group not inoculated with the strain, the tip of the rice leaf was slightly dry and disease did not develop. In addition, In the negative control inoculated with the strain and treated with distilled water instead of the acid compound, it turned out to be completely yellow from the end of the rice leaf and it was confirmed that it developed. On the contrary, the rice plants treated with the acid compounds at 300, 600 and 900 ppm, respectively, developed disease, but the onset was decreased compared with the negative control, 7].

FIG. 8 is a graph showing the length of the onset area. The length of the lesion on the rice leaf of the positive control group was 0.07 cm and the length of the onset was 15.52 cm in the negative control group. In contrast, achene compounds were found to be 12.38 and 7.73 cm in the treated group at 300 and 600 ppm, respectively, and 7.34 ppm in the treated group at 900 ppm, indicating that the acid compound inhibits the disease caused by rice blast fungus Or delaying the onset of the disease.

Example 10 Toxicity Test of Acetic Compound Using Daphnia

To assess the acute swimming toxicity of daphnia to daphnia, the National Institute of Environmental Research 2009-57 (Nov. 24, 2009), 「Annex 5 of the Regulation on Designation of Chemical Hazardous Test Research Institutions」 ] "Acute Toxicity Test for Daphniae" and "OECD Guidelines for Testing of Chemicals" in Chapter 2, Section 2. 202 " Daphnia sp., Acute Immobilization Test (adopted: April 13, 2004).

The water temperature, pH and dissolved oxygen of the test solution were measured at all concentrations at which no 100% lethal effects were observed before and after exposure of the daphnia. The results were summarized as average values. Daphnia magna , which was used for the toxicity test, Children younger than 24 hours were used in the breeding room of the Safety Evaluation Laboratory and cultured over 3 broods. The medium used for the dilution of water fleas was 245 mg / l (CaCO ₃ ), 43.4 mg / l (CaCO ₃ ) for hardness, M4 medium prepared according to the OECD Guideline (1996) .

As the test solution, the test compounds were diluted with diluted water to prepare 100, 50, 25, 12.5 and 6.25 mg / l, respectively. Dilution was used in the control group. During the test period, the test solution was replaced with a static system. The test solution was 100 ml of the beaker with a volume of 100 ml, and 4 replicates of 5 daphnia per test concentration Treated and exposed for 48 hours, followed by lethal or affected population for 24 hours and 48 hours. During the experimental period, the photoperiod was adjusted to 16 hours for light condition and 8 hours for dark condition, and the illuminance of the laboratory was maintained at about 520-660 lux. The observation method was as follows: After lightly stirring the test solution, Those who failed were regarded as affected. Statistical analysis was performed using CETIS v1.7.0 (Tidepool, California, USA) and the toxicity values EC ₅₀ and 95% confidence limits were calculated using linear regression. As a result esin compounds were able to determine that there is 16.74 g. In the EC _50, EC ₅₀ and after 48 hours after 24 hours, the scope that can be used as a sterilizing agent exhibits a 11.93 The results are shown in Table 4 below.

Exposure time Escin (mg / l) 24 hours 48 hours EC ₅₀ values 16.74 11.93 95% confidence limits 13.47 ~ 20.71 9.99 to 14.24

Claims (4)

A plant controlling agent for a plant disease caused by a phytopathogenic Gram-negative bacterium, which comprises a compound represented by the following formula (1) as an active ingredient.
[Chemical Formula 1]

delete [2] The plant disease control agent according to claim 1, wherein the plant disease is soybean flour disease, tomato stain disease, rice grain blight, red pepper disease, rice blast fungus disease or pepper spotty fungus disease.
The plant disease control agent according to claim 1, which shows an effect of inhibiting the secretory system of phytopathogenic Gram-negative bacteria.

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