KR100891720B1 - 4-carbamoylphenylacetic acid from Pseudomonas chlororapis O6 which induces resistance to plant diseases and uses thereof - Google Patents

Abstract

The present invention relates to plant diseases, particularly Pseudomonas syringae pv. The other Bridge (Pseudomonas syringae pv. Tabaci) mediated wildfire disease (wild fire) chloro, Pseudomonas inducing resistance to O6 lapis (Pseudomonas 4-carbamoylphenylacetic acid derived from chlororaphis O6), a plant disease resistance inducer containing the substance, and a plant disease resistance induction method using the substance.

Description

4-carbamoylphenylacetic acid from Pseudomonas chlororaphis O6 inducing systemic resistance against plant diseases, and uses according to Pseudomonas chlororapis O6 inducing resistance to plant diseases

The present invention relates to novel substances and their use for inducing resistance to plant diseases, and more particularly, to Pseudomonas syringae pv. 4-carbamoylphenylacetic acid [(4-carbamoylphenyl) derived from Pseudomonas chlororaphis O6, which induces resistance to Pseudomonas syringae pv.tabaci mediated wild fire. ) acetic acid], a plant disease resistance inducer containing the substance, and a plant disease resistance induction method using the substance.

Plants are exposed to the attack of various microorganisms that cause disease throughout their growth. When a plant becomes diseased, its growth and growth are inhibited and show several forms of disease. This results in the death of some or all of the plants and, in the case of crops, productivity is drastically reduced. As an environmentally friendly technique that prevents or treats plant diseases microbiologically without using chemical synthetic pesticides, a method using microorganisms that produce antibiotics (Folman, LB et al. (2004) Biol Control 31, 145-154 ]), Methods using microorganisms that produce enzymes (Chae, DH et al. (2006) Biocontrol 51, 339-351), methods of promoting plant development (Zdor, R. et al. ( 1992) Plant Soil 140, 99-107]. There is also a preventive method in which plants are treated with microorganisms that produce phenazine in advance before the plant disease occurs, thereby making the plant resistant to the disease and preventing the disease (Pierson, LS et al. (1992) Mol Plant-Microbe Interact 5, 330-339]. In addition to phenazine, acibenzolar-S-methyl (Kunz W. et al. (1997) J Pestic Sci 50, 275-282), 2,6-dichloroisonicotinic acid (document (Metraux JP et al. (1991) Mol Plant-Microbe Interact 1, 432-439), salicylic acid (Sticher L. et al. (1997) Annu Rev Plant Pathol 35, 235-270), probenazole (probenazole) (Watanabe T. (1997) J Pestic Sci 2, 394-404), beta-aminobutyric acid (Tosi et al. (1998) J Phytopathol 146, 259-280), and the like. It is known to induce resistance to.

The inventors isolated and identified Pseudomonas Chlorophyll O6 strain (Accession No. KACC 91054), a plant rhizome microorganism having the ability to induce systemic resistance and drought resistance to plant pathogens from soil, and isolated the strain itself and the strain. Application No. 3 for the method of inducing systemic resistance to plant pathogens, especially pepper bacterial wilt, cucumber streptococcus, bacterial spot pattern germ, cucumber mosaic virus, tobacco soft germ, tobacco wild germ, mysterious fungus and soil fungus The patent was filed in 2003-62692 and patented under the registration number 521,744. Pseudomonas chlorolapis O6 is known to have a function of preventing plants from making them resistant to disease (Spencer, M., et al. (2003) Physiol Mol Plant Pathol 63, 27-34). Has been reported to induce plant disease resistance irrespective of existing plant disease resistance inducers such as phenazine (Han, SH, et al. (2006) Moi Plant-Microbe Interact 19, 924-930). ). However, until now, no metabolites of Pseudomonas chlorolapis O6 function to induce tobacco wildfire resistance. Wildfire disease is one of the tobacco diseases, and Pseudomonas syringe pv. It is caused by a parasitic of a bacterium called Tabashi. A leaf has a small, dark yellow spot on the needle, and later a clear yellow cluster around the lesion. The lesion grows in conjunction with the adjacent lesion and eventually leaves the forks torn. The disease is called wildfire because it spreads rapidly and causes great damage, especially after a storm.

The present inventors conducted an ongoing study to determine which metabolites of Pseudomonas chlorolapis O6 are involved in inducing wildfire resistance. As a result, the present invention was completed by finding out that 4-carbamoylphenylacetic acid, a novel substance, is an active substance that induces plant disease resistance and biologically verifying that the substance actually exhibits activity that induces resistance to tobacco wildfire. It came to the following.

It is therefore an object of the present invention to provide plant diseases, in particular Pseudomonas syringe pv. To provide a novel substance, 4-carbamoylphenylacetic acid, derived from Pseudomonas chlorolapis O6, which induces resistance to Tabashi mediated wildfire.

Another object of the present invention is to give Pseudomonas syringae pv. Of plant diseases, especially tobacco, containing said 4-carbamoylphenylacetic acid. To provide a resistance inducer to Tabashi mediated wildfire.

Another object of the present invention is to use the 4-carbamoylphenylacetic acid, plant diseases, in particular Pseudomonas syringe pv. To provide a method for inducing resistance to Tabashi-mediated wildfire.

First, the present invention relates to 4-carbamoylphenylacetic acid of formula (1).

[Formula 1]

The material is a plant disease, in particular Pseudomonas syringae pv. It is a novel substance derived from Pseudomonas chlorolapis O6, which induces resistance to Tabashi mediated wildfire.

Second, the present invention relates to an inducer resistant to plant diseases, containing 4-carbamoylphenylacetic acid as an active ingredient. The plant disease is preferably Pseudomonas syringe pv. It is Tabashi mediated wildfire.

Third, the present invention relates to a method of inducing resistance to plant diseases, comprising treating 4-carbamoylphenylacetic acid on plant roots. The plant disease is preferably Pseudomonas syringe pv. It is Tabashi mediated wildfire.

The present invention is known to induce resistance to plant diseases (eg, tobacco wildfire disease), but the mechanism and active substance of Pseudomonas chlororapis O6 strain, which is not known, produces 4-carbamoylphenylacetic acid in plant diseases. As the first to induce resistance to 4-carbamoylphenylacetic acid is a novel substance different from the known plant disease resistance inducer. Using the present invention, the plant (for example, tobacco) exhibits the effect of preventing the plant disease by making it resistant to plant diseases (for example, Pseudomonas syringe pv. In addition to the known plant disease resistance inducers can be effectively used as a plant disease prevention and control, and when applied to crop cultivation can increase the productivity of the crop. In addition, the material of the present invention is biologically obtained, not chemically synthesized, and may be applied for plant disease control and prevention in environmentally friendly agriculture.

Hereinafter, the present invention will be described in detail.

In the present invention, the metabolite produced by the Pseudomonas chlorolapis O6 strain known to induce resistance to plant diseases (eg, tobacco wildfire disease) is isolated and identified, and this substance actually induces plant disease resistance. The activity was verified. That is, the culture solution of Pseudomonas chlorolapis O6 was extracted and chromatographically purified using various solvents, and the tobacco wild-fire resistance resistance-inducing activity was tested for each fraction of solvent and chromatographic purification. The chemical structure of the isolated active material was analyzed using a mass spectrometer and a nuclear magnetic resonance analyzer to identify 4-carbamoylphenylacetic acid, and biologically verified that the material actually has wild-induced disease-inducing activity.

In the present invention, the metabolite showing tobacco wild-blotting resistance inducing activity is contained in the butanol extract of the culture medium of Pseudomonas chlorolapis O6 strain. The butanol extract may be obtained by extracting a culture solution of Pseudomonas chlorolapis O6 strain with ethyl acetate to obtain an aqueous solution layer, and extracting the aqueous solution layer with butanol at pH 2 to 4.5, particularly pH 2 conditions. The metabolite is contained in an aqueous solution eluate containing 40% methanol when the extract is purified by ODS column chromatography. For this reason, the extract and the eluate also exhibit tobacco wild fire disease resistance induction activity.

In addition, the metabolite was purified by the column chromatography, and the C-18 solid phase column was attached thereto, and then, using a high performance liquid chromatography (HPLC) equipped with a PDA detector, a pH 2 condition of trifluoroacetic acid was added at a wavelength of 270 nm. Elution with 20% acetonitrile aqueous solution mixed mobile phase, contained in the extract extracted from the main chromatogram. For this reason, this fraction also exhibits tobacco wildfire resistance induction activity.

In the present invention, the chromatograms obtained from the above repeated liquid chromatography analysis were separated into single substances, and then the tobacco wild fire resistance resistance inducing activity test was performed for each isolated material, and the tobacco wild fire disease resistance inducing activity was shown. The isolated material was identified as 4-carbamoylphenylacetic acid by mass spectrometry and nuclear magnetic resonance analyzer.

In the present invention, in order to determine whether the substance actually induces tobacco wildfire resistance, the isolated substance was treated at the root of the tobacco and then infected with the wild fire bacteria to investigate the symptoms of tobacco. In addition, the reference chemicals (salicylic acid and nanosilica), which are known to induce resistance to tobacco wild infestation, were selected as a comparison target and the wild-induced disease-inducing activity was investigated.

4-carbamoylphenylacetic acid according to the present invention is dissolved in water and treated in advance to the root of tobacco at 68 mM level, Pseudomonas syringae pv. In the case of treatment with Tabashi, it was confirmed that the activity of inhibiting the development of wildfire disease was 80% or more. This effect is similar to that of salicylic acid and nanosilica used as reference compounds. Therefore, 4-carbamoylphenylacetic acid of the present invention may have an effect of preventing wildfire disease by making tobacco resistant to wildfire disease. 4-carbamoylphenylacetic acid can be dissolved in water-soluble solvents, including water, and treated in tobacco or its habitat at a level of 50-100 mM, preferably by treatment with tobacco roots.

In addition, Pseudomonas Chlorophyll O6 strains have activity that induces systemic resistance to drought and bacterial resistance to drought, cucumber streptococcus, bacterial streaks, cucumber mosaic virus, tobacco purifying bacteria, mysterious fungi and soil fungi. As such, 4-carbamoylphenylacetic acid derived from Pseudomonas chlorolapis O6 strain may induce resistance to such pathogens and may be used for the prevention and control of plant diseases mediated by it and for the reduction of crop damage by drought.

Hereinafter, the present invention will be described in more detail with reference to Examples, which are intended to aid the understanding of the present invention but are not intended to limit the scope of the present invention in any way.

Example 1: Cultivation of Microorganisms, and Extraction and Purification of Culture Solution

Pseudomonas chlorolapis O6 strain (Accession No. KACC 91054) was incubated for 60 hours at 27 ° C. and 150 rpm in a liquid medium containing Luria Broth (LB) and antibiotics Ampicillin and Streptomycin 100 mg / l, respectively.

Pseudomonas chlorophylla O6 strain culture obtained above was centrifuged for 15 minutes at 8000 rpm. The supernatant obtained after centrifugation was subjected to solvent extraction with ethyl acetate corresponding to twice the volume of the supernatant. The obtained aqueous solution layer was adjusted to 2 with 1 N hydrochloric acid (HCl), and then extracted with butanol corresponding to twice the volume. The butanol layer was collected, concentrated under reduced pressure, redissolved in 30% methanol, and purified using an ODS gel (Wakosil C18, Japan) column chromatography method. The ODS gel was slurry filled with methanol in a glass column (5 cm × 50 cm, horizontal × vertical), and then eluted and fractionated by increasing the methanol ratio by 10% with the water-methanol solvent system as the mobile phase. The fraction obtained from the 40% methanol elution fraction was separated and fractionated again by high performance liquid chromatography (HPLC) equipped with a C-18 solid column and a PDA detector. The preparative and analysis conditions of the high performance liquid chromatography were as follows.

① Instrument Model: Dionex p680 HPLC

② Detector: PDA-100 Photodiode Array Detector

③ Analysis wavelength: 270 nm

④ Preparative column: Watersμ - Bondapak ™ prep column (C18 10 μm 125 Å 19 × 300 mm)

⑤ Mobile phase: 20% acetonitrile / water at pH 2 condition with trifluoroacetic acid

⑥ mobile phase flow rate: 4 ml / min

The results are shown in FIG. As shown in FIG. 1, the finally separated material was identified as a single material through high performance liquid chromatography analysis.

Example 2 Structure Determination of Isolates

The chemical structure of the single material obtained in Example 1 was characterized by high resolution mass spectrometry, liquid chromatography mass spectrometry, and nuclear magnetic resonance analysis.

The molecular weight of the isolated material was 179.0602, and the molecular formula was estimated to be C ₇ H ₁₀ O ₂ N ₂ . The liquid chromatography mass spectrometry showed that the (M + H) ⁺ value was 180, which produced ions of 163, 136, and 119. The mass spectra of the daughter ions were m / z 163 for C ₂ H ₇ O ₃ , m / z 136 for C ₇ H ₆ NO, and m / z 119 for C ₈ H ₇ O ₂ (FIG. 2).

In addition, the results of nuclear magnetic resonance analysis are shown in Table 1 below.

As shown in Table 1, the analysis results of ¹ H-NMR (500 MHz, CD ₃ OD) showed that at δ2.15 (s, 3H), δ7.65 (d, 2H), and δ 7.95 (d, 2H). The signal was observed. Seven carbon signals were observed through ¹³ C-NMR (500 MHz, CD ₃ OD), one SP ³ carbon (δ22.14), one carbonyl carbon (δ172.11), and two quaternary carbons ( It was confirmed that the compound was composed of δ 127.51, δ 144.52), and two kinds of olefin methine carbons (δ 120.18 and 131.90).

The material isolated from the above mass spectrometry and nuclear magnetic resonance analysis was identified as 4-carbamoylphenylacetic acid (FIG. 3), which was a novel compound not reported so far.

Example 3: Biological Verification of Tobacco Wild Disease Resistance Induction Activity

Tobacco seeds were surface sterilized with 70% ethanol and hypochlorous acid (HClO ₄ ), followed by incubation for 3 days in a culture dish containing vitamins, sugars, and nutrients, and the seeds were germinated. The culture composition is as follows.

4.4 g / l Murashige & Skoog medium with vitamin

0.5 g / l 2-morpholinoethanesulfonic acid monohydrate

Sucrose 30 g / ℓ

Phytogel Agar 5 g / L

The germinated seeds were transferred to a tissue culture dish and planted, and then tobacco was incubated at a place where light and dark conditions were 14 and 10 hours, respectively. After about two weeks, when up to 5-6 tobacco leaves were grown, the wild ill-resistant inducer, 4-carbamoylphenylacetic acid, isolated in Example 2 was dissolved in water and irrigated to tobacco roots at 68 mM level. After 5 days of treatment, the tobacco wildfire pathogen Pseudomonas syringe pv. Tabashi was treated on tobacco leaves at the level of 1.0 × 10 ⁸ cells. The incidence of wildfire disease observed in tobacco leaves after 3 days of pathogen treatment was investigated. The control group was treated with distilled water, and the reference group was treated with 1 mM salicylic acid and 80 mM nanosilica.

The results are shown in FIG. As shown in Figure 4, 4-carbamoylphenylacetic acid of the present invention showed about 80% level of tobacco wild fire disease inhibition effect compared to the control. This was similar to the effect of inhibiting the onset of the reference compounds, silicic acid and nanosilica. In other words, even when the Pseudomonas chloro-lapis O6 culture stock was treated before the separation of a single substance, the incidence of wildfire disease was remarkably suppressed compared to the control group. Inhibitory effect was shown. This was a similar effect as the treatment of the reference compound.

The results indicate that 4-carbamoylphenylacetic acid of the present invention is a major metabolite that causes Pseudomonas chlorolapis O6 to exhibit plant disease resistance inducing activity in tobacco.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows the results of liquid chromatography analysis of plant disease resistance inducer 4-carbamoylphenylacetic acid isolated from Pseudomonas chlorolapis O6 strain culture medium;

FIG. 2 is a diagram showing the results of liquid chromatography mass spectrometry of plant disease resistance inducer 4-carbamoylphenylacetic acid isolated from Pseudomonas chlorolapis O6 strain culture medium; FIG.

Figure 3 is a view showing the chemical structure of the plant disease resistance inducer 4-carbamoylphenylacetic acid isolated from Pseudomonas Chlorophyll O6 strain culture medium;

Figure 4 is a graph showing the plant disease resistance inhibitory effect of plant disease resistance inducer 4-carbamoylphenylacetic acid in the biological assay experiment;

Figure 5 is a photograph showing the degree of infection of phytopathogens of tobacco after treatment with plant disease resistance inducer 4-carbamoylphenylacetic acid.

Claims (7)

delete Pseudomonas syringae pv. Of tobacco containing 4-carbamoylphenylacetic acid of formula (1) as an active ingredient. Induced resistance to Pseudomonas syringae pv. Tabaci mediated wildfire. [Formula 1]

delete According to claim 2, 4-carbamoyl phenylacetic acid is obtained by extracting the culture medium of Pseudomonas chlororaphis O6 (KACC 91054) strain with ethyl acetate to obtain an aqueous layer, the aqueous layer is butanol at pH 2 ~ 4.5 conditions Resistance inducing agent contained in the butanol layer obtained by extraction. Tobacco or its habitat comprising the step of treating 4-carbamoylphenylacetic acid of formula 1, Pseudomonas syringae pv. Method of inducing resistance to Pseudomonas syringae pv. Tabaci mediated wildfire. [Formula 1]

delete The method according to claim 5, wherein 4-carbamoylphenylacetic acid is obtained by extracting a culture solution of Pseudomonas chlorolapis O6 (KACC 91054) strain with ethyl acetate to obtain an aqueous solution layer, the aqueous solution layer is extracted with butanol at pH 2 ~ 4.5 conditions Resistance induction method that is contained in the butanol layer.

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