JP7688943B2 - Method for inducing plant resistance, plant resistance inducer and biostimulant - Google Patents

Description

The present invention relates to a method for inducing plant resistance, a plant resistance inducer, and a biostimulant.
This application claims priority based on Japanese Patent Application No. 2021-046236, filed on March 19, 2021, the contents of which are incorporated herein by reference.

Plants are invaded and attacked by a variety of pathogenic microorganisms, including filamentous fungi, bacteria, and viruses. To combat these, plants have developed defense mechanisms. There are various stages in the defense mechanism. First, before the pathogen invades, the invasion of the pathogen is prevented by physical barriers created by cell walls and by opening and closing the stomata in the leaves. Even after the pathogen invades, plant cells have a mechanism to recognize the pathogen invasion and inhibit the progress of infection by accumulating polysaccharides at the site of invasion. Furthermore, plants have a mechanism to strengthen resistance not only at the site of pathogen infection, but also at non-infected sites by transmitting signals from the infected site to the entire body. This mechanism involves plant hormones and the expression of numerous genes.

FIG. 1 is a diagram illustrating the signal transduction pathway involved in induction of plant resistance.
Systemic acquired resistance (SAR), a mechanism in which the plant hormone salicylic acid (SA) is involved in signal transduction, has been studied in recent years as a systemic resistance induction mechanism. It has become clear that the SAR defense response can counter a wide range of pathogens by strengthening the plant's own disease resistance.

SA is known to primarily induce resistance against "parasitic pathogens," which are pathogens that obtain nutrients from living cells. Parasitic pathogens absorb nutrients from plant cells and often coexist with the plant. Examples of parasitic pathogens include rice blast fungus and anthracnose fungus.

Compounds with SAR-inducing activity have been put to practical use as resistance inducers or plant activators, and are mainly used as effective materials for disease control of rice in Japan. For example, probenazole (PBZ, product name Oryzemate) has annual sales of about 10 billion yen, even though it has been more than 40 years since its development. In addition to propenazole, there are several other resistance inducers or plant activators with SAR-inducing activity, including acibenzolar-S-methyl (ASM), palidamycin A (VMA), benzothiadiazole (BTH), thiadinil (TDL), and isotianil.

On the other hand, a mechanism for disease resistance expression that works through a different mechanism of action from SAR is also known. Unlike SAR, induced systemic resistance (ISR) is not dependent on SA, but is known to be induced through a pathway for disease resistance expression that is dependent on the plant hormone jasmonic acid (JA) (see Figure 1). It has been found that the defense response genes induced by ISR and the types of pathogens against which it is effective are also different from those of SAR.

JA primarily induces resistance to saprophytic pathogens (also known as necrotic parasites), which are pathogens that obtain nutrients from dead cells, and a defense response against damage such as feeding damage by pests. A representative saprophytic pathogen is Botrytis cinerea. Botrytis cinerea infects almost all plants, and drug-resistant strains are highly susceptible to the development of such pathogens. However, compounds with SAR-inducing activity are poorly effective in controlling saprophytic pathogens, and existing plant activators such as probenazole are ineffective against saprophytic pathogens such as Botrytis cinerea.

Therefore, if there were a compound that had the activity of inducing the ISR system, it could potentially be used as a new pest control material that would be effective against diseases that cannot be treated with existing SAR resistance inducers. However, research to date has not found any low-molecular-weight compounds with such activity to a commercially viable level. Bestatin has been reported as a compound that specifically activates the JA signal (Non-Patent Document 1).

In addition to the JA signaling system, a pathway for disease resistance expression dependent on the plant hormone ethylene (ET) is known, which induces a defense response against "necropsy parasites" in the same way as JA. To date, compounds known to induce defense activation through the JA/ET signaling system include hexanoic acid, arachidonic acid, and N-acylamides (alkamides) (Non-Patent Documents 2-4). All of these induce the expression of JA-responsive genes, including PDF1.2 and VSP2, and have been shown to be effective in suppressing the formation of lesions caused by Botrytis cinerea using Arabidopsis thaliana. In addition, similar inhibition of lesion formation has been observed in tomatoes treated with hexanoic acid and arachidonic acid (Non-Patent Document 5). This indicates that JA-based resistance inducers are effective in controlling Botrytis cinerea. However, there are problems with the effectiveness of these agents, such as the need for high-concentration treatment.

Since resistance inducers act on plants and have no bactericidal activity in themselves, they are less likely to cause the emergence of drug-resistant bacteria and have been attracting attention in recent years as drugs with low environmental impact. However, the only resistance inducers currently in practical use are PBZ and ASM, which act on the SA signaling pathway, and no resistance inducers that act on the JA/ET signaling pathway have been put to practical use.

The inventors' previous research has provided a resistance inducer that acts on the SA signaling pathway (Patent Document 1) and a resistance inducer that acts on the JA signaling pathway (Patent Document 2).

JP 2017-197456 A International Publication No. 2016/006351

Zheng W, Zhai Q, Sun J, Li CB, Zhang L, Li H, Zhang X, Li S, Xu Y, Jiang H, Wu X, Li C. Bestatin, an inhibitor of aminopeptidases, provides a chemical genetics approach to dissect jasmonate signaling in Arabidopsis. Plant Physiol. (2006) 141, 1400-1413. Kravchuk Z, Vicedo B, Flors V, Camanes G, Gonzalez-Bosch C, Garcia-Agustin P (2011) Priming for JA-dependent defenses using hexanoic acid is an effective mechanism to protect Arabidopsis against B. cinerea. J Plant Physiol 168: 359-366 Mendez-Bravo A, Calderon-Vazquez C, Ibarra-Laclette E, Raya-Gonzalez J, Ramirez-Chavez E, Molina-Torres J, Guevara-Garcia AA, Lopez-Bucio J, Herrera-Estrella L (2011) Alkamides activate jasmonic acid biosynthesis and signaling pathways and confer resistance to Botrytis cinerea in Arabidopsis thaliana. PLoS One 6 e27251 Savchenko T, Walley JW, Chehab EW, Xiao Y, Kaspi R, Pye MF, Mohamed ME, Lazarus CM, Bostock RM, Dehesh K (2010) Arachidonic acid: an evolutionarily conserved signaling molecule modulates plant stress signaling networks. Plant Cell 22: 3193-205 Vicedo B, Flors V, de la O Leyva M, Finiti I, Kravchuk Z, Real MD, Garcia-Agustin P, Gonzalez-Bosch C (2009) Hexanoic acid-induced resistance against Botrytis cinerea in tomato plants. Mol Plant Microbe Interact 22:1455-65

As described above, several compounds that activate the JA signal have been reported, but none have been put to practical use. In addition, since activation of the SA signaling system usually suppresses the JA/ET signaling system, no resistance inducer that activates both the SA signaling system and the JA/ET signaling system and has excellent plant resistance induction control activity is known.
The present invention has been made in view of the above circumstances, and an objective of the present invention is to provide a method for inducing plant resistance and a plant resistance inducer that are excellent in plant resistance induction activity.
Another object of the present invention is to provide a biostimulant.

As a result of intensive research aimed at solving the above-mentioned problems, the inventors discovered that it is possible to activate both the SA signaling system and the JA/ET signaling system, thereby inducing extremely excellent resistance, and thus completed the present invention.
That is, the present invention has the following aspects.

［式（１）中、
Ｒ^１、Ｒ^２及びＲ^３は、それぞれ独立して、炭素数１～４の直鎖状若しくは分岐鎖状のアルキル基、水酸基、ハロゲン原子、又は炭素数２～４の直鎖状若しくは分岐鎖状のアルケニル基を表し、
Ｒ^４は、水素原子、炭素数１～４の直鎖状若しくは分岐鎖状のアルキル基又は炭素数２～４の直鎖状若しくは分岐鎖状のアルケニル基を表し、
Ｒ^５及びＲ^６は、それぞれ独立して、水素原子、炭素数１～４の直鎖状若しくは分岐鎖状のアルキル基、炭素数２～４の直鎖状若しくは分岐鎖状のアルケニル基、又はハロゲン原子を表す。
ｎ１はＲ^１の数を表し、０～５のいずれかの整数であり、ｎ１が２以上のとき、Ｒ^１同士は互いに同一でも異なっていてもよい。ｎ２はＲ^２の数を表し、０～５のいずれかの整数であり、ｎ２が２以上のとき、Ｒ^２同士は互いに同一でも異なっていてもよい。ｎ３はＲ^３の数を表し、０～３のいずれかの整数であり、ｎ３が２以上のとき、Ｒ^３同士は互いに同一でも異なっていてもよい。］
［３］活物寄生菌及び死物寄生菌の両方に対する植物抵抗性を誘導する、前記［１］又は［２］に記載の方法。
［４］活物寄生菌及び死物寄生菌に対する抗菌活性を有さない、前記［１］～［３］のいずれか一つに記載の方法。
［５］前記施用が、前記植物抵抗性誘導剤の有効量を植物の根に接触させることを含む、前記［１］～［４］のいずれか一つに記載の方法。
［６］下記一般式（１）で表される化合物又はその塩を有効成分として含有する、植物抵抗性誘導剤。

［式（１）中、
Ｒ^１、Ｒ^２及びＲ^３は、それぞれ独立して、炭素数１～４の直鎖状若しくは分岐鎖状のアルキル基、水酸基、ハロゲン原子、又は炭素数２～４の直鎖状若しくは分岐鎖状のアルケニル基を表し、
Ｒ^４は、水素原子、炭素数１～４の直鎖状若しくは分岐鎖状のアルキル基又は炭素数２～４の直鎖状若しくは分岐鎖状のアルケニル基を表し、
Ｒ^５及びＲ^６は、それぞれ独立して、水素原子、炭素数１～４の直鎖状若しくは分岐鎖状のアルキル基、炭素数２～４の直鎖状若しくは分岐鎖状のアルケニル基、又はハロゲン原子を表す。
ｎ１はＲ^１の数を表し、０～５のいずれかの整数であり、ｎ１が２以上のとき、Ｒ^１同士は互いに同一でも異なっていてもよい。ｎ２はＲ^２の数を表し、０～５のいずれかの整数であり、ｎ２が２以上のとき、Ｒ^２同士は互いに同一でも異なっていてもよい。ｎ３はＲ^３の数を表し、０～３のいずれかの整数であり、ｎ３が２以上のとき、Ｒ^３同士は互いに同一でも異なっていてもよい。］
［７］下記一般式（１）で表される化合物又はその塩を有効成分として含有する、バイオスティミュラント。

［式（１）中、
Ｒ^１、Ｒ^２及びＲ^３は、それぞれ独立して、炭素数１～４の直鎖状若しくは分岐鎖状のアルキル基、水酸基、ハロゲン原子、又は炭素数２～４の直鎖状若しくは分岐鎖状のアルケニル基を表し、
Ｒ^４は、水素原子、炭素数１～４の直鎖状若しくは分岐鎖状のアルキル基又は炭素数２～４の直鎖状若しくは分岐鎖状のアルケニル基を表し、
Ｒ^５及びＲ^６は、それぞれ独立して、水素原子、炭素数１～４の直鎖状若しくは分岐鎖状のアルキル基、炭素数２～４の直鎖状若しくは分岐鎖状のアルケニル基、又はハロゲン原子を表す。
ｎ１はＲ^１の数を表し、０～５のいずれかの整数であり、ｎ１が２以上のとき、Ｒ^１同士は互いに同一でも異なっていてもよい。ｎ２はＲ^２の数を表し、０～５のいずれかの整数であり、ｎ２が２以上のとき、Ｒ^２同士は互いに同一でも異なっていてもよい。ｎ３はＲ^３の数を表し、０～３のいずれかの整数であり、ｎ３が２以上のとき、Ｒ^３同士は互いに同一でも異なっていてもよい。］ [1] A method for inducing resistance in a plant, comprising the steps of:
The method comprises applying a plant resistance inducer to a plant to activate a salicylic acid signaling system and a jasmonic acid and/or ethylene signaling system;
A method for inducing plant resistance, wherein the active ingredient of the plant resistance inducer is a low molecular weight compound, and one type of the active ingredient activates the salicylic acid signaling system, and the jasmonic acid and/or the ethylene signaling system.
[2] The method according to [1] above, wherein the plant resistance inducer contains a compound represented by the following general formula (1) or a salt thereof as an active ingredient:

[In formula (1),
R ¹ , R ² and R ³ each independently represent a linear or branched alkyl group having 1 to 4 carbon atoms, a hydroxyl group, a halogen atom, or a linear or branched alkenyl group having 2 to 4 carbon atoms;
R ⁴ represents a hydrogen atom, a linear or branched alkyl group having 1 to 4 carbon atoms, or a linear or branched alkenyl group having 2 to 4 carbon atoms;
^R5 and ^R6 each independently represent a hydrogen atom, a linear or branched alkyl group having 1 to 4 carbon atoms, a linear or branched alkenyl group having 2 to 4 carbon atoms, or a halogen atom.
n1 represents the number of ^R1 and is an integer of 0 to 5, and when n1 is 2 or more, ^R1 may be the same or different from each other. n2 represents the number of ^R2 and is an integer of 0 to 5, and when n2 is 2 or more, ^R2 may be the same or different from each other. n3 represents the number of ^R3 and is an integer of 0 to 3, and when n3 is 2 or more, ^R3 may be the same or different from each other.
[3] The method according to [1] or [2], which induces plant resistance against both live and dead parasitic fungi.
[4] The method according to any one of [1] to [3] above, which has no antibacterial activity against live parasitic bacteria and non-live parasitic bacteria.
[5] The method according to any one of [1] to [4], wherein the applying comprises contacting an effective amount of the plant resistance inducer with a root of a plant.
[6] A plant resistance inducer comprising, as an active ingredient, a compound represented by the following general formula (1) or a salt thereof:

[In formula (1),
R ¹ , R ² and R ³ each independently represent a linear or branched alkyl group having 1 to 4 carbon atoms, a hydroxyl group, a halogen atom, or a linear or branched alkenyl group having 2 to 4 carbon atoms;
R ⁴ represents a hydrogen atom, a linear or branched alkyl group having 1 to 4 carbon atoms, or a linear or branched alkenyl group having 2 to 4 carbon atoms;
^R5 and ^R6 each independently represent a hydrogen atom, a linear or branched alkyl group having 1 to 4 carbon atoms, a linear or branched alkenyl group having 2 to 4 carbon atoms, or a halogen atom.
n1 represents the number of ^R1 and is an integer of 0 to 5, and when n1 is 2 or more, ^R1 may be the same or different from each other. n2 represents the number of ^R2 and is an integer of 0 to 5, and when n2 is 2 or more, ^R2 may be the same or different from each other. n3 represents the number of ^R3 and is an integer of 0 to 3, and when n3 is 2 or more, ^R3 may be the same or different from each other.
[7] A biostimulant comprising a compound represented by the following general formula (1) or a salt thereof as an active ingredient:

[In formula (1),
R ¹ , R ² and R ³ each independently represent a linear or branched alkyl group having 1 to 4 carbon atoms, a hydroxyl group, a halogen atom, or a linear or branched alkenyl group having 2 to 4 carbon atoms;
R ⁴ represents a hydrogen atom, a linear or branched alkyl group having 1 to 4 carbon atoms, or a linear or branched alkenyl group having 2 to 4 carbon atoms;
^R5 and ^R6 each independently represent a hydrogen atom, a linear or branched alkyl group having 1 to 4 carbon atoms, a linear or branched alkenyl group having 2 to 4 carbon atoms, or a halogen atom.
n1 represents the number of ^R1 and is an integer of 0 to 5, and when n1 is 2 or more, ^R1 may be the same or different from each other. n2 represents the number of ^R2 and is an integer of 0 to 5, and when n2 is 2 or more, ^R2 may be the same or different from each other. n3 represents the number of ^R3 and is an integer of 0 to 3, and when n3 is 2 or more, ^R3 may be the same or different from each other.

According to the present invention, there can be provided a method for inducing plant resistance and a plant resistance inducer having excellent plant resistance induction activity.
Furthermore, according to the present invention, a biostimulant can be provided.

FIG. 1 is a diagram illustrating the signal transduction pathway involved in resistance induction in plants. This shows the results of measuring JA-responsive gene expression in Arabidopsis seedlings carrying VSP1::Fluc after treatment with compound X. This shows the results of measuring JA-responsive gene expression in mature Arabidopsis plants carrying VSP1::Fluc after treatment with compound X. This shows the results of measuring SA-responsive gene expression in Arabidopsis seedlings carrying PR-1a::Fluc after treatment with compound X. This shows the results of measuring ET-responsive gene expression in Arabidopsis seedlings carrying PDF1.2::Fluc after treatment with compound X. 1 shows the results of evaluating the control effect of compound X against Botrytis cinerea. 1 shows the results of evaluating the control effect of compound X against anthrax bacteria. This shows the results of measuring the time course of JA-responsive gene expression following treatment with compound X in Arabidopsis seedlings. This shows the results of measuring the time course of changes in expression of SA-responsive genes following treatment with compound X in Arabidopsis seedlings. This shows the results of measuring the time course of changes in the expression of JA-responsive genes in mature Arabidopsis plants following treatment with compound X. 1 shows the results of measuring the time course of changes in expression of SA-responsive genes in mature Arabidopsis plants following treatment with compound X. This shows the results of measuring the time course of changes in the expression of ET responsive genes in Arabidopsis seedlings following treatment with compound X. 1 shows the results of measuring the time course of changes in expression of ET responsive genes in mature Arabidopsis plants following treatment with compound X. 1 shows the results of evaluating the antibacterial activity of compound X against Botrytis cinerea. 1 shows the results of evaluating the antibacterial activity of compound X against anthrax bacteria.

Below, embodiments of the plant resistance induction method, plant resistance inducer, and biostimulant of the present invention are described.

<Method of inducing plant resistance>
The method for inducing plant resistance according to the embodiment is a method for inducing resistance in a plant, comprising:
The method comprises applying a plant resistance inducer to a plant to activate a salicylic acid signaling system and a jasmonic acid and/or ethylene signaling system;
The active ingredient of the plant resistance inducer is a low molecular weight compound, and one type of the active ingredient activates the salicylic acid signaling system, and the jasmonic acid and/or ethylene signaling system.

According to the mechanism of action classification of drugs by FRAC (Fungicide Resistance Action Committee), plant resistance inducers are classified as "P Induction of resistance in host plants". This is clearly distinguished from general fungicides (including bactericides) that act on the biosynthetic pathway of pathogens. Resistance inducers themselves do not have fungicidal activity. Therefore, they have the advantage that drug-resistant bacteria are less likely to appear. Note that only salicylic acid signaling is presented as a classification of the above resistance inducers.
Hereinafter, the plant resistance inducer may be simply referred to as the "resistance inducer."

Figure 1 is a diagram explaining the signal transduction pathway involved in the induction of resistance in plants. As shown in Figure 1, the salicylic acid signal transduction system (also called the "SA system") includes a signal transduction pathway that continues from SA to NPR1 gene expression and PR1 gene expression. The SA system is capable of inducing resistance against living parasitic fungi.

The jasmonic acid signaling system (also referred to as the "JA system") includes a signaling pathway that starts with JA, followed by JAR1 gene expression and VSP1 gene expression. The JA system can induce resistance to necrotic parasites and/or injury responses. Examples of injury responses include pest resistance and drought tolerance.

The ethylene signal transduction system (also referred to as "ET system") includes a signal transduction pathway that continues from ET to EIN2 gene expression and PDF1.2 gene expression. The ET system can induce resistance to necrotic parasites.
Because signals from JAR1 also induce PDF1.2 gene expression, jasmonic acid can activate the JA and/or ET systems.

On the other hand, it has been widely known that the JA and SA systems are in an antagonistic relationship, and it was not known that resistance inducers could activate both of them and induce resistance.

However, according to the method of the embodiment, the application of one active ingredient can activate the SA system as well as the JA system and/or the ET system, a finding that overturns the conventional concept of resistance induction.

According to the method of the embodiment, it is possible to exert resistance induction activity equal to or greater than that of ASM and PBZ, which are representative resistance inducers that activate only the SA system. It is presumed that this excellent effect is achieved by a novel mechanism of action that activates the SA system, as well as the JA system and/or the ET system. And, according to the method of the embodiment, it is possible to activate the SA system, as well as the JA system and/or the ET system, with at least one active ingredient.

The reason why the SA system, JA system, and/or ET system can be activated is not entirely clear. However, in the examples described below, the JA system is activated early and the SA system is activated late, so it is presumed that even antagonistic resistance induction pathways can be activated with a time lag.

Among the resistances in plants, the expression of disease resistance can be determined, for example, by any one or more of the following indicators "1" and "2".
"1" Using the expression of a gene induced in the SA, JA, or ET system as an indicator, plants treated with a resistance inducer are compared with untreated plants (control plants), and if the expression of the gene is significantly improved in the plants treated with the resistance inducer, the expression of disease resistance can be determined.
The expression of resistance to living parasitic bacteria can be indicated by the expression of genes whose expression is induced in the SA system.
The expression of resistance to necrotrophic parasites can be indicated by the expression of genes whose expression is induced by the JA system and/or the ET system.
"2" Using the severity of the plant disease as an indicator, plants treated with a resistance inducer can be compared with untreated plants (control plants), and if the pathological condition of the plant disease is significantly improved in the plants treated with the resistance inducer, the expression of disease resistance can be determined.

The expression of pest resistance in a plant can be determined, for example, by any one or more of the following indicators "3" to "5".
"3" Using the expression of a gene induced in the JA system as an indicator, plants treated with a resistance inducer are compared with untreated plants (control plants), and if the expression of the gene is significantly improved in the plants treated with the resistance inducer, the expression of pest resistance can be determined.
"4" The degree of feeding damage to the plant body is used as an indicator, and by comparing plants treated with a resistance inducer with untreated plants (control plants), the expression of pest resistance can be determined if the condition of feeding damage to the plant body is improved in the plants treated with the resistance inducer.
"5" The occurrence of pests and other organisms in the area treated with the resistance inducer is used as an indicator, and by comparing plants treated with the resistance inducer with untreated plants (control plants), the expression of pest resistance can be determined if the occurrence of pests and other organisms in the area treated with the resistance inducer is low in the plants treated with the resistance inducer.

The method of the embodiment is excellent in activating the salicylic acid signaling system, and the jasmonic acid and/or ethylene signaling system, and allows the plant to exhibit excellent resistance. The following values are preferable as values representing the degree of activation of the SA system, the JA system and/or the ET system compared to a control plant.
In the method of the embodiment, it is preferable to activate the SA system by more than two-fold compared to a control plant, using the expression level of the VSP1 gene as an indicator, and it is preferable to activate the JA system by more than two-fold using the expression level of the PR1 gene as an indicator, and/or it is preferable to activate the ET system by more than two-fold using the expression level of the PDF1.2 gene as an indicator.

The activation of the SA system is preferably 2-fold or more, more preferably 2- to 100-fold, even more preferably 3- to 60-fold, and particularly preferably 4- to 10-fold.
The activation of the JA system is preferably 2-fold or more, more preferably 2- to 10-fold, and even more preferably 3- to 8-fold.
The activation of the ET system is preferably 2-fold or more, more preferably 2- to 150-fold, even more preferably 3- to 60-fold, and particularly preferably 4- to 50-fold.

The above VSP1, PR1, and PDF1.2 genes are genes (homologs) corresponding to each gene of the plant in which the resistance inducer is used.

The VSP1 gene includes genes encoding proteins selected from the group consisting of the following (a1) to (c1):
(a1) a protein having an amino acid sequence represented by SEQ ID NO: 1; (b1) a protein having an amino acid sequence represented by SEQ ID NO: 1 in which one to several amino acids have been substituted, deleted, inserted, or added, and having acid phosphatase activity; (c1) a protein having an amino acid sequence that has a sequence identity of 80% or more with the amino acid sequence represented by SEQ ID NO: 1, and having acid phosphatase activity.

The amino acid sequence represented by sequence number 1 is the amino acid sequence of Arabidopsis thaliana VSP1 (AT5G24780.1).

The PR1 gene includes genes encoding proteins selected from the group consisting of the following (a2) to (c2):
(a2) a protein having an amino acid sequence represented by SEQ ID NO: 2; (b2) a protein having an amino acid sequence represented by SEQ ID NO: 2 in which one to several amino acids have been substituted, deleted, inserted, or added, and which induces resistance in plants; (c2) a protein having an amino acid sequence that has a sequence identity of 80% or more with the amino acid sequence represented by SEQ ID NO: 2, and which induces resistance in plants.

The amino acid sequence represented by sequence number 2 is the amino acid sequence of Arabidopsis thaliana PR1 (AT2G14610.1).

The PDF1.2 gene includes a gene encoding a protein selected from the group consisting of the following (a3) to (c3):
(a3) a protein having an amino acid sequence represented by SEQ ID NO: 3; (b3) a protein having an amino acid sequence represented by SEQ ID NO: 3 in which one to several amino acids have been substituted, deleted, inserted, or added, and which induces resistance in plants; (c3) a protein having an amino acid sequence that has a sequence identity of 80% or more with the amino acid sequence represented by SEQ ID NO: 3, and which induces resistance in plants.

The amino acid sequence represented by sequence number 3 is the amino acid sequence of Arabidopsis thaliana PDF1.2 (AT5G44420.1).

The "one to several" bases may be, for example, 1 to 30, 1 to 20, 1 to 10, 1 to 5, or 1 to 3 bases.
The sequence identity may be 80% or more but less than 100%, for example, 85% or more, 90% or more, 95% or more, or 98% or more.
The sequence identity between amino acid sequences can be calculated using known sequence alignment algorithms such as BLAST (Basic Local Alignment Search Tool) or blastp.

The plant resistance inducer used in the method of the embodiment is a low molecular weight compound, and preferred plant resistance inducers are described in detail below. In this specification, a "low molecular weight compound" refers to a compound with a molecular weight of 10,000 or less, and may have a molecular weight of 100 to 10,000, 130 to 1,000, or 150 to 500, for example. Methods for applying the plant resistance inducer include various methods exemplified below as methods for contacting a plant with a plant resistance inducer.

According to the method of the embodiment, a resistance inducer is used to activate the JA system and/or the ET system, and a wide range of resistance, including resistance to live and non-live parasitic bacteria, can be induced. In addition, the method does not have antibacterial activity against bacteria including live and non-live parasitic bacteria, and can be implemented as a production method with low environmental impact, with reduced damage to environmental microorganisms.

Examples of plant resistance inducers that can be used in the method of the embodiment include the following plant resistance inducers:

≪Plant resistance inducers≫

The plant resistance inducer of the embodiment contains a compound represented by the following general formula (1) or a salt thereof as an active ingredient.

[In formula (1),
R ¹ , R ² and R ³ each independently represent a linear or branched alkyl group having 1 to 4 carbon atoms, a hydroxyl group, a halogen atom, or a linear or branched alkenyl group having 2 to 4 carbon atoms;
R ⁴ represents a hydrogen atom, a linear or branched alkyl group having 1 to 4 carbon atoms, or a linear or branched alkenyl group having 2 to 4 carbon atoms;
^R5 and ^R6 each independently represent a hydrogen atom, a linear or branched alkyl group having 1 to 4 carbon atoms, a linear or branched alkenyl group having 2 to 4 carbon atoms, or a halogen atom.
n1 represents the number of ^R1 and is an integer of 0 to 5, and when n1 is 2 or more, ^R1 may be the same or different from each other. n2 represents the number of ^R2 and is an integer of 0 to 5, and when n2 is 2 or more, ^R2 may be the same or different from each other. n3 represents the number of ^R3 and is an integer of 0 to 3, and when n3 is 2 or more, ^R3 may be the same or different from each other.

Examples of the linear or branched alkyl group having 1 to 4 carbon atoms for R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group and a tert-butyl group.
The linear or branched alkenyl group having 2 to 4 carbon atoms for R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ preferably has 2 to 3 carbon atoms. Examples of the alkenyl group include an ethenyl group (vinyl group) and a 2-propenyl group (allyl group).

The halogen atoms of R ¹ , R ² , R ³ , R ⁵ and R ⁶ are elements belonging to Group 17 of the periodic table, such as F, Cl, Br, I, etc.

In the compound represented by the general formula (1), when R ⁴ to R ⁶ are hydrogen atoms, examples of the compound represented by the following general formula (1-1) include:

[In formula (1-1), R ¹ , R ² , R ³ , n1, n2 and n3 are the same as those in general formula (1).]

In the compound represented by the general formula (1-1), R ¹ , R ² and R ³ may each independently represent a linear or branched alkyl group having 1 to 4 carbon atoms.

In the compound represented by general formula (1-1), when n1 to n3 are 0, examples include the compound represented by the following formula (1-2).

The compound represented by the general formula (1) may be a salt, and the salt is preferably an agriculturally acceptable salt. Depending on the type of the substituent, it may form an acid addition salt or a salt with a base. Specifically, it may be an acid addition salt with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, etc., or an organic acid such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, mandelic acid, tartaric acid, dibenzoyltartaric acid, ditoluoyltartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, aspartic acid, glutamic acid, or an inorganic base such as sodium, potassium, magnesium, calcium, or aluminum, or methylamine, ethylamine, ethanolamine, Examples of the salt include salts with organic bases such as lysine and ornithine, salts of various amino acids and amino acid derivatives such as acetylleucine, and ammonium salts. Preferred are acid addition salts with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, mandelic acid, tartaric acid, dibenzoyltartaric acid, ditoluoyltartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, aspartic acid, and glutamic acid.

Furthermore, the active ingredient also includes various hydrates and solvates of the compound of the general formula (1) and its salts, as well as crystalline polymorphic substances. The active ingredient also includes compounds labeled with various radioactive or non-radioactive isotopes.

The compounds of the general formula (1) and their salts may be commercially available compounds and their salts. The compounds of the general formula (1) and their salts may be produced by applying known synthesis methods to various known compounds, taking advantage of the characteristics based on the basic structure or the type of substituent. In this case, depending on the type of functional group, it may be effective in terms of production technology to replace the functional group with an appropriate protecting group well known to those skilled in the art at the stage from the raw material to the intermediate.

As used herein, induction of plant resistance includes inducing, strengthening, promoting, and maintaining disease resistance or pest resistance in a plant.
The plant resistance inducer of the embodiment induces disease resistance, and therefore can also be provided as a plant disease control agent.
Since the plant resistance inducer of the embodiment induces pest resistance, it can also be provided as a pest control agent.

Inducing, strengthening, and promoting disease resistance or pest resistance in a plant means significantly improving the expression of plant resistance or pest resistance in a plant treated with the plant resistance inducer of the embodiment, when compared to a plant treated with the plant resistance inducer of the embodiment and an untreated plant.

Maintaining disease resistance or pest resistance in a plant means that the expression of plant resistance or pest resistance is sustained significantly longer in a plant treated with the plant resistance inducer of the embodiment compared to a plant that has been treated with the plant resistance inducer of the embodiment and an untreated plant.

According to the embodiment, the plant resistance inducer can activate the SA system, as well as the JA system and/or the ET system, which are involved in plant resistance induction.
The resistance inducer of the embodiment has been demonstrated in the examples described below to have the same or higher resistance induction activity as the existing resistance inducers ASM and PBZ. It is presumed that this excellent effect is achieved by a novel mechanism of action related to signal transduction. According to the resistance inducer of the embodiment, the SA system and the JA system and/or the ET system can be activated by one active ingredient.

The type of plant to which the resistance inducer is applied is not particularly limited as long as it is a plant that can acquire resistance by inducing the SAR (i.e., SA system) and the ISR (i.e., JA system and/or ET system), and may be a terrestrial plant or an aquatic plant. As a terrestrial plant, angiosperms or gymnosperms are preferred, and may be herbaceous or woody. As angiosperms, Rosaceae, Rutaceae, Vitaceae, Asteraceae, Orchidaceae, Liliaceae, Fabaceae, Poaceae, Rubiaceae, Euphorbiaceae, Cyperaceae, Umbelliferae, Lamiaceae, Cucurbitaceae, Solanaceae, and Brassicaceae are more preferred, and Brassicaceae are even more preferred.

An example of the Liliaceae plant is onion. An example of the Leguminosae plant is soybean. An example of the Apiaceae plant is carrot. An example of the Gramineae plant is rice, corn, barley, and wheat. An example of the Cucurbitaceae plant is melon, watermelon, winter melon, cucumber, and pumpkin. An example of the Solanaceae plant is tobacco, tomato, potato, eggplant, and green pepper. An example of the Brassicaceae plant is shepherd's purse, rapeseed, cabbage, kale, Chinese cabbage, turnip, radish, wasabi, and mustard.
Preferred plants for which the resistance inducer of the embodiment can be used include tomato, tobacco, cucumber, shepherd's purse, and rapeseed.

The resistance inducer of the embodiment may be mixed with an agriculturally acceptable carrier, filler, etc., as necessary, and provided in the form of a powder, tablet, granule, microgranule, etc. Alternatively, it may be mixed with an agriculturally acceptable solvent, surfactant, emulsifier, dispersant, etc., and provided in the form of an emulsion, liquid, suspension, wettable powder, water-soluble powder, oil, etc.

The solvent in which the plant resistance inducer is dissolved may be selected appropriately depending on the plant resistance inducer and the type of plant, but preferred examples of hydrophilic solvents include sulfoxide compounds such as dimethyl sulfoxide (DMSO) and amide compounds such as N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc) and N-methylpyrrolidone (NMP).

The resistance inducer of the embodiment may be provided in a formulation that can be used in combination with other agricultural and horticultural agents.
For example, the resistance inducer of the embodiment may be provided in the form of a combination drug, a combined preparation, or the like, containing the resistance inducer of the embodiment together with other JA-based resistance inducers such as bestatin, hexanoic acid, arachidonic acid, N-acylamide, etc.
Furthermore, for example, the resistance inducer of the embodiment may be provided in the form of a combination drug, a combined preparation, or the like, together with other SA-based resistance inducers such as probenazole, acibenzolar-S-methyl, or the like.

The pathogen to be protected against by use of the resistance inducer of the embodiment is not particularly limited, but is preferably a pathogen that induces SAR or ISR induction or that can be protected against by the induction of SAR or ISR. Or, it is preferably a pathogen that induces SAR or ISR induction and can be protected against by the induction of SAR or ISR. From this perspective, it is more preferable that the pathogen to be protected against by the resistance inducer of the embodiment is a living parasitic pathogen (also simply referred to as a "living parasitic fungus") or a saprophytic pathogen (also referred to as a "necrophytic parasitic fungus").

According to the embodiment, the resistance inducer can induce plant resistance against both live parasitic fungi and necrotic parasitic fungi. In addition, the resistance inducer according to the embodiment preferably does not have antibacterial activity against live parasitic fungi and necrotic parasitic fungi.
Examples of parasitic fungi include various parasitic plant pathogenic fungi (such as Colletotrichum higginsianum, which causes anthracnose in the Brassicaceae family, Puccinia graminis, Erysiphe graminis, Ustilago maydis, and Magnaporthe grisea), various parasitic plant pathogenic bacteria, and various plant viruses.

In particular, fungi of the genus Colletotrichum are suitable examples of pathogens that can be protected against by using the resistance inducer of the embodiment.

Mortality-infesting fungi include Botrytis cinerea, Colletotrichum atramentarium, Colletotrichum lagenarium, Phytophthora infestans, Gaeumannomyces graminis, Erwinia carotovora, Diplocarpon rosae, Valsa ceratosperma, Cryphonectria parasitica, Claviceps purpurea, Alternaria alternata, Mycosphaerella pinodes, Cochliobolus miyabeanus, Stemphylium Examples of pathogenic fungi include Sclerotinia sclerotiorum, Monilinia sp., Passalora fulva, Fusarium oxysporum f. sp. cucumerinum, Fusarium oxysporum, Raffaelea quercivora, Penicillium digitatum, Penicillium italicum, Xanthomonas campestris pv. citri, and Ralstonia solanacearum. Among them, Alternaria alternata and Botrytis cinerea are representative examples of parasitic fungi.

Among them, as pathogens to be protected by using the resistance inducer of the embodiment, preferred examples include fungi of the genus Alternaria, which are necroparasitic fungi, and fungi of the genus Botrytis, such as Botrytis cinerea, Botrytis byssoidea, Botrytis squamosa, and Botrytis allii, which are necroparasitic fungi, and fungi of the genus Botrytis are more preferred. Among them, preferred examples include Alternaria alternata of the genus Alternaria and Botrytis cinerea of the genus Botrytis, and particularly preferred examples include tomato gray mold fungus (Botrytis cinerea) and cucumber gray mold fungus (Botrytis cinerea).
Necrotrophic parasites also include saprophytic pathogens that become conditionally saprophytic depending on the surrounding environment.

There are no particular limitations on the pests and other living organisms that are the target of control using the resistance inducer of the embodiment. As described above, the ISR system induces a defense response against "damage" such as feeding damage by pests. Therefore, the resistance inducer of the embodiment can be applied as a control agent to a wide range of pests and other living organisms, such as insects and mites that feed on plants.

Examples of living organisms such as pests that can be controlled by using the resistance inducer of the embodiment include Coleoptera pests such as the azuki bean weevil (Callosobruchus chinensis), Lepidoptera pests such as the diamondback moth (Plutella xylostella) and the cabbage white butterfly (Pieris rapae), Diptera pests such as the house fly (Musca domestica) and the melon fly (Dacus cucurbitae), Hemiptera pests such as the green stink bug (Nezara antennata), Thripida pests such as the western flower thrips (Frankliniella occidentalis), Orthoptera pests such as the migratory locust (Locusta migratoria), Blattella pests such as the German cockroach (Blattella germanica), and Dermatophagoides farinae (Dermatophagoides These include various agricultural pests such as acarina pests such as Acarina farinae, and nematodes such as Meloidogyne incognita. Examples of forest pests include Coleoptera pests, such as Platypus quercivorus and other Platypodidae pests, and Scolytidae pests such as Tomicus piniperda, as well as Mochamus pests, which include Mochamus alternatus and M. saltuaris.

As one embodiment, the present invention provides a method for inducing resistance, which comprises contacting a target plant with a compound represented by the above general formula (1) or a salt thereof.
As one embodiment, the present invention provides a compound represented by the above general formula (1) or a salt thereof for inducing resistance in a plant.
As one embodiment, the present invention provides use of a compound represented by the above general formula (1) or a salt thereof for inducing resistance in a plant.
As one embodiment, the present invention provides use of a compound represented by the above general formula (1) or a salt thereof for producing a plant defense inducer.

The resistance inducer can induce resistance in a target plant by contacting the plant with an effective amount of the resistance inducer.
As a method for applying the resistance inducer, the method for contacting an effective amount with a plant may be the same as that for known inducers, and examples thereof include a method for spraying the resistance inducer on the soil in which the plant is growing, a method for mixing the resistance inducer with the soil, a method for irrigating the soil, a method for applying or spraying a resistance inducer solution in which the resistance inducer is dissolved on the plant, a method for growing the plant in the resistance inducer solution, a method for mixing the resistance inducer into a hydroponic solution, etc. Among these, a method for contacting an effective amount of the resistance inducer with the roots of the plant is preferred, and a treatment method for applying the resistance inducer to the soil in which the plant is grown or the hydroponic solution in which the plant is grown is exemplified, and irrigation treatment is preferred.

In the plant resistance induction method of the embodiment, the part of the plant body to which the resistance inducer is treated or administered is not particularly limited. For example, the resistance inducer may be sprayed on all of the leaves, stems, and roots of the plant body, or only on some of the leaves, stems, or roots. Even if the entire plant body is not sprayed, secondary metabolites produced in the sprayed part may reach the necessary parts of the plant body, and resistance may be acquired even in the parts that are not sprayed. Resistance may also be acquired by infiltrating the plant body from the root system by soil treatment, immersion treatment, irrigation treatment, etc.

The amount of resistance inducer used can be adjusted appropriately depending on the type of resistance inducer and plant. When treating the soil by spraying, mixing or irrigating the resistance inducer, the amount of active ingredient used per application can be, for example, 1-20 kg/10a, 1-10 kg/10a, or 1-1.3 kg/10a, and the agent can be used once a year or multiple times as necessary during the period from when the plant germinates to when it is harvested. When used multiple times, it is preferable to use the agent 2-6 times a year, or 1-3 times a month.

In addition, when the solution of the resistance inducer is applied or sprayed onto the stems and leaves of the plant, the concentration of the compound represented by the general formula (1) or its salt contained in the solution of the resistance inducer is preferably 0.1 to 500 μM, more preferably 1 to 500 μM, more preferably 1 to 300 μM, more preferably 1 to 100 μM, more preferably 1 to 10 μM, even more preferably 1 to 5 μM, and in another aspect, 10 to 50 μM is preferable. For example, the amount of the solution of the resistance inducer having a concentration of 0.1 to 500 μM or 1 to 500 μM used per time is 1 to 1000 μL per leaf, and it can be used once a year or multiple times as necessary during the period from when the plant germinates to when it is harvested. When used multiple times, it is preferable to use it 2 to 6 times a year, or 1 to 3 times a month.

When the plant is grown in a solution of the resistance inducer, such as by irrigation or hydroponics, the concentration of the compound represented by the general formula (1) or its salt contained in the solution of the resistance inducer is preferably 0.1 to 500 μM, more preferably 1 to 500 μM, more preferably 1 to 300 μM, even more preferably 1 to 100 μM, more preferably 1 to 10 μM, even more preferably 1 to 5 μM, and in another aspect, 10 to 50 μM is preferable. For example, the amount of a solution of the resistance inducer having a concentration of 0.1 to 500 μM or 1 to 500 μM used per time is 1 to 1000 μL per plant body, and the solution can be used once a year or multiple times as necessary during the period from when the plant germinates to when it is harvested. When used multiple times, it is preferable to use the solution 2 to 6 times a year, or 1 to 3 times a month.

The timing of application of the resistance inducer can be any time when the plant is sown, transplanted, or planted. It can also be applied at any stage of growth, be it a seed, sprout, young plant, or mature individual. For example, it can be applied 1 to 3 times between 20 days after germination and 14 days before harvest.

The resistance inducer of the embodiment may be used in combination with other agricultural and horticultural agents. The resistance inducer and the other agricultural and horticultural agents may be used simultaneously or separately.
For example, the plant resistance inducer of the embodiment may be used in combination with other JA-based plant resistance inducers such as bestatin, hexanoic acid, arachidonic acid, and N-acylamide.
Furthermore, for example, the resistance inducer of the embodiment may be used in combination with other SAR resistance inducers such as probenazole and acibenzolar-S-methyl.

In this specification, "combined use" means applying multiple types of pesticides to the same plant individual.

The resistance inducer may be contacted with the plant body after the emergence of pests or the onset of plant disease. The resistance inducer may also be used prophylactically, and may be contacted with the plant body before the emergence of pests or the onset of plant disease.

Compared to conventional JA-based resistance inducers, the resistance inducer of the embodiment is capable of inducing good resistance by applying the active ingredient at a low concentration.

According to the embodiment of the resistance inducer, the SA system, JA system and/or ET system involved in the induction of plant resistance can be activated. The resistance inducer of the embodiment can be suitably used in the method for inducing plant resistance described in the above embodiment.

<Biostimulant>
The plant resistance inducer of the embodiment can also be used as a biostimulant. The biostimulant of the embodiment contains a compound represented by the following general formula (1) or a salt thereof as an active ingredient.

[In formula (1),
R ¹ , R ² and R ³ each independently represent a linear or branched alkyl group having 1 to 4 carbon atoms, a hydroxyl group, a halogen atom, or a linear or branched alkenyl group having 2 to 4 carbon atoms;
R ⁴ represents a hydrogen atom, a linear or branched alkyl group having 1 to 4 carbon atoms, or a linear or branched alkenyl group having 2 to 4 carbon atoms;
^R5 and ^R6 each independently represent a hydrogen atom, a linear or branched alkyl group having 1 to 4 carbon atoms, a linear or branched alkenyl group having 2 to 4 carbon atoms, or a halogen atom.
n1 represents the number of ^R1 and is an integer of 0 to 5, and when n1 is 2 or more, ^R1 may be the same or different from each other. n2 represents the number of ^R2 and is an integer of 0 to 5, and when n2 is 2 or more, ^R2 may be the same or different from each other. n3 represents the number of ^R3 and is an integer of 0 to 3, and when n3 is 2 or more, ^R3 may be the same or different from each other.

As used herein, the term "biostimulant" refers to a substance that has the effect of increasing the production and/or accumulation of secondary metabolites in a plant when applied to the plant.
The examples described below suggest that the compound represented by the above general formula (1) or a salt thereof contributes to the activation of multiple plant hormone synthesis pathways. That is, the compound represented by the above general formula (1) or a salt thereof can activate a wide range of biosynthetic pathways and improve the synthesis of various secondary metabolites.

Secondary metabolites include alkaloids and terpenoids, which can be used as active ingredients in herbal medicines and traditional Chinese medicine preparations.

According to the embodiment of the biostimulant, for example, by applying it to a raw material plant of a herbal medicine preparation or a herbal medicine preparation, it is possible to increase the production and/or accumulation amount of the active ingredient, which is a target secondary metabolite.
The raw plant material is not particularly limited, but includes plants such as liquorice, ginseng, ginger, and cinnamon.

The methods of use and formulations of biostimulants can be similar to those described above for the plant resistance inducer, so detailed explanation will be omitted.

The biostimulant of the embodiment may be provided in a formulation that can be used in combination with other agricultural and horticultural agents.
For example, the biostimulant of the embodiment may be provided in the form of a combination drug, a combined preparation, or the like, containing the biostimulant of the embodiment and another known compound having a biostimulant effect.

The biostimulant of the embodiment may be used in combination with other agricultural and horticultural agents. The biostimulant and the other agricultural and horticultural agents may be used simultaneously or separately.
For example, the biostimulant of the embodiment may be used in combination with other known compounds having a biostimulant effect.

The biostimulant of the embodiment has an effect of improving the production and/or accumulation of secondary metabolites. The manifestation of the biostimulant effect in plants can be determined, for example, by the following indexes.
The amount of secondary metabolites produced in the plant body is used as an indicator, and by comparing plants treated with the biostimulant of the embodiment with untreated plants, the manifestation of the biostimulant effect can be determined if the amount of secondary metabolites produced in the plant body is significantly increased in the plants treated with the biostimulant of the embodiment.

In one embodiment, the present invention provides a method for promoting the production of secondary metabolites, comprising contacting a plant with a biostimulant containing, as an active ingredient, a compound represented by the above general formula (1) or a salt thereof. Such a method can be exemplified by the same method as that described in the method for contacting a plant with a plant resistance inducer, and therefore a description thereof will be omitted.

As one embodiment, the present invention provides a method for producing a secondary metabolite, which comprises contacting a compound represented by the above general formula (1) or a salt thereof with a target plant.
As one embodiment, the present invention provides a method for improving the production of a secondary metabolite, which comprises contacting a compound represented by the above general formula (1) or a salt thereof with a target plant.

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

<Selection of compounds that induce VSP1 gene expression>
First, in accordance with the results shown in the literature (Utsugi et al. (1998) Plant Mol Biol 38:565-576; Guerineau et al. (2003) J Exp Bot 54:1153-1162), the promoter sequence of the Vegetative Storage Protein 1 (VSP1) gene was amplified by PCR from the genomic DNA of Arabidopsis thaliana (Arabidopsis thaliana ecotype Columbia). The promoter sequence of the VSP1 gene was linked upstream of Fluc in a plasmid (pBI221-Fluc) carrying the gene sequence of a reporter gene (firefly luciferase gene (FLuc)), to obtain the pBI121-VSP1::Fluc plasmid. The plasmid was introduced into Arabidopsis thaliana via Agrobacterium tumefaciens LBA4404 to obtain a transformed Arabidopsis thaliana having VSP1::Fluc. Seeds of the transformed Arabidopsis thaliana were sown in a multi-well plate and germinated in an aqueous solution of luciferin.

A dimethyl sulfoxide (DMSO) solution of each compound from a commercially available compound library was prepared, and the solution was added to each well to give a compound concentration of 30 μM, and the transgenic Arabidopsis seedlings were treated with the solution. The plants were grown at 22°C under a photoperiod of 12 hours dark/12 hours light (70 μmol m ^-2 S ^-1 ).

A photocounting device (ARGUS system, Hamamatsu Photonics) and software (AQUACOSMOS, Hamamatsu Photonics) were used to measure the luminescence intensity in each well and the expression level of the reporter Fluc, thereby evaluating the VSP1 gene expression-inducing activity of each compound.

As a result, it was revealed that the expression of Fluc protein was induced in the transformed Arabidopsis thaliana VSP1::Fluc by treatment with the compound represented by the formula (1-2) (molecular weight 356.4). The compound represented by the formula (1-2), 2-{2-[(1,3-dioxaindan-5-yl)methylidene]hydrazin-1-ylidene}-1,2-diphenylethan-1-one, is hereinafter referred to as "compound X".
In the following, compound X used was a compound commercially available from Enamine.

<Evaluation of the effect of Compound X on the JA line of 1-week-old Arabidopsis thaliana plants>
The same experiment as the above selection experiment was performed on 1-week-old Arabidopsis plants (n = 24). Compound X was prepared in DMSO, and each solution was added to each well so that the concentrations of compound X were 1 μM, 5 μM, 30 μM, and 100 μM, respectively, and evaluation was performed on a group treated with transformed Arabidopsis (compound X group) and a group treated with DMSO only instead of the DMSO solution of compound X (DMSO group). In addition to the compound X group and DMSO group, evaluation was also performed on a group treated with 10 μM MeJA in DMSO instead of the DMSO solution of compound X (MeJA group).

The results are shown in Figure 2. In the transformed Arabidopsis VSP1::Fluc plants treated with compound X, a significant increase in the luminescence intensity of the Fluc protein was confirmed by 72 hours after treatment. On the other hand, in the Arabidopsis VSP1::Fluc plants treated with DMSO, no luminescence from the Fluc protein was detected. This suggests that compound X has the activity of inducing the expression of the VSP1 gene even in 1-week-old Arabidopsis plants.
In addition, when comparing compound X and MeJA, the effect of compound X on VSP1 gene expression activity was exerted slightly later than that of the MeJA group. However, the effect of compound X on VSP1 gene expression activity continued for a longer period than that of MeJA. Furthermore, compound X was able to induce high VSP1 gene expression activity at all concentrations, and showed high activity even at very low concentrations. In other words, compound X was found to have an excellent resistance induction effect.

<Evaluation of the effect of Compound X on the JA line of 4-week-old Arabidopsis thaliana plants>
Solid media were prepared by mixing compound X to a concentration of 50 μM with MS medium, or by mixing only DMSO with MS medium instead of compound X. Four-week-old Arabidopsis thaliana VSP1::Fluc individuals (n = 24) grown on MS solid medium were replanted therein and allowed to absorb compound X through the roots. The time immediately after replanting was set as 0 hours, and VSP1 activity was observed over a time course from 24 to 168 hours after replanting in the same manner as above.

The results are shown in Figure 3. A significant increase in the luminescence intensity of Fluc protein was confirmed by application of compound X, and it was confirmed that compound X exhibits VSP1 gene expression-inducing activity even in mature individuals. It was also suggested that compound X is highly efficiently absorbed from the roots, suggesting that irrigation is a suitable application method.

<Evaluation of the effect of compound X on the SA system and ET system in 4-week-old Arabidopsis thaliana plants>
In accordance with the contents of the literature (Ono S, Kusama M, Ogura R, Hiratsuka K (2011) Evaluation of the use of the tobacco PR-1a promoter to monitor defense gene expression by the luciferase bioluminescence reporter system. Biosci Biotechnol Biochem 75: 1796-1800), a plasmid was obtained that had a fusion gene (PR-1a::F-luc) in which the firefly luciferase gene (F-luc) was linked downstream of the tobacco-derived Pathogenesis-related gene 1a (PR-1a) gene promoter. The plasmid was introduced into Arabidopsis thaliana via Agrobacterium tumefaciens LBA4404, and transformed Arabidopsis thaliana PR-1a::F-luc carrying PR-1a::F-luc was obtained.

In addition, in accordance with the contents of the literature (Minami T, Tanaka T, Takasaki S, Kawamura K, Hiratsuka K, In vivo bioluminescence monitoring of defense gene expression in response to treatment with yeast cell wall extract. Plant biotechnol. 28, 481-484, 2011), a plasmid was obtained that had a fusion gene (PDF1.2::F-luc) in which the firefly luciferase gene was linked downstream of the Arabidopsis thaliana-derived PDF1.2 gene promoter. The plasmid was introduced into Arabidopsis thaliana via Agrobacterium (Agrobacterium tumefaciens LBA4404), and transformed Arabidopsis thaliana PDF1.2::F-luc was obtained.

Solid medium was prepared by mixing compound X to a concentration of 50 μM with MS medium, or by mixing only DMSO with MS medium instead of compound X. Four-week-old Arabidopsis thaliana PR-1a::F-luc or PDF1.2::F-luc individuals (n = 24) grown on MS solid medium were replanted therein and allowed to absorb compound X through the roots. The time immediately after replanting was set as 0 hours, and VSP1 activity was observed over time from 24 to 168 hours after replanting in the same manner as above.

The results are shown in Figures 4A and 4B. A significant increase in the luminescence intensity of Fluc protein was confirmed by the application of compound X, suggesting that compound X acts not only on the JA system, but also on the SA and ET systems.

<Verification of disease suppression effect against gray mold>
To investigate the usefulness of the compound as a resistance inducer, we examined its effect of suppressing the onset of Botrytis cinerea in mature Arabidopsis plants. Botrytis cinerea is a necrotic parasite.
A DMSO solution of Compound X (50 μM), a DMSO solution of MeJA (50 μM), and DMSO were poured onto the soil surface of 4-week-old wild-type Arabidopsis plants (n=6) grown in cultivation soil. After 120 to 144 hours, a spore suspension (1×10 ⁵ spores/mL) was dripped onto the leaves of the Arabidopsis plants, and lesions were measured 72 hours later.

The results are shown in Figure 5. The graph shows the maximum lesion diameter 72 hours after inoculation. Only when Compound X was applied by irrigation, a high inhibitory effect on Botrytis cinerea was observed.

<Verification of the disease suppression effect against anthrax bacteria>
In addition, the inhibitory effect on anthracnose of Compound X was examined by applying the same method as that for the inhibitory effect on gray mold described above. Anthracnose is a parasitic fungus.
A DMSO solution of Compound X (50 μM), a DMSO solution of ASM (50 μM), and DMSO were poured onto the soil surface of 4-week-old wild-type Arabidopsis plants (n=6) grown in cultivation soil. Then, 96 to 144 hours later, a spore suspension (1×10 ⁵ spores/mL) was dripped onto the leaves of the Arabidopsis plants, and lesions were measured 144 hours later.

The results are shown in Figure 6. The graph shows the maximum lesion diameter 144 hours after inoculation. Compound X showed an inhibitory effect similar to or greater than that of ASM, a representative resistance inducer, demonstrating excellent effectiveness in inhibiting the infection and proliferation of anthrax bacteria.

<Verification of the difference in induction patterns between JA and SA systems>
Many papers have reported that the JA and SA systems act antagonistically to each other. However, the above examples of the present invention suggest that compound X acts not only on the JA system but also on the SA system.
We therefore investigated the pattern in which these multiple pathways are induced by compound X. Using wild-type Arabidopsis seedlings and mature plants, the expression levels (mRNA levels) of the JA-based VSP1 gene and the SA-based PR1 gene were quantified by real-time PCR.

Unless otherwise specified, the treatment with Compound X was carried out in the same manner as above, by replanting Arabidopsis thaliana on solid medium prepared by mixing Compound X with MS medium, and the time immediately after replanting was designated as 0 hours of treatment. The same was true for the treatment of the other control groups (MeJA group and DMSO group).

The base sequences of the real-time PCR primer set used for quantifying the expression level of the Arabidopsis VSP1 gene are as follows.
F: 5'-TCGAAGTTGACGCAAGTGGT-3' (SEQ ID NO: 4)
R: 5'-GGGGACAATGCCATGAAGAT-3' (SEQ ID NO: 5)

The base sequences of the real-time PCR primer set used for quantifying the expression level of the Arabidopsis PR1 gene are as follows:
F: 5'-CTAACTACAACTACGCTGCGAAC-3' (SEQ ID NO: 6)
R: 5'-TTCATTAGTATGGCTTCTCGTTCA-3' (SEQ ID NO: 7)

The results for seedlings are shown in Figures 7A-B. Expression of VSP1 increased 48 hours after treatment with compound X, peaked at 96 hours, and then showed an induction pattern in which activity decreased. On the other hand, PR1 was highly expressed 144 hours after treatment.

The results for mature individuals are shown in Figures 8A and 8B. VSP1 was highly expressed 48 hours after treatment. On the other hand, PR1 expression increased from 48 hours after treatment and reached a peak at 96 hours.
These results suggested that although there was a time lag between the peak expression in seedlings and mature plants, at both growth stages, compound X activated the JA system at an early stage and activated the SA system at a later stage while the activity of the JA system decreased.

Because compound X has this JA-SA induction pattern, it is believed to have exhibited excellent control effects against both Botrytis cinerea and Anthracnose fungus.

<Verification of induction patterns of the ET system>
Similarly, expression of the PDF1.2 gene of the ET system was quantified by real-time PCR.

The base sequences of the real-time PCR primer set used for quantifying the expression level of the Arabidopsis thaliana PDF1.2 gene are as follows:
F: 5'-TTTGCTTTCGACGCAC-3' (SEQ ID NO: 8)
R: 5'-CGCAAACCCCTGACCATG-3' (SEQ ID NO: 9)

The results are shown in Figures 9A and 9B. The graph in Figure 9A shows the results for seedlings, and the graph in Figure 9B shows the results for mature individuals. At both growth stages, PDF1.2 was confirmed to be highly expressed 144 hours after treatment with Compound X. This suggests that Compound X activates the ET system at a later stage.

<Evaluation of antibacterial activity of compound X>
In order to confirm the antibacterial activity of compound X itself, an inhibition zone method was used to evaluate the antibacterial activity against Botrytis cinerea and Anthracnose fungus. Botrytis cinerea is a representative saprophytic pathogen, and Anthracnose fungus is a representative parasitic pathogen.

100μl (3.0× ¹⁰⁵ spores/ml) of gray mold fungus (B. cinerea) were applied to PSA medium, and then treated with 10μl (100mM) of compound X solution. 10μl (100mM) of hygromycin was used as a positive control, and DMSO was used as a negative control. This was cultured at 24℃ for 5 days.

100μl (1.0× ¹⁰⁵ spores/ml) of C. higginsianum were applied to PDA medium and treated with 10μl (100mM) of compound X solution. 10μl (100mM) of hygromycin was used as a positive control, and DMSO was used as a negative control. This was cultured in the dark at 24℃ for 10 days.

The results are shown in Figures 10A-B. The test results revealed that compound X did not exhibit antibacterial activity against Botrytis cinerea and Anthracnose fungus, even when treated at a high concentration of 100 mM. Compounds that do not have antibacterial activity are less likely to produce drug-resistant bacteria. Therefore, compound X in the plant resistance inducer of the embodiment is excellent in that it is less likely to produce drug-resistant bacteria and can be expected to be used for a long period of time.

Compound X, unlike existing resistance inducers, activated not only the JA system but also the SA and ET systems, and exerted clear resistance to both Botrytis cinerea (a necrotic parasite) and Bacillus anthracnose (a live parasite), demonstrating that compound X can induce resistance to a wide range of pathogens.
Furthermore, compound X was able to effectively induce resistance even when applied at a low concentration.

The effectiveness of Compound X was equal to or greater than that of ASM and PBZ, which are representative resistance inducers, demonstrating its extremely high effectiveness and usefulness as a resistance inducer.

The configurations and combinations thereof in each embodiment are merely examples, and additions, omissions, substitutions, and other modifications of the configurations are possible without departing from the spirit of the present invention. Furthermore, the present invention is not limited to each embodiment, but is limited only by the scope of the claims.

Claims (7)

A method for inducing resistance in a plant, comprising the steps of:
The method comprises applying a plant resistance inducer to a plant to activate a salicylic acid signaling system and a jasmonic acid and/or ethylene signaling system;
The plant resistance inducer comprises, as an active ingredient, a compound represented by the following general formula (1) or a salt thereof:

[In formula (1),
R ¹ , R ² and R ³ each independently represent a linear or branched alkyl group having 1 to 4 carbon atoms, a hydroxyl group, a halogen atom, or a linear or branched alkenyl group having 2 to 4 carbon atoms;
R ⁴ represents a hydrogen atom, a linear or branched alkyl group having 1 to 4 carbon atoms, or a linear or branched alkenyl group having 2 to 4 carbon atoms;
^R5 and ^R6 each independently represent a hydrogen atom, a linear or branched alkyl group having 1 to 4 carbon atoms, a linear or branched alkenyl group having 2 to 4 carbon atoms, or a halogen atom.
n1 represents the number of ^R1 and is an integer of 0 to 5, and when n1 is 2 or more, ^R1 may be the same or different from each other. n2 represents the number of ^R2 and is an integer of 0 to 5, and when n2 is 2 or more, ^R2 may be the same or different from each other. n3 represents the number of ^R3 and is an integer of 0 to 3, and when n3 is 2 or more, ^R3 may be the same or different from each other. (delete) The method of claim 1, which induces plant resistance to both live and necroparasitic fungi. The method according to claim 1 or 3, which has no antibacterial activity against live parasitic bacteria and non-live parasitic bacteria. 5. The method of claim 1, 3 or 4, wherein said applying comprises contacting a root of a plant with an effective amount of said plant resistance inducer. A plant resistance inducer comprising, as an active ingredient, a compound represented by the following general formula (1) or a salt thereof:

[In formula (1),
R ¹ , R ² and R ³ each independently represent a linear or branched alkyl group having 1 to 4 carbon atoms, a hydroxyl group, a halogen atom, or a linear or branched alkenyl group having 2 to 4 carbon atoms;
R ⁴ represents a hydrogen atom, a linear or branched alkyl group having 1 to 4 carbon atoms, or a linear or branched alkenyl group having 2 to 4 carbon atoms;
^R5 and ^R6 each independently represent a hydrogen atom, a linear or branched alkyl group having 1 to 4 carbon atoms, a linear or branched alkenyl group having 2 to 4 carbon atoms, or a halogen atom.
n1 represents the number of ^R1 and is an integer of 0 to 5, and when n1 is 2 or more, ^R1 may be the same or different from each other. n2 represents the number of ^R2 and is an integer of 0 to 5, and when n2 is 2 or more, ^R2 may be the same or different from each other. n3 represents the number of ^R3 and is an integer of 0 to 3, and when n3 is 2 or more, ^R3 may be the same or different from each other. A biostimulant comprising a compound represented by the following general formula (1) or a salt thereof as an active ingredient:

[In formula (1),
R ¹ , R ² and R ³ each independently represent a linear or branched alkyl group having 1 to 4 carbon atoms, a hydroxyl group, a halogen atom, or a linear or branched alkenyl group having 2 to 4 carbon atoms;
R ⁴ represents a hydrogen atom, a linear or branched alkyl group having 1 to 4 carbon atoms, or a linear or branched alkenyl group having 2 to 4 carbon atoms;
^R5 and ^R6 each independently represent a hydrogen atom, a linear or branched alkyl group having 1 to 4 carbon atoms, a linear or branched alkenyl group having 2 to 4 carbon atoms, or a halogen atom.
n1 represents the number of ^R1 and is an integer of 0 to 5, and when n1 is 2 or more, ^R1 may be the same or different from each other. n2 represents the number of ^R2 and is an integer of 0 to 5, and when n2 is 2 or more, ^R2 may be the same or different from each other. n3 represents the number of ^R3 and is an integer of 0 to 3, and when n3 is 2 or more, ^R3 may be the same or different from each other.

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