JP5807955B2 - Resistance inducer for plants, method for inducing plant resistance, and method for preventing plant diseases - Google Patents

Description

  The present invention relates to a plant resistance inducer that suppresses the growth of fungi that cause plant diseases, a method for inducing plant resistance, and a method for preventing plant diseases.

  Conventionally, agricultural chemicals and horticultural plants have used agricultural chemicals to reduce diseases caused by pathogenic bacteria. However, in order to reduce the risk to human health and environmental burden, there is a demand for the use of pesticides with higher safety by reducing the amount of conventional pesticides. Development of plant resistance inducers is underway as agrochemicals that meet this social demand.

The plant resistance inducer itself has almost no action of killing pathogens, and has an action of urging the plant itself to reject the pathogens by activating the defense mechanism inherent in the plant. For example, salicylic acid (SA), probenazole, validamycin A, acibenzoral S methyl (ASM) and the like are known. These plant resistance inducers activate a defense mechanism known as the plant's systemic acquired resistance (SAR) model. Specifically, when a part of a plant senses attack by a pathogen, the concentration of salicylic acid in the plant increases, and then a signal transduction pathway that expresses PR genes via the transcriptional regulator NPR1 leads to the pathogen. Resistance is acquired throughout the body of the plant (see FIG. 1). The above plant resistance inducers have already been put into practical use and are used all over the world.
In addition, in order to search for new plant resistance inducers, development of screening methods using a PR-1α gene expression monitoring system based on the SAR model has been actively developed (Non-patent Documents 1 and 2).

Evaluation of the Use of the Tobacco PR-1α Promoter to Monitor Defense Gene Expression by the Luciferase Bioluminescence Reporter System, Biosci. Biotechnol. Biochem., 75 (9), 1796-1800 (2011) Non-destructive bioluminescence detection system for monitoring defense gene expression in tobacco BY-2 cells, Plant Biotechnology, 28, 295-301 (2011)

Conventional plant resistance inducers have the problem of inhibiting plant growth.
The present invention has been made in view of the above circumstances, and uses a plant resistance inducer that can increase plant resistance and reduce plant growth inhibition, and the plant resistance inducer. It is an object of the present invention to provide a method for inducing plant resistance and a method for preventing plant diseases using the method for inducing resistance.

Plant resistance inducing agent according to claim 1 of the present invention is characterized in that it comprises a compound or a salt thereof as an active ingredient represented by the following general formula (C).

（一般式（C）中、X_１はハロゲンを表す。）

(In general formula (C), X ₁ represents halogen.)

Plant resistance inducing agent according to claim 2 of the present invention, in claim 1, characterized in that it comprises a compound or a salt thereof as an active ingredient represented by the following general formula (B).

（一般式（B）中、X_２はハロゲンを表す。）

(In the general formula (B), X ₂ represents halogen.)

Plant resistance inducing agent according to claim 3 of the present invention is characterized in that it comprises a compound or a salt thereof as an active ingredient represented by the following general formula (A).

（一般式（A）中、X_３，X_４は各々独立にハロゲンを表し、R_１は炭素原子数１〜６の直鎖又は分岐鎖状のアルキレン基を表す。）

(In general formula (A), X ₃ and X ₄ each independently represent halogen, and R ₁ represents a linear or branched alkylene group having 1 to 6 carbon atoms.)

Plant resistance inducing agent according to claim 4 of the present invention, in any one of claims 1 to 3, characterized in that it induces an antimicrobial substance.
Plant resistance inducing agent according to claim 5 of the present invention, in any one of claims 1-4, characterized in that induces resistance to pathogenic fungi.
Plant resistance inducing agent according to claim 6 of the present invention, in any one of claims 1-5, characterized in that it is used for cruciferous plants.
Resistance induction method of the plant according to claim 7 of the present invention, a plant for resistance inducing agent according to any one of claims 1 to 6, characterized in that exposing the plant.
The plant disease prevention method according to claim 8 of the present invention is characterized by using the plant resistance induction method according to claim 7.
The plant disease prevention method according to claim 9 of the present invention is characterized in that in claim 8, disease caused by infection with pathogenic filamentous fungi is prevented.
As used herein, the term “plant resistance inducer” means “plant resistance inducer”.

  According to the plant resistance inducer according to the present invention, it is possible to reduce plant diseases and to suppress a decrease in plant growth rate. Further, according to the plant resistance inducing method according to the present invention, it is possible to reduce plant diseases and reduce the growth rate of plants by a simple method of exposing a plant resistance inducing agent to a target plant. Can be suppressed. In addition, this method can prevent the target plant from being infected with pathogenic bacteria.

It is a schematic diagram showing the whole body acquisition model (SAR) of a plant. It is the schematic diagram showing the prevention method of the plant disease concerning this invention. It is an example of the result of investigating the induction transition of PR-1α gene expression of the plant resistance inducer according to the present invention. It is the arrangement | sequence of the primer used by RT-PCR of the Example. It is an example of the result of having investigated the infection inhibitory activity of the plant resistance inducer concerning this invention. It is an example of the result of having investigated the influence which the plant resistance inducer concerning this invention has on the growth of a plant.

Hereinafter, although embodiment of this invention is described, this invention is not limited to this embodiment.
<First embodiment>
1st embodiment of the plant resistance inducer of this invention contains the compound or its salt represented by the following general formula (C) as an active ingredient.

（一般式（C）中、Xはハロゲンを表す。）

(In the general formula (C), X represents halogen.)

X _{1 in the} general formula (C) is an element belonging to Group 17 in the periodic table such as Cl, Br, I, etc., preferably Cl or Br, and more preferably Cl.
When X ₁ is Cl, the compound represented by the general formula (C) is
6-Chloro-4-hydroxycoumarin (IUPAC Name: 6-chloro-2-hydroxychromen-4-one).
Hereinafter, X ₁ is a compound of Cl as compound C1.

<Second embodiment>
1st embodiment of the plant resistance inducer of this invention contains the compound or its salt represented by the following general formula (B) as an active ingredient.

（一般式（B）中、X_２はハロゲンを表す。）

(In the general formula (B), X ₂ represents halogen.)

X _{2 in the} general formula (B) is an element belonging to Group 17 in the periodic table such as Cl, Br, I, etc., preferably Cl or Br, and more preferably Br.
When X ₂ is Br, the compound represented by the general formula (B) is
5-Bromonicotinic acid (IUPAC Name: 5-bromopyridine-3-carboxylic acid).
Hereinafter, X ₂ is a compound of Br as Compound B1.

<Third embodiment>
3rd embodiment of the plant resistance inducer of this invention contains the compound represented by the following general formula (A), or its salt as an active ingredient.

（一般式（A）中、X_３，X_４は各々独立にハロゲンを表し、R_１は炭素原子数１〜６の直鎖又は分岐鎖状のアルキレン基を表す。）

(In general formula (A), X ₃ and X ₄ each independently represent halogen, and R ₁ represents a linear or branched alkylene group having 1 to 6 carbon atoms.)

X ₃ and X _{4 in} the general formula (A) are each independently an element belonging to Group 17 in the periodic table such as Cl, Br, I, etc., preferably Cl or Br, and more preferably Cl. X ₃ and X ₄ may be the same or different.
R ₁ is preferably a linear alkylene group having 1 to 6 carbon atoms, more preferably a linear alkylene group having 1 to 5 carbon atoms, and a linear alkylene group having 1 to 4 carbon atoms. Further, a linear alkylene group having 1 to 3 carbon atoms is particularly preferable, and a methylene group (—CH ₂ —) or an ethylene group (—CH ₂ —CH ₂ —) is most preferable.
When X ₃ and X ₄ are both Cl and R ₁ is an ethylene group, the compound represented by the general formula (A) is
3,5-dichloro-2-hydroxy benzaldehyde N- (2-hydroxyethyl) hydrazone
(IUPAC Name: 2,4-dichloro-6-[(1E)-[2- (2-hydroxyethyl) hydrazin-1-ylidene] methyl] phenol). Hereinafter, a compound in which X ₃ and X ₄ are both Cl and R ₁ is an ethylene group is referred to as a compound A1.

The compound represented by the general formula (A), (B), (C) may be a salt, and the salt is preferably an agriculturally acceptable salt. That is, among the salts, the form of the cation is known, and is acceptable for forming an agricultural or horticultural salt, for example, a salt with a cation such as Na ⁺ or K ⁺ It is preferable. The salt is preferably water-soluble.

  The compounds represented by the general formulas (A), (B), and (C) according to the present invention or salts thereof are conventional plant resistance inducers in target plants, as shown in the Examples below. It can induce the expression of PR-1α gene more than ASM. Moreover, the degree of plant growth inhibition is less than that of ASM. Therefore, a drug containing one or more of the compounds represented by the general formulas (A), (B) and (C) or a salt thereof as an active ingredient is an excellent plant resistance inducer.

  Since the plant resistance inducer according to the present invention enhances the expression of the PR-1α gene in the plant body that has been infected with the pathogen, the plant resistance inducer according to the present invention is the SA, NPR1 shown in FIG. It is considered that the signal transduction pathway of the PR-1α gene is positively regulated (activated) to impart systemic acquired resistance to the target plant.

  The plant resistance inducer according to the present invention can induce or promote the expression of PR proteins (pathogenesis-related proteins) and the like by activating the defense response of plants. Here, the PR protein is a general term for proteins that themselves have antibacterial activity and proteins that can produce other antibacterial substances.

  In general, PR proteins produced by plants have a very broad antimicrobial spectrum. Since the plant resistance inducer according to the present invention induces or promotes the target plant to produce PR protein, the antimicrobial spectrum of the plant resistance inducer according to the present invention is the PR of the plant. It becomes very wide like the antibacterial spectrum of proteins. For example, it is effective against pathogenic filamentous fungi (for example, the genus Colletotrichum) and bacteria (for example, the genus Pseudomonas). In addition, induction of systemic acquired resistance is considered to be effective for various viral diseases.

  Examples of the pathogenic filamentous fungi include blue mold disease, red blight disease, groove rot, red coat disease caused by fungal fungus and rust fungus, red star disease, gray mold disease, red burning disease, yellow patch, yellowing disease, Dwarf disease, powdery mildew, purple mold disease, ringworm disease, ash spot disease, horn spot disease, brown rot caused by fungal fungi, brown round spot disease, brown round spot disease, brown spot disease, brown spot disease, perforation Filamentous fungi that cause brown spot disease, browning disease, brown spot disease, strain rot, etc. are preferred, filamentous fungi of the genus Colletotrichum are more preferred, and Colletotrichum higginsianum (hereinafter referred to as C. higginsianum) is more preferred. Bacteria causing black rot, soft rot, spotted bacterial disease and the like are preferred as bacterial diseases.

  The type of plant to be used for the plant resistance inducer according to the present invention is not particularly limited as long as it is a plant that can acquire resistance by the SAR model, and may be a land plant or an aquatic plant. . As land plants, angiosperms and gymnosperms are suitable, asteraceae, orchidaceae, lilies, families, legumes, gramineae, rhesidaceae, euphorbiaceae, cyperaceae, sericidae, perilla, cucurbitaceae, eggplant Family and Brassicaceae are more preferred, and Eggplant and Brassicaceae are more preferred.

  An example of the lily family plant is onion. An example of the leguminous plant is soybean. An example of the celery family plant is carrot. Examples of the gramineous plant include rice, corn, and wheat. Examples of the cucurbitaceae plant include melon, watermelon, winter melon, cucumber, and pumpkin. Examples of the solanaceous plant include tobacco, tomato, potato, eggplant and bell pepper. Examples of the Brassicaceae plants include, for example, Nazuna, Brassica, Cabbage, Kale, Chinese cabbage, turnip, Japanese radish, Wasabi and mustard.

  In Examples described later, expression of PR-1α gene, which is an index of resistance to diseases, was improved in Arabidopsis thaliana plants. Therefore, it is particularly preferred that the plant resistance inducer of the present invention is used against cruciferous plants. For the same reason, it is particularly preferable that the method for inducing plant resistance of the present invention is also carried out on Brassicaceae plants.

  The plant resistance inducing method according to the present invention comprises a plant resistance inducing agent containing one or more compounds represented by the general formulas (A), (B), (C) or a salt thereof as an active ingredient. It is a method of exposing to the target plant. By this method, resistance to diseases can be induced in the target plant. In addition, preparation (preparation) of the said plant resistance inducer can be performed by a well-known method similarly to the conventional plant resistance inducer. The solvent used for the preparation is not particularly limited as long as it can dissolve the plant resistance inducer according to the present invention at a concentration capable of becoming an active ingredient, and a known solvent can be used. The solvent is preferably one that has no or little harm to plants. The concentration that can be the active ingredient can be determined by examining, for example, the plant resistance induction method described in the examples described later, and can be examined in a concentration range of 0.1 μM to 100 mM, for example. However, it is not limited to this concentration range.

  The specific operation and means for exposing the plant resistance inducer according to the present invention to the target plant are not particularly limited. For example, a chemical solution prepared by dissolving one or more of the compounds represented by the general formulas (A), (B), and (C) or a salt thereof at a concentration capable of becoming an active ingredient is prepared, and the chemical solution is sprayed on the plant. Spraying the body, bringing gauze soaked with the chemical into close contact with the plant, injecting the chemical into the stem of the plant with a syringe, or instilling the chemical into the root of the plant using an infusion device The operation to do can be illustrated.

  In the plant resistance inducing method according to the present invention, the site of the plant body sprayed with the chemical solution is not particularly limited. For example, it may be sprayed on all the leaves and stems of the plant body, or may be sprayed on only some leaves and some stems. Even when the whole plant body is not sprayed, the secondary metabolite produced in the sprayed site reaches the necessary site of the plant body, and resistance to the disease can be obtained even in the non-sprayed site.

  FIG. 2 is a schematic diagram showing a method for preventing plant diseases according to the present invention. The prevention method is a method in which primary stimulation is performed on a plant body (priming treatment is performed) by the plant resistance inducer and the resistance induction method of the present invention before the target plant body is infected with a pathogen. . Thereafter, when the plant body is invaded or attacked by pathogenic bacteria, this becomes a secondary stimulus, and a more prompt and powerful defense response can be generated compared to the case where the priming treatment is not performed.

  The type of disease and the type of pathogenic bacteria targeted by the prevention method are not particularly limited. In the examples described later, in Arabidopsis thaliana, which is a cruciferous plant, when an anthrax fungus that is a pathogenic filamentous fungus is infected, the expression of the PR-1α gene, which is an index for acquiring resistance of the plant, was improved. Therefore, the method for preventing plant diseases according to the present invention is preferably performed for the purpose of preventing diseases caused by infection with pathogenic filamentous fungi.

[Reference test]
Using a dimethyl sulfoxide aqueous solution (about 3%) as a solvent, a chemical solution a1 containing Compound A1 at 100 μM, a chemical solution b1 containing Compound B1 at 100 μM, and a chemical solution c1 containing Compound C1 at 100 μM were prepared. A filter paper containing 10 μl of each drug solution was placed on a PDA medium seeded so that C. higginsianum had a spore concentration of 10 ⁵ spores / single dish, and allowed to stand at 24 ° C. for about 2 weeks.
As a result, since C. higginsianum grew around the filter paper containing any chemical solution, none of compounds A1, B1, C1 may have direct bactericidal activity against C. higginsianum. confirmed. In addition, the growth of C. higginsianum was remarkably suppressed around the filter paper containing Hygromycin, which was a positive control performed in the same manner.

  Using Arabidopsis thaliana ecotype Columbia as a target plant and exposing compounds A1, B1, and C1 to acquire resistance to the pathogenic fungus Colletotrichum higginsianum (NIAS Genebanke: MAFF305635) Examined. As a control compound, ASM, which is a known plant resistance inducer, was used.

<Evaluation of priming effect>
[Example 1]
Arabidopsis seeds introduced with a plasmid construct (PR-1a :: Fluc ⁺ ) linked with a firefly luciferase gene downstream of the PR-1a gene promoter were sown in a 96-well plate with 100 μl of sterile water and subjected to vernalization for 5 days. Later, a final concentration of 0.1 mM luciferin was added and placed in a Biotron (light condition 12 hours, dark condition 12 hours, relative humidity 100%). After 24 hours, a dimethyl sulfoxide (DMSO) solution containing 2 mM of compound A1 was added to each well to adjust the final concentration of compound A1 to about 28 μM. 48 hours after adding compound A1, in order to verify the effect of priming, each well was added with salicylic acid (SA) to a final concentration of 2 mM to give secondary stimulation, and PR-1a gene expression inducing activity was obtained. It was examined by measuring luciferase activity. At this time, DMSO not containing Compound A1 was used as a negative control, and a DMSO solution of ASM used at the same concentration as Compound A1 was used as a positive control. The result is shown in the graph of FIG.
As can be seen from the graph, compound A1 has the same amount of luminescence as ASM, and therefore compound A1 has the same priming effect as ASM.

[Example 2]
The test was conducted in the same manner as in Example 1 except that Compound B1 was used instead of Compound A1. The result is shown in the graph of FIG.
As is apparent from the graph, although compound B1 is inferior to ASM, it exhibits a significantly higher luminescence amount than the negative control, so compound B1 has a priming effect.

[Example 3]
The test was conducted in the same manner as in Example 1 except that Compound C1 was used instead of Compound A1. The result is shown in the graph of FIG.
As is apparent from the graph, from the first day (48 hours after compound treatment) to the fifth day, compound C1 showed higher light emission than ASM, and during this period, the priming effect was superior to ASM. Have Further, in the period after the ninth day, the light emission amount is smaller than that of ASM, but it can be said that the priming effect is almost the same as that of ASM.

  From the results of Examples 1 to 3, each compound A1, B1, and C1 can activate the signal transduction pathway related to systemic acquired resistance (SAR) and exert a priming effect, and thus is useful for prevention of plant diseases. Can induce resistance to plant diseases.

<Evaluation of resistance to pathogenic bacteria>
[Example 4]
To evaluate the ability of compound A1 to induce resistance to pathogens, RT-PCR quantifies the expression levels of At-CBP20 gene in Arabidopsis thaliana and Ch-ACT gene in C. higginsianum. By determining the expression level of the Ch-ACT gene, the growth of C. higginsianum, that is, the infection suppression activity of anthrax was evaluated. At this time, the specific methods described in the following references were appropriately referred to. Each gene is a housekeeping gene.
(Reference: Monitoring fungal viability and development in plants infected with Colletorichum higginsianum by quantitative reverse transcription-polymerase chain reaction. J. Gen. Plant Pathol., 76: 1-6 (2010))

8 days after germination, mature Arabidopsis thaliana plant (Col-0) is spray-sprayed with 100 μM compound A1 using DMSO aqueous solution (about 3%), 48 hours later, spore concentration is adjusted to 10 ⁶ spores / ml The sterilized water containing C. higginsianum was inoculated by spray. Four days after the inoculation, the above-ground part of the plant was collected, and quantitative RT-PCR was performed by a known method using cDNA prepared from the extracted RNA. Primers having the sequence shown in FIG. 4 were used.
In addition, DMSO aqueous solution not containing Compound A1 was used as negative control (indicated as “Control” in the figure), and ASM DMSO aqueous solution used at the same concentration as Compound A1 was used as positive control (indicated as “ASM” in the figure). ). These results are shown in the graph of FIG. In FIG. 5, the symbols “a, b” indicate that there is no significant difference between bars with the same symbol.
As is apparent from the graph, Compound A1 showed an infection-suppressing activity equivalent to that of ASM.

[Example 5]
The test was conducted in the same manner as in Example 4 except that Compound B1 was used at a concentration of 300 μM instead of Compound A1, and the ASM concentration was changed to 300 μM. The result is shown in the graph of FIG.
As is apparent from the graph, Compound B1 showed an infection-suppressing activity equivalent to that of ASM.

[Example 6]
The test was conducted in the same manner as in Example 4 except that Compound C1 was used at a concentration of 300 μM instead of Compound A1, and the ASM concentration was changed to 300 μM. The result is shown in the graph of FIG.
As is apparent from the graph, Compound C1 exhibited an infection-suppressing activity equivalent to or better than ASM.

<Evaluation of plant growth inhibitory activity 1>
[Example 7]
8 days after germination, mature Arabidopsis thaliana plant (Col-0) is spray-sprayed with 100 μM of compound A1 using DMSO aqueous solution (about 3%), 48 hours later, spore concentration is 10 ² spores / ml The sterilized water containing C. higginsianum was inoculated by spray. 14 days after the inoculation, the above-ground part of the plant body was collected, and the raw weight of the above-ground part of the plant body was measured. In addition, the case where a DMSO aqueous solution not containing Compound A1 was used as a negative control (indicated as “Control” in the figure), and the case where a DMSO aqueous solution containing ASM at the same concentration was used as a positive control (in the figure, “ ASM ”). The result is shown in the graph (left) of FIG. In FIG. 6, the symbols “A, B, C, D, a, b” indicate that there is no significant difference between bars with the same symbol.
As is apparent from the graph, Compound A1 had a lower degree of growth inhibition than ASM. That is, Arabidopsis thaliana grew larger when Compound A1 was used.

[Example 8]
The test was conducted in the same manner as in Example 7 except that Compound B1 was used at a concentration of 300 μM instead of Compound A1, and the ASM concentration was changed to 300 μM. The result is shown in the graph (left) of FIG.
As is apparent from the graph, Compound B1 had a lower degree of growth inhibition than Compounds A1 and ASM. That is, Arabidopsis thaliana grew larger when Compound B1 was used.

[Example 9]
The test was conducted in the same manner as in Example 7, except that Compound C1 was used at a concentration of 300 μM instead of Compound A1, and the ASM concentration was changed to 300 μM. The result is shown in the graph (left) of FIG.
As is apparent from the graph, Compound C1 had a lower degree of growth inhibition than Compound A1, Compound B1 and ASM. That is, Arabidopsis thaliana grew larger when Compound C1 was used.

<Evaluation of plant growth inhibitory activity 2>
[Example 10]
A mature Arabidopsis thaliana plant (Col-0) 8 days after germination was sprayed with Compound A1 prepared to 100 μM using an aqueous DMSO solution (about 3%), and sterilized water was sprayed 48 hours later. 14 days later, the above-ground part of the plant body was collected, and the raw weight of the above-ground part of the plant body was measured. In addition, the case where a DMSO aqueous solution not containing Compound A1 was used as a negative control (indicated as “Control” in the figure), and the case where a DMSO aqueous solution containing ASM at the same concentration was used as a positive control (in the figure, “ ASM ”). The result is shown in the graph (right) of FIG.
As is apparent from the graph, Compound A1 showed growth inhibition equivalent to ASM.

[Example 11]
The test was conducted in the same manner as in Example 10 except that Compound B1 was used at a concentration of 300 μM instead of Compound A1, and the ASM concentration was changed to 300 μM. The result is shown in the graph (right) of FIG.
As is apparent from the graph, Compound B1 showed growth inhibition equivalent to that of Compounds A1 and ASM.

[Example 12]
The test was conducted in the same manner as in Example 10 except that Compound C1 was used at a concentration of 300 μM instead of Compound A1, and the ASM concentration was changed to 300 μM. The result is shown in the graph (right) of FIG.
As is apparent from the graph, Compound C1 had a lower degree of growth inhibition than Compound A1, Compound B1 and ASM. In addition, it is clear that Compound C1 does not substantially inhibit plant growth because Compound C1 has the same growth inhibitory activity as when sprayed with a DMSO aqueous solution containing no compound.

  From the results of Examples 4 to 12, each compound A1, B1, and C1 is excellent in that it induces resistance to diseases at a level equivalent to that of conventional ASM and that the degree of plant growth inhibition is less than that of conventional ASM. It is clear that it is a plant resistance inducer.

Claims (9)

Plant resistance inducing agent which comprises a compound or a salt thereof as an active ingredient represented by the following general formula (C).

(In general formula (C), X ₁ represents halogen.) Plant resistance inducing agent which comprises a compound or a salt thereof as an active ingredient represented by the following general formula (B).

(In the general formula (B), X ₂ represents halogen.) Plant resistance inducing agent which comprises a compound or a salt thereof as an active ingredient represented by the following general formula (A).

(In general formula (A), X ₃ and X ₄ each independently represent halogen, and R ₁ represents a linear or branched alkylene group having 1 to 6 carbon atoms.) Plant resistance inducing agent according to any one of claims 1 to 3, characterized in that induces antimicrobial substance. The resistance inducer for plants according to any one of claims 1 to 4, which induces resistance to pathogenic filamentous fungi. Plant resistance inducing agent according to any one of claims 1 to 5, characterized in that used for cruciferous plants. Resistance induction method of the plant, characterized in that the plant resistance inducing agent according to any one of claims 1 to 6 is exposed to the plant. A method for preventing plant diseases, which comprises using the plant resistance induction method according to claim 7. The method for preventing plant diseases according to claim 8, wherein diseases caused by infection with pathogenic filamentous fungi are prevented.

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